Kidney pericyte hypoxia-inducible factor regulates erythropoiesis but not kidney fibrosis
Authors: Szu-Yu Pan, Pei-Zhen Tsai, Yu-Hsiang Chou, Yu-Ting Chang, Fan-Chi Chang, Yen-Ling Chiu, Wen-Chih Chiang, Tien Hsu, Yung-Ming Chen, Tzong-Shinn Chu, Shuei-Liong Lin
Journal: Kidney International (2021)

Prolyl hydroxylase domain enzyme (PHD) inhibitors are effective in the treatment of chronic kidney disease (CKD)-associated anemia by stabilizing hypoxia inducible factor (HIF), thereby increasing erythropoietin and consequently erythropoiesis. However, concern for CKD progression needs to be addressed in clinical trials. Although pre-clinical studies showed an anti-inflammatory effect in kidney disease models, the effect of PHD inhibitors on kidney fibrosis was inconsistent probably because the effects of HIF are cell type and context dependent. The major kidney erythropoietin-producing cells are pericytes that produce erythropoietin through HIF-2α-dependent gene transcription. The concern for the impact of HIF in pericytes on kidney fibrosis arises from the fact that pericytes are the major precursor cells of myofibroblasts in CKD. Since cells expressing Gli1 fulfill the morphologic and anatomic criteria for pericytes, we induced Gli1+ cell-specific HIF stabilization or knockout to study the impact of HIF in pericytes on kidney pathology of mice with or without fibrotic injury induced by unilateral ureteral obstruction. Compared with the littermate controls, mice with pericyte-specific HIF stabilization due to von Hippel-Lindau protein or PHD2 knockout showed increased serum erythropoietin and polycythemia rather than a discernible difference in kidney fibrosis. Compared with Gli1+ pericytes sorted from littermate controls, Gli1+ pericytes sorted from PHD2 knockout mice showed increased erythropoietin gene expression rather than discernible changes in Col1a1 or Acta2 expression. Furthermore, pericyte-specific knockout of HIF-1α or HIF-2α did not affect kidney fibrosis. Thus, our study supports the absence of negative effects of PHD inhibitors on kidney fibrosis of mice despite HIF stabilization in pericytes. Prolyl hydroxylase domain enzyme (PHD) inhibitors are effective in the treatment of chronic kidney disease (CKD)-associated anemia by stabilizing hypoxia inducible factor (HIF), thereby increasing erythropoietin and consequently erythropoiesis. However, concern for CKD progression needs to be addressed in clinical trials. Although pre-clinical studies showed an anti-inflammatory effect in kidney disease models, the effect of PHD inhibitors on kidney fibrosis was inconsistent probably because the effects of HIF are cell type and context dependent. The major kidney erythropoietin-producing cells are pericytes that produce erythropoietin through HIF-2α-dependent gene transcription. The concern for the impact of HIF in pericytes on kidney fibrosis arises from the fact that pericytes are the major precursor cells of myofibroblasts in CKD. Since cells expressing Gli1 fulfill the morphologic and anatomic criteria for pericytes, we induced Gli1+ cell-specific HIF stabilization or knockout to study the impact of HIF in pericytes on kidney pathology of mice with or without fibrotic injury induced by unilateral ureteral obstruction. Compared with the littermate controls, mice with pericyte-specific HIF stabilization due to von Hippel-Lindau protein or PHD2 knockout showed increased serum erythropoietin and polycythemia rather than a discernible difference in kidney fibrosis. Compared with Gli1+ pericytes sorted from littermate controls, Gli1+ pericytes sorted from PHD2 knockout mice showed increased erythropoietin gene expression rather than discernible changes in Col1a1 or Acta2 expression. Furthermore, pericyte-specific knockout of HIF-1α or HIF-2α did not affect kidney fibrosis. Thus, our study supports the absence of negative effects of PHD inhibitors on kidney fibrosis of mice despite HIF stabilization in pericytes. Translational