Luděk Červenka

Essential Role of AT1A Receptor in the Development of 2K1C Hypertension

Abstract—The aims of this study were to delineate the relative contribution of angiotensin II (ANG II) subtype 1A (AT1A) and 1B (AT1B) receptors to the development of two-kidney, one-clip (2K1C) Goldblatt hypertension in mice, to examine if increased nitric oxide synthase (NOS) activity counteracts the vasoconstrictor influences of ANG II in 2K1C hypertensive mice, and to determine the role of ANG II type 2 (AT2) receptors in 2K1C hypertension in mice. AT1A ANG II receptor knockout (AT1A−/−) and wild-type (AT1A+/+) mice underwent clipping of the right renal artery. Systolic blood pressure (SBP) was significantly lower in AT1A−/− compared with AT1A+/+ mice, and neither clip placement nor AT2 receptor blockade with PD 123319 (PD) altered SBP in AT1A−/− mice. A significant and sustained rise in SBP from 119±5 to 163±6 mm Hg was observed in the 2K1C AT1A+/+ mice from day 10 to day 26. Chronic PD infusion did not alter the course of hypertension in 2K1C/AT1A+/+. Acute PD infusion did not alter mean arterial pressure (MAP) in AT1A+/+, PD/AT1A+/+, 2K1C/AT1A+/+, PD/2K1C/AT1A+/+, AT1A−/−, PD/AT1A−/−, and PD/2K1C/AT1A−/− mice compared with basal levels. In contrast, acute PD infusion caused significant increases in MAP in 2K1C/AT1A−/− mice. The subsequent acute NOS inhibition caused greater increases in MAP in 2K1C/AT1A+/+ and PD/2K1C/AT1A+/+ mice than in AT1A+/+ and PD/AT1A+/+ mice. These results support the essential role of AT1A receptors in mediating 2K1C hypertension and support the hypothesis that augmented NO production serves as a counteracting system in this model of hypertension.

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.

Impairment of the angiotensin-converting enzyme 2-angiotensin-(1-7)-Mas axis contributes to the acceleration of two-kidney, one-clip Goldblatt hypertension.

Objective Recent studies have shown that the heptapeptide angiotensin-(1-7) [Ang-(1-7)] exerts important vasoactive actions and can act as an endogenous physiological antagonist of angiotensin II (Ang II) within the renin–angiotensin system (RAS). The present study was performed to evaluate the effects, first, of chronic increases of Ang-(1-7) levels, second, of [7-D-Ala], an Ang-(1-7) receptor antagonist, and, third, of an angiotensin-converting enzyme 2 (ACE2) inhibitor on the course of hypertension and of renal function of the nonclipped kidney in two-kidney, one-clip (2K1C) Goldblatt hypertensive rats. Methods Blood pressure (BP) was monitored by radiotelemetry. Elevation of the effect of circulating Ang-(1-7) levels was achieved either by chronic subcutaneous infusion of Ang-(1-7) through osmotic minipumps or by employing transgenic rats that express an Ang-(1-7)-producing fusion protein [Ang-(1-7)TGR+/+] (and its control Ang-(1-7)TGR−/−). [7-D-Ala] was also infused subcutaneously and the ACE2 inhibitor was administrated in drinking water. On day 25 after clipping, rats were anesthetized and renal function was evaluated. Results Chronic infusion of Ang-(1-7) did not modify the course of 2K1C hypertension and did not alter renal function as compared with saline vehicle-infused 2K1C rats. Chronic infusion of [7-D-Ala] or treatment with the ACE2 inhibitor worsened the course of hypertension and elicited decreases in renal hemodynamics. [Ang-(1-7)TGR+/+] and [Ang-(1-7)TGR−/−] rats exhibited a similar course of hypertension. Conclusion The present data support the notion that Ang-(1-7) serves as an important endogenous vasodilator and natriuretic agent and its deficiency might contribute to the acceleration of 2K1C Goldblatt hypertension.

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 by cis-4-(4-(3-adamantan-1-ylureido)cyclohexyl- oxy)benzoic acid exhibits antihypertensive and cardioprotective actions in transgenic rats with angiotensin II-dependent hypertension

