Salt Sensitivity Research Articles

Role for clock protein PER1 in cardiac adaption to salt-sensitive hypertension in male rats

The clock protein PERIOD1 (PER1) is known to regulate the transcription of many genes, impacting subsequent physiological responses and working with other proteins to establish rhythmicity in processes such as blood pressure regulation. Global PER1 knock-out (KO) in male rats is associated with increased severity of the salt-sensitive (SS) hypertensive phenotype in Dahl SS rats. Male PER1 KO SS rats demonstrated reduced amplitude of heart rate as well as altered renal electrolyte and fluid balance with accompanying changes in the expression of relevant genes in the kidney. However, little is known regarding changes in gene expression in cardiac tissue which may contribute to this phenotype. The goal of this study was to test the hypothesis that knockout of PER1 in SS rats results in altered gene expression in the heart. We selected the following genes due to their role in the clock protein complex and circadian control over blood pressure: Basic Helix-Loop-Helix ARNT Like 1 ( Bmal1), Cryptochrome 1 and 2 ( Cry1, Cry2), and Period 2 ( Per2). Given its role in compensatory natriuretic responses to pressure overload, we also assessed the gene Nppa which encodes atrial natriuretic peptide (ANP). In order to test this hypothesis, hearts were collected at 2 am (during the rat active period) from male Dahl SS control and PER1 KO rats following 3 weeks of high salt (4% NaCl) diet (n=5-6). Changes in expression of candidate genes were measured using quantitative real time RT-PCR. Genotype effects were assessed using unpaired student’s t test. PER1 KO SS male rats on high salt diet exhibited significantly decreased cardiac expression of in the clock genes Bmal1 (p<0.0001), Cry1 (p=0.0007), and Cry2 (p=0.0094). Changes in Per2 expression were not apparent (p=0.1152). In contrast, PER1 KO rats exhibited significantly increased expression of Nppa (p=0.0017). Global PER1 knock-out in male SS rats results in hypertension associated with disrupted rhythms in heart rate. These changes are associated with reduction in expression of the genes encoding regulatory clock proteins Bmal1, Cry1, and Cry2, but not Per2. The decreased circadian gene expression in PER1 KO Dahl SS rats may contribute to the disrupted rhythms in heart rate. Increased ANP gene expression in the hearts of PER1 KO SS rats could serve as a compensatory mechanism to induce natriuresis. Thus, the data support the hypothesis that knockout of PER1 in SS rats alters gene expression in the heart. Together, these data illuminate a possible circadian-regulated pathway through which the heart and kidney communicate to respond to fluid and electrolyte imbalance in salt-sensitive hypertension. This work was supported by the National Institutes of Health grants R35 HL135789 (to AS), R01 DK109570 (to MLG and AS), R56DK128271 (to MLG), and Department of Veteran Affairs grants I01 BX004024 (to AS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

SODIUM-MEDIATED BLOOD PRESSURE RISE IS ASSOCIATED WITH A LOWER VASODILATION RESPONSE TO NITROGLYCERIN IN HEALTHY CONTROLS, BUT NOT IN PATIENTS WITH CKD

Objective: Chronic kidney disease (CKD) is characterized by endothelial dysfunction, that is partly caused by reduced bioavailability of NO. In addition to volume overload, endothelial dysfunction may contribute to hypertension in CKD. High sodium consumption affects NO levels, potentially leading to impaired vasorelaxation and a salt-sensitive blood pressure (BP) response. Whether salt-sensitivity in CKD is related to impaired NO bioavailability remains unknown. In this randomized-crossover-study, the vasoresponse to the NO donor nitroglycerin (NTG) during a high sodium diet in both CKD and healthy controls (HC) was assessed. Design and method: CKD patients (KDIGO stage G2-3A, proteinuria >0.5 g/d) and HC followed a 7-day low sodium control diet (LSD, <50mmol/d) and high sodium diet (HSD, >200mmol/d) in random order. After each diet, the hemodynamic profile was assessed by daytime BP and cardiac output (CO) measurements, by which systemic vascular resistance (SVR) was calculated. Nitroglycerin was administered to assess SVR changes. Results: We included 15 CKD patients (mean age, 49 yrs; median (IQR) proteinuria 0.7 (0.6-1.4) g/d; and plasma creatinine 129 (105-160) μmol/l) and 16 HC (41 yrs). Dietary salt intervention was comparable in the CKD and HC groups. After HSD, mean (SD) systolic BP increased in CKD by 9.7 mmHg (9.8; p<0.01) as compared to 3.1 mmHg ((6.0), p=0.07)) in HC (between-group difference p=0.04). After HSD, changes in body weight and CO did not differ between groups. Median (IQR) SVR change after HSD was 163.0 (-34.9-275.7) in CKD and -13.8 ((-299.4–61.4),p=0.03) dyn/sec/cm-5 in HC. In HC, salt-mediated blood pressure change was positively associated with a NTG-induced SVR change during HSD (r= 0.70, p= 0.01). This association was not present in CKD (r=-0.24, p=0.34) and significantly different from that in HC (p<0.01). Conclusions: CKD patients have a higher sodium-mediated BP response with comparable change of fluid status but a different SVR change as compared to HC. In HC, impaired sensitivity to the NO donor nitroglycerin associates with higher BP response to sodium. No such association was found in CKD patients, suggesting that persistent endothelial dysfunction is pivotal for salt sensitivity in CKD.

