Title

Expression of Matrix Gla Protein in MDCK cells exposed to Oxalates, Calcium Oxalate Monohydrate and Hydroxyapatite crystals

Document Type

Article

Department

Biological and Biomedical Sciences

Abstract

Introduction and Objectives: A number of proteins, characterized as the i) causators, ii) effectors and iii) bystanders, involved in the pathogenesis of kidney stones and expressed in response to oxalate (Ox.) and/or calcium oxalate monohydrate (COM)/hydroxyapatite (HA) crystals. The involvement of Matrix Gla Protein (MGP), a well-known mineralization inhibitor, has not yet been fully explored in stone formation. MGP is a 10–12 kD protein expressed at high levels in heart, lungs and kidneys. Objective: This study was designed to determine the role of MGP in kidney stone formation by investigating the expression of MGP in MDCK cells exposed to various concentrations of Ox, COM crystals or HA. Expression and production of a number of proteins with role in stone formation has been previously shown to be influenced by the exposure to Ox, COM or HA crystals. Material and Methods: MGP detection was carried out by using western blotting, after cells were exposed to 0.3, 0.5 and 1 mM Ox, or 33, 66 and 133 mg/cm2 of COM/HA crystals for 3 hrs, 6 hrs and 24 hrs. Since oxalate as well as crystal exposure leads to development of oxidative stress as well as cell injury we also investigated LDH release to determine cell injury; Super Oxide Dismutase (SOD) activity to estimate oxidative stress and cell death by Trypan Blue Exclusion (TEB) assay. For these studies, cells were exposed to the same doses of oxalate and crystals for duration of 15, 30, 60, 120 and 180 minutes. Results: Expression of MGP increased in a concentration and time dependent manner. It was significantly increased after 24 hrs of exposure to Ox. Exposure to COM and/or HA crystals resulted in significant, concentration and time dependent, upregulation of MGP at 3, 6 and 24 hrs. Exposures to Ox, COM and/or HA produced oxidative stress, caused cell injury (LHD release) and cell death (TBE exclusion assay). Conclusions: The data indicate that MGP is expressed by the MDCK cells and expression is increased by exposure to both the oxalate as well as COM and/or HA crystals. There are however significant differences in the expression. Crystal exposure leads to significant increases within 3 hours while Ox produces similar increases after a longer exposure of 24 hours. The increase in expression is associated with development of oxidative stress. Significant numbers of cells are injured and died as a result of various exposures.

Publication

European Urology Supplements