High-salt Diet Research Articles

High salt diet induces cognitive impairment and is linked to the activation of IGF1R/mTOR/p70S6K signaling.

A high-salt diet (HSD) has been associated with various health issues, including hypertension and cardiovascular diseases. However, recent studies have revealed a potential link between high salt intake and cognitive impairment. This study aims to investigate the effects of high salt intake on autophagy, tau protein hyperphosphorylation, and synaptic function and their potential associations with cognitive impairment. To explore these mechanisms, 8-month-old male C57BL/6 mice were fed either a normal diet (0.4% NaCl) or an HSD (8% NaCl) for 3 months, and Neuro-2a cells were incubated with normal medium or NaCl medium (80 mM). Behavioral tests revealed learning and memory deficits in mice fed the HSD. We further discovered that the HSD decreased autophagy, as indicated by diminished levels of the autophagy-associated proteins Beclin-1 and LC3, along with an elevated p62 protein level. HSD feeding significantly decreased insulin-like growth factor-1 receptor (IGF1R) expression in the brain of C57BL/6 mice and activated mechanistic target of rapamycin (mTOR) signaling. In addition, the HSD reduced synaptophysin and postsynaptic density protein 95 (PSD95) expression in the hippocampus and caused synaptic loss in mice. We also found amyloid β accumulation and hyperphosphorylation of tau protein at different loci both in vivo and in vitro. Overall, this study highlights the clinical significance of understanding the impact of an HSD on cognitive function. By targeting the IGF1R/mTOR/p70S6K pathway or promoting autophagy, it may be possible to mitigate the negative effects of high salt intake on cognitive function.

High-Salt Diet Inhibits the Expression of Bmal1 and Promotes Atrial Fibrosis and Vulnerability to Atrial Fibrillation in Dahl Salt-Sensitive Rats.

Hypertension is a risk factor for atrial fibrillation (AF), and brain and muscle arnt-like protein 1 (Bmal1) regulate circadian blood pressure and is implicated in several fibrotic disorders. Our hypothesis that Bmal1 inhibits atrial fibrosis and susceptibility to AF in salt-sensitive hypertension (SSHT) and our study provides a new target for the pathogenesis of AF induced by hypertension. The study involved 7-week-old male Dahl salt-sensitive that were fed either a high-salt diet (8% NaCl; DSH group) or a normal diet (0.3% NaCl; DSN group). An experimental model was used to measure systolic blood pressure (SBP), left atrial ejection fraction (LAEF), left atrial end-volume index (LAEVI), left atrial index (LAFI), AF inducibility, AF duration, and atrial fibrosis pathological examination and the expression of Baml1 and fibrosis-related proteins (TNF-α and α-SMA) in left atrial tissue. DSH increased TNF-α and α-SMA expression in atrial tissue, level of SBP and LAESVI, atrial fibrosis, AF induction rate, and AF duration, and decreased Bmal1 expression in atrial tissue, the circadian rhythm of hypertension, and level of LAEF and LAFI. Our results also showed that the degree of atrial fibrosis was negatively correlated with Bmal1 expression, but positively correlated with the expression of TNF-α and α-SMA. We demonstrated that a high-salt diet leads to circadian changes in hypertension due to a reduction of Bmal1 expression, which plays a crucial role in atrial fibrosis and increased susceptibility to AF in SSHT rats.

Modulatory Effects of the Kuwanon-Rich Fraction from Mulberry Root Bark on the Renin-Angiotensin System.

In this study, we investigated the anti-hypertensive properties of mulberry products by modulating the renin-angiotensin system (RAS). Comparative analysis showed that the ethyl acetate fractions, particularly from the Cheongil and Daeshim cultivars, contained the highest levels of polyphenols and flavonoids, with concentrations reaching 110 mg gallic acid equivalent (GE)/g and 471 mg catechin equivalent (CE)/g of extract, respectively. The ethyl acetate fraction showed superior angiotensin-converting enzyme (ACE) inhibitory activity, mainly because of the presence of the prenylated flavonoids kuwanon G and H. UPLC/Q-TOF-MS analysis identified kuwanon G and H as the primary active components, which significantly contributed to the pharmacological efficacy of the extract. In vivo testing of mice fed a high-salt diet showed that the ethyl acetate fraction substantially reduced the heart weight and lowered the serum renin and angiotensinogen levels by 34% and 25%, respectively, highlighting its potential to modulate the RAS. These results suggested that the ethyl acetate fraction of mulberry root bark is a promising candidate for the development of natural ACE inhibitors. This finding has significant implications for the management of hypertension through RAS regulation and the promotion of cardiovascular health in the functional food industry.

High-salt diet induces microbiome dysregulation, neuroinflammation and anxiety in the chronic period after mild repetitive closed head injury in adolescent mice.

