Inverse Salt Sensitivity of Blood Pressure: Mechanisms and Potential Relevance for Prevention of Cardiovascular Disease

Abstract

Purpose of Review

To review the etiology of inverse salt sensitivity of blood pressure (BP).

Recent Findings

Both high and low sodium (Na⁺) intake can be associated with increased BP and cardiovascular morbidity and mortality. However, little is known regarding the mechanisms involved in the increase in BP in response to low Na⁺ intake, a condition termed inverse salt sensitivity of BP, which affects approximately 15% of the adult population. The renal proximal tubule is important in regulating up to 70% of renal Na⁺ transport. The renin-angiotensin and renal dopaminergic systems play both synergistic and opposing roles in the regulation of Na⁺ transport in this nephron segment. Clinical studies have demonstrated that individuals express a “personal salt index” (PSI) that marks whether they are salt-resistant, salt-sensitive, or inverse salt-sensitive. Inverse salt sensitivity results in part from genetic polymorphisms in various Na⁺ regulatory genes leading to a decrease in natriuretic activity and an increase in renal tubular Na⁺ reabsorption leading to an increase in BP.

Summary

This article reviews the potential mechanisms of a new pathophysiologic entity, inverse salt sensitivity of BP, which affects approximately 15% of the general adult population.

Introduction

Blood pressure (BP) can be reduced in individuals with hypertension by reducing dietary Na⁺ intake [1, 2••, 3••, 4]. Many studies have demonstrated a sustained increase in blood pressure (BP) in response to an increase in dietary Na⁺ intake [1, 5–16], as well as an increase in cardiovascular events and mortality [14–19]. Salt sensitivity (SS) of BP, defined as a rise in BP in response to an increase in Na⁺ intake, is present in approximately 50% of hypertensive adults. However, about 25% of individuals with normal BP are also salt-sensitive (SS) [14, 20]. Past controlled Na⁺ intake studies have used either low or high Na⁺ diets, relative to average daily consumption by Americans, and variable cutoffs for classifying individuals as SS or salt-resistant (SR). A long-term clinical study has shown that SS normotensives have the same mortality rates as hypertensives [14]. However, little is known about individuals at the opposite end of the SS spectrum, those who are inverse salt-sensitive (ISS), defined as those demonstrating an increase in BP in response to low Na⁺ intake. ISS, which was recently discovered as masquerading within the broad SR category, has a prevalence of approximately 15% of the general population. [21–26]. Figure 1 shows a model of the effect of sustained diets of low (10 mmol/day Na⁺), intermediate (140 mmol/day Na⁺), and high Na⁺ (300 mmol/day Na⁺) on BP in hypertensive, normotensive, SS, and ISS individuals in the University of Virginia (UVA) Salt Study. Table 1 facilitates the conversion between the concentrations of Na⁺ mentioned in this review and other popular units used in the literature and lay press. ISS individuals are those with ≥ 7 mm Hg decrease in mean arterial pressure (MAP) when transitioning from normal to high Na⁺ diet and an increase in MAP ≥ 7 mm Hg when transitioning from normal to low Na⁺ diet (Fig. 2). Hypertensive adults are now classified as having sustained BP ≥ 130/80 mm Hg [1], and the prevalence of hypertension has been predicted to rise to approximately 45.6% in the American adult population [27], of which approximately 15% may have ISS hypertension.

Fig. 1.

This model shows the relationship between dietary Na⁺ and BP (BP) in SR normotensive, SS, ISS, and hypertensive individuals. The daily dietary Na⁺ intake used in the UVA Salt Studies are 10 mmol/day (low Na⁺ intake), 140 mmol/day (moderate Na⁺ intake), and 300 mmol/day (high Na⁺ intake). SR normotensive individuals maintain their BP at or below the ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults (< 130/80 mm Hg systolic/diastolic) at both ends of the high and low Na⁺ dietary spectrum (blue symbols). SR hypertensive individuals have elevated BP, which is relatively unaffected by Na⁺ intake, but some individuals can reduce their elevated BP to some degree on a low Na⁺ intake (red triangle). SS individuals have normal BP on normal Na⁺ intake but have elevated BP on a high Na⁺ intake, which can be ameliorated by low Na⁺ intake (green square). ISS individuals have a response to dietary Na⁺ intake that is opposite to SS individuals (orange circle). Very low Na⁺ intake increases BP, which can be ameliorated by increasing their Na⁺ intake. ISS individuals are those with ≥ 7 mm Hg decrease in MAP when transitioning from normal to high Na⁺ intake and an increase in MAP ≥ 7 mm Hg when transitioning from normal to low Na⁺ intake, as shown in Fig. 2

Table 1.

