Angiotensin II-induced Hypertension Research Articles

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.

HUMAN AMNION EPITHELIAL CELLS REDUCE BLOOD PRESSURE AND PREVENT AORTIC INFLAMMATION AND COLLAGEN EXPRESSION DURING EXPERIMENTAL HYPERTENSION

Objective: Vascular inflammation and fibrosis are hallmarks of hypertension and contribute to the development of cardiovascular disease. Current treatments for hypertension may reduce blood pressure but do not target the associated pathological changes and end-organ damage. Cell therapy has great therapeutic potential for many conditions as, unlike single pharmacological agents, cells can deliver multiple mediators which could more effectively target complex disease mechanisms. Human amnion epithelial cells (hAECs) have many properties (eg. anti-inflammatory, anti-fibrotic, regenerative and immunologically inert) that make them attractive candidates for a cell-based therapy for vascular pathology. This study aimed to test the potential of hAECs to treat vascular pathology in angiotensin II-induced hypertension. Design and method: Male C57Bl6 mice (8–12 weeks) were administered vehicle (saline; n = 35) or angiotensin II (0.7 mg/kg/d, n = 37) for 14 d via a subcutaneous osmotic minipump. After minipump implantation, a subset of mice were injected with 10^6 of hAECs intravenously. Systolic blood pressure was measured using tail-cuff; inflammation was assessed using flow cytometry and markers of fibrosis using quantitative PCR. Results: Angiotensin II infusion increased systolic blood pressure and promoted accumulation of aortic leukocytes, specifically macrophages and monocytes, which were elevated by ∼3-fold compared to vehicle-infused mice (n = 9–11, P &lt; 0.05). Angiotensin II also increased aortic mRNA expression of collagen type 1 alpha 1 (Col1a1) by ∼4-fold and collagen type 5 alpha 1 (Col5a1) by ∼7-fold compared to vehicle (n = 6–8, P &lt; 0.05). Co-administration of hAECs limited the development of hypertension by angiotensin II (185 ± 5 mmHg vs 165 ± 4 mmHg; n = 9–11, P &lt; 0.05), as well as the aortic infiltration of macrophages and monocytes (n = 9–11, P &lt; 0.05) and expression of Col1a1 and Col5a1 (n = 6–8, P &lt; 0.05). Conclusions: Intravenous administration of hAECs blunted angiotensin II-induced hypertension, aortic inflammation and collagen expression in male mice. This study suggests that hAECs or their cellular products could be explored as treatments for vascular pathology during hypertension.

VARIANT GAMMA 6+ GAMMA-DELTA T CELLS PLAY A ROLE IN ANGIOTENSIN II-INDUCED HYPERTENSION

Objective: We previously demonstrated that a small subpopulation of T cells considered (innate-like), expressing the γδ T-cell receptor (TCR) plays a key role in hypertension and vascular injury. γδ T cells can be subdivided according to the TCR variant (V) subtype that is generally specific for a tissue. A subpopulation of lungs and skin γδ T cells that are Vγ6+ and produce interleukin (IL)-17A was shown to respond promptly to pneumococcal infection and skin inflammation. However, γδ T cell Vγ subtypes involved in hypertension are still unknown. We hypothesized that the Vγ6+ γδ T cell may play a role in angiotensin (Ang) II-induced hypertension and vascular injury. Design and method: Eleven- to 13-week-old C57BL/6J male mice were infused or not with Ang II (490 ng/kg/min, SC) for 14 days, (n = 5–14). The γδ T cell Vγ subtypes were profiled and markers of activation of Vγ6+ γδ T cells were determined by flow cytometry. Results: In spleen and mesenteric lymph nodes (MLNs) the most abundant γδ T cells Vγ were Vγ1/2+ and Vγ4+ followed by Vγ6+,Vγ5+ and Vγ7+. In thoracic aortic (TA) perivascular adipose tissue (PVAT), the most abundant γδ T cell Vγ was Vγ6+ followed by Vγ4+, Vγ1/2+, Vγ5+ and Vγ7+. In mesenteric artery (MA) PVAT, the most abundant γδ T cell Vγ subtype was Vγ6+ followed by Vγ4+, Vγ7+, Vγ5+ and Vγ1/2+. Ang II increased the frequency of Vγ6+ γδ T cells in the spleen (1.5-fold, P &lt; 0.01) and TA PVAT (1.6-fold, P &lt; 0.01), whereas it only tended to increase them in MA PVAT. The frequency of IL-17 producing effector memory (CCR6 + CXCR3–CD44 + CD69 + ) Vγ6+ γδ T cells were increased in spleen (1.7-fold, P &lt; 0.01) and trended to be elevated in mesenteric PVAT in Ang II-infused mice compared to control mice. Conclusions: Vγ6+ γδ T cells were the most abundant Vγ subtype in PVAT. IL-17 producing effector memory Vγ6+ γδ T cells may play a role in Ang II-induced hypertension. Targeting a γδ T cell variant subtype could be a therapeutic approach to reduce inflammation in hypertension.

