Gpr97 Exacerbates AKI by Mediating Sema3a Signaling

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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 increased 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

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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 responses 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 activity, 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 data showing the protein levels of Gpr97 in the kidneys of mice with renal IRI. (H) Representative photomicrographs of 3,3’-diaminobenzidine (DAB) immunohistochemical staining of Gpr97 in the kidneys of mice with IRI. (I) Representative photomicrographs of 3,3’-diaminobenzidine (DAB) immunohistochemical staining of Gpr97 in the kidneys of patients with biopsy-proven acute tubular necrosis (ATN). Normal kidney tissues were obtained from the healthy kidney poles of individuals who underwent tumor nephrectomies without renal disease. *P,0.05 versus sham-operated wild-type mice (n=8).

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Figure 2. Gpr97 deﬁciency ameliorated renal IRI in mice. (A) Serum creatinine (SCr) concentration in different groups of mice. (B) BUN levels in different groups of mice. (C) Representative micrographs showing the morphology by hematoxylin and eosin (H&E) staining of the kidney from different groups of mice. (D) Quantitative assessment of tubular damage. (E) In situ terminal deoxynucleotidyl transferase–mediated UTP nick- end labeling (TUNEL) assays were performed to assess renal cell death. Nuclei were revealed using 4’,6-diamidino-2-phenylindole (DAPI) staining. (F) Quantitative assessment of the number of cell death (numbers per high-power ﬁeld [HPF]). *P,0.05 versus sham-operated wild- type mice (n=8); #P,0.05 versus ischemic wild-type mice at the same experimental conditions.

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Figure 3. Gpr97 deficiency reduced inflammatory responses in the kidney after renal IRI in mice. (A) The mRNA levels of proinflammatory mediators including IL-1b, IL-6, TNF-a, and monocyte chemoattractant protein-1 (MCP-1) in the kidney from different groups of mice. (B) Representative sections of kidney stained for macrophages from different groups of mice. (C) Data analysis of macrophage infiltrates in the kidney (numbers per high-power field [HPF]) from different groups of mice. (D) Representative sections of kidney stained for neutrophils from different groups of mice. (E) Data analysis of neutrophil infiltrates in the kidney (numbers per HPF) from different groups of mice. *P,0.05 versus sham-operated wild-type mice; #P,0.05 versus ischemic wild-type mice at the same experimental conditions (n=8).

bone marrow by mRNA (Figure 1A) and Western blot (Figure Whole Mouse Genome Oligo Microarray for global gene ex- 1B) analyses (Supplemental Tables 1 and 2). In the kidney, pression analysis, we found the upregulation of Gpr97 in the Gpr97 was expressed in various types of renal parenchymal kidneys of mice with renal IRI (Figure 1E), which was further cells, including rat proximal tubule epithelial cells (NRK-52E), confirmed by mRNA (Figure 1F), Western blot (Figure 1G), rat glomerular mesangial cells, rat glomerular endothelial and immunohistochemical (Figure 1H) analyses. To define the cells, and murine podocytes (Figure 1, C and D). Using Agilent tubular segment-specificity of Gpr97 expression in the kidney,

J Am Soc Nephrol 29: 1475–1489, 2018 Gpr97 and AKI 1479 BASIC RESEARCH www.jasn.org we used double immunostaining for Gpr97 (green) and var- and BUN (Figure 2B), improved morphologic injury (Figure ious tubular markers (red) in the kidney. As shown in Supple- 2, C and D), and reduced cell death as demonstrated by ter- mental Figure 1, Gpr97 was mainly expressed and induced in minal deoxynucleotidyl transferase–mediated digoxigenin- proximal tubules and distal tubules after renal IRI. Moreover, deoxyuridine nick-end labeling staining (Figure 2, E and F). compared with kidney tissues obtained from patients who underwent tumor nephrectomies without other renal disease, Gpr97 Deficiency Reduced Inflammatory Responses in Gpr97 was also upregulated in the kidneys of patients with the Kidney after Renal IRI in Mice biopsy-proven acute tubular necrosis, which presents with Gpr97 deficiency reduced inflammatory responses by decreas- AKI and is one of the most common causes of AKI (Figure 1I). ing the levels of proinflammatory mediators (Figure 3A), and macrophage (Figure 3, B and C) and neutrophil (Figure 3, D Gpr97 Deficiency Ameliorated Renal IRI in Mice and E) infiltration in the kidneys of mice with IRI. 2 2 Gpr97 / mice were phenotypically normal and had no ap- preciable defects in renal morphology and function. However, Gpr97 Mediated Sema3A Expression Gpr97 deficiency significantly ameliorated renal IRI in mice, Using microarray analysis, we found that IRI-induced Sema3A as evidenced by the reduction of serum creatinine (Figure 2A) expression was significantly abolished by Gpr97 deficiency in

