Journal: American Journal of Physiology - Renal Physiology
Article Title: Cutaneous exposure to lewisite causes acute kidney injury by invoking DNA damage and autophagic response
doi: 10.1152/ajprenal.00277.2017
Figure Lengend Snippet: Cutaneous exposure to lewisite or PAO induces autophagy, DNA damage, and apoptosis responses in the kidney. A and B: immunohistochemical analysis showing enhanced nuclear staining of phosphorylated (p)-H2A.X (red arrows) in the tubules of the kidney tissue of lewisite-treated Ptch1+/−/SKH-1 mice or PAO-treated C57BL/6 mice compared with vehicle-treated controls. Bars = 25 µm. C and D: representative Western blot analysis shows enhanced phosphorylation-dependent activation of checkpoint kinase (CHK) 1 and H2A.X in the kidney tissue of lewisite (C)- and PAO (D)-treated mice compared with their respective control tissues. E and F: representative Western blot analysis of autophagy-related gene (ATG) 7 and LC3A/B-II in the kidney tissue lysates of lewisite-treated Ptch1+/−/SKH-1 mice (E) and beclin-1, ATG7, and LC3A/B-II in PAO-treated C57BL/6 mice (F) compared with their respective controls. G: transmission electron microscopy (TEM) analysis of kidney tissues of vehicle- and PAO-treated C57BL/6 mice (n = 3/group). Bars = 150 nm. Arrows indicate autophagosomes with damaged organelles and mitochondrial cristae loss. H and I: terminal deoxyribonucleotide-transferase-mediated dUTP nick-end labeling (TUNEL) staining of the kidney sections showing frequent presence of green TUNEL-positive cells in lewisite-treated (H, bars = 25 µm) and PAO-treated (I, bars = 12.5 µm) animals compared with vehicle-treated control animals. Apoptotic cells (green positive nuclei) were present in the tubules of the kidney. J: representative Western blot analysis showing induction of proapoptotic proteins, BAX and cleaved caspase-3, and reduced expression of anti-apoptotic BCL2 in the kidneys of lewisite-treated mice. β-Actin was used as a loading control. Western blot data shown in C, E, and J represent pooled kidney lysates for each group (n = 5, control vs. lewisite).
Article Snippet: The membrane was probed with various primary antibodies [kidney injury molecule-1 (KIM-1, 1:1,000; Abcam), BAX (1:1,000; Cell Signaling), BCL 2 (1:1,000; Cell Signaling), cleaved caspase-3 (1:1,000; Cell Signaling), phosphorylated (p)- checkpoint kinase (CHK)-1 (1:1,000; Cell Signaling), p-CHK-2 (1:1,000; Cell Signaling), total CHK1 (1:1,000; Cell Signaling), p-γH 2 A.X (1:1,000; Cell Signaling), beclin-1 (1:1,000; Cell Signaling), autophagy-related gene (ATG) 7 (1:1,000; Cell Signaling), ATG5 (1:1,000; Cell Signaling), LC3A/B (1:1,000; Cell Signaling), or β-actin (1:5,000; Sigma)] overnight at 4°C or for 2 h at room temperature.
Techniques: Immunohistochemical staining, Staining, Western Blot, Phospho-proteomics, Activation Assay, Control, Transmission Assay, Electron Microscopy, End Labeling, TUNEL Assay, Expressing
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