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The role of endogenous H2S production during hibernation and forced hypothermiaDugbartey, George Johnson

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The role of endogenous H2S production during hibernation and forced hypothermia:

towards safe cooling and rewarming in clinical practice

George Johnson Dugbartey

Studies presented in this thesis were financially supported by

Groningen University Institute for Drug Exploration (GUIDE)University Medical Centre Groningen (UMCG)

Printing of this thesis was financially supported by

University of Groningen University Medical Centre Groningen (UMCG)

Groningen University Institute for Drug Exploration (GUIDE)

Cover Design and thesis lay out

MidasMentink.nl

Printed by Gildeprint – Enschede, The Netherlands

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ISBN (printed): 978-90-367-7741-4

ISBN (digital): 978-90-367-7740-7

Copyright © 2015 by George Dugbartey

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted,

in any form or by any means without prior written permission of the author.

The role of endogenous H2S production during hibernation and forced hypothermia:

towards safe cooling and rewarming in clinical practice

PhD thesis

to obtain the degree of PhD at the

University of Groningen

on the authority of the

Rector Magnificus Prof. E. Sterken

and in accordance with

the decision by the College of Deans.

This thesis will be defended in public on

Wednesday 1 July 2015 at 11.00 hours

By

George Johnson Dugbartey

born on 3 April 1982

in Tema, Ghana

Supervisor:

Prof. R.H. Henning

Co-supervisor:

Dr. H.R. Bouma

Assessment committee:

Prof. H. van Goor

Prof. B.A. Yard

Prof. E.A. van der Zee

Paranymphs:

M.N. Tolouee

V.A. Reitsema

This book is lovingly dedicated to the man and woman who gave me life (Jonathan and Hannah)

the man and woman who kept my feet on the path of knowledge (Ray and Joycelyn)my twin sister with whom I came into this world (Georgina)

my brother who has always been there for me (Hayford)

CONTENTS

Chapter 1 Introduction and aims of the thesis 9

Chapter 2 Reduction of body temperature governs neutrophil retention in hibernating and non-hibernating animals by margination

27

Chapter 3 Endogenous H2S production is crucial in maintaining torpor-arousal cycles and preserving renal integrity in the hibernating Syrian hamster

45

Chapter 4 Induction of a torpor-like state by 5’-AMP: a role for endogenous H2S production?

65

Chapter 5 Dopamine treatment attenuates renal injury via production of H2S in a rat model of deep hypothermia and rewarming

79

Chapter 6 General discussion 101

Chapter 7 Nederlandse samenvatting 113

Acknowledgements 121

Bibliography 127

1Introduction

10

Hypothermia and its clinical applicationsHypothermia is a life-threatening condition in which core body temperature (Tb) drops below the desired temperature range maintained by thermoregulation. As a proper body temperature is required for normal metabolism and homeostasis, prolonged and servere hypothermia may lead to death (Fritsch, 1995; Jurkovich, 2007; Yoon et al., 2014). Hypothermia may be induced intentionally as a therapeutic approach to specific clinical situations or occur accidentally. Depending on its severity, hypothermia can be classified as mild (32-35 °C), moderate (28-32 °C), severe (20-28 °C) and deep (< 20 °C ) (Darocha et al., 2014; Ding et al., 2014; Jarosz et al., 2014; Turtiainen et al., 2014). Intentional hypothermia (also called therapeutic hypothermia) is induced as a therapeutic intervention aimed at preserving cells or organs and maximizing their function. It is very often encountered in transplantation, during which the donor organ is preserved in a hypothermic solution during transportation prior to transplantation. Therapeutic hypothermia is also applied in major surgery, post myocardial infarction, and the intensive care unit. Several human and animal studies reported beneficial effects of therapeutic hypothermia by reducing complications and conferring neuroprotection in patients with traumatic brain injury, ischemic stroke and neonates with hypoxic-ischemic encephalopathy, and in patients with myocardial infarction, hence improving human survival (Marion et al., 1997; Margarit et al., 2000; Kozaki et al., 2002; Pokorny et al., 2000; van der Worp et al., 2007; Polderman, 2008; van der Worp et al., 2010; Faridar et al., 2011; Granja et al., 2011; Polderman and Andrews, 2011; Jomaa et al., 2013).

On the other hand, accidental hypothermia is unintentional drop in Tb below 35 °C as a result of unanticipated cold exposure (Yoshitomi et al., 1998; Udayaraj et al., 2004; Turtiainen et al., 2014; Yoon et al., 2014). It can be subclassified as acute (also called immersion hypothermia) as a result of sudden exposure to cold such as immersion in cold water or a person caught in a winter storm. There is also exhaustion accidental hypothermia in which the cold exposure is associated with lack of food and exhaustion such that the body’s thermoregulatory system is impaired and unable to generate heat. Chronic hypothermia is another type of accidental hypothermia mainly affecting the elderly, and it is often encountered over days or weeks (Guly, 2011). Since ancient times, death from accidental hypothermia has been recognised but the clinical syndrome of hypothermia was only recognized in the mid-20th century and only in extreme conditions such as in acute accidental hypothermia. In the United Kingdom, for example, hypothermia in less extreme conditions was only recognised in the 1960s (Guly, 2011).

1.1. Cellular mechanisms of hypothermic injuryWhile hypothermia extends organ life, there is mounting evidence that it also induces cellular injury. Strom and Suthanthiran (1996) reported that hypothermic organ preservation inhibits

11

1hypothalamic enzymes, lowers pH and disrupts osmolarity, leading to phase transition of lipids and changes in membrane stability. Other authors also reported that exposure of organs ex vivo to hypothermic storage leads to marked disturbances in cellular ion homeostasis due to increase in membrane fluidity and permeability, and alters the function of membrane-bound enzymes (Hochachka, 1986; Belzer and Southard, 1988; Zachariassen, 1991). Hypothermia impairs proper functioning of ionic cell membrane pumps such as the Na+/K+-ATPase due to lack of ATP or excessive production of H+, which can contribute to disruption of cellular ion homeostasis (figure 1) (Priebe et al., 1996; Rolfe and Brown, 1997). This leads to an influx of Na+ and Cl- followed by an osmotic shift of water into the cell and efflux of K+, leading to cell swelling (Hochchka, 1986; Belzer and Southard, 1988; Marsh et al., 1989; Blankensteijn and Terpstra, 1991; Clavien et al., 1992; figure 1). Progressive cellular swelling is associated with necrotic cell death (Stahl and Gattone, 1985; Oberbauer et al., 1999; Castaneda et al., 2003; Jani et al., 2011). In addition, cellular swelling leads to interstitial expansion and edema, capillary compression and poor tissue perfusion (Belzer and Southard, 1988). If the hypothermia-induced Na+ accumulation continues unabated, this increase will eventually result in membrane depolarization and rapid influx of Ca2+ (Hochachka, 1986). The increase in cytosolic Ca2+ is deleterious and might lead to cellular apoptosis (White and Somero, 1982; Hochachka, 1986).

Recent studies indicate that hypothermic storage of organs is characterized by impairment of mitochondrial function also results in depletion of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), the major sources of cellular metabolic energy, due to reduction in the activity of mitochondrial enzymes (Boutilier, 2001; Salahudeen, 2004). This leads to a mismatch between ATP supply and demand. As levels of ATP decrease, this leads to failure of ATPases, followed by membrane depolarization and an uncontrolled influx of Ca2+ through voltage-gated Ca2+ channels. The rise in free cytosolic intracellular Ca2+ concentration is a major feature of cellular injury, and results in the activation of Ca2+-dependent phospholipases and proteases that further hasten the rate of membrane depolarization, leading to uncontrolled cellular swelling (figure 1) and, ultimately, to cell necrosis (Hochachka, 1986; Rolfe and Brand, 1996; Priebe et al., 1996; Rolfe and Brown, 1997; Plesnila et al., 2000). The Ca2+ overload leads to opening of mitochondrial permeability transition pore (MPTP), resulting in an increase in intracellular Ca2+, which increases further and more abruptly during rewarming (Piper et al., 1993; Salahudeen et al., 2003; Green and Kroemer, 2004).

A key feature of the hypothermia-induced mitochondrial swelling is the trigger of a number of pro-apoptotic events such as translocation of cytochrome c and increase in Bax-Bcl-2 ratio as well as increase in caspase-3 activity (Salahudeen et al., 2003; Green and Kroemer, 2004). Lactic acid generation during anaerobic metabolism due to hypothermia leads to

12

(intracellular) acidosis, which can result in lysosomal instability and might further impair mitochondrial function (Belzer and Southard, 1988). In summary, hypothermia induced impairment of ionic cell membrane pumps is also associated with mitochondrial dysfunction and activation of the mitochondrial apoptotic machinery.

1.2. Effects of hypothermia on organsHypothermia primarily affects all organs and organ systems. Recent studies indicate that hypothermia compromises the endothelial barrier function leading to further endothelial dysfunction (Jomaa et al., 2013). Hypothermia-induced damage negatively affects the vascular and barrier function of the endothelium by activating endothelial cells leading to expression of adhesion molecules which in turn enhances influx of leukocytes and thus, inflammation and tissue destruction (Kukan and Haddad, 2001; Scheinichen et al., 2003). In the transplantation setting, for example, this may lead to a decline in organ function and predispose the organ to functional impairment after transplantation, thereby jeopardizing a successful transplant. Transplantation of an injured allograft prior to implantation, may lead to both acute and chronic graft failure (Broman and Kallskog, 1995; Kukan and Haddad, 2001; Scheinichen et al., 2003). Thus, limiting organ injury by hypothermia during transplantation not only improves short-term graft survival, but also positively affects chronic rejection. Therefore, optimization of organ preservation might reduce the induction of graft injury during transplantation procedure and furthermore, might allow longer preservation times.

1.2.1. Kidney as a typical example of a vulnerable organ to hypothermia In the renal system, hypothermic preservation of kidneys for transplantation, for example, is associated with an increase in preglomerular vasoconstriction leading to a decrease in renal blood flow (RBF) and reduced glomerular filtration rate (GFR). Renal function is eventually decreased during hypothermia due to the effect of the cold on the kidney metabolism itself. As the RBF decreases, renal vascular resistance (RVR) rises, leading to a further decrease in RBF and a subsequent decrease in GFR (Broman and Kallskog, 1995; Li et al., 2013). During hypothermia, renal oxygen consumption is more rapidly reduced relative to other organs such as the liver, heart, brain, skeletal muscle, and skin (Mori et al., 2011). At the ionic level, potassium moves into the cells as Tb drops and patients may experience hypokalemia (Polderman et al., 2001). Other imbalances that have been described during hypothermia include hypomagnesemia, hypocalcemia, and hypophosphatemia (Polderman et al., 2001; Arpino and Greer, 2008).

Studies have shown that reduction in Tb to 28°C is accompanied by decreases in RBF and GFR by 50% and an increase in renal vascular resistance in anesthetized rats (Broman and Kallskog, 1995; Li et al., 2013), and in other species including man (Boylon and Hong, 1966).

13

1At these lowered temperatures, there is a corresponding reduction in the tubular secretion and reabsorption of ions and organic solutes along the nephron, as these are energy requiring processes and therefore temperature sensitive (Boylon and Hong, 1966). This, together with a decrease in tubular reabsorption of water, leads to hypothermia-induced natriuresis and diuresis. Thus, hypothermia leads to dehydration. The mechanisms for this cold-induced diuresis remain unclear. However, available data suggest that hypothermia-induced diuresis is an autoregulatory response of the kidney to a relative central hypervolemia induced by peripheral vasoconstriction (Chapman et al., 1975; Withey et al., 1976; Broman and Kallskog, 1995). Owing to a volume overload, the secretion and release of antidiuretic hormone (ADH) is suppressed (Broman et al., 1998). The subsequent hypothermia-induced diuresis decreases the blood volume so that a progressive hemoconcentration develops (Chapman et al., 1975). Hypothermia-induced diuresis is a major concern. For example, accidental hypothermia by cold water immersion has been shown to increase urinary output by 3.5-fold, and the subsequent decrease in body water may be a factor contributing to the ‘rewarming shock’ that occurs following active vasodilation induced by rewarming treatments (Cupples et al., 1980).

1.3. Reperfusion injury following hypothermiaRestoration of blood supply after hypothermic preservation or cold exposure of an organ or whole organism induces a cascade of events that further aggravates the hypothermia-induced injury. This key event is referred to as reperfusion injury (Perico et al., 2004; Tapuria et al., 2008; Zaouali et al., 2010). On reperfusion, oxygen becomes available and metabolism increases rapidly, resulting in a sudden production of reactive oxygen species (ROS) such as superoxide anions, hydroxyl radicals and hydrogen peroxide (Haugen and Nath, 1999). The cellular pathways to scavenge oxygen free-radicals are overwhelmed and cellular injury ensues. Thereby, restoration of blood supply results in oxygen supply in excess of the requirements, which (amongst others) leads to activation of macrophages in the vasculature and generation of ROS, which results in apoptotic cell death (Castaneda et al., 2003). Production of ROS leads to endothelial injury (Tapuria et al., 2008). Inflammatory cytokines and chemokines released during ROS generation, together with increased expression of adhesion molecules on the cell surface, attract neutrophils and monocytes, which in turn, release additional ROS and further potentiate the damage (Haugen and Nath, 1999; DeVries et al., 2003). Thus, reperfusion after hypothermic exposure of an organ or organism is associated with increased injury.

1.4. How nature deals with hypothermia and reperfusion: lessons from mammalian

hibernatorsDuring the hibernation season, often associated with winter, when food is scarce, hibernating

14

mammals cycle through phases of suppressed metabolism with low body temperature (torpor) that are interrupted intermittently by short periods of complete restoration of metabolism and body temperature (arousal) (Dausmann et al., 2004; Andrews, 2007; Bouma et al., 2010; Bouma et al., 2011; Talaei et al., 2011; figure 1). Torpor may occur daily with a reduction of body temperature to ~18-30°C for several hours (Geiser, 2004), or may be deep, during which body temperature of a typical hibernator might be reduced to temperatures as low as -2.9°C, lasting for several days to a month (depending on the ambient temperature and the species) (Andjus et at., 1964; Barnes, 1989; Kenagy et al., 1989; Hut et al., 2002; Heldmaier, 2004). Interestingly, available data from hibernating animals demonstrate that this repetitive cycles and the stress of torpor-arousal with a significantly reduced cardiac output and ventilation during torpor does not seem to cause significant organ damage in hibernators, as demonstrated by Zancanaro et al. (1999) and Talaei et al. (2012). Meanwhile, cooling and rewarming of organs of non-hibernating animals including humans generally results in substantial damage. Therefore, hibernation could represent a unique natural model of hypothermic organ preservation and rewarming in specific clinical situations such as in transplantation.

A.

B.

K+

ATP ATP

K+

Na+

H2O

Cl-

Cell swelling

Hypothermic storage

Ca2+

+Hypothermia

Na+ H+

Na+ Na+

Na+

K+

K+

ATP

Na+

Na+

K+

K+ Na+

Na+ K+

Na+ H+

ATP

+Torpor

Torpid state

No cellswelling

Cl-

Ca2+

H2O

Figure 1. Model of the hypothermic response in the cells of non-hibernating (A) and hibernating animals (B).(A) Hypothermic preservation impairs the Na+/K+-ATPase and also interrupts the normal balance between K+ efflux and the equivalent Na+ influx to the cell. This results in a net accumulation of Na+ and subsequent influx of Cl-, which creates a local increase intracellular osmolarity driving an inflow of water. Cellular and subcellular swelling ensues, followed by cellular edema. If left unabated, the accumulated intracellular [Na+] will eventually lead to membrane depolarization and subsequent opening of voltage-dependent Ca2+ channels and a rapid influx of Ca2+, which is deleterious to the cell. (B) Hibernating animals maintain cellular ion homeostasis in the cell during torpor. Therefore, cellular and subcellular swelling is absent.

15

1

1.5.0. Mechanisms of resistance against cooling-associated damage in hibernatorsWhereas the Na+/K+-ATPase is impaired during hypothermic preservation of organs from non-hibernating animals including humans, Willis et al. (1969) observed that the activity of the Na+/K+-ATPase is fully preserved during ex vivo cooling of organs taken from hibernating hamsters. In the kidney, for example, the number of Na+/K+-ATPase subunits and its hydrolytic activity remained unaffected in most segments of the nephron during deep torpor in the greater Egyptian jerboa (Bennis et al., 1995). Kamm et al. (1979) also reported that incubation of vascular smooth muscle cells from rats for 48 h at 7°C leads to a reduction in intracellular [K+] and a significant increase in cytosolic [Na+], thus suggesting dysfunction of Na+/K+-ATPase. In contrast, smooth muscle cells from hibernating ground squirrels were able to maintain their ionic integrity when incubated under the same conditions (Belzer and Southard, 1998). Wang et al. (1997) also observed increase in Ca2+ in cardiomyocytes of rats as temperature was lowered from 25°C to 7–12 °C. However, they observed a depressed Ca2+ influx in cardiac cells of the hibernating ground squirrel under the same condition, which evidently helped to prevent hypothermic Ca2+ overload and subsequently prevented cardiac injury. Thus, maintenance of the activity of Na+/K+-ATPase during torpor and arousal accounts for the reduced cell swelling and hence better cell survival in hibernating animals (Kamm et al., 1979; Wang et al., 1997).

 

Time (months)

Body

tem

pera

ture

( o C)

5

10

15

20

25

30

35

40

Dec Jan Feb Mar AprNov May

Summer  active Torpor Arousal

   

Figure 2. Diagram showing torpor-arousal cycle of Syrian golden hamster with respect to appearance and core body temperature (Tb). The graph shows a simulation of Tb tracings (solid line) of a Syrian hamster inside a climate controlled chamber. The dashed line represents the ambient temperature, which is lowered from 21°C to 5°C to induce hibernation and back to 21°C to end the torpor-arousal cycle. Tb dropped from ~37°C to ~7°C as ambient temperature was lowered. Periodic arousals between each torpor bout are associated with restoration of euthermic temperature of ~37°C despite constant ambient temperature. Photographs of hamsters at three different time points during the hibernation are shown above the graph.

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1.5.1. Mitochondria in hibernating animalsActivation of specific anti-apoptotic pathways might allow hibernators to preclude cell death despite the cellular stresses of torpor and arousal that are known to activate pro-apoptotic proteins in non-hibernating animals (e.g. nutrient deprivation, low body temperature, ischemia/reperfusion, oxidative damage etc.). Jani et al. (2011) observed no difference in caspase-3 activity in the kidneys of hibernating and summer active thirteen-lined ground squirrels. The number of mitochondria in torpid dormice remained unchanged as compared to summer active animals (Soria-Milla and Coca-Garcia, 1986). Whereas the synthesis and activity of mitochondrial cytochrome c oxidase subunit 1 (COX1) and other genes are reported to be markedly decreased in warm ischemia/perperfusion injury (IRI) of rat brain as well as in a cardiac model of IRI (Lesnefsky et al., 2004; Recay et al., 2009), protein expressions of COX1 and mitochondrial ATP synthase subunits 6 and 8 are significantly upregulated in the kidneys of thirteen-lined ground squirrels during torpor and return to euthermic levels upon arousal from torpor (Hittel and Storey, 2002; Smeek et al., 2011). The specific dynamics in COX1 and mitochondrial ATP synthase subunits in hibernators may thus confer mitochondrial tolerance and protection during hypothermia and reperfusion. Also, Carey et al. (2005) reported a larger increase in anti-apoptotic Bcl-xL expression (12-fold) relative to that of pro-apoptotic Bax (2-fold) in intestinal mucosa of 13-lined ground squirrels. This suggests that pro-apoptotic influences during hibernation are counteracted by increased expression of anti-apoptotic proteins, resulting in suppression, rather than activation of apoptotic pathway. Hence, it is seems likely that hibernating animals employ diverse mitochondrial anti-apoptotic mechanisms to counteract the pro-apoptotic machinery observed during hypothermic storage and reperfusion in non-hibernating animals.

1.5.2. Oxidative stress and antioxidant defense system during hibernationDespite the repetitive cycling between torpor and arousal with significant changes in tissue oxygenation and oxygen consumption, hibernators do not show gross signs of organ damage. Likely, hibernators activate an effective antioxidant defense system to combat injurious effects of oxidative stress upon arousal. Several reports demonstrate increased activity of antioxidant enzymes, including glutathione peroxidase, superoxide dismutase (Buzadzic et al., 1990), peroxiredoxin 3/6 and glutathione reductase in organs from hibernating thirteen-lined ground squirrels (Jani et al., 2012). Other authors also reported increased levels of extracellular catalase in the blood of hamsters (Ohta et al. (2006)) and ascorbate in plasma from 13-lined ground squirrels during torpor (Drew et al., 2002). In addition, Jani et al. (2012) reported an increase in the cytoprotective enzyme heme oxygenase-1 in the kidneys of torpid 13-lined ground squirrels. Taken together, the increase in the levels of antioxidant enzymes during hibernation suggests that hibernators employ substantial antioxidant defenses to protect cells and tissues against the damaging effects of oxidative stress under conditions of

17

1hypothermia, hypoperfusion and reperfusion.

1.6.0. Pharmacological considerations of hibernationSeveral pharmacological compounds can induce an artificial and reversible torpor-like state in non-hibernating animals, a condition generally referred to as suspended animation. Pharmacologically reducing the demand for oxygen is a promising strategy to minimize unavoidable hypoxia-induced injury such as ischemia-reperfusion injury during transplantation and may hold an enormous promise in diverse fields, including transplantation and major surgeries. However, the induction of a suspended animation-like state by drugs is still a considerable challenge and is associated with a dramatically increased rate of cardiac arrest in non-hibernators (Zhang et al., 2006). It has been observed that during hibernation, erythrocyte and organs have significantly lower ATP levels (Doherty et al., 1993; English and Storey, 2000). In particular, the ATP level of erythrocytes from hibernating thirteen-lined ground squirrels is ~50% of the level observed in a euthermic state (Doherty et al., 1993). Such a large decrease in ATP level in the erythrocyte would significantly compromise its function in regulating oxygen/carbon dioxide molecules necessary for maintaining high metabolic activity of the major organs, and therefore result in metabolic suppression of these organs. This implies that the metabolic pathways (e.g. anaerobic glycolysis, oxidative phosphorylation, fatty acid oxidation, etc.) that produce ATP are inhibited during hibernation. Under such conditions, it is highly possible that the metabolic rate of organs will slow down, and heat loss from the body to the environment will not be adequately controlled, hence hypothermia ensues. This reasoning is consistent with the hypothesis that decreased ATP production or utilization is involved in hibernating behavior (English and Storey, 2000; Heldmaier et al., 2004).

1.6.1. Hydrogen sulfide (H2S) induces a reversible hibernation-like stateFor several centuries, hydrogen sulfide (H2S) was known for its toxicity with a characteristic rotten-egg smell. It is the second cause of gas-related mortalities in industrial accidents after carbon monoxide. However, H2S can also be made at home by mixing bath additive and toilet detergent. In Japan, for example, Morii et al. (2008) reported 220 cases of home-made H2S poisoning within three months with 208 deaths. Contrary to the widely known toxicity of H2S, Kimura et al. (1996) discovered its biological importance as a signaling molecule. Since then, several researchers have reported the biological usefulness and therapeutic properties of H2S. H2S is a colorless, flammable and water-soluble gas. It is a specific and reversible inhibitor of cytochrome c oxidase, a key enzyme in the mitochondrial respiratory complex IV (Beauchamp et al., 1984). Inhibitors of the mitochondrial respiratory chain are toxic to mammals because they disrupt ATP production by oxidative phosphorylation. Inhibition of oxidative phosphorylation reversibly induces a state of profound hypometabolism (Padilla

18

and Roth, 2001; Nystul and Roth, 2004). H2S is produced primarily by two cytosolic enzymes (cystathionine-β-synthase and cystathionine-γ-lyase), one mitochondrial enzyme (3-mecaptopyruvate sulfurtransferase) and as disclosed more recently by a peroxisomal enzyme (D-amino acid oxidase). At a low dose of 80 ppm, inhaled H2S induced a hibernation-like state in mice for 6 h characterized by hypometabolism and consequent hypothermia at 15°C (Blackstone et al., 2005). No behavioral or functional differences were observed after recovery following the 6 h of exposure (Blackstone et al., 2005). Also, Beauchamp (1984) reported no long-term health effects of H2S at the 80 ppm concentration. Johansson (1996) proposed that a Tb of 20°C in non-hibernating animals will lead to cardiac fibrillation. Moreover, the lowest Tb recorded in mice during fasting-induced torpor was about 23°C even when the ambient temperature was maintained at 5°C (Bouma et al., 2011). Thus, the ability to reversibly lower Tb to 15°C in a non-hibernating animal by administration of H2S without major side-efects is a major step forward. It suggests that non-hibernating animals are fully capable of withstanding extreme hypometabolism and hypothermia in the presence of protective pharmacological agents.

