jpet#128603 human renal cortical and medullary...

JPET#128603

1

HUMAN RENAL CORTICAL AND MEDULLARY UDP-

GLUCURONOSYLTRANSFERASES (UGT): IMMUNOHISTOCHEMICAL

LOCALIZATION OF UGT2B7 AND UGT1A ENZYMES AND KINETIC

CHARACTERIZATION OF S-NAPROXEN GLUCURONIDATION

Paraskevi Gaganis, John O. Miners, James S. Brennan, Anthony Thomas, and Kathleen M.

Knights

Department of Clinical Pharmacology, Flinders University (PG, JOM, KMK) and Flinders

Medical Centre (JOM); Department of Anatomical Pathology, Flinders Medical Centre (JSB,

AT), Bedford Park, Adelaide, Australia 5042

JPET Fast Forward. Published on August 14, 2007 as DOI:10.1124/jpet.107.128603

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on August 14, 2007 as DOI: 10.1124/jpet.107.128603

at ASPE

T Journals on June 10, 2020

jpet.aspetjournals.orgD

ownloaded from

http://jpet.aspetjournals.org/

JPET#128603

2

Running Title: Renal UGT localization and naproxen glucuronidation kinetics

Corresponding Author:

Professor John O Miners

Department of Clinical Pharmacology, Flinders Medical Centre and Flinders University

Bedford Park, South Australia, Australia

Tel: +61-8-8204-4131 ; Fax: +61-8-8204-5114

Email address: [email protected]

Number of text pages: 31

Number of Tables: 1

Number of Figures: 4

Number of References: 41

Number of words in Abstract: 236

Number of words in Introduction: 727

Number of words in Discussion: 1499

Abbreviations: CD: collecting duct; CLint: intrinsic clearance; DAB: 3,3’-diaminobenzidine

tetra hydrochloride; DCT: distal convoluted tubule: HKCM: human kidney cortical

microsomes; HKMM: human kidney medullary microsomes; HRP: horseradish peroxidase;

LOH: thin loop of Henle; Km: apparent Michaelis constant; MM: Michaelis-Menten;

NSAIDs: non-steroidal anti-inflammatory drugs; NGS: normal goat serum; NRIgG: normal

rabbit immunoglobulin-G; PCT: proximal convoluted tubule; TALH: thick ascending loop of

Henle; TBS: Tris-buffered saline; UDP: uridine diphosphate; UGT: UDP-

glucuronosyltransferase; UDPGA: UDP-glucuronic acid; Vmax: maximal velocity.

Section: Metabolism, transport and pharmacogenomics


at ASPE



ownloaded from


JPET#128603

3

Abstract

There is currently little information regarding the localization of UGTs in human renal cortex

and medulla and the functional contribution of renal UGTs to drug glucuronidation remains

poorly defined. Using human kidney sections and kidney cortical (HKCM) and medullary

microsomes (HKMM) we combined immunohistochemistry to investigate UGT1A and

UGT2B7 expression with in vitro microsomal studies to determine the kinetics of S-naproxen

acyl glucuronidation. With the exception of the glomerulus, Bowman’s capsule and renal

vasculature UGT1A proteins and UGT2B7 were expressed throughout the proximal and

distal convoluted tubules, the loops of Henle and the collecting ducts. Additionally, UGT1A

and UGT2B7 expression was demonstrated in the macula densa, supporting a potential role

of UGTs in regulating aldosterone. Consistent with the immunohistochemical data, S-

naproxen acyl glucuronidation was catalyzed by HKCM and HKMM. Kinetic data were well

described by the two-enzyme Michaelis-Menten equation. Km values for the high-affinity

components were 34±14µM (HKCM) and 45±14µM (HKMM). Fluconazole inhibited the

high-affinity component establishing UGT2B7 as the enzyme responsible for S-naproxen

glucuronidation in cortex and medulla. The low-affinity component was relatively unaffected

by fluconazole (<15% inhibition), supporting the presence of other UGTs with S-naproxen

glucuronidation capacity (e.g. UGT1A6 and UGT1A9) in cortex and medulla. We postulate

that the ubiquitous distribution of UGTs in mammalian kidney may buffer physiological

responses to endogenous mediators, but at the same time competitive xenobiotic-endobiotic

interactions may provide an explanation for the adverse renal effects of drugs including

NSAIDs.


at ASPE



ownloaded from


JPET#128603

4

Introduction

Enzymes of the UDP-glucuronosyltransferase (UGT) superfamily catalyze the covalent

linkage of glucuronic acid, derived from UDP-glucuronic acid (UDPGA), to typically

lipophilic substrates containing a carboxylic acid, hydroxyl or amine functional group

(Miners and Mackenzie, 1991). Thus glucuronidation is an important elimination mechanism

for numerous drugs, environmental chemicals and endogenous compounds (e.g. bilirubin,

fatty acids, eicosanoids and hydroxy-steroids) in humans (Miners and Mackenzie, 1991;

Tsoutsikos et al., 2004). UGTs have been classified into two families, UGT1 and UGT2, and

of the human UGT proteins identified to date, sixteen have the capacity to catalyze the

glucuronidation of endogenous compounds and/or xenobiotics; UGT 1A1, 1A3, 1A4, 1A5,

1A6, 1A7, 1A8, 1A9, 1A10, 2A1, 2B4, 2B7, 2B10, 2B15, 2B17, and 2B28 (Miners et al.,

2004). Individual UGTs exhibit distinct but overlapping substrate selectivities and differ in

terms of regulation of expression. Age, diet, disease states, induction and inhibition by

chemicals, ethnicity, genetic polymorphism and hormonal factors are known to influence

UGT activity (Miners and Mackenzie, 1991; Miners et al., 2004). Tissue-specific expression

is also a feature of UGTs and, while most UGTs are expressed in liver, only UGT 1A3, 1A6,

1A9, 2B4, 2B7, 2B10, 2B11, 2B15, and 2B17 have been identified in kidney (Tukey and

Strassburg, 2000).

