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The anionic matrix at the rat glomerular endothelial surface.

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THE ANATOMICAL RECORD 220:258-266 (1988)
The Anionic Matrix at the Rat Glomerular
Endothelial Surface
Research Service, VA. Medical Center and Department of Medicine, University of New
Mexico School of Medicine, Albuquerque, New Mexico 87108
The anionic macromolecules at the glomerular endothelial cell surface are visualized only when stained with cationic stains. We investigated the
arrangement and composition of this anionic matrix at the luminal surface. Rat
kidneys were perfused with anionic ferritin (PI 4.5), ferritin (PI 7.41, or cationized
ferritin (CF, PI 8.3). Anionic ferritin (PI4.5) did not bind to the capillary wall, ferritin
(PI7.4) bound discontinuously only to the laminae rarae of the basement membrane,
but cationized ferritin (CF, PI 8.3) bound as a thick continuous layer to the cell
plasmalemma and bound to the anionic matrix in the fenestral spaces. These observations show that a n anionic matrix lines the entire capillary lumen surface, fills
the fenestrae, and is interposed between the blood and the basement membrane a t
the fenestrae.
The anionic constituents at the capillary luminal surface were identified by in
vivo digestion with specific enzymes. Absence of CF binding following digestion with
specific enzymes was taken to indicate the presence of the particular glycoprotein
known to be susceptible to the enzyme used. Neuraminidase digestion revealed that
anionic sites over the surface plasmalemma are mainly from sialoproteins. In contrast, the matrix in fenestral channels contains heparan sulfate, hyaluronic acid,
and sialoproteins. Papain digestion showed no glycolipids at the luminal surface.
The functions of this continuous anionic layer located a t the luminal surface of
glomerular capillaries have not yet been established.
For ultrafiltration of water and macromolecules across
the glomerular capillary wall, the major pathway appears to be through the endothelial fenestrae, across the
three layers of glomerular basement membrane, and
finally across the epithelial cell slit diaphragm (Shea
and Morrison, 1975). The lamina densa of the glomerular basement membrane has been shown to function as
the size-selective barrier for the neutral macromolecules
traversing the glomerular capillary wall (Batsford et al.,
1987; Caulfield and Farquhar, 1974). The glomerular
capillary wall functions both as a charge-selective barrier and as a size-selective barrier (Bennett et al., 1976
Bohrer et al., 1978).Anionic macromolecules have been
found to be particularly concentrated in both the laminae rarae of the basement membrane (Caulfield and
Farquhar, 1976; Kanwar and Farquhar, 1979; Rennke
et al., 1975). In addition, the epithelial cell glycocalyx
and possibly the slit diaphragms also have anionic sialoproteins (Kerjaschki et al., 1984; Blau and Michael,
1971). The anionic barrier function of the glomerular
capillary wall has been ascribed to all these structures
but the precise location of the anionic barrier remains
uncertain (Kanwar et al., 1980; Kanwar and Jakubowski, 1984; Vernier et al., 1983;Fishman and Karnovsky, 1985). Fenestrations in the glomerular endothelial
cell cytoplasm in adult rats are mainly circular holes
that traverse the entire cell cytoplasm and have a mean
diameter of 75 nm (Avasthi et al., 1980).These fenestrae
0 1988 ALAN R. LISS, INC
do not have a diaphragm and on routine electron microscopy appear to be mostly empty. In other blood vessels,
endothelial cells have been shown to have a layer of
anionic macromolecules that is attached to their plasmalemma and extends into the vascular lumen. Although invisible when unstained, a 10- to 20-nm-thick
fuzzy layer of anionic macromolecules is visualized a t
the vascular endothelial surfaces when stained with cationic electron-dense tracers (Luft, 1966; Copley, 1974).A
similar layer of anionic macromolecular glycocalyx has
been noted at the luminal surface of glomerular endothelial cells (Latta and Johnston, 1976). Owing to the
fenestrations in the cytoplasm of this cell, the distribution of glycocalyx on the plasmalemma of this cell is
expected to be quite different. The purpose of this study
was to map the distribution of glycocalyx on the plasmalemma of the fenestrated glomerular endothelial cell
and to ascertain whether the fenestral spaces are occupied by a n anionic fiber matrix that may be invisible by
the routine electron microscopy. Furthermore, some of
the constituents of this glycocalyx were histochemically
Received May 27, 1987; accepted July 23, 1987
Female Sprague-Dawley rats (150-200 gm body
weight) were utilized in these experiments. To minimize
variability, if any, due to sex and age, only the female
rats in a narrow range of body weight were utilized.
