| BackgroundRenal interstitial fibrosis is a common pathological path of a variety of kindey disease includes renal graft disease develepment for end-stage renal failure. The main cause of chronic kidney disease include primary glomerulonephritis, hypertensive nephropathy, secondary glomerulonephritis, diabetic nephropathy, tubulointerstitial disease, hereditary kidney disease, in developed countries, diabetic nephropathy and hypertensive nephropathy has become the leading cause of CKD,but in China, in a variety of causes, the above two diseases incidence rate is still lower than the primary glomerulonephritis, there was a marked increase trend in recent years. Since the past 10 years,the epidemiological studies of CKD is very concerned about the international, in the United States and other developed countries show, CKD is a common chronic disease, CKD morbidity rate in the adult population is 10.2%-13.0%. In China, led by Professor Wang Haiyan, "China epidemiology of chronic kidney disease" uses a multi-stage stratified sampling design, a total of 47,204 people is gets to represent the Chinese adult population surveyed, evaluates survey results show that morbidity rate of Chinese people over the age of 18 CKD was 10.8%,accordingly estimates, China currently has CKD patients will reach 120 million cases. According to statistics in developed countries, about 2% CKD patients will develop end-stage renal failure stage,they need kidney transplant or dialysis to sustain life. According to each uremic patients dialysis costs 100,000 yuan/year China will pay for these dialysis patients 240 billion yuan each year. In addition, CKD presences complex interactions with other common chronic diseases,for example, a follow-up study of 100 million people showed that compared with normal renal function, cardiovascular events increased by 40%, mortality increased by 20% in renal function decline patients,and with the deterioration of renal function,the risk increases linearly trend. So chronic kidney disease has become one of the most serious public health problems in China.With the development of medical technology, organ transplantation has become one of the significant progress of the medicine field in the 20th century, renal transplant technology has become the best alternative treatment methods for end-stage renal disease, although recent graft survival was significantly improved, however, the late survival rate has not increased correspondingly, the main reason is chronic allograft nephropathy for long-term renal graft failure,which always occurs after transplatation few months to several years, the major pathological changes of CAN includes interstitial fibrosis,tubular atrophy and glomerular sclerosis, In the above pathological changes, renal tubular atrophy and interstitial fibrosis is the most critical factor affecting long-term prognosis of renal graft. Banff2005 had been deleted the concept of CAN, replaced by interstitial fibrosis and tubular atrophy,and two new types of pathology has been Introduced, chronic T-cell mediated rejection(CTMR) and chronic antibody-mediated rejection(CAMR).The development of renal interstitial fibrosis has made a thorough study in China and abroad,in particular,signaling pathways in renal interstitial fibrosis research has been made remarkable achievements. A large number of studies have shown that TGF-β1/Smad signaling pathway plays a major role in the development of organ fibrosis, and some studies reported that a variety of downstream microRNAs may be regulated by TGF-β1/Smad signaling pathway, thus affecting the development of fibrosis in various organs. MicroRNAs (miRNAs) are noncoding small RNAs, which are widely found in animal and plant genome, most miRNAs composed of 21 to 23 nucleotides,can bind to the target gene mRNA specific sites, and induce mRNA degradation or protein synthesis,thus miRNAs participate in the expression and regulation of various genes.In the current level of medical technology stage,when appear elevated serum creatinine, proteinuria, and other symptoms after chronic kidney disease and renal transplantation, tissue biopsy is still the gold standard for diagnosis of the cause, but whether in situ or renal graft biopsy puncture, we have to face the risk of bleeding, infection.Few studies about the role miRNA-21 in RIF mechanisms currently, in this study, by measuring the expression of miRNA-21 in plasma and fibrosis kidney tissue of UUO rats, preliminary explore mechanisms of miRNA-21 take part in TGF-β1/Smad3 signaling pathway in renal interstitial fibrosis,then look for a non-invasive detection of specific biomarkers of RIF.Objective(1)Detect the expression of MicroRNA-21 (miRNA-21) in kidney tissue and plasma of normal rats and unilateral ureteral obstruction (UUO) rats, observate and analysis of different expression of miRNA-21 in both groups.(2) Preliminary explore mechanisms of miRNA-21 take part in TGF-β1/Smad3 signaling pathway in renal interstitial fibrosis (RIF).Methods(1 Established UUO rats model:Totally 20 adult male SD rats were randomly divided into UUO model group and Sham operation group,the left ureter of rats in UUO group were ligated, the rats in sham group were dissociated left ureter only, but not to be obstructed. Collect the left kidney tissue and blood at the 3rd、7th days after surgery in two groups.(2)Detection of miRNA-21:Real-time quantitative PCR analysed the expression of miRNA-21 in the kidney and plasma of two group rats:According to Trizol reagent instructions to extract kindey tissue’ RNA and plasma’ RNA of UUO group and Sham group at the 3rd、7th days after surgery, and using an ultraviolet absorption to detect the purity and concentration of extracted RNA, synthesis cDNA by using RNA extracted from kindey tissue,then underwent RT PCR amplification reaction after preparation of RT reagents,each sample miRNA and U6 underwent Real-time PCR reactions, data were analyzed using 2-△△CT method, Sham group’2-△△CT was 1 as the reference correction。 