Ang II-dependent Hypertension 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.

Impaired renal autoregulation and pressure-natriuresis: any role in the development of heart failure in normotensive and angiotensin II-dependent hypertensive rats?

The aim of the present study was to assess the autoregulatory capacity of renal blood flow (RBF) and of the pressure-natriuresis characteristics in the early phase of heart failure (HF) in rats, normotensive and with angiotensin II (ANG II)-dependent hypertension. Ren-2 transgenic rats (TGR) were employed as a model of ANG II-dependent hypertension. HF was induced by creating the aorto-caval fistula (ACF). One week after ACF creation or sham-operation, the animals were prepared for studies evaluating in vivo RBF autoregulatory capacity and the pressure-natriuresis characteristics after stepwise changes in renal arterial pressure (RAP) induced by aortic clamping. In ACF TGR the basal mean arterial pressure, RBF, urine flow (UF), and absolute sodium excretion (UNaV) were all significantly lower tha n in sham-operated TGR. In the latter, reductions in renal arterial pressure (RAP) significantly decreased RBF whereas in ACF TGR they did not change. Stepwise reductions in RAP resulted in marked decreases in UF and UNaV in sham-operated as well as in ACF TGR, however, these decreases were significantly greater in the former. Our data show that compared with sham-operated TGR, ACF TGR displayed well-maintained RBF autoregulatory capacity and improved slope of the pressure-natriuresis relationship. Thus, even though in the very early HF stage renal dysfunction was demonstrable, in the HF model of ANG II-dependent hypertensive rat such dysfunction and the subsequent HF decompensation cannot be simply ascribed to impaired renal autoregulation and pressure-natriuresis relationship.

Interaction of Angiotensin II AT1 Receptors with Purinergic P2X Receptors in Regulating Renal Afferent Arterioles in Angiotensin II-Dependent Hypertension.

In angiotensin II (Ang II)-dependent hypertension, Ang II activates angiotensin II type 1 receptors (AT1R) on renal vascular smooth muscle cells, leading to renal vasoconstriction with eventual glomerular and tubular injury and interstitial inflammation. While afferent arteriolar vasoconstriction is initiated by the increased intrarenal levels of Ang II activating AT1R, the progressive increases in arterial pressure stimulate the paracrine secretion of adenosine triphosphate (ATP), leading to the purinergic P2X receptor (P2XR)-mediated constriction of afferent arterioles. Thus, the afferent arteriolar tone is maintained by two powerful systems eliciting the co-existing activation of P2XR and AT1R. This raises the conundrum of how the AT1R and P2XR can both be responsible for most of the increased renal afferent vascular resistance existing in angiotensin-dependent hypertension. Its resolution implies that AT1R and P2XR share common receptor or post receptor signaling mechanisms which converge to maintain renal vasoconstriction in Ang II-dependent hypertension. In this review, we briefly discuss (1) the regulation of renal afferent arterioles in Ang II-dependent hypertension, (2) the interaction of AT1R and P2XR activation in regulating renal afferent arterioles in a setting of hypertension, (3) mechanisms regulating ATP release and effect of angiotensin II on ATP release, and (4) the possible intracellular pathways involved in AT1R and P2XR interactions. Emerging evidence supports the hypothesis that P2X1R, P2X7R, and AT1R actions converge at receptor or post-receptor signaling pathways but that P2XR exerts a dominant influence abrogating the actions of AT1R on renal afferent arterioles in Ang II-dependent hypertension. This finding raises clinical implications for the design of therapeutic interventions that will prevent the impairment of kidney function and subsequent tissue injury.

Cardioprotective Effects of Sodium Glucose Cotransporter 2 Inhibition in Angiotensin II-Dependent Hypertension Are Mediated by the Local Reduction of Sympathetic Activity and Inflammation.

