Leukotriene B4 (LTB4) is a potent inflammatory mediator, derived from arachidonic acid, which plays a prominent role in ischemia-reperfusion injury and inflammation of the kidney. The enzyme CYP4F3 has specialized properties which enable it to inactivate LTB4: it has a uniquely low Km for LTB4 as a substrate, and it has a restricted localization to leukocytes which mediate LTB4-dependent responses. The hypothesis of this proposal is that CYP4F3 negatively regulates inflammation by attenuating the activities of LTB4. To test this hypothesis, three specific aims are proposed: (1) to investigate the regulation of LTB4-dependent responses by CYP4F3 in leukocytes in vitro;(2) to investigate the mechanism by which CYP4F3 regulates inflammation;and (3) to investigate the role of the mouse homologue of CYP4F3 (CYP4F18) in renal disease in vivo.
In Specific Aim 1, CYP4F3 enzyme activity in leukocytes will be inhibited using a specific chemical inhibitor, a lentiviral- delivered siRNA approach, and a mouse genetic knockout, and the ability of LTB4 to stimulate cell chemoattraction and secretion will be measured using in vitro assays. The uniqueness of CYP4F3 function will be assessed by comparing other enzymes which metabolize LTB4 with higher Km values.
In Specific Aim 2, receptor studies will be performed to determine if the product of LTB4 inactivation (20-OH LTB4) promotes LTB4 receptor (BLT1) down-regulation, thus providing a doubly efficient attenuation mechanism. The integration of CYP4F3 and BLT1 activity will be further investigated by determining the effects of CYP4F3:BLT1 expression ratio on LTB4 responsiveness.
In Specific Aim 3, a mouse model of renal ischemia-reperfusion injury will be used to determine if targeted deletion of the CYP4F18 gene leads to increased inflammation and cell injury in vivo. These studies are relevant to public health because ischemic injury and inflammation are primary pathophysiological mechanisms leading to disorders of the kidney, such as renal failure and transplant rejection, which are associated with a high mortality rate. The significance of the research is that CYP4F3 provides a natural (non-toxic) anti-inflammatory target that might be selectively modulated in specific tissues such as the kidney. The long-term objectives are to correlate changes in CYP4F3 activity with inflammatory disease states in humans, and to identify and manipulate the regulatory pathways that control CYP4F3 gene expression and activity.
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