Deficient dopamine D2 receptor function causes renal inflammation independently of high blood pressure.

Renal dopamine receptors participate in the regulation of blood pressure. Genetic factors, including polymorphisms of the dopamine D(2) receptor gene (DRD2) are associated with essential hypertension, but the mechanisms of their contribution are incompletely understood. Mice lacking Drd2 (D(2)-/-) have elevated blood pressure, increased renal expression of inflammatory factors, and renal injury. We tested the hypothesis that decreased dopamine D(2) receptor (D(2)R) function increases vulnerability to renal inflammation independently of blood pressure, is an immediate cause of renal injury, and contributes to the subsequent development of hypertension. In D(2)-/- mice, treatment with apocynin normalized blood pressure and decreased oxidative stress, but did not affect the expression of inflammatory factors. In mouse RPTCs Drd2 silencing increased the expression of TNFα and MCP-1, while treatment with a D(2)R agonist abolished the angiotensin II-induced increase in TNF-α and MCP-1. In uni-nephrectomized wild-type mice, selective Drd2 silencing by subcapsular infusion of Drd2 siRNA into the remaining kidney produced the same increase in renal cytokines/chemokines that occurs after Drd2 deletion, increased the expression of markers of renal injury, and increased blood pressure. Moreover, in mice with two intact kidneys, short-term Drd2 silencing in one kidney, leaving the other kidney undisturbed, induced inflammatory factors and markers of renal injury in the treated kidney without increasing blood pressure. Our results demonstrate that the impact of decreased D(2)R function on renal inflammation is a primary effect, not necessarily associated with enhanced oxidant activity, or blood pressure; renal damage is the cause, not the result, of hypertension. Deficient renal D(2)R function may be of clinical relevance since common polymorphisms of the human DRD2 gene result in decreased D(2)R expression and function.

Renal dopaminergic defect in C57Bl/6J mice.

The C57Bl/6J mouse strain, the genetic background of many transgenic and gene knockout models, is salt sensitive and resistant to renal injury. We tested the hypothesis that renal dopaminergic function is defective in C57Bl/6J mice. On normal NaCl (0.8%, 1 wk) diet, anesthetized and conscious (telemetry) blood pressures were similar in C57Bl/6J and SJL/J mice. High NaCl (6%, 1 wk) increased blood pressure (approximately 30%) in C57Bl/6J but not in SJL/J mice and urinary dopamine to greater extent in SJL/J than in C57Bl/6J mice. Absolute and fractional sodium excretions were lower in SJL/J than in C57Bl/6J mice. The blood pressure-natriuresis plot was shifted to the right in C57Bl/6J mice. Renal expressions of D(1)-like (D(1)R and D(5)R) and angiotensin II AT(1) receptors were similar on normal salt, but high salt increased D(5)R only in C57Bl/6J. GRK4 expression was lower on normal but higher on high salt in C57Bl/6J than in SJL/J mice. Salt increased the excretion of microalbumin and 8-isoprostane (oxidative stress marker) and the degree of renal injury to a greater extent in SJL/J than in C57Bl/6J mice. A D(1)-like receptor agonist increased sodium excretion whereas a D(1)-like receptor antagonist decreased sodium excretion in SJL/J but not in C57Bl/6J mice. In contrast, parathyroid hormone had a similar natriuretic effect in both strains. These results show that defective D(1)-like receptor function is a major cause of salt sensitivity in C57Bl/6J mice, decreased renal dopamine production might also contribute. The relative resistance to renal injury of C57Bl/6J may be a consequence of decreased production of reactive oxygen species.

Inhibitory effect of ETB receptor on Na(+)-K(+) ATPase activity by extracellular Ca(2+) entry and Ca(2+) release from the endoplasmic reticulum in renal proximal tubule cells.

