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

Inhibitory effect of ETB receptor on Na\u3csup\u3e+\u3c/sup\u3e-K\u3csup\u3e+\u3c/sup\u3e ATPase activity by extracellular Ca\u3csup\u3e2+\u3c/sup\u3e entry and Ca\u3csup\u3e2+\u3c/sup\u3e 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\u3c0.01). 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 × 10-3M15 min-1), Ca2+ channel blocker (10-6M15 min-1 nicardipine) and PI3 kinase inhibitor (10-7M per wortmannin). An inositol 1,4,5-trisphosphate (IP3) receptor blocker (2-aminoethyl diphenyl borate; 10-4M15 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\u3c0.01). The calcium channel agonist (BAY-K8644; 10-6M15 min-1) inhibited Na+-K+ ATPase activity, an effect that was blocked by a phosphatidylinositol-3 kinase inhibitor (10-7M15 min-1 wortmannin). In rat RPT cells, activation of the ETB receptor inhibits Na+ -K+ ATPase activity by facilitating extracellular Ca2+ entry and Ca2+ release from endoplasmic reticulum

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 NHE3\u27s half-life (USP48 siRNA t1/2=6.1 h vs. vehicle t 1/2=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 kidneyrestricted 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 D 3R and USP48 in the regulation of NHE3 expression and function. © FASEB

Sorting nexin 1 loss results in D\u3csub\u3e5\u3c/sub\u3e dopamine receptor dysfunction in human renal proximal tubule cells and hypertension in mice

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. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc

Expression of chemokines/cytokines in renal cortex and urine of D₂−/− mice. A

<p>. Expression of Ltα, MCP-2, and NFkB1 mRNA was quantified by qRT-PCR; results were corrected for expression of GAPDH mRNA and expressed as fold change in comparison to their expression in D₂+/+ mice. *P<0.03 vs. D₂+/+ mice. <b>B</b>. Protein expression of MCP-1 (17 kDa) and TNFα protein (25 kDa) was semi-quantified by immunoblotting. Inset shows one set of immunoblots. Results were corrected for expression of actin and expressed as percentage of the expression in D₂+/+ mice, *P<0.02 vs. D₂+/+ mice, n = 5/group. <b>C.</b> Protein expression of IL-6 (25 kDa) and IL-10 (20 kDa) protein semi-quantified by immunoblotting. Results were corrected for expression of actin and expressed as percentage of the expression in D₂+/+, <b>*</b> P<0.05 vs. D₂+/+ mice, n = 5/group Urinary excretion of IL-6 and IL-10 was quantified by ELISA. *P<0.02 vs. D₂+/+ mice, n = 5/group.</p

Effect of selective renal silencing of D₂R in the remaining kidney of uni-nephrectomized mice on blood pressure and expression on inflammatory factors in the kidney and liver.

<p>Renal cortical <i>Drd2</i> was silenced by the renal subcapsular infusion for seven days of <i>Drd2</i> siRNA, via an osmotic minipump in uni-nephrectomized adult male C57BL/6J mice (see Methods). A. Expression of D₂R protein (55 kDa band) in renal cortex and liver was semi-quantified by immunoblotting. Results were corrected for GAPDH and expressed as % of non-silencing siRNA treated kidneys. * P<0.05 vs non-silencing (NS) siRNA; n = 5/group. B. Systolic blood pressure measured under anesthesia in mice before and seven days after <i>Drd2</i> siRNA infusion. * P<0.05 vs, NS siRNA; n = 5/group. C. Renal cortical expression of TNFα, Ltα, NFkB1, MCP-1, MCP-2, IL-10, IL-11 osteopontin, and Col 1α1 mRNA was quantified by qRT-PCR, results corrected for expression of GAPDH mRNA, and expressed as fold change in comparison to their expression in mice treated with NS siRNA. *P<0.05 vs. NS; n = 5/group.</p

Renal inflammation and injury in D₂−/− mice.

