Targeting succinate dehydrogenase with malonate ester prodrugs decreases renal ischemia reperfusion injury

Abstract

Renal ischemia reperfusion (IR) injury leads to significant patient morbidity and mortality, and its amelioration is an urgent unmet clinical need. Succinate accumulates during ischemia and its oxidation by the mitochondrial enzyme succinate dehydrogenase (SDH) drives the ROS production that underlies IR injury. Consequently, compounds that inhibit SDH may have therapeutic potential against renal IR injury. Among these, the competitive SDH inhibitor malonate, administered as a cell-permeable malonate ester prodrug, has shown promise in models of cardiac IR injury, but the efficacy of malonate ester prodrugs against renal IR injury have not been investigated. Here we show that succinate accumulates during ischemia in mouse, pig and human models of renal IR injury, and that its rapid oxidation by SDH upon reperfusion drives IR injury. We then show that the malonate ester prodrug, dimethyl malonate (DMM), can ameliorate renal IR injury when administered at reperfusion but not prior to ischemia in the mouse. Finally, we show that another malonate ester prodrug, diacetoxymethyl malonate (MAM), is more potent than DMM because of its faster esterase hydrolysis. Our data show that the mitochondrial mechanisms of renal IR injury are conserved in the mouse, pig and human and that inhibition of SDH by 'tuned' malonate ester prodrugs, such as MAM, is a promising therapeutic strategy in the treatment of clinical renal IR injury.

Keywords: Ischemia reperfusion injury; Kidney; Malonate; Mitochondria; Succinate; Succinate dehydrogenase.

Graphical abstract

Fig. 1

Inhibition of Succinate Dehydrogenase During Ischemia Reperfusion Injury with Malonate. (a) During ischemia, succinate accumulates due to reversal of succinate dehydrogenase (SDH). The accumulated succinate acts as a store of electrons capable of driving reverse electron transport (RET) and reactive oxygen species (ROS) production when rapidly re-oxidised on reperfusion. SDH reversal and succinate accumulation may be inhibited during ischemia by the competitive inhibitor of SDH, malonate. (b) On reperfusion, accumulated succinate is rapidly re-oxidised by SDH leading to reverse electron transport (RET) and the production of superoxide (O₂˙) from the flavin mononucleotide site of Complex I (CI). Rapid re-oxidation of succinate by SDH on reperfusion may also be inhibited by the competitive inhibitor of SDH, malonate, which prevents RET and ROS production. Intermembrane space (IMS). Inner mitochondrial membrane (IMM).

Fig. 2

Malonate Ester Prodrugs Used to Target Succinate Dehydrogenase In Vivo. (a) Malonate ester prodrugs and particularly acyloxymethyl ester prodrugs are hydrolysed by intracellular carboxylesterases (CES1 and CES2) to release malonate. (b) Chemical structure of succinate, malonate, dimethyl malonate (DMM) and diacetoxymethyl malonate (MAM).

Fig. 3

Metabolic Changes During Ischemia Reperfusion in the Mouse Kidney. (a) Serum creatinine concentration (n = 3–4) at 24 h reperfusion following bilateral renal ischemia for the indicated durations. Control values represent serum creatinine concentration in age-matched mice that have not undergone bilateral renal IR injury. (b) Model used to investigate the metabolic changes during renal IR injury in the mouse. Mice underwent bilateral renal ischemia for the indicated durations, at the end of which one kidney was removed and rapidly clamp frozen in LN₂ using Wollenburger clamps (End-Isc). The second kidney was reperfused for 10 min and then removed and rapidly clamp frozen in LN₂ using Wollenberger clamps also (Rep). (c) Tissue succinate concentration (n = 4), (d) ATP/ADP ratio (n = 3–4) and (e) sum of the ATP and ADP concentrations (n = 3–4) at the end of ischemia (End-Isc) and at 10 min reperfusion (Rep). Control kidneys (0 min) were removed from the mouse under conditions of normoxia. (f) Relative increase in MitoP/MitoB ratio on reperfusion (n = 3–4) calculated from the MitoP/MitoB ratio in the kidney at 10 min reperfusion (Rep) over the MitoP/MitoB ratio at the end of ischemia (End-Isc). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P values were calculated by one-way ANOVA with Dunnett's multiple comparison test. Data are mean ± SEM.

