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A Newly Discovered Antifibrotic Pathway Regulated by Two Fatty Acid Receptors

GPR40 and GPR84
Open AccessPublished:February 15, 2018DOI:https://doi.org/10.1016/j.ajpath.2018.01.009
      Numerous clinical conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiological pathways involved in fibrosis. GPR40 and GPR84 are G protein–coupled receptors with free fatty acid ligands and are associated with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiological processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, respectively, we uncovered an antifibrotic pathway involving these receptors. In experiments using Gpr40- and Gpr84-knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity observed in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising molecular targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compound that may be effective for managing inflammatory and fibrosis-related diseases.
      Fibrosis is characterized by the excessive accumulation of extracellular matrix in damaged or inflamed tissues, and it is the common pathological outcome of many inflammatory and metabolic diseases. Numerous clinical conditions can lead to organ fibrosis and functional failure; in many disorders, acute or persistent inflammation is crucial to trigger the fibrotic response. The production of various profibrotic cytokines and growth factors by innate inflammatory cells results in the recruitment and activation of extracellular matrix–producing myofibroblasts.
      • Wynn T.A.
      • Ramalingam T.R.
      Mechanisms of fibrosis: therapeutic translation for fibrotic disease.
      There is currently a great need for therapies that could effectively target pathophysiological pathways involved in fibrosis.
      Free fatty acids (FFAs) are essential nutrients that exert various biological effects and have been implicated in many diseases, playing protective or harmful roles depending on the context. Besides their effects on intracellular metabolism and nuclear receptors, studies in the past 15 years have shown that FFAs can activate several cell surface G protein–coupled receptors, including FFA receptor 1 (GPR40) and GPR84. GPR40 and GPR84 show distinct characteristics in both fatty acid binding and biological effects. GPR40 is activated by both medium-chain FFAs (eg, decanoic acid) and long-chain FFAs (eg, linoleic acid)
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      Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40.
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      In addition, GPR40 and GPR84 exhibit distinct tissue distribution profiles. GPR40 is abundantly expressed in pancreatic β cells, where it enhances glucose-mediated insulin secretion.
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      Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40.
      Accordingly, several GPR40 agonists have advanced to clinical trials for type 2 diabetes.
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      TAK-875 versus placebo or glimepiride in type 2 diabetes mellitus: a phase 2, randomised, double-blind, placebo-controlled trial.
      However, the actions of GPR40 may not be limited to insulin secretion. GPR40 is also expressed in enteroendocrine cells of the gastrointestinal tract and may mediate release of glucagon-like peptide-1 and cholecystokinin secretion.
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      The G-protein-coupled receptor GPR40 directly mediates long-chain fatty acid-induced secretion of cholecystokinin.
      In addition, GPR40 is expressed in murine skin and may serve to limit and attenuate inflammation.
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      A GPR40 agonist GW9508 suppresses CCL5, CCL17, and CXCL10 induction in keratinocytes and attenuates cutaneous immune inflammation.
      Recent studies have also involved GPR40 in regulation of pain perception
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      Taste preference for fatty acids is mediated by GPR40 and GPR120.
      Finally, GPR40 has been shown to be expressed in the rat kidney
      • Ma S.K.
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      Activation of G-protein-coupled receptor 40 attenuates the cisplatin-induced apoptosis of human renal proximal tubule epithelial cells.
      and in a subset of murine kidney tubules, including the cortical collecting duct.
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      • Chen J.K.
      Overexpression of G-protein-coupled receptor 40 enhances the mitogenic response to epoxyeicosatrienoic acids.
      Moreover, in human renal proximal tubule epithelial HK-2 cells, activation of GPR40 with the synthetic agonist GW9508 reduced cisplatin-induced apoptosis.
      • Ma S.K.
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      • Kim S.W.
      Activation of G-protein-coupled receptor 40 attenuates the cisplatin-induced apoptosis of human renal proximal tubule epithelial cells.
      Studies of human and mouse GPR84, as ascertained by mRNA levels in various tissues, have determined that GPR84 is highly expressed on bone marrow cells, splenic T and B cells,
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      The G-protein coupled receptor, GPR84 regulates IL-4 production by T lymphocytes in response to CD3 crosslinking.
      and circulating granulocytes/monocytes/macrophages.
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      • Ling L.
      Medium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84.
      In the latter cells, mRNA expression of GPR84 is up-regulated only under inflammatory conditions. GPR84 is also expressed in brain, heart, muscle, colon, thymus, spleen, kidney, liver, intestine, placenta, lung, and leukocytes.
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      • Ling L.
      Medium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84.
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      An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors.
      GPR84 may be a mediator of the relationship between obesity and diabetes. Indeed, as adipocytes release fatty acids in the presence of macrophages, increased GPR84 expression and stimulation prevent the release of regulating hormones/adiponectin.
      • Nagasaki H.
      • Kondo T.
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      • Ozaki N.
      • Arima H.
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      • Oiso Y.
      • Hamada Y.
      Inflammatory changes in adipose tissue enhance expression of GPR84, a medium-chain fatty acid receptor: TNFalpha enhances GPR84 expression in adipocytes.
      GPR84 is expressed in the gastric corpus mucosa, and this receptor can be an important luminal sensor of food intake; it is most likely expressed on enteroendocrine cells, where it stimulates the release of peptide hormones, including incretins glucagon-like peptides 1 and 2.
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      • Sachs G.
      Selective gene expression by rat gastric corpus epithelium.
      Although both receptors have been associated with metabolic regulation and inflammation, they have not been previously linked to organ fibrosis. In this study, we demonstrate that PBI-4050, a synthetic ligand of GPR40 and GPR84, acts on cells involved in the fibrotic pathway: macrophages, fibroblasts, and epithelial cells. Moreover, PBI-4050 reduces fibrosis in animal models of kidney, lung, heart, liver, pancreas, and skin fibrosis. We also demonstrate that both receptors are modulated in models of fibrotic diseases and show that mice with a deletion in Gpr40 have increased renal interstitial fibrosis in response to ischemia, unilateral ureteral obstruction (UUO), and adenine-induced nephropathy models, whereas Gpr84 knockout mice have reduced kidney fibrosis in a model of adenine-induced nephropathy.

      Materials and Methods

      Compound

      PBI-4050, a first-in-class compound synthesized by Prometic BioSciences Inc. (Laval, QC, Canada) from a family of low-molecular-weight orally active new molecular entities, is an analog of medium-chain fatty acids. This lead drug candidate was prepared in five steps using modified Sonogashira coupling, as follows: 3-bromophenylacetic acid was converted to the corresponding ester and then reacted with palladium catalyst under Sonogashira condition to give the pentyne derivative. This compound was reduced over palladium, hydrolyzed to the acid, and treated with a base to yield the expected product PBI-4050.

      Cell Culture and Transfections

      Normal human dermal fibroblasts (NHDFs) from adult donors (Clonetics, East Rutherford, NJ) were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). HK-2 human epithelial proximal tubule cells (HK-2; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium/F12 medium with 10% FBS. NHDF and HK-2 cells were starved overnight in medium with 0.5% (NHDF) or 0.2% (HK-2) FBS and treated with or without recombinant human transforming growth factor (TGF)-β1 (10 ng/mL; R&D Systems, Minneapolis, MN) and PBI-4050 for 24 hours. Mouse peritoneal macrophages were isolated as previously described.
      • Zhang X.
      • Goncalves R.
      • Mosser D.M.
      The isolation and characterization of murine macrophages.
      Briefly, 6- to 8-week–old BALB/c mice were injected intraperitoneally with 1 mL of 3% Brewer thioglycollate medium. After 4 days, mice were euthanized and peritoneal exudate cells were recovered in 10 mL of cold phosphate-buffered saline. The cells were centrifuged, resuspended in Dulbecco's modified Eagle's medium/F12 medium + 10% FBS, and seeded in a cell culture plate. Macrophages were allowed to adhere for 2 hours in a cell incubator, after which nonadherent B and T cells were removed by gently washing three times with warm phosphate-buffered saline. Mouse peritoneal macrophages were pretreated for 30 minutes with PBI-4050 or vehicle before activation. Classically activated (M1) macrophages were obtained by priming with recombinant mouse interferon-γ (10 ng/mL; Fisher Scientific, Ottawa, ON, Canada) for 18 hours and adding lipopolysaccharide (LPS) 055:B5 (50 ng/mL; Sigma-Aldrich, Oakville, ON, Canada) for an additional 6 hours, whereas alternatively activated (M2) macrophages were generated by treating with recombinant mouse IL-4 (10 ng/mL; R&D Systems) for 24 hours. Human podocytes were obtained as previously described.
      • Keir L.S.
      • Firth R.
      • May C.
      • Ni L.
      • Welsh G.I.
      • Saleem M.A.
      Generating conditionally immortalised podocyte cell lines from wild-type mice.
      Briefly, conditionally immortalized podocytes were allowed to proliferate at 33°C in RPMI 1640 medium supplemented with 10% newborn calf serum, 1% penicillin/streptomycin, and 10 U/mL of interferon-γ (permissive conditions). Cells were passaged, counted, and seeded in RPMI 1640 medium supplemented with 2% newborn calf serum and 1% penicillin/streptomycin at a density of 1 × 105 cells per 10-cm dish and were thermoshifted to 37°C to allow for terminal differentiation 10 to 14 days later. After overnight serum starvation (0.1% newborn calf serum), podocytes were treated for 24 hours with 50 ng/mL LPS or 10 ng/mL TGF-β1, with or without a 30-minute preincubation with 100 μmol/L PBI-4050. Human embryonic kidney (HEK) 293 cells (Sigma-Aldrich) were cultured in Eagle's minimum essential medium (Wisent, Saint-Jean-Baptiste, QC, Canada) supplemented with 2 mmol/L l-glutamine, 10% FBS (Wisent), and 1% nonessential amino acids (Sigma-Aldrich). Transient HEK293 transfections were performed using the polyethylenimine (Polysciences, Warrington, PA) method.
      • Longo P.A.
      • Kavran J.M.
      • Kim M.S.
      • Leahy D.J.
      Transient mammalian cell transfection with polyethylenimine (PEI).
      HEK293-GPR84 and HEK293-GPR40 cell lines were obtained by selecting cells stably expressing 3xHA N-terminally tagged human GPR84 and GPR40 receptors.

