Role of osteopontin in early phase of renal crystal formation: immunohistochemical and microstructural comparisons with osteopontin knock-out mice

Osteopontin (OPN) is an important matrix protein of renal calcium stone. However, the function of OPN in the early phase of renal crystal formation is not well defined. In this study, we examined OPN expression in the early phase of renal crystal formation with ultra-microstructural observations and immuno-TEM (transmission electron microscopy) in control and OPN knock-out (OPN-KO) mice. Glyoxylate (100 mg/kg) was intra-abdominally administered to male wild-type mice (C57BL/6, 8 weeks of age) and OPN-KO mice (C57BL/6, 8 weeks of age). Kidney was collected before and 6, 12, and 24 h after administration. We examined the relation between renal crystal formation and microstructural OPN location using TEM and immunohistochemical staining of OPN as well as western blotting and quantitative RT-PCR for OPN. OPN protein expression gradually increased in the renal cortex-medulla junction after glyoxylate administration, and OPN mRNA was increased until 12 h, but decreased at 24 h. In ultra-microstructural observation, OPN began to appear on the luminal side of renal distal tubular cells at 6 h and was gradually detected in the tubular lumen at 12 h. OPN was present in the crystal nuclei and collapsed mitochondria in the tubular lumen. In the OPN-KO mice, collapsed mitochondria were present, but no crystal nuclei formation were detected at 24 h. Based on the results this study proposed that the appearance of organelles, such as mitochondria and microvilli, in the tubular lumen after cell injury may be the starting point of crystal nucleus formation due to the aggregation ability of OPN.