Fig. 4

Metabolic Changes in Mouse, Pig and Humans During Renal Ischemia. (a) A tissue wedge biopsy was taken from the kidney of anaesthetised pigs under conditions of normoxia and rapidly clamp frozen in LN₂ using Wollenburger clamps. The renal vessels were then tied and divided, and the pig kidney was exposed to 30 min ischemia in the abdomen of the pig maintained at the physiological of temperature 38 °C [27]. A second wedge biopsy was taken at the end of 30 min ischemia. (b) Human kidneys retrieved for transplantation but subsequently offered for research were reperfused with oxygenated ABO-matched blood for 1 h using ex vivo normothermic perfusion (EVNP). A wedge biopsy was taken from the kidney at the end of 1 h to act as a normoxic control. Human kidneys were then exposed to 30 min ischemia at a temperature of 36 °C. At the end of 30 min ischemia, a second wedge biopsy was taken. (c) Schematic of the EVNP circuit. (d) Succinate concentration (n = 4), (e) ATP/ADP ratio (n = 4) and (f) sum of the ATP and ADP concentration (n = 4) in the kidneys of mice, pigs and humans following 30 min ischemia (End-Isc) relative to normoxia. (g) Absolute tissue succinate concentration in the kidneys of mice, pigs and humans during normoxia and following 30 min ischemia (End-Isc). Mouse kidneys were exposed to 30 min ischemia as previously described in Fig. 3b. **P < 0.01, ***P < 0.001, ****P < 0.0001. P values were calculated by two-way ANOVA with Sidak's multiple comparisons test. Data are mean ± SEM.

Fig. 5

Targeting Succinate Accumulation with Dimethyl Malonate during Ischemia in the Mouse Kidney. (a) DMM was administered at the indicated doses in 160 μL saline as an infusion (rate 16 μL/min) 10 min prior to the onset of ischemia. At the end of the infusion, immediately prior to ischemia, one kidney was removed and rapidly clamp frozen in LN₂ using Wollenberger clamps (Pre-Isc). The second kidney was exposed to 20 min warm ischemia and then rapidly clamp frozen (End-Isc). Control kidneys were given a 160 μL infusion of saline only. (b & c) Tissue malonate (n = 4) and succinate (n = 4) concentration at the end of the infusion (Pre-Isc) and following 20 min ischemia (End-Isc). (d) Serum creatinine concentration (n = 4) at 24 h reperfusion following infusion of saline or DMM prior to ischemia as described in (a) followed by 20 min bilateral renal ischemia. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P values were calculated by two-way ANOVA with Sidak's multiple comparison test (b & c) and one-way ANOVA with Dunnett's multiple comparison test (d). Data are mean ± SEM. Of note, 640 mg/kg DMM administered prior to ischemia was toxic to the mice and the serum creatinine concentration at 24 h reperfusion could not be measured.

Fig. 6

Targeting Succinate Oxidation on Reperfusion with Dimethyl Malonate and Diacetoxymethyl Malonate in the Mouse Kidney. (a) Mice underwent 20 min bilateral renal ischemia. At the end of ischemia, one kidney was removed and rapidly clamp frozen in LN₂ using Wollenburger clamps (End-Isc). The second kidney was reperfused for 10 min and then removed and rapidly clamp frozen (Rep). An infusion of 160 μL 160 mg/kg dimethyl malonate (DMM) or 16 mg/kg diacetoxymethyl malonate in 1% DMSO (MAM) was given at a rate of 16 μL/min starting 5 min prior to the onset of reperfusion. Control kidneys were given an infusion of saline or 1% DMSO only. (b) Tissue malonate (n = 4) concentration at the end of 20 min ischemia (End-Isc) and at 10 min reperfusion (Rep) in mice infused with 160 mg/kg DMM. (c) Serum creatinine concentration (n = 6) at 24 h reperfusion following 20 min bilateral renal ischemia and infusion of saline or 160 mg/kg of DMM as described in (a). Sham mice were infused with 160 μL saline at a rate 16 μL/min but did not undergo bilateral renal ischemia. (d) Tissue malonate (n = 4) concentration at the end of 20 min ischemia (End-Isc) and at 10 min reperfusion (Rep) in mice infused with 16 mg/kg MAM. (e) Serum creatinine concentration (n = 6) at 24 h reperfusion following 20 min bilateral renal ischemia and infusion of 1% DMSO or MAM as described in (a). *P < 0.05, **P < 0.01, ****P < 0.0001. P values were calculated by two-way ANOVA with Sidak's multiple comparison test (b & d), one-way ANOVA with Tukey's multiple comparison test (c) and an unpaired, two-tailed Student's t-test assuming equal variance (e). Data are mean ± SEM.