Salt-sensitive Hypertension Research Articles

Sympathoinhibitory electroacupuncture (EA) interacts positively with anti-inflammatory EA alleviating blood pressure in hypertensive rats.

Elevated sympathetic activity and chronic inflammation are known contributory factors observed in hypertension. We have observed that sympathoinhibitory electroacupuncture (SI-EA) at acupoints ST36-37 alleviates sympathetic activity and hypertension. Additionally, EA at acupoints SP6-7 exerts anti-inflammatory (AI-EA) effects. However, it is not known whether simultaneous stimulation of this combination of acupoints attenuates or enhances individual effects. A 2 × 2 factorial design was used to test the hypothesis that combining SI-EA and AI-EA (cEA) leads to greater reduction of hypertension by decreasing sympathetic activity and inflammation in hypertensive rats than either set of acupoints alone. Dahl salt-sensitive hypertensive (DSSH) rats were treated with four EA regimens including cEA, SI-EA, AI-EA, and sham-EA twice weekly for five weeks. A group of normotensive (NTN) rats served as control. Systolic and diastolic BP (SBP and DBP) and heart rate (HR) were measured non-invasively by tail-cuff. Plasma norepinephrine (NE), high-sensitivity C-reactive protein (hs-CRP) and interleukin 6 (IL-6) concentrations were determined with ELISA at the completion of treatments. DSSH rats on high salt diet progressively developed moderate hypertension within five weeks. DSSH rats treated with sham-EA showed continuous increase in SBP and DBP and elevations in plasma NE, hs-CRP, and IL-6 levels relative to NTN control. Both SI-EA and cEA decreased SBP and DBP, and had corresponding changes in biomarkers (NE, hs-CRP, and IL-6) compared with sham-EA. AI-EA prevented SBP and DBP elevation and decreased IL-6 and hs-CRP relative to sham-EA. Importantly in DSSH rats that received repetitive cEA treatment, SI-EA interacted positively with AI-EA leading to greater reduction of SBP, DBP, NE, hs-CRP, and IL-6 than SI-EA or AI-EA alone. These data suggest that by targeting both elevated sympathetic activity and chronic inflammation, cEA regimen results in a greater reduction of BP effects in treating hypertension compared to using individual SI-EA or AI-EA alone.

Indole Propionic Acid Increases T Regulatory Cells and Decreases T Helper 17 Cells and Blood Pressure in Mice with Salt-Sensitive Hypertension.

Hypertension affects over a billion adults worldwide and is a major risk factor for cardiovascular disease. Studies have reported that the microbiota and its metabolites regulate hypertension pathophysiology. Recently, tryptophan metabolites have been identified to contribute to and inhibit the progression of metabolic disorders and cardiovascular diseases, including hypertension. Indole propionic acid (IPA) is a tryptophan metabolite with reported protective effects in neurodegenerative and cardiovascular diseases; however, its involvement in renal immunomodulation and sodium handling in hypertension is unknown. In the current study, targeted metabolomic analysis revealed decreased serum and fecal IPA levels in mice with L-arginine methyl ester hydrochloride (L-NAME)/high salt diet-induced hypertension (LSHTN) compared to normotensive control mice. Additionally, kidneys from LSHTN mice had increased T helper 17 (Th17) cells and decreased T regulatory (Treg) cells. Dietary IPA supplementation in LSHTN mice for 3 weeks resulted in decreased systolic blood pressure, along with increased total 24 h and fractional sodium excretion. Kidney immunophenotyping demonstrated decreased Th17 cells and a trend toward increased Treg cells in IPA-supplemented LSHTN mice. In vitro, naïve T cells from control mice were skewed into Th17 or Treg cells. The presence of IPA decreased Th17 cells and increased Treg cells after 3 days. These results identify a direct role for IPA in attenuating renal Th17 cells and increasing Treg cells, leading to improved sodium handling and decreased blood pressure. IPA may be a potential metabolite-based therapeutic option for hypertension.

High-Salt Diet Aggravates Endothelial-to-Mesenchymal Transition in Glomerular Fibrosis in Dahl Salt-Sensitive Rats.

Both diabetic and hypertensive nephropathy eventually progress to glomerulosclerosis. Previous studies revealed a potential role of endothelial-to-mesenchymal transition (EndMT) in the pathophysiology of glomerulosclerosis in diabetic rats. Therefore, we hypothesized that EndMT was also involved in the development of glomerulosclerosis in salt-sensitive hypertension. We aimed to explore the effects of high-salt diet on endothelial-to-mesenchymal transition (EndMT) in glomerulosclerosis in Dahl salt-sensitive (Dahl-SS) rats. Eight-week-old male rats were fed high-salt (8%NaCl; DSH group) or normal salt (0.3%NaCl; DSN group) for eight weeks, with systolic blood pressure (SBP), serum creatinine, urea, 24-hour urinary protein/sodium, renal interlobar artery blood flow, and pathological examination measured. We also examined endothelial-(CD31) and fibrosis-related protein(α-SMA) expressions in glomeruli. High-salt diet increased SBP (DSH vs. DSN, 205.2 ± 8.9 vs. 135.4 ± 7.9 mm Hg, P < 0.01), 24-hour urinary protein (132.55 ± 11.75 vs. 23.52 ± 5.94 mg/day, P < 0.05), urine sodium excretions (14.09 ± 1.49 vs. 0.47 ± 0.06 mmol/day, P < 0.05), and renal interlobar artery resistance. Glomerulosclerosis increased (26.1 ± 4.6 vs. 7.3 ± 1.6%, P < 0.05), glomerular CD31 expressions decreased while α-SMA expression increased in DSH group. Immunofluorescence staining showed that CD31 and α-SMA co-expressed in glomeruli of the DSH group. The degree of glomerulosclerosis negatively correlated with CD31 expressions (r = -0.823, P < 0.01) but positively correlated with α-SMA expressions (r = 0.936, P < 0.01). We demonstrated that a high-salt diet led to glomerulosclerosis involving the EndMT process, which played an essential role in glomerulosclerosis in hypertensive Dahl-SS rats.

Renal Medullary Overexpression of Sphingosine-1-Phosphate Receptor 1 Transgene Attenuates Deoxycorticosterone Acetate (DOCA)-Salt Hypertension.

