Changes in Mitochondrial Proteome of Renal Tubular Cells Induced by Calcium Oxalate Monohydrate Crystal Adhesion and Internalization Are Related to Mitochondrial Dysfunction
journal contributionposted on 20.02.2016, 20:41 authored by Sakdithep Chaiyarit, Visith Thongboonkerd
Calcium oxalate monohydrate (COM) crystals, the major crystalline compound in kidney stones, have been suggested to induce oxidative stress by overproduction of reactive oxygen species (ROS) and renal tubular cell injury. Our present study aimed to examine changes in mitochondrial proteome in distal renal tubular cells induced by COM crystals (100 μg of crystals/mL of culture medium). Adhesion and internalization of COM crystals by MDCK cells were examined by fluorescent and laser-scanning confocal microscopy. Moreover, the internalized COM crystals were quantified by flow cytometry. Thereafter, mitochondria were isolated from controlled and COM-treated cells, and mitochondrial proteins were subjected to 2-DE-based comparative proteomic analysis, which revealed 15 differentially expressed proteins. These significantly altered proteins were identified by Q-TOF MS and MS/MS analyses, including those involved in several biological processes, e.g., cellular structure, carbohydrate metabolism, and energy metabolism. 2-D Western blot analysis confirmed the increase of ezrin and decrease of β-actin. Global protein network analysis was then performed to obtain additional functional significance of the identified proteins and to guide for subsequent functional analysis. The results implicated that the altered proteins were involved in energy production and might contribute to mitochondrial dysfunction. The loss of ROS regulation by mitochondria was finally confirmed by OxyBlot assay, which demonstrated markedly increased levels of the oxidatively modified mitochondrial proteins in the COM-treated cells in a dose-dependent manner. Our data may lead to a better understanding of molecular mechanisms of mitochondrial dysfunction underlying the overt oxidative stress induced by COM crystals in kidney stone disease.