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Impact of Biological Gender and Soluble Guanylate Cyclase Stimulation on Renal Recovery After Relief of Unilateral Ureteral Obstruction
Impact of Biological Gender and Soluble Guanylate Cyclase Stimulation on Renal Recovery After Relief of Unilateral Ureteral Obstruction Yingrui Wang-Rosenke, Alice Mika, Dymtro Khadzhynov, Tanja Loof, Hans-Hellmut Neumayer and Harm Peters* From the Department of Nephrology and Center of Cardiovascular Research, Campus Charité Mitte, Charité-Universitätsmedizin Berlin, Berlin, Germany
Abbreviations and Acronyms
␣-SMA ⫽ ␣-smooth muscle actin cGMP ⫽ cyclic guanosine monophosphate eNOS ⫽ endothelial NO synthase GAPDH ⫽ glyceraldehyde-3phosphate dehydrogenase NO ⫽ nitric oxide PAI-1 ⫽ plasminogen activator inhibitor-1 PCNA ⫽ proliferating cell nuclear antigen PCR ⫽ polymerase chain reaction r-UUO ⫽ UUO relief sGC ⫽ soluble guanylate cyclase TGF-1 ⫽ transforming growth factor- UUO ⫽ unilateral ureteral obstruction Submitted for publication September 3, 2011. Study received approval from local authorities. Supported by Grant PE 558/2-3 from the Deutsche Forschungsgemeinschaft, Bonn, and the Sonnenfeldstiftung, Berlin, Germany. * Correspondence: Department of Nephrology, Campus Charité Mitte, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany (telephone: ⫹49-30-450-514 013; FAX: ⫹49-30450-514 902; e-mail: [email protected]).
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Purpose: Gender difference and nitric oxide deficiency contribute to the progression of many chronic kidney diseases. In a model of unilateral ureteral obstruction relief we analyzed the impact of biological gender and nitric oxide/cyclic guanosine monophosphate signaling stimulation on renal disease severity and restoration. Materials and Methods: Female and male rats underwent sham surgery or unilateral ureteral obstruction. After 5-day unilateral ureteral obstruction female and male rats were assigned to obstruction relief alone or obstruction relief plus 7-day treatment with the soluble guanylate cyclase stimulator BAY 41-8543. Results: Compared to male rats with obstruction relief renal disease was less severe in female rats, which had significantly less tubulointerstitial matrix accumulation and tubular atrophy. In each gender group ␣1 and 1-soluble guanylate cyclase was comparably and significantly increased but female rats produced significantly more cyclic guanosine monophosphate after treatment with the soluble guanylate cyclase stimulator. In each group BAY 41-8543 treatment was associated with significant amelioration of renal matrix protein expansion, macrophage infiltration, tubular apoptosis and atrophy. Conclusions: Female gender is protective for unilateral ureteral obstruction relief. This was linked to higher sensitivity of the soluble guanylate cyclase enzyme and cyclic guanosine monophosphate production in response to BAY 41-8543. In these female and male rats enhancing the signaling of nitric oxide/ cyclic guanosine monophosphate with BAY 41-8543 significantly accelerated the restoration of renal architecture after obstruction relief and largely ameliorated the differences in disease severity due to the gender disparity. Key Words: kidney, ureteral obstruction, BAY 41-8543, female, male SEVERAL studies show that biological gender differentiation affects the incidence, prevalence and progression of many renal diseases. A recent multicenter meta-analysis documented that renal disease in women progresses at a slower rate than in men with similar concomitant diseases.1 This is in line with results in various experimental animal models.2–5 The molecular and
cellular mechanisms underlying these findings have not been clearly identified. A key pathway is the enhancing action of estrogens on NO generation, which is linked to an antihypertensive effect, and to cell and organ damage protection.6 – 8 The most physiologically relevant action of NO, produced mainly by eNOS, is the activation of heme con-
0022-5347/12/1881-0316/0 THE JOURNAL OF UROLOGY® © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 188, 316-323, July 2012 Printed in U.S.A. DOI:10.1016/j.juro.2012.02.2552
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RESEARCH, INC.
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taining sGC and the subsequent conversion of guanosine triphosphate into the intracellular second messenger cGMP. Research in the last decade has shown that endothelial NO deficiency is critically involved in pathological matrix production and accumulation in the kidney as well as in other organ systems.9 –12 NO deficiency has been documented in numerous experimental and human renal diseases, such as diabetic and hypertensive nephropathy, chronic glomerulosclerosis, obstructive nephropathy and chronic interstitial nephritis, as well as renal diseases due to cyclosporin A and radio contrast media.11 Thus, therapeutic strategies to counteract NO deficiency are considered to be renoprotective for many renal diseases. We recently reported that correcting NO deficiency by sGC stimulation improved renal recovery in a male rat model with relief of transient 5-day UUO.13 In UUO decreased renal blood flow and glomerular filtration rate are mediated in part by stimulating renal angiotensin II production, which activates TGF- in a cascade of events culminating in tubulointerstitial inflammation and fibrosis. The r-UUO model provides the opportunity to study in detail the molecular and cellular changes that occur during recovery from ureteral obstruction. This corresponds to the frequent clinical situation in which obstruction is corrected by surgery. The current study was designed to characterize the impact of gender difference and involvement of the NO/cGMP signaling pathway on recovery from ureteral obstruction by comparing the male study13 with matching groups of contemporaneously examined female animals. Thus, we determined disease parameters in the r-UUO model of each gender. The novel, orally applicable compound BAY 41-8543 was given to specifically stimulate sGC activity and cGMP production.
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and encased in a Silastic® sheath, which was compressed with a single hemostatic Hemoclip® ligating clip using a Weck Applier (Pilling Weck Chirurgische Produkte, Karlstein, Germany). On the day of relief the Hemoclip was located and removed with the sheath. The control group underwent sham operation.
BAY 41-8543 Administration BAY 41-8543 was given with the food at a daily dose of 10 mg/kg body weight, as described previously.15 The dose was based on previous reports showing that it decreased matrix expansion and progression in rats with acute and chronic anti-thy1 glomerulonephritis.16,17 BAY 41-8543 is a sGC stimulator that markedly amplifies cGMP production of sGC in the absence of NO or synergizes with given amounts of NO.18 In vivo BAY 41– 8543 has vasodilating capacity, anti-proliferative properties in vascular smooth muscle cells and anti-aggregator effects in platelets.19
Experimental Groups and Design On the day of relief after 5 days of obstruction 10 rats each were randomly assigned to groups, including group 1—female r-UUO without treatment, group 2—male r-UUO without treatment, group 3—female r-UUO plus BAY 418543 and group 4 —male r-UUO plus BAY 41-8543. Four female and 4 male control rats with sham surgery were included. BAY 41-8543 treatment began directly after r-UUO. Seven days after r-UUO we determined the actions of the sGC stimulator BAY 41-8543 on NO/cGMP signaling and disease parameters. Systolic blood pressure was assessed 7 days after r-UUO in conscious rats using tail cuff plethysmography, as previously described.16
Experiment Termination Rats were anesthetized, as previously described.13 After a midline abdominal incision was made 5 to 10 ml blood were drawn from the abdominal aorta into ethylenediaminetetraacetic acid coated tubes. The kidneys were perfused with 40 ml ice-cold phosphate buffered saline. Materials and tissues were subsequently processed as described.
