Increases in Renal ε-(γ-Glutamyl)-Lysine Crosslinks Result from Compartment-Specific Changes in Tissue Transglutaminase in Early Experimental Diabetic Nephropathy: Pathologic Implications

Abstract

SUMMARY:Diabetic nephropathy (DN) is characterized by an early, progressive expansion and sclerosis of the glomerular mesangium leading to glomerulosclerosis. This is associated with parallel fibrosis of the renal interstitium. In experimental renal scarring, the protein cross-linking enzyme, tissue transglutaminase (tTg), is up-regulated and externalized causing an increase in its crosslink product, ⑀-(␥-glutamyl)-lysine, in the extracellular space. This potentially contributes to the extracellular matrix (ECM) accumulation central to tissue fibrosis by increasing deposition and inhibiting breakdown. We investigated if a similar mechanism may contribute to the ECM expansion characteristic of DN using the rat streptozotocin model over 120 days. Whole kidney ⑀-(␥-glutamyl)-lysine (HPLC analysis) was significantly increased from Day 90 (ϩ337%) and peaked at Day 120 (ϩ650%) (p Ͻ 0.05). Immunofluorescence showed this increase to be predominantly extracellular in the peritubular interstitial space, but also in individual glomeruli. Total kidney transglutaminase (Tg) was not elevated. However, using a Tg in situ activity assay, increased Tg was detected in both the extracellular interstitial space and glomeruli by Day 60, with a maximal 53% increase at Day 120 (p Ͻ 0.05). Using a specific anti-tTg antibody, immunohistochemistry showed a similar increase in extracellular enzyme in the interstitium and glomeruli. To biochemically characterize glomerular changes, glomeruli were isolated by selective sieving. In line with whole kidney measurement, there was an increase in glomerular ⑀-(␥-glutamyl) lysine (ϩ361%); however, in the glomeruli this was associated with increases in Tg activity (ϩ228%) and tTg antigen by Western blotting (ϩ215%). Importantly, the ratio of glomerular ⑀-(␥-glutamyl) lysine to hydroxyproline increased by 2.2-fold. In DN, changes in the kidney result in increased translocation of tTg to the extracellular environment where high Ca 2ϩ and low GTP levels allow its activation. In the tubulointerstitium this is independent of increased tTg production, but dependent in the glomerulus. This leads to excessive ECM cross-linking, contributing to the renal fibrosis characteristic of progressive DN. (Lab Invest 2001, 81:705-716).

Tissue Transglutaminase and the Progression of Human Renal Scarring

“…The tubular and peritubular distribution of tTg within diseased kidneys is reminiscent of our observations in the remnant kidneys of rats submitted to subtotal nephrectomy (8,9) and those with streptozotocin-induced diabetic nephropathy (26). It suggests similar mechanisms of action of tTg during the course of human renal scarring compared with that of experimental animals, including intracellular upregulation of the enzyme, its extracellular translocation, and its crosslinking of substrates in a peritubular and periglomerular distribution suggestive of an ECM target.…”

“…Previous studies conducted in experimental models of diabetic (26) and nondiabetic (8,9) renal scarring have implicated this enzyme in the crosslinking of the renal ECM, accelerating deposition and conferring resistance to the proteolytic activity of renal metalloproteinases (8). In addition, we have previously suggested that this enzyme may also contribute through intracellular cytoplasmic crosslinking to tubular cell death (9).…”

Matrix changes induced by transglutaminase 2 lead to inhibition of angiogenesis and tumor growth

“…Our studies demonstrating that in TG2 wild-type mice the enzyme is found upregulated in the host tissue surrounding the B16-F1 melanoma is supported by an immunohistochemical study in human mammary tumors, in which TG2 was also shown to be associated with the host's ECM surrounding the tumor. 18 Our studies in vitro using the coculture model of angiogenesis and in vivo demonstrating TG2 involvement in tissue fibrosis 9,21,31 indicate that TG2 can indeed regulate the balance between ECM degradation and accumulation. Moreover, preliminary studies undertaken on the R3230Ac tumor implanted into a skin flap window chamber indicated that the topical addition of TG2 could limit tumor growth with a concomitant decrease in angiogenesis.…”

A Crucial Sequence for Transglutaminase Type 2 Extracellular Trafficking in Renal Tubular Epithelial Cells Lies in Its N-terminal β-Sandwich Domain

“…The pathological role of TG2 in fibrosis is tightly associated with its trafficking to the extracellular environment (5,10,18) where it has both enzymatic (post-translational ECM modification and activation of cytokines (19,20)) and non-enzymatic roles (cell migration, adhesion (21), and growth (1)). However, the mechanism by which TG2 is trafficked to the extracellular space remains unknown.…”