Peroxiredoxin-4 and Dopamine D5 Receptor Interact to Reduce Oxidative Stress and Inflammation in the Kidney.

Aims: Reactive oxygen species are highly reactive molecules generated in different subcellular compartments. Both the dopamine D5 receptor (D5R) and endoplasmic reticulum (ER)-resident peroxiredoxin-4 (PRDX4) play protective roles against oxidative stress. This study is aimed at investigating the interaction between PRDX4 and D5R in regulating oxidative stress in the kidney. Results: Fenoldopam (FEN), a D1R and D5R agonist, increased PRDX4 protein expression, mainly in non-lipid rafts, in D5R-HEK 293 cells. FEN increased the co-immunoprecipitation of D5R and PRDX4 and their colocalization, particularly in the ER. The efficiency of Förster resonance energy transfer was increased with FEN treatment measured with fluorescence lifetime imaging microscopy. Silencing of PRDX4 increased hydrogen peroxide production, impaired the inhibitory effect of FEN on hydrogen peroxide production, and increased the production of interleukin-1ß, tumor necrosis factor (TNF), and caspase-12 in renal cells. Furthermore, in Drd5-/- mice, which are in a state of oxidative stress, renal cortical PRDX4 was decreased whereas interleukin-1ß, TNF, and caspase-12 were increased, relative to their normotensive wild-type Drd5+/+ littermates. Innovation: Our findings demonstrate a novel relationship between D5R and PRDX4 and the consequent effects of this relationship in attenuating hydrogen peroxide production in the ER and the production of proinflammatory cytokines. This study provides the potential for the development of biomarkers and new therapeutics for renal inflammatory disorders, including hypertension. Conclusion: PRDX4 interacts with D5R to decrease oxidative stress and inflammation in renal cells that may have the potential for translational significance. Antioxid. Redox Signal. 38, 1150-1166.

HIV-1 Vpr suppresses expression of the thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubule.

HIV-associated nephropathy (HIVAN) impairs functions of both glomeruli and tubules. Attention has been previously focused on the HIVAN glomerulopathy. Tubular injury has drawn increased attention because sodium wasting is common in hospitalized HIV/AIDS patients. We used viral protein R (Vpr)-transgenic mice to investigate the mechanisms whereby Vpr contributes to urinary sodium wasting. In phosphoenolpyruvate carboxykinase promoter-driven Vpr-transgenic mice, in situ hybridization showed that Vpr mRNA was expressed in all nephron segments, including the distal convoluted tubule. Vpr-transgenic mice, compared with wild-type littermates, markedly increased urinary sodium excretion, despite similar plasma renin activity and aldosterone levels. Kidneys from Vpr-transgenic mice also markedly reduced protein abundance of the Na+-Cl- cotransporter (NCC), while mineralocorticoid receptor (MR) protein expression level was unchanged. In African green monkey kidney cells, Vpr abrogated the aldosterone-mediated stimulation of MR transcriptional activity. Gene expression of Slc12a3 (NCC) in Vpr-transgenic mice was significantly lower compared with wild-type mice, assessed by both qRT-PCR and RNAScope in situ hybridization analysis. Chromatin immunoprecipitation assays identified multiple MR response elements (MRE), located from 5 kb upstream of the transcription start site and extending to the third exon of the SLC12A3 gene. Mutation of MRE and SP1 sites in the SLC12A3 promoter region abrogated the transcriptional responses to aldosterone and Vpr, indicating that functional MRE and SP1 are required for the SLC12A3 gene suppression in response to Vpr. Thus, Vpr attenuates MR transcriptional activity and inhibits Slc12a3 transcription in the distal convoluted tubule and contributes to salt wasting in Vpr-transgenic mice.

Isolation of Lipid Rafts by the Detergent-Based and Non-detergent-Based Methods for Localization of GPCRs with Immunoblotting and Laser Scanning Confocal Microscopy.

The understanding of how biological membranes are organized and how they function has constantly been evolving over the past decades. Instead of just serving as a medium in which specific proteins are located, certain parts of the lipid bilayer contribute to platforms that assemble signaling complexes by providing a microenvironment that facilitates 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. Lipid rafts are essential 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 methodologies to study lipid rafts, in general, and of the workings of GPCRs in lipid rafts, in particular, and the inescapable 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 appears to be the best way for drawing conclusions with regard to the distribution and activity of GPCRs in lipid rafts.

