The Role Of Gpr97 In The Pathogensis Of Acute Kidney Injury

ObjectiveAcute kidney injury(AKI)is a severe clinical syndrome characterized by sudden decline in renal function and continuous accumulation of nitrogen wastes.Although more and more studies have gradually uncovered the pathogenesis of AKI,there is still no effective treatment for AKI in clinical except dialysis.G protein-coupled receptor(GPCR)family is the largest membrane protein family.They are the targets for over 30%drugs in clinic.Therefore,to explore the GPCR which plays a key role in the pathogenesis of AKI may provide a new effective therapeutic strategy for AKI patients.Gpr97 is an orphan adhesive G protein coupled receptor.Studies have shown that the number of mature B220+B cells in Gpr97 gene knockout mice is significantly reduced,but it is also reported that Gpr97 does not affect the maturation of B cells and T cells.In obese mice induced by high fat diet,lacking of Gpr97 can decrease the infiltration of M1 macrophages and increase the infiltration of M2 macrophages,and then decrease the expression of pro-inflammatory cytokines and increase the expression of anti-inflammatory cytokines in adipose tissues.But in obese mice induced by high-fat diet,same effects did not be found in the liver and in the kidney.More and more studies have shown that the accumulation of inflammatory responses plays an important role in the pathogenesis of AKI.The expression of Gpr97 is significantly increased in renal ischemia-reperfusion injury and in cisplatin-induced acute kidney injury in mice,and it is important to further study of the role of Gpr97 in AKI.MethodsPart I The role of Gpr97 in the pathogensis of renal ischemia/reperfusion injury1 Expression pattern of Gpr97 in renal ischemia/reperfusion injury model1.1 Wild type(WT)male mice were sacrificed to detect the expression pattern of Gpr97 in heart,liver,spleen,lung,kidney,brain and bone marrow by agarose gel electrophoresis and western blot(WB).Renal parenchymal cells were cultured in vitro to detect the expression pattern of Gpr97 by agarose gel electrophoresis and WB.1.2 WT male mice were selected to establish animal model.Bilateral renal pedicles were clipped for 30 minutes with microaneurysm clamps.Gpr97 was detected by WB,Real-time RT-PCR and immunohistochemical(IHC)methods.1.3 Renal biopsies from patients with acute tubual necrosis(ATN)were detected by IHC to explore the expression pattern of Gpr97.1.4 Double immunofluorescence(IF)was used to detecte the distribution of Gpr97 in kidney.1.5 Immortalized rat proximal tubule epithelial cells(NRK-52E)were cultured to detect the expression pattern of Gpr97 under different hypoxia conditions.2 Functions of Gpr97 in renal ischemia/reperfusion injury modelGpr97-/-mice were used to detect the function of Gpr97 in renal ischemia/reperfusion injury model.Concentrations of serum creatinine(SCr)and blood urea nitrogen(BUN)were detected.Renal injury and cell death were measured by HE and TUNEL.Effects of Gpr97 deficiency in kidney inflammatory responses were measured by Real-time-RT-PCR.IF was carried out to detect the markers of neutrophil and macrophage.Part II Gpr97 exacerbates acute kidney injury via mediating Sema3A signaling1 Effects of Gpr97 on the expression of Sema3A1.1 The microarray experiments were performed by Shanghai Biotechnology Corporation to detect the expression level of Semaphorin family in the kidney of WT and Gpr97-/-mice.The expression level of Sema3A in the kidney and the content of Sema3A in the serum were detected.1.2 Effects of Gpr97 and Sema3A on the expression level of inflammatory factors in NRK-52E cells induced by hypoxia were detected by Real-time RT-PCR.Effects of Gpr97 and Sema3A on the apoptosis were detected by flow cytometry.Effects of Gpr97 and Sema3A on the expression level of activated Caspase-3 was detected by ELISA.2 Gpr97 regulates Sema3A through HuRWB was performed to detect the protein level of HuR in the kidney of WT and Gpr97-/-mice and to detect the effects of Gpr97 on the expression level of HuR under hypoxia conditions.The localization and transposition of HuR in NRK-52E cells were detected by IF assay.Effects of HuR on the expression of Sema3A were detected by WB.Effects of HuR on the half-life of Sema3A mRNA were detected by Real-time RT-PCR.WB was performed to detect the effects of Gpr97 and HuR on the expression of Sema3A.3 HuR regulates the stability of Sema3A mRNAThe ARE sequences of Sema3A mRNA 3 'UTR region of different species were compared and analyzed.The binding efficiency of different ARE sequences in Sema3A mRNA 3'UTR region and HuR was verified by RNA-EMSA tests.Part III The role of Gpr97 in the pathogensis of AKI induced by cisplatin 1 The expression level of Gpr97 in AKI induced by cisplatinWB and Real-time RT-PCR were used to detect the expression level of Gpr97 in mice induced by cisplatin.WB was used to detect the expression level of Gpr97 in NRK-52E cells induced by cisplatin.2 Effects of Gpr97 deficiency on cisplatin induced AKIWT and Gpr97-/-mice were used to detect the function of Gpr97 in AKI induced by cisplatin.High-performance liquid chromatography(HPLC)was used to measure the content of SCr and BUN.HE and TUNE were used to detect the renal injury and cell death.Effects of Gpr97 deficiency in kidney inflammatory responses were measured by Real-time RT-PCR.IF was