Study On Regulatory Mechanisms Of Fluvastatin On P27^kip1 And CTGF Expression In Diabetic Rat Kidney

Diabetic nephropathy (DN) is not only one of the major microvascular complications of diabetes mellitus(DM), but also the leading cause of the end stage renal disease (ESRD). It is very important to investigate the pathogenesis of DN and seek effective drugs to ameliorate its progression. DN is characterized by glomerular and tubular hypertrophy. Glomerular hypertrophy is related to the glomerular sclerosis that occurs in the late stage DN. p27^kip1 plays an important role in the pathogenesis of glomerular hypertrophy, and CTGF is also involved in the pathogenesis of DN. It is well known that cyclin dependence kinase(CDK) inhibitor, p27^kip1, and mitogen activated protein kinase(MAPK) activation are related to the kidney cell hypertrophy.Kidney size is increased in DN, its possible mechanism is mitogen-induced entry into cell cycle arrest at the G1/S interphase, cell cycle is arrested at G1 phase, the DNA synthesis is inhibited, while protein synthesis is increased. In vivo and in vitro studys indicated that although some low-grade proliferation of glomerular cells is present in the early phase, such as renal tubulointerstitial cells, but most of cells especially mesangial cells and renal tubular epithelial cells are arrested at late stage of G1, followed by cell cycle arrest and hypertrophy. Cyclin dependent kinase inhibitor(CKI) is a kind of negative regulatory protein of cell cycle, which combine with cyclin-CDK complex, inhibits CDK activity, and affects the function of downstream target protein, negatively control G1/S and G2/M transition, inhibit cell cycle. For example, cell becomes hypertrophy when cell cycle is arrested in G1 late stage. P27^kip1 is a non-specific CKI, which can inhibit CDK complex and arrest cell cycle in G1 phase. The level of p27^kip1 is the important for whether cell is in resting stage or propagation stage. Generally, cell stayed in resting stage, when the level of p27^kip1 is high. On the contrary, cell will be in the division and proliferation stage when the level of p27^kip1 is low. p27^kip1 increment is related to the kidney cell hypertrophy in diabetic nephropathy. Furthermore, activation of MAPK that can inhibit CKI degradation contributes cell hypertrophy in DN conditions.Pathological changes of DN include cell hypertrophy, ECM accumulation, glomerular sclerosis and tubulointerstitial fibrosis. ECM accumulation is due to the increment of collagenⅣ, fibronectin, glucoprotein synthesis and decrement of its synthesis.It is known that TGF-βis the most important cytokine which can induce renal fibrosis. However, its biological activities are complex, it has other multiple biological effects, for example, it has anti-proliferation and anti-imflammation effects. Blocking TGF-βfor a long time might be harmful to health. CTGF is a downstream effector of TGF-β, which mediate fibrosis induced by TGF-β. CTGF participates in cell proliferation and extracellular matrix synthesis, but does not have anti-imflammation effect. It plays an important role in pathogenesis of renal fibrosis in DN. Hyperglycemia and hemodynamic abnormality that related with DN progression can enhance expression and secretion of CTGF. When CTGF antisense oligonucleotide is added in mesangial cell stimulated by high glucose, and the level of the expression of CTGF is decreased more than 90%, the level and synthesis of FN mRNA is decreased. It means that CTGF is an important progression of glomerular sclerosis in diabetes. CTGF induce mesangial cell hypertrophy through arresting cell cycle in G1/S interphase.DN is reversible in early stage. How to attenuate the progression of DN in early stage? The key point is inhibiting glomerular hypertrophy and ECM accumulation.Nowdays, many studies indicate that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (HCRI) has renoprotective effects independent of its lipid-decreasing effect. Fluvastatin is one of representative drugs of HCRI, which is synthetical statins, and also is common lipid-lowering agents. It can reduce renal cell proliferation, hypertrophy, and ECM deposition, can decrease the urinary protein excretion and cytokine expression. It is not clear whether fluvastatin can inhibit ERK1/2 activation and decrease the expressions of p27^kip1 and CTGF that related to the glomerular hypertrophy, and ECM accumulation.In order to clarify the effect of fluvastation on the progression of DN, this study investigated the effects of fluvastation on ERK1/2 activation on p27^kip1, CTGF, FN and ColⅣexpression using STZ-diabetic rat and high glucose stimulated mesangial cell.The male Wistar rats were used in the present study. 