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Two-photon excitation fluorescence imaging of the living
juxtaglomerular apparatus
János
Peti-Peterdi1,2,
Shigeru
Morishima1,3,
P.
Darwin
Bell1,2, and
Yasunobu
Okada1,3
1 Core Research for Evolutional Science and
Technology, Japan Science and Technology Corporation, and
3 Department of Cell Physiology, National Institute for
Physiological Sciences, Okazaki 444-8585, Japan; and
2 Nephrology Research and Training Center, Division of
Nephrology, Department of Medicine, University of Alabama at
Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
Recently, multiphoton excitation
fluorescence microscopy has been developed that offers important
advantages over confocal imaging, particularly for in vivo
visualization of thick tissue samples. We used this state-of-the-art
technique to capture high-quality images and study the function of
otherwise inaccessible cell types and complex cell structures of the
juxtaglomerular apparatus (JGA) in living preparations of the kidney.
This structure has multiple cell types that exhibit a complex array of
functions, which regulate the process of filtrate formation and renal
hemodynamics. We report, for the first time, on high-resolution
three-dimensional morphology and Z-sectioning through isolated,
perfused kidney glomeruli, tubules, and JGA. Time-series images show
how alterations in tubular fluid composition cause striking changes in
single-cell volume of the unique macula densa tubular epithelium in
situ and how they also affect glomerular filtration through alterations
in associated structures within the JGA. In addition, calcium imaging of the glomerulus and JGA demonstrates the utility of this system in
capturing the complexity of events and effects that are exerted by the
specific hypertensive autacoid angiotensin II. This imaging approach to
the study of isolated, perfused live tissue with multiphoton microscopy
may be applied to other biological systems in which multiple cell types
form a functionally integrated syncytium.
multiphoton excitation; fluorescence microscopy; real-time imaging; macula densa
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INTRODUCTION |
THE PROCESS OF
GLOMERULAR filtration and its control by local hemodynamics and
the hormonal milieu involve the complex interaction of a number of
different cell types. Regulation of glomerular filtration occurs at the
juxtaglomerular apparatus (JGA), a complex structure that consists of a
number of different cell types, including vascular smooth muscle cells,
secretory granular epitheloid cells, vascular endothelium, mesangial
cells, and macula densa (MD) tubular epithelial cells (2).
MD cells, in the distal nephron, function as specific sensor cells that
detect changes in tubular fluid osmolality and/or salt concentration
via specific transport mechanisms (3) and send signals
through mesangial cells in the JGA. These signals effect afferent
arteriolar smooth muscle cells and renin granular cells to control
preglomerular vascular resistance [tubuloglomerular feedback (TGF)]
and renin release, respectively (1-3, 13). It has
been difficult to study these cellular interactions within the JGA in
living preparations, given the constraints of existing technologies.
Recently, multiphoton excitation laser scanning fluorescence
microscopy has been developed (8, 14) that is particularly applicable to deep optical sectioning of living tissue samples. This
technique offers a tremendous increase in image resolution vs.
conventional confocal microscopy. Importantly, it can optically section
through an entire glomerulus (glomerular diameter
100 µm), thus
providing the ability to directly study structures and cellular
components that lie deep within the glomerulus. This new technology was
used in the present work to visualize the living perfused JGA.
