Diabetic nephropathy affects 30 -40% of diabetics leading to end-stage kidney failure through progressive scarring and fibrosis. Previous evidence suggests that tissue transglutaminase (tTg) and its protein cross-link product ⑀(␥-glutamyl)lysine contribute to the expanding renal tubulointerstitial and glomerular basement membranes in this disease. Using an in vitro cell culture model of renal proximal tubular epithelial cells we determined the link between elevated glucose levels with changes in expression and activity of tTg and then, by using a highly specific site directed inhibitor of tTg (1,3-dimethyl-2[(oxopropyl)thio]imidazolium), determined the contribution of tTg to glucose-induced matrix accumulation. Exposure of cells to 36 mM glucose over 96 h caused an mRNA-dependent increase in tTg activity with a 25% increase in extracellular matrix (ECM)-associated tTg and a 150% increase in ECM ⑀(␥-glutamyl)lysine cross-linking. This was paralleled by an elevation in total deposited ECM resulting from higher levels of deposited collagen and fibronectin. These were associated with raised mRNA for collagens III, IV, and fibronectin. The specific site-directed inhibitor of tTg normalized both tTg activity and ECMassociated ⑀(␥-glutamyl)lysine. Levels of ECM per cell returned to near control levels with non-transcriptional reductions in deposited collagen and fibronectin. No changes in transforming growth factor ␤1 (expression or biological activity) occurred that could account for our observations, whereas incubation of tTg with collagen III indicated that cross-linking could directly increase the rate of collagen fibril/gel formation. We conclude that Tg inhibition reduces glucose-induced deposition of ECM proteins independently of changes in ECM and transforming growth factor ␤1 synthesis thus opening up its possible application in the treatment other fibrotic and scarring diseases where tTg has been implicated.

Transglutaminase type 2 (TG2) is an extracellular matrix crosslinking enzyme with a pivotal role in kidney fibrosis. The interaction of TG2 with the heparan sulfate proteoglycan syndecan-4 (Sdc4) regulates the cell surface trafficking, localization, and activity of TG2 in vitro but remains unstudied in vivo. We tested the hypothesis that Sdc4 is required for cell surface targeting of TG2 and the development of kidney fibrosis in CKD. Wild-type and Sdc4-null mice were subjected to unilateral ureteric obstruction and aristolochic acid nephropathy (AAN) as experimental models of kidney fibrosis. Analysis of renal scarring by Masson trichrome staining, kidney hydroxyproline levels, and collagen immunofluorescence demonstrated progressive fibrosis associated with increases in extracellular TG2 and TG activity in the tubulointerstitium in both models. Knockout of Sdc-4 reduced these effects and prevented AAN-induced increases in total and active TGF-b1. In wild-type mice subjected to AAN, extracellular TG2 colocalized with Sdc4 in the tubular interstitium and basement membrane, where TG2 also colocalized with heparan sulfate chains. Heparitinase I, which selectively cleaves heparan sulfate, completely abolished extracellular TG2 in normal and diseased kidney sections. In conclusion, the lack of Sdc4 heparan sulfate chains in the kidneys of Sdc4-null mice abrogates injury-induced externalization of TG2, thereby preventing profibrotic crosslinking of extracellular matrix and recruitment of large latent TGF-b1. This finding suggests that targeting the TG2-Sdc4 interaction may provide a specific interventional strategy for the treatment of CKD.

Background: Genetically modified mice are used to investigate disease and assess potential interventions. However, research into kidney fibrosis is hampered by a lack of models of chronic kidney disease (CKD) in mice. Recently, aristolochic acid nephropathy (AAN), characterised by severe tubulointerstitial fibrosis, has been identified as a cause of end stage kidney disease and proposed as a model of CKD. Published studies have used various dosing regimens, species and strains, with variable outcomes. Therefore, we aimed to develop a standardised protocol to develop tubulointerstitial fibrosis using pure aristolochic acid I (AAI) in C57BL/6 mice. Methods: AAI dose optimisation was performed by intraperitoneal injection of AAI at varying dose, frequency and duration. Kidney function was assessed by serum creatinine. Fibrosis was quantified by hydroxyproline levels and Masson’s Trichrome staining. Specific collagens were measured by immunofluorescent staining. Results: Single doses of AAI of >10 mg/kg caused acute kidney failure and death. Lower doses of 2.5 mg/kg needed to be administrated more than weekly to cause significant fibrosis. 3 mg/kg once every 3 days for 6 weeks followed by a disease development time of 6 weeks after AAI led to reduced kidney weight and function. Substantial tubulointerstitial fibrosis occurred, with males more severely affected. Increased deposition of collagen I, III and IV contributed to fibrosis, with collagen III and IV higher in males. Conclusions: AAN can be induced in C57BL/6 mice. The regimen of 3 mg/kg every 3 days for 6 weeks followed by 6 weeks of disease development time gives substantial tubulointerstitial fibrosis with lesions similar to those in humans.