Basic Research
Open Access

Loss of the Endothelial Glycocalyx Links Albuminuria and Vascular Dysfunction

Andrew H.J. Salmon, Joanne K. Ferguson, James L. Burford, Haykanush Gevorgyan, Daisuke Nakano, Steven J. Harper, David O. Bates and Janos Peti-Peterdi
JASN August 2012, 23 (8) 1339-1350; DOI: https://doi.org/10.1681/ASN.2012010017

Abstract

Patients with albuminuria and CKD frequently have vascular dysfunction but the underlying mechanisms remain unclear. Because the endothelial surface layer, a meshwork of surface-bound and loosely adherent glycosaminoglycans and proteoglycans, modulates vascular function, its loss could contribute to both renal and systemic vascular dysfunction in proteinuric CKD. Using Munich-Wistar-Fromter (MWF) rats as a model of spontaneous albuminuric CKD, multiphoton fluorescence imaging and single-vessel physiology measurements revealed that old MWF rats exhibited widespread loss of the endothelial surface layer in parallel with defects in microvascular permeability to both water and albumin, in both continuous mesenteric microvessels and fenestrated glomerular microvessels. In contrast to young MWF rats, enzymatic disruption of the endothelial surface layer in old MWF rats resulted in neither additional loss of the layer nor additional changes in permeability. Intravenous injection of wheat germ agglutinin lectin and its adsorption onto the endothelial surface layer significantly improved glomerular albumin permeability. Taken together, these results suggest that widespread loss of the endothelial surface layer links albuminuric kidney disease with systemic vascular dysfunction, providing a potential therapeutic target for proteinuric kidney disease.

Albuminuria and CKD are potent indicators of long-term vascular health and survival. Progressive albuminuria and deteriorating renal function both independently predict increased risk of cardiovascular disease1,2 and are associated with widespread vascular dysfunction, such as impaired macrovascular flow-dependent dilation,3–5 and increased microvascular fluid6 and albumin7 flux. Both flow-dependent dilation and microvascular permeability depend upon intact endothelial cell function, and generalized endothelial dysfunction has been proposed to underlie the widespread vascular complications apparent in those with CKD and albuminuria.8 Generalized endothelial dysfunction is also considered important in the development of atherosclerosis,9,10 which contributes to increased cardiovascular disease risk in these individuals.11 Mechanisms underlying the relation between renal disease and systemic endothelial cell dysfunction remain incompletely understood,12,13 but a defect in the endothelial surface layer (ESL), which is common to all blood vessels, has been suggested as a mechanistic link between widespread vascular dysfunction and albuminuric kidney disease.12–14

Glycosaminoglycans, proteoglycans, and adsorbed plasma components form a surface-bound (“endothelial glycocalyx”) and loosely adherent matrix covering the luminal surface of all vascular endothelial cells, termed the ESL.15 As the primary interface between blood and vessel walls, the ESL regulates vital endothelial cell functions under physiologic and pathophysiologic circumstances. In large conduit vessels, ESL removal limits nitric oxide bioavailability,16,17 resulting in blunted shear-stress–dependent vasodilation16 that resembles the impaired flow-dependent dilation observed in albuminuric individuals.5 In addition, inhibition of hyaluronan synthesis diminishes ESL depth and accelerates atherosclerosis.18 ESL loss may therefore result in, as well as result from,19 large vessel dysfunction and disease.

The ESL also regulates microvascular function, including mechanotransduction, leukocyte adhesion, and the permeability of exchange vessels (capillaries and postcapillary venules).20,21 The ESL is a cross-linked matrix that provides resistance to fluid movement, thereby contributing to the water permeability (hydraulic conductivity: LP) of the vessel wall.20 In addition, the regular arrangement22 and negative charge23 of endothelial glycocalyx fibers permits sieving of macromolecules (e.g., albumin), such that the ESL also contributes to the proportion of albumin molecules “reflected” by the vessel wall back into plasma (reflection coefficient).24 Because the ESL covers the major pathways for fluid and solute movement in vessels with continuous endothelium (e.g., luminal opening of intercellular clefts in mesenteric microvessels) and fenestrated endothelium (e.g., fenestrae of glomerular capillaries), the ESL is a shared regulator of permeability coefficients in these vessel types that are otherwise very different.25 Selective removal of ESL components with a variety of enzymes (e.g., neuraminidase26) increases flux across glomerular endothelial cell monolayers,26 intact glomerular capillary walls,27,28 coronary arterioles,29 and mesenteric microvessels.30,31 The ESL therefore regulates glomerular and extrarenal microvessel permeability, and ESL changes result in quantifiable changes in microvascular permeability.

Acknowledging the difficulties of measuring ESL in humans,32 widespread ESL loss has been reported in patients with type 133 and type 234 diabetes, and the development of microalbuminuria in patients with diabetes results in additional reductions in whole body ESL volume.33 ESL loss from mouse glomerular capillaries occurs shortly after adriamycin administration, in association with albuminuria and increased glomerular albumin clearance.35 However, both hyperglycemia36 and some nephrotoxic agents37 directly suppress ESL constituent synthesis by endothelial cells. The possibility that the ESL is lost in spontaneous, chronic, albuminuric kidney disease, and the potential importance of this loss in mediating the widespread vascular dysfunction that frequently occurs in albuminuria and kidney disease, have not been assessed.

Given that vascular dysfunction after both ESL removal and development of kidney disease are strikingly similar, and given the prognostic importance of widespread vascular dysfunction in albuminuria and renal impairment, we sought to test the hypothesis that spontaneous development of proteinuric kidney disease is associated with ESL loss and deranged vascular function (microvascular permeability) in both renal and extrarenal microvessels. We therefore examined Munich-Wistar-Fromter (MWF) rats, an inbred strain that develop proteinuria, hypertension, and renal failure as they age.

Results

Older MWF rats, compared with both healthy age-matched Wistar rats and healthy young MWF rats, displayed typical features of proteinuric kidney disease,38 including increased urine protein/creatinine and albumin/creatinine ratios, hypertension, elevated plasma creatinine (Table 1), FSGS, glomerulomegaly, and flattened tubular epithelium (Supplemental Figure 1).

View this table:
Table 1.

Animal phenotype

Increased Microvascular Permeability in Older MWF Rats

Mesenteric microvessel hydraulic conductivity (LP) (Figure 1A) and glomerular Kf (LPA) (Figure 1B) were both approximately 2.3-fold higher in older (proteinuric) MWF rats (both P<0.05; unpaired t test). Both glomerular volume–corrected LPA (LPA/Vi [min−1 · mmHg−1] 1.5±0.1 versus 1.0±0.1, P<0.01, unpaired t test) and surface area–corrected LPA (LPA/A [cm · min−1 · mmHg−1] 4.8±0.4 versus 2.7±0.3; P<0.001; unpaired t test) remained higher in older MWF rats, suggesting elevated LP of glomerular and mesenteric capillary walls in older MWF rats. Higher mesenteric LP and glomerular LPA/Vi values were significantly correlated (Pearson r=0.58; P<0.01) (Figure 1C).

Figure 1.
Figure 1.

Widespread increase in microvascular permeability in old MWF rats with proteinuric kidney disease. (A) Mean (±SEM) hydraulic conductivity (LP) of mesenteric microvessels was significantly elevated in MWF rats (black bar; n=7 animals and vessels), compared with healthy Wistar rats (open bar; n=15 animals and vessels). (B) Mean (±SEM) glomerular Kf, normalized for glomerular volume (LPA/Vi) was significantly elevated in glomeruli from MWF rats (black bar; n=58 glomeruli from 10 animals) compared with glomeruli isolated from healthy Wistar rats (open bar; n=24 glomeruli from 6 animals). (C) Glomerular LPA/Vi and mesenteric LP values from the same animal were positively correlated (Pearson r=0.58; P<0.05) (open circles, Wistar rats; filled circles, MWF rats). (D) There was a significant, negative relation between mesenteric microvessel reflection coefficient (mesenteric σ) and urinary protein/creatinine ratio in MWF rats (Spearman r=−0.71; P<0.05). (E) Glomerular albumin sieving coefficient (Θalbumin) was low in healthy young MWF rats (n=11 glomeruli in 3 animals), and significantly elevated in old MWF rats with proteinuric kidney disease (n=20 glomeruli in 5 animals). *P<0.05 (unpaired t test). **P<0.01 (unpaired t test).