The associations between human concussions and subsequent sequelae of chronic neuropsychiatric and cardiovascular diseases such as hypertension have been reported; however, little is known about the underlying biological processes. We hypothesized that dietary changes, including a high-salt diet, disrupt the bidirectional gut-brain axis, resulting in worsening neuroinflammation and emergence of cardiovascular and behavioural phenotypes in the chronic period after repetitive closed head injury in adolescent mice. Adolescent mice were subjected to three daily closed head injuries, recovered for 12 weeks and then maintained on a high-salt diet or a normal diet for an additional 12 weeks. Experimental endpoints were haemodynamics, behaviour, microglial gene expression (bulk RNA sequencing), brain inflammation (brain tissue quantitative PCR) and microbiome diversity (16S RNA sequencing). High-salt diet did not affect systemic blood pressure or heart rate in sham or injured mice. High-salt diet increased anxiety-like behaviour in injured mice compared to sham mice fed with high-salt diet and injured mice fed with normal diet. Increased anxiety in injured mice that received a high-salt diet was associated with microgliosis and a proinflammatory microglial transcriptomic signature, including upregulation in interferon-gamma, interferon-beta and oxidative stress-related pathways. Accordingly, we found upregulation of tumour necrosis factor-alpha and interferon-gamma mRNA in the brain tissue of high salt diet-fed injured mice. High-salt diet had a larger effect on the gut microbiome composition than repetitive closed head injury. Increases in gut microbes in the families Lachnospiraceae, Erysipelotrichaceae and Clostridiaceae were positively correlated with anxiety-like behaviours. In contrast, Muribaculaceae, Acholeplasmataceae and Lactobacillaceae were negatively correlated with anxiety in injured mice that received a high-salt diet, a time-dependent effect. The findings suggest that high-salt diet, administered after a recovery period, may affect neurologic outcomes following mild repetitive head injury, including the development of anxiety. This effect was linked to microbiome dysregulation and an exacerbation of microglial inflammation, which may be physiological targets to prevent behavioural sequelae in the chronic period after mild repetitive head injury. The data suggest an important contribution of diet in determining long-term outcomes after mild repetitive head injury.

HIGH SALT DIET EFFECTS ON CEREBRAL AND SYSTEM HEMODYNAMICS IN RESPONSE TO BREATH HOLDING TEST AND HYPERVENTILATION

Objective: High salt (HS) diet impairs endothelium-dependent vasodilation in many vascular beds, including cerebral microcirculation, and may affect vascular resistance. The aim of present study was to determine if 7-day HS diet affected cerebral and system hemodynamics in response to breath holding test (BHT) and hyperventilation in healthy young persons. Design and method: In this controlled intervention study 27 persons participated (F:21, M:6, age range 19-24). Participants consumed 7-day low salt (LS) diet (<2.3g NaCl/day) and afterwards 7-day of HS diet (>11.2g NaCl/day). Blood and urine analysis and anthropometric measurements were performed after each diet. Arterial blood pressure, heart rate and heart rate variability (HRV), cerebral and systemic hemodynamic parameters were recorded simultaneously with transcranial Doppler ultrasound and the Task Force® Monitor in response to BHT and hyperventilation. Results: The subjects remained normotensive during both protocols. After hyperventilation test, HS diet did not affect parameters of cerebral or systemic hemodynamics. HRV was more decreased after HS diet, while baroreceptor sensitivity (BRS) decreased in both LS and HS diet. Similar lack of effects of HS diet on cerebral and system hemodynamics was observed in BHT. Urine concentration of noradrenaline and vanillylmandelic acid was significantly decreased by HS diet. Conclusions: Results suggest that HS diet suppresses sympathetic activity. However, physiological hemodynamic responses to BHT and hyperventilation are preserved, which could be contributed to salt-resistant phenotype of participants and some, still unexplained (chemoreceptors-mediated) compensatory mechanisms preserving cerebral and systemic blood flow.

NADPH Oxidase 4 Knockout in Dahl Salt-Sensitive Rats Impacts Kidney O2 Consumption and UCP2 Responses to a High Salt Diet

