AT1A Receptor Research Articles

Deletion of AT1a Receptors Selectively in Renomedullary Interstitial Cells Attenuates Basal, Angiotensin II-, or Vasopressin-induced Vasoactive Factors' mRNA Expression in The Mouse Kidney Medulla

We recently shown that deletion of the vasoactive peptide angiotensin II (Ang II) AT1a receptors selectively in renomedullary interstitial cells (RMICs) of the kidney medulla significantly lowered basal blood pressure and impaired urine concentration in mice. However, the underlying signaling mechanisms involved remains poorly understood. In the present study, AT1a receptors were selectively deleted in the RMICs of the mouse kidney medulla using tamoxifen-inducible Tenascin-C-CreER2/Agtr1af/f approach. Three groups of adult male wild-type (WT) and RMIC- Agtr1a−/− mice (n=6-10) were pretreated with or without tamoxifen (150 mg/kg, i.p., for 5 days) before Ang II was infused to induce Ang II-dependent hypertension (~0.75 mg/kg/day, i.p., for 2 weeks). Systolic, diastolic, and mean arterial blood pressure and urine osmolality were determined before and after Ang II treatment. At the end of the experiment, the kidneys were harvested, and the inner and outer medulla were dissected out for the measurement of the mRNA expression of key vasoactive factors that are associated with the regulation of blood pressure and urine concentration using digital droplet PCR. Deletion of AT1a receptors in RMICs significantly decreased AT1a receptor mRNA expression by 41% (WT: 554 ± 49 vs. RMIC- Agtr1a−/−: 327 ± 10 copies/ng RNA; P<0.01) and bradykinin B2 receptor mRNA expression by 52% (WT: 10.10 ± 0.75 vs. RMIC- Agtr1a−/−: 4.90 ± 0.56 copies/ng RNA; P<0.01), without altering the expression of endothelin ETB (WT: 466 ± 21 vs. RMIC- Agtr1a−/−: 393 ± 24 copies/ng RNA; n.s.) and vasopressin V2 receptor mRNAs in the kidney medulla (WT: 580 ± 60 vs. RMIC- Agtr1a−/−: 586 ± 61 copies/ng RNA; n.s.). Deletion of AT1a receptors in RMICs also significantly decreased the expression of eNOS (WT: 87.0 ± 3.0 vs. RMIC- Agtr1a−/−: 61.2 ± 2.8 copies/ng RNA; P<0.01) and aquaporin 2 (AQP2) mRNAs (WT: 12463 ± 2015 vs. RMIC- Agtr1a−/−: 6613 ± 692 copies/ng RNA; P<0.01). Interestingly, deletion of AT1a receptors in RMICs did not alter the mRNA expression of cyclooxygenase-2 (COX-2, n.s.), prostaglandin E2 (PGE2, n.s.), epithelial sodium channel (ENaC, n.s.) or prorenin receptors (PRR, n.s.) in the kidney medulla. In response to Ang II infusion, deletion of AT1a receptors in RMICs significantly attenuated Ang II-induced expression of bradykinin B2 receptors, COX-2, eNOS, and AQP2 mRNAs ( P<0.01), respectively. Furthermore, deletion of AT1a receptors in RMICs also significantly attenuated vasopressin-induced bradykinin B2 receptor, eNOS, and AQP2 mRNA expression in the kidney medulla ( P<0.01). We concluded that AT1a receptors in RMICs in the kidney medulla play an important role in maintaining physiological blood pressure homeostasis and urine concentration process likely via AT1a receptor/bradykinin B2 receptor/eNOS/AQP2-mediated signaling pathways. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

DNA demethylation in the hypothalamus promotes transcription of Agtr1a and Slc12a2 and hypertension development

Increased expression of the angiotensin II AT1A receptor (encoded by Agtr1a) and Na+-K+-Cl− cotransporter-1 (NKCC1, encoded by Slc12a2) in the hypothalamic paraventricular nucleus (PVN) contributes to the development of hypertension. However, little is known about transcriptional control of Agtr1a and Slc12a2 in the PVN in hypertension. DNA methylation is a critical epigenetic mechanism that regulates gene expression. Here, we determined whether transcriptional activation of Agtr1a and Slc12a2 results from altered DNA methylation in spontaneously hypertensive rats (SHR). Methylated DNA immunoprecipitation and bisulfite sequencing-PCR showed that CpG methylation at Agtr1a and Slc12a2 promoters in the PVN was progressively diminished in SHR compared with normotensive Wister-Kyoto rats (WKY). Chromatin immunoprecipitation-qPCR revealed that enrichment of DNA methyltransferases (DNMT1 and DNMT3A) and MeCP2, a DNA methylation reader protein, at Agtr1a and Slc12a2 promoters in the PVN was profoundly reduced in SHR compared with WKY. By contrast, the abundance of ten-eleven translocation enzymes (TET1-3) at Agtr1a and Slc12a2 promoters in the PVN was much greater in SHR than in WKY. Furthermore, microinjecting of RG108, a selective DNMT inhibitor, into the PVN of WKY increased arterial blood pressure and correspondingly potentiated Agtr1a and Slc12a2 mRNA levels in the PVN. Conversely, microinjection of C35, a specific TET inhibitor, into the PVN of SHR markedly reduced arterial blood pressure, accompanied by a decrease in Agtr1a and Slc12a2 mRNA levels in the PVN. Collectively, our findings suggest that DNA hypomethylation resulting from the DNMT/TET switch at gene promoters in the PVN promotes transcription of Agtr1a and Slc12a2 and hypertension development.

