Downregulation of SPARC expression is mediated by nitric oxide in rat mesangial cells and during endotoxemia in the rat.
Nitric oxide (NO) has been implicated in several forms of glomerulonephritis. In this study, a low stringency reversed transcription/PCR protocol was used to evaluate the action of NO on the mRNA expression pattern in rat mesangial cells (MC). To mimic the state of glomerular inflammation, MC were stimulated by exposure to the cytokines interleukin-1beta and tumor necrosis factor-alpha into producing high levels of NO via expression of inducible nitric oxide synthase (NOS). To detect NO-mediated effects, the resulting expression pattern was compared to that of MC stimulated by the cytokines in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA). Computer analysis of a differentially expressed cDNA fragment resulted in a 100% homology to the recently characterized mRNA of SPARC (secreted protein acidic and rich in cysteine). Further characterization of SPARC regulation revealed a cytokine- and cAMP-dependent decrease in SPARC mRNA and protein levels. Blocking NO formation by L-NMMA reversed the effects of cytokines and cAMP on SPARC expression, suggesting an NO-mediated mechanism. The NO donors S-nitroso-N-acetyl-penicillamine and diethylenetriamine/NO further reduced SPARC expression in cytokine-treated MC as well as in controls. Moreover, downregulation of SPARC mRNA and protein expression in whole kidneys obtained from rats treated with endotoxin was observed. This downregulation of SPARC was reversed by treatment with L-N6-l (iminoethyl) lysine dihydrochloride, a potent and highly selective inhibitor of inducible NOS. These data characterize SPARC as an NO-regulated gene. This observation may be important in the context of tissue remodeling in chronic inflammatory kidney diseases.
Walpen
Sebastian
S
Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Beck
Karl-Friedrich
KF
Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Eberhardt
Wolfgang
W
Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Apel
Martina
M
Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Chatterjee
Prabal K
PK
The William Harvey Research Institute, St. Bartholomew's and The Royal London School of Medicine and Dentistry, London, United Kingdom.
Wray
Gillian M H
GMH
The William Harvey Research Institute, St. Bartholomew's and The Royal London School of Medicine and Dentistry, London, United Kingdom.
Thiemermann
Christoph
C
The William Harvey Research Institute, St. Bartholomew's and The Royal London School of Medicine and Dentistry, London, United Kingdom.
Pfeilschifter
Josef
J
Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
D000818:Animals; D000242:Cyclic AMP / Q000502:physiology; D016207:Cytokines / Q000502:physiology; D015536:Down-Regulation; D019446:Endotoxemia / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005920:Glomerular Mesangium / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005921:Glomerulonephritis / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism; D008297:Male; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000502:physiology*; D015676:Osteonectin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019323:omega-N-Methylarginine / Q000494:pharmacology
D016207:Cytokines; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D015676:Osteonectin; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D000242:Cyclic AMP