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MATERIALS AND METHODS |
Isolated, perfused afferent arteriole-cortical thick ascending
limb preparations were used as previously described (10, 11). Preparations were visualized using a two-photon laser
scanning fluorescence microscope (MRC1024MP, Bio-Rad) composed of a
mode-locked titanium-sapphire laser (Tsunami, Spectra Physics), and
a photo-diode pump laser (Millennium, Spectra Physics). Individual
preparations were transferred to a thermoregulated Lucite chamber
mounted on a Zeiss Axiolab inverted microscope and visualized using a
×40 water-immersion lens. The fluorescent membrane-staining dye
1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH; 5 µM,
Dojin) or the Ca2+-sensitive fluorophore indo
1-acetoyxmethyl ester (indo 1-AM; 5 µM, Molecular Probes, Eugene, OR)
was added to the tubular and/or arteriolar perfusion solutions (Table
1) for a loading period of 5 min to
visualize cellular structures or monitor intracellular Ca2+
concentration ([Ca2+]i). After the loading
period, TMA-DPH or indo 1-AM was removed. TMA-DPH fluorescence was
captured using a band-width emission filter at 430 ± 15 nm in
response to a two-photon excitation wavelength of 755 nm. Indo 1 fluorescence was measured at emission wavelengths of 453 and 405 nm in
response to two-photon excitation wavelength of 720 nm. A 405 ± 17-nm band-pass filter, placed before detection by a photomultiplier
tube (PMT; channel 2), was used to select indo 1 emissions
that increase with increasing [Ca2+], whereas a 453 ± 2-nm band-pass filter, placed before a second PMT (channel
1) was used to select isosbestic emissions of indo 1 that are
independent of [Ca2+]. After ~15-min incubation with
control perfusion solutions, fluorescence intensities for both
wavelengths stabilized at a constant level. Ratiometric (405 nm/453 nm)
images were collected and analyzed using a time series function of the
LaserSharp1.2 software (Bio-Rad) with the most frequently used
application of cycle time 10-s, for 30 cycles.
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RESULTS |
Glomerular and JGA morphology.
We examined three-dimensional (3D) morphology of the perfused, living
glomerulus with attached tubular segments using optical Z-sectioning.
Figure 1 represents midlevel sections (a
complete Z-series in 2-µm steps is available in a supplementary
file), using a cell membrane marker (TMA-DPH) or an intracellular
fluorophore (indo 1). Both morphology and cytosolic parameters, e.g.,
[Ca2+]i of individual cells that comprise the
glomerulus can be studied with high resolution. Afferent and efferent
arterioles, glomerular capillary loops, the proximal tubule and
cortical thick ascending limb, and MD cells can be identified and
visualized in striking detail. In addition, it was also possible to
visualize the process of filtration across the capillary wall into the
Bowman's capsule. In terms of the MD cells, we show for the first time
(Fig. 2), 3D imaging of a perfused MD
plaque, consisting of 24 individual cells.

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Fig. 1.
Visualization of various glomerular and juxtaglomerular apparatus
(JGA) structures in situ. a: Glomerulus perfused through the
afferent arteriole (AA) with attached cortical thick ascending limb
(cTAL) and macula densa (MD). Tissue was stained with TMA-DPH. Note the
dilation of Bowman's capsule (CAP) and exit of glomerular filtrate via
the proximal tubule (PT). b: Ca2+ image of the
double-perfused AA and cTAL containing the MD. Tissue was loaded with
indo 1. G, glomerulus; EA, efferent ateriole. Bar = 10 µm.
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Fig. 2.
Sectioning of the MD (arrowheads) plaque stained with
1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) in the
longitudinal (a), sagittal (b), and horizontal
(c) planes. Bar = 10 µm.
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Real-time imaging of the JGA during TGF.
We visualized MD cells as well as the entire JGA area during activation
of the TGF mechanism. Images are shown in Figs.
3 and 4
[videos (time-series images taken every 10 s) of the same preparation are available in supplementary files]. Increasing tubular
perfusate osmolality and NaCl concentration ([NaCl]), which mimics
normal physiological TGF activation, produced a remarkably fast and
reversible MD cell swelling. MD cell swelling is most likely due to a
high level of NaCl transport into these cells. It should also be noted
that a reversible blebbing or expansion of the MD apical membrane
occurred during TGF activation (Fig. 3b). Thus two-photon
imaging has revealed what may be a novel rapid process of membrane
insertion + retrieval that occurs in MD cells during TGF. Figures
4 and 5 demonstrate morphological changes
in the JGA during TGF activation. Parallel with MD cell swelling,
swelling/contraction of cells in the final part of the afferent
arteriole was observed that caused an almost complete collapse of the
arteriolar lumen and a shrinkage of capillary loops and the entire
glomerulus. This is the first evidence for a "sphincter-like"
response of the terminal intraglomerular afferent arteriole. These
intraglomerular morphological changes are absent when the efferent
arteriole is perfused, suggesting the lack of efferent arteriolar
vasoconstriction during TGF.

