Transglutaminase Inhibition Reduces Fibrosis and Preserves Function in Experimental Chronic Kidney Disease

Abstract

Progressive tissue fibrosis is involved in debilitating diseases that affect organs including the lungs, liver, heart, skin, and kidneys. Recent evidence suggests that tissue transglutaminase, an enzyme that crosslinks proteins, may be involved in tissue fibrosis by crosslinking and stabilizing the extracellular matrix or by recruiting and activating the large latent transforming growth factor (TGF)-β1 complex. We treated rats that had undergone 5/6-nephrectomy with two different irreversible inhibitors of transglutaminase and found that both prevented a decline in kidney function and reduced the development of glomerulosclerosis and tubulointerstitial fibrosis by up to 77% and 92%, respectively. Treatment reduced the accumulation of collagen I and collagen III, with the primary mechanism of action being direct interference with the crosslinking of extracellular matrix rather than altered regulation of TGFβ1. We conclude that inhibition of transglutaminase offers a potential therapeutic option for chronic kidney disease and other conditions that result from tissue fibrosis. Tissue remodeling leading to scarring and fibrosis is associated with a number of debilitating diseases affecting major organs. Pathologic changes that are associated with scarring are often highly conserved between organs despite varying initial stimuli. The proliferation of fibroblasts and overproduction/accumulation of extracellular matrix (ECM) proteins is well characterized, with an array of growth factors and cytokines implicated in driving the disease; however, despite an extensive knowledge of the cellular and biochemical changes, there remains no effective therapy. One enzyme that increasingly is associated with ECM is the protein–cross-linking enzyme tissue transglutaminase (TG2). TG2 is a member of the transglutaminase family of enzymes that have the capacity to cross-link proteins irreversibly through ε(γ-glutamyl) lysine iso-peptide bonds.1 Although changes in TG2 have been described in scarring of the lung,2 liver,3 and heart4 as well as in atherosclerosis,5 the enzyme has been most extensively characterized in the kidney, where a persistent upregulation and cellular release of TG2 occurs in animal models and human biopsies.6,7 In human kidney disease, there is strong correlation between levels of TG2 (R2 = 0.92), ε(γ-glutamyl) lysine iso-peptide crosslinking (R2 = 0.86) and the development of tissue scarring in all diseases investigated.6 Chronic kidney disease (CKD) affects up to 10% of the population8 and contributes considerably to their morbidity and mortality. The progressive nature of CKD is associated with a relentless loss of renal tissue and its replacement by ECM, culminating in organ fibrosis and failure. Evidence suggests that TG2 has a multifunctional role in the progression of disease.7,9 TG2 is trafficked to the extracellular environment, where the high Ca2+ levels result in activation of TG2,10 leading to ECM cross-linking. This cross-linking can facilitate inappropriate deposition of ECM proteins11,12 and confer resistance to the action of matrix metalloproteinases.9 High intracellular levels of TG2 have also been associated with cell death by the cross-linking of cytoplasmic proteins after cell trauma and loss of Ca2+ homeostasis, possibly acting as an alternative to apoptosis in limiting necrosis.13,14 This role of TG2 is thought to be independent of its proposed roles in apoptosis.15 Cell deletion and tubular atrophy are major components of kidney scarring and contribute to progressive kidney insufficiency.16 TG2 also has a key role in the matrix storage and activation of TGF-β1, through the cross-linking of the large latent TGF-β1–binding protein to the ECM.17,18 It is interesting that TGF-β1 has also been shown to upregulate TG2 transcription.19,20 Matrix-associated TG2 may also have a cell adhesion/migration role independent of its transamidating activity that is important in the wound response.14,21 Despite this accumulating evidence to implicate TG2 as a key enzyme in the scarring process, a causative role for the enzyme still has not been established. Here we applied two specific site-directed, irreversible inhibitors of TG by direct intrarenal delivery to the five sixths subtotal nephrectomy (SNx) model of progressive kidney scarring and fibrosis with the aim of determining whether TG inhibition can reduce scarring and preserve kidney architecture and function. For ascertainment of whether total or predominantly extracellular inhibition of the enzyme leads to comparable outcomes, compounds with different characteristics were chosen. 