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Abstract: TH-PO329

Claudin-10b Role in the Basolateral Infoldings of the Thick Ascending Limb

Session Information

Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders

  • 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic

Authors

  • Quintanova, Catarina, Christian-Albrechts-Universitat zu Kiel, Kiel, Schleswig-Holstein, Germany
  • Himmerkus, Nina, Christian-Albrechts-Universitat zu Kiel, Kiel, Schleswig-Holstein, Germany
  • Mutig, Kerim, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
  • Müller, Dominik, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
  • Breiderhoff, Tilman, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
  • Günzel, Dorothee, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
  • Bleich, Markus, Christian-Albrechts-Universitat zu Kiel, Kiel, Schleswig-Holstein, Germany
Background

The thick ascending limb (TAL) of the loop of Henle is responsible for salt reabsorption and thus for urine concentration. The paracellular pathway is determined by the combination of claudin expression in the tight junctions (TJ). In the TAL there is a mosaic expression of either claudin-10b or claudin-3/claudin-16/claudin-19 in a complex. Claudin-10b TJ confer a sodium (Na+) permeability. Remarkably, claudin-10b is also expressed extra-junctional in the infoldings of the basolateral membrane.

Methods

Freshly isolated single murine TAL segments of C57Bl6 and kidney specific (Ksp-Cre) Claudin-10 knockout (cKO) mice were investigated by immunofluorescence (IF) or western blot (WB). Dissected TAL were transferred into bath solution (37 °C) and microperfused under constant pressure and measured under different basolateral solutions to assess infolding accessibility by the Na+/K+-ATPase (NKA) inhibitor ouabain or by fluorescein. To loosen the intermembrane protein contacts, we removed calcium (Ca2+) from the basolateral bath solution.

Results

In single TAL segments, we performed triple staining with claudin-10, NKA and the chloride channel subunit Barttin. The localization of all proteins co-localized in the infoldings and NKA and Barttin expression were not changed in IF or WB by cKO. The relative speed of ouabain inhibition was higher in the cKO and in Ca2+-free conditions. Further, we tested the diffusion of fluorescein into the infoldings. In the WT, upon re-addition of Ca2+ we were able to trap more fluorescein when compared to the cKO.

Conclusion

Claudin-10b KO has no direct influence on the expression and localization of other important basolateral transport proteins. However, the increased speed of ouabain inhibition and the decrease of fluorescein trapping in the absence of Claudin-10b and Ca2+-free situations suggests a role of claudin-10b in the stabilization of the infoldings by forming a complex between neighboring invaginated membranes.