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The role of the glomerular endothelium

inalbumin handling
Simon Satchell
Abstract | The unique permeability characteristics of the glomerular capillary wall depend on its three-layer
structure, consisting of endothelial cells, the basement membrane and podocytes. These components form
the glomerular filtration barrier (GFB). That albuminuria may occur in the absence of changes in podocyte foot
processes suggests that GFB components other than podocytes have essential roles in albumin handling. The
endothelium forms the first part of the GFB and is characterized by fenestrationstranscellular holes that
are filled with endothelial glycocalyx, a hydrated mesh principally comprised of proteoglycans. The glycocalyx
and adsorbed plasma constituents form the endothelial surface layer (ESL). Human and animal studies have
shown that the glomerular ESL restricts macromolecule passage and ensures that plasma albumin is largely
excluded from the GFB. The glomerular endothelium is also likely to indirectly influence glomerular albumin
handling by modifying podocyte behaviour. These modifications may occur physiologically through soluble
mediators and/or pathologically through increased exposure of podocytes to plasma components as a
consequence of ESL dysfunction. The importance of the glomerular endothelium and ESL in albumin handling
also sheds light on the relationship between albuminuria and vascular disease. The therapeutic potential
that this relationship offers will become evident with better understanding of the structure, composition and
regulation of the glycocalyx.
Satchell, S. Nat. Rev. Nephrol. advance online publication 1 October 2013; doi:10.1038/nrneph.2013.197

Introduction
The glomerulus is a highly specialized capillary bed in
which the capillary walls are uniquely adapted enabling
them to function as a biological sievethe glomerular filtration barrier (GFB). The GFB is comprised of
glomerular endothelial cells, the glomerular basement
membrane, and podocytes or, more specifically, their
interdigitating foot processes and the slit diaphragms
that span the gaps between them. The subpodocyte
space is a downstream component of the GFB.1 The
human GFB is highly selective: it is freely permeable to
water and small molecules, but has an estimated sieving
coefficient of albumin of 0.00008 (meaning that normally
only 0.008% of plasma albumin passes through the GFB
to the urinary space).2
Comparison with systemic capillaries indicates
that the low permeability of the glomerular capillary
to albumin is not particularly unusual, whereas its
permeability to water is orders of magnitude higher
than in other capillary beds.3 Insights into the importance of proteins expressed at the podocyte slit diaphragm have led to a focus on the role of podocytes
in the barrier to albumin.4 However, the dimensions
of podocyte filtration slits are not consistent with the
observed permeability properties of the GFB, suggesting
that the podocyte slit diaphragm alone does not explain
glomerular permselectivity. 5 The GFB is increasingly
Competing interests
The author declares no competing interests.

understood to function as a whole entity, both in biophysical terms and with respect to the importance of
cell-to-cell communication in maintaining its function.6 In this Review I describe the contribution of the
glomerular endothelium to the permeability properties
of the GFB and the evidence for a role of glomerular
endothelial cells in albumin handling. I also discuss
the role of cell-to-cell communication in glomerular
homeostasis and the therapeutic potential of targeting
the endothelial surface layer (ESL) in glomerular and
vascular disease.

The glomerular endothelium

Evidence of an albumin barrier function
The glomerular endothelium is the first part of the GFB
that is encountered by plasma and the first structure that
provides a barrier to albumin. In their classic work, Ryan
and Karnovsky used immunolabelling to study the location of albumin within glomeruli that had been rapidly
fixed insitu under conditions of normal filtration.7
They found that during normal blood flow albumin is
largely excluded from the GFB, whereas if blood flow
stops albumin can penetrate the glomerular basement
membrane and enter the urinary space (Figure1).7
Unsurprisingly, the highest density of albumin was
present in the capillary lumen, whereas a marked
decrease in albumin density at the endothelium confirmed that this structure is the first effective barrier to
albumin. Interestingly, no decrease in albumin density