1.6.2. 5’-Adenosine monophosphate (5’-AMP) as an inducer of a hibernation-like stateAnother pharmacological compound that has been found recently to induce a torpor-like state in non-hibernating mammals is 5’-Adenosine monophosphate (5’-AMP), a metabolite of ATP hydrolysis. Injection of 5’-AMP induces profound and transient hypometabolism and hypothermia in rodents (Zhang et al., 2006; Zhang et al., 2009; Bouma et al., 2013; de Vrij et al., 2014). Also, high plasma levels of 5’-AMP were detected in fasting-induced torpid mice. All these studies suggest that 5’-AMP may be used to manage metabolic rate and Tb in humans during major surgery and trauma. The mechanisms for this 5’-AMP-induced hypometabolism and consequent hypothermia remain controversial. One hypothesis suggests that 5’-AMP induces a torpor-like state by activating adenosine A1 receptors (A1Rs) on temperature-sensitive hypothalamic neurons, thereby suppressing the thermoregulatory responses that maintain Tb (Muzzi et al., 2012). Others hypothesized that uptake of 5’-AMP activates AMP-activated protein kinase (AMPK), a central enzyme involved in cellular energy homeostasis of which the downstream signaling pathways lead to decreased metabolism (Lindsley and Rutter, 2004; Lee, 2008; Melvin and Andrews, 2009). Thus, the induction of hypometabolism and subsequently hypothermia.

Taken together, induction of a hibernation-like state by pharmacological agents could be harnessed to clinical cases such as IRI encountered in transplantation and other clinical conditions. Thus, a full understanding of the mechanisms behind hibernation might lead to the development of pharmacological strategies to protect organs during several clinical cases including hypothermic storage for transplantation.

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1AIMS OF THE THESIS

The aim of this thesis is to identify the underlying protective mechanisms in mammalian hibernators against hypothermia-rewarming injury and to explore whether such mechanisms may benefit subjects during forced cooling and rewarming. To this end, we studied the effects of hypothermia-rewarming on neutrophil dynamics and the kidneys of hibernating and non-hibernating animals with or without administration of pharmacological compounds. In chapter 2, we investigated the mechanism by which neutrophilic granulocytes disappear from the circulation during hibernation in Syrian hamsters and forced hypothermia in rats. Thereafter, we demonstrated in chapter 3 the role of H2S in the induction of natural hibernation and the protection against kidney injury. We described in detail activation of the H2S system as a major underlying mechanism of renal preservation and protection during hibernation. Next, we explored pharmacological induction of a torpor-like state in natural hibernators using 5’-AMP and investigated the influence of 5’-AMP on the renal H2S system (chapter 4). Chapter 5 focused on the beneficial effect of dopamine in protecting kidney from injury in an in vivo rat model of deep hypothermia and rewarming, particularly addressing its effects on the renal H2S system.

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1

2Reduction of body temperature governs neutrophil

retention in hibernating and non-hibernating animals by

margination.

Hjalmar R. Bouma1, George J. Dugbartey1, Ate S. Boerema2, Fatimeh Talaei1, Annika Herwig3, Maaike Goris1, Azuwerus van Buiten1, Arjen M. Strijkstra2,

Hannah V. Carey5, Robert H. Henning1, Frans G.M. Kroese4

1Department of Clinical Pharmacology, University of Groningen, University Medical Center Groningen, The Netherlands

2Centre for Behavior and Neuroscience, Departments of Chronobiology and Molecular Neurobiology, University of Groningen, The Netherlands

3Zoological Institute, University of Hamburg, Germany4Department of Rheumatology and Clinical Immunology, University of Groningen, University Medical

Center Groningen, The Netherlands5Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin,

Madison, United States

Key words: neutrophils, hibernation, hypothermia, margination, torpor

Published in Journal of Leuckocyte BiologyReference: J Leukoc Biol. 2013 Sep;94(3):431-7

Digital object identifier (DOI): 10.1189/jlb.0611298.

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ABSTRACT

Hibernation consists of periods of low metabolism called torpor that are interspersed by euthermic arousal periods. During both deep and daily (shallow) torpor, the number of circulating leukocytes decreases, while circulating cells are restored to normal numbers upon arousal. Here we show that neutropenia during torpor is due solely to lowering of body temperature, since a reduction of circulating also occurred following forced hypothermia in summer euthermic hamsters and rats that do not hibernate. Splenectomy had no effect on reduction in circulating neutrophils during torpor. Margination of neutrophils to vessel walls appears to be the mechanism responsible for reduced numbers of neutrophils in hypothermic animals because the effect is inhibited by pretreatment with dexamethasone. In conclusion, low body temperature in species that natural use torpor or in non-hibernating species under forced hypothermia leads to a decrease of circulating neutrophils due to margination. These findings may be of clinical relevance as they could explain, at in least part, the benefits and drawbacks of therapeutic hypothermia as employed in trauma patients and during major surgery.

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2

INTRODUCTION

Hibernation is an energy-conserving mechanism that promotes survival of endothermic animals during periods of scarce food supply. The hibernation season is characterized by periods of reduced metabolism and body temperature known as torpor bouts, which are interspersed by short (<24 h) arousal periods to normal (euthermic) body temperatures. During bouts of deep torpor, which last for several days to a month, body temperature can fall as lows as low as ~ -3°C, depending on species and ambient temperature (1-5). A second form of metabolic depression is “daily torpor”, in which minimum body temperatures typically reach only ~18-30°C and torpor bout duration does not exceed 24 h (6). In addition to a suite of physiological changes that occur during torpor (e.g., depressed heart and ventilation rates (7)), immune function is significantly reduced (8). Both the innate and the adaptive arms of the immune system are affected, of which the underlying mechanisms remain to be unraveled. At least part of the reduced immune function in vivo can be explained by a substantial drop in numbers of circulating leukocytes (leucopenia) during torpor as demonstrated for all species of hibernating animals studied thus far (7;9-14). Previously, we demonstrated that low body temperature affects the number of circulating lymphocytes during torpor through a reduced plasma level of sphingosine-1-phosphate (S1P), resulting in impaired lymphocyte egress from lymphoid tissues (10).

The explanation for the decrease in number of circulating neutrophils during torpor is less clear. One mechanism could be retention of neutrophils in lungs, because numbers of neutrophils in lungs increase during torpor in hibernating hedgehogs (Erinaceus europaeus L.) (15). Studies with mongrel dogs (Canis familiaris) also revealed that hypothermia induced a reversible leucopenia due to adherence of cells to the endothelium of mesenteric venules (16). However, whether this mechanism leads to a reduced number of circulating neutrophils during torpor is not known. Therefore, in the present study we determined whether transient neutropenia during torpor (and hypothermia) is due to temporary attachment to vessel walls, or whether alternative mechanisms are involved. We performed experiments in naturally hibernating and in non-hibernating (forced hypothermic) animals to better identify the underlying mechanisms that lead to transient neutropenia during torpor.

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MATERIALS AND METHODS

Induction of deep torpor in Syrian hamsters Male and female Syrian hamsters (Mesocricetus auratus) were bred in the animal facilities of the University of Groningen. Animals were housed at an ambient temperature of 21 ± 1 °C and summer photoperiod (L:D-cycle 14:10 h). Torpor was induced by first shortening the L:D-cycle to 8:16 h for ~10 weeks followed by housing at continuous dim light (<5 Lux) at an ambient temperature of 5°C; animals at this stage that did not show signs of torpor behavior were called “winter euthermic”. During hibernation, Syrian hamsters exhibit deep torpor bouts of 3-6 days alternating with arousal periods of approximately 23 hours (17). Torpor-arousal patterns were monitored by movement detectors connected to a computer. Hamsters were sacrificed during winter euthermia, torpor or during interbout arousal (referred to as “arousal”). Experiments with Syrian hamsters were approved by the Institutional Animal Care and Use Committee of the University Medical Center Groningen.

Induction of “daily torpor” in Djungarian hamsters Male and female Djungarian hamsters were bred in the animal facilities of the Rowett Institute for Nutrition and Health at the University of Aberdeen. All animal work was licensed under the Animals (Scientific Procedures) Act of 1986. Prior to the experiments animals were housed at an ambient temperature of 21 ± 1 °C and a summer-like photoperiod (LD 16:8). “Daily torpor” was induced by shortening the LD cycle to LD8:16 for ~14 weeks at an ambient temperature of 21°C. Visual inspection of torpid behavior (absence of activity and typical torpor posture) was carried out in the middle of the light phase, which is the usual torpor time for Djungarian hamsters. Animals were sacrificed during winter euthermia (time-matched euthermic control for torpid animals), torpor or arousal (± 12 hours following a bout of “daily torpor”).

RatsMale Wistar rats (Rattus norvegicus) weighing 350 – 400 grams were obtained from Harlan Netherlands B.V. Experiments in rats were approved by the Institutional Animal Care and Use Committee of the University Medical Center Groningen.

Forced hypothermiaSummer euthermic Syrian hamsters housed at a LD-cycle of 14:10 h and Wistar rats housed at a L:D-cycle of 12:12 h were anesthetized by intraperitoneal injection of 200 mg/kg ketamine and 2 mg/kg diazepam intraperitoneally. After induction of anesthesia, a catheter was inserted into the jugular vein for blood sampling. Rectal temperature and heart rate (ECG) were monitored continuously (Cardiocap S/5, Datex Ohmeda, USA). Following cannulation, an initial blood draw and installation of the monitoring devices, animals were cooled by

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2

applying ice-cold water on the fur until they reached a body temperature of ~10-15°C. These animals could be rewarmed without ventilatory support. Rats had to be ventilated throughout the procedure to allow safe cooling to a body temperature of ~15°C and subsequent rewarming. Therefore, following intubation, rats were ventilated mechanically (Amsterdam Infant Ventilator; HoekLoos). The tidal volume was set to achieve normocapnia, as verified by capnography and arterial blood gas analysis, with O2/air (1:2) at a ventilation rate of 50 min-1 (0.5 s inspiration time). After reaching the desired low body temperature, animals were immediately rewarmed using a water-based heating mattress. Cooling and rewarming rates were ~1°C per 3 minutes. Blood samples were taken at several body temperatures during cooling, when animals reached the desired low body temperature, during rewarming and at least ten minutes after a euthermic temperature (>35°C) was reached. In another experiment, summer euthermic Syrian hamsters were pretreated 4 hours preceding forced hypothermia with 10 mg/kg dexamethasone or saline intraperitoneally and a baseline blood sample was drawn by orbital puncture. They were then cannulated, cooled and rewarmed as described above, and blood samples were drawn at the induction of anesthesia, upon reaching the desired low body temperature and following rewarming. In all experiments, blood (~200 µl) was collected via the jugular vein catheter in EDTA-coated cups (minicollect EDTA-K3; Greiner Bio-One, Alphen a/d Rijn, The Netherlands) for automated hematological analysis using a Sysmex XE-2100 (Sysmex, Etten-Leur, The Netherlands) (18;19).

SplenectomySplenectomies were performed on summer euthermic and torpid Syrian hamsters. After induction of anesthesia with isoflurane (2-2.5 % in O2), flunixin-meglumin was given subcutaneously (4 mg/kg) for analgesia. A small abdominal midline incision was made, the arteries and veins towards and from the spleen were ligated and the spleen was resected followed by wound closure. Splenectomized summer euthermic animals were allowed to recover at ~20°C (LD-cycle 14 h:10 h) for at least a week before induction of torpor. They were sacrificed during their third torpor bout, which was 60.3 ± 8.1 days following splenectomy. Torpid animals were splenectomized during their third torpor bout. These animals were kept <10°C using ice-packs. Animals recovered in a climate-controlled room at an ambient temperature of ~5°C (continuous dim light) and were sacrificed directly after reaching euthermia (>33°C) following arousal. The perioperative handling of animals induced the onset of arousal, which started immediately followed weaning from anesthesia.

Sacrification and sample collectionSyrian hamsters were sacrificed by intraperitoneal injection of an overdose of pentobarbital followed by decapitation. Djungarian hamsters were sacrificed with CO2 and then decapitated. Rats were sacrificed after the last blood sample was drawn by decapitation

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while being anesthetized. After euthanasia (but before decapitation), body temperature was measured rectally and blood (~200 µl) was collected via cardiac puncture in EDTA-coated cups (minicollect EDTA-K3). This blood sample was analyzed with an automated hematology analyzer (Sysmex XE-2100) (18;19). Differential leukocyte counts were validated manually using Wright-Giemsa stained blood smears. Spleens from torpid and aroused Syrian hamsters were removed, snap-frozen in liquid nitrogen and stored at -80°C.

Statistical analysis and data presentationData are presented as mean ± standard error of the mean (SEM). Group comparisons were performed using a two-tailed independent samples Student’s T-test in the case of two groups. In the case of more than two groups, a One-Way ANOVA with post-hoc Tukey on normally distributed variables (based on Homogeneity of Variances) or a Kruskal-Wallis test followed by a Mann-Whitney U test in the case of non-normally distributed variables was performed. Statistical differences were calculated using SPSS 20.0, where p < 0.05 was considered significantly different.

RESULTS

Deep torpor and “daily torpor” are associated with a reduced number of circulating

leukocytes Body temperatures of Syrian hamsters during winter euthermia and deep torpor were 34.0 ± 0.5°C (n = 8) and 6.2 ± 0.1°C (n = 8), respectively (p < 0.01). Numbers of circulating leukocytes were 2.92 ± 0.71 (x 106/ml) during winter euthermia and 0.12 ± 0.04 (x 106/ml) in deep torpor (p < 0.01). After 5-6 days of torpor, animals aroused spontaneously and the number of leukocytes increased rapidly to 2.72 ± 0.34 (x 106/ml; n = 8) (p < 0.01), i.e. to the same level as seen in winter euthermic animals. The numbers of erythrocytes in the blood were similar in winter euthermic hamsters (8.96 ± 0.42 x 109/ml), torpid hamsters (9.05 ± 0.26 x 109/ml) and in aroused animals (7.71 ± 0.49 x 109/ml).

Body temperatures of Djungarian hamsters during winter euthermia and “daily torpor” were 35.0 ± 0.2°C (n = 6) and 25.2 ± 1.3°C (n = 8), respectively (p < 0.01). The number of circulating leukocytes declined from 9.50 ± 0.85 (x 106/ml) in winter euthermia to 5.02 ± 0.41 (x 106/ml) in torpor (p < 0.01), and then rose again to winter euthermic levels 8.22 ± 0.96 (x 106/ml) upon arousal (n = 5) (p < 0.01). Erythrocyte counts in Djungarian hamsters remained stable during hibernation, being 9.45 ± 0.33 (x 109/ml) in euthermia, 8.80 ± 0.31 (x 109/ml) during “daily torpor” and 9.67 ± 0.15 (x 109/ml) upon arousal. There results suggest that leucopenia is not restricted to deep torpor, but also occurs in animals exhibiting “daily torpor”.

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Deep and “daily torpor” are associated with neutropenia Leucopenia during torpor is due to a decrease in numbers of all major classes of leukocytes, including neutrophils, lymphocytes and monocytes (7;9-14). In Syrian hamsters the number of circulating neutrophils decreases during entry into deep torpor as body temperature falls, and numbers are correlated strongly with the body temperature (P = 0.679; n = 79; p < 0.01). The number of circulating neutrophils is 0.92 ± 0.31 (x 106/ml; Figure 1A) during winter euthermia and 0.02 ± 0.00 (x 106/ml) (p < 0.05; Figure 1B) during torpor. Numbers of circulating neutrophils are restored to 1.50 ± 0.35 (x 106/ml) (p < 0.01; Figure 1C) upon arousal. Interestingly, arousal from torpor is associated with a greater number of circulating neutrophils compared with animals that had not yet entered torpor (i.e. winter euthermic animals) or following rewarming from forced hypothermia (see below). During “daily torpor” in the Djungarian hamster, the number of circulating neutrophils falls significantly during torpor as compared to winter euthermic animals and is followed by full restoration upon arousal (Figure 1D).

Figure 1. Low body temperature governs a decrease in the number of circulating neutrophils, which is unaffected by splenectomy. Normal number of circulating neutrophils in winter euthermic Syrian hamsters (A); the number of circulating neutrophils decreases gradually during either entrance into deep torpor or forced hypothermia in nonhibernating Syrian hamsters (B); blood neutrophil counts rise upon arousal from torpor and rewarming following forced hypothermia in Syrian hamsters (C); the number of circulating neutrophils is reduced during “daily torpor” followed by restoration upon arousal in the Djungarian hamster (D); splenectomy preceding hibernation, in the summer season, does not affect clearance of circulating neutrophils during torpor (E); splenectomy during torpor leads to an increased number of circulating neutrophils upon arousal in the Syrian hamster (F). Bars represent means ± SEM of n = 4-8 animals per group. * represents significantly different at p < 0.05.

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Forced hypothermia induces neutropenia in hamsters The effect of low body temperature on the number of circulating neutrophils was further demonstrated by cooling anesthetized summer euthermic Syrian hamsters (“forced hypothermia”). Cooling to a body temperature of 9.1 ± 0.8°C induces a decrease in the number of circulating neutrophils as observed in deep torpor: from 0.54 ± 0.09 (x 106/ml; n = 5) to 0.02 ± 0.01 (x 106/ml; n = 5) (p < 0.01; Figures 1B, C). Thus, this finding indicates that simply lowering the body temperature leads to the induction of neutropenia.

Figure 2. Neutropenia induced by low body temperature in a hibernator is due to margination of neutrophils. Forced hypothermia of anesthetized summer active Syrian hamsters lead to a profound reduction in body temperature that was unaffected by dexamethasone pretreatment intraperitoneally (A); lowering of body temperature is associated with a severe drop in heart rate, both in saline as well as in dexamethasone pretreated hamsters (B); although hypothermia did not affect erythrocyte counts, the number of neutrophils was transiently reduced by hypothermia, which was precluded by pretreatment with dexamethasone (p < 0.05) (C). Dexa = dexamethasone (10mg/kg) pretreated animals. Graphs represent means ± SEM (shown as grey shaded areas or by caps, respectively) of n = 4 (saline) and n = 6 (dexamethasone) animals per group. * represents significantly different at p < 0.05.

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The spleen is not the only organ involved in induction or restoration of neutropeniaTo evaluate a potential role of spleen in storage of circulating neutrophils during torpor, we examined the effect of splenectomy on the numbers of circulating neutrophils. Syrian hamsters were splenectomized either before entering hibernation or during torpor. Removal of the spleen before hibernation did not affect the number of circulating neutrophils during torpor, i.e. neutrophil numbers in splenectomized torpid hamsters are similar to those in non-splenectomized animals (Figure 1F). In addition, splenectomy during torpor did not prevent the rise in the number of circulating neutrophils upon arousal (Figure 1G). However, following removal of the spleen during torpor, the number of circulating neutrophils was significantly higher in aroused animals that were splenectomized during torpor compared to non-splenectomized animals (p < 0.05; Figure 1G). Thus, the spleen is not essential for retention or storage of large numbers of neutrophils during torpor.

The percentage of circulating immature granulocytes is not altered during hibernationWe speculate that if massive apoptosis of neutrophils occurs during torpor, then substantial release of (newly produced) neutrophils from the bone marrow should occur upon arousal to restore the number of circulating cells. To obtain information about the number of newly produced neutrophils, we measured the number of immature granulocytes in the Syrian hamster. Numbers of immature granulocytes are very low in summer, winter euthermic and hibernating (torpid and aroused) animals (detection limit of 0.001 x 106 cells/ml). Although this low number hampers exact quantification of immature granulocytes, it does rule out significant release of immature (neutrophilic) granulocytes from the bone marrow upon arousal. Hence, massive apoptosis of neutrophils seems unlikely to be the sole explanation for neutropenia during torpor.

Low body temperature also drives neutropenia in non-hibernating species (rats)We found that the number of circulating neutrophils is reduced during deep and “daily torpor” in hibernating hamsters as well as during forced hypothermia in non-hibernating hamsters. To assess whether the induction of neutropenia by low body temperature is a general mechanism not restricted to hibernating species, we induced forced hypothermia in rats (Rattus norvegicus), a non-hibernating species. Lowering the body temperature results in a concomitant decrease in the number of circulating leukocytes, which is due to reduced numbers of neutrophils, lymphocytes, monocytes and eosinophils, but not basophils (Table 1). However, the number of circulating erythrocytes remains stable during forced hypothermia. In contrast to hamsters, rats have a higher number of circulating immature granulocytes. Cooling the rats reduced the number of immature granulocytes to values below the detection threshold, although not significantly different from the number prior to cooling (i.e. < 0.001 x 106/ml) (Table 1). Following rewarming, the number of immature granulocytes

36

Table 1. Forced hypothermia in a non-hibernating animal, the rat, leads to a reduced number of circulating neutrophils, lymphocytes and monocytes. Table shows the body temperature (°C), the number of circulating erythrocytes (x109/ml), leukocytes (x106/ml) and differentiated leukocyte counts (x106/ml) during euthermia (prior to experiment), forced hypothermia and following full rewarming in the Wistar rat. Shown are means ± SEM of n = 5 animals per group. Significant differences (p < 0.05) between groups are indicated by the printed superscripts (HT = hypothermia, EU = euthermia, REW = rewarming).

Wistar rat Euthermia Hypothermia Rewarming

( n = 5) ( n = 5) ( n = 5)Body temperature 37.0 ± 0.0 HT 15.4 ± 0.4 EU;REW 37.0 ± 0.0 HT

Erythrocytes 7.81 ± 0.20 6.84 ± 0.72 7.48 ± 0.49Leukocytes 8.82 ± 1.07 HT 0.57 ± 0.20 EU;REW 7.68 ± 1.53 HT

Neutrophils 1.63 ± 0.49 HT 0.15 ± 0.04 EU;REW 2.59 ± 0.75 HT

Lymphocytes 6.76 ± 0.96 HT 0.39 ± 0.19 EU;REW 4.79 ± 1.37 HT

Monocytes 0.34 ± 0.08 HT 0.03 ± 0.01 EU 0.21 ± 0.11Eosinophils 0.08 ± 0.03 0.00 ± 0.00 0.08 ± 0.03Basophils 0.01 ± 0.01 0.00 ± 0.00 0.01 ± 0.01

Immature Granulocytes 0.012 ± 0.005 0.00 ± 0.00 0.012 ± 0.005

rose to values that were not significantly different from pre-cooling levels (Table 1). The absence of a significant rise in the number of immature granulocytes following hypothermia as compared to baseline values suggests that the restoration of circulating neutrophils upon rewarming is likely not due to release of immature cells from the bone marrow.

Pretreatment with dexamethasone prevents neutropenia during forced hypothermia. Since apoptosis and reduced release from the bone marrow are unlikely explanations for the kinetics of neutrophils during torpor and arousal, we explored whether temporary retention of cells might lead to neutropenia during torpor. Although the spleen is not involved in the induction of neutropenia during torpor, an alternative explanation for transient neutropenia during torpor and hypothermia is temporary retention of neutrophils in organs due to margination of cells to vessel walls. Thus, we injected summer euthermic Syrian hamsters prior to forced hypothermia with 10mg/kg dexamethasone intraperitoneally to inhibit margination of neutrophils (20-22). As shown in Figures 2A and B, dexamethasone pretreatment did not affect the induction of hypothermia or the associated drop in heart rate. However, it abolished the neutropenia that normally occurs during hypothermia (Figure 2B), but had no effect on the number of circulating erythrocytes (Figure 2C). Since dexamethasone can also stimulate release of neutrophils from the bone marrow in addition to demargination of cells (20-22), we counted the number of immature granulocytes in blood. The number of circulating immature granulocytes was comparable to that during hibernation, around the detection limit of 0.001 x 106 cells/ml. These results support the hypothesis that low body temperature governs neutropenia by margination.

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DISCUSSION

In all hibernating animals studied so far, the number of circulating leukocytes decrease after entrance into torpor followed by rapid restoration upon arousal (8). Several studies demonstrated that leucopenia during torpor is due to a reduction in the number of circulating lymphocytes, neutrophils and monocytes (9-12;14;23). Previously, we showed that the drop in lymphocytes during hibernation is driven by low body temperature that directly results in lower sphingosine-1-phosphate (S1P) levels in the blood (10). In the current study, we demonstrate that the reduction in circulating neutrophils is also driven by the decrease in body temperature during torpor, because neutropenia occurs in deep hibernators as well as during daily torpor in the Djungarian hamster. Moreover, neutropenia is also induced by forced hypothermia in summer euthermic Syrian hamsters and in rats, a non-hibernating species.

