Inhibition of transforming growth factor β1 signaling in resident interstitial cells attenuates profibrotic gene expression and preserves erythropoietin production during experimental kidney fibrosis in mice

Kidney fibrosis is characterized by the development of myofibroblasts originating from resident kidney and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various cytokines. Among these, transforming growth factor β1 (TGFβ1) is considered a central trigger for kidney fibrosis. We found a highly upregulated expression of TGFβ1 and TGFβ receptor 2 (TGFβ-R2) mRNAs in kidney interstitial cells in experimental fibrosis. Here, we investigated the contribution of TGFβ1 signaling in resident kidney interstitial cells to organ fibrosis using the models of adenine induced nephropathy and unilateral ureteral occlusion in mice. For this purpose TGFβ1 signaling was interrupted by inducible deletion of the TGFβ-R2 gene in interstitial cells expressing the fibroblast marker platelet derived growth factor receptor-β. Expression of profibrotic genes was attenuated up to 50% in kidneys lacking TGFβ-R2 in cells positive for platelet derived growth factor receptor-β. Additionally, deletion of TGFβ-R2 prevented the decline of erythropoietin production in ureter ligated kidneys. Notably, fibrosis associated expression of α-smooth muscle actin as a myofibroblast marker and deposits of extracellular collagens were not altered in mice with targeted deletion of TGFβ-R2. Thus, our findings suggest an enhancing effect of TGFβ1 signaling in resident interstitial cells that contributes to profibrotic gene expression and the downregulation of erythropoietin production, but not to the development of myofibroblasts during kidney fibrosis. Kidney fibrosis is characterized by the development of myofibroblasts originating from resident kidney and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various cytokines. Among these, transforming growth factor β1 (TGFβ1) is considered a central trigger for kidney fibrosis. We found a highly upregulated expression of TGFβ1 and TGFβ receptor 2 (TGFβ-R2) mRNAs in kidney interstitial cells in experimental fibrosis. Here, we investigated the contribution of TGFβ1 signaling in resident kidney interstitial cells to organ fibrosis using the models of adenine induced nephropathy and unilateral ureteral occlusion in mice. For this purpose TGFβ1 signaling was interrupted by inducible deletion of the TGFβ-R2 gene in interstitial cells expressing the fibroblast marker platelet derived growth factor receptor-β. Expression of profibrotic genes was attenuated up to 50% in kidneys lacking TGFβ-R2 in cells positive for platelet derived growth factor receptor-β. Additionally, deletion of TGFβ-R2 prevented the decline of erythropoietin production in ureter ligated kidneys. Notably, fibrosis associated expression of α-smooth muscle actin as a myofibroblast marker and deposits of extracellular collagens were not altered in mice with targeted deletion of TGFβ-R2. Thus, our findings suggest an enhancing effect of TGFβ1 signaling in resident interstitial cells that contributes to profibrotic gene expression and the downregulation of erythropoietin production, but not to the development of myofibroblasts during kidney fibrosis. Translational StatementResident renal interstitial platelet-derived growth factor receptor β–positive cells are capable of producing erythropoietin and are key cells for the development of fibrosis in states of kidney disease. We found that interruption of transforming growth factor β1 signaling in platelet-derived growth factor receptor β–positive cells attenuates profibrotic gene expression and prevents the downregulation of erythropoietin expression in mouse models of kidney fibrosis. These findings suggest a crucial role of transforming growth factor β1 signaling in insufficient erythropoietin production in fibrotic kidneys independent of myofibroblast formation. Better understanding of the mechanisms suppressing renal erythropoietin production could lead to novel targeted therapeutic approaches in treating anemia of patients with chronic kidney failure. Resident renal interstitial platelet-derived growth factor receptor β–positive cells are capable of producing erythropoietin and are key cells for the development of fibrosis in states of kidney disease. We found that interruption of transforming growth factor β1 signaling in platelet-derived growth factor receptor β–positive cells attenuates profibrotic gene expression and prevents the downregulation of erythropoietin expression in mouse models of kidney fibrosis. These findings suggest a crucial role of transforming growth factor β1 signaling in insufficient erythropoietin production in fibrotic kidneys independent of myofibroblast formation. Better understanding of the mechanisms suppressing renal erythropoietin production could lead to novel targeted therapeutic approaches in treating anemia of patients with chronic kidney failure. Development and progression of kidney fibrosis is a hallmark of chronic kidney disease. It is commonly thought of as the result of an increased production and deposition of extracellular matrix (ECM) proteins such as collagens, fibronectin, or tenascins.1Schrimpf C. Duffield J.S. Mechanisms of fibrosis: the role of the pericyte.Curr Opin Nephrol Hypertens. 