The Role And Mechanism Of MFACR/miR-652-3p/MTFP1 Pathway In Ischemia/reperfusion-induced Acute Kidney Injury

Objective: Mitochondrial injury is involved in the pathogenesis of various kidney diseases,and is a key issue in the progression of acute tubular injury.Recently,numerous studies have found that circular RNAs(circRNAs)are involved in the pathogenesis of a variety of diseases.It has been reported in a literature that mitochondrial fission and apoptosis-related circRNA MFACR acts as sponge of miR-652-3p to inhibit its activity,thus up-regulating MTFP1 and aggravating myocardial cell damage in the mouse model of myocardial infarction,which suggests that MFACR is involved in mitochondrial function regulation in myocardial infarction models,but MFACR has not been reported in acute kidney injury(AKI).The present study aimed at exploring whether MFACR is involved in ischemia/reperfusion(I/R)-induced AKI and whether MFACR/ mir-652-3p /MTFP1 pathway is a possible mechanism of renal injury in AKI.Methods: Male Balb/c mice were subjected to bilateral ischemia for 35 minutes followed by removing the atraumatic microvascular clips to achieve reperfusion.Body weight was measured 48 hours later,and plasma was collected under anesthesia for the detection of blood urea nitrogen.After being perfused with PBS for removing the remaining blood cells in the renal tissue,kidneys were collected and weighed.Paraffin-embedded blocks were prepared from one 1/2 kidney.And paraffin sections were stained with Periodic Acid-Schiff stain(PAS)to evaluate renal histopathological damage,with TUNEL staining to analyze the degree of apoptosis in renal tissue,and with immunofluorescence staining to detect localization of mitochondrial protein MTFP1.Another 1/2 kidney was made into OCT-embedded tissue blocks,and electron microscope specimens were set aside for future use.The rest of the renal tissues were stored at-80? for extraction of RNA and proteins for molecular biological detection.Immunoblotting was used to detect changes in protein levels,including apoptotic indicators Bax and bcl-2.Mitochondrial related indexes included PGC-1?,TFAM and MTFP1.RT-qPCR was used to detect changes in RNA/mRNA expression including MFACR,miR-652-3p,Mtfp1,Bax,Bcl-2,Pgc-1? and Tfam.Then we used bioinformatics techniques,CircInteractome and miRbase,to predict whether there were binding sites between MFACR and miR-652-3p as well as miR-652-3p and MTFP1.In addition,we used fluorescence in situ hybridization(FISH)to analyze whether there were co-localized expressions of MFACR,miR-652-3p and MTFP1 in kidneys of I/R-induced AKI mice.Finally,this study retrospectively screened paraffin sections of patients with clinical renal biopsy pathological diagnosis of AKI.FISH was used to conduct triple co-staining of MFACR,miR-652-3p and MTFP1 in renal tissues of AKI patients,to analyze the expression changes of MFACR/ miR-652-3p /MTFP1.Results: 48 h post I/R,kidney weight was significantly increased(1.24 ± 0.13 vs.1.00 ± 0.05,P<0.05)and blood urea nitrogen level was increased [(16.65 ± 6.02)mmol/L vs.(5.25 ± 0.76)mmol/L,P<0.05] in I/R-induced AKI mice.PAS staining showed characteristics of acute tubular injury,including loss of brush border,tubular lumen dilation,tubular cast formation,tubular cell vacuolization,tubular epithelial cells detachment into the tubular lumen or necrosis.And tubular injury score evaluated by semi-quantification increased in I/R-induced AKI mice(3.58 ± 0.31 vs.0.33 ± 0.20,P<0.05).Bax/Bcl-2 ratio increased at mRNA and protein levels,and apoptotic cells were significantly increased in TUNEL staining.Expression of mitochondrial biogenesis related genes PGC-1? and TFAM decreased at mRNA and protein levels.The match seed between MFACR and miR-652-3p as well as between miR-652-3p and MTFP1 were predicted.Results of RT-qPCR showed that MFACR was down-regulated,miR-652-3p was up-regulated and MTFP1 was down-regulated in I/R-induced AKI mice.Immunoblot showed that MTFP1 decreased at protein levels.In addition,results of FISH showed that MFACR,miR-652-3p and MTFP1 were co-localized in renal tubules of AKI mice and patients.And MFACR was down-regulated,miR-652-3p was up-regulated,and MTFP1 was down-regulated.Conclusion: 1)Renal tubular necrosis and apoptosis were significantly increased in the kidney tissues of I/R-induced AKI mice,and mitochondrial biogenesis related genes were decreased.2)Mitochondrial fission and apoptosis-related circRNA(MFACR)was down-regulated,miR-652-3p was up-regulated,and MTFP1 was down-regulated in I/R-induced AKI mice.And MFACR,miR-652-3p and MTFP1 were co-localized in renal tubules of AKI mice.3)The expression changes of MFACR/mi R-652-3p/MTFP1 pathway in AKI patients were consistent with that in I/R-induced AKI mice.4)The results of this study suggested that MFACR/miR-652-3p/MTFP1 pathway may be involved in the pathogenesis of AKI and play an important role.