Endogenous Ouabain Research Articles

Role of Na+-K+ ATPase Alterations in the Development of Heart Failure.

Na+-K+ ATPase is an integral component of cardiac sarcolemma and consists of three major subunits, namely the α-subunit with three isoforms (α1, α2, and α3), β-subunit with two isoforms (β1 and β2) and γ-subunit (phospholemman). This enzyme has been demonstrated to transport three Na and two K ions to generate a trans-membrane gradient, maintain cation homeostasis in cardiomyocytes and participate in regulating contractile force development. Na+-K+ ATPase serves as a receptor for both exogenous and endogenous cardiotonic glycosides and steroids, and a signal transducer for modifying myocardial metabolism as well as cellular survival and death. In addition, Na+-K+ ATPase is regulated by different hormones through the phosphorylation/dephosphorylation of phospholemman, which is tightly bound to this enzyme. The activity of Na+-K+ ATPase has been reported to be increased, unaltered and depressed in failing hearts depending upon the type and stage of heart failure as well as the association/disassociation of phospholemman and binding with endogenous cardiotonic steroids, namely endogenous ouabain and marinobufagenin. Increased Na+-K+ ATPase activity in association with a depressed level of intracellular Na+ in failing hearts is considered to decrease intracellular Ca2+ and serve as an adaptive mechanism for maintaining cardiac function. The slight to moderate depression of Na+-K+ ATPase by cardiac glycosides in association with an increased level of Na+ in cardiomyocytes is known to produce beneficial effects in failing hearts. On the other hand, markedly reduced Na+-K+ ATPase activity associated with an increased level of intracellular Na+ in failing hearts has been demonstrated to result in an intracellular Ca2+ overload, the occurrence of cardiac arrhythmias and depression in cardiac function during the development of heart failure. Furthermore, the status of Na+-K+ ATPase activity in heart failure is determined by changes in isoform subunits of the enzyme, the development of oxidative stress, intracellular Ca2+-overload, protease activation, the activity of inflammatory cytokines and sarcolemmal lipid composition. Evidence has been presented to show that marked alterations in myocardial cations cannot be explained exclusively on the basis of sarcolemma alterations, as other Ca2+ channels, cation transporters and exchangers may be involved in this event. A marked reduction in Na+-K+ ATPase activity due to a shift in its isoform subunits in association with intracellular Ca2+-overload, cardiac energy depletion, increased membrane permeability, Ca2+-handling abnormalities and damage to myocardial ultrastructure appear to be involved in the progression of heart failure.

A novel mouse model recapitulates the effects of rs2254524 variant in the lanosterol synthase gene on salt sensitivity and organ damage.

The blood pressure (BP) response to salt intake (salt sensitivity) shows great variability among individuals and is more frequent in hypertensive patients. Elevated levels of the steroid hormone Endogenous Ouabain (EO) are associated with hypertension (HT) and salt sensitivity. The lanosterol synthase gene ( LSS ) plays a key role in the biosynthesis of steroids and its rs2254524 variant (Val642Leu) is linked to salt sensitivity in humans. This study aims to investigate the pathophysiological significance of the Lss missense variation in a new knock-in mouse model of salt-sensitive HT onset. We generated a mouse model carrying the murine homolog (Val643Leu) of the human LSS variant. C57BL/6N LssV643L/V643L mice were fed different NaCl diets (low-salt, LSD; normal-salt, NSD; high-salt, HSD) and were characterized at functional, histological, and molecular levels. At baseline, mutant mice showed an enlarged kidney compared to the wild-type (WT) counterpart, but the Lss V643L variant did not affect EO biosynthesis nor systolic BP at 3 and 12 months. In HSD, we observed an increased systolic BP only in 12-month-old LssV643L/V643L mice, compared to NSD. Moreover, only the HSD LssV643L/V643L mice showed cardiac hypertrophy and a higher incidence of cardiac fibrosis compared to WT at 12 months. Finally, the Lss mRNA level was differentially regulated by HSD in the adrenal gland, liver, and heart of LssV643L/V643L mice compared to WT. The novel Lss mouse model resembles the salt-sensitive HT phenotype observed in hypertensive patients and provides a good model of salt-sensitive HT and HT-mediated organ damage.

#1954 Effects of lanosterol synthase (LSS) gene on the development of acute kidney injury after cardiac surgery

