Relationships among endogenous ouabain, α-adducin polymorphisms and renal sodium handling in primary hypertension

doi:10.1097/HJH.0b013e3282f5315f

J Hypertens. Author manuscript; available in PMC 2012 December 10.

Published in final edited form as:

J Hypertens. 2008 May; 26(5): 914–920.

doi: 10.1097/HJH.0b013e3282f5315f

PMCID: PMC3518487

NIHMSID: NIHMS425357

Relationships among endogenous ouabain, α-adducin polymorphisms and renal sodium handling in primary hypertension

Paolo Manunta,^a Marc Maillard,^b Cristina Tantardini,^a Marco Simonini,^a Chiara Lanzani,^a Lorena Citterio,^a Paola Stella,^a Nunzia Casamassima,^a Michel Burnier,^b John M. Hamlyn,^c and Giuseppe Bianchi^a

^aDivision of Nephrology, Dialysis and Hypertension, Scientific Institute San Raffaele, Uiversity ‘Vita-Salute’ San Raffaele, Milan, Italy

^bDivision of Nephrology, CHUV, Lausanne, Switzerland

^cDepartment of Physiology, School of Medicine, University of Maryland, Baltimore, Maryland, USA

Correspondence to: Paolo Manunta, MD, Nephrology, Dialysis and Hypertension, Scientific Institute San Raffaele, Università ‘Vita-Salute’ San Raffaele, Chair of Nephrology, Via Olgettina 60, 20132 Milan, Italy, Tel: +39 022 6433891; fax: +39 022 6432384; Email: manunta.paolo/at/hsr.it

The publisher's final edited version of this article is available at J Hypertens

See other articles in PMC that cite the published article.

Abstract

Objective

The basolateral Na pump drives renotubular reabsorption. In cultured renal cells, mutant adducins, as well as sub-nanomolar ouabain concentrations, stimulate the Na–K pump.

Methods

To determine whether these factors interact and affect Na handling and blood pressure (BP) in vivo, we studied 155 untreated hypertensive patients subdivided on the basis of their plasma endogenous ouabain or α-adducin genotype (ADD1 Gly460Trp-rs4961).

Results

Under basal conditions, proximal tubular reabsorption and plasma Na were higher in patients with mutated Trp ADD1 or increased endogenous ouabain (P = 0.002 and 0.05, respectively). BPs were higher in the high plasma endogenous ouabain group (P = 0.001). Following volume loading, the increment in BP (7.73 vs. 4.81 mmHg) and the slopes of the relationship between BP and Na excretion were greater [0.017 ± 0.002 vs. 0.009 ± 0.003 mmHg/(μEq min)] in ADD1 Trp vs. ADD1 Gly carriers (P < 0.05). BP changes were similar, whereas the slopes of the relationship between BP and Na excretion were lower [0.016 ± 0.003 vs. 0.008 ± 0.002 mmHg/(μEq min)] in patients with low vs. high endogenous ouabain (P < 0.05). In patients with high endogenous ouabain, volume loading increased the BP in the ADD1 Trp group but not in the Gly group (P < 0.05). Thus, patients with ADD1 Trp alleles are sensitive to salt and tubular Na reabsorption remains elevated after volume expansion.

Conclusion

With saline loading, BP changes are similar in high and low endogenous ouabain patients, whereas tubular Na reabsorption increases in the high endogenous ouabain group. Saline loading unmasks differences in renal Na handling in patients with mutant adducin or high endogenous ouabain and exposes an interaction of endogenous ouabain and Trp alleles on BP.

Keywords: functional genomic, high blood pressure, Na pump inhibitors, Na transport, ouabain-like

Introduction

Abnormalities in renal sodium handling have been proposed as a major cause of primary hypertension. The pivotal function of the kidney in regulating body sodium and blood pressure (BP) is achieved via the interactions among various hormones (angiotensin II, aldosterone, vasopressin, endogenous ouabain, and dopamine, etc.) and the genetically determined constitutive capacity of the renal tubules to reabsorb Na. The latter is affected by the polymorphisms of the genes coding for various proteins involved in the regulation of Na transport in the renal plasma membranes [1–3]. Therefore, the individual long-term body sodium and BP set points reflect the normal coordination between these intrinsic and extrinsic mechanisms. In humans, the specific molecular mechanisms that mediate the link between salt intake, body sodium, and BP remain unclear.

