Alan C. Pao

Journal Articles

• Prostaglandin E2 induces chloride secretion through crosstalk between cAMP and calcium signaling in mouse inner medullary collecting duct cells. American journal of physiology. Cell physiology Rajagopal, M., Thomas, S. V., Kathpalia, P. P., Chen, Y., Pao, A. C. 2014; 306 (3): C263-78

  Abstract

  Under conditions of high dietary salt intake, prostaglandin E2 (PGE2) production is increased in the collecting duct and promotes urinary sodium chloride (NaCl) excretion; however, the molecular mechanisms by which PGE2 increases NaCl excretion in this context have not been clearly defined. We used the mIMCD-K2 cell line to characterize mechanisms underlying PGE2-regulated NaCl transport. When epithelial Na(+) channels were inhibited, PGE2 exclusively stimulated basolateral EP4 receptors to increase short-circuit current (Isc(PGE2)). We found that Isc(PGE2) was sensitive to inhibition by H-89 and CFTR-172, indicating that EP4 receptors signal through protein kinase A to induce Cl- secretion via Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Unexpectedly, we also found that IscPGE2 was sensitive to inhibition by BAPTA-AM (Ca(2+) chelator), 2-APB (IP3 receptor blocker), and FFA (Ca(2+) activated Cl(-) channel (CACC) inhibitor), suggesting that EP4 receptors also signal through Ca(2+) to induce Cl(-) secretion via CACC. Additionally, we observed that PGE2 stimulated an increase in Isc through crosstalk between cAMP and Ca(2+) signaling: BAPTA-AM or 2-APB inhibited a component of Isc(PGE2) that was sensitive to CFTR-172 inhibition; H-89 inhibited a component of Isc(PGE2) that was sensitive to FFA inhibition. Together, our findings indicate that PGE2 activates basolateral EP4 receptors and signals through both cAMP and Ca(2+) to stimulate Cl(-) secretion in IMCD-K2 cells. We propose that these signaling pathways, and the crosstalk between them, may provide a concerted mechanism for enhancing urinary NaCl excretion under conditions of high dietary NaCl intake.

  View details for DOI 10.1152/ajpcell.00381.2012

  View details for PubMedID 24284792

• Fulvene-5 inhibition of Nadph oxidases attenuates activation of epithelial sodium channels in A6 distal nephron cells AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY Trac, D., Liu, B., Pao, A. C., Thomas, S. V., Park, M., Downs, C. A., Ma, H., Helms, M. N. 2013; 305 (7): F995-F1005

  Abstract

  Nadph oxidase 4 is an important cellular source of reactive oxygen species (ROS) generation in the kidney. Novel antioxidant drugs, such as Nox4 inhibitor compounds, are being developed. There is, however, very little experimental evidence for the biological role and regulation of Nadph oxidase isoforms in the kidney. Herein, we show that Fulvene-5 is an effective inhibitor of Nox-generated ROS and report the role of Nox isoforms in activating epithelial sodium channels (ENaC) in A6 distal nephron cells via oxidant signaling and cell stretch activation. Using single-channel patch-clamp analysis, we report that Fulvene-5 blocked the increase in ENaC activity that is typically observed with H2O2 treatment of A6 cells: average ENaC NPo values decreased from a baseline level of 1.04 ± 0.18 (means ± SE) to 0.25 ± 0.08 following Fulvene-5 treatment. H2O2 treatment failed to increase ENaC activity in the presence of Fulvene-5. Moreover, Fulvene-5 treatment of A6 cells blocked the osmotic cell stretch response of A6 cells, indicating that stretch activation of Nox-derived ROS plays an important role in ENaC regulation. Together, these findings indicate that Fulvene-5, and perhaps other classes of antioxidant inhibitors, may represent a novel class of compounds useful for the treatment of pathological disorders stemming from inappropriate ion channel activity, such as hypertension.

