Salt Sensitivity Research Articles

Abstract 33: CD4+ T Cell-Induced Renal Damage and Salt-Sensitive Hypertension is Not Modified by CD8+ T Cells

Salt-Sensitive (SS) hypertension and renal damage in the Dahl SS rat mirrors human disease with T cell infiltration in the kidney accompanying the development of SS hypertension and renal damage. Deletion of T cells on the Dahl SS genetic background (SS CD247-/- ) attenuates hypertension and renal damage, and subsequent replacement of CD4+ T cells alone restores the disease phenotype while the transfer of only CD8+ T cells had no significant effect on the disease phenotype. The present study tested the hypothesis that CD4+ and CD8+ T cells will synergistically act to enhance salt-sensitive hypertension and renal damage in the Dahl SS compared to the injection of only CD4+ T cells. This hypothesis was tested by adoptive transferring purified CD4+ T cells from Dahl SS (~5 million, CD4+ SS→), a combination of ~5 million CD4+ T cells with ~5 million CD8+ T cells from Dahl SS (CD4+CD8+ SS→) or PBS vehicle (PBS→) into the SS CD247-/- rat on postnatal day 5. Animals were instrumented with radiotelemeters at 8 weeks of age. After a week of recovery, baseline measurements of blood pressure and albumin excretion were obtained while rats were fed a low salt (LS, 0.4% NaCl) diet. Rats were then switched to a high salt (HS, 4.0% NaCl) diet for 21 days, with continuous blood pressure measurements and urine collections every 7 days. On day 21, the rats were euthanized, and their kidneys were perfused and collected for immune cell analysis by flow cytometry. During the LS baseline period there was no significant difference observed in mean arterial pressure (MAP) between the CD4+ SS→, CD4+CD8+ SS→, or PBS→ groups (121±2, 122±2, 119±1 mmHg; n=9, 10, 6, respectively). After three weeks of HS, both CD4+ SS→ and CD4+CD8+ SS→ rats demonstrated an equivalent amplification of SS hypertension (176±6 and 179±5 mmHg) and albuminuria (261±53 and 285±37 mg/day, respectively) which was significantly greater than the MAP (159±5 mmHg) and albuminuria (169±29 mg/day) in PBS→. Finally, a flow cytometric analysis at the end of the experiment confirmed the absence of CD3+ T cells in the PBS→ and documented the reconstitution of equal numbers of CD4+ cells in the blood and kidney of the rats receiving CD4+ cells or the combined CD4+ CD8+ cell transfer. We also confirmed the CD4+CD8+ was the only group that had reconstituted CD8+ T cells. These studies demonstrated that CD4+ T cells amplify salt-sensitive hypertension and renal damage in the Dahl SS independently from CD8+ T cells.

Abstract MP34: Muscle Sodium Content and Isolevuglandin Formation in Antigen Presenting Cells in Human Salt Sensitive Hypertension

Background: Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular mortality. However, the mechanisms by which salt exposure affects blood pressure in SSBP are still unclear. We have shown that under high sodium (Na + ) concentrations, Na + enters antigen-presenting cells (APCs) via epithelial Na + channel (ENaC), which leads to isolevuglandin (IsoLG) production and hypertension. Baseline %IsoLG+ APC correlates with SSBP. While the exact location where APCs are activated by salt and trigger an inflammatory response remains to be elucidated, muscle and skin are proposed as potential sites. We hypothesize that muscle and skin Na + levels are associated with SSBP. Methods: We assessed SSBP in 30 hypertensive subjects using an inpatient salt loading and depletion protocol. Flow cytometry was used to identify IsoLG-containing APCs; dendritic cells (DCs), classic (CD14+16-), non-classic (CD14-16+), and intermediate (CD14+16+) monocytes. Salt loading was achieved through saline infusion and a high-salt diet (460mmol/24 hours), while salt depletion was achieved via diuretic administration (furosemide 40mg x3) and a low-salt diet (10mmol/24hours). Baseline muscle and skin Na + levels were assessed with the 23 Na MRI technique. Spearman's correlation was used to analyze the associations. Demographic data are presented with mean and standard error of the mean. Results: Participants’ age was 53 ± 1.34 years, 53% female, baseline blood pressure was 140.2 ± 2.62 / 87.30 ± 1.77 mmHg, and salt sensitivity index was 3.72 ± 1.4 mmHg. We found an inverse correlation between baseline muscle Na + levels and the decrease in IsoLGs from salt loading to depletion (ΔIsoLGs) in DCs (r=-0.50, p=0.007), intermediate (r=-0.42 p=0.0284) and non-classic monocytes (r=-0.39 p=0.0432), but not in classical monocytes. Skin Na + levels were not correlated with any of these measures. There was a decreasing trend of salt sensitivity index with increasing muscle Na + , although this correlation did not reach statistical significance (r=-0.35 p=0.0686) Conclusion: This highlights the potential importance of muscle Na + in SSBP, such that patients with lower muscle Na + exhibited a higher trend of salt sensitivity index and increased variability in IsoLG+ APCs. Moreover, baseline tissue Na + assessment may emerge as a potential tool for evaluating the degree of salt sensitivity and IsoLG activation in SSBP.

