Mechanism Of CircZNF609 In Focal Segmental Glomerulosclerosis

Objective:Focal segmental glomerulosclerosis(FSGS)is one of the main causes of end-stage renal disease(ESRD).It’s very important to study the pathogenesis of FSGS,find new treatment methods or specific targeted drugs.Recently,circZNF609 has been reported to participate in the occurrence and development of some human diseases via sponging its downstream micro RNAs(mi R)with its binding sites.However,pathophysiological function of circZNF609 in FSGS remains unknown.This study aims to explore whether circZNF609 in circular RNA plays a"sponge"role in FSGS and its molecular mechanism and to provide a theoretical basis for determining whether circZNF609 can be used as a potential new target for the treatment of FSGS.Methods:1.A total of 90 patients with primary FSGS approved by biopsy were enrolled in this study.The pathological classification included non-otherwise special(NOS,n=30),Tip(n=30)and cellular(n=30).The following clinical information of patients was collected:the percentage of sclerotic glomeruli in renal biopsy pathology in total glomeruli,estimated glomerular filtration rate(e GFR)based on CKD-EPI formula,24-hour urinary total protein(UTP),serum albumin(ALB),total cholesterol(TC),serum creatinine(Scr),and blood urea nitrogen(BUN).Thirty control subjects matching the gender and age of the FSGS group were collected.Normal control kidney tissue was taken from patients with urinary system tumors after total nephrectomy.Kidney tissue more than 5 cm from the tumor was selected and confirmed as normal kidney tissue by a nephrology pathologist as a normal control group.The kidney tissue was stained with Periodic Acid-Schiff staining(PAS)and Masson trichromatic staining(Masson)to observe kidney pathological injuries,and immunofluorescence(IF)was used to evaluate the expression of podocyte injury-specific protein Wilms tumor protein 1(WT1)and pro-fibrotic protein-typeⅠcollagenα1(COL1A1).Fluorescence-labeled in situ hybridization(FISH)was used to determine the localization and expression changes of circZNF609 in kidney tissue,and to analyze the correlation between expression intensity of circZNF609 in kidney tissue and traditional clinical biochemical indicators.2.Forty-eight male 11-week-old BALB/c mice were randomly classified into two groups:normal control with saline injection(NC group,n=24)and ADR-induced FSGS group(n=24).The FSGS mouse model was made by intravenous injection of 10.5 mg/kg adriamycin(ADR)via the penis,and the normal control group was injected with the same volume of normal saline.Five days,2 weeks,4 weeks,and 6 weeks after ADR intravenous injection,the urine of mice was collected to detect the mice urine albumin and urine creatinine,and the urine albumin/urinary creatinine(UACR)ratio was calculated.Mice peripheral venous blood was collected to detect Scr,BUN,ALB,TG,TC and serum low-density lipoprotein(LDL)of mice.Blood and urine at the above four time points were collected,respectively,carbon dioxide anesthesia was given to 6 mice of NC group and 6of ADR-induced FSGS group.The blood cells were removed by renal perfusion with normal saline at 4℃until the kidney turned from red to white,and then the kidney tissue was collected for histological and molecular biological examination.One quarter of the kidney was fixed with 4%paraformaldehyde for 48 hours,and was used as pathologically embedded sections to make paraffin sections for PAS and Masson staining to clarify the pathomorphological changes of FSGS.PAS and Masson staining were performed to confirm whether FSGS occurred,immunohistochemistry(IHC)was used to detect podocyte protein(Podocin)and transforming growth factor-β1(TGF-β1),and FISH was used to detect expression changes in circZNF609 and mi R-615-5p which was confirmed as one mi RNA with perfect binding sites with circZNF609,in paraffin sections of kidney tissue.Proteins were extracted from 1/4 of the kidney tissue,and the protein expression levels of