Rationale: The effects of a high salt (HS) diet upon renal metabolism and O2 utilization have not been extensively studied and there is a significant gap in our understanding of the role of oxidative stress in the effciency of energy production and substrate metabolism. The present study compared the effects of a HS diet (4% NaCl) on changes in O2 consumption and transcriptomic profiles in Dahl salt-sensitive (SS) rats to SS rats with a global knockout of NADPH oxidase 4 (NOX4) gene (SS Nox4−/−). Methods: Male SS (n=9) and SS Nox4−/− (n=10) rats were fed a 0.4% NaCl (LS) diet. At 7-8 weeks of age, the rats were implanted with a renal artery ultrasonic flow probe (Transonic) together with a femoral arterial catheter and a renal venous catheter. Renal blood flow (RBF) and mean arterial pressure (MAP) were measured continuously (24/7) and arterial (A) and renal venous (Vr) blood was sampled intermittently when rats were fed the LS and on days 7, 14, and 21 after switching to the HS diet. A and Vr blood O2 content was immediately measured by radiometer. From separate groups of rats subjected to the same protocol, the renal cortical tissue (Cx) and outer medulla (OM) were removed for mRNAseq analysis (Novogene, Inc). Results: When switching to the HS diet, the average 24-hour MAP of SS rats rose from 122 ± 2 to 140 ± 2 on HS7 to 164 ± 5 mmHg by HS21. In contrast, MAP in SS Nox4−/− rats increased from 121 ± 2 at LS to 132 ± 1 at HS7 to 152 ± 3 mmHg at HS21 (p<0.05, compared to SS). RBF, normalized by changes of kidney weights obtained in another group of SS and SS Nox4−/− rats fed the same HS diet, SS rats exhibited a significant reduction of RBF over the 3 weeks of the HS diet averaging LS 7.2 ± 0.6, HS7 5.1 ± 0.5 and HS21 4.6 ± 0.7 mL/min/g kidney weight (gkw). This reduction of RBF was not observed in SS Nox4−/− rats (LS 6.8 ± 0.8, HS7 6.6 ± 0.6, HS21 6.0 ± 0.6 mL/min/gkw). The kidney O2 extraction ratio at LS tended to be lower in SS Nox4−/− (11.2 ± 1.7%) compared to SS (14.0 ± 1.1%) (p=0.10). When switched to the HS diet, O2 extraction was significantly reduced in the SS Nox4−/− rats reaching 8.3 ± 1.0% at HS21 (p<0.05), while it did not change significantly in SS (12.7 ± 1.4%) at HS21. Notably, as determined by RNAseq analysis, Ucp2 expression in cortical tissue was significantly greater in SS fed HS than in SS Nox4−/−rats at HS21 . A gene enrichment analysis of the mRNAseq cortical tissue data (GSEA-KEGG pathways) focused on metabolic pathways indicated that knockout of Nox4 resulted in reduced expression of linoleic acid, lysine, and histidine metabolism pathways at LS when compared to SS. Those differences were not observed after HS feeding. However, upregulation of oxidative phosphorylation pathway was observed at HS21 in SS Nox4−/− when compared to SS rats. In the OM, a greater expression of genes associated with fatty acid degradation and carbon metabolism was observed in SS Nox4−/− rats when fed LS which was observed also at HS21. Conclusion: The preliminary results of this study suggest that reduced extraction of O2 in the SS Nox4−/− rats compared to SS rats may be partially explained by the differential response of Ucp2 to salt. Further studies are needed to determine whether the effciency of ATP production is enhanced in SS Nox4−/− as consequence of reduction of reactive oxygen species (e.g., H2O2) and the related downstream pathways such as phosphorylation of mTORC1. R01 HL151587, AHA 23POST1008714. 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.

Sex-specific role for the clock protein PER1 in renal ET-1 regulation in salt-sensitive hypertension

Global knockout (KO) of the clock protein PERIOD1 (PER1) in male, but not female, C57BL/6J mice displayed both blunted blood pressure and urinary endothelin-1 (ET-1) rhythms. More recently, we showed that Dahl salt-sensitive (SS) Per1 KO male rats had exacerbated SS hypertension, alongside increased renal injury, renal ET-1 gene ( Edn1) expression, and ET-1 peptide expression from kidneys collected during the rat’s inactive period (2 PM). Additionally, we see increased long non-coding RNA EDN1-AS, which has been suggested to modulate ET-1 production. However, it is unknown whether PER1 regulates rhythms of ET-1 in SS rats and whether PER1 regulates ET-1 in a sex-dependent way. The study objective was to test the hypothesis that PER1 negatively regulates renal EDN1-AS and Edn1 to modulate ET-1 in a sex- and time-dependent manner. To test this hypothesis, kidneys were collected during rat’s active period (2 AM) from male and female SS Per1 KO and SS control rats on a normal salt (0.4% NaCl) or 3 weeks high salt (4% NaCl) diet ( N=5-6). Renal ET-1 protein levels were measured by ELISA and semi- and quantitative PCR was performed to quantify the levels of EDN1-AS and Edn1. Renal ET-1 levels were significantly higher at 2 AM than our previously reported data at 2 PM in both SS Per1 KO and control male rats ( p<0.0001). Renal ET-1 levels were significantly increased in male SS Per1 KO rats following a high salt diet ( p=0.0362) but not in females ( p=0.9522). This was also reflected in the Edn1 mRNA levels (male, p=0.0005; female, p=0.0814). EDN1-AS levels were also increased in males ( p=0.0123) but not in females. Interestingly, significant sex differences were seen in EDN1-AS levels, where EDN1-AS levels were higher in males than females following both low and high salt diets, suggesting a potential novel sex-dependent mechanism for ET-1 regulation. Together, these data suggest sex-specific action of PER1 in the regulation of ET-1 in a model of salt-sensitive hypertension. Future work aims to investigate blood pressure and renal injury in female SS Per1 KO rats and whether this apparent negative regulation of ET-1 by PER1 contributes to the SS hypertensive and renal injury phenotype seen in male SS Per1 KO rats. This work was supported by the National Institutes of Health grants R35 HL135749 (to A.S.), R01 342 DK109570 (to M.G. and A.S.), and Department of Veteran Affairs grants I01 BX004024 (to A.S.). 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.

