Effect Of Simvastatin And Xuezhikang On Renal Cell Apoptosis In Diabetic Rats And Its Mechanism

The prevalence of diabetes is increasing at an alarming rate, regardless of the population studied. Due to rapid economic growth, an increase in life expectancy and changes in lifestyle, diabetes and its complications have become great threats to global health in the 21st century. Diabetic nephropathy, common but severe microangiopathy in diabetes, is becoming one of the major reasons that lead to end stage renal dysfunction. The mechanism of diabetic nephropathy is not clear yet. Researchers have hooked renal cell apoptosis with diabetic nephropathy recently, as the studies on cell apoptosis going deeper, suggesting renal cell apoptosis an early manifestation of diabetic nephropathy.Statins, HMG-CoA reductase inhibitors, are a class of drugs used to lower plasma cholesterol levels. Statins are used widely in clinical works, especially in diabetic patients, since diabetic patients often suffer from lipid dysfunction and the criterion of lipid control is stricter for diabetic patients. Studies have demonstrated a protective effect of statins on renal function that statins could ameliorate the progress of diabetic nephropathy. Simvastatin, used widely clinically, is confirmed to attenuate the decline of glomerular filtration rate in diabetic patients according to large scale clinical trials. Xuezhikang is a natural statin extract from red yeast rice, including thirteen natural statins and homologues of statin. Xuezhikang is suggested to have effect on oxidative stress, endothelial cell function and inflammation according to basic and clinical researches. Meta analysis also demonstrated that xuezhikang could lower urinary protein and albumin extretion.Streptozocin (STZ) induced diabetic rats were used in this study, giving simvastatin and xuezhikang treatment after establishment of diabetic model. Renal histological changes and renal cell apoptosis in each group (control group, diabetic group, diabetic group with simvastatin treatment and diabetic group with xuezhikang treatment) were observed, and the possible mechanism how statins affect cell apoptosis was also investigated.1 Materials and methods1.1 Reagents and materialsSTZ was from Sigma, simvastatin from Merck and Co. China, xuezhikang from Beijing WBL Peking University Biotech Co., PAS and Masson staining kits from Baso Diagnostics Inc. Zhuhai, TUNEL apoptosis assay kit from Roche, Caspase-9 and Bax antibodies from Santa cruz, Bcl-2 antibody from Boster (Wuhan), and cytochrome c antibody from BD Inc.1.2 Induction of diabetes and experimental groupsNinety male Sprague-Dawley (SD) rats, of 180-220g, were purchased from the Experimental Animal Center of Zhejiang University and raised with standard food, adequate water and normal light/dark cycle. Diabetes was induced by a single intraperitoneal injection of 1% STZ (in cold 0.1M citrate buffer. pH 4.2) at 60mg/kg body weight. Blood samples for glucose measurements were taken from the tail vein 48-72 hours after STZ injection and rats with blood glucose of 16.7mmol/L or higher were considered diabetic rats. The control rats were injected with equal volume of citrate buffer instead. The experimental groups included (1) normal control rats (group SD); (2) diabetic control group (group DM); (3) diabetic rats treated with simvastatin (20mg/kg body weight gastric perfusion everyday, group SIM); (4) diabetic rats treated with xuezhikang (1200mg/kg body weight gastric perfusion everyday, group XZK). Treatments started 7 days after STZ injection. Rats from group SD and DM received equal volume of saline instead.1.3 Detection of clinical measurementsHalf the rats from each group were sacrificed after three months'treatments and the rest after six months'treatments. Twenty-four hours'urine was collected using metabolic cages and urinary albumin excretion was measured by rate nephelometry (Beckman Coulter Immage800). After weighing the body weight, blood sample was collected from abdominal aorta for glucose and lipid detection by Beckman Coulter LX20 biochemical analyzer.1.4 Histological examination and apoptosis detectionAfter lavaging the abdominal aorta by cold saline, both kidneys were taken out. The ratio of right kidney weight to body weight was recorded as kidney index. The right kidney was cut into two pieces, half of which was fixed by formalin and embedded by paraffin. Paraffin-embedded tissue section (3μm thick) were deparaffinized. rehydrated. and stained with PAS and Masson's trichrome for saccharides and collagen detection, and TUNEL for apoptosis detection. 1.5 Expression of Bax, Bcl-2, Caspase-9 and Cytochrome cRenal cortex was separated and stored in-80℃. After extracting the total protein, Bax, Bcl-2 and active Caspase-9 expression in renal cortex were detected by western blotting. Half of the right kidney was used for detection of cytochrome c expression after removing mitochondria. Bax and Bcl-2 expression were also detected by immunohistochemistry.1.6 Statistical analysisAll data were presented as mean±SD of the mean. When multiple comparisons were made, statistical significance was determined using one-way ANOVA followed by Bonferroni's test. All statistical analyses were performed using the software package SPSS 16.0 and P<0.05 was considered statistically significant.2 Results2.1 General dataSeventy SD rats underwent STZ injection, sixty-six of which had blood glucose of 