Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Augmented cytoplasmic Smad4 induces acceleration of TGF-β1 signaling in renal tubulointerstitial cells of hereditary nephrotic ICGN mice with chronic renal fibrosis; possible role for myofibroblastic differentiation


The Institute of Cancer Research (ICR)-derived glomerulonephritis (ICGN) mouse is a hereditary model animal for nephrotic syndrome with chronic renal tubulointerstitial fibrosis. In most fibrotic diseases, myofibroblastic differentiation is considered to play crucial roles in pathogenesis of fibrosis and is dominantly regulated by the transforming growth factor (TGF)-β1 signaling system. To reveal the pathogenic mechanism of chronic renal fibrosis in ICGN mice, we examined the expression and localization of TGF-β1 signal transducer proteins (TGF-β receptor-I and -II, Smad2/3 and Smad4) in kidney sections and in primarily cultured tubulointerstitial fibroblasts (TIFs). In kidneys of ICGN mice, many tubulointerstitial cells were differentiated to myofibroblastic cells and were α-smooth muscle actin (αSMA)-positive. The numbers of αSMA-positive TIFs prepared from kidneys of ICGN mice (ICGN-TIFs), but not those of ICR control mice (ICR-TIFs), increased during cell culture. No significant differences in production or activation of TGF-β1 between ICGN-TIFs and ICR-TIFs were seen by enzyme-linked immunosorbent assay. In vitro transcriptional reporter assay for TGF-β1 and Western immunoblotting for TGF-β1 signal transducers showed no notable differences in the expression levels of TGF-β receptor-I or -II or Smad2/3 between these TIFs. However, augumented cytoplasmic Smad4 protein in ICGN-TIFs, but not ICR-TIFs, seemed to cause hypersensitivity against TGF-β1, and the eventual nuclear localization of Smad2/3-Smad4 complex was increased in ICGN-TIFs. Thus, the abnormal cytoplasmic augmentation of Smad4 induces acceleration of TGF-β1 signaling in the renal tubulointerstitial cells of ICGN mice.

This is a preview of subscription content, log in to check access.

Fig. 1A–E
Fig. 2A, B
Fig. 3A–F
Fig. 4A–F
Fig. 5A–G
Fig. 6A, B
Fig. 7A–G
Fig. 8


  1. Alvarez RJ, Sun MJ, Haverty TP, Iozzo RV, Myers JC, Neilson EG (1992) Biosynthetic and proliferative characteristics of tubulointerstitial fibroblasts probed with paracrine cytokines. Kidney Int 41:14–23

  2. Blobe GC, Schiemann WP, Lodish HF (2000) Role of transforming growth factor β in human disease. N Engl J Med 342:1350–1358

  3. Border WA, Ruoslahti E (1992) Transforming growth factor-β in disease: the dark side of tissue repair. J Clin Invest 90:1–7

  4. Bottinger EP, Kopp JB (1998) Lessons from TGF-β transgenic mice. Miner Electrolyte Metab 24:154–160

  5. Chacko BM, Qin B, Correia JJ, Lam SS, Caestecker MP de, Lin K (2001) The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization. Nat Struct Biol 8: 248–253

  6. Chuang YH, Chuang WL, Chen SS, Huang CH (2000) Expression of transforming growth factor-β1 and its receptors related to the ureteric fibrosis in a rat model of obstructive uropathy. J Urol 163:1298–1303

  7. Desmouliére A, Geinoz A, Gabbiani F, Gabbiani G (1993) Transforming growth factor-β1 induces α-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 122:103–111

  8. Dooley S, Streckert M, Delvoux B, Gressner AM (2001) Expression of Smads during in vitro transdifferentiation of hepatic stellate cells to myofibroblasts. Biochem Biophys Res Commun 283:554–562

  9. Evans RA, Tian YC, Steadman R, Phillips AO (2003) TGF-β1-mediated fibroblast-myofibroblast terminal differentiation—the role of smad proteins. Exp Cell Res 282:90–100

  10. Feng XH, Derynck R (1996) Ligand-independent activation of transforming growth factor (TGF) β signaling pathways by heteromeric cytoplasmic domains of TGF-β receptors. J Biol Chem 271:13123–13129

  11. Fukuda K, Yoshitomi K, Yanagida T, Tokumoto M, Hirakata H (2001) Quantification of TGF-β1 mRNA along rat nephron in obstructive nephropathy. Am J Physiol Renal Physiol 281:513–521

  12. Gabbiani G (1996) The cellular derivation and the life span of the myofibroblast. Pathol Res Pract 192:708–711

  13. Gorsch SM, Memoli VA, Stukel TA, Gold LI, Arrick BA(1992) Immunohistochemical staining for transforming growth factor β1 associates with disease progression in human breast cancer. Cancer Res 52:6949–6952

  14. Guo Y, Kyprianou N (1998) Overexpression of transforming growth factor (TGF) β1 type II receptor restores TGF-β1 sensitivity and signaling in human prostate cancer cells. Cell Growth Differ 9:185–193