The mechanism underlying the reduction of neutrophils in blood might be explained by lower production of neutrophils, increased apoptosis and/or temporary retention of cells during torpor. The half-life of neutrophils is estimated to be 14 hours (as measured in mice) (24;25). Therefore, we speculate that if a reduced release from the bone marrow is the sole explanation for neutropenia during torpor, a period of approximately three to five days is required to lead to the low number of circulating neutrophils as observed during torpor. However, the number of circulating leukocytes correlates closely with the reduction in body temperature. Furthermore, ~95% of the leukocytes are cleared from the circulation as soon as the animals approach their torpid body temperature, which is well within 14 hours. Thus, it is unlikely that a lower production rate of neutrophils by the bone marrow contributes significantly to the drop in cell numbers observed during torpor. Neutropenia during torpor is also not explained by massive apoptosis of neutrophils, because there is no difference in the number of immature circulating neutrophils during arousal from deep torpor or rewarming from forced hypothermia in Syrian hamsters or rats, and the fraction of immature granulocytes remains very low. Thus, a reduced production of cells and/or apoptosis cannot explain the rapid induction of leucopenia during torpor or forced hypothermia. Furthermore restoration of circulating neutrophils upon arousal or following rewarming from hypothermia does not appear to be due to rapid regeneration in the bone marrow.

An alternative explanation is that neutrophils are temporarily retained at certain sites during torpid periods and are released again during arousal. Villalobos et al. demonstrated a comparable phenomenon to occur in deep hypothermic dogs leading to leucopenia and a reduction in the number of platelets (26). In line with our results, rewarming of the dogs induced restoration of the number of circulating leukocytes and platelets. Comparison of the number of circulating platelets in blood samples derived from aorta, upper and lower vena

38

cava and capillaries (of the tongue) did not reveal specific retention in the arterial, capillary or venous vascular system. Splenectomizing and hepatectomizing dogs prior to hypothermia reduced the extent of trombopenia. However, neither splenectomy, nor hepatectomy or splenectomy combined with hepatectomy, precluded trombopenia induced by hypothermia. Thus, hypothermia leads to reversible clearance of circulating leukocytes and platelets, which does not seem to be confined to arteries, capillaries or veins and in which the spleen and liver do not play a key role.

Our findings also indicate that the spleen does not play a significant role in storage neutrophils during torpor. Splenectomy preceding the hibernation season does not affect the reduction in the number of circulating neutrophils, nor did splenectomy of torpid animals prevent restoration of normal numbers of circulating neutrophils during subsequent arousal to euthermia. Although these results suggest that the spleen does not play an essential role, they do not completely rule out a role in the induction or restoration of neutropenia during hibernation. In contrast, our data support the hypothesis that neutrophils stop circulating during torpor due to reversible adherence to endothelial cells of blood vessel walls (margination), because neutropenia induced by low body temperature was fully blocked by dexamethasone pretreatment in our model of forced hypothermia. Margination depends on the interaction between leukocytes and endothelial cells, due to expression of integrins, selectins and adhesion molecules and detaching forces, such as shear forces due to blood flow velocity (27;28). Studies in rabbits show that under normal circumstances, a substantial number of neutrophils are marginated: the marginated fraction can be as high as ~61% of the number of circulating neutrophils (20). Dexamethasone reduces margination of neutrophils through mechanisms that are not fully understood yet (20-22). Possibly, reduced expression of L-selectin and CD18 on neutrophils (29;30) and endothelial-leukocyte adhesion molecule-1 (ELAM-1) and intracellular adhesion molecule-1 (ICAM-1) on endothelial cells (31), observed after dexamethasone treatment of cells, plays a role in demargination of neutrophils. In addition, glucocorticoids can increase the half-life of neutrophils and stimulate release from the bone marrow through G-CSF (21;22). The number of circulating immature granulocytes remains low after injection of dexamethasone, which suggests that the effect of dexamethasone on hypothermia-induced neutropenia is not due to stimulation of bone marrow release. Thus, low body temperature governs neutropenia by margination, which likely explains the neutropenia associated with torpor.

The mechanism by which low body temperature induces margination is not yet fully understood. In general, margination of neutrophils is promoted by adhering forces such as the expression of adhesion molecules and integrins and is reduced by detaching forces such as increased shear stress by blood flow. Direct effects of lowered body temperature are unlikely to cause neutropenia by means of margination since low temperature reduces

39

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the expression of ICAM-1 in lung in vivo (32) and E-selectin on endothelial cells in vitro (33;34). However, these effects might be compensated for by indirect effects of low body temperature in vivo, such as a reduced cardiac output and the subsequent drop in blood flow velocity that might increase leukocyte-endothelium adhesion. Our data support the hypothesis that lowered body temperature induces margination, an effect which is not restricted to hibernating species, as we also observed this phenomenon in the rat. However, the existence of additional effects, specific for hibernators, cannot be excluded. Yasuma

et al., (35) showed that incubation of rat endothelial cells at 37°C in vitro with plasma from hibernating, but not from euthermic thirteen-lined ground squirrels leads to upregulation of ICAM-1 expression and increased monocyte adhesion to these cells. These findings suggest that in addition to lowered body temperature, temperature-independent effects of torpor might affect the expression of adhesion molecules, which could potentially promote margination of neutrophils during torpor in vivo. In summary, adherence of neutrophils to vessel walls increases when body temperature falls. This finding may explain the reduced number of circulating cells during deep torpor, “daily torpor” and forced hypothermia.

Neutropenia during torpor may also contribute to the diminished function of the innate immune system during torpor. A reduced innate immune function has been demonstrated by intraperitoneal injection of lipopolysaccharide (LPS) during deep torpor, which does not lead to a febrile response as long as the animals remain torpid. However, injection of LPS during arousal induced a febrile response and prolonged the duration of arousal (36). Possibly, reversible reduction of the innate immune system by margination of cells allows conservation of energy while maintaining the potential of rapid reactivation in case of infection. Understanding the mechanisms involved in the reduced function of the immune system may be relevant to the pathophysiological consequences of therapeutic hypothermia (37). Although hypothermia is used clinically to stabilize trauma patients and during major surgery to suppress metabolic activity and limit organ injury during periods of low oxygen supply, it is associated with the occurrence of serious side effects such as fatal thrombosis (38-40), neurogical deficits (41), renal injury (42) and increased blood loss (43). Organ injury might be exaggerated by inflammation with influx of neutrophils, in which adhesion (margination) of neutrophils is an important initial step. Indeed, experimental blockade of margination reduces the extent of pulmonary injury following normothermic cardiopulmonary bypass in a sheep model (44). Interestingly, hypothermia in humans leads to the induction of a reversible leucopenia, comparable to hibernating animals and hypothermic rats (45). Possibly, the increased resistance to ischemia/reperfusion and hypothermia of hibernating animals (46-53) prevents the occurrence of cellular injury and the subsequent onset of an inflammatory response, as might occur in humans during treatment with cardiopulmonary bypass (42;51;54-56). Thus, margination of neutrophils induced by low body temperature might be involved in the etiology of organ injury following hypothermia in humans.

40

CONCLUSION

Low body temperature induces a decrease in the number of circulating neutrophils, which is not restricted to hibernating species. Our data support the hypothesis that neutropenia due to low body temperature is caused by reversible margination of cells, rather than being caused by apoptosis of cells, a reduced release from the bone marrow or retention in the spleen. Despite the fact that hibernators regularly cycle through periods with an extremely reduced body temperature followed by rapid rewarming, no gross signs of organ injury are evident. Translating the underlying mechanisms that govern increased resistance to hypothermic injury may therefore be of major clinical relevance.

CONFLICT OF INTEREST DISCLOSURE

The authors declare no conflict of interest.

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3Endogenous H2S Production is Crucial in Maintaining

Torpor-arousal Cycles and Preserving Renal Integrity in the

Hibernating Syrian Hamster

George J. Dugbartey1, Arjen M. Strijkstra2, , Edwin L. de Vrij1, Ate S. Boerema2,3, Maaike Goris1, Hjalmar R. Bouma1,4, Robert H. Henning1,*

1Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, 9700 RB Groningen, The Netherlands

2Departments of Chronobiology and Molecular Neurobiology, Groningen Institute for Evolutionary Life Sciences, University of Groningen, 9700 RB Groningen, The

Netherlands

3Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

4Department of Rheumatology and Clinical Immunology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

Manuscript in preparation

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ABSTRACT

Background: Mammalian hibernation is characterized by repetitive cycle of profound reduction in metabolism and core body temperature known as torpor followed by restoration to euthermic level (arousal). Despite cycling through the stress of torpor and arousal, no sign of organ injury has been reported upon arousal from torpor. Hydrogen sulfide (H2S) has recently been implicated in inducing a hibernation-like state in small mammals without organ injury or behavioral changes upon full recovery. The present study seeks to investigate the involvement of endogenous H2S and its role in natural hibernation.

Methods: Syrian golden hamsters (Mesocricetus auratus) were housed in a climate controlled chamber at 5°C under dim red light to induce torpor. Movement of all animals was continuously monitored with passive infrared detectors. Some groups of hamsters were euthanized during torpor and arousal phases. Other groups of torpid hamsters were implanted with osmotic mini-pumps i.p. filled with saline or a non-specific H2S inhibitor, amino-oxyacetic acid (AOAA; 100mg/kg/day) after receiving an i.p. bolus injection of AOAA 70mg/kg or saline, causing arousal in all animals. Summer euthermic- and winter euthermic animals underwent a similar protocol. Hamsters were euthanized in the normothermic state ~ 4 days following the implantation by an overdose of pentobarbital. Blood and kidney samples were collected.

Results: Core body temperature dropped from 36.5 ± 0.5°C in summer euthermic animals to 10.0 ± 2.0°C during torpor and returned to euthermic temperature during arousal. Despite this significant reduction in temperature, renal function as determined by serum creatinine levels, renal morphology and ROS production, is not different from summer euthermic, torpor and arousal. Also, endogenous H2S production is increased, potentially through upregulation of renal CBS, CSE and 3-MST during torpor. H2S production was restored to near euthermic level upon arousal from torpor. Further, Pharmacological inhibition of H2S production using AOAA precluded torpid hamsters to enter the subsequent torpor bouts, whereas torpid hamsters infused with saline re-entered torpor following pump implantation. Moreover, torpid animals infused with AOAA showed substantial renal damage, as indicated by profound changes in renal morphology and increased expressions of KIM-1, α-SMA, ED-1, HIS-48, high levels of serum creatinine and renal ROS production, decreased expression levels of H2S-producing enzymes as well as a hundred-fold decrease in endogenous H2S production. Also, renal damage was observed in both winter euthermic groups. However, renal morphology remained well preserved during hibernation in the saline and non-hibernating summer control groups (± AOAA) and correlated positively with upregulation of H2S-producing enzymes and subsequent endogenous H2S production.

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Conclusion. Our results demonstrate that renal H2S system seems crucial in maintaining torpor-arousal cycle in Syrian hamsters and preserving their kidney morphology and function through regulation of H2S-producing enzymes under these physiological extremes. These findings might be relevant for a number of clinical conditions such as therapeutic hypothermia (e.g. during cardiac and brain surgery and following cardiac arrest) or organ preservation for transplantation medicine.

INTRODUCTION

During hibernation, mammals undergo repetitive cycles of metabolic depression and reduced core body temperature (Tb) known as ‘torpor’ and restoration of metabolism and Tb known as ‘arousal’. Typically, in small rodents such as European and golden-mantled ground squirrels, oxygen consumption decreases by 97-98% of euthermic value (Barnes, 1989; Boyer and Barnes, 1999) and Tb drops to 2-10°C (Kenagy et al., 1989; Hut et al., 2002). In addition, heart rate drops substantially, e.g. in the marmot (Marmota flaviventris) from 200–300 bpm in the summer euthermic state to 3–10 bpm during torpor (Zatzman, 1984). Despite cycling through extreme physiological situations during hibernation, organ injury is absent in the kidney, (Zancanaro et al., 1999; Jani et al., 2011), brain (Drew et al., 2001; Zhou et al., 2001), the gut and liver (Kurtz et al., 2006) and the heart (Camici et al., 2008) after the hibernation season. However, organs of torpid animals undergo major changes in architecture and/or the expression of specific molecular markers resembling those observed in chronic diseases as found in brain (Popov and Bocharova 1992, Arends et al., 2011) and lung (Talaei et al., 2011). Nevertheless, such changes are quickly reversed during arousal bouts, thus preventing permanent organ injury and ensuring proper organ function after hibernation. In fact, it has been speculated that arousals principally serve to reverse organ changes contracted during torpor (Fisher, 1964; French, 1982a, Daan et al., 1991). In addition to its reversal, hibernators may be intrinsically protected from substantial organ damage because of metabolic adaptations (Pan et al., 2013; Stenvinkel et al., 2013; Bogren et al., 2014), and/or suppression of the immune response (Bouma et al., 2010; Bouma et al., 2011).

The molecular mechanisms that convey the rapid repair and/or intrinsic protection of torpid tissues in hibernators are essentially unknown, although hibernators show changes in gene and protein expression. Recently, we identified the production of endogenous hydrogen sulfide (H2S) to be a crucial player in the restoration of lung architecture during arousal (Talaei et al., 2012), thus extending the previously disclosed protection of cells and organs from cooling and rewarming injury by the CBS/H2S system (Talaei et al., 2011). Moreover,

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Revsbech et al. (2014) also reported high levels of unbound free sulfide in the blood of hibernating Scandinavian brown bears. These observations suggest that H2S mechanism may form an integral part of hibernation. The potential involvement of H2S in hibernation is further supported by its ability to induce a hibernation-like state in non-hibernating rodents such as mice for several hours without negative behavioral changes upon reversal (Blackstone et al., 2005; Blackstone and Roth 2007; Aslami et al., 2009; Bos et al., 2009). Moreover, H2S has emerged as an endogenous molecule with potent cyto- and organoprotective properties (Kimura and Kimura 2004; Whiteman et al., 2004; Elrod et al., 2007; Bos et al., 2009; Szabo et al., 2011; Lobb et al., 2014).

Therefore, we hypothesized that endogenous H2S is involved in entrance into natural hibernation and in preserving organ integrity during the repetitive cycle and stress of torpor-arousal. We first examined the endogenous H2S system and organ damage in kidney - an organ highly vulnerable to hypothermia and hypoxia – in natural hibernation and found H2S-producing enzymes to be substantially upregulated, particularly in torpor. In a second experiment, endogenous H2S production was blocked during torpor and the subsequent arousal using continuous infusion of amino-oxyacetic acid (AOAA), a non-specific inhibitor of H2S producing enzymes (Asimakopoulou et al., 2013). Effects of AOAA on the torpor-arousal cycle and kidney damage were assessed.

MATERIALS AND METHODS

Induction of hibernation Hibernation was induced and monitored in male Syrian hamsters (Mesocricetus auratus) as described previously (Bouma et al., 2011). Periods with ≥ 24h of inactivity were considered to be torpid phases. Hamsters were euthanized on day 1 upon entering torpor (TE: torpor early; n = 4), on day_ 3 during deep torpor (TL: torpor late; n = 4), 1.5 h after onset of arousal (AE: arousal early; n =5) and 8 h after reaching full euthermia at ~37°C (AL: arousal late; n = 5). In a second experiment, TL animals were implanted with osmotic mini-pumps filled with saline (0.9%, n = 5) or amino-oxyacetic acid (AOAA; 100 mg/kg/day, n = 6) under 2.5% isoflurane anesthesia. As AOAA precluded animals to return to torpor, all animals were euthanized during at euthermia ~4 days following implantation, which required induction of arousal 4 h prior to euthanization in animals which re-entered torpor following pump implantation. Summer euthermic hamsters (SE, 14h:10h light:dark cycle, ambient temperature of 21°C) and winter euthermic animals (WE, complete darkness, ambient temperature of 5°C) implanted with or without mini-pumps filled with AOAA served as controls. Summer euthermic and winter euthermic animals did not show any torpor behavior. Animals were euthanized by 1.5 ml 6% sodium pentobarbital i.p.. Blood samples were obtained and kidneys tissue was

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either snap-frozen in liquid nitrogen and stored at –80°C or fixated in 4% paraformaldehyde for histopathological analysis. The experiments were approved by the Animal Experiments Committee of the University of Groningen (DEC5456I).

Histopathology Paraffin sections (4 µm) were dewaxed and stained with periodic acid-Schiff (PAS) reagent and counterstained briefly with Meyer’s hematoxylin. Renal sections were examined blindly by 2 independent observers (Gross et al., 2006). Glomerular damage was scored semiquantitativily in 100 glomeruli from 0 to 4 (El Nahas et al., 1991) and tubulointerstitial damage was quantified on the basis of tubular dilatation, atrophy of epithelial cells and widening of tubular lumen (Gross et al., 2006).

ImmunohistochemistrySections were stained for kidney injury molecule (KIM-1, diluted 1:50), a marker for renal tubular damage (goat polyclonal, Santa Cruz, Te Huissen, Netherlands), ED-1 (diluted 1:500), a marker for macrophages (mouse monoclonal, Serotec Ltd, Oxford, UK), HIS-48, a marker for neutrophils (mouse monoclonal, generously provided Prof. F.G.M. Kroese, ProHisto foundation, Groningen, The Netherlands), α-SMA (diluted 1:100), a marker for fibrosis (mouse monoclonal, Dako, Glostrup, Denmark) as previously described (Sandovici et al., 2006).

Western BlotThe expression of renal CBS, CSE and 3-MST in all groups of hamsters was analyzed by western blot (Talaei et al., 2011). In brief, proteins on the nitrocellulose membranes were detected with CBS, CSE and 3-MST antibodies (1:1000) overnight at 4°C, washed three times with Tris buffered saline with 0.004% Tween and treated with the respective secondary antibody (1:1000, 2 h at room temperature). Protein bands were visualized using a Gene Genome and band intensities were quantified using Gene Tools software (Westburg B.V., Leusden, The Netherlands).

ROS MeasurementReactive oxygen species (ROS) were determined by spectrophotometrical measurement of MDA levels in renal tissue (Kim and Vaziri, 2010) following homogenization with PBS containing butylatedhydroxytoluene (BHT, Cell Biolabs, Inc, Te Huissen, Netherlands) and expressed as micromoles of MDA per milligram of tissue.

H2S Measurement in BloodSulfide antioxidant buffer was prepared from 25 g of sodium salicylate, 6.5 g of ascorbic acid and 8.5 g of sodium hydroxide in 100 mL of distilled water at pH 13. Next, 100µL

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of the sulfide antioxidant buffer was added to 100 µL whole blood. A sulfide ion selective electrode was immersed into the mixture and the electrode potential was measured. The sulfide ion concentration of the serum was calculated using a standard curve of Na2S.9H2O in the sulfide antioxidant buffer, according to the manufacturer’s instructions (Lazar Research Laboratories, Inc. Los Angeles, CA, 90036 USA).

Statistical Analysis and Data PresentationVariables are expressed as mean ± SEM. Differences between groups were tested for significance using a One-Way ANOVA with Bonferroni Post-Hoc testing for normally distributed values or a Kruskal-Wallis ANOVA on ranks. P-values < 0.05 were considered statistically significant (SPSS version 22).

RESULTS

Renal function is preserved during hibernationHibernating Syrian hamsters (Mesocricetus auratus) underwent repetitive cycles of torpor and arousal, while summer euthermic hamsters remained fully active (supplemental figure S1). Renal function was preserved during torpor-arousal cycle, as shown by unchanged serum creatinine levels throughout the torpor-arousal cycle in comparison with summer euthermic controls (figure 1A; p > 0.05). In accord, (immune)histochemistry of renal tissue showed no changes in morphological features, and the absence of markers for tubular damage (KIM-1), influx of immune cells (ED-1, HIS-48) and fibrosis (α-SMA). and preservation of mitochondrial structure in glomerular and proximal tubular cells throughout the hibernation phases (supplementary figure S1).

Blood H2S and renal H2S-producing enzymes are increased during torpor To investigate the role of H2S during hibernation, blood H2S level and the renal expression of 3 H2S-producing enzymes (CBS, CSE and 3-MST) were measured. The blood H2S level increased about 2-fold during early torpor and increased even further towards the end of torpor, followed by a substantial decrease during arousal close to summer euthermic levels (figure 1B). In kidney, CBS expression increased about 2-fold during early and late torpor compared to summer euthermic hamsters, reduced during early arousal and increased again during late arousal (figure 1C,D). Unlike CBS, CSE expression was significantly upregulated only during late torpor (figure 1C,E). Finally, 3-MST expression followed a pattern similar to CBS and increased 3-fold during early and late torpor, normalized during early arousal and increased two-fold during late arousal (figure 1C,F). Thus, the blood H2S level and expression of H2S-producing enzymes are increased during torpor and decreased upon arousal.

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Inhibition of H2S production precludes subsequent torpor bouts To further substantiate the involvement of H2S during natural hibernation, summer euthermic (SE), winter euthermic (WE) and hibernating animals (HIB) were i.p. implanted with mini-pumps delivering AOAA, a non-selective inhibitor of H2S production. In HIB, AOAA was infused from the start of arousal. Importantly, AOAA precluded hibernating hamsters to re-enter torpor in contrast to saline-infused HIB animals (figure 2A, S2E,F; p < 0.001). Hence we decided to euthanize animals at similar body temperatures, i.e. 4 hours after induction of arousal in saline-infused hamsters. Blood H2S levels were similar in saline-infused HIB and SE, but were reduced below detection in saline-infused WE (figure 2E; p < 0.01). Blocking H2S production by AOAA dramatically reduced blood H2S, ranging from being non-detectable in HIB to a 6-fold reduction in SE. Thus, AOAA strongly reduced blood H2S levels during euthermia and precluded HIB animals from entering a subsequent torpor bout.

Inhibition of H2S production lowers renal expression of all H2S-producing enzymes and

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Figure 1. Renal function and expression of H2S-synthesizing enzymes in hibernation. Unchanged levels of serum creatinine (A) and renal MDA (ROS) formation (B) throughout the various phases of hibernation. However, endogenous H2S in the blood (C) and its synthesizing enzymes in the kidney (D-F) increased significantly in TE and TL compared to SE and decreased in AE and AL. Data are presented as mean ± SEM. */ # P < 0.05/0.01. SE = Summer Euthermic; TE = Torpor Early; TL = Torpor Late; AE = Arousal Early; AL = Arousal Late.

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Figure 2. Inhibition of endogenous H2S production precludes torpor bouts and induces renal injury. (A) AOAA infusion in hibernating animals reduced torpor duration as animals failed to enter subsequent torpor bouts. (B) Representative sections of the kidney (magnification x400) from all groups showing PAS, ED-1, α-SMA KIM-1, and HIS-48 stainings. Arrows point to positively stained areas (in brown). (C,D) Significant increase in serum creatinine level and renal MDA in WE and AOAA-infused HIB animals compared to SE animals. (E-H) H2S inhibition by AOAA resulted in a substantial decrease in blood H2S and its synthesizing enzymes in SE, WE and AOAA-infused HIB animals compared to saline-infused HIB animals. (I-M) quantification of immuno(histochemical) stainings of PAS, ED-1, α-SMA, KIM-1 and HIS-48. PAS= Periodic Acid Schiff; ED-1 = Macrophage marker; α-SMA = fibrosis marker; KIM-1 = Kidney Injury Molecule; HIS-48 = Neutrophil marker. SE = Summer Euthermic; WE = Winter Euthermic; HIB = Hibernating.

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Next, we substantiated whether H2S protects the kidney from damage during natural hibernation. In saline-infused groups, the renal expression of CBS, CSE and 3-MST was similar in HIB and SE, but strongly reduced in WE (figure 2F-H). AOAA infusion dramatically reduced the renal expression of all three H2S-producing enzymes in SE and HIB to levels also found in WE, with much larger downregulation in CSE expression compared to CBS and 3-MST (figure 2F-H). Expression of H2S-producing enzymes was mirrored by kidney damage, as determined by levels of serum creatinine and kidney damage markers. Creatinine and damage markers were considerably increased in saline-infused WE compared to HIB and SE (figure 2C,D,I-M). AOAA infusion strongly increased these parameters in HIB to levels similar to those found in AOAA-treated WE. In contrast, no increase in creatinine, ROS or damage markers was found in AOAA-infused SE. Thus, AOAA infusion strongly reduced renal expression of H2S-producing enzymes and produced renal damage in hibernating hamsters.

