Project: Research project


This proposal studies cellular injury and repair in renal epithelial
cells using a model called chemical anoxia to deplete the intracellular
ATP stores. We have developed a reversible model of ATP depletion using
chemical inhibitors of oxidative phosphorylation and glycolysis.
Rotenone, an inhibitor of oxidative phosphorylation, and 2-deoxyglucose,
ad competitive inhibitor of glycolysis, are added to renal epithelial
cells grown on membrane filer supports. Following the application of the
inhibitors, there is a rapid, reproducible fall in intracellular ATP.
When a perfusion" media is added to the cells the intracellular ATP
levels rapidly rise. At 2.5 hours following the addition or the
perfusion media, the intracellular ATP levels are equivalent to controls.
This new, reversible, chemical anoxia model will be used to study the
nature of cellular injury and repair in two renal epithelial cell lines,
MDCK (Madin-Darby canine kidney) and JTC (monkey proximal tubule cell
line) in three ways. 1. The MDCK and JTC cell lines will be subjected to ATP depletion
followed by restoration of their intracellular ATP reserves. This will
allow us to test the hypothesis that renal cell recapitulate their normal
developmental steps following the recovery from cellular injury (Bacallao
and Fine, 1991). In these studies, the subcellular organization of the
actin cytoskeleton, focal adherens, tight junction and adherens junctions
will be examined by confocal laser scanning fluorescence microscopy. In
addition we will study the recovery of the tight junction barrier
function (Mandel, Bacallao and Zampighi, Nature, 1993) by measuring the
transepithelial resistance across the epithelial monolayer at various
times after recovery from chemical anoxia. 2. We will also test the hypothesis that cellular injury caused by
chemical anoxia disrupts the accuracy of the protein sorting machinery
of renal epithelial cells. This leads to loss in functional polarity.
We suggest that the altered polarity of the injured cells will not be
corrected until the protein sorting machinery is repaired. This
hypothesis will be tested by quantifying the kinetics and stoichiometry
of membrane assembly for a variety of endogenous membrane proteins.
These studies may uncover significant different in the way proteins are
sorted, especially during the recovery from the injury. For example the
protein targeting mechanism of cytoskeletal linked proteins may be very
sensitive to the effects of chemical anoxia, while other plasma membrane
proteins may exhibit no change in their polarized delivery. These
studies could potentially delineate different pathways of protein
sorting. 3. Additionally, we will examine the effects of chemical anoxia on the
protein salvage pathway. This pathway identifies mis-sorted proteins,
removes them from the plasma membrane and delivers the proteins to the
correct plasma membrane domain. This pathway will be examined by low pH
fusion of vesicular stomatitis virus G protein in the apical membrane of
the cells and determining the kinetics of delivery to the basolateral
Effective start/end date5/1/946/30/04


  • National Institutes of Health: $65,089.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $208,452.00
  • National Institutes of Health: $69,054.00
  • National Institutes of Health: $103,280.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $67,043.00


Wounds and Injuries
Adenosine Triphosphate
Protein Transport
Epithelial Cells
Cell Membrane
Membrane Proteins
Chemical Models
Tight Junctions
Oxidative Phosphorylation
Adherens Junctions
Cell Line
Actin Cytoskeleton
Blood Proteins


  • Medicine(all)