Martin Opočenský

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.

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.

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.

Inappropriately high circulating and intrarenal angiotensin II levels during dietary salt loading exacerbate hypertension in Cyp1a1-Ren-2 transgenic rats.

Objective We evaluated the effects of salt restriction and of increasing dietary salt loading on blood pressure and the renin–angiotensin system in transgenic rats with inducible hypertension. Methods Hypertension was induced in Cyp1a1–Ren-2 rats through dietary administration of the natural xenobiotic indole-3-carbinol (0.3%), which activates the renin gene. Rats were fed either a normal-salt diet (0.6% NaCl), three different high-salt diets (2, 4 and 8% NaCl) or a low-salt diet (<0.04% NaCl). Blood pressure was monitored by radiotelemetry. Angiotensin II (ANG II) levels were determined by radioimmunoassay. Results Induction of the renin gene by administration of indole-3-carbinol resulted in normal-salt diet fed animals in the development of severe hypertension that was accompanied by marked increases in plasma and kidney ANG II levels. Feeding the low-salt diet substantially attenuated the development of hypertension. Treatment with the 2 and 4% high-salt diet did not worsen the course of hypertension and did not alter ANG II levels when compared with rats on the normal salt diet. Feeding the 8% high-salt diet exacerbated the course of hypertension and was associated with further strong increases in plasma and kidney ANG II levels. Conclusion Our results demonstrate that after induction of the renin gene in Cyp1a1–Ren-2 transgenic rats inappropriate increases in plasma and kidney ANG II levels in response to very high dietary salt intake are responsible for the development of severe hypertension in this model of inducible renin transgenic rats.

Blockade of Endothelin Receptors Attenuates End-Organ Damage in Homozygous Hypertensive Ren-2 Transgenic Rats

Background/Aims: A growing body of evidence suggests that the interplay between the endothelin (ET) and the renin-angiotensin systems (RAS) plays an important role in the development of the malignant phase of hypertension. The present study was performed to evaluate the role of an interaction between ET and RAS in the development of hypertension and hypertension-associated end-organ damage in homozygous male transgenic rats harboring the mouse Ren-2 renin gene (TGRs) under conditions of normal-salt (NS, 0.45% NaCl) and high-salt (HS, 2% NaCl) intake. Methods: Twenty-eight-day-old homozygous male TGRs and age-matched transgene-negative male normotensive Hannover Sprague-Dawley (HanSD) rats were randomly assigned to groups with NS or HS intake. Nonselective ETA/B receptor blockade was achieved with bosentan (100 mg/kg/day). Systolic blood pressure (BP) was measured in conscious animals by tail plethysmography. Rats were placed into metabolic cages to determine proteinuria and clearance of endogenous creatinine. At the end of the experiment the final arterial BP was measured directly in anesthetized rats. Kidneys were taken for morphological examination. Results: All male HanSD fed either the NS or HS diet exhibited a 100% survival rate until 180 days of age (end of experiment). The survival rate in untreated homozygous male TGRs fed the NS diet was 41%, which was markedly improved by treatment with bosentan to 88%. The HS diet reduced the survival rate in homozygous male TGRs to 10%. The survival rate in homozygous male TGRs on the HS diet was significantly improved by bosentan to 69%. Treatment with bosentan did not influence either the course of hypertension or the final levels of BP in any of the experimental groups of HanSD rats or TGRs. Although the ET-1 content in the renal cortex did not differ between HanSD rats and TGRs, ET-1 in the left heart ventricle of TGRs fed the HS diet was significantly higher compared with all other groups. Administration of bosentan to homozygous male TGRs fed either the NS or HS diet markedly reduced proteinuria, glomerulosclerosis and attenuated the development of cardiac hypertrophy compared with untreated TGR. Conclusions: Our data show that nonselective ETA/B receptor blockade markedly improves the survival rate and ameliorates end-organ damage in homozygous male TGRs without significantly lowering BP.

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 Dietary Salt Load and Salt Depletion on the Course of Hypertension and Angiotensin II Levels in Male and Female Heterozygous Ren-2 Transgenic Rats

Background: In the present study we evaluated plasma and kidney angiotensin II (ANG II) levels in female and male Ren-2 transgenic rats (TGR) in comparison to age-matched female and male normotensive Hannover Sprague-Dawley rats. Methods: The rats were maintained on a normal sodium (NS) diet (0.6% NaCl) or fed a high sodium (HS) diet (2% NaCl) for 4 days or were sodium depleted by administration of 40 mg furosemide per liter drinking water overnight followed by 3 days of low sodium diet (0.01% NaCl) (LS + F). ANG II levels were determined by radioimmunoassay. Results: Female TGR at the age of 38 days were already hypertensive and had developed cardiac hypertrophy, whereas male TGR at this age still exhibited a normotensive phenotype. HS diet increased the blood pressure (BP) but did not alter the ANG II levels in TGR at any age. LS + F decreased the BP without significant change in ANG II concentrations in TGR. Female TGR responded to salt loading and salt depletion by more pronounced changes in BP than male TGR. Conclusions: Female TGR develop hypertension more rapidly and the salt-sensitive component of hypertension is more pronounced in female than in male TGR.

Roles of nitric oxide and oxidative stress in the regulation of blood pressure and renal function in prehypertensive Ren-2 transgenic rats.

Aims: The present study was performed to evaluate the role of nitric oxide (NO) and its interaction with superoxide anion (O2–) in the regulation of blood pressure (BP) and renal function during the developmental phase of hypertension in Ren-2 transgenic rats (TGR). The first aim was to compare BP and renal functional responses to acute NO synthase (NOS) inhibition achieved by intravenous (i.v.) infusion of Nω-nitro-L-arginine-methyl ester (L-NAME) in prehypertensive heterozygous TGR and in transgene-negative Hannover Sprague-Dawley (HanSD) rats. The second aim was to evaluate whether scavenging of O2– by infusion of the superoxide dismutase mimetic tempol increases NO bioavailability which therefore should augment BP and renal functional responses to L-NAME. Methods: Rats were anesthetized, prepared for clearance experiments and BP and renal functional responses were evaluated in response to i.v. L-NAME administration (20 µg·100 g–1·min–1) without or with tempol pretreatment (i.v., 300 µg·100 g–1·min–1). In renal cortical tissue, nitrotyrosine protein expression was assessed by immunoblotting as marker of O2– production and urinary 8-epi-PGF2α excretion as marker of intrarenal oxidative stress was assessed by enzyme immunoassay. Results: BP, glomerular filtration rate (GFR), renal plasma flow (RPF) and sodium excretion were similar in TGR and HanSD. L-NAME infusion induced greater increases in BP in TGR than in HanSD (+42 ± 4 vs. +25 ± 3 mm Hg, p < 0.05). In the absence of a significant change in GFR, L-NAME caused similar decreases in RPF (–32 ± 6 and –25 ± 4%, p < 0.05) in TGR and HanSD. Despite significantly higher renocortical expression of nitrotyrosine and urinary 8-epi-PGF2α excretion in TGR than in HanSD, pretreatment with tempol did not augment the rise in BP and the decrease in RPF induced by L-NAME. Conclusions: The greater BP response to L-NAME in TGR suggests that prehypertensive TGR exhibit an enhanced NO activity in the systemic vasculature as compared with HanSD. Despite increased intrarenal oxidative stress in TGR, the dependency of the intrarenal vascular tone on NO appears to be similar in TGR and HanSD. The lack of a compensatory increase in renal NO activity may partially account for the enhanced renal vascular response to ANG II present in TGR.

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.