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.

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.

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.

Genetic polymorphisms associated with reactive oxygen species and blood pressure regulation.

Hypertension is the most prevalent cause of cardiovascular disease and kidney failure, but only about 50% of patients achieve adequate blood pressure control, in part, due to inter-individual genetic variations in the response to antihypertensive medication. Significant strides have been made toward the understanding of the role of reactive oxygen species (ROS) in the regulation of the cardiovascular system. However, the role of ROS in human hypertension is still unclear. Polymorphisms of some genes involved in the regulation of ROS production are associated with hypertension, suggesting their potential influence on blood pressure control and response to antihypertensive medication. This review provides an update on the genes associated with the regulation of ROS production in hypertension and discusses the controversies on the use of antioxidants in the treatment of hypertension, including the antioxidant effects of antihypertensive drugs.

The emerging role of sorting nexins in cardiovascular diseases.

The sorting nexin (SNX) family consists of a diverse group of cytoplasmic- and membrane-associated phosphoinositide-binding proteins that play pivotal roles in the regulation of protein trafficking. This includes the entire endocytic pathway, such as endocytosis, endosomal sorting, and endosomal signaling. Dysfunctions of SNX pathway are involved in several forms of cardiovascular disease (CVD). Moreover, SNX gene variants are associated with CVDs. In this review, we discuss the current knowledge on SNX-mediated regulatory mechanisms and their roles in the pathogenesis and treatment of CVDs.

Non-additive effects of ACVR2A in preeclampsia in a Philippine population.

BACKGROUND: Multiple interrelated pathways contribute to the pathogenesis of preeclampsia, and variants in susceptibility genes may play a role among Filipinos, an ethnically distinct group with high prevalence of the disease. The objective of this study was to examine the association between variants in maternal candidate genes and the development of preeclampsia in a Philippine population. METHODS: A case-control study involving 29 single nucleotide polymorphisms (SNPs) in 21 candidate genes was conducted in 150 patients with preeclampsia (cases) and 175 women with uncomplicated normal pregnancies (controls). Genotyping for the GRK4 and DRD1 gene variants was carried out using the TaqMan Assay, and all other variants were assayed using the Sequenom MassARRAY Iplex Platform. PLINK was used for SNP association testing. Multilocus association analysis was performed using multifactor dimensionality reduction (MDR) analysis. RESULTS: Among the clinical factors, older age (P <  1 × 10-4), higher BMI (P <  1 × 10-4), having a new partner (P = 0.006), and increased time interval from previous pregnancy (P = 0.018) associated with preeclampsia. The MDR algorithm identified the genetic variant ACVR2A rs1014064 as interacting with age and BMI in association with preeclampsia among Filipino women. CONCLUSIONS: The MDR algorithm identified an interaction between age, BMI and ACVR2A rs1014064, indicating that context among genetic variants and demographic/clinical factors may be crucial to understanding the pathogenesis of preeclampsia among Filipino women.

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.

VEGF-A and VEGFR1 SNPs associate with preeclampsia in a Philippine population.

The vascular endothelial growth factor (VEGF) family is important for establishing normal pregnancy, and related single nucleotide polymorphisms (SNPs) are implicated in abnormal placentation and preeclampsia. We evaluated the association between preeclampsia and several VEGF SNPs among Filipinos, an ethnically distinct group with high prevalence of preeclampsia. The genotypes and allelic variants were determined in a case-control study (191 controls and 165 preeclampsia patients) through SNP analysis of VEGF-A (rs2010963, rs3025039) and VEGF-C (rs7664413) and their corresponding receptors VEGFR1 (rs722503, rs12584067, rs7335588) and VEGFR3 (rs307826) from venous blood DNA. VEGF-A rs3025039 C allele has been shown to associate with preeclampsia (odds ratio of 1.648 (1.03-2.62)), while the T allele bestowed an additive effect for the maintenance of normal, uncomplicated pregnancy and against the development of preeclampsia (odds ratio of 0.62 (0.39-0.98)). VEGFR1 rs722503 is associated with preeclampsia occurring at or after the age of 40 years. The results showed that genetic variability of VEGF-A and VEGFR1 are important in the etiology of preeclampsia among Filipinos.

Dopamine D3 receptor inhibits the ubiquitin-specific peptidase 48 to promote NHE3 degradation.