1,Dimethyl-2[(oxopropyl)thio]imidazolium (NTU283), originally synthesized as a factor XIIIa inhibitor,22,23 is highly cell soluble and readily enters the intracellular compartment. The other compound, N-benzyloxycarbonyl-l-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine (NTU281), has poor transfer across the cell membrane, thereby limiting its action to the extracellular compartment.24 We demonstrate that TG inhibition when applied from the onset of disease is effective at preventing tissue scarring and maintaining kidney function with beneficial effects through direct ECM modification. RESULTS General Observations Rats that were treated with TG inhibitors showed no obvious phenotypic changes. Measurement of heart and liver enzymes demonstrated no deleterious effect of TG inhibition during the experimental period. Blood clot stability as measured by solubility in 5 M urea indicated that fibrin cross-linking was not affected. Rats that underwent SNx and were treated with TG inhibitors gained body weight (Table 1) at a slightly greater rate than untreated rats, but this achieved significance only for NTU281 at day 84 and was less than that in normal controls. Compensatory renal growth after SNx was not significantly altered by either inhibitor, although the mean values were less with both inhibitors (Table 1). TG activity increased in SNx kidneys during the 84 d (Figure 1A). Inhibitor NTU283 decreased activity levels to below that of control rats (P ≤ 0.05) at 7 d after SNx and to control levels by 28 d. At 84 d, activity in NTU283-treated rats rose to just under half that of untreated SNx rats (Figure 1A). NTU281 has limited cell membrane permeability; therefore, its inhibitory activity is more accurately measured using a TG in situ activity assay optimized for measuring extracellular TG activity.9 Extracellular TG activity was four-fold greater in remnant kidneys of SNx rats when compared with controls (Figure 1B). NTU281 prevented any increase in extracellular TG activity until 28 d and reduced the elevated level in SNx rats at 84 d by >60%. Both inhibitors were highly effective in preventing the seven-fold increase in the TG-mediated ε(γ-glutamyl) lysine cross-link associated with kidney scarring (Figure 1C). Importantly ε(γ-glutamyl) lysine cross-link levels were not significantly higher than those in control rats with either inhibitor at 84 d. Kidney Scarring and Function By 84 d after surgery, all rats that had undergone SNx had developed significant glomerulosclerosis (Figures 2 and 3A) associated with advanced tubulointerstitial scarring (Figures 2 and 3B) as assessed by Masson's trichrome staining. There was considerable flattening of the tubular epithelium, leading to significant tubular atrophy (Figure 2). Rats receiving TG inhibitors had far less advanced disease throughout the remnant kidney, with the exception of one rat that received NTU281 and had an area of heavily scarred and fibrosed tissue. Although improved, the renal histology of treated rats did not seem completely normal. Most had areas of slightly thickened tubular basement membrane, and some had an enlarged tubular lumen, although minimal tubular atrophy was seen. There was a visible reduction in interstitial cell numbers (inflammatory and noninflammatory) in both treated groups. Masson's trichrome staining quantified by multiphase image analysis for assessment of collagen deposition demonstrated a significant improvement in the level of both glomerulosclerosis (Figure 3A) and tubulointerstitial scarring (Figure 3B) in both TG inhibitor–treated groups. Measurement of kidney function by creatinine clearance showed a 50% reduction in the SNx group 7 d after SNx (Figure 4A). This increased slightly during the first month in response to compensatory renal growth but fell sharply between 28 and 84 d as the remnant kidney scarred. Both treated groups showed comparable reductions in creatinine clearance to the untreated rats until 28 d as a result of the reduction in renal mass associated with this model. After this, rats receiving inhibitors maintained renal function at approximately 50% of normal, whereas in untreated kidneys renal function fell below 20% of controls. Normalizing creatinine clearance for the degree of resection (i.e., correcting for the surviving nephron number [Figure 4B]) indicated marked hyperfiltration in both treated and untreated remnant kidneys up to 28 d. After this, treated groups maintained this level of hyperfiltration, whereas the untreated rats had a dramatic reduction in the creatinine clearance. All clearance values were consistent, with TG inhibition improving serum creatinine levels (Figure 4C). Albuminuria in untreated SNx kidneys was 85-fold that of control rats by day 84 (Figure 4D), but treatment with NTU281 and NTU283 reduced this increase by 61 and 72%, respectively. Changes in ECM Levels Measurement of whole kidney collagen using hydroxyproline