6 nephrectomized rats

original article

http://www.kidney-international.org & 2006 International Society of Nephrology

see commentary on page 1889

Gender differences in renal nuclear receptors and aryl hydrocarbon receptor in 5/6 nephrectomized rats H Lu1, X Lei1 and C Klaassen1 1

Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, USA

This study was aimed at delineating molecular pathways essential in gender-different pathogenesis of chronic kidney diseases (CKD). Renal transcripts of nuclear receptors and metabolic enzymes in male and female kidneys from 5/6 nephrectomized (Nx) rats 7 weeks post-Nx were examined using branched DNA signal amplification assay. Nx-males had marked kidney injury coupled with anemia and malnutrition. Nx-females had moderate renal injury, and were free of albuminuria, anemia, and malnutrition. Nx-males had systemic and renal inflammation, which were largely absent in Nx-females. Blood 17b-estradiol, testosterone, and corticosterone did not change, whereas urinary testosterone decreased in both genders. Compared to males, female kidneys had higher androgen receptor (AR) and aryl hydrocarbon receptor (AhR) but lower estrogen receptor a (ERa). Compared to Nx-males, female remnant kidneys had less decreases in ERa and peroxisome proliferator-activated receptor a (PPARa), had no induction of AR and decrease of acyl-CoA oxidase, whereas had induction of cytochrome P450 4a1 (Cyp4a1) but decrease of AhR. Renal protein expression of a 52-kDa isoform of Wilm’s tumor 1 (WT1), transcription factor critical in nephrogenesis, decreased dramatically in Nx-males but largely preserved in Nx-females. In conclusion, gender divergences in basal expression and alteration of ERa, AR, AhR, WT1, and PPARa/Cyp4a1 during CKD may explain gender differences in CKD progression and outcome of renal transplantation. Kidney International (2006) 70, 1920–1928. doi:10.1038/sj.ki.5001880; published online 20 September 2006 KEYWORDS: chronic kidney disease; gender; nuclear receptor; steroid hormone

Correspondence: C Klaassen, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, Kansas 66160-7417, USA. E-mail: [email protected] Received 30 December 2005; revised 14 August 2006; accepted 15 August 2006; published online 20 September 2006 1920

Chronic kidney disease (CKD) is a major social and economic burden worldwide.1 Currently, there is no curative pharmacological therapy; the most effective drugs for CKD, angiotensin-converting enzyme inhibitors, decrease CKD progression by less than 50%.2 Many studies have disclosed various pathophysiological factors affecting CKD, such as sex hormones, glucocorticoids, and inflammation. Nuclear receptors are attractive therapeutic targets due to their powerful physiological functions and accessibility by small molecules. It is well known that the expression of a given nuclear receptor is critical in determining its physiological function. However, previous studies were mainly focused on receptor ligands, with little known about alterations in renal expression of these nuclear receptors during CKD. An intriguing reality of CKD is the gender disparity in the progression of kidney diseases; men display a faster rate of progression of certain CKD. Such a gender difference has been verified in various animal models, which conclude that androgens exaggerate kidney disease, whereas estrogens confer renal protection.3–6 In women, the deterioration of renal disease markedly accelerates after menopause, further confirming the renal protective effects of estrogens.3 The peroxisome proliferator-activated receptor a (PPARa) is highly expressed in the liver and kidney. In the kidney, PPARa and its target gene cytochrome P450 4a1 (Cyp4a1) are primarily expressed in proximal tubules.7,8 PPARa is not only essential in fatty acid metabolism, but important in antiinflammatory reactions.9–12 Acyl-CoA oxidase (AOX) and Cyp4a1, two well-characterized PPARa-regulated genes, are vital in the metabolism of fatty acids, important sources of energy supply and signaling molecules. During acute renal failure induced by ischemia/reperfusion, rat kidneys have downregulation of PPARa, AOX, and Cyp4a1 mRNA, whereas PPARa-null mice have exacerbated renal injury.13 Moreover, PPARa ligand WY-14643 protected wild-type, but not PPARa-null mice, from cisplatin-induced acute renal failure through inhibiting inflammatory response and apoptosis.14 However, there was no report on alteration of renal PPARa signaling during CKD. The importance of aryl hydrocarbon receptor (AhR) in normal renal function is clearly demonstrated by the abnormity of kidney observed in AhR-null mice and mice Kidney International (2006) 70, 1920–1928

