Transglutaminase inhibition ameliorates experimental diabetic nephropathy

Diabetic nephropathy is characterized by excessive extracellular matrix accumulation resulting in renal scarring and end-stage renal disease. Previous studies have suggested that transglutaminase type 2, by formation of its protein crosslink product ε-(γ-glutamyl)lysine, alters extracellular matrix homeostasis, causing basement membrane thickening and expansion of the mesangium and interstitium. To determine whether transglutaminase inhibition can slow the progression of chronic experimental diabetic nephropathy over an extended treatment period, the inhibitor NTU281 was given to uninephrectomized streptozotocin-induced diabetic rats for up to 8 months. Effective transglutaminase inhibition significantly reversed the increased serum creatinine and albuminuria in the diabetic rats. These improvements were accompanied by a fivefold decrease in glomerulosclerosis and a sixfold reduction in tubulointerstitial scarring. This was associated with reductions in collagen IV accumulation by 4 months, along with reductions in collagens I and III by 8 months. This inhibition also decreased the number of myofibroblasts, suggesting that tissue transglutaminase may play a role in myofibroblast transformation. Our study suggests that transglutaminase inhibition ameliorates the progression of experimental diabetic nephropathy and can be considered for clinical application. Diabetic nephropathy is characterized by excessive extracellular matrix accumulation resulting in renal scarring and end-stage renal disease. Previous studies have suggested that transglutaminase type 2, by formation of its protein crosslink product ε-(γ-glutamyl)lysine, alters extracellular matrix homeostasis, causing basement membrane thickening and expansion of the mesangium and interstitium. To determine whether transglutaminase inhibition can slow the progression of chronic experimental diabetic nephropathy over an extended treatment period, the inhibitor NTU281 was given to uninephrectomized streptozotocin-induced diabetic rats for up to 8 months. Effective transglutaminase inhibition significantly reversed the increased serum creatinine and albuminuria in the diabetic rats. These improvements were accompanied by a fivefold decrease in glomerulosclerosis and a sixfold reduction in tubulointerstitial scarring. This was associated with reductions in collagen IV accumulation by 4 months, along with reductions in collagens I and III by 8 months. This inhibition also decreased the number of myofibroblasts, suggesting that tissue transglutaminase may play a role in myofibroblast transformation. Our study suggests that transglutaminase inhibition ameliorates the progression of experimental diabetic nephropathy and can be considered for clinical application. Diabetic nephropathy (DN) is the most common cause of end-stage renal disease1.Parving H.H. Diabetic nephropathy: prevention and treatment.Kidney Int. 2001; 60: 2041-2055Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar,2.Remuzzi G. Schieppati A. Ruggenenti P. Clinical practice. Nephropathy in patients with type 2 diabetes.N Engl J Med. 2002; 346: 1145-1151Crossref PubMed Scopus (515) Google Scholar accounting for 50% of cases requiring renal replacement therapy in the United States.3.Lameire N. Diabetes and diabetic nephropathy – a worldwide problem.Acta Diabetol. 2004; 41: S3-S5Crossref PubMed Scopus (10) Google Scholar This is due to the increasing prevalence of type 2 diabetes and the reduced mortality of DN patients resulting from better management. Diabetic patients now live longer and patients with diabetic end-stage renal disease are now being accepted for treatment in dialysis programs where formerly they might have been excluded. Clinically, the progression of DN is accompanied by the development of proteinuria and early glomerular hyperfiltration followed by a decline of glomerular filtration rate. Morphologically, it is characterized by excessive renal extracellular matrix (ECM) accumulation in the glomeruli and tubulointerstitial space, causing glomerulosclerosis and tubulointerstitial fibrosis. This ultimately leads to end-stage renal disease. Transglutaminase type 2 (TG2) is a calcium-dependent enzyme that catalyzes an acyl-transfer reaction (EC 2.3.2.13) between the γ-carboxamide group of peptide-bound glutamine and the ε-amino group of peptide-bound lysine. This leads to the formation of a stable and proteolytic-resistant ε-(γ-glutamyl)lysine dipeptide bond, resulting in intra- or intermolecular crosslinks in protein. A number of extracellular proteins including fibronectin,4.Mosher D.F. Cross-linking of fibronectin to collagenous proteins.Mol Cell Biochem. 