Cyclic strain promotes MCP-1 expression in Dahl Salt Sensitive (SS) rat vascular smooth muscle cells (VSMCs) through the TGF-β pathway

Prior studies have suggested a role for TGF-β in the generation of hypertension in SS rats. We have shown that production of MCP-1 (CCL-2) and TGF-β1 increase in the vasculature of hypertensive SS rats. This study aims to explore novel mechanisms of hypertension development. We hypothesize that TGF-β1 regulated the production of MCP-1 by VSMCs in the setting of cyclic strain. Primary cultures of SS aortic VSMCs in early (<6) passages were grown in standard fashion in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum. Cultured cells were identified as smooth muscle cells by their characteristic morphology and positive immunostaining for α-smooth muscle actin. In the first study, VSMCs were treated with recombinant active TGF-β1 (1 ng/mL and 10 ng/mL) for 6 h, then harvested for MCP-1 mRNA quantification by SYBR green qPCR. Medium was also collected to determine MCP-1 levels using ELISA. TGF-β1 increased MCP-1 mRNA expression by 2.0- and 3.1-fold in VSMC incubated in medium containing 1 ng/mL and 10 ng/mL of TGF-β1, respectively. In a second series of studies, VSMCs were seeded onto 6-well BioFlex plates. Some cells were pre-treated with 10 μmol/mL of an ALK4/5 inhibitor. Cyclic strain, an in vitro model of hypertension, was performed using the FlexCell 6000 and a standardized regimen (8 h, 15% elongation @ 1 Hz). Cells and medium were then harvested for mRNA analyses and determination of MCP-1 levels. When compared with expression by VSMCs maintained under static condition, cyclic strain increased (p<0.05) MCP-1 mRNA expression 4.2-fold and TGF-β1 mRNA 1.93-fold. Cyclic strain also increased (p<0.01) MCP-1 protein in the medium (static condition vs cyclic strain, 112.6 ± 7.1 pg/mL vs 222.5 ± 6.5 pg/mL, n=6). Pre-treatment of VSMC with the ALK4/5 inhibitor reduced mRNA expression of MCP-1 mediated by cyclic strain (Cyclic strain vs Cyclic strain + AKL4/5 inhibitor, 4.5 ± 1.06 vs 2.88 ± 0.47, n=6, P < 0.01). Thus, cyclic strain promotes MCP-1 production by VSMCs through the TGF-β pathway. The data support the need for further study of this interaction as well as a potential role in arterial hypertension in this rodent model. Department of Veterans Affairs Basic Sciences R&D (BSRD) Service (1 I01 BX005640) National Institutes of Health (P30 DK079337). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Kidney disease associated with pre-pubertal obesity initiates pathological alterations in the brain of Dahl salt-sensitive rats

Childhood/pre-pubertal obesity (PPO) is implicated in the development of chronic kidney disease (CKD), and there is evidence that PPO also increases the risk of developing Alzheimer disease (AD) later in life. In the current study, we aim to investigate the correlation between CKD and the development of AD-like pathology in a rat model of CKD associated with PPO. We hypothesize that systemic inflammation due to PPO exacerbates CKD and induces AD-like pathology in the brain at older age. In our preliminary studies, we utilized the Dahl salt sensitive leptin receptor mutant rat (SSLepRmutant), which is a novel model of PPO. 6-week-old Dahl salt-sensitive (SS) (control, n=3) and SSLepRmutant rats (n=5) were monitored for proteinuria every 3 weeks, for a 12-week period. Throughout the study, we observed significantly elevated proteinuria in SSLepRmutant rats (Week-6: 337±81 mg/day, Week-9: 371±147 mg/day, Week-18: 1920±318 mg/day) compared to SS rats (Week-6: 25±7 mg/day, Week-9: 35±39 mg/day, Week-18: 35±33 mg/day). At 18-week, creatinine levels were significantly higher in SSLepRmutant rats (1.5±0.5 mg/dL) versus SS rats (0.4±0.03 mg/dL) indicating CKD. Additionally, we found significant elevation of triglycerides in SSLepRmutant rats (5134±1211 mg/dL) compared to SS rats (79±21 mg/dL). To investigate the brain pathology, we performed immunochemical analysis (dot-blot assay) using A11 antibody to determine the levels of Amyloid-beta oligomers (Aβo). Our results show that there was tendency for Aβo levels to be increased in the brains from obese SSLepRmutant rats compared to SS. Finally, we tested the permeability of the blood-brain barrier to Evans blue dye and observed that the brain of SSLepRmutant rats was more permeable to the dye compared to SS rats as early as 8 weeks of age. Overall, these results indicate that PPO obesity not only increases the susceptibility to develop CKD but also causes Alzheimer-like pathology during the prepubescent stage. NIDDK (DK109133) and AG (AG057842) awarded to Jan M. Williams. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of Fasting During Time Restricted Feeding on Mitochondrial Beta-Oxidation Enzyme Expression in the Kidney Cortex of Adult Male Salt-Sensitive Dahl Rats