Abstract P430: Primary Neuronal Cilium As A Novel Mediator Of Angiotensin II And Hypertension

Cilia are membrane-bound, microtubular projections emanating from the cell surface and present on virtually all cell types including neurons. Cilia abnormalities (ciliopathies) have been linked to a broad range of diseases including hypertension. Moreover, we previously reported that DOCA-salt hypertension is associated with alterations in cilia in select nuclei involved in cardiovascular regulation and fluid homeostasis such as the supraoptic nucleus, but the underlying mechanisms are not known. Given the well-established role of the brain renin-angiotensin system in DOCA-salt hypertension, we hypothesized that Angiotensin II (Ang II) contribute to neuronal cilia abnormalities in hypertension. We began by examining the effect of Ang II on cilia in two different neuronal cell lines, mouse hypothalamic N47 and Cath.a. Ang II treatment (100 nM for 4 hrs) caused a significant increase in cilia length of N47 cells that were transiently transfected with a Flag-tagged AT1a receptor (AT1aR) compared to non-transfected cells (3.83±0.15 μm vs 2.25±0.06 μm, P<0.05). No change in cilia length was observed in AT1aR positive (2.71±0.09 μm) or negative cells (2.55±0.07 μm) treated with vehicle. Moreover, Ang II had no effect on cilia in N47 cells transfected with a Flag-tagged AT2R. Ang II also significantly increased cilia length in Cath.a cells which express the endogenous AT1aR. Truncation of AT1aR at position 318 resulted in loss of Ang II induced cilia elongation in N47 cells, indicating that the C-terminus of the receptor is important for Ang II regulation of cilia. Next, we evaluated the effect of Ang II infusion with minipumps (480ng/kg/min) on neuronal cilia in C57BL/6 mice. Mice infused with Ang II for 5 days which have elevated blood pressure displayed abnormally elongated cilia in the supraoptic nucleus (8.28±0.12 μm vs 6.37± 0.11 μm, P<0.05). Interestingly, Ang II-induced cilia elongation preceded the development of hypertension as it was observed in mice infused with Ang II for 2 days when no change in blood pressure was detected. These findings point to Ang II as an important regulator of neuronal cilia via AT1aR. This represents a novel mechanism for cardiovascular regulation by the renin-angiotensin system.

Manipulation of components of the renin angiotensin system in renal proximal tubules fails to alter atherosclerosis in hypercholesterolemic mice.

Whole body manipulation of the renin-angiotensin system (RAS) consistently exerts profound effects on experimental atherosclerosis development. A deficit in the literature has been a lack of attention to the effects of sex. Also, based on data with gene-deleted mice, the site of RAS activity that influences lesion formation is at an unknown distant location. Since angiotensin (AngII) concentrations are high in kidney and the major components of the RAS are present in renal proximal tubule cells (PTCs), this study evaluated the role of the RAS in PTCs in atherosclerosis development. Mice with an LDL receptor -/- background were fed Western diet to induce hypercholesterolemia and atherosclerosis. We first demonstrated the role of AT1 receptor antagonism on atherosclerosis in both sexes. Losartan, an AngII type 1 (AT1) receptor blocker, had greater blood pressure-lowering effects in females than males, but equivalent effects between sexes in reducing atherosclerotic lesion size. To determine the roles of renal AT1a receptor and angiotensin-converting enzyme (ACE), either component was deleted in PTCs after weaning using a tamoxifen-inducible Cre expressed under the control of an Ndrg1 promoter. Despite profound deletion of AT1a receptor or ACE in PTCs, the absence of either protein did not influence development of atherosclerosis in either sex. Conversely, mice expressing human angiotensinogen and renin in PTCs or expressing human angiotensinogen in liver but human renin in PTCs did not change atherosclerotic lesion size in male mice. Whole-body AT1R inhibition reduced atherosclerosis equivalently in both male and female mice; however, PTC-specific manipulation of the RAS components had no effects on hypercholesterolemia-induced atherosclerosis.

Angiotensin AT1A receptor signal switching in Agouti-related peptide neurons mediates metabolic rate adaptation during obesity

Resting metabolic rate (RMR) adaptation occurs during obesity and is hypothesized to contribute to failed weight management. Angiotensin II (Ang-II) type 1 (AT1A) receptors in Agouti-related peptide (AgRP) neurons contribute to the integrative control of RMR, and deletion of AT1A from AgRP neurons causes RMR adaptation. Extracellular patch-clamp recordings identify distinct cellular responses of individual AgRP neurons from lean mice to Ang-II: no response, inhibition via AT1A and Gαi, or stimulation via Ang-II type 2 (AT2) receptors and Gαq. Following diet-induced obesity, a subset of Ang-II/AT1A-inhibited AgRP neurons undergo a spontaneous G-protein "signal switch," whereby AT1A stop inhibiting the cell via Gαi and instead begin stimulating the cell via Gαq. DREADD-mediated activation of Gαi, but not Gαq, in AT1A-expressing AgRP cells stimulates RMR in lean and obese mice. Thus, loss of AT1A-Gαi coupling within the AT1A-expressing AgRP neuron subtype represents a molecular mechanism contributing to RMR adaptation.

