Tryptophan Metabolites Regulate Neuropentraxin 1 Expression in Endothelial Cells

Abstract

In patients with chronic kidney disease (CKD) and in animal models of CKD, the transcription factor Aryl Hydrocabon Receptor (AhR) is overactivated. In addition to the canonical AhR targets constituting the AhR signature, numerous other genes are regulated by this factor. We identified neuronal pentraxin 1 (NPTX1) as a new AhR target. Belonging to the inflammatory protein family, NPTX1 seems of prime interest regarding the inflammatory state observed in CKD. Endothelial cells were exposed to tryptophan-derived toxins, indoxyl sulfate (IS) and indole-3-acetic acid (IAA). The adenine mouse model of CKD was used to analyze NPTX1 expression in the burden of uremia. NPTX1 expression was quantified by RT-PCR and western blot. AhR involvement was analyzed using silencing RNA. We found that IS and IAA upregulated NPTX1 expression in an AhR-dependent way. Furthermore, this effect was not restricted to uremic indolic toxins since the dioxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) do the same. In CKD mice, NPTX1 expression was increased in the aorta. Therefore, NPTX1 is a new target of AhR and further work is necessary to elucidate its exact role during CKD.

Keywords: aryl hydrocarbon receptor; indole-3-acetic acid; indolic uremic toxins; indoxyl sulfate; neuropentraxin 1.

Figure 1

Effects of IS and IAA on NPTX1 expression. Cells were incubated with IS, IAA or with their respective controls at the same concentration [IS or KCl, 200 µM; IAA or ethanol (Eth), 50 µM] for the indicated time. (A) NPTX1 mRNA levels were quantified by RT-PCR (n = 5). (B) Western blots were performed with 40 µg total protein and revealed with antibodies against NPTX1 or against actin (n = 5). (C) NPTX1 protein amounts were quantified by ImageJ software. Wilcoxon matched-pairs signed rank test was used to compare toxins to their controls, and a p value < 0.05 was considered significant (*).

Figure 2

Effects of IS (A) and IAA (B) concentrations on NPTX1 mRNA levels. Cells were incubated with IS, IAA or their respective controls (KCl for IS and ethanol (Eth) for IAA) at the indicated concentrations during 4h. NPTX1 mRNA levels were quantified by RT-PCR (n = 5). Values are expressed as mean ± SD. Wilcoxon matched-pairs signed rank test was used and a p value < 0.05 was considered significant (*).

Figure 3

Role of AhR in the induction of NPTX1 by IS and IAA. HUVEC were transfected by si RNA controls (siControls) or directed against AhR (siAhR). 48H after transfection, HUVEC were incubated during 4h with IS (200 µM), IAA (50 µM), KCl or ethanol. (A) NPTX1 mRNA levels were quantified by RT-PCR (n = 4). (B) 40 µg of total protein were loaded on SDS PAGE and revealed using antibodies against AhR, NPTX1 and actin (n = 5). (C) NPTX1 and AhR protein amounts were quantified by the ImageJ software. For (A,C), values are expressed as mean ± SD. Wilcoxon matched-pairs signed rank test was used and a p value < 0.05 was considered significant (*).

Figure 4

XRE sequences in the Promotor of the NPTX1gene. The first 5000 bases upstream of the first exon were presented. The three long canonical XRE sequences (5′GCGTGNNA/TNNNC/G3′) of AhR binding and the four short (5′GCGTG3′) are displaying with their respective position from the first exon.

Figure 5

Effects of well known agonists of AhR on NPTX1 mRNA levels. Cells were incubated during 4 hours with IS (200 µM), IAA (50 µM), FICZ (10 nM), and TCDD (30 nM), or their respective controls at the same concentrations. Culture medium did not contain growth factor nor serum but 0.5% human albumin. NPTX1 mRNA levels were quantified by RT-PCR (n = 5). Values are expressed as mean ± SD. Wilcoxon matched-pairs signed rank test was used and a p value < 0.05 was considered significant (*).

Figure 6

Effects of CKD of Nptx1 mRNA levels. Nptx1 mRNA levels in heart and Aorte of mice with normal diet (ND) or adenin diet (AD). Values are expressed as mean ± SD. Mann Whitney test was used and a p value < 0.05 was considered significant (*).