Renoprotective Effect Of The Combination Of Irbesartan With Clopidogrel On 5/6 Nephrectomized Rats

Background and ObjectiveDespite significant advances in the understanding of chronic kidney disease (CKD), the incidence of end-stage renal failure (ESRF) continues to increase. Possible mechanisms of progressive renal damage include hemodynamic factors and chronic inflammation. Regardless of the initial insult, the natural history of CKD is relentless progression to ESRF. Following a progressively downhill course, the residential nephrons are adapted with compensatory hypertrophy and hyperfiltration partially for nephron losses. It is of significance to further elucidate the mechanism and to explore a way to arrest the established renal injury. Angiotensin Ⅱ (Ang Ⅱ) can regulate cell growth and fibrosis through the production of several mediators, it plays an active role in the progression of renal injury. Platelet releases a variety of growth factors and proinfalmmatory cytokines, which participates in the pathogenesis of CRD progression. Ang Ⅱ and platelet participate in the pathogenesis of chronic nephropathy, including glomerular extracellular matrix (ECM) accumulation and inflammatory reaction. Blockade of Ang Ⅱ or platelet activation delays the progress of renal damage. However, little is known about the combination effect of blocking both Ang Ⅱ and platelet simultaneously. Platelet expresses Ang Ⅱ type 1 receptors on their surface. AngⅡ can promote platelet aggregation and releasing products, it also can participate in the thrombotic processes. Sowe hypothesized that the combination effect of blocking both Ang II and platelet simultaneously could delay the progress of renal damage more effectively. Irbesartan is Angll type 1 receptor antagonist (ATiRA), clopidogrel specially inhibits ADP-dependent platelet activation. The study observed renoprotective effect of combination therapy of both irbesartan and clopidogrel on 5/6 nephrectomized rats. To study the possible mechanism of the nephroprotective effects, Histological change, platelet relative index, some cytokines were detected.MethodsForty male Wistar rats were subjected to 5/6 nephrectomy and were randomly assigned to 5 groups (n=10): untreated group, irbesartan group (20mg/kg/d), clopidogrel group (20mg/kg/d), and irbesartan plus clopidogrelgroup, and the sham-operated rats served as the control group (n=10). The drugs and distilled water were given by drenching for successive 12 weeks. Rats were monitored and sacrificed at 12th weeks. Blood, urine and tissue samples were harvested. Twenty-four hours urinary protein (UP) and serum creatinine (SCr) were measured. Plasma GMP-140 was assessed by enzyme-linked immunosorbent assay (ELISA). Histological changes in the kidney were evaluated at the end of the study. Direct immunofluorescence was applied to detect the expression of fibrinogen (Fib); fibroblast growth factor-2 (FGF-2), intercellular adhesion molecule-1 (ICAM-1), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) protein and mRNA levels were examined with immunohistochemistry, Western blot and Northern blot.Results1. The body weight, UP and SCr: The body weights of rats in sham-operated or treated groups were significantly higher than those of untreated group at each time point (P<0.05). There was no prominent difference between the treated groups (P>0.05). The UP and SCr of untreated group were higher than those of the sham-operated group (P<0.01). Irbesartan or clopidogrel reduced UP and SCr, Irbesartan was more effective than clopidogrel (P<0.05). Compared to single irbesartan or clopidogrel, combinationtherapy was more effective in reducing UP and SCr (P<0.05).2. Histological change: Glomenilar ECM of untreated group was higher than that of the sham-operated group (P<0.01). Irbesartan or clopidogrel decreased glomenilar ECM accumulation. Irbesartan was more effective than clopidogrel (P<0.05). Compared to single irbesartan or clopidogrel, combination therapy was more effective in reducing glomenilar ECM accumulation (P<0.05).3. Platelet relative index: GMP-140 and the accumulation of Fib of untreated group were higher than those of the sham-operated group (P<0.01). Clopidogrel significantly reduced GMP-140 and Fib deposition (P<0.05). Irbesartan reduced GMP-140 and Fib in a far less pronounced way. Compared to single irbesartan or clopidogrel, combination therapy was more effective in reducing Fib deposition(P<0.05), but there was no significant difference in plasma GMP-140 between combination therapy and single clopidogrel therapy.4. Immunohistochemistry: The expression of FGF-2, ICAM-1, PAI-1, and TIMP-1 of untreated group were higher than those of the sham-operated group (0.1 + 0.01 vs. 0.01 + 0.002; 0.28 + 0.01 vs. 0.03 + 0.005; 0.21 + 0.016 vs. 0; 0.24 + 0.008 vs. 0.017 + 0.007. P<0.01). Irbesartan or clopidogrel reduced the expressions of FGF-2, ICAM-1, PAI-1, and TIMP-l(0.03 +0.005 vs. 0.1+0.01, 0.06 + 0.007 vs. 0.1 + 0.01; 0.08+0.01 vs. 0.28 + 0.01, 0.12±0.011 vs. 0.28 + 0.01; 0.07±0.014 vs. 0.21 + 0.016, 0.09+0.01 vs. 0.21 ±0.016; 0.07 + 0.008 vs. 0.24 + 0.008, 0.12 + 0.012 vs. 0.24 + 0.008. P<0.05). Irbesartan was more effective than clopidogrel (0.03+0.005 vs. 0.06 + 0.007; 0.08+0.01 vs. 0.12 + 0.011; 0.07 + 0.014 vs. 0.09 + 0.01; 0.07 + 0.008 vs. 0.12 + 0.012. P<0.05). Compared to single irbesartan or clopidogrel, combination therapy was more effective in reducing the expression of FGF-2, ICAM-1, PAI-1, and TIMP-1 (0.02 + 0.003 vs. 0.03 + 0.005, 0.02 + 0.003 vs. 0.06+0.007; 0.06±0.012 vs. 0.08±0.01, 0.06±0.012 vs. 0.12+0.011; 0.04+ 0.01 vs. 0.07 + 0.014, 0.04+0.01 vs. 0.09 + 0.01; 0.05 + 0.009 vs. 0.07 + 0.008, 0.05 + 0.009 vsO.12 +0.012. P<0.05).5. Western blot and Northern blot: The protein and mRNA levels of FGF-2, ICAM-1, PAI-1, and TIMP-1 of untreated group were higher than those of thesham-operated group (P<0.01). Irbesartan or clopidogrel reduced the protein and mRNA levels of FGF-2, ICAM-1, PAI-1, and TIMP-1. Irbesartan was more effective than clopidogrel (P<0.05). Compared to single irbesartan or clopidogrel, combination therapy was more effective in reducing the protein and mRNA levels of FGF-2, ICAM-1, PAI-1, and TIMP-1 (P<0.05).Conclusion1. It demonstrates that Single irbesartan and/or clopidogrel therapy have renoprotective effect on 5/6 nephrectomized rats. Compared to single irbesartan or clopidogrel, combination therapy was more effective.2. Renoprotective effect of the combination of irbesartan with clopidogrel relates to the inhibition renal glomerular ECM accumulation and inflammatory reaction.