Figure 1.
Figure 1. NLRX1 deficiency increases tubular apoptosis during IRI. (A) Transcription analysis of NLRX1 in kidneys from WT animals obtained the indicated time points during IRI (n = 5 per time point). (B) Confocal microscopic imaging of kidney tissue from respectively control WT, ischemic (day 1) WT, and NLRX1 KO mice stained for NLRX1 (red), F actin (green) and nuclei (blue). (C) Agarose gel electrophoresis for NLRX1 transcription of WT and KO BM, total BM-derived macrophages (Mφ), and ex vivo differentiated M1 and M2 Mφ, granulocytes, primary cultured TECs and the proximal tubular epithelial IM-PTEC cell line, amplicon sizes: NLRX1, 90 bp and TBP, 89 bp. (D–J) Analysis of unilateral IRI outcome in WT mice (white bars, n = 8 per time point) and NLRX1 KO mice (black bars, n = 8 per time point). (D) Semiquantitative histological scoring of tubular necrosis of kidney sections; representative periodic acid-Schiff with digestion images are shown. (E) Plasma LDH levels expressed in units per liter. Immunohistochemistry was performed on kidney sections for (F) cleaved caspase-3, (G) Gr-1, and (I) F4/80; representative images are shown for each staining. Positive (F) TECs or (G) Gr-1 expressing cells were counted per high-power field (HPF), and (I) F4/80 expressing cells were quantified by digital image analysis and expressed as percentage positive staining per HPF. ELISA was performed on kidney homogenates for (H) CXCL1 and (J) CCL2. Chemokine levels are corrected for total protein contents of the homogenates. (K) Analysis of bilateral IRI outcome in WT mice (white bars, n = 2 in sham group, n = 8 in IRI group) and NLRX1 KO mice (black bars, n = 2 in sham group, n = 8 in IRI group). Plasma creatinine levels of mice subjected to bilateral injury and sacrificed at day 1. (L) Survival of WT (n = 8) and NLRX1 KO (n = 8) mice at day 5 after bilateral ischemia; white area represents live animals, black area represents dead animals. (M–O) Analysis of bilateral IRI outcome in BM chimeric animals: WT BM into WT recipients (WT → WT, white bars, n = 7), WT BM into KO recipients (WT → KO, black bars, n = 7), and KO BM into WT recipients (KO → WT, gray bars, n = 7). Analysis of (M) semiquantitative histological scoring of tubular necrosis of kidney sections, (N) plasma LDH levels, and (O) immunohistochemical detection of cleaved caspase-3 stained TECs per HPF. Bars, 50 µm (B); 100 µm (D, F, G, I, M, and O). All data are expressed as mean ± SEM and were analyzed using a Mann-Whitney U test (*, P < 0.05; **, P < 0.01). a.u., arbitrary units; contr, contralateral kidney; NS, not significant.

NLRX1 deficiency increases tubular apoptosis during IRI. (A) Transcription analysis of NLRX1 in kidneys from WT animals obtained the indicated time points during IRI (n = 5 per time point). (B) Confocal microscopic imaging of kidney tissue from respectively control WT, ischemic (day 1) WT, and NLRX1 KO mice stained for NLRX1 (red), F actin (green) and nuclei (blue). (C) Agarose gel electrophoresis for NLRX1 transcription of WT and KO BM, total BM-derived macrophages (Mφ), and ex vivo differentiated M1 and M2 Mφ, granulocytes, primary cultured TECs and the proximal tubular epithelial IM-PTEC cell line, amplicon sizes: NLRX1, 90 bp and TBP, 89 bp. (D–J) Analysis of unilateral IRI outcome in WT mice (white bars, n = 8 per time point) and NLRX1 KO mice (black bars, n = 8 per time point). (D) Semiquantitative histological scoring of tubular necrosis of kidney sections; representative periodic acid-Schiff with digestion images are shown. (E) Plasma LDH levels expressed in units per liter. Immunohistochemistry was performed on kidney sections for (F) cleaved caspase-3, (G) Gr-1, and (I) F4/80; representative images are shown for each staining. Positive (F) TECs or (G) Gr-1 expressing cells were counted per high-power field (HPF), and (I) F4/80 expressing cells were quantified by digital image analysis and expressed as percentage positive staining per HPF. ELISA was performed on kidney homogenates for (H) CXCL1 and (J) CCL2. Chemokine levels are corrected for total protein contents of the homogenates. (K) Analysis of bilateral IRI outcome in WT mice (white bars, n = 2 in sham group, n = 8 in IRI group) and NLRX1 KO mice (black bars, n = 2 in sham group, n = 8 in IRI group). Plasma creatinine levels of mice subjected to bilateral injury and sacrificed at day 1. (L) Survival of WT (n = 8) and NLRX1 KO (n = 8) mice at day 5 after bilateral ischemia; white area represents live animals, black area represents dead animals. (M–O) Analysis of bilateral IRI outcome in BM chimeric animals: WT BM into WT recipients (WT → WT, white bars, n = 7), WT BM into KO recipients (WT → KO, black bars, n = 7), and KO BM into WT recipients (KO → WT, gray bars, n = 7). Analysis of (M) semiquantitative histological scoring of tubular necrosis of kidney sections, (N) plasma LDH levels, and (O) immunohistochemical detection of cleaved caspase-3 stained TECs per HPF. Bars, 50 µm (B); 100 µm (D, F, G, I, M, and O). All data are expressed as mean ± SEM and were analyzed using a Mann-Whitney U test (*, P < 0.05; **, P < 0.01). a.u., arbitrary units; contr, contralateral kidney; NS, not significant.

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