Role In Salt-sensitive Hypertension Research Articles

Deletion of ADAM17 on microglia attenuates salt-sensitive hypertension

Previous work from our group identified ADAM17-mediated shedding as a major mechanism responsible for ACE2 shedding, thus impairing ACE2 compensatory activity and contributing to the development of salt sensitive hypertension. While microglia have been shown to contribute to salt sensitive hypertension, the mechanisms responsible are unknown. Accordingly, we hypothesize that ADAM17 promotes microglia priming, sympatho-excitation and neuro-inflammation in salt sensitive hypertension, and thus targeting ADAM17 or its maturation, via iRhom2, provides a multi-pronged strategy to normalize BP. Here, we aimed to evaluate whether deletion of ADAM17 on microglia attenuates hypertension. Uninephrectomized male and female mice (14–16-week-old, n=10/group) with selective deletion of ADAM17 in microglia (Cx3CR1-ADAM17tom) or with global deletion of iRhom2 (iRhom2-KO) and littermate controls (Cx3CR1-Cretom) mice were used to monitor blood pressure (BP telemetry, DSI) at baseline and following the initiation of salt sensitive hypertension (DOCA 1mg/g body weight sc + 1% saline po for 3 weeks). Following the establishment of hypertension (7 days post DOCA implantation) mice were injected with tamoxifen (200 nl ICV) and monitored for 2 more weeks. At the end of the treatment, mice were euthanized, and their brains were collected. Baseline mean arterial pressure (MAP) was not different in Cx3CR1-ADAM17tom, iRhom2-KO compared to controls (102±1, 106±1 and 104±1 mmHg, respectively). Surprisingly, all mice developed hypertension following DOCA implantation, including iRhom2 KO (Cx3CR1-ADAM17tom: 131±1, iRhom2 KO: 131±1, control: 126±1 mmHg, respectively. p<0.05). Interestingly, following tamoxifen injection Cx3CR1-ADAM17tom mice showed a blunted hypertension compared to the control group (MAP: 122±0.2 vs. 146±0.5 mmHg, one way ANOVA, p<0.05) suggesting that ADAM17 in microglia contributes to salt sensitive hypertension. In vitro, human microglia cells (iCell microglia — hIPSC) were shown to express lower ACE2 protein (0.03 ± 0.1 vs. 1.18 ± 0.3, Student’s t test, p<0.05) when exposed to Ang-II (100 ng) with a trend for ADAM17 upregulation (1.5 ± 0.3 vs. 1.1 ± 0.1, p<0.05). Together, our data suggest that overactivity of the renin-angiotensin system is associated with overexpression of ADAM17 in microglia and a simultaneous downregulation of ACE2. In addition, knockdown of ADAM17 specifically on microglia blunted the development of hypertension. We conclude that ADAM17 expression in microglia plays a pivotal role in salt sensitive hypertension. This work was supported in part by a research grant from the National Institutes of Health (HL163588). 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.

Global Deletion of DPP4 Prevents the Development of Salt-sensitive Hypertension in Obese Mice via Regulation of Sodium and Water Excretion

Salt-sensitivity of hypertension (HTN) occurs in up to 50% of hypertensive patients and is more common in the older population, Blacks and the obesity/diabetes/hypertension subsets. The renin-angiotensin aldosterone system (RAAS), in particular Angiotensin II (Ang II) and aldosterone known to regulate the Na+/H+ exchanger isoform 3 (NHE3) and ENaC respectively, is thought to be activated in the obesity/diabetes/hypertension subsets and thereby plays an important role in salt-sensitive HTN. RAAS blockade is an acceptable treatment for salt-sensitive HTN along with the use of diuretics. However, not every patient can tolerate RAAS blockade. Recently, dipeptidyl peptidase 4 (DPP4) has been shown to bind to NHE3, colocalizes to the microvillar fractions with active NHE3 and DPP4 inhibition reduces sodium uptake into proximal tubule cells and isolated rat proximal tubules. In addition, DPP4 may act on more distal transporters. Furthermore, data from our lab has revealed that DPP4 knockout (DPP4KO) mice have a lower sodium balance both under conditions of high salt (1.23%) and low salt (0.02%) ingestion suggesting that DPP4 regulates sodium transport and/or balance under physiological conditions. However, whether DPP4 has any effects on salt-sensitive HTN and the underlying mechanisms remain unknown. Therefore, we hypothesized that the absence of DPP4 could mitigate the development of salt-sensitive HTN in an obesity model, by improving the natriuretic and diuretic response mediated by NHE3 and/or distal mechanisms. To this end, 6-8wk old DPP4KO and C57Bl/6 littermate wild-type (WT) mice were fed a control (CD) or Western diet (WD, high in refined fats and sugars) to induce obesity. Circulating (175%) and kidney (125%) DPP4 activity were both elevated in the obese WT mice compared to the CD-fed WT mice (100%) and DPP4KO mice had minimal DPP4 activity as expected. The lack of DPP4 prevented the increase in systolic blood pressure (~15 mmHg) in the obese animals, and the absence of the development of cardiac hypertrophy in this group corroborated the pressure effect. The development of salt-sensitive HTN in the obese WT mice was associated with a lower natriuretic and diuretic response assessed by the salt overload experiment, suggesting higher tubular reabsorption of sodium and water by the nephrons. In turn, the lack of DPP4 prevented the lowering of natriuretic and diuretic response in obese mice, indicating that the beneficial effect of the absence of DPP4 on the development of salt-sensitive HTN is closely related to effects on renal tubule transport. Importantly, a high salt diet increased sodium balance and blood pressure in the obese WT mice and DPP4 deletion mitigated this increase. Furthermore, on low salt diet, the absence of DPP4 led to “salt wasting” in the obese mice similar to the CD-fed mice. Surprisingly, gene and protein expression measurements showed involvement of distal tubule sodium transporters and aquaporins and not NHE3. In contrast, assessment of the DPP4 interactome in obese mice showed binding to Myosin 6 and APN, which may regulate proximal tubule transporters and hypertension. Transdermal measurements showed no changes in the glomerular filtration rate. In conclusion, deletion of DPP4 protects against the development of salt-sensitive hypertension in obese mice by improving the natriuretic and diuretic response likely via regulation of sodium and water transporters. FAPESP, Brazil NIH K08DK115886, USA. 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.

