| Part1 Expression of microRNAs in the urinary supernatant of adult patients with primary focal segmental glomerulosclerosisAbstract Objective:We aimed to examine the microRNA expression profile of urine supernatant in patients with FSGS and to analyse the correlation between the level of miRNAs and disease severity or the treatment effect.Methods:Random urine specimen was collected from 98 FSGS-A patients(urine protein≥3.5g/24h),94 FSGS-NA patients(urine protein≤0.4g/24h)and 94 normal controls.Urine samples before treatment and after treatment of 41 patients who accepted 60mg glucocorticosteroid treatment,and urine samples of 55 MN and 50 DN patients were also collected.Samples were centrifuged at 1500rpm for 10 minutes at 4℃,and supernatant was used for qRT-PCR.We quantified the mature miRNAs by real-time PCR using probe acquired from appliedbiosystems.Nonparametric Mann-Whitney U-tests were used to compare differences in urine miRNAs concentrations between the groups.Results:The microarray showed the urinary expression of 22 miRNAs in FSGS-A group was higher than other two groups.Quantitative real-time PCR showed urinary expression of miR-196a and miR-30a were significantly elevated in FSGS-A group(P<0.001),and were not elevated in FSGS-NA group compared with normal group.Urinary expression of miR-155 and miR-490 were significantly elevated in FSGS-A group compared to the FSGS-NA group and normal control(P<0.001),and were significantly in FSGS-NA group elevated compared to the normal control(P<0.05).The fold change of miRNAs was correlated with proteinuria,serum albumin level and total cholesterol level of FSGS patients.The Urinary expression of these four miRNAs after treatment significantly came down compared to they were before treatment in those 26 patients(P<0.05),but had no difference in the other fifteen patients(P>0.05).We also compared urinary free miRNAs levels in FSGS patients with membranous nephropathy(MN)and diabetic nephropathy(DN).The expression level of miR-196a,miR-30a and miR-490 were both up-regulated in avtive and non-active patients(P<0.05),while the changing trend of miR-155 was the same as FSGS patients.Conclusion:Urinary expression of miR-155,miR-196a,miR-30a and miR-490 were up-regulated in active patients with FSGS,and the expression level was correlated with disease severity,and the changing trend of miRNAs were different from MN and DN patients.The results suggested that miR-196a,miR-30a and miR-490 might be the new urinary biomarker for FSGS.Part2 Expression of microRNAs in the kidney tissue of adult patients with primary focal segmental glomerulosclerosisAbstract Objective:We aimed to examine the microRNA expression profile of kidney tissue in patients with FSGS and to analyse the correlation between the level of miRNAs and disease severity.Methods:kidney tissue was collected from 18 FSGS patients and 6 patients with Renal cell carcinoma.We tested the 754 miRNAs by Taqman microRNAarray and quantified the mature miRNAs by real-time RT-PCR using probe acquired from appliedbiosystems.Nonparametric Mann-Whitney U-tests were used to compare differences in urine miRNAs concentrations between the groups.Results:microRNAarray showed that the expression level of 170 miRNAs were elevated and 9 miRNAs were reduced.Quantitative RT-PCR showed that miR-155,miR-194,miR-30a,miR-146a,miR-196a,miR-21,miR-27b and miR-29c expression level in kidney tissue of FSGS patients were significantly elevated compared with renal tissues adjacent to cancer,the fold change were over twofold.The fold change of miR-194,miR-155,miR-146a,miR-196a and miR-21 were positive correlated with proteinuria.miR-155,miR-30a and miR-196a level were inversely correlated with the serum albumin level.Conclusion:The intrarenal expression level of miR-155,miR-194,miR-30a,miR-146a,miR-196a,miR-21,miR-27b and miR-29c were up-regulated in patients with FSGS.The expression level of miR-155,miR-196a,miR-30a was correlated with disease severity,and may be important in the pathogenesis and progression of FSGS. |
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