Equivalent Na⁺ concentrations in mmol, milligrams (mg), grams (g), and teaspoon (tsp)

Sodium (mmol)	Sodium (mg)	Salt (g)	Salt (tsp)
10	230	0.585	0.1
140	3220	8.2	1.39
300	6897	17.55	3.08

1 mol sodium = 23 g

1 mol sodium in salt (NaCl) = 58.5 g

milliequivalents = millimoles

Fig. 2.

Data are shown from three independent salt study cohorts placed on one week of high and low Na⁺ intakes. The ranked magnitude of directional change in BP from each volunteer participant is shown and graphed in rank order on the X-axis, with the Y-axis being proportional to the increase, lack of change, or decrease in mean arterial BP (ΔMAP) when transitioning between the two diets. Since the order of the diets from low to high or high to low was randomized to rule out directional bias, we graphed the directional change in the ΔMAP. ISS subjects are those having a negative change in MAP (≥ − 7 mm) when transitioning from low to high Na⁺ intake or a positive change in ΔMAP (≥ 7 mm) when transitioning from high to low Na.⁺ intake (4 to 16%). Data are from the University of Virginia’s (UVA) Salt Study, Harvard’s Hyperpath Study, and the University of Maryland’s HAPI salt study, as noted on the graph. SS individuals (approximately 18%) are on the extreme right-hand side of this graph and are not the subject of this review

Cardiovascular death, stroke, myocardial infarction, and congestive heart failure hospitalization are increased in some individuals who consume low amounts of Na⁺ (< 1.2–3 g/day) [15, 18, 22–24, 28, 29, 30•]. There is still considerable debate as to why a low Na⁺ intake could lead to increased mortality. Cappuccio et al. [3••] demonstrated in clinical studies that the use of the Kawasaki formula to estimate 24-h urinary Na⁺ excretion could lead to an overestimation of Na⁺ consumption at the lowest levels of intake and underestimation of Na⁺ consumption at the highest levels of intake due to systematic bias. The overestimation is such that it could falsely raise the negative outcomes at the lowest Na⁺ intake levels. By contrast, the estimations of Na⁺ intakes are more accurate with 24-h urine collections. Other studies that used 24-h urine collections to estimate Na⁺ consumption confirm the increased mortality on low Na⁺ diets [15, 18, 31]. We hypothesize that individuals with ISS hypertension could be the reason for the more negative cardiovascular sequelae that result from low Na⁺ diets mentioned above. A randomly selected study cohort showed about 15% of hypertensive participants have ISS hypertension in our study [5], as well as studies by Overlack et al. [22, 24] and Longworth et al. [32], and a prevalence of as high as 40% as reported by Castiglioni et al. [33••]. While this review does not address the long-term consequences (e.g., cardiovascular disease and death) of ISS hypertension, it does address the biological, genetic, and epigenetic mechanisms associated with the paradoxical increase in BP in adults consuming a low Na⁺ diet. When approached from a mechanistic viewpoint, future epidemiological studies will be better equipped to stratify SS and ISS cohorts in order to appropriately determine if clinical outcomes are related to the known mechanisms of BP dysregulation found in ISS hypertension.

The dietary Na⁺ recommendation of the Institute of Medicine/American College of Cardiology (ACC)/American Heart Association (AHA) [1, 19] is intended to be a generalized recommendation for any population. Recently, the Center for Disease Control (CDC) and the Food and Drug Administration (FDA) both recommended a daily intake of 2300 mg NaCl. However, it is becoming clear that each individual retains or eliminates Na⁺ in an individual manner, for which we have coined the term “Personal Salt Index” (PSI) [34] and SS-index by Castiglioni et al. [33••] that is genetically programmed but has substantial environmental and behavioral influences [35]. Thus, further research is needed to understand how both low and high Na⁺ intakes affect cardiovascular disease and death. Three NHLBI-funded studies [5, 36] have accumulated data demonstrating agreement that the prevalence of ISS hypertension (aka counter-regulators) constitutes approximately 15% of the adult population, suggesting that ISS hypertension needs to be better understood and accepted [22–29, 30•, 32, 33••, 34]. The ranked magnitude response of each participant on high and low Na⁺ intake from 3 different cohorts, the UVA Salt Study [5], the University of Maryland HAPI study [36], and the Harvard HyperPATH study, using a MAP cutoff value of 7 mm Hg [unpublished] is depicted in Fig. 2. In other studies, three definitions of ISS (aka counter-regulator [23]) were used: an increase in ambulatory BP (≥ 5 mm Hg MAP, or systolic BP ≥ 4 mm Hg) [23] on a low Na⁺ intake (< 1.4 g NaCl/day (< 551 mg sodium)) or a decrease in MAP ≥ 5 mm Hg or systolic BP ≥ 4 mm Hg on a high Na⁺ intake (> 10 g NaCl/day (> 3,934 mmol sodium)) [22, 24, 30•]. Castiglioni et al. used the term “sodium sensitivity index” for the ratio of the change in MAP and change in urinary Na⁺ (ΔMAP/ΔUNaV) expressed in mm Hg/mol Na⁺/day (> 15 mm Hg/mol Na⁺/day for SS individuals and ≤ 15 mm Hg/mol Na⁺/day for ISS individuals) [33••].

