Splenectomy exacerbates lung injury after ischemic acute kidney injury in mice

Abstract

Patients with acute kidney injury (AKI) have increased serum proinflammatory cytokines and an increased occurrence of respiratory complications. The aim of the present study was to examine the effect of renal and extrarenal cytokine production on AKI-mediated lung injury in mice. C57Bl/6 mice underwent sham surgery, splenectomy, ischemic AKI, or ischemic AKI with splenectomy and kidney, spleen, and liver cytokine mRNA, serum cytokines, and lung injury were examined. The proinflammatory cytokines IL-6, CXCL1, IL-1β, and TNF-α were increased in the kidney, spleen, and liver within 6 h of ischemic AKI. Since splenic proinflammatory cytokines were increased, we hypothesized that splenectomy would protect against AKI-mediated lung injury. On the contrary, splenectomy with AKI resulted in increased serum IL-6 and worse lung injury as judged by increased lung capillary leak, higher lung myeloperoxidase activity, and higher lung CXCL1 vs. AKI alone. Splenectomy itself was not associated with increased serum IL-6 or lung injury vs. sham. To investigate the mechanism of the increased proinflammatory response, splenic production of the anti-inflammatory cytokine IL-10 was determined and was markedly upregulated. To confirm that splenic IL-10 downregulates the proinflammatory response of AKI, IL-10 was administered to splenectomized mice with AKI, which reduced serum IL-6 and improved lung injury. Our data demonstrate that AKI in the absence of a counter anti-inflammatory response by splenic IL-10 production results in an exuberant proinflammatory response and lung injury.

Fig. 1.

mRNA expression of proinflammatory cytokines in the kidney, spleen, and liver after acute kidney injury (AKI). The mRNA expression of kidney, spleen, and liver IL-6, CXCL1, IL-1β, and TNF-α was measured by QPCR at baseline and at 2, 4, and 6 h after AKI or sham operation in C57/B6 mice. A: IL-6, CXCL1, and IL-1β mRNA expressions were highly upregulated in the kidney only 2 h after AKI vs. sham-operated mice. IL-6, CXCL1, and IL-1β mRNA expressions were elevated after AKI vs. sham operation in the spleen (B) and liver (C), 2 and 6 h post-AKI. Changes in TNF-α mRNA expression were found at 2 h post-AKI in the liver and 6 h post-AKI in the kidney and spleen. All the graphs are expressed as arbitrary units corrected by the loading control β-actin. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 3–5).

Fig. 2.

Lung inflammation and lung capillary leak 4 h after AKI plus splenectomy. Lung myeloperoxidase (MPO) activity, lung CXCL1, and lung capillary leak were assessed 4 h after sham operation (Sham), splenectomy (Splnx), ischemic AKI (AKI), and ischemic AKI plus Splnx (AKI+Splnx). A: MPO activity significantly increased in AKI compared with sham or Splnx and was further increased in AKI plus Splnx compared with AKI alone. B: lung CXCL1 increased in AKI compared with sham or Splnx and was further increased in AKI plus Splnx compared with AKI alone. C: capillary leak, as assessed by extravasation of Evans blue dye (EBD) into the lungs, was increased in AKI plus Splnx, indicating increased capillary leak (n = 6–10).

Fig. 3.

Lung inflammation and lung capillary leak 24 h after AKI plus Splnx. Lung MPO activity, lung CXCL1, and lung capillary leak were assessed 24 h after sham operation (Sham), Splnx, ischemic AKI, and AKI+Splnx. A: lung MPO activity was increased after AKI plus Splnx compared with sham. B: lung CXCL1 was increased after AKI plus Splnx compared with sham, Splnx, and AKI. C: lung EBD content was increased after AKI plus Splnx compared with AKI alone (n = 5). (n = 6–10).

Fig. 4.

