Increases in renal epsilon-(gamma-glutamyl)-lysine crosslinks result from compartment-specific changes in tissue transglutaminase in early experimental diabetic nephropathy: Pathologic implications

Diabetic nephropathy (DN) is characterized by an early, progressive expansi on and sclerosis of the glomerular mesangium leading to glomerulosclerosis. This is associated with parallel fibrosis of the renal interstitium. In ex perimental renal scarring, the protein cross-linking enzyme, tissue transgl utaminase (tTg), is up-regulated and externalized causing an increase in it s crosslink product, epsilon-(gamma -glutamyl)-lysine, in the extracellular space. This potentially contributes to the extracellular matrix (ECM) accu mulation central to tissue fibrosis by increasing deposition and inhibiting breakdown. We investigated if a similar mechanism may contribute to the EC M expansion characteristic: of UN using the rat streptozotocin model over 1 20 days. Whole kidney epsilon-(gamma -glutamyl)-lysine (HPLC analysis) was significantly increased from Day 90 (+337%) and peaked at Day 120 (+650%) ( p < 0.05). Immunofluorescence showed this increase to be predominantly extr acellular in the peritubular interstitial space, but also in individual glo meruli. Total kidney transglutaminase (Tg) was not elevated. However, using a Tg in situ activity assay, increased Tg was detected in both the extrace llular interstitial space and glomeruli by Day 60, with a maximal 53%, incr ease at Day 120 (p < 0.05). Using a specific anti-tTg antibody, immunohisto chemistry showed a similar increase in extracellular enzyme in the intersti tium and glomeruli. To biochemically characterize glomerular changes, glome ruli were isolated by selective sieving. In line with whole kidney measurem ent, there was an increase in glomerular epsilon-(gamma -glutamyl) lysine ( +361%); however, in the glomeruli this was associated with increases in Tg activity (+228%) and tTg antigen by Western blotting (+215%). Importantly, the ratio of glomerular epsilon-(gamma -glutamyl) lysine to hydroxyproline increased by 2.2-fold. In DN, changes in the kidney result in increased tra nslocation of tig to the extracellular environment where high Ca2+ and low GTP levels allow its activation. In the tubulointerstitium this is independ ent of increased tTg production, but dependent in the glomerulus. This lead s to excessive ECM cross-linking, contributing to the renal fibrosis charac teristic of progressive DN.