Acute renal depletion of sorting nexin 1 (SNX1) in mice results in blunted natriuretic response and hypertension due to impaired dopamine D-5 receptor (D5R) activity. We elucidated the molecular mechanisms for these phenotypes in Snx1(-/-) mice. These mice had increased renal expressions of angiotensin II type 1 receptor (AT(1)R), NADPH oxidase (NOX) subunits, D5R, and NaCl cotransporter. Basal reactive oxygen species (ROS), NOX activity, and blood pressure (BP) were also higher in Snx1(-/-) mice, which were normalized by apocynin, a drug that prevents NOX assembly. Renal proximal tubule (RPT) cells from hypertensive (HT) Euro-American males had deficient SNX1 activity, impaired D5R endocytosis, and increased ROS compared with cells from normotensive (NT) Euro-American males. siRNA-mediated depletion of SNX1 in RPT cells from NT subjects led to a blunting of D5R agonist-induced increase in cAMP production and decrease in Na+ transport, effects that were normalized by over-expression of SNX1. Among HT African-Americans, three of the 12 single nucleotide polymorphisms interrogated for the SNX1 gene were associated with a decrease in systolic BP in response to hydrochlorothiazide (HCTZ). The results illustrate a new paradigm for the development of hypertension and imply that the trafficking protein SNX1 may be a crucial determinant for hypertension and response to antihypertensive therapy.

Abnormalities of the D2R gene (DRD2) play a role in the pathogenesis of human essential hypertension; variants of the DRD2 have been reported to be associated with hypertension. Disruption of Drd2 (D2−/−) in mice increases blood pressure. The hypertension of D2−/− mice has been related, in part, to increased sympathetic activity, renal oxidative stress, and renal endothelin B receptor (ETBR) expression. We tested in D2−/− mice the effect of etamicastat, a reversible peripheral inhibitor of dopamine-β-hydroxylase that reduces the biosynthesis of norepinephrine from dopamine and decreases sympathetic nerve activity. Blood pressure was measured in anesthetized D2−/− mice treated with etamicastat by gavage, (10 mg/kg), conscious D2−/− mice, and D2+/+ littermates, and mice with the D2R selectively silenced in the kidney, treated with etamicastat in the drinking water (10 mg/kg per day). Tissue and urinary catecholamines and renal expression of selected G protein-coupled receptors, enzymes related to the production of reactive oxygen species, and sodium transporters were also measured. Etamicastat decreased blood pressure both in anesthetized and conscious D2−/− mice and mice with renal-selective silencing of D2R to levels similar or close to those measured in D2+/+ littermates. Etamicastat decreased cardiac and renal norepinephrine and increased cardiac and urinary dopamine levels in D2−/− mice. It also normalized the increased renal protein expressions of ETBR, NADPH oxidase isoenzymes, and urinary 8-isoprostane, as well as renal NHE3 and NCC, and increased the renal expression of D1R but not D5R in D2−/− mice. In conclusion, etamicastat is effective in normalizing the increased blood pressure and some of the abnormal renal biochemical alterations of D2−/− mice.

Overactivity of the sympathetic nervous system is one of the mechanisms involved in the pathogenesis of human essential hypertension. However, treating hypertension by inhibiting the sympathetic nervous system is associated with adverse side effects. Reducing the biosynthesis of norepinephrine (NE) by inhibiting the enzyme that catalyzes the conversion of dopamine to NE, dopamine β‐hydroxylase (DβH), in the peripheral but not the central sympathetic nervous system may be a good drug to treat hypertension; BIA5‐1058 is such a drug. Therefore, we used BIA5‐1058 to treat the hypertension of human GRK4γ 142V transgenic (142V) mice. 142V mice and non‐transgenic (NT) littermates (7 mo old, n=4/group) were gavaged (10:00 AM) with BIA5‐1058 (30 mg/kg) or vehicle, daily for 1 week. The mice were placed in metabolic cages for 24 hr urine collection before blood pressure (BP), under anesthesia, was measured, via the femoral artery on day 7. In additional studies, BP was also monitored by telemetry in conscious mice. Food and water intake, body weight, heart rate, urine flow and sodium and serum Na + , K + , and Cl − were similar in the 4 groups. Compared with NT mice, 142V mice had increased systolic (142v mice: 120±1.5 vs. NT mice: 98.8±2.7, mm Hg) and diastolic BPs (94.8±2.7 vs. 66.5±5.1) that were normalized by BIA5‐1058 (SBP: 97±2 vs. 97.3±0.6; DBP: 70.3±4.8 vs. 70.8±2.5). In conscious mice, 30 mg/kg but not 3 and 10 mg/kg BIA5‐1058, also normalized the BPs of 142V mice. Renal protein abundance of type 1 angiotensin II receptor (AT 1 R) was greater in 142V than NT mice (265±56 vs. 100±18%), similar to our previous report. BIA5‐1058 also decreased renal AT 1 R protein in 142V but not NT mice (27±6 vs. 100±14%). Our data suggest that inhibition of DβH normalizes the BP of 142V mice, not only by decreasing the synthesis of NE but also renal AT 1 R protein expression. Support or Funding Information P01HL074940 P01HL068686 R01HL092196 R37HL023081 R01DK039308

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.

Bacterial endotoxin lipopolysaccharide (LPS) activates inflammatory pathways, induces cytokine expression in the endothelium, augments reactive oxygen species (ROS) production in the vascular wall, and induces endothelial dysfunction. The aim of the present study was to analyze the effects of peroxisome proliferator-activated receptor (PPAR)β/δ activation on LPS-induced inflammation, oxidative stress and endothelial dysfunction and to determine whether uncoupling protein-2 (UCP2) plays a role in these effects. In vivo, the PPARβ/δ agonist GW0742 treatment prevented the LPS-induced reduction in aortic relaxation, the increase in vascular ROS production, the upregulation of NOX1, NOX2, p47phox, and p22phox mRNA levels, and the endoplasmic reticulum (ER) stress markers in mice. We show that in mouse aortic endothelial cells (MAECs), GW0742 prevented the decreased A23187-stimulated nitric oxide (NO) production, and the increased intracellular ROS levels caused by exposure to LPS in vitro. The PPARβ/δ antagonist GSK0660 abolished all these in vivo and in vitro protective effects induced by GW0742. This agonist also restored the reduced expression of UCP2 and mitofusin-2 induced by LPS. The effects of GW0742 on NO and ROS production in MAEC exposed to LPS were abolished by the UCP2 inhibitor genipin or by siRNA targeting UCP-2. Genipin also suppressed the expressional changes on NADPH oxidase and ER stress markers induced by GW0742. In conclusion, PPARβ/δ activation restored the LPS-induced endothelial dysfunction by upregulation of UCP2, with the subsequent alleviation of ER stress and NADPH oxidase activity, thus reducing intracellular ROS production and increasing NO bioavailability.

