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. 1997 Jun 15;99(12):2950–2960. doi: 10.1172/JCI119490

The role of transglutaminase in the rat subtotal nephrectomy model of renal fibrosis.

T S Johnson 1, M Griffin 1, G L Thomas 1, J Skill 1, A Cox 1, B Yang 1, B Nicholas 1, P J Birckbichler 1, C Muchaneta-Kubara 1, A Meguid El Nahas 1
PMCID: PMC508147  PMID: 9185519

Abstract

Tissue transglutaminase is a calcium-dependent enzyme that catalyzes the cross-linking of polypeptide chains, including those of extracellular matrix (ECM) proteins, through the formation of epsilon-(gamma-glutamyl) lysine bonds. This crosslinking leads to the formation of protein polymers that are highly resistant to degradation. As a consequence, the enzyme has been implicated in the deposition of ECM protein in fibrotic diseases such as pulmonary fibrosis and atherosclerosis. In this study, we have investigated the involvement of tissue transglutaminase in the development of kidney fibrosis in adult male Wistar rats submitted to subtotal nephrectomy (SNx). Groups of six rats were killed on days 7, 30, 90, and 120 after SNx. As previously described, these rats developed progressive glomerulosclerosis and tubulo-interstitial fibrosis. The tissue level of epsilon-(gamma-glutamyl) lysine cross-link (as determined by exhaustive proteolytic digestion followed by cation exchange chromatography) increased from 3.47+/- 0.94 (mean+/-SEM) in controls to 13.24+/-1.43 nmol/g protein 90 d after SNx, P </= 0.01. Levels of epsilon-(gamma-glutamyl) lysine cross-link correlated well with the renal fibrosis score throughout the 120 observation days (r = 0.78, P </= 0.01). Tissue homogenates showed no significant change in overall transglutaminase activity (14C putrescine incorporation assay) unless adjusted for the loss of viable tubule cells, when an increase from 5.77+/-0.35 to 13.93+/-4.21 U/mg DNA in cytosolic tissue transglutaminase activity was seen. This increase was supported by Western blot analysis, showing a parallel increase in renal tissue transglutaminase content. Immunohistochemistry demonstrated that this large increase in epsilon-(gamma-glutamyl) lysine cross-link and tissue transglutaminase took place predominantly in the cytoplasm of tubular cells, while immunofluorescence also showed low levels of the epsilon-(gamma-glutamyl) lysine cross-link in the extracellular renal interstitial space. The number of cells showing increases in tissue transglutaminase and its cross-link product, epsilon-(gamma-glutamyl) lysine appeared greater than those showing signs of typical apoptosis as determined by in situ end-labeling. This observed association between tissue transglutaminase, epsilon-(gamma-glutamyl) lysine cross-link, and renal tubulointerstitial scarring in rats submitted to SNx suggests that tissue transglutaminase may play an important role in the development of experimental renal fibrosis and the associated loss of tubule integrity.