<p>Masson stained sections of D₂+/+ mouse kidney (<b>A</b> and <b>B</b>) and D₂−/− mouse kidney (<b>C</b> and <b>D</b>). H-E stained sections of D₂+/+ mouse kidney (<b>E</b>) and D₂−/− mouse kidney (<b>F</b>). G: glomerulus. PCT: proximal convoluted tubule. Proteinaceous casts are marked with arrows (<b>F</b>). Sections from 3 mouse kidneys per group were studied. <b>G</b> and <b>H</b>: Inflammatory cell infiltration. Kidney sections from D₂+/+ (<b>G</b>) and D₂−/− (<b>H</b>) mice were immunostained for the presence of macrophages and monocytes (arrows). The number of positive cells in 10 randomly selected fields was greater in D2−/− (68±3) than in D2+/+ (15±1, P<0.01) mice. Sections from 3 mouse kidneys per group were studied. <b>I</b>. Renal cortical expression of Col 1α1 mRNA determined by qRT-PCR. Results were corrected for expression of GAPDH mRNA and expressed as fold change in comparison to their expression in D₂+/+ mice. *P<0.05 vs D₂+/+; n = 5/group. <b>J</b>. Urinary microalbuminuria. Urine samples were collected for 24 h from mice in metabolic cages. Albumin was measured by ELISA. *P<0.04 vs. D₂+/+; n = 5/group. Magnification: A and C: 100X; B, D, G and H: 400X; E-F: 200X.</p

D₂R function in moue renal proximal tubule cells A.

<p>Effect of silencing of D₂R on the expression of pro-inflammatory cytokines/chemokines in mouse RPTCs. Cells were cultured to 60–70% confluence and transfected with non-silencing (NS siRNA) or <i>Drd2</i> siRNA. After 48 h the cells were washed and lysed. Protein expression of D₂R (55 kDa), TNFα (25 kDa), and MCP-1(17 kDa) was semi-quantified by immunoblotting. Inset shows one set of immunoblots. NFkB activation was analyzed via the transient expression of a NFkB-luciferase reporter system by reverse transfection Results are expressed as percentage of NS siRNA or fold activation compared to NS siRNA. *P<0.05 vs. NS (non-silencing) siRNA, n = 4/group. <b>B</b>. Effects of Ang II and D₂R stimulation on TNFα and MCP-1 in mouse RPTCs. Cells were serum starved for 2 h before treatment for 24 h in serum-free medium with vehicle (PBS) or 100 nM Ang II, in the presence or absence of 1 μM quinpirole (D₂R/D₃R agonist) or 1 μM quinpirole plus 1 μM L-741,262 (D₂R antagonist). Expression of TNFα (25 kDa) and MCP-1 (17 kDa) protein was semi-quantified by immunoblotting. Inset shows one set of immunoblots. Results were corrected for actin and expressed as % of vehicle. * P<0.05 vs. vehicle; n = 6/group.</p

Effect of selective renal silencing of D₂R in one kidney of mice without uni-nephrectomy on blood pressure and expression of inflammatory factors in the kidney and liver.

<p>Renal cortical D₂R was silenced by the renal subcapsular infusion in the left kidney for seven days of <i>Drd2</i> siRNA, via an osmotic minipump in adult male C57BL/6J mice (see Methods). A. Expression of D₂R protein (55 kDa band) in renal cortex and liver was semi-quantified by immunoblotting. Results were corrected for GAPDH and expressed as % of NS siRNA treated kidneys. * P<0.05 vs non-silencing NS siRNA; n = 4/group. B. Systolic blood pressure measured under anesthesia in mice before and seven days after <i>Drd2</i> siRNA infusion; n = 5/group. C. Renal cortical expression of TNFα, Ltα, NFkB1, MCP-1, MCP-2, IL-10, IL-11, osteopontin and collagen 1α1 mRNA was quantified by qRT-PCR, results corrected for expression of GAPDH mRNA, and expressed as fold change in comparison to their expression in mice treated with NS siRNA. *P<0.05 vs. NS; n = 5/group.</p

Effect of apocynin on renal cortical expression of TNFα, MCP-1, and IL-6, and urinary excretion of IL-6.

<p> Expression of TNFα (25 kDa) and MCP-1 (17 kDa) protein in renal cortex was semi-quantified by immunoblotting. Inset shows one set of immunoblots. Results were corrected for expression of GAPDH and expressed as percentage of D₂+/+ mice treated with vehicle, *P<0.05 vs. vehicle or apocynin treated D₂+/+; n = 5/group. Renal expression, semi-quantified by immunoblotting (25 kDa), and urinary excretion of IL-6 quantified by ELISA in 24 h urine samples. Results are expressed as percentage of D₂+/+ mice treated with vehicle. *P<0.05 vs. vehicle or apocynin treated D₂+/+; n = 5/group.</p