Expression Of GLUT1 And Activity Of ERK In Renal Cortex Of Diabetic Rats And Effects Of AngⅡ Receptor Antagonist As Well As HMG-CoA Reductase Inhibitior

With the increase of the living standards of the people and aging of population,the incidence of diabetes mellitus is going up year by year, followed with rising of number of the patients with diabetic nephropathy(DN). DN, as a serious chronic implication of diabetes, has become one of the main etiological factors resulting in renal failure.It is very important to clarify the pathogenesis for seeking the methods to prevent and treat DN. Recent reports have indicated that many factors, including metabolic abnormality induced by hyperglycemia, changes of glomerular hemodynamics, cytokines, growth factors and genetic factors,are involved in the pathogenesis of DN.Hyperglycemia is the basic biochemical feature of diabetes and has been believed to play major role in the development of diabetes and diabetic complications.Glucose transporter located in cytoplasm is the main carrier mediating glucose intake, and expression and activity of it are important for glucose intake of cells. Isoforms of glucose transporter have seven kinds,which are distributed in different tissues. GLUT1 is the main isoform of glucose transporter located in mesangial cells of kidneys,which has a close affinity to glucose and is not regulated by insulin.The regulating effect of GLUT1 for intracellular glucose metabolism is independent of density of blood sugar and has been followed with interest.In diabetic state, the signal transduction pathways can be activated by various extracellular stimuli, including abnormal glucose metabolism, changes of glomerular hemodynamics and various cytokines, thus inducing and accelerating the occurrenc and progression of DN. Extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) cascade, plays an essential role in signal transduction cascades,which can transfer extracellular signals into inside of nuclei and induce various cellular reactions. TGF-? is a important profibrogenic cytokine in the pathogenesis of diabetic nephropathy. Recently, experimental and clinical investigations indicated that both angiotensinâ…¡receptor antagonist and HMG-CoA reductase inhibitior have protective effect for the renal function of diabetic rats and diabetic patients,but the mechanism remains to be studied.In the present study,the expression of GLUT1, ERK ,TGF β₁ and type â…£collagen was observed and the interrelation between these factors and the development of renal alterations was analysed in the renal tissues of experimental diabetic rats and mesangial cells exposed to high glucose.In addition, the possible mechanism for angiotensin â…¡receptor antagonist and HMG-CoA reductase inhibitior to protect renal function was also investigated,in order to provide theoretical basis for the prevention and treatment of DN. 1 The expression of GLUT1 in the renal cortex of diabetic rats and the effect of AngII receptor antagonist valsartan on it Forty five healthy male Sprague-dawley rats were randomly divided to 3 groups: normal control group (A), diabetic group (B) and valsartan-treated diabetic group (C). The rats of group B and C received a signal intraperitoneal injection of STZ(dissolved in 0.1mol/L citrate buffer pH 4.5) at a dose of 60mg/kg WT and the rats of group A only received an injection of the same volume of sodium citrate. The diabetic model was considered to be successful when the blood glucose was 16.7mmol/L and urinary glucose (+++)-(++++) after 48 hours of STZ injection. The rats of group C were treated with valsartan (10mg·kg-1·d-1) and the rats of group A and B were treated with the same volume of tap water.All rats were allowed free access to food and water during the experiment,whereas insulin and other hypoglycemic drugs were not supplied.At 4 and 6 weeks after the onset of diabetes, mean arterial pressure(MAP), blood glucose (BG), kidney mass/body mass ratio, serum creatinine(Scr) ,urinary creatinine(Ucr),24 urinary albumin excretion rate (UAER) and urinary NAG were measured, then seven or eight rats were sacrificed for each group respectively.The renal cortical tissues were obtainedand used for light and electron microscopy. Mean glomerular transverse sectional area and mean glomerular volume were calculated by analysis system of the image . The expression values of GLUT1 and TGFβ1mRNA in renal cortices were semi-quantitatively measured with RT-PCR. Results: At week 4 and week 6, kidney mass/body mass ratio,Ccr, UAER and urinary NAG were higher in group B than in group A(P<0.01) and these parameters were significantly decreased in group C(P<0.01). There was no significant difference observed in values of MAP in the three groups (P>0.05). Mean glomerular transverse sectional area and mean glomerular volume were significantly larger in group B than in group A,smaller in group C than in group B (P<0.01). In diabetic rats of group B,light microscopy showed that PAS-positive area of glomeruli was increased. Electron microscopically,irregular thickening of glomerular basement membrane, mesangial expansion and fusion of foot processes were observed at week 4 and these alterations became apparent at week 6. The above morphological changes of glomeruli were markedly alleviated in group C.RT-PCR analysis showed that mRNA expression of GLUT1 and TGFβ₁ in renal cortex was obviously increased in group B.Compared with that in group B mRNA expression of GLUT1 and TGFβ₁ was decreased in group C(P<0.01). 