Embryonic origin and lineage of juxtaglomerular cells
Maria Luisa S.
Sequeira Lopez,
Ellen
S.
Pentz,
Barry
Robert,
Dale R.
Abrahamson, and
R. Ariel
Gomez
Department of Pediatrics, University of Virginia School of
Medicine, Charlottesville, Virginia 22908; and Department of Anatomy
and Cell Biology, University of Kansas Medical Center, Kansas City,
Kansas 66160
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ABSTRACT |
To define
the embryonic origin and lineage of the juxtaglomerular (JG) cell,
transplantation of embryonic kidneys between genetically marked and
wild-type mice; labeling studies for renin, smooth muscle, and
endothelial cells at different developmental stages; and single cell
RT-PCR for renin and other cell identity markers in prevascular kidneys
were performed. From embryonic kidney day 12 to day
15 (E12 to E15), renin cells did not yet express smooth muscle or endothelial markers. At E16 renin
cells acquired smooth muscle but not endothelial markers, indicating that these cells are not related to the endothelial lineage, and that
the smooth muscle phenotype is a later event in the differentiation of
the JG cell. Prevascular genetically labeled E12 mouse
kidneys transplanted into the anterior chamber of the eye or under the kidney capsule of adult mice demonstrated that renin cell progenitors originating within the metanephric blastema differentiated in situ to
JG cells. We conclude that JG cells originate from the metanephric
mesenchyme rather than from an extrarenal source. We propose that renin
cells are less differentiated than (and have the capability to give
rise to) smooth muscle cells of the renal arterioles.
renin; differentiation; mouse; vessels; kidney
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INTRODUCTION |
THE JUXTAGLOMERULAR
(JG) CELL is one of the components of the JG apparatus.
This cell is located in the wall of the afferent arteriole at the
entrance to the glomerulus (15, 33). JG cells synthesize
and release renin from storage granules (18, 21, 33).
Renin is a hormone enzyme that initiates the enzymatic cascade that
generates the angiotensin peptides that regulate blood pressure, renal
hemodynamics, and electrolyte balance. In addition to renin, adult JG
cells also contain myofilaments, peroxisomes, small electron dense
vesicles, and few mitochondria (33). They are connected to
arteriolar smooth muscle cells, endothelial cells, and other JG cells
by gap and myoendothelial junctions. JG cells are round, plump, and
epithelioid in nature (33). Although renin has been the
characteristic marker of JG cells, other markers have been cloned such
as Zis (Zinc finger Splicing
factor), which is a developmentally regulated gene expressed in JG
cells (19).
It has been postulated that JG cells derive from smooth muscle cells
because in the adult mammal they contain myofilaments (33); however, no studies have been performed to determine
the lineage of these cells.
In the fetal kidney of mammals, renin cells are widely
distributed along the walls of large renal arteries and afferent
arterioles (7, 13, 26, 28), in contrast to the typical
adult JG localization (4, 34). An association between
renin cells and the branching of renal arterioles has been described,
suggesting that these cells play a role in the development of the
kidney vasculature (27). We have observed that in the
fetal rat at embyronic day 14 (E14) renin cells
are also present in the kidney interstitium before vessel formation has occurred.
Although it has been suggested that glomerular capillaries develop from
an intrinsic precursor (30), the origin of the renal arteriolar endothelium, the smooth muscle of the whole kidney vasculature, and the renin cells is unknown.
It is well known that embryonic kidneys (E12 mouse,
E14 rat) in culture systems undergo nephrogenesis,
developing tubules and glomeruli. Unfortunately, under the usual
culture conditions, in this otherwise excellent model, there is no
vessel formation and therefore renin cells do not assemble into
arterioles, remaining dispersed in the interstitium. Although
interstitial and glomerular capillaries may develop in vitro under
certain specific conditions such as exposure to vascular endothelial
growth factor (VEGF) (35) or to a low oxygen concentration
(3% O2) (36), renal arterioles do not form
uniformly. Therefore, we chose a transplantation model of prevascular
embryonic kidneys to define the embryonic origin of JG cells.
Furthermore, we utilized single cell PCR and double immunostaining
combined with lineage markers to define the lineage of the JG cell.
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MATERIALS AND METHODS |
Animals
To study both the lineage and the embryonic site of origin of
the JG cell, we utilized several mouse strains expressing clearly identifiable markers such as LacZ or green
fluorescent protein (GFP) in specific cell types as detailed in Table
1.