NATURE REVIEWS | NEPHROLOGY

University of Bristol,
Academic Renal Unit,
Learning and Research,
Southmead Hospital,
Bristol BS10 5NB, UK.
s.c.satchell@
bristol.ac.uk

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Key points
The glomerular filtration barrier (GFB) functions as a whole with each layer
making an important contribution
Albuminuria can occur in the absence of changes in podocyte foot processes,
confirming that other GFB components have essential roles in albumin handling
The glomerular endothelium is characterized by fenestrations (transcellular
holes essential for filtration function) and a surface glycocalyx that extends over
the fenestrations
The glomerular endothelial surface layer (ESL) consists of a surface-bound
glycocalyx and adsorbed plasma components; dysfunction of the ESL
contributes to increased glomerular permeability in disease
Cell-to-cell communication has an important role in glomerular homeostasis;
glomerular endothelial cell-to-podocyte communication is likely to regulate the
podocyte contribution to glomerular albumin handling in health and disease
Dysfunction of the glomerular endothelial glycocalyx is an attractive therapeutic
target that links albuminuria to systemic vascular disease and potentially helps
to explain why albuminuria is a risk factor for cardiovascular disease

b
P

U
P

GBM
GBM
L

Figure 1 | Under normal haemodynamic conditions albumin is largely excluded from

the glomerular filtration barrier. a | Superficial glomeruli in anaesthetised Munich
Wistar Frmter rats were rapidly fixed insitu and endogenous albumin was labelled
and visualized using an ultrastructural peroxidise localization procedure. Albumin
(the black reaction product) is visible on the luminal side of the endothelium
(highlighted in yellow) and is largely confined to the capillary lumen with only small
amounts visible in the lamina rarainterna. No albumin is visible deeper in the
basement membrane or upstream of the podocyte slit diaphragm. b | After routine
immersion fixation of glomeruli, large amounts of endogenous albumin are visible
within the GBM and the urinary space. Abbreviations: E, glomerular endothelium;
GBM, glomerular basement membrane; L, lumen; P,podocyte; R, red blood cells in
capillary lumen; U, urinary space. Reproduced with permission from Nature
Publishing Group Ryan, G.B. & Karnovsky, M.J. Kidney Int. 9, 3645 (1976).

was observed at the level of podocyte foot processes.

These results are consistent with subsequent observations of the relationship between glomerular filtration
rate (GFR) and the sieving coefficient. If a substantial
decrease in albumin concentration across the podocyte slit diaphragm occurred, this decrease would
be expected to be accentuated by increases in GFR.
Increased flow rate would result in a greater concentration of solute upstream of the slit diaphragm as water
passes through the barrier and leaves the solute behind.
This concentration polarization would in turn lead to
an increase in the concentration gradient of solute and
hence the diffusion of increased amounts of albumin.
However, such an increase in albumin diffusion does
not occur in response to increases in GFR in experimental models, suggesting that a structure upstream
of the slit diaphragm in the GFB has a greater role in
restricting the passage of albumin, particularly on the
basis of charge.810

Although proteinuria is often accompanied by ultrastructural changes in podocyte foot processes, many
examples exist where this is not the case. In experi
mental animals, blockade of circulating vascular endo
thelial growth factor (VEGF) results in proteinuria
without changes in foot processes.11,12 Similar effects are
observed in response to reactive oxygen species (ROS)13
and hypertonic saline,14 both of which disrupt the ESL.
Podocyte-specific overexpression of angiopoietin-2
results in apoptosis of glomerular endothelial cells and
albuminuria, but podocytes remain structurally intact.15
In addition, Munich Wistar Frmter rats develop
proteinuria with age without alteration of podocyte foot
processes.16 In humans, proteinuria in the absence of
podocyte changes may be observed in pre-eclampsia,17
in early diabetes18 and in a rare familial nephrotic syndrome. 19 Indeed, podocyte foot process effacement
generally correlates poorly with proteinuria in human
glomerular disease.20 Hence, it is clear that podocyte foot
process abnormalities are not necessary for proteinuria,
indicating that dysfunction of another part of the GFB
must be responsible in some cases. Podocyte foot process
effacement may be seen as a response to injury and glomerular dysfunction rather than necessarily the cause of
this dysfunction.21
Finally, endothelial cells are the key regulators of
vascular permeability in microcirculations in which
no podocytes are present. 3,22 If the glomerulus is a
specialized capillary bed, we might expect the barrier
function of the endothelium to be adapted in the glomerulus rather than replaced with a different cell type.
The fact that various conditions (such as, diabetes,
sepsis and inflammatory bowel disease) in which systemic endothelial dysfunction can be demonstrated are
associated with microalbuminuria or proteinuria also
provides strong circumstantial evidence that a glomerular endothelial defect may contribute to proteinuria.23 To
understand how the glomerular endothelium provides a
barrier to albumin, the various structural features of the
endothelium must be considered.