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H Lu et al.: Gender differences in nuclear receptors during CKD

treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin, the prototypical AhR ligand.15,16 AhR is constitutively activated by unknown endogenous ligand(s).17 AhR activation has been associated with a decrease in estrogen receptor a (ERa),18 disruption of Wilms’ tumor 1 (WT1) homeostasis, antagonism of hypoxia signaling pathway, and induction of oxidative stress,19–22 all of which are important in renal pathophysiology. Interestingly, protein expression of Cyp1a2, the AhR target gene, is induced in both the liver and kidney of male CKD rats,23 suggesting that AhR may be activated in CKD males. However, there was no report on alteration of renal AhR expression during CKD. Aiming at identifying key molecular targets for novel therapeutic modalities through combination therapy, this study is an in vivo integrative study on the contribution of certain nuclear receptors to gender-divergent CKD progression. Without genetic defects or nephrotoxin treatment, the 5/6-nephrectomized (Nx) rat is considered the best animal model mimicking human CKD. A previous study demonstrates that CKD progression is slower in female Nx rats.24 In this study, branched DNA (bDNA) signal amplification assay, a high-throughput and quantitative technique for mRNA determination, was used to examine gender-divergent renal mRNA expression of ERa, androgen receptor (AR), glucocorticoid receptor (GR), AhR, and PPARa, as well as AOX and Cyp4a1. These genes were selected due to their aforementioned demonstrated importance in renal function and their potential as drug targets. Profound gender differences in renal basal expression and alteration of these genes during CKD were identified, and their associations with CKD severity were examined.

RESULTS Gender difference in the progression of CKD in Nx rats

Nx-male rats displayed significant kidney disease, indicated by increased blood urea nitrogen, decreased creatinine clearance (CrCl), proteinuria, and albuminuria (Table 1). In contrast, Nx-females had much milder kidney injury: only blood urea nitrogen was elevated and glomerular filtration rate (GFR) was decreased; urinary protein and blood creatinine tended to increase slightly. Males had higher basal GFR than females; however, Nx-males only retained 34%, whereas Nx-females maintained 60% of normal GFR. Moreover, Nx-males had an immune defect, anemia, and malnutrition, manifested by splenomegaly, decreased blood hemoglobin/hematocrit, and decreased blood albumin, respectively; these pathological changes were absent in Nxfemales (Table 1). Gender difference in renal activities of caspase-3

In Nx-males, renal caspase-3 activity increased 210%, indicating increased apoptosis. In five of seven Nx-females, caspase-3 activity remained similar to that in sham females, but two of seven Nx-females displayed a striking elevation of caspase-3 activity, resulting in a marked increase of mean caspase-3 activity similar to that in Nx-males; however, such an increase was statistically insignificant (Figure 1). Gender difference in renal mRNA expression of inflammation-related genes

Inflammation is essential in inducing apoptosis and renal diseases. Nx-males had 76, 350, and 130% higher interleukin1beta (IL-1b), IL-6, and IL-10 transcripts than sham males, respectively; whereas elevations of these cytokines were

Table 1 | Gender difference in CKD in 5/6 Nx rats Sham male

Nx-male

Sham female

Nx-female

Right kidney (mg/100 g body weight) Spleen (mg/100 g body weight)

452721 16078

479720 23378a

387711 21479

422717 221710

Blood Urea nitrogen (mg/dl) Creatinine (mg/dl) Hemoglobin (g/dl) Hematocrit (%) Albumin (g/dl)

22.172.3 0.7370.08 14.670.36 45.071.3 3.870.1

73.3713.1a 1.4270.20a 11.970.72a 35.872.1a 3.270.1a

17.571.2 0.5670.06 14.870.29 46.471.0 4.470.04c

33.672.9a,b 0.7870.10b 14.570.19b 45.371.0b 4.270.1b

Urine 24 h urine (ml) Urea nitrogen (mg/24 h) Albumin (mg/24 h) Protein (mg/24 h)

26.274.1 520738 7.170.82 10.471.2

39.773.8a 374735a 12.171.4a 61.5717.5a

16.271.8 305722c 5.270.57 6.471.3

18.171.5b 290720 3.870.37b 12.673.6b

Systemic Creatinine clearance (ml/min) Glomerular filtration rate (ml/min)

1.0270.11 1.3670.08

0.4770.08a 0.4670.07a

0.8370.08 1.0670.09c

0.6470.13 0.6470.09a

CKD=chronic kidney disease; Nx=nephrectomized. N=6–7. Mean7s.e. a Po0.05 compared to corresponding sham control. b Po0.05 compared to Nx-male rats. c Po0.05 compared to sham male.

Kidney International (2006) 70, 1920–1928

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1000

a 10

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IL-1
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Figure 1 | Renal enzyme activities of caspase-3 in 5/6 Nx rats. Kidney samples of male and female rats were collected 7 weeks after Nx. Renal caspase-3 activity was determined with a diagnostic kit. N ¼ 6–7. Error bars indicate s.e.m. SM, sham-operated male rats; NxM, 5/6 nephrectomized male rats; SF, sham-operated female rats; NxF, 5/6 nephrectomized female rats. *Po0.05 compared to corresponding sham-operated group.