Mesenteric microvessel reflection coefficients (σ) in older MWF rats (0.95±0.04; n=7) and healthy, age-matched Wistar rats (0.93±0.02; n=7) were not significantly different, but older MWF rats with greater proteinuria had lower σ (Spearman r=–0.71; P<0.05) (Figure 1D). As an index of glomerular macromolecular permeability, the glomerular albumin sieving coefficient (Θalbumin), measured with multiphoton microscopy,39–41 averaged 0.00065±0.00003 in healthy young MWF rats, and was significantly elevated in older MWF rats (0.0015±0.00016; P<0.01; unpaired t test) (Figure 1E). Animals with more albuminuria had higher glomerular Θalbumin (Pearson r=0.83; P<0.05).

Altered ESL in Nonrenal Microvessels in Older MWF Rats

An intense, linear FITC-WGA lectin signal was present along the luminal border of mesenteric microvessel walls in healthy young MWF rats (Figure 2A). Neuraminidase, which enzymatically removes the ESL,26 removed this WGA signal (Figure 2B). This linear, marginal FITC-WGA lectin signal in mesenteric microvessels was substantially reduced in older MWF rats (Figure 2C). This baseline pattern in older MWF rats (Figure 2C) resembled the pattern after enzymatic ESL removal in healthy rats (Figure 2B). Furthermore, neuraminidase did not alter the pattern of FITC-WGA lectin labeling of mesenteric microvessels in older MWF rats (Figure 2D). These findings indicate loss of ESL from nonrenal microvessels in these animals with proteinuric kidney disease.

Figure 2.
Figure 2.

ESL of mesenteric microvessels is compromised in old MWF rats. Confocal microscopy images of FITC-WGA lectin (green) labeling ESL (esl), alongside simultaneous DIC imaging of microvessel anatomy in a healthy young MWF rat (A and B) and an old MWF rat (C and D). Linear labeling of ESL at the vessel margin in a young MWF rat (Ai and Aii) is removed by neuraminidase (Bi and Bii). ESL is not apparent in mesenteric microvessels of old MWF rats before (Ci and Cii) or after (Di and Dii) neuraminidase. Under baseline conditions (open bars), ESL covers most of wall of mesenteric microvessels (E), and is approximately 0.6 µm deep (F), in healthy rats (old Wistar rats, n=111 vessels in 3 rats; young MWF rats, n=47 vessels in 3 rats). ESL coverage and depth are both reduced by neuraminidase (black bar) in these healthy rats. In contrast, ESL is significantly sparser (E) and shallower (F) in old MWF rats under baseline conditions (n=165 vessels in 4 rats), and is not reduced any further by neuraminidase. *P<0.05 versus both healthy rat groups (one-way ANOVA, Bonferroni); #P<0.05 versus baseline values (one-way ANOVA, Bonferroni); ns, not significant versus preneuraminidase values (one-way ANOVA, Bonferroni). esl, ESL; vl, vessel lumen; i, interstitium; vw, vessel wall. Scale bar, 10 microns.

Mesenteric microvessel ESL coverage (Wistar rats: 90%±1%; young MWF rats: 83%±11%) (Figure 2E) and depth (Wistar rats: 0.65±0.03 µm; young MWF rats: 0.57±0.07 µm) (Figure 2F) in healthy animals were significantly greater than in older MWF rats (detectable coverage: 25%±9%; depth: 0.20±0.05 µm; both P<0.001). In healthy animals, neuraminidase significantly reduced mesenteric microvessel ESL depth and coverage (all P<0.05; ANOVA) (Figure 2, E and F), but did not alter mesenteric microvessel ESL depth and coverage in older MWF rats. Reduced ESL depth and coverage is compatible with increased permeability of mesenteric microvessels in older MWF rats.

Altered Glomerular ESL in Older MWF Rats

Similar patterns of ESL labeling were also observed in glomerular capillaries of healthy young MWF rats (Figure 3), with linear FITC-WGA labeling of the margin of glomerular capillaries under baseline conditions (Figure 3A). WGA labeled the ESL, instead of (or in combination with) labeling of other GAG- and sialoprotein-rich components of the glomerular capillary wall, as demonstrated by alexa-594-WGA labeling at the luminal border of lucifer yellow-loaded glomerular endothelial cells (Figure 3B). Neuraminidase removed the linear FITC-WGA label at the margin of glomerular capillaries (Figure 3C) covering the luminal surface of endothelial cells (Figure 3D). Neuraminidase reduced glomerular ESL coverage (83%±2% to 31%±5%; P<0.005; paired t test) (Figure 3E). As with mesenteric microvessels, glomerular capillaries in older MWF rats did not exhibit linear FITC-WGA labeling of the ESL (Figure 3F), exhibited less coverage with glomerular ESL (P<0.05 versus young MWF; one-way ANOVA, Bonferroni), and no significant changes were observed after neuraminidase treatment (Figure 3G) (baseline coverage 39%±1%, postneuraminidase 32%±2%; P>0.05; paired test) (Figure 3H). Animals with proteinuric kidney disease therefore exhibit ESL loss from both renal and nonrenal microvessels.

Figure 3.
Figure 3.

ESL of glomerular capillaries is compromised in older MWF rats. (A) Under baseline conditions, multiphoton microscopy imaging reveals a linear label (white arrows) of FITC-WGA lectin (green) at the margin of superficial glomerular capillaries containing fluorescently labeled albumin (red). (B) Again under baseline conditions, linear labeling with the same WGA lectin (but tagged with the red fluorophore alexa-594) is on the luminal surface of lucifer yellow-labeled glomerular endothelial cells (green), confirming that the WGA lectin labels glomerular ESL. (Bii) High-magnification image of the rectangular region outlined in Bi. (C) After neuraminidase treatment, the majority of glomerular capillary wall sites do not exhibit linear glomerular ESL label (open arrowheads), although some linear label is still apparent (white arrows). (D) Imaging of the same optical section of the same glomerulus shown in C after neuraminidase treatment. Most of the ESL label has been removed as observed at low magnification (open arrowheads in Di) and high magnification (broken lines in Di). (E) Under baseline conditions (open circles), the majority of glomerular capillary wall sites in young MWF rats exhibit ESL labeling. Neuraminidase treatment of these healthy animals (black circles) significantly reduces glomerular ESL labeling. (F) In old MWF rats, multiphoton microscopy imaging of FITC-WGA lectin (green) reveals very little linear labeling (white arrow) at the margin of superficial glomerular capillaries containing fluorescently labeled albumin (red). The majority of glomerular capillary wall sites do not display linear labeling (open arrowhead). (G) After neuraminidase treatment of an old MWF rat, there is little change in the pattern of WGA lectin labeling (labeling of the brush border of tubular epithelium confirms the presence of detectable FITC-WGA lectin). (H) ESL coverage in old MWF rats under baseline conditions (open circles) is less than in young rats (compared with 3E), and is not further reduced by neuraminidase (closed circles. Each circle represents mean ± SEM values from 4 to 10 glomeruli in individual animals. $P<0.05 versus baseline (one-way ANOVA, Bonferroni); #P<0.05 (one-way ANOVA, Bonferroni); ns, P>0.05 versus baseline (one-way ANOVA, Bonferroni). gc, glomerular capillaries; ec, endothelial cell; esl, ESL; *, extracapsular capillary; pus, peripheral urinary space; P, podocyte (negatively labeled by lucifer yellow); t, tubular epithelium. Scale bar, 10 µm in A, C, F, and G; 5 µm in Bi, Bii, Di, and Dii.

ESL Loss Contributes to Altered Permeability in Older MWF Rats

To assess the functional relevance of ESL loss in older MWF rats, we examined changes in glomerular Θalbumin after neuraminidase-induced ESL removal, because these measurements can uniquely be coupled with real-time ESL imaging, before and after neuraminidase, in multiple vessels in the same animal. Low values of Θalbumin in healthy young MWF rats were significantly elevated by application of neuraminidase (0.00040±0.00010 to 0.00096±0.00020; P<0.05; paired t test) (Figure 4A), mirroring neuraminidase-induced glomerular ESL removal in these animals (Figure 3E). Θalbumin in healthy young MWF rats after neuraminidase was similar to Θalbumin in older MWF rats before neuraminidase (Figure 4A).