  View details for DOI 10.1152/ajprenal.00098.2013

  View details for Web of Science ID 000325353900010

  View details for PubMedID 23863470

• Novel diuretic targets AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY Denton, J. S., Pao, A. C., Maduke, M. 2013; 305 (7): F931-F942

  Abstract

  As the molecular revolution continues to inform a deeper understanding of disease mechanisms and pathways, there exist unprecedented opportunities for translating discoveries at the bench into novel therapies for improving human health. Despite the availability of several different classes of antihypertensive medications, only about half of the 67 million Americans with hypertension manage their blood pressure appropriately. A broader selection of structurally diverse antihypertensive drugs acting through different mechanisms would provide clinicians with greater flexibility in developing effective treatment regimens for an increasingly diverse and aging patient population. An emerging body of physiological, genetic, and pharmacological evidence has implicated several renal ion-transport proteins, or regulators thereof, as novel, yet clinically unexploited, diuretic targets. These include the renal outer medullary potassium channel, ROMK (Kir1.1), Kir4.1/5.1 potassium channels, ClC-Ka/b chloride channels, UTA/B urea transporters, the chloride/bicarbonate exchanger pendrin, and the STE20/SPS1-related proline/alanine-rich kinase (SPAK). The molecular pharmacology of these putative targets is poorly developed or lacking altogether; however, recent efforts by a few academic and pharmaceutical laboratories have begun to lessen this critical barrier. Here, we review the evidence in support of the aforementioned proteins as novel diuretic targets and highlight examples where progress toward developing small-molecule pharmacology has been made.

  View details for DOI 10.1152/ajprenal.00230.2013

  View details for Web of Science ID 000325353900001

  View details for PubMedID 23863472

• Landscape of ENaC regulation in the kidney AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY Pao, A. C. 2012; 303 (9): F1287-F1288

  View details for DOI 10.1152/ajprenal.00518.2012

  View details for Web of Science ID 000310644600004

  View details for PubMedID 22993072

• Prostaglandin E-2 mediates proliferation and chloride secretion in ADPKD cystic renal epithelia AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY Liu, Y., Rajagopal, M., Lee, K., Battini, L., Flores, D., Gusella, G. L., Pao, A. C., Rohatgi, R. 2012; 303 (10): F1425-F1434

  Abstract

  Prostaglandin E(2) (PGE(2)) contributes to cystogenesis in genetically nonorthologous models of autosomal dominant polycystic kidney disease (ADPKD). However, it remains unknown whether PGE(2) induces the classic features of cystic epithelia in genetically orthologous models of ADPKD. We hypothesized that, in ADPKD epithelia, PGE(2) induces proliferation and chloride (Cl(-)) secretion, two archetypal phenotypic features of ADPKD. To test this hypothesis, proliferation and Cl(-) secretion were measured in renal epithelial cells deficient in polycystin-1 (PC-1). PC-1-deficient cells increased in cell number (proliferated) faster than PC-1-replete cells, and this proliferative advantage was abrogated by cyclooxygenase inhibition, indicating a role for PGE(2) in cell proliferation. Exogenous administration of PGE(2) increased proliferation of PC-1-deficient cells by 38.8 ± 5.2% (P < 0.05) but inhibited the growth of PC-1-replete control cells by 49.4 ± 1.9% (P < 0.05). Next, we tested whether PGE(2)-specific E prostanoid (EP) receptor agonists induce intracellular cAMP and downstream ?-catenin activation. PGE(2) and EP4 receptor agonism (TCS 2510) increased intracellular cAMP concentration and the abundance of active ?-catenin in PC-1-deficient cells, suggesting a mechanism for PGE(2)-mediated proliferation. Consistent with this hypothesis, antagonizing EP4 receptors reverted the growth advantage of PC-1-deficient cells, implicating a central role for the EP4 receptor in proliferation. To test whether PGE(2)-dependent Cl(-) secretion is also enhanced in PC-1-deficient cells, we used an Ussing chamber to measure short-circuit current (I(sc)). Addition of PGE(2) induced a fivefold higher increase in I(sc) in PC-1-deficient cells compared with PC-1-replete cells. This PGE(2)-induced increase in I(sc) in PC-1-deficient cells was blocked by CFTR-172 and flufenamic acid, indicating that PGE(2) activates CFTR and calcium-activated Cl(-) channels. In conclusion, PGE(2) activates aberrant signaling pathways in PC-1-deficient epithelia that contribute to the proliferative and secretory phenotype characteristic of ADPKD and suggests a therapeutic role for PGE(2) inhibition and EP4 receptor antagonism.

