Two-Photon Intravital Fluorescence Lifetime Imaging of the Kidney Reveals Cell-Type Specific Metabolic Signatures

Abstract

In the live animal, tissue autofluorescence arises from a number of biologically important metabolites, such as the reduced form of nicotinamide adenine dinucleotide. Because autofluorescence changes with metabolic state, it can be harnessed as a label-free imaging tool with which to study metabolism Here, we used the combination of intravital two-photon microscopy and frequency-domain fluorescence lifetime imaging microscopy (FLIM) to map cell-specific metabolic signatures in the kidneys of live animals. The FLIM images are analyzed using the phasor approach, which requires no prior knowledge of metabolite species and can provide unbiased metabolic fingerprints for each pixel of the lifetime image. Intravital FLIM revealed the metabolic signatures of S1 and S2 proximal tubules to be distinct and resolvable at the subcellular level. Notably, S1 and distal tubules exhibited similar metabolic profiles despite apparent differences in morphology and autofluorescence emission with traditional two-photon microscopy. Time-lapse imaging revealed dynamic changes in the metabolic profiles of the interstitium, urinary lumen, and glomerulus-areas that are not resolved by traditional intensity-based two-photon microscopy. Finally, using a model of endotoxemia, we present examples of the way in which intravital FLIM can be applied to study kidney diseases and metabolism. In conclusion, intravital FLIM of intrinsic metabolites is a bias-free approach with which to characterize and monitor metabolism, and offers the unique opportunity to uncover dynamic metabolic changes in living animals with subcellular resolution.

Exogenous Gene Transmission of Isocitrate Dehydrogenase 2 Mimics Ischemic Preconditioning Protection

“…The cell signaling systems responsible for these changes in mitochondria composition are still unclear. Preliminary work by our laboratory failed to identify any changes in endogenous metabolites observed by Hato et al 41 in response to endotoxin, and we were not able to observe an increase in ROS that may account for altered mitochondria protein gene transcription. 42 Our findings suggest that more work is needed to identify the adaptive signaling in response to IPC.…”

Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis

“…31 Recent studies have also reported variation in transcriptomes 32 and metabolic autofluorescence signals along the PCT. 33 However, although differences in the ultrastructure of the ELS between S1 and S2 cells have been described, 13 and could be observed in our mice by EM, these are fairly subtle. Both segments have a highly developed apical brush border and express megalin, and the vast majority of studies of endocytosis have considered the PCT as a single entity.…”

The archaeal Dps nanocage targets kidney proximal tubules via glomerular filtration

“…Male mouse strain C57BL/6J was obtained from the Jackson Laboratory. The endothelial-specific CreERT2 transgenic mice were generated by crossbreeding Tie2-CreER mice with Rosa26-TD-Tomato transgenic mice, as we described previously (48). Tamoxifen was injected i.p.…”