Sorting nexin 5 (SNX5) belongs to the SNX family, which is composed of a diverse group of proteins that mediate trafficking of plasma membrane proteins, receptors, and transporters. SNX5 is important in the resensitization of the dopamine D1-like receptor (D1R). D1R is uncoupled from its effector proteins in hypertension and diabetes, and treatment of diabetes restores D1R function and insulin receptor (IR) expression. We tested the hypothesis that the D1R and SNX5 regulate IR by studying the expression, distribution, dynamics, and functional consequences of their interaction in human renal proximal tubule cells (hRPTCs). D1R, SNX5, and IR were expressed and colocalized in the brush border of RPTs. Insulin promoted the colocalization of SNX5 and IR at the perinuclear area of hRPTCs. Unlike SNX5, the D1R colocalized and coimmunoprecipitated with IR, and this interaction was enhanced by insulin. To evaluate the role of SNX5 and D1R on IR signaling, we silenced via RNA interference the endogenous expression of SNX5 or the D1R gene DRD1 in hRPTCs. We observed a decrease in IR expression and abundance of phosphorylated IR substrate and phosphorylated protein kinase B, which are crucial components of the IR signal transduction pathway. Our data indicate that SNX5 and D1R are necessary for normal IR expression and activity. It is conceivable that D1R and SNX5 may interact to increase the sensitivity to insulin via a positive regulation of IR and insulin signaling.

DJ-1 exerts a protective role against oxidative stress. We reported that DJ-1 mediates, in part, the antioxidant properties of the dopamine 2 receptor; renal-selective silencing of DJ-1 decreased Nrf2 activity and increased blood pressure (BP). This study determined the mechanisms involved in the oxidative stress-mediated hypertension with DJ-1 depletion. DJ-1-/- mice, relative to wild-type (WT) littermates, had decreased expression of Nrf2 (46.8±6.8%), NQO1 (19.4±2%), and GST (51±12%), but increased systolic BP (130±3%). There were no differences in the Na+ excretion between DJ-1-/- and WT mice (n=5). High Na+ diet (one week) did not affect the BP of DJ-1-/- mice (n=3). H&E and picro sirius red staining did not show any abnormalities in renal morphology of DJ-1-/- mice. The renal mRNA expressions (qPCR) of ER stress markers GRP94, ATF-4, ATF-6, Sxbp-1, CHOP, caspase-12, and caspase-3 were not different between DJ-1-/- and WT (n=7) mice. Renal NADPH oxidase activity (n=5) was also not different between these two mouse strains. However, the renal expression of nitro-tyrosine was increased in DJ-1-/- mice, relative to WT littermates (176.8±31%, n=5). Tempol, a superoxide dismutase mimetic, and bardoxolone, an Nrf2 inducer, normalized the BP (tempol:118±2% vs 100±1%, bardoxolone:118±3.9% vs 95±3.97%, vs WT, n=4) and renal malondialdehyde production (Tempol:160±23% vs 109±15%, bardoxolone 140±6%: vs 76±9% vs WT, n=4, by ELISA) in DJ-1-/- mice but had no effect in their WT littermates. In conclusion, DJ-1-/- mice have hypertension that is mediated by superoxide anion production, independent of sodium homeostasis, ER stress, or NADPH oxidase activity

The kidney plays an important role in the long-term control of blood pressure and is the major organ involved in the regulation of sodium homeostasis.1 The inappropriate sodium retention in hypertension results from enhanced renal sodium transport per se or a failure to respond appropriately to signals that decrease renal sodium transport in the face of increased sodium intake. Humans with polygenic essential hypertension have increased renal sodium transport that is not properly regulated by natriuretic and antinatriuretic hormones and humoral factors, including dopamine and angiotensin II (Ang II). Dopamine and Ang II exert their effects via G protein-coupled receptors (GPCRs).1–3 GPCRs constitute by far the largest receptor family in mammals, which are encoded by >800 genes in the human genome and play a vital role in the regulation of most cellular and physiological functions in the body.4 On ligand binding, GPCRs regulate and modulate a variety of cell functions by coupling to heterotrimeric G proteins and regulating downstream effectors, such as adenylyl cyclases, phospholipases, protein kinases, and ion channels.5 Activation of renal GPCRs, including dopamine and Ang II receptors, leads to either natriuresis (sodium excretion) or antinatriuresis (sodium retention), keeping a normal sodium balance, resulting in the maintenance of a normal blood pressure.2,3 GPCR kinases (GRKs) constitute a family of 7 serine/threonine protein kinases characterized by their ability to specifically recognize and phosphorylate agonist-activated GPCRs.6 GRK-mediated receptor phosphorylation is one of the well-characterized mechanisms for GPCR desensitization. In the process of GPCR desensitization, GRKs phosphorylate agonist-bound receptors, leading to the translocation and binding of arrestins to the receptors and inhibition of subsequent receptor activation by blocking GPCR-G protein coupling. In particular, GRK4 seems to play a vital role in regulating dopamine-mediated natriuresis and renin–angiotensin system (RAS)–mediated antinatriuresis.7 Increasing …