Our previous studies showed that renal medullary sphingosine-1-phosphate receptor 1 (S1PR1) mediated sodium excretion, high salt intake increased S1PR1 level, deoxycorticosterone acetate (DOCA) blocked high salt-induced S1PR1 in the renal medulla, and that conditional knockout of S1PR1 in the collecting duct aggravated DOCA-salt hypertension. The present study tested the hypothesis that overexpression of S1PR1 transgene in the renal medulla attenuates the sodium retention and hypertension in DOCA-salt mouse model. Male C57BL/6J mice received renal medullary transfection of control or S1PR1-expressing plasmids and then DOCA-salt treatment. Renal sodium excretion and arterial pressure were compared between control and S1PR1-overexpressed mice in response to high salt loading or pressure natriuresis. S1PR1-transfected mice showed significantly enhanced urinary sodium excretion in response to acute sodium loading (0.93 ± 0.27 in control vs. 4.72 ± 1.12 µmol/min/gKW in S1PR1-overexpressed mice, P < 0.05) and the pressure natriuresis (3.58 ± 1.77 vs. 9.52 ± 1.38, P < 0.05), less positive sodium balance in response to chronic high-salt intake (3.05 ± 0.39 vs. 1.65 ± 0.39 mmol/72 hr, P < 0.05), and consequently, the attenuation of DOCA-salt hypertension (134.2 ± 6.79 vs. 109.8 ± 3.54 mm Hg, P < 0.05). The αENaC protein amount in the renal medulla was not changed, however, the βENaC was significantly decreased and the γENaC was significantly increased in S1PR1-overexpressed mice. The immunostaining showed apical membrane translocation of γENaC, while no change of αENaC and βENaC in control mice, and that the apical membrane translocation of γENaC was blocked in S1PR1-treasffected mice. These results suggested that activation of S1PR1 in the renal medulla attenuates DOCA-induced sodium retention and salt-sensitive hypertension associated with inhibition of ENaC.

Signaling crosstalk between Dipeptidyl Peptidase 4 and Angiotensin II-AT1R plays a role in proximal tubule NHE3 regulation and salt sensitive hypertension

Background: Salt sensitivity to hypertension occurs in 50-60% of all hypertensive patients and is more common in overweight/obese, females and aging population. In salt sensitive hypertension, the renin-angiotensin system (RAS) is known to play a major role and in particular, Angiotensin II (Ang II) regulation of the sodium hydrogen exchanger (NHE3) in the proximal tubule may mediate the pathophysiology. Dipeptidyl peptidase 4 (DPP4), an exopeptidase expressed abundantly on the brush border, binds to NHE3 and DPP4 inhibition reduces NHE3-mediated sodium reabsorption in proximal tubule cells. Interestingly, we observed both in vitro and in vivo that Ang II stimulates DPP4 activity. However, the mechanisms mediating the interplay between Ang II and DPP4 and its role in NHE3 regulation remain unknown. Therefore, we hypothesized that Ang II stimulation of DPP4 activity via crosstalk between AT1R and DPP4 may account for NHE3 modulation and, consequently, salt-sensitive hypertension. Methods: To address this hypothesis, we stimulated OK-AT1R (stable expressing rat AT1R) proximal tubule cells with Ang II (10 -8 to 10 -10 M), OKP cells with Ang II 10 -8 M and microperfused rat nephrons (Ang II 10 -10 M). In addition, sodium balance studies were conducted on DPP4-/- mice with high salt (1% sodium choride in drinking water) and low salt (0.02% in diet) Results: In OK-AT1R cells, we observed a dose-dependent increase in DPP4 activity, which was inhibited with AT1R blockade, and a battery of kinase inhibitors, including those for Src, PI3K, mTOR, S6K, ERK, and EGFR. Blockade with the DPP4 inhibitor MK-0626 not only suppressed DPP4 activity but partially suppressed Ang II-mediated signaling via EGFR, src, PI3K, mTOR and S6K. Further dissection of the pathways revealed that the blockade of src, which binds to both AT1R and DPP4, mimicked many of the effects of AT1R and DPP4 blockade. Mechanistically, blockade of motor proteins myosin II and myosin VI, which have been shown to redistribute with DPP4 and NHE3 along the microvilli microdomains, did not inhibit Ang II-induced DPP4 activity suggesting other mechanisms (phosphorylation) may underlie Ang II-DPP4 crosstalk in proximal tubule cells. Importantly, we found that DPP4 inhibition blocked Ang II-mediated stimulation of NHE3 activity (pHi) in vitro (OKP cells) and JHCO 3 - in vivo (rat nephrons). In addition, we observed that DPP4 gene-deleted mice lose more salt under both high and low salt ingestion and that DPP4 deficiency lowers BP under conditions of high salt, suggesting that DPP4 may regulate salt-sensitive hypertension. In conclusion, Ang II-AT1R and DPP4 likely crosstalk via binding to src and/or PI3-K, and inhibition of DPP4 suppresses Ang II-AT1R signaling and downstream effectors, including NHE3. Moreover, inhibition of signaling downstream of AT1R can suppress DPP4 activity, suggesting that AT1R and DPP4 act in concert but not via subcellular redistribution via motor proteins. The Sao Paulo Research Foundation - FAPESP (2021/14534-3), Sao Paulo, SP, Brazil to Flavia Martins and Dr. Adriana Girardi; NIH/NIDDK - K08 DK115886 to Dr. Ravi Nistala This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

T-cells regulate albuminuria but not blood pressure or tissue damage in the Dahl SSCD247+/+ rat model of hypertension

Previous studies have demonstrated a key role for renal inflammation in the progression of salt-sensitive hypertension (SS-HTN). Pharmacological and genetic inhibition of inflammation blunts the progression of hypertension in several models, including Dahl salt-sensitive (SS) rats. Traditionally, it has been difficult to separate the pathological effects of renal T-cells from those of exposure of the kidney to elevated pressure since the inhibition of renal inflammation results in a reduction in blood pressure, and vice versa. Therefore, improved renal function and reduced injury after the suppression of inflammation may be the result of reduced renal T-cells, reduced blood pressure, or both. In these studies, we took advantage of a loss of protection from SS-HTN in SS rats lacking T-cells, SS CD247-/- rats, to isolate the pathological effects of renal T-cells from those of hypertension on the kidney.Studies were performed on male SS CD247+/+ and SS CD247-/- rats obtained from heterozygous breeders. From birth rats were maintained on a purified control salt diet (CS, 0.4% NaCl) and switched to a high-salt diet (HS, 4.0% NaCl) during experiments.In contrast to previous studies, we found that MAP following 21-days of HS was not significantly different between SS CD247+/+ and SS CD247-/- rats (SS CD247+/+ : 156 ± 2.2 mmHg, n=16 vs SS CD247-/- : 151 ± 2.2mmHg, n=14, p=0.05). Using flow-cytometry we confirmed that the loss of protection from SS-HTN was not due to the reconstitution of T-cells in the SS CD247-/- rats. T-cells were absent in the kidneys of SS CD247-/- rats after 21-days of HS, comprising 0.06 ± 0.015% of live renal cells, compared to 26.08 ± 2.13% in SS CD247+/+ rats (p= 0.006, n=4+7).Despite the equivalent levels of SS-HTN, we found that the SS CD247-/- rats demonstrated significantly lower levels of albuminuria than their wild-type littermates (528 ± 73 mg/g, n=5 vs 963 ± 156 mg/g, n=7, p=0.008). In contrast to the albuminuria, we found that kidney injury, specifically tubular protein casts and glomerular injury, were not significantly different between SS CD247+/+ and SS CD247-/- rats, while there was a significant correlation between these factors and MAP at tissue collection. RNA-sequencing data confirmed this correlation between injury and MAP – only 99 genes differed significantly in the outer medulla of SS CD247-/- and SS CD247+/+ rats, while &gt;4000 genes differed significantly across the range of pressures evaluated.These findings demonstrate that the pathological effects of renal T-cells are primarily manifested in albuminuria and that the effects of pressure substantially outweigh the effects of T-cell infiltration on histologic injury and transcriptomics of the kidney in SS-HTN. R01HL152166. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

