Crystal-cell interaction and apoptosis in oxalate-associated injury of renal epithelial cells

Abstract

Two renal epithelial cell lines, LLC-PK1 and Madin-Darby canine kidney (MDCK), were grown in monolayers and exposed to oxalate (Ox) and/or calcium oxalate (CaOx) crystals to investigate cellular responses to these challenges. In addition, LLC-PK1 cells were exposed to high concentrations of Ox for various time periods to investigate the role of apoptosis in Ox-associated cell injury. Both cell types showed signs of damage when exposed to Ox. However, LLC-PK1 cells appeared more sensitive than MDCK cells. There was a significant increase in release of lactate dehydrogenase into the medium and decrease in trypan blue exclusion by cells in the monolayer. Most noticeable was the detachment of cells from the substrate. Exposure of cells to CaOx crystals resulted in their attachment to cell surfaces followed by internalization. Using flow cytometry for quantification of apoptotic cells, transmission electron microscopy for morphology, and electrophoresis for DNA laddering detection, we observed significant apoptotic changes including condensation and margination of nuclear chromatin, DNA fragmentation, and migration of phosphatidylserine of the plasma membrane from inside to the cell surface. However, these cells also showed some necrotic changes such as loss of plasma membrane integrity and release of lactate dehydrogenase, indicating that the apoptotic process was interrupted.