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Abstract: FR-PO979

Intracellular Trafficking Pathway of Albumin in Glomerular Epithelial Cells

Session Information

Category: Pathology and Lab Medicine

  • 1601 Pathology and Lab Medicine: Basic

Authors

  • Moriyama, Takahito, Tokyo Women's Medical University, Tokyo, Japan
  • Nitta, Kosaku, Tokyo Women's Medical University, Tokyo, Japan
Background

Recently, intracellular trafficking pathway of albumin through caveolae in glomerular epithelial cells (podocytes) has been suspected to new etiological hypothesis of albuminuria in addition to pathway through gap between foot processes. In this analysis, we analyzed albumin endocytosis, subsequent transcytosis in cytoplasm and exocytosis.

Methods

Alexa flour 488 labeled bovine serum albumin (AF488-BSA) were incubated with Podocytes for 30, 60, and 120 minutes, and analyzed co-localization with caveolin-1(which was main structural component of caveolae), clathrin, and FC receptors (FcRn) as endocytosis, with several organelles, such as early endosome, Golgi apparatus (GA), endoplasmic reticulum (ER), lysosome, and proteasome, and with cytoskeletons such as microtubules and actins as transcytosis by immunofluorescence analysis (IF). In western blotting (WB) and IF, methyl beta cyclodextrin (MBCD) were preincubated with podocytes, then incubated with AF488-BSA or human serum albumin (HSA), and the amount of intracellular albumin through caveolae was analyzed. HAS were incubated with full confluent podocytes on transwells plate with or without MBCD, and concentration of HAS in inside and outside medium between transwells plate were evaluated to analyze exocytosis.

Results

At first, AF488-BSA were colocalized with Cav-1 and FcRn, but not with clathrin. Then AF488-BSA were colocalized with actin cytoskeleton, but not with microtubules, and colocalized with early endosome, lysosome, and proteasome, but not with ER and GA. MBCD treatment significantly decrease the Cav-1 and decreased the 488-BSA in IF and HAS in WB. The amount of HAS were gradually decreased in inside medium over podocytes on transwells plate and gradually increased in outside medium under transwells plate, and MBCD interfered that phenomenon. All these results indicated that albumin entered into Podocytes through Caveolae or FcRn but not clathrin, moved along with actin but not with microtunules, and reached to early endosome. At endosome, some of them were sorted to be degraded in lysosome or proteasome, and others were bypassed GA and ER and transported to the other side of cells as exocytosis.

Conclusion

In this study, we have shown intracellular trafficking pathway of albumin, and this pathway may be a new etiological hypotheis of urinay albumin excretion.