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Fig. 3.
MD
cell (arrowheads) swelling in response to increasing tubular NaCl
concentration from 25 [osmolality 210 mosmol/kgH2O, NaCl
substituted by N-methyl-D-glucamine (NMDG);
a] to 135 mM (osmolality 300 mosmol/kgH2O; b). Tissue was stained with
TMA-DPH. Note that balloon-like blebbing occurred in
several MD cells. Bar = 10 µm.
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Fig. 4.
Changes in JGA morphology during increasing tubular NaCl
concentration from 25 (osmolality 210 mosmol/kgH2O, NaCl
substituted by NMDG; a) to 135 mM (osmolality 300 mosmol/kgH2O; b). MD cell (arrowhead) swelling
and parallel swelling/contraction of cells in the final part of the AA
that causes an almost complete closure of the arteriolar lumen
(arrows), collapse of capillary loops (CAP), and shrinkage of the
entire glomerulus (G) are shown. Tissue was stained with TMA-DPH. *,
Mesangial cells. Bar = 10 µm.
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Fig. 5.
Comparison of changes in afferent arteriolar diameter in intra- and
extraglomerular segments. Tubular NaCl concentration was
increased from 25 (osmolality 210 mosmol/kgH2O, NaCl
substituted by NMDG; a) to 135 mM (osmolality 300 mosmol/kgH2O; b). Tissue was stained with
TMA-DPH. Bar = 10 µm.
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Effects of ANG II.
In another effort to apply this new technology to the JGA, we measured
morphological and dimensional changes in arterioles and glomerular
capillary loops in response to the vasoactive peptide ANG II in the
presence or absence of the type 1 ANG II receptor (AT1)
blockade with candesartan. We also tested the effects of another
vasoconstrictor hormone, norepinephrine (Fig.
6). ANG II (10
8 M), added
to the arteriolar perfusate, significantly constricted afferent (AA)
and efferent arterioles (EA). These vascular effects were prevented by
coadministration of 10
6 M candesartan. ANG II also caused
a significant reduction in the glomerular capillary loop diameter (Fig.
6). Vascular reactivity was well preserved in these studies, because
addition of 10
6 M norepinephrine at the end of each
experiment significantly constricted both AA and EA.

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Fig. 6.
Effects of ANG II alone or with AT1 receptor
blockade candesarten (Cand) and norepinephrine (NE) on AA, EA, and
glomerular capillary loop (GCL) internal diameter (ID).
*P < 0.05, nonsignificant compared with control (ctrl)
in each group (n = 5 each).
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Figure 7 demonstrates changes in
glomerular morphology and [Ca2+]i in response
to ANG II. ANG II (10
8 M) added to the perfusate
significantly increased [Ca2+]i in AA, EA,
mesangial cells, and podocytes. In the case of intraglomerular mesangial cells, there was a 35.2 ± 11.7% increase in the indo 1 fluorescence ratio (n = 5) with addition of ANG II that
had been antagonized by 10
6 M candesartan.

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Fig. 7.
Ca2+ image of the perfused AA-glomerulus (G)-EA complex
before (a) and 2 min after (b) the
addition of ANG II to the lumen. Tissue was loaded with indo 1. Bar = 10 µm.