Because many Na⁺ sensitivity studies use arbitrary BP cutoffs for defining SS, SR, and ISS, we performed statistical analysis of our UVA Salt Study utilizing the K-means clustering method (Squared Euclidean Distance), shown in Fig. 3. The UVA Salt Study data on individual BP variation caused by a change in Na⁺ intake had a Gaussian distribution in SR subjects and non-Gaussian distribution in SS and ISS subjects. We hypothesize that the non-Gaussian distribution in ISS and SS participants is the consequence of the influence of several genes [37] that independently regulate renal tubular Na⁺ transport, the sum of which is related to the BP phenotype. This is similar to the report of Ji and Lifton et al. that several independent gene variants involved in the regulation of renal tubular Na⁺ and K⁺ transport can cause a decrease in BP that would protect an individual from hypertension [38].

Fig. 3.

Statistical justification for the cutoff points between the ISS, SR, and SS groups was determined from the UVA Salt Study as follows. We plotted all 306 delta MAP (ΔMAP) (dots) from the UVA Salt Study and then used the K-Means Clustering method (Squared Euclidean Distance), which shows that the data may be categorized into the three groups, namely ISS (inverse salt-sensitive) on the left, SS (salt-sensitive) on the right, and SR (salt-resistant) in the middle. The centroids of the three clusters were mapped to ΔMAP inverse salt sensitivity, salt resistance, and salt sensitivity values: − 8.142, 0.138, and 9.137, respectively. The boundary points of ΔMAP between the adjacent clusters are − 4 and 4.6, respectively. By assuming the control group as ΔMAP between − 7 and 7 mm Hg, the data of the control group were fitted by the linear regression (Y = 0.0594*X − 8.537, R² = 0.9951, n = 219) (straight line). The overall data were fitted to the 3rd-order polynomial curve (Y = − 14.38 ⁺ 0.219*X − 0.001208*X^{2 +} (2.685e − 6)X³, R.2 = 0.9887, n = 306) (see curved line). Allowing a ± 5% variation, the linear regression curve of the control group may have the cross points at the boundary (ISS N = 52, − 5.8) and (SR N = 249, 6.65). Performing a post hoc power check, the powers between the clusters (± 7 mm Hg boundary) are all around 1. We hypothesize that the non-Gaussian nature of the ISS ΔMAP may be due to additional genes variants, as yet to be discovered

Alternatives to Dietary Protocols Using Surrogate Markers for Determining PSI

Surrogate markers of BP have been studied in an attempt to obviate the need for measuring Na⁺ intake and excretion [6]. We isolated freshly excreted renal proximal tubule cells (RPTCs) from our Na⁺ study participants to measure Na⁺ regulated cellular receptor pathways. Specifically, we measured the ability of RPTCs to recruit dopamine type-1 receptors (D₁R) to the cell membrane in response to an increase in intracellular Na⁺ by monensin (a Na⁺ ionophore) and the intracellular calcium response to angiotensin II (Ang II) which are impaired in SS individuals [21]. The inverse linear relationship between these 2 parameters and the magnitude of the change in BP [ΔMAP] support the hypothesis that the impairment of renal tubular Na⁺ transport by these 2 pathways may define an individual’s PSI. Importantly, the characterization of D₁R recruitment and Ang II-stimulated Ca⁺⁺ signaling was performed 5 years after dietary Na⁺ phenotyping, indicating independence of the marker from the current Na⁺ intake of the subject. Additionally, 45 micro-RNAs (miRNAs) were measured in the urine of adults who express ISS, SR, or SS phenotype [39, 40]. The miRNAs only originate from the renal tissue because exosomes cannot cross the glomerular filtration barrier and many miRNAs found in urine have not been measured in serum [40, 41]. Some of these miRNAs are known to participate in the regulation of signaling pathways involved in hypertension (e.g., peroxisome proliferator-activated receptor-γ, epidermal growth factor receptor, transforming growth factor-β1, phosphatase and tensin homolog/phosphoinositide 3-kinase) [6, 42]. These preliminary clinical studies hold promise for cost-effective methods to screen for SS [6] and ISS phenotypes [21, 34, 39, 40, 42].

