Development Of ANG II-dependent Hypertension Research Articles

Intratubular, Intracellular, and Mitochondrial Angiotensin II/AT1 (AT1a) Receptor/NHE3 Signaling Plays a Critical Role in Angiotensin II-Induced Hypertension and Kidney Injury.

Hypertension is well recognized to be the most important risk factor for cardiovascular diseases, stroke, and end-stage kidney failure. A quarter of the world’s adult populations and 46% of the US adults develop hypertension and currently require antihypertensive treatments. Only 50% of hypertensive patients are responsive to current antihypertensive drugs, whereas remaining patients may continue to develop cardiovascular, stroke, and kidney diseases. The mechanisms underlying the poorly controlled hypertension remain incompletely understood. Recently, we have focused our efforts to uncover additional renal mechanisms, pathways, and therapeutic targets of poorly controlled hypertension and target organ injury using novel animal models or innovative experimental approaches. Specifically, we studied and elucidated the important roles of intratubular, intracellular, and mitochondrial angiotensin II (Ang II) system in the development of Ang II-dependent hypertension. The objectives of this invited article are to review and discuss our recent findings that (a) circulating and intratubular Ang II is taken up by the proximal tubules via the (AT1) AT1a receptor-dependent mechanism, (b) intracellular administration of Ang II in proximal tubule cells or adenovirus-mediated overexpression of an intracellular Ang II fusion protein selectively in the mitochonria of the proximal tubules induces blood pressure responses, and (c) genetic deletion of AT1 (AT1a) receptors or the Na+/H+ exchanger 3 selectively in the proximal tubules decreases basal blood pressure and attenuates Ang II-induced hypertension. These studies provide a new perspective into the important roles of the intratubular, intracellular, and mitochondrial angiotensin II/AT1 (AT1a) receptor signaling in Ang II-dependent hypertensive kidney diseases.

New Insights into the Critical Importance of Intratubular Na+/H+ Exchanger 3 and Its Potential Therapeutic Implications in Hypertension.

The sodium (Na+) and hydrogen (H+) exchanger 3 (NHE3), known as solute carrier family 9 member 3 (SLC9A3), mediates active transcellular Na+ and bicarbonate reabsorption in the small intestine of the gut and proximal tubules of the kidney. The purpose of this article is to review and discuss recent findings on the critical roles of intestinal and proximal tubule NHE3 in maintaining basal blood pressure (BP) homeostasis and their potential therapeutic implications in the development of angiotensin II (Ang II)-dependent hypertension. Recently, our and other laboratories have generated or used novel genetically modified mouse models with whole-body, kidney-specific, or proximal tubule-specific deletion of NHE3 to determine the critical roles and underlying mechanisms of NHE3 in maintaining basal BP homeostasis and the development of Ang II-induced hypertension at the whole-body, kidney, or proximal tubule levels. The new findings demonstrate that NHE3 contributes to about 10 to 15 mmHg to basal blood pressure levels, and that deletion of NHE3 at the whole-kidney or proximal tubule level, or pharmacological inhibition of NHE3 at the kidney level with an orally absorbable NHE3 inhibitor AVE-0657, attenuates ~ 50% of Ang II-induced hypertension in mice. The results support the proof-of-concept hypothesis that NHE3 plays critical roles in physiologically maintaining normal BP and in the development of Ang II-dependent hypertension. Our results also strongly suggest that NHE3 in the proximal tubules of the kidney may be therapeutically targeted to treat poorly controlled hypertension in humans.

