Crisanto S. Escano

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.

Role of Renal DJ-1 in the Pathogenesis of Hypertension Associated With Increased Reactive Oxygen Species Production

The D2 dopamine receptor (D2R) is important in the pathogenesis of essential hypertension. We have already reported that systemic deletion of the D2R gene in mice results in reactive oxygen species (ROS)-dependent hypertension, suggesting that the D2R has antioxidant effects. However, the mechanism of this effect is unknown. DJ-1 is a protein that has antioxidant properties. D2R and DJ-1 are expressed in the mouse kidney and colocalize and coimunoprecipitate in mouse renal proximal tubule cells. We hypothesized that D2Rs regulate renal ROS production in the kidney through regulation of DJ-1 expression or function. Heterozygous D2+/− mice have increased blood pressure, urinary 8-isoprostanes, and renal Nox 4 expression, but decreased renal DJ-1 expression. Silencing D2R expression in mouse renal proximal tubule cells increases ROS production and decreases the expression of DJ-1. Conversely, treatment of these cells with a D2R agonist increases DJ-1 expression and decreases Nox 4 expression and NADPH oxidase activity, effects that are partially blocked by a D2R antagonist. Silencing DJ-1 expression in mouse renal proximal tubule cells increases ROS production and Nox 4 expression. Selective renal DJ-1 silencing by the subcapsular infusion of DJ-1 siRNA in mice increases blood pressure, renal Nox4 expression, and NADPH oxidase activity. These results suggest that the inhibitory effects of D2R on renal ROS production are at least, in part, mediated by a positive regulation of DJ-1 expression/function and that DJ-1 may have a role in the prevention of hypertension associated with increased ROS production.

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.

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 D2 receptor gene (DRD2) are associated with essential hypertension, but the mechanisms of their contribution are incompletely understood. Mice lacking Drd2 (D2−/−) have elevated blood pressure, increased renal expression of inflammatory factors, and renal injury. We tested the hypothesis that decreased dopamine D2 receptor (D2R) 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 D2−/− 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 D2R 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 D2R 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 D2R function may be of clinical relevance since common polymorphisms of the human DRD2 gene result in decreased D2R expression and function.

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.

Upregulation of Renal Sodium Transporters in D5 Dopamine Receptor–Deficient Mice

D5 dopamine receptor (D5R)-deficient (D5−/−) mice have hypertension that is aggravated by an increase in sodium intake. The present experiments were designed to test the hypothesis that a dysregulation of renal sodium transporters is related to the salt sensitivity in D5−/− mice. D5R was expressed in the renal proximal tubule, thick ascending limb, distal convoluted tubule, and cortical and outer medullary collecting ducts in D5+/+ mice. On a control Na+ diet, renal protein expressions of NKCC2 (sodium-potassium-2 chloride cotransporter), sodium chloride cotransporter, and &agr; and &ggr; subunits of the epithelial sodium channel were greater in D5−/− than in D5+/+ mice. Renal renin abundance and urine aldosterone levels were similar but renal angiotensin II type 1 receptor (AT1R) protein expression was increased in D5−/− mice. An elevated Na+ diet increased further the elevated blood pressure of D5−/− mice but did not affect the normal blood pressure of D5+/+ mice. The increased levels of NKCC2, sodium chloride cotransporter, and &agr; and &ggr; subunits of the epithelial sodium channel persisted with the elevated Na+ diet and unaffected by chronic AT1R blockade (losartan) in D5−/− mice. The expressions of proximal sodium transporters NHE3 (sodium hydrogen exchanger type 3) and NaPi2 (sodium phosphate cotransporter type 2) were increased by the elevated Na+ diet in D5−/− mice; the increased expression of NHE3 but not NaPi2 was abolished by AT1R blockade. Our findings suggest that the increased protein expression of sodium transporters/channels in distal nephron segments may be the direct consequence of the disruption of D5R, independent of the renin–angiotensin aldosterone system.

Lack of Renal Dopamine D5 Receptors Promotes Hypertension

Disruption of the dopamine D(5) receptor gene in mice increases BP and causes salt sensitivity. To determine the role of renal versus extrarenal D(5) receptors in BP regulation, we performed cross-renal transplantation experiments. BP was similar between wild-type mice and wild-type mice transplanted with wild-type kidneys, indicating that the transplantation procedure did not affect BP. BP was lower among D(5)(-/-) mice transplanted with wild-type kidneys than D(5)(-/-) kidneys, demonstrating that the renal D(5) receptors are important in BP control. BP was higher in wild-type mice transplanted with D(5)(-/-) kidneys than wild-type kidneys but not significantly different from syngenic transplanted D(5)(-/-) mice, indicating the importance of the kidney in the development of hypertension. On a high-salt diet, all mice with D(5)(-/-) kidneys excreted less sodium than mice with wild-type kidneys. Transplantation of a wild-type kidney into a D(5)(-/-) mouse decreased the renal expression of AT(1) receptors and Nox-2. Conversely, transplantation of a D(5)(-/-) kidney into a wild-type mouse increased the expression of both, suggesting that both renal and extrarenal factors are important in the regulation of AT(1) receptor and Nox-2 expression. These results highlight the role of renal D(5) receptors in BP homeostasis and the pathogenesis of hypertension.

