ANG II-dependent Malignant Hypertension Research Articles

20-Hydroxyeicosatetraenoic acid antagonist attenuates the development of malignant hypertension and reverses it once established: a study in Cyp1a1-Ren-2 transgenic rats.

We hypothesized that vascular actions of 20-hydroxyeicosatetraenoic acid (20-HETE), the product of cytochrome P450 (CYP450)-dependent ω-hydroxylase, potentiate prohypertensive actions of angiotensin II (ANG II) in Cyp1a1-Ren-2 transgenic rats, a model of ANG II-dependent malignant hypertension. Therefore, we evaluated the antihypertensive effectiveness of 20-HETE receptor antagonist (AAA) in this model. Malignant hypertension was induced in Cyp1a1-Ren-2 transgenic rats by activation of the renin gene using indole-3-carbinol (I3C), a natural xenobiotic. Treatment with AAA was started either simultaneously with induction of hypertension or 10 days later, during established hypertension. Systolic blood pressure (SBP) was monitored by radiotelemetry, indices of renal and cardiac injury, and kidney ANG II levels were determined. In I3C-induced hypertensive rats, early AAA treatment reduced SBP elevation (to 161 ± 3 compared with 199 ± 3 mmHg in untreated I3C-induced rats), reduced albuminuria, glomerulosclerosis index, and cardiac hypertrophy (P<0.05 in all cases). Untreated I3C-induced rats showed augmented kidney ANG II (405 ± 14 compared with 52 ± 3 fmol/g in non-induced rats, P<0.05) which was markedly lowered by AAA treatment (72 ± 6 fmol/g). Remarkably, in TGR with established hypertension, AAA also decreased SBP (from 187 ± 4 to 158 ± 4 mmHg, P<0.05) and exhibited organoprotective effects in addition to marked suppression of kidney ANG II levels. In conclusion, 20-HETE antagonist attenuated the development and largely reversed the established ANG II-dependent malignant hypertension, likely via suppression of intrarenal ANG II levels. This suggests that intrarenal ANG II activation by 20-HETE is important in the pathophysiology of this hypertension form.

Abstract P426: Suppression of Intrarenal Ras With a 20-hydroxyeicosatetraenoic Acid Antagonist Attenuates Ang II-dependent Malignant Hypertension and Reverses Established Ang-II-dependent Hypertension in Cyp1a1-ren-2 Transgenic Rats

Rationale and Objective: Interplay of 20-HETE and ANG II might be a factor in pathophysiology of many forms of experimental hypertension. We hypothesized that intrarenal 20-HETE potentiates prohypertensive actions of ANG II in Cyp1a1-Ren-2 transgenic rats (TGR), a model of ANG II-dependent malignant hypertension. Therefore, we evaluated the antihypertensive effectiveness of a new, orally active 20-HETE antagonist (SOLA) in this model. Methods: Treatment with SOLA (10 mg.kg -1 .day - 1 in drinking water) was started either simultaneously with induction of hypertension (early treatment) by indol-3-carbinol or 10 days later, during established hypertension (late treatment). Systolic blood pressure (SBP, measured by radiotelemetry) was monitored continuously and indices of renal and cardiac injury, and kidney 20-HETE and ANG II levels were determined at the end of experiments. Results: In TGR with induced hypertension, early SOLA treatment reduced SBP elevation (to 161±3 vs. 199±3 mmHg in induced TGR, p&lt;0.001), reduced albuminuria (16±2 vs. 35±3 mg/24 h, p&lt;0.002), glomerulosclerosis index (0.12±0.01 vs. 0.32±0.02, p&lt;0.001) and cardiac hypertrophy (left ventricle weight (mg)/tibial length ratio: 16.9±0.4 vs 20.6±0.5, p&lt;0.02). TGR with induced hypertension exhibited elevated intrarenal 20-HETE levels (15.2±0.03 vs. 9.2±0.02 μg/g in noninduced rats, p&lt;0.01); however, the elevation was not altered by SOLA treatment. Hypertensive TGR showed also augmented kidney ANG II levels (405±30 vs. 51±2 fmol/g in noninduced rats, p&lt;0.001). SOLA treatment significantly lowered kidney ANG II (95±19). Remarkably, in TGR with established hypertension, late SOLA treatment also decreased SBP (from 187±4 to 158±4 mmHg, p&lt;0.002) and exhibited cardio- and renoprotective effects in addition to a marked suppression of kidney ANG II levels (72±12 fmol/g). Conclusion: In hypertensive TGR the new orally active 20-HETE antagonist attenuated the development and largely reversed the established ANG II-dependent experimental malignant hypertension, likely via suppression of intrarenal ANG II levels. This suggests that intrarenal RAS activation by 20-HETE is important in pathophysiology of this hypertension form

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

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. 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. 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 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. 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.

