miR-29a基因敲除对C57BL/6小鼠肾小球结构和功能的影响

Effects of miR-29a knockout on glomerular structure and function in C57BL/6 mice

目的:观察微小RNA-29a(miR-29a)基因敲除对小鼠肾小球功能和结构的影响。方法:应用CRISPR/Cas9技术,选择C57BL/6 miR-29a基因敲除型(miR-29a knockout,miR-29a KO)小鼠作为实验组,未敲除型小鼠作为野生型(wild-type,WT)对照组(n=30)。分别在第3、12和24周收集小鼠24 h尿液。采用考马斯亮蓝染色法进行尿蛋白测定;ELISA测定尿白蛋白和尿肌酐水平,并计算尿白蛋白/肌酐比值(urinary albumin/creatinine ra‐tio,UACR)。选取3、12和24周的小鼠,采用过碘酸雪夫氏(periodic acid-schiff,PAS)染色法观察肾脏组织的病理变化;通过透射电镜以及冷冻蚀刻电镜观察肾小球超微结构。另取2组24周的小鼠,一组小鼠腹腔麻醉后用Dyna‐beads™M-450 Tosylactivated微磁珠经心脏灌流后分离肾小球,用于RNA和蛋白的提取;另一组小鼠肾脏用于免疫荧光染色。采用RT-qPCR、免疫荧光染色以及Western blot检测小鼠肾小球足细胞标志性蛋白nephrin和podocin以及肾小球IV型胶原(COL4α3、COL4α4和COL4α5)和层粘连蛋白β2(lamininβ2,LAMB2)的表达。结果:考马斯亮蓝染色法实验结果显示,miR-29a KO组小鼠出现尿白蛋白,WT组小鼠无蛋白尿。UACR结果显示3周时,miR-29a KO组与WT组相比,数据无统计学差异;12周时,miR-29a KO组的UACR比值与WT组相比显著增高(P<0.01);24周时,UACR比值差异更加显著(P<0.01)。PAS染色显示,3周时,miR-29a KO组小鼠肾小球出现轻微的细胞外基质沉积和系膜增生,随着周龄的增加,miR-29a KO组小鼠肾小球出现胶原纤维沉积,系膜扩张和外周血管壁增厚。透射电镜显示,miR-29a KO组小鼠肾小球超微结构在3周开始出现改变,包括足突排列紊乱,轻微融合等现象;12周时足突数量减少、足突融合加重,肾小球基底膜增厚的程度加重;24周时,上述变化进一步加重。与WT组小鼠相比,miR-29a KO组小鼠足细胞标志性蛋白nephrin的表达在mRNA水平显著下调(P<0.05);podocin表达在mRNA水平也呈下降趋势,但结果无统计学意义;COL4α3/α4/α5以及LAMB2表达在mRNA水平明显上调(P<0.05)。West‐ern blot结果显示,与WT组小鼠相比,miR-29a KO组小鼠nephrin和podocin蛋白表达显著降低(P<0.05);COL4α3/α4/α5以及LAMB2表达显著增加(P<0.01)。肾小球免疫荧光结果和Western blot结果一致。结论:miR-29a基因敲除会导致小鼠出现蛋白尿、肾小球损伤及纤维化,随着小鼠的老龄化损伤程度加重。

AIM:To observe the effect of miR-29a knockout on the function and structure in mouse glomerular.METHODS:Using CRISPR/Cas9 technology,C57BL/6 miR-29a knockout(miR-29a KO)mice were selected as the experimental group,and non-knockout littermates were selected as the wild-type(WT)control group.The 24 h urine of the mice were collected on the 3rd,12th and 24th week respectively.Coomassie brilliant blue staining was used for the determination of urine protein.The levels of urinary albumin and creatinine were measured by ELISA,and the urinary albumin/creatinine ratio(UACR)was calculated.The pathological changes of kidney tissue were observed by periodic acid-schiff(PAS)staining at week 3,week 12 and week 24.Transmission electron microscope and cryo-etching electron microscope were used to observe the glomerular ultrastructure.Another two groups of mice at 24 weeks were selected.Dynabeads™M-450 Tosylactivated micromagnetic beads were used to separate the glomeruli of mice in one group through cardiac perfusion,then RNA and protein were extracted.Another group of mouse kidneys were used for immunofluorescence staining.RT-qPCR,immunofluorescence staining and Western blot were used to detect the marker proteins,such as nephrin and podocin,as well as glomerular type IV collagen(COL4α3,COL4α4 and COL4α5)and lamininβ2(LAMB2).RESULTS:Urine albumin was observed in miR-29a KO mice.The UACR results showed that there was no statistical difference between the miR-29a KO group and the WT group at week 3.Howerver,the UACR of the miR-29a KO group was significantly higher than that of the WT group at week 12(P<0.01),and lasted for another 12 weeks(P<0.01).PAS staining showed that slight extracellular matrix deposition and mesangial hyperplasia were observed in the glomeruli of the miR-29a KO mice on the 3rd week.Collagen fiber deposition,mesangial expansion and peripheral blood vessel wall thickening were observed in the glomeruli in the miR-29a KO group with increasing age.Transmission electron microscopy showed that the glomerular ultrastructure of the miR-29a KO group mice began to change on the 3rd week,including disordered foot process arrangement and slight fusion.At 12 weeks,the number of foot processes decreased,the foot process fusion increased,and glomerular basement membrane thickening increased.The above changes further increased at 24 w.Compared with the mice in WT group,the expression of podocyte marker protein nephrin in miR-29a KO group was significantly down-regulated at the mRNA level(P<0.05),the expression of podocin also decreased at the mRNA level,but the results were not statistically significant,and COL4α3/α4/α5 and LAMB2 expressions were significantly up-regulated at the mRNA level(P<0.05).Western blot results showed that compared with the WT group,the protein expressions of nephrin and podocin in the miR-29a KO group were significantly decreased(P<0.05),the protein expressions of COL4α3/α4/α5 and LAMB2 were significantly increased(P<0.01).The glomerular immunofluorescence results were consistent with the Western blot results.CONCLUSION:miR-29a gene knockout induces proteinuria,glomerular damage and fibrosis in mice.