Abstract Background and Aims Acute kidney injury (AKI) is a common post-cardiac surgery complication, significantly affecting morbidity and mortality. Recent studies reported that high plasma levels of endogenous ouabain (EO), a stress hormone secreted by the adrenal glands, are associated to worse kidney outcomes after cardiac surgery. Our group demonstrated that EO activity is affected by a missense variant (rs2254524, C -> A, Val642Leu) in Lanosterol Synthase (LSS) gene, an enzyme implicated in EO synthesis. The aim of this work is to analyze the relationship between this LSS polymorphism and the development of AKI after cardiac surgery. Method 1237 patients undergoing elective cardiac surgery were enrolled in the study. Preoperative biological samples were collected (Table 1). LSS genotypes were determined for each patient. The primary outcome was AKI development, according to KDIGO 2012 guidelines. Results 21.4% of patients developed AKI. Different preoperative clinical variables were analyzed, identifying five independent risk factors significantly correlated to AKI development in multivariate logistic regression analysis: age (p < 0.001), FE (p = 0.005), NYHA class (p < 0.001), reoperation (p = 0.002) and complex surgical intervention (p < 0.001), Table 2. No significant differences were observed between preoperative EO plasma levels and allelic variants of LSS gene and patients with the less common A allele were not associated to a more severe preoperative clinical presentation, expressed as EuroScore II value (AA 2.54 ± 3.86 vs AC 2.15 ± 2.52 vs CC 2.04 ± 2.73; p = ns). LSS minor variants turned out significantly associated with the incidence of AKI (AA = 29.3%; AC = 25.3%; CC = 17.2%; X2 p < 0.001—Fig. 1). This evidence remains significant after correction for covariates associated to AKI previously reported (Log regression p = 0.002; RR for AA variant: 2.09 (IC95% 1.15-3.78)) Conclusion Patients with at least one mutated allele of LSS variant have a higher probability of developing AKI after cardiac surgery. We think these results could be of interest to further understand cellular mechanisms underlying AKI development, especially in the cardiac surgery field.

#1463 Assessment of salt-sensitive hypertension in a novel Lanosterol Synthase knock-in mouse model

Abstract Background and Aims The diversity in blood pressure (BP) response to various salt intakes, known as Salt Sensitivity (SS), exhibits significant differences among individuals within the hypertensive subjects relative to the general population. Hypertension (HT) and SS are correlated to elevated levels of plasma Endogenous Ouabain (EO). We aimed at delineating the role of the missense variation rs2254524 (Val642Leu; C>A) within the Lanosterol Synthase gene (LSS), encoding for an enzyme involved in steroid biosynthesis. Individuals with the AA LSS variant, adherent to a low-salt diet, had a more significant BP reduction compared to those with CC genotype. Method Utilising CRISPR-Cas9, we established a knock-in mouse model carrying the analogous LssV643L/V643L. Male mice were subjected to a normal-salt diet (0.5% NaCl), high-salt diet (4% NaCl), or low-salt diet (<0.03% Na) and BP was monitored every two days using a tail-cuff system for 10 days. Blood and urine were collected separately in metabolic cages. Results Homozygous mice for Lss mutation demonstrated normal viability and healthy phenotype, undistinguishable from the WT. At baseline, the Lss AA mice were affected by a significant augmentation in kidney weight at 3 and 12 months of age, relative to WT. The Lss V643L does not influence EO levels or systolic BP (SBP), specifically at 3 and 12 months, but affects the SBP response to dietary salt. AA mice showed a high SBP in a high-salt diet against control diet at the same time intervals. Moreover, the Lss variant impacted the pressure-natriuresis relationship, with the mutated AA strain showing a less steep curve both in control diet versus the high-salt diet and in control diet versus the low-salt diet, displaying the SS phenotype of the AA mice. The 12-month-old mice in high-salt diet displayed cardiac hypertrophy with the AA genotype exhibiting an elevated prevalence of cardiac fibrosis. The level of Lss mRNA decreased in the adrenal glands in response to a high-salt diet. RNASeq analysis identified distinct patterns in several Slc genes, Cbr2 and Arhgap26 within the kidneys of these mice, in control or high-salt dietary conditions. Conclusion The established LssV643L/V643L mouse model effectively mirrors the SS-HT phenotype. Our findings highlight the significance of the Lss in modulating BP in response to salt stimuli suggesting a potential organ damage pathway requiring further exploration in older mice.

Sensational site: the sodium pump ouabain-binding site and its ligands.

Cardiotonic steroids (CTS), used by certain insects, toads, and rats for protection from predators, became, thanks to Withering's trailblazing 1785 monograph, the mainstay of heart failure (HF) therapy. In the 1950s and 1960s, we learned that the CTS receptor was part of the sodium pump (NKA) and that the Na+/Ca2+ exchanger was critical for the acute cardiotonic effect of digoxin- and ouabain-related CTS. This "settled" view was upended by seven revolutionary observations. First, subnanomolar ouabain sometimes stimulates NKA while higher concentrations are invariably inhibitory. Second, endogenous ouabain (EO) was discovered in the human circulation. Third, in the DIG clinical trial, digoxin only marginally improved outcomes in patients with HF. Fourth, cloning of NKA in 1985 revealed multiple NKA α and β subunit isoforms that, in the rodent, differ in their sensitivities to CTS. Fifth, the NKA is a cation pump and a hormone receptor/signal transducer. EO binding to NKA activates, in a ligand- and cell-specific manner, several protein kinase and Ca2+-dependent signaling cascades that have widespread physiological effects and can contribute to hypertension and HF pathogenesis. Sixth, all CTS are not equivalent, e.g., ouabain induces hypertension in rodents while digoxin is antihypertensinogenic ("biased signaling"). Seventh, most common rodent hypertension models require a highly ouabain-sensitive α2 NKA and the elevated blood pressure is alleviated by EO immunoneutralization. These numerous phenomena are enabled by NKA's intricate structure. We have just begun to understand the endocrine role of the endogenous ligands and the broad impact of the ouabain-binding site on physiology and pathophysiology.

