Familial FSGS Screening Pathogenic Gene STUB1,ADCK1,TCF7L1 Expression In Adriamycin-mouse Kidney Tissue And Its Significance

Objectives: Focal segmental glomerulosclerosis(FSGS)is associated with gene mutation.Previous studies concluded 19 genes that may be associated with familial focal segmental glomerulosclerosis.The purpose of this experiment is to investigate the expression and significance of STUB1,TCF7L1 and ADCK1 in doxorubicin-induced FSGS mouse model.Methods:1.21 healthy SPF grade 8-week-old male Balb / c mice were randomly divided into control group(9 mice)and model group(12 mice)after adaptive feeding for 7 days,and 3 mice in each group were randomly selected to receive 24 hours urine.After that,mice in model group were given a single tail vein injection of 10.5 mg / kg Adriamycin(ADR)to make FSGS model,and mice in control group were given a single tail vein injection of equivalent volume normal saline.2.At the end of the 7th day and the 21 st day after modeling,3 mice randomly selected from each group and the remaining mice at the end of the 35 th day,were taken for 24-hour urine and blood samples.The blood samples were collected by eyelid sampling and the mice were sacrificed.The formaldehyde was fixed in one side of the kidney,and the other side of the kidney was frozen in liquid nitrogen.3.24-hour urinary protein was measured at each time point;serum creatinine and blood urea nitrogen levels were measured in blood samples;renal tissue was stained with Peri-acid Schiff(PAS)and pathological changes were observed under light microscope.4.RNA was extracted from the renal tissue of model and control mice on day 35,and the expression of STUB1,TCF7L1 and ADCK1 was detected by RT-PCR after reverse transcription.5.The expression of STUB1,TCF7L1 and ADCK1 in renal tissue was detected by immunohistochemical staining and Western blotting.6.SPSS 23.0 software was used for statistical analysis of the results.P <0.05 was statistically significant.Results:1.Construction of FSGS mice model: The model group showed that 24-hour urinary protein began to rise after the 7th day.On the 21 st day,it continued to increase,and it was significantly higher than the control group(P<0.01).It peaked on the 35 th day and was still significantly higher than the control group(P<0.05).Creatinine and urea nitrogen showed no significant difference between the model group and the control group on the 7th,21 st,and 35 th day.PAS staining was observed under the light microscope:On the 7th day,there was no significant change in the glomerulus.On the 21 st day,glomerular mesangial cells and mesangial matrix proliferated,and protein cast was testified as well.Mesangial cells and mesangial matrix appeared on the 35 th day.Hyperplasia,segmental aggravation,segmental sclerosis of the glomeruli,and a large number of protein casts were observed in the renal tubules.There was no change in the glomeruli and renal tubules in the control group,suggesting that the mouse FSGS nephropathy model was successfully constructed.2.mRNA levels of STUB1,TCF7L1,ADCK1: Compared with the control group,the expression of mRNA of STUB1 in model group was significantly decreased 35 days after injection of doxorubicin(P <0.05);the mRNA expression of TCF7L1 was not significantly different from that in control group,which is the same as ADCK1.3.Protein levels of STUB1,TCF7L1,ADCK1(1)Immunohistochemical staining: The expression of STUB1 in the model group was mainly in glomerular epithelial cells and renal tubules,but almost no expression in the mesangial area.The control group had a small amount of scattered expression in glomerular epithelial cells and mesangial area.,but strong expression in renal tubular epithelial cells.Compared with the control group,the expression of STUB1 in the model group was significantly reduced(P<0.001);No positive expression of TCF7L1 was observed in eihter model group or control group;ADCK1 was strongly expressed in glomerular epithelial cells,mesangial area,and basement membrane and weakly expressed in the renal tubules in model group.And the expression was in the same position with the control group.There was no significant difference in the expression level between the model group and the control group.(2)Western blotting: The expression of STUB1 protein in the kidney of the model group mice was significantly reduced 35 days after the injection of adriamycin(P<0.001).Compared with the control group,there was no significant difference in the expression of TCF7L1 and ADCK1 protein in model group.Conclusions:1.STUB1 participated in the pathogenesis of adriamycin-induced FSGS in mice.And its down-regulated expression in glomerular epithelial cells and renal tubules suggested that it may be involved in the pathological changes of podocyte and the development of edema.But the pathogenicity of its mutation needs to be further validated in a large number of people with FSGS and their families.2.There was no direct correlation between either TCF7L1 and FSGS or ADCK1 and FSGS.Whether or not it controls the pathogenesis of other genes remains to be further studied.