MiR-150 Inhibitor Ameliorates Adriamycin-induced Focal Segmental Glomerulosclerosis

Objective:Focal segmental glomerulosclerosis(FSGS)is the most common cause of adult nephrotic syndrome.Its mechanism remains unclear and the effective treatment lack and podocyte damage plays an important role in the pathogenesis of FSGS.Adriamycin can induce FSGS in mice and can be used as an ideal animal model to study the disease.microRNAs are small non-coding RNAs involved in the development of kidney and pathogenesis of kidney disease.We previously reported that upregulation of microRNA(miR)-150 in human podocytes increases profibrotic proteins and decreases anti-fibrotic suppressor of cytokine signaling 1(SOCS1).We aimed to clarify whether miR-150 inhibitor can ameliorate glomerular injury and to identify its corresponding mechanisms in adriamycin-induced FSGS mice.We observed the changes of ACR in urine at different time points,serum albumin,blood lipid,renal function and pathology at6 weeks,and determined that the treatment of LNA-anti-microRNA-150 improved the clinical and pathological changes of FSGS.To investigate the effect of LNA-anti-microRNA-150 on the expression of anti-fibrosis factor and the decrease of anti-fibrosis factor by inhibiting the expression of microRNA-150 in the kidney of FSGS.The increased expression of microRNA-150 in FSGS was verified in renal biopsy specimens of small samples of FSGS patients.To explore whether microRNA-150 can be a new target for clinical treatment of FSGS,and to provide theoretical basis for the treatment of FSGS by LNA-anti-microRNA-150.Methods:The male Balb/c mice with age 12 weeks were introvaneously injected with Adriamycin.After ADR injection the 5 days,2 weeks,4 weeks and 6 weeks,urine samples were collected.The concentration of urinary albumin and creatinine was measured by ELISA companion kit.The urinary ACR was expressed as mg/g for FSGS mice.At 6weeks,peripheral blood was collected.The serum albumin,total cholesterol,low density lipoprotein cholesterol,renal function including Scr and BUN were measured.miR-150 expression in ADR induced FSGS were measured using RT-qPCR.PAS and Masson-trichrome used for evaluation of histology changes under light microscopy.Renal biopsies were from new onset FSGS patients(n=8)enrolled between January 2016 and October 2017 in The Department of Nephrology.All kidney biopsies were obtained before the treatment with corticosteroid and immunosuppressants.Two individual nephrology pathologists provided the pathological diagnosis of FSGS.The normal control(NC)kidney tissues were from 7 patients with kidney tumor from The Department of Urology.NC kidney tissues were at least 5 cm from the edge of renal tumor.The age,gender,pathology type,estimated glomerular filtration rate based on CKD-EPI formula,urinary total proteinuria per day,serum albumin,serum total cholesterol and sclerotic glomeruli/total glomeruli were collected in FSGS patients.The age,gender,estimated glomerular filtration rate based on CKD-EPI formula,proteinuria,gravity and serum albumin were collected in renal tumor patients as normal control.miR-150 expression in renal biopsies of FSGS patients were measured using RT-qPCR.FISH(fluorescence in situ hybridization)was used for studying whether miR-150expression in glomeruli or not.2.The male Balb/c mice with age 12 weeks were subcutaneously injected with FAM-labeled LNA-anti-miR-150 at two doses of 2mg/kg or 4mg/kg.The kidneys were harvested 6 h after the LNA or vehicle injection.The kidneys were embedded into OCT(Sakura Finetek,CA,USA)for confirming the absorption of the LNA by immunofluorescence microscopy.The dose of 2mg/kg was decided to use in all treatment with LNA-anti-miR-150 and the scrambled LNA.The male Balb/c mice with age 12weeks were injected intravenously with adriamycin(10.5 