Immunohistochemical and in vitro studies on endolymphatic sodium chloride homeostasis by the endolymphatic sac

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Disturbances of endolymphatic fluid balance in the inner ear lead to a decrease (collapse) or an increase of endolymph volume (hydrops) and cause severe vestibular and auditory dysfunction, as observed in Menières disease. As the major site of endolymphatic fluid regulation, the endolymphatic sac (ES) has been proposed to actively secrete and resorb endolymphatic fluid and thereby maintains a constant endolymph volume. Defective endolymph secretion and/or resorption in the ES presumably underlie the pathogenesis of endolymphatic hydrops in Menières disease. However, the molecular mechanisms of the secretory and resorptive processes in the ES-epithelium have not been elucidated. In the ES-epithelium, the presence of ion- and water-transporting mechanisms that regulate whole body fluid volume in the kidney tubular epithelium has been suggested, based on morphological and functional similarities between the ES-epithelial cells and kidney tubular cells. A major pathway of fluid regulation in the kidney tubule is the anti-diuretic hormone (ADH)/aldosterone-regulated resorption of sodium chloride (NaCl) that enables osmotic water retention. Here, it is proposed that these renal NaCl-transporting mechanisms are present in the ES-epithelium where they control endolymphatic Na+ and Cl- concentrations. Transepithelial NaCl-fluxes across the ES-epithelium potentially enable passive water-movements between endolymph and the extracellular fluid around the ES, and therefore contribute to endolymphatic volume homeostasis. A failure of transepithelial NaCl transport in the ES-epithelium might be causative for the pathogenesis of endolymphatic hydrops in Menières disease. The purpose of this proposal is to examine the presence and function of renal ADH- and aldosterone-regulated NaCl-transporting mechanisms by ENaC, NKCC2, Na+/K+-ATPase, Pendrin, ClC-KB and NDCBE in the ES-epithelium. Therefore, i) the expression of ENaC, NKCC2, Na+/K+-ATPase, Pendrin, ClC-KB and NDCBE, ADH (V1 and V2)- and aldosterone-receptors in the ES-epithelium is examined in the mouse ES by immunohistochemistry. ii) The expression of these proteins is examined immunohistochemically in post mortem ES-tissue of Menières patients and healthy controls. iii) Na+ uptake and release in mouse ES-epithelial cells is investigated in vitro using sodium-binding fluorescent indicators (SBFI) and confocal laser scanning microscopy (cLSM). iv) The influence of ADH and aldosterone on epithelial Na+-uptake and release is investigated in vitro using SBFI and cLSM.