Osteopontin gene expression and immunolocalization in the rabbit urinary tract

Purpose: Osteopontin is a highly phosphorylated, calcium binding sialoprotein characterized by a conserved arginine-glycine-aspartate sequence. Vitronectin receptor (alphavbeta3 integrin) and hyaluronan receptor (CD44) are documented as receptors for osteopontin and their expression has been established in the bladder. Based on that finding and the fact that osteopontin protein is present in urine we hypothesized that osteopontin is expressed in the lower urinary tract. Materials and Methods: Osteopontin messenger (m)RNA and protein were analyzed in 5 adult urinary tracts and 5 neonatal bladders of New Zealand White rabbits using reverse transcriptase-polymerase chain reaction and immunohistochemical testing. Analysis of mRNA expression and localization of osteopontin receptors, alphavbeta3 integrin and CD44 were also performed in adult bladders and primary cultures of detrusor myocytes. Results: Adult renal pelvis, ureter, bladder and urethra, and neonatal bladders contained significant levels of osteopontin mRNA. Immunohistochemical staining revealed osteopontin expression in all layers of the transitional epithelium of the bladder, co-localizing with alphavbeta3 integrin mainly in the superficial layers and with CD44 mainly in the basal layers. Osteopontin was detected within the cytoplasm of smooth muscle cells, while alphavbeta3 integrin was located closer to the plasmalemma. Furthermore, primary cultured detrusor myocytes expressed osteopontin mRNA in stable fashion for up to 4 passages. Treating bladder myocyte cultures with insulin-like growth factor-1 and 17beta-estradiol resulted in up-regulation and down-regulation of osteopontin mRNA, respectively. Conclusions: Adult and neonatal rabbit detrusors are a prominent source of osteopontin in vivo and in vitro. Epithelial osteopontin may be a source of osteopontin in urine. The co-localization of osteopontin in the bladder epithelium with alphavbeta3 integrin and CD44 suggests a role in maintaining the integrity of the transitional epithelium by providing the sealing and adhesiveness needed for the impermeable state of the bladder.