Intrarenal alterations of the angiotensin-converting enzyme type 2/angiotensin 1-7 complex of the renin-angiotensin system do not alter the course of malignant hypertension in Cyp1a1-Ren-2 transgenic rats.

The role of the intrarenal renin-angiotensin system (RAS) in the pathophysiology of malignant hypertension is not fully understood. Accumulating evidence indicates that the recently discovered vasodilator axis of the RAS, angiotensin-converting enzyme (ACE) type 2 (ACE2)/angiotensin 1-7 (ANG 1-7), constitutes an endogenous system counterbalancing the hypertensiogenic axis, ACE/angiotensin II (ANG II)/AT1 receptor. This study aimed to evaluate the role of the intrarenal vasodilator RAS axis in the pathophysiology of ANG II-dependent malignant hypertension in Cyp1a1-Ren-2 transgenic rats. ANG II-dependent malignant hypertension was induced by 13days' dietary administration of indole-3-carbinol (I3C), a natural xenobiotic that activates the mouse renin gene in Cyp1a1-Ren-2 transgenic rats. It was hypothesized that pharmacologically-induced inhibition of the ACE2/ANG 1-7 complex should aggravate, and activation of this axis should attenuate, the course of ANG II-dependent malignant hypertension. Blood pressure (BP) was monitored by radiotelemetry. ACE2 inhibitor (DX 600, 0.2μg/day) and ACE2 activator (DIZE, 1mg/day) were administrated via osmotic minipumps. Even though ACE2 inhibitor significantly decreased and ACE2 activator increased intrarenal ANG 1-7 concentrations, the course of BP, as well as of albuminuria, cardiac hypertrophy and renal glomerular damage, were not altered. It was shown that intrarenal alterations in the ACE2/ANG 1-7 complex did not significantly modify the course of malignant hypertension in I3C-induced Cyp1a1-Ren-2 transgenic rats. Thus, in our experimental setting alterations of this intrarenal vasodilator complex of the RAS do not significantly modify the form of malignant hypertension that clearly depends on the inappropriately increased activity of the ACE/ANG II/AT1 receptor axis.

Different mechanisms of acute versus long-term antihypertensive effects of soluble epoxide hydrolase inhibition: studies in Cyp1a1-Ren-2 transgenic rats.

Recent studies have shown that the long-term antihypertensive action of soluble epoxide hydrolase inhibition (sEH) in angiotensin-II (AngII)-dependent hypertension might be mediated by the suppression of intrarenal AngII levels. To test this hypothesis, we examined the effects of acute (2 days) and chronic (14 days) sEH inhibition on blood pressure (BP) in transgenic rats with inducible AngII-dependent hypertension. AngII-dependent malignant hypertension was induced by 10 days' dietary administration of indole-3-carbinol (I3C), a natural xenobiotic that activates the mouse renin gene in Cyp1a1-Ren-2 transgenic rats. BP was monitored by radiotelemetry. Acute and chronic sEH inhibition was achieved using cis-4-(4-(3-adamantan-1-yl-ureido)cyclohexyloxy) benzoic acid, given at doses of 0.3, 3, 13, 26, 60 and 130 mg/L in drinking water. At the end of experiments, renal concentrations of epoxyeicosatrienoic acids, their inactive metabolites dihydroxyeicosatrienoic acids and AngII were measured. Acute BP-lowering effects of sEH inhibition in I3C-induced rats was associated with a marked increase in renal epoxyeicosatrienoic acids to dihydroxyeicosatrienoic acids ratio and acute natriuresis. Chronic treatment with cis-4-(4-(3-adamantan-1-yl-ureido)cyclohexyloxy) benzoic acid in I3C-induced rats elicited dose-dependent persistent BP lowering associated with a significant reduction of plasma and kidney AngII levels. Our findings show that the acute BP-lowering effect of sEH inhibition in I3C-induced Cyp1a1-Ren-2 transgenic rats is mediated by a substantial increase in intrarenal epoxyeicosatrienoic acids and their natriuretic action without altering intrarenal renin-angiotensin system activity. Long-term antihypertensive action of cis-4-(4-(3-adamantan-1-yl-ureido)cyclohexyloxy) benzoic acid in I3C-induced Cyp1a1-Ren-2 transgenic rats is mediated mostly by suppression of intrarenal AngII concentration.

