Renal Protective Mechanism Of Valsartan And Fluvastatin For Diabetic Nephropathy

ObjectiveDiabetic Nephropathy(DN) is one important diabetic microangiopathy characterized by glomerular sclerosis and renal interstitial fibrosis.The current treatment for DN focuses on the comprehensive treating method contains lowering blood glucose and pressure and regulating blood lipid. The blocker of AngiotensinⅡreceptorⅠ(ARB) and the inhibitor of 3-hydroxy-3-methyl glutaryl coenzymeA(HMG-COA) reductase(Statins) have important effects on lowering blood pressure and regulating blood lipid.Valsartan is an ARB with high selectivity.Valsartan has renal protective property acts on angiotensin receptors as function of anti-hypertension. Fluvastatin also has renal protective function and effectively reduced the synthesis of intracellular cholesterol as function of anti-atherosclerosis. Valsartan and fluvastatin were reported to protect kidney by suppressing proliferation of mesangial cells,anti-inflammation and immuno-regulation that are not related with anti-hypertension and regulating blood lipid.But the mechanisms are still controversial.Mitogen-activated protein kinase(MAPK) pathway plays a critical role on cells for signal transduction and proliferation.p38MAPK is named as MAPK stress-signal pathway.p38MAPK is a intersection of the common signal pathways which contain a variety of risk factors for diabetes,and p38MAPK also involves in the onset and progression of renal fibrosis.The renal fibrosis may be the results from the transforming growth factors-β1(TGF-β1),AngiotensinⅡ,transformations of phenotype of the renal resident cells and apoptosis of renal tubule epithelial cells.There are abundant of TGF-β1 in kidney.TGF-β1,as a volume of factors in kidney,is a cytokine which can strongly induce renal fibrosis by synthesizing extracellular matrix(ECM) and stimulating ECM secretion by glomerular mesangial cells.The p38MAPK was reported to play an important role in renal fibrosis which is caused by TGF-β1.But seldom downstream signal pathway of TGF-β1 receptors was identified. Thep38MAPK may be a downstream pathway of TGF-β1 system and lead to glomerular sclerosis by mediating TGF-β1.This study is designed:1)To assess the effects of valsartan and fluvastatin on the proliferation, oxidative stress,morphological changes and expression of p38MAPK, TGF-β1 proteins of the cultured rat mesangial cells under high glucose condition in vitro.2)To investigate the changes of TGF-β1 in patients with early diabetic nephropathy treated by valsartan and the relationship between the combined modality therapy and the changes of p38MAPK pathway or TGF-β1.3)To illuminate the mechanism of renal protection.4)To evaluate the actions of renal protection between using single medication and combined medications.5)To identify useful means and theory evidence for the treatment of diabetic nephropathy.Methods1.Culture and classification of HBZY-1 cellsThe HBZY-1 cells were cultured in the Dulbecco's modified Eagle's medium(DMEM) with 10% fetal bovine serum under the condition of 37℃, 5%CO2.The 3rd to 5th generations of the cells were used.TheHBZY-1 cells in logarithmic growth phase were collected and randomly classified into following six groups of:normal control(contains 5.5mmol/L glucose),high glucose (contains 30mmol/L glucose),mannitol control(5.5 mmol/L glucose and 24.5mmol/L mannitol),high glucose plus vlsartan(valsartan practical concentration is 10μmol/L),high glucose plus fluvastatin(fluvastatin practical concentration is 10μmol/L),high glucose plus valsartan and fluvastatin (valsartan and fluvastatin practical concentration is 10μmol/L),high glucose plus p38MAPK blocker(30mmol/L glucose and blocker SB203580 20μmol/L).2.Observation for morphology of mesangial cells and identification for the index of oxidative stressAfter 24 hours of cultivation, the morphology under microscope was observed and the survival rate of cells by MTT was measured.The contents of SOD,NO and MDA from different groups of cells by biochemical way were also assessed.3.Exploration for the expression of p38MAPK and TGF-β1 on mRNA and protein levelThe expression of p38MAPK mRNA and TGF-β1 mRNA by RT-PCR were measured.The expression of p38MAPK protein and TGF-β1 protein by immunocytochemistry,immunofluorescense and western blot from different groups were evaluated.4.Assessment for the level of serum TGF-β1 of patients with early DNA total of 66 patients with early DN were classified into groups of normal albuminuria groupl(NA 1 group),UMA/Cre<10μg/mg, normal albuminuria group 2(NA2 group),UMA/Cre10-30μg/mg and microalbu-minuria group(MA group),UMA/Cre30-300μg/mg respectively. Each group includes valsartan and non-valsartan administration group.The serum TGF-β1 was measured by ELISA for all subjects.The data were compared with that of 30 normal control group.Results1.The effect of proliferation in group of high glucose and groups treated with drugA high level of glucose can stimulate the proliferation of HBZY-1 and improveOD values and cell survival rate. The proliferation of mesangial cells was inhibited in the groups of high glucose treated with drug. In these groups, the group of high glucose treated with valsartan and fluvastatin is critical.2.The oxidative stress index for group of high glucose and groups treated with drugCompared with group of normal control, the activity of SOD and NO reduced,but the content of MDA increased in the group of high glucose. Compared with group of high glucose, the activity of SOD and NO was ascended, the content of MDA was depressed in the group of high glucose treated with medicine.In these groups, the group of high glucose treated with valsartan and fluvastatin is the most prominent.3.The expression of p38MAPK mRNA,TGF-β1 mRNA, p38MAPK protein and TGF-β1 protein in high glucose group and groups treated with drug.The expression of p38MAPK mRNA,TGF-β1 mRNA,p38MAPK protein and TGF-β1 protein was increased under high level of glucose.But the expression was reduced in the groups of high glucose treated with drug.the level of TGF-β1 protein was decreased significantly in high glucose group treated with valsartan and fluvastatin.4.The level of serum TGF-β1 in the patients with early DN.Compared with normal control group,the level of serum TGF-β1 was increased in normal albuminuria group1,normal albuminuria group2 and microalbuminuria group.5.Effect of Valsartan on serum TGF-β1 levels in early diabetic nephropathy.The serum TGF-β1 was significantly decreased in the group 1 of normal albuminuria with valsartan administration compared to that of without valsartan administration.But there was no statistical significance in the serum TGF-β1 level in normal albuminuria group2 and microalbuminuria group with or without valsartan administration.Conclusions1.The proliferation of HBZY-1 could be stimulated by a high level of glucose, which is independent of osmotic pressure.Valsartan and Fluvastatin lowered proliferation of cells and the mesangial cells were protected by anti-oxidative-stress.2.Valsartan and fluvastatin have renal protective property that is associated with p38MAPK pathway and dependent on its suppression on the expression of TGF-β1.3.Combined treatment of valsartan and fluvastatin could further protect kidney.4. A positive relationship between the serum TGF-β1 and the amount of microalbuminuria in early diabetic nephropathy was observed. 5.The serum TGF-β1 level can be one of index for early diagnosis of diabetic nephropathy.