The Role of Superoxide in Sex Differences in Angiotensin II‐induced Hypertensive and Natriuretic Responses in Mice with Proximal Tubule‐Specific Deletion of Mitochondrial Protein Sirtuin 3 in The Kidney

Sirtuin 3 (SIRT3), aNAD-dependent deacetylase, is a major mitochondrial protein in the matrix that is encoded by the SIRT3 gene. The key functions of SIRT3 in the mitochondria include anti-oxidative, anti-aging, anti-inflammation, and blood pressure-regulating effects by decreasing reactive oxygen species/superoxide production. However, the role of SIRT3 via superoxide in the proximal tubules (PT) of the kidney in angiotensin II-induced hypertensive or natriuretic responses remains poorly understood. To test our hypothesis, five groups of adult male and female wild-type (WT) and PT-SIRT3-/- mice were treated without (Control) or with Ang II (~0.65 mg/kg/day, i.p.) treated with 2% NaCl diet or tempol, a membrane-permeable radical scavenger and superoxide dismutase-mimetic (50 mg/kg/day, p.o.) for 2 weeks. Under basal conditions, systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP) were significantly lower in adult male and female PT-SIRT3-/- mice than male and female wild-type mice (~13 ± 3 mmHg, P<0.01). Although there were no sex differences in male and female WT mice, SBP and MAP were significantly lower in female than male PT-SIRT3-/- mice (P<0.01). Urinary excretion of electrolytes including sodium (Na+), potassium (K+), chloride (Cl-), and lithium (Li+) were also significantly lower in female PT-SIRT3-/- mice (P<0.01). In response to Ang II infusion and 2% high salt treatment, SBP was increased similarly to 136 ± 7 mmHg in male and 135 ± 7 mmHg in female PT-SIRT3-/- mice (P<0.01). Urinary Na+, K+, Cl-, and Li+ excretion were all significantly increased in male and female PT-SIRT3-/- mice without significant sex differences in these responses to Ang II-induced hypertension. There were also no sex differences found in response to Ang II infusion alone. Tempol treatment alone had no significant effects on blood pressure and urinary electrolyte excretory responses compared with their basal levels. Interestingly, however, tempol treatment of Ang II-infused and 2% NaCl-fed male and female PT-SIRT3-/- mice revealed significant sex differences in these responses (P<0.01). SBP and MAP increased further in male (P<0.01), but not in female PT-SIRT3-/- mice (n.s.), whereas the natriuretic response was significantly greater in female PT-SIRT3-/- mice (P<0.01). Taken together, we concluded that SIRT3 in the mitochondria of the proximal tubules plays an important role in the development of ANG II-dependent hypertension, and that superoxide may be implicated in sex differences in Ang II-induced hypertensive and natriuretic responses in PT-SIRT3-/- mice.

SAT-109 NEW INSIGHTS INTO THE NOVEL ROLES OF ANGIOTENSIN AT1A RECEPTORS IN THE PROXIMAL TUBULES OF THE KIDNEY IN ANGIOTENSIN II-INDUCED HYPERTENSION AND KIDNEY INJURY