FBLN2 Abates Epigenetic-mediated Salt-sensitive Hypertension Through Regulation of NCC Trafficking

Epigenetics play a role in the development of hypertension. However, the precise mechanisms of how histone modification in renal tubule cells regulates blood pressure is largely undiscovered. In this study, we investigated the role of renal Kdm6a in salt-sensitive hypertension using a kidney tubular cell-specific Kdm6a gene knockout (Kdm6a cKO) mouse model. First, we demonstrated that knockout of Kdm6a in the renal tubular cells resulted in an increase of H3K27me3 associated with the development of hypertension in both male and female Kdm6a cKO mice. To explore the mechanism of H3K27me3-mediated hypertension, we performed a ChIP seq to identify the target genes of H3K27me3 in the renal tubular cells. Data showed that H3K27me3 regulated several physiological pathways in the renal tubular cells, including sodium transport in proximal convoluted tubule (PCT), and blood pressure regulation in distal convoluted tubule (DCT). Indeed, we found that the expressions of NCC, NKCC2, and AQP2 were upregulated in kidneys of Kdm6a cKO mice. Furthermore, ChIP seq data reveals that FBLN2, an extracellular matrix protein, appears to be a target of H3K27me3, which was downregulated in the kidney of Kdm6a cKO mice determined by real-time PCR, Western, and IHC. Notably, we demonstrated that FBLN2 abated high salt-induced hypertension through downregulation of NCC, NKCC2, and AQP2 in the tubular cells of Kdm6a cKO mice. Interestingly, pre-treatment of FBLN2 prevented salt-sensitive hypertension in these mice. Additionally, we have provided evidence that FBLN2 may regulate blood pressure through mediating the NCC cell membrane trafficking. Using a super high-resolution microscopy, we demonstrated that FBLN2 reduced levels of cell surface NCC in DCT cells cultured in hypotonic medium. Here, we have first demonstrated that FBLN2 may be a target gene of H3K27me3 in epigenetic-mediated hypertension. FBLN2 treatment may prevent and treat salt-sensitive hypertension. NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Enhanced Intrarenal pro-renin and salt sensitive hypertension in a mouse model of Type-1 diabetes

Hypertension affects up to 50% of patients with type 1 diabetes. Despite aggressive anti-hypertensive therapy, at least 30% of diabetic patients develop chronic kidney disease. Renin is the rate-limiting enzyme in the activation of the RAS and is essential for blood pressure control. Renin is expressed in Juxtaglomerular (JG) cells, as an immature pro-peptide (pro-renin), and then stored as active renin. In diabetic humans, plasma pro-renin is enhanced while renin remains unchanged or low. We hypothesize that intrarenal renin and pro-renin are enhanced in type 1 diabetes, which could potentially contribute to hypertension and diabetic nephropathy. We used the Akita mice strain, one of the few mouse models of diabetes mellitus that shows features of the human disease including hypertension and kidney damage. We found that Akita mice had higher blood pressure as measured by tailcuff (mmHg: C57=109±2.8; Aki=120.6±2.5;p&lt;0.05, n=6), plasma glucose (in mM: C57=7.71±23.9; Aki=23.2±2.3; p&lt;0.01; n=4) and proteinuria (mg/24hrs: C57 18.2±3.1; Aki: 29.4±3.3; p&lt;0.05; n=5) when compared to C57 (control). In addition, plasma renin was not changed in Akita mice (n=6; p= NS). Histological staining of kidney cuts with a renin antibody (recognizes both renin and pro-renin) showed overall decreased total renin expression in Akita mice (O.D C57: 46.6±1.5; Akita=30.7±3.5). Surprisingly, Western Blots of renal cortical lysates and JG cells showed that pro-renin (43-45 kDa) expression was 79±21% higher in male Akita diabetic mice (p&lt;0.05), demonstrating increased intra-renal pro-renin in Akita. The mechanism driving the increase in pro-renin is unknown. We previously showed that ROS potently stimulate renin release from JG cells. We found that Akita mice have increased urinary isoprostanes, supporting higher kidney ROS (ng/24hrs: C57:3.5±0.6 Aki=6.0±0.8 p&lt;0.05; n=5). We measured H202 levels by transducing primary JG cells in culture with the fluorescent H202 protein sensor HyPer and found that JG cells isolated from Akita mice had higher baseline levels of H202 (C57= 100, Akita=117±7%, p&lt;0.05). An activated intrarenal RAS contributes to salt sensitive hypertension, and at least one third of diabetic patients are salt-sensitive. It is not known whether diabetic Akita mice are salt sensitive. Therefore, we tested the effect of a high salt diet on blood pressure in Akita and C57 mice measured by radiotelemetry. High salt increased systolic blood pressure (SBP) in Akita (from 109±5 to 138±6 mmHg, p&lt;0.001) but not in control C57 (from 104±2 to 117±6 mmHg, NS). We conclude that intrarenal renin and pro-renin is enhanced in Akita mice which also exhibit salt sensitive hypertension. The mechanism may involve elevated superoxide and H202 levels within JG cells stimulating pro-renin expression and release. AHA-SDG; NIH-NIDDK-RO3 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Divergent Sex-Specific Effects of Toll-like Receptor 4 and Cluster of Differentiation 14 in Dahl Salt-Sensitive Hypertension