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.

Mice lacking angiotensin AT1A receptors in neurons exhibit a behavioral preference for decreased ambient temperature

The local tissue renin-angiotensin system (RAS) within the brain is implicated in cardiometabolic control, including core temperature and resting metabolic rate (RMR). Previously we demonstrated a critical role for the angiotensin (ANG) type 1A receptor (AT1R), localized to neurons of the hypothalamic arcuate nucleus that express Agouti-related peptide (AgRP), in the control of RMR. Further, we demonstrated that mice deficient for melanocortin type 4 receptors (MC4R) for AgRP resist stimulation of RMR by ANG in the brain. Given the known projections of AgRP neurons and RMR-regulating networks within the forebrain and hypothalamus, we hypothesize that ANG in the brain acts to sensitize the animal to the sensation of cold, rather than to directly stimulate pre-autonomic functions. To test this hypothesis, we examined the consequences of genetically deleting the AT1R gene from all neurons upon ambient temperature preference, as mice with inhibited sensation of cold should prefer a colder temperature, while mice with inhibited stimulation of pre-autonomic areas should prefer a warmer temperature. We constructed a thermal gradient that consisted of four parallel lanes with opaque white polycarbonate walls (58.4 x 7.5 cm, 20.5 cm tall) and an aluminum floor. Temperature-controlled circulating water baths supplied heating/cooling blocks on opposite ends of lanes to generate a linear temperature gradient as assessed using infrared imaging (FLIR A700: gradient range 40-15°C; T=38.40-0.04091✕D, R2=0.99, where D=distance from warmed end of lane, in mm) that was similar between lanes (within 0.87±0.31°C). Mice were placed in the center of the gradient and mouse position in the gradient was analyzed continuously for 2h by video (AnyMaze, Stoelting) during the middle of the light phase. Mice with AT1R deleted from neurons ( Syn1-Cre+/- x Agtr1a F/F, n=3m+4f) and littermate controls ( Agtr1a F/F, n=3m) on the C57BL/6 background exhibited similar decaying locomotor activity for the first 90 min before reaching a nadir in activity between 90-120 min (23±13 cm/min, p=ns between groups). Between 90-120 min, the knockout mice localized to a significantly lower ambient temperature than controls (28.1±1.0 vs 31.9±0.3°C, p=0.04). Validating our approach, others have previously reported 32-33°C as the preferred light-phase ambient temperature for adult mice. Together, these findings demonstrate our fabrication and validation of a thermal gradient system for mice and support the working hypothesis that the brain RAS functions to modify the integration of cold stimuli, ultimately to influence energy homeostasis. HL134850, HL084207, DK133121, UL1TR001436, 18EIA33890055 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

AT1a receptor knockout selectively in renomedullary interstitial cells of the kidney lowers basal blood pressure, impairs urine concentration, and attenuates angiotensin II-induced hypertension in RMIC-Agtr1a-/- mice

We previously reported that global deletion of angiotensin II (Ang II) AT1a receptors significantly lowered basal blood pressure and impaired urine concentration in mice. As we also demonstrated that renomedullary interstitial cells (RMICs) of the kidney expressed a high level of AT1 (AT1a) receptors, we hypothesized that AT1a receptors in RMICs may play an important physiological role in maintaining basal blood pressure and urine concentration homeostasis. To test the hypothesis, we generated a novel mouse model with deletion of AT1a receptors selectively in the RMICs of the kidney medulla using inducible Tenascin-C-CreER2/Agtr1af/f recombination approach (RMIC- Agtr1a-/- ) in the present study. Adult male wildtype (WT) and RMIC- Agtr1a-/- mice (n=10-18 per group) were phenotyped under basal conditions and treated with or without a slow pressor dose of Ang II infusion via an osmotic minipump (~500 μg/kg/day, i.p.) for 2 weeks to determine the roles of AT1a receptors in RMICs in maintaining basal blood pressure and urine concentration homeostasis and in the development of Ang II-induced hypertension. Under basal conditions, telemetry systolic blood pressure was significantly lower in adult male RMIC- Agtr1a-/- than WT mice (WT: 122 ± 4 mmHg vs. RMIC-Agtr1a-/-: 102 ± 3 mmHg, P<0.01). 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 even though 24 h urine output was lowered (WT: 1724 ± 335 mOsm/Kg H2O vs. RMIC- Agtr1a-/- : 1128 ± 102 mOsm/Kg H2O, P<0.01). In response to Ang II infusion, telemetry systolic blood pressure increased to 148 ± 5 mmHg in WT mice, but it increased only to 129 ± 8 mmHg in RMIC- Agtr1a-/- mice ( P<0.01). Furthermore, Ang II infusion for 2 weeks failed to alter urine osmolality in RMIC- Agtr1a-/- mice (1226 ± 110 mOsm/Kg H2O, n.s.). In conclusion, we demonstrated for the 1st time that AT1a receptors in RMICs of the kidney medulla play a key role in physiologically maintaining basal blood pressure homeostasis and urine concentration, as well as in the development of Ang II-induced hypertension. R01DK123144, R01DK067299, R01DK102429 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sepsis modulates aortic AT1 and P2Y6 receptors to produce vascular hyporeactivity in mice

