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Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis

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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.

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Figure 1: Genetic targeting of EMT reduces renal fibrosis and improves TEC health.
Figure 2: Inhibition of the EMT prevents loss of TEC-associated solute and solvent transporters.
Figure 3: An EMT program is associated with deregulated expression and functionality of TEC solute and solvent transporters in humans with kidney disease.
Figure 4: Inhibition of an EMT reduces immune infiltration in kidney fibrosis.
Figure 5: EMT program G2 cell cycle arrest.
Figure 6: p21 controls the EMT program G2 cell cycle arrest.

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References

  1. 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).

    Article  CAS  PubMed  Google Scholar 

  2. Grams, M.E. et al. Lifetime incidence of CKD stages 3–5 in the United States. Am. J. Kidney Dis. 62, 245–252 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Sugimoto, H. et al. Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis. Nat. Med. 18, 396–404 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. LeBleu, V.S. et al. Origin and function of myofibroblasts in kidney fibrosis. Nat. Med. 19, 1047–1053 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Bechtel, W. et al. Methylation determines fibroblast activation and fibrogenesis in the kidney. Nat. Med. 16, 544–550 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Iwano, M. et al. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin. Invest. 110, 341–350 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zeisberg, M. & Kalluri, R. Fibroblasts emerge via epithelial-mesenchymal transition in chronic kidney fibrosis. Front. Biosci. 13, 6991–6998 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Zeisberg, E.M. et al. Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition. J. Am. Soc. Nephrol. 19, 2282–2287 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Lamouille, S., Xu, J. & Derynck, R. Molecular mechanisms of epithelial-mesenchymal transition. Nat. Rev. Mol. Cell Biol. 15, 178–196 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 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).

    Article  CAS  PubMed  Google Scholar 

  11. Zeisberg, M. et al. Renal fibrosis: collagen composition and assembly regulates epithelial-mesenchymal transdifferentiation. Am. J. Pathol. 159, 1313–1321 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zeisberg, M. et al. Renal fibrosis. Extracellular matrix microenvironment regulates migratory behavior of activated tubular epithelial cells. Am. J. Pathol. 160, 2001–2008 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zeisberg, M. & Kalluri, R. The role of epithelial-to-mesenchymal transition in renal fibrosis. J. Mol. Med. (Berl) 82, 175–181 (2004).

    Article  Google Scholar 

  14. 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).

    Article  CAS  PubMed  Google Scholar 

  15. Teng, Y., Zeisberg, M. & Kalluri, R. Transcriptional regulation of epithelial-mesenchymal transition. J. Clin. Invest. 117, 304–306 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 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).

    Article  CAS  PubMed  Google Scholar 

  17. Strutz, F. et al. Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation. Kidney Int. 61, 1714–1728 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Kalluri, R. & Weinberg, R.A. The basics of epithelial-mesenchymal transition. J. Clin. Invest. 119, 1420–1428 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hertig, A. et al. Early epithelial phenotypic changes predict graft fibrosis. J. Am. Soc. Nephrol. 19, 1584–1591 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Boutet, A. et al. Snail activation disrupts tissue homeostasis and induces fibrosis in the adult kidney. EMBO J. 25, 5603–5613 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Rastaldi, M.P. Epithelial-mesenchymal transition and its implications for the development of renal tubulointerstitial fibrosis. J. Nephrol. 19, 407–412 (2006).

    CAS  PubMed  Google Scholar 

  22. Kriz, W., Kaissling, B. & Le Hir, M. Epithelial-mesenchymal transition (EMT) in kidney fibrosis: fact or fantasy? J. Clin. Invest. 121, 468–474 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zeisberg, M. & Duffield, J.S. Resolved: EMT produces fibroblasts in the kidney. J. Am. Soc. Nephrol. 21, 1247–1253 (2010).

    Article  PubMed  Google Scholar 

  24. Liu, Y. New insights into epithelial-mesenchymal transition in kidney fibrosis. J. Am. Soc. Nephrol. 21, 212–222 (2010).

    Article  CAS  PubMed  Google Scholar 

  25. Yang, L. et al. Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury. Nat. Med. 16, 535–543 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 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).

    Article  CAS  PubMed  Google Scholar 

  27. 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).

    Article  CAS  PubMed  Google Scholar 

  28. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 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).

    Article  CAS  PubMed  Google Scholar 

  30. 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).

    Article  CAS  Google Scholar 

  31. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Köttgen, A. et al. New loci associated with kidney function and chronic kidney disease. Nat. Genet. 42, 376–384 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Martini, S. et al. Integrative biology identifies shared transcriptional networks in CKD. J. Am. Soc. Nephrol. 25, 2559–2572 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Reich, H.N. et al. A molecular signature of proteinuria in glomerulonephritis. PLoS ONE 5, e13451 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Schmid, H. et al. Modular activation of nuclear factor-κB transcriptional programs in human diabetic nephropathy. Diabetes 55, 2993–3003 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Neusser, M.A. et al. Human nephrosclerosis triggers a hypoxia-related glomerulopathy. Am. J. Pathol. 176, 594–607 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Doherty, J.R. & Cleveland, J.L. Targeting lactate metabolism for cancer therapeutics. J. Clin. Invest. 123, 3685–3692 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Stern, R. et al. Lactate stimulates fibroblast expression of hyaluronan and CD44: the Warburg effect revisited. Exp. Cell Res. 276, 24–31 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. 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).

    Article  CAS  PubMed  Google Scholar 

  41. Toyohara, T. et al. SLCO4C1 transporter eliminates uremic toxins and attenuates hypertension and renal inflammation. J. Am. Soc. Nephrol. 20, 2546–2555 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Wynn, T.A. Cellular and molecular mechanisms of fibrosis. J. Pathol. 214, 199–210 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Vega, S. et al. Snail blocks the cell cycle and confers resistance to cell death. Genes Dev. 18, 1131–1143 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wynn, T.A. Fibrosis under arrest. Nat. Med. 16, 523–525 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Rowe, R.G. et al. Hepatocyte-derived Snail1 propagates liver fibrosis progression. Mol. Cell. Biol. 31, 2392–2403 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. LeBleu, V.S. et al. Identification of human epididymis protein-4 as a fibroblast-derived mediator of fibrosis. Nat. Med. 19, 227–231 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Smyth, G.K. Limma: linear models for microarray data. In Bioinformatics and Computational Biology Solutions using R and Bioconductor. (Springer, 2005).

  53. 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).

    Article  CAS  PubMed  Google Scholar 

  54. Yang, J. et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117, 927–939 (2004).

    Article  CAS  PubMed  Google Scholar 

  55. Olive, P.L. & Banath, J.P. The comet assay: a method to measure DNA damage in individual cells. Nat. Protoc. 1, 23–29 (2006).

    Article  CAS  PubMed  Google Scholar 

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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.

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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.

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Correspondence to Raghu Kalluri.

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J.P.A. is an inventor of intellectual property owned by the University of California, Berkeley, and licensed to Bristol Meyers–Squibb.

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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

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