Inhibition of indoleamine 2, 3-dioxygenase-mediated tryptophan catabolism accelerates crescentic glomerulonephritis

Abstract

Immunomodulatory enzyme indoleamine 2, 3-dioxygenase (IDO) is one of the initial and rate-limiting enzymes involved in the catabolism of the essential amino acid tryptophan. Via catalysing tryptophan degradation, IDO suppresses adaptive T cell-mediated immunity and plays an important role in various forms of immune tolerance. Its role in T helper type 1 (Th1)-directed, cell-mediated crescentic glomerulonephritis (GN) is still unclear. Therefore, we investigated the activity and role of IDO in crescentic GN using a model of nephrotoxic serum nephritis (NTN), and IDO activity was inhibited by 1-methyl-tryptophan (1-MT) in vivo. Our results showed that activity of IDO, as determined by high performance liquid chromatography analysis of the kynurenine/tryptophan ratio, was increased markedly in the serum and renal tissue of NTN mice, and immunohistochemistry revealed that expression of IDO was up-regulated significantly in glomeruli and renal tubular epithelial cells during NTN. Treatment with 1-MT resulted in significantly exacerbated kidney disease with increased glomerular crescent formation, accumulation of CD4(+)T cells and macrophages in renal tissue, and augmented renal injury compared with phosphate-buffered saline-treated NTN mice, which was associated with enhanced Th1 responses and intrarenal cellular proliferation. These findings suggest that the development of NTN was regulated negatively by increased IDO activity, and IDO might play an important role in the pathogenesis of crescentic GN.

Fig. 1

High performance liquid chromatography analysis of indoleamine 2, 3-dioxygenase (IDO) activity. Kyurinine/tryptophan (kyn/trp) ratio in the serum and kidney of non-immunized normal mice and nephrotoxic serum nephritis (NTN) mice treated with 1-methyl-tryptophan (1-MT) or phosphate-buffered saline. Non-immunized mice (n = 15) had a low-level (kyn/trp) ratio (µmol/mmol), but IDO activity was increased significantly in the serum (a) and kidney (b) of NTN mice (n = 15) at the end of the experiments (day 19), and 1-MT blockade (n = 15) effectively decreased the IDO activity during NTN. *P < 0·01 compared with non-immunized normal mice; **P < 0·01 compared with 1-MT treated NTN mice.

Fig. 3

Renal injury, as assessed by percentage of crescent formation (a), proteinuria (b) and serum creatinine (c) in 1-methyl-tryptophan (1-MT) treated nephrotoxic serum nephritis (NTN) mice (n = 15) and phosphate-buffered saline (PBS)-treated NTN mice (n = 15). Percentage of crescent formation and serum creatinine on day 19 and urinary protein excretion during the last 24 h of experiments were both increased prominently in the 1-MT treated NTN mice (*P < 0·01, n = 15) compared with PBS-treated NTN mice (n = 15).

Fig. 2

Renal histology of non-immunized mice and nephrotoxic serum nephritis (NTN) mice treated with 1-methyl-tryptophan (1-MT) or phosphate-buffered saline (PBS) on day 19. Representative glomeruli are shown from the three groups of mice; the non-immunized mice did not develop crescentic glomerulonephritis (GN) (a), while the other mice groups did (b,c). Crescents were assessed by periodic acid-Schiff staining on paraffin-embedded kidney sections. PBS-treated NTN mice developed proliferative crescentic GN (b), which was exacerbated by 1-MT blockade (c) (magnification: ×400).

Fig. 4

Intrarenal accumulation of CD4⁺ T cells and macrophages 1-methyl-tryptophan-treated nephrotoxic serum nephritis (NTN) mice (n = 15) and phosphate-buffered saline-treated NTN mice (n = 15) on day 19.

Fig. 5

Immunohistochemical staining for indoleamine 2, 3-dioxygenase (IDO). Non-immunized normal mice showed no IDO expression (a). However, the expression of IDO was up-regulated strongly in glomeruli of nephrotoxic serum nephritis mice, and the morphology of IDO-positive cells was similar to the infiltrated macrophages (b); IDO expression was also found in tubular epithelial cells (magnification: ×400).

Fig. 6

Intrarenal cells proliferation in 1-methyl-tryptophan (1-MT)-treated nephrotoxic serum nephritis (NTN) mice (n = 15) and phosphate-buffered saline-treated NTN mice (n = 15). Proliferating cells were detected in renal tissue by immunohistochemistry on paraffin-embedded kidney section using rat anti-mouse proliferating cell nuclear antigen monoclonal antibody. 1-MT treatment during NTN increased cellular proliferation significantly within diseased glomeruli (a, *P < 0·01, cells/glomerulus) and interstitial (b, *P < 0·01, cells/high-power field).

Fig. 7

Effect of 1-methyl-tryptophan (1-MT) blockade on splenocyte cytokine production in vitro. Cytokine levels were measured by enzyme-linked immunosorbent assay on supernatants that were collected from cultured splenocytes. Two control groups had low levels of interferon (IFN)-γ and interleukin (IL)-4, but levels of those cytokines were both increased significantly after antigen was restimulated (a,b, *P < 0·01 compared with control groups; n = 15). After antigen was restimulated, the production of IFN-γ (a) by splenocytes from 1-MT treated nephrotoxic serum nephritis (NTN) mice was increased prominently compared with phosphate-buffered saline (PBS)-treated NTN mice (**P < 0·01; n = 15). However, the production of IL-4 (b) by splenocytes from 1-MT-treated NTN mice was decreased markedly compared with PBS-treated NTN mice (**P < 0·01; n = 15).

Fig. 8

Circulating antigen-specific immunoglobulin (Ig)G1 and IgG2a levels in non-immunized normal mice (n = 15) and nephrotoxic serum nephritis (NTN) mice treated with 1-methyl-tryptophan (1-MT) (n = 15) or phosphate-buffered saline (PBS) (n = 15). Serum antigen-specific Ig subclass levels were measured by enzyme-linked immunosorbent assay and tested at five dilutions (1:100 to 1:1600) at the end of the experiments. Baseline represents Ig subclass levels in non-immunized normal C57BL/6 mice. (a)IgG1 levels were reduced significantly in 1-MT-treated NTN mice compared with PBS-treated NTN mice (P < 0·01 at all dilutions; while (b) IgG2a levels rose markedly in 1-MT treated NTN mice compared with PBS-treated NTN mice (P < 0·01 at all dilutions).