Abstract
Kidney fibrosis is marked by an epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs). Here we find that, during renal fibrosis, TECs acquire a partial EMT program during which they remain associated with their basement membrane and express markers of both epithelial and mesenchymal cells. The functional consequence of the EMT program during fibrotic injury is an arrest in the G2 phase of the cell cycle and lower expression of several solute and solvent transporters in TECs. We also found that transgenic expression of either Twist1 (encoding twist family bHLH transcription factor 1, known as Twist) or Snai1 (encoding snail family zinc finger 1, known as Snail) expression is sufficient to promote prolonged TGF-β1–induced G2 arrest of TECs, limiting the cells' potential for repair and regeneration. In mouse models of experimentally induced renal fibrosis, conditional deletion of Twist1 or Snai1 in proximal TECs resulted in inhibition of the EMT program and the maintenance of TEC integrity, while also restoring cell proliferation, dedifferentiation-associated repair and regeneration of the kidney parenchyma and attenuating interstitial fibrosis. Thus, inhibition of the EMT program in TECs during chronic renal injury represents a potential anti-fibrosis therapy.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Zeisberg, M. & Kalluri, R. Cellular mechanisms of tissue fibrosis. 1. Common and organ-specific mechanisms associated with tissue fibrosis. Am. J. Physiol. Cell Physiol. 304, C216–C225 (2013).
Grams, M.E. et al. Lifetime incidence of CKD stages 3–5 in the United States. Am. J. Kidney Dis. 62, 245–252 (2013).
Sugimoto, H. et al. Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis. Nat. Med. 18, 396–404 (2012).
LeBleu, V.S. et al. Origin and function of myofibroblasts in kidney fibrosis. Nat. Med. 19, 1047–1053 (2013).
Bechtel, W. et al. Methylation determines fibroblast activation and fibrogenesis in the kidney. Nat. Med. 16, 544–550 (2010).
Iwano, M. et al. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin. Invest. 110, 341–350 (2002).
Zeisberg, M. & Kalluri, R. Fibroblasts emerge via epithelial-mesenchymal transition in chronic kidney fibrosis. Front. Biosci. 13, 6991–6998 (2008).
Zeisberg, E.M. et al. Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition. J. Am. Soc. Nephrol. 19, 2282–2287 (2008).
Lamouille, S., Xu, J. & Derynck, R. Molecular mechanisms of epithelial-mesenchymal transition. Nat. Rev. Mol. Cell Biol. 15, 178–196 (2014).
Lee, K. & Nelson, C.M. New insights into the regulation of epithelial-mesenchymal transition and tissue fibrosis. Int. Rev. Cell Mol. Biol. 294, 171–221 (2012).
Zeisberg, M. et al. Renal fibrosis: collagen composition and assembly regulates epithelial-mesenchymal transdifferentiation. Am. J. Pathol. 159, 1313–1321 (2001).
Zeisberg, M. et al. Renal fibrosis. Extracellular matrix microenvironment regulates migratory behavior of activated tubular epithelial cells. Am. J. Pathol. 160, 2001–2008 (2002).
Zeisberg, M. & Kalluri, R. The role of epithelial-to-mesenchymal transition in renal fibrosis. J. Mol. Med. (Berl) 82, 175–181 (2004).
Burns, W.C., Kantharidis, P. & Thomas, M.C. The role of tubular epithelial-mesenchymal transition in progressive kidney disease. Cells Tissues Organs 185, 222–231 (2007).
Teng, Y., Zeisberg, M. & Kalluri, R. Transcriptional regulation of epithelial-mesenchymal transition. J. Clin. Invest. 117, 304–306 (2007).
Kida, Y. et al. Twist relates to tubular epithelial-mesenchymal transition and interstitial fibrogenesis in the obstructed kidney. J. Histochem. Cytochem. 55, 661–673 (2007).
Strutz, F. et al. Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation. Kidney Int. 61, 1714–1728 (2002).
Kalluri, R. & Weinberg, R.A. The basics of epithelial-mesenchymal transition. J. Clin. Invest. 119, 1420–1428 (2009).
Hertig, A. et al. Early epithelial phenotypic changes predict graft fibrosis. J. Am. Soc. Nephrol. 19, 1584–1591 (2008).
Boutet, A. et al. Snail activation disrupts tissue homeostasis and induces fibrosis in the adult kidney. EMBO J. 25, 5603–5613 (2006).
Rastaldi, M.P. Epithelial-mesenchymal transition and its implications for the development of renal tubulointerstitial fibrosis. J. Nephrol. 19, 407–412 (2006).
