Am J Physiol Renal Physiol 306: F664–F671, 2014.
First published January 29, 2014; doi:10.1152/ajprenal.00522.2013.
Three-dimensional reconstruction of the rat nephron
Erik I. Christensen, Birgitte Grann, Inger B. Kristoffersen, Elisabeth Skriver, Jesper S. Thomsen,
and Arne Andreasen
Department of Biomedicine, Anatomy, Aarhus University, Aarhus, Denmark
Submitted 24 September 2013; accepted in final form 20 January 2014
rat kidney morphology; three-dimensional structural analysis; digital
tracing
years been the target of a large
number of functional and morphological investigations (10 –
13, 17) and was recently reviewed (5). In recent years, especially the rat renal medulla has been the subject of intensive
comparative structural-functional studies and computer modeling [see e.g., Refs. 15 and 20 and very recently a very
comprehensive study on modeling calcium transport in the rat
nephron was published (28)]. A prerequisite for conducting
these kinds of studies is that the detailed morphology of the
kidney is known. However, such a detailed three-dimensional
(3D) analysis enabling identification of structural changes at a
precisely defined distance from the glomerulus of the different
segments along the nephron and collecting duct (CD) in the
kidney is currently not available. We have previously described the 3D organization and ultrastructural segmental variation of the mouse kidney nephrons and CDs (29, 32). To
describe the variation in kidney morphology in different species, we decided to study the morphology of the rat kidney in
THE RAT KIDNEY HAS FOR MANY
Address for reprint requests and other correspondence: E. I. Christensen,
Dept. of Biomedicine, Anatomy Section of Cell Biology, Univ. of Aarhus,
DK-8000 Aarhus C, Denmark (e-mail: [email protected]).
F664
detail. We have developed a combined histological and computerized technique, which enables the reconstruction of a
large number of rat nephrons in 3D space. Consequently, the
present study gives a detailed 3D analysis of rat nephrons, both
short-loop nephrons (SLNs) and long-loop nephrons (LLNs),
including a description of the course of individual nephrons,
the spatial orientation, the interrelation between nephrons, the
exact lengths of the different nephron segments and a structural
analysis at well-defined intervals along different segments of
the nephrons. These findings not only serve to describe the
morphology of the rat kidney, but can also be used as a
morphological basis for functional studies and for making
computer simulation models of the rat kidney. Finally, together
with our previous work on the morphology of the mouse
kidney, the present study can provide insight into morphological and related functional differences between mouse and rat
kidneys.
MATERIALS AND METHODS
Tissue preparation. The kidneys from three 3-mo-old male Wistar
rats were fixed by retrograde perfusion through the abdominal aorta
with 1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4.
The tissue blocks were cut perpendicular to the longitudinal axis of
the kidney and extended from the surface through the papilla. The
tissue blocks were fixed overnight in the same fixative and postfixed
for 1 h in 1% OsO4 in 0.1 M sodium cacodylate buffer, én bloc stained
with uranyl acetate, dehydrated in ethanol, and embedded in flat
molds in Epon 812 (TAAB, Aldermaston, Berks, UK). From each of
the three kidneys, 4,252-, 4,384-, and 4,541 2.5-␮m-thick serial
sections were cut from the surface to the papillary tip using a Reichert
Ultracut S microtome (Reichert, Vienna, Austria) and a Diatome
histoknife (Diatome, Biel, Switzerland). The sections were mounted
on microscope slides and stained with toluidine blue.
All animal experiments were carried out in accordance with the
provisions for the animal care license provided by the Danish National
Animal Experiments Inspectorate.
Image acquisition. The histological sections were digitized using
an Olympus AX 70 microscope (Olympus, Tokyo, Japan) equipped
with an Olympus DP 50 digital camera attached to a standard PC. The
images were recorded using a ⫻3 objective lens system (a ⫻4
objective lens in combination with a ⫻0.5 distance ring and a ⫻1.5
zoom lens).
