Renal Dopamine Research Articles

Gastrin stimulates renal dopamine production by increasing the renal tubular uptake of l-DOPA.

Gastrin is a peptide hormone that is involved in the regulation of sodium balance and blood pressure. Dopamine, which is also involved in the regulation of sodium balance and blood pressure, directly or indirectly interacts with other blood pressure-regulating hormones, including gastrin. This study aimed to determine the mechanisms of the interaction between gastrin and dopamine and tested the hypothesis that gastrin produced in the kidney increases renal dopamine production to keep blood pressure within the normal range. We show that in human and mouse renal proximal tubule cells (hRPTCs and mRPTCs, respectively), gastrin stimulates renal dopamine production by increasing the cellular uptake of l-DOPA via the l-type amino acid transporter (LAT) at the plasma membrane. The uptake of l-DOPA in RPTCs from C57Bl/6J mice is lower than in RPTCs from normotensive humans. l-DOPA uptake in renal cortical slices is also lower in salt-sensitive C57Bl/6J than in salt-resistant BALB/c mice. The deficient renal cortical uptake of l-DOPA in C57Bl/6J mice may be due to decreased LAT-1 activity that is related to its decreased expression at the plasma membrane, relative to BALB/c mice. We also show that renal-selective silencing of Gast by the renal subcapsular injection of Gast siRNA in BALB/c mice decreases renal dopamine production and increases blood pressure. These results highlight the importance of renal gastrin in stimulating renal dopamine production, which may give a new perspective in the prevention and treatment of hypertension.

PP.22.09] RENAL NATRIURETRIC SYSTEMS ALTERATION IN FRUCTOSE-OVERLOADED HYPERTENSIVE RATS

Objective: Metabolic and hemodynamic changes induced by a high fructose diet could be associated with an impairment of renal natriuretic systems, which contributes to sodium retention and hypertension. There is little evidence about renal dopamine (DA) and type-A natriuretic peptide (ANP) interaction on blood pressure elevation in this model. The aim of this study was to evaluate both natriuretic systems in fructose-overloaded rats. Design and method: Male Sprague Dawley rats were randomly assigned: a) Controls (C, tap water to drink) and b) Fructose overloaded: (F, 10% w/v of fructose to drink), treated during 4, 8 and 12 weeks (n = 8 rats/group/period). Urinary L-dopa and DA (HPLC); diuresis, creatinine and sodium were measured in 24 hour urine samples; and ANP (ELISA), triglycerides, glycemia, cholesterol, insulinemia, creatinine and sodium in plasma. Systolic blood pressure (SBP) was determined by tail-cuff and renal tissue protein expression of L-dopa transporter (LAT-2), D1R and NPR-A receptors by Western blot. Results: Fructose overload increased SBP (mmHg, C4: 121 ± 8 vs. F4: 145 ± 1*; C8: 130 ± 4 vs. F8: 161 ± 10#; C12: 133 ± 5 vs. F12: 163 ± 4#), which positively correlated (R2 = 0.78; p < 0.002) to urinary L-dopa/DA index (C4: 0.49 ± 0.05 vs. F4: 1.9 ± 0.09#; C8: 0.53 ± 0.06 vs. F8: 2.35 ± 0.10#; C12: 0.54 ± 0.07 vs. F12: 2.57 ± 0.20#). An elevation in cortex LAT-2 expression (arbitrary units) (C4: 1.00 ± 0.02 vs. F4: 1.30 ± 0.04*; C8: 1.00 ± 0.05 vs. F8: 1.23 ± 0.09*; C12: 1.00 ± 0.06 vs. F12: 1.40 ± 0.01*) and medulla (C8: 1.00 ± 0.01 vs. F8: 1.42 ± 0.02*; C12: 1.00 ± 0.01 vs. F12: 1.46 ± 0.04*); and a decrease D1R in cortex (C4: 1.00 ± 0.05 vs. F4: 0.72 ± 0.08*; C8: 1.00 ± 0.05 vs. F8: 0.75 ± 0.07*) were found. Plasma ANP decrease (ng/ml: C8: 1.61 ± 0.10 vs. F8: 1.30 ± 0.08*; C12: 1.65 ± 0.32 vs. F12: 1.04 ± 0.16*) along with a lower NPR-A expression in medulla (C4: 1.00 ± 0.05 vs. F4: 0.81 ± 0.02*; C8: 1.00 ± 0.04 vs. F8: 0.80 ± 0.03*; C12: 1.00 ± 0.03 vs. F12: 0.81 ± 0.01*) were also observed (*p < 0.05, #p < 0.01 vs. C). Conclusions: Fructose overload leads to an increase in the urinary L-dopa/DA index from 4th week of treatment that correlates positively with blood pressure elevation. Furthermore the decrease in the natriuretic systems DA and ANP seems to be related to the physiopathology of hypertension in this model.

