Figure 6. Glutamine affects the expression of apoptosis- and oxidative stress–related proteins in renal TECs on the transcriptional and proteomic level.

TECs were identified in kidney homogenates by FACS based on the cell-specific marker prominin-1. RNA was isolated and sequenced. Hierarchical clustering heatmaps of RNA-Seq indicate differentially expressed genes (rows) between TECs of glutamine-treated mice after IRI and glutamine treatment compared with vehicle control (A, n = 3). Blue color indicates downregulation and red color indicates upregulation. Volcano plots generated to compare glutamine versus saline treatment after IRI induction identify 481 differently expressed genes in renal TECs (B). GO pathway enrichment analyses specify the fold enrichment of the transcriptome sequencing relative to the Mus musculus genome in TECs (C). GO terms representing molecular function are presented in red, cellular component in green, and biological processes in blue (Fisher’s exact test, P value < 0.05). TECs were isolated from murine kidneys and exposed to TNF-α for 18 hours or hypoxia for 24 hours. Additionally, TECs were treated with glutamine or saline as well as Tgm2 inhibitor ERW1041E or vehicle control, respectively. Caspase-3 activity was detected after TNF-α (n = 5) or hypoxia stimulation (n = 3, D), and Tgm2 levels were determined by Western blotting (E and F). Tgm2 activity in kidneys was assessed (G, n = 4). Immuno- and coimmunoprecipitated proteins were separated by SDS-PAGE and blotted using the indicated antibodies. Whole-cell lysate (INPUT) was used as protein control (H, n = 3). Mean ± SEM; 1-way ANOVA *P < 0.05; **P < 0.005; ***P < 0.001.