Native ferritin (horse spleen, isoelectric point, PI 4.55.5) from Sigma Chemical Company, St. Louis, and cationized ferritin (PI 8.2-8.4, CF) from Miles Laboratory,
Elkhart, IN, were purchased. Neutral ferritin (PI 7.4)
was prepared by the method of Danon et al. (1972). All
batches of ferritin were checked for the stated PI by
isoelectric focusing (Righetti and Drysdale, 1974), and
monomolecular dispersion was checked by electron microscopy. All ferritins were dialyzed against 0.15 M NaCl
at 4°C for 24 hr. Dulbecco's phosphate-buffered saline
and minimum essential medium were purchased from
Gibco Laboratories (Grand Island Biological Company,
Grand Island, NY). The final perfusion solution (DPBS)
contained Dulbecco's phosphate-buffered saline (pH 7.27.4) with 5% minimum essential medium and 14 mM
glucose. Mannitol was obtained from Invenox, Chagrin
Falls, OH, and lissamine green from Serva Entwicklunglabor, Heidelberg.
Experimental Protocol
Rats were anesthetized by intramuscular injection of
ketamine HCl(87 m g k g body weight) and xylazine (13
m g k g body weight). Laparatomy was performed; the
abdominal aorta was isolated, and a polyethylene catheter (18-to 22-gauge) was positioned so that the catheter
tip lay adjacent to the left renal artery. Two ligatures,
one above and the other below the left renal artery, were
tied on the aorta, so as to perfuse only the left kidney.
Open-circuit perfusion was carried out by making a n
opening in the left renal vein. With a n occlusive roller
pump molter pump, model No. RL 175 110, International Medical Corporation, Englewood, CO), the renal
vasculature was cleared of blood by a 2- to 7-min perfusion of 10-15 ml of DPBS at 37"C, gassed with 0 2 .
Through a side-arm, perfusion pressures were continuously monitored with a Statham pressure gauge and a
calibrated electronic pressure monitor (Electronics for
Medicine, White Plains, NY). Following the washing out
of blood from the vasculature, ferritin was perfused (7
mg/100 gm body weight in 2 ml of 0.15 M saline) through
the renal vasculature. Unbound ferritin was removed
by one more minute of DPBS infusion followed by perfusion of fixative. Details of the sequence of perfusions
for all the experiments are given in Table 1. Except for
the period of fixative perfusion, throughout the entire
experiment, perfusion pressures were generally 80-150
Tissue Fixation and Processing for Electron Microscopy
Tissues were fixed by intrarenal arterial perfusion of
30 ml of Karnovsky's fixative in 10 min. Kidneys were
removed, and 1-mm cubes of renal cortex were cut and
further fixed in Karnovsky's fixative overnight (12-18
hr), and were stored in 0.1 M sodium cacodylate buffer
(pH 7.4) till further processed. The tissues were postfixed
in osmium tetroxide, dehydrated in acetone, and embed-
ded in LX 112 resin (Ladd, Burlington, VA). Thin sections ( 60 nm) were stained with lead citrate. Viewing
and photography were done on a Hitachi HU 11-B EM
(75 kV). From each rat, a minimum of three glomeruli
were studied. The results were qualitatively assessed by
comparing the number of ferritin particles on the experimental rat capillary luminal surface to the appropriate
To resolve the issue whether CF was completely filling
the fenestrae or was attached only to its walls, first,
three serial thin sections (gray) of one glomerulus were
mounted on a single-slotted formvar-coated grid. Several
sets of sequential sections were photographed to assess
the pattern of CF attachment in the fenestrae. In addition, by means of a Hitachi 600 electron microscope with
a tilt stage, paired photomicrographs of glomerular capillary walls were taken at a + l o " and a -10" tilt and
were viewed through a stereoscopic viewer to obtain
three-dimensional images. These procedures permitted
resolution of the question whether ferritin was filling
the entire fenestrae. Next, we considered the possibility
that CF may have initially attached to the laminae rara
interna, and then owing to the ongoing water filtration,
additional CF may have accumulated in the fenestrae
passively. To settle this question, we perfused CF in
three rats with nonfiltering kidneys. Nonfiltering kidneys were produced by intrasaphenous vein injection of
mannitol (0.1 gm per 100 gm rat weight) and by tying
the left ureter (Holdaas et al., 1981). Lack of filtration
by such maneuvers was ascertained by the injection of 1
ml of lissamine green dye (0.17 gm% in 0.3 M NaC1) in
the tail vein of a similarly prepared rat. Dye in renal
tubules was visualized through a dissecting scope. The
dye was readily seen in the tubules on the unligated
side but not on the side with the ureteric ligation, which
confirmed the lack of filtration. In three rats with such
nonfiltering kidneys, CF was injected and tissues were
prepared and examined as outlined earlier.