Structure
The most obvious characteristic of glomerular endo
thelial cells is the presence of numerous transcellular
holes or fenestrations of 6080nm in diameter that
occupy 3040% of the cell surface. 24 These holes are
arranged in sieve plates that are separated by ridges of
cytoplasm and the majority do not possess diaphragms.25
Fenestrations are an adaptation that facilitates the high
water permeability of the glomerular endothelium,
which is essential for filtration function. 26 They are
also found in other capillary beds where high rates of
exchange between intravascular and extravascular
compartments arerequired.
Cell-to-cell junctions in the glomerular endothelium
have not been characterized in detail. However, glomerular endothelial cells have been shown to express
typical endothelial junction markers (including platelet endothelial cell adhesion molecule and cadherin5)
invivo and invitro,27,28 suggesting a predominance of

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adherens junctions over tight junctions. Caveolae, vacuoles and vesicularvacuolar organelles form a group of
subcellular structures characterized by diaphragms. In
some endothelial cells, these structures have an important role in transendothelial molecular transport. In
glomerular endothelium, caveolae are prominent 25 but
their physiological role is not clear. Deletion of caveolin1, which leads to a loss of caveolae, does not result in
any obvious filtration abnormality (such as proteinuria)
or in loss of fenestrations.29
Attention has now turned to the endothelial glyco
calyx, which forms a polyanionic hydrated mesh on the
surface of glomerular endothelial cells. This fragile structure spans the fenestral and interfenestral domains.3032
As specialized tissue fixation and labelling techniques
are required to visualize the glycocalyx using electron
microscopy, the majority of published micrographs
do not show this structure and its significance has not
always been appreciated.

Permeability
Detailed analyses of the permeability of fenestrated capillaries indicate that fenestrations provide the major route
for water movement across the endothelial cell layer.33
A strong linear relationship exists between the density
of fenestrations and water permeability. Moreover, the
fractional area of fenestrations is much higher than that
of the intercellular clefts, suggesting that the contribution of these clefts to water permeability is insignificant.
Fenestrations are also likely to provide the major route
for albumin passage. They are far too big to provide
substantial restriction to the passage of albumin molecules (which have an approximate radius of 36 or
3.6nm) across the glomerular endothelium. This observation led to the long-held assumption that the glomerular endothelium does not contribute to the barrier
toalbumin.
However, biophysical analyses indicate that fenestrations are not as permeable to water or small solutes as
would be expected if they were empty, and the permea
bility of fenestrated capillaries to macromolecules is
not higher than that of nonfenestrated capillaries.3,33
These observations imply the presence of an additional
barrier within fenestrationsthey are not simply empty
holes. The endothelial glycocalyx covers the fenestrations and has molecular and charge characteristics that
could restrict albumin passage. Strong evidence suggests
that the endothelial glycocalyx restricts macromolecular flux in systemic microvessels 3,22,34 and biophysical
models predict that this is also the case in glomerular
capillaries.6,26 These models also provide another important insight; as mentioned earlier the GFB functions as
a whole, with each layer making an important contribution to selective permeability. Hydraulic resistances
are essentially additive, whereas sieving coefficients are
multiplicative.26 This relationship means that a change in
one component affects the overall protein permeability
by the same proportion; for example, a 10% change in the
permeability of any one layer will produce a 10% change
in the overall permeability of the GFB.

