Mechanismes Of Mirnas Induced Proteinuria Via Triggering Nephrin Phosphorylation In Puromycin Aminonucleoside Nephrosis Rats And Intervention Of Tripterygium Wilfordii Hook. Preparation

Background and objective:Proteinuria is a dangerous factor of chronic kidney disease (CKD). Podocyte injury is not only the key link of proteinuria, but also closely related with CKD progress. Some microRNAs may induce slit diaphragm (SD) molecules expression via triggering nephrin phosphorylation to reduce proteinuria. Tripterygium glucosides can be proved to improve SD molecules expression, protect podocyte and reduce proteinuria.but its molecular mechanism is not clear. The hypothesis is that Tripterygium glucosides can improve SD molecules expression via triggering nephrin phosphorylation to reduce proteinuria. With SD molecules expression as target, and miRNAs as regulatory factors, the mechnisme that miRNA regulate SD molecules expression and Tripterygium preparation induce SD molecules expression via miRNA is explored to provide experiment basis for delying CKD progress, by observing morphology change of PAN nephrosis, expression of miRNA, SD molecules (nephrin and podocin) Nucleic acids and proteins with intervention of Tripterygium preparation.Methods:Eighty male wistar rats were randomly divided into eight groups, including control group, model group, leizhi capsule high-dosage group, leizhi capsule low-dosage group, Tripterygium glucosides high-dosage group, Tripterygium glucosides low-dosage group, triptolid group and valsartan group(with each grou ten rats). PAN nephrosis medel was made by jugular vein injection of PAN (100mg/kg body weight, dissolve, in physiological saline),while control group rats were made by jμgular vein injection of physiological saline with equal volume. By irrigation stomach once a day for ten days, All rats had been given medicines as follows:physiological saline 2 ml for control group and model group, Tripterygium glucosides 2mg dissolved in Erzhi(containing and Droμght Ephraim grass) solusion lml or lmg dissolved in Erzhi(containing and Droμght Ephraim grass) solusion 0.5ml for Leizhi capsule high-dosage or low-dose group, Tripterygium glucosides lmg/kg/d orlmg/200g/d for Tripterygium glucosides high-dosage or low-dose group, triptolid 2mg/200g/d for triptolid group and valsartan valsartan 1.5 mg/200g/d for valsartan group. The blood and urine samples were collected, and renal tissues were processed after killed. The 24h urinary protein excretion and blood biochemistry parameters were measured by routine methods. The glomerular morphology and podocyte ultrastructure were observed by light microscopy and transmission electron microscopy respectively. The foot processwidth was examind by morphometric method. The nephrin, podocin, dicer and synaptopodin expression and distribution change were determined by indirect immunofluorescence staining. TUNEL dyeing was for detecting apoptosis podocytes.Nephrin, podocin,dicer and synaptopodin mRNA and proteins were detected by RT PCR and Western Blotting respectively.miRNA expression profile was detected by Exiqon miRNA Array, including:prepare the RNA Sample and RNA Sample QC, miRNA labeling, miRNA array hybridization, miRNA array scanning and analysis. Real time RT-PCR analysis for mature miRNAs was used to validate 4 differentially expressed miRNAs between control and model group in microRNA microarray assaysResults:1. PAN nephrosis rats were made successfully by jμgular vein injection of PAN (100mg/kg body weight). In day 5, model rats were in low spirits, with decreased urine volume, ascites, malnutrition and wight loss. From day 7 to day 10, the nephrotic syndromes were worst in model rats, but without skin edema. Some rats died of serious ascites, the mortality is 30%(3/10).2. Morphologic changes in light microscope include epithelial cells degenerationrenal tubular and transparent cast, but there are no obvious changes in glomerulus and renal interstitial.Meanwhile, the degree of podocte processes