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Dose-dependent movement of cationic molecules across the glomerular wall.

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THE ANATOMICAL RECORD 212223-231 (1985)
Dose-Dependent Movement of Cationic Molecules
Across the Glomerular Wall
PETER M. ANDREWS AND SALLY B. BATES
Department of Anatomy, Georgetown University Schools of Medicine and Dentistry,
Washington, DC 20007
ABSTRACT
Different concentrations of the polycation polyethyleneimine (PET)
were administered by single intravenous injections or by constant vascular perfusion
to the kidneys of Sprague-Dawley rats. At a fixed time interval after administration
of PEI, the kidneys were fixed and the distribution of PEI in the glomerular wall
was evaluated by electron microscopy. At the lower concentrations (e.g., 0.005%),
PEI bound only to the glomerular endothelial glycocalyx and preferentially to
microvillous projections on this endothelium. At higher concentrations (e.g., 0.05%),
PEI also bound to discrete anionic sites in the lamina rara interna (LRD but was
rarely seen in the lamina rara externa (LRE). As the concentration of PEI was
further increased (e.g., 0.5%),PEI moved deeper into the glomerular basement
membrane (GBM) and bound extensively to discrete anionic sites in the lamina rara
externa. Although anionic sites in the LRI and LRE appeared nearly saturated
following infusion of 0.5% PEI, this cationic molecule was rarely seen to cross
filtration slits and pass into the urinary space. At still higher concentrations (e.g.,
2%), however, PEI moved freely across the filtration slits, bound extensively to the
glomerular epithelial glycocalyx, and induced a narrowing of the filtration slits.
When PEI was mechanically perfused through the kidney vasculature for 3 minutes,
PEI binding to the epithelial glycocalyx caused very extensive adherence of adjacent
podocyte processes and the narrowing and loss of filtration slits. Also in these latter
samples, discrete anionic sites in the LRE were no longer apparent and a dense
band of PEI was seen under the foot processes. Addition of PEI to culture media
containing intact glomeruli revealed that even in the absence of hemodynamic
driving forces, the PEI used in the above studies will gradually move across the
glomerular basement membrane and bind to anionic sites throughout the glomerular wall. The above observations suggest that anionic sites in the glomerular wall
may trap cationic molecules and thereby prevent low concentrations of these molecules from passing deeper into the glomerular wall and gaining entrance to the
urinary space.
The permselectivity of the glomerular wall has been
studied extensively and discussed in a number of excellent reviews Varquhar, 1975; Brenner et al., 1977; Venkatachalam and Rennke, 1977; Karnovsky, 1979;
Kanwar, 1984). Briefly, molecular size, configuration,
charge, and hemodynamic factors all play important
roles in determining the degree to which a given molecule can penetrate and/or cross the glomerular wall.
With respect to charge, it has been demonstrated that
cationic molecules penetrate deeper into and/or transverse the glomerular wall easier than similarly sized
neutral or anionic molecules (Chang et al., 1975;Rennke
et al., 1975; Rennke and Venkatachalam, 1977; Bohrer
et al., 1978; Rennke et al., 1978; Purtell et al., 1979). It
has also been shown that experimental neutralization
or removal of anionic barriers results in the increased
ability of anionic molecules to penetrate and pass across
the glomerular wall (Kelly and Cavallo, 1978; Kanwar
et al., 1980; Hunsicker et al., 1981; Rosenzweig and
Kanwar, 1982; Barnes et al., 1984).
Q 1985 ALAN R. LISS, INC.
Polyethyleneimine (PEI) is a cationic molecule which
has been used to label anionic sites in the glomerular
basement membrane (GBM) (Schurer et al., 1977, 1978).
In this investigation, we found that the extent to which
this cationic molecule can penetrate the glomerular wall
is concentration dependent and that only at high concentrations does this molecule appear t o cross the filtration
slits and enter the urinary space. These observations
suggest that for low concentrations of cationic molecules, anionic sites in the glomerular wall may behave
as barriers which can trap these molecules and prevent
them from passing into the urinary space.
