Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C125
Multiphoton imaging of mitochondrial function in the isolated perfused rat kidney
A. Hall1, C. Crawford2, R. J. Unwin1, M. R. Duchen3, C. M. Peppiatt-Wildman2
1. Centre for Nephrology, UCL, London, United Kingdom. 2. Royal Veterinary College, London, United Kingdom. 3. Cell and Developmental Biology, UCL, London, United Kingdom.
Abnormal mitochondrial function is central to the pathogenesis of a number of kidney diseases, including ischaemic acute kidney injury (AKI). We demonstrated recently that mitochondrial function can be imaged in live slices of rat kidney using multiphoton fluorescence microscopy (1). However, as metabolism in tubular cells is tightly coupled to solute transport, we wanted to devise a method for measuring mitochondrial function in intact viable organs. Imaging the rodent kidney in vivo has been limited by difficulties in achieving adequate intracellular loading of fluorescent dyes; so we investigated whether sufficient dye loading could be achieved to image mitochondrial function in tubular cells in the isolated perfused rat kidney (IPK) preparation. Adult male Sprague-Dawley rats were anaesthetised with intra-peritoneal pentobarbitone (50-100mg/kg). The right renal artery was cannulated using an established technique (2). Perfusion was commenced immediately with a HEPES-buffered solution at 37C. Once the cannula was securely in place, the preparation was quickly moved to a custom built imaging chamber. Dyes were loaded for 20-30 min using a re-circulating perfusion system. Kidneys were imaged with a Zeiss LSM 510 NLO axiovert microscope, coupled to a tunable Coherent Chameleon laser. Using the approach described above, dyes were successfully loaded into cells of the renal tubules. Cell structure and viability was demonstrated using the dye calcein. Tubular and vascular compartments were identified using fluorescently labelled dextrans of differing sizes that either do or do not cross the glomerular filtration barrier. Various aspects of mitochondrial function were imaged using endogenous or exogenous fluorophores, including: NADH autofluorescence, mitochondrial membrane potential (ΔΨm - tetramethyl rhodamine methyl ester [TMRM]), reactive oxygen species production (dihydroethidium) and glutathione levels (monochlorobimane). Furthermore, by stopping and then restarting the perfusion, changes in mitochondrial function and cell structure in response to ischaemia-reperfusion injury could be observed in real time. Active uptake of low-molecular weight proteins by receptor-mediated endocytosis from the glomerular filtrate is a major function of the proximal tubule; we found that apical uptake of Cy2-or Cy3-labelled insulin could be co-imaged with basolateral mitochondrial NADH or TMRM signals in this nephron segment. We have demonstrated that mitochondrial function in renal tubular cells can be imaged in the IPK using multiphoton microscopy and a re-circulating perfusion system. Furthermore, signals can be related to solute transport, and changes can be followed in real-time in a model of ischaemic AKI. We believe that this is a powerful approach with much potential for future nephrological research.
Where applicable, experiments conform with Society ethical requirements