Image aligning. Initially, the digitized images were aligned using a
custom-made computer program written in C running under Linux as
previously described in detail (18, 25). In brief, the program iteratively estimated the translation dx and dy and the rotation ␪ between
two adjacent images until the difference between the two images was
as small as possible. The transformation values between two adjacent
images were summarized into a set of absolute transformation values.
The absolute transformation values underwent a high-pass filtration to
avoid potential distortions of the image stacks as a result of small but
accumulating “trends.” Then, the images were transformed according
to these absolute transformation values (1–3, 32).
However, after this initial image aligning it turned out that this
classic image-aligning method was not sufficient to align the sections,
1931-857X/14 Copyright © 2014 the American Physiological Society
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Christensen EI, Grann B, Kristoffersen IB, Skriver E, Thomsen JS, Andreasen A. Three-dimensional reconstruction of the rat
nephron. Am J Physiol Renal Physiol 306: F664 –F671, 2014. First
published January 29, 2014; doi:10.1152/ajprenal.00522.2013.—This
study gives a three-dimensional (3D) structural analysis of rat
nephrons and their connections to collecting ducts. Approximately
4,500 2.5-␮m-thick serial sections from the renal surface to the
papillary tip were obtained from each of 3 kidneys of Wistar rats.
Digital images were recorded and aligned into three image stacks and
traced from image to image. Short-loop nephrons (SLNs), long-loop
nephrons (LLNs), and collecting ducts (CDs) were reconstructed in
3D. We identified a well-defined boundary between the outer stripe
and the inner stripe of the outer medulla corresponding to the transition of descending thick limbs to descending thin limbs and between
the inner stripe and the inner medulla, i.e., the transition of ascending
thin limbs into ascending thick limbs of LLNs. In all nephrons, a
mosaic pattern of proximal tubule (PT) cells and descending thin limb
(DTL) cells was observed at the transition between the PT and the
DTL. The course of the LLNs revealed tortuous proximal “straight”
tubules and winding of the DTLs within the outer half of the inner
stripe. The localization of loop bends of SLNs in the inner stripe of the
outer medulla and the bends of LLNs in the inner medulla reflected
the localization of their glomeruli; i.e., the deeper the glomerulus, the
deeper the bend. Each CD drained approximately three to six
nephrons with a different pattern than previously established in mice.
This information will provide a basis for evaluation of structural
changes within nephrons as a result of physiological or pharmaceutical intervention.
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RECONSTRUCTION OF THE RAT NEPHRON
Nephron length measurements. Each nephron was subdivided into
eight segments: proximal convoluted tubule (PCT), PST, mosaic part
(mosaic pattern of PST and DTL), DTL, ascending thin limb (ATL),
thick ascending limb (TAL), distal convoluted tubule (DCT), and
connecting tubule (CNT). The length of each segment was calculated
as the sum of the Euclidian distance between two subsequent markers
in the course of the nephrons. To reduce measurement noise arising
from the alignment procedures and the manual placement of the
markers, the path was smoothed using a triangular moving average
window, in such a way that the previous point in the path was
weighted with ¼, the current point with ½, and the next point in the
path with ¼.
Glomerular volume estimates. The glomeruli were not truly spherical, but more oval, round bodies. The margin of the glomeruli was
therefore manually outlined by a polygon using the tracing program.
The glomerular volume was then estimated using Cavalieri’s principle
(8), i.e., as the area of the polygons times the section thickness.
RESULTS
Figure 1 depicts a screen dump from the tracing program
showing a cortical section (573 ␮m from the renal surface).
The red numbers identify which nephron the tubular cross
sections belong to.
Figure 2A shows a 3D plot of all CDs (pink) and CNTs
(blue) traced in one of the three kidneys. The CDs start to join
with each other and with other CDs (white) at the transition
from the outer medulla (OM) to the IM.
