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Activation of the renal D 1 R (dopamine D 1 -like receptor) or AT 2 R (angiotensin II type-2 receptor), individually or both, simultaneously, is necessary in the normal regulation of renal sodium (Na + ) transport and blood pressure. However, little is known regarding the precise mechanism of this interaction. Pharmacological stimulation, membrane biotinylation, and cell surface immunofluorescence were used to study the effect of the D 1 R/AT 2 R interaction in human renal proximal tubule cells. D 1 R activation of Gα S stimulates AC (adenylyl cyclase) and induces apical plasma membrane recruitment of AT 2 Rs. We now show for the first time the reciprocal reaction, AT 2 R stimulation with Ang III (angiotensin III) leads to the apical plasma membrane recruitment of the D 1 R. The cell-permeable second messenger analogs of cAMP (8-Br-cAMP) or cGMP (8-Br-cGMP) induce translocation of both D 1 R and AT 2 R to the plasma membrane. Inhibition of PKA (protein kinase A) with Rp-cAMPS and PKG (protein kinase G) with Rp-8-CPT-cGMPS blocks D 1 R and AT 2 R recruitment, respectively, indicating that both PKA and PKG are necessary for D 1 R and AT 2 R trafficking. Both 8-Br-cAMP and 8-Br-cGMP activate PP2A (protein phosphatase 2A), which is necessary for both plasma membrane recruitment of D 1 R and AT 2 R and the inhibition of sodium hydrogen exchanger 3-dependent Na + transport. These studies provide insights into the D 1 R/AT 2 R transregulation mechanisms that play a crucial role in maintaining Na + and ultimately blood pressure homeostasis.

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 Na/K ratio is considered to be a useful index, the monitoring of which allows an effective Na reduction and K increase, because practical methods (self-monitoring devices and reliable individual estimates from spot urine) are available for assessing these levels in individuals. An intervention trial for lowering the Na/K ratio has demonstrated that a reduction of the Na/K ratio mainly involved Na reduction, with only a small change in K. The present study aimed to clarify the relationship between dietary Na intake and the urinary Na/K molar ratio, using standardized low- and high-salt diets, with an equal dietary K intake, to determine the corresponding Na/K ratio. Fourteen healthy young adult volunteers ingested low-salt (3 g salt per day) and high-salt (20 g salt per day) meals for seven days each. Using a portable urinary Na/K meter, participants measured their spot urine at each voiding, and 24-h urine was collected on the last day of each diet period. On the last day of the unrestricted, low-salt, and high-salt diet periods, the group averages of the 24-h urine Na/K ratio were 4.2, 1.0, and 6.9, while the group averages of the daily mean spot urine Na/K ratio were 4.2, 1.1, and 6.6, respectively. The urinary Na/K ratio tracked changes in dietary salt intake, and reached a plateau approximately three days after each change in diet. Frequent monitoring of the spot urine Na/K ratio may help individuals adhere to an appropriate dietary Na intake., Competing Interests: Toshiyuki Iwahori is an employee of Omron Healthcare Co., Ltd. Hirotsugu Ueshima serves as a consultant for Omron Healthcare Co., Ltd. Katsuyuki Miura received a research fund from Omron Healthcare Co. Ltd.

The sodium bicarbonate cotransporter (NBCe2, aka NBC4) was originally isolated from the human testis and heart (Pushkin et al. IUBMB Life 50:13-19, 2000). Subsequently, NBCe2 was found in diverse locations where it plays a role in regulating sodium and bicarbonate transport, influencing intracellular, extracellular, interstitial, and ultimately plasma pH (Boron et al. J Exp Biol. 212:1697-1706, 2009; Parker and Boron, Physiol Rev. 93:803-959, 2013; Romero et al. Mol Asp Med. 34:159-182, 2013). NBCe2 is located in human and rodent renal-collecting duct and proximal tubule. While much is known about the two electrogenic sodium bicarbonate cotransporters, NBCe1 and NBCe2, in the regulation of sodium homeostasis and pH balance in the rodent kidney, little is known about their roles in human renal physiology. NBCe2 is located in the proximal tubule Golgi apparatus under basal conditions and then disperses throughout the cell, but particularly into the apical membrane microvilli, during various maneuvers that increase intracellular sodium. This review will summarize our current understanding of the distribution and function of NBCe2 in the human kidney and how genetic variants of its gene, SLC4A5, contribute to salt sensitivity of blood pressure.