NFAT5 Is Involved in GRP-Enhanced Secretion of GLP-1 by Sodium.

Gastrin, secreted by G-cells, and glucagon-like peptide-1 (GLP-1), secreted by L-cells, may participate in the regulation of sodium balance. We studied the effect of sodium in mice in vivo and mouse ileum and human L-cells, on GLP-1 secretion, and the role of NFAT5 and gastrin-releasing peptide receptor (GRPR) in this process. A high-sodium diet increases serum GLP-1 levels in mice. Increasing sodium concentration stimulates GLP-1 secretion from mouse ileum and L-cells. GRP enhances the high sodium-induced increase in GLP-1 secretion. High sodium increases cellular GLP-1 expression, while low and high sodium concentrations increase NFAT5 and GRPR expression. Silencing NFAT5 in L-cells abrogates the stimulatory effect of GRP on the high sodium-induced GLP-1 secretion and protein expression, and the sodium-induced increase in GRPR expression. GLP-1 and gastrin decrease the expression of Na+-K+/ATPase and increase the phosphorylation of sodium/hydrogen exchanger type 3 (NHE3) in human renal proximal tubule cells (hRPTCs). This study gives a new perspective on the mechanisms of GLP-1 secretion, especially that engendered by ingested sodium, and the ability of GLP-1, with gastrin, to decrease Na+-K+/ATPase expression and NHE3 function in hRPTCs. These results may contribute to the better utilization of current and future GLP-1-based drugs in the treatment of hypertension.

Transplantation of Apoptosis-Resistant Endothelial Progenitor Cells Improves Renal Function in Diabetic Kidney Disease.

Background Diabetic kidney disease is associated with glomerulosclerosis and poor renal perfusion. Increased capillary formation and improved perfusion may help to halt or reverse the injury. Transplanting apoptosis-resistant p53-silenced endothelial progenitor cells (p53sh-EPCs) may help improve vascularization and renal perfusion and could be more beneficial than another stem cell such as the mouse mesenchymal stromal cell (mMSC). Methods and Results Hyperglycemia and proteinuria were confirmed at 8 to 10 weeks in streptozotocin-induced type1 diabetic C57Bl/6 mice, followed by transplantation of 0.3 million p53sh-EPCs, Null-EPCs (control), or mMSC under each kidney capsule. Urine was collected weekly for creatinine and protein levels. Blood pressure was measured by direct arterial cannulation and renal perfusion was measured by renal ultrasound. The kidneys were harvested for histology and mRNA expression. Reduction of protein/creatinine (AUC) was observed in p53sh-EPC-transplanted mice more than null-EPC (1.8-fold, P=0.03) or null-mMSC (1.6-fold, P=0.04, n=4) transplanted mice. Markers for angiogenesis, such as endothelial nitric oxide synthase (1.7-fold, P=0.06), were upregulated post p53sh-EPC transplantation compared with null EPC. However, vascular endothelial growth factor-A expression was reduced (7-fold, P=0.0004) in mMSC-transplanted mice, compared with p53sh-EPC-transplanted mice. Isolectin-B4 staining of kidney section showed improvement of glomerular sclerosis when p53sh-EPC was transplanted, compared with null-EPC or mMSC. In addition, mean and peak renal blood velocity (1.3-fold, P=0.01, 1.4-fold, P=0.001, respectively) were increased in p53sh-EPC-transplanted mice, relative to null-EPC transplanted mice. Conclusions Apoptosis-resistant p53sh EPC transplantation could be beneficial in the treatment of diabetic kidney disease by decreasing proteinuria, and improving renal perfusion and glomerular architecture.

SNX-PXA-RGS-PXC Subfamily of SNXs in the Regulation of Receptor-Mediated Signaling and Membrane Trafficking.