      Plasmids

      The cDNA clones for human GPR40 and GPR84 receptors, human β-arrestin 2, Gαi2, Gαq, Gα13, Gβ1, Gγ1, and Gγ2 were obtained from the cDNA Resource Center (http://www.cdna.org). Green fluorescent protein (GFP) 10 (F64L, S147P, S202F, and H231L variant of Aequorea victoria GFP) gBlocks gene fragments (Integrated DNA Technologies, Coralville, IA) and linker were inserted in frame at the N-terminus of human Gγ1 and Gγ2 or at the C-terminus of GPR40. Rluc8
      • Loening A.M.
      • Fenn T.D.
      • Wu A.M.
      • Gambhir S.S.
      Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output.
      gBlocks fragment was inserted with linkers in the coding sequence of Gαi2, Gαq, and Gα13, as described previously.
      • Sauliere A.
      • Bellot M.
      • Paris H.
      • Denis C.
      • Finana F.
      • Hansen J.T.
      • Altie M.F.
      • Seguelas M.H.
      • Pathak A.
      • Hansen J.L.
      • Senard J.M.
      • Gales C.
      Deciphering biased-agonism complexity reveals a new active AT1 receptor entity.
      All generated constructs were confirmed by sequencing.

      BRET Measurement

      Transiently transfected HEK293 cells were seeded in 96-well white clear bottom Costar microplates (Fisher Scientific) coated with poly-d-lysine (Sigma-Aldrich) and left in culture for 24 hours. Cells were washed once with Tyrode's buffer (140 mmol/L NaCl, 1 mmol/L CaCl2, 2.7 mmol/L KCl, 0.49 mmol/L MgCl2, 0.37 mmol/L NaH2PO4, 5.6 mmol/L glucose, 12 mmol/L NaHCO3, and 25 mmol/L HEPES, pH 7.5) and the Rluc8 substrate coelenterazine 400A (Prolume, Lakeside, AZ) added at a final concentration of 5 μmol/L in Tyrode's buffer. Ligands were incubated with cells at room temperature for 8 minutes (G protein) or 15 minutes (β-arrestin) before reading bioluminescence resonance energy transfer (BRET) signal. BRET readings were collected using an Infinite M1000 microplate reader (Tecan, Morrisville, NC). BRET2 readings between Rluc8 and GFP10 were collected by sequential integration of the signals detected in the 370 to 450 nm (Rluc8) and 510 to 540 nm (GFP10) windows. The BRET signal was calculated as the ratio of light emitted by acceptor (GFP10) over the light emitted by donor (Rluc8). The values were corrected to net BRET by subtracting the background BRET signal obtained in cells transfected with Rluc8 constructs alone. For the β-arrestin assays, ligand-promoted net BRET values were calculated by subtracting vehicle-induced net BRET from ligand-induced net BRET. For the G protein activation biosensor assays, ligand-promoted net BRET values were calculated by first subtracting vehicle-induced net BRET from ligand-induced net BRET (ΔBRET), and then subtracting the ΔBRET values obtained in cells cotransfected with pcDNA3 from the ΔBRET values obtained in cells cotransfected with GPR40 or GPR84 receptors.

      Western Blot Analysis

      HEK293-GPR40, HEK293-GPR84, and untransfected HEK293 cells were treated with ligands for 7 minutes. Lysed cell extracts were separated by standard SDS-PAGE techniques and immunoblotted with anti–phosphorylated extracellular signal–regulated kinase (ERK) 1/2 antibody (Cell Signaling Technology, Danvers, MA). Chemiluminescence was revealed with a ChemiDoc MP imaging system (Bio-Rad, Mississauga, ON, Canada), and densitometric analyses of Western blot were performed using ImageLab version 5.2.1 (Bio-Rad). Phospho-ERK1/2 signal was normalized on total protein lane (MemCode protein stain kit; Fisher Scientific).

      qPCR

      RNA was extracted from cultured cells and homogenized tissue using the Qiagen RNEasy minikit or TRIzol reagent (Fisher Scientific) and treated with TURBO-DNA free DNase (Fisher Scientific), as per manufacturer's instructions. Extracted RNA was converted to cDNA using GoScript Reverse Transcriptase or the High-Capacity cDNA Reverse Transcription kit (Fisher Scientific) with 500 to 1000 ng starting material per reaction. Real-time quantitative PCR was performed on an AB-7900HT real-time cycler using TaqMan gene expression assays (Fisher Scientific), or for podocytes using an ABI Prism 7000 Sequence Detection System with SYBR Advantage qPCR Premix (Clontech, Mountain View, CA). qPCR data were analyzed using the ΔΔCt method, using glyceraldehyde-3-phosphate dehydrogenase as normalization control.

      In Situ Hybridization

      In situ hybridization was performed to localize GPR40 (official name FFAR1) and GPR84 mRNA expression in formalin-fixed, paraffin-embedded mouse kidney sections using the RNAscope 2.5 Duplex HD Detection Kit (Advanced Cell Diagnostics, Hayward, CA). Briefly, paraffin-embedded kidney sections were cut at 4 μm, air dried overnight, baked at 60°C for 1 hour, dewaxed, and air dried before pretreatments. Standard pretreatment protocol was used according to manufacturer's instructions. RNAscope probes for GPR40 (Mm-Ffar1-O1; catalog number 464311) and GPR84 (Mm-Gpr84-O1-C2; catalog number 447031) were used. Detection of probe binding was performed using the RNAscope Detection Kit based on horseradish peroxidase–based Green and alkaline phosphatase (diaminobenzidine)–based Fast Red chromogens for GPR40 and GPR84, respectively.

      Microdissected Mouse Tubule Preparations

      Three-month–old wild-type (WT; healthy) C57BL/6 mice were anesthetized under isoflurane, and the left kidney was quickly removed and placed in ice-cold sterile phosphate-buffered saline (pH 7.4). Three or four coronal slices (1 to 2 mm thick) were placed into chilled (4°C) dissection medium (105 mmol/L NaCl, 25 mmol/L NaHCO3, 10 mmol/L C2H3NaO2, 2.3 mmol/L Na2HPO4, 5 mmol/L KCl, 1.8 mmol/L CaCl2,1 mmol/L MgSO4, 8.3 mmol/L glucose, and 5 mmol/L alanine; osmolality, 300 mOsm/kg H2O) for freehand microdissection with forceps. Microdissected proximal tubules, thick ascending limb, and cortical collecting duct slices (1.0 to 2.2 mm thick) were transferred to RNAqueous-Micro Total RNA Isolation solution or stored at −80°C until required. RNA from microdissected kidney tubular segments was isolated using the Ambion RNAqueous microkit (Fisher Scientific).