carried out to detect the markers of neutrophil and macrophage.3 Effects of Gpr97 on the expression of Sema3AThe expression level of Sema3A in the kidney and the content of Sema3A in the serum were detected.ResultsPart I The role of Gpr97 in the pathogensis of renal ischemia/reperfusion injury1 Gpr97 was significantly induced in the kidney from mice with renal IRI1.1 The expression of Gpr97 has been detected in different mice tissues and in renal parenchyma cells.1.2 Real-time RT-PCR and WB results showed that the expression of Gpr97 were up-regulated in the kidney from mice with renal IRI,which was further confirmed by IHC staining.1.3 IHC results showed a significant increase in the expression level of Gpr97 in the patients with ATN.1.4 IF results showed that Gpr97 was mainly expressed in proximal tubules and distal tubules in the kidney.1.5 WB results showed that all the approaches of mimicing hypoxia conditions could increase the level of Gpr97 in NRK-52E cells.2 Gpr97 deficiency alleviates renal ischemia/reperfusion injuryCompared with WT mice,Gpr97-/-mice after I/R showed lower concentration of SCr and BUN,less severe morphological injury and decreased cell death.Gpr97 deficiency decreased the levels of pro-inflammatory mediators by Real-time RT-PCR.Consistently,neutrophil and macrophage accumulation was further decreased in the kidney from ischemic Gpr97-/-mice,indicating that Gpr97 exacerbates renal injury.Part ? Gpr97 exacerbates acute kidney injury via mediating Sema3A signaling1 Effects of Gpr97 on the expression of Sema3A in the kidney from mice with renal IRI 1.1 Real-time RT-PCR,WB and IHC results showed that the increase of Sema3A was alleviated in Gpr97-/-mice compared with the WT mice during ischemia-reperfusion injury.ELISA results showed that the content of Sema3A in the serum of Gpr97-/-mice was lower than that of WT mice and was positively correlated with the content of SCr and BUN.1.2 Gpr97 deficiency reduces the expression of inflammatory factors,cell death,and the expression of activated Caspase-3 in NRK-52E cells induced by hypoxia.WB results showed that Gpr97 deficiency significantly increase the phosphorylation level of Stat3 and upregulate the expression of anti-apoptotic protein Survivin.The addition of exogenous Sema3A recombinant protein reversed these effects.It is further confirmed that Gpr97 deficiency can reduce the expression level of inflammatory factors and cell death.All these effects are mediated by Sema3A signaling.2 Gpr97 regulats the expression of Sema3A by affecting the expression and activation of HuRGpr97 deficiency reduces the expression level of HuR in the kidney of mice with IRI and reduces the cytoplasmic translocation of HuR.HuR deficiency reduces the expression of Sema3A and shortens the half-life of Sema3A mRNA.The expression of Sema3A was restored by the addition of recombinant HuR overexpression virus.3 Effects of HuR on the stability of Sema3A mRNASequences of 3 '-UTR of Sema3A from various species showed that three AU-rich elements(AREs)with canonical AUUUA sequences that may serve as determinants for Sema3A mRNA stability are highly conserved.HuR interacted with the 3'-UTR of Sema3A mRNA and was involved in the hypoxia-mediated Sema3 A mRNA stabilityPart III The role of Gpr97 in the pathogensis of AKI induced by cisplatin 1 Gpr97 was significantly upregulated in cisplatin-induced AKIReal-time RT-PCR and WB results showed that Gpr97 were upregulated in cisplatin-induced AKI.In cisplatin stimulated NRK-52E cells,WB results showed that the expression of Gpr97 was upregulated significantly after stimulation.2 Gpr97 deficiency alleviates AKI induced by cisplatinCompared with WT mice,Gpr97_-/-mice showed lower concentration of SCr and BUN and less severe morphological injury.Gpr97 deficiency decreased the levels of pro-inflammatory mediators by Real-time RT-PCR.Consistently,neutrophil and macrophage accumulation was further decreased in the kidney from Gpr97-/-mice,indicating that Gpr97 exacerbates AKI induced by cisplatin.3 Effects of Gpr97 on the expression of Sema3A in cisplatin-induced AKIReal-time RT-PCR and WB results showed that the increase of Sema3A was alleviated in Gpr97-/-mice compared with the wild-type mice during AKI.ELISA results showed that the content of Sema3A in the serum of Gpr97-/-mice was lower than that of WT mice.Conclusions and innovations1 This study reveals the expression pattern of Gpr97 in AKI for the first time.It is found that the expression level of Gpr97 is significantly elevated after renal IRI.Gpr97 deficiency alleviates renal ischemia/reperfusion injury.These results were confirmed in AKI induced by cisplatin.This study provides the important experimental basis and theoretical basis for the design and development of Gpr97 related drugs.2 We found that Gpr97 can regulate Sema3A signaling for the first time.It is proved that Gpr97 renal protection effects are at least partially dependent on its regulation on Sema3A signaling.This study is helpful to further elucidate the role of GPCR in AKI and to provide the possibility of a novel approach for the treatment of patients with AKI.3 We first discovered that Gpr97 is the upstream of RNA binding protein HuR.Considering HuR can regulate the expression of many disease related molecules,our research provides the possibility of Gpr97 as a drug target for other diseases.