Relationship Between Adhesive Properties And Expression Of Adhesion Molecules On TECs And GECs Following Ischemia/reperfusion Injury

The mechanism of ischemic acute renal failure (IARF) is complicated and incompletely understood. Many studies postulated that changes of distribution of integrins on tubular epithelial cells(TEC) and expression of adhesion molecules on Glomerular endothelial cells(GEC) after ischemia/reperfusion injury are involved in the pathophysiology of IARF. Altered polar distribution of integrins is related to TEC detachment and tubular obstruction. Increased expression of adhesion molecules on microvascular endothelium in kidney were related to infiltration of polymorphonuclear leukocytes(PMN). But all these conclusions are based on the results of immunohistochemistry, fluorescence-activated cell sorting, gene knockout, antagonists of adhesion molecules and so on, but lack direct measurement of adhesive forces of cell-matrix and cell-cell to elucidate the relationship between cell attachment and expression of adhesion molecules on cells. In this study we perform the following researches.Cultured human tubular epithelial cells (TECs) and primary cultured glomerular endothelial cells (GEC) were exposed to 0.1 u mol/L antimycin A and 0.1mmol/L H2O2 respectively to induce chemical anoxia and oxidative stress. Micropipette aspiration technique was used to measure the force required to separate TEC or PMN from basal surface composed of different substrate or adherent cells directly; the expression and distribution of P-selectin, E-selectin, ICAM-1, a 3 integrins on GECs and TECs were studied with immunofluorescence, fluorescence activated cell sorting, in situhybridization; F-actin were revealed with phalloidin. In vivo expression of adhesion molecules in kidney were revealed with immunohistochemistry, immunogold- silver staining, in situ hybridization after rats renal ischemia/ reperfusion injury.The main results and conclusions were as follows:1. We use micropipette aspiration technique to direct measure the adhesive forces of TECs and GECs after anoxia and oxidative stress for the first time in the world, and testified the speculation of TEC and GEC adhesion changes after anoxia and oxidative stress by other traditional methods. The results evidence experimental proof which shows that the changes of adhesive properties of GECs and TECs were involved in the pathophysiology of IARF.2.The adhesive forces between TEC-matrix decreased markedly but adhesive forces between TEC-TEC increased after chemical anoxia/ oxidative stress. Simultaneously the polar distribution of a 3-integrins on TECs were impaired and a 3-integrins tend to diffuse to lateral and apical membrane. Antibody to a 3-integrin decreased the TEC-TEC adhesive forces, the result showed that altered distribution of integrins on TECs lead to changed adhesive forces of TEC-matrix and TEC-TEC.3. The adhesive forces between TEC and matrix were greater than that between TEC and glass, and different matrix lead to different adhesive forces, which showed that various matrix have corresponding affinity with integrins on TEC.4. For the first time, we discoverd immunofluorescence of a 3 integrins at the lateral surface of TEC after anoxia damage. It was postulated that for the low membrane permeability of living cells to integrin-antibody, so only the cellular membrane exposed to culture media could react with antibody of a 3 integrins. This result further proved the lateral diffusion of a 3-integrins on TECs after anoxia injury.5. Anoxia and oxidative stress have different effects on the distribution of F-actin cytoskeleton of TECs. because the polar distribution of integrins on TECs was depend on normal cytoskeletion function, so different mechanism may be involved to mediate the expression of adhesion molecules after anoxia and oxidative stress.6.The adhesive forces of GEC-PMN increased after anoxia/oxidative stress, with increasing expression of P-selectin, E-selectin, ICAM-1. The increased adhesive forces were inhibited by the use of ICAM-1 antibody. Our results showed that many kind of adhesion molecules were involved in the altered adhesive p...