16.7mmol/L or higher were considered diabetic rats and divided into three groups randomly. Except those died during experiment and blood glucose lower than 16.7mmol/L, the final number of rats in each group was as follows:SD (3 months). n=10; SD (6 months), n=10; DM (3 months), n=9; DM (6 months), n=10; SIM (3 months), n=10; SIM (6 months), n=9; XZK (3 months), n=9; XZK (6 months), n=8.The blood glucose was less than 7mmol/L in control group and increased dramatically (≥16.7mmol/L) 48-72 hours after STZ injection; neither simvastatin or xuezhikang treatment showed significant differences from group DM in blood glucose level. The STZ-induced diabetic rats showed classic diabetic symptoms of polyuria, polydipsia and weight loss:simvastatin and xuezhikang did not help to retrieve the body weight. No significant difference was found in lipid level among each group.2.2 Kidney index and 24 hours'urinary albumin excretionKidney index of group DM increased significantly after three and six months(P< 0.01). Simvastatin and xuezhikang treatment could decrease kidney index compare to that of group DM at each time point (P< 0.05或P< 0.01), but kidney indexes of group SIM and XZK were higher compare to that of group SD (P<0.01). There was no significant difference between the two drugs.Urinary albumin excretion was low in group SD, which increased in group DM after three months and progressed after six months (P< 0.01). Three and six months' treatment of simvastatin and xuezhikang could reduce urinary albumin excretion compare to that of group DM (P< 0.01或P< 0.05). There was no difference between group SIM and group XZK in urinary albumin excretion.2.3 Results of PAS and Masson stainPathological sections stained with PAS showed that control rats had normal glomerulus and renal tubule, with tubular cells arraying in order. However, diabetic rats showed dilation of glomerular mesangial region, irregular thickening of tubular basement, as well as increased deposition of PAS positive substance after three and six months (P< 0.01). Simvastatin treatment could totally alleviate the deposition of PAS positive substance in diabetic kidney (P<0.01). PAS positive substance was reduced significantly after xuezhikang treatment (P< 0.01), but more than that in control rats at each time point (P< 0.05). There was difference between group SIM and group XZK at three months (P< 0.05), which disappeared six months later.Masson's trichrome stain showed increased accumulation of collagen in glomerular mesangial region and basement as well as in tubular basement in diabetic rats after three and six months (P< 0.01). Treatment of simvastatin decreased collagen accumulation in diabetic rats at each time point (P< 0.01). Xuezhikang treatment could decrease collagen deposition in diabetic kidney after six months (P< 0.01), but no difference was observed after three months'treatment. Neither simvastatin nor xuezhikang treatment could totally reverse collagen deposition compared to control rats (P< 0.01 or P< 0.05). Collagen deposition was less in group SIM compare to that in group XZK at both time points (P< 0.01 or P< 0.05).2.4 Renal cell apoptosisTUNEL apoptosis detection found few apoptotic cells in kidney of control rats, mainly distal tubular epithelial cells. More apoptotic cells were observed in diabetic rats (P< 0.01), mainly proximal and distal tubular cells, as well as some glomerular cells, the number of which was fewer. Simvastatin and xuezhikang treatment could reduce renal cell apoptosis compared to diabetic rats at each point (P< 0.01), but not completely as compared to control rats (P< 0.05).2.5 Expression of Caspase-9 and cytoplasmic Cytochrome cExpression of renal Caspase-9 and cytochrome c were detected by western blotting. Active Caspase-9 and cytochrome c expression were low in control rats. Western blotting revealed that expression of active Caspase-9 and cytoplasmic cytochrome c increased significantly in diabetic rats, which was ameliorated by both simvastatin and xuezhikang treatment, but still higher than that in control group.2.6 Expression of Bax and Bcl-2Renal expression of Bax and Bcl-2 were detected by both immunohistochemisty and western blotting. Bax and Bcl-2 expression was low in control group. Bax expression was higher in diabetic group and progressed as diabetic duration prolonged (P< 0.01), while Bcl-2 expression increased a little after three months, without significant difference, but decreased significantly after six months (P< 0.01), thus Bax/Bcl-2 ratio increased dramatically in diabetic rats (P<0.01). Both simvastatin and xuezhikang treatment could suppress the increment of Bax and up-regulate the expression of Bcl-2, leading to the suppression of Bax/Bcl-2 ratio (P< 0.01).3 Conclusions3.1 Obvious renal cell apoptosis was observed in diabetic kidney, which progressed as diabetic duration prolonged. Simvastatin and xuezhikang treatment could attenuate pathological changes in diabetic kidney, reduce renal cell apoptosis. The effect of statins did not depend on their cholesterol-lowering effect.3.2 Simvastatin and xuezhikang could reduce the activation of Caspase-9 and the expression of cytochrome c in cytoplasm, suggesting that suppression of mitochondria pathway was involved in statins' effect against cell apoptosis.3.3 Simvastatin and xuezhikang could suppress Bax/Bcl-2 ratio, suggesting that statins could reduce cell apoptosis by regulating the expression of Bcl-2 family.