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 α-smooth muscle actin (α-SMA) expression. NTU281 treatment reduced glomerular collagen I accumulation, Hic-5 and α-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 α-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.

Do changes in transglutaminase activity alter latent transforming growth factor beta activation in experimental diabetic nephropathy?

BACKGROUND: Diabetic nephropathy is the leading cause of end-stage kidney failure worldwide. It is characterized by excessive extracellular matrix accumulation. Transforming growth factor beta 1 (TGF-ß1) is a fibrogenic cytokine playing a major role in the healing process and scarring by regulating extracellular matrix turnover, cell proliferation and epithelial mesanchymal transdifferentiation. Newly synthesized TGF-ß is released as a latent, biologically inactive complex. The cross-linking of the large latent TGF-ß to the extracellular matrix by transglutaminase 2 (TG2) is one of the key mechanisms of recruitment and activation of this cytokine. TG2 is an enzyme catalyzing an acyl transfer reaction leading to the formation of a stable ε(γ-glutamyl)-lysine cross-link between peptides. METHODS: To investigate if changes in TG activity can modulate TGF-ß1 activation, we used the mink lung cell bioassay to assess TGF-ß activity in the streptozotocin model of diabetic nephropathy treated with TG inhibitor NTU281 and in TG2 overexpressing opossum kidney (OK) proximal tubular epithelial cells. RESULTS: Application of the site-directed TG inhibitor NTU281 caused a 25% reduction in kidney levels of active TGF-ß1. Specific upregulation of TG2 in OK proximal tubular epithelial cells increased latent TGF-ß recruitment and activation by 20.7% and 19.7%, respectively, in co-cultures with latent TGF-ß binding protein producing fibroblasts. CONCLUSIONS: Regulation of TG2 directly influences the level of active TGF-ß1, and thus, TG inhibition may exert a renoprotective effect by targeting not only a direct extracellular matrix deposition but also TGF-ß1 activation and recruitment.

Modulation of tissue transglutaminase in tubular epithelial cells alters extracellular matrix levels: a potential mechanism of tissue scarring.

The up-regulation and trafficking of tissue transglutaminase (TG2) by tubular epithelial cells (TEC) has been implicated in the development of kidney scarring. TG2 catalyses the crosslinking of proteins via the formation of highly stable epsilon(gamma-glutamyl) lysine bonds. We have proposed that TG2 may contribute to kidney scarring by accelerating extracellular matrix (ECM) deposition and by stabilising the ECM against proteolytic decay. To investigate this, we have studied ECM metabolism in Opossum kidney (OK) TEC induced to over-express TG2 by stable transfection and in tubular cells isolated from TG2 knockout mice. Increasing the expression of TG2 led to increased extracellular TG2 activity (p<0.05), elevated epsilon(gamma-glutamyl) lysine crosslinking in the ECM and higher levels of ECM collagen per cell by (3)H-proline labelling. Immunofluorescence demonstrated that this was attributable to increased collagen III and IV levels. Higher TG2 levels were associated with an accelerated collagen deposition rate and a reduced ECM breakdown by matrix metalloproteinases (MMPs). In contrast, a lack of TG2 was associated with reduced epsilon(gamma-glutamyl) lysine crosslinking in the ECM, causing reduced ECM collagen levels and lower ECM per cell. We report that TG2 contributes to ECM accumulation primarily by accelerating collagen deposition, but also by altering the susceptibility of the tubular ECM to decay. These findings support a role for TG2 in the expansion of the ECM associated with kidney scarring.

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 epsilon-(gamma-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.

Reduction of ischemia-reperfusion injury in the rat kidney by FTY720, a synthetic derivative of sphingosine.