Assessment of Mitochondrial Function in a Mouse Model of Mild Chronic Kidney Disease Induced by High-Fat and High-Salt Diet

Intro: Kidneys are known for high energy demands involved in active transport and are thus highly oxidative organs, consuming the second-highest amount of oxygen per gram of tissue at rest (only exceeded by the heart). Chronic kidney disease (CKD) is a complex disorder that presents substantial challenges in understanding its pathophysiological mechanisms, including mitochondrial alterations. However, there are no established mouse models that recapitulate the CKD phenotype observed in humans.Hypothesis: We hypothesized that a high-fat, high-salt diet would induce a mild CKD phenotype and mitochondrial dysfunction in mice. Methods: We subjected C57Bl/6 mice to a high-fat (42%) and high-salt (8% NaCl) diet (HF/HNaCl) or standard chow diet for 16 weeks (n=4 per group). Additionally, we conducted comprehensive investigations into mitochondrial function, focusing on state 3 mitochondrial oxygen consumption (Oroboros O2K) and adenosine triphosphate (ATP) production (Horiba Fluoromax). Differences between groups were determined using Student’s t-tests. Results: HF/HNaCl feeding resulted in polyurea indicated by an increase in 24-hour urine output (Chow: 1.5 ± 0.2 mL; HF/HNaCl: 3.7 ± 0.8 mL; p=0.05) and a 30% reduction in glomerular filtration rate compared to chow-fed animals (Chow: 911.2 ± 107.8 μL/min/100g; Hf/HNaCl: 639.5 ± 25.9 μL/min/100g; p=0.08), indicative of the development of mild kidney disease. Although subtle changes were observed in mitochondrial oxygen consumption and ATP production, the alterations were not statistically significant. Discussion: Our findings suggest that the induced mild CKD state via the dietary regimen led to moderate renal impairment without substantial impacts on mitochondrial respiratory capacity and ATP synthesis. The establishment of this mouse model provides a valuable platform for further investigations into the complex interplay between renal dysfunction and mitochondrial dynamics in the context of mild CKD, offering insights that may pave the way for the development of targeted therapeutic interventions for this prevalent and debilitating condition. This project was supported by grants from the National Institutes of Health. 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.

Impact of high-salt diet in health and diseases and its role in pursuit of cancer immunotherapy by modulating gut microbiome.

Cancer chemotherapy remains an area of concern, as many of the therapies are uncomfortable involving side effects and unpleasant experiences. These factors could further reduce patient's quality of life, and even endanger their life. Many therapeutic strategies have been tried to reduce the unpleasant side effects and increase the treatment effectiveness; however, none have shown to have promising effects. One of the main hindrances to cancer therapy is the escape strategies by tumor cells to the immune attack. Promoting inflammation in the tumor microenvironment is the cornerstone and key therapeutic target in cancer chemotherapy. High-salt diet (HSD) intake, though it has deleterious effects on human health by promoting chronic inflammation, is found to be advantageous in the tumor microenvironment. Studies identified HSD favors an increased abundance of Bifidobacterium species in the tumor environment due to gut barrier alteration, which, in turn, promotes inflammation and favors improved response to cancer chemotherapy. A review of the literature was carried out to find out the effects of an HSD on health and diseases, with special mention of its effect on cancer chemotherapy. Studies emphasized HSD would block the myeloid-derived suppressor cells which will enhance the tumor immunity. Exploration of the precise mechanism of simple HSD regime/ingestion of specific bacterial species as probiotics will be effective and essential to formulate the game-changing cancer chemotherapy. With the modern era of healthcare moving toward precision medicine where the physician can choose the treatment option suitable for the individual, HSD regime/ingestion of specific bacterial species can be considered.