MATERIALS AND METHODS
In Vivo Studies
Female Sprague-Dawley rats weighing 200-250 gm
were used in these investigations. Each rat was weighed,
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Received September 26,1984; accepted February 6,1985.
224
P.M. ANDREWS AND S.B. BATES
anesthetized with 35 mgkg of pentobarbital sodium injected intraperitoneally, and secured to a dissecting table. Different concentrations of polyethyleneimine (M.W.
30-40,000) (purchased from Sigma) were made up in
0.9% NaCl and the pH adjusted to 7.3 with dilute HCl.
Exactly 0.2 cc of either a 2%, a 0.5%, a 0.05%, or a
0.005% solution of PEI was injected into the femoral
vein. Fifteen minutes following injection of PEI, a laparotomy was performed and the right kidney was excised
and cut into slices while immersed in a fixative consisting of 2% glutaraldehyde and 2%phosphotungstic acid
in 0.1 M cacodylate buffer. At the same time, the left
kidney was fixed by in situ retrograde vascular perfusion of the same fixative using methods described in
detail in a recent paper (Andrews and Coffey, 1984).
Following vascular perfusion fixation, the left kidney
was excised, cut into slices, and fixed by immersion for
another 2 hours.
In another series of experiments, the kidneys of
Sprague-Dawley rats were perfused in situ for 3 minutes
with a pH-adjusted (i.e., pH 7.3) saline solution containing either 0.001%, 0.01%, or 0.1% PEI. Immediately following vascular perfusion of the kidneys with PEI via
the abdominal aorta, the kidneys were fixed by vascular
perfusion, excised, and fixed by immersion for another 2
hours.
Slices from both immersion- and vascular-perfusionfixed kidneys were rinsed in cacodylate buffer, postfixed
in 1%osmium tetroxide, stained en bloc in 2% uranyl
acetate, dehydrated through graded acetones, and
embedded in Spurr medium. Thin sections (50-60 nm)
were poststained with uranyl acetate and lead citrate
and examined in a JEOL 100s transmission electron
microscope operating at 80 kV.
In Vitro Studies
Details describing the in vitro technique used in this
investigation have been described in detail in a previous
manuscript (Andrews and Coffey, 1980). Briefly,
Sprague-Dawley rats were anesthetized with 35 m g k g
pentobarbital sodium injected intraperitoneally, secured
to a dissecting board, a laparotomy was performed, and
the kidneys were cleared of blood by retrograde vascular
perfusion of culture medium 199. One-millimeter-thick
kidney slices with glomeruli exposed on their cut surfaces were immediately placed in Petri dishes containing pH adjusted 0.1% PEI in medium 199. At timed
intervals of 5 seconds, 30 seconds, and 1minute, kidney
slices were removed from the PEI solutions and fixed by
immersion in the same glutaraldehyde-phosphotungstic
acid fixative as used in the in vivo studies described
above. The kidney slices were then postfixed in osmium,
stained en bloc, and processed for transmission electron
microscopy in the same manner as described above.
untreated controls. Only rarely was PEI seen to penetrate deeper into the glomerular basement membrane
(GBM) than the LRI. Intravenous injection of 0.5% PEI
resulted in PEI binding extensively to anionic sites in
the lamina rara externa &RE) as well as to anionic sites
in the LRI (Figs. 3, 4), revealing a pattern of binding
similar to that observed by other investigators using
this same concentration of PEI (Schurer et al., 1977,
1978). In general, anionic sites in the LRE appeared to
be considerably more numerous than similar sites in the
LRI. In these glomeruli, PEI rarely appeared to cross
the slit diaphragms and enter the urinary space. When
2% PEI was injected, however, large amounts of PEI
crossed the filtration slits and were seen binding to the
glomerular epithelial (podocyte) glycocalyx (Figs. 5 , 6).
In these samples, there was a narrowing of the filtration
slits (from 40 nm to approximately 20 nm or less) and
dense deposits to PEI were seen around the slit diaphragms (Fig. 5).