The cardioprotective effects of sodium glucose cotrasponter 2 (SGLT2) inhibitors seem to be independent from the effects on glycemic control, through little-known mechanisms. In this study, we investigate whether the cardioprotective effects of empagliflozin, a SGLT2 inhibitor, may be associated with myocardial sympathetic activity and inflammatory cell infiltration in an experimental model of angiotensin II-dependent hypertension. Angiotensin II (Ang II), Ang II plus Empagliflozin, physiological saline, or physiological saline plus empagliflozin were administered to Sprague Dawley rats for two weeks. Blood pressure was measured by plethysmographic method. Myocardial hypertrophy and fibrosis were analysed by histomorphometry, and inflammatory cell infiltration and tyrosine hydroxylase expression, implemented as a marker of sympathetic activity, were evaluated by immunohistochemistry. Ang II increased blood pressure, myocardial hypertrophy, fibrosis, inflammatory infiltrates and tyrosine hydroxylase expression, as compared to the control group. Empagliflozin administration prevented the development of myocardial hypertrophy, fibrosis, inflammatory infiltrates and tyrosine hydroxylase overexpression in Ang II-treated rats, without affecting blood glucose and the Ang II-dependent increase in blood pressure. These data demonstrate that the cardioprotective effects of SGLT2 inhibition in Ang II-dependent hypertension may result from the myocardial reduction of sympathetic activity and inflammation and are independent of the modulation of blood pressure and blood glucose levels.

Inappropriate activation of the renin-angiotensin system improves cardiac tolerance to ischemia/reperfusion injury in rats with late angiotensin II-dependent hypertension.

The aim of the study was to clarify the role of the interplay between hypertension and the renin-angiotensin system (RAS) in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury. We hypothesized that in the late phase of hypertension with already developed signs of end-organ damage, inappropriate RAS activation could impair cardiac tolerance to I/R injury. Experiments were performed in male Cyp1a1-Ren-2 transgenic rats with inducible hypertension. The early phase of ANG II-dependent hypertension was induced by 5days and the late phase by the 13days dietary indole-3-carbinol (I3C) administration. Noninduced rats served as controls. Echocardiography and pressure-volume analysis were performed, angiotensins' levels were measured and cardiac tolerance to ischemia/reperfusion injury was studied. The infarct size was significantly reduced (by 50%) in 13days I3C-induced hypertensive rats with marked cardiac hypertrophy, this reduction was abolished by losartan treatment. In the late phase of hypertension there are indications of a failing heart, mainly in reduced preload recruitable stroke work (PRSW), but only nonsignificant trends in worsening of some other parameters, showing that the myocardium is in a compensated phase. The influence of the RAS depends on the balance between the vasoconstrictive and the opposed vasodilatory axis. In the initial stage of hypertension, the vasodilatory axis of the RAS prevails, and with the development of hypertension the vasoconstrictive axis of the RAS becomes stronger. We observed a clear effect of AT1 receptor blockade on maximum pressure in left ventricle, cardiac hypertrophy and ANG II levels. In conclusion, we confirmed improved cardiac tolerance to I/R injury in hypertensive hypertrophied rats and showed that, in the late phase of hypertension, the myocardium is in a compensated phase.

PS-B01-11: GENERATION OF TRANSGENIC MICE WITH PROXIMAL TUBULE-SPECIFIC OVEREXPRESSION OF ATRAP AND ANALYSIS OF PROXIMAL TUBULE ATRAP IN BLOOD PRESSURE REGULATION