showed a 275% increase in untreated SNx kidneys by day 84 compared with controls (Figure 3C). In comparison, the hydroxyproline levels in remnant kidneys that were treated with TG inhibitors increased by only 33% with NTU281 (not significantly different from controls) and by just 70% with NTU283 (Figure 3C). Immunohistochemical analysis (Figure 5) quantified by multiphase image analysis indicated significant increases in collagens I, III, and IV in the untreated remnant kidneys at day 84 (Figure 6A, i through iii). Collagen 1 changes were seen extensively in the glomeruli (Figure 5, row 1) but also in the tubulointerstitium (Figure 5, row 2). Collagen III changes were mainly confined to the interstitial space (Figure 5, row 3), whereas collagen IV was elevated both periglomerular and in the tubular basement membrane (Figure 5, row 4). Both inhibitors prevented or significantly reduced levels of immunoreactive collagens I and III. Only compound NTU281 was able to lower collagen IV. Fibronectin was also reduced by both inhibitors (Figure 6Aiv). Measurement of mRNA levels of major ECM components and processing enzymes by Northern blot analysis (Figure 6B) indicated that SNx caused a significant induction in collagens I, III, and IV; fibronectin; matrix metalloproteinase-1; and tissue inhibitor of metalloproteinase-1 mRNA levels. Application of TG inhibitors, despite some variation in the mean values, caused no significant change in mRNA levels for any collagen measured compared with the untreated SNx. However, NTU281 significantly increased fibronectin and reduced tissue inhibitor of Matrix metalloproteinase-1 mRNA levels (Figure 6B, x and xii). Interstitial Cells Examination of Masson's trichrome–stained sections suggested that fewer interstitial cells were present in TG inhibitor–treated kidneys; however, staining for myofibroblasts using α-smooth muscle actin (α-SMA) showed that levels in the inhibitor-treated groups were still elevated and comparable to untreated remnant kidneys (Figure 6Av). Staining with ED1, a monocytic cell marker in rat, also showed no reduction with NTU283 but was reduced with NTU281 at 84 d (Figure 6Avi). At 28 d, both TG inhibitors elevated ED1-positive staining to levels greater than SNx alone. A five-fold increase over controls was observed in cells showing ED1 staining in the SNx, which was increased to 21- and 13-fold normal levels when treated with compounds NTU283 and NTU281, respectively (P < 0001). TGF-β Activation Remnant kidney scarring was associated with a significant 30% increase in biologically active renal TGF-β from day 28 d onward (Figure 7A). This increase in active TGF-β was associated with a 2.5-fold increase in total TGF-β protein (Figure 7B) that was mRNA dependent (data not shown), although the percentage of the available latent TGF-β actually activated was one third lower in the untreated SNx (Figure 7C) compared with control rats. Treatment with TG inhibitors did not reduce the levels of active TGF-β1, with levels on the whole being slightly raised throughout the time course and significantly elevated with NTU283 from 28 d onward. TG inhibition did not consistently alter total TGF-β levels compared with untreated SNx rats. After 84 d of treatment, TG inhibition led to a higher conversion rate of latent TGF-β than in the untreated SNx rats (Figure 6C). Blood Pressure For assessment of whether NTU283 and NTU281 could affect BP, a separate group of SNx rats were left for 60 d to develop hypertension (Table 2). After application of both TG inhibitors at day 60, after 7 d of infusion, there was no significant difference in BP between treated and untreated SNx rats (Table 2) within time-matched groups; however, after 30 d of treatment, there was a tendency for rats that were treated with NTU283 to have a higher BP, although this was significant only with diastolic BP (Table 2). Correlation Analysis Across all time points, the TG product, ε(γ-glutamyl) lysine cross-link, correlated well with levels of tubulointerstitial scarring (Masson's trichrome = < levels also well (P < 0.05) with collagen I = collagen IV = and collagen III = when measured of collagen by hydroxyproline a with immunoreactive collagen I = < than either collagen III = < 0.05) or collagen IV = < collagen 1 to be the clearance correlated with scarring = < and collagen III = < with a to cross-link = < ED1 staining showed no correlation to cross-link levels when compound NTU283 were = although there was with scarring = < protein cross-linking has been associated with the progression of kidney disease and in experimental models and in using the SNx model of chronic renal we demonstrate that two irreversible TG inhibitors the in the enzyme active both are able to reduce intrarenal TG this reduction in TG activity is by a reduction in the of the the