DISCUSSION

In the present study we identified an important role for H2S in the induction of torpor and preservation of renal integrity during hibernation in the Syrian hamster. Natural hibernation was characterized by an increase both in circulating H2S and in the renal expression of all 3 H2S-producing enzymes during torpor, and by the absence of kidney damage in all phases of hibernation. In addition, pharmacological inhibition of H2S by AOAA lowered serum H2S, precluded hibernating animals from re-entering a torpor bout and induced renal injury, as shown by an increase in serum creatinine levels, renal ROS formation and increased levels of markers of kidney damage. The AOAA-induced increase in renal injury also attenuated the upregulation of H2S-producing enzymes observed in saline-infused hibernating animals. Thus, endogenous H2S and its synthesizing enzymes are crucial in the induction of torpor as well as in preserving renal integrity during the torpor-arousal cycle.

Endogenous H2S is pivotal in induction of torpor during hibernationOur data strongly implicate H2S as a key player in natural hibernation. A first major finding of this study is that H2S seems to be of pivotal importance in the induction of torpor. Blood levels of H2S are substantially regulated during the hibernation cycle, with the highest values found in the torpor phase. Moreover, blockade of endogenous H2S production by infusion of the non-specific inhibitor, AOAA, substantially lowered blood H2S levels below detection limits, precluded hibernating animals from re-entering torpor. Induction of torpor is characterized by an active and rapid inhibition of metabolism with subsequent lowering of body temperature towards the ambient temperature (Heldmaier et al., 2004). H2S is a good candidate to convey the initial suppression of metabolism in torpor, since the compound suppresses metabolism

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by reversibly inhibiting oxidative phosphorylation through inhibition of mitochondrial complex IV (cytochrome c oxidase), the terminal enzyme in the mitochondrial electron transport chain (Beauchamp et al., 1984, reviewed in Szabo et al., 2014), particularly under normoxic conditions (such as at the entrance of torpor). However, H2S actually supports mitochondrial function at the level of coenzyme Q (Goubern et al., 2007) and may help in maintaining ATP levels under hypoxic conditions (Fu et al., 2012, Modis et al., 2013; reviewed in Szabo et al., 2014), as encountered during the torpor bout. The decreasing rate of H2S oxidation as tissue oxygen level drops during torpor may further assist in the maintenance of biologically active H2S in the tissue (Olson, 2008). Thus, H2S seems a prime candidate to manage both facets of torpor as increased levels during torpor may both assist in torpor induction and in the maintenance of tissue viability during torpor by safeguarding ATP production.

The importance of H2S in natural torpor is in keeping with observations in non-hibernating mouse kept under low ambient temperatures, in which the exogenous administration of H2S gas induces a hibernation-like state (Blackstone et al., 2005; Blackstone and Roth, 2007). Curiously, the level of blood H2S of torpid hamster is of the same order of magnitude as the one reported in H2S-treated mice, i.e. both in the low µM range. Importantly, reversal of the artificial hibernation-state produced by exogenous H2S is dependent on increasing the ambient temperature to induce rewarming in the animal. In contrast, rewarming in natural hibernation is initiated from within by heat generated by uncoupled oxidative phosphorylation in brown fat (Zivadinovic et al., 2005), leaving open the option that the induction of arousals in natural hibernation may be primarily depend on the initiation of brown fat oxidation.

Endogenous H2S is produced from L-cysteine in various mammalian tissues by a variety of biochemical pathways catalyzed by two cytosolic enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) (Wang, 2002; Kimura, 2011) and a mitochondrial enzyme, 3-mecaptopyruvate sulfurtransferase (3-MST) (Olson et al., 2010; Kimura, 2011). All 3 H2S-generating enzymes are highly expressed in the kidneys, mainly in the renal proximal tubules (Ishii et al., 2004; Li et al., 2006; Kimura, 2011). These observations are in agreement with previous reports in which lung H2S and CBS increase during torpor in hibernating Syrian hamsters (Talaei et al., 2012) and with the reported elevation of unbound free sulfide with high cysteine levels in the blood of hibernating Scandinavian brown bears (Revsbech et al. 2014).

Blood H2S levels The 2-3 fold increase in the expression levels of H2S-producing enzymes in kidney during torpor closely matches the 2-3 fold increase in endogenous H2S production, which level was also reduced following arousal. Consequently, H2S levels in blood throughout hibernation closely match those of the expression of H2S-producing enzymes in kidney. It is still not clear

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what constitutes the source of blood H2S. Apart from the kidney, H2S-producing enzymes are also abundantly expressed in the brain, liver and the cardiovascular system (Wang, 2002; Szabo, 2007; Kimura, 2011). Further, H2S is also produced by CSE in erythrocytes and released into circulation (Bearden et al., 2010). Therefore, one could surmise that the H2S-producing enzymes in these sources may contribute to the high levels of circulating H2S during torpor.

Renal H2S formation and kidney damageA second obvious finding of the current study implicates H2S as a key player in precluding organ damage during the torpor-arousal cycle. The expression of all 3 H2S-producing enzymes in the kidney is strongly increased during torpor. During arousal, the expression patterns differ substantially; whereas CSE is normalized to SE levels, expression of CBS – and to a lesser extent that of 3-MST – remain increased compared to SE. These observations in kidney are consistent with our previous findings in lung tissue, where both CBS expression and lung H2S production is increased during torpor and normalized upon arousal (Talaei et al., 2012). Further, blocking the H2S production with AOAA significantly reduced the expression levels of all 3 H2S-producing enzymes in kidney, which was accompanied by a marked increase in serum creatinine, renal production of ROS, pathological changes in glomerular and tubular structures, increased expression of the damage markers such as α-SMA and KIM-1, and the influx of neutrophils and macrophages. The observed renal protection by H2S is in agreement with the organoprotective property of exogenously applied H2S in several animal studies (Esechie et al., 2008; Bos et al., 2009; Bos et al., 2013; Lobb et al., 2014). The mechanism by which H2S induces organ protection is not completely understood. Blocking endogenous H2S during torpor in the present study resulted in a substantial increase in the level of ROS production, suggesting that H2S may act an antioxidant to inhibit ROS production during torpor-arousal cycle and prevent oxidative stress in the kidney. Alternatively, increased H2S may recruit additional antioxidant pathways e.g. through induction of the expression of various antioxidant genes upon activation of nuclear factor erythroid 2-related factor (nrf2) (Calvert et al., 2009). Therefore, both H2S antioxidant properties or activation of endogenous antioxidant pathways may protect the kidney during hibernation.

A third and remarkable finding in the current study is that WE hamsters show very low levels of blood H2S and reduced expression of all renal H2S-producing enzymes. Moreover, in contrast to hibernating hamsters, WE hamsters displayed a substantial increase in serum creatinine, increased renal ROS production and, importantly, kidney damage. Curiously, these phenomena were not observed during (late) arousal in hibernating animals, which are at similar body temperature (and presumably metabolic rate) as WE. Possibly, this difference is related to the duration of the housing under 5ºC ambient temperature, lasting only about 4

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days for arousals, whereas amounting several weeks in WE, exhausting defense mechanisms and provoking damage in the latter. Further, AOAA infusion induces (excess) renal damage in WE and HIB and decreases the expression of H2S-producing enzymes in the kidney. In contrast, AOAA infusion in SE did not provoke kidney damage, although blood H2S level and expression of renal H2S-producing enzymes were significantly reduced. Important to note is that HIB and WE animals have a substantial higher metabolic rate compared to SE due to low ambient temperatures. Taken together, these data suggest that H2S provides renal protection at high metabolic demand, possibly by fueling ATP production (Modis et al., 2013; reviewed in Szabo et al., 2014), whereas it is of minor significance under baseline metabolic conditions.

Potential applications of H2S-induced hibernation-like state in biomedicineThe induction of torpor by H2S and the cell protection by upregulation of its synthesizing enzymes holds a substantial promise in human medicine. H2S has been found to protect cells and organs against inflammatory responses in animal models of inflammatory diseases (Whiteman and Winyard 2011). More recently is the observation of cyto- and organoprotective properties of H2S in the field of hypoxic- and ischemia/reperfusion injury (Lobb et al., 2012; Lobb et al., 2014). Therefore, elucidating the exact mechanisms of H2S-induced hibernation-like state will add to novel therapeutic approaches biomedicine.

In conclusion, we have demonstrated for the first time that the H2S system plays a pivotal role in the induction of torpor and the regulation of the torpor-arousal cycle in mammalian hibernators. Moreover, it is of major importance to protect kidney under extreme physiological conditions. Threats encountered during hibernating conditions resemble specific clinical situations, such as accidental hypothermia, hypothermic organ preservation for transplantation and ischemia/reperfusion injuries. Therefore, hibernation may represent a unique natural model providing strategies to cope with such conditions. Hence, unraveling the exact molecular mechanisms involved in the induction of torpor, the timing of arousal and the preservation of organ function may lead to novel pharmacological strategies relevant to human medicine.

CONFLICT OF INTEREST STATEMENT

There are no conflicts of interest.

ACKNOWLEDGEMENTS

This research was funded by Groningen University Institute for Drug Exploration (GUIDE). We thank Prof. Frans Kroese and Prof. Harry van Goor (University Medical Center Groningen, Netherlands) for kindly providing the HIS-48 and CSE antibody respectively.

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SUPPLEMENTARY MATERIAL

SE TE TL AE AL

PAS

KIM-1

ED-1

HIS-48

α-SMA

Supplementary figure (S1). Renal integrity is preserved during torpor-arousal cycle. Representative sections of the kidney (magnification x400) from hamsters in various stages of hibernation showing PAS, KIM-1, α-SMA, ED-1, and HIS-48 stainings showing preservation of the glomerular and interstitial compartments of the kidney during the torpor-arousal cycle. PAS= Periodic Acid Schiff; ED-1 = Macrophage marker; α-SMA = fibrosis marker; KIM-1 = Kidney Injury Molecule; HIS-48 = Neutrophil marker. SE = Summer Euthermic; TE = Torpor Early; TL = Torpor Late; AE = Arousal Early; AL = Arousal Late.

A

CBS CSE 3-MST

63kDa 45kDa 33kDa

SE TE TL AE AL

B

CBS CSE 3-MST

63kDa 45kDa 33kDa

SE WE HIB

AOAA + - - + - +

Supplementary figure (S2). Full immunoblots of the renal expression of the three H2S-producing enzymes. Expression was measured for CBS, CSE and 3-MST in the kidney. (A) The expression levels of all three H2S-producing enzymes increased during torpor and reduced following arousal. (B) AOAA infusion decreased the expression levels of all three H2S-producing enzymes in SE, WE and HIB animals. SE = Summer Euthermic; TE = Torpor Early; TL = Torpor Late; AE = Arousal Early; AL = Arousal Late; WE = Winter Euthermic; HIB = Hibernating.

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4Induction of a Torpor-like state by 5’-AMP: a Role for

Endogenous H2S Production?

George J. Dugbartey1, Hjalmar R. Bouma1,2, Arjen M. Strijkstra3, Ate S. Boerema3,4, Robert H. Henning1,*

1Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

2Department of Rheumatology and Clinical Immunology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

3Departments of Chronobiology and Molecular Neurobiology, Groningen Institute for Evolutionary Life Sciences, University of Groningen, 9700 RB Groningen, The

Netherlands

4Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

Submitted

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ABSTRACT

Introduction: Therapeutic hypothermia is used to reduce ischemia/reperfusion injury (IRI) during organ transplantation and major surgery, but does not fully prevent organ injury. Interestingly, hibernating animals undergo repetitive periods of low body temperature called ‘torpor’ without signs of organ injury. Recently, we identified an essential role of H2S in entrance into torpor and preservation of kidney integrity during hibernation. A torpor-like state can be induced pharmacologically by injecting 5’-AMP. The mechanism by which 5’-AMP leads to the induction of a torpor-like state, and the role of H2S herein, remains to be unraveled.

Methods: To study the role of H2S on the induction of torpor, AOAA was administered before injection with 5’-AMP to block endogenous H2S production in Syrian hamster. To assess the role of H2S on maintenance of torpor induced by 5’-AMP, additional animals were injected with AOAA during torpor.

Results: During the torpor-like state induced by 5’-AMP, the expression of H2S synthesizing enzymes and plasma levels of H2S were increased. Blockade of these enzymes inhibited the rise in the plasma level of H2S, but neither precluded torpor nor induced arousal. Remarkably, blockade of endogenous H2S production was associated with increased renal injury.

Conclusion: Induction of a torpor-like state by 5’-AMP does not depend on H2S, although production of H2S seems to attenuate renal injury. Unraveling the mechanisms by which 5’-AMP reduces the metabolism without organ injury may allow optimization of current strategies to limit (hypothermic) IRI and improve outcome following organ transplantation, major cardiac and brain surgery.

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INTRODUCTION

Therapeutic hypothermia is a commonly used technique to prevent ischemia/reperfusion injury (IRI) during major cardiac and neuronal surgery and following cardiopulmonary resuscitation. Although hypothermia reduces ischemia by lowering the metabolism, therapeutic hypothermia does not completely preclude organ injury. The generation of reactive oxygen species is the major culprit in IRI (Carden and Granger, 2000). Interestingly, hibernating animals cycle through a state of lowered metabolism with a profoundly reduced body temperature called ‘torpor’ and periods of euthermia called ‘arousal’, without gross signs of organ injury (Zancanaro et al., 1999; Talaei et al., 2011; Jani et al., 2011; Carey et al., 2013). The duration of a torpor bout depends on the species and varies from several days to a month. The body temperature during torpor may be reduced towards freezing point, and is typically close to the ambient temperature (Barnes, 1989; Talaei et al., 2011; Talaei et al., 2012; Bouma et al., 2013a; Bouma et al., 2013b; de Vrij et al., 2014). Recently, we demonstrated that the induction of torpor and the prevention of kidney injury depend on the endogenous formation of hydrogen sulfide (H2S) in the Syrian hamster (see Chapter 3). Moreover, lung tissue H2S are increased during torpor in the Syrian hamster (Talaei et al., 2012) and plasma levels of unbound free sulfur are augmented in hibernating brown bears (Revsbech et al., 2014). Endogenous H2S can be produced by cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate-sulfurtransferase (MST). During torpor in the Syrian hamster, CBS expression is increased in pulmonary tissue (Talaei et al., 2012), while the expression of all three enzymes increases in kidney (see Chapter 3).

A torpor-like state can be induced pharmacologically in non-hibernating animals through inhalation of H2S or injection of 5’-adenosine monophosphate (5’-AMP), thereby mimicking natural torpor (Zhang et al., 2006; Swoap et al., 2007; Blackstone et al., 2005; Bouma et al., 2010). Fasting of mice housed under constant darkness, stimulates torpor behavior which is associated with increased levels of 5’-AMP in plasma (Zhang et al., 2006). Interestingly, H2S governs protection against lethal hypoxia in mice (Blackstone et al., 2007). Infusion of 5’-AMP activates the molecular energy sensor adenosine monophosphate kinase (AMPK), which mediated the protective effects of ischemic preconditioning on IRI (Bouma et al., 2010). Further, infusion of 5’-AMP in rats limits activation of mitogen-activated protein kinases (MAP-kinases) and NFkB and pulmonary inflammation in models of endotoxemia (Miao et al., 2012; Wang et al., 2014). The mechanisms underlying 5’-AMP mediated induction of a torpor-like state remain to be unraveled. Given the similarity of 5’-AMP and H2S on the induction of this torpor-like state and the preservation of organ integrity, we hypothesized that 5’-AMP may mediate its effects through stimulation of H2S production. To study whether the induction of a torpor-like state and preservation of kidney integrity by 5’-AMP depends

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on H2S, we measured the effect of 5’-AMP on activity, body temperature, kidney function and morphology in Syrian hamsters that were co-infused with either saline or the non-specific inhibitor of H2S production, amino-oxyacetic acid (AOAA). To exclude the influence of interspecies differences, we studied involvement of H2S in 5’-AMP induced torpor and the prevention of kidney injury in a natural hibernator, i.e. Syrian hamster, the same species in which we revealed the essential role of H2S in the induction of natural torpor and preservation of renal integrity (see Chapter 3).

MATERIALS AND METHODS

AnimalsPrior to experiments, Syrian hamsters (Mesocricetus auratus) were fed ad libitum using standard animal lab chow and animals were housed under normal light/dark conditions (L: D-cycle 12: 12 hours) at an ambient temperature of 20-25°C. Animals were randomly assigned to one of four groups, being control (n = 7), 5’-AMP with saline (n = 6), 5’-AMP with AOAA prior to torpor (n = 6; AOAA early) and 5’-AMP followed by AOAA during torpor (n = 6; AOAA late). The Animal Experimental Committee of the University of Groningen, The Netherlands, approved the animal experiments.

ExperimentThree weeks before the experiment, i-Button temperature loggers (Maxim Direct, France, DS1920 model) sealed in paraffin, were implanted intraperitoneally under isoflurane and analgesia with flunixin/meglumine (2 mg/kg). A blood sample (300 microliter) was obtained for baseline creatinine and H2S levels. One day before start of the experiment, animals were housed individually in a climate-controlled room at 5°C. After 24 hours, animals were injected intraperitoneally with saline or AOAA (100 mg/kg) followed by 3 µmol/g 5’-AMP (in 0.9% saline, pH 7.5; Sigma Aldrich, The Netherlands) to induce a torpor-like state. Animals were euthanized by injecting an overdose of pentobarbital intraperitoneally 10 hours after injection of 5’-AMP. Next, a blood sample was drawn by cardiac puncture. Kidney samples were snap-frozen in liquid nitrogen and fixated in formaldehyde.

(Immune)histochemistryKidney samples were fixated in 4% paraformaldehyde for 3 hours at room temperature followed by 4°C for 24 hours. Next, samples were dehydrated using a decreasing series of ethanol for 12 hours and embedded in paraffin. Four µm thick sections were deparaffinized in xylene (twice 5 minutes), followed by rehydration in a decreasing series of ethanol and distilled water. To evaluate changes in glomerular and tubular morphology, the kidney sections were stained with hematoxylin/eosin. Renal sections were examined blindly by two independent

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observers (Gross et al., 2006). Glomerular damage was scored semiquantitativily in 100 glomeruli from 0 to 4 (El Nahas et al., 1991) and tubulointerstitial damage was quantified on the basis of tubular dilatation, atrophy of epithelial cells and widening of tubular lumen (Gross et al., 2006). To evaluate the renal damage, sections were stained for kidney injury molecule (KIM-1, diluted 1: 50 v/v), a marker for renal tubular damage (Santa Cruz, The Netherlands), ED-1, a marker for macrophages (CD68, diluted 1: 500 v/v, Serotec Ltd, United Kingdom). Secondary and tertiary antibodies used are Horse Radish Peroxidase (HRP)-linked polyclonal rabbit anti-mouse IgG (diluted 1: 100 v/v), HRP-linked polyclonal rabbit anti-goat IgG (diluted 1: 100 v/v), and HRP-linked polyclonal goat anti-rabbit IgG (diluted 1: 100 v/v). Kidney sections were subjected to antigen retrieval in 0.1M Tris/HCl buffer (pH 9.0) by overnight incubation at 80°C. Next, sections were washed in PBS and blocked in 500 µl of 30% H2O2 for 30 minutes followed by incubation with the appropriate primary antibody for 60 minutes at room temperature. Following an additional washing step with PBS, samples were incubated for 30 minutes at room temperature with the appropriate secondary antibody and then with tertiary antibody at room temperature for 30 minutes. Finally, following a last washing step, samples were incubated with either DAB or AEC for 10-20 minutes and covered in either Depex mounting medium or DAKO Faramount aqueous mounting medium, and cover slips were applied.

Western BlottingFrozen kidney tissue samples (~500 mg) were homogenized in 400 µl RIPA buffer, consisting of 40 µl protease inhibitor cocktail (prepared according to the manufacturer’s instructions, Roche, The Netherlands), 2.5 mM sodium orthovanadate (Sigma Aldrich, The Netherlands) and 10 mM β-mercaptoethanol (Sigma Aldrich, The Netherlands). After 30 minutes incubation on ice, the homogenized samples were centrifuged at 14.000 g at 4°C for 20 minutes. Supernatants were collected and protein concentrations were determined using a Bradford protein assay, according to the manufacturer’s prescriptions (Bio-Rad, Germany). Samples were boiled for 5 minutes. SDS-polyacrylamide gel electrophoresis was run using 40 µg of protein per slot at 100V for 60 minutes. Proteins were then wet blotted onto nitrocellulose membranes (Bio-Rad, Germany) using a transfer buffer solution containing 0.25mM Tris (pH 8.5), 192 mM glycine and 10% v/v methanol at 4°C for 60 minutes at 0.3 mA. Next, the nitrocellulose membranes were blocked for 30 minutes in TBS + Tween-20 (50mM Tris-HCl, pH 6.8, 150mM NaCl, 0.05% v/v Tween-20) supplemented with 5% w/v skim milk. After decantation of the blocking buffer, membranes were incubated overnight at 4°C with the primary antibody diluted 1: 1000 v/v in 3% BSA/TBST (anti-CBS and anti-3-MST, Santa Cruz, The Netherlands; anti-CSE, Abnova, USA). Subsequently, membranes were washed three times in TBS buffer and incubated with HRP-linked polyclonal rabbit anti-goat IgG secondary antibody (1: 1000 v/v dilution) in TBS + Tween-20 supplemented with 3% BSA (w/v) for

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60 minutes. Blots were developed using the SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, USA) according to the manufacturer’s protocol. Protein bands were visualized using the Gene Genome system (Westburg B.V., The Netherlands) and band intensities were quantified using Gene Tools software (Westburg B.V., The Netherlands). β-actin was used as a house-keeping protein to normalize protein concentrations.

Plasma H2S MeasurementSulfide antioxidant buffer was prepared from 25 g of sodium salicylate, 6.5 g of ascorbic acid and 8.5 g of sodium hydroxide in 100 mL of distilled water and pH adjusted to ≥ 13. Next, 100 µL of the sulfide antioxidant buffer was added to 100 µL plasma samples. A sulfide sensitive electrode (Lazer Research Laboratories Inc., USA) was immersed into the mixture and the electrode potential was monitored and the stabilized mV reading was recorded. The sulfide ion concentration of the plasma was calculated using the electrode standardization curve prepared from 10 mL of the sulfide antioxidant buffer and 24 mg of Na2S.9H2O, according to the manufacturer’s guide.

Statistical AnalysisAll values are expressed as mean ± standard error of the mean (SEM). Differences between groups were tested for significance using a One-Way ANOVA. P-values < 0.05 were considered statistically significant. Significant differences were calculated with SPSS version 22 and graphs were produced using Sigmaplot version 12.5 for Windows.

RESULTS

Endogenous H2S is not essential for the induction of a torpor-like state by 5’-AMPInjection of 5’-AMP in summer euthermic hamsters induced a torpor-like state as characterized by inactivity and marked drop in core body temperature from 37°C to 7°C, which lasted at least 10 hours (figure 1A). At the time of euthanization, all animals were in the torpor-like state. To determine the involvement of H2S in 5’-AMP-induced torpor, we measured the plasma levels of H2S and blocked endogenous production either before or during torpor by AOAA. 5’-AMP-induced torpor significantly increased endogenous H2S level in the plasma by 47.7% as compared to summer euthermic control animals (figure 1B; p < 0.05). Although the increased plasma level of H2S suggests a role for H2S in the induction of torpor, blocking endogenous H2S production by AOAA prior to 5’-AMP injection did not prevent the 5’-AMP-induced hypothermia (figure 1A), although it substantially decreased plasma H2S level at 10 hours following injection of 5’-AMP to 11.4% of control animals (figure 1B; p < 0.01). Further, injection of AOAA during the torpor-like state, 4 hours after injection of 5’-AMP, reduced the plasma level of H2S to 22.7% at 10 hours following injection of 5’-AMP (figure 1B; p < 0.01).

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Despite the effect of AOAA on the plasma level of H2S, blockade of H2S production did not induce arousal. To determine whether the increased plasma level of H2S is due to changes in the amount of H2S producing enzymes, we measured the amount of CBS, CSE and 3-MST in the kidney by Western Blot. As expected based on the effect of 5’-AMP on plasma H2S levels, administration of 5’-AMP resulted in a substantial upregulation of CBS, and smaller upregulation of CSE and 3-MST, as compared to control animals (figure 1C-E; p < 0.05). Further, injection of AOAA, either prior to or during torpor, resulted in a significant lower amount of CBS, CSE and 3-MST as compared to control animals (figure 1C-E; p < 0.05). Thus, injection of 5’-AMP induces a torpor-like state in hamsters, which is not precluded by blocking H2S production, although 5’-AMP increases the plasma level of H2S, potentially due to an increased amount of CBS, CSE and 3-MST.

