Regulation Of Sodium Transport Research Articles

New insights into sodium transport regulation in the distal nephron: Role of G-protein coupled receptors.

The renal handling of Na(+) balance is a major determinant of the blood pressure (BP) level. The inability of the kidney to excrete the daily load of Na(+) represents the primary cause of chronic hypertension. Among the different segments that constitute the nephron, those present in the distal part (i.e., the cortical thick ascending limb, the distal convoluted tubule, the connecting and collecting tubules) play a central role in the fine-tuning of renal Na(+) excretion and are the target of many different regulatory processes that modulate Na(+) retention more or less efficiently. G-protein coupled receptors (GPCRs) are crucially involved in this regulation and could represent efficient pharmacological targets to control BP levels. In this review, we describe both classical and novel GPCR-dependent regulatory systems that have been shown to modulate renal Na(+) absorption in the distal nephron. In addition to the multiplicity of the GPCR that regulate Na(+) excretion, this review also highlights the complexity of these different pathways, and the connections between them.

The role of dexamethasone in scorpion venom-induced deregulation of sodium and water transport in rat lungs.

BackgroundSevere scorpion envenomation can evolve to lung injury and, in some cases, death. The lung injury could be attributed to acute left ventricular failure and increased pulmonary vascular permeability secondary to the release of inflammatory mediators. In clinical practice, corticosteroids have been administered to reduce the early side effects of the anti-venom. We propose to study the effects of Tityus serrulatus venom and dexamethasone on pulmonary expression of sodium and water transporters, as well as on the inflammatory response.MethodsWistar rats were injected intraperitoneally with saline (control group), dexamethasone, and saline (2.0 mg/kg body weight—60 min before saline injection; dexamethasone + saline group), venom (T. serrulatus venom—3.8 mg/kg body weight), or dexamethasone and venom (2.0 mg/kg body weight—60 min before venom injection; dexamethasone + venom group). At 60 min after venom/saline injection, experiments were performed in ventilated and non-ventilated animals. We analyzed sodium transporters, water transporters, and Toll-like receptor 4 (TLR4) by Western blotting, macrophage infiltration by immunohistochemistry, and serum interleukin (IL) by cytokine assay.ResultsIn the lung tissue of non-ventilated envenomed animals, protein expression of the epithelial sodium channel alpha subunit (α-ENaC) and aquaporin 5 (AQP5) were markedly downregulated whereas that of the Na-K-2Cl cotransporter (NKCC1) and TLR4 was elevated although expression of the Na,K-ATPase alpha 1 subunit was unaffected. Dexamethasone protected protein expression of α-ENaC, NKCC1, and TLR4 but not that of AQP5. We found that IL-6, IL-10, and tumor necrosis factor alpha were elevated in the venom and dexamethasone + venom groups although CD68 expression in lung tissue was elevated only in the venom group. Among the ventilated animals, both envenomed groups presented hypotension at 50 min after injection, and the arterial oxygen tension/fraction of inspired oxygen ratio was lower at 60 min than at baseline.ConclusionsOur results suggest that T. serrulatus venom and dexamethasone both regulate sodium transport in the lung and that T serrulatus venom regulates sodium transport via the TLR4 pathway.

Vasopressin regulation of sodium transport in the distal nephron and collecting duct.

Arginine vasopressin (AVP) is released from the posterior pituitary gland during states of hyperosmolality or hypovolemia. AVP is a peptide hormone, with antidiuretic and antinatriuretic properties. It allows the kidneys to increase body water retention predominantly by increasing the cell surface expression of aquaporin water channels in the collecting duct alongside increasing the osmotic driving forces for water reabsorption. The antinatriuretic effects of AVP are mediated by the regulation of sodium transport throughout the distal nephron, from the thick ascending limb through to the collecting duct, which in turn partially facilitates osmotic movement of water. In this review, we will discuss the regulatory role of AVP in sodium transport and summarize the effects of AVP on various molecular targets, including the sodium-potassium-chloride cotransporter NKCC2, the thiazide-sensitive sodium-chloride cotransporter NCC, and the epithelial sodium channel ENaC.