  View details for DOI 10.1152/ajprenal.00010.2012

  View details for Web of Science ID 000311208200004

  View details for PubMedID 22933297

• SGK regulation of renal sodium transport CURRENT OPINION IN NEPHROLOGY AND HYPERTENSION Pao, A. C. 2012; 21 (5): 534-540

  Abstract

  The serum and glucocorticoid regulated kinase (SGK) family of protein kinases shares similar biochemical and hormonal signaling properties; however, the SGK kinases also exhibit distinct differences in regulating renal sodium (Na(+)) transport. This review will highlight recent advances in our understanding of the specificity of SGK kinase signaling and regulation of renal Na(+) transport.Differential expression of SGK kinases at the cellular and subcellular levels contributes to signaling specificity. New evidence indicates that SGK1 associates with the apical cell membrane of cortical collecting duct cells to regulate open probability of the epithelial Na(+) channel (ENaC). Scaffold proteins can also recruit SGK1 to multiprotein complexes for regulation of ENaC expression in the apical membrane. Recent SGK1 knockout models have implicated the NaCl co-transporter (NCC) as another target of SGK1 regulation. Less is known about the function of SGK2 or SGK3, but both kinases can regulate Na(+)/H(+) exchanger 3 (NHE3) activity.The SGK kinases assume distinct roles in regulating Na transport in both proximal and distal elements of the kidney tubule. Future examination of the molecular mechanisms by which the SGK kinases regulate specific substrates will inform our understanding of how these kinases contribute to the physiology of renal Na(+) transport.

  View details for DOI 10.1097/MNH.0b013e32835571be

  View details for Web of Science ID 000307649300012

  View details for PubMedID 22691875

• Differential effects of extracellular ATP on chloride transport in cortical collecting duct cells AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY Rajagopal, M., Kathpalia, P. P., Widdicombe, J. H., Pao, A. C. 2012; 303 (4): F483-F491

  Abstract

  Extracellular ATP in the cortical collecting duct can inhibit epithelial sodium channels (ENaC) but also stimulate calcium-activated chloride channels (CACC). The relationship between ATP-mediated regulation of ENaC and CACC activity in cortical collecting duct cells has not been clearly defined. We used the mpkCCD(c14) cortical collecting duct cell line to determine effects of ATP on sodium (Na(+)) and chloride (Cl(-)) transport with an Ussing chamber system. ATP, at a concentration of 10(-6) M or less, did not inhibit ENaC-mediated short-circuit current (I(sc)) but instead stimulated a transient increase in I(sc). The macroscopic current-voltage relationship for ATP-inducible current demonstrated that the direction of this ATP response changes from positive to negative when transepithelial voltage (V(te)) is clamped to less than -10 mV. We hypothesized that this negative V(te) might be found under conditions of aldosterone stimulation. We next stimulated mpkCCD(c14) cells with aldosterone (10(-6) M) and then clamped the V(te) to -50 mV, the V(te) of aldosterone-stimulated cells under open-circuit conditions. ATP (10(-6) M) induced a transient increase in negative clamp current, which could be inhibited by flufenamic acid (CACC inhibitor) and BAPTA-AM (calcium chelator), suggesting that ATP stimulates Cl(-) absorption through CACC. Together, our findings suggest that the status of ENaC activity, by controlling V(te), may dictate the direction of ATP-stimulated Cl(-) transport. This interplay between aldosterone and purinergic signaling pathways may be relevant for regulating NaCl transport in cortical collecting duct cells under different states of extracellular fluid volume.

  View details for DOI 10.1152/ajprenal.00062.2012

  View details for Web of Science ID 000307791200001

  View details for PubMedID 22647633

• Metabolic syndrome, insulin resistance and kidney function in non-diabetic individuals NEPHROLOGY DIALYSIS TRANSPLANTATION Johns, B. R., Pao, A. C., Kim, S. H. 2012; 27 (4): 1410-1415