NO/cGMP Signaling
MATERIALS AND METHODS Materials and Animals Unless otherwise indicated, materials, chemicals and cell culture medium were obtained from Sigma-Aldrich®. Female and male Sprague-Dawley® rats with a body weight of 150 to 180 gm were housed in a constant temperature room with a 12-hour dark/12-hour light cycle. Animals were fed a standard 22.5% protein diet (Altromin, Lage, Germany) and had free access to water. Body weight, and food and water intake were monitored daily throughout the experiment. Animal care and treatment conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the local authorities.
r-UUO Model Induction The female and male Sprague-Dawley rats were subjected to complete UUO.13,14 Briefly, the left ureter was exposed
Plasma cGMP was measured by enzyme-linked immunosorbent assay (Amersham™ Pharmacia™ Biotech) according to manufacturer instructions.16 Tubulointerstitial mRNA expression of the ␣1 and 1-sGC subunits, and eNOS was measured using reverse transcriptase-PCR.
Light and Immunohistochemical Microscopy All histological examinations were done in blinded fashion using computer based morphometric analysis, as described previously.20 All histological parameters were evaluated using cortical tissue fixed in Carnoy’s solution except the TUNEL assay, for which buffered, formalin fixed tissue was used. The relative degree of tubulointerstitial lesions, ie matrix deposition, tubular atrophy and dilatation, inflammation and cell proliferation, was calculated in 10 randomly selected cortical areas per rat at 400⫻ magnification and expressed as the area affected in relation to the total area analyzed.
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Assessment Tubulointerstitial fibrosis. We evaluated renal fibrosis by measuring tubulointerstitial total collagen deposition, interstitial volume, collagen IV deposition and ␣-SMA expression. Total collagen deposition was evaluated by aniline blue staining. Interstitial volume was determined by a point counting technique on tissue sections stained by collagen IV using a 10 ⫻ 10 orthographic grid overlaid on digital images, as previously described.13 Collagen IV deposition and ␣-SMA expression were measured by positive collagen IV and ␣-SMA staining, respectively, in relation to select cortical sections, as previously described.13 The antifibrotic mechanism was further analyzed by determining mRNA expression of the key fibrosis marker and mediator TGF-1, the matrix protein fibronectin as an indicator of matrix protein synthesis, and tissue PAI-1 as a marker of matrix protein degradation. Tubular atrophy and apoptosis. Tubular atrophy was determined by measuring tubular diameter on cortical sections stained with collagen IV. Tubular apoptosis was evaluated with the TUNEL assay using the Apoptosis Detection Kit (Chemicon®), as described by the manufacturer. The number of TUNEL positive tubular cells was divided by the total number of tubular cells to indicate tubular apoptosis. Tubulointerstitial inflammation and cell proliferation. Tubulointerstitial macrophage infiltration and cell proliferation were evaluated by ED1 and PCNA positive cells in selected cortical areas using immunohistochemistry, as previously described.16,17
mRNA Expression Cortical mRNA expression was determined by 2-step reverse transcriptase-PCR, as described previously.16,21 The primer pairs of eNOS, ␣1 and 1-sGC, TGF-1, fibronectin and PAI-1 with GAPDH as the housekeeping gene were used as previously described.17
Statistical Analysis
each p not significant, fig. 1, A). BAY 41-8543 significantly lowered systolic blood pressure a mean of 117 ⫾ 3 mm Hg in group 3 and 114 ⫾ 4 in group 4 (vs groups 1 and 2, respectively, each p ⬍0.01). NO/cGMP Signaling Ureteral obstruction induced a significant increase in ␣1-sGC mRNA in groups 1 and 2 vs controls (mean 187% ⫾ 22% vs 101% ⫾ 8% and 169% ⫾ 14% vs 101% ⫾ 8%, respectively, each p ⬍0.01, fig. 2). It also induced a significant increase in 1-sGC mRNA in groups 1 and 2 vs controls (mean 188% ⫾ 22% vs 100% ⫾ 5% and 185% ⫾ 19% vs 101% ⫾ 8%, respectively, each p ⬍0.01). Plasma cGMP remained unaltered in groups 1 and 2 vs controls (mean 503 ⫾ 40 vs 528 ⫾ 66 and 479 ⫾ 29 vs 545 ⫾ 129 fmol per well, respectively, each p not significant). In contrast, there was a significant increase in plasma cGMP in groups 3 and 4 vs 1 and 2 (412% and 104%, respectively, each p ⬍0.01, fig. 1, B). BAY 41-8543 induced significantly higher plasma cGMP in group 3 than in group 4 (2,573 ⫾ 509 vs 977 ⫾ 113 fmol per well, p ⬍0.05). mRNA expression of the ␣1 and 1-sGC subunits was normalized after treatment with the sGC stimulator. mRNA expression of eNOS was not significantly affected by gender, r-UUO or BAY 41-8543. Tubulointerstitial Matrix Accumulation Figures 3 and 4 show representative histological views of tubulointerstitial changes in the r-UUO model, and the effects of gender and BAY 41-8543. Histomorphometric analysis indicated that the severity of tubulointerstitial matrix expansion and fibrosis was significantly less in group 1 than in group 2. Mean tubulointerstitial volume was 15.4% ⫾ 0.7% vs 19.3% ⫾ 0.5% in group 1 vs 2 (p ⬍0.001, fig. 5).
Results are shown as the mean ⫾ SEM. Statistical analysis between groups was performed by 1-way ANOVA and a subsequent t test with p ⬍0.05 considered significant.
