Abstract-Transgenic rats overexpressing both human renin and angiotensinogen genes (dTGR) develop hypertension, inflammation, and renal failure. We tested the hypothesis that these pathological features are associated with changes in renal P450-dependent arachidonic acid (AA) metabolism. Samples were prepared from 5-and 7-week-old dTGR and from normotensive Sprague-Dawley (SD) rats, ie, before and after the dTGR developed severe hypertension and albuminuria. At both stages, dTGR showed significantly lower renal microsomal AA epoxygenase and hydroxylase activities that reached 63% and 76% of the control values at week 7. Furthermore, the protein levels of several potential AA epoxygenases (CYP2C11, CYP2C23, and CYP2J) were significantly reduced. Immunoinhibition studies identified CYP2C23 as the major AA epoxygenase, both in dTGR and SD rats. Immunohistochemistry showed that CYP2C23 was localized in cortical and outer medullary tubules that progressively lost this enzyme from week 5 to week 7 in dTGR. CYP2C11 expression occurred only in the outer medullary tubules and was markedly reduced in dTGR compared with age-matched SD rats. These findings indicate site-specific decreases in the availability of AA epoxygenase products in the kidney of dTGR. In contrast to renal microsomes, liver microsomes of dTGR and SD rats showed no change in the expression and activity of AA epoxygenases and hydroxylases. We conclude that hypertension and end-organ damage in dTGR is associated with kidney-specific downregulation of P450-dependent AA metabolism. Because the products of AA epoxygenation have anti-inflammatory properties, this alteration may contribute to uncontrolled renal inflammation, which is a major cause of renal damage in dTGR. Key Words: rats Ⅲ kidney Ⅲ cytochrome P450 Ⅲ arachidonic acid Ⅲ angiotensin II Ⅲ inflammation Ⅲ end-organ damage C ytochrome P450 enzymes (P450/CYP) generate 2 major classes of arachidonic acid (AA) metabolites in the kidney, namely epoxyeicosatrienoic acids (5,6-, 8,9-, 11,12-, and 14,15-EETs) and hydroxyeicosatetraenoic acids (19-and 20-HETE). 1,2 In rat kidney, EET formation is catalyzed by P450 isoforms belonging to the CYP2C and CYP2J subfamilies. 3-5 CYP4A subfamily members produce 20-HETE. 6,7 Both EETs and 20-HETE contribute to the regulation of renal vascular tone and tubular sodium and water transport. 5,8,9 Impaired renal hemodynamics and increased salt retention was associated with altered EET-and 20-HETE-generating P450 expression in a number of hypertensive rat and mouse models. 10 -17 A recent study indicates that EETs may be important by acting as anti-inflammatory mediators in endothelial cells. 18 EETs were found to inhibit cytokine-induced activation of the nuclear transcription factor B (NF-B) and may thus prevent vascular inflammation at a very early stage. We have studied a double transgenic rat (dTGR) model, which harbors the human genes for renin and angiotensinogen. 19,20 dTGR develop hypertension associated with impaired renal hemodynamics and tubular sodium reabsorp...
Abstract-We tested the hypothesis that endothelin-converting enzyme (ECE) inhibition ameliorates end-organ damage in rats harboring both human renin and human angiotensinogen genes (dTGR). Hypertension develops in the animals, and they die by age 7 weeks of heart and kidney failure. Three groups were studied: dTGR (nϭ12) receiving vehicle, dTGR receiving ECE inhibitor (RO0687629; 30 mg/kg by gavage; nϭ10), and Sprague-Dawley control rats (SD; nϭ10) receiving vehicle, all after week 4, with euthanasia at week 7. Systolic blood pressure was not reduced by ECE inhibitor compared with dTGR (205Ϯ6 versus 206Ϯ6 mm Hg at week 7, respectively). In contrast, ECE inhibitor treatment significantly reduced mortality rate to 20% (2 of 10), whereas untreated dTGR had a 52% mortality rate (7 of 12). ECE inhibitor treatment ameliorated cardiac damage and reduced left ventricular ECE activity below SD levels. Echocardiography at week 7 showed reduced cardiac hypertrophy (4.8Ϯ0.2 versus 5.7Ϯ0.2 mg/g, PϽ0.01) and increased left ventricular cavity diameter (5.5Ϯ0.3 versus 3.1Ϯ0.1 mm, PϽ0.001) and filling volume (0.42Ϯ0.04 versus 0.16Ϯ0.06 mL, PϽ0.05) after ECE inhibitor compared with untreated dTGR. ECE inhibitor treatment also reduced cardiac fibrosis, tissue factor expression, left ventricular basic fibroblast growth factor mRNA levels, and immunostaining in the vessel wall, independent of high blood pressure. In contrast, the ECE inhibitor treatment showed no renoprotective effect. These data are the first to show that ECE inhibition reduces angiotensin II-induced cardiac damage. Key Words: angiotensin II Ⅲ enzymes Ⅲ fibrosis Ⅲ hypertrophy A ngiotensin (Ang) II-related vascular effects are partially mediated by endothelin-1 (ET-1). Long-term Ang II infusion induces preproendothelin mRNA expression. 1 In rats transgenic for both the human renin and human angiotensinogen genes (dTGR), hypertension as well as severe heart and kidney damage develop, largely independent of blood pressure elevation. The rats die by age 7 weeks. 2 The ET-1 A and B (ETA/B) receptor blocker bosentan inhibits the activation of both nuclear factor-kappa B (NF-B) and transcription factor activator protein (AP)-1 in the kidney and the heart, independent of blood pressure reduction in these rats. 3 Bohlender et al 4 studied the same rat strain and showed that a specific ETA receptor blocker is effective, particularly when combined with an Ang II receptor blocker. ET-1 is a 21-amino acid peptide that was first isolated from porcine endothelial cells. 5 Two structurally related peptides differing by 2 (ET-2) and 6 (ET-3) amino acids were subsequently identified. The endothelin precursors are processed by 2 proteases that create mature active forms, termed preproendothelins. The preproendothelins are cleaved at dibasic sites by furin-like endopeptidases to produce inactive intermediates termed big endothelins. Big endothelins are cleaved to form the final products. A family of membrane-bound zinc metalloproteases from the neprilysin superfamily conducts the last...
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