Abstract 19133: Sodium Induced-RGMa Plays a Role in Salt-Sensitive Hypertension

Salt sensitivity of blood pressure, characterized by blood pressure fluctuations that mirror dietary sodium (Na + ) intake, is an independent risk factor for cardiovascular morbidity and mortality in people with and without hypertension. We previously found that elevated Na + uptake via the amiloride-sensitive epithelial sodium channel (ENaC) in antigen-presenting cells leads to the upregulation of isoleuvoglandins and proinflammatory cytokines ultimately leading to T cell activation and salt-sensitive hypertension. In addition, we also showed that SMAD3 activation plays a role in inflammation and salt-sensitive hypertension. In previous studies, RGMa has been shown to be upstream of SMAD3. Furthermore, previous studies have also shown that knockdown of RGMa leads to reduced levels of TGFβ1-induced SMAD3 phosphorylation. The exact mechanism by which elevated extracellular Na + leads to increased blood pressure remains unknown. We hypothesized that RGMa complexes with TGFβ1R1 to promote SMAD3 phosphorylation ultimately contributing to inflammation in salt-sensitive hypertension. To test our hypothesis, we isolated monocytes from human participants (N =11) and treated them with either high (190 mMol/L) or normal (150 mMol/L) Na + in vitro for 72 hours and subsequently performed bulk RNA sequencing. We found that TGFβ1R1 (8837 ± 780.9 vs. 9688 ± 942.5, p =0.350) and TGFβ1R2 (7012 ± 717.0 vs. 7176 ± 633.9, p = 0.059) were being similarly expressed between high or normal salt treatment. Interestingly, RGMa (2253 ± 441.0 vs. 516 ± 84.1, p <0.001) and TGFβ1 (5118 ± 353.9 vs. 9067 ± 826.4, p = 0.073) were significantly upregulated in human monocytes after high Na + treatment compared to normal salt. To further investigate, we performed an in vivo high Na + treatment by utilizing a rigorous salt-loading/depletion protocol on human participants (N = 9) and conducted single-cell transcriptomic analyses. We found a positive correlation between the changes in pulse pressure and RGMa (r = 0.5154, p = 0.1911). Our data suggest that RGMa mediates Na + -induced inflammation and salt-sensitive hypertension which may reveal a novel therapeutic target.

Abstract 082: Dysregulated Microbiota-indole Axis In Male, Not Female Ace2 Deficient Salt-sensitive Hypertension

Introduction: Aberrant tryptophan (Trp) absorption may promote hypertension (HTN) due to gut microbiota converting Trp to pro-hypertensive indoles. Intestinal Ace2 regulates Trp homeostasis, but its role in salt-sensitive HTN is unknown. Further, lack of Ace2 can affect males and females differently because Ace2 is transcribed from the X-chromosome in a genomic region which escapes X-inactivation. Therefore, we hypothesized that sex-specific intestinal ACE2 dependent transport of Trp contributes to salt-sensitive HTN via the microbiota-indole axis. Methodology: 8-10-week-old Dahl salt-sensitive (S) rats and newly generated CRISPR/Cas9 Ace2 deficient S rats of both sexes (N= 7 rats/group) were maintained on either a low (0.3 % NaCl, LS) or high (2 % NaCl, HS) salt diet for 21 days. BP was measured by radiotelemetry for 24 hours on day 21. Serum and cecal content were collected for indole measurement and microbiota analysis. Results: Mean arterial BP of S Ace2 deficient rats on LS was elevated in females by 38 mmHg (S Ace2 -/- 170 ± 10 vs. S 132 ± 3; P =0.0028) and in males by 18 mmHg (S Ace2 -/Y 140 ± 6 vs. S 122 ± 1.5; P =0.01), and further elevated on HS by 68 mmHg in females (S Ace2-/- 211 ± 14 vs. S 143 ± 4.5; P =0.0003) and by 40 mmHg in males (S Ace2-/Y 182 ± 9 vs. S 142 ± 3; P =0.0009). These were associated with elevated serum indole levels mainly in male S Ace2 -/Y rats, compared to S rats on both LS (1.2 ± 0.2 vs. 0.3 ± 0.2 μM; P =0.02) and HS (1.5 ± 0.25 vs. 0.2 ± 0.03 μM; P =0.0001). Microbiota analysis revealed (1) significant shifts in β-diversity in Ace2 deficient rats of both sexes with a higher R value and a lower P value in HS males; (2) Ace2 deficiency resulted in higher abundance of Trp-utilizing, indole-producing bacteria Escherichia - Shigella in males on both LS ( P <0.05) and HS ( P <0.0001). Strikingly, none of these differences were found in females. Conclusion: Lack of Ace2 led to increased mean BP regardless of sex and salt intake. In males, Ace2 deficiency led to increased indole levels with higher abundance of Escherichia - Shigella . Overall microbial changes by salt and ACE2 deficiency were found mainly in males, suggesting a more resilient microbiota in females. Thus, the microbiota-indole axis may be a potential therapeutic target for BP control in males with altered Trp metabolism.

