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.