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Fig. 1. Rac1 expression and activity after kidney I/R injury. Mice were subjected to either 25 min of ischemia or sham operation. Kidneys and blood were harvested 4 or 24 h after reperfusion. (A) Normal whole kidney sections were subjected to immunohistochemical staining using an anti-Rac1 antibody. Hematoxylin staining was used to visualize the nuclei of cells. (B) Kidney sections were stained with Periodic Acid-Schiff (PAS) reagent as described in the Methods. Scale bar = 50 µm. (C, D) Kidney sections were subjected to immunohistochemical staining using an anti-F4/80 antibody. Hematoxylin was used to visualize the nuclei of cells. Images were obtained from the outer medulla. (D) F4/80-positive cells were counted in the outer medulla. Insert is at high magnification of the dash-lined rectangle. Arrowheads indicate F4/80-positive cells (brown). Scale bar = 50 µm. (E) Plasma creatinine (PCr) levels were measured. (F) Kidney sections were subjected to immunohistochemical staining using an anti-Rac1 antibody. Arrowheads indicate Rac1-positive cells in the interstitium. Scale bar = 50 µm. (G) Rac1-GTP activity was analyzed by GTP-bound Rac1 pulldown assay. GAPDH was used as a loading control. (H) Kidney serial sections were subjected to immunohistochemical staining using anti-F4/80 and -Rac1 antibodies, respectively. Arrowheads indicate Rac1-positive cells. Asterisk indicates the same tubule. Scale bar = 30 µm. Results are expressed as mean ± SEM (n = 4). *p < 0.05. Rac1, Ras-related C3 botulinum toxin substrate 1; I/R, ischemia/reperfusion; CD, collecting duct; DT, distal tubule; G, glomerulus; TL, thin limb; IM, inner medulla; OM, outer medulla; S1–2 PT, segment 1–2 in the proximal tubule; S3 PT, segment 3 in proximal tubule; N, negative control; Isch, ischemia.
Korean J Physiol Pharmacol 2023;27:257-265 https://doi.org/10.4196/kjpp.2023.27.3.257
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