The Exon 2 Deletion of the COMMD1 Causing Copper Toxicosis in Bedlington Terriers in Korea

This study was performed to survey prevalence of Copper metabolism domain containing 1 (COMMD1) mutation using molecular diagnostic method in a population of Bedlington terriers in Korea. COMMD1 gene (formerly MURR1) functions as a regulator of sodium transport and copper metabolism. The deletion of exon 2 of the COMMD1 gene causes copper toxicosis in Bedlington terriers. Bedlington terriers with this autosomal recessive disorder were shown to have the elevated liver copper levels due to genetic derangement in the biliary copper excretion pathway. DNA samples were extracted from whole blood collected from 257 Bedlington terriers (109 males, 148 females) of pet dog clubs in Korea. A multiplex PCR was carried out to detect of exon 2 deletion of COMMD1 gene. In this study, it was possible to know the existence and prevalence of exon 2 deletion of COMMD1 in Bedlington terriers in Korea. Of the 257 samples, 131 (51%) were wild type homozygous for the normal COMMD1 gene, 108 (42%) were heterozygous, having both normal and mutated copy of the COMMD1 gene. The eighteen (7%) were mutant type homozygous. The results of genetic analysis could help establish proper management strategy and selective breeding program to prevent COMMD1 mutation in Bedlington terriers in Korea.

Atrial natriuretic peptide reduces the α-subunit of the epithelial sodium channel mRNA expression in the mouse stria vascularis.

Atrial natriuretic peptide (ANP) is extensively expressed in the cochlea, including the strial vascularis (StV). ANP may participate in the regulation of the water-electrolyte balance. However, the functional significance of ANP in the cochlea is less understood and little is known regarding the exact mechanisms. Studies suggest that the epithelial sodium channel (ENaC) is important for regulating sodium transport across epithelia. ENaC may be involved in the clearance of endolymphatic Na+ and maintenance of a K-rich and Na-poor composition in the endolymph. Whether ANP has a regulatory effect on the Na+ channel in the StV remains unknown. The aim of the present study was to evaluate whether ANP affects the expression of the α-subunit of the ENaC (α-ENaC) mRNA in the mouse StV, using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technique. The mouse StV tissues were incubated with 10-6 mol/1 ANP for different times (2, 6, 12, 24 and 48 h) and were subsequently harvested. α-ENaC mRNA was extracted for RT-qPCR analysis of the expression. The study demonstrated the existence of α-ENaC in the mouse StV. Tissues treated with ANP (10-6 mol/1) showed a significant reduction in α-ENaC mRNA expression (n=3, P<0.05). A maximum effect was reached at 2 h after treatment. The present results indicate that ANP may regulate cochlear ion transport and endolymph fluid balance in the inner ear via reducing expression of the α-ENaC mRNA in the mouse StV.

STE20/SPS1-related proline/alanine-rich kinase (SPAK) is critical for sodium reabsorption in isolated, perfused thick ascending limb.

SPAK [STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase] kinase consists of a full-length (FL-) and an alternatively spliced kidney-specific (KS-) isoform. SPAK regulates the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). The relative abundance and role of FL- vs. KS-SPAK in regulating Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) in thick ascending limb (TAL) are not completely understood. Here, we report that FL-SPAK mRNA was the most abundant in medullary TAL (mTAL), followed by cortical TAL (cTAL) and DCT. KS-SPAK mRNA abundance was relatively lower than FL-SPAK. The ratios of FL-SPAK to KS-SPAK in mTAL, cTAL, and DCT were 12.3, 12.5, and 10.2, respectively. To examine the role of SPAK in the regulation of sodium transport in TAL, we used in vitro microperfusion of mTAL and cTAL isolated from wild-type (WT) and SPAK knockout mice (SPAK-KO) that lack both FL- and KS-SPAK. The rates of sodium absorption in cTAL and mTAL of SPAK-KO mice were 34.5 and 12.5% of WT tubules, respectively. The mRNA levels of related OSR1 kinase and SPAK protease Dnpep in SPAK-KO tubules were not significantly different from WT tubules. We next examined the role of SPAK in the regulation of sodium reabsorption by vasopressin in TAL. Vasopressin increased sodium reabsorption by ∼80% in both mTAL and cTAL from WT mice. While baseline sodium reabsorption was lower in SPAK-KO tubules, vasopressin increased sodium reabsorption over twofold. In conclusion, the combined net effect of SPAK isoforms on sodium reabsorption in TAL is stimulatory. SPAK is not essential for vasopressin stimulation of sodium reabsorption in TAL.