Previous studies in cultured renal cells have shown that the renal Na–K pump activity, the key ion transporter regulating the driving force for tubular Na reabsorption is modulated by at least two hypertension-promoting factors. One is a hormonal factor, the endogenous ouabain [4], whereas the other is genetic, the cytoskeletal protein adducin [5]. Both human and rat mutated variants of adducin produce similar effects on the Na–K pump, even though the missense mutations occur on different coding sites in the two species [6,7]. Long-term exposure of renal cells to nanomolar ouabain [8] or to cell transfection with the mutated adducin variants from either rats or humans enhances the expression and activity of the renal Na–K pump in the basolateral membrane [5,9,10]. Augmentation of Na pump activity increases the constitutive capacity of the tubular cells to transport Na and is expected to enhance renal Na retention [11].

In view of the similar effect of ouabain and the mutated adducin on the renal cell Na–K pump [5–7,8,12,13], we investigated the extent to which these factors affected BP and/or renal Na handling in patients with primary hypertension under basal conditions and after an acute saline load.

Methods

Clinical measurements

The study was conducted in 155 patients with untreated mild hypertension who were referred to the ‘Outpatient Clinic for Hypertension’ of San Raffaele Hospital of Milan. The Ethics Committee of the San Raffaele Hospital approved the study and informed consent was obtained from each individual. Patients underwent clinical examination, routine biochemistry, and secondary hypertension was excluded by routine methods.

To avoid the large day-to-day variation in salt intake, the patients were asked to follow a controlled diet with a known content of about 150 mmol per day for at least 15 days before the study period. A 24-h urine collection was obtained to verify dietary compliance.

All patients underwent 24-h ambulatory BP monitoring (24-h ABPM; Spacelab 90207; Spacelab Medical Inc., Redmond, Washington, USA). Blood samples for Na, creatinine, renin activity (PRA), aldosterone (Aldo), endogenous ouabain, genotype characterization (ADD1 Gly460Trp-rs4961) [11], and 24-h urine collections for Na, K, and creatinine were obtained the day before the 24-h ABPM recording.

Acute protocol saline infusion

A subset of the whole study group of 124 hypertensive patients, who had given signed informed consent, were included in this study. The acute Na loading protocol used was similar to that previously reported [14].

Biochemical and renal parameters

Serum and urinary sodium were measured by flame photometry, serum and urinary creatinine by an automated enzymatic method, and plasma renin activity and plasma aldosterone by radioimmunoassay (RIA; Medical System, Genova, Italy). Plasma endogenous ouabain was determined by RIA on C-18 extracted samples using a specific antiserum as previously described [15]. Endogenous trace lithium was determined with an electrothermal atomic absorption spectrophotometer (model 1100B) with a HGA-700 graphite furnace (Perkin-Elmer Inc., Boston, Massachusetts, USA) [9].

Clearances (C) were calculated as C_x = U_x × V/P_x, where U_x and P_x are the urinary and plasma concentrations of the solute x, respectively, and V is the urine flow rate measured in milliliter per minute. Fractional excretion of sodium (FENa) and lithium (FELi) were calculated as P_x × U_crea/P_crea × U_x, where U_x and P_x are the urinary and plasma concentrations of the solute x, respectively, and U_crea and P_crea are the urinary and plasma concentrations of creatinine, respectively [9].

The slope of the relationship between BP and Na or lithium excretion [mmHg/(μEeq min)] was calculated by plotting the lithium or Na excretion on the Y-axis as a function of mean blood pressure (MBP), on the X-axis, observed both under basal conditions and after 2 h of saline infusion, as discussed elsewhere [14].