  Abstract

  Metabolic syndrome has been recently identified as a risk factor for chronic kidney disease (CKD). Since the five individual components of the metabolic syndrome have also been identified as risk factors for CKD, the metabolic syndrome diagnosis may represent an aggregate of CKD risk factors. On the other hand, the components of the metabolic syndrome are also associated with insulin resistance, which may directly mediate the increased CKD risk.This study was a cross-sectional evaluation of the relationship between metabolic syndrome, insulin resistance and estimated glomerular filtration rate (eGFR) in 574 non-diabetic individuals. Insulin resistance was directly quantified using the insulin suppression test, and the metabolic syndrome components were measured. eGFR was calculated using the three validated estimation equations: the Chronic Kidney Disease Epidemiology Collaboration equation, the Mayo quadratic equation and the Modification of Diet in Renal Disease study equation.While CKD prevalence was higher and mean eGFR was lower in individuals who met the metabolic syndrome criteria compared with those who did not, we did not observe a significant relationship between insulin resistance and eGFR. Of all of the components of the metabolic syndrome, only hypertension was significantly associated with CKD prevalence [odds ratio (95% confidence interval), 3.5 (1.2-10.1), P=0.02].Although CKD is more common among individuals with the metabolic syndrome, insulin resistance is not a common factor.

  View details for DOI 10.1093/ndt/gfr498

  View details for Web of Science ID 000302310700022

  View details for PubMedID 21908415

• Epithelial Sodium Channel Regulation by Cell Surface-associated Serum- and Glucocorticoid-regulated Kinase 1 JOURNAL OF BIOLOGICAL CHEMISTRY Thomas, S. V., Kathpalia, P. P., Rajagopal, M., Charlton, C., Zhang, J., Eaton, D. C., Helms, M. N., Pao, A. C. 2011; 286 (37): 32074-32085

  Abstract

  Serum- and glucocorticoid-regulated kinase 1 (sgk1) participates in diverse biological processes, including cell growth, apoptosis, and sodium homeostasis. In the cortical collecting duct of the kidney, sgk1 regulates sodium transport by stimulating the epithelial sodium channel (ENaC). Control of subcellular localization of sgk1 may be an important mechanism for modulating specificity of sgk1 function; however, which subcellular locations are required for sgk1-regulated ENaC activity in collecting duct cells has yet to be established. Using cell surface biotinylation studies, we detected endogenous sgk1 at the apical cell membrane of aldosterone-stimulated mpkCCD(c14) collecting duct cells. The association of sgk1 with the cell membrane was enhanced when ENaC was co-transfected with sgk1 in kidney cells, suggesting that ENaC brings sgk1 to the cell surface. Furthermore, association of endogenous sgk1 with the apical cell membrane of mpkCCD(c14) cells could be modulated by treatments that increase or decrease ENaC expression at the apical membrane; forskolin increased the association of sgk1 with the apical surface, whereas methyl-?-cyclodextrin decreased the association of sgk1 with the apical surface. Single channel recordings of excised inside-out patches from the apical membrane of aldosterone-stimulated A6 collecting duct cells revealed that the open probability of ENaC was sensitive to the sgk1 inhibitor GSK650394, indicating that endogenous sgk1 is functionally active at the apical cell membrane. We propose that the association of sgk1 with the apical cell membrane, where it interacts with ENaC, is a novel means by which sgk1 specifically enhances ENaC activity in aldosterone-stimulated collecting duct cells.

  View details for DOI 10.1074/jbc.M111.278283

  View details for Web of Science ID 000294726800017

  View details for PubMedID 21784856

• Activation of P2Y(1) and P2Y(2) receptors induces chloride secretion via calcium-activated chloride channels in kidney inner medullary collecting duct cells AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY Rajagopal, M., Kathpalia, P. P., Thomas, S. V., Pao, A. C. 2011; 301 (3): F544-F553