Purpose Hyporeactivity to vasopressors leading to multiple organ failure is a serious clinical implication in sepsis. Though the regulatory role of purinoceptors in inflammation is reported, their involvement in sepsis-induced vasoplegia is still unknown. Thus we investigated the effect of sepsis on vascular AT1 and P2Y6 receptors. Materials and methods Polymicrobial sepsis was induced by cecal ligation and puncture in mice. Vascular reactivity was assessed by organ bath study and aortic mRNA expression of AT1 and P2Y6 was quantified by qRT-PCR. Results Both angiotensin-II and UDP produced higher contractions in the absence of endothelium as well as following inhibition of nitric oxide synthase. Angiotensin-II mediated aortic contraction was antagonized by losartan (AT1 antagonist), but not by PD123319 (AT2 antagonist) whereas UDP-induced aortic contraction was significantly inhibited by MRS2578 (P2Y6 antagonist). In addition, MRS2578 significantly inhibited the contractile response of Ang-II. Compared to SO mice, angiotensin-II and UDP-induced maximum contraction were found to be significantly attenuated in sepsis. Accordingly, aortic mRNA expression of AT1a receptors was significantly down-regulated while that of P2Y6 receptors was significantly increased in sepsis. 1400 W (a selective iNOS inhibitor) significantly reversed angiotensin-II-induced vascular hyporeactivity in sepsis without affecting UDP-induced hypo-reactivity. Conclusion Sepsis-induced vascular hyporeactivity to angiotensin-II is mediated by enhanced expression of iNOS. Moreover, AT1R-P2Y6 cross talk/heterodimerization could be a novel target for regulating vascular dysfunction in sepsis.

S-27-5: DELETION OF AT1A RECEPTORS IN THE PROXIMAL TUBULES AUGMENTS NATRIURETIC RESPONSE TO ATRIAL NATRIURETIC PEPTIDE VIA NPR/CGMP/NO SIGNALING

Objective: The proximal tubules of the kidney play a critical role in maintaining normal body salt and fluid balance and blood pressure homeostasis in part by interactions between two key vasoactive hormone receptors. In the proximal tubules, angiotensin II (Ang II) binds and activate AT1a receptors to stimulate proximal tubule Na+ reabsorption, whereas atrial natriuretic peptide (ANP) binds and activates NPRA receptors to inhibit Ang II-induced proximal tubule Na+ reabsorption. These two vasoactive systems play important counteracting roles in the proximal tubules to control proximal tubule Na+ reabsorption in the kidney and maintain blood pressure homeostasis. However, it is not known whether the hypotensive and natriuretic responses to ANP may be augmented by deletion of AT1 (AT1a) receptors selectively in the proximal tubules of the kidney. Design and Methods: 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 deletion of AT1a receptors selectively in the proximal tubules indeed augments the hypotensive and natriuretic responses to ANP. Four groups (n = 9–12 per group) of adult male wild-type and PT-Agtr1a -/- mice were infused without (served as time controls) or with ANP via osmotic minipump (10 ng/min, i.p.) continuously for 2 weeks. Results: Basal blood pressure was about 16 ± 3 mmHg lower (P < 0.01), fractional proximal tubule Na+ reabsorption was significantly lower (P < 0.05), whereas 24 h urinary Na+ excretion was significantly higher in PT-Agtr1a -/- mice (P < 0.01), compared with age-matched wild-type mice. ANP infusion significantly decreased blood pressure and increased the natriuretic response in PT-Agtr1a -/- mice further by inhibiting proximal tubule Na+ reabsorption, compared with wild-type controls (P < 0.01). The augmented hypotensive and natriuretic responses to ANP infusion in PT-Agtr1a -/- mice was associated with increased plasma and kidney cGMP levels (P < 0.01), kidney cortical NPRA and NPRC mRNA expression (P < 0.05), total endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS proteins (P < 0.01), and urinary nitric oxide (NO) excretion (P < 0.01), respectively. Conclusions: Taken together, the results of the present study provide further new insights into the important physiological roles for intratubular Ang II/AT1a and ANP/NPRA in the proximal tubules to regulate proximal tubule Na+ reabsorption and help maintain blood pressure homeostasis. Supported by 2R01DK067299-10A1, 2R01DK102429-03A1, and 1 R01DK123144-01.