ε(γ-glutamyl) lysine the progressive that between and is significantly in reduced and interstitial scarring in the treated groups. inhibitor caused a reduction in inhibition of protein cross-linking by TG renal function at a level that is associated with the reduction in renal mass in this model and not whereas the reduction in glomerulosclerosis and is in with of the major ECM proteins as well as matrix metalloproteinase-1 are not significantly different from untreated levels using either This suggests that the major driving and that TG inhibition is to be with ECM deposition and in the kidneys. This is with cell using NTU283 in an in model of using tubular ECM accumulation was reduced by reductions ECM levels or the of available active demonstrated that of TG2 with both collagens 1 and III leads to an increased rate of and that both collagen or a cell matrix by TG2 more to In cell matrix also demonstrated that the increased of TG2 leads to a reduced rate of matrix deposition and both inhibitors the accumulation of collagens I and III, only NTU281 is able to reduce collagen IV. The in of and the far in only as to the the action of the inhibitors on collagen IV. It may be a of the intrarenal and Both compounds have comparable when measured but NTU281 has a greater mass than NTU283 has poor cell the direct intrarenal delivery is that the extracellular levels of NTU281 are higher than NTU283 and NTU281 is more able to the tubular basement This is by the that when inhibition is measured by in situ TG activity assay in NTU283 is approximately 30% less effective at the (data not important with TG inhibitors is whether their effects on matrix accumulation are through inhibition of cross-linking of matrix proteins, whether is an effect inhibition of role in the activation of suggested a role for TG2 in the activation of the large latent TGF-β1 complex. any action be through reduction in either active or total from the elevated levels after to evidence for a in this model. The that increased TGF-β1 any inhibition of activation by TG2 was no increased of TGF-β1 mRNA in treated groups was It is to changes in TGF-β1 activation as a mechanism for TG2 action in this model. The of TG inhibitors to TGF-β1 activation is in with in A lower interstitial cell number in the two inhibitor-treated groups by the of the time course was however, using as a marker indicated a level of staining in the treated and untreated groups. ED1 staining for monocytic cells high when cells were treated with the of an alternative interstitial cell that is lower in the treated groups. NTU281, the number of ED1-positive cells was reduced at 84 d, which in for the lower interstitial cell One for the reduction in monocytic cells by the NTU281 inhibitor is the inhibition of TG2 cross-linking in the activation of the of of the of reduce the release of from the cell membrane during which is the in the of by by thereby the The of the two inhibitors when in an in model the of the TG far characterized by and It is that both inhibitors all although with varying however, only two TG are to be associated with the TG2 and factor In the initial of wound we any effects on factor although this enzyme is to be less important in the of ECM deposition by NTU281 is predominantly found in the extracellular which is by using tissue and tissue in which the of NTU281 is only when TG activity in which predominantly extracellular NTU281 is as effective as this that the beneficial effect of both inhibitors is cell of NTU281 effects on other TG or other potential enzymes in the intracellular and the of other enzyme in the extracellular space is NTU283 its was originally factor but is effective and and has undergone extensive in to its the active of The of the NTU283 family of compounds a of both and has also indicated no enzymes when up to We that other intracellular may be by however, the of both NTU283 and NTU281 key the has shown no effect on when at up to 1 (data not both compounds have renal activity when we to both compounds to the kidney an intrarenal from an This major this less compound, which is an important is not on a the direct delivery of the by or effects after delivery an and This delivery has been extensively of the some remodeling to which has to be from any but using and showed that throughout the kidney is and of any major The a significant improvement in both kidney function and of renal remodeling in kidneys that were treated with site-directed, irreversible inhibitors of TG thereby the of TG2 in disease progression and as a potential therapeutic for the of tissue Although we that key still to be as the of TG inhibition on disease, the of TG inhibition and the effects of of this of compound, this for the first time a and effective that offers the potential to reduce tissue scarring in a of SNx of Scarring rats to were