The Dahl salt-sensitive (SS) rat is a well-characterized model of SS hypertension and renal disease that exhibits enhanced Na+ reabsorption in the kidney. Prior research has shown that SS rats experience a reduction in fatty acid beta oxidation (FAO) in the kidney cortex when placed on a high salt (HS) diet. Time restricted feeding (TRF), a type of intermittent fasting in which food consumption is restricted to <10 hrs/day, has been shown to activate FAO. Since the proximal tubule is responsible for the highest amount of Na+ reabsorption, we are interested in understanding if intermittent salt intake via TRF can mitigate dysfunctional cortical metabolism and slow progression of renal damage. We hypothesized that fasting during TRF would activate FAO throughout the body, as well as in the kidney cortex. To study this, 10-week-old male SS rats were provided ad libitum access to 4.0% NaCl (high salt; HS) or 0.4% (low salt; LS) diet for one week. Rats were then placed into a Promethion Core system (Sable Systems International) for metabolic phenotyping, where some continued ad libitum feeding, and others began time restricted feeding (TRF) for an additional week (n=7-9/group). During TRF, food was provided for 8 hours during the active cycle (2200hrs-0600hrs). Rats were euthanized in the last two hours of the final fasting period. We found that during the last hour of the fasting period, when compared to ad libitum rats, both the LS TRF (0.824 ± 0.01 vs 0.986 ± 0.008) and HS TRF (0.830 ± 0.01 vs 0.980 ± 0.006) rats had a significant reduction in their respiratory exchange ratio (2-way ANOVA with Tukey multiple comparisons, p<0.05, mean ± SEM). This suggests that the TRF protocol induced a net whole-body shift in fuel utilization from primarily carbohydrates to a mix of fuel sources by the end of the 16-hour fasting period, regardless of diet. To investigate the effect of fasting during TRF on the kidney cortex, we performed western blot analysis of acyl-CoA dehydrogenase long chain (ACADL) and acetyl-CoA acyltransferase 2 (ACAA2), enzymes involved in initial and final steps of mitochondrial beta-oxidation, respectively. We found that HS rats had lower ACADL and ACAA2 expression than the LS rats (2-way ANOVA; main effect of HS, p<0.05), and there was no difference between ad libitum and TRF groups within LS or HS groups (2-way ANOVA; main effect of TRF, p>0.05). This data suggests that, even though whole-body FAO may be stimulated by TRF fasting, HS-induced suppression of mitochondrial FAO proteins ACADL and ACAA2 was not restored by TRF. Future studies will investigate the effects of the shift in fuel utilization on other metabolic pathways in the cortex and proximal tubules. Supported by the Advancing a Healthier Wisconsin Endowment, the Medical College of Wisconsin, and NIH 5T32HL007852-25. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of SGL2 Prevents Albumin Induced Proximal Tubule Inflammation

Albuminuria is an independent risk factor for cardiovascular disease and renal damage in hypertension. It is associated with a higher risk of cardiovascular morbidity and mortality, and with declining kidney function. The Na+-glucose cotransporter-2 (SGLT2) plays a crucial role in glucose and sodium reabsorption in the proximal tubule. Clinical trials have shown that SGLT2 inhibitors can reduce cardiovascular events, lower blood pressure, and decrease the risk of end-stage kidney disease in individuals with type 2 diabetes mellitus. These trials have also demonstrated the potential of SGLT2 inhibitors to provide additional benefits beyond their glucose control, including decreased albuminuria. In this study, we hypothesized that exposure to elevated albumin causes proximal tubule injury and the release of cytokines from the basolateral membrane. We propose that by reducing transport in the proximal tubule, SGLT2 inhibitors may alleviate kidney damage by reducing cytokine production and subsequently renal inflammation. To test our hypothesis, we conducted experiments using Dahl salt-sensitive (SS) rats and immortalized human proximal tubule cells (RPTEC-TERT1). To determine how early albuminuria and proximal injury occur in the development of salt-sensitive hypertension, 24-hour urine was collected from SS rats fed either a control salt (CS, 0.4% NaCl, n=10) or a high salt (HS, 4.0% NaCl, n=11) diet for 7 days. Significant increases in albuminuria were observed after 1 day of HS (CS: 25±7 vs. HS: 59±13 mg/day p=0.016). This was followed by significant increases in urinary kidney injury molecule-1 (Kim-1) after 2 days of HS (CS: 13±2 vs. HS: 23±3 pg/day p=0.021). Preliminary analysis found substantial increases in urinary CCL2 after 4 days of HS (CS: 58±7 vs. HS: 135±25 pg/mg, n=5). To investigate whether the renoprotective effects of SGLT2 inhibitors are in part due to their ability to reduce inflammation in the proximal tubule, we exposed the apical membrane of RPTEC-TERT1 cells to human serum albumin (HSA, 10mg/ml, n=6) with and without Dapagliflozin (Dapa, 15ng/ml, n=5) for 24-hours and measured the levels of CCL2 in the basolateral media. HSA caused a significant increase in CCL2 levels relative to control cells (Control: 42.5±5.6 vs. HSA: 84.1±8.7 pg/mg, p=0.00). This increase was prevented by co-treatment with Dapa and CCL2 levels were equivalent to those in the control cells (HSA+Dapa: 44.0±4.5 pg/mg, p=0.988). In conclusion, our findings support the hypothesis that albuminuria precedes proximal tubule injury and may contribute to the development of renal inflammation in salt-sensitive hypertension. The ability of Dapa to reduce prevent albumin induced CCL2 production in RPTEC-TERT1 cells suggests that SGLT2 inhibitors may reduce renal inflammation in salt-sensitive hypertension and is an area of ongoing studies. Studies funded by R01HL152166 to LE. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