Abstract P337: Angiotensin 1-7 Mitigates Oxidative Stress, Protects Renal Dopamine D1 Receptor Function and Prevents Development of Hypertension

The role of angiotensin in etiology of cardiovascular diseases especially in hypertension is well established. Renin-angiotensin-aldosterone contributes to the development and maintenance of hypertension directly by increases in vascular tone and renal sodium reabsorption or indirectly by increasing oxidative stress and inflammation. Contrary to this pathological arm, angiotensin (Ang) 1-7 via Mas receptors has been reported to protect the cardiovascular function although the exact mechanism is not yet clear. We have previously shown that oxidative stress leads to renal dopamine D1 receptor (D1R) dysfunction which could disrupt sodium regulation and subsequently lead to hypertension. In here we wanted to test whether chronic administration of Ang 1-7 in mice could mitigate oxidative stress, protect renal D1R function and prevent development of hypertension. Mice (C57BL) were implanted with telemetry probes and concomitantly treated with L-buthionine sulfoximine (BSO, in drinking water) and Ang 1-7 (via jugular vein by osmotic pumps). Control (C, no treatment) and shams (implanted with saline filled pumps) exhibited similar behavioral and physiological parameters. Mice treated with BSO alone exhibited increased oxidative stress and high BP as compared to controls. Ang 1-7 treatment did not affect oxidative stress and BP in control mice but prevented the increase in BP and oxidative milieu in BSO treated mice. Mean arterial pressure (mmHg), C: 78.5 ± 2.3*; BSO: 97.3 ± 3.8; Ang 1-7: 80.1* ± 4.1; BSO+Ang 1-7: 83.2 ± 3.4*, *P &lt;0.05 vs BSO. SKF38393, a D1R agonist, increased urine and sodium excretion in control mice but failed to induce diuresis or natriuresis in BSO-treated mice. Treatment with Ang 1-7 protected D1R function as both natriuresis and diuresis was observed in mice treated with BSO plus Ang 1-7. Chronic Ang 1-7 had no effect on D1R function in the absence of BSO. These data show that oxidative stress leads to hypertension by disrupting renal D1R dependent sodium regulation. Ang 1-7 mitigates oxidative stress, protects renal D1R function and prevents increase in BP. This study provides a new insight on how beneficial arm of Ang system could protect renal D1R-mediated sodium regulation and prevent development of hypertension during oxidative stress.

Gastrorenal Axis.

Gastrorenal Axis.

Mycophenolate mofetil and curcumin provide comparable therapeutic benefit in experimental chronic kidney disease: role of Nrf2-Keap1 and renal dopamine pathways

Increased oxidative stress and inflammation have an important role in the pathophysiology of chronic kidney disease (CKD). On the other hand, more affordable therapeutic alternatives for treating this disease are urgently needed. Therefore, we compared the therapeutic efficacy of curcumin and mycophenolate mofetil (MMF) in 5/6 nephrectomy (5/6 Nx) model of CKD. Also, we evaluated whether both compounds provide benefit through the preservation of similar antioxidant mechanisms. Four groups of male Wistar were studied over a period of 4 wk. Control sham group (n= 12), 5/6 Nx (n = 12), 5/6 Nx + MMF (30 mg/k BW/day, n = 11) and 5/6 Nx + Curcumin (120 mg/k BW/day, n = 12). Renal function and markers of oxidative stress and inflammation were evaluated. Also Nrf2-Keap1 and renal dopamine, antioxidant pathways were assessed. 5/6 Nx induced an altered renal autoregulation response, proteinuria, and hypertension; these effects were in association with increased oxidative stress, endothelial dysfunction and renal inflammation. The mechanisms associated with these alterations included a reduced nuclear translocation of Nrf2 and hyperphosphorylation of dopamine D1 receptor with a concurrent overactivation of renal NADPH oxidase. Treatments with MMF and curcumin provided equivalent therapeutic efficacy as both prevented functional renal alterations as well as preserved antioxidant capacity and avoided renal inflammatory infiltration. Moreover, both treatments preserved Nrf2-Keap1 and renal dopamine antioxidant pathways. In summary, therapeutic strategies aimed to preserve renal antioxidant pathways can help to retard the progression of CKD.

The Synergistic Roles of Cholecystokinin B and Dopamine D5 Receptors on the Regulation of Renal Sodium Excretion.

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.

Regulation of Dopamine Uptake by Vasoactive Peptides in the Kidney.

