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Intravital multi-photon microscopy reveals several levels of heterogeneity in endocytic uptake by mouse renal proximal tubules.

Caplanusi A, Parreira KS, Lima WR, Marien B, Van Der Smissen P, de Diesbach P, Devuyst O, Courtoy PJ - J. Cell. Mol. Med. (2007)

Bottom Line: Understanding renal function requires one to integrate the structural complexity of kidney nephrons and the dynamic nature of their cellular processes.Multi-photon fluorescence microscopy is a state-of-the-art imaging technique for in vivo analysis of kidney tubules structure and function in real time.This study presents visual evidence for several levels of heterogeneity of proximal tubular endocytic uptake in the superficial renal mouse cortex and illustrates the potential of multi-photon microscopy for providing a comprehensive and dynamic portrayal of renal function.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Unit (CELL), Université catholique de Louvain Medical School and de Duve Institute, Brussels, Belgium.

ABSTRACT
Understanding renal function requires one to integrate the structural complexity of kidney nephrons and the dynamic nature of their cellular processes. Multi-photon fluorescence microscopy is a state-of-the-art imaging technique for in vivo analysis of kidney tubules structure and function in real time. This study presents visual evidence for several levels of heterogeneity of proximal tubular endocytic uptake in the superficial renal mouse cortex and illustrates the potential of multi-photon microscopy for providing a comprehensive and dynamic portrayal of renal function.

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Fine tissular heterogeneity of fluorescent dextrans uptake by cortical proximal tubules. This image was taken at 60 min. after the intravenous injection of a mixture of Alexa568- (red signal at panel A) and fluorescein-dextran 10 kDa (green signal at panel B), together with the cell-permeant DNA-intercalating dye, Hoechst 33342, to label the nuclei of renal epithelial cells (blue signal at panel C), using a 63×/NA 1.2 water immersion objective. The fluorophores were simultaneously excited at 800 nm, the emitted fluorescence was collected by separate photomultipliers, with channels centered at 600, 525 and 450 nm, respectively, and the multi-color image was generated by superimposition of the three channels (C). Different levels of Alexa568- and fluorescein-dextran within endocytic vesicles is evidenced at C by a range of colours from red (only Alexa568-dextran) to green (only fluorescein-dextran), with orange to yellow as intermediates. Alexa568-dextran shows rapid ultrafiltration and high uptake in most nephron profiles. Fluorescein dextran shows delayed ultrafiltration (not shown) and preferential uptake by different nephron profiles. The two asterisks indicate proximal tubular profiles with exclusive Alexa568-dextran uptake. Small arrowheads collectively delineate another profile, presumably more distal, still containing fluorescein-dextran in the tubular lumen, and no detectable Alexa568-dextran endocytosis. Paired large arrowheads at the upper left of panel C show a sharp boundary between two continuous segments of a proximal tubule with predominant uptake of either Alexa568-dextran (upper part) or fluorescein-dextran (lower part). The lower right part of panel C shows heterogeneity of uptake at the single cell level, with scattered preference for Alexa568- (single arrows) or fluorescein-dextran uptake (double arrows). Scale bar, 20 μm.
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fig02: Fine tissular heterogeneity of fluorescent dextrans uptake by cortical proximal tubules. This image was taken at 60 min. after the intravenous injection of a mixture of Alexa568- (red signal at panel A) and fluorescein-dextran 10 kDa (green signal at panel B), together with the cell-permeant DNA-intercalating dye, Hoechst 33342, to label the nuclei of renal epithelial cells (blue signal at panel C), using a 63×/NA 1.2 water immersion objective. The fluorophores were simultaneously excited at 800 nm, the emitted fluorescence was collected by separate photomultipliers, with channels centered at 600, 525 and 450 nm, respectively, and the multi-color image was generated by superimposition of the three channels (C). Different levels of Alexa568- and fluorescein-dextran within endocytic vesicles is evidenced at C by a range of colours from red (only Alexa568-dextran) to green (only fluorescein-dextran), with orange to yellow as intermediates. Alexa568-dextran shows rapid ultrafiltration and high uptake in most nephron profiles. Fluorescein dextran shows delayed ultrafiltration (not shown) and preferential uptake by different nephron profiles. The two asterisks indicate proximal tubular profiles with exclusive Alexa568-dextran uptake. Small arrowheads collectively delineate another profile, presumably more distal, still containing fluorescein-dextran in the tubular lumen, and no detectable Alexa568-dextran endocytosis. Paired large arrowheads at the upper left of panel C show a sharp boundary between two continuous segments of a proximal tubule with predominant uptake of either Alexa568-dextran (upper part) or fluorescein-dextran (lower part). The lower right part of panel C shows heterogeneity of uptake at the single cell level, with scattered preference for Alexa568- (single arrows) or fluorescein-dextran uptake (double arrows). Scale bar, 20 μm.