The endothelial surface layer

Structure
The glycocalyx is a complex structure, comprised of cellsurface-anchored proteoglycans, glycoproteins and sialic
acids,35,36 which cover the luminal surface of endothelial
cells throughout the vasculature.37 Secreted molecules,
including hyaluronan, and circulating molecules, including albumin and orosomucoid (also known as 1-acid
glycoprotein) are adsorbed onto this layer in dynamic
equilibrium with the plasma. 37 Together the glyco
calyx and adsorbed components are known as the ESL
(Figure2). The term glycocalyx was originally used to
describe the thin noncellular coat that can be visualized
by electron microscopy using various dyes, whereas the
ESL explains the much thicker layer that can be observed
using intravital microscopy.6,37
Proteoglycans consist of a core protein, such as a syndecan, glypican or biglycan, with a number of covalently
bound glycosaminoglycan (GAG) side chains. These
polys accharide chains consist of distinct repeating
disaccharide units. Some GAGs, including heparan sulphate and chondroitin sulphate, are highly sulphated,
resulting in a strong negative charge.36,37 Heparan sulphate is thought to be abundant in the endothelial glyco
calyx together with smaller amounts of chondroitin
sulphate and the other sulphated GAGs.35 It should be
noted, however, that this assertion is based on historical
cell culture studies3840 and the proportions of the various
GAGs in the endothelial glycocalyx have not been examined in detail. The nonsulphated GAG, hyaluronan, is
also abundantly present in the glycocalyx and contributes
to its gel-like structure and volume. Hyaluronan forms
long chains that are either tethered to the cell surface
through interactions with receptors (for example, CD44
or hyaluronan-mediated motility receptor), bound to
hyaluronan synthase or more loosely adsorbed.35
Few studies have examined the glycocalyx of glomerular endothelial cells invivo. In rats, glomerular
endothelial cells express sialoproteins, heparan sulphate
and hyaluronan.41 Human glomerular endothelial cells
invitro express a range of proteoglycan core proteins
as well as heparan sulphate, chondroitin sulphate and
hyaluronan GAGs. 4244 Evidence exists of an under
lying formal structure that is consistent across capillary beds. 45,46 Some studies have suggested that the
depth and composition of the endothelial glycocalyx
varies between different capillary beds, 4749 but some
of this reported variation is likely attributable to differences in the fixation and staining of various samples.49
However, variation does exist between the composition
of the fenestral and interfenestral regions of glomerular endothelial cells. The glycocalyx in the fenestrae has
a higher ratio of heparan sulphate and hyaluronan to
sialoproteins.41 A similar predominance of heparan sulphate in fenestrations has been observed in other capillaries.50 As the glycocalyx forms a mesh that is capable
of restricting the movement of macromolecules on the
basis of size, charge and steric hindrance, the particular
composition of the glycocalyx in the fenestrae is likely
to be crucial for its permeabilityproperties.

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a
Plasma flow exerts shear stress

Plasma

Albumin
ESL
Fenestrated
endothelium
Glomerular
basement
membrane
Podocyte slit
diaphragm

Proteoglycan
VEGFR2
VEGF-A

? NO and other
mediators

VEGFR3 Tie2
VEGF-C

Ang1

Podocyte
foot process

Podocyte
cell body

b
Albumin
Orosomucoid, lumican or
other plasma component
Hyaluronan
Heparan
sulphate