Objective: Proximal tubule angiotensin II type 1 receptor (AT1R) is involved in blood pressure (BP) regulation (Li XC, et al. Hypertension, 77:1285-1298, 2021). AT1 receptor-associated protein (ATRAP) interacts with AT1R and promotes constitutive internalization of AT1R, to inhibit pathological activation of its downstream signaling (Tamura K, et al. Hypertens Res, 45:32–39, 2022). Endogenous ATRAP is abundantly expressed along kidney tubules in the kidney, and our previous investigations using systemic ATRAP knockout and distal tubule-dominant ATRAP transgenic mice revealed that ATRAP suppressed Ang II-induced hypertension mainly through a distal tubule-mediated mechanism (Wakui H, et al. Hypertension, 61:1203–10, 2013; Ohsawa M, et al. Kidney Int, 86:570–81, 2014). On the other hand, proximal tubule-specific ATRAP knockout mice exhibited similar baseline BP and pressor response to Ang II compared with wild-type mice (Kinguchi S, et al. J Am Heart Assoc, 8(8):e012395, 2019). The present study was performed to investigate the effects of enhancement of proximal tubule ATRAP on Ang II-mediated hypertension, using proximal tubule-specific ATRAP transgenic mice. Design and method: [Experiment 1] Using mouse ATRAP cDNA linked to the type II sodium-dependent phosphate cotransporters (NPT2) promoter, we generated transgenic mice (NPT2-Tg mice) with proximal tubule-specific overexpression of ATRAP. ATRAP mRNA expression in the proximal tubule was quantified by a laser capture microdissection and quantitative reverse transcription-polymerase chain reaction analysis. [Experiment 2] Male NPT2-Tg and wild-type littermate control (LC) mice were divided into Ang II (1000 ng/kg/min) and vehicle groups. BP was measured using a tail-cuff method before and 14 days after treatment, and then mice were sacrificed and their organs were collected. Results: [Experiment 1] 5 of 12 lines of ATRAP Tg mice exhibited kidney overexpression of the ATRAP-transgene in comparison with LC mice. Kidney ATRAP expression at the protein level revealed the highest expression level of ATRAP in lines 23, approximately 10-fold higher compared to LC mice. Therefore, we chose line 23 (NPT2-Tg23) for further analysis. ATRAP mRNA expression was increased approximately 9-fold in the proximal tubules of NPT2-Tg23 mice compared with LC mice. On the other hand, ATRAP mRNA expression in the distal tubules was comparable between NPT2-Tg23 and LC mice. [Experiment 2] We are currently conducting the experiments and will present our findings at this conference. Conclusions: We succeeded in generating transgenic mice with proximal tubule-specific overexpression of ATRAP. We plan to present the results of the impact of enhancement of proximal tubular ATRAP on Ang II-dependent hypertension at this conference.

Immunosuppression by Mycophenolate Mofetil Mitigates Intrarenal Angiotensinogen Augmentation in Angiotensin II-Dependent Hypertension

Augmentation of intrarenal angiotensinogen (AGT) leads to further formation of intrarenal angiotensin II (Ang II) and the development of hypertensive kidney injury. Recent studies demonstrated that macrophages and the enhanced production of pro-inflammatory cytokines can be crucial mediators of renal AGT augmentation in hypertension. Accordingly, this study investigated the effects of immunosuppression by mycophenolate mofetil (MMF) on intrarenal AGT augmentation. Ang II (80 ng/min) was infused with or without daily administration of MMF (50 mg/kg) to Sprague-Dawley rats for 2 weeks. Mean arterial pressure (MAP) in Ang II infused rats was slightly higher (169.7 ± 6.1 mmHg) than the Ang II + MMF group (154.7 ± 2.0 mmHg), but was not statistically different from the Ang II + MMF group. MMF treatment suppressed Ang II-induced renal macrophages and IL-6 elevation. Augmentation of urinary AGT by Ang II infusion was attenuated by MMF treatment (control: 89.3 ± 25.2, Ang II: 1194 ± 305.1, and Ang II + MMF: 389 ± 192.0 ng/day). The augmentation of urinary AGT by Ang II infusion was observed before the onset of proteinuria. Elevated intrarenal AGT mRNA and protein levels in Ang II infused rats were also normalized by the MMF treatment (AGT mRNA, Ang II: 2.5 ± 0.2 and Ang II + MMF: 1.5 ± 0.1, ratio to control). Ang II-induced proteinuria, mesangial expansion and renal tubulointerstitial fibrosis were attenuated by MMF. Furthermore, MMF treatment attenuated the augmentation of intrarenal NLRP3 mRNA, a component of inflammasome. These results indicate that stimulated cytokine production in macrophages contributes to intrarenal AGT augmentation in Ang II-dependent hypertension, which leads to the development of kidney injury.

Effect of Empagliflozin on Sphingolipid Catabolism in Diabetic and Hypertensive Rats.