The structural diversity of the human kidney underlies its multiple functions. These include

excretion of polar chemicals and metabolites, and the regulation of fluid, electrolytes, blood

pressure and hormone secretion. Subdivided anatomically into the cortex and medulla, the

functional unit of the kidney is the nephron, which consists of the renal corpuscle

(glomerulus and Bowman’s capsule), proximal convoluted tubule (PCT), loop of Henle, and

the distal convoluted tubule (DCT). Although incorrect in a strict anatomic sense the term

‘nephron’ commonly also includes the collecting duct (CD). This heterogeneity has made


at ASPE



ownloaded from


JPET#128603

5

defining the cellular localization of UGT in human kidney difficult and consequently studies

are limited. UGTs have been reported as occurring exclusively in epithelial cells of the PCT

(Murray and Burke 1995) but absent in the glomerulus, DCT, vasculature and collecting

tubules of the renal cortex (Peters et al., 1987).

In addition to endogenous synthetic and metabolic capability, the human kidney metabolizes

a variety of drugs including NSAIDs (Bowalgaha and Miners, 2001; McGurk et al., 1998;

Soars et al., 2001). Metabolism is the principal route of NSAID elimination in vivo and only

a small fraction of the administered dose is excreted unchanged in urine (Murray and Brater,

1993). Like other carboxylic acids, NSAIDs are metabolized largely as glucuronides, either of

the parent drug or oxidative metabolites. Of the UGTs identified in the kidney, UGT1A9,

UGT2B7 and, to a lesser extent, UGT1A3 are the predominant NSAID glucuronidating forms

(Sakaguchi et al., 2004; Kuehl et al., 2005). NSAID acyl-glucuronides are electrophilic and

bind covalently to plasma and tissue proteins both in vitro and in vivo (Spahn-Langguth and

Benet, 1992).

It is generally assumed that the metabolic clearance of drugs is determined principally, if not

exclusively, by hepatic metabolism and that the kidneys, with comparatively smaller organ

mass, contribute minimally to systemic clearance. Interestingly, the intrinsic clearances of the

glucuronidated substrates 4-methylumbelliferone and mycophenolic acid by the human

kidney are comparable to those of human liver (Tsoutsikos et al., 2004; Bowalgaha and

Miners, 2001; Olyaei et al., 2003). In addition, glucuronidation of the anesthetic propofol is

fourfold greater by human kidney than human liver (McGurk et al., 1998). However, there is

little information on the localization of UGTs in human renal cortex and medulla, and the

contribution of individual renal UGTs to drug (including NSAID) glucuronidation remains

poorly defined. This lack of knowledge precludes a complete understanding of NSAID


at ASPE



ownloaded from


JPET#128603

6

glucuronidation and the relationship between renal drug glucuronidation and target organ

toxicity.

Here we report the immunohistochemical localization of UGT1A proteins and UGT2B7 in

cortical and medullary sections of normal human kidney, and characterization of the kinetics

of S-naproxen glucuronidation by human kidney cortical and medullary microsomes to

ascertain the relative contribution of each to renal metabolism. S-Naproxen was chosen as the

probe substrate since; glucuronidation is the principal route of elimination in humans and is

estimated to be responsible for 60% of in vivo clearance (Upton et al., 1980, Vree et al.,

1993); it is a substrate for both UGT2B7 and UGT1A enzymes (Bowalgaha et al., 2005); it

forms an acyl glucuronide; and it has been implicated in the development of renal toxicity

(Whelton et al., 2003).


at ASPE



ownloaded from


JPET#128603

7

MATERIALS AND METHODS

Materials

Polyclonal anti-peptide antibodies, raised in rabbits, to human UGT1A proteins and

UGT2B7, and human UGT1A1 and UGT2B7 ‘standards’ were obtained from BD

Biosciences, (Sydney, Australia). The antibodies (20µL aliquots) were treated initially with

sodium azide (0.1% v/v) and stored at -70°C. Normal rabbit immunoglobulin (NRIgG) and

the EnVision+ peroxidase (goat anti-rabbit) detection system were purchased from DAKO

Corporation (Glostrup, Denmark), and S-naproxen and normal goat serum (NGS) from

Sigma-Aldrich (Sydney, Australia). All other chemicals and reagents were of the highest

analytical grade available.

Human kidney tissue collection and processing

Human kidney cortical and medullary tissue from ten male subjects (K2-K11) undergoing

radical nephrectomy for malignant disease was obtained from the joint Flinders Medical

Centre/Repatriation General Hospital Tissue Bank, Adelaide, South Australia. Approvals for

the collection, use of the kidney tissue and kidney donor details were provided by the

Research and Ethics Committee of the Repatriation General Hospital and the Flinders

Clinical Research Ethics Committee of Flinders Medical Centre. Renal tissue distant to the

primary tumor was collected immediately following surgery and sectioned into cortex and

medulla prior to either: (a) fixation overnight in 10% neutral phosphate buffered formalin

(1:10 dilution of 40% formaldehyde in aqueous 0.03M sodium dihydrogen orthophosphate

and 0.045M di-sodium hydrogen orthophosphate, pH 7.0), or (b) immediate use for sub-

cellular fractionation and isolation of microsomal protein, or (c) stored at -70°C for later use.

Representative formalin-fixed and paraffin-embedded tissue samples were stained with

haematoxylin and eosin and examined by a specialist histopathologist (AT). Typical for the


at ASPE



ownloaded from


JPET#128603

8

age of the donors (43-83 years) only age related benign nephrosclerosis and hypertensive-like

vascular changes were observed. All sections were confirmed as solely cortex or medulla.

Human kidney cortical microsomes (HKCM n=6, K6-K11) and human kidney medullary

microsomes (HKMM n=5, K6, K7, K9-K11) were prepared using a standard differential

ultracentrifugation technique as reported by Tsoutsikos et al., (2004). Microsomal protein

was resuspended in Na2HPO4 (pH 7.4)/20% (w/v) glycerol, analyzed for protein

concentration (Lowry et al., 1951), and stored at -70°C prior to use.

Immunohistochemistry studies

Tissue processing and epitope retrieval

Formalin-fixed kidney tissue (n=6, K2-K7) was embedded in paraffin following a standard

overnight tissue processing schedule (Leica TP 1050, Leica Microsystems, Germany). Serial,

parallel sections (4µm) were cut, mounted onto positively-charged Superfrost Plus

microscope slides (Menzel-Glaser, Germany), stored at 37°C and used within 30 days.