Increased renal generation of reactive oxygen species (ROS) is important in the pathogenesis of hypertension caused by absent or dysfunctional dopamine receptor subtype. Germline deletion of the dopamine 2 receptor in mice increases renal NADPH oxidase (NOX) activity and decreases expression paraoxonase 2 (PON2) and results in ROS-dependent hypertension. We determined if microRNA (miR) is involved in PON2-mediated regulation of NOX. Silencing PON2 in human renal proximal tubules cells decreased PON2 (-60±4%, n=3,*P<0.05) and increased NOX2 (110±15%, n=3,*P<0.05) and NOX4 (80±10%, n=3,*P<0.05) proteins, NOX activity (50±6%, n=3,*P<0.05), and ROS production (57±3%, n=4,*P<0.05). Inhibition of NOX activity by diphenyleneiodonium normalized the increase in ROS caused by PON2 silencing. Renal-selective silencing of Pon2 in mice by the renal subcapsular infusion of Pon2 siRNA decreased PON2 (~50%), and increased NOX2 (191±11%,n=3, P<0.05), NOX4 (60±4%,n=3, P<0.05), NOX activity (94±23%, n=3, P<0.05), and blood pressure (BP) (+41±6 mmHg, n=3, P<0.05). Pon2-/- mice also had higher BP than wild-type littermates (+15±2 mmHg,n=3/4,*P<0.05) but less than observed with renal-selective silencing indicating extrarenal compensation. Renal NOX2 (220±64%, n=3/4,*P<0.05) and NOX activity (195±77%, n=3,*P<0.05) were also increased in Pon2-/- mice. However, the renal expression of NOX4 was similar in Pon2-/- and wild-type littermates. The renal expressions of miR-23b, miR-34a and miR-155 (reported to regulate NOX expression) were also similar in Pon2-/- mice and wild-type littermates. However, renal miR-146a expression was decreased (-25±4%, n=3/4,*P<0.05) while miR-204 (150±12%, n=3/4,*P<0.05) and NFAT expressions (21±7%, n=3/4,*P<0.05) were increased in Pon2-/- mice. The increase in miR-204 could be a compensatory response because miR-204 has been shown to decrease NFAT expression. It is known that NFAT and NOX2 can positively regulate each other’s expression while miR-146a negatively regulates NOX4 expression and inflammation. We conclude that PON2 by increasing miR-146a and decreasing NFAT expression negatively regulates NOX activity and reduce ROS production that would contribute to the maintenance of normal BP.

The objective of this study was to elucidate whether a Western diet was associated with nonalcoholic steatohepatitis (NASH), and the relationship between NASH, autophagy and endoplasmic reticulum (ER) stress.Four-month-old Lee–Sung minipigs were randomly assigned to two groups: control diet (C) and Western diet (W), for a 5-month experimental period.Feeding a Western diet produced a body composition with more fat, less lean and a greater liver weight. Compared with C pigs, W pigs also exhibited an elevated level of plasma insulin and free fatty acid. The W pigs displayed glucose intolerance, lower circulation antioxidant capacity and greater hepatic oxidative stress. Furthermore, pig fed the W diets had increased collagen accumulation in the liver and elevated systemic inflammation [tumor necrosis factor α and interleukin (IL)-6]. Compared with C pigs, W pigs had higher hepatic ER stress-related protein expression of GRP94, CHOP and caspase-12. The W pigs also had greater hepatic autophagy-related protein expression of p62 and LC3II. In an obesity antibody array analysis, W pigs had higher type 2 diabetes mellitus- (insulin-like growth factor 1, osteoprotegerin and resistin), atherosclerosis- (vascular endothelial growth factor, platelet-derived growth factor-AA and plasminogen activator inhibitor-I) and inflammation- [IL-1, macrophage-stimulating protein alpha, X-linked ectodermal dysplasia receptor and serum amyloid A (SAA)] related protein expressions. In addition, W pigs had greater plasma SAA concentration than C pigs and plasma SAA level was highly associated with IL-6.We successfully established a NASH pig model, and our findings suggested an association of NASH with ER stress and autophagy. The SAA has potential as a novel plasma biomarker for nonalcoholic fatty liver disease pigs.

Humans have dopamine D5 receptors (hD5R) with single-nucleotide polymorphisms and a diminished function. We generated hD5F173L cDNA that has a decreased response to D5R agonist-mediated increase in cAMP production and increased production of reactive oxygen species, relative to wild-type hD5R (hD5WT) cDNA expressed in Chinese hamster ovary cells. To investigate the role of hD5F173L in the pathogenesis of salt-sensitive hypertension, we generated transgenic mice overexpressing hD5F173L or hD5WT and fed them normal (0.8% NaCl) or high (4% NaCl) salt diet. On normal salt diet, the blood pressure, and renal NADPH oxidase activity and angiotensin type 1 receptor (AT1R) expression were higher in hD5F173L than hD5WT transgenic mice. After 2 weeks on high salt diet, the blood pressure and renal NADPH oxidase activity, but not AT1R expression, were increased in hD5F173L but not in hD5WT transgenic mice. Candesartan, an AT1R antagonist, decreased the blood pressure and NADPH oxidase activity in hD5F173L but not in hD5WT transgenic mice. We suggest that the ability of the hD5R to negatively regulate the renal NADPH oxidase activity and AT1R function may have important implications in the pathogenesis of salt-sensitive blood pressure. However, the mechanisms involved in regulating the balance of renal D5R and AT1R function in the oxidative stress-mediated salt-sensitive blood pressure remain to be determined.