RESULTS Body Weight, and Food and Drug Intake At the end of the experiments body weight did not differ among groups of the same gender but body weight in females was significantly lower than in males (mean 190 ⫾ 5, 185 ⫾ 3 and 185 ⫾ 2 gm in female controls, and groups 1 and 3 vs 228 ⫾ 7, 218 ⫾ 4 and 219 ⫾ 4 in male controls, and groups 2 and 4, respectively, each female vs male group p ⬍0.001). Systolic Blood Pressure Systolic blood pressure was slightly increased in groups 1 and 2 on day 7 after r-UUO vs controls (mean 145 ⫾ 8 vs 131 ⫾ 6 and 146 ⫾ 8 vs 122 ⫾ 7 mm Hg,
Figure 1. Effects of biological gender and BAY 41-8543 (⫹Bay) 7 days after r-UUO in groups 3 (open bars) and 4 (black bars). A, systolic blood pressure was measured in conscious rats by tail cuff method. Asterisks indicate p ⬍0.01 vs same gender group 1 or 2. B, plasma cGMP was measured by enzyme-linked immunosorbent assay. Asterisk indicates p ⬍0.05 vs same gender group 1 or 2. Pound sign indicates p ⬍0.05 vs group 4.
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Figure 2. Effects of biological gender and BAY 41-8543 (⫹Bay) 7 days after r-UUO in groups 3 (open bars) and 4 (black bars) on mRNA expression analyzed by real-time PCR with GAPDH as housekeeping gene, shown as measured mRNA/GAPDH. A, ␣1-sGC mRNA. Curleys indicate ⬍0.01 vs same gender control. Single asterisk indicates p ⬍0.05 vs same gender group 1 or 2. Double asterisks indicate p ⬍0.01 vs same gender group 1 or 2. B, 1-sGC mRNA. C, eNOS mRNA.
Aniline staining showed that mean matrix protein expansion was 6.9% ⫾ 0.9% vs 10.3 ⫾ 1% in group 1 vs 2 while mean collagen IV deposition was 3.8% ⫾ 0.5% vs 5.4% ⫾ 0.5% (each p ⬍0.05, fig. 5).
BAY 41-8543 plus r-UUO significantly reduced tubulointerstitial volume ⫺29% in group 3 and ⫺38% in group 4 (vs groups 1 and 2, respectively, each p ⬍0.001). Histological tubulointerstitial fibrosis was more effectively limited in group 4 than in
Figure 3. Representative renal sections show histological collagen IV protein deposition 7 days after r-UUO. ⫹Bay, BAY 418543. Collagen IV staining, reduced from ⫻400.
Figure 4. Representative histological ␣-SMA protein expression 7 days after r-UUO. ⫹Bay, BAY 41-8543. ␣-SMA staining, reduced from ⫻400.
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group 3, including matrix protein expansion ⫺60% vs ⫺16%, collagen IV deposition ⫺52% vs ⫺19% and ␣-SMA expression ⫺69% vs ⫺49% (group 4 vs 2, respectively, each p ⬍0.05). In line with the histological analysis group 1 showed lower renal mRNA expression than group 2, including TGF-1 mRNA (mean 203% ⫾ 30% vs 300% ⫾ 42%, p ⫽ 0.07), fibronectin mRNA (mean 448% ⫾ 73% vs 714% ⫾ 207%, p not significant) and PAI-1 mRNA (mean 81% ⫾ 22% vs 574% ⫾ 195%, p ⬍0.05, fig. 6). BAY 41-8543 lowered mRNA expression in group 3, although more effectively in group 4, compared to groups 1 and 2, including TGF-1 ⫺45% in group 3 (vs group 1 p ⬍0.05) and ⫺52% in group 4 (vs group 2 p ⬍0.01), fibronectin ⫺74% (p ⬍0.01) and ⫺77% (p ⬍0.05), and PAI-1 no decrease and ⫺80% (p ⬍0.05), respectively.
Figure 5. Computer based morphometric analysis of effects of biological gender and BAY 41-8543 (⫹Bay) 7 days after relief of UUO in groups 3 (open bars) and 4 (black bars). A, tubulointerstitial matrix accumulation measured by aniline blue staining. Single asterisk indicates p ⬍0.05 vs same gender group 1 or 2. Single pound sign indicates p ⬍0.05 vs group 2. Single curley indicates p ⬍0.05 vs control. Double curleys indicate p ⬍0.01 vs control. B, interstitial volume determined by point counting technique on tissue sections stained for collagen type IV. Triple asterisks indicate p ⬍0.001 vs same gender group 1 or 2. Triple pound signs indicate p ⬍0.001 vs group 2. Triple curleys indicate p ⬍0.001 vs control. C, collagen IV deposition. D, ␣-smooth muscle actin. Double asterisks indicate p ⬍0.01 vs same gender group 1 or 2.
Tubular Atrophy and Apoptosis In group 1 tubular diameter was remarkably less dilated than in group 2 (mean 36.3 ⫾ 0.5 vs 41.2 ⫾ 0.3 m, p ⬍0.001, fig. 7, A). sGC stimulation with BAY 41-8543 improved tubular dilatation in groups 3 and 4 (mean 30.4 ⫾ 0.2 and 33.0 ⫾ 0.7 m, vs groups 1 and 2, respectively, each p ⬍0.001). Ureteral obstruction induced a significant increase in the number of apoptotic cells in groups 1 and 2 vs controls (mean 21% ⫾ 4.0% vs 1% ⫾ 0.2% and 18% ⫾ 2.5% vs 2% ⫾ 0.5%, respectively, each p ⬍0.01, fig. 7, B). BAY 41-8543 significantly decreased tubular apoptosis ⫺76% in group 3 and ⫺67% in group 4 (vs groups 1 and 2, respectively, each p ⬍0.05). Renal Macrophage Infiltration and Cell Proliferation Untreated r-UUO was associated with a significantly higher number of tubulointerstitial ED1 positive cells, indicating macrophages, in groups 1 and
Figure 6. Effects of biological gender and BAY 41-8543 (⫹Bay) 7 days after r-UUO in groups 3 (open bars) and 4 (black bars) on mRNA expression analyzed by real-time PCR with GAPDH as housekeeping gene, shown as measured mRNA/GAPDH. A, TGF-1 mRNA. Single asterisk indicates p ⬍0.05 vs same gender group 1 or 2. Double asterisks indicate p ⬍0.01 vs same gender group 1 or 2. Single curley indicates p ⬍0.05 vs control. Double curleys indicate p ⬍0.01 vs control. B, fibronectin mRNA. C, PAI-1 mRNA. Pound sign indicates p ⬍0.05 vs group 2.
IMPACT OF GENDER AND SOLUBLE GUANYLATE CYCLASE STIMULATION ON RENAL RECOVERY
Figure 7. Effects of biological gender and BAY 41-8543 (⫹Bay) 7 days after r-UUO in groups 3 (open bars) and 4 (black bars) in computer based morphometric analysis. A, tubular diameter was used to measure tubular atrophy. Asterisks indicate p ⬍0.001 vs same gender group 1 or 2. Pound signs indicate p ⬍0.001 vs group 2. Curleys indicate p ⬍0.001 vs control. B, tubular apoptosis was measured by TUNEL assay. Single asterisk indicates p ⬍0.05 vs same gender group 1 or 2. Double asterisks indicate p ⬍0.01 vs same gender group 1 or 2. Double curleys indicate p ⬍0.01 vs control. C, tubulointerstitial macrophage infiltration. Asterisk indicates p ⬍0.05 vs same gender group 1 or 2. Single curley indicates p ⬍0.05 vs same gender control. Double curleys indicate p ⬍0.01 vs same gender control. Primary ED1 antibody, reduced from ⫻400. D, cell proliferation. Primary PCNA antibody, reduced from ⫻400.