Timed-pregnant Sprague-Dawley rats were purchased from Hilltop Farms
(Scottdale, PA). Embryos at 14 days of gestation were the source of
fetal kidneys used to aspirate single cells and perform single cell
RT-PCR to monitor the expression of cell identity markers. Time-dated
pregnant mice and rats were mated overnight, and the females were
checked for vaginal plugs the following morning. The day of detection
of a vaginal plug was regarded as day 0 of gestation. All
mice were fed regular mouse chow (Prolab 2000, PMI Feeds, St. Louis,
MO) and tap water ad libitum and housed in a temperature-controlled
(22 ± 2°C) environment with a 12-h light/dark cycle. All
procedures were performed in accordance with the guidelines of the
American Physiological Society and were approved by the University of
Virginia Animal Care Committee.
Grafting of Embryonic Kidneys
Grafting into the anterior chamber of the eye.
Allografts (n = 11) of fetal kidneys into the anterior
eye chamber were performed as described previously (1).
Briefly, adult C57 Bl6/6J hosts were anesthetized by intraperitoneal
injection of a ketamine-xylazine combination (100 and 15 mg,
respectively, per kg body wt), and then tropicamide was applied to the
mouse eye to dilate the iris. The cornea was incised with a 27-gauge needle, and the incision was extended 2 mm with Vannas scissors. Freshly harvested embryonic (E12) prevascular kidneys were
placed into the anterior eye chamber of a host mouse via the corneal incision and positioned over the iris. Antibiotic (neomycin and polymyxin B sulfates, and bacitracin zinc) ophthalmic ointment was
applied to the eye, and grafts were allowed to develop in oculo for 8 days. After the animals were killed, grafts were removed, fixed, and
embedded in paraffin as previously described (14, 37), and
processed for immunohistochemistry as described in Immunohistochemistry.
Grafting under the kidney capsule.
To define whether vascular progenitors originating within the
metanephric mesenchyme differentiate in situ to JG cells, smooth muscle
cells, and endothelial cells, we cross-transplanted kidneys between
wild-type and transgenic mice expressing LacZ in endothelial cells (Flk1+/
LacZ mice) or all cells (Rosa 26 mice) and GFP in renin cells (see Table 1). Metanephric
(E12) kidneys were grafted under the kidney capsule of adult
mice (host: Rosa 26 or Flk1+/
, donor: E12
kidneys from C57 Bl/6J and vice versa; and host: C57 Bl/6J, donor:
E12 kidneys from Ren-GFP) (see Table 2). Donor and host mice were anesthetized
by intraperitoneal injection of tribromoethanol (300 mg/kg)
(11). Metanephric (E12) kidneys were dissected
aseptically at 37°C in serum-free organ culture medium [DMEM/F-12
(GIBCO-BRL no. 430-2500EG) with 10 mM HEPES (Sigma H9136), 1.1 mg/ml
NaHCO3, 50 U/ml penicillin, 50 U/ml nystatin,
insulin-transferrin-selenite (Sigma I1884; 5 µg/ml insulin and
transferrin, 2.8 nM selenite), 25 ng/ml PGE1, and 32 pg/ml
triiodothyronine (T3)]. In the host, an incision was made
along the dorsal lumbar side above the kidney, the muscle layers
overlaying the kidney were dissected, the left kidney was exteriorized,
and a small incision was made in the renal capsule. A blunt 20-gauge
needle was gently inserted into the incision to create a 1-cm
subcapsular tunnel towards the upper pole of the kidney and another one
towards the lower pole where the embryonic kidneys (E12)
were placed with forceps. Usually, from two to three embryonic kidneys
were transplanted in this fashion. The host kidney was replaced into
the abdomen, and the muscle layers and the skin were sutured
separately. The animals were allowed to recover from the anesthesia on
a heating pad at 37°C. Subcapsular grafts were allowed to undergo
nephrovascular development for 7-8 days.
5-Bromo-4-Chloro-3-Indolyl
-D-Galactopyranoside
Reaction
The relationship of the JG cell with the endothelial lineage was
defined using mice expressing LacZ in endothelial cells
(Flk1+/
LacZ and
Tie2-LacZ mice) (see Table 1).
Kidneys from mice carrying transplants (left kidneys with grafts and
right kidneys as controls) performed between Rosa 26 or
Flk1+/
and C57 Bl/6J mice (Rosa 26
B6 and
Flk1+/
B6), and kidneys from Flk1+/
and
Tie2 mice were harvested from anesthetized mice as described
above, decapsulated, sectioned in 2-mm slices, and fixed for 15 min in
3.7% formaldehyde. After being washed 3 times for 15 min each in
detergent rinse (0.1 M phosphate buffer, pH 7.4, containing 2 mM
MgCl2, 0.01% sodium deoxycholate, and 0.02% tergitol
NP-40), the tissue was placed in staining solution [detergent rinse, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/ml
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-Gal;
Fisher Biotech) in dimethylformamide] overnight in the dark at 37°C.