to SNx by and of the left kidney using a and lower of the kidney were to a loss of five sixths of the renal rats were to a of TG and were synthesized to was by and mass Inhibitor was renal TG by application of inhibitors at and to a 20% kidney with activity measured using the Application of TG A was at 1 and a 2 from the The was to a At the was through one of the remnant that the area was within the renal The was and through the muscle to the on the This has been shown to delivery throughout the remnant kidney by both and were with either or of NTU281 or were as TG inhibitor was on using a that completely the increase in TG activity after SNx. Rats were maintained at and on a and to rat and All were under to by groups = 5 to group time of SNx, and rats were at and 84 d after SNx. A and were The remnant kidney was One was in and and the was in and Albuminuria was measured using the rat as the Scarring sections were with Masson's trichrome and For of tubulointerstitial at were using a For glomeruli were Scarring was assessed using analysis and using Analysis total in of The tubulointerstitial scarring was by the area of by the cellular correcting for cell number and tubular was by with the area to of changes in and Measurement of TG in Kidney TG activity in kidney was measured using the assay as described are to protein 1 at In situ sections were with the TG primary as described was using a and with a were using at and the level of was using multiphase analysis as This was to the TG inhibitory effects of of were to with and levels in the were measured by using an on an using a of a as described Measurement of mRNA was using from rat in the and to Northern blot analysis as described using Northern were to and were quantified by using a and values were for using with the and analysis were in on separate for although from a Immunohistochemical Staining was on sections using a After were applied as collagen III IV High and Collagen 1 was on sections using a image analysis was as with staining to Analysis Kidney were in M at for and were using the analysis to the The and hydroxyproline was measured by using a and TGF-β TGF-β activity was measured using the cell using the TGF-β1 response on the up to the Kidney were at and the was through were in and cells in a After cells were with A total of of cell was with of and a The of NTU283 and NTU281 at up to to of normal and SNx did not alter values TGF-β1 was measured after activation using for 1 at of on BP For assessment of whether TG inhibitors BP, rats were to SNx and to a after SNx, significant hypertension had all SNx rats had an intrarenal were with with NTU281, and with NTU283 from Both and diastolic BP were measured by with rats in SNx, 60 d after SNx, and 7 and 30 d after of TG At and mean BP were from an of five between experimental groups was using either the with or as A of (P ≤ 0.05) was as TG TG activity after SNx was assessed in a renal using the assay for NTU283 and an in situ activity assay on for NTU281 This was by of TG ε(γ-glutamyl) lysine cross-link levels at 84 d after SNx are = 5 to rats for experimental group time with in on separate a < < of TG inhibition on kidney scarring at 84 d after SNx. Masson's trichrome–stained kidney sections from rats, SNx rats, SNx rats that were treated with NTU281, and SNx rats that were treated with NTU283 at 84 d after SNx. of renal The level of glomerulosclerosis and tubulointerstitial scarring was assessed by multiphase image analysis of Masson's trichrome–stained glomeruli or of were for rat on at This was by of total renal hydroxyproline levels day 84 are = 5 to rats for experimental group time a < < of kidney function and Kidney function was assessed by of creatinine clearance which was for nephron number after resection and serum creatinine Albuminuria was to are = 5 to rats for experimental group time a < < Collagen sections for collagen I 1 and collagen III and collagen IV at 84 d after SNx. Changes in protein and mRNA levels of implicated in the scarring at 84 d after SNx. Kidney levels of collagen I collagen III collagen IV and fibronectin protein were assessed by multiphase analysis of animal on a of Levels of myofibroblasts and monocytic cells were assessed using and ED1 as respectively. mRNA levels of collagen I collagen III collagen fibronectin matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 were assessed by Northern blot analysis using for using are = 5 to rats for experimental group time was in on of TG inhibition on TGF-β1 TGF-β was assessed in kidney using the in cells TGF-β was by activating the and the level of conversion of total to active are = 5 to rats for experimental group time kidney was in on separate body and kidney weight in TG inhibitor–treated SNx BP in TG inhibitor–treated SNx was by the of a Kidney to with from the Kidney and