Nitric oxide induces MIP-2 transcription in rat renal mesangial cells and in a rat model of glomerulonephritis.
Nitric oxide is a crucial mediator of several forms of glomerulonephritis. We examined the effects of NO on the mRNA expression pattern in glomerular mesangial cells by using a low-stringency reverse transcriptase-polymerase chain reaction method and detected a cDNA fragment that was induced by interleukin 1b (IL-1b) and further up-regulated by the NO donor diethylenetriamine-nitric oxide (DETA-NO). Each respective cDNA fragment was found to match with the cDNAs of rat macrophage inflammatory protein 2 (MIP-2) and GRO/cytokine-induced neutrophil chemoattractant 2b (CINC-2b). Further characterization of MIP-2 regulation by Northern blot analysis confirmed an NO- and IL-1b-dependent increase in MIP-2 mRNA levels. Moreover, inhibition of IL-1b-induced endogenous NO formation by the NO-synthase (NOS) inhibitor L-NMMA markedly attenuated MIP-2 protein expression. We cloned 770 bp of the 5'-flanking region of rat MIP-2 and fused this fragment to a luciferase reporter gene. Transfection of the construct into mesangial cells resulted in a 3.5-fold increase in luciferase activity in cells treated with DETA-NO when compared to controls, suggesting a transcriptional mechanism for NO-induced MIP-2 expression. Deletion and mutational analysis identified critical nuclear factor (NF)-kB and NF-IL-6 binding sites required for NO regulation of MIP-2. In vivo, inhibition of NO synthesis in the Thy-1.1 model of mesangioproliferative glomerulonephritis by the specific inducible-NOS inhibitor L-NIL resulted in a marked reduction of MIP-2 mRNA expression. Furthermore, infiltration of neutrophils into the glomerulus was dramatically attenuated in L-NIL-treated rats.
*Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany;, daggerAbteilung für Innere Medizin, Universität Münster, Münster, Germany.
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S
Walpen
K F
KF
Beck
L
L
Schaefer
I
I
Raslik
W
W
Eberhardt
R M
RM
Schaefer
J
J
Pfeilschifter
D000578:Amidines / Q000494:pharmacology; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D054426:Chemokine CXCL2; D018925:Chemokines / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D005921:Glomerulonephritis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D007375:Interleukin-1 / Q000378:metabolism; D007678:Kidney Glomerulus / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000578:Amidines; D001596:Benzylamines; D054426:Chemokine CXCL2; D018925:Chemokines; C404915:Cxcl2 protein, mouse; D004791:Enzyme Inhibitors; D007375:Interleukin-1; C090663:N(6)-(1-iminoethyl)lysine; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D011993:Recombinant Fusion Proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D008239:Lysine