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Methylation determines fibroblast activation and fibrogenesis in the kidney

Abstract

Fibrogenesis is a pathological wound repair process that fails to cease, even when the initial insult has been removed. Fibroblasts are principal mediators of fibrosis, and fibroblasts from fibrotic tissues fail to return to their quiescent stage, including when cultured in vitro. In a search for underlying molecular mechanisms, we hypothesized that this perpetuation of fibrogenesis is caused by epigenetic modifications. We demonstrate here that hypermethylation of RASAL1, encoding an inhibitor of the Ras oncoprotein, is associated with the perpetuation of fibroblast activation and fibrogenesis in the kidney. RASAL1 hypermethylation is mediated by the methyltransferase Dnmt1 in renal fibrogenesis, and kidney fibrosis is ameliorated in Dnmt1+/− heterozygous mice. These studies demonstrate that epigenetic modifications may provide a molecular basis for perpetuated fibroblast activation and fibrogenesis in the kidney.

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Figure 1: 5′-azacytidine ameliorates experimental renal fibrosis.
Figure 2: RASAL1 hypermethylation in fibrotic kidney fibroblasts.
Figure 3: Absence of Rasal1 hypermethylation in kidneys that do not become fibrotic upon injury.
Figure 4: Rasal1 silencing causes renal fibroblast activation via Ras hyperactivity.
Figure 5: Dnmts in the mouse model of folic acid–induced nephropathy.
Figure 6: TGF-β1–induced methylation of Rasal1 in kidney fibroblasts is mediated by Dnmt1.

References

  1. 1

    Eddy, A.A. Molecular insights into renal interstitial fibrosis. J. Am. Soc. Nephrol. 7, 2495–2508 (1996).

    CAS  PubMed  Google Scholar 

  2. 2

    Kuncio, G.S., Neilson, E.G. & Haverty, T. Mechanisms of tubulointerstitial fibrosis. Kidney Int. 39, 550–556 (1991).

    CAS  Article  Google Scholar 

  3. 3

    Strutz, F. & Zeisberg, M. Renal fibroblasts and myofibroblasts in chronic kidney disease. J. Am. Soc. Nephrol. 17, 2992–2998 (2006).

    CAS  Article  Google Scholar 

  4. 4

    Kalluri, R. & Zeisberg, M. Fibroblasts in cancer. Nat. Rev. Cancer 6, 392–401 (2006).

    CAS  Article  Google Scholar 

  5. 5

    Müller, G.A. & Rodemann, H.P. Characterization of human renal fibroblasts in health and disease: I. Immunophenotyping of cultured tubular epithelial cells and fibroblasts derived from kidneys with histologically proven interstitial fibrosis. Am. J. Kidney Dis. 17, 680–683 (1991).

    Article  Google Scholar 

  6. 6

    Rodemann, H.P. & Müller, G.A. Characterization of human renal fibroblasts in health and disease: II. In vitro growth, differentiation, and collagen synthesis of fibroblasts from kidneys with interstitial fibrosis. Am. J. Kidney Dis. 17, 684–686 (1991).

    CAS  Article  Google Scholar 

  7. 7

    Weber, K.T. & Brilla, C.G. Factors associated with reactive and reparative fibrosis of the myocardium. Basic Res. Cardiol. 87, Suppl 1, 291–301 (1992).

    PubMed  Google Scholar 

  8. 8

    Hinz, B. et al. The myofibroblast: one function, multiple origins. Am. J. Pathol. 170, 1807–1816 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Lafyatis, R. Targeting fibrosis in systemic sclerosis. Endocr. Metab. Immune Disord. Drug Targets 6, 395–400 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Lin, J. et al. Kielin/chordin-like protein, a novel enhancer of BMP signaling, attenuates renal fibrotic disease. Nat. Med. 11, 387–393 (2005).

    CAS  Article  Google Scholar 

  11. 11

    Santini, V., Kantarjian, H.M. & Issa, J.P. Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann. Intern. Med. 134, 573–586 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Bird, A.P. & Wolffe, A.P. Methylation-induced repression—belts, braces, and chromatin. Cell 99, 451–454 (1999).

    CAS  Article  Google Scholar 

  13. 13

    Feinberg, A.P. & Vogelstein, B. Alterations in DNA methylation in human colon neoplasia. Semin. Surg. Oncol. 3, 149–151 (1987).