2 The activity of ERK in renal cortex of diabetic rats and the effect of HMG-CoA reductase inhibitior fluvastatin on it Forty five SD rats were randomly divided into 3 groups : normal control group (A), diabetic group (B) and fluvastatin-treated diabetic group (C). The method to induce diabetic model was the same as part 1.The rats of group C were treated with fluvastatin(2mg·kg^-1·d^-1). The rats were respectively sacrificed at week 4 and 6. Serum lipids, including total cholesterol(TC), triglyceride (TG) and low-density lipoprotein(LDL), blood glucose(BG), kidney mass/body mass ratio, creatinine clearance rate (Ccr) and 24 urinary albumin excretion rate (UAER) were measured and calculated. The renal samples were used for light and electron microscopy, and immunohistochemistry to observe the expression of transforming growthfactor-β₁ and type â…£collagen in renal cortex. The expression of ERK1,ERK2 and phospho-ERK1/2 protein in renal cortex were observed with western blotting. Results: Three serum lipid indexes, UAER ,Ccr and kidney mass/body mass ratio were higher in the rats of group B than in the rats of group A at week 4,and the difference became apparent further at week 6 (P <0.01).These indexes were significantly lower in group C than in group B(TC,UAER,Ccr and kidney mass/body mass ratio P <0.01, TG,LDL P <0.05),though there was no difference of blood glucose level observed between group B and C (P >0.05). Light and electron microscopically, the lesions of glomeruli, including glomerular hypertrophy, mesangial expansion, glomerular basement membrane thickening and fusion of foot processes ,became increasingly apparent with duration of DM in the rats of group B, but these changes were alleviated in the rats of group C. Semi-quantitative analysis for immunohistochemical staining showed that the protein expression of TGF-β1 and collagen â…£was significantly increased in glomeruli of group B and this change was partly reversed in the glomeruli of group C (p<0.01). Western blotting showed that the expression of total ERK1and ERK2 in the three groups were similar(P>0.05),but the expression of pERK1/2 was higher in group B than group A(P <0.01),lower in group C than in group B(P <0.01). 3 The effect of high glucose on the expression of GLUT1 and pERK in glomerular mesangial cells Glomerular mesangial cells were obtained from the glomeruli of male Sprague-Dawley rats weighting 130-150g.The rats were anesthetized by intraperitoneal injection with 10% chloral hydrate,then the renal tissue was surgically removed .After the cortex was minced to broken bits,glomeruli were separated from the bits by sequential mechanical sieving. The obtained glomeruli were digested at 37°C with collagenase, then cultured at 37°C, 5% CO2 in RPMI-1640 media supplemented with 20% fetal bovine serum.The media were replaced after four to six days.After the cells reached confluence (about two weeks),they were passaged using 0.25%trypsin/ 0.02%EDTA andreseeded into new flasks for further growth and expansion.Cultured cells were identified as mesangial cells by morphological and biochemical characters.Cells between passages 4 and 10 were used for the experiments,which were cultured separately in normal glucose( D-glucose 5.5mmol/L) or high glucose ( D-glucose 30mmol/L) medium. The expression of GLUT1 and pERK1/2 protein as well as changes of GLUT1 mRNA and TGF-β1mRNA were determined with western blotting and RT-PCR respectively at 12hour , 24hour, 48hour and 72hour after the beginning of culture. Immunocytochemical technique was used for the localization and semi-quantitative analysis of pERK1/2 in mesangial cells.Radioimmunoassay was used to measure the content of type â…£collagen in the supernatant. Results:The expressions of GLUT1 protein and mRNA were all up-regulated and reached top level at 48h and kept up a steady high level to 72h in the measangial cells exposed to high glucose(p<0.01), with a coincident tendency for protein and mRNA. Western blotting showed that the content of pERK1/2 was increased in the mesangial cells exposed to high glucose from 12h and gradually increased to 72h(p<0.01).Immunohistochemically, only weakly-positive staining for pERK1/2 was observed in cytoplasm of mesangial cells exposed to normal glucose. In mesangial cells exposed to high glucose strongly positive staining for pERK1/2 was observed not only in cytoplasm, but also in nuclei at 12h,with an increasing tendency to 72h. The level of TGF-β1mRNA was also increased in mesangial cells exposed to high glucose from 12h ,then gradually increased(p<0.01).The content of type â…£collagen in the supernatant of high glucose group was detected to be high at 48h(p<0.05) and became higher markedly at 72h(p<0.01). 4 The effect of valsartan and fluvastatin on the expression of GLUT1 and pERK in glomerular mesangial cells cultured in high glucose medium Glomerular mesangial cells between passages 4 and 10 were used for the experiments.The cells were randomly divided into four groups and cultured for 48 hours.The groups were as follows: normal glucose group( D-glucose 5.5mmol/L, group NG), high glucose group ( D-glucose 30mmol/L ,groupHG) , 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). GLUT1 mRNA and protein levels in mesangial cells cultured under different conditions were semi-quantitatively analysed with RT-PCR and western blotting. The expression of pERK1/2 protein was detected by western blotting and immunocytochemical technique. RT-PCR and radioimmunoassay were respectively used to measure the expression of TGF-β₁ mRNA in mesangial cells and the content of type â…£collagen in the supernatant. Results: The expression of GLUT1 mRNA and protein , pERK1/2 protein and TGF-β₁ mRNA in the mesangial cells and the content of type â…£collagen in the supernatant were higher in group HG than in group NG(p<0.01).Compared with those in group HG, above parameters were significantly down-regulated both in group HG+val and group HG+flu(p<0.01),whereas there was no difference between group HG+val and group HG+flu(p>0.05). Conclusions: 1 The expressions of GLUT1 and pERK1/2 were up-regulated both in renal cortex of diabetic rats and in the mesangial cells exposed to high glucose medium,with increase of TGF-β₁ expression and extracellular matrix components,suggesting that over-expression of GLUT1 and activation of ERK signal transduction pathways maybe take part in the onset and progression of DN. 2 AngII receptor antagonist valsartan could inhibite the over-expression of GLUT1 and TGFβ₁ in the renal cortex and reduce the degree of urinary albumin, urinary NAG and the renal pathological changes in diabetic rats. 3 HMG-CoA reductase inhibitior fluvastatin could inhibit the activation of ERK and down-regulate the expression of TGFβ₁ and type â…£collagen,thus ameliorating the abnormality of renal function and morphology in diabetic rats. 4 Both Valsartan and fluvastatin could inhibit the expression of GLUT1 and activation of ERK signal transduction pathways,further inhibit the expression of TGFβ₁ and the synthesis of type â…£collagen, which suggested that the protective effects of Valsartan and...