A TLR5 agonist inhibits acute renal ischemic failure

Abstract

Reperfusion of ischemic organs induces a potent inflammatory response initiated by the generation of reactive oxygen species that directly damage tissue and promote leukocyte infiltration and activation that also mediate tissue injury. We recently found that radiation-induced tissue injury, which is caused by radiation-induced reactive oxygen species, is attenuated by administration of CBLB502, a pharmacologically optimized derivative of the TLR5 agonist flagellin. Therefore, we tested the ability of CBLB502 to attenuate injury in a murine model of acute ischemic renal failure. CBLB502 given 30 min before imposition of bilateral renal pedicle occlusion provided marked protection against the renal dysfunction and inflammation that follows reperfusion of ischemic kidneys, including marked decreases in leukocyte infiltration, proinflammatory cytokine production, and tubular injury. Importantly, CBLB502 given within 30 min after ischemic kidney reperfusion reproduced the protective effects of pretreatment with the TLR5 agonist, indicating a window following reperfusion in which CBLB502 administration abrogates acute renal ischemic failure. Bone marrow-reconstituted chimeras were used to show that the protective effects of CBLB502 could be delivered by intact MyD88 signaling on renal parenchymal cells. Consistent with this, Ab staining of kidney sections indicated that cells lining the renal vasculature expressed TLR5. Overall, these results indicate the use of TLR5 agonists as mitigators and protectants of acute renal ischemic failure.

FIGURE 1

CBLB502 given prior to imposition of renal ischemia protects against acute renal ischemic injury. Groups of 5 C57BL/6 mice were subjected to bilateral occlusion of renal pedicles for 45 minutes with one group serving as the sham control. Groups of the mice were given PBS or the indicated dose of CBLB502 i.v. 30 minutes before imposing renal ischemia. (a) The viability of the animals was monitored following reperfusion. (b) The serum creatinine levels at 24 hours post-reperfusion was determined. *P < 0.05

FIGURE 2

CBLB502 attenuates leukocytic infiltration and tubular necrosis when given prior to imposition of renal ischemia. Groups of animals were given PBS or 0.5 μg CBLB502 i.v. 30 minutes prior to imposition of bilateral renal pedicle occlusion. A group of animals was also given 0.5 μg CBLB502 and then sham operated. After 45 minutes, the ischemic kidneys were reperfused and were harvested 7 days later. Prepared sections were stained with hematoxylin and representative sections are shown. Magnification, x100.

FIGURE 3

CBLB502 given prior to imposition of renal ischemia attenuates neutrophil infiltration into ischemic kidneys during reperfusion. Groups of C57BL/6 mice were given PBS or 0.5 μg CBLB502 i.v. 30 minutes prior to imposition of bilateral renal pedicle occlusion. A group of animals was also given PBS and then sham operated. After 45 minutes, the ischemic kidneys were reperfused and were harvested 9 and 24 hours later. Prepared sections were stained with anti-Gr1 mAb to detect neutrophils and representative sections are shown. Magnification, x200.

Figure 4

CBLB502 given prior to imposition of renal ischemia attenuates neutrophil but not macrophage infiltration into ischemic kidneys during reperfusion. Groups of 5 C57BL/6 mice were given PBS or 0.5 μg CBLB502 i.v. 30 minutes prior to imposition of bilateral renal pedicle occlusion for 45 minutes. (a) Kidneys were harvested 24 hours after reperfusion from each animal, were digested, and the infiltrating leukocyte populations were quantitated following antibody staining and flow cytometry analyses to detect neutrophils, macrophages, CD4 T cells and CD8 T cells. (b) Kidneys were harvested 9 and 24 hours after reperfusion and prepared tissue homogenates were tested for levels of myeloperoxidase protein. *P < 0.05

FIGURE 5

CBLB502 given prior to imposition of renal ischemia attenuates production of neutrophil chemoattractant chemokines during reperfusion. Groups of 5 C57BL/6 mice were given PBS or 0.5 μg CBLB502 i.v. 30 minutes prior to imposition of bilateral renal pedicle occlusion or sham operation. After 45 minutes, the ischemic kidneys were reperfused and were harvested 9 and 24 hours later and tissue homogenates prepared. In the upper panels, whole cell RNA was isolated and tested by qRT/PCR for expression levels of neutrophil (CXCL1 and CXCL2) and macrophage (CCL2) chemoattractant chemokines. In the lower panels, aliquots of tissue homogenates were tested for levels of chemokine protein by ELISA.

Figure 6

Effect of CBLB502 given prior to imposition of renal ischemia on expression of acute phase cytokines during reperfusion. Groups of 5 C57BL/6 mice were given PBS or 0.5μg CBLB502 i.v. 30 minutes prior to imposition of bilateral renal pedicle occlusion for 45 minutes. Whole cell RNA was isolated from individual kidneys 1, 9 and 24 hours post-reperfusion and tested by qRT/PCR for expression levels of the acute phase cytokines TNFα, IL-1βand IL-6. *P < 0.05.

FIGURE 7

CBLB502 given after initiation of reperfusion of ischemic kidneys protects against acute renal ischemic injury. Groups of 5 C57BL/6 mice were subjected to bilateral occlusion of renal pedicles for 45 minutes with one group serving as the sham control. Groups of the mice were given PBS or 0.5 μg of CBLB502 i.v. at the indicated time before or after reperfusion. (a) The viability of the animals was monitored following reperfusion. (b) The serum creatinine levels at 24 hours post-reperfusion was determined.

Figure 8

CBLB502 given after initiation of reperfusion of ischemic kidneys attenuates the expression of inflammatory components. Groups of 5 C57BL/6 mice were subjected to bilateral occlusion of renal pedicles for 45 minutes with groups given PBS or 0.5 μg of CBLB502 i.v. 30 minutes after reperfusion. (a) Kidneys were harvested 9 and 24 hours after reperfusion and aliquots of tissue homogenates tested for levels of CXCL1 and CXCL2 protein by ELISA. *p < 0.05. (b) The ischemic kidneys from groups of 5 mice given PBS or 0.5 μg CBLB502 30 minutes after reperfusion were harvested 9 hours later and whole cell RNA was isolated and tested by qRT/PCR for expression levels of HO-1 and IL-10. **P < 0.01.

FIGURE 9

Reciprocal bone marrow reconstituted chimeras were generated between wild-type C57BL/6 and B6.MyD88^−/− mice. Six weeks after the bone marrow transplants, groups of 4 chimeras were subjected to bilateral occlusion of renal pedicles for 45 minutes with two groups serving as sham controls. Groups of the mice were given PBS or 0.5 μg of CBLB502 i.v. 30 minutes after reperfusion of the ischemic kidneys. The ischemic kidneys were harvested 9 hours after reperfusion, whole cell RNA was isolated, and expression levels of CXCL1 and CXCL2 were tested by qRT/PCR.

FIGURE 10

Expression of TLR5 in cells lining the renal vasculature. Kidney sections from C57BL/6, BALB/c and TLR5-deficient MOLF mice were stained to detect TLR5. Representative sections are shown. Magnification, x200.