It is well-recognized that the renin-angiotensin system plays powerful endocrine, paracrine, and intracellular roles in the physiological regulation of blood pressure, cardiovascular and kidney function, as well as in the development of hypertension and kidney diseases. The present study tested the hypothesis that genetic deletion of AT1a receptors selectively in the proximal tubules of the kidney attenuates angiotensin II (ANG II)-induced hypertension and renal ischemia-reperfusion injury using global and proximal tubule-specific AT1a receptor knockout mouse models. Global and proximal tubule-specific AT1a receptor-knockout mice (PT-AT1a-KO) were bred, or generated in this laboratory using the standard Cre/LoxP recombination approach. The hypothesis was tested in the following experimental protocols. Three groups (n=12 per group) of age- and body wt.-matched adult male C57BL/6J (WT), global AT1a-KO (Agtr1a-/-), and PT-AT1a-KO mice were first used to measure basal systolic, diastolic, and mean arterial blood pressure using the telemetry and tail-cuff techniques. Another two groups of WT, AT1a-KO, and PT-AT1a-KO mice (n=8-12 per group) were infused with saline (control) or a pressor dose of ANG II at 1.5 mg/kg/day, i.p., for 2 weeks. Additional three groups of WT, AT1a-KO and PT-AT1a-KO mice (n=8-12 per group) were treated with sham surgery (control) or 45 min bilateral renal ischemia, followed by reperfusion for 24 h or 7 days. Blood pressure and renal functional responses, and glomerular, tubuointerstitial, and perivascular injury were determined accordingly. Under basal conditions, systolic, diastolic and mean arterial blood pressure was 23 ± 3 mmHg lower in global AT1a-KO (p<0.01 vs. WT), and 12 ± 3 mmHg lower in PT-AT1a-KO mice (p<0.01 vs. WT). 24 h urine output and urinary sodium excretion were significantly higher in AT1a-KO and PT-AT1a-KO than in WT mice (p<0.01). Urine osmolality was significantly lower in AT1a-KO mice (p<0.01), but to a much less extent in PT-AT1a-KO mice (p<0.05). The kidney wt. to body wt. ratio was smaller in AT1a-KO mice (p<0.01), but was not different between WT and PT-AT1a-KO mice. In response to ANG II infusion, systolic, diastolic and mean arterial blood pressure were significantly increased by 45 ± 5 mmHg (p<0.01 vs. baseline) in WT mice, but only by 25 ± 3 mmHg in PT-AT1a-KO mice (p<0.01 vs. baseline). The ANG II-induced hypertension was completely prevented in global AT1a-KO mice (n.s. vs. baseline). In response to induction of renal ischemia-reperfusion injury, WT mice developed significant glomerular, cortical tubulointerstitial and perivascular injury 24 h or 7 days after renal ischemia-reperfusion was induced (p<0.01), as expected. However, both acute and chronic ischemia-reperfusion renal injury was not attenuated in global AT1a-KO mice (n.s. vs. WT). Surprisingly, however, acute and chronic ischemia-reperfusion renal injury was significantly attenuated in PT-AT1a-KO mice 24 h or 7 days after renal ischemia-reperfusion was induced (p<0.01 vs. WT or AT1a-KO). The results of the present study provide the direct evidence for an important role of ANG II and AT1a receptors in the proximal tubules of the kidney in maintaining physiological blood pressure homeostasis, and in the full development of ANG II-dependent Hypertension and renal ischemia-reperfusion injury in mice.

Abstract 011: An Orally Absorbable Potent NHE3 Inhibitor Attenuates Angiotensin II-induced Hypertension Primarily by Inhibiting NHE3 in the Proximal Tubule of the Kidney

We have recently shown that angiotensin (ANG II)-induced hypertension was attenuated in mice with global ( Nhe3 -/- ) and Nhe3 -/- mice with transgenic rescue of the NHE3 gene selectively in small intestines (tg Nhe3 -/- ), suggesting an important role of NHE3 in the development of ANG II-dependent hypertension. In this study, we specifically tested whether the pharmacological inhibition of NHE3 mainly in the proximal tubules of the kidney attenuates ANG II-dependent hypertension induced by a low and slow pressor dose of ANG II supplemented with a high salt diet. Overall, 9 groups (n=5-12) of adult male C57BL/6J mice were infused with or without ANG II (500 μg/kg/day, i.p. via minipump) and supplemented with or without a 2% NaCl diet to slowly and moderately increase systolic blood pressure (SBP) in 2 weeks. ANG II alone increased SBP from 116 ± 2 mmHg to 140 ± 2 mmHg ( p &lt;0.01), and supplement of ANG II with a 2% NaCl diet further increased SBP to 147 ± 4 mmHg ( p &lt;0.05). Concurrent treatment with an orally active, absorbable NHE3 inhibitor AVE0657 (Sanofi-Aventis; 20 mg/kg/day, p.o.) significantly decreased SBP to 125 ± 4 mmHg in ANG II-infused mice ( p &lt;0.01), and to 134 ± 6 mmHg in ANG II-infused mice supplemented with 2% NaCl ( p &lt;0.01), respectively. Further treatment with AVE0657 and losartan, an AT 1 receptor blocker (20 mg/kg/day, p.o.), completely normalize SBP in mice treated with ANG II and 2% NaCl to control (115 ± 5 mmHg, p &lt;0.01). In the kidney, AVE0657 significantly increased 24h urinary Na + excretion from 157.1 ± 6.7 to 207.7 ± 8.1 μmol/24h ( p &lt;0.01) without altering 24h urine excretion or SBP. Furthermore, AVE0657 did not significantly alter 24 h fecal Na + excretion in non ANG II-infused (4.99 ± 0.37 μmol/24h, n.s.) or ANG II-infused mice (4.19 ± 0.67 μmol/24h, n.s.), compared with control (4.02 ± 0.20 μmol/24h, n.s. ) or global Nhe3 -/- mice (50.8 ± 0.8 μmol/24h, p &lt;0.01). Since small intestines in the gut and the proximal tubules of the kidney express the vast majority of NHE3 in the body, these results provide preclinical evidence and perspectives that orally absorbable NHE3 inhibitors may be pharmacologically beneficial to prevent and treat hypertension induced by ANG II and a high salt, mainly by inhibiting NHE3 in the proximal tubule of the kidney.