StatementProlyl hydroxylase domain enzyme (PHD) inhibitors are effective in the treatment of anemia in chronic kidney disease (CKD) through stabilizing hypoxia-inducible factor (HIF), thereby increasing erythropoietin (EPO) and erythropoiesis. Renal pericytes produce EPO through HIF-2α in normal physiology, but they differentiate to scar-producing myofibroblasts in CKD. It is not clear whether HIF expression in renal pericytes impacts renal fibrosis. By preclinical murine models of HIF manipulation and renal fibrosis, we show that Gli1+ pericyte-specific HIF manipulation affects EPO production and erythropoiesis but not renal fibrosis. Our study endorses the absence of negative effect of PHD inhibitors on renal fibrosis. Prolyl hydroxylase domain enzyme (PHD) inhibitors are effective in the treatment of anemia in chronic kidney disease (CKD) through stabilizing hypoxia-inducible factor (HIF), thereby increasing erythropoietin (EPO) and erythropoiesis. Renal pericytes produce EPO through HIF-2α in normal physiology, but they differentiate to scar-producing myofibroblasts in CKD. It is not clear whether HIF expression in renal pericytes impacts renal fibrosis. By preclinical murine models of HIF manipulation and renal fibrosis, we show that Gli1+ pericyte-specific HIF manipulation affects EPO production and erythropoiesis but not renal fibrosis. Our study endorses the absence of negative effect of PHD inhibitors on renal fibrosis. The mainstay of treatment for anemia in patients with chronic kidney disease (CKD) are erythropoiesis-stimulating agents and iron supplement.1Kidney Disease: Improving Global Outcomes (KDIGO) Anemia Work GroupKDIGO clinical practice guideline for anemia in chronic kidney disease.Kidney Int Suppl. 2012; 2: 279-335Abstract Full Text Full Text PDF Scopus (594) Google Scholar,2Astor B.C. Muntner P. Levin A. et al.Association of kidney function with anemia: the Third National Health and Nutrition Examination Survey (1988-1994).Arch Intern Med. 2002; 162: 1401-1408Crossref PubMed Scopus (560) Google Scholar Recently, the prolyl hydroxylase domain enzyme (PHD) inhibitor emerges as a promising therapeutic agent for the CKD-associated anemia principally through stabilizing the hypoxia-inducible factor (HIF) and thereby increasing erythropoietin (EPO) production.3Maxwell P.H. Eckardt K.U. HIF prolyl hydroxylase inhibitors for the treatment of renal anaemia and beyond.Nat Rev Nephrol. 2016; 12: 157-168Crossref PubMed Scopus (154) Google Scholar, 4Chen N. Hao C. Peng X. et al.Roxadustat for anemia in patients with kidney disease not receiving dialysis.N Engl J Med. 2019; 381: 1001-1010Crossref PubMed Scopus (206) Google Scholar, 5Chen N. Hao C. Liu B.C. et al.Roxadustat treatment for anemia in patients undergoing long-term dialysis.N Engl J Med. 2019; 381: 1011-1022Crossref PubMed Scopus (208) Google Scholar The clinical application of PHD inhibitors or HIF stabilizers in patients with CKD is imminent in the foreseeable future if the concerns regarding possible angiogenesis, tumor growth, abnormal glucose metabolism, and accelerated decline of kidney function are to be addressed in the clinical trials. HIF is the most important known cellular oxygen–sensing machinery.6Schodel J. Ratcliffe P.J. Mechanisms of hypoxia signalling: new implications for nephrology.Nat Rev Nephrol. 2019; 15: 641-659Crossref PubMed Scopus (86) Google Scholar,7Pan S.Y. Chiang W.C. Chen Y.M. The journey from erythropoietin to 2019 Nobel Prize: focus on hypoxia-inducible factors in the kidney.J Formos Med Assoc. 