Novel Targets for Hypertension Drug Discovery.

Despite the availability of various medications and prescribing combination therapies, uncontrolled blood pressure and resistance are observed in more than 40% of patients. The purpose of this review is to discuss emerging novel approaches for the treatment of hypertension and propose future research and clinical directions. Hypertension is a common disease of the cardiovascular system which may arise solely or as a comorbidity of other disorders. It is a crucial risk factor for cardiovascular diseases such as coronary artery disease, myocardial infarction, congestive heart failure, renal failure, and stroke. The results from current literature regarding the novel approaches showed several targets that could be explored as potential therapeutic options. These include toll-like receptor 4, a critical regulator of angiotensin II-induced hypertension; protease-activated receptor 2, which promotes collagen deposition and inflammatory responses; chemerin, which causes metabolic and obesity-associated hypertension; apelin receptor; transient receptor potential melastatin; urotensin-II; and Tie2 receptor. This review discusses various targets and pathways that could be emerging pharmacological therapies for hypertension.

Calpastatin prevents Angiotensin II–mediated podocyte injury through maintenance of autophagy

The strong predictive value of proteinuria in chronic glomerulopathies is firmly established as well as the pathogenic role of angiotensin II promoting progression of glomerular disease with an altered glomerular filtration barrier, podocyte injury and scarring of glomeruli. Here we found that chronic angiotensin II-induced hypertension inhibited autophagy flux in mouse glomeruli. Deletion of Atg5 (a gene encoding a protein involved autophagy) specifically in the podocyte resulted in accelerated angiotensin II-induced podocytopathy, accentuated albuminuria and glomerulosclerosis. This indicates that autophagy is a key protective mechanism in the podocyte in this condition. Angiotensin-II induced calpain activity in podocytes inhibits autophagy flux. Podocytes from mice with transgenic expression of the endogenous calpain inhibitor calpastatin displayed higher podocyte autophagy at baseline that was resistant to angiotensin II-dependent inhibition. Also, sustained autophagy with calpastatin limited podocyte damage and albuminuria. These findings suggest that hypertension has pathogenic effects on the glomerular structure and function, in part through activation of calpains leading to blockade of podocyte autophagy. These findings uncover an original mechanism whereby angiotensin II-mediated hypertension inhibits autophagy via calcium-induced recruitment of calpain with pathogenic consequences in case of imbalance by calpastatin activity. Thus, preventing a calpain-mediated decrease in autophagy may be a promising new therapeutic strategy for nephropathies associated with high renin-angiotensin system activity.

KV 1.3 channels are novel determinants of macrophage-dependent endothelial dysfunction in angiotensin II-induced hypertension in mice.

