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.