Figure 4. Gpr97 mediated Sema3A expression. (A) Representative heatmap of gene expression levels in the kidneys of mice with IRI by multiplex qRT-PCR array. (B) Summarized data showing the mRNA levels of Sema3A in the kidneys of mice with renal IRI. (C) Representative Western blot gel documents and summarized data showing the protein levels of Sema3A in the kidneys of mice with renal IRI. (D) Rep- resentative photomicrographs of Sema3A 3,3’-diaminobenzidine (DAB) immunohistochemical staining in the kidneys of mice with renal IRI. (E) Summarized data showing the serum levels of Sema3A in mice with renal IRI. (F) Positive correlation between the serum levels of Sema3A and serum creatinine (SCr) in all mice. (G) Positive correlation between the serum levels of Sema3A and BUN in all mice. *P,0.05 versus sham-operated wild-type mice; #P,0.05 versus ischemic wild-type mice at the same experimental conditions (n=8).

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Figure 5. Gpr97 deﬁciency protected against AKI induced by cisplatin. (A) Relative mRNA levels of Gpr97 in the kidney from different groups of mice. (B) Representative Western blot gel documents and summarized data showing the levels of Gpr97 in the kidney from different groups of mice. (C) Serum creatinine (SCr) concentration in different groups of mice. (D) BUN levels in different groups of mice. (E) Representative micrographs showing the morphology by hematoxylin and eosin (H&E) stainingand and quantitative assessment of tubular damage from different groups of mice. (F) Summarized data showing the expression levels of Sema3A in the kidney from different groups of mice. (G) Summarized data showing the serum levels of Sema3A in different groups of mice. *P,0.05 versus sham-operated wild-type mice; #P,0.05 versus wild-type mice with cisplatin treatment (n=8).

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Figure 6. Gpr97 contributed to hypoxia-induced production of proinflammatory mediators and cell death in NRK-52E cells via Sema3A. (A) Representative Western blot gel documents and summarized data showing the protein levels of Gpr97 in NRK-52E cells under OGD conditions (cultured in a hypoxic environment for 90 minutes at 0.1% O2, followed by reoxygenation at different time points [6, 12, 24, or 48 hours]). (B) Representative Western blot gel documents and summarized data showing the gene silencing efficiency of Gpr97 by siRNA-Gpr97 transfection. (C) The effect of Gpr97 on the mRNA levels of proinflammatory mediators in NRK-52E cells under OGD conditions (cultured in a hypoxic environment for 90 minutes at 0.1% O2, followed by reoxygenation at 24 hours). (D) Summarized data showing the overall percentage of cell death, including the amount of apoptotic and necrotic cells determined by flow cytometric analysis in NRK-52E cells with different treatments. (E) Summarized data showing the caspase-3 activity in NRK-52E