Profound Sex Differences in the Kidney Expression of Mitochondria-Related Genes in Endothelin-1 Receptor B (ETB) Deficient Rats Fed a High Salt Diet

Elevated levels of endothelin-1 (ET-1), overactivation of the ETA receptor and oxidative stress are critical in the development of hypertensive kidney disease. Females are more resistant than males to kidney damage; however, it is unclear if overactivation of the ETA receptor disrupts mitochondrial homeostasis similarly in both sexes. With these studies we aimed to determine if overactivation of the ET-1/ETA axis has a differential effect on mitochondrial homeostasis in the male and female kidney during high salt consumption. Male and female ETB deficient (ETB def) and transgenic (TG) control rats were placed on a high salt diet (4.0% NaCl) for 3 weeks. Kidneys were collected and the expression of 84 mitochondria-related genes in renal cortex and outer medulla was assessed with qRT-PCR arrays. In response to high salt, male ETB def rats displayed cortical overexpression of 13 genes when compared to male TG controls, while only 7 genes were upregulated in cortex of female ETB def rats compared to female TG controls. Genes upregulated in male ETB def rats are involved in apoptosis, mitochondrial biogenesis and transport, energy metabolism and ROS handling (selected genes, male ETB def vs. TG controls, n=3/group: Ucp1 (19-fold increase, p=0.03), Bnip3 and Scl25a21 (6-fold, p=0.003 and p=0.004, respectively), and Mfn1 (5-fold, p=0.03). Genes upregulated in females are involved in apoptosis, ROS handling and mitochondrial transport (selected examples, female ETB def vs. TG control, n=3/group: 2-fold increase in Sod1 (p=0.03), Akt1 (p=0.003) and Grpel1 (p=0.019). No common genes were changed between the sexes. When comparisons were made in the outer medulla, we found that deficiency of the ETB receptor in males resulted in a ≥30% reduction in the expression of 29 genes involved in apoptosis, mitochondrial biogenesis and transport, energy metabolism, ROS handling and other mitochondrial functions (p<0.05). On the other hand, the expression of 8 genes involved in mitochondrial transport and apoptosis was reduced by ≥80% in the female ETB def rat outer medulla vs. TG controls, while 3 genes involved in mitochondrial transport and energy metabolism had a 2-fold overexpression (p<0.05). Altogether, our results demonstrate that salt-sensitive hypertension affects mitochondrial homeostasis differentially in distinct regions of the kidney, and that the male and female kidney respond to salt in a disparate manner: with overactivation of the ET-1/ETA axis resulting in greater dysregulation of mitochondria-related genes in the male versus the female kidney during a high salt diet. Funded by UAB-UCSD O’Brien Center Summer Research Pathway for Students P30 DK079337 and KURE R25 DK115353 to BSM, NIGMS UAB CORD BBRT to GJC, and NIH K01HL145324 and Sapphire North America Return to Science Research grant to CDM. 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 Diet Regulates Intrarenal Sex Steroid Biosynthesis Pathway in a Sex-Specific Manner

Hypertension is more prevalent in men than age-matched pre-menopausal women. Sodium (Na+) homeostasis plays a major role in the control of blood pressure. Renal medullary estrogenic signaling promotes Na+ excretion in female, but not male Sprague Dawley (SD) rats implicating estrogen as a potential female-specific natriuretic factor. Recently we showed that the components of estrogen biosynthesis pathway are expressed in rat kidney suggesting the kidneys can produce estrogen locally to invoke this natriuretic response. It is unknown how dietary Na+ impacts biosynthesis of sex hormones. We hypothesize that a high salt (HS) diet enhances intrarenal estrogen biosynthesis pathway, and this enhancement is more pronounced in females compared to males. To test this hypothesis, we harvested the renal cortex from 14 week old male and female SD rats maintained on either a normal salt (NS 0.3% NaCl) or HS (4% NaCl) diet for 5 days. Given that cholesterol is the main precursor of steroid hormones, we measured the mRNA expression of HMG-CoA reductase (HMGCR), the key enzyme for cholesterol synthesis; and the steroidogenic acute regulatory protein (StAR), which transports cholesterol into the mitochondria for steroid commitment. We also assessed the mRNA expression of steroid 5α reductase (SRD5A), which converts testosterone to the non-aromatizable androgen, dihydrotestosterone. Further, we assessed aromatase, the key enzyme in estrogen biosynthesis, via immunohistochemistry. We found that renal HMGCR was more abundant in female compared to male rats maintained on a NS diet (P=0.0467). Increasing dietary salt intake upregulated renal HMGCR expression in male (P=0.0020), but not female rats (NS male; 100.0 ± 26.8 vs. NS female; 235.3 ± 23.2 vs. HS male 294.6 ± 42.0 vs HS female 314.6 ± 21.0 relative expression % NS male n=6-7/group). On a NS diet, renal StAR was more abundant in female compared to male rats (NS male; 100.0 ± 25.3 vs. NS female; 217.2 ± 36.5 relative expression % NS male P=0.0255 n=5-7/group). Increasing dietary salt did not alter expression of StAR in either sex. Similarly, renal SRD5A expression was higher in female compared to male (P=0.0141) rats. In addition, a HS diet increased the expression of SRD5A in female rats (0.0093) and tended to increase SRD5A expression in male (P=0.0849) rats (NS male; 100.0 ± 11.6 vs. NS female; 297.8 ± 37.7 vs. HS male 243.0 ± 39.2 vs HS female 498.8 ± 44.8 relative expression % NS male n=6-7/group). We also revealed that aromatase was expressed at a comparable level in male and female renal cortex, regardless of the diet (NS male; 2.4 ± 0.3 vs. NS female; 2.6 ± 0.3 vs. HS male 2.8 ± 0.3 vs HS female 1.9 ± 0.1 Average intensity x area mM2 n=6-9/group). Together, this data suggests that (i) the female kidney may be more primed for steroid biosynthesis and (ii) there is a sex-specific upregulation of the intrarenal sex steroid biosynthesis pathway in response to increasing dietary salt intake. Additional work is required to investigate the potential role of the kidney in androgen and estrogen production. A better understanding of this steroidogenic pathway could reveal novel sex-specific therapeutics for hypertension. T32DK007569-34 Carl Gottschalk Grant. 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.