Abstract P182: Antigen Presenting Cell-specific Keap1-Nrf2 pathway Mediates Salt-Sensitive Hypertension in Humans

Background: Hypertension remains the leading cause of mortality worldwide and is a primary risk factor for cardiovascular disease. Of the 1.3 billion people globally affected by hypertension, over 50% exhibit salt-sensitivity of blood pressure (SSBP), which leads to higher rates of mortality, morbidity, and kidney damage compared to those with salt-resistant hypertension. Previously our research has shown that high salt intake activates antigen-presenting cells (APCs), causing inflammation and hypertension. This pro-inflammatory response is partly driven by the epithelial sodium channel (ENaC), which facilitates sodium ion transport into APCs. While the Keap1-Nrf2 pathway is known for its role in antioxidant defense and inflammatory responses, its contribution to salt-sensitive hypertension remains unclear. We hypothesize that ENaC-dependent sodium entry triggers inflammation and activates the Sp1-MALAT1-Keap1-Nrf2 signaling axis in APCs, thereby contributing to SSBP. Method: We utilized a well-established inpatient protocol to phenotype participants for salt sensitivity of blood pressure. Additionally, we conducted RNA sequencing (RNAseq) on human monocytes exposed to elevated sodium levels in vitro and performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) analysis on peripheral blood mononuclear cells (PBMCs). Results: We found that whereas high salt did not significantly increases expression of Sp1 (5380.63 ± 359.77 vs 7414.54 ± 434.19, q=0.829), MALAT1 (86888.54 ±12200.12 vs 219429.90 ± 109994.16, q=0.717), Keap1 (1586.54 ± 132.94 vs 2203.72 ± 177.48, q=0.884), it significantly increased Nrf2 expression (5080.36 ± 416.64 vs 5443.54 ± 432.78, q=0.019) in RNAseq analysis of human high salt compared to normal salt treated monocyte. Furthermore, the results of the CITE-seq experiment indicated that changes in the expression of the Sp1 correlate with SSBP (r=0.5205, p=0.038). Conclusion: These preliminary findings reveal a physiological link between inflammation and salt-sensitive hypertension through the Sp1-MALAT1-Keap1-Nrf2 signaling axis in APCs. The activation of APCs mediated by Sp1 could potentially serve as a diagnostic marker for salt sensitivity in blood pressure.

Abstract 49: Activator protein 1 (AP-1) Complex in Antigen Presenting Cells contributes to Salt-Sensitive Blood Pressure in Humans

Salt-sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular morbidity and mortality. The exact mechanism by which salt intake increases blood pressure is complex and not fully understood. We previously found that sodium entry into antigen-presenting cells (APCs) via the epithelial sodium channel (ENaC) activates proinflammatory cytokines to activate T cells and modulate salt-sensitive hypertension. The activator protein 1 (AP-1) transcriptional factors (FOS/JUN) have been implicated in activating the pro-inflammatory pathway, but its role in SSBP is unknown. We hypothesized that high salt activates the AP-1 signaling pathway and inflammation in APCs and contributes to SSBP. Our bulk RNA-sequencing data in human monocytes, demonstrated that high salt increases the expression of the AP-1 gene family, FOS (2,378.1 ± 480.7 vs 6,494.0 ± 945.5, p= 0.0009), and JUN (7,313.9 ± 984.9 vs11,370.0 ± 1,286.3, p= 0.0015) compared to normal salt-treated monocytes. In additional experiments, we enrolled patients with hypertension and phenotype them for SSBP using an established inpatient protocol of salt-loading/depletion and performed single-cell transcriptomic analyses on peripheral blood mononuclear cells (PBMCs). We observed expression of the e FOS (r= 0.5041, p= 0.0464) and JUN (r= 0.7083, p= 0.0021) genes changes in concert with blood pressure in salt-sensitive (SS) but not in salt-resistant (SR) patients. To confirm whether genes expression translates to protein expression, we cultured total splenocytes from salt-sensitive SV/129 mice for 24 hours in either normal (150 mM) or high salt media (190 mM) and performed flow cytometry. Compared to normal salt, high salt-induced a significant increase in the expression of FOS-JUN genes in monocytes of the spleen. We also adoptively transferred PBMCs from salt-sensitive (SS) and salt-resistant (SR) hypertensive individuals into immunodeficient NSG mice, treated with the high salt diet for three weeks and performed flow cytometry on immune cells of the kidney, aorta, and spleen. We found that immune cells from SS people demonstrated a higher propensity to infiltrate mouse tissues with increased expression of FOS-JUN genes than immune cells from SR people. These preliminary findings disclose the role of the AP-1 gene family in salt-sensitive hypertension and may provide a potential therapeutic target for the treatment and diagnosis of SSBP.

Abstract P205: Single-nucleus Sequencing Reveals Endothelial Cell-specific Dysregulation in the Kidney in Multiple Models of Hypertension

The kidney plays a crucial role in the onset and progression of hypertension. To investigate the roles of different renal cell types in hypertension, we applied single-nucleus sequencing and spatial transcriptome sequencing to kidney samples from three well-established hypertension models: C57BL/6 mice treated with Ang II, the Dahl salt-sensitive (SS) rat on high-salt diets, and the spontaneously hypertensive rat (SHR), along with their respective controls including Sprague-Dawley (SD) and Wistar Kyoto (WKY) rats. Our dataset comprised 179,637 nuclei from 24 samples across 12 experimental conditions, identifying 17 major cell types including various epithelial cells, endothelial cells, vascular smooth muscle cells, fibroblasts, and immune cells. At baseline, differentially expressed genes between the SS and SD groups were specifically enriched in endothelial cell pathways related to vascular development. This included expression deficiencies in genes involved in angiogenesis in SS rats, such as Arhgap24 and Nox4. Compensatory expression changes in these genes were observed in the SS rats after a high-salt diet and in 26-week-old SHR, but not in their respective controls. These genes were also associated with multiple blood pressure-associated single-nucleotide polymorphisms (SNPs). Furthermore, changes in endothelial cell communication were most prominent upon stimuli, with impaired PTPRM (Protein Tyrosine Phosphatase Receptor Type M) signaling being a robust feature in hypertension models, involving angiogenic endothelial cells in the SS model and proliferating capillary endothelial cells in the SHR model. Our study highlights the critical role of renal endothelial cell angiogenic dysfunction in the pathogenesis of hypertension and suggests a link between specific renal endothelial cell subtype dysregulation and hypertension development.

