Development Of ANG II-induced Hypertension Research Articles

DELETION OF BRADYKININ TYPE 2 RECEPTORS SELECTIVELY IN THE RENOMEDULLARY INTERSTITIAL CELLS OF THE KIDNEY MEDULLA ATTENUATES ANGIOTENSIN II-INDUCED HYPERTENSION IN MICE

Objective: The present study tested the hypothesis that deletion of vasoactive peptide bradykinin B2 receptors selectively in the renomemdullary interstitial cells (RMICs) of the kidney medulla attenuates angiotensin II-induced hypertension in mice. Design and method: To test the hypothesis, we employed inducible Tenascin-C-CreER2 and bradykinin B2 receptor-floxed recombination approach to generate a novel mouse model with deletion of B2 receptors selectively in the RMICs of the kidney medulla (RMIC-B2R-/-). Basal blood pressure and urine osmolality and their responses to angiotensin II (Ang II, ∼500 μg/kg/day, i.p., for 2 weeks) were determined in adult male wildtype (WT) and RMIC-B2R-/- mice (n=10 per group), respectively. Results: Mice with deletion of B2 receptors selectively in RMICs of the kidney medulla developed normally without structural abnormalities in the kidney medulla. However, basal telemetry systolic, diastolic, and mean arterial blood pressure were significantly lower in RMIC-B2R-/- than WT mice (WT: 123 ± 3 mmHg vs. RMIC-B2R-/-: 106 ± 3 mmHg, P<0.01). The hypotensive phenotype in RMIC-B2R-/- mice was associated with significant decreases in 24 h urine output (WT: 1.86 ± 0.15 mL/24 h vs. RMIC-B2R-/-: 1.09 ± 0.15 mL/24 h, P<0.05) and urine osmolality (WT: 1893 ± 441 mOsm/Kg H2O vs. RMIC-B2R-/-: 1311 ± 78 mOsm/Kg H2O, P<0.01). In response to Ang II infusion (∼0.5 mg/kg/day, i.p., for 2 weeks), systolic blood pressure increased to 166 ± 6 mmHg in WT mice (P<0.01 vs. Basal), but it increased only to 139 ± 5 mmHg in RMIC-B2R-/- mice (P<0.01 vs. WT). By comparison, infusion of DDAVP (1-deamino-8-D-arginine vasopressin, ∼0.5 mg/kg/day, i.p., for 2 weeks) did not significantly alter blood pressure in WT and RMIC-B2R-/- mice (n.s.). However, infusion of Ang II for 2 weeks significantly increased urine osmolality in WT mice (P<0.01), and restored urine osmolality in RMIC-B2R-/- mice to the WT basal level (1995 ± 315 mOsm/Kg H2O, P<0.01). Conclusions: In conclusion, the present study demonstrates for the 1st time that bradykinin B2 receptors in the RMICs of the kidney medulla plays an important role in maintaining basal blood pressure and urine osmolality homeostasis and the development of Ang II-induced hypertension.

Abstract P433: Evidence For An Important Role Of AT1a Receptors In The Renomedullary Interstitial Cells In Basal Blood Pressure Homeostasis And The Development Of Angiotensin Ii-induced Hypertension

The renomedullary interstitial cells (RMICs) are highly specialized lipid-laden interstitial fibroblasts, which form characteristic "ladder-like" structural associations with adjacent microvessels and renal tubules in the kidney medulla. We have previously reported that RMICs express abundant AT 1 (AT 1a ) receptors in vitro and in vivo , but the physiological role of AT 1 (AT 1a ) receptors in RMICs has not been determined. In the present study, we generated and phenotyped for the 1 st time a new mutant mouse model with RMIC-selective deletion of AT 1a receptors in the kidney medulla, RMIC- Agtr1a -/- , using an innovative inducible Tenascin-C-CreER2/Agtr1a f/f approach. Deletion of AT 1a receptors selectively in RMICs did not alter the development and cellular structures in the kidney medulla of RMIC- Agtr1a -/- mice. The heart wt. to body wt. ratio was lower ( P <0.01), whereas the kidney wt. to body wt. ratio was significantly higher ( P <0.01) in RMIC- Agtr1a -/- than wild-type (WT) mice without altering heart rate. Compared with WT mice, basal systolic, diastolic, and mean arterial blood pressure, as measured by the telemetry technique, were significantly lower in adult male RMIC- Agtr1a -/- mice (WT: 122 ± 3 mmHg vs. RMIC- Agtr1a -/- : 103 ± 4 mmHg, P <0.01). The decreases in basal blood pressure in RMIC- Agtr1a -/- mice was associated with significantly lower urine excretion (WT: 1.56 ± 0.15 mL/24 h vs. RMIC- Agtr1a -/- : 0.98 ± 0.13 mL/24 h, P <0.01), urinary sodium (WT: 207.9 ± 7.3 μmol/24 h vs. RMIC- Agtr1a -/- : 145.2 ± 5.4 μmol/24 h, P <0.01), potassium excretion (WT: 221.0 ± 6.7 μmol/24 h vs. RMIC- Agtr1a -/- : 160.8 ± 10.6 μmol/24 h, P <0.01), and markedly decreased urine osmolality in RMIC- Agtr1a -/- mice (WT: 1724 ± 335 mOsm/Kg H 2 O vs. RMIC- Agtr1a -/- : 1128 ± 102 mOsm/Kg H 2 O, P <0.01). Ang II infusion (0.5 mg/kg/day, i.p., 2 weeks) increased blood pressure to 148 ± 5 mmHg in WT mice, as expected, but it increased only to 129 ± 8 mmHg without further altering urine osmolality from controls in RMIC- Agtr1a -/- mice ( P <0.01). In conclusion, the present study provides new evidence for the 1st time for an important role of AT 1a receptors in RMICs in maintaining basal blood pressure homeostasis and the development of Ang II-induced hypertension.

