cGMP-Dependent Protein Kinase 1 Polymorphisms Underlie Renal Sodium Handling ImpairmentNovelty and Significance
Defective pressure-natriuresis related to abnormalities in the natriuretic response has been associated with hypertension development. A major signaling pathway mediating pressure natriuresis involves the cGMP-dependent protein kinase 1 (PRKG1) that, once activated by Src kinase, inhibits renal Na+ reabsorption via a direct action on basolateral Na-K ATPase and luminal Na–H exchanger type 3, as shown in renal tubuli of animals. Because a clear implication of PRKG1 in humans is still lacking, here we addressed whether PRKG1 polymorphisms affect pressure-natriuresis in patients. Naive hypertensive patients (n=574), genotyped for PRKG1 rs1904694, rs7897633, and rs7905063 single nucleotide polymorphisms (SNPs), underwent an acute Na+ loading, and the slope of the pressure–natriuresis relationship between blood pressure and Na+ excretion was calculated. The underlying molecular mechanism was investigated by immunoblotting protein quantifications in human kidneys. The results demonstrate that the PRKG1 risk haplotype GAT (rs1904694, rs7897633, rs7905063, respectively) associates with a rightward shift of the pressure–natriuresis curve (0.017±0.004 μEq/mm Hg per minute) compared with the ACC (0.0013±0.003 μEq/mm Hg per minute; P=0.001). In human kidneys, a positive correlation of protein expression levels between PRKG1 and Src (r=0.83; P<0.001) or α1 Na-K ATPase (r=0.557; P<0.01) and between α1 Na-K ATPase and Na–H exchanger type 3 (r=0.584; P<0.01) or Src (r=0.691; P<0.001) was observed in patients carrying PRKG1 risk GAT (n=23) but not ACC (n=14) variants. A functional signaling complex among PRKG1, α1 Na-K ATPase, and Src was shown by immunoprecipitation from human renal caveolae. These findings indicate that PRKG1 risk alleles associate with salt-sensitivity related to a loss of the inhibitory control of renal Na+ reabsorption, suggestive of a blunt pressure–natriuresis response.
- PRKG1 protein, human, kidney, natriuresis
- salt-sensitive hypertension
- sodium reabsorption
Kidneys exert a pivotal role in the modulation of blood pressure (BP) by regulating salt and water excretion and by controlling peripheral vascular tone via several neurohormonal mechanisms.1 Kidney function is influenced by salt intake and may adapt rapidly to salt variations by modulating pressure-natriuresis (PNat).2 However, kidney adaptability to Na+ differs among individuals and is influenced by genetic and environmental factors. In particular, salt-sensitive individuals respond to a high-salt diet with a large rise in BP, indicative of a blunted PNat relationship.3,4 Several inter-related pathways involving tubular Na+ and Cl− transports along the nephron are implicated in the maintenance of PNat. Any disturbance of these mechanisms, including gain- or loss-of-function mutations in single genes, may lead to hypertension.3,4 Recently, intrarenal NO–cGMP has been indicated as a candidate mediator of PNat that occurs via activation of cGMP-dependent protein kinase 1 (PRKG1).5 As shown in rats, PRKG1 promotes high BP-mediated natriuresis by inhibiting the basolateral α1 Na-K ATPase and the luminal Na–H exchanger type 3 (NHE3) localized in renal proximal tubuli.5
Interestingly, a genome-wide genotyping study performed on a selected population of whites with mild hypertension who were undergoing an acute salt load led to the identification of a cluster of SNPs in the PRKG1 gene involved in BP response to an acute salt load.6
However, the role of PRKG1 in the PNat mechanism has not yet been demonstrated in humans. Therefore, here we investigated the effects of an acute saline load in an expanded cohort of newly discovered never-treated (naive) hypertensive patients on the PNat relationship as function of PRKG1 polymorphisms. To understand the molecular pathways regulating this association, we analyzed the influence of these polymorphisms on the expression of target proteins implicated in PRKG1 activation in human renal specimens.
Studies in Patients
We enrolled 574 consecutive, newly discovered naive hypertensive patients in the Outpatient Clinic for Hypertension of San Raffaele Hospital, Milan. The Ethics Committee of the San Raffaele Hospital approved the study, and informed consent was obtained from each individual. Patients underwent clinical examination and routine biochemistry. Secondary hypertension was excluded by routine methods. Women taking contraceptive pill were not enrolled in the study protocol. All patients underwent 24-hour ambulatory BP monitoring (Spacelab 90207; Spacelab Medical Inc, Redmond, WA). Blood samples for renin activity, aldosterone, and endogenous ouabain and 24-hour urine collections for Na+ and K+ were obtained the day before the 24-hour ambulatory BP monitoring recording.