2 vs controls (mean 38% ⫾ 6% vs 1% ⫾ 0.3% and 46% ⫾ 7% vs 5% ⫾ 0.2%, respectively, each p ⬍0.01, fig. 7, C). Untreated r-UUO was also associated with significantly more PCNA positive cells, indicating cell proliferation, in groups 1 and 2 vs controls (mean 5.2% ⫾ 0.5% vs 0.4% ⫾ 0.1% and 6.4% ⫾ 1.7% vs 1.3% ⫾ 0.1%, respectively, each p ⬍0.05, fig. 7, D). BAY 41-8543 decreased tubulointerstitial macrophage infiltration vs r-UUO alone ⫺50% in group 3 and ⫺65% in group 4 (group 4 vs 2 p ⬍0.05). It also decreased cell proliferation ⫺35% and ⫺44%, respectively.
DISCUSSION Ureteral obstruction is an important cause of renal failure in children and adults. Recovery of obstructive nephropathy after the relief of urinary obstruction is an especially important clinical issue. There
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were several major findings in this animal model of r-UUO. 1) Significant renal fibrotic, apoptotic and inflammatory changes were detectable 7 days after the relief of transient UUO. 2) Female rats with r-UUO showed less tubulointerstitial fibrosis and tubular atrophy than male rats. 3) The gender difference was associated with a more sensitive NO/cGMP signaling pathway in female rats. 4) In rats of each gender treatment with the sGC stimulator BAY 41-8543 significantly increased endogenous cGMP production and decreased tubulointerstitial fibrosis and inflammation as well as tubular atrophy and apoptosis. 5) In male r-UUO rats sGC stimulation was significantly more effective for renal fibrosis and cell infiltration parameters, which largely ameliorated the differences in disease severity due to the gender disparity. In our study group 1 showed significantly less tubular dilatation, tubulointerstitial volume expansion, collagen IV deposition and TGF-1 mRNA expression than group 2. This impact of biological gender on outcome is in line with human data. Studies suggest that female rat mesangial cells inherently show fewer profibrotic and proinflammatory characteristics than male cells.22 The relaxation of mesenteric arterial rings by the exogenous NO donor is greater in spontaneously hypertensive female rats than in the corresponding males.23 Male rats are more susceptible than female rats to the vascular and renal injury induced by fructose feeding or hypercholesterolemia, which are states associated with NO deficiency.24,25 These studies indicate that the male represents an inherent gender phenotype for profibrotic, proinflammatory characteristics, blunt reactivity to NO and the subsequent increased sensitivity to NO deficiency. In the kidney NO produced by endothelial cells is vasodilatory and inhibits mesangial cell growth, matrix production, cell proliferation and inflammatory cell infiltration. Arterial walls and mesangial cells are main sites of sGC expression, representing the physiological receptor of NO and mediating the downstream renoprotective effects of endothelial NO production.26 Thus, different sGC sensitivities and activities may well be involved in the gender difference of the mesangial cell reaction to vascular and renal injury. In our series renal sGC mRNA expression was significantly up-regulated in the untreated r-UUO rats, which may serve as a biological self-protective pathway to potentially enhance NO/cGMP signaling. However, this increase was not strong enough to lead to correspondingly increased plasma cGMP or counteract pathogenetic factors. Overall this indicates relative insufficiency in NO/cGMP signaling. However, BAY 41-8543 treatment was highly effective in stimulating sGC activity and increasing
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cGMP production in group 3 as well as group 4. After 1 week of therapy disease severity, eg tubular apoptosis, tubulointerstitial fibrosis and inflammation, was decreased to a comparable degree in groups 3 and 4. The plasma cGMP level stimulated by BAY 41-8543 in group 3 was much higher than in group 4, indicating lower sGC sensitivity in male rats. This might be an important reason for the gender difference in NO deficiency and obstructive nephropathy. In groups 3 and 4 significantly increased plasma cGMP and decreased systolic blood pressure indicate the pharmacological properties of the sGC stimulator and the vasodilative effects of enhanced NO/cGMP signaling. BAY 41-8543 significantly increased endogenous cGMP production, thus, attenuating tubulointerstitial fibrosis in each biological gender by decreasing collagen deposition, ␣-SMA expression and tubulointerstitial volume. The molecular antifibrotic mechanism may be related to decreased expression of the key profibrotic mediator TGF-1, matrix protein fibronectin and the matrix degradation inhibitor PAI-1. The second key renoprotective effect of BAY 418543 was associated with decreased tubulointerstitial macrophage infiltration. As an important source of TGF- production under pathological conditions, less white cell infiltration may have an important role in the renoprotective effect of sGC stimulation. Furthermore, BAY 41-8543 treatment lowered tubular atrophy, as shown by the decreased tubular diameter and positive apoptotic cells in male and female rats in our r-UUO model. Thus, BAY 41-8543 showed antifibrotic, anti-inflammatory and antiapoptotic effects, and potentiated disease recovery in the r-UUO model via increased cGMP production.
A decrease in the expression or activity of vascular sGC rather than eNOS/NO was recently reported in several diseases characterized by a functional deficiency in NO/cGMP signaling.27 This included rats with anti-thy1-induced renal disease, spontaneous hypertension, aging, myocardial infarction, angiotensin II infusion and lead induced hypertension, and diabetic Goto-Kakizaki rats. In cases of insensitive sGC and insufficient NO/cGMP signaling, as in the r-UUO model, therapy that primarily increases NO production might have no or only a limited effect. Also, compounds that enhance NO/cGMP signaling by interaction with the sGC enzyme have a number of therapeutic advantages over L-arginine, NO donors and gender hormone therapy, which have been previously used to overcome impaired NO production. sGC stimulators amplify the cGMP signal exactly at that subcellular location and avoid potentially unwanted side effects via unrelated pathways.
CONCLUSIONS In this study female rats showed less tubulointerstitial fibrosis and tubular atrophy in the UUO model after r-UUO. Data indicate that female associated renal protection is linked to higher sensitivity of the sGC enzyme and the downstream cGMP signaling pathway. sGC stimulation by BAY 41-8543 increased cGMP production in rats with r-UUO, and ameliorated disease severity and the gender difference in renal fibrosis and apoptosis. Findings suggest that pharmacological sGC stimulation may be a novel approach to slow the progression of obstructive nephropathy in males and females.
