Role of Lipid Domains in Renal Phosphate Transport

脂质结构域在肾磷酸盐转运中的作用

• 批准号: 6649685
• 负责人: HUBERT K ZAJICEK
• 金额: $ 12.42万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-21 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6649685
• 关键词: apical membrane; caveolins; chemical structure function; cholesterol; clathrin; dynamin; fluorescence resonance energy transfer; fluorescence spectrometry; immunofluorescence technique; membrane lipids; membrane proteins; phosphates; renal tubular transport; sodium; tissue /cell culture; transport proteins

DESCRIPTION (provided by applicant): Disorders of extracellular inorganic phosphate (Pi) concentration and impairment in Pi reabsorption are common clinical problems in the general population. Aging, diabetes mellitus, malignancy, alcoholism, post-organ transplantation, AIDS, and several therapeutic drugs are well known to cause or to be associated with hypophosphatemia or hyperphosphatemia, mainly by affecting renal tubular Pi transport. The kidney plays a critical role in the regulation of Pi homeostasis. Pi is freely filtered across the glomerulus and most of the Pi is reabsorbed along the proximal tubule via a sodium gradient-dependent process (Na/Pi cotransport). The Na/Pi cotransporter is located on the apical brush border membrane (BBM) of the proximal tubule. The evidence to date indicates that regulation of the overall renal tubular Pi transport by dietary, hormonal, or metabolic factors occurs at the level of the proximal tubular BBM Na/Pi cotransport system. The specific aims of this proposal are to determine: 1) the role of lipid microdomains/ lipid rafts, alterations in the diffusion and/or clustering of Na/Pi proteins in the regulation of Na/Pi cotransport activity, 2) the role of clathrin, caveolae and dynamin in trafficking; specifically, internalization of Na/Pi protein from the apical membrane, 3) the role of SNAREs in trafficking, especially targeting of Na/Pi protein to the apical membrane and 4) the effect of direct alterations in cholesterol content on these processes of lateral and vertical diffusion of Na/Pi Protein. The models utilized will consist of Pi-adaptation and modification of cholesterol content, in Oppossum Kidney Cells (OK), a cell culture line. The methods employed will range from well-known biochemical approaches of transport rate, protein measurements and biotinylation experiments to newer biophysical techniques of imaging and co-localization such as Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) and Confocal Immunofluorescence Microscopy.

描述（由申请人提供）： 细胞外无机磷酸盐（PI）浓度和PI重吸收障碍的疾病是普通人群中常见的临床问题。众所周知，衰老，糖尿病，恶性，酒精中毒，酗酒，酗酒，酗酒，艾滋病和几种治疗药物是众所周知的，主要是通过影响肾管PI转运而引起或与降磷血症或高磷酸血症有关。肾脏在PI稳态调节中起着至关重要的作用。 PI在肾小球上自由过滤，大多数PI通过钠梯度依赖性过程（Na/pi cotransport）沿近端小管重新吸收。 Na/pi共转运蛋白位于近端小管的顶端刷边膜（BBM）上。迄今为止的证据表明，在饮食，荷尔蒙或代谢因子中对整个肾小管PI转运的调节发生在近端肾小管BBM NA/PI共转运系统的水平上。 该提案的具体目的是确定：1​​）脂质微域/脂质筏的作用，Na/Pi蛋白在Na/pi cotransport活性调节中的扩散和/或聚类的改变和/或聚类的作用，2）2）网状蛋白，caveolae，caveolae的作用以及在运输中的动力蛋白；具体而言，从根尖膜上内部化Na/pi蛋白的内在化，3）SNARE在运输中的作用，尤其是将Na/Pi蛋白靶向顶端膜，以及4）胆固醇含量直接改变胆固醇含量对Na/Pi蛋白横向和垂直扩散过程的影响。 所使用的模型将包括PI-ADACT和修饰胆固醇含量，在Oppossum肾细胞中（OK），一种细胞培养线。所采用的方法将从众所周知的运输速率生化方法，蛋白质测量和生物素化实验到成像和共定位的新生物物理技术，例如荧光共振能量转移（FRET）和荧光相关光谱（FCS）和共荧光荧光荧光法。