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; N

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

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

Renal nerves and D\u3csub\u3e1\u3c/sub\u3e-dopamine receptor-mediated natriuresis

The resistance of the spontaneously hypertensive rat (SHR) kidney to the natriuretic effect of dopamine and D1 agonists may be due to increased renal nerve activity. Therefore, we compared the effects of the intrarenal arterial infusion of the D1 agonist, SKF 38383, into the denervated (DNX) kidney of saline-loadedanesthetized SHR and its control, the Wistar-Kyoto (WKY) rat. In both WKY and SHR, DNX of the left kidney slightly decreased urine flow (UV) and absolute (UNaV) and fractional sodium excretion (FENa) in the innervated right kidney; neither vehicle nor D1 agonist infusion exerted any effect. In the left kidney, denervation increased UV, UNaV, and FENa to a similar degree in WKY and SHR (2-fold), without affecting renal blood flow, glomerular filtration rate, or blood pressure. In WKY but not in SHR, after DNX, the D1 agonist dose-dependently increased UV, UNaV, and FENa in the denervated kidney. We conclude that the decreased natriuretic effect of D1 agonists in the SHR is not due to increased renal nerve activity. These data support our previous studies implicating a defect of the D1 receptor or its regulation in the kidney in genetic hypertension

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

© 2019 the American Physiological Society. 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

Regulation of blood pressure by D\u3csub\u3e5\u3c/sub\u3e dopamine receptors

Dopamine receptors have been identified in a number of organs and tissues, which include the central and peripheral nervous systems, various vascular beds, the heart, the gastrointestinal tract, and the kidney. Dopamine receptors are classified into D1- and D2-like subtypes based on their structure and pharmacology; during conditions of moderate sodium balance, more than 50% of renal sodium excretion is regulated by D1-like receptors. Most of the knowledge on the actions of dopamine has been focused on the D1 dopamine receptor. The D5 dopamine receptor also belongs to the D1-like receptor subfamily. Disruption of the D5 receptor results in hypertension. However, unlike the D1 receptor, the hypertension in D5 receptor null mice is caused by the increased activity of the sympathetic nervous system, apparently due to activation of oxytocin, V1 vasopressin, and non-NMDA receptors in the central nervous system. In this paper, we review the physiological action of D5 receptor on the central and peripheral nervous systems, and discuss the possible mechanisms by which hypertension develops when the D5 receptor function is perturbed. © 2007 Bentham Science Publishers Ltd

A splice variant of the myosin phosphatase regulatory subunit tunes arterial reactivity and suppresses response to salt loading

© 2016 the American Physiological Society. The cGMP activated kinase cGK1α is targeted to its substrates via leucine zipper (LZ)-mediated heterodimerization and thereby mediates vascular smooth muscle (VSM) relaxation. One target is myosin phosphatase (MP), which when activated by cGK1α results in VSM relaxation even in the presence of activating calcium. Variants of MP regulatory subunit Mypt1 are generated by alternative splicing of the 31 nt exon 24 (E24), which, by changing the reading frame, codes for isoforms that contain or lack the COOH-terminal LZ motif (E24+/LZ−; E24−/LZ+). Expression of these isoforms is vessel specific and developmentally regulated, modulates in disease, and is proposed to confer sensitivity to nitric oxide (NO)/cGMP-mediated vasorelaxation. To test this, mice underwent Tamoxifen-inducible and smooth muscle-specific knockout of E24 (E24 cKO) after weaning. Deletion of a single allele of E24 (shift to Mypt1 LZ+) enhanced vasorelaxation of first-order mesenteric arteries (MA1) to diethylamine-NONOate (DEA/NO) and to cGMP in permeabilized and calcium-clamped arteries and lowered blood pressure. There was no further effect of deletion of both E24 alleles, indicating high sensitivity to shift of Mypt1 isoforms. However, a unique property of MA1s from homozygous E24 cKOs was significantly reduced force generation to α-adrenergic activation. Furthermore 2 wk of high-salt (4% NaCl) diet increased MA1 force generation to phenylephrine in control mice, a response that was markedly suppressed in the E24 cKO homozygotes. Thus Mypt1 E24 splice variants tune arterial reactivity and could be worthy targets for lowering vascular resistance in disease states

Redox signaling and splicing dependent change in myosin phosphatase underlie early versus late changes in NO vasodilator reserve in a mouse LPS model of sepsis.