Abstract P308: Leucine Rich Repeat Containing 8A Anion Channels Modulate Vasodilation In Angiotensin II-induced Hypertensive Mice

Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E) comprise Volume-regulated anion channels (VRACs) which are activated by tumor necrosis factor alpha (TNFα) and angiotensin II (AngII). We have previously investigated vascular reactivity in mesenteric arteries from male wild type (WT) and smooth muscle-specific LRRC8A knockout (KO) mice. KO vessels displayed enhanced responsiveness to both endothelium-dependent (acetylcholine, ACh), and independent (sodium nitroprusside, SNP) vasodilators. We hypothesized that LRRC8A KO mice would be protected from AngII-induced hypertension. Blood pressure (BP) was measured by radiotelemetry. Resting BP was unaltered compared to WT and AngII infusion by osmotic minipump (alzet, 90 ng/min for 14 days) induced a similar degree of hypertension in awake mice (nighttime) but the difference between day and night pressures was significantly larger in KO mice. Mesenteric arteries were isolated from WT and KO mice after 14 days and vascular reactivity was quantified by wire myograph. Relaxation responses to both ACh and SNP relaxation were impaired in AngII hypertensive compared to normotensive WT mice. However, KO vessels relaxed better to both vasodilators in rings from both vehicle and AngII-treated mice (ACh LogEC 50 , WT+AngII -5.3 ± 0.27 vs. KO+AngII -6.5 ± 0.19, p < 0.05, n=4-6; SNP LogEC 50 , WT+AngII -7.4 ± 0.10 vs. KO+AngII -8.1 ± 0.10, p < 0.05, n=3-6). These results demonstrate a dissociation between elevated blood pressure and impaired vasodilator function. The data suggest that LRRC8A anion channels play a complex role in the development of AngII-induced hypertension.

GENETIC DELETION OF AT1A RECEPTORS SELECTIVELY IN THE RENOMEDULLARY INTERSTITIAL CELLS OF THE KIDNEY MEDULLA ATTENUATES ANGIOTENSIN II-INDUCED HYPERTENSION

Objective: The present study tested the hypothesis that genetic deletion of angiotensin II (Ang II) AT1 (AT1a) receptors selectively in the renomemdullary interstitial cells (RMICs) of the kidney medulla attenuates the development of Ang II-induced hypertension in mice. Design and method: To test the hypothesis, we used inducible Tenascin-C-CreER2 and Agtr1a-floxed recombination approach to generate a novel mouse model with deletion of AT1a receptors selectively in the RMICs of the kidney (RMIC-Agtr1a-/-). Adult male wildtype (WT) and RMIC-Agtr1a-/- mice (n = 10 per group) were implanted with a telemetry probe and infused with or without Ang II via an osmotic minipump (∼500 μg/kg/day, i.p.) for 2 weeks. Results: Mice with deletion of AT1a receptors selectively in RMICs of the kidney medulla were fertile and grew normally without apparent structural or histological abnormality in the kidney medulla. However, telemetry systolic, diastolic, and mean blood pressure were significantly lower in adult male RMIC-Agtr1a-/- than WT mice (WT: 122 ± 4 mmHg vs. RMIC-Agtr1a-/-: 102 ± 3 mmHg, P < 0.01). Interestingly, this hypotensive phenotype in RMIC-Agtr1a-/- mice was associated with significant decreases in 24 h urine output (WT: 1.56 ± 0.15 mL/24 h vs. RMIC-Agtr1a-/-: 0.98 ± 0.13 mL/24 h, P < 0.01), urinary sodium (WT: 207.9 ± 7.3 μmol/24 h vs. RMIC-Agtr1a-/-: 145.2 ± 5.4 μmol/24 h, P < 0.01), and potassium excretion (WT: 221.0 ± 6.7 μmol/24 h vs. RMIC-Agtr1a-/-: 160.8 ± 10.6 μmol/24 h, P < 0.01), respectively. Moreover, urine osmolality was markedly decreased in RMIC-Agtr1a-/-mice (WT: 1724 ± 335 mOsm/Kg H2O vs. RMIC-Agtr1a-/-: 1128 ± 102 mOsm/Kg H2O, P < 0.01). Ang II infusion significantly increased telemetry systolic blood pressure to 148 ± 5 mmHg in WT mice, but it increased systolic blood pressure only to 129 ± 8 mmHg in RMIC-Agtr1a-/- mice (P < 0.01). Furthermore, infusion of Ang II for 2 weeks failed to restore urine osmolality in RMIC-Agtr1a-/- mice (1226 ± 110 mOsm/Kg H2O, n.s.). Conclusions: In conclusion, the results of the present study support the hypothesis that AT1a receptors in the RMICs of the kidney medulla plays a critical role in maintaining basal blood pressure homeostasis and the development of Ang II-induced hypertension.

Angiotensin II-induced a steeper blood pressure elevation in IL-23 receptor-deficient mice: Role of interferon-γ-producing T cells.