HomeHypertensionVol. 65, No. 6G Protein-Coupled Receptor Kinase 4 Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBG Protein-Coupled Receptor Kinase 4Role in Hypertension Jian Yang, Van Anthony M. Villar, John E. Jones, Pedro A. Jose and Chunyu Zeng Jian YangJian Yang From the Departments of Cardiology (J.Y., C.Z.) and Nutrition (J.Y.), Daping Hospital, The Third Military Medical University, Chongqing, People's Republic of China; and Division of Nephrology, Department of Medicine (V.A.M.V., J.E.J., P.A.J.) and Department of Physiology (P.A.J.), University of Maryland School of Medicine, Baltimore. , Van Anthony M. VillarVan Anthony M. Villar From the Departments of Cardiology (J.Y., C.Z.) and Nutrition (J.Y.), Daping Hospital, The Third Military Medical University, Chongqing, People's Republic of China; and Division of Nephrology, Department of Medicine (V.A.M.V., J.E.J., P.A.J.) and Department of Physiology (P.A.J.), University of Maryland School of Medicine, Baltimore. , John E. JonesJohn E. Jones From the Departments of Cardiology (J.Y., C.Z.) and Nutrition (J.Y.), Daping Hospital, The Third Military Medical University, Chongqing, People's Republic of China; and Division of Nephrology, Department of Medicine (V.A.M.V., J.E.J., P.A.J.) and Department of Physiology (P.A.J.), University of Maryland School of Medicine, Baltimore. , Pedro A. JosePedro A. Jose From the Departments of Cardiology (J.Y., C.Z.) and Nutrition (J.Y.), Daping Hospital, The Third Military Medical University, Chongqing, People's Republic of China; and Division of Nephrology, Department of Medicine (V.A.M.V., J.E.J., P.A.J.) and Department of Physiology (P.A.J.), University of Maryland School of Medicine, Baltimore. and Chunyu ZengChunyu Zeng From the Departments of Cardiology (J.Y., C.Z.) and Nutrition (J.Y.), Daping Hospital, The Third Military Medical University, Chongqing, People's Republic of China; and Division of Nephrology, Department of Medicine (V.A.M.V., J.E.J., P.A.J.) and Department of Physiology (P.A.J.), University of Maryland School of Medicine, Baltimore. Originally published13 Apr 2015https://doi.org/10.1161/HYPERTENSIONAHA.115.05189Hypertension. 2015;65:1148–1155Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2015: Previous Version 1 The kidney plays an important role in the long-term control of blood pressure and is the major organ involved in the regulation of sodium homeostasis.1 The inappropriate sodium retention in hypertension results from enhanced renal sodium transport per se or a failure to respond appropriately to signals that decrease renal sodium transport in the face of increased sodium intake. Humans with polygenic essential hypertension have increased renal sodium transport that is not properly regulated by natriuretic and antinatriuretic hormones and humoral factors, including dopamine and angiotensin II (Ang II). Dopamine and Ang II exert their effects via G protein-coupled receptors (GPCRs).1–3GPCRs constitute by far the largest receptor family in mammals, which are encoded by >800 genes in the human genome and play a vital role in the regulation of most cellular and physiological functions in the body.4 On ligand binding, GPCRs regulate and modulate a variety of cell functions by coupling to heterotrimeric G proteins and regulating downstream effectors, such as adenylyl cyclases, phospholipases, protein kinases, and ion channels.5 Activation of renal GPCRs, including dopamine and Ang II receptors, leads to either natriuresis (sodium excretion) or antinatriuresis (sodium retention), keeping a normal sodium balance, resulting in the maintenance of a normal blood pressure.2,3GPCR kinases (GRKs) constitute a family of 7 serine/threonine protein kinases characterized by their ability to specifically recognize and phosphorylate agonist-activated GPCRs.6 GRK-mediated receptor phosphorylation is one of the well-characterized mechanisms for GPCR desensitization. In the process of GPCR desensitization, GRKs phosphorylate agonist-bound receptors, leading to the translocation and binding of arrestins to the receptors and inhibition of subsequent receptor activation by blocking GPCR-G protein coupling. In particular, GRK4 seems to play a vital role in regulating dopamine-mediated natriuresis and renin–angiotensin system (RAS)–mediated antinatriuresis.7 Increasing number of studies show that GRK4 is associated with hypertension and blood pressure response to antihypertensive medicines and adverse cardiovascular outcomes of antihypertensive treatment.8–12 In this report, we review our evolving understanding of the role of GRK4 in the regulation of dopamine and Ang II receptor function, which advances our understanding of the role of GRK4 in the control of blood pressure and highlights potential and novel strategies for the prevention and treatment of hypertension.Physiological Role of Intrarenal Dopamine and RASDopamine, via 5 subtypes of receptors, plays an important role in the control of blood pressure by regulating epithelial sodium transport, vascular smooth muscle contractility, inflammation, and production of reactive oxygen species and by interacting with the RAS and sympathetic nervous system.1,3,7 Dopamine receptors are classified into D1- and D2-like receptor subtypes: D1-like receptors (D1R and D5R) couple to stimulatory G protein GαS and stimulate adenylyl cyclases activity, whereas D2-like receptors (D2R, D3R, and D4R) couple to inhibitory G protein Gαi/Gαo and inhibit adenylyl cyclases activity. Disruption of any of the dopamine receptor genes in mice results in hypertension, the pathogenesis of which is specific for each receptor subtype.13The RAS is classically known as a coordinated hormonal cascade regulating blood pressure, as well as electrolyte and fluid homeostasis.1,2 Ang II is a biologically active octapeptide that is considered the main mediator of classic RAS. Ang II exerts its action through 2 major receptor subtypes, namely type 1 (AT1R) and type 2. AT1R mediates the vast majority of cardiovascular and renal actions of Ang II, including vasoconstriction, renal tubule sodium reabsorption, reactive oxygen species generation, and inflammation.1,14,15 In contrast, activation of the Ang II receptor type 2 induces vasodilatation, promotes natriuresis, and lowers blood pressure.16The activity of dopamine receptors and AT1R is regulated by phosphorylation/dephosphorylation, which is mediated by GRKs and protein phosphatases, respectively. Basal protein phosphatase 2A activity in renal proximal tubules (RPTs) is not different between the normotensive Wistar–Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs), which have impaired D1R function.17 D1-like receptor agonist treatment of RPT membranes from SHRs failed to increase protein phosphatase 2A activity; an impaired ability to increase protein phosphatase 2A activity would result in continued phosphorylation and desensitization of the D1R.18 However, the GRKs have received, by far, the most attention in the regulation of renal dopamine and Ang II receptors in hypertension.GPCR Kinase FamilyClassification of GRKsThere are over 800 known GPCRs in the human genome, but only 7 GRKs have been identified. All GRKs have a similar general structure: a highly conserved central protein kinase domain, inserted in the regulator of G protein signaling homology domain that keeps the ability of the kinase domain to phosphorylate activated GPCRs. The first 20 or so amino acids of GRKs are highly conserved, whereas the carboxy tail region is GRK subtype-specific: prenylated in the GRK1 subfamily; binds to Gβγ and contains a pleckstrin homology domain in the GRK2 subfamily; and has a C-terminal helix/palmitoylation site in the GRK4 subfamily.19 The GRK1 subfamily (opsin kinase family) consists of GRK1 and GRK7; GRK2-like subfamily (β-adrenergic receptor kinase family) consists of GRK2 and GRK3; and the GRK4-like subfamily consists of GRK4, GRK5, and GRK6. GRK1 and GRK7 are found almost exclusively in the retina and modulate opsins. GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed, whereas GRK4 is expressed in only a few organs (vide infra).20–22GRKs and HypertensionGRKs have multiple physiological effects on the regulation of blood pressure. Vascular smooth muscle overexpression of GRK2 in transgenic mice attenuates β-adrenergic receptor–induced vasodilation and increases resting blood pressure.23 GRK2 expression and GRK activity are increased in both the lymphocytes and vascular smooth muscles of patients with essential hypertension and in SHRs.24 GRK2 hemizygous knockout mice have increased nitric oxide bioavailability that protects against Ang II–induced hypertension.25 The transgenic mice with vascular smooth muscle–specific GRK5 overexpression are hypertensive.26 By contrast, GRK3 expression in human lymphocytes has been reported to be inversely correlated with blood pressure, suggesting a protective role for GRK3 in the regulation of blood pressure that is supported by the findings in transgenic mice.27 Overexpression of GRK2, GRK3, and GRK5 in human embryonic kidney (HEK293) cells desensitizes the D1R.28 Inhibition of GRK6 prevents intestinal D1R desensitization.29 However, renal GRK6 levels are lower in hypertensive subjects and SHRs than their normotensive controls.30Role of GRK4 in HypertensionGRK4 IsoformsGRK4 has some inherent characteristics. For example, GRK4 has constitutive activity under basal conditions, which may, in part, be because of its ability to bind to inactive GαS and Gα13 subunits.31 It is the only GRK subtype that is capable of phosphorylating unstimulated GPCRs.32 GRK4 is expressed in a limited number of tissues, for example, artery, bone, cerebellum, heart, kidney, myometrium, small intestines, and testes, unlike GRK2, GRK3, GRK5, and GRK6, which are ubiquitously expressed. Moreover, 4 splice variants (GRK4α, β, γ, and δ) of