The Interaction of Kidneys and Gut in Development of Salt-Sensitive Hypertension.

The incidence of salt-sensitive hypertension is quite common and varies between 30-60% in hypertensive patients. Regarding the causal role of high salt intake in the development of salt-sensitive hypertension, recent evidence has demonstrated that the gut through its microbiota plays a significant role in its genesis. Besides the gut, the kidneys also play important role in salt-sensitive hypertension and there is clinical and experimental evidence of an interrelationship between the gut and the kidneys in the development of salt-sensitive hypertension through the so-called "gastro-renal axis." The gut besides being an absorptive organ, it is also a hormonal secretory organ involving the secretion of gastrin, dopamine, norepinephrine, angiotensin, and aldosterone which through their action with the kidneys are involved in the development of salt-sensitive hypertension. In addition, the kidneys exert a protective role against the development of hypertension through the secretion of prostaglandins and their vasodilatory action. To assess the current evidence on the role of high salt intake and the interplay of the gut and kidneys in its development, a Medline search of the English literature was contacted between 2012 and 2022, and 46 pertinent papers were selected. These papers together with collateral literature will be discussed in this review.

EMPAGLIFLOZIN IMPROVES IMPAIRED SENSITIVITY OF AFFERENT NEURONS WITH RENAL AXONS

Objective: Afferent renal nerve pathways likely play a role in salt sensitive hypertension. We recently reported that high salt diet (HS) impairs these afferent renal pathways in rats. Now we tested the hypothesis that during HS a decrease in sensitivity of renal afferent neurons is prevented by the SGLT2 inhibitor empagiflozin. Design and method: Respective groups of rats were put on HS containing 8% NaCl or a normal diet. Two groups (HS, controls) received empagiflozin 20 mg/kg BW/day orally. Renal neurons were retrogradely labeled with DiI. In culture, labeled dorsal root ganglion neurons (DRG Th11-L2) with renal afferents were investigated electrophysiologically using current clamp mode to assess action potential generation during current injection. Neurons were characterized as tonic highly active ( less than 5 action potentials, AP) and phasic less active neurons (more than 5 AP upon stimulation). Results: In neurons from rats on HS, the relation of tonic highly active neurons to less active phasic neurons shifted consistently towards phasic units (63,8% tonic neurons in controls vs. 42%* on HS, *p < 0.05, z-test). However, continuous treatment with empagiflozin preserved the proportion of tonic neurons as in controls (67,9% on HS with concomitant administration of empagiflozin). In controls, empagiflozin did not affect the proportion of tonic to phasic neurons (63,8% tonic neurons in controls vs. 67,9% on HS & empagliflozin, p = 0.7, z-test). Blood pressure and heart rate were not altered by HS and/or treatment with any chosen dose of empagiflozin. Conclusions: In rats, chronically elevated sodium intake (8% NaCl) reduced the sensitivity and stimulability of renal afferent DRG neurons. Under these circumstances, concomitant treatment with the SGLT2 inhibitor empagiflozin preserved the function of renal afferent DRG neurons. SGLT 2 inhibitors may help to treat dysfunction of renal innervation in cardiovascular disease.

Abstract 074: Empagliflozin Improves Impaired Sensitivity Of Afferent Neurons With Renal Axons

Afferent renal nerve pathways likely play a role in salt sensitive hypertension. We recently reported that high salt diet (HS) impairs these afferent renal pathways in rats. Now we tested the hypothesis that during HS a decrease in sensitivity of renal afferent neurons is prevented by the SGLT2 inhibitor empagiflozin.Respective groups of rats were put on HS containing 8% NaCl or a normal diet. Two groups (HS, controls) received empagiflozin 20 mg/kg BW/day orally. Renal neurons were retrogradely labeled with DiI. In culture, labeled dorsal root ganglion neurons (DRG Th11-L2) with renal afferents were investigated electrophysiologically using current clamp mode to assess action potential generation during current injection. Neurons were characterized as tonic highly active (> 5 action potentials, AP) and phasic less active neurons (≤ 5 AP upon stimulation. )In neurons from rats on HS, the relation of tonic highly active neurons to less active phasic neurons shifted consistently towards phasic units (63,8% tonic neurons in controls vs. 42%* on HS, *p<0.05, z-test). However, continuous treatment with empagiflozin preserved the proportion of tonic neurons as in controls (67,9% on HS with concomitant administration of empagiflozin). In controls, empagiflozin did not affect the proportion of tonic to phasic neurons (63,8% tonic neurons in controls vs. 67,9% on HS & empagliflozin, p=0.7, z-test). Blood pressure and heart rate were not altered by HS and/or treatment with any chosen dose of empagiflozin.In rats, chronically elevated sodium intake (8% NaCl) reduced the sensitivity and stimulability of renal afferent DRG neurons. Under these circumstances, concomitant treatment with the SGLT2 inhibitor empagiflozin preserved the function of renal afferent DRG neurons. SGLT 2 inhibitors may help to treat dysfunction of renal innervation in cardiovascular disease.

