Inhibition of collagen I accumulation reduces glomerulosclerosis by a Hic-5-dependent mechanism in experimental diabetic nephropathy

Glomerulosclerosis of any cause is characterized by loss of functional glomerular cells and deposition of excessive amounts of interstitial collagens including collagen I. We have previously reported that mesangial cell attachment to collagen I leads to upregulation of Hic-5 in vitro, which mediates mesangial cell apoptosis. Furthermore, glomerular Hic-5 expression was increased during the progression of experimental glomerulosclerosis. We hypothesized that reducing collagen I accumulation in glomerulosclerosis would in turn lower Hic-5 expression, reducing mesangial cell apoptosis, and thus maintaining glomerular integrity. We examined archive renal tissue from rats undergoing experimental diabetic glomerulosclerosis, treated with the transglutaminase-2 inhibitor NTU281. Untreated animals exhibited increased glomerular collagen I accumulation, associated with increased glomerular Hic-5 expression, apoptosis, and mesangial myofibroblast transdifferentiation characterized by a-smooth muscle actin (a-SMA) expression. NTU281 treatment reduced glomerular collagen I accumulation, Hic-5 and a-SMA expression, and apoptosis. Proteinurea and serum creatinine levels were significantly reduced in animals with reduced Hic-5 expression. In vitro studies of Hic-5 knockdown or overexpression show that mesangial cell apoptosis and expression of both a-SMA and collagen I are Hic-5 dependent. Together, these data suggest that there exists, in vitro and in vivo, a positive feedback loop whereby increased levels of collagen I lead to increased mesangial Hic-5 expression favoring not only increased apoptosis, but also mesangial myofibroblast transdifferentiation and increased collagen I expression. Prevention of collagen I accumulation interrupts this Hic-5-dependent positive feedback loop, preserving glomerular architecture, cellular phenotype, and function. © 2013 USCAP, Inc All rights reserved.

Increases in renal ε-(γ-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 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, e-(γ-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 e-(γ-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 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 <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 e-(γ-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 e-(γ-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 Ca2+ 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.