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

Abstract

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.

Osteopontin protects against high phosphate-induced nephrocalcinosis and vascular calcification

“…This is particularly noteworthy as clinical studies show a strong correlation between kidney stone formation and VC, and pathogenic links between nephrocalcinosis and VC have been hypothesized 1, 36 . Also, previous in vivo calcification models utilizing OPN KO animals have induced calcification in various ways such as graft implantation or oxalate administration (given orally or via injection) 37–39 . While both of these scenarios are of clinical importance, our study is the first to show significant calcification with oral phosphate administration.…”

Osteopontin: A novel regulator at the cross roads of inflammation, obesity and diabetes

“…Hence, OPN −/− bones unlike those from wild type mice are hypermineralized and more fragile [4,43]. Furthermore, OPN is not only critical for bone mineralization, but is also strongly upregulated at sites of ectopic, pathologic calcification – such as vascular calcification [44], valvular calcification [45], renal crystal formation [46], and gallstone formation [47].…”

Biomimetic Randall’s plaque as an in vitro model system for studying the role of acidic biopolymers in idiopathic stone formation

“…In fact, RP as well as the stones themselves contain various amounts of proteins and polysaccharides, including albumin, globulin, Tamm-Horsfall glycoprotein (THP), prothrombin fragment 1, inter-a-trypsin inhibitor (bikunin), osteopontin (OPN), and calprotectin, all of which could conceivably contribute to forming an amorphous phase, or even a PILP-like phase, as demonstrated for various acidic polymers in our group [66–68]. These components are termed crystal matrix proteins, and constitute 1 5% of RP and urinary stones, implying that they may have some influence in the stone-formation process [10, 31, 34, 36, 38, 39, 41, 50, 51, 55, 69–71]. …”

“…This group forms calcium oxalate renal stones without any systemic disorder and make up about 80% of all stone-formers [8, 16, 24, 31, 33, 39, 41, 42, 48–51]. The lack of a known cause (such as various urinary imbalances) for the majority of patients who develop stones also contributes to the difficulty of understanding and treating this painful and recurring disease.…”

“…A protein of great interest is osteopontin (OPN or uropontin, the urinary form of OPN), which has been shown to be important in the pathogenesis of renal stones and, as mentioned, has been identified as one of the proteins in the matrix layer [4, 7–9, 14, 27, 31, 36, 39, 41–43, 46]. It also has been shown to be present in the concentric laminations found in RP (where it was immunostained)[9].…”