devil’s triangle in kidney diseases: oxidative stress...

International Journal of Nephrology

Devil’s Triangle in Kidney Diseases: Oxidative Stress, Mediators, and Inflammation

Guest Editors: Ays e Balat, Halima Resic, Guido Bellinghieri, and Ali Anarat

Devil’s Triangle in Kidney Diseases: OxidativeStress, Mediators, and Inflammation

International Journal of Nephrology

Devil’s Triangle in Kidney Diseases: OxidativeStress, Mediators, and Inflammation

Guest Editors: Ayse Balat, Halima Resic, Guido Bellinghieri,and Ali Anarat

Copyright © 2012 Hindawi Publishing Corporation. All rights reserved.

This is a special issue published in “International Journal of Nephrology.” All articles are open access articles distributed under theCreative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided theoriginal work is properly cited.

Editorial Board

Anil K. Agarwal, USAZiyad Al-Aly, USAAlessandro Amore, ItalyFranca Anglani, ItalyNuket Bavbek, TurkeyRichard Fatica, USASimin Goral, USAW. H. Horl, Austria

Kamel S. Kamel, CanadaKazunari I. Kaneko, JapanDavid B. Kershaw, USAAlejandro Martın-Malo, SpainTej Mattoo, USATibor Nadasdy, USAFrank Park, USAJochen Reiser, USA

Laszlo Rosivall, HungaryMichael J. Ross, USAJames E. Springate, USAVladimır Tesar, Czech RepublicGreg Tesch, AustraliaSuresh C. Tiwari, IndiaJaime Uribarri, USA

Contents

Devil’s Triangle in Kidney Diseases: Oxidative Stress, Mediators, and Inflammation, Ayse Balat,Halima Resic, Guido Bellinghieri, and Ali AnaratVolume 2012, Article ID 156286, 2 pages

Urotensin-II: More Than a Mediator for Kidney, Ayse Balat and Mithat BuyukcelikVolume 2012, Article ID 249790, 7 pages

Antioxidants in Kidney Diseases: The Impact of Bardoxolone Methyl, Jorge Rojas-Rivera, Alberto Ortiz,and Jesus EgidoVolume 2012, Article ID 321714, 11 pages

Induction of Oxidative Stress in Kidney, Emin OzbekVolume 2012, Article ID 465897, 9 pages

Inflammation and Oxidative Stress in Obesity-Related Glomerulopathy, Jinhua Tang, Haidong Yan,and Shougang ZhuangVolume 2012, Article ID 608397, 11 pages

Reactive Oxygen Species Modulation of Na/K-ATPase Regulates Fibrosis and Renal Proximal TubularSodium Handling, Jiang Liu, David J. Kennedy, Yanling Yan, and Joseph I. ShapiroVolume 2012, Article ID 381320, 14 pages

Hindawi Publishing CorporationInternational Journal of NephrologyVolume 2012, Article ID 156286, 2 pagesdoi:10.1155/2012/156286

Editorial

Devil’s Triangle in Kidney Diseases: Oxidative Stress,Mediators, and Inflammation

Ayse Balat,1 Halima Resic,2 Guido Bellinghieri,3 and Ali Anarat4

1 Division of Pediatric Nephrology, Faculty of Medicine, University of Gaziantep, PK 34, 27310 Gaziantep, Turkey2 Division of Nephrology, Clinical Center University of Sarajevo, Sarajevo, Bosnia and Herzegovina3 Nephrology and Dialysis Unit and Postgraduate School of Nephrology, Messina University, Via Consolare Pompea No. 1867 Ganzirri,98165 Messina, Italy

4 Department of Pediatric Nephrology, Faculty of Medicine, University of Cukurova, 01330 Adana, Turkey

Correspondence should be addressed to Ayse Balat, [email protected]

Received 13 November 2012; Accepted 13 November 2012

Copyright © 2012 Ayse Balat et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

This issue of the International Journal of Nephrology focusedon kidney diseases within a devil’s triangle, oxidative stress(OS), mediators, inflammation, specifically relating to theclinical significance of identification, and prevention.

Every creature in need of oxygen faces OS. It has a criticalrole in the molecular mechanisms of renal injury in severalkidney diseases, and many complications of these diseasesare mediated by OS, mediators, and inflammation. Thereis a complex relationship between these three; mostly theyinduce each other. While some of the diseases themselves cancontribute to OS, reactive oxygen species (ROS) producedby activated leukocytes and endothelial cells in sites ofinflammation cause tissue damage. Although inflammationlooks dangerous for the organism, it is a normal reactionof organs and tissues to protect themselves against severalinvasion(s). It enables the immune system to remove theinjurious stimuli and initiate the healing process of tissues.However, the interactions between OS, mediators, andinflammation may result in glomerular damage, proteinuria,electrolyte, and volume instabilities which cause nephronloss, on the long view. Detailed studies on this topic areincluded in this issue.

The kidney can easily be damaged by ROS, due to therich structure of long-chain polyunsaturated fatty acids.The article by E. Ozbek summarizes the induction of OSwithin kidney in several conditions, including diabetes,hypertension, hypercholesterolemia, obesity, aging, urinaryobstruction, environmental toxins, and molecular mecha-nisms of these inductions in the light of existing literaturedata.

Diabetic nephropathy is one of the most commonmicrovascular complications of type 1 and type 2 diabetesmellitus and the leading cause of end-stage renal diseaseworldwide [1].

Rojas-Rivera et al. reviewed the biological bases ofoxidative stress and its role especially on diabetic nephropa-thy, as well as the role of the Keap1-Nrf2 pathway, andrecent clinical trials targeting this pathway with bardoxolonemethyl, a novel synthetic triterpenoid with antioxidant andanti-inflammatory properties.

Obesity continues to be a public health problem through-out the world. Epidemiologic studies have shown that 66% ofadults and 16% of children and adolescents are overweight orobese [2]. Obesity-related glomerulopathy is an increasingcause of end-stage renal diseases. J. Tang et al. stressed thechronic low-grade systemic inflammation in obesity anddiscussed the roles of inflammation and oxidative stressin the progression of obesity-related glomerulopathy andpossible treatment modalities to prevent kidney injury inobesity, such as the usage of anti-IL-6 receptor antibody,TNF-α antagonist, adiponectin, nutritional and surgicalinterventions to reduce OS.

Hypertension is an another important global healthissue both in adults and children. It is one of the majorrisk factors for the progression of kidney diseases. Therelationship between blood pressure and dietary sodiumand salt sensitivity has been well known, and renal sodiumhandling is a key determinant of long-term blood pressureregulation [3]. There is a limited knowledge in the literatureregarding the role of ROS-mediated fibrosis and renal

2 International Journal of Nephrology

proximal tubule sodium reabsorption through the Na/K-ATPase. S. Liu et al. reviewed the possible role of ROSin the regulation of Na/K-ATPase activity. The authorsemphasized the importance of further researches whetherROS signaling is a link between the Na/K-ATPase/c-Srccascade and NHE3 regulation and how OS, stimulated byhigh salt and cardiotonic steroids, regulates Na/K-ATPase/c-Src signaling in renal sodium handling and fibrosis.

Urotensin-II is the most potent mammalian vasocon-strictor identified to date, almost tenfold more potent thanendothelin-I [4]. A. Balat and M. Buyukcelik discussedthe role of urotensin-II on renal hemodynamics and itspossible role on several kidney diseases, such as the minimalchange nephrotic syndrome. The article includes a detaileddiscussion of urotensin-II immunoreactivity in renal biopsyspecimens of children with membranoproliferative glomeru-lonephritis, membranous nephropathy, IgA nephropathy,Henoch-Schonlein nephritis, and focal segmental glomeru-losclerosis. Because of its complex relation with OS andother mediators, authors describe it as “more than amediator” in glomerular diseases. They briefly mention fromthe effectiveness of U-II antagonism, as a new promisingpharmacological treatment target in some kidney diseases.

Given the potential impact of OS, mediators, andinflammation trio, the importance of prevention has comeinto question. Strong evidence indicates the importance ofnew molecules that are able to diminish them which inturn may help to decrease the prevalence and/or progressionof several kidney diseases. Therefore, further researches areneeded to the better understanding of the molecular andclinical mechanisms of this triad. They may help to providenew therapeutical strategies to control several complicationsin patients with kidney diseases.

Ayse BalatHalima Resic

Guido BellinghieriAli Anarat

References

[1] A. A. Lopes, “End-stage renal disease due to diabetes inracial/ethnic minorities and disadvantaged populations.,” Eth-nicity & Disease, vol. 19, supplement 1, no. 1, pp. S47–S51, 2009.

[2] Y. Wang and M. A. Beydoun, “The obesity epidemic inthe United States–gender, age, socioeconomic, racial/ethnic,and geographic characteristics: a systematic review and meta-regression analysis,” Epidemiologic Reviews, vol. 29, no. 1, pp.6–28, 2007.

[3] A. C. Guyton, “Blood pressure control–special role of thekidneys and body fluids,” Science, vol. 252, no. 5014, pp. 1813–1816, 1991.

[4] J. J. Maguire and A. P. Davenport, “Is urotensin-II the newendothelin?” British Journal of Pharmacology, vol. 137, no. 5,pp. 579–588, 2002.


Review Article

Urotensin-II: More Than a Mediator for Kidney

Ayse Balat1, 2 and Mithat Buyukcelik1

1 Division of Pediatric Nephrology, Faculty of Medicine, University of Gaziantep, 27310 Gaziantep, Turkey2 Division of Pediatric Nephrology and Rheumatology, Faculty of Medicine, University of Gaziantep, 27310 Gaziantep, Turkey

Correspondence should be addressed to Ayse Balat, [email protected]

Received 30 November 2011; Accepted 6 September 2012

Academic Editor: Ali Anarat

Copyright © 2012 A. Balat and M. Buyukcelik. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Human urotensin-II (hU-II) is one of the most potent vasoconstrictors in mammals. Although both hU-II and its receptor, GPR14,are detected in several tissues, kidney is a major source of U-II in humans. Recent studies suggest that U-II may have a possibleautocrine/paracrine functions in kidney and may be an important target molecule in studying renal pathophysiology. It has severaleffects on tubular transport and probably has active role in renal hemodynamics. Although it is an important peptide in renalphysiology, certain diseases, such as hypertension and glomerulonephritis, may alter the expression of U-II. As might be expected,oxidative stress, mediators, and inflammation are like a devil’s triangle in kidney diseases, mostly they induce each other. Sincethere is a complex relationship between U-II and oxidative stress, and other mediators, such as transforming growth factor β1 andangiotensin II, U-II is more than a mediator in glomerular diseases. Although it is an ancient peptide, known for 31 years, it lookslike that U-II will continue to give new messages as well as raising more questions as research on it increases. In this paper, wemainly discuss the possible role of U-II on renal physiology and its effect on kidney diseases.

1. Introduction

Although urotensin-II was firstly identified in a neurohemalorgan of fish in 1980s [1, 2], only recently it became a majorfocus of clinical and experimental researches [3].

Human urotensin-II (hU-II) is a cyclic peptide of 11amino acids cleaved from a larger prepro-U-II precursorpeptide of about 130 amino acids [1, 3, 4]. The gene encodingthis peptide is located at 1p36 and contains 5 exons [5]. It isa ligand for the orphan G-protein-coupled receptor, knownas GPR14 [3, 6, 7].

Although human prepro-U-II mRNA is expressed mainlyin the brain and spinal cord [4, 8], both hU-II and itsreceptor are also detected in other organs and tissues, such askidney, spleen, smooth muscle, endothelium, small intestine,thymus, prostate, pituitary, and adrenal gland [1, 9–11].

Being almost tenfold more potent than endothelin-I(ET), it is the most potent mammalian vasoconstrictoridentified to date [3, 12]. It circulates in the plasma of healthyindividuals and acts as a circulating vasoactive hormoneand as a locally acting paracrine or autocrine factor incardiovascular regulation [4, 13].

Although it mainly has a vasoconstrictor effect, regionaldifferences may be seen in its effects in various vascularbeds and blood vessels of some species. For example, it hasa vasodilatory effect on the small arteries of rats [14, 15],and on the resistance arteries of humans, through release ofendothelium-derived hyperpolarizing factor (EDHF), nitricoxide (NO) [14–16].

It has been shown that the potent vasoconstrictoractions of U-II is mediated by Ca+2 mobilization throughactivation of a number of signaling pathways including Ca+2

channels, tyrosine kinase, p38 mitogen-activated proteinkinase (p38MAPK), and extracellular signal-regulated kinase1 and 2 (ERK1/2) [17, 18].

Since U-II and its receptor have been demonstrated inmouse, monkey [19], and human kidneys [20, 21], it isacceptable to consider that U-II is synthesized, secreted, andcleared by the kidneys [6, 22–24].

Interestingly, Mosenkis et al. [25] showed that hU-II wasalso present in 2 surgically anephric subjects. Although thisfinding is inconsistent with the conclusion that the kidneysare the primary source for production of U-II, as the authorsstressed, the high density of U-II and its receptor in renal


tissues suggest that U-II is metabolically active in the kidneyeven though it is produced in outside of the kidneys.

In this paper, we mainly discuss the possible role of U-IIon renal physiology and its effect on kidney diseases.

2. Effect of U-II on Renal Hemodynamics

Because of its potent vasoconstrictor effect, U-II attractedthe interest of researchers in general hemodynamy. In fact,hemodynamic responses to U-II show regional heterogeneityin relation to its receptor localization, even in the differencesof functional state of the endothelium [26].

However, in contrast to animal studies, Wilkinson et al.found no vasoactive responses to hU-II in vivo in man[27]. They injected hU-II intra-arterially to healthy malevolunteers, and despite the high-circulating hU-II levels,no change was seen in systemic hemodynamics, ECGs ofsubjects, and hU-II had no effect on hand vein diameter.

However, another study published in the same year [28]demonstrated that U-II produces potent vasoconstrictionin humans in vivo. They showed that U-II induced dose-dependent reduction in forearm blood flow (FBF) of healthyvolunteers, and FBF returned to baseline values within30 min.

Known data in the literature show that the kidney is amajor source of U-II in humans [29], primates, mice [19],and rats [11]. It has been found in the urine of humans[22, 24] and rats [11] at a concentration far exceeding thatof plasma. In humans the renal clearance of U-II has foundto be greater than that of creatinine, suggesting that urinaryU-II is derived primarily from the kidney [22]. An animalstudy also revealed that there was an arteriovenous con-centration gradient for U-II across the renal circulation inanaesthetised sheep [30]. As well as U-II, its receptor has alsobeen localized to the mammalian kidneys, such as in human[22], monkey, mouse [19], and rat kidneys [11]. It has beenshown that the medulla, especially tubular component of thekidney, is the principal site of U-II receptor expression in therat kidney [11, 31].

Shenouda et al. [20] demonstrated that U-II was mostlypresent in the epithelial cells of tubules and ducts, withgreater intensity in the distal convoluted tubules in normalhuman kidneys. Moderate U-II immunoreactivity was seenin the endothelial cells of renal capillaries, but only focalimmunoreactivity was found in the endothelial cells of theglomeruli. We also observed similar results in kidneys ofchildren [21], and these findings suggest that hU-II maycontribute to the pathophysiology of human kidneys (Figure1(a)) [21, 32].

Expression of U-II mostly in tubules may suggest itsprobable active role in renal hemodynamics. Loretz andBern [33] demonstrated that U-II stimulated Na transport inthe teleost urinary bladder, while Ovcharenko et al.’s study[34] indicated that short-term adminisration of U-II didnot influence sodium (Na) handling by the kidney in rats.However, Zhang et al. [35] observed that infusion of U-IIdirectly into the rat renal artery increased renal blood flow(RBF), associated with a diuresis and natriuresis. In contrast,

Song et al. [11] reported that U-II caused an antinatriuresisand antidiuresis when administered as an i.v. bolus doseand stressed that this was associated with and driven byrenal hemodynamic effects leading to a marked reduction inglomerular filtration rate (GFR).

Two years later from the above study, Abdel-Razik et al.[36] searched the potential direct tubular action of urotensinin rats. They observed dose-dependent changes in GFR andurinary electrolytes. The hemodynamic effects were predom-inated at higher doses and caused a profound reduction inGFR which was accompanied by an antidiuresis and anti-natriuresis. When a lower infusion rate of rat U-II wasemployed, a tubular action to reduce electrolyte reabsorptionbecame apparent through an increase in fractional excretionof Na and potassium (K) [36].

However, in children with minimal change nephroticsyndrome (MCNS), and their healthy controls, we couldnot find any relationship between the U-II level and Na/Kexcretion [24]. Although this differences may be partiallyrelated to different biological effects of U-II in differentspecies, contradictory observations in same species underlinethe complex influence of U-II on renal hemodynamics.

It is not clear enough whether the effect of U-IIon tubular transport is direct or mediated by secondarymechanisms. However, considering the expression of U-IIreceptor in the thin ascending limb of Henle’s loop andthe inner medullary collecting duct [11] and the greaterU-II receptor mRNA and protein expression in the medullacompared with the cortex [36], together with the abundantexpression of hU-II in the proximal and distal tubules ofchildren (patients and controls) [21], it may be suggestedthat U-II may have a direct action on tubular electrolytetransport.

3. The Effect of Urotensin-II on Kidney Diseases

Since U-II and its receptor, GPR14, are expressed abundantlyin cardiorenal system [10, 11], most of the researches on it arerelated to cardiovascular and renal diseases.

Although some studies have been investigated the circu-lating levels of U-II in several diseases, such as hypertension[37], congestive heart failure [38], renal failure [3], MCNS[24], and preeclampsia-eclampsia [39], little is known aboutthe actions of this important peptide within the kidney.Some studies suggest that renal dysfunction affects theU-II levels, since the plasma U-II level has been foundelevated in renal failure [3], congestive heart failure [38], andsystemic hypertension [37], and it was found to be an inversepredictor of overall and cardiovascular mortality in patientswith moderate-to-severe chronic kidney disease (CKD)[40].