In Fig. 2B, all the SLNs (red) and LLNs (blue) traced in the
same kidney are shown together with their CDs. A unique
pattern of mosaic transitions from the proximal tubule (PT) to
the DTL (mixture of PT and DTL cells; see also later, yellow)
takes place inside a very narrow region (⬃2,800 ␮m from the
renal surface, mean of the 3 kidneys), marking the border
between the outer stripe of the outer medulla (OSOM) and the
inner stripe of the outer medulla (ISOM). Similarly, the transitions from the ATL to the TAL, corresponding to the loop
Fig. 1. Screen dump from the interactive
tracing computer program showing a section
through the renal cortex (573 ␮m from the
renal surface) illustrating all the tubules
traced in that area (nephron numbers shown
in red). Bar ⫽ 100 ␮m.
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as the sectioning process had induced distortions in the relatively large
plastic-embedded sections. Therefore, an additional image-aligning
procedure was applied to the aligned sections (27) to compensate for
the distortions induced by the sectioning process. In brief, five
landmarks (e.g., CDs) were identified in two adjacent sections. Each
section was subdivided into four polygons, each with four corners.
Then, a nonlinear image transformation similar to the one used for
image morphing was applied to each polygon, thus aligning the two
adjacent sections. This refined image aligning was also carried out
using custom-made software running under Linux.
After both image-aligning procedures, the images were 2,750 ⫻
2,500 pixels large, with an isotropic pixel size of 1.53 ␮m.
Digital tracing and 3D presentation. The tracing of the nephron
paths was performed interactively using custom-made software on the
Linux system (32). Calculation of the length of nephron segments and
glomerular volume (see below) were integrated features of this software. In addition, the nephron path could be exported from the tracing
software to the free 3D plotting program PlotMTV. The tracing was
performed by manually placing a marker in the transversal tubule
lumen along the course of the tubule. For each marker, the x-, y-, and
z-coordinates were recorded in a data file, which thus documented the
paths of all traced nephrons in three dimensions. In addition, the
transitions between each segment of the nephrons were recorded
manually in a separate data file. The tubular profiles and the transitions between different segments were identified from the structure in
the digital images.
The nephron tracing was conducted in so-called “families” (32). A
“family” is defined as a CD and the nephrons that drain into the CD
by the connecting tubules (CNTs) at the cortical level. The nephron
tracing started at the end of the CNTs draining into a CD located in the
center of the section, and ended at the glomerulus. The CD was then
traced from the cortex toward the inner medulla (IM), and then these
two paths were merged.
3D reconstruction and visualization of histological sections from
the mosaic pattern of the proximal straight tubule (PST) and the
descending thin limb (DTL) were performed with the visualization
software Amira (version 4.1.2, Mercury Computer Systems) on the
Linux system.
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RECONSTRUCTION OF THE RAT NEPHRON
bends for the SLNs, also take place inside a very narrow region
(4,270 – 4,541 ␮m, mean of the 3 kidneys), marking the border
between the ISOM and the IM very distinctly (see below).
The schematic drawing in Fig. 3 illustrates the location of
the glomeruli in the renal cortex and the location of the
corresponding loop bends in the medulla in one of the three
kidneys (a total of 56 traced nephrons). The size of the filled
circles in the figure indicates the glomerular volume. The
numbers next to the circles give the distance from the bend to
the renal surface. The largest glomeruli with their corresponding longest nephrons are located between or even more deeply
than the arcuate vessels, in the OSOM. The arcuate vessels,
marking the border between cortex and the OSOM, are located
1,360 –2,975 ␮m from the renal surface (mean of the 3 kidneys). Nephrons from the outer 30% of the cortex (yellow)
have their bend located in a very narrow region [4,000 – 4,350
␮m from the renal surface (in this kidney)], i.e., at the border
between the ISOM and IM. The nephrons in the midcortical
region (30 – 60%, red) have their bends located progressively
more deeply in the IM, the deeper the location of the glomerulus. The juxtamedullary nephrons, i.e., the nephrons starting
at the inner 40% of the cortex (blue), have their loop bend
located progressively even more deeply, the deeper the location of the glomerulus. In Fig. 3, the nephrons are grouped in
Fig. 3. Drawing illustrating 56 traced nephrons from
1 kidney. The size of the filled circles indicates the
glomerular volume. The numbers besides the circles
give the location of the bend (in ␮m) from the renal
surface. Yellow circles indicate location of glomeruli in outer 30% of the cortex, red circles indicate
midcortical nephrons (30 – 60%), and blue circles
indicate juxtamedullary nephrons of the inner 40%
of the cortex. The nephrons are grouped in “families” (aligned vertically). In the figure, 14 families
are shown. The y-axis gives the glomerular distance
(in ␮m) from the renal surface.