1482 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1475–1489, 2018 www.jasn.org BASIC RESEARCH mice (Figure 4A), which was further confirmed by mRNA protect against AKI and aid the functional and structural re- (Figure 4B), Western blot (Figure 4C), and immunohisto- covery of the kidney from AKI,23 and STAT3 phosphorylation chemical (Figure 4D) analyses. Moreover, the serum levels of is downregulated by Sema3A,24,25 we further investigated 2 2 Sema3A were also decreased in Gpr97 / mice with renal IRI whether Gpr97 regulates the STAT3-survivin signaling path- (Figure 4E), which were positively correlated with serum cre- way by Sema3A. Compared with controls, STAT3 phosphory- atinine (Figure 4F) and BUN (Figure 4G), individually. lation and survivin had a compensatory increase in NRK-52E cells under OGD conditions, which was further induced by Gpr97 Deficiency Also Protected against AKI Induced Gpr97 deficiency (Figure 6F). In addition, gene silencing of by Cisplatin Gpr97 reduced toll-like receptor 4 (TLR4) mRNA levels, a ma- To further confirm the implications of Gpr97 signaling in the jor target of Sema3A for the regulation of inflammatory re- kidney, we investigated whether Gpr97 also plays a detrimental sponses (Figure 6G). Importantly, all these effects of Gpr97 role in mice with AKI induced by cisplatin. It was found that knockdown could be counteracted by recombinant mouse Gpr97 expressionwas alsoincreasedin the kidneysof miceafter Sema3A proteins (Fc tagged, 100 ng/ml) in NRK-52E cells. cisplatin injection (Figure 5, A and B). Gpr97 deficiency ame- The upregulation of Gpr97 was also confirmed in NRK-52E liorated renal dysfunction (Figure 5, C and D), histologic le- cells with cisplatin treatment (Supplemental Figure 5). sions (Figure 5E), and reduced the levels of proinflammatory mediators and macrophage and neutrophil infiltration (Sup- Gpr97 Induced Sema3A Expression by Mediating HuR plemental Figure 2). Gpr97 deficiency reduced the levels of Expression and Activation Sema3A both in the kidney (Figure 5F) and in the serum As shown in Figure 7A, IRI-induced HuR expression in the 2 2 (Figure 5G) from mice with cisplatin treatment. Together, kidney was significantly attenuated in Gpr97 / mice. In vitro, these results indicate that Gpr97-mediated Sema3A signaling we also found that gene silencing of Gpr97 inhibited hypoxia- may be a common response in the kidney after AKI. induced HuR expression (Figure 7B). A marked accumulation of cytoplasmic HuR was also observed in NRK-52E cells under Gpr97 Contributed to Hypoxia-Induced Production of OGD treatment by immunofluorescence analysis, which was Proinflammatory Mediators and Cell Death in Rat inhibited by Gpr97 knockdown (Figure 7C). Moreover, gene Proximal Tubular Epithelial (NRK-52E) Cells via silencing of HuR (Figure 7D) attenuated hypoxia-induced Sema3A Sema3A expression, even at the basal level in NRK-52E cells In vitro, we used several approaches to produce hypoxic con- (Figure 7E). To determine whether HuR-mediated Sema3A ditions: oxygen–glucose deprivation (OGD, Figure 6A) via expression results from the regulation of mRNA stability, ac- chemical anoxia/recovery induced by incubating NRK-52E tinomycin D, a transcription inhibitor, was applied to block cells in glucose-free medium with antimycin A/2-deoxyglucose Sema3A transcription 12 hours after OGD treatment. It was for ATP depletion (60 minutes, anoxia), and then in glucose- found that siRNA-HuR transfected NRK-52E cells had a replete complete growth medium (recovery) or cobalt chloride shorter t1/2 of Sema3A mRNA under hypoxia conditions (Fig- treatment (Supplemental Figure 3). All manipulations signifi- ure 7F), indicating that HuR contributes to the regulation of cantly induced Gpr97 expression. Gene silencing of Gpr97 Sema3A mRNA stability. Consistently, overexpression of HuR (Figure 6B) inhibited hypoxia-induced levels of proinflamma- by recombinant HuR-expressing adenoviral plasmid transfec- tory mediators (Figure 6C). Using flow cytometric analysis, we tion (Figure 7G) counteracted Gpr97 deficiency-reduced found that the overall percentage of cell death and the number Sema3A expression (Figure 7H). of apoptotic and necrotic cells was significantly decreased by gene silencing of Gpr97 in NRK-52E cells under hypoxia con- HuR Interacted with the 39-UTR of Sema3A mRNA and ditions (Figure 6D). We also found that gene silencing of Gpr97 Was Involved in Hypoxia-Mediated Sema3A mRNA reduced hypoxia-induced caspase-3 activity, further confirm- Stability ing that Gpr97 mediates cell apoptosis (Figure 6E). Similar Sequence alignment of 39-UTR of Sema3A from various spe- results were also obtained from gene silencing of Sema3A cies showed that three AU-rich elements (AREs) with canon- (Supplemental Figure 4). At the molecular level, Gpr97 ical AUUUA sequences that may serve as determinants for deficiency inhibited hypoxia-induced Sema3A expression. Sema3A mRNA stability are highly conserved (Figure 8A). Considering that STAT3 phosphorylation and subsequent up- Thus, RNA-EMSA was performed to explore whether HuR regulation of survivin in renal proximal tubule epithelial cells regulates Sema3A expression via directly binding to its

cells with different treatments. (F) Representative Western blot gel documents and summarized data showing the effect of Gpr97 on the levels of STAT3 phosphorylation and survivin in NRK-52E cells with different treatments. (G) Summarized data showing the mRNA levels of TLR4 in NRK-52E cells with different treatments. *P,0.05 versus control; #P,0.05 versus vehicle of NRK-52E cells under OGD conditions (n=6).

