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α-Ketoglutarate (α-KG) is a citric acid cycle intermediate and a glutamine catabolism product. It is also the natural ligand of 2-oxoglutarate receptor 1 (OXGR1), a G q protein-coupled receptor expressed on the apical membrane of intercalated cells. In the cortical collecting duct (CCD), Cl - /[Formula: see text] exchange increases upon α-KG binding to the OXGR1. To determine the signaling pathway(s) by which α-KG stimulates Cl - absorption, we examined α-KG-stimulated Cl - absorption in isolated perfused mouse CCDs. α-KG increased electroneutral Cl - absorption in CCDs from wild-type mice but had no effect on Cl - absorption in pendrin knockout mice. Because G q protein-coupled receptors activate PKC, we hypothesized that α-KG stimulates Cl - absorption through PKC. If so, PKC agonists should mimic, whereas PKC inhibitors should abolish, α-KG-stimulated Cl - absorption. Like α-KG, PKC agonist (phorbol-12,13-dibutyrate, 500 nM) application increased Cl - absorption in wild-type but not in pendrin null CCDs. Moreover, PKC inhibitors (2.5 mM GF109203X and 20 nM calphostin C), Ca 2+ chelators (BAPTA, 10-20 μM), or PKC-α or -δ gene ablation eliminated α-KG-stimulated Cl - absorption. We have shown that STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) gene ablation increases urinary α-KG excretion, renal pendrin abundance, and CCD Cl - absorption. However, in SPAK null CCDs, Cl - absorption was not activated further by luminal α-KG application nor was Cl - absorption reduced with the PKC inhibitor GF109203 . Thus SPAK gene ablation likely acts through a PKC-independent pathway to produce a chronic adaptive increase in pendrin function. In conclusion, α-KG stimulates pendrin-dependent Cl - /[Formula: see text] exchange through a mechanism dependent on PKC and Ca 2+ that involves PKC-α and PKC-δ.

Purpose of Review: This review describes the recent discoveries about a powerful electroneutral NaCl reabsorption mechanism in intercalated cells, and its regulation by an intrarenal metabolite paracrine, α-ketoglutartate, and the G-protein coupled receptor, Oxgr1., Recent Findings: The distal nephron fine-tunes sodium, chloride, potassium, hydrogen, bicarbonate and water transport to maintain electrolyte homeostasis and blood pressure. Intercalated cells have been traditionally viewed as the professional regulators of acid-base balance, but recent studies reveal that a specific population of intercalated cells, identified by the pendrin-transporter, have a surprising role in the regulation of salt balance. The pendrin-positive intercalated cells (PP-ICs) facilitate electroneutral NaCl reabsorption through the cooperative activation of multitransport protein network. α-Ketoglutartate is synthesized and secreted into the proximal tubule lumen in the combined state of metabolic alkalosis and intravascular volume contraction to activate Oxgr1 in PP-IC, which in turn activates the multitransport protein network to drive salt reabsorption and bicarbonate secretion by these cells., Summary: Recent studies identify a novel salt transport pathway in intercalated cells that is activated by an intrarenal paracrine system, α-ketoglutartate/Oxgr1. Activation of the paracrine system and transport pathway, particularly during alkalosis and volume contraction, mitigates deleterious salt wasting while restoring acid-base balance.

