Axial Flow Effects in Proximal Tubule

描述（由申请人提供）：近端小管中流动激活的盐和水转运的生理重要性已被认识超过40年，然而，这种调节的机制仍然没有得到很好的定义。最近，我们已经证明，a）灌注-吸收平衡存在于小鼠的离体灌注近端小管中。B）管腔膜NHE 3和H-ATP酶均受流量调节; c）紧密连接通透性的变化在流量调节的转运中不起作用。我们已经开发了一个理论计算的力和扭矩上的微绒毛，并证明，流量引起的变化的近端小管吸收的扭矩依赖性，和一个完整的肌动蛋白细胞骨架是必需的，将信号传递给细胞。实验数据和模拟计算提供了有力的证据，刷状缘微绒毛功能的流量传感器在近端小管。然而，初级纤毛是否也起流量传感器的作用，以及管周离子转运蛋白是否也可以通过轴向流进行调节还没有研究。本论文拟通过体外微灌注实验、免疫荧光和Western印迹等方法，研究以下三个方面的问题：1）通过比较模型预测值和流动诱导的野生型、绒毛、纤毛、肌球蛋白和纤毛缺陷小鼠Na ~+和HCO ~ 3 ~-吸收的变化，探讨中央纤毛和微绒毛是否是近端小管的流动传感器; 2）研究流动诱导的肌动蛋白细胞骨架重组在调节小鼠近端小管转运蛋白运输和功能中的作用; 3）研究第二信使、钙信号、cAMP和PKA调节机制以及多巴胺在流动依赖性近端小管转运中的作用。本研究的独特之处在于：1）比较了小鼠和基因敲除动物完整肾小管中依赖于流动的近端小管转运; 2）将重吸收通量表示为微绒毛和纤毛上的流体动力学力和扭矩的函数;和3）评估流动诱导的肌动蛋白细胞骨架重组，离子转运蛋白定位，和近端小管运输的数学模型内的功能变化。这些研究将提供新的信息，肾小球肾小管平衡（GTB）的机制和方面的肾液和HCO 3-运输的生理和病理生理条件。公共卫生相关性：全身血压的维持取决于肾脏内钠重吸收的速率，沿着整个肾单位，其中约2/3发生在近端小管。这些研究将为近端小管钠转运的调控提供直接的信息。可能的益处包括鉴定靶分子，其可以被阻断或修饰以调节肾脏的钠重吸收。

DESCRIPTION (provided by applicant): The physiological importance of flow-activated salt and water transport in proximal tubules has been recognized for more than four decades, however the mechanism of this regulation is still not well defined. Recently we have demonstrated that a) Perfusion-absorption balance is present in the isolated perfused proximal tubule of the mouse. b) Both luminal membrane NHE3 and H-ATPase are regulated by flow; c) Changes in tight junction permeabilities do not play a role in flow-modulated transport. We have developed a theory for calculating the forces and torques on the microvilli, and demonstrated that flow-induced changes of proximal tubule absorption are torque dependent, and that an intact actin cytoskeleton is required to transduce the signal to the cell. Experimental data and modeling calculations provide strong evidence that brush border microvilli function as flow sensors in the proximal tubule. However, whether the primary cilium also functions as flow sensor and whether peritubular ion transporters can also be regulated by axial flow has not been examined. In the work proposed, in vitro microperfusion experiments, immunofluorescence, and Western blotting will be conducted with the following three aims: 1) To study the controversy of whether central cilia or microvilli are the flow sensors in proximal tubules by comparing model predictions with flow-induced changes on Na+ and HCO3- absorption in wild-type, and villin, fimbrin, myosins and cilia-deficient mice; 2) To study the role of flow-induced actin cytoskeletal reorganization in modulating transporter trafficking and function in mouse proximal tubules; 3) To examine the role of second messengers, calcium signals, cAMP- and PKA- modulated mechanisms and the role of dopamine in flow-dependent proximal tubule transport. The unique features of our proposed collaboration are: 1) the comparison of flow-dependent proximal tubule transport in intact tubules in mice and in knockout animals; 2) the representation of reabsorptive fluxes as a function of the hydrodynamic forces and torques on microvilli and cilia; and 3) the assessment of flow-induced actin cytoskeletal reorganization, ion transporter localization, and functional changes within a mathematical model of proximal tubule transport. These studies will provide new information on mechanisms of glomerulotubular balance (GTB) and aspects of renal fluid and HCO3- transport in physiological and pathophysiological conditions. PUBLIC HEALTH RELEVANCE: The maintenance of systemic blood pressure depends upon the rate of sodium reabsorption within the kidney, along the entire nephron, and about 2/3 of this occurs in the proximal tubule. The proposed studies will provide direct information on the regulation of proximal tubule sodium transport. Possible benefits include identification of target molecules, which may be blocked or modified in order to modulate sodium reabsorption by the kidney.