Serum IL-6, 4 and 24 h after AKI plus Splnx. Serum IL-6 was determined at 4 and 24 h after sham operation, Splnx, AKI, and AKI plus Splnx in wild-type mice. A: serum IL-6 was elevated after AKI compared with sham or Splnx (P < 0.05) and was further increased after AKI plus Splnx compared with AKI (P < 0.04). B: no significant difference was found in serum IL-6 after AKI compared with sham or Splnx, but a significant increase in serum IL-6 was observed after AKI plus Splnx compared with sham, Splnx, and AKI (P < 0.01; n = 8–10).

Fig. 5.

Renal function and IL-6 mRNA production in kidney and liver after AKI plus Splnx. Serum creatinine (A) and blood urea nitrogen (BUN; B) were measured at 4 and 24 h postprocedure and were elevated after AKI and AKI plus Splnx compared with sham or Splnx. No change was found in serum creatinine and BUN in AKI plus Splnx compared with AKI alone (n = 8–10). mRNA expression of kidney and liver IL-6 was measured by QPCR at 4 h after sham operation, Splnx, AKI, and AKI plus Splnx. No increase in IL-6 mRNA was found in the kidney (C) or in the liver (D) in mice with AKI plus Splnx compared with AKI alone. (n = 3–4).

Fig. 6.

Splenic, hepatic, and renal IL-10 mRNA production after AKI. Splenic, hepatic, and renal IL-10 mRNA was measured at baseline and at 2, 4, and 6 h after sham operation or ischemic AKI. A: splenic IL-10 mRNA was highly upregulated by 2 h after AKI compared with sham-operated mice. B: hepatic IL-10 was upregulated by 2 h but to a lesser extent than splenic IL-10. C: only basal levels were found in the kidney of mice after AKI or sham operation. *P < 0.05, ***P < 0.001 (n = 3–5).

Fig. 7.

Splenic IL-10 production is increased 4 h after AKI. Immunofluorescence in OCT-embedded spleens from wild-type mice was analyzed by confocal microscopy 4 h after sham operation or AKI. A: IL-10 (in green) colocalized (merge in yellow) with CD11b, CD4, and B220 but not with Ly6G (all in red) in the spleens of wild-type mice 4 h after AKI. IL-10-positive cells (in green) were found to be positive for F4/80, but they did not colocalize. CD11b is a monocyte/macrophage and neutrophil marker, F4/80 is a macrophage marker, Ly6G is a neutrophil marker, CD4 is a T cell marker, and B220 is a B cell marker. B: no IL-10 was found in the spleen of wild-type mice after sham operation (n = 5–6).

Fig. 8.

Macrophage programming to an M1 or M2 phenotype did not occur in the spleen 4 h after AKI. A: splenic macrophages were analyzed by flow cytometry 4 h after sham operation or AKI. Expressions of inducible nitric oxide synthase (iNOS; M1 marker) and CD206 (M2) were analyzed in F4/80-positive, CD11b-positive, and Ly6G-negative cells. No difference in iNOS or CD206 was found 4 h after AKI compared with sham-operated mice (n = 3–5). B: no difference in splenic iNOS protein expression was found by Western blot 4 h after AKI compared with sham operation (n = 4–6).

Fig. 9.

Administration of IL-10 ameliorates lung inflammation and reduces the levels of serum IL-6 after ischemic AKI plus Splnx. A: lung MPO activity was reduced 4 h after AKI plus Splnx in IL-10-treated compared with vehicle-treated mice (P < 0.05). B: lung CXCL1 was reduced 4 h after AKI plus Splnx in IL-10-treated compared with vehicle-treated mice (P < 0.03). C: serum IL-6 was reduced 4 h after AKI plus Splnx in IL-10-treated compared with vehicle-treated mice (P < 0.02; n = 6–10). D: IL-6 mRNA levels were measured in kidney and liver of splenectomized mice treated with recombinant murine IL-10 or vehicle. Administration of IL-10 blocked the increase in kidney (P < 0.03) and liver (P < 0.04) mRNA levels after AKI plus Splnx compared with vehicle-treated animals (n = 6–10).