The profile of sphingomyelin and its metabolites shows changes in the plasma, organs, and tissues of patients with cardiovascular, renal, and metabolic diseases. The objective of this study was to investigate the effect of empagliflozin on the levels of sphingomyelin and its metabolites, as well as on the activity of acid and neutral sphingomyelinase (aSMase and nSMase) and neutral ceramidase (nCDase) in the plasma, kidney, heart, and liver of streptozotocin-induced diabetic and Angiotensin II (Ang II)-induced hypertension rats. Empagliflozin treatment decreased hyperglycemia in diabetic rats whereas blood pressure remained elevated in hypertensive rats. In diabetic rats, empagliflozin treatment decreased sphingomyelin in the plasma and liver, ceramide in the heart, sphingosine-1-phosphate (S1P) in the kidney, and nCDase activity in the plasma, heart, and liver. In hypertensive rats, empagliflozin treatment decreased sphingomyelin in the plasma, kidney, and liver; S1P in the plasma and kidney; aSMase in the heart, and nCDase activity in the plasma, kidney, and heart. Our results suggest that empagliflozin downregulates the interaction of the de novo pathway and the catabolic pathway of sphingolipid metabolism in the diabetes, whereas in Ang II-dependent hypertension, it only downregulates the sphingolipid catabolic pathway.

Effects of Renal Denervation on the Enhanced Renal Vascular Responsiveness to Angiotensin II in High-Output Heart Failure: Angiotensin II Receptor Binding Assessment and Functional Studies in Ren-2 Transgenic Hypertensive Rats.

Detailed mechanism(s) of the beneficial effects of renal denervation (RDN) on the course of heart failure (HF) remain unclear. The study aimed to evaluate renal vascular responsiveness to angiotensin II (ANG II) and to characterize ANG II type 1 (AT1) and type 2 (AT2) receptors in the kidney of Ren-2 transgenic rats (TGR), a model of ANG II-dependent hypertension. HF was induced by volume overload using aorto-caval fistula (ACF). The studies were performed two weeks after RDN (three weeks after the creation of ACF), i.e., when non-denervated ACF TGR enter the decompensation phase of HF whereas those after RDN are still in the compensation phase. We found that ACF TGR showed lower renal blood flow (RBF) and its exaggerated response to intrarenal ANG II (8 ng); RDN further augmented this responsiveness. We found that all ANG II receptors in the kidney cortex were of the AT1 subtype. ANG II receptor binding characteristics in the renal cortex did not significantly differ between experimental groups, hence AT1 alterations are not responsible for renal vascular hyperresponsiveness to ANG II in ACF TGR, denervated or not. In conclusion, maintained renal AT1 receptor binding combined with elevated ANG II levels and renal vascular hyperresponsiveness to ANG II in ACF TGR influence renal hemodynamics and tubular reabsorption and lead to renal dysfunction in the high-output HF model. Since RDN did not attenuate the RBF decrease and enhanced renal vascular responsiveness to ANG II, the beneficial actions of RDN on HF-related mortality are probably not dominantly mediated by renal mechanism(s).

Renin-Angiotensin System Induced Secondary Hypertension: The Alteration of Kidney Function and Structure

Long-term hypertension is known as a major risk factor for cardiovascular and chronic kidney disease (CKD). The Renin-angiotensin system (RAS) plays a key role in hypertension pathogenesis. Angiotensin II (Ang II) enhancement in Ang II-dependent hypertension leads to progressive CKD and kidney fibrosis. In the two-kidney one-clip model (2K1C), more renin is synthesized in the principal cells of the collecting duct than juxtaglomerular cells (JGCs). An increase of renal Ang I and Ang II levels and a decrease of renal cortical and medullary Ang 1–7 occur in both kidneys of the 2K1C hypertensive rat model. In addition, the activity of the angiotensin-converting enzyme (ACE) increases, while ACE2's activity decreases in the medullary region of both kidneys in the 2K1C hypertensive model. Also, the renal prolyl carboxypeptidase (PrCP) expression and its activity reduce in the clipped kidneys. The imbalance in the production of renal ACE, ACE2, and PrCP expression causes the progression of renal injury. Intrarenal angiotensinogen (AGT) expression and urine AGT (uAGT) excretion rates in the unclipped kidney are greater than the clipped kidney in the 2K1C hypertensive rat model. The enhancement of Ang II in the clipped kidney is related to renin secretion, while the elevation of intrarenal Ang II in the unclipped kidney is related to stimulation of AGT mRNA and protein in proximal tubule cells by a direct effect of systemic Ang II level. Ang II-dependent hypertension enhances macrophages and T-cell infiltration into the kidney which increases cytokines, and AGT synthesis in proximal tubules is stimulated via cytokines. Accumulation of inflammatory cells in the kidney aggravates hypertension and renal damage. Moreover, Ang II-dependent hypertension alters renal Ang II type 1 & 2 receptors (AT1R & AT2R) and Mas receptor (MasR) expression, and the renal interstitial fluid bradykinin, nitric oxide, and cGMP response to AT1R, AT2R, or BK B2-receptor antagonists. Based on a variety of sources including PubMed, Google Scholar, Scopus, and Science-Direct, in the current review, we will discuss the role of RAS-induced secondary hypertension on the alteration of renal function.