Small artery remodeling and stiffening in deoxycorticosterone acetate-salt hypertensive rats involves the interaction between endogenous ouabain/Na + K + -ATPase/cSrc signaling.

Endogenous ouabain (EO) increases in some patients with hypertension and in rats with volume-dependent hypertension. When ouabain binds to Na + K + -ATPase, cSrc is activated, which leads to multieffector signaling activation and high blood pressure (BP). In mesenteric resistance arteries (MRA) from deoxycorticosterone acetate (DOCA)-salt rats, we have demonstrated that the EO antagonist rostafuroxin blocks downstream cSrc activation, enhancing endothelial function and lowering oxidative stress and BP. Here, we examined the possibility that EO is involved in the structural and mechanical alterations that occur in MRA from DOCA-salt rats. MRA were taken from control, vehicle-treated DOCA-salt or rostafuroxin (1 mg/kg per day, for 3 weeks)-treated DOCA-salt rats. Pressure myography and histology were used to evaluate the mechanics and structure of the MRA, and western blotting to assess protein expression. DOCA-salt MRA exhibited signs of inward hypertrophic remodeling and increased stiffness, with a higher wall:lumen ratio, which were reduced by rostafuroxin treatment. The enhanced type I collagen, TGFβ1, pSmad2/3 Ser465/457 /Smad2/3 ratio, CTGF, p-Src Tyr418 , EGFR, c-Raf, ERK1/2 and p38MAPK protein expression in DOCA-salt MRA were all recovered by rostafuroxin. A process combining Na + K + -ATPase/cSrc/EGFR/Raf/ERK1/2/p38MAPK activation and a Na + K + -ATPase/cSrc/TGF-1/Smad2/3/CTGF-dependent mechanism explains how EO contributes to small artery inward hypertrophic remodeling and stiffening in DOCA-salt rats. This result supports the significance of EO as a key mediator for end-organ damage in volume-dependent hypertension and the efficacy of rostafuroxin in avoiding remodeling and stiffening of small arteries.

#4375 LANOSTEROL SYNTHASE RS2254524 POLYMORPHISM MODULATES SALT SENSITIVITY AND RENAL EXPRESSION IN A NOVEL MICE MODEL, LSSV643L/V643L

Abstract Background and Aims The blood pressure (BP) response to different salt intakes (salt sensitivity-SS), shows variability among individuals, with more frequency in hypertensives compared to the general population. Elevated levels of the Endogenous Ouabain (EO) have been associated with hypertension (HT) and SS. We characterized the missense variation rs2254524 (Val642Leu; C>A) of the Lanosterol Synthase gene (LSS) coding for an enzyme in steroid biosynthesis, since AA patients on a low-salt diet showed a greater reduction in BP compared to the LSS CC, with only CC having an increase in plasma EO. Moreover, AKI incidence following cardiovascular surgery was greater among those with the LSS rs2254524 AA genotype than in those with the CC genotype. We aim at dissecting the functional correlation between LSS polymorphism, EO, SS-HT, and renal function. Method We generated by CRISPR-Cas9 a knock-in mouse model carrying the Lss V643L (LssV643L/V643L), homologous to V642L to dissect the functional correlation between LSS polymorphism, EO and salt-sensitive hypertension, and kidney expression. Male mice were fed with a normal-salt diet (0.5% NaCl), high-salt diet (4% NaCl), or low-salt diet (<0.03% Na) and BP was measured every two days by the tail-cuff system. Results LssV643L/V643L mice were viable, healthy, and undistinguishable phenotypically from WT. At baseline, the Lss AA affected kidney weight that was significantly enlarged at 3 (p = 0.02) and 12 months of age, compared to WT (kidney p = 0.04; liver p = 0.003). The Lss V643L mutation did not affect EO and SBP at 3 and 12 months, per se, but affects SBP responsiveness to salt intake. Indeed, we observed an increased SBP upon a high-salt diet only in LssV643L/V643L mice 12 months old compared to the control diet (p = 0.01). Moreover, the 12-month-old LssV643L/V643L mice in high-salt diet showed cardiac hypertrophy and showed a higher incidence of heart fibrosis. At 12 months, in the adrenal gland of LssV643L/V643L mice, Lss mRNA level was reduced upon high-salt diet (p = 0.03), while RNA-seq analysis of renal differentially expressed genes revealed a different regulation of multiple Slc genes, both in control and high-salt diet, but also of Cbr2, and Arhgap26. Conclusion The new LssV643L/V643L mouse model resembles the SS-HT phenotype together with EO non-responsiveness observed in HT patients, thus providing a good model of SS-HT. Our results reveal a role of Lss gene in the regulation of BP upon salt stimulus, and its influence at renal level at 3 and 12 months of age.