Genetic Determinants of Inverse Salt Sensitivity of BP

Many genetic determinants of SS are known, and the current consensus is that combinations of gene variants determine one’s PSI [5, 34, 43–46]. Variants of the G protein-coupled receptor kinase type 4 (GRK4) are associated with SS in humans [34, 44, 47–50] and cause SS or SR [51] hypertension when expressed in mice, which is dependent on the GRK4 variant and genetic background of the mice in which it is expressed [51–53]. Variants in the human SLC4A5 gene that encodes for the Na⁺ bicarbonate electrogenic cotransporter type 2 (NBCe2) are strongly associated with the SS hypertensive phenotype [5, 34, 54]. The angiotensin (Ang) type 1 receptor (AT₁R) is an antinatriuretic G protein-coupled receptor that increases calcium signaling via Ang II in ISS [21]. A model of various renin-angiotensin system (RAS) components contributing to Na⁺ balance is shown in Fig. 4. Enzymes that convert RAS peptides into downstream mediators of Na⁺ balance may also play a role in the pathogenesis of ISS, such as aminopeptidase N (APN) which converts Ang III into angiotensin IV (Ang IV) and reduces the levels of Ang III, the preferred endogenous agonist of the natriuretic Ang II type 2 receptor (AT₂R) [55]. APN, per se, positively regulates cytokine activity and thus can be pro-inflammatory [56], but has variable effects on BP. The intracerebroventricular infusion of APN decreases BP; APN has also been reported to inhibit Na⁺/K⁺/ATPase activity in LLC-PKI cells, an epithelial cell line derived from pig kidneys [57]. By contrast, we have reported that the inhibition of APN by preventing the degradation of Ang III to Ang IV restores the ability of AT₁R blockers to induce natriuresis in the spontaneously hypertensive rat [58]. Overlack et al. found a steeper increase in plasma renin activity in ISS when compared with SR and SS humans placed on a low Na⁺ intake, indicating that ISS subjects may have an overstimulated RAS [22]. Additional work is required not only to quantify the RAS peptides but also to determine the relative activities of the enzymes involved in the production of RAS peptides under various amounts of Na⁺ intake to which the kidney is exposed.

Fig. 4.

Mechanistic model of our hypothesis that the D₂R is at least one etiology of inverse salt sensitivity of blood pressure. The top line shows the model of a normally operating renin-angiotensin system (RAS) in a salt-resistant individual. Various RAS peptides result from successive proteolytic catalysis of angiotensinogen (AGT) into shorter forms of AGT named Ang I-IV. The RAS proteolytic sequential enzyme cascade consists of renin, angiotensin converting enzyme (ACE), aminopeptidase A (APA), and aminopeptidase N (APN). There are other members of RAS but are beyond the scope of this review. “Normal” salt-resistant individuals maintain a homeostatic balance between the antinatriuretic angiotensin type 1 receptor (AT₁R) and the natriuretic angiotensin type 2 receptor (AT₂R). In inverse salt-sensitive (ISS) study participants (lower panel), the response to low Na⁺ intake is shown on the left column and the response to high Na⁺ intake on the right column. APN is increased in ISS on low Na⁺ intake and is hypothesized to decrease under high Na⁺ intake. The low Na⁺ intake stimulation of APN is hypothesized to cause a depletion of Ang III, thus lowering the natriuresis caused by Ang III, via the AT₂R. The D₂R wild type is shown in the 2nd row on the left, which normally suppresses AT₁R-mediated antinatriuresis to maintain Na⁺ intake in balance. Human D₂R gene (DRD2) variants associated with inverse salt sensitivity have a reduced capacity to keep the AT₁R in check (low Na⁺ intake response in inverse salt sensitivity) and thus leads to increased AT₁R-mediated renal tubular Na⁺ reabsorption. The high Na⁺ intake response, in contrast to the low Na⁺ intake response, to DRD2 variants is hypothesized to cause a decrease in the activity of APN, leading to increased Ang III (agonist for the AT₂R) levels, causing an increase in Na.⁺ excretion and a lowering of BP