An Ex Vivo Approach to Studying Renin Dynamics

Hypertension is the leading risk factor for cardiovascular disease and impacts one in three adults globally. One non-coding mechanism that may affect blood pressure regulation and alter hypertension risk in the general population is the disruption of chromatin conformation. CCCTCF-binding factor (CTCF) has many important functions including regulating the conformation of chromatin by binding to specific DNA sequences. Renin has multiple CTCF motifs surrounding the gene and cellular expression of CTCF is critical for proper Renin expression. Using CRISPR-Cas9 in the Dahl salt-sensitive (SS) rat, we have developed three mutant rat models with one or more mutations to the CTCF motif surrounding the Renin gene. CTCF binding was confirmed to be disrupted in all three models at the CRISPR-targeted locations. Plasma renin activity (PRA) was measured in wild-type (WT) and mutant rats fed a 0.1%, 0.4% or 4.0% NaCl diet for four days. PRA in WT males responded as expected to dietary NaCl (17.52±0.81, 12.87±1.00, and 7.13±0.19 ng/mL; p<0.05 for 0.1% and 4.0% vs. 0.4%). 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 0.1% NaCl). Despite this, no differences in Renin mRNA were detected. We hypothesize Renin transcriptional response to salt depletion is delayed in the mutant models compared to WT. To test this, we developed an ex vivo approach to culture kidney slices and evaluate Renin expression to a variety of stimuli. Kidneys from SS rats fed low salt (LS; 0.4% NaCl) or high salt (HS) diet (4.0% NaCl) for 24 hours were harvested, sliced, and cultured in basal media or media containing increasing salt. Renin expression was examined at 1, 2, 4, 8 and 24 hrs of incubation. Kidney slices from SS rats fed HS that were incubated basal media stimulated Renin expression as soon as 2 hrs and continued to increase until 8 hrs. This was the first time for establishing an ex vivo approach for motoring the dynamic changes in Renin expression in response to a stimulus by culturing kidney slices. This method could be used to evaluate other stimuli, such as therapeutics, and could decrease the number of animals needed for a study. Funded by NIH 1HL149620. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High Salt Induced Alterations in Substrate Metabolism, Respiratory Activity, and Acidification in Isolated Proximal Tubules from Dahl Salt-Sensitive Rats

Rationale: The renal nephron actively reabsorbs nearly 99% of Na+ from the glomerular filtrate to maintain fluid and solute homeostasis, roughly 70% of which occurs in the proximal tubules (PT), where mitochondria consume a great amount of O2 for ATP production to meet the energy demand. However, the effect of a high salt diet upon the PT substrate metabolism and respiratory activity towards energy production and associated cellular acidification is not well-known. Method: To determine alterations in the PT substrate metabolism, respiratory activity, and acidification with a high salt diet, the PT of adult Dahl salt-sensitive (SS) rats were isolated by collagenase digestion and sieving with the rats fed either a 0.4% (LS) or a 4.0% (HS) NaCl diet for 7, 14 and 21 days (age-matched). Responses of the PT O2 consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using a high-throughput Agilent Seahorse XF96 Extracellular Flux Analyzer and a modified mitochondrial stress test protocol, which combined a substrate addition step to the mitochondrial stress reagent addition steps. Basal OCR and ECAR were assessed with only amino acids (basic Seahorse medium) used as fuel for respiration. Subsequently, OCR and ECAR responses were determined by adding different circulating substrates followed by perturbations to the electron transport chain (ETC) in sequential order with oligomycin to inhibit ATP synthase, FCCP to uncouple oxidative phosphorylation, and rotenone + antimycin A to inhibit complexes I and III of the ETC. From these measurements, different OCR and ECAR parameters were derived for each substrate and each salt load condition. Results: The OCR data show that the PT of SS rats preferred to use lactate, glutamine, and palmitate for ATP production under the LS diet. However, the HS diet resulted in significantly reduced OCR when supplied with the same substrates. Interestingly, the basal OCR which occurred with amino acids as metabolic substrates was increased in the PT obtained from HS fed SS rats starting on day 7. These alterations in substrate metabolism were also apparent from the same parametric analysis of the ECAR data. An increased mitochondrial proton leak induced respiration was also deduced form both OCR and ECAR data under the HS diet. Since it is known that uncoupling proteins (UCP) and ADP/ATP translocase (ANT) enhance proton leak, gene expression was quantified from the isolated PT segments. Increased Ucp2 gene expression was found in response to the HS diet (days 7, 14, 21) compared to the LS fed SS rats but no changes were observed in the expression of Slc25A4 (ANT1) or Slc25A5 (ANT2). Summary: HS diet resulted in significant metabolic changes in the PT segments, including substrate metabolism, respiratory activity, and cellular acidification. HS diet also increased PT basal respiration and proton leak, which was associated with an increase of Ucp2 gene expression. Considering the increased ATP demand in the PT segments with a HS diet, these changes would result in increased O2 consumption and increased reactive oxygen species production. Together with the physiological changes on renal hemodynamics that we have observed, we propose that the observed metabolomic changes on the PT upon HS diet contribute to the onset of associated regional ischemia, tissue inflammation, and renal injury observed in hypertensive SS rats. NIH R01-HL151587. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deletion of Kcnj16 altered transcriptomic and metabolomic profiles of Dahl salt-sensitive rats

