Activation of nuclear factor-κB by podocytes in the autologous phase of passive Heymann nephritis

Activation of nuclear factor-κB by podocytes in the autologous phase of passive Heymann nephritis. The present study examined whether activation of nuclear factor-κB (NF-κB) occurs within podocytes in passive Heymann nephritis (PHN) and contributes to the pathogenesis of proteinuria. Electrophoretic mobility shift assays (EMSAs) were used to detect NF-κB activation, and supershift assays were used to determine the subunits involved. Localization of the activated NF-κB subunit p50 was performed by immunohistochemistry. Expression of the NF-κB–dependent genes interleukin-1β (IL-1β) and matrix metalloproteinase-9 (MMP-9) were determined by reverse transcription-polymerase chain reaction and, for IL-1β, immunohistochemistry. To inhibit activation of NF-κB in vivo, pyrrolidone dithiocarbamate (PDTC) was administered for 10 days following induction of PHN. Glomerular nuclear extracts from rats with PHN showed increased NF-κB binding activity in comparison to normal rats. The major Rel/NF-κB proteins in these activated complexes were p65 and p50. Immunohistochemistry showed that nuclear translocation of p50 occurred predominantly within podocytes. IL-1β mRNA was increased in the PHN rats, and increased IL-1β protein was localized predominantly to podocytes by immunohistochemistry. To investigate whether activation of NF-κB is involved in the pathogenesis of proteinuria, PDTC was administered to rats with PHN. Electrophoretic mobility shift assays of glomerular nuclear extracts showed a significant reduction in NF-κB binding activity in the PDTC-treated rats with a striking reduction in MMP-9 mRNA. Compared with control rats, there was a significant reduction in albuminuria at days 15 (P < 0.001) and 20 (P < 0.001) when PHN was induced with a suboptimal dose of anti-Fx1A antiserum. There was no detectable difference in the systemic immune response to sheep Ig in the treated rats. These data show that NF-κB is activated within podocytes in PHN and suggest that it contributes to autologous phase proteinuria. The critical genes regulated by NF-κB in the podocyte have not yet been determined, but may include MMP-9.