P0017ESTIMATION OF KIDNEY MITOCHONDRIA TOLERANCE VIA FLUORESCENCE MICROSCOPY
Authors: Plotnikov Egor , Popkov Vasily , Tvorogova Anna , Andrianova Nadezda , Buyan Marina , Zorov Dmitry 
Published: June 2020

Abstract

Background and Aims
The functioning of mitochondria is a key parameter that determines the normal activity of kidney cells and triggers life/death transition in pathological conditions. The main pathological event in the mitochondria is the opening of a permeability transition pore, observed in renal ischemia and other acute nephrological pathologies. The development of non-specific mitochondrial permeability leads to a burst of ROS generation and the release of proapoptotic factors.
Assessment of mitochondrial resistance to induction of permeability transition is an important characteristic for the analysis of kidney tolerance to damaging factors, such as ischemia. In this study, we developed a method for evaluating mitochondrial permeability transition in renal tubular cell culture using fluorescence microscopy.

Method
The primary renal tubular epithelial cells were loaded with a fluorescent probe, TMRE, which accumulates in the mitochondria depending on the transmembrane potential. Cells in the culture were analyzed on a fluorescent microscope for 180 seconds under constant exposure with exciting light and the video was recorded at a frequency of 1 frame/sec. To analyze the images, a Python algorithm was created that allocated the individual mitochondria in the image and analyzed the shape, size, and dynamics of the TMRE fluorescence of each mitochondrion. In total, more than 10^5 mitochondria were analyzed in each experiment.

Results
The initial state of mitochondrial transmembrane potential was evaluated by the intensity of TMRE fluorescence in the first frame in each series when phototoxicity of the dye was not manifested yet. Population analysis revealed that the total distribution of mitochondria by TMRE fluorescence intensity was a single-modal. In this case, the histogram of the distribution had a wide arm of mitochondria with greater TMRE fluorescence (that means high transmembrane potential). This distribution suggests that renal cells have mitochondria with high and low TMRE fluorescence values. This can be interpreted as the presence of mitochondria in cells with higher transmembrane potential values.
Analysis of the dynamics of TMRE fluorescence intensity revealed that some mitochondria after a certain time demonstrated a sharp drop in the fluorescence intensity, which we interpret as the opening of the mitochondrial pore (Fig.1). However, this drop did not occur in all kidney cells mitochondria, but only in about 15% of the cells. A small number of mitochondria had several repeated falls/rises in TMRE fluorescence. This can be interpreted as the closing and subsequent opening of the permeability transition pores.
Analysis of a subpopulation of mitochondria with low TMRE values revealed that low-energized mitochondria have a later pore opening time. It is important to note that mitochondria with higher TMRE fluorescence are more susceptible to photodynamic effects when the probe is excited by light.

Conclusion
Renal epithelial cells happen to be very heterogeneous in terms of mitochondrial potential and the time of the induction of mitochondrial permeability transition. Correlations were found between the value of the mitochondrial potential (TMRE accumulation) and the cell's resistance to induction of non-specific mitochondrial pore.