podocyte-specific proteins WT1,Podocin and pro-fibrotic proteins COL1A1 and TGF-β1 were examined by Western blotting.One quarter of kidney tissue was used for total RNA extraction.Renal RNA expression levels of circZNF609 and mi R-615-5p in the kidney were analyzed by real time quantitative polymerase chain reaction(RT-q PCR).One quarter of kidney tissue was used to make frozen sections for immunofluorescence staining to detect the location and protein expression levels of WT1 and COL1A1.The correlations between circZNF609 and mi R-615-5p and UACR,biochemical indicators including Scr,BUN,ALB,TG,TC,LDL,podocyte injury biomarkers and fibrosis-related indicators in kidney tissue.3.FSGS is a type of glomerular disease initialized by podocyte injury.Massive proteinuria can be toxic to renal tubular cells,leading to damage to renal tubular epithelial cells,which causes glomerulosclerosis and tubular interstitial fibrosis and eventually progresses to ESRD.Therefore,we selected human podocytes and renal tubular epithelial cell line HK2cell for cell culture and study.Podocyte culture and transfection with circZNF609 were not succeeded.Therefore,we focused on studying the circZNF609’s damage mechanisms of proteinuria on renal tubular epithelial cells during the development of FSGS.We selected the classic renal tubular epithelial cell line HK2 cells for materials of in vitro study.FISH and IF were used to detect whether circZNF609,mi R-615-5p,and COL1A1 were co-expressed after HK2 cell slides.The specific method was as follows:the HK2 cell culture density was 1.0×10~5cells/ml.After the cells attached on the bottom of the dishes,the HK2 cells were cultured in DMEM/F12 medium without 10%fetal bovine serum for12 hours,and then the HK2 cells were randomly divided into the following two groups:1)control group in which HK2 cells were cultured in DMEM/F12 medium;2)bovine serum albumin(BSA)group with BSA stimulation to mimic the toxic effect of proteinuria filtered after podocyte injury:HK2 cells were cultured in DMEM/F12 medium containing 4 mg/ml BSA.Twenty-four hours later,the cells were harvested to extract total RNA and protein to examine expression changes in circZNF609 and mi R-615-5p as well as fibrosis indicators COL1A1 and TGF-β1,and analyze the correlation between the levels of circZNF609 and mi R-615-5p,as well as the correlation between circZNF609,mi R-615-5p and fibrosis indicators COL1A1 and TGF-β1,respectively.The culture density of HK2cells was 1.0×10~5 cells/ml.When the cells were 60-70%attached,the circZNF609 plasmid and the negative control plasmid were transfected,and the cells’total RNA and protein were extracted to detect circZNF609 and mi R-615-5p and fibrosis-related indicators including changes in the expression of COL1A1 and TGF-β1.Results:1.FSGS patients clinically manifested as nephrotic syndrome including massive proteinuria,hypoalbuminemia,and hyperlipidemia,often accompanied by decreased e GFR.Renal biopsy PAS and Masson staining indicated that the pathological changes corresponded to those of FSGS.Compared with the normal control group,IF detected that the expression of the podocyte injury-specific protein WT1 decreased and that of the fibrosis-related protein COL1A1 increased in the renal tissue of FSGS patients.FISH showed that circZNF609 was localized in both the glomerulus and renal tubules,and the expression of circZNF609 increased in kidney tissue of FSGS patients.circZNF609 was positively correlated with TC and Scr,and negatively correlated with ALB and e GFR.2.In vivo study,5 days after ADR injection,BALB/c mice developed proteinuria which increased with the prolonged observation time.At 6 weeks,hypoalbuminemia,hyperlipidemia,and elevated BUN were seen in BALB/c mice while Scr had no significant changes.Pathological changes of PAS and Masson staining accorded with FSGS,which confirmed FSGS mouse model was successfully established.Western blotting showed that the expression of podocyte-specific proteins WT1 and Podocin