OSTEOPONTIN ANTISENSE OLIGONUCLEOTIDE INHIBITS ADHESION OF CALCIUM-OXALATE CRYSTALS IN MADIN-DARBY CANINE KIDNEY-CELL

Purpose: We previously suggested that osteopontin (OPN) plays an impor tant role in the process of deposited calcium crystals adhesion to cel ls in the early stages of urolithiasis. To further confirm this theory , we tried to inhibit OPN expression at the translational level and ex amined its cellular biological consequence on the formation and adhesi on process of crystals. Materials and Methods: We synthesized antisens e and sense oligonucleotide corresponding to an appropriate part of th e coding sequence for OPN in Madin Darby canine kidney (MDCK) cells. W ith the aid of lipofection reagent DOTAP, antisense and sense oligonuc leotide 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 int o the stone and visualized by scanning electron microscopy (SEM). Resu lts: Antisense oligonucleotide at concentrations higher than 20 mu M i nhibited synthesis of OPN. Incorporation of 45Ca into the calculus sto ne was inhibited by the addition of oligonucleotide in a concentration dependent manner in a range above 20 mu M. More than 90% of incorpora tion was inhibited at 50 mu M as compared to control. Inhibition of ca lcium crystal formation was confirmed by SEM. Conclusions: OPN was sho wn as a major component in the extracellular matrix involving the form ation and adhesion of calcium crystals in the distal renal tubular cel ls, suggesting that OPN plays an important role in stimulating deposit ion and adhesion of calculus crystals to cells in the early stages of urolithiasis.