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

内淋巴囊内淋巴氯化钠稳态的免疫组织化学和体外研究

• 批准号: 241878539
• 负责人: Dr. Andreas Eckhard
• 金额: --
• 依托单位: Massachusetts Eye and Ear
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/241878539?language=en
• 关键词: Immunohistochemical; vitro; studies; endolymphatic; sodium

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.

内耳内淋巴液平衡的紊乱会导致内淋巴容量减少（塌陷）或增加（水肿），并导致严重的前庭和听觉功能障碍，如梅尼埃病中所观察到的那样。作为内淋巴液调节的主要部位，内淋巴囊（ES）被认为能够主动分泌和再吸收内淋巴液，从而维持恒定的内淋巴体积。 ES 中的内淋巴分泌和/或吸收缺陷可能是梅尼埃病内淋巴积水的发病机制的基础。然而，ES 上皮分泌和吸收过程的分子机制尚未阐明。基于ES上皮细胞和肾小管细胞之间的形态和功能相似性，有人提出在ES上皮中存在调节肾小管上皮中全身液体体积的离子和水输送机制。肾小管液体调节的主要途径是抗利尿激素 (ADH)/醛固酮调节的氯化钠 (NaCl) 重吸收，从而实现渗透水潴留。在此，有人提出这些肾脏 NaCl 转运机制存在于 ES 上皮中，它们控制内淋巴 Na+ 和 Cl- 浓度。穿过 ES 上皮的跨上皮 NaCl 通量可能使 ES 周围的内淋巴和细胞外液之间的被动水运动成为可能，因此有助于内淋巴容量稳态。 ES 上皮中跨上皮 NaCl 转运的失败可能是梅尼埃病内淋巴积水发病机制的原因。本提案的目的是检查 ES 上皮中 ENaC、NKCC2、Na+/K+-ATPase、Pendrin、ClC-KB 和 NDCBE 肾 ADH 和醛固酮调节的 NaCl 转运机制的存在和功能。因此，i) 通过免疫组织化学在小鼠 ES 中检查 ES 上皮细胞中 ENaC、NKCC2、Na+/K+-ATPase、Pendrin、ClC-KB 和 NDCBE、ADH（V1 和 V2）- 和醛固酮受体的表达。 ii) 在梅尼埃病患者和健康对照的死后 ES 组织中通过免疫组织化学方法检查这些蛋白质的表达。 iii) 使用钠结合荧光指示剂 (SBFI) 和共聚焦激光扫描显微镜 (cLSM) 在体外研究小鼠 ES 上皮细胞中 Na+ 的吸收和释放。 iv) 使用 SBFI 和 cLSM 在体外研究 ADH 和醛固酮对上皮 Na+ 吸收和释放的影响。