Abstract P426: Reduced Nfatc1 Expression Results in Enhanced Pressure Natriuresis and Resistance to Salt-Sensitive Hypertension

Background: Nuclear factor of activated T cells-cytoplasmic 1 (NFATc1) has recently been shown to have a non-canonical role in chronic kidney disease in humans by a trans-ethnic meta-analysis of genome-wide associate studies (GWAS). Follow-up studies in a salt-sensitive mouse model showed that Nfatc1 mRNA expression was suppressed in the kidneys when animals were fed a high-salt diet. Here, we further explore the link between NFATc1 and salt-sensitive (SS) hypertension. Methods: Adult male wild-type (WT) and heterozygous Nfatc1 knockout (Het) mice (2-5 months of age) on the SS 129S6 background were started on standard chow (0.8% NaCl, NSD) and then switched to a high-salt diet (6% NaCl, HSD). Systolic blood pressure (SBP) was measured by either tail cuff manometry with at least 2 weeks of training or implantable radiotelemetry transmitters with at least 2 weeks of post-surgical recovery. Immediately before and for 2-3 days after the change in diet, 24 h urine was collected using metabolic cages, and urinary sodium was measured by flame photometry. Sigmoid pressure-natriuresis curves were generated and the Hill slope was compared between genotypes. Results: WT mice showed a significant increase in SBP between NSD and after 3 weeks on HSD (127.8±9.9 vs 143.4±8.9, p = 0.0089, n = 9). In contrast, Het mice had no change in SBP (125.8±8.7 mmHg vs. 125.7±10.6 mmHg, p = 1.0, n = 9). During the initial switch to the HSD, SBP was not significantly different between WT and Het mice (133.2±2.9 vs. 134.4±3.0, p = 0.45, n = 7/group). However, WT mice had a shallower pressure-natriuresis curve (Hill slope of 0.58 vs. 0.93, 7 mice/group, 25/26 data points). Conclusion: Our data illustrates that decreasing Nfatc1 expression in a mouse model is protective against SS hypertension. While acute increases in SBP in response to HSD remained in Het mice, sodium excretion was increased and SBP remained unchanged after 3 weeks, indicating that NFATc1 has the potential to be a therapeutic target against SS hypertension.

Abstract P206: Renal hemodynamic and transcriptomic changes in response to salt in Dahl salt-sensitive rats

Introduction: Continuous measurements (24/7) of arterial pressure (AP) and renal blood flow (RBF), along with intermittent sampling of arterial and renal venous blood and urine in freely moving rats, have enabled the determination of sequential changes in global substrate metabolism and their association with physiological responses in the kidney. It was found that salt loading in Sprague-Dawley (SD) rats resulted in an increase in RBF and renal O 2 consumption with minimal changes in AP. At the same time, TCA cycle pathway was downregulated with reduced metabolism of free fatty acids and amino acids, while glycolysis was upregulated, in the kidney cortex (Cx). In contrast, no significant changes were observed in metabolism-related pathways in the outer medulla (OM). (PMID: 37575482). Here, we report transcriptomic responses of Dahl salt-sensitive (SS) rats to the HS diet as compared to the salt-resistant SD rats. Method: Male SS rats were fed a 0.4% NaCl (LS) diet. At 8 weeks of age, the rats were implanted with an RBF probe together with an arterial and a renal venous catheter. RBF and AP were measured continuously, and arterial and renal venous blood and urine were sampled when rats were fed the LS and on days 7, 14, and 21 after switching to the 4.0% NaCl (HS) diet. From separate groups of rats subjected to the same protocol, Cx and OM were removed for mRNAseq analysis (Novogene, Inc). Results: When switching to the HS diet, mean AP of SS rats rose from 122 ± 2 to 164 ± 5 mmHg by HS21. RBF (mL/min/g kidney weight) fell significantly over the 3 weeks of the HS diet averaging 7.2 ± 0.6 (LS) to 4.6 ± 0.7 (HS21). The gene set enrichment analysis (GSEA) of the mRNAseq data revealed that only a few metabolic pathways were enriched in HS7 in Cx such as glycosphingolipid biosynthesis, while metabolic pathways such as linolenic acid and arachidonic acid metabolism and glycosphingolipid biosynthesis were enriched in OM. Downregulation of the lysine degradation and glutathione metabolic pathways was observed in both Cx and OM in HS21. In general, fewer metabolic pathways responded to HS in SS rats compared to SD in Cx (22 in SD vs 7 in SS). Conclusion: The Cx of SS rats appear less capable of adjusting to the increased metabolic needs imposed by a HS diet compared to SD rats in contrast to the OM. The functional implications of this are being examined and the metabolomic analysis of blood, tissue and urine samples is currently in progress at the JAX lab.

Abstract P177: High salt modulates Antigen Presenting Cell TNF-alpha production in an Isolevuglandin-dependent manner in Hypertensive Humans