Epitope retrieval was performed on deparaffinized and rehydrated cortical and medullary

sections by boiling (4min) then simmering (10min) in sodium citrate buffer (0.01M, pH 6.0),

followed by cooling to room temperature (15min). The sections were rinsed in water and

placed in Tris-buffered saline (TBS; 0.1M Tris-HCl, pH 7.6 in isotonic NaCl) prior to

incubation with either of the primary UGT antibodies or with the negative control media.

Antibody adsorption

Aliquots (20µL) of either the UGT2B7 or UGT1A antibody were thawed, diluted in normal

goat serum (2% v/v in TBS containing sodium azide 0.1% v/v) and stored at 4°C. The

UGT1A and UGT2B7 primary antibodies (diluted 1:1500) were incubated with human

UGT1A1 protein (1µg and 10µg) and UGT2B7 protein (1µg, 10µg and 25µg), respectively.

Incubations (final volume 1.5mL) were carried out sequentially at three temperatures: 37ºC


at ASPE



ownloaded from


JPET#128603

9

(2h), room temperature (overnight), and 4ºC (2h), followed by centrifugation at 10,410 x g

(10min). The supernatant fraction was applied to human kidney cortical and medullary

sections in place of the primary antibody (see following section).

Immunostaining and quantification

Preliminary immunoblotting experiments confirmed binding of the UGT1A and UGT2B7

antibodies to HKCM and HKMM, and a lack of binding of NRIgG to UGT1A1 and

UGT2B7. In addition to the supplier’s specification of lack of cross reactivity between the

UGT2B7 antibody and UGT 1A subfamily enzymes and UGT2B15, we further established no

cross reactivity with UGT2B4 and UGT2B17 (data not shown). The UGT1A antibody

recognizes all UGT1A proteins, but not UGT 2B4, 2B7, 2B15 and 2B17 (supplier’s

specification and Uchaipichat et al., 2004).

Sections were incubated overnight with either negative control media (NGS or NRIgG diluted

1:2250 in NGS) or UGT1A or UGT2B7 primary antibodies (diluted 1:1500 in NGS).

Endogenous peroxidase activity was quenched using hydrogen peroxide (1% v/v, 10min).

Sections were incubated (1h) using the EnVision+ peroxidase detection system (goat anti-

rabbit) HRP-labeled antibody, followed by incubation with the substrate solution comprising

the chromogen 3,3’-diaminobenzidine tetra hydrochloride (DAB) and hydrogen peroxide

(0.1% v/v, 5min). The color reaction was terminated by rinsing with water. Sections were

counterstained in Harris’ haematoxylin, briefly differentiated in acidified alcohol (0.5% HCl

in 70% (v/v) ethanol) and ‘blued’ in a saturated solution of lithium carbonate. Sections were

dehydrated in absolute ethanol, cleared in xylol, mounted using DePeX mounting medium,

and dried overnight (60°C).

Labeled UGT enzymes were visualized by the presence of an insoluble reddish-brown

precipitate. The intensity of staining, which was readily observed by light microscopy, was

scored by two investigators (AT and PG) according to a semi-quantitative method (Okonogi


at ASPE



ownloaded from


JPET#128603

10

et al., 2001; Saarikoski et al., 2005): (−) absence of positive staining, (1+) weak, (2+)

moderate, (3+) intense staining.

Image capture

Sections were photographed using the Video Pro 32 image analysis system (Leading Edge

P/L, Adelaide, Australia) comprising an Olympus BX40 microscope with a 2.5x camera

eyepiece and a continuous interference filter monochromator for the enhancement of contrast

between haematoxylin (blue nuclear counter-stain) and DAB. Images were captured with a

color digital CCD video camera (Panasonic model GP-KR222) with the automatic gain

disabled. Correction of non-uniform illumination was standardized using a bright field image

of the background illumination. Images were photographed using either a 10x or 20x

objective.

Kinetics of S-naproxen acyl glucuronidation

S-Naproxen acyl glucuronide formation by HKCM (n=6) and HKMM (n=5) was measured

using a modification of the method of Bowalgaha et al. (2005). Preliminary studies

established linearity of product formation with respect to incubation time and microsomal

protein concentration, and less than 10% consumption of substrate occurred during the

course of incubations. The incubation mixture (0.2mL for HKCM or 0.1mL for HKMM)

contained MgCl2 (4mM), UDPGA (5mM), S-naproxen (5-2000µM), and HKCM (100µg) or

HKMM (50µg) in phosphate buffer (0.1M, pH 7.4). Incubations, which were performed in

duplicate, were conducted for 30min (HKCM) or 60min (HKMM) at 37°C. Reactions were

terminated by the addition of an equal volume of glacial acetic acid in methanol (4% v/v) and

cooling on ice. Following centrifugation (5,000 x g, 10min, 10°C), an aliquot of the

supernatant fraction (30µL) was injected onto the HPLC column. The HPLC system

comprised an Agilent 1100 series instrument with a solvent delivery system, auto injector and


at ASPE



ownloaded from


JPET#128603

11

variable wavelength UV-VIS detector (Agilent Technologies, Sydney, Australia) fitted with a

Novapak C18 analytical column (4µm particle size, 3.9mm x 150mm; Waters, Sydney,

Australia). The analytes were detected by UV absorption at 230nm. The mobile phase

comprised water: acetonitrile: glacial acetic acid (700:300:1.2 v/v), delivered at a flow rate of

1.5mL/min. S-Naproxen acyl glucuronide and S-naproxen eluted at 2.4min and 11.4min,

respectively. The S-naproxen acyl glucuronide peak was confirmed by reference to an

authentic standard and by hydrolysis with β-glucuronidase. However, standard curves were

prepared using S-naproxen, 0.25-5µM, since S-naproxen acyl glucuronide is hygroscopic and

unsuitable for use as the prime standard (Bowalgaha et al., 2005). Thus Vmax (and CLint)

values should be considered ‘apparent’. S-Naproxen standard curves were linear over the

range 0.25-5µM (r2 > 0.99). The lower limit of quantification of the assay was 0.02µM, and

the overall assay inter-day coefficient of variation was 1.9% (n=7). Incubation samples were

analyzed within 12 hr; serial injection of incubation samples (containing high, medium and

low substrate concentrations) treated with glacial acetic acid in methanol (4% v/v)

demonstrated that the S-naproxen acyl glucuronide product was stable (< 5% decrease in

peak height) to at least 12 hr.