Abstract 11: Single Cell Eccite-seq Analysis Reveal An Important Role Of Monocyte-specific Nlrp3 Inflammasome For Salt-sensitivity Of Hypertension In Humans

Salt sensitivity of blood pressure is an independent predictor of death due to cardiovascular events. Diagnosis of salt-sensitivity is not feasible in the clinic, making it difficult to investigate therapeutic strategies. We hypothesized that NLRP3-inflammasome and IL-1β production in monocytes plays a role in salt-sensitive hypertension. We phenotyped salt-sensitivity of blood pressure using an acute inpatient Weinberger protocol of an isocaloric high salt diet and rapid intravenous salt-loading, followed by low salt diet and furosemide-induced salt-depletion. Ambulatory blood pressure was continuously monitored and averaged for the days of salt-loading and salt-depletion. Blood samples were obtained at baseline, salt-loading, and after salt-depletion. Median age was 54 years (44-55), 3 of the 5 subjects were female, screening systolic blood pressure was 140 mmHg (134-148), diastolic blood pressure was 88 mmHg (84-99), and BMI was 35 kg/m 2 (30-39). Using cell hashing and ECCITE-seq analysis, we profiled transcriptomes in multiple immune cell types using antibody-derived tags (ADTs). UMAP clustering of different cell types were identified by ADTs including monocyte markers CD14 and CD16. Interestingly, UMAP visualization of CD14+ and CD16+ clusters indicated a greater decrease in CD14+ clusters after salt-depletion in the salt-resistant subjects than the salt-sensitive; however the salt-sensitive subjects had a greater decrease in CD16+ clusters than the salt-resistant group after both salt-loading and salt-depletion. These data were confirmed using flow cytometry. Unlike in salt-resistant participants, we found that within monocyte clusters, salt-sensitivity was associated with down regulation of the inflammasome components NLRP3 (0.386 ± 1.18 vs. 0.197 ± 0.778) and IL-1β (0.858 ± 2.32 vs. 0.159 ± 0.925) following salt-depletion. Using flow cytometry, we found Δ% isoLG+ CD14+/CD16+ monocytes correlated with salt-sensitivity of blood pressure (r=0.88, 95% CI, p=0.05). These results suggest that the inflammasome and monocyte activation are dynamically regulated by dietary salt in vivo and can serve as a potential diagnostic biomarker for salt-sensitivity of blood pressure.

Tumor Necrosis Factor‐Alpha Receptor Type‐1 Protein Level Decreases in Renal Cortical But Not in Medullary Tissue During High Salt Intake in Nitric Oxide Deficient Mice

Tumor necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine that is involved in renal injury associated with salt-sensitive hypertension (SSH). In the kidney, TNFα receptor type 1 (TNFR1) activation induces natriuresis, suggesting its’ counter-regulatory role in SSH opposing salt retention. We hypothesize that TNFR1 activity in the kidney is reduced in nitric oxide (NO) deficient condition to facilitate induction of SSH. Experiments were performed in intact (C57BL6 strain) and in 2 models of NO deficient mice: a) chronically treated with NO synthase inhibitor, nitro-L-arginine methyl ester (L-NAME; 0.05 mg/min/g by osmotic mini-pump), b) eNOS gene knockout (KO) mice, which were fed either normal (NS; 0.4% NaCl) or HS (4% NaCl) containing diet for 4 weeks (n=6-7 in each group). Systemic blood pressure (SBP) was measured using tail-cuff plethysmography. The kidneys were isolated and processed for tissue analysis at the end of the experiment. The protein expression of TNFR1 in renal (cortical and medullary) tissues collected from these experimental mice was analyzed using the Western Blot technique. Protein concentration was determined (BCA Assay) using appropriate TNFR1 and GAPDH antibodies. The blot image was quantified with Image J software. HS intake alone did not alter mean SBP in intact mice (NS, 76 ±2.1 vs HS, 78 ±1.4 mmHg) but increased in L-NAME treated mice (104 ±2.8 mmHg) and in eNOSKO mice (107±3.6 mmHg). The quantitative immunoblot analysis showed that TNFR1 protein level (measured as band intensity density with Image -J) was lower in renal cortical (43.9±9.0 vs 110.7±29.8; P<0.05) but not in medullary tissues (114.4±15.5 vs 128.4±97.1; P=n.s.) in L-NAME treated mice compared to untreated control mice during HS intake condition. The intensity of TNFR1 immunoblot analysis in eNOSKO mice also showed showed the similar results (cortical, 23.7 ± 5.0 vs 110.7±29.8; P<0.001 and medullary, 73.6 ±20.8 vs 128.4±97.1; P=n.s). These data indicate that TNFR1 activity is downregulated in the renal cortical tissues in the condition of NO deficiency. Such downregulation of TNFR1 activity in the renal tubules endowed in cortical tissues (proximal and distal convoluted tubules) would minimize sodium excretion during HS intake leading to the development of SSH in NO deficient conditions.

