Peiying Yu

Dopamine-1 Receptor Coupling Defect in Renal Proximal Tubule Cells in Hypertension

The ability of the dopamine-1 (D1)-like receptor to stimulate adenylyl cyclase (AC) and phospholipase C (PLC), inhibit sodium transport in the renal proximal tubule (RPT), and produce natriuresis is attenuated in several rat models of hypertension. Since the inhibitory effect of D1-like receptors on RPT sodium transport is also reduced in some patients with essential hypertension, we measured D1-like receptor coupling to AC and PLC in cultures of human RPT cells from normotensive (NT) and hypertensive (HT) subjects. Basal cAMP concentrations were the same in NT (n=6) and HT (n=4). However, the D1-like receptor agonist fenoldopam increased cAMP production to a greater extent in NT (maximum response=67+/-1%) than in HT (maximum response=17+/-5%), with a potency ratio of 105. Dopamine also increased cAMP production to a greater extent in NT (32+/-3%) than in HT (14+/-3%). The fenoldopam-mediated increase in cAMP production was blocked by SCH23390 (a D1-like receptor antagonist) and by antisense D1 oligonucleotides in both HT and NT, indicating action at the D1 receptor. The stimulatory effects of forskolin and parathyroid hormone-related protein of cAMP accumulation were not statistically different in NT and HT, indicating receptor specificity and an intact G-protein/AC pathway. The fenoldopam-stimulated PLC activity was not impaired in HT, and the primary sequence and expression of the D1 receptor were the same in NT and HT. However, D1 receptor serine phosphorylation in the basal state was greater in HT than in NT and was not responsive to fenoldopam stimulation in HT. These studies demonstrate the expression of D1 receptors in human RPT cells in culture. The uncoupling of the D1 receptor in both rats (previously described) and humans (described here) suggests that this mechanism may be involved in the pathogenesis of hypertension; the uncoupling may be due to ligand-independent phosphorylation of the D1 receptor in hypertension.

Activation of D3 Dopamine Receptor Decreases Angiotensin II Type 1 Receptor Expression in Rat Renal Proximal Tubule Cells

The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D3 dopamine receptor gene in mice produces renin-dependent hypertension. In rats, D2-like receptors reduce angiotensin II binding sites in renal proximal tubules (RPTs). Because the major D2-like receptor in RPTs is the D3 receptor, we examined whether D3 receptors regulate angiotensin II type 1 (AT1) receptors in rat RPT cells. The effect of D3 receptors on AT1 receptors was studied in vitro and in vivo. The D3 receptor agonist PD128907 decreased AT1 receptor protein and mRNA in WKY RPT cells and increased it in SHR cells. PD128907 increased D3 receptors in WKY cells but had no effect in SHR cells. D3/AT1 receptors colocalized in RPT cells; D3 receptor stimulation decreased the percent amount of D3 receptors that coimmunoprecipitated with AT1 receptors to a greater extent in WKY than in SHR cells. However, D3 receptor stimulation did not change the percent amount of AT1 receptors that coimmunoprecipitated with D3 receptors in WKY cells and markedly decreased the coimmunoprecipitation in SHR cells. The D3 receptor also regulated the AT1 receptor in vivo because AT1 receptor expression was increased in kidneys of D3 receptor–null mice compared with wild type littermates. D3 receptors may regulate AT1 receptor function by direct interaction with and regulation of AT1 receptor expression. One mechanism of hypertension may be related to increased renal expression of AT1 receptors due decreased D3 receptor regulation.

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.

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 p22phox and the small GTPase Rac1 are expressed in LRs in human renal proximal tubule cells. The D1-like receptor agonist, fenoldopam (1 &mgr;mol/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-β-cyclodextrin [βCD]) 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 βCD (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, βCD-induced reactive oxygen species production was reversed by knocking down either Nox2 (81.0±5.1% versus βCD: 162.0±2.0%) or Nox4 (108.0±10.8% versus βCD: 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.

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 D3 receptor. G protein-coupled receptor kinase 4 (GRK4), whose gene locus (4p16.3) is linked to essential hypertension, desensitizes the D1 receptor, another dopamine receptor. This study evaluated the role of GRK4 on D3 receptor function in human proximal tubule cells. D3 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 D3 receptor and GRK4 at the cell membrane and promoted it intracellularly, presumably en route to endosomal trafficking. Of the four GRK4 splice variants, GRK4-γ and GRK4-α mediated a 3- and 2-fold increase in the phosphorylation of agonist-activated D3 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 D3 receptor stimulation. These data demonstrate that GRK4, specifically the GRK4-γ and GRK4-α isoforms, phosphorylates the D3 receptor and is crucial for its signaling in human proximal tubule cells.