The SNX-PXA-RGS-PXC subfamily of sorting nexins (SNXs) belongs to the superfamily of SNX proteins. SNXs are characterized by the presence of a common phox-homology (PX) domain, along with other functional domains that play versatile roles in cellular signaling and membrane trafficking. In addition to the PX domain, the SNX-PXA-RGS-PXC subfamily, except for SNX19, contains a unique RGS (regulators of G protein signaling) domain that serves as GTPase activating proteins (GAPs), which accelerates GTP hydrolysis on the G protein α subunit, resulting in termination of G protein-coupled receptor (GPCR) signaling. Moreover, the PX domain selectively interacts with phosphatidylinositol-3-phosphate and other phosphoinositides found in endosomal membranes, while also associating with various intracellular proteins. Although SNX19 lacks an RGS domain, all members of the SNX-PXA-RGS-PXC subfamily serve as dual regulators of receptor cargo signaling and endosomal trafficking. This review discusses the known and proposed functions of the SNX-PXA-RGS-PXC subfamily and how it participates in receptor signaling (both GPCR and non-GPCR) and endosomal-based membrane trafficking. Furthermore, we discuss the difference of this subfamily of SNXs from other subfamilies, such as SNX-BAR nexins (Bin-Amphiphysin-Rvs) that are associated with retromer or other retrieval complexes for the regulation of receptor signaling and membrane trafficking. Emerging evidence has shown that the dysregulation and malfunction of this subfamily of sorting nexins lead to various pathophysiological processes and disorders, including hypertension.

Elucidating the Role of Lipid Rafts on G Protein-Coupled Receptor Function in the Mouse Kidney: An In Vivo Approach.

Numerous G protein-coupled receptors (GPCRs) and GPCR-signaling molecules reside in lipid rafts and thus, are inherently regulated in these microdomains. However, the limitations of current methods to investigate lipid raft biology and GPCR activity in situ have hindered the complete understanding of the molecular underpinnings of GPCR trafficking and signaling, especially in the whole organism. This book chapter details an innovative in vivo approach to study the crucial role of lipid rafts on the workings of GPCRs in the mouse kidney. This protocol involves the use of a modified mini osmotic pump to deliver an agent that selectively disrupts the lipid raft in the kidney.

Lipid Rafts and Dopamine Receptor Signaling.

The renal dopaminergic system has been identified as a modulator of sodium balance and blood pressure. According to the Centers for Disease Control and Prevention, in 2018 in the United States, almost half a million deaths included hypertension as a primary or contributing cause. Renal dopamine receptors, members of the G protein-coupled receptor family, are divided in two groups: D1-like receptors that act to keep the blood pressure in the normal range, and D2-like receptors with a variable effect on blood pressure, depending on volume status. The renal dopamine receptor function is regulated, in part, by its expression in microdomains in the plasma membrane. Lipid rafts form platforms within the plasma membrane for the organization and dynamic contact of molecules involved in numerous cellular processes such as ligand binding, membrane sorting, effector specificity, and signal transduction. Understanding all the components of lipid rafts, their interaction with renal dopamine receptors, and their signaling process offers an opportunity to unravel potential treatment targets that could halt the progression of hypertension, chronic kidney disease (CKD), and their complications.

ND-13, a DJ-1-Derived Peptide, Attenuates the Renal Expression of Fibrotic and Inflammatory Markers Associated with Unilateral Ureter Obstruction.

DJ-1 is a redox-sensitive chaperone with reported antioxidant and anti-inflammatory properties in the kidney. The 20 amino acid (aa) peptide ND-13 consists of 13 highly conserved aas from the DJ-1 sequence and a TAT-derived 7 aa sequence that helps in cell penetration. This study aimed to determine if ND-13 treatment prevents the renal damage and inflammation associated with unilateral ureter obstruction (UUO). Male C57Bl/6 and DJ-1-/- mice underwent UUO and were treated with ND-13 or vehicle for 14 days. ND-13 attenuated the renal expression of fibrotic markers TGF-ß and collagen1a1 (Col1a1) and inflammatory markers TNF-α and IL-6 in C57Bl/6 mice. DJ-1-/- mice treated with ND-13 presented similar decreased expression of TNF-α, IL-6 and TGF-ß. However, in contrast to C57Bl/6 mice, ND-13 failed to prevent renal fibrosis or to ameliorate the expression of Col1a1 in this genotype. Further, UUO led to elevated urinary levels of the proximal tubular injury marker neutrophil gelatinase-associated lipocalin (NGAL) in DJ-1-/- mice, which were blunted by ND-13. Our results suggest that ND-13 protects against UUO-induced renal injury, inflammation and fibrosis. These are all crucial mechanisms in the pathogenesis of kidney injury. Thus, ND-13 may be a new therapeutic approach to prevent renal diseases.

Sorting nexin 1 loss results in increased oxidative stress and hypertension.

Acute renal depletion of sorting nexin 1 (SNX1) in mice results in blunted natriuretic response and hypertension due to impaired dopamine D5 receptor (D5 R) activity. We elucidated the molecular mechanisms for these phenotypes in Snx1-/- mice. These mice had increased renal expressions of angiotensin II type 1 receptor (AT1 R), NADPH oxidase (NOX) subunits, D5 R, 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 D5 R 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 D5 R 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.