BACKGROUND: The current shortage of organ donors has led many centers to use marginal and nonheart-beating donors (NHBDs). Recent research has implicated the infiltration of lymphocytes as an important mediator of ischemia-reperfusion injury (IRI). FTY720 is an immunosuppressant that promotes lymphocyte sequestration into lymph nodes. The purpose of this study was to examine the potential for FTY720 to abrogate IRI when subjected to increasing ischemic times. METHODS: Male Sprague-Dawley rats underwent bilateral flank incision with removal of the right kidney and clamping of the left hilum. Groups were divided into ischemia times of 45, 55, and 65min; each group was further divided into a control group (IRI only), IRI+FTY720 (1 mg/kg/d), and IRI+cyclosporine (15 mg/kg/d), n=4 per group. RESULTS: Thre days after 45 min of ischemia, serum creatinine in the ischemia only (477+/-37 micromol/L) and cyclosporine groups (698+/-32 micromol/L) was significantly increased compared with the FTY720-treated animals (194+/-66 micromol/L). The beneficial effect of FTY720 was also observed at 55 and 65 min; indeed, FTY720-treated animals demonstrated signs of recovery from 65 min of ischemia whereas control and cyclosporine-treated animals required sacrifice between days 3 and 5. Treatment with FTY720 reduced renal damage assessed histologically and also reduced apoptosis and increased cell proliferation. CONCLUSION: Treatment with FTY720 reduced IRI and prevented unrecoverable acute renal failure after significant ischemic injury. This study suggests that FTY720 may help improve the quality of grafts from NHBD and marginal donors by abrogating the IRI insult.

Inhibiting ERK Activation with CI-1040 Leads to Compensatory Upregulation of Alternate MAPKs and Plasminogen Activator Inhibitor-1 following Subtotal Nephrectomy with No Impact on Kidney Fibrosis.

Extracellular-signal regulated kinase (ERK) activation by MEK plays a key role in many of the cellular processes that underlie progressive kidney fibrosis including cell proliferation, apoptosis and transforming growth factor ß1-mediated epithelial to mesenchymal transition. We therefore assessed the therapeutic impact of ERK1/2 inhibition using a MEK inhibitor in the rat 5/6 subtotal nephrectomy (SNx) model of kidney fibrosis. There was a twentyfold upregulation in phospho-ERK1/2 expression in the kidney after SNx in Male Wistar rats. Rats undergoing SNx became hypertensive, proteinuric and developed progressive kidney failure with reduced creatinine clearance. Treatment with the MEK inhibitor, CI-1040 abolished phospho- ERK1/2 expression in kidney tissue and prevented phospho-ERK1/2 expression in peripheral lymphocytes during the entire course of therapy. CI-1040 had no impact on creatinine clearance, proteinuria, glomerular and tubular fibrosis, and α-smooth muscle actin expression. However, inhibition of ERK1/2 activation led to significant compensatory upregulation of the MAP kinases, p38 and JNK in kidney tissue. CI-1040 also increased the expression of plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasmin-dependent matrix metalloproteinases. Thus inhibition of ERK1/2 activation has no therapeutic effect on kidney fibrosis in SNx possibly due to increased compensatory activation of the p38 and JNK signalling pathways with subsequent upregulation of PAI-1.

Cyclosporine A sensitizes the kidney to tubulointerstitial fibrosis induced by renal warm ischemia.

BACKGROUND: Renal warm ischemic injury and immunosuppression with cyclosporin A (CsA) may contribute to chronic allograft nephropathy after cadaveric transplantation. This study establishes whether CsA can sensitize the kidney to injury and fibrosis induced by renal warm ischemia (RWI). METHODS: The left kidney of Sprague-Dawley rats was subjected to 30 min of warm ischemia and/or intraperitoneal CsA (15 mg/kg/d) for 30 days (n=6 per group). Renal injury and fibrosis were assessed histologically together with immunohistochemistry for collagen III, transforming growth factor (TGF)-beta1, ED1 (macrophage marker), and alpha-smooth muscle actin. Renal mRNAs for collagen III, TGF-beta 1, matrix metalloproteinase (MMP)-2, and tissue inhibitor of metalloproteinase-1 together with MMP enzyme activity were also determined. RESULTS: RWI or CsA alone produced only minor effects on renal injury and fibrosis. However, in CsA-treated rats, RWI produced a marked increase in tubulointerstitial fibrosis, as shown by the potentiation of collagen III and TGF-beta1 determined by immunochemistry and mRNA analysis. The up-regulation of tissue inhibitor of metalloproteinase-1 mRNA was associated with a decrease in MMP enzyme activity. In CsA-treated rats, RWI was also associated with an increase in inflammatory infiltrates, elevated immunostain for ED1 (indicating extensive macrophage influx), and elevated immunostain for alpha-smooth muscle actin (indicating myofibroblast activation). CONCLUSIONS: In the rat, CsA can sensitize the kidney to fibrosis induced by renal warm ischemia. In renal transplantation, when cadaveric donor kidneys have been subjected to a period of warm ischemia, CsA may be an inappropriate choice for immunosuppressive therapy.