      Gpr40 and Gpr84 KO Mice

      The Gpr40-targeted knockout (KO) mice (a gift from Dr. Stephen Wank, NIH, Bethesda, MD) used in the UUO and ischemia-reperfusion models were developed, as previously described,
      • Liou A.P.
      • Lu X.
      • Sei Y.
      • Zhao X.
      • Pechhold S.
      • Carrero R.J.
      • Raybould H.E.
      • Wank S.
      The G-protein-coupled receptor GPR40 directly mediates long-chain fatty acid-induced secretion of cholecystokinin.
      by replacing the Gpr40 coding region (except for the first 51 nucleotides) with a DNA fragment that included a hemagglutinin antigen, enhanced GFP, and neomycin genes. Gpr40−/− and Gpr84−/− mice used in the adenine-induced chronic kidney disease (CKD) model were generated by Deltagen (San Mateo, CA) by replacing 152 (Gpr40) or 257 (Gpr84) bp of genomic DNA within the receptor coding region with a mutant Neo (Gpr40) or Lac0-SA-IRES-lacZ-WT Neo/Kan (Gpr84) cassette. The KO mice were backcrossed for at least five generations against a C57BL/6N genetic background, and WT littermates on the same background were used as controls in the experiments.

      Adenine-Induced CKD Mouse Model

      The impact of both PBI-4050 treatment and GPR40 or GPR84 receptor deletion on the progression of tubulointerstitial injury was determined using the previously described
      • Tamura M.
      • Aizawa R.
      • Hori M.
      • Ozaki H.
      Progressive renal dysfunction and macrophage infiltration in interstitial fibrosis in an adenine-induced tubulointerstitial nephritis mouse model.
      adenine-induced mouse model of CKD. WT C57BL/6 (Charles River, Saint-Constant, QC, Canada), Gpr40−/−, or Gpr84−/− mice were fed either a standard rodent chow (Teklad 2018) or a diet supplemented with 0.25% adenine (Envigo, Madison, WI) for 4 weeks ad libitum. After 1 week of adenine administration, mice were given either vehicle (water) or PBI-4050 (200 mg/kg per day) by gastric gavage for 3 weeks. In separate studies, Gpr40−/− or Gpr84−/− mice or WT littermates were subjected to 4 weeks of standard or adenine-supplemented diet, as described earlier in this section. Kidney sections were stained with Masson's trichrome for histologic evaluation of tubulointerstitial fibrosis and cystic lesions scores.

      5/6-Nx Rat Model of CKD

      Six-week–old Sprague-Dawley male rats were subjected to 5/6 nephrectomy (5/6-Nx) or sham operations. Under ketamine anesthesia (60 to 100 mg/kg, i.p.), two-thirds of the left kidney was removed on day 0, followed by the right total nephrectomy on day 7. Sham-operated rats underwent exposition of the kidneys and removal of the perirenal fat and were used as controls. Animals that underwent the sham operation were treated with vehicle (water) and used as controls. On day 21, rats were allocated on the basis of glomerular filtration rate results. 5/6-Nx animals were divided into two groups (n = 8). One group received vehicle, and the other group was treated with once-daily oral administration of PBI-4050 (200 mg/kg), by gavage. Animals were treated from day 21 to 189 and sacrificed on day 190. Animals that died before day 190 were not used in the results analysis. Tissue sections of the kidney were prepared and stained with Masson's trichrome for the evaluation of glomerular and tubular lesions and collagen deposition. Serum and urinary (24-hour) creatinine were measured to determine creatinine clearance (glomerular filtration rate), and serum blood pressure was determined by tail-cuff method.

      Doxorubicin Mouse Model of Nephropathy

      Nephrotoxicity was induced by i.v. injection of doxorubicin (doxorubicin; Novopharm, Mirabel, QC, Canada; 10 mg/kg) on day 0 in BALB/c male mice (6 to 10 weeks of age). Vehicle (water) or PBI-4050 (200 mg/kg per day) was administered from day −3 to −1 and day 1 to day 10 (histology) or 13 (qPCR), and mice were sacrificed on the following day. Kidneys were prepared for histologic assessment of glomerular and tubular lesions with hematoxylin and eosin staining.

      Ischemia-Reperfusion Injury Model

      Male 8-week–old WT and Gpr40−/− mice on a mixed C56BL/6/129 background were used. The mice were subjected to contralateral nephrectomy, followed by 29 minutes of unilateral clamping of the renal pedicle to reduce variability of results. These animals were sacrificed after 3 weeks, and kidney sections were stained with picrosirius red for histologic quantification of fibrosis.

      UUO Fibrotic Model

      Male 8-week–old WT and Gpr40−/− mice on a mixed C56BL/6/129 background were used for these studies. The mice were subjected to the left ureter ligation for 4 days and then sacrificed for analysis. Kidney sections were stained with picrosirius red for histologic quantification of fibrosis.

      Carbon Tetrachloride–Induced Liver Fibrosis

      Liver fibrosis was induced in male C57BL/6 mice by i.p. administration of 2 mL/kg of CCl4 (10% in olive oil, twice a week for 58 days, time that corresponds to the fibrotic phase). Mice were treated with vehicle (water) or PBI-4050 at 200 mg/kg from day 1 to day 58 and euthanized at day 59. Liver was prepared for histologic assessment of fibrosis with Masson's trichrome staining.

      Suprarenal Abdominal Aorta Constriction Model

      Suprarenal abdominal aorta constriction was performed on adult male Sprague-Dawley rats (9 to 11 weeks old; Charles River), using a 21-gauge needle, as previously described.
      • Tardif K.
      • Hertig V.
      • Duquette N.
      • Villeneuve L.
      • El-Hamamsy I.
      • Tanguay J.F.
      • Calderone A.
      Nestin upregulation characterizes vascular remodeling secondary to hypertension in the rat.
      Suprarenal abdominal aorta constriction leads to concentric hypertrophy and the secondary development of interstitial fibrosis.
      • Tardif K.
      • Hertig V.
      • Duquette N.
      • Villeneuve L.
      • El-Hamamsy I.
      • Tanguay J.F.
      • Calderone A.
      Nestin upregulation characterizes vascular remodeling secondary to hypertension in the rat.
      Sham and suprarenal aorta constricted rats were randomly allocated and treated with vehicle (water) or PBI-4050 (200 mg/kg per day, oral gavage) from day 14 to day 28. On day 28, heart was excised and processed for histologic assessment of interstitial fibrosis with Masson's trichrome staining, as previously described.
      • Tardif K.
      • Hertig V.
      • Duquette N.
      • Villeneuve L.
      • El-Hamamsy I.
      • Tanguay J.F.
      • Calderone A.
      Nestin upregulation characterizes vascular remodeling secondary to hypertension in the rat.

      Bleomycin-Induced Mouse Model of Pulmonary Fibrosis

      Intratracheal instillation of bleomycin (0.025 U per mouse; Calbiochem, San Diego, CA) was performed in C57BL/6 10-week–old female mice (Charles River). Mice were allocated according to their bleomycin-induced body weight loss on day 7, and animals were treated with PBI-4050 (200 mg/kg per day) or vehicle (water) from day 7 to day 20 via gastric gavage. On day 21, lungs were prepared for histologic assessment of lesions (disrupted lung architecture, thickness of alveolar wall, and fibrosis) with Masson's trichrome staining.

      Fibrillin 1–Mutant Mouse Model of Systemic Scleroderma

      B6.Cg-Fbn1Tsk/J breeders were purchased from The Jackson Laboratory (Bar Harbor, ME). Both male and female heterozygotes were treated with vehicle (water) or PBI-4050 at 5 weeks of age (200 mg/kg per day, oral administration). The mice were sacrificed at 15 weeks of age (10 weeks of treatment). Before euthanasia, the back hair was shaved and a square or a rectangle on the back was marked, then the marked skin was removed, weighed, and stained with picrosirius red.

      db/db eNOS−/− Mouse Model of Diabetic Kidney Disease

      db/db eNOS−/− male mice (16 weeks old; n = 21) were randomized into two groups. Both groups were treated starting at 16 weeks of age and continuing until 30 weeks of age. One group was treated with PBI-4050 (100 mg/kg per day), and one group received vehicle (water). Mice were euthanized at 30 weeks of age, when pancreatic fibrosis is well established. Tissue sections of the pancreas were prepared for histologic assessment (picrosirius red staining).

      Histologic Image Analysis

      Renal and pulmonary injury was assessed in a blinded manner by a trained pathologist (A.G.). On the basis of the distinctive density and color of staining in digital images, the area of collagen in the tissue was quantified using Image-Pro Premier 9.1 for Masson's trichrome and with the BIOQUANT true-color Windows system (R & M Biometrics, Nashville, TN) for picrosirius red. Sections from at least four regions of each organ were analyzed, and the average was used as data from one animal sample.

      Statistical Analysis

      Data are expressed as means ± SEM for each treatment compared with control. For each independent experiment treatment, values were expressed as a percentage or fold difference of controls, and statistical analysis was then performed on the data from different experiments. Statistical analysis was performed using either one-way analysis of variance with Tukey's or Dunnett's post test for multiple comparisons or t-test (two tailed) when comparing two groups (P < 0.05 was taken as significant). Nonlinear fit analysis was used for dose-response curves. All data were analyzed using GraphPad Prism version 7 for Windows (GraphPad, San Diego, CA).

