Altered Proteins in MDCK Renal Tubular Cells in Response to Calcium Oxalate Dihydrate Crystal Adhesion: A Proteomics Approach

Abstract

The interaction between crystals and renal tubular cells has been proposed to be a crucial event that elicits subsequent cellular responses, leading to kidney stone formation. Nevertheless, the molecular mechanisms of these cellular responses remain poorly understood. We performed a gel-based differential proteomics study to examine cellular responses (as determined by altered protein expression) in Madin-Darby canine kidney (MDCK) cells, which were derived from dog kidney and exhibited distal renal tubule phenotype, during calcium oxalate dihydrate (COD) crystal adhesion. MDCK cells were grown in a medium without or with COD crystals (100 microg/ml) for 48 h. Crystal adhesion was illustrated by phase-contrast and scanning electron microscopy. Flow cytometry using annexin V/propidium iodide double staining showed that the percentage of cell death did not significantly differ between cells with and without COD crystal adhesion. Cellular proteins were then extracted, resolved with two-dimensional gel electrophoresis (2-DE), and visualized by SYPRO Ruby staining ( n = 5 gels per group). Quantitative intensity analysis revealed 11 significantly altered proteins, 10 of which were successfully identified by quadrupole time-of-flight peptide mass fingerprinting (MS) and/or tandem MS (MS/MS), including metabolic enzymes, cellular structural protein, calcium-binding protein, adhesion molecule, protein involved in RNA metabolism, and chaperone. An increase in annexin II was confirmed by 2-D Western blot analysis. These data may lead to better understanding of the cellular responses in distal renal tubular cells during COD crystal adhesion.