Lipid rafts are required for effective renal D1 dopamine receptor function.

Effective receptor signaling is anchored on the preferential localization of the receptor in lipid rafts, which are plasma membrane platforms replete with cholesterol and sphingolipids. We hypothesized that the dopamine D1 receptor (D1 R) contains structural features that allow it to reside in lipid rafts for its activity. Mutation of C347 palmitoylation site and Y218 of a newly identified Cholesterol Recognition Amino Acid Consensus motif resulted in the exclusion of D1 R from lipid rafts, blunted cAMP response, impaired sodium transport, and increased oxidative stress in renal proximal tubule cells (RPTCs). Kidney-restricted silencing of Drd1 in C57BL/6J mice increased blood pressure (BP) that was normalized by renal tubule-restricted rescue with D1 R-wild-type but not the mutant D1 R 347A that lacks a palmitoylation site. Kidney-restricted disruption of lipid rafts by ß-MCD jettisoned the D1 R from the brush border, decreased sodium excretion, and increased oxidative stress and BP in C57BL/6J mice. Deletion of the PX domain of the novel D1 R-binding partner sorting nexin 19 (SNX19) resulted in D1 R partitioning solely to non-raft domains, while silencing of SNX19 impaired D1 R function in RPTCs. Kidney-restricted silencing of Snx19 resulted in hypertension in C57BL/6J mice. Our results highlight the essential role of lipid rafts for effective D1 R signaling.

Renal Hydrogen Peroxide Production Prevents Salt-Sensitive Hypertension.

Background The regulation of sodium excretion is important in the pathogenesis of hypertension and salt sensitivity is predictive of cardiovascular events and mortality. C57Bl/6 and BALB/c mice have different blood pressure sensitivities to salt intake. High salt intake increases blood pressure in some C57Bl/6J mouse strains but not in any BALB/c mouse strain. Methods and Results We determined the cause of the difference in salt sensitivity between C57Bl/6 and BALB/c mice. Basal levels of superoxide and H2O2 were higher in renal proximal tubule cells (RPTCs) from BALB/c than C57Bl/6J mice. High salt diet increased H2O2 production in kidneys from BALB/c but C57Bl/6J mice. High sodium concentration (170 mmol/L) in the incubation medium increased H2O2 levels in BALB/c-RPTCs but not in C57Bl/6J-RPTCs. H2O2 (10 µmol/L) treatment decreased sodium transport in RPTCs from BALB/c but not C57Bl/6J mice. Overexpression of catalase in the mouse kidney predisposed BALB/c mice to salt-sensitive hypertension. Conclusions Our data show that the level of salt-induced H2O2 production negatively regulates RPTC sodium transport and determines the state of salt sensitivity in 2 strains of mice. High concentrations of antioxidants could prevent H2O2 production in renal proximal tubules, which would result in sodium retention and increased blood pressure.

Dopamine D2 receptor modulates Wnt expression and control of cell proliferation.

The Wnt/ß-catenin pathway is one of the most conserved signaling pathways across species with essential roles in development, cell proliferation, and disease. Wnt signaling occurs at the protein level and via ß-catenin-mediated transcription of target genes. However, little is known about the underlying mechanisms regulating the expression of the key Wnt ligand Wnt3a or the modulation of its activity. Here, we provide evidence that there is significant cross-talk between the dopamine D2 receptor (D2R) and Wnt/ß-catenin signaling pathways. Our data suggest that D2R-dependent cross-talk modulates Wnt3a expression via an evolutionarily-conserved TCF/LEF site within the WNT3A promoter. Moreover, D2R signaling also modulates cell proliferation and modifies the pathology in a renal ischemia/reperfusion-injury disease model, via its effects on Wnt/ß-catenin signaling. Together, our results suggest that D2R is a transcriptional modulator of Wnt/ß-catenin signal transduction with broad implications for health and development of new therapeutics.

Uncoupling Protein 2 Increases Blood Pressure in DJ -1 Knockout Mice.

