Dataset Information

Alternative channels for urea in the inner medulla of the rat kidney.

ABSTRACT: The ascending thin limbs (ATLs) and lower descending thin limbs (DTLs) of Henle's loop in the inner medulla of the rat are highly permeable to urea, and yet no urea transporters have been identified in these sections. We hypothesized that novel, yet-unidentified transporters in these tubule segments could explain the high urea permeability. cDNAs encoding for Na(+)-glucose transporter 1a (SGLT1a), Na(+)-glucose transporter 1 (NaGLT1), urea transporter (UT)-A2c, and UT-A2d were isolated and cloned from the Munich-Wistar rat inner medulla. SGLT1a is a novel NH2-terminal truncated variant of SGLT1. NaGLT1 is a Na(+)-dependent glucose transporter primarily located in the proximal tubules and not previously described in the thin limbs. UT-A2c and UT-A2d are novel variants of UT-A2. UT-A2c is truncated at the COOH terminus, and UT-A2d has one exon skipped. When rats underwent water restriction for 72 h, mRNA levels of SGLT1a increased in ATLs, NaGLT1 levels increased in both ATLs and DTLs, and UT-A2c increased in ATLs. [(14)C]urea uptake assays performed on Xenopus oocytes heterologously expressing these proteins revealed that despite having structural differences from their full-length versions, SGLT1a, UT-A2c, and UT-A2d enhanced urea uptake. NaGLT1 also facilitated urea uptake. Uptakes were Na(+) independent and inhibitable by phloretin and/or phloridzin. Our data indicate that there are several alternative channels for urea in the rat inner medulla that could potentially contribute to the high urea permeabilities in thin limb segments.

SUBMITTER: Nawata CM

PROVIDER: S-EPMC4669356 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Alternative channels for urea in the inner medulla of the rat kidney.

Nawata C Michele CM Dantzler William H WH Pannabecker Thomas L TL

American journal of physiology. Renal physiology 20150930 11

The ascending thin limbs (ATLs) and lower descending thin limbs (DTLs) of Henle's loop in the inner medulla of the rat are highly permeable to urea, and yet no urea transporters have been identified in these sections. We hypothesized that novel, yet-unidentified transporters in these tubule segments could explain the high urea permeability. cDNAs encoding for Na(+)-glucose transporter 1a (SGLT1a), Na(+)-glucose transporter 1 (NaGLT1), urea transporter (UT)-A2c, and UT-A2d were isolated and clone ...[more]

PMID: 26423860

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Project description:UNLABELLED:The UT-A1 urea transporter is crucial to the kidney's ability to generate concentrated urine. Native UT-A1 from kidney inner medulla (IM) is a heavily glycosylated protein with two glycosylation forms of 97 and 117 kDa. In diabetes, UT-A1 protein abundance, particularly the 117 kD isoform, is significantly increased corresponding to an increased urea permeability in perfused IM collecting ducts, which plays an important role in preventing the osmotic diuresis caused by glucosuria. However, how the glycan carbohydrate structure change and the glycan related enzymes regulate kidney urea transport activity, particularly under diabetic condition, is largely unknown. In this study, using sugar-specific binding lectins, we found that the carbohydrate structure of UT-A1 is changed with increased amounts of sialic acid, fucose, and increased glycan branching under diabetic conditions. These changes were accompanied by altered UT-A1 association with the galectin proteins, ?-galactoside glycan binding proteins. To explore the molecular basis of the alterations of glycan structures, the highly sensitive next generation sequencing (NGS) technology, Illumina RNA-seq, was employed to analyze genes involved in the process of UT-A1 glycosylation using streptozotocin (STZ)-induced diabetic rat kidney. Differential gene expression analysis combining with quantitative PCR revealed that expression of a number of important glycosylation related genes were changed under diabetic conditions. These genes include the glycosyltransferase genes Mgat4a, the sialylation enzymes St3gal1 and St3gal4 and glycan binding protein galectin-3, -5, -8, and -9. In contrast, although highly expressed in kidney IM, the glycosyltransferase genes Mgat1, Mgat2, and fucosyltransferase Fut8, did not show any changes. CONCLUSIONS:In diabetes, not only is UT-A1 protein abundance increased but the protein's glycan structure is also significantly changed. UT-A1 protein becomes highly sialylated, fucosylated and branched. Consistently, a number of crucial glycosylation related genes are changed under diabetic conditions. The alteration of these genes may contribute to changes in the UT-A1 glycan structure and therefore modulate kidney urea transport activity and alleviate osmotic diuresis caused by glucosuria in diabetes.

Project description:CLC-K channels belong to the CLC gene family, which comprises both Cl(-) channels and Cl(-)/H(+) antiporters. They form homodimers which additionally co-assemble with the small protein barttin. In the kidney, they are involved in NaCl reabsorption; in the inner ear they are important for endolymph production. Mutations in CLC-Kb lead to renal salt loss (Bartter's syndrome); mutations in barttin lead additionally to deafness. CLC-K channels are interesting potential drug targets. CLC-K channel blockers have potential as alternative diuretics, whereas CLC-K activators could be used for the treatment of patients with Bartter's syndrome. Several small organic acids inhibit CLC-K channels from the outside by binding to a site in the external vestibule of the ion conducting pore. Benzofuran derivatives with affinities better than 10 ?M have been discovered. Niflumic acid (NFA) exhibits a complex interaction with CLC-K channels. Below ?1?mM, NFA activates CLC-Ka, whereas at higher concentrations NFA inhibits channel activity. The co-planarity of the rings of the NFA molecule is essential for its activating action. Mutagenesis has led to the identification of potential regions of the channel that interact with NFA. CLC-K channels are also modulated by pH and [Ca(2+)](ext). The inhibition at low pH has been shown to be mediated by a His-residue at the beginning of helix Q, the penultimate transmembrane helix. Two acidic residues from opposite subunits form two symmetrically related intersubunit Ca(2+) binding sites, whose occupation increases channel activity. The relatively high affinity CLC-K blockers may already serve as leads for the development of useful drugs. On the other hand, the CLC-K potentiator NFA has a quite low affinity, and, being a non-steroidal anti-inflammatory drug, can be expected to exert significant side effects. More specific and more potent activators will be needed and it will be important to understand the molecular mechanisms that underlie NFA activation.

Dataset Information

Alternative channels for urea in the inner medulla of the rat kidney.

Publications

Alternative channels for urea in the inner medulla of the rat kidney.

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