Salt intake is a predisposing factor to high blood pressure in people with salt sensitivity, but the mechanisms are not fully understood. Previously, we found that the epithelial sodium channel facilitates sodium entry into antigen-presenting cells (APCs), leading to the production of Isolevuglandins (IsoLGs) and hypertension via proinflammatory cytokines. TNF-alpha is a major proinflammatory cytokine that is produced in response to oxidative stress. We hypothesize that high salt intake-associated increased production of IsoLGs activates TNF-alpha-dependent mechanisms to contribute to salt-sensitive hypertension. To test this hypothesis, we conducted bulk RNA sequencing on isolated human monocytes with and without high salt. We also performed in vivo studies on humans living with hypertension using the rigorous Weinberger salt loading and depletion protocol at baseline (B), salt loading (SL), and salt depletion (SD) timepoints. We found that in vitro high salt treatment increased human monocyte expression of TNF-α, TNFRSF1A and TNFRSF1B (117.09 ± 23.71 vs 357.82 ± 59.62, p=0.001; 4560 ± 430.02 vs 6189 ± 461.11, p=0.0014 and 43824.73 ± 6462.55 vs 54585.64 ± 5383.58, p=0.0336 respectively, [n=11]) when compared to normal salt. In vivo studies on 16 hypertensive subjects reveal mean blood pressures as baseline (SBP: 137.7 ± 3.1, DBP: 85±2.2, MAP 104.7±2.1 [mmHg]), salt-loading (SBP: 141.3 ± 2.9, DBP: 86.1±2.3, MAP 105.6±2.2 [mmHg]), and salt-depletion (SBP: 138.7 ± 2.9, DBP: 87±2.4, MAP 104.7±2.1 [mmHg]). Using flow cytometry, we determined the buildup of Isolevuglandin adducts in APCs as follows: B: 57.6±6.4, SL: 59.8±4.6, SD: 46.0±7.1 (dendritic cells); B: 78.6±5.8, SL: 77.3±5.7, SD: 61.0±6.9 (CD14 + cells); B: 83.2±4.9, SL: 74.0±6.4, SD: 63.3±8.2 (CD14 + 16 + cells) and B: 63.3± 5.7, SL: 54.2±6.1, SD: 46.8±7.9 (CD16+ cells). In response to salt loading, patient plasma levels of TNF-α were B: 13.8±0.5 pg/mL, SL: 14.1±0.6 pg/mL, and SD: 15.3±1.2 pg/mL respectively. Finally, in vivo single-cell transcriptomic analysis in peripheral blood mononuclear cells revealed that in response to high salt treatment, TNF expression changes dynamically with blood pressure in salt-sensitive, but not salt-resistant patients. These preliminary findings suggest that TNF-α plays a role in salt-sensitive hypertension, possibly following IsoLG-driven reactive oxygen species generation. Further studies are needed to delineate specific dependent mechanisms by which this occurs.

Abstract P391: Role of Renal Dopamine D2 Receptor in Inverse Salt Sensitivity and Salt Sensitivity of Blood Pressure: Insights from Nephron Segment-Specific Deletion Models

The renal dopamine D2 receptor (D2R) is essential in renal and blood pressure (BP) homeostasis. In mice, global Drd2 deletion or renal-selective Drd2 silencing increases the renal expression of proinflammatory factors, renal injury markers, and BP. The D2R is expressed in several nephron segments, including the proximal and distal convoluted tubules. To study the differential effects of D2R in these segments, we generated two mouse models: one with Drd2 deletion only in the S1 and S2 segments of the proximal tubule P SGLT2 ::Cre+ Drd2 fl/fl ( Sglt2 cKO ) and their littermates as controls, P SGLT2 ::Cre- Drd2 fl/fl (Sglt2 WT) and another with deletion of the receptor in the whole nephron P CDHR16 ::Cre+ Drd2 fl/fl ( Cdhr16 cKO ) and their littermates as controls, P CDHR16 ::Cre- Drd2 fl/fl (Cdhr16 WT). In male Sglt2 cKO mice, compared with Sglt2 WT mice, D2R expression in the renal cortex was decreased by 25% (P<0.05, n=5-6/group). In Cdhr16 cKO mice, compared with Cdhr16 WT mice, D2R expression was decreased by 30% (P<0.05; n=3/group). In Sglt2 cKO mice, tubular lumen expansion, an indication of tubular damage, was higher on low salt (0.09%NaCl; LS) than on normal salt (0.4%NaCl; NS) (25.5±.8 vs. 11.4±.0.3; P<0.001) diet. In Cdhr16 cKO mice tubular lumen expansion was also higher on LS than NS (13.9±0.4 vs 8.7±0.2; P<0.001) diet. In Sglt2 cKO mice, BP (carotid artery under anesthesia) was higher on LS (130±2 mm Hg) than on NS (107±2 mm Hg) or HS (4% NaCl) (100±3 mm Hg) (n=10/group) (P<0.05) diet. In Cdhr16 cKO mice, BP (tail cuff, CODA under anesthesia, confirmed by telemetry) was higher on LS (110±8 mm Hg, P<0.02; n=4-5/group) and HS (119±9 mm Hg, P<0.05; n=4/group) diets than on NS diet (84±5 mmHg). On LS diet, UNaV was lower in Sglt2 cKO than Sglt2 WT (1±0.3 vs 7±2 µEq/24h; P< 0.05; n=4-6/group) mice. On LS diet, UNaV was also lower in Cdhr16 cKO than Cdhr16 WT (5±0.9 vs 34±14 µEq/24h; P<0.05; n=3-4/group) mice. These results indicate that a decrease in D2R expression only in the proximal tubule results in inverse salt sensitivity of BP, i.e., BP higher on LS than NS or HS diets, while downregulation of its expression in the whole nephron results in inverse salt sensitivity, as well as salt sensitivity.

O37 LINK BETWEEN NMDA RECEPTOR GluN2C DEFICIENCY AND HYPERTENSION IN MICE

N-methyl-D-aspartate (NMDA) receptors are glutamate receptors located in the CNS and peripheral tissues. We previously demonstrated that kidney NMDA receptors in the connecting/collecting ducts (CNT/CCD) participate in renal autoregulation and kidney vasodilation. Additionally, we found that the use of non-specific NMDA inhibitors increases blood pressure. This study aims to identify which NMDA receptor subtypes are associated with hypertension. We evaluated the RNA transcription of the Grin2C gene (GluN2C subunit) and the Grin2D gene (GluN2D subunit) in the CNT/CCD using in-situ hybridization RNA scope, along with the presence of the CNT/CCD marker AQP-2. Blood pressure was evaluated in C57B mice and global genetic ablated mice on a C57B background: GluN2C-KO and GluN2D-KO. Blood pressure was measured using the tail-cuff method three times a week for two weeks following a three-session training period. Both male and female mice were used. Animals were fed regular and high salt diets (4%). RNA expression of NMDA subunits 2C and 2D was found in AQP2-positive regions of mouse kidneys, consistent with CNT/CCD regions. Females had higher Grin2C transcription levels than males (p<0.01). The average systolic blood pressure (SBP) for C57B control mice was 105.6±10.5 mmHg. SBP in GluN2C-KO mice was 114.6±7.3 mmHg, significantly elevated compared to the matched C57B control mice (p<0.01). SBP in GluN2D-KO mice was 111.6±10.4 mmHg, with no significant differences from the matched control (111.1±4.6 mmHg; p=0.93). GluN2C-KO female tends to have higher SBP tan males, however the difference was not statistical significant. Following a high salt diet challenge, blood pressure did not change in any of the three groups. NMDA receptor subunits GluN2C and GluN2D are expressed in the CNT/CCD region. The genetic absence of GluN2C is associated with increased blood pressure, whereas the lack of GluN2D did not affect SBP. No salt sensitivity was observed in any of the genetically ablated mice. Future research will explore the effects of these subunits on renal hemodynamics.

