Santiago Cuevas

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

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

Renal Dopamine Receptors, Oxidative Stress, and Hypertension

Dopamine, which is synthesized in the kidney, independent of renal nerves, plays an important role in the regulation of fluid and electrolyte balance and systemic blood pressure. Lack of any of the five dopamine receptor subtypes (D1R, D2R, D3R, D4R, and D5R) results in hypertension. D1R, D2R, and D5R have been reported to be important in the maintenance of a normal redox balance. In the kidney, the antioxidant effects of these receptors are caused by direct and indirect inhibition of pro-oxidant enzymes, specifically, nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase, and stimulation of anti-oxidant enzymes, which can also indirectly inhibit NADPH oxidase activity. Thus, stimulation of the D2R increases the expression of endogenous anti-oxidants, such as Parkinson protein 7 (PARK7 or DJ-1), paraoxonase 2 (PON2), and heme oxygenase 2 (HO-2), all of which can inhibit NADPH oxidase activity. The D5R decreases NADPH oxidase activity, via the inhibition of phospholipase D2, and increases the expression of HO-1, another antioxidant. D1R inhibits NADPH oxidase activity via protein kinase A and protein kinase C cross-talk. In this review, we provide an overview of the protective roles of a specific dopamine receptor subtype on renal oxidative stress, the different mechanisms involved in this effect, and the role of oxidative stress and impairment of dopamine receptor function in the hypertension that arises from the genetic ablation of a specific dopamine receptor gene in mice.

Deficient Dopamine D2 Receptor Function Causes Renal Inflammation Independently of High Blood Pressure

Renal dopamine receptors participate in the regulation of blood pressure. Genetic factors, including polymorphisms of the dopamine D2 receptor gene (DRD2) are associated with essential hypertension, but the mechanisms of their contribution are incompletely understood. Mice lacking Drd2 (D2−/−) have elevated blood pressure, increased renal expression of inflammatory factors, and renal injury. We tested the hypothesis that decreased dopamine D2 receptor (D2R) function increases vulnerability to renal inflammation independently of blood pressure, is an immediate cause of renal injury, and contributes to the subsequent development of hypertension. In D2−/− mice, treatment with apocynin normalized blood pressure and decreased oxidative stress, but did not affect the expression of inflammatory factors. In mouse RPTCs Drd2 silencing increased the expression of TNFα and MCP-1, while treatment with a D2R agonist abolished the angiotensin II-induced increase in TNF-α and MCP-1. In uni-nephrectomized wild-type mice, selective Drd2 silencing by subcapsular infusion of Drd2 siRNA into the remaining kidney produced the same increase in renal cytokines/chemokines that occurs after Drd2 deletion, increased the expression of markers of renal injury, and increased blood pressure. Moreover, in mice with two intact kidneys, short-term Drd2 silencing in one kidney, leaving the other kidney undisturbed, induced inflammatory factors and markers of renal injury in the treated kidney without increasing blood pressure. Our results demonstrate that the impact of decreased D2R function on renal inflammation is a primary effect, not necessarily associated with enhanced oxidant activity, or blood pressure; renal damage is the cause, not the result, of hypertension. Deficient renal D2R function may be of clinical relevance since common polymorphisms of the human DRD2 gene result in decreased D2R expression and function.

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

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

Localization and signaling of GPCRs in lipid rafts

The understanding of how biological membranes are organized and how they function has evolved. Instead of just serving as a medium in which certain proteins are found, portions of the lipid bilayer have been demonstrated to form specialized platforms that foster the assembly of signaling complexes by providing a microenvironment that is conducive for effective protein-protein interactions. G protein-coupled receptors (GPCRs) and relevant signaling molecules, including the heterotrimeric G proteins, key enzymes such as kinases and phosphatases, trafficking proteins, and secondary messengers, preferentially partition to these highly organized cell membrane microdomains, called lipid rafts. As such, lipid rafts are crucial for the trafficking and signaling of GPCRs. The study of GPCR biology in the context of lipid rafts involves the localization of the GPCR of interest in lipid rafts, at the basal state and upon receptor agonism, and the evaluation of the biological functions of the GPCR in appropriate cell lines. The lack of standardized methodology to study lipid rafts, in general, and of the workings of GPCRs in lipid rafts, in particular, and the inherent drawbacks of current methods have hampered the complete understanding of the underlying molecular mechanisms. Newer methodologies that allow the study of GPCRs in their native form are needed. The use of complementary approaches that produce mutually supportive results appear to be the best way for drawing conclusions with regards to the distribution and activity of GPCRs in lipid rafts.

Single-Nucleotide Polymorphisms of the Dopamine D2 Receptor Increase Inflammation and Fibrosis in Human Renal Proximal Tubule Cells

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-&agr; and the profibrotic transforming growth factor-&bgr;1 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-&bgr;1, 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.