1984; 58: 63-68Crossref PubMed Scopus (54) Google Scholar collagen,5.Kleman J.P. Aeschlimann D. Paulsson M. et al.Transglutaminase-catalyzed cross-linking of fibrils of collagen V/XI in A204 rhabdomyosarcoma cells.Biochemistry. 1995; 34: 13768-13775Crossref PubMed Scopus (103) Google Scholar fibrinogen,6.Martinez J. Rich E. Barsigian C. Transglutaminase-mediated cross-linking of fibrinogen by human umbilical vein endothelial cells.J Biol Chem. 1989; 264: 20502-20508Abstract Full Text PDF PubMed Google Scholar osteopontin,7.Kaartinen M.T. Pirhonen A. Linnala-Kankkunen A. et al.Cross-linking of osteopontin by tissue transglutaminase increases its collagen binding properties.J Biol Chem. 1999; 274: 1729-1735Crossref PubMed Scopus (137) Google Scholar laminin, and nidogen8.Aeschlimann D. Paulsson M. Cross-linking of laminin-nidogen complexes by tissue transglutaminase. A novel mechanism for basement membrane stabilization.J Biol Chem. 1991; 266: 15308-15317Abstract Full Text PDF PubMed Google Scholar are TG2 substrates. When TG2 is released from the cell, the high extracellular Ca2+ and low GTP (which modulates Ca2+ activation of the enzyme) activates the enzyme, enabling crosslinking of these ECM proteins at the cell surface and in the surrounding matrix. Crosslinking of collagen by TG2 has been associated with stabilization of the collagen fibril independently of lysyl oxidase,5.Kleman J.P. Aeschlimann D. Paulsson M. et al.Transglutaminase-catalyzed cross-linking of fibrils of collagen V/XI in A204 rhabdomyosarcoma cells.Biochemistry. 1995; 34: 13768-13775Crossref PubMed Scopus (103) Google Scholar accelerated ECM deposition,9.Fisher M. Huang L. Hau Z. et al.Over-Expression of Tissue Transglutaminase (tTg) in Proximal Tubular Epithelial (PTEC) Cells Affects ECM Accumulation In Vitro. Renal Association, Belfast, Northern Ireland, UK2005: 246Google Scholar reduced proteolytic breakdown of the ECM,10.Johnson T.S. Skill N.J. El Nahas A.M. et al.Transglutaminase transcription and antigen translocation in experimental renal scarring.J Am Soc Nephrol. 1999; 10: 2146-2157Crossref PubMed Google Scholar and ultimately lower ECM turnover.11.Gross S.R. Balklava Z. Griffin M. Importance of tissue transglutaminase in repair of extracellular matrices and cell death of dermal fibroblasts after exposure to a solarium ultraviolet A source.J Invest Dermatol. 2003; 121: 412-423Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Thus, TG2 action shifts the ECM deposition–degradation balance toward accumulation12.Fisher M. Jones R.A. Huang L. et al.Modulation of tissue transglutaminase in tubular epithelial cells alters extracellular matrix levels: a potential mechanism of tissue scarring.Matrix Biol. 2009; 28: 20-31Crossref PubMed Scopus (42) Google Scholar and has subsequently been linked to a range of fibrogenic conditions. A role for TG2 in the pathogenesis of DN has been reported in both the streptozotocin (STZ)-induced model of type 1 diabetes13.Skill N.J. Griffin M. El Nahas A.M. et al.Increases in renal epsilon-(gamma-glutamyl)-lysine crosslinks result from compartment-specific changes in tissue transglutaminase in early experimental diabetic nephropathy: pathologic implications.Lab Invest. 2001; 81: 705-716Crossref PubMed Scopus (48) Google Scholar and human diabetic kidney disease,14.El Nahas A.M. Abo-Zenah H. Skill N.J. et al.Elevated epsilon-(gamma-glutamyl)lysine in human diabetic nephropathy results from increased expression and cellular release of tissue transglutaminase.Nephron Clin Pract. 