KV 1.3 channels are expressed in vascular smooth muscle cells (VSMCs), where they contribute to proliferation rather than contraction and participate in vascular remodelling. KV 1.3 channels are also expressed in macrophages, where they assemble with KV 1.5 channels (KV 1.3/KV 1.5), whose activation generates a KV current. In macrophages, the KV 1.3/KV 1.5 ratio is increased by classical activation (M1). Whether these channels are involved in angiotensin II (AngII)-induced vascular remodelling, and whether they can modulate the macrophage phenotype in hypertension, remains unknown. We characterized the role of KV 1.3 channels in vascular damage in hypertension. We used AngII-infused mice treated with two selective KV 1.3 channel inhibitors (HsTX[R14A] and [EWSS]ShK). Vascular function and structure were measured using wire and pressure myography, respectively. VSMC and macrophage electrophysiology were studied using the patch-clamp technique; gene expression was analysed using RT-PCR. AngII increased KV 1.3 channel expression in mice aorta and peritoneal macrophages which was abolished by HsTX[R14A] treatment. KV 1.3 inhibition did not prevent hypertension, vascular remodelling, or stiffness but corrected AngII-induced macrophage infiltration and endothelial dysfunction in the small mesenteric arteries and/or aorta, via a mechanism independent of electrophysiological changes in VSMCs. AngII modified the electrophysiological properties of peritoneal macrophages, indicating an M1-like activated state, with enhanced expression of proinflammatory cytokines that induced endothelial dysfunction. These effects were prevented by KV 1.3 blockade. We unravelled a new role for KV 1.3 channels in the macrophage-dependent endothelial dysfunction induced by AngII in mice which might be due to modulation of macrophage phenotype.

Endogenous SO2-dependent Smad3 redox modification controls vascular remodeling

Sulfur dioxide (SO2) has emerged as a physiological relevant signaling molecule that plays a prominent role in regulating vascular functions. However, molecular mechanisms whereby SO2 influences its upper-stream targets have been elusive. Here we show that SO2 may mediate conversion of hydrogen peroxide (H2O2) to a more potent oxidant, peroxymonosulfite, providing a pathway for activation of H2O2 to convert the thiol group of protein cysteine residues to a sulfenic acid group, aka cysteine sulfenylation. By using site-centric chemoproteomics, we quantified >1000 sulfenylation events in vascular smooth muscle cells in response to exogenous SO2. Notably, ~42% of these sulfenylated cysteines are dynamically regulated by SO2, among which is cysteine-64 of Smad3 (Mothers against decapentaplegic homolog 3), a key transcriptional modulator of transforming growth factor β signaling. Sulfenylation of Smad3 at cysteine-64 inhibits its DNA binding activity, while mutation of this site attenuates the protective effects of SO2 on angiotensin II-induced vascular remodeling and hypertension. Taken together, our findings highlight the important role of SO2 in vascular pathophysiology through a redox-dependent mechanism.

Differential biomechanical responses of elastic and muscular arteries to angiotensin II-induced hypertension

Elastic and muscular arteries are distinguished by their distinct microstructures, biomechanical properties, and smooth muscle cell contractile functions. They also exhibit differential remodeling in aging and hypertension. Although regional differences in biomechanical properties have been compared, few studies have quantified biaxial differences in response to hypertension. Here, we contrast passive and active changes in large elastic and medium- and small-sized muscular arteries in adult mice in response to chronic infusion of angiotensin over 14 days. We found a significant increase in wall thickness, both medial and adventitial, in the descending thoracic aorta that associated with trends of an increased collagen:elastin ratio. There was adventitial thickening in the small-sized mesenteric artery, but also significant changes in elastic lamellar structure and contractility. An increased contractile response to phenylephrine coupled with a reduced vasodilatory response to acetylcholine in the mesenteric artery suggested an increased contractile state in response to hypertension. Overall reductions in the calculated gradients in pulse wave velocity and elastin energy storage capability from elastic-to-muscular arteries suggested a possible transfer of excessive pulsatile energy into the small-sized muscular arteries resulting in significant functional consequences in response to hypertension.

Deletion of Orphan G Protein-Coupled Receptor GPR37L1 in Mice Alters Cardiovascular Homeostasis in a Sex-Specific Manner.

