Macrophages mediate lung inflammation in a mouse model of ischemic acute kidney injury

Abstract

Serum IL-6 is increased in acute kidney injury (AKI) and inhibition of IL-6 reduces AKI-mediated lung inflammation. We hypothesized that circulating monocytes produce IL-6 and that alveolar macrophages mediate lung inflammation after AKI via chemokine (CXCL1) production. To investigate systemic and alveolar macrophages in lung injury after AKI, sham operation or 22 min of renal pedicle clamping (AKI) was performed in three experimental settings: 1) systemic macrophage depletion via diphtheria toxin (DT) injection to CD11b-DTR transgenic mice, 2) DT injection to wild-type mice, and 3) alveolar macrophage depletion via intratracheal (IT) liposome-encapsulated clodronate (LEC) administration to wild-type mice. In mice with AKI and systemic macrophage depletion (CD11b-DTR transgenic administered DT) vs. vehicle-treated AKI, blood monocytes and lung interstitial macrophages were reduced, renal function was similar, serum IL-6 was increased, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In wild-type mice with AKI administered DT vs. vehicle, serum IL-6 was increased. In mice with AKI and alveolar macrophage depletion (IT-LEC) vs. AKI with normal alveolar macrophage content, blood monocytes and lung interstitial macrophages were similar, alveolar macrophages were reduced, renal function was similar, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In conclusion, administration of DT in AKI is proinflammatory, limiting the use of the DTR-transgenic model to study systemic effects of AKI. Mice with AKI and either systemic mononuclear phagocyte depletion or alveolar macrophage depletion had reduced lung inflammation and lung CXCL1, but increased lung capillary leak; thus, mononuclear phagocytes mediate lung inflammation, but they protect against lung capillary leak after ischemic AKI. Since macrophage activation and chemokine production are key events in the development of acute lung injury (ALI), these data provide further evidence that AKI may cause ALI.

Fig. 1.

Blood monocytes in CD11b-DTR transgenic mice with ischemic acute kidney injury (AKI). CD11b-DTR transgenic mice were injected with intravenous diphtheria toxin (DT) or vehicle (Veh) 18 h before ischemic AKI. Blood monocytes as assessed by flow cytometry are completely depleted in CD11b-DTR transgenic mice administered DT (n = 4–5).

Fig. 2.

Serum creatinine, blood urea nitrogen (BUN), and serum IL-6 after ischemic AKI in CD11b-DTR transgenic mice. Serum creatinine, BUN, and serum IL-6 were determined 4 h after sham operation or ischemic AKI in vehicle-injected (Veh) or DT-injected (DT) CD11b-DTR transgenic mice. DT injection to CD11b-DTR transgenic mice results in systemic mononuclear phagocyte depletion (MØ−). Serum creatinine (A) and BUN (B) were similarly increased in ischemic AKI with mononuclear phagocytes (Veh) and with mononuclear phagocyte depletion (DT; n = 4–9). C: serum IL-6 was significantly increased in ischemic AKI with mononuclear phagocyte depletion (DT) vs. ischemic AKI with mononuclear phagocytes (Veh; n = 7–9).

Fig. 3.

Lung CXCL1, lung myeloperoxidase (MPO) activity, and lung histology in CD11b-DTR transgenic mice. Lung CXCL1 and lung MPO activity were determined 4 h after sham operation (Sham) or ischemic AKI (AKI) in vehicle-injected (Veh) or DT-injected (DT) CD11b-DTR transgenic mice. DT injection to CD11b-DTR transgenic mice results in MØ−. CXCL1 is a neutrophil chemokine; lung MPO activity is a marker of lung neutrophil infiltration. Lung CXCL1 (A) and lung MPO activity (B) were significantly reduced in mice with mononuclear phagocytes (Veh) vs. mononuclear phagocyte-depleted (DT) mice after either sham operation or ischemic AKI (n = 4–18). C: lung histology was characterized by septal edema and an increased cellular/neutrophil infiltration in AKI with mononuclear phagocytes (AKI + Veh) vs. AKI with mononuclear phagocyte depletion (AKI + DT).

Fig. 4.

Lung capillary leak in CD11b-DTR transgenic mice. Lung capillary leak was determined by bronchoalveolar lavage (BAL) fluid protein content and Evans blue dye extravasation in separate groups of mice 4 h after ischemic AKI (AKI) in Veh or DT CD11b-DTR transgenic mice. DT injection to CD11b-DTR transgenic mice results in MØ−. Increased BAL fluid protein content and increased lung Evans blue dye indicate increased lung capillary leak. BAL fluid protein content (A) and lung Evans blue dye extravasation (B) were significantly increased in mononuclear phagocyte-depleted (DT) mice with ischemic AKI vs. ischemic AKI with mononuclear phagocytes (Veh) (n = 3–7).