New Findings: What is the topic of this review? Sensing the amount of ingested sodium is one mechanism by which sodium balance is regulated. This review describes the role of gastrin in the cross-talk between the stomach and the kidney following the ingestion of sodium. What advances does it highlight? Neural mechanisms and several gut hormones, including cholecystokinin and uroguanylin, have been suggested to mediate the natriuresis after an oral sodium load. It is proposed that gastrin produced by G-cells via its receptor, cholecystokinin B receptor, interacts with renal D1 -like dopamine receptors to increase renal sodium excretion. Hypertension develops with chronically increased sodium intake when sodium that accumulates in the body can no longer be sequestered, extracellular fluid volume is expanded, and compensatory neural, hormonal and pressure-natriuresis mechanisms fail. Sensing the amount of ingested sodium, by the stomach, is one mechanism by which sodium balance is regulated. The natriuresis following the ingestion of a certain amount of sodium may be due to an enterokine, gastrin, secreted by G-cells in the stomach and duodenum and released into the circulation. Circulating gastrin levels are 10- to 20-fold higher than those for cholecystokinin. Of all the gut hormones circulating in the plasma, gastrin is the one that is reabsorbed to the greatest extent by renal tubules. Gastrin, via its receptor, the cholecystokinin type B receptor (CCKBR), is natriuretic in mammals, including humans, by inhibition of renal sodium transport. Germline deletion of gastrin (Gast) or Cckbr gene in mice causes salt-sensitive hypertension. Selective silencing of Gast in the stomach and duodenum impairs the ability to excrete an oral sodium load and also increases blood pressure. Thus, the gastrorenal axis, mediated by gastrin, can complement pronatriuretic hormones, such as dopamine, to increase sodium excretion after an oral sodium load. These studies in mice may be translatable to humans because the chromosomal loci of CCKBR and GAST are linked to human essential hypertension. Understanding the role of genes in the regulation of renal function and blood pressure may lead to the tailoring of antihypertensive treatment based on genetic make-up., (© 2016 The Authors. Experimental Physiology © 2016 The Physiological Society.)

Renal dopamine D1-like receptors (D1R and D5R) and the gastrin receptor (CCKBR) are involved in the maintenance of sodium homeostasis. The D1R has been found to interact synergistically with CCKBR in renal proximal tubule (RPT) cells to promote natriuresis and diuresis. D5R, which has a higher affinity for dopamine than D1R, has some constitutive activity. Hence, we sought to investigate the interaction between D5R and CCKBR in the regulation of renal sodium excretion. In present study, we found D5R and CCKBR increase each other's expression in a concentration- and time-dependent manner in the HK-2 cell, the specificity of which was verified in HEK293 cells heterologously expressing both human D5R and CCKBR and in RPT cells from a male normotensive human. The specificity of D5R in the D5R and CCKBR interaction was verified further using a selective D5R antagonist, LE-PM436. Also, D5R and CCKBR colocalize and co-immunoprecipitate in BALB/c mouse RPTs and human RPT cells. CCKBR protein expression in plasma membrane-enriched fractions of renal cortex (PMFs) is greater in D5R-/- mice than D5R+/+ littermates and D5R protein expression in PMFs is also greater in CCKBR-/- mice than CCKBR+/+ littermates. High salt diet, relative to normal salt diet, increased the expression of CCKBR and D5R proteins in PMFs. Disruption of CCKBR in mice caused hypertension and decreased sodium excretion. The natriuresis in salt-loaded BALB/c mice was decreased by YF476, a CCKBR antagonist and Sch23390, a D1R/D5R antagonist. Furthermore, the natriuresis caused by gastrin was blocked by Sch23390 while the natriuresis caused by fenoldopam, a D1R/D5R agonist, was blocked by YF476. Taken together, our findings indicate that CCKBR and D5R synergistically interact in the kidney, which may contribute to the maintenance of normal sodium balance following an increase in sodium intake.

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., (Copyright © 2015 the American Physiological Society.)

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.

