Effects Of MiR-29a Gene Knockout On The Structure And Function Of C57BL/6 Mouse Glomerulus

miRNA-29（miR-29）is a type of micro RNA,which is highly expressed in tissues such as the kidney.This study observed the changes of miR-29 a mRNA in the glomeruli of mice with diabetes and explored the effects of miR-29 a gene knockout on the function and structure of mouse glomeruli.Methods: The type I diabetes model was induced by unilateral nephrectomy add streptozotocin（STZ）injection in C57BL/6 mice.The db/db and db/m mice of 24 weeks were selected as the type 2 diabetes model.After successful induction by STZ,the glomeruli were separated by cardiac perfusion with Dynabeads^TM M-450 Tosylactivated micromagnetic beads,and the changes of miR-29 a mRNA in the glomeruli were detected by real-time fluorescence quantitative PCR（RT-qPCR）.The miR-29 a gene was knocked out using CRISPR/Cas9 technology in C57BL/6 mice,and the genotype of newborn mice was detected by genetic identification after hybridization with wild mice of the same species.Mi R-29 a knockout mice were selected as the experimental group（miR-29 a knockout,miR-29 a KO）,non-knockout mice served as wild type control（wide type,WT）.The 24 h urine and retrobulbar venous blood of mice were collected on the 3 rd,12 th,and 24 th week respectively.Coomassie brilliant blue staining method was used to qualitatively analyze whether mice had proteinuria;enzyme-linked immunosorbent assay（ELISA）method was used to determine urine albumin,urine creatinine and blood urea nitrogen（BUN）levels,and to calculate urine albumin/ Creatinine ratio（urinary albumin/creatinine ratio,UACR）.The mice were selected on the 3 rd,12 th,and 24 th week respectively,one kidney was used for hematoxylin-eosin staining（HE）,periodic acid-schiff（PAS）staining and Masson’s three-color staining to observe the pathological changes of the tissue.Other side of the kidney was used to observe the glomerular ultrastructure by transmission electron microscope and cryo-etching electron microscope.The thickness of glomerular basement membrane,the width and number of podocyte were analyzed quantitatively in 24 w mouse.Another two groups of 24 w mice were selected,one group of mice were anaesthetized by intraperitoneal injection of chloral hydrate.The glomeruli was isolated by Dynabeads^TM M-450 Tosylactivated micromagnetic beadsafter cardiac perfusion,to extract RNA and protein.another group of the mice were directly euthanized.The kidney was taken for OCT embedding,sectioning,and immunofluorescence staining of the glomerulus.RT-qPCR,immunofluorescence and Western Blot（WB）techniques were used to detect the markers of mouse glomerular podocytes,Nephrin,Podocin,glomerular type IV collagen（COL4α3,COL4α4,COL4α5）and laminin β2（laminin β2,LAMB2）.Results: RT-qPCR results showed that,compared with the control group,the mRNA levels of miR-29 a in the glomeruli of mice with type I and type II diabetes was significantly down-regulated（p <0.01）,which was 58% or 57% of the control group respectively.Results of renal function: Coomassie brilliant blue staining qualitative experiment showed that,the mice in miR-29 a KO group had urine albumin,while there was no proteinuria in WT group.The UACR results had no significant changes in miR-29 a KO mice at 3w,it,and the data was not statistically different compared with that in WT mice（p>0.05）.At 12 w,the UACR increased significantly（1.77±0.14）,and there was a significant difference compared with that in WT group（1.00±0.07）（p <0.01）.At 24 w,the UACR increased more significantly（2.50±0.25）compared with that in WT mice（p <0.001）.There was no significant changes of BUN in miR-29 a KO mice compared with that in WT mice at 3 w,12 w,and 24 w（p >0.05）.Results of kidney histological staining: At 3 w,the glomeruli of mice in miR-29 a KO group had slight extracellular matrix increase and mesangial matrix proliferation.With the increase of age,the fibrous collagen deposition,mesangial matrix expansion and basement membrane thickening appeared in the glomeruli of miR-29 a knockout mice.At 24 w,the above-changes were more significant.PAS staining showed that the glomerular basement membrane and mesangial of mice were purple-red.Masson staining showed that the kidney collagen fibers were blue.Compared with that in the WT group,the glomeruli of mice in the miR-29 a KO group were deep purple-red in PAS staining and deep blue in Masson staining.Results of glomerular ultrastructure observation: Transmission electron microscopy showed that the glomerular ultrastructure of mice in the miR-29 a KO group began to change at 3 w,including the disorder of foot process arrangement and slight fusion.At 12 w,the number of foot processes were less,while the foot process fusion and glomerular basement membrane were thicker.At 24 w,the above changes were further aggravated.Quantitative analysis at 24 w mice showed that compared with the WT group,the thickness of basement membrane and the width of foot process in the miR-29 a KO group increased significantly,the number of foot processes decreased significantly（p <0.05）.Results of glomerular podocytes and basement membrane markers: RT-qPCR results showed that compared with the WT group,the expression of the podocyte marker Nephrin in the miR-29 a KO group was significantly down-regulated at the mRNA level（p <0.05）,Podocin expression also showed a downward trend in mRNA levels,but the results were not statistically significant（p >0.05）.Compared with the WT group,the expression of glomerulus COL4α3,COL4α4,COL4α5 and LAMB2 in the miR-29 a KO group was significantly up-regulated at the mRNA level（p <0.05）.WB results showed that,compared with the WT group,the protein expression of Nephrin and Podocin in the miR-29 a KO group was significantly reduced（p <0.05）;the expression of COL4α3/α4/α5 and LAMB2 were significantly increased（p <0.01）.The glomerular immunofluorescence results were consistent with the WB results: The expressions of Nephrin and Podocin in the glomeruli of mice in the miR-29 a KO group were all down-regulated,and the expressions of COL4α3/α4/α5 and LAMB2 were all up-regulated.Conclusion: 1.The expression of miR-29 a mRNA is down-regulated in the glomeruli of diabetic mice.2.Mi R-29 a gene knockout can cause proteinuria,glomerular structure damage and fibrosis in mice.With the aging of mice,the degree of proteinuria and glomerular structural damage is aggravated.