GPR87 Promotes Renal Tubulointerstitial Frbrosis By Accelerating Glycolysis And Mitochondrial Dysfunction

BackgroundChronic kidney disease(CKD)has been recognized as one of the leading causes of death worldwide.Renal tubulointerstitial fibrosis is the common final outcome of almost all progressive CKD and is also a major determinant of renal failure.Therefore,novel therapeutic targets to either slow down or reverse CKD progression are urgently needed.G protein-coupled receptors(GPCRs)are a family of membrane receptors that respond to extracellular signaling and participate in a comprehensive variety of physiological processes in various organs.Although GPCRs constitute about 30%ofdrug targets,the majority of the family members remain unexplored in therapies or clinical trials.Moreover,most of GPCRs are largely unknown in the kidney.Therefore,further identification of key GPCRs involved in the pathogenesis of renal fibrosis may lead to more effective treatment approaches for CKD.Lysophosphatidic acid(LPA)is a small ubiquitous phospholipid and acts as an extracellular signaling molecule involved in numerous physiological and pathological processes,including cellular survival,proliferation,migration,and contraction.Thus,it is critical for the progress of fibrosis in various organs.Meanwhile,as a ligand for GPCRs,the biological functions of LPA are mediated through some GPCRs,which are also called LPA receptors.Therefore,targeting these LPA receptors may be a novel therapeutic strategy for the treatment of various organ fibrosis.Among GPCRs,GPR87 is identified as a novel LPA receptor and regulate the progression of numerous tumors,including bladder,lung,pancreatic,skin,and cervical cancers.Therefore,GPR87 is attracting attention as a promising cancer therapeutic target.Notably,a previous proteomics analysis revealed that GPR87 expression was dramatically upregulated in the kidney from mice with aristolochic acid nephropathy(AAN).Meanwhile,one of our published RNA-seq data(The accession number in GEO website:GSE185226)showed that GPR87 was remarkably induced in podocytes with adriamycin treatment.These results suggested that GPR87 may play an important role in the development of CKD.Objective1.Detect the expression patterns of GPR87 in chronic kidney disease and the models of renal tubulointerstitial fibrosis.2.Explore the role of GPR87 in the process of renal tubulointerstitial fibrosis.3.Explore the mechanisms of GPR87 in renal tubulointerstitial fibrosis,provide some research basis for the mechanisms of renal tubulointerstitial fibrosis,and provide new possible therapeutic strategies and target for the treatment of CKD.Methods and Results1.The expression patterns of GPR87 in renal tubulointerstitial fibrosisWe collected 51 biopsies from patients with various CKD.Compared with the normal kidney tissues,the expression of GPR87 was significantly enhanced in renal biopsies from CKD patients,including IgA nephropathy,LN,HTN,and DN.We further confirmed that GPR87 was predominantly localized in the tubules rather than fibroblasts in the kidney from patients with DN.Notably,the protein level of GPR87 was negatively correlated with eGFR in all subjects.It was found that the expression of GPR87 was significantly induced in the kidney from UUO,AAN and DN mice by RTqPCR,WB,and IHC analyses.2.The role of GPR87 in renal tubulointerstitial fibrosisWe generated Gpr87-/-mice and Cre+/Gpr87fl/fl mice which was confirmed by tail genotyping,RT-qPCR and WB analyses to evaluate the contribution of GPR87 to renal fibrosis.WT mice displayed remarkable tubular atrophy and extracellular matrix deposition after UUO,whereas less renal injuries were observed in Gpr87-/-and and Cre+/Gpr87fl/fl UUO mice.At the molecular level,GPR87 deletion reduced the upregulation of FN1,α-SMA,Vimentin and Collagen I in the kidney from UUO mice by WB and IHC.3.The mechanisms of GPR87 in promoting glycolysis and mitochondrial injuryBy analyzing the ECAR,we found that gene silencing of GPR87 markedly reduced the level of glycolysis in HK-2 cells with TGF-β1 treatment,indicating that GPR87 is a crucial regulator of glycolysis.We used 2-DG,an inhibitor of glycolysis,to treat HK-2 cells and found that 2-DG could reduce FN1 and Vimentin expression in HK-2 cells with TGF-β1 treatment.However,gene silencing of GPR87 could not further reduce fibrosis,as evidenced by the expression levels of FN1 and Vimentin in 2-DG treated HK-2 cells,suggesting that glycolysis is an important mediator for the function of GPR87 in renal fibrosis.Moreover,2-DG could decrease TGF-β1-induced mitochondrial injury,as evidenced by PGC-1α expression.Consistently,gene silencing of GPR87 could