关键词: 肾病综合征, 透明形成素相关蛋白1, 阿霉素, Diaph1基因敲除小鼠

Abstract: Objective To explore the key role of diaphanous-related formin 1(Diaph1)gene in the kidneys of nephropathy model mice. Methods Wild-type(WT),heterozygous(Diaph1^+/-),and homozygous(Diaph1^-/-)mice were used to establish the control group and the adriamycin model group,respectively,with 7 mice in each group.Mice in the adriamycin model group received adriamycin via tail vein injection,while those in the control group were administered an equal volume of normal saline.The Diaph1 gene was globally knocked out in mice using the CRISPR/Cas9 system,and homozygous mice were obtained by breeding heterozygous littermates.Genotyping was performed by polymerase chain reaction(PCR).Hematoxylin and eosin(HE)staining was used to observe renal morphology in each group.Immunofluorescence staining was applied to detect the expression of Diaph1 in glomeruli and surrounding renal tubules.Serum and urinary albumin levels were measured using a microplate reader method,and creatinine levels were determined by the sarcosine oxidase method.Western blotting was performed to assess Diaph1 protein expression in the renal cortex.Quantitative real-time polymerase chain reaction(qRT-PCR)was conducted to detect the mRNA expression levels of Podocin,CD2-associated protein(Cd2ap),Nephrin,and kin of IRRE-like protein 1(Neph1)in mouse kidneys. Results In wild-type mice,compared with the control group,the adriamycin model group showed significantly decreased body weight(P<0.05)and narrowed glomerular capsule spaces.Biochemical assays revealed elevated urinary albumin(P<0.05)and serum creatinine(P<0.05),along with reduced urinary creatinine(P<0.05).At the molecular level,both mRNA and protein expression of Diaph1 were significantly upregulated in the adriamycin model group(P<0.05).In the Diaph1-knockout mice,compared with adriamycin-treated wild-type mice,adriamycin-treated heterozygous(Diaph1^+/-)and homozygous(Diaph1^-/-)mice exhibited aggravated glomerulosclerosis and further narrowed glomerular capsule spaces.Moreover,renal mRNA expression of Podocin,Cd2ap,and Nephrin was significantly decreased in adriamycin-treated Diaph1^-/- mice(P<0.05),and biochemical analysis showed significantly reduced serum albumin and serum creatinine levels in these mice(P<0.05). Conclusion The Diaph1 gene exerts a critical protective role in renal podocytes and tubular epithelial cells,suggesting that Diaph1 is a potential functional gene in the adriamycin-induced nephropathy model.This provides new insights and directions for the treatment and future research of nephrotic syndrome.

Key words: nephrotic syndrome, diaphanous-related formin 1, adriamycin, Diaph1 gene knockout mice