The Study About The Effect Of Valsartan And Fluvastatin On PDGF And VEGF Activity In Glomerular Mesangial Cells Exposed To High Glucose

Objective: In western developed countries, Diabetic nephropathy (DN) has become one of the main etiological factors resulting in end stage of renal failure. Nowadays, with the rising incidence of diabets in our country, the number of diabetic nephropathy patients has also increased. So there is a great need to seek the methods of preventing and treating DN for nephrologist. But the precise pathogenesis is unclear. Glomerular mesangial cells are the most activity cells in the glomerulus, which are important for the normal structure and physiologic function. Most kinds of renal disease indicated the dysfunction of glomerular mesangial cells.Recent researches indicate that many factors, such as TGF-β, PDGF, VEGF and so on, can promote cells proliferation and secrete extracellular matrix (ECM). In early diabetic nephropathy, glomerular mesangial cells (GMC) show the state of proliferation and hypertrophy, with the accumulating of extracellular matrix. Some studies have confirmed that TGF-βparticipates in the whole pathologic process, but the relationship between PDGF, VEGF and the excrescent expression of the GMC is not clear, which can reveal the pathogenesy of DN all the better.Hyperglycemia, one of the dangerous factors that induces afferent complications of diabetes, can activate mitogen- activated protein kinases (MAPKs) through the signal transduction pathways PKC. Mitogen-activated protein kinases (MAPKs) belong to the family of serine/threonine kinases that have been shown to play a key role in transduction extracellular signals to cellular responses. In mammalian cells, four subfamilies of MAPKs have been characterized: extracellular signal-regulated kinases1/2(ERK1/2), c-Jun N-terminal kinases (JNKs), p38-MAPKs and ERK5. MAPK pathways relay, amplify and integrate signals from a diverse range of stimuli and elicit an appropriate physiological response . ERK has been the best characteried MAPK and usually mediated the celluar responses of hormone and growth factors, that is, hormone and growth factors activate ERK through Ras-dependent, then the activated ERKs translocate to the nucleus, transactivate transcription factors and change gene expression to promote growth, differentiation or mitosis. For example, Hyperglycemia, promoting the abnormal express of PDGF mRNA and VEGF mRNA, evokes the cells proliferation and differentiation. In addition, MC in high glucose can autocrine most kinds of growth factors, such as TGF-βand AngⅡ, which can increase the expressing of VEGF and PDGF.PDGF and VEGF, belonging to the family of somatomedin, are related to the growth of tissue and cells. PDGF, an important growth factor that can promote cell growing, is generated by blood platelet. VEGF, a kind of mitogen, mainly affect in vascular endothelial cells. Some literatures report that GMC can excrete them and there are many receptors in GMC membrane. The studies of immunohistochemistry confirm PDGF and VEGF express more in the mesangial region of DM patients and animal models. Hyperglycaemia, the basic biochemistry character of DM, probably prompts PDGF and VEGF to express abnormally , then induce biological effect and participate in the development of DN through GMC's specific receptor.AT1Ra is used extensively in the hypertension therapy. It inhibits angiotensinⅡ(AngⅡ) from combining with its receptor . Thus, AT1Ra inhibits the biologic effect of AngⅡ. It has been reported that MC in high glucose can autocrine AngⅡ, which can bind with AT1 receptors in MC membance. So, some paths are activated, some growth factors express abnormally and MCs proliferate finally. 3-hydroxy-3- methyglutary1 coenzyme A(HMG-CoA) reductase(HRI) is a well-known lipid-lowing agent. Recent reports indicate that HRI has the direct glomerular protective effect independent of the reduction in circulating lipids. HRI can inhibit the synthesis of FPP and GGpp, that Ras (small GTP protein) can not locate in the membrane, and the signal transmission is inhibited. Valsartan (one kind of AT1Ra) and Fluvastatin (one kind of HRI) is used for interfering GMC in hyperglycaemia. In this course, we study the PDGF and VEGF, in order to illuminate the mechanism and search the method of preventing DN.Methods:The GMC(HBZY-1) of SD rat was bought from CCTCC and cultured conventionally. After reached confluence, the cells were passaged using 0. 25% trypsin/0. 02% EDTA and reseeded into new flasks for further growth and expansion. Cultured cells were identified as mesangial cells by morphological characters. When cells grew enough, they were used for the experiments and cultured separately in normal glucose( D-glucose 5. 5mmol/L, group NG ) ,Mannitol-treated normal glucose group (D-glucose 5. 5mmol/L+ Mannitol 24. 5mmol/L, group MG), high glucose ( D-glucose 30mmol/L, group HG), Valsartan-treated high glucose group(D-glucose 30mmol/L+ Valsartan 10μmol/L, group HG+val) and fluvastatin-treated high glucose group ( D-glucose 30mmol/L+fluvastatin 10μmol/L, group HG+flu). NG , MG, and HG were harvasted at 3 hours, 6 hours, 24 hours, 48 hours and 72 hours respectively while HG+val , HG+flu was at 3, 48 hours after intervention. Reversed transcriptive polymerase chain reaction (RT-PCR) and Western blot was applied in determining the activity of PDGF, VEGF mRNA and protein. Enzyme linked immunosorbent assay (ELISA) was conducted for the content of typeⅣcollagen , LN and FN in the supernatant.Result: 1 GMC shows fusiform, irregular stellar, trianglar or branch-shaped under the microscope. The cells can growth overlapping, the endochylema has some exphyma and a number of microfilament-liking structure. The nucilus which are round or elliptic lies in the center. 2 The expression of PDGF mRNA and protein in GMC in response to high glucose of 24 hours was higher than that to normal glucose in 24 hours (P<0. 01), and with the extending of time, PDGF mRNA and protein express increasing gradually (P<0. 01). These effects could be inhibited by Valsartan and fluvastatin (P<0. 01). At the same time, The expression of PDGF mRNA and protein in GMC in response to Mannitol group was higher than that to normal glucose(P<0. 01) ; The expression of VEGF mRNA and protein in GMC in response to high glucose of 3 hours was higher than that to normal glucose in 3 hours(P<0. 01), and with the extending of time, VEGF mRNA and protein express decreasing gradually, after 24 hours, the expression of VEGF mRNA and protein in these two group have no difference(P>0. 05). These effects could be inhibited by Valsartan and fluvastatin in 3 hours (P<0. 01). At the same time, the expression of VEGF mRNA and protein in GMC between Mannitol group and normal group have no difference (P>0. 05) ; 3 In 3 hours, 6 hours and 24 hours, TypeⅣcollagen(except 24 hour ,P<0. 05) , LN and FN in the supernatant were not different between high glucose group and normal group(P>0. 05), but they were higher than normal glucose group in 48 hours and 72 hours(P<0. 01, P<0. 01, P<0. 05) . These effects could be inhibited by Valsartan and fluvastatin. (P<0. 01) . Mannitol group and normal group have no difference (P>0. 05).Conclusion: The super-expressing of PDGF and VEGF maybe take part in the onset and progression of DN. Maybe these abnormal expressing are related with the activation of MAPKs. Valsartan and Fluvastation could down-regulate the expression of mRNA, protein of PDGF, VEGF and the synthesis of typeⅣcollagen, FN, LN through inhibiting the super-expressing of PDGF and VEGF, suggesting that Valsartan and fluvastatin has the renal protective effect.