A subset of interleukin (IL)-17A-producing γδ T cells called γδT17 cells may contribute to progression of hypertension. γδT17 cell development is in part dependent upon IL-23 receptor (IL-23R) stimulation. We hypothesized that angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular injury would be blunted in Il23r knock-in (Il23rgfp/gfp) mice deficient in functional IL-23R. To test this hypothesis, we infused wild-type (WT) and Il23rgfp/gfp mice with Ang II (490 ng/kg/min, SC) for 7 or 14 days. We recorded BP by telemetry, assessed vascular function and remodeling using pressurized myography, and profiled T cell populations and cytokine production by flow cytometry. An additional set of Il23rgfp/gfp mice was infused with Ang II for 7 days and injected with interferon (IFN)-γ-neutralizing or control antibodies. Il23rgfp/gfp mice had smaller and stiffer mesenteric arteries and were not protected against Ang II-induced BP elevation. BP was higher in Il23rgfp/gfp mice than WT mice from day 3 until day 9 of Ang II infusion. Il23rgfp/gfp mice had less γδT17 cells and more IFN-γ-producing γδ, CD4+, and CD8+ T cells than WT mice. Seven days of Ang II infusion led to increased IFN-γ-producing γδ, CD4+, and CD8+ T cells in Il23rgfp/gfp mice, whereas only IFN-γ-producing γδ T cells were increased in WT mice. Blocking IFN-γ with a neutralizing antibody reduced the pressor response to 7 days of Ang II infusion in Il23rgfp/gfp mice. Functional IL-23R deficiency was associated with increased IFN-γ-producing T cells and exaggerated initial development of Ang II-induced hypertension, which was in part mediated by IFN-γ.

Protection against angiotensin II-induced hypertension in mice lacking sphingosine kinase 1 in vascular smooth muscle cells

Abstract Background Sphingosine-1-phosphate (S1P), a lipid mediator produced by sphingosine kinases (Sphks), significantly contributes to the maintenance of cardiovascular homeostasis. Mice lacking Sphk1 globally are protected against angiotensin II-induced hypertension, yet, due to pleiotropic action of S1P, it has been challenging to identify a specific cell type responsible for this protection. Purpose The aim of this study was to investigate the role of Sphk1 expressed in vascular smooth muscle cells (VSMCs) in the development of angiotensin II (AngII)-induced hypertension in mice. Methods Male, 12-week-old C57BL/6J wild type (WT) or VSMC-specific, Cre-mediated, Sphk1 knockout (Sphk1VSMC−/−) mice underwent either Sham or AngII (490 ng/min/kg s.c.) treatment for 2 weeks, using a surgically implanted osmotic minipump. Blood pressure was measured by the tail-cuff method. Vascular structure and function were examined ex vivo using wire (vasoconstrictor/vasorelaxant responses) and pressure myography (myogenic tone, structural and elasticity parameters). Gene set enrichment analysis (GSEA) was performed using transcriptomic profiles of the isolated mesenteric arteries (MA, RNASeq). Expression of proteins was analyzed by Western blot. Results Deletion of Sphk1 in VSMCs resulted in 5x and 40x lower expression of Sphk1 mRNA in MA and aorta compared to WT mice respectively. A 2-week infusion of AngII resulted in a development of higher systolic blood pressure (SBP) in WT mice compared to Sphk1VSMC−/− mice (mean SBP ± SEM: 168±4.3 mmHg vs. 132±4.4 mmHg respectively, p<0.05). Studies of ex vivo mesenteric artery (MA) function revealed diminished both, depolarization-induced (in response to KCl), as well as G protein-coupled receptor-elicited (in response to phenylephrine and thromboxane A2 analogue) contraction of MA derived from hypertensive Sphk1VSMC−/− mice compared to WT mice. In the same time, significantly impaired endothelial vasorelaxation was observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice. Moreover, MA from Sphk1VSMC−/− mice were characterized by the significantly reduced myogenic response evoked by intravascular pressure elevation (maximal myogenic response ± SEM: 19.7±1.8% for WT normotensive, 26.2±1.8% for WT AngII vs. 19.0±2.5% for Sphk1VSMC−/− AngII mice), and an increased stiffness compared to WT mice. GSEA found that the lack of Sphk1 gene in VSMCs significantly downregulated molecular pathways related to smooth muscle contraction. In line with transcriptomic studies, significantly reduced level of the Rho-associated protein kinase (Rock) has been observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice using Western blot. Conclusions This study revealed that mice lacking Sphk1 gene in VSMCs are protected against the development of AngII-induced hypertension likely due to the lowered myogenic tone in resistance arteries, which may be mediated by autocrine effects of Sphk1 on Rock proteins. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The National Science Centre, Poland

The Na+/H+ Exchanger 3 in the Intestines and the Proximal Tubule of the Kidney: Localization, Physiological Function, and Key Roles in Angiotensin II-Induced Hypertension.

The sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3) is one of the most important Na+/H+ antiporters in the small intestines of the gastrointestinal tract and the proximal tubules of the kidney. The roles of NHE3 in the regulation of intracellular pH and acid–base balance have been well established in cellular physiology using in vitro techniques. Localized primarily on the apical membranes in small intestines and proximal tubules, the key action of NHE3 is to facilitate the entry of luminal Na+ and the extrusion of intracellular H+ from intestinal and proximal tubule tubular epithelial cells. NHE3 is, directly and indirectly, responsible for absorbing the majority of ingested Na+ from small and large intestines and reabsorbing >50% of filtered Na+ in the proximal tubules of the kidney. However, the roles of NHE3 in the regulation of proximal tubular Na+ transport in the integrative physiological settings and its contributions to the basal blood pressure regulation and angiotensin II (Ang II)-induced hypertension have not been well studied previously due to the lack of suitable animal models. Recently, novel genetically modified mouse models with whole-body, kidney-specific, or proximal tubule-specific deletion of NHE3 have been generated by us and others to determine the critical roles and underlying mechanisms of NHE3 in maintaining basal body salt and fluid balance, blood pressure homeostasis, and the development of Ang II-induced hypertension at the whole-body, kidney, or proximal tubule levels. The objective of this invited article is to review, update, and discuss recent findings on the critical roles of intestinal and proximal tubule NHE3 in maintaining basal blood pressure homeostasis and their potential therapeutic implications in the development of angiotensin II (Ang II)-dependent hypertension.