Enzyme Digestion Experiments
Enzymes were obtained from Sigma Chemical Company and Miles Laboratory. The purity, specificity, and
enzyme activity of the enzymes used in this study are
shown in Table 2. The enzymes used to remove the
specific proteoglycans from the capillary lumen surface,
their source, and the conditions utilized to optimize in
situ enzyme action are shown in Table 3. For each enzyme experiment, enzyme solutions were prepared just
before perfusion to preserve maximal activity. Optimal
conditions for enzyme action were taken from the references shown in the extreme right-hand column of Table
3 (Pino et al., 1982; De Bruyn et al., 1978; Kanwar and
Farquhar, 1980; Underhill and Toole, 1979; Gamse et
al., 1978; Buonassisi and Root, 1975; Linker and Hovingh, 1972; Gill et al., 1981; Yamagata et al., 1968).
Renal temperatures were maintained as required for the
individual experiment (Table 3) by dripping saline (0.15
M) warmed to the required temperature over the kidney
surface during the entire experiment. Also, all perfusion
solutions were warmed to the required temperatures.
Control rats for each enzyme were perfused with a
similar volume of enzyme diluent (Table 3). Thus, each
control rat underwent a perfusion sequence that was
exactly similar to its corresponding enzyme experiment
TABLE 1. Sequence of perfusion steps for native ferritin, for cationized ferritin, and for the enzymes
used for in situ digestion of luminal anionic sites
No. of rats
ferritin (NF)
wash, min
Sequence of perfusions
Tracer (PI)
(4-5 ml), min wash, min (7 mg/100 g, bw) wash, min (Karnovsky's), ml
NF (4.5)
CF (7.4)
CF (8.3)
CF (8.3)
CF (8.3)
CF (8 * 3)
CF (8.3)
CF (8.3)
'Control rats were perfused, with diluent without the enzyme. All non-enzyme-controlshad identical CF binding; hence the number of controls
for individual enzymes is small.
TABLE 2. Purity, specificity and specific activity of enzymes
Proteolytic activity
Not detected
< .002 U/mg protein
Not detected
Chondroitinase ABC
Not detected
Substrate specificity
Specific activity
Degrades only hyaluronic acid
No 0-galactosidase
< 1%a8ainst other
Degrades hyaluronic
acid and
2,000 TRUImg protein'
160 U/mg protein
260 U/mg protein
37.5 pWmg protein
'TRU, Turbidity reducing units.
~ glycosaminoglycans.
TABLE 3. Conditions and enzymes used for removing the anionic sites from the
luminal surface of glomerular capillaries
Temp., "C
Time, min
5-10 mglml
Pino et al. (1982)
De Bruyn et al.
Kanwar and Farquhar (1980)
Underhill and Toole
Buonassisi and Root
Gamse et al. (1978)
Gill et al. (1981)
Linker and Hovingh
Yamagata et al.
Papaya laytex
(Sigma, Type
C1. perfringens
(Sigma, Type
Strep. hyaluronyticus (Miles)
F1. heparinum
Proteus vulgaris
2 Ulml
'Enzymes were diluted in 0.1 M sodium chloride-acetate to the concentrations specified for each enzyme. Conditions used
were taken from the references given.