The tissue was then washed three times for 15 min each in PBS,
postfixed in 3.7% formaldehyde at 4°C overnight, dehydrated in
graded alcohols to xylenes, and embedded in paraffin. On the X-Gal
reaction thus performed, cells expressing
-galactosidase turn blue.
After the X-Gal reaction, the tissues were subjected to
immunohistochemistry for renin and
-smooth muscle actin (
-SMA). Coincidences or discrepancies among blue cells and cells immunostained for those markers were evaluated.
Fluorescence Microscopy
Ren-GFP
B6 transplanted kidneys were harvested as
described above and fixed overnight in 4% paraformaldehyde, then
cryoprotected in 30% sucrose at 4°C for 24 h, placed in an
optimal cutting temperature compound (OCT; Miles, Elkhart, IN), and
stored at
20°C as previously described (15). Then
10-µm frozen sections were observed with a fluorescence
microscope. With this technique, renin-expressing cells fluoresce
bright green in a light green background.
Immunohistochemistry
To define whether renin cells express smooth muscle proteins,
immunohistochemical detection for
-SMA and renin was performed on
consecutive sections of kidneys carrying embryonic transplants (Rosa 26
B6, B6
Rosa26, Flk1+/
B6, B6
Flk1+/
), and on Flk1+/
mice kidneys as
described previously (14, 31). Briefly, 5-µm
kidney tissue sections were deparaffinized in xylenes and graded
alcohols. Endogenous peroxidase activity was quenched by incubation
with 0.3% hydrogen peroxide, and sections were incubated with a
specific anti-rat-renin polyclonal antibody made in goat (dilution
1:10,000; kind gift of Dr. T. Inagami, Nashville,TN) or a monoclonal
anti-
-SMA-specific antibody (isotype IgG2a, dilution 1:10,000; clone
1A4, lot no. 076H4843, Sigma, St. Louis, MO). After addition of the
secondary biotinylated antibody (biotin-conjugated anti-goat IgG
for renin staining and biotin-conjugated anti-mouse IgG for
-SMA
staining, both from Vector Lab, Burlingame, CA), the sections were
incubated with avidin-biotinylated horseradish peroxidase complex
(Vectastain ABC kit, Vector Laboratories) and then exposed to 0.1%
diaminobenzidine tetrahydrochloride and 0.02% hydrogen peroxide as a
source of peroxidase substrate. Each section was counterstained with
nuclear fast red (Vector Laboratories), dehydrated through graded
alcohols to xylenes, and mounted with Permount. As negative controls,
the primary antibody was replaced by 3% BSA in PBS.
Double immunostaining for both renin and
-SMA on the same tissue
section was performed on kidney sections from mice at different embryonic and postnatal (N) ages (E14-E16, E18,
N1, N5, N10, N21, N45, and N70, n = 3 to 5 animals for each age). This procedure was performed as described
above through the peroxidase immunohistochemistry reaction for renin.
After the first reaction, the sections were microwaved (3 cycles, 1 min
each) in antigen retrieval solution (0.01 M sodium citrate buffer, pH
6), and then a second immunodetection was performed by the method
described above for
-SMA using a peroxidase substrate, which
generates a different color reaction product (VIC purple, Vector Lab).
The tissue was not counterstained, and was directly dehydrated through
graded alcohols to xylenes and mounted with Permount. Using this
procedure, renin-containing cells are purple and smooth muscle cells
will be brown or vice versa depending on which antibody was used first.
Single Cell RT-PCR of Cells Aspirated From Embryonic Kidneys
Embryonic kidneys at 14 days of gestation were harvested
from Sprague-Dawley timed-pregnant rats. The kidneys were
placed on top of a membrane placed in an organ culture dish over 1.5 ml
of organ culture medium, as described in grafting of embryonic kidneys.
Then the filter with the kidneys was removed from the culture dish and
transferred to a 35-mm petri dish, and 100-200 µl of organ
culture medium were added over the kidneys and under the
membrane. The embryonic kidney was viewed using an inverted microscope
(Nikon Diaphot 300), and the cells were aspirated individually into a
borosilicate capillary pipette backfilled with 2 µl of lysis buffer
(2.5% Triton X-100, 5 mM dithiothreitol, and 1.2 U/µl RNAsin in
RNAse-DNAse-free water) using a Nikon Narishige Micromanipulator
attached to a PLI-100 Pico-Injector (Medical System, Greenvale, NY).