Figure 1. 5’-AMP induces torpor in natural hibernators and increases plasma H2S and renal expression of H2S synthesizing enzymes. (A) Administration of 5’-AMP (at t=0) resulted in a drop in core body temperature from 37 °C to ~7 °C in all three experimental groups after 10 h of 5’-AMP injection, which was not affected by early or late administration of AOAA. (B) Administration of 5’-AMP (at t=0) significantly increased plasma H2S level compared to control animals, while AOAA injection prior to or 4 h after 5’-AMP administration reduced plasma H2S level. (C-E) Administration of 5’-AMP (at t=0) upregulated the renal expression of CBS, CSE and 3-MST, while expression was decreased by AOAA injection prior to or 4 h after 5’-AMP administration. C=control animals */**, p < 0.05/0.01 compared to control. Data are presented as mean ± SEM.

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Blocking H2S production markedly increases plasma creatinine in 5’-AMP induced torpor In order to assess kidney function, we measured the plasma level of creatinine in all hamsters. During the torpor-like state induced by 5’-AMP, the level of creatinine in plasma is slightly increased as compared to control animals (figure 2B; p < 0.05). Blocking endogenous H2S production with AOAA, either prior to the induction or during the torpor-like state, profoundly increased the plasma creatinine level, reaching levels around threefold higher as compared to control animals (figure 2B; p < 0.01). Thus, induction of a torpor-like state by 5’-AMP leads to slightly elevated plasma creatinine level, which is augmented upon inhibition of endogenous H2S production. Potentially, H2S mediates preservation of the kidney function during the torpor-like state induced by 5’-AMP.

Blocking H2S production is associated with glomerular and tubulointerstitial injury Induction of torpor by 5’-AMP did not affect the morphology of glomeruli (figure 2A,C p > 0.05), but was associated with minor signs of tubulointerstitial injury associated with influx of a low number of macrophages into the renal interstitium as compared to the control group (figure 2A,C,E; p < 0.05). Further, injection of 5’-AMP resulted in a slight increase in the amount of KIM-1 protein in the renal tubules as compared to control group (figure 2A,D; p < 0.05). To further substantiate the role of endogenous H2S production on renal morphology, renal sections from animals treated with AOAA were analyzed. Blocking endogenous H2S production with AOAA, either prior to or during 5’-AMP-induced torpor, lead to pronounced glomerular, tubular and interstitial damage that was associated with a substantial influx of macrophages in the renal interstitium as compared to control animals (figure 2A,C,E; p < 0.01). The higher level of renal injury during torpor is reflected by an increased amount of KIM-1 following blockade of H2S production (figure 2A,D; p < 0.01). There was no significant difference in KIM-1 expression between early and late AOAA groups (figure 2D; p > 0.05). Hence, 5’-AMP is associated with minor signs of tubulointerstitial injury. Although H2S is not essential for the induction of torpor, blockade of endogenous H2S production leads to pronounced glomerular and tubulointerstitial injury, thus suggesting a protective role of H2S against renal injury.

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Figure 2. Blocking H2S production during 5’-AMP-induced torpor provokes kidney injury. (A) Representative photographs of kidney tissue; magnification x400. (B) Administration of 5’-AMP significantly increased plasma creatinine level compared to control animals, which was further elevated by AOAA injection prior to or 4 h after 5’-AMP administration. (C-E) Quantification of renal injury and markers, demonstrating modest renal injury in 5’-AMP induced torpor, which is grossly amplified by AOAA administration. HE = hematoxylin eosin staining; KIM-1 = Kidney Injury Molecule 1; ED-1 = antibody against CD68 specific for macrophages. */** represents p < 0.05/0.01 compared to control.

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DISCUSSION

H2S is not essential for the induction of a torpor-like state by 5’-AMP, but seems to play a

key role in preserving kidney function and integrityIn the current study, we reveal that the induction of a torpor-like state by 5’-AMP in natural hibernators is not dependent on production of endogenous H2S. Blocking H2S production by AOAA, did not preclude torpor and did not induce an arousal. Remarkably, the torpor-like state induced by 5’-AMP is associated with increased plasma levels of H2S. The increased amount of CBS, but not CSE and 3-MST, may account for the higher levels of H2S. Pharmacological induction of torpor by 5’-AMP leads to a slight increase in the plasma creatinine level and minor signs of tubulointerstitial injury, associated with a small influx of macrophages. Blockade of endogenous H2S production reveals an essential role in the preservation of kidney function and renal integrity, since infusion of AOAA either before or during torpor lead to a profound increase in plasma creatinine level that was associated with glomerular and tubulointerstial damage with influx of macrophages into the kidney. Thus, in line with the role of endogenous H2S in preserving renal integrity during natural torpor (see Chapter 3) and consistent with the renal protection during exogenously applied H2S in mouse (Bos et al., 2009; Bos et al., 2013; Lobb et al., 2014), H2S seem to play a key role in mediating kidney preservation during pharmacologically induced torpor by 5’-AMP. However, H2S is not involved in the induction or maintenance of torpor induced by 5’-AMP.

The mechanisms underlying 5’-AMP induction of torpor remain to be unraveled As described, our data demonstrate that H2S does not play an essential role in the induction of torpor by 5’-AMP. As an alternative explanation, activation of adenosine receptors, adenosine monophosphate protein kinase (AMPK) and adenylate kinase may lead to the induction of a torpor-like state. Swoap et al. (2007) suggested that activation of adenosine receptors following dephosphorylation of 5’-AMP to adenosine may lead to lowering of the body temperature secondary to a reduction in cardiac output. This hypothesis is supported by the observation that not only (5’-)AMP, but also ATP, ADP and adenosine can induce a torpor-like state in mice and that lowering of the body temperature is blunted by co-treatment with an adenosine receptor antagonist (Swoap et al., 2007). The second hypothesis describes a role for AMPK, a key enzyme that plays a role in cellular energy homeostasis, which can be activated by depletion of cellular ATP (and consequently elevate AMP), and switches off energy consuming energetically demanding metabolic pathways (Peralta et al., 2001; Adams et al., 2004; Lindsley and Rutter, 2004; Swoap et al., 2007). Activation of signaling pathways downstream of AMPK promote a shift from anabolic towards catabolic processes and thereby reduce energy expenditure of the cells. However, it is unclear whether this leads to torpor-like behavior of the animal. Furthermore, activation of AMPK by intracerebroventricular

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infusion of AICAR (a specific AMPK-activator) in yellow-bellied marmots (Marmota flaviventris) during interbout arousal does not induce torpor, but lead to increased food intake and even prevents the return to torpor (Florant et al., 2010). As a third hypothesis, relatively high levels of AMP lead to activation of adenylate kinase, which converts (5’-)AMP together with ATP to ADP. Injection of 5’-AMP may thereby lead to a relative ATP-depletion, which is implicated to reduce metabolism as observed during entrance into torpor (Lee et al., 2008). Hence, the mechanism by which 5’-AMP induces a torpor-like state, and potentially natural torpor as well, remain to be unraveled. We reveal that pharmacological induction of a torpor-like state by 5’-AMP does not depend on H2S.

ConclusionTaken together, we demonstrate that 5’-AMP induces a torpor-like state in natural hibernators, leading to a lowering of the body temperature that is independent of the activation of H2S system. Although H2S does not seem to play an essential role in the induction of a torpor-likes state by 5’-AMP, endogenous production of H2S seems to play an essential role in precluding glomerular and tubulointerstitial renal injury and maintaining renal function. The exact mechanism(s) through which 5’-AMP induces a torpor-like state is not yet understood. Unraveling these molecular mechanisms may lead to the development of novel pharmacological therapies to safely reduce the metabolism to limit (hypothermic) IRI and thereby improve the outcome following organ transplantation and major cardiac/brain surgery.

FINANCIAL & COMPETING INTERESTS DISCLOSURE

The authors declare that no competing financial interests exist.

ACKNOWLEDGEMENTS

This study was supported by a grant from Groningen University Institute for Drug Exploration (GUIDE). We are grateful to Prof. Harry van Goor (University Medical Center Groningen, Netherlands) for generously providing the CSE antibody.

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Bos EM, Wang R, Snijder PM, et al. Cystathionine γ-lyase protects against renal ischemia/reperfusion by modulating oxidative stress. J Am Soc Nephrol. 2013; 24(5): 759-70.

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5Dopamine Treatment Attenuates Renal Injury via

Production of H2S in a Rat Model of Deep Hypothermia and

Rewarming

George J. Dugbartey1, Fatemeh Talaei1, Martin C. Houwertjes2, Maaike Goris1, Anne H. Epema2, Hjalmar R. Bouma1,3, Robert H. Henning1

1Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

2Department of Anesthesiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

3Department of Rheumatology and Clinical Immunology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

Submitted

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ABSTRACT

Background: Hypothermia and rewarming produces organ injury through the production of reactive oxygen species (ROS). We previously found that cellular uptake of dopamine prevents hypothermia and rewarming-induced apoptosis by H2S formation through upregulation of the enzyme cystathionine β-Synthase (CBS) in cultured cells. Here, we investigate whether dopamine protects via H2S pathway in an in vivo rat model of deep hypothermia and rewarming, examining the kidney as a highly vulnerable organ.

Method: In 4 groups of Wistar rats (n=5 each) anesthetized with ketamine, body temperature was decreased to 15°C at a rate of 1°C per 3 minutes, maintained at this temperature for 3 h followed by rewarming at 1°C per 2 minutes. Rats were treated throughout the procedure with vehicle or dopamine infusion, and in the presence or absence of a non-specific inhibitor of H2S production, amino-oxyacetic acid (AOAA) and compared to non-cooled rats.

Results: Hypothermia and rewarming substantially lowered serum H2S level, the renal expression of H2S producing enzymes (CBS, CSE, 3-MST) and induced renal damage as demonstrated by pathological changes in glomeruli and tubuli, high KIM-1 expression and substantial influx of macrophages and neutrophils. AOAA further decreased serum H2S levels and H2S-producing enzymes, and aggravated renal damage and white blood cell influx. Infusion of dopamine increased serum H2S and the expression of H2S-producing enzymes, and fully protected kidneys from hypothermia and rewarming injury.

Conclusion: Dopamine infusion maintained H2S production and fully preserved renal integrity during deep hypothermia and rewarming in the rat.

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INTRODUCTION

Hypothermia represents a condition in which core body temperature drops below 35°C,1 interfering with normal metabolism and body function. Hypothermia can either be accidental or intentional, and can be further classified by severity. Deep hypothermia is clinically applied to preserve organs for transplantation and in major cardiac surgery to preserve organ integrity and function.2,3,4 Irrespective of its clinical usefulness, deep hypothermia is not a panacea. Hypothermia negatively affects oxygen and nutrient availability and the accumulation of metabolic waste, leading to organ injury, of which production of reactive oxygen species (ROS) is an important meditator.5,6 Further, restoration of blood supply upon rewarming results in excessive generation of ROS.7 Specifically in the kidney, hypothermia may result in acute tubular necrosis as a result of renal vasoconstriction and ischemia.8

Dopamine is a central and peripheral nervous system biogenic monoamine neurotransmitter, which can be also synthesized in non-neuronal tissues including proximal renal tubular cells by decarboxylation of L-DOPA.9,10 Dopamine has been reported to reduce damage following hypothermic storage of donor kidney transplant in a rat model11 and in human transplantation.12,13 We previously identified in cell culture and whole tissue slices that the mechanism of action of dopamine constitutes of an increased synthesis of the endogenous production of hydrogen sulfide (H2S) by cystathionine β-synthase (CBS).14 CBS is a cytosolic enzyme, which comprises the first and the rate-limiting step in the transsulfuration pathway that leads to production of H2S.15,16 In addition to CBS, endogenous H2S production may also be catalyzed by another cytosolic enzyme, cystathionine γ-lyase (CSE), and a mitochondrial enzyme, 3-mecaptopyruvate sulfurtransferase (3-MST).

To examine whether dopamine protects against kidney injury in the whole animal, we examined its actions in a rat model of deep hypothermia and rewarming under general anesthesia. Thus, we examined dopamine effects on kidney damage, blood H2S levels and expression of H2S producing enzymes in the absence and presence of amino-oxyacetic acid (AOAA), a non-specific inhibitor of endogenous H2S production.17

MATERIALS AND METHODS

AnimalsThe experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Groningen (DEC5920). Male Wister rats (Charles River, The Netherlands) with a weight of 400 ± 41g were used for this study. Rats were housed under standard conditions (standard rat chow; Hope Farms, Woerden, The Netherlands) and drinking water provided ad libitum throughout the study period.

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Experimental GroupsWistar rats were allocated to one of 5 experimental groups, being healthy non-cooled control (n=4, euthanized following brief isoflurane anesthesia), or one of the 4 experimental groups that underwent forced cooling and rewarming. Rats in the experimental groups (n=5 each) were treated with vehicle or dopamine, with and without administration of AOAA.

Experimental DesignFigure 1 shows a schematic representation of the experiment. Anesthesia was induced with 2.5% isoflurane in O2/air (1:1) followed by intubation and mechanical ventilation (Amsterdam Infant Ventilator; Hoekloos, Amsterdam, The Netherlands). Tidal volume was set to achieve normocapnia at a ventilation rate of 50 min-1 (0.5 sec inspiration time). Body temperature was measured rectally. Next, the carotid artery and jugular vein were cannulated to continuously monitor arterial blood pressure and heart rate, and take blood samples.45 Following this initial preparative phase, isoflurane was stopped and anesthesia was maintained by i.v. infusion of ketamine (5 µg/kg/min) combined with single bolus dose of pancuronium (1.5 mg/kg). Animals were maintained normothermic at 37.0 ± 0.3°C. In this phase, infusion of dopamine (5 µg/kg/min) was initiated at a constant infusion rate and maintained throughout the cooling and rewarming phase in the corresponding experimental groups. Animals treated with AOAA were injected intravenously with 20 mg/kg prior to cooling. Following this normothermic phase, hypothermia was induced by applying icepacks externally, producing an average decrease of body temperature of 1°C per 3 min. Upon reaching a body temperature of 25°C, the infusion rate of ketamine and dopamine were reduced by 50%. When the body temperature reached 15°C, AOAA treated groups received another i.v. bolus injection of 4 mg/kg. Three hours after reaching a body temperature of 15°C, rats were rewarmed at a rate of 1°C per 2 min until they reached a body temperature of 37 ± 0.3°C. Subsequently, rats were maintained normothermic for 60 min followed by euthanization under anesthesia. Arterial blood samples were taken directly after the preparative phase ended at 37°C, just prior to cooling after one h of normothermia at 37°C, upon reaching 15°C, 1 h after reaching 15°C, just prior to rewarming, and 1 h after rewarming to 37°C (figure 1). Plasma samples were collected in heparinized tubes. Tissue samples were snap-frozen in liquid nitrogen and stored at -80°C until further analysis or fixated followed by embedding in paraffin.

HistologyKidney samples were fixated in zinc solution (0.1M Tris buffer, pH 7.4 with 0.5g calcium acetate, 5.0g zinc acetate and 5.0g zinc chloride) for 6 hours at room temperature followed by storage at 4°C for 24 hours.14 Next, samples were dehydrated in xylene and a decreasing series of ethanol from 100% to 70% for 12 hours and then embedded in paraffin. Five µm thick sections were deparaffinized in xylene and hydrated in decreasing series of ethanol

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from 100% to 70% and distilled water. To evaluate glomerular and interstitial damage, the hydrated kidney samples were oxidized in 0.5% periodic acid solution (PAS) and stained with Schiff reagent and then counterstained with hematoxylin for 1 minute. Kidney sections were examined blindly by two independent observers for contraction of glomerular tuft, mesangial disorganization and shrinkage of glomerular content. The degree of glomerular damage was characterized at a magnification of 400x in 100 glomeruli by a semiquantitative scoring system with scores ranging from 0 to 4.44 Tubulointerstitial damage was examined and quantified on the basis of tubular dilatation, atrophy of epithelial cells and widening of tubular lumen using a scoring system described by Gross et al.9

ImmunohistochemistrySections were stained for kidney injury molecule (KIM-1), a marker for renal tubular damage (goat polyclonal, diluted 1:50 v/v, Santa Cruz), HIS-48, a marker for neutrophils (mouse monoclonal, generously provided by Prof. F.G.M. Kroese, Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, Groningen, The Netherlands), ED-1, a marker for macrophages (mouse monoclonal, diluted 1:500 v/v, Serotec Ltd, Oxford, UK). In addition, samples were stained for cystathionine beta-synthase (CBS, diluted 1:100 v/v in 1% phosphate buffered saline/bovine serum albumen (PBS/BSA). Secondary and tertiary antibodies used are horseradish peroxidase (HRP)-linked polyclonal rabbit anti-mouse IgG (1:100 v/v, Thermo Scientific, Etten-Leur, Netherlands), HRP-linked polyclonal rabbit anti-goat IgG (1:100 v/v), and HRP-linked polyclonal goat anti-rabbit IgG (1:100 v/v, Thermo Scientific, Etten-Leur, Netherlands). Paraffin sections were dewaxed using a xylol and a decreasing series of ethanol from 100% to 70%, and then subjected to antigen retrieval in 0.1M Tris/HCl buffer, pH 9.0, by overnight incubation at 80°C. Next, sections were washed three times in PBS and blocked in 500 µl of 30% H2O2 and 50µl PBS for 30 minutes followed by incubation with the appropriate primary antibody for 60 minutes at room temperature. Following an additional three times washing step with PBS, samples were incubated for 30 minutes at room temperature with the appropriate secondary antibody after which they were washed three times in PBS and then with tertiary antibody at room temperature for 30 minutes. In the CBS staining however, samples were incubated with secondary and tertiary antibodies for 60 minutes. Finally, following a last washing step, samples were incubated with AEC high sensitivity substrate chromogen for 10-20 min and covered in DAKO Faramount (Dako, Heverlee, Belgium) aqueous mounting medium, and cover slip was applied over the mounting medium.

Western BlottingFrozen kidney tissue samples (~500mg) were homogenized in 0.4 ml of RIPA buffer containing 100 mM protease cocktail inhibitor, 100 mM sodium orthovanadate and 10 mM

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mercaptoethanol. After 30 minutes of incubation on ice, the homogenized samples were centrifuged at 14000 rpm at 4°C for 20 minutes. The supernatants were collected and soluble protein concentrations were determined using a Bradford protein assay. Samples were boiled for 5 min, cooled and then frozen at -20°C until use. Sodium dodecyl sulfate -polyacrylamide gel electrophoresis was run using 40 µg of protein per slot at 100V for 60 minutes. Proteins were then wet blotted onto nitrocellulose membranes with a pore size of 0.45 µm (Bio-Rad Laboratories, Inc, Germany) using a transfer buffer solution that consisted of 0.25mM Tris (pH 8.5), 192mM glycine and 10% v/v methanol at 4°C for 60 min at 0.3mA. Following blotting, the nitrocellulose membranes were blocked for 30 minutes in TBS + Tween 20 (50mM Tris-HCl, pH 6.8, 150mM NaCl, 0.5% v/v Tween 20, Sigma-Aldrich, USA) with 5% w/v powdered skim milk (Sigma-Aldrich, Switzerland). After decantation of the blocking buffer, membranes were incubated overnight at 4°C with the following primary antibodies at a 1:1000 v/v dilution in 3% BSA/TBST: CBS (mouse monoclonal, Santa Cruz, Te Huissen, Netherlands), CSE (mouse monoclonal, Abnova, Walnut, CA, USA) and 3-MST (rabbit polyclonal, Santa Cruz, Te Huissen, Netherlands). Subsequently, membranes were incubated with HRP-linked polyclonal rabbit anti-goat IgG secondary antibody (1:1000 v/v dilution) in 3% BSA/TBST for 60 minutes and then blots were developed using the SuperSignal West Dura Extended Duration Substrate (IL, USA) according to the manufacturer’s protocol. Protein bands were visualized using Gene Genome (Westburg B.V., Leusden, The Netherlands) and band intensities were quantified using the associated Gene Tools software (Westburg B.V., Leusden, The Netherlands). β-actin was used as a house-keeping protein to normalize protein concentrations.

Measurement of Reactive Oxygen SpeciesTo determine the levels of reactive oxygen species (ROS) in renal tissue, samples from the left kidney were homogenized with 100 ml PBS containing butylatedhydroxytoluene (BHT, Cell Biolabs, Inc, Netherlands) and centrifuged at 10.000g for 5 minutes at 4°C. 50µl of SDS-Lysis solution (Cell Biolabs, Inc, Netherlands) was added to 50 µl of the supernatant and malondialdehyde (MDA) standards in separate microcentrifuge tubes followed by 5 minutes incubation at room temperature. Next, 125 µl of thiobarbituric acid (TBA) Reagent (Cell Biolabs, Inc, Netherlands) was added to each supernatant and standard and incubated at 95°C for 60 minutes. Following this incubation, the samples were cooled to room temperature in an ice bath for 5 minutes and then centrifuged at 1000 g for 15 minutes. The supernatants were transferred to another tube and 150 µl of 2-butanol (Merck, Darmstadt, Germany) was added, vortexed vigorously for 2 minutes and centrifuged for 5 minutes at 10,000g. The level of lipid peroxidation in the tissue sample was estimated by measuring the optical density at 532 nm on a spectrophotometer in 200 µl of the butanol fraction. Using the standard curve, readings were transformed into µM MDA per milligram of tissue.

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Serum H2S MeasurementSulfide antioxidant buffer was prepared from 25 g of sodium salicylate, 6.5 g of ascorbic acid and 8.5 g of sodium hydroxide in 100 mL of distilled water and pH adjusted to ≥ 13. One hundred µl of the sulfide antioxidant buffer was added to 100 µl serum samples. A sulfide sensitive electrode (Lazar Research Laboratories Inc., CA, USA) was immersed into the mixture and the electrode potential was monitored and the stabilized mV reading was recorded. The sulfide ion concentration of the serum was calculated using a standard curve prepared from a stock of 10 mL of the sulfide antioxidant buffer and 24 mg of Na2S.9H2O, according to the manufacturer’s instructions.

Statistical Analysis and Data Presentation All variables are expressed as mean ± standard error of the mean (SEM). Differences between groups were tested for significance using a One-Way ANOVA (Bonferroni Post-Hoc testing) and Repeated Measures ANOVA (Tukey HSD Post-Hoc testing). P-values < 0.05 were considered statistically significant (SPSS version 22).

RESULTS

Dopamine is largely without effect on hemodynamic changes in hypothermia and rewarmingThe cardiovascular effect of hypothermia and rewarming was assessed by continuous measurement of heart rate, blood pressure and arterial blood gas analyses. In all groups,

Figure 1. The hypothermia and rewarming model in the rat. Body temperature of ketamine anesthetized rats was lowered from 37°C using externally applied icepacks, at an average rate of ~1 °C per 3 minutes to reach a minimum body temperature of 15°C, which was then maintained for 3 h. Rats were then rewarmed at a rate of 1°C per 2 minutes until they reached a body temperature of 37 °C, which was maintained for 60 minutes until euthanization. Arrows indicate blood sampling at different time points.

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heart rate was reduced to about 75% of normothermic baseline values upon reaching 15°C (p < 0.01; figure 2). In addition, animals treated with dopamine experienced a significant increase in heart rate of about 15% during the normothermic phase (p < 0.05; figure 2), while dopamine-treated animals showed a similar drop in heart rate during hypothermia as compared to the other groups (figure 2). Heart rate was not affected by AOAA at any of the time points as compared to the other groups (figure 2).

Forced hypothermia resulted in a significant drop in basal systolic blood pressure (p < 0.001; figure 2), while rewarming restored systolic blood pressure to baseline values in all groups (figure 2). In addition, dopamine treatment was associated with a small increase in systolic blood pressure amounting about 9.1 ± 5.8 and 25.5 ± 11.0 % in normothermia, respectively before induction of hypothermia and after rewarming (p < 0.05; figure 2). AOAA did not affect

Figure 2. Hypothermia induced reduction in heart rate and blood pressure. Induction of hypothermia to 15 ºC resulted in a significant drop in (A) heart rate and (B) systolic blood pressure (closed symbols) and diastolic blood pressure (open symbols) in all groups compared to baseline values. Rewarming after 3 h of hypothermia (time = 6 h) restored heart rate and blood pressure to baseline values. Heart rate and blood pressure in dopamine-treated animals was significantly higher during the normothermic period prior to the induction of hypothermia (time = 1 h) and after the rewarming phase compared to other groups (p < 0.05). Timepoints t = 2 - 5 h represent the hypothermic period. Data are presented as mean ± SEM. V,D,A: P < 0.05; curve differs significantly from vehicle treated (V), dopamine treated (D) or AOAA treated (A), RM ANOVA with Kruksal-Tukey correction.