The promoting role of Cx43 on the proliferation and migration of arterial smooth muscle cells for angiotensin II-dependent hypertension

BackgroundRecent studies have shown that endothelin-1 and angiotensin II (AngII) can increase gap junctional intercellular communication (GJIC) by activating Mitogen-activated protein kinases (MAPKs) pathway. However, not only the precise interaction of AngII with Connexin43(Cx43) and the associated functions remain unclear, but also the regulatory role of Cx43 on the AngII-mediated promotion proliferation and migration of VSMCs is poorly understood. Material and methodsOur research applicated pressure myography measurements, immunofluorescence and Western blot analyses to investigate the changes in physiological indicators in spontaneously hypertensive rats (SHRs) and AngII-stimulated proliferation and migration of A7r5 SMCs(Rat vascular smooth muscle cells). The aim was to elucidate the role of CX43 in hypertension induced by AngII. ResultsChronic ramipril (angiotensin converting enzyme inhibitor) management for SHRs significantly attenuated blood pressure and blood vessel wall thickness, also reduced contraction rate in the cerebral artery. The cerebral artery contraction rates, mRNA and protein expression of Cx43, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) protein expression in the SHR + ramipril and SHR + ramipril + carbenoxolone (CBX, Cx43 specific blocker) groups were significantly lower than those in the SHR group. Cx43 protein expression and Ser368 phosphorylated Cx43 protein levels increased significantly in AngII-stimulated A7r5 cells. However, the levels of phosphorylated Cx43 decreased after pre-treatment with candesartan (AT1 receptor blocker), GF109203X (protein kinase C (PKC) blocker) and U0126 (mitogen-activated protein kinases/extracellular signal-regulated kinase1/2(MEK/ERK1/2)-specific blocker) in AngII-stimulated A7r5 cells. Cx43 was widely distributed in the cell membrane, nucleus, and cytoplasm of the SMCs. Furthermore, pre-treatment of the AngII- stimulated A7r5 cells with Gap26 (Cx43 blocker) significantly inhibited cell migration and decreased the expression levels of MEK1/2, ERK1/2, P-MEK1/2, and P-ERK1/2. ConclusionOur research confirms that Cx43 plays an important role in the regulation of proliferation and migration of VSMCs via MEK/ERK and PKC signal pathway in AngII-dependent hypertension.

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.

Increased Endogenous Activity of the Renin-Angiotensin System Reduces Infarct Size in the Rats with Early Angiotensin II-dependent Hypertension which Survive the Acute Ischemia/Reperfusion Injury.

We investigated the role of the interaction between hypertension and the renin-angiotensin system in the pathophysiology of myocardial ischemia/reperfusion injury. We hypothesized that in the early phase of angiotensin II (ANG II)-dependent hypertension with developed left ventricular hypertrophy, cardioprotective mechanism(s) are fully activated. The experiments were performed in transgenic rats with inducible hypertension, noninduced rats served as controls. The early phase of ANG II-dependent hypertension was induced by five-days (5 days) dietary indole-3-carbinol administration. Cardiac hypertrophy, ANG II and ANG 1–7 levels, protein expression of their receptors and enzymes were determined. Separate groups were subjected to acute myocardial ischemia/reperfusion injury, and infarct size and ventricular arrhythmias were assessed. Induced rats developed marked cardiac hypertrophy accompanied by elevated ANG levels. Ischemia/reperfusion mortality was significantly higher in induced than noninduced rats (52.1 and 25%, respectively). The blockade of AT1 receptors with losartan significantly increased survival rate in both groups. Myocardial infarct size was significantly reduced after 5 days induction (by 11%), without changes after losartan treatment. In conclusion, we confirmed improved cardiac tolerance to ischemia/reperfusion injury in hypertensive cardiohypertrophied rats and found that activation of AT1 receptors by locally produced ANG II in the heart was not the mechanism underlying infarct size reduction.