Certain diseases, such as hypertension and glomeru-lonephritis, may alter the expression of U-II. It has beenshown that both U-II and its receptor mRNA expression lev-els were up to threefold higher in spontaneously hypertensiverat (SHR) tissue compared to control Wistar-Kyoto (WKY)rats, taking into consideration that SHR is more sensitivethan WKY to the effect of U-II [41].

International Journal of Nephrology 3

(a) (b)

(c) (d)

Figure 1: (a) Localization of urotensin-II (U-II) immunoreactivity (brown color) in the normal human kidney. Weak immunostaining inglomerulus, abundant expression of U-II in tubules. Thick arrows: weak immunostaining and black arrow: strong immunostaining [21].(b) Urotensin-II immunoreactivity in membranoproliferative glomerulonephritis. Immunostaining in glomerular basement membrane,mesangium, Bowman capsule, and tubules. GBM: glomerular basement membrane, MPGN: membranoproliferative glomerulonephritis,BC: Bowman capsule, M: mesangium, PT: proximal tubule, and DT: distal tubule [21]. (c) Localization of urotensin-II immunoreactivity ina crescent [21]. (d) Urotensin-II immunoreactivity in focal segmental glomerulosclerosis. Notice the abundant expression of U-II in scleroticarea [32].

In the literature, there are no enough data on the levelof this vasoactive peptide in glomerular diseases. Recently,we firstly demonstrated that U-II was present in plasma andurine samples of 26 children with MCNS [24]. It showedimportant changes in relapse and remission periods. PlasmaU-II concentrations during relapse were significantly lowerthan in remission and in controls, whereas urinary U-II levelswere higher in relapse than in remission [24]. The plasmaU-II level showed a significant positive correlation with theplasma albumin concentration during remission. However,there was no correlation between the amount of proteinuriaand plasma/urinary U-II levels, and we could not detectany relationship between U-II levels and other clinical andlaboratory parameters (such as the age at onset of disease,number of relapses, time to remission, blood pressure, serumcreatinine, and hematological parameters). We suggestedthat the important changes in plasma and urinary U-II levelsduring relapse may be the result of heavy proteinuria ratherthan playing a role in mediating the clinical and laboratorymanifestations of MCNS. After this, it would be interestingto search the possible role(s) of this peptide in childrenwith glomerular diseases other than MCNS. Therefore, weexamined the urotensin-II immunoreactivity in renal biopsyspecimens of children with several renal diseases, includingmembranoproliferative glomerulonephritis (MPGN), mem-branous nephropathy (MGN), IgA nephropathy (IgAN),Henoch-Schonlein nephritis (HSN), and focal segmental

glomerulosclerosis (FSGS) [21, 32]. In normal human kid-ney, there was weak expression of human U-II in glomerulus,while abundant expressions were seen in proximal, distaltubules, and collecting ducts (Figure 1(a)) [21], similar to aprevious study [20].

We observed different expression pattern of U-II indifferent glomerular diseases. In MPGN and FSGS, differentfrom the normal kidneys, more dense U-II immunoreactivitywas seen in the glomerular basement membrane (GBM),glomerular mesangium, Bowman capsule (BC), and tubules(Figures 1(b), and 1(d)) [21, 32]. Interestingly, we alsoobserved U-II immunoreactivity in crescents (Figure 1(c)),and sclerotic areas in FSGS (Figure 1(d)) [21, 32].

Systolic blood pressure (BP) was positively correlatedwith mesangial expression of U-II (r = 0.418, P = 0.042),while diastolic BP was correlated with endothelial U-IIexpression in MPGN (r = 0.469, P = 0.021) [21].

In children with MGN, U-II was mostly seen in GBMand BC. We observed more dense U-II immunoreactivity indistal tubules (P = 0.030), endothelium (P = 0.009), andmesangium (P = 0.002) in children with MPGN than inMGN. Diastolic BP was positively correlated with the expres-sion of U-II in BC in children with MGN (r = 1, P = 0.000)[21].

There is no enough data about the precise role of hU-II inrenal diseases, and that was the first report demonstrating thepresence of U-II by immunohistochemically in children with


several renal diseases, suggesting that hU-II may contributeto the pathophysiology of human kidneys.

The positive correlation between BP and intensity ofU-II expression in mesangium and endothelium in MPGN,and BC in MGN was noteworthy. Considering the literaturedata about U-II, as an important physiological mediator ofvascular tone and blood pressure in humans [16], and alsoan extremely potent constrictor of renal blood vessels fromprimates [6], it is reasonable to suggest that U-II may play animportant role in the regulation of BP in MPGN and MGN.

As it has been known, mainly, two basic mechanismsare feasible in glomerulonephritis: antibody interaction withantigens in situ within the glomerulus and antibody bindingto soluble antigens in the circulation, followed by immune-complex deposition within the glomeruli [42]. The sec-ondary immune mechanisms of glomerular injury are thecascade of inflammatory mediators that are recruited topropagate renal damage following the primary glomerularattack. Some of these mediators play essential roles, whereasothers may aggravate the glomerular lesion [42]. Most of thesecondary mediators include cytokines, growth factors, reac-tive oxygen metabolites, bioactive lipids (platelet-activatingfactor and eicosanoids), proteases, and vasoactive substances,such as ET and NO [42].

Since U-II is abundantly expressed in the glomeruli inMPGN and MGN, it is reasonable to suggest that U-II mayplay a role in this mechanism, probably in the secondaryimmune mechanisms of glomerular injury, by a paracrine oran endocrine action [21]. Djordjevic et al. [43] demonstratedthat hU-II increases the levels of NADPH oxidase-derivedreactive oxygen species, leading to the activation of mitogen-activated protein kinases and protein kinase B (akt), followedby enhanced plasminogen activator inhibitor-1 expressionand increased proliferation of pulmonary arterial smoothmuscle cells. It has been also shown that exposure of the ratproximal tubular epithelial cells (NRK-52E) to transforminggrowth factor β1 (TGF-β1) or angiotensin II (Ang II)increased U-II and GPR14 mRNA expressions [44], andU-II acts synergistically with Ang II [45, 46]. As might beexpected, oxidative stress, mediators, and inflammation arelike a devil’s triangle in kidney diseases, mostly they induceeach other. Since there is a complex relationship betweenU-II and oxidative stress [43], and other mediators, suchas TGF-β1 and Ang II [44–46], U-II is probably more thana mediator in glomerular diseases and takes place in animportant part of this devil’s triangle.

Interestingly, we observed abundant U-II immunoreac-tivity in crescents and sclerotic areas in FSGS [21, 32]. Cres-cents are composed of large swollen cells arising from bothmacrophages of hematogenous origin and native parietalepithelial cells [47]. As time elapses, the cellular crescents areprogressively replaced by fibroblasts, and in more advancedstages, the fibroblastic component is entirely replaced bycollagenous lamellar materials with a few remnant cells [48].Recent reports have shown a mitogenic role for U-II throughinduction of smooth muscle cell proliferation [49, 50], andadditionally, it has been shown to induce collagen depositionby fibroblasts [51]. Zhang et al. [52] showed that U-IIcould stimulate the phenotypic conversion, migration, and

collagen synthesis in adventitial fibroblasts. Additionally, itmay act as autocrine/paracrine growth stimulators in tumorcells [53].

The pathogenesis of glomerulosclerosis is still unknown.Several factors, cytokines and growth factors, hyperlipidemiaand platelet activation, lead to an increase of mesangialmatrix production by resident cells. Several data demon-strate that abnormal glomerular growth is associated withglomerular sclerosis [54].

Since hU-II stimulates cell proliferation in adrenaltumors [55], renal epithelial cells [23], and vascular smoothmuscle cells [49] and it has been found elevated in carotidand aortic atherosclerotic plaques [56], the abundant expres-sion of U-II in crescents and sclerotic areas suggests thatU-II may also play a role in the progression of crescents andglomerular sclerosis, probably as a growth factor or as aninflammatory peptide. This hypothesis must be searched andtested in future.

MGN is an antibody-mediated disease of uncertain andimprecise pathogenesis. However, the hypotheses that it is anautoimmune disease of the kidney and that the subepithelialimmune deposits are formed in situ with an endogenousglomerular antigen are attractive [57]. The electron-densedeposits are generally located at the site of the slit diaphragm,and subepithelial space, while no electron-dense depositsare seen in the subendothelial space or in the mesangium,and hypertension at onset is associated with a less favorableoutcome in MGN [57]. In our study, hU-II expressionwas mostly seen in GBM and BC, and there was a strongpositive correlation between diastolic BP and intensity ofU-II expression in BC. These findings may increase twointeresting questions: may U-II play a role in the formationof these deposits as a mitogenic factor, as we mentionedpreviously, and may it have any role in the clinical course ofMGN by regulating the BP? However, it is difficult to answerthese questions with that study, and these hypothesis must beclarified by further detailed studies.

In kidneys of children with HSN and IgAN, similar toeach other, more dense U-II immunoreactivity was seen inGBM, glomerular mesangium, BC, proximal/distal tubules,and also in crescents [21, 32].

Although the pathogenesis of IgAN and HSN is not wellknown [58], animal studies have shown the key role ofcytokines and growth factors (particularly platelet-derivedgrowth factor and TGF-β) in the induction and resolutionof mesangial injury, and there is some evidence that these arealso involved in IgAN [58]. The similar expression patternof U-II in HSN and IgAN has been considered that U-IImay have a role in mesangial inflammation and crescentformation in these disorders [32].

Different expression pattern of U-II in several renaldiseases may give rise to thought whether the effect ofU-II gene polymorphism. Recently, we performed a prelim-inary study, and firstly investigated the possible associationbetween a coding single nucleotide polymorphism of UT-IIgene, T21M (T/C), in 87 children with childhood nephroticsyndrome (NS), 16 children with acute poststreptococcalglomerulonephritis (APSGN), and 10 children with HSN[59]. We found higher TC genotype of U-II gene in NS


(56.3% versus 38.9%, P = 0.025), higher TT genotype inAPSGN (50.0% versus 25.9%, P < 0.001), and a positivecorrelation between TT polymorphism and the presence ofmacroscopic hematuria in APSGN (r = 0.51, P = 0.04).This study considered that urotensin-II may be an importantmediator in pathophysiology of the childhood glomeru-lonephritis, and Turkish children with TC genotype may havea higher genetic susceptibility to NS, while TT genotype ofU-II may increase the risk of APSGN.

4. Urotensin-II Antagonists as a New PromisingPharmacological Treatment Target

Several influences of U-II in cardiovascular/renal system,and the presence of its receptor in the heart, lungs, bloodvessels, kidneys, and brain, led the researchers to investigatethe role of U-II antagonists in various diseases. The mostknown U-II receptor antagonists are palosuran and urantide.Sidharta et al. [60] investigated whether palosuran, a potent,selective, and competitive antagonist of the U-II receptor,had effects in macroalbuminuric, diabetic patients who areprone to the development of renal disease. They observedan overall clinically significant reduction of 24.3% in the 24-hour urinary albumin excretion rate.

In an experimental study, it has been shown that long-term treatment of streptozotocin-induced diabetic rats withpalosuran improved survival, increased insulin, and slowedthe increase in glycemia, glycosylated hemoglobin, andserum lipids. Furthermore, palosuran increased renal bloodflow and delayed the development of proteinuria and renaldamage [61].

These two researches suggest that U-II receptor antago-nism might be a new therapeutic approach for the treatmentand/or prevention of diabetic nephropathy.

The tolerability and safety, pharmacokinetics, and phar-macodynamics of palosuran were evaluated in also healthyyoung men with a double-blinded placebo-controlled singleascending dose designed study [62]. It has been shown thatpalosuran was well tolerated, and no serious adverse eventsor dose-related adverse events were reported. However, as theauthors stressed, the results of this entry-into-humans studywarrant further investigation of the therapeutic potential ofpalosuran.

Recently, Nitescu et al. [63] examined the effects ofanother selective U-II receptor antagonist, urantide, on renalhemodynamics, oxygenation, and function in endotoxemicrats. However, they found that urantide had no statisticallysignificant effects on any of the investigated variables (kidneyfunction, renal blood flow, cortical and outer medullaryperfusion, and oxygen tension) in these rats.

In spite of different results about the effectiveness of U-IIantagonism, it appears that the therapeutic potential of U-IIantagonists may be the focus of research interest in the nearfuture.

In summary, U-II may be an important mediator, in factprobably more than a mediator, in kidney diseases. Whetherthe observed findings which are primary or secondaryto these pathological conditions still remain unclear, they

suggest a possible role of U-II in the pathophysiology ofseveral kidney diseases. It looks like that U-II will continueto give new messages as well as raising more questions asresearch on it increases. Further, detailed studies are neededto address the exact role(s) of this peptide in renal diseases.

References

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Review Article

Antioxidants in Kidney Diseases:The Impact of Bardoxolone Methyl

Jorge Rojas-Rivera, Alberto Ortiz, and Jesus Egido

Laboratory of Renal and Vascular Pathology, Division of Nephrology and Hypertension, IIS Fundacion Jimenez Dıaz,Autonoma University of Madrid, 28040 Madrid, Spain

Correspondence should be addressed to Jorge Rojas-Rivera, [email protected]

Received 15 September 2011; Revised 2 April 2012; Accepted 10 April 2012

Academic Editor: Ali Anarat

Copyright © 2012 Jorge Rojas-Rivera et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Drugs targeting the renin-angiotensin-aldosterone system (RAAS) are the mainstay of therapy to retard the progression ofproteinuric chronic kidney disease (CKD) such as diabetic nephropathy. However, diabetic nephropathy is still the first causeof end-stage renal disease. New drugs targeted to the pathogenesis and mechanisms of progression of these diseases beyond RAASinhibition are needed. There is solid experimental evidence of a key role of oxidative stress and its interrelation with inflammationon renal damage. However, randomized and well-powered trials on these agents in CKD are scarce. We now review the biologicalbases of oxidative stress and its role in kidney diseases, with focus on diabetic nephropathy, as well as the role of the Keap1-Nrf2pathway and recent clinical trials targeting this pathway with bardoxolone methyl.

1. Background

Chronic kidney disease (CKD) is a serious public healthproblem, which carries a high morbidity and mortality[1]. CKD is characterized by a progressive loss of renalfunction, chronic inflammation, oxidative stress, vascular re-modeling, and glomerular and tubulointerstitial scarring.CKD treatment still represents a clinical challenge. Diabeticnephropathy (DN) is the leading cause of CKD and end-stage renal disease (ESRD) [2]. The renin-angiotensin-aldosterone system (RAAS) is a major pathway involvedin the pathogenesis and progression of DN [3, 4], andRAAS blockade is an effective therapeutic strategy to reduceproteinuria and slow progression of diabetic and nondiabeticCKD. However targeting the system sets off compensatorymechanisms that may increase angiotensin II, aldosterone,or renin, and partial RAAS blockade does not preventprogression in all CKD patients. Angiotensin II (AT II) is thekey mediator of the RAAS [5–7]. Animal models of experi-mental diabetes, clinical trials, and metanalysis have clearlydemonstrated the effectiveness of angiotensin-converting

enzyme inhibitors (ACEIs) or angiotensin receptor blockers(ARBs) therapy to improve glomerular/tubulointerstitialdamage, reduce proteinuria, and decrease CKD progression,independently of blood pressure (BP) control [8–13]. DualRAAS blockade with ACEI plus ARB inhibits compensatoryAT II activity resulting from ACE-independent pathwaysand limits compensatory AT production induced by AT1receptor blockade. This combination reduced proteinuriaby 25–45% in DN [14–16]. Results are worse for DN withdiminished kidney function or nonproteinuric CKD withischemic renal injury, probably due to advanced structuralrenal changes [13, 17, 18] and adverse effects; such acutedeterioration of renal function or hyperkalemia is morefrequent. The aldosterone antagonists spironolactone andeplerenone reduce albuminuria by 30–60% and slow CKDprogression in experimental models [19–21] and clinicalstudies [22–25] in DN. These agents abrogated the “aldos-terone breakthrough” phenomenon and its proinflammatoryand profibrotic effects. ACEI/ARB therapy increases renin.Aliskiren, a direct renin inhibitor, was beneficial in animalmodels of diabetic/hypertensive nephropathy [26, 27] and


reduced albuminuria in clinical DN [28]. In a multicenterand double-blind, randomized clinical trial in hyperten-sive type 2 DM patients with nephropathy, aliskiren pluslosartan at maximal dose was 20% more effective thanlosartan/placebo to reduce albuminuria without adverseeffects, independent of BP control [29].

A number of other strategies have been tried. AdequateBP and glucose control are part of standard care of DNpatients. Intensive glucose control has more impact on GFRif early instituted in patients with type 1 DM but this maynot necessarily apply to patients with type 2 DM or withadvanced CKD [30]. A trial of the vitamin D activatorparicalcitol missed the primary endpoint of albuminuriareduction in DN and caused a transient decrease in eGFR[31]. The nephroprotective effect of statins on CKD foundin experimental models has not been conclusively provenin clinical studies [32]. A 1-year dose-ranging study ofpirfenidone suggested better preservation of eGFR by pir-fenidone in a small number of diabetic nephropathy patients[33]. The selective endothelin antagonist atrasentan reducedalbuminuria in a short-term (8 weeks) study in a smallnumber of diabetic patients while receiving RAS inhibitorsbut did not assess long-term renal function [34]. Heartfailure patients or with peripheral edema were excluded.

In spite of all this experimental and clinical evidence,there are 35–40% of patients with DN that progress toadvanced renal disease or ESRD. The risk of progressionto ESRD is still clinically relevant in other proteinuricnephropathies [35, 36]. Novel therapeutic targets are neededin CKD that are based on a clear understanding of thepathogenesis of CKD progression beyond the RAAS.

2. Oxidative Stress and Kidney Disease

Oxidative stress and inflammation promote kidney andvascular injury [37–42]. Several factors induce ROS in renalcells, such as inflammatory cytokines, Toll-like receptors,Angiotensin II, bradykinin, arachidonic acid, thrombin,growth factors, and mechanical pressure. NADPH oxidases,now renamed Nox enzymes, are key ROS generators inresponse to these stimuli [43, 44].

