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Gpr97 Exacerbates AKI by Mediating Sema3a Signaling

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Gpr97 Exacerbates AKI by Mediating Sema3a Signaling BASIC RESEARCH www.jasn.org Gpr97 Exacerbates AKI by Mediating Sema3A Signaling Wei Fang,1 Ziying Wang,1 Quanxin Li,1 Xiaojie Wang,1 Yan Zhang,1 Yu Sun,1 Wei Tang,2 Chunhong Ma,3 Jinpeng Sun,4 Ningjun Li,5 and Fan Yi1,6 1Departments of Pharmacology, 2Pathogenic Biology, 3Immunology, and 4Biochemistry and Molecular Biology, The Key Laboratory of Infection and Immunity of Shandong Province, School of Basic Medical Science, and 6The State Key Laboratory of Microbial Technology, Shandong University, Jinan, China; and 5Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia ABSTRACT Background G protein-coupled receptors (GPCRs) participate in a variety of physiologic functions, and several GPCRs have critical physiologic and pathophysiologic roles in the regulation of renal function. We investigated the role of Gpr97, a newly identified member of the adhesion GPCR family, in AKI. Methods AKI was induced by ischemia–reperfusion or cisplatin treatment in Gpr97-deficient mice. We assessed renal injury in these models and in patients with acute tubular necrosis by histologic examination, and we conducted microarray analysis and in vitro assays to determine the molecular mechanisms of Gpr97 function. Results Gpr97 was upregulated in the kidneys from mice with AKI and patients with biopsy-proven acute tubular necrosis compared with healthy controls. In AKI models, Gpr97-deficient mice had significantly less renal injury and inflammation than wild-type mice. Gpr97 deficiency also attenuated the AKI-induced expression of semaphorin 3A (Sema3A), a potential early diagnostic biomarker of renal injury. In NRK- 52E cells subjected to oxygen–glucose deprivation, siRNA-mediated knockdown of Gpr97 further in- creased the expression of survivin and phosphorylated STAT3 and reduced toll-like receptor 4 expression. Cotreatment with recombinant murine Sema3A protein counteracted these effects. Finally, additional in vivo and in vitro studies, including electrophoretic mobility shift assays and luciferase reporter assays, showed that Gpr97 deficiency attenuates ischemia–reperfusion-induced expression of the RNA-binding protein human antigen R, which post-transcriptionally regulates Sema3A expression. Conclusions Gpr97 is an important mediator of AKI, and pharmacologic targeting of Gpr97-mediated Sema3A signaling at multiple levels may provide a novel approach for the treatment of AKI. J Am Soc Nephrol 29: 1475–1489, 2018. doi: https://doi.org/10.1681/ASN.2017080932 AKIis a serious clinical complicationwith high morbidity and mortality. Renal ischemia–reperfusion injury (IRI), nephrotoxic agents, and infection leading to sepsis are Significance Statement 1 the major causes of AKI. Despite substantial progress in AKI is a serious clinical complication with high morbidity and mortality. Despite substantial progress in understanding the mechanisms contributing to the pathophysiology of AKI, Received August 25, 2017. Accepted February 15, 2018. there is no effective therapy available to treat or prevent AKI. This article elucidates the role of Gpr97, an orphan adhesion Published online ahead of print. Publication date available at G protein-coupled receptor, in the pathogenesis of AKI. In www.jasn.org. two independent murine AKI models, ischemia–reperfusion Correspondence: Prof. Fan Yi, Department of Pharmacology, injury and cisplatin-induced AKI, Gpr97 exacerbates AKI by The Key Laboratory of Infection and Immunity of Shandong the regulation of semaphorin 3A, a potential early di- Province, School of Basic Medical Science, Shandong University, agnostic biomarker of renal injury, thereby contributing to 44 Wenhua Xi Road, Jinan, Shandong 250012, China. Email: fa- inflammatory responses and tubular epithelial cell death. [email protected] This study indicates that targeting Gpr97 may be an in- novative therapeutic strategy for patients with AKI. Copyright © 2018 by the American Society of Nephrology J Am Soc Nephrol 29: 1475–1489, 2018 ISSN : 1046-6673/2905-1475 1475 BASIC RESEARCH www.jasn.org Figure 1. Gpr97 was significantly induced in the kidneys of mice with renal IRI. (A) RT-PCR analysis of Gpr97 mRNA levels in selected murine tissues, including heart, liver, spleen, lung, kidney, brain, and bone marrow (BM). (B) Western blot analysis of the relative protein levels of Gpr97 in selected murine tissues. (C) RT-PCR analysis of Gpr97 mRNA levels in renal cells including rat proximal tubule epithelial cells (NRK-52E), rat glomerular mesangial cells, rat glomerular endothelial cells, and murine podocytes. (D) Western blot analysis of the relative protein levels of Gpr97 in renal cells. (E) Representative heatmap of gene expression levels of adhesion GPCRs inthekidneysofmicewithIRIbymultiplexqRT-PCRarray.