#4677 EARLY MARKERS OF POSTOPERATIVE ACUTE KIDNEY INJURY

Abstract Background and Aims Acute kidney injury (AKI) is a common clinical condition among patients who undergo cardiac surgery, significantly affecting morbidity and mortality[1]. To date, preoperative biomarkers and solid predictive models for AKI are not yet currently available in clinical practice. Recent studies reported endogenous ouabain (EO), a stress hormone secreted by the adrenal glands, as associated to worse kidney outcomes after cardiac surgery[2]. The aim was to validate the use of EO as biomarker of individual susceptibility for AKI after cardiac surgery and to create a new powerful score for postoperative AKI risk. Method 1174 patients undergoing elective cardiac surgery were enrolled in the study. Preoperative biological samples were collected and analysed. The primary outcome was AKI development, according to KDIGO 2012 guidelines. Results 21.6% of patients developed AKI (9% developed severe AKI, stage≥2). AKI confirmed strong correlation to postoperative death: all patients dead within 90 days for postsurgical complication developed AKI and each AKI stage was associated to a 10 times higher mortality risk (p < 0.001). Different preoperative clinical variables were analyzed, identifying five independent risk factors significantly correlated to AKI and severe AKI: age, FE, NYHA class, reoperation and complex surgical intervention (p < 0.001 for all of them). Preoperative EO levels turned out significantly associated to the incidence of AKI (p < 0.001) and clinical complications. Specifically, patients with higher EO levels had greater incidence of AKI (p < 0.001) and worse cardiac and kidney basal function (p = 0.005 and p = 0.003, respectively). Finally, a simple score was developed on the base of those preoperative clinical values significantly associated to AKI (AUC for severe AKI = 0.79, p <0,001). Adding preoperative EO to the model significantly improved the predictive capability (AUC for severe AKI = 0.82, p < 0.001; Δ-AUC +0.0229, p = 0.026). Conclusion We confirmed in a large population the role of preoperative plasma level of EO as an important early predictor for post-surgical AKI and we built a powerful clinical model for postoperative AKI risk, exclusively using few simple preoperative clinical factors and a single biohumoral marker.

#4434 IMPACT OF GENETIC FACTORS FOR SALT-SENSITIVE HYPERTENSION AND RENAL DAMAGE ON HOSPITALIZED COVID-19 PATIENTS

Abstract Background and Aims The coronavirus SARS-CoV-2 infects patients by binding human angiotensin-converting enzyme 2. Also, the ATP1A1 subunit of the plasma membrane Na-K-pump is critical in supporting the entry of SARS-CoV-2 into cells. Targeting of the ouabain binding site on the Na-K-pump by gene silencing or low concentrations of ouabain blocks viral infection in murine model. A recent finding demonstrated a more effective antiviral activity of digoxin and ouabain against SARS-CoV-2 infection in vitro than previously approved antiviral agents such as chloroquine and remdesivir. Moreover, cardiotonic steroids can promote renal inflammation and oxidative stress through the Na-K-pump α-1 and Src kinase signaling complex in both renal epithelial and immune cells. Arterial hypertension is one of the most common comorbidities associated with COVID-19, especially in patients with severe clinical involvement and at risk of death. The aim of this study is to evaluate the genetic aspects of pathways related to hypertension and renal failure, and to Na-K-pump activity such as endogenous ouabain and RAAS system, in order to partly dissect the wide clinical spectrum of the disease in hospitalized patients infected by SARS-CoV-2, with various degrees of symptoms. Method We investigated the relationship between three outcomes and genetic determinants in the COVID-BioB study (ClincalTrials.gov NCT04318366), a characterization of an Italian cohort of about 500 patients, a SARS-CoV-2 positive population recruited during the first wave of pandemic at San Raffaele Scientific Institute with biological samples and clinical assessment data available in an internal biobank. Targeted DNA genotyping was performed by custom arrays on TaqMan OpenArray system (ThermoFisher) for single nucleotide polymorphisms (SNPs). The genetic variants were selected as candidate for salt-sensitive hypertension and renal failure. Associations with genetic markers and outcomes were carried out with logistic regression analysis for outcome absence/presence comparison. Results COVID-19 patients were all hospitalized, with mean age 67.4±13.5 (30.2% female), pneumonia 96.9%, hypertension 51.8%, coronary arterial disease 26.8%, emergency department AKI stage 1- 11.1%, AKI stage 2- 0.4%, CKD stage>3 11.5%, Chronic Obstructive Pulmonary Disease 8.2%, creatinine 1.19±0.70, all at emergency department admission, in-hospital AKI 37.8%, Intensive Care unit admission 18.3%, and in-hospital death 20.6%. The main outcomes for the analysis of SNPs were in-hospital death, AKI and onset of proteinuria. The main findings for the SNP analysis concerned different genetic markers, each specific for different outcomes. A SNP in renin gene (REN), rs10900555, was associated with the in-hospital death (OR 3.84 [95%CI 1.15;12.85], P = .029). PRKG1 gene coding for the protein kinase cGMP-Dependent reported association with AKI for two different SNPs (rs1904694, OR 2.78 [95%CI 1.63;4.72], P = .0002; rs7905063, OR 2.71 [95%CI 1.72;4.23], P = .00002), with the same risk alleles previously linked to salt-sensitive hypertension. TT genotype (at risk for salt-sensitivity) in uromodulin gene (UMOD, SNP rs4293393) increases the risk of proteinuria development (OR 1.86 [95%CI 1.02;3.40], P = .044). Conclusion This genetic analysis, firstly reported on the COVID-BioB cohort, showed an intriguing relation between some polymorphisms previously associated with salt-sensitive hypertension and worst outcome or renal damage, during COVID-19. This genetic stratification may help to identify patients at risk AKI, and (directly or not) for death and renal damage (proteinuria) during COVID-19. Moreover, it may explain, at least in part, the debated relationship between hypertension and severity of COVID-19.