Dynamic Shift Towards Increased O2 Consumption and Reduced Lactate Uptake During Acute Volume Expansion in Sprague Dawley (SD) Rats

Rationale: In a recent study (PMID: 37575482), we established a method to assess alterations in O2 and substance metabolism in the kidneys in conscious free moving rat. This involved the repeated collection of renal arteriovenous blood and urine, alongside continuous measurements (24/7) of renal blood flow (RBF) and blood pressure (BP). Our previous studies demonstrated that in SD rats, there is an augmentation in RBF and GFR following chronic salt loading. Additionally, total renal O2 consumption (VO2) increases while there is a decrease in the net consumption of lactate with a tendency for its increased production. We hypothesize that these changes are not exclusive to the chronic phase of salt-loading but can also manifest during rapid increases of RBF and GFR. The primary aim of this study was to establish an experimental model reflecting these dynamics. Methods: SD rat (male, 10 wk age fed 0.4 NaCl (LS) diet, n=8) were studied. The jugular vein and femoral vein and artery were catheterized together with the renal vein. FITC-inulin (2 mg/mL) dissolved in 2%BSA saline was infused at 1.0 mL/100 g body wt/h from jugular vein. RBF was measured with an ultrasonic flow probe (Transonic, Inc) on the left renal artery and BP was measured through an arterial catheter. Whole blood, collected from donor rats, was diluted by 20% with saline, and a 6 mL volume expansion was performed with this solution over 1 hour. Blood was collected from both the renal venous and arterial catheters and urine from a ureteral catheter before and after volume expansion. O2 content was measured by radiometer and plasma lactate measured with a high sensitivity L-lactate fluorescent assay kit (ab169557, abcam). The same experiment was performed without volume expansion (i.e., sham study). O2 and lactate delivery and consumption were calculated as follows: Delivery = arterial content (A) x RBF; Consumption = (A − renal venous content (RV)) x RBF; Extraction = (A-RV) / A. Results: BP increased from 101 ± 3 to 111 ± 4 mmHg (p<0.05), RBF increased from 5.4 ± 0.5 to 7.9 to 0.6 mL/min/g kidney weight (gkw) (p<0.05) by volume expansion. GFR increased from 0.94 ± 0.06 to 1.19 ± 0.08 mL/min/gkw (p<0.05). O2 delivery increased from 1.06 ± 0.09 to 1.61 ± 0.12 mL/min/gkw (p<0.05). O2 consumption (VO2) increased from 0.06 ± 0.01 to 0.08 ± 0.01 mL/min/gkw (p<0.05). The O2 extraction ratio remained unchanged. Both lactate consumption (0.94 ± 0.11 to 0.47 ± 0.11 mM/gkw, p<0.05) and extraction ratio (0.18 ± 0.01 to 0.11 ± 0.02, p<0.05) were reduced by volume expansion. Conclusion: The increase of O2 consumption and reduction of lactate consumption with acute volume expansion model appear to be similar to those that we observed when SD rats were subjected to a chronic step increase of salt intake (from 0.4 to 4.0% NaCl). In contrast, however, an increase in O2 extraction was not observed with acute volume expansion as was the case with a high salt diet in which our studies have indicated that a prolonged tubular reabsorptive workload leads to enhanced oxidative stress with an uncoupling of oxidative phosphorylation. R01 HL151587, AHA 23POST1008714. 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.