Little is known regarding how the kidney shifts from a sodium and water reclaiming state (antinatriuresis) to a state where sodium and water are eliminated (natriuresis). In human renal proximal tubule cells, sodium reabsorption is decreased by the dopamine D(1)-like receptors (D(1)R/D(5)R) and the angiotensin type 2 receptor (AT(2)R), whereas the angiotensin type 1 receptor increases sodium reabsorption. Aberrant control of these opposing systems is thought to lead to sodium retention and, subsequently, hypertension. We show that D(1)R/D(5)R stimulation increased plasma membrane AT(2)R 4-fold via a D(1)R-mediated, cAMP-coupled, and protein phosphatase 2A-dependent specific signaling pathway. D(1)R/D(5)R stimulation also reduced the ability of angiotensin II to stimulate phospho-extracellular signal-regulated kinase, an effect that was partially reversed by an AT(2)R antagonist. Fenoldopam did not increase AT(2)R recruitment in renal proximal tubule cells with D(1)Rs uncoupled from adenylyl cyclase, suggesting a role of cAMP in mediating these events. D(1)Rs and AT(2)Rs heterodimerized and cooperatively increased cAMP and cGMP production, protein phosphatase 2A activation, sodium-potassium-ATPase internalization, and sodium transport inhibition. These studies shed new light on the regulation of renal sodium transport by the dopaminergic and angiotensin systems and potential new therapeutic targets for selectively treating hypertension.

The present study was designed to determine the capability of human renal proximal tubule (RPT) to generate and export guanosine cyclic 3', 5' monophosphate (cGMP) in response to direct stimulation of soluble guanylyl cyclase by nitric oxide (NO) donors. In addition, we investigated whether cGMP extrusion from human RPT cells is required for inhibition of cellular sodium uptake. RPT cells were cultured from fresh human kidneys (normotensive subjects, n=4, mean age 65+/-4.7 years, 3 men, 1 woman; hypertensive patients, n=6, mean age 64+/-6.1 years, 4 men, 2 women) after unilateral nephrectomy. The fluorescence dye Sodium Green was employed to determine cytoplasmic Na+ concentration. In the presence of the Na+/K+ ATPase inhibitor ouabain, fluorescence was monitored at the appropriate wavelength (excitation 485 nm, emission 535 nm). Nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-4) M), increased both intracellular and extracellular cGMP (from 1.26+/-0.21 to 88.7+/-12.6 pmol/mg protein and from 0.58+/-0.10 to 9.24+/-1.9 pmol/mL, respectively, P<0.01) and decreased cellular Na+ uptake by 37.4+/-6.8% (P<0.05) compared with control. The effects of SNAP on cGMP production were similar in normotensive and hypertensive subjects. The increases in intracellular and extracellular cGMP concentration because of SNAP were blocked completely by soluble guanylyl cyclase inhibitor ODQ (1-H-[1,2,4] oxadiazolo [4,2-alpha] quinoxalin-1-one). Probenecid, an organic anion transport inhibitor, augmented the SNAP (10(-6) M)-induced increase in intracellular cGMP accumulation (from 4.9+/-0.9 to 9.8+/-1.5 pmol/mg protein, P<0.05), abrogated the SNAP-induced increase in extracellular cGMP extrusion (from 1.07+/-0.4 to 0.37+/-0.1 pmol/L, P<0.05) and blocked the SNAP-induced reduction in cellular Na+ uptake. Neither intracellular nor extracellular cGMP were influenced by l-arginine, the metabolic precursor of NO, or N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase. After exogenous administration of cGMP (10(-5) M) or its membrane-permeable analogue 8-Br-cGMP (10(-5) M), only 8-Br-cGMP crossed the cell membrane to increase intracellular cGMP (from 1.36+/-0.19 to 289.7+/-29.4 pmol/mg protein, P<0.01). However, both cGMP and 8-Br-cGMP were effective in decreasing cellular Na+ uptake. In conclusion, human RPT cells contain soluble guanylyl cyclase and are able to generate and export cGMP in response to NO. Because human RPT cells do not themselves contain constitutive NO synthase, the NO-generating cGMP must be derived from sources outside the human RPT. The cGMP cellular export system is critical in the regulation of RPT cellular Na+ absorption in humans.