Background The redox-sensitive chaperone DJ -1 and uncoupling protein 2 are protective against mitochondrial oxidative stress. We previously reported that renal-selective depletion and germline deletion of DJ -1 increases blood pressure in mice. This study aimed to determine the mechanisms involved in the oxidative stress-mediated hypertension in DJ -1 -/- mice. Methods and Results There were no differences in sodium excretion, renal renin expression, renal NADPH oxidase activity, and serum creatinine levels between DJ -1 -/- and wild-type mice. Renal expression of nitro-tyrosine, malondialdehyde, and urinary kidney injury marker-1 were increased in DJ -1 -/- mice relative to wild-type littermates. mRNA expression of mitochondrial heat shock protein 60 was also elevated in kidneys from DJ -1 -/- mice, indicating the presence of oxidative stress. Tempol-treated DJ -1 -/- mice presented higher serum nitrite/nitrate levels than vehicle-treated DJ -1 -/- mice, suggesting a role of the NO system in the high blood pressure of this model. Tempol treatment normalized renal kidney injury marker-1 and malondialdehyde expression as well as blood pressure in DJ -1 -/- mice, but had no effect in wild-type mice. The renal Ucp2 mRNA expression was increased in DJ -1 -/- mice versus wild-type and was also normalized by tempol. The renal-selective silencing of Ucp2 led to normalization of blood pressure and serum nitrite/nitrate ratio in DJ -1 -/- mice. Conclusions The deletion of DJ -1 leads to oxidative stress-induced hypertension associated with downregulation of NO function, and overexpression of Ucp2 in the kidney increases blood pressure in DJ -1 -/- mice. To our knowledge, this is the first report providing evidence of the role of uncoupling protein 2 in blood pressure regulation.

Alpha2B-Adrenergic Receptor Overexpression in the Brain Potentiate Air Pollution-induced Behavior and Blood Pressure Changes.

Fine ambient particulate matter (PM2.5) is able to induce sympathetic activation and inflammation in the brain. However, direct evidence demonstrating an essential role of sympathetic activation in PM2.5-associated disease progression is lacking. We assess the contribution of α2B-adrenergic receptor (Adra2b) in air pollution-associated hypertension and behavioral changes in this study. Wild-type mice and Adra2b-transgenic mice overexpressing Adra2b in the brain (Adra2bTg) were exposed to concentrated PM2.5 or filtered air for 3 months via a versatile aerosol concentrator exposure system. Mice were fed with a high salt diet (4.0% NaCl) for 1 week at week 11 of exposure to induce blood pressure elevation. Intra-arterial blood pressure was monitored by radio-telemetry and behavior changes were assessed by open field, light-dark, and prepulse inhibition tests. PM2.5 exposure increased Adra2b in the brain of wild-type mice. Adra2b overexpression enhanced the anxiety-like behavior and high salt diet-induced blood pressure elevation in response to air pollution but not filtered air exposure. Adra2b overexpression induced upregulation of inflammatory genes such as TLR2, TLR4, and IL-6 in the brain exposed to PM2.5. In addition, there were increased frequencies of activated effector T cells and increased expression of oxidative stress-related genes, such as SOD1, NQO1, Nrf2, and Gclm in Adra2bTg mice compared with wild-type mice. Our results provide new evidence of distinct behavioral changes consistent with anxiety and blood pressure elevation in response to high salt intake and air pollution exposure, highlighting the importance of centrally expressed Adra2b in the vulnerability to air pollution exposure.

G protein-coupled receptor 37L1 regulates renal sodium transport and blood pressure.

G protein-coupled receptors (GPCRs) in the kidney regulate the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Abnormalities in renal epithelial ion transport play important roles in the pathogenesis of essential hypertension. The orphan G protein-coupled receptor 37L1 (GPR37L1), also known as endothelin receptor type B-like protein (ETBR-LP2), is expressed in several regions in the brain, but its expression profile and function in peripheral tissues are poorly understood. We found that GPR37L1 mRNA expression is highest in the brain, followed by the stomach, heart, testis, and ovary, with moderate expression in the kidney, pancreas, skeletal muscle, liver, lung, and spleen. Immunofluorescence analyses revealed the expression of GPR37L1 in specific regions within some organs. In the kidney, GPR37L1 is expressed in the apical membrane of renal proximal tubule cells. In human renal proximal tubule cells, the transient expression of GPR37LI increased intracellular sodium, whereas the silencing of GPR37LI decreased intracellular sodium. Inhibition of Na+/H+ exchanger isoform 3 (NHE3) activity abrogated the GPR37L1-mediated increase in intracellular sodium. Renal-selective silencing of Gpr37l1 in mice increased urine output and sodium excretion and decreased systolic and diastolic blood pressures. The renal-selective silencing of GPR37L1 decreased the protein expression of NHE3 but not the expression of Na+-K+-ATPase or sodium-glucose cotransporter 2. Our findings show that in the kidney, GPR37L1 participates in renal proximal tubule luminal sodium transport and regulation of blood pressure by increasing the renal expression and function of NHE3 by decreasing cAMP production. The role of GPR37L1, expressed in specific cell types in organs other than the kidney, remains to be determined.

