Claudin-4 forms paracellular chloride channel in the kidney and requires claudin-8 for tight junction localization

Abstract

Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. The paracellular pathway in the collecting duct of the kidney is a predominant route for transepithelial chloride reabsorption that determines the extracellular NaCl content and the blood pressure. However, the molecular mechanisms underlying the paracellular chloride reabsorption in the collecting duct are not understood. Here we showed that in mouse kidney collecting duct cells, claudin-4 functioned as a Cl -channel. A positively charged lysine residue at position 65 of claudin-4 was critical for its anion selectivity. Claudin-4 was observed to interact with claudin-8 using several criteria. In the collecting duct cells, the assembly of claudin-4 into TJ strands required its interaction with claudin-8. Depletion of claudin-8 resulted in the loss of paracellular chloride conductance, through a mechanism involving its recruitment of claudin-4 during TJ assembly. Together, our data show that claudin-4 interacts with claudin-8 and that their association is required for the anion-selective paracellular pathway in the collecting duct, suggesting a mechanism for coupling chloride reabsorption with sodium reabsorption in the collecting duct.C hloride is the predominant extracellular ionic constituent and thereby determines extracellular fluid volume (ECFV) and blood pressure (1-3). Although only responsible for the reabsorption of 2-3% filtered chloride, the aldosterone-sensitive distal nephron (ASDN) plays a vital regulatory role in renal handling of salt, ECFV control, and managing blood pressure (4). The ASDN comprises the distal convoluted tubule (DCT), the connecting tubule (CNT), and the collecting duct. The collecting duct is characterized by a heterogeneous epithelium-the principal cells and intercalated cells (5). Sodium reabsorption in the collecting duct is an active process driven by the basolateral Na + /K + -ATPase; acts through the apical epithelial sodium channel (ENaC) in the principal cell (6); and is responsible for generating the lumen-negative transepithelial potential. This electrogenic transport step creates a favorable electrical driving force for luminal reabsorption of chloride and secretion of potassium and proton. Chloride is transported by two major mechanisms (1). Chloride is actively reabsorbed by an electroneutral Cl − /HCO − exchanger (Slc26a4: pendrin) localized to the apical membrane of the β-type intercalated cell (7). Choride exits this cell via a basolateral Cl − channel (2) and diffuses passively down electrochemical gradients via the paracellular channel in the tight junction (TJ).The TJ is the most apical member of the junctional complex found in vertebrate epithelia responsible for the barrier to movement of ions and molecules between apical and basal compartments, the paracellular pathway (8). TJs are composed of three transmembrane proteins: occludin, claudins, and junctional adhesion molecule (JAM). The claudins (CLDNs) are a 28-member family of tetraspan proteins that range in m...

The Cap1–claudin-4 regulatory pathway is important for renal chloride reabsorption and blood pressure regulation

“…We have previously identified the molecular component underlying paracellular Cl − permeation, claudin-4, from the CD cells using RNA interference (11). Next, we asked whether cap1 acted through claudin-4.…”

“…Because cap1 was secreted into the tubular lumen (16), its expression in renal tubules proximal to the CD would suggest a paracrine role, whereas its expression in the CD per se would suggest an autocrine role. To reveal the autocrine function of cap1 in the CD, we generated transgenic M-1 and mIMCD3 cells expressing small interference RNA (siRNA) molecules against cap1 mRNA, as described by Hou et al (11). The siRNA molecules were designed by the siDESIGN center at Thermo Scientific, and each contained a unique complementary 19-nucleotide sequence within the coding region of the mouse cap1 gene.…”

“…Claudins associate by cis interactions within the plasma membrane of the same cell followed by trans interactions between neighboring cells to assemble them in the TJ. Previously, we have observed the cis interaction between claudin-4 and claudin-8 and demonstrated that their interaction was required for TJ assembly (11). How claudins trans interact, on the other hand, is poorly understood.…”

“…However, such an electroneutral pathway is not able to couple Cl − reabsorption with epithelial sodium channel (ENaC)-based Na + reabsorption that takes place in the principal cells (PCs) of CNT and CD (8), despite many efforts to connect ENaC and pendrin gene expression through endocrine or paracrine mechanisms (9,10). We and others have previously demonstrated the presence of a paracellular Cl − pathway or "chloride shunt" in vitro in the CNT and CD cells (11,12). The paracellular Cl − channel is made of a key claudin molecule, claudin-4, within the tight junction (TJ) (11).…”

“…We and others have previously demonstrated the presence of a paracellular Cl − pathway or "chloride shunt" in vitro in the CNT and CD cells (11,12). The paracellular Cl − channel is made of a key claudin molecule, claudin-4, within the tight junction (TJ) (11). Here, using a tissuespecific KO approach, we have provided conclusive evidence that claudin-4 is required for renal reabsorption of Cl − ; loss of claudin-4 in a transgenic mouse model claudin-4 flox/flox /Aqp2 Cre caused significant renal wasting of chloride, sodium, and volume.…”