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In blood, sham females had higher corticosterone and 17bestradiol (E2) concentrations than sham males (Figure 3a and b), whereas sham males had much higher testosterone (Figure 3c). In Nx rats, corticosterone remained unchanged, whereas testosterone and E2 tended to decrease (P40.05) in males and females, respectively (Figure 3a–c). Testosterone concentrations in urine were B6-fold higher in sham females than sham males, and decreased in both Nx-females and Nxmales (Figure 3d). In Nx-males, urinary excretion of testosterone correlated positively with GFR (r ¼ 0.78). 17b-Estradiol was not detectable in urine. Gender difference in renal expression of steroid hormone receptors

Sham females had 50% higher AR transcripts and slightly (15%) higher GR transcripts than males (Figure 4a and b), whereas sham males had 150% higher ERa transcripts than sham females (Figure 4c). Renal GR transcripts was decreased to a higher degree in Nx-females (40%) than Nx-males (28%) (Figure 4a). Nx rats displayed marked gender differences in renal sex hormone receptors: AR transcripts increased by 40% in Nx-males but tended to decrease (P ¼ 0.06) in Nx-females (Figure 4b), whereas ERa transcripts decreased 45% in Nx-males, but decreased slightly (17%) in Nx-females (Figure 4c). Western blot data (Figure 4d) confirmed that basal protein expression of ERa was higher in sham males than sham females, and ERa protein 1922

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largely absent in Nx-females (Figure 2a–c). In Nx rats, renal transcripts of Fas, a key death receptor mediating inflammation-induced apoptosis, was elevated similarly in both genders (Figure 2d); however, mRNA of FasL, the ligand activating Fas, was only increased in Nx-males (Figure 2e). Renal Fas mRNA correlated with proteinuria (r ¼ 0.80) and caspase-3 activity (r ¼ 0.80) in Nx-males and Nx-females, respectively. Thus, Nx-males had both systemic (splenomegaly) and renal inflammation, which is largely absent in Nx-females.

IL-10 mRNA

c 0.4

*

8 #

6 4 2 0 SM

NxM

SF

NxF

Figure 2 | Renal mRNA expression of inflammation-related genes in 5/6 Nx rats. Kidney samples of male and female rats were collected 7 weeks after Nx. The mRNA expression of (a) IL-1b), (b) IL-6, (c) IL-10, (d) Fas, and (e) Fas ligand (FasL) in kidney total RNA was quantified with the bDNA signal amplification assay. N ¼ 6–7. Y axis represents renal mRNA of target genes expressed as RLU per 4 mg total RNA. Error bars indicate s.em. SM, sham-operated male rats; NxM, 5/6 nephrectomized male rats; SF, sham-operated female rats; NxF, 5/6 nephrectomized female rats. *Po0.05 compared to corresponding sham-operated group; #Po0.05 compared to corresponding male group.

expression decreased more dramatically in Nx-males than Nx-females. In Nx-males, renal GR transcripts correlated with GFR (Figure 4e) and CrCl (r ¼ 0.82), and ERa transcripts correlated with multiple markers of kidney disease, such as GFR (Figure 4f), CrCl (r ¼ 0.83), and proteinuria (r ¼ 0.91). Renal AR did not correlate with any CKD parameters. Kidney International (2006) 70, 1920–1928

original article

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Renal PPARa mRNA decreased markedly in Nx-males (44%), but moderately in Nx-females (24%), resulting in a lower expression in Nx-males (Figure 5a). Renal AOX decreased 42% in Nx-males, but was maintained in Nxfemales, resulting in lower expression in Nx-males (Figure 5b). Importantly, in both Nx-males and Nx-females, renal PPARa and AOX correlated with proteinuria (Figure 5c and d). Renal basal expression of Cyp4a isoforms was gender divergent: females had twofold higher Cyp4a1 (Figure 5e),

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30 25 20 15 10

40

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0

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200 50

g 160

Figure 4 | Renal expression of steroid hormone receptors in 5/6 Nx rats. Kidney samples of male and female rats were collected 7 weeks after Nx. Renal mRNA expression of ERa, AR, and GR was quantified with the bDNA signal amplification assay. Renal protein expression of ERa was determined by Western blot. Y axis represents renal mRNA of target genes expressed as RLU per 4 mg total RNA. N ¼ 6–7. Error bars indicate s.e.m. (a) GR mRNA, (b) AR mRNA, (c) ERa mRNA, (d) ERa protein, (e) positive correlation of renal GR mRNA with GFR in Nx-males, and (f) positive correlation of renal ERa mRNA with GFR in Nx-males. SM, sham-operated male rats; NxM, 5/6 nephrectomized male rats; SF, sham-operated female rats; NxF, 5/6 nephrectomized female rats. *Po0.05 compared to corresponding sham-operated group; #Po0.05 compared to corresponding male group. Kidney International (2006) 70, 1920–1928

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PPAR mRNA

Figure 3 | Blood and/or urine levels of E2, testosterone, and corticosteroid in 5/6 Nx rats. Blood and urine samples of male and female rats were collected 7 weeks after Nx. Blood and urine levels of steroid hormones were determined with radioimmunoassay kits. N ¼ 6–7. Error bars indicate s.e.m. (a) Plasma levels of corticosterone, (b) plasma levels of E2, (c) plasma levels of testosterone, and (d) urinary excretion of testosterone. SM, sham-operated male rats; NxM, 5/6 nephrectomized male rats; SF, sham-operated female rats; NxF, 5/6 nephrectomized female rats. *Po0.05 compared to corresponding sham-operated group; #Po0.05 compared to corresponding male group.