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systolic blood pressure. Diastolic blood pressure in all groups followed a similar profile as systolic blood pressure (figure 2). Taken together, dopamine slightly increased heart rate and blood pressure under normothermia, but was without effect on the marked reduction in these parameters during hypothermia.

Dopamine prevents metabolic effects of hypothermia and rewarmingArterial blood gas analyses revealed a significant drop in pH upon hypothermia as compared to baseline values at the start of the experiment (p < 0.01; Table 1). Whereas pH increased upon subsequent rewarming, it did not fully restore to baseline level (p < 0.05). Remarkably, dopamine treatment fully prevented the change in pH induced by hypothermia and rewarming (Table 1). Infusion of AOAA attenuated the normalization of blood pH by dopamine during hypothermia and rewarming periods (p < 0.05; Table 1). Thus, dopamine infusion precluded the acidosis induced by forced hypothermia.

Forced hypothermia led to a slight increase in partial arterial carbon dioxide pressure (PaCO2), substantial increase in serum lactate and a decrease in bicarbonate level (HCO3

-) (p < 0.05; Table 1). Rewarming partially restored lactate levels, although still significantly higher than baseline values (p < 0.05; Table 1). Infusion of dopamine prevented these changes (Table 1). Addition of AOAA in animals infused with dopamine partially precluded the beneficial effect of dopamine on HCO3

- and lactate values, while sole administration of AOAA amplified the effects of hypothermia and rewarming (Table 1). These data show that hypothermia is associated with metabolic lactate acidosis, which was fully attenuated by dopamine treatment.

The lactate acidosis upon hypothermia is likely due to hypoperfusion or poor oxygenation. Hypothermia substantially reduced the partial arterial pressure of oxygen (PaO2), which was restored upon rewarming, while dopamine treatment precluded changes in PaO2 throughout the experiment (p < 0.01; Table 1). Thus, infusion with dopamine prevented the decrease in PaO2 level upon hypothermia.

Dopamine prevents hypothermia and rewarming-induced kidney dysfunction and damageAs a marker of kidney function, we measured serum creatinine levels in all experimental groups throughout the procedure. Serum creatinine level increased in the vehicle-treated group during hypothermia. In addition, rewarming induced a further rise of the serum creatinine level (p < 0.05; figure 4B) as compared to the pre-procedure value. Infusion of dopamine prevented the increase in serum creatinine levels during both hypothermia and rewarming (p < 0.01; figure 4B). Dopamine maintenance of serum creatinine levels was unchanged by administration of AOAA (p > 0.05; figure 4B). However, AOAA treatment alone increased the level of serum creatinine to a level significantly higher than the vehicle-treated

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Table 1. Arterial blood gas analysisBody temp (°C)

Time from start (h)

Vehicle Dopamine AOAA Dopamine + AOAA

pH 37 0 7.38 ± 0.01 7.39 ± 0.01 7.39 ± 0.03# 7.38 ± 0.0137 1 7.38 ± 0.01 7.39 ± 0.02 7.38 ± 0.02 7.37 ± 0.0315 2 7.29 ± 0.02 7.41 ± 0.01 7.23 ± 0.02* 7.20 ± 0.02*

15 3 7.26 ± 0.04 7.30 ± 0.01 7.15 ± 0.02 7.19 ± 0.0315 4 7.22 ± 0.01 7.36 ± 0.02 7.13 ± 0.02 7.17 ± 0.0215 5 7.20 ± 0.01 7.37 ± 0.01 7.02 ± 0.01# 7.16 ± 0.0137 6 7.29 ± 0.03 7.39 ± 0.02 7.27 ± 0.02* 7.29 ± 0.02*

PaO2 37 0 23.1 ± 0.1 23.3 ± 0.3 23.4 ± 0.2 23.1 ± 0.237 1 23.1 ± 0.4 23.5 ± 0.2 23.0 ± 0.2 23.4 ± 0.3 15 2 12.2 ± 0.2* 23.0 ± 0.3 17.4 ± 0.5* 27.0 ± 0.215 3 12.2 ± 0.1 24.5 ± 0.1 14.8 ± 0.3 30.4 ± 0.215 4 12.2 ± 0.4 24.5 ± 0.3 14.3 ± 0.1 30.1 ± 0.315 5 12.2 ± 0.3 24.5 ± 0.2 13.5 ± 0.2 27.0 ± 0.137 6 21.6 ± 0.4* 25.2 ± 0.2 20.9 ± 0.4* 25.9 ± 0.2

PaCO2 37 0 5.1 ± 0.2 5.1 ± 0.1 5.1 ± 0.1 5.1 ± 0.337 1 5.1 ± 0.1 5.7 ± 0.1 6.1 ± 0.1 6.8 ± 0.215 2 6.0 ± 0.3* 5.2 ± 0.1* 6.0 ± 0.3* 8.1 ± 0.1*

15 3 6.0 ± 0.2 5.2 ± 0.3 5.7 ± 0.2 8.5 ± 0.415 4 6.1 ± 0.4 4.9 ± 0.2 5.5 ± 0.4 7.6 ± 0.115 5 6.1 ± 0.3 4.7 ± 0.1 5.4 ± 0.1 7.6 ± 0.337 6 4.4 ± 0.2* 3.6 ± 0.3* 4.8 ± 0.2* 4.6 ± 0.2

HCO3- 37 0 24.5 ± 0.1 24.9 ± 0.2 24.6 ± 0.2 24.7 ± 0.3

37 1 24.5 ± 0.1 24.2 ± 0.1 24.6 ± 0.2 24.4 ± 0.215 2 24.9 ± 0.3* 22.2 ± 0.1 24.1 ± 0.1* 23.9 ± 0.2*

15 3 18.1 ± 0.2 20.2 ± 0.3 17.8 ± 0.4 21.1 ± 0.115 4 17.7 ± 0.1 23.6 ± 0.1 16.1 ± 0.2 20.4 ± 0.215 5 17.1 ± 0.3 24.8 ± 0.2 15.0 ± 0.3 18.6 ± 0.337 6 16.0 ± 0.4* 25.8 ± 0.1 15.4 ± 0.5* 19.5 ± 0.4*

Glu 37 0 12.5 ± 0.1 12.7 ± 0.3 12.6 ± 0.1 12.6 ± 0.337 1 12.4 ± 0.2 10.8 ± 0.2 13.3 ± 0.1 12.9 ± 0.115 2 15.6 ± 0.3* 12.8 ± 0.3 23.5 ± 0.1* 16.4 ± 0.215 3 19.5 ± 0.2 15.2 ± 0.3 24.0 ± 0.3 16.8 ± 0.215 4 19.7 ± 0.3 15.7 ± 0.2 24.4 ± 0.2 17.2 ± 0.415 5 19.7 ± 0.1 16.8 ± 0.1 23.0 ± 0.1 17.8 ± 0.137 6 20.8 ± 0.2* 11.7 ± 0.2 19.0 ± 0.3* 10.6 ± 0.3

Lac 37 0 1.9 ± 0.3 1.9 ± 0.2 1.8 ± 0.4 1.8 ± 0.137 1 1.9 ± 0.1 1.8 ± 0.1 2.7 ± 0.1 4.3 ± 0.215 2 6.6 ± 0.2* 2.1 ± 0.3 6.6 ± 0.1* 5.6 ± 0.115 3 4.2 ± 0.2 2.0 ± 0.1 7.5 ± 0.2 3.5 ± 0.315 4 4.0 ± 0.1 1.8 ± 0.3 7.9 ± 0.1 3.3 ± 0.115 5 3.9 ± 0.3 1.5 ± 0.2 8.4 ± 0.3 3.1 ± 0.337 6 3.1 ± 0.1* 2.3 ± 0.1 12.3 ± 0.2* 5.9 ± 0.1

Data are mean ± SEM. Interventions are compared to vehicle using Repeated Measures ANOVA#P < 0.01: time point 1 vs 5; *P <0.05: time point 2 vs 6.

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Figure 3. Dopamine infusion prevents hypothermia and rewarming induced kidney injury. (A) Representative sections of the kidney (magnification x400) from all groups showing PAS, KIM-1, ED-1, HIS-48 and CBS stainings. Arrows point to positively stained areas (in brown). (B-F) panels show the quantification of immuno(histochemical) stainings of (B) PAS, (C) ED-1, (D) HIS-48, (E) KIM-1 and (F) CBS. PAS= Periodic Acid Schiff, ED-1 = Macrophage marker, HIS-48 = Neutrophil marker, KIM-1 = Kidney Injury Molecule, CBS = Cystathionine β-synthase. NC = Non-cooled; veh = Vehicle; dopa = Dopamine; A0AA = A0AA; dopa+AOAA = Dopamine+AOAA. Data are presented as mean ± SEM. */** P < 0.05/0.01 compared to non-cooled.

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group (p < 0.05; figure 4B). Thus, dopamine prevented the hypothermia-induced increase in serum creatinine, which was further increased by blockade of H2S-producing enzymes with AOAA in the absence of dopamine.

To further evaluate the effect of hypothermia on the kidney, we examined histopathological changes in kidney tissues obtained at one hour after rewarming. Hypothermia is associated with substantial glomerular and tubular injury as assessed by PAS- and KIM-1 stains compared to the untreated group (p < 0.01; figure 3A, 3B, 3E). Dopamine infusion fully prevented these morphological changes (figure 3A, 3B, 3E), while AOAA induced excess renal injury as compared to vehicle-treated group (p < 0.05; figure 3A, 3B, 3E). Furthermore, the protection from kidney damage offered by dopamine infusion was unaffected by AOAA (p < 0.05; figure 3A, 3B, 3E). Thus, dopamine treatment reduces morphological changes and the expression of markers of tubular damage induced by hypothermia and rewarming.

Further, hypothermia and rewarming induced a substantial influx of macrophages and neutrophils in the kidney in hypothermia and rewarmed animals as compared to non-cooled control animals (p < 0.01; figure 3A, 3C, 3D). Similarly to damage markers, administration of dopamine fully prevented the increase in influx of these cells, which was unaffected by co-administration of AOAA (p > 0.05; figure 3A, 3C, 3D). Treatment with AOAA on the other hand, resulted in an excess influx of macrophages and neutrophils as compared to vehicle-treated animals (p < 0.05; figure 3A, 3C, 3D).

Finally, ROS production in kidney tissue was assessed by measurement of MDA levels. Hypothermia and rewarming induced a 5-fold increase in MDA (p < 0.01; figure 4C). Treatment with dopamine fully prevented the increase in MDA (p > 0.05; figure 4C), which was not affected by AOAA administration in these animals (figure 4C, p > 0.05). Also, MDA levels were higher in animals treated with AOAA compared to vehicle treated animals (p < 0.05; figure 4C). Thus, dopamine reduces renal ROS production associated with hypothermia and rewarming in parallel to changes observed in serum H2S levels. Collectively, these data demonstrate that dopamine attenuates kidney injury following hypothermia and rewarming.

Dopamine maintains renal expression of H2S-producing enzymes and serum levels of

endogenous H2S in hypothermia and rewarmingTo explore the potential mechanism by which dopamine protects from hypothermia and rewarming damage in kidney, we determined the renal expression of the three different H2S-producing enzymes (CBS, CSE and 3-MST). Hypothermia and rewarming resulted in significant decrease in the expression of CBS, CSE and 3-MST as compared to non-cooled animals (p < 0.05; figure 4D). Notably, dopamine infusion maintained renal CBS expression at the level of non-cooled animals both in immunostaining and western blot (p

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> 0.05; figure 3F, 4D). Addition of AOAA did not affect the maintenance of CBS expression in dopamine-treated animals (p > 0.05; figure 4D). Similar to CBS, renal CSE expression was substantially downregulated following hypothermia and rewarming as compared to non-cooled animals (p < 0.01; figure 4E). However, dopamine treatment not only restored, but markedly upregulated CSE expression by 210% compared to non-cooled animals (p < 0.01; figure 3E). Administration of AOAA, both in vehicle or dopamine treated animals, strongly downregulated CSE expression level below the detection limit (figure 4E). The expression of renal 3-MST was markedly reduced in hypothermic and rewarmed rats compared to non-cooled animals (p < 0.05; figure 4F), which was prevented by dopamine (p > 0.05; figure 4F). Addition of AOAA in either vehicle-treated or dopamine-treated animals, did not affect

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Figure 4. Dopamine prevents metabolic effects of hypothermia and rewarming and maintains the expression of H2S-producing enzymes. (A) Dopamine treatment maintained blood pH during hypothermia and rewarming, while other groups show a significant decrease compared to their baseline values (p < 0.05). (B) Dopamine infusion maintains serum H2S level during hypothermia near levels found in non-cooled animals, irrespective of AOAA co-administration. Vehicle- and AOAA-treated groups show substantial decrease in serum H2S, which is not restored after rewarming (p < 0.01). (C) Dopamine infusion attenuated the increase in renal MDA found in vehicle, irrespective of AOAA co-administration (p > 0.05). (D) Dopamine maintained renal CBS expression both in the absence and presence of AOAA treatment (p < 0.01). (E) Dopamine strongly upregulates renal CSE expression, which was fully annihilated by AOAA (p < 0.01). (F) Dopamine maintains renal 3-MST expression, which was blocked by co-treatment with AOAA. */** represents p < 0.05/0.01; RM ANOVA (A,B) or ANOVA (C-F). Data are presented as mean ± SEM.

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the expression levels of 3-MST (figure 4F). Thus, hypothermia and rewarming substantially downregulated the expressions of all H2S-producing enzymes in the kidney, which was fully attenuated by dopamine infusion.

Next, we investigated whether changes in the expression of H2S producing enzymes in the kidney is paralleled by changes in serum levels of H2S. Hypothermia induced a strong reduction in serum H2S levels that was maintained throughout hypothermia compared to baseline value (p < 0.01; figure 4A). Remarkably, subsequent rewarming did not restore serum H2S levels (figure 4A). Dopamine infusion slightly increased baseline serum H2S level (p < 0.05; figure 4A) and completely attenuated its decrease during the subsequent hypothermia and rewarming phases (figure 4A). Administration of AOAA to animals infused with dopamine partly prevented the effect of dopamine on the serum H2S level upon hypothermia (p < 0.05; figure 4A), while administration of AOAA alone further reduced the H2S level (p < 0.05; figure 4A). Thus, dopamine prevented the hypothermia induced decrease in serum H2S.

DISCUSSION

In the present study, we demonstrate the protective effect of dopamine infusion against renal injury caused by whole body hypothermia and rewarming in the rat. Our data implicate the maintenance of production of H2S as a major mechanism of action of dopamine, as dopamine prevented the drop in serum H2S levels upon hypothermia. Further, dopamine preserved the expression of the H2S producing enzymes CBS and 3-MST and even increased expression of CSE in the kidney following hypothermia and rewarming. Further evidence for the importance of H2S production in hypothermia and rewarming-induced renal damage is derived from the group infused with AOAA, a non-specific blocker of H2S production,17 in which renal damage was substantially aggravated. Remarkably, the detrimental effects of AOAA on kidney damage and ROS production in hypothermia and rewarming were fully overcome by dopamine co-infusion. However, dopamine only maintained a normal expression of CBS in the kidney of AOAA treated animals, but did not rescue the AOAA invoked downregulation of CSE and 3-MST. Thus, our data extend our findings in cell culture and organ slices,14 by showing that the beneficial effect of dopamine maintains endogenous H2S production during hypothermia and rewarming in the whole animal,

In vivo hypothermic and rewarmed rat as a model of hypothermiaTo the best of our knowledge, this study is the first demonstrating the beneficial action of dopamine in hypothermia and rewarming injury in vivo, thus extending previous in vitro observations in cells13,14 and isolated organs.15 Although the in vitro hypothermia and rewarming protocol we used is less extensive in magnitude and duration than usually applied in the transplantation setting, its effects are remarkably similar. Particularly, the increase in

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renal ROS production, expression of KIM-1 and influx of immune cells in the renal interstitium we found in our model are also common findings in 4 ºC cold-preserved kidney transplants.18,19 In addition, the downregulation of CBS following hypothermia and rewarming was previously observed in cultured cells and organ slices.14 Thus, our model bears great resemblance to transplant cooling, and may explain the beneficial effect of dopamine pretreatment of donors on kidney outcome following transplantation.20 Moreover, our model seems to closely match the clinical situation in deep hypothermia in which there is unavoidable (compensated) metabolic acidosis,21 elevated level of serum creatinine, and drastic fall in cardiovascular parameters such as heart rate and blood pressure.21 Thus, the hypothermic and rewarmed rat represents an adequate model for examining hypothermia and rewarming-induced renal injury in the in vivo situation.

Influence of hypothermia and rewarming on H2S biologyIn developing our model, we identified a profound impact of hypothermia and rewarming on H2S biology. Hypothermia induced a substantial decrease in serum H2S level, which did not restore during the rewarming. Further, hypothermia and rewarming substantially reduced the renal expression of all three H2S-producing enzymes, extending previous observations in cells and tissue slices in which hypothermia down regulated CBS expression.14 While the source of plasma H2S is currently unknown, there are speculations that it is derived from circulating enzymes22 or rapidly diffuses from tissues following local production.23,24 The concomitant decrease in serum H2S and the reduction in renal expression of H2S-producing enzymes in hypothermic and rewarmed animals treated with vehicle or AOAA seems compatible with the last option. To further substantiate the involvement of H2S, animals were treated with AOAA, a non-specific inhibitor of H2S-producing enzymes.17 Administration of AOAA reduced serum H2S levels both at baseline and throughout the hypothermic and rewarming periods, and resulted in a substantial increase in ROS production and renal damage. Moreover, the decrease in renal expression of all three H2S-forming enzymes by hypothermia and rewarming was amplified by AOAA treatment. Therefore, the decrease in H2S and/or H2S-producing enzymes during hypothermia may represent the mechanism inducing the observed renal damage following hypothermia and rewarming. As H2S has been reported to be renoprotective in several animal models of ischemia/reperfusion injury via its anti-apoptotic and anti-inflammatory properties,25,26,27 such properties may have conferred a similar renoprotection under hypothermia and rewarming.

Dopamine increases the endogenous production of H2S Dopamine treatment increased baseline serum H2S level and maintained this level during the entire hypothermia and rewarming procedure, giving support to findings in cells where dopamine increased H2S production.14 Dopamine infusion maintained serum H2S even

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during the hypothermic phase, most likely reflecting increased H2S production, as reported previously in vitro during hypothermia at 3°C.14 Moreover, dopamine preserved (CBS, 3-MST) or even increased (CSE) expression of H2S-producing enzymes in the kidney following hypothermia and rewarming. Our data show that much of the beneficial effect of dopamine was maintained under co-infusion of AOAA, including the maintenance of serum H2S levels, albeit at a slightly lower level than observed in dopamine-treated animals. Further, in the presence of AOAA, dopamine reduced renal ROS production and expression of damage markers, and fully preserved the expression of CBS. However, dopamine infusion failed to maintain the renal expression of 3-MST and particularly of CSE in hypothermic and rewarmed animals treated with AOAA. This observation suggests that preservation of kidney integrity is most dependent on the maintenance of CBS expression, rather than CSE or 3-MST. Such notion is in accord with previous data showing that selective down-regulation of CBS by siRNA abrogates the beneficial action of dopamine during hypothermia and rewarming.14 Also, Sen et al. observed sodium hydrosulfide (an H2S donor) to prevent chronic kidney injury in uninephrectomized CBS-knockout mice.28 However, also in CSE-knockout mice, high vulnerability to kidney injury following ischemia-reperfusion injury (IRI) was reported.26,29 Taken together, our data imply a substantial effect of dopamine on serum H2S levels and expression of H2S-producing enzymes, which is highly consistent with its action observed in cells and organs. Therefore, the protective effects of dopamine during hypothermia and rewarming may well result from maintenance of H2S level.

Additional Dopamine effects in preservation of kidney function and integrityDopamine has been reported to maintain renal blood flow (RBF) and glomerular filtration rate (GFR) in both animal and human studies,30,31,32,33 which has been attributed to the activation of specific dopamine receptors.34,35,36,37 Alternatively, preservation of H2S levels by dopamine, either systemically or locally, may induce H2S mediated vasodilation,38 resulting in an increase RBF and GFR, as found in L-cysteine infused rat.39 Our results further demonstrate dopamine to convey a potent antioxidant action in kidney tissue, which likely protects the organ in hypothermia and rewarming. This action of dopamine may be explained by 2 mechanisms. First, dopamine may protect cells from the damaging effect of ROS through increased formation of H2S.14 Protection from ROS through enhanced H2S availability is supported by several in vivo and in vitro studies demonstrating that exogenous administration of H2S increases the production and activity of antioxidant enzymes and protects against oxidative stress.40,41,42,43 Second, dopamine itself also possesses ROS-scavenging properties, which is thought to prevent injury by cold-preservation and ischemia-reperfusion in cells.11,13,37 In accord, dopamine-infused animals co-treated with AOAA still show a reduced ROS formation following hypothermia and rewarming compared to vehicle-treated animals. However, dopamine infusion also produces high serum H2S level and maintains expression of CBS in

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AOAA co-treated animals. Thus, dopamine attenuation of ROS production and subsequent kidney damage may originate from antioxidant effects either through the maintenance of H2S production or its scavenging properties. Our data are, however, consistent with the view that maintenance of H2S by dopamine limits ROS production.

ConclusionIn summary, we have shown for the first time that dopamine preserves kidney function and integrity in whole body cooling and rewarming in the rat. A major mechanism involved seems to consist of the dopamine-induced increase in the endogenous H2S production, possibly through its maintenance of the expression of H2S-producing enzymes in rat kidney. Therefore, implementing dopamine treatment in clinical conditions, which require hypothermia and rewarming, may have the potential to ameliorate renal injury.

ACKNOWLEDGEMENTS

This study was supported by grants from Groningen University Institute for Drug Exploration (GUIDE). The University of Groningen together with University Medical Centre Groningen (UMCG, Netherlands) provided the laboratory and all materials and equipment for this study. We thank Prof. Frans Kroese and Prof. Harry van Goor (UMCG, Netherlands) for kindly providing the HIS-48 and CSE antibody, respectively.

