Losartan Promotes Survival And Repairs Podocyte Injury In5/6Nephrectomy Rats

Background and ObjectiveEpidemiological evidence suggests that chronic kidney disease (CKD) has become one of the worldwide public health problems. Our previous study indicated that the prevalence of CKD was13.57%in urban adult population of Zhengzhou. Glomerulosclerosis is the final outcome of all kinds of chronic kidney injury no matter what is the cause. Recent data from both animals and humans have documented rennin-angiotensin system (RAS) plays an important role in the progression of kidney disease. Activating of RAS can increase bloodpressure and accelerate glomerulosclerosis via hemodynamic change, however, Angiotensin II (Ang II) also involves in glomerulosclerosis via nonhemodynamic effect. The application of RAS inhibitor-Angiotensin receptor antagonist (ARB) is the best way in the clinical therapy. Recent studies by Dr. Fogo’s group and others indicate that regression of glomerulosclerosis is achievable by high dose of RAS inhibition in animal models, linked to inhibition of plasminogen activator inhibitor-1(PAI-1). Our new findings suggest that PAI-1is not only an important molecule in inhibiting matrix turnover, but is also expressed in podocytes, having a direct role in modulating podocyte differentiation and cytoskeletal rearrangements. Podocyte injury/repair has been the focus of investigation of kidney injury because it plays an indispensable role as a filtration barrier for maromolecules in the glomerulus. Podocyte injury induces proteinuria and glomerulosclerosis, some current reports suggests parietal epithelial cell (PEC) has potiential ability to recover injury podocyte. However, whether long-term RAS inhibition induces even greater regression of glomerulosclerosis and whether RAS inhibition increases long-term survival of CKD rats are unknown. In this study we investigated whether RAS inhibition promoted long-term survival taking advantage of5/6nephrectomy (Nx) rats and the possible mechanism of protective effect of ARB on podocyte injury, this would provide clinical experimaental basis for using ARB to slow down CKD progression and promote CKD long-term survival.Therefore, the study was further divided into two parts.Part Ⅰ:ARB promotes long-term survival and protects podocyte injury in5/6Nx rats.We took advantage of5/6Nx rats and assessed the glomerulosclerosis index at eight weeks after Nx by renal biopsy, and investigated the long term effects on survival and the possible mechanism.Part Ⅱ:The protective effect of ARB on Ang Ⅱ induced podocyte injury.We cultured podocyte from wild type and PAJ-1-/-mouse kidneys then exposed to Ang Ⅱ and detected the effect of ARB on the podocyte injury.Part Ⅰ:ARB promotes long-term survival and protects podocyte injury in5/6Nx rats.Materials and MethodsForty-one adult male Sprague Dawley rats (250to300g; Charles River, Nashville, TN, USA) were studied. Rats underwent5/6nephrectomy (Nx). Severity of glomerulosclerosis was assessed by sclerosis index (SI;0-4scale) scoring from open renal biopsy (Bx) performed8wks later. Rats were then divided into two groups with equal average systolic blood pressure (SBP),24-h urinary protein (Uprot) and biopsy glomerulosclerosis index. Control animals received no further treatment (CONT; n=21). The ARB group received losartan [ARB;200mg/L losartan in drinking water (DW); n=20]. Body weight, SBP,24-h urinary protein, serum creatinine were measured at baseline and week8,16,24and30. Animals were monitored till30weeks. Kidneys were harvested at30wks after5/6Nx or before death. Change of glomerulosclerosis index was assessed by PAS staining. PAI-1expression was assessed by Western blot. Podocyte injury and parietal epithelial cell (PEC) to podocyte transition were assessed by immunohistochemistry and immunofluorescence staining. The time occurrence of death (weeks) was assessed by Kaplan-Meier cumulative survival functions. Time-to-event (death) was calculated in weeks. Log-rank test was used to exam if the survival time were different between different treatments. We used log-rank test to look for association between survival time and variate at8weeks. Cox proportional hazards model was used for multivariate survival analysis, Time depended AUC is used to find the cut-off point of sclerosis index to separate rats into two groups with most different survival curve. P<0.05was considered to be