Mentions: Heterogeneity between tubular profiles and among adjacent cells of a given profile in C57BL mice kidneys is illustrated in both Figures 1 and 2. Since the analysis was limited to the superficial cortical zone, the marked heterogeneity between tubular profiles for accessibility to, and endocytic labelling by, distinct fluorescent dextrans cannot be due to the differences between cortical and juxtamedullary nephrons, since the latter do not reach the region analysed [11]. Our data therefore primarily reflect intranephron segmental heterogeneity. An abrupt boundary in tracer uptake preference between continuous nephron segments is indeed evidenced in favourable sections (large arrowheads in Fig. 1, right and Fig. 2C, left). Intranephron segmental heterogeneity has already been documented for albumin uptake [12]. As a second level of heterogeneity, clear-cut differences in fluorescent dextran preference between adjacent cells in a random, scattered fashion is also evident in some tubular profiles (Fig. 2C, single versus double arrows). On the top of these two documented levels of structural heterogeneity, functional differences in regional blood flow and/or glomerular filtration may add to the complexity of tubular endocytosis, but this level of heterogeneity has not been addressed here. Finally, the strikingly different handling of two different fluorescent dextran preparations with presumably superimposable size distribution points to an effect of charge density on ultrafiltration and/or endocytosis efficiency [10].


Intravital multi-photon microscopy reveals several levels of heterogeneity in endocytic uptake by mouse renal proximal tubules.

Caplanusi A, Parreira KS, Lima WR, Marien B, Van Der Smissen P, de Diesbach P, Devuyst O, Courtoy PJ - J. Cell. Mol. Med. (2007)

Fine tissular heterogeneity of fluorescent dextrans uptake by cortical proximal tubules. This image was taken at 60 min. after the intravenous injection of a mixture of Alexa568- (red signal at panel A) and fluorescein-dextran 10 kDa (green signal at panel B), together with the cell-permeant DNA-intercalating dye, Hoechst 33342, to label the nuclei of renal epithelial cells (blue signal at panel C), using a 63×/NA 1.2 water immersion objective. The fluorophores were simultaneously excited at 800 nm, the emitted fluorescence was collected by separate photomultipliers, with channels centered at 600, 525 and 450 nm, respectively, and the multi-color image was generated by superimposition of the three channels (C). Different levels of Alexa568- and fluorescein-dextran within endocytic vesicles is evidenced at C by a range of colours from red (only Alexa568-dextran) to green (only fluorescein-dextran), with orange to yellow as intermediates. Alexa568-dextran shows rapid ultrafiltration and high uptake in most nephron profiles. Fluorescein dextran shows delayed ultrafiltration (not shown) and preferential uptake by different nephron profiles. The two asterisks indicate proximal tubular profiles with exclusive Alexa568-dextran uptake. Small arrowheads collectively delineate another profile, presumably more distal, still containing fluorescein-dextran in the tubular lumen, and no detectable Alexa568-dextran endocytosis. Paired large arrowheads at the upper left of panel C show a sharp boundary between two continuous segments of a proximal tubule with predominant uptake of either Alexa568-dextran (upper part) or fluorescein-dextran (lower part). The lower right part of panel C shows heterogeneity of uptake at the single cell level, with scattered preference for Alexa568- (single arrows) or fluorescein-dextran uptake (double arrows). Scale bar, 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823495&req=5