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Figure 7. Gpr97 induced Sema3A expression by mediating HuR expression and activation. (A) Representative Western blot gel documents and summarized data showing the protein levels of HuR in the kidneys of mice with renal IRI. *P,0.05 versus sham-operated wild-type mice; #P,0.05 versus ischemic wild-type mice (n=8). (B) Representative Western blot gel documents and summarized data showing the protein levels of HuR in NRK-52E cells under OGD conditions. *P,0.05 versus control; #P,0.05 versus vehicle of NRK-52E cells under OGD conditions (n=6). (C) Representative microscopic images for immunoﬂuorescence staining showing the distribution of HuR in NRK-52E cells

1484 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1475–1489, 2018 www.jasn.org BASIC RESEARCH transcript. Different RNA oligonucleotides each encompassed CKD, including FSGS, minimal change disease, and membra- conserved putative AREs from the 39-UTR of rat Sema3A nous GN (Supplemental Figure 7), suggesting that Gpr97 may mRNA, denominated as Sema3A-ARE1, Sema3A-ARE2, and be a common predictor for renal injury. Sema3A-ARE3, respectively. A complex was formed when Regarding the regulatory mechanism of Gpr97 expression Sema3A-AREs were applied for the binding reaction in in the kidney under hypoxia conditions, we speculate that NRK-52E cells (Figure 8B), as well as in the cytoplasmic ex- Gpr97 upregulation may be mediated by transcription factor tracts from the mouse kidney and human proximal tubular hypoxia-inducible factor 1a (HIF-1a). It is known that HIF- epithelial (HK-2) cells (Supplemental Figure 6). Using the 1a is induced in mammalian cells in response to hypoxia con- SEMA3A-ARE1 probe, we found that the binding ability was ditions or hypoxia-mimetic agents such as cobalt chloride. strongly increased in extracts from NRE-52E cells under OGD Emerging evidence has confirmed that some GPCRs are conditions, which was abolished by Gpr97 deficiency (Figure HIF-1a target genes. HIF-1a is a mediator of heregulin-in- 8C). In addition, the probe Sema3A-ARE1-HuR complex was duced CXCR4 upregulation in breast cancer cells.28 HIF-1a is supershifted by anti-HuR antibody. Recombinant human also required for both the transactivation of the Gpr30 pro- HuR protein was used as a positive control to confirm the moter reporter gene as well as for the upregulation of Gpr30 interaction between HuR and Sema3A-ARE1 (Figure 8D). expression upon hypoxia.29 Therefore, considering that hyp- To further confirm that the 39-UTR promotes Sema3A oxia induces Gpr97 expression, Gpr97 may be induced by mRNA expression, various plasmids containing the 39-UTR HIF-1a. On the other hand, certain ligand-activated GPCRs and luciferase reporter gene were transfected into NRK-52E can also induce HIF-1a expression. The recruitment of tran- cells individually (Figure 8E). In the absence of the Sema3A- scription factors to the promoter sequence of HIF-1a as well ARE1 sequence or with a mutation in ARE1, hypoxia had no as the stabilization of HIF-1a protein levels may occur upon effect on the enzyme activity (Figure 8F). However, hypoxia activation of GPCRs by endothelin-1 or b-adrenoceptor ago- significantly induced enzyme activity in the pmirGLO-Luciferase- nists.30–32 Therefore, further study will be needed to deter- Sema3A-ARE1 plasmid-transfected group, which was attenuated mine whether HIF-1a induces Gpr97 expression and by siRNA-HuR. Similar results were also found the interaction whether a feedforward mechanism exists between Gpr97 between HuR and Sema3A-ARE2 or Sema3A-ARE3 (data not and HIF-1a–mediated signaling. shown). Although the endogenous ligands of Gpr97 are unknown, Gpr97 is an orphan adhesion GPCR with high homology to the better characterized Gpr56. Like Gpr56, Gpr97 possesses both DISCUSSION an exceptionally long extracellular region, characteristic of cell adhesion proteins, and an intracellular region reminiscent of GPCRs such as dopamine-1 receptor, angiotensin II receptors, other GPCRs. Recently, some specific ligands of Gpr56 have and endothelin-1 receptors are widely expressed in the kid- been identified. Collagen III33 and tissue transglutaminase 234 ney,26,27 and are known to play vital roles in the regulation of interact with the N-terminal extended extracellular domain renal function, with dysregulation of GPCR signaling closely fragment of Gpr56. In addition to these ligands, which pre- associated with kidney disease and systemic disorders, but the sumably interact with Gpr56 in trans,somecis-acting binding role of Gpr97 in the kidney remains unknown. In this study, partners have also been found, such as tetraspanins CD9 and using two separate murine AKI models of IRI and cisplatin- CD81.35 Considering the similarity between Gpr56 and induced AKI, we found that Gpr97 deficiency markedly Gpr97, it is possible that Gpr97 may share ligands with ameliorated renal dysfunction, histologic lesions, and inflam- Gpr56, and the cellular functions of Gpr97 may be mediated matory responses in mice. Importantly, we confirmed that the by interacting with specific protein ligands on the cell surface expression of Gpr97 was also induced in the kidneys of pa- and/or in the extracellular matrix. tients with biopsy-proven acute tubular necrosis in the retro- In this study, a positive correlation between the levels of spective cohort study. In addition, we also observed the Gpr97 and Sema3A was observed in the kidney. The sema- upregulation of Gpr97 in renal biopsies from patients with phorin family members are divided into eight classes on the

