Central to the vectorial transport function of renal epithelium is the polarized distribution of surface membrane proteins in tubule cells. Recent evidence indicates that the spectrin based cortical cytoskeleton, which is often associated with certain integral membrane proteins, is also highly polarized, may play a fundamental role in maintaining and guiding topographic membrane assembly, and is disrupted following even sublethal renal tubule ischemia. The overall goal of the proposed studies will be to understand how the cortical cytoskeleton achieves its polarized distribution, the relationship of this process to the sorting of basolaterally restricted integral membrane proteins, and the aberrations in this process that typify various pathologic states. Specifically, research will focus on the factors that target the assembly of the spectrin skeleton to the lateral margins of kidney epithelial cells, regulate its interactions with the integral membrane proteins such as E- cadherin and Na+,K+-ATPase, and mediate the changes in the spectrin skeleton that accompany ischemia. The cytoskeletal proteins to be examined include the erythroid like and non-erythroid isoforms of spectrin , ankyrin, adducin, and stomatin. The interaction between these proteins will be measured by in vitro assays; their distribution and rates of assembly into the cortical cytoskeleton will be measured in cultured renal epithelial (MDCK) cells as a function of tight junction formation, epithelial monolayer formation, and in vitro ischemic injury. Recombinate DNA techniques will be used to characterize unique isoforms of spectrin and adducin in renal cells. Functional sites in these proteins will be identified by deletional analysis in vitro and by the effects of expressed recombinant proteins in cultured epithelial cells. Sequence specific antibodies, Northern blot analysis, and in situ hybridization will be used to monitor changes in the distribution, composition, processing, and synthesis of these components after in vivo and in vitro ischemia.
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