Renal inflammation accompanies hypertension and renal damage in Dahl Salt-Sensitive rats fed a high salt diet (HS, 4.0% NaCl). Via a transcriptomics approach, we previously found cluster of differentiation 14 (CD14) and toll-like receptor 4 (TLR4), partner molecules involved in innate immune system activation, to be upregulated in renal medullary tissue of Dahl SS rats after a HS challenge. A subsequent study demonstrated that both CD14 and TLR4 are upregulated in macrophages infiltrating the kidneys of Dahl SS fed HS. Interestingly, CD14 deletion on the Dahl SS background confers an amplification of hypertension and renal damage in females but has no effect in males. Given this finding, we examined sex-specific effects of TLR4 deletion in the pathogenesis of Dahl SS hypertension. The current study tested the hypothesis that TLR4 knockout differentially modulates salt-sensitivity in males versus females. A mutation was generated in the TLR4 gene on the Dahl SS background using CRISPR-Cas9 technology to create a 1-basepair frameshift mutation resulting in a premature stop codon. SS TLR4+/+ and SS TLR4-/- males (n=10) and females (n=6), maintained on a low salt chow (LS, 0.4% NaCl), underwent telemeter implantation at approximately 7 weeks of age for continuous monitoring of blood pressure. Following recovery and baseline blood pressure recordings, animals were switched to a HS chow for 3 weeks. Urine samples were collected once during the LS period and weekly throughout the HS challenge. Kidneys were flushed and harvested for analysis. Mean arterial pressure (MAP) was not different between the groups during the baseline period (117.3 ± 1, 116.9 ± 1, 117.3 ± 1, 116.9 ± 2 mmHg). Throughout the HS challenge, MAP was significantly reduced in the male SS TLR4-/- compared to male SS TLR4+/+ (day 19: 150.8 ± 4 vs. 162.5 ± 5 mmHg, p=0.010), but MAP was unchanged in female SS TLR4-/- compared to SSTLR4+/+ (151.6 ± 4 vs 155.3 ± 4 mmHg, p=0.554). Similarly, urinary albumin excretion was reduced in the male SS TLR4-/- versus SS TLR4+/+ (170.3 ± 27 vs 321.3 ± 63 mg/day, p=0.023), indicative of reduced renal injury in these animals. Albuminuria was not observed to be different between female SS TLR4-/- and SS TLR4+/+ (149.7 ± 24 vs 145.9 ± 14, p=0.448). At the end of the HS challenge, immune cells were isolated from the kidneys of these animals and characterized by flow cytometric analysis. We observed no difference in either the circulating or infiltrating immune cell profile between wildtypes and knockouts. These results indicate a male-specific effect of TLR4 deletion and suggest that TLR4 promotes salt-sensitive hypertension and renal damage in males. This finding contrasts with the effect of TLR4’s partner molecule, CD14, which attenuates SS hypertension in females. Further studies are needed to elucidate the molecular mechanisms of TLR4/CD14 activation and signaling during salt-sensitive hypertension. HL161231 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The effect of time-restricted feeding on kidney pathology in salt sensitive hypertension

Salt-sensitivity (SS) continues to be a large contributor to hypertension and increases the risk of developing chronic kidney disease. Treatment options remain limited to consumption of a low-sodium diet and administration of different pharmaceuticals and yet some individuals remain hypertensive. The goal of this study was to examine whether time-restricted feeding (TRF) can lessen renal damage secondary to SS hypertension. TRF has been previously shown to improve inflammation, dyslipidemia, and hypertension in cardiometabolic disorders, however it has not been well studied in SS. When fed a high salt (HS) diet, the Dahl SS rat, a well-known animal model of SS hypertension, experiences enhanced Na+ reabsorption and increased blood pressure followed by exacerbated renal pathology, mimicking what is seen clinically. Increased Na+ reabsorption requires high amounts of ATP, which is normally produced via fatty acid beta oxidation (FAO). Published and preliminary studies have revealed that SS rats experience a decrease in renal FAO and associated lipotoxicity. We hypothesized that TRF would improve renal FAO and slow the progression of renal injury seen in SS hypertension. Male SS rats (10-wk-old) were divided into either the HS ad libitum or the HS TRF group (n=8/group) and underwent a two-week protocol. During week one, all rats had ad libitum access to the HS diet to develop hypertension. During week two, TRF was implemented by providing food for 8 hours during the active cycle. Prior to endpoint, but immediately after the feeding period, anesthetized blood pressure was measured via carotid artery catheterization. HS TRF rats experienced a significant increase in systolic and diastolic blood pressure compared to the HS ad libitum group (SBP 175.56 ± 11.43 mmHg vs 141.27 ± 8.68 mmHg; DBP 118.92 ± 7.37 mmHg vs 95.14 ± 6.01 mmHg; mean ± SEM; p &lt; 0.05, t-test). Despite an increase in blood pressure, analysis of renal fibrosis revealed that HS TRF rats had a significant decrease in cortical and outer medullary (OM) fibrosis compared to the ad libitum fed groups (cortical: 2.58 ± 0.068% vs 5.21± 0.44%; OM: 3.09 ± 0.31% vs 6.04 ± 0.81%; mean ± SEM; p &lt; 0.05, t-test). Assessment of renal lipid accumulation revealed that HS TRF rats appear to have reduced lipid droplets compared to the HS ad libitum rats. Further, HS TRF rats had decreased body weight adjusted ventricle weight compared to the HS ad libitum group (0.31 ± 0.0058% vs 0.35 ± 0.012%; mean ± SEM; p &lt; 0.05, t-test). Together these data suggest the elevated blood pressure measured in the HS TRF group may be transient, resulting from the salt load following the feeding period. This study revealed that TRF may have the potential to reduce renal pathology seen in SS hypertension. Future studies will address how blood pressure changes with timing of the salt load and how TRF impacts renal FAO. Support provided by NIH Grant T32HL007852 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex-specific splenic inflammation underlies sexual dimorphic effects of ACE2 in hypertension