#4720 SEPSIS, AKI AND MORTALITY. SEARCHING FOR NEW MARKERS: OUR EXPERIENCE WITH ENDOGENOUS OUABAIN

Abstract Background and Aims Sepsis and septic shock are quite common events in the hospital setting. The classification of these events has changed a lot over the years, to ensure better sensitivity in recognizing a situation that, if not managed properly and quickly, leads to the death of the patient. In our study we focused on the search for predictors of acute kidney injury and mortality during the evolution of this pathology, with particular attention to the role of the Endogenous Ouabain hormone. Method We enrolled 177 patients diagnosed with sepsis or septic shock, under an ordinary hospital stay. We standardized patients according to the latest classification criteria (Sepsis-III), we focused our attention specifically on SOFA score and the relationship with acute kidney injury and mortality, including its relationship with other laboratory parameters, especially lactates. In a subgroup of patients was also dosed the level of Endogenous Ouabain (EO - hormone produced by the adrenal glands but which has an important impact on renal hemodynamics), through a venous sampling in 3 different times (at the beginning of the septic state, 24 and 48 hours later). Subsequently the variation of EO and the correlation with laboratory parameters was investigated in this period, to assess its impact on the development of kidney damage and mortality. Results The 70.1% of septic patients in our study develop AKI and, of this percentage, 75% in a severe form (stage 2-3 KDIGO). Both the presence and severity of AKI are unable to predict mortality in our sample (p-value 0.13). The presence of an impaired hemodynamics status (expressed ad MAP < 70 mmHg) has a strong impact in the severe stages of AKI but not in the mild form (p-value 0.006; ExpB 1.81 vs p-value 0.105; ExpB 1.46). The other elements related to the development of AKI were found to be the high levels of white blood cells, respiratory distress and the SOFA value at the presentation, while we found no correlation with CRP and lactate levels. Lactate levels well correlate with SOFA (p-value <0.0001 and Pearson's index 0.407) and mortality, particularly when the initial values are > 2mg/dL (p < 0.0001; ExpB 5.31, IC95% [2.07-13.57]). In the subgroup of patients in which EO was dosed, there was a strong correlation of the basal levels of this hormone with mortality even after correction for lactate levels and SOFA (p-value 0.009) but not with the development of AKI. However, patients in AKI stage-3 show persistently higher levels of EO both at 24 and 48 h (p = 0.015). Conclusion The presence of high levels of lactates at the onset is the most important predictor of mortality, followed by EO levels at presentation and the SOFA value. Elevation of Endogenous Ouabain levels is indicative of an acute state of stress but is not a parameter influenced by the SOFA score, nor by the general condition of patient. It seems non directly related with the development of AKI, tissue hypoperfusion or activation of the inflammatory response but is associated with an inability to rapidly recover renal function. The EO could be very useful in predicting mortality in patients with sepsis and septic shock as it seems to be an earlier, more specific, and sensitive than the other laboratory parameters used so far.

PRELIMINARY DATA ON THE ENDOGENOUS OUABAIN IN IDIOPATHIC PULMONARY ARTERIAL HYPERTENSION PATIENTS

Objective: The aim is to detect the endogenous ouabain (EO) in idiopathic pulmonary arterial hypertension (IPAH) patients. Design and method: EO is a steroid hormone secreted by the adrenal glands associated with adverse cardiovascular outcomes. However, EO plays other roles as brain protection against traumatic injury and seems involved in the adaptive response to hypoxia. Recently, we detected, for the first time, EO in a healthy human group of acute hypoxia (elite apnea divers) and in diving animals (common bottlenose dolphins, phocids and otariids, and loggerhead sea turtles). This study complements the above as we considered a human model of chronic hypoxia. We collected blood samples from five Caucasian males and measured EO with a Scintillation Proximity Assay. Results: We found that IPAH patients had higher plasma concentrations levels of EO than control subjects. In addition, EO plasma concentrations were negatively correlated with the mean pulmonary arterial pressure and total pulmonary vascular resistance (p-values <0.05). A positive correlation trend was observed with the cardiac index, a marker of healthy cardiac output. The results could suggest that high concentrations of EO are predictive of a better adaptation of the right ventricular afterload. Conclusions: Although these are preliminary results, they match data coming from other studies on clinical conditions that frequently occur in comorbidity with IPAH. It can represent a helpful hint for future investigations involving other pathological conditions for possible therapeutic and diagnostic approaches.