GPR37L1 is a family A orphan G protein-coupled receptor (GPCR) with a putative role in blood pressure regulation and cardioprotection. In mice, genetic ablation of Gpr37l1 causes sex-dependent effects; female mice lacking Gpr37l1 (GPR37L1−/−) have a modest but significant elevation in blood pressure, while male GPR37L1−/− mice are more susceptible to cardiovascular dysfunction following angiotensin II-induced hypertension. Given that this receptor is highly expressed in the brain, we hypothesize that the cardiovascular phenotype of GPR37L1−/− mice is due to changes in autonomic regulation of blood pressure and heart rate. To investigate this, radiotelemetry was employed to characterize baseline cardiovascular variables in GPR37L1−/− mice of both sexes compared to wildtype controls, followed by power spectral analysis to quantify short-term fluctuations in blood pressure and heart rate attributable to alterations in autonomic homeostatic mechanisms. Additionally, pharmacological ganglionic blockade was performed to determine vasomotor tone, and environmental stress tests were used to assess whether cardiovascular reactivity was altered in GPR37L1−/− mice. We observed that mean arterial pressure was significantly lower in female GPR37L1−/− mice compared to wildtype counterparts, but was unchanged in male GPR37L1−/− mice. GPR37L1−/− genotype had a statistically significant positive chronotropic effect on heart rate across both sexes when analyzed by two-way ANOVA. Power spectral analysis of these data revealed a reduction in power in the heart rate spectrum between 0.5 and 3 Hz in female GPR37L1−/− mice during the diurnal active period, which indicates that GPR37L1−/− mice may have impaired cardiac vagal drive. GPR37L1−/− mice of both sexes also exhibited attenuated depressor responses to ganglionic blockade with pentolinium, indicating that GPR37L1 is involved in maintaining sympathetic vasomotor tone. Interestingly, when these mice were subjected to aversive and appetitive behavioral stressors, the female GPR37L1−/− mice exhibited an attenuation of cardiovascular reactivity to aversive, but not appetitive, environmental stimuli. Together, these results suggest that loss of GPR37L1 affects autonomic maintenance of blood pressure, giving rise to sex-specific cardiovascular changes in GPR37L1−/− mice.

Cardiac and renal protective effects of 2,5-dimethylcelecoxib in angiotensin II-induced hypertension model mice

Cardiac and renal protective effects of 2,5-dimethylcelecoxib in angiotensin II-induced hypertension model mice

TLR2 regulates angiotensin II-induced vascular remodeling and EndMT through NF-κB signaling.

Excessive vascular remodeling has been shown in hypertensive patients. In experimental models of hypertensive vascular injury, such as angiotensin II (Ang II) challenged mice, toll like receptor 2 (TLR2) initiates inflammatory responses. More recently, studies have reported atypical endothelial to mesenchymal transition (EndMT) in vascular injuries and inflammatory conditions. Here, we aimed to investigate whether TLR2 mediates Ang II-induced vascular inflammation and initiates EndMT. In a mouse model of angiotensin II-induced hypertension, we show that aortas exhibit increased medial thickening, fibrosis, and features of EndMT. These alterations were not observed in TLR2 knockout mice in response to Ang II. TLR2 silencing in cultured endothelial cells confirmed the essential role of TLR2 in Ang II-induced inflammatory factor induction, and EndMT-associated phenotypic change. Mechanistically, we found Ang II activates nuclear factor-κB signaling, inducing pro-inflammatory cytokine production, and mediates EndMT in both cultured endothelial cells and in mice. These studies illustrate a novel role of TLR2 in regulating Ang II-induced deleterious vascular remodeling through the induction of EndMT. The studies also suggest that TLR2 may be targeted to alleviate hypertension-associated vascular injury.

Site-1 Protease-Derived Soluble (Pro)Renin Receptor Contributes to Angiotensin II-Induced Hypertension in Mice.

Activation of PRR ([pro]renin receptor) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during Ang II (angiotensin II) infusion. The goal of the present study was to test whether such action of PRR was mediated by sPRR (soluble PRR), generated by S1P (site-1 protease), a newly identified PRR cleavage protease. F1 B6129SF1/J mice were infused for 6 days with control or Ang II at 300 ng/kg per day alone or in combination with S1P inhibitor PF-429242 (PF), and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated Ang II-induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histidine-tagged sPRR termed as sPRR-His. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mouse cortical collecting duct cell line cells exposed for 24 hours to Ang II, Ang II + PF, or Ang II + PF + sPRR-His. Ang II-induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that S1P-derived sPRR mediates Ang II-induced hypertension through enhancement of intrarenal renin level and activation of ENaC.