The Dopamine Type 2 Receptor (D₂R) and ISS

The dopamine type 2 receptor (D₂R) has been reported to interact negatively with the AT₁R in several tissues, including the kidney [59–61]. The D₂R and their other members of the D₂-like receptor subfamily, D₃R and D₄R, are expressed in the kidney [62] and are involved with Na⁺ homeostasis and the regulation of inflammation [63, 64]. The D₂R decreases reactive oxygen species production through stimulation of sestrin 2 and paraoxonase 2 (PON2), and inhibition of NADPH [65] oxidase activity, keeping BP in the normal range [66]. Single nucleotide polymorphisms (SNPs) of the DRD2 decrease D₂R expression and activity. DRD2 rs6276 (1347G > A) at 3’UTR (MAF 0.484) has decreased D₂R function, and DRD2 rs6277 (957C > T exon 7 synonymous mutation, MAF = 0.273) has decreased DRD2 mRNA stability [67]. These DRD2 variants increase inflammation and fibrosis in human RPTCs [67]. Deficient dopamine D₂R function causes renal inflammation independently of high BP [68]. Renal-selective rescue of D₂R function reverses the renal injury and high BP caused by renal Drd2 deficiency in mice [69]. DRD2 variant-mediated dysregulation of the RAS may contribute to the pathogenesis of ISS hypertension [70].

Catecholaminergic Regulation of BP

The D₂R interacts with other cardiovascular regulating systems to keep the BP normal during high, normal, and low Na⁺ intake, via a negative interaction with antinatriuretic AT₁R and/or α1-adrenoceptor. For example, decreasing sympathetic nerve activity with etamicastat normalizes the increased blood pressure of mice with germline deletion of Drd2 or mice with selective renal silencing of Drd2 [119]. The D₂R can also antagonize the stimulatory effects of Ang II on inflammation [68]. D2-like receptor stimulation inhibits Ang II-mediated stimulation of Na⁺K⁺/ATPase activity and inhibition of cAMP production in renal proximal tubules [59]. In ISS with compromised D₂R the RAS system fails to be inhibited and thus leads to ISS hypertension. For example, AT₁R may be responsible for the decreased renal expression of natriuretic D₁R and D₂R in obese Zucker rats [136].

The salt resistance in SJL and BALB/c mice may be related to the increase in renal dopamine production and normal D₁R and D₅R function with the increase in Na⁺ intake [77–79]. In the salt-resistant Wistar rat [80], D₃R and AT₂R interact to increase Na⁺ excretion [76]. We propose that a similar mechanism occurs in SR mice normally expressing the wild-type D₂R. When Na⁺ intake is high, circulating norepinephrine [81–83], systemic norepinephrine spillover [84], urinary norepinephrine [85], and renal norepinephrine overflow [86] are decreased, relative to when Na⁺ intake is low, keeping BP in the normal range [87]. High Na⁺ intake also causes the phosphorylation of NHE-3, and redistribution of NHE-3, NaPi2, NKCC2, β-ENaC, α1 and β 1 Na⁺-K⁺/ATPase, and AT₂R from plasma membrane fractions to intracellular fractions in RPTs, resulting in a decrease in RPT Na⁺ transport [88]. The opposite occurs when Na⁺ intake is low, and in addition, circulating norepinephrine [81, 82, 85], systemic norepinephrine spillover [84], urinary norepinephrine [85], and renal sympathetic activity [86] are increased, relative to what occurs with Na⁺ intake is high [86]. With low Na⁺ intake, the inhibitory effect of D₂Rs on RPT Na⁺ transport is minimized by the increased activity of the renal RAS and adrenergic system [87], similar to the increase in RAS activity that we reported for the D₁R [89]. Total renal [90] and plasma membrane [91] expressions of NCC and α-ENaC but not NHE-3 or NKCC2 increase in rats fed a low NaCl diet. However, in mice [92], low Na⁺ intake increased renal tubular total and cell surface NHE3, NKCC2, and gamma-ENaC, as observed in rats. In mice, renal tubular NHE-3 is required to maintain Na⁺ and water homeostasis and BP [93].