The Mineralocorticoid Receptor in Salt-Sensitive Hypertension and Renal Injury.

Hypertension and its comorbidities pose a major public health problem associated with disease-associated factors related to a modern lifestyle, such high salt intake or obesity. Accumulating evidence has demonstrated that aldosterone and its receptor, the mineralocorticoid receptor (MR), have crucial roles in the development of salt-sensitive hypertension and coexisting cardiovascular and renal injuries. Accordingly, clinical trials have repetitively shown the promising effects of MR blockers in these diseases. We and other researchers have identified novel mechanisms of MR activation involved in salt-sensitive hypertension and renal injury, including the obesity-derived overproduction of aldosterone and ligand-independent signaling. Moreover, recent advances in the analysis of cell-specific and context-dependent mechanisms of MR activation in various tissues-including a classic target of aldosterone, aldosterone-sensitive distal nephrons-are now providing new insights. In this review, we summarize recent updates to our understanding of aldosterone-MR signaling, focusing on its role in salt-sensitive hypertension and renal injury.

Transcriptome Sequencing to Detect the Potential Role of Long Noncoding RNAs in Salt-Sensitive Hypertensive Rats.

Backgrounds Long noncoding RNAs (lncRNAs) play an important role in various biological processes. However, their functions in salt-sensitive hypertension are largely unknown. In this study, the lncRNA-seq technique was employed to compare the expression profiles of lncRNAs and mRNAs in salt-sensitive hypertensive rats. Methods Blood pressure, serum sodium, and urinary creatinine were texted in salt-sensitive and salt-insensitive rats fed with different salt concentrations. High-throughput sequencing was used to detect the expression of lncRNAs and mRNA in the renal medulla of the two groups. Results Blood pressure and urinary sodium/creatinine of high-salt diets of the sensitive group were significantly higher than that in the control group. Serum sodium has no significant difference between the two groups in high-salt diets. NONRATG007131.2 and NONRATG012674.2 were the most different lncRNAs in the high salt-sensitive group. Correlation analysis reveals that Matn1, Serpinb12, Anxa8, and Hspa5 may play an important role in salt-sensitive hypertension. Conclusion This study analyzed the difference in lncRNA and mRNA between salt-sensitive and salt-insensitive rats with different salt diets by high-throughput sequencing. Salt sensitivity and salt concentration were two key factors for the induction of hypertension. We found some potential genes that play an important role in salt-sensitive hypertension.

Abstract P219: Site-1 Protease-Derived Soluble (Pro)Renin Receptor Contributes to Salt-Sensitive Hypertension via Activation of the Intrarenal RAS

Dahl salt-sensitive (SS) hypertensive rats exhibit increased expression of renal (pro)renin receptor (PRR) and aberrant activation of the intrarenal renin-angiotensin system (RAS) with unclear functional implication. Recent evidence demonstrates that soluble PRR (sPRR) serves as a potential regulator of tubular transport via activating Wnt/ β-catenin pathway in the distal nephron and is derived from the cleavage by site-1 protease (S1P). The present study attempted to define the role of sPRR during salt-sensitive hypertension in SS rats. The effect of S1P inhibitor PF-429242 (PF at 20 mg/kg/d via mini-pump) on blood pressure, proteinuria, and urinary renin were tested in SS rats during high salt loading. In a separate group, a recombinant sPRR (sPRR-His at 20 μg/kg/d) was infused via catheterization of jugular vein to SS rats during PF treatment. In High sodium (HS)-loaded SS rats, administration of PF attenuated the hypertension development (MAP on day 10, HS+PF group: 124.5 ± 4.6 mmHg vs. HS group: 144.1 ± 8.2 mmHg) as assessed by radiotelemetry and proteinuria (HS+PF group: 50.9 ± 5.5 μg/24h vs. HS group: 105.3 ± 7.4 μg/24h). PF treatment reduced HS-stimulated urinary sPRR excretion by 65% in SS rats. Supplement of sPRR restored HS-induced hypertension during PF treatment (MAP on day 10, HS+PF+sPRR-His group: 138.7 ± 6.1 vs. HS+PF group: 124.5 ± 4.6 mmHg) and proteinuria (HS+PF+sPRR-His group: 79.0 ± 5.8 μg/24h vs. HS+PF group: 50.9 ± 5.5 μg/24h) in SS rats. Concurrently, PF treatment reduced HS-stimulated urinary renin activity (by 80%), aldosterone excretion (by 36%) and renal medullary prorenin and renin (by 60%), which were all restored by sPRR-His infusion (1.5- to 3-fold). Indices of Wnt/β-catenin pathway including protein levels of renal cytosolic Axin-2 and nuclear β-catenin as assessed by immunoblotting were elevated by HS (3.4-fold, 2.2-fold respectively) in SS but not SR rats, which were all suppressed by S1P inhibition (by 80% and 50% respectively) and restored by sPRR-His infusion. Given β-catenin signaling as a known regulator of intrarenal RAS, our results suggest that S1P-derived sPRR plays a pathogenic role in salt-sensitive hypertension in SS rats via β-catenin/intrarenal RAS pathway.