significant.Results1. SBP was increased at8weeks after5/6Nx vs baseline in all rats (.P>0.05CONT vs ARB) and continued to increase in CONT, while ARB had significantly lower SBP at week16,24(P<0.05). Uprot was increased equally in two groups at8weeks after5/6Nx and was less at16wks,24wks and30wks in ARB than CONT (P<0.05). Renal function assessed by serum creatinine at24wk and30wk was significantly improved in ARB treated rats (P<0.05).2. ARB treatment resulted in significantly higher30wks survival rate vs non-treated CONT5/6Nx rats (median survival time27.1wks vs19.7wks, ARB vs CONT, P<0.05). Univariate model analysis showed that biopsy SI, protein uria and treatment, are three independent predictors for survival time. Multivariate Cox model analysis indicated treatment and Bx SI are two key factors with significant effects on survival time. The hazard ratio of uremic death in rats without treatment was3.5(P=0.0019) with hazard ratio2.9per unit increase in starting sclerosis at biopsy (P=0.0006). Then we subgrouped the rats by the cut-off point of biopsy SI, we found that ARB promoted survival in two different SI group (the rats with SI<1.1, median survival time>30weeks vs20.6weeks, ARB vs CONT, P=0.0284; the rats with SI>1.1, median survival time22weeks vsl7.3weeks, ARB vs CONT, P=0.0153).3. At endpoint, the ARB treated rats had lower average SI than CONT rats (P<0.05). The average sclerosis progression rate was significantly decreased by ARB treatment (P<0.05).4. At endpoint, PAI-1expression in CONT rats was lowered than the ARB treated rats(P<0.05).5. At biopsy, WT1-positive podocyte numbers were decreased vs normal baseline kidney while claudin1-positive PECs were increased at biopsy vs baseline. ARB significantly maintained WT-1-positive podocyte numbers vs non-treated CONT (P<0.05). Interestingly, claudin1immunostaining was increased in capillary loop area at sacrifice (P<0.05). ARB-treated rats showed more glomerular cells with positivity for both synaptopodin (podocyte marker)and claudin1(a PECs marker) by performing immunofluorescence double staining, indicating ARB could promote PECs to podocyte transition.Part II:The protective effect of ARB on Ang Ⅱ induced podocyte injury.Materials and MethodsWe cultured podocyte from wild type (WT) and PAI-1-/-(PAI-1KO) mouse kidneys. Podocyte identity was confirmed by WT-1, nephrin and synaptopodin staining. then exposed to Ang Ⅱ and detected the effect of ARB on the podocyte injury. To determine whether regulated apoptosis in podocyte and PAI-1deficiency protect Ang Ⅱ induced podocyte apoptosis, podocytes (from WT vs PAI-1KO) were treated as follws:exposed to AngⅡ (10-7M,48h) and control cells without Ang II. To assess effects of ARB on collegen IV expression in podocytes, podocytes (from WT vs PAI-1KO) were treated as follws:Ang II group:podocytes were exposed to AngⅡ (10-7M,48h), AngⅡ+ARB group:podocytes were preincubated with ARB (10-5M,24h) then exposed to AngⅡ (10-7M,48h), and control cells without Ang II or ARB. Collagen al/2(IV) accumulation in the supermatant was assessed by ELISA.Results1. Podocytes were identified by immunofluorescence staining for podocyte specific marker WT1, nephrin and synaptopodin. Resulting cultures were>95%podocytes.2. In wild type podocytes, Ang Ⅱ stimulated secreted PAI-1activity1.22fold (P<0.05). Ang Ⅱ increased WT podocyte apoptosis90.91%, with no change in PAI-1-/-podocytes. Bcl-xl was reduced in WT induced by Ang Ⅱ, compared to no change in PAI-1-/-podocytes induced by Ang Ⅱ.3. In wild type podocytes, but not PAI-1-/-podocytes, Ang Ⅱ significantly increased collagen1/2(IV) in supernatant measured by ELISA compared to control. ARB completely blocked this response in wild type podocytes.Conclusions1. ARB treatment promoted30weeks survival in5/6Nx rats, the rats with milder sclerosis at biopsy had a longer survial.2. ARB treatment decreased SBP, Uprot, improved renal function and slow glomerulosclerosis progression.3. ARB protect renal injury induced by PAI-1decrased.4. We postulate that the beneficial effects of ARB on survival are in part linked to maintained podocyte numbers contributed to by parietal epithelial cell to podocyte transition.5. PAI-1deficiency protected Ang Ⅱ induced podocyte apoptosis.6. Ang Ⅱ increased collagen content in podocyte supernatant. ARB completely blocked this response in wild type podocytes. PAI-1deficiency decreased collagen content in Ang Ⅱ induced podocyte.