Aberrant activation of with no lysine (WNK) kinases causes familial hyperkalemic hypertension (FHHt). Thiazide diuretics treat the disease, fostering the view that hyperactivation of the thiazide-sensitive sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT) is solely responsible. However, aberrant signaling in the aldosterone-sensitive distal nephron (ASDN) and inhibition of the potassium-excretory renal outer medullary potassium (ROMK) channel have also been implicated. To test these ideas, we introduced kinase-activating mutations after Lox-P sites in the mouse Stk39 gene, which encodes the terminal kinase in the WNK signaling pathway, Ste20-related proline-alanine-rich kinase (SPAK). Renal expression of the constitutively active (CA)-SPAK mutant was specifically targeted to the early DCT using a DCT-driven Cre recombinase. CA-SPAK mice displayed thiazide-treatable hypertension and hyperkalemia, concurrent with NCC hyperphosphorylation. However, thiazide-mediated inhibition of NCC and consequent restoration of sodium excretion did not immediately restore urinary potassium excretion in CA-SPAK mice. Notably, CA-SPAK mice exhibited ASDN remodeling, involving a reduction in connecting tubule mass and attenuation of epithelial sodium channel (ENaC) and ROMK expression and apical localization. Blocking hyperactive NCC in the DCT gradually restored ASDN structure and ENaC and ROMK expression, concurrent with the restoration of urinary potassium excretion. These findings verify that NCC hyperactivity underlies FHHt but also reveal that NCC-dependent changes in the driving force for potassium secretion are not sufficient to explain hyperkalemia. Instead, a DCT-ASDN coupling process controls potassium balance in health and becomes aberrantly activated in FHHt., (Copyright © 2017 by the American Society of Nephrology.)

Nearly all members of the inwardly rectifying potassium (Kir) channel family share a cytoplasmic domain structure that serves as an unusual AP-1 clathrin adaptor-dependent Golgi export signal in one Kir channel, Kir2.1 (KCNJ2), raising the question whether Kir channels share a common Golgi export mechanism. Here we explore this idea, focusing on two structurally and functionally divergent Kir family members, Kir2.3 (KCNJ4) and Kir4.1/5.1 (KCNJ10/16), which have ∼50% amino identity. We found that Golgi export of both channels is blocked upon siRNA-mediated knockdown of the AP-1 γ subunit, as predicted for the common AP-1-dependent trafficking process. A comprehensive mutagenic analysis, guided by homology mapping in atomic resolution models of Kir2.1, Kir2.3, and Kir4.1/5.1, identified a common structure that serves as a recognition site for AP-1 binding and governs Golgi export. Larger than realized from previous studies with Kir2.1, the signal is created by a patch of residues distributed at the confluence of cytoplasmic N and C termini. The signal involves a stretch of hydrophobic residues from the C-terminal region that form a hydrophobic cleft, an adjacent cluster of basic residues within the N terminus, and a potential network of salt bridges that join the N- and C-terminal poles together. Because patch formation and AP-1 binding are dependent on proper folding of the cytoplasmic domains, the signal provides a common quality control mechanism at the Golgi for Kir channels. These findings identify a new proteostatic mechanism that couples protein folding of channels to forward trafficking in the secretory pathway., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The NaCl cotransporter (NCC) of the renal distal convoluted tubule is stimulated by low-K(+) diet by an unknown mechanism. Since recent work has shown that the STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) can function to stimulate NCC by phosphorylation of specific N-terminal sites, we investigated whether the NCC response to low-K(+) diet is mediated by SPAK. Using phospho-specific antibodies in Western blot and immunolocalization studies of wild-type and SPAK knockout (SPAK(-/-)) mice fed a low-K(+) or control diet for 4 days, we found that low-K(+) diet strongly increased total NCC expression and phosphorylation of NCC. This was associated with an increase in total SPAK expression in cortical homogenates and an increase in phosphorylation of SPAK at the S383 activation site. The increased pNCC in response to low-K(+) diet was blunted but not completely inhibited in SPAK(-/-) mice. These findings reveal that SPAK is an important mediator of the increased NCC activation by phosphorylation that occurs in the distal convoluted tubule in response to a low-K(+) diet, but other low-potassium-activated kinases are likely to be involved., (Copyright © 2015 the American Physiological Society.)

