Abstract 470: Development of Control Cells in a Cell-Based Assay for Salt Sensitivity

Abstract

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.