GRK4 have been identified in humans.21 Alternative splicing generates 4 isoforms of human GRK4 mRNA that differ in the presence or absence of exon 2 and exon 15: GRK4α is the longest isoform and contains all of the 16 exons; GRK4β, which lacks exon 2, has a 32-codon- deletion that encompasses the phosphatidylinositol bisphosphate–binding domain near the amino terminus; GRK4γ, which lacks exon 15, has a 46-codon-deletion near the carboxyl terminus; and GRK4δ lacks both exons 2 and 15.21 The human GRK4 gene locus at 4p16.3 is linked to hypertension.33 Numerous studies show that abnormal GRK4 function has the potential to affect GPCR (such as D1R, D3R, and AT1R)-regulated biological responses in many physiological and pathological conditions, such as hypertension,1,3,7,13,20,22,33–38 which makes GRK4 as an attractive candidate for a genetic determinant for essential hypertension.Distribution of GRK4 in Kidney and ArteryGRK4 is expressed in the rat renal cortex.34 In both WKY and SHRs, GRK4 is expressed in the subapical membranes of the RPT, thick ascending limb of Henle, and renal artery, with much less expression in the glomerulus. Renal cortical GRK4 expression is increased in SHRs compared with WKY rats, whereas cardiac GRK4 expression is similar in the 2 rat strains, indicating that the increased GRK4 expression in hypertension has organ specificity.34 In mice, the renal expression of GRK4 is strain-dependent and influenced by salt intake, for example, lower on normal but higher on high-salt diet in C57BL/6J than in SJL/J mice. C57BL/6 mice are salt-sensitive and have an impaired ability to excrete a NaCl load that is associated with an increase in blood pressure, whereas SJL/J mice are salt-resistant.35 All 4 GRK4 isoforms are expressed abundantly in human RPT cells; GRK4 is localized at the RPT cell surface membrane and cytoplasm and internalized after stimulation of dopamine receptors.20,36GRK4 activity is increased in kidneys of hypertensive humans.20 Antisense GRK4 oligonucleotides completely blocked the constitutive serine phosphorylation of the D1R and restored the ability of the D1-like receptor agonist, fenoldopam, to stimulate cAMP accumulation in RPT cells from hypertensive subjects, which suggests that the major GRK involved in the phosphorylation of the D1R in hypertension is GRK4 and not the other GRKs.20 However, there are no differences in the expression of the GRK4 isoforms (α/β,γ/δ) in kidneys or cultured RPT cells between hypertensive and normotensive subjects.20 Therefore, we assume that the increased activity of GRK4 in the kidney of hypertensive subjects is not caused by increased renal GRK4 protein expression but rather by constitutively active variants of GRK4.In Sprague–Dawley rats and C57BL/6J mice, GRK4 is well-expressed in large and small arterial vessels, including the carotid arteries, thoracic aorta, superior mesenteric artery, and renal artery.22 In the aorta, GRK4 is expressed in the tunica media and adventitia. However, removal of the adventitia does not affect the Ang II–mediated vasoconstriction, suggesting that GRK4 in the adventitia does not participate in Ang II–mediated vasoconstriction.22Regulation of GRK4As a regulator of GPCRs, GRK4 per se is regulated by transcription factors and signaling molecules. The GRK4 promoter region, containing 1851 bp of the 5′-flanking region and 275 bp of the 5′-untranslated region, is reported to be highly active.39 The GRK4 core promoter resides in the first 1851 bp upstream of its transcription start site,39 suggesting that the complex DNA–protein and protein–protein interaction patterns at this portion may affect the transcriptional and expression capacities of GRK4. The transcription factor c-Myc, binding to the promoter of GRK4, positively regulates GRK4 protein expression in human RPT cells, which connects aberrant Ang II activation to D1R–adenylyl cyclases uncoupling.40 The GRK4 subfamily, including GRK4, is potently inhibited by ubiquitous calcium-binding protein calmodulin, which has little or no effect on members of other GRK subfamilies.41 Sorting nexins are involved in receptor endocytosis and trafficking through the endosomes. Sorting nexin 5 directly interacts with GRK4 and prevents GRK4 from targeting the phosphorylation sites of the D1R, which is enhanced after D1R activation. In contrast, depletion of sorting nexin 5 markedly increases the ability of GRK4 to constitutively phosphorylate D1R in human RPT cells, consistent with the in vivo studies showing that renal sorting nexin 5 depletion increases blood pressure and decreases D1R-mediated sodium excretion.42GRK4 Regulation of D1R and AT1RStudies have provided direct evidence of a crucial role of renal GRK4 in the D1R-mediated control of sodium excretion and blood pressure in genetic hypertension. GRK4 is more effective than GRK2 in attenuating the D1-like agonist-induced desensitization, suggesting a greater role for GRK4 in the D1R homologous desensitization in human RPT cells.43 Increased GRK4 activity causes an impairment of renal D1R function in hypertension. Both basal GRK4 and serine-phosphorylated D1R levels are much higher in renal cortical membranes of SHRs than WKY rats. Silencing of renal cortical GRK4 decreases serine-phosphorylated D1R to a greater extent in SHRs than WKY rats.34 Depletion of renal cortical GRK4 also increases urine flow and sodium excretion and attenuates the increased blood pressure in SHRs, but does not affect the blood pressure in WKY rats.34In vitro studies also support the regulation of D1R by GRK4. GRK4 constitutively phosphorylates the D1R in the absence of agonist activation, whereas depletion of GRK4 blunts the D1R desensitization.43,44 Moreover, increased activity of GRK4 because of constitutively active GRK4 variants, not GRK4 protein abundance, causes the decrease in D1R function in RPT cells from hypertensive subjects, which is restored by GRK4 depletion.20 All 4 GRK4 isoforms are expressed in RPT cells. GRK4α and not the other GRK4 isoforms can phosphorylate D1R in certain HEK293 (eg, HEK293T) cells,44 whereas GRK4γ can phosphorylate D1R in Chinese hamster ovary cells20 and HEK293 that do not stably express the SV40 large T antigen (J.J. Gildea, P.A. Jose, R.A. Felder, unpublished data, 2015). The phosphorylation of D3R is regulated by both GRK4α and GRK4γ (GRK4γ>GRK4α) isoforms in human RPT cells.36 These results indicate that the regulatory activity of GRK4 isoforms on the desensitization of dopamine receptors is cell-specific. It should also be noted that the effect of human GRK4α variant on blood pressure in mice has not been studied, whereas the effect of human GRK4γ wild-type and variants on blood pressure has been reported in transgenic mice.20,45Similar to D1Rs, the AT1R is also regulated by GRK4. Our previous studies have shown that AT1R expression and actions are enhanced in both kidney and artery in transgenic mice expressing GRK4 variants (vide infra).22,46 The increased renal GRK4 expression in old rats is also associated with increased renal AT1R expression and function.47 Interestingly, the renal-selective depletion of AT1R in SHRs does not decrease blood pressure because the interruption of the renin–angiotensin negative feedback loop results in increased circulating renin and Ang II.48 However, combined renal-selective silencing of both GRK4 and AT1R decreases blood pressure, as well as plasma renin activity and Ang II levels in both rat strains,49 which can explain, in part, the crucial role of GRK4 in the regulation of sodium excretion and blood pressure.Regulation of Blood Pressure by GRK4 VariantsThree missense single nucleotide polymorphisms in the coding region of GRK4γ impair D1R function (Table 1). They are nucleotide 448, CGT to CTT (amino acid 65R>L, rs2960306); nucleotide 679, GCC to GTC (amino acid 142A>V, rs1024323); and nucleotide 1711, GCG to GTG (amino acid 486A>V, rs1801058). GRK4γ single nucleotide polymorphisms (65R>L, 142A>V, 486A>V) markedly impair D1R-mediated cAMP accumulation that is not caused by differences in the quantity of the expression of either D1R or GRK4γ.20 Human GRK4γ (hGRK4γ) 142V transgenic mice have higher blood pressures and greater heart weights than hGRK4γ wild-type transgenic mice.22,45 Infusion of fenoldopam, a D1-like receptor agonist, increases urine flow and sodium excretion in hGRK4γ wild-type mice, but not in the hypertensive hGRK4γ 142V transgenic mice.20 Additional studies showed that the higher blood pressure in hGRK4γ 142V transgenic mice is not because of the transgene integration sites, flanking genes, or copy numbers, but due mainly to the effect of hGRK4γ 142V transgene acting via D1R.45 This is confirmed by in vitro studies: in the transfected Chinese hamster ovary cells, GRK4γ 142V increases GRK activity and causes D1R phosphorylation, which may explain, in part, the decreased responsiveness of the D1R in hypertensive hGRK4γ 142V transgenic mice.20 There is, however, cell specificity of the ability of GRK4γ to regulate D1R function; GRK4γ regulates D1R function in human RPT, Chinese hamster ovary cells,20 and HEK293 cells (vide supra), whereas it is GRK4α in HEK293T cells.44 We also found that the function of D3R is also impaired in the hGRK4γ 142V-transfected human RPT cells (J. Yang, V.A.M. Villar, and P.A. Jose, unpublished data, 2015).Table 1. GRK4 Variant Transgenic Mice and In Vitro StudiesGRK4 VariantsTransgenic MiceVariants Transduced Cell StudiesMouse PhenotypeFunctional Deficit(s)Receptor Defect(s)Cell LineReceptor Defect(s)R65LSalt-sensitive hypertension (high-salt diet)Not determinedNot determinedCHOIncreased renal D1R phosphorylation and impaired renal D1R function, R65L alone, or combined with 486V variant20A142VHypertension (normal-salt diet)Decreased urine flow and sodium excretion20Decreased renal expression and responsiveness of D1R20CHOIncreased