effacement was obvious in model groups cample in electronic microscope.3. miRNA array detection shows 106 miRNA upregulated and 62 miRNA down regulated in PAN nephrosis rat. Fold change (model vs. control group) vary from 1.8 to 7.0. For leizhi capsule high-dose group and model sample, therr are 90 miRNA differentially expressed, with 65 up and 25 down. The most important finding in our study is the discovery of the specific miRNA related to PAN nephrosis (rno-miR23a, rno-miR-24, rno-miR-30c and rno-miR-300-3p, which haved been validated by Real time RT-PCR analysis.4. Compared with control sample, miRNA mature key enzyme dicer up- regulated in PAN nephrosis rats. Expression profile of nephrin, podocin and synaptopodin mRNA and protein reduced in model sample. In addition, apoptosis of podocyte increase in PAN sample. So dicer is not only closely related with SD molecules expression (negative regulation), but also correlates well with scaffolding proteins synaptopodin and podocyte apoptosis via miRNAs.5.Compared with model sample, Immune Fluorescence intensity of dicer, expression profile of nephrin, podocin and synaptopodin mRNA and protein decrease in samples treated with Tripterygium wilfordii Hook. preparation. This sμggests that dicer correlates with SD molecules and scaffolding proteins synaptopodin expression with intervention of Tripterygium wilfordii Hook. preparation. Tripterygium glucosides improve SD molecules expression and reduce proteinuria by triggering dicer-miRNA. Protection effect on podocyte of Tripterygium preparation is related with miRNA induceing podocyte apotosis.6. The nephrotic syndrome of PAN rats treated with Tripterygium preparation was significantly improved compared with that in PAN nephrosis control. Their urinary protein excretion was decreased, plasma albumin was increased, and high cholesterol/ triglyceride levels were remission, and anemia improced. Serum creatinine increased a little (p<0.05). In light microscop, epithelial cells degenerationrenal of renal tubular and transparent cast improved inn both leizhi capsule and Tripterygium glucosides samples(p<0.05).Meanwhile, the degree and area of podocte processes effacement was significantly reduced in both leizhi capsule and Tripterygium glucosides groups in electronic microscope(p<0.05).Conclusion:1. PAN nephrosis rats can be made successfully by jμgular vein injection of PAN (100mg/kg body weight).2. miRNA array detection shows 106 miRNA upregulated and 62 miRNA down regulated in PAN nephrosis rat, among these up-regulated miRNAs,two miRNA clusters on two different chromosome:one cluster(rno-miR-24 and rno-miR-23a) in chromosome 19,the other cluster (rno-miR-300,rno-miR-381, rno-miR-487b, rno-miR-376c and rno-miR-495) in chromosome 6. This sμggests PAN nephrosis correlates with chromosome 6 and chromosome 19. miRNAs of PAN nephrosis were screening. Up-regulated miRNAs(rno-miR-23a. rno-miR-300-3p) may trigger podocyte injury and proteinuria, while down-regulated miRNAs(rno-miR-24,rno-miR-30c) may be protective factors by anti-apoptosis.3. Dicer may regulate negatively expression profile of nephrin, podocin and synaptopodin mRNA and protein, participating in regulate podocyte apoptosis via miRNA. It is sμggested that dicer and these miRNAs are probably key regulated targets.4. Dicer can be down-regulated in PAN induced rats treated with Tripterygium preparation. Expression profile of nephrin, podocin and synaptopodin mRNA and protein decrease in samples treated with Tripterygium wilfordii Hook by triggering dicer-miRNA, which play the podocyte protection role by inhibit podocyte apotosis.so dicer and these miRNAs(rno-miR-24,rno-miR-30c,rno-miR-23a) may be are probably key molecules therapeutic targets.5. Leizhi capsule(Erzhi pil plus Tripterygium glucosides) can reduce toxity and increase effects of Tripterygium glucosides by reducing proteinuria, improving anemia, lowering AST, improving podocte processes effacement, and reducing epithelial cells degenerationrenal of renal tubular.