Production of gastric intrinsic factor, transcobalamin, and haptocorrin inopossum kidney cells

Opossum kidney epithelial cells were shown previously to synthesize and sec rete two cobalamin (Cbl)-binding proteins, presumed to be haptocorrin (Hc) and transcobalamin II (TCII). The present study examines the hypothesis tha t renal tubular cells also produce intrinsic factor (IF), and this producti on provides an explanation for the presence of IF in urine. By using antise ra raised against human IF and against TCII, the presence of TCII was confi rmed, and that of IF discovered in the media of opossum kidney (OK) cells i n culture. The apparent molecular weight of IF and TCII was 68 and 43 kDa, respectively. Immunoreactivity on Western blot of the putative IF protein w as blocked by recombinant human IF. When proteins secreted into the media w ere separated electrophoretically under nondenaturing conditions after bind ing with [Co-57]Cbl, a broad major band migrated at a relative front indepe ndently of recombinant IF or TCII, and probably represents Hc, as the Cbl b inding is blocked by cobinamide. Small amounts of bound [Co-57]Cbl migrated in the position of both IF and TCII, when cobinamide was present. The pres ence of IF and TCII in OK cells was confirmed by immunohistology. Specific reactivity for IF (blocked by recombinant IF) was found in proximal tubules of opossum kidney, but not in other portions of the nephron, confirming th e ability of anti-human IF antiserum to detect opossum IF. A 732-bp fragmen t of IF, nearly identical in sequence to rat IF, was isolated by RT-PCR fro m opossum kidney mRNA, and Western blot confirmed the presence of IF protei n. The presence of IF was also documented in rat kidney by isolation of an RT-PCR fragment, immunocytochemistry, and Western blot. IF should be added to the list of renal (proximal) tubular antigens that are shared by other e pithelia.