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Fig. 2. A: 3-dimensional (3D) plot showing the course
of 14 collecting ducts (CDs; pink) and their connecting
tubules (CNTs; blue) in one rat kidney. The CDs starts
to merge with each other at the transition from the outer
medulla (OM) to the inner medulla (IM). Further fusions with other CDs are marked with white lines and
arrows. In B, 56 short-loop nephrons (SLNs; red) and
long-loop nephrons (LLNs; blue) are added; white,
glomeruli; vellow, mosaic zone. The bends of the longest LLNs are located in the innermost region of the
papilla, whereas the bends of the SLNs are located in a
very narrow region at the transition between the OM
and the IM. See also the numbers besides the glomerular
markings in Figs. 3 and 5A. The ordinate gives the
distance in millimeters from the renal surface. The renal
zones are indicated on the right.
RECONSTRUCTION OF THE RAT NEPHRON
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“families” (aligned vertically), i.e., the nephrons that drain into
the same cortical collecting duct (CCD). In general, each
family consists of three to six nephrons, two to four SLNs, and
one to three “LLNs.” However, a few families consisted of 7,
8, or even 11 nephrons. All three kidneys showed the same
family pattern.
There is a linear correlation between the glomerular volume
and the localization of the glomerulus (Fig. 4A) so that the
deeper the location, the larger the glomerulus, r2 ⫽ 0.61.
Similarly, there is a linear correlation between the glomerular
volume and the nephron length (Fig. 4B), r2 ⫽ 0.66; between
the glomerular volume and the PT length (Fig. 4C), r2 ⫽ 0.67
(see also below concerning very large glomeruli); and between
the glomerular localization (depth) and the localization of the
bend (Fig. 4D), r2 ⫽ 0.92.
The lengths of the different segments in all 159 traced
nephrons are shown in Fig. 5A and in Table 1, subdividing the
nephrons into cortical (outer 30%), midcortical (next 30%),
and juxtamedullary nephrons (inner 40% of the cortex). Figure
5B illustrates the length of the various nephron segments
relative to the length of the whole nephron. The length measurements demonstrate that the length of the PT (⬃13.7 mm) is
surprisingly similar for the three different types of nephrons.
The difference in length of the whole nephron between the
cortical and midcortical, ⬃28 mm, vs. the juxtamedullary, ⬃37
mm, is mainly due to the long DTLs and ATLs of the
juxtamedullary nephrons.
The location of the bends is also indicated in Fig. 5. For the
SLNs, which also include some of the outer midcortical
nephrons, most bends are located at the transition between the
DTL and the TAL, i.e., they have no ATL. In a few nephrons,
the transition from DTL to TAL takes place before the bend,
and a few SLNs have a very short ATL. It is striking that the
bend is localized at ⬃60% of the whole nephron length
regardless of the type of nephron (Fig. 5B).
The length of the mosaic pattern at the transition from the PT
to the TDL (see also below) was highly variable, but the
mosaic pattern was nevertheless found in all nephrons, as seen
in Fig. 5. The length of the CNT appears to be similar for the
superficial, midcortical, and juxtamedullary nephrons. However, these lengths were measured from the end of the DCT to
the first fusion with either the CCD or another CNT from the
same family, forming an arcade. In all traced families (35 in the
3 kidneys), juxtamedullary and midcortical nephrons formed
arcades, as demonstrated for the family present in Fig. 6,
consisting of five nephrons. In general, all CNTs or arcades
connected to the CCDs in the outer cortex, and from then on,
there was no joining of CDs until the IM, where the medullary
collecting ducts (MCDs) started to join.