Considering the key role of renal dopamine in tubular sodium handling, we hypothesized that c-type natriuretic peptide (CNP) and Ang-(1-7) may regulate renal dopamine availability in tubular cells, contributing to Na+, K+-ATPase inhibition. Present results show that CNP did not affect either 3H-dopamine uptake in renal tissue or Na+, K+-ATPase activity; meanwhile, Ang-(1-7) was able to increase 3H-dopamine uptake and decreased Na+, K+-ATPase activity in renal cortex. Ang-(1-7) and dopamine together decreased further Na+, K+-ATPase activity showing an additive effect on the sodium pump. In addition, hydrocortisone reversed Ang-(1-7)-dopamine overinhibition on the enzyme, suggesting that this inhibition is closely related to Ang-(1-7) stimulation on renal dopamine uptake. Both anantin and cANP (4-23-amide) did not modify CNP effects on 3H-dopamine uptake by tubular cells. The Mas receptor antagonist, A-779, blocked the increase elicited by Ang-(1-7) on 3H-dopamine uptake. The stimulatory uptake induced by Ang-(1-7) was even more pronounced in the presence of losartan, suggesting an inhibitory effect of Ang-(1-7) on AT1 receptors on 3H-dopamine uptake. By increasing dopamine bioavailability in tubular cells, Ang-(1-7) enhances Na+, K+-ATPase activity inhibition, contributing to its natriuretic and diuretic effects.

Abstract 100: Sorting Nexin 1: Molecular Mechanisms and Pharmacogenomics

Sorting nexin 1 (SNX1) plays a pivotal role for the normal activity of renal dopamine D5 receptor (D5R). Kidney-restricted, Snx1-siRNA depletion of SNX1 results in impaired natriuretic response to salt load and hypertension in mice. Genetic ablation of the Snx1 gene (Snx1-/-) resulted in increased oxidative stress, impaired sodium excretion, and elevated systolic blood pressure (SBP, 131.3±6.4 mm Hg, n=5) in mice. The D5R has antioxidant properties by negatively regulating the expression of the NADPH oxidase (NOX). We found that NOX1, NOX2, and p47phox, as well as the antioxidant PON2, conceivably as compensation, were increased in Snx1-/- mice compared with wild-type littermates. Snx1-/- mice had higher ROS (218.6±7.7%), NOX activity (43.9±3.3 AU/mg protein/min vs. 25.98±3.5), and other markers of oxidative stress, e.g., malonyldialdehyde (32.5±3.4 pmol/mg protein vs. 17.2±2.1) and 3-nitrotyrosine (128.3±4%), which were all normalized by 10-day renal infusion of apocynin, a drug that prevents NOX assembly. The SBP in Snx1-/- mice was also normalized by apocynin (131.3±4.8 mm Hg to 105.7±0.3). Compared with human renal proximal tubule cells obtained from normotensive Caucasian males (NT cells), those from hypertensive subjects (HT cells) had reduced expression of SNX1 (160±2.1%, n=4=5/group), increased ROS (182±10.5%), and blunted cAMP response (110.8±35.3%) and sodium transport inhibition (101.2±1.9%) in response to D1-like receptor stimulation. These observations were corroborated by results in siRNA-induced gene silencing in NT cells or “genetic rescue” in HT cells. We also evaluated 12 SNPs in the SNX1 gene as possible genetic predictors of BP response to monotherapy with HCTZ among hypertensive patients enrolled in the Pharmacogenomic Evaluation of Antihypertensive Responses study (n=768). Three of the 12 SNPs (rs12591947, rs11854249, and rs11635627) associated with poor BP response to thiazide ([[Unable to Display Character: &amp;#8710;]] SBP of -1.8 mm Hg vs. 113.5) among blacks. An SNX1 SNP (rs1802376) was associated with essential hypertension in a Caucasian population (n=502). Our data demonstrate the novelty and relevance of SNX1 in human pathology and pharmacogenomics of essential hypertension.