Abstract 318: The Role of PKC-alpha in NCC-Mediated Hypertension

The sodium-chloride cotransporter (NCC) is an essential regulator of sodium transport in the distal convoluted tubule (DCT); functional aberrations will lead to dysregulation of blood pressure. Although this role is well-accepted, the mechanisms accounting for increased distal nephron sodium reabsorption is not completely understood. Previously published data from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) Study has demonstrated that a polymorphism in PKC-alpha may lead to an increased sensitivity to thiazide diuretics. To investigate whether PKC-alpha may be altering blood pressure via NCC, PKC-alpha KO (PKC KO) and PKC Control (Ctl) mice were used to measure blood pressure via radiotelemetry after normal and high salt (HS, 4%) dietary manipulations and Western blot analysis performed using kidney lysates. Using radiotelemetry (DSI) measurements obtained over an 18 hour ‘active’ time period, we report a significant increase in mean arterial pressure (MAP) in the PKC KO mice as compared to control (123.2±1.1 mmHg vs. 102.0±0.77 mmHg, p&lt;0.001). With HS feeding, MAP increased in both groups; in addition MAP remained significantly higher in the PKC KO mice as compared to the Ctl after 21 days (139.5±0.3 mmHg vs. 124.7±1.1 mmHg, p&lt;0.01). To determine a possible mechanism for this phenomenon, whole kidney lysate was used to assay for total NCC protein. Total NCC protein was almost 3 fold higher in the PKC KO mice as compared to Ctl (in arbitrary units, 208±16 vs. 61±30, p&lt;0.01). Herein we report that lack of PKC-alpha leads to an increase in MAP, which may be accounted for by increased total NCC expression and a subsequent increase in distal nephron reabsorption. We believe that PKC-alpha may be negative regulator of NCC protein expression, and that aberrations in this pathway will lead to hypertension.

Aldosterone regulates microRNAs in the cortical collecting duct to alter sodium transport.

A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells. Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport. Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein. A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3' untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.

A Role for the Circadian Clock Protein Per1 in the Regulation of the NaCl Co-transporter (NCC) and the with-no-lysine Kinase (WNK) Cascade in Mouse Distal Convoluted Tubule Cells

It has been well established that blood pressure and renal function undergo circadian fluctuations. We have demonstrated that the circadian protein Per1 regulates multiple genes involved in sodium transport in the collecting duct of the kidney. However, the role of Per1 in other parts of the nephron has not been investigated. The distal convoluted tubule (DCT) plays a critical role in renal sodium reabsorption. Sodium is reabsorbed in this segment through the actions of the NaCl co-transporter (NCC), which is regulated by the with-no-lysine kinases (WNKs). The goal of this study was to test if Per1 regulates sodium transport in the DCT through modulation of NCC and the WNK kinases, WNK1 and WNK4. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of NCC and WNK1 but increased expression of WNK4 in the renal cortex of mice. These findings were confirmed by using Per1 siRNA and pharmacological blockade of Per1 nuclear entry in mDCT15 cells, a model of the mouse distal convoluted tubule. Transcriptional regulation was demonstrated by changes in short lived heterogeneous nuclear RNA. Chromatin immunoprecipitation experiments demonstrated interaction of Per1 and CLOCK with the promoters of NCC, WNK1, and WNK4. This interaction was modulated by blockade of Per1 nuclear entry. Importantly, NCC protein expression and NCC activity, as measured by thiazide-sensitive, chloride-dependent (22)Na uptake, were decreased upon pharmacological inhibition of Per1 nuclear entry. Taken together, these data demonstrate a role for Per1 in the transcriptional regulation of NCC, WNK1, and WNK4.