Other Genes Associated with ISS

Two Na⁺/myoinositol cotransporters, SMIT1 (SLC5A3) and SMIT2 (SLC5A11, SGLT6), are expressed in the kidney, but SMIT2 is responsible for the apical myoinositol transport in the RPT [94]. In mice (0.025–0.05% NaCl) also increases the renal expression of SLCA511 (unpublished), aiding the increase in renal Na⁺ reabsorption (Fig. 5), as described for NHE3; an interaction between NHE3 and SLC5A11 remains to be determined. In mice, the D₂R is important in the negative regulation of SLCA511 expression in the kidney because deletion of D₂R only in the RPT increases renal SLCA511 protein expression (unpublished). In humans, the CC genotype in SLC5A11 is associated with lower serum myoinositol concentration, relative to the normal TT genotype [95], which presumably is due to an increase in its (myoinositol) excretion. However, normally D₂R expression/function inhibits SLC5A11 activity so that a negative Na⁺ balance should not occur. When Na⁺ intake is low, the expression of the wild-type sodium/myoinositol transporter (SLCA511 (T/T)) increases, aiding the increase in Na⁺ reabsorption and a positive Na⁺ balance ensues until the ECF volume is normalized and BP remains normal (Fig. 5). When Na⁺ intake is high, the presence of SLC5A11 containing the SNPs (T/C) or SLC5A11 (C/C) decreases sodium/myoinositol cotransporter activity [95] which results in an increase in Na⁺ excretion. However, normally, D₂R expression/function inhibits SLC5A11 activity so that a negative Na⁺ balance should not occur. When Na⁺ intake is low, the natriuretic effect of SLC5A11 (T/C), SLC5A11 (C/C) [95] persists, but the increase circulating norepinephrine [82, 83], systemic norepinephrine spillover [84], urinary norepinephrine [85], and renal norepinephrine overflow [86] also persists, relative to what occurs when Na⁺ intake is high [87]. When Na⁺ intake is low, the inhibitory effect of D₂Rs on RPT transport is minimized by the increased activity of the renal RAS and adrenergic system, similar to the increase in RAS activity that we reported for the D₁R [89]. Ang II and norepinephrine increase NHE3 activity [96]. As stated above, in mice, renal tubular NHE3 is required to maintain Na⁺ and water homeostasis and BP [92]. The increase in norepinephrine activity causes a redistribution of NHE-3 to the RPT plasma membrane, increasing its activity and, thus, Na⁺ transport [97]. Ang II via AT₁R also promotes the dephosphorylation of NHE-3, increasing its activity [98]. A positive Na⁺ balance ensues until a normal BP is achieved (Fig. 6).

Fig. 5.

The normal D₂R negatively interacts with AT₁R and α₁-adrenergic receptors and positively interacts with the AT₂R to regulate sodium transport in the RPT, via NHE3 and SLC5A11 (SGLT6). Normal D₂R and SLC5A11 (T/T) regulate renal sodium transport in the RPT properly, and therefore, the BP is normal. A dashed red line (- - -) indicates inhibition, while a continuous green line (–––) indicates stimulation

Fig. 6.

The normal D₂R negatively interacts with AT₁R and α₁-adrenergic receptors and positively interacts with the AT₂R to inhibit sodium transport in the RPT, via NHE3 and SLC5A11 (SGLT6). The SLC5A11 heterozygous (T/C) and homozygous variants (C/C) decrease rather than increase Na.⁺/myoinositol cotransport. However, the presence of a normal D₂R keeps RPT transport and BP normal. A dashed red line (- - - -) indicates inhibition, while a continuous green line (–––) indicates stimulation

Other Pathways Potentially Associated with ISS

The non-osmotic storage of Na ⁺ in the body’s extracellular space such as in blood vessels and skin could act as a buffer that would have minimal effect on extracellular volume [99, 100]. It has been hypothesized that negative charges in endothelium glycocalyx neutralize the positive charge on Na⁺ to enable non-osmotic storage. In ISS, the effect of high salt diets may not lead to salt sensitivity if the ISS individuals have a greater glycocalyx Na⁺ capacity than SR or SS individuals, such is found in men vs. women [101].

Low D₂R Expression/Activity and Presence of SLC5A11 (C/C) in ISS (Fig. 7)

Fig. 7.