2004; 97: c108-c117Crossref PubMed Scopus (25) Google Scholar with increased TG2-mediated ε-(γ-glutamyl)lysine crosslink formation in diabetic kidneys tightly associated with both glomerular and tubulointerstitial ECM expansion. Currently, there remains no viable mechanism for interfering with this ECM buildup. Subsequently, the development of therapeutic approaches directly targeting this process may provide an effective approach to the prevention of DN as well as numerous other fibrotic conditions. The inhibition of TG activity in proximal tubular epithelial cells in culture has already been shown to reduce glucose-induced ECM accumulation,15.Skill N.J. Johnson T.S. Coutts I.G. et al.Inhibition of transglutaminase activity reduces extracellular matrix accumulation induced by high glucose levels in proximal tubular epithelial cells.J Biol Chem. 2004; 279: 47754-47762Crossref PubMed Scopus (82) Google Scholar providing strong support for the in vivo application of such compounds in the treatment for DN. Therefore, in this study, the TG site-directed irreversible inhibitor N-benzyloxycarbonyl-L-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine (NTU281) has been applied by direct intrarenal infusion into the kidneys of rats receiving STZ injection and by uninephrectomy over an 8-month treatment period. NTU281 is a benzyloxycarbonyl phenylalanyl analog containing a dimethylsulfonium group that binds the cysteine residue in the active site of TG to instigate an acetonylation reaction leading to permanent noncompetitive inhibition of the enzyme.16.Fisher M. Jones R. Griffin M. et al.Primary Tubular Epithelial Cells Isolated From The Tissue Transglutaminase Knockout Mouse Deposit Less Extracellular Matrix Than Wild Type Cells. Renal Associate, Harrogate, UK2006: 393Google Scholar Whereas the STZ model is not a direct representative of DN in humans as it lacks the characteristic histological lesions, it is a useful surrogate for the impact of sustained hyperglycemia on changes in ECM turnover in the kidney. Uninephrectomy has been used to accelerate diabetic kidney changes.17.Bower G. Brown D.M. Steffes M.W. et al.Studies of the glomerular mesangium and the juxtaglomerular apparatus in the genetically diabetic mouse.Lab Invest. 1980; 43: 333-341PubMed Google Scholar,18.Steffes M.W. Buchwald H. Wigness B.D. et al.Diabetic nephropathy in the uninephrectomized dog: microscopic lesions after one year.Kidney Int. 1982; 21: 721-724Abstract Full Text PDF PubMed Scopus (31) Google Scholar We report that TG inhibition can preserve kidney function, reduce albuminuria, and ameliorate the progression of the histological changes associated with the formation of scar tissue in DN. To ascertain if intrarenal cannulation was achieving uniform drug delivery throughout the kidney, we prepared a dansyl-labeled version of NTU281. Using a kidney that was cannulated 28 days previously (to allow the fibrous coat around the cannula to develop) and had received phosphate-buffered saline from implant, we infused 50 mM dansyl-labeled NTU281 for 24 h. Cryostat sections viewed under a fluorescent microscope showed a uniform distribution of dansyl-NTU281 in both the longitudinal and the transverse planes in comparison with the contralateral kidney where no fluorescence was visible (Figure 1a). Import of images into multianalyst image analysis software allowed densitometric profile assessment of the fluorescence, which confirmed equal distribution in both planes (Figure 1b). Four experimental groups were used. Normal, uninephrectomy (UNx), UNx+Streptozotocin (STZ) (referred to as diabetic or DM), and UNx+STZ+Transglutaminase inhibitor NTU281 (referred to as diabetic treated). Calculation of osmotic pump delivery rates from residual pump volumes showed a consistent drug delivery throughout the treatment period (Figure 2a). Examination of cannulas at termination indicated that all had remained in situ and there was no evidence of cannula leakage. Compared with normal and UNx animals, both diabetic groups (that is, treated and untreated with NTU281) had higher levels of blood glucose throughout the experimental period (Figure 2c) and within the 10–25 mmol/l target range using a comparable insulin dose (Figure 2b), thus showing an equal metabolic burden in treated and untreated rats. In hyperglycemic animals by 8 months post-STZ, body weight gain was approximately twofold lower (Figure 2d) than