The essential role of inwardly rectifying K+ Channel Kir5.1 (encoded by the Kcnj16 gene) in renal salt handling and blood pressure control has been demonstrated in both human and animal models. However, the underlying mechanisms are still not fully understood. Here, we aimed to integrate transcriptomics and metabolomics to profile the comprehensive changes of genes and metabolites under the deletion of Kcnj16 in the Dahl salt-sensitive (SS) rat background to identify novel mechanisms. For the transcriptomics profiling, we performed RNA-sequencing on kidney cortex tissue of Kcnj16 knockout (KO) and wild-type (WT) SS rats (N=5 per group). For the metabolomics profiling, we performed untargeted metabolomics of kidney cortex tissue, plasma, and urine of KO (N=6) and WT (N=5) rats. Transcriptomics analysis revealed over 6,000 differentially expressed genes in KO rats compared to WT rats, with renin Ren (the gene encoding renin) being the most upregulated gene. Consistent with the phenotype observed in the KO rats, ingenuity pathway analysis (IPA) based on the transcriptomic profile predicted reduced blood pressure and kidney damage and increased ion transport. Canonical pathway analysis suggested activation of metabolic-related pathways [e.g., xenobiotic metabolism, tricarboxylic acid (TCA) cycle, phenylalanine degradation, etc.] and suppression of immune responses-related pathways [e.g., pathogen-induced cytokine storm signaling pathway, macrophage classical activation signaling pathway, nuclear factor of activated T cells (NFAT) pathway, etc.] in KO rats. Untargeted metabolomic analysis suggested distinct metabolic profiles of plasma, urine, and kidneys between WT and KO rats, with 219, 790, and 31 differentially expressed metabolites identified in plasma, urine, and kidney in KO rats compared to WT rats. Canonical pathway analysis identified spermine and spermidine degradation pathways in both plasma and urine and taurine biosynthesis pathways in both urine and kidney. Integration analysis based on transcriptomic and metabolomic profiles suggested an altered TCA cycle, amino acid, and reactive oxygen species metabolism. In conclusion, besides significantly altered ion transport, our transcriptomics and metabolomics data suggest significant suppressed immune responses-related and altered metabolic-related pathways in SS rats lacking Kir5.1, which provides new insights into the underlying mechanisms of the regulation of blood pressure and renal function by Kir5.1. This work is supported by National Institutes of Health Grants R35 HL135749 and R01 DK135644 (to A.S.), Department of Veteran Affairs grant I01 BX004024 (to A.S.), the American Physiological Society Postdoctoral Fellowship (to B.X.) and the American Society of Nephrology Dimitrios G. Oreopoulos Research Fellowship Award (to L.V.D.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus Induce 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) to activate T cells and produce cytokines that promote sodium retention, kidney damage, salt sensitivity of blood pressure, and SLE. We hypothesized that high salt intake increases both blood pressure and the proinflammatory milieu in mice humanized with the immune cells of patients with SLE. We humanized female NSG/MHCI/MHCII−/− immunodeficient mice with peripheral blood mononuclear cells (PBMCs) isolated from patients with SLE (n=5) and controls without autoimmune disease (n=4). The blood pressure was measured with radiotelemetry, immune phenotyping was performed with flow cytometry, and vascular reactivity was assessed in mesenteric arteries using wire myography after two weeks of a 4% high salt diet. High salt treatment significantly increased systolic blood pressure in lupus mice compared to the controls (135.5 vs. 123.8 mmHg, p=0.014). APC infiltration (macrophages, monocytes, dendritic cells), their activation (CD86) and proinflammatory markers (IL-23 p19, TNF-alpha), and oxidative stress (Isolevuglandins, Nox2) were significantly higher in the kidneys of lupus mice. Moreover, the signaling molecule p-STAT3 was significantly higher in myeloid cells in the kidneys of lupus mice. The 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 exhibited vascular dysfunction (p<0.05), suggesting reduced vascular compliance. We measured urine albumin and found that lupus mice tended to excrete more albumin than the controls (17.24 vs. 25.89 μg/ml p=0.142), suggesting greater kidney injury. In conclusion, our findings suggest that immune cells from patients with SLE, in response to high salt intake, increase blood pressure and the proinflammatory milieu in the kidney, aorta, and spleen; impair vascular compliance; and worsen albuminuria in immunodeficient mice. 1R01HL144941-01A1 1R03HL155041 23CDA1053072. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dietary effects on blood pressure control in primary aldosteronism using a cardiorenal model

Primary aldosteronism (PA) is associated with hypertension (HTN), and these patients have significant lowering of blood pressure (BP) and improved hypokalemia when moving to a DASH dietary intervention (≤65 mEq/day Na+ and ≥120 mEq/day K+). However, current guidelines for PA treatment briefly recommend lowering Na+ intake and make no specific mention of supplementing K+ intake or using DASH in PA. Additionally, high levels of aldosterone increase the risk of developing cardiovascular and chronic kidney disease. However, the impact of DASH diet on hyperkalemia risk and the extent of BP reduction in PA patients with renal dysfunction are unknown. Because continuous monitoring of cardiovascular, neural, and hormonal changes during PA and its treatment cannot be achieved experimentally, we used the mathematical model of human physiology, HumMod, to test the responses to a DASH diet during PA. We hypothesized that both low Na+ and high K+ (DASH) have additive benefits on natriuresis and BP control during PA. We added known renal physiological effects of K+ in HumMod using data from David Young’s work in dogs and further validated the model’s response to changes in Na+ intake based off of chronic salt sensitivity experiments in PA patients. Starting from baseline conditions with normal Na+/K+ intake (150 and 40 mmol/day, respectively), we simulated an aldosterone secreting tumor to gradually increase plasma aldosterone to 20 ng/dL over 3 years. Baseline PA was associated with hypertension (149/85 mmHg), very low endogenous renin and aldosterone secretion, hypokalemia (2.6 mmol/L K+), and tumor aldosterone secretion of 360 μg/day. We simulated a chronic change in dietary intake: 1) Low Na+ diet- Na+ intake fixed at 65 mmol/day with normal K+ intake, 2) High K+ diet - 120 mmol/day K+ intake with normal Na+ intake, or 3) DASH diet- Low Na+ intake and high K+ intake. Lowering Na+ intake without or with high K+ intake (DASH) were associated with lower systolic BP over the first 4 weeks and persisted for 2 years despite continued activation of the renin-angiotensin system and increasing tumor aldosterone secretion. Simulating mineralocorticoid blockade (MCB) was associated with reductions in systolic BP (-17 mmHg), decrease in left ventricular mass, and correction of hypokalemia (~4.0 mmol/L), but BP still remained uncontrolled (134/81 mmHg), partly due to the compensatory increase in renin secretion. Adding DASH to MCB therapy completely normalized BP without inducing hyperkalemia (4.8 mmol/L). Simulating chronic kidney disease by reducing nephron number in the model amplified BP sensitivity to changes in both Na+ and K+ but increased plasma K+ near the hyperkalemic range. These current simulations may help better understand the sequence of physiological events in PA and yield new insights into its diagnosis and management in patients with different baseline renal function. The physiological effects of the DASH diet desperately need to be tested in patients with PA with various baseline renal function as our simulations suggest beneficial effects beyond current treatment guidelines. Supported by the National Institute on Minority Health and Health Disparities (R00 MD014738). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

