Differential trafficking of albumin and IgG facilitated by the neonatal Fc receptor in podocytes in vitro and in vivo.

Abstract

Proteinuria is strongly associated with kidney disease progression but the mechanisms underlying podocyte handling of serum proteins such as albumin and IgG remain to be elucidated. We have previously shown that albumin and IgG are transcytosed by podocytes in vitro. In other epithelial cells, the neonatal Fc receptor (FcRn) is required to salvage albumin and IgG from the degradative pathway thereby allowing these proteins to be transcytosed or recycled.Here we directly examine the role of FcRn in albumin and IgG trafficking in podocytes by studying handling of these proteins in FcRn knockout (KO) podocytes in vitro and in a podocytespecific FcRn knockout mice in vivo. In vitro, we find that knockout of FcRn leads to IgG accumulation in podocytes but does not alter albumin trafficking. Similarly, in vivo, podocytespecific knockout of FcRn does not result in albumin accumulation in podocytes in vivo as measured by mean albumin fluorescence intensity whereas these mice demonstrate significant intraglomerular accumulation of IgG over time. In addition we find that podocyte-specific FcRn KO mice demonstrate mesangial expansion as they age and activation of mesangial cells as demonstrated by increased expression of α-smooth muscle actin. Taken together, these results suggest that trafficking pathways for albumin and IgG differ in podocytes and that sustained disruption of trafficking of plasma proteins alters glomerular structure.

Podocyte-specific knockout of the neonatal Fc receptor (FcRn) results in differential protection depending on the model of immune-mediated kidney disease

“…Cell culture: Generation of conditionally immortalized WT and FcRn KO podocytes: Podocytes were isolated from WT or global FcRn KO mice and then underwent immortalization using a thermosensitive polyomavirus simian virus 40 (SV40) T antigen as previously described. (35)(36)(37) Primary podocytes were allowed to replicate at 33 o C. To induce differentiation, podocytes were placed at 37 o C for 8 -10 days. To verify expression of podocyte markers, podocytes were stained with podocin or WT1 and synaptopodin expression was checked by Western blot.…”

“…Conditionally immortalized WT or KO podocytes were allowed to differentiate at 37 °C for 9 -10 days prior to being used in experiments. Harbor, Maine) as previously described (36) days later by an intravenous injection of 0.2 mg/g of rabbit anti-mouse GBM antibody provided by Dr. Gabriela Garcia. Mice were sacrificed at 3 or 8 days after GBM antibody injection.…”

“…Podocytes were isolated from wild type (WT) and global FcRn KO (KO) mice and immortalized using the thermosensitive polyomavirus simian virus (SV40) T antigen as described previously (36,37). Since podocytes are not known to express MHCII at baseline, we used interferon gamma (IFN to stimulate MHCII expression.…”