Ischemia is a major cause of acute renal failure (ARF), and also an important cause of dysfunction in other epithelial organs such as liver and intestine, especially after transplantation. The ischemic injury in epithelial cells has complex multiple effects. However, there is a consensus in that loss of plasma membrane polarity resulting from the disorganization of the cytoskeleton is one of the major pathophysiological mechanisms for epithelial function failure. Most recent research in this area has focused in the effects on the actin cytoskeleton, although it has been reported that microtubules are also disorganized during reperfusion. The applicant's lab has recently shown that the apical network of intermediate filaments plays a substantial role in the organization of the apical pole, by maintaining the stability of F-actin microvillus cores and the organization of microtubules. In this regard, he has just reported that the apical IF network maintains the position of Microtubule Organizing Centers (MTOC) that bind it. The preliminary data indicate that the IF network is stable during ATP depletion and that MTOC detach from it during ATP depletion. Furthermore, using a transgenic knock-out of the CK8 gene, he have demonstrated that the terminal web is indeed required for apical polarity. These data provide the foundation for a hypothesis where the apical IF network is responsible for the re-localization of MTOC after ATP depletion through the function of IF-associated proteins, or, alternatively, directly through binding to the cytokeratins. To test this hypothesis, the specific aims are: 1) Study the specificity of IF proteins to re-polarize the cytoskeleton after ATP depletion. 2) Characterize the proteins involved in regaining microtubule polarity after an ischemic injury in epithelia cells and their molecular changes during ATP depletion. The long term goals of this project are to identify the molecular mechanisms mediating the detachment of MTOC from intermediate filaments to define possible therapeutic alternatives to block them and, therefore, protect epithelial cells from ischemic injury or promote their recovery.