Angiotensin II and AT1a Receptors in the Proximal Tubules of the Kidney: New Roles in Blood Pressure Control and Hypertension.

Contrary to public perception, hypertension remains one of the most important public health problems in the United States, affecting 46% of adults with increased risk for heart attack, stroke, and kidney diseases. The mechanisms underlying poorly controlled hypertension remain incompletely understood. Recent development in the Cre/LoxP approach to study gain or loss of function of a particular gene has significantly helped advance our new insights into the role of proximal tubule angiotensin II (Ang II) and its AT1 (AT1a) receptors in basal blood pressure control and the development of Ang II-induced hypertension. This novel approach has provided us and others with an important tool to generate novel mouse models with proximal tubule-specific loss (deletion) or gain of the function (overexpression). The objective of this invited review article is to review and discuss recent findings using novel genetically modifying proximal tubule-specific mouse models. These new studies have consistently demonstrated that deletion of AT1 (AT1a) receptors or its direct downstream target Na+/H+ exchanger 3 (NHE3) selectively in the proximal tubules of the kidney lowers basal blood pressure, increases the pressure-natriuresis response, and induces natriuretic responses, whereas overexpression of an intracellular Ang II fusion protein or AT1 (AT1a) receptors selectively in the proximal tubules increases proximal tubule Na+ reabsorption, impairs the pressure-natriuresis response, and elevates blood pressure. Furthermore, the development of Ang II-induced hypertension by systemic Ang II infusion or by proximal tubule-specific overexpression of an intracellular Ang II fusion protein was attenuated in mutant mice with proximal tubule-specific deletion of AT1 (AT1a) receptors or NHE3. Thus, these recent studies provide evidence for and new insights into the important roles of intratubular Ang II via AT1 (AT1a) receptors and NHE3 in the proximal tubules in maintaining basal blood pressure homeostasis and the development of Ang II-induced hypertension.

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.

Genetic Deletion of AT 1a Receptors Selectively in The Proximal Tubules of The Kidney Attenuates Angiotensin II‐induced Hypertensive and Glomerular Filtration Responses in Aging PT‐ Agtr1a ‐/‐ Mice

The intratubular renin-angiotensin system (RAS) via the angiotensinogen/renin/ACE/Ang II/AT1 (AT1a) receptor signaling pathways in the proximal tubules of the kidney play a critical role in the physiological regulation of blood pressure and the development of hypertension. Whether intratubular RAS continues to exert significant impacts on blood pressure and glomerular filtration responses to Ang II remains unknown. In the present study, we tested the hypothesis that deletion of AT1a receptors selectively in the proximal tubules of the kidney attenuates Ang II-induced hypertensive and glomerular filtration responses (GFR) in aging PT-Agtr1a-/- mice. To test the hypothesis, 6 groups (n=8-12 per group) of 20- to 24-month-old male wild-type (WT) and PT-Agtr1a-/- mice were infused with or without a slow pressor dose of Ang II (~0.65 mg/kg/day, i.p.), supplemented with a 2% Na+ diet for 2 weeks. Systolic blood pressure was determined by telemetry and tail-cuff techniques, whereas GFR was measured by the transdermal GFR monitoring technique with FITC-sinistrin, and the pressure-natriuresis responses was determined before and during Ang II infusion, respectively. Basal systolic blood pressure remained significantly lower in aging PT-Agtr1a-/- (104 ± 4 mmHg) than in aging WT mice (120 ± 4 mmHg, P<0.01), whereas basal GFR was significantly higher in aging PT-Agtr1a-/- mice (196.4 ± 14.7 µl/min) than in aging WT mice (142.6 ± 11.6 µl/min, P<0.01). Furthermore, the pressure-natriuresis response was significantly augmented in aging PT-Agtr1a-/-mice (P<0.05). Ang II infusion significantly induced hypertension (152 ± 3 mmHg, P<0.01) and decreased GFR in aging WT mice (122.4 ± 3.3 µl/min, P<0.01), as expected, but the hypertensive (135 ± 3 mmHg, P<0.01) and GFR responses were significantly improved in aging PT-Agtr1a-/-mice (155.9 ± 10.1 µl/min, P<0.01), respectively. Finally, proximal tubule-specific deletion of AT1a receptors significantly augmented the pressure-natriuresis response and natriuretic responses to Ang II infusion in aging PT-Agtr1a-/-mice (P<0.01). Taken together, the results of the present study suggest that intratubular Ang II and AT1a receptors in the proximal tubules continue to play a key role in the development of Ang II-induced hypertension in aging WT mice and that deletion of AT1a receptors selectively in the proximal tubules attenuates Ang II-induced hypertension and improves glomerular filtration in aging PT-Agtr1a-/- mice.