Response Gene to Complement 32 Is Essential for Fibroblast Activation in Renal Fibrosis

Background: TGF-β plays an important role in kidney fibrogenesis, but the downstream target genes remain largely unknown. Results: Response gene to complement 32 (RGC-32), a TGF-β target, is involved in myofibroblast activation in renal fibrosis. Conclusion: RGC-32 is a fibrogenic factor contributing to the kidney fibrogenesis. Significance: Targeting RGC-32 may be a novel strategy to repair kidney injury. Response gene to complement 32 (RGC-32) is a downstream target of transforming growth factor-β (TGF-β). TGF-β is known to play a pathogenic role in renal fibrosis. In this study, we investigated RGC-32 function in renal fibrosis following unilateral ureteral obstruction (UUO) in mice, a model of progressive tubulointerstitial fibrosis. RGC-32 is normally expressed only in blood vessels of mouse kidney. However, UUO induces RGC-32 expression in renal interstitial cells at the early stage of kidney injury, suggesting that RGC-32 is involved in interstitial fibroblast activation. Indeed, expression of smooth muscle α-actin (α-SMA), an indicator of fibroblast activation, is limited to the interstitial cells at the early stage, and became apparent later in both interstitial and tubular cells. RGC-32 knockdown by shRNA significantly inhibits UUO-induced renal structural damage, α-SMA expression and collagen deposition, suggesting that RGC-32 is essential for the onset of renal interstitial fibrosis. In vitro studies indicate that RGC-32 mediates TGF-β-induced fibroblast activation. Mechanistically, RGC-32 interacts with Smad3 and enhances Smad3 binding to the Smad binding element in α-SMA promoter as demonstrated by DNA affinity assay. In the chromatin setting, Smad3, but not Smad2, binds to α-SMA promoter in fibroblasts. RGC-32 appears to be essential for Smad3 interaction with the promoters of fibroblast activation-related genes in vivo. Functionally, RGC-32 is crucial for Smad3-mediated α-SMA promoter activity. Taken together, we identify RGC-32 as a novel fibrogenic factor contributing to the pathogenesis of renal fibrosis through fibroblast activation.

Sorting nexin 1 loss results in D5 dopamine receptor dysfunction in human renal proximal tubule cells and hypertension in mice

Background: SNX1 is a protein involved in the trafficking of internalized receptors. Results: Inhibition of SNX1 expression leads to failure of D5R endocytosis and signaling. Conclusion: Depletion SNX1 function results in D5R dysfunction and high blood pressure. Significance: Loss of SNX1 expression may be a novel mechanism for the development of hypertension. The peripheral dopaminergic system plays a crucial role in blood pressure regulation through its actions on renal hemodynamics and epithelial ion transport. The dopamine D5 receptor (D5R) interacts with sorting nexin 1 (SNX1), a protein involved in receptor retrieval from the trans-Golgi network. In this report, we elucidated the spatial, temporal, and functional significance of this interaction in human renal proximal tubule cells and HEK293 cells stably expressing human D5R and in mice. Silencing of SNX1 expression via RNAi resulted in the failure of D5R to internalize and bind GTP, blunting of the agonist-induced increase in cAMP production and decrease in sodium transport, and up-regulation of angiotensin II receptor expression, of which expression was previously shown to be negatively regulated by D5R. Moreover, siRNA-mediated depletion of renal SNX1 in C57BL/6J and BALB/cJ mice resulted in increased blood pressure and blunted natriuretic response to agonist in salt-loaded BALB/cJ mice. These data demonstrate a crucial role for SNX1 in D5R trafficking and that SNX1 depletion results in D5R dysfunction and thus may represent a novel mechanism for the pathogenesis of essential hypertension.

Dopamine D3 receptor inhibits the ubiquitin-specific peptidase 48 to promote NHE3 degradation

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 NHE3s 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.—Armando, I., Villar, V. A. M., Jones J. E., Lee, H., Wang, X., Asico L. D., Yu, P., Yang, J., Escano, C. S. Jr., Pascua‐Crusan, A. M., Felder, R. A., Jose, P. A. Dopamine D3 receptor inhibits the ubiquitin‐specific peptidase 48 to promote NHE3 degradation. FASEB J. 28, 1422–1434 (2014). www.fasebj.org