Rationale Salt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure. Objective To characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5. Methods and results The expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte nuclear factor type 4A (HNF4A) to DNA was increased in hRPTCs carrying HV SLC4A5 rs7571842 but not rs10177833. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was abolished by HNF4A antagonists. Conclusion NBCe2 activity is stimulated by an increase in intracellular sodium and is hyper-responsive in hRPTCs carrying HV SLC4A5 rs7571842 through an aberrant HNF4A-mediated mechanism.

The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure (BP), which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT), another site of bicarbonate transport. The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by siRNA and Western blot, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and HEK293 cells. We immune-localized (peptide pre-absorbable) NBCe2 protein in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and in isolated RPTC apical membrane. NBCe2 was primarily found in the Golgi while NBCe1, another electrogenic member of the same family, was primarily found at the basolateral membrane, under basal conditions. Following a short-term increase in intracellular sodium, NBCe2 expression increased at the apical membrane in cultured slices of human kidney (1.75±0.15 fold increase over basal, N=3, p<0.05) and polarized, immortalized RPTCs (0.08±0.015 vs 0.03±0.006 control, NBCe2/CD13 RFU, N=3, p<0.05), while the basolateral localization of NBCe1 was decreased (30.7±5.4% over basal, N=3, p<0.05). DIDS (electrogenic sodium bicarbonate inhibitor, 500 μM, 10 min) inhibitable bicarbonate dependent pH recovery in the presence of EIPA (NHE inhibitor,100 nM, 10 min) after ammonium chloride prepulse (20 mM, 5 min) was higher in the cells cultured under high salt conditions (26.7±7.2% over basal, N=3, p<0.05). Thus, NBCe2 appears to be important in apical sodium and bicarbonate cotransport under high salt conditions. Future studies will examine the role of NBCe2 in mediating increased renal sodium transport in human salt sensitivity of BP.

The sodium bicarbonate cotransporter NBCe2 (encoded by SLC4A5) partially regulates renal tubular sodium bicarbonate transport. Hypothesis: since SLC4A5 single nucleotide polymorphisms (SNPs, rs10177833 and rs7571842) are associated with salt sensitivity of blood pressure, the gene product, NBCe2, would be involved with the etiology of human salt sensitivity. NBCe2 was localized in freshly fixed renal tissue and in primary and immortalized RPT cell (RPTC) cultures from tissue or isolated from urine. Basal expression of NBCe2 mRNA and protein was not different between RPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, total transcellular sodium transport, NHE3 protein expression, and Cl-/HCO3- exchanger activity were higher in SLC4A5 HV than WT RPTCs (WT: 8.6±1.2 mM NaCl n=6, 5207.1±386.4 RFU n=36, 0.265±0.006 pH unit/min, n=33 respectively; VS HV: 14.75±0.7 mM NaCl n=4, 6946.2±500.4 RFU n=48, 0.314±0.018 pH unit/min n=35 respectively, p<0.01). Aberrant sodium transport was even more evident after increasing intracellular sodium, which resulted in increased NBCe2 mRNA, NBCe2 protein and bicarbonate transport in HV RPTCs compared to WT (WT 146% ± 24% , 109% ± 4.7%, 89% ± 4.5%, respectively, VS HV 214% ± 23%, 128% ± 5.7%, 141% ± 4.8%, respectively N=8-12, p<0.05). RPTCs carrying HV variants showed increased binding of HNF4A to SLC4A5 DNA, which was blocked by two HNF4A antagonists. Assays in RPTCs isolated from urine showed increased bicarbonate-dependent pH recovery in RPTCs from salt-sensitive subjects who are HV for SLC4A5. NBCe2 under high sodium is hyper-responsive in RPTCs carrying SLC4A5 HV through an aberrant HNF4A-mediated mechanism.

The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and Western blot analysis, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and human embryonic kidney 293 (HEK293) cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi, while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA, but not by NBCe1 shRNA, and blocked by 2,2'-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid]. NBCe2 could be important in apical sodium and bicarbonate cotransport under high-salt conditions; the implication of the ex vivo studies to the in vivo situation when salt intake is increased remains unclear. Therefore, future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake.

