The proximal tubule contains the highest concentration of cP450 within the mammalian kidney, and does not express cyclooxygenase or lipogenase activity. Our hypothesis is that hormonally mediated release of arachidonic acid in the proximal tubule leads to cP450 mediated generation of epoxyeicosatrienoic acid and w/w-1 HETES, and these metabolites serve as second messengers in regulation of proximal tubule function and growth. There is suggestive evidence that these compounds are involved in regulation of proximal tubule transport function, including regulation of sodium influx, Na/K ATPase activity and phosphate transport. These metabolites have also been implicated as second messengers mediating alterations in transport by angiotensin II, PTH and dopamine, and regulation of growth by EGF. Since the proximal tubule is a major site of volume and solute reabsorption, we hypothesize that cP450 arachidonate metabolites are important mediators of the actions of systematically and locally acting hormones in the proximal tubule and participate in integrated glomerulo-tubular responses that serve to maintain the constancy of glomerular filtration rate and plasma volume in the face of acute and chronic changes in intravascular pressure and volume. In addition, the proximal tubule is the nephron segment undergoing the most damage in acute ischemic and toxic nephropathies and a major target for injury in chronic inflammatory and non-inflammatory tubulointerstitial diseases. Abnormal growth and dedifferentiation of the proximal tubules has been implicated in the maintenance and progression of chronic progressive renal injury, as well as in renal adenocarcinoma.
Specific aim #1 will characterize the effects of addition of exogenous stable analogs of EETs and HETEs to isolated perfused proximal tubules and to cultured renal epithelial cells. These studies will examine both transport and cell growth and cytoprotection.
Specific Aim #2 will characterize in greater detail signaling pathways of selected EETs and/or HETEs and will investigate whether specific receptors exist in renal epithelial cells.
Specific aim #3 will examine the role of these compounds as second messengers of hormones and growth factors in cultured cells and isolated perfused tubules will utilize selective epoxygenase and w hydroxylase inhibitors, genetically engineered """"""""knockout"""""""" animals and expression of cP450 mutated to express stereoselective EETs or HETEs. We will also examine the effect of inhibition of arachidonic acid release upon hormonal action in the proximal tubule. These integrated perfused tubule and cell culture studies should definitively delineate the role of these compounds as intracellular second messengers and define their signaling pathways, information that will allow further exploration into the role of cP450 AA metabolites in renal pathophysiology.
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