Activation of the renal D1R (dopamine D1-like receptor) or AT2R (angiotensin II type-2 receptor), individually or both, simultaneously, is necessary in the normal regulation of renal sodium (Na+) transport and blood pressure. However, little is known regarding the precise mechanism of this interaction. Pharmacological stimulation, membrane biotinylation, and cell surface immunofluorescence were used to study the effect of the D1R/AT2R interaction in human renal proximal tubule cells. D1R activation of GαS stimulates AC (adenylyl cyclase) and induces apical plasma membrane recruitment of AT2Rs. We now show for the first time the reciprocal reaction, AT2R stimulation with Ang III (angiotensin III) leads to the apical plasma membrane recruitment of the D1R. The cell-permeable second messenger analogs of cAMP (8-Br-cAMP) or cGMP (8-Br-cGMP) induce translocation of both D1R and AT2R to the plasma membrane. Inhibition of PKA (protein kinase A) with Rp-cAMPS and PKG (protein kinase G) with Rp-8-CPT-cGMPS blocks D1R and AT2R recruitment, respectively, indicating that both PKA and PKG are necessary for D1R and AT2R trafficking. Both 8-Br-cAMP and 8-Br-cGMP activate PP2A (protein phosphatase 2A), which is necessary for both plasma membrane recruitment of D1R and AT2R and the inhibition of sodium hydrogen exchanger 3-dependent Na+ transport. These studies provide insights into the D1R/AT2R transregulation mechanisms that play a crucial role in maintaining Na+ and ultimately blood pressure homeostasis. [ABSTRACT FROM AUTHOR]

The dopamine D3 receptor (D3R) is crucial in the regulation of blood pressure and sodium balance, in that Drd3 gene ablation in mice results in hypertension and failure to excrete a dietary salt load. The mechanism responsible for the renal sodium retention in these mice is largely unknown. We now offer and describe a novel mechanism by which D3R decreases sodium transport in the long term by inhibiting the deubiquitinylating activity of ubiquitin-specific peptidase 48 (USP48), thereby promoting Na+-H+ exchanger (NHE)-3 degradation. We found that stimulation with the D3R-specific agonist PD128907 (1 µM, 30 min) promoted the interaction and colocalization among D3R, NHE3, and USP48; inhibited USP48 activity (-35±6%, vs. vehicle), resulting in increased ubiquitinylated NHE3 (+140±10%); and decreased NHE3 expression (-50±9%) in human renal proximal tubule cells (hRPTCs). USP48 silencing decreased NHE3's half-life (USP48 siRNA t1/2=6.1 h vs. vehicle t1/2=12.9 h), whereas overexpression of USP48 increased NHE3 half-life (t1/2=21.8 h), indicating that USP48 protects NHE3 from degradation via deubiquitinylation. USP48 accounted for ~30% of the total deubiquitinylating activity in these cells. Extending our studies in vivo, we found that pharmacologic blockade of D3R via the D3R-specific antagonist GR103691 (1 µg/kg/min, 4 d) in C57Bl/6J mice increased renal NHE3 expression (+310±15%, vs. vehicle), whereas an innovative kidney-restricted Usp48 silencing via siRNA (3 µg/d, 7 d) increased ubiquitinylated NHE3 (+250±30%, vs. controls), decreased total NHE3 (-23±2%), and lowered blood pressure (-24±2 mm Hg), compared with that in control mice that received either the vehicle or nonsilencing siRNA. Our data demonstrate a crucial role for the dynamic interaction between D3R and USP48 in the regulation of NHE3 expression and function. [ABSTRACT FROM AUTHOR]

Gastrin, secreted by stomach G cells in response to ingested so dium, stimulates the renal cholecystokinin B receptor (CCKBR) to increase renal sodium excretion. It is not known how dietary sodium, independent of food, can increase gastrin secretion in human G cells. However, fenofibrate (FFB), a peroxisome proliferator-activated receptor-α (PPAR-α) agonist, increases gastrin secretion in rodents and several human gastrin-secreting cells, via a gastrin transcriptional promoter. We tested the following hypotheses: (1.) the sodium sensor in G cells plays a critical role in the sodiu m-mediated increase in gastrin expression/secretion, and (2.) dopamine, via the D1R and PPAR-α, is involved. Intact human stomach antrum and G cells were compared with huma n gastrin-secreting gastric and ovarian adenocarcinoma cells. When extra-or intracellular sodium was increased in human antrum, human G cells, and adenocarcinoma cells, gastrin mRNA and protein expression/secretion were increased. In human G cells, the PPAR-α agonist FFB increased gastrin protein expression that was blocked by GW6471, a PPAR-α antagonist, and LE300, a D1-like receptor antagonist. LE300 prevented the ability of FFB to increase gastrin protein expression in human G cells via the D1R, because the D5R, the other D1- like receptor, is not expressed in human G cells. Human G cells also express tyrosine hydroxylase and DOPA decarboxylase, enzymes needed to synthesize dopamine. G cells in the stomach may be the sodium sensor that stimulates g astrin secretion, which enables the kidney to eliminate acutely an oral sodium load. Do pamine, via the D1R, by interacting with PPAR-α, is involved in this process. [ABSTRACT FROM AUTHOR]