Figure 4.
Figure 4.

Loss of glomerular ESL contributes to increased glomerular albumin sieving coefficient, and modification of ESL improves glomerular albumin sieving coefficient, in old MWF rats. (A) In healthy young MWF rats, glomerular albumin sieving coefficient values (Θalbumin) of individual glomeruli (open circles; open square represents mean ± SEM) were elevated after exposure to neuraminidase in every glomerulus (black circles; black square represents mean ± SEM). #P<0.05 versus baseline (paired t test). In contrast, higher Θalbumin values in old MWF rats (open circles; open square represents mean ± SEM) were not significantly elevated any further after neuraminidase exposure (black circles; black square represents mean ± SEM) ns, P>0.05 versus baseline (paired t test). (B) Binding of WGA lectin to the ESL (hashed bars) significantly reduced Θalbumin in old MWF rats (black bar). A similar, nonsignificant trend toward reduced Θalbumin after lectin binding was also observed in young MWF rats (open bar). *P<0.05; ns, P>0.05 versus baseline (one-way ANOVA, Bonferroni). (C) Θalbumin for individual glomeruli (gray circles, young MWF; black circles, old MWF) were significantly correlated with the percentage of glomerular capillary wall sites with ESL cover in the same glomerulus (Pearson r=−0.59; P<0.005).

In contrast, Θalbumin in older MWF rats (0.00082±0.00016) was not significantly different after neuraminidase treatment (0.00097±0.00012; P>0.05; paired t test) (Figure 4A). Coupled with observations that neuraminidase does not exacerbate the loss of ESL already apparent in older MWF rats (Figure 3H), this indicates that loss of ESL contributes to altered Θalbumin in these animals with proteinuric kidney disease.

Moreover, modification of the ESL, by adsorption of acutely intravenously injected WGA lectin, reduced glomerular albumin permeability in older MWF rats. Glomerular Θalbumin decreased from 0.0015±0.0002 in older MWF rats under baseline conditions to 0.00092±0.00007 in older MWF rats treated with WGA lectin (Figure 4B) (P<0.05; one-way ANOVA, Bonferroni). A similar pattern of reduced Θalbumin after lectin binding to the ESL, although not statistically significant, was also observed in young MWF rats (lectin binding: 0.00043±0.00009; baseline conditions: 0.00065±0.00004; P>0.05; one-way ANOVA).

Coupled examinations also revealed that the degree of albuminuria in individual animals correlated with the degree of ESL cover of both mesenteric (Pearson r=−0.87; P<0.005) and glomerular (Pearson r=–0.94; P<0.001) capillaries in the same animal: higher degrees of albuminuria were associated with significantly sparser coverage of microvessel walls with ESL. In individual glomeruli, ESL coverage and Θalbumin were also significantly correlated: glomeruli with less ESL cover had higher Θalbumin (Figure 4C).

Discussion

We report widespread disruption of microvascular permeability and ESL in MWF rats with spontaneous established proteinuric kidney disease, using physiologic and multiphoton microscopy measurements of single microvessels in vivo, which allow unparalleled resolution of the structure and function of the microcirculation.39,42 Changes in both permeability and ESL in these animals mirrored the consequences of enzymatic removal of ESL in healthy animals. In addition, application of the same enzyme (neuraminidase) that successfully removed labeled ESL in healthy animals was ineffective in altering either the structure or function of glomerular capillaries in animals with proteinuric kidney disease. Modification of the ESL in old MWF rats with proteinuric kidney disease significantly reduced glomerular albumin permeability. These findings indicate that ESL loss contributes to increased microvascular permeability in this model of proteinuric kidney disease.

Widespread ESL loss has been reported in individuals with diabetes,33,34 albeit recognizing the difficulties of measuring the ESL in humans.32 Acute hyperglycemia directly suppresses ESL component synthesis36 and reduces ESL volume in healthy individuals.43 ESL loss may therefore represent a specific consequence of diabetes. However, individuals with diabetes and albuminuria exhibit greater loss of ESL (despite equivalent glycemic control33 and the predicted additional elevations in systemic microvessel permeability,44 suggesting that the development of renal dysfunction may also result in widespread ESL damage. These reports are in accord with our findings of widespread loss of ESL and permeability defects in a model of proteinuric kidney disease, and support previous contentions12–14 that alterations in ESL may provide a functional link between kidney disease and extrarenal manifestations of vascular dysfunction.

Increased permeability of nonrenal vessels (Figure 1) was considered the most likely explanation for the increased limb capillary filtration coefficient (product of LP and available surface area) demonstrated in patients with nephrotic syndrome,6 and may contribute to the development and perpetuation of edema in nephrotic states.6,45 We and others have shown that enzymatic removal of ESL in mesenteric microvessels of healthy animals increases LP,30,31 as observed in older MWF rats with reduced ESL depth and cover, although undoubtedly the endothelial glycocalyx is only one important layer of the composite barriers presented by capillary walls.46 Widespread, functionally relevant loss of ESL in albuminuric conditions may also result in large blood vessel dysfunction and disease. Both removal of ESL and albuminuria result in a nitric-oxide–dependent disruption of flow-dependent vasodilation5,16; and loss of ESL in proteinuric kidney disease may contribute to this aspect of vascular dysfunction. In atheroma-prone vessels, ESL limits LDL entry into the vessel wall,47 is thinner at atherosclerosis-prone sites,48 and is lost in response to maneuvers that accelerate atherosclerosis (e.g., high-fat diet).48 Pharmacologic disruption of ESL accelerates atherosclerosis.18 We speculate that disruption of ESL in proteinuric kidney disease also contributes to the accelerated atherosclerosis that affects these individuals. Techniques for examining the ESL in humans are being developed,32,33 and identifying whether the phenomenon demonstrated here also occurs in humans with proteinuric kidney disease and low-grade albuminuria is an important question.

Our observations confirm previous reports that the ESL is a functionally important layer of the healthy glomerular filtration barrier.26–28,49 Jeansson et al. recently reported an 80% reduction in glomerular ESL depth, coupled with a 3.5-fold increase in Θalbumin, shortly after induction of adrimaycin nephropathy in mice.35 These changes are larger than in our study, which is in keeping with our finding that greater loss of glomerular ESL results in a larger increase in Θalbumin. Moreover, we report that glomerular ESL remains diminished 10–20 weeks after the spontaneous onset of albuminuria in older MWF rats,50 in the context of a number of clinical features reminiscent of human proteinuric kidney disease. In addition, our demonstration that neuraminidase was incapable of removing more ESL or elevating Θalbumin further in older MWF rats suggests that this loss of ESL continues to make an important contribution to increased Θalbumin in these animals. In addition to the observed changes in the ESL, however, it is likely that damage to other layers of the glomerular filtration barrier is present in these glomeruli with FSGS, and occurs in response to neuraminidase treatment,51 thereby contributing to the functional defects of this multilayered barrier in these settings. However, modification of the ESL, through adsorption of lectins and other ligands (e.g., ferritin), alters microvascular function in other organs,52,53 and the observation that binding of WGA lectin to the ESL significantly improved glomerular albumin permeability in old MWF rats provides a preliminary indication that modification of the ESL may be an effective therapeutic strategy to reduce albumin leak across the glomerular capillary wall in kidney disease. Because the ESL also regulates endothelial responses to hemodynamic stimuli and regulates nitric oxide bioavailability,54 and supplementation of the ESL can be achieved with endogenous and therapeutic agents,31,55 modification of the ESL may improve a number of aspects of glomerular function in kidney disease.

This study extends previous reports of multiphoton imaging of ESL56,57 to the intravital setting, allowing real-time examination of the ESL, including before and after intervention, in multiple organs of the same animal, and coupling these measurements with assessment of physiology. The approach appears sufficiently sensitive to detect changes in the ESL induced by enzymatic degradation and disease states. The depth of ESL in healthy animals (0.57–0.65 µm) is comparable with results obtained with electron microscopy,58–60 cell exclusion,61 lectin binding,62 and particle velocimetry,63 although significant variability within and between techniques, and between organ beds, is reported.64 The dimensions of the ESL approach the optical resolution of fluorescence microscopy, however, and current limitations precluded coupling of microvascular physiology and glycocalyx anatomy assessments in real time in cannulated vessels (required for measurement of mesenteric permeability coefficients), in contrast to our ability to couple measurements of structure and function before and after neuraminidase in glomeruli.