GENETIC VARIANCE IN HEPARAN SULFATION IS ASSOCIATED WITH SALT-SENSITIVITY: A GENE-ENVIRONMENT ANALYSIS

Objective: The high heritability of salt sensitivity suggests an essential role for genetic variants in the relationship between sodium intake and blood pressure (BP). Candidate gene studies have mainly focused on renal and vascular pathways, but the role of glycosaminoglycan (GAG) genes, which are crucial for salinity tolerance, remains to be elucidated. Design and method: Interactions between 54,126 variants in 130 GAG genes and daily sodium excretion on BP were explored in 20,420 EPIC-Norfolk subjects. UK Biobank (n=414,132) and the multi-ethnic HELIUS study (n=2,239) were used for validation. In HELIUS sodium intake was estimated with Food Frequency Questionnaires (FFQ). Urinary GAG composition in 57 Dutch origin HELIUS participants stratified by genotype were analyzed to assess the association between SNP and GAG difference. Also, urinary GAG composition in 12 healthy volunteers after a sodium intervention (7-day low (50 mmol Na+/day) and 7-day high (200 mmol Na+/day) diet) were studied to assess if differences in genotype are associated with different GAG expression. Results: The intron variant rs2892799, mapped to NDST3, showed the strongest interaction with sodium on mean arterial pressure (MAP) (FDR 0.03), and the intron variant rs9654628 mapped to HS3ST5 with sodium on systolic BP (FDR 0.03). These interactions were multi-ethnically validated (Fig 1A, B). When stratified for rs2892799 genotype, T-allele carriers had a significantly higher urinary expression of N-sulfated heparan sulfate epitope D0S0 (Fig1C). Conversely, upon dietary salt loading, urinary D0S0 expression differed substantially between salt-sensitive and salt-resistant individuals (Fig1D). Sodium-induced effects on this epitope were opposite in salt-sensitive in salt-resistant individuals. Conclusions: Genetic variants associated with NDST3 and HS3ST5 interact with relation between sodium consumption and BP. rs2892799 (NDST3) genotype results in a different expression of the N-sulfated D0S0 epitope. In turn, D0S0 expression is associated with salt-mediated BP increase, indicating that genetic GAG variation and metabolic GAG adaptation to a high salt environment might be important for human BP regulation.

High Salt Diet Induces Aldosterone Synthase Elevation in Adrenal and Visceral Adipose Tissue - A Potential Culprit for Unsuppressed Aldosterone Levels in Female BALB/c Mice

Women are more salt-sensitive than men, irrespective of age, menopausal status, or ethnic background. Nonetheless, the mechanisms predisposing women to salt sensitivity of blood pressure (SSBP) are poorly understood, notably due to the diffculty in finding appropriate animal models. Previously, we demonstrated that female BALB/c mice spontaneously develop SSBP, likely through an inability to reduce the expression of adrenal aldosterone synthase (CYP11B2) appropriately, which leads to unsuppressed aldosterone levels in response to high salt intake. Herein, we aim to decipher the mechanism involved in SSBP in female BALB/c mice by comparing them to female salt-resistant C57BL/6 mice. Eleven-week-old female BALB/c and C57BL/6J mice were fed either a normal (0.4%, NSD) or a high salt diet (4%, HSD) for seven days. HSD significantly reduced adrenal aldosterone synthase transcript and protein expression in female C57BL/6 mice compared to those on a NSD. Conversely, HSD substantially increased CYP11B2 levels in BALB/c female mice when compared to strain-matched NSD and C57BL/6 NSD mice (2-way ANOVA, p<0.05). HSD induced a more significant aldosterone drop in C57BL/6 versus BALB/c mice (C57BL/6: 72% vs. BALB/c: 49%). Remarkably, HSD led to an increase in CYP11B2 in the visceral adipose tissue (VAT) of BALB/c mice in comparison to strain-matched NSD mice, while no such alteration was observed between C57BL/6 NSD and HSD. We previously showed that leptin regulates CYP11B2 expression. We report here that HSD increased leptin receptor (lepR) expression in adrenal and VAT from BALB/c mice in comparison to both strain-matched NSD and C57BL/6 NSD. Baseline levels of leptin were similar in C57BL/6 and BALB/c mice. However, BALB/c mice on HSD displayed increased levels of leptin compared to its strain-matched NSD mice, while no such alteration was observed in C57BL/6 mice. Furthermore, female BALB/c mice on HSD displayed elevated endothelial cell-specific mineralocorticoid receptor (ECMR) expression compared to female C57BL/6 NSD and HSD mice (2-way ANOVA, p<0.05). Endothelium-dependent relaxation to acetylcholine was significantly reduced in the female BALB/c mice on HSD compared to strain-matched NSD and C57BL/6 NSD (n = 3, 2-way ANOVA, P < 0.05). Interestingly, on NSD, C57BL/6 mice present a better endothelial function than BALB/c, which was not altered by HSD. Collectively, our data supports an overactivation of the leptin-CYP11B2-aldosterone axis in both adrenals and VAT, leading to inappropriate aldosterone levels, which, associated with higher ECMR expression, induce endothelial dysfunction in BALB/c mice. Together, these mechanisms are likely the cause of SSBP in female BALB/c mice. Funding: R01HL130301, R01HL155265 (E.J.B.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CwJAZ4/9 negatively regulates jasmonate-mediated biosynthesis of terpenoids through interacting with CwMYC2 and confers salt tolerance in Curcuma wenyujin.