PS-BPB08-1: ANGIOTENSIN II AT1A RECEPTORS IN THE PROXIMAL TUBULES PLAY A CRITICAL ROLE IN THE DEVELOPMENT OF TWO-KIDNEY, ONE-CLIP GOLDBLATT HYPERTENSION

Objective: It is well recognized that the activation of the renin-angiotensin system (RAS) in the clipped kidney plays a critical role in the development of two-kidney, one-clip Goldblatt hypertension (2K1C) in animal models, but the roles of intratubular angiotensin II (Ang II) AT1 (AT1a) receptors and its downstream target, Na+/H+ exchanger 3 (NHE3) in the proximal tubules have not been determined previously. In the present study, we tested the hypothesis that genetic deletion of AT1a receptors selectively in the proximal tubules of the kidney attenuates the development of 2K1C Goldblatt hypertension via AT1a receptor-mediated, Na+/H+ exchanger 3 (NHE3)-dependent mechanisms. Design and Methods: To test the hypothesis, 2K1C Goldblatt hypertension was induced by placing a silver clip, 0.12 mm internal diameter, on the left renal artery for 4 weeks in adult male and female wild-type (WT), global AT1areceptor knockout (Agtr1a-/- ), proximal tubule (PT)-specific Agtr1a-/- (PT-Agtr1a-/- ), or PT-Nhe3-/- mice, respectively. Results: Systolic blood pressure increased in a time-dependent manner reaching a maximal response by Week 2–3 in wild-type mice (Basal: 112 ± 2 vs. 2K1C: 149 ± 4 mmHg, n = 12, P < 0.01). 2K1C Goldblatt hypertension in WT mice was associated with significant increases in renin mRNA expression in the clipped kidney (Control: 2066 ± 255 vs. Clipped: 3144 ± 569 copies/ng RNA, P < 0.01), and decreases in renin mRNA expression in the nonclipped right kidney (1738 ± 341 copies/ng RNA, P < 0.05). Plasma Ang II levels were significantly increased in WT mice with 2K1C Goldblatt hypertension (Control: 50.2 ± 7.2 vs. 2K1C: 109.7 ± 17.2 pg/ml, P < 0.05). Glomerular and tubulointerstitial fibrotic responses were also significantly increased in the clipped kidney (P < 0.01). In contrast to WT mice, the development of 2K1C hypertension was completely attenuated in global Agtr1a-/- (Basal: 88 ± 4 vs. 2K1C: 92 ± 2 mmHg, n = 9, n.s.), PT-Agtr1a-/- mice (Basal: 101 ± 2 vs. 2K1C: 104 ± 4 mmHg, n = 12, n.s.), or PT-Nhe3-/- mice (Basal: 103 ± 3 vs. 109 ± 5 mmHg, n.s.). Renin mRNA expression was not different in clipped and nonclipped kidney of Agtr1a-/- mice, but it was decreased in the nonclipped kidney of PT-Agtr1a-/- mice (P < 0.05). Conclusions: Taken together, our results suggest that the activation of intratubular RAS in the proximal tubules and increase of NHE3-mediated proximal tubular Na+ reabsorption play a key role in the development of 2K1C Goldblatt hypertension in mice.

Genetic Deletion of AT1a Receptor or Na+/H+ Exchanger 3 Selectively in the Proximal Tubules of the Kidney Attenuates Two-Kidney, One-Clip Goldblatt Hypertension in Mice.

The roles of angiotensin II (Ang II) AT1 (AT1a) receptors and its downstream target Na+/H+ exchanger 3 (NHE3) in the proximal tubules in the development of two-kidney, 1-clip (2K1C) Goldblatt hypertension have not been investigated previously. The present study tested the hypothesis that deletion of the AT1a receptor or NHE3 selectively in the proximal tubules of the kidney attenuates the development of 2K1C hypertension using novel mouse models with proximal tubule-specific deletion of AT1a receptors or NHE3. 2K1C Goldblatt hypertension was induced by placing a silver clip (0.12 mm) on the left renal artery for 4 weeks in adult male wild-type (WT), global Agtr1a−/−, proximal tubule (PT)-specific PT-Agtr1a−/− or PT-Nhe3−/− mice, respectively. As expected, telemetry blood pressure increased in a time-dependent manner in WT mice, reaching a maximal response by Week 3 (p < 0.01). 2K1C hypertension in WT mice was associated with increases in renin expression in the clipped kidney and decreases in the nonclipped kidney (p < 0.05). Plasma and kidney Ang II were significantly increased in WT mice with 2K1C hypertension (p < 0.05). Tubulointerstitial fibrotic responses were significantly increased in the clipped kidney (p < 0.01). Whole-body deletion of AT1a receptors completely blocked the development of 2K1C hypertension in Agtr1a−/− mice (p < 0.01 vs. WT). Likewise, proximal tubule-specific deletion of Agtr1a in PT-Agtr1a−/− mice or NHE3 in PT-Nhe3−/− mice also blocked the development of 2K1C hypertension (p < 0.01 vs. WT). Taken together, the present study provides new evidence for a critical role of proximal tubule Ang II/AT1 (AT1a)/NHE3 axis in the development of 2K1C Goldblatt hypertension.

Deletion of AT1a Receptors Selectively in the Proximal Tubules of the Kidney Augments Glomerular Filtration in Male and Female PT-Agtr1a-/- Mice

Deletion of AT1a Receptors Selectively in the Proximal Tubules of the Kidney Augments Glomerular Filtration in Male and Female PT-Agtr1a-/- Mice

Abstract 114: Metabolic Adaptation During Obesity Is Associated With G Protein Switching By The Angiotensin At1a Receptor In Agrp Neurons