In acute kidney injury (AKI) induced by ischemic-reperfusion injury, sepsis or acute rejections ROS contributeof to endothelial and tubular injury [45, 46]. In murinemodels of AKI, bardoxolone methyl decreased functionaland structural renal injury and increased the expressionof protective genes (Nrf2, PPARγ, HO-1) on glomerularendothelium, cortical peritubular capillaries, tubules, andinterstitial leukocytes [47].

ROS contribute to hypertension-induced kidney andvascular injury [41, 43, 48, 49]. The chronic complications ofdiabetes are characterized by a defect in Nrf2 signaling andits adaptive response to oxidative stress. This is a potentialmechanism for cellular stress hypersensitivity and tissuedamage [50]. Diabetic nephropathy is characterized by initialhyperfiltration, albuminuria and subsequent loss of renalfunction, thickening of basement membranes, expansionof mesangial matrix and interstitial fibrosis, and podocytes

and renal cell damage [51]. ROS production in response tohyperglycemia, protein kinase C (PKC), advanced glycosy-lation end products (AGEs), free fatty acids, inflammatorycytokines, and TGF-beta1 contributes to these changes [43,44, 52–54]. These stimuli activate the NADPH/NADPHoxidase system in renal cells. Oxidative stress induced byhyperglycemia or glucose degradation products may causeleukocyte or renal cell apoptosis and release of extracellularmatrix [52, 55–59]. PKC activates NF-kappaB, extending theinflammatory response [60]. TGF-beta signaling is key tothe excessive matrix formation [61, 62]. The activation ofNrf2 is increased in diabetic nephropathy and can amelioratemesangial damage via partial inhibition of TGF-beta1 andreduction of extracellular matrix deposition [63]. ACEinhibitors lower TGF-beta in urine from DN patients. Inrat DN glomerular HO-1 is increased, evidencing oxidativestress [52, 64]. ROS can activate several transcription factorssuch as NF-kappaB, AP-1, Sp1, which in turn affect theexpression of mediators of inflammation, fibrosis, and celldeath [52, 65] (Figure 1(a)).

ROS also contribute to renal injury in experimentalglomerulonephritis. In experimental anti-Thy 1 glomeru-lonephritis, ROS enhance cell proliferation and matrixaccumulation and fibrosis and this is improved by theantioxidant alpha-lipoic acid [66, 67]. ROS also regu-late the immune response [68]. In nephrotoxic nephritis,neutrophils promote glomerular TNF-alpha expression viaH2O2 production [69]. TNF-alpha is a key mediator ofglomerular injury [70]. Interstitial inflammatory leukocytesin proliferative glomerulonephritis locally generate ROS andcontribute to sodium retention [71]. Angiotensin II pro-motes ROS-mediated F-actin cytoskeleton rearrangement,resulting in podocyte injury [72]. In cultured podocytesAT1R signaling activates Rac-1 and NADPH oxidase toproduce additional ROS and downregulates the antioxidantprotein peroxiredoxin (Prdx2) [73]. In experimental passiveHeymann nephritis, a model of membranous nephropa-thy, C5b-9 activation promotes ROS-mediated injury inglomerular cells [74, 75]. In this regard, evidence foroxidative stress, the glomerular neoexpression of aldosereductase (AR) and SOD2, and the appearance of anti-AR and anti-SOD2 autoantibodies was recently reported inhuman membranous nephropathy suggesting that oxidativestress may generate new autoimmune targets [76].

In lupus nephritis, multiple abnormalities in T and B cellslead to autoimmune renal inflammation and ROS produc-tion [77]. Nrf2-knockout mice showed impaired antioxidantactivity, increased oxidative stress, and a lupus-like autoim-mune nephritis with glomerular injury, impaired kidneyfunction, and a shortened lifespan. Thus, Nrf2 deficiencycould lead to systemic autoimmune inflammation withenhanced lymphoproliferation [78]. In antineutrophil cyto-plasmic antibodies (ANCAs-) associated vasculitis, ANCA-activated neutrophils and monocytes release MPO andgenerate ROS, producing endothelial and tissue damage [79,80]. ANCAs also promote ROS-dependent dysregulation ofneutrophil apoptosis [81] (Figure 1(a)).

In proteinuric nephropathies and independently of eti-ology, the presence of albumin in urine activates proximal


AGE-RAGE

Hyperglycemia

RAAS activation

Increased vasoconstrictors

JAK/STAT

signaling

Albuminuria

Loss of kidney function

NADPH

oxidase

Tubulointerstitial

fibrosis

PKCGrowth factors

Cytokines

Inflammatory

cells

Podocyte, mesangial, endothelial,

and tubular cell damage

Chronic immunological disorder

Antibody production

(auto-anti-Abs, ANCAs)

Complement system

activation

Cytokines

Tubulointerstitial

inflammationGlomerulosclerosisTubular atrophy

Salt retention

Salt-sensitive

hypertension

Vasoconstriction

Ischemia

Progression to

ESRD

↑ ROS

↑ TGF-β1

↑ ECM/EMT

(a)

Figure 1: Continued.


Albuminuria

Activation of PKC

ROS

Tubulointerstitialinflammation and fibrosis

Release of chemokines (MCP-1)and attraction of macrophages

Loss of

kidney function

Progression to

ESRD

Oxidation of albumin

RCG

Autooxidation offatty acids bounds

to albumin

RCG

Activation of

NADPH oxidase

Activation ofNF-κB in PTC

(b)

Figure 1: Overview of interrelation of ROS with other key pathogenic factors in kidney disease. (a) Role of ROS in diabetic nephropathyand immune-mediated glomerulonephritis. ROS are induced in renal cells in response to high glucose, AGE, and cytokines. PKC, NADPHoxidase, and mitochondrial metabolism are key to ROS generation. ROS activate signal transduction cascade and transcription factors,leading to upregulation of genes and proteins involved in renal cell injury, glomerular and interstitial extracellular matrix deposition, andrecruitment of inflammatory cells, promoting albuminuria and progression of chronic kidney disease. (b) Role of albuminuria and ROS intubular damage and progression of CKD. Albuminuria injures PTC and activates them to release chemokines that attract macrophages andpromote tubulointerstitial fibrosis. Membrane NADPH oxidase is the main source of the ROS. It is possible the generation of other reactivespecies, as carbonyl groups derived from abnormal oxidation of albumin and fatty acids bound to albumin. Abs: antibodies, AGE: advancedglycation end products, ANCA: antineutrophil cytoplasmic antibodies, ECM: extracellular matrix, EMT: epithelial-mesenchymal transition,ESRD: end-stage renal disease, MCP-1: monocyte chemoattractant protein-1, NADPH: nicotinamide adenine dinucleotide phosphate, NF-kappa B: nuclear factor kappa B, PKC: protein kinase C, PTC: proximal tubular cell, RAAS: renin-angiotensin-aldosterone system, ROS:reactive oxygen species, RCG: reactive carbonyl groups, TGF-β1: transforming growth factor beta 1.


tubules to release chemokines that promote interstitialinflammation [82]. Albumin uptake by tubular cells activatesPKC and ROS generation via NADPH oxidase which acti-vates NF-kappaB and production of inflammatory mediators[83, 84] (Figure 1(b)).

Accelerated atherogenesis is common in early and ad-vanced stages of CKD [40, 85]. Oxidative stress contributesto the Immune inflammation-Renal injury-Atherosclerosiscomplex (IRA paradigm), a condition present in AKI and inCKD, and accelerated atherogenesis [86, 87].

A simplified overview of several components of oxidativestress and its interrelations with other key elements ofpathogenesis and progression in kidney disease is shown inFigure 1(a).

3. Oxidative Stress and the Keap1-Nrf2 Pathway

Reactive oxygen species (ROS) include superoxide anion(SOA), hydrogen peroxide (H2O2), and hydroxyl radical. ROSare formed continuously as by-product of aerobic metab-olism. Sources of ROS include the mitochondrial electrontransport chain, metabolism of arachidonate by cyclooxyge-nases or lipoxygenases, cytochrome P450 enzymes, NADPHoxidases, or nitric oxide synthetases [88]. ROS contributeto killing bacteria, and genetic defects of NADPH oxidasecause chronic granulomatosis [89]. However, ROS may causechemical damage to DNA, proteins, and unsaturated lipidsand lead to cell death. ROS contribute to multiple pathologicprocesses [48, 90]. In this regard, homeostasis is maintainedthrough a complex set of antioxidants mechanisms that pre-vent oxidative stress-induced injury. The main mechanismsare enzymes that catalyze antioxidant reactions: glutathioneperoxidase, superoxide dismutase, catalase, Hem-oxygenase(HO-1), NADPH-quinone oxidoreductase and glutamate-cysteine ligase. These enzymes are encoded by stress-responsegenes or phase 2 genes that contain antioxidant responseelements (AREs) in their regulatory regions [88, 91]. Nrf2is the principal transcription factor that binds to the AREpromoting transcription. Actin-tethered Keap1 is a cytosolicrepressor that binds to and retains Nrf2 in the cytoplasm,promoting its proteasomal degradation. Inducers of phase2 genes modify specific cysteine residues of Keap1 resultingin conformational changes that render Keap1 unable torepress Nrf2. Consequently, Nrf2 activates the transcriptionof phase 2 genes. Oleanolic acid activates the ARE-Keap1-Nrf2 pathway, resulting in reduced proinflammatory activityof the IKK-beta/NF-kappaB pathway, increases productionof antioxidant/reductive molecules, and decreases oxidativestress, thereby restoring redox homeostasis in areas ofinflammation. In various cell lines, this results in inhibitionof proliferation, promotion of differentiation and apoptosisinduction [91–93]. Synthetic analogues of oleanolic acid,named triterpenoids, are potent anti-inflammatory agentsthat activate the ARE-Keap1-Nrf2 pathway [91]. Bardox-olone methyl, also known as CDDOMe, is a triterpenoidwhose nephroprotective action has been recently explored inhumans.

4. Antioxidant Agents in Kidney Disease

Epidemiological studies have demonstrated associationbetween inflammatory and oxidative stress markers withcardiovascular and renal outcomes in CKD and ESRD [40,94–96]. Experimental data in animal models of renal diseasesuggest beneficial effects of antioxidants agents, but results inhuman studies are limited and controversial.

In early experimental diabetes mellitus in hypertensiverats, the administration of tempol, an antioxidant SODmimetic, corrected the oxidative imbalance and improvedoxidative stress-induced renal injury, decreasing albuminuriaand fibrosis [97]. Similar protection was afforded by theantioxidants N-acetyl-L-cysteine (thiol) and kallistatin inDahl salt-sensitive rats [98, 99]. In spontaneously hyper-tensive rats, a lifelong antioxidant-rich diet diminished theseverity of hypertension, improved oxidative stress and ame-liorated abnormalities of antioxidant enzyme expressionsand activities in contrast to regular diet [100]. In sum-mary, in models of hypertensive rats, synthetic and naturalantioxidants induced renal and endothelial protection withreduction of oxidative stress. In a model of ischaemia reper-fusion and cyclosporin toxicity after unilateral nephrectomy,the blockage of the mitochondrial enzymes monoamineoxidases with pargyline 28 days following surgery preventedH2O2 production and improved renal function and renalinflammation (lower IL-1β and TNF-α gene expression)[101]. Pargyline administrated before ischemia reperfusionsignificantly reduced apoptosis, necrosis, and fibrosis. Thiseffect was associated to decreased expression of TGF-β1, col-lagen types I, III, and IV and to the normalization of SOD1,catalase, and inflammatory gene expression. In models ofrenal chronic failure (5/6 nephrectomy rats) [102], AST-120,an oral carbonic adsorbent, improved the oxidative stress inendothelial cells, measured as oxidized/unoxidized albuminratio. This effect was reached reducing the blood levels ofindoxyl sulfate, an uremic toxin that induces ROS. In anothermodel of remnant kidney, the administration of omega-3fatty acids, an effective compound in mitigating atheroscle-rosis, significantly lowered several components of oxidativestress and markers of inflammatory and fibrotic response.Furthermore, it attenuated tubulointerstitial fibrosis andinflammation in the remnant kidney [103]. In anti-Thy1glomerulonephritis, the treatment with parthenolide, ananti-inflammatory agent related to the triterpenoid family,diminished renal inflammation via NF-kappaB inhibition,decreased MCP-1 and iNOS, and improved proteinuria,tubular, and glomerular damage [104]. The beneficial effectof exogenous antioxidants shown in animal models withhypertension or chronic renal failure has not been demon-strated in people with clinical hypertension [96, 105] orCKD.

5. Nephroprotection by Bardoxolone Methyl

Bardoxolone was initially described as an agent that pro-tected cells from radiation-induced damage (radiation mit-igator) through Nrf2-dependent and -independent pathways[106]. In humans, its potential antineoplasic activity was


RAASoveractivation

BARD

ACEI/ARB or

MRA/DRI therapy

CKDprogression

Increased antioxidant response

Decreased ROS generation

Decreased inflammation

Nrf2 activation

Other mechanisms

Aldosterone leakage phenomenon

Prorenin effects

Transcription of genes with

ARE sequences in promoters

Keap1

+

↑ Cell injury

↑ TGF-β1

↑ NFκB

↑ ROS

Figure 2: Overview of RAAS blockers and bardoxolone in pathogenic pathways in kidney diseases. RAAS blockers target several pathogenicpathways in kidney injury, including those generating ROS. However, there are several escape mechanisms (aldosterone breakthrough,increased prorenin effects) as well as less sensitive lesions (significant loss of kidney function, ischemic disease, persistent immune activity).BARD promotes activation of the Nrf2 transcription factor, that is released of the inhibitory Keap1 protein and migrates to the nucleuswhere it regulates transcription of genes containing ARE sequences in their promoters. These phase 2 response genes are collectivelyinvolved in the reduction of ROS and inhibition of NF-kappaB. Thus, BARD could promote renal protection through antioxidants and anti-inflammatory effects be promoting the activity of the Nrf2 transcription factor and inhibiting the activity of the NF-kappaB transcriptionfactor. ACE/ACEIs: angiotensin converting enzyme/angiotensin converting enzyme inhibitors, ARBs: angiotensin receptor blockers, AREs:antioxidant response elements, BARD: bardoxolone methyl, CKD: chronic kidney disease, DRI: direct renin inhibitor, mineralocorticoidreceptor antagonists, Keap1: Kelch-like ECH-associated protein 1, MRA: mineralocorticoid receptor antagonists, NF-kappaB: nuclear factorkappa B, Nrf2: nuclear factor (erythroid derive 2)-like 2, RAAS: renin-angiotensin-aldosterone system, ROS: reactive oxygen species, TGFβ-1:transforming growth factor beta 1.

evaluated. In phase 1 trials in oncologic patients, bar-doxolone unexpectedly improved kidney function, assessedas serum creatinine and creatinine clearance, especially inpatients with previous CKD. These findings lead to evaluatepotential nephroprotective actions in patients with CKD and

type 2 DM, first in an exploratory phase II open-label trialand then in a larger randomized clinical trial. In the first trial[107], 20 patients older than 18 years, with moderate-severediabetic CKD, were evaluated after 8 weeks of bardoxoloneat increasing oral doses of 25 to 75 mg/day. Notably, there


was a significant increase in estimated GFR at 4 weeks(+2.8 mL/min/1.73 m2) with 25 mg/day and at 8 weeks(+7.2 mL/min/1.73 m2) with 75 mg/day. Serum creatinineand BUN decreased and creatinine clearance increased,without changes in total excretion or tubular secretion ofcreatinine. Unfortunately, GFR was not measured. Bloodpressure did not change, and albuminuria had a small albeitnot statistically significant increase. Markers of vascularinjury and inflammation were improved by treatment withbardoxolone, suggesting a potential beneficial effect onendothelial injury. There were not changes in urine NGALor NAG adjusted for creatinine concentration, suggestinglack of significant renal toxicity associated to bardoxolone.There were few adverse effects, mainly muscle spasms anda self-limited increase of hepatic enzymes without a truehepatic toxicity. A short followup and an open-label designwithout control group are major limitations of this study,and they do not allow drawing solid conclusions about theefficacy and long-term safety of this drug on relevant renaloutcomes.

The beneficial effect on eGFR was confirmed in a larger,multicenter, double-blind, randomized trial [108]. This trialrandomized 227 patients with moderate-severe CKD andtype 2 DM, with stable treatment with ACEI/ARB, to bardox-olone 25, 75 or 150 mg/day or placebo for 52 weeks. Patientswere categorized by GFR, urinary albumin-creatinine ratio(UACR), and HbA1c. Patients with hepatic dysfunction orrecent cardiovascular events were excluded. A significantimprovement in the primary endpoint (change of GFR at 24weeks) was observed in all bardoxolone groups (+8.2, +11.4and +10.4 mL/min/1.73 m2 resp.) versus 0 mL/min/1.73m2

in the placebo group. The secondary endpoint (changeof GFR at 52 weeks) also was significantly improved inbardoxolone groups (+5.8, +10.5 and +9.3 mL/min/1.73 m2

resp. versus 0). More patients in the placebo group had a GFRdecrease ≥25% with respect to baseline value at 24 and 52weeks. Additionally, serum BUN, phosphorus, and uric acidwere significantly lower at 24 and 52 weeks in all bardoxolonegroups when compared to placebo.

Potential unwanted effects included a mild but significantincrease of UACR and decreased serum magnesium. UACRincreased in patients receiving 75 or 150 mg/day bardoxoloneversus placebo. This was observed at 24 and 52 weeks oftreatment, but UACR decreased when patients stopped thetherapy, suggesting that this effect is reversible. There wasan inverse correlation between changes in serum BUN,phosphorus, uric acid, magnesium, and changes in eGFR,as well as a direct correlation between changes in eGFR andchanges in UACR, suggesting that changes in eGFR may bethe basis for the other observed changes. Interestingly, therewas a trend toward higher systolic BP values in the 75 mgbardoxolone group, which was observed despite weight lossand that will merit close attention in further trials. The mainadverse effects were muscle spasms (63% of patients in the75 mg group) and nausea (25%).