After aspiration, the tip of the pipette containing the cell was
immediately broken off into a 0.6-ml microcentrifuge tube containing 8 µl of lysis buffer. The samples were snap-frozen in liquid nitrogen
and immediately stored at
80°C. Reverse transcription was
performed as follows: 1 µl (0.5 µg) oligo (dT) (Promega,
Madison, Wisconsin) was added to the cell aspirate, and the solution
was heated for 5 min at 65°C and chilled on ice to anneal the primer. The reverse transcription reaction (20-µl final volume) containing cell lysate+oligo (dT), 1× RT buffer, 0.25 mM dNTP, and 400 U Moloney murine leukemia virus RT (Promega, Madison, Wisconsin) was
incubated for 10 min at 23°C, 60 min at 42°C, and 10 min at 94°C,
and stored at
20°C.
Nested PCR was performed on the RT reactions to test for the presence
of the lineage marker mRNAs:
-SMA (6, 24) and myosin
heavy chain (MHC) (3) for smooth muscle cells, Ets1 (20) and vimentin (5, 16) for mesenchymal
cells, and tenascin (2, 9) for interstitial cells. Each
individual sample was subjected to two PCR reactions: one for renin and
the second for a lineage marker. The basic PCR reaction (using either
outer primers or nested primers) contained 1× PCR buffer, 0.1 mM
dNTPs, and 1.5 units Taq DNA polymerase (Promega, Madison,
Wisconsin) in a volume of 50 µl, and PCR amplification was carried
out for 40 cycles. The volume of template was added, and the
concentration of MgCl2 and primers and the cycling
parameters were adjusted for the marker in question. (See Table
3 for the primers, specific PCR
conditions, and volume of template used.) Depending on the abundance of
the mRNA to be detected, 2 to 20 µl of the RT reaction were used as a
template in the first PCR reaction. Twenty microliters of the first PCR
reaction were used as a template in the second PCR reaction with the
nested primers.
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RESULTS |
Lineage of the JG Cell
Staining for lineage markers.
Double immunostaining of mouse kidneys for both renin and
-SMA at
different embryonic and postnatal ages (E14-E18, N1, N5, N10, N21, N45, N70) showed that at early embryonic ages
(E14, E15) renin-expressing cells are large,
either round or oval shaped, and found among undifferentiated
mesenchymal cells usually as single isolated cells or in small groups
of two to three cells (Fig. 1). These
cells are distributed in the mesenchyme throughout the entire kidney.
They can be found close to forming vessels but definitely separated
from smooth muscle cells (Fig. 1, inset). They are also seen
inside the forming glomeruli (Fig. 1). At E16 we can
identify two populations of renin-expressing cells: some are still
isolated but others are found in groups close to the forming vessels
and glomeruli (not shown). Some renin-expressing cells within the
vessels contain
-SMA (Fig. 2). Thus at
this developmental stage three types of cells expressing renin and/or
-SMA can be found: one type expressing only renin, another
expressing only
-SMA, and a third cell type expressing both markers.
By 18 days of gestation, isolated cells expressing solely renin can no
longer be found. As shown in Fig. 3, by
E18, renin-expressing cells are mostly associated with the
vasculature. However, they can still be found inside some glomeruli and
in the interstitium. Renin cells at this embryonic age are found mainly
in large arteries, whereas in the adult kidney they are found in their
classic JG localization (Fig. 4). The
above findings are supported by immunohistochemistry for renin or
-SMA performed individually on consecutive sections at the same
embryonic and postnatal ages as the double immunostaining referred to
above. These experiments demonstrated that renin-expressing cells begin
to express
-SMA at E16, and expression of both proteins is maintained thereafter in the mature JG cells. As described below,
the lack of coincidence between smooth muscle and renin expression in
embryonic renal cells was confirmed by single cell RT-PCR experiments
performed in rat embryonic kidneys at E14, a time where no
arterioles are present in the rat kidney.

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Fig. 1.
Embryonic day 15 (E15) mouse kidney double
immunostained for renin and -smooth muscle actin ( -SMA). Renin
cells (brown, arrows) are distributed in the mesenchyme as single
isolated cells. They are present inside the forming glomeruli and close
to the vessels (identified by -SMA staining in purple).
Inset: higher magnification of renin cell close to a forming
vessel. g, Glomerulus. Bars: 100 µm; inset: 25 µm.
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Fig. 2.
E16 mouse kidney
double immunostained for renin and -SMA. At this stage, renin cells
(brown) that have become part of a newly assembled vessel express
either only renin (arrow) or both renin and -SMA (arrowheads). Bar:
25 µm.
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Fig. 3.
E18 mouse kidney immunostained for renin and -SMA.
Double immunostaining for renin (brown, arrow) and -SMA (purple).
Renin cells are mainly associated with the vasculature (arrows).
Inset left: higher magnification showing the colocalization
of renin and -SMA. Inset right: consecutive section
immunostained only for renin (brown). Renin is present in the vessel at
the same location as -SMA. Bars: 50 µm; inset left:
25 µm; inset right: 50 µm.
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Fig. 4.