PROXIMAL TUBULE-SPECIFIC DELETION OF AT1A RECEPTORS ATTENUATES CIRCULATING AND INTRATUBULAR ANGIOTENSIN II-INDUCED HYPERTENSION IN MICE

Objective: The present study used a novel mouse model with proximal tubule-specific knockout of AT1a receptors in the kidney, PT-Agtr1a-/-, to test the hypothesis that intratubular angiotensin II (Ang II) and AT1a receptors in the proximal tubules are required for maintaining normal blood pressure and the development of Ang II-induced hypertension. Design and method: To test our hypothesis, eight groups (n = 7–15 per group) of adult male wild-type, global Agtr1a-/-, and PT-Agtr1a-/-, and PT-Nhe3-/- mice were infused with Ang II (1.5 mg/kg/day, i.p.), or overexpressed an intracellular Ang II fusion protein (Ad-sglt2-ECFP/Ang II) in the proximal tubules of the kidney for 2 weeks. Basal blood pressure and the pressor responses, glomerular filtration rate (GFR) and 24 h urinary natriuretic response, and the pressure-natriuresis response were determined. Results: Basal telemetry blood pressure were ∼15 ± 3 mmHg lower in PT-Agtr1a-/- than wild-type mice and ∼13 ± 3 mmHg higher than Agtr1a-/- mice (P &lt; 0.01). Basal glomerular filtration was ∼23.9% higher (P &lt; 0.01), whereas fractional proximal tubule Na+ reabsorption was lower in PT-Agtr1a-/- mice (P &lt; 0.01). Deletion of AT1a receptors in the proximal tubules augmented the pressure-natriuresis response (P &lt; 0.01), and natriuretic responses to salt loading or Ang III infusion (P &lt; 0.01). Ang II induced hypertension in wild-type, PT-Agtr1a-/- and PT-Nhe3-/- mice, but the pressor response was ∼16 ± 2 mmHg lower in PT-Agtr1a-/- and PT-Nhe3-/-mice (P &lt; 0.01). Deletion of AT1a receptors or NHE3 in the proximal tubules attenuated ∼50% of Ang II-induced hypertension in wild-type mice (P &lt; 0.01), but blocked intracellular Ang II fusion protein-induced hypertension in PT-Agtr1a-/- mice (P &lt; 0.01). Conclusions: Taken together, the results of the present study provide new insights into the critical role of intratubular Ang II/AT1 (AT1a)/NHE3 pathways in the proximal tubules in normal blood pressure control and the development of Ang II-induced hypertension.

PROXIMAL TUBULE-SPECIFIC DELETION OF AT1A RECEPTORS ATTENUATES ANGIOTENSIN II-INDUCED HYPERTENSION BY INCREASING GLOMERULAR FILTRATION AND THE PRESSURE-NATRIURESIS RESPONSE

Objective: The present study tested the hypothesis that Ang II and AT1a receptors in the proximal tubules are required for normal blood pressure homeostasis and full development of Ang II-induced hypertension. Design and method: Six groups (n = 7–12 per group) of adult male wild-type (WT) and PT-Agtr1a-/- mice were infused with or without Ang II for 2 weeks (1.5 mg/kg/day, i.p.). Glomerular filtration rate (GFR) was measued using fluorescein-labeled sinistrin, and natriuretic responses were determined under basal conditions and after Ang II infusion. Results: Basal systolic blood pressure were ∼13 ± 3 mmHg lower in PT-Agtr1a-/- than WT mice (P &lt; 0.01). However, basal GFR was higher in PT-Agtr1a-/- mice (WT: 160.4 ± 7.0 μl/min vs. PT-Agtr1a-/-: 186.0 ± 10 μl/min, P &lt; 0.01). Basal 24 h urinary Na+ excretion (UNaV) was higher in PT-Agtr1a-/- than WT mice (P &lt; 0.01). In response to Ang II infusion, both WT and PT-Agtr1a-/- mice developed hypertension with similar pressor response to Ang II in WT (43 ± 3 mmHg, P &lt; 0.01) and PT-Agtr1a-/- mice (39 ± 5 mmHg, P &lt; 0.01). However, systolic blood pressure was still 16 ± 3 mmHg lower in PT-Agtr1a-/- mice (P &lt; 0.01). Ang II decreased GFR to 132.2 ± 7.0 μl/min in WT mice (P &lt; 0.01), and to 129.4 ± 18.6 μl/min in PT-Agtr1a-/- mice (P &lt; 0.01), respectively. In WT mice, UNaV increased from 139.3 ± 22.3 μmol/24 h in the control group to 196.4 ± 29.6 μmol/24 h in the Ang II-infused group (P &lt; 0.01). In PT-Agtr1a-/- mice, UNaV increased from 172.0 ± 10.2 μmol/24 h in the control group to 264.7 ± 35.4 μmol/24 h in the Ang II-infused group (P &lt; 0.01). The pressor response to Ang II was attenuated, while the natriuretic response was augmented by losartan in WT and PT-Agtr1a-/- mice (P &lt; 0.01). Finally, proximal tubule-specific deletion of AT1a receptors significantly augmented the pressure-natriuresis response and natriuretic responses to acute saline infusion (P &lt; 0.01) or a 2% high salt diet for 2 weeks (P &lt; 0.01). Conclusions: The present study supports the hypothesis that Ang II and AT1a receptors in the proximal tubules are required for normal blood pressure homeostasis and the development of Ang II-induced hypertension.

