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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 permeabil ity 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 endo thelium confirmed that this structure is the first effective barrier to albumin. Interestingly, no decrease in albumin density

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

a
P U

P E GBM GBM R L E

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).

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 endo thelial 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.

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
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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 facili tates 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.

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 poly s accharide chains consist of distinct repeating di saccha ride 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 endo thelial 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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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.
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a
Plasma Plasma flow exerts shear stress

Albumin ESL Fenestrated endothelium Glomerular basement membrane Podocyte slit diaphragm Proteoglycan VEGFR2 VEGF-A VEGFR3 Tie2 VEGF-C Ang1 ? NO and other mediators

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 hya luronan 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 albumin u ria in rodent models. 59,60 In apolipo proteinE-deficient mice, continuous administration of hyaluronidase via osmotic pumps resulted in a reduction in systemic glyco calyx 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, protein uria was not observed, perhaps because the normal levels of apolipo proteinE 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 albumin u ria. In isolated perfused murine kidneys, cooling prevents this energy- d ependent re uptake 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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Podocyte foot process

Podocyte cell body

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

Syndecan

Hyaluronan Chondroitin receptor sulphate Fenestration

Glypican

Glycoprotein

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
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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
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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 microalbumin uria66,73,74 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 angio poietin 1 increases the depth of the endo thelial glyco calyx in systemic vessels invivo and reduces their permeability to albumin. Angiopoietin1 protects against enzyme-induced increases in water permea bility 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 endo thelial cell glycocalyx may be controlled by crosstalk with podocytes. The developmentof endo thelial 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. 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 permeabil ity 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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Regulation of glomerular cells If podocytes regulate the behaviour of glomerular endothelial cells, the possibility exists that glomeru larendo thelial 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 endo thelial 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 suscepti bility 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 co culture model, we have now provided direct evidence that glomerular endothelial cells modulate podocyte
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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 glyco calyx 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
1. Neal, C.R., Crook, H., Bell, E., Harper, S.J. & Bates, D.O. Three-dimensional reconstruction of glomeruli by electron microscopy reveals a distinct restrictive urinary subpodocyte space. J.Am. Soc. Nephrol. 16, 12231235 (2005). Norden, A.G. etal. Glomerular protein sieving and implications for renal failure in Fanconi syndrome. Kidney Int. 60, 18851892 (2001). Michel, C.C. & Curry, F.E. Microvascular permeability. Physiol. Rev. 79, 703761 (1999). Pavenstadt, H., Kriz, W. & Kretzler, M. Cell biology of the glomerular podocyte. Physiol. Rev. 83, 253307 (2003). Haraldsson, B. & Sorensson, J. Why do we not all have proteinuria? An update of our current understanding of the glomerular barrier. News Physiol. Sci. 19, 710 (2004). Haraldsson, B., Nystrom, J. & Deen, W.M. Properties of the glomerular barrier and mechanisms of proteinuria. Physiol. Rev. 88, 451487 (2008). Ryan, G.B. & Karnovsky, M.J. Distribution of endogenous albumin in the rat glomerulus: role of hemodynamic factors in glomerular barrier function. Kidney Int. 9, 3645 (1976). Lund, U. etal. Glomerular filtration rate dependence of sieving of albumin and some neutral proteins in rat kidneys. Am. J. Physiol. Renal Physiol. 284, F1226F1234 (2003). Rippe, C., Asgeirsson, D., Venturoli, D., Rippe, A. & Rippe, B. Effects of glomerular filtration rate on Ficoll sieving coefficients () in rats. Kidney Int. 69, 13261332 (2006). Rippe, B. What is the role of albumin in proteinuric glomerulopathies? Nephrol. Dial. Transplant. 19, 15 (2004). Sugimoto, H. etal. Neutralization of circulating vascular endothelial growth factor (VEGF) by antiVEGF antibodies and soluble VEGF receptor 1 (sFlt1) induces proteinuria. J. Biol. Chem. 278, 1260512608 (2003). Maynard, S.E. etal. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J. Clin. Invest. 111, 649658 (2003). Yoshioka, T., Ichikawa, I. & Fogo, A. Reactive oxygen metabolites cause massive, reversible proteinuria and glomerular sieving defect without apparent ultrastructural abnormality. J. Am. Soc. Nephrol. 2, 902912 (1991). Friden, V. etal. The glomerular endothelial cell coat is essential for glomerular filtration. Kidney Int. 79, 13221330 (2011). Davis, B. etal. Podocyte-specific expression of angiopoietin2 causes proteinuria and apoptosis of glomerular endothelia. J. Am. Soc. Nephrol. 18, 23202329 (2007). Macconi, D. etal. Effect of angiotensinconverting enzyme inhibition on glomerular

sophisticated biochemical analyses, will provide answers to these questions.

Review criteria
A search for original articles published between 1990 and 2013 was performed in PubMed. The search terms used were endothelial, glycocalyx, glomerulus, albumin, proteoglycan and glycosaminoglycan in various combinations. All articles identified were Englishlanguage, full-text papers. Reference lists of identified articles were searched for further relevant papers.
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