Adult (P70) mouse kidney double immunostained for renin
and -SMA. Renin cells (brown) are found in the typical
juxtaglomerular location (arrows). Some of these cells also express
-SMA (purple, arrowheads). Bar: 25 µm.
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Next, the lineage relationship between endothelial cells and renin
cells was investigated. For this purpose, kidneys from Flk1+/
mice (expressing
-galactosidase in endothelial
cells and their precursors during development, and maintained only in glomerular and peritubular capillaries in the adult; Ref.
29) and Tie2-LacZ mice (expressing
-galactosidase in all endothelial cells throughout life) were first
subjected to the X-Gal reaction and then immunostained for renin. No
coincidence between blue endothelial cells and renin immunostained
cells was found (Fig. 5). To study the
lineage relationship between smooth muscle and endothelial cells, the
same Flk1+/
mice kidneys were immunostained for
-SMA,
and Fig. 6A shows no
coincidence between blue endothelial cells and smooth muscle cells
stained in purple. Similar results were obtained using
Tie2-LacZ mice kidneys. Figure 6B
shows the distribution of
-SMA and
-galactosidase expression in
the adult kidney. Clearly, there is no coincidence between endothelial
cells and smooth muscle cells, in agreement with the studies shown
above using Flk1+/
mice. Confirming all these findings,
triple labeling for renin (brown),
-SMA (purple), and Flk1 (blue)
showed that renin cells at 5 days of postnatal life also contain
-SMA, but neither renin cells nor smooth muscle cells contain the
endothelial cell marker Flk1 (Fig. 7).

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Fig. 5.
Endothelial cells and renin cells in 5-day-old
Flk1+/ mouse kidney. The tissue was subjected first to the
5-bromo-4-chloro-3-indolyl -D-galactopyranoside (X-Gal)
reaction and then immunostained for renin (brown). The
immunolocalization of renin does not coincide with the blue endothelial
cells. Bar: 25 µm.
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Fig. 6.
Endothelial cells and smooth muscle cells in 5-day-old
Flk1+/ (A) and adult Tie2
(B) mouse kidneys. The tissue was subjected first to the
X-Gal reaction and then immunostained for -SMA. A: N5
Flk1+/ mouse kidney showing the immunolocalization of
-SMA (purple) not coinciding with the blue endothelial cells.
B: adult Tie2 mouse kidney immunostained for
-SMA (brown) showing no coincidence with the blue endothelial cells.
Bars: 25 µm.
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Fig. 7.
Triple labeling of endothelial cells, renin cells, and
smooth muscle cells in 5-day-old Flk1+/ mouse kidney. The
tissue was subjected first to the X-Gal reaction and then double
immunostained for renin (brown) and -SMA (purple). Blue endothelial
cells do not coincide with either renin- or -SMA-containing cells.
Some renin cells coexpress the -SMA protein (arrowheads).
Peritubular cells at this developmental age still express -SMA
protein. Bar: 50 µm.
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In addition, triple labeling of Flk1+/
B6 transplants
grown for 7 days under the kidney capsule showed coincidence of some renin cells with the
-smooth muscle marker in arterioles but showed
no coincidence of endothelial cells staining (blue) with renin cells
(Fig. 8).

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Fig. 8.
Triple labeling of endothelial cells, renin cells, and
smooth muscle cells in E12 Flk1+/ mouse kidney
transplanted under the kidney capsule of an adult wild-type host for 7 days. The tissue was subjected first to the X-Gal reaction and then
double immunostained for renin (brown) and -SMA (purple). Blue
endothelial cells are derived from the grafted
Flk1+/ embryonic kidney, and they do not costain with
either renin or -SMA. However, some renin cells also contain -SMA
(arrowheads). Bar: 50 µm.
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Overall, using different approaches, the results show that there is no
coincidence of endothelial markers with renin cells.
Single cell RT-PCR.
To study the lineage of the JG cell we performed single cell RT-PCR of
potential JG cell precursors from rat embryonic kidney cells at
day 14 of gestation. Distribution and morphology of these cells resembled distribution of the renin cell. The potential JG cell
precursors were selected by their large size and granular morphology as
revealed by staining with the vital dye neutral red (Fig.
9). By immunostaining with renin antibody
in the whole prevascular metanephric kidney, we previously found that
some but not all of these large granulated cells contained renin.
Therefore, these cells were chosen for microaspiration, and some of
them did express renin (Table 4). As
shown in Table 4, the experiments (1-5) were designed
to test, in each single cell, the expression of renin and one of the
following cell markers:
-SMA, MHC, Ets1, vimentin, or tenascin. At
this prevascular stage of kidney development, all the markers were
already present in the metanephric kidney in a variety of cell types.