was down-regulated in kidney tissue of mice in ADR-induced FSGS group,and that of fibrosis-related proteins COL1A1 and TGF-β1 was up-regulated.Immunostaining showed that the change in expression of WT1 and Podocin as well as of COL1A1 and TGF-β1 were consistent with the directions of expression changes on Western blotting.RT-q PCR detected that the expression of circZNF609 increased in the kidney of FSGS mice,while that of mi R-615-5p decreased.FISH detected that circZNF609 and mi R-615-5p were co-expressed in the glomeruli and renal tubules,and the expression of circZNF609 increased in kidney tissue of FSGS mice induced by ADR,and that of mi R-615-5p decreased,and the two were negatively correlated.circZNF609 was positively correlated with UACR,TC,TG,BUN,and negatively correlated with ALB,but had no obvious correlation with Scr.circZNF609was negatively correlated with podocyte injury-specific proteins WT1 and Podocin,and was positively correlated with fibrosis-related protein TGF-β1,but had no obvious correlation with COL1A1.mi R-615-5p was positively correlated with podocyte injury-specific protein WT1,and negatively correlated with fibrosis-related protein COL1A1,but had no obvious correlation with Podocin and TGF-β1.3.In vitro study,HK2 cell slides,circZNF609,mi R-615-5p and COL1A1 were co-expressed in the cytoplasm on FISH and IF.After treating HK2 cells with 4 mg/ml BSA for 24 hours,the expression of fibrosis-related proteins COL1A1 and TGF-β1 increased.More importantly,the expression of circZNF609 was up-regulated and mi R-615-5p expression was down-regulated.In addition,negative correlation was seen between circZNF609 and mi R-615-5p.The overexpression of circZNF609 with transfection of circZNF609 down-regulated the expression of mi R-615-5p,and up-regulated expression of fibrosis-related proteins COL1A1 and TGF-β1.In two methods of stimulation to HK2cells with either BSA or circZNF609 plasmid,circZNF609 in treated HK2 cells was negatively correlated with mi R-615-5p,circZNF609 was positively correlated with COL1A1 and TGF-β1,and mi R-615-5p was negatively correlated with COL1A1 and TGF-β1 according to correlation analysis.Conclusion:1.circZNF609 is up-regulated in the kidney tissue of FSGS patients compared to normal kidney tissue.In addition,renal circZNF609 is positively correlated with TC and Scr,and negatively correlated with serum ALB and e GFR.This suggests that circZNF609was involved in the development and progress of FSGS.2.ADR can induce FSGS in male BALB/c mice.The renal expression of circZNF609increases in ADR-induced FSGS mice with in control mice.This is consistent with the findings in kidney tissue of FSGS and control subjects and simulates the expression changes of clinical FSGS.In addition,renal expression of circZNF609 and mi R-615-5p is negatively correlated,which suggests that circZNF609 and mi R-615-5p are jointly involved in the pathogenesis and progression of FSGS.The renal expression of fibrosis-related proteins COL1A1 and TGF-β1 increases in ADR mice.These results further suggest that circZNF609 participates in the progression of renal fibrosis of FSGS by down-regulating mi R-615-5p.3.Although the roles of the circZNF609 and its transfection in podocytes are not successfully completed,the sponging role of the circZNF609 to mi R-615-5p and direct down-regulation of mi R-615-5p to COL1A1 are confirmed in HK2 cells treated with BSA stimulation and are consistent with those of animal experiments.They suggest that circZNF609,mi R-615-5p,and COL1A1 participate in renal tubular damage caused by massive proteinuria after FSGS podocyte injury,and promote fibrosis;and after HK2 cells transfect circZNF609,the expression of mi R-615-5p is down-regulated,and that of COL1A1 is up-regulated.These in vitro results further provide more evidence of circZNF609 acting as a"sponge"of the downstream target gene mi R-615-5p,which in turn regulate the downstream target protein of COL1A1,an important production of renal fibrosis.