| ObjectivesDiabetes Mellitus (DM) and its complications are diseases which severely jeopardize health and quality of life. Diabetic nephropathy (DN) is one of the most severe complications of DM and is also an important cause of death. The earlier manifestations of DN are hypertension, hypertransfusion and hyperfiltration of renal glomerulus, and further the glomerular basement membrane (GBM) thickening of glomerular capillary loop and the increasing of extracellular matrix(ECM), eventually, the glomerular sclerosis.The pathogenesis of DN is extremely complex. Many factors can act on the Glomerular mesangial cell(GMC) and podocyte and result in disfunction in DN. The concentration of angiotensinⅡ(AngⅡ) in kidney increases during DN and the high Ang II leads to the disorder of series of cytokines in the body. This disorder will promote the further progress of DN. AngⅡ, transforming growth factor-betal(TGF-β1), plasminogen activator inhibitor-1(PAI-1) and so on are very important factor in the onset of DN. Especially, the TGF-β1 plays a key role in development of glomerular sclerosis and fibrosis and is the main factor promoting DN.Type IV collagen(Col-Ⅳ) and laminin(LN) are major components of ECM. The quantity of Con-Ⅳor LN in the glomerulus is the main index for judging ECM accumulation. Matrix metalloproteinase (MMPs), tissue inhibitor of metalloproteinase-1(TIMP-1) and PAI-1, is a major component of fibrinolytic system and responsible for the degradation of ECM. Moreover, monocyte chemoattractant protein-1(MCP-1),oxidative stress and antioxidative system are closely related to the DN.Valsartan (Val) is an antagonist of angiotensinⅡ, low molecular weight heparin (LMWH) posseses the effects of anticoagulation and antithrombogenesis, and the both are used in the treatment of DN. The present study was to investigate the renoprotective effects of valsartan and low molecular weight heparin(LMWH) when was used in single or in combination on diabetic rats induced by uninephrectomy and streptozotocin(STZ). Meanwhile, the effects of both valsartan and LMWH on the expression of cytokins, metabolism of matrix and antioxidation in glomerular mesangial cells and podocytes were investigated.MethodsPart one:Effects of valsartan and LMWH on diabetic nephropathy ratsRight kidney of rat was removed by unilateral nephrectomy and STZ (45mg/kg) was injected by intraperitoneal injection for 2 weeks after the removal. Qualified rats were divided into DN model group, valsartan group (valsartan 20mg/kg/d), LMWH group(LMWH 600IU/kg/d) and combination group(combine valsartan 10mg/kg/d with LMWH 300IU/kg/d). The sham group was established at the same time. The blood glucose and 24h urine protein were measured by biochemical methods. The contents of serum TGF-β1 and PAI-1 were analyzed by enzyme linked immunosorbent assay(ELISA). The expression of Col-Ⅳand LN in renal cortex were detected by immunohistochemistry method. The area of GBM was measured.Part two:Effects of valsartan and LMWH on glomerular mesangial cells of ratGlomerular mesangial cells stimulated by high glucose were investigated in vitro. Cells were divided into normal(control) group, Valsartan group(Val 10-6mol/L), LMWH group(LMWH 25IU/L) and Val+LMWH combination group(Val:10-6 mol/L +LMWH:25IU/L). The proliferation of GMCs induced by high glucose was measured by MTT method. The activities of MMP-2 and MMP-9 were measured by gelatin zymography. The expression of LN, Col-Ⅳ, MCP-1, MMP-2 and TIMP-1was detected by immuocytochemical method. The mRNA contents of MMP-2,TIMP-1, TGF-β1 and MCP-lwere detected by RT-PCR. The level of TGF-β1 and PAI-1 in cell culture fluid was measured by ELISA.Part three:Effects of valsartan and LMWH on glomerular endothelial cells(GECs)ofmiceGlomerular endothelial cells stimulated by high glucose were investigated in vitro. Cells were divided into normal(control) group, valsartan group(Val 10-6mol/L), LMWH group(LMWH 25IU/L) and combination of val and LMWH group(Val:10-6 mol/L+LMWH:25IU/L). The levels of TGF-β1 and PAI-1 in cell culture fluid were measured by ELISA. The activities of MMP-2 and MMP-9 were measured by gelatin zymography.Results1.1 Effect of valsartan and LMWH on blood glucose of DN ratsThe blood glucose level of DN rats was significantly increased compared with that of sham rats. In three treated groups (Val, LMWH, Val+LMWH), the blood glucose level was similar to those in DN group.1.2 Effect on 24h urinary protein excretion in DN ratsThe 24h urinary protein excretion of DN model rats was significantly increased compared with that of sham rats. The increased 24h urinary protein excretion was significantly attenuated by both valsartan and LMWH compared with DN model group. Furthermore, Val+LMWH treatment is more effective compared with Val group.1.3 Effect of valsartan and LMWH on serum TGF-β1 in DN ratsThe serum TGF-β1 level of DN model rats was significantly increased compared with that of sham rats. The increased serum TGF-β1was significantly attenuated by three treated groups, but there were no significant differences among three treated groups.1.4 Effect of valsartan and LMWH on serum PAI-1 in DN ratsThe serum PAI-1 level of DN model rats was significantly increased compared with that of sham rats. The increased PAI-1 was significantly attenuated by three treated groups, but there were no significant differences among three treated groups.1.5 Effects of valsartan and LMWH on the expression of collagenⅣand LN in the renal cortex of DN ratsThe expressions of collagenⅣand LN in DN groups were