Axial Flow Effects in Proximal Tubule

描述(申请人提供)：近端小管中流动激活的盐和水运输的生理重要性已经被认识到40多年了，然而这一调节的机制仍然没有很好地确定。最近，我们已经证明：a)在小鼠灌流的近端小管上存在灌流-吸收平衡。B)管腔膜NHE3和H-ATPase均受流量调节；c)紧密连接通透性的变化在流量调节转运中不起作用。我们已经发展了一个计算微绒毛上的力和扭矩的理论，并证明了近端小管吸收的流动诱导的变化是扭矩相关的，并且需要一个完整的肌动蛋白细胞骨架来将信号传递给细胞。实验数据和模拟计算提供了强有力的证据，证明刷状边缘微绒毛在近端小管中起着流量传感器的作用。然而，初级纤毛是否也起到流量传感器的作用，以及管周离子转运体是否也可以受到轴向血流的调节，还没有得到研究。在拟议的工作中，体外微灌流实验、免疫荧光和Western blotting将出于以下三个目的：1)通过比较模型预测和流动诱导的变化，研究中心纤毛或微绒毛是否是近端小管的流量传感器；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体转运和功能中的作用；3)研究第二信使、钙信号、cAMP和PKA调节机制的作用以及多巴胺在流量依赖的近端小管运输中的作用。我们提出的合作的独特特征是：1)比较小鼠和敲除动物的完整小管中依赖流动的近端小管运输；2)表示作为流体动力和扭矩对微绒毛和纤毛的函数的重吸收通量；以及3)在近端小管运输的数学模型中评估流动诱导的肌动蛋白细胞骨架重组、离子转运体定位和功能变化。这些研究将提供有关肾小管平衡(GTB)机制的新信息，以及生理和病理生理条件下肾液和HCO3-转运方面的信息。公共卫生相关性：全身血压的维持取决于沿整个肾单位的肾脏内钠重吸收的速率，其中约2/3发生在近端小管。拟议的研究将提供有关近端小管钠转运调节的直接信息。可能的好处包括识别目标分子，这些分子可能被阻断或修饰，以调节肾脏对钠的重吸收。

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.