In experiments 1 and 2, none of the cells that
expressed smooth muscle markers (either
-SMA or MHC) were positive
for renin, and cells expressing renin tested negative for smooth muscle
markers. Experiment 3 showed that 50% of renin-expressing cells coexpressed the Ets1 marker and 50% did not. The transcriptional factor Ets1, known to be present in most mesenchymal cells, was widely
distributed among these embryonic cells, with more than one-half of all
the studied cells (25/41) expressing Ets1. In experiment 4,
1 out of 8 cells expressing tenascin was also positive for renin, and
in experiment 5, cells positive for vimentin did not express
renin. Overall, the number of cells expressing renin for the combined
five experiments were 18 out of 123 cells picked, which represents
~15%. These results confirmed the presence of progenitors of renin
cells, as well as other cell types identified by different cell
markers. In fact, they demonstrate the presence of vascular precursors
for all cell types of the renal arteriole. Furthermore, renin cell
progenitors at this prevascular stage of kidney development did not
express smooth muscle markers. However, many of them did express the
transcriptional factor Ets1, and there was one renin cell that also
contained the interstitial marker tenascin. Among the markers studied,
renin cells show a clear lineage relationship with mesenchymal cells in
early development.

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Fig. 9.
E14 rat kidney incubated with the vital dye
neutral red. Large granulated cells that stained red with the dye were
chosen for microaspiration to perform single cell RT-PCR.
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Origin and Formation of Renal Arterioles
To study the participation of renin cells in blood vessel
formation, and to determine whether these cells adopt the appropriate position in the blood vessels, E12 mouse kidneys were
transplanted into the anterior chamber of the eye and under the kidney
capsule of adult mice. Renin and
-SMA immunostaining of these
transplanted kidneys demonstrated that JG cell precursors, smooth
muscle cells, and endothelial cells assembled into preglomerular
arterioles in a normal pattern resembling the pattern found in the
intact fetal kidney (Fig. 10,
A and B).

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Fig. 10.
E12 mouse kidney transplanted into the anterior eye
chamber of an adult mouse for 7 days. A: immunostaining for
renin (brown) showing a normal distribution of renin cells in
juxtaglomerular areas. B: immunostaining for -SMA
(brown). The vessels developed in a similar pattern as the one found in
the intact 19-day-old embryo. Bars: 25 µm (A); 100 µm
(B).
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Origin of Renin-Expressing, Smooth Muscle, and Endothelial
Cells
To define whether the metanephric blastema contains vascular
progenitors that are capable of differentiating in situ to
renin-expressing cells, arteriolar smooth muscle cells and endothelial
cells, embryonic wild-type kidneys (E12) were transplanted
under the kidney capsule of Rosa 26 mice (B6
Rosa 26) and vice
versa (Rosa 26
B6), and between Flk1+/
and wild-type
mice. After the X-Gal reaction, as shown in Fig.
11, A and D, the
wild-type embryonic kidneys were completely white and did not seem to
be invaded by host vessels, whereas the Rosa 26 kidneys were completely
blue. Immunostaining for renin and
-SMA showed that JG cells and
arteriolar smooth muscle cells within the graft were of intrinsic
kidney origin. As shown in Fig. 11, B and C,
wild-type E12 embryonic kidneys grafted under the kidney
capsule of Rosa 26 mice (B6
Rosa 26) had no blue staining in
renin-positive cells. On the other hand, when Rosa 26 E12
kidneys were transplanted under the kidney capsule of a wild-type host
(Rosa 26
B6), renin cells detected by dark brown renin
immunostaining also expressed the
-galactosidase enzyme turning blue
on the X-Gal reaction (Fig. 11E). Similar results were
obtained when these embryonic kidneys were stained for
-SMA as shown
in Fig. 11F. The endothelial cells of the arterioles are also blue in the transplanted Rosa 26 embryonic kidneys (Fig. 11F). These results demonstrate that all kidney arteriolar
cells originate from the grafted kidney. In addition,
Flk1+/
B6 and B6
Flk1+/
transplants
showed that endothelial cells derive from the embryonic kidney as
previously described by Robert et al. (30).

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Fig. 11.
Transplants of E12 kidneys under the kidney capsule of
adult mice. Tissues were subjected to the X-Gal reaction and to
immunohistochemistry for renin (B, C, and
E) and -SMA (F). A: E12
wild-type embryonic kidney under the kidney capsule of a Rosa 26 host.
After the X-Gal reaction, the wild-type embryonic kidney, in white, was
not invaded by host vessels, whereas all host cells were completely
blue. B and C: renin immunostaining of
transplanted E12 wild type Rosa 26 showing renin
distribution along the arterioles (B) and in the
juxtaglomerular area and inside the glomerulus (arrows, C). None of
these cells are blue, indicating the intrinsic origin of the
juxtaglomerular (JG) cells. D: E12 Rosa 26 embryonic kidney under the kidney capsule of a wild-type host. After
the X-Gal reaction, the transplanted kidney turned completely blue.
