Nitric oxide induces MIP‐2 transcription in rat renal mesangial cells and in a rat model of glomerulonephritis

Abstract

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.

Requirement of Inducible Nitric-oxide Synthase in Lipopolysaccharide-mediated Src Induction and Macrophage Migration

“…However, Src did not appear on their list as one of the NO targets. Because MIF-2, the prominent NO-responsive gene in mesangial cells (49), was also elusive in this microarray assay (29), we believed that induction of Src by NO might depend on the type of tissues studied.…”

Inducible Nitric Oxide Synthase–Derived Nitric Oxide Promotes Glomerular Angiogenesis via Upregulation of Vascular Endothelial Growth Factor Receptors

“…It is 23-fold more selective for iNOS versus nNOS and 49-fold more selective for iNOS versus eNOS. It has no other known pharmacologic actions apart from competition with L-arginine for cellular uptake, and it has been used widely to probe the effects of iNOS inhibition (21)(22)(23)(24). The in vivo dose of L-NIL (60 mg/kg) was chosen to resemble directly a previous study in the same model for reasons of comparability (21).…”

Nitric Oxide Inhibits Glomerular TGF-β Signaling via SMOC-1

“…This point in time closely correlates with the expression of iNOS and with the maximal inflammatory response in the glomerulus. 11 Using a specific ELISA, we could confirm the expression of SMOC-1 in normal glomeruli on the protein level as well ( Figure 8B). Anti-Thy-1 GN caused a marked reduction of SMOC-1 in isolated glomeruli ( Figure 8B); however, this effect was completely prevented when nephritic rats were treated with the iNOS-specific inhibitor L-NIL ( Figure 8B), demonstrating the importance of NO in modulating SMOC-1 expression in vivo.…”

“…18 -20 In contrast, the expression of the chemokine macrophage inflammatory protein 2 is upregulated by NO in rat mesangial cells in a cGMP-independent manner, and blocking of iNOS activity in a rat model of anti-Thy-1 GN by L-NIL resulted in a clear reduction of glomerular neutrophil infiltration accompanied by reduced levels of macrophage inflammatory protein 2 mRNA and protein expression. 11 A protective role of the iNOS/cGMP pathway in vivo was further corroborated by reports that described an adverse effect of NOS inhibition and a beneficial effect of Bay 41-2272, an activator of the sGC in the rat models of anti-Thy-1 GN. 13,21,22 We compared mRNA expression patterns of rat glomerular mesangial cells treated with the NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) or vehicle using the RAP-PCR method.…”

“…These results clearly suggest a detrimental role for NO also in vivo. 11,12 In contrast, as exemplified in a chronic model of GN, administration of the iNOS-specific inhibitor L-N 6 -(1-iminoethyl)-lysine (L-NIL) markedly enhanced proteinuria and fibronectin deposition, indicating that activity of iNOS may also exert protective effects during inflammation. 13 These seemingly conflicting results might be explained by the high diversity of molecular targets of NO that trigger further downstream deleterious or protective signaling pathways.…”