The dopamine D3 receptor (D3R) is crucial in the regulation of blood pressure and sodium balance, in that Drd3 gene ablation in mice results in hypertension and failure to excrete a dietary salt load. The mechanism responsible for the renal sodium retention in these mice is largely unknown. We now offer and describe a novel mechanism by which D3R decreases sodium transport in the long term by inhibiting the deubiquitinylating activity of ubiquitin-specific peptidase 48 (USP48), thereby promoting Na(+)-H(+) exchanger (NHE)-3 degradation. We found that stimulation with the D3R-specific agonist PD128907 (1 µM, 30 min) promoted the interaction and colocalization among D3R, NHE3, and USP48; inhibited USP48 activity (-35±6%, vs. vehicle), resulting in increased ubiquitinylated NHE3 (+140±10%); and decreased NHE3 expression (-50±9%) in human renal proximal tubule cells (hRPTCs). USP48 silencing decreased NHE3's half-life (USP48 siRNA t1/2=6.1 h vs. vehicle t1/2=12.9 h), whereas overexpression of USP48 increased NHE3 half-life (t1/2=21.8 h), indicating that USP48 protects NHE3 from degradation via deubiquitinylation. USP48 accounted for ∼30% of the total deubiquitinylating activity in these cells. Extending our studies in vivo, we found that pharmacologic blockade of D3R via the D3R-specific antagonist GR103691 (1 µg/kg/min, 4 d) in C57Bl/6J mice increased renal NHE3 expression (+310±15%, vs. vehicle), whereas an innovative kidney-restricted Usp48 silencing via siRNA (3 µg/d, 7 d) increased ubiquitinylated NHE3 (+250±30%, vs. controls), decreased total NHE3 (-23±2%), and lowered blood pressure (-24±2 mm Hg), compared with that in control mice that received either the vehicle or nonsilencing siRNA. Our data demonstrate a crucial role for the dynamic interaction between D3R and USP48 in the regulation of NHE3 expression and function.

Sorting nexin 1 loss results in D5 dopamine receptor dysfunction in human renal proximal tubule cells and hypertension in mice.

The peripheral dopaminergic system plays a crucial role in blood pressure regulation through its actions on renal hemodynamics and epithelial ion transport. The dopamine D5 receptor (D(5)R) interacts with sorting nexin 1 (SNX1), a protein involved in receptor retrieval from the trans-Golgi network. In this report, we elucidated the spatial, temporal, and functional significance of this interaction in human renal proximal tubule cells and HEK293 cells stably expressing human D(5)R and in mice. Silencing of SNX1 expression via RNAi resulted in the failure of D(5)R to internalize and bind GTP, blunting of the agonist-induced increase in cAMP production and decrease in sodium transport, and up-regulation of angiotensin II receptor expression, of which expression was previously shown to be negatively regulated by D(5)R. Moreover, siRNA-mediated depletion of renal SNX1 in C57BL/6J and BALB/cJ mice resulted in increased blood pressure and blunted natriuretic response to agonist in salt-loaded BALB/cJ mice. These data demonstrate a crucial role for SNX1 in D(5)R trafficking and that SNX1 depletion results in D(5)R dysfunction and thus may represent a novel mechanism for the pathogenesis of essential hypertension.

Dopamine D1-like receptors regulate the α1A-adrenergic receptor in human renal proximal tubule cells and D1-like dopamine receptor knockout mice.

The homeostatic control of blood pressure hinges upon the delicate balance between prohypertensinogenic and antihypertensinogenic systems. D1-like dopamine receptors [dopamine D1 and D5 receptors (D1Rs and D5Rs, respectively)] and the α1A-adrenergic receptor (α1A-AR) are expressed in the renal proximal tubule and engender opposing effects on Na(+) transport, i.e., natriuresis (via D1Rs and D5Rs) or antinatriuresis (via α1A-ARs). We tested the hypothesis that the D1R/D5R regulates the α1A-AR. D1-like dopamine receptors coimmunoprecipitated, colocalized, and cofractionated with α1A-ARs in lipid rafts in immortalized human renal proximal tubule cells. Long-term treatment with the D1R/D5R agonist fenoldopam resulted in decreased D1R and D5R expression but increased α1A-AR abundance in the plasma membrane. Short-term fenoldopam treatment stimulated the translocation of Na(+)-K(+)-ATPase from the plasma membrane to the cytosol that was partially reversed by an α1A-AR agonist, which by itself induced Na(+)-K(+)-ATPase translocation from the cytosol to the plasma membrane. The α1A-AR-specific agonist A610603 also minimized the ability of fenoldopam to inhibit Na(+)-K(+)-ATPase activity. To determine the interaction among D1Rs, D5Rs, and α1A-ARs in vivo, we used phenylephrine and A610603 to decrease Na(+) excretion in several D1-like dopamine receptor knockout mouse strains. Phenylephrine and A61603 treatment resulted in a partial reduction of urinary Na(+) excretion in wild-type mice and its abolition in D1R knockout, D5R knockout, and D1R-D5R double-knockout mice. Our results demonstrate the ability of the D1-like dopamine receptors to regulate the expression and activity of α1A-AR. Elucidating the intricacies of the interaction among these receptors is crucial for a better understanding of the crosstalk between anti- and pro-hypertensive systems.

D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus.

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: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-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 D1ßR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1ßR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1ßR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-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 D1ßR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1ßR 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.