Renal angiotensin-converting enzyme and blood pressure control.

This review presents novel findings regarding the renal angiotensin-converting enzyme (ACE) and its role in blood pressure (BP) control. The textbook flow diagram of the renin-angiotensin system (RAS) shows the pulmonary endothelium as the main source of the ACE that converts angiotensin I to angiotensin II. However, ACE is made in large quantities by the kidneys, which raises the important question of what precisely is the function of renal ACE? Recent studies in gene-targeted mice indicates that renal ACE plays a dominant role in regulating the response of the kidney to experimental hypertension. In particular, renal ACE and locally generated angiotensin II affect the activity of several key sodium transporters and the induction of sodium and water retention resulting in the elevation of BP. New experimental data link the renal ACE/angiotensin II pathway and the local regulation of sodium transport as key elements in the development of hypertension.

Abstract 626: Caveolin-1 (CAV1) Levels Determine the Cell Surface Expression of the Dopamine D1-like Receptors

In human renal proximal tubule cells, the dopamine-1 receptor (D1R) increases cAMP production and negatively regulates sodium transport. Defects in D1R signaling have been associated with SNPs in G-protein coupled receptor kinase type 4 (GRK4), which hyperphosphorylates and decreases D1R function, leading to sodium retention. We have previously shown that knockdown of CAV1 in proximal tubule cells impairs sodium transport. In this report we show that CAV1 reduction, knockdown or knockout leads to decreased dopamine D1-like receptor binding at the proximal tubule cell surface. Dopamine D1-like brush border and cell surface binding was measured using Bodipy-FL conjugated SKF83566 . WT and Caveolin 1 knockout (CAV1KO) kidney cortex brush border membrane isolation was performed by magnetic lotus tetragonolobus agglutinin affinity purification and fluorescent SKF83566 binding by microplate fluorometry. CAV1KO had a 35.8±3.3% decrease in binding vs WT (p&lt;0.01, n=6 per group, WT level 18,492 RFU above background). In hTERT immortalized human renal proximal tubule cell lines, lentiviral shRNAi CAV1 stable knockdown cell lines had a 76.4±2.5%, p&lt;0.001 n=4, reduction in CAV1 expression by western blot. Cell surface D1-like receptor binding was reduced by 34.0±1.3% (p&lt;0.001, n=4) in the CAV1 knockdown cell lines (scrambled control levels 117193 RFU). We previously have shown that CAV1 expression is dramatically reduced in the hypertensive SHR proximal tubule cell line vs the normotensive WKY proximal tubule cell line. The SHR cell line had a 57.5±1.2% (p&lt;0.001, n=7) reduction in D1-like receptor binding (WKY 116114 RFU). Re-expression of rat CAV1 alpha or CAV1 beta isoform in the SHR cell line by stable lentiviral infection increased binding by 51.6±2.1%, and 63.3±1.6% respectively (p&lt;0.01, n=14). Sodium transport was decreased by CAV1 alpha and CAV1 beta expression in SHR cell lines. The average number of domes per 10x objective fields of view was 9.5±1.6 in SHR cell, 4.5±0.3 in SHR CAV1alpha (p&lt;0.05, n=4 vs SHR) and in SHR CAV1beta 5.0±0.3 (p&lt;0.05, n=4 vs SHR). In mouse, rat, and human renal proximal tubule cells, CAV1 is necessary for dopamine D1-like receptor cell surface localization and for normal dopaminergic inhibition of sodium transport.

