Solutes and water move across epithelial layers either through the epithelial cells (transcellular) or between cells through the tight junction (paracellular). Maintenance of the paracellular permeability barrier is essential for normal function of renal epithelia. During renal ischemia/reperfusion (I/R) injury, the paracellular permeability barrier is disrupted and contributes to loss of renal functional capacity. Src Family Kinase (SFK) activity has been implicated in mediating I/R-induced loss of barrier function but this is controversial and the roles of individual SFKs are unknown. Based on previous studies and our preliminary results, we hypothesize that renal epithelial cell paracellular permeability barrier is maintained through interaction of one SFK, c-yes, with the tight junction protein occludin. Renal I/R injury, mediated in part by localized increase in H2O2, causes dissociation of c-yes from occludin and association of c-src, another SFK, with occludin. This leads to a decreased occludin residency time in the tight junction which increases paracellular permeability. The first series of experiments will test the hypothesis that c-yes activity decrease and c-src activity increases renal epithelial cell paracellular permeability. Experiments will employ genetic techniques to manipulate the activities of these two SFKs and will monitor two components of paracellular permeability, the pore pathway (high capacity, small size - monitored by measuring TransEpithelial Resistance (TER)) and the leak pathway (low capacity, large size - monitored by measuring the flux of calcein and fluorescein-dextran 4000). The second series of experiments will define the effect of H2O2 on the interaction of occludin with c-src and c-yes. Experiments will utilize genetic techniques to manipulate SFK activities and occludin content. Occludin interaction with c-src and c-yes as well as localization to the tight junction structure will be monitored by confocal microscopy. The third series of experiments will test directly the hypothesis that H2O2 treatment produces a decrease in the residency time of occludin at the tight junction structure and that this decrease is mediated by c-src activity. Thes experiments will employ confocal microscopic imaging of live cells to monitor occludin protein dynamic behavior. The fourth series of experiments will determine if a similar mechanism is operative in a rat renal I/R injury model. Individual SFK contents will be manipulated using a unique lentivirus delivery system to target constructs to the proximal tubule segment. I/R injury severity will be monitored by measuring parameters reflecting renal functional capacity, glomerular filtration rate (serum/urinary creatinine ratio), and proximal tubule cell structural integrity, Na+-K+-ATPase polarity. These studies will define the mechanism underlying renal I/R-induced loss of renal epithelial cell paracellular permeability barrier and will provide the bais for development of therapeutic interventions to minimize renal I/R injury.
Movement of compounds across epithelial cell layers through the paracellular pathway (between the cells) is important for control of body solute and water balance. The experiments described in this proposal will define the molecular mechanism by which this pathway is disrupted during a pathogenic condition, renal ischemia/reperfusion injury, that contributes substantially to sickness and death in renal transplant patients and others.