The volume of the glomeruli is indicated on Fig. 3. The
equivalent diameters of the glomeruli (calculated from the
glomerular volume assuming that the glomeruli can be represented as spheres) was very similar for the cortical, 134 ⫾ 2.6
␮m, midcortical, 133 ⫾ 1.0 ␮m, and juxtamedullary, 145 ⫾
1.7 ␮m nephrons. However, a few of the juxtamedullary
glomeruli were very large; five of them were ⬎160 ␮m in
diameter, the largest being 178 ␮m. Their nephrons were also
longer than the other juxtamedullary nephrons, ranging from
⬃44 to 50 mm compared with a mean of 37 mm for the
juxtamedullary nephrons (Table 1). This was in part due to the
long thin limbs of these nephrons as they bent very deeply into
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Fig. 4. Relationship between different morphological nephron parameters. A:
glomerular volume vs. glomerular depth; r2 ⫽ 0.61. B: nephron length vs.
glomerular volume; r2 ⫽ 0.66. C: proximal tubule length vs. glomerular
volume; r2 ⫽ 0.67. D: location of bend vs. glomerular depth; r2 ⫽ 0.92.
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RECONSTRUCTION OF THE RAT NEPHRON
Fig. 5. A: absolute lengths of the individual segments of all traced nephrons for
the 3 kidneys. B: length of the individual segments relative to the total length
of the nephrons for the 3 kidneys. Top: cortical nephrons; middle, midcortical
nephrons; bottom, juxtamedullary nephrons. Dark blue, proximal convoluted
tubule (PCT); light blue, proximal straight tubule (PST); yellow, mosaic zone;
dark green, descending thin limb (DTL); light green, ascending thin limb
(ATL); red, thick ascending limb (TAL); pink, distal convoluted tubule (DCT);
blue, CNT. The black lines show the bend location.
the IM, and in part due to longer PT, from 16.3 to 17.5 mm
compared with a mean of 13.9 mm for the juxtamedullary
nephrons (Table 1). Similarly, the DCTs were longer, from 2.7
to 3.8 mm, compared with a mean of 2.2 mm for the juxtamedullary nephrons (Table 1).
In the 3D plot presented in Fig. 7, the course of a SLN and
a LLN is shown. Specific colors (compare with Fig. 5), representing seven well-defined segments along the nephron, were
used in the 3D plot: PCT (dark blue), PST (light blue), mosaic
zone (yellow), DTL (dark green), ATL (light green), TAL
(red), DCT (pink), and the CNT (blue). The transition from one
segment to another was readily recognized during the tracing.
However, as we have previously demonstrated in the mouse
(29), a straight part of the PT of LLNs was often difficult to
define as it was convoluted along nearly its entire length.
Similarly, the initial part of the DTL of LLNs demonstrated a
tortuous course for a certain distance within the ISOM (Fig. 7),
as also seen in the mouse (32).
Table 1. Lengths of segments of cortical, midcortical, and juxtamedullary nephrons in 3 rat kidneys
Proximal tubule, ␮m
Convoluted portion, ␮m
Straight portion, ␮m
Mosaic, ␮m
Descending thin limb, ␮m
Ascending thin limb, ␮m
Thick ascending limb, ␮m
Distal convoluted tubule, ␮m
Connecting tubule, ␮m
Total nephron length, ␮m
Cortical
Midcortical
Juxtamedullary
14,724 ⫾ 1,331
9,515 ⫾ 748
5,209 ⫾ 674
233 ⫾ 59
2,633 ⫾ 253
71 ⫾ 115
7,613 ⫾ 448
2,051 ⫾ 196
1,760 ⫾ 419
29,085 ⫾ 2,020
12,494 ⫾ 357
7,606 ⫾ 782
4,888 ⫾ 571
307 ⫾ 90
4,348 ⫾ 906
1,232 ⫾ 588
6,739 ⫾ 486
1,659 ⫾ 149
1,073 ⫾ 206
27,854 ⫾ 1,163
13,927 ⫾ 674
9,790 ⫾ 651
4,137 ⫾ 589
476 ⫾ 123
8,785 ⫾ 1,028
5,318 ⫾ 765
5,239 ⫾ 381
2,243 ⫾ 373
1,178 ⫾ 456
37,162 ⫾ 616
Values are means ⫾ SE.