      Study Approval

      All animal studies were reviewed and approved by the animal care and use committee of the National Institute of Scientific Research, INRS-Institut-Armand-Frappier Center (Laval, QC, Canada), Vanderbilt University (Nashville, TN), Ottawa Hospital Research Institute (Ottawa, ON, Canada), or Montreal Heart Institute (Montreal, QC, Canada).

      Results

      PBI-4050 (3-pentylbenzeneacetic acid sodium salt) (Figure 1A) , a synthetic analog of decanoic acid, was designed to rigidify the fatty acid structure and reduce the β-oxidation of decanoate, the sodium salt of decanoic acid, a medium-chain fatty acid. As decanoic acid can activate the free fatty acid receptors GPR40
      • Briscoe C.P.
      • Tadayyon M.
      • Andrews J.L.
      • Benson W.G.
      • Chambers J.K.
      • Eilert M.M.
      • Ellis C.
      • Elshourbagy N.A.
      • Goetz A.S.
      • Minnick D.T.
      • Murdock P.R.
      • Sauls Jr., H.R.
      • Shabon U.
      • Spinage L.D.
      • Strum J.C.
      • Szekeres P.G.
      • Tan K.B.
      • Way J.M.
      • Ignar D.M.
      • Wilson S.
      • Muir A.I.
      The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids.
      • Kotarsky K.
      • Nilsson N.E.
      • Flodgren E.
      • Owman C.
      • Olde B.
      A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs.
      and GPR84,
      • Wang J.
      • Wu X.
      • Simonavicius N.
      • Tian H.
      • Ling L.
      Medium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84.
      • Southern C.
      • Cook J.M.
      • Neetoo-Isseljee Z.
      • Taylor D.L.
      • Kettleborough C.A.
      • Merritt A.
      • Bassoni D.L.
      • Raab W.J.
      • Quinn E.
      • Wehrman T.S.
      • Davenport A.P.
      • Brown A.J.
      • Green A.
      • Wigglesworth M.J.
      • Rees S.
      Screening beta-arrestin recruitment for the identification of natural ligands for orphan G-protein-coupled receptors.
      • Suzuki M.
      • Takaishi S.
      • Nagasaki M.
      • Onozawa Y.
      • Iino I.
      • Maeda H.
      • Komai T.
      • Oda T.
      Medium-chain fatty acid-sensing receptor, GPR84, is a proinflammatory receptor.
      the signaling properties of PBI-4050 on these receptors were verified.
      Figure thumbnail gr1
      Figure 1PBI-4050 is an agonist of GPR40. A: Chemical structure of PBI-4050. B: β-Arrestin 2 recruitment to activated GPR40 receptor was monitored in human embryonic kidney (HEK) 293 cells transfected with RLuc8-β-arrestin 2 and GPR40–green fluorescent protein (GFP) 10. Cells were exposed to increasing concentrations of sodium decanoate or PBI-4050, and bioluminescence resonance energy transfer (BRET) variation compared with vehicle was measured. C and D: Cells transfected with GPR40 and Gαq (C) or Gαi (D) activation biosensor were exposed to increasing concentrations of sodium decanoate or PBI-4050, and BRET variation compared with vehicle (dotted lines) was measured. E and F: Western blot of phosphorylated extracellular signal–regulated kinase (ERK) 1 and phosphorylated ERK2 in HEK293-GPR40 (E) and parental untransfected HEK293 (F) cells stimulated with the indicated ligands for 7 minutes. The bar graph represents the densitometric analysis. Data are expressed as means ± SEM (B–F). n = 4 to 7 experiments (A and B); n = 4 to 6 experiments (C and D); n = 3 independent experiments (E and F). P < 0.05, ∗∗∗P < 0.001 (one-way analysis of variance, followed by Tukey's multiple comparisons test).

      PBI-4050 Is an Agonist of GPR40

      It was recently shown that binding of the FFAs linoleate, palmitate, and oleate, and of the synthetic agonist TAK-875 to GPR40 promotes recruitment of β-arrestins 1 and 2 to the receptor.
      • Mancini A.D.
      • Bertrand G.
      • Vivot K.
      • Carpentier E.
      • Tremblay C.
      • Ghislain J.
      • Bouvier M.
      • Poitout V.
      beta-Arrestin recruitment and biased agonism at free fatty acid receptor 1.
      • Qian J.
      • Wu C.
      • Chen X.
      • Li X.
      • Ying G.
      • Jin L.
      • Ma Q.
      • Li G.
      • Shi Y.
      • Zhang G.
      • Zhou N.
      Differential requirements of arrestin-3 and clathrin for ligand-dependent and -independent internalization of human G protein-coupled receptor 40.
      A BRET-based assay that allows the monitoring of Rluc8-tagged β-arrestin 2 recruitment to GFP10-tagged GPR40 in living HEK293 cells was, therefore, used to assess GPR40 activation. PBI-4050 and sodium decanoate both concentration-dependently promoted β-arrestin 2 recruitment to GPR40 (pEC50: 2.98 ± 0.12 and 3.56 ± 0.08 mol/L, respectively) (Figure 1B). GPR40 has been described as coupling mainly to the Gα protein subunit of the Gq family,
      • Briscoe C.P.
      • Tadayyon M.
      • Andrews J.L.
      • Benson W.G.
      • Chambers J.K.
      • Eilert M.M.
      • Ellis C.
      • Elshourbagy N.A.
      • Goetz A.S.
      • Minnick D.T.
      • Murdock P.R.
      • Sauls Jr., H.R.
      • Shabon U.
      • Spinage L.D.
      • Strum J.C.
      • Szekeres P.G.
      • Tan K.B.
      • Way J.M.
      • Ignar D.M.
      • Wilson S.
      • Muir A.I.
      The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids.
      • Itoh Y.
      • Hinuma S.
      GPR40, a free fatty acid receptor on pancreatic beta cells, regulates insulin secretion.
      and partially to Gi.
      • Itoh Y.
      • Hinuma S.
      GPR40, a free fatty acid receptor on pancreatic beta cells, regulates insulin secretion.
      • Hardy S.
      • St-Onge G.G.
      • Joly E.
      • Langelier Y.
      • Prentki M.
      Oleate promotes the proliferation of breast cancer cells via the G protein-coupled receptor GPR40.
      • Yonezawa T.
      • Katoh K.
      • Obara Y.
      Existence of GPR40 functioning in a human breast cancer cell line, MCF-7.
      Hence, a previously described
      • Sauliere A.
      • Bellot M.
      • Paris H.
      • Denis C.
      • Finana F.
      • Hansen J.T.
      • Altie M.F.
      • Seguelas M.H.
      • Pathak A.
      • Hansen J.L.
      • Senard J.M.
      • Gales C.
      Deciphering biased-agonism complexity reveals a new active AT1 receptor entity.
      • Gales C.
      • Van Durm J.J.
      • Schaak S.
      • Pontier S.
      • Percherancier Y.
      • Audet M.
      • Paris H.
      • Bouvier M.
      Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes.
      BRET biosensor was used to directly monitor GPR40-mediated activation of Gα in living HEK293 cells. The Gα biosensor consists of an Rluc8-tagged Gαq or Gαi2 subunit, a GFP10-tagged Gγ1 subunit, and an untagged Gβ1. Agonist stimulation and receptor activation trigger a physical separation between the Rluc8-Gα donor and the GFP10-Gγ1 acceptor, resulting in a decrease in BRET signal, whose amplitude is correlated to ligand efficacy.
      • Gales C.
      • Van Durm J.J.
      • Schaak S.
      • Pontier S.
      • Percherancier Y.
      • Audet M.
      • Paris H.
      • Bouvier M.
      Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes.
      Treatment with increasing concentrations of either PBI-4050 or sodium decanoate resulted in a BRET signal decrease for both biosensors, indicative of Gαq (pEC50: 3.54 ± 0.16 and 3.99 ± 0.43 mol/L, respectively) (Figure 1C) and Gαi (pEC50: 4.53 ± 0.18 and 4.79 ± 0.25 mol/L, respectively) (Figure 1D) activation. It was also verified if PBI-4050 could trigger GPR40-mediated activation of G13 or Gs; both PBI-4050 and sodium decanoate had no effect on the BRET signal of Gα13 activation biosensor (Supplemental Figure S1A), nor did they induce cAMP production (Gαs pathway) in CHO-K1-GPR40 cells (Supplemental Figure S1B). GPR40 activation can also result in ERK1/2 phosphorylation.
      • Hara T.
      • Hirasawa A.
      • Sun Q.
      • Koshimizu T.A.
      • Itsubo C.
      • Sadakane K.
      • Awaji T.
      • Tsujimoto G.
      Flow cytometry-based binding assay for GPR40 (FFAR1; free fatty acid receptor 1).
      • Panse M.
      • Gerst F.
      • Kaiser G.
      • Teutsch C.A.
      • Dolker R.
      • Wagner R.
      • Haring H.U.
      • Ullrich S.
      Activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) by free fatty acid receptor 1 (FFAR1/GPR40) protects from palmitate-induced beta cell death, but plays no role in insulin secretion.
      Stimulation of HEK293-GPR40 cells with PBI-4050 increased ERK1/2 phosphorylation, but to a lesser extent than sodium decanoate at equivalent concentrations (Figure 1E). This increase of ERK1/2 phosphorylation was specific to GPR40 activation, because it was absent in untransfected HEK293 cells (Figure 1F).

