title: 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 µg 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.
authors: Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, 467-8601, Japan.
authors: Shuzo
authors: S
authors: Hamamoto
authors: Takahiro
authors: T
authors: Yasui
authors: Atsushi
authors: A
authors: Okada
authors: Masahito
authors: M
authors: Hirose
authors: Yutaka
authors: Y
authors: Matsui
authors: Shigeyuki
authors: S
authors: Kon
authors: Fumihiko
authors: F
authors: Sakai
authors: Yoshiyuki
authors: Y
authors: Kojima
authors: Yutaro
authors: Y
authors: Hayashi
authors: Keiichi
authors: K
authors: Tozawa
authors: Toshimitsu
authors: T
authors: Uede
authors: Kenjiro
authors: K
authors: Kohri
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000097:blood; D002460:Cell Line; D003460:Crystallization; D004285:Dogs; D000939:Epitopes / Q000276:immunology*; D005260:Female; D006801:Humans; D007669:Kidney Calculi / Q000473:pathology* / Q000648:ultrastructure; D007684:Kidney Tubules / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D053495:Osteopontin / Q000737:chemistry* / Q000172:deficiency / Q000276:immunology*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism
chemical_list: D000906:Antibodies; D000939:Epitopes; D053495:Osteopontin; D013917:Thrombin