Abstract 40: Sodium-mediated Blood Pressure Increase in Chronic Kidney Disease and Diabetic Kidney Disease Patients Coincides With Vasodysfunction.

Background: Chronic kidney disease (CKD) and diabetic kidney disease (DKD) patients are advised to limit salt intake, because of its detrimental effects on blood pressure (BP) and albuminuria. The salt-sensitive BP response in these patients may be attributed to impaired renal ability to excrete sodium (Na+) resulting in fluid overload and/or vasodysfunction. To define which factor relates to BP sensitivity in both CKD and DKD, we studied the Na+-mediated BP response in these patients as compared to healthy volunteers (HV). Methods: In this randomized crossover study, CKD patients (KDIGO stage G2/3A, proteinuria >0.5 g/d), Type 2 DKD patients (KDIGO stage G2/3A, proteinuria 0.2 - 0.5 g/d) and HV followed a 7-day low sodium diet (LSD, <50mmol/d) and high sodium diet (HSD, >200mmol/d), respectively. After each diet, assessment of the hemodynamic profile included daytime BP (Mobil-O-Graph) and cardiac output (CO) measurements (using NexfinTM), by which SVR was calculated accordingly. Extracellular fluid volume (ECV) change was assessed with multi-frequency body impedance spectroscopy (Fresenius). Results: We included 20 CKD (mean age 48 yrs, median (IQR) proteinuria 0.7 (0.6-1.6) g/d, mean (SD) eGFR 60.7 (16.3) ml/min/1.73m2) and 8 DKD patients (age 68 yrs, proteinuria 0.3 (0.2-0.4) g/d, eGFR 59.0 (15.9) ml/min/1.73m2), and 16 HV (age 41 yrs). Dietary salt intervention was successful with mean difference in urine Na+ excretion of 185, 168, and 209 mmol/day in CKD, DKD and HV respectively. After HSD, mean (SD) systolic BP change was 9.3 mmHg (8.9; p<0.001) in CKD and 13.8 mmHg (10.4; p<0.01) in DKD, while systolic BP change (3.1 mmHg (6)) in HV was not significant. After HSD, we observed significant ECV expansion in all groups without differences between groups (CKD 1.6 L, DKD 1.6 L, HV 1.4 L, p=0.27), while no effects on CO were observed. After HSD, the mean (SD) SVR in CKD increased with 125.7 dyn*sec*cm-5 (247.9, p=0.04) and in DKD with 125.5 dyn*sec*cm-5 (142.6, p=0.04), while in HC a non-significant decline of -109.2 dyn*sec*cm-5 was observed (358.2, p=0.24). Conclusion: The Na+-mediated BP rise in both CKD and DKD coincides with an increase in SVR. The absence of differences in ECV and CO between the three groups indicates that salt sensitivity in both CKD and DKD is not completely based on fluid overload but might be due to an inability to induce vasodilation.

O67 URINARY PROTEOMIC PROFILING REVEALS MOLECULAR CUES UNDERLYING HIGHER HYPERTENSION SUSCEPTIBILITY IN BLACKS THAN WHITES

Background and Objective: Compared with Whites, Blacks are more prone to hypertension and salt-sensitivity. The objective was to investigate whether sequenced urinary peptide fragments derived from proteins with a key role in cardiovascular or renal structure or function have different levels in sub-Saharan Blacks compared with Whites. Methods: This individual-participant meta-analysis included individuals enrolled in African-PREDICT (476 Blacks and 483 Whites), FLEMENGHO (720 Whites), PROVALID-Austria (467 Whites) and UPRIGHT-HTM (106 Nigerian Blacks and 61 Polish and Slovenian Whites). Urinary peptides were quantified by capillary electrophoresis combined with mass spectrometry. Results: Among 2313 participants (49.7% women), median age was 40.9 years. Of 513 urinary peptides with 70% prevalence, 300 had Bonferroni-corrected significantly different levels among Black and White South Africans sharing the same environment. Analyses contrasting 582 Blacks vs 1731 Whites, sub-Saharan Blacks (582) vs European Whites (1248) replicated the findings. Combining these results with information from public databases, we identified COL4A1, COL4A2, CD99, FAT1, FGA, FXYD2, MGP, MYOCD, PCDH7, PROC, and UMOD as most likely candidates underlying the different susceptibility to hypertension and salt sensitivity between Blacks and Whites. The most relevant enriched pathways were related to hemostasis, platelet activity, collagens and biology of the extracellular matrix, and protein digestion and absorption. Disease Ontology terms comprised small-vessel disease and coronary and macrovascular atherosclerotic disease. Conclusions: MGP and MYOCD being involved in cardiovascular function, FGA in clot formation, FYXD2 and UMOD in salt homeostasis, and COL4A1 and COL4A2 as major component of the glomerular basement membrane are proteins deserving further exploration in molecular and human studies as potential targets for intervention to reduce the excess hypertension risk and associated complications in Blacks.

