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MULTI-PHOTON IMAGING OF LIVE RAT KIDNEY SLICES REVEALS
DIFFERENCES IN MITOCHONDRIAL FUNCTION BETWEEN PROXIMAL AND
DISTAL SEGMENTS OF THE NEPHRON IN RESPONSE TO ANOXIA.
Hall AM¹ ², Unwin R J¹ ², Duchen M R¹.
1
Department of Physiology, UCL, UK. 2Centre for Nephrology, UCL, UK.
INTRODUCTION: Mitochondrial dysfunction (MD) has been implicated in a range of renal
diseases, including ischaemia-reperfusion injury (IR) and Fanconi syndrome. Different segments of
the nephron appear to be differentially sensitive to MD: clinically, the proximal tubule (PT) appears
particularly vulnerable. Using multi-photon microscopy, we have identified differences in
mitochondrial function between the PT and other nephron segments in live rat kidney tissue, which
might explain this clinical finding.
METHODS: Live 200μm slices of rat kidney were produced using a Micron 650V tissue slicer.
The slices were imaged using a Zeiss LSM 510 upright multi-photon microscope coupled to a
Coherent Chameleon tunable laser. An onstage perfusion system was used to superfuse fluorescent
dyes and mitochondrial reagents. To model in vivo ischaemia, slices were superfused with a
hypoxic buffer to which cyanide (CN) was added to produce chemical anoxia. Fluorescence of
mitochondrial NADH was excited at 720nm and emitted light collected between 435-485nm.
Mitochondrial membrane potential was measured by allowing equilibration of the lipophilic
fluorescent probes Tetramethyl rhodamine methyl ester (TMRM) or rhodamine 123, which were
excited either at 860nm or 800nm respectively.
RESULTS: Resting mitochondrial NADH redox state was measured by expressing the resting
signal relative to the signal when maximally reduced (in the presence of CN) and maximally
oxidized (in the presence of uncoupler). The resting redox state was not significantly different
between PT and distal tubules (DT), and both were predominantly in an oxidized state. We have
previously reported that uptake of the mitochondrial membrane potential (Δψm) dependent dye
TMRM is greater in DT than PT cells, and we have since found the same uptake pattern when using
another Δψm dependent dye, Rhodamine 123. Furthermore, these differences were not affected by
inhibitors of either the multi-drug resistance protein (verapamil) or of the organic cation transporter
(cimetidine), suggesting that they are not due to dye loss via these efflux pathways.
In response to chemical anoxia, Δψm in cells of the PT showed a rapid decrease and PT
mitochondria were almost completely depolarized after 30 minutes. In contrast, Δψm was
maintained to a much greater extent in mitochondria of DT cells for up to 60 minutes of anoxia.
This difference was obliterated by the addition of oligomycin, an inhibitor of the F 1F0-ATP
synthase, showing that mitochondrial membrane potential is maintained by the proton pumping
activity of the mitochondrial ATP synthase operating in ‘reverse’ mode.
Using slices loaded with dihydroethidium, a sensor for superoxide generation, we observed a burst
of reactive oxide species (ROS) production in response to CN, which appeared greater in the PT
than DT.
CONCLUSIONS: Using multi-photon imaging of live slices of rat kidney we have demonstrated
differences in mitochondrial function, both at rest and in response to toxic stimuli, between the PT
and more distal nephron segments. These differences may be important in understanding the nature
and localization of renal cell injury in a range of diseases that affect mitochondria, such as IR.