Statistical analysis

For database management and statistical analysis, we used the SPSS software package (SAS Institute, Cary, North Carolina, USA), version 11. In basal conditions, quartile display values correspond to the 25th, 50th, and 75th percentiles. The two lower and the two higher quartiles were grouped in the subset of patients that carried out the acute Na load because of the smaller size of this cohort. We performed analysis of variance to compare means between groups. Our statistical methods also included single and multiple linear regressions. We searched for possible covariates of the arterial and renal phenotypes; age, BMI and sex were considered for entry into the model.

Results

Baseline conditions

Table 1 summarizes the baseline clinical characteristics of the 155 untreated patients with mild hypertension who were subdivided into two groups according to their ADD1 genotypes (Table 1a) and into four groups according to their plasma endogenous ouabain quartiles (Table 1b). Only PRA and diastolic blood pressure (DBP) differed in the ADD1 and endogenous ouabain groups, respectively. Systolic BP showed a similar trend without reaching statistical significance due to the small group size. All other parameters measured were similar among the groups.

Table 1

Characteristics of the 155 hypertensive patients grouped by their ADD1 genotype and quartiles of endogenous ouabain

As shown in Fig. 1, the patients with the highest levels of plasma endogenous ouabain or those who carried the Trp ADD1 allele had higher DBP, plasma Na, and proximal tubular reabsorption (detected by the decrease of FELi) compared with the low (<157, and 157–236) endogenous ouabain groups. Similarly, patients with one or more ADD1 Trp alleles have higher plasma Na and augmented proximal tubular reabsorption of lithium than those with ADD1 Gly alleles. In Fig. 1, statistical significance was achieved for the parameters shown except for the DBP between Trp and Gly ADD1 carriers where the trend was evident but not significant (P = 0.09). In addition, under baseline conditions, we observed no significant interaction or additive effect between the ADD1 genotypes and plasma endogenous ouabain on either BP or tubular Na reabsorption when analyzed as described below (data not shown).

Fig. 1

Baseline values of various factors. (a) Baseline values of diastolic blood pressure (DBP), plasma Na, and FELi in patients according to their plasma endogenous ouabain level quartiles. (b) Baseline values DBP, plasma Na, and FELi in patients according (more ...)

Acute Na loading protocol and renal Na handling

Of the 155 patients studied, 124 entered the sodium loading protocol. Due to the relatively lower size of this cohort, the patients were subdivided into two groups according to their ADD1 genotypes (Table 2a) and their endogenous ouabain levels. Only three patients were homozygous for Trp so they were included in the Gly/Trp subgroup. For endogenous ouabain, we grouped the patients in the lower two endogenous ouabain quartiles as the low group and those in the higher endogenous ouabain quartiles as the high group (Table 2b).

Table 2

Impact of an acute Na load: changes (Δ) from baseline

The saline load increased BP significantly in all patient subgroups (P <0.001). The BP increase was similar in the high and low endogenous ouabain groups; whereas those patients who carried the mutated Trp ADD1 allele displayed a larger increase in DBP and MBP (not shown) compared with the Gly ADD1 homozygotes. The difference in the BP response to the saline load was not explained by variations in the Na or lithium excretion rates.

Following 2-l infusion of saline, the renal electrolyte handling can be affected by changes in blood and kidney perfusion pressures. Thus, a more appropriate evaluation of the influence of the ADD1 genotype or endogenous ouabain levels on renal electrolyte handling should ideally consider the simultaneous changes in BP by calculating the slope of the relationship between BP and Na (or Li) excretion both under basal conditions and at the end of the infusion period. As shown in Table 2, the slopes of the pressure–natriuresis relationship were higher for Na and Li in patients who carried mutated Trp ADD1 alleles than in control patients that carried the Gly ADD1 homozygote. This indicates that a larger increase in BP was needed in the Trp carriers to excrete the same amount of Na and Li. Conversely, the slope of the pressure–natriuresis relationship was significantly lower in the patient group with high endogenous ouabain compared to those with low endogenous ouabain levels. Thus, during saline loading, patients with high endogenous ouabain seem to excrete more Na at any given level of BP.