Plant JASMONATE ZIM-DOMAIN (JAZ) genes play crucial roles in regulating the biosynthesis of specialized metabolites and stressful responses. However, understanding of JAZs controlling these biological processes lags due to numerous JAZ copies. Here, we found that two leaf-specific CwJAZ4/9 genes from Curcuma wenyujin are strongly induced by methyl-jasmonate (MeJA) and negatively correlated with terpenoid biosynthesis. Yeast two-hybrid, luciferase complementation imaging and in vitro pull-down assays confirmed that CwJAZ4/9 proteins interact with CwMYC2 to form the CwJAZ4/9-CwMYC2 regulatory cascade. Furthermore, transgenic hairy roots showed that CwJAZ4/9 acts as repressors of MeJA-induced terpenoid biosynthesis by inhibiting the terpenoid pathway and jasmonate response, thus reducing terpenoid accumulation. In addition, we revealed that CwJAZ4/9 decreases salt sensitivity and sustains the growth of hairy roots under salt stress by suppressing the salt-mediated jasmonate responses. Transcriptome analysis for MeJA-mediated transgenic hairy root lines further confirmed that CwJAZ4/9 negatively regulates the terpenoid pathway genes and massively alters the expression of genes related to salt stress signaling and responses, and crosstalks of multiple phytohormones. Altogether, our results establish a genetic framework to understand how CwJAZ4/9 inhibits terpenoid biosynthesis and confers salt tolerance, which provides a potential strategy for producing high-value pharmaceutical terpenoids and improving resistantC. wenyujin varieties by a genetic approach.

Lysine and salt-sensitive hypertension.

Salt-sensitive (SS) hypertension and its associated kidney damage have been extensively studied, yet proper therapeutic strategies are lacking. The interest in altering the metabolome to affect renal and cardiovascular disease has been emerging. Here, we discuss the effect and potential mechanism behind the protective effect of lysine, an essential amino acid, on the progression of SS hypertension. We have recently demonstrated that administering lysine in an SS rodent model can control the progression of hypertension. Both the animal and pilot human studies showed that lysine can efficiently inhibit tubular reabsorption of albumin and protect the kidneys from further damage. In addition, we conducted multilevel omics studies that showed increased lysine conjugation and excretion, leading to the depletion of harmful metabolites and an increase in useful ones. Lysine's twofold action involves both mechanically flushing protein from proximal tubules to shield the kidneys and initiating metabolic adaptations in the kidneys. This results in a net positive impact on SS hypertension. While further research is necessary to apply the current findings in clinical settings, this study offers some evidence suggesting that lysine supplementation holds promise as a therapeutic approach for hypertensive kidney disease.

Arabidopsis class II TPS controls root development and confers salt stress tolerance through enhanced hydrophobic barrier deposition.

The mechanism of conferring salt tolerance by AtTPS9 involves enhanced deposition of suberin lamellae in the Arabidopsis root endodermis, resulting in reduction of Na+ transported to the leaves. Members of the class I trehalose-6-phosphate synthase (TPS) enzymes are known to play an important role in plant growth and development in Arabidopsis. However, class II TPSs and their functions in salinity stress tolerance are not well studied. We characterized the function of a class II TPS gene, AtTPS9, to understand its role in salt stress response and root development in Arabidopsis. The attps9 mutant exhibited significant reduction of soluble sugar levels in the leaves and formation of suberin lamellae (SL) in the endodermis of roots compared to the wild type (WT). The reduction in SL deposition (hydrophobic barriers) leads to increased apoplastic xylem loading, resulting in enhanced Na+ content in the plants, which explains salt sensitivity of the mutant plants. Conversely, AtTPS9 overexpression lines exhibited increased SL deposition in the root endodermis along with increased salt tolerance, showing that regulation of SL deposition is one of the mechanisms of action of AtTPS9 in conferring salt tolerance to Arabidopsis plants. Our data showed that besides salt tolerance, AtTPS9 also regulates seed germination and root development. qRT-PCR analyses showed significant downregulation of selected SNF1-RELATED PROTEIN KINASE2 genes (SnRK2s) and ABA-responsive genes in the mutant, suggesting that AtTPS9 may regulate the ABA-signaling intermediates as part of the mechanism conferring salinity tolerance.

Exploring Natural Variations in Arabidopsis thaliana: Plant Adaptability to Salt Stress.

The increase in soil salinization represents a current challenge for plant productivity, as most plants, including crops, are mainly salt-sensitive species. The identification of molecular traits underpinning salt tolerance represents a primary goal for breeding programs. In this scenario, the study of intraspecific variability represents a valid tool for investigating natural genetic resources evolved by plants in different environmental conditions. As a model system, Arabidopsis thaliana, including over 750 natural accessions, represents a species extensively studied at phenotypic, metabolic, and genomic levels under different environmental conditions. Two haplogroups showing opposite root architecture (shallow or deep roots) in response to auxin flux perturbation were identified and associated with EXO70A3 locus variations. Here, we studied the influence of these genetic backgrounds on plant salt tolerance. Eight accessions belonging to the two haplogroups were tested for salt sensitivity by exposing them to moderate (75 mM NaCl) or severe (150 mM NaCl) salt stress. Salt-tolerant accessions were found in both haplogroups, and all of them showed efficient ROS-scavenging ability. Even if an exclusive relation between salt tolerance and haplogroup membership was not observed, the modulation of root system architecture might also contribute to salt tolerance.

