Characterization and distribution of albumin binding protein in normal rat kidney

Abstract

The mechanism by which proteins that pass through the glomerular basal lamina are taken up by proximal tubule cells is incompletely characterized. Past work has identified the kinetics of albumin binding to renal brush-border membrane. We have now purified and characterized albumin binding protein (ABP) and shown its distribution in renal proximal tubular cells. ABP was purified from rat renal proximal tubular cell brush-border membrane by affinity chromatography with rat serum albumin-Sepharose. The resulting ABP had two apparent molecular masses (55 and 31 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies to ABP were raised in rabbits and checked by immunoassay and immunoblotting. Light-microscopic immunohistochemistry showed ABP all along the proximal tubule in the pars convoluta and pars recta. Electron-microscopic immunohistochemistry showed labeling on microvilli and in apical endocytic vacuoles, dense apical tubules, and lysosomes. These results indicate that ABP is involved in proximal tubule endocytosis.

Cubilin is an albumin binding protein important for renal tubular albumin reabsorption

“…The present study shows strong cross reactivity between antibodies raised against these low-molecular-weight binding proteins and cubilin in addition to a very similar cellular distribution in the proximal tubule cells. Furthermore, the anti-cubilin antibody identified the same low-molecular-weight protein bands as the antibody raised by Cessac-Guillemet et al (22), strongly indicating that these low-molecular-weight proteins are in fact fragments of cubilin. When performing albumin affinity chromatography similar to the protocol described by Cessac-Guillemet et al (22), we were able to purify significant amounts of cubilin, which however, were not identified in their study.…”

“…Furthermore, the anti-cubilin antibody identified the same low-molecular-weight protein bands as the antibody raised by Cessac-Guillemet et al (22), strongly indicating that these low-molecular-weight proteins are in fact fragments of cubilin. When performing albumin affinity chromatography similar to the protocol described by Cessac-Guillemet et al (22), we were able to purify significant amounts of cubilin, which however, were not identified in their study. This may be explained by their use of 12% polyacrylamide separating gels to identify purified proteins, not allowing the migration of high-molecular-weight proteins such as cubilin.…”

“…Receptors of similar molecular weight were purified from kidney cortex by albumin affinity chromatography and identified by immunoblotting and immunocytochemistry (22). The present study shows strong cross reactivity between antibodies raised against these low-molecular-weight binding proteins and cubilin in addition to a very similar cellular distribution in the proximal tubule cells.…”

“…Furthermore, the labeling by the antibody against the low-molecular-weight albumin binding proteins could be strongly inhibited by preincubation with IF-B 12 affinity purified cubilin ( Figure 5, d and e). Finally, immunoblotting using the anti-cubilin antibody on the albumin binding protein samples purified by Cessac-Guillemet et al (22) revealed the same low-molecular-weight bands as identified by the antibody against the low-molecular-weight albumin binding proteins, suggesting that these protein bands represent fragments of cubilin (data not shown).…”

“…The solution was lyophilized and dissolved in 10 mM HEPES, 0.14 M NaCl, and 2 mM CaCl 2 (pH 7.4) before use. Samples of possible low-molecular-weight albumin binding proteins was generated by CessacGuillemet et al as described previously (22).…”