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Selected References

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  1. Aeschlimann D., Kaupp O., Paulsson M. Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate. J Cell Biol. 1995 May;129(3):881–892. doi: 10.1083/jcb.129.3.881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aeschlimann D., Paulsson M. Cross-linking of laminin-nidogen complexes by tissue transglutaminase. A novel mechanism for basement membrane stabilization. J Biol Chem. 1991 Aug 15;266(23):15308–15317. [PubMed] [Google Scholar]
  3. Ando T., Okuda S., Tamaki K., Yoshitomi K., Fujishima M. Localization of transforming growth factor-beta and latent transforming growth factor-beta binding protein in rat kidney. Kidney Int. 1995 Mar;47(3):733–739. doi: 10.1038/ki.1995.112. [DOI] [PubMed] [Google Scholar]
  4. Basile D. P., Rovak J. M., Martin D. R., Hammerman M. R. Increased transforming growth factor-beta 1 expression in regenerating rat renal tubules following ischemic injury. Am J Physiol. 1996 Mar;270(3 Pt 2):F500–F509. doi: 10.1152/ajprenal.1996.270.3.F500. [DOI] [PubMed] [Google Scholar]
  5. Billett H. H., Puszkin E. G. The red cell membrane contains calmodulin-regulated crosslinking and proteolytic activity. Hematol Pathol. 1991;5(4):185–193. [PubMed] [Google Scholar]
  6. Birckbichler P. J., Upchurch H. F., Patterson M. K., Jr, Conway E. A monoclonal antibody to cellular transglutaminase. Hybridoma. 1985 Summer;4(2):179–186. doi: 10.1089/hyb.1985.4.179. [DOI] [PubMed] [Google Scholar]
  7. Birkedal-Hansen H., Moore W. G., Bodden M. K., Windsor L. J., Birkedal-Hansen B., DeCarlo A., Engler J. A. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med. 1993;4(2):197–250. doi: 10.1177/10454411930040020401. [DOI] [PubMed] [Google Scholar]
  8. Border W. A., Noble N. A. Cytokines in kidney disease: the role of transforming growth factor-beta. Am J Kidney Dis. 1993 Jul;22(1):105–113. doi: 10.1016/s0272-6386(12)70175-0. [DOI] [PubMed] [Google Scholar]
  9. Bowness J. M., Folk J. E., Timpl R. Identification of a substrate site for liver transglutaminase on the aminopropeptide of type III collagen. J Biol Chem. 1987 Jan 25;262(3):1022–1024. [PubMed] [Google Scholar]
  10. Bowness J. M., Tarr A. H. Lipoprotein binding of crosslinked type III collagen aminopropeptide and fractions of its antigen in blood. Biochem Biophys Res Commun. 1990 Jul 31;170(2):519–525. doi: 10.1016/0006-291x(90)92122-g. [DOI] [PubMed] [Google Scholar]
  11. Bowness J. M., Tarr A. H., Wiebe R. I. Transglutaminase-catalysed cross-linking: a potential mechanism for the interaction of fibrinogen, low density lipoprotein and arterial type III procollagen. Thromb Res. 1989 May 15;54(4):357–367. doi: 10.1016/0049-3848(89)90094-7. [DOI] [PubMed] [Google Scholar]
  12. Bowness J. M., Tarr A. H., Wong T. Increased transglutaminase activity during skin wound healing in rats. Biochim Biophys Acta. 1988 Nov 17;967(2):234–240. doi: 10.1016/0304-4165(88)90014-1. [DOI] [PubMed] [Google Scholar]
  13. Bowness J. M., Venditti M., Tarr A. H., Taylor J. R. Increase in epsilon(gamma-glutamyl)lysine crosslinks in atherosclerotic aortas. Atherosclerosis. 1994 Dec;111(2):247–253. doi: 10.1016/0021-9150(94)90099-x. [DOI] [PubMed] [Google Scholar]
  14. Cai D., Ben T., De Luca L. M. Retinoids induce tissue transglutaminase in NIH-3T3 cells. Biochem Biophys Res Commun. 1991 Mar 29;175(3):1119–1124. doi: 10.1016/0006-291x(91)91681-2. [DOI] [PubMed] [Google Scholar]
  15. Campbell R. A. Polyamines and uremia. Adv Exp Med Biol. 1987;223:47–54. doi: 10.1007/978-1-4684-5445-1_6. [DOI] [PubMed] [Google Scholar]
  16. Cohen G. M., Sun X. M., Fearnhead H., MacFarlane M., Brown D. G., Snowden R. T., Dinsdale D. Formation of large molecular weight fragments of DNA is a key committed step of apoptosis in thymocytes. J Immunol. 1994 Jul 15;153(2):507–516. [PubMed] [Google Scholar]