Abstract 559: Augmented Renal Levels of PDGFß And PDGFß Receptors in Cyp1a1-Ren2 Transgenic Rats With Angiotensin II-Dependent Malignant Hypertension

PDGF receptor antagonism with imatinib mesylate prevents the renal injury, proteinuria and augmented urinary ANG II excretion independent of changes in blood pressure that occur in Cyp1a1-Ren2 rats with ANG II-dependent malignant hypertension. These findings suggest that arterial pressure-dependent increases in PDGFβ protein levels and PDGFβ receptor activation contribute importantly to the marked renal functional and morphological derangements that occur in ANG II-dependent malignant hypertension. To address this issue, the present study was performed to determine the protein levels of PDGFβ and PDGFβ receptors in renal cortical and medullary tissue from kidneys of hypertensive Cyp1a1-Ren2 transgenic rats. Male Cyp1a1-Ren2 rats were fed a diet containing indole-3-carbinol (I3C; 0.3%) for 10 days to induce malignant hypertension (n=5) or a non-I3C containing diet (controls, n=6). Rats induced with I3C developed malignant hypertension and exhibited increases in PDGFβ protein levels in both the renal cortex (0.046±0.002 vs.0.026±0.003 AU, P&lt;0.05) and renal medulla (0.054±0.007 vs.0.026±0.003 AU, P&lt;0.05), and elevated PDGFβ receptor levels in both renal cortex (0.26±0.03 vs.0.12±0.02 AU, P&lt;0.05) and renal medulla (0.60±0.06 vs.0.21±0.02 AU, P&lt;0.05). In a separate group (n=5), rats were chronically treated with the PDGF receptor antagonist, imatinib mesylate, by oral gavage (60 mg/kg/d) starting 3 days before initiating I3C induction and maintained on imatinib for the 10 day duration of I3C administration. Chronic PDGF receptor blockade prevented the increase in PDGFβ protein levels in both renal cortical and medullary tissues (0.027±0.004 vs. 0.026±0.003 and 0.029±0.005 vs. 0.026±0.003 AU, respectively) but did not influence the elevated PDGFβ receptor levels in either renal cortex (0.23±0.02 vs. 0.26±0.02 AU) or renal medulla (0.46±0.03 vs.0.60±0.06 AU). These data demonstrate that both PDGFβ protein and PDGFβ receptor levels are elevated in both renal cortex and medulla in ANG II-dependent malignant hypertension. Such elevated levels may contribute to the renal injury and the increased urinary ANG II excretion in ANG II-dependent malignant hypertension.

Renal Nitric Oxide Synthase and Antioxidant Preservation in Cyp1a1-Ren-2 Transgenic Rats With Inducible Malignant Hypertension

Dietary administration of 0.30% indole-3-carbinol (I3C) to Cyp1a1-Ren2 transgenic rats (TGRs) generates angiotensin II (ANG II)-dependent malignant hypertension (HTN) and increased renal vascular resistance. However, TGRs with HTN maintain a normal or slightly reduced glomerular filtration rate. We tested the hypothesis that maintenance of renal function in hypertensive Cyp1a1-Ren2 TGRs is due to preservation of the intrarenal nitric oxide (NO) and antioxidant systems. Kidney cortex, kidney medulla, aortic endothelial (e) and neuronal (n) nitric oxide synthase (NOS), superoxide dismutases (SODs), and p22phox (nicotinamide adenine dinucleotide phosphate-oxidase subunit) protein abundances were measured along with kidney cortex total antioxidant capacity (TAC) and NOx. TGRs were fed a normal diet that contained 0.3% I3C or 0.3% I3C + candesartan (AT1 receptor antagonist; 25mg/L in drinking water) (n = 5-6 per group) for 10 days. Blood pressure increased and body weight decreased in I3C-induced TGRs, while candesartan blunted these responses. Abundances of NOS, SOD, and p22phox as well as TAC were maintained in the kidney cortex of I3C-induced TGRs with and without candesartan, while kidney cortex NOx production increased in both groups. Kidney medulla eNOS and extracellular (EC) SOD decreased and nNOS were unchanged in both groups of I3C-induced TGRs. In addition, a compensatory increase occurred in kidney medulla Mn SOD in I3C-induced TGRs + candesartan. Aortic eNOS and nNOS∝ fell and p22phox and Mn SOD increased in hypertensive I3C-induced TGRs; all changes were reversed with candesartan. The preservation of renal cortical NO and antioxidant capacity is associated with preserved renal function in Cyp1a1-Ren2 TGRs with ANG II-dependent malignant HTN.

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

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. 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 [Nω-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. 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. 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.