Cellular toxicity of catechin analogues containing gallate in opossum kidney proximal tubular (OK) cells.

A variety of catechins exist in green tea and Chinese rhubarb. It is also known that the green tea tannin mixture and its individual tannin components such as (-)-epicatechin 3-O-gallate (ECG) and (-)-epigallocatechin 3-O-gallate (EGCG) suppress renal failure in animals and inhibit the growth of mesangial cells. In addition, gallic acid (GA), a structural constituent of these catechins, induces apoptosis in tumor cell lines. However, the effects of catechins on renal tubular cells have not been investigated. In this experiment, the growth of opossum kidney proximal tubular (OK) cells was inhibited by GA (36.9 +/- 9.5%, p < 0.01) and EGCG (48.6 +/- 16.7%, p < 0.01) at 50 microM, and was almost completely inhibited by these compounds at concentrations over 100 microM. Furthermore, ECG inhibited the growth of OK cells at concentrations over 200 microM (52.6 +/- 16.5%, p < 0.01). The numbers of in situ end-labeled (ISEL) cells in cultures treated with GA at 25 and 50 microM, ECG at 100 and 200 microM, or EGCG at 25 microM were significantly less than those in the cultures treated with high-concentration EGCG (50 microM). In addition, exposure to 50 microM EGCG or 400 microM GA for 24 hr led to a significant increase in fragmented DNA, but ECG did not significantly induce DNA fragmentation compared to the control. These results suggest that EGCG induces mostly apoptosis in OK cells, but the cellular toxicity of GA involves both apoptosis and other mechanisms. Finally, ECG inhibits the growth largely via some mechanism other than apoptosis. This chemical-specific difference of cytotoxic pattern may be dependent on the combination of GA and basal catechin structures, or NADH oxidase (NOX) activity on the OK cell surface.

Atorvastatin improving renal ischemia reperfusion injury via direct inhibition of active caspase-3 in rats.

Caspase-3 is a key molecule involved in the inflammation and apoptosis of ischemia reperfusion (IR) injury. Statins are known to inhibit IR injury, but the mechanism of action remains uncertain. In the present study, the effect and underlying mechanism of ischemia alone, and reperfusion with or without atorvastatin (AT) as a timed intervention were examined, since clinically the kidney is only exposed to drug delivery during reperfusion. Male Sprague-Dawley rats were subjected to 45-min clamping of the left renal hilus followed by four hours reperfusion with a right nephrectomy. AT 10 mg/kg was intravenously administered after clamping the renal hilus, but prior to kidney reperfusion. Ischemia alone did cause tubulointerstitial damage (TID), protein carbonylation and caspase-3 activation with an increase in 12 kDa subunit, while reperfusion further enhanced TID, monocyte (ED-1+ cell) infiltration, apoptosis and necrosis together with caspase-3 activity and 17 kDa subunit, but reversed protein carbonylation. AT significantly reduced TID (26%), ED-1+ cell infiltration (74%), tubular apoptosis (47%) and necrosis (73%), and interstitial apoptosis (64%), as well as caspase-3 activity (26%), but did not change serum creatinine and cholesterol. Importantly, without affecting either caspase-3 active protein cleavage or S-nitrosylation, AT directly inhibited caspase-3 active enzyme in a dose-dependent manner in vitro. In conclusions, IR and AT exerted opposing effects on caspase-3 activity by differing mechanisms, with IR stimulating caspase-3 proteolytic cleavage and AT inhibiting active caspase-3 enzyme. This new inhibitory mechanism of AT may improve reperfusion tolerance in ischemic kidneys and benefit transplant recipients.