Introduction: Sex differences in salt-sensitive hypertension and pharmacotherapy are commonly recognized. However, the mechanisms are largely unknown. ACE inhibitor is less effective in females than males, suggesting a better protective mechanism of the renin angiotensin system (RAS) in female hypertension. Angiotensin converting enzyme (ACE) 2, located on the X chromosome, is thereby a central candidate of sex differences in hypertension and pharmacotherapy. Thus, our objective is to test the sex specific protective role of ACE2 in salt hypertension. Since the females have a more regulatory RAS than males, we hypothesized that absence of ACE2 resulted in a higher blood pressure and more inflammation in females than males. Methods: ACE2 exon 2 was globally deleted using CRISPR/Cas9, resulting in a novel rat model in the Dahl salt-sensitive (S) background: male Ace2-/y and female Ace2-/+, Ace2-/-. Blood pressure was measured by telemetry in male (S n=7; S Ace2-/y n=6) and female (S n=7; Ace2-/+ n=7; Ace2-/- n=5) rats on 3 weeks high salt diets. Serum ACE2 activity was measured using ELISA. Spleen as the harbor of systemic immunity was studied for Inflammatory markers by gene expression analysis and flow cytometry. Results: Compared to sex-matched S rats, (1) serum ACE2 activity was decreased in female Ace2-/- (0.28 fold, p&lt;0.0001) and male Ace2-/y (0.3 fold, p&lt;0.0001); (2) systolic blood pressure was increased in female Ace2-/- (+67.89 mmHg, p&lt;0.0001) and male Ace2-/y (+48.64 mmHg, p&lt;0.0001) rats. Importantly, a sex-specific upregulation of the pro-inflammatory cytokines were found exclusively in the female Ace2-/- rat spleens: Il6 (1.8 fold, p&lt;0.05), Il1b (2.1 fold, p&lt;0.05) and Il17rc (2.3 fold, p&lt;0.01). In contrast, male Ace2-/y rats showed no difference in Il6 and Il1b, but downregulation of the splenic Il17rc (0.78 fold, p&lt;0.05), Th17 cells (IL17a on CD4+ cells, 0.76 fold, p&lt;0.01), regulatory T cells (FOXP3 on CD4+ cells, 0.49 fold, p&lt;0.01), CD68+ macrophage (0.76 fold, p&lt;0.01) and CD11bc+ dendritic cells (0.89 fold, p&lt;0.001).Furthermore, compared with female S rats, heterozygous female Ace2-/+ rats had decreased serum ACE2 activity (0.6 fold, p&lt;0.001), rescued blood pressure increase (+7.5 mmHg, p&lt;0.0001) and no increases in splenic inflammatory cytokines Il6, Il1b and Il17rc. Conclusions: Despite significantly lower serum ACE2 activity in heterozygous female Ace2-/+ rats, the protection in blood pressure and splenic inflammation was mainly preserved. This suggests a protective role of ACE2, beyond RAS, in inflammation-associated high blood pressure. Moreover, this novel protective role of ACE2 is exclusively in female salt hypertension, evidenced by greater increases in systolic blood pressure and splenic inflammation in female only. NIH R21AI164449 and R21AG079357. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cellular and Molecular Mechanisms Regulating the Normal Physiological Function of ENaC in Salt-Sensitive Hypertension

High blood pressure is a significant risk factor for cardiovascular disease. Many details about the molecular mechanisms that regulate blood pressure are unknown. The Epithelial Na + Channel (ENaC) is the final arbiter of Na + excretion in the kidneys. Appropriate renal sodium excretion is a key factor in the regulation of blood pressure. Abnormalities in ENaC function have been directly linked to several human diseases of blood pressure. The kinase, casein kinase II (CKII), phosphorylates ENaC but the physiological importance of this is obscure. The CKII phosphorylation site within ENaC resides within a canonical “anchor” ankyrin binding motif. Phosphorylation of Nav and KCNQ channels by CKII acts as a molecular “switch” favoring the binding of ankyrin-3 (Ank-3). The binding of Ank-3 facilitates the proper membrane localization of these channels increasing their activity. Here, we tested the premise that phosphorylation of ENaC by CKII within “anchor” motifs is necessary and sufficient for Ank-3 binding to the channel, which is required for normal channel locale and function, and the proper regulation of renal Na+ excretion and blood pressure. We tested this hypothesis using endpoint measurements including analyses of ENaC activity and trafficking to the plasma membrane, and quantification of CKII-dependent ENaC-Ank3 binding. This was addressed by combining total internal reflection fluorescence microscopy with fluorescence recovery after photo bleaching (TIRF/FRAP) to follow movement of ENaC toward the plasma membrane in living cells and electrophysiology of ENaC activity in split-open collecting ducts from principal cell-specific CKII and Ank3 knockout mice. We also used whole animal physiological studies of sodium excretion and blood pressure in knockout mice to understand the physiological consequences of CKII and Ank3 regulation of ENaC. In addition, scanning mutagenesis was used to identify key residues in the overlapping CKII and Ank3 sites in ENaC. Furthermore, we tested whether CKII influences Ank-3 binding to β-ENaC subunit using fluorescence resonance energy transfer (FRET). Findings showed that disrupting CKII signaling and abrogation of the CKII phosphorylation and Ank3 binding site within ENaC decreases channel trafficking toward the plasma membrane and activity, promotes improper sodium excretion, and decreases blood pressure in KO mice. These results showed how and when CKII phosphorylation of ENaC functions as a “switch” to favor Ank-3 binding to increase channel activity to include having a detailed understanding of the mechanisms mediating this regulation, and a rich appreciation of the physiological consequences of such regulation. This work was supported by National Institutes of Health (NIH) grants R01 HL139841 and R01 DK113816. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A novel small molecule inhibitor of vascular endothelin signaling and hypertension

Augmented endothelin-1 (ET-1) signaling causes profound vasoconstriction, smooth muscle proliferation, endothelial dysfunction, oxidative stress, and vascular inflammation and sensitivity, which are often present in resistant hypertension, salt-sensitive hypertension and hypertension among African Americans. Here, we sought to examine the role of indole-3 acetic acid (IAA) as a novel inhibitor of vascular ET-1 signaling and hypertension. IAA, popularly known as auxin, is the most potent plant growth hormone, which is also produced in the human body via tryptophan metabolism by gut microbiota. Although the role of IAA as a potent plant growth hormone has been characterized, little is known about the biological activity of this compound in humans. Importantly, studies on the role of IAA as a regulator of arterial contractility, vascular inflammation and hypertension remain completely unexplored. Our data demonstrates that IAA inhibits ET-1-evoked vasoconstriction in resistance mesenteric arteries, ROS production in human aortic SMCs, expression of adhesion molecule VCAM-1 on EC surface, leukocyte adhesion, and ECE-1 activity. IAA dose-dependently inhibits the activity of a purified human endothelin converting enzyme-1 (ECE-1) that converts the precursor big ET to its active form ET-1 and is upregulated in hypertension. Consistent with these findings, we show that treatment of Dahl salt-sensitive (DSS) hypertensive rats, a model of augmented ET-1 signaling, with an oral extended-release, in situ gel-based formulation of IAA (IAA-oragel) reduces blood pressure (BP) elevation, suggesting a key role for IAA in BP regulation. Taken together, this study presents a novel inquiry into regulation of ET-1 function by IAA and could identify IAA as the first dual action inhibitor of the ET system. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CD8+ T cells stimulate renal sodium transporters contributing to salt-sensitive hypertension