that in normal rats, reaching 355±12.8 and 377±14.3 g in diabetic and NTU281-treated diabetic rats, respectively, compared with 546±22.6 g in normal (Table 1).Table 1Final body weight and terminal kidney weight.TimeFinal body weight (g)Terminal kidney weight (g)Groups1 Month4 Months8 Months1 Month4 Months8 MonthsNormal361±11.7472±8.6546±22.61.0±0.01.4±0.131.4±0.13UNx389±7.2a447±14.2514±15.81.4±0.08a1.5±0.041.7±0.10UNx+DM319±13.4a,b356±20.0a,b355±12.8a,b1.7±0.17a3.1±0.30a,b5.7±0.91a,bUNx+DM+NTU281320±6.7a,b350±10.3a,b377±14.3a,b2.3±0.25a,b,c2.8±0.36a,b3.3±0.25a,b,cData represent mean±s.e.m., n=4–7 per group.Significance (P<0.05) is indicated in comparison with normal (a), UNx (b), and untreated DM (c). Open table in a new tab Data represent mean±s.e.m., n=4–7 per group. Significance (P<0.05) is indicated in comparison with normal (a), UNx (b), and untreated DM (c). Kidney weight following UNx was greater than that in normal, but only significantly at 1 month post-STZ (Table 1). Imposing hyperglycemia caused a marked increase in kidney weight, being more than threefold greater by 8 months; however, TG inhibition reduced increased kidney weight by 42% at this point (Table 1). Although blood pressure was raised in hyperglycemic animals, there was no significant difference in systolic blood pressure between the four groups throughout the study (not shown). TG in situ activity assays showed a sixfold increase in extracellular TG activity in the untreated diabetic animals by 8 months (Figure 3i). Increased extracellular TG activity was present both in the glomeruli and in the tubulointerstitial compartments (Figure 3ii). In comparison, the extracellular TG activity in the NTU281-treated diabetic rats remained similar to that in both normal and UNx controls throughout (Figure 3i). In agreement with less extracellular TG activity in treated animals were lower levels of catalyzed ε-(γ-glutamyl)lysine crosslinking (P<0.05), which were unaltered compared with the normal and UNx controls throughout the experimental period (Figure 3iii), showing continuous effective TG inhibition. Kidney function was assessed using serum creatinine. In untreated diabetic animals, serum creatinine rose throughout the experimental period, with more than a 3.5-fold increase by 8 months compared with that in controls (Figure 2e). Although a small increase in serum creatinine (P<0.05) was detected in the NTU281-treated diabetic rats by 8 months, this was 68.0% lower than that in those receiving no treatment (P<0.05). Measurement of 24-h albumin excretion showed substantial increases in both treated and untreated diabetic groups by 4 months (P<0.05), which progressively increased at 8 months (P<0.05). However, in NTU281-treated animals, the increase in 24-h albumin excretion was lowered by approximately 80% at the end point (Figure 2f), suggesting that TG inhibition slows down the deterioration of glomerular structure and function. After 1 month of hyperglycemia, there was no substantial difference morphologically between the four experimental groups (Figure 4a–d). By 4 months, kidney hypertrophy was well established in the UNx, treated, and untreated diabetic kidneys, with the largest increment occurring in the diabetic groups (Figure 4e–h). Furthermore, in the untreated diabetic kidney, both tubular atrophy and peritubular fibrosis were noted (Figure 4g). No evidence of interstitial fibrosis was seen at 8 months in the normal (Figure 4i) and UNx (Figure 4j) kidneys. In comparison, in the untreated diabetic kidney (Figure 4k), there was extensive epithelial flattening, tubular atrophy, and interstitial expansion with severe tubulointerstitial scarring. Nearly all glomeruli showed advanced glomerulosclerosis, with Bowman's space filled with collagen. There was extensive expansion of the mesangial matrix, the capillary network had collapsed, and the glomeruli were extensively vacuolated. In comparison, all these changes were dramatically reduced in the NTU281-treated diabetic kidneys (Figure 4l), with only mild focal tubular epithelial flattening and a minor expansion of the tubular basement membrane. Transmission electron microscopy of glomeruli in 8-month kidneys showed significant widespread changes