Fluconazole has previously been demonstrated to be a selective inhibitor of UGT2B7

(Uchaipichat et al., 2006). Using the incubation and chromatography conditions described

above, inhibition of the high- and low- affinity components of HKCM- and HKMM-

catalyzed S-naproxen acyl glucuronidation (see Results) by fluconazole (2.5mM) was

investigated to assess the contribution of UGT2B7 to these reactions. Given the limited

availability of renal tissue, inhibition experiments were performed with microsomes from

three kidneys only.

Data analysis


at ASPE



ownloaded from


JPET#128603

12

Data points represent the mean of duplicate determinations, except where indicated. The

kinetic parameters Km and Vmax were derived from fitting untransformed data to the single-

and two- enzyme Michaelis-Menten (MM) equations using an extended least squares

modeling program (EnzFitter version 2.0.18.0, Biosoft, Cambridge, UK). The goodness of fit

was determined by comparison of statistical parameters (95% confidence intervals for the

curve fit and F-statistic). Kinetic data are reported as the mean ± SD. In vitro intrinsic

clearance (CLint) was calculated as Vmax/ Km.


at ASPE



ownloaded from


JPET#128603

13

RESULTS

Antibody adsorption

Non-specific binding of the primary UGT antibodies to non-UGT epitopes was excluded by

antibody preadsorption. Compared to the control (no UGT1A1 protein added, Figure 1A),

UGT1A antibody labeling was reduced by preadsorption with of 1µg of UGT1A1 (Figure 1B)

and prevented by 10µg of UGT1A1 protein (Figure 1C). Similarly, compared to the control

(Figure 1D, no UGT2B7 protein added) UGT2B7 immunoreactivity was decreased by

preadsorption with 10µg of UGT2B7 microsomal protein (Figure 1E) and was completely

prevented by 25µg UGT2B7 (Figure 1F).

Negative controls

Two negative control sections employing either normal goat serum or a commercial

preparation of pre-immune serum of NRIgG in place of the primary antibody were included

routinely. When using normal goat serum, there was no evidence of positive staining in the

cortical (Figure 2A) or medullary sections (Figure 2E). However, incubation with NRIgG

resulted in a slight pink ‘blush’ indicative of non-specific protein binding homogeneously

distributed in the interstitium, glomeruli and tubules of the renal cortex (Figures 2B), and

interstitium, loops and collecting ducts of the renal medulla (Figure 2F). Subsequent grading

of UGT1A and UGT2B7 immunoreactivity was determined relative to the non-specific

background staining observed using NRIgG.

Immunolocalization of UGT1A proteins and UGT2B7 in the renal cortex

There was no evidence of UGT1A and UGT2B7 immunoreactivity in the glomeruli and

Bowman’s capsule and the associated cortical vasculature including glomerular capillaries,

arteries and afferent/efferent arterioles and veins (Figures 2C-2D). In contrast, the epithelial

cells of the PCT exhibited strong localized ‘pockets’ of intense staining (3+, n=6) with the


at ASPE



ownloaded from


JPET#128603

14

UGT1A antibody (Figure 2C) while UGT2B7 immunoreactivity was highly variable ranging

from weak to moderate staining (1+ to 2+, n=6) (Figure 2D and Table 1). Similar variability

in staining was observed in the DCT for UGT1A and UGT2B7 (Table 1 and Figures 2C and

2D). Although the pattern of immunoreactivity for UGT1A and UGT2B7 in the DCT was

similar, the intensity of staining for UGT1A proteins and UGT2B7 was generally more

pronounced on the apical or luminal side of the cytoplasmic membrane.

The macula densa, identified as columnar cells adjacent to the glomerulus, exhibited

equivalent staining to that of the associated DCT, with weak immunoreactivity (1+) for

UGT1A proteins (Figure 3A) and moderate staining (2+) for UGT2B7 (Figure 3B). UGT

immunoreactivity was not detected in the interstitial cells of the juxta-glomerular apparatus.

There was positive staining of two of the identifiable components comprising medullary rays;

the thick ascending loop of Henle (TALH) and the cortical collecting ducts. The third

component, the proximal straight tubule, was difficult to distinguish from PCT and none

were identified with certainty. Variable immunoreactivity was observed across the TALH and

CD with both the UGT1A and UGT2B7 antibodies (Table 1).

Immunolocalization of UGT1A proteins and UGT2B7 in the renal medulla

There was absence of positive immunoreactivity for UGT1A and UGT2B7 in medullary veins

and arteries. The collecting ducts (CD) were grouped as either small or large CD. UGT1A

and UGT2B7 immunoreactivity in the small CDs ranged from weak (1+) to moderate (2+) in

five of the six kidneys studied (Figure 2G) with the exception of K7, which exhibited intense

(3+) staining with the UGT1A antibody. Intensity of staining was similar in the large CDs for

UGT1A proteins and UGT2B7 ranging from weak (1+) to moderate (2+) (Figure 2G and 2H),

respectively. Variable immunoreactivity (1+ to 2+) was observed in epithelial cells of the thin

loop of Henle (LOH) and the TALH for both UGT1A and UGT2B7 (Figures 2G and 2H). An

exception was K4 where the TALH stained intensely (3+) for UGT1A proteins. Interestingly,


at ASPE



ownloaded from


JPET#128603

15

intense staining was observed for UGT1A proteins on the luminal side of the TALH in a

proportion of the UGT positive cells.

Within epithelial cells of the PCT, DCT and TALH continuous granular staining was

observed in the nuclear envelope, and focal granular staining within nuclei for UGT1A

proteins. A similar pattern of immunoreactivity was observed for UGT2B7. Positive staining

for UGT1A proteins and UGT2B7 was not observed in the nuclear envelope and nuclei of

epithelial cells of the CDs and thin LOH.

Kinetics of S-naproxen acyl glucuronidation by HKCM and HKMM

The glucuronidation kinetics of S-naproxen were investigated using HKCM and HKMM

from six kidneys over the substrate concentration range 5 to 2000µM. S-Naproxen acyl

glucuronide formation by HKCM and HKMM exhibited ‘biphasic’ kinetics (Figures 4A-4B).