      PBI-4050 Inhibits GPR84 Signaling

      GPR84 has been reported to couple primarily to the pertussis toxin–sensitive Gαi/o family of G proteins,
      • Wang J.
      • Wu X.
      • Simonavicius N.
      • Tian H.
      • Ling L.
      Medium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84.
      • Zhang Q.
      • Yang H.
      • Li J.
      • Xie X.
      Discovery and characterization of a novel small-molecule agonist for medium-chain free fatty acid receptor G protein-coupled receptor 84.
      which decrease intracellular cAMP levels. HEK293 cells cotransfected with GPR84 and the Gαi activation BRET biosensor were used to monitor the effect of PBI-4050 on this signaling pathway. Stimulation of HEK293 cells with increasing concentrations of sodium decanoate or the GPR84 agonist embelin
      • Southern C.
      • Cook J.M.
      • Neetoo-Isseljee Z.
      • Taylor D.L.
      • Kettleborough C.A.
      • Merritt A.
      • Bassoni D.L.
      • Raab W.J.
      • Quinn E.
      • Wehrman T.S.
      • Davenport A.P.
      • Brown A.J.
      • Green A.
      • Wigglesworth M.J.
      • Rees S.
      Screening beta-arrestin recruitment for the identification of natural ligands for orphan G-protein-coupled receptors.
      resulted in a concentration-dependent BRET signal decrease, indicative of Gi activation (Figure 2A) (pEC50 ± SEM sodium decanoate: 4.83 ± 0.17 mol/L; embelin: 5.01 ± 0.09 mol/L). The sodium decanoate–induced Gi activation was blocked in cells pretreated with pertussis toxin and was absent in cells in which empty vector was cotransfected in place of GPR84 (Figure 2B), demonstrating that the measured BRET decrease specifically reflected GPR84-mediated activation of Gαi2. PBI-4050 treatment did not induce Gαi activation (Figure 2, A and B); however, cotreatment of sodium decanoate–stimulated cells with increasing concentrations of PBI-4050 led to a concentration-dependent inhibition of Gαi activation (pIC50 ± SEM: 3.40 ± 0.06 mol/L) (Figure 2C). A similar inhibition of Gαi activation by PBI-4050 was also observed in cells stimulated with embelin (pIC50 ± SEM: 3.68 ± 0.28 mol/L) (Figure 2D). Although GPR84 has not been reported to couple to other families of Gα, it was verified if PBI-4050, sodium decanoate, or embelin could activate these signaling pathways. The three tested compounds neither had any effect on the BRET signal of Gαq (Supplemental Figure S2A) or Gα13 (Supplemental Figure S2B) activation biosensors in GPR84-transfected cells, nor did they induce cAMP production in CHO-K1-GPR84 cells (Gαs pathway) (Supplemental Figure S2C). GPR84 has been shown to activate the ERK1/2 pathway.
      • Zhang Q.
      • Yang H.
      • Li J.
      • Xie X.
      Discovery and characterization of a novel small-molecule agonist for medium-chain free fatty acid receptor G protein-coupled receptor 84.
      Indeed, stimulation of HEK293-GPR84 cells with sodium decanoate or embelin led to ERK1/2 phosphorylation (Figure 2E). In contrast, treatment with PBI-4050 decreased the basal ERK1/2 phosphorylation level. In accordance with results obtained with the Gαi activation biosensor, cotreatment of cells with PBI-4050 together with either sodium decanoate or embelin resulted in a significant reduction of ERK1/2 phosphorylation induced by these agonists, further confirming that PBI-4050 can inhibit GPR84-mediated signal transduction. In contrast, in untransfected HEK293 cells, ERK1/2 phosphorylation was not induced by GPR84 agonist sodium decanoate or embelin (Figure 1F). However, a slight reduction of basal ERK1/2 phosphorylation was observed after PBI-4050 treatment in these cells.
      Figure thumbnail gr2
      Figure 2PBI-4050 inhibits GPR84-mediated signaling. A–D: Gi pathway activation was monitored in living human embryonic kidney (HEK) 293 cells transfected with GPR84 and the Gαi2 activation biosensor. A: Cells were exposed to increasing concentrations of sodium decanoate, embelin, or PBI-4050, and bioluminescence resonance energy transfer (BRET) variation compared with vehicle (dotted line) was measured. B: Ligand-promoted BRET variation in the presence or absence of cotransfected GPR84 receptor, pretreated or not with 100 ng/mL pertussis toxin (18 hours, 37°C). One-way analysis of variance, followed by Dunnett's multiple comparisons test (compared with cells transfected with GPR84 and treated with sodium decanoate), was performed. C and D: Cells treated with 125 μmol/L sodium decanoate (C) or 30 μmol/L embelin (D) were cotreated with increasing concentrations of PBI-4050, and BRET variation compared with vehicle (dotted lines) was measured. E: Western blot of phosphorylated extracellular signal–regulated kinase (ERK) 1 and phosphorylated ERK2 in HEK293-GPR84 cells stimulated with the indicated ligands for 7 minutes. The column bar graph represents the densitometric analysis. One-way analysis of variance, followed by Tukey's multiple comparisons test, was performed. Data are expressed as means ± SEM (A–E). n = 4 to 7 experiments (A); n = 3 to 8 experiments (B); n = 4 to 5 experiments (C and D); n = 3 to 4 independent experiments (E). ∗∗P < 0.01, ∗∗∗P < 0.001 versus cells transfected with GPR84 and treated with sodium decanoate (Dunnett's test); ††P < 0.01, †††P < 0.001. GFP, green fluorescent protein.

      Effect of PBI-4050 on Fibroblasts, Epithelial Cells, Macrophages, and Podocytes

      To elucidate the role of GPR40 and GPR84 on cells involved in inflammation and fibrosis, the expression of both receptors and the effect of PBI-4050 was evaluated using cells stimulated under inflammatory (LPS) or fibrotic (TGF-β) conditions. In NHDFs expressing only GPR84, GPR84 mRNA levels increased significantly under fibrotic conditions (Figure 3A). Moreover, stimulation of NHDFs with TGF-β led to a strong increase in the expression of α-smooth muscle actin (α-SMA; myofibroblast marker) as well as profibrotic (connective tissue growth factor) and fibrotic (collagen I) markers, which were all significantly and concentration-dependently reduced by PBI-4050 treatment. In human epithelial proximal tubule cells (HK-2) expressing only GPR40, GPR40 mRNA levels were down-regulated (3.6-fold) under fibrotic conditions; in these cells, PBI-4050 also significantly inhibited the TGF-β–induced overexpression of collagen I (Figure 3B). In addition, peritoneal mouse macrophages showed both GPR40 and GPR84 expression. Activation of these macrophages to a classic, or M1 phenotype, with interferon-γ and LPS led to a robust increase (254-fold) of GPR84 expression and a significant reduction of GPR40 expression (2.5-fold). This strong up-regulation of GPR84 was accompanied by a significant increase in the expression of the proinflammatory cytokines monocyte chemoattractant protein-1, IL-6, and IL-12(p40), all of which were down-regulated in the presence of PBI-4050 (Figure 3C). Interestingly, both receptor expression levels were not modulated by activation of macrophages to an alternative, or M2 phenotype, with IL-4; however, mRNA expression of resistin-like molecule (RELM)α (Retnla), a macrophage alternative activation marker that has also been shown to promote the differentiation and survival of myofibroblasts,
      • Chung M.J.
      • Liu T.
      • Ullenbruch M.
      • Phan S.H.
      Antiapoptotic effect of found in inflammatory zone (FIZZ)1 on mouse lung fibroblasts.
      was increased (124-fold) by IL-4 stimulation, and significantly down-regulated in a concentration-dependent manner with PBI-4050 (Figure 3D). Finally, our results confirmed that LPS and TGF-β induced GPR84 expression in cultured human podocytes (Figure 3E), a critical cell type in maintaining the kidney's glomerular filtration barrier integrity. LPS stimulation in podocytes was accompanied with an increase in IL6 and IL8 mRNA expression and profibrotic and inflammatory biomarkers, which were down-regulated by treatment with PBI-4050.
      Figure thumbnail gr3
      Figure 3PBI-4050 regulates fibrotic and inflammatory markers in fibroblasts, proximal tubule epithelial cells, macrophages, and podocytes. mRNA expression of GPR84 and GPR40 (official name FFAR1) receptors and profibrotic and proinflammatory markers was determined in transforming growth factor (TGF)-β1–stimulated normal human dermal fibroblast (A) and HK-2 human epithelial proximal tubule (B) cells, in interferon (INF)-γ–primed and lipopolysaccharide (LPS)–stimulated (C) or IL-4–stimulated (D) murine peritoneal macrophages, and in TGF-β1– or LPS-treated human podocytes (E). Data are expressed as means ± SEM (A–E). n = 3 to 7 experiments (A and B); n = 2 (C, PBI-4050, 250 µmol/L); n = 3 to 5 experiments (C, all other groups); n = 3 to 6 experiments (E). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (t-test or one-way analysis of variance, followed by Tukey's multiple comparisons test).

