Abstract
Objective: microRNAs (miRNAs) are regulatory RNAs that act as important players in diverse biologic and pathologic processes. Under circumstance as podocye-injury triggering proteinuria, which miRNAs are up-regulated or down-regulated? This experiment aims at detecting miRNAs changes in PAN nephrosis rats based on miRNA arrays and exploring the therapeutic targets of Leizhi capsule. Methods: Fifty male wistar rats were randomly divided into five groups, including control group, model group, leizhi capsule group, Tripterygium glucosides group, and valsartan group. PAN nephrosis models were made by jugular vein injection of PAN (100 mg/kg body weight, dissolve in physiological saline), while control group rats were made by jugular vein injection of physiological saline with equal volume. Other groups rats had been given medicines by irrigating stomach once a day for ten days. Blood and urine samples were collected, and renal tissues were processed after rats being euthanasised. The 24 h urinary protein excretion and blood biochemistry parameters were measured by routine methods. The glomerular morphology and podocyte ultrastructure were observed with light microscopy and transmission electron microscopy respectively. miRNA expression profile was detected by Exiqon miRNA Array. Real time RT-PCR analysis for mature miRNAs was used to validate differentially expressed miRNAs. Results: 1) In day 3 - 5, model rats had decreased urine volume, ascites, malnutrition and wight loss. From day 7 to day 10, the nephrotic syndromes were worst in model rats, but which had no skin edema. Some rats died in serious ascites, the mortality is 3/10. 2) miRNA array detection shows 106 miRNAs up regulated and 62 miRNAs down regulated in PAN nephrosis rats. Fold change (model vs. control group) varies from 1.8 to 7.0. For leizhi capsule group and model sample, there are 90 miRNAs differentially expressed, with 65 miRNAs up and 25 miRNAs down. The most important finding in our research is the discovery of the specific miRNAs related to PAN nephrosis (rno-miR23a, rno-miR-24, rno-miR-30c and rno-miR-300-3p), which have been validated by Real time RT-PCR analysis. 3) Compared with control sample, immune fluorescence intensity of dicer, expression profile of nephrin, podocin and synaptopodin mRNA and protein decrease in PAN nephrosis rats. After treated with Leizhi Capsule, immune fluorescence intensity of the above molecules improved. Conclusion: 1) Characteristic 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. 2) Dicer and these miRNAs (rno-miR-24, rno-miR-30c, rno-miR-23a) may be are probably key molecules therapeutic targets of Leizhi capsule.
Highlights
MicroRNAs are a group of non-coding small RNA that are present in lower through higher organism and function to regulate gene expression by translation repression or transcript degradation of target genes
The most important finding in our study is the discovery of the specific miRNA related to PAN nephrosis
Our results show that dicer correlates well with proteinuria, but correlating negatively with nephrin and podocin
Summary
MicroRNAs (miRNA) are a group of non-coding small RNA (approximately 22-nt) that are present in lower through higher organism and function to regulate gene expression by translation repression or transcript degradation of target genes. Dicer is a key enzyme in miRNA biosynthetic, by which pri-miRNAs are processed into approximately 22-nt mature microRNA [1] [2]. Dicer is involved in the immediate downstream effecter steps of this pathway, whereby it serves as an essential component of miRNA-containing catalytic enzyme complexes, such as the RNA-induced silencing complex. As a key enzyme of miRNA biosynthesis, dicer probabaly have a role on proteinuria of CKD. Podocyte-selective deletion of dicer induces proteinuria and glomerulosclerosis [1] [7], alter cytoskeletal dynamics and causes glomerular disease [1] [2] [8], leading to rapid glomerular and tubular injury [3] [9]. How dicer enzyme have effects on podocyte in PAN (puromycin aminonucleoside, PAN) nephrosis pathlogy? So the aim of our experiment is to explore how dicer has effects on podocyte apotosis and cytoskeleton
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