IL-33 exacerbates acute kidney injury

Abstract

Inflammation contributes to the pathogenesis of acute kidney injury (AKI). IL-33 is a proinflammatory cytokine, but its role in AKI is unknown. Here we observed increased protein expression of full-length IL-33 in the kidney following induction of AKI with cisplatin. To determine whether IL-33 promotes injury, we administered soluble ST2 (sST2), a fusion protein that neutralizes IL-33 activity by acting as a decoy receptor. Compared with cisplatin-induced AKI in untreated mice, mice treated with sST2 had fewer CD4 T cells infiltrate the kidney, lower serum creatinine, and reduced acute tubular necrosis (ATN) and apoptosis. In contrast, administration of recombinant IL-33 (rIL-33) exacerbated cisplatin-induced AKI, measured by an increase in CD4 T cell infiltration, serum creatinine, ATN, and apoptosis; this did not occur in CD4-deficient mice, suggesting that CD4 T cells mediate the injurious effect of IL-33. Wildtype mice that received cisplatin and rIL-33 also had higher levels of the proinflammatory chemokine CXCL1, which CD T cells produce, in the kidney compared with CD4-deficient mice. Mice deficient in the CXCL1 receptor also had lower serum creatinine, ATN, and apoptosis than wildtype mice following cisplatin-induced AKI. Taken together, IL-33 promotes AKI through CD4 T cell-mediated production of CXCL1. These data suggest that inhibiting IL-33 or CXCL1 may have therapeutic potential in AKI.

Figure 1.

IL-33 in serum. Serum IL-33, measured by Enzyme-linked immunosorbent assay (ELISA), was significantly increased on day 2 of cisplatin-induced AKI compared with vehicle (Veh), days 1 and 3. *P < 0.01 versus vehicle (Veh), days 1 and 3.

Figure 2.

Immunofluorescence staining for IL-33 in vehicle-treated mouse kidneys. (A) Staining for lectin (green) and IL-33 (red) demonstrates IL-33 (red) in glomeruli and in the peritubular spaces. Staining for von Willebrand factor (red) and IL-33 (green) demonstrates colocalization of vWF and IL-33 (yellow-orange) in glomeruli and in peritubular capillaries (arrows) in cortex (B) and outer stripe of outer medulla (C). (D) Staining for vascular α-smooth muscle actin (green) and IL-33 (red) demonstrates IL-33 staining on the endothelial surface of blood vessels.

Figure 3.

Cisplatin increases IL-33. (A) On ELISA of whole kidney, IL-33 was increased on days 1, 2, and 3 of cisplatin-induced AKI. *P < 0.05 compared with vehicle-treated (Veh); **P < 0.01 versus Veh. (B) On immunoblot of whole kidney, there was an increase in both the full-length (34 kD) and cleaved (22 kD) IL-33 on days 2 and 3 of cisplatin-induced AKI. (C) On ELISA of nuclear extracts of whole kidney, IL-33 was increased on days 1, 2, and 3 of cisplatin-induced AKI. *P < 0.001 compared with vehicle-treated (Veh). (D) On immunoblot of nuclear extracts of whole kidney, there was an increase in the full-length (34 kD) IL-33 on day 1, 2, and 3 and cleaved (22 kD) IL-33 on day 3 of cisplatin-induced AKI. Representative immunoblots of at least three independent experiments are demonstrated. Equal protein loading was confirmed by staining the same membrane for actin (43 kD) or the nuclear protein Lamin A (69 kD).

Figure 4.

In cisplatin-treated endothelial cells, there is release of the full-length IL-33 into the medium. Microvascular endothelial cells were treated with 10 μM cisplatin that we have described to induce apoptosis and 50 μM cisplatin that produces necrosis. On immunoblot of the medium of the cisplatin-treated endothelial cells, there was an increase in the full-length (34 kD) IL-33 with 50 μM cisplatin compared with vehicle-treated. Pos = positive control: recombinant cleaved IL-33 (22 kD). *P < 0.05 versus vehicle (Veh) and Cisplatin 10 μM (Cis 10); n = 3 per group.

Figure 5.

sST2 injection protects against cisplatin-induced AKI. Injection of cisplatin plus sST2 (Cis+sST2) resulted in a significant decrease in (A) creatinine, (B) ATN score, and (C) number of apoptotic tubular cells compared with cisplatin plus vehicle for sST2 (Cis+Veh). *P < 0.001 versus Veh; **P < 0.01 versus Cis+Veh; NS versus Veh; n = 5 to 11 per group.

Figure 6.

IL-33 injection aggravates cisplatin-induced AKI. Injection of low-dose cisplatin (15 mg/kg) plus rIL-33 in wildtype mice (WT Cis + IL-33) resulted in a threefold increase in serum creatinine compared with cisplatin alone (WT Cis). The increase in (A) creatinine, (B) ATN score, and (C) apoptotic tubular cells with cisplatin plus rIL-33 in wildtype mice (WT Cis + IL-33) was not seen in cisplatin plus rIL-33 in CD4-T-cell-deficient mice (CD4−/− Cis +IL-33). *P < 0.001 versus WT Cis and CD4 −/− Cis+IL-33; n = 3 to 6 per group.