The low D₂R expression/function cannot negatively regulate SLC5A11 (C/C), adding to the increase in sodium excretion on high salt intake. On low salt intake, the natriuretic effect of SLC5A11 (C/C) remains increased caused by the decreased D₂R expression/function. However, the increase in both RAS and α₁-adrenergic nervous system increase renal sodium transport (via NHE3 mainly, as well as NCC and Na⁺, K⁺/ATPase). Sodium retention occurs, and eventually, BP increases, a case of inverse salt sensitivity of blood pressure. A dashed red line (- - -) indicates inhibition, while a continuous green line (–––) indicates stimulation

When Na⁺ intake is high, catecholamine and renin levels decrease and NHE3 activity decreases. However, the low D₂R expression/function cannot prevent the ability of SLC5A11 (C/C) to decrease Na⁺ and myoinositol transport [95], adding to the increase in Na⁺ excretion. The persistence of the negative Na⁺ balance eventually results in a decrease in BP.

By contrast, when Na⁺ intake is low, the low D₂R expression results in overactivity of NHE3 (plus NCC and Na⁺K⁺/ATPase), facilitated by increased Ang II and α-adrenergic action (see above), accompanied by impaired natriuretic activity (decreased plasma membrane expression of AT₂R) [89]. The natriuretic effect of SLC5A11 (C/C) should persist because of decreased D₂R expression/function. However, on a low salt diet, the marked increase in both RAS and α-adrenergic activities [102–106] that may be caused in part by the inability of abnormal D₂R to inhibit them would result in an inappropriate increase in renal Na⁺ transport. Na⁺ retention occurs, and eventually, BP increases, a case of ISS hypertension.

Contributing to these changes in BP is the circaseptan Na⁺ excretion rhythm which is a 7-day rhythm around which many biological processes revolve [107]. A recent meta-analysis on the effect of the dose and duration of dietary Na⁺ studies on the activation of the RAS and sympathetic nervous system may not lead to circaseptan rhythms if the dietary studies are conducted with longer interventions (> 14 days) [108].

Association of Single Nucleotide Polymorphisms (SNPs) in the D₂R and ISS

DRD2 is encoded on chromosome 11q23.2 and expressed as D₂ (short), D₂ (long), and D₂(X1) isoforms [109]. The two DRD2 variants, rs6276 and rs6277, were found to be associated with a rise in BP on the low Na⁺ (10 mmol/day) diet of a two-week controlled Na⁺ BP study [5]. The quantitative assessment found that the DRD2 variants were associated with ISS (rs6276 and rs6267 have P values of 1.0 × 10−2 and 3.8 × 10−2 with odds ratios of 0.32 and 0.48 in unadjusted regression models, respectively) (unpublished data). The rise in MAP on a low Na⁺ diet correlated with the number of SNPs in DRD2, achieving significance with 2 or more DRD2 SNPs. There was a 1 mm Hg increase in MAP per DRD2 SNP (p < 0.01, y = 1.077 × 0.074, R² = 0.8517, n = 174) (Fig. 8). Plasma membrane D₂R expression was measured using an extracellular epitope-specific rabbit anti-D₂R antibody and found to be reduced by 25.3 ± 0.5% in RPTCs from ISS subjects (p < 0.01, N = 5/group), relative to RPTCs from SR subjects (unpublished data). To date, the D₂R is the only gene with SNPs associated with the ISS phenotype. However, additional contributors to the ISS phenotype undoubtedly exist because some ISS individuals have wild-type DRD2.

Fig. 8.

The number of DRD2 SNPs at both rs6276 and is6277 is graphed vs. the delta MAP measured during the transition from low to a high Na⁺ intake. (A) There is a linear relationship between the number of allelic variants and the change (Δ) in MAP (y = 0.73X − 0.06, R.2 = 0.83, p < 0.05, n = 280). (B) The cohort is divided into two groups, those with two or less DRD2 SNPs and those with 3 or 4 DRD2 SNPs. ΔMAP is significantly different between the two groups (0–2 SNPs: − 1.23 ± 0.46, n = 164; 3–4 SNPs: 0.74 ± 0.64, n = 116; p < 0.05)

