Local Inhibition of Indoleamine 2,3-Dioxygenase Mitigates Renal Fibrosis

Abstract

Chronic kidney disease (CKD) is a major global health concern and renal fibrosis is an integral part of the pathophysiological mechanism underlying disease progression. In CKD patients, the majority of metabolic pathways are in disarray and perturbations in enzyme activity most likely contribute to the wide variety of comorbidities observed in these patients. To illustrate, catabolism of tryptophan by indoleamine 2,3-dioxygenase (IDO) gives rise to numerous biologically active metabolites implicated in CKD progression. Here, we evaluated the effect of antagonizing IDO on renal fibrogenesis. To this end, we antagonized IDO using 1-methyl-D-tryptophan (1-MT) and BMS-98620 in TGF-β-treated murine precision-cut kidney slices (mPCKS) and in mice subjected to unilateral ureteral obstruction (UUO). The fibrotic response was evaluated on both the gene and protein level using qPCR and western blotting. Our results demonstrated that treatment with 1-MT or BMS-985205 markedly reduced TGF-β-mediated fibrosis in mPCKS, as seen by a decreased expression of collagen type 1, fibronectin, and α-smooth muscle actin. Moreover, IDO protein expression clearly increased following UUO, however, treatment of UUO mice with either 1-MT or BMS-986205 did not significantly affect the gene and protein expression of the tested fibrosis markers. However, both inhibitors significantly reduced the renal deposition of collagen in UUO mice as shown by Sirius red and trichrome staining. In conclusion, this study demonstrates that IDO antagonism effectively mitigates fibrogenesis in mPCKS and reduces renal collagen accumulation in UUO mice. These findings warrant further research into the clinical application of IDO inhibitors for the treatment of renal fibrosis.

Keywords: 1-methyl-tryptophan; BMS-98620; indoleamine 2,3-dioxygenase; precision-cut kidney slices; renal fibrosis.

Figure 1

1-methyl-D-tryptophan and BMS-986205 mitigate TGF-β-induced fibrosis in murine PCKS. PCKS were exposed to 10 ng/mL TGF-β in the absence or presence of 1-methyl-D-tryptophan (1 mM; 1-MT) or BMS-986205 (5 nM) for 48 h. (A) Relative mRNA expression of the fibrosis markers collagen 1A1 (COL1), collagen 3A1 (COL3), fibronectin (FN), and α-smooth muscle actin (αSMA) normalized to GAPDH. n = 4–10. (B) Western blotting was used to study the protein expression of fibronectin (FN) and α-smooth muscle actin (αSMA). n = 7. (C) Relative mRNA expression of the inflammation markers tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1β) normalized to GAPDH. n = 6–7. (D) Viability of mPCKS after treatment, assessed by ATP content of the slices. n = 4–10. Data are presented as mean ± SEM. * p < 0.05.

Figure 2

BMS-986205 does not alter canonical TGF-β signaling in murine PCKS. PCKS were exposed to 10 ng/mL TGF-β in the absence or presence of BMS-986205 (5 nM) for 48 h. (A) Relative mRNA expression of plasminogen activator inhibitor-1 (PAI-1) normalized to GAPDH. n = 3–7. (B) Western blotting was used to study the protein expression of total and phosphorylated Smad2. n = 6. Data are presented as mean ± SEM. * p < 0.05.

Figure 3

Cortical IDO protein expression increases following UUO. Mice were subjected to 7 days of unilateral ureteral obstruction (UUO). Afterward, indoleamine 2,3-dioxygenase (IDO) protein expression was studied using western blot (n = 8–10). Data are presented as mean ± SEM. * p < 0.05.

Figure 4

1-Methyl-D-tryptophan solely reduced collagen 3A1 gene expression in UUO mice. Mice were subjected to 7 days of unilateral ureteral obstruction (UUO) and treated with 1-methyl-D-tryptophan (10 mg/mL). (A) Relative mRNA expression of the fibrosis markers collagen 1A1 (COL1), collagen 3A1 (COL3), fibronectin (FN), and α-smooth muscle actin (αSMA), and the inflammation markers tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1β), all normalized to 18S. n = 6–11. (B) Protein expression of fibronectin (FN) and α-smooth muscle actin (αSMA) was studied using western blot, corrected for total protein levels. n = 6–11. Data are presented as mean ± SEM. * p < 0.05.

Figure 5

BMS-986205 does not reduce the gene and protein expression of fibrosis markers in UUO mice. Mice were subjected to 7 days of unilateral ureteral obstruction (UUO) and treated with BMS-986205 (10 mg/mL). (A) Relative mRNA expression of the fibrosis markers collagen 1A1 (COL1), collagen 3A1 (COL3), fibronectin (FN), and α-smooth muscle actin (αSMA), and the inflammation markers tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1β), all normalized to 18S. n = 4–7. (B) Protein expression of fibronectin (FN) and α-smooth muscle actin (αSMA) was studied using western blot, corrected for total protein levels. n = 4–9. Data are presented as mean ± SEM. * p < 0.05.

Figure 6

1-Methyl-D-tryptophan and BMS-986205 markedly reduce collagen deposition in UUO mice. Mice were subjected to 7 days of unilateral ureteral obstruction (UUO) and treated with either 1-methyl-D-tryptophan (10 mg/mL) or BMS-986205 (10 mg/mL). (A) Representative images of Picrosirius red staining. (B) Representative images of Masson’s trichrome staining. (C) Quantification of Picrosirius red and Masson’s trichrome staining as a percentage of the total area of the tissue from Sham and UUO mice. Magnification 20×. Scale bar: 20 μm. n = 3–5. Data are presented as mean ± SEM. * p < 0.05.