Beta1 integrin-mediated adhesion between renal tubular cells after anoxic injury.

beta 1 integrin-mediated adhesion between renal tubular cells after anoxic injury. This study examined the effect of sublethal injury, induced by ATP depletion (5 mM cyanide in the absence of dextrose), on the distribution and function of beta 1 integrins in primary cultures of mouse proximal tubular (MPT) cells. It was shown in this study that sublethal injury results in loss of focal contacts present in uninjured MPT cells, and that the beta 1 integrin molecule becomes redistributed to the apical membrane domain of sublethally injured cells. Polystyrene beads coated with Arg-Gly-Asp (RGD)-containing peptide adhere to the surface of sublethally injured MPT cells but not to control, dextrose-treated cells, indicating that the beta 1 integrins present on the apical surface of the cell remain functional. The presence of an excess of free RGD-containing peptide reduces binding of RGD-coated beads to sublethally injured MPT cells by approximately 50%. It was also demonstrated that adherence of MPT cells in suspension to cyanide-treated monolayers is increased more than 300% above adhesion to control, uninjured monolayers. This abnormal cell-cell adhesion is ameliorated by the presence of an excess of RGD-containing peptide and is reversed if cyanide-treated cells are allowed to recover for 1 h. It was concluded that the beta 1 integrin becomes expressed on the apical surface of MPT cells after sublethal injury. These apically expressed integrins remain functional and mediate aberrant adhesion between MPT cells.

Functional and cytoskeletal changes induced by sublethal injury in proximal tubular epithelial cells.

Mouse proximal tubular (MPT) cells in culture were subjected to ATP depletion by incubating them with cyanide in the absence of dextrose for 1 h. This insult resulted in marked alterations in the actin cytoskeleton. These changes were not associated with a decrease in cell viability and thus reflected sublethal injury. The effect of sublethal injury on the functional integrity of the intercellular tight junction (TJ) was then examined in MPT cell monolayers grown on permeable supports. During chemical anoxia, monolayer permeability to the paracellular marker mannitol progressively increased to 297 +/- 62% of baseline after 1 h. Chemical anoxia also caused a reversible loss in cell-substrate adhesion when MPT cells were studied as confluent monolayers or as single cells. Thus disruption of the actin cytoskeleton in nonlethally injured cells results in important reversible alterations in renal epithelial function characterized by impairment of the "gate" function of the TJ as well as impaired cell-substrate adhesion. We hypothesize that sublethal epithelial cell injury without accompanying necrosis may contribute to the decrement in renal function characteristic of ischemic renal injury.

Renal mouse proximal tubular cells are more susceptible than MDCK cells to chemical anoxia.

To elucidate the mechanisms responsible for the resistance of continuous cell lines to anoxic injury, we have compared the effects of ATP depletion induced by chemical anoxia on primary cultures of mouse proximal tubular (MPT) cells and on Madin-Darby canine kidney (MDCK) cells. Inhibition of ATP production by cyanide and 2-deoxyglucose (CN+DOG) in the absence of dextrose reduced cell ATP content to < 5% of control values in MPT cells and caused progressive deterioration in mitochondrial function as well as loss of cell viability in these cells. Cell free fatty acid (FFA) content rose from 4.3 +/- 0.9 to 23.7 +/- 2.0 micrograms/mg of total lipid weight after 4 h of CN + DOG (P < 0.05). The mitochondrial injury and cell death induced by CN + DOG in MPT cells was ameliorated by the addition of fatty acid-free bovine albumin to the cell medium, which reduced cell FFA content during chemical anoxia from 25.0 +/- 3.0 to 10.4 +/- 2.0 micrograms/mg (P < 0.05). The phospholipase A2 (PLA2) inhibitor, mepacrine, also resulted in functional protection and reduction of cell FFA content from 20.2 +/- 2.3 to 15.9 +/- 1.7 micrograms/mg (P < 0.05). These data suggest a role for phospholipase activation and accumulation of toxic lipid metabolites in the pathophysiology of MPT cell injury. We then compared cell injury induced by CN + DOG in MPT and MDCK cells. Despite comparable reduction in cell ATP content in the two cell types, injury was far more severe in MPT than MDCK cells.(ABSTRACT TRUNCATED AT 250 WORDS)