The highly sialylated vascular endothelial surface undergoes changes in sialylation upon adopting the migratory/angiogenic phenotype. We recently established endothelial cell (EC) expression of NEU1 sialidase (Cross, A. S., Hyun, S. W., Miranda-Ribera, A., Feng, C., Liu, A., Nguyen, C., Zhang, L., Luzina, I. G., Atamas, S. P., Twaddell, W. S., Guang, W., Lillehoj, E. P., Puché, A. C., Huang, W., Wang, L. X., Passaniti, A., and Goldblum, S. E. (2012) NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia. NEU1 restrains endothelial cell migration whereas NEU3 does not. J. Biol. Chem. 287, 15966-15980). We asked whether NEU1 might regulate EC capillary-like tube formation on a Matrigel substrate. In human pulmonary microvascular ECs (HPMECs), prior silencing of NEU1 did not alter tube formation. Infection of HPMECs with increasing multiplicities of infection of an adenovirus encoding for catalytically active WT NEU1 dose-dependently impaired tube formation, whereas overexpression of either a catalytically dead NEU1 mutant, NEU1-G68V, or another human sialidase, NEU3, did not. NEU1 overexpression also diminished EC adhesion to the Matrigel substrate and restrained EC migration in a wounding assay. In HPMECs, the adhesion molecule, CD31, also known as platelet endothelial cell adhesion molecule-1, was sialylated via α2,6-linkages, as shown by Sambucus nigra agglutinin lectin blotting. NEU1 overexpression increased CD31 binding to Arachis hypogaea or peanut agglutinin lectin, indicating CD31 desialylation. In the postconfluent state, when CD31 ectodomains are homophilically engaged, NEU1 was recruited to and desialylated CD31. In postconfluent ECs, CD31 was desialylated compared with subconfluent cells, and prior NEU1 silencing completely protected against CD31 desialylation. Prior CD31 silencing and the use of CD31-null ECs each abrogated the NEU1 inhibitory effect on EC tube formation. Sialyltransferase 6 GAL-I overexpression increased α2,6-linked CD31 sialylation and dose-dependently counteracted NEU1-mediated inhibition of EC tube formation. These combined data indicate that catalytically active NEU1 inhibits in vitro angiogenesis through desialylation of its substrate, CD31.

The renal outer medullary K(+) (ROMK) channel plays a critical role in renal sodium handling. Recent genome sequencing efforts in the Framingham Heart Study offspring cohort (Ji W, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State MW, Levy D, and Lifton RP. Nat Genet 40: 592-599, 2008) recently revealed an association between suspected loss-of-function polymorphisms in the ROMK channel and resistance to hypertension, suggesting that ROMK activity may also be a determinant of blood pressure control in the general population. Here we examine whether these sequence variants do, in fact, alter ROMK channel function and explore the mechanisms. As assessed by two-microelectrode voltage clamp in Xenopus oocytes, 3/5 of the variants (R193P, H251Y, and T313FS) displayed an almost complete attenuation of whole cell ROMK channel activity. Surface antibody binding measurements of external epitope-tagged channels and analysis of glycosylation-state maturation revealed that these variants prevent channel expression at the plasmalemma, likely as a consequence of retention in the endoplasmic reticulum. The other variants (P166S, R169H) had no obvious effects on the basal channel activity or surface expression but, instead, conferred a gain in regulated-inhibitory gating. As assessed in giant excised patch-clamp studies, apparent phosphotidylinositol 4,5-bisphosphate (PIP(2)) binding affinity of the variants was reduced, causing channels to be more susceptible to inhibition upon PIP(2) depletion. Unlike the protein product of the major ROMK allele, these two variants are sensitive to the inhibitory affects of a G protein-coupled receptor, which stimulates PIP(2) hydrolysis. In summary, we have found that hypertension resistance sequence variants inhibit ROMK channel function by different mechanisms, providing new insights into the role of the channel in the maintenance of blood pressure.

Renal outer medullary potassium (ROMK) channels are exquisitely regulated to adjust renal potassium excretion and maintain potassium balance. Clathrin-dependent endocytosis plays a critical role, limiting urinary potassium loss in potassium deficiency. In renal disease, aberrant ROMK endocytosis may contribute to potassium retention and hyperkalemia. Previous work has indicated that ROMK endocytosis is stimulated by with-no-lysine (WNK) kinases, but the endocytotic signal and the internalization machinery have not been defined. Here, we found that ROMK bound directly to the clathrin adaptor molecule autosomal recessive hypercholesterolemia (ARH), and this interaction was mediated by what we believe to be a novel variant of the canonical "NPXY" endocytotic signal, YxNPxFV. ARH recruits ROMK to clathrin-coated pits for constitutive and WNK1-stimuated endocytosis, and ARH knockdown decreased basal rates of ROMK endocytosis, in a heterologous expression system, COS-7 cells. We found that ARH was predominantly expressed in the distal nephron where it coimmunoprecipitated and colocalized with ROMK. In mice, the abundance of kidney ARH protein was modulated by dietary potassium and inversely correlated with changes in ROMK. Furthermore, ARH-knockout mice exhibited an altered ROMK response to potassium intake. These data suggest that ARH marks ROMK for clathrin-dependent endocytosis, in concert with the demands of potassium homeostasis.

