Interaction between osteopontin on Madin Darby canine kidney cell membraneand calcium oxalate crystal

We recently reported that the addition of the protein osteopontin (OPN) res ulted in an increase in the deposition of calcium oxalate (CaOx) crystals o n the surface of Madin Darby canine kidney (MDCK) cells. To determine the d egree to which this increased deposition is caused by OPN, we investigated the extent to which the CaOx crystal deposition produced by the expression of OPN at the cell surface was suppressed by 4 different methods prior to t he determination of the level of CaOx crystal binding. MDCK cells (2 x 10(6 ) cells/well) were cultured to a confluent state, and the binding of OPN to the cellular surface was then inhibited by adding one of the following 4 s ubstances: human OPN polyclonal antibody, thrombin, cyclic Arg-Gly-Asp (RGD ) peptides and tunicamycin. The cells were cultured for 24 h. We then used a fluorescent antibody technique with an OPN polyclonal antibody to determi ned whether the expression of OPN at the cell surface was inhibited, and we measured the degree of CaOx crystal deposition using the isotope Ca-45. Th e degree of CaOx crystal deposition was inhibited by 80% or more in the ant ibody-treated group, by 50-80% in the thrombin-treated group, by 60-80% in the cyclic RGD-treated group, and by 50-60% in the tunicamycin-treated grou p. These results suggest that OPN in the extracellular matrix is the main c ause of CaOx crystal deposition on the surface of MDCK cells.