under OGD conditions. (D) Representative Western blot gel documents and summarized data showing the gene silencing efﬁciency of HuR by siRNA-HuR transfection. (E) Representative Western blot gel documents and summarized data showing the effect of HuR on the Sema3A protein in NRK-52E cells under OGD conditions. *P,0.05 versus control; #P,0.05 versus vehicle of NRK-52E cells under OGD conditions (n=6). (F) Summarized data showing the effect of HuR on the regulation of Sema3A mRNA stability. After NRK-52E cells were transfected with siRNA-HuR or scramble, the cells were treated without (control) or with OGD conditions for 12 hours before addition of actinomycin D (ActD). (G) Representative Western blot gel documents and summarized data showing the overexpression of HuR by adenovirus transfection (ORF of ELAVL1 [HuR] in adenoviral vector pAd with C-terminal Flag and His tag; Adenovirus-HuR) in NRK-52E cells. *P,0.05 versus control (n=6). (H) Representative Western blot gel documents and summarized data showing that HuR overexpression counteracted Gpr97 deﬁciency-reduced Sema3A expression. *P,0.05 versus control; #P,0.05 versus vehicle of NRK-52E cells under OGD conditions (n=6).

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Figure 8. HuR interacted with the 39-UTR of Sema3A mRNA and was involved in hypoxia-induced Sema3A mRNA stability. (A) Se- quence homology analysis of 39UTR of Sema3A mRNA among different species. In rats, 22 canonical ARE sites were labeled. (B) The cytoplasmic extracts from NRK-52E cells were prepared for RNA-EMSA analysis. Complexes were formed when Sema3A-ARE1, Sema3A-ARE2, and Sema3A-ARE3 were applied for the binding reaction. (C) RNA-EMSA assay showing that the binding ability was strongly increased in extracts from NRE-52E cells under OGD conditions, which was abolished by gene silencing of Gpr97 using Sema3A-ARE1. (D) Recombinant human HuR proteins (rhHuR) were used as positive controls to conﬁrm the interaction between HuR and Sema3A-ARE1 by RNA-EMSA. (E) A schematic representation of the construction of various plasmids containing luciferase and 39- UTR of Sema3A-ARE1 mRNA. (F) The luciferase activity was quantiﬁed by in NRE-52E cells transfected with different plasmids. *P,0.05 versus control (n=6).

1486 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1475–1489, 2018 www.jasn.org BASIC RESEARCH basis of their structural features and distributions among dif- downstream targets of Sema3A. It was found that Gpr97 me- ferent phyla. Classes 1 and 2 contain invertebrate semaphorins diated TLR4 expression by Sema3A, which was consistent with whereas classes 3–7 contain the 22 vertebrate semaphorins. the previous studies showing that Sema3A induced TLR4 sig- Class 8 contains viral semaphorins. The semaphorins not naling, which could foster nuclear NF-kB activation and only play essential roles in the development and function of inflammation.15,40 In addition, Gpr97 reduced STAT3 many different physiologic systems, but also contribute to the phosphorylation and subsequent downregulated survivin ex- pathology of various diseases. In this study, we observed the pression in renal proximal tubule epithelial cells, thereby pre- changes in semaphorins by microarray analysis in the kidneys venting the functional and structural recovery of the kidney of ischemic mice. Among them, Sema3A was significantly in- from AKI. Taken together, these data clearly suggest that duced, which has been demonstrated to play a vital role on the Sema3A signaling is one of the critical signal transduction regulation of renal function. Sema3A is of importance in pat- pathways that link Gpr97 to AKI. terning the developing kidney by modulating ureteric bud Finally,weprovidethefirstdirectevidencethatHuRactsasakey branching, vascularization, podocyte differentiation, and post-transcriptional regulatorof Gpr97 mediated-Sema3Aexpres- maintaining the integrity of the glomerular filtration bar- sion in AKI. HuR is a member of the embryonic lethal abnormal rier.36–38 Moreover, dysregulation of Sema3A is known to ex- vision (ELAV)-like family of RNA-binding proteins, which regu- acerbate renal injury in adult kidney models of both chronic lates mRNA cargos that typically contain AREs in the 39-UTR.41 and acute kidney disease. Sema3A gain-of-function in podo- Although HuR is normally localized to the nucleus, the stability of cytes leads to proteinuric glomerular disease in mice and the ARE-containing mRNAs in response to stress conditions cor- excess Sema3A causes foot process effacement, glomerular relates with the translocation of HuR to the cytoplasm. HuR has basement lamination, and endothelial damage.39 In AKI, been demonstrated to be involved in the regulation of renal ho- genetic inactivation of Sema3A protected mice from IRI-in- meostasis and the adaptive responses contributing to the patho- duced AKI, improved tissue histology, reduced neutrophil genesis of various renal disease, such as ischemia, fibrosis, and infiltration, and inhibited epithelial cell apoptosis.15 Recent diabetic nephropathy.42–44 Although Sema3A mRNA from differ- studies have also demonstrated that Sema3A is an early, pre- ent species bear long 39-UTR with various AREs, the mechanisms dictive biomarker in experimental and pediatric AKI.13 In this involved in the post-transcriptional regulation of Sema3A are study, we found that Gpr97 deficiency markedly inhibited IRI- poorly understood. Using RNA-EMSA assay, we demonstrated induced Sema3A expression both in the kidney and in the that Sema3A mRNA is a target of the mRNA-stabilizing protein serum. We further found that Gpr97 induced inflammatory HuR. It should be noted that although our studies indicate a causal responses and tubular epithelial cell death through mediation link between Gpr97 and HuR, we did not focus on the regulatory of Sema3A. Although we did not attempt to explore the mo- mechanisms of Gpr97 on HuR, and recent studies have shown lecular mechanisms by which Gpr97-mediated Sema3A sig- that inactivation of Gpr97 would lead to an increase in cAMP naling contributes to AKI in detail, we also detected the known levels.45 cAMP is a negative regulator of HuR,46 therefore it is