Increased neuronal activity in the OVLT of Cyp1a1-Ren2 transgenic rats with inducible Ang II-dependent malignant hypertension

The contribution of angiotensin II (Ang II) to the pathophysiology of hypertension is established based on facts that high levels of circulating Ang II increase vasoconstriction of peripheral arteries causing a rise in blood pressure (BP). In addition, circulating Ang II has various effects on the central nervous system, including the osmosensitive neurons in the organum vasculosum of the lamina terminalis (OVLT). Osmosensitive neurons in the OVLT transduce hypertonicity via the activation of the nonselective cation channel known as transient receptor potential vanilloid 1 (TRPV1), causing membrane depolarization, followed by increased action potential discharge. This effect is absent in mice lacking expression of the TRPV1 gene. Most observations related to the importance of the OVLT in cardiovascular control are mainly based on models of lesion of the entire preoptic periventricular tissue. However, it remains unclear whether neuronal activity and TRPV1 protein expression levels alter in the OVLT of Cyp1a1-Ren2 transgenic rats with inducible Ang II-dependent malignant hypertension. C-fos was used as a marker of neuronal activity. Immunostaining was used to demonstrate distribution of c-fos positive neurons in the OVLT of Cyp1a1Ren2 transgenic rats. Western blot analysis showed increased c-fos and TRPV1 total protein expression levels in the OVLT of hypertensive rats. The present findings demonstrate increased c-fos and TRPV1 expression levels in the OVLT of Cyp1a1-Ren2 transgenic rats with Ang II-dependent malignant hypertension.

Renal Functional Responses to Intrarenal Arterial Administration of the Direct Renin Inhibitor Aliskiren in Cyp1a1‐Ren2 Transgenic Rats with ANG II‐Dependent Malignant Hypertension

The present study was performed to determine the effects of selective intrarenal administration of the direct renin inhibitor, aliskiren, on whole kidney hemodynamics and excretory function in Cyp1a1-Ren2 transgenic rats with ANG II-dependent malignant hypertension in the absence of the confounding influence of associated reductions in arterial blood pressure. Male Cyp1a1-Ren2 rats (n=5) were fed a normal diet containing indole-3-carbinol (0.3%, wt/wt) for 10 days to induce malignant hypertension. MAP and renal hemodynamics and excretory function were measured in pentobarbital-anesthetized rats during control conditions and following intrarenal administration of aliskiren (0.01 mg/kg). Intrarenal administration of aliskiren increased urine flow (10.47±1.7 to 12.96±2.6 μl/min, P<0.05) and urinary sodium excretion (0.46±0.15 to 1.16±0.26 μEq/min, P<0.05) without affecting MAP, GFR, or RPF. The present findings demonstrate that selective renal renin inhibition elicits diuretic and natriuretic effects in Cyp1a1-Ren2 rats with ANG II-dependent malignant hypertension. The data also indicate that de novo intrarenal ANG II generation may contribute to an augmented sodium reabsorptive capability and, thereby, to the elevated arterial blood pressure in ANG II-dependent malignant hypertension.

Chronic Renin Inhibition Normalizes Blood Pressure in Cyp1a1‐Ren2 Transgenic Rats with ANG II‐Dependent Malignant Hypertension

The present study was performed to determine if chronic renin inhibition can normalize blood pressure in Cyp1a1-Ren2 transgenic rats with ANG II-dependent malignant hypertension. Adult male Cyp1a1-Ren2 rats (n=6) were fed a normal diet containing 0.3% indole-3-carbinol (I3C) for 10 days to induce ANG II-dependent malignant hypertension. Subsequently, the rats were treated chronically for 10 days with the renin inhibitor, aliskiren (60 mg/kg/day, sc), while maintaining dietary I3C administration. Systolic blood pressure (SBP) was measured daily by tail-cuff plethysmography and rats were placed in metabolic cages for determination of 24-hr urine flow and electrolyte excretion. Dietary I3C increased SBP (135±3 to 207±10 mmHg, P<0.001) and urine flow (5.6±0.7 to 37.2±10.8 ml/day, P<0.001). Subsequent chronic aliskiren administration normalized SBP within 1 day (108±1 mmHg, P<0.001). SBP remained at normotensive levels throughout the 10 days of chronic aliskiren treatment (125±7 mmHg, P<0.001). Urine flow and sodium excretion increased (from 37.2±10.8 to 56.3±6.1 ml/day, P<0.05, and from 0.89±0.28 to 2.06±0.32 mmol/day, P<0.01, respectively) by day 10 of aliskiren treatment. These findings demonstrate that chronic renin inhibition with aliskiren is highly effective in normalizing the elevated arterial pressure, and increases renal excretory function in ANG II-dependent malignant hypertension.