Effects of Salt Stress on Salt-Repellent and Salt-Secreting Characteristics of Two Apple Rootstocks.

The effects of NaCl-induced salinity on biomass allocation, anatomical characteristics of leaves, ion accumulation, salt repellency, and salt secretion ability were investigated in two apple rootstock cultivars (Malus halliana '9-1-6' and Malus baccata), which revealed the physiological adaptive mechanisms of M. halliana '9-1-6' in response to salt stress factors. This experiment was conducted in a greenhouse using a nutrient solution pot. Salt stress was simulated by treating the plants with a 100 mM NaCl solution, while 1/2 Hoagland nutrient solution was used as a control (CK) instead of the NaCl solution. The results showed that the two rootstocks responded to salt environments by increasing the proportion of root biomass allocation. According to the stress susceptibility index, '9-1-6' exhibits a lower salt sensitivity index and a higher salt tolerance index. The thickness of the leaf, upper and lower epidermis, palisade tissue, and mesophyll tissue compactness (CTR) of the two rootstocks were significantly decreased, while the thickness of sponge tissue and mesophyll tissue looseness (SR) were significantly increased, and the range of '9-1-6' was smaller than that of M. baccata. With an extension of stress time, the accumulation of Na+ increased significantly, and the accumulation of K+ decreased gradually. The stem and leaves of '9-1-6' showed a lower accumulation of Na+ and a higher accumulation of K+, and the roots displayed a higher ability to reject Na+, as well as young and old leaves showed a stronger ability to secrete Na+. In conclusion, within a certain salt concentration range, the '9-1-6' root part can maintain lower salt sensitivity and a higher root-to-shoot ratio by increasing the proportion of root biomass allocation; the aerial part responds to salt stress through thicker leaves and a complete double-layer fence structure; the roots and stem bases can effectively reduce the transportation of Na+ to the aerial parts, as well as effectively secrete Na+ from the aerial parts through young and old leaves, thereby maintaining a higher K+/Na+ ratio in the aerial parts, showing a strong salt tolerance.

Changes in Photosystem II Complex and Physiological Activities in Pea and Maize Plants in Response to Salt Stress.

Salt stress significantly impacts the functions of the photosynthetic apparatus, with varying degrees of damage to its components. Photosystem II (PSII) is more sensitive to environmental stresses, including salinity, than photosystem I (PSI). This study investigated the effects of different salinity levels (0 to 200 mM NaCl) on the PSII complex in isolated thylakoid membranes from hydroponically grown pea (Pisum sativum L.) and maize (Zea mays L.) plants treated with NaCl for 5 days. The data revealed that salt stress inhibits the photochemical activity of PSII (H2O → BQ), affecting the energy transfer between the pigment-protein complexes of PSII (as indicated by the fluorescence emission ratio F695/F685), QA reoxidation, and the function of the oxygen-evolving complex (OEC). These processes were more significantly affected in pea than in maize under salinity. Analysis of the oxygen evolution curves after flashes and continuous illumination showed a stronger influence on the PSIIα than PSIIβ centers. The inhibition of oxygen evolution was associated with an increase in misses (α), double hits (β), and blocked centers (SB) and a decrease in the rate constant of turnover of PSII reaction centers (KD). Salinity had different effects on the two pathways of QA reoxidation in maize and pea. In maize, the electron flow from QA- to plastoquinone was dominant after treatment with higher NaCl concentrations (150 mM and 200 mM), while in pea, the electron recombination on QAQB- with oxidized S2 (or S3) of the OEC was more pronounced. Analysis of the 77 K fluorescence emission spectra revealed changes in the ratio of the light-harvesting complex of PSII (LHCII) monomers and trimers to LHCII aggregates after salt treatment. There was also a decrease in pigment composition and an increase in oxidative stress markers, membrane injury index, antioxidant activity (FRAP assay), and antiradical activity (DPPH assay). These effects were more pronounced in pea than in maize after treatment with higher NaCl concentrations (150 mM-200 mM). This study provides insights into how salinity influences the processes in the donor and acceptor sides of PSII in plants with different salt sensitivity.

Salt-Sensitive Hypertension and the Kidney.

Salt-sensitive hypertension (SS-HT) is characterized by blood pressure elevation in response to high dietary salt intake and is considered to increase the risk of cardiovascular and renal morbidity. Although the mechanisms responsible for SS-HT are complex, the kidneys are known to play a central role in the development of SS-HT and the salt sensitivity of blood pressure (SSBP). Moreover, several factors influence renal function and SSBP, including the renin-angiotensin-aldosterone system, sympathetic nervous system, obesity, and aging. A phenotypic characteristic of SSBP is aberrant activation of the renin-angiotensin system and sympathetic nervous system in response to excessive salt intake. SSBP is also accompanied by a blunted increase in renal blood flow after salt loading, resulting in sodium retention and SS-HT. Obesity is associated with inappropriate activation of the aldosterone mineralocorticoid receptor pathway and renal sympathetic nervous system in response to excessive salt, and mineralocorticoid receptor antagonists and renal denervation attenuate sodium retention and inhibit salt-induced blood pressure elevation in obese dogs and humans. SSBP increases with age, which has been attributed to impaired renal sodium handling and a decline in renal function, even in the absence of kidney disease. Aging-associated changes in renal hemodynamics are accompanied by significant alterations in renal hormone levels and renal sodium handling, resulting in SS-HT. In this review, we focus mainly on the contribution of renal function to the development of SS-HT.