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DISCUSSION |
Despite the key role that the JGA plays in the regulation of renin
release, TGF, and control of glomerular filtration and blood flow
(1, 2, 13), it has been very difficult to examine single
cells of the JGA even with conventional videomicroscopy (4-7). However, we now report that with two-photon
microscopy, one can visualize single cells of the living JGA and
glomerulus in real time and in striking detail.
With simultaneous increases in tubular osmolality and [NaCl], MD
cells produced a significant cell swelling. This was primarily due to
an increase in cell height (Fig. 3), consistent with an earlier work
(4). However, in contrast to the studies of Gonzalez et
al. (4) we found much larger increases in MD cell volume. Remarkably, these cells are capable of almost doubling their initial cell volume within 30 s despite a concomitant increase in luminal osmolality. This cell swelling is primarily due to NaCl entry into the
cell, because the same change in the tubular perfusate osmolality at
constant [NaCl] did not produce any significant change in cell volume.
We also examined TGF activation on intraglomerular elements. For the
first time, we observed a sphincter-like constriction of the afferent
arteriole in the terminal intraglomerular segment of this vessel.
During TGF, the vascular diameter of this sphincter decreased to ~0.
Compared with this strong vasoconstrictor response, we observed only
minor changes in vascular diameter in more proximal extraglomerular
segments of the afferent arteriole (Fig. 5). These studies indicate
that the principal effector site for TGF occurs in the
terminal-intraglomerular afferent arteriole. These studies also suggest
that measurements of proximal afferent arteriolar diameter, as used by
others (5, 12), may not accurately reflect TGF responses.
One can further speculate that the minor vasoconstriction seen in the
proximal afferent arteriole may be, at least in part, a myogenic
response that is a consequence of the terminal-TGF-mediated sphincter
activity. Also, the TGF-mediated reduction in intraglomerular afferent
arteriolar diameter is more consistent with the magnitude of in vivo
TGF responses obtained with micropuncture (9). Further studies are needed to identify the cell type in the afferent arteriole that constituents this sphincter-like structure.
In other experiments, we tested the effects of ANG II on glomerular and
JGA morphology, because ANG II is a well-known modulator of renal
hemodynamics and kidney function (9). In addition to the
expected arteriolar vasoactive effects, to our knowledge this is the
first study that demonstrates an increase in mesangial cell
Ca2+ in the intact JGA. Thus these studies suggest that ANG
II, perhaps through activation of mesangial cells and podocytes, may
modify the glomerular capillary filtration surface area and that these effects are inhibited by AT1 receptor blockade with
candesartan. This approach has allowed us to identify a novel
intraglomerular sphincter-like effector site for TGF. It has also
permitted us to begin to define the intraglomerular effects of ANG II.
In summary, multiphoton excitation fluorescence microscopy in
combination with isolated perfused JGA offers a powerful new tool for
investigating the structural and cellular components that regulate the
process of glomerular filtrate formation and renal hemodynamics.
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ACKNOWLEDGEMENTS |
We thank M. Ohara for excellent technical assistance and T. Okayasu
and M. Yeager for secretarial help.
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FOOTNOTES |
This work was supported by grants from Core Research for Evolutional
Science and Technology (to Y. Okada), the National Institute of
Diabetes and Digestive and Kidney Diseases (DK-32032 to P. D. Bell), the American Heart Association SDG 0230074N, and Aventis Pharma
Deutschland (to J. Peti-Peterdi). J. Peti-Peterdi was a National Kidney
Foundation Postdoctoral Research Fellow during the conduct of some of
these studies.
Supplementary digital video files are available at
http://ajprenal.physiology.org/cqi/content/full/283/1/F197/DC1.
Address for reprint requests and other correspondence: J. Peti-Peterdi, 865 Sparks Ctr., 1720 Seventh Ave., South, Birmingham, AL
35294 (E-mail: petjan{at}uab.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 29, 2002;10.1152/ajprenal.00356.2001
Received 6 December 2001; accepted in final form 16 January 2002.
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