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.
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.
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