Background Salt sensitivity (SS) of blood pressure (BP) affects 25% of adults, shares comorbidity with hypertension, and has no convenient diagnostic test. We tested the hypothesis that urine-derived exfoliated renal proximal tubule cells (RPTCs) could diagnose the degree of an individual's SS of BP. Methods Subjects were selected who had their SS of BP determined 5 y prior to this study (salt-sensitive: ≥7 mm Hg increase in mean arterial pressure (MAP) following transition from a random weekly diet of low (10 mmol/day) to high (300 mmol/day) sodium (Na+) intake, N = 4; inverse salt-sensitive (ISS): ≥7 mm Hg increase in MAP transitioning from a high to low Na+ diet, N = 3, and salt-resistant (SR): <7 mm Hg change in MAP transitioned on either diet, N = 5). RPTC responses to 2 independent Na+ transport pathways were measured. Results There was a negative correlation between the degree of SS and dopamine-1 receptor (D1R) plasma membrane recruitment (y = −0.0107x + 0.68 relative fluorescent units (RFU), R2 = 0.88, N = 12, P < 0.0001) and angiotensin II-stimulated intracellular Ca++ (y = −0.0016x + 0.0336, R2 = 0.7112, P < 0.001, N = 10) concentration over baseline. Conclusions Isolating RPTCs from urine provides a personalized cell-based diagnostic test of SS index that offers advantages over a 2-week controlled diet with respect to cost and patient compliance. Furthermore, the linear relationship between the change in MAP and response to 2 Na+ regulatory pathways suggests that an individual's RPTC response to intracellular Na+ is personalized and predictive.

Salt-sensitivity of blood pressure (BP) is a cardiovascular risk that affects 25% of the world’s population due to its resulting hypertension, although independent of BP. Salt-sensitivity is detected with a two week controlled diet, which is difficult to administer in the clinical setting. We therefore developed a rapid method of diagnosis based on exfoliated renal proximal tubule cells (RPTC) in urine. Subjects were divided into 3 salt sensitivity index categories: High-Salt-Sensitive (HSS; ≥ 7 mmHg increase in mean arterial pressure (MAP) on a high salt diet of 300 mEq of sodium, 17%prevalence), Low-Salt-Sensitive (LSS; ≥ 7 mmHg increase in MAP on a low salt diet of 10 mEq of sodium, 11% prevalence) and Salt Resistant (SR; ≤ 7 mmHg increase in MAP on both high and low salt diets, 72% prevalence) (Carey et al., in review). Three individuals were analyzed in each category on a minimum of 3 separate occasions. Cells were isolated from urine using centrifugation and measured for dopamine-1 receptor (D1R) plasma membrane recruitment using fluorescently-labeled antibodies under a confocal microscope as well as in a flow cytometer. Confocal microscopy analysis (total of 100 RPTCs for the 9 subjects) showed a negative correlation between salt-sensitivity index and D1R surface recruitment in RPTCs in their response to salt stimulation (y = -0.0073x + 0.5248, p = 0.0159). Flow cytometry analysis (total of 4938 RPTCs for the 9 subjects) also demonstrated a negative correlation between salt-induced D1R recruitment and salt-sensitivity (y = -2.547x + 239.97, p < 0.0001). Flow cytometry analysis showed a greater degree of separation amongst the subjects than confocal microscopy analysis, and would allow for a rapid diagnostic use of exfoliated renal cells in urine. Cryopreserved RPTCs (viability = 57.16% ± 9.15%, n = 12) compare favorably with cell viability from freshly voided urine cells and were still capable of eliciting intracellular sodium-mediated D1R recruitment. Cryopreservation thus enables batch collection, transport and processing of specimens between sites. We expect these procedures to provide a novel and convenient method of diagnosing the salt-sensitivity index in humans.