Increased glomerular LP in old MWF rats with proteinuric kidney disease has also been noted in micropuncture studies.50 Low Θalbumin values in healthy, nonalbuminuric, young MWF rats in this report (approximately 0.0006) are also comparable with Θalbumin values obtained with other techniques in healthy animals in vivo,27,65,66 but are significantly lower than previous estimates made with multiphoton microscopy.40,41,67 Although the magnitude of Θalbumin varies with technical factors,39,41,68 it was possible to detect differences in Θalbumin between experimental groups (young versus older MWF rats), before and after ESL disruption (neuraminidase) and binding (lectin), and to discern significant relations between increasing Θalbumin and the magnitude of albuminuria, and between Θalbumin and ESL coverage. However, we note that Θalbumin doubled in older MWF rats, whereas final urine albumin concentrations were approximately 10-fold higher. Focal areas of high glomerular clearance of albumin, either from a small number of individual glomeruli or from leaky areas within a single glomerulus,42 could substantially increase the amount of albumin in final urine. In addition, although the magnitude of tubular handling of albumin has been extensively debated,69 renal tubules do reabsorb significant quantities of filtered albumin.70,71 We note flattened tubular epithelium in these older MWF rats (Supplemental Figure 1), and altered glomerular and tubular handling of albumin may coexist in this model of proteinuric kidney disease.

In summary, we have extended recent advances in multiphoton imaging techniques, in combination with accurate measurements of microvascular permeability, to demonstrate widespread loss of ESL and altered permeability in an animal model of proteinuric kidney disease. We have also shown that modification of the ESL can improve glomerular albumin permeability in this model of proteinuric kidney disease. These findings have important implications for understanding the mechanisms of both altered glomerular permeability and widespread vascular dysfunction in proteinuric kidney disease.

Concise Methods

Animal Models

Male MWF rats, examined at 4–6 months of age (“older MWF”), were compared with either healthy age-matched male Wistar rats (mesentery studies) or healthy young (aged 4–5 weeks) male MWF rats (“young MWF”). Two groups of control rats were included to accommodate potential strain-related (Wistar versus older MWF rats) and age-related (young MWF versus older MWF rats) differences in ESL. In addition, although young MWF exhibit the superficial glomeruli required for multiphoton imaging of Θalbumin and glomerular ESL, their small size before the development of albuminuria (90±13 g) precluded mesenteric microvessel cannulation studies. Conversely, age-matched Wistar rats were suitable for mesenteric microvessel cannulation studies, but these rats do not have superficial glomeruli for renal imaging studies. In total, 29 MWF rats (7 young; 22 older) and 21 Wistar rats were included in this study.

Wistar and MWF rats were purchased from Harlan; MWF rats were subsequently bred in-house at the University of Southern California (USC) (Los Angeles, CA). Rats were kept under environmentally controlled conditions with a 12-hour/12-hour light/dark cycle at 20–22°C, were fed a standard rat diet, and had free access to food and water. Permeability studies (other than Θalbumin) were conducted at the University of Bristol (UB) (Bristol, UK), and imaging studies were conducted at USC. All experiments were carried out in accordance with national regulations, including the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH) as well as UK Home Office legislation. Local approval was obtained from the USC Institutional Animal Care and Use Committee as well as the UB local ethical committee.

Phenotype Analyses

Rats were housed in metabolic cages (Techniplast) for collection of random urine samples. Urine albumin was measured with a rat-specific albumin ELISA (Exocell, Philadelphia, PA), urine protein with spectrophotometric analysis of pyrogallol molybdenum-protein complexes (Thermo Fisher Scientific, Waltham, MA), and urine glycosaminoglycans by spectrophotometry of Alcian blue-urine glycosaminoglycan complexes formed in acidic solution.72 To account for differences in urine volume, urine creatinine concentration was determined in the same sample using either an enzymatic colorimetric assay (UB) (Konelab T-Series 981845; Thermo Fisher Scientific, Finland), or ELISA (USC). Results obtained with the two techniques were identical (UB: 65.49±7.28 mg/dl; USC 65.51±7.06 mg/dl; P>0.99, unpaired t test). Systolic BP was measured using a noninvasive specialized tail cuff and pulse transducer (ADInstruments; Visitech Systems).

Structural Analyses

Kidney samples from Wistar and older MWF rats were formalin fixed, embedded in paraffin, sectioned (5 µm), mounted onto glass slides, dewaxed (Histoclear; RA Lamb, Eastbourne, UK), and rehydrated through graded ethanol solutions (100%, 90%, and 70% vol/vol). Mounted sagittal kidney sections were then stained with hematoxylin and eosin and imaged using a Nikon Eclipse E600FN Microscope, Coolpix 995 digital camera, and a DN-100 digital imaging system (Nikon Instruments). Average glomerular volume (VMEAN) in each animal was determined using the method described by Pagtulanan et al.73 Glomerular images were replicated in Adobe Photoshop CS3 (Adobe Systems Inc, San Jose, CA) and the area (Aμm2) calculated using Image J software (NIH, Bethesda, MD). In each animal glomerular area (A) was measured in at least 30 profiles from at least three different sections. Glomerular volume was calculated from the area measurements using Equation 1:Embedded Imagewhere β (the shape coefficient for spheres: the idealized shape of glomeruli) equals 1.38, and k (the size distribution coefficient) equals 1.1.73,74

Microvascular Permeability

Mesenteric Permeability

The hydraulic conductivity (Lp – water permeability) and the effective oncotic pressure (σΔπ − protein permeability) were measured in individually perfused rat mesenteric microvessels in Wistar and older MWF rats using a well characterized24 modification of the Landis technique reported by Michel et al.75 Anesthesia was induced by intramuscular injection of fentanyl, fluanisone, and midazolam, with maintenance subcutaneous doses titrated to maintain suppression of reflex responses. The mesentery was teased out over a transparent quartz pillar and transilluminated, and images were recorded on videotape via an inverted bright-field microscope (Leica DM IL HC Fluo). The mesentery was continuously superfused with warmed (37°C) physiologic mammalian Ringer solution (all in mm): NaCl 132, KCl 4.6, MgSO4 1.27, CaCl2 2, NaHCO3 25, d-glucose 5.5, Hepes acid 3.07, and 1.9 Hepes sodium salt, pH corrected to7.45±0.02 with 0.115 m NaOH if required.

Individual mesenteric microvessels were cannulated using a beveled glass micropipette, pressurized (30–70 cmH20) via an attached, adjustable manometer and perfused with physiologic Ringer solution containing BSA at a concentration (C) of 50 mg · ml−1 (solution oncotic pressure [πc] calculated as 27.2 cmH2O76), and containing rat erythrocytes as flow markers. For each pressure, the microvessel was transiently occluded downstream of the cannulation site. No vessel was occluded >5 times in the same site to avoid measurements being made on damaged portions of capillary. The rate of movement of erythrocytes within the occluded portion of vessel represents fluid flux across the vessel wall between the erythrocyte and the occluder. The radius of the vessel (r), velocity of the marker cells approximately 2 seconds after occlusion (dL/dt) and the distance between the marker cell and the occlusion site (L) were measured from recorded images off-line using Apple iMovie and Image J software. The rate of fluid efflux per unit area (Jv/A) was calculated using Equation 2:Embedded ImageThe rate of fluid efflux during each occlusion (Jv/A) was plotted against the imposed capillary hydrostatic pressure, and a linear regression line applied to the data points. Assuming the capillary hydrostatic pressure was equal to that in the manometer, and the interstitial hydrostatic pressure was zero,77 the slope of the regression line describes Lp, and the abscissal intercept describes the effective oncotic pressure of the luminal solution, σΔπ.78 The reflection coefficient of the vessel wall to albumin (σ) was calculated from the relation σΔπ = σ2πc.78