DISCLOSURES

None

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26. Bos EM, Wang R, Snijder PM, Boersema M, Damman J, Fu M, Moser J, Hillebrands JL, Ploeg RJ, Yang G, Leuvenink HJ, van Goor H. Cystathionine γ-lyase protects against renal ischemia/reperfusion by modulating oxidative stress. J Am Soc Nephrol 24(5):759-70, 2013

27. Lobb I, Zhu J, Liu W, Haig A, Lan Z, Sener A. Hydrogen sulfide treatment ameliorates long-term renal dysfunction resulting from prolonged warm

renal ischemia-reperfusion injury. Can Urol Assoc J 8(5-6):E413-8, 2014

28. Sen U, Basu P, Abe OA, Giwimani S, Tyagi N, Metreveli N, Shah KS, Passmore JC, Tyagi SC. Hydrogen sulfide ameliorates hyperhomocysteinemia-associated chronic renal failure. Am J Physiol Renal Physiol 297(2):F410-9, 2009

29. Tripatara P, Patel NS, Collino M, Gallicchio M, Kieswich J, Castiglia S, Benetti E, Stewart KN, Brown PA, Yagoob MM, Fantozzi R, Thiemermann C. Generation of endogenous hydrogen sulfide by cystathionine gamma-lyase limits renal ischemia/reperfusion injury and dysfunction. Lab Invest 88(10):1038-48, 2008

30. Fink MP, Nelson R, Roethel R. Low-dose dopamine preserves renal blood flow in endotoxin shocked dogs treated with ibuprofen. J Surg Res 38(6):582-91, 1985

31. McGrath BP, Qin WX. Dopamine: clinical applications. Cardiovascular. Aust Prescr 17:44-5, 1994

32. Olsen NV. Effects of dopamine on renal hemodynamics tubular function and sodium extraction in normal humans. Dan Med Bull 45(3):282-97, 1998

33. Abay MC, Everts K, Wisser J. Current literature questions the routine use of low-dose dopamine. AANA J 75(1):57-63, 2007

34. Mathur VS, Swan SK, Lambrecht LJ, Anjum S, Fellmann J, McGuire D, Epstein M, Luther RR. The effects of fenoldopam, a selective dopamine receptor agonist, on systemic and renal hemodynamics in normotensive subjects. Crit Care Med 27(9):1832-7, 1999

35. Narkar V, Kunduzova O, Hussain T, Cambon C, Parini A, Lokhandwala M. Dopamine D2-like receptor agonist bromocriptine protects against ischemia/reperfusion injury in rat kidney. Kidney Int 66(2):633-40, 2004

36. Fontana I, Germi MR, Beatini M, Fontana S, Bertocchi M, Porcile E, Saltalamacchia L, Ornis S, Ghinolfi D, Bonifazio L, Valente U. Dopamine

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37. Li HZ, Guo J, Gao J, Han LP, Jiang CM, Li HX, Bai SZ, Zhang WH, Li GW, Wang LN, Li H, Zhao YJ, Lin Y, Tian Y, Yang GD, Wang R, Wu LY, Yang BF, Xu CQ. Role of dopamine D2 receptors in ischemia/reperfusion induced apoptosis of cultured neonatal rat cardiomyocytes. J Biomed Sci 18:18, 2011

38. Hosoki R, Matsuki N, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem Biophys Res Commun 237:527-531, 1997

39. Xia M, Chen L, Muh RW, Li PL, Li N. Production and actions of hydrogen sulfide, a novel gaseous bioactive substance in the kidneys. J Pharmacol Exp Ther 329(3):1056-62, 2009

40. Kimura Y, Kimura H. Hydrogen sulfide protects neurons from oxidative stress. FASEB J 18(10):1165-7, 2004

41. Liu H, Bai XB, Shi X, Cao YX. Hydrogen sulfide protects from intestinal ischemia-reperfusion injury in rats. J Pharm Pharmacol 61(2):207-12, 2009

42. Calvert JW, Jha S, Gundewar S, Elrod JW, Ramachandran A, Pattillo CB, Kevil CG, Lefer DJ. Hydrogen sulfide mediates cardioprotection through Nrf2 Signaling. Circ Res 105(4):365-74, 2009

43. Yang G, Zhao K, Ju Y, Mani S, Cao Q, Puukila S, Khaper N, Wu L, Wang R. Hydrogen sulfide protect against cellular senescence via S-sulfhydration of Keap1 and activation of Nrf2. Antioxid Redox Signal 18(15):1906-19, 2013

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6General Discussion & Future Perspectives

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GENERAL DISCUSSION

Therapeutic hypothermia is widely used to reduce ischemic injury to cells, tissues and organs before transplantation, after cardiac arrest or a cerebrovascular accident and during major cardiac and brain surgery to minimize complications and improve survival of patients (Polderman, 2004). However, hypothermia is associated with a wide range of physiological changes that negatively affect cells, organs, organ systems and ultimately the whole organism. The negative effect of hypothermia is attributed mainly mediated to excessive production of reactive oxygen species (ROS) during rewarming, when blood and oxygen supply are restored to the hypothermic and hypoperfused tissue (Carden and Granger, 2000). Excessive ROS production leads to oxidative stress and subsequent cell injury and cell death (Carden and Granger, 2000). Interesting, hibernating animals undergo repetitive cycles of torpor (hypothermia) and arousal (rewarming) without signs of organ injury (Frerichs et al., 1994; Kurtz et al., 2006; Zancanaro et al., 1999; Camici et al., 2008). Bogren et al. (2014) also reported absence of systemic inflammation in hibernating arctic ground squirrel. Therefore, hibernators seem to be ideal organisms to study the natural resistance to hypothermia and rewarming as applied in specific clinical situations including organ transplantation, treatment of stroke and cardiac arrest. Recently, there are reports indicating a reduction in circulating leukocytes (leukopenia) during torpor (Bouma et al., 2011).

To identify mechanisms that confer resistance to hypothermia, we studied (1) the mechanism underlying transient reduction in neutrophil number (neutropenia) during torpor and forced hypothermia in hibernating and non-hibernating animals, (2) the role of the H2S pathway in natural hibernation/organ protection, (3) its role in pharmacologically induced hypothermia and (4) the effectiveness of interventions aimed at maintaining a patent H2S pathway in in vivo hypothermia and rewarming in non-hibernating animals. Unraveling the molecular mechanisms underlying hibernation and organ protection may have major relevance for therapeutic hypothermia. Also, pharmacological metabolic suppression resembling natural hibernation may raise the possibility of safe procedures to allow for prolonged cardiac or neurosurgery, even under complete circulatory arrest.

In chapter 1, hypothermia and rewarming are introduced together with its therapeutic applications. Moreover, the negative effects of hypothermia and rewarming on cells and organs are discussed, using the kidney as a typical organ with high vulnerability to injury. This is followed by a comprehensive review on natural hibernation as a natural model for resistance to hypothermia/rewarming injury, which is characterized by repetitive cycle of torpor and arousal, and mechanisms of resistance against hypothermia/rewarming-induced damage including maintenance of the activity of Na+/K+-ATPase during torpor and arousal and activation of specific anti-apoptotic and antioxidant pathways. This section was followed

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by a pharmacological consideration of hibernation and its potential applications in human medicine with specific reference to hydrogen sulfide (H2S) and 5’-adenosine monophosphate (5’-AMP).

In chapter 2, we determined whether transient reduction in neutropenia during torpor (and hypothermia) in hibernating animals and forced hypothermia in non-hibernating animals is due to reversible margination or whether other mechanisms are involved, and how this might affect organ integrity. First, we demonstrate that neutrophils disappear from circulation during hypothermia, but are restored to normal levels following rewarming in both hibernating and non-hibernating animals. As the spleen is a major storage site of neutrophils, we surgically removed the spleen (splenectomy) to determine if neutropenia during torpidity is due to storage in the spleen and release into circulation following arousal. However, splenectomy, either prior to entering into hibernation or during torpor, had no effect on neutropenia as neutrophil numbers in splenectomized animals were similar to those in non-splenectomized animals, thereby ruling out the involvement of the spleen. We also reveal that the mechanism underlying this reversible neutropenia is unlikely to be due to apoptosis-and-replacement as the number of circulating immature neutrophils remained low during arousal, which rules out the involvement of bone marrow as the sole underlying mechanisms that governs neutropenia during hypothermia and torpor. Neutropenia during low body temperature seems to be due to the attachment of the neutrophils to the vessel wall (margination). By pretreatment with dexamethasone, an anti-inflammatory drug, we demonstrate the role of margination. Thus, indirect data suggest that margination is the mechanism underlying reversible clearance of circulating neutrophils during torpor and forced hypothermia. Potentially, this may be due to increased expression of adhesion molecules and integrins as margination is controlled by adhesion molecules on the neutrophils interacting with complementary receptors on the endothelium (Johnson et al., 1995; Le Deist et al., 1995; Hidax et al., 1996; Kira et al., 2005). However, direct evidence is lacking. Therefore, studies should be done to demonstrate the involvement of these adhesion molecules in margination. Since organ injury is aggravated by activation and recruitment of inflammatory mediators (Tapuria et al., 2008), in which margination of neutrophils is an important early event, blocking margination of neutrophils (and other proinflammatory mediators) may reduce the degree of organ injury during clinical interventions requiring hypothermia and rewarming. Indeed, experimental blockade of margination reduced the extent of pulmonary injury following normothermic cardiopulmonary bypass in a sheep model (Friedman et al., 1996). Interestingly, induction of hypothermia at 27.1°C in patients undergoing cardiopulmonary bypass surgery leads to the induction of a reversible leukopenia (Le Deist et al., 1995), comparable with hibernating animals in torpor at 6.2°C (Bouma et al., 2013) and hypothermic rats at 9.1°C (Shenaq et al., 1986; Bouma et al., 2013). Possibly, the increased resistance to ischemia/reperfusion injury and

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hypothermic injury in hibernating animals (Lindell et al., 2005; Kurtz et al., 2006; Bogren et al., 2014) prevents the occurrence of cellular injury and subsequent onset of inflammation, as might occur in specific clinical situations requiring hypothermia and rewarming. Thus, margination of neutrophils induced by hypothermia might be involved in the etiology of organ injury following hypothermia in humans and therefore, preventing margination might limit organ injury in specific clinical situations such as cardiopulmonary bypass.

To further unravel the mechanisms that underlie the induction of torpor and consequent organ protection during hibernation, we studied the H2S system since exogenously administered H2S inhibits leukocyte adherence to vascular endothelium (Fiorucci et al., 2005), suppresses the expression of adhesion molecules on leukocytes (Fiorucci et al., 2005; Zanardo et al., 2006) and reduces LPS-induced accumulation of neutrophils in rat liver and lungs (Li et al., 2007), leading to organ protection. Moreover, H2S has been implicated in the induction of a hibernation-like state in non-hibernating rodents (Blackstone et al., 2005; Blackstone and Roth, 2007; Bos et al., 2009) without behavioural changes or organ injury after full recovery. Therefore, in chapter 3, we investigated the role of endogenous H2S and its synthesizing enzymes (CBS, CSE and 3-MST) in regulating torpor-arousal cycle and organ protection during hibernation. We show that H2S production seems to be of critical importance in the induction of torpor as blood levels of H2S are substantially regulated during the hibernation cycle, with the highest values found during torpor. Moreover, the expression levels of all 3 H2S-producing enzymes in kidney increased substantially during torpor phase. The involvement of H2S in the induction of torpor is elucidated by administration of a non-specific inhibitor of H2S production during torpor, which precludes a return into torpor. Next, we reveal that H2S plays an essential role in protection of the kidneys throughout the torpor-arousal cycle, since pharmacological inhibition of the H2S system during torpor did not only preclude re-entrance into torpor but also resulted in substantial renal injury characterized by neutrophil and macrophage infiltration as well as high levels of serum creatinine, KIM-1 protein and ROS formation. Moreover, winter euthermic hamsters show very low levels of blood H2S and reduced expression of all renal H2S-producing enzymes. In contrast to hibernating hamsters, winter euthermic hamsters displayed a substantial increase in serum creatinine, increased renal ROS production and, importantly, kidney damage, which was further aggravated following H2S inhibition. Curiously, these phenomena were not observed during (late) arousal in hibernating animals, which are at similar body temperature (and presumably metabolic rate) as winter euthermic hamsters. Possibly, this difference is related to the duration of the housing under 5ºC ambient temperature, lasting only about 4 days for arousals, whereas amounting several weeks in winter euthermic, exhausting defense mechanisms and provoking damage in the latter. However, other organs such as the brain, liver and the cardiovascular system, in which specific H2S-producing enzymes are abundantly expressed, need to be

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examined to confirm these findings. Our findings so far suggest that H2S provides renal protection at high metabolic demand, possibly by maintaining adenosine triphosphate (ATP) production (Modis et al., 2013; reviewed in Szabo et al., 2014) during torpor/hypothermia. Unravelling the mechanisms that lead to induction of natural torpor and regulation of the torpor-arousal cycle, may have major clinical relevance for the prevention of acute organ injury upon therapeutic hypothermia and ischemia/reperfusion. Although the mechanisms by which H2S induces hibernation-like state in smaller mammals are not completely understood, an attempt to reproduce a similar hibernation-like state in larger mammals failed (Haouzi et al., 2008), and therefore H2S might preclude therapeutic hypothermia or its therapeutic use should be cautioned. There are also reports that H2S competes with oxygen and reversibly inhibits mitochondrial complex IV (Beauchamp et al., 1984; Khan et al., 1990), the terminal enzyme in the mitochondrial electron transport chain, thereby leading to hypometabolism and consequent hypothermia in small mammals. Since induction of a similar hypometabolic- and hypothermic state by H2S failed in larger mammals, this poses the question of whether H2S-induced hibernation-like state in smaller mammals exists in larger mammals. It might be that H2S failed to inhibit the mitochondrial complex IV in these animals perhaps due to a smaller window of opportunity to compromise oxidative phosphorylation in larger mammals (and humans) (Szabo, 2007). It might also be that a higher concentration of H2S and longer exposure time are required to induce such a hibernation-state in larger mammals. However, one possible drawback might be H2S toxicity at a higher concentration and longer exposure time in larger mammals.

As H2S seems to be a key player in induction of torpor and preserving renal integrity during torpor-arousal cycle in hamster, we hypothesized that pharmacological induction of torpor using 5’-AMP might occur through the H2S pathway. 5’-AMP has been previously used in other studies to induce a hibernation-like state in non-hibernating rodents (Zhang et al., 2006; Swoap et al., 2007; Strijkstra et al., 2012; Bouma et al., 2013; de Vrij et al., 2014). Therefore, in chapter 4, we induced pharmacologically induced hypothermia, resembling natural torpor by using 5’-AMP and investigated whether 5’-AMP influences the renal H2S system in naturally hibernating animals and protects organs from hypothermic injury as observed in chapter 3. Our results show that 5’-AMP-induced hypothermia is independent of activation of the H2S since inhibition of H2S prior to and during 5’-AMP-induced hypothermia did not preclude hypothermia. However, 5’-AMP seems to activate the renal H2S system and attenuated renal hypothermic injury. As 5’-AMP-induced hypothermia confers organ protection in non-hibernating species (Zhenyin et al. 2011; Miao et al., 2012; Wang et al., 2014), pharmacological inhibition of the H2S system prior to and during 5’-AMP-induced hypothermia resulted in substantial renal injury, suggesting that H2S plays a role in organ protection during 5’-AMP-induced hypothermia. Therefore, 5’-AMP might be an alternative

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approach to induction of hypothermia in patients who require therapeutic hypothermia. This approach, however, is novel and may need further investigations.

The mechanisms underlying 5’-AMP-induced hypothermia are unclear. Previously, some studies reported that 5’-AMP induces a torpor-like state through dephosphorylation to adenosine, which activates adenosine receptors in the temperature-sensitive hypothalamic nucleus (Zhang et al., 2006), thereby suppressing the thermoregulatory responses that maintain core body temperature. This subsequently results in a reduced cardiac output and thereby in hypothermia. However, there are reports that ATP and adenosine diphosphate (ADP) equally induce a torpor-like state, with even a much longer duration than 5’-AMP (Swoap et al., 2007), thereby downplaying the involvement of adenosine. The mechanism by which these adenine nucleotides induce such a torpor-like state remains largely unknown. Whereas it is speculated that adenosine activates adenosine A1 receptor in the sino-atrial and atrial-ventricular nodes of the heart, resulting in hyperpolarization of cardiomyocytes and consequent bradycardia as observed during the torpor-like state (Evans et al., 1982; Pelleg et al., 1987), we also demonstrated in chapter 4, that 5’-AMP-induced hypothermia seems to activate renal H2S system, a system that induces hypometabolism (and hypothermia), and attenuated renal hypothermic injury. However, the mechanism by which 5’-AMP influences the renal H2S system leading to reduced renal injury is unknown and needs to be explored.

The identification of organ protection by H2S during natural hibernation and 5’-AMP-induced hypothermia has broken the ground for in vivo experiments in non-hibernating mammals. Therefore, in chapter 5, we mimicked the effects of hypothermia/rewarming during torpor-arousal by inducing forced hypothermia in rats (non-hibernating animals) under anesthesia with or without dopamine co-infusion followed by rewarming to normothermia and investigated the role of the renal H2S system. Similar to previous data in cultured smooth muscle aortic cells (Talaei et al., 2011), dopamine infusion in the whole rat increased blood H2S level and maintained expression levels of all 3 H2S-producing enzymes thereby precluding hypothermia/rewarming-induced renal injury. Nevertheless, most effects of dopamine were insensitive to blockade of H2S production with AOAA, a non-specific inhibitor of H2S enzymes (Asimakopoulou et al., 2013), suggesting that H2S does not play a part in the observed dopamine effects. However, it should be noted that of all the 3 H2S-producing enzymes, dopamine infusion maintained the renal CBS expression, but did not maintain the renal expression of 3-MST and particularly of CSE in hypothermic and rewarmed rats treated with AOAA. This observation suggests that preservation of kidney integrity is most dependent on the maintenance of CBS expression, rather than CSE or 3-MST. Such a notion is in accord with previous data showing that selective down-regulation of CBS by siRNA abrogates the beneficial action of dopamine during hypothermia and rewarming (Talaei et al., 2011).

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In conclusion, we showed that regulation of the H2S pathway is a crucial underlying mechanism in the induction of torpor and preservation of organ integrity during the physiological extremes of torpor and arousal. Also, the H2S system seems to play a major role in attenuating renal hypothermic injury during pharmacologically induced torpor as it is activated by 5’-AMP. Finally, we demonstrated that the H2S system is also essential in protecting organs (kidney) during forced hypothermia and rewarming in non-hibernating animals under anesthesia. Therefore, H2S treatment could offer a novel therapeutic approach in treating clinical conditions, which require hypothermia and rewarming.

FUTURE PERSPECTIVES

While hibernators undergo repetitive cycles of torpor and arousal without signs of organ injury, a similar phenomenon in non-hibernating mammals including humans would be expected to result in apoptosis or necrosis, similar to what is observed following ischemia-reperfusion injury (IRI) due to interruption of cellular homeostasis. Although the underlying mechanisms of organ protection during torpor and arousal are not completely understood, hibernation could pave the way for in vivo experiments in non-hibernating animals and may lead to novel pharmacological strategies relevant to human medicine. This, therefore, raises the thought that humans can undergo a significant hypometabolic state analogous to natural hibernation. However, this might just be a science fiction as a search for pharmacological agents that induce hypometabolism, let alone hibernation-on-demand, in humans remains elusive. The recent discovery of pharmacological agents capable of inducing a safe hypometabolic, hypothermic state in small non-hibernating mammals with no apparent detrimental outcome was widely publicized as holding great promises and could be adopted as a routine clinical tool in the future. However, as discussed previously, recent reports indicate unsuccessful attempts to induce a similar hypometabolic, hypothermic state in large mammals (Haouzi et al., 2008). Thus, the proposed idea of the therapeutic use of exogenously administered H2S in human medicine should be reconsidered.

The most common clinical example of hypothermia and rewarming is encountered in organ transplantation, which is comparable to torpor and arousal (Green, 2000). So far there is no possible clinical approach to safely cool and rewarm organs without inflicting damage to the organs. There exists experimental evidence from in vitro, ex vivo and in vivo studies suggesting that dopamine protects against various pathologic stimuli during hypothermic storage (Schnuelle et al., 2004; Yard et al., 2004; Gottman et al., 2006; Talaei et al., 2011). Potential pathways by which dopamine is protective during ischemia/reperfusion-associated alterations comprise the reduction of proteolysis, a mechanism shown in cold-stored liver grafts (Koetting et al., 2010), as well as the maintenance of vascular endothelial cell integrity through mitigation of ROS induced cell damage (Koetting et al., 2010; Minor et

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al., 2011). Therefore, addition of dopamine to hypothermic preservation solution in both static and machine perfusion may limit the unavoidable IRI during transplantation and might improve organ function after transplantation. Also, as a pharmacological intervention to maintain structural and functional integrity of an allograft between the time of harvest, transportation and final implantation into the recipient, inclusion of H2S donor molecules and/or H2S-releasing drugs to the preservation solution may improve standard methods of preparing for organ transplant, and may protect the graft from cold ischemic injury as well as reperfusion injury. Some groups have shown promising effects in experimental models in which exogenously administered H2S mitigated renal graft IRI during transplantation and prolonged cold storage, improving early graft function and recipient survival in a clinically applicable model of renal transplantation (Hosgood SA, Nicholson ML, 2010; Lobb et al., 2012). This, therefore, indicates a role in H2S-based therapeutics in these settings.

Since the physiological benefits and therapeutic potentials of H2S have been discovered by Hideo Kimura’s group in 1996, there has been an explosive increase in the knowledge and properties of this rotten-egg smelling gaseous signaling molecule. Unlike nitric oxide (the first gaseous signaling molecule), whose introduction was met with skepticism by the scientific community, H2S has been unreservedly accepted, and substantial efforts are currently being made to imbue it into the medical and pharmaceutical fields. Although research in the H2S field is still in its infancy, a plethora of evidence indicates that H2S has significant pharmacological and physiological benefits. As a young and rapidly growing field, there are problems and challenges that need to be addressed to enhance further H2S research. Currently, there are no selective inhibitors of H2S-producing enzymes and available inhibitors of H2S synthesis have additional off-target effects (Asimakopoulou et al., 2013). Moreover, there are no sustained and controlled-releasing H2S donors that function in both in vivo and in vitro situations (Fiorucci and Santucci, 2011; Zhao et al., 2011; Ali et al., 2014), although a slow-releasing H2S-generating compound has been newly synthesized (Robinson and Wray, 2012). In addition, finding an effective and quick method to measure H2S concentration, especially in a non-invasive way is another challenge (Wu et al., 2006). Unfortunately, there are no techniques with the sensitivity, selectivity, and real-time capability to measure intracellular H2S (Wu et al., 2006). Furthermore, H2S has been implicated in specific diseases in which its concentration varies from one disease to another. In sepsis, for example, H2S is overproduced while its level is insufficient in Alzheimer’s disease (Gong et al., 2011). This suggests that the mechanism controlling the actual concentration of H2S in certain tissues could pose another problem in H2S research. Taken together, H2S seems to have considerable therapeutic potentials, and research in the coming years will very likely unravel several therapeutic feasibility of this gaseous signaling molecule. Moreover, to the best of our knowledge, we are the first to identify dopamine as a main modulator of endogenous H2S

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production and its synthesizing enzymes in preservation of kidney function and integrity in in vivo whole body cooling and rewarming, and therefore, could be implemented in specific clinical conditions which require cooling and rewarming.

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Szabo C. Hydrogen sulfide and its therapeutic potential. Nat Rev Drug Discov. 2007; 6(11):917-35.

Szabo C, Ransy C, Modis K, et al. Regulation of mitochondrial bioenergetic function by hydrogen sulfide. Part I. Biochemcal and physiological mechanisms. Br J Pharmacol. 2014; 171(8):2099-122.

Talaei F, Bouma HR, van der Graaf AC, Strijkstra AM, Schmidt M, Henning RH. Serotonin and dopamine protect from hypothermia/rewarming damage through the CBS/H2S pathway. PLoS One. 2011; 6(7):e22568.

Tapuria N, Kumar Y, Habib MM, Amara MA, Seifalian AM, Davidson BR. Remote ischemic preconditioning: A novel protective method from ischemia reperfusion injury. J Surg Res. 2008; 150: 304-330.

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Yard B, Beck G, Schnuelle P, et al. Prevention of cold-preservation injury of cultured endothelial cells by catecholeamines and related compounds. Am J Transplant. 2004; 4:22-30.

Zanardo RC, Brancaleone V, Distrutti E, Fiorucci S, Cirino G, Wallace JL. Hydrogen sulfide as an endogenous modulator of leukocyte-mediated inflammation. FASEB J. 2006; 20(12):2118-20.

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7Nederlanse Samenvatting

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In hoofdstuk 1 worden onderkoeling en heropwarming geïntroduceerd met hun therapeutische toepassingen. Verder worden de negatieve effecten van onderkoeling en heropwarming op cellen en organen bediscussieerd. Hierbij wordt de nier gebruikt als een voorbeeld van een orgaan met een hoge kwetsbaarheid voor schade.

Dit wordt gevolgd door een uitgebreide review over natuurlijke winterslaap als een natuurlijk model in bescherming tegen schade veroorzaakt door onderkoeling en heropwarming. Dit model wordt gekenmerkt door een zich herhalende cyclus van periodes met inactiviteit (torpor) en activiteit (arousal) en beschermingsmechanismen tegen schade veroorzaakt door hypothermie en opwarming. Deze beschermingsmechanismen bestaan uit het in stand houden van de Na+/K+-ATPase activiteit gedurende torpor en arousal en het activeren van specifieke anti-apoptotische en anti-oxidatieve routes. Deze sectie wordt gevolgd door de farmacologische overweging van winterslaap en zijn potentiele toepassingen in de geneeskunde, waarbij specifieke aandacht wordt besteed aan waterstofsulfide (H2S) en 5’-adenosine monofosfaat (5’-AMP).