Data were well fitted to the two-enzyme Michaelis-Menten equation. Derived kinetic

constants (mean ± SD) for the high- and low-affinity components using HKCM as the

enzyme source were: Km1 34±14µM, Vmax1 134±98pmol/min.mg protein, Km2 627±541µM,

and Vmax2 133±71pmol/min.mg protein. Intrinsic clearances (CLint), calculated as Vmax/Km,

for the high- and low-affinity components of S-naproxen acyl glucuronidation by HKCM

were 5.1± 4.2µL/min.mg and 0.73±0.73µL/min.mg respectively. Derived kinetic constants

for the high- and low-affinity components of S-naproxen acyl glucuronidation by HKMM

were Km1 45±14µM, Vmax1 48±19 pmol/min.mg protein, Km2 654±488µM and Vmax2 45±7

pmol/min.mg protein. CLint for the respective high- and low- affinity components were

1.2±0.6µL/min.mg and 0.09±0.03µL/min.mg. At S-naproxen concentrations of 10µM and

1000µM, fluconazole inhibited cortical S-naproxen acyl glucuronidation by 73% and 8%,

respectively, and medullary S-naproxen acyl glucuronidation by 68% and 12%, respectively.


at ASPE



ownloaded from


JPET#128603

16

Discussion

This study is the first to report the cellular localization and semi-quantitative expression of

UGT1A proteins and UGT2B7 in cortical and medullary segments of the human nephron.

Moreover, we provide the first evidence of UGT1A and UGT2B7 expression in the macula

densa, loops of Henle and collecting ducts. Exclusion of antibody cross reactivity, inclusion

of negative controls using NRIgG and normal goat serum, and attenuation of labeling by

antibody preadsorption supports the conclusion that the renal tissue antigens detected were

UGTs. Although a comprehensive understanding of the role of UGTs in the kidney is lacking,

their presence throughout the nephron suggests a role for UGT enzymes in renal metabolic

elimination processes.

Comparatively little variability with respect to UGT expression was observed in tissue from

the six kidneys. Similar to other studies (Girard et al., 2003; Peters et al., 1987) UGT1A and

UGT2B7 immunoreactivity was not demonstrated in Bowman’s capsule or the glomerulus.

Moderate to intense staining was evident for UGT2B7 and UGT1A proteins in epithelial cells

of the PCT. Previous studies in animals have reported that UGT activity is primarily localized

to the cortical region (Rush and Hook, 1984) and that rates of glucuronidation are highest in

cells of the PCT (Lohr et al., 1998; Schaaf et al., 2001). It is not surprising that UGTs are

expressed in the PCT in humans, which is located adjacent to the glomerulus, and is the

initial site of exposure to the glomerular ultrafiltrate. The capacity of the PCT for active

transport of organic compounds further increases the propensity for exposure to high

concentrations of potentially nephrotoxic xenobiotics (Schaaf et al., 2001). In addition,

localization in epithelial cells of the PCT is consistent with a role of UGTs in limiting the

biological activity of mineralocorticoids and glucocorticoids (Girard et al., 2003).

In contrast to the study of Peters et al., (1987), who reported absence of UGT in the DCT, our

study has shown that UGT1A proteins and UGT2B7 are present in the epithelial cells of the


at ASPE



ownloaded from


JPET#128603

17

DCT and, importantly, in the macula densa. Expression in the macula densa has not been

reported previously and is consistent with the hypothesis that glucuronidation terminates the

biological reactivity of endogenous molecules involved in regulating renal hemodynamics

(Knights et al., 2005). The macula densa has a role in regulating intravascular renal tone via

the renin-angiotensin-aldosterone pathway. Available evidence indicates that UGT2B7 is the

predominant enzyme responsible for aldosterone glucuronidation (Girard et al., 2003) and

hence UGT2B7 in the macula densa may play a role in regulating aldosterone. It is

noteworthy that the majority of aldosterone 18β-glucuronide excreted in urine is thought to

be formed in extra-hepatic tissues, particularly the kidney (Bledsoe et al., 1966).

Two components of the medullary ray were identified; the thick ascending loop of Henle and

the cortical collecting ducts. In both of these segments of the nephron UGT1A and UGT2B7

expression was variable with absence of immunoreactivity for UGT2B7 in one kidney and

weak to moderate staining with both antibodies in the other five kidneys. UGT protein

expression in collecting ducts has been previously reported as absent in human renal tissue

(Peters et al., 1987) and ‘weak’ in cortical collecting ducts of monkey kidney (Girard et al.,

2003). In the present study UGT1A proteins and UGT2B7 were identified in the medullary

collecting ducts and loops of Henle. The former is not surprizing given the role of aldosterone

in the DCT and collecting ducts and evidence of aldosterone glucuronidation by UGT2B7

(Girard et al., 2003).

Evidence of expression of UGT1A proteins and UGT2B7 in the loops of Henle is noteworthy.

It is conceivable that intrarenal glucuronidation terminates the biological activity of

eicosanoids, including prostaglandins, synthesized locally in the loops of Henle. This view is

supported by evidence that UGT2B7 and UGT1A enzymes glucuronidate a variety of

eicosanoids including prostaglandins B1 and E2 (Little et al., 2004). PGE2 is the major

prostaglandin produced along all segments of the nephron (Bonvalet et al., 1987).


at ASPE



ownloaded from


JPET#128603

18

Additional observations from our study include expression of UGT1A proteins and UGT2B7

in cell nuclei and the nuclear envelope of epithelial cells within PCT, DCT and TALH. These

data support previous reports of UGT expression in the nuclei of human prostate epithelial

cells (Barbier et al., 2000), the nuclear envelope of rat liver hepatocytes and epithelial cells of

the jejunum, renal PCT and adrenal glands (Chowdhury et al., 1985; Elmamlouk et al., 1981)

and nuclear membranes of human hepatocytes (Radominska-Pandya et al., 2002). Nuclear

expression of UGT1A and UGT2B7 in epithelial cells of the renal tubules may reflect an

evolutionary mechanism to control nuclear receptor ligand interactions (Radominska-Pandya

et al., 2002).

A substantial body of research has provided evidence for renal xenobiotic metabolism. Using

HKCM and HKMM, formation of S-naproxen acyl glucuronide exhibited biphasic kinetics

characteristic of the involvement of high- and low-affinity enzymes in this pathway. The

mean Km value for the high-affinity component determined from fitting to the two-enzyme

MM equation was 18- and 15-fold lower than the Km values for the low-affinity reaction in

cortex and medulla, respectively. Similarly, the microsomal intrinsic clearances of the high-

and low-affinity components of the cortex and medulla differed 7- and 12-fold, respectively.