Epidermal growth factors in the kidney and relationship to hypertension

Members of the epidermal growth factor (EGF)-family bind to ErbB (EGFR)-family receptors that play an important role in the regulation of various fundamental cell processes in many organs including the kidney. In this field, most of the research efforts are focused on the role of EGF-ErbB axis in cancer biology. However, many studies indicate that abnormal ErbB-mediated signaling pathways are critical in the development of renal and cardiovascular pathologies. The kidney is a major site of the EGF-family ligands synthesis, and it has been shown to express all four members of the ErbB receptor family. The study of kidney disease regulation by ErbB receptor ligands has expanded considerably in recent years. In vitro and in vivo studies have provided direct evidence of the role of ErbB signaling in the kidney. Recent advances in the understanding of how the proteins in the EGF-family regulate sodium transport and development of hypertension are specifically discussed here. Collectively, these results suggest that EGF-ErbB signaling pathways could be major determinants in the progress of renal lesions, including its effects on the regulation of sodium reabsorption in collecting ducts.

Renin knock out modulates sodium reabsorption in the Dahl salt‐sensitive rats

Dahl salt‐sensitive (SS) rats fed a high salt diet exhibit increased blood pressure and renal damage. We have shown recently that ENaC expression and activity were significantly enhanced in SS rats fed a high salt diet. The renin‐angiotensin‐aldosterone system (RAAS) regulates blood pressure and water electrolyte homeostasis, and renin is a critical component of this system. Ren−/− rats were used to further investigate the role of the renin and RAAS in regulation of sodium transport in salt‐sensitive hypertension. Knock out rats were created using zinc‐finger nucleases designed to target the renin gene in the SS rat. As it has been shown previously, Ren−/− rats have lower body weight and blood pressure whereas no difference is observed in plasma and urine Na+ concentration compared to WT littermates. Here we demonstrate that aldosterone level is significantly lower in Ren−/− rats compared to WT littermates. Aldosterone level is also suppressed in SS rats fed high salt diet compared to animals fed low salt. Single‐channel patch clamp analysis of the freshly isolated collecting ducts revealed decreased ENaC activity in Ren−/− rats. Expression of ENaC subunits was also assessed by immunohistochemistry and Western blotting. We conclude that renin knock out causes low blood pressure, decrease in aldosterone level and suppression of ENaC activity in the aldosterone‐sensitive distal nephron. Supported by AHA, ADA and NIH.

The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes

Cellular directional migration in an electric field (galvanotaxis) is one of the mechanisms guiding cell movement in embryogenesis and in skin epidermal repair. The epithelial sodium channel (ENaC), in addition to its function of regulating sodium transport in kidney, has recently been found to modulate cell locomotory speed. Here we tested whether ENaC has an additional function of mediating the directional migration of galvanotaxis in keratinocytes. Genetic depletion of ENaC completely blocks only galvanotaxis and does not decrease migration speed. Overexpression of ENaC is sufficient to drive galvanotaxis in otherwise unresponsive cells. Pharmacologic blockade or maintenance of the open state of ENaC also decreases or increases, respectively, galvanotaxis, suggesting that the channel open state is responsible for the response. Stable lamellipodial extensions formed at the cathodal sides of wild-type cells at the start of galvanotaxis; these were absent in the ENaC knockout keratinocytes, suggesting that ENaC mediates galvanotaxis by generating stable lamellipodia that steer cell migration. We provide evidence that ENaC is required for directional migration of keratinocytes in an electric field, supporting a role for ENaC in skin wound healing.