Macrophage-derived IL-6 contributes to ANG II-mediated angiotensinogen stimulation in renal proximal tubular cells.

The development of ANG II-dependent hypertension involves increased infiltration of macrophages (MΦ) and T cells into the kidney and the consequent elevation of intrarenal cytokines including IL-6, which facilitates the progression of hypertension and associated kidney injury. Intrarenal renin-angiotensin system (RAS) activation, including proximal tubular angiotensinogen (AGT) stimulation, has also been regarded as a cardinal mechanism contributing to these diseases. However, the interaction between immune cells and intrarenal RAS activation has not been fully delineated. Therefore, the present study investigated whether ANG II-treated MΦ induce AGT upregulation in renal proximal tubular cells (PTCs). MΦ were treated with 0-10(-6) M ANG II for up to 48 h. PTCs were incubated with the collected medium from MΦ. In ANG II-treated MΦ, IL-6 mRNA and protein levels were increased (1.86 ± 0.14, protein level, ratio to control); moreover, IL-6 levels were higher than TNF-α and IL-1β in culture medium isolated from ANG II-treated MΦ. Elevated AGT expression (1.69 ± 0.04, ratio to control) accompanied by phosphorylated STAT3 were observed in PTCs that received culture medium from ANG II-treated MΦ. The addition of a neutralizing IL-6 antibody to the collected medium attenuated phosphorylation of STAT3 and AGT augmentation in PTCs. Furthermore, a JAK2 inhibitor also suppressed STAT3 phosphorylation and AGT augmentation in PTCs. These results demonstrate that ANG II-induced IL-6 elevation in MΦ enhances activation of the JAK-STAT pathway and consequent AGT upregulation in PTCs, suggesting involvement of an immune response in driving intrarenal RAS activity.

Endoplasmic reticulum and oxidant stress mediate nuclear factor-κB activation in the subfornical organ during angiotensin II hypertension.

Endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) generation in the brain circumventricular subfornical organ (SFO) mediate the central hypertensive actions of Angiotensin II (ANG II). However, the downstream signaling events remain unclear. Here we tested the hypothesis that angiotensin type 1a receptors (AT1aR), ER stress, and ROS induce activation of the transcription factor nuclear factor-κB (NF-κB) during ANG II-dependent hypertension. To spatiotemporally track NF-κB activity in the SFO throughout the development of ANG II-dependent hypertension, we used SFO-targeted adenoviral delivery and longitudinal bioluminescence imaging in mice. During low-dose infusion of ANG II, bioluminescence imaging revealed a prehypertensive surge in NF-κB activity in the SFO at a time point prior to a significant rise in arterial blood pressure. SFO-targeted ablation of AT1aR, inhibition of ER stress, or adenoviral scavenging of ROS in the SFO prevented the ANG II-induced increase in SFO NF-κB. These findings highlight the utility of bioluminescence imaging to longitudinally track transcription factor activation during the development of ANG II-dependent hypertension and reveal an AT1aR-, ER stress-, and ROS-dependent prehypertensive surge in NF-κB activity in the SFO. Furthermore, the increase in NF-κB activity before a rise in arterial blood pressure suggests a causal role for SFO NF-κB in the development of ANG II-dependent hypertension.