Enhancement of renin and prorenin receptor in collecting duct of Cyp1a1-Ren2 rats may contribute to development and progression of malignant hypertension

To determine whether in the transgenic rat model [TGR(Cyp1a1Ren2)] with inducible ANG II-dependent malignant hypertension changes in the activation of intrarenal renin-angiotensin system may contribute to the pathogenesis of hypertension, we examined the gene expression of angiotensinogen (AGT) in renal cortical tissues and renin and prorenin receptor [(P)RR] in the collecting duct (CD) of the kidneys from Cyp1a1Ren2 rats (n = 6) fed a normal diet containing 0.3% indole-3-carbinol (I3C) for 10 days and noninduced rats maintained on a normal diet (0.6% NaCl diet; n = 6). Rats induced with I3C developed malignant hypertension and exhibited alterations in the expression of renin and (P)RR expressed by the CD cells. In the renal medullary tissues of the Cyp1a1Ren2 transgenic rats with malignant hypertension, renin protein levels in CD cells were associated with maintained renin content and lack of suppression of the endogenous Ren1c gene expression. Furthermore, these tissues exhibited increased levels of (P)RR transcript, as well as of the protein levels of the soluble form of this receptor, the s(P)RR. Intriguingly, although previous findings demonstrated that urinary AGT excretion is augmented in Cyp1a1Ren2 transgenic rats with malignant hypertension, in the present study we did not find changes in the gene expression of AGT in renal cortical tissues of these rats. The data suggest that upregulation of renin and the s(P)RR in the CD, especially in the renal medullary tissues of Cyp1a1Ren2 transgenic rats with malignant hypertension, along with the previously demonstrated increased availability of AGT in the urine of these rats, may constitute a leading mechanism to explain elevated formation of kidney ANG II levels in this model of ANG II-dependent hypertension.

Enhanced Urinary Angiotensinogen Excretion in Cyp1a1-Ren2 Transgenic Rats With Inducible ANG II-Dependent Malignant Hypertension

IntroductionPrevious studies have demonstrated that the urinary excretion of angiotensinogen is significantly increased in ANG II-infused hypertensive rats, which is associated with an augmentation of intrarenal ANG II levels. These findings suggest that urinary angiotensinogen excretion rates provide an index of intrarenal ANG II levels in ANG II-dependent hypertensive states. However, little information is available regarding the urinary excretion of angiotensinogen in ANG II-dependent malignant hypertension. MethodsThis study was performed to determine if urinary angiotensinogen excretion is increased in Cyp1a1-Ren2 transgenic rats [strain name: TGR(Cyp1aRen2)] with inducible ANG II-dependent malignant hypertension. Adult male Cyp1a1-Ren2 rats (n=6) were fed a normal diet containing 0.3% indole-3-carbinol (I3C) for 10days to induce ANG II-dependent malignant hypertension. ResultsRats induced with I3C exhibited pronounced increases in systolic blood pressure (208 ± 7 versus 127 ± 3mm Hg; P < 0.001), marked proteinuria (29.4 ± 3.6 versus 5.9 ± 0.3mg/d; P < 0.001) and augmented urinary angiotensinogen excretion (996 ± 186 versus 241 ± 31 ng/d; P < 0.01). Chronic administration of the AT1 receptor antagonist, candesartan (25mg/L in drinking water, n=6), prevented the I3C-induced increases in systolic blood pressure (125 ± 5mm Hg; P < 0.001), proteinuria (7.3 ± 1.0mg/d; P < 0.001) and urinary angiotensinogen excretion (488 ± 51 ng/d, P < 0.01). ConclusionsThese data demonstrate that the urinary excretion of angiotensinogen is markedly augmented in ANG II-dependent malignant hypertension. Such increased urinary angiotensinogen excretion may contribute to augmented intrarenal ANG II levels and, thereby, to the increased blood pressure in Cyp1a1-Ren2 transgenic rats with inducible ANG II-dependent malignant hypertension.

Transient Induction of ANG II-Dependent Malignant Hypertension Causes Sustained Elevation of Blood Pressure and Augmentation of the Pressor Response to ANG II in CYP1A1-REN2 Transgenic Rats