in both the glomerular basement membrane (GBM) and the podocytes in untreated diabetic kidneys (Figure 5a). There was a visible thickening of the GBM and most noticeably an effacement/loss of the podocytes, especially visible at a high power (Figure 5b). Tg inhibition prevented the effacement/loss of podocytes, whereas computerized morphometric assessment of GBM thickness showed that the diabetic-induced GBM thickening was prevented with TG inhibition (Figure 5c). Computerized multiphase image analysis was used to assess the degree of kidney scarring on Masson's trichrome–stained sections. Assessment of glomerulosclerosis (Figure 6a) showed a significant increase in the level of fibrous tissue present by 4 months post-hyperglycemia that progressed significantly by 8 months in untreated diabetic kidneys, being fivefold that in control groups. NTU281-treated kidneys showed no significant increase in glomerulosclerosis compared with normal and UNx glomeruli over the 8-month period. Tubulointerstitial fibrosis showed a similar trend to glomerulosclerosis, with twofold increase in scarring at 4 months rising to sixfold by 8 months in untreated diabetic animals (Figure 6b). NTU281 significantly reduced scarring in diabetic kidneys, with no detectable change compared with control at 4 months and only a twofold increase by 8 months. Whole kidney collagen analysis was undertaken by measuring hydroxyproline in hydrolyzed kidney homogenates. By 8 months post-STZ, the hydroxyproline constituted 0.89±0.04% of total amino acids in untreated hyperglycemic kidneys and double those in normal (0.42±0.03%) and UNx animals (0.48±0.03%). In NTU281-treated diabetic kidneys, hydroxyproline rose by just half that in the untreated diabetics, reaching 0.67±0.03%. Changes in collagen type I, III, and IV were investigated by immunofluorescence. In early experimental DN (1 and 4 months), there was no significant change in either collagen I (Figure 7a) or III (Figure 7b) staining in the kidney between the four experimental groups. By 8 months, both collagen I (Figure 7a) and collagen III (Figure 7b) were elevated in the untreated diabetic kidney (P<0.05). Increased collagen I was mainly found in sclerotic glomeruli (Figure 8c), whereas elevated collagen III occurred in the expanded interstitium and in periglomerular areas (Figure 8g). In contrast, levels of both collagens in the NTU281-treated diabetic kidney remained similar to those in normal. Collagen IV was significantly increased by 4 months in the untreated diabetic kidney (P<0.05) and continued to increase predominantly in peritubular and periglomerular areas (Figure 8k) at 8 months (P<0.05). NTU281-treated diabetic kidneys had collagen IV staining levels that were comparable with the normal and UNx animals throughout experimental period (Figure 7c).Figure 8Immunofluorescent staining of collagens I, III, IV, and α-SMA in kidneys 8 months post-hyperglycemia. Collagen I staining (a–d) was performed on cryostat sections with collagen III (e–h), collagen IV (i–l), and α-SMA (m–p) on paraffin sections. Fields were acquired at × 200 magnification on an Olympus BX61 fluorescent microscope using FITC and DAPI filter sets. DAPI, 4’,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; α-SMA, α-smooth muscle actin.View Large Image Figure ViewerDownload (PPT) Levels of kidney scarring significantly correlated with changes in accumulation of collagen I (r=0.515, P<0.01), collagen III (r=0.718, P<0.01), and collagen IV (r=0.570, P<0.01). To detect if the TG inhibition had an effect on collagen synthesis, northern blotting was performed using 4- and 8-month kidneys. At 4 months, collagen I mRNA levels in the treated diabetic kidney were similar to those in normal, but higher than those in the UNx and untreated diabetic kidneys (Figure 9a). By 8 months, collagen I mRNA levels in diabetic kidneys were significantly higher than those in the normal and UNx (Figure 9a) kidneys. The NTU281-treated kidney had levels not significantly different from those in the untreated diabetic kidney (Figure 9a). The collagen III mRNA levels in the untreated diabetic kidney reached fivefold higher than those in the normal and UNx by 8 months (Figure 9b). In contrast, the