An interaction between plasma endogenous ouabain and the ADD1 Trp genotype was found to cause changes in DBP after an acute Na load (Fig. 2, panel a; P = 0.028). The difference in the BP increase between carriers of the mutated Trp ADD1 allele and Gly/Gly homozygotes was observed only in the presence of high circulating endogenous ouabain. Moreover, the hypertensive effect of the adducin Trp allele and plasma endogenous ouabain combination was not explained by differences in renal Na excretion; FELi and FENa were not different between the two subgroups of patients with the lowest and largest increase in BP (Fig. 2).

Fig. 2

Interaction between ADD1 genotypes and circulating endogenous ouabain in hypertensive patients after an acute Na load. For this comparison, patients were subdivided into two subgroups according to the level of endogenous ouabain below or above the median (more ...)

Discussion

The major new results of the present study are as follows. First, under basal conditions, untreated patients with mild hypertension who carry the mutated ADD1 or have elevated circulating levels of endogenous ouabain show significantly higher DBP, higher plasma Na concentrations, and increased proximal tubular reabsorption compared with their relevant controls (Table 1; Fig. 1). Previous studies [7,16] from our laboratory using a larger sample of untreated hypertensive patients showed a statistically different DBP between ADD1 genotypes. Although evident (Fig. 1), the same trend was not significant in the present study.

Second, we found no evidence for additive effects between endogenous ouabain and the ADD1 genotype among the patients at baseline for the renal function parameters measured. Following saline infusion, however, the slopes of the pressure natriuresis (MBP/Na or Li excretion) relationship were positive in all groups (Table 2). Moreover, there were significant differences in the slopes for patients who carried the ADD1 Trp allele compared with those with high circulating endogenous ouabain. For example, the tendency to retain sodium was persistent in the Trp carriers. In contrast, sodium excretion in patients with high endogenous ouabain was higher at any given BP compared with those with low circulating endogenous ouabain.

Third, following the saline load, significant BP effects were found that reflect the interaction between the ADD1 Trp genotype and circulating endogenous ouabain (P = 0.028). Among those patients with high circulating endogenous ouabain, saline infusion produced a greater hypertensive response in the Trp allele carriers (Fig. 2) and the pressor response was clearly unrelated to renal cation excretion as determined from the similar values for FELi and FENa.

One intriguing observation of our study is the significantly higher plasma Na concentration among patients under basal conditions with either high circulating endogenous ouabain levels or the mutated ADD1 (Fig. 1). This concordance is striking especially because both endogenous ouabain and Trp alleles have been proposed to augment renal Na pump activity and have anti-natriuretic effects. Previous studies [17–21] have observed significant changes in plasma sodium following an increase in dietary sodium or between hypertensive and normotensive subjects. Moreover, the changes in plasma sodium may have a number of functional consequences including activation of sympathetic nerve activity [21]. Sustained changes in plasma sodium imply an alteration in the relationship between cation balance and water homeostasis.

Increased proximal tubular reabsorption has been proposed as an independent determinant of the BP response to salt [22,23]. The molecular mechanisms that link proximal tubular reabsorption with increased levels of BP and plasma Na concentration, however, remain elusive. Humoral inhibitors of the vascular Na pump have been implicated as a link between volume retention and increased arterial tone [24,25]. Inhibition of the vascular sodium pump promotes calcium influx via the Na–Ca exchanger [26] and recent studies [4,26,27] show that the vascular Na–Ca exchanger and the vascular α-2 Na pump are key mediators of the increase in vascular tone in salt and ouabain-dependent hypertension.

Although inhibition of the vascular Na pump by ouabain or endogenous ouabain can raise BP, the net effects of these humoral factors on the renal tubular Na pump appear to be more complex. For example, high concentrations of ouabain inhibit the Na–K pump, whereas lower nanomolar concentrations augment the Na pump abundance in the tubular basolateral membrane and increase the pump activity [28–30]. At low concentrations, endogenous ouabain and ouabain, therefore, appear to stimulate sodium retention, whereas at higher concentrations, the direct inhibitory actions of ouabain may augment sodium excretion. This biphasic effect of ouabain has been demonstrated in cultured renal tubular cells [31] and can be detected in isolated membranes [12,32]. Therefore, it is difficult to predict in the whole organism whether a given concentration may exhibit an inhibitory or stimulatory effect on the Na pump in any given tissue. Moreover, the effect of a given concentration of ouabain on the Na pump may be modulated by a variety of factors, some of which may be relevant to the present results. For example, the ouabain inhibitory potency of Na–K ATPase varies according to the α-isoform, extracellular K, lipid composition of the cell membrane, intracellular Na and ROS, cytoskeleton proteins status, NOS and/or ANP levels, among others. Therefore, the same concentration may exhibit opposite effects according to the modification of the modulatory factors mentioned above. We suggest that modification of one or more modulatory factors may explain the switch from endogenous ouabain-stimulated Na retention in the basal state to the endogenous ouabain facilitated Na excretion following saline infusion in the high endogenous ouabain subgroup.

