Abinash C. Mistry

Identification of intestinal bicarbonate transporters involved in formation of carbonate precipitates to stimulate water absorption in marine teleost fish

Marine teleost fish precipitate divalent cations as carbonate deposits in the intestine to minimize the potential for excessive Ca2+ entry and to stimulate water absorption by reducing luminal osmotic pressure. This carbonate deposit formation, therefore, helps maintain osmoregulation in the seawater (SW) environment and requires controlled secretion of HCO3(-) to match the amount of Ca2+ entering the intestinal lumen. Despite its physiological importance, the process of HCO3(-) secretion has not been characterized at the molecular level. We analyzed the expression of two families of HCO3(-) transporters, Slc4 and Slc26, in fresh-water- and SW-acclimated euryhaline pufferfish, mefugu (Takifugu obscurus), and obtained the following candidate clones: NBCe1 (an Na+-HCO3(-) cotransporter) and Slc26a6A and Slc26a6B (putative Cl(-)/HCO3(-) exchangers). Heterologous expression in Xenopus oocytes showed that Slc26a6A and Slc26a6B have potent HCO3(-)-transporting activity as electrogenic Cl(-)/nHCO3(-) exchangers, whereas mefugu NBCe1 functions as an electrogenic Na+-nHCO3(-) cotransporter. Expression of NBCe1 and Slc26a6A was highly induced in the intestine in SW and expression of Slc26a6B was high in the intestine in SW and fresh water, suggesting their involvement in HCO3(-) secretion and carbonate precipitate formation. Immunohistochemistry showed staining on the apical (Slc26a6A and Slc26a6B) and basolateral (NBCe1) membranes of the intestinal epithelial cells in SW. We therefore propose a mechanism for HCO3(-) transport across the intestinal epithelial cells of marine fish that includes basolateral HCO3(-) uptake (NBCe1) and apical HCO3(-) secretion (Slc26a6A and Slc26a6B).

Phosphorylation of UT-A1 urea transporter at serines 486 and 499 is important for vasopressin-regulated activity and membrane accumulation

The UT-A1 urea transporter plays an important role in the urine concentrating mechanism. Vasopressin (or cAMP) increases urea permeability in perfused terminal inner medullary collecting ducts and increases the abundance of phosphorylated UT-A1, suggesting regulation by phosphorylation. We performed a phosphopeptide analysis that strongly suggested that a PKA consensus site(s) in the central loop region of UT-A1 was/were phosphorylated. Serine 486 was most strongly identified, with other potential sites at serine 499 and threonine 524. Phosphomutation constructs of each residue were made and transiently transfected into LLC-PK1 cells to assay for UT-A1 phosphorylation. The basal level of UT-A1 phosphorylation was unaltered by mutation of these sites. We injected oocytes, assayed [14C]urea flux, and determined that mutation of these sites did not alter basal urea transport activity. Next, we tested the effect of stimulating cAMP production with forskolin. Forskolin increased wild-type UT-A1 and T524A phosphorylation in LLC-PK1 cells and increased urea flux in oocytes. In contrast, the S486A and S499A mutants demonstrated loss of forskolin-stimulated UT-A1 phosphorylation and reduced urea flux. In LLC-PK1 cells, we assessed biotinylated UT-A1. Wild-type UT-A1, S486A, and S499A accumulated in the membrane in response to forskolin. However, in the S486A/S499A double mutant, forskolin-stimulated UT-A1 membrane accumulation and urea flux were totally blocked. We conclude that the phosphorylation of UT-A1 on both serines 486 and 499 is important for activity and that this phosphorylation may be involved in UT-A1 membrane accumulation.

Structure, properties and enhanced expression of galactose-binding C-type lectins in mucous cells of gills from freshwater Japanese eels (Anguilla japonica).

Using a Japanese-eel (Anguilla japonica) gill cDNA subtraction library, two novel beta-d-galactose-binding lectins were identified that belong to group VII of the animal C-type lectin family. The eel C-type lectins, termed eCL-1 and eCL-2, are simple lectins composed of 163 amino acid residues, including a 22-residue signal peptide for secretion and a single carbohydrate-recognition domain (CRD) of approximately 130 residues typical of C-type lectins. The galactose specificity of the CRD was suggested by the presence of a QPD motif and confirmed by a competitive binding assay. Using Ruthenium Red staining, the lectins were shown to bind Ca(2+) ions. SDS/PAGE showed that native eCL-1 and eCL-2 have an SDS-resistant octameric structure (a tetramer of disulphide-linked dimers). Northern and Western blot analyses demonstrated high-level expression of eCL-1 and eCL-2 mRNAs and their protein products in gills from freshwater eels, which decreased markedly when the eels were transferred from freshwater to seawater. Immunohistochemistry showed that the eel lectins are localized in the exocrine mucous cells of the gill.