Impact of Renin Angiotensin System Inhibition on Renal Function in Fructose-induced Salt Sensitive Blood Pressure

Combined dietary intake of fructose with high salt at levels comparable to that in the upper quintile of western population increases blood pressure, decreases glomerular filtration rate, and results in albuminuria and insulin resistance. We tested the hypothesis that chronic treatment with either an angiotensin converting enzyme inhibitor (enalapril) or angiotensin receptor blocker (losartan) will preserve glomerular filtration rate (GFR) and reduce albuminuria. Sprague Dawley rats of both sexes were instrumented with telemetry. After a baseline period on glucose+0.4% NaCl, rats were assigned to the following groups: 20% glucose+0.4%NaCl treated with vehicle (GNS+V) or 20% fructose+4% NaCl and treated with either vehicle (FHS+V), losartan 0.42 mg/kg/hr by osmotic minipump (FHS+L) or enalapril 0.21 mg/kg/hr (FHS+E). The doses of losartan and enalapril were chosen to achieve MAP similar to the control GNS+V group. Body weights did not differ among the groups upon entry into the protocol and increased similarly among the groups. Non-fasting blood glucose was elevated in all groups in both male and female rats. Mean arterial pressure (MAP) was 108±2 mmHg in GNS+V but was elevated in the FHS+V male rats, 118±2 mmHg ( P<0.01). FHS+L and FHS+E displayed MAP similar GNS+V rats: 104±2 and 106±2 mmHg, respectively. GFR assessed by transdermal FITC-sinistrin elimination was similar across all groups: 0.85±0.04 (GNS+V), 0.86±0.08 (FHS+V), 0.94±0.10 (FHS+L), 1.13±0.12 ml/min/100 g bw (FHS+E). Female rats did not display elevated MAP: 108±1 mmHg (FHS+V) vs 105±2 mmHg (GNS+V). Losartan and enalapril did not change MAP, but GFR was higher in female FHS+L rats, 1.14± 0.05, vs GNS+V 0.75±0.06 ( P < 0.05). Thus, male but not female fructose-fed rats on high salt diet display significantly higher blood pressure. Short-term fructose high salt feeding did not change GFR in either male or female rats. RAS inhibition did not impact GFR in male rats, but losartan increased GFR in female rats on high fructose plus high salt diet compared with glucose-fed female rats on normal salt diet. R01 HL163844. 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.

Adrenal circadian clock protein BMAL1 regulates aldosterone and renal excretory function in a sex- and time-dependent manner

BMAL1 is a core circadian clock protein that is important for circadian rhythms of physiological function, as well as maintaining physiological homeostasis, including aspects of kidney function. Global Bmal1 knockout (KO) male mice exhibit loss of diurnal rhythm of renal sodium (Na) excretion in response to a normal or high salt diet. However, renal Na excretion rhythm remained intact in kidney-specific Bmal1 KO male mice. The adrenal gland synthesizes hormones, such as aldosterone, which influence renal electrolyte handling. The role of adrenal BMAL1 in the regulation of renal function remains unknown. The study objective was to test the hypothesis that adrenal BMAL1 is required for normal rhythms of aldosterone and renal Na handling. Male and female adrenal-specific aldosterone synthase Cre positive (AS) Bmal1 KO and littermate Cre negative, floxed control mice ( n=5-9/genotype) were acclimated to metabolic cages for 3 days then samples were collected for three days with mice on a normal salt diet followed by 7 days on a low salt diet to challenge the mice to stimulate aldosterone production. Twelve-hour food intake was measured, and urine samples were collected to measure night/day patterns in renal Na balance. Urinary aldosterone excretion was measured by ELISA. Both day and night urinary aldosterone excretion were increased following a low salt diet in control and KO male mice (ANOVA main effect of diet, p<0.05). Male AS Bmal1 KO mice displayed a significant increase in day urinary aldosterone excretion following a normal and low salt diet (ANOVA main effect of genotype, p=0.0230), with a trend for an increase in night urinary aldosterone excretion ( p=0.0571). Interestingly, these genotype effects were not seen in female AS Bmal1 KO mice. Urinary aldosterone was increased following a low salt diet in female mice, but only during the nighttime in control and KO mice ( p=0.0205). Sex differences were also observed in renal Na balance, where Na balance was more positive during the day in male, but not female, AS Bmal1 KO vs. control mice (ANOVA main effect of genotype, p=0.0282). Together, these data suggest adrenal BMAL1 is required for normal rhythms of aldosterone and renal Na balance in a sex-dependent manner. Future work will investigate whether the changes in aldosterone contribute to changes in renal Na balance. This work was supported by the American Heart Association (Postdoctoral FellowshipP0240441 to H.C.; Established Investigator Award 19EIA34660135 to M.G), theRobert and Mary Cade Professorship in Physiology (to M.G.) and the NIH(F32DK121424 to G.R.C.). 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.