  15. Hahn SA, Hoque AT, Moskaluk CA, Costa LT da, Schutte M, Rozenblum E, Seymour AB, Weinstein CL, Yeo CJ, Hruban RH, Kern SE (1996) Homozygous deletion map at 18q21.1 in pancreatic cancer. Cancer Res 56:490–494

  16. Hao J, Ju H, Zhao S, Junaid A, Scammell-La Fleur T, Dixon IM (1999) Elevation of expression of Smads 2, 3, and 4, decorin and TGF-β in the chronic phase of myocardial infarct scar healing. J Mol Cell Cardiol 31:667–678

  17. Hong SW, Isono M, Chen S, Iglesias-De La Cruz MC, Han DC, Ziyadeh FN (2001) Increased glomerular and tubular expression of transforming growth factor-β1, its type II receptor, and activation of the Smad signaling pathway in the db/db mouse. Am J Pathol 158:1653–1663

  18. Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG (2002) Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 110:341–350

  19. Jones CL, Buch S, Post M, McCulloch L, Liu E, Eddy AA (1991) Pathogenesis of interstitial fibrosis in chronic purine aminonucleoside nephrosis. Kidney Int 40:1020–1031

  20. Kawabata M, Inoue H, Hanyu A, Imamura T, Miyazono K (1998) Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors. EMBOJ 17:4056–4065

  21. Khalil N, Parekh TV, O’Connor R, Antman N, Kepron W, Yehaulaeshet T, Xu YD, Gold LI (2001) Regulation of the effects of TGF-β1 by activation of latent TGF-β1 and differential expression of TGF-β receptors (Tβ R-I and Tβ R-II) in idiopathic pulmonary fibrosis. Thorax 56:907–915

  22. Liu C, Gaca MD, Swenson ES, Vellucci VF, Reiss M, Wells RG (2003) Smads 2 and 3 are differentially activated by TGF-β in quiescent and activated hepatic stellate cells: constitutive nuclear localization of Smads in activated cells is TGF-β independent. J Biol Chem 278: 11721–11728

  23. Martin M, Lefaix J, Delanian S (2000) TGF-β1 and radiation fibrosis: a master switch and a specific therapeutic target? Int J Radiat Oncol Biol Phys 47:277–290

  24. Massague J (1990) The transforming growth factor-β family. Annu Rev Cell Biol 6:597–641

  25. Massague J (1998) TGF-β signal transduction. Annu Rev Biochem 67:753–791

  26. Miyaki M, Kuroki T (2003) Role of Smad4 (DPC4) inactivation in human cancer. Biochem Biophys Res Commun 306:799–804

  27. Mori Y, Chen SJ, Varga J (2000) Modulation of endogenous Smad expression in normal skin fibroblasts by transforming growth factor-β. Exp Cell Res 258:374–383

  28. Moses HL, Yang EY, Pietenpol JA (1990) TGF-β stimulation and inhibition of cell proliferation: new mechanistic insights. Cell 63:245–247

  29. Ogura A, Asano T, Matsuda J, Noguchi Y, Yamamoto Y, Takano K, Nakagawa M (1989a) Development of nephritic ICGN mice: the origin, reproduction ability, and incidence of glomerulonephritis. Exp Anim 38:349–352

  30. Ogura A, Asano T, Matsuda J, Takano K, Nakagawa M, Fukui M (1989b) Characteristics of mutant mice (ICGN) with spontaneous renal lesion: a new model for human nephrotic syndrome. Lab Anim 23:169–174

  31. Ogura A, Asano T, Matsuda J, Takano K, Koura M, Nakagawa M, Kawaguchi H, Yamaguchi Y (1990) An electron microscopic study of glomerular lesions in hereditary nephrotic mice (ICGN strain). Virchows Archiv [A] 417:223–228

  32. Ogura A, Asano T, Matsuda J, Fujimura H (1991) Evolution of glomerular lesions in nephrotic ICGN mice: serial biopsy study with electron microscopy. J Vet Med Sci 53:513–515

  33. Ogura A, Asano T, Suzuki O, Yamamoto Y, Noguchi Y, Kawaguchi H, Yamaguchi Y (1994) Hereditary nephrotic syndrome with progression to renal failure in a mouse model (ICGN strain): clinical study. Nephron 68:239–244

  34. Ogura A, Fujimura H, Asano T, Koura M, Naito I, Kobayashi Y (1995) Early ultrastructural glomerular alterations of neonatal nephrotic mice (ICGN strain). Vet Pathol 32:321–323

  35. Okamoto M, Oyasu R (1997) Overexpression of transforming growth factor β type I receptor abolishes malignant phenotype of a rat bladder carcinoma cell line. Cell Growth Differ 8:921–926

  36. Park IS, Kiyomoto H, Abboud SL, Abboud HE (1997) Expression of transforming growth factor-β and type IV collagen in early streptozotocin-induced diabetes. Diabetes 46:473–480