Renal Protein Phosphatase 2A Activity and Spontaneous Hypertension in Rats

The impaired renal paracrine function of dopamine in spontaneously hypertensive rats (SHR) is caused by hyperphosphorylation and desensitization of the renal D1 dopamine receptor. Protein phosphatase 2A (PP2A) is critical in the regulation of G-protein–coupled receptor function. To determine whether PP2A expression and activity in the kidney are differentially regulated in genetic hypertension, we examined the effects of a D1-like agonist, fenoldopam, in renal cortical tubules and immortalized renal proximal tubule cells from normotensive Wistar-Kyoto rats (WKY) and SHR. In cortical tubules and immortalized proximal tubule cells, PP2A expression and activities were greater in cytosol than in membrane fractions in both WKY and SHR. Although PP2A expressions were similar in WKY and SHR, basal PP2A activity was greater in immortalized proximal tubule cells of SHR than WKY. In immortalized proximal tubule cells of WKY, fenoldopam increased membrane PP2A activity and expression of the regulatory subunit PP2A-B56&agr;, effects that were blocked by the D1-like antagonist SCH23390. Fenoldopam had no effect on cytosolic PP2A activity but decreased PP2A-B56&agr; expression. In contrast, in immortalized proximal tubule cells of SHR, fenoldopam decreased PP2A activity in both membranes and cytosol but predominantly in the membrane fraction, without affecting PP2A-B56&agr; expression; this effect was blocked by the D1-like antagonist SCH23390. We conclude that renal PP2A activity and expression are differentially regulated in WKY and SHR by D1-like receptors. A failure of D1-like agonists to increase PP2A activity in proximal tubule membranes may be a cause of the increased phosphorylation of the D1 receptor in the SHR.

D1-Like Receptors Regulate NADPH Oxidase Activity and Subunit Expression in Lipid Raft Microdomains of Renal Proximal Tubule Cells

NADPH oxidase (Nox)–dependent reactive oxygen species production is implicated in the pathogenesis of cardiovascular diseases, including hypertension. We tested the hypothesis that oxidase subunits are differentially regulated in renal proximal tubules from normotensive and spontaneously hypertensive rats. Basal Nox2 and Nox4, but not Rac1, in immortalized renal proximal tubule cells and brush border membranes were greater in hypertensive than in normotensive rats. However, more Rac1 was expressed in lipid rafts in cells from hypertensive rats than in cells from normotensive rats; the converse was observed with Nox4, whereas Nox2 expression was similar. The D1-like receptor agonist fenoldopam decreased Nox2 and Rac1 protein in lipid rafts to a greater extent in hypertensive than in normotensive rats. Basal oxidase activity was 3-fold higher in hypertensive than in normotensive rats but was inhibited to a greater extent by fenoldopam in normotensive (58±3.3%) than in hypertensive rats (31±5.2%; P<0.05; n=6 per group). Fenoldopam decreased the amount of Nox2 that coimmunoprecipitated with p67phox in cells from normotensive rats. D1-like receptors may decrease oxidase activity by disrupting the distribution and assembly of oxidase subunits in cell membrane microdomains. The cholesterol-depleting reagent methyl–β-cyclodextrin decreased oxidase activity and cholesterol content to a greater extent in hypertensive than in normotensive rats. The greater basal levels of Nox2 and Nox4 in cell membranes and Nox2 and Rac1 in lipid rafts in hypertensive rats than in normotensive rats may explain the increased basal oxidase activity in hypertensive rats.

D3 Dopamine Receptor Directly Interacts With D1 Dopamine Receptor in Immortalized Renal Proximal Tubule Cells

D3 receptors act synergistically with D1 receptors to inhibit sodium transport in renal proximal tubules; however, the mechanism by which this occurs is not known. Because dopamine receptor subtypes can regulate and interact with each other, we studied the interaction of D3 and D1 receptors in rat renal proximal tubule (RPT) cells. The D3 agonist PD128907 increased the immunoreactive expression of D1 receptors in a concentration- and time-dependent manner; these effects were blocked by the D3 antagonist U99194A. PD128907 also transiently (15 minutes) increased the amount of cell surface membrane D1 receptors. Laser confocal immunofluorescence microscopy showed that D3 receptor and D1 receptor colocalized in RPT cells more distinctly in Wistar-Kyoto rats than in spontaneously hypertensive rats (SHRs). In addition, D3 and D1 receptors could be coimmunoprecipitated, and this interaction was increased after D3 receptor agonist stimulation for 24 hours in Wistar-Kyoto rats but not in SHRs. We propose that the synergistic effects of D3 and D1 receptors may be caused by a D3 receptor–mediated increase in total, as well as cell surface membrane D1 receptor expression, and direct D3 and D1 receptor interaction, both of which are impaired in SHRs.