Glomerular Kf

The glomerular Kf (hydraulic conductivity-surface area product: LpA) was determined using an oncometric technique initially reported by Savin and Terreros79 and modified by Salmon et al.80 After measurement of mesenteric microvessel permeability, rats were culled by cervical dislocation and kidneys removed. Glomeruli were isolated in precooled mammalian Ringer solution supplemented with 10 mg · ml−1 BSA using a standard sieving technique. Individual glomeruli were loaded onto the tip of a suction micropipette within a flow controlled closed system. Initial glomerular volume (Vi) was determined off-line from the glomerular profile. Swift exchange of the surrounding solution from 10 mg · ml−1 BSA to 80 mg · ml−1 BSA sets up an oncotic pressure gradient across the glomerular capillary wall, creating an absorptive force that draws fluid out of the glomerulus, resulting in a reduction in glomerular volume. The rate of glomerular volume change represents the rate at which fluid moves across the glomerular filtration barrier (Jv). Glomerular volume measurements were made from individual video images immediately before and after exchange of bathing solution. Glomerular LpA (nl · min−1 · mmHg−1) was calculated from the quotient of the rate of glomerular volume change and applied oncotic pressure, as shown in Equation 3:

Embedded Image

Microvascular Imaging Preparation

Animals were anesthetized for imaging studies with thiobutabarbital (Inactin: 65 mg/ml intraperitoneally), and the right carotid artery and trachea of older MWF rats were cannulated with PE-50 and PE-160 tubing, respectively (PE-10 and PE-90 were used in young MWF rats). Core body temperature was maintained at 36±0.5°C throughout the procedure with a homeothermic device, and BP was maintained at postinduction baseline values throughout the experiment with an infusion of 0.9% saline solution, supplemented with the volume required for delivery of fluorophores. The mesentery was exteriorized via a left flank incision, and imaged through a no. 1 glass coverslip during continuous superfusion with physiologic Ringer solution. After mesenteric microvessel imaging, the mesentery was carefully returned to the abdominal cavity, and the left kidney exteriorized through the same incision. When necessary, a ligature was applied to one end of the incision to maintain the external position of the kidney without compromising the blood supply of the kidney. The kidney was stabilized in a custom-designed holder placed above the same coverslip. Images of mesenteric microvessels after neuraminidase were obtained by reversing the order of imaging (i.e., kidney then mesentery). All images were acquired using an inverted microscope (Leica TCS SP5 AOBS MP confocal microscope system; Leica Microsystems, Heidelberg, Germany) using an HCX PL APO 63×/1.3NA glycerol CS objective (Leica).

Glomerular Albumin Sieving Coefficient

Rat serum albumin (A6414; Sigma, St. Louis, MO) was labeled with atto-550 NHS Ester (51146; Sigma) according to the manufacturer’s instructions. Labeled albumin was separated from free dye by gel permeation chromatography (PD-10 column; Sigma), followed by repeated and extensive centrifugation across a 30-kD molecular weight cutoff membrane (Pall, Ann Arbor, MI).

Fluorescence excitation at 820 nm wavelength was achieved with a Chameleon Ultra-II multiphoton laser (Coherent, Santa Clara, CA), and the emitted, nondescanned fluorescent light was detected by two external photomultipliers (green and red channels) after passage through a FITC/TRITC filter block (Leica). Photodetector gain (red: 775V), amplifier offset (−25%), and system output (16-bit) were fixed for all images in which measurements of Θalbumin were made. The relative positions of up to seven glomeruli per animal were recorded, and background images were obtained, before delivery of labeled albumin, such that individual glomeruli could be identified for repeated imaging before and after intervention (e.g., infusion of neuraminidase).

Labeled albumin (approximately 1 ml/kg body wt of approximately 1.5 mg/ml solution; selected after preliminary experiments with the quick look-up table setting to achieve plasma fluorescence intensity [IFplasma] that did not saturate the detection system) was delivered via the carotid cannula, and allowed to equilibrate for approximately 10 minutes before imaging. Up to 20 sequential images were collected over 15–60 s from superficial optical sections of surface glomeruli, to minimize the consequences of light scattering in deeper renal tissue. An area of the urinary space was recorded as the locus for urinary space fluorescence intensity measurements (IFUS) after confirming that there were no capillaries (from which out-of-focus fluorescence could be collected) in the same x-y position but at different depths.

Image analysis was performed with LAS AF software (Leica). Mean IFUS was recorded in a region of interest in the previously identified urinary space area. The region of interest was created as large as possible to obtain a representative average value of IFUS in a single optical section. The average value of these readings across the 20 sequential frames was recorded, from which background values were subtracted. For IFplasma measurements, obtaining a representative measurement is most reliably achieved by recording IFplasma at the margin of glomerular capillaries, where scattering and absorption of both exciting and emitted light by erythrocytes is minimized. IFplasma was recorded at the margin of four glomerular capillaries in a single optical section, selected from the 20 sequential images because of equal intensity across capillaries and minimal interference from erythrocytes, and background values were again subtracted. The lower limit of detection of the system was calculated as 4.65 × SD of IFUS in all background images, divided by mean IFplasma in all experimental images.41 The albumin sieving coefficient was calculated as IFUS/ IFplasma, and the mean ± SEM values for an individual glomerulus were calculated from the four estimates of IFplasma made in that glomerulus.

ESL Imaging

FITC-labeled wheat germ agglutinin (FITC-WGA) lectin (from Triticum vulgaris; 6.25 mg/kg body wt; L4895; Sigma, MO), administered via the carotid cannula approximately 30 minutes before imaging, was used to label N-acetyl glucosamine (a component of heparan sulfate and hyaluronan glycosaminoglycans) and N-acetyl neuraminic acid (a major sialic acid present on the endothelial cell surface) oligosaccharide moieties on the endothelial cell surface.26 N-acetyl glucosamine residues are present in both the surface-bound “glycocalyx” region and loosely adherent “cell coat” region of the ESL.15 For mesenteric imaging studies, Argon (488 nm; 38% power) and He/Ne (543 nm; 62% power) lasers were used for single photon excitation. Emitted fluorescent signal was collected with 512×512-pixel resolution in 148×148-µm images with 16-bit intensity output, using an internal (de-scanned) detector (gain 950 V, offset –6%) with bandwidth set at 500–535 nm (FITC) using an AOBS bandwidth modification system (Leica), alongside differential interference contrast (DIC) detection. With these settings, the optical (approximately 0.24–0.29 µm) and digital (0.29 µm) resolution of the system were matched. For glomerular studies, FITC-WGA lectin was excited at the same 820 nm wavelength used for excitation of labeled albumin, by a Chameleon Ultra-II multiphoton laser (Coherent, Santa Clara, CA), and the emitted, nondescanned fluorescent light emitted by FITC-WGA and atto-550-albumin were detected simultaneously by two external photomultipliers (green: gain 1100 V, offset –2.5%, other settings and red channel as above) with the help of a FITC/TRITC filter block (Leica). For both kidney and mesenteric tissue, all identifiable microvessel networks were imaged.

Images were analyzed with LAS AF software (Leica). For mesenteric studies, DIC images of microvessels were used to identify the line of greatest contrast between the endothelial cell border (dark) and apposed vessel lumen (bright) as the luminal border of endothelial cells (Supplemental Figure 2). To determine mean ± SD background FITC fluorescence signal, 10 regions of interest were placed within each of the vessel lumen and mesenteric interstitium. A rectangular region of interest (1 μm perpendicular to vessel wall × 2–5 μm parallel to vessel wall) was placed at the edge of the vessel lumen (i.e., with one edge abutting the luminal border of endothelial cells), as defined above. FITC fluorescence signal intensity in this vessel margin region that was greater than the mean + 2 SDs of both the plasma and interstitial signal was taken to indicate the presence of FITC-WGA labeling material at the vessel wall, interpreted as presence of ESL. Ten replicate measurements were made in each vessel.

Depth of the ESL in mesenteric microvessels was determined by placing a line of interest across the vessel wall. The position of the pixels with the greatest difference in DIC signal intensity was identified as the luminal border of the endothelial cell (as defined above). To ensure consistency, the darkest pixel (endothelial cell) was used as the vessel wall reference point. The distance between this reference point and the position of the peak FITC signal was taken as the depth of the ESL.

For glomerular studies, only ESL coverage could be measured, because no glomerular endothelial cell label was routinely available. Glomerular anatomy precluded DIC imaging, and the use of two fluorophores for coupled structure-function studies required both available photodetectors and precluded routine use of a fluorescent label for endothelial cells. The presence of ESL was determined as described above (vessel wall signal exceeds mean + 2 SDs of vessel lumen signal) at four equally spaced loci around the margin of three randomly selected capillary loops in every identifiable glomerulus.