  17. Dolynchuk K. N., Bendor-Samuel R., Bowness J. M. Effect of putrescine on tissue transglutaminase activity in wounds: decreased breaking strength and increased matrix fucoprotein solubility. Plast Reconstr Surg. 1994 Mar;93(3):567–573. [PubMed] [Google Scholar]
  18. Dudek S. M., Johnson G. V. Transglutaminase catalyzes the formation of sodium dodecyl sulfate-insoluble, Alz-50-reactive polymers of tau. J Neurochem. 1993 Sep;61(3):1159–1162. doi: 10.1111/j.1471-4159.1993.tb03636.x. [DOI] [PubMed] [Google Scholar]
  19. Eddy A. A., Giachelli C. M. Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria. Kidney Int. 1995 Jun;47(6):1546–1557. doi: 10.1038/ki.1995.218. [DOI] [PubMed] [Google Scholar]
  20. Fesus L., Davies P. J., Piacentini M. Apoptosis: molecular mechanisms in programmed cell death. Eur J Cell Biol. 1991 Dec;56(2):170–177. [PubMed] [Google Scholar]
  21. Fesus L., Thomazy V., Autuori F., Ceru M. P., Tarcsa E., Piacentini M. Apoptotic hepatocytes become insoluble in detergents and chaotropic agents as a result of transglutaminase action. FEBS Lett. 1989 Mar 13;245(1-2):150–154. doi: 10.1016/0014-5793(89)80210-8. [DOI] [PubMed] [Google Scholar]
  22. Fesus L., Thomazy V., Falus A. Induction and activation of tissue transglutaminase during programmed cell death. FEBS Lett. 1987 Nov 16;224(1):104–108. doi: 10.1016/0014-5793(87)80430-1. [DOI] [PubMed] [Google Scholar]
  23. Floege J., Johnson R. J., Gordon K., Yoshimura A., Campbell C., Iruela-Arispe L., Alpers C. E., Couser W. G. Altered glomerular extracellular matrix synthesis in experimental membranous nephropathy. Kidney Int. 1992 Sep;42(3):573–585. doi: 10.1038/ki.1992.321. [DOI] [PubMed] [Google Scholar]
  24. Folk J. E., Finlayson J. S. The epsilon-(gamma-glutamyl)lysine crosslink and the catalytic role of transglutaminases. Adv Protein Chem. 1977;31:1–133. doi: 10.1016/s0065-3233(08)60217-x. [DOI] [PubMed] [Google Scholar]
  25. Folk J. E. Transglutaminases. Annu Rev Biochem. 1980;49:517–531. doi: 10.1146/annurev.bi.49.070180.002505. [DOI] [PubMed] [Google Scholar]
  26. George M. D., Vollberg T. M., Floyd E. E., Stein J. P., Jetten A. M. Regulation of transglutaminase type II by transforming growth factor-beta 1 in normal and transformed human epidermal keratinocytes. J Biol Chem. 1990 Jul 5;265(19):11098–11104. [PubMed] [Google Scholar]
  27. Greenberg C. S., Achyuthan K. E., Borowitz M. J., Shuman M. A. The transglutaminase in vascular cells and tissues could provide an alternate pathway for fibrin stabilization. Blood. 1987 Sep;70(3):702–709. [PubMed] [Google Scholar]
  28. Greenberg C. S., Birckbichler P. J., Rice R. H. Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. FASEB J. 1991 Dec;5(15):3071–3077. doi: 10.1096/fasebj.5.15.1683845. [DOI] [PubMed] [Google Scholar]
  29. Griffin M., Smith L. L., Wynne J. Changes in transglutaminase activity in an experimental model of pulmonary fibrosis induced by paraquat. Br J Exp Pathol. 1979 Dec;60(6):653–661. [PMC free article] [PubMed] [Google Scholar]
  30. Griffin M., Wilson J. Detection of epsilon(gamma-glutamyl) lysine. Mol Cell Biochem. 1984;58(1-2):37–49. doi: 10.1007/BF00240603. [DOI] [PubMed] [Google Scholar]
  31. Hornyak T. J., Shafer J. A. Interactions of factor XIII with fibrin as substrate and cofactor. Biochemistry. 1992 Jan 21;31(2):423–429. doi: 10.1021/bi00117a017. [DOI] [PubMed] [Google Scholar]
  32. Howie A. J., Gunson B. K., Sparke J. Morphometric correlates of renal excretory function. J Pathol. 1990 Mar;160(3):245–253. doi: 10.1002/path.1711600311. [DOI] [PubMed] [Google Scholar]
  33. Jeong J. M., Murthy S. N., Radek J. T., Lorand L. The fibronectin-binding domain of transglutaminase. J Biol Chem. 1995 Mar 10;270(10):5654–5658. doi: 10.1074/jbc.270.10.5654. [DOI] [PubMed] [Google Scholar]
  34. Johnson T. S., Knight C. R., el-Alaoui S., Mian S., Rees R. C., Gentile V., Davies P. J., Griffin M. Transfection of tissue transglutaminase into a highly malignant hamster fibrosarcoma leads to a reduced incidence of primary tumour growth. Oncogene. 1994 Oct;9(10):2935–2942. [PubMed] [Google Scholar]