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H Lu et al.: Gender differences in nuclear receptors during CKD

40

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9 12 Hemoglobin (g/dl)

15

Figure 5 | Renal mRNA expression of PPARa and its downstream genes in 5/6 Nx rats. Kidney samples of male and female rats were collected 7 weeks after Nx. Renal mRNA expression of PPARa, AOX, Cyp4a1, and Cyp4a2/3 was quantified with the bDNA signal amplification assay. Y axis represents renal mRNA of target genes expressed as RLU per 4 mg total RNA. N ¼ 6–7. Error bars indicate s.e.m. (a) PPARa, (b) AOX, (c) negative correlation of renal PPARa mRNA with proteinuria in Nx-males and -females, (d) negative correlation of renal AOX mRNA with proteinuria in Nx-males and -females; (e) Cyp4a1; (f) Cyp4a2/3; (g) negative correlation of renal Cyp4a1 mRNA with renal caspase-3 activity in Nx-females; and (h) positive correlation of renal Cyp4a1 mRNA with blood levels of hemoglobin in Nx-males. SM, sham-operated male rats; NxM, 5/6 nephrectomized male rats; SF, sham-operated female rats; NxF, 5/6 nephrectomized female rats. *Po0.05 compared to corresponding sham-operated group; #Po0.05 compared to corresponding male group. 1923

original article

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H Lu et al.: Gender differences in nuclear receptors during CKD

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62 kDa 52 kDa

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62 kDa 52 kDa

0 SM

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Figure 6 | Renal expression of AhR and WT1 in 5/6 Nx rats. Kidney samples of male and female rats were collected 7 weeks after Nx. Renal mRNA expression of AhR and WT1 was quantified with the bDNA signal amplification assay. Renal protein expression of WT1 was determined by Western blot. Y axis represents renal mRNA of target genes expressed as RLU per 4 mg total RNA. N ¼ 6–7. Error bars indicate s.e.m. (a) AhR mRNA, (b) negative correlation of renal AhR mRNA with GFR in Nx-females, (c) WT1 mRNA, and (d) WT1 protein. SM, sham-operated male rats; NxM, 5/6 nephrectomized male rats; SF, sham-operated female rats; NxF, 5/6 nephrectomized female rats. *Po0.05 compared to corresponding sham-operated group; #Po0.05 compared to corresponding male group.

whereas males had twofold higher Cyp4a2/3 (Figure 5f). Renal Cyp4a1 mRNA was further increased by 48% in Nxfemales, but remained unchanged in Nx-males, resulting in a threefold higher Cyp4a1 expression in Nx-females than Nxmales (Figure 5e). Renal Cyp4a2/3 tended to decrease in both genders of Nx rats (Figure 5f). In Nx-females, Cyp4a1 expression correlated negatively with renal caspase-3 activity (Figure 5g) as well as inflammatory cytokines and death receptors, including IL-1b (r ¼ 0.76), IL-10 (r ¼ 0.82), Fas (r ¼ 0.85), and Fas Ligand (r ¼ 0.84). In Nx-males, renal Cyp4a1 correlated with blood hemoglobin (Figure 5h), and tended to correlate with CrCl (r ¼ 0.72) and caspase-3 activity (r ¼ 0.61). Gender difference in renal expression of AhR and WT1

The basal renal mRNA expression of AhR was 150% higher in females than in males (Figure 6a). Renal AhR transcripts decreased 37% in Nx-females, but remained unchanged in Nx-males (Figure 6a). Renal AhR transcripts correlated negatively with urinary excretion of creatinine (r ¼ 0.77) and GFR (Figure 6b) in Nx-males and Nx-females, respectively. Moreover, in both genders, renal AhR tended to correlate negatively with CrCl. Renal WT1 mRNA was similarly decreased in both genders of Nx rats (Figure 6c). However, the AhR ligand benzo[a]pyrene disrupts nephrogenesis by markedly decreasing a 52-kDa WT1 isoform via increasing the ratio of KTS/ þ KTS (lysine-threonineserine) WT1 mRNA variants, without affecting renal levels of total WT1 mRNA.15 Thus, we hypothesized that differential AhR expression in Nx rats results in differential expression of the 52-kDa WT1 isoform. As shown in Figure 6d, the 62-kDa WT1 protein remained unchanged in both 1924

genders of Nx rats. However, the 52-kDa WT1 isoform almost completely disappeared in Nx-males, but remained at high levels in Nx-females.