In conclusion, under basal conditions, our findings are in agreement with that of earlier studies [8–10,12,26,27,30], showing that both ouabain and Trp adducin may activate the renal Na–K pump in cultured renal tubular cells and increase BP and tubular reabsorption at the whole organism level. After a saline load, carriers of the Trp adducin continued to reabsorb more Na at any given level of BP compared with their wild adducin counterparts. Conversely, in the high endogenous ouabain subgroup, the tubular reabsorption capacity for any given level of BP is decreased compared with the low endogenous ouabain subgroup.

Saline infusion unmasked a pressor interaction between endogenous ouabain and ADD1 Trp allele on BP as shown in Fig. 2. The mechanism of this interaction is not clear but may involve augmented sensitivity of the vascular sodium pump that may be most significant in the high endogenous ouabain patients. Clearly, the different BP responses to the saline load in patients with wild adducin compared with those with mutated adducin could not be explained by variations in the renal Na handling alone.

The patients in this study had a relatively recent onset of hypertension, were not previously treated, and may be especially suitable to unravel the pathophysiological and molecular mechanisms underlying their hypertension without the interference of previous antihypertensive therapies or secondary vascular changes that occur in the later stages of hypertension. Moreover, these patients were under a controlled Na diet to reduce the ordinarily wide variability of Na intake.

The striking similarity between the changes in proximal tubular reabsorption and endogenous ouabain in humans and those produced in isolated cells by ouabain and the mutated ADD1 strengthens the notion that these two molecules trigger a similar mechanism within the kidney (i.e., activation of the basolateral Na–K pump).

Nevertheless, we recognize the following limitations: the lithium clearance data indicate an increased proximal tubular reabsorption triggered by high endogenous ouabain or the mutated ADD1. The basic assumption for renal clearance is the steady state of all factors involved that, in our study, is disturbed by the saline infusion. This deviation from the steady state should apply equally to all subgroups, but there are no means to determine whether this goal was met.

A residual if remote possibility is that human endogenous ouabain is an isomer of ouabain. Extensive studies have been performed to validate the RIA for endogenous ouabain in plasma [17,33] and the immunoassay we used does not distinguish endogenous ouabain from ouabain. Moreover, among 21 patients, a positive correlation (P <0.001) has been observed between plasma endogenous ouabain measured by both HPLC mass spectrometry and RIA (P. Manunta, M. Ferrandi, J.M. Hamlyn, unpublished observation). Therefore, measurements made with our RIA are not likely to be compromised by other materials (e.g., digoxin, marinobufagenin) that may be in the circulation and are structurally distinct from ouabain.

Acknowledgments

This work was supported in part by grants from Ministero Università e Ricerca Scientifica of Italy (PRIN Grant 2006065339_01 to G.B.) and the European Union (grant LSHM-CT-2006-037093 In Genious Hyper Care) and in part by the grants USPHS HL75584 and HL078870 to J.M.H.