Enzymatic Removal of ESL

Neuraminidase (N2876 from Clostridium perfringens [C. welchii], type V, lyophilized powder; molecular mass of approximately 65 kD), an enzyme that cleaves sialic acid residues of oligosaccharides and glycoproteins and has been shown to remove the ESL from the surface of glomerular endothelial cells,26,81 was prepared and stored according to the manufacturer’s instructions to maintain activity. Neuraminidase was administered via the carotid cannula over 5–10 minutes at a low dose of 1.6 IU/kg body wt.81,82 Studies in healthy control rats and older MWF rats were interspersed to ensure consistency of enzyme action.

Statistical Analyses

Unpaired t tests and one-way ANOVA were used to compare permeability coefficient values of ESL measurements between groups; reflection coefficient values cannot exceed 1 and were therefore analyzed with nonparametric statistics (Mann–Whitney test). Correlations were assessed with Pearson’s (parametric) or Spearman’s (nonparametric) method. Multiple vessels were analyzed in each animal: results from individual vessels were pooled for each animal, and the number of animals studied was used for statistical comparisons. Glomerular LPA measurements were the exception to this rule (as justified in Salmon et al.80): the number of glomeruli studied was compared between groups. Where permeability coefficient or ESL measurements were made in the same vessel before and after neuraminidase, paired t tests were used for comparisons. A P value <0.05 was considered statistically significant.

Disclosures

None.

Acknowledgments

This work was supported by the British Heart Foundation (FS/05/114/19959 to J.K.F.), Medical Research Council and Academy of Medical Sciences (G0802829 to A.H.J.S.) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK64324 (to J.P.P.).

Part of this work was presented in abstract form at the American Society of Nephrology Kidney Week 2011, held November 8–13, 2011, in Philadelphia, Pennsylvania.

