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. 2022 Nov 8;7(21):e163161. doi: 10.1172/jci.insight.163161

Figure 3. Glutamine administration functionally attenuates renal tubular cell apoptosis.

Figure 3

WT mice were subjected to sham or IRI surgery and received glutamine or saline. Paraffin-embedded tissue sections were prepared and TUNEL staining was performed (A, TUNEL-positive cells [%]; B, representative TUNEL-stained cortex and medulla tissue) (n = 4; scale bar: 50 μm). Caspase-3 activity was detected in kidney lysates (C, n = 4). Western blotting was performed to assess the expression level of HSP70 (D and E), p-JNK (D and F), 14-3-3ζ and p–14‑3‑3ζ (Thr232) (D and G), Bad and p-Bad (Ser136) (D and H), caspase-3 (D and I), as well as apoptosis signal-regulating kinase (Ask1) and p-Ask1 (D and J). TECs were transfected with Ask1 siRNA and control siRNA using the Lipofectamine RNAiMAX Reagent. Knockdown efficiency determined by Western blot analysis was ~10% protein expression. Transfected TECs were treated with glutamine or saline, then subjected to hypoxia. TEC lysates were analyzed by Western blotting to assess the expression levels of HSP70 (K and L, n = 3) and caspase-3 (K and M, n = 3). MALDI imaging mass spectrometry (MALDI-IMS) of kidney sections was performed to analyze protein distribution of HSP70, 14-3-3ζ, and Bad (N). The scale represents the relative intensity of the protein (m/z, mass-to-charge ratio). Mean ± SEM, 1-way ANOVA *P < 0.05; **P < 0.005; ***P < 0.001.