Drug Screening Using Normal Cell and Cancer Cell Mixture in an Automated 3D Cell Culture System.

Three-dimensional (3D) cell culture creates a more physiologically relevant environment for enhanced drug screening capabilities using microcarriers. An automated 3D system that integrates robotic manipulators, liquid handling systems, sensors, and environment control systems has the capacity to handle multiple samples in parallel, perform repetitive tasks, and provide real-time monitoring and analysis. This chapter describes a potential 3D cell culture drug screening model by combining renal proximal tubule cells as a representative normal cell line with cancer cell lines. This combination is subjected to drug screening to evaluate the drug's efficacy in suppressing cancer cells while minimizing impact on normal cells with the added benefit of having the ability to separate the two cell types by magnetic isolation for high content screens including mass spectrometry-based proteomics. This study presents advancements in 3D cell culture techniques, emphasizing the importance of automation and the potential of microcarriers in drug screening and disease modeling.

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.

Bridging the gaps in newborn screening programmes: Challenges and opportunities to detect haemoglobinopathies in Africa.

Background: Haemoglobinopathies, including sickle cell disease and ß-thalassaemia, are monogenic disorders with a relatively higher prevalence among malaria-endemic areas in Africa. Despite this prevalence, most African countries lack the necessary resources for diagnosing and managing these debilitating conditions. Aim: This study provides a critical review of newborn screening for detecting haemoglobinopathies in Africa, highlighting challenges and proposing strategies for improved diagnosis and management. Methods: A literature search on haemoglobinopathies in Africa was conducted in PubMed, Google Scholar and ScienceDirect, using specific keywords and Boolean operators, including articles published from January 1981 to December 2022. Results: The data show that sickle cell disease is prevalent among populations in Central and West Africa; however, ß-thalassaemia is prevalent among people in the northern parts of Africa. Newborn screening pilot initiatives for haemoglobinopathies were being implemented in Angola, Nigeria, Ghana, the Democratic Republic of Congo and the Republic of Benin. The cost of testing, lack of sufficient and accessible medical records, and inadequacy in healthcare infrastructure pose significant challenges in bridging the gaps in newborn screening. Furthermore, the stigmatisation and lack of awareness of haemoglobinopathies and access to newborn screening programmes pose additional challenges. Conclusion: This review highlights the challenges associated with haemoglobinopathy testing, effective strategies for mitigating these challenges, and future perspectives for expanding efforts toward detecting and managing these disorders across Africa. Providing affordable diagnostic tools, mobile clinics, government subsidies, education campaigns, and the implementation of electronic medical records systems could help bridge the gaps in newborn screening in Africa. What this study adds: The study presents a comprehensive view of newborn screening of haemoglobinopathies in Africa, provides a detailed outline of the challenges faced by newborn screening for haemoglobinopathies in Africa, and offers strategies for better diagnosis and care.

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.

Inverse Salt Sensitivity of Blood Pressure Is Associated with an Increased Renin-Angiotensin System Activity.

High and low sodium diets are associated with increased blood pressure and cardiovascular morbidity and mortality. The paradoxical response of elevated BP in low salt diets, aka inverse salt sensitivity (ISS), is an understudied vulnerable 11% of the adult population with yet undiscovered etiology. A linear relationship between the number of single nucleotide polymorphisms (SNPs) in the dopamine D2 receptor (DRD2, rs6276 and 6277), and the sodium myo-inositol cotransporter 2 (SLC5A11, rs11074656), as well as decreased expression of these two genes in urine-derived renal proximal tubule cells (uRPTCs) isolated from clinical study participants suggest involvement of these cells in ISS. Insight into this newly discovered paradoxical response to sodium is found by incubating cells in low sodium (LS) conditions that unveil cell physiologic differences that are then reversed by mir-485-5p miRNA blocker transfection and bypassing the genetic defect by DRD2 re-expression. The renin-angiotensin system (RAS) is an important counter-regulatory mechanism to prevent hyponatremia under LS conditions. Oversensitive RAS under LS conditions could partially explain the increased mortality in ISS. Angiotensin-II (AngII, 10 nmol/L) increased sodium transport in uRPTCs to a greater extent in individuals with ISS than SR. Downstream signaling of AngII is verified by identifying lowered expression of nuclear factor erythroid 2-related factor 2 (NRF2), CCCTC-binding factor (CTCF), and manganese-dependent mitochondrial superoxide dismutase (SOD2) only in ISS-derived uRPTCs and not SR-derived uRPTCs when incubated in LS conditions. We conclude that DRD2 and SLC5A11 variants in ISS may cause an increased low sodium sensitivity to AngII and renal sodium reabsorption which can contribute to inverse salt-sensitive hypertension.