Kriz, W., Kaissling, B. & Le Hir, M. Epithelial-mesenchymal transition (EMT) in kidney fibrosis: fact or fantasy? J. Clin. Invest. 121, 468–474 (2011).
Zeisberg, M. & Duffield, J.S. Resolved: EMT produces fibroblasts in the kidney. J. Am. Soc. Nephrol. 21, 1247–1253 (2010).
Liu, Y. New insights into epithelial-mesenchymal transition in kidney fibrosis. J. Am. Soc. Nephrol. 21, 212–222 (2010).
Yang, L. et al. Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury. Nat. Med. 16, 535–543 (2010).
Canaud, G. & Bonventre, J.V. Cell cycle arrest and the evolution of chronic kidney disease from acute kidney injury. Nephrol. Dial. Transplant. 30, 575–583 (2015).
Kang, H.M. et al. Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development. Nat. Med. 21, 37–46 (2015).
Rajasekaran, S.A. et al. Na,K-ATPase subunits as markers for epithelial-mesenchymal transition in cancer and fibrosis. Mol. Cancer Ther. 9, 1515–1524 (2010).
Ito, S. et al. Reduction of indoxyl sulfate by AST-120 attenuates monocyte inflammation related to chronic kidney disease. J. Leukoc. Biol. 93, 837–845 (2013).
Lui, T. et al. Changes in expression of renal Oat1, Oat3 and Mrp2 in cisplatin-induced acute renal failure after treatment of JBP485 in rats. Toxicol. Appl. Pharmacol. 264, 423–430 (2012).
Hills, C.E., Willars, G.B. & Brunskill, N.J. Proinsulin C-peptide antagonizes the profibrotic effects of TGF-β1 via up-regulation of retinoic acid and HGF-related signaling pathways. Mol. Endocrinol. 24, 822–831 (2010).
Köttgen, A. et al. New loci associated with kidney function and chronic kidney disease. Nat. Genet. 42, 376–384 (2010).
Martini, S. et al. Integrative biology identifies shared transcriptional networks in CKD. J. Am. Soc. Nephrol. 25, 2559–2572 (2014).
Reich, H.N. et al. A molecular signature of proteinuria in glomerulonephritis. PLoS ONE 5, e13451 (2010).
Schmid, H. et al. Modular activation of nuclear factor-κB transcriptional programs in human diabetic nephropathy. Diabetes 55, 2993–3003 (2006).
Neusser, M.A. et al. Human nephrosclerosis triggers a hypoxia-related glomerulopathy. Am. J. Pathol. 176, 594–607 (2010).
Hodgin, J.B. et al. A molecular profile of focal segmental glomerulosclerosis from formalin-fixed, paraffin-embedded tissue. Am. J. Pathol. 177, 1674–1686 (2010).
Doherty, J.R. & Cleveland, J.L. Targeting lactate metabolism for cancer therapeutics. J. Clin. Invest. 123, 3685–3692 (2013).
Stern, R. et al. Lactate stimulates fibroblast expression of hyaluronan and CD44: the Warburg effect revisited. Exp. Cell Res. 276, 24–31 (2002).
Maeda, T. et al. Mechanism of the regulation of organic cation/carnitine transporter 1 (SLC22A4) by rheumatoid arthritis-associated transcriptional factor RUNX1 and inflammatory cytokines. Drug Metab. Dispos. 35, 394–401 (2007).
Toyohara, T. et al. SLCO4C1 transporter eliminates uremic toxins and attenuates hypertension and renal inflammation. J. Am. Soc. Nephrol. 20, 2546–2555 (2009).
Wynn, T.A. Cellular and molecular mechanisms of fibrosis. J. Pathol. 214, 199–210 (2008).
Witzgall, R. et al. Localization of proliferating cell nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney. Evidence for a heterogenous genetic response among nephron segments, and a large pool of mitotically active and dedifferentiated cells. J. Clin. Invest. 93, 2175–2188 (1994).
Duffield, J.S. et al. Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells. J. Clin. Invest. 115, 1743–1755 (2005).
Wu, 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. 182, 118–131 (2013).
Vega, S. et al. Snail blocks the cell cycle and confers resistance to cell death. Genes Dev. 18, 1131–1143 (2004).
Megyesi, J. et al. The lack of a functional p21(WAF1/CIP1) gene ameliorates progression to chronic renal failure. Proc. Natl. Acad. Sci. USA 96, 10830–10835 (1999).
Cooke, V.G. et al. Pericyte depletion results in hypoxia-associated epithelial-to-mesenchymal transition and metastasis mediated by met signaling pathway. Cancer Cell 21, 66–81 (2012).
