Role of Mitochondrial DNA in Septic AKI via Toll-Like Receptor 9

Abstract

Toll-like receptor 9 (TLR9) contributes to the development of polymicrobial septic AKI. However, the mechanisms that activate the TLR9 pathway and cause kidney injury during sepsis remain unknown. To determine the role of mitochondrial DNA (mtDNA) in TLR9-associated septic AKI, we established a cecal ligation and puncture (CLP) model of sepsis in wild-type (WT) and Tlr9-knockout (Tlr9KO) mice. We evaluated systemic circulation and peritoneal cavity dynamics and immune response and tubular mitochondrial dysfunction to determine upstream and downstream effects on the TLR9 pathway, respectively. CLP increased mtDNA levels in the plasma and peritoneal cavity of WT and Tlr9KO mice in the early phase, but the increase in the peritoneal cavity was significantly higher in Tlr9KO mice than in WT mice. Concomitantly, leukocyte migration to the peritoneal cavity increased, and plasma cytokine production and splenic apoptosis decreased in Tlr9KO mice compared with WT mice. Furthermore, CLP-generated renal mitochondrial oxidative stress and mitochondrial vacuolization in the proximal tubules in the early phase were reversed in Tlr9KO mice. To elucidate the effects of mtDNA on immune response and kidney injury, we intravenously injected mice with mitochondrial debris (MTD), including substantial amounts of mtDNA. MTD caused an immune response similar to that induced by CLP, including upregulated levels of plasma IL-12, splenic apoptosis, and mitochondrial injury, but this effect was attenuated by Tlr9KO. Moreover, MTD-induced renal mitochondrial injury was abolished by DNase pretreatment. These findings suggest that mtDNA activates TLR9 and contributes to cytokine production, splenic apoptosis, and kidney injury during polymicrobial sepsis.

Keywords: Immunology and pathology; acute renal failure; mitochondria; renal injury.

Figure 1.

The systemic circulating mtDNA increased at 2, 6 and 24 hours after CLP of WT mice. (A–C). Plasma Nd1, CytB, and Cox3. *P<0.05 versus sham (n=8–14 per group). All bar graphs represent mean±SEM.

Figure 2.

Tlr9KO increased peritoneal but not circulating mtDNA in the early phase after CLP. (A–C) Plasma Nd1, CytB, and Cox3 (n=7–12 per CLP group; n=4–9 per sham group). (D–F) Nd1, CytB, and Cox3 in the peritoneal cavity. *P<0.05 versus each sham group; ^†P<0.05 versus WT (n=6–8 per CLP group; n=4–5 per sham group). All bar graphs represent mean±SEM.

Figure 3.

Tlr9KO promoted leukocyte migration to peritoneal cavity from 2 hours and reduced bacteremia at 24 hours after CLP. (A) Leukocytes in peritoneal cavity (n=4–6 per group). (B) Bacterial count in blood. (C) Bacterial count in peritoneal fluid (n=4–7 per group). *P<0.05 versus each sham group; ^†P<0.05 versus WT. All bar graphs represent mean±SEM.

Figure 4.

Tlr9KO attenuated splenic apoptosis from 2 to 24 hours and reduced plasma cytokines at 2 hours after CLP. (A and B) Active caspase 3- and TUNEL-positive cells by immunohistochemical staining (n=3 per group). (C–E) Plasma TNF-α, IFN-γ, and IL-12 (n=8–10 per group). *P<0.05 versus each sham group; ^†P<0.05 versus WT. HPF, high-power field. All bar graphs represent mean±SEM.

Figure 5.

Tlr9KO reduced renal dysfunction and histologic change at 24 hours after CLP. (A) BUN. (B) Plasma creatinine (n=8–12 per group). (C) Histologic features of the cortex; original magnification, ×400. (D) Tubular damage score in the cortex (n=4–8 per group). *P<0.05 versus each sham group; ^†P<0.05 versus WT. PAS, periodic acid-Schiff. All bar graphs represent mean±SEM.

Figure 6.

Tlr9KO reduced kidney ATP depletion, mitochondrial oxidative stress and disruption in tubules at 2 hours after CLP. (A) Representative images (original magnification, ×200) of MitoSOX fluorescence microscopy of the proximal tubules after sham or CLP in WT or Tlr9KO mice. (B and C) Electron microscope images of the proximal tubules in sham-operated WT and KO mice showing elongated mitochondria with densely packed cristae membranes. The proximal tubules in WT mice after CLP showing scattered and swollen mitochondria (arrows) with damaged cristae or without cristae (arrowheads). These findings are representative of four mice per group. (D) Kidney ATP (n=4–8 per group). *P<0.05 versus each sham group; ^†P<0.05 versus WT. All bar graphs represent mean±SEM.

Figure 7.

MTD caused slight but significant renal dysfunction and production of plasma IL-12 and TNFα. (A and B) BUN and plasma creatinine. (C–E) Plasma TNF-α, IFN-γ, and IL-12 (n=4–8 per group). *P<0.05 versus vehicle.

Figure 8.

Tlr9KO reduced splenic apoptosis and production of plasma IL-12 at 2 hours after MTD administration. (A and B) active caspase 3- and TUNEL-positive cells by immunohistochemistry (n=4 per group). (C–E) Plasma TNF-α, IFN-γ, and IL-12 (n=4–8 per group). *P<0.05 versus each vehicle group; ^†P<0.05 versus WT. HPF, high-power field. All bar graphs represent mean±SEM.

Figure 9.

MTD administration caused kidney ATP depletion, mitochondrial oxidative stress and disruption in tubules at 2 hours, which were reduced by Tlr9KO. (A) Representative images (original magnification, ×200) of MitoSOX fluorescence microscopy of the proximal tubules are shown. (B and C) Electron microscopy images of the proximal tubules after vehicle administration to WT or Tlr9KO mice, showing elongated mitochondria with densely packed cristae membranes. Proximal tubules in WT mice after MTD showed scattered and swollen mitochondria (arrows) with damaged cristae or without cristae (arrowheads). This mitochondrial damage was not observed in Tlr9KO mice after MTD administration. These findings are representative of four mice per group. (D) Kidney ATP (n=4–8 per group). *P<0.05 versus each vehicle group; ^†P<0.05 versus WT. All bar graphs represent mean±SEM.

Figure 10.

Pretreatment with DNase reduced production of plasma IL-12 and kidney ATP depletion, mitochondrial oxidative stress and disruption in tubules, which were caused by MTD. (A) Plasma IL-12. (B) Kidney ATP (n=4–8 per group). (C) Representative images (original magnification, ×200) of MitoSOX fluorescence microscopy of the proximal tubules. (D and E) Electron microscopy images of the proximal tubules after vehicle with DNase pretreatment. Proximal tubules after MTD showed scattered and swollen mitochondria (arrows) with damaged cristae or without cristae (arrowheads). This mitochondrial damage was hardly observed after administration of MTD with DNase pretreatment. These findings are representative of 4–6 mice per group. *P<0.05 versus vehicle; ^‡P<0.05 versus MTD. All bar graphs represent mean±SEM.