Assuming an unbound fraction in plasma of 0.018 (Vree et al., 1993) and a maximum

unbound plasma S-naproxen concentration of 5-10µM (Vree et al., 1993, Runkel et al.,

1976), substitution of the mean kinetic data in the two enzyme MM equation indicates that

the low-affinity cortical and medullary UGT(s) would be expected to contribute <25% and

<5%, respectively to naproxen renal glucuronidation in humans taking therapeutic doses

(250-1000mg daily).

The range of Km values for the high affinity component of S-naproxen acyl glucuronidation

by both cortical (20-63µM) and medullary (26-62µM) microsomes is similar to that reported


at ASPE



ownloaded from


JPET#128603

19

for the high affinity component of human liver microsomal S-naproxen glucuronidation (14-

49µM) and recombinant UGT2B7 (72µM) (Bowalgaha et al., 2005). In this study

involvement of UGT2B7 was confirmed by use of the selective UGT2B7 inhibitor

fluconazole (Uchaipichat et al., 2006). Fluconazole inhibited the high-affinity component of

S-naproxen glucuronidation in cortex and medulla by 73% and 68%, respectively, similar to

the 70% inhibition of S-naproxen glucuronidation observed with human liver microsomes

(Bowalgaha et al., 2005). Collectively, the results indicate that in both the cortex and medulla

UGT2B7 is the high-affinity enzyme involved in renal S-naproxen glucuronidation in

humans. As the low-affinity component was relatively unaffected by fluconazole (<15%

inhibition), it is likely that the low-affinity component comprises several enzymes with

naproxen glucuronidation capacity. UGT1A6 and UGT1A9 have been identified in human

kidney (Sutherland et al., 1993) and both have Km or S50 values (855-1036µM) of similar

order to the low-affinity component (~650µM) of human renal microsomal S-naproxen

glucuronidation. Consistent with other reports describing the renal medulla as having lower

UGT activity than cortex (Rush and Hook, 1984; Hjelle et al., 1986; Yue et al., 1988) in this

study S-naproxen glucuronidation activity differed 2.7-fold between cortex and medulla.

The kidney receives 20-25% of resting cardiac output (Saker, 2000) and perfusion of

different regions of the kidney is estimated at 90% to the cortex, 6-10% to the medulla, and

1-2% to the papillae (Goldstein and Schnellmann, 1996). In ‘normal’ individuals renal

perfusion is largely prostaglandin independent. However, in circumstances of ‘prostaglandin-

dependent’ renal perfusion (e.g. congestive heart failure) NSAIDs can cause acute

deterioration of renal function and in some instances renal ischemia. It may be speculated

that renal glucuronidation normally plays an important ‘local’ role in modulating intrarenal

suppression of cyclooxygenase activity by NSAIDS, including naproxen, thereby buffering


at ASPE



ownloaded from


JPET#128603

20

perturbation of renal hemodynamics. However, in circumstances where cortical and

medullary perfusion is compromised intrarenal concentrations of NSAIDs and/or NSAID acyl

glucuronides may become elevated increasing the potential for acylation of renal proteins by

NSAID acyl glucuronides. Accumulation of reactive NSAID acyl glucuronides coupled with

ischemia of the medullary papillae is a possible precipitating factor in the development of

NSAID-induced renal papillary necrosis (Whelton et al., 2003). Hence, depending on the

pathophysiological circumstances, intrarenal glucuronidation of NSAIDs may be either

beneficial or deleterious.

In summary, we report that with the exception of the glomerulus, Bowman’s capsule and

renal vasculature, UGT1A proteins and UGT2B7 are expressed throughout cortical and

medullary segments of the human nephron. This pattern of expression is particularly

interesting in view of the widespread synthesis of prostaglandins along the nephron and

evidence of their metabolism by UGTs. Furthermore, it is noteworthy that UGT1A and

UGT2B7 were localized in the macula densa consistent with an important role for UGT in the

regulation of the mineralocorticoid aldosterone. Consistent with the immunohistochemistry

data, S-naproxen was glucuronidated by both HKCM and HKMM. Of the UGTs present in

human kidney, UGT2B7 appears to be the principal enzyme responsible for S-naproxen acyl

glucuronide formation in both renal cortex and medulla. Finally, we suggest that the

ubiquitous distribution of UGTs in mammalian kidneys may buffer physiological responses

to multiple endogenous mediators but at the same time specific competitive intrarenal

xenobiotic-endobiotic interactions may provide an explanation for the adverse renal effects of

some drugs including NSAIDs.


at ASPE



ownloaded from


JPET#128603

21

Acknowledgments

The authors are grateful to Dr Alan Stapleton and Ms Virginia Papangelis for coordinating

renal tissue collection, and Mr David Elliot and Ms Kushari Bowalgaha for their assistance

with the HPLC assay for S-naproxen acyl glucuronidation.


at ASPE



ownloaded from


JPET#128603

22

References

Barbier O, Lapointe H, El Alfy M, Hum DW and Belanger A (2000) Cellular localization of

uridine diphosphoglucuronosyltransferase 2B enzymes in the human prostate by in situ

hybridization and immunohistochemistry. J Clin Endocrinol Metab 85:4819-4826.

Bledsoe T, Liddle GW, Riondel A, Island DP, Bloomfield D and Sinclair-Smith B (1966)

Comparative fates of intravenously and orally administered aldosterone: evidence for

extrahepatic formation of acid-hydrolyzable conjugate in man. J Clin Invest 45:264-269.

Bonvalet JP, Pradelles P and Farman N (1987) Segmental synthesis and actions of

prostaglandins along the nephron. Am J Physiol 253:F377-F38.

Bowalgaha K, Elliot DJ, Mackenzie PI, Knights KM, Swedmark S and Miners JO (2005) S-

Naproxen and desmethylnaproxen glucuronidation by human liver microsomes and

recombinant human UDP-glucuronosyltransferases (UGT): role of UGT2B7 in the

elimination of naproxen. Br J Clin Pharmacol 60:423-433.