  Abstract

  Dysregulation of urinary sodium chloride (NaCl) excretion can result in extracellular fluid (ECF) volume expansion and hypertension. Recent studies demonstrated that urinary nucleotide excretion increases in mice ingesting a high-salt diet and that these increases in extracellular nucleotides can signal through P2Y(2) receptors in the kidney collecting duct to inhibit epithelial Na(+) channels (ENaC). However, under conditions of ECF volume expansion brought about by high-dietary salt intake, ENaC activity should already be suppressed. We hypothesized that alternative pathways exist by which extracellular nucleotides control renal NaCl excretion. We used an inner medullary collecting duct (mIMCD-K2) cell line in an Ussing chamber system as a model to study additional ion transport pathways that are regulated by extracellular nucleotides. When ENaC was inhibited, the addition of adenosine triphosphate (ATP) to the basal side of cell sheets activated both P2Y(1) and P2Y(2) receptors, inducing a transient increase in short-circuit current (I(sc)); addition of ATP to the apical side activated only P2Y(2) receptors, inducing first a transient and then a sustained increase in I(sc). The ATP-induced increases in I(sc) were blocked by pretreatment with a phospholipase C (PLC) inhibitor, a calcium (Ca(2+)) chelator, or Ca(2+)-activated Cl(-) channel (CACC) inhibitors, suggesting that ATP signals through both PLC and intracellular Ca(2+) to activate CACC. We propose that P2Y(1) and P2Y(2) receptors operate in tandem in IMCD cells to provide an adaptive mechanism for enhancing urinary NaCl excretion in the setting of high-dietary NaCl intake.

  View details for DOI 10.1152/ajprenal.00709.2010

  View details for Web of Science ID 000294551400011

  View details for PubMedID 21653634

• The natriuretic mechanism of Gamma-Melanocyte-Stimulating Hormone PEPTIDES Kathpalia, P. P., Charlton, C., Rajagopal, M., Pao, A. C. 2011; 32 (5): 1068-1072

  Abstract

  Gamma-Melanocyte Stimulating Hormone (Gamma-MSH) regulates sodium (Na(+)) balance and blood pressure through activation of the melanocortin receptor 3 (MC3-R). The mechanism of the natriuretic effect is proposed to involve binding of MC3-R either in the kidney to directly inhibit tubular Na(+) transport or in the brain to inhibit central neural pathways that control renal tubular Na(+) absorption. This study aimed to clarify the mechanism involved in the natriuretic effect of Gamma-MSH on MC3-R in kidney cells. In Ussing chamber studies, we observed no effects of Gamma-MSH on NaCl transport in the mouse inner medullary collecting duct cell line (mIMCD-K2). We also found that neither MC3-R protein nor mRNA was expressed in mouse kidney, suggesting that renal Gamma-MSH action may not be mediated through direct effects on tubular Na(+) transport but rather through effects on central neural pathways that innervate the kidney.

  View details for DOI 10.1016/j.peptides.2011.02.006

  View details for Web of Science ID 000291286200032

  View details for PubMedID 21335042

• Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY Pao, A. C., Bhargava, A., Di Sole, F., Quigley, R., Shao, X., Wang, J., Thomas, S., Zhang, J., Shi, M., Funder, J. W., Moe, O. W., Pearce, D. 2010; 299 (6): F1496-F1506

  Abstract

  Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na(+)) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na(+)/H(+) exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na(+)/H(+) exchange activity by Na(+)-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na(+)/H(+) exchange activity by >30%. Moreover, the sgk2-mediated increase in Na(+)/H(+) exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na(+) transport through NHE3 in the proximal tubule.

  View details for DOI 10.1152/ajprenal.00075.2010

  View details for Web of Science ID 000285084700030

  View details for PubMedID 20926631

• Adenosine Activates A2b Receptors and Enhances Chloride Secretion in Kidney Inner Medullary Collecting Duct Cells HYPERTENSION Rajagopal, M., Pao, A. C. 2010; 55 (5): 1123-U89