levels in the NTU281-treated kidney were normalized at this stage, although they had been higher than those in the untreated diabetic kidney at the earlier time point (Figure 9b). The collagen IV mRNA levels in both treated and untreated diabetic groups were similar and higher than those in the UNx group at 4 months post-STZ administration (Figure 9c). By 8 months, it was significantly increased in the untreated diabetic kidney compared with that in the other three experimental groups, with the levels more than double those in normal (Figure 9c; P<0.05). Although the treated group had levels higher than those in the normal, it was not significant (Figure 9c). Overall, the above results suggested that the TG inhibitor, NTU281, had a minimal effect on collagen I mRNA levels, but reduced the collagen III and IV mRNA levels by the end of the study. Less interstitial cells were observed on Masson's trichrome-stained sections in the TG inhibitor-treated kidney than that in the untreated. ECM-producing myofibroblasts have been reported to be associated with the progression of fibrosis in DN.19.Essawy M. Soylemezoglu O. Muchaneta-Kubara E.C. et al.Myofibroblasts and the progression of diabetic nephropathy.Nephrol Dial Transplant. 1997; 12: 43-50Crossref PubMed Scopus (177) Google Scholar,20.Goumenos D.S. Tsamandas A.C. Oldroyd S. et al.Transforming growth factor-beta(1) and myofibroblasts: a potential pathway towards renal scarring in human glomerular disease.Nephron. 2001; 87: 240-248Crossref PubMed Scopus (70) Google Scholar Therefore, the abundance of these cells was determined using α-smooth muscle actin (SMA) as a marker. At 1 month, there were no substantial changes in α-SMA staining between any of the experimental groups, although significant increases in α-SMA staining were detected in tubulointerstitial space of both treated and untreated diabetic kidneys by 4 months (Figure 7d). This continued to increase at 8 months (Figure 7d) in the untreated group (Figure 8o). However, levels in the treated diabetic group were 67% lower than those in the untreated diabetic kidney (P<0.05), suggesting that TG inhibition reduced the accumulation of myofibroblasts in advanced DN. All current TG inhibitors block not only TG2 activity, but also other TGs such as factor XIIIa, TG1, and TG3. Hence, there is the potential for nonspecific effects that could complicate data interpretation. For example, loss of keratinocyte TG (TG1) activity has been reported to cause parakeratosis and psoriasis-like symptoms when applied to skin composites,21.Wolf R. Lo Schiavo A. Lombardi M.L. et al.The in vitro effect of hydroxychloroquine on skin morphology and transglutaminase.Int J Dermatol. 1997; 36: 704-707Crossref PubMed Scopus (18) Google Scholar,22.Harrison C.A. Gossiel F. Bullock A.J. et al.Investigation of keratinocyte regulation of collagen I synthesis by dermal fibroblasts in a simple in vitro model.Br J Dermatol. 2006; 154: 401-410Crossref PubMed Scopus (59) Google Scholar whereas blocking factor XIIIa may potentially lead to systemic effects such as hemorrhage and bleeding. To overcome this, we employed NTU281, an inhibitor designed to act on the outside of the cell,23.Griffin M. Mongeot A. Collighan R. et al.Synthesis of potent water-soluble tissue transglutaminase inhibitors.Bioorg Med Chem Lett. 2008; 18: 5559-5562Crossref PubMed Scopus (62) Google Scholar limiting its targets to TG2 and factor XIIIa, which are the only two forms of mammalian TG known to be secreted into the extracellular space. Other potential targets such as cysteine proteases are in the main intracellular in nature, whereas concentrations up to 1 mmol/l of NTU281 do not inhibit other important cysteine-containing enzymes such as caspase-3. In addition, osmotic minipumps were employed to enable direct delivery of TG inhibitor into the kidney to minimize dose and hence systemic effects. Pressures generated by these pumps ensure that blockage is unlikely, which is supported by stable drug delivery volumes over the 8 months. Application of dansyl-labeled NTU281 showed a uniform distribution of the drug within the receiving kidney by this method. Administration of 50 mmol/l NTU281 from an implantable osmotic pump at 