Renin immunostaining (dark brown, arrows, E) shows
coincidence with blue, and -SMA immunostaining (dark brown,
arrowheads, F) along the arterioles also coincides with the
blue reaction product. The endothelial cells inside the immunostained
vessels are also blue, confirming the intrinsic embryonic origin of all
these cells. Magnification: (A and D) ×16. Bars:
100 µm (B); 25 µm (C and F); 12.5 µm (E).
|
|
Transplants of E12 Ren-GFP kidneys (both
homozygous and heterozygous) grown under the kidney capsule of
wild-type mice (Ren-GFP
B6) showed expression of
GFP-labeled renin cells in the interstitium and along kidney
microarterioles, confirming their intrinsic metanephric blastema
embryonic origin (Fig. 12, A
and B).

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Fig. 12.
Transplant of E12 ren-green fluorescence protein (GFP)
under the kidney capsule of a wild-type host. A:
fluorescence microscopy (darkfield) showing green fluorescent cells
labeling renin cells derived from the embryonic kidney. B:
brightfield of A showing green cells are distributed along
the arterioles and inside the glomerulus (arrows). g, Glomerulus; a,
arterioles. Bars: 25 µm.
|
|
 |
DISCUSSION |
In this study, we examined the embryonic origin and lineage of JG
cells and their relationship with smooth muscle and endothelial cells.
The results showed that all arteriolar cell precursors, including JG
cells, are already present in the metanephric blastema at
E11 and E12 before vessel development has
occurred. Our transplantation experiments demonstrated that renin
precursor cells are capable of assembling to the appropriate vessel
type and segment (i.e., afferent arteriole). Finally, those
transplantation experiments provided the first experimental evidence
indicating that JG cells and renal vascular smooth muscle cells
originate within the metanephric blastema rather than from an
extrarenal source. As we determined that JG cell precursors are present
in the embryonic rat kidney, at E14 before vascular
structures are formed, we also confirmed that other cell markers such
as
-SMA, MHC, Ets1, vimentin, and tenascin are present at this
prevascular stage of kidney development. These results demonstrate that
before the vasculature has developed, the metanephric blastema possess
renin cell progenitors as well as precursors for many other cell types.
The present study agrees with those of others regarding the origin of
endothelial cells (17, 22, 30). Using specific cell
markers for endothelial cells, such as two of the receptors for VEGF
(VEGF-R1 or Flt1 and VEGF-R2 or Flk1) involved in the commitment and
differentiation of the endothelial cells (25) and
Tie1 Rc (22), several investigators have identified
the presence of endothelial cell precursors in the rodent kidney
(17, 23, 30). We have previously shown that smooth muscle
precursors are present in the primitive interstitium of fetal rat
kidneys at 14 days of gestation (6) as well as Flk1- and
Flt1-positive cells in the E12 mouse kidney
(35). These results suggest that at the time that the
ureteric bud begins its induction of the metanephric mesenchyme
(E11 and E12) a variety of cell progenitors are
already present, and contribute to both nephrogenesis and
vasculogenesis. The molecular signals that define whether an
undifferentiated mesenchymal cell follows one lineage pathway or
another require further work.
In addition to demonstrating the intrinsic origin of JG, smooth muscle,
and endothelial cells, our cross-transplantation experiments showed
that JG cell progenitors were capable of assembling into preglomerular
arterioles in a normal pattern. Interestingly, embryonic kidneys grown
in vitro develop nephrons but they do not develop blood vessels, and
renin cells remain dispersed in the interstitium. However, if these
same embryonic kidneys are grown under the kidney capsule or in oculo,
blood vessels (containing renin cells, smooth muscle, and endothelial
cells) develop properly. Celio and collaborators (8)
described that renin-containing cells were present in kidney transplants grown in the anterior eye chamber. However, in those studies rat E17-E19 kidneys were used, and
arterial blood vessels containing renin-expressing cells were already
developed at the time of transplant. Although no clear conclusions can
be ascertained regarding the origin of those structures, it seems clear
that the anterior eye chamber microenvironment provided the appropriate signals for the maintenance of the vascular structures and for renin
cell localization. Our transplantation experiments using prevascular
embryonic kidneys clearly suggest that signals from the environment
provided the appropriate positional information for JG cell
localization and arteriolar development. In addition, these experiments
rendered further support to the hypothesis that JG cells, smooth
muscle, and endothelial cells all originate from within the metanephric
mesenchyme. The cross-transplantation studies of embryonic kidneys
under the kidney capsule of adult mice (between Rosa 26 and C57
Bl/6J-strain mice) demonstrated that the JG cells, smooth muscle, and
endothelial cells found within the grafted tissue developed in situ
from the metanephric blastema and not from invading host cells,
suggesting that JG cell precursors have the capability to, and in
effect do, differentiate into JG cells. This finding can be related to
those of Hyink et al. (17) who demonstrated that
glomerular capillaries and mesangial cells originate in situ within the
metanephric blastema. These data reveal that all vascular precursor
cells are already present within the metanephric blastema. Further
studies are needed to define the molecular mechanisms governing the
lineage of kidney vascular cells.