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.

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.

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.

Shared and Distinct Cellular and Molecular Landscapes of Cardiac Left Ventricle Injury in Three Models of Hypertension: Insights from Single-nucleus RNA Sequencing

High blood pressure is the leading risk factor of heart diseases, affecting millions of patients who remain hypertensive despite taking three or more anti-hypertensive medications. Understanding the cellular and molecular basis of heart damage at cell type-specific level in different types of hypertension is crucial for developing targeted therapies. In this study, we applied 10x Genomics' single-nucleus RNA sequencing (snRNA-seq) technology to cardiac left ventricle samples from three well-established hypertension models: C57BL/6 mice treated with Ang II, the Dahl salt-sensitive rat, and the spontaneous hypertensive rat, along with their respective controls. Our dataset included 166,489 nuclei from 18 samples across 9 experimental conditions. Our analysis revealed a shared set of cellular and molecular changes across these hypertensive models. For instance, we observed extracellular matrix remodeling in fibroblasts in salt-sensitive and spontaneous hypertension, as indicated by increased collagen production. Endothelial cells showed an increased proportion in all three models, and showed higher levels of injury based on expression patterns of increased proliferation and migration. Both AngII-induced and spontaneous hypertension showed cardiomyocyte dysfunction, affecting cardiac muscle contraction and ATP metabolic process. Notably, impaired ATP generation was a common response in AngII-induced hypertension, involving not only cardiomyocytes but also endothelial cells and fibroblasts. During the progression of salt-sensitive hypertension, a notable increase in inflammation occurred, marked by steadily increasing immune cell infiltration, eventually reaching a 1.5-fold increase in immune cells. T cells were particularly elevated during the high-salt diet and actively engaged in the CD45 signaling network in the late stage of spontaneous hypertension, where an elevated interaction between cardiomyocyte and endothelial cells through vascular endothelial growth factor was observed. In conclusion, the utilization of snRNA-seq identified shared and distinct cellular and molecular changes associated with left ventricular injury in various models of hypertension. NIH grant HL149620. 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-glucose exchanger 2 inhibitor canagliflozin promotes mitochondrial metabolism and alleviates salt-induced cardiac hypertrophy via preserving SIRT3 expression

IntroductionExcess salt intake is not only an independent risk factor for heart failure, but also one of the most important dietary factors associated with cardiovascular disease worldwide. Metabolic reprogramming in cardiomyocytes is an early event provoking cardiac hypertrophy that leads to subsequent cardiovascular events upon high salt loading. Although SGLT2 inhibitors, such as canagliflozin, displayed impressive cardiovascular health benefits, whether SGLT2 inhibitors protect against cardiac hypertrophy-related metabolic reprogramming upon salt loading remain elusive. ObjectivesTo investigate whether canagliflozin can improve salt-induced cardiac hypertrophy and the underlying mechanisms. MethodsDahl salt-sensitive rats developed cardiac hypertrophy by feeding them an 8% high-salt diet, and some rats were treated with canagliflozin. Cardiac function and structure as well as mitochondrial function were examined. Cardiac proteomics, targeted metabolomics and SIRT3 cardiac-specific knockout mice were used to uncover the underlying mechanisms. ResultsIn Dahl salt-sensitive rats, canagliflozin showed a potent therapeutic effect on salt-induced cardiac hypertrophy, accompanied by lowered glucose uptake, reduced accumulation of glycolytic end-products and improved cardiac mitochondrial function, which was associated with the recovery of cardiac expression of SIRT3, a key mitochondrial metabolic regulator. Cardiac-specific knockout of SIRT3 not only exacerbated salt-induced cardiac hypertrophy but also abolished the therapeutic effect of canagliflozin. Mechanistically, high salt intake repressed cardiac SIRT3 expression through a calcium-dependent epigenetic modifications, which could be blocked by canagliflozin by inhibiting SGLT1-mediated calcium uptake. SIRT3 improved myocardial metabolic reprogramming by deacetylating MPC1 in cardiomyocytes exposed to pro-hypertrophic stimuli. Similar to canagliflozin, the SIRT3 activator honokiol also exerted therapeutic effects on cardiac hypertrophy. ConclusionCardiac mitochondrial dysfunction caused by SIRT3 repression is a critical promotional determinant of metabolic pattern switching underlying salt-induced cardiac hypertrophy. Improving SIRT3-mediated mitochondrial function by SGLT2 inhibitors-mediated calcium handling would represent a therapeutic strategy against salt-related cardiovascular events.