Abstract 15061: Essential Hypertension is Linked to Acetylation of Mitochondrial Superoxide Dismutase and Deacetylation Mimetic Mutation of Lysine 68 to Arginine Protects From Oxidative Stress and Hypertension

Almost one-half of adults have hypertension, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs, likely due to mechanisms contributing to blood pressure elevation that are not affected by current treatments. Hypertension is linked to oxidative stress; however, common antioxidants are ineffective. We found that hypertension is associated with inactivation of key mitochondrial antioxidant, superoxide dismutase 2 (SOD2), due to acetylation of lysine residues at the catalytic center. The role of specific SOD2 lysine residues in hypertension, however, has not been defined. Hypothesis: We proposed that inactivation of key intrinsic antioxidant, SOD2, in hypertension is linked to acetylation of Lysine 68, and mutation of K68 to Arginine mimics SOD2 deacetylation, inhibits vascular oxidative stress and attenuates angiotensin II-induced hypertension. To test this hypothesis, we have investigated SOD2 acetylation in arterioles from patients with essential hypertension and developed a new deacetylation mimic SOD2 mutant K68R mice (SOD2-K68R). Western blot of arterioles isolated from human mediastinal fat showed 3-fold increase in SOD2 acetylation in hypertensive patients compared with normotensive subjects while SOD2 levels were not affected. To define the functional significance of K68 acetylation we performed studies in vivo in SOD2-K68R mice using angiotensin II model of vascular dysfunction and hypertension. Angiotensin II infusion in wild-type C57Bl/6J mice induced vascular inflammation and oxidative stress, and increased blood pressure to 160 mm Hg. Mutation of Lysine 68 to Arginine in SOD2-K68R mice completely prevented the increase in mitochondrial superoxide and significantly attenuated the angiotensin II induced hypertension (135 mm Hg). Angiotensin II and TNFα co-operatively induce SOD2 acetylation and hypertension. Treatment of wild-type aortas with angiotensin II and TNFα in organoid culture increased mitochondrial superoxide by 2-fold which was completely prevented in aortas isolated from SOD2-K68R mice. Conclusions: These data support an important role of SOD2-K68 acetylation in hypertension and targeting Sirt3-mediated deacetylation of SOD2 may have therapeutic potential.

Predator Scent-Induced Sensitization of Hypertension and Anxiety-like Behaviors.

Post-traumatic stress disorder (PTSD), ananxiety-related syndrome, is associated with increased risk for cardiovascular diseases. The present study investigated whether predator scent (PS) stress, a model of PTSD, induces sensitization of hypertension and anxiety-like behaviors and underlying mechanisms related to renin-angiotensin systems (RAS) and inflammation. Coyote urine, as a PS stressor, was used to model PTSD. After PS exposures, separate cohorts of rats were studied for hypertensive response sensitization (HTRS), anxiety-like behaviors, and changes in plasma levels and mRNA expression of several components of the RAS and proinflammatory cytokines (PICs) in the lamina terminalis (LT), paraventricular nucleus (PVN), and amygdala (AMY). Rats exposed to PS as compared to control animals exhibited (1) a significantly greater hypertensive response (i.e., HTRS) when challenged with a slow-pressor dose of angiotensin (ANG) II, (2) significant decrease in locomotor activity and increase in time spent in the closed arms of a plus maze as well as general immobility (i.e., behavioral signs of increased anxiety), (3) upregulated plasma levels of ANG II and interleukin-6, and (4) increased expression of message for components of the RAS and PICs in key brain nuclei. All the PS-induced adverse effects were blocked by pretreatment with either an angiotensin-converting enzyme antagonist or a tumor necrosis factor-α inhibitor. The results suggest that PS, used as an experimental model of PTSD, sensitizes ANG II-induced hypertension and produces behavioral signs of anxiety, probably through upregulation of RAS components and inflammatory markers in plasma and brain areas associated with anxiety and blood pressure control.