2021; 120: 60-67Crossref PubMed Scopus (4) Google Scholar The functional HIF consists of α and β subunits. HIF-α is an oxygen labile subunit that is rapidly degraded at a normoxic condition in the presence of cofactors. HIF-β is not regulated by oxygen tension. The degradation of HIF-α is preceded by proline hydroxylation and polyubiquitination. The proline hydroxylation is catalyzed by PHD in the presence of oxygen, iron, and 2-oxoglutarate as cofactors. The polyubiquitination is then catalyzed by an E3 ubiquitin ligase composed of von Hippel-Lindau protein (pVHL)-Elongin BC-CUL2 complex. The requirement of iron, PHD, and pVHL for HIF degradation makes it possible to stabilize HIF in the normoxic condition with inhibition or decrease of any of these key components. Renal pericytes are embedded within microvascular basement membrane and in close contact with endothelial cells to support microvasculature and regulate blood flow.8Lin S.L. Kisseleva T. 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Pan S.Y. et al.DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys.J Clin Invest. 2016; 126: 721-731Crossref PubMed Scopus (49) Google Scholar,17Souma T. Nezu M. Nakano D. et al.Erythropoietin synthesis in renal myofibroblasts is restored by activation of hypoxia signaling.J Am Soc Nephrol. 2016; 27: 428-438Crossref PubMed Scopus (96) Google Scholar Our studies have shown that renal fibrosis can be attenuated by interruption of pericyte-myofibroblast transition through pharmacologic blockade of transforming growth factor-β,19Wu C.F. Chiang W.C. Lai C.F. et al.Transforming growth factor beta-1 stimulates profibrotic epithelial signaling to activate pericyte-myofibroblast transition in obstructive kidney fibrosis.Am J Pathol. 2013; 182: 118-131Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar platelet-derived growth factor,20Chen Y.T. Chang F.C. 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Kida Y. et al.LRP-6 is a coreceptor for multiple fibrogenic signaling pathways in pericytes and myofibroblasts that are inhibited by DKK-1.Proc Natl Acad Sci U S A. 2013; 110: 1440-1445Crossref PubMed Scopus (137) Google Scholar Regarding the concern for accelerated decline of kidney function in patients under the treatment with PHD inhibitors, little is known about the effects of HIF activity in pericytes or myofibroblasts on renal fibrosis.23Kawakami T. Mimura I. Shoji K. et al.Hypoxia and fibrosis in chronic kidney disease: crossing at pericytes.Kidney Int Suppl. 2014; 4: 107-112Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar In contrast, the effects of HIF overexpression or knockout on renal fibrosis have been extensively studied, either nonselectively24Kapitsinou P.P. Jaffe J. 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Li C. et al.Loss of the tumor suppressor Vhlh leads to upregulation of Cxcr4 and rapidly progressive glomerulonephritis in mice.Nat Med. 2006; 12: 1081-1087Crossref PubMed Scopus (166) Google Scholar,32Ding M. Coward R.J. Jeansson M. et al.Regulation of hypoxia-inducible factor 2-a is essential for integrity of the glomerular barrier.Am J Physiol Renal Physiol. 2013; 304: F120-F126Crossref PubMed Scopus (10) Google Scholar However, the results were inconsistent probably because the effects of HIF are cell type and context dependent. In vitro studies using isolated human renal fibroblasts33Norman J.T. Clark I.M. Garcia P.L. Hypoxia promotes fibrogenesis in human renal fibroblasts.Kidney Int. 2000; 58: 2351-2366Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar and rat renal medullary interstitial cells34Wang Z. Tang L. Zhu Q. et al.Hypoxia-inducible factor-1alpha contributes to the profibrotic action of angiotensin II in renal medullary interstitial cells.Kidney Int. 2011; 79: 300-310Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar suggested a profibrotic role of HIF-1α. However, Souma et al.17Souma T. Nezu M. Nakano D. et al.Erythropoietin synthesis in renal myofibroblasts is restored by activation of hypoxia signaling.J Am Soc Nephrol. 2016; 27: 428-438Crossref PubMed Scopus (96) Google Scholar reported that concomitant knockout of PHD1, PHD2, and PHD3 in REPCs of Tg(Epo-Cre) mice who express Cre recombinase under the control of Epo promoter does not affect renal fibrosis in the model of unilateral ureteral obstruction (UUO). However, renal Epo expression is low and the pool size of REPCs is small in a steady state without hypoxia or anemia,35Koury M.J. Haase V.H. Anaemia in kidney disease: harnessing hypoxia responses for therapy.Nat Rev Nephrol. 