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 (; ). Previous studies [7
] from our laboratory using a larger sample of untreated hypertensive patients showed a statistically different DBP between ADD1 genotypes. Although evident (), 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 (). 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 () 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 (). 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
] 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
]. 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
]. Inhibition of the vascular sodium pump promotes calcium influx via the Na–Ca exchanger [26
] and recent studies [4
] 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
]. 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
]. 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
], 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 . 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
] 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.