Saline Load Test
The slope of the PNat relationship between BP and Na+ excretion (μEq/mm Hg per minute) was calculated for each patient by plotting Na+ excretion on the y axis as a function of MBP on the x axis, observed both under basal conditions (T0) and after 120 minutes of saline infusion (T120), as previously reported.4,7 MBP was calculated as one-third differential BP plus diastolic BP.
Experimental Studies in Human Kidney Specimens
The study of human kidney samples was approved by the San Raffaele Scientific Institute Ethical Committee. Written informed consent was obtained from all patients. Human kidney samples, derived from nephrectomy because of tumors, were stored at the Pathology Department of San Raffaele Hospital. The kidney portions used were verified to be histologically normal before analysis. Total RNA was isolated from PRKG1 ACC and GAT homozygous samples, as described.6
Renal Microsome Preparation and Western Blotting
Human kidney specimens were homogenized in 250 mmol/L sucrose, 30 mmol/L histidine, and 1 mmol/L EDTA (pH 7.2) and microsomes were prepared, as published.8 Western blot analysis on microsomes (15 μg protein/lane) followed a published procedure.9 Antibodies used were as follows: anti-PRKG1 (Cell Signaling); anti-α1 Na-K ATPase and anti-Src (Millipore); anti-Na-H exchanger type 3 (NHE3) (Millipore); anti-actin (Sigma).
Caveolae Isolation and Coimmunoprecipitation Experiments
Caveolae-enriched microdomains have been purified from human renal specimens according to a detergent-free procedure, as described.10 Human renal caveolae were then used for immunoprecipitation experiments with anti-Src antibody, as shown.10
The Hardy–Weinberg equilibrium was calculated for PRKG1 rs1904694, rs7897633, and rs7905063 using χ2 with 1 degree of freedom. The effects of each PRKG1 genotype and haplotype on BP variations and PNat relationship were analyzed with a general linear model covariated for body mass index (BMI), age, and sex. Each SNP was then fitted in regression analysis as a linear term: the effect (β) on BP in mm Hg and on PNat in μEq/mm Hg per minute of the risk allele for Na+ sensitivity was assumed to be additive. Covariates considered for entry into the model were sex, age, and BMI. SPSS (version 19 for MacOS X; SPSS Inc, Chicago, IL) software was used for general statistical analysis.
Normalized gene expression data were derived from automatic analysis with RQ Manager 1.2 software (Applied Biosystems). ANOVA was used to compute P value in univariate models for evaluation of the effect of SNP genotype on PRKG1 expression. Data from immunoblotting were reported as mean±SEM or mean±SD, as specified. P<0.05 was considered statistically significant.
Studies in Patients
The clinical characteristics of the naive hypertensive cohort are presented in Table 1. The 574 participants included 100 (17.4%) female and 474 (82.6%) male naive hypertensive patients.
As expected, daytime systolic BP, BMI, and urinary Na+ and K+ were lower in women. MBP changes after saline load were found to be normally distributed with an average value of +2.19 mm Hg (SD, 6.61), with a wide range of values (minimum, −18.3 mm Hg and maximum +28.4 mm Hg). No deviation from Hardy–Weinberg equilibrium in the 3 SNPs was observed.
As shown in Table 2, all the 3 PRKG1 SNPs were significantly associated with PNat, and the relative risk alleles for rs1904694, rs7897633, and rs7905063 SNPs resulted in G, A, and T, respectively. Particularly, the risk rs7905063 genotype variant (TT) showed a significant rightward shift of the PNat relationship at T120 (TT=0.0132±0.002 versus CT+CC=0.0051±0.002; P=0.0046 adjusted for sex, age, and BMI), as shown in Figure 1 and Table 2.
When the homozygous state of haplotypes was considered, PRKG1 GAT was the combination at higher risk for PNat than the ACC haplotype. Even if the number of each haplotype group was substantially reduced compared with that of genotypes, we observed a significant association with PNat (GAT=0.017±0.004 versus ACC=0.013±0.003; P=0.001 adjusted for sex, age, and BMI) along with a change in diastolic BP after Na+ load (GAT=4.5±1 versus ACC=0.11±0.7; P=0.0001 adjusted for sex, age, and BMI; Table 3).
Experimental Studies in Human Kidney Specimens
The PRKG1 mRNA expression level showed no difference between the CC group (units±SEM, 1.22±0.18 arbitrary; n=12) and the risk TT group (1.17±0.15; n=9). A Western blot analysis for PRKG1, Src, α1 Na-K ATPase, and NHE3 protein content was performed on human renal microsomes (PRKG1 GAT, n=23 and ACC, n=14). The average of the densitometric analysis, which is expressed as arbitrary units and normalized for actin protein level, did not show any significant difference in protein expression between PRKG1 GAT and ACC groups (Figure S1 in the online-only Data Supplement; Table S1). However, only in PRKG1 GAT (n=23), but not ACC (n=14) carriers, a positive correlation was observed between protein expressions for α1 Na-K ATPase and PRKG1 (GAT: r=0.557, P<0.01; ACC: r=0.24, nonsignificant; Figure 2A) or NHE3 (GAT: r=0.584, P<0.01; ACC: r=0.28, nonsignificant; Figure 2B) and between Src and PRKG1 (GAT: r=0.84, P<0.001; ACC: r=0.48, nonsignificant; Figure 2C) or α1 Na-K ATPase (GAT: r=0.69, P<0.001; ACC: r=0.11, nonsignificant; Figure 2D).
