Ischemic Actin-Cytoskeleton Alterations in Renal Cells

Chen, Jing

Abstract

The applicant proposes to test the hypothesis actin is sequestered by specific, as yet unknown proteins during hypoxia. The applicant proposes that the actin may be sequestered by short F-actin polymers and/or by G-actin monomers. The objective of this grant proposal is to identify and to ultimately sequence the protein(s) that supposedly sequester actin during hypoxic injury to renal tubular epithelial cells. The applicant also plans to determine the subcellular localization of these actin sequestering proteins using immunofluorescence. Also the ATP threshold for sequestration and release of actin following hypoxia-reoxygenation will be examined.
The Specific Aims of the proposal are:
Specific Aim 1. To demonstrate that actin is sequestered with the cell cytoskeleton in renal proximal tubular cells during ATP depletion.
Specific Aim 2. To identify the protein or proteins that sequester and release actin during ATP depletion.
Specific Aim 3. To demonstrate that ATP depletion induces colocalization of actin and the identified sequestering proteins within proximal tubular cells. The applicant's hypothesis is based on an interesting and potentially important finding presented as preliminary data by the applicant. The applicant has demonstrated that when normal renal tubular tubules are extracted sequentially by digitonin, calcium-Triton-X and then by a calcium free extraction buffer, the fraction remaining contains no detectable actin. In contrast, tubules subjected to hypoxia have a substantial amount of actin in the insoluble fraction following this extraction procedure. The applicant has also demonstrated that the insoluble pellet from hypoxic cells that contains actin, also contains a number of other proteins. The applicant has demonstrated that when the insoluble pellet remaining after extraction of hypoxic sample were extracted again in ATP-containing buffer and resolved on an SDS-PAGE gel a number of proteins other than actin were observed on silver stained gels. In addition to actin (which represents the major band) there are at least 4 or 5 other protein bands, two most abundant in the ~100kDa and 200kDa size. The applicant hypothesizes that amongst these proteins are one or more proteins responsible for sequestering actin after hypoxic injury. The major goal of this application is to identify and characterize these putative proteins. Finally the applicant will generate antibodies against the actin sequestering proteins using the purified proteins or a minor sequencing of the proteins using antibody they would demonstrate ATP depletion.
The Specific Aims are: 1) To demonstrate sequestration of actin during ATP-depletion in PT; 2) To identify the cytoskeletal protein(s) that sequester and release actin during hypoxia/reoxyg; 3) To demonstrate ATP-depletion induced co-localization of actin and actin sequestering proteins.