Fig. 1. Monomolecular dispersion of cationized ferritin (CF) in 0.15
M saline, PI 8.3. X225,OOO.
Fig. 2. Capillary wall of a rat infused with neutral ferritin (PI 7.4).
Ferritin is sparingly bound in clusters within the laminae rarae. CL,
capillary lumen; BS, Bowman’s space. x49,OOO.
Fig. 3. Attachment of CF (PI 8.3) to the entire luminal front of a
normal rat glomerular capillary wall. CF is attached to the endothelial
glycocalyx, which fills the majority of the fenestrae. CL, capillary
lumen; BS, Bowman’s space. ~58,000.
Fig. 4. Composite of three sequential sections from a glomerular
capillary loop. Many fenestrae have been sequentially cut but none
show a central CF-free area. CL, capillary lumen. X35,OOO.
Fig. 5. Paired photomicrographs of the same section taken at +lo" the entire fenestral spaces, including the center. CL, capillary lumen.
and -10" stage tilt. Stereoscopic viewing reveals attachment of CF in X50,OOO.
except that the enzymes were omitted from the diluent
in controls. It should be noted that ferritins were not
allowed to remain stationary or dwell in the renal vasculature, but were perfused through the vasculature.The number of rats, duration of perfusions, and
sequence of perfusions for the experimental and control
groups are given in Table 1.In enzyme perfusion experiments, the total enzyme quantity to be perfused was
administered and each enzyme was retained in the renal
vasculature for the indicated time (Table 1) by clamping
the renal vessels. Tissue fixation and microscopy were
done as outlined earlier.
Ferritins were dispersed as single particles for every
batch of tracer utilized, as is shown in Figure 1. The PI
of each batch of tracer was checked. For native ferritin
the PI ranged between 4.4 and 5.5; neutral ferritins had
a PI of 7.2-7.5 and cationized ferritin PI > 8, with major
bands at 8.2 and 8.4.
Binding of the Tracer
Rat kidneys perfused with native ferritin (PI 4.5)
showed no binding of the tracer to any component of the
A clear zone between endothelial plasmalemma and basement membrane indicates the lack of binding sites parallel to the abluminal
plasmalemma. Some binding of CF to fenestral floor, though decreased, is still seen (compare with Fig. 10)and is situated luminal to
lamina rara interna (arrows). CL, capillary lumen; BS, Bowman’s
Fig. 7. Detached endothelial and epithelial cells in a papain-treated space. ~79,000.
kidney. No ferritin is bound at the luminal surface. CF is bound to
Fig. 9. CF binding following hyaluronidase. CF binding in some of
epithelial cell surface and a few ferritin particles are in the basement
membrane. CL, capillary lumen; BS, Bowman’s space. x 58,000.
the fenestrae is reduced (arrowheads), but only a minimal reduction is
seen at plasmalemma glycocalyx. CL, capillary lumen; BS, Bowman’s
Fig. 8. CF binding following 10-min neuraminidase treatment. space. ~ 4 0 , 0 0 0 .
Marked reduction in CF binding to the endothelial glycocalyx is seen.
Fig. 6. Glomerular capillary wall from a nonfiltering kidney. CF is
attached to the entire luminal front, the ferritin in the fenestrae is
seen up to the capillary luminal (CL) level, and the pattern of CF
binding is the same as in Figure 3. ~ 4 3 , 0 0 0 .
capillary wall. Neutral ferritin (PI7.4) was found in both
laminae rarae of the glomerular basement membrane
in discontinuous clusters (Fig. 2). This is in agreement
with results reported by others (Rennke et al., 1975;
Kanwar and Farquhar, 1979).
Kidneys perfused with CF (PI 8.3) showed a layer of
ferritin three to four particles thick attached to the
entire luminal surface of the endothelium. This band of
ferritin lined the luminal surface of endothelial cells,
bound to plasmalemma at the sides of the fenestrae,
filled the fenestral channels, and blended with ferritin
bound to the lamina rarae interna (Fig. 3). CF filled the
majority of the fenestrae up to the level of luminal
plasmalemma. Thus, the endothelial glycocalyx forms a
continuous layer at the luminal surface, covering the
luminal plasmalemma and spanning the fenestrae.