Sestrin2 Decreases Renal Oxidative Stress, Lowers Blood Pressure, and Mediates Dopamine D2 Receptor–Induced Inhibition of Reactive Oxygen Species Production

The dopamine D2 receptor (D2R) decreases renal reactive oxygen species (ROS) production and regulates blood pressure, in part, via positive regulation of paraoxonase 2. Sestrin2, a highly conserved antioxidant protein, regulates intracellular ROS level by regenerating hyperoxidized peroxiredoxins. We hypothesized that sestrin2 may be involved in preventing excessive renal ROS production and thus contribute to the maintenance of normal blood pressure. Moreover, the D2R may decrease ROS production, in part, through the regulation of sestrin2. Renal sestrin2 expression was lower (−62±13%) in D2R−/− than in D2R+/+ mice. Silencing D2R in human renal proximal tubule cells decreased sestrin2 expression (−53±3%) and increased hyperoxidized peroxiredoxins (2.9-fold). Stimulation of D2R in renal proximal tubule cells increased sestrin2 expression (1.6-fold), decreased hyperoxidized peroxiredoxins (−61±3%), and reduced ROS production (−31±4%). Silencing sestrin2 in renal proximal tubule cells increased hyperoxidized peroxiredoxins (2.1-fold) and ROS production (1.3-fold). Silencing sestrin2 also abolished D2R-induced decrease in peroxiredoxin hyperoxidation and partially prevented the inhibitory effect of D2R stimulation on ROS production. Silencing paraoxonase 2 increased sestrin2 ubiquitinylation (2.8-fold), decreased sestrin2 expression (−30±3%), and increased ROS production (1.3-fold), peroxiredoxin hyperoxidation (2.9-fold), and lipid peroxidation (2.3-fold), and blocked the increase in sestrin2 that occurs with D2R stimulation. In vivo renal selective silencing of sestrin2 by the renal subcapsular infusion of sestrin2 small interfering RNA (3 &mgr;g/day; 7 days) in mice increased renal oxidative stress (1.3-fold) and blood pressure. These results suggest that the D2R, via paraoxonase 2 and sestrin2, keeps normal renal redox balance, which contributes to the maintenance of normal blood pressure.

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.

Dopamine D2 receptors' effects on renal inflammation are mediated by regulation of PP2A function

Lack or downregulation of the dopamine D2 receptor (D2R) results in increased renal expression of injury markers and proinflammatory factors that is independent of a blood pressure increase. This study aimed to determine the mechanisms involved in the regulation of renal inflammation by D2Rs. Silencing D2Rs in mouse renal proximal tubule cells increased the expression of the proinflammatory TNF-α, monocyte chemoattractant protein-1 (MCP-1), and IL-6. D2R downregulation also increased Akt phosphorylation and activity, and glycogen synthase kinase-3β (GSK3β) phosphorylation and cyclin D1 expression, downstream targets of Akt; however. phosphatidylinositol 3-kinase (PI3K) activity was not affected. Conversely, D2R stimulation decreased Akt and GSK3β phosphorylation and cyclin D1 expression. Increased phospho-Akt, in the absence of increased PI3K activity, may result from decreased Akt dephosphorylation. Inhibition of protein phosphatase 2A (PP2A) with okadaic acid reproduced the effects of D2R downregulation on Akt, GSK3β, and cyclin D1. The PP2A catalytic subunit and regulatory subunit PPP2R2C coimmunoprecipitated with the D2R. Basal phosphatase activity and the expression of PPP2R2C were decreased by D2R silencing that also blunted the increase in phosphatase activity induced by D2R stimulation. Similarly, silencing PPP2R2C also increased the phosphorylation of Akt and GSK3β. Moreover, downregulation of PPP2R2C resulted in increased expression of TNF-α, MCP-1, and IL-6, indicating that decreased phosphatase activity may be responsible for the D2R effect on inflammatory factors. Indeed, the increase in NF-κB reporter activity induced by D2R silencing was blunted by increasing PP2A activity with protamine. Our results show that D2R controls renal inflammation, at least in part, by modulation of the Akt pathway through effects on PP2A activity/expression.

Nephron segment-specific gene expression using AAV vectors.

AAV9 vector provides efficient gene transfer in all segments of the renal nephron, with minimum expression in non-renal cells, when administered retrogradely via the ureter. It is important to restrict the transgene expression to the desired cell type within the kidney, so that the physiological endpoints represent the function of the transgene expressed in that specific cell type within kidney. We hypothesized that segment-specific gene expression within the kidney can be accomplished using the highly efficient AAV9 vectors carrying the promoters of genes that are expressed exclusively in the desired segment of the nephron in combination with administration by retrograde infusion into the kidney via the ureter. We constructed AAV vectors carrying eGFP under the control of: kidney-specific cadherin (KSPC) gene promoter for expression in the entire nephron; Na+/glucose co-transporter (SGLT2) gene promoter for expression in the S1 and S2 segments of the proximal tubule; sodium, potassium, 2 chloride co-transporter (NKCC2) gene promoter for expression in the thick ascending limb of Henles loop (TALH); E-cadherin (ECAD) gene promoter for expression in the collecting duct (CD); and cytomegalovirus (CMV) early promoter that provides expression in most of the mammalian cells, as control. We tested the specificity of the promoter constructs in vitro for cell type-specific expression in mouse kidney cells in primary culture, followed by retrograde infusion of the AAV vectors via the ureter in the mouse. Our data show that AAV9 vector, in combination with the segment-specific promoters administered by retrograde infusion via the ureter, provides renal nephron segment-specific gene expression.