Věra Čertíková Chábová

Late-Onset Endothelin-A Receptor Blockade Reduces Podocyte Injury in Homozygous Ren-2 Rats Despite Severe Hypertension

We have recently found in male homozygous hypertensive Ren-2 transgenic rats (TGRs) fed a high-salt diet that early onset selective endothelin (ET) A (ETA) or nonselective ETA/ET B (ETB) receptor blockade improved survival rate and reduced proteinuria, glomerulosclerosis, and cardiac hypertrophy, whereas selective ETA receptor blockade also significantly attenuated the rise in blood pressure. Because antihypertensive therapy in general is known to be more efficient when started at early age, our study was performed to determine whether onset of ET receptor blockade at a later age in animals with established hypertension will have similar protective effects as does early-onset therapy. Male homozygous TGRs and age-matched normotensive Hannover Sprague–Dawley rats were fed a high-salt diet between days 51 and 90 of age. TGRs received vehicle (untreated), the selective ETA receptor blocker atrasentan (ABT-627), or the nonselective ETA/ETB receptor blocker bosentan. Survival rates in untreated and bosentan-treated TGRs were 50% and 64%, respectively, whereas with atrasentan, survival rate of TGR was 96%, thus, similar to 93% in Hannover Sprague–Dawley rats. From day 60 on, systolic blood pressure in atrasentan-treated TGRs was transiently lower (P<0.05) than in untreated or bosentan-treated TGRs. Glomerular podocyte injury was substantially reduced with atrasentan treatment independent of severe hypertension and strongly correlated with survival (P<0.001). Our data indicate that in homozygous TGR ET receptors play an important role also in established hypertension. Selective ETA receptor blockade not only reduces podocyte injury and end-organ damage but also improves growth and survival independently of hypertension.

Pivotal role of angiotensin II receptor subtype 1A in the development of two-kidney, one-clip hypertension: study in angiotensin II receptor subtype 1A knockout mice.

Objective The present study was performed to examine in two-kidney, one-clip (2K1C) Goldblatt hypertensive mice: first, the relative contribution of angiotensin II receptor subtypes 1A (AT1A) and 1B (AT1B); second, the role of angiotensin II type 2 (AT2) receptors in the development of hypertension in wild-type (AT1A+/+) and AT1A receptor knockout (AT1A−/−) mice; and third, the role of increased nitric oxide synthase activity in counteracting the hypertensinogenic action of angiotensin II in this model. Methods AT1A+/+ and AT1A−/− mice underwent clipping of one renal artery and were infused with either saline vehicle or selective AT2 receptor agonist CGP-42112A (CGP). Blood pressure was monitored by radiotelemetry. Blood pressure responses to the nitric oxide synthase inhibitor nitro-L-arginine-methyl-ester were evaluated. Results AT1A+/+ mice responded to clipping by a rise in blood pressure that was not modified by CGP infusion. Clip placement caused a slight increase in blood pressure in AT1A−/− mice that remained significantly lower than in AT1A+/+ mice. Acute nitric oxide synthase inhibition caused greater increase in blood pressure in 2K1C/AT1A+/+ than in AT1A+/+ mice. Conclusion The present data support the critical role of AT1A receptors in the development of 2K1C hypertension, whereas AT1B receptors play only a minor role in blood pressure regulation in this model of angiotensin II-dependent hypertension. Activation of AT2 receptors does not play an antagonistic role in the AT1 receptor-mediated hypertensinogenic actions of angiotensin II in this model. Finally, enhanced nitric oxide synthase activity plays a protective role by counteracting the vasoconstrictor influences of angiotensin II in 2K1C hypertensive mice.