Figure 9. Schematic representation showing that Gpr97 exacerbates AKI via mediating Sema3A signaling. In this model, Gpr97 contributed to the development of postischemic renal inﬂammation and promoted tubular epithelial cell death, which was associated with the regulation of Sema3A. Gpr97-induced Sema3A expression and mRNA stability were regulated by HuR.

J Am Soc Nephrol 29: 1475–1489, 2018 Gpr97 and AKI 1487 BASIC RESEARCH www.jasn.org possible that Gpr97 regulates the expression of HuR via cAMP- 10. Shi J, Zhang X, Wang S, Wang J, Du B, Wang Z, et al.: Gpr97 is dis- mediated signaling. Further studies are needed to confirm these pensable for metabolic syndrome but is involved in macrophage in- fl findings in the kidney. ammation in high-fat diet-induced obesity in mice. Sci Rep 6: 24649, 2016 In summary, we demonstrate that Gpr97 exacerbates AKI by 11. Shi JP, Li XN, Zhang XY, Du B, Jiang WZ, Liu MY, et al.: Gpr97 is dis- regulation of the Sema3A signaling pathways, and further identify pensable for inflammation in OVA-induced asthmatic mice. PLoS One that Gpr97-mediated HuR acts as a key post-transcriptional reg- 10: e0131461, 2015 ulator of Sema3A expression (Figure 9). Our results suggest that 12. Jang HR, Ko GJ, Wasowska BA, Rabb H: The interaction between is- Gpr97 is an important mediator of AKI, and that pharmacologic chemia-reperfusion and immune responses in the kidney. J Mol Med (Berl) 87: 859–864, 2009 targeting of Gpr97-mediated Sema3A signaling pathways at mul- 13. Jayakumar C, Ranganathan P, Devarajan P, Krawczeski CD, Looney S, tiple levels may provide a novel approach for the treatment of Ramesh G: Semaphorin 3A is a new early diagnostic biomarker of ex- patients with AKI. perimental and pediatric acute kidney injury. PLoS One 8: e58446, 2013 14. Aggarwal PK, Veron D, Thomas DB, Siegel D, Moeckel G, Kashgarian M, et al.: Semaphorin3a promotes advanced diabetic nephropathy. – ACKNOWLEDGMENTS Diabetes 64: 1743 1759, 2015 15. Ranganathan P, Jayakumar C, Mohamed R, Weintraub NL, Ramesh G: Semaphorin 3A inactivation suppresses ischemia-reperfusion-induced F.Y. designed the study; W.F., Z.W., Q.L., X.W., and Y.S. carried out inflammation and acute kidney injury. Am J Physiol Renal Physiol 307: experiments; C.M., J.S., and N.L. analyzed the data; W.F., W.T., and Y. F183–F194, 2014 Z. made the figures; F.Y. and W.F. drafted and revised the manuscript; 16.KimM,HamA,KimJY,BrownKM,D ’Agati VD, Lee HT: The volatile fl 9 all authors approved the final version of the manuscript. anesthetic iso urane induces ecto-5 -nucleotidase (CD73) to protect against renal ischemia and reperfusion injury. Kidney Int 84: 90–103, This study was supported by the National Science Fund for Dis- 2013 tinguished Young Scholars (grant 81525005 to F.Y.), The National 17. Wei Q, Dong Z: Mouse model of ischemic acute kidney injury: Tech- Nature Science Foundation of China (grants 91642204 and 81470958 nical notes and tricks. Am J Physiol Renal Physiol 303: F1487–F1494, to F.Y., 81600570 to X.W., 81670629 to W.T., 81770726 to Y.Z., and 2012 81400730 to Z.W.). 