Abstract P179: The Role of Sodium Induced-RGMa 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 isolevuglandins 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. In additional experiments, 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. Our bulk RNA sequencing analyses revealed there was no difference in expression of TGFβ1R1 (8837 ± 780.9 vs. 9688 ± 942.5, p =0.350) and TGFβ1R2 (7012 ± 717.0 vs. 7176 ± 633.9, p = 0.059). 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. However, single-cell transcriptomic analyses found no correlation between the changes in pulse pressure and RGMa (r = 0.5154, p = 0.1911). Our data suggest that RGMa mediates Na-induced inflammation but may not play a role in salt-sensitive hypertension.

Abstract P147: Inappropriate Activation of the Leptin-Aldosterone-Endothelial Mineralocorticoid Receptor Axis Promotes Salt Sensitivity of Blood Pressure in Young Female Mice

While compelling clinical evidence indicates that women are more salt-sensitive than men, the mechanisms of the onset of women’s salt-sensitive blood pressure (SSBP) remain largely elusive. Herein, we employed salt-sensitive (SS)-Balb/C and salt-resistant (SR)-C57Bl/6 mice to investigate the etiopathology of SSBP in females. We hypothesized that a high-salt diet alters the leptin-aldosterone-endothelial mineralocorticoid receptor axis (ECMR), leading to SSBP and vascular dysfunction in young SS-female mice. Eleven-week-old female mice were fed a normal (0.4%, NSD) or a high salt diet (4%, HSD) for 7-days. We implanted radiotelemeters for BP monitoring at baseline, HSD, and in response to the leptin receptor (LepR) blockade combined with HSD. Following euthanasia, mesenteric arteries are isolated to measure vascular function via wire myography. HSD elevated mean arterial pressure in Balb/C (NSD: 98.4±2.3 vs. HSD: 105.1±0.9 mmHg) but not in C57 mice (NSD: 99.3±0.9 vs. HSD: 101.3±0.5 mmHg). However, SS-Balb/C mice showed superior natriuretic function in response to salt loading experiments (NSD: 13.3% vs. HSD: 42.3%) as opposed to C57 mice (NSD: 25.8% vs. HSD: 35.1%) (N=3). HSD feeding notably blunted endothelial relaxation to acetylcholine in Balb/c (NSD: 96.5% vs. HSD: 75.4%, N=7), an effect not seen in C57. SS-Balb/C mice failed to suppress aldosterone and adrenal aldosterone synthase (CYP11B2) levels (protein and transcript) compared to C57 mice. This concomitated a 1.8-fold increase in adrenal steroidogenic acute regulatory (StAR) mRNA in SS-Balb/C mice. HSD raised CYP11B2 protein levels in the visceral adipose tissue (VAT) of Balb/C mice. Leptin regulating CYP11B2 expression; we measured fat mass, leptin, and LepR expression. HSD enhanced fat accumulation (C57: +0.7% vs. Balb/c: +26.5%, N=12), leptin levels, and LepR transcript in the adrenal and VAT only in Balb/C mice. LepR blockade reduced BP in Balb/C mice fed a HSD (HSD: 105.1±0.9 vs. HSD+Allo-aca: 100.3±1.9 mmHg). HSD elevated ECMR expression only in Balb/C mice (N=6). To test the potential role of aldosterone-mediated ECMR activation in SSBP, we employed mice with selective overexpression of MR in endothelial cells (ECMR-OE). ECMR-OE young female mice exhibited impaired endothelial function (N=7), reproducing the effects of HSD. In conclusion, SSBP involves an over-activation of the leptin-aldosterone axis in both adrenal and adipose tissue, along with an escalated ECMR expression in females.

Abstract P214: Targeted disruption of CTCF binding in the Dahl salt-sensitive rat reveals Renin transcriptional dynamics

Changes in chromatin conformation caused by common genetic variations may impact blood pressure regulation and hypertension risk. A major function of CCCTCF-binding factor (CTCF) is to bind to DNA to regulate and maintain chromatin conformation, and thereby gene expression. Cellular expression of CTCF is critical for proper Renin expression and the Renin gene is surrounded by multiple CTCF-binding motifs . In the Dahl salt-sensitive (SS) rat, CRISPR-Cas9 was utilized to create three models harboring mutations to the CTCF motif surrounding the Renin gene. CTCF binding was confirmed to be disrupted in all three models. The mutants and wild-type (WT) littermates were then placed on a 0.1%, 4.0% or maintained on a 0.4% NaCl diet for four days, and Renin mRNA and plasma renin activity (PRA) was examined. mRNA analysis showed no differences in Renin expression on any diet for any model compared to WT. Interestingly, two of our male mutant models failed to increase their PRA on the 0.1% NaCl diet (CTCF1=13.87±0.86; CTCF2=13.95±0.59; p<0.05 compared to WT=17.52±0.81 on 0.1% NaCl). We hypothesized that Renin dynamic transcriptional response to salt depletion is delayed in the mutant models compared to WT. To test this, we have utilized an ex vivo kidney slice culture approach. Kidneys were excised from SS rats fed a 4.0% NaCl diet for 1-2 days, sliced to ~1-3 mms, and cultured for 24 hrs. Renin expression was evaluated at 0, 1, 2, 4, 8, 12, 16, 20 and 24 hrs of incubation in basal media. Renin expression was elevated in SS rats at 8 hrs (3.82±0.38 compared to 0 hr = 0.93±0.32) and continued to increase until 12 hrs (7.05±1.87) and reduced at 16 hrs (1.42±0.70). CTCF1 mutant Renin expression was significantly reduced at 12 hrs (3.77±0.58 compared to WT=7.05±1.87), while CTCF2 mutants showed no differences in Renin expression compared to WT. Thus, we established an ex vivo approach for monitoring the dynamic changes in Renin expression by culturing kidney slices. This method could be used to evaluate other stimuli, such as therapeutics.