not further enhance PGC-1α expression in HK-2 cells with 2-DG treatment.Gene silencing of GPR87 decreased the level of lactate in the cultured medium of HK-2 cells with TGF-β1 treatment.We further treated NRK-49F cells with the cultured medium of HK-2 cells to examine the role of lactate produced by HK-2 cells on the profibrotic effect of NRK-49F cells.It was found that the medium from GPR87-deficient HK-2 cells had a lower profibrotic effect on NRK-49F cells,as evidenced by the reduced expressions of α-SMA,Vimentin,and FN1.By RNA-seq analysis,we revealed that hexokinase-2 is one of the most significantly altered glycolysis-related enzymes in Gpr87-/-mice with UUO nephropathy,which was further validated in the kidney from both Gpr87-/-and Cre+/Gpr87fl/fl mice with UUO nephropathy by WB and IHC analysis.In vitro,gene silencing of GPR87 reduced the expression of hexokinase-2 in TGF-β1 treated HK-2 cells.To test whether hexokinase-2 promotes renal fibrosis,we treated UUO mice with 3-bromopyruvate(3-BrPA),an inhibitor of hexokinase-2.Our results showed that 3BrPA dramatically alleviated renal fibrosis in WT UUO mice.However,3-BrPA treatment did not further inhibit the UUO-induced renal fibrosis in Cre+/Gpr87fl/fl mice.We overexpressed GPR87 in HK-2 cells.By using MitoTracker dye to label mitochondria in HK-2 cells,we found that TGF-β1 treatment reduced mitochondrial length,which was exacerbated by overexpression of GPR87.In addition,3-BrPA treatment could reverse the effect of GPR87 overexpression in TGF-β1 treated HK-2 cells,indicating that 3-BrPA could ameliorate GPR87-induced mitochondrial dynamics perturbation.Consistently,3-BrPA treatment attenuated GPR87-induced mitochondrial depolarization as evidenced by JC-1 staining.Meanwhile,3-BrPA treatment recovered GPR87-mediated PGC-1α reduction.We further assessed oxidative stress in HK-2 cells by DHE and MitoSOX.Our results revealed that overexpression of GPR87 increased the production of intracellular ROS and mtROS induced by TGF-β1.In addition,3BrPA treatment could attenuated the effect of GPR87 overexpression in TGF-β1 treated HK-2 cells.In vivo,tubule-specific GPR87 deletion significantly recovered the expressions of the complexes of OXPHOS proteins,mitochondrial associated proteins including PGC-1α,MFN2,and TFAM in UUO mice.We also found that tubule-specific GPR87 deletion decreased ROS accumulation in the kidney from UUO mice by DHE staining.To further validate the role of hexokinase-2 in renal fibrosis,we tested the effect of 3-BrPA in Cre+/Gpr87fl/fl UUO mice.WB analysis showed that 3-BrPA dramatically recovered mitochondrial injury in WT UUO mice as evidenced by the changes of PGC-1α,MFN2,and TFAM expression.However,3-BrPA treatment did not further recover UUO-induced mitochondrial injury in Cre+/Gpr87fl/fl mice.Meanwhile,the DHE staining analysis revealed that 3-BrPA treatment did not further reduce the UUO-induced ROS accumulation in Cre+/Gpr87fl/fl mice.We found that YAP expression rather than TAZ was significantly induced in the kidney from UUO mice.WB and IHC analysis showed that tubule-specific GPR87 deletion significantly reduced YAP expression in the kidney from UUO mice.In vitro,GPR87 silencing attenuated TGF-β1-induced YAP expression.We further found that GPR87 overexpression enhanced TGF-β1-induced hexokinase-2 expression,which was recovered by gene silencing of YAP.Moreover,gene silencing of YAP significantly decreased GPR87-mediated glycolysis,as evidenced by the decreased lactate concentration in cultured medium of HK-2 cells.We also found that GPR87 overexpression further exacerbated the TGF-β1-induced PGC-1α reduction,which was recovered by gene silencing of YAP.Consistently,gene silencing of YAP reduced GPR87-mediated ROS accumulation which was detected by DHE and MitoSOX in HK-2 cells.Gene silencing of YAP ameliorated GPR87-mediated renal tubulointerstitial fibrosis,as evidenced by the decreased of FN1,Vimentin and α-SMA expression levels in HK-2 cells.Conclusion1.GPR87 was significantly increased in the renal biopsies from CKD patients and the mice models with renal fibrosis,and was mainly located in the tubule area.2.Our study revealed for the first time that GPR87 difficiency significantly ameliorated renal tubulointerstitial fibrosis induced by UUO.3.Our study demonstrated for the first time that GPR87 promotes glycolysis and mitochondrial damage in renal tubular epithelial cells.4.GPR87 accelerated glycolysis and mitochondrial injury at least in part by YAPhexokinase-2 signaling,which could result in renal fibrosis.Inhibition of GPR87 and its downstream signaling pathways may provide novel therapeutic strategies for the treatment of CKD.