The Role And Mechanism Of CircSAMD4 In Iodinated Contrast-induced Acute Kidney Injury

Background: Iodinated contrast-induced acute kidney injury(CI-AKI)is one of the leading causes of acute kidney injury in hospitalized patients,increasing the risk of death and accelerating the progression of chronic kidney disease.The pathogenesis of CI-AKI has not been fully clarified.Previous studies have reported that iodinated contrast media cause renal injury by altering renal hemodynamics and/or directly acting on renal tubular epithelial cells leading to cell injury.Further investigating the molecule mechanism of CI-AKI will aid in developing new prevention and treatment protocols.Circular RNAs(circ RNAs)are a class of endogenously expressed non-coding RNAs with single-stranded closed loop structures.Emerging data have shown that the abnormal expression of circ RNAs participated in the development of AKI,but the reports about the role of circ RNAs in CI-AKI are rare.Objective: To explore the differential circ RNAs of CI-AKI and verify their functions,and further explore their upstream regulatory mechanisms.Methods:1.Screening and validation of differentially expressed circ RNAs: To establish CI-AKI mouse model,unilateral nephrectomy was conducted in male C57BL/6J mice.Three weeks after the surgery,the mice were deprived of water for 24?h and then injected with furosemide via tail vein.Twenty minutes after furosemide injection,the mice were administrated with iohexol via tail vein.We divided the animals into normal control group,model control group,and CI-AKI group.Twenty-four hours after iohexol injection,the serum of mice was collected to detect blood urea nitrogen and creatinine levels.Hematoxylin and eosin staining was performed to evaluate renal pathological changes.To identify the differential expression profiles of circ RNAs in CI-AKI,the kidney tissue samples of the model control group and the CI-AKI group were subjected to circ RNA microarray detection.The differential circ RNAs highly conserved between human and mouse were screened by BLAST,circ Base and circ Bank websites.The expression and localization of circ Samd4 were detected by q RT-PCR and RNA fluorescence in situ hybridization(FISH),respectively.Human renal tubular epithelial cells(HK-2 cells)were treated with 200 mg I/m L iohexol for 6 hours to establish an iodinated contrast medium-induced renal tubular epithelial cell injury model.The expression changes of circ SAMD4 were detected by q RT-PCR;after verifying the ring structure of circ SAMD4,FISH was used to detect the subcellular localization of circ SAMD4.2.Functional analysis: HK-2 cells were treated with circ SAMD4 small interfering RNA(si RNA)and circ SAMD4 overexpression plasmid,respectively,and after intervention with iohexol,apoptosis-related indicators were analyzed by western blot and flow cytometry.3.Upstream mechanism investigation: The CATRAPID website was used to predict the interaction between RNA-binding protein(RBP)serine/arginine-rich splicing factor 3(SRSF3)and circ SAMD4.The expression changes of SRSF3 were detected by western blot in vivo and in vitro.After HK-2 cells were treated with SRSF3 si RNA,the expression changes of circ SAMD4 were detected by q RT-PCR.We performed the RNA-binding protein immunoprecipitation assays to validate the interaction between SRSF3 and circ SAMD4 in 293 T cells.HK-2 cells were treated with SRSF3 si RNA,and after intervention with iohexol,apoptosisrelated indicators were analyzed by western blot and flow cytometry.Results:1.Using circ RNA microarray analysis,we identified 236 circ RNAs transcripts with significant dysregulation in the iohexol group relative to model group,of which 120 were up-regulated and 116 were downregulated.2.We further characterized the conserved circ Samd4 produced by the Samd4 gene was markedly up-regulated in CI-AKI mouse model and predominantly located in the renal tubular epithelial cells.Its conserved human homolog circ SAMD4 was also up-expressed in HK-2 cells exposed to iohexol and mainly located in cytoplasm.3.Functional studies in vitro revealed that silencing circ SAMD4 alleviated apoptosis in HK-2 cells treated by iohexol.Consistently,overexpressing circ SAMD4 aggravated apoptosis in HK-2 cells exposed to iohexol,supporting the notion that circ SAMD4 acted as an apoptotic inducer in iohexol-treated HK-2 cells.4.Mechanistically,the RBP SRSF3 could bind to circ SAMD4.We demonstrated suppression of SRSF3 protein contributed critically to the upregulation of circ SAMD4 expression,thereby exacerbating the apoptosis of HK-2 cells exposed to iohexol.Conclusion: In renal tubular epithelial cells of CI-AKI,iodinated contrast media can induce the expression of circ SAMD4 by downregulating SRSF3.Upregulation of circ SAMD4 exacerbates apoptosis in renal tubular epithelial cells of CI-AKI.