Abstract P628: Angiotensin 1-7 Mas Receptor and Dopamine D1 receptor Interaction as a Novel Natriuretic Mechanism in Rat Kidneys

Evidence to date suggests that a positive interaction between natriuretic factors promotes sodium excretion to maintain sodium homeostasis and blood pressure. Although the involvement of renal dopamine D1 receptor (D1R) in promoting sodium excretion is well established; the role of Angiotensin (Ang) 1-7 Mas receptor (MasR) is not clear. Here we provide evidence for a functional interaction between these two renal G protein-coupled receptors which suggests that natriuretic response to Ang 1-7 via MasR is dependent on D1R activation. Male Sprague Dawley rats of comparable weight and age were infused with Ang 1-7, MasR antagonist DPro, D1R agonist SKF38393 and D1R antagonist SCH23390. Blood pressure was monitored throughout the experimental procedure and none of the infused drugs affected the pressure. Animals infused with saline alone served as controls. Infusion of Ang1-7 caused significant natriuresis and robust diuresis compared to saline. SKF38393 infusion also induced significant natriuresis and diuresis when compared to saline infusion. Both natriuretic and diuretic response to Ang 1-7 was blocked by Dpro and SCH23390. However, Dpro failed to block SKF38393 response. Concomitant infusion of SKF38393 and Ang 1-7 did not show a cumulative natriuretic or diuretic effect when compared to SKF38393 or Ang 1-7 infusion alone. FENa (%), control (saline): 0.30 ± 0.09; Ang 1-7: 1.03 ± 0.21; Ang 1-7 plus Dpro: 0.49 ± 0.11; Ang 1-7 plus SCH23390: 0.36 ± 0.10; SKF38393: 0.83 ± 0.16; SKF38393 plus SCH23390: 0.41 ± 0.09; SKF38393 plus Dpro: 0.82 ± 0.17; Ang 1-7 plus SKF38393: 1.06 ± 0.21. These data suggest that Ang 1-7 via MasR causes natriuresis which is dependent on D1R activation. On the other hand, renal D1R-mediated sodium excretion is independent of MasR. This study is a paradigm shift as these data identify a novel functional unidirectional interaction between renal MasR and D1R which deviates from commonly known receptor-receptor interaction.

PP.28.13

Objective: Oxidative stress plays an important role in the development of hypertension. It is reported that renal dopamine D1 receptor (D1R), a major contributor to sodium homeostasis and BP regulation, is negatively regulated by oxidative stress. Antioxidants can reduce BP and redox-sensitive transcription factor Nrf2 could provide important clues for antihypertensive properties of antioxidants. We investigated the signaling molecules responsible for redox-sensitive renal proximal tubular D1R dysfunction and the role of renal specific Nrf2 in preserving D1R function. Design and method: Wild type (WT) and renal proximal tubular specific Nrf2 knockout (KO) mice were treated with 10 mM L-buthionine-sulfoximine (BSO, a pro-oxidant) without and with sulforaphane (S, an antioxidant; 5 μM daily by gavage) for 5 weeks. Human kidney proximal tubular (HK2) cells were also treated with BSO with and without sulforaphane. Results: In WT mice, BSO treatment increased oxidative stress and BP, reduced renal tubular D1R expression and attenuated SKF38393 (a D1R agonist)-induced Na/K-ATPase inhibition and sodium excretion. BSO also activated transcription factors activator protein (AP) 1 and SP3. However, the onset and severity of hypertension as well as oxidative stress, activation of AP1 and SP3 and D1R dysfunction were more robust in BSO-treated Nrf2 KO compared to WT mice. Also, sulforaphane activated Nrf2, reduced oxidative stress, normalized AP1 and SP3 activation, rescued DIR function and lowered BP in BSO-treated WT mice but not in Nrf2 KO mice. BSO also caused oxidative stress and D1R dysfunction in HK2 cells. BSO-treated HK2 cells exhibited increased AP1 (c-fos) and SP3 nuclear expression. Sulforaphane per se had no effect on oxidative milieu or D1R expression, but it mitigated BSO-induced oxidative stress, AP1—SP3 up-regulation and D1R down-regulation and dysfunction. Down-regulation of SP3 had no effect on BSO-mediated AP1 activation, however, AP1 siRNA blocked BSO-dependent SP3 up-regulation. In cells transfected with Nrf2 siRNA, sulforaphane failed to block BSO-induced AP1—SP3 activation and D1R dysfunction. Conclusions: Oxidative stress via AP1-SP3 pathway down-regulates D1R expression and function which leads to sodium retention and hypertension. Antioxidants, via Nrf2 activation, reduce oxidative stress, normalize AP1-SP3 signaling, protect renal D1R function and prevent development of hypertension.

Defective renal dopamine function and sodium-sensitive hypertension in adult ovariectomized Wistar rats: role of the cytochrome P-450 pathway.