2015; 11: 394-410Crossref PubMed Scopus (147) Google Scholar suggesting that Epo-Cre transgene only defines a small fraction of total REPCs. Recently, renal Gli1-expressing cells are shown to be pericytes and differentiate to myofibroblasts on kidney injury.11Kramann R. Schneider R.K. DiRocco D.P. et al.Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis.Cell Stem Cell. 2015; 16: 51-66Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar,36Fabian S.L. Penchev R.R. St-Jacques B. et al.Hedgehog-Gli pathway activation during kidney fibrosis.Am J Pathol. 2012; 180: 1441-1453Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar Ablation of Gli1+ cells ameliorates UUO-induced renal fibrosis.11Kramann R. Schneider R.K. DiRocco D.P. et al.Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis.Cell Stem Cell. 2015; 16: 51-66Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar Here we used mice with Cre recombinase under the control of Gli1 promoter/enhancer to study the effects of pericyte-specific HIF stabilization or knockout on renal fibrosis. Gli1CreERT2/+, Tg(UBC-CreERT2), VhlF/F, ROSA26fstdTomato/fstdTomat, Hif1aF/F, and Hif2aF/F mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Egln1F/F mice were kindly provided by Professor Guo-Hua Fong (University of Connecticut Health Center, Farmington, CT). All studies were carried out under a protocol approved by Institutional Animal Care and Use Committee, National Taiwan University College of Medicine (IACUC 20160499). The procedure had been detailed in a previous study.14Chang Y.T. Yang C.C. Pan S.Y. et al.DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys.J Clin Invest. 2016; 126: 721-731Crossref PubMed Scopus (49) Google Scholar In short, in adult mice 7 to 9 weeks old under anesthesia with ketamine/xylazine (ketamine 100 mg/kg, xylazine 10 mg/kg), the left ureters of the mice were exposed and ligated with nylon suture through an incision at left flank. The date of UUO surgery was identified as day 0 of the experiment. Data were expressed as mean + SD. Statistical analyses were carried out using GraphPad Prism (GraphPad Software, San Diego, CA). The statistical significance was determined by Student t test or Mann-Whitney U test as shown in the captions. Full methods including mice, UUO, tamoxifen administration, quantitative polymerase chain reaction, immunoblot, immunohistochemistry, RNA in situ hybridization, microscopy, flow cytometry, and isolation of renal Gli1+ cells are in the Supplementary Methods. To demonstrate that Gli1+ pericytes produced EPO before we headed to study the effect of pericyte-specific HIF manipulation on renal fibrosis, we generated Gli1CreERT2/+;Egln1F/F mice to induce PHD2 knockout in Gli1+ pericytes conditionally (Figure 1a). Compared with littermate Egln1F/F mice, Gli1CreERT2/+;Egln1F/F mice showed higher level of serum EPO (Figure 1b), heavier splenic weight (Figure 1c), more TER-119+ erythroid lineage in splenocytes by day 14 after tamoxifen (Figure 1d), but similar body weight change after tamoxifen administration (Supplementary Figure S1A). Hematocrit (Hct) was higher in Gli1CreERT2/+;Egln1F/F mice at day 21 (Figure 1e). We analyzed renal Epo mRNA and peripheral blood count 14 days after tamoxifen. Compared with littermate Egln1F/F mice, Gli1CreERT2/+;Egln1F/F mice had increased renal Epo expression (Figure 1f) and peripheral reticulocytes (Figure 1g), whereas peripheral white blood cell count and platelet count were not different (Supplementary Figure S1B and C). To identify renal cells responsible for Epo expression, we performed in situ hybridization of Epo on kidney sections from the Egln1F/F and Gli1CreERT2/+;Egln1F/F mice (Supplementary Figure S2A–F). Increased Epo-expressing interstitial cells could be identified mainly in the corticomedullary