Obesity represents the single greatest impediment to continued improvements in hypertension and cardiovascular disease. Prolonged obesity is associated with “Resting Metabolic Rate (RMR) adaptation” (ie, reduced RMR relative to body composition), and RMR adaptation is proposed to explain resistance to weight loss / weight maintenance after obesity is established. Previously we showed that angiotensin II (ANG) Agtr1a (AT1A) receptors expressed in Agouti-related peptide (AgRP) neurons of the hypothalamic arcuate nucleus (ARC) are required for integrative control of RMR. We hypothesize that changes in AT1A signaling in AgRP cells underlie the development of RMR adaptation. ARC slices from mice expressing tdTomato in AgRP cells ( Agrp -Cre x Ai9) were used for cell-attached voltage clamp recording of AgRP neurons. ANG inhibited ~33% of AgRP neurons via an AT1A-dependent mechanism (deemed a “Type 1i” response, which was inhibitable by losartan (LOS) or genetic deletion of AT1A; Firing rate; aCSF:0.85 ± 0.03 vs ANG:0.20 ± 0.04 vs ANG+LOS:0.91 ± 0.11 Hz, p&lt;0.05) that was also blocked by the Gαi inhibitor, pertussis toxin (PTX), but not by the Gαq inhibitor, BIM-46187 (Δ Firing rate: PTX: -0.39 ± 0.09 Hz, p&lt;0.05 vs BIM: 0.10 ± 0.09 Hz). After 10 weeks of high-fat feeding, roughly half of Type 1 cells exhibited a spontaneous switch from the ANG/AT1A-mediated inhibitory (Type 1i) response to an ANG/AT1A-mediated stimulatory response (“Type 1s”; Firing rate; aCSF: 0.86 ± 0.03 vs ANG:1.10 ± 0.12 vs ANG+LOS:0.82 ± 0.03 Hz, p&lt;0.05), which was blocked by BIM-46187, but not by PTX (Δ Firing rate: PTX: 0.07 ± 0.06 Hz vs BIM: 0.49 ± 0.06 Hz, p&lt;0.05). AAV delivery of Cre-inducible DREADDs into the ARC of lean Agtr1a -Cre mice was then used to study the effects of Gαi vs Gαq signaling in Type 1 AgRP cells upon RMR control. Activation of the Gαi-coupled hM4Di DREADD in AT1A-expressing cells of the ARC was sufficient to increase RMR (+25% from baseline, n=8-10, p&lt;0.05), but activation of a Gαq-coupled DREADD had no effect. We conclude (i) that the AT1A receptor is expressed by a unique subtype of AgRP neuron; (ii) that inhibitory AT1A-Gαi signaling in the Type 1 AgRP cell stimulates RMR; and (iii) that loss of AT1A-Gαi coupling in these cells is sufficient to explain obesity-associated RMR adaptation.

Abstract P250: Targeting The Thrombospondin-CD47 Axis Attenuates Blood Pressure And Renal Injury In Female Hypertensive (mRen2)27 Rats

Alterations in the extracellular matrix (ECM) can influence vascular stiffness and compromise organ function contributing to hypertension. A key component of the ECM is the type I integral membrane receptor CD47 that regulates the ECM by binding to the matricellular protein thrombospondin-1(TSP1). Increased circulating levels of TSP1 and CD47 activation are associated with cardiac and renal pathologies. Thus, the objective of the current study determined the efficacy of targeting CD47 with an anti-sense morpholinos to reduce blood pressure and renal injury in 20-week old hypertensive (mRen2)27 female rats. The CD47 morpholino (Morph) or the missense probe (Control) were dissolved in sterile PBS and administered by tail vein injection twice weekly for 6 weeks. Systolic blood pressures (SBPs) were monitored in conscious rats by a tail cuff auscultation and expression of RAS components assessed by RT-qPCR; all data are means ± SEM with n=8-10 per group. Prior to treatment, the baseline SBPs did not differ between the Control and Morph groups [196 ± 5 vs.197 ± 4 mmHg; p=0.87]. However, as early as one week of treatment, the CD47 Morph group exhibited a significant reduction in SBPs compared to the Control transgenics [185 ± 5 vs. 204 ± 3 mmHg; p&lt;0.01]. At the end of the 6 week treatment period, SBPs remained significantly lower in the CD47 Morph group compared to the Controls [185 ± 4 vs.199 ± 3 mmHg; p&lt;0.02]. In addition, CD47 blockade was associated with reduced proteinuria in the CD47 Morph compared to Controls [7 ± 1 vs. 26 ± 7 mg protein/24 hr excretion; p&lt;0.05]. Regarding the mechanism of CD47 blockade to reduce blood pressure and proteinuria, mRNA analysis revealed lower renal renin mRNA levels in the CD47 Morph versus the Control group [0.54 ± 0.11 vs.1.04 ± 0.11; p&lt;0.01]. Moreover, CD47 Morph treatment significantly reduced the mRNA levels of renal ACE [0.78 ± 0.13 vs.1.05 ± 0.14; p&lt;0.05] and the renal AT1A receptor [0.74 ± 0.07 vs.1.05 ± 0.12; p&lt;0.05], however, renal angiotensinogen mRNA levels were not different among the CD47 Morph and Controls [1.04.± 0.10 vs. 1.16 ± 0.20, p=0.83]. We conclude that silencing CD47 attenuates hypertension and renal injury in the female (mRen2)27 potentially by reducing expression of the renin-ACE-AT1 receptor axis within the kidney.