Lack of effect of IGF-I on the glomerular filtration rate in non-diabetic patients with advanced chronic kidney disease.

Recombinant human insulin-like growth factor I (rhIGF-I) acutely increases the glomerular filtration rate (GFR) in human volunteers and patients with advanced chronic kidney disease (CKD). However, on chronic administration, rhIGF-I induces tolerance to its renal effects attributed to a fall in serum IGF-binding protein 3 (IGFBP-3) enhancing its systemic clearance. Tolerance may be avoided by the use of an intermittent dosage regimen of rhIGF-I. A randomised, double-blind, placebo-controlled study was undertaken in non-diabetic patients with advanced CKD to establish whether intermittent subcutaneous injections of rhIGF-I (50 microg/kg, four days/week) could increase GFR over a 24 week period and thereby have the potential to delay the onset of renal replacement therapy. Twenty-seven patients were randomised into rhIGF-I/placebo groups using a 2:1 treatment ratio. GFR was determined by inulin clearance. RhIGF-I therapy produced a sustained increase serum total and free IGF-I elevating IGFBP-1 without decreasing IGFBP-3. Inulin clearance however, was not increased after either four weeks or over the 24 week observation period. Only 4/18 rhIGF-I treated patients compared to 6/9 placebo patients completed the study, the major reason being the requirement for dialysis. Compared with healthy volunteers, advanced CKD patients had elevated serum levels of IGFBP-1, IGFBP-2, tumour necrosis factor-alpha and asymmetric dimethylarginine, all factors proposed to mediate IGF-I resistance. In conclusion, although intermittent rhIGF-I therapy elevated serum total IGF-I and prevented any fall in serum IGFBP-3, it failed to increase GFR in non-diabetic patients with advanced CKD. The lack of efficacy was attributed to the presence of renal IGF-I resistance in CKD.

(-)-Epigallocatechin 3-O-gallate (EGCG)-induced apoptosis in normal rat kidney interstitial fibroblast (NRK-49F) cells.

(-)-Epigallocatechin 3-O-gallate (EGCG), a major catechin in green tea, suppresses renal failure in animals, and inhibits the growth of mesangial cells and opossum kidney proximal tubular cells. In addition, gallic acid, a structural constituent of this catechin, induces apoptosis in tumor cell lines. However, the effects of catechins on renal fibroblastic cells have not been investigated. In this experiment, the growth of normal rat kidney interstitial fibroblast (NRK-49F) cells was significantly inhibited by EGCG at concentrations higher than 6.25 microM, and almost completely inhibited at concentrations over 200 microM. The numbers of in situ end-labeled (ISEL) cells in cultures treated with EGCG at 6.25 to 200 microM increased dose-dependently. Furthermore, exposure to 6.25 to 50 microM EGCG for 24 hr led to a significant increase in caspase-3 activity compared to the control. These results suggest that EGCG induces apoptosis in NRK-49F cells.

Endothelin B receptor blockade accelerates disease progression in a murine model of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease that causes kidney failure and accounts for 10% of all patients who are on renal replacement therapy. However, the marked phenotypic variation between patients who carry the same PKD1 or PKD2 mutation suggests that nonallelic factors may have a greater influence on the cystic phenotype. Endothelin-1 (ET-1) transgenic mice have been reported to develop profound renal cystic disease and interstitial fibrosis without hypertension. The hypothesis that ET-1 acts as a modifying factor for cystic disease progression was tested in an orthologous mouse model of ADPKD (Pkd2(WS25/-)). Four experimental groups (n = 8 to 11) were treated from 5 to 16 wk of age with the highly selective orally active receptor antagonists ABT-627 (ETA) and A-192621 (ETB) singly or in combination. Unexpected, ETB blockade led to accelerated cystic kidney disease. Of significance, this was associated with a reduction in urine volume and sodium excretion and increases in urine osmolarity and renal cAMP and ET-1 concentrations. The deleterious effect of chronic ETB blockade was neutralized by simultaneous ETA blockade. ETA blockade alone resulted in a significant increase in tubular cell proliferation but did not alter the cystic phenotype. It is concluded that the balance between ETA and ETB signaling is critical for maintaining tubular structure and function in the cystic kidney. These results implicate ET, acting via vasopressin-dependent and independent pathways, as a major modifying factor for cystic disease progression in human ADPKD.