Our previous work demonstrated that there is an association between
renin-expressing cells and the branching of renal arterioles (27). In fact, inactivation of various components of the
renin-angiotensin system using gene targeting results in aberrant renal
arteriolar branching, suggesting that renin, acting through local
generation of angiotensin, regulates renal vascular development. It
remains to be determined whether JG cells, independent of renin, can
contribute to vascular development. Furthermore, whether the assembling
vessel contributes to differentiation of the JG cell and the signals involved in that process remains to be investigated.
Using single cell RT-PCR, we demonstrate that during early embryonic
life, renin-expressing cells are not related to smooth muscle cells. In
addition, immunostaining studies also showed that renin cells in early
stages of kidney development (before E15) are unrelated to
cells that express
-SMA. Analysis at later ages (E16 to
N70) revealed that some JG cells contained
-SMA, indicating that acquisition of a smooth muscle phenotype is a later
event in the differentiation of the JG cell. Although it has been
suggested for many years that renin cells are derived from smooth
muscle cells (33), this assertion has never been tested
experimentally. The current experiments, however, suggest a different
scenario in which at this stage there are at least two distinct
populations of cells expressing either renin or smooth muscle markers
but not both. Subsequently, the subpopulation of renin-expressing cells
acquires the capacity to express smooth muscle markers. This finding
suggests that renin cells are capable of giving rise to smooth muscle
cells (arteriolar smooth muscle cells and very likely to other smooth
muscle-like cells such as the interstitial pericyte and the glomerular
mesangial cell), rather than originating from them. Interestingly, not
all smooth muscle cells seem to originate from renin cells, suggesting
that smooth muscle cells can also originate from another nonrenin
precursor, including a distinct embryonic smooth muscle cell
progenitor. Smooth muscle cells that have descended from renin
precursors are very likely the ones that undergo metaplasia to renin
cells when homeostasis is threatened with a need for more renin to
preserve it (12). By contrast, during aging and long-term
diabetes there are less JG cells, probably due to a retransformation of
JG cells to smooth muscle cells (10). A brief
conceptualization of the lineage of JG cells is shown in Fig.
13.

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Fig. 13.
Conceptualization of the lineage of the JG cell.
Metanephric mesenchymal cells (MCs) give origin to angioblasts, which
in turn give origin to endothelial cells. MCs also give rise to smooth
muscle cells and to renin precursor cells. During ontogeny the renin
precursor has the capability to give rise to JG cells and to a subset
of arteriolar smooth muscle cells. Smooth muscle cells that have
descended from renin precursors are very likely the ones that undergo
metaplasia to renin cells when the body needs more renin to preserve
homeostasis. By contrast, during aging and long-term diabetes there are
less JG cells, probably due to a transformation of JG cells to smooth
muscle cells.
|
|
In summary, our data show that JG cells originate in situ within the
metanephric kidney from mesenchymal cells unrelated to the endothelial
or smooth muscle lineages. Interestingly, as they differentiate, they
acquire smooth muscle markers that are maintained throughout adulthood.
The mechanisms that direct JG cell development, and their acquisition
of smooth muscle characteristics, remain to be determined.
 |
ACKNOWLEDGEMENTS |
We thank Barbara Thornhill, Alice Chang, and Marjorie Garmey for
advice regarding surgical techniques. The technical contribution of
Laxmi Chekuri, Madeline Hann, and Vasantha Reddi is greatly appreciated. M. L. S. Sequeira Lopez is a Howard Hughes Medical Institute Physician Postdoctoral Fellow.
 |
FOOTNOTES |
This work was supported by the Center of Excellence in Pediatric
Nephrology (National Institute of Diabetes and Digestive and Kidney
Diseases Grant DK-52612), the Child Health Research Center and
the Organogenesis Center, University of Virginia.
Address for reprint requests and other correspondence: R. Ariel
Gomez, Dept. of Pediatrics, Univ. of Virginia Health Sciences Center,
300 Lane Rd., MR4 Bldg., Rm. 2001, Charlottesville, VA 22908 (E-mail:
rg{at}virginia.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.
Received 27 February 2001; accepted in final form 11 April 2001.
 |
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