Abstract P123: Angiotensin Ii Type 1a Receptor Expressed Insmooth Muscle Cells Is Required Forangiotensin Ii-induced Vascular Remodeling But Not For Cardiac Hypertrophy

Angiotensin II Type 1a Receptor (AT1a) has been shown to play a critical role in cardiovascular diseases. We have demonstrated that ADAM17 mediates cardiovascular remodeling induced by angiotensin II (AngII) by using an SM22α Cre reporter, which expresses in smooth muscle cells (SMC) and cardiomyocytes. It is conceivable that AT1 signaling in SMC specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. Deficiency of SMC AT1a receptors via endogenous SM22α promoter knock-in (KISMKO) results in diminished hypertension and cardiac hypertrophy induced by AngII. However, we have limited understanding whether SMC AT1a receptors affect vascular fibrosis and cardiac hypertrophy. Thus, this study was designed to elucidate the roles of the SMC AT1a receptor in cardiovascular remodeling using two distinct SMC AT1a receptor deficient mice. One line is the KISMKO, and another line is Tamoxifen inducible SMMHC-Cre AT1aflox/flox mouse (SMMHCKO) silencing in SMC but not cardiac myocytes. 8 to 12-week-old male mice were infused with AngII (1 μg/kg/min) for 2 weeks. Cardiac hypertrophy was assessed by heart-to-body weight ratios and found to be reduced in KISMKO mice compared with control mice (6.04 mg/g versus 4.89 mg/g, p=0.032), whereas ratios were not altered in SMMHCKO mice (5.31 mg/g versus 5.47 mg/g, p=0.941). Histological assessment demonstrated that perivascular fibrosis in coronary arteries was increased in control mice and attenuated in both KISMKO and SMMHCKO mice (1.27 vs. 0.22, p&lt;0.0001 and 0.70 vs. 0.16, p&lt;0.0001, respectively.). Medial thickness of descending aorta was increased in control mice and these phenotypic changes were attenuated in both KISMKO and SMMHCKO mice (97 vs. 56 μm, p&lt;0.0001 and 98 vs. 74 μm, p&lt;0.0001, respectively.). In conclusion, SMC AT1a receptors could mediate AngII-induced vascular remodeling including perivascular fibrosis but not cardiac hypertrophy. Cardiac myocyte AT1a appears critical for cardiac hypertrophy. Our data together with a past manuscript demonstrate a protective effect of SMC AT1a silencing in overall vascular remodeling. However, the exact relationship between hypertension and vascular remodeling remains to be elucidated.

Distinct Contribution of Global and Regional Angiotensin II Type 1a Receptor Inactivation to Amelioration of Aortopathy in Tgfbr1 M318R/+ Mice.

Angiotensin II (Ang II) type 1 receptor (AT1R) signaling controls both physiological and pathogenetic responses in the vasculature. In mouse models of Loeys-Dietz syndrome (LDS), a hereditary disorder characterized by aggressive aortic aneurysms, treatment with angiotensin receptor blockers (ARBs) prevents aortic root dilation and associated histological alterations. In this study we use germline and conditional genetic inactivation of Agtr1a (coding for the AT1a receptor) to assess the effect of systemic and localized AT1R signaling attenuation on aortic disease in a mouse model of LDS (Tgfbr1M318R/+). Aortic diameters and histological features were examined in control and Tgfbr1M318R/+ mice with either germline or Mef2CSHF-Cre mediated genetic inactivation of Agtr1a, the latter resulting in deletion in second heart field (SHF)-derived lineages in the aortic root and proximal aorta. Both systemic and regional AT1R signaling attenuation resulted in reduction of diameters and improvement of tissue morphology in the aortic root of LDS mice; these outcomes were associated with reduced levels of Smad2/3 and ERK phosphorylation, signaling events previously linked to aortic disease in LDS. However, regional AT1a inactivation in SHF-derived lineages resulted in a more modest reduction in aortic diameters relative to the more complete effect of germline Agtr1a deletion, which was also associated with lower blood pressure. Our findings suggest that the therapeutic effects of AT1R antagonisms in preclinical models of aortic disease depend on both regional and systemic factors and suggest that combinatorial approaches targeting both processes may prove beneficial for aneurysm mitigation.

Enalapril Diminishes the Diabetes-Induced Changes in Intestinal Morphology, Intestinal RAS and Blood SCFA Concentration in Rats.

Evidence suggests that microbiota-derived metabolites, including short-chain fatty acids (SCFAs) and trimethylamine-oxide (TMAO), affect the course of diabetic multiorgan pathology. We hypothesized that diabetes activates the intestinal renin–angiotensin system (RAS), contributing to gut pathology. Twelve-week-old male rats were divided into three groups: controls, diabetic (streptozotocin-induced) and diabetic treated with enalapril. Histological examination and RT-qPCR were performed to evaluate morphology and RAS expression in the jejunum and the colon. SCFA and TMAO concentrations in stools, portal and systemic blood were evaluated. In comparison to the controls, the diabetic rats showed hyperplastic changes in jejunal and colonic mucosa, increased plasma SCFA, and slightly increased plasma TMAO. The size of the changes was smaller in enalapril-treated rats. Diabetic rats had a lower expression of Mas receptor (MasR) and angiotensinogen in the jejunum whereas, in the colon, the expression of MasR and renin was greater in diabetic rats. Enalapril-treated rats had a lower expression of MasR in the colon. The expression of AT1a, AT1b, and AT2 receptors was similar between groups. In conclusion, diabetes produces morphological changes in the intestines, increases plasma SCFA, and alters the expression of renin and MasR. These alterations were reduced in enalapril-treated rats. Future studies need to evaluate the clinical significance of intestinal pathology in diabetes.