SALT SENSITIVITY IS MODULATED BY LANOSTEROL SYNTHASE RS2254524 POLYMORPHISM

Objective: The blood pressure (BP) response to different salt intakes (salt sensitivity-SS), shows variability among individuals, with more frequency in hypertensives compared to the general population. Elevated levels of the Endogenous Ouabain (EO) have been associated with hypertension (HT) and SS. We characterized the missense variation rs2254524 (Val642Leu; C>A) of the Lanosterol Synthase gene (LSS) coding for an enzyme in steroid biosynthesis, since AA patients on a low-salt diet showed a greater reduction in BP compared to the LSS CC, with only CC having an increase in plasma EO. We aim at dissecting the functional correlation between LSS polymorphism, EO and SS-HT. Design and method: We generated by CRISPR-Cas9 a knock-in mouse model carrying the Lss V643L (LssV643L/V643L), homologous to V642L. Male mice were fed with a normal-salt diet (0.5% NaCl), high-salt diet (4% NaCl), or low-salt diet (<0.03% Na) and BP was measured every two days by the tail-cuff system. Results: LssV643L/V643L mice were viable, healthy, and undistinguishable phenotypically from WT. At baseline, the Lss AA affected kidney weight that was significantly enlarged at 3 and 12 months of age, compared to WT. The Lss V643L mutation did not affect EO and SBP at 3 and 12 months, per se, but affects SBP responsiveness to salt stimulus. We observed a decreased SBP after 10 days of a low-salt diet and increased SBP upon a high-salt diet only in LssV643L/V643L mice compared to the control diet at 3 and 12 months, respectively. Moreover, 12-month-old mice in high-salt diet showed cardiac hypertrophy, and LssV643L/V643L showed a higher incidence of heart fibrosis. In the adrenal gland of LssV643L/V643L mice, Lss mRNA level was reduced upon high-salt diet. At 12 months, RNASeq analysis of renal differentially expressed genes revealed a different regulation of multiple Slc genes, both in control and high-salt diet, but also of Cbr2, and Arhgap26. Conclusions: The new LssV643L/V643L mouse model resembles the SS-HT phenotype together with EO non-responsiveness observed in HT patients, thus providing a good model of SS-HT. Our results reveal a role of Lss gene in the regulation of BP upon salt stimulus.

Na, K-ATPase α1 cooperates with its endogenous ligand to reprogram immune microenvironment of lung carcinoma and promotes immune escape.

Dysregulated endocrine hormones (EHs) contribute to tumorigenesis, but how EHs affect the tumor immune microenvironment (TIM) and the immunotherapy of non-small cell lung cancer (NSCLC) is still unclear. Here, endogenous ouabain (EO), an adrenergic hormone, is elevated in patients with NSCLC and closely related to tumor pathological stage, metastasis, and survival. EO promotes the suppression of TIM in vivo by modulating the expression of immune checkpoint proteins, in which programmed cell death protein ligand 1 (PD-L1) plays a major role. EO increases PD-L1 transcription; however, the EO receptor Na- and K-dependent adenosine triphosphatase (Na, K-ATPase) α1 interacts with PD-L1 to trigger the endocytic degradation of PD-L1. This seemingly contradictory result led us to discover the mechanism whereby EO cooperates with Na, K-ATPase α1 to finely control PD-L1 expression and dampen tumoral immunity. In conclusion, the Na, K-ATPase α1/EO signaling facilitates immune escape in lung cancer, and manipulation of this signaling shows great promise in improving immunotherapy for lung adenocarcinoma.

Exploratory study on the endogenous ouabain in idiopathic pulmonary arterial hypertension patients.

Endogenous ouabain (EO) is a steroid hormone secreted by the adrenal glands associated with adverse cardiovascular outcomes. However, EO plays other roles as brain protection against traumatic injury and seems involved in the adaptive response to hypoxia. Recently, we detected, for the first time, EO in a healthy human group of acute hypoxia and diving animals. This study complements the above as we considered a human model of chronic hypoxia. The aim is to detect EO in five idiopathic pulmonary arterial hypertension patients. We found that these patients had higher plasma concentrations of EO than control subjects. In addition, EO plasma concentrations were negatively correlated with the mean pulmonary arterial pressure and total pulmonary vascular resistance. The results could suggest that high concentrations of EO are predictive of better adaptation of the right ventricular afterload. Although the results are preliminary, they can represent a helpful hint for future investigations for possible therapeutic and diagnostic approaches.