The finding of filling of fenestrae by CF could have
been due to the CF attached to the rim of the fenestrae
in the section even if CF did not bind to the center of the
fenestrae. To examine this possibility, we obtained thin
sequential sections. If CF was attached only to the walls,
a CF-free area would be found in some of the sequentially cut fenestrae. Of the many sequentially cut fenestrae, none showed a central CF-free area (Fig. 4).
Furthermore, the three-dimensional images obtained
from the steroscopic paired micrographs of glomerular
capillary wall sections (Fig. 5) demonstrate CF in the
entire fenestral space. If CF were attached only to the
fenestral walls, three-dimensional constructs would have
revealed CF attached to a curved rim and a central area
without CF. Instead, CF was found to be filling the
entire fenestral spaces. These observations support our
interpretation that CF attaches to the anionic fiber matrix that fills fenestrae completely up to the capillary
lumen level.
Next, we dealt with the possibility that following initial attachment of CF to the fenestral floor, additional
CF may have accumulated passively due to ongoing
water filtration. Nonfiltering kidneys were utilized to
answer this question with the idea that if passive accumulation of CF in the fenestrae were occurring because
of the water filtration, CF would bind only to the fenestral floor and would not be seen in the fenestral channel
in the absence of filtration. Figure 6 shows that the
pattern of CF binding in nonfiltering glomeruli was
identical to that of the normal rat kidney. These observations are in agreement with the hypothesis that the
anionic fiber matrix of endothelial glycocalyx in the
fenestrae is made visible by the active binding of CF.
Enzyme Digestion Experiments
In these experiments, we tested the effects of a protease and a series of specific glycoprotein degrading
enzymes on the anionic constituents at the luminal surface of the capillary wall. Papain digestion resulted in a
complete loss of CF binding from the entire luminal
front. Both the endothelial and the epithelial cells were
detached from the basement membrane in places. CF
was found in Bowman’s space and was bound to the
surface of the epithelial cells only in the papain-digested
kidneys (Fig. 7). Thus, papain increased the permeability of the barrier, which allowed CF to penetrate across
the capillary wall. These results indicate that glycolipids are not a major constituent of the anionic sites at
the luminal front of glomerular capillaries, since papain
is expected to spare the glycolipids. Neuraminidase
digestion erased the binding of CF from the plasmalemma glycocalyx at the luminal surface of endothelial
cells (Fig. 8). It should be noted that CF failed to bind
along the abluminal plasmalemma (Fig. 8). The extent
of loss of anionic sites depended on the time allowed for
the enzyme action. The loss was patchy following a 5min digestion; more extensive loss was observed after a
long period of digestion. These results suggest that the
anionic sites at the plasmalemma glycocalyx consist of
sialoglycoproteins and are consistent with the findings
reported previously (Latta et al., 1975). However, some
residual binding of CF to the fenestral floor is still seen
(Fig. 8). There was no detachment of endothelial or epithelial cells from the basement membrane in these neuraminidase-treated glomeruh, perhaps because of the
short duration of enzyme action. These findings indicate
that a majority of the anionic sites at the plasmalemmal
glycocalyx are from neuraminic acid residues. Also, in
the fenestral glycocalyx, a majority of anionic sites are
removed with the removal of sialic acid. Digestion with
hyaluronidase shows only a partial reduction in CF
binding in some of the fenestrae (Fig. 9). Digestion with
heparitinase decreased the binding of CF preferentially
within the fenestrae, whereas the anionic sites in the
endothelial glycocalyx at plasmaIemma were only minimally decreased (Fig. 10). These observations suggest
that in the fenestrae the anionic sites of endothelial
glycocalyx also contain hyaluronic acid and heparan
sulfate glycosaminoglycans in addition to the sialoproteins (Figs. 9,lO). A 5-min digestion with chondroitinase
ABC did not reduce CF binding to the luminal surface.
At 10 min, some of the fenestrae had reduced CF binding, but others showed no change (Fig. 11).As chondroitinase ABC can degrade both chondroitin sulfate and
hyaluronic acid (Yamagata et al., 1968), perhaps the
relatively low activity of the enzyme against hyaluronic
acid may have been responsible for these findings.