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 colocalize in renal proximal tubule cells and physically interact, as determined by co-immunoprecipitation and fluorescent resonance energy transfer 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 fluorescent resonance energy transfer 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, whereas 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, whereas the D1R/D5R heteromer modulates the D1R effect through a PLC pathway.

The kidney is a highly heterogeneous organ that is responsible for fluid and electrolyte balance. Much interest is focused on determining the function of specific renal epithelial cells in humans, which can only be accomplished through the isolation and growth of nephron segment-specific epithelial cells. However, human renal epithelial cells are notoriously difficult to maintain in culture. This chapter describes the isolation, growth, immortalization, and characterization of the human renal proximal tubule cell. In addition, we describe new paradigms in 3D cell culture which allow the cells to maintain more in vivo-like morphology and function.

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), R(2)=0.88, N=12, P<0.0001) and angiotensin II-stimulated intracellular Ca(++) (y=-0.0016x+0.0336, R(2)=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.

SLC4A5 is a sodium-bicarbonate co-transporter involved with sodium homeostasis. Based on unpublished data, two SLC4A5 single nucleotide polymorphisms (SNPs rs1017783 and rs7571842) have been highly associated with an individual’s salt-sensitivity status. Since the renal proximal tubule (RPT) regulates a large percentage of renal sodium transport, we investigated whether SLC4A5 was present in this nephron segment. Using confocal immunofluorescence microscopy, we found expression of SLC4A5 in human RPT cell plasma membrane and intracellular membrane vesicles. We then examined the physiologic implications of the SLC4A5 SNPs in human RPT cells. Using immunoblotting and RT-PCR, we found no significant differences in basal SLC4A5 expression in RPT cells between individuals that are homozygous variant at both SNPs and individuals that are wild-type (WT) for both alleles. Stimulation of the dopaminergic system with 1μM fenoldopam, or the renin-angiotensin system with 10 nM angiotensin II or 10 nM angiotensin III (n=18 per treatment) over 3 and 24 hours did not significantly alter SLC4A5 protein or 24 hour mRNA expression. These data indicate that SLC4A5 is not directly regulated by either the renal dopaminergic or renin-angiotensin system. However, 24 hour stimulation with the sodium ionophore monensin (MON, 1μM) significantly increased overall mRNA expression of SLC4A5 by 182±0.098% over vehicle (VEH) (ΔCq VEH=0.283±0.035; n=18, p<0.001). There was also a significant increase in SLC4A5 mRNA in three cell lines homozygous variant for both alleles compared to three WT cell lines following MON treatment at both 3 hours (138±0.10%; ΔCq WT MON = 0.5±0.052; n=9, p<0.05) and 24 hours (161±0.11%; ΔCq WT MON = 0.39±0.066; n=9, p<0.02). Three but not 24 hour stimulation with MON also significantly increased overall expression of SLC4A5 protein (137±0.00041%; RFU VEH=0.0030±0.00022; n=18, p<0.01). MON, by allowing salt to enter a cell, may be activating an enhancer that leads to increased transcription of SLC4A5 mRNA that is more effective in homozygous variant cell lines. These novel observations demonstrate that SNPs located in a non-promoter DNA intron are associated with enhanced promoter activity that is regulated by altered intracellular sodium.

