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This study examines the role of the actin cytoskeleton and integrin expression in the recovery of cell adhesion in the proximal tubule cell line JTC-12 after peroxide injury. The cells were exposed to 10, 20, or 50 mM hydrogen peroxide for 10 min and then allowed to recover. Viability measurements by trypan blue exclusion confirmed that the injury was largely nonlethal with 85% viability at 1 h even at 50 mM peroxide. ATP levels fell immediately after the peroxide incubation in all groups to approximately 10% of normal, but already showed some recovery by 1 h and full recovery in the 10 and 20 mM groups by 24 h. Cell adhesion to extracellular matrix immediately after injury was depressed at 20 and 50 mM peroxide, but by 12 h was abnormal only at 50 mM peroxide and at 24 h was essentially normal at all peroxide concentrations. Immediately after exposure to 10 mM peroxide, there were subtle abnormalities in the actin cytoskeleton (thickening of fibrils) as assessed by phalloidin staining, with more pronounced effects at 20 and 50 mM. At 1 h, many cells showed collapse of the actin cytoskeleton to the periphery. There was some recovery at 4 h; by 12 h, the actin cytoskeleton showed further recovery, although was still abnormal (coarsened microfilaments), especially at 20 and 50 mM peroxide. By 24 h, the actin cytoskeleton showed only subtle coarsening. Integrin surface expression was assessed by flow cytometry. The alpha6 subunit on cells exposed to 20 mM peroxide was unchanged at 1 h and 4 h, but by 12 h had increased to 118.5+/-4.5% and by 24 h to 146+/-13.4% of control levels. The expression of the beta1 and alphaVbeta3 integrins remained unchanged. Thus, despite coarsening of the actin cytoskeleton and depressed ATP levels, cell adhesion recovered from oxidant stress. Abnormal cell adhesion after injury was not a consequence of a decrease in integrin expression, and recovery of cell adhesion was not a consequence of the modest and selective increase in integrin expression.

We have characterized the integrins present on cultured tubule epithelial cells from human renal cortexes, enriched for proximal cells, using fluorescence microscopy, immunoprecipitation, and cell adhesion assays. By immunofluorescence, the alpha 3-integrin subunit stained most intensely and was present on all cells predominantly at cell-cell contacts. The alpha 6-subunit was present on all cells in a pattern consistent with extracellular matrix contacts. The alpha 5-subunit was present on most cells in a cell-matrix contact pattern; alpha V-subunit was weakly positive and occasionally seen in cell-matrix contacts. The alpha 2-subunit was present on clusters of distal tubule cells, predominantly at cell-cell contacts. Immunoprecipitation revealed the predominant integrin to be alpha 3 beta 1 with some alpha 2 beta 1, presumably contributed by distal cells. The alpha 5 beta 1-, alpha 6 beta 1-, alpha 6 beta 4-, and alpha V beta 3-integrins, as well as trace amounts of alpha 1 beta 1-integrins, were also present. The alpha 4 beta 1-integrin was not detected. Initial attachment to fibronectin was mediated by alpha V beta 3- and alpha 5 beta 1-integrins; initial attachment to laminin was mediated by the alpha 6 beta 1- and alpha 3 beta 1- integrins and, in some preparations, by an unidentified integrin; and initial attachment to collagen type IV was mediated by alpha V beta 3-integrin and an unidentified beta 1-integrin. After extensively immunodepleting membrane extracts with anti-alpha 1, -alpha 2, -alpha 3, -alpha 4, -alpha 5, -alpha 6, and -alpha V antibodies, an anti-beta 1 antibody still precipitated an integrin. Its electrophoretic mobility differs from the laminin-binding alpha 7 beta 1-integrin. Thus we have identified many of the integrins on cortical tubule cells and their role in mediating initial attachment to extracellular matrix. However, the cell adhesion assays and immunoprecipitations suggest the presence of an unidentified beta 1-integrin that may mediate renal tubule cell attachment to laminin and collagen.

Many cell adhesion molecules probably contribute to the pathogenesis of acute renal failure. This review focuses on the potential importance of integrins. Integrins are a family of molecules that mediate cell-matrix and cell-cell adhesion, and are found on almost all cells. After acute renal injury, tubule epithelial cell-cell contacts (mediated by many molecules including uvomorulin and possibly integrins) are disrupted, as is the cytoskeleton. Detachment of viable tubule epithelial cells from the basement membrane has also been documented following acute renal injury. Such detachment would lead to back-leak of glomerular filtrate and could also be important in the production of cellular casts. As the attachment of the cytoskeleton to integrin is critical to cell-matrix adhesion, it is likely that cytoskeletal disruption is one of the mechanisms contributing to cell detachment. Integrins are also likely to be important in repair, as integrins will probably participate in migration along the basement membrane and may also influence cell function during the redifferentiation process. Finally, leukocyte integrins will probably play a role in the migration of leukocytes into areas of injury and also be critically important to their function.