Expression And Significance Of Plasma MiRNA-29c In Unilateral Ureteral Obstruction Of Rats

BackgroundChronic Kidney Disease (CKD) is a progressive disease caused by many causes,Most no clinical symptoms in the disease early, due to renal functional reserve,serum creatinine often maintained at the normal range in the early, once clinical symptoms was appeared or serum creatinine level was increased significantly, The disease was often development to middle-late stage. If can’t diagnosed of chronic kidney disease in early, To remove the cause or be intervention treatment, Eventually develop into End-stage renal diasease(ESRD), That uremia, In the aspect of treatment, Just renal replacement therap:Peritoneal dialysis(PD), hemodialysis(HD)or Kidney transplant. Because the kidneys have endocrine,maintain a stable internal environment and other functions, once kidney disease progression to ESRD, All functions will be lost of kidney, patients will appear anemia, body electrolyte imbalance, sodium and water retention, hypertension, heart failure and so on,long-term dialysis appears hyperphosphatemia, metastatic calcification and other complications, these complications can reduce the quality of life of patients, shorten the life.At present, china suffering from uremia and need to renal transplant patients approximately 1.5 million, and 10-15 million per year rate increased.In recent years, chronic kidney disease gradually tend to be younger, the minimum patients less than 10 years old, this is related to the future development of a country. And because of the shortage of kidneys, renal transplantation each year only about 10,000 cases, far short of demand, in addition, renal transplant recipients must long-term take anti-rejection drugs, induced immune function in patients with lower than normal, prone to infection and other complications, even life-threatening, meanwhile, the vast majority of patients with renal transplantation can not bear the high cost of treatment and postoperative. The high cost of renal replacement therapy has become a huge financial burden for the whole family and society, chronic kidney disease has become a threat to human health an important public health problem.Therefore, in patients with chronic kidney disease early diagnosis and give treatment to be, extend the life of the kidney,postpone or avoid row renal replacement therapy,is to improve the quality of life of patients, prolong life, the key to reduce complications of kidney replacement therapy, is also an important measure to reduce the burden on the family and society.During the development and progression of renal disease process,renal interstitial fibrosis (RIF) is the main pathological changes in kidney tissue,Is the common pathway of many cause leading to renal failure, the main factor which the result of damage and repair factors continue a role in disease progression, its pathological changes mainly that generated extracellular matrix components (ECM) and degradation imbalance, resulting in an increase of ECM components,its complex mechanism. In recent years,micro RNA (miRNA) family become a hot in research organ fibrosis, research shows that miRNA families may be associated with liver, heart, lungs, kidneys, skin and other organs fibrosis, in the circulation, tissue, urine or other body fluids the miRNA can be stable in exist, therefore, many researchers began to explore the likelihood that miRNA as a biomarker to reflect certain diseases and the severity,especially prominent in the study of organ fibrosis, and tried from mechanisms of organ fibrosis to seeking a new treatment target for fibrotic diseases. Study confirms,transforming growth factor-β (TGF-β)/Samds signaling pathways involved in the occurrence of RIF by regulating miRNA.Objective:In this study, by detecting the expression levels of miR-29c in plasma and operative side kidney tissue after model 3,7 days of the sham operation group(Sham) group and unilateral ureteral obstruction (unilateral urethral obstruction, UUO) group, the degree of renal tissue RIF, fibrosis related indicators(E-cadherin, col-Ⅰ,α-SMA),TGF-β1,Smad3 protein expression, By correlation analysis the relationships between miR-29c and RIF, E-cadherin,col-Ⅰ,α-SMA,TGF-β1,Samd3.To investigate the potential of miR-29c as a hematology diagnostic marker for RIF;and further elucidate the pathogenesis of RIF, provide appropriate theories and new therapeutic targets for prevention and treatment of RIF.Methods:1.Establish UUO model:20 SD rats were randomly divided into 2 groups,namely Sham group and UUO group,each group 10.3% pentobarbital sodium solution by 0.5ml/100g intraperitoneal injection of anesthesia,take the left kidney area about 2-3cm abdominal incision into the abdominal cavity,find the left iliac veins, according to the anatomic landmarks that ureters crosses the iliac vessels,find left ureter and free to nearly pelvis.UUOgroup,left ureter was ligated by silk NO.0 in place near the renal pelvis and ureter 1/3,however,Sham group,only free but not ligated,sutured abdominal wall, the rats were placed in a clean, dry environment postoperative,freedom to eat food and drink,rats were reared under the same conditions.2. Obtain samples of blood and kidneys of rats:In the first three and seven days after surgery,from the two groups of rats were randomly selected five rats,3% pentobarbital sodium solution by 0.5ml/100g intraperitoneal injection of anesthesia,satisfaction after anesthesia,cut the abdominal and chest,exposing the heart,blood collection needle was inserted into the left ventricle from the apex, Blood was collected 2-3ml and stored in EDTA tubes,plasma was separated after centrifugation(1200r/min), placed in -80℃ refrigerator spare,blood had been completed,used ice saline infusion, until both kidneys became pale,then removed the left side kidney,get kidney tissue, were placed in 4% paraformaldehyde solution and RNAlater storage solution,stored in -80℃ refrigerator spare.3. Histopathology of rats kidney tissue3.1 Masson and HE staining:RIF extent observed of kidney tissues,which were preserved by 4% paraformaldehyde solution, that by Masson and HE staining