Antihypertensive effect of etamicastat in dopamine D2 receptor-deficient mice.

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-ß-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.

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 Henle's 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.

Loss of renal SNX5 results in impaired IDE activity and insulin resistance in mice.

AIMS/HYPOTHESIS: We hypothesised that renal sorting nexin 5 (SNX5) regulates the insulin-degrading enzyme (IDE) and, thus, circulating insulin levels. We therefore studied the dynamic interaction between SNX5 and IDE in human renal proximal tubule cells (hRPTCs), as well as in rat and mouse kidneys. METHODS: The regulation of IDE by SNX5 expressed in the kidney was studied in vitro and in vivo. Snx5 or mock siRNA was added to immortalised hRPTCs (passage <20) in culture or selectively infused, via osmotic mini-pump, into the remnant kidney of uninephrectomised mice and rats. RESULTS: SNX5 co-localised with IDE at the plasma membrane and perinuclear area of hRPTCs and in the brush border membrane of proximal tubules of human, rat, and mouse kidneys. Insulin increased the co-localisation and co-immunoprecipitation of SNX5 and IDE in hRPTCs. Silencing SNX5 in hRPTCs decreased IDE expression and activity. Renal-selective silencing of Snx5 (SNX5 protein: 100 ± 25 vs 29 ± 10, p < 0.05 [% of control]) in C57Bl/6J mice decreased IDE protein (100 ± 13 vs 57 ± 6, p < 0.05 [% of control]) and urinary insulin excretion, impaired the responses to insulin and glucose, and increased blood insulin and glucose levels. Spontaneously hypertensive rats (SHRs) had increased blood insulin and glucose levels and decreased renal SNX5 (100 ± 27 vs 29 ± 6, p < 0.05 [% of control]) and IDE (100 ± 5 vs 75 ± 4, p < 0.05 [% of control]) proteins, compared with normotensive Wistar-Kyoto (WKY) rats. Kidney Snx5-depleted WKY rats also had increased blood insulin and glucose levels. The expression of SNX5 and IDE was decreased in RPTCs from SHRs and hypertensive humans compared with cells from normotensive volunteers, indicating a common cause for hyperinsulinaemia and hypertension. CONCLUSIONS/INTERPRETATION: Renal SNX5 positively regulates IDE expression and function. This study is the first to demonstrate the novel and crucial role of renal SNX5 in insulin and glucose metabolism.

Primary Pediatric Hypertension: Current Understanding and Emerging Concepts.

The rising prevalence of primary pediatric hypertension and its tracking into adult hypertension point to the importance of determining its pathogenesis to gain insights into its current and emerging management. Considering that the intricate control of BP is governed by a myriad of anatomical, molecular biological, biochemical, and physiological systems, multiple genes are likely to influence an individual's BP and susceptibility to develop hypertension. The long-term regulation of BP rests on renal and non-renal mechanisms. One renal mechanism relates to sodium transport. The impaired renal sodium handling in primary hypertension and salt sensitivity may be caused by aberrant counter-regulatory natriuretic and anti-natriuretic pathways. The sympathetic nervous and renin-angiotensin-aldosterone systems are examples of antinatriuretic pathways. An important counter-regulatory natriuretic pathway is afforded by the renal autocrine/paracrine dopamine system, aberrations of which are involved in the pathogenesis of hypertension, including that associated with obesity. We present updates on the complex interactions of these two systems with dietary salt intake in relation to obesity, insulin resistance, inflammation, and oxidative stress. We review how insults during pregnancy such as maternal and paternal malnutrition, glucocorticoid exposure, infection, placental insufficiency, and treatments during the neonatal period have long-lasting effects in the regulation of renal function and BP. Moreover, these effects have sex differences. There is a need for early diagnosis, frequent monitoring, and timely management due to increasing evidence of premature target organ damage. Large controlled studies are needed to evaluate the long-term consequences of the treatment of elevated BP during childhood, especially to establish the validity of the current definition and treatment of pediatric hypertension.