Footnotes

References

  1. ↵
    1. Matsushita K,
    2. van der Velde M,
    3. Astor BC,
    4. Woodward M,
    5. Levey AS,
    6. de Jong PE,
    7. Coresh J,
    8. Gansevoort RT,
    9. Chronic Kidney Disease Prognosis Consortium
    : Association of estimated glomerular filtration rate and albuminuria with all-cause and cardiovascular mortality in general population cohorts: A collaborative meta-analysis. Lancet 375: 2073–2081, 2010pmid:20483451
    OpenUrlCrossRefPubMed
  2. ↵
    1. Hillege HL,
    2. Fidler V,
    3. Diercks GF,
    4. van Gilst WH,
    5. de Zeeuw D,
    6. van Veldhuisen DJ,
    7. Gans RO,
    8. Janssen WM,
    9. Grobbee DE,
    10. de Jong PE,
    11. Prevention of Renal and Vascular End Stage Disease (PREVEND) Study Group
    : Urinary albumin excretion predicts cardiovascular and noncardiovascular mortality in general population. Circulation 106: 1777–1782, 2002pmid:12356629
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Tatematsu S,
    2. Wakino S,
    3. Kanda T,
    4. Homma K,
    5. Yoshioka K,
    6. Hasegawa K,
    7. Sugano N,
    8. Kimoto M,
    9. Saruta T,
    10. Hayashi K
    : Role of nitric oxide-producing and -degrading pathways in coronary endothelial dysfunction in chronic kidney disease. J Am Soc Nephrol 18: 741–749, 2007pmid:17267746
    OpenUrlAbstract/FREE Full Text
    1. Clausen P,
    2. Jensen JS,
    3. Jensen G,
    4. Borch-Johnsen K,
    5. Feldt-Rasmussen B
    : Elevated urinary albumin excretion is associated with impaired arterial dilatory capacity in clinically healthy subjects. Circulation 103: 1869–1874, 2001pmid:11294805
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Stehouwer CD,
    2. Henry RM,
    3. Dekker JM,
    4. Nijpels G,
    5. Heine RJ,
    6. Bouter LM
    : Microalbuminuria is associated with impaired brachial artery, flow-mediated vasodilation in elderly individuals without and with diabetes: Further evidence for a link between microalbuminuria and endothelial dysfunction—the Hoorn Study. Kidney Int Suppl 92: S42–S44, 2004pmid:15485416
    OpenUrlPubMed
  6. ↵
    1. Lewis DM,
    2. Tooke JE,
    3. Beaman M,
    4. Gamble J,
    5. Shore AC
    : Peripheral microvascular parameters in the nephrotic syndrome. Kidney Int 54: 1261–1266, 1998pmid:9767542
    OpenUrlCrossRefPubMed
  7. ↵
    1. Jensen JS,
    2. Borch-Johnsen K,
    3. Jensen G,
    4. Feldt-Rasmussen B
    : Microalbuminuria reflects a generalized transvascular albumin leakiness in clinically healthy subjects. Clin Sci (Lond) 88: 629–633, 1995pmid:7634745
    OpenUrlPubMed
  8. ↵
    1. Deckert T,
    2. Feldt-Rasmussen B,
    3. Borch-Johnsen K,
    4. Jensen T,
    5. Kofoed-Enevoldsen A
    : Albuminuria reflects widespread vascular damage. The Steno hypothesis. Diabetologia 32: 219–226, 1989pmid:2668076
    OpenUrlCrossRefPubMed
  9. ↵
    1. Davignon J,
    2. Ganz P
    : Role of endothelial dysfunction in atherosclerosis. Circulation 109[Suppl 1]: III27–III32, 2004pmid:15198963
    OpenUrlCrossRefPubMed
  10. ↵
    1. Ochodnicky P,
    2. Henning RH,
    3. van Dokkum RP,
    4. de Zeeuw D
    : Microalbuminuria and endothelial dysfunction: Emerging targets for primary prevention of end-organ damage. J Cardiovasc Pharmacol 47[Suppl 2]: S151–S162; discussion S172–176, 2006
  11. ↵
    1. Go AS,
    2. Chertow GM,
    3. Fan D,
    4. McCulloch CE,
    5. Hsu CY
    : Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. N Engl J Med 351: 1296–1305, 2004pmid:15385656
    OpenUrlCrossRefPubMed
  12. ↵
    1. Amann K,
    2. Wanner C,
    3. Ritz E
    : Cross-talk between the kidney and the cardiovascular system. J Am Soc Nephrol 17: 2112–2119, 2006pmid:16825329
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Stehouwer CD,
    2. Smulders YM
    : Microalbuminuria and risk for cardiovascular disease: Analysis of potential mechanisms. J Am Soc Nephrol 17: 2106–2111, 2006pmid:16825333
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Singh A,
    2. Satchell SC
    : Microalbuminuria: Causes and implications. Pediatr Nephrol 26: 1957–1965, 2011pmid:21301888
    OpenUrlCrossRefPubMed
  15. ↵
    1. Haraldsson B,
    2. Nyström J,
    3. Deen WM
    : Properties of the glomerular barrier and mechanisms of proteinuria. Physiol Rev 88: 451–487, 2008pmid:18391170
    OpenUrlCrossRefPubMed
  16. ↵
    1. Kumagai R,
    2. Lu X,
    3. Kassab GS
    : Role of glycocalyx in flow-induced production of nitric oxide and reactive oxygen species. Free Radic Biol Med 47: 600–607, 2009pmid:19500664
    OpenUrlCrossRefPubMed
  17. ↵
    1. Mochizuki S,
    2. Vink H,
    3. Hiramatsu O,
    4. Kajita T,
    5. Shigeto F,
    6. Spaan JA,
    7. Kajiya F
    : Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release. Am J Physiol Heart Circ Physiol 285: H722–H726, 2003pmid:12730059
    OpenUrlCrossRefPubMed
  18. ↵
    1. Nagy N,
    2. Freudenberger T,
    3. Melchior-Becker A,
    4. Röck K,
    5. Ter Braak M,
    6. Jastrow H,
    7. Kinzig M,
    8. Lucke S,
    9. Suvorava T,
    10. Kojda G,
    11. Weber AA,
    12. Sörgel F,
    13. Levkau B,
    14. Ergün S,
    15. Fischer JW
    : Inhibition of hyaluronan synthesis accelerates murine atherosclerosis: Novel insights into the role of hyaluronan synthesis. Circulation 122: 2313–2322, 2010pmid:21098434
    OpenUrlCrossRefPubMed
  19. ↵
    1. Grundmann S,
    2. Schirmer SH,
    3. Hekking LH,
    4. Post JA,
    5. Ionita MG,
    6. de Groot D,
    7. van Royen N,
    8. van den Berg B,
    9. Vink H,
    10. Moser M,
    11. Bode C,
    12. de Kleijn D,
    13. Pasterkamp G,
    14. Piek JJ,
    15. Hoefer IE
    : Endothelial glycocalyx dimensions are reduced in growing collateral arteries and modulate leucocyte adhesion in arteriogenesis. J Cell Mol Med 13[9B]: 3463–3474, 2009pmid:19438808
    OpenUrlCrossRefPubMed
  20. ↵
    1. Michel CC,
    2. Curry FE
    : Microvascular permeability. Physiol Rev 79: 703–761, 1999pmid:10390517
    OpenUrlPubMed
  21. ↵
    1. Weinbaum S,
    2. Tarbell JM,
    3. Damiano ER
    : The structure and function of the endothelial glycocalyx layer. Annu Rev Biomed Eng 9: 121–167, 2007pmid:17373886
    OpenUrlCrossRefPubMed
  22. ↵
    1. Squire JM,
    2. Chew M,
    3. Nneji G,
    4. Neal C,
    5. Barry J,
    6. Michel C
    : Quasi-periodic substructure in the microvessel endothelial glycocalyx: A possible explanation for molecular filtering? J Struct Biol 136: 239–255, 2001pmid:12051903
    OpenUrlCrossRefPubMed
  23. ↵
    1. Stace TM,
    2. Damiano ER
    : An electrochemical model of the transport of charged molecules through the capillary glycocalyx. Biophys J 80: 1670–1690, 2001pmid:11259282
    OpenUrlCrossRefPubMed
  24. ↵
    1. Adamson RH,
    2. Lenz JF,
    3. Zhang X,
    4. Adamson GN,
    5. Weinbaum S,
    6. Curry FE
    : Oncotic pressures opposing filtration across non-fenestrated rat microvessels. J Physiol 557: 889–907, 2004pmid:15073281
    OpenUrlCrossRefPubMed
  25. ↵
    1. Rippe B,
    2. Davies S
    : Permeability of peritoneal and glomerular capillaries: What are the differences according to pore theory? Perit Dial Int 31: 249–258, 2011pmid:21555410
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Singh A,
    2. Satchell SC,
    3. Neal CR,
    4. McKenzie EA,
    5. Tooke JE,
    6. Mathieson PW
    : Glomerular endothelial glycocalyx constitutes a barrier to protein permeability. J Am Soc Nephrol 18: 2885–2893, 2007pmid:17942961
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Jeansson M,
    2. Haraldsson B
    : Glomerular size and charge selectivity in the mouse after exposure to glucosaminoglycan-degrading enzymes. J Am Soc Nephrol 14: 1756–1765, 2003pmid:12819235
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Jeansson M,
    2. Haraldsson B
    : Morphological and functional evidence for an important role of the endothelial cell glycocalyx in the glomerular barrier. Am J Physiol Renal Physiol 290: F111–F116, 2006pmid:16091582
    OpenUrlCrossRefPubMed
  29. ↵
    1. Huxley VH,
    2. Williams DA
    : Role of a glycocalyx on coronary arteriole permeability to proteins: Evidence from enzyme treatments. Am J Physiol Heart Circ Physiol 278: H1177–H1185, 2000pmid:10749712
    OpenUrlPubMed
  30. ↵
    1. Adamson RH
    : Permeability of frog mesenteric capillaries after partial pronase digestion of the endothelial glycocalyx. J Physiol 428: 1–13, 1990pmid:2231409
    OpenUrlCrossRefPubMed
  31. ↵
    1. Salmon AH,
    2. Neal CR,
    3. Sage LM,
    4. Glass CA,
    5. Harper SJ,
    6. Bates DO
    : Angiopoietin-1 alters microvascular permeability coefficients in vivo via modification of endothelial glycocalyx. Cardiovasc Res 83: 24–33, 2009pmid:19297368
    OpenUrlCrossRefPubMed
  32. ↵
    1. Michel CC,
    2. Curry FR
    : Glycocalyx volume: A critical review of tracer dilution methods for its measurement. Microcirculation 16: 213–219, 2009pmid:19184776
    OpenUrlCrossRefPubMed
  33. ↵
    1. Nieuwdorp M,
    2. Mooij HL,
    3. Kroon J,
    4. Atasever B,
    5. Spaan JA,
    6. Ince C,
    7. Holleman F,
    8. Diamant M,
    9. Heine RJ,
    10. Hoekstra JB,
    11. Kastelein JJ,
    12. Stroes ES,
    13. Vink H
    : Endothelial glycocalyx damage coincides with microalbuminuria in type 1 diabetes. Diabetes 55: 1127–1132, 2006pmid:16567538
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Broekhuizen LN,
    2. Lemkes BA,
    3. Mooij HL,
    4. Meuwese MC,
    5. Verberne H,
    6. Holleman F,