Abstract 564: Differential Expression and Regulation of Angiotensinogen in Renal Proximal Tubule Cells From S1, S2 and S3 Segments

In angiotensin II (Ang II)-dependent hypertension, intrarenal angiotensinogen (AGT) augmentation induced by Ang II and associated pathogenic factors including interleukin 6 (IL-6) cause further elevation of intratubular Ang II production, leading to the progression of hypertension and kidney injury. Recent studies have suggested that renal proximal straight tubules (S3 segment) are the main source of intrarenal AGT and that S1 and S2 segments do not express AGT mRNA under normal conditions. However, AGT expression and its regulation by Ang II and/or IL-6 in each proximal tubule segment have not been demonstrated an in vitro setting. The availability of specific cell lines derived from mouse S1, S2 and S3 segments provided an opportunity to decisively determine each segments’ capability to express AGT and respond to stimuli. Thus, this study was performed to determine AGT expression and its response to stimulation with Ang II and IL-6 in S1, S2 and S3 cell line. Basal AGT mRNA and protein levels were detected by RT-PCR and western blot analysis. Basal levels of Ang II type 1 receptor (AT1R) and STAT3, which is a transcription factor in IL-6 signaling pathway, were also measured. In addition, the cells were incubated with 100 nM Ang II and/or 400 nM IL-6 for 24 h. Basal AGT levels in S1 and S3 cells were lower than in mouse whole kidney (0.09-fold and 0.33-fold compared with mouse whole kidney). S2 cells exhibited the highest basal AGT levels (4.15-fold) among these cells. In S1 cells, AGT expression was stimulated by IL-6 (1.89 ± 0.32, ratio to control) and co-stimulation with Ang II and IL-6 (1.85 ± 0.28) although Ang II alone did not alter AGT levels. In S2 cells, only the co-stimulation increased AGT expression (1.35 ± 0.01). No changes were observed by similar treatments in S3 cells. Basal AT1R levels were lower in S3 than in S1 and S2 cells (0.97 ± 0.09 in S2, 0.32 ± 0.07 in S3, ratio to S1). S1 cells showed the highest basal levels of STAT3. Basal STAT3 levels in S3 cells were lower than that in S1 and S2 cells. These results indicate that S2 cells are main source of intrarenal AGT which can be augmented by Ang II and IL-6 during the development of Ang II-dependent hypertension. Furthermore, low basal levels of AT1R and STAT3 in S3 cells explain why these cells do not respond to Ang II and IL-6.

Augmented intratubular renin and prorenin expression in the medullary collecting ducts of the kidney as a novel mechanism of angiotensin II-induced hypertension

Augmented intratubular renin and prorenin expression in the medullary collecting ducts of the kidney as a novel mechanism of angiotensin II-induced hypertension

Chronic Direct Renin Inhibition with Aliskiren Prevents the Development of Hypertension in Cyp1a1‐Ren2 Transgenic Rats with Inducible ANG II‐Dependent Hypertension

The present study was performed to determine the effects of chronic direct renin inhibition on blood pressure and renal hemodynamics in Cyp1a1-Ren2 transgenic rats with inducible ANG II-dependent hypertension. Male Cyp1a1-Ren2 rats (n=6) were fed a normal diet containing 0.15% indole-3-carbinol (I3C) for 16 days to induce slowly progressive ANG II-dependent hypertension. Conscious systolic blood pressure (SBP) was measured daily using tail-cuff plethysmography. The rats were then anesthetized with pentobarbital sodium and surgically prepared for the measurement of mean arterial pressure (MAP) and renal hemodynamic function. In rats induced with I3C, SBP increased from 130±7 to 191±6 mmHg (P<0.001) by day 16. In a separate group of rats (n=6), chronic administration of aliskiren (30 mg/kg/day, sc) prevented the development of hypertension (113±5 vs. 114±5 mmHg, NS). Rats treated with aliskiren exhibited lower MAP (138±4 vs. 201±6 mmHg, P<0.001) and RVR (23±4 vs. 38±3 mmHg/ml/min.g, P<0.01), and higher RPF (4.22±0.23 vs. 2.56±0.21 ml/min.g, P<0.01) and GFR (1.19±0.07 vs. 0.78±0.08 ml/min.g, P<0.01) compared with induced rats not treated chronically with aliskiren. The present findings demonstrate that chronic direct renin inhibition with aliskiren prevents the development of ANG II-dependent hypertension and the associated decrease in renal hemodynamic function in Cyp1a1-Ren2 transgenic rats.