We have previously shown that ovariectomy in adult Wistar rats under normal sodium (NS) intake results in an overexpression of the total Na(+)-K(+)-ATPase (NKA) α1-subunit (Di Ciano LA, Azurmendi PJ, Toledo JE, Oddo EM, Zotta E, Ochoa F, Arrizurieta EE, Ibarra FR. Clin Exp Hypertens 35: 475-483, 2013). Upon high sodium (HS) intake, ovariectomized (oVx) rats developed defective NKA phosphorylation, a decrease in sodium excretion, and an increment in mean blood pressure (MBP). Since NKA phosphorylation is modulated by dopamine (DA), the aim of this study was to compare the intracellular response of the renal DA system leading to NKA phosphorylation upon sodium challenge in intact female (IF) and oVx rats. In IF rats, HS caused an increase in urinary DA and sodium, in NKA phosphorylation state, in cytochrome P-4504A (CYP4A) expression, and in 20-HETE production, while MBP kept normal. Blockade of the D1 receptor (D1R) with the D1-like receptor antagonist SCH 23390 in IFHS rats shifted NKA into a more dephosphorylated state, decreased sodium excretion by 50%, and increased MBP. In oVxNS rats, D1R expression was reduced and D3R expression was increased, and under HS intake sodium excretion was lower and MBP higher than in IFHS rats (both P < 0.05), NKA was more dephosphorylated than in IFHS, and CYP4A expression or 20-HETE production did not change. Blockade of D1R in oVxHS rats changed neither NKA phosphorylation state nor sodium excretion or MBP. D2R and PKCα expression did not vary among groups. The alteration of the renal DA system produced by ovariectomy could account for the defective NKA phosphorylation, the inefficient excretion of sodium load, and the development of salt-sensitive hypertension.

Role of nuclear factor erythroid 2-related factor 2 in the oxidative stress-dependent hypertension associated with the depletion of DJ-1.

Renal dopamine 2 receptor dysfunction is associated with oxidative stress and high blood pressure (BP). We have reported that DJ-1, an oxidative stress response protein, is positively regulated by dopamine 2 receptor in the kidney. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of several antioxidant genes. We tested the hypothesis that Nrf2 is involved in the renal DJ-1-mediated inhibition of reactive oxygen species production. We have reported that silencing dopamine 2 receptor in mouse renal proximal tubule cells decreases the expression of DJ-1. We now report that silencing DJ-1 or dopamine 2 receptor in mouse proximal tubule cells and mouse kidneys decreases Nrf2 expression and activity and increases reactive oxygen species production; BP is also increased in mice in which renal DJ-1 or dopamine 2 receptor is silenced. DJ-1(-/-) mice have decreased renal Nrf2 expression and activity and increased nitro-tyrosine levels and BP. Silencing Nrf2 in mouse proximal tubule cells does not alter the expression of DJ-1 or dopamine 2 receptor, indicating that Nrf2 is downstream of dopamine 2 receptor and DJ-1. An Nrf2 inducer, bardoxolone, normalizes the systolic BP and renal malondialdehyde levels in DJ-1(-/-) mice without affecting them in their wild-type littermates. Because Nrf2 ubiquitination is increased in DJ-1(-/-) mice, we conclude that the protective effect of DJ-1 on renal oxidative stress is mediated, in part, by preventing Nrf2 degradation. Moreover, renal dopamine 2 receptor and DJ-1 are necessary for normal Nrf2 activity to keep a normal redox balance and BP.

Transcriptional regulation of renal dopamine D1 receptor function during oxidative stress.

There exists a strong link between oxidative stress, renal dopaminergic system, and hypertension. It is reported that reactive oxygen species attenuate renal proximal tubular dopamine receptor (D1R) function, which disrupts sodium regulation and leads to hypertension. However, the mechanisms for renal D1R dysfunction are not clear. We investigated the role of redox-sensitive transcription factors AP1 and SP3 in transcriptional suppression of D1R gene and subsequent D1R signaling. Human kidney proximal tubular cells were treated with a pro-oxidant l-buthionine sulfoximine (BSO) with and without an antioxidant tempol. In human kidney cells, BSO caused oxidative stress and reduced D1R mRNA and membrane receptor expression. Incubation of human kidney cells with SKF38393, a D1R agonist, caused a concentration-dependent inhibition of Na/K-ATPase. However, SKF38393 failed to inhibit Na/K-ATPase in BSO-treated cells. BSO increased AP1 and SP3 nuclear expression. Transfection with AP1- or SP3-specific siRNA abolished BSO-induced D1R downregulation. Treatment of rats with BSO for 4 weeks increased oxidative stress and SP3-AP1 expression and reduced D1R numbers in renal proximal tubules. These rats exhibited high blood pressure, and SKF38393 failed to inhibit proximal tubular Na/K-ATPase activity. Control rats were kept on tap water. Tempol per se had no effect on D1R expression or other signaling molecules but prevented BSO-induced oxidative stress, SP3-AP1 upregulation, and D1R dysfunction in both human kidney cells and rats. These data show that oxidative stress via AP1-SP3 activation suppresses D1R transcription and function. Tempol mitigates oxidative stress, blocks AP1-SP3 activation, and prevents D1R dysfunction and hypertension.