2.5 μl/h to the remnant kidney in the 5/6th nephrectomy (SNx) model has previously shown to halt increases in kidney TG activity without affecting blood clot stability or TG activity in other organs such as the heart, liver, and skin (unpublished data). Therefore, the same dose of NTU281 was used in this study and proved equally effective in reducing TG crosslinking activity in the diabetic kidney throughout the study as shown by the reduction in both TG in situ activity and crosslink levels, which were uniform across the treated kidney. TG inhibition resulted in preservation of kidney function and a reduction in albuminuria as a consequence of lower levels of both glomerulosclerosis and tubulointerstitial fibrosis. On commencing this study, we expected that the underlying mechanism would be related to changes in ECM accumulation by blocking the post-translational processing of the ECM by extracellular TG, given that in vitro studies have repeatedly shown that TG2 alters ECM homeostasis by accelerating the rate of collagen deposition12.Fisher M. Jones R.A. Huang L. et al.Modulation of tissue transglutaminase in tubular epithelial cells alters extracellular matrix levels: a potential mechanism of tissue scarring.Matrix Biol. 2009; 28: 20-31Crossref PubMed Scopus (42) Google Scholar and conferring the ECM with resistance to MMP proteolysis.10.Johnson T.S. Skill N.J. El Nahas A.M. et al.Transglutaminase transcription and antigen translocation in experimental renal scarring.J Am Soc Nephrol. 1999; 10: 2146-2157Crossref PubMed Google Scholar In keeping with this, we see marked reductions in mature deposited collagen I, III, and IV levels in the NTU281-treated diabetic kidneys. The levels of collagen I mRNA remained high, with levels between untreated and NTU281-treated groups identical. In contrast, levels of collagen IV mRNA were 37% lower in the treated group and those of collagen III mRNA similar to normal animals by 8 months The collagen type I mRNA data clearly support a post-transcriptional mechanism (as predicted) in the presence of a continual fibrotic stimuli for collagen I, but the additional, unanticipated, and interesting effect of NTU281 in suppressing type III and IV collagen mRNA levels is suggestive of an additional mechanism in lowering protein levels of these collagens. This study was not designed to isolate particular mechanisms, and it therefore remains unclear from the data presented whether these improvements are a direct result of TG inhibition or a secondary effect caused by the reduction in fibrous tissue expansion (and thus fibrotic stimuli) brought about by lowering TGase activity. However, one could hypothesize as to two mechanisms that could lead to lower levels of collagen III and IV mRNA. It has been reported that TG2 is able to activate the fibrogenic cytokine transforming growth factor-β1 (TGF-β1) by recruiting large latent TGF-β1 in the ECM before proteolytic release of the active dimer.24.Kojima S. Nara K. Rifkin D.B. Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells.J Cell Biol. 1993; 121: 439-448Crossref PubMed Scopus (277) Google Scholar,25.Nunes I. Gleizes P.E. Metz C.N. et al.Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta.J Cell Biol. 1997; 136: 1151-1163Crossref PubMed Scopus (347) Google Scholar Therefore, TG inhibition may affect collagen synthesis by simply altering latent TGF-β1 activation, and this may be the case for collagen III and IV although other in vivo (unpublished data) and in vitro15.Skill N.J. Johnson T.S. Coutts I.G. et al.Inhibition of transglutaminase activity reduces extracellular matrix accumulation induced by high glucose levels in proximal tubular epithelial cells.J Biol Chem. 2004; 279: 47754-47762Crossref PubMed Scopus (82) Google Scholar studies using TG inhibitors have failed to show lower active TGF-β1 levels. Alternatively, tubular ischemia caused by ECM expansion reducing blood flow to renal tubules may be less pronounced following NTU281 treatment. The reduction in levels of tubulointerstitial fibrosis in the treated diabetic kidney may therefore allow a better perfusion of the tubular epithelium, lowering the hypoxia-inducible factor-mediated induction of ECM proteins.26.Norman J.T. O