Abstract 093: Salt Resistance and Renal Gpr83 Function

The G protein coupled receptor 83 (Gpr83) is an orphan receptor that has been associated with essential hypertension. Gpr83 is expressed in the kidney but its function is unknown. We found that Gpr83 is expressed in mouse renal proximal and distal convoluted tubules, as well as in human renal proximal tubule cells (hRPTCs). In C57Bl/6J mice on normal salt diet, the lack of one (Gpr83+/-) or both Gpr83 (Gpr83-/-) alleles resulted in an increase in systolic blood pressure (SBP, ~20 mm Hg (P&lt;0.05; n=4/group, measured under anesthesia) compared with Gpr83+/+ littermates, suggesting that Gpr83 is needed to keep a normal BP. Renal specific Gpr83 silencing by the renal subcapsular infusion of Gpr83 siRNA (3 μg/day; 7 days) increased SBP in C57Bl/6J mice on a normal salt diet, relative to mice treated with nonsilencing siRNA (120±5 vs 98±6 mmHg; P&lt;0.05; n=4/group). A high salt (4% NaCl) diet increased renal Gpr83 transcription by 2 fold (P&lt;0.05; n=4/group) in SJL/J and BALB/c salt resistant mice, relative to C57Bl/6J salt sensitive mice. Gpr83 gene down-regulation in mouse and human renal proximal tubule cells decreased about 50% the expression of nuclear factor of activated T cells 5 (NFAT5). The renal expressions of NFAT5 and the NFAT5 targets UT-A3, Tau T and BGT1 were increased by a high salt diet in salt resistant mice. The neuropeptide PEN (PEN), a ligand of Gpr83, is also expressed in hRPTCs (0.43±0.03 mRNA PEN/mRNA GAPDH). We found that Gpr83 stimulation with PEN (1 μM, 3 hr) decreased the transcription of NFAT5 (0.59±0.05 vs 1.0±0.2fold, P&lt;0.05; n=3-4/group), which was abolished by Gpr83 silencing. Our results suggest that Gpr83 may protect against the development of salt sensitivity. Gpr83 mediated regulation of NFAT5 may play a role in salt-sensitive hypertension.

Abstract P284: Neuron Specific (Pro)renin Receptor Knockout Reduces Sodium Appetite and Attenuates the Development of DOCA-salt Induced Hypertension

The (pro) renin receptor (PRR) is a key component of the renin-angiotensin system that is highly expressed in the brain. We previously showed that the neuronal PRR deletion attenuates deoxycorticosterone acetate (DOCA)-salt induced hypertension. However, the mechanism underlying remains unclear. To test our hypothesis that PRR is involved in the regulation of sodium appetite during DOCA-salt hypertension, we used a neuron-specific PRR knockout (PRRKO) mouse model generated using the Cre-LoxP system. The PRRKO and their wildtype controls (WT) were implanted with 50 mg DOCA pellet with free access to regular water and 0.9 % saline as the drinking solution. Blood pressure (BP) was monitored by telemetry system in conscious free moving mice. The fluid intake and urine output were monitored along 21 days of DOCA-Salt treatment. The BP is significantly lower in PRRKO compared with WT mice following 21 days of DOCA-salt treatment (112 ± 2 vs. 134 ± 7 mmHg, P= 0.0186). Interestingly, we found that saline intake (27.8 ± 1.8 vs. 15.9 ± 1.2 ml/day, P=0.0007) and total fluid intake (31.1 ± 1.9 vs. 21.1 ± 1.4 ml/day, P=0.003) were higher; while the regular water intake was lower (3.4 ± 0.6 vs. 5.2 ± 0.3 ml/day, P=0.03) in WT compared to PRRKO mice. PRR deletion in the neurons reduced sodium appetite presented as the ratio of saline intake over total fluid intake (0.75 ± 0.016 vs. 0.89 ± 0.019, P=0.0005), as well as total sodium intake (2.45 ± 0.19 vs. 4.28 ± 0.28 mmol/day, P=0.0007) compared with WT mice at the end of the protocol. In addition, the urinary sodium excretion was lower (13.3±1.17 vs. 20±1.17 mmol/day, P&lt;0.0001), but not potassium excretion (0.64 ± 0.028 vs. 0.56 ± 0.05 mmol/day, P=0.1291) in PRRKO compared with WT mice; however, there is no difference in urine sodium and potassium concentrations. Furthermore, plasma vasopressin level (19.0 ± 2.7 vs. 33.6 ± 2.7 pg/ml, P=0.0037) is lower in the PRRKO compared with WT mice at the end of DOCA-salt treatment. In summary, PRR deletion in the neurons reduced sodium appetite, circulating vasopressin level, and attenuated the development of DOCA-salt induced hypertension. Taken together, the present findings suggest that PRR regulates the BP and plays a key role in salt-sensitive hypertension, at least in part, by modulating sodium appetite.