The present studies demonstrate a clear gender difference in CKD progression in rats. Nx-males have markedly elevated blood urea nitrogen and decreased CrCl, develop significant proteinuria, albuminuria, anemia, and malnutrition; in contrast, Nx-females have only moderate kidney injury, and are free of albuminuria, anemia, and malnutrition. Nx-males also have systemic and renal inflammation, which are largely absent in Nx-females. The present studies provide the first evidence of gender divergences in renal expression of sex hormone receptors (Figure 4) and metabolism of steroid hormones (Figure 3). Many CKD patients progress to end-stage renal disease and require kidney transplantation. Chronic rejection of renal transplants is a major obstacle for long-term success; the risk of chronic rejection is particularly high when female kidney is transplanted into a male recipient.25 Both sex hormones and donor gender affect long-term outcome of renal transplantation.26,27 Numerous studies have shown that estrogens are essential for maintaining function of female kidneys, whereas male kidneys benefit from the absence of testosterone in rats.24,26–28 For the first time, this study demonstrates remarkable gender divergences in renal expression of ERa and AR. The very low blood testosterone in female Sprague–Dawley rats is consistent with a recent report.29 Moreover, male kidney is a site of testosterone catabolism, whereas female kidney is a site of testosterone production, consistent with human female kidney being an androgen-producing organ.30 Thus, female kidneys rely on high estrogen and glucocorticoid as well as low androgen concentrations in the blood to maintain homeostasis of steroid hormones. Transplantation of a female kidney to a male recipient will result in low estrogen and ERa, low glucocorticoid, but high testosterone and AR, markedly disrupting renal homeostasis of steroid signaling, leading to accelerated renal failure.25 Postmenopausal CKD women, who may have intrinsically lower renal expression of ERa, suffer from the marked decrease of blood E2 and resultant accelerated deterioration of renal function. Among 13 mouse tissues, kidney has the third largest number of E2-regulated genes.31 Many studies have shown that cellular levels of hormone receptors play a key role in determining local hormonal responses. The current study, for the first time, demonstrates that in Nx-male rats, renal ERa transcripts, rather than circulatory estrogens, correlate with multiple markers of kidney disease. Similarly, renal expression of ERa, rather than circulatory E2, controls renal function in female mice genetically prone to glomerulosclerosis.32 Our study is the first report indicating in males, without any genetic defect, renal ERa expression may be a key determinant of CKD severity. Moreover, this study provides the first evidence of renal induction of AR in Nx-males. In Kidney International (2006) 70, 1920–1928

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H Lu et al.: Gender differences in nuclear receptors during CKD

rats transfected with the renin gene, the AR antagonist flutamide ameliorated kidney damage.33 Thus, an AR antagonist may be promising in treating CKD. Glucocorticoids protect endothelial and epithelial cells from stress-induced apoptosis, but induce apoptosis of mature lymphocytes, exhibiting potent anti-inflammatory effects.34,35 Dexamethasone ameliorated angiotensin II-induced renal damage.36 11b-Hydroxysteroid dehydrogenase-1 is an enzyme that biotransforms inactive cortisone to bioactive cortisol in proximal tubules. Both GR (Figure 4a) and 11b-hydroxysteroid dehydrogenase-1 (data not shown) decrease in Nx rats, with more dramatic decreases observed in Nx-females. However, only in Nx-males do the decreases correlate with kidney disease, suggesting that a mechanism exists in Nx-females to counteract decreases of GR and 11b-hydroxysteroid dehydrogenase-1. Moreover, both blood levels of E2 (Figure 3b) and renal ERa (Figure 4c) tend to decrease in Nx-females. Apparently, other mechanisms besides estrogens and glucocorticoids exist, contributing to less inflammation and renal injury in Nx-females. PPARa has a pivotal role in inflammation control.37,38 This study provides the first evidence of a considerable gender difference in renal expression of PPARa, AOX, and Cyp4a during CKD. Moreover, renal PPARa and AOX correlate with multiple markers of CKD in both genders. AOX is crucial in fatty acid metabolism;39 downregulation of AOX will not only impair energy supply, but lead to the accumulation of fatty acyl-CoA, which may cause lipotoxicity. The development of monogenic hypertension in Cyp4a14null mice clearly demonstrates the physiological importance of Cyp4a.40 Moreover, polymorphism of CYP4A11, present in proximal tubules,41 is associated with human hypertension.42 Interestingly, both rat Cyp4a1 and mouse Cyp4a1440 are female-predominant in kidneys. The femalepredominant renal expression of Cyp4a1 is further increased in Nx rats (Figure 5e). Among the four isoforms of rat Cyp4a, Cyp4a1 has the highest capacity to metabolize saturated fatty acids (e.g. lauric acid and palmitic acid) and w/w-1-hydroxylation of arachidonic acid and epoxyeicosatrienoic acids.43,44 20Hydroxyeicosatetraenoic acid is a major w/w-1-hydroxylation product of arachidonic acid. Depending on the site of generation, 20-hydroxyeicosatetraenoic acid plays a dual role in regulating blood pressure via its ability to induce contraction, as well as to inhibit sodium reabsorption.45,46 Renal expression of Cyp4a1 is predominant in proximal tubules,7 where both 20-HETE and epoxyeicosatrienoic acids inhibit sodium reabsorption, increase sodium excretion, and inhibit the development of hypertension.47 In contrast, Cyp4a2 and 4a3 are detected in all nephron segments.48 Moreover, renal expression of Cyp4a1, but not Cyp4a2, is induced by the PPARa ligand clofibrate.49 Interestingly, in Nx-females, Cyp4a1 expression correlates negatively with various inflammatory cytokines and apoptosis, suggesting an important role of Cyp4a1 upregulation in preventing Kidney International (2006) 70, 1920–1928