Parvalbumin: calcium and magnesium buffering in the distal nephron

Parvalbumin (PV) is a classical member of the EF-hand protein superfamily that has been described as a Ca(2+) buffer and Ca(2+) transporter/shuttle protein and may also play an additional role in Mg(2+) handling. PV is exclusively expressed in the early part of the distal convoluted tubule in the human and mouse kidneys. Recent studies in Pvalb knockout mice revealed a role of PV in the distal handling of electrolytes: the lack of PV was associated with a mild salt-losing phenotype with secondary aldosteronism, salt craving and stronger bones compared with controls. A link between the Ca(2+)-buffering capacity of PV and the expression of the thiazide-sensitive Na(+)-Cl(-) cotransporter was established, which could be relevant to the regulation of sodium transport in the distal nephron. Variants in the PVALB gene that encodes PV have been described, but their relevance to kidney function has not been established. PV is also considered a reliable marker of chromophobe carcinoma and oncocytoma, two neoplasms deriving from the distal nephron. The putative role of PV in tumour genesis remains to be investigated.

The epithelial sodium channel in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

Epithelial sodium channel (ENaC) is a Na(+)-selective, aldosterone-stimulated ion channel involved in sodium transport homeostasis. ENaC is rate-limiting for Na(+) absorption in the epithelia of osmoregulatory organs of tetrapods. Although the ENaC/degenerin gene family is proposed to be present in metazoans, no orthologues or paralogues for ENaC have been found in the genome databases of teleosts. We studied full-length cDNA cloning and tissue distributions of ENaCα, β and γ subunits in the Australian lungfish, Neoceratodus forsteri, which is the closest living relative of tetrapods. Neoceratodus ENaC (nENaC) comprised three subunits: nENaCα, β and γ proteins. The nENaCα, β and γ subunits are closely related to amphibian ENaCα, β and γ subunits, respectively. Three ENaC subunit mRNAs were highly expressed in the gills, kidney and rectum. Amiloride-sensitive sodium current was recorded from Xenopus oocytes injected with the nENaCαβγ subunit complementary RNAs under a two-electrode voltage clamp. nENaCα immunoreactivity was observed in the apical cell membrane of the gills, kidney and rectum. Thus, nENaC may play a role in regulating sodium transport of the lungfish, which has a renin-angiotensin-aldosterone system. This is interesting because there may have been an ENaC sodium absorption system controlled by aldosterone before the conquest of land by vertebrates.

Acid base and fluid electrolyte disturbances in Chronic Kidney Disease

Abstract The kidneys maintain homeostasis in the body avoiding significant alterations in the balance of fluid electrolyte or acid–base balance until the Glomerular filtration rates (GFR) declines to below 25 ml/min due to a series of adaptive changes, both Renal and extra renal. With progressive decline in renal function these mechanisms are overwhelmed resulting in disturbances in water metabolism contributing to hyponatremia and hypernatremia. The altered regulation of sodium transport causes disturbed volume status including volume overload and depletion. The incidence of Hyperkelemia and metabolic acidosis is more frequent in Chronic Kidney Disease (CKD) with GFR below 10 ml/min. In this review article we will attempt to review the renal and extra renal adaptation mechanisms maintaining fluid, electrolyte and acid base balance in CKD along with factors which cause failure of these mechanisms. The article will also highlight the common fluid electrolyte and acid base disorders in CKD and their treatment.

Abstract 616: Caveolin-1 (CAV1) inhibits G Protein Coupled Receptor Kinase Type 4 (GRK4) kinase activity