Whither digitalis? What we can still learn from cardiotonic steroids about heart failure and hypertension.

Cloning of the "Na+ pump" (Na+,K+-ATPase or NKA) and identification of a circulating ligand, endogenous ouabain (EO), a cardiotonic steroid (CTS), triggered seminal discoveries regarding EO and its NKA receptor in cardiovascular function and the pathophysiology of heart failure (HF) and hypertension. Cardiotonic digitalis preparations were a preferred treatment for HF for two centuries, but digoxin was only marginally effective in a large clinical trial (1997). This led to diminished digoxin use. Missing from the trial, however, was any consideration that endogenous CTS might influence digitalis' efficacy. Digoxin, at therapeutic concentrations, acutely inhibits NKA but, remarkably, antagonizes ouabain's action. Prolonged treatment with ouabain, but not digoxin, causes hypertension in rodents; in this model, digoxin lowers blood pressure (BP). Furthermore, NKA-bound ouabain and digoxin modulate different protein kinase signaling pathways and have disparate long-term cardiovascular effects. Reports of "brain ouabain" led to the elucidation of a new, slow neuromodulatory pathway in the brain; locally generated EO and the α2 NKA isoform help regulate sympathetic drive to the heart and vasculature. The roles of EO and α2 NKA have been studied by EO assay, ouabain-resistant mutation of α2 NKA, and immunoneutralization of EO with ouabain-binding Fab fragments. The NKA α2 CTS binding site and its endogenous ligand are required for BP elevation in many common hypertension models and full expression of cardiac remodeling and dysfunction following pressure overload or myocardial infarction. Understanding how endogenous CTS impact hypertension and HF pathophysiology and therapy should foster reconsideration of digoxin's therapeutic utility.

Short-Term Mild Hypoxia Modulates Na,K-ATPase to Maintain Membrane Electrogenesis in Rat Skeletal Muscle.

The Na,K-ATPase plays an important role in adaptation to hypoxia. Prolonged hypoxia results in loss of skeletal muscle mass, structure, and performance. However, hypoxic preconditioning is known to protect against a variety of functional impairments. In this study, we tested the possibility of mild hypoxia to modulate the Na,K-ATPase and to improve skeletal muscle electrogenesis. The rats were subjected to simulated high-altitude (3000 m above sea level) hypobaric hypoxia (HH) for 3 h using a hypobaric chamber. Isolated diaphragm and soleus muscles were tested. In the diaphragm muscle, HH increased the α2 Na,K-ATPase isozyme electrogenic activity and stably hyperpolarized the extrajunctional membrane for 24 h. These changes were accompanied by a steady increase in the production of thiobarbituric acid reactive substances as well as a decrease in the serum level of endogenous ouabain, a specific ligand of the Na,K-ATPase. HH also increased the α2 Na,K-ATPase membrane abundance without changing its total protein content; the plasma membrane lipid-ordered phase did not change. In the soleus muscle, HH protected against disuse (hindlimb suspension) induced sarcolemmal depolarization. Considering that the Na,K-ATPase is critical for maintaining skeletal muscle electrogenesis and performance, these findings may have implications for countermeasures in disuse-induced pathology and hypoxic therapy.

Inverse salt sensitivity: an independent risk factor for cardiovascular damage in essential hypertension.

Salt sensitivity is a powerful risk factor for cardiovascular (CV) disease and mortality in both normotensive and hypertensive patients. We investigated the predictive value of the salt sensitivity phenotype in the development of CV events and hypertensive target organ damage (TOD) among essential hypertensive patients. Eight hundred forty-four naive hypertensive patients were recruited and underwent an acute saline test during which blood pressure (BP) displayed either no substantial variation (salt-resistant, SR individuals), an increase (salt-sensitive, SS), or a paradoxical decrease (inverse salt-sensitive, ISS). Sixty-one patients with the longest monitored follow-up (median 16 years) for blood pressure and organ damage were selected for the present study. A clinical score for TOD development based on the severity and the age of onset was set up by considering hypertensive heart disease, cerebrovascular damage, microalbuminuria, and vascular events. CV events were significantly higher among SS and ISS than in SR patients. The relative risk of developing CV events was 12.67 times higher in SS than SR and 5.94 times higher in ISS than SR patients. The development of moderate to severe TOD was 10-fold higher in SS and over 15-fold higher in ISS than in SR patients. Among the three phenotypes, changes in plasma endogenous ouabain were linked with the blood pressure effects of saline. Salt sensitivity and inverse salt sensitivity appear to be equivalent risk factors for CV events. The response to an acute saline test is predictive of CV damage for newly identified ISS individuals.

