Osteopontin antisense oligonucleotide inhibits adhesion of calcium oxalate crystals in Madin-Darby canine kidney cell

Purpose: We previously suggested that osteopontin (OPN) plays an important role in the process of deposited calcium crystals adhesion to cells in the early stages of urolithiasis. To further confirm this theory, we tried to inhibit OPN expression at the translational level and examined its cellular biological consequence on the formation and adhesion process of crystals. Materials and Methods: We synthesized antisense and sense oligonucleotide corresponding to an appropriate part of the coding sequence for OPN in Madin Darby canine kidney (MDCK) cells. With the aid of lipofection reagent DOTAP, antisense and sense oligonucleotide were introduced into MDCK cells grown in a confluent monolayer. After further incubation, inhibition of OPN expression in the cells was assessed by immunofluorescence photomicrography, and formation of calcium oxalate crystals was quantitated by incorporation of Ca-45 into the stone and visualized by scanning electron microscopy (SEM). Results: Antisense oligonucleotide at concentrations higher than 20 mu M inhibited synthesis of OPN. Incorporation of 45Ca into the calculus stone was inhibited by the addition of oligonucleotide in a concentration dependent manner in a range above 20 mu M. More than 90% of incorporation was inhibited at 50 mu M as compared to control. Inhibition of calcium crystal formation was confirmed by SEM. Conclusions: OPN was shown as a major component in the extracellular matrix involving the formation and adhesion of calcium crystals in the distal renal tubular cells, suggesting that OPN plays an important role in stimulating deposition and adhesion of calculus crystals to cells in the early stages of urolithiasis.