significantly increased compared with that of sham group. The increased expression were significantly attenuated in three treated groups compared with DN model group. Furthermore, combination of Val and LMWH is more effective compared with either compound alone.1.6 Effect on glomerular morphology of DN ratsThe area of glomerular mesangium and glomerular basement membrane was determined by PAS staining. The results showed that the percentage of areas of glomerular mesangium and glomerular basement membrane in DN model groups was significantly increased compared with that of sham group. The increased percentages in three treated groups (Val, LMWH, Val+LMWH) were significantly attenuated compared with DN model group. There were no significantly differences among three groups.Part two:Effects of valsartan and LMWH on glomerular mesangial cells of rat2.1 Effect of valsartan and LMWH on the proliferation of glomerular mesangial cells induced by high glucoseBoth valsartan and LMWH could inhibit the proliferation of glomerular mesangial cells induced by high glucose, and the combination of Val and LMWH is more effective compared with either compound alone.2.2 Effects of valsartan and LMWH on the expression of LN and Col-Ⅳof GMCs induced by high glucoseHigh glucose level could promote the expressions of LN and Col-IV. LMWH could inhibit the Col-Ⅳexpression. Valsartan had no effects on LN and Col-Ⅳ. The expressions of LN and Col-Ⅳcould be inhibited in the combination of Val and LMWH group. 2.3 Effect of valsartan and LMWH on the activity of MMPs of GMC induced by high glucoseHigh glucose level could inhibit MMPs activities and LMWH treated group and combination treated group could increase the activities. Valsartan had no effect on MMPs activities.2.4 Effect of valsartan and LMWH on the expression of MMP-2/TIMP-1 of GMCs induced by high glucoseHigh glucose level could decrease MMP-2 mRNA expression and protein secretion, and increase the TIMP-1 mRNA expression and protein secretion. After treatment with valsartan or LMWH, MMP-2 was promoted and TIMP-1 was inhibited. Furthermore, combination of Val and LMWH is more effective on mRNA expression but not on protein secretion compared with either compound alone.2.5 Effect of valsartan and LMWH on serum PAI-1 of GMCs induced by high glucoseHigh glucose level could increase PAI-1 secretion. The increased PAI-1 was significantly attenuated by three treated groups, and combination of Val and LMWH is more effective compared with either compound alone.2.6 Effect of valsartan and LMWH on TGF-β1 of GMCs induced by high glucoseHigh glucose level could increase TGF-β1 mRNA expression and protein secretion. The increased TGF-β1 was significantly attenuated by three treatment groups, and it was more effective in combination of Val and LMWH group compared with Val or LMWH alone.2.7 Effect of valsartan and LMWH on MCP-1 of GMCs induced by high glucoseHigh glucose level could increase MCP-1 mRNA expression and protein secretion. The increased MCP-1 was significantly attenuated by three treatment groups, and it was more effective in combination of Val and LMWH group compared with Val or LMWH alone.2.8 Effect of valsartan and LMWH on the antioxidation system of GMCs induced by high glucoseStimulated by high glucose, the activities of SOD, GSH-Px decreased and the content of MDA increased compared with the normal group. These indexes could be reversed when treated with valsartan, LMWH, or Val+LMWH treated group, and it was more effective in combination of Val and LMWH group compared with Val or LMWH alone. The oxidative stress state could be inhibited by valsartan and LMWH.Part three:Effects of valsartan and LMWH on glomerular endothelial cells(GECs) of mice3.1 Effect of valsartan and LMWH on the secretion of MMP-2/TIMP-1 induced by high glucoseHigh glucose could inhibit MMP-2 secretion and increase TIMP-1 secretion. MMP-2 secretion could be increased and TIMP-1 secretion could be decreased significantly by three treatment groups, and it was more effective in combination of Val and LMWH group compared with Val or LMWH alone.3.2 Effect of valsartan and LMWH on the activity of MMPs of GECs induced by high glucoseHigh glucose could inhibit MMPs activities. The increased activities was significantly increased by three treated groups, and it was more effective in combination of Val and LMWH group compared with Val or LMWH alone.3.3 Effect of valsartan and LMWH on the antioxidative system of GECs induced by high glucoseStimulated by high glucose, the activities of SOD, GSH-PX decreased and the content of MDA increased compared with the normal group. These indexes could be reversed when treated with valsartan, LMWH, or Val+LMWH treated group. And it was more effective in the combination of Val and LMWH group compared with Val or LMWH alone. The oxidative stress state could be inhibited by valsartan and LMWH.Conclusions 1. Combination valsartan with LMWH effectively decreases the urine protein and prevents renal injury in diabetic nephropathy rats with synergy effect. 2. The renoprotective mechanism of valsartan and LMWH is concerned with the inhibition on mesangial matrix synthesis and the promotion on mesangial matrix degration, the inhibition of synthesis or scretion on MCP-1 and TGF-β1.3. The renoprotective effect of valsartan and LMWH is concerned with the antioxidative effects. Valsartan and LMWH can increase the activities of antioxidases in mesangial cells and podocytes stimulated by high glucose. |
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