inflammation and apoptosis in Nx-females. Release of palmitic acid-derived free fatty acids was shown to be a key early event in Fas-induced apoptosis.50 Thus, Cyp4a1 may inhibit apoptosis through decreasing cellular concentrations of palmitic acid. Moreover, Cyp4a1 catalyzes the formation of omega-alcohols of epoxyeicosatrienoic acids, high-affinity PPARa activators.44 Inhibition of nuclear factor-kB activation through inducing IkBa is a putative mechanism of antiinflammation by PPARa ligands.51 Moreover, the PPARa activator fenofibrate induces renal expression of Cyp2c23, a major enzyme in producing the anti-inflammatory epoxyeicosatrienoic acids.52 Hence, upregulation of Cyp4a1 might play a key role in activating PPARa and thus preventing the reduction of AOX (Figure 5b), and meanwhile induce important anti-inflammatory genes. Taken collectively, this study strongly suggests a critical protective role of PPARa and its downstream genes AOX and Cyp4a1 against CKD progression. Correspondingly, the PPARa ligand clofibrate is renoprotective in Nx rats.53 Currently, the mechanism of renal induction of Cyp4a1 in Nx-females remains unknown. Although Cyp4a1 is a wellcharacterized PPARa-regulated gene, renal Cyp4a1 expression is also regulated negatively by androgens.54 Thus, despite a slight downregulation of PPARa, a putative decrease in renal androgen (Figure 3d) and AR may contribute to renal induction of Cyp4a1 in Nx-females. This study provides the first evidence of gender-divergent renal expression of AhR in rats. In both Nx males and females, renal AhR expression correlates positively with CKD severity, suggesting a pathogenic role of AhR activation in CKD. The critical role of WT1 in kidney development is established by the lack of kidney in WT1-null mice, the abnormality of kidney in WT1 heterozygous mice, and the existence of WT1 mutation in WT.55,56 Heterozygous mice with decreased levels of þ KTS WT1 variant develop glomerulosclerosis and are a model for Frasier syndrome.57 Our novel finding of a selective dramatic decrease of the 52kDa WT1 protein in Nx-males is remarkably similar to that in kidneys of mice treated with the AhR ligand benzo[a]pyrene,15 and a marked decrease of renal AhR in Nx-females is associated with better GFR and preservation of the 52-kDa WT1 protein. Therefore, in Nx-females, a marked decrease of renal AhR might cause an increase of þ KTS variants, preventing the decrease of the 52-kDa WT1 protein and CKD pathogenesis. In fact, resveratrol, a natural AhR antagonist, inhibited proteinuria, hypoalbuminemia, and hyperlipidemia in nephritic rats.58 Conclusion

This study identifies key molecular pathways contributing to the marked gender difference in CKD progression: ERa, AR, PPARa/AOX/Cyp4a1, AhR, and WT1. These pathways are relatively independent; however, apparent interactions between these pathways exist. The beneficial alterations of these pathways in Nx-females may function in concert to protect female kidneys during CKD. Future combination therapy 1925

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H Lu et al.: Gender differences in nuclear receptors during CKD

simultaneously targeting these pathways, mimicking beneficial adaptive responses in female Nx rats, may achieve a much better efficacy than the current modalities in treating CKD. MATERIALS AND METHODS Materials Bradford reagent and diagnostic kits for creatinine and urea nitrogen were purchased from Sigma Inc. (St Louis, MO, USA). Diagnostic kits for albumin and glucose were obtained from Stanbio Laboratory (Boerne, TX, USA). Radioimmunoassay kits for corticosterone, E2, and testosterone were purchased from ICN Pharmaceuticals Inc. (Orangeburg, NY, USA).