Impaired salt handling by the kidneys acts as one source of hypertension. However, the exact identity of this kidney defect remains unknown. As established recently, the adaptive immunity, T cells in particular, acts as an important contributor to the pathogenesis of hypertension. In this regard, our laboratory has reported that CD8+ T cells (CD8Ts), when adoptively transferred from salt-sensitive hypertensive mice to normotensive mice, stimulate Sodium Chloride Cotransporter (NCC). Through this stimulation, sodium retention occurs, and salt-sensitive hypertension develops. However, whether other sodium transporters within the nephron are affected by hypertensive CD8Ts and contribute to this pathogenesis of hypertension remains uncharacterized. We hypothesize hypertensive CD8Ts will also have an impact on other sodium channels. Here, we demonstrate the impact of hypertensive CD8Ts on sodium-potassium-co-transporter (NKCC) and epithelial sodium channel (ENaCs) in the kidney and the potential of these regulations contributing to salt-sensitive hypertension.Through in vivo studies, we employed a CD8T-adoptive transfer model as described in our previous publications. The kidneys from mice receiving adoptive transfer of hypertensive CD8Ts were harvested and protein levels of NKCC, αENaC, βENaC, and γENaC were analyzed by western blot. With in vitro studies, we co-cultured mouse CD8Ts with Collecting Duct epithelial cells (CDs, M1 cells) to examine the direct interaction between CD8Ts and CDs in media containing various levels of corticosteroids. Similar western blots were performed after this co-culture, as well as measurement of sodium retention through an intracellular sodium indicator and flow cytometry, as reported previously.In the results of in vivo studies, we found that the kidneys of mice receiving adoptive transfer of hypertensive CD8Ts demonstrated higher expression of NKCC, βENaC and γENaC, but no change in αENaC. In the results from in vitro studies, we further found that in charcoal-stripped co-culture conditions, pre-activated CD8Ts stimulate higher expression of ENaC subunits and ENaC-mediated sodium retention in M1 cells. However, these effects are masked in culture conditions with physiological hormone levels or with additional corticosteroids.Overall, our results suggest that, as a potential kidney defect, hypertensive CD8Ts strongly stimulate NCC and NKCC in the kidney to mediate excessive salt retention. Although CD8Ts themselves can increase ENaC, this effect might be masked in physiologic conditions. NIH R01-HL146713 (Mu), AHA 15BGIA25730047 (Mu), and SPaT T32-GM10699 (Deck) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alpha-7 Nicotinic Acetylcholine Receptors Contribute to the Combined Electroacupuncture Modulation of Hypertension in Salt-Sensitive Rats

Elevated sympathetic activities and low-grade chronic inflammation are observed in hypertensive (HTNive) patients and animals including dahl salt-sensitive hypertensive (DSSH) rats. We have shown that stimulation of the deep peroneal nerve and the tibial nerve by combined electroacupuncture (cEA) at the combination of two sets of acupoints ST36-37 and SP6-7 decreased blood pressure (BP) and proinflammatory cytokines in HTNive subjects through sympathoinhibition and anti-inflammation. Acetylcholine (ACh), the primary neurotransmitter of parasympathetic nerves, is known to participate in modulation of inflammation. We hypothesized that α-7 nicotinic acetylcholine receptors (α7nAChR) play an important role in the BP lowering effects of cEA in DSSH rats. DSS rats were fed with 4% salt diet and randomly allocated into three groups (4 rats/group). Once they developed moderate hypertension (SBP 160±5 mmHg), one group was treated with Sham-EA (no electrical stimulations), and two groups were treated with cEA. Both cEA and Sham-EA groups were given twice weekly for eight weeks. Methyllycaconitine (MLA) is a α7nAChR antagonist. MLA (2.4 mg/kg) was injected intraperitoneally (ip) daily into one group of cEA treated rats (n=4) during the last three weeks with cEA treatments. In a similar manner, the other cEA group and the Sham-EA group received daily saline ip injection for three weeks. Another four DSS rats were fed with standard rat chow and treated as the normotensive (NTN) controls. Systolic/diastolic BP (SBP/DBP) of rats were measured with Tail-cuff via CODA system once a week. At the end of the experiment, compared to Sham-EA rats (207.8±2.5 mmHg, DBP 144.6±3.2 mmHg), cEA decreased SBP (138.8±0.8 mmHg) by 33.2% and DBP (98.6±1.7 mmHg) by 24.25%. Blockade of α7nAChR with MLA partially reversed the cEA BP lowering effects (SBP 193.4±2.6 mmHg and DBP 136.5±2.4 mmHg), and BPs in cEA+MLA treated rats were still significantly lower than Sham-EA-treated rats after the reveral. BPs of NTNive rats remained in the normal BP ranges throughout the 13 weeks of the experiment (from 135.9±2.8 to 134.2±1.1 mmHg). In conclusion, systemic antagonism of α7nAChR with MLA partially reversed the BP lowering effects of cEA in DSSH rats. National Institute of Health National Center for Complementary &amp; Integrative Health Grant (1RO1AT011306). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of SGLT2 Inhibition on the transcriptomic and proteomic profile of non-diabetic Dahl SS Rats

