Korean J Physiol Pharmacol.  2023 May;27(3):257-265. 10.4196/kjpp.2023.27.3.257.

Rac1 inhibition protects the kidney against kidney ischemia/ reperfusion through the inhibition of macrophage migration

• Park YR
• Kong MJ
• Noh MR
• Park KM

• KMID: 2541637
• DOI: http://doi.org/10.4196/kjpp.2023.27.3.257

Abstract

Kidney ischemia/reperfusion (I/R) injury, a common cause of acute kidney injury (AKI), is associated with the migration of inflammatory cells into the kidney. Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of the Rho family of small GTPase, plays an important role in inflammatory cell migration by cytoskeleton rearrangement. Here, we investigated the role of Rac1 on kidney I/R injury and macrophage migration. Male mice were subjected to either 25 min of bilateral ischemia followed by reperfusion (I/R) or a sham operation. Some mice were administrated with either NSC23766, an inhibitor of Rac1, or 0.9% NaCl (vehicle). Kidney damage and Rac1 activity and expression were measured. The migration and lamellipodia formation of RAW264.7 cells, mouse monocyte/macrophage, induced by monocyte chemoattractant protein-1 (MCP-1, a chemokine) were determined using transwell migration assay and phalloidin staining, respectively. In sham-operated kidneys, Rac1 was expressed in tubular cells and interstitial cells. In I/R-injured kidneys, Rac1 expression was decreased in tubule cells in correlation with the damage of tubular cells, whereas Rac1 expression increased in the interstitium in correlation with an increased population of F4/80 cells, monocytes/macrophages. I/R increased Rac1 activity without changing total Rac1 expression in the whole kidney lysates. NSC23766 administration blocked Rac1 activation and protected the kidney against I/R-induced kidney damage and interstitial F4/80 cell increase. NSC23766 suppressed monocyte MCP-1-induced lamellipodia and filopodia formation and migration of RAW 264.7 cells. These results indicate Rac1 inhibition protects the kidney against I/R via inhibition of monocytes/macrophages migration into the kidney.

Keyword

Acute kidney injury; Ischemia; Macrophages; Macrophage migration; Rac1-GTPase

Figure

• 

  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.

• 

  Fig. 2 Effect of NSC23766 on Rac1 activity in kidneys after I/R. Mice were subjected to either 25 min of ischemia or sham operation. Some mice were administered either NSC23766 (10 mg/kg body weight) or 0.9% NaCl (vehicle) daily beginning 3 days before the operation. Kidneys were harvested 24 h after I/R. (A) Kidneys were subjected to Rac1-GTP pulldown assay as described in the Methods. (B) The densities of bands were measured using ImageJ software. Results are expressed as mean ± SEM (n = 4). *p < 0.05. Rac1, Ras-related C3 b otulinum toxin substrate; I/R, ischemia/reperfusion; ns, no significance; N, negative control; Isch, ischemia.

• 

  Fig. 3 Effect of Rac1 inhibition on kidney I/R. Mice were subjected to either 25 min of ischemia or sham operation. Some mice were administered either NSC23766 (10 mg/kg body weight) or 0.9% NaCl (vehicle) daily beginning 3 days before the operation. Kidneys and blood were harvested 4 and 24 h after the operation. (A-D) Twenty-four hours after the operation, kidney sections were subjected to immunohistochemical staining using anti-F4/80 (A, B) and -Rac1 (C, D) antibodies. Hematoxylin was used to visualize the nuclei of cells. Insert is at high magnification of the dash-lined rectangle. Arrows indicate F4/80- or Rac1-positive cells. Images were obtained from the outer medulla. F4/80-positive cells were counted in the outer medulla. Scale bar = 50 µm. (E, F) Kidney sections taken 24 h after the operation were stained with Periodic Acid-Schiff (PAS) and damage was scored. Scale bar = 50 µm. (G) Plasma creatinine (PCr) levels were measured at indicated times. Results are expressed as mean ± SEM (n = 4–5). *p < 0.05. Rac1, Ras-related C3 botulinum toxin substrate 1; I/R, ischemia/reperfusion; OM, outer medulla; ns, no significance; Isch, ischemia; NSC, NSC23766; V, vehicle.

• 

  Fig. 4 Effect of Rac1 inhibition on the MCP-1-induced migration and lamellipodia and filopodia formation of Raw264.7 cells. (A, B) RAW264.7 cells grown on transwell filter chambers were treated with 100 µM NSC23766 for 1 h and then with 2 nM MCP-1 for 4 h. Migrated cells were H&E-stained after removing the non-migrated cells. Scale bar = 20 µm. (B) Migrated cells were counted. Blank arrowheads indicate migrated cells. (C) RAW264.7 cells grown on slide glass were treated for 1 h with 100 µM NSC23766 and then with 2 nM MCP-1 for 4 h. Cells were subjected to phalloidin (F-actin, red) staining. White and green arrowheads indicate filopodia and lamellipodia, respectively. DAPI was used to visualize the nucleus (blue). Scale bar = 10 µm. Results are expressed as mean ± SEM (n = 3). *p < 0.05. Rac1, Ras-related C3 botulinum toxin substrate 1; MCP-1, monocyte che moattractant protein-1.

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