renal D1R phosphorylation; Impaired renal D1R function20Increased renal AT1R expression and responsiveness46Human RPT cellsImpaired D3R functionIncreased AT1R-mediated vasoconstriction22Higher arterial AT1R expression22Vascular smooth muscle cellsIncreased AT1R expression; decreased AT1R phosphorylation; decreased AT1R protein degradation; increased AT1R function22Increased AT1R-mediated blood pressure response to Ang II infusion and AT1R blockade22A486VSalt-sensitive hypertension (high-salt diet)Impaired pressure-natriuresis plot50Increased AT1R expression50CHOIncreased renal D1R phosphorylation and impaired renal D1R function20AT1R indicates angiotensin type 1 receptor; CHO, Chinese hamster ovary; D1R, D1-like receptors; GRK4, GPCR kinase 4; and RPT, renal proximal tubules.The hypertension in hGRK4γ 142V transgenic mice is also associated with increased AT1R expression and function in both kidney and artery.22,46 GRK4γ 142V transgenic mice have increased renal AT1R expression and function, for example, increased blood pressure response to Ang II infusion and AT1R blockade. By contrast, GRK4γ 142V transgenic mice that are deficient of AT1R have normal blood pressure.46 We have reported that both AT1R expression and AT1R-mediated vasoconstriction are higher in the aorta of hGRK4γ 142V transgenic mice.22 Moreover, infusion of Ang II causes a greater increase in blood pressure, whereas infusion of the AT1R antagonist candesartan causes a greater decrease in blood pressure in hGRK4γ 142V transgenic mice than GRK4 wild-type transgenic mice.46 AT1R mRNA and protein expression and function are higher in hGRK4γ142V than in GRK4γ wild-type cells, but the opposite is true for AT1R phosphorylation and degradation,22 indicating that the regulation of AT1R expression by hGRK4γ occurs at both transcriptional and post-translational levels.Depending on the genetic background and sodium intake, hGRK4γ 486V transgenic mice may develop increased blood pressure. hGRK4γ 486V transgenic mice have increased renal AT1R expression and develop hypertension only after an increase in sodium intake, in contrast to GRK4γ 142V transgenic mice, which have increased AT1R expression and develop hypertension even on a normal-salt diet.46,50 Depending on the genetic background of the mouse, hGRK4γ wild-type prevents salt-sensitive hypertension, whereas hGRK4γ 486V converts a salt-resistant phenotype to a salt-sensitive phenotype.50 hGRK4γ 65L transgenic mice, similar to hGRK4γ 486V transgenic mice, have normal blood pressure on a normal-salt diet, but have increased blood pressure on high-salt diet (L.D. Asico and P.A. Jose, unpublished data, 2015). In vitro studies showed that in single (65L or 486V) or double variant GRK4 (65L/486V)-transfected Chinese hamster ovary cells, there is an increase in basal D1R phosphorylation and impairment of D1R-mediated cAMP production.20 The mechanism for the increase in blood pressure in GRK4γ 65L or 486V transgenic mice only when salt intake is increased remains unclear.GRK4 Polymorphisms and HypertensionThe GRK4 gene polymorphisms have different allele frequencies among different populations. GRK4 486V is more frequent in Asians and less frequent in blacks than in other populations (Hispanics and whites).51 The GRK4 locus on human chromosome 4p16.3 is linked to essential hypertension and salt sensitivity.33 The first report in 2002 by Bengra et al found a significant association between GRK4 A486V variant and an Italian population of mildly hypertensive patients.52 Subsequent studies showed that GRK4 gene variants R65L, A142V, and A486V are each associated with essential hypertension in several ethnic groups (Table 2). In Euro-Australians, GRK4 486V is associated with essential hypertension, whereas the 65L and 142V variants track with elevation in diastolic blood pressure only in male hypertensives.58 In a Chinese Han population, GRK4 A486V is also associated with hypertension in additive, dominant, and recessive model, whereas GRK4 142V is associated with hypertension in an additive model only.59,60 However, there are reports that do not show the association between GRK4 variants and hypertension.63,64 In a population of blacks 18 to 49 years of age, GRK4 A486V variant was found to be negatively associated with hypertension.61 Although the reasons leading to the differences among studies are not known, the negative studies could be the consequence of not taking into account salt sensitivity (particularly for GRK4 R65L and A486V) or assessing the role of GRK4 in conjunction with other single nucleotide polymorphisms of GRK457 and other genes.53,60,65Salt-sensitive hypertension is associated with GRK4 gene variants. The Italian patients whose hypertension is associated with GRK4 A486V are actually salt-sensitive.52 In a Japanese population, the GRK4 variants (R65L, A142V, and A486V) are more frequent in salt-sensitive than salt-resistant hypertensive patients.57 A genetic model of GRK4 R65L, A142V, and A486V is 94.4% predictive of salt sensitivity. By contrast, the single-locus model with only GRK4 A142V is 78.4% predictive, whereas a 2-locus model of GRK4 A142V and aldosterone synthase CYP11B2 is 77.8% predictive of low-renin hypertension.57 The ability to excrete a salt load is inversely related to the number of GRK4 variant alleles (R65L, A142V, and A486V) in hypertensive Japanese.57 GRK4 variants are also associated with salt sensitivity in normotensive subjects.55–57 In black normotensive adolescents, the GRK4 65L allele is associated with a reduced urinary sodium excretion in response to stress.56 In young normotensive twins, GRK4 65L was associated with the steepest increase in blood pressure; the GRK4 65L-142V-A486V haplotype had a 1.05 mm Hg steeper increase in systolic blood pressure per year increase in age, relative to those with GRK4 R65, A142, and A486 haplotype.54 Therefore, genetic variations of GRK4 may contribute to variations of blood pressure in normotensive individuals, potentially influencing the development of hypertension.Table 2. Association Studies of GRK4 Variants in Hypertensive or Normotensive SubjectsGRK4 VariantsHypertensive SubjectsNormotensive SubjectsEthnic Group (Year)Single or Multilocus AnalysesHypertension PhenotypeEthnic Group (Year)Single or Multilocus AnalysesBlood Pressure or Sodium ExcretionR65L (rs2960306)Ghanaian (2004)R65L and ACENot classified53Euro- and African American (2006)Single-locusIncreased SBP54European ancestry (2012)Single-locusSalt sensitivity55Euro- and African American (2006)Single-locusStress-induced UNaV reduction56A142V (rs1024323)Japanese (2006)Single-locusLow-renin hypertension57Japanese (2006)R65L, A142V, and A486VImpaired natriuretic response to dopaminergic stimulation57Japanese (2006)A142V and CYP11B2Low-renin hypertension57Japanese (2006)R65L, A142V, and A486VSalt-sensitive hypertension57A486V (rs1801058)Italian (2002)Single-locusMild salt-sesitive hypertension52Euro-Australian (2004)Single-locusNot classified58Chinese (2006)Single-locusNot classified59Chinese (2006)Single-locusNot classified60African American (2010)Single-locusNegative association61African-derived Brazilian (2012)A486V and NOS3Not classified62ACE indicates angiotensin-converting enzyme; GRK4, GPCR kinase 4; NOS, nitric oxide synthase; SBP, systolic blood pressure; and UNaV, urinary sodium excretion.As indicated earlier, GRK4 variants interact with other genes in the pathogenesis of hypertension. Multilocus analyses have shown association between GRK4 variants and other gene variants with high blood pressure. In an African population from Ghana, the combination of angiotensin-converting enzyme and GRK4 R65L is the best genetic model to predict hypertension (70.5% prediction of hypertension).53 Among Japanese subjects, the best combination that is predictive of hypertension, not classified according to salt sensitivity, is GRK4, angiotensin-converting enzyme, and CYP11B2, with an estimated prediction success of 63%; however, for low-renin hypertension in Japanese, the single best genetic model includes only GRK4 A142V and CYP11B2, with an estimated prediction success of 77.8%.57,66 Normotensive Japanese with GRK4 polymorphisms were reported to have increased serum N-terminal pro-B-type natriuretic peptide levels.65 A recent study among African-derived Brazilian populations reported that an interaction between GRK4 A486V and endothelial nitric oxide synthase is associated with increased diastolic blood pressure.62There are currently 2 meta-analyses on the associations of GRK4 polymorphisms with hypertension risk. Our previous meta-analysis showed that GRK4 486V increases the risk for essential hypertension with an odds ratio of 1.5 (95% confidence interval, 1.2–1.9).7 A more recent meta-analysis showed that GRK4 486V is inversely associated with hypertension among East Asians (odds ratio =0.39, 95% confidence interval, 0.28–0.55), but positively associated with hypertension among Europeans (odds ratio =2.38, 95% confidence interval,1.38–4.10); GRK4 65L was also associated with hypertension among Europeans.11GRK4 and Pharmacogenomics of Antihypertensive MedicinesHypertension is a condition involving the interaction between genetics and environment that includes diet and lifestyle, among others. Although it is generally believed that the heritability of blood pressure is ≈30% to 55%, genome-wide association studies (GWAS) have identified <5% of the genetic factors believed to be involved in the pathogenesis of hypertension.67 Epigenetics, ethnicity, and low frequency of the variants (eg, SLC12A3 [thiazide-sensitive sodium/chloride cotransporter], SLC12A1 [sodium-potassium 2 chloride cotransporter]) are some reasons.68 Many of the genes identified in GWAS are also found in noncoding regions. Another limitation of GWAS is the absence of some genes in the chips. For example, GRK4 was not