Another significant effect was loss of body weight. Thisappears to be related to decreased appetite and/or nauseaand may be a welcome addition to the therapeutic arma-mentarium for patients with increased body mass index

(BMI). Indeed, weight loss was more evident in patients withhigher (>35 kg/m2) BMI (mean change −10 kg). However,and perhaps worryingly, it was also observed in patients withnormal BMI (−3 kg over 52 weeks).

The increased eGFR and effects on systolic BP and albu-minuria are interesting results on surrogates renal variableswhich requires more long-term studies. These parametersand, more importantly, cardiac and renal hard end-points(cardiovascular death and progression to ESRD) will bestudied at 2 years of followup in an ongoing randomizedclinical trial in 1600 patients older than 18 years withadvanced CKD (stage 4) and type 2 DM [109]. This study willcompare bardoxolone versus placebo in patients receivingstandard of care.

6. Conclusions and Recommendations

RAAS blockade is the mainstay of current therapy to slowprogression of diabetic and nondiabetic CKD, but thisstrategy is frequently not enough. Consequently, an impor-tant number of patients progress to ESRD. There is solidexperimental evidence for a key role of ROS and oxidativestress and their interplay with RAAS and inflammation, inthe pathogenesis of CKD. Bardoxolone methyl, a novel syn-thetic triterpenoid with antioxidant and anti-inflammatoryproperties, has shown to improve kidney function in patientswith advanced DN already receiving RAAS blockers, withfew adverse events. This may be a welcomed addition to thetherapeutic armamentarium if data are confirmed in larger,longer trials (Figure 2). However, the relative importanceand eventual management of the observed influence ofbardoxolone on UACR, magnesium, and body weight mustbe further studied.

Acknowledgments

This paper is supported by the following Grants: ISCIIIand FEDER funds CP04/00060, FIS PS09/00447, SociedadEspanola de Nefrologia, ISCIII-RETIC REDinREN/RD06/0016, Comunidad de Madrid/FRACM/S-BIO0283/2006,S2010/BMD-2378, Programa Intensificacion Actividad In-vestigadora (ISCIII/Agencia Laın-Entralgo/CM) to AO, andISCIII-Redes RECAVA (RD06/0014/0035), ISCIII fundsPI10/00072, EUS2008/03565 to JE and Fundacion Lilly,cvREMOD.

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Review Article

Induction of Oxidative Stress in Kidney

Emin Ozbek

Department of Urology, Okmeydani Research & Education Hospital, Sisli, 34384 Istanbul, Turkey

Correspondence should be addressed to Emin Ozbek, [email protected]

Received 1 December 2011; Revised 27 January 2012; Accepted 6 February 2012

Academic Editor: Ayse Balat

Copyright © 2012 Emin Ozbek. This is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Oxidative stress has a critical role in the pathophysiology of several kidney diseases, and many complications of these diseasesare mediated by oxidative stress, oxidative stress-related mediators, and inflammation. Several systemic diseases such ashypertension, diabetes mellitus, and hypercholesterolemia; infection; antibiotics, chemotherapeutics, and radiocontrast agents;and environmental toxins, occupational chemicals, radiation, smoking, as well as alcohol consumption induce oxidative stress inkidney. We searched the literature using PubMed, MEDLINE, and Google scholar with “oxidative stress, reactive oxygen species,oxygen free radicals, kidney, renal injury, nephropathy, nephrotoxicity, and induction”. The literature search included only articleswritten in English language. Letters or case reports were excluded. Scientific relevance, for clinical studies target populations, andstudy design, for basic science studies full coverage of main topics, are eligibility criteria for articles used in this paper.

1. Introduction

Free radicals are chemical species with a single unpairedelectron, which is highly reactive as it seeks to pair with anew free electron, and as a result of these reactions, otherfree radicals or paired electrons occur and radical featuremay be lost. If newly formed free radical occurs, it is alsounstable and it can react with another molecule to produceanother free radical or a nonradical molecule occurs becauseof the paired electrons of the newly formed molecule. Thus,a chain reaction of free radicals occurs, leading to damagingbiological systems and tissues. In aerobic conditions, allbiological systems are exposed to oxidative stress (OS), eithergenerated internally or as by-products. The great majorityof these free radicals are mainly oxygen radicals and otherreactive oxygen species (ROS) [1].

The well-known ROS are superoxide ion (O2 • −), hy-drogen peroxide (H2O2), and peroxyl radical (OH•), and thereactive nitrogen species (RNS) are nitric oxide (NO) andperoxynitrite (ONOO−). Peroxynitrite generates from rapidchemical interaction between NO and O2•−. Main sites ofROS produced in living organisms are mitochondrial elec-tron transport system, peroxisomal fatty acid, cytochromeP-450, and phagocytic cells [2–6]. Several extracellular andintracellular factors such as hormones, growth factors,

proinflammatory cytokines, physical environmental factors(like ultraviolet irradiation), nutrient metabolism, and thedetoxification of various xenobiotics affect production of OS[7–13].

In physiological conditions, ROS produced in the courseof normal conditions are completely inactivated by cellularand extracellular defence mechanisms. This means thatnormally there is a balance between prooxidant (or oxidant)and antioxidant defence systems. In certain pathologicalconditions, increased generation of ROS and/or depletionof antioxidant defence system leads to enhanced ROSactivity and OS, resulting tissue damage. OS causes tissuedamage by different mechanisms including promoting lipidperoxidation, DNA damage, and protein modification. Theseprocesses have been implicated in the pathogenesis of severalsystemic diseases including kidney.

Several systemic diseases such as hypertension, diabetesmellitus, metabolic syndrome, and hypercholesterolemia; in-fection; antibiotics and chemotherapeutic agents, and radio-contrast agents mainly excreted from kidney; and envi-ronmental toxins especially heavy metals such as lead andmercury, occupational chemicals such as urban fine particles,radiation, smoking, as well as alcohol consumption inducerenal OS. The kidney is an organ highly vulnerable to


damage caused by ROS, likely due to the abundance of long-chain polyunsaturated fatty acids in the composition of renallipids. In recent years, OSs have become one of the mostpopular topics in research of molecular mechanism of renaldiseases. The aim of this paper is to summarize the condi-tions inducing OS in kidney and molecular mechanisms ofthis induction and kidney damage.

2. Diabetes Mellitus and Induction ofOxidative Stress in Kidney

Recent years, diabetes and diabetic kidney disease continueto increase worldwide. In the USA, diabetes-associated kid-ney disease is a major cause of all new cases of end-stagekidney disease. All diabetic patients are considered to be atrisk for nephropathy. Today we have not specific markersto expect development of end-stage renal disease. Clinicallycontrol of blood sugar level and blood pressure regulationsare important two parameters to the prevention of diabeticnephropathy [14, 15].

There are huge amount of in vitro and in vivo studiesregarding explanation of mechanism of diabetes-mellitus-induced nephropathy. All of these mechanisms are a conse-quence of uncontrolled elevation of blood glucose level. Cur-rently the proposed mechanism is the glomerular hyperfiltra-tion/hypertension hypothesis. According to this hypothesis,diabetes leads to increased glomerular hyperfiltration anda resultant increased glomerular pressure. This increasedglomerular pressure leads to damage to glomerular cellsand to development of focal and segmental glomeruloscle-rosis [16, 17]. Angiotensin II inhibitors reduce glomerularpressure and prevent albuminuria. Increased angiotensin IIlevel induces OS through activation of NADPH oxidase,stimulating inflammatory cytokines, and so forth . . . [18, 19].

The mechanism by which hyperglycemia causes freeradical generation thus causes OS to be complex. Increasedblood glucose promotes glycosylation of circulator andcellular protein and may initiate a series of autooxidationreactions that culminate in the formation and accumulationof advanced glycosylation end-products (AGEs) in tissues.The AGEs have oxidizing potential and promote tissuedamage by oxygen-free radicals [20].

In experimental studies, formation of OS increases be-cause of high level of blood glucose. Sadi et al. showed thatin diabetic rat kidney antioxidant enzyme, namely, catalase(CAT) and glutathion peroxidase (GSHPx), activities werefound to be reduced; however, ∝-lipoic acid and vitamin Cadministration increased these antioxidant enzyme activities[21]. Increased OS is the common finding in tissues effectingfrom diabetes, including kidney. Reddi et al. showed thattransforming growth factor β1 (TGF-β1) is prooxidant andSe (selenium) deficiency increases OS via this growth factor.In addition Se deficiency may simulate hyperglycemic con-ditions. Se supplementation to diabetic rats prevents forformation of OS and renal structural injury [22]. Chen etal. showed that nitrosative stress increases in diabetic ratmodel [23]. These results show the induction of oxidativeand nitrosative stress in rat kidney. These may have a role

in pathophysiology of diabetes-induced morphological andfunctional changes of kidney.

3. Hypertension, Hypercholesterolemia,Obesity, and Aging Induce OxidativeStress in Kidney

Hypertension is one of the major causes of development ofrenal failure. Key regulator of this pathology is OS. Renalartery stenosis is the most common cause of secondaryhypertension and may lead to deterioration of renal functionand ischemic nephropathy. Chade et al. showed that across-talk between hypoperfusion and atherosclerosis tointeractively increased OS, inflammation, and tubular injuryin the stenotic kidney [24]. In experimental atheroscleroticrenovascular disease (simulated by concurrent hypercholes-terolemia and renal artery stenosis), it was reported thatthe activity of both CuZn and MnSOD isoforms wassignificantly decreased; however, protein expression of boththe NAD (P)H-oxidase subunits p67phox and p47phox,nitrotyrosine, inducible nitric oxide synthase (iNOS), andnuclear factor kappa-B (NFκB) increased. Furthermore,tubular and glomerular protein expression of nitrotyrosineis significantly elevated [25]. Chronic blockade of OS withantioxidant improves OS in kidney. All of these molecularabnormalities suggest increased OS in rat kidney.

Noeman et al. showed that high-fat diet-induced obesityis accompanied by increased hepatic, cardiac, and renal tissueOS, which is characterized by reduction in the antioxidantenzymes activities and glutathione levels, that correlate withthe increase in MDA and protein carbonyl (PCO) levels [26].Increased cytokine release (inflammation-related cytokinessuch as tumor necrosis factor-∝ and adiponectin) and renalmacrophage infiltration have been shown to contribute torenal injury in models of obesity. Chow et al. reportedthat monocyte chemoattractant protein-1 (MCP-1) is apotent stimulator of macrophage recruitment. It is increasedin adipose tissue during obesity and in diabetic kidneys,suggesting that inflammation of these tissues may be MCP-1 dependent [27]. Knight et al. also showed an increase inrenal macrophage-specific CD68-positive staining in a modelof obesity and hypertension [28]. From these results, wecan say that macrophages are the source of increased OSand renal injury in diabetes and obesity-induced renalinjury. Aging is associated with increased OS. Most of age-dependent changes in the kidney such as excessive fibrosis, ageneral lack of regenerative ability, and an increase in apop-tosis in cells that determine healthy renal functions are oftenrelated to excess OS [29]. At a molecular level, with agingincreased mutations in nuclear and mitochondrial DNA(mtDNA), increased lipofuscin and AGEs, increased OS, andincreased apoptosis have been observed. Proximal tubularcells contain large numbers of mitochondria and are the mostreliant upon oxidative phosphorylation and most susceptibleto oxidant-induced apoptosis and mutations [30]. Recentstudies showed that antiaging gen, klotho, is important inrenal aging and OS-induced renal damage. The klotho geneencodes the klotho protein, a single transmembrane protein


of the beta-glycosidase family [31]. Mice overexpressingklotho exhibit an extension in lifespan and resistance to oxi-dative injury. Klotho is predominantly expressed in thekidney, with its highest expression in cells of the distal con-voluted tubule [32, 33]. Overexpression of klotho has beenfound to enhance resistance to OS through the upregulationof manganese superoxide dismutase (MnSOD). Yamamotoet al. found that klotho modulates MnSOD in a FoxO-dependent process [34]. MnSOD is found within the mito-chondria where it acts as primary scavenger of oxidants.

4. Urinary Obstruction, Urolithiasis,Infection, Ischemia Reperfusion Injury,Transplantation of Kidney, and Inductionof Oxidative Stress in Kidney

Most clinical and experimental studies have shown that OSis increased in kidney and systemic circulation. Huang et al.reported that the activities of catalase and manganesesuperoxide dismutase were elevated in early stage of ethyleneglycol-induced urolithiasis model in rats; however, on day42 almost all antioxidant enzyme activities were attenuatedexcept those of CAT. In this experiment, the possible mecha-nism that causes free radical elevation in the kidney may bedifferent in the course of ethylene glycol-induced urolithiasis.Initially systemic circulation may bring the toxic substancesto the kidney, and eventually these substances cause to pro-duce free radicals. In the late stage progressive accumulationof leucocytes and defective antioxidant enzyme activitiesmay cause kidney to remain under huge amount of OS[35, 36]. In our experimental urolithiasis studies, we showeddecreased antioxidant enzyme activities and involvement ofNFκB and p38-MAPK (mitogen-activated protein kinase)signaling pathways, related to OS in rat kidney [37–40]. Inin vitro cell culture studies using proximal tubular origin linederived from pig proximal tubules (LLC-PK1), and collectingduct origin Madin-Darby canine kidney (MDCK) cell lines,it has been reported that calcium phosphate crystals causecellular injury by increasing ROS [41].

Today, extra corporeal shock wave lithotripsy (ESWL)is widely used for the treatment of renal stones in selectedrenal cases. In our work, we showed increased expression ofinducible NO synthase (iNOS) and NFκB, indirect evidenceof increased OS [42]. Recently, Gecit et al. showed thatESWL treatment produces OS and causes impairment in theantioxidant and trace element levels in the kidneys of rats[43]. ESWL is associated with greater prevalence of hyper-tension [44]. Ischemia insult and increased renal OS andconsequent endothelial dysfunction may be possible mech-anism of hypertension after ESWL.

Urinary obstruction, especially ureteral obstruction dueto urolithiasis, is a common urological problem seen inurology practice. Unilateral ureteral obstruction (UUO)leads to decreased renal MnSOD and CAT protein expressionin a time-dependent manner. Increased 4-hydroxynoneal (4-HNE) stain for ROS products in the renal tubulointerstitialcompartment occurred after 4 hr of UUO in the kidney. Theauthors explain renal tubular apoptosis in UUO rat model

explained by increasing ROS in this study [45]. Variousmarkers of OS increase in UUO rat kidneys such as the oxida-tively damaged protein product Ne-carboxymethyl-lysine(CML); the marker of DNA oxidant damage, 8-hydroxy-2′′-deoxyguanosine (8-OHdG)); and lipid peroxidation mark-ers such as malondialdehyde (MDA), 8-iso prostaglandinF2∝ (8-iPGF2∝), and 4-HNE or 4-hydroxy-hexenal (4-HHE). OS response molecules like heat shock protein-70(HSP-70), heat-shock protein-27, and heme oxygenase-1(HO-1) [46–51] are strongly expressed after UUO. Micethat are genetically deficient endogenous antioxidant enzymeCAT are more susceptible to UUO-induced renal damagethan normal wild type mice. Furthermore, increased renalconcentrations of ROS have been observed in obstructedkidneys, together with decreased activities of the majorprotective antioxidant enzymes SOD, CAT, and glutathioneperoxidase [52, 53]. UUO-induced nephrotoxicity and renalfibrosis is thought to be secondary to increased OS inkidney. In the literature there is some information about theamelioration of antioxidant and reactive oxygen scavengeragents against UUO-induced renal damage [54, 55]. For anexcellent detailed review, please refer to [56].

Infection is another inducer of OS in kidney. There area lot of experiments showing the increased oxidative stressand decreased antioxidant defence mechanism, antioxidantenzyme systems in kidney due to infection [57–60].

ROS are important mediators exerting toxic effectson various organs, including kidney during ischemia-reperfusion (IR) injury. A large body of evidences indicatethe role of increased OS in the kidney and protective roleof antioxidants and ROS scavengers in IR injury-inducednephropathy in the literature [61–63]. OS also has a role asa mediator of injury in chronic allograft tubular atrophy andinterstitial fibrosis in rat kidney [64]. Renal transplantationis another OS inducer in kidney in human and animals.OS increases in transplanted kidney because of pretransplantand posttransplant conditions. If there is preexisting diseasessuch as chronic kidney failure, inflammation, and diabetesmellitus, kidneys are more sensitive to OS during reperfusioninjury. Postoperative immunosuppressive agents are amongmany risk factors inducing OS in kidney [65].

5. Antibiotics, Antineoplastic Agents,Immunosuppressants, Analgesics,Nonsteroidal Antiinflammatory Drugs,and Radiocontrast Agents InduceOxidative Stress in Kidney

Antibiotics, commonly used aminoglycosides, are nephro-toxic agents. Their nephrotoxicity is mainly attributed toinduction of OS and depletion of antioxidat enzyme ac-tivities in kidney. In our experiments, we showed thatiNOS/NFκB/p38MAPK pathway, OS taking place in thisaxis, is involved in gentamicin-induced nephrotoxicity [66,67]. Our and other studies showed the protective effect ofanti oxidants and reactive oxygen scavenger agents againstgentamicin-induced nephrotoxicity [68–70].


Antineoplastic agents are commonly used for the treat-ment of metastatic cancers. Some of these are nephrotoxic.Excess ROS production and depressed antioxidant defencemechanism are responsible from nephrotoxicity. Cisplatin isthe well-known and commonly used antineoplastic andnephrotoxic agent. Other nephrotoxic anticancer agents arecarboplatin, methotrexate, doxorubicin, cyclosporine, andadriamycin. Immunosuppressant such as sirolimus and cy-closporine leads to nephrotoxicity via OS [71–80].

Cisplatin is one of the commonly used potent antitumordrugs and cisplatin-based combination protocols are used asfront-line therapy for several human malignancies. Cisplatinis toxic to the renal proximal tubules and dose dependent[81, 82]. Several studies have reported the role of OS regard-ing cisplatin-induced nephrotoxicity. Cisplatin is known toaccumulate in mitochondria of renal tubular epithelial cellstogether with ROS; renal tubular cell mitochondrial dysfunc-tion is also important in cisplatin-induced nephrotoxicity.Mitochondria also continuously scavenge ROS via the actionof antioxidant enzymes such as SOD, GSHPx, CAT, andglutathione S-transferase. Studies have demonstrated thatcisplatin induces ROS in renal epithelial cells primarilyby decreasing the activity of antioxidant enzymes andby depleting intracellular concentrations of GSH. In vitrostudies using LLC-PK1 cells, which is characteristic of renalproximal tubular epithelium, also showed the role of ROS incisplatin nephrotoxicity [83–89].