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Fig. 6. In all the 35 traced families, the juxtamedullary and midcortical
nephrons formed arcades. In general, all CNTs or arcades connected to the
CCDs in the outer cortex and from then on, there was no joining of CDs until
the IM, where the medullary collecting ducts (MCDs) started to join. Light red,
thick ascending limb of Henle’s loop (TAL): white, macula densa; red, DCT;
blue, arcades; yellow, CD.
RECONSTRUCTION OF THE RAT NEPHRON
The unique mosaic pattern of PT cells and DTL cells in the
initial DTL is shown as a 3D reconstruction in Fig. 8. This
mosaic pattern was found in all traced nephrons, and the length
of this mosaic pattern varied between ⬃40 and 1,000 ␮m (see
also Fig. 5A, Table 1, and Fig. 2). The length of the mosaic
pattern was not related to the type of nephron (SLN or LLN).
DISCUSSION
The present study gives the results of histological analyses
and computer-assisted tracing and 3D reconstruction of 159
nephrons and 35 collecting ducts from 3 rat kidneys. This
combination of histological and computer techniques provides
an enhanced understanding of the exact spatial structure of
individual nephrons, the interrelation of the nephrons, the exact
lengths of nephron segments, and a morphological analysis at
well-defined segments along the course of the nephrons. This
information will provide the basis for the evaluation of structural changes within the nephron as a result of physiological
and pharmaceutical intervention.
The glomeruli were relatively constant in size, ⬃133 ␮m in
diameter for cortical and midcortical nephrons and 146 ␮m in
diameter for juxtamedullary nephrons, with the exception of
some very large (up to 178 ␮m) juxtamedullary glomeruli. The
nephron segments PT, thin limbs, and DCT in these nephrons
were also significantly longer than the mean for juxtamedullary
nephrons, indicating that the increased glomerular filtration in
these individual nephrons is reflected in an increased nephron
length (4). In the human kidney, there seems not to be a cortical
zonal difference in glomerular size (24), and therefore it is
difficult to predict a further pathophysiological importance for
our finding of the very large juxtamedullary glomeruli, which
to our knowledge has not been described in the human kidney.
The segmentation of the rat PT has been described in
detail by Maunsbach (16), subdividing it into S1, S2, and
S3, although in the present study we chose to only subdivide
the PT into a convoluted and a straight portion. However,
our subdivision into nephrons originating from three cortical
zones revealed that there was no difference in total length of
the PTs regardless of whether they originated from the
cortical, midcortical, or juxtamedullary zone (Fig. 5 and
Table 1). This is in accordance with results in rats from
Dørup and Maunsbach (7) and from Sperber (26), but in
contrast to our findings in mice (29).
The relatively short, straight portion of the PT of the juxtamedullary nephrons is likely due to the fact that the straight
portion may be difficult to define since it was convoluted along
nearly the entire length, as we have also previously described
in the mouse (29).
The mosaic pattern of PT cells and DTL cells in the initial
DTL has, to the best of our knowledge, never been described
before. What made this observation possible in the rat is the
serial section technique applied in the present study. In contrast, we did not observe a similar mosaic pattern in the mouse
using the same technique (32). The mosaic pattern was seen in
all nephrons studied and is probably a developmental feature,
and it is difficult to see any functional implications. This
mosaic pattern may be similar to the features seen, e.g., at the
CNT with a mixture of CNT cells, principal cells, and intercalated cells, as recently reviewed by Christensen et al. (5).