Combined inhibition of 20-hydroxyeicosatetraenoic acid formation and of epoxyeicosatrienoic acids degradation attenuates hypertension and hypertension-induced end-organ damage in Ren-2 transgenic rats

Recent studies have shown that the renal CYP450 (cytochrome P450) metabolites of AA (arachidonic acid), the vasoconstrictor 20-HETE (20-hydroxyeicosatetraenoic acid) and the vasodilator EETs (epoxyeicosatrienoic acids), play an important role in the pathophysiology of AngII (angiotensin II)-dependent forms of hypertension and the associated target organ damage. The present studies were performed in Ren-2 renin transgenic rats (TGR) to evaluate the effects of chronic selective inhibition of 20-HETE formation or elevation of the level of EETs, alone or in combination, on the course of hypertension and hypertension-associated end-organ damage. Both young (30 days of age) prehypertensive TGR and adult (190 days of age) TGR with established hypertension were examined. Normotensive HanSD (Hannover Sprague-Dawley) rats served as controls. The rats were treated with N-methylsulfonyl-12,12-dibromododec-11-enamide to inhibit 20-HETE formation and/or with N-cyclohexyl-N-dodecyl urea to inhibit soluble epoxide hydrolase and prevent degradation of EETs. Inhibition in TGR of 20-HETE formation combined with enhanced bioavailability of EETs attenuated the development of hypertension, cardiac hypertrophy, proteinuria, glomerular hypertrophy and sclerosis as well as renal tubulointerstitial injury. This was also associated with attenuation of the responsiveness of the systemic and renal vascular beds to AngII without modifying their responses to noradrenaline (norepinephrine). Our findings suggest that altered production and/or action of 20-HETE and EETs plays a permissive role in the development of hypertension and hypertension-associated end-organ damage in this model of AngII-dependent hypertension. This information provides a basis for a search for new therapeutic approaches for the treatment of hypertension.

Inhibition of soluble epoxide hydrolase improves the impaired pressure-natriuresis relationship and attenuates the development of hypertension and hypertension-associated end-organ damage in Cyp1a1-Ren-2 transgenic rats.

Objective In the present study, we compared the effects of treatment with the novel soluble epoxide hydrolase (sEH) inhibitor (c-AUCB) with those of the AT1 receptor antagonist losartan on blood pressure (BP), autoregulation of renal blood flow (RBF) and on glomerular filtration rate (GFR) and the pressure–natriuresis relationship in response to stepwise reduction in renal arterial pressure (RAP) in Cyp1a1-Ren-2 transgenic rats. Methods Hypertension was induced in Cyp1a1-Ren-2 rats through dietary administration for 11 days of the natural xenobiotic indole-3-carbinol (I3C) which activates the renin gene. Treatment with c-AUCB and losartan was started 48 h before initiating administration of the diet containing I3C. Rats were prepared for renal functional studies to evaluate in-vivo renal autoregulatory efficiency when RAP was gradually decreased by an aortic clamp. Results I3C administration resulted in the development of severe hypertension which was associated with markedly lower basal RBF and GFR and substantially impaired autoregulatory efficiency as well as a suppression of the pressure–natriuresis relationship when compared with noninduced rats. Treatment with c-AUCB significantly decreased BP, improved autoregulatory efficiency of RBF and GFR and the slope of pressure–natriuresis relationship. Treatment with losartan completely prevented the impaired autoregulation and pressure–natriuresis relationship as well as the development of hypertension in I3C-induced rats. Conclusion Our present findings indicate that chronic treatment with the sEH inhibitor c-AUCB substantially attenuates the development of malignant hypertension in I3C-induced rats likely via improvement of the renal autoregulatory efficiency and the pressure–natriuresis relationship.

Similar renoprotection after renin‐angiotensin‐dependent and ‐independent antihypertensive therapy in 5/6‐nephrectomized Ren‐2 transgenic rats: are there blood pressure‐independent effects?

1. Hypertension plays a critical role in the progression of chronic kidney disease (CKD) to end‐stage renal disease (ESRD), but it has also been postulated that antihypertensive drugs that block the renin‐angiotensin system (RAS) show class‐specific renoprotective actions beyond their blood pressure (BP)‐lowering effects.