Renal dopaminergic system: Pathophysiological implications and clinical perspectives

Fluid homeostasis, blood pressure and redox balance in the kidney are regulated by an intricate interaction between local and systemic anti-natriuretic and natriuretic systems. Intrarenal dopamine plays a central role on this interactive network. By activating specific receptors, dopamine promotes sodium excretion and stimulates anti-oxidant and anti-inflammatory pathways. Different pathological scenarios where renal sodium excretion is dysregulated, as in nephrotic syndrome, hypertension and renal inflammation, can be associated with impaired action of renal dopamine including alteration in biosynthesis, dopamine receptor expression and signal transduction. Given its properties on the regulation of renal blood flow and sodium excretion, exogenous dopamine has been postulated as a potential therapeutic strategy to prevent renal failure in critically ill patients. The aim of this review is to update and discuss on the most recent findings about renal dopaminergic system and its role in several diseases involving the kidneys and the potential use of dopamine as a nephroprotective agent.

ASSA14-03-11 Role of GRK4γ variant 142V in the regulation of renal ETB receptor in hypertension

Background The endothelin receptor B (ETBR) regulates blood pressure and water and electrolyte balance by engendenring natriuresis. In hypertensive states, the ETBR mediated diureis and natriuresis is impaired. However, the underlying mechanisms are not clear. G protein-coupled receptor kinase 4 (GRK4), whose gene locus 4p16.3 is linked to essential hypertension, cause sodium retention and increase blood pressure via impairment of renal dopamine receptor and enhancement of renin-angiotensin system functions. Due to the higher activity of GRK4 in kidney from spontaneously hypertensive rats (SHRs) and hypertensive patients, we hypothesise that GRK4 might be the cause of ETBR impairment in hypertension. Method Experiments were carried out in male anaesthetic spontaneously hypertensive rats (SHR)and in normotensive Wistar-Kyoto (WKY) rats. The ETB receptor agonist, BQ-3020 (0.1, 0.5, 1.0 ug/kg/min) were infused via supra-renal artery at a rate of 0.04 ml/min for 40 min. The same experiments were conducted in GRK4 A142V and GRK4 Wild Type transgenic mice. The ETBR function were also checked in the wild-type and A142V transfected renal proximal tubule (RPT) cells from mice. Results We found that diuresis and natriuresis of ETBR agonist, BQ3020, in Wistar-Kyoto (WKY) rats, which was impaired in SHRs. The GRK4 expression was higher in renal cortex from SHRs as compared with WKY rats. In GRK4 A142V transgenic mice, it resulted that ETBR-mediated diuresis and natriuresis was impaired compared with Wild type. In wild-type transfected cells, activation of ETBR inhibited Na + -K + -ATPase activity; while in A142V transfected cells, the inhibitory effect was lost. There are co-localization and co-immunoprecipitation between ETBR and GRK4 in RPT cells. The linkage of ETBR/GRK4 was higher in wilde-type cells than in A142V cells. Similar phenomenon was found in the kidney from WKY and SHRs, SHRs had higher ETBR/GRK4 linkage, accompanied with higher ETBR phosphorylation, which might account for the impaired ETBR function in hypertension. Conclusion This study provides a mechanism by which GRK4, via regulation of renal ETBR function, participates in the pathogenesis of hypertension.

Renalase regulates peripheral and central dopaminergic activities.

Renalase is a recently identified FAD/NADH-dependent amine oxidase mainly expressed in kidney that is secreted into blood and urine where it was suggested to metabolize catecholamines. The present study evaluated central and peripheral dopaminergic activities in the renalase knockout (KO) mouse model and examined the changes induced by recombinant renalase (RR) administration on plasma and urine catecholamine levels. Compared with wild-type (WT) mice, KO mice presented increased plasma levels of epinephrine (Epi), norepinephrine (NE), and dopamine (DA) that were accompanied by increases in the urinary excretion of Epi, NE, DA. In addition, the KO mice presented an increase in urinary DA-to-l-3,4-dihydroxyphenylalanine (l-DOPA) ratios without changes in renal tubular aromatic-l-amino acid decarboxylase (AADC) activity. By contrast, the in vivo administration of RR (1.5 mg/kg sc) to KO mice was accompanied by significant decreases in plasma levels of Epi, DA, and l-DOPA as well as in urinary excretion of Epi, DA, and DA-to-l-DOPA ratios notwithstanding the accompanied increase in renal AADC activity. In addition, the increase in renal DA output observed in renalase KO mice was accompanied by an increase in the expression of the L-type amino acid transporter like (LAT) 1 that is reversed by the administration of RR in these animals. These results suggest that the overexpression of LAT1 in the renal cortex of the renalase KO mice might contribute to the enhanced l-DOPA availability/uptake and consequently to the activation of the renal dopaminergic system in the presence of renalase deficiency.

