Correspondence: Dr. Masayuki Yamamoto, Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. Email: pj.ca.ukohot.dem@otomamayisam

Received 2013 Jan 8; Accepted 2013 Apr 24.

Abstract

CKD progresses with fibrosis and erythropoietin (Epo)-dependent anemia, leading to increased cardiovascular complications, but the mechanisms linking Epo-dependent anemia and fibrosis remain unclear. Here, we show that the cellular phenotype of renal Epo-producing cells (REPs) alternates between a physiologic Epo-producing state and a pathologic fibrogenic state in response to microenvironmental signals. In a novel mouse model, unilateral ureteral obstruction–induced inflammatory milieu activated NFκB and Smad signaling pathways in REPs, rapidly repressed the Epo-producing potential of REPs, and led to myofibroblast transformation of these cells. Moreover, we developed a unique Cre-based cell-fate tracing method that marked current and/or previous Epo-producing cells and revealed that the majority of myofibroblasts are derived from REPs. Genetic induction of NFκB activity selectively in REPs resulted in myofibroblastic transformation, indicating that NFκB signaling elicits a phenotypic switch. Reversing the unilateral ureteral obstruction–induced inflammatory microenvironment restored the Epo-producing potential and the physiologic phenotype of REPs. This phenotypic reversion was accelerated by anti-inflammatory therapy. These findings demonstrate that REPs possess cellular plasticity, and suggest that the phenotypic transition of REPs to myofibroblasts, modulated by inflammatory molecules, underlies the connection between anemia and renal fibrosis in CKD.

Abstract

Renal erythropoietin (Epo)-producing cells (REPs) are fibroblast-like cells that express neural markers and produce Epo, an indispensable erythropoietic hormone.¹3 REPs control Epo gene expression primarily through the prolyl hydroxylases/von Hippel-Lindau protein/hypoxia-inducible factor (HIF) pathway.³4 Production of Epo at cellular level in REPs is thought to be either “on” or “off,” hence the total Epo production from kidney is regulated by the number of Epo-producing REPs, which changes markedly responding to hypoxia and/or anemia.²56

Renal fibrosis is considered a final common pathway of CKD leading to ESRD.⁷8 Because fibrotic kidneys have a limited Epo-producing ability at any given anemic stimuli,⁹10 renal anemia develops along with the progression of fibrosis, leading to cardiovascular events.⁷910 Interestingly, REPs in the fibrotic kidney express markers of myofibroblasts, such as desmin and α smooth muscle actin (αSMA),¹¹12 suggesting an etiological link between renal anemia and fibrosis by damaged REPs.

Myofibroblasts are the culprit of renal fibrosis, and their origin has been debated.⁸13 Recent cell-fate mapping studies revealed that myofibroblasts are mainly derived from P0 (myelin protein 0)-Cre–labeled cells,¹² or FoxD1-Cre–labeled pericytes and perivascular fibroblasts.¹⁴ However, there remains uncertainty, as these Cre-labeled cells are heterogeneous because of the shared developmental program.¹⁴ Moreover, in terms of Epo-producing ability, REPs are only <10% of P0-Cre–labeled cells, and approximately 20% of REPs are not fate-mapped by P0-Cre.⁶12 Thus, precise contribution of REPs to renal fibrosis is still unclear.

There are many unanswered important questions related to REPs and renal fibrosis: (1) whether the majority of REPs contribute to total myofibroblast population or REPs rather disappear through cell death upon renal damages, (2) how REPs change their phenotype to myofibroblasts and lose their Epo-producing ability, and (3) whether this phenotypic conversion is reversible. In this regard, because CKD is characterized by sterile chronic inflammation caused by activated resident renal cells and infiltrating leukocytes,¹⁵17 we surmised that the phenotypic changes of REPs in diseased kidneys may be attributable to inflammatory signals generated in renal microenvironments.

In this study, we utilized two novel mouse lines that manifest severe anemia (inherited super anemic mice [ISAM]) and that express Cre enzyme under the regulatory influences of Epo gene (Epo-Cre mouse) to monitor phenotypic changes of individual REPs. Utilizing unilateral ureteral obstruction (UUO), a commonly used inflammatory fibrogenic method,¹⁸19 we analyzed how activated inflammatory signaling affects the phenotype of REPs. Through these analyses, we have discovered that REPs are the major source of myofibroblasts and myofibroblast-transformed REPs recover their normal Epo-producing potential upon removal of inflammatory stresses. These results indicate that a profound cellular plasticity resides in REPs and REPs may be ideal targets for CKD treatment.

All indices were not statistically significant (one-way ANOVA). Data are shown as mean ± SD.

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Acknowledgments

The authors thank Professors Hozumi Motohashi and Mitchell Halperin for their invaluable advice, Ms. Maggie Patient for editing our manuscript, and Ms. Kiyomi Kisu for helping with immunoelectron microscopy. The authors also thank Biomedical Research Core and Center for Laboratory Animal Research of Tohoku University Graduate School of Medicine for technical support, and Chugai Pharmaceutical Co Ltd for comments.

This work was supported in part by JST, CREST (to M.Y.); Grants-in-Aid for Creative Scientific Research (to M.Y.), for Scientific Research (to M.Y.), and for JSPS fellows (to T.S. and S.Y.) from the JSPS; Grants-in-Aid for Scientific Research on Priority Areas (to M.Y.) from the MEXT; and a research grant from the Kidney Foundation, Japan (JKFB12-1 to T.S.).

Acknowledgments

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2013010030/-/DCSupplemental.

Footnotes

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