Effects of Combined Endothelin A Receptor and Renin-Angiotensin System Blockade on the Course of End-Organ Damage in 5/6 Nephrectomized Ren-2 Hypertensive Rats

Our previous studies in rats with ablation nephrectomy have shown similar cardiorenal protective effects of renin-angiotensin system (RAS)-dependent treatment (combination of angiotensin-converting enzyme inhibitor and angiotensin II receptor blocker) and RAS-independent treatment (combination of α- and β-adrenoreceptor antagonist and diuretics). Moreover, selective blockade of endothelin (ET) receptor type A (ETA) improved survival rate and attenuated hypertension and organ damage in Ren-2 transgenic rats. Therefore, we were interested in whether ETA receptor blockade could have additive effects to the classical blockade of the RAS. Transgenic rats underwent 5/6 renal ablation at the age of 2 months and were treated for 20 weeks with RAS blockers alone (angiotensin II receptor blocker – losartan, and angiotensin-converting enzyme inhibitor – trandolapril), ETA receptor blocker alone (atrasentan) or with the combination of RAS and ETA receptor blockade. RAS blockade normalized blood pressure and improved survival. It decreased cardiac hypertrophy and proteinuria as well as tissue angiotensin II and ET-1 levels. In contrast, ETA receptor blockade only partially improved survival rate, reduced blood pressure, attenuated the development of cardiac hypertrophy and transiently reduced proteinuria. However, no additive cardio- and renoprotective effects of ETA and RAS blockade were noted at the end of the study.

Intrarenal cytochrome P-450 metabolites of arachidonic acid in the regulation of the nonclipped kidney function in two-kidney, one-clip Goldblatt hypertensive rats.

Objective The contribution of epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) as cytochrome P-450 metabolites of arachidonic acid in the regulation of the nonclipped kidney function in two-kidney, one-clip (2K1C) Goldblatt hypertensive rats was investigated during the phases of initial and stable hypertension, that is, 7 or 27 days after clipping, respectively. Methods Male Hannover Sprague–Dawley rats had the right renal artery clipped or underwent sham operation. Urinary excretion of EETs, their inactive metabolites dihydroxyeicosatrienoic acids and of 20-HETE was measured. Intrarenal cytochrome P-450 protein expression and the activities of epoxygenase, ω-hydroxylase and soluble epoxide hydrolase were also determined. The responses of renal hemodynamics and electrolyte excretion of the nonclipped kidney to left renal artery infusions of inhibitors of EETs or 20-HETE formation (MS-PPOH and DDMS, respectively) were measured. Results In 2K1C rats, urinary excretion of EETs was significantly lower and that of 20-HETE was higher than that in sham-operated animals only on day 27 after clipping. Intrarenal inhibition of EETs significantly decreased renal hemodynamics and sodium excretion in sham-operated but not in 2K1C rats. Intrarenal inhibition of 20-HETE decreased sodium excretion in sham-operated rats but elicited increases in renal hemodynamics and sodium excretion in 2K1C rats. Conclusion Our results indicate that the nonclipped kidney of Goldblatt 2K1C rats in the phase of sustained hypertension exhibits decreased intrarenal EETs and elevated 20-HETE levels as compared with the kidney of sham-operated animals. This suggests that altered production and action of cytochrome P-450-derived metabolites during this stable phase contributes to the mechanism of Goldblatt 2K1C hypertension.

Late-onset endothelin receptor blockade in hypertensive heterozygous REN-2 transgenic rats

