Proceedings of The Physiological Society

Mitochondria: Form and function (London, UK) (2017) Proc Physiol Soc 38, C05

Oral Communications

Components of the innate immune system evoke mitochondrial membrane hyperpolarisation, increases in mitochondrial reactive oxygen species via Nox and induce cell death in the renal medulla.

K. D. Taylor1, S. Wildman1, C. M. Peppiatt-Wildman1

1. Medway School of Pharmacy, University of Kent, Kent, United Kingdom.

Background: The innate immune system, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) and cell death are all thought to be involved in the pathogenesis of acute kidney injury (AKI) [1]. How these processes are linked however, remains unclear. AKI is sudden damage to the kidneys, which in the absence of specific therapy can lead to severe kidney damage and mortality [2]. Understanding the cellular mechanisms involved in AKI may help improve diagnostics and provide novel therapeutic targets for the treatment of AKI after ischemic injury. Method: A rat ‘live' kidney slice model [3] was used in combination with multiphoton microscopy to investigate the effect of components of the innate immune system on mitochondrial membrane potential, mitochondrial ROS production and cell death in the renal medulla. Kidney slices were loaded with the appropriate fluorescent dye; tetramethylrhodamine methyl ester (TMRM), mitoSOX and propidium iodide (PI), respectively prior to exposure to C5a, IL-33, TNF-α or IL-18 for 10 minutes. Real time changes in fluorescence was recorded throughout experiments and analysed off line. All data are mean ± S.E.M, n≥6 animals. Significance was determined using Students' t-test and all p ≤ 0.05. Results: C5a evoked a significant increase in TMRM signal in endothelial (E; 34.73% ± 4.56%,), pericyte (P; 40.87% ± 8.34%) and tubule cells (T; 34.40% ± 7.12%). As did IL-33 (E: 26.25% ± 2.68%, P: 29.27% ± 8.82, T: 20.79% ± 2.61%), IL-18 (E: 31.61% ± 4.76%, P: 32.66% ± 2.95%, T: 23.94% ± 1.57%) and TNF-α (E: 44.72% ± 3.72%, P: 52.64% ± 4.29%, T: 54.75% ± 6.14%). C5a also evoked a significant increase in MitoSOX signal in endothelial (E; 24.96% ± 5.87%), pericyte (P; 31.41% ± 7.70%,) and tubule (T; 50.14% ± 7.69%) cells. As did IL-33 (E: 44.59% ± 8.59%, P: 30.92% ± 9.07%, T: 26.25% ± 3.37%), IL-18 (E: 54.05% ± 3.46%, P: 56.56% ± 9.75%, T: 69.60% ± 5.17%) and TNF-α (E: 61.10% ± 9.72%, P: 51.51% ± 12.08%, T: 70.41% ± 9.25%). When tissue was exposed to innate immune components in combination with a Nox inhibitor apocynin, the innate immune component-induced increases in MitoSOX fluorescence was significantly attenuated in all cases. C5a, IL-33, IL-18 and TNF-α all induced significant increases (22.74% ± 4.80%, 15.58% ± 3.81%, 11.79% ± 1.93%, 17.02% ± 4.78%) in dead cells labelled with PI across the kidney slice. Conclusion: Innate immune system components C5a, IL-33, IL-18 and TNF-α all act at medullary endothelial, pericyte and tubule cells to cause mitochondrial hyperpolarisation and increases Nox-dependent mitochondrial ROS, that are linked to renal cell death. This data suggest alterations in mitochondria function are likely to be key in immune-mediated pathophysiology associated with AKI.

Where applicable, experiments conform with Society ethical requirements