Co-Regulated Pendrin and Aquaporin 5 Expression and Trafficking in Type-B Intercalated Cells under Potassium Depletion

Abstract

Background: We recently reported that aquaporin 5 (AQP5), a water channel never identified in the kidney before, co-localizes with pendrin at the apical membrane of type-B intercalated cells in the kidney cortex. Since co-expression of AQP5 and pendrin in the apical membrane domain is a common feature of several other epithelia such as cochlear and bronchial epithelial cells, we evaluated here whether this strict membrane association may reflect a co-regulation of the two proteins. To investigate this possibility, we analyzed AQP5 and pendrin expression and trafficking in mice under chronic K⁺ depletion, a condition that results in an increased ability of renal tubule to reabsorb bicarbonate, often leads to metabolic alkalosis and is known to strongly reduce pendrin expression. Methods: Mice were housed in metabolic cages and pair-fed with either a standard laboratory chow or a K⁺-deficient diet. AQP5 abundance was assessed by western blot in whole kidney homogenates and AQP5 and pendrin were localized by confocal microscopy in kidney sections from those mice. In addition, the short-term effect of changes in external pH on pendrin trafficking was evaluated by fluorescence resonance energy transfer (FRET) in MDCK cells, and the functional activity of pendrin was tested in the presence and absence of AQP5 in HEK 293 Phoenix cells. Results: Chronic K⁺ depletion caused a strong reduction in pendrin and AQP5 expression. Moreover, both proteins shifted from the apical cell membrane to an intracellular compartment. An acute pH shift from 7.4 to 7.0 caused pendrin internalization from the plasma membrane. Conversely, a pH shift from 7.4 to 7.8 caused a significant increase in the cell surface expression of pendrin. Finally, pendrin ion transport activity was not affected by co-expression with AQP5. Conclusions: The co-regulation of pendrin and AQP5 membrane expression under chronic K⁺-deficiency indicates that these two molecules could cooperate as an osmosensor to rapidly detect and respond to alterations in luminal fluid osmolality.

Functional Testing of SLC26A4 Variants—Clinical and Molecular Analysis of a Cohort with Enlarged Vestibular Aqueduct from Austria

“…For testing the function of pendrin variants, the influx of iodide was measured in cells expressing wild type or mutated pendrin and in control cells. The functional test was performed as already described [ 9 , 59 , 72 , 73 ], with adaptations allowing for the use of a multiplate reader [ 27 , 60 , 74 , 75 , 76 ]. Shortly, cells were seeded into black 96-well plates, grown overnight and co-transfected with 0.1 μg of a plasmid encoding for EYFP H148Q;I152L (an EYFP variant with substantially improved sensitivity for iodide [ 77 ]) and 0.1 μg of pTARGET plasmid bearing the cDNA of wild type or mutated pendrin.…”

SPAK Sensitive Regulation of the Epithelial Na⁺ Channel ENaC

“…Mice were placed individually in metabolic cages (Techniplast, Hohenpeissenberg, Germany) and they were maintained on a standard diet and had free access to tap water before the experiment [28,43]. They were allowed a two day habituation period.…”

Carbonic anhydrase 12 mutation modulates membrane stability and volume regulation of aquaporin 5

“…Stimulation of the salivary gland cells results in bulk water efflux and can be observed as cell shrinkage 15 , whereas water influx results in cell swelling. AQP5 has been known as an osmosensor 16 . To evaluate the volume regulation of AQP5 in salivary glands, isolated SMG acinar cells were stimulated in a hypotonic (215 mOsm) solution, and the volume change was measured using the calcein-AM fluorescent technique.…”