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.

Inverse Salt Sensitivity of Blood Pressure: Mechanisms and Potential Relevance for Prevention of Cardiovascular Disease.

PURPOSE OF REVIEW: To review the etiology of inverse salt sensitivity of blood pressure (BP). RECENT FINDINGS: Both high and low sodium (Na+) intake can be associated with increased BP and cardiovascular morbidity and mortality. However, little is known regarding the mechanisms involved in the increase in BP in response to low Na+ intake, a condition termed inverse salt sensitivity of BP, which affects approximately 15% of the adult population. The renal proximal tubule is important in regulating up to 70% of renal Na+ transport. The renin-angiotensin and renal dopaminergic systems play both synergistic and opposing roles in the regulation of Na+ transport in this nephron segment. Clinical studies have demonstrated that individuals express a "personal salt index" (PSI) that marks whether they are salt-resistant, salt-sensitive, or inverse salt-sensitive. Inverse salt sensitivity results in part from genetic polymorphisms in various Na+ regulatory genes leading to a decrease in natriuretic activity and an increase in renal tubular Na+ reabsorption leading to an increase in BP. This article reviews the potential mechanisms of a new pathophysiologic entity, inverse salt sensitivity of BP, which affects approximately 15% of the general adult population.

Epithelial Sodium Channel Alpha Subunit (αENaC) Is Associated with Inverse Salt Sensitivity of Blood Pressure.

Salt sensitivity of blood pressure (BP) refers to an increase in BP following an increase in dietary salt, which is associated with increased incidence of cardiovascular disease and early death. However, decreased sodium intake also increases mortality and morbidity. Inverse salt sensitivity (ISS), defined as a paradoxical increase in BP on a low-salt diet, about 11% of the population, may be the cause of this phenomenon. The epithelial sodium channel (ENaC) is a major regulator of sodium reabsorption in the kidney. In this study, human renal tubular epithelial cells (hRTC) were cultured from the urine of phenotyped salt study participants. αENaC expression was significantly lower in ISS than salt resistant (SR) hRTC, while ENaC-like channel activity was dramatically increased by trypsin treatment in ISS cells analyzed by patch clamp. αENaC expression was also decreased under high-salt treatment and increased by aldosterone treatment in ISS cells. Moreover, the αENaC variant, rs4764586, was more prevalent in ISS. In summary, αENaC may be associated with ISS hypertension on low salt. These findings may contribute to understanding the mechanisms of ISS and low salt effect on morbidity and mortality.

GRK4, A Potential Link between Hypertension and Breast Cancer.

Hypertension and breast cancer are two common diseases occurring in women. Clinical studies have shown increased breast cancer incidence in hypertensive women. Several lines of evidence demonstrate that G protein-coupled Receptor Kinase 4 (GRK4) could be a common risk factor for hypertension and breast cancer. This article reviews our current understanding of molecular mechanisms of GRK4 in hypertension and breast cancer.

Comparative microsomal proteomics of a model lung cancer cell line NCI-H23 reveals distinct differences between molecular profiles of 3D and 2D cultured cells.

Lung cancer is the leading cause of cancer-related deaths in the USA and worldwide. Yet, about 95% of new drug candidates validated in preclinical phase eventually fail in clinical trials. Such a high attrition rate is attributed mostly to the inability of conventional two-dimensionally (2D) cultured cancer cells to mimic native three-dimensional (3D) growth of malignant cells in human tumors. To ascertain phenotypical differences between these two distinct culture conditions, we carried out a comparative proteomic analysis of a membrane fraction obtained from 3D- and 2D-cultured NSCLC model cell line NCI-H23. This analysis revealed a map of 1,166 (24%) protein species regulated in culture dependent manner, including differential regulation of a subset of cell surface-based CD molecules. We confirmed exclusive expression of CD99, CD146 and CD239 in 3D culture. Furthermore, label-free quantitation, targeting KRas proteoform-specific peptides, revealed upregulation of both wild type and monoallelic KRas4BG12C mutant at the surface of 3D cultured cells. In order to reduce the high attrition rate of new drug candidates, the results of this study strongly suggests exploiting base-line molecular profiling of a large number of patient-derived NSCLC cell lines grown in 2D and 3D culture, prior to actual drug candidate testing.

The Hypertension Related Gene G-Protein Coupled Receptor Kinase 4 Contributes to Breast Cancer Proliferation.