Prenatal lipopolysaccharide exposure results in dysfunction of the renal dopamine D1 receptor in offspring

Adverse environment in early life can modulate the adult phenotype, including blood pressure. Lipopolysaccharide (LPS) exposure in utero results in increased blood pressure in the offspring, but the exact mechanisms are not clear. Studies have shown that the renal dopamine D1 receptor (D1R) plays an important role in maintaining sodium homeostasis and normal blood pressure; dysfunction of D1R is associated with oxidative stress and hypertension. In this study, we determined if dysfunction of the renal D1R is involved in fetal-programmed hypertension, and if oxidative stress contributes to this process. Pregnant Sprague–Dawley (SD) rats were intraperitoneally injected with LPS (0.79mg/kg) or saline at gestation days 8, 10, and 12. As compared with saline-injected (control) dams, offspring of LPS-treated dams had increased blood pressure, decreased renal sodium excretion, and increased markers of oxidative stress. In addition, offspring of LPS-treated dams had decreased renal D1R expression, increased D1R phosphorylation, and G protein-coupled receptor kinase type 2 (GRK2) and type 4 (GRK4) protein expression, and impaired D1R-mediated natriuresis and diuresis. All of the findings in the offspring of LPS-treated dams were normalized after treatment with TEMPOL, an oxygen free radical scavenger. In conclusion, prenatal LPS exposure, via an increase in oxidative stress, impairs renal D1R function and leads to hypertension in the offspring. Normalization of renal D1R function by amelioration of oxidative stress may be a therapeutic target of fetal programming of hypertension.

Abstract 572: Transcriptional Regulation Of Renal Dopamine D1 Receptor Function During Oxidative Stress

There exists a strong link between Oxidative stress, renal dopaminergic system and hypertension. It is reported that reactive oxygen species attenuate renal proximal tubular dopamine receptor (D1R) function which disrupts sodium regulation and leads to hypertension. The mechanisms for renal D1R dysfunction, however are not clear. Here in, we investigated the role of redox sensitive transcription factors AP1 and SP3 in transcriptional suppression of D1R gene function and subsequent D1R signaling. Human kidney proximal tubular ( HK2 ) cell were treated with a pro-oxidant L-buthionine sulfoximine ( BSO , 50 mM for 72 hr) with and without a polyphenol resveratrol (250 μM). Treatment of HK2 cells with BSO caused oxidative stress (malondialdehyde [MDA], nmol/mg protein: control: 0.13 ± 0.01, BSO: 0.20 ± .02) and significantly reduced D1R mRNA and membrane receptor number (fmol/mg protein; control—140.1 ± 5.6, BSO—81.7 ± 4.4). Incubation of HK2 cells with SKF38393, a D1R agonist, caused a concentration dependent inhibition of Na/K-ATPase ( 86 Rb uptake, nmol/mg protein, control: basal—9.2 ± 0.2, SKF (1 μM)—6.3 ± 0.1). However, SKF38393 failed to inhibit Na/K-ATPase in BSO-treated cells (BSO: basal—9.5 ± 0.3, SKF—8.6 ± 0.2). Cells incubated with BSO exhibited increased AP1 (c-fos) and SP3 nuclear expression. Resveratrol per se had no effect on oxidative milieu (MDA: 0.11 ± 0.01) or D1R expression (143.3 ± 4.4), but it mitigated BSO-induced oxidative stress (MDA: 0.13 ± 0.02), AP1—SP3 up-regulation and D1R down-regulation (133.6 ± 6.2) and dysfunction (resveratrol + BSO [ 86 Rb uptake]: basal—9.7 ±0.3, SKF—6.7 ± 0.4). Incubation of cells with AP1 or SP3 specific siRNA reduced the expression of respective protein by ~85% and abolished BSO-induced D1R dysfunction. Down-regulation of SP3 had no effect on BSO-mediated AP1 activation, however, AP1 siRNA blocked BSO-dependent SP3 up-regulation. In cells transfected with Nrf2 siRNA, resveratrol failed to block BSO-induced AP1—SP3 activation and D1R dysfunction. These data shows that oxidative stress via AP1—SP3 activation suppresses D1R transcription which leads to D1R dysfunction. Resveratrol activates Nrf2, mitigates oxidative stress, blocks AP1—SP3 signaling and prevents D1R down-regulation and dysfunction.

