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运输的生理重要性已经被认识了四十多年，但是这种调节的机制仍然没有很好地定义。最近我们已经证明a)小鼠离体灌注近端小管存在灌注-吸收平衡。b)腔膜NHE3和h - atp酶均受血流调节；c)紧结渗透率的变化在流量调制输运中不起作用。我们已经开发了一种计算微绒毛上的力和扭矩的理论，并证明了流体诱导的近端小管吸收变化是扭矩依赖的，并且需要一个完整的肌动蛋白细胞骨架将信号传递给细胞。实验数据和模型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具有流量传感器的功能。然而，初级纤毛是否也具有流量传感器的功能，以及小管周围离子转运体是否也能受到轴向流的调节，尚未得到研究。本研究拟开展体外微灌注实验、免疫荧光和Western blotting，目的有三个：1)通过比较模型预测结果与野生型、绒毛蛋白、纤蛋白、肌球蛋白和纤毛缺陷小鼠的Na+和HCO3-吸收变化，研究近端小管中究竟是中央纤毛还是微绒毛作为流量传感器的争议；2)研究血流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转运及其功能中的作用；3)研究第二信使、钙信号、cAMP-和PKA-调节机制以及多巴胺在血流依赖性近端小管运输中的作用。我们提出的合作的独特之处在于：1)在小鼠和敲除动物的完整小管中比较流量依赖的近端小管运输；2)重吸收通量作为微绒毛和纤毛上流体动力力和扭矩的函数的表示；3)在近端小管运输的数学模型中评估血流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。这些研究将为生理和病理生理条件下肾小球-小管平衡（GTB）的机制以及肾液和HCO3-转运方面提供新的信息。公共卫生相关性：全身血压的维持取决于钠在肾脏内沿整个肾元的再吸收率，其中约2/3发生在近端肾小管。拟议的研究将提供关于近端小管钠运输调节的直接信息。可能的好处包括识别目标分子，这些目标分子可能被阻断或修饰，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.