In human renal proximal tubule cells, the dopamine-1 receptor (D1R) has been shown to increase cAMP production and negatively regulate sodium transport. Defects in D1R signaling have been associated with SNPs in G-protein-coupled receptor kinase type 4 (GRK4) which hyperphosphorylates and decreases D1R function, and the D1R uncoupling phenotype is fully reverted by reducing GRK4 expression with siRNA. We have also shown that CAV1 co-immunoprecipitates with GRK4, and that the normal D1R inhibition of the sodium transporter, NaKATPase alpha, can be blocked using siRNA to CAV1. We therefore hypothesized that CAV1 directly physically interacts with GRK4 and inhibits its kinase activity. Using purified human GRK4 and CAV1 proteins and an in-vitro peptide homogeneous kinase assay, we investigated the effects of caveolin-1 (CAV1) on the kinase activity of GRK4. By overexpressing and isolating a tandem affinity purified protein (TAP tagged) functional version of human CAV1 isolated from a human renal proximal tubule cell line, we were able to ensure that all of the post-translational modifications of the protein were present. Utilizing a peptide Ser/Thr kinase activity assay, we demonstrated that with an excess of CAV1, the kinase activity of GRK4 is inhibited by 37.8±1.3% (n=4, p&lt;0.05). Using this same assay, we showed that purified human calcium calmodulin 1 (CALM1), a known inhibitor of GRK4 when using a complete cell lysate, does not significantly decrease the kinase activity of purified GRK4 using a CALM1 concentration over 100 times the published Kd value. This suggests that there is a missing co-factor or factors required for CALM1 to inhibit GRK4. In summary, we report for the first time that CAV1 inhibits the kinase activity of GRK4, and that a known inhibitor of GRK4, CALM1, does not directly inhibit GRK4 kinase activity using purified proteins in an in-vitro kinase activity assay.

Sodium reabsorption in the distal convoluted tubule is enhanced in the 11β‐hydroxysteroid dehydrogenase type 2 knockout mouse

Mice lacking 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2) exhibit hypertension and renal sodium retention that has been attributed to increased activation of the epithelial sodium channel (ENaC) in the connecting tubule and collecting duct. We tested the hypothesis that increased activation of the sodium chloride co‐transporter (NCC) in the distal convoluted tubule (DCT) also contributes to this phenotype.We compared male 11β‐HSD2 knockout mice at 55 – 65 days of age to wild‐type controls in experiments designed to assess NCC expression and function.Knockout mice exhibited hyperplasia in the DCT (indentified using an anti‐NCC antibody in immunohistochemistry on formalin‐fixed tissue). NCC protein expression was upregulated in knockout mice (immunoblot of whole kidney homogenates; n = 8, p &lt; 0.01) as was expression of the gene encoding NCC, Slc12a3 (Q‐PCR using whole kidney homogenates; n = 8; p &lt; 0.01). The functional consequences of this were assessed by measuring the effect of an acute hydrochlorothiazide bolus on the fractional excretion of sodium in anaesthetised mice.Our results suggest that enhanced sodium chloride reabsorption in the DCT contributes to the hypertensive phenotype in this mouse model of apparent mineralocorticoid excess and provide an insight into the role played by adrenal steroids in the regulation of sodium transport in the distal nephron.

Ankyrin 3 is regulated by aldosterone and mircoRNAs, and alters ENaC‐mediated sodium transport

The role of microRNAs (miRs) in the physiologic regulation of sodium transport has yet to be fully appreciated. miRs are small non‐coding RNAs that selectively target and downregulate mRNAs to inhibit protein expression. Our previous studies indicated that aldosterone altered miR expression to directly modulate Na+ transport via the epithelial sodium channel (ENaC). In silico predicted protein targets were screened in cultured mouse cortical collecting duct (mCCD) cells to test for their involvement in ENaC regulation. One of the candidate miR‐regulated targets was ankyrin 3 (ank3). Ank3 expression increased in mCCD cells stimulated with 50nM aldosterone for 24hrs. By inhibiting the expression of miRs directly we increased ank3 protein levels indicating that ank3 was a miR target. To demonstrate the regulation of ank3 by miRs we transiently transfected the 3’‐untranslated region (UTR) of ank3 linked to a dual luciferase reporter into mCCD cells. Both aldosterone and miRs altered luciferase expression confirming the ank3‐UTR as a miR target. Knockdown of ank3 reduced ENaC short‐circuit currents (53.9 ± 5.13%, n=22) and overexpression of ank3 raised ENaC currents (140.6 ± 9.75%, n=12), independent of aldosterone or miR regulation, thereby linking expression of this protein with Na+ transport. Ank3 represents a novel aldosterone‐induced and miR‐regulated protein involved in altering Na+ transport in the CCD.