Phosphorylated claudin-16 interacts with Trpv5 and regulates transcellular calcium transport in the kidney.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) was previously considered to be a paracellular channelopathy caused by mutations in the claudin-16 and claudin-19 genes. Here, we provide evidence that a missense FHHNC mutation c.908C>G (p.T303R) in the claudin-16 gene interferes with the phosphorylation in the claudin-16 protein. The claudin-16 protein carrying phosphorylation at residue T303 is localized in the distal convoluted tubule (DCT) but not in the thick ascending limb (TAL) of the mouse kidney. The phosphomimetic claudin-16 protein carrying the T303E mutation but not the wildtype claudin-16 or the T303R mutant protein increases the Trpv5 channel conductance and membrane abundance in human kidney cells. Phosphorylated claudin-16 and Trpv5 are colocalized in the luminal membrane of the mouse DCT tubule; phosphomimetic claudin-16 and Trpv5 interact in the yeast and mammalian cell membranes. Knockdown of claudin-16 gene expression in transgenic mouse kidney delocalizes Trpv5 from the luminal membrane in the DCT. Unlike wildtype claudin-16, phosphomimetic claudin-16 is delocalized from the tight junction but relocated to the apical membrane in renal epithelial cells because of diminished binding affinity to ZO-1. High-Ca2+ diet reduces the phosphorylation of claudin-16 protein at T303 in the DCT of mouse kidney via the PTH signaling cascade. Knockout of the PTH receptor, PTH1R, from the mouse kidney abrogates the claudin-16 phosphorylation at T303. Together, these results suggest a pathogenic mechanism for FHHNC involving transcellular Ca2+ pathway in the DCT and identify a molecular component in renal Ca2+ homeostasis under direct regulation of PTH.

ILDR1 is important for paracellular water transport and urine concentration mechanism.

Whether the tight junction is permeable to water remains highly controversial. Here, we provide evidence that the tricellular tight junction is important for paracellular water permeation and that Ig-like domain containing receptor 1 (ILDR1) regulates its permeability. In the mouse kidney, ILDR1 is localized to tricellular tight junctions of the distal tubules. Genetic knockout of Ildr1 in the mouse kidney causes polyuria and polydipsia due to renal concentrating defects. Microperfusion of live renal distal tubules reveals that they are impermeable to water in normal animals but become highly permeable to water in Ildr1 knockout animals whereas paracellular ionic permeabilities in the Ildr1 knockout mouse renal tubules are not affected. Vasopressin cannot correct paracellular water loss in Ildr1 knockout animals despite normal effects on the transcellular aquaporin-2-dependent pathway. In cultured renal epithelial cells normally lacking the expression of Ildr1, overexpression of Ildr1 significantly reduces the paracellular water permeability. Together, our study provides a mechanism of how cells transport water and shows how such a mechanism may be exploited as a therapeutic approach to maintain water homeostasis.

Inducible Expression of Claudin-1 in Glomerular Podocytes Generates Aberrant Tight Junctions and Proteinuria through Slit Diaphragm Destabilization.

The tight junction (TJ) has a key role in regulating paracellular permeability to water and solutes in the kidney. However, the functional role of the TJ in the glomerular podocyte is unclear. In diabetic nephropathy, the gene expression of claudins, in particular claudin-1, is markedly upregulated in the podocyte, accompanied by a tighter filtration slit and the appearance of TJ-like structures between the foot processes. However, there is no definitive evidence to show slit diaphragm (SD) to TJ transition in vivo Here, we report the generation of a claudin-1 transgenic mouse model with doxycycline-inducible transgene expression specifically in the glomerular podocyte. We found that induction of claudin-1 gene expression in mature podocytes caused profound proteinuria, and with deep-etching freeze-fracture electron microscopy, we resolved the ultrastructural change in the claudin-1-induced SD-TJ transition. Notably, immunolabeling of kidney proteins revealed that claudin-1 induction destabilized the SD protein complex in podocytes, with significantly reduced expression and altered localization of nephrin and podocin proteins. Mechanistically, claudin-1 interacted with both nephrin and podocin through cis- and trans-associations in cultured cells. Furthermore, the rat puromycin aminonucleoside nephrosis model, previously suspected of undergoing SD-TJ transition, exhibited upregulated expression levels of claudin-1 mRNA and protein in podocytes. Together, our data attest to the novel concept that claudins and the TJ have essential roles in podocyte pathophysiology and that claudin interactions with SD components may facilitate SD-TJ transition that appears to be common to many nephrotic conditions.

Capturing Rare Conductance in Epithelia with Potentiometric-Scanning Ion Conductance Microscopy.

Tight junctions (TJs) are barrier forming structures of epithelia and can be described as tightly sealed intercellular spaces. Transport properties have been extensively studied for bicellular TJs (bTJs). Knowledge of the barrier functions of tricellular junctions (tTJs) are less well understood, due largely to a lack of proper techniques to locally measure discrete tTJ properties within a much larger area of epithelium. In this study, we use a nanoscale pipet to precisely locate tTJs within epithelia and measure the apparent local conductance of tTJs with a technique termed potentiometric scanning ion conductance microscopy (P-SICM). P-SICM shows the ability to differentiate transport through tTJs and bTJs, which was not possible with previous techniques and assays. We describe P-SICM investigations of both wild type and tricellulin overexpression Madin-Darby Canine Kidney (strain II, MDCKII) cells.

Biochemical and biophysical analyses of tight junction permeability made of claudin-16 and claudin-19 dimerization.

The molecular nature of tight junction architecture and permeability is a long-standing mystery. Here, by comprehensive biochemical, biophysical, genetic, and electron microscopic analyses of claudin-16 and -19 interactions--two claudins that play key polygenic roles in fatal human renal disease, FHHNC--we found that 1) claudin-16 and -19 form a stable dimer through cis association of transmembrane domains 3 and 4; 2) mutations disrupting the claudin-16 and -19 cis interaction increase tight junction ultrastructural complexity but reduce tight junction permeability; and 3) no claudin hemichannel or heterotypic channel made of claudin-16 and -19 trans interaction can exist. These principles can be used to artificially alter tight junction permeabilities in various epithelia by manipulating selective claudin interactions. Our study also emphasizes the use of a novel recording approach based on scanning ion conductance microscopy to resolve tight junction permeabilities with submicrometer precision.