The influence of a single gene on the pathogenesis of essential hypertension may be difficult to ascertain, unless the gene interacts with other genes that are germane to blood pressure regulation. G-protein–coupled receptor kinase type 4 ( GRK4 ) is one such gene. We have reported that the expression of its variant hGRK4γ 142V in mice results in hypertension because of impaired dopamine D 1 receptor. Signaling through dopamine D 1 receptor and angiotensin II type I receptor (AT 1 R) reciprocally modulates renal sodium excretion and blood pressure. Here, we demonstrate the ability of the hGRK4γ 142V to increase the expression and activity of the AT 1 R. We show that hGRK4γ 142V phosphorylates histone deacetylase type 1 and promotes its nuclear export to the cytoplasm, resulting in increased AT 1 R expression and greater pressor response to angiotensin II. AT 1 R blockade and the deletion of the Agtr1a gene normalize the hypertension in hGRK4γ 142V mice. These findings illustrate the unique role of GRK4 by targeting receptors with opposite physiological activity for the same goal of maintaining blood pressure homeostasis, and thus making the GRK4 a relevant therapeutic target to control blood pressure.

Previous work from our laboratory indicates that the dopamine D2 receptor (D2R) in the kidney has a direct role in regulating renal inflammation and injury and blood pressure. Some common single nucleotide polymorphisms (D2R SNPs; rs 6276, 6277, and 1800497) in the human DRD2 gene are associated with decreased D2R expression and function. Immortalized renal proximal tubule cells (RPTCs) from subjects carrying D2R SNPs (RPTC-D2R SNPs) express less D2Rs than RPTCs carrying no D2R SNPs (RPTC-D2R WT) (62±4 vs 100±6%; P<0.04) and a pro-inflammatory and pro-fibrotic phenotype with markers of epithelial mesenchymal transition. RPTC-D2R SNPs showed increased apoptosis compared with RPTC-D2R WT (11± 0.8 vs 2.3±0.4% TUNEL positive cells, P<0.01, n=5/group). We hypothesized that the D2R regulates renal cell survival through effects on Wnt signaling. We found that Wnt3 expression was increased in RPTC-D2R SNPs compared with RPTC-D2R WT (mRNA: 2.6±0.35 vs 1±0.11 fold; P<0.05; protein: 133±4 vs 100±5%; P<0.05). RPTC-D2R SNPs showed activated Wnt3/β-catenin signaling pathway demonstrated by decreased β-catenin phosphorylation (64±4 vs 100±8%; P<0.05) and increased expression of downstream pro-apoptotic factors Bax (136±4.6 vs 100±5%, P<0.05) and FasL (128±5.6 vs 100±6.5%, P<0.05). Silencing D2R in RPTC-D2R WT (siRNA; 0.30±0.02 vs 1±0.07 fold, P<0.05) increased Wnt 3 expression, decreased β-catenin phosphorylation, and increased expression of Bax and FasL. By contrast, treatment of RPTC-D2R WT with a D2R agonist (quinpirole,1μM, 24h) or transfection of RPTC-D2R SNPs with a DRD2 which restored D2R expression decreased Wnt3 expression, increased β-catenin phosphorylation, and decreased Bax and FasL expression. Moreover, Wnt3 silencing (siRNA) in RPTC-D2R SNPs increased β-catenin phosphorylation (132±5 vs100±9%, P<0.05), decreased Bax and FasL expression, and reduced the number of apoptotic cells (6±1.0 vs12± 0.9 % TUNEL positive cells, P<0.01). Our results indicate that D 2 R function is important in the regulation of the Wnt pathway and that the alterations in D 2 R function result in modifications in the pathway potentially leading to fibrosis, cell death, and hypertension. These results may have clinical relevance for subjects bearing D2R SNPs.