junction, and to a lesser extent in the inner medulla and cortex in the Gli1CreERT2/+;Egln1F/F mice. These results suggested that Gli1+ pericyte-specific PHD2 knockout induced renal EPO production and erythropoiesis. To determine whether PHD2 regulates EPO production in cell types and organs other than pericytes in kidney, we generated Tg(UBC-CreERT2);Egln1F/F mice to induce global PHD2 knockout (Supplementary Figure S3A). After tamoxifen administration, knockout of Egln1 gene in kidney, liver, spleen, heart, and lung could be detected (Supplementary Figure S4). In kidney, the expression of Egln1 mRNA reduced to 4%, indicating high efficiency of PHD2 knockout (Supplementary Figure S3B). As in Gli1CreERT2/+;Egln1F/F mice, Tg(UBC-CreERT2);Egln1F/F mice showed progressive increase of Hct compared with littermate Egln1F/F mice (Supplementary Figure S3C). Notably, Tg(UBC-CreERT2);Egln1F/F mice showed decrease of body weight (Supplementary Figure S3D). Remarkably high level of serum EPO, around 130,000 pg/ml, was found in Tg(UBC-CreERT2);Egln1F/F mice, which was much higher than that achieved by phlebotomy (less than 5,000 pg/ml) in wild-type mice (Supplementary Figure S3E). To identify the source of serum EPO, we checked Epo mRNA levels in different organs from Tg(UBC-CreERT2);Egln1F/F and littermate control mice at day 15. In littermate control mice, Epo mRNA was hardly detected in any organ. In 5 major organs (kidney, liver, lung, heart, spleen) of Tg(UBC-CreERT2);Egln1F/F mice, only kidney showed remarkable increase of Epo mRNA, implying kidney as a major source of serum EPO (Supplementary Figure S3F and G). In situ hybridization of Epo mRNA on kidney sections revealed that renal cells with Epo expression were interstitial cells with pericyte morphology in Tg(UBC-CreERT2);Egln1F/F mice (Supplementary Figure S5). Therefore, these data confirmed that renal Gli1+ pericytes produced EPO on PHD2 knockout, thereby increasing erythropoiesis and Hct. Consistent with previous reports,11Kramann R. Schneider R.K. DiRocco D.P. et al.Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis.Cell Stem Cell. 2015; 16: 51-66Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar,36Fabian S.L. Penchev R.R. St-Jacques B. et al.Hedgehog-Gli pathway activation during kidney fibrosis.Am J Pathol. 2012; 180: 1441-1453Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar increased Gli1+ pericytes were shown in the corticomedullary junction and medulla after UUO injury in Gli1CreERT2/+;ROSA26fstdTomato/fstdTomato reporter mice (Supplementary Figure S6), suggesting that Gli1+ pericytes proliferate and contribute considerably to myofibroblasts during renal fibrosis. We hence studied the effect of HIF stabilization or knockout in Gli1+ pericytes on renal fibrosis. We used mice with Gli1+ pericyte-specific knockout of pVHL, not PHD2 because of possible redundancy in 3 different PHDs. To investigate long-term effect of pericyte-specific HIF stabilization on kidneys in mice without CKD, Gli1CreERT2/+;VhlF/F mice and VhlF/F littermates were administered tamoxifen at 6 weeks of age and then observed until 30 weeks of age (Supplementary Figure S7A). One unexpected death in Gli1CreERT2/+;VhlF/F mice took place at 25 weeks of age (14.3% mortality rate), but there were no deaths in VhlF/F littermates. Weakness and decreased body weight gain was found in Gli1CreERT2/+;VhlF/F mice as early as 16 weeks of age (Supplementary Figure S7B). Because of emaciation and unexpected death, some mice were euthanized before 30 weeks of age. Pathology revealed diffuse severe erythropoiesis in the spleen and congestion of red blood cells in liver, lung, and kidney at 18 weeks of age (Supplementary Figure S8). Except for red blood cell congestion, no discernible fibrosis was observed in major organs including kidneys. The levels of Hct and serum EPO increased to 87 ± 2.6% and 1604 ± 141 pg/ml at 30 weeks of age (i.e., 24 weeks after tamoxifen) (Supplementary Figure S9A and B). Because of no discernible renal fibrosis in mice with Gli1+ pericyte-specific pVHL knockout, we induced UUO in mice and assessed the severity of renal fibrosis 7 or 14 days later (Figure 2a). Deleted Vhl alleles in Gli1CreERT2/+;VhlF/F mice were detected in kidney genomic DNA by polymerase chain reaction, indicating knockout of pVHL in pericytes (Supplementary Figure S10). Hct was increased in Gli1CreERT2/+;VhlF/F mice even after UUO surgery (Figure 2b). Notably, the expression of Epo mRNA was increased in both contralateral and UUO kidneys of Gli1CreERT2/+;VhlF/F mice (Figure 2c), suggesting HIF stabilization could activate Epo transcription in both pericytes and myofibroblasts. However, no difference in renal fibrosis between Gli1CreERT2/+;VhlF/F and littermate VhlF/F mice could be detected, as measured by picrosirius red–stained area (Figures 2d and 3a and b ) and expression of Col1a1 mRNA (Figure 2e) in kidney. Besides, mRNA and protein levels of α-smooth muscle actin were not different in kidney (Figure 2f and g and Supplementary Figure S11). To confirm that myofibroblasts retained Epo expression ability after pVHL knockout, we performed in situ hybridization of Epo and Acta2 on kidney sections (Supplementary Figures S12 and S13). Renal Acta2 expression increased drastically and comparably after UUO injury in both Gli1CreERT2/+;VhlF/F and VhlF/F mice. More interstitial Epo-expressing cells could be observed in Gli1CreERT2/+;VhlF/F mice than in VhlF/F mice, regardless of the presence of UUO injury. In addition, cells with concomitant expression of Acta2 and Epo could be identified in UUO kidney from Gli1CreERT2/+;VhlF/F mice but not VhlF/F mice. In fact, we could hardly identify any Epo-expressing cell in UUO kidney from VhlF/F mice. These results suggested that Gli1+ pericyte-restricted pVHL knockout promoted Epo expression but not myofibroblast differentiation or collagen production even after UUO injury.Figure 3Pericyte-specific von Hippel-Lindau protein knockout does not affect renal fibrosis. Representative images of picrosirius red–stained formalin-fixed paraffin-embedded kidney sections from VhlF/F (shown as +/+) and Gli1CreERT2/+;VhlF/F (shown as Cre/+) mice at days 7 (a) and 14 (b) after unilateral ureteral obstruction (UUO) surgery. Bars = 100 μm. CLK, contralateral kidney. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because UUO injury results in damage of proximal tubular cells and recruitment of macrophages,37Lin S.L. Castano A.P. Nowlin B.T. et al.Bone marrow Ly6Chigh monocytes are selectively recruited to injured kidney and differentiate into functionally distinct populations.J Immunol. 2009; 183: 6733-6743Crossref PubMed Scopus (246) Google Scholar mRNA levels of Havcr1 (encoding kidney injury molecule-1) (Figure 4a) and Adgre1 (encoding endothelial growth factor -like module-containing mucin-like hormone receptor-like 1, also known as F4/80) (Figure 4b) increased in UUO kidneys, but no difference could be detected between Gli1CreERT2/+;VhlF/F and littermate VhlF/F mice. The mRNA levels of Vegfa, Hmox1, Egln1, and Egln3 (encoding vascular endothelial cell growth factor-A, heme oxygenase-1, PHD2, and PHD3, respectively) in kidneys were not changed (Figure 4c–f), probably reflecting that these transcripts were not dominantly produced by Gli1+ pericytes or not changed after Gli1+ pericyte-restricted VHL knockout. We assessed tubulointerstitial injury in periodic acid–Schiff–stained kidney sections, and no difference was detected between Gli1CreERT2/+;VhlF/F and littermate VhlF/F mice (Figures 4g and 5a and b ). Because tamoxifen is reported to attenuate fibrosis,38Delle H. Rocha J.R. Cavaglieri R.C. et al.Antifibrotic effect of tamoxifen in a model of progressive renal disease.J Am Soc Nephrol. 2012; 23: 37-48Crossref PubMed Scopus (54) Google Scholar,39Falke L.L. Broekhuizen R. Huitema A. et al.Tamoxifen for induction of Cre-recombination may confound fibrosis studies in female mice.J Cell Commu