  35. Kleman J. P., Aeschlimann D., Paulsson M., van der Rest M. Transglutaminase-catalyzed cross-linking of fibrils of collagen V/XI in A204 rhabdomyosarcoma cells. Biochemistry. 1995 Oct 24;34(42):13768–13775. doi: 10.1021/bi00042a007. [DOI] [PubMed] [Google Scholar]
  36. Knight C. R., Rees R. C., Elliott B. M., Griffin M. The existence of an inactive form of transglutaminase within metastasising tumours. Biochim Biophys Acta. 1990 Jun 12;1053(1):13–20. doi: 10.1016/0167-4889(90)90019-a. [DOI] [PubMed] [Google Scholar]
  37. Knight C. R., Rees R. C., Griffin M. Apoptosis: a potential role for cytosolic transglutaminase and its importance in tumour progression. Biochim Biophys Acta. 1991 Jun 5;1096(4):312–318. doi: 10.1016/0925-4439(91)90067-j. [DOI] [PubMed] [Google Scholar]
  38. Knight R. L., Hand D., Piacentini M., Griffin M. Characterization of the transglutaminase-mediated large molecular weight polymer from rat liver; its relationship to apoptosis. Eur J Cell Biol. 1993 Feb;60(1):210–216. [PubMed] [Google Scholar]
  39. Kojima S., Nara K., Rifkin D. B. Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells. J Cell Biol. 1993 Apr;121(2):439–448. doi: 10.1083/jcb.121.2.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  41. Lorand L., Conrad S. M. Transglutaminases. Mol Cell Biochem. 1984;58(1-2):9–35. doi: 10.1007/BF00240602. [DOI] [PubMed] [Google Scholar]
  42. Lorand L., Conrad S. M., Velasco P. T. Inhibition of beta-crystallin cross-linking in the Ca2+-treated lens. Invest Ophthalmol Vis Sci. 1987 Jul;28(7):1218–1222. [PubMed] [Google Scholar]
  43. Peten E. P., Striker L. J., Carome M. A., Elliott S. J., Yang C. W., Striker G. E. The contribution of increased collagen synthesis to human glomerulosclerosis: a quantitative analysis of alpha 2IV collagen mRNA expression by competitive polymerase chain reaction. J Exp Med. 1992 Dec 1;176(6):1571–1576. doi: 10.1084/jem.176.6.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rice R. H., Green H. The cornified envelope of terminally differentiated human epidermal keratinocytes consists of cross-linked protein. Cell. 1977 Jun;11(2):417–422. doi: 10.1016/0092-8674(77)90059-9. [DOI] [PubMed] [Google Scholar]
  45. Riemenschneider T., Mackensen-Haen S., Christ H., Bohle A. Correlation between endogenous creatinine clearance and relative interstitial volume of the renal cortex in patients with diffuse membranous glomerulonephritis having a normal serum creatinine concentration. Lab Invest. 1980 Aug;43(2):145–149. [PubMed] [Google Scholar]
  46. Schulze-Osthoff K., Bauer M. K., Vogt M., Los M. Role of ICE-related and other proteases in Fas-mediated apoptosis. Cell Death Differ. 1996 Apr;3(2):177–184. [PubMed] [Google Scholar]
  47. Selkoe D. J., Abraham C., Ihara Y. Brain transglutaminase: in vitro crosslinking of human neurofilament proteins into insoluble polymers. Proc Natl Acad Sci U S A. 1982 Oct;79(19):6070–6074. doi: 10.1073/pnas.79.19.6070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sugiyama H., Kashihara N., Makino H., Yamasaki Y., Ota A. Apoptosis in glomerular sclerosis. Kidney Int. 1996 Jan;49(1):103–111. doi: 10.1038/ki.1996.14. [DOI] [PubMed] [Google Scholar]
  49. Upchurch H. F., Conway E., Patterson M. K., Jr, Maxwell M. D. Localization of cellular transglutaminase on the extracellular matrix after wounding: characteristics of the matrix bound enzyme. J Cell Physiol. 1991 Dec;149(3):375–382. doi: 10.1002/jcp.1041490304. [DOI] [PubMed] [Google Scholar]
  50. el Alaoui S., Legastelois S., Roch A. M., Chantepie J., Quash G. Transglutaminase activity and N epsilon (gamma glutamyl) lysine isopeptide levels during cell growth: an enzymic and immunological study. Int J Cancer. 1991 May 10;48(2):221–226. doi: 10.1002/ijc.2910480212. [DOI] [PubMed] [Google Scholar]
  51. el Nahas A. M., Bassett A. H., Cope G. H., Le Carpentier J. E. Role of growth hormone in the development of experimental renal scarring. Kidney Int. 1991 Jul;40(1):29–34. doi: 10.1038/ki.1991.175. [DOI] [PubMed] [Google Scholar]

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