An IGF-I antagonist does not inhibit renal fibrosis in the rat following subtotal nephrectomy.

Insulin-like growth factor I (IGF-I) has been proposed as a mediator of kidney scarring, although no interventional studies on the role of IGF-I in models of chronic kidney disease have been reported. The effect of a peptide IGF-I receptor antagonist (JB3) has been examined on kidney fibrosis and function in the rat following 5/6 subtotal nephrectomy (SNx). Male Wistar rats were anesthetized with halothane and subjected to SNx. JB3 was delivered by subcutaneous infusion using Alzet osmotic minipumps. In vitro studies showed JB3 to displace (125)I-IGF-I binding to isolated rat glomeruli and to inhibit IGF-I-induced receptor phosphorylation in renal tubular cells in culture. In the 7-day SNx rats, IGF-I immunostain was present in collecting tubules and JB3 inhibited compensatory renal growth, the maximum effect occurring at 10 microg. kg(-1).day(-1). After 90 days, the SNx rats developed proteinuria, hypertension, and a fall in glomerular filtration rate. IGF-I immunostain was present in the tubulointerstitial space of the remnant kidney together with marked tubulointerstitial fibrosis. Treatment with JB3 at a dose of 10 microg. kg(-1).day(-1) had no effect on the renal fibrosis measured by Masson's trichrome staining or immunostain for collagen III and collagen IV. The proteinuria, hypertension, and lower creatinine clearance all remained unchanged. The remnant kidney was associated with a 50% decrease in renal IGF-I mRNA, which was partially restored by treatment with JB3. Thus an interventional study with an IGF-I receptor antagonist does not support a role for IGF-I in the development of renal fibrosis in the SNx rat, although IGF-I does make an important contribution to compensatory kidney growth.

Haploinsufficiency of Pkd2 is associated with increased tubular cell proliferation and interstitial fibrosis in two murine Pkd2 models.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited human kidney disease and is caused by germline mutations in PKD1 (85%) or PKD2 (15%). It has been estimated that around 1% of tubular cells give rise to cysts, and cell hyperproliferation has been noted to be a cardinal feature of cystic epithelium. Nevertheless, it is uncertain whether the increase in proliferative index observed is an early or late feature of the cystic ADPKD kidney. METHODS: Two Pkd2 mouse mutants (WS25 and WS183) have been recently generated as orthologous models of PKD2. To determine the effect of Pkd2 dosage on cell proliferation, cyst formation and renal fibrosis, we studied renal tissue from Pkd2(WS25/WS25) and Pkd2(+/-) mice by histological analysis. We also examined the proliferative index in archival nephrectomy tissue obtained from patients with ADPKD and normal controls. RESULTS: The proliferative index of non-cystic tubules in Pkd2 mutant mice as assessed by proliferating cell nuclear antigen and Ki67-positive nuclei was between 1-2%, values 5-10 times higher than control tissue. Similarly, the proliferative index of non-cystic tubules in human ADPKD kidneys was 40 times higher than corresponding controls. In Pkd2 mutant mice, significant correlations were found between the fibrosis score and the mean cyst area as well as with the proliferative index. Of significance, proliferating tubular cells were uniformly positive for polycystin-2 expression in Pkd2(+/-) kidney. CONCLUSION: These results suggest that an increase in cell proliferation is an early event preceding cyst formation and can result from haploinsufficiency at Pkd2. The possible pathogenic link between tubular cell proliferation, interstitial fibrosis and cyst formation is discussed.