Abstract P041: Proximal Tubule-specific Deletion Of Angiotensin Ii Type 1a Receptors In The Kidney Lowers Basal Blood Pressure And Attenuates Angiotensin Ii-induced Hypertension By Increasing Glomerular Filtration And The Pressure-natriuresis Response

The present study tested the hypothesis that intratubular angiotensin II (Ang II) and AT 1a receptors in the proximal tubules of the kidney plays an important role in basal blood pressure control and in the development of Ang II-induced hypertension. Mutant mice with proximal tubule-specific deletion of AT 1a receptors in the kidney, PT- Agtr1a -/- , were generated to test the hypothesis. Eight groups (n=7-12 per group) of adult male wild-type (WT) and PT- Agtr1a -/- mice were infused with or without Ang II for 2 weeks (1.5 mg/kg, i.p.). Basal systolic, diastolic, and mean arterial pressures were ~13 ± 3 mmHg lower in PT- Agtr1a -/- than WT mice ( P &lt;0.01). Basal glomerular filtration rate (GFR), as measured using transdermal FITC-sinistrin, was significantly higher in PT- Agtr1a -/- mice (WT: 160.4 ± 7.0 μl/min vs. PT- Agtr1a -/- : 186.0 ± 6.0 μl/min, P &lt;0.05). Basal 24 h urinary Na + excretion (U Na V) was significantly higher in PT- Agtr1a -/- than WT mice ( P &lt;0.01). In response to Ang II infusion, both WT and PT- Agtr1a -/- mice developed hypertension, and the magnitude of the pressor response to Ang II was similar in WT (Δ43 ± 3 mmHg, P &lt;0.01) and PT- Agtr1a -/- mice (Δ39 ± 5 mmHg, P &lt;0.01). However, the absolute blood pressure level was still 16 ± 3 mmHg lower in PT- Agtr1a -/- mice ( P &lt;0.01). Ang II significantly decreased GFR to 132.2 ± 7.0 μl/min in WT mice ( P &lt;0.01), and to 129.4 ± 18.6 μl/min in PT- Agtr1a -/- mice ( P &lt;0.01), respectively. In WT mice, U Na V increased from 139.3 ± 22.3 μmol/24 h in the control group to 196.4 ± 29.6 μmol/24 h in the Ang II-infused group ( P &lt;0.01). In PT- Agtr1a -/- mice, U Na V increased from 172.0 ± 10.2 μmol/24 h in the control group to 264.7 ± 35.4 μmol/24 h in the Ang II-infused group ( P &lt;0.01). The pressor response to Ang II was attenuated, while the natriuretic response was augmented by losartan in WT and PT- Agtr1a -/- mice ( P &lt;0.01). Finally, proximal tubule-specific deletion of AT 1a receptors significantly augmented the pressure-natriuresis response and natriuretic responses to acute saline infusion ( P &lt;0.01) or a 2% high salt diet ( P &lt;0.01). We concluded that deletion of AT 1a receptors selectively in the proximal tubules lowers basal blood pressure and attenuates Ang II-induced hypertension by increasing GFR and promoting the natriuretic response in PT- Agtr1a -/- mice.

Abstract P033: PRESSOR, NATRIURETIC, AND RENAL RESPONSES IN ANGIOTENSIN II-INDUCED HYPERTENSION IN MALE AND FEMALE WILD-TYPE AND PROXIMAL TUBULE-SPECIFIC AT 1A RECEPTOR KNOCKOUT MICE

Dysregulation of intrarenal renin-angiotensin system is one of the key factors of human hypertension, but the mechanisms involved remain incompletely understood. To determine the roles of AT 1a receptors in the proximal tubules of the kidney, we infused angiotensin II (Ang II) for 2 weeks (40 ng / min, i.p.) in adult male and female wild-type C57BL/6J and mutant mice with deletion of AT 1a receptors in the proximal tubules (PT- Agtr1a -/- ), and treated with or without the AT 1 receptor blocker losartan (20 mg/kg/day, p.o.) (n=8 per group). The pressor response, 24 h urinary Na + excretion, glomerular and tubulointerstitial injury were compared between male and female wild-type and PT- Agtr1a -/- mice. Basal systolic, diastolic, and mean arterial blood pressure were about 13 ± 3 mmHg lower in male and female PT- Agtr1a -/- mice ( P &lt;0.01), but no differences were observed between male and female wild-type or PT- Agtr1a -/- mice. In response to Ang II, both male and female wild-type and PT- Agtr1a -/- mice developed hypertension ( P &lt;0.01), and the net pressor response were similar in male and female wild-type and PT- Agtr1a -/- mice (n.s.). However, absolute blood pressure was about 12 ± 3 mmHg lower in male and female PT- Agtr1a -/- mice ( P &lt;0.01 vs. wild-type). Ang II-induced hypertension increased the natriuretic response in both male and female wild-type and PT- Agtr1a -/- mice ( P &lt;0.01), but there were no significant differences between male and female wild-type and PT- Agtr1a -/- mice (n.s). Losartan did not increase the natriuretic responses further in all animals. Furthermore, Ang II-induced hypertension was associated with significant increases in glomerular and tubulointerstitial injury in male and female wild-type mice ( P &lt;0.01), which were attenuated in male and female PT- Agtr1a -/- mice ( P &lt;0.01). LOS treatment attenuated Ang II-induced hypertension and decreased Ang II-induced glomerular and tubulointerstitial injury in male and female wild-type and PT- Agtr1a -/- mice ( P &lt;0.01). Taken together, we demonstrated that intratubular AT 1 (AT 1a ) receptors in the proximal tubules of the kidney plays a key role in maintaining basal blood pressure homeostasis and overall body salt and fluid balance, and the development of Ang II-induced hypertension and kidney injury.