Abbreviations


ABPM	ambulatory blood pressure monitoring

ADD1	alpha adducing

BMI	body mass index

DBP	diastolic blood pressure

EO	endogenos ouabain

FELi	fractional excretion of lithium

FENa	Fractional excretionm of sodium

MBP	mean blood pressure

PRA	plasma rennin activity

Press/Nat	pressure natriuresis relationship

RIA	radioimmunoassay

UK	urinary K excretion

UNa	urinary Na excretion

References

1. Lifton RP, Gharavi AG, Geller DS. Molecular mechanisms of human hypertension. Cell. 2001;104:545–556. [PubMed]

2. Kahle KT, Rinehart J, Ring A, Gimenez I, Gamba G, Hebert SC, et al. WNK protein kinases modulate cellular Cl⁻ flux by altering the phosphorylation state of the Na–K–Cl and K–Cl. Physiology. 2006;21:326–335. [PubMed]

3. Bianchi G. Genetic variations of tubular sodium reabsorption leading to ‘primary’ hypertension: from gene polymorphism to clinical symptoms. Am J Physiol Regul Integr Comp Physiol. 2005;289:R1536–R1549. [PubMed]

4. Blaustein MP, Zhang J, Chen L, Hamilton BP. How does salt retention raise blood pressure? Am J Physiol Regul Integr Comp Physiol. 2006;290:R514–R523. [PubMed]

5. Tripodi G, Valtorta F, Torielli L, Chieregatti E, Salardi S, Trusolino L, et al. Hypertension-associated point mutations in the adducin alpha and beta subunits affect actin cytoskeleton and ion transport. J Clin Invest. 1996;97:2815–2822. [PMC free article] [PubMed]

6. Bianchi G, Tripodi G, Casari G, Salardi S, Barber BR, Garcia R, et al. Two point mutations within the adducin genes are involved in blood pressure variation. Proc Natl Acad Sci U S A. 1994;91:3999–4003. [PubMed]

7. Cusi D, Barlassina C, Azzani T, Casari G, Citterio L, Devoto M, et al. Polymorphisms of alpha-adducin and salt sensitivity in patients with essential hypertension. Lancet. 1997;349:1353–1357. [PubMed]

8. Ferrari P, Torielli L, Ferrandi M, Padoani G, Duzzi L, Florio M, et al. PST2238: a new antihypertensive compound that antagonizes the long-term pressor effect of ouabain. J Pharmacol Exp Ther. 1998;285:83–94. [PubMed]

9. Efendiev R, Krmar RT, Ogimoto G, Zwiller J, Tripodi G, Katz AI, et al. Hypertension-linked mutation in the adducin alpha-subunit leads to higher AP2-mu2 phosphorylation and impaired Na+, K+-ATPase trafficking in response to GPCR signals and intracellular sodium. Circ Res. 2004;95:1100–1108. [PubMed]

10. Ferrandi M, Salardi S, Tripodi G, Barassi P, Rivera R, Manunta P, et al. Evidence for an interaction between adducin and Na⁺ -K⁺ -ATPase: relation to genetic hypertension. Am J Physiol. 1999;277:H1338–H1349. [PubMed]

11. Manunta P, Burnier M, D’Amico M, Buzzi L, Maillard M, Barlassina C, et al. Adducin polymorphism affects renal proximal tubule reabsorption in hypertension. Hyertension. 1999;33:694–697. [PubMed]

12. Ferrandi M, Molinari I, Barassi P, Minotti E, Bianchi G, Ferrari P. Organ hypertrophic signaling within caveolae membrane subdomains triggered by ouabain and antagonized by PST 2238. J Biol Chem. 2004;279:33306–33314. [PubMed]

13. Ferrari P, Ferrandi M, Valentini G, Bianchi G. Rostafuroxin: an ouabain antagonist that corrects renal and vascular Na+-K+ ATPase alterations in ouabain and adducin-dependent hypertension. Am J Physiol Regul Integr Comp Physiol. 2006;290:R529–R535. [PubMed]

14. Manunta P, Cusi D, Barlassina C, Righetti M, Lanzani C, D’Amico M, et al. Alpha-adducin polymorphisms and renal sodium handling in essential hypertensive patients. Kidney Int. 1998;53:1471–1478. [PubMed]

15. Ferrandi M, Manunta P, Balzan S, Hamlyn JM, Bianchi G, Ferrari P. Ouabain-like factor quantification in mammalian tissues and plasma: comparison of two independent assays. Hypertension. 1997;30:886–896. [PubMed]