Sodium-Glucose Cotransporter-2 (SGLT2) inhibitors are new, highly effective drugs for treating type 2 diabetes mellitus. SGLT2 inhibitors also substantially reduced the risk of hospitalization for heart failure. Previous studies in rodents and humans have shown that SGLT2 inhibitors lower blood pressure levels, cardiovascular risk events, and kidney diseases in subjects with and without diabetes mellitus. However, the mechanisms behind those effects are still unknown. Our previous study showed that SGLT2 inhibition blunted the development of salt-sensitive (SS) hypertension and caused glucosuria and natriuresis in Dahl SS rats without affecting the expression and activity of Na + channels and changes in the renin-angiotensin-aldosterone system. Here, we aim to investigate the effect of SGLT2 treatment on non-diabetic SS rats using multi-omics analysis, which may provide insights into the underlying mechanisms. 8-week-old male SS rats were treated with or without SGLT2 inhibitor (dapagliflozin, 2 mg/kg/day in drinking water) for 3 weeks. Kidney cortex and medulla tissue were collected for RNA-Seq analysis. Proteomics analysis was performed on kidney cortex tissue and plasma. The data were further processed by Ingenuity Pathway Analysis (IPA).RNA-Seq analysis indicated that despite SLGT2 mainly being expressed in the kidney cortex, dapagliflozin treatment altered the expression of more genes in the medulla (855 in the cortex vs 2244 differentially expressed genes (DEGs) in the medulla). Dapagliflozin treatment also caused significant changes in multiple genes related to inflammation and hypertension, with IPA analysis predicting the downregulation of both functions. In hypertension network Adrb3, Retn, Foxn4 were the most upregulated genes, and Ptgs1, Nos1, Oxtr – downregulated. In addition, disease and biological function enrichment analysis revealed that the altered genes were mainly related to the following functions: lipid metabolism, small molecule biochemistry, amino acid metabolism, cell cycle and morphology, and molecular transport. Interestingly, lipid metabolism was predicted to be downregulated in the cortex but upregulated in the medulla.Proteomic analysis identified 450 differentially expressed proteins (DEPs) in the kidney cortex and 22 in plasma. Disease and biological function enrichment analysis on proteomics data revealed mostly the same biological functions as RNA-Seq data. In addition, canonical pathways analysis based on RNA-Seq and proteomics data revealed that endothelin-1 and oxytocin signaling pathways were the most downregulated pathways in the kidney cortex and medulla under the dapagliflozin treatment. Urea cycle and glucocorticoid receptor signaling were the most changed canonical pathway in proteomic data. In conclusion, our results indicated that inhibition of SGLT2 by dapagliflozin altered various genes and proteins in the kidney of Dahl SS rats, which may provide insights for future mechanistic studies. This research was supported by National Institutes of Health grants R35 HL135749 (to AS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deletion of PREX1 (Phosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor 1) worsens salt sensitive hypertension and kidney damage in Dahl S rats

Prex1 (Phosphatidylinositol 3,4,5-trisphosphate-dependent RAC exchanger 1) is a Rac-specific guanine nucleotide exchanging factor and an integrator of signaling between Rac1 and protein kinase A (PKA). Both Rac1 and PKA are involved in pathways associated with salt sensitive hypertension. However, the roe of Prex1 in renal function and salt sensitive hypertension has not been studied. To investigate the role of Prex1 in salt sensitive hypertension. We used a Prex1 knockout (KO) rat on the Dahl salt sensitive genetic background generated by the RGERC at the Medical College of Wisconsin. Genotyping showed that the Prex1 gene was deleted. We studied the expression and localization of Prex1 in kidney sections using immunolabeling and confocal microscopy. Prex1 protein was mainly located in renal tubules, infiltrating inflammatory cells, and endothelial cells of large vessels. We then studied if deletion of Prex1 altered salt sensitivity of blood pressure. We found that both male and female WT Dahl S and Prex1 KO developed salt sensitivity after 2 weeks of 4% high salt diet. However, the increase in blood pressure was higher in male Prex1 KO Dahl S rats (Prex1 KO: 33± 7, vs male WT: 19± 2 mmHg, p&lt;0.05). In females Prex1 KO, high salt increased BP by 31± 3 mmHg whereas the increase was lower in female WT Dahl S (17± 1 mmHg, p&lt;0.05). Rats were placed in metabolic cages for urine collection. Male rats had increased albuminuria after high salt diet for 2 weeks that was not different between WT and Prex1 KO. Female rats had a smaller increase in albuminuria which was not different between WT and KO. Given the expression of Prex1 in inflammatory cells we quantified macrophage infiltration in the kidney after high salt diet. Male Prex1 KO rats had significantly higher macrophage infiltration compared to WT (WT: 141±10 vs Prex1 KO: 337± 18, p&lt;0.05). Female Prex1 KO rats also had higher macrophage infiltration after high salt diet, but the total number of macrophages was lower than males (103 ± 7 female WT vs 208 ± 18 female Prex1 KO). We conclude that Prex1 protects against salt sensitive hypertension and inflammatory cell infiltration in Dahl salt sensitive rats such that its deletion worsens both phenotypes. Females Dahl S were somewhat protected against salt induced hypertension and this response was not worsened in Prex1 KO. Future experiments will study the role of Prex1 in renal NaCl transport. NIDDK-R56DK131114 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Development of maternal syndrome during pregnancy enhances the severity of post-partum salt-sensitive hypertension and renal damage in the Dahl SS rat

Preeclampsia (PE) is a pregnancy-specific disorder that is characterized by hypertension and proteinuria (maternal syndrome). Our group has demonstrated that Dahl salt-sensitive rats maintained on a low salt casein-based diet (SSC) develop maternal syndrome, while Dahl SS rats maintained on a low salt wheat gluten-based diet (SSWG) are protected. Population-based data demonstrated woman that experience a preeclamptic event are at an increased risk for developing hypertension and kidney disease after the pregnancy relative to woman who have a normotensive pregnancy. Since the SSC are susceptible to developing maternal syndrome, we tested the hypothesis that following a single pregnancy SSC rats would exhibit greater salt sensitive hypertension and renal damage relative to SSWG rats in response to a postpartum high salt (HS) challenge. To test this hypothesis SSC and SSWG rats were exposed to a single pregnancy, allowed to deliver naturally and then pups were weaned at 3 weeks. Following a postpartum urinalysis, dams recovered for one week before a two week HS diet challenge. Blood pressure and proteinuria were tracked throughout the study and immune cell infiltration was assessed at the end of the HS challenge. Despite exhibiting comparable baseline MAP measured via telemetry (135±6 vs 126±4mmHg, p&gt;0.05) and urinary protein excretion levels (42.3±5 vs 26.7.1±7 mg/day, p&gt;0.05, n=4-5/group), the SSC rats exhibit significantly elevated postpartum MAP (147±7 vs 131±2 mmHg, p&lt;0.05) and proteinuria (230±40 vs 75±11 mg/day, p&lt;0.05) relative to the SSWG mated rats. Additionally, when these animals were challenged with a postpartum HS diet, the SSC mated rats exhibited an exacerbated response in both MAP (ΔMAP: 22±5 vs 6±3, p&lt;0.05) and proteinuria (300±25 vs 122±20 mg/day, p&lt;0.05) that is accompanied with a significant reduction in creatinine clearance (CC: 0.31±0.08 vs 0.63±0.02 mL/min/g, p&lt;0.05) relative to the SSWG mated rats. These findings are specific to pregnancy in the SSC rats since diet-matched virgin controls only exhibited a slight ΔMAP (SSC Virgin: 10±4mmHg, SSWG Virgin: 9±1mmHg) in response to a HS diet and comparable CC to the SSWG mated rats (SSC Virgin: 0.48±0.07, SSWG Virgin: 0.63±0.06 mL/min/g, p&gt;0.05). These renal damage phenotypes observed in the mated SSC are associated with a two-fold increase in infiltrating immune cells including monocytes/macrophages, T and B cells relative to all other groups. These data suggest the renal demand of an adverse pregnancy that occurs in maternal syndrome is exacerbated with a secondary insult such as a high salt diet. Future studies will investigate the mechanisms behind these pregnancy-induced renal damage phenotypes and how an adverse pregnancy can lead to worsened health outcomes for both mother and offspring. HL157549, HL161231 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The mechanism of CD8+ T cell activation in promoting salt-sensitive hypertension