A new model of the distal convoluted tubule

The Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney is a key determinant of Na(+) balance. Disturbances in NCC function are characterized by disordered volume and blood pressure regulation. However, many details concerning the mechanisms of NCC regulation remain controversial or undefined. This is partially due to the lack of a mammalian cell model of the DCT that is amenable to functional assessment of NCC activity. Previously reported investigations of NCC regulation in mammalian cells have either not attempted measurements of NCC function or have required perturbation of the critical without a lysine kinase (WNK)/STE20/SPS-1-related proline/alanine-rich kinase regulatory pathway before functional assessment. Here, we present a new mammalian model of the DCT, the mouse DCT15 (mDCT15) cell line. These cells display native NCC function as measured by thiazide-sensitive, Cl(-)-dependent (22)Na(+) uptake and allow for the separate assessment of NCC surface expression and activity. Knockdown by short interfering RNA confirmed that this function was dependent on NCC protein. Similar to the mammalian DCT, these cells express many of the known regulators of NCC and display significant baseline activity and dimerization of NCC. As described in previous models, NCC activity is inhibited by appropriate concentrations of thiazides, and phorbol esters strongly suppress function. Importantly, they display release of WNK4 inhibition of NCC by small hairpin RNA knockdown. We feel that this new model represents a critical tool for the study of NCC physiology. The work that can be accomplished in such a system represents a significant step forward toward unraveling the complex regulation of NCC.

The UT-A1 Urea Transporter Interacts with Snapin, a SNARE-associated Protein

The UT-A1 urea transporter mediates rapid transepithelial urea transport across the inner medullary collecting duct and plays a major role in the urinary concentrating mechanism. To transport urea, UT-A1 must be present in the plasma membrane. The purpose of this study was to screen for UT-A1-interacting proteins and to study the interactions of one of the identified potential binding partners with UT-A1. Using a yeast two-hybrid screen of a human kidney cDNA library with the UT-A1 intracellular loop (residues 409–594) as bait, we identified snapin, a ubiquitously expressed SNARE-associated protein, as a novel UT-A1 binding partner. Deletion analysis indicated that the C-terminal coiled-coil domain (H2) of snapin is required for UT-A1 interaction. Snapin binds to the intracellular loop of UT-A1 but not to the N- or C-terminal fragments. Glutathione S-transferase pulldown experiments and co-immunoprecipitation studies verified that snapin interacts with native UT-A1, SNAP23, and syntaxin-4 (t-SNARE partners), indicating that UT-A1 participates with the SNARE machinery in rat kidney inner medulla. Confocal microscopic analysis of immunofluorescent UT-A1 and snapin showed co-localization in both the cytoplasm and in the plasma membrane. When we co-injected UT-A1 with snapin cRNA in Xenopus oocytes, urea influx was significantly increased. In the absence of snapin, the influx was decreased when UT-A1 was combined with t-SNARE components syntaxin-4 and SNAP23. We conclude that UT-A1 may be linked to the SNARE machinery via snapin and that this interaction may be functionally and physiologically important for urea transport.

Aldosterone acutely stimulates NCC activity via a SPAK-mediated pathway

Hypertension is a leading cause of morbidity and mortality worldwide, and disordered sodium balance has long been implicated in its pathogenesis. Aldosterone is perhaps the key regulator of sodium balance and thus blood pressure. The sodium chloride cotransporter (NCC) in the distal convoluted tubule of the kidney is a major site of sodium reabsorption and plays a key role in blood pressure regulation. Chronic exposure to aldosterone increases NCC protein expression and function. However, more acute effects of aldosterone on NCC are unknown. In our salt-abundant modern society where chronic salt deprivation is rare, understanding the acute effects of aldosterone is critical. Here, we examined the acute effects (12-36 h) of aldosterone on NCC in the rodent kidney and in a mouse distal convoluted tubule cell line. Studies demonstrated that aldosterone acutely stimulated NCC activity and phosphorylation without affecting total NCC abundance or surface expression. This effect was dependent upon the presence of the mineralocorticoid receptor and serum- and glucocorticoid-regulated kinase 1 (SGK1). Furthermore, STE20/SPS-1-related proline/alanine-rich kinase (SPAK) phosphorylation also increased, and gene silencing of SPAK eliminated the effect of aldosterone on NCC activity. Aldosterone administration via a minipump in adrenalectomized rodents confirmed an increase in NCC phosphorylation without a change in NCC total protein. These data indicate that acute aldosterone-induced SPAK-dependent phosphorylation of NCC increases individual transporter activity.

The sodium chloride cotransporter (NCC) and epithelial sodium channel (ENaC) associate

The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α- and γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.