16. Lanzani C, Citterio L, Jankaricova M, Sciarrone MT, Barlassina C, Fattori S, et al. Role of the adducin family genes in human essential hypertension. J Hypertens. 2005;23:543–549. [PubMed]

17. Manunta P, Hamilton BP, Hamlyn JM. Salt intake and depletion increase circulating levels of endogenous ouabain in normal men. Am J Physiol Regul Integr Comp Physiol. 2006;290:R553–R559. [PubMed]

18. He FJ, Markandu NM, Sagnella GA, deWardener HE, MacGregor GA. Plasma sodium. Ignored and underestimated. Hypertension. 2005;45:98–102. [PubMed]

19. Gasowski J, Manunta P, Bianchi G, Staessen JA. Ouabain and serum sodium. Hypertension. 2005;45:e16. [PubMed]

20. Meneton P, Jeunemaitre X, de Wardener HE, MacGregor GA. Links between dietary salt intake, renal salt handling, blood pressure, and cardiovascular diseases. Physiol Rev. 2005;85:679–715. [PubMed]

21. Huang BS, Amin MS, Leenen FH. The central role of the brain in salt-sensitive hypertension. Curr Opin Cardiol. 2006;21:295–304. [PubMed]

22. Chiolero A, Maillard M, Nussberger J, Brunner HR, Burnier M. Proximal sodium reabsorption: an independent determinant of blood pressure response to salt. Hypertension. 2000;36:631–637. [PubMed]

23. Burnier M, Bochud M, Maillard M. Proximal tubular function and salt sensitivity. Curr Hypertens Rep. 2006;8:8–15. [PubMed]

24. Haddy FJ, Overbeck HW. The role of humoral agents in volume expanded hypertension. Life Sci. 1976;19:935–947. [PubMed]

25. Blaustein MP. Sodium ions, calcium ions, blood pressure regulation, and hypertension: a reassessment and a hypothesis. Am J Physiol. 1977;232:C165–C173. [PubMed]

26. Iwamoto T, Kita S, Zhang J, Blaustein MP, Arai Y, Yoshida S, et al. Salt-sensitive hypertension is triggered by Ca2+ entry via Na+/Ca2+ exchanger type-1 in vascular smooth muscle. Nat Med. 2004;10:1193–1199. [PubMed]

27. Dostanic-Larson I, Van Huysse JW, Lorenz JN, Lingrel JB. The highly conserved cardiac glycoside binding site of Na, K-ATPase plays a role in blood pressure regulation. Proc Natl Acad Sci U S A. 2005;102:15845–15850. [PubMed]

28. Godfraind T, Ghysel-Burton J. Binding sites related to ouabain-induced stimulation or inhibition of the sodium pump. Nature. 1977;265:165–166. [PubMed]

29. Gao J, Wymore RS, Wang Y, Gaudette GR, Krukenkamp IB, Cohen IS, et al. Isoform-specific stimulation of cardiac Na/K pumps by nanomolar concentrations of glycosides. J Gen Physiol. 2002;119:297–312. [PMC free article] [PubMed]

30. Schoner W, Scheiner-Bobis G. Endogenous and exogenous cardiac glycosides: their roles in hypertension, salt metabolism, and cell growth. Am J Physiol Cell Physiol. 2007;293:C509–C536. [PubMed]

31. Nguyen AN, Wallace DP, Blanco G. Ouabain binds with high affinity to the Na, K-ATPase in human polycystic kidney cells and induces extracellular signal-regulated kinase activation and cell proliferation. J Am Soc Nephrol. 2007;18:46–57. [PubMed]

32. Hamlyn JM, Cohen N, Zyren J, Blaustein MP. Activating effects of low dose cardiotonic steroids on dog kidney Na, K ATPase activity: role of endogenous inhibition. In: Glynn Ellory., editor. The sodium pump. Cambridge, UK: The Company of Biologists Ltd; 1985. pp. 667–673.

33. Pitzalis MV, Hamlyn JM, Messaggio E, Iacoviello M, Forleo C, Romito R, et al. Independent and incremental prognostic value of endogenous ouabain in idiopathic dilated cardiomyopathy. Eur J Heart Fail. 2006;8:179–186. [PubMed]