Activation of nuclear factor-kappa B by podocytes in the autologous phase of passive Heymann nephritis.
BACKGROUND
The present study examined whether activation of nuclear factor-kappa B (NF-kappa B) occurs within podocytes in passive Heymann nephritis (PHN) and contributes to the pathogenesis of proteinuria.


METHODS
Electrophoretic mobility shift assays (EMSAs) were used to detect NF-kappa B activation, and supershift assays were used to determine the subunits involved. Localization of the activated NF-kappa B subunit p50 was performed by immunohistochemistry. Expression of the NF-kappa B-dependent genes interleukin-1 beta (IL-1 beta) and matrix metalloproteinase-9 (MMP-9) were determined by reverse transcription-polymerase chain reaction and, for IL-1 beta, immunohistochemistry. To inhibit activation of NF-kappa B in vivo, pyrrolidone dithiocarbamate (PDTC) was administered for 10 days following induction of PHN.


RESULTS
Glomerular nuclear extracts from rats with PHN showed increased NF-kappa B binding activity in comparison to normal rats. The major Rel/NF-kappa B proteins in these activated complexes were p65 and p50. Immunohistochemistry showed that nuclear translocation of p50 occurred predominantly within podocytes. IL-1 beta mRNA was increased in the PHN rats, and increased IL-1 beta protein was localized predominantly to podocytes by immunohistochemistry. To investigate whether activation of NF-kappa B 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-kappa B 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.


CONCLUSIONS
These data show that NF-kappa B is activated within podocytes in PHN and suggest that it contributes to autologous phase proteinuria. The critical genes regulated by NF-kappa B in the podocyte have not yet been determined, but may include MMP-9.
Mudge
S J
SJ
Department of Nephrology, St. Vincent's Hospital, Melbourne, Victoria, Australia.
Paizis
K
K
Auwardt
R B
RB
Thomas
R J
RJ
Power
D A
DA
D000419:Albuminuria / Q000652:urine; D000818:Animals; D005921:Glomerulonephritis / Q000473:pathology / Q000503:physiopathology* / Q000652:urine; D007136:Immunoglobulins / Q000276:immunology / Q000378:metabolism; D007375:Interleukin-1 / Q000378:metabolism; D007678:Kidney Glomerulus / Q000473:pathology / Q000503:physiopathology*; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D016328:NF-kappa B / Q000502:physiology*; D011759:Pyrrolidines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012756:Sheep / Q000276:immunology; D013859:Thiocarbamates / Q000494:pharmacology
D007136:Immunoglobulins; D007375:Interleukin-1; D016328:NF-kappa B; D011759:Pyrrolidines; D013859:Thiocarbamates; C020972:pyrrolidine dithiocarbamic acid; D020780:Matrix Metalloproteinase 9