Objectives: The aim of this study was to compare the single-dose effects of thiazide-type diuretics cicletanine and hydrochlorothiazide (HCTZ), on natriuresis and kaliuresis in prehypertensive and treatment-naive, stage 1 hypertensive patients and to explore the impact of GRK4 gene polymorphisms on thiazide-induced urinary electrolyte excretion.Methods: The study was a randomized, double-blind, placebo-controlled, three-period, four-treatment, balanced incomplete block, cross-over study in male patients assigned to treatment sequences consisting of placebo, cicletanine 50 mg, cicletanine 150 mg, and HCTZ 25 mg, doses used to treat hypertension. Cumulative urine samples were collected predosing and over 24 h after dosing in each period to compare urine electrolyte excretion profiles of potassium (UKV), sodium (UNaV), magnesium, calcium, phosphate, chloride, and pH among groups. Each treatment was administered to 18 different patients in each period, and an equal number of patients had less than and at least three GRK4 allele variants.Results: Compared with placebo, mean UKV was significantly increased with HCTZ 25mg (12.7 mmol/day; P <= 0.001), cicletanine 50mg (4.6 mmol/day; P = 0.026), and cicletanine 150mg (5.5 mmol/day; P = 0.011), and mean UNaV was significantly increased with HCTZ 25mg (102.2 mmol/day; P <= 0.001), cicletanine 50mg (21.7 mmol/day; P = 0.005), and cicletanine 150mg (57.9 mmol/day; P < 0.001).Conclusion: All treatments had more natriuresis, diuresis, and kaliuresis than placebo, and both doses of cicletanine had less kaliuresis than HCTZ. These findings suggest that cicletanine is a favorable and well tolerated option for the treatment of hypertension with an improved safety profile compared with HCTZ.

Previous studies have demonstrated that single nucleotide polymorphisms (SNPs) of the sodium-bicarbonate co-transporter gene ( SLC4A5 ) are associated with hypertension. We tested the hypothesis that SNPs in SLC4A5 are associated with salt sensitivity of blood pressure in 185 whites consuming an isocaloric constant diet with a randomized order of 7 days of low Na + (10 mmol/d) and 7 days of high Na + (300 mmol/d) intake. Salt sensitivity was defined as a ≥7-mm Hg increase in mean arterial pressure during a randomized transition between high and low Na + diet. A total of 35 polymorphisms in 17 candidate genes were assayed, 25 of which were tested for association. Association analyses with salt sensitivity revealed 3 variants that associated with salt sensitivity, 2 in SLC4A5 ( P <0.001) and 1 in GRK4 ( P =0.020). Of these, 2 SNPs in SLC4A5 (rs7571842 and rs10177833) demonstrated highly significant results and large effects sizes, using logistic regression. These 2 SNPs had P values of 1.0×10 −4 and 3.1×10 −4 with odds ratios of 0.221 and 0.221 in unadjusted regression models, respectively, with the G allele at both sites conferring protection. These SNPs remained significant after adjusting for body mass index and age ( P =8.9×10 −5 and 2.6×10 −4 and odds ratios 0.210 and 0.286, respectively). Furthermore, the association of these SNPs with salt sensitivity was replicated in a second hypertensive population. Meta-analysis demonstrated significant associations of both SNPs with salt sensitivity (rs7571842 [ P =1.2×10 −5 ]; rs1017783 [ P =1.1×10 −4 ]). In conclusion, SLC4A5 variants are strongly associated with salt sensitivity of blood pressure in 2 separate white populations.

HK-2 human renal proximal tubule cells (RPTC) are commonly used in the in vitro study of “normal” RPTCs. We discovered recently that HK-2 cells are uncoupled from dopamine 1 receptor (D 1 R) adenylyl cyclase (AC) stimulation. We hypothesized that G protein–coupled receptor kinase type 4 (GRK4) single nucleotide polymorphisms may be responsible for the D 1 R/AC uncoupling in HK-2. This hypothesis was tested by genotyping GRK4 single nucleotide polymorphisms, measuring D 1 -like receptor agonist (fenoldopam)-stimulated cAMP accumulation, quantifying D 1 R inhibition of sodium transport, and testing the ability of GRK4 small interfering RNA to reverse the D 1 R/AC uncoupling. We compared HK-2 with 2 normally coupled human RPTC cell lines and 2 uncoupled RPTC cell lines. The HK-2 cell line was found to have 4 of 6 potential GRK4 single nucleotide polymorphisms known to uncouple the D 1 R from AC (namely, R65L, A142V, and A486V). AC response to fenoldopam stimulation was increased in the 2 normally coupled human RPTC 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 2 uncoupled or HK-2 cell lines. GRK4 small interfering RNA 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 uncoupled RPTC cell lines (n=6) but was observed in the 2 normally coupled human RPTC cell lines (−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.