Animals The 5/6 Nx and sham-operated adult male and female Sprague–Dawley rats were purchased from Charles River Laboratories Inc. (Wilmington, MA, USA). Rats (175–225 g) were randomly allotted to 5/6 nephrectomy or sham-operated groups. Five-sixth nephrectomy of seven male and seven female rats was performed by surgical resection of the 2/3 of the right kidney, followed by removal of the left kidney 1 week later. Six males and seven females underwent sham operation as controls. Following nephrectomy or sham operation, rats were allowed 1 week to recover and then transferred to an American Animal Associations Laboratory Animal Careaccredited facility at our institution. Rats were housed in an ambient temperature of 221C with alternating 12-h light/dark cycles, and allowed water and rat chow ad libitum (Teklad; Harlan,

Table 2 | Oligonucleotide probes for the analysis of rat mRNA by bDNA signal amplification assay Targeta

Functionb

ERa 418–434 493–513 514–532 620–639 640–665 396–417 435–451 472–492 533–554 577–598 599–619 666–690 691–712 452–471 555–576

CE CE CE CE CE LE LE LE LE LE LE LE LE BL BL

AR 629–647 648–670 760–779 851–870 910–933 954–974 671–694 718–737 780–797 798–816 832–850 871–887 934–953 975–996 695–717 738–759 817–831 888–909

CE CE CE CE CE CE LE LE LE LE LE LE LE LE BL BL BL BL

Targeta

Functionb

GR 1546–1566 1591–1614 1704–1725 1886–1908 1523–1545 1615–1636 1752–1771 1772–1792 1793–1818 1819–1839 1867–1885 1567–1590 1637–1657 1658–1677 1678–1703 1726–1751 1840–1866

CE CE CE CE LE LE LE LE LE LE LE BL BL BL BL BL BL

AhR 2373–2389 2410–2431 2451–2472 2535–2552 2614–2636 2309–2330 2331–2351 2352–2372 2432–2450 2473–2494 2495–2513 2575–2593 2594–2613 2390–2409 2514–2534 2553–2574

CE CE CE CE CE LE LE LE LE LE LE LE LE BL BL BL

Targeta

Functionb

PPARa 743–760 806–827 908–932 956–978 1066–1088 761–781 782–805 828–848 868–887 888–907 933–955 979–999 1047–1065 1089–1112 1113–1133 849–867 1000–1021 1022–1046

CE CE CE CE CE LE LE LE LE LE LE LE LE LE LE BL BL BL

WT1 463–480 481–497 554–569 632–648 570–589 590–610 611–631 649–668 669–692 693–713 498–514 515–537 538–553

CE CE CE CE LE LE LE LE LE LE BL BL BL

Targeta

Functionb

Cyp4a1 362–379 551–569 668–693 873–895 312–337 338–361 380–399 400–419 420–442 505–527 528–550 597–623 646–667 694–714 786–806 830–853 854–872 443–462 463–480 481–504 570–596 624–645 715–735 736–761 762–785 807–829

CE CE CE CE LE LE LE LE LE LE LE LE LE LE LE LE LE BL BL BL BL BL BL BL BL BL

Fas 832–853 895–917 937–957 980–1004 1051–1072 854–876 877–894 918–936 959–979 1005–1028 1073–1091 1092–1112 1029–1050

CE CE CE CE CE LE LE LE LE LE LE LE BL

Targeta

Functionb

AOX 3247–3272 3446–3471 3522–3546 3613–3634 3273–3298 3299–3318 3365–3386 3387–3405 3406–3424 3425–3445 3472–3499 3500–3521 3547–3571 3572–3593 3594–3612 3635–3659 3660–3683 3319–3342 3343–3364

CE CE CE CE LE LE LE LE LE LE LE LE LE LE LE LE LE BL BL

FasL 785–804 968–994 1017–1038 1173–1194 1219–1240 805–830 832–861 862–887 888–913 915–943 944–967 1063–1084 1107–1129 1130–1149 1195–1218 995–1016 1039–1062 1085–1106

CE CE CE CE CE LE LE LE LE LE LE LE LE LE LE BL BL BL

AOX=acyl-CoA oxidase; AhR=aryl hydrocarbon receptor; AR=androgen receptor; bDNA=branched DNA; Cyp4a1=cytochrome P450 4a1; ERa=estrogen receptor a; FasL=Fas ligand; GR=glucocorticoid receptor; PPARa=peroxisome proliferator-activated receptor a; WT1=Wilm’s tumor 1. a Target refers to the sequence of the mRNA transcript as enumerated in the GenBank file. Genebank ID: ERa, 6978814; AR, 6978534; GR, 204271; AhR, 6978474; PPARa, 6981381; AOX, 8394148; Cyp4a1, 28460697; WT1, 13928719; Fas, 59624976; FasL, 6978524. b Function refers to the use of the oligonucleotide probe in the bDNA assay (CE, capture extender; LE, label extender; BL, blocker probe). An oligonucleotide of TTTTTctcttggaaagaaagt is linked to the 30 -end of each CE. An oligonucleotide of TTTTTaggcataggacccgtgtct is linked to the 30 -end of each LE.