Syndecan

Hyaluronan Chondroitin
receptor
sulphate

Glypican

Glycoprotein

Fenestration

Figure 2 | The role of the ESL in the glomerular filtration barrier. a | The ESL
comprises the cell-surface-anchored glycocalyx and adsorbed plasma constituents,
and covers the luminal surface of the endothelium, extending over and into
thefenestrae. The ESL forms the first barrier to albumin passage across the
glomerular filtration barrier and ensures that albumin is largely confined to
thecapillary lumen. Behaviour of the glomerular endothelium is in part regulated
bysoluble mediators including VEGFA, VEGFC and Ang1, which are produced by
podocytes and act on their cognate receptors expressed on the endothelium.
Flowing plasma exerts shear stress, which is transduced by the glycocalyx to
modulate endothelial cell behaviour, including release of mediators such as NO,
which are likely to have reciprocal actions on podocytes. b | Detail of the ESL
showing its heterogeneous structure. The cell-surface-anchored proteoglycan core
proteins include glypicans and syndecans, which have anionic glycosaminoglycan
side chains. Glypicans have heparan sulphate chains, whereas syndecans also
have chondroitin sulphate chains. Hyaluronan, which is often present in very long
chains, binds to cell-surface receptors including CD44 and may also be more
loosely adsorbed onto cell-surface anchored molecules along with other plasma
components. Glycoproteins may have short carbohydrate side chains and terminal
sialic acids residues. The ESL, therefore, forms a negatively charged barrier to the
passage of albumin. Abbreviations: Ang1, angiopoietin-1; ESL; endothelial surface
layer; NO, nitric oxide; Tie2, angiopoietin 1 receptor; VEGF, vascular endothelial
growth factor; VEGFR, vascular endothelial growth factor receptor.

Circulating molecules have an important role in modifying the barrier properties of the endothelial surface.
For example, binding of albumin to proteoglycans in
the glycocalyx 51 is important for maintaining the barrier
to water in glomerular capillaries.52 Maintenance of

the barrier to macromolecules requires other plasma

components. Orosomucoid seems to be particularly
important in this respect. This molecule, which can
be eluted from the renal microvascular ESL exvivo,14
binds to the endothelial cell surface53 and adds negative charge to the vessel wall, reducing its permeability
tomacromolecules.54

Evidence for a role in the barrier to albumin

Invivo and exvivo studies
In culture, conditionally immortalised human glomerular
endothelial cells have a 200nm glycocalyx.42 Enzymatic
removal of particular components of this glycocalyx (for
example removal of heparan sulphate by heparanase42
or of sialic acid residues by hemopexin55) increased the
rate of passage of albumin across glomerular endothelial
cell monolayers. Similarly, long-term (14days) exposure of glomerular endothelial cell monolayers to a high
glucose concentration reduced the amount of cell-surface
sulphated GAGs by approximately 50% and resulted in
an increase in the passage of albumin, consistent with
a loss of glycocalyx.43 ROS also cause shedding of glomerular endothelial cell glycocalyx components and a
corresponding increase in the transmonolayer passage
of labelled albumin.56
In cooled isolated perfused murine kidneys, enzym
atic removal of heparan sulphate markedly reduced
GFB anionic sites and increased albumin excretion
exvivo.57 Similarly, removal of chondroitin sulphate
or hyaluronan reduced the thickness of the glomerular ESL and increased albumin excretion.57,58 Removal
of sialic acid residues in the glomerulus also resulted
in loss of anionic sites on glomerular endothelial cells
and albuminu ria in rodent models. 59,60 In apolipo
proteinE-deficient mice, continuous administration of
hyaluronidase via osmotic pumps resulted in a reduction in systemic glycocalyx volume and an increase in
urinary protein excretion.61 In another mouse study,
hyaluronidase treatment resulted in loss of glomerular
endothelial cell glycocalyx and immunostaining showed
the presence of endogenous albumin downstream of the
glomerular basement membrane on the podocyte cell
membrane.32 However, proteinuria was not observed,
perhaps because the normal levels of apolipoproteinE
in these mice resulted in a less severe insult. The appearance of albumin on the podocytes suggests that reuptake
of albumin by podocytes and elsewhere in the nephron
reduced albuminu ria. In isolated perfused murine
kidneys, cooling prevents this energy-d ependent
reuptake of albumin so all of the filtered albumin is
excreted in the urine.57
A limitation of the invivo and exvivo studies discussed
above is that a contribution of the effects of enzymes on
structures other than the ESL to the observed increases
in albuminuria cannot be excluded. Also chondroitinase
and hyaluronidase can cross-react with hyaluronan and
chondroitin sulphate, respectively. The careful dose titration that is necessary to exclude such cross-reactivity 44
is difficult invivo, meaning that hyaluronidase, for
example, may remove both hyaluronan and chondroitin