Abstract P453: Dissecting tunica responsibility in arterial stress relaxation: Smooth muscle need not apply

Perivascular adipose tissue (PVAT) is being recognized as an important arterial tunica. Stress relaxation, a gradual reduction in stress over time while under a constant strain, is considered a property of smooth muscle (ex. intestine, uterus, bladder). While aortic PVAT (aPVAT) contains a microvasculature, it does not contain organized smooth muscle and isolated PVAT stress relaxes to a greater extent than the vessel itself. This raised the idea that other tunicas lacking smooth muscle may also stress relax. Here we hypothesize that appropriately oriented and functional smooth muscle was not necessary for stress relaxation to occur. To test this, we used histochemical staining and isometric contractility of thoracic aorta from the male Dahl salt-sensitive (SS) rat. Some rings of aorta were separated into adventitia (no muscle), media (muscle), and PVAT (no muscle), and responses of these isolates compared to the intact aortic ring (muscle + integration of tunicas). Masson Trichrome (MT) and Verhoeff Van Gieson (VVG) staining validated the dissection of the different tunicas with minimal contamination of cells from other tunicas. Stress relaxation was measured after increasing, cumulative amounts of passive tension (6 grams total) was applied in the presence or absence of Ca 2+ , essential for smooth muscle contraction. Each step was followed by challenge with a maximum concentration of the a 1 adrenergic agonist phenylephrine (PE, 10 -5 M). Stress relaxation occurred in all tunicas at each passive tension, whether or not Ca 2+ was present (figures). In fact, loss of Ca 2+ improved stress relaxation in adventitia and PVAT. Importantly, in the presence of Ca 2+ , PE contraction was observed at each passive tension in tissues containing smooth muscle: media (min, 250 mg PT: 26.67 +/- 8.51 mg; max, 6000 mg PT: 589.59 +/- 114.53 mg) and aorta + PVAT (min, 250 mg PT: 562.61 +/- 238.45 mg; max, 6000 mg PT: 1322.91 +/- 352.73mg). Little to no measurable contraction was recorded in the adventitia and aPVAT. There was little to no measurable contraction in tunicas in the absence of Ca 2+ . Taken together, these data suggest that all tunicas of the rat thoracic aorta stress relax and can do so without the presence of organized smooth muscle. Of all tunicas, PVAT demonstrated the greatest ability to stress relax. Collectively, these findings force reconsideration of individual responsibilities of arterial tunicas, all of which participate in the (dys)function of an artery.

Abstract 45: Deletion of a 4-bp noncoding genomic segment orthologous to human rs1882961 results in sex-divergent changes in salt-induced hypertension in Dahl salt-sensitive rats

Most single nucleotide polymorphisms (SNPs) associated with human blood pressure (BP) are in noncoding regions of the genome and have small effect sizes in the general population. Functional studies in a controlled model system are essential to ascertain the effect of these noncoding loci on blood pressure. Such direct testing is challenging and largely absent, contributing to a critical gap between genetic discoveries and physiological understanding. We reasoned that physiological studies in genetically engineered and phenotypically permissive animal models would help to address this critical gap. We tested the hypothesis by studying the noncoding SNP rs1882961. The T allele of rs1882961, which has a frequency of approximately 30% in the European population, is associated with 0.23 mmHg higher systolic blood pressure in humans. We mapped the human rs1882961 SNP to the rat genome and used CRISPR/Cas9 to delete a 4-bp segment encompassed the rs1882961 orthologous site in the genome of the Dahl salt-sensitive (SS) rat (SS-Δrs1882961). Male SS-Δrs1882961 -/- rats exhibited significantly higher mean arterial pressure (MAP) and systolic and diastolic blood pressures (SBP and DBP) than wild-type littermates after the rats were fed a high-salt (4% NaCl) diet for 14 days (156.6 ± 5.9 vs 149.3 ± 5.1 mmHg for MAP, 179.4 ± 7.8 vs 171.9 ± 6.0 mmHg for SBP, and 133.2 ± 4.6 vs 125.1 ± 4.5 mmHg for DBP; n=7 for SS-Δrs1882961 -/- and 9 for wild-type, p<0.05). In contrast, MAP, SBP, DBP, and pulse pressure of female SS-Δrs1882961 -/- rats were significantly lower than wild-type littermates on a high-salt diet (139.8 ± 1.8 vs 149.5 ± 4.7 mmHg for MAP, 159.3 ± 2.5 vs 172.3 ± 6.0 mmHg for SBP, 116.8 ± 1.5 vs 124.5 ± 3.7 mmHg for DBP, and 42.5 ± 1.6 vs 47.7 ± 2.3 mmHg for pulse pressure; n=8 for SS-Δrs1882961 -/- and 7 for wild-type, p<0.05). The difference in systolic blood pressure reached more than 10 mmHg in female rats. Urinary albumin and protein levels of SS-Δrs1882961 -/- rats after 14 days on a high-salt diet tended to be higher in male and lower in female compared with wild-type littermates. The remarkable effects on BP as well as the sex-divergent changes resulted from a 4-bp noncoding segment deletion around rs1882961 orthologous site indicate that noncoding genomic segment may have profound impact on phenotypes on permissive genomic background and with informative stressors.

Abstract P183: Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus Induce Inflammation and Salt Sensitivity of Blood Pressure in Humanized Mice

Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular disease (CVD), even in normotensive people. Systemic lupus erythematosus (SLE) is also a risk factor for CVD and hypertension. SLE and CVD are on the rise, and it is not known if this is attributable to the increased dietary salt in the modern diet and SSBP. Isolevuglandins (IsoLGs) adduct self-proteins forming neoantigens in antigen-presenting cells (APC), activate T cells, and produce cytokines that promote sodium retention, kidney damage, SLE, and SSBP. We hypothesized the adoptive transfer of peripheral blood mononuclear cells (PBMCs) from patients with SLE into immunodeficient mice (NSG/MHCI/MHCII-/-) would increase inflammation and SSBP after high salt feeding. In vitro studies were performed using PBMCs from patients with SLE and treated with and without high salt, and myeloid cell activation and cytokine expression were measured with flow cytometry. We humanized mice with PBMCs from patients with SLE (n=7) and controls without autoimmune disease (n=7) in animal studies. The mice were fed a 4% high-salt diet for two weeks. Blood pressure was measured with radiotelemetry, immune phenotyping was performed with flow cytometry, and vascular reactivity was assessed in mesenteric arteries using wire myography. A high salt diet significantly increased systolic blood pressure in lupus mice compared to the controls (133.0 vs.116.4 mmHg, p=0.001). APC infiltration (macrophages, monocytes, dendritic cells), proinflammatory markers, and oxidative stress (Isolevuglandins, Nox2) were significantly higher in the kidneys of lupus mice. In lupus mice, classical monocyte and proinflammatory markers were significantly elevated in the spleen lymphocytes, and proinflammatory markers were increased in the kidney, spleen, and aorta in lupus mice. These mice also exhibited mesenteric artery vascular dysfunction (p<0.05). Lupus mice tended to excrete more urinary albumin than the controls (25.89 vs. 17.24 µg/ml p=0.142), suggesting kidney injury. High salt treatment in vitro increased intermediate and nonclassical monocytes with increased CD86, IsoLG, and TNF-alpha expression compared to normal salt-treated cells. In conclusion, our findings suggest that immune cells from patients with SLE, in response to high salt, increase SSBP and the proinflammatory milieu in the kidney, aorta, and spleen; impair vascular function; and worsen albuminuria in immunodeficient mice.

Abstract 66: Early-Life Sodium Restriction Programs Salt-Sensitivity of Autonomics and Blood Pressure in Adult C57BL/6J Mice

Extremely premature birth is a risk factor for sodium (Na) depletion due to renal immaturity and urine Na losses. We have proposed that Na depletion in preterm infants mechanistically contributes to their increased risk for cardiovascular and metabolic disease observed in adulthood, potentially due to changes during hypothalamic development. In humans, the hypothalamus matures between 20-25 weeks of gestation; in mice, this developmental window occurs between postnatal day (PD) 21 and PD42. Previously, we demonstrated in mice that dietary Na restriction from PD21 to PD42 induced long-lasting increases in energy expenditure via exaggerated autonomic activity. Therefore, we hypothesized that restricting dietary Na from PD21 to PD42 would similarly induce programmed changes in cardiovascular control via changes in autonomic and neurohypophyseal functions. Male C57BL/6J mice were provided a customized low (0.04%, n=7) or supplemented (0.30%, n=8) Na version of the soy protein-free Teklad 2920x diet from PD21 to PD42, after which they were returned to the standard (0.15% Na) 2920x diet. Radiotelemetric blood pressure (BP) transducers were implanted at 7-8 weeks of age. At 10 weeks of age, no differences in BP or heart rate (HR) were noted between groups. All animals were switched to a high (1%) Na diet at 12 weeks of age. At 16 weeks of age, systolic BP was not altered in the 0.30% Na group but significantly increased in the 0.04% Na group compared to the week 10 baseline (24h avg: +6±2 mmHg, p<0.05). Ganglionic block with chlorisondamine or β-adrenergic block with propranolol reduced heart rate (HR) to a greater extent (each p<0.05) in the 0.04% group. Conversely, muscarinic block with atropine resulted in a smaller increase in HR in this group (p<0.05). Urine elimination rates of aldosterone (0.30%: 1.96±0.44 vs 0.04%: 2.92±0.064 ng/d) and copeptin (0.30%: 0.16±0.03 vs. 0.04%: 0.14±0.02 ng/d) were similar between groups. Finally, at 18 weeks of age, all mice received the angiotensin AT 1 R antagonist losartan in drinking water. This caused a greater BP reduction in the 0.04% group at 20 weeks (24h avg: -12±2 vs -19±2 mmHg, p<0.05). Together, these data indicate that dietary Na restriction during hypothalamic development programs increased salt sensitivity of BP via a mechanism involving AT 1 R and characterized by increased sympathetic yet decreased parasympathetic activity.

Abstract P200: Immune memory contributes to chronic hypertension recurrence

Keeping blood pressure under control is a challenge in hypertension treatment because high blood pressure quickly returns with a momentary lapse in treatment or a high-salt diet. We have previously reported the CD8+ T cells (CD8Ts) in the kidney can drive aberrant sodium handling to aggravate blood pressure. Recently, our new findings suggest that these CD8Ts remain long-term within the kidney. Here, we propose these CD8Ts have developed into a resident memory (Trm) population with the aid of TGFβ signaling, and this residency continually aggravates and leads to the recurrence of hypertension . To explore this hypothesis, we utilized two mouse models of hypertension, including a classic DOCA-salt model and a novel Angiotensin II (Ang II) induced salt memory model, with continuous blood pressure monitored by radiotelemetry. In the Ang II-induced salt memory model, the mice were administered Ang II and high salt for a week before a resting period to restore normal blood pressure. Thereafter, the mice were given a second high salt challenge to model the high salt-driven recurrence of hypertension. Mouse strains include both wild-type (WT) and a T-cell specific TGFβRII KO (Tsp-TGFβR KO), which has T cells unable to develop into Trm due to the reliance of Trm on TGFβ signaling. T cells were analyzed with flow cytometry. In the DOCA-salt model, WT mice developed a significant CD8Trm population within the kidney. In contrast, TSP-TGFβR KO mice, which lack the ability to develop Trm, had an attenuated development of hypertension with DOCA-salt treatment. Testing the capability for hypertension to recur, both WT and Tsp-TGFβR KO mice experienced a similar degree of hypertension with Ang II-salt treatment. However, only the WT had a significant expansion of Trm and demonstrated the clinical manifestation of recurring hypertension with the rapid increase of blood pressure with a high salt challenge. Isolating the T cells from the kidneys confirmed that the significant CD8Trm population development in the WT was not present in the Tsp-TGFβR KO. In summary, our findings suggest that the memory T cell population developed in the kidney during hypertension instills a perpetual memory for salt sensitivity in hypertension. Without the ability to develop this memory population, mice are protected against this recurrence of high-salt-induced hypertension.