Bowalgaha K and Miners JO (2001) The glucuronidation of mycophenolic acid by human

liver, kidney and jejunum microsomes. Br J Clin Pharmacol 52:605-609.

Chowdhury JR, Novikoff PM, Chowdhury NR and Novikoff AB (1985) Distribution of

UDPglucuronosyltransferase in rat tissue. P Natl Acad Sci USA 82:2990-2994.

Elmamlouk TH, Mukhtar H and Bend JR (1981) The nuclear envelope as a site of

glucuronyltransferase in rat liver: properties of and effect of inducers on enzyme activity. J

Pharmacol Exp Ther 219:27-34.

Girard C, Barbier O, Veilleux G, El-Alfy M and Belanger A (2003) Human uridine

diphosphate-glucuronosyltransferase UGT2B7 conjugates mineralocorticoid and

glucocorticoid metabolites. Endocrinology 144:2659-2668.


at ASPE



ownloaded from


JPET#128603

23

Goldstein RS and Schnellmann RG (1996) Toxic responses of the kidney, in Casarett and

Doull’s Toxicology: the basic science of poisons (Klaassen CD ed) pp 471-442, McGraw-

Hill, USA.

Hjelle JT, Hazelton GA, Klaassen CD and Hjelle JJ (1986) Glucuronidation and sulfation in

rabbit kidney. J Pharmacol Exp Ther 236:150-156.

Knights KM, Tsoutsikos P and Miners JO (2005) Novel mechanisms of nonsteroidal anti-

inflammatory drug-induced renal toxicity. Expert Opin Drug Metab Toxicol 1:399-408.

Kuehl GE, Lampe JW, Potter JD and Bigler J (2005) Glucuronidation of nonsteroidal anti-

inflammatory drugs: identifying the enzymes responsible in human liver microsomes. Drug

Metab Dispos 33:1027-1035.

Little JM, Kurkela M, Sonka J, Jantti S, Ketola R, Bratton S, Finel M and Radominska-

Pandya A (2004) Glucuronidation of oxidized fatty acids and prostaglandins B1 and E2 by

human hepatic and recombinant UDP-glucuronosyltransferases. J Lipid Res 45:1694-1703.

Lohr JW, Wilsky GR and Acara MA (1998) Renal drug metabolism. Pharmacol Rev 50:107-

141.

Lowry OH, Rosebrough NJ, Farr AL and Randall RJ (1951) Protein measurement with the

Folin phenol reagent. J Biol Chem 193:265–275.

McGurk KA, Brierley CH and Burchell B (1998) Drug glucuronidation by human renal

UDP-glucuronosyltransferases. Biochem Pharmacol 55:1005-1012.

Miners JO and Mackenzie PI (1991) Drug glucuronidation in humans. Pharmac Ther

51:347-369.

Miners JO, Smith PA, Sorich MJ, McKinnon RA, Mackenzie PI (2004) Predicting human

drug glucuronidation parameters: Application if in vitro and in silico modeling approaches.

Annu Rev Pharmacol Toxicol 44:1-25.


at ASPE



ownloaded from


JPET#128603

24

Murray GI and Burke MD (1995) Immunohistochemistry of drug-metabolizing enzymes.

Biochem Pharmacol 50:895-903.

Murray MD and Brater DC (1993) Renal toxicity of the nonsteroidal anti-inflammatory

drugs. Annu Rev Pharmacol Toxicol 32:435-465.

Okonogi H, Nishimura M, Utsunomiya Y, Hamaguchi K, Tsuchida H, Miura Y, Suzuki S,

Kawamura T, Hosoya T and Yamada K (2001) Urinary type IV collagen excretion reflects

renal morphological alterations and type IV collagen expression in patients with type 2

diabetes mellitus. Clin Nephrol 55:357-364.

Olyaei AJ, Demattos AM and Bennett WM (2003) Drug dosage in renal failure, in Clinical

Nephrotoxins, 2nd Ed (De Broe ME, Porter GA, Bennett WM and Verpooten GA eds) pp

667-679, Kluwer Academic Publishers, Dordrecht.

Peters WH, Allebes WA, Jansen PL, Poels LG and Capel PJ (1987) Characterization and

tissue specificity of a monoclonal antibody against human uridine 5’-diphosphate-

glucuronosyltransferase. Gastroenterology 93:162-169.

Radominska-Pandya A, Pokrovskaya ID, Xu J, Little JM, Jude AR, Kurten RC and Czernik

PJ (2002) Nuclear UDP-glucuronosyltransferases: identification of UGT2B7 and UGT1A6

in human liver nuclear membranes. Arch Biochem Biophys 399:37-48.

Runkel R, Chaplin MD, Sevelius H, Ortega E and Segre E (1976) Pharmacokinetics of

naproxen overdoses. Clin Pharmacol Ther 20:269-277

Rush GF and Hook JB (1984) Characteristics of renal UDP-glucuronyltransferase.

Life Sci 35:145-153.

Saarikoski ST, Wikman HA, Smith G, Wolff CH and Husgafvel-Pursiainen K (2005)

Localization of cytochrome P450 CYP2S1 expression in human tissues by in situ

hybridization and immunohistochemistry. J Histochem Cytochem 53:549-556.


at ASPE



ownloaded from


JPET#128603

25

Sakaguchi K, Green M, Stock N, Reger TS, Zunic J and King C (2004) Glucuronidation of

carboxylic acid containing compounds by UDP-glucuronosyltransferase isoforms. Arch

Biochem Biophys 424:219-225.

Saker BM (2000) Everyday drug therapies affecting the kidneys. Aust Presc 23:17-19.

Schaaf GJ, de Groene EM, Maas RF, Commandeur JN and Fink-Gremmels J (2001)

Characterization of biotransformation enzyme activities in primary rat proximal tubular

cells. Chem-Biol Interact 134:167-190.

Soars MG, Riley RJ, Findlay KA, Coffey MJ and Burchell B (2001) Evidence for significant

differences in microsomal drug glucuronidation by canine and human liver and kidney.

Drug Metab Dispos 29:121-126.

Spahn-Langguth H and Benet LZ (1992) Acyl glucuronides revisited: is the glucuronidation

process a toxification as well as a detoxification mechanism? Drug Metab Rev 24:5-48.