    CAS  Article  Google Scholar 

  14. 14

    Herman, J.G. & Baylin, S.B. Gene silencing in cancer in association with promoter hypermethylation. N. Engl. J. Med. 349, 2042–2054 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Jones, P.A. & Baylin, S.B. The epigenomics of cancer. Cell 128, 683–692 (2007).

    CAS  Article  Google Scholar 

  16. 16

    Clark, S.J., Statham, A., Stirzaker, C., Molloy, P.L. & Frommer, M. DNA methylation: bisulphite modification and analysis. Nat. Protoc. 1, 2353–2364 (2006).

    CAS  Article  Google Scholar 

  17. 17

    Gius, D. et al. The epigenome as a molecular marker and target. Cancer 104, 1789–1793 (2005).

    CAS  Article  Google Scholar 

  18. 18

    Downward, J. Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 11–22 (2003).

    CAS  Article  Google Scholar 

  19. 19

    Mitin, N., Rossman, K.L. & Der, C.J. Signaling interplay in Ras superfamily function. Curr. Biol. 15, R563–R574 (2005).

    CAS  Article  Google Scholar 

  20. 20

    Walker, S.A. et al. Identification of a Ras GTPase-activating protein regulated by receptor-mediated Ca2+ oscillations. EMBO J. 23, 1749–1760 (2004).

    CAS  Article  Google Scholar 

  21. 21

    Barbacid, M. Ras genes. Annu. Rev. Biochem. 56, 779–827 (1987).

    CAS  Article  Google Scholar 

  22. 22

    Bos, J.L. Ras oncogenes in human cancer: a review. Cancer Res. 49, 4682–4689 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Arun, D. & Gutmann, D.H. Recent advances in neurofibromatosis type 1. Curr. Opin. Neurol. 17, 101–105 (2004).

    CAS  Article  Google Scholar 

  24. 24

    Kolfschoten, I.G. et al. A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell 121, 849–858 (2005).

    CAS  Article  Google Scholar 

  25. 25

    Jin, H. et al. Epigenetic silencing of a Ca2+-regulated Ras GTPase-activating protein RASAL defines a new mechanism of Ras activation in human cancers. Proc. Natl. Acad. Sci. USA 104, 12353–12358 (2007).

    CAS  Article  Google Scholar 

  26. 26

    Gazin, C., Wajapeyee, N., Gobeil, S., Virbasius, C.M. & Green, M.R. An elaborate pathway required for Ras-mediated epigenetic silencing. Nature 449, 1073–1077 (2007).

    CAS  Article  Google Scholar 

  27. 27

    Lloyd, C.M. et al. RANTES and monocyte chemoattractant protein-1 (MCP-1) play an important role in the inflammatory phase of crescentic nephritis, but only MCP-1 is involved in crescent formation and interstitial fibrosis. J. Exp. Med. 185, 1371–1380 (1997).

    CAS  Article  Google Scholar 

  28. 28

    Zeisberg, M. et al. BMP-7 counteracts TGF-β1–induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat. Med. 9, 964–968 (2003).

    CAS  Article  Google Scholar 

  29. 29

    Witzgall, R., Brown, D., Schwarz, C. & Bonventre, J.V. 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).

    CAS  Article  Google Scholar 

  30. 30

    Oliver, J.A., Maarouf, O., Cheema, F.H., Martens, T.P. & Al-Awqati, Q. The renal papilla is a niche for adult kidney stem cells. J. Clin. Invest. 114, 795–804 (2004).

    CAS  Article  Google Scholar 

  31. 31

    Hendry, B.M. & Sharpe, C.C. Targeting Ras genes in kidney disease. Nephron 93, e129–e133 (2003).

    CAS  PubMed  Google Scholar 

  32. 32

    Li, E., Beard, C. & Jaenisch, R. Role for DNA methylation in genomic imprinting. Nature 366, 362–365 (1993).

    CAS  Article  Google Scholar 

  33. 33

    Okano, M., Bell, D.W., Haber, D.A. & Li, E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–257 (1999).

    CAS  Article  Google Scholar 

  34. 34

    Li, E., Bestor, T.H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926 (1992).

    CAS  Article  Google Scholar 

  35. 35

    Bottinger, E.P. & Bitzer, M. TGF-β signaling in renal disease. J. Am. Soc. Nephrol. 13, 2600–2610 (2002).

    Article  Google Scholar 

  36. 36

    Border, W.A. & Noble, N.A. Targeting TGF-β for treatment of disease. Nat. Med. 1, 1000–1001 (1995).

