(A) Western blot probing for IDO1 (left) and quantification (right) using WT and Pkd1^RC/RC kidney homogenates, highlighting upregulation of IDO1 in Pkd1^RC/RC kidneys compared with WT (n = 3 males/3 females, 9 months old). (B) Western blot probing for IDO1 (left) and quantification (right) of cell lysates obtained from normal renal cortical epithelial cells (RCTE, WT for PKD1) or 9-12 cells (null for PKD1) with and without IFN-γ stimulation, confirming overexpression of IDO1 in PKD-relevant human cell lines compared with control. IDO1 expression levels were further increased by the cytokine IFN-γ, which is known to be upregulated in PKD kidneys (n = 4 per condition). Data are presented as mean ± SEM. *P < 0.05 by unpaired t test (A) or 2-way ANOVA (Kruskal-Wallis P = 0.0373) with FDR Benjamini and Hochberg multiple-comparison test (B). Comparisons with nonsignificant statistics are not shown. (C) Western blot probing for IDO1 levels in epithelial cells obtained from individual cysts of ADPKD patients (each cyst was derived from a different patient; PKD genotype unknown). Most tested cysts showed high levels of IDO1 relative to IDO1 levels in RCTE or 9-12 cells (exposure time to detect IDO1 in RCTE or 9-12 cells was insufficient but IDO1 is expressed in these cell lines — see panel B). This provides direct clinical relevance for dysregulation of the tryptophan pathway in ADPKD patient kidneys. (D) Immunofluorescence images probing for IDO1 (red), E-cadherin (ECAD, green, epithelial cells), and DAPI (blue, nuclei). IDO1 is sparsely expressed in WT kidneys but upregulated in kidney cystic epithelial cells and interstitial cells of Pkd1^RC/RC kidneys. Ido1-knockout animals served as negative control. *Indicates IDO1-positive interstitial cells in WT or Pkd1^RC/RC;Ido1^+/+ kidneys; #1 and #2 indicate 2 different Pkd1^RC/RC;Ido1^+/+ animals. Scale bars: 50 μm.