Novel role of sorting nexin 5 in renal D(1) dopamine receptor trafficking and function: implications for hypertension.

The D1 dopamine receptor (D1R) is widely expressed in the kidney and plays a crucial role in blood pressure regulation. Although much is known about D1R desensitization, especially through G-protein-coupled receptor kinase 4 (GRK4), comparatively little is known about other aspects of D1R trafficking and the proteins involved in the process. We now report the discovery of a dynamic interaction between sorting nexin 5 (SNX5), a component of the mammalian retromer, and D1R in human renal epithelial cells. We show that internalization of agonist-activated D1R is regulated by both SNX5 and GRK4, and that SNX5 is critical to the recycling of the receptor to the plasma membrane. SNX5 depletion increases agonist-activated D1R phosphorylation (>50% at basal condition), prevents D1R internalization and cAMP response, and delays receptor recycling compared to mock siRNA-transfected controls. Moreover, renal restricted subcapsular infusion of Snx5-specific siRNA (vs. mock siRNA) decreases sodium excretion (Δ=-0.2±0.005 mEq/mg creatinine) and further elevates the systolic blood pressure (Δ=48±5 mm Hg) in spontaneously hypertensive rats, indicating that SNX5 depletion impairs renal D1R function. These studies demonstrate an essential role for SNX5 in regulating D1R function, which may have important diagnostic, prognostic, and therapeutic implications in the management of essential hypertension.

Renal autocrine neuropeptide FF (NPFF) signaling regulates blood pressure.

The kidney and brain play critical roles in the regulation of blood pressure. Neuropeptide FF (NPFF), originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the roles of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, are not known. In this study, we found that the transcripts and proteins of NPFF and its receptors, NPFF-R1 and NPFF-R2, were expressed in mouse and human renal proximal tubules (RPTs). In mouse RPT cells (RPTCs), NPFF, but not RF-amide-related peptide-2 (RFRP-2), decreased the forskolin-stimulated cAMP production in a concentration- and time-dependent manner. Furthermore, dopamine D1-like receptors colocalized and co-immunoprecipitated with NPFF-R1 and NPFF-R2 in human RPTCs. The increase in cAMP production in human RPTCs caused by fenoldopam, a D1-like receptor agonist, was attenuated by NPFF, indicating an antagonistic interaction between NPFF and D1-like receptors. The renal subcapsular infusion of NPFF in C57BL/6 mice decreased renal sodium excretion and increased blood pressure. The NPFF-mediated increase in blood pressure was prevented by RF-9, an antagonist of NPFF receptors. Taken together, our findings suggest that autocrine NPFF and its receptors in the kidney regulate blood pressure, but the mechanisms remain to be determined.

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells.

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R.

Renal Dopamine Receptors and Oxidative Stress: Role in Hypertension.

Significance: The kidney plays an important role in the long-term control of blood pressure. Oxidative stress is one of the fundamental mechanisms responsible for the development of hypertension. Dopamine, via five subtypes of receptors, plays an important role in the control of blood pressure by various mechanisms, including the inhibition of oxidative stress. Recent Advances: Dopamine receptors exert their regulatory function to decrease the oxidative stress in the kidney and ultimately maintain normal sodium balance and blood pressure homeostasis. An aberration of this regulation may be involved in the pathogenesis of hypertension. Critical Issues: Our present article reviews the important role of oxidative stress and intrarenal dopaminergic system in the regulation of blood pressure, summarizes the current knowledge on renal dopamine receptor-mediated antioxidation, including decreasing reactive oxygen species production, inhibiting pro-oxidant enzyme nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and stimulating antioxidative enzymes, and also discusses its underlying mechanisms, including the increased activity of G protein-coupled receptor kinase 4 (GRK4) and abnormal trafficking of renal dopamine receptors in hypertensive status. Future Directions: Identifying the mechanisms of renal dopamine receptors in the regulation of oxidative stress and their contribution to the pathogenesis of hypertension remains an important research focus. Increased understanding of the role of reciprocal regulation between renal dopamine receptors and oxidative stress in the regulation of blood pressure may give us novel insights into the pathogenesis of hypertension and provide a new treatment strategy for 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.

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.

Recent trends in pediatric hypertension research.

Hypertension in the young is increasingly being recognized as an emerging critical healthcare problem, not only because of its increasing prevalence in recent years but also of its significant impact on the health and well-being of children and adolescents and tracking into adult life. A wealth of epidemiological studies has allowed the formulation of guidelines and recommendations on the diagnosis and classification of hypertension in the pediatric group based on normative data. Significant advances in the field of medicine and genetics have paved the way for the identification and demonstration of recent trends in hypertension research in children and adults. This article highlights both established and emerging concepts in order to provide a quick yet panoramic view of the current understanding on pediatric hypertension.