Detect the expression of miRNA-21 in rats plasma:using TRI Reagent BD extracted plasma RNA, remain detection steps as fomer method.(3)Pathological examination of renal tissue in ratsThe kidney tissue samples were embedder with paraffin wax and producted kidney tissue sections, masson staining,HE staining and immunohislochemistry staining were used to evaluate the degree of RIF, expression of TGF-β1 Smad3, α-smooth muscle actin (α-SMA)、 and collagen Ⅰ (Col-Ⅰ) were examined by immunohislochemistry staining.(4)Statistical methodsStatistical analysis was performed by SPSS for Windows software version 19.0,measurement data between groups were presented as mean±SD, and analyzed using one-way ANOVA,homogeneity of variance between groups were compared using LSD-t test, heterogeneity of variance using Dunnett’s T3,correlation analysis using Spearman method,a p value of <0.05 was considered as statistically significant.Results(1)MicroR-21 expression changes of two groups ratsThe expression of miRNA-21 in kidney tissue and plasma was higher at 3rd、7th days after surgery in UUO groups than Sham group(P=0.000), the expression in UUO group at 7th days was higher than 3rd days after surgery (P=0.008,P=0.024), but compared with the tissue,the expression levels of plasma was not significantly.(2)Kidney tissue pathology observation of the two groups of ratsHE staining was observed that the renal unit size,morphology were normal in Sham group,and mild renal tubular dilation,tubule vacuolar degeneration,interstitial edema and scattered inflammatory cells wsa observed in UUO group at 3rd days after surgery. Apparent renal interstitial widening, increased collagen deposition, tubular atrophy and inflammatory cell infiltration were observed in UUO group at 7th day after surgery, UUO rats model is successfully established.There wsa no difference in masson staining kidney tissue sections in Sham group at 3rd and 7th days after surgery,the renal units and interstitial tissue were normal. We can observe that dilated tubules,interstitial edema widened,monocytes and lymphocytes extensive infiltration in UUO group at 7th day after surgery,and also observed interstitial collagen fibers progressive increased,collagen fibers collagen fiber area and blue area increased significantly. There were significant differences in collagen positive area score with masson staining among UUO 3rd days group,7th days group and Sham 3rd days group,7th days (F=194.228, P=0.000),the fibrosis score of UUO at 3rd days and 7th days was significant higher than Sham group at the same time (P=0.000 and P=0.000),the fibrosis area of 7th days UUO group was more than 3rd days UUO group (P=0.000)TGF-β1 protein slightly expressed in renal tubular epithelial cells at 3rd、7th days after model establishment in Sham group, no expression in interstitial and no significant changes at each time point.Compared with the Sham group, TGF-β1 protein expression area in renal tubular epithelial cells and interstitial tissue gradually increased over time,and positive staining gradually deepened at 3rd、7th days in UUO group. There were significant statistical differences in TGF-β1 protein positive area between groups at 3rd、7th in UUO and Sham group(F=423.844,.P=0.000), there were significant statistical differences of TGF-β1 protein positive area in 3rd、7th UUO group compared to Sham group at the same time (P=0.000 and P=0.000).The TGF-β1 protein positive area of 7th days UUO group was more than 3rd days UUO group (P=0.000)There were no apparent changes of Smad3 protein expression in kidney tissue of Sham group at 3rd days and 7th days, only slightly expression of Smad3 protein in renal tubular epithelial.Compared to Sham group,Smad3 protein positive area of UUO group in renal tubular epithelial cells and interstitial tissue gradually increased,and strong expressed in 7th UUO group. There were significant statistical differences in Smad3 protein positive area between groups at 3r、7th UUO and 3rd、 7th Shman group (F=768.544,P=0.000).There were significant statistical differences of Smad3 protein positive area in 3rd、7th UUO group compared to Sham group at the same time(P=0.000 and P=0.000). The Smad3 protein positive area of 7th days UUO group was more than 3rd days UUO group (P=0.000)Col-Ⅰ protein slightly expressed in renal tubular epithelial in Sham group, compared to the same time Sham group, Col-Ⅰ protein positive area of UUO group gradually increased.There were significant statistical differences in Col-Ⅰ protein positive area among groups (F=1108.079, P=0.000), there were significant statistical differences of Col-Ⅰ protein positive area in 3 rd、7th UUO group compared to Sham group at the same time (P=0.000), the Col-Ⅰ protein positive area of 7th days UUO group was more than 3rd days UUO group (P=0.000)a-SMA protein slightly expressed in Sham group, a-SMA protein positive area of UUO group in renal interstitial tissue gradually increased over time,and strong expressed in 7th UUO group, here were significant statistical differences in Col-Ⅰ protein positive area among groups (F=292.363, P=0.000),pairwise comparisons among groups,a-SMA protein protein positive area of UUO group was more than that in Sham group (P=0.000), thea-SMA protein positive area of 7th days UUO group was more than 3rd days UUO group (P=0.000)(3) Correlation analysis of microR-21 and tissue fibrosisThe expressions of miRNA-21 in kidney tissue and plasma were positive correlations with the fibrosis score(r=0.816、r=0.907,P=0.000); The expressions of miRNA-21 in kidney tissue was positive correlations with the protein TGF-β1、 Smad3、 Col-Ⅰ、α-SMA (r=0.79、r=0.849、r=0.888,r=0.882, P=0.000),and expressions of miRNA-21 in plasma was also positive correlations with those protein (r=0.816、r=0.875、r=0.904、r=0.905, P=0.000)Conclusions(1)The expression of miRNA-21 was upregulated in UUO kidney tissue and plasma after model establishment, the more severe the RIF degree,the higher the expression level.(2)TGF-β1/Smad3 signaling pathway was possible positively regulate miRNA-21 to mediate rats RIF. |
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