  Abstract

  In the kidney, defects in the regulation of urine salt excretion can result in extracellular fluid volume expansion, leading to salt-sensitive hypertension. Previous studies have demonstrated that, when rats are maintained on a high sodium chloride (NaCl) diet, adenosine production increases in the renal medulla with parallel changes in adenosine receptor expression. These studies suggest that adenosine signaling in the kidney can respond to high NaCl loading; however, the functional consequences of these changes in adenosine signaling are not clear. We used the immortalized cell line mIMCD-K2, a murine model system for the renal inner medullary collecting duct, to study the direct effects of adenosine on NaCl transport across the inner medullary collecting duct epithelium with an Ussing chamber system. When epithelial Na(+) channels were inhibited, the addition of adenosine to the apical side of mIMCD-K2 cell sheets stimulated short-circuit current in a dose-dependent manner. This increase in short-circuit current was inhibited by a cystic fibrosis transmembrane conductance regulator Cl(-) channel inhibitor. Pharmacological studies with a panel of adenosine receptor agonists and antagonists demonstrated that adenosine activates apical A2b adenosine receptors to enhance the short-circuit current. Furthermore, adenosine application to mIMCD-K2 cell sheets increased intracellular cAMP, whereas inhibition of protein kinase A completely blocked the adenosine response. Together, our findings indicate that adenosine stimulates Cl(-) secretion through the cystic fibrosis transmembrane conductance regulator in mIMCD-K2 cells by activating apical A2b receptors and signaling through cAMP/protein kinase A. We propose that this adenosine receptor pathway may provide one mechanism for enhancing urine NaCl excretion in the setting of high dietary NaCl intake.

  View details for DOI 10.1161/HYPERTENSIONAHA.109.10404

  View details for Web of Science ID 000276672500013

  View details for PubMedID 20308611

• Melamine nephrotoxicity: an emerging epidemic in an era of globalization KIDNEY INTERNATIONAL Bhalla, V., Grimm, P. C., Chertow, G. M., Pao, A. C. 2009; 75 (8): 774-779

  Abstract

  Recent outbreaks of nephrolithiasis and acute kidney injury among children in China have been linked to ingestion of milk-based infant formula contaminated with melamine. These cases provide evidence in humans for the nephrotoxicity of melamine, which previously had been described only in animals. The consequences of this outbreak are already severe and will likely continue to worsen. Herein we summarize the global impact of the melamine milk contamination, the reemergence of melamine-tainted animal feed, and potential mechanisms of melamine nephrotoxicity. Large-scale epidemiologic studies are necessary to further characterize this disease and to assess its potential long-term sequelae. This epidemic of environmental kidney disease highlights the morbidity associated with adulterated food products available in today's global marketplace and reminds us of the unique vulnerability of the kidney to environmental insults. Melamine is the latest in a growing list of diverse potentially toxic compounds about which nephrologists and other health-care providers responsible for the diagnosis and management of kidney disease must now be aware.

  View details for DOI 10.1038/ki.2009.16

  View details for Web of Science ID 000264747900005

  View details for PubMedID 19212415

• NH2 terminus of serum and glucocorticoid-regulated kinase 1 binds to phosphoinositides and is essential for isoform-specific physiological functions AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY Pao, A. C., McCormick, J. A., Li, H., Siu, J., Govaerts, C., Bhalla, V., Soundararajan, R., Pearce, D. 2007; 292 (6): F1741-F1750

  Abstract

  Serum and glucocorticoid regulated kinase 1 (SGK1) has been identified as a key regulatory protein that controls a diverse set of cellular processes including sodium (Na(+)) homeostasis, osmoregulation, cell survival, and cell proliferation. Two other SGK isoforms, SGK2 and SGK3, have been identified, which differ most markedly from SGK1 in their NH(2)-terminal domains. We found that SGK1 and SGK3 are potent stimulators of epithelial Na(+) channel (ENaC)-dependent Na(+) transport, while SGK2, which has a short NH(2) terminus, is a weak stimulator of ENaC. Further characterization of the role of the SGK1 NH(2) terminus revealed that its deletion does not affect in vitro kinase activity but profoundly limits the ability of SGK1 either to stimulate ENaC-dependent Na(+) transport or inhibit Forkhead-dependent gene transcription. The NH(2) terminus of SGK1, which shares sequence homology with the phosphoinositide 3-phosphate [PI(3)P] binding domain of SGK3, binds phosphoinositides in protein lipid overlay assays, interacting specifically with PI(3)P, PI(4)P, and PI(5)P, but not with PI(3,4,5)P(3). Moreover, a point mutation that reduces phosphoinositide binding to the NH(2) terminus also reduces SGK1 effects on Na(+) transport and Forkhead activity. These data suggest that the NH(2) terminus, although not required for PI 3-kinase-dependent modulation of SGK1 catalytic activity, is required for multiple SGK1 functions, including stimulation of ENaC and inhibition of the proapoptotic Forkhead transcription factor. Together, these observations support the idea that the NH(2)-terminal domain acts downstream of PI 3-kinase-dependent activation to target the kinase to specific cellular compartments and/or substrates, possibly through its interactions with a subset of phosphoinositides.