      Expression of GPR40 and GPR84 in Kidney Fibrosis

      To elucidate further the involvement of both GPR40 and GPR84 receptors in the fibrotic process, several renal disease models associated with glomerular and/or tubulointerstitial fibrosis were studied. Interestingly, although the expression profile of GPR40 and GPR84 has not been completely described in the kidney, GPR40 is expressed in epithelial cells of the proximal tubule
      • Ma S.K.
      • Joo S.Y.
      • Choi H.I.
      • Bae E.H.
      • Nam K.I.
      • Lee J.
      • Kim S.W.
      Activation of G-protein-coupled receptor 40 attenuates the cisplatin-induced apoptosis of human renal proximal tubule epithelial cells.
      • Ma S.K.
      • Wang Y.
      • Chen J.
      • Zhang M.Z.
      • Harris R.C.
      • Chen J.K.
      Overexpression of G-protein-coupled receptor 40 enhances the mitogenic response to epoxyeicosatrienoic acids.
      and the cortical collecting duct in microdissected mouse tubule preparations (Figure 4A), whereas it is not detected in other segments, including the thick ascending limb. Our results have also confirmed GPR40 and GPR84 expression in various kidney disease models. Indeed, GPR84 mRNA levels increased in 5/6-Nx remnant kidney (Figure 4B), doxorubicin-induced nephropathy (Figure 4C), and adenine-associated nephropathy (Figure 4D). Although basal GPR40 expression is relatively unaltered in healthy kidneys and in several CKD models, it was up-regulated in models involving direct renal epithelial cell injury [ie, doxorubicin (Figure 4E) and adenine injury models (Figure 4F)]. Furthermore, in situ hybridization showed low-level renal expression of GPR40 and GPR84 in healthy mice, but was considerably up-regulated in adenine-induced nephropathy (Figure 4G).
      Figure thumbnail gr4
      Figure 4GPR40 and GPR84 expression is up-regulated in models of kidney injury. A: In kidney, GPR40 (official name FFAR1) mRNA is expressed in microdissected mouse proximal tubules (PTs) and cortical collecting ducts (CCDs), but is not detected in thick ascending limb (TAL). B–D: GPR84 mRNA is overexpressed in various acute and chronic kidney models: 5/6-nephrectomy (Nx)–induced chronic kidney disease (B), doxorubicin [doxorubicin (DOX)]–induced nephropathy (C), and adenine-induced tubulointerstitial injury (D). E and F: GPR40 (FFAR1) mRNA is overexpressed in epithelial injury models: doxorubicin-induced nephropathy (E) and adenine diet (F). G: GPR40 and GPR84 mRNA in situ hybridization was performed on kidney tissue from normal diet and adenine-fed mice(blue, GPR40; red, GPR84). Data are expressed as means ± SEM (B–F). n = 5 (B, sham, and C, E, and F, control); n = 8 (B, 5/6-Nx, and D, adenine); n = 4 (C and E, doxorubicin, and D, control); n = 6 (F, adenine). P < 0.05, ∗∗P < 0.01 (t-test). Scale bars: 100 μm (G, left column); 50 μm (G, right column). CTRL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

      PBI-4050 Reduces Kidney Fibrosis and Lesions in Renal Disease Rodent Models

      The effect of PBI-4050 on kidney fibrosis and lesions in the aforementioned renal disease models was next studied. In the 5/6-Nx CKD/chronic renal failure model, 5/6-Nx rats showed severe histologic renal lesions (interstitial fibrosis and inflammation, tubular dilation, and glomerulosclerosis), which were reduced by PBI-4050 treatment (Figure 5, A and B ). The glomerular filtration rate on day 190 was significantly reduced (P < 0.01) in 5/6-Nx animals compared with sham-operated controls, and showed an increased trend in PBI-4050–treated rats (5/6-Nx: 0.51 ± 0.22 mL/minute, n = 4; 5/6-Nx + PBI-4050: 1.13 ± 0.44 mL/minute, n = 4; sham: 3.37 ± 0.44 mL/minute, n = 2). Blood pressure was significantly increased in 5/6-Nx rats and showed a reduced trend with PBI-4050 (5/6-Nx: 192.6 ± 4.5 mm Hg, n = 4; 5/6-Nx + PBI-4050: 181.6 ± 2.6, n = 4; sham: 138.3 ± 3.3, n = 2). PBI-4050 did not significantly change the survival of 5/6-Nx animals compared with vehicle control. Furthermore, doxorubicin-induced nephrotoxicity, a mouse model of acute kidney injury, resulted in glomerulosclerosis, intratubular casts, and tubular dilation, which were collectively reduced by PBI-4050 (Figure 5, C and D). Tubulointerstitial fibrosis and cystic lesions were also reduced by PBI-4050 in the adenine-induced mouse model of CKD (Figure 5, E and F). In summary, and in accordance with GPR40 and GPR84 receptor expression data, treatment with PBI-4050 reduced fibrosis and kidney lesions in 5/6 nephrectomy, doxorubicin-induced nephropathy, and adenine-associated nephropathy models.
      Figure thumbnail gr5
      Figure 5PBI-4050 reduces fibrotic lesions in multiple models of kidney injury. A: Masson's trichrome stain of kidney sections from sham-operated, 5/6-nephrectomy (Nx), and PBI-4050–treated 5/6-Nx rats. B: Quantification of glomerulus and tubular injury score (interstitial fibrosis and inflammation, tubular dilation, and glomerulosclerosis). C: Hematoxylin and eosin staining of kidney sections from doxorubicin [doxorubicin (DOX)] mice treated with vehicle or PBI-4050; arrows indicate tubular dilation, and arrowheads indicate intratubular casts. D: Evaluation of histologic lesion (glomerulosclerosis, intratubular casts, and tubular dilation) score. E: Masson's trichrome stain of kidney sections from normal or adenine diet–fed mice treated with vehicle or PBI-4050. F: Evaluation of interstitial fibrosis and cystic lesion score. n = 4 (B, 5/6-Nx); n = 5 (B, 5/6-Nx + PBI-4050); n = 9 (D, doxorubicin); n = 8 (D, doxorubicin + PBI-4050); n = 6 per group (F). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (t-test). Scale bars: 400 μm (A, top row); 100 μm (A, bottom row, and E); 200 μm (C, top row); 50 μm (C, bottom row).