Microcirculatory dysfunction may cause tissue malperfusion and progression to organ failure in the later stages of sepsis, but the role of smooth muscle contractile dysfunction is uncertain. Mice were given intraperitoneal LPS, and mesenteric arteries were harvested at 6-h intervals for analyses of gene expression and contractile function by wire myography. Contractile (myosin and actin) and regulatory [myosin light chain kinase and phosphatase subunits (Mypt1, CPI-17)] mRNAs and proteins were decreased in mesenteric arteries at 24 h concordant with reduced force generation to depolarization, Ca(2+), and phenylephrine. Vasodilator sensitivity to DEA/nitric oxide (NO) and cGMP under Ca(2+) clamp were increased at 24 h after LPS concordant with a switch to Mypt1 exon 24− splice variant coding for a leucine zipper (LZ) motif required for PKG-1α activation of myosin phosphatase. This was reproduced by smooth muscle-specific deletion of Mypt1 exon 24, causing a shift to the Mypt1 LZ+ isoform. These mice had significantly lower resting blood pressure than control mice but similar hypotensive responses to LPS. The vasodilator sensitivity of wild-type mice to DEA/NO, but not cGMP, was increased at 6 h after LPS. This was abrogated in mice with a redox dead version of PKG-1α (Cys42Ser). Enhanced vasorelaxation in early endotoxemia is mediated by redox signaling through PKG-1α but in later endotoxemia by myosin phosphatase isoform shifts enhancing sensitivity to NO/cGMP as well as smooth muscle atrophy. Muscle atrophy and modulation may be a novel target to suppress microcirculatory dysfunction; however, inactivation of inducible NO synthase, treatment with the IL-1 antagonist IL-1ra, or early activation of α-adrenergic signaling did not suppressed this response

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

GPR83 function contributes to salt resistance

The G protein-coupled receptor (GPCR) 83 (Gpr83) is an orphan receptor belonging to the rhodopsin-like family of GPCRs. Gpr83 was originally identified as a glucocorticoid-induced transcript in a murine T cell line and referred to as glucocorticoid-induced receptor. Gpr83 is expressed in brain hypothalamic nuclei relevant to energy metabolism control and has a role in the central regulation of energy metabolism. Gpr83 is also expressed in the kidney but its function is unknown. We found that Gpr83 is expressed in mouse renal proximal and distal convoluted tubules, as well as in human renal proximal tubule cells (hRPTCs). High salt diet increased Gpr83 transcription by 2-fold (P\u3c0.05; n=4/group) in Swiss Jim Lambert (SJL/J) and Bagg Albino (BALB/c) salt-resistant mice, relative to C57 Black (C57Bl/6J) salt-sensitive mice. In C57Bl/6J mice on normal salt diet, the lack of one (Gpr83+/-) or both Gpr83 (Gpr83-/-) alleles resulted in an increase in systolic blood pressure (SBP, ~20 mm Hg (P\u3c0.05; n=4/group, measured under anesthesia) compared with Gpr83+/+ littermates, suggesting that Gpr83 is needed to keep a normal BP. Renal-specific Gpr83 silencing by the renal subcapsular infusion of Gpr83 siRNA (3 µg/day; 7 days) increased SBP in C57Bl/6J mice on a normal salt diet, relative to mice treated with non-silencing siRNA (120±5 vs 98±6 mmHg; P\u3c0.05; n=4/group). In hRPTCs, forskolin (10 µM, 30 min) increased Gpr83 mRNA (3.5±0.06 vs 1.0±0.12-fold; P\u3c0.05; n=4-5/group), the effect of which was blocked by the protein kinase A (PKA) inhibitor H-89 (20 µM, 1 h). In hRPTCs, phorbol myristate acetate (200 ng/mL, 30 min) which activates protein kinase C (PKC) decreased Gpr83 mRNA (0.43±0.2 vs 1.0±0.04-fold, P\u3c0.05; n=4-5/group), effect that was partially blocked by the PKC inhibitor GF109203x (1µM, 1h). Stimulation of hRPTCs with ZnCl2 (100 µM, 1 h), an activator of Gpr83, increased AKT (2.5±0.5 vs 1.0±0.06-fold; P\u3c0.05; n=4-5/group) and ERK1/2 (1.4±0.1 vs 1.0±0.08-fold; P\u3c0.05; n=4-5/group) phosphorylation and decreased p-38 mitogen-activated protein kinase (MAPK) phosphorylation (0.1±0.05 vs 1.0±0.1-fold; P\u3c0.05; n=4-5/group). Our results suggest that Gpr83 may protect against the development of salt sensitivity. PKA positively while PKC negatively regulates Gpr83 expression. Gpr83 function may be mediated by the phosphorylation of AKT/ERK1/2 and dephosphorylation of MAPK. Thus, several pathways are involved in the Gpr83-mediated regulation of salt-sensitive hypertension