Polycystin-2 expression is increased following experimental ischaemic renal injury.

BACKGROUND: Mutations in PKD2 account for 15% of patients with autosomal dominant polycystic kidney disease. Expression of the PKD2 protein, polycystin-2, is developmentally regulated, suggesting a major role for this protein during nephrogenesis. However, the regulation of polycystin-2 expression in the adult kidney has not been previously explored. METHODS: We have utilized an established model of renal ischaemic injury to study polycystin-2 expression in adult rat kidney for up to 120 h following ischaemia. RESULTS: Our results indicate that polycystin-2 expression is increased in the post-ischaemic kidney by up to 5-fold, with a peak in expression at 48 h reperfusion. This time course mirrored the increase in cell proliferation observed. In the non-ischaemic kidney, polycystin-2 expression was highest in distal nephron segments but faint proximal tubular staining was also observed. No expression was seen in glomeruli. In the ischaemic kidney, polycystin-2 expression was greatly increased but the increase in expression was not restricted to segments with the highest number of proliferating cells. Moreover, polycystin-2 was detectable mainly intracellularly following ischaemia. Consistent with this, polycystin-2 was completely sensitive to endoglycosidase H during renal recovery, suggesting that it remains largely retained within the endoplasmic reticulum under these conditions. CONCLUSIONS: Our results provide the first evidence that polycystin-2 is increased following renal ischaemia, but show that this increase is not restricted to actively proliferating cells. The increase in polycystin-2 may relate instead to the process of cellular repair or differentiation following injury.

Matrix metalloproteinases and their inhibitions in experimental renal scarring.

Renal fibrosis is characterised by an excessive accumulation of extracellular matrix (ECM) proteins. Evidence suggests that this results from both increased ECM synthesis and a reduced degradation. Here, we determine changes in the matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP) in relation to ECM production and the progression of renal fibrosis in subtotally nephrectomized (SNx) rats. Groups of 4-6 SNx or sham-operated male Wistar rats were sacrificed between days 7 and 120 following surgery. Total RNA was analysed by Northern blotting. Messenger RNA for collagens I (+710%), III (+674%), and IV (+358%) were significantly (p < 0.05) raised by day 7 and remained elevated over the 120 days. Significant (p < 0.05) increase in fibronectin, laminin and heparan sulfate proteoglycan mRNAs occurred latter at days 60 (+224%), 120 (+210%), and 120 (+256%), respectively. Increases (p < 0.05) in mRNA for MMP-1 (+360%) and MMP-2 (+239%) occurred from day 7 with MMP-1 reaching +881% by day 120. MMP-3 and -9 showed no change. Zymography on day 90 remnant kidneys showed mRNA changes were translated into active MMP-1 (+1,700%) and MMP-2 (+440%), p < 0.05. TIMP-1 mRNA was also raised (+548%, p < 0.05) by day 7 and remained elevated, while TIMP-2 mRNA levels only reached significance by day 120 (+165%). In contrast, TIMP-3 mRNA was decreased by day 30 (p > 0.05) and dropped to 27% of control by day 120. However, Western blot analysis of TIMPs 1 and 3 at day 90 showed a 5- and 4-fold increase respectively, while TIMP-2 levels were not significantly altered. Measurements of overall collagenase activities in remnant kidney homogenates were reduced. Using collagen I and IV substrates, proteolytic activity in remnant kidneys dropped to 40 and 27% of controls (p < 0.01), respectively. This data suggests that reduced MMP activity may contribute towards renal scarring, however this is not a result of reduced MMP transcription or activation, but likely to be due to the inhibition by TIMPs.

Effects of caspase inhibition on the progression of experimental glomerulonephritis.