The kidney is important in the long-term regulation of blood pressure and sodium homeostasis. Stimulation of ETB receptors in the kidney increases sodium excretion, in part, by decreasing sodium transport in the medullary thick ascending limb of Henle and in collecting duct. However, the role of ETB receptor on Na(+)-K(+) ATPase activity in renal proximal tubule (RPT) cells is not well defined. The purpose of this study is to test the hypothesis that ETB receptor inhibits Na(+)-K(+) ATPase activity in rat RPT cells, and investigate the mechanism(s) by which such an action is produced. In RPT cells from Wistar-Kyoto rats, stimulation of ETB receptors by the ETB receptor agonist, BQ3020, decreased Na(+)-K(+) ATPase activity, determined by ATP hydrolysis (control=0.38+/-0.02, BQ3020=0.26+/-0.03, BQ788=0.40+/-0.06, BQ3020+BQ788=0.37+/-0.04, n=5, P<0.01). The ETB receptor-mediated inhibition of Na(+)-K(+) ATPase activity was dependent on an increase in intracellular calcium, because this effect was abrogated by a chelator of intracellular-free calcium (BAPTA-AM; 5 x 10(-3) M 15 min(-1)), Ca(2+) channel blocker (10(-6) M 15 min(-1) nicardipine) and PI3 kinase inhibitor (10(-7) M per wortmannin). An inositol 1,4,5-trisphosphate (IP3) receptor blocker (2-aminoethyl diphenyl borate; 10(-4) M 15 min(-1)) also blocked the inhibitory effect of the ETB receptor on Na(+)-K(+)ATPase activity (control=0.39+/-0.06, BQ3020=0.25+/-0.01, 2-APB=0.35+/-0.05, BQ3020+ 2-APB=0.35+/-0.06, n=4, P<0.01). The calcium channel agonist (BAY-K8644; 10(-6) M 15 min(-1)) inhibited Na(+)-K(+) ATPase activity, an effect that was blocked by a phosphatidylinositol-3 kinase inhibitor (10(-7) M 15 min(-1) wortmannin). In rat RPT cells, activation of the ETB receptor inhibits Na(+)-K(+) ATPase activity by facilitating extracellular Ca(2+) entry and Ca(2+) release from endoplasmic reticulum.

The regulation of proximal tubular salt transport in hypertension: an update.

PURPOSE OF REVIEW: Renal proximal tubular sodium reabsorption is regulated by sodium transporters, including the sodium glucose transporter, sodium amino acid transporter, sodium hydrogen exchanger isoform 3 and sodium phosphate cotransporter type 2 located at the luminal/apical membrane, and sodium bicarbonate cotransporter and Na+/K+ATPase located at the basolateral membrane. This review summarizes recent studies on sodium transporters that play a major role in the increase in blood pressure in essential/polygenic hypertension. RECENT FINDINGS: Sodium transporters and Na+/K+ATPase are segregated in membrane lipid and nonlipid raft microdomains that regulate their activities and trafficking via cytoskeletal proteins. The increase in renal proximal tubule ion transport in polygenic hypertension is primarily due to increased activity of NHE3 and Cl/HCO3 exchanger at the luminal/apical membrane and a primary or secondary increase in Na+/K+ATPase activity. SUMMARY: The increase in renal proximal tubule ion transport in hypertension is due to increased actions by prohypertensive factors that are unopposed by antihypertensive factors.

G protein-coupled receptor kinase 4 (GRK4) regulates the phosphorylation and function of the dopamine D3 receptor.

During conditions of moderate sodium excess, the dopaminergic system regulates blood pressure and water and electrolyte balance by engendering natriuresis. Dopamine exerts its effects on dopamine receptors, including the dopamine D(3) receptor. G protein-coupled receptor kinase 4 (GRK4), whose gene locus (4p16.3) is linked to essential hypertension, desensitizes the D(1) receptor, another dopamine receptor. This study evaluated the role of GRK4 on D(3) receptor function in human proximal tubule cells. D(3) receptor co-segregated in lipid rafts and co-immunoprecipitated and co-localized in human proximal tubule cells and in proximal and distal tubules and glomeruli of kidneys of Wistar Kyoto rats. Bimolecular fluorescence complementation and confocal microscopy revealed that agonist activation of the receptor initiated the interaction between D(3) receptor and GRK4 at the cell membrane and promoted it intracellularly, presumably en route to endosomal trafficking. Of the four GRK4 splice variants, GRK4-gamma and GRK4-alpha mediated a 3- and 2-fold increase in the phosphorylation of agonist-activated D(3) receptor, respectively. Inhibition of GRK activity with heparin or knockdown of GRK4 expression via RNA interference completely abolished p44/42 phosphorylation and mitogenesis induced by D(3) receptor stimulation. These data demonstrate that GRK4, specifically the GRK4-gamma and GRK4-alpha isoforms, phosphorylates the D(3) receptor and is crucial for its signaling in human proximal tubule cells.

