Ischemia-induced activation of MT1-MMP leads to cleavage of cadherins in normal rat kidney cells.

摘要

Ischemia is the leading cause of acute renal failure (ARF), a disease associated with high morbidity and mortality. Disruption of intercellular adhesion in the proximal tubules is linked to ARF, although the molecular mechanism(s) remain unclear. In these studies, we adapted a model of in vitro ischemia to normal rat kidney (NRK) cells and examined the cadherin/catenin complex, the predominant regulator of cell-cell adhesion. Ischemia was induced by applying a thin layer of phosphate buffered saline (PBS) solution supplemented with calcium and magnesium and a layer of mineral oil, restricting exposure to oxygen. Hypoxia was detected by an increase in pimonidazole adducts, as well as an increase in GLUT-1 protein levels. Ischemia did not decrease cell number, but there was a decrease in ATP levels. In addition, there was no evidence of cleaved caspase 3 or 9 during 6 hr of ischemia. Ischemic cells exhibited an extracellular 80 kDa and intracellular 40 kDa E-cadherin fragment after 4--6 hr of ischemia. While no fragments of N-cadherin were observed at any time point, the detectable level of this protein decreased during ischemia. E-cadherin cleavage and loss of N-cadherin was associated with decreased cadherin function as assessed by cell aggregation. The effects of ischemia on E- and N-cadherin were independent of de novo mRNA and protein synthesis. The MMP inhibitors, GM6001 and TAPI-O, inhibited cleavage and/or loss of E- and N-cadherin protein expression. TIMP-3 and to a lesser extent TIMP-2, but not TIMP-1, inhibits ischemic cleavage and/or loss of E- and N-cadherin, suggesting the involvement of a membrane-bound MMP. Ischemic cells expressed increased levels of active MT1-MMP, and blocking MT1-MMP activity with a blocking antibody, antisense, and shRNA constructs fully protects full-length E- and N-cadherin. Using shRNA constructs to suppress MT1-MMP expression, ischemia-induced disruption of cadherin function is ablated, and cell-cell contacts are preserved. These results demonstrate that ischemia induces activation of latent MT1-MMP and subsequent disruption of cadherin/catenin complexes, implying that MT1-MMP plays a role in ischemia-induced ARF.