Tamm-Horsfall glycoprotein interacts with renal outer medullary potassium channel ROMK2 and regulates its function

Abstract

Tamm-Horsfall glycoprotein (THGP) or Uromodulin is a membrane protein exclusively expressed along the thick ascending limb (TAL) and early distal convoluted tubule (DCT) of the nephron. Mutations in the THGP encoding gene result in Familial Juvenile Hyperuricemic Nephropathy (FJHN), Medullary Cystic Kidney Disease type 2 (MCKD-2), and Glomerulocystic Kidney Disease (GCKD). The physicochemical and biological properties of THGP have been studied extensively, but its physiological function in the TAL remains obscure. We performed yeast two-hybrid screening employing a human kidney cDNA library and identified THGP as a potential interaction partner of the renal outer medullary potassium channel (ROMK2), a key player in the process of salt reabsorption along the TAL. Functional analysis by electrophysiological techniques in Xenopus oocytes showed a strong increase in ROMK current amplitudes when co-expressed with THGP. The effect of THGP was specific for ROMK2 and did not influence current amplitudes upon co-expression with Kir2.x, inward rectifier potassium channels related to ROMK. Single channel conductance and open probability of ROMK2 were not altered by co-expression of THGP, which instead increased surface expression of ROMK2 as determined by patch clamp analysis and luminometric surface quantification, respectively. Despite preserved interaction with ROMK2, disease-causing THGP mutants failed to increase its current amplitude and surface expression. THGP(-/-) mice exhibited increased ROMK accumulation in intracellular vesicular compartments when compared with WT animals. Therefore, THGP modulation of ROMK function confers a new role of THGP on renal ion transport and may contribute to salt wasting observed in FJHN/MCKD-2/GCKD patients.

FIGURE 1.

ROMK2 interacts with THGP. A, membrane yeast two hybrid (m-YTH) constructs used in this study. ROMK2 fused to the C-terminal half of ubiquitin (Cub) along with an artificial transcriptional factor (TF) is in-frame to the upstream and downstream reporter cassette respectively were used as baits (left). Y2H assays showing interaction between ROMK2 and THGP. Shown are plates with selective medium (SD) lacking leucine and tryptophan (−LW), indicating the transforming of both bait and prey vectors; with SD−LWHA, indicating the expression of reporter genes HIS3 and ADE2; and with β-galactosidase as a second reporter assay, indicating positive interaction. ROMK2 was also co-transformed with negative (pDL2-Alg5) and positive (pAI-Alg5) control preys (right). B, fluorescence (upper panel) and confocal (lower panel) images show immunohistochemical co-localization of THGP and ROMK in the medullary thick ascending limb (TAL) of mouse kidney revealing double immunostaining for THGP (red) and ROMK (green). Merging THGP and ROMK staining revealed partial overlap of the signals within the mouse TAL cells, indicated by arrows. All images are supplemented with scale bars (10 μm). C, total kidney lysates of wild-type and THGP^−/− mice were prepared and subjected to immunoprecipitation using anti-THGP antibody and the blots were probed with anti-ROMK antibody. D, Flp-In-293/ROMK2 stable cells were transfected with GFP-tagged THGP or empty GFP constructs. Cell lysates were immunoprecipitated with anti-THGP antibody, and immunoblotted with anti-ROMK antibody. HEK-293 cell lysate was used to verify the specificity of anti-ROMK antibody.

FIGURE 2.

Effect of THGP on ROMK currents. A, representative whole cell currents from Xenopus oocytes injected with ROMK2 (left) or THGP (middle) or both (right) are shown. B, macroscopic current-voltage relationships and C, currents activated at −100mV were plotted. D, Xenopus oocytes injected with HA-tagged ROMK2 alone or together with THGP were assayed for surface expression, as determined by a cell surface luminescence assay. E, corresponding Western blots of HA-tagged protein in total oocyte lysates. Water-injected oocytes were taken as control to determine antibody specificity (n = 15, ***, p < 0.0001 versus ROMK2).

FIGURE 3.

Effect of THGP on Kir2. x inward rectifier potassium channels. A, representative whole cell currents from Xenopus oocytes co-injected with Kir2.1 and THGP are shown. B, corresponding macroscopic current-voltage relationships and C, currents activated at −100mV were plotted for three different Kir2.x (2.1, 2.3, and 2.4) potassium channels. n = 10, ***, p < 0.0001 versus ROMK2, n.s: not significant.

FIGURE 4.

THGP mutants interact with ROMK2. A, schematic representation of THGP domain organization. I-IV are the epidermal growth factor-like domains; D8C, domain containing the eight conserved cysteine residues; ZP, zona pellucida domain responsible for the polymerization of extracellular proteins; GPI, site of GPI anchor attachment. The positions of the five THGP mutations that were analyzed in this study are shown. B, Y2H assays showing interaction between ROMK2 and THGP WT and mutants. Shown are plates with selective medium lacking leucine and tryptophan (−LW), indicating the transforming of both bait and prey vectors; with SD−LWH and SD−LWHA, indicating the expression of reporter genes HIS3 and ADE2; and with β-galactosidase as a second reporter assay. Growth of yeast cells in SD−LWH, SD−LWHA and β-gal plates indicate positive interaction.

FIGURE 5.

Effect of THGP mutants on ROMK2. A, macroscopic whole cell current-voltage relationships and B, currents activated at −100 mV were plotted for Xenopus oocytes co-injected with ROMK2 (R) and THGP (T) mutants. C, Xenopus oocytes co-injected with HA-tagged ROMK2 and THGP (WT or mutants) were assayed for surface expression, as measured by a cell surface luminescence assay. The inset contains the corresponding Western blots of HA-tagged protein in the total oocyte lysates. n = 15, ***, p < 0.0001 versus ROMK2+THGP.

FIGURE 6.

ROMK expression in THGP^−/− mice. A, Western blots from the TAL cell extracts of WT and THGP^−/− mice (n = 6 animals each) show specific bands for ROMK in the plasma membrane- and vesicle-enriched fractions. Corresponding control flotillin-1 blots for plasma membrane- and vesicle-enriched fractions are represented. B, representative Western blots from kidney homogenates of WT mouse (n = 6 animals) show specific bands for α-tubulin, β-actin, flotillin-1, and HSP-70 in the plasma membrane (PM; 17,000 × g, pellet), vesicle and cytosol- (Ves+Cyt; 17,000 × g, supernatant) cytosol (Cyt; 200,000 × g, supernatant) and vesicle-enriched fractions (200,000 × g, pellet). Note the nearly complete to absolute absence of signal for cytoskeleton (α-tubulin, β-actin) and cytosolic (HSP-70) proteins in membrane-enriched fractions, whereas membrane resident protein Flotillin-1 was mainly distributed in membrane-enriched fractions clearly demonstrating the purity of plasma membrane and vesicle preparations. C, densitometric analysis of Western blots with intensity values normalized to flotillin for plasma membrane- and vesicle-enriched fractions are plotted for both strains (n = 6, *, p < 0.01; **, p < 0.001 versus WT). D, confocal images show a sharp membrane staining of ROMK in the wild-type mice, whereas THGP^−/− mice show a diffuse staining pattern of ROMK in the TAL tubules. TAL cells (*) were identified by co-staining for NKCC2, a sodium-potassium-chloride transporter specifically expressed along the TAL. ROMK staining in CCD cells (×) lacking expression of THGP and NKCC2 serves as a negative control. Phase contrast images of TAL and CCD for both WT and THGP^−/− mice are provided in supplemental Fig. S3). All images are supplemented with scale bars (10 μm).