Petr Kujal

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.

Persistent antihypertensive effect of aliskiren is accompanied by reduced proteinuria and normalization of glomerular area in Ren-2 transgenic rats

The effects of the human renin inhibitor aliskiren on blood pressure (BP), end-organ damage, proteinuria, and tissue and plasma angiotensin (ANG) II levels in young and adult heterozygous Ren-2 transgenic rats (TGR) were evaluated and compared with the effect of the ANG type 1 (AT(1)) receptor blocker losartan during treatment and after 12 days after the withdrawal of drug treatments. BP was monitored by telemetry from the age of 32 days on (young rats) and at 100 days (adult rats). Aliskiren (10 mg·kg(-1)·day(-1) in osmotic minipumps) or losartan (5 mg·kg(-1)·day(-1) in drinking water) treatment was applied for 28 days in young rats and for 70 days in adult rats. In young untreated TGR, severe hypertension rapidly evolved. Adult untreated TGR exhibited stable established hypertension. Both aliskiren and losartan fully prevented the development of hypertension and cardiac hypertrophy in young TGR and normalized BP and cardiac hypertrophy in adult TGR. After cessation of aliskiren treatment in both young and adult TGR BP and cardiac hypertrophy were persistently reduced, while after losartan withdrawal BP and cardiac hypertrophy rapidly increased. In adult aliskiren-treated rats proteinuria was significantly reduced compared with losartan (the effect persisting after withdrawal of treatment), and this decrease strongly correlated with normalization of glomerular size in these animals. In conclusion, aliskiren and losartan had similar antihypertensive effects during chronic treatment, but the antihypertensive and organoprotective effects of aliskiren were persistent even after the 12-day washout period. The durable effect on proteinuria can possibly be attributed to the normalization of glomerular morphology.

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.

Inhibition of soluble epoxide hydrolase is renoprotective in 5/6 nephrectomized Ren-2 transgenic hypertensive rats

The aim of the present study was to test the hypothesis that increasing kidney tissue concentrations of epoxyeicosatrienoic acids (EETs) by preventing their degradation to the biologically inactive dihydroxyeicosatrienoic acids (DHETEs) using blockade of soluble epoxide hydrolase (sEH) would attenuate the progression of chronic kidney disease (CKD). Ren‐2 transgenic rats (TGR) after 5/6 renal mass reduction (5/6 NX) served as a model of CKD associated with angiotensin (Ang) II‐dependent hypertension. Soluble epoxide hydrolase was inhibited using cis‐4‐[4‐(3‐adamantan‐1‐yl‐ureido)cyclohexyloxy]benzoic acid (c‐AUCB; 3 mg/L drinking water) for 20 weeks after 5/6 NX. Sham‐operated normotensive transgene‐negative Hannover Sprague‐Dawley (HanSD) rats served as controls. When applied in TGR subjected to 5/6 NX, c‐AUCB treatment improved survival rate, prevented the increase in blood pressure, retarded the progression of cardiac hypertrophy, reduced proteinuria and the degree of glomerular and tubulointerstitial injury and reduced glomerular volume. All these organ‐protective actions were associated with normalization of the intrarenal EETs : DHETEs ratio, an index of the availability of biologically active EETs, to levels observed in sham‐operated HanSD rats. There were no significant concurrent changes of increased intrarenal AngII content. Together, these results show that 5/6 NX TGR exhibit a profound deficiency of intrarenal availability of active epoxygenase metabolites (EETs), which probably contributes to the progression of CKD in this model of AngII‐dependent hypertension, and that restoration of intrarenal availability of EETs using long‐term c‐AUCB treatment exhibits substantial renoprotective actions.

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.

Soluble epoxide hydrolase inhibition exhibits antihypertensive actions independently of nitric oxide in mice with renovascular hypertension

Objective: The present study was performed to examine whether the blood pressure (BP)-lowering effects of soluble epoxide hydrolase (sEH) inhibition in two-kidney, one-clip (2K1C) Goldblatt hypertension are nitric oxide (NO) dependent. Methods: Mice lacking the endothelial NO synthase (eNOS) gene (eNOS–/–) and their wild-type controls (eNOS+/+) underwent clipping of one renal artery. BP was monitored by radiotelemetry and the treatment with the sEH inhibitor cis-4-[4-(3-adamantan-1-yl-ureido)cyclohex-yloxy]-benzoic acid (c-AUCB) was initiated on day 25 after clipping and lasted for 14 days. Renal concentrations of epoxyeicosatrienoic acids (EETs) and their inactive metabolite dihydroxyeicosatrienoic acids (DHETs) were measured in the nonclipped kidney. Renal NO synthase (NOS) activity was determined by measuring the rate of formation of L-[14C]citruline from L-[14C]arginine. Results: Treatment with the sEH inhibitor elicited similar BP decreases that were associated with increases in daily sodium excretion in 2K1C eNOS+/+ as well as 2K1C eNOS–/– mice. In addition, treatment with the sEH inhibitor increased the ratio of EETs/DHETs in the nonclipped kidney of 2K1C eNOS+/+ as well as 2K1C eNOS–/– mice. Treatment with the sEH inhibitor did not alter renal NOS activity in any of the experimental groups. Conclusions: Collectively, our present data suggest that the BP-lowering effects of chronic sEH inhibition in 2K1C mice are mainly associated with normalization of the reduced availability of biologically active EETs in the nonclipped kidney and their direct natriuretic actions.

