Fig. 6. SOCE mediates crystal induced Ca²⁺ mobilization.

Crystal internalization induces upregulation of STIM and ORAI channel expression. Control (noncrystal) CaP, CaOx, and CaP + CaOx (mixed) crystals were introduced into HK2 cells for 24 h. a mRNA expression levels of STIM1, 2, and ORAI channels were analyzed by PCR. ORAI and STIM expression is mediated by SOCE in HK2 cells. HK2 cells were incubated and representative blots were obtained, relative mRNA expression was quantified and represented as bar diagram. b To confirm knockdown following siRNA (10 nM), b STIM1; c STIM2; and d ORAI3 transcript were determined by PCR, representative blots were obtained and quantified as bar diagrams. e To ascertain the role of STIM12 and ORAI3 channel in crystal internalization, STIM1, 2, and ORAI3 expression was knocked down using siRNA, cells were then introduced with or without CaP, CaOx, and mixed (CaP + CaOx) crystals for 24 h. Following crystal internalization, cells were loaded with fura-2AM and Ca²⁺ influx was determined and represented as d [Ca²⁺] rise and e Ca²⁺ influx was determined and represented as reduction in [Ca²⁺] response. Two-tailed t-test was used for statistical comparison. Statistically significant differences are indicated (mean ± SEM) from three different experiments. Levels of significance are indicated as *p < 0.05; **p < 0.01 as shown in the bar diagrams