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27. Wang-Rosenke Y, Neumayer HH and Peters H: NO signaling through cGMP in renal tissue fibrosis and beyond: key pathway and novel therapeutic target. Curr Med Chem 2008; 15: 1396.
Abbreviations and Acronyms
␣-SMA ⫽ ␣-smooth muscle actin cGMP ⫽ cyclic guanosine monophosphate eNOS ⫽ endothelial NO synthase GAPDH ⫽ glyceraldehyde-3phosphate dehydrogenase NO ⫽ nitric oxide PAI-1 ⫽ plasminogen activator inhibitor-1 PCNA ⫽ proliferating cell nuclear antigen PCR ⫽ polymerase chain reaction r-UUO ⫽ UUO relief sGC ⫽ soluble guanylate cyclase TGF-1 ⫽ transforming growth factor- UUO ⫽ unilateral ureteral obstruction Submitted for publication September 3, 2011. Study received approval from local authorities. Supported by Grant PE 558/2-3 from the Deutsche Forschungsgemeinschaft, Bonn, and the Sonnenfeldstiftung, Berlin, Germany. * Correspondence: Department of Nephrology, Campus Charité Mitte, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany (telephone: ⫹49-30-450-514 013; FAX: ⫹49-30450-514 902; e-mail: [email protected]).
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www.jurology.com
Purpose: Gender difference and nitric oxide deficiency contribute to the progression of many chronic kidney diseases. In a model of unilateral ureteral obstruction relief we analyzed the impact of biological gender and nitric oxide/cyclic guanosine monophosphate signaling stimulation on renal disease severity and restoration. Materials and Methods: Female and male rats underwent sham surgery or unilateral ureteral obstruction. After 5-day unilateral ureteral obstruction female and male rats were assigned to obstruction relief alone or obstruction relief plus 7-day treatment with the soluble guanylate cyclase stimulator BAY 41-8543. Results: Compared to male rats with obstruction relief renal disease was less severe in female rats, which had significantly less tubulointerstitial matrix accumulation and tubular atrophy. In each gender group ␣1 and 1-soluble guanylate cyclase was comparably and significantly increased but female rats produced significantly more cyclic guanosine monophosphate after treatment with the soluble guanylate cyclase stimulator. In each group BAY 41-8543 treatment was associated with significant amelioration of renal matrix protein expansion, macrophage infiltration, tubular apoptosis and atrophy. Conclusions: Female gender is protective for unilateral ureteral obstruction relief. This was linked to higher sensitivity of the soluble guanylate cyclase enzyme and cyclic guanosine monophosphate production in response to BAY 41-8543. In these female and male rats enhancing the signaling of nitric oxide/ cyclic guanosine monophosphate with BAY 41-8543 significantly accelerated the restoration of renal architecture after obstruction relief and largely ameliorated the differences in disease severity due to the gender disparity. Key Words: kidney, ureteral obstruction, BAY 41-8543, female, male SEVERAL studies show that biological gender differentiation affects the incidence, prevalence and progression of many renal diseases. A recent multicenter meta-analysis documented that renal disease in women progresses at a slower rate than in men with similar concomitant diseases.1 This is in line with results in various experimental animal models.2–5 The molecular and
cellular mechanisms underlying these findings have not been clearly identified. A key pathway is the enhancing action of estrogens on NO generation, which is linked to an antihypertensive effect, and to cell and organ damage protection.6 – 8 The most physiologically relevant action of NO, produced mainly by eNOS, is the activation of heme con-
0022-5347/12/1881-0316/0 THE JOURNAL OF UROLOGY® © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 188, 316-323, July 2012 Printed in U.S.A. DOI:10.1016/j.juro.2012.02.2552
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RESEARCH, INC.
IMPACT OF GENDER AND SOLUBLE GUANYLATE CYCLASE STIMULATION ON RENAL RECOVERY
taining sGC and the subsequent conversion of guanosine triphosphate into the intracellular second messenger cGMP. Research in the last decade has shown that endothelial NO deficiency is critically involved in pathological matrix production and accumulation in the kidney as well as in other organ systems.9 –12 NO deficiency has been documented in numerous experimental and human renal diseases, such as diabetic and hypertensive nephropathy, chronic glomerulosclerosis, obstructive nephropathy and chronic interstitial nephritis, as well as renal diseases due to cyclosporin A and radio contrast media.11 Thus, therapeutic strategies to counteract NO deficiency are considered to be renoprotective for many renal diseases. We recently reported that correcting NO deficiency by sGC stimulation improved renal recovery in a male rat model with relief of transient 5-day UUO.13 In UUO decreased renal blood flow and glomerular filtration rate are mediated in part by stimulating renal angiotensin II production, which activates TGF- in a cascade of events culminating in tubulointerstitial inflammation and fibrosis. The r-UUO model provides the opportunity to study in detail the molecular and cellular changes that occur during recovery from ureteral obstruction. This corresponds to the frequent clinical situation in which obstruction is corrected by surgery. The current study was designed to characterize the impact of gender difference and involvement of the NO/cGMP signaling pathway on recovery from ureteral obstruction by comparing the male study13 with matching groups of contemporaneously examined female animals. Thus, we determined disease parameters in the r-UUO model of each gender. The novel, orally applicable compound BAY 41-8543 was given to specifically stimulate sGC activity and cGMP production.
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and encased in a Silastic® sheath, which was compressed with a single hemostatic Hemoclip® ligating clip using a Weck Applier (Pilling Weck Chirurgische Produkte, Karlstein, Germany). On the day of relief the Hemoclip was located and removed with the sheath. The control group underwent sham operation.
BAY 41-8543 Administration BAY 41-8543 was given with the food at a daily dose of 10 mg/kg body weight, as described previously.15 The dose was based on previous reports showing that it decreased matrix expansion and progression in rats with acute and chronic anti-thy1 glomerulonephritis.16,17 BAY 41-8543 is a sGC stimulator that markedly amplifies cGMP production of sGC in the absence of NO or synergizes with given amounts of NO.18 In vivo BAY 41– 8543 has vasodilating capacity, anti-proliferative properties in vascular smooth muscle cells and anti-aggregator effects in platelets.19
Experimental Groups and Design On the day of relief after 5 days of obstruction 10 rats each were randomly assigned to groups, including group 1—female r-UUO without treatment, group 2—male r-UUO without treatment, group 3—female r-UUO plus BAY 418543 and group 4 —male r-UUO plus BAY 41-8543. Four female and 4 male control rats with sham surgery were included. BAY 41-8543 treatment began directly after r-UUO. Seven days after r-UUO we determined the actions of the sGC stimulator BAY 41-8543 on NO/cGMP signaling and disease parameters. Systolic blood pressure was assessed 7 days after r-UUO in conscious rats using tail cuff plethysmography, as previously described.16
Experiment Termination Rats were anesthetized, as previously described.13 After a midline abdominal incision was made 5 to 10 ml blood were drawn from the abdominal aorta into ethylenediaminetetraacetic acid coated tubes. The kidneys were perfused with 40 ml ice-cold phosphate buffered saline. Materials and tissues were subsequently processed as described.
