Role of cyclooxygenase isoforms in prostacyclin biosynthesis and murine prehepatic portal hypertension.

Portal hypertension (PHT) is a common complication of liver cirrhosis and significantly increases morbidity and mortality. Abrogation of PHT using NSAIDs has demonstrated that prostacyclin (PGI(2)), a direct downstream metabolic product of cyclooxygenase (COX) activity, is an important mediator in the development of experimental and clinical PHT. However, the role of COX isoforms in PGI(2) biosynthesis and PHT is not fully understood. Prehepatic PHT was induced by portal vein ligation (PVL) in wild-type, COX-1(-/-), and COX-2(-/-) mice treated with and without COX-2 (NS398) or COX-1 (SC560) inhibitors. Hemodynamic measurements and PGI(2) biosynthesis were determined 1-7 days after PVL or sham surgery. Gene deletion or pharmacological inhibition of COX-1 or COX-2 attenuated but did not ameliorate PGI(2) biosynthesis after PVL or prevent PHT. In contrast, treatment of COX-1(-/-) mice with NS398 or COX-2(-/-) mice with SC560 restricted PGI(2) biosynthesis and abrogated the development of PHT following PVL. In conclusion, either COX-1 or COX-2 can mediate elevated PGI(2) biosynthesis and the development of experimental prehepatic PHT. Consequently, PGI(2) rather then COX-selective drugs are indicated in the treatment of PHT. Identification of additional target sites downstream of COX may benefit the >27,000 patients whom die annually from cirrhosis in the United States alone.

Elevated epsilon-(gamma-glutamyl)lysine in human diabetic nephropathy results from increased expression and cellular release of tissue transglutaminase.

INTRODUCTION: Diabetic nephropathy (DN) is the leading cause of chronic kidney failure, however the mechanisms underlying the characteristic expansion of the extracellular matrix (ECM) in diabetic kidneys remain controversial and unclear. In non-diabetic kidney scarring the protein crosslinking enzyme tissue transglutaminase (tTg) has been implicated in this process by the formation of increased epsilon-(gamma-glutamyl)lysine bonds between ECM components in both experimental and human disease. Studies in db+/db+ diabetic mice and in streptozotocin-treated rats have suggested a similar mechanism, although the relevance of this to human disease has not been addressed. METHODS: We have undertaken a retrospective analysis of renal biopsies from 16 DN patients with type 2 diabetes mellitus using an immunohistochemical and immunofluorescence approach, with tTg and epsilon-(gamma-glutamyl)lysine crosslink quantified by confocal microscopy. RESULTS: Immunofluorescent analysis of human biopsies (confocal microscopy) showed increases in levels of tTg (+1,266%, p < 0.001) and epsilon-(gamma-glutamyl)lysine (+486%, p < 0.001) in kidneys with DN compared to normal. Changes were predominantly in the extracellular periglomerular and peritubular areas. tTg staining correlated with epsilon-(gamma-glutamyl)lysine (r = 0.615, p < 0.01) and renal scarring (Masson's trichrome, r = 0.728, p < 0.001). Significant changes in epsilon-(gamma-glutamyl)lysine were also noted intracellularly in some (< or =5%) tubular epithelial cells. This is consistent with cells undergoing a novel transglutaminase-mediated cell death process in response to Ca2+ influx and subsequent activation of intracellular tTg. CONCLUSION: Changes in tTg and epsilon-(gamma-glutamyl)lysine occur in human DN. Cellular export of tTg may therefore be a factor in the perpetuation of DN by crosslinking and stabilisation of the ECM, while intracellular activation may lead to cell death contributing towards tubular atrophy.

Increases in renal epsilon-(gamma-glutamyl)-lysine crosslinks result from compartment-specific changes in tissue transglutaminase in early experimental diabetic nephropathy: pathologic implications.

Diabetic nephropathy (DN) is characterized by an early, progressive expansion and sclerosis of the glomerular mesangium leading to glomerulosclerosis. This is associated with parallel fibrosis of the renal interstitium. In experimental renal scarring, the protein cross-linking enzyme, tissue transglutaminase (tTg), is up-regulated and externalized causing an increase in its crosslink product, epsilon-(gamma-glutamyl)-lysine, in the extracellular space. This potentially contributes to the extracellular matrix (ECM) accumulation central to tissue fibrosis by increasing deposition and inhibiting breakdown. We investigated if a similar mechanism may contribute to the ECM expansion characteristic of DN using the rat streptozotocin model over 120 days. Whole kidney epsilon-(gamma-glutamyl)-lysine (HPLC analysis) was significantly increased from Day 90 (+337%) and peaked at Day 120 (+650%) (p < 0.05). Immunofluorescence showed this increase to be predominantly extracellular in the peritubular interstitial space, but also in individual glomeruli. Total kidney transglutaminase (Tg) was not elevated. However, using a Tg in situ activity assay, increased Tg was detected in both the extracellular interstitial space and glomeruli by Day 60, with a maximal 53% increase at Day 120 (p < 0.05). Using a specific anti-tTg antibody, immunohistochemistry showed a similar increase in extracellular enzyme in the interstitium and glomeruli. To biochemically characterize glomerular changes, glomeruli were isolated by selective sieving. In line with whole kidney measurement, there was an increase in glomerular epsilon-(gamma-glutamyl) lysine (+361%); however, in the glomeruli this was associated with increases in Tg activity (+228%) and tTg antigen by Western blotting (+215%). Importantly, the ratio of glomerular epsilon-(gamma-glutamyl) lysine to hydroxyproline increased by 2.2-fold. In DN, changes in the kidney result in increased translocation of tTg to the extracellular environment where high Ca(2+) and low GTP levels allow its activation. In the tubulointerstitium this is independent of increased tTg production, but dependent in the glomerulus. This leads to excessive ECM cross-linking, contributing to the renal fibrosis characteristic of progressive DN.

Transglutaminase transcription and antigen translocation in experimental renal scarring.

It was recently demonstrated that renal tissue transglutaminase (tTg) protein and its catalytic product the epsilon(gamma-glutamyl) lysine protein cross-link are significantly increased in the subtotal (5/6) nephrectomy model (SNx) of renal fibrosis in rats. It was proposed that the enzyme had two important physiologic functions in disease development; one of stabilizing the increased extracellular matrix (ECM) by protein cross-linking, the other in a novel form of tubular cell death. This study, using the same rat SNx model, demonstrates first by Northern blotting that expression of tTg mRNA when compared with controls is increased by day 15 (+70% increase, P < 0.05), then rises steadily, peaking at day 90 (+391%, P < 0.01), and remains elevated at 120 d (+205%, P < 0.05) when compared with controls. In situ hybridization histochemistry demonstrated that the tubular cells were the major site of the additional tTg synthesis. Immunohistochemistry on cryostat sections revealed a sixfold increase (P < 0.001) in ECM-bound tTg antigen at 90-d post-SNx, whereas in situ transglutaminase activity demonstrated by the incorporation of fluorescein cadaverine into cryostat sections indicated a 750% increase (P < 0.001) on day 90 in SNx animals. This increased activity was extracellular and predominantly found in the peritubular region. These results indicate that increased tTg gene transcription by tubular cells underlies the major changes in renal tTg protein reported previously in SNx rats, and that the presence of the epsilon(gamma-glutamyl) lysine cross-links in the extracellular environment is the result of the extracellular action of tTg. These changes may be in response to tubular cell injury during the scarring process and are likely to contribute to the progressive expansion of the ECM in renal fibrosis.