HK-2 human renal proximal tubule cells (RPTC) are commonly used in the in vitro study of “normal” RPTCs. We recently discovered that HK-2 cells are uncoupled from dopamine-1 receptor (D1R) adenylyl cyclase (AC) stimulation. We hypothesized that G protein coupled receptor kinase type 4 (GRK4) single nucleotide polymorphisms (SNPs) may be responsible for the D1R/AC uncoupling in HK-2. This hypothesis was tested by genotyping GRK4 SNPs, measuring D1-like receptor agonist (fenoldopam)stimulated cAMP accumulation, quantifying D1R inhibition of sodium transport, and testing the ability of GRK4 siRNA to reverse the D1R/AC uncoupling. We compared HK-2 to 2 normally coupled human RPTC cell lines (nRPTC) and 2 uncoupled RPTC cell lines (uRPTC). The HK-2 cell line was found to have 4 out of 6 potential GRK4 SNPs known to uncouple the D1R from AC (namely R65L, A142V, and A486V). AC response to fenoldopam stimulation was increased in the two nRPTC cell lines (FEN 2.02±0.05-fold and 2.33±0.19-fold over control, P<0.001, N=4), but not in the two uncoupled or HK-2 cell lines. GRK4 siRNA rescued the fenoldopam-mediated AC stimulation in the uncoupled cells, including HK-2. The expected fenoldopam -mediated inhibition of sodium hydrogen exchanger type 3 was absent in HK-2 (N=6) and uRPTCs (N=6), but was observed in the two nRPTCs (−25.41±4.7% and −27.36±2.70% (P<0.001, N=6)), which express wild-type GRK4. Despite the fact that HK-2 cells retain many functional characteristics of RPTCs, they are not normal from the perspective of dopaminergic function.

Determining the individual roles of the two dopamine D1-like receptors (D1R and D5R) on sodium transport in the human renal proximal tubule has been complicated by their structural and functional similarity. Here we used a novel D5R-selective antagonist (LE-PM436) and D1R or D5R-specific gene silencing to determine second messenger coupling pathways and heterologous receptor interaction between the two receptors. D1R and D5R co-localized in renal proximal tubule cells and physically interact, as determined by co-immunoprecipitation and FRET microscopy. Stimulation of renal proximal tubule cells with fenoldopam (D1R/D5R agonist) led to both adenylyl cyclase and phospholipase C (PLC) activation using real-time FRET biosensors ICUE3 and CYPHR, respectively. Fenoldopam increased cAMP accumulation and PLC activity and inhibited both NHE3 and NaKATPase activities. LE-PM436 and D5R siRNA blocked the fenoldopam-stimulated PLC pathway but not cAMP accumulation, while D1R siRNA blocked both fenoldopam-stimulated cAMP accumulation and PLC signaling. Either D1R or D5R siRNA, or LE-PM436 blocked the fenoldopam dependent inhibition of sodium transport. Further studies using the cAMP-selective D1R/D5R agonist SKF83822 and PLC-selective D1R/D5R agonist SKF83959 confirmed the cooperative influence of the two pathways on sodium transport. Thus, D1R and D5R interact in the inhibition of NHE3 and NaKATPase activity, the D1R primarily by cAMP, while the D1R/D5R heteromer modulates the D1R effect through a PLC pathway.

Background: Financial penalties rendered by the Centers for Medicare and Medicaid Services have brought about new challenges for safety net hospitals that serve a vulnerable patient population with risk factors associated with high readmission rates. Our goal was to determine the 1-year trajectory of unplanned readmissions in post-myocardial infarction (MI) patients, and to identify factors associated with readmission. Methods: A total of 261 acute MI patients admitted from April 2015 to April 2016 were evaluated in a multidisciplinary cardiology clinic within 10 days of hospital discharge and baseline characteristics and medical comorbidities were collected. Readmission and mortality data were obtained at 1 year through chart review and telephone follow-up. Results: At 1 year, there were 90 (34%) unplanned readmissions of which half were for noncardiac diagnoses. Of these, 69 patients (77%) were readmitted once, 16 (18%) were readmitted twice, 2 (2%) were readmitted 3 times, and 3 (3%) were readmitted 4 times over the subsequent year. Cardiac causes of 1-year readmission included recurrent MI in 23 (9%) and decompensated heart failure in 18 (7%) patients. Depressed left ventricular systolic function (hazard ratio, 2.23; 95% confidence interval, 2.00–2.44; P = 0.0003) and diabetes mellitus (hazard ratio, 1.60; 95% confidence interval, 1.38–1.82; P = 0.029) were associated with a significantly higher risk of readmission at 1 year. Conclusion: Following acute MI, patients are readmitted for cardiac and noncardiac diagnoses well beyond the 30-day mark. This is likely a function of the vulnerability of the patient population rather than a reflection of the medical care provided. More frequent surveillance may attenuate this problem.