Abstract 573: Uncovering the Molecular Mechanisms Underlying the Hypertension in Snx1 Knockout Mice

We have reported that sorting nexin 1 (SNX1) is crucial for renal dopamine D 5 receptor (D 5 R) trafficking, signal transduction, and function in human renal epithelial cells and in C57Bl/6J and BALB/cJ mice, as shown by the development of hypertension and impaired natriuretic response to agonist stimulation after an acute SNX1 depletion in the kidney. Thus, we elucidated the renal molecular mechanisms for these phenotypes in Snx1 -/- mice, which have congenital absence of SNX1. These mice have increased expression of glycosylated AT 1 R (123.8±2.1 vs . 100±2.0% in wild-type littermates, P&lt;0.05, Student’s t -test, n=5/group), a receptor with pro-oxidant and hypertensinogenic effects. We next determined the expression profiles of the components of the NADPH oxidase (NOX), an enzyme complex that is a major source of reactive oxygen species (ROS). We found an increased expression of renal NOX1 (153.4±12.2% vs . 100±4.1%, P&lt;0.05), NOX2 or gp91 phox (129.9±5.5% vs . 100±7.7%, P&lt;0.05), and p47 phox (118.2±2.7% vs . 100±5.0%, P&lt;0.05), suggesting increased oxidative stress in these mice. Interestingly, the Snx1 null mice have elevated renal D 5 R (142.9±4.7% vs . 100±6.8%, P&lt;0.05) and D 3 R (134.3±5.3% vs . 100 ± 1.6%, P&lt;0.05), receptors with anti-oxidant activity, as well as the antioxidant paraoxonase 2, perhaps as compensatory mechanisms; the loss of SNX1 impairs the function of D 5 R. To corroborate our findings, we treated the Snx1 -/- mice and controls with a 10-day renal infusion of apocynin, a drug that blocks NOX assembly by preventing p47 phox translocation to NOX2. Apocynin treatment resulted in the amelioration of systolic blood pressure (SBP) in Snx1 -/- mice (131.3±4.8 mm Hg to 105.7±1 mm Hg, P&lt;0.05). There was no difference in the SBP with vehicle treatment in both strains, or with apocynin in control mice. Basal NOX activity was higher in Snx1 -/- mice (169±12.8 units/mg protein/min vs . 100±13.3 in controls, P&lt;0.05), which was normalized by apocynin (99.4±16.5), while basal ROS levels were 2-fold higher in the Snx1 -/- mice (218.6±7.7 units/mg protein vs . 100±17.9, P&lt;0.05), which was also normalized by apocynin (125.8±20.4). Our data indicate that the hypertension in Snx1 -/- mice is due to impaired D 5 R activity, higher NOX expression and activity, and increased AT 1 R.

Abstract 349: Gastrin and D1 Dopamine Receptor Interact to Induce Natriuresis and Diuresis

Oral NaCl produces a greater natriuresis and diuresis than the intravenous infusion of the same amount of NaCl. Gastrin is the major gastrointestinal hormone taken up by renal proximal tubule (RPT) cells. We hypothesized that renal gastrin and dopamine receptors interact to synergistically increase sodium excretion, an impaired interaction of which may be involved in the pathogenesis of hypertension. In Wistar-Kyoto (WKY) rats, infusion of gastrin induced natriuresis and diuresis, which was abrogated in the presence of a gastrin (CCK B R; CI-988) or D 1 -like receptor antagonist (SCH23390). Similarly, the natriuretic and diuretic effects of fenoldopam, a D 1 -like receptor agonist, were blocked by SCH23390, as well as by CI-988. However, the natriuretic effects of gastrin and fenoldopam were not observed in spontaneously hypertensive rats (SHRs). The gastrin/D 1 -like receptor interaction was also confirmed in RPT cells. In RPT cells from WKY but not SHRs, stimulation of either D 1 -like or gastrin receptor inhibited Na + -K + -ATPase activity, an effect that was blocked in the presence of SCH23390 or CI-988. In RPT cells from WKY and SHRs, CCK B R and D 1 receptor (D 1 R) co-immunoprecipitated, which was increased after stimulation of either D 1 R or CCK B R in RPT cells from WKY rats; stimulation of one receptor increased RPT cell membrane expression of the other receptor, effects that were not observed in SHRs. These data suggest that there is a synergism between CCK B R and D 1 -like receptors to increase sodium excretion. An aberrant interaction between the renal CCK B R and D 1 -like receptors (e.g., D 1 R) may play a role in the pathogenesis of hypertension.