The long term objective of this laboratory is to obtain a detailed understanding of the cellular and molecular events underlying the cellular dysfunctions induced by renal ischemia. Past and continually expanding experience regarding the relationship between tubular metabolism and function as well as novel techniques will be used toward this goal. Two major specific aims are proposed far the next 5 years: In terms of the first aim, much experimental evidence suggests that a breakdown in the interactions between the plasma membrane and the scaffolding-like cytoskeletal structure may precede cell injury. The effects of ischemia on these interactions will be studied in various renal preparations, each one selected to address a different set of molecular mechanisms. The intact rat kidney will be used to study alterations in brush border structure caused by ischemia/reoxygenation, particularly investigating changes in actin, villin, and the 105kDa protein believed to link the actin filamentous bundle to the plasma membrane. At the same time, changes in the proximal tubule basolateral membrane structure will be studied, especially the role of ankyrin in this process. To this end, other experiments will seek to isolate the cDNAs encoding the isoform expressed in proximal renal tubules of the cytoskeletal linkage protein ankyrin, with the intention of cloning it and raising antibodies against this protein. Once these antibodies are available, they will be used to study the fate of this protein during ischemia and the role it plays in the structural changes that occur with oxygen deprivation. Freshly isolated tubular fragments will be used to focus on the plasma membrane- cytoskeletal breakdown that leads to membrane bleb formation during energy depletion. These studies will use a combination of video-enhanced differential interference contrast microscopy and fluorescence. Cultured renal cell lines will be used as models for specific aspects of ischemic injury, emphasizing the role of cytoskeletal and plasma membrane proteins and possible reversibility of function.
The second aim deals with the adaptation of renal energy metabolism and transport to the culture environment. The search will continue for culture conditions that optimize oxidative metabolism and Na,K-ATPase activity, especially examining the expression of this enzyme.
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