Smooth Muscle At1a Receptor Mediates Perivascular Fibrosis in Angiotensin II‐infused Mice

Angiotensin II (Ang II) signaling via AT1 receptor has been shown to play a critical role in the pathogenesis of hypertension, cardiovascular hypertrophy and fibrosis. We have demonstrated that ADAM17 expressed in vascular smooth muscle cells (VSMC) mediates EGF receptor activation and promotes cardiac hypertrophy and perivascular fibrosis induced by Ang II. It is conceivable that Ang II signaling in VSMCs specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. In a recent study, deficiency of smooth muscle AT1a receptors results in diminished hypertension and protection from cardiac hypertrophy induced by Ang II. However, we have limited understanding whether smooth muscle AT1a receptors affects hypertensive fibrosis in vasculature. Thus, this study was designed to elucidate the roles of the AT1a receptor in VSMCs in cardiovascular remodeling including fibrosis during Ang II stimulation using VSMC AT1a receptor deficient mice. To delete the AT1a receptor from VSMCs, we crossed C57BL/6 transgenic mouse lines expressing Cre recombinase under the control of the endogenous sm22α promoter (Kock‐In, KIsm22α‐Cre). Male AT1a flox/flox KIsm22α‐Cre+/‐ (SMKO) and control (AT1a flox/flox KIsm22α‐Cre‐/‐) mice were infused with Ang II (1 µg/kg/min) for 2 weeks via osmotic mini‐pump. In control mice, Ang II infusion for 2 weeks induced cardiac hypertrophy indicated by heart‐to‐body weight ratio and echocardiogram. After 2 weeks of Ang II infusion, heart‐to‐body weight ratios were significantly increased in control mice compared with AT1a SMKO mice (6.04 versus 4.89, respectively, p=0.032). Cardiac wall hypertrophy was seen in control mice after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKO mice. Control mice (n=5) showed vascular medial hypertrophy and perivascular fibrosis in coronary arteries, whereas these phenotypic changes were attenuated in SMKO mice (96.8 µm vs. 56.4 µm, respectively, p&lt;0.0001). In conclusion, AT1a receptors expressed in VSMC could mediate Ang II‐induced cardiovascular hypertrophy and perivascular fibrosis. Whether the data can be fully explained by the prevention of hypertension remains to be determined. Our data also contrast with the past manuscript showing a protective effect in AT1a flox/flox S100A4‐Cre+/‐ (fibroblast silencing) but not in AT1a flox/flox sm22α‐Cre+/‐ mice with Ang II infusion.

Abstract 374: Smooth Muscle Cell-specific Deletion Of At1a Receptor Attenuates Vascular Fibrosis In Angiotensin Ii-infused Mice

Angiotensin II (Ang II) AT1 receptor has been shown to play a critical role in several cardiovascular diseases. We have demonstrated that ADAM17 expressed in vascular smooth muscle cells (VSMC) mediates cardiac hypertrophy and perivascular fibrosis induced by Ang II. It is conceivable that Ang II signaling in VSMCs specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. Deficiency of smooth muscle AT1a receptors results in diminished hypertension and protection from cardiac hypertrophy induced by Ang II. However, we have limited understanding whether smooth muscle AT1a receptors affect hypertensive fibrosis. Thus, this study was designed to elucidate the roles of the AT1a receptor in VSMCs in cardiovascular remodeling including fibrosis using VSMC AT1a receptor deficient mice. To delete the AT1a receptor from VSMCs, we crossed C57BL/6 transgenic mouse lines expressing Cre recombinase under the control of the endogenous sm22α promoter (Kock-In, KIsm22α-Cre). Male AT1a flox/flox KIsm22α-Cre+/- (SMKO) and control (AT1a flox/flox Cre-/-) mice were infused with Ang II (1 μg/kg/min) for 2 weeks. In control mice, Ang II infusion induced cardiac hypertrophy indicated by heart-to-body weight ratio and echocardiogram. After 2 weeks of Ang II infusion, heart-to-body weight ratios were significantly decreased in AT1a SMKO mice compared with control mice (6.04 versus 4.89, p=0.032). Cardiac hypertrophy was seen in control mice after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKO mice. Control mice showed vascular medial hypertrophy and perivascular fibrosis in coronary arteries, whereas these phenotypic changes were attenuated in SMKO mice (96.8 μm vs. 56.4 μm, p&lt;0.0001). Control mice with Ang II infusion showed increased area of perivascular collagen III immunostaining in aorta, which was attenuated in AT1a SMKO mice. In conclusion, AT1a receptors expressed in VSMC could mediate Ang II-induced perivascular fibrosis. Our data together with the past manuscript showing a protective effect of fibroblast AT1a silencing suggest the importance of both VSMC and fibroblast AT1a in fibrosis. However, whether our data can be fully explained by the prevention of hypertension remains to be determined.