Multiphoton Imaging Reveals Differences in Metabolic Auto‐fluorescence Signals Between Early and Late Proximal Tubule Segments of the Kidney

Abstract

The kidney emits a large amount of auto‐fluorescence due to the existence of endogenous fluorophores. These include NAD(P)H and flavoproteins (such as FAD), important mitochondrial redox co‐factors essential for metabolic processes such as the citric acid cycle (TCA), beta oxidation and the electron transport chain (ETC). Imaging of metabolic auto‐fluorescence signals could therefore provide new insights into kidney physiology.Multiphoton microscopy (MP) is a powerful tool with high penetrance and reduced phototoxicity that can be used to image dynamic changes in cell structure and function in living tissue. We have used this approach to investigate auto‐fluorescence signals in proximal tubules (PTs) as readouts of cellular metabolism using freshly prepared murine kidney cortical slices. The peak emission of NAD(P)H (excited at 720 nm) was at 460 nm, as previously reported, whereas the peak emission of FAD (excited at 900 nm) was in the range of 560–610 nm, slightly red shifted compared to previous studies using single photon excitation. NAD(P)H signal showed expected changes in intensity in response to inhibition or uncoupling of the ETC. In contrast, no such changes were observed in FAD signal, suggesting that it is not redox sensitive. Striking differences in baseline NAD(P)H and FAD signals were observed between early (S1) and later (S2) sections of the PT, implying metabolic differences between these segments. These differences were also observed in intact kidneys using intravital microscopy, suggesting they are not artefacts of the slicing process.To further investigate these findings, we assessed how NAD(P)H and FAD signals change in real time in response to different single substrates metabolized by the PT. NAD(P)H was increased in both PT segments by lactate and the short chain fatty acid (FA) hexonate, and decreased in response to gutamine. FAD signal was also decreased by glutamine, but was increased by the long chain FA palmitate. Fluorescence lifetime microscopy (FLIM) of the NAD(P)H signal revealed 2 principal lifetimes, most likely corresponding to free and bound forms of the molecule. A single lifetime was identified for mitochondrial FAD in both PT segments, implying that differences in intensity are likely to be explained by differences in abundance of the same metabolite.In summary, using MP and FLIM we found that there are striking previously unknown differences in baseline mitochondrial auto‐fluorescence signals between S1 and S2 PT segments, implying underlying differences in metabolism. Moreover, the intensity of signals is dependent not only on mitochondrial redox state, but also on metabolic substrate supply. These findings may help to explain axial patterns of injury typically observed in certain kidney diseases.Support or Funding InformationJRM is a IKPP2 fellow funded by the European Union's Seventh Framework Programme for research, technological development and demonstration under the grant agreement no 608847This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.