In hoofdstuk 2 hebben we onderzocht of voorbijgaande vermindering in neutropenie tijdens torpor (en onderkoeling) in dieren in winterslaap en geforceerde onderkoeling in dieren die geen winterslaap houden, veroorzaakt wordt door de omkeerbare marginatie of dat andere mechanisme een rol spelen en hoe dit het orgaan behoud kan beïnvloeden. Ten eerste hebben we aangetoond dat neutrofielen verdwijnen in de circulatie gedurende de onderkoelen, maar herstellen naar normale waarden gedurende de heropwarming in zowel dieren met als zonder winterslaap. Aangezien de milt een grote opslagplaats is voor neutrofielen, hebben we de milt operatief verwijderd (splenectomie) om te bepalen of the neutropenie gedurende de torpor-stadium het gevolg is van opslag in de milt en vrijlaten in de circulatie gedurende arousal. Echter, splenectomie, zowel voorafgaand aan de winterslaap als gedurende torpor had geen effect op de neutropenie aangezien de neutrofielenaantallen in zowel dieren wel of geen splenectomie hebben ondergaan, gelijk waren. Hiermee is de rol van de milt uitgesloten. We kunnen ook aantonen dat het mechanisme dat ten grondslag ligt aan deze omkeerbare neutropenie waarschijnlijk niet komt door apoptose-en-vervanging aangezien het aantal circulerende immature neutrofielen laag bleef gedurende arousal, wat de betrokkenheid van beenmerg als enig onderliggend mechanisme dat neutropenie tijdens onderkoeling en torpor leidt, uitsluit. Het lijkt alsof neutropenie gedurende lage lichaamstemperaturen komt door de aanhechting van neutrofielen aan de vaatwand (marginatie). Door voorbehandeling met dexamethason, een anti-inflammatoir middel, hebben we aangetoond wat de rol van marginatie is. Daarmee suggereert indirecte data that marginatie het onderliggende mechanisme is van de omkeerbare verdwijning van circulerende neutrofielen gedurende torpor en geforceerde onderkoeling. Mogelijk komt dit door een verhoogde expressie van adhesie moleculen en integrinen aangezien marginatie gecontroleerd wordt door adhesie moleculen op de neutrofielen die interageren met complementaire receptoren op het endotheel. (Johnson et al., 1995; Le Deist et al., 1995; Hidax et al., 1996; Kira et al., 2005). Echter, direct bewijs ontbreekt. Daarom zouden er studies gedaan moeten worden om de betrokkenheid van deze adhesie moleculen in marginatie aan te tonen. Aangezien orgaanbeschading verergerd door activatie en verzamelen van

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inflammatoire mediatoren (Tapuria et al., 2008), in welke marginatie van neutrofielen een belangrijk vroege consequentie is, kan het blokkeren van de marginatie van neutrofielen (en andere pro-inflammatoire medicatioren) de mate van orgaanbeschadiging verminderen gedurende klinische interventies die afkoeling en heropwarming vereisen. Inderdaad, experimentele blokkade van de marginatie verminderde de omvang van longbeschadiging volgend op normoterme cardiopulmonale bypass in een schaapmodel (Friedman et al., 1996). Interessant is dat de inductie van onderkoeling naar 27.1°C in patienten die een cardiopumonale bypass operatie ondergaan, leidt tot de inductie van omkeerbare leukopenie (Le Deist et al., 1995), vergelijkbaar met winterslaap houdende dieren in een torpor van 6.2°C (Bouma et al., 2013) en onderkoelde ratten van 9.1°C (Shenaq et al., 1986; Bouma et al., 2013). Het is mogelijk dat de verhoogde weerstand voor ischemie-/reperfusieschade en schade door onderkoeling in winterslaap houdende dieren (Lindell et al., 2005; Kurtz et al., 2006; Bogren et al., 2014) het ontstaan van cellulaire schade en een daaropvolgend ontstaan van ontsteking voorkomt, zoals wellicht voorkomt in specifieke klinische situaties die onderkoeling en heropwarming vereisen. Dus marginatie van neutrofielen veroorzaakt door onderkoeling kan betrokken zijn in de etiologie van orgaanbeschadiging volgend op onderkoeling in mensen. Daarom kan het voorkomen van marginatie orgaanbeschadiging beperken in specifieke klinische situaties zoals cardiopulmonale bypass.

Om de onderliggende mechanismen van de inductie van torpor en de resulterende orgaan bescherming tijdens de winterslaap verder te onderzoeken, hebben we het H2S systeem verder onderzocht. Exogeen toegediend H2S remt de adhesie van leukocyten aan het vasculaire endotheel (Fiorucci et al., 2005), onderdrukt de expressie van adhesie moleculen aan leukocyten ((Fiorucci et al., 2005; Zanardo et al., 2006) en vermindert de LPS-geïnduceerde accumulatie van neutrofielen in de lever en longen van de rat. Tezamen leidt dit tot orgaan bescherming. Bovendien is H2S toegepast in het induceren van een winterslaap-achtige staat bij knaagdieren welke geen winterslaap houden (Blackstone et al., 2005; Blackstone and Roth, 2007; Bos et al., 2009), zonder dat dit na volledig herstel gedragsveranderingen of orgaanschade tot gevolg heeft gehad.

Daarom hebben we in hoofdstuk 3 de rol van endogeen H2S en zijn producerende enzymen (CBS, CSE en 3-MST) in het reguleren van de torpor-arousal cyclus en orgaanbescherming tijdens winterslaap onderzocht. We laten zien dat de H2S productie van cruciaal belang lijkt te zijn in de inductie van torpor, aangezien bloedspiegels van H2S substantieel gereguleerd zijn tijdens winterslaap, waarbij de hoogste waardes tijdens torpor gevonden worden. Bovendien zijn de waardes van alle drie de H2S-producerende enzymen in de nier substantieel verhoogd tijdens de torpor fase. De betrokkenheid van H2S in de inductie van torpor is opgehelderd door de toediening van een non-specifieke inhibitor van H2S tijdens torpor, wat een terugkeer in torpor inluidt. Vervolgens onthullen we dat H2S een essentiële rol speelt in de bescherming van de nieren gedurende de torpor-arousal cyclus, aangezien farmacologische remming van het H2S-systeem gedurende torpor niet alleen herintreding in de torpor-fase verhinderde, maar ook resulteerde in substantiële nierschade, welke werd gekarakteriseerd door infiltratie van neutrofielen en macrofagen en hoge waardes van serum creatinine, KIM-1 proteïne en ROS formatie. Bovendien hebben hamsters die geen

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winterslaap houden zeer lage bloedspiegels van H2S en verminderde expressie van alle renale H2S producerende enzymen. In tegenstelling tot winterslaap houdende hamsters, lieten hamsters die geen winterslaap houden een substantiële stijging in serum creatinine, renale ROS productie en – zeer belangrijk- nierschade zien, wat verder verhevigd werd door H2S inhibitie. Merkwaardig genoeg werden deze fenomenen niet gezien tijdens (late) arousal in winterslaap houdende dieren, welke dan dezelfde lichaamstemperatuur (en vermoedelijk hetzelfde metabolisme) hebben als niet winterslaap houdende hamsters. Mogelijkerwijs is dit verschil gerelateerd aan de duur van de blootstelling aan een omgevingstemperatuur onder de 5ºC, welke slechts ongeveer vier dagen is voor arousals, maar meerdere weken kan aanhouden in niet winterslaap houdende dieren. Dit put het verdedigingsmechanisme in de niet winterslaap houdende dieren uit, wat schade tot gevolg heeft.

Echter, andere organen waarin de expressie van specifieke H2S-producerende enzymen overvloedig is, zoals de brein, lever en het cardiovasculaire systeem, dienen te worden onderzocht om deze bevindingen te bevestigen. Onze bevindingen tot nu toe suggereren dat H2S bescherming van de nieren biedt bij een hoog metabolisme, mogelijk door het behoud van adenosine trifosfaat (ATP) productie (Modis et al., 2013; reviewed in Szabo et al., 2014) gedurende torpor/onderkoeling. Het ontrafelen van de mechanismen die tot de inductie van natuurlijke torpor en de regulatie van de torpor-arousal cyclus leiden, kan van grote klinische relevantie zijn voor de preventie van acute orgaan schade bij therapeutische onderkoeling en ischemie/reperfusie. Hoewel de mechanismen hoe H2S een winterslaap-achtige staat in kleine zoogdieren induceert niet volledig begrepen worden, is een poging om een soortgelijke winterslaap-achtige staat bij grotere zoogdieren te reproduceren mislukt (Haouzi et al., 2008). En daarom zou H2S een therapeutische onderkoeling kunnen verhinderen en zou het therapeutische gebruik van H2S met voorzichtigheid gepaard moeten gaan. Er zijn ook berichten dat H2S concurreert met zuurstof en reversibele mitochondriële complex IV – het terminale enzym in de mitochondriale electronen transport keten – remt (Beauchamp et al., 1984; Khan et al., 1990). Dit leidt tot hypometabolisme en als gevolg daarvan onderkoeling in kleine zoogdieren. Aangezien de inductie van een soortgelijke hypometabole en hypotherme staat in grote zoogdieren is mislukt, ontstaat de vraag of een H2S-geïnduceerde winterslaap-achtige staat zoals in kleine zoogdieren wel bestaat in grotere zoogdieren. Mogelijk faalt H2S er bij deze dieren in om het mitochondriële complex IV te remmen doordat er minder mogelijkheden zijn om de oxidatieve fosforylering in grotere zoogdieren (en mensen) te compromitteren (Szabo, 2007). Het is ook mogelijk dat een hogere concentratie H2S en een langere blootstelling noodzakelijk zijn om een dergelijke winterslaap-achtige staat in grotere zoogdieren te induceren. Echter, een mogelijke nadeel zou H2S toxiciteit in grotere zoogdieren kunnen zijn bij een hogere concentratie of een langere blootstelling. Omdat H2S een hoofdrol lijkt te spelen in de inductie van torpor en het behoud van de nierfunctie tijdens de torpor-arousal cyclus in hamsters was onze hypothese dat farmacologische inductie van torpor met behulp van 5’-AMP op kan treden door de H2S route. 5’-AMP is eerder gebruikt in andere onderzoeken om een winterslaapachtige staat te induceren in knaagdieren die geen winterslaap houden (Zhang et al., 2006; Swoap et al., 2007; Strijkstra et al., 2012; Bouma et al., 2013; de Vrij et al., 2014). Daarom hebben we onderkoeling in

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hoofdstuk 4 farmacologisch geïnduceerd, wat lijkt op natuurlijke verdoving met behulp van 5’-AMP en hebben we onderzocht of 5’-AMP het renale H2S systeem beïnvloed in dieren in winterslaap en of het organen beschermt tegen hypothermisch letsel zoals geobserveerd in hoofdstuk 3. Onze resultaten laten zien dat 5’-AMP-geïnduceerde onderkoeling onafhankelijk is van de activatie van H2S omdat remming van H2S voorafgaand aan en tijdens de 5’-AMP-geïnduceerde onderkoeling de onderkoeling niet verhindert. 5’-AMP lijkt echter het renale H2S systeem te activeren en het renale hypothermische letsel te verzwakken. 5’-AMP-geïnduceerde onderkoeling beschermt organen in diersoorten die geen winterslaap houden (Zhenyin et al. 2011; Miao et al., 2012; Wang et al., 2014). Farmacologische remming van het H2S systeem voorafgaand aan en tijdens 5’-AMP-geïnduceerde onderkoeling resulteerde in substantiële nierschade, wat suggereert dat H2S een rol speelt in orgaan bescherming tijdens 5’-AMP-geïnduceerde onderkoeling.

Daarom is 5’-AMP mogelijk een alternatieve benadering om onderkoeling te induceren in patiënten die therapeutische onderkoeling nodig hebben. Deze benadering is echter nieuw en moet verder onderzocht worden. De mechanismen onderliggende aan 5’-AMP-geïnduceerde onderkoeling zijn niet duidelijk. Eerder is in sommige studies gerapporteerd dat 5’-AMP een torpor-achtige staat induceren door defosforylering naar adenosine wat adenosine receptoren activeert in de temperatuurgevoelige hypothalamus nucleus (Zhang et al., 2006), en daarbij de thermoregulatoire reacties die de kerntemperatuur van het lichaam handhaven onderdrukken. Dit resulteert vervolgens in een verminderde cardiale output en daardoor in onderkoeling. Er zijn echter ook berichten dat ATP en adenosinedifosfaat (ADP) in gelijke mate een torpor-achtige staat induceren van zelfs een veel langere duur dan 5’-AMP (Swoap et al., 2007) waardoor de rol van adenosine wordt gebagatelliseerd. Het mechanisme waardoor deze adenine nucleotiden zo’n torpor-achtige staat induceren blijft grotendeels onbekend. Aangezien er wordt gespeculeerd dat adenosine de adenosine A1 receptor in de sino-atriale en de atriale-ventriculaire knopen van het hart induceert, wat resulteert in hyperpolarisatie van hartspiercellen en daaropvolgend bradycardie zoals waargenomen gedurende de torpor-achtige staat (Evans et al., 1982; Pelleg et al., 1987), hebben we in hoofdstuk 4 ook aangetoond dat 5’-AMP-geïnduceerde onderkoeling het renale H2S systeem, een systeem dat the hypometabolisme (en onderkoeling) induceert, lijkt te activeren en hypotermische nierschade lijkt te verminderen. Het mechanisme waarbij 5’-AMP het renale H2S systeem beïnvloedt wat leid tot een verminderde nierschade is echter niet bekend en moet onderzocht worden. De identificatie van orgaanbescherming door H2S gedurende natuurlijke winterslaap en 5’-AMP-geinduceerde onderkoelen heeft de deuren op gezet voor in vivo experimenten in niet-winterslaap houdende zoogdieren. Daarom, hebben we in hoofdstuk 5 de effecten van onderkoeling/heropwarming gedurende torpor-arousal geïmiteerd door het induceren van geforceerde onderkoeling bij ratten (niet-winterslaap houdende dieren) onder anesthesie met en zonder dopamine co-infusie gevolgd door opwarming naar normothermie. De rol van het renale H2S systeem werd hierbij onderzocht. Vergelijkbaar met eerdere data in gekweekte gladde spiercellen van de aorta (Talaei et al., 2011), verhoogde dopamine infusie in de

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gehele rat de H2S bloedspiegel en handhaafde expressie levels van alle 3 H2S-producerende enzymen en verhinderd daardoor renale schade als gevolg van onderkoeling/heropwarming. Niettemin, de meeste effecten van dopamine waren ongevoelig voor de blokkade van H2S productie met AOAA, a niet-specifieke remmer van H2S enzymen (Asimakopoulou et al., 2013), wat suggereert dat H2S geen rol speelt in de geobserveerde dopamine effecten. Echter, het dient te worden opgemerkt dat van alle H2S-producerende enzymen, dopamine infusie de renale CBS expressie handhaafde, maar dat de renale expressie van 3-MST en in het bijzonder van CSE in onderkoelde en heropgewarmde ratten behandeld met AOAA, niet handhaafde. Deze observatie suggereert dat behoud van de nieren voornamelijk afhankelijk is van de handhaving van de CBS expressie, in plaats van CSE of 3-MST. Een dergelijk idee is in overeenstemming met eerdere data die aantonen dat selectieve down-regulatie van CBS bij siRNA de gunstige werking van dopamine gedurende onderkoeling en heropwarming opheft (Talaei et al., 2011).

In conclusie, wij hebben aangetoond dat de regulatie van de H2S route een cruciaal, onderliggend mechanisme is in de inductie van torpor en de instandhouding van organen gedurende de fysiologische extremen van torpor en arousal. Daarnaast lijkt het H2S systeem een grote rol te spelen in het verminderen van de hypotermisch, renaal letsel gedurende farmacologisch geïnduceerde torpor aangezien dit is geactiveerd door 5’-AMP. Ten slotte hebben we aangetoond dat het H2S systeem ook essentieel is in het beschermen van organen (nieren) gedurende geforceerde onderkoeling en heropwarming in niet-winterslaaphoudende dieren onder anesthesie. Daarom kan H2S behandeling een nieuw therapeutische aanpak kunnen bieden in klinische situaties die onderkoeling en heropwarming vereisen.

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ACKNOWLEDGEMENTS - Dankwoord

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ACKNOWLEDGEMENTS

After a 4-year scientific adventure of exploring the secrets of nature to the benefit of human medicine, the journey has come to an end. As great as it feels to see all the chapters of this thesis finished, I feel even much better to look back and reflect over how the journey of my life started in Ghana, West Africa, the good and the bad times, the challenges that seemed insurmountable and some great individuals who inspired me along the journey when I felt like giving up. I want to thank my biological parents for bringing me into this world. May your souls rest in perfect peace. I know that you are very proud of me wherever you are now. I express my heartiest gratitude to Rev. Dr. Ray Johnson and Rev. Dr. Joycelyn Johnson, who raised me and kept my feet on the path of knowledge. I cannot imagine coming this far without the firm foundation and the right training you gave me. Thank you for your love and for believing in me. I am eternally indebted to you. I love you very much. I also thank my auntie Mrs. Angela Evans and Crenshaw Christian Center for their support especially during my undergraduate years.

I am also thankful to Hayford Nti Awere, who has always been there for me. Some friends stick closer than a brother. You are more than a friend. You are a brother - my brother from another mother. That is who you are to me. I wish you success in all your endeavours.

Many thanks to The Netherlands government, who saw the potential in me and invested to develop it to the benefit of human medicine. I couldn’t have come this far without your huge financial investment in the form of a prestigious scholarship from the level of my top master degree program to the end of my PhD program. You have given me a great opportunity to have an enviable career and to serve humanity through medical science research. I am grateful for choosing me out of many equally-deserving candidates and giving me such an opportunity.

I wish to also express my sincere gratitude to my promoter, Prof. Dr. Robert Henning. I am very thankful to you for giving me the opportunity to work with you, and refining me in the process to suit my future medical science career. I have learned a lot of qualities from you both personally and professionally. Each time I had a meeting with you and shared ideas on our projects, one of the many things that almost immediately comes into my mind is that ‘I would like to coach students with your style of coaching’. I admire your personality, your hardwork, your friendly nature, your dedication to your work, your simplicity about life, and how you coach your students. You infused me with such an overwhelming enthusiasm that I was able to finish my PhD thesis within the shortest possible time. Through you I came to realize that enthusiasm is all about believing in yourself and believing in what you do, and the willingness to achieve your goals in the phase of challenges. I enjoyed working with you and I would like to keep collaboration with you. I thank you very much.

To my co-promoter, Dr. Hjalmar Bouma, I would like to thank you for co-supervising my work. I learned

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some secrets from your success as a PhD student. In the early years of my PhD, I saw that you are a gifted writer and I was fortunate to be your first PhD student to learn that attribute from you. You also infused me with a sense of urgency as you were so quick in everything you do. There were times when things were not working well for me and I seemed to have lost courage but you encouraged me to go on. I am grateful to you for your encouragement and guidance and helping to improve my planning and organizational skills.

I wish to thank Dr. Fatemah Talaei, whose idea and work served as a starting point for my PhD research. I wish you well in all your endeavours.

To the technicians, Martin, Marry, Maaike, Azuweris, Femke, I would like to say a big thank you for teaching me most of the techniques needed to execute all the projects I undertook in the Department. Thank you for your warmness. It was a pleasure working with you all.

My gratitude also goes to the ‘hibernation team’, who has been of tremendous support to me for the past four years. I will like to single out Dr. Arjen Strijkstra and Dr. Ate Boerema for sharing with me their experience in the hibernation field and the technical support they gave me throughout the period of my research.

To Martijn Salomons and the animal care takers at the CvL and CDP, I say thank you for taking care of my experimental animals and for all other supports during my hibernation and forced hypothermia experiments especially at times when I was absent.

I would also like to thank Prof. Ulrich Eisel, Prof. Wilfred den Dunnen, Dr. Pieter Naude and Dr. Kay Seidel. You were my project supervisors during my master degree programme. Coming from a background with insufficient laboratory experience, you were patient with me and guided me through the two projects I undertook under your supervision. Working with you has reshaped my perception about career in medical science and gave me a new look into a brighter future. In the end you gave me sufficient knowledge to start a PhD research.

Dear Dr. Mate Siakwa, you taught me some medical courses and also supervised my undergraduate project. Through your warmness and kindness, you inspired me and gave me a better focus during my undergraduate years. Interacting with you shaped my career path and affirmed my decision to choose a career in medical science.

To my colleagues, Mahdi, Sjoerd, Nagesh, Arash, Deli, Edwin, Vera, Jojanneke, Marit, all members of the Clinical Pharmacology Department including Ardy, Alexandra, Bianca, Leo, Hendrik, I wish to thank you all for your support and being a part of my success story over the last four years. It was a pleasure knowing you and I enjoyed working with you all.

Acknowledging all members of the Department would not be complete without mentioning a great

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friend, Adriaan van Doorn. I drew a lot of inspiration from your wisdom. You are a sexagenarian, yet you look so young and you exude so much youthfulness and enthusiasm. I wish to look young like you when I get to your age. You have always reminded me that a person’s skin color or his religion or background doesn’t matter, and that we are the world, we are the children of the world. It all has to do with how we process information in our brain. Your words built confidence in me. I always remember your words ‘Let’s change the world with pharmacology’. I am glad I met you on the journey of my life.

To my best Iranian friend and colleague and also my paranymph, Marziyeh Tolouee Nodolaghi, and Rick Meijer, her boyfriend, I thank you for your friendship and warmth. I won’t forget those emotional supports you gave me when I needed it most.

A special thanks to my landlord, Jan Rozenveld. You have made my stay in Groningen an enjoyable one. Living in your house was like a home away from home. You have been very nice to me. You were not only a landlord but also a friend. There were times I came home tired after a long day’s work. You cooked for me and served me tea. We shared many jokes and laughter together though you understood your own jokes much better. Through you I came to understand Dutch culture and the philosophy of ‘live simply so others may simply live’. You were very supportive when you offered me some side jobs to earn extra money. I really appreciate your support. I will miss you but remember that you have a room in my house anytime you visit Ghana. It’s a small round world. Living in your house will not be complete without acknowledging Harrit Lamster and his parents, who were so thoughtful that they always sent me Christmas cards in each Christmas season for all the 6 years I stayed in the house. Thank you Harrit, for being a good housemate to me. You were so polite and also kind to me.

To my neuroscience friends, Inge (the alpha female), Melanie (Mama Mela), Karen, Sygrid, Jennifer, Amarins, Erin and Martin. You were my classmates and friends when I first arrived in Groningen. All the lecturers attested that our N-track was the best and the most united compared to all the batches before us. This was due to your warmness and friendliness. You organized trips each year during which I learn a lot of lessons, and today such trips have formed the basis for my adventurous life, having covered several countries across the globe.

To a wonderful person, Brenda. Thank you for all the pieces of advice and support especially during the last two years of my PhD. To Sarah, Lucy, Thembile, Bright, Nana Aba, Anani, Fahimeh, Paul, Fany, Adwoa, Marshall, Misghina, Gifty, George, Sieta, Lisa, Joshua, Juliana, Fareeba, Peter, Maggie, Diana, Filip, Andrea, Rita and all my friends across the globe. Thank you all for your warm friendship.

Thank you, Motsum, Medaase, Akpe, Dankjewel

George

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2. Hjalmar R. Bouma, George J. Dugbartey, Ate S. Boerema, Fatemeh Talaei, Annika Herwig, Maaike Goris, Azuwerus van Buiten, Arjen M. Strijkstra, Hannah V. Carey, Robert H. Henning, Frans G. M. M. Kroese. Reduction of body temperature governs neutrophil retention in hibernating and non-hibernating animals by margination. Journal of Leucocyte Biology 2013. 94(3):431-7

3. Edwin de Vrij, Peter Vogelaar, Annika Herwig, Maaike Goris, Houwertjes M, George Dugbartey, Ate Boerema, Arjen M. Strijkstra, Hjalmar Bouma, Robert H. Henning. Platelet dynamcs during natural natural and pharmacologically induced torpor. PloS One 2014. 10;9(4)

4. George J. Dugbartey, Fatemah Talaei, Martin C. Houwertjes, Maaike Goris, Anne H. Epema, Hjalmar R. Bouma, Robert H. Henning. Dopamine treatment attenuates renal injury via H2S production in a rat model of deep hypothermia and rewarming. (Submitted)

5. George J. Dugbartey, Arjen M. Strijkstra, Ate S. Boerema, Hjalmar R. Bouma, Robert H. Henning. Induction of a torpor-like state by 5’-AMP: A role for endogenous H2S production. (Submitted)

6. George J. Dugbartey, Anouk H.G. Wolters, Edwin L. de Vrij, Arjen M. Strijkstra et al. H2S system is crucial in maintaining torpor-arousal cycle and preserving renal integrity in the hibernating Syrian hamster. (Manuscript in preparation)