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sulphate from the ESL. It is intriguing, however, that
damage to different components of the glomerular
endothelial cell ESL has similar effects in disturbing the
filtration barrier and increasing albumin passage. This
finding provides further evidence that the ESL functions
as an integrated whole and compromise of one element
affects its overall barrier properties.
Evidence of a role of noncovalently bound ESL components in the barrier to albumin comes from experiments in which injection of hypertonic saline into the
renal arteries of rats was used to displace these components.14 This displacement led to a 12-fold increase in
the fractional clearance of albumin without any apparent
ultrastructural changes in the GFB, further demonstrating the importance of the ESL in the albumin barrier.
Interestingly, displacement of the noncovalently bound
ESL components did not reduce the thickness of the ESL,
suggesting that the gross structure of the ESL does not
depend on these components. Mass spectrometry of the
eluate from the hypertonic solution showed the presence
of orosomucoid, lumican and other molecules that might
have a direct role in ESL barrier properties.
Disease models
The role of the glomerular ESL in the albumin barrier
has also been investigated in disease models. The effects
of adriamycin (which induces proteinuria) on the GFB
were investigated in an isolated perfused kidney system.62
Adriamycin treatment reduced glomerular ESL dimensions to 20% of that of controls, and this reduction was
associated with an increase in the clearance of albumin.
Although flattening of podocyte foot processes was
observed in this study, loss of the ESL likely contributed
to the observed permeability changes.
Munich Wistar Frmter rats develop albuminuria and
chronic kidney disease spontaneously with age.16,63 Using
invivo multiphoton microscopy, Salmon and colleagues
showed loss of endothelial glycocalyx in the glomerular and mesenteric microvessels of aged Munich Wistar
Frmter rats.48 This loss was accompanied by systemic
defects in capillary permeability and by an increase in
the glomerular sieving coefficient of albumin. A similar
increase could be induced in young rats by glycocalyx
removal using neuraminidase, but capillary permea
bility could not be increased further in old rats using
this enzyme. Importantly, the increased albumin
permeability in older rats could be reduced by intra
venous administration of wheat germ agglutinin lectin,
which apparently bound to the remaining glycocalyx
and restored its barrier properties. These data provide
strong evidence of a role of the glomerular endothelial
glycocalyx in the glomerular permeability barrier to
albumin and also directly link albuminuria to systemic
vasculardysfunction.
Zucker fatty rats also spontaneously develop albumin
uria as they age. This albuminuria is accompanied by a loss
of glomerular ESL, hyperfiltration of macromolecules, and
loss of glomerular expression of syndecan1.64 Similarly, in
a rat sepsis model, increased urinary albumin excretion
was accompanied by severe disruption of the glomerular