  View details for DOI 10.1152/ajprenal.00027.2007

  View details for Web of Science ID 000247942000010

  View details for PubMedID 17356130

• Disinhibitory pathways for control of sodium transport: regulation of ENaC by SGK1 and GILZ AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY Bhalla, V., Soundararajan, R., Pao, A. C., Li, H., Pearce, D. 2006; 291 (4): F714-F721

  Abstract

  Regulation of ENaC occurs at several levels. The principal hormonal regulator of ENaC, aldosterone, acts through the mineralocorticoid receptor to modulate ENaC-mediated sodium transport, and considerable attention has focused on defining the components of the early phase of this response. Two genes, SGK1 and GILZ, have now been implicated in this regulation. While the functional significance of SGK1 in mediating aldosterone effects is well established, new evidence has enhanced our understanding of the mechanisms of SGK1 action. In addition, recent work demonstrates a novel role for GILZ in the stimulation of ENaC-mediated sodium transport. Interestingly, both SGK1 and GILZ appear to negatively regulate tonic inhibition of ENaC and thus use disinhibition to propagate the rapid effects of aldosterone to increase sodium reabsorption in tight epithelia.

  View details for DOI 10.1152/ajprenal.00061.2006

  View details for Web of Science ID 000240313000002

  View details for PubMedID 16720863

• Serum- and glucocorticoid-regulated kinase 1 regulates ubiquitin ligase neural precursor cell-expressed, developmentally down-regulated protein 4-2 by inducing interaction with 14-3-3 MOLECULAR ENDOCRINOLOGY Bhalla, V., Daidie, D., Li, H. Y., Pao, A. C., LaGrange, L. P., Wang, J., VANDEWALLE, A., Stockand, J. D., Staub, O., Pearce, D. 2005; 19 (12): 3073-3084

  Abstract

  Serum- and glucocorticoid-regulated kinase 1 (SGK1) is an aldosterone-regulated early response gene product that regulates the activity of several ion transport proteins, most notably that of the epithelial sodium channel (ENaC). Recent evidence has established that SGK1 phosphorylates and inhibits Nedd4-2 (neural precursor cell-expressed, developmentally down-regulated protein 4-2), a ubiquitin ligase that decreases cell surface expression of the channel and possibly stimulates its degradation. The mechanistic basis for this SGK1-induced Nedd4-2 inhibition is currently unknown. In this study we show that SGK1-mediated phosphorylation of Nedd4-2 induces its interaction with members of the 14-3-3 family of regulatory proteins. Through functional characterization of Nedd4-2-mutant proteins, we demonstrate that this interaction is required for SGK1-mediated inhibition of Nedd4-2. The concerted action of SGK1 and 14-3-3 appears to disrupt Nedd4-2-mediated ubiquitination of ENaC, thus providing a mechanism by which SGK1 modulates the ENaC-mediated Na(+) current. Finally, the expression pattern of 14-3-3 is also consistent with a functional role in distal nephron Na(+) transport. These results demonstrate a novel, physiologically significant role for 14-3-3 proteins in modulating ubiquitin ligase-dependent pathways in the control of epithelial ion transport.

  View details for DOI 10.1210/me.2005-0193

  View details for Web of Science ID 000233460500015

  View details for PubMedID 16099816

• SGK1: A rapid aldosterone-induced regulator of renal sodium reabsorption PHYSIOLOGY McCormick, J. A., Bhalla, V., Pao, A. C., Pearce, D. 2005; 20: 134-139

  Abstract

  Recently, substantial progress has been made in understanding the mechanisms by which aldosterone rapidly stimulates sodium transport in the distal nephron and other tight epithelia. Serum- and glucocorticoid-regulated kinase 1 (SGK1) has been identified as an important mediator of this process. Its physiological relevance has been revealed through heterologous expression in cultured cells and generation of SGK1 knockout mice.

  View details for DOI 10.1152/physiol.00053.2004

  View details for Web of Science ID 000227958500008

  View details for PubMedID 15772302