IntroductionTransgenic rats with inducible expression of the mouse Ren2 renin gene [strain name: TGR(Cyp1a1Ren2)] allow induction of various degrees of ANG II-dependent hypertension. Dietary administration of the aryl hydrocarbon indole-3-carbinol (I3C) at a dose of 0.15% induces a slowly developing form of ANG II-dependent hypertension, whereas dietary administration of a higher dose (0.3%) of I3C results in the development of ANG II-dependent malignant hypertension. Cessation of administration of 0.15% I3C results in the normalization of blood pressure, indicating the reversibility of hypertension induced by this dose of I3C. The present study was performed to determine if ANG II-dependent malignant hypertension is similarly reversible following cessation of dietary administration of 0.3% I3C. MethodsCyp1a1-Ren2 rats (n=6) were fed a normal diet containing 0.3% I3C for 11days to induce malignant hypertension. ResultsCyp1a1-Ren2 rats induced with I3C exhibited pronounced increases in systolic blood pressure (SBP) (132 ± 3–229 ± 11mm Hg, P < 0.001) and marked decreases in body weight (303 ± 4–222 ± 2 g, P < 0.001). When I3C administration was terminated, SBP decreased to 167 ± 4mm Hg (P < 0.01) and body weight increased to normal levels (309 ± 2 g, P < 0.01) within 12days. However, SBP remained significantly elevated (172 ± 1mm Hg, P < 0.01) for up to 3 weeks after termination of dietary administration of 0.3% I3C. In addition, the magnitude of the blood pressure response to intravenous bolus administration of 50 ng of ANG II (50 μL in volume) 3 weeks after cessation of dietary I3C administration was substantially higher than that observed in normotensive control rats (134 ± 1mm Hg, n=6) not previously induced with 0.3% I3C (53 ± 2 versus 38 ± 3mm Hg, P < 0.05). ConclusionsThe present findings demonstrate that transient induction of ANG II-dependent malignant hypertension results in prolonged elevations of arterial blood pressure and marked augmentation of the magnitude of the pressor response to ANG II in Cyp1a1-Ren2 transgenic rats.

Enhanced Urinary Angiotensinogen Excretion in Cyp1a1‐Ren2 Transgenic Rats with Inducible ANG II‐Dependent Malignant Hypertension

The present study was performed to determine if urinary angiotensinogen excretion is increased in Cyp1a1‐Ren2 transgenic rats [strain name: TGR(Cyp1aRen2)] with inducible ANG II‐dependent malignant hypertension. Adult male Cyp1a1‐Ren2 rats (n=6) were fed a normal diet containing 0.3% indole‐3‐carbinol (I3C) for 10 days to induce ANG II‐dependent malignant hypertension. Rats induced with I3C exhibited pronounced increases in systolic blood pressure (SBP) (208±7 vs. 127±3 mmHg, P&lt;0.001), marked proteinuria (29.4±3.6 vs. 5.0±0.4 mg/day, P&lt;0.01), and augmented urinary angiotensinogen excretion (996±186 vs. 199±19 mg/day, P&lt;0.05). Chronic administration of the AT1 receptor antagonist, candesartan (25 mg/L in drinking water, n=6), prevented the I3C‐induced increases in SBP (125±5, P&lt;0.001), proteinuria (7.3±1.5 mg/day, p&lt;0.001) and urinary angiotensinogen excretion (488±51 mg/day, P&lt;0.05). The present findings confirm that AT1 receptor activation contributes to the increase in SBP and proteinuria in Cyp1a1‐Ren2 transgenic rats with ANG II‐dependent malignant hypertension. These data also demonstrate that the urinary excretion of angiotensinogen is markedly augmented in ANG II‐dependent malignant hypertension. Such increased urinary angiotensinogen excretion likely reflects an AT1 receptor‐mediated stimulation of intrarenal angiotensinogen production which may contribute to augmented intrarenal ANG II levels and, thereby, to the increased blood pressure in Cyp1a1‐Ren2 transgenic rats with inducible ANG II‐dependent malignant hypertension.

Direct Inhibition with Aliskiren Normalizes Blood Pressure in Cyp1a1‐Ren2 Transgenic Rats with Inducible ANG II‐Dependent Malignant Hypertension

The present study was performed to determine the effects of acute direct renin inhibition with aliskiren on blood pressure and renal hemodynamics in Cyp1a1‐Ren2 transgenic rats with ANG II‐dependent malignant hypertension. Male Cyp1a1‐Ren2 rats (n=6) were fed a normal diet containing 0.3% I3C for 9–11 days to induce malignant hypertension. Mean arterial pressure (MAP) and renal hemodynamics were measured in pentobarbital‐anesthetized male Cyp1a1‐Ren2 rats during control conditions and following administration of the renin inhibitor, aliskiren (10 mg/kg, iv). Rats induced with I3C had higher MAP (196±4 vs. 142±3 mmHg, P&lt;0.01) and lower renal plasma flow (RPF; 1.98±0.32 vs. 3.86±0.26 ml/min.g, P&lt;0.01) than non‐induced rats (n=6). There were no differences in glomerular filtration rate (GFR) between the two groups (1.22±0.13 vs. 0.85±0.15 ml/min.g). Aliskiren administration decreased MAP (196±4 to 126±6 mmHg, P&lt;0.001) and increased RPF (1.98±0.32 to 2.67±0.46 ml/min.g, P&lt;0.05) in the hypertensive rats. GFR remained unaltered following renin inhibition in the hypertensive rats. Aliskiren administration did not alter MAP or GFR, but increased RPF (3.86±0.26 to 4.53±0.33 ml/min.g, P&lt;0.05) in the normotensive rats. The present data demonstrate that acute renin inhibition with aliskiren normalizes MAP and improves RPF in Cyp1a1‐Ren2 transgenic rats with malignant hypertension. The normalization of MAP and the increased RPF following acute renin inhibition with aliskiren indicate that renin generated as a consequence of expression of the Ren2 gene is responsible for the development of malignant hypertension and the associated reduction of renal hemodynamic function in Cyp1a1‐Ren2 transgenic rats.