Antihypertensive and renoprotective actions of soluble epoxide hydrolase inhibition in ANG II-dependent malignant hypertension are abolished by pretreatment with L-NAME

Objective: The present study was performed to investigate in a model of malignant hypertension if the antihypertensive actions of soluble epoxide hydrolase (sEH) inhibition are nitric oxide (NO)-dependent. Methods: ANG II-dependent malignant hypertension was induced through dietary administration for 3 days of the natural xenobiotic indole-3-carbinol (I3C) in Cyp1a1-Ren-2 transgenic rats. Blood pressure (BP) was monitored by radiotelemetry and treatment with the sEH inhibitor [cis-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyl-oxy]-benzoic acid (c-AUCB)] was started 48 h before administration of the diet containing I3C. In separate groups of rats, combined administration of the sEH inhibitor and the nonspecific NO synthase inhibitor [N&ohgr;-nitro-L-arginine methyl ester (L-NAME)] on the course of BP in I3C-induced and noninduced rats were evaluated. In addition, combined blockade of renin–angiotensin system (RAS) was superimposed on L-NAME administration in separate groups of rats. After 3 days of experimental protocols, the rats were prepared for renal functional studies and renal concentrations of epoxyeicosatrienoic acids (EETs) and their inactive metabolites dihydroxyeicosatrienoic acids (DHETEs) were measured. Results: Treatment with c-AUCB increased the renal EETs/DHETEs ratio, attenuated the increases in BP, and prevented the decreases in renal function and the development of renal damage in I3C-induced Cyp1a1-Ren-2 rats. The BP lowering and renoprotective actions of the treatment with the sEH inhibitor c-AUCB were completely abolished by concomitant administration of L-NAME and not fully rescued by double RAS blockade without altering the increased EETs/DHETEs ratio. Conclusion: Our current findings indicate that the antihypertensive actions of sEH inhibition in this ANG II-dependent malignant form of hypertension are dependent on the interactions of endogenous bioavailability of EETs and NO.

Epoxyeicosatrienoic acid analog attenuates the development of malignant hypertension, but does not reverse it once established: A study in Cyp1a1-Ren-2 transgenic rats

Objective: We evaluated the therapeutic effectiveness of a new, orally active epoxyeicosatrienoic acid analog (EET-A) in rats with angiotensin II (ANG II)-dependent malignant hypertension. Methods: Malignant hypertension was induced in Cyp1a1-Ren-2 transgenic rats by activation of the renin gene using indole-3-carbinol (I3C), a natural xenobiotic. EET-A treatment was started either simultaneously with I3C induction process (early treatment) or 10 days later during established hypertension (late treatment). Blood pressure (BP) (radiotelemetry), indices of renal and cardiac injury, and plasma and kidney levels of the components of the renin–angiotensin system (RAS) were determined. Results: In I3C-induced hypertensive rats, early EET-A treatment attenuated BP increase (to 175 ± 3 versus 193 ± 4 mmHg, P < 0.05, on day 13), reduced albuminuria (15 ± 1 versus 28 ± 2 mg/24 h, P < 0.05), and cardiac hypertrophy as compared with untreated I3C-induced rats. This was associated with suppression of plasma and kidney ANG II levels (48 ± 6 versus 106 ± 9 and 122 ± 19 versus 346 ± 11 fmol ml−1 or g, respectively, P < 0.05) and increases in plasma and kidney angiotensin (1–7) concentrations (84 ± 9 versus 37 ± 6 and 199 ± 12 versus 68 ± 9 fmol/ml or g, respectively, P < 0.05). Remarkably, late EET-A treatment did not lower BP or improve renal and cardiac injury; indices of RAS activity were not affected. Conclusion: The new, orally active EET-A attenuated the development of experimental ANG II-dependent malignant hypertension, likely via suppression of the hypertensiogenic axis and augmentation of the vasodilatory/natriuretic axis of RAS.

Orally active epoxyeicosatrienoic acid analog does not exhibit antihypertensive and reno- or cardioprotective actions in two-kidney, one-clip Goldblatt hypertensive rats.

This study examined the effects of a novel orally active 14,15-epoxyeicosatrienoic acid analog (EET-A) on blood pressure (BP) and myocardial infarct size (IS) in two-kidney, one-clip (2K1C) Goldblatt hypertensive rats during sustained phase of hypertension. Between days 31 and 35 after clip placement the rats were treated with EET-A and BP was monitored by radiotelemetry; sham-operated normotensive rats were used as controls. Tissue concentrations of epoxyeicosatrienoic acids served as a marker of production of epoxygenase metabolites. The rats were subjected to acute myocardial ischemia/reperfusion (I/R) injury and IS was determined. We found that EET-A treatment did not lower BP in 2K1C rats and did not alter availability of biologically active epoxygenase metabolites in 2K1C or in sham-operated rats. The myocardial IS was significantly smaller in untreated 2K1C rats as compared with normotensive controls and EET-A reduced it in controls but not in 2K1C rats. Our findings suggest that during the phase of sustained hypertension 2K1C Goldblatt hypertensive rats exhibit increased cardiac tolerance to I/R injury as compared with normotensive controls, and that in this animal model of human renovascular hypertension short-term treatment with EET-A does not induce any antihypertensive and cardioprotective actions.