In this era, analgesics, especially paracetamol andacetaminophen (APAP), and nonsteroidal antiinflammatorydrugs (NSAIDs) are widely used throught the world. Parac-etamol and APAP are nephrotoxic drugs. Several in vitroand in vivo studies showed that analgesics nephrotoxicity iscaused by increased ROS in kidney. Zhao et al. showed theincreased ROS, nitric oxide, and MDA levels, together withdepleted glutathione (GSH) concentration in the kidney ofrats. However, rhein, Chinese herb, can attenuate APAP-in-duced nephrotoxicity in a dose-dependent manner [90]. Weshowed in our experiment a significant increase in MDAand decreases in GSHPx, CAT, and SOD activities in APAP-treated rat kidneys. These findings support the inductionof OS in rat kidney by APAP. Significant beneficial changeswere noted in serum and tissue OS indicators in rats treatedwith strong antioxidant pineal hormone melatonin andcurcumin [91, 92]. Ghosh et al. reported increased OSand TNF-alpha production in rat tissues [93]. Efrati et al.reported that diclofenac (NSAID) leads to nephrotoxicity byincreasing intrarenal ROS in rat kidney, and antioxidant, N-acetylcysteine, prevents kidney damage [94].

Contrast-induced nephropathy (CIN) is a major clinicalconcern, particularly with imaging procedures. CIN is thethird most common cause of acute kidney injury in hospi-talized patients [95]. Experimental findings in vitro and invivo illustrate enhanced hypoxia and the formation of ROSwithin the kidney following the administration of iodinatedcontrast media, which may play a role in the developmentof CIN. Studies indeed support this possibility, suggestinga protective effect of ROS scavenging or reduced ROSformation with the administration of N-acetyl cysteine andbicarbonate infusion, respectively [96–99].

6. Alcohol, Smoking, Environmental Toxins,Radiation, and Mobile Phones InduceOxidative Stress in Kidney

Ethanol and its metabolites are excreted into urine, and itscontent in the urine is higher than that of the blood andthe liver. Chronic alcohol administration decreases the renaltubular reabsorption and reduces renal function. Functionalabnormalities of renal tubules may be associated withethanol-induced changes in membrane composition andlipid peroxidation. Because of high content of long-chain-polyunsaturated fatty acids, kidney is highly sensitive to OSdamage [100].

Recently it is reported that ethanol administration causeda significant decrease in the levels of antioxidant enzymeCAT, SOD, and GSHPx activities and increases MDA inkidney of the rats [101]. Shankar et al. showed that renalmetabolism of ethanol via Cytochrome P450 2E1 (CYP2E1)and antidiuretic hormone-1 led to production of renal OS,and activation of MAPK induces CYP24A1 resulting inreducing circulating 1,25 (OH)2 D3 concentrations [102].Pathogenesis of aldosterone/salt-induced renal injury simi-larly is attributed to increased ROS and activation of MAPKin rat kidney [103]. In other studies, authors showed thatchronic ethanol administration and cigarette smoke expo-sure may cause renal injury by increasing oxidative andnitrosative stress in rat kidney [104, 105].

Epidemiological studies have shown that smoking is anaccelerating risk factor for the development of nephropathy,in which TGFβ1 plays a role in diabetic patients. Cell culturestudies using mesangial cell showed that smoking couldincrease TGFβ1, probably due to increased oxidative stressand PKCβ (protein kinase C beta) activation. This findingsupports the concept that smoking is a risk factor fordevelopment of diabetic nephropathy by increasing OS inkidney [106]. Similarly smoking and alcohol together in-crease OS and suppress antioxidant defence mechanism inkidney [104, 105].

In modern era, especially industrialised countries envi-ronmental toxins such as air pollutions, substances in storedfoods, radiations, as well as heavy metals in waters especiallyin underdeveloped countries are major health problem.Ochratoxin A (OTA), a mycotoxin, produced by fungiof improperly stored food products, has been linked tothe genesis of several disease states in both animals andhumans. It has been reported as nephrotoxic, carcinogenic,teratogenic, immunotoxic, and hepatotoxic in laboratoryand domestic animals [107–109]. In primary rat kidney cellsand in vivo experiments, it has been shown that OTA inducesOS and depletes antioxidant systems. These events mightrepresent pivotal factors in the chain of cellular events leadinginto nephrotoxicity of OTA. Cadmium (Cd) is known tobe a widespread environmental contaminant and a potentialtoxin that may adversely affect public health. Cigarette smokeand food (from contaminated soil and water) are importantnonindustrial sources of exposure to Cd. Cd accumulatesin the kidney because of its preferential uptake by receptor-mediated endocytosis of freely filtered and metallothionein


bound Cd (Cd-MT) in the proximal tubule. Internalised Cd-MT is degraded in endosomes and lysosomes, releasing freeCd into the cytosol, where it can generate ROS and activatecell death pathways [110].

Another environmental nephrotoxic agent is diazi-non (O,O-diethyl-O-[2-isopropyl-6-methyl-4-pyrimidinyl]phosphorothioate). It is an organophosphate insecticide andhas been used worldwide in agriculture and domestic for sev-eral years. Shah et al. showed that diazinon exposure depletesantioxidant enzymes and induces OS in rat kidney [111].

Increased air pollution as a result of industry is anotherlife threatening health problem. Boor et al. showed renal,vascular, and cardiac fibrosis in rats exposed to passive smok-ing and industrial dust fibre amosite. Authors explain thesechanges by increased OS in these tissues [112]. Nanosizedfraction of particulate air pollutions have been reported totranslocate from the airways into the bloodstream and acton different organs such as lungs, heart, liver, and kidneys.Nemmar et al. examined the distribution and the patholog-ical changes of diesel exhaust particles (DEPs) on systolicblood pressure (SBP), systemic inflammation, oxidativestress, and morphological alterations in lungs, heart, liver,and kidneys in Wistar rats. They showed that DEPs causeinflammation especially in lungs and pulmonary tissue, andthese pathological changes are attributed to increased OSand inflammatory cytokines in these tissues [113]. Lead andcadmium nephrotoxicity are also related to increased OS inkidney [114, 115].

Radiation is an important inducer of OS. For diagnosticand therapeutic purposes, radiation is commonly used.Chronic OS after total body irradiation is thought to be thecause of radiation nephropathy in rats. Authors looked forevidence of OS after total-body irradiation in a rat model;focusing on the period before that there is physiologicallysignificant renal damage. No statistically significant increasein urinary 8-isoprostane (a marker of lipid peroxidation) orcarbonylated proteins (a marker of protein oxidation) wasfound over the first 42 days after irradiation, while a small butstatistically significant increase in urinary 8-hydroxydeoxy-guanosine (a marker of DNA oxidation) was detected at 35–55 days. In renal tissues, they found no significant increasein either DNA or protein oxidation products over the first89 days after irradiation. They suggest that if chronic OSis a part of the pathogenesis of radiation nephropathy,it does not leave widespread or easily detectable evidencebehind. Emre et al. investigated the effect of extremely low-frequency electromagnetic field (ELF-EMF) with pulse trainsexposure on lipid peroxidation and hence oxidative stress inthe rat liver and kidney tissue. They found increases in thelevels of oxidative stress indicators, and the flow cytometricdata suggested a possible relationship between the exposureto magnetic field and the cell death; however, there weresignificantly lower necrotic cell percentages in experimentalanimals compared to either unexposed or sham controlgroups [116]. These results suggest the inductive effect ofradiation on OS in kidney.

For the last two decades, a large number of studieshave investigated the effects of mobile phone radiation onthe human and animal. Cellular target and tissue damage

are different. Male reproductive system is among the mostaffected system [117, 118]. Increased OS plays a centralrole in radiofrequency-electromagnetic-waves- (RF-EMW-)induced tissue damage. Devrim et al. examined the effect ofRF-EMW on oxidant and antioxidant status in erythrocytesand kidney, heart, liver, and ovary tissues from rats andpossible protective role of vitamin C. It was observed thatMDA level, xanthine oxydase (XO), and GSH-Px activitiessignificantly increased in the EMR group as comparedwith those of the control group in the erythrocytes. Inthe kidney tissues, it was found that MDA level and CATactivity significantly increased, whereas XO and adenosinedeaminase (ADA) activities decreased in the cellular phonegroup as compared with those of the control group. However,in the heart tissues, it was observed that MDA level, ADA,and XO activities significantly decreased in the cellular phonegroup as compared with those of the control group. Theyconcluded that RF-EMR at the frequency generated by a cellphone causes OS and peroxidation in the erythrocytes andkidney tissues from rats. In the erythrocytes, vitamin C seemsto make partial protection against the OS [119]. Ozguneret al. reported similar results in rat experiments and theyalso reported that preventive effect of Caffeic acid phenethylester (CAPE), a flavonoid-like compound, is one of the majorcomponents of honeybee propolis and melatonin against RF-EMW-induced nephrotoxicity [120–122].

7. Conclusion

There is huge amount of literature concerning the linkbetween the OS and renal diseases. Systemic diseases such ashypertension, diabetes mellitus, and hypercholesterolemia;infection; antibiotics, chemotherapeutics, and radiocontrastagents; and environmental toxins, occupational chemicals,radiation, smoking, as well as alcohol consumption inducerenal OS. The kidney is a highly vulnerable organ to damagecaused by ROS, due to the abundance of long-chain-poly-unsaturated fatty acids. Antioxidant and reactive oxygenscavengers have been shown to be effective in animals forprotecting kidney, but it is hard to translocate these results tohumans. This may be due to short duration of animal studies,dose differences between animals and humans, and differentpathophysiologic processes between animals and humans.For understanding these steps, drugs will be developed toalter the main process(es) responsible for increased OS. Inthis paper, the conditions inducing OS in kidney andmolecular mechanisms of this induction and kidney damagehave been summarized. I hope that this paper will aid inthe understanding of this complex system and directing newresearch effort.

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Research Article

Inflammation and Oxidative Stress inObesity-Related Glomerulopathy

Jinhua Tang,1, 2 Haidong Yan,1 and Shougang Zhuang1, 2

1 Department of Nephrology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China2 Department of Medicine, Alpert Medical School of Brown University, Rhode Island Hospital-Middle House 301,593 Eddy Street, Providence, RI 02903, USA

Correspondence should be addressed to Haidong Yan, [email protected] and Shougang Zhuang, [email protected]

Received 2 September 2011; Accepted 6 February 2012


Copyright © 2012 Jinhua Tang et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Obesity-related glomerulopathy is an increasing cause of end-stage renal disease. Obesity has been considered a state of chroniclow-grade systemic inflammation and chronic oxidative stress. Augmented inflammation in adipose and kidney tissues promotesthe progression of kidney damage in obesity. Adipose tissue, which is accumulated in obesity, is a key endocrine organ thatproduces multiple biologically active molecules, including leptin, adiponectin, resistin, that affect inflammation, and subsequentderegulation of cell function in renal glomeruli that leads to pathological changes. Oxidative stress is also associated with obesity-related renal diseases and may trigger the initiation or progression of renal damage in obesity. In this paper, we focus oninflammation and oxidative stress in the progression of obesity-related glomerulopathy and possible interventions to preventkidney injury in obesity.

1. Introduction

Obesity has become a heavy public health problem in theUnited States, with a prevalence among adults increasing to32% from 13% between the 1960s and 2004 [1]. Currently,66% of adults and 16% of children and adolescents are over-weight or obese [1]. Although obesity has long been recog-nized as an independent risk factor for cardiovascular dis-eases and diabetes mellitus, newer research points to obesityas an important risk factor for chronic kidney diseases(CKDs) [2–4]. In 1974, Weisinger et al. [5] firstly reportedthat massive obese patients developed nephrotic-range pro-teinuria. Subsequent studies confirmed that obesity couldinduce renal injury, namely, obesity-related glomerulopathy(ORG) [6–8]. A large-scale clinicopathologic study including6818 renal biopsies from 1986 to 2000 revealed a progressiveincrease in biopsy incidence of ORG from 0.2% in 1986–1990to 2.0% in 1996–2000 [8]. The tenfold increase in incidenceof ORG over 15 years suggests a newly emerging epidemic[8].

The clinical characteristics of subjects with ORG typi-cally manifest with nephrotic or subnephrotic proteinuria,

accompanied by renal insufficiency [8–10]. Histologically,ORG presents as focal segmental glomerulosclerosis (FSGS)and glomerular hypertrophy or glomerular hypertrophyalone and relatively decreased podocyte density and numberand mild foot process fusion [8, 11, 12]. Clinically, it isdistinguished from idiopathic FSGS (I-FSGS) by its lowerincidence of nephrotic syndrome, more benign course, andslower progression of proteinuria and renal failure [8, 11].

ORG is an increasing cause of end-stage renal disease(ESRD). The pathophysiology of ORG remains incomplete-ly understood. Potential mechanisms by which obesity af-fects renal physiology include altered renal hemodynam-ics, insulin resistance, hyperlipidemia, activation of renin-angiotensin-aldosterone system (RAAS), inflammation, andoxidative stress. Increases in both glomerular filtration rate(GFR) and renal plasma flow (RPF) were observed inobese subjects and animals [13, 14]. This likely occurs be-cause of afferent arteriolar dilation as a result of proximalsalt reabsorption, coupled with efferent renal arteriolar vaso-constriction as a result of elevated angiotensin II (AngII)[15]. These effects may contribute to hyperfiltration, glom-erulomegaly, and later focal glomerulosclerosis [8, 9]. Insulin


resistance can raise the transcapillary pressure gradient andcause hydrostatic pressure and hyperfiltration by reducingnorepinephrine-induced efferent arteriolar constriction [16],leading to glomerular hypertrophy and sclerosis. Hyperin-sulinemia also has been shown to stimulate the synthe-sis of growth factors such as insulin-like growth factor-(IGF-) 1 and IGF-2 and transforming growth factor-β1(TGF-β1), which accelerate production of extracellularmatrix and promote glomerular hypertrophy and sclerosis[17, 18]. Hyperlipidemia may promote glomerulosclerosisthrough mechanisms that involve engagement of low-densitylipoprotein receptors on mesangial cells, direct podocytetoxicity, oxidative cellular injury, macrophage chemotaxis,and increase renal expression of sterol regulatory element-binding proteins (SREBP-1 and SREBP-2), resulting in therenal accumulation of cholesterol and triglycerides and to-gether with significant renal increase of fibrogenic cytokines[19, 20]. Adipose tissue is the major source of the com-ponents of RAAS. Obese subjects usually have increasesin plasma renin activity, angiotensinogen, angiotensin-con-verting enzyme activity, and circulating AngII, which triggeror promote renal damage by renal hemodynamic changesand nonhemodynamic pathways such as hyperinsulinemia,oxidative stress, and inflammation [21–23]. Inflammatoryabnormalities and oxidative stress are characteristic findingsof obesity and play important roles in the renal damage as-sociated with obesity, which will be discussed in detail in thefollowing.

2. Inflammation in Obesity-RelatedGlomerulopathy

Recent studies have demonstrated that obesity causes chroniclow-grade systemic inflammation and thus contributes tothe development of systemic metabolic dysfunction that isassociated with obesity-related disorders and renal disease[24–27]. Levels of some inflammatory markers and cytokinessuch as C-reactive protein (CRP), tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6), and macrophage migrationinhibitory factor (MIF) are elevated, whereas concentra-tions of adiponectin, a protein hormone that exerts anti-inflammatory activities, are reduced in obesity [28–34].

2.1. Leptin. Leptin is a 16-kDa-peptide hormone encoded byobese (ob) gene that is mainly produced by adipose tissue.Leptin serves as a regulator of energy balance by binding tothe full-length leptin receptors—obese receptor b (Ob-Rb)in the hypothalamus, leading to reduction in food intakeand elevation in temperature and energy expenditure [35].Leptin receptors can be classified as secreted-forms (Ob-Re),short-forms (ob-Ra, c, d, and f) mainly expressed in periph-eral tissue, and long-forms (ob-Rb) predominantly expressedin hypothalamus. The kidney expresses abundant concentra-tions of the truncated isoform of the leptin receptor Ob-Ra, but only a small amount of the full-length receptorOb-Rb [36]. Leptin production is associated with increasedsize of adipocytes and is positively correlated with thebody mass index (BMI) [37]. Increased circulating leptin, a

marker of leptin resistance, is common in obesity. Obesity-induced leptin resistance injures numerous peripheral tissuesincluding kidney, liver, myocardium, and vasculature [36,38]. Leptin results in the development of renal disease bybinding to its specific receptors in renal endothelial cells andmesangial cells. In glomerular endothelial cells, leptin stimu-lates cellular proliferation, TGF-β1 synthesis, and type IV col-lagen production [36, 39]. In mesangial cells, leptin upregu-lates synthesis of the TGF-β type II receptor, but not TGF-β1, and stimulates glucose transport and type I collagen pro-duction through signal transduction pathways involvingphosphatidylinositol-3-kinase [40]. Leptin also stimulateshypertrophy, but not proliferation in cultured rat mesangialcells [41]. However, both those cell types increase their ex-pression of extracellular matrix in response to leptin. Trans-genic mice with leptin overexpression demonstrated an in-crease in collagen type IV and fibronectin mRNA in thekidney [41]. Leptin is involved in the development ofglomerulosclerosis through a paracrine TGF-β pathway (be-tween glomerular endothelial and mesangial cells) that pro-motes the deposition of extracellular matrix, proteinuria,and, eventually, glomerulosclerosis [39]. Infusion of leptininto normal rats for 3 weeks fosters the development of focalglomerulosclerosis and proteinuria [36].