Angiotensin II Elicits Intracellular Ca 2+ Responses in Rat Cortical Collecting Ducts

Regulation of sodium transport in the cortical collecting duct (CCD) is pivotal for the long-term blood pressure control and excessive Na+ retention in this nephron segment is a known pathogenic determinant of hypertension. Sodium reabsorption in the CCD cells can be stimulated by volume-regulatory hormones, such as angiotensin II (AngII). AngII reportedly binds to AT1 receptors and increases the activity of the epithelial sodium channel (ENaC) via generation of reactive oxygen species (ROS). Yet, our understanding of the exact molecular determinants mediating AngII-induced intracellular signaling in the CCD cells remains limited. Numerous studies show that AngII induces intracellular Ca2+ mobilization in the vasculature, cardiomyocytes and glomerular podocytes. Here, we used ratiometric Fura-2 Ca2+-imaging to test if AngII employs Ca2+ as a second messenger in the CCDs freshly isolated from male and female Sprague-Dawley (SD) rats. We found that application of 500 nM of AngII to a split-opened CCD results in a transient elevation of [Ca2+]i in the CCD cells. The amplitude of the observed [Ca2+]i increase is about 150 nM and is not significantly different in the CCDs isolated from male and female rats. AngII-dependent [Ca2+]i signal is not affected by removal of Ca2+ from the extracellular bathing solution, but is practically abolished, when the tubules are pre-incubated in 3 μM thapsigargin, indicative of Ca2+ release from the intracellular stores. Inhibition of PLC with 10 μM U73122 markedly diminishes AngII-induced [Ca2+]i transient. Chronic AngII infusion moderately impairs [Ca2+]i responses to AngII in the CCDs isolated from male rats by approximately 30%. In contrast, AngII-induced [Ca2+]i elevation is attenuated by 70% in the CCDs from AngII-infused females. We have previously reported a stronger antihypertensive effect of aldosterone antagonism in AngII-infused females, when compared to males. Here, we found that systemic administration of eplerenone prevents the decline of AngII-stimulated [Ca2+]i response in the CCDs from female rats with AngII-dependent hypertension, but not in males. Overall, our data demonstrate that AngII triggers Ca2+ release from the intracellular stores of the CCD cells in a PLC-dependent manner. This AngII-activated [Ca2+]i mobilization is impaired in AngII-infused SD rats of both sexes, but the attenuation is remarkably more pronounced in females. Systemic aldosterone antagonism with eplerenone restores the AngII-mediated [Ca2+]i response in the CCDs from female rats with AngII-dependent hypertension. Future studies are granted to investigate the interplay between AngII-dependent [Ca2+]i signal and ROS generation in the CCD cells.

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.

Angiotensin II Stimulates Basolateral Conductance in Intercalated but Not Principal Cells of the Collecting Duct by Promoting Generation of Reactive Oxygen Species