Abstract P011: Novel Roles Of Global, Intestinal, And Kidney Proximal Tubule Sodium And Hydrogen Exchanger 3 In Angiotensin Ii-induced Hypertension

The present study used global ( Nhe3 -/- ), kidney-selective (tg Nhe3 -/- ), and proximal tubule-specific Na + /H + exchanger 3 (NHE3)-deficient mice (PT- Nhe3 -/- ) to test the hypothesis that NHE3 is required for the full development of angiotensin II (Ang II)-induced hypertension in mice. Four groups of adult male, age-matched wild-type (WT), global Nhe3 -/- , kidney-selective tg Nhe3 -/- and proximal tubule-specific Nhe3 -/- mice were infused with: a) saline; b) Ang II (10 pmol/min, i.v.); Ang II via an osmotic minipump for 2 weeks (1.5 mg/kg/day, i.p.); or treated with Ang II and losartan concurrently for 2 weeks (20 mg/kg/day, p.o.). Under basal conditions, global Nhe3 -/- , kidney-selective tg Nhe3 -/- and proximal tubule-specific Nhe3 -/- mice all showed significantly lower systolic, diastolic, and mean arterial pressure than wild-type mice (~15 ± 3 mmHg, P &lt;0.01). The hypotensive phenotype in both global Nhe3 -/- and kidney-selective tg Nhe3 -/- mice was associated with abnormal intestinal structures, diarrhea, increased 24 h fecal Na + excretion, and salt wasting ( P &lt;0.01). By contrast, there were no differences in intestinal structures and fecal Na + excretion between wild-type and PT- Nhe3 -/- mice. PT- Nhe3 -/- mice showed significant diuretic and natriuretic responses compared with wild-type mice ( P &lt;0.01). Acute infusion of Ang II markedly increased arterial blood pressure in a time-dependent manner in wild-type mice, as expected ( P &lt;0.01), but the pressure response was attenuated in global Nhe3 -/- , kidney-selective tg Nhe3 -/- , and PT- Nhe3 -/- mice ( P &lt;0.01). Furthermore, the chronic pressor response to 2-week Ang II infusion was also significantly attenuated in Nhe3 -/- , tgNhe3 -/- , and PT- Nhe3 -/- mice, compared with wild-type mice ( P &lt;0.01). Finally, concurrent treatment with losartan completely blocked the acute and chronic pressor responses to Ang II in wild-type, Nhe3 -/- , tg Nhe3 -/- , and PT- Nhe3 -/- mice (p&lt;0.01). Taken together, these data support the proof of concept that NHE3 in the small intestines and the proximal tubules of the kidney is required for maintaining basal blood pressure homeostasis and for the development of Ang II-induced hypertension. Supported by NIH grants, 2R01DK102429-03A1, 1R56HL130988-01, and 2R01DK067299-10A1.

Abstract 077: Proximal Tubule-Specific Deletion of the NHE3 (na + /h + Exchanger 3) in the Kidney Attenuates Angiotensin II-Induced Hypertension in Mice

The Na + /H + exchanger 3 (NHE3), a ~85 kDa protein encoded by the SLC9A3 gene, is the most important Na + transporter in the proximal tubules of the kidney. Angiotensin II (ANG II) is well-recognized to increase proximal tubule Na + reabsorption by stimulating NHE3 expression and activity in the proximal tubules. However, a direct cause and effect relationship between ANG II and NHE3 in the proximal tubules in ANG II-induced hypertension has not been determined previously. The present study directly tested the hypothesis that NHE3 in the proximal tubules of the kidney is required for the development of ANG II-induced hypertension using proximal tubule-specific NHE3 knockout mice (PT- Nhe3 -/- ). Specifically, PT- Nhe3 -/- mice were generated using the SGLT2-Cre / Nhe3 loxlox approach, whereas ANG II-induced hypertension was studied in 12 groups (n=5-12 per group) of adult male and female wild-type (WT) and PT- Nhe3 -/- mice. Under basal conditions, systolic (SBP), diastolic (DBP), and mean arterial blood pressure (MAP) were significantly lower in male and female PT- Nhe3 -/- than WT mice ( P &lt;0.01). A high pressor, 1.5 mg/kg/day, i.p., or a slow pressor dose of ANG II, 0.5 mg/kg/day, i.p., for 2 weeks significantly increased SBP, DBP, and MAP in male and female WT mice ( P &lt;0.01), but the hypertensive response to ANG II was markedly attenuated in male and female PT- Nhe3 -/- mice ( P &lt;0.01). ANG II impaired the pressure-natriuresis response in WT mice, whereas proximal tubule-specific deletion of NHE3 improved the pressure-natriuresis response in ANG II-infused PT- Nhe3 -/- mice ( P &lt;0.01). AT 1 receptor blocker losartan completely blocked ANG II-induced hypertension in both WT and PT- Nhe3 -/- mice ( P &lt;0.01). However, inhibition of nitric oxide synthase with L-NAME had no effect on ANG II-induced hypertension in WT or PT- Nhe3 -/- mice (n.s.). Furthermore, ANG II-induced hypertension was significantly attenuated by an orally absorbable NHE3 inhibitor AVE0657. In conclusion, NHE3 in the proximal tubules of the kidney is required for the full development of ANG II-induced hypertension. Our results suggest that NHE3 in the proximal tubules may be therapeutically targeted to treat hypertension induced by ANG II or associated with increased NHE3 expression in the proximal tubules.

SAT-110 THE SODIUM AND HYDROGEN EXCHANGER 3 (NHE3) IS REQUIRED FOR THE FULL DEVELOPMENT OF ANGIOTENSIN II-INDUCED HYPERTENSION IN GLOBAL, KIDNEY-, AND PROXIMAL TUBULE-SPECIFIC NHE3-KO MICE

The sodium (Na+) and hydrogen (H+) exchanger 3 (NHE3) plays a key role in the regulation of sodium (Na+), fluid, and bicarbonate reabsorption in the proximal tubules of the kidney, but its roles in the full development of angiotensin II (ANG II)-induced hypertension remain incompletely understood. In this study, we used global (Nhe3-/-), kidney-selective (tgNhe3-/-), and proximal tubule-specific NHE3-deficient mice (PT-Nhe3-/-) to test the hypothesis that NHE3 is required for the full development of ANG II-induced hypertension in mice.