Leptin also has proinflammatory actions through itsinteraction with mediators of innate and adaptive immunityand CRP [38]. Leptin regulates components of innate andadaptive immunity, including T lymphocytes and mono-cytes/macrophages [42, 43]. Central leptin administrationin ob/ob mice accelerates renal macrophage infiltrationthrough the melanocortin system [44]. Leptin stimulatescentral T-cell production and a peripheral shift in favor ofT helper (Th) 1 adaptive immune responses (proinflamma-tory) as opposed to Th2 responses (anti-inflammatory) [38].Leptin has been shown to modulate adaptive immunity byenhancing T-cell survival and stimulating production of pro-inflammatory cytokines such as IFN-γ and IL-2 [45]. Leptinalso has structural and functional resemblance to proinflam-matory cytokines, such as IL-6 [42], and may modulate CRP,a leptin-interacting protein [46].

Therefore, these direct and indirect effects of leptin onthe kidney, including stimulating cellular proliferation andhypertrophy, increasing extracellular matrix expression, andexhibiting proinflammatory activities, may partially explainobesity-related kidney disease.

2.2. Adiponectin. Adiponectin is a 30 kDa adipocyte-derivedprotein hormone encoded by the adipose most abundantgene transcript 1 (APM1), [47] which plays a role in thesuppression of inflammation-associated metabolic disorders.Two distinct adiponectin receptors, AdipoR1 and R2, havebeen cloned [48]. AdipoR1 is expressed ubiquitously whileAdipoR2 is most abundant in the liver. Adiponectin is highlyabundant in human serum, but its level is decreased inmost obese animal and human subjects, particularly in thosewith visceral obesity [49–51]. Recent clinical studies show anegative association of adiponectin in obese patients, [52,53] suggesting that adiponectin may play a key role in the


development of obesity-related albuminuria and alterationof renal function.

Studies with the adiponectin knockout mouse provideevidence that adiponectin can regulate podocyte functionand thus contribute to the initial development of albumin-uria [37, 53]. Sharma et al. showed that knockout of adipo-nectin in mice increased albuminuria and caused fusionof podocyte foot processes [53]. In cell culture studieswith podocytes, incubation with adiponectin potently de-creased permeability to albumin, induced translocation ofzona occludens-1 (ZO-1) to the plasma membrane, and re-duced the renal predominant NADPH oxidase Nox 4,largely via a 5′-AMP-activated-protein kinase- (AMPK-)dependent pathway. Treatment of the adiponectin knockoutmice with exogenous adiponectin was able to decrease albu-minuria and improve podocyte morphology [53]. Chronichyperadiponectinemia significantly alleviated the progres-sion of proteinuria in early-stage diabetic nephropathy byseveral mechanisms. It led to an increase in nephrin expres-sion, improvement of the endothelial dysfunction due todecreases in endothelin 1 (ET-1) and plasminogen activatorinhibitor 1 (PAI-1), and an increase in endothelial nitricoxide synthase (eNOS) expression in the renal cortex [54].

Recent studies suggest that adiponectin exerts anti-in-flammatory effects by suppressing TNF-α-induced activationof nuclear factor-κB (NF-κB) in human aortic endothelialcells and aortic smooth muscle cells through inhibition ofIκB phosphorylation [55, 56] and inhibition of vascular celladhesion molecule 1 (VCAM-1) and intercellular adhesionmolecule 1 (ICAM-1) expression [57], thereby reducing mo-nocyte adhesion and macrophage-induced cytokine produc-tion [58] and CRP expression in human adipose tissue [59].Human adipose tissue expressed CRP, which was negativelycorrelated with adiponectin expression in adipose tissue [59].Low levels of adiponectin are associated with higher levelsof highly-sensitive C-reactive protein (hs-CRP) and IL-6[49], two inflammatory mediators that are involved in theinitiation and progression of atherosclerosis and renal dis-ease. Therefore, hypoadiponectinemia contributes to devel-opment of a low-grade systemic chronic inflammation state,suggesting that hypoadiponectinemia may play a causativerole in the systemic and vascular inflammation commonlyfound in obesity and obesity-related disorders, including re-nal injury, through its proinflammatory effects.

2.3. Resistin. Resistin, also known as adipocyte-specificsecretory factor (ADSF) or as found in inflammatory zone(Fizz), is a cysteine-rich 12.5-kDa polypeptide that belongsto a small family called resistin-like molecules (RELMs) [60,61]. In rodents, resistin is secreted from white adipocytes [62,63]. In human, it is produced largely by macrophages andexpressed in adipose tissue predominantly by nonadipocyteresident inflammatory cells [64–66]. Current evidence sug-gests that resistin has been variably associated with obesity,insulin resistance, inflammation, and renal dysfunction.Resistin levels are elevated in both genetic and diet-inducedanimal models of obesity [62, 67]. Studies of obese subjectshave frequently noted higher serum levels of resistin as wellas direct correlations between resistin level and adiposity as

measured by BMI [68, 69]. There has been a link betweencirculating resistin and low-grade inflammation that accom-pany obesity [70]. Resistin is associated with elevated CRPand white blood cells, suggesting that the role of resistinmay be a component of obesity-related inflammation [70].It has recently been found that resistin involves in theregulation of proinflammatory cytokine expression. Resistinstrongly upregulates IL-6 and TNF-α in human peripheralblood mononuclear cells (PBMCs) via NF-κB pathway [71].Human resistin enhanced secretion of proinflammatory cy-tokines, TNF-α and IL-12 in macrophages by NF-κB-de-pendent pathway [72]. Studies also show that increased levelsof resistin in patients with CKD are associated with declinedrenal function and inflammation [73, 74]. This suggests thatresistin may play an important role in obesity and obesity-as-sociated disease by triggering the release of other proinflam-matory cytokines.

2.4. Inflammatory Markers. A growing body of evidence in-dicates that obesity-related glomerulopathy is associatedwith upregulation of inflammatory mediators [75]. Obesityleads to adipose tissue macrophage infiltration in white adi-pose tissue and increased levels in proinflammatory cyto-kines. Several inflammatory mediators released from adipo-cytes and macrophages, such as TNF-α, IL-6, IL-1β, CRP,monocyte chemoattractant protein 1 (MCP-1), PAI-1, andMIF, contribute to a low level of chronic inflammatory statein obesity and may be responsible for renal injury in obesity-associated glomerulopathy. An emerging pattern of geneexpression was observed in adipose tissue in mice fed highfat-fed diets, indicating a shift toward global upregulationof inflammatory genes, including TNF-α, IL-6, and MCP-1[76].

TNF-α, a proinflammatory cytokine, is predominantlyproduced by macrophages infiltrating adipose tissue [77, 78]and can also be produced by the kidney [79]. This cytokineis involved in the genesis of inflammation and contributesto obesity-associated insulin resistance [80–82]. Within thekidney, AngII, advanced glycation end-products (AGEs), andoxidized low-density lipoprotein (LDL) can stimulate TNF-αsynthesis from renal cells to initiate local damage [83–86]. Arecent study demonstrates that TNF-α reduces the expressionof Klotho, a protein expressed by renal cells, through an NF-κB-dependent mechanism, which contribute to renal injury[87]. TNF-α enhances the expression of PAI-1 in human adi-pose tissue and plasma PAI-1 levels in obesity subjects andis responsible for reduced fibrinolysis and also a componentof extracellular matrix, leading to renal fibrosis and terminalrenal failure [88–90]. TNF-α also has been shown to inducethe expression of MCP-1 via p38 mitogen-activated proteinkinase (MAPK) signaling pathway in renal mesangial cells[91]. MCP-1, a key regulator in recruiting monocytes to theglomeruli, may also contribute to renal damage at a laterstage of kidney disease in obesity.

IL-6 is another important proinflammatory mediatorsystemically secreted from adipose tissue and locally pro-duced in the kidney. Studies have demonstrated the positiverelationship between BMI and plasma IL-6 concentrations[92–94]. Studies also suggest that IL-6 plays a key role in


the development of renal disease. Endogenous IL-6 enhancesthe degree of renal injury, dysfunction, and inflammationcaused by ischemia/reperfusion by promoting the expressionof adhesion molecules and subsequent oxidative stress [95].Transgenic knockout of IL-6 ameliorates renal injury asmeasured by serum creatinine and histology [96]. Blockingthe IL-6 receptor prevents progression of proteinuria andrenal lipid deposit as well as the mesangial cell proliferationassociated with severe hyperlipoproteinemia [97]. IL-6 alsostimulates the synthesis of CRP [98], which is well known asboth a marker and important risk factor of atherosclerosis inthe general population and CKD patients [99].

MIF was initially described as an immunomodulatoryfactor isolated from the supernatants of T lymphocytes andwas found to inhibit the random migration of macrophages[100]. Subsequent studies have indicated that MIF acts asa proinflammatory cytokine and pituitary-derived hormonethat potentiates endotoxemia [101]. MIF also plays a path-ogenic role in kidney disease. In a rat model of immuno-logically induced crescentic antiglomerular basement mem-brane glomerulonephritis, treatment with anti-MIF anti-body reduced proteinuria, prevented the loss of renal func-tion, attenuated histological damage including glomerularcrescent formation, inhibited renal leukocytic infiltrationand activation, and reduced IL-1β expression by both intrin-sic kidney cells and macrophages [102]. Thus, MIF is a keymediator of the inflammatory and immune response andplays a pathological role in immune-mediated renal injury.

3. Oxidative Stress inObesity-Related Glomerulopathy

3.1. Obesity and Oxidative Stress. Oxidative stress is causedby an imbalance between increased production of reactiveoxygen species (ROS) and/or reduced antioxidant activity,leading to oxidative damage to cells or tissue including lip-ids, proteins and DNA. It is known that oxidative stress isinvolved in pathological processes of various diseases, suchas cancer, diabetes mellitus, hypertension, and cardiovasculardisease. Studies have suggested that obesity is associated withincreased oxidative stress [103, 104]. Analysis of oxidativemarkers in obesity subjects indicates that oxidative damageis associated with increased BMI and percentage of body fat[105, 106]. Conversely, parameters of antioxidant capacityare inversely related to the amount of body fat and centralobesity [107, 108]. The possible mechanisms of obesity-related oxidative stress include increased oxygen consump-tion and subsequent production of free radicals derivedfrom the increase in mitochondrial respiration, diminishedantioxidant capacity, fatty acid oxidation, lipid oxidizability,and cell injury causing increased rates of free radical form-ation [104, 109, 110]. It is also reported that the increasein obesity-associated oxidative stress is due to the presenceof excessive adipose tissue accumulation [111]. Accumulatedadipose tissue generates an immune response leading to thesecretion of proinflammatory cytokines, including TNF-α,IL-1, and IL-6, which lead to increased generation of ROS.Excessive fat accumulation also stimulates nicotinamide

adenine dinucleotide phosphate (NADPH) oxidase activity,which contributes to ROS production [112]. ROS, in return,augmented the expressions of NADPH oxidase (NOX) sub-units, including NOX4 and PU.1 in adipocytes, establishing avicious cycle that augments oxidative stress in white adiposetissue and blood [112].

3.2. Oxidative Stress and Inflammation. Oxidative stress inadipocyte seems to be responsible for the low-grade proin-flammatory state commonly observed in obesity [113, 114].Oxidative stress is increasingly viewed as a major upstreamcomponent in cell-signaling cascades involved in inflamma-tory responses, stimulating the expression of proinflamma-tory cytokines. ROS activate redox-sensitive transcriptionfactors, particularly NF-κB, inducing the release of proin-flammatory cytokines and the expression of adhesion mole-cules and growth factors, including TNF-α, IL-6, IL-1β, TGF-β1, connective tissue growth factor, IGF-1, platelet-derivedgrowth factor, and VCAM-1 [115, 116]. H2O2 stimulates IL-4 and IL-6 gene expression and cytokine secretion by anapurinic/apyrimidinic-endonuclease/redox-factor-1- (APE/Ref-1-) dependent pathway [117]. ROS increased the expres-sion levels of PAI-1, IL-6, and MCP-1 through NADPHoxidase pathway [112]. Oxidized high-density lipoprotein(HDL) enhances proinflammatory properties such as TNF-α and MCP-1 in renal mesangial cells partly via CD36and LDL receptor-1 and via MAPK and NF-κB pathways[118]. Oxidized LDL can stimulate TNF-α synthesis fromrenal cells and initiate local effects of renal damage [85].Increased ROS production and MCP-1 secretion fromaccumulated fat may cause infiltration of macrophages andinflammation in adipose tissue of obesity [112]. Moreover,enhanced macrophage migration induces the release ofproinflammatory cytokines, which further stimulates thegeneration of ROS [119, 120]. Therefore, oxidative stress-induced cytokine production is likely to further increaseoxidative stress levels, setting a vicious cycle [121] that maypromote the progression of kidney damage in obesity.

3.3. Oxidative Stress Leads to Renal Injury in Obesity. Oxida-tive stress has been commonly identified in obesity-relatedrenal diseases and may be the mechanism underlying theinitiation or progression of renal injury in obesity [122, 123].Previous studies suggest that oxidative stress triggers, at anearly age, the onset of kidney lesions and functional im-pairment in Zucker obese (ZO) fa/fa rats, a good model ofobesity-related renal disease, in absence of hyperglycaemia,hypertension, and inflammation [124]. ROS play a crucialrole in mediating renal injury [125–127]. ROS are highlyreactive molecules that oxidize lipids and proteins, cause cell-ular injury, and promote glomerular and renal tubule injuryand associated proteinuria [128].

ROS are produced by various cells, such as vascularcells, inflammatory cells, and renal cells, and have distinctfunction on different types of cells, such as endothelial dys-function, inflammatory gene expression, and renal tubuleion transport. A major source for vascular and renal ROS is aNox family of nonphagocytic NAD(P)H oxidases, includingthe prototypic Nox2 homolog-based NAD(P)H oxidase, as


well as other NAD(P)H oxidases, such as Nox1 and Nox4[129]. Numerous reports indicate that within the kidney,NAD(P)H oxidase, an enzyme that produces superoxide(O2

•−) by transferring electrons from NADH/NADPH tomolecular oxygen and thereby forming O2

−, H+, andNAD+/NADP+, is capable of modulating renal epithelial iontransport [130, 131]. NAP(D)H oxidase-derived ROS canalter renal pressure natriuresis and blood pressure regulationthrough its effects on renal hemodynamics and renal tubularsodium transport. Recent data suggest that NADPH oxidase-mediated oxidative injury to the proximal tubule, like thatseen in the glomerulus, contributes to proteinuria in insulin-resistant states [128].

Oxidative stress also plays an important role in the path-ogenesis of renal damage through its effects on vascular bio-logy. ROS are generated by all types of vascular cells, in-cluding endothelial, smooth muscle, and adventitial cells.ROS influence vascular cell growth, migration, proliferation,and activation [132, 133]. Physiologically, ROS can mediatecellular function, receptor signals, and immune responseson vascular cells. In pathophysiological condition, ROS con-tribute to progressive vascular dysfunction and remodel-ing through oxidative damage caused by decreased nitricoxide (NO) bioavailability, impaired endothelium-depend-ent vasodilatation and endothelial cell growth, apoptosis oranoikis, endothelial cell migration, and activation of adhe-sion molecules and inflammatory reactions [134, 135].

4. Potential Interventions toPrevent Renal Injury

4.1. Anti-Inflammation Therapy. Obesity accelerates the pro-gression of renal injury, associated with augmented inflam-mation in adipose and kidney tissues [136]. Anti-inflam-mation therapy might be a potential treatment for renaldamage. IL-6 is a key inflammatory molecule in renaldiseases. Studies in IL-6 transgenic mice suggested that highconcentrations of IL-6 contribute to development of renal in-jury [137]. Treatment with anti-IL-6 receptor antibodyMR16-1 prevented progression of proteinuria, renal lipiddeposit, and the mesangial cell proliferation in hypercho-lesterolemia-induced renal injury [97]. TNF-α is anotherimportant proinflammatory cytokine in the development ofrenal diseases. Inhibition of TNF-α by etanercept, a TNF-α antagonist, also decreased blood pressure and protectedthe kidney through reduction of renal NF-κB, oxidativestress, and inflammation [138]. TNF-α blockade increasesrenal Cyp2c23 expression and slows the progression of renaldamage in salt-sensitive hypertension [139]. TNF-α inhibi-tion also reduces renal injury in deoxycorticosterone-acetate-(DOCA-) salt hypertensive rats via suppression of renal cor-tical NF-κB activity [140]. Adiponectin is an adipose-secret-ed hormone with anti-inflammatory properties. Treatmentof adiponectin-knockout mice with adenovirus-mediatedadiponectin results in amelioration of albuminuria, glom-erular hypertrophy, and tubulointerstitial fibrosis and redu-ces the elevated levels of VCAM-1, MCP-1, TNF-α, TGF-β1,collagen type I/III, and NADPH oxidase components [141].Adiponectin prevents glomerular and tubulointerstitial

injury through modulating inflammation and oxidativestress [141].

Excessive fat accumulation contributes to macrophageinfiltration in adipose tissue and increased production ofproinflammatory cytokines, such as TNF-α, IL-8, and IL-6 [142–145]. Consequently, it is possible that weight lossmay be a potential method to reduce inflammation. Evidenceindicates that weight loss induced by nutritional interventionor gastric surgery markedly improves the systemic and adi-pose tissue inflammatory states linked to obesity [143, 146,147]. Studies by gene profiling analysis have shown thatcaloric restriction-induced weight reduction leads to the reg-ulation of a wide variety of inflammation-related moleculesin human adipose tissue [148]. Weight loss globally improvesthe inflammatory profile of obese subjects through a de-crease of proinflammatory factors and an increase of anti-inflammatory molecules in white adipose tissue [148]. Roux-en-Y-Gastric-Bypass- (RYGB-) induced weight loss has beenshown to reduce MCP-1, IL-18, IL-6, and TNF-α concentra-tions [149, 150]. A longer-term weight reduction induced byRYGB in corpulence also prevails in regulating circulatingcytokine concentrations [151]. Weight loss also amelioratesthe low-grade inflammation state that leads to glomerulardysfunction in obesity. In morbidly obese individuals withglomerular hyperfiltration, weight loss by surgical inter-ventions normalizes glomerular filtration rate (GFR) andreduces blood pressure and microalbuminuria [152]. Weightloss improves renal function as shown by reduced levels ofserum creatinine and improved creatinine clearance [153].