Blocking of interleukin-1 suppresses both angiotensin II-induced renal inflammation and hypertension

Abstract Background Clinical hypertension is associated with renal inflammation and elevated circulating levels of proinflammatory cytokines. IL-1 receptor antagonist (IL-1Ra) is one of the most important anti-inflammatory cytokines and plays a crucial role in inflammation. Inhibition of IL-1 may contribute to modulation of the Angiotensin II (AngII)-induced hypertension response. This study aimed to elucidate the effects of IL-1Ra and anti-IL-1beta antibody (01BSUR) on AngII-induced hypertension and renal inflammation. Methods and results To determine the contribution of IL-1Ra to AngII-induced renal inflammation, male wild-type (WT) and IL-1Ra-deficient (IL-1Ra−/−) mice were infused with AngII (1000ng/kg/min) using subcutaneous osmotic pumps for 14 days. We checked blood pressure, histological change, and several mRNA expressions 14 days after infusion. Fourteen days after infusion, systolic blood pressure (197±5 vs 169±9 mmHg, p&amp;lt;0.05) in IL-1Ra−/− mice significantly increased compared with WT mice. Furthermore, on day 14 of AngII infusion, plasma IL-6 was 5.9-fold higher in IL-1Ra−/− versus WT mice (p&amp;lt;0.001); renal preproendothelin-1 mRNA expression was also significantly higher in IL-1Ra−/− mice (p&amp;lt;0.05). To examine renal function, we analyzed 24-hour urinary protein excretion and serum levels of blood urea nitrogen, creatinine, and uric acid in IL-1Ra−/− and WT mice. On day 14 of Ang II infusion, all levels increased significantly in IL-1Ra−/− mice compared with WT mice, suggesting that IL-1Ra deficiency reduced renal function following Ang II infusion. In addition, renal histology revealed that glomerular injury (Figure upper panels: PAS staining) and tubulointerstitial fibrosis (Figure lower panels: Elastica Masson staining) increased significantly in Ang II-infused IL-1Ra−/− versus Ang II-infused WT mice. Finally, we administrated 01BSUR to both IL-1Ra−/− and WT mice, and 01BSUR treatment decreased AngII-induced hypertension (162±17 vs 204±6 mmHg, p&amp;lt;0.05) and renal damage (glomerular injury and fibrosis of the tubulointerstitial area) in both IL-1Ra−/− and WT mice compared with IgG2a treatment. These findings suggest that 01BSUR suppresses Ang II-induced inflammation and renal injury. Conclusions Inhibition of interleukin-1 by both endogenous IL-1Ra and exogenous 01BSUR decreased AngII-induced hypertension and renal damage in mice, suggesting suppression of IL-1 may provide an additional strategy to protect against renal damage in hypertensive patients. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): JSPS KAKENHI

Oral nitrite treatment increases S-nitrosylation of vascular protein kinase C and attenuates the responses to angiotensin II

Nitrate and nitrite supplement deficient endogenous nitric oxide (NO) formation. While these anions may generate NO, recent studies have shown that circulating nitrite levels do not necessarily correlate with the antihypertensive effect of oral nitrite administration and that formation of nitrosylated species (RXNO) in the stomach is critically involved in this effect. This study examined the possibility that RXNO formed in the stomach after oral nitrite administration promotes target protein nitrosylation in the vasculature, inhibits vasoconstriction and the hypertensive responses to angiotensin II. Our results show that oral nitrite treatment enhances circulating RXNO concentrations (measured by ozone-based chemiluminescence methods), increases aortic protein kinase C (PKC) nitrosylation (measured by resin-assisted capture SNO-RAC method), and reduces both angiotensin II-induced vasoconstriction (isolated aortic ring preparation) and hypertensive (in vivo invasive blood pressure measurements) effects implicating PKC nitrosylation as a key mechanism for the responses to oral nitrite. Treatment of rats with the nitrosylating compound S-nitrosoglutathione (GSNO) resulted in the same effects described for oral nitrite. Moreover, partial depletion of thiols with buthionine sulfoximine prevented PKC nitrosylation and the blood pressure effects of oral nitrite. Further confirming a role for PKC nitrosylation, preincubation of aortas with GSNO attenuated the responses to both angiotensin II and to a direct PKC activator, and this effect was attenuated by ascorbate (reverses GSNO-induced nitrosylation). GSNO-induced nitrosylation also inhibited the increases in Ca2+ mobilization in angiotensin II-stimulated HEK293T cells expressing angiotensin type 1 receptor. Together, these results are consistent with the idea that PKC nitrosylation in the vasculature may underlie oral nitrite treatment-induced reduction in the vascular and hypertensive responses to angiotensin II.