endothelial glycocalyx and loss of glomerular syndecan1,

hyaluronan and sialic acid residues.65 Loss of podocyte
foot processes also occurred in this model. Studies using
non-obese diabetic mice have shown modification of the
renal cortical expression of proteoglycan core proteins and
an increase in the fractional clearance of albumin in these
animals.66 Similarly, loss of GAGs within the glomerular
capillary wall has been demonstrated in streptozotocininduced diabetic rats with albuminuria67 and reduced
levels of glomerular sialic acid and heparan sulphate and
an increased urinary albumin-to-creatinine ratio have
been observed in lipopolysaccharide-treated mice. 68
However, these changes were not precisely localized to
the glomerular endothelial glycocalyx.
The importance of glomerular heparan sulphate
in the barrier to albumin passage across the GFB
is further suggested by the fact that transgenic diabetic mice that lackheparanase (the only endogenous
enzymethatdegrades heparan sulphate) do not develop
albuminuria.69 Moreover, albuminuria was reduced by
heparanase inhibition in wildtype diabetic mice. However,
the endothelial glycocalyx was not specifically investigated
in this study and the GFB component that was affected by
loss of heparan sulphate was not identified.
Human studies
In humans, direct study of the glomerular endothelial
glycocalyx is not possible. However, a reduction in the
systemic endothelial glycocalyx volume (measured
using a tracer dilution technique) has been associated
with increased urinary protein excretion in patients with
type1 diabetes mellitus.70 Although not directly shown to
be associated with increased urinary albumin excretion,
widespread loss of the glycocalyx also occurs in healthy
individuals rendered hyperglycaemic,71 and glycocalyx
thickness is reduced in sublingual capillaries in patients
with type2 diabetes mellitus. 72 These observations
further suggest that glomerular endothelial glycocalyx
damage is likely and may contribute to albuminuria in
diabetic patients. The loss of glomerular charge selectivity in individuals with type166 or type267 diabetes
mellitus and microalbuminuria66,73,74 is consistent with
dysfunction of the glomerular endothelial glycocalyx.
Taken together, the data described above provide
compelling evidence that the ESL makes a substantial
contribution to the glomerular permeability barrier
to albumin. They are also commensurate with the
increasing evidence that the ESL has important roles
in the pathophysiology of diseases, such as ischaemia
reperfusion injury and sepsis, that affect organ systems
other than the kidneys.34,75

Glomerular homeostasis
Regulation of the glycocalyxAlthough a variety of factors
(including high glucose concentrations,43,76 ROS,56,64,77
endogenous enzymes, 42,75 and inflammatory medi
ators75,78,79) are known to damage the ESL, little is known
about its physiological regulation. The complexity of the
structure means that making progress in understanding this area will be challenging. As well as the multiple

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protein constituents, the carbohydrate components of the
glycocalyx (GAGs and sialic acids) present a major challenge because of their structural complexity and the multiple biosynthetic and degradative enzymes that control
their production, modification and degradation.8083
The endogenous endot helial g rowt h fac tor
angiopoietin1 increases the depth of the endothelial
glycocalyx in systemic vessels invivo and reduces their
permeability to albumin. Angiopoietin1 protects
against enzyme-induced increases in water permeability
by restoring the glycocalyx at the cell surface.84 We have
shown differential regulation of GAGs by the endothelial
growth factors VEGFA and VEGFC in cultured glomerular endothelial cells.44 Both of these factors increasethe
synthesis of hyaluronan; however, VEGFC increases
theoverall charge on sulphated GAGs and VEGFA
induces shedding of charged GAGs. Angiopoietin1,
VEGFA and VEGFC are produced by podocytes,
whereas glomerular endothelial cells express their cognate
receptors and respond to these factors with changes in
their permeability to water and albumin. 27,8589 These
observations suggest that regulation of the glomerular
endothelial cell glycocalyx may be controlled by crosstalk with podocytes. The developmentof endothelial
damage and proteinuria in the presence of comparatively
preserved podocyte foot processes in micewithpodocyte-specific knockout of VEGF 90 andin mice with
podocyte-specific overexpression of angiotensin2,15 also
suggests a role of podocyte cross-talk in the regulation of
glomerular endothelial cell glycocalyx.