The present study was undertaken to evaluate the effects of chronic treatment with c-AUCB {cis-4-[4-(3-adamantan-1-ylureido)cyclohexyl-oxy]benzoic acid}, a novel inhibitor of sEH (soluble epoxide hydrolase), which is responsible for the conversion of biologically active EETs (epoxyeicosatrienoic acids) into biologically inactive DHETEs (dihydroxyeicosatrienoic acids), on BP (blood pressure) and myocardial infarct size in male heterozygous TGR (Ren-2 renin transgenic rats) with established hypertension. Normotensive HanSD (Hannover Sprague-Dawley) rats served as controls. Myocardial ischaemia was induced by coronary artery occlusion. Systolic BP was measured in conscious animals by tail plethysmography. c-AUCB was administrated in drinking water. Renal and myocardial concentrations of EETs and DHETEs served as markers of internal production of epoxygenase metabolites. Chronic treatment with c-AUCB, which resulted in significant increases in the availability of biologically active epoxygenase metabolites in TGR (assessed as the ratio of EETs to DHETEs), was accompanied by a significant reduction in BP and a significantly reduced infarct size in TGR as compared with untreated TGR. The cardioprotective action of c-AUCB treatment was completely prevented by acute administration of a selective EETs antagonist [14,15-epoxyeicosa-5(Z)-enoic acid], supporting the notion that the improved cardiac ischaemic tolerance conferred by sEH inhibition is mediated by EETs actions at the cellular level. These findings indicate that chronic inhibition of sEH exhibits antihypertensive and cardioprotective actions in this transgenic model of angiotensin II-dependent hypertension.

Role of cytochrome P-450 metabolites in the regulation of renal function and blood pressure in 2-kidney 1-clip hypertensive rats

Alterations in renal function contribute to Goldblatt two-kidney, one-clip (2K1C) hypertension. A previous study indicated that bioavailability of cytochrome P-450 metabolites epoxyeicosatrienoic acids (EETs) is decreased while that of 20-hydroxyeicosatetraenoic acids (20-HETE) is increased in this model. We utilized the inhibitor of soluble epoxide hydrolase cis-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (c-AUCB) and HET-0016, the inhibitor of 20-HETE production, to study the role of EETs and 20-HETE in the regulation of renal function. Chronic c-AUCB treatment significantly decreased systolic blood pressure (SBP) (133 ± 1 vs. 163 ± 3 mmHg) and increased sodium excretion (1.23 ± 0.10 vs. 0.59 ± 0.03 mmol/day) in 2K1C rats. HET-0016 did not affect SBP and sodium excretion. In acute experiments, renal blood flow (RBF) was decreased in 2K1C rats (5.0 ± 0.2 vs. 6.9 ± 0.2 ml·min(-1)·g(-1)). c-AUCB normalized RBF in 2K1C rats (6.5 ± 0.6 ml·min(-1)·g(-1)). HET-0016 also increased RBF in 2K1C rats (5.8 ± 0.2 ml·min(-1)·g(-1)). Although RBF and glomerular filtration rate (GFR) remained stable in normotensive rats during renal arterial pressure (RAP) reductions, both were significantly reduced at 100 mmHg RAP in 2K1C rats. c-AUCB did not improve autoregulation but increased RBF at all RAPs and shifted the pressure-natriuresis curve to the left. HET-0016-treated 2K1C rats exhibited impaired autoregulation of RBF and GFR. Our data indicate that c-AUCB displays antihypertensive properties in 2K1C hypertension that are mediated by an improvement of RBF and pressure natriuresis. While HET-0016 enhanced RBF, its anti-natriuretic effect likely prevented it from producing a blood pressure-lowering effect in the 2K1C model.

Intrarenal Infusion of Angiotensin-(1–7) Modulates Renal Functional Responses to Exogenous Angiotensin II in the Rat

In the present study we investigated the possible role of angiotensin-(1–7) [Ang-(1–7)] in modulating renal functional responses to intrarenal (i.e.) infusion of angiotensin II (ANG II) in normotensive anesthetized rats. ANG II (6 ng/min, n = 14) decreased glomerular filtration rate (GFR), renal plasma flow (RPF), absolute and fractional sodium excretion by –24 ± 5, –25 ± 6, –44 ± 6 and –28 ± 7%, respectively (p < 0.05). i.r. infusion of Ang-(1–7) (50 ng/min, n = 13) did not significantly alter GFR (+6 ± 4%) but reduced RPF by –19 ± 7% (p < 0.05). Ang-(1–7) increased absolute and fractional sodium excretion by +36 ± 6 and +37 ± 8%, respectively (p < 0.05). Infusion of Ang-(1–7) did not prevent the decreases in GFR and RPF but completely blunted the decreases in absolute (–2 ± 2%) and fractional sodium excretion (–4 ± 4%) induced by ANG II (n = 11). Blockade of the Ang-(1–7) receptor by [7-D-Ala]-Ang-(1–7) (5 µg/min, n = 11) significantly decreased GFR, RPF, absolute and fractional sodium excretion by –28 ± 7, –20 ± 5, –32 ± 7 and –24 ± 4%, respectively (p < 0.05), suggesting that the action of endogenous ANG II is unopposed by compensatory effect of endogenous Ang-(1–7). i.r. infusion of Ang-(1–7) (n = 10) did not alter the effect of Ang-(1–7) receptor blockade on RPF (–21 ± 6%) but blunted its effects on GFR (+4 ± 3%) and absolute (+7 ± 5%) and fractional (+6 ± 4%) urinary sodium excretion probably by displacing the receptor blocker. While exogenous ANG II during blockade of the Ang-(1–7) receptor and the AT2 receptor (by PD 123319; 1 µg/min i.r., n = 9) resulted in the same decreases in absolute and fractional sodium excretion (–39 ± 8 and –38 ± 6%, respectively, p < 0.05) as did ANG II in the absence of Ang-(1–7) receptor blockade. These results suggest that in normotensive rats high i.r. Ang-(1–7) concentration attenuates the tubular, i.e. sodium reabsorptive effect, but not the vascular effect of exogenous i.r. ANG II. Results obtained during blockade of Ang-(1–7) and of AT2 receptors imply that AT2 receptors play a role in tubular sodium reabsorption in the presence of high ANG II concentration