The general structure of the rat kidney, including location of
loop bends for different types of nephrons, has previously been
described (see e.g., Refs. 5, 10, 12, 13, and 26). Similarly, the
medulla has also been studied in detail (see e.g., Ref. 14).
However, the exact localization of nephron segments and exact
measurements of lengths for such a large number of whole
nephrons as performed in the present study has, to the best of
our knowledge, not been carried out before.
With respect to the CNT formation of arcades, the rat has
generally been supposed to be intermediate (10) between e.g.,
rabbits and pigs, in which virtually all CNTs with the exception
of the most superficial nephrons form arcades (21) and Psammomus obesus, in which only the deepest nephrons form
arcades (9). In a study in rats by Dørup (6) with emphasis on
superficial nephrons, he presented five families, of which two
did not form arcades. In the present study, we found that all 35
traced families formed arcades, which in our opinion would
place the rat in the same category as the rabbit and the pig with
respect to arcades. As suggested by Kaissling (9), the formation of arcades in the cortical labyrinth probably serves to
prevent addition of dilute urine from midcortical and juxtamedullary nephrons to CDs in the deep cortex.
As also discussed by Kaissling (9), in species with a scarcity
of arcades like P. obesus and humans, a proper fluid concentration in CDs is ensured by principal cells with antidiuretic
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Fig. 7. 3D plot of a SLN and a LLN. Dark blue, proximal tubule (PCT⫹PST);
yellow, mosaic zone; dark green, DTL⫹ATL; red, TAL; pink, DCT⫹CNT.
Ordinate gives the distance (in mm) from the renal surface.
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RECONSTRUCTION OF THE RAT NEPHRON
hormone sensitivity/aquaporin-2 expression and other channels
and transporters also in the intercalated cells in the CNTs.
In summary, due to the large number of nephrons analyzed
in detail, the present study gives an excellent platform for
future studies (see also, e.g. Ref. 19 on rat renal function giving
the exact 3D interrelations of nephrons and the localization and
lengths of distinct nephron segments in the different zones of
the rat kidney). The results presented here and the material
used will form the basis for future studies on the exact location
of different transporters and other membrane proteins as well
as ultrastructural studies at exact locations in the nephrons as
we have already done in the mouse (22, 23, 29 –32), of great
importance for an understanding of renal function.
ACKNOWLEDGMENTS
Portions of this work were presented at the 2009 annual meeting of the
American Society of Nephrology and were published in abstract form (abstract
no. SA-PO2155: J Am Soc Nephrol 20: 604A, 2009).
GRANTS
This work was supported by a grant from The Danish Council for Independent Research, Medical Sciences (FSS 11-104255 to E. I. Christensen) and
by “Maskinfabrikant Jochum Jensen og hustru Mette Marie Jensen,” “F.
Poulsens Mindelegat,” “Søster og Verner Lipperts Fond,” and “Bagenkop
Nielsens Myopi-Fond” (to A. Andreasen). The Amira visualization system was
donated by the Toyota Foundation (to A. Andreasen).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: E.I.C., J.S.T., and A.A. provided conception and
design of research; E.I.C., B.G., I.B.K., E.S., J.S.T., and A.A. performed
experiments; E.I.C., B.G., I.B.K., E.S., J.S.T., and A.A. analyzed data; E.I.C.,
B.G., I.B.K., E.S., J.S.T., and A.A. interpreted results of experiments; E.I.C.,
B.G., I.B.K., J.S.T., and A.A. prepared figures; E.I.C., J.S.T., and A.A. drafted
manuscript; E.I.C., B.G., I.B.K., E.S., J.S.T., and A.A. edited and revised
manuscript; E.I.C., B.G., I.B.K., E.S., J.S.T., and A.A. approved final version
of manuscript.
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