Abstract P285: Hydralazine Attenuates the Development of Hypertension in the Female Dahl Salt-sensitive Rat in a T Cell-independent Manner

Introduction: Several studies in Dahl salt-sensitive ( DS ) rats suggest that T cells play a role in salt-sensitive hypertension. To further investigate the role of T cells, we compared T cell profiles in hypertensive DS and normotensive Dahl salt-resistant ( DR ) rats as well as in DS rats treated with hydralazine ( HYD ) to attenuate the development of hypertension. Methods: Mean arterial pressure ( MAP ) was measured by telemetry in DS rats (n=13) from 1 to 4.5 months ( mo ) of age. At 1.5 mo, all of the DR (n=8) and half of the DS rats were treated with vehicle (VEH, n=7). The other half of the DS rats (n=6) received HYD (25 mg/kg/day) in the drinking water. At 4.5 mo, renal T helper ( Th ) and cytotoxic ( Tc ) cells were assessed by multicolor flow cytometry. Results: In the DS kidney, the frequency of CD4 + Th cells [(%): DS-VEH, 76±1.2* vs. DR-VEH, 55±0.7; *p&lt;0.0001; n=7-8/group] was higher while the frequency of CD8 + Tc cells [(%): DS-VEH, 14±1.2* vs. DR-VEH, 35±1; *p&lt;0.0001; n=7-8/group] was lower compared to DR rats. 10 weeks of HYD treatment attenuated the age-associated increase in MAP observed in DS rats [p&lt;0.0001, Two-Way ANOVA (time, treatment); MAP (mmHg): DS-VEH, 157±4 vs. DS-HYD, 133±3; *p&lt;0.0004; n=6-7/group]. HYD had no effect on the frequency of CD4 + [(%):77±1.5] or CD8 + [(%):15.5±0.9] T cells in the kidney of DS rats [(CD4 + ): DS-VEH vs. DS-HYD, p=0.83; (CD8 + ): DS-VEH vs. DS-HYD, p=0.5; n=7-8/group]. In summary, the ratio of Th (CD4 + ) to Tc (CD8 + ) cells is higher in the kidney of DS compared to DR rats and HYD had no effect on the T cell profile in the DS rat kidney under conditions in which the MAP was attenuated by 20 mm Hg. Conclusions: These findings indicate the DS rat has more active Th cells in the kidney compared to the DR rat. Our study also suggests that vasodilators can attenuate the development of hypertension in the DS rat in a Th- and Tc-independent manner.

Increased activity of the orexin system in the paraventricular nucleus contributes to salt-sensitive hypertension.

The orexin system is involved in arginine vasopressin (AVP) regulation, and its overactivation has been implicated in hypertension. However, its role in salt-sensitive hypertension (SSHTN) is unknown. Here, we tested the hypothesis that hyperactivity of the orexin system in the paraventricular nucleus (PVN) contributes to SSHTN via enhancing AVP signaling. Eight-week-old male Dahl salt-sensitive (Dahl S) and age- and sex-matched Sprague-Dawley (SD) rats were placed on a high-salt (HS; 8% NaCl) or normal-salt (NS; 0.4% NaCl) diet for 4 wk. HS intake did not alter mean arterial pressure (MAP), PVN mRNA levels of orexin receptor 1 (OX1R), or OX2R but slightly increased PVN AVP mRNA expression in SD rats. HS diet induced significant increases in MAP and PVN mRNA levels of OX1R, OX2R, and AVP in Dahl S rats. Intracerebroventricular infusion of orexin A (0.2 nmol) dramatically increased AVP mRNA levels and immunoreactivity in the PVN of SD rats. Incubation of cultured hypothalamus neurons from newborn SD rats with orexin A increased AVP mRNA expression, which was attenuated by OX1R blockade. In addition, increased cerebrospinal fluid Na+ concentration through intracerebroventricular infusion of NaCl solution (4 µmol) increased PVN OX1R and AVP mRNA levels and immunoreactivity in SD rats. Furthermore, bilateral PVN microinjection of the OX1R antagonist SB-408124 resulted in a greater reduction in MAP in HS intake (-16 ± 5 mmHg) compared with NS-fed (-4 ± 4 mmHg) anesthetized Dahl S rats. These results suggest that elevated PVN OX1R activation may contribute to SSHTN by enhancing AVP signaling.NEW & NOTEWORTHY To our best knowledge, this study is the first to investigate the involvement of the orexin system in salt-sensitive hypertension. Our results suggest that the orexin system may contribute to the Dahl model of salt-sensitive hypertension by enhancing vasopressin signaling in the hypothalamic paraventricular nucleus.

Targeting Interleukin-1 beta to Suppress Sympathoexcitation in Hypothalamic Paraventricular Nucleus in Dahl Salt-Sensitive Hypertensive Rats.

Findings from our laboratory indicate that expressions of some proinflammatory cytokines such as tumor necrosis factor, interleukin-6 and oxidative stress responses are increased in the hypothalamic paraventricular nucleus (PVN) and contribute to the progression of salt-sensitive hypertension. In this study, we determined whether interleukin-1 beta (IL-1β) activation within the PVN contributes to sympathoexcitation during development of salt-dependent hypertension. Eight-week-old male Dahl salt-sensitive (S) rats received a high-salt diet (HS, 8% NaCl) or a normal-salt diet (NS, 0.3% NaCl) for 6weeks, and all rats were treated with bilateral PVN injection of gevokizumab (IL-1β inhibitor, 1μL of 10μg) or vehicle once a week. The mean arterial pressure (MAP), heart rate (HR) and plasma norepinephrine (NE) were significantly increased in high-salt-fed rats. In addition, rats with high-salt diet had higher levels of NOX-2, NOX-4 [subunits of NAD (P) H oxidase], IL-1β, NLRP3 (NOD-like receptor family pyrin domain containing 3), Fra-LI (an indicator of chronic neuronal activation) and lower levels of IL-10 in the PVN than normal-diet rats. Bilateral PVN injection of gevokizumab decreased MAP, HR and NE, attenuated the levels of oxidative stress and restored the balance of cytokines. These findings suggest that IL-1β activation in the PVN plays a role in salt-sensitive hypertension.

