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 in vivo 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 in vivo, and offers the unique opportunity to uncover dynamic metabolic changes in living animals with subcellular resolution.

Keywords: endothelium; metabolism; podocyte; tubules.

Figure 1.

Intravital fluorescence lifetime imaging (FLIM) distinguishes the metabolic signatures of S1 and S2 tubules. (A) Representative S1 and S2 proximal tubules identified by traditional intensity based intravital two-photon microscopy (red, green, and blue channels combined). (B) FLIM blue channel intensity shown in grayscale. (C) The phasor distribution of lifetimes collected from the area shown in (B). Note that lifetimes decrease moving from left to right in the phasor plot. (D) Four lifetime regions are selected in the phasor plot (yellow, beige, orange, and brown circles). (E) The gated lifetime regions are back plotted with corresponding colors into the blue channel (grayscale) image. These lifetime regions are characteristic of S1 tubules. (F and G) Three lifetime regions characteristic of S2 tubules are shown in blue, red, and pink.

Figure 2.

Intravital FLIM identifies renal structures that are not well resolved with conventional two-photon microscopy. (A) The urinary lumen, interstitium, and glomeruli exhibit weak autofluorescence and are not resolved with intravital two-photon microscopy. The distal tubules also have a very weak autofluorescence signal. (B and C) With FLIM, distal tubules and S1 (but not S2) possess similar metabolic signatures (yellow and orange). The urinary lumen, especially in the distal tubule, exhibits metabolites with long lifetimes (pseudocolored in dark green and light green). (D–F) The blood compartment shows species with short lifetimes (light green). Interstitial structures are highlighted in pink and violet and the endothelium in blue. (G and H) Dynamic changes in metabolic signatures of the urinary lumen and interstitium were captured with time-lapse intravital FLIM. See Supplemental Video 1. DT, distal tubule.

Figure 3.

Endothelial specific tdTomato fluorescence confirms FLIM mapping of endothelial cells. (A) Endothelial specific CreERT2 transgenic mouse kidney imaged with conventional two-photon microscopy. The structure of peritubular capillary endothelium is delineated with red fluorescence (tdTomato). (B and C) Red and blue channel FLIM intensity images are shown. (D and E) The phasor distributions of red and blue channel lifetimes obtained from the area shown in (B) and (C). (F and G) Lifetime regions gated on the phasor plots are back-mapped into the intensity images. Note the overlap of the FLIM blue channel and conventional two-photon imaging of tdTomato signal. Arrow points to the endothelium that was not induced by tamoxifen.

Figure 4.

Podocyte specific DsRed fluorescence confirms FLIM mapping of podocytes. (A) Conventional two-photon intravital imaging of wild-type Munich–Wistar–Frömter rat glomerulus. (B) Podocyte-DsRed Munich–Wistar–Frömter rat glomerulus imaged with conventional two-photon microscopy. (C–H) FLIM imaging of red and blue channels of the glomerulus shown in (B). Note that the FLIM mapping of the podocyte in the blue channel [red overlay; (H)] compares well with the DsRed lifetime mapping in the red channel (G) and the podocyte fluorescence by conventional two-photon microscopy (B). In (H), we also show the glomerular capillary endothelium (turquoise) and blood compartment (light green). DT, distal tubule; Glom, glomerulus; PT, proximal tubule; WT, wild-type.

Figure 5.

Endotoxin alters the S2 metabolic signature. (A and B) Regular two-photon intravital imaging reveals that systemically-administered endotoxin (Alexa-LPS; red) is filtered and internalized primarily by S1 proximal tubules. Oxidative stress (reflected by strong H₂DCFDA green fluorescence) occurs in S2 proximal tubules. (C and D) FLIM image of control mouse kidney before endotoxin exposure. S1 FLIM signature is highlighted in orange and yellow, S2 in blue and red. (E and F) FLIM revealed the loss of S2 signature in the kidney 24 hours after endotoxin administration. DT, distal tubule.

Figure 6.

Endotoxin treatment increases the NADH/NAD+ ratio in S2 tubules. The phasor lifetime distributions of control (Figure 5D) and endotoxin-treated mice (Figure 5F) were superimposed onto phasor plots of standard metabolites as indicated in (A). Additional metabolites mapped are shown in Supplemental Figure 2. When a pixel has two lifetime components, the phasor location of the mixture falls onto a straight line between the two individual lifetime points (see Concise Methods). (A) The alteration of S2 signature after endotoxin treatment can be explained by an increase in the NADH/NAD⁺ ratio as well as decreases in FAD and its precursor riboflavin. (B–D) Metabolite levels of kidney tissues measured with liquid chromatography/mass spectrometry are shown. Endotoxin treatment reduced the levels of NAD⁺, FAD, and riboflavin, consistent with the S2 signature changes observed with FLIM. Horizontal bars represent mean values.