To further support the role of PRKG1 in modulating renal Na+ reabsorption via Src kinase, a score matrix–assisted ligand identification analysis was conducted. The results indicated that PRKG1 contains SH2 consensus sequences for several tyrosine kinases, including Src (Figure S2). This analysis predicts that PRKG1 may be phosphorylated in Y566 by Src.
Because NO–cGMP–PRKG1 and Src–Na-K ATPase signaling pathways are both functionally compartmentalized within the plasma membrane caveolae subdomains in several tissues, mainly derived from rats,9–12 we hypothesized that PRKG1 may be part of a unique signaling complex with Src and α1 Na-K ATPase in renal caveolae and that it could be immunoprecipitated by an anti-Src antibody. Caveolae isolated from human renal tissues were found to be enriched in PRKG1 with Src, α1 Na-K ATPase, and caveolin 1, the specific marker of caveolae (Figure 3A–3C), and a PRKG1–Src–α1 Na-K ATPase complex could be immunoprecipitated by an anti-Src antibody (Figure 3D).
Collectively, the present findings suggest that PRKG1 forms a functional signaling complex with Src and α1 Na-K ATPase in human renal caveolae and that PRKG1 polymorphisms may modulate, via Src kinase, the α1 Na-K ATPase activity in renal tubuli, thus affecting renal Na+ reabsorption.
PNat is a major homeostatic control mechanism designed to protect against a long-term rise in arterial BP causing diuresis and natriuresis.2,13 Evidences indicate that the increase in renal interstitial hydrostatic pressure favors the release of cGMP compartmentalized within the renal proximal tubule and mediates PNat via activation of Src and PRKG1-dependent signaling pathways responsible for the inhibition of renal Na+ reabsorption. This occurs mainly by the inhibition of the basolateral α1 Na-K ATPase and the coupled luminal NHE3.5 However, this protective mechanism is defective in clinical and experimental forms of hypertension.3,4 Here, we show that, after an acute saline load on mild hypertensive patients, those carrying the risk PRKG1 GAT alleles presented a rightward shift of the PNat relationship compared with those carrying ACC. Thus, PRKG1 risk alleles seem to associate with a salt-sensitive phenotype related to a loss of the inhibitory control of renal Na+ reabsorption.
In analogy with the results described here, a recent article reported an association analysis of the same PRKG1 intronic SNPs examined in the present study with the diastolic BP response to salt load.6 As hydrostatic pressure is one of the main determinants of glomerular filtration, in the present study, we preferred to use the MBP instead of diastolic BP because the former is considered the real pressure present in capillary bed.2 This adjustment allowed us to manage a more accurate PNat phenotype. It is worth mentioning that the PRKG1 rs7897633, a risk allele for salt-sensitivity, reflected the ancestral status, whereas the protective C allele that was present exclusively in humans and was more frequent in non-African populations reflected a possible signature of positive natural selection.6,14 A similar trend of frequency was observed for PRKG1 rs7905063. Thus, an association study of these PRKG1 SNPs with PNat might be verified in naive hypertensive Africans because they represent a population with a high frequency of salt-sensitivity. Another PRKG1 association study in relation to essential hypertension was assessed in a Chinese population. Particularly, a 4-gene interaction model of association that includes genes from the contractile pathway of vascular smooth muscle cells was found to affect the risk for hypertension.15
The putative target for cGMP and PRKG1 kinase in renal tubular cells is the α1 Na-K ATPase, which works as a receptor linked to the Src signaling pathway.10,12 PRKG1 by itself is a substrate of Src and may be phosphorylated and activated by this kinase.5 Accordingly, score matrix–assisted ligand identification analysis of PRKG1 identified in its protein the presence of SH2 consensus sequences for several tyrosine kinases, including Src.