Aldosteroneʼs mechanism of action

Aldosterone's functions and mechanisms of action are different depending on the tissue and the environmental condition. The mineralocorticoid receptor is present in tissues beyond epithelial cells, including the heart and vessels. Furthermore, aldosterone has direct adverse effects by both genomic and rapid/nongenomic actions not only through a nuclear receptor but also through caveolae-mediated intracellular events. Also, multiple environmental-genetic interactions play an important role in salt-sensitive hypertension (SSH) and aldosterone modulation. These findings have reshaped our vision of aldosterone's role in cardiovascular pathophysiology. This review describes new mediators of aldosterone's mechanisms of action: lysine-specific demethylase 1 (LSD1), caveolin 1 (cav-1) and striatin. LSD1, an epigenetic regulator, is involved in the pathogenesis of SSH in both humans and rodents. In addition, cav-1, the main component of caveolae, plays a substantial role in mediating aldosterone pathways of SSH. The mineralocorticoid receptor interacts with cav-1 and is modulated by sodium intake. Finally, striatin, a scaffolding protein, mediates a novel interaction between signalling molecules and mineralocorticoid receptor's rapid effects in the cardiovascular system. Substantial progress in aldosterone's functions and mechanisms of action should facilitate the study of cardiovascular diseases and the role of sodium intake in aldosterone-induced damage.

Hydrogen Sulfide Prevents Hydrogen Peroxide-Induced Activation of Epithelial Sodium Channel through a PTEN/PI(3,4,5)P3 Dependent Pathway

Sodium reabsorption through the epithelial sodium channel (ENaC) at the distal segment of the kidney plays an important role in salt-sensitive hypertension. We reported previously that hydrogen peroxide (H2O2) stimulates ENaC in A6 distal nephron cells via elevation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) in the apical membrane. Here we report that H2S can antagonize H2O2-induced activation of ENaC in A6 cells. Our cell-attached patch-clamp data show that ENaC open probability (PO) was significantly increased by exogenous H2O2, which is consistent with our previous finding. The aberrant activation of ENaC induced by exogenous H2O2 was completely abolished by H2S (0.1 mM NaHS). Pre-treatment of A6 cells with H2S slightly decreased ENaC PO; however, in these cells H2O2 failed to elevate ENaC PO. Confocal microscopy data show that application of exogenous H2O2 to A6 cells significantly increased intracellular reactive oxygen species (ROS) level and induced accumulation of PI(3,4,5)P3 in the apical compartment of the cell membrane. These effects of exogenous H2O2 on intracellular ROS levels and on apical PI(3,4,5)P3 levels were almost completely abolished by treatment of A6 cells with H2S. In addition, H2S significantly inhibited H2O2-induced oxidative inactivation of the tumor suppressor phosphatase and tensin homolog (PTEN) which is a negative regulator of PI(3,4,5)P3. Moreover, BPV(pic), a specific inhibitor of PTEN, elevated PI(3,4,5)P3 and ENaC activity in a manner similar to that of H2O2 in A6 cells. Our data show, for the first time, that H2S prevents H2O2-induced activation of ENaC through a PTEN-PI(3,4,5)P3 dependent pathway.

Immune reactivity to heat shock protein 70 expressed in the kidney is cause of salt-sensitive hypertension

Hypertension affects one-third of the adult population of the world. The causes of hypertension are incompletely understood, but relative impairment of sodium excretion is central to its pathogenesis. Immune cell infiltration in the kidney is a constant finding in hypertension that in association with local angiotensin and oxidants causes a defect in sodium excretion. However, it is unclear if the T cell influx into the kidney responds to nonspecific chemokine cues or is due to antigen-driven immune attraction. We found that T cells in experimentally induced salt-driven hypertension present a CD4 clonal response to heat shock protein 70 (HSP70) that is overexpressed in the kidney. We used a highly preserved amino acid sequence within the HSP molecule to induce immune tolerance associated with the generation of IL-10 producing regulatory T cells. Immune tolerant rats to HSP70 developed minimal renal inflammation and were protected from the development of salt-sensitive hypertension. Adoptive transfer of T lymphocytes isolated from spleen of tolerized rats also reversed hypertension. HSP70 gene delivery to the renal vein of the kidneys of rats sensitized to HSP70 caused an increment in blood pressure in response to a high-salt diet. The HSP70 peptide used in this work induces a strong proliferative response in peripheral blood lymphocytes of patients with essential hypertension. These studies provide evidence that autoimmunity plays a role in salt-sensitive hypertension and identifies HSP70 expressed in the kidney as one key antigen. These findings raise the possibility of novel approaches to the treatment of this condition.