NO/cGMP Signaling
MATERIALS AND METHODS Materials and Animals Unless otherwise indicated, materials, chemicals and cell culture medium were obtained from Sigma-Aldrich®. Female and male Sprague-Dawley® rats with a body weight of 150 to 180 gm were housed in a constant temperature room with a 12-hour dark/12-hour light cycle. Animals were fed a standard 22.5% protein diet (Altromin, Lage, Germany) and had free access to water. Body weight, and food and water intake were monitored daily throughout the experiment. Animal care and treatment conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the local authorities.
r-UUO Model Induction The female and male Sprague-Dawley rats were subjected to complete UUO.13,14 Briefly, the left ureter was exposed
Plasma cGMP was measured by enzyme-linked immunosorbent assay (Amersham™ Pharmacia™ Biotech) according to manufacturer instructions.16 Tubulointerstitial mRNA expression of the ␣1 and 1-sGC subunits, and eNOS was measured using reverse transcriptase-PCR.
Light and Immunohistochemical Microscopy All histological examinations were done in blinded fashion using computer based morphometric analysis, as described previously.20 All histological parameters were evaluated using cortical tissue fixed in Carnoy’s solution except the TUNEL assay, for which buffered, formalin fixed tissue was used. The relative degree of tubulointerstitial lesions, ie matrix deposition, tubular atrophy and dilatation, inflammation and cell proliferation, was calculated in 10 randomly selected cortical areas per rat at 400⫻ magnification and expressed as the area affected in relation to the total area analyzed.
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Assessment Tubulointerstitial fibrosis. We evaluated renal fibrosis by measuring tubulointerstitial total collagen deposition, interstitial volume, collagen IV deposition and ␣-SMA expression. Total collagen deposition was evaluated by aniline blue staining. Interstitial volume was determined by a point counting technique on tissue sections stained by collagen IV using a 10 ⫻ 10 orthographic grid overlaid on digital images, as previously described.13 Collagen IV deposition and ␣-SMA expression were measured by positive collagen IV and ␣-SMA staining, respectively, in relation to select cortical sections, as previously described.13 The antifibrotic mechanism was further analyzed by determining mRNA expression of the key fibrosis marker and mediator TGF-1, the matrix protein fibronectin as an indicator of matrix protein synthesis, and tissue PAI-1 as a marker of matrix protein degradation. Tubular atrophy and apoptosis. Tubular atrophy was determined by measuring tubular diameter on cortical sections stained with collagen IV. Tubular apoptosis was evaluated with the TUNEL assay using the Apoptosis Detection Kit (Chemicon®), as described by the manufacturer. The number of TUNEL positive tubular cells was divided by the total number of tubular cells to indicate tubular apoptosis. Tubulointerstitial inflammation and cell proliferation. Tubulointerstitial macrophage infiltration and cell proliferation were evaluated by ED1 and PCNA positive cells in selected cortical areas using immunohistochemistry, as previously described.16,17
mRNA Expression Cortical mRNA expression was determined by 2-step reverse transcriptase-PCR, as described previously.16,21 The primer pairs of eNOS, ␣1 and 1-sGC, TGF-1, fibronectin and PAI-1 with GAPDH as the housekeeping gene were used as previously described.17
Statistical Analysis
each p not significant, fig. 1, A). BAY 41-8543 significantly lowered systolic blood pressure a mean of 117 ⫾ 3 mm Hg in group 3 and 114 ⫾ 4 in group 4 (vs groups 1 and 2, respectively, each p ⬍0.01). NO/cGMP Signaling Ureteral obstruction induced a significant increase in ␣1-sGC mRNA in groups 1 and 2 vs controls (mean 187% ⫾ 22% vs 101% ⫾ 8% and 169% ⫾ 14% vs 101% ⫾ 8%, respectively, each p ⬍0.01, fig. 2). It also induced a significant increase in 1-sGC mRNA in groups 1 and 2 vs controls (mean 188% ⫾ 22% vs 100% ⫾ 5% and 185% ⫾ 19% vs 101% ⫾ 8%, respectively, each p ⬍0.01). Plasma cGMP remained unaltered in groups 1 and 2 vs controls (mean 503 ⫾ 40 vs 528 ⫾ 66 and 479 ⫾ 29 vs 545 ⫾ 129 fmol per well, respectively, each p not significant). In contrast, there was a significant increase in plasma cGMP in groups 3 and 4 vs 1 and 2 (412% and 104%, respectively, each p ⬍0.01, fig. 1, B). BAY 41-8543 induced significantly higher plasma cGMP in group 3 than in group 4 (2,573 ⫾ 509 vs 977 ⫾ 113 fmol per well, p ⬍0.05). mRNA expression of the ␣1 and 1-sGC subunits was normalized after treatment with the sGC stimulator. mRNA expression of eNOS was not significantly affected by gender, r-UUO or BAY 41-8543. Tubulointerstitial Matrix Accumulation Figures 3 and 4 show representative histological views of tubulointerstitial changes in the r-UUO model, and the effects of gender and BAY 41-8543. Histomorphometric analysis indicated that the severity of tubulointerstitial matrix expansion and fibrosis was significantly less in group 1 than in group 2. Mean tubulointerstitial volume was 15.4% ⫾ 0.7% vs 19.3% ⫾ 0.5% in group 1 vs 2 (p ⬍0.001, fig. 5).
Results are shown as the mean ⫾ SEM. Statistical analysis between groups was performed by 1-way ANOVA and a subsequent t test with p ⬍0.05 considered significant.
RESULTS Body Weight, and Food and Drug Intake At the end of the experiments body weight did not differ among groups of the same gender but body weight in females was significantly lower than in males (mean 190 ⫾ 5, 185 ⫾ 3 and 185 ⫾ 2 gm in female controls, and groups 1 and 3 vs 228 ⫾ 7, 218 ⫾ 4 and 219 ⫾ 4 in male controls, and groups 2 and 4, respectively, each female vs male group p ⬍0.001). Systolic Blood Pressure Systolic blood pressure was slightly increased in groups 1 and 2 on day 7 after r-UUO vs controls (mean 145 ⫾ 8 vs 131 ⫾ 6 and 146 ⫾ 8 vs 122 ⫾ 7 mm Hg,
Figure 1. Effects of biological gender and BAY 41-8543 (⫹Bay) 7 days after r-UUO in groups 3 (open bars) and 4 (black bars). A, systolic blood pressure was measured in conscious rats by tail cuff method. Asterisks indicate p ⬍0.01 vs same gender group 1 or 2. B, plasma cGMP was measured by enzyme-linked immunosorbent assay. Asterisk indicates p ⬍0.05 vs same gender group 1 or 2. Pound sign indicates p ⬍0.05 vs group 4.