Paraoxonase 2 decreases renal reactive oxygen species production, lowers blood pressure, and mediates dopamine D2 receptor-induced inhibition of NADPH oxidase

The dopamine D(2) receptor (D(2)R) regulates renal reactive oxygen species (ROS) production, and impaired D(2)R function results in ROS-dependent hypertension. Paraoxonase 2 (PON2), which belongs to the paraoxonase gene family, is expressed in various tissues, acting to protect against cellular oxidative stress. We hypothesized that PON2 may be involved in preventing excessive renal ROS production and thus may contribute to maintenance of normal blood pressure. Moreover, D(2)R may decrease ROS production, in part, through regulation of PON2. D(2)R colocalized with PON2 in the brush border of mouse renal proximal tubules. Renal PON2 protein was decreased (-33±6%) in D(2)(-/-) relative to D(2)(+/+) mice. Renal subcapsular infusion of PON2 siRNA decreased PON2 protein expression (-55%), increased renal oxidative stress (2.2-fold), associated with increased renal NADPH oxidase expression (Nox1, 1.9-fold; Nox2, 2.9-fold; and Nox4, 1.6-fold) and activity (1.9-fold), and elevated arterial blood pressure (systolic, 134±5 vs 93±6mmHg; diastolic, 97±4 vs 65±7mmHg; mean 113±4 vs 75±7mmHg). To determine the relevance of the PON2 and D(2)R interaction in humans, we studied human renal proximal tubule cells. Both D(2)R and PON2 were found in nonlipid and lipid rafts and physically interacted with each other. Treatment of these cells with the D(2)R/D(3)R agonist quinpirole (1μM, 24h) decreased ROS production (-35±6%), associated with decreased NADPH oxidase activity (-32±3%) and expression of Nox2 (-41±7%) and Nox4 (-47±8%) protein, and increased expression of PON2 mRNA (2.1-fold) and protein (1.6-fold) at 24h. Silencing PON2 (siRNA, 10nM, 48h) not only partially prevented the quinpirole-induced decrease in ROS production by 36%, but also increased basal ROS production (1.3-fold), which was associated with an increase in NADPH oxidase activity (1.4-fold) and expression of Nox2 (2.1-fold) and Nox4 (1.8-fold) protein. Inhibition of NADPH oxidase with diphenylene iodonium (10μM/30 min) inhibited the increase in ROS production caused by PON2 silencing. Our results suggest that renal PON2 is involved in the inhibition of renal NADPH oxidase activity and ROS production and contributes to the maintenance of normal blood pressure. PON2 is positively regulated by D(2)R and may, in part, mediate the inhibitory effect of renal D(2)R on NADPH oxidase activity and ROS production.

Unique role of NADPH oxidase 5 in oxidative stress in human renal proximal tubule cells

NADPH oxidases are the major sources of reactive oxygen species in cardiovascular, neural, and kidney cells. The NADPH oxidase 5 (NOX5) gene is present in humans but not rodents. Because Nox isoforms in renal proximal tubules (RPTs) are involved in the pathogenesis of hypertension, we tested the hypothesis that NOX5 is differentially expressed in RPT cells from normotensive (NT) and hypertensive subjects (HT). We found that NOX5 mRNA, total NOX5 protein, and apical membrane NOX5 protein were 4.2±0.7-fold, 5.2±0.7-fold, and 2.8±0.5-fold greater in HT than NT. Basal total NADPH oxidase activity was 4.5±0.2-fold and basal NOX5 activity in NOX5 immunoprecipitates was 6.2±0.2-fold greater in HT than NT (P=<0.001, n=6–14/group). Ionomycin increased total NOX and NOX5 activities in RPT cells from HT (P<0.01, n=4, ANOVA), effects that were abrogated by pre-treatment of the RPT cells with diphenylene-iodonium or superoxide dismutase. Silencing NOX5 using NOX5-siRNA decreased NADPH oxidase activity (−45.1±3.2% vs. mock-siRNA, n=6–8) in HT. D1-like receptor stimulation decreased NADPH oxidase activity to a greater extent in NT (−32.5±1.8%) than HT (−14.8±1.8). In contrast to the marked increase in expression and activity of NOX5 in HT, NOX1 mRNA and protein were minimally increased in HT, relative to NT; total NOX2 and NOX4 proteins were not different between HT and NT, while the increase in apical RPT cell membrane NOX1, NOX2, and NOX4 proteins in HT, relative to NT, was much less than those observed with NOX5. Thus, we demonstrate, for the first time, that NOX5 is expressed in human RPT cells and to greater extent than the other Nox isoforms in HT than NT. We suggest that the increased expression of NOX5, which may be responsible for the increased oxidative stress in RPT cells in human essential hypertension, is caused, in part, by a defective renal dopaminergic system.