  37. Powell DW, Mifflin RC, Valentich JD, Crowe SE, Saada JI, West AB (1999) Myofibroblasts. I. Paracrine cells important in health and disease. Am J Physiol 277:C1–C9

  38. Reisdorf P, Lawrence DA, Sivan V, Klising E, Martin MT (2001) Alteration of transforming growth factor-β1 response involves down-regulation of Smad3 signaling in myofibroblasts from skin fibrosis. Am J Pathol 159:263–272

  39. Rieder H, Armbrust T, Meyer zum Buschenfelde KH, Ramadori G (1993) Contribution of sinusoidal endothelial liver cells to liver fibrosis: expression of transforming growth factor-β1 receptors and modulation of plasmin-generating enzymes by transforming growth factor-β1. Hepatology 18:937–944

  40. Ronnov-Jessen L, Petersen OW (1993) Induction of α-smooth muscle actin by transforming growth factor-β1 in quiescent human breast gland fibroblasts: implications for myofibroblast generation in breast neoplasia. Lab Invest 68:696–707

  41. Schreiber E, Matthias P, Muller MM, Schaffner W (1989) Rapid detection of octamer binding proteins with “mini-extracts”, prepared from a small number of cells. Nucleic Acids Res 17:6419

  42. Serini G, Gabbiani G (1999) Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res 250:273–283

  43. Terrell TG, Working PK, Chow CP, Green JD (1993) Pathology of recombinant human transforming growth factor-β1 in rats and rabbits. Int Rev Exp Pathol. 34:43–67

  44. Tsuji T, Okada F, Yamaguchi K, Nakamura T (1990) Molecular cloning of the large subunit of transforming growth factor type β masking protein and expression of the mRNA in various rat tissues. Proc Natl Acad Sci USA 87:8835–8839

  45. Uchio K, Manabe N, Kinoshita A, Tamura K, Miyamoto M, Ogura A, Yamamoto Y, Miyamoto H (1999) Abnormalities of extracellular matrices and transforming growth factor β1 localization in the kidney of the hereditary nephrotic mice (ICGN strain). J Vet Med Sci 61:769–776

  46. Uchio K, Manabe N, Tamura K, Miyamoto M, Yamaguchi M, Ogura A, Yamamoto Y, Miyamoto H (2000) Decreased matrix metalloproteinase activity in the kidneys of hereditary nephrotic mice (ICGN strain). Nephron 86:145–151

  47. Uchio-Yamada K, Manabe N, Yamaguchi M, Akashi N, Goto Y, Yamamoto Y, Ogura A, Miyamoto H (2001) Localization of extracellular matrix receptors in ICGN mice, a strain of mice with hereditary nephrotic syndrome. J Vet Med Sci 63:1171–1178

  48. Vries CJ de, Boer J de, Joore J, Strahle U, Achterberg TA van, Huylebroeck D, Verschueren K, Miyazono K, Eijnden-van Raaij AJ van den, Zivkovic D (1996) Active complex formation of type I and type II activin and TGF β receptors in vivo as studied by overexpression in zebrafish embryos. Mech Dev 54:225–236

  49. Westergren-Thorsson G, Hernnas J, Sarnstrand B, Oldberg A, Heinegard D, Malmstrom A (1993) Altered expression of small proteoglycans, collagen, and transforming growth factor-β1 in developing bleomycin-induced pulmonary fibrosis in rats. J Clin Invest 92:632–637

  50. Wolf G, Killen PD, Neilson EG (1990) Cyclosporin A stimulates transcription and procollagen secretion in tubulointerstitial fibroblasts and proximal tubular cells. J Am Soc Nephrol 1:918–922

  51. Yang J, Liu Y (2001) Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis. Am J Pathol 159:1465–1475

  52. Zonneveld AJ van, Curriden SA, Loskutoff DJ (1988) Type 1 plasminogen activator inhibitor gene: functional analysis and glucocorticoid regulation of its promoter. Proc Natl Acad Sci USA 85:5525–5529

Download references

Author information

Correspondence to N. Manabe.

Additional information

This work was supported by Grant-in-Aid for Creative Scientific Research (13GS0008) and Grant-in-Aid for Scientific Research on Priority Areas (A) (13027241) to N.M. from the Ministry of Education, Culture, Sports, Science, and Technology, Japan

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Goto, Y., Manabe, N., Uchio-Yamada, K. et al. Augmented cytoplasmic Smad4 induces acceleration of TGF-β1 signaling in renal tubulointerstitial cells of hereditary nephrotic ICGN mice with chronic renal fibrosis; possible role for myofibroblastic differentiation. Cell Tissue Res 315, 209–221 (2004). https://doi.org/10.1007/s00441-003-0824-z

Download citation


  • Transforming growth factor-β1
  • Renal tubulointerstitial fibrosis
  • Myofibroblast
  • Smad proteins
  • Mouse (hereditary nephrotic ICGN)