      A Protective Role for GPR40 and a Deleterious Role for GPR84 in Renal Fibrosis

      To elucidate the role of GPR40 in renal fibrosis, UUO, long-term post-acute ischemic injury, and adenine-induced nephropathy models were used in WT and Gpr40−/− mice. Fibrosis in these models was quantitated using picrosirius red (UUO, ischemic injury) or Masson's trichrome (adenine) histochemistry. Histologic examination of the kidneys revealed that Gpr40−/− mice displayed increased fibrosis after injury in all three models. Indeed, 4 days after ureteral ligation, Gpr40−/− mice showed a 2.8-fold increase in interstitial fibrosis compared with WT mice, for which only moderate collagen deposition was observed (Figure 6, A and B ). Likewise, a 4.2-fold increase of fibrosis in Gpr40−/− mice was observed as a long-term (21-day) response to severe acute renal ischemic injury (Figure 6, C and D). Finally, adenine-induced nephropathy also resulted in increased fibrosis (but unchanged cystic lesions) in Gpr40−/− mice compared with WT mice (Figure 6, E and F). Collectively, these studies are indicative of a protective role in renal fibrosis for GPR40.
      Figure thumbnail gr6
      Figure 6Gpr40 deletion increases renal fibrosis in unilateral ureteral obstruction (UUO), ischemia-reperfusion injury, and adenine-induced nephropathy models. A and C: Picrosirius red staining (collagen deposition) of kidney cortex (top row) and medulla (bottom row) sections of wild-type (WT) and Gpr40−/− mice subjected to UUO for 4 days (A) or to long-term post-acute ischemia-reperfusion injury (C). B and D: Digital image analysis of interstitial fibrosis area. E: Kidney sections of adenine-fed WT and Gpr40−/− mice stained with Masson's trichrome. F: Evaluation of interstitial fibrosis and cystic lesion score. n = 4 per group (B and D); n = 6 (F, WT); n = 8 (F, Gpr40−/−). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (t-test). Scale bars: 100 μm (A, C, and E, top row); 50 μm (Ebottom row).
      To validate the role of GPR84 in fibrosis, the adenine-induced CKD mouse model was used. Histologic examination of the kidneys revealed that Gpr84−/− mice showed significantly reduced (by half) adenine-induced interstitial fibrosis and cyst formation (Figure 7), uncovering a detrimental role of GPR84 in renal nephropathy.
      Figure thumbnail gr7
      Figure 7GPR84 contributes to kidney fibrosis and cystic lesions in adenine-induced nephropathy. A: Kidney sections of adenine-fed wild-type (WT) and Gpr84−/− mice stained with Masson's trichrome; arrows indicate cystic lesions. B: Evaluation of interstitial fibrosis and cystic lesion score. n = 4 per group (B). P < 0.05, ∗∗∗P < 0.001 (t-test). Scale bars: 100 μm (A, top row); 50 μm (A, bottom row).
      To further establish the link between the antifibrotic effects of PBI-4050 and the role of GPR40 and GPR84 in fibrosis, adenine-fed Gpr40−/− and Gpr84−/− mice were treated with PBI-4050 or vehicle. Treatment of Gpr40−/− mice with PBI-4050 resulted in only a slight decrease (13%) of fibrosis compared with vehicle, whereas in Gpr84−/− mice PBI-4050 significantly reduced fibrosis (by 36%) (Figure 8). Interestingly, the combined reduction of fibrosis induced by PBI-4050 in both knockouts was equivalent to the 50% reduction of fibrosis induced by PBI-4050 in the WT adenine-fed mice (Figure 5F). In contrast, cystic lesions were not affected by PBI-4050 treatment in the Gpr84−/− mice, but trended to be lower in the Gpr40−/− mice compared with vehicle. These results suggest an effect of PBI-4050 treatment predominantly via GPR40 for tubulointerstitial fibrosis reduction and mostly through GPR84 for decreased cystic lesions in the adenine-induced CKD model.
      Figure thumbnail gr8
      Figure 8PBI-4050 treatment of Gpr40−/− and Gpr84−/− mice in adenine-induced nephropathy. A and C: Kidney sections of adenine-fed wild-type and Gpr40−/− (A) or Gpr84−/− (C) mice stained with Masson's trichrome. B and D: Evaluation of interstitial fibrosis and cystic lesions score. n = 6 per group (B and D). P < 0.05 (t-test). Scale bars = 100 μm (A and C).

      Effect of PBI-4050 on Multiple Organ Tissue Fibrosis

      To evaluate the capacity of PBI-4050 to attenuate tissue fibrosis in other organs, multiple animal models were used. PBI-4050 significantly reduced liver fibrosis induced by carbon tetrachloride hepatotoxicity (31% reduction) (Figure 9, A and B ). In addition, the level of aspartate aminotransferase (a marker of liver damage) was increased by carbon tetrachloride and significantly reduced by PBI-4050 (results not shown; B.G., unpublished data). PBI-4050 treatment also resulted in reduced interstitial fibrosis in the rat heart after suprarenal aorta constriction (almost complete abrogation of fibrosis) (Figure 9, C and D), bleomycin-induced pulmonary fibrosis (47% reduction of histologic lesions) (Figure 9, E and F), skin fibrosis in the fibrillin 1–mutant mouse model of systemic scleroderma (reduction of skin weight by 40%) (Figure 9, G and H), and pancreas fibrosis that develops in the db/db eNOS−/− type II diabetic nephropathy mouse model (no fibrosis detected in PBI-4050–treated mice) (Figure 9, I and J).
      Figure thumbnail gr9
      Figure 9PBI-4050 treatment is protective in multiple organ fibrosis models. A: Masson's trichrome staining of liver sections of mice that received administration of olive oil (sham), carbon tetrachloride, or carbon tetrachloride and PBI-4050. B: Quantitation of interstitial fibrosis area. C: Masson's trichrome staining of rat heart sections that underwent suprarenal abdominal aorta constriction (SAC), treated with vehicle or PBI-4050, and of sham-operated rats. D: Quantitation of interstitial fibrosis area. E: Masson's trichrome staining of lung sections of control, bleomycin (bleo)-instilled, and PBI-4050–treated bleomycin-instilled mice. F: Scoring of histologic lesions (disrupted lung architecture, alveolar shrinking, and wall thickening). G: Picrosirius red staining of skin sections from wild-type (WT) and fibrillin 1–mutant mice (FbnTsk) treated with vehicle or PBI-4050. H: Quantitation of skin weight. I: Picrosirius red staining of pancreas sections from db/db eNOS−/− mice treated with vehicle or PBI-4050. Arrows show fibrotic areas. J: Quantitation of islet fibrosis area. n = 10 (B, D, SAC, and H, PBI-4050); n = 6 (D, SAC + PBI-4050 and sham); n = 8 (F, bleomycin); n = 4 (F, bleomycin + PBI-4050, and J); n = 5 (H, WT); n = 17 (H, vehicle). ∗∗P < 0.01, ∗∗∗P < 0.001 (t-test or one-way analysis of variance, followed by Dunnett's multiple comparisons test). Scale bars: 100 μm (A and I); 200 μm (E). Original magnification: ×40 (C); ×25 (G).