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Indianapolis, IN, USA). Seven weeks after 5/6 nephrectomy or sham operation, each rat was individually housed in a metabolic cage and given food and water ad libitum for 24 h. Urine of each rat was collected, quantified, and then stored at 801C. After pentobarbital anesthesia, rat blood samples were drawn by cardiac puncture into glass tubes containing ethylenediaminetetraacetic acid. Rat blood sample (1 ml) was analyzed for complete blood count by Physicians Reference Laboratory (Overland Park, KS, USA). The remaining blood samples were centrifuged and the plasma was aliquoted and stored at 801C. The remnant kidney and spleen were collected, weighed, and snap-frozen in liquid nitrogen for storage at 801C. Blood and urine levels of creatinine, urea nitrogen, and albumin were analyzed with diagnostic kits following the manufacturers’ instructions. The formula to calculate CrCl was as follows: urine creatinine (mg/dl)  urine volume (ml)/blood creatinine (mg/dl)/ time (min). The formula for the calculation of GFR was: GFR ¼ (urea clearance þ CrCl)/2. RNA extraction Total tissue RNA was extracted using RNA-Bee reagent (Tel-Test Inc., Friendswood, TX, USA) according to the manufacturer’s protocol. RNA pellets were redissolved in diethyl pyrocarbonatetreated water and quantified by ultraviolet absorbance at 260 nm. RNA integrity was confirmed by agarose gel electrophoresis of 5 mg of total RNA and visualization of the intact 18S and 28S bands by ethidium bromide staining.

ethylenediaminetetraacetic acid, 10% glycerol, 1 mM dithiothreitol, 0.1 mg/ml phenylmethylsulfonyl fluoride, 0.5 M KCl, and 1  protease inhibitor cocktail). After being incubated on ice for 1 h, the homogenates were centrifuged at 100 000 g for 30 min at 41C. The resultant supernatant was stored at 801C. Protein concentrations were determined with Bradford reagent. Protein expression of ERa and WT1 was determined by Western blot.62 Briefly, protein samples were boiled for 2 min and loaded to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. After electrophoresis, proteins were transferred to polyvinylidine difluoride membranes and filters probed with an ERa (sc-542) or WT1 (sc-846) polyclonal antibody (Santa Cruz Biotechnologies Inc). Signals were visualized with horseradish peroxidase-conjugated secondary antibody. Statistics Data are presented as mean7s.e. of 6–7 kidney tissues. Differences between groups were determined by analysis of variance, followed by Duncan’s multiple-range test with significance set at Po0.05. Correlation analysis was conducted with Correlation Matrices within Statistica for Windows release 4.5 (StatSoft Inc., Tulsa, OK, USA). ACKNOWLEDGMENTS

This work was supported in part by NIH Grants ES-09649 and ES-09716. REFERENCES

bDNA signal amplification assay The mRNA of genes examined was quantified using the bDNA assay (Quantigene bDNA signal amplification kit; Genospectra, Fremont, CA, USA) with modifications.59 Sequences of target genes were accessed from GenBank (www.ncbi.nlm.nih.gov/Genbank). The probes of rat Cyp4a2/3,60 IL-1b, IL-6, and IL-1061 have been reported previously. The names and Genebank accession number for probes of other rat genes are given in Table 2. All reagents for analysis were supplied in the Quantigene bDNA signal amplification kit. Total RNA (4 mg/well) was added to each well of a 96-well plate. Subsequent incubation, washing, and hybridization steps were carried out following the manufacturer’s protocol. Luminescence of the 96-well plate was determined with a Quantiplex 320 bDNA luminometer interfaced with Quantiplex Data Management software version 5.02 for data analysis. The luminescence for each well is reported as relative light units (RLU) per 4 mg of total RNA. Determination of caspase-3 activity in kidney Renal caspase-3 activity was quantified as an indicator of apoptosis. Kidney samples were homogenized in cell lysis buffer (5% (wt/vol)) provided in the caspase-3 kit (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Protein levels of kidney homogenates were determined with Bradford reagent. A 20 ml aliquot of 5% kidney homogenates was used to determine caspase-3 activity following the manufacturer’s instructions. The fluorescent product was detected at 505 nm upon excitation at 400 nm by an LS50B luminescence spectrometer (Perkin-Elmer Inc., Boston, MA, USA). Caspase-3 activity was expressed as RLU per mg protein.

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