Hypertension places a major burden on individual and public health, but the genetic basis of this complex disorder is poorly understood. We conducted a genome-wide association study of systolic and diastolic blood pressure (SBP and DBP) in Amish subjects and found strong association signals with common variants in a serine/threonine kinase gene, STK39. We confirmed this association in an independent Amish and 4 non-Amish Caucasian samples including the Diabetes Genetics Initiative, Framingham Heart Study, GenNet, and Hutterites (meta-analysis combining all studies: n = 7,125, P < 10(-6)). The higher BP-associated alleles have frequencies > 0.09 and were associated with increases of 3.3/1.3 mm Hg in SBP/DBP, respectively, in the Amish subjects and with smaller but consistent effects across the non-Amish studies. Cell-based functional studies showed that STK39 interacts with WNK kinases and cation-chloride cotransporters, mutations in which cause monogenic forms of BP dysregulation. We demonstrate that in vivo, STK39 is expressed in the distal nephron, where it may interact with these proteins. Although none of the associated SNPs alter protein structure, we identified and experimentally confirmed a highly conserved intronic element with allele-specific in vitro transcription activity as a functional candidate for this association. Thus, variants in STK39 may influence BP by increasing STK39 expression and consequently altering renal Na(+) excretion, thus unifying rare and common BP-regulating alleles in the same physiological pathway.

Loss-of-function mutations in the inward rectifier potassium channel, Kir2.1, cause Andersen-Tawil syndrome (ATS-1), an inherited disorder of periodic paralysis and ventricular arrhythmias. Here, we explore the mechanism by which a specific ATS-1 mutation (V302M) alters channel function. Val-302 is located in the G-loop, a structure that is believed to form a flexible barrier for potassium permeation at the apex of the cytoplasmic pore. Consistent with a role in stabilizing the G-loop in an open conformation, we found the V302M mutation specifically renders the channel unable to conduct potassium without altering subunit assembly or attenuating cell surface expression. As predicted by the position of the Val-302 side chain in the crystal structure, amino acid substitution analysis revealed that channel activity and phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity are profoundly sensitive to alterations in the size, shape, and hydrophobicity of side chains at the Val-302 position. The observations establish that the Val-302 side chain is a critical determinant of potassium conduction through the G-loop. Based on our functional studies and the cytoplasmic domain crystal structure, we suggest that Val-302 may influence PIP2 gating indirectly by translating PIP2 binding to conformational changes in the G-loop pore.

ICa(TTX) is a sodium current component, functionally distinct from the main body of sodium current, seen in cardiac and other cells. To determine if ICa(TTX) channels are a separate isoform from the classical cardiac sodium channels, we exposed rat ventricular cells in primary culture to an antisense oligonucleotide (AON) directed against rH1 (rNav1.5): 5'-CTCCTCATACCCTCT-3'. The homologous human sequence has been identified (and confirmed by us on HEK 293 cells) as effective against hH1 expressed heterologously. Scrambled sequence (5'-CCCCCCTTATCTACT-3') controls were also included. The AON (10 microM; day 2 of exposure) reduced the classical sodium current by 69.6 % compared to untreated and 60.8 % compared to scrambled sequence (10 microM; day 2 of exposure) controls (mean +/- S.E.M. maximum peak inward current density of -8.23 +/- 0.60 pA pF-1, 18 cells, for untreated; -6.37 +/- 0.79 pA pF-1, 16 cells, for scrambled sequence; and -2.50 +/- 0.31 pA pF-1, 18 cells, for AON-treated cells). The two control groups are not significantly different from each other, but are both significantly different from the AON-treated group (P < 0.001). The inhibition was specific for sodium channels, with no significant AON effect on the L-type calcium current. This confirms that H1 generates the classical cardiac sodium current. This same AON at the same concentration and time of exposure had no significant effect on ICa(TTX) (mean of -4.72 +/- 0.55, 15 cells; -5.47 +/- 0.53, 13 cells; and -5.04 +/- 0.63 pA pF-1, 15 cells, for untreated controls, scrambled controls and AON treated, respectively). Hence, ICa(TTX), which is functionally distinct from the classical cardiac sodium current, is encoded by a distinct gene.

