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.
Highlights
It is localized in the luminal cell surface of the thick ascending limb (TAL) of Henle’s loop and early distal convoluted tubules of the nephron [1]
Full-length Tamm-Horsfall glycoprotein (THGP) fused to the N-terminal ubiquitin (Nub) gave a positive interaction signal in the split-ubiquitin yeast two-hybrid assay upon expression with both the ROMK baits, yielding a hisϩ/adeϩ/lacZϩ phenotype in the yeast NMY51 strain (Fig. 1A, right)
We observed a strong THGP signal in the TAL of wild-type mouse and absence of THGP signal in the THGPϪ/Ϫ mouse
Summary
Molecular Cloning and Mutagenesis—Full-length hROMK2 was cloned into the pcDNA5/FRT/V5-His-TOPO TA cloning vector (Invitrogen) according to the manufacturer’s protocol. The yeast strains expressing the baits were transformed with human kidney cDNA library (MoBiTec Molecular Biotechnology, Gottingen, Germany) for screening or with prey vectors for direct interaction studies. Following the removal of the large plasma membrane enriched pellet at 17,000 ϫ g for 1 h, the supernatant was subsequently centrifuged at 200,000 ϫ g for 1 h to obtain a vesicle-enriched pellet (Ves) and a supernatant containing cytoplasmic components (Cyt). Pellets containing the plasma membrane-enriched and vesicle-enriched fractions were dissolved in buffer-I and protein concentrations were measured using the BCA protein assay kit (Pierce). After electrophoretic transfer of the proteins, polyvinylidene fluoride membranes were incubated with the primary antibody against ROMK (1:500, Alomone) or flotillin-1 (1:1000, BD Biosciences) (Following stripping), each for 1 h at room temperature, followed by overnight incubation at 4 °C and subsequent exposure to HRP-conjugated secondary antibodies for 2 h at room temperature. Statistically significant differences to control values are marked by asterisks (*, p Ͻ 0.01; **, p Ͻ 0.001; and ***, p Ͻ 0.0001); n.s indicates non-significant differences (p Ͼ 0.05)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