Experimental model of congestive heart failure induced by transverse aortic constriction in BALB/c mice

IntroductionMurine transverse aortic constriction (TAC) is a frequently used model of pressure overload-induced left ventricular (LV) remodeling. However, there is considerable variability in disease progression to overt heart failure (HF) development in the most commonly used strain of mice (i.e., C57BL/6J). Studies have shown that C57BL/6J mice are more resistant than BALB/c mice to congestive HF development following myocardial infarction or angiotensin II-induced hypertension. Therefore, we tested the hypothesis that BALB/c mice may be a better research model to study TAC-induced progressive HF. MethodsFollowing sham or TAC surgery in both C57BL/6J (n = 29) and BALB/c (n = 32) mice, we evaluated cardiac dimensions and function by echocardiography at 2, 4, 8, and 12 weeks and monitored survival throughout the study. In a separate cohort of BALB/c mice, we repeated the study in the presence of the angiotensin converting enzyme inhibitor enalapril or a vehicle initiated 2 weeks post-TAC and administered for 6 weeks. At the end of the studies, we assessed the heart weight, lung weight, and plasma brain natriuretic peptide (BNP) concentration. ResultsFollowing comparable TAC, both C57BL/6J and BALB/c mice showed significant LV remodeling compared with the sham control mice. BALB/c mice progressively developed systolic dysfunction, LV dilation, lung congestion, and significant mortality, whereas C57BL/6J mice did not. In the separate cohort of BALB/c TAC mice, enalapril significantly reduced the heart weight, lung weight, and plasma BNP concentration and improved survival compared with the vehicle control. DiscussionBALB/c mice uniformly developed congestive HF post-TAC. Enalapril was effective in improving survival and reducing lung congestion in this model. The data suggest that BALB/c mice may be a better research tool than C57BL/6J mice to study TAC-induced disease progression to HF and to evaluate novel therapies for the treatment of chronic HF with reduced ejection fraction.

A new mechanism for the sex differences in angiotensin II-induced hypertension: the role of macula densa NOS1β-mediated tubuloglomerular feedback.

Females are protected against the development of angiotensin II (ANG II)-induced hypertension compared with males, but the mechanisms have not been completely elucidated. In the present study, we hypothesized that the effect of ANG II on the macula densa nitric oxide (NO) synthase 1β (NOS1β)-mediated tubuloglomerular feedback (TGF) mechanism is different between males and females, thereby contributing to the sexual dimorphism of ANG II-induced hypertension. We used microperfusion, micropuncture, clearance of FITC-inulin, and radio telemetry to examine the sex differences in the changes of macula densa NOS1β expression and activity, TGF response, natriuresis, and blood pressure (BP) after a 2-wk ANG II infusion in wild-type and macula densa-specific NOS1 knockout mice. In wild-type mice, ANG II induced higher expression of macula densa NOS1β, greater NO generation by the macula densa, and a lower TGF response in vitro and in vivo in females than in males; the increases of glomerular filtration rate, urine flow rate, and Na+ excretion in response to an acute volume expansion were significantly greater and the BP responses to ANG II were significantly less in females than in males. In contrast, these sex differences in the effects of ANG II on TGF, natriuretic response, and BP were largely diminished in knockout mice. In addition, tissue culture of human kidney biopsies (renal cortex) with ANG II resulted in a greater increase in NOS1β expression in females than in males. In conclusion, macula densa NOS1β-mediated TGF is a novel and important mechanism for the sex differences in ANG II-induced hypertension.

Sodium butyrate ameliorates deoxycorticosterone acetate/salt-induced hypertension and renal damage by inhibiting the MR/SGK1 pathway.