4.2. Antioxidant Intervention. Since ROS play a key role inthe pathogenesis of renal injury such as glomerulosclerosisand tubulointerstitial fibrosis, approaches to reduce oxidativestress by antioxidants supplementation, nutritional and sur-gical interventions may have renoprotective effects. Garciniaprotects against obesity-induced nephropathy by attenuatingoxidative stress through reduced lipid peroxidation and levelsof oxidized LDL [154]. The obese Zucker rat is a goodmodel for studying obesity-related kidney disease because itdevelops proteinuria, glomerular hypertrophy, and focal seg-mental glomerulosclerosis [155–157]. Using these rats, it hasbeen demonstrated that nephropathy is associated with oxi-dative stress, and supplementation with an antioxidantebselen improved kidney damage by ameliorating protein-uria and renal focal and segmental sclerosis [158]. Chronicebselen therapy also improved vasculopathy with lipiddeposits, tubulointerstitial scarring, and inflammation [158].Other antioxidants also have renoprotective effects. For ex-ample, administration of grape seed proanthocyanidin ex-tract (GSPE), an efficient phytochemical antioxidant, canprotect against the nephrotoxicity effects induced by cisplatinand gentamicin [159, 160] and reverse experimental myo-globinuric acute renal failure [161]. Quercetin, a flavonoidthat exhibits antioxidant properties in many diseases, couldalso protect the rat kidney against lead-induced injury andimprove renal function [162]. Thus, antioxidants may be apotential therapeutic to prevent the renal damage in ORG.

Nutritional and surgical interventions are additional ap-proaches to reduce oxidative stress and prevent kidney injury


in obesity. Caloric restriction and protein restriction reducefree radicals and ROS formation and inhibit accumulationof oxidative biomarkers in animal models [163]. In geneti-cally obese animals, diet restriction can prevent or greatlydelay the onset of specific degenerative lesions, in particularglomerulonephritis associated with obesity [164]. Sinceadipose tissue mass in obesity contributes to oxidative stress,bariatric surgery-induced weight loss also results in decreas-ing systemic oxidative stress in adiposity [111]. Weight lossinduced by diet restriction or bariatric surgery not only im-proves inflammation state but also reduces oxidative stressstate in obesity, which may protect renal function in obesity-related glomerulopathy.

5. Conclusion

Obesity causes chronic low-grade inflammation and sys-temic and local oxidative stress, which may play a pivotal rolein the initiation or progression of obesity-associated glomer-ulopathy. Elevated inflammation in obesity is the result ofthe production of adipokines and increased inflammatorycytokines and decreased anti-inflammatory factors. Oxida-tive stress is triggered by an imbalance between increasedproduction of ROS and/or reduced antioxidant activity. Bothinflammation and oxidative stress induce damage to renaltubule and glomerulus and result in endothelial dysfunctionin the kidney. Therefore, anti-inflammation and antioxidantinterventions may be the potential therapies to prevent andtreat obesity-related renal diseases.

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Review Article

Reactive Oxygen Species Modulation of Na/K-ATPase RegulatesFibrosis and Renal Proximal Tubular Sodium Handling

Jiang Liu,1 David J. Kennedy,2 Yanling Yan,1, 3 and Joseph I. Shapiro1

1 Department of Medicine, College of Medicine University of Toledo, 3000 Arlington Avenue, Toledo, OH 43614, USA2 Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA3 Institute of Biomedical Engineering, Yanshan University, Qinhuangdao 066004, China

Correspondence should be addressed to Joseph I. Shapiro, [email protected]

Received 7 October 2011; Accepted 7 November 2011


Copyright © 2012 Jiang Liu et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The Na/K-ATPase is the primary force regulating renal sodium handling and plays a key role in both ion homeostasis and bloodpressure regulation. Recently, cardiotonic steroids (CTS)-mediated Na/K-ATPase signaling has been shown to regulate fibrosis,renal proximal tubule (RPT) sodium reabsorption, and experimental Dahl salt-sensitive hypertension in response to a high-saltdiet. Reactive oxygen species (ROS) are an important modulator of nephron ion transport. As there is limited knowledge regardingthe role of ROS-mediated fibrosis and RPT sodium reabsorption through the Na/K-ATPase, the focus of this review is to examinethe possible role of ROS in the regulation of Na/K-ATPase activity, its signaling, fibrosis, and RPT sodium reabsorption.

1. Introduction

According to the American Heart Association (AHA), over70 million people in the United States aged 20 and olderhave high blood pressure (BP). The cause of 90–95 percentof the cases of high BP is unknown, yet in the last decadethe associated morbidity and mortality from high BP hasincreased precipitously. In the 2008 AHA Scientific State-ment [1], excessive dietary salt intake is listed as one ofthe major lifestyle factors which significantly contributesto the development of hypertension and tends to be morepronounced in typical salt-sensitive patients. Modest dietarysalt restriction and diuretic therapy, therefore, are recom-mended for treatment of resistant hypertension, especiallyin the salt-sensitive subgroup [1, 2]. Renal sodium handlingis a key determinant of long-term BP regulation [3]. Therelationship between dietary sodium, salt sensitivity, andBP has been established on an epidemiological and clinicalbasis. It is estimated that hypertension affects 25% to 35%of the world population aged 18 and older [4], and morehypertensive subjects (∼50%) are significantly salt sensitivethan normotensive subjects (∼25%) [5]. In the DASH-Sodium clinical trial, BP reduction was correlated withsodium restriction in the salt-sensitive subjects regardless

of diet [6]. Interestingly, animal renal cross-transplantationexperiments [7–10] and studies of human renal transplanta-tion [11] demonstrate that BP levels “travel with the donor’skidney,” providing compelling evidence for the role of renalfunction in the pathogenesis of hypertension. In clinical andexperimental models, renal proximal tubule (RPT) sodiumhandling accounts for over 60% reabsorption of filteredsodium and is an independent determinant of BP responseto salt intake, playing a critical role in the pathogenesisof salt-sensitive hypertension. Recently, accumulating dataindicate that cardiotonic steroids (CTS) signaling throughthe Na/K-ATPase contribute to RPT sodium handling andsalt sensitivity [12–18].

2. CTS and Na/K-ATPase Signaling

CTS (also known as endogenous digitalis-like substances)include plant-derived digitalis drugs, such as digoxin andouabain, and vertebrate-derived aglycones such as bufalinand marinobufagenin (MBG) [16, 18]. Recent studies haveidentified both ouabain and MBG as endogenous steroidswhose production, and secretion are regulated by stimuliincluding angiotensin II (Ang II) [18–21]. The Na/K-ATPase


belongs to the P-type ATPases family and consists of twononcovalently linked α and β subunits [22–24]. Several αand β isoforms, expressed in a tissue-specific manner, havebeen identified and functionally characterized [22–25]. Theα1 subunit contains multiple structural motifs that interactwith soluble, membrane and structural proteins includingSrc, caveolin-1, phospholipase C-γ, PI3 kinase, IP3 receptor,BiP, calnexin, cofilin, and ankyrin [26–36]. Binding to theseproteins not only regulates the ion-pumping function ofthe enzyme, but it also conveys signal-transducing functionsto the Na/K-ATPase [18, 32, 37–39]. In LLC-PK1 cells,the Na/K-ATPase α1 subunit and Src form a functionalreceptor in which the binding of CTS to the α1 subunitactivates Src and consequent signaling cascade [39]. Thesignaling function of the Na/K-ATPase regulates numerouscell functions in various types of organs and cells includingcell motility, cell proliferation, cancer, endothelin release,glycogen synthesis, apoptosis, hypertension, intracellularcalcium signaling, cardiac hypertrophy, cardiac remodeling,renal remodeling, epithelial cell tight junction, vascular tonehomeostasis, and sodium homeostasis [26, 40–59]. The topicof CTS-Na/K-ATPase signaling and its downstream patho-physiological implications has been extensively reviewed inthe last few years [12–16, 18, 21, 32, 39, 59–64], and we willnot discuss them in detail in this review.

3. CTS and Sodium Homeostasis

Endogenous CTS were first proposed many years ago tofunction as a natriuretic hormone. Although their patho-physiological significance has been a subject of debate formany years [65], the concept of a natriuretic hormone issupported by the experimental observations in animal mod-els of ouabain-induced natriuresis [66, 67]. Recently, severalelegant reports have confirmed this concept with differentapproaches. Gene replacement studies have unequivocallydemonstrated an important role of endogenous CTS inregulation of renal sodium excretion and BP [68, 69]. Intransgenic mice expressing ouabain-sensitive Na/K-ATPaseα1 subunit, a significant observation is an augmentednatriuretic response to both acute salt load and ouabaininfusion, indicating that the ouabain-binding site of Na/K-ATPase α1 subunit participates in the natriuretic response tosalt load by responding to endogenous ouabain. Moreover,the augmented natriuretic response in the ouabain-sensitiveα1 isoform mice can be blocked with administration ofan anti-digoxin antibody fragment [68, 69]. In normalmale Wistar rats, endogenous circulatory ouabain hasphysiological roles controlling vasculature tone and sodiumhomeostasis, showing endogenous ouabain regulates renalsodium excretion in normal animals [70]. A significantinhibition of natriuresis is observed in rats that were passivelyimmunized with anti-ouabain antibody and in rats thatwere actively immunized with ouabain-albumin antigen, inwhich endogenous ouabain levels were reduced in both cases.Like ouabain, MBG has both natriuretic and vasoconstrictoreffects [12, 71, 72]. High salt intake induced an initial tran-sient stimulation of ouabain and a subsequent progressive

increase of MBG both in Dahl salt-sensitive (S) rats and inhumans [12, 73]. In Dahl S rats, the increase in natriuresisstimulated by an acute salt loading is prevented by admin-istration of both an anti-ouabain antibody and an anti-MBG antibody [72]. Furthermore, endogenous CTS havealso been implied in age-related increases in salt sensitivityof BP in human normotensive subjects [73]. It is estimatedthat approximately 50% of humans with untreated essentialhypertension have significantly elevated levels of endogenousouabain [74] which is involved in the regulation of vasculartone homeostasis through stimulation of interaction betweenthe Na/K-ATPase and sodium/calcium exchanger [59]. Innormotensive human males, both a high salt diet and system-atic sodium depletion (by hydrochlorothiazide) significantlyincrease plasma ouabain concentration [75].

Release of endogenous ouabain from the brain hypotha-lamus stimulated by a high salt diet leads to inhibition ofthe Na/K-ATPase activity and central sympathetic activation(reviewed in [76, 77]), which plays a crucial role in the pres-sor effects of high salt intake in spontaneously hypertensiverats (SHR) and Dahl S rats. In Dahl S rats, elevation in brainouabain increased MBG secretion from the adrenal cortex,and this effect was blocked by the AT1 receptor antagonistlosartan [78, 79]. The data suggest that the observed CTS-induced natriuretic effect might be a result of the combinedeffects of ouabain and MBG [60]. Furthermore, CTS notonly induced hypertension in rats but also caused significantcardiovascular remodeling and natriuresis independent oftheir effect on BP [49, 51, 70, 80, 81].

4. CTS and Oxidative Stress in Cardiac andRenal Fibrosis

CTS binding to the Na/K-ATPase induces cellular reactiveoxygen species (ROS) production and its downstream effects,such as cardiac and renal fibrosis, and these effects can beprevented by ROS scavenging [80, 82–84]. In kidney andheart, the central role of CTS in the development of fibrosishas been demonstrated both in vivo, in the partial (5/6th)nephrectomy model, and in vitro cell culture, includingcardiac and renal fibroblasts. 5/6th nephrectomy increasescirculating levels of MBG and stimulates cardiac fibrosisin both rat and mouse [80, 84, 85]. Rats subjected to5/6th nephrectomy develop systemic oxidant stress that issimilar to that seen in rats subjected to MBG infusion asevidenced by significant elevation of plasma carbonylatedprotein. Active immunization against MBG and reductionof circulating levels of MBG by adrenalectomy substan-tially attenuate 5/6th nephrectomy and MBG infusion-mediated cardiac fibrosis and oxidant stress, an effect thatis independent of BP [80, 84]. In primary culture of ratcardiac and human dermal fibroblasts as well as a cellline derived from rat renal fibroblasts, MBG and ouabainstimulate [3H]proline incorporation as well as gene andprotein expression of collagen [86]. MBG induced a PLC-dependant translocation of PKC-δ to the nucleus, resultingin the phosphorylation and degradation of transcriptionfactor Friend leukemia integration-1 (Fli-1), a negative


Partial (5/6th) nephrectomy

Fli-1-p

ROS

Na/K-ATPase/c-Src

Shift of cardiac and renal function curve

CTS

PLC

Fli-1

Fibrosis

EMT

Collagen production

AntioxidantsImmunizationSpironolactoneCanrenone

dermal fibroblastsRenal cortexRenal proximal tubules

PKC-δ

Snail

Cardiac, renal, and

Figure 1: Schematic illustration of the effect of CTS on fibrosis.

regulator of collagen synthesis [86]. Both CTS-inducedNa/K-ATPase signaling and oxidative stress are necessary inthe pathogenesis of cardiac and renal fibrosis as evidencedby CTS-stimulated phosphorylation of both Src and MAPKwhich is effectively blocked not only by ROS scavengingand Src inhibition but also through possible competitiveinhibition of CTS binding to Na/K-ATPase by spironolactoneand canrenone [80, 84, 87, 88]. MBG infusion causes renalfibrosis mainly in the cortex of the kidney by stimulationof the transcription factor Snail expression and its nuclearlocalization in the tubular epithelia, which is associated withepithelial-to-mesenchymal transition (EMT) during renalfibrosis [89]. This EMT phenomenon is also demonstratedin LLC-PK1 cells, indicating that MBG may cause damageof renal proximal tubules. CTS-induced fibrosis in heart andkidney might shift the cardiac and renal function curve tofavor a higher set point of pressure natriuresis (Figure 1).

5. Na/K-ATPase Signaling and RPTSodium Handling

It has been postulated for decades that endogenous CTSstimulated by increased sodium intake increases natriuresisand diuresis by directly inhibiting renal tubular Na/K-ATPase to prevent renal reabsorption of filtered sodium [90–92]. There is accumulating evidence supporting this ideaunder conditions such as high salt intake, chronic renalsodium retention, renal ischemia, uremic cardiomyopathy,and volume expansion in various animal models and humanbeings [12, 13, 15, 60, 62, 75, 93–107]. Although thedirect inhibition of the Na/K-ATPase enzymatic activityand sodium reabsorption in RPTs by CTS has not beenvalidated, recent observations indicate that ligand-mediatedRPT sodium reabsorption via Na/K-ATPase/c-Src signalingcounterbalances the sodium retention-mediated increasesin BP, such as that seen in salt-sensitive hypertension

[108–114]. Ouabain, a ligand of the Na/K-ATPase, acti-vates the Na/K-ATPase/c-Src signaling pathway and sub-sequently redistributes basolateral Na/K-ATPase and apicalsodium/hydrogen exchanger isoform 3 (NHE3) in RPTs,leading to reduced RPT sodium reabsorption and increasedurinary sodium excretion.

In LLC-PK1 cells, ouabain activates Na/K-ATPase/c-Srcsignaling pathways and reduces cell surface Na/K-ATPase andNHE3, leading to a significant inhibition of active transcel-lular 22Na+ flux from the apical to basolateral compartment[108–112]. MBG, an important CTS species, and depro-teinated extract of serum (derived from patients with chronicrenal failure) also induce Na/K-ATPase endocytosis andinhibition of active transepithelial 22Na+ flux. However, it isstill not clear how ouabain-activated Na/K-ATPase signalingregulates NHE3, since, at concentrations of ouabain used,no significant change in intracellular Na+ concentration wasobserved [108]. Interestingly, this phenomenon is either notobserved or is much less significant in MDCK cells (a caninerenal distal tubule cell line). These in vitro data suggest thatCTS-Na/K-ATPase signaling has a profound effect on RPTsodium handling, but not in distal tubules.

Different species of endogenous CTS show differences inthe kinetics and tissue actions in response to salt loading inboth animal models and in human salt-sensitive hypertensivepatients [16, 18, 60, 75, 79, 105]. The Dahl salt-resistant(R) and salt-sensitive (S) strains were developed by selectivebreeding of the outbred Sprague-Dawley rat strain forresistance or susceptibility to the hypertensive effects of highdietary sodium [115]. RPT sodium handling is a criticaldeterminant of the different BP responses in these strains[7, 116–118], and there is no Na/K-ATPase α1 gene (Atp1a1)difference between R and S rats [119]. In comparison toDahl S rats that eliminate excessive sodium mainly throughpressure natriuresis at the expense of an elevated systolicBP, a major response to salt loading in the Dahl R rats is agreater reduction in renal sodium reabsorption to eliminateexcessive sodium without raising BP. Our recent in vivoobservation [114] is in agreement with this hypothesis andour in vitro observations in LLC-PK1 cells. Specifically inisolated RPTs, both a high salt diet and ouabain are ableto activate Na/K-ATPase/c-Src signaling pathways, leading tothe redistribution of Na/K-ATPase and NHE3 in the Dahl Rbut not in the S rats. The R rats show significant increases intotal urinary sodium excretion and RPT-mediated fractionalsodium excretion, without BP elevation [114]. While theBP response to salt loading in R and S rats involves manyregulatory factors [117], our data indicate that impairmentof the RPT Na/K-ATPase/c-Src signaling contributes to saltsensitivity. Since it failed to confirm the possible differenceof Na/K-ATPase α1 gene (Atp1a1) between R and S rats[119], other factor(s) must be present to prevent activationof Na/K-ATPase/c-Src signaling in the S rats. The possibleeffect of CTS on RPT Na/K-ATPase signaling and sodiumreabsorption is summarized in Figure 2.