PURPOSE: Clinical studies have shown that breast cancer risk is increased in hypertensive women. The underlying molecular mechanism remains undetermined. The current study tests our hypothesis that G protein coupled receptor kinase 4 (GRK4) is a molecule that links hypertension and breast cancer. GRK4 plays an important role in regulation of renal sodium excretion. Sustained activation of GRK4 as in the circumstances of single nucleotide polymorphism (SNPs) causes hypertension. Expression of GRK4 in the kidney is regulated by cMyc, an oncogene that is amplified in breast cancer. METHODS: Western analysis was used to evaluate GRK4 protein expression in seven breast cancer cell lines. GRK4 gene single nucleotide polymorphism in breast cancer cell lines and in breast cancer cDNA arrays were determined using TaqMan Genotyping qPRC. The function of GRK4 was evaluated in MCF-7 cells with cMyc knock-down and GRK4 re-expression and in MDA-MB-468 cells expressing inducible GRK4 shRNA lentivirus constructs. Nuclei counting and 5-Bromo-2'-deoxy-uridine (BrdU) labeling were used to evaluate cell growth and proliferation. RESULTS: Genotyping of GRK4 SNPs in breast cancer cDNA arrays (n = 94) revealed that the frequency of carrying two hypertension related SNPs A142 V or R65 L is threefold higher in breast cancer patients than in healthy people (P = 7.53E-11). GRK4 protein is expressed in seven breast cancer cell lines but not the benign mammary epithelial cell line, MCF-10A. Three hypertension related SNPs in the GRK4 gene were identified in the breast cancer cell lines. Except for BT20, all other breast cancer lines have 1-3 GRK4 SNPs of which A142 V occurs in all 6 lines. MDA-MB-468 cells contain homozygous A142 V and R65 L SNPs. Knocking down cMyc in MCF-7 cells significantly reduced the growth rate, which was rescued by re-expression of GRK4. We then generated three stable GRK4 knock-down MDA-MB-468 lines using inducible lentiviral shRNA vectors. Doxycycline (Dox) induced GRK4 silencing significantly reduced GRK4 mRNA and protein levels, growth rates, and proliferation. As a marker of cell proliferation, the percentage of BrdU-labeled cells decreased from 45 ± 3% in the cells without Dox to 32 ± 5% in the cells treated with 0.1 µg/mL Dox. CONCLUSIONS: GRK4 acts as an independent proliferation promotor in breast cancer. Our results suggest that targeted inhibition of GRK4 could be a new therapy for both hypertension and breast cancer.

Dopamine D5 receptor-mediated decreases in mitochondrial reactive oxygen species production are cAMP and autophagy dependent.

Overproduction of reactive oxygen species (ROS) plays an important role in the pathogenesis of hypertension. The dopamine D5 receptor (D5R) is known to decrease ROS production, but the mechanism is not completely understood. In HEK293 cells overexpressing D5R, fenoldopam, an agonist of the two D1-like receptors, D1R and D5R, decreased the production of mitochondria-derived ROS (mito-ROS). The fenoldopam-mediated decrease in mito-ROS production was mimicked by Sp-cAMPS but blocked by Rp-cAMPS. In human renal proximal tubule cells with DRD1 gene silencing to eliminate the confounding effect of D1R, fenoldopam still decreased mito-ROS production. By contrast, Sch23390, a D1R and D5R antagonist, increased mito-ROS production in the absence of D1R, D5R is constitutively active. The fenoldopam-mediated inhibition of mito-ROS production may have been related to autophagy because fenoldopam increased the expression of the autophagy hallmark proteins, autophagy protein 5 (ATG5), and the microtubule-associated protein 1 light chain (LC)3-II. In the presence of chloroquine or spautin-1, inhibitors of autophagy, fenoldopam further increased ATG5 and LC3-II expression, indicating an important role of D5R in the positive regulation of autophagy. However, when autophagy was inhibited, fenoldopam was unable to inhibit ROS production. Indeed, the levels of these autophagy hallmark proteins were decreased in the kidney cortices of Drd5-/- mice. Moreover, ROS production was increased in mitochondria isolated from the kidney cortices of Drd5-/- mice, relative to Drd5+/+ littermates. In conclusion, D5R-mediated activation of autophagy plays a role in the D5R-mediated inhibition of mito-ROS production in the kidneys.

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.

The inositol pyrophosphate 5-InsP7 drives sodium-potassium pump degradation by relieving an autoinhibitory domain of PI3K p85α.