fig02: Fine tissular heterogeneity of fluorescent dextrans uptake by cortical proximal tubules. This image was taken at 60 min. after the intravenous injection of a mixture of Alexa568- (red signal at panel A) and fluorescein-dextran 10 kDa (green signal at panel B), together with the cell-permeant DNA-intercalating dye, Hoechst 33342, to label the nuclei of renal epithelial cells (blue signal at panel C), using a 63×/NA 1.2 water immersion objective. The fluorophores were simultaneously excited at 800 nm, the emitted fluorescence was collected by separate photomultipliers, with channels centered at 600, 525 and 450 nm, respectively, and the multi-color image was generated by superimposition of the three channels (C). Different levels of Alexa568- and fluorescein-dextran within endocytic vesicles is evidenced at C by a range of colours from red (only Alexa568-dextran) to green (only fluorescein-dextran), with orange to yellow as intermediates. Alexa568-dextran shows rapid ultrafiltration and high uptake in most nephron profiles. Fluorescein dextran shows delayed ultrafiltration (not shown) and preferential uptake by different nephron profiles. The two asterisks indicate proximal tubular profiles with exclusive Alexa568-dextran uptake. Small arrowheads collectively delineate another profile, presumably more distal, still containing fluorescein-dextran in the tubular lumen, and no detectable Alexa568-dextran endocytosis. Paired large arrowheads at the upper left of panel C show a sharp boundary between two continuous segments of a proximal tubule with predominant uptake of either Alexa568-dextran (upper part) or fluorescein-dextran (lower part). The lower right part of panel C shows heterogeneity of uptake at the single cell level, with scattered preference for Alexa568- (single arrows) or fluorescein-dextran uptake (double arrows). Scale bar, 20 μm.
Mentions: Heterogeneity between tubular profiles and among adjacent cells of a given profile in C57BL mice kidneys is illustrated in both Figures 1 and 2. Since the analysis was limited to the superficial cortical zone, the marked heterogeneity between tubular profiles for accessibility to, and endocytic labelling by, distinct fluorescent dextrans cannot be due to the differences between cortical and juxtamedullary nephrons, since the latter do not reach the region analysed [11]. Our data therefore primarily reflect intranephron segmental heterogeneity. An abrupt boundary in tracer uptake preference between continuous nephron segments is indeed evidenced in favourable sections (large arrowheads in Fig. 1, right and Fig. 2C, left). Intranephron segmental heterogeneity has already been documented for albumin uptake [12]. As a second level of heterogeneity, clear-cut differences in fluorescent dextran preference between adjacent cells in a random, scattered fashion is also evident in some tubular profiles (Fig. 2C, single versus double arrows). On the top of these two documented levels of structural heterogeneity, functional differences in regional blood flow and/or glomerular filtration may add to the complexity of tubular endocytosis, but this level of heterogeneity has not been addressed here. Finally, the strikingly different handling of two different fluorescent dextran preparations with presumably superimposable size distribution points to an effect of charge density on ultrafiltration and/or endocytosis efficiency [10].

Bottom Line: Understanding renal function requires one to integrate the structural complexity of kidney nephrons and the dynamic nature of their cellular processes.Multi-photon fluorescence microscopy is a state-of-the-art imaging technique for in vivo analysis of kidney tubules structure and function in real time.This study presents visual evidence for several levels of heterogeneity of proximal tubular endocytic uptake in the superficial renal mouse cortex and illustrates the potential of multi-photon microscopy for providing a comprehensive and dynamic portrayal of renal function.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Unit (CELL), Université catholique de Louvain Medical School and de Duve Institute, Brussels, Belgium.

ABSTRACT
Understanding renal function requires one to integrate the structural complexity of kidney nephrons and the dynamic nature of their cellular processes. Multi-photon fluorescence microscopy is a state-of-the-art imaging technique for in vivo analysis of kidney tubules structure and function in real time. This study presents visual evidence for several levels of heterogeneity of proximal tubular endocytic uptake in the superficial renal mouse cortex and illustrates the potential of multi-photon microscopy for providing a comprehensive and dynamic portrayal of renal function.

Show MeSH
Related in: MedlinePlus