(F)Relative 1476 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1475–1489, 2018 www.jasn.org BASIC RESEARCH understanding the mechanisms contributing to the pathophysiology Animal Studies 2 2 of AKI, there is no effective therapy available to treat or prevent AKI.2 Twelve-week-old male Gpr97-deficient (Gpr97 / )mice The G protein-coupled receptor (GPCR) family is the largest (B6.129-Adgrg3tm1Smoc,20–25 g body wt) were obtained membrane protein family; members participate in a variety of from Shanghai Model Organisms Center (Shanghai, China). physiologic functions and are major targets of pharmaceutical Induction of AKI induced by renal IRI or cisplatin was as drugs. Although GPCRs make up about 30% of targets for drugs previously described.16–18 All protocols were approved by In- on the market and areone of thelargest families in clinical trials, the stitutional Animal Care and Use Committee of Shandong majority are still unexploited in therapies or trials. Therefore, University (permit no. 201402037) and conducted in accor- identifying the key GPCRs involved in the pathogenesis of AKI dance with the National Institutes of Health Guide for the anddevelopmentofselectiveligandstargetingGPCRsmay provide Care and Use of Laboratory Animals. novel and effective treatment strategies for patients with AKI. The GPCR superfamily is grouped into five major families: glutamate, Cell Culture and Treatments rhodopsin, adhesion, frizzled, and secretin. The adhesion GPCRs Immortalized rat renal proximal tubule (NRK-52E) cells were are a recently defined subfamily of GPCRs containing .30 mem- cultured as described.19 bers in the human genome, combining characteristics of adhesion molecules and seven transmembrane receptors.3 Adhesion GPCRs Flow Cytometry are receiving increased attention because many human diseases are Cell apoptosis was determined by propidium iodide-annexin V linked to adhesion GPCRs mutations and genetic deletions produce staining as described.20 intriguing phenotypes.4,5 Among them, Gpr97 is a newly identified adhesion GPCR that was originally identified in mouse intestinal RNA Electrophoretic Mobility Shift Assay lymphatic endothelium. It regulates migration of human lymphatic Electrophoretic mobility shift assays (EMSAs) and supershift endothelial cells.6 Gpr97 is also expressed in murine pre-B cells and analysis with protein extracts were performed as described.21 thymocytes.7 Although several studies8–9 have demonstrated that adhesion GPCRs are modulators of immune cell function, the role Luciferase Reporter Assay and Transfection of Gpr97 on the regulation inflammatory responses continues to be Luciferase assays were performed for functional analysis of the controversial. Studies have shown that Gpr97 is involved in mac- 39-UTR of Sema3A mRNA, using a luciferase reporter assay rophage inflammation in high fat diet–induced obesity in mice.10 system (Promega, Madison, WI) as described.22 However, GPR97 is not required for the development of airway inflammation in ovalbumin-induced allergic asthma in mice,11 in- Data Availability dicating that the regulation of inflammation via Gpr97 may vary Microarray data sets have been deposited to the Gene Ex- depending on the pathophysiologic conditions. Considering that pression Omnibus database under accession code Gpr97 expression was significantly induced in the kidneys of mice GSE108195. with renal IRI in our preliminary studies and inflammatory re- sponses elicited by ischemia are of importance in the functional Statistical Analyses deterioration of the kidney,12 this study was designed to elucidate Data are expressed as means6SEM. The significance of the the role of Gpr97 in AKI. Our results demonstrate for the first time differences in mean values between and within multiple that Gpr97 exacerbated AKI by regulation of semaphorin 3A groups was examined by one-way ANOVA followed by Dun- (Sema3A), a potential early diagnostic biomarker of renal in- can multiple range tests. P,0.05 was considered statistically jury,13–15 thereby contributing to the inflammatory responses and significant. tubular epithelial cell death. We further found that Gpr97 induced RNA-binding protein human antigen R (HuR) expression and ac- tivity, thereby increasing Sema3A mRNA stability. RESULTS Gpr97 Was Significantly Induced in the Kidneys of Mice METHODS with Renal IRI Tissues examined were adult murine heart, liver, spleen, lung, An extended Material and Methods section can be found on- kidney, brain, and bone marrow; Gpr97 was present in all these line in the Supplemental Material. tissues with a relative high expression in the heart, kidney, and mRNA levels of Gpr97 in the kidneys of mice with renal IRI. (G) Representative Western blot gel documents and summarized
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