Sodium/potassium-transporting adenosine triphosphatase (Na+/K+-ATPase) is one of the most abundant cell membrane proteins and is essential for eukaryotes. Endogenous negative regulators have long been postulated to play an important role in regulating the activity and stability of Na+/K+-ATPase, but characterization of these regulators has been elusive. Mechanisms of regulating Na+/K+-ATPase homeostatic turnover are unknown. Here, we report that 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7), generated by inositol hexakisphosphate kinase 1 (IP6K1), promotes physiological endocytosis and downstream degradation of Na+/K+-ATPase-α1. Deletion of IP6K1 elicits a twofold enrichment of Na+/K+-ATPase-α1 in plasma membranes of multiple tissues and cell types. Using a suite of synthetic chemical biology tools, we found that 5-InsP7 binds the RhoGAP domain of phosphatidylinositol 3-kinase (PI3K) p85α to disinhibit its interaction with Na+/K+-ATPase-α1. This recruits adaptor protein 2 (AP2) and triggers the clathrin-mediated endocytosis of Na+/K+-ATPase-α1. Our study identifies 5-InsP7 as an endogenous negative regulator of Na+/K+-ATPase-α1.

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.

Stomach gastrin is regulated by sodium via PPAR-α and dopamine D1 receptor.

Gastrin, secreted by stomach G cells in response to ingested sodium, stimulates the renal cholecystokinin B receptor (CCKBR) to increase renal sodium excretion. It is not known how dietary sodium, independent of food, can increase gastrin secretion in human G cells. However, fenofibrate (FFB), a peroxisome proliferator-activated receptor-α (PPAR-α) agonist, increases gastrin secretion in rodents and several human gastrin-secreting cells, via a gastrin transcriptional promoter. We tested the following hypotheses: (1.) the sodium sensor in G cells plays a critical role in the sodium-mediated increase in gastrin expression/secretion, and (2.) dopamine, via the D1R and PPAR-α, is involved. Intact human stomach antrum and G cells were compared with human gastrin-secreting gastric and ovarian adenocarcinoma cells. When extra- or intracellular sodium was increased in human antrum, human G cells, and adenocarcinoma cells, gastrin mRNA and protein expression/secretion were increased. In human G cells, the PPAR-α agonist FFB increased gastrin protein expression that was blocked by GW6471, a PPAR-α antagonist, and LE300, a D1-like receptor antagonist. LE300 prevented the ability of FFB to increase gastrin protein expression in human G cells via the D1R, because the D5R, the other D1-like receptor, is not expressed in human G cells. Human G cells also express tyrosine hydroxylase and DOPA decarboxylase, enzymes needed to synthesize dopamine. G cells in the stomach may be the sodium sensor that stimulates gastrin secretion, which enables the kidney to eliminate acutely an oral sodium load. Dopamine, via the D1R, by interacting with PPAR-α, is involved in this process.

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.

The Dopamine D1 Receptor and Angiotensin II Type-2 Receptor are Required for Inhibition of Sodium Transport Through a Protein Phosphatase 2A Pathway.

Activation of the renal D1R (dopamine D1-like receptor) or AT2R (angiotensin II type-2 receptor), individually or both, simultaneously, is necessary in the normal regulation of renal sodium (Na+) transport and blood pressure. However, little is known regarding the precise mechanism of this interaction. Pharmacological stimulation, membrane biotinylation, and cell surface immunofluorescence were used to study the effect of the D1R/AT2R interaction in human renal proximal tubule cells. D1R activation of GαS stimulates AC (adenylyl cyclase) and induces apical plasma membrane recruitment of AT2Rs. We now show for the first time the reciprocal reaction, AT2R stimulation with Ang III (angiotensin III) leads to the apical plasma membrane recruitment of the D1R. The cell-permeable second messenger analogs of cAMP (8-Br-cAMP) or cGMP (8-Br-cGMP) induce translocation of both D1R and AT2R to the plasma membrane. Inhibition of PKA (protein kinase A) with Rp-cAMPS and PKG (protein kinase G) with Rp-8-CPT-cGMPS blocks D1R and AT2R recruitment, respectively, indicating that both PKA and PKG are necessary for D1R and AT2R trafficking. Both 8-Br-cAMP and 8-Br-cGMP activate PP2A (protein phosphatase 2A), which is necessary for both plasma membrane recruitment of D1R and AT2R and the inhibition of sodium hydrogen exchanger 3-dependent Na+ transport. These studies provide insights into the D1R/AT2R transregulation mechanisms that play a crucial role in maintaining Na+ and ultimately blood pressure homeostasis.

Sodium bicarbonate cotransporter NBCe2 gene variants increase sodium and bicarbonate transport in human renal proximal tubule cells.

RATIONALE: Salt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure. OBJECTIVE: To characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5. METHODS AND RESULTS: The expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte nuclear factor type 4A (HNF4A) to DNA was increased in hRPTCs carrying HV SLC4A5 rs7571842 but not rs10177833. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was abolished by HNF4A antagonists. CONCLUSION: NBCe2 activity is stimulated by an increase in intracellular sodium and is hyper-responsive in hRPTCs carrying HV SLC4A5 rs7571842 through an aberrant HNF4A-mediated mechanism.