Study Of Effect Of Telmisartan On ERS-related Kidney Cells Apoptosis In Diabetic Rats

Background and ObjectiveDiabetic nephropathy (DN), a common and serious diabetes complication, is one of the important factors to lead end-stage renal disease (ESRD). Recent studies have found that apoptosis, as a primary and independent factor, plays an essential role in the pathogenesis of DN. Endoplasmic reticulum stress (ERS) induced apoptosis pathway is a newly discovered one. It may play an important role in the development of diabetic. Therefore, the anti-ERS treatment has naturally become the new choice of treatment of diabetic complications. AngiotensinⅡreceptor antagonists (ARB) are widely used in the treatment of DN. They are used to delay the development and deterioration of DN by lowering blood pressure and reducing proteinuria. However, the specific mechanisms remain unclear.In this experiment, we established diabetic rats model by intraperitoneal injection of streptozotocin and observed the general biochemical changes in each group rats and the expression of ERS-induced apoptosis pathway-related factors GRP78, CHOP, and caspasel2 in renal tissue. And then we discussed the possible effect of ERS-induced apoptosis in DN and the effect of ARB telmisartan on ERS-related kidney cells apoptosis in diabetic rats.Materials and MethodsAmong 48 healthy male SD rats,10 rats was used as the normal control group (NC group) (n=10 rats),38 rats were intraperitoneally injected STZ(60mg/kg) for diabetes animal model.8 unsuccessful rats were removed. Successful model rats were divided into two groups:diabetic group (DM group) (n=15), telmisartan treatment group (DT group) (n=15). Each group rats were given normal diet. DT rats were intragastrically administered with telmisartan (10mg/kg, dissolved in distilled water) every morning, while NC and DM group were only given the equivalent distilled water for 12 weeks. After the experiment, the weight of the rats and right kidney, the level of 24h urinary albumin, blood glucose, serum insulin, and serum creatinine were measured; the right amout of kidney tissues were kept to do immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) and detect the level of GRP78, caspase12 and CHOP protein and gene expression; Terminal deoxynucleotidy ltransferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) staining was used to detect renal cell apoptosis. All data were statistically analyzed, withα=0.05 as a statistically significant.Results1. After establishing DM model, six of them are removed because of the decrease of the blood sugar. In this experiment, two rats of DM group died and one of DT group died.There are thirty-one rats completing the final experiment. Compared to NC, the glucose of DM rats is greatly increasing (P<0.05). With hemaoxylin-eosin (HE) staining, we found that glomerular hypertrophy, lumen pressured, basement membrane thicken, msangial broaden, extracellular matrix increased, some glomerular volume reduced and occurred sclerosis in DM group. With Masson staining, we found that the collagen in kidney tissues of DM rats significantly increased. Coarse collagen fibers connected with each other to a mesh, disorganized and distributed unevenly in DM group.24h urine protein of DM rats was significantly higher (P<0.05). Serum creatinine level was significantly higher in the DM group (P<0.05).The results suggested that establishment of type 1 diabetes DN model is successful.2. TUNEL staining assay of renal cell apoptosis showed that positive staining signals were located in kidney cell nuclei, stained in brown, the size of positive staining cell nuclei is equal to or less than the normal nucleus. Apoptosis was can occasionally be seen in NC group. NC apoptotic index is (2.71±0.19)%. DN apoptotic index is (25.75±2.72)%. The apoptotic index of DM group was significantly higher than that of the NC group (P<0.05). The related analysis showed that 24h urinary protein and renal cell apoptosis index was significantly positive correlation in DM group and NC group (all P<0.05).3. Compared with the NC group, expression of GRP78, caspasel2 and CHOP protein and mRNA expression were all increased in DM group (P<0.05). Meanwhile, the expression of GRP78, caspasel2, CHOP mRNA and proteins had significant positive correlation with kidney apoptosis index in the DM group and NC group. (all P<0.05).4. By telmisartan treatment, pathological changes of glomerular and tubular fibrosis and collagen in DT group rats significantly reduced compared with DM group,24h urine protein and serum creatinine value were significantly less than that of DM group (all P<0.05). With TUNEL staining assay, renal cells apoptosis of DT group decreased compared with DM group (P<0.05). Meanwhile, after Telmisartan treatment, GRP78, Casepes12, CHOP protein and mRNA expression levels were significantly lower compared with DM group (P<0.05). In addition, there is a positive relation between 24h urine protein and kidney apoptosis index in DT group (P<0.05). The expression of GRP78, CHOP, caspase12 mRNA and proteins had significant positive correlation with kidney apoptosis index on DT group (P<0.05).Conclusions1. The abnormal increase of GRP78, CHOP, Caspasel2 protein and mRNA expression in kidney of DM rats had close correlation with renal cell apoptosis index, and renal cell apoptosis index had significant positive correlation with 24h urinary protein. It suggested that ERS-related apoptosis might play an important role on the occurrence and development of DN.2. Telmisartan could delay the occurrence and development of DN by reducing renal cell apoptosis through decreasing ERS-related apoptosis factor GRP78, CHOP, Caspasel2 mRNA and protein expression in the kidneys of diabetic rats.