Vascular perfusion of PEI in concentrations of 0.001%
and above results in labeling of anionic sites throughout
the GBM, movement of PEI across the filtration slits,
and binding of PEI to the podocyte glycocalyx (Figs. 79). At concentrations of 0.01% and higher, vascular perfusion of PEI results in extensive adherence of adjacent
podocyte cell bodies and major and foot processes and
the resultant closing of filtration slits (Figs. 7-9). In
these glomeruli, discrete anionic sites in the LRE can
no longer be discerned by PEI binding. Instead, a dense
band of PEI is seen located immediately subjacent to the
bases of the foot processes (Fig. 7).
In vitro incubation of intact (i.e., uncut) glomeruli on
the surfaces of kidney slices in medium 199 containing
0.1% PEI results in the movement of PEI across the
glomerular wall and its binding to anionic sites in the
LRE, LRI, and endothelial glycocalyx wigs. 10, 11).Podocytes on the surface of exposed glomeruli exhibit extensive binding of PET, adherence of adjacent processes,
and the resultant loss of filtration slits (Fig. 11).As in
the in situ studies, discrete sites in the LRE are no
longer discernible in samples exposed to such high concentrations of PEI (Fig. 11).Podocytes located deeper in
the glomerular tuft and which were therefore not exposed to as high concentrations of PEI, exhibit considerably less binding of PEI and a gradual movement of the
PEI across the glomerular wall (Fig. 10). Even intact
glomeruli which were still enclosed in the thick basement membrane of the surrounding parietal epithelium
exhibit extensive binding to PEI throughout the glomerular wall, demonstrating that the parietal epithelial
basement membrane is also highly permeable to PEI.
DISCUSSION
In recent years significant insights have been gained
into the roles that charge plays in the movement of
OBSERVATIONS
molecules across the glomerular wall. Among other
Intravenous injection of 0.005% PEI results in the things, it has come to be generally accepted that while
binding of PEI to the endothelial glycocalyx and espe- anionic molecules are inhibited from crossing the glocially to the glycocalyx covering endothelial free surface merular wall, the movement of cationic molecules across
microprojections (Fig. 1).Injection of higher concentra- the GBM appears to be enhanced (Chang et al., 1975;
tions of PEI (i.e., 0.05%)results in binding to both the Rennke et al., 1975; Rennke and Venkatachalam. 1977:
endothelial dvcocalvx and to lamina rara interna (LRI) Rennke et al.', 1978; Bohrer et al., 1978; Purtell'et al.;
anionic siteg@'ig. 2 i the latter not normally detected in 1979). As a result of the present investigation, however,
GLOMERULAR PERMEABILITY TO POLYCATIONS
Fig. 1. Cross section through the glomerular wall 15 minutes after intravenous injection of
0.005%PEI. PEI is bound mainly to the endothelial glycocalyx (small arrows) and is especially
abundant on endothelial microprojections (large arrows). C, capillary lumen; U, urinary space.
X75,OOO.
Fig. 2. Cross section through the glomerular wall 15 minutes after intravenous injection of
0.05% PEI. The PEI (arrows) is mainly seen binding to the lamina rara interna of the glomerular basement membrane. x 75,000.
225
226
P.M. ANDREWS AND S.B. BATES
Figs. 3, 4. Cross (Fig. 3) and tangential (Fig. 4) sections through the glomerular wall 15
minutes after intravenous injection of 0.5% PEL The PEI is evident along discrete anionic sites
in the lamina rara externa (arrows) as well as along the lamina rara interna. EM, glomerular
basement membrane. Figure 3, X75,OOO; Figure 4, X44,OOO.
GLOMERULAR PERMEABILITY TO POLYCATIONS
Figs. 5, 6. Cross (Fig. 5 ) and tangential (Fig. 6) sections through the glomerular wall 15
minutes after intravenous injection of 2.0% PEI. Note that the PEI has bound extensively to
the filtration slit diaphragms (large arrows) and has crossed these diaphragms and bound to
the glomerular epithelial Le., podocyte) glycocalyx (small arrows). Figure 5, x 75,000; Figure
6, ~44,000.
