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 ⑀ -( ␥ -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 ⑀ -( ␥ -glutamyl) lysine cross-link (as determined by exhaustive proteolytic digestion followed by cation exchange chromatography) increased from 3. 47

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Transglutaminase type 2 (TG2) is both a protein cross-linking enzyme and a cell adhesion molecule with an elusive unconventional secretion pathway. In normal conditions, TG2-mediated modification of the extracellular matrix modulates cell motility, proliferation and tissue repair, but under continuous cell insult, higher expression and elevated extracellular trafficking of TG2 contribute to the pathogenesis of tissue scarring. In search of TG2 ligands that could contribute to its regulation, we characterized the affinity of TG2 for heparan sulfate (HS) and heparin, an analogue of the chains of HS proteoglycans (HSPGs). By using heparin/HS solid-binding assays and surface plasmon resonance we showed that purified TG2 has high affinity for heparin/HS, comparable to that for fibronectin, and that cell-surface TG2 interacts with heparin/HS. We demonstrated that cell-surface TG2 directly associates with the HS chains of syndecan-4 without the mediation of fibronectin, which has affinity for both syndecan-4 and TG2. Functional inhibition of the cell-surface HS chains of wild-type and syndecan-4-null fibroblasts revealed that the extracellular cross-linking activity of TG2 depends on the HS of HSPG and that syndecan-4 plays a major but not exclusive role. We found that heparin binding did not alter TG2 activity per se. Conversely, fibroblasts deprived of syndecan-4 were unable to effectively externalize TG2, resulting in its cytosolic accumulation. We propose that the membrane trafficking of TG2, and hence its extracellular activity, is linked to TG2 binding to cell-surface HSPG.

Administration of active TG2 to two different in vitro angiogenesis assays resulted in the accumulation of a complex extracellular matrix (ECM) leading to the suppression of endothelial tube formation without causing cell death. Matrix accumulation was accompanied by a decreased rate of ECM turnover, with increased resistance to matrix metalloproteinase-1. Intratumor injection of TG2 into mice bearing CT26 colon carcinoma tumors demonstrated a reduction in tumor growth, and in some cases tumor regression. In TG2 knockout mice, tumor progression was increased and survival rate reduced compared to wild-type mice. In wild-type mice, an increased presence of TG2 was detectable in the host tissue around the tumor. Analysis of CT26 tumors injected with TG2 revealed fibrotic-like tissue containing increased collagen, TG2-mediated crosslink and reduced organized vasculature. TG2-mediated modulation of cell behavior via changes in the ECM may provide a new approach to solid tumor therapy.

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.

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.

Abstract. Experimental renal scarring indicates that tissue transglutaminase (tTg) may be associated with the accumulation of extracellular matrix (ECM), both indirectly via TGF-␤1 activation and directly by the formation of ⑀(␥-glutamyl) lysine dipeptide bonds within the ECM. The latter potentially accelerates deposition and confers the ECM with resistance to proteolytic digestion. Studied were 136 human renal biopsy samples from a range of chronic renal diseases (CRD) to determine changes in tTg and ⑀(␥-glutamyl) lysine crosslinking. Immunofluorescence for insoluble tTg showed a 14-fold increase in the kidneys of CRD patients (5.3 Ϯ 0.5 versus 76 Ϯ 54 mV/cm 2 ), which was shown to be active by a similar 11-fold increase in the ⑀(␥-glutamyl) lysine crosslink (1.8 Ϯ 0.2 versus 19.3 Ϯ 14.2 mV/cm 2 ). Correlations were obtained with renal function for tTg and crosslink. In situ hybridization for tTg mRNA showed that tubular epithelial cells were the major source of tTg; however, both mesangial and interstitial cells also contributed to elevated levels in CRD. This mRNA pattern was consistent with immunohistochemistry for soluble tTg. Changes in renal tTg and its product, the ⑀(␥-glutamyl) lysine crosslink, occur in progressive renal scarring in humans independently of the original etiology and in a similar manner to experimental models. tTg may therefore play a role in the pathogenesis of renal scarring and fibrosis in patients with CRD and can therefore be considered a potential therapeutic target.

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.