Therefore, we are unable to decide from these observations whether chondroitin sulfate is also present in endothelial glycocalyx.
Although a glycocalyx has been previously noted at
the glomerular endothelial surface, details of its distribution and composition have not been explored (Latta
and Johnston, 1976; Rennke et al., 1975). The arrangement of macromolecules at the plasmalemmal surface is
detailed here, and these observations show that the glomerular endothelial cell fenestrae are not empty holes
as they often appear to be by routine electron microscopy. Instead of a fenestral diaphragm, these fenestrae
are occupied by an anionic matrix that is visualized only
following the binding of an electron-densetracer. In this
respect, the ultrastructure of the matrix in the fenestrae
is similar to the glycocalyx at the external surface of
cells, which also remains invisible in unstained preparations. It appears that the glycocalyx attached to the
endothelial plasmalemma at the fenestral rim extends
into and fills the fenestral channels. We excluded the
possibility of passive accumulation of ferritin at this
location a) by the failure of the anionic or the neutral
ferritin to similarly accumulate in the fenestrae and b)
by the identical binding of CF in the nonfltering kidneys. Inclusion of a part of the fenestral rim within the
Fig. 10. CF binding following heparitinase. Endothelial glycocalyx
Fig. 11. CF binding following chondroitinase ABC. Slightly reduced
over the plasmalemma shows a minimal reduction in attachment of binding in some fenestrae (arrowheads) is seen but the majority of
CF. Majority of fenestrae do not bind CF. CL, capillary lumen; BS, fenestrae remain normal. CL, capillary lumen; BS, Bowman’s space.
Bowman’s space. x 30,000.
thickness of the tissue section could also have mimicked
these findings. This possibility was excluded by examining sequential sections and by stereoscopic evaluation.
By both these methods ferritin was seen in the fenestral
center, which excluded the possibility that the ferritin
was attached only to the rim of the fenestrae. Finally,
an uncertainty remained that initially the cationized
ferritin may have bound to the surface of the basement
membrane and that the resultant clogging of the passage may have allowed passive accumulation of cationized ferritin in the fenestrae. This possibility was
excluded by the finding of an identical binding pattern
for cationized ferritin in the kidneys without water filtration. Thus, our findings demonstrate that the glomerular endothelial fenestrae are occupied by a fiber matrix
of the macromolecules that are attached to the plasmalemma around the fenestrae and extend into the fenestral channels to fill the fenestral spaces.
The functional significance of this anionic fiber matrix
in the filtration pathway cannot be assessed from these
mo~holo@calobservations’ Other observers have proposed that the fiber matrix in the endothelial transcellular channels in general may determine the molecular
sieving across the capillary walls (Curry and Michel,
1980).Also, theoretical considerations favor the location
of the anionic barrier at the very luminal surface rather
than inside the layers Of the
1970). However, conflicting results have been obtained
regarding the location of the charge-selective barrier in
studies utilizing different tracers
and Karnovsky,
1976; Farquhar et al., 1961; Masri et al., 1985). Therefore, despite the demonstration of a series of anionic
layers in the filtration pathway, the precise location of
the barrier in the glomerular capillary wall for serum
anionic macromolecules (albumin) remains uncertain.
Histochemical characterization of anionic sites in the
endothelial glycocalyx reveals the following features: 1)
A complete loss of CF binding following digestion with
papain indicates that glycolipids are not likely contributors to the anionic sites. 2) The glycocalyx at the luminal surface derives a majority of its anionic groups
from sialic acid residues, since digestion with neuraminidase preferentially erases the binding of CF from the
glycocalyx covering the luminal and the abluminal plasmalemma. 3) Anionic sites in the glycocalyx within the
fenestrae are from heparan sulfate proteoglycans, hyaluronic acid, and sialyl residues. The fenestral glycocalyx
has a greater concentration of heparan sulfate proteoglycans.
we acknowledge the excellent technical
Paul E. Clark and Judy Davis. The help of Drs. John
Trotter, R. Chiovetti, and K.D. Gardner is acknowledged
Karin Woods provided excellent secretarial
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endothelial, glomerular, matrix, anionic, surface, rat
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