Salt sensitivity of blood pressure (BP) affects 25% of the world’s population and has a cardiovascular risk that is similar to hypertension, but can be present in normotensive individuals. Salt sensitivity can be detected with a two week controlled diet, which is difficult to administer in the clinical setting. Our previous method of determining an individual’s salt sensitivity status through the use of confocal microscopy is challenging as a clinical assay due to its complexity. We therefore developed a rapid method of diagnosis based on exfoliated renal proximal tubule cells (RPTC) in urine. Our streamlined approach made use of an Accuri™ C6 (BD Biosciences) flow cytometer to measure monensin (MON)-stimulated dopamine-1 receptor (D1R) and angiotensin-2 receptor (AT2R) recruitment to the plasma membrane. In order to control for method-induced variability and biological variation, we developed external controls. Two different immortalized cell lines, i22 (D1R coupled) and i19 (D1R uncoupled) were chosen for their consistent level of response to stimulation by MON, a sodium ionophore. These cell lines were each labeled with D1R or AT2R antibodies directly conjugated to R-Phycoerythrin (RPE) (Ex: 535nm, Em: 575nm) to obtain a basal level of surface receptor. Afterwards, the cells were stimulated with MON (1μM) to elicit a Na+ -induced receptor recruitment response. Finally, the cells were stained with directly labeled D1R or AT2R antibodies conjugated to Allophycocyanin (APC) (Ex: 650 nm, Em: 660 nm), after which they were read on the Accuri. Normally coupled i22 cells showed a significantly higher (p<0.005) recruitment ratio (Stimulated RFU (APC) / Basal RFU (RPE)) for D1R of 0.42 (SEM ± 0.012, n=7) than did uncoupled i19 cells, which showed a recruitment ratio of 0.36 (SEM = ± 0.006, n=7). Normally coupled i22 cells showed a significantly higher (p<0.05) recruitment ratio for AT2R of 0.45 (SEM ± 0.018, n=6) than did uncoupled i19 cells, which showed a recruitment ratio of 0.39 (SEM = ± 0.013, n=6). This controlled cell-based assay will facilitate testing in large cohorts to validate its use as a means to determine each individual’s salt sensitivity index.

Salt-sensitivity of blood pressure is an inappropriate increase in blood pressure following high salt intake. Subjects in our clinical study were typed according to their salt-sensitivity status into 3 categories: High-Salt-Sensitive (HSS; ≥ 7 mmHg increase in mean arterial pressure (MAP) on a high salt diet of 300 mEq of sodium, 17% prevalence), Low-Salt-Sensitive (LSS:, who paradoxically showed a ≥ 7 mmHg increase in MAP on a low salt diet of 10 mEq of sodium, 11% prevalence), and Salt-Resistant (SR, individuals who showed no significant increase in blood pressure on either diet, 72% prevalence). We previously demonstrated that LSS subjects show increased recruitment of the natriuretic dopamine-1 receptor (D1R) to the plasma membrane following a salt stimulation as compared to HSS subjects. Stimulation of the D1R in RPTC with fenoldopam (dopaminergic agonist) results in recruitment of the natriuretic angiotensin type-2 receptor (AT2R) to the cell surface. We hypothesized that LSS individuals may also demonstrate an enhanced AT2R RPTC membrane recruitment compared to HSS individuals when challenged with fenoldopam. In order to gain access to fresh RPTC from each subject, we isolated exfoliated RPTC from randomly voided urine from SR, LSS, and HSS subjects from our clinical study. We measured three subjects from each category with a minimum of three voids for each subject. We counted individual cells as independent events using both the confocal microscope (n=245) and the flow cytometer (n=5344). We found an inverse correlation between AT2R recruitment and the degree of salt-sensitivity of blood pressure. Fenoldopam stimulated AT2R recruitment as measured by confocal microscopy (y = -0.0047x + 0.4966, R2 = 0.2488, P<0.0001) and flow cytometry (y =-0.057x + 1.5645, R2=0.2912, P=0.0185). Flow cytometry provided a more sensitive diagnostic for LSS than HSS subjects. AT2R recruitment was more predictive of LSS than HSS. AT2R recruitment may be used as a rapid method to test for LSS individuals who need to be identified and encouraged to increase their sodium intake in order to avoid paradoxical hypertension.