      Discussion

      The data presented herein clearly demonstrate the contribution of two fatty acid receptors, GPR40 and GPR84, in the regulation of cells involved in fibrosis. Although GPR40 is minimally detectable in immune cells, with the exception of monocytes,
      • Briscoe C.P.
      • Tadayyon M.
      • Andrews J.L.
      • Benson W.G.
      • Chambers J.K.
      • Eilert M.M.
      • Ellis C.
      • Elshourbagy N.A.
      • Goetz A.S.
      • Minnick D.T.
      • Murdock P.R.
      • Sauls Jr., H.R.
      • Shabon U.
      • Spinage L.D.
      • Strum J.C.
      • Szekeres P.G.
      • Tan K.B.
      • Way J.M.
      • Ignar D.M.
      • Wilson S.
      • Muir A.I.
      The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids.
      GPR84 is abundantly found in bone marrow, T cells, B cells, granulocytes, microglia, neutrophils, and monocytes/macrophages,
      • Yonezawa T.
      • Kurata R.
      • Yoshida K.
      • Murayama M.A.
      • Cui X.
      • Hasegawa A.
      Free fatty acids-sensing G protein-coupled receptors in drug targeting and therapeutics.
      • Nicol L.S.
      • Dawes J.M.
      • La Russa F.
      • Didangelos A.
      • Clark A.K.
      • Gentry C.
      • Grist J.
      • Davies J.B.
      • Malcangio M.
      • McMahon S.B.
      The role of G-protein receptor 84 in experimental neuropathic pain.
      • Bouchard C.
      • Page J.
      • Bedard A.
      • Tremblay P.
      • Vallieres L.
      G protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditions.
      and has been shown to work as a proinflammatory mediator.
      • Suzuki M.
      • Takaishi S.
      • Nagasaki M.
      • Onozawa Y.
      • Iino I.
      • Maeda H.
      • Komai T.
      • Oda T.
      Medium-chain fatty acid-sensing receptor, GPR84, is a proinflammatory receptor.
      Herein, we report that GPR40 is also expressed in human and mouse epithelial cells (proximal tubule and cortical collecting duct), and GPR84 is expressed in human fibroblasts/myofibroblasts and human podocytes. To date, there have only been limited reports indicating that GPR40 is expressed and modulated in kidney,
      • Ma S.K.
      • Joo S.Y.
      • Choi H.I.
      • Bae E.H.
      • Nam K.I.
      • Lee J.
      • Kim S.W.
      Activation of G-protein-coupled receptor 40 attenuates the cisplatin-induced apoptosis of human renal proximal tubule epithelial cells.
      • Ma S.K.
      • Wang Y.
      • Chen J.
      • Zhang M.Z.
      • Harris R.C.
      • Chen J.K.
      Overexpression of G-protein-coupled receptor 40 enhances the mitogenic response to epoxyeicosatrienoic acids.
      and to our knowledge, there is no report that GPR84 is modulated in kidney diseases. Our data confirm the overexpression of GPR40 and GPR84 in different chronic and acute kidney injury models. Interestingly, although GPR84 is weakly expressed under normal conditions in renal tissue, the data indicate that it is overexpressed in various kidney disease models, whereas GPR40 is overexpressed after epithelial injury. Also, LPS-treated podocytes and macrophages, as well as TGF-β–treated fibroblasts and podocytes, show significant GPR84 up-regulation.
      Our data suggest that through the binding to GPR40 and/or GPR84, PBI-4050 reduces fibrosis via the regulation of macrophages, fibroblasts/myofibroblasts, and epithelial cells. PBI-4050 inhibits activation of fibroblasts to profibrotic myofibroblasts, as demonstrated by abrogation of α-SMA expression in fibroblasts, and subsequent accumulation of extracellular matrix protein deposition and fibrosis. Furthermore, PBI-4050 reduced macrophage activation and the expression of proinflammatory markers (monocyte chemoattractant protein-1, IL-8, and IL-6) and profibrotic markers (connective tissue growth factor and IL-6). PBI-4050 treatment decreases F4/80 immunostaining (marker of macrophages) in the kidneys and pancreas islets of db/db eNOS−/− mice (M.-Z.Z. and R.C.H., unpublished data). Therefore, the protective actions of PBI-4050 via GPR40 and GPR84 may reside in modulating such infiltrating cells, and in reducing proliferation/activation of myofibroblasts and epithelial-mesenchymal transition in the injured tissue. Ma et al
      • Ma S.K.
      • Joo S.Y.
      • Choi H.I.
      • Bae E.H.
      • Nam K.I.
      • Lee J.
      • Kim S.W.
      Activation of G-protein-coupled receptor 40 attenuates the cisplatin-induced apoptosis of human renal proximal tubule epithelial cells.
      have shown that pretreatment of human renal proximal tubule epithelial (HK-2) cells with the GPR40 agonist GW9508 attenuated cisplatin-induced apoptosis, an effect that could also contribute to the protective action of PBI-4050 in our renal injury models.
      PBI-4050 is an agonist of GPR40 and acts as an antagonist or inverse agonist of GPR84. It cannot be excluded that other targets besides GPR40 and GPR84 could be implicated in the mechanism of action of PBI-4050 and could be explored in future studies. However, the present study, and in particular the receptor KO models, strongly supports GPR40 and GPR84 as major mediators in pathologic fibrotic pathways and as the targets of the antifibrotic effects of PBI-4050. Our data show that PBI-4050 significantly attenuated fibrosis in a variety of injury contexts, as evidenced with the antifibrotic activity observed in kidney, liver, lung, heart, pancreas, and skin fibrosis models. Given our findings with both Gpr40 and Gpr84 KO mice, both receptors appear to be involved in the fibrotic pathways. Considering their expression along the nephron, within the glomerulus, and in numerous bone marrow–derived cell types, it is likely that GPR40 and GPR84 modulate profibrotic, inflammatory, and epithelial-mesenchymal transition processes. Therefore, GPR40 may partially protect against development of fibrosis, whereas GPR84 may induce the promotion and stimulation of fibrosis, as observed with the significant increase in fibrosis in Gpr40−/− mice and reduction of fibrosis in Gpr84−/− mice. The dual modulator PBI-4050 reinforces the involvement of both GPR40 and GPR84 in multiple models of fibrosis. In the context of the adenine-induced CKD model, treatment of Gpr40 and Gpr84 KO mice with PBI-4050 suggests its antifibrosis effects to be mainly mediated by GPR40 activation, whereas inhibition of GPR84 mostly accounts for reduction of cystic lesions. The relative role of each receptor may vary depending on the type of insult, pathology, and organ. Future work will aim to elucidate the precise intracellular signaling pathways used by both GPR40 and GPR84 to regulate fibrotic events in the pathogenesis of disease.
      The inhibition of fibrotic and inflammatory markers by PBI-4050, found in human proximal tubule epithelial cells, podocytes, and primary fibroblasts (Figure 3), suggests that the attenuation of fibrosis that we have shown in various rodent fibrosis models could translate to human disease. Moreover, significant clinical activity observed in recent phase 2 clinical trials in type 2 diabetes subjects with metabolic syndrome

      Laurin P, Grouix B, Laverdure A, Zacharie B, Gagnon L: PBI-4050 reduces cardiovascular and renal biomarkers in type II diabetic patients with metabolic syndrome. Chicago, IL: American Society of Nephrology Kidney Week 2016, Poster #FR-PO829

      and in idiopathic pulmonary fibrosis patients

      Parker J, Sawtell R, Gagnon L, Hagerimana A, Laurin P, Kolb M, Cantin A, Moran J: PBI-4050 is safe and well tolerated and shows evidence of benefit in idiopathic pulmonary fibrosis [abstract #A7606]. May 19–24, 2017, Washington, DC: American Thoracic Society International Conference 2017

      confirm translation of pharmacologic activity of PBI-4050 in humans. Of interest, PBI-4050 was well tolerated and demonstrated a good safety profile in these two early-phase clinical trials.
      Taken together, our data show the involvement and validation at a mechanistic level of GPR40 and GPR84 as bona fide modulators of fibrotic disease progression. This suggests that drugs, such as PBI-4050, acting on these therapeutic targets may delay and/or prevent fibrotic diseases progression and organ failure in human. Its unique novel mechanism of action and effectiveness in various preclinical models support the development of PBI-4050 for the treatment of fibrosis-inducing conditions.

      Acknowledgments

      We thank Dr. Stephen Wank for Gpr40−/− mice.
      L.Ga., B.G., M.L., J.-F.T., and P.L. conceived and designed the project; L.Ga. and M.L. wrote the manuscript; J.-F.T., M.L., F.S.-B., W.G., L.Ge., LGer., K.H., M.T., J.O., J.R., A.F., A.L., S.L., M.-P.C., F.A.L., and J.-C.S. performed and analyzed all experiments, except for those indicated below; S.D.A., C.P., J.-S.D., and B.Z. designed and synthesized PBI-4050; M.-Z.Z. and R.C.H. provided the Gpr40−/−, B6.Cg-Fbn1Tsk/J, and db/db eNOS−/− mice, and designed, performed, and analyzed the experiments with these mice; the Gpr40−/− and Gpr84−/− adenine model experiments and the microdissected mouse tubule preparations were done in the laboratories of C.R.J.K. and R.L.H.; C.E.H. and E.K. provided technical assistance with the cultured podocytes and adenine mice experiments; J.D., A.C., and Q.T.N. designed, performed, and analyzed the suprarenal abdominal aorta constriction experiments; A.G. performed histologic assessments.

      Supplemental Data

      • Supplemental Figure S1

        PBI-4050 does not activate GPR40-mediated Gα13 or Gαs signaling. A:13 pathway activation was monitored in living human embryonic kidney 293 cells transfected with GPR40 and the Gα13 activation biosensor. Cells were exposed to sodium decanoate or PBI-4050, and bioluminescence resonance energy transfer (BRET) variation compared with vehicle was measured. B: cAMP production was determined in CHO-K1 cells (ATCC) stably transfected with 3xHA-human GPR40 treated for 30 minutes at 37°C with the indicated compounds, using the HitHunter cAMP assay for small molecules (DiscoveRx, Fremont, CA). Forskolin (Cayman Chemical, Ann Arbor, MI) treatment served as a positive control. Data are expressed as means ± SEM. n = 2 to 5 experiments (A); n = 2 experiments (B). GFP, green fluorescent protein; RLU, relative light units.

      • Supplemental Figure S2

        PBI-4050 does not activate GPR84-mediated Gαq, Gα13, or Gαs signaling. A and B: Cells transfected with GPR84 and Gαq (A) or Gα13 (B) activation biosensor were exposed to sodium decanoate, embelin, or PBI-4050, and bioluminescence resonance energy transfer (BRET) variation compared with vehicle was measured. C: cAMP production was determined in CHO-K1 cells stably transfected with 3xHA-human GPR84 treated for 30 minutes at 37°C with the indicated compounds, using the HitHunter cAMP assay for small molecules. Forskolin treatment served as a positive control. Data are expressed as means ± SEM. n = 3 to 6 experiments (A and B); n = 2 experiments (C). GFP, green fluorescent protein; RLU, relative light units.

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      1. Laurin P, Grouix B, Laverdure A, Zacharie B, Gagnon L: PBI-4050 reduces cardiovascular and renal biomarkers in type II diabetic patients with metabolic syndrome. Chicago, IL: American Society of Nephrology Kidney Week 2016, Poster #FR-PO829

      2. Parker J, Sawtell R, Gagnon L, Hagerimana A, Laurin P, Kolb M, Cantin A, Moran J: PBI-4050 is safe and well tolerated and shows evidence of benefit in idiopathic pulmonary fibrosis [abstract #A7606]. May 19–24, 2017, Washington, DC: American Thoracic Society International Conference 2017