Increased Collecting Duct Renin Protein Expression in Cyp1a1‐Ren2 Transgenic Rats with Inducible Ang II‐dependent Malignant Hypertension.

In angiotensin II (AngII)‐dependent hypertension, renin is upregulated in collecting ducts (CD) despite suppression of plasma renin activity and renin in juxtaglomerular (JG) cells. The transgenic rat model with insertion of the mouse Ren2 gene [Cyp1a1‐Ren2 strain] exhibits renin expression only in the liver, enhancement of PRA, high circulating and intrarenal Ang II levels, and an inducible form of AngII‐dependent malignant hypertension by dietary administration of the aryl hydrocarbon, indole‐3‐carbinol (I3C). Although increased intrarenal AngII content in Cyp1a1‐Ren2 rats can be partially due to internalization via AT1R, de novo intrarenal Ang II formation is suspected in this model. This study determined the renin protein expression in the distal nephron segments as a potential pathway to explain the increased intrarenal AngII content in Cyp1a1‐Ren2 rats. Body weights decreased in hypertensive rats (248±0.2g) compared to controls (349±0.1g). Renin immunostaining was not suppressed in JG cells of Cyp1a1‐Ren2 rats (0.7±0.01 vs. 0.7±0.01DU). In contrast, CD renin immunoreactivity and renal medullary renin protein levels measured by Western blot were 3.8‐fold and 2.6‐fold higher in hypertensive rats than controls. Augmentation of CD renin may contribute to increase intrarenal Ang II content and, thereby, to the increased blood pressure in Cyp1a1‐Ren2 with AngII‐dependent malignant hypertension.

Renoprotective effects of neuronal NOS-derived nitric oxide and cyclooxygenase-2 metabolites in transgenic rats with inducible malignant hypertension

The present study was performed to determine the effects of neuronal nitric oxide synthase (nNOS) and cyclooxygenase-2 (COX-2) inhibition on blood pressure and renal hemodynamics in transgenic rats with inducible ANG II-dependent malignant hypertension [strain name: TGR(Cyp1a1Ren2)]. Male Cyp1a1-Ren2 rats (n = 7) were fed a normal diet containing indole-3-carbinol (I3C; 0.3%) for 6-9 days to induce malignant hypertension. Mean arterial pressure (MAP) and renal hemodynamics were assessed in pentobarbital sodium-anesthetized Cyp1a1-Ren2 rats before and during intravenous infusion of the nNOS inhibitor S-methyl-l-thiocitrulline (l-SMTC; 1 mg/h). In hypertensive Cyp1a1-Ren2 rats, l-SMTC increased MAP from 169 +/- 3 to 188 +/- 4 mmHg (P < 0.01), which was a smaller increase than in noninduced rats (124 +/- 9 to 149 +/- 9 mmHg, P < 0.01, n = 5). Additionally, l-SMTC decreased renal plasma flow (RPF) to a similar extent (-34 +/- 13 vs. -35 +/- 12%) in the hypertensive and normotensive rats (4.1 +/- 0.2 to 2.7 +/- 0.5 and 3.1 +/- 0.3 to 2.0 +/- 0.3 ml x min(-1) x g(-1), respectively, P < 0.01) but did not alter glomerular filtration rate (GFR) in either group. In additional experiments, administration of the COX-2 inhibitor, nimesulide (3 mg/kg i.v.), during simultaneous infusion of l-SMTC decreased MAP in both hypertensive and noninduced rats (182 +/- 2 to 170 +/- 3 mmHg and 153 +/- 3 to 140 +/- 3 mmHg, respectively, P < 0.01). Nimesulide also decreased RPF (1.9 +/- 0.2 to 0.8 +/- 0.1 ml x min(-1) x g(-1), P < 0.01) and GFR (0.9 +/- 0.1 to 0.4 +/- 0.1 ml x min(-1) x g(-1), P < 0.01) in hypertensive rats but did not alter RPF or GFR in noninduced rats. The present findings demonstrate that both nNOS-derived NO and COX-2 metabolites exert pronounced renal vasodilator influences in hypertensive Cyp1a1-Ren2 rats. The data also indicate that the renal vasodilator effects of COX-2-derived prostanoids in hypertensive Cyp1a1-Ren2 rats are not dependent on nNOS activity.