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Figure 2. Effects of biological gender and BAY 41-8543 (⫹Bay) 7 days after r-UUO in groups 3 (open bars) and 4 (black bars) on mRNA expression analyzed by real-time PCR with GAPDH as housekeeping gene, shown as measured mRNA/GAPDH. A, ␣1-sGC mRNA. Curleys indicate ⬍0.01 vs same gender control. Single asterisk indicates p ⬍0.05 vs same gender group 1 or 2. Double asterisks indicate p ⬍0.01 vs same gender group 1 or 2. B, 1-sGC mRNA. C, eNOS mRNA.
Aniline staining showed that mean matrix protein expansion was 6.9% ⫾ 0.9% vs 10.3 ⫾ 1% in group 1 vs 2 while mean collagen IV deposition was 3.8% ⫾ 0.5% vs 5.4% ⫾ 0.5% (each p ⬍0.05, fig. 5).
BAY 41-8543 plus r-UUO significantly reduced tubulointerstitial volume ⫺29% in group 3 and ⫺38% in group 4 (vs groups 1 and 2, respectively, each p ⬍0.001). Histological tubulointerstitial fibrosis was more effectively limited in group 4 than in
Figure 3. Representative renal sections show histological collagen IV protein deposition 7 days after r-UUO. ⫹Bay, BAY 418543. Collagen IV staining, reduced from ⫻400.
Figure 4. Representative histological ␣-SMA protein expression 7 days after r-UUO. ⫹Bay, BAY 41-8543. ␣-SMA staining, reduced from ⫻400.
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group 3, including matrix protein expansion ⫺60% vs ⫺16%, collagen IV deposition ⫺52% vs ⫺19% and ␣-SMA expression ⫺69% vs ⫺49% (group 4 vs 2, respectively, each p ⬍0.05). In line with the histological analysis group 1 showed lower renal mRNA expression than group 2, including TGF-1 mRNA (mean 203% ⫾ 30% vs 300% ⫾ 42%, p ⫽ 0.07), fibronectin mRNA (mean 448% ⫾ 73% vs 714% ⫾ 207%, p not significant) and PAI-1 mRNA (mean 81% ⫾ 22% vs 574% ⫾ 195%, p ⬍0.05, fig. 6). BAY 41-8543 lowered mRNA expression in group 3, although more effectively in group 4, compared to groups 1 and 2, including TGF-1 ⫺45% in group 3 (vs group 1 p ⬍0.05) and ⫺52% in group 4 (vs group 2 p ⬍0.01), fibronectin ⫺74% (p ⬍0.01) and ⫺77% (p ⬍0.05), and PAI-1 no decrease and ⫺80% (p ⬍0.05), respectively.
Figure 5. Computer based morphometric analysis of effects of biological gender and BAY 41-8543 (⫹Bay) 7 days after relief of UUO in groups 3 (open bars) and 4 (black bars). A, tubulointerstitial matrix accumulation measured by aniline blue staining. Single asterisk indicates p ⬍0.05 vs same gender group 1 or 2. Single pound sign indicates p ⬍0.05 vs group 2. Single curley indicates p ⬍0.05 vs control. Double curleys indicate p ⬍0.01 vs control. B, interstitial volume determined by point counting technique on tissue sections stained for collagen type IV. Triple asterisks indicate p ⬍0.001 vs same gender group 1 or 2. Triple pound signs indicate p ⬍0.001 vs group 2. Triple curleys indicate p ⬍0.001 vs control. C, collagen IV deposition. D, ␣-smooth muscle actin. Double asterisks indicate p ⬍0.01 vs same gender group 1 or 2.
Tubular Atrophy and Apoptosis In group 1 tubular diameter was remarkably less dilated than in group 2 (mean 36.3 ⫾ 0.5 vs 41.2 ⫾ 0.3 m, p ⬍0.001, fig. 7, A). sGC stimulation with BAY 41-8543 improved tubular dilatation in groups 3 and 4 (mean 30.4 ⫾ 0.2 and 33.0 ⫾ 0.7 m, vs groups 1 and 2, respectively, each p ⬍0.001). Ureteral obstruction induced a significant increase in the number of apoptotic cells in groups 1 and 2 vs controls (mean 21% ⫾ 4.0% vs 1% ⫾ 0.2% and 18% ⫾ 2.5% vs 2% ⫾ 0.5%, respectively, each p ⬍0.01, fig. 7, B). BAY 41-8543 significantly decreased tubular apoptosis ⫺76% in group 3 and ⫺67% in group 4 (vs groups 1 and 2, respectively, each p ⬍0.05). Renal Macrophage Infiltration and Cell Proliferation Untreated r-UUO was associated with a significantly higher number of tubulointerstitial ED1 positive cells, indicating macrophages, in groups 1 and
Figure 6. Effects of biological gender and BAY 41-8543 (⫹Bay) 7 days after r-UUO in groups 3 (open bars) and 4 (black bars) on mRNA expression analyzed by real-time PCR with GAPDH as housekeeping gene, shown as measured mRNA/GAPDH. A, TGF-1 mRNA. Single asterisk indicates p ⬍0.05 vs same gender group 1 or 2. Double asterisks indicate p ⬍0.01 vs same gender group 1 or 2. Single curley indicates p ⬍0.05 vs control. Double curleys indicate p ⬍0.01 vs control. B, fibronectin mRNA. C, PAI-1 mRNA. Pound sign indicates p ⬍0.05 vs group 2.
IMPACT OF GENDER AND SOLUBLE GUANYLATE CYCLASE STIMULATION ON RENAL RECOVERY
Figure 7. Effects of biological gender and BAY 41-8543 (⫹Bay) 7 days after r-UUO in groups 3 (open bars) and 4 (black bars) in computer based morphometric analysis. A, tubular diameter was used to measure tubular atrophy. Asterisks indicate p ⬍0.001 vs same gender group 1 or 2. Pound signs indicate p ⬍0.001 vs group 2. Curleys indicate p ⬍0.001 vs control. B, tubular apoptosis was measured by TUNEL assay. Single asterisk indicates p ⬍0.05 vs same gender group 1 or 2. Double asterisks indicate p ⬍0.01 vs same gender group 1 or 2. Double curleys indicate p ⬍0.01 vs control. C, tubulointerstitial macrophage infiltration. Asterisk indicates p ⬍0.05 vs same gender group 1 or 2. Single curley indicates p ⬍0.05 vs same gender control. Double curleys indicate p ⬍0.01 vs same gender control. Primary ED1 antibody, reduced from ⫻400. D, cell proliferation. Primary PCNA antibody, reduced from ⫻400.
