The Effect Of Fluvastatin On The Expression Of CTGF In Kidney Of The Diabetic Nephropathy

一. Study backgrounds and objectionsDiabetic nephropathy (Diabetic nephropathy, DN) is the most common complication of diabetes, and also is one of important reasons which leads to the chronic renal insufficiency.Early pathological changes in the DN are the main volume of glomerular hypertrophy, as the condition for the progress of the development of glomerular sclerosis slowly and fibrosis, and eventually enter the period of chronic renal insufficiency, until the period of urinemia. Recent studies show that connective tissue growth factor (Connective tissue growth factor, CTGF) is a newly discovered important factor to induce fibrosis, mainly to the increasing proliferation rate of kidney disease, but the specific mechanism is not very clear so far.Statin lipid-lowering drugs except for this role, recent studies show that it can through other ways to achieve non-lipid-lowering kidney protection. Statin drugs can decrease expression of CTGF in the kidney, achieve renal protective effect, in this respect at home and abroad there is less reports, Through observating the expression of the CTGF in the diabetic nephropathy patient's blood and changes in the concentration of CTGF in DN rat kidney and high glucose-induced glomerular mesangial cells CTGF expression in this experiment, so as to find a new drugs to delay the progress of DN, which provide a theoretical basis.二. Experimental materials and methodsPartⅠ(animals Experiment)1. Access to materials rats with diabetic nephropathy: Choose 40 weight (200-250g)Wister male. 30 were randomly selected 12-hour fasting, streptozotocin (STZ) dissolved in the cold of 0.05 M sodium citrate solution (PH = 4.5) by intraperitoneal injection of STZ 55mg/kg, tail vein blood glucose measurement, after three days glucose> 16.7 mmol/l, which indicates DM models are successful, Unsuccessful removed.24 of the experimental model, were randomly divided into two groups:①DM + fluvastatin group (13), 5mg/kg/d Fluvastatin to gavage.②DM control group (11), the same volume of distilled water to gavage. At the same time the remaining animals for the normal control group (10), the same volume of distilled water to gavage.2. Obtaining samples: gavaging twelve weeks after treatment in rats are as follows:①By regularly collectling the 24-hour urine and serum, measureing the 24-hour urinary albumin excretion rate and serum glucose.②Ether anesthesia thoracic, cardiac blood exposure, to 5000/min, 5 min blood centrifugation, the supernatant collecting blood biochemical parameters measured by ELISA method of detecting biochemical indicators.③After the removal of renal capsule weighing, kidney hypertrophy index with renal weight/body weight, on average.④The renal pathology staining (PAS, PAM), and the SP detecting the expression of CTGF in kidney.⑤with RT-PCR and Western blot detecting the expression of CTGF in glomerular mesangial cells.Part II (Cell Experiment)Glomerular mesangial cells cultured were divided into 4 groups: 1, the normal control group: (NG, 5.6mmol/L D-glucose); 2, the normal group+ fluvastatin group (NG+ F); 3, high glucose groups: (HG, 30mmol/L D-glucose); 4, high sugar group+fluvastatin group (HG+ F); RT-PCR and Western blot were used to detect the expression of CTGF.Part III (clinical trials)Clinical diagnosis of diabetic nephropathy (24-hour urine protein quantitation of 150-500 mg) of the patients, according to drugs in use were divided into three groups: diabetic nephropathy + ARB therapy group(DA), diabetic nephropathy+Fluvastatin treatment group(DF), diabetic nephropathy Fluvastatin+ARBtherapy group(DAF). Observeing a period of three months. Finally monitoring the 24-hour urinary albumin excretion rate and serum CTGF concentration, and the treatment group compared before and after treatment.Part IV Statistical analysisMeasurement data was performed by using mean±standard deviation (±s), using SPSS12.0 software analysis, groups compared with the single-factor analysis of variance, P <0.05, P <0.01 for the difference between statistical significance.三. Results1. Animal experiments: weight-growth in DN, DF groups was significantly (P <0.05). the 24-hour urinary albumin excretion rate and kidney hypertrophy index in the DN, DF groups were more significantly increasing than the NC gourp (P <0.01), DF gourp was lower than the DN group (P <0.05).DN group, DF and DN groups of blood cholesterol and triglycerides increased much more than NC group, but there are not significant difference (P> 0.05).the Ccr of DN Group was higher than the NC group (P <0.01), the Ccr of DF Group was higher than the NC group (P <0.05), but the Ccr of DF Group was lower than the DN group (P <0.05).The Scr of DN Group and DF group have a trend to increase comprared wiht NC group, but not statistically significant (P>0.05). Immunohistoch- emistry technology semi-quantitative analysis showed that the expression of CTGF in the DN, DF group rat of kidney cells inherent was more than NC group (P <0.01), the DN group was significantly higher than the DF group (P <0.05). RT-PCR and Western blot product of the relative quantitative analysis showed that the expression of CTGF in the DN, DF group rat of kidney cells inherent was more than NC group (P <0.01),the DN group was significantly higher than the DF group (P <0.05). Pathological changes in the kidney showed that DN group and DF group in early diabetic nephropathy change, DF group lighter than DN group.2. Cell experiments: RT-PCR and Western blot product of the relative quantitative analysis showed that the expression of CTGF in high-sugar and high-sugar group + fluvastatin group was significantly higher than the control group and the control group + fluvastatin group (P <0.01), and high Sugar group was higher than in high glucose group + fluvastatin group (P <0.05), but the control group and the control+fluvastatin group were no significant statistical difference (P> 0.05).3. Clinical trials: The the 24-hour urinary albumin excretion rate in the DA group, DF group and DAF group was significantly reduced than the former (P <0.01), but the 24-hour urinary albumin excretion rate in the DAF group was significantly reduced than the the DA group (P <0.05). ELISA method showed that the expression of CTGF in the DA group, DF group and DAF group was significantly reduced than the former (P <0.01), but the expression of CTGF in the DAF group was significantly reduced than the the DA group (P <0.05).四. Conclusions1. Fluvastatin intervention in this animal experiment was given at once after the model success, the results showed that the 24-hour urinary albumin excretion rate ,the Serum concentration of CTGF and the expression of CTGF in the renal of the fluvastatin intervention on the diabetes groups were significantly reduced than in rats with diabetes, but still more than normal rats,which indicate that the Fluvastatin not completely blocked diabetic nephropathy, but can significantly delay the progress of their disease.2. The fluvastatin dose (5 mg/kg/d) in use in this animal experiment does not cause blood lipid to obviously change, which implys the protective Effect of fluvastatin does not rely on its classic role.3. The expression of CTGF in Glomerular mesangial cells cultured in high glucose increased significantly. 4. Experimental observation of the clinical effects of fluvastatin in patients with diabetic nephropathy can reduce the 24-hour urinary albumin excretion rate and blood CTGF expression, and the ARB and the effects of the drug combination more obvious, but not completely blocked the occurrence of diabetic nephropathy, a significant delay the progress of their disease.5. We find that non-lipid-lowering doses of fluvastatin used in the diabetic nephropathy can retard the progress of the disease through detecting the expression of CTGF in kindey.