Abstract
► Bilateral renal ischemia/reperfusion (IR) induced kidney injury in mice. ► IR activated Toll-like receptors (TLRs). ► TLRs induced inflammatory cytokine production. ► Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) modulated TLRs. ► PACAP38 protected kidney even when treatment was started 24h after IR.
We investigated whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) ameliorates kidney injury after ischemia/reperfusion (IR) by modulating Toll-like receptor (TLR)-associated signaling pathways. Male C57BL/6 mice were subjected to bilateral renal ischemia for 45min. PACAP38, 20μg in 100μl of saline, was administered i.p. at 24 and 48h after IR, and mice were euthanized at 72h. In IR mice, PACAP38 maintained serum creatinine near control levels (0.81±0.08 vs. 0.69±0.17mg/dl in controls, p=NS, vs. 1.8±0.03 in saline-treated IR mice, p<0.01) and significantly reduced the expression of kidney injury biomarkers. PACAP38 significantly reduced the levels of apoptosis and neutrophil infiltration, and protected against tubular damage. With PCR arrays, 59 of 83 TLR-related genes significantly changed their expression after IR. TLR2 increased 162 fold, followed by Fas-associated death domain (37 fold) and TLR6 (24 fold), while ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) decreased 55 fold. PACAP38 given 24 and 48h after IR injury significantly reversed these changes in 56 genes, including TLR2, TLR3, TLR4, TLR6, and genes in the NF-κB pathways. The alterations in TLR2, TLR3, TLR6, and UBE2V1 were confirmed by RT-PCR. After IR, PACAP38 also suppressed protein levels of TLR-associated cytokines. PACAP38 reversed the changes in IR-activated TLR-associated NF-κB signaling pathways even when treatment was delayed 24h. Therefore, PACAP38 could be an effective therapeutic for unexpected IR-mediated renal injury. The prominently IR-induced TLR-related genes identified in this study could be novel drug targets.