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells.

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R.

H2O2 stimulation of the Cl-/HCO3- exchanger by angiotensin II and angiotensin II type 1 receptor distribution in membrane microdomains.

The present study tested the hypothesis that angiotensin II (Ang II)-induced oxidative stress and Ang II-stimulated Cl(-)/HCO(3)(-) exchanger are increased and related to the differential membrane Ang II type 1 (AT(1)) receptor and reduced nicotinamide-adenine dinucleotide phosphate oxidase expression in immortalized renal proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) relative to its normotensive control (Wistar Kyoto rat [WKY]). The exposure of cells to Ang II increased Cl(-)/HCO(3)(-) exchanger activity with EC(50)s of 0.10 and 12.2 nmol/L in SHR and WKY PTE cells, respectively. SHR PTE cells were found to overexpress nicotinamide-adenine dinucleotide phosphate oxidase 2 and 4 and were endowed with an enhanced ability to generate H(2)O(2). The reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor apocynin reduced the production of H(2)O(2) in SHR PTE cells and abolished their hypersensitivity to Ang II. The expression of the glycosylated form of the AT(1) receptor in both lipid and nonlipid rafts were higher in SHR cells than in WKY PTE cells. Pretreatment with apocynin reduced the abundance of AT(1) receptors in both microdomains, mainly the glycosylated form of the AT(1) receptor in lipid rafts, in SHR cells but not in WKY PTE cells. In conclusion, differences between WKY and SHR PTE cells in their sensitivity to Ang II correlate with the higher H(2)O(2) generation that provokes an enhanced expression of glycosylated and nonglycosylated AT(1) receptor forms in lipid rafts.

Lipid rafts keep NADPH oxidase in the inactive state in human renal proximal tubule cells.

Recent studies have indicated the importance of cholesterol-rich membrane lipid rafts (LRs) in oxidative stress-induced signal transduction. Reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases, the major sources of reactive oxygen species, are implicated in cardiovascular diseases, including hypertension. We tested the hypothesis that NADPH oxidase subunits and activity are regulated by LRs in human renal proximal tubule cells. We report that a high proportion of p22(phox) and the small GTPase Rac1 are expressed in LRs in human renal proximal tubule cells. The D(1)-like receptor agonist, fenoldopam (1 micromol/L per 20 minutes) dispersed Nox subunits within LRs and non-LRs and decreased oxidase activity (30.7+/-3.3%). In contrast, cholesterol depletion (2% methyl-beta-cyclodextrin [beta CD]) translocated NADPH oxidase subunits out of LRs and increased oxidase activity (154.0+/-10.5% versus control, 103.1+/-3.4%), which was reversed by cholesterol repletion (118.9+/-9.9%). Moreover, NADPH oxidase activation by beta CD (145.5+/-9.0%; control: 98.6+/-1.6%) was also abrogated by the NADPH oxidase inhibitors apocynin (100.4+/-3.2%) and diphenylene iodonium (9.5+/-3.3%). Furthermore, beta CD-induced reactive oxygen species production was reversed by knocking down either Nox2 (81.0+/-5.1% versus beta CD: 162.0+/-2.0%) or Nox4 (108.0+/-10.8% versus beta CD: 152.0+/-9.8%). We have demonstrated for the first time that disruption of LRs results in NADPH oxidase activation that is abolished by antioxidants and silencing of Nox2 or Nox4. Therefore, in human renal proximal tubule cells, LRs maintain NADPH oxidase in an inactive state.