Knockout of Angiotensin 1–7 Receptor Mas Worsens the Course of Two-Kidney, One-Clip Goldblatt Hypertension: Roles of Nitric Oxide Deficiency and Enhanced Vascular Responsiveness to Angiotensin II

Aims: The present study was performed to evaluate the effects of target disruption of the G-protein-coupled receptor Mas for angiotensin 1–7 [Ang(1–7)] in knockout mice on the course of two-kidney, one-clip (2K1C) Goldblatt hypertension. Methods: Knockout and wild-type mice underwent clipping of one renal artery. Blood pressure (BP) was monitored by radiotelemetry. The mice were either untreated or chronically treated with the superoxide (O2–) scavenger tempol (400 mg/l) or the inhibitor of NADPH oxidase apocynin (1 g/l) administered in drinking water. Results: Knockout mice responded to clipping by accelerated increases in BP and the final BP was significantly higher than that in wild-type mice. Chronic treatment with tempol or apocynin elicited similar antihypertensive effects in 2K1C/knockout as in 2K1C/wild-type mice. Acute nitric oxide synthase inhibition caused greater BP increases in 2K1C/wild-type than in 2K1C/knockout mice. Conclusion: Our present findings support the notion that the angiotensin-converting enzyme 2-Ang(1–7)-Mas axis serves as an important endogenous physiological counterbalancing mechanism that partially attenuates the hypertensinogenic actions of the activated renin-angiotensin system. The impairment in this axis may contribute to the deterioration of the course of 2K1C Goldblatt hypertension.

Effects of Anesthesia on Plasma and Kidney ANG II Levels in Normotensive and ANG II-Dependent Hypertensive Rats

Background: Previous studies have implicated that normotensive rats with normal renal renin activity respond to anesthesia and surgery with greater increases in plasma and kidney angiotensin II (ANG II) concentrations than ANG II-dependent hypertensive rats with intrarenal renin depletion. In the present study, we therefore compared plasma and kidney ANG II levels in anesthetized and conscious normotensive and ANG II-dependent hypertensive rats. Methods: Salt-replete Hannover-Sprague-Dawley rats (HanSD) served as controls. As models of ANG II-dependent hypertension we used: 1st, transgenic rats harboring the Ren-2 renin gene (TGR); 2nd, two-kidney, one-clip (2K1C) Goldblatt hypertensive rats, and, 3rd, ANG II-infused hypertensive rats. As additional model with enhanced renin-angiotensin system (RAS) activity, salt-depleted HanSD and TGR were employed. Results: In anesthetized salt-repleted HanSD, plasma and kidney ANG II levels were higher than in salt-repleted TGR, ANG II-infused and 2K1C rats. Salt depletion caused marked increases in ANG II levels in HanSD but did not alter them in TGR. In contrast, in conscious animals immediately after decapitation plasma and kidney ANG II levels were similar in salt-repleted and salt-depleted TGR, in ANG II-infused rats, in the clipped kidney of 2K1C rats and in salt-depleted HanSD and in all these groups they were significantly higher than in salt-repleted HanSD. Conclusions: These findings indicate that anesthesia increases plasma and kidney ANG II levels in HanSD to a greater degree than in ANG II-dependent models of hypertension. Therefore, the results from studies employing anesthetized animals must be interpreted with caution.

Renal mechanisms contributing to the antihypertensive action of soluble epoxide hydrolase inhibition in Ren‐2 transgenic rats with inducible hypertension

Arachidonic acid metabolites called epoxyeicosatrienoic acids (EETs) influence vascular tone and renal tubular sodium and water transport and thus have been implicated in the control of blood pressure. Inhibition of the enzyme soluble epoxide hydrolase (sEH), which reduces EET degradation to the corresponding diols, leads to substantial attenuation of malignant hypertension in a transgenic rat strain harbouring the mouse renin gene particularly via an improvement of renal function. The observed antihypertensive and renoprotective effects of this novel pharmacological approach provide a potentially new direction in antihypertensive therapy.