Inhibition of bilirubin metabolism induces moderate hyperbilirubinemia and attenuates ANG II-dependent hypertension in mice

Population studies indicate that moderate hyperbilirubinemia is associated with reduced incidence of cardiovascular diseases, including hypertension. Despite this correlative evidence, no studies have directly tested the hypothesis that moderate increases in plasma bilirubin levels can attenuate the development of hypertension. This hypothesis was tested by treating mice with Indinavir, a drug that competes with bilirubin for metabolism by UDP-glucuronosyltransferase 1A1 (UGT1A1). Treatment of mice with Indinavir (500 mg x kg(-1) x day(-1), gavage) resulted in a twofold increase in plasma unconjugated bilirubin levels. Next, we determined the effect of Indinavir-induced changes in plasma bilirubin on the development of ANG II-dependent hypertension. Moderate hyperbilirubinemia was induced 3 days before the implantation of an osmotic minipump that delivered ANG II at a rate of 1 microg x kg(-1) x min(-1). ANG II infusion increased mean arterial pressure (MAP) by 20 mmHg in control mice but by only 6 mmHg in mice treated with Indinavir (n = 6). Similar to Indinavir treatment, direct infusion of bilirubin (37.2 mg x kg(-1) x day(-1) i.v.) resulted in a twofold increase in plasma bilirubin levels and also attenuated the development of ANG II-dependent hypertension. Moderate hyperbilirubinemia resulted in an increase in plasma nitrate/nitrite levels, which averaged 36 +/- 2 vs. 50 +/- 7 microM in ANG II vehicle vs. Indinavir-treated mice (n = 5). Moderate hyperbilirubinemia resulted in attenuation of vascular oxidative stress as determined by dihydroethidium staining of aortic segments. These results indicate that moderate hyperbilirubinemia prevents ANG II-dependent hypertension by a mechanism that may involve decreases in vascular oxidative stress.

Impaired pressure‐natriuresis relationship precedes the development of hypertension in Cyp1a1‐Ren‐2 transgenic rats

ObjectiveThe present study was performed to characterize the autoregulatory efficiency of renal blood flow (RBF) and glomerular filtration rate (GFR) and the pressure‐natriuresis relationship in transgenic rats with inducible hypertension (Cyp1a1‐Ren‐2 rats).MethodsThe renin gene was induced in Cyp1a1‐Ren‐2 rats trough dietary administration of the natural xenobiotic indole‐3‐carbinol (I3C, 0.3 %) for 12 hours. Non‐induced rats served as controls. Anesthetized rats were prepared for renal function studies and an aortic clamp was placed on the aorta to regulate the level of renal arterial pressure. ANG II levels were measured by radioimmunoassay.ResultsAdministration of I3C resulted in progressive increases in ANG II levels as compared with non‐induced rats. I3C‐induction for 12 hours did not cause significant changes in blood pressure as compared with non‐induced rats. 12 hours I3C‐induction caused an impairment of the autoregulatory efficiency of RBF and GFR and of the pressure‐natriuresis relationship as compared with non‐induced rats.ConclusionsOur findings indicate that an impairment of the pressure‐natriuresis mechanism precedes the development of ANG II‐dependent hypertension in Cyp1a1‐Ren‐2 transgenic rats.