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: ∆]] 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.

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<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<0.05), NOX2 or gp91 phox (129.9±5.5% vs . 100±7.7%, P<0.05), and p47 phox (118.2±2.7% vs . 100±5.0%, P<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<0.05) and D 3 R (134.3±5.3% vs . 100 ± 1.6%, P<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<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<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<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.

The dopamine D2 receptor (D2R) negatively regulates inflammation in mouse renal proximal tubule cells (RPTCs), and lack or downregulation of the receptor in mice increases the vulnerability to renal inflammation independent of blood pressure. Some common single-nucleotide polymorphisms (SNPs; rs6276, rs6277, and rs1800497) in the human DRD2 gene are associated with decreased D2R expression and function, as well as high blood pressure. We tested the hypothesis that human RPTCs (hRPTCs) expressing these SNPs have increased expression of inflammatory and injury markers. We studied immortalized hRPTCs carrying D2R SNPs and compared them with cells carrying no D2R SNPs. RPTCs with D2R SNPs had decreased D2R expression and function. The expressions of the proinflammatory tumor necrosis factor-α and the profibrotic transforming growth factor-β1 and its signaling targets Smad3 and Snail1 were increased in hRPTC with D2R SNPs. These cells also showed induction of epithelial mesenchymal transition and production of extracellular matrix proteins, assessed by increased vimentin, fibronectin 1, and collagen I a1. To test the specificity of these D2R SNP effects, hRPTC with D2R SNPs were transfected with a plasmid encoding wild-type DRD2. The expression of D2R was increased and that of transforming growth factor-β1, Smad3, Snail1, vimentin, fibronectin 1, and collagen I a1 was decreased in hRPTC with D2R SNPs transfected with wild-type DRD2 compared with hRPTC-D2R SNP transfected with empty vector. These data support the hypothesis that D2R function has protective effects in hRPTCs and suggest that carriers of these SNPs may be prone to chronic renal disease and high blood pressure.

Dopamine-mediated regulation of Na + -K + -ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D 1 - and D 2 -like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D 1 -like receptor is expressed in the blue crab Callinectes sapidus and regulates Na + -K + -ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D 5 receptor (D 5 R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na + -K + -ATPase and D1βR in the basolateral membrane. To determine the effect of D 1 -like receptor stimulation on Na + -K + -ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D 1 -like receptor agonist fenoldopam, with or without the D 1 -like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na + -K + -ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na + -K + -ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na + -K + -ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis.