Crucial role of the cryptic epitope SLAYGLR within osteopontin in renal crystal formation of mice

Abstract

Osteopontin plays a crucial role in the formation of renal calcium crystals, which are primarily induced by renal tubular cell injury, especially mitochondrial damage. We have previously shown that the impaired Arg-Gly-Asp (RGD) sequence of osteopontin inhibits renal crystal formation by using OPN-transgenic mice and OPN-knockout (OPN-KO) mice. Here, we investigated the effects of an antimurine osteopontin antibody (35B6-Ab) that specifically reacts with the 162 SLAYGLR 168 sequence, which is exposed by thrombin cleavage and is located adjacent to the RGD sequence, on renal crystal formation. Renal crystals induced by daily administration of glyoxylate over 9 days (from days 1 to 9) in a murine model were sporadically detected in the renal tubular cells at the corticomedullary junction, where thrombin-cleaved osteopontin expression was also coincidentally detected. On days 0, 3, 6, and 9, 35B6-Ab administration inhibited renal crystal formation and induced significant morphological changes in a dose-dependent manner (250, 500, and 1000 mg per mouse). Scanning electron microscopy showed that the crystals in 35B6-Ab-treated mice were aberrantly formed and their density was low; in contrast, the crystals in untreated mice that were not administered 35B6-Ab had a radial pattern of growth (rosette petal-like crystals), and their density was high. Microstructure analysis of renal tubular cells by transmission electron microscopy revealed that untreated mice showed collapsed mitochondria in the flattened cytoplasm of renal tubular cells, unlike the corresponding structures in 35B6-Ab-treated mice, in which renal tubular cell injury was inhibited. In vitro, 35B6-Ab was found to inhibit the attachment of 14 C-labeled crystals to renal tubular culture cells and reduce morphological damage to these cells. We conclude that thrombin-cleaved osteopontin plays an important role in formation of renal calcium crystals and that 35B6-Ab contributes to the remarkable inhibition of early-stage renal crystal formation by preventing renal tubular cell injury and crystal-cell attachment. ß

A CD153+CD4+ T Follicular Cell Population with Cell-Senescence Features Plays a Crucial Role in Lupus Pathogenesis via Osteopontin Production

“…Mouse anti-OPN Ab (35B6) (22) or isotype-matched mouse IgG (Sigma-Aldrich) was administered i.p. at 500 mg per mouse twice per week.…”

A New Integrin-Binding Site on a Transglutaminase-Catalyzed Polymer

“…OPN is crosslinked to fibronectin to increase matrix integrity (Beninati et al 1994) and is polymerized to enhance its collagen binding activity (Kaartinen et al 1999). OPN is also covalently linked to bone matrix (Kaartinen et al 2002) and urinary stones (Kohri et al 1992;Hamamoto et al 2011). In the mineralized compartment of intramembranous rat bone there are three different transglutaminase substrates: bone sialoprotein, α2 HS-glycoprotein, and OPN.…”

The potential role of Osteopontin in the maintenance of commensal bacteria homeostasis in the intestine

“…Therefore, to examine whether Opn contributes to IEL survival, we sorted TCRαβ + and TCRγδ + IELs from WT and Opn KO mice and cells were cultured in the presence of anti-Opn blocking antibody (35B6) [3] for 12 hours. As shown in Fig 6, treatment with anti-Opn antibody inhibited the survival of TCRαβ + and TCRγδ + IELs.…”

“…Twenty-thousand cells of each type were seeded into 96-well round-bottom plate (FALCON) in 10% FCS/RPMI. Cells were cultured for 12 hours in the presence of anti-murine Opn antibody (35B6) [3] or control antibody (10 μg/ml). Surviving cells were counted using a conventional counting method with Trypan Blue and Cell Titer Glo (Promega) according to the manufactures’ instructions.…”

“…Under physiologic conditions, expression of Opn is known to be restricted to tissues such as bone and kidney. In these organs, Opn has been shown to be involved in various physiological functions including biomineralization of bone [1,2] and the regulation of renal crystal formation [3]. In contrast, Opn is upregulated in inflamed and injured tissues, and is implicated in the pathogenesis of various inflammatory disorders [4], tissue remodeling [5], wound healing [6], tumor invasion [7], and metastasis [8].…”