Multiphoton imaging reveals axial differences in metabolic autofluorescence signals along the kidney proximal tubule

Am J Physiol Renal Physiol. 2018 Dec 1;315(6):F1613-F1625. doi: 10.1152/ajprenal.00165.2018. Epub 2018 Aug 22.


Kidney proximal tubules (PTs) are densely packed with mitochondria, and defects in mitochondrial function are implicated in many kidney diseases. However, little is known about intrinsic mitochondrial function within PT cells. Here, using intravital multiphoton microscopy and live slices of mouse kidney cortex, we show that autofluorescence signals provide important functional readouts of redox state and substrate metabolism and that there are striking axial differences in signals along the PT. Mitochondrial NAD(P)H intensity was similar in both PT segment (S)1 and S2 and was sensitive to changes in respiratory chain (RC) redox state, whereas cytosolic NAD(P)H intensity was significantly higher in S2. Mitochondrial NAD(P)H increased in response to lactate and butyrate but decreased in response to glutamine and glutamate. Cytosolic NAD(P)H was sensitive to lactate and pyruvate and decreased dramatically in S2 in response to inhibition of glucose metabolism. Mitochondrial flavoprotein (FP) intensity was markedly higher in S2 than in S1 but was insensitive to changes in RC redox state. Mitochondrial FP signal increased in response to palmitate but decreased in response to glutamine and glutamate. Fluorescence lifetime decays were similar in both S1 and S2, suggesting that intensity differences are explained by differences in abundance of the same molecular species. Expression levels of known fluorescent mitochondrial FPs were higher in S2 than S1. In summary, substantial metabolic information can be obtained in kidney tissue using a label-free live imaging approach, and our findings suggest that metabolism is tailored to the specialized functions of S1 and S2 PT segments.

Keywords: autofluorescence; kidney; mitochondria; multiphoton imaging; proximal tubule.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • Energy Metabolism*
  • In Vitro Techniques
  • Kidney Tubules, Proximal / cytology
  • Kidney Tubules, Proximal / metabolism*
  • Male
  • Mice, Inbred C57BL
  • Microscopy, Fluorescence, Multiphoton*
  • Mitochondria / metabolism*
  • NADP / metabolism
  • Oxidation-Reduction


  • Biomarkers
  • NADP