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

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Abstract
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. 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 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 of45 Ca into the stone and visualized by scanning electron microscopy (SEM). Antisense oligonucleotide at concentrations higher than 20 micro M inhibited synthesis of OPN. Incorporation of45 Ca into the calculus stone was inhibited by the addition of oligonucleotide in a concentration dependent manner in a range above 20 micro M. More than 90% of incorporation was inhibited at 50 micro M as compared to control. Inhibition of calcium crystal formation was confirmed by SEM. 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.