    CAS  Article  Google Scholar 

  37. 37

    Strutz, F. et al. TGF-β1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor (FGF-2). Kidney Int. 59, 579–592 (2001).

    CAS  Article  Google Scholar 

  38. 38

    Basile, D.P. et al. Identification of persistently altered gene expression in the kidney after functional recovery from ischemic acute renal failure. Am. J. Physiol. Renal Physiol. 288, F953–F963 (2005).

    CAS  Article  Google Scholar 

  39. 39

    Forbes, J.M., Leaker, B., Hewitson, T.D., Becker, G.J. & Jones, C.L. Macrophage and myofibroblast involvement in ischemic acute renal failure is attenuated by endothelin receptor antagonists. Kidney Int. 55, 198–208 (1999).

    CAS  Article  Google Scholar 

  40. 40

    Villanueva, S., Cespedes, C. & Vio, C.P. Ischemic acute renal failure induces the expression of a wide range of nephrogenic proteins. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R861–R870 (2006).

    CAS  Article  Google Scholar 

  41. 41

    Ding, M. et al. Loss of the tumor suppressor Vhlh leads to upregulation of Cxcr4 and rapidly progressive glomerulonephritis in mice. Nat. Med. 12, 1081–1087 (2006).

    CAS  Article  Google Scholar 

  42. 42

    Rothenpieler, U.W. & Dressler, G.R. Pax-2 is required for mesenchyme-to-epithelium conversion during kidney development. Development 119, 711–720 (1993).

    CAS  PubMed  Google Scholar 

  43. 43

    Khwaja, A., Sharpe, C.C., Noor, M., Kloog, Y. & Hendry, B.M. The inhibition of human mesangial cell proliferation by S-trans, trans-farnesylthiosalicylic acid. Kidney Int. 68, 474–486 (2005).

    CAS  Article  Google Scholar 

  44. 44

    Zaidi, N. et al. A new approach for distinguishing cathepsin E and D activity in antigen-processing organelles. FEBS J. 274, 3138–3149 (2007).

    CAS  Article  Google Scholar 

  45. 45

    Zeisberg, E.M. et al. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat. Med. 13, 952–961 (2007).

    CAS  Article  Google Scholar 

  46. 46

    Zeisberg, E.M., Potenta, S.E., Sugimoto, H., Zeisberg, M. & Kalluri, R. Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition. J. Am. Soc. Nephrol. 19, 2282–2287 (2008).

    Article  Google Scholar 

  47. 47

    Chatterjee, P.K. et al. Calpain inhibitor-1 reduces renal ischemia/reperfusion injury in the rat. Kidney Int. 59, 2073–2083 (2001).

    CAS  Article  Google Scholar 

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Acknowledgements

This study was partially funded by research grants R01DK081576 (M.Z.), R03DK081687 (M.Z.), R01DK30932 (D.J.S.), R01DK55001 (R.K.), R01CA125550 (R.K.), Mentored Clinical Scientist Development Awards 1K08 CA129204 (E.M.Z.) and K08 DK074558 (M.Z.) from the National Institutes of Health, the American Society of Nephrology Carl W. Gottschalk Scholar Grant (M.Z.), American Heart Association Scientist Development Grant SDG0735602T, Deutsche Forschungsgemeinschaft-Stipendium BE4211/1-1 (W.B.), Champalimaud Metastasis Research Program (R.K.) and research funds from the Beth Israel Deaconess Medical Center for the Division of Matrix Biology. We thank A. Lau and T. Zhong Hu for technical assistance. FSP-1–specific antibodies were a gift from E. Neilson (Vanderbilt University).

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W.B. performed and designed experiments, analyzed data and edited the manuscript. S.M. performed experiments and analyzed data. E.M.Z. advised, performed experiments, analyzed data and edited the manuscript. G.A.M. and C.A.M. characterized and provided human fibroblasts. H.K. generated Rasal1 antibodies. D.J.S. provided nephrotoxic serum and edited the manuscript. R.K. advised and edited the manuscript. M.Z. designed, performed and supervised experiments, analyzed data and wrote the manuscript.

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Correspondence to Michael Zeisberg.

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Bechtel, W., McGoohan, S., Zeisberg, E. et al. Methylation determines fibroblast activation and fibrogenesis in the kidney. Nat Med 16, 544–550 (2010). https://doi.org/10.1038/nm.2135

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