Figure 7.

Flow cytometry for CD4 T cells in kidney. The number of CD4 + cells as a percentage of total live cells in the kidney is demonstrated in panel A. In wildtype kidneys, CD4 T cells were increased with cisplatin (25 mg/kg)-treatment on days 1, 2, and 3 (D1, D2, and D3) compared with vehicle treatment alone (Veh). Injection of rIL-33 plus cisplatin (15 mg/kg; Cis 15 + IL-33) resulted in a significant increase in CD4 T cells in kidney compared with cisplatin (15 mg/kg; Cis 15) alone. CD4 T cells in the kidney were absent in CD4-deficient mice treated with cisplatin (15 mg/kg) plus rIL-33 (Cis 15 + IL-33 CD4 −/−) and significantly decreased in wildtype mice treated with cisplatin (25 mg/kg) plus sST2 injection (Cis 25+sST2). *P < 0.05 versus vehicle-treated (Veh); **P < 0.05 versus cisplatin 15 mg/kg (Cis 15) or cisplatin 25 mg/kg (Cis 25); n = 3 to 6 per group. Representative pictures of flow cytometry are demonstrated in panels B to I. In Figures B to I, CD4 T cells are represented by Quadrant 2 (Q2), which includes cells strongly positive for CD3 and CD4. B, vehicle-treated; C, cisplatin 25 mg/kg day 1; D, cisplatin 25 mg/kg day 2; E, cisplatin 25 mg/kg day 3; F, cisplatin 15 mg/kg; G, cisplatin 15 mg/kg plus rIL-33 injection; H, cisplatin 15 mg/kg plus rIL-33 injection in CD4-deficient mice; I = cisplatin 25 mg/kg plus sST2 injection.

Figure 8.

Depletion of CD4 T cells protects against cisplatin-induced AKI. CD4 −/− mice treated with cisplatin (CD4 −/− Cis) had a significantly lower (A) creatinine and (C) apoptosis score than wildtype mice treated with cisplatin (WT Cis). (B) The ATN score in CD4 −/− mice treated with cisplatin (CD4 −/− Cis) was lower than wildtype mice treated with cisplatin (WT Cis) but did not reach statistical significance. (D) Mice treated with GK1.5 antibody plus cisplatin (GK1.5 Cis) had a significantly lower creatinine than mice treated with cisplatin plus vehicle (Veh Cis). *P < 0.001 versus Veh; **P < 0.05 versus WTCis or VehCis; n = 4 to 8 per group.

Figure 9.

CXCR2 −/− mice are protected against cisplatin-induced AKI. (A) CXCL1 was increased in the kidney by day 2 after cisplatin (Cis day 2). *P < 0.01 versus vehicle (Veh) and day 1 after cisplatin (Cis day 1). (B) rIL-33 injection in wildtype mice (WT + IL-33) did not increase CXCL1 compared with vehicle injection in wildtype mice (WT Veh). CXCL1 was significantly higher in wildtype mice treated with low-dose cisplatin (15 mg/kg; WT Cis) compared with wildtype mice treated with the vehicle for cisplatin (WT Veh). CXCL1 was increased in wildtype mice treated with low-dose cisplatin (15 mg/kg) plus rIL-33 (WT Cis IL33) compared with wildtype mice treated with low-dose cisplatin (15 mg/kg) alone (WT Cis). The increase in CXCL1 seen in wildtype mice treated with low-dose cisplatin (WT Cis) and wildtype mice treated with low-dose cisplatin plus rIL-33 (WT Cis IL33) was not seen in CD4 T cell −/− mice treated with low-dose cisplatin (CD4 −/− Cis) or low-dose cisplatin plus rIL-33 (CD4 −/− Cis IL-33). **P < 0.001 versus all other groups; *P < 0.01 versus Veh. (C) The increase in CXCL1 in cisplatin-treated (25 mg/kg) wildtype mice AKI (WT Cis) was decreased in cisplatin-treated CD4 T cell −/− mice (CD4 −/− Cis). *P < 0.001 versus vehicle-treated (Veh) and CD4−/− Cis. (D–F) CXCR2 −/− mice (CR2 −/−) that lack the receptor for CXCL1 have significantly lower creatinine, ATN score, and apoptosis in tubular cells compared with cisplatin-treated wildtype mice (WT Cis).*P < 0.001 versus Veh; **P < 0.01 versus WT Cis; n = 4 per group.

Figure 10.

Time sequence of events in cisplatin-induced AKI. There is ATN and tubular apoptosis on day 2 and ATN and renal failure on day 3 of cisplatin-induced AKI. There is increased neutrophil infiltration on day 3 but not on days 1 and 2 of cisplatin-induced AKI. There is increased macrophage infiltration on day 2. In the present study, we demonstrate increased IL-33 in the kidney on days 1, 2, and 3; increased CD4 T cell infiltration in the kidney on days 1, 2, and 3; and increased CXCL1 in the kidney on days 2 and 3. The increase in IL-33 on day 1 precedes the ATN, tubular apoptosis, inflammation, and renal failure. Mφ, macrophage; BUN, blood urea nitrogen; SCr, serum creatinine.