2 vs controls (mean 38% ⫾ 6% vs 1% ⫾ 0.3% and 46% ⫾ 7% vs 5% ⫾ 0.2%, respectively, each p ⬍0.01, fig. 7, C). Untreated r-UUO was also associated with significantly more PCNA positive cells, indicating cell proliferation, in groups 1 and 2 vs controls (mean 5.2% ⫾ 0.5% vs 0.4% ⫾ 0.1% and 6.4% ⫾ 1.7% vs 1.3% ⫾ 0.1%, respectively, each p ⬍0.05, fig. 7, D). BAY 41-8543 decreased tubulointerstitial macrophage infiltration vs r-UUO alone ⫺50% in group 3 and ⫺65% in group 4 (group 4 vs 2 p ⬍0.05). It also decreased cell proliferation ⫺35% and ⫺44%, respectively.
DISCUSSION Ureteral obstruction is an important cause of renal failure in children and adults. Recovery of obstructive nephropathy after the relief of urinary obstruction is an especially important clinical issue. There
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were several major findings in this animal model of r-UUO. 1) Significant renal fibrotic, apoptotic and inflammatory changes were detectable 7 days after the relief of transient UUO. 2) Female rats with r-UUO showed less tubulointerstitial fibrosis and tubular atrophy than male rats. 3) The gender difference was associated with a more sensitive NO/cGMP signaling pathway in female rats. 4) In rats of each gender treatment with the sGC stimulator BAY 41-8543 significantly increased endogenous cGMP production and decreased tubulointerstitial fibrosis and inflammation as well as tubular atrophy and apoptosis. 5) In male r-UUO rats sGC stimulation was significantly more effective for renal fibrosis and cell infiltration parameters, which largely ameliorated the differences in disease severity due to the gender disparity. In our study group 1 showed significantly less tubular dilatation, tubulointerstitial volume expansion, collagen IV deposition and TGF-1 mRNA expression than group 2. This impact of biological gender on outcome is in line with human data. Studies suggest that female rat mesangial cells inherently show fewer profibrotic and proinflammatory characteristics than male cells.22 The relaxation of mesenteric arterial rings by the exogenous NO donor is greater in spontaneously hypertensive female rats than in the corresponding males.23 Male rats are more susceptible than female rats to the vascular and renal injury induced by fructose feeding or hypercholesterolemia, which are states associated with NO deficiency.24,25 These studies indicate that the male represents an inherent gender phenotype for profibrotic, proinflammatory characteristics, blunt reactivity to NO and the subsequent increased sensitivity to NO deficiency. In the kidney NO produced by endothelial cells is vasodilatory and inhibits mesangial cell growth, matrix production, cell proliferation and inflammatory cell infiltration. Arterial walls and mesangial cells are main sites of sGC expression, representing the physiological receptor of NO and mediating the downstream renoprotective effects of endothelial NO production.26 Thus, different sGC sensitivities and activities may well be involved in the gender difference of the mesangial cell reaction to vascular and renal injury. In our series renal sGC mRNA expression was significantly up-regulated in the untreated r-UUO rats, which may serve as a biological self-protective pathway to potentially enhance NO/cGMP signaling. However, this increase was not strong enough to lead to correspondingly increased plasma cGMP or counteract pathogenetic factors. Overall this indicates relative insufficiency in NO/cGMP signaling. However, BAY 41-8543 treatment was highly effective in stimulating sGC activity and increasing
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cGMP production in group 3 as well as group 4. After 1 week of therapy disease severity, eg tubular apoptosis, tubulointerstitial fibrosis and inflammation, was decreased to a comparable degree in groups 3 and 4. The plasma cGMP level stimulated by BAY 41-8543 in group 3 was much higher than in group 4, indicating lower sGC sensitivity in male rats. This might be an important reason for the gender difference in NO deficiency and obstructive nephropathy. In groups 3 and 4 significantly increased plasma cGMP and decreased systolic blood pressure indicate the pharmacological properties of the sGC stimulator and the vasodilative effects of enhanced NO/cGMP signaling. BAY 41-8543 significantly increased endogenous cGMP production, thus, attenuating tubulointerstitial fibrosis in each biological gender by decreasing collagen deposition, ␣-SMA expression and tubulointerstitial volume. The molecular antifibrotic mechanism may be related to decreased expression of the key profibrotic mediator TGF-1, matrix protein fibronectin and the matrix degradation inhibitor PAI-1. The second key renoprotective effect of BAY 418543 was associated with decreased tubulointerstitial macrophage infiltration. As an important source of TGF- production under pathological conditions, less white cell infiltration may have an important role in the renoprotective effect of sGC stimulation. Furthermore, BAY 41-8543 treatment lowered tubular atrophy, as shown by the decreased tubular diameter and positive apoptotic cells in male and female rats in our r-UUO model. Thus, BAY 41-8543 showed antifibrotic, anti-inflammatory and antiapoptotic effects, and potentiated disease recovery in the r-UUO model via increased cGMP production.
A decrease in the expression or activity of vascular sGC rather than eNOS/NO was recently reported in several diseases characterized by a functional deficiency in NO/cGMP signaling.27 This included rats with anti-thy1-induced renal disease, spontaneous hypertension, aging, myocardial infarction, angiotensin II infusion and lead induced hypertension, and diabetic Goto-Kakizaki rats. In cases of insensitive sGC and insufficient NO/cGMP signaling, as in the r-UUO model, therapy that primarily increases NO production might have no or only a limited effect. Also, compounds that enhance NO/cGMP signaling by interaction with the sGC enzyme have a number of therapeutic advantages over L-arginine, NO donors and gender hormone therapy, which have been previously used to overcome impaired NO production. sGC stimulators amplify the cGMP signal exactly at that subcellular location and avoid potentially unwanted side effects via unrelated pathways.
CONCLUSIONS In this study female rats showed less tubulointerstitial fibrosis and tubular atrophy in the UUO model after r-UUO. Data indicate that female associated renal protection is linked to higher sensitivity of the sGC enzyme and the downstream cGMP signaling pathway. sGC stimulation by BAY 41-8543 increased cGMP production in rats with r-UUO, and ameliorated disease severity and the gender difference in renal fibrosis and apoptosis. Findings suggest that pharmacological sGC stimulation may be a novel approach to slow the progression of obstructive nephropathy in males and females.
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