Regulation of glomerular cells

If podocytes regulate the behaviour of glomerular
endothelial cells, the possibility exists that glomeru
larendothelial cells reciprocally regulate the behaviour
of podocytes and other glomerular cells and, therefore, indirectly affect glomerular albumin handling.
In the systemic circulation, mediators produced by
endothelial cells (for example nitric oxide; NO), have
important roles in regulating the behaviour of vascular wall cells.91,92 Evidence of an important role of NO
produced by glomerular endothelial cells in maintenance of the GFB is accumulating. Knockout studies
have shown that deletion of endothelial NO synthase
(eNOS; the gene NOS3) leads to distinct glomerular
lesions93 and markedly increases susceptibility to diabetic glomerular disease.9496 Conversely, maintenance
of endothelial levels of the essential eNOS cofactor tetra
hydrobiopterin ameliorates diabetic nephropathy.97 In
humans, loss of glomerular expression of eNOS has been
identified in glomerulonephritis, further suggesting a
protective role of the enzyme.98100 In addition, polymorphisms in NOS3 are associated with more advanced
diabeticnephropathy.101
Although these studies suggest that NO produced
byglomerular endothelial cells is important in regulating glomerular biology, they do not conclusively identify the podocyte as the target cell. Using a novel invitro
coculture model, we have now provided direct evidence
that glomerular endothelial cells modulate podocyte

behaviour, including barrier properties. 102 Inthese

studies, we found that the regulation of glomerular endo
thelial cellpodocyte communication was dependent on
shear-stress (that is, the force exerted on the endothelial
surface by flowing liquid). Shear-sensing is dependent
on the endothelial glycocalyx,103 suggesting an additional
important role for the glomerular endothelial glycocalyx
in glomerularphysiology.
Another potentially important pathway through
which glomerular endothelial cells might modulate the
behaviour of other glomerular cells is via the increased
permeability of the glomerular endothelium, and particularly the ESL, in disease states such as diabetes.34
Increased delivery of macromolecules to podocytes as a
result of this increased permeability has been proposed
to cause their dysfunction.23,104 The presence of albumin
on the podocyte surface when ESL function is disturbed
by interruption of blood flow 7 or by enzymatic degrada
tion32 demonstrates increased podocyte exposure to
this potentially adverse stimulus. Damage to podocytes
via protein overload, which results in the saturation of
physiological clearance mechanisms, has been directly
demonstrated in animal models.105,106 In mice, genetic
deletion of the glomerular endothelial cell-restricted
endosomal recycling regulator, EHdomain containing
protein 3, resulted in proteinuria and foot-process effacement, further demonstrating that podocyte dysfunction
can occur as a consequence of an endothelial lesion.107

Conclusions
The evidence that the glomerular endothelium, and particularly the ESL, contributes to the glomerular barrier
to albumin is overwhelming. This conclusion has two
important corollaries. Firstly, ESL dysfunction may
provide the missing link between increased urinary
albumin excretion and disease elsewhere in the vasculature, and hence may help to explain why increased
urinary albumin excretion is an important risk factor
for cardiovascular disease.108,109 This hypothesis suggests
that both albuminuria and vascular diseases are a con
sequence of ESL damage. Secondly, the ESL is a target for
therapies that could potentially impact not only glomerular disease, but also other vascular diseases. The fact that
the endothelial glycocalyx can be manipulated by exo
genous agents invitro,44,84 in animals models34,84,110 and in
humans72 gives credence to this therapeuticpotential.111
However, many questions remain to be answered
before this potential can be realised. Evidence is emerging
of consistent structural features of the glycocalyx,45,46 but
how these structures relate to its chemical components
and to what extent, if any, they interact 51 is poorly understood. Similarly, although the composition of the glycocalyx is known in broad terms, the disaccharide sequence
of GAGs are likely to be important for glycocalyx function
but have not yet been characterized. The glycocalyx has
multiple functions in addition to its permeability barrier
properties, including regulation of clotting, complement
activation and leukocyteendothelial cell interactions,
growth-factor binding and shear-sensing.35,36 Which of
the multiple components are important for each of these

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REVIEWS
functions and for albumin handling is not yet clear. To
design therapies intelligently, identification of the components that need to be preserved or restored and understanding of how they are regulated will be necessary.
Simple labelling of glycocalyx components on glomerular tissue sections68,97 does not provide robust information about the composition of the glomerular endothelial
glycocalyx as the labelled molecules might not be specific to the endothelial glycocalyx.32 The development of
specific techniques for the study of the glomerular endo
thelial glycocalyx invivo,48 in conjunction with transgenic approaches to manipulate specific components and
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