Wynn, T.A. Fibrosis under arrest. Nat. Med. 16, 523–525 (2010).
Rowe, R.G. et al. Hepatocyte-derived Snail1 propagates liver fibrosis progression. Mol. Cell. Biol. 31, 2392–2403 (2011).
LeBleu, V.S. et al. Identification of human epididymis protein-4 as a fibroblast-derived mediator of fibrosis. Nat. Med. 19, 227–231 (2013).
Smyth, G.K. Limma: linear models for microarray data. In Bioinformatics and Computational Biology Solutions using R and Bioconductor. (Springer, 2005).
Haverty, T.P. et al. Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis. J. Cell Biol. 107, 1359–1368 (1988).
Yang, J. et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117, 927–939 (2004).
Olive, P.L. & Banath, J.P. The comet assay: a method to measure DNA damage in individual cells. Nat. Protoc. 1, 23–29 (2006).
Acknowledgements
This work was primarily supported with funds from the University of Texas M.D. Anderson Cancer Center (UT MDACC), partially supported by the Cancer Prevention and Research Institute of Texas and funding from the University of Texas System Science and Technology Acquisition and Retention (STARS) awards to RK and VSL. The research in R.K. laboratory is also supported by the US National Institutes of Health (NIH) (grants CA-155370, CA-151925, DK-081576, DK-55001) and the Metastasis Research Center at the M.D. Anderson Cancer Center (P30CA016672). V.S.L. is supported by the NIH under award number P30CA016672 and the Khalifa Bin Zayed Al Nahya Foundation. This research was performed in the Flow Cytometry & Cellular Imaging Facility at UT MDACC, which is supported in part by the NIH through MDACC Support grant CA–016672. This work was in part supported by the Deutsche Forschungsgemeinschaft (equipment grant INST1525/16–1 FUGG). SnailloxP/loxP mice were kindly provided by S.J. Weiss, University of Michigan, and Twist1loxP/loxP mice were kindly provided by R.R. Behringer, UT MDACC via the Mutant Mouse Regional Resource Center (MMRRC) repository. pcDNA3-Twist plasmid was kindly provided by R. Maestro, Centro di Riferimento Oncologico National Cancer Institute, Italy. Nephrotoxic serum was a kind gift from D.J. Salant, Boston University. MCT cells were a gift from E.G. Neilson, Northwestern University School of Medicine. We thank E. Lawson for technical help with immunostaining, E. Chang for help with digital microscopy scanning of tissue histology slides and L. Gibson for help with breeding and genotyping mice.
Author information
Authors and Affiliations
Contributions
R.K. conceptually designed the strategy for this study, participated in discussions, provided intellectual input, supervised the studies and wrote the manuscript. V.S.L. designed the study, provided intellectual input, supervised and conducted the studies, designed and performed experiments, generated the figures and wrote the manuscript. S.L. designed and performed experiments, collected the data, generated the figures and participated in writing the manuscript. S.L., V.S.L., B.T., H.S., K.V., J.L.C., C.-C.W.,Y.H., B.C.B., T.P.-H., and H.N. performed some experiments and collected data. The data was analyzed by S.L., V.S.L., B.T., J.L.C., C.-C.W. and T.P.-H. J.P.A. and M.Z. participated in discussions, provided intellectual input, supervised the studies and edited the manuscript.
Corresponding author
Ethics declarations
Competing interests
J.P.A. is an inventor of intellectual property owned by the University of California, Berkeley, and licensed to Bristol Meyers–Squibb.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–15 (PDF 7322 kb)
Rights and permissions
About this article
Cite this article
Lovisa, S., LeBleu, V., Tampe, B. et al. Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis. Nat Med 21, 998–1009 (2015). https://doi.org/10.1038/nm.3902
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nm.3902
This article is cited by
-
Dysregulated lipid metabolism is associated with kidney allograft fibrosis
Lipids in Health and Disease (2024)
-
WNT-dependent interaction between inflammatory fibroblasts and FOLR2+ macrophages promotes fibrosis in chronic kidney disease
Nature Communications (2024)
-
Circulating exosomal miR-16-5p and let-7e-5p are associated with bladder fibrosis of diabetic cystopathy
Scientific Reports (2024)
-
Sodium glucose cotransporter 2 inhibitor suppresses renal injury in rats with renal congestion
Hypertension Research (2024)
-
Single-copy Snail upregulation causes partial epithelial-mesenchymal transition in colon cancer cells
BMC Cancer (2023)