Transgenic RNAi Depletion of Claudin-16 and the Renal Handling of Magnesium

Jianghui Hou,Qixian Shan,Tong Wang,Antonio S Gomes,Qingshang Yan,David L Paul,Markus Bleich,Daniel A Goodenough

doi:10.1074/jbc.m700632200

Abstract

Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a human disorder caused by mutations in the tight junction protein claudin-16. However, the molecular mechanisms underlining the renal handling of magnesium and its dysfunction causing FHHNC are unknown. Here we show that claudin-16 plays a key role in maintaining the paracellular cation selectivity of the thick ascending limbs of the nephron. Using RNA interference, we have generated claudin-16-deficient mouse models. Claudin-16 knock-down (KD) mice exhibit chronic renal wasting of magnesium and calcium and develop renal nephrocalcinosis. Our data suggest that claudin-16 forms a non-selective paracellular cation channel, rather than a selective Mg(2+)/Ca(2+) channel as previously proposed. Our study highlights the pivotal importance of the tight junction in renal control of ion homeostasis and provides answer to the pathogenesis of FHHNC. We anticipate our study to be a starting point for more sophisticated in vivo analysis of tight junction proteins in renal functions. Furthermore, tight junction proteins could be major targets of drug development for electrolyte disorders.

Highlights

Gene family that encodes essential structural components of the tight junction, the principal regulator of paracellular permeability
It is generated by the electrogenic NaCl reabsorption and, as a direct consequence of the tubular fluid dilution, it is mainly a diffusion potential between luminal and basolateral extracellular spaces if they are separated by cation-selective tight junctions
Lentivirus-directed Transgenesis—To characterize the efficiency of lentiviral transgenesis, we used lentivirus based on the pFUGW vector in which the ubiquitin-C promoter drives expression of GFP (Fig. S1A of supplemental information; Ref. 11)

Summary

EXPERIMENTAL PROCEDURES

Antibodies, Cell Lines, and Animals—The following antibodies were used in this study: rabbit polyclonal anti-CLDN16 (Zymed Laboratories); fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin G (Chemicon). 293T cells (a kind gift from Dr Joan Brugge, Harvard Medical School) were cultured in Dulbecco’s modified Eagle’s medium supplemented. To increase the yield of embryo collection following superovulation in female mice and to facilitate the rapid generation of transgenic animals, we utilized a common hybrid mouse strain (B6D2F1: F1 cross between DBA/2 male and C57BL/6 female) as the donor stain [14]. Transverse CT slices were acquired at the femoral mid-shaft (diaphysis) using 12-␮m slice increment (50 ␮CT slices per specimen) For this cortical region, we assessed the total cross-sectional area, cortical bone area and medullary area (TA, BA, and MA, respectively, mm2), bone area fraction (BA/TA, %), and cortical thickness (␮m) [18]. For viewing CLDN16 expression and localization, fresh cryostat sections (10 ␮m) were fixed with cold methanol at Ϫ20 °C, followed by blocking with PBS containing 10% fetal bovine serum, incubation with primary antibodies (CLDN16, Zymed Laboratories Inc.; 1:300) and fluorescein isothiocyanate (FITC)-labeled secondary antibodies (1:200). Values were expressed as mean Ϯ S.E. unless otherwise stated

RESULTS

PMgc g mmHg mM

Whole body bone Distal femur

DISCUSSION