Mechanisms of angiotensin II stimulation of NCC are time-dependent in mDCT15 cells

Angiotensin II (ANG II) increases thiazide-sensitive sodium-chloride cotransporter (NCC) activity both acutely and chronically. ANG II has been implicated as a switch that turns WNK4 from an inhibitor of NCC into an activator of NCC, and ANG IIs effect on NCC appears to require WNK4. Chronically, ANG II stimulation of NCC results in an increase in total and phosphorylated NCC, but the role of NCC phosphorylation in acute ANG II actions is unclear. Here, using a mammalian cell model with robust native NCC activity, we corroborate the role that ANG II plays in WNK4 regulation and clarify the role of Ste20-related proline alanine-rich kinase (SPAK)-induced NCC phosphorylation in ANG II action. ANG II was noted to have a biphasic effect on NCC, with a peak increase in NCC activity in the physiologic range of 10(-11) M ANG II. This effect was apparent as early as 15 min and remained sustained through 120 min. These changes correlated with significant increases in NCC surface protein expression. Knockdown of WNK4 expression sharply attenuated the effect of ANG II. SPAK knockdown did not affect ANG II action at early time points (15 and 30 min), but it did attenuate the response at 60 min. Correspondingly, NCC phosphorylation did not increase at 15 or 30 min, but increased significantly at 60 min. We therefore conclude that within minutes of an increase in ANG II, NCC is rapidly trafficked to the cell surface in a phosphorylation-independent but WNK4-dependent manner. Then, after 60 min, ANG II induces SPAK-dependent phosphorylation of NCC.

Functional characterization of the central hydrophilic linker region of the urea transporter UT-A1: cAMP activation and snapin binding.

Of the three major protein variants produced by the UT-A gene (UT-A1, UT-A2, and UT-A3) UT-A1 is the largest. It contains UT-A3 as its NH(2)-terminal half and UT-A2 as its COOH-terminal half. When being part of UT-A1, UT-A3 and UT-A2 are joined by a segment, Lp, whose central part, Lc, is not part of UT-A3 or UT-A2 but is present only in UT-A1. Lc contains the phosphorylation sites S486 and S499 that are involved in protein kinase A-dependent activation, as well as the binding site for snapin, a protein involved in soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-mediated vesicle trafficking and fusion to the plasma membrane. We attached Lc to UT-A2 and UT-A3 to test how these phosphorylation sites influenced their urea transport activity. Adding Lc to UT-A2 conferred stimulation by cAMP to the cAMP-unresponsive UT-A2, and adding Lc to UT-A3 did not further enhance its already existing cAMP response. These findings suggest that the responsiveness to vasopressin that is observed with UT-A1 can be introduced into the unresponsive UT-A2 variant through the Lc segment that is unique to UT-A1. In UT-A3, however, the Lc segment plays no significant role in its activation by cAMP. In addition, the Lc segment also gave UT-A2 the ability to bind snapin and, in Xenopus oocytes, to be stimulated in its urea transport activity by snapin and syntaxins 3 and 4, in the same way as UT-A1.

Transgenic Restoration of Urea Transporter A1 Confers Maximal Urinary Concentration in the Absence of Urea Transporter A3

Urea has a critical role in urinary concentration. Mice lacking the inner medullary collecting duct (IMCD) urea transporter A1 (UT-A1) and urea transporter A3 (UT-A3) have very low levels of urea permeability and are unable to concentrate urine. To investigate the role of UT-A1 in the concentration of urine, we transgenically expressed UT-A1 in knockout mice lacking UT-A1 and UT-A3 using a construct with a UT-A1 gene that cannot be spliced to produce UT-A3. This construct was inserted behind the original UT-A promoter to yield a mouse expressing only UT-A1 (UT-A1(+/+)/UT-A3(-/-)). Western blot analysis demonstrated UT-A1 in the inner medulla of UT-A1(+/+)/UT-A3(-/-) and wild-type mice, but not in UT-A1/UT-A3 knockout mice, and an absence of UT-A3 in UT-A1(+/+)/UT-A3(-/-) and UT-A1/UT-A3 knockout mice. Immunohistochemistry in UT-A1(+/+)/UT-A3(-/-) mice also showed negative UT-A3 staining in kidney and other tissues and positive UT-A1 staining only in the IMCD. Urea permeability in isolated perfused IMCDs showed basal permeability in the UT-A1(+/+)/UT-A3(-/-) mice was similar to levels in wild-type mice, but vasopressin stimulation of urea permeability in wild-type mice was significantly greater (100% increase) than in UT-A1(+/+)/UT-A3(-/-) mice (8% increase). Notably, basal urine osmolalities in both wild-type and UT-A1(+/+)/UT-A3(-/-) mice increased upon overnight water restriction. We conclude that transgenic expression of UT-A1 restores basal urea permeability to the level in wild-type mice but does not restore vasopressin-stimulated levels of urea permeability. This information suggests that transgenic expression of UT-A1 alone in mice lacking UT-A1 and UT-A3 is sufficient to restore urine-concentrating ability.