BACKGROUND: Caspase-3 has a central role in the execution of apoptosis. In a nephrotoxic nephritis (NTN) model, we previously demonstrated an up-regulation of caspase-3 that was associated with inappropriate renal apoptosis, inflammation, tubular atrophy, and renal scarring. METHODS: We applied a pan caspase inhibitor, Boc-Asp (OMe)-fluoro-methyl-ketone (B-D-FMK), directly to rat NTN kidney using an intrarenal cannula fed from an osmotic pump. Animals were treated either for the first 7 days (acutely) to determine the effects on renal inflammation (ED-1 staining) and apoptosis (in situ end labeling of fragmented DNA), or for 28 days commencing 15 days after NTN (chronically) to observe the effects on cell death and renal fibrosis. Changes of caspase-3 and caspase-1 activity were detected by fluorometric substrate cleavage assay. Changes in caspase-3 and caspase-1, interleukin-1 beta (IL-1 beta), and collagen I, III, and IV proteins and mRNA were detected by Western blotting and Northern blotting, respectively. RESULTS: In both treated groups, caspase-3 activity was inhibited, and 17 and 24 kD active caspase-3 proteins were reduced significantly. A compensatory increase of caspase-3 mRNA occurred in the acutely treated group, but decreased in the chronically treated group (P < 0.05). Although there were no significant changes in caspase-1 activity and its active protein, the observed decrease in its precursor in the chronic group was increased by treatment (P < 0.05). Further, IL-1 beta precursor and its mRNA were significantly reduced by treatment only in the chronically treated group. Apoptosis was decreased in the glomeruli of acutely treated rats, and in the tubules and interstitium of chronically treated animals (P < 0.05). Glomerular inflammation was decreased only in the acutely treated group, whereas tubulointerstitial inflammation was lowered in both treated groups (P < 0.05). Glomerulosclerosis was reduced in both inhibitor groups, with a reduction in tubulointerstitial fibrosis and collagen I, III, and IV mRNA restricted to chronically treated animals (P < 0.05). Proteinuria was significantly decreased with caspase inhibition in both treated groups, but not serum creatinine level. CONCLUSION: This study clearly indicates that caspase inhibition reduces renal apoptosis, ameliorates inflammation and fibrosis, and improves proteinuria in experimental glomerulonephritis, which may mainly be related to changes in the caspase enzymatic system.

Expression of apoptosis-related genes and proteins in experimental chronic renal scarring.

Apoptosis has been proposed to play an important role in the progression of renal scarring. The mechanisms that determine whether a cell enters the apoptotic program are complex. Bax and Bcl-2 are recognized modulators of this event; their relative levels determine the fate of cells. A role for apoptosis in the progression of renal scarring in the remnant kidneys of rats submitted to subtotal nephrectomy (SNx) has been described. This study investigated the expression (protein and mRNA) of Bax and Bcl-2 in remnant kidneys between day 7 and day 120 post-SNx. Northern blot analysis showed that bax mRNA was increased in remnant kidneys from day 7 (150% of control; P: < 0.05), whereas bcl-2 mRNA was decreased from day 15 (23% of control; P: < 0.05) resulting in a 14-fold increase in the ratio of bax to bcl-2 mRNA by day 120. Western blot analysis showed similar changes in Bax and Bcl-2 protein in remnant kidneys, resulting in a 147-fold increase in the ratio of Bax to Bcl-2 on day 120. Immunohistochemistry showed increases in Bax to be located predominantly in tubules in SNx kidneys. Interestingly, Bcl-2 immunostaining increased in some epithelial cells within atrophic tubules despite the overall decrease in Bcl-2 protein and mRNA. The overall renal apoptotic cells correlated closely with the ratio of bax to bcl-2 at both the mRNA and protein levels (r = 0.594 and 0.308, respectively; P: < 0.05). Furthermore, tubular apoptosis correlated positively with the mRNA level of bax (r = 0.471; P: < 0.01) and negatively with the mRNA and protein levels of bcl-2 (r = -0.443 and -0.607, respectively; P: < 0.01). The increase in the ratio of the death inducer (Bax) to the death repressor (Bcl-2) at the mRNA and protein levels may control the apoptosis associated with the progression of tubular atrophy and chronic renal fibrosis within remnant rat kidneys. These observations may have prognostic and therapeutic implications in chronic renal failure.