In additional studies, it has been shown that Src forms a functional signaling complex with α1 Na-K ATPase10,12 that consequently becomes phosphorylated in tyrosine residues and becomes functionally activated.10 The activation of the Src–Na-K ATPase signaling pathway in the basolateral membrane of renal epithelial cells is paralleled by an increase in the luminal NHE3, thereby favoring an overall increase in renal tubular Na+ reabsorption. It seems that the activation of Src is necessary for PRKG1 modulation and, consequently, for the full regulation of PNat mechanism.5 Therefore, we investigated whether the molecular mechanisms underlying the changes in the PNat slope could involve alterations of PRKG1, Src, α1 Na-K ATPase, and NHE3 protein expressions in human renal specimens. We showed that PRKG1 mRNA and protein expression indicated no quantitative difference in renal specimens as function of PRKG1 polymorphisms. However, in the presence of the PRKG1 risk GAT, but not in ACC variants, a positive correlation was observed in microsomes between protein levels of PRKG1 and Src or α1 Na-K ATPase and between α1 Na-K ATPase and NHE3 or Src. This finding suggests that PRKG1 GAT polymorphisms blunt the inhibition of renal tubular Na+ reabsorption and cause a loss of the PNat protective control in a Src–α1 Na-K ATPase dependent manner. Because the luminal NHE3 is strictly functionally coupled to the basolateral alteration of the α1 Na-K ATPase, it may be postulated that at least part of the enhanced renal Na+ reabsorption occurs in the proximal tubuli.
It is well known that the characteristics of the PNat relationship differ according to the mechanisms involved.16 In fact, the increase in tubular Na+ reabsorption produces a decrease in the slope of this relationship as observed in the salt-sensitive form of hypertension. Interestingly, we have recently reported that α-adducin polymorphism also induces the activation of the Src–Na-K ATPase signaling pathway in the basolateral membranes of renal epithelial cells associated with an increase in renal Na+ reabsorption and PNat modulation.4,9,17
PRKG1 is a kinase with a wide spectrum of physiological functions because it has been detected, besides kidney, in lungs, adrenals, brain, cardiomyocytes, and vasculature and at low levels in pulmonary artery smooth muscle cells.18–20 In the cardiovascular system, PRKG1 regulates cardiac contractility, vascular tone, platelet function, cardiac and vascular hypertrophy, and remodeling by the modulation of its numerous target proteins.18,19
Kidney and heart functions are inter-related and implicated in maintaining the hemodynamic stability and the organ perfusion via a strict relationship that controls the volume status, the vascular tone, and the cardiac output.21 The cardiorenal connection between these 2 organs ensures that subtle physiological changes in 1 system are compensated by the other through the activation of a cascade of mediators and pathways.21 As a consequence, any perturbation in cardiac or renal functions may impair the ability of the other organ to compensate.
According to this view, the impairment of renal function because of PRKG1 polymorphisms may result in a cardiac dysfunction and an increased cardiovascular risk. Thus, the possible mechanisms connecting PRKG1 to salt-sensitive hypertension may be relevant to vascular22 and cardiac complications. To address this point, Kuznetsova et al23 investigated the role of PRKG1 polymorphisms in cardiac left ventricular dysfunctions in a cohort of a general population.
In naive hypertensive patients, genetic variants in cGMP-dependent PRKG1 have been shown to affect the PNat relationship. The underlying molecular mechanism seems to depend on the activation of the PRKG1–α1 Na-K ATPase–Src signaling pathway within human renal tubular cells. In the presence of risk PRKG1 polymorphisms, a blunt PNat response that causes a loss of inhibitory control of renal Na+ reabsorption occurs. Thus, patients carrying PRKG1 risk alleles result in a salt-sensitive phenotype. In view of the widespread distribution of PRKG1 in different tissues of the cardiovascular system, we suggest that these polymorphisms may induce other organ alterations as a consequence of the renal function impairment. This aspect warrants more investigations.
We acknowledge the technical assistance of Cinzia Scotti.
Sources of Funding
This study was supported in part by the Italian Ministry of Health RF-FSR-2008-1141719 (P. Manunta) and the Italian Ministry of University and Scientific Research 2008W5AZEC_001 (P. Manunta).
This paper was sent to Toshiro Fujita, Consulting editor, for review by expert referees, editorial decision, and final disposition.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.113.01628/-/DC1.
- Received April 30, 2013.
- Revision received May 21, 2013.
- Accepted August 22, 2013.
- © 2013 American Heart Association, Inc.
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Novelty and Significance
What Is New?
This is the first study that addresses the role of cGMP-dependent protein kinase 1 (PRKG1) polymorphisms on the pressure–natriuresis relationship, that is, salt-sensitivity, in humans.
In human kidneys, PRKG1 participates in the functional signaling complex with Src and α1 Na-K ATPase in caveolae subdomains.
What Is Relevant?
PRKG1 risk alleles associate with a salt-sensitive phenotype related to a loss of the inhibitory control of renal Na+ reabsorption.
Because of its wide spectrum of physiological functions, PRKG1 is implicated in hypertension organ damage.
PRKG1 polymorphisms are related to salt-sensitive hypertension and modulate renal tubular sodium reabsorption, causing a loss of pressure–natriuresis control in a Src–α1 Na-K ATPase dependent manner.