2011; 20: 297-305Crossref PubMed Scopus (121) Google Scholar, 2Neelisetty S. Alford C. 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This may also explain some of the controversial reports in recent years.11Yang Q. Ren G. Wei B. et al.Conditional knockout of TGF-βRII /Smad2 signals protects against acute renal injury by alleviating cell necroptosis, apoptosis and inflammation.Theranostics. 2019; 9: 8277-8293Crossref PubMed Scopus (36) Google Scholar,14Nlandu-Khodo S. Neelisetty S. Phillips M. et al.Blocking TGF-β and β-catenin epithelial crosstalk exacerbates CKD.J Am Soc Nephrol. 2017; 28: 3490-3503Crossref PubMed Scopus (30) Google Scholar,25Gewin L. Bulus N. Mernaugh G. et al.TGF-β receptor deletion in the renal collecting system exacerbates fibrosis.J Am Soc Nephrol. 2010; 21: 1334-1343Crossref PubMed Scopus (37) Google Scholar TGFβ1 has also been reported to promote kidney fibrosis by enhancing the infiltration of macrophages, which can transdifferentiate into myofibroblasts.11Yang Q. Ren G. 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Ehinger M. et al.Induced disruption of the transforming growth factor beta type II receptor gene in mice causes a lethal inflammatory disorder that is transplantable.Blood. 2002; 100: 560-568Crossref PubMed Scopus (162) Google Scholar For lineage tracing, a double fluorescent reporter construct mT/mG (B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J; JAX stock #007676) was bred into TGFβ-R2ko mice.48Muzumdar M.D. Tasic B. Miyamichi K. et al.A global double-fluorescent Cre reporter mouse.Genesis. 2007; 45: 593-605Crossref PubMed Scopus (1993) Google Scholar Genotyping was performed using the primers listed in Table 1. Littermates negative for Cre were used as control animals. Animals were maintained on standard rodent chow (0.6% NaCl; Ssniff) with free access to tap water. All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and approved by the local ethics committee.Table 1Primer sequences used for genotyping of miceGenotypeSequence (5′ to 3′), fwdSequence (5′ to 3′), revPDGFR-βCreERT2/+gaactgtcaccggcaggaaggcaaattttggtgtacggTGFβ-R2 floxgcaggcatcaggacccagtttgatccagagtgaagccgtggtaggtgagcttgTGFβ-R2 deltaaacaaggtccggagcccaagagtgaagccgtggtaggtgagcttgmT/mGctctgctgcctcctggcttctcgaggcggatcacaagcaataccaatgggcgggggtcgttdel, deletion; fwd, forward; PDGFR-β, platelet-derived growth factor receptor β; rev, reverse; TGFβ-R2, transforming growth factor β receptor 2. Open table in a new tab del, deletion; fwd, forward; PDGFR-β, platelet-derived growth factor receptor β; rev, reverse; TGFβ-R2, transforming growth factor β receptor 2. Cre recombination was induced by feeding a diet containing 400 mg of tamoxifen citrate per kilogram (A115T00404, Ssniff) for 4 weeks followed by a 3-week period with standard chow. Excision of the floxed TGFβ-R2 gene was confirmed by polymerase chain reaction.49Chytil A. Magnuson M.A. Wright C.V.E. Moses H.L. Conditional inactivation of the TGF-β type II receptor using Cre:Lox.Genesis. 2002; 32: 73-75Crossref PubMed Scopus (211) Google Scholar,50Boneva S.K. Characterization of the neuroprotective function of the TGF-β signaling pathway in the retina [e-pub ahead of print]. Thesis of the University of Regensburg (PhD).https://doi.org/10.5283/epub.35581Google Scholar Adenine-induced fibrosis was generated in adult mice.29Buchtler S. Grill A. Hofmarksrichter S. et al.Cellular origin and functional relevance of collagen I production in the kidney.J Am Soc Nephrol. 2018; 29: 1859-1873Crossref PubMed Scopus (40) Google Scholar,33Klinkhammer B.M. Djudjaj S. Kunter U. et al.Cellular and molecular mechanisms of kidney injury in 2,8-dihydroxyadenine nephropathy.J Am Soc Nephrol. 2020; 31: 799-816Crossref PubMed Scopus (17) Google Scholar,44Rahman A. Yamazaki D. Sufiun A. et al.A novel approach to adenine-induced chronic kidney disease associated anemia in rodents.PLoS One. 2018; 13e0192531Crossref PubMed Scopus (28) Google Scholar,51Jia T. Olauson H. Lindberg K. et al.A novel model of adenine-induced tubulointerstitial nephropathy in mice.BMC Nephrol. 2013; 14: 116Crossref PubMed Scopus (90) Google Scholar After tamoxifen treatment and the following rest period, male mice were fed an adenine-containing diet (0.2%) continuously for 3 weeks. Experiments were performed after exactly 3 weeks (3-week adenine). Under inhalation anesthesia, ureteral ligation was performed close to the right kidney through a small abdominal incision.42Chevalier R.L. Forbes