Epigenetics and ISS Hypertension

Epigenetics may also be involved in the etiology of ISS hypertension. The epigenetic regulation of gene transcription and translation can be influenced by diet [110–112]. An increase or decrease in oxidative stress caused by diet can also provoke changes in epigenetics [112, 113]. Thus, increased Na⁺ intake can increase oxidative stress and can also influence epigenetics [114–116]. The renal D₂R and D₅R receptor, among others, regulate several redox enzymes [52, 66, 117–122]. D₅R influences redox signaling by inhibiting NADPH oxidase via phospholipase D2 and stimulating superoxide dismutase (SOD), glutathione peroxidase, glutamyl cysteine transferase, and heme oxygenase-1 (HO-1) [123]. The D₂R also influences redox signaling by inhibiting the pro-oxidant NADPH oxidase [65] and activating antioxidant enzymes DJ-1, PON2, HO-2, glutathione, catalase, and SOD [117, 119, 120, 123–125]. The dopaminergic system opposes RAS signaling, at least, in part, through specific receptor signaling pathways [126]. The D₃R, D₄R, and D₅R oppose RAS signaling by inhibiting the expression of AT₁R, a known mediator of pro-oxidant activity [127–129]. The D₂R opposes aldosterone production, another pro-oxidant component of the renin–angiotensin–aldosterone system, yet blocking aldosterone binding with spironolactone does not block ROS production [120].

Micro-RNAs (miRNAs), which can regulate the transcription of DNA, are epigenetic markers for various diseases. Micro-RNAs in urinary exosomes and their association with an individual’s BP response to dietary Na⁺ intake have been studied [130]. A wide microarray containing 1898 probes was used to examine the human urinary exosomal miRNome. We extended these studies, and as previously described, of the 194 miRNAs found in the subjects tested, 45 miRNAs had significant associations with SS or ISS [40]. Although not differentially expressed, miR-485-5p is one of the 194 miRNAs found and binds to the DRD2 rs6276, reducing D₂R expression [131]. Furthermore, a miRNA mimic that behaved just as the endogenous mir-485-5p miRNA reduced the expression of D₂R, but only in the RPTCs from the urine of individuals with ISS hypertension. A miRNA blocker inhibited endogenous miRNAs, resulting in normalizing the expression of the D₂R (unpublished data). Fourteen additional miRNAs were found to be associated with ISS hypertension [40]. Among these 14 miRNAs, miRNA 30c-1-3p is pro-inflammatory [132], but its role in the inflammation that occurs with decreased D₂R expression [67, 68, 70, 117, 133–135] remains to be determined.

Conclusions

ISS individuals have a paradoxically elevated BP, even to hypertensive levels, when consuming low levels of Na⁺ (essentially equivalent to the strict vegan diets that are gaining in popularity for health and ethical reasons). At least one pathway involved in the pathogenesis of inverse salt sensitivity is the D₂R pathway; reduced D₂R expression or function has been shown to contribute to the pathogenesis of ISS. There are likely many other pathways, such as the Na⁺/myoinositol pathway described above. An ISS individual’s magnitude of BP rise will likely be determined by the summation of pathways that cause an increase in renal tubular Na⁺ reabsorption that is mediated by increased activity of RAS and the adrenergic nervous system-mediated renal Na⁺ transport. The differential diagnosis of hypertension should include ISS that can be ameliorated by increasing Na⁺ intake, thus avoiding the overuse of antihypertensive medications with potential side effects. What was not reviewed here is the role of the non-osmotic extracellular glycosaminoglycans (GAGs) Na⁺ storage buffer on ISS which may play a significant role in ISS hypertension.

Novelty and Significance.

1. What is new?

• Both high and low Na⁺ intakes associate with increased cardiovascular morbidity and mortality, but the etiology of the high mortality in individuals with low Na⁺ intake is unknown.

• 15% of the adult population may have “inverse salt sensitivity” which is an increase in BP in response to low Na⁺ intake.

2. What is relevant?

• ISS hypertension is due, at least in part, to DRD2 variants, associated with decreased renal proximal tubule cell expression of D₂R, that increases renal tubular Na⁺ reabsorption under low Na⁺ intake.

• High blood pressure may be reduced to the normotensive range by increasing Na⁺ consumption in individuals with ISS hypertension.

3. Conclusion

• Inverse salt sensitivity, which is an increase in blood pressure induced by low Na⁺ intake, is associated with an overactive RAS and adrenergic system caused by a decrease in D₂R receptor expression in the kidney due to DRD2 gene variants. D₂R function is required to suppress the RAS- and adrenergic-mediated stimulation of renal tubular Na⁺ transport.

Abbreviations

Ang

Angiotensin

AT₁R

Angiotensin II type 1 receptor

AT₂R

Angiotensin II type 2 receptor

BP

Blood pressure

D₂R

Dopamine type 2 receptor

DRD2

Dopamine type 2 receptor gene

GRK4

G protein-coupled receptor kinase type 4

MAP

Mean arterial pressure

ISS

Inverse salt-sensitive and inverse salt sensitivity

SR

Salt-resistant

SS

Salt-sensitive

uRPTCs

Urine renal proximal tubule cells