SALT SENSITIVITY IS MODULATED BY LANOSTEROL SYNTHASE RS2254524 POLYMORPHISM

Objective: The blood pressure (BP) response to different salt intake is associated with hypertension (HT). Elevated levels of the steroid hormone Endogenous Ouabain (EO) have been also associated with HT and salt sensitivity. We characterized the missense variation rs2254524 (V642: CC variant; L642: AA variant) of the Lanosterol Synthase (LSS), a key enzyme in steroid biosynthesis, because AA patients on a low salt diet showed a greater reduction in BP compared to the LSS CC and only CC patients had an increase in plasma EO after the low salt protocol. Hence, we hypothesized that LSS could affect salt-sensitive HT by regulation of EO biosynthesis. Design and method: We generated a knock-in mouse model carrying the variation ubiquitously. Male mice were fed with Normal Salt (NSD; 0.5% NaCl), High Salt (HSD; 4% NaCl) or Low Salt Diet (LSD; 0.03 % Na) for 15 days. BP was measured by the tail-cuff system, on 5 consequent days in conscious trained mice. Results: LSS AA mice were viable, healthy, and undistinguishable phenotypically from LSS CC. The LSS mRNA and protein levels were reduced in the adrenal gland of LSS AA mice at 3 months. At baseline, the LSS did not affect kidney function, while at 9 and 12 months the Systolic BP (SBP) in LSS CC mice showed a progressive increase with age; this increase was not observed in LSS AA mice. At 3 months, we observed an increase in SBP in both genotypes upon HSD compared to those on an NSD, however only the AA mice showed a significant SBP reduction after an LSD (Fig.1). The same response of BP upon HSD has also been observed in AA e CC mice at 12 months but only AA mice showed statistically significant cardiac hypertrophy (Fig. 2), a common feature of hypertensives. Conclusions: The new LSS mouse model resembles what was observed in hypertensive patients and provides a good model to prove our hypothesis. In the perspective of clinical practice, these findings allow the identification of patients whose SBP can seriously benefit from a low salt diet as a lifestyle modification.

Endogenous Ouabain in Human and Animal Models of Hypoxia

Endogenous ouabain (EO) is a steroid hormone secreted by the adrenal glands, usually associated with adverse cardiovascular effects. However, recent studies have highlighted its possible role in blood pressure control and in cardio-renal damage, and it seems to be involved in the adaptive response to hypoxia. The aim of this study is to detect the EO in human and animal models of hypoxia. We collected blood samples from seven competitive elite apnea divers, 11 noncompetitive elite apnea divers, and 26 healthy control subjects. Animal blood samples were collected from 16 common bottlenose dolphins (Tursiops truncatus), two phocids and two otariids kept under human care, and 11 wild loggerhead sea turtles (Caretta caretta) hosted in two rescue centers. We measured EO plasma concentrations with a Scintillation Proximity Assay. In elite apnea divers and healthy control subjects, EO plasma concentrations were positively correlated to weight (p < 0.05). Elite apnea divers showed statistically significant (p < 10-6) higher EO plasma concentrations compared to healthy subjects without any diving experience or experience in other sports activities involving breath-holding. In dolphins, EO plasma concentrations were positively correlated to age, total length, and weight (p < 0.05). In loggerhead sea turtles, EO plasma concentrations were negatively correlated to total length and weight (p < 0.05). In pinnipeds, correlation analysis was not performed due to the small number of animals. Herein, we demonstrate, for the first time, that different taxa, phylogenetically distant from each other and which perform apneas without reporting neurological damages, express EO. Our findings, although preliminary, are in line with the recently emerging hypothesis on a possible role of EO in the adaptive response to hypoxia and represent a helpful hint for future investigations aimed to identify novel molecules useful to treat very disabling pathological conditions such as idiopathic pulmonary arterial hypertension or obstructive sleep apnea.

Sleep deprivation is associated with increased circulating levels of endogenous ouabain: Potential role in bipolar disorder

Endogenously produced cardiac glycosides, like endogenous ouabain (EO), are putative hormones that have been implicated in the pathophysiology of bipolar disorder. Individuals with bipolar disorder appear to be unable to sufficiently upregulate production of EO in situations of increased need. This study was performed to determine the effect of sleep deprivation on the circulating levels of EO. Plasma EO concentrations were measured by ouabain-radioimmunoassay in heterozygote Na,K-ATPase a2 knockout (KO) mice, which have been used as an animal model of mania, and wildtype siblings at baseline and after sleep fragmentation utilizing the moving bar method. a2 KO animals had elevated endogenous ouabain concentrations compared to wild type controls (0.82 ± SD 0.22 nM vs 0.26 ± 0.02, P = 0.03). Sleep fragmentation increased ouabain concentrations in wild type mice (0.53 ± 0.08 nM sleep fragmentation vs 0.26 ± 0.02 nM baseline, P = 0.04), but not in a2 KO mice (0.60 ± 0.07 nM sleep fragmentation vs 0.82 ± 0.22 nM baseline, P > 0.05). These studies demonstrate that sleep disturbance can increase EO in control mice but animals that exhibit some manic behaviors are unable to increase EO production.