Changes in Mitochondrial
Proteome of Renal Tubular
Cells Induced by Calcium Oxalate Monohydrate Crystal Adhesion and
Internalization Are Related to Mitochondrial Dysfunction
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Abstract
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