The SHR rat is an established model of human essentialhypertension with the characteristic of vascular resistance.SHR rat develops hypertension spontaneously at the ageof 7–15 weeks, regardless of salt loading, mainly through


ROS

RPT Na/K-ATPase

c-Src ROS

Redistribution ofNa/K-ATPase and NHE3

RPT sodium reabsorption

Desensitization ofNa/K-ATPase

CTS

Antioxidants

High-salt diet

Salt sensitivity

Figure 2: Schematic illustration of the effects of ROS and Na/K-ATPase signaling on RPT sodium reabsorption. CTS: cardiotonicsteroids; RPT: renal proximal tubules.

increase in peripheral vascular resistance [120] includingrenal vascular resistance. Interestingly, either reduction ofdietary vitamin E or caloric restriction without sodiumrestriction prevents the development of hypertension [121,122]. Like Dahl S rats, SHR rats eliminate excessive sodiumvia a pressure-natriuresis mechanism but have significantlower BP response to a high salt diet and higher systolic BPto eliminate the same amount of sodium when comparedto Dahl S rats [123–126]. Most interestingly, comparingto control Wistar-Kyoto (WKY) rats, regulation of theNa/K-ATPase and NHE3 with both aging and oxidativestress have been shown contributed to the developmentand maintenance of hypertension in the SHR [127–135].In renal cortical (proximal) tubules, activity and proteinlevels of NHE-3 are significantly higher in SHR than age-matched WKY rats at all stages during the developmentand maintenance of hypertension. When NHE3 function isdetermined by the rate of bicarbonate reabsorption by invivo stationary microperfusion in RPT, young SHR rats showhigher NHE3 activity than adult SHR and WKY rats, andthis is accompanied by changes in NHE3 phosphorylationand distribution. In young SHR rats, the RPT Na/K-ATPaseactivity is significantly higher than in age-matched WKY,which can be prevented by treatment of the diuretic drughydrochlorothiazide. However, the Na-K-ATPase activity inmedullary thick ascending limb, cortical thick ascendinglimb, and distal tubule is significantly lower in young SHRrats than in age-matched WKY rats. There were no signif-icant differences in Na/K-ATPase activity in these nephronsegments in adult SHR and WKY rats, nor in collecting ductsegments of young and adult SHR and age-matched WKYrats. Interestingly, however, the Na/K-ATPase α1 subunitgene expression is lower in both young and adult SHR ratsthan age-matched WKY rats, indicating a posttranscriptionalregulation.

Recent study indicates that SHR rats have enhancedrenal superoxide generation and NADPH oxidase (NOX)expression in both vascular and renal tissue before and

after development of hypertension [136]. Elevated basal levelof superoxide inhibits proximal tubule NHE3 activity andfluid reabsorption in SHR rats in comparison to WKY rats.This effect is prevented by the NOX inhibitor apocyninor knockdown of the critical NOX subunit p22phox withsmall interfering RNA, indicating that increased basal levelof superoxide impairs RPT function [137]. Furthermore,in Sprague-Dawley rats, oxidative stress impairs Ang II-mediated regulation of NHE3 [138].

6. Oxidative Stress and Regulationof Na/K-ATPase

Many stimuli including hypoxia and dopamine induce acell- and tissue-specific endocytosis and exocytosis of Na/K-ATPase and a change in Na/K-ATPase activity [33, 61, 139].Ouabain-stimulated ROS generation functions as a secondmessenger in ouabain-activated Na/K-ATPase signaling inisolated rat cardiac myocytes [82, 140–142]. Binding ofouabain to the Na/K-ATPase activates Src kinase, which inturn transactivates EGFR, leading to activation of the Ras-Raf-MEK-ERK pathway [32, 39, 63]. Ras activation leads tothe activation of MAPK and increase in [Ca2+]i which resultin opening of mitochondrial ATP-sensitive K+ channels[142] and generation of mitochondrial ROS [82, 141]. ROSsubsequently activate NF-κB [141, 143] and slow [Ca2+]i

oscillations at nanomolar ouabain concentrations [40].Additionally, ouabain-induced generation of ROS in neona-tal myocytes is antagonized by overexpression of a dominantnegative Ras as well as myxothiazol/diphenyleneiodonium,indicating a mitochondrial origin of the Ras-dependent ROSgeneration [82]. Ouabain also stimulates ROS generation inother cell types [144–146]. Conversely, oxidative stress canactivate the Na/K-ATPase signaling. Both a bolus of H2O2

and exogenously added glucose oxidase (which generates asustained low level of H2O2 by consuming glucose) activatesNa/K-ATPase signaling in cardiac myocytes [140]. Pretreat-ment with the antioxidant N-acetyl cysteine (NAC) preventsouabain-Na/K-ATPase signaling and its downstream effects.Moreover, infusion of CTS causes ROS generation andprotein oxidation in experimental animals [80, 147].

The redox sensitivity of the Na/K-ATPase was first dem-onstrated in electric eels with treatment of H2O2 [148]. Thisphenomenon was further observed in a wide range of animalspecies, tissues, and other species of ROS such as hypochlor-ous anion, hydroxyl radicals, superoxide, hyperchloriteanions, and singlet oxygen. It has been shown that the Na/K-ATPase in skeletal muscle is redox-sensitive and infusionof NAC attenuated ROS-mediated inhibition of maximalpump activity [149]. In dog kidney, oxidative modificationof kidney Na/K-ATPase by H2O2 was accompanied by adecrease in the amount of sulfhydryl (SH) groups [150] and,importantly, oxidative modification can result in formationof Na/K-ATPase oligomeric structure [151]. The differencesin the number, location, and accessibility of SH groups inNa/K-ATPase isozymes might predict their oxidative stability[152]. Different antioxidant enzymes, natural or syntheticantioxidants, and some inhibitors of oxidase activity can


attenuate ROS mediated inhibition of Na/K-ATPase activity.ROS are known to inhibit Na/K-ATPase activity in differenttypes of cells as well. Interestingly, the Na/K-ATPase in ratcerebellar granule cells are redox-sensitive with an “optimalredox potential range,” where ROS levels out of this “optimalrange” are capable of inhibiting Na/K-ATPase activity [153].While the Na/K-ATPase does not contain heme groups, itdoes contain cysteine residues located in α subunit cytosolicloops which may determine the redox sensitivity of the αsubunit. The sensitivity of the Na/K-ATPase to redox andoxygen status, the regulatory factors which govern theseinteractions, and the implied molecular mechanism wererecently reviewed [154].

Increases in ROS can oxidize the Na/K-ATPase α/βsubunits and its independent regulator FXYD proteins. Thisoxidation inhibits its activity and promotes its susceptibilityto degradation by proteasomal and endosomal/lysosomalproteolytic pathways in different cell types including cardiacmyocytes, vascular smooth muscle cells, and RPTs [155–166]. It appears that the oxidized modification of the Na/K-ATPase is a reversible, redox-sensitive modification. How-ever, purified enzyme has also been shown to be irreversiblyinhibited upon exposure to hydrogen peroxide, the super-oxide anion, and the hydroxyl radical [158]. The regulationof renal Na/K-ATPase α1 by oxidants is not dependent onthe ouabain sensitivity of the α1 subunit per se. It appearsthat the α2 and α3 subunits are more sensitive to oxidantsthan the α1 subunit, and ouabain-sensitive α1 (canine)and insensitive-α1 (rat) have similar sensitivity to oxidants,suggesting the regulation of α1 by ROS is not species specific.Furthermore, ROS accelerates degradation of the oxidativelydamaged Na/K-ATPase and the α2 and α3 subunits, whichalso appear to be more susceptible to degradation than the α1subunit [159]. These studies indicate that differential oxidantsensitivities of the Na/K-ATPase subunits are dictated by theprimary sequences of different subunits and different subunitcompositions of the various tissues may contribute to theirrelative susceptibilities to oxidant stress. In the RPT cellline originated from WKY rats, cadmium, a ROS generator,stimulates ROS production and causes a toxic oxidativedamage of the Na/K-ATPase. Oxidative damage increasesNa/K-ATPase degradation through both the proteasomaland endo-/lysosomal proteolytic pathways [163]. In purifiedrenal Na/K-ATPase, peroxynitrite (ONOO−) causes tyrosinenitration and cysteine thiol group modification of the Na/K-ATPase, but only cysteine thiol group modification is impliedin the inhibition of the enzyme activity since glutathione isunable to reverse the inhibition [167]. In isolated rat renalRPTs, peroxynitrite and its signaling participates in Ang II-induced regulation of renal Na/K-ATPase activity [168].

Ang II inhibits the Na/K-ATPase via PKC-dependentNOX activation. The dependence of Ang II-induced Na/K-ATPase inhibition on NOX and superoxide, as well asreversible oxidative modification of the Na/K-ATPase,strongly suggests a role for redox signaling [164–166]. Incardiac myocytes and pig kidney, oxidative stress inducesglutathionylation of the β1 subunit of the Na/K-ATPase.In purified pig renal Na/K-ATPase, peroxynitrite inhibitsthe enzymatic activity by stabilization of the enzyme in an

E2-prone conformation. At the same time, FXYD proteinsreverse oxidative stress-induced inhibition of the Na/K-ATPase by facilitating deglutathionylation of the β1 subunit.Moreover, both tyrosine kinase c-Src and cell membranestructural component lipid rafts, which are critical in Na/K-ATPase/c-Src signaling, are also critical in redox signalingplatform formation [169–172]. These observations, alongwith the fact that c-Src is redox-sensitive [173] and itsactivation regulates NOX-derived superoxide generation[174], suggests a redox-sensitive Na/K-ATPase/c-Src signal-ing cascade and its possible role in ROS regulation, althoughthe mechanism is not clear. Our unpublished data suggestthat certain basal levels of ROS might be required for theinitiation of ouabain-Na/K-ATPase/c-Src signaling. In LLC-PK1 cells, pretreatment with higher concentrations of NAC(5 and 10 mM for 30 min), but not with lower concentrationof 1 mM, can prevent ouabain-stimulated c-Src activationand the redistribution of Na/K-ATPase and NHE3. Thissuggests that ROS may stabilize Na/K-ATPase in a certainconformational status in order to facilitate ouabain bindingto the Na/K-ATPase α1 subunit and favor ouabain-Na/K-ATPase/c-Src signaling. Some pertinent questions remainto be resolved, such as how ROS interacts with and influ-ences the Na/K-ATPase/c-Src signaling cascade and whetherouabain-induced ROS boosts the Na/K-ATPase signaling bya positive feedback mechanism and chronically desensitizesthe signaling cascade by stimulating Na/K-ATPase/c-Srcendocytosis (Figure 2).

7. Oxidative Stress and Regulationof Renal Function

ROS function as important intracellular and extracellularsecond messengers to modulate many signaling molecules.Physiological concentrations of ROS play an importantrole in normal redox signaling, while pathological levelsof ROS contribute to renal and vascular dysfunction andremodeling through oxidative damage [175–177]. Geneticfactor(s) partially contribute to high basal ROS levels and thedevelopment of hypertension [178, 179].

Oxidative stress has been shown to regulate BP andsodium handling in various animal models. High saltintake increases oxidative stress, and this has importantimplications for the regulation of cardiovascular and renalsystems. An increase in oxidative stress is both a cause andconsequence of hypertension [177, 180–183]. Renal NOX ispresent in the renal cortex, medulla, and vasculature. NOX,the major source of superoxide in the kidney, is of particularinterest because of its prominent expression and implicationin pathophysiology. In the kidney, increased oxidative stressinfluences a number of physiologic processes including renalsodium handling in the proximal tubule [137, 168, 184, 185]and thick ascending limb [186], renal medulla blood flow[183, 187–190], descending vasa recta contraction [191, 192]in addition to interactions with other regulatory systemssuch as the dopamine signaling pathway [193]. Increasedoxidative stress has been shown to contribute to saltsensitivity [194, 195]. In macula densa cells, NOX isoform


Nox2 is responsible for salt-induced superoxide generation,while Nox4 regulates basal ROS [196]. In the medullar thickascending limb of the loop of Henle, both exogenous andendogenous superoxides stimulate sodium absorption [197].Also, in this nephron segment, NOX-induced generation ofsuperoxide enhances sodium absorption by reduction of thebioavailability of nitric oxide (NO) to prevent NO-inducedreduction of NaCl absorption [198], which contributes tosalt-sensitive hypertension observed in Dahl salt-sensitiverats [190].

In PRTs, in particular, increases in ROS inhibit the Na/K-ATPase as well as the apical NHE3 and sodium/glucosecotransporter, in order to promote RPT sodium excretionunder certain circumstances [137, 168, 184, 185]. Whileelevated basal level of superoxide inhibits proximal tubuleNHE3 activity and fluid reabsorption in SHR rats in com-parison to WKY rats [137], peroxynitrite and its signalingparticipates in Ang II-induced regulation of renal Na/K-ATPase activity in isolated rat renal RPTs [168]. In thepathogenesis of diabetic nephropathy, a high level of glucose-induced ROS generation induced by stimulation of mito-chondrial metabolism and NOX activity in RPT primarycultures leads to inhibition of the expression and activityof the sodium/glucose cotransport system [185]. In maleWistar rats, a high salt diet (3% NaCl for 2 weeks) promotessodium/water excretion and urinary 8-isoprostane excretion,a marker of oxidative stress, which can be attenuated bytreatment with apocynin, an NOX inhibitor. The salt loadingleads to increased generation of ROS and a state of oxidativestress in the cortex but not to such a degree in the medulla[199].

RPT sodium and/or fluid absorption in the normal rat isreduced by inhibition of NO synthesis, while NO promotesRPT Na+ and/or fluid reabsorption [200]. In immortalizedand freshly isolated RPTs from the WKY and SHR rats,the basal level of membrane NOX activity is greater inSHRs [201]. Moreover, NOX-induced superoxide genera-tion inhibits RPT fluid reabsorption in SHRs [137]. InSprague-Dawley rats treated with the oxidant L-buthioninesulfoximine, Ang II overstimulates RPT Na/K-ATPase andNOX and leads to increased sodium reabsorption, whichis prevented by administration of the superoxide scavengerTempol [202].

High salt diet, which is well documented for its stim-ulation of systematic oxidative stress, regulates the activityand distribution of the Na/K-ATPase and NHE3 in differentanimal models [203–207]. PRT sodium reabsorption signif-icantly affects water and sodium homeostasis by regulatingredistribution of ion transporters in response to high saltintake [208]. Regulation of NHE3 activity and distribution aswell as PRT fluid reabsorption contribute to the developmentand maintenance of hypertension in young and adult SHRrats [127, 137, 209].

It has become clear that antioxidant agents such asTempol and enzymes such as heme oxygenase-1 (HO-1)exhibit a beneficial and protective effect on BP in variousanimal models of hypertension. As an example, inhibitionof HO activity reduces renal medullary blood flow [84,210, 211], total renal blood flow (RBF) [212], glomerular

filtration rate (GFR), and renal production of nitric oxide[213]. Inhibition of HO-1 increases mean arterial pressurein Sprague-Dawley rats [214, 215] and SHR rats [216].Induction of HO-1 reduces BP in SHR rats [217–220]. Theeffect of HO-1 on BP is presumably through the carbonmonoxide (CO)/HO system and depression of cytochromep-450-derived 20-HETE. In the Dahl salt-dependent modelof systemic hypertension, induction of HO-1 occurred in thevasculature and is accompanied by endothelial dysfunction[221, 222]. In Dahl salt-sensitive rats, a high salt dietincreases HO-1 expression and CO generation in aorta andcoronary arteries [221, 223], and, in Dahl salt-sensitive ratsfed low salt diet, induction of HO-1 expression attenuatesoxidative stress and reduces proteinuria and renal injury[224]. However, most studies examining the contributionof HO-1 to BP regulation have focused on the vasculature,that is, pressure-natriuresis and arterial BP, and so the roleof HO-1 in RPT-mediated sodium handling is still poorlyunderstood. Nevertheless, increased HO expression in RPTscould result in an increased ability to buffer locally producedoxidants, leading to their neutralization.

The beneficial effect of antioxidants is controversial andnot seen in most clinic trials with administration of antioxi-dants (reviewed in [177, 225]). The Dietary Approaches toStop Hypertension (DASH) study and subsequent studieshave demonstrated that lower BP associated with reduceddietary salt intake may be related to reductions in oxidativestress [6, 226–228]. Interestingly, however, while a com-bination antioxidant supplement (with an ascorbic acid,synthetic vitamin E, and β-carotene) had no improvementon BP after 5-year treatment [229], another combinationantioxidant supplement (zinc, ascorbic acid, α-tocopherol,and β-carotene) did result in a significant reduction insystolic BP [230]. Other studies have also shown that certainantioxidants, such as glutathione and vitamin C, have ablood-pressure-lowering effect [231, 232]. However, antiox-idant supplementation may be ineffective or even dangerous[233] due to the possible “over-antioxidant-buffering” effectof excessive antioxidant supplementation. In this scenario,excess antioxidants might become pro-oxidants (by pro-viding H+) if they cannot promptly be reduced by thefollowing antioxidant in the biological antioxidant chain.Thus, it appears that the balance of the ROS status, withina physiological range, may be more important to maintainbeneficial ROS signaling.

8. Perspective

Renal sodium handling is a key determinant of blood pres-sure. ROS status, among others, is an important regulatorof vasculature and sodium handling. However, the effectof ROS and Na/K-ATPase, especially of their interaction,on RPT sodium reabsorption has only been explored in alimited fashion. Coordinated regulation of two major iontransporters, the basolateral Na/K-ATPase and the apicalNHE3, has been implicated in the counterbalancing of highsalt intake (volume expansion) mediated blood pressureincrease. The Na/K-ATPase/c-Src signaling regulates this


coordinated regulatory mechanism and impairment of thissignaling cascade contributes to experimental Dahl salt-sensitive hypertension. Both the Na/K-ATPase (α and βsubunit) and its proximal signaling partner c-Src are redoxsensitive, suggesting a redox-sensitive Na/K-ATPase/c-Srcsignaling complex. However, the mechanisms remain largelyto be elucidated since the available data is limited [184,234]. Nevertheless, some pertinent questions remain to beaddressed. In the future, it will be important to explorewhether ROS signaling is a link between the Na/K-ATPase/c-Src cascade and NHE3 regulation and how oxidative stressstimulated by high salt and CTS regulates Na/K-ATPase/c-Src signaling in renal sodium handling and fibrosis.

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