Sutherland L, Ebner T and Burchell B (1993) The expression of UDP-

glucuronosyltransferases of the UGT1 family in human liver and kidney and in response to

drugs. Biochem Pharmacol 45:295-301.

Tsoutsikos P, Miners JO, Stapleton A, Thomas A, Sallustio BC and Knights KM (2004)

Evidence that unsaturated fatty acids are potent inhibitors of renal UDP-

glucuronosyltransferases (UGT): kinetic studies using human kidney cortical microsomes

and recombinant UGT1A9 and UGT2B7. Biochem Pharmacol 67:191-199.

Tukey RH and Strassburg CP (2000) Human UDP-glucuronosyltransferases: metabolism,

expression, and disease. Annu Rev Pharmacol Toxicol 40:581-616.

Uchaipichat V, Mackenzie PI, Guo X-H, Gardner-Stephen D, Galetin A, Houston JB and

Miners JO (2004) Human UDP-glucuronosyltransferases: Isoform selectivity and kinetics


at ASPE



ownloaded from


JPET#128603

26

of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and

inhibition by diclofenac and probenecid. Drug Metab Dispos 32:413-423.

Uchaipichat V, Winner LK, Mackenzie PI, Elliot DJ, Williams JA and Miners JO (2006)

Quantitative prediction of in vivo inhibitory interactions involving glucuronidated drugs

from in vitro data: the effect of fluconazole on zidovudine glucuronidation. Br J Clin

Pharmacol 61:427-439.

Upton RA, Buskin JN, Williams RL, Holford NH and Riegelman S (1980) Negligible

excretion of unchanged ketoprofen, naproxen, and probenecid in urine.

J Pharm Sci 69:1254-7.

Vree TB, van den Biggelaar-Martea M, Verwey-van Wissen CP, Vree JB and Guelen PJ

(1993) Pharmacokinetics of naproxen, its metabolite O-desmethylnaproxen, and their acyl

glucuronides in humans. Biopharm Drug Dispos 14:491-502.

Whelton A, Sturmer T and Porter GA (2003) Non-steroidal anti-inflammatory drugs, in

Clinical Nephrotoxins, 2nd Ed (De Broe ME, Porter GA, Bennett WM and Verpooten GA

eds) pp 279-306, Kluwer Academic Publishers, Dordrecht.

Yue Q, Odar-Cederlof I, Svensson JO and Sawe J (1988) Glucuronidation of morphine in

human kidney microsomes. Pharmacol Toxicol 63:337-341.


at ASPE



ownloaded from


JPET#128603

27

Footnotes

a) This research was supported by grants-in aid from the National Health & Medical

Research Council of Australia and the Faculty of Health Sciences, Flinders

University.

b) Associate Professor Kathleen Knights, Department of Clinical Pharmacology, Flinders

Medical Centre, Flinders Drive, Bedford Park, South Australia, Australia 5042.

Email: [email protected]


at ASPE



ownloaded from


JPET#128603

28

Legends for figures

Figure 1

Adsorption of rabbit anti-UGT antibodies by UGT1A1 and UGT2B7

Human kidney cortical sections were incubated with either: (A) UGT1A antibody (1:1500, no

preadsorption) or UGT1A antibody preadsorbed with (B) 1µg or (C) 10µg human UGT1A1

microsomal protein or (D) UGT2B7 antibody (1:1500, no preadsorption) or UGT2B7

antibody preadsorbed with (E) 10µg or (F) 25µg human UGT2B7 microsomal protein.

Magnification, 10x objective with 2.5x camera eyepiece. Bar = 100µm.

Figure 2

Immunohistochemistry of human kidney cortex and medulla

Human kidney cortical sections incubated with either: (A) NGS, (B) NRIgG (1:2250), (C)

UGT1A antibody (1:1500), or (D) UGT2B7 antibody (1:1500).

g: glomerulus; d: distal convoluted tubule; p: proximal convoluted tubule

Human kidney medullary sections incubated with either: (E) NGS, (F) NRIgG (1:2250), (G)

UGT1A antibody or (H) UGT2B7 antibody.

c: collecting duct; LOH: loop of Henle; t: Thick ascending limb.

Magnification, 20x objective with 2.5x camera. Bar = 50µm.

Figure 3

Immunohistochemistry of macula densa

Human kidney cortical sections were incubated with either: (A) UGT1A antibody (1:1500),

or (B) UGT2B7 antibody (1:1500).

Arrows indicate positive staining of columnar epithelial cells of the macula densa.

Magnification, 20x objective with 2.5x camera eyepiece. Bar = 20µm.


at ASPE



ownloaded from


JPET#128603

29

Figure 4

Representative Eadie-Hofstee plots (K9) of S-naproxen acyl-glucuronide formation by HKCM (A)

and HKMM (B). Data points represent the mean of experimentally determined values and lines

show the computer generated curves of best fit.


at ASPE



ownloaded from


JPET#128603

30

Table 1 Staining intensity for UGT1A proteins and UGT2B7 in Human Kidney Cortex and

Medulla

Human Kidney Staining Intensitya

UGT1A UGT2B7

Cortex

Vasculatureb − −

Bowman’s capsule/Glomeruli − −

Proximal Convoluted Tubule 3+ − to 2+

Distal Convoluted Tubule 1+ to 2+ 1+ to 2+

Macula Densa 1+ to 2+ − to 2+

Medullary Ray - Thick ascending limb loop of Henle 1+ to 2+c − to 2+ c

Medullary Ray - Collecting duct 1+ to 2+ − to 2+

Medullary Ray - Proximal straight tubule n.i. n.i.

Medulla

Vasculatureb − −

Collecting Ducts - Small 1+ to 3+ 1+ to 3+

Collecting Ducts - Large 1+ to 2+ 1+ to 2+

Loop of Henle - Thin limb 1+ to 2+ 1+ to 2+ c

Loop of Henle - Thick ascending limb 1+ to 3+ 1+ to 2+ c


at ASPE



ownloaded from


JPET#128603

31

a Staining intensity graded relative to NRIgG control (minimum to maximum observed, n =

6): (−) absence of positive staining; (1+) weak; (2+) moderate; and (3+) intense staining.

b Vasculature: veins, arteries, arterioles

c n=5 kidneys.

n.i. None identified


at ASPE



ownloaded from



at ASPE



ownloaded from


jpet#128603 human renal cortical and medullary...

Documents