Knockout of the neonatal Fc receptor in cultured podocytes alters IL-6 signaling and the actin cytoskeleton

Abstract

The neonatal Fc receptor (FcRn) has been shown to be required for antigen presentation in dendritic cells, and global knockout of FcRn attenuates immune-mediated kidney disease. Podocytes express interleukin-6 (IL-6) receptor and produce IL-6 under proinflammatory conditions. Here we examined the role of FcRn in the IL-6-mediated inflammatory response in podocytes. We examined IL-6 production by ELISA and expression by qPCR in wild type (WT) and FcRn knockout (KO) podocytes after treatment with proinflammatory stimuli as well as IL-6-mediated signaling via the JAK/STAT pathway. We also examined podocyte motility in cultured WT and KO podocytes after a proinflammatory challenge. We found that FcRn KO podocytes produced minimal amount of IL-6 after treatment with albumin, IgG, or immune complexes whereas WT podocytes had a robust response. FcRn KO podocytes also had minimal expression of IL-6 compared with WT. By Western blotting, there was significantly less phosphorylated STAT3 in KO podocytes after treatment with IFNγ or immune complexes. In a scratch assay, FcRn KO podocytes showed increased motility comparted KO, suggesting a defect in actin dynamics. Cultured FcRn KO podocytes also demonstrated abnormal stress fibers compared with WT and the defect could be rescued by IL-6 treatment. This study shows that in podocytes, FcRn modulates the IL-6 mediated response to proinflammatory stimuli and regulates podocytes actin structure, motility and synaptopodin expression.

Regulation of the Actin Cytoskeleton in Podocytes

“…FcRn KO podocytes are also hypermotile at baseline and have significantly shorter stress fibers. These defects can be rescued by treatment with IL-6 [ 205 ]. FcRn-mediated modulation of the actin cytoskeleton via IL-6 provides further evidence that protein trafficking in podocytes is linked to actin cytoskeletal dynamics.…”

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

“…The exact mechanisms by which the absence of FcRn prevents crescent formation may have to do with impaired podocyte mobility, alterations in actin dynamics or impaired intracellular signaling. The mechanism underlying how FcRn may affect these pathways is unknown but we have found that knockout of FcRn KO in cultured podocytes alters mobility and actin dynamics (37). In addition, when comparing the relative quantity of the crescents between the two models, we found significantly fewer glomeruli in the anti-GBM had crescents compared to the NTS model, which is suggestive that crescent formation is more dependent on the autologous phase than the heterologous phase 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.…”

“…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.…”

“…In order to determine whether podocyte-specific knockout of FcRn would protect against induction of immune-mediated kidney disease, we generated podocyte-specific FcRn KO (podFcRn KO) mice by crossing podocin-Cre mice with FcRn floxed mice to create FcRn fl/fl:Podocin-Cre/+ mice ( Figure 3) as previously described (37). FcRn floxed mice lacking the Cre transgene (FcRn fl/fl;+/+) served as littermate controls.…”