Coexpression of proximal tubule claudins‐2 and ‐10a increases conductance and decreases permselectivity in a renal epithelial cell line

Abstract

The mammalian nephron is composed of a series of tubule segments with distinct patterns of tight junction morphology, ion selectivity, and protein expression. Paracellular permeability of epithelial tissues is determined in part by claudins, a family of tight junction membrane proteins. The most abundantly expressed claudins in the proximal tubule (PT) are claudin‐2 and ‐10a. In general, claudin‐2 is expressed in leaky epithelia and functions as a cation‐permeable paracellular channel, whereas claudin‐10a has been shown to form anion‐selective pores. We hypothesized that coexpression of these PT claudins leads to distinct paracellular permeability properties, and contribute to ion transport in this segment.We established a double‐inducible, titratable gene expression system using Madin‐Darby canine kidney cells (MDCK I). Utilizing tetracycline‐controlled transcriptional activation (Tet‐Off), we developed stable cell lines which expressed claudin‐2 in the absence of tetracycline antibiotics. From this parent line, we generated stable transfectants expressing claudin‐10a under the control of a cumate‐inducible switch (SBI). Induction of claudin‐2, ‐10a, or both isoforms, all reduced TER compared to cells with no claudin overexpression (p<0.0001 using 2‐way ANOVA). We found a marked increase in PCl in cells induced to express claudin‐10a alone (17.4 ± 1.7 * 106 cm/s vs. 0.55 ± 0.06 * 106 cm/s with no induction), and a marked increase in PNa in cells induced to express claudin‐2 alone (12.2 ± 0.6 * 106 cm/s vs. 0.59 ± 0.07 * 106 cm/s with no induction). When both claudin‐2 and ‐10a were expressed, PCl and PNa were both increased (to 9.5 ± 1.5 * 106 cm/s and 6.2 ± 0.4 * 106 cm/s, respectively) and PNa/PCl was unchanged (0.76 ± 0.10 vs. 1.07 ± 0.05 with no induction). Calcium transport is believed to occur via a paracellular route in the PT, and so we measured the effect of PT claudin overexpression on unidirectional 45calcium radiotracer flux. Claudin‐2 expression increased calcium flux by over 7‐fold (p<0.0001), while claudin‐10a had no effect compared with no expression (p=0.99). No difference was detected in calcium flux between claudin‐2 expression and claudin‐2 and ‐10a in combination (p=0.89). These results further support claudin‐2 as the mediator of calcium reabsorption in the PT, and suggest that an interaction between PT claudin isoforms reduces the permselectivity of the PT to sodium and chloride.Support or Funding InformationSupported in part by NIH grant F30DK109605This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.