Fig. 5.

Serum creatinine, BUN, serum IL-6, lung CXCL1, and lung MPO in DT-injected wild-type mice. Serum creatinine, BUN, serum IL-6, lung CXCL1, and lung MPO activity were determined 4 h after sham operation (Sham) or ischemic AKI (AKI) in vehicle-injected (Veh) or DT-injected (DT) wild-type BALB/c mice. Serum creatinine (A) and BUN (B) were similarly increased in vehicle-injected and DT-injected mice with AKI vs. sham (n = 4–6). C: serum IL-6 was significantly increased in vehicle-injected AKI vs. sham and was significantly increased in DT-injected AKI vs. vehicle-injected AKI (n = 4–6). D: lung CXCL1 was significantly increased in vehicle-injected ischemic AKI vs. sham and was significantly increased in DT-injected AKI vs. vehicle-injected AKI (n = 4–6). E: lung MPO activity was similarly increased in vehicle-injected AKI and DT-injected AKI vs. vehicle-injected sham operation (n = 4).

Fig. 6.

Interstitial and alveolar macrophages in CD11b-DTR transgenic mice with AKI. Lung interstitial macrophages and alveolar macrophages were determined in lung digests and BAL fluid, respectively, by flow cytometry in vehicle-injected (Veh) or DT-injected CD11b-DTR transgenic mice 4 h after ischemic AKI. The percent (A) and total number (B) of lung interstitial macrophages were reduced in mice after DT injection to CD11b-DTR transgenic mice (n = 4–5). The percent (C) and total number (D) of alveolar macrophages are similar in Veh- and DT-injected mice with AKI (n = 14–16).

Fig. 7.

Lung interstitial and alveolar macrophages in wild-type mice administered intratracheal (IT) liposome-encapsulated clodronate (LEC) with AKI. Lung interstitial macrophages and alveolar macrophages were determined in lung digests and BAL fluid, respectively, by flow cytometry in IT vehicle-treated (Veh) or IT LEC-treated wild-type C57Bl/6 mice 4 h after ischemic AKI. The percent (A) and total number (B) of interstitial macrophages were similar in IT Veh- and IT LEC-treated mice (n = 10). The percent (C) and total number (D) of alveolar macrophages were reduced in IT LEC-treated mice (n = 10).

Fig. 8.

Blood monocytes in wild-type mice administered IT LEC with AKI. Blood monocytes were assessed 4 h after AKI in mice treated with IT vehicle or IT LEC. Blood monocytes as assessed by flow cytometry are similar in IT vehicle (3.7%) and IT LEC (4.4%); thus, IT LEC does not affect circulating monocytes (P = NS, n = 5).

Fig. 9.

Serum creatinine, BUN, and serum IL-6 in wild-type mice administered IT LEC. Serum creatinine, BUN, and serum IL-6 were determined 4 h after sham operation (Sham) or ischemic AKI (AKI) in IT vehicle-treated (Veh) (Alveolar MØ+) or IT LEC-treated (LEC) (Alveolar MØ−) wild-type mice. IT LEC administration specifically reduces alveolar macrophages. Serum creatinine (A), BUN (B), and serum IL-6 (C) were similarly increased in IT Veh- and IT LEC-treated mice with AKI vs. sham (n = 8–16).

Fig. 10.

Lung CXCL1, lung MPO activity, and lung histology in wild-type mice administered IT LEC. Lung CXCL1 and lung MPO activity were determined 4 h after sham operation (Sham) or ischemic AKI (AKI) in IT vehicle-treated (Veh) or IT LEC-treated (LEC) wild-type mice. IT LEC administration specifically reduces alveolar macrophages. CXCL1 is a neutrophil chemokine; lung MPO activity is a marker of lung neutrophil infiltration. Lung CXCL1 (A) and lung MPO activity (B) were significantly increased in IT-Veh with AKI vs. sham and were significantly decreased in IT-LEC with AKI vs. IT-Veh with AKI (n = 8–14). C: lung histology was characterized by septal edema and an increased cellular/neutrophil infiltration in AKI with alveolar macrophages (AKI + Veh) vs. alveolar macrophage depletion (LEC).

Fig. 11.

Lung capillary leak in wild-type mice administered IT LEC. Lung capillary leak was determined by BAL fluid protein content and Evans blue dye extravasation in separate groups of mice 4 h after ischemic AKI (AKI) in IT vehicle-treated (Veh) or IT LEC-treated (LEC) wild-type mice. IT LEC administration specifically reduces alveolar macrophages. BAL fluid protein content (A) and lung Evans blue dye extravasation (B) were significantly increased in alveolar macrophage-depleted (IT LEC) mice with ischemic AKI (n = 5–9).