BAM15 treats mouse sepsis and kidney injury, linking mortality, mitochondrial DNA, tubule damage, and neutrophils
Naoko Tsuji, Takayuki Tsuji, Tetsushi Yamashita, Naoki Hayase, Xuzhen Hu, Peter S.T. Yuen, Robert A. Star
Naoko Tsuji, Takayuki Tsuji, Tetsushi Yamashita, Naoki Hayase, Xuzhen Hu, Peter S.T. Yuen, Robert A. Star
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Research Article Nephrology

BAM15 treats mouse sepsis and kidney injury, linking mortality, mitochondrial DNA, tubule damage, and neutrophils

Abstract

Sepsis pathogenesis is complex and heterogeneous; hence, a precision-medicine strategy is needed. Acute kidney injury (AKI) following sepsis portends higher mortality. Overproduction of mitochondrial ROS (mtROS) is a potential mediator of sepsis and sepsis-induced AKI. BAM15, a chemical uncoupler, dissipates mitochondrial proton gradients without generating mtROS. We injected BAM15 into mice at 0, 6, or 12 hours after cecal ligation and puncture (CLP), and these mice were treated with fluids and antibiotics. BAM15 reduced mortality, even after 12 hours, when mice were ill, and BAM15 reduced kidney damage and splenic apoptosis. Serial plasma and urinary mitochondrial DNA (mtDNA) levels increased after CLP and decreased after BAM15 administration (at 0 or 6 hours). In vitro septic serum proportionately increased mtROS overproduction and mtDNA release from kidney tubule cells, which BAM15 prevented. BAM15 decreased neutrophil apoptosis and mtDNA release; neutrophil depletion counteracted BAM15 benefits. Further, mtDNA injection in vivo replicated inflammation and kidney injury, which was prevented by BAM15. A large dose of exogenous mtDNA reversed protection by BAM15. We conclude that BAM15 is an effective preventive and therapeutic candidate in experimental sepsis and that BAM15 and mtDNA, a potential drug-companion diagnostic/drug-efficacy pair for clinical sepsis, are mechanistically linked via mtROS.

Authors

Naoko Tsuji, Takayuki Tsuji, Tetsushi Yamashita, Naoki Hayase, Xuzhen Hu, Peter S.T. Yuen, Robert A. Star

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Figure 6

BAM15 effect on kidney injury and systemic inflammation caused by mtDNA as DAMPs in vivo.

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BAM15 effect on kidney injury and systemic inflammation caused by mtDNA ...
(A) Circulating mtDNA levels after injection of mtDNA (400 ng, 2,000 ng, or 8,000 ng) into naive mice. n = 3–4 each. Data are represented as mean ± SEM. *P < 0.05, mtDNA, 8,000 ng versus control; #P < 0.05, mtDNA, 2,000 ng versus control; †P < 0.05, mtDNA, 400 ng versus control, unpaired t test. (B) IL-6 levels in serum at 3 hours after injection of mtDNA (400, 2,000, or 8,000 ng) into naive mice. n = 3–5 each. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Tukey’s multiple-comparison test following 1-way ANOVA. (C–I) BAM15 effect (5 mg/kg, i.p., at 0 hours) at 3 hours after injection of mtDNA (8,000 ng) into naive mice on serum IL-6 level (C), BUN (D), PAS staining (E and F), and scoring (G) of kidney cortex, and mtDNA level in plasma (H) and urine (I). Original magnification, ×400. n = 3–5 each. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001, Šidák’s multiple-comparison test following 1-way ANOVA. (J–L) Serum IL-6 levels (J), BUN (K), and PAS staining and scoring (L) in WT, TLR9-KO, cGAS-KO, and AIM2-KO mice at 3 hours after injection of mtDNA (8,000 ng). n = 4 each for IL-6 and PAS staining. n = 6 for BUN. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Šidák’s multiple-comparison test following 1-way ANOVA. (M) Study design for survival study of mtDNA (8,000 ng) injection at 0, 3, and 6 hours after CLP surgery. (N) Kaplan-Meier curves of CLP mice treated for 7 days with vehicle or BAM15 (5 mg/kg, at 0 hours) following injection of mtDNA (8,000 ng) or control buffer. n = 15 each. log-rank test. *P < 0.05, CLP+vehicle+control versus CLP+BAM15+control; #P < 0.05, CLP+BAM15+mtDNA versus CLP+BAM15+control buffer.