The renal collecting duct plays a critical role in setting urinary volume and composition by performing Na+ and K+ transport by principal cells and Cl- reabsorption via intercalated cells. Published evidence implies Angiotensin II (Ang II) is a potent activator of the apically localized epithelial Na+ channel (ENaC) and Cl-/HCO3- exchanger, pendrin, in principal and intercalated cells, respectively, particularly during hypovolemic states. However, virtually nothing is known about Ang II actions on the basolateral conductance of the collecting duct cells. To address this gap, we employed macroscopic and single channel patch clamp recordings from the basolateral membrane of freshly isolated mouse collecting duct principal and intercalated cells, pharmacological tools to delineate Ang II signaling pathway, and fluorescence-base real-time detection of Ang II-induced generation reactive oxygen species. We found that nanomolar concentrations of Ang II acutely stimulate the basolateral Cl- conductance and specifically ClC-K2 Cl- channel in intercalated cells. In contrast, similar concentration of Ang II did not affect the basolateral conductance and Kir4.1/5.1 potassium channel activity in principal cells. Inhibition of AT1 receptors with losartan abolished stimulatory actions of Ang II on ClC-K2. Deletion of AT1 receptors dramatically decreased renal ClC-K2 expression further indicating its major role in regulation of ClC-K2 function. Inhibition of either phospholipase C (PLC), phospholipase A2 (PLA2), or phosphoinositide 3 kinase (PI3-K) did not affect stimulation of ClC-K2 activity by Ang II. In contrast, we show that activation of NADPH oxidases (NOX) is the major signaling pathway downstream of Ang II-AT1R in stimulation of ClC-K2. Application of Ang II resulted in a rapid production of reactive oxygen species in both principal and intercalated cells within 5 min, which is consistent with the time course of single channel ClC-K2 activation. Overall, this study demonstrates that physiologically relevant Ang II levels stimulate ClC-K2 activity and by extension trans-cellular Cl- reabsorption in intercalated cells by triggering AT1 receptor-NOX-ROS signaling pathway. We speculate that upregulation of ClC-K2 by Ang II is critical for protection of circulatory volume during hypovolemic states, whereas over-activation of ClC-K2 might contribute to the pathophysiology of Ang II-dependent hypertension.

The evolving complexity of the collecting duct renin-angiotensin system in hypertension.

The intrarenal renin-angiotensin system is critical for the regulation of tubule sodium reabsorption, renal haemodynamics and blood pressure. The excretion of renin in urine can result from its increased filtration, the inhibition of renin reabsorption by megalin in the proximal tubule, or its secretion by the principal cells of the collecting duct. Modest increases in circulating or intrarenal angiotensin II (ANGII) stimulate the synthesis and secretion of angiotensinogen in the proximal tubule, which provides sufficient substrate for collecting duct-derived renin to form angiotensin I (ANGI). In models of ANGII-dependent hypertension, ANGII suppresses plasma renin, suggesting that urinary renin is not likely to be the result of increased filtered load. In the collecting duct, ANGII stimulates the synthesis and secretion of prorenin and renin through the activation of ANGII type 1 receptor (AT1R) expressed primarily by principal cells. The stimulation of collecting duct-derived renin is enhanced by paracrine factors including vasopressin, prostaglandin E2 and bradykinin. Furthermore, binding of prorenin and renin to the prorenin receptor in the collecting duct evokes a number of responses, including the non-proteolytic enzymatic activation of prorenin to produce ANGI from proximal tubule-derived angiotensinogen, which is then converted into ANGII by luminal angiotensin-converting enzyme; stimulation of the epithelial sodium channel (ENaC) in principal cells; and activation of intracellular pathways linked to the upregulation of cyclooxygenase 2 and profibrotic genes. These findings suggest that dysregulation of the renin-angiotensin system in the collecting duct contributes to the development of hypertension by enhancing sodium reabsorption and the progression of kidney injury.

ENHANCED PRODUCTIONS OF INTRARENAL NLRP-3 INFLAMMASOME AND ANGIOTENSINOGEN IN ANGIOTENSIN II-DEPENDENT HYPERTENSION ARE PREVENTED BY IMMUNOSUPPRESSANT TREATMENT

Objective: Activated inflammasomes enhance maturation of pro-inflammatory cytokines, which facilitates the development of kidney injury. NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), one of major subunits in the inflammasome complex, plays a crucial role in innate immunity and inflammation. NLRP3 inflammasome is activated by ATP-P2X7 axis and reactive oxygen species. Expression of pro-NLRP3 is promoted by activated NF-κB by cytokines or PAMPs/DAMPs. In angiotensin II (Ang II)-dependent hypertension, there is augmentation of renal immune cells leading to enhanced production of cytokines and consequent activation of NF-κB. Therefore, we hypothesized that intrarenal NLRP3 levels are increased in Ang II-dependent hypertension in association with the kidney injury, and treatment with mycophenolate mofetil (MMF), an immunosuppressant, prevents the augmentation of NLRP3 and attenuates the consequent progression of kidney injury. Design and method: Ang II (80 ng/min) was infused with/without daily MMF administration (50 ng/kg) to male rats for 2 weeks. mRNA levels of intrarenal NLRP3 and AIM2, which forms another type of inflammasome complex by viral or bacterial infections, were measured by droplet digital PCR. Furthermore, levels of intrarenal angiotensinogen (AGT), a critical contributor to activation of intrarenal renin-angiotensin system leading to the development of hypertension and associated kidney injury, were evaluated. Histological analyses of kidney injury were also performed. Results: MMF prevented increases in intrarenal macrophage/monocyte and IL-6 levels induced by Ang II infusion, confirming immunosuppression by the drug. Ang II infusion significantly increased intrarenal NLRP3 levels (control group: 4.12 ± 1.1 copies/ng RNA vs. Ang II-infused group: 9.96 ± 1.8 copies). The elevated NLRP3 expression was attenuated by MMF (6.24 ± 1.4 copies). In contrast, intrarenal AIM2 levels were not altered by Ang II infusion or MMF. Urinary protein and AGT levels were elevated in Ang II-infused rats, which were prevented by MMF. Histological analyses showed mesangial expansion and tubulointerstitial fibrosis observed in Ang II-infused group, but these injury markers were in normal ranges in the group receiving Ang II + MMF. Conclusions: These results demonstrate activation of the NLRP3 inflammasome in Ang II dependent hypertension and indicate that immunosuppression by MMF mitigates the inflammasome activation, which contributes to attenuation of the kidney injury.

Abstract P035: Prostaglandin Ep4 Receptors In Kidney Epithelial Cells Promote Resistance To Hypertension

A role for prostanoids in blood pressure control is demonstrated by clinical studies showing that use of nonsteroidal anti-inflammatory drugs, which block all the prostanoid production, is associated with hypertension. Prostaglandin E 2 (PGE 2 ) is a major prostanoid produced by the kidney and its actions are mediated by four E-prostanoid (EP) receptors: EP1-EP4. In previous studies, we found that conditional deletion of EP4R from all tissues in adult mice dramatically exaggerates Ang II-dependent hypertension, suggesting a powerful effect of EP4R to resist blood pressure elevation. We also found that elimination of EP4R from vascular smooth muscle cells did not affect severity of hypertension, indicating other cellular targets of PGE 2 must mediate this anti-hypertensive effect. Here we report studies examining actions of EP4R in two additional candidate cell lineages: macrophages and kidney epithelial cells, which have both been implicated in hypertension pathogeneses. Cell-specific deletion of EP4R was accomplished by crossing mice bearing a conditional EP4 allele ( EP4 flox ) with mouse lines carrying a LyzM-Cre + transgene expressing in macrophages (MACKOs) or tetracycline-inducible Pax8-rtTA + TetO-Cre + transgene driving expression in renal tubular epithelial cells (RTKOs). Elimination of EP4R from macrophages in MACKOs had minimal effects on baseline BP and salt sensitivity, and responses to chronic Ang II infusion were virtually identical in MACKOs and controls (138±3 vs 133±2 mm Hg; n=14/group). By contrast, absence of EP4R from kidney epithelia in RTKOs caused significant augmentation of Ang II-induced hypertension compared to Controls (average MAP: 145±4 vs. 135±3 mmHg, p=0.03; MAP increase from baseline: 31±2 vs. 20±3 mmHg, p=0.02). The increased severity of hypertension in RTKOs was associated with exaggerated cardiac hypertrophy relative to controls (heart:body weight ratio 8.6±1.5 vs. 7.3±1.2 mg/g; p=0.03). Our findings suggest that kidney epithelial cells, but not macrophages, are critical targets of PGE 2 acting via EP4R to oppose the development of hypertension. As collecting duct is the major site of EP4R expression in renal epithelia, this may reflect actions of EP4R to modulate key sodium transporters in this nephron segment.