mg/kg)to induce FSGS.FSGS mice were treatment of the scrambled LNA or LNA-anti-miR-150 for 6 weeks.The urinary ACR,serum albumin,total cholesterol,low density lipoprotein cholesterol,renal function including Scr and BUN,liver function including alanine aminotransferase(ALT)and weight were measured.miR-150 expression in each group were measured using RT-qPCR Il6,Inflammatory responses gene Il6,Ifnγ,Tnfαexpression were studied.Profibrotic proteins TGFβ1,αSMA,FN and antifibrotic SOCS1 expression and the infiltration of T lymphocytes were detected by western blotting,RT-qPCR and immunofluorescence method.PAS and Masson-trichrome specimen of electron microscope used for evaluation of histology changes.Results:1.The proteinuria was progressively elevated;hypoalbuminemia,hyperlidemia,podocyte detachment,and glomerulosclerosis were seen in ADR-induced FSGS mice at six weeks after the ADR injection.We found that the renal miR-150 levels were increased at 6weeks after the ADR injection.We found that miR-150 increased in renal biopsies from Chinese FSGS patients without the treatment.Primary FSGS was diagnosed based on typical detachment and vacuolization of podocytes,presence of segmental glomerulosclerosis on PAS and Masson staining,and diffuse foot process effacement on electronic microscopy.As matching diagnostic criteria of nephrotic syndrome,massive proteinuria,hypoalbuminemia,and hyperlidemia presented.For NC kidney tissues,the tumor patients showed normal urinalysis,serum albumin level,and eGFR.miR-150expression in glomeruli on FISH(fluorescence in situ hybridization)in renal biopsies from FSGS patients.WT-1 and SOCS1 decreased in glomeruli of FSGS patients.2.The renal absorption of FAM-labeled LNA-anti-miR-150 showed a dose dependent manner at 6h after the injection.We chose 2mg/kg of LNA-anti-miR-150(twice weekly)as the treatment dose and frequency for the studied mouse models to conform the inhibiting effect of renal miR-150.We found that the renal miR-150 levels were reduced by 6 weeks treatment of LNA-anti-miR-150 compared to the scrambled LNA in ADR-induced FSGS mice.No damages showed in liver and loss of animal body weight after the 6 weeks the treatment in FSGS mice with LNA-anti-miR-150 compared to the scrambled LNA.We found that proteinuria,hypoalbuminemia,hypercholesteremia,blood urea nitrogenemia(BUN),and glomerulosclerosis on PAS and Masson staining were all ameliorated by 6 weeks of LNA-anti-miR-150 treatment.WT-1 decreased in FSGS kidney and LNA-anti-miR-150 reduced the loss of WT-1 on western blotting and IF staining.mRNA levels of profibrotic genes including transforming growth factor-β1(Tgfβ1),α-smooth muscle antibody(αSma),and fibronectin(Fn)and the consistent protein were increased in FSGS mice compared to normal mice.And the increment of these three genes was ameliorated by LNA-anti-miR-150 compared to the scrambled LNA.The mRNA level of Socs1 and the renal SOCS1 protein level was remarkably upregulated in the kidneys of FSGS mice by LNA-anti-miR-150 compared to the scrambled LNA.mRNA levels of Ifnγ,Il6,and Tnfαwere increased in the kidneys of ADR-induced FSGS mice compared to the normal mice.The increments of three cytokines were reduced by LNA-anti-miR-150 compared to the scrambled LNA.LNA-anti-miR-150 also decreased the infiltration of CD3~+T cells compared to the scrambled LNA on immunostaining.Conclution:1.ADR induced the mouse model of FSGS,and in which miR-150 expression increased in kidney.2.miR-150 expression in renal biopsies from FSGS patients,especially high expression in glomeruli.3.SOCS1 expression in glomeruli from FSGS patients decreased.4.LNA-anti-miR-150 may be mediated by a combination of decrease in the infiltrated T lymphocytes,pro-inflammatory cytokines and profibrotic proteins,and increase in antifibrotic SOCS1 in the kidney.