18. Zhou M, Tang W, Fu Y, Xu X, Wang Z, Lu Y, et al.: Progranulin protects against renal ischemia/reperfusion injury in mice. Kidney Int 87: 918– 929, 2015 19. Sasaki K, Doi S, Nakashima A, Irifuku T, Yamada K, Kokoroishi K, DISCLOSURES et al.: Inhibition of SET Domain-Containing Lysine Methyl- None. transferase 7/9 Ameliorates Renal Fibrosis. J Am Soc Nephrol 27: 203–215, 2016 20. Crowley LC, Marfell BJ, Scott AP, Waterhouse NJ: Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, REFERENCES and flow cytometry. Cold Spring Harb Protoc 2016: 953–957, 2016 21. Klöss S, Rodenbach D, Bordel R, Mülsch A: Human-antigen R (HuR) 1. Jang HR, Rabb H: Immune cells in experimental acute kidney injury. expression in hypertension: Downregulation of the mRNA stabilizing Nat Rev Nephrol 11: 88–101, 2015 protein HuR in genetic hypertension. Hypertension 45: 1200–1206, 2. Rewa O, Bagshaw SM: Acute kidney injury-epidemiology, outcomes 2005 and economics. Nat Rev Nephrol 10: 193–207, 2014 22. Joassard OR, Bélanger G, Karmouch J, Lunde JA, Shukla AH, Chopard 3. Paavola KJ, Sidik H, Zuchero JB, Eckart M, Talbot WS: Type IV collagen A, et al.: HuR mediates changes in the stability of AChR b-subunit is an activating ligand for the adhesion G protein-coupled receptor mRNAs after skeletal muscle denervation. JNeurosci35: 10949– GPR126. Sci Signal 7: ra76, 2014 10962, 2015 4. Hamann J, Aust G, Araç D, Engel FB, Formstone C, Fredriksson R, et al.: 23. Chen J, Chen JK, Conway EM, Harris RC: Survivin mediates renal International Union of Basic and Clinical Pharmacology. XCIV. Adhe- proximal tubule recovery from AKI. J Am Soc Nephrol 24: 2023–2033, sion G protein-coupled receptors. Pharmacol Rev 67: 338–367, 2015 2013 5. Kishore A, Hall RA: Versatile signaling activity of adhesion GPCRs. 24. Movassagh H, Tatari N, Shan L, Koussih L, Alsubait D, Khattabi M, et al.: Handb Exp Pharmacol 234: 127–146, 2016 Human airway smooth muscle cell proliferation from asthmatics is 6. Valtcheva N, Primorac A, Jurisic G, Hollmén M, Detmar M: The orphan negatively regulated by semaphorin3A. Oncotarget 7: 80238–80251, adhesion G protein-coupled receptor GPR97 regulates migration of 2016 lymphatic endothelial cells via the small GTPases RhoA and Cdc42. J 25. Teng Y, Yin Z, Li J, Li K, Li X, Zhang Y: Adenovirus-mediated delivery of Biol Chem 288: 35736–35748, 2013 Sema3A alleviates rheumatoid arthritis in a serum-transfer induced 7. Sleckman BP, Khan WN, Xu W, Bassing CH, Malynn BA, Copeland NG, mouse model. Oncotarget 8: 66270–66280, 2017 et al.: Cloning and functional characterization of the early-lymphocyte- 26. Rudomanova V, Blaxall BC: Targeting GPCR-Gbg-GRK2 signaling as a specificPb99gene.Mol Cell Biol 20: 4405–4410, 2000 novel strategy for treating cardiorenal pathologies. Biochim Biophys 8. Zugasti O, Bose N, Squiban B, Belougne J, Kurz CL, Schroeder FC, Acta 1863: 1883–1892, 2017 et al.: Activation of a G protein-coupled receptor by its endogenous 27. Weiss RH: G protein-coupled receptor signalling in the kidney. Cell ligand triggers the innate immune response of Caenorhabditis Signal 10: 313–320, 1998 elegans. Nat Immunol 15: 833–838, 2014 28. Lopez-Haber C, Barrio-Real L, Casado-Medrano V, Kazanietz MG: 9. Causton B, Ramadas RA, Cho JL, Jones K, Pardo-Saganta A, Rajagopal Heregulin/ErbB3 signaling enhances CXCR4-driven Rac1 activation J, et al.: CARMA3 Is Critical for the Initiation of Allergic Airway In- and breast cancer cell motility via hypoxia-inducible factor 1a. Mol Cell flammation. JImmunol195: 683–694, 2015 Biol 36: 2011–2026, 2016