Our recent work demonstrates that infusion of sodium butyrate (NaBu) into the renal medulla blunts angiotensin II-induced hypertension and improves renal injury. The present study aimed to test whether oral administration of NaBu attenuates salt-sensitive hypertension in deoxycorticosterone acetate (DOCA)/salt-treated rats. Uninephrectomized male Sprague-Dawley (SD) rats were treated with DOCA pellets (150 mg/rat) plus 1% NaCl drinking water for 2 weeks. Animals received oral administration of NaBu (1 g/kg) or vehicle once per day. Our results showed that NaBu administration significantly attenuated DOCA/salt-increased mean arterial pressure from 156 ± 4 mmHg to 136 ± 1 mmHg. DOCA/salt treatment markedly enhanced renal damage as indicated by an increased ratio of kidney weight/body weight, elevated urinary albumin, extensive fibrosis, and inflammation, whereas kidneys from NaBu-treated rats exhibited a significant reduction in these renal damage responses. Compared to the DOCA/salt group, the DOCA/salt-NaBu group had ~30% less salt water intake and decreased Na+ and Cl- excretion in urine but no alteration in 24-h urine excretion. Mechanistically, NaBu inhibited the protein levels of several sodium transporters stimulated by DOCA/salt in vivo, such as βENaC, γENaC, NCC, and NKCC-2. Further examination showed that NaBu downregulated the expression of mineralocorticoid receptor (MR) and serum and glucocorticoid-dependent protein kinase 1 (SGK1) in DOCA/salt-treated rats or aldosterone-treated human renal tubular duct epithelial cells. These results provide evidence that NaBu may attenuate DOCA/salt-induced hypertension and renal damage by inhibiting the MR/SGK1 pathway.

Abstract P132: A Kidney-Targeted Nanoparticle To Augment Renal Lymphatic Density Decreases Blood Pressure In Mice With L-NAME- And Angiotensin II-Induced Hypertension

Chronic interstitial inflammation and renal infiltration of activated immune cells play an integral role in hypertension. Lymphatic vessels attenuate inflammation by trafficking activated immune cells and excess fluid from the interstitial space to lymph nodes. Previously, our laboratory demonstrated that genetically inducing renal lymphangiogenesis could treat hypertension in three different mouse models. In an effort to translate these findings into a clinical treatment, we hypothesized that a targeted nanoparticle could deliver the pro-lymphangiogenic factor VEGF-C156S to the kidney, induce lymphangiogenesis, and lower blood pressure in hypertensive mice. A micellar nanoparticle was developed with the capacity to deliver protein to the kidney, as demonstrated through delivery trials. This nanoparticle was loaded with VEGF-C156S and injected into mice with LNAME-induced hypertension (LHTN) or angiotensin II-induced hypertension (AIIHTN) via tail vein every 3 days. Compared to hypertensive mice injected with VEGF-C156S only (no nanoparticle) every 3 days, nanoparticle-treated mice exhibited a significantly lower systolic blood pressure (SBP) after 4 injections (LHTN SBP: 160±5 vs. 120±3 mmHg, p&lt;0.001; AIIHTN SBP: 150±8 vs. 126±6 mmHg, p=0.03). Immunolabeled kidney sections from nanoparticle-treated LHTN mice showed a significant increase in podoplanin+ pixels, corresponding to an increase in lymphatic vessel density (p&lt;0.01). A 5-fold increase in renal gene expression of podoplanin in nanoparticle-treated LHTN mice further supported this finding (p=0.01). Flow cytometric analysis of the nanoparticle-treated LHTN mice showed decreased renal CD45+F4/80+CD11c- cells, while AIIHTN mice revealed decreased levels of renal CD45+CD3e+, CD45+CD4+CD8-, and CD45+F4/80+CD11c+ cells (p&lt;0.01, p=0.03, and p&lt;0.001, respectively) when compared to their respective hypertensive groups. These data support our previous findings that expanding the renal lymphatic vasculature can treat existing hypertension by reducing renal immune cells. The results of this study may provide clinicians with a renal lymphatic-targeted therapeutic for treating hypertensive patients.