| Objective: Renal interstitial fibrosis is the final performance of chronic kidney disease(CKD), which is inevitable progress to end-stage renal disease(ESRD), the treatment of it including dialysis and renal replacement therapy. The main performances of renal interstitial fibrosis are renal tubular interstitial fibrosis and glomerular sclerosis, which molecular mechanism is the excessive deposition of extracellular matrix(ECM), and pathology is often characterized by the activation of cells, such as: epithelial mesenchymal transition, fibroblasts and fiber cells, myofibroblast, monocyte/macrophage infiltration, etc. Cell apoptosis and the activation of signaling molecules, such as transforming growth factor beta(TGF- beta), angiotensin-Ⅱ(Ang- Ⅱ) etc. With the progress of chronic kidney disease(CKD), the increase of survival and mortality rate, which are harm to human survival. 3- hydroxy- 3- methyl glutaric acyl coenzyme A reductase(HMG Co A reductase) also known as statins, which block the rate-limiting step of cholesterol biosynthesis. and its inhibitors is a classic drugs for the treatment of dyslipidemia. In widespread clinical use, statins have proven safe and effective for both primary prevention of coronary heart disease and secondary prevention of coronary events, and its pleiotropic effect are as follows: inhibit inflammatory cells infiltration, resistance to oxidative stress, relieve extracellular matrix deposition, at the same time it has the renal protective effection. Relevant research proves that: lysophospholipids acid receptor 1(LPA1R) expression is increased in the process of renal interstitial fibrosis, thus induced connective tissue growth factor(CTGF) and Collagen Type Ⅲ(Collagen Type Ⅲ) expression also increased, whether this progress through small G proteins Rho A has yet to have a clear research, statins can through its isoprene effect to reduce Rho A protein expression. Through the intervention of rosuvastatin, this experiment detect the expression of LPA1 R, Rho A, CTGF and Collagen type Ⅲ in renal interstitial fibrosis rats, studying whether small G protein Rho A can control this pathway that LPA1 R induce RIF, exploring how to reduce and delay the renal interstitial fibrosis, possible mechanisms of renal protection effect, to delay the progress of chronic kidney disease(CKD) and provide new therapeutic targets.Method: 54 SPF level healthy male SD rats, body weight 180±20 g, after 1 week adaptive feeding, were randomly divided into three groups: normal control group(group A), model group(group B), rosuvastatin group(group C), with 18 rats in each group. All groups would not provide food before 12 hours in the operation day. The rats in every group received Chloral hydrate(0.1ml/kg) intraperitoneal injection of anesthesia, as well as the 2% lidocaine hydrochloride subcutaneous injection of anesthesia. Conduct the surgery under sterile conditions. Left ureter was exposed via a left abdominal incision.The mid-ureter was obstructed by two point ligation with 4-0 silk sutures, and then cut the ureter between the ligated two points to avoid retrograding infection. The sham operated rat underwent the same surgery procedure except for the obstruction and cut of the left ureter. Each group administrated orally one day before the surgery, once a day, reveal rat’s weight per week, and adjust the administration according to their weight. The treatment group were given rosuvastatin 10 mg/kg/d orally, the control group and model group were gavaged with isometric saline. The rats were allowed to eat and drink free. Randomly select 6 rats from each group respectively, and harvest their obstructed kidneys on day 3, 7, 14 after the UUO surgery. Slice the kidneys and place the slices into fixation solution, embed them in paraffin. The pathological changes were determined in the kidney tissues underwent operation(HE) staining, Masson staining, as well as immunohistochemistry to detect the expression of LPA1 R, Rho A, CTGF and Collagen Type Ⅲ. The result of immunohistochemistry would be analysed by semi-quantitative analysis. Statistical analysis software SPSS 20.0 was applied for statistical analysis of experimental data which was analyzed with one-way ANOVA, and displayed as mean ± standard deviation, P <0.05 was considered statistically significant.Results:1 Pathomorphological observation of renal tissueHE staining under light microscopy:no change in group A, 3 days after UUO, a small amount of inflammatory cells infiltration and mild expansion of renal tubules were found in group B, as well as after 7 days, except for the changes of renal tubules dilated and tubular epithelial cells were more degeneration and necrosis, large quantities of inflammatory cells infiltration occurred in renal interstitium, furthermore interstitial width was significantly increased. On the 14 th day, visible tubular atrophy was more, diffuse thickening of tubular basement membrane, shrinkage, with varying degrees of fault occurred in group B; in group C, changes became worse as time went by, however, they were still better than those in group B, but they were worse than those in group A.Masson staining under light microscopy: in group A, there was no abnormal signs; slight tubular expansion and increased collagenous fibers presented on the 3rd day after UUO in group B,and these phenomenon got worse as time went by; in group C, changes became worse after the 7th day, however, they were still better than those in group B, but they were worse than those in group A.2 The immunohistochemical results of LPA1 R, Rho A, CTGF and Col-Ⅲ in renal tissue:LPA1R:in group A, only trace in amount of LPA1 R expression was detected in mesangial cell and tubular epithelial cells; however, the expression of LPA1 R were largely detected in proximal tubular epithelial cells and renal interstitial in group B, the expression level of LPA1 R went up in a time dependent manner, and there was a significant difference(P<0.05) between group A and group B on the same time point; in group C, the expression level of LPA1 R was not as much as that in group B(P<0.05), but it was still much higher than that of group A(P<0.05).Rho A: in group A, the expression of Rho A was detected in the tubular epithelial cells only bassic trace expression and no expression on glomerulus; compared to the normal control group, the expression of group B was increased in a time dependent manner, and there was a significant difference(P<0.05);the level of group C was less than group B, and there was a significant difference(P<0.05) between group A and group B on the same time point, but there was no statistically significant difference(P>0.05)between 3 days and 7 days after UUO compared with group B,the expression level on 14 th day was lower than group B(P<0.05).CTGF: in group A,there was no expression on glomerulus, but only trace expression in tubular epithelial cells; in group B, CTGF significantly increased on the 3rd and the 7th day compared to the group A(P<0.05), while the expression level on 7th day was higher than that on the 3rd day(P<0.05), and then it was increased highest in the 14 th day after UUO, there was statistically significant difference(P<0.05); the level of group C was less than group B and more than group A,and there was a significant difference(P<0.05).Col-Ⅲ: in group A,there was no expression on glomerulus, but only trace expression in tubular epithelial cells; in group B, Col-Ⅲ significantly increased with the obstruction, but there was no significant difference(P>0.05) between the 3rd and the 7th day, while the expression level on 14 th day was higher than that on the 3rd day and 7th day(P<0.05), there was statistically significant difference(P<0.05)compared to group A; the level of group C was less than group B and more than group A, and there was a significant difference(P<0.05),but between the 3rd and the 7th day, there was no significant difference(P>0.05), while the expression level on 14 th day was higher than that on the 3rd day and 7th day(P<0.05).Conclusion: The expression of LPA1 R,Rho A,CTGF and Col-Ⅲ were increased in obstruction kidney after UUO, and they are relaying on each other. While the level of Rho A was decreased by rosuvastatin intervention under the fibrosis factor LPA1 R increasing, the expression of CTGF and Col-Ⅲ was decreased in kidney. It maybe reduce and put off renal interstitial fibrosis. By this experiment, it maybe infer that one of the mechanism of rosuvastatin inhibited renal interstitial fibrosis should be adjust the level of small G protein to reduce RIF induced by LPA1 R,which may protect renal by its isoprene effect to reduce Rho A protein expression. |
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