The effect of inactin on kidney mitochondrial function and production of reactive oxygen species

Abstract

Inactin is a long lasting anesthetic agent commonly used in rat studies, but is also shown to exert physiological effects such as reducing renal blood flow, glomerular filtration rate and depressing tubular transport capacity. The effect of inactin on isolated kidney mitochondria is unknown and may be important when studying related topics in anaesthetized animals. The aim of this study was to determine whether inactin exerts effects on mitochondrial function and production of reactive oxygen species. Kidney mitochondrial function and production of reactive oxygen after acutely (5 min) or longer (1.5 hour) anesthetizing rats with inactin was evaluated using high-resolution respirometry. The results demonstrate that inactin significantly improves respiratory control ratio, inhibits complex I in the mitochondrial respiratory chain, reduce both unregulated proton leak and time dependently reduce the regulated proton leak via uncoupling protein-2 and adenine nucleotide translocase. Inactin also contributes to increased mitochondrial hydrogen peroxide production. In conclusion, inactin exerts persistent effects on mitochondrial function and these profound effects on mitochondrial function should to be considered when studying mitochondria isolated from animals anesthesized with inactin.

Fig 1. Mitochondrial respiratory function evaluated using high resolution respirometry.

(A) Maximal complex I (CI) mediated OXPHOS capacity (state 3) respiration was determined in the presence of pyruvate, malate and ADP, and (B) CI + CII state 3 after addition of succinate. (C) Succinate control ratio (SCR) was calculated by dividing (CI + CII) state 3 with CI mediated state 3 respiration. (D) Respiratory control ratio was defined as state 3 respiration (pyruvate, malate) divided by state 2 respiration. (E) State 2 respiration was measured in the presence of pyruvate and malate and (F) LEAK respiration by inhibiting ATP synthase with oligomycin.

Fig 2. Mitochondrial LEAK respiration measured by high resolution respirometry.

(A) Unregulated LEAK respiration was calculated by subtracting GDP and CAT dependent respiration from the total LEAK respiration. (B) Regulated LEAK respiration is the sum of GDP and CAT dependent respiration. (C) GDP dependent respiration was determined by adding GDP in LEAK respiration and calculating the change in respiration. (D) Similarly, CAT dependent respiration was evaluated by adding CAT during LEAK respiration.

Fig 3

(A) Amplex UltraRed was used to spectrofluorometrically measure mitochondrial hydrogen peroxide production in CI mediated state 2 respiration. (B) Mitochondrial apparent k_M for oxygen (P50_mito) was determined by allowing mitochondria respire until anoxia and calculate the hyperbolic function. (C) Mitochondrial P/O-ratio was evaluated by steady state infusion of ADP corresponding to approximately half-maximal CI mediated state 3 respiration assuming all infused ADP was converted to ATP.