Background and Hypothesis: T cells play a critical role in hypertension; however, the molecules driving this interaction were unclear. We recently showed that CD8Ts are activated during the initial phase of blood pressure elevation and directly interact with the distal convoluted tubule (DCT) through the interferon gamma (IFNγ)-PDL1 pathway. Here we identified that these CD8Ts are activated during this initial phase via pro-inflammatory extracellular ATP stimulating their P2X7 receptors, leading to enhanced IFNγ production and increased sodium retention in tubular cells that interacted with these CD8Ts. We predict the CD8T P2X7 receptor is contributing to hypertension and abrogating this receptor on CD8Ts will block CD8T-tubular interaction and lower blood pressure. Methods: To determine whether CD8Ts activation in hypertension requires T cell receptor (TCR) stimulation, we used transgenic OT1 mice and induced hypertension via the DOCA + salt (DOCA) model, measuring blood pressure and confirming enhanced CD8T activation despite no exposure to ovalbumin. In in-vitro studies, we co-cultured DCTs with pre-stimulated CD8Ts and measured PDL1 expression and sodium retention in DCTs via flow cytometry. CD8Ts were obtained from WT or P2X7 KO mice and stimulated with BZATP or ATP using calcium influx (intracellular calcium indicator Fluo-4), IFNγ transcription, and glycolytic activity (Seahorse) as activation endpoints. To confirm in-vivo, the activation profiles of CD8Ts obtained from sham or DOCA treated WT and P2X7 KO mice were measured using rt-qPCR and flow cytometry. To confirm the role of P2X7 receptors on CD8Ts, we performed adoptive transfer of DOCA CD8Ts (WT or P2X7 KO) to the alternative model and transferred naive WT CD8Ts to P2X7 KO mice receiving DOCA treatment. All blood pressure was measured via radio-biotelemetry. Results: We found OT1 CD8Ts become activated to the DOCA presser, produce more IFNγ, and contribute to salt-sensitive hypertension without antigen-mediated TCR stimulation. P2X7 receptor agonists BZATP and ATP drove CD8T calcium influx, increased IFNγ transcription, and increased glycolytic activity in CD8Ts – indicative of T cell activation; as such, CD8Ts pre-stimulated with the P2X7 agonist BZATP increased tubular PDL1 expression and sodium retention more than naive CD8Ts in our co-culture model. DOCA treated P2X7 KO CD8Ts were unable to increase blood pressure in our adoptive transfer model; however, transferring sham WT CD8Ts to DOCA P2X7 KO mice restored blood pressure response to the presser to WT levels. Conclusion: Stimulation of the P2X7 receptor contributes to CD8T activation in the pathogenesis of hypertension, resulting in enhanced sodium retention. Deficiency of this receptor on CD8Ts prevents activation by ATP and results in blunted blood pressure elevation in two models of hypertension; as such, the CD8T P2X7 receptor may be a promising target for improving blood pressure management. NIH R01-HL146713, AHA 15BGIA25730047, SPaT 5T32GM106999-09 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CAMP stimulated NKCC2 phosphorylation at Thr-96,101 is in part independent from SPAK: a Role for TNIK

Abnormally enhanced NaCl reabsorption by the thick ascending limb of the loop of Henle (TAL) contributes to the development of salt-sensitivity. NaCl reabsorption by the TAL is mediated by the apical Na/K/2Cl cotransporter NKCC2. NKCC2 activity can be stimulated by phosphorylation at Thr 96,101 by upstream kinases SPAK or OSR1. We found that Thr 96,101 phosphorylation is enhanced in TALs of Dahl Salt Sensitive rats (Dahl SS) on normal or high salt diet. Genetic deletion of SPAK in Dahl SS lowers NKCC2 phosphorylation by 40% and blunts salt-sensitive hypertension but does not completely restore these to baseline. Other kinases may be involved in NKCC2 phosphorylation. Using a targeted proteomics approach, we identified TNIK (Traf2 and NCK interacting kinase) as a kinase that binds and phosphorylates Thr 96,101 in NKCC2 in rats and mice (manuscript under revision). In addition, we and others found that cAMP is a potent stimulus for NKCC2 Thr-96,101 phosphorylation. We hypothesize that TNIK is in part responsible for cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. First, we tested the ability of a novel TNIK inhibitor (NCB-0846) to decrease baseline NKCC2 phosphorylation in Dahl SS rats. Treating TALs from Dahl SS with NCB-0846 (0.1 μM) for 25 min decreased NKCC2 Thr 96,101 phosphorylation by 21 ± 2% compared to the vehicle group (p&lt;0.0001, n=3). Next, we measured NKCC2 phosphorylation in rats with genetic deletion of SPAK in a Dahl SS background. In SPAK KO rats, the cAMP analogue db-cAMP (500 μM) increased NKCC2 phosphorylation at Thr 96,101 by 626 ± 46% (p&lt;0.0001, n=3) and the TNIK inhibitor NCB-0846, blunted cAMP-stimulated NKCC2 phosphorylation by 56 ± 4.3% (p=0.0013, n=3). There was no significant decrease in phosphorylation at phospho-Ser 126 NKCC2 (cAMP=46 ± 7-fold vs cAMP+NCB= 31±5-fold, n.s). These data indicate that in the absence of SPAK, TNIK mediates cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. To further support this, we studied TALs from whole animal TNIK knock-out mice (TNIK KO). db-cAMP (500 μM) increased NKCC2 phosphorylation by 1330 ± 268% in wild-type mice (p&lt;0.01) whereas cAMP-stimulated NKCC2 phosphorylation was blunted by 49 ± 15% in TNIK KO TALs (p&lt;0.05, n=3). No significant difference was found in cAMP-stimulated NKCC2-Ser 126 phosphorylation in TNIK KO (WT=23 ± 12-fold vs TNIK KO=15 ± 4-fold, n=3). We conclude that enhanced NKCC2 phosphorylation at baseline in Dahl SS TALs is in part caused by TNIK and that cAMP-stimulated NKCC2 Thr-96,101 phosphorylation in TALs is in part independent from SPAK and involves TNIK. Our data point to potential role for TNIK in renal ion transport, blood pressure regulation and urine concentration. AHA15GRNT25710369 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.