methods. Observe changes in the organizational structure of rat kidney by HE staining. Masson staining was to assess the extent of RIF, calculation of renal interstitial fibrosis index (%), renal interstitial fibrosis index based grading, press Banff07RIF score on renal tissue fibrosis semiquantitative score:Each specimen randomly selected 10 non-repetition of the renal cortex in the light microscope (×200), to take pictures, use IPP6.0 (Image-Pro Plus 6.0) image analysis software to calculate the percentage of the area of fibrosis per field vision accounts, calculation per field fibrosis score according to the following criteria:No damage, less than 5% of the area of fibrosis,0 score; Mild injury, fibrotic area accounts for 6%-25%,1 score; Moderate damage, fibrotic area accounts for 26%-50%,2 scores; Severe damage, fibrosis area greater than 50%,3 scores, the score of 10 fields are added and averaged, get the slice fibrosis score.3.2 Immunohistochemical staining of kidney tissue:Use EliVision method,that is the paraffin(3μm thickness) dewaxing to water, boiling water of citrate buffer cook 20 min to repair antigen,3% hydrogen peroxide to inactivate endogenous enzyme,then the following antibodies were added:Rabbit anti-human Col-Ⅰ monoclonal antibody(was diluted 200-fold), rabbit anti-human E-cadherin monoclonal antibody(was diluted 200-fold),rabbit anti-human α-SMA monoclonal antibody(was diluted 200-fold),rabbit anti-human TGF-β1 polyclonal antibody(was diluted 200-fold),rabbit anti-human Smad3 polyclonal antibody(was diluted 200-fold), incubated overnight at 4℃,dropping anti-mouse HRP/rabbit polymer after dropping the polymer enhancer, DAB color,control color reaction under the microscope; mounted with neutral gum after hematoxylin,.Using image analysis software IPP6.0, each sample randomly selected 10 non-repetition vision(×200), all part of the antibody expression is yellow,calculate the percentage of the positive area in each field.4. Determination of miR-29c in In plasma and kidney tissues:The samples RNA were extracted in plasma and kidney through the following steps,that homogenization, two-phase separation,RNA precipitation,RNA washing,the RNA pellet was redissolved; RNA as a template,RT mixture was the reaction liquid,RT reaction was carried out by using real-time quantitative PCR(qRT-PCR) amplification,cDNA was synthesized.The reaction conditions for 16℃30mim、42℃40min、85℃5min,all cDNA samples were arranged in qRT-PCR reaction system,the system configuration is as follows:2×Master Mix is 5μl,the specific primers F is 0.5μl and 0.5μl R,water was added to a total volume 8μl,finally,mixed solution,briefly centrifuge(5000r/min).The 8ul mixture solution was added to each well of the 384-PCR plate,then add the corresponding 2μlcDNA,glue sealing membrane and mixed briefly centrifuged,the 384-PCR platewas placed on the qRT-PCR instrument and PCR amplification reaction, melting curve analysis of the PCR product after completion of the reaction,with U6 as internal control, in the control group of three days as a reference,the results are shown using 2-△△CT.5. Statistical analysis:Using SPSS 13.0 software system analysis.All measurement data using x±s representation,between the two groups were compared using t test ofcompletely randomized design,correlation analysis using spearman correlation coefficient analysis.The difference was consider ststistically significant when P<0.05.Results:1.Sham group,kidneys were no changes in the 3rd and 7th days after surgery: Kidneys no increases, no hydronephrosis;UUO group,left kidneys wre significantly increased in the 3rd and 7th days after surgery:Significant hydronephrosis.Compared with the 3rd day after surgery,the 7th more serious of hydronephrosis.2.HE staining was observed:In Sham group:the nephron size and shape were normal.But in UUO group:mild renal tubular dilation,tubule vacuolar degeneration,interstitial edema and scattered inflammatory cells in the 3rd day after surgery.Renal interstitial widened significantly increased collagen deposition,tubular atrophy and inflammatory cell infiltration in the 7th day after surgery.3. Masson staining was observed:ln Sham group:the nephron size and interstitial were normal.But in UUO group:tubular lumen was dilated,a small amount of light blue collagen deposition in part of interstitial in the 3rd day after surgery. Tubules are dilated,interstitial edema widened significantly with monocytes and lymphocytes extensive infiltration,interstitial collagen fiber were increased.4. UUO group RIF semiquantitative score was significantly higher than that of Sham group in the 3rd and 7th days after surgery(P<0.01),the expression levels of miR-29c in plasma and kidney tissue were lower than the Sham group,this is also the E-cadherin in kidney tissue(P<0.01);the expression levels of Col-Ⅰ, α-SMA, TGF-β1 and Smad3 protein were higher than the Sham group (P<0.01).5.In UUO group,the 7th day is compared with the 3rd after surgery:RIF semiquantitative score was significantly increased(P<0.01),the expression levels of miR-29c in plasma and kidney tissue and E-cadherin in kidney tissue were decreased (P<0.01), the expression levels of Col-Ⅰ, α-SMA, TGF-β1 and Smad3 protein in kidney tissue were increased(P<0.01),however, these indicators were no change in Sham group.6. Correlation analysis:The expression of miR-29c decline consistency in plasma and tissue of UUO group,positively correlation with decreased of E-cadherin in tissue(r=0.633,0.672,P<0.01),negative correlation with RIF degree,Col-Ⅰ,α-SMA, TGF-β1 and Smad3 protein(r=-0.581,-0.712;-0.812,-0.712;-0.774,-0.781;-0.741,-0.862;-0.695,-0.594,P<0.01).Conclusions:1.The modeling was successful by observing the appearance of the kidneys, kidney tissue HE staining, Masson staining, immunohistochemistry staining.2.miR-29 may be regulated by TGF-β1/Smad3 signaling pathway to participate in the development and progression of RIF,there are likely to be non-invasive markers of Hematology to diagnose RIF.3. TGF-β1/Smad3 signaling pathway may be involved in RIF through down miRNA-29c, and provide a theoretical basis for new therapeutic targets for prevention and treatment of RIF.