1. The effect of fluvastatin on the levels of urinary albumin excretion rate, liver function, renal function, blood sugar and lipid, and kidney hypertrophy index were investigated using diabetic rats induced by a single intraperitoneal injection of streptozotocin. The activation of ERK1/2, the expressions of p27^kip1, CTGF, FN and ColⅣin kidney tissue were observed by Western blot, RT-PCR, and immunohistochemistry. 2. The effects of fluvastatin and ERK1/2 inhibitor PD98059 on the activation of ERK1/2, expressions of p27Kip1 and CTGF protein and mRNA, were investigated using glomerular mesangial cells stimulated by high glucose. Intracellular total protein level was measured by Coomassie brilliant blue method, fibronectin concentration of the mediam was detected by ELISA.The main results of this study are as follows:The animal experiment showed that fluvastatin attenuated the urine protein excretion, glomerular high filtration status and the kidney hypertrophy index, but did not influence on liver function, blood sugar and lipid levels. The activation of ERK1/2, expressions of p27^kip1, CTGF, FN and ColⅣwere increased obviously in DN group, but decreased after fluvastatin administration. The in vitro study indicated that expressions of p27^kip1, CTGF and ERK1/2 activation in mesangial cells induced by high glucose were increased significantly, but decreased obviously by fluvastatin and PD98059 treatment. Addition of fluvastatin and PD98059 obviously inhibited the expression of protein and mRNA of p27^kip1, CTGF as well as ERK1/2 activation protein, also decreased cell total protein and FN excretion.The conclusions of this study are as follows:1. The activation of ERK1/2, the expressions of p27^kip1, CTGF mRNA and protein in the renal cortex of diabetic rats are increased significantly, which accompanying with the increment of urinary albumin excretion rate, GFR, and kidney hypertrophy. The expressions of FN and ColⅣare increased markedly. This means ERK1/2, p27^kip1 and CTGF may be involved in the development of glomerular hypertrophy and ECM accumulation. 2. Activation of ERK1/2, expressions of p27^kip1 and CTGF in cultured glomerular mesangial cells stimulated by high glucose, the total protein and FN are increased significantly. This indicates that high glucose can induce glomerular hypertrophy and ECM accumulation.3. ERK1/2 inhibitor PD98059 significantly inhibits the activity of ERK1/2 and mRNA and protein expression of p27^kip1and CTGF induced by high glucose in mesangial cells, also reduces the content of total protein and FN, which indicates that the activity of ERK1/2 is related to the expressions of p27^kip1 and CTGF. This indicates p27^kip1 and CTGF expression are mediated by ERK1/2 activation.4. Fluvastatin can reduce urinary albumin excretion and kidney hypertrophy, and also inhibit the activation of ERK1/2, expressions of p27^kip1 and CTGF mRNA and protein in the renal cortex of diabetic rats. The fluvastatin can reduce the activation of ERK1/2, the expressions of p27^kip1 and CTGF, the content of total protein and FN, that induced by high glucose in mesangial cells. These results indicate that fluvastatin can ameliorate the progression of glomerular hypertrophy and ECM accumulation via inhibition of activation of ERK1/2 and expressions of p27^kip1 and CTGF.Taken together, the characteristics of DN is glomerular hypertrophy and ECM accumulation, it is related to the progression of DN. The results of the present study show that fluvastatin can attenuate glomerular hypertrophy and ECM accumulation via reducing p27^kip1 and CTGF expression that mediated by ERK1/2 activation in DN conditions through in vitro and in vivo study.