227
228
P.M. ANDREWS AND S.B. BATES
Figs. 7, 8 . Cross (Fig. 7) and tangential (Fig. 8 ) sections through the glomerular wall 3
minutes following in situ perfusion of 0.01% PEI. PEI (large arrows) has bound heavily to the
endothelial and podocyte glycocalyx and caused adherence of adjacent foot processes. Note that
PEI is diffusely distributed in the glomerular basement membrane except in dense bands just
8,
subjacent to the bases of podocyte foot processes (small arrows). Figure 7, ~ 7 5 , 0 0 0Figure
;
x 37,000.
GLOMERULAR PERMEABILITY TO POLYCATIONS
229
Fig. 9. Section through a portion of a glomerulus which was perfused in situ with 0.01% PEI
for 3 minutes. Note the extensive adherence of adjacent podocytes and their processes (arrows).
x 15,000.
the latter view may have to be modified to consider the
concentrations of cationic molecules t o which the glomerular wall is exposed. Our results suggest that until
certain anionic sites in the glomerular wall become saturated with cationic molecules, these sites may attract
and apparently trap such molecules. The first anionic
barriers involved in this trapping phenomenon appear
to be the endothelial glycocalyx and anionic sites in the
LRI. Only after these sites become saturated does PEI
appear t o move across the lamina densa and bind to the
extensive array of anionic sites in the LRE. Once all
these anionic sites in the glomerular wall are saturated,
cationic molecules appear to pass across the filtration
slits and enter the urinary space. Our observations are
in part supported by a previous report that the buildup
of the cationic molecule hexadimethrine in the glomerular basement membrane and its movement across the
filtration slits depend upon the length of time that this
molecule is infused (Hunsicker et al., 1981).The results
of our in vitro study of intact glomeruli indicate that the
glomerular wall is highly permeable to PEI and that
even in the absence of hemodynamic driving forces PEI
will freely filter across the wall and gradually bind to
anionic sites in a manner similar to that seen after in
vivo injection of this molecule.
Although it appears that the gradual neutralization of
glomerular anionic sites may be important in the subsequent movement of cationic molecules across the glomerular wall, alterations in the gel-like matrix of the
GBM may also play a role. This latter possibility was
recently suggested by Barnes et al. (1984), who noted
that tagging of anionic sites in the GBM by PEI results
in an increased permeability of the glomerular wall to
cationic as well as anionic ferritin. These investigators
suggested that this increased permeability to polycations may have resulted from a distortion of the GBM
gel structure similar to structural changes induced by
hexadimethrine in polyacrylamide-polyacrylic acid gels
(Bertolatus and Hunsicker, 1982). Our finding that discrete anionic sites in the LRE are no longer evident in
glomeruli exposed to an excess of cationic molecules,
and that these sites may be replaced by a band of cationic binding subjacent t o the base of the foot processes
in these samples, may reflect such a change in the structure of the GBM matrix.
Finally, the present study signals that caution should
be used when cationic tags such as PEI are used to
demonstrate anionic sites in the GBM. The use of too
much or too little of such tracers can affect where such
tracers become localized in the glomerular wall, whether
230
P.M. ANDREWS AND S.B. BATES
Fig. 10. Cross section through the glomerular wall of an inner glomerular loop after in vitro incubation in 0.1% PEI for 1 minute. The
PEI has bound to the epithelial glycocalyx, filtration slits, and to
discrete anionic sites in the lamina rara externa (arrows). X75,OOO.
Fig. 11. Cross section through an outer glomerular loop after in vitro
incubation in 0.1% PEI for 1minute. PEI (arrows) binding has caused
adherence of adjacent foot processes and has crossed the glomerular
basement membrane and bound extensively t o the endothelial glycocalyx. Note that like after in situ infusion, treatment with excess PEI
has resulted in less discrete visualization of anionic sites in the glomerular basement membrane. x 75,000.
231
GLOMERULAR PERMEABILITY TO POLYCATIONS
such molecules pass across the filtration slits, and even
whether there is a possible reorganization of anionic
sites in the GBM due to infusion of a n excess of cationic
molecules.
ACKNOWLEDGMENTS
This investigation was supported by National Institues of Health grants AM31111 and AM31177.
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