I025 Smooth muscle specific deletion of the RhoA exchange factor Arhgef1 protects against Ang II-dependent hypertension

Hypertension is one of the most frequent pathology in the industrialized world. Although it is recognized to be dependent on a combination of genetic and environmental factors, its molecular basis remains elusive. A possible candidate is the monomeric G protein RhoA which activates Rho kinase. However, how it is activated and whether it has a causative role in hypertension is still unknown. By in vitro experiments, we provide evidence that Arhgef1 is the RhoA guanine exchange factor specifically responsible for Angiotensin II (Ang II)-induced RhoA/Rho kinase activation in arterial smooth muscle cells. To analyze in vivo the role of Arhgef1, we generated mice lacking Arhgef1 specifically in smooth muscle cells (SM-Arhgef1-KO). We used Arhgef1lox/lox mice, which were mated to SMMHC-CreERT2 to produce SMMHC-CreERT2 ; Arhgef1lox/lox mice (SM-Arhgef1lox/lox). SM-Arhgef1-KO mice were then obtained by treating SM-Arhgef1lox/lox mice with tamoxifen. Remarkably, SM-Arhgef1-KO mice were resistant to hypertension induced by chronic Ang II infusion (sub-cutaneous osmotic pump, 1 μg/kg/min). Furthermore, Arhgef1 deletion after induction of hypertension by Ang II restored normal arterial pressure level. Basal arterial pressure and responses to vasoconstrictors other than Ang II (phenylephrine, U46619, endotheline) were not modified in SM-Arhgef1-KO mice. Our results show that Arhgef1 activation and its downstream RhoA/Rho kinase signaling are central to the development of Ang IIdependent hypertension. We identify Arhgef1 as an alternative potential target for the treatment of hypertension.

Impairment of the autoregulation of renal hemodynamics and of the pressure-natriuresis relationship precedes the development of hypertension in Cyp1a1-Ren-2 transgenic rats

The present study was performed to characterize the autoregulatory efficiency of renal blood flow and glomerular filtration rate and the pressure-natriuresis relationship in transgenic rats with inducible angiotensin II (ANG II)-dependent hypertension (Cyp1a1-Ren-2 rats). The renin gene was induced in Cyp1a1-Ren-2 rats through dietary administration of the natural xenobiotic indole-3-carbinol (I3C, 0.3%) for 12 and 24 h, respectively. Noninduced rats served as controls. Anesthetized rats were prepared for renal function studies and an aortic clamp was placed above the junction of the left renal artery to regulate the level of renal arterial pressure. Plasma renin activity, ANG II and aldosterone levels were measured at the end of the experiment by radioimmunoassay. Administration of I3C resulted in progressive increases in plasma renin activity and plasma and kidney ANG II levels; however, it did not significantly alter aldosterone levels as compared with those in noninduced rats. I3C induction for 12 h did not cause significant changes in blood pressure as compared with those in noninduced rats. I3C induction for 24 h elicited a significant rise in blood pressure. Twelve-hour I3C induction caused an impairment of the autoregulatory efficiency of renal blood flow and glomerular filtration rate and of the pressure-natriuresis relationship as compared with that in noninduced rats. In addition, 24 h I3C induction of the renin gene resulted in a marked reduction in renal blood flow and glomerular filtration rate and a further impairment of the pressure-natriuresis mechanism as compared with that in noninduced rats. Our findings indicate that an impairment of the pressure-natriuresis mechanism precedes the development of ANG II-dependent hypertension in Cyp1a1-Ren-2 transgenic rats.

Angiotensin Type 2 Receptor‐Dependent Vasodilation

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Angiotensin Type 2 Receptor-Dependent Vasodilation

Angiotensin II (Ang II) signaling is mediated by two receptor subtypes, type 1 (AT(1)) and type 2 (AT(2)). The activation of AT(1) receptors is responsible for the development of Ang II-dependent hypertension, whereas the activation of AT(2) receptor is thought to play a counter-regulatory protective role in the regulation of blood pressure that opposes the AT(1) receptor-mediated vasoconstriction. However, the precise mechanisms by which increased numbers of AT(2) receptors counterbalance the AT(1)-mediated actions of Ang II are unknown. We have demonstrated that the abdominal aortic banding in mice and rats and the 2-kidney, 1-clip Goldblatt model of hypertension in mice induces up-regulation of AT(2) receptors in the pressure-overloaded thoracic aorta. In these hypertensive animals, the AT(1)-receptor antagonists but not calcium antagonist abolish up-regulation of the aortic AT(2) receptor as well as blood pressure elevation, suggesting that the pressure-overload up-regulates the aortic AT(2) receptor by Ang II via the activation of AT(1) receptor. Ang II binding to up-regulated AT(2) receptors induces vasodilation in these aortas through bradykinin B(2)-receptor-mediated phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser(633) and Ser(1177) via a protein kinase A-dependent signaling pathway, resulting in sustained production of nitric oxide. These studies provide evidence that the vascular AT(2) receptor is up-regulated in the course of hypertension through the activation of AT(1) receptor, thereby activating a vasodilatory pathway in vessels through the AT(2) receptor via the bradykinin/nitric oxide/cGMP. This issue is important because the antihypertensive effect of AT(1)-receptor blockers is, at least in part, dependent on AT(2)-receptor activation.

