Transforming Growth Factor β-1 Stimulates Profibrotic Epithelial Signaling to Activate Pericyte-Myofibroblast Transition in Obstructive Kidney Fibrosis

Abstract

Pericytes have been identified as the major source of precursors of scar-producing myofibroblasts during kidney fibrosis. The underlying mechanisms triggering pericyte-myofibroblast transition are poorly understood. Transforming growth factor β-1 (TGF-β1) is well recognized as a pluripotent cytokine that drives organ fibrosis. We investigated the role of TGF-β1 in inducing profibrotic signaling from epithelial cells to activate pericyte-myofibroblast transition. Increased expression of TGF-β1 was detected predominantly in injured epithelium after unilateral ureteral obstruction, whereas downstream signaling from the TGF-β1 receptor increased in both injured epithelium and pericytes. In mice with ureteral obstruction that were treated with the pan anti-TGF-β antibody (1D11) or TGF-β receptor type I inhibitor (SB431542), kidney pericyte-myofibroblast transition was blunted. The consequence was marked attenuation of fibrosis. In addition, epithelial cell cycle G2/M arrest and production of profibrotic cytokines were both attenuated. Although TGF-β1 alone did not trigger pericyte proliferation in vitro, it robustly induced α smooth muscle actin (α-SMA). In cultured kidney epithelial cells, TGF-β1 stimulated G2/M arrest and production of profibrotic cytokines that had the capacity to stimulate proliferation and transition of pericytes to myofibroblasts. In conclusion, this study identified a novel link between injured epithelium and pericyte-myofibroblast transition through TGF-β1 during kidney fibrosis.

DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys

“…In cancer treatment, it is emerging that low-dose demethylating agents can effectively deplete DNMTs, while high doses induce cell cycle arrest and cytotoxicity (36)(37)(38). Of the well-recognized profibrotic cytokines, we have previously shown that TGF-β1 induces pericyte-myofibroblast transition during kidney fibrosis (28)(29)(30). Here, our data further support that TGF-β1 was one of factors that led to upregulation of DNMTs and methylation and repression of Epo in kidney pericytes.…”

“…Moreover, TGF-β1, a well-recognized cytokine inducing pericytemyofibroblast transition (29,30), can induce Rasal1 methylation through DNA methyltransferase 1 (DNMT1), thereby leading to perpetuation of fibroblast activation and kidney fibrosis (32). We propose that TGF-β1-induced methylation of Epo 5′-regulatory elements may provide a molecular basis for a decreased Epo response of REPCs to anemic stimulation in CKD.…”

“…These pericytes deserve attention due to their potential to produce EPO in healthy kidneys, and, as these cells stand at the junction between the circulation and the kidney, they are primed to sense the change of oxygenation and hemoglobin concentration (21)(22)(23)(24)(25)(26)(27). During fibrogenic injury, the pericytes proliferate and differentiate into myofibroblasts that produce pathogenic extracellular collagenous matrix, which leads to kidney fibrosis and function failure (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). In accordance with previous studies that refer to REPCs as fibroblast-like cells that might transit to myofibroblasts and contribute to kidney fibrosis (10,11,14), FOXD1…”

“…Primary antibodies against the following proteins were used for immunolabeling: αSMA-Cy3 (C6198, clone 1A4, Sigma-Aldrich), DNMT1 (5032, Cell Signaling Technology), DNMT3b (70-205, Cosmo Bio Co. LTD.), DNMT3a (sc-20703), nidogen (sc-33706, Santa Cruz Biotechnology), p75 NGF receptor (ab8875, Abcam), CD73 (550738, BD Biosciences), and PDGFRβ (a gift from William Stallcup, Burnham Institute, La Jolla, California, USA). Fluorescence-conjugated secondary antibody labeling (111-165-144, 112-165-167, 112-605-167, Jackson Immunoresearch Laboratories), DAPI staining, VECTASHIELD (Vector Laboratories) mounting, and image capture and processing were carried out as previously described (29). Quantification of specific cells in tissue sections was carried out as follows.…”

“…Mouse tissues were prepared and stained as previously described (29). Primary antibodies against the following proteins were used for immunolabeling: αSMA-Cy3 (C6198, clone 1A4, Sigma-Aldrich), DNMT1 (5032, Cell Signaling Technology), DNMT3b (70-205, Cosmo Bio Co. LTD.), DNMT3a (sc-20703), nidogen (sc-33706, Santa Cruz Biotechnology), p75 NGF receptor (ab8875, Abcam), CD73 (550738, BD Biosciences), and PDGFRβ (a gift from William Stallcup, Burnham Institute, La Jolla, California, USA).…”