Renal vascular and tubulointerstitial inflammation and proliferation in Cyp1a1-Ren2 transgenic rats with inducible ANG II-dependent malignant hypertension

Transgenic rats with inducible ANG II-dependent malignant hypertension [TGR(Cyp1a1Ren2)] were generated by inserting the mouse Ren2 renin gene into the genome of the rat. The present study was performed to assess renal morphological changes occurring during the development of ANG II-dependent malignant hypertension in these rats. Male Cyp1a1-Ren2 rats (n = 10) were fed normal rat food containing indole-3-carbinol (I3C; 0.3%) for 10 days to induce malignant hypertension. Rats induced with I3C had higher mean arterial pressures (173 +/- 9 vs. 112 +/- 11 mmHg, P < 0.01) than noninduced normotensive rats (n = 9). Glomerular damage was evaluated by determination of the glomerulosclerosis index (GSI) in tissue sections stained with periodic acid-Schiff. Kidneys of hypertensive rats had a higher GSI than normotensive rats (21.3 +/- 5.6 vs. 3.5 +/- 1.31 units). Quantitative analysis of macrophage ED-1-positive cells and proliferating cell nuclear antigen using immunohistochemistry demonstrated increased macrophage numbers in the renal interstitium (106.4 +/- 11.4 vs. 58.7 +/- 5.0 cells/mm(2)) and increased proliferating cell number in cortical tubules (37.8 +/- 5.7 vs. 24.2 +/- 2.1 cells/mm(2)), renal cortical vessels (2.2 +/- 0.5 vs. 0.13 +/- 0.07 cells/vessel), and the cortical interstitium (33.6 +/- 5.7 vs. 4.2 +/- 1.4 cells/mm(2)) of hypertensive rat kidneys. These findings demonstrate that the renal pathological changes that occur during the development of malignant hypertension in Cyp1a1-Ren2 rats are characterized by inflammation and cellular proliferation in cortical vessels and tubulointerstitium.

Enhanced tubuloglomerular feedback in Cyp1a1-Ren2 transgenic rats with inducible ANG II-dependent malignant hypertension

The present study was performed to evaluate tubuloglomerular feedback responses in transgenic rats [TGR(Cypa1a1Ren2)] with inducible malignant hypertension and to determine the degree to which feedback responsiveness is modulated by ANG II in these rats. Male Cyp1a1-Ren2 rats were fed a normal diet containing the aryl hydrocarbon indole-3-carbinol (I3C; 0.3%), for 5-6 days to stimulate expression of the Cyp1a1-Ren2 transgene and, thereby, to induce malignant hypertension. Stop-flow pressure (SFP) feedback responses to a late proximal perfusion rate of 40 nl/min were assessed in pentobarbital sodium-anesthetized rats during control conditions and after administration of the AT(1) receptor antagonist candesartan (0.1 mg/kg iv). Rats induced with I3C (n = 6) exhibited elevated mean arterial pressure and increased maximal SFP feedback responses compared with noninduced rats (n = 4; 163 +/- 4 vs. 130 +/- 2 mmHg, P < 0.01 and 16.3 +/- 1.4 vs. 11.7 +/- 0.5 mmHg, P < 0.05, respectively). Systemic candesartan decreased arterial pressure (to 98 +/- 7 and to 101 +/- 5 mmHg, respectively, P < 0.001) and attenuated SFP feedback responses (to 2.0 +/- 0.4 and to 3.3 +/- 0.9 mmHg, respectively, P < 0.01) in both hypertensive and normotensive rats. In additional experiments, peritubular capillary infusion of 10(-3) M candesartan did not alter arterial pressure but attenuated feedback responses in both hypertensive (19.3 +/- 1.4 to 8.8 +/- 0.9 mmHg, P < 0.01, n = 9) and normotensive Cyp1a1-Ren2 rats (9.0 +/- 0.8 to 4.7 +/- 0.6 mmHg, P < 0.01, n = 7). The present findings indicate that Cyp1a1-Ren2 rats with ANG II-dependent malignant hypertension exhibit augmented tubuloglomerular feedback responses. The data also show that AT(1) receptor activation by ANG II contributes to the enhanced feedback responsiveness in Cyp1a1-Ren2 rats with malignant hypertension.