Remote effects of lower limb ischemia-reperfusion: impaired lung, unchanged liver, and stimulated kidney oxidative capacities

Abstract

Remote organ impairments are frequent and increase patient morbidity and mortality after lower limb ischemia-reperfusion (IR). We challenged the hypothesis that lower limb IR might also impair lung, renal, and liver mitochondrial respiration. Two-hour tourniquet-induced ischemia was performed on both hindlimbs, followed by a two-hour reperfusion period in C57BL6 mice. Lungs, liver and kidneys maximal mitochondrial respiration (V(max)), complexes II, III, and IV activity (V(succ)), and complex IV activity (V(TMPD)) were analyzed on isolated mitochondria. Lower limb IR decreased significantly lung V(max) (29.4 ± 3.3 versus 24 ± 3.7 μmol O2/min/g dry weight, resp.; P = 0.042) and tended to reduce V(succ) and V(TMPD). IR did not modify liver but increased kidneys mitochondrial respiration (79.5 ± 19.9 versus 108.6 ± 21.4, P = 0.035, and 126 ± 13.4 versus 142.4 ± 10.4 μmol O2/min/g dry weight for V(max) and V(succ), resp.). Kidneys mitochondrial coupling was increased after IR (6.5 ± 1.3 versus 8.8 ± 1.1, P = 0.008). There were no histological changes in liver and kidneys. Thus, lung mitochondrial dysfunction appears as a new early marker of hindlimb IR injuries in mice. Further studies will be useful to determine whether enhanced kidneys mitochondrial function allows postponing kidney impairment in lower limb IR setting.

Figure 1

(a) Experimental design. C: control; IR: ischemia-reperfusion. (b) Schematic representation of the mitochondrial respiratory chain with specific substrates and inhibitors. C_I: complex I (NADH-CoQ reductase), C_II: complex II (succinate-CoQ reductase), C_III: complex III (CoQH2-c reductase), C_IV: complex IV (cytochrome c oxidase, COX), and TMPD: N, N, N′, N′-tetramethyl-p-phenylenediamine dihydrochloride. Schematic oxygraph trace showing oxygen consumption by the permeabilized skeletal myofibers, using indicated substrates and inhibitors: V ₀, before ADP; V _max⁡, complexes I, III, and IV activities, using glutamate and malate; V _succ, complexes II, III, and IV activities, using succinate; V _TMPD/asc, complex IV activity using TMPD/Ascorbate.

Figure 2

Effects of lower limb ischemia/reperfusion on lung mitochondrial respiratory chain complexes activities. (A) V _max⁡, complexes I, III, and IV activities, using glutamate and malate; (B) V _succ, complexes II, III, and IV activities, using succinate; (C) V _TMPD/asc, complex IV activity using TMPD/Ascorbate, as mitochondrial substrates. Data are presented in Sham and after ischemia-reperfusion (IR). Results are expressed as means ± SEM; *P < 0.05.

Figure 3

Effects of lower limb ischemia/reperfusion on liver mitochondrial respiratory chain complexes activities. (A) V _max⁡, complexes I, III, and IV activities, using glutamate and malate. (B) V _succ, complexes II, III, and IV activities, using succinate. (C) V _TMPD/asc, complex IV activity using TMPD/Ascorbate, as mitochondrial substrates. Data are presented in Sham and after ischemia-reperfusion (IR). Results are expressed as means ± SEM.

Figure 4

Effects of lower limb ischemia/reperfusion on kidney mitochondrial respiratory chain complexes activities. (A) V _max⁡, complexes I, III, and IV activities, using glutamate and malate. (B) V _succ, complexes II, III, and IV activities, using succinate. (C) V _TMPD/asc, complex IV activity using TMPD/Ascorbate, as mitochondrial substrates. Data are presented in Sham and after ischemia-reperfusion (IR). Results are expressed as means ± SEM; *P < 0.05.

Figure 5

Effects of lower limb ischemia/reperfusion on lung, liver, and kidney mitochondrial coupling. ACR: acceptor complex ration (V _max⁡/V ₀). Data are presented in Sham and after ischemia-reperfusion (IR). Results are expressed as means ± SEM; *P < 0.05.