Transepithelial chloride and fluid secretion by many types of epithelia involves activation of a conductive K(+) pathway that serves to support the electrochemical driving force for Cl(-) secretion. This study sought to determine if such a pathway is involved in Cl(-) and fluid secretion by the cystic epithelia in autosomal dominant polycystic kidney disease (ADPKD). Primary cultures of cells derived from the cysts of patients with ADPKD were used. Confluent monolayers of these cells, mounted in Ussing chambers, were stimulated to secrete Cl(-) by application of the adenylyl cyclase agonist, forskolin. The effects of various K(+) channel blockers on the increase in short-circuit current (I(sc)) generated by active Cl(-) secretion were determined. Charybdotoxin, an inhibitor of Ca(2+)-sensitive K(+) channels exerted no effect. Similarly, the chromanole 293B, an inhibitor of cAMP-induced K(+) conductance, exerted no effect on cAMP-dependent anion secretion. Glibenclamide, an inhibitor of ATP-sensitive K(+) channels and the cystic fibrosis transmembrane conductance regulator (CFTR), modestly inhibited the forskolin-stimulated current when applied to the apical surface of the monolayers, suggesting a relatively weak effect on CFTR. Basolateral application of glibenclamide inhibited I(sc) to a greater extent. This latter effect may be due to inhibition of a K(+)-conductive transport step. Glibenclamide exerted little effect on the I(sc) of nonstimulated monolayers. Cyst growth in ADPKD is driven by cell proliferation and Cl(-) and fluid secretion. The effect of glibenclamide on the growth of cysts formed within a collagen gel by cultured ADPKD cells was tested. Addition of glibenclamide to the media bathing the cysts inhibited their growth. Glibenclamide also blocked the formation of cysts when it was added to the media at the time the cells were seeded within the collagen gel. Glibenclamide was also found to inhibit the proliferation of ADPKD cells. RT-PCR analysis demonstrated that the ATP-sensitive K(+) channel, K(ir) 6.2, is expressed in cultured ADPKD cells and in normal human kidney. These results suggest that ATP-sensitive K(+) channel blockers should be investigated as possible therapeutic agents to inhibit cyst growth in ADPKD.

ROMK channels are present in the cortical collecting ducts of kidney and are responsible for K(+) secretion in this nephron segment. Recent studies suggest that endocytosis of ROMK channels is important for regulation of K(+) secretion in cortical collecting ducts. We investigated the molecular mechanisms for endocytosis of ROMK channels expressed in Xenopus laevis oocytes and cultured Madin-Darby canine kidney cells. When plasma membrane insertion of newly synthesized channel proteins was blocked by incubation with brefeldin A, ROMK currents decreased with a half-time of ~6 h. Coexpression with the Lys44-->Ala dominant-negative mutant dynamin, but not wild-type dynamin, reduced the rate of reduction of ROMK in the presence of brefeldin A. Mutation of Asn371 to Ile in the putative NPXY internalization motif of ROMK1 abolished the effect of the Lys44-->Ala dynamin mutant on endocytosis of the channel. Coimmunoprecipitation study and confocal fluorescent imaging revealed that ROMK channels associated with clathrin coat proteins in Madin-Darby canine kidney cells. These results provide compelling evidence for endocytosis of ROMK channels via clathrin-coated vesicles.