Interleukin-18 binding protein transgenic mice are protected against ischemic acute kidney injury

Abstract

IL-18 function is neutralized in IL-18 binding protein transgenic (IL-18BP Tg) mice. First, we determined whether IL-18BP Tg mice are protected against ischemic acute kidney injury (AKI). Ischemic AKI was induced by bilateral renal pedicle clamping. IL-18BP Tg mice were functionally and histologically protected against ischemic AKI as determined by blood urea nitrogen, serum creatinine, and acute tubular necrosis score. We have demonstrated that the injurious effect of IL-18 in the kidney is independent of neutrophils and lymphocytes. Thus the effect of IL-18 inhibition on renal macrophage infiltration was determined. The number of macrophages was significantly reduced in IL-18BP Tg compared with wild-type kidneys. To determine the cytokines and chemokines that are dependent on IL-18, we performed flow cytometry based assays. Multiple chemokines/cytokines, IL-3, IL-6, IL-15, IL-18, leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2, granulocyte-macrophage colony-stimulating factor, and monocyte chemotactic protein-1 were significantly increased in AKI vs. sham kidneys. Only CXCL1 (also known as KC or IL-8) was significantly increased in AKI vs. sham kidneys and significantly reduced in IL-18BP Tg AKI vs. wild-type AKI kidneys. To determine whether macrophages are the source of CXCL1 in the kidney, we depleted macrophages with liposomal encapsulated clodronate. CXCL1 was significantly decreased in macrophage-depleted vs. control AKI mice. In summary, in ischemic AKI in mice, 1) IL-18BP Tg mice are functionally and histologically protected, 2) macrophage infiltration in the kidney and CXCL1 are significantly reduced in IL-18BP Tg mice, and 3) macrophage depletion significantly reduces CXCL1 in the kidney. In conclusion, protection against ischemic AKI in IL-18BP Tg mice is associated with less macrophage infiltration and less production of CXCL1 in the kidney.

Fig. 1.

Demonstration of human IL-18 binding protein isoform a (hIL-18BPa) in kidney of IL-18BP transgenic (Tg) mice. A: on RT-PCR analysis, human IL-18BPa mRNA (295 bp) was present in kidneys from IL-18BP Tg mice but not from wild-type (WT) mice. GAPDH (452 bp) was included as an internal control. B: on immunoblot analysis, hIL-18BPa protein (40 kDa) was present in kidneys from IL-18BP Tg mice but not in kidneys from WT mice.

Fig. 2.

Renal function. IL-18BP Tg mice were functionally protected against ischemic acute kidney injury (AKI). IL-18 BP Tg mice with AKI had a significantly lower serum creatinine (A) and blood urea nitrogen (BUN; B) than WT with AKI. *P < 0.001 vs. sham. **P < 0.01 vs. WT AKI.

Fig. 3.

Acute tubular necrosis (ATN) score. IL-18BP Tg mice were histologically protected against ischemic AKI. IL-18 BP Tg mice with AKI had a significantly lower ATN score than WT mice with AKI (A). *P < 0.001 vs. sham. **P < 0.05 vs. WT AKI. Representative images of the renal histology of WT AKI mice (B) and IL-18 BP Tg AKI mice (C) are shown. No tubular necrosis was seen in sham-operated mice. In WT AKI mice most of the renal tubules were necrotic (B; arrows) compared with less tubular necrosis in IL-18 BP Tg AKI mice (C; arrows).

Fig. 4.

Macrophage infiltration. The number of CD11b+ cells in the outer stripe of the outer medulla in ischemic AKI was significantly reduced in IL-18BP Tg kidneys compared with WT kidneys (A). *P < 0.001 vs. sham. **P < 0.01 vs. WT AKI. Representative images of CD11b-positive staining in macrophages (arrows) in sham-operated mice (B), WT mice with AKI (C), and IL-18BP Tg mice with AKI (D) are shown.

Fig. 5.

Effect of macrophage depletion on CXCL1 (KC). To determine whether macrophages are the source of CXCL1 in ischemic AKI, we measured CXCL1 in the kidney in macrophage-depleted mice using 2 bead-based multiplex cytokine kits. The increase in CXCL1 in ischemic AKI was virtually completely inhibited by macrophage depletion with liposomal encapsulated clodronate (LEC). *P < 0.01 vs. sham. **P < 0.05 vs. AKI + vehicle (empty liposomes).

Fig. 6.

Immunofluorescence staining for CXCL1 in mouse kidneys. CXCL1 staining (green) is demonstrated in macrophages in the outer stripe of the outer medulla (A; arrows) as well as in a proximal tubule (T) (B; arrows). No glomerular staining was seen. Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue).