Effect Of Steroids On FSGS And The Role Of Autophagy And MCP-1 In COL4A3 Related Nephropathy

Objective: To investigate the effect of steroids on FSGS patients with moderate proteinuria;to explore the role of autophagy in the pathogenesis of COL4A3-related nephropathy;to evaluate the clinical significance of MCP-1 as a urine molecule marker in assessing the clinical outcome of COL4A3-related nephropathy and FSGS.Methods: Primary FSGS patients with urine protein between 1.0-3.5g/d were recruited from 2006 to 2016.No decline in urine protein>50% was observed after 2 months of run-in ACEI/ARB treatment.Patients were assigned to study group(glucocorticoids with ACEI/ARB)or control group(ACEI/ARB without glucocorticoids).Variables including 24-hour urinary protein,albumin and creatinine during the trial were recorded.Remission was defined as proteinuria<0.3g/24 h or declined>50%,and our composite end point as >30% decrease of eGFR or eGFR<30ml/min.In the second part,Col4a3 p.Arg1631 Stop mouse model was constructed by CRISPR/Cas9.The phenotypes of mice were observed by urine routine and biochemical tests and pathological examination.The expression of autophagy related molecules were analyzed by Westernblot.In the third part,we investigated the expression of MCP-1 by RT-qPCR and Westernblot in both cell and mouse models.The levels of MCP-1 in the supernatants of COL4A3 mutant HP cell lines,as well as urine of mouse models and FSGS patients and healthy controls were detected by ELISA.The correlation between urinary MCP-1 level and proteinuria,serum creatinine was analyzed.Results: In the first part,a total of 102 patients were enrolled(Study group N=52,Control group N=50),and the median follow-up time was 36(12-117)months without significant difference between groups.During the 12-month follow-up,the remission rate was significantly higher in study group [73.1% vs 50.0%(P=0.01)],and the initial median response time was 3 months in the study group while 6 in the control group.The end point was reached by 22.2% cases in study group,and 42.0% in control.The medium survival times were study group 72 months and control 57(P=0.03).Minor adverse reactions were observed in 10 patients(Study group N=8,Control group N=2).In the second part,Col4a3 p.Arg1631 Stop mouse model homozygous group showed significant hematuria and proteinuria with elevated serum creatinine from 6 weeks,and renal pathology presented with FSGS changes.The expressions of autophagy-related proteins LC3-I/LC3-II,Atg16L1,Beclin-1 and CHOP in renal cortex of homozygous mice were significantly up-regulated.Significantly increased mumber of autophagosomes was observed by electron microscopy in homozygous mouse.In the third part,we found that the expression of MCP-1 was up-regulated in renal cortex of Col4a3 p.Arg1631 Stop mice.MCP-1 levels were elevated in urine of homozygous mouse and FSGS patients as well as supernatants of HP cell with severe COL4A3 mutant.Serum creatinine levels were positively correlated with urinary MCP-1 levels in FSGS patients(P<0.01,Pearson correlation coefficient 0.426).Conclusions: Additional glucocorticoids therapy is more efficacious compared to ACEI/ARB drugs alone in the treatment of patients with pFSGS and moderate proteinuria,with good safety and absence of major side effects.Severe renal pathological changes(FSGS)presented in homozygous Col4a3 p.Arg1631 Stop mice,and over activation of autophagy may be an important mechanism.MCP-1 is expected to be a new molecular indicator of COL4A3-associated nephropathy and FSGS.