1488 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1475–1489, 2018 www.jasn.org BASIC RESEARCH

29. Recchia AG, De Francesco EM, Vivacqua A, Sisci D, Panno ML, Andò S, differentiation during glomerular development. Development 136: et al.: The G protein-coupled receptor 30 is up-regulated by hypoxia- 3979–3989, 2009 inducible factor-1alpha (HIF-1alpha) in breast cancer cells and car- 38. Tufro A, Teichman J, Woda C, Villegas G: Semaphorin3a inhibits ure- diomyocytes. JBiolChem286: 10773–10782, 2011 teric bud branching morphogenesis. Mech Dev 125: 558–568, 2008 30. Lappano R, Rigiracciolo D, De Marco P, Avino S, Cappello AR, Rosano 39. Tufro A: Semaphorin3a signaling, podocyte shape, and glomerular C, et al.: Recent advances on the role of G protein-coupled receptors in disease. Pediatr Nephrol 29: 751–755, 2014 hypoxia-mediated signaling. AAPS J 18: 305–310, 2016 40. Wen H, Lei Y, Eun SY, Ting JP: Plexin-A4-semaphorin 3A signaling is 31. Caprara V, Scappa S, Garrafa E, Di Castro V, Rosanò L, Bagnato A, et al.: required for Toll-like receptor- and sepsis-induced cytokine storm. J Endothelin-1 regulates hypoxia-inducible factor-1a and -2a stability Exp Med 207: 2943–2957, 2010 through prolyl hydroxylase domain 2 inhibition in human lymphatic 41. Brennan CM, Steitz JA: HuR and mRNA stability. Cell Mol Life Sci 58: endothelial cells. Life Sci 118: 185–190, 2014 266–277, 2001 32. Hu HT, Ma QY, Zhang D, Shen SG, Han L, Ma YD, et al.: HIF-1alpha links 42. Ibrahim H, Lee YJ, Curthoys NP: Renal response to metabolic acidosis: beta-adrenoceptor agonists and pancreatic cancer cells under nor- Role of mRNA stabilization. Kidney Int 73: 11–18, 2008 moxic condition. Acta Pharmacol Sin 31: 102–110, 2010 43. Pullmann R Jr., Rabb H: HuR and other turnover- and translation-regulatory 33. Luo R, Jeong SJ, Jin Z, Strokes N, Li S, Piao X: G protein-coupled re- RNA-binding proteins: Implications for the kidney. Am J Physiol Renal ceptor 56 and collagen III, a receptor-ligand pair, regulates cortical Physiol 306: F569–F576, 2014 development and lamination. Proc Natl Acad Sci U S A 108: 12925– 44. Shang J, Wan Q, Wang X, Duan Y, Wang Z, Wei X, et al.: Identiﬁcation 12930, 2011 of NOD2 as a novel target of RNA-binding protein HuR: Evidence from 34. Effendi K, Yamazaki K, Fukuma M, Sakamoto M: Overexpression of NADPH oxidase-mediated HuR signaling in diabetic nephropathy. leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) Free Radic Biol Med 79: 217–227, 2015 represents a typical Wnt/b-catenin pathway-activated hepatocellular 45. Gupte J, Swaminath G, Danao J, Tian H, Li Y, Wu X: Signaling property study carcinoma. Liver Cancer 3: 451–457, 2014 of adhesion G-protein-coupled receptors. FEBS Lett 586: 1214–1219, 2012 35. Little KD, Hemler ME, Stipp CS: Dynamic regulation of a GPCR-tetraspanin-G 46. Klöss S, Srivastava R, Mülsch A: Down-regulation of soluble guanylyl protein complex on intact cells: Central role of CD81 in facilitating GPR56- cyclase expression by cyclic AMP is mediated by mRNA-stabilizing Galpha q/11 association. MolBiolCell15: 2375–2387, 2004 protein HuR. Mol Pharmacol 65: 1440–1451, 2004 36. Serini G, Valdembri D, Zanivan S, Morterra G, Burkhardt C, Caccavari F, et al.: Class 3 semaphorins control vascular morphogenesis by inhibit- ing integrin function. Nature 424: 391–397, 2003 37. Reidy KJ, Villegas G, Teichman J, Veron D, Shen W, Jimenez J, et al.: This article contains supplemental material online at http://jasn.asnjournals. Semaphorin3a regulates endothelial cell number and podocyte org/lookup/suppl/doi:10.1681/ASN.2017080932/-/DCSupplemental.

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