Our previous studies in heterozygous Ren-2 transgenic rats (TGR) have shown that early treatment with selective endothelin (ET)(A) receptor blockade is superior to nonselective ET(A/B) receptor blockade. The aim of this study was to evaluate the role of the ET system in male heterozygous TGR with established hypertension (late-onset treatment). TGR and control Hannover Sprague-Dawley (HanSD) rats were fed a high-salt diet and were treated concomitantly with the nonselective ET(A/B) receptor blocker bosentan or the selective ET(A) receptor blocker atrasentan from day 52 of age on. Survival rate was partly increased by bosentan and fully normalized with atrasentan. Bosentan transiently decreased blood pressure (BP), whereas atrasentan significantly reduced BP as early as one week after the start of the treatment. This effect persisted for the whole experimental period. Atrasentan also substantially reduced cardiac hypertrophy, proteinuria, glomerulosclerosis and left ventricle ET-1 content. Bosentan improved and atrasentan almost restored podocyte architecture and reversed changes in podocyte phenotype represented by the expression of CD 10, desmin and vimentin. Our results demonstrate that selective ET(A) receptor blockade has more favorable effects than nonselective ET(A/B) receptor blockade and, unlike observed in homozygous TGR, ET(A) receptor blockade has similar effects in heterozygous rats with established hypertension as in young animals with developing hypertension.

The roles of intrarenal 20-hydroxyeicosatetraenoic and epoxyeicosatrienoic acids in the regulation of renal function in hypertensive Ren-2 transgenic rats.

Background: The present study was performed in hypertensive Ren-2 transgenic rats (TGR) and in normotensive Hannover Sprague-Dawley (HanSD) rats. First, the intrarenal protein expression of CYP4A, the enzyme catalyzing the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), and of CYP2C23, the enzyme responsible for epoxyeicosatrienoic acid (EET) production, was evaluated. Second, the renal functional responses to inhibition of the intrarenal formation of 20-HETE and EETs were investigated. Methods: Renal hemodynamics and electrolyte excretion were evaluated in response to the administration of inhibitors of 20-HETE and EET formation into the renal artery. In renal cortical tissue, CYP4A and CYP2C23 protein expression was assessed by Western blot analysis. Urinary concentrations of 20-HETE and EETs were measured using a fluorescent HPLC assay. Results: TGR have higher kidney CYP4A protein expression and urinary 20-HETE excretion but significantly lower CYP2C23 protein expression and urinary EET excretion than HanSD. Intrarenal inhibition of 20-HETE and EET formation decreased sodium excretion in HanSD, whereas inhibition of 20-HETE increased urinary excretion of sodium in TGR without altering renal hemodynamics. Conclusions: Our data suggest that in TGR, deficient intrarenal synthesis of EETs combined with increased synthesis of 20-HETE with its stimulation of tubular sodium absorption may contribute to the development of hypertension in TGR.

Endothelin receptor blockade does not affect blood pressure or angiotensin II levels in CYP1A1-Ren-2 transgenic rats with acutely induced hypertension

We found previously that selective blockade of endothelin ETA receptors is superior to nonselective ET(A)/ET(B) in attenuating hypertension and survival rate in Ren-2 transgenic rats (TGR). In the present pilot study, we were interested in whether similar effects will be found in TGR with inducible malignant hypertension (iTGR; official strain name Cyp1A1-Ren-2rats), which were derived from the original Ren-2 transgenic rat strain. Studies were performed in three-month old male iTGR. Treatment with either bosentan, a non-selective ET(A)/ET(B), or with atrasentan, a selective ET(A) receptor blocker, was started on day 2 of the experiment. Feeding with indole-3-carbinole (13C; 03% in rat chow), a natural xenobiotic which activates the Cyplal promoter of the mouse Ren-2 gene, began on day 3 and lasted for 4 days until day 6. Systolic BP, body weight, plasma ANG II and tissue ANG II and ET-1 concentrations were determined daily. Severe hypertension developed as early as 1 day after beginning of 13C feeding which was accompanied by a significant reduction in body weight and by increases in plasma and tissue ANG II and left ventricle ET-1 concentrations. Atrasentan or bosentan had no effects on the rise in BP or plasma and tissue ANG II concentrations but prevented the rise in heart ventricle ET-1 concentration. Our data show that blockade of the ET system does not prevent or attenuate the rapid development of severe hypertension in iTGR; a long-term protective effect of ET blockade on cardiac (and renal) damage, however, cannot be excluded and awaits further investigations.