CD59 or C3 are not requred for angiotensin II‐dependent hypertension or hypertrophy in mice

Complement is a major pro-inflammatory innate immune system whose serum activity correlates with systolic blood pressure in humans. To date, no studies using in vivo models have directly examined the role of individual complement components in regulating vessel function, hypertension and cardiac hypertrophy. Herein, in vivo responses to angiotensin (ang) II were characterized in mice deficient in CD59a or C3. CD59a(-/-) mice had slightly but significantly elevated systolic blood pressure (107.2 +/- 1.7 mmHg versus 113.8 +/- 1.31 mmHg, P < 0.01, for wild-type and CD59a(-/-), respectively). Aortic rings from CD59a(-/-) mice showed significantly less platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, with elevated deposition of membrane attack complex. However, acetylcholine- and sodium nitroprusside-dependent dilatation, plasma nitrate/nitrite and aortic cyclic guanosine monophosphate levels were unchanged from wild-type. Also, in vivo infusion with either ang II or noradrenaline caused similar hypertension and vascular hypertrophy to wild-type. Mice deficient in C3 had similar basal blood pressure to wild type and showed no differences in hypertension or hypertrophy responses to in vivo infusion with ang II. These data indicate that CD59a deficiency is associated with some vascular alterations that may represent early damage occurring as a result of increased complement attack. However, a direct role for CD59a or C3 in modulating development of ang II-dependent hypertension or hypertrophy in vivo is excluded and we suggest caution in development of complement intervention strategies for hypertension and heart failure.

Renal Medullary Infusion of CoPP Prevents Angiotensin‐II Dependent Hypertension in Mice

Several studies have established systemic induction of Heme Oxygenase-1 (HO-1) as a viable means of attenuating the development of hypertension in several experimental models. However, the precise mechanism behind the anti-hypertensive actions of HO-1 induction is still unclear. In this study, we tested the hypothesis that kidney specific induction of HO-1 can prevent the development of Ang-II dependent hypertension. To test this hypothesis, renal medullary interstitial catheters were implanted into the left kidney of uninephrectomized mice. Infusion of a known inducer of HO-1, cobalt protoporphyrin (CoPP; 250 μg/ml; at 50 μl/hr for 48hrs), resulted in significant induction of HO-1 mainly in the renal medulla when examined 2 weeks after the infusion with no induction observed in other organs such as the brain, heart, and liver. Next, we examined the effect of this approach of inducing of HO-1 on the development of Ang II dependent hypertension. CoPP or Vehicle (0.1 M NaOH, pH 8.3) was infused as indicated above 2 days prior to implantation of an osmotic minipump which delivered Ang II at a rate of 1 μg/kg/min. Mean arterial pressure (MAP) was measured in conscious, unrestrained mice for 3 consecutive days starting 7 days after the implantation of the Ang II minipumps. MAP averaged 112 ± 4, 121 ± 4, 162 ± 2 and 126 ± 6 mmHg in Vehicle + Vehicle, CoPP + Vehicle, Vehicle + Ang II and CoPP + Ang II treated mice respectively (n=6). These results suggest that renal medullary infusion of CoPP to induce HO-1 specifically in the kidneys can prevent the development of Ang II dependent hypertension in mice. Induction of HO-1 in the renal medulla may be the mechanism by which systemic delivery of CoPP lowers blood pressure in Ang-II dependent hypertension.