Genetic and genomic evidence for an important role of the Na+/H+ exchanger 3 in blood pressure regulation and angiotensin II-induced hypertension.

The sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3) and sodium-potassium adenosine triphosphatase (Na+/K+-ATPase) are two of the most important Na+ transporters in the proximal tubules of the kidney. On the apical membrane side, NHE3 primarily mediates the entry of Na+ into and the exit of H+ from the proximal tubules, directly and indirectly being responsible for reabsorbing ~50% of filtered Na+ in the proximal tubules of the kidney. On the basolateral membrane side, Na+/K+-ATPase serves as a powerful engine driving Na+ out of, while pumping K+ into the proximal tubules against their concentration gradients. While the roles of NHE3 and Na+/K+-ATPase in proximal tubular Na+ transport under in vitro conditions are well recognized, their respective contributions to the basal blood pressure regulation and angiotensin II (ANG II)-induced hypertension remain poorly understood. Recently, we have been fortunate to be able to use genetically modified mouse models with global, kidney- or proximal tubule-specific deletion of NHE3 to directly determine the cause and effect relationship between NHE3, basal blood pressure homeostasis, and ANG II-induced hypertension at the whole body, kidney and/or proximal tubule levels. The purpose of this article is to review the genetic and genomic evidence for an important role of NHE3 with a focus in the regulation of basal blood pressure and ANG II-induced hypertension, as we learned from studies using global, kidney- or proximal tubule-specific NHE3 knockout mice. We hypothesize that NHE3 in the proximal tubules is necessary for maintaining basal blood pressure homeostasis and the development of ANG II-induced hypertension.

Abstract P155: B Cells Are Not Necessary for the Developmentof Ang II-induced Hypertension and End-Organ Damage

Chronic inflammation characterized by vascular T cell and macrophage infiltration is associated with angiotensin II (Ang II)-induced hypertension and end-organ damage. However, the role of B cells in the development of hypertension is not well established. Elevated levels of serum immunoglobulins have been observed in experimental and human hypertension. Prior studies demonstrated that B-cell-activating factor receptor-deficient (BAFF-R -/- ) mice, which lack mature B cells, are protected from Ang II-induced hypertension and vascular remodeling. However, BAFF-R can be expressed on non-B cells as well. Thus, to further investigate the role of B cells in hypertension, we studied mice with targeted disruption of the membrane exon of the immunoglobulin μ heavy chain gene (μMT -/- ) that encodes the constant region of the IgM isotype. In these mice, B cells are arrested at the pre-B cell maturation stage. Serum immunoglobulins were undetectable at a 1:30,000 dilution in μMT -/- mice. Interestingly, μMT -/- mice exhibited similar blood pressure increase in response to 4 weeks of Ang II infusion compared to age-matched wild type (WT) mice (p=0.675). Flow cytometry of aortae from Ang II-treated μMT -/- mice demonstrated fewer CD45+ cells (p=0.047), similar levels of CD8+ and CD4+ T cells (p=0.631 and p=0.721 respectively), a trend for more F4/80+ monocytes/macrophages (p=0.2567), and virtually no CD19+ B cells (p=0.002) compared to WT mice. Endothelium-dependent vasodilatation to acetylcholine was comparable between μMT -/- mice and WT mice after Ang II treatment (p=0.223), however μMT -/- mice displayed enhanced endothelium-independent vasodilatation to sodium nitroprusside than WT mice (p=0.024). Urinary albumin/creatinine ratio was similar between Ang II-treated μMT -/- mice and WT mice (p=0.488), indicating similar renal damage. Taken together, μMT -/- mice are not protected from Ang II-induced hypertension, suggesting that B cells and immunoglobulin production are not critical for hypertension and the associated end-organ damage.

Increased mitochondrial superoxide in the brain, but not periphery, sensitizes mice to angiotensin II-mediated hypertension

Angiotensin II (AngII) elicits the production of superoxide (O2•−) from mitochondria in numerous cell types within peripheral organs and in the brain suggesting a role for mitochondrial-produced O2•− in the pathogenesis of hypertension. However, it remains unclear if mitochondrial O2•− is causal in the development of AngII-induced hypertension, or if mitochondrial O2•− in the absence of elevated AngII is sufficient to increase blood pressure. Further, the tissue specific (i.e. central versus peripheral) redox regulation of AngII hypertension remains elusive. Herein, we hypothesized that increased mitochondrial O2•− in the absence of pro-hypertensive stimuli, such as AngII, elevates baseline systemic mean arterial pressure (MAP), and that AngII-mediated hypertension is exacerbated in animals with increased mitochondrial O2•− levels. To address this hypothesis, we generated novel inducible knock-down mouse models of manganese superoxide dismutase (MnSOD), the O2•− scavenging antioxidant enzyme specifically localized to mitochondria, targeted to either the brain subfornical organ (SFO) or peripheral tissues. Contrary to our hypothesis, knock-down of MnSOD either in the SFO or in peripheral tissues was not sufficient to alter baseline systemic MAP. Interestingly, when mice were challenged with chronic, peripheral infusion of AngII, only the MnSOD knock-down confined to the SFO, and not the periphery, demonstrated an increased sensitization and potentiated hypertension. In complementary experiments, over-expressing MnSOD in the SFO significantly decreased blood pressure in response to chronic AngII. Overall, these studies indicate that mitochondrial O2•− in the brain SFO works in concert with other AngII-dependent factors to drive an increase in MAP, as elevated mitochondrial O2•− alone, either in the SFO or peripheral tissues, failed to raise baseline blood pressure.