    7. Schlingemann RO,
    8. Nieuwdorp M,
    9. Stroes ES,
    10. Vink H
    : Effect of sulodexide on endothelial glycocalyx and vascular permeability in patients with type 2 diabetes mellitus. Diabetologia 53: 2646–2655, 2010pmid:20865240
    OpenUrlCrossRefPubMed
  35. ↵
    1. Jeansson M,
    2. Björck K,
    3. Tenstad O,
    4. Haraldsson B
    : Adriamycin alters glomerular endothelium to induce proteinuria. J Am Soc Nephrol 20: 114–122, 2009pmid:19073829
    OpenUrlFREE Full Text
  36. ↵
    1. Singh A,
    2. Fridén V,
    3. Dasgupta I,
    4. Foster RR,
    5. Welsh GI,
    6. Tooke JE,
    7. Haraldsson B,
    8. Mathieson PW,
    9. Satchell SC
    : High glucose causes dysfunction of the human glomerular endothelial glycocalyx. Am J Physiol Renal Physiol 300: F40–F48, 2011pmid:20980411
    OpenUrlCrossRefPubMed
  37. ↵
    1. Björnson A,
    2. Moses J,
    3. Ingemansson A,
    4. Haraldsson B,
    5. Sörensson J
    : Primary human glomerular endothelial cells produce proteoglycans, and puromycin affects their posttranslational modification. Am J Physiol Renal Physiol 288: F748–F756, 2005pmid:15585670
    OpenUrlCrossRefPubMed
  38. ↵
    1. Remuzzi A,
    2. Puntorieri S,
    3. Battaglia C,
    4. Bertani T,
    5. Remuzzi G
    : Angiotensin converting enzyme inhibition ameliorates glomerular filtration of macromolecules and water and lessens glomerular injury in the rat. J Clin Invest 85: 541–549, 1990pmid:1688888
    OpenUrlCrossRefPubMed
  39. ↵
    1. Peti-Peterdi J,
    2. Sipos A
    : A high-powered view of the filtration barrier. J Am Soc Nephrol 21: 1835–1841, 2010pmid:20576805
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Russo LM,
    2. Sandoval RM,
    3. McKee M,
    4. Osicka TM,
    5. Collins AB,
    6. Brown D,
    7. Molitoris BA,
    8. Comper WD
    : The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: Retrieval is disrupted in nephrotic states. Kidney Int 71: 504–513, 2007pmid:17228368
    OpenUrlCrossRefPubMed
  41. ↵
    1. Tanner GA
    : Glomerular sieving coefficient of serum albumin in the rat: A two-photon microscopy study. Am J Physiol Renal Physiol 296: F1258–F1265, 2009pmid:19211688
    OpenUrlCrossRefPubMed
  42. ↵
    1. Peti-Peterdi J,
    2. Burford JL,
    3. Hackl MJ
    : The first decade of using multiphoton microscopy for high-power kidney imaging. Am J Physiol Renal Physiol 302: F227–F233, 2012pmid:22031850
    OpenUrlCrossRefPubMed
  43. ↵
    1. Nieuwdorp M,
    2. van Haeften TW,
    3. Gouverneur MC,
    4. Mooij HL,
    5. van Lieshout MH,
    6. Levi M,
    7. Meijers JC,
    8. Holleman F,
    9. Hoekstra JB,
    10. Vink H,
    11. Kastelein JJ,
    12. Stroes ES
    : Loss of endothelial glycocalyx during acute hyperglycemia coincides with endothelial dysfunction and coagulation activation in vivo. Diabetes 55: 480–486, 2006pmid:16443784
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Jaap AJ,
    2. Shore AC,
    3. Tooke JE
    : Differences in microvascular fluid permeability between long-duration type I (insulin-dependent) diabetic patients with and without significant microangiopathy. Clin Sci (Lond) 90: 113–117, 1996pmid:8829880
    OpenUrlPubMed
  45. ↵
    1. Doucet A,
    2. Favre G,
    3. Deschênes G
    : Molecular mechanism of edema formation in nephrotic syndrome: Therapeutic implications. Pediatr Nephrol 22: 1983–1990, 2007pmid:17554565
    OpenUrlCrossRefPubMed
  46. ↵
    1. Salmon AH,
    2. Satchell SC
    : Endothelial glycocalyx dysfunction in disease: Albuminuria and increased microvascular permeability. J Pathol 226: 562–574, 2012pmid:22102407
    OpenUrlCrossRefPubMed
  47. ↵
    1. van den Berg BM,
    2. Spaan JA,
    3. Vink H
    : Impaired glycocalyx barrier properties contribute to enhanced intimal low-density lipoprotein accumulation at the carotid artery bifurcation in mice. Pflugers Arch 457: 1199–1206, 2009pmid:18839207
    OpenUrlCrossRefPubMed
  48. ↵
    1. van den Berg BM,
    2. Spaan JA,
    3. Rolf TM,
    4. Vink H
    : Atherogenic region and diet diminish glycocalyx dimension and increase intima-to-media ratios at murine carotid artery bifurcation. Am J Physiol Heart Circ Physiol 290: H915–H920, 2006pmid:16155109
    OpenUrlCrossRefPubMed
  49. ↵
    1. Fridén V,
    2. Oveland E,
    3. Tenstad O,
    4. Ebefors K,
    5. Nyström J,
    6. Nilsson UA,
    7. Haraldsson B
    : The glomerular endothelial cell coat is essential for glomerular filtration. Kidney Int 79: 1322–1330, 2011pmid:21412215
    OpenUrlCrossRefPubMed
  50. ↵
    1. Remuzzi A,
    2. Puntorieri S,
    3. Mazzoleni A,
    4. Remuzzi G
    : Sex related differences in glomerular ultrafiltration and proteinuria in Munich-Wistar rats. Kidney Int 34: 481–486, 1988pmid:3199667
    OpenUrlCrossRefPubMed
  51. ↵
    1. Andrews PM
    : Glomerular epithelial alterations resulting from sialic acid surface coat removal. Kidney Int 15: 376–385, 1979pmid:513496
    OpenUrlCrossRefPubMed
  52. ↵
    1. Michel CC,
    2. Phillips ME
    : The effects of bovine serum albumin and a form of cationised ferritin upon the molecular selectivity of the walls of single frog capillaries. Microvasc Res 29: 190–203, 1985pmid:3872988
    OpenUrlCrossRefPubMed
  53. ↵
    1. Turner MR,
    2. Clough G,
    3. Michel CC
    : The effects of cationised ferritin and native ferritin upon the filtration coefficient of single frog capillaries. Evidence that proteins in the endothelial cell coat influence permeability. Microvasc Res 25: 205–222, 1983pmid:6843373
    OpenUrlCrossRefPubMed
  54. ↵
    1. Tarbell JM
    : Shear stress and the endothelial transport barrier. Cardiovasc Res 87: 320–330, 2010pmid:20543206
    OpenUrlCrossRefPubMed
  55. ↵
    1. Chappell D,
    2. Jacob M,
    3. Hofmann-Kiefer K,
    4. Bruegger D,
    5. Rehm M,
    6. Conzen P,
    7. Welsch U,
    8. Becker BF
    : Hydrocortisone preserves the vascular barrier by protecting the endothelial glycocalyx. Anesthesiology 107: 776–784, 2007pmid:18073553
    OpenUrlCrossRefPubMed
  56. ↵
    1. Megens RT,
    2. Reitsma S,
    3. Schiffers PH,
    4. Hilgers RH,
    5. De Mey JG,
    6. Slaaf DW,
    7. oude Egbrink MG,
    8. van Zandvoort MA
    : Two-photon microscopy of vital murine elastic and muscular arteries. Combined structural and functional imaging with subcellular resolution. J Vasc Res 44: 87–98, 2007pmid:17192719
    OpenUrlCrossRefPubMed
  57. ↵
    1. Reitsma S,
    2. oude Egbrink MG,
    3. Vink H,
    4. van den Berg BM,
    5. Passos VL,
    6. Engels W,
    7. Slaaf DW,
    8. van Zandvoort MA
    : Endothelial glycocalyx structure in the intact carotid artery: A two-photon laser scanning microscopy study. J Vasc Res 48: 297–306, 2011pmid:21273784
    OpenUrlCrossRefPubMed
  58. ↵
    1. Chappell D,
    2. Jacob M,
    3. Paul O,
    4. Rehm M,
    5. Welsch U,
    6. Stoeckelhuber M,
    7. Conzen P,
    8. Becker BF
    : The glycocalyx of the human umbilical vein endothelial cell: An impressive structure ex vivo but not in culture. Circ Res 104: 1313–1317, 2009pmid:19423849
    OpenUrlAbstract/FREE Full Text
    1. Hjalmarsson C,
    2. Johansson BR,
    3. Haraldsson B
    : Electron microscopic evaluation of the endothelial surface layer of glomerular capillaries. Microvasc Res 67: 9–17, 2004pmid:14709398
    OpenUrlCrossRefPubMed
  60. ↵
    1. van den Berg BM,
    2. Vink H,
    3. Spaan JA
    : The endothelial glycocalyx protects against myocardial edema. Circ Res 92: 592–594, 2003pmid:12637366
    OpenUrlAbstract/FREE Full Text
  61. ↵
    1. Vink H,
    2. Duling BR
    : Identification of distinct luminal domains for macromolecules, erythrocytes, and leukocytes within mammalian capillaries. Circ Res 79: 581–589, 1996pmid:8781491
    OpenUrlAbstract/FREE Full Text
  62. ↵
    1. Gao L,
    2. Lipowsky HH
    : Composition of the endothelial glycocalyx and its relation to its thickness and diffusion of small solutes. Microvasc Res 80: 394–401, 2010pmid:20600162
    OpenUrlCrossRefPubMed
  63. ↵
    1. Potter DR,
    2. Damiano ER
    : The hydrodynamically relevant endothelial cell glycocalyx observed in vivo is absent in vitro. Circ Res 102: 770–776, 2008pmid:18258858
    OpenUrlAbstract/FREE Full Text
  64. ↵
    1. Ebong EE,
    2. Macaluso FP,
    3. Spray DC,
    4. Tarbell JM
    : Imaging the endothelial glycocalyx in vitro by rapid freezing/freeze substitution transmission electron microscopy. Arterioscler Thromb Vasc Biol 31: 1908–1915, 2011pmid:21474821
    OpenUrlAbstract/FREE Full Text
  65. ↵
    1. Rippe C,
    2. Rippe A,
    3. Torffvit O,
    4. Rippe B
    : Size and charge selectivity of the glomerular filter in early experimental diabetes in rats. Am J Physiol Renal Physiol 293: F1533–F1538, 2007pmid:17699552
    OpenUrlCrossRefPubMed
  66. ↵
    1. Tojo A,
    2. Endou H
    : Intrarenal handling of proteins in rats using fractional micropuncture technique. Am J Physiol 263: F601–F606, 1992pmid:1415732
    OpenUrlPubMed
  67. ↵
    1. Russo LM,
    2. Sandoval RM,
    3. Campos SB,
    4. Molitoris BA,
    5. Comper WD,
    6. Brown D
    : Impaired tubular uptake explains albuminuria in early diabetic nephropathy. J Am Soc Nephrol 20: 489–494, 2009pmid:19118149
    OpenUrlAbstract/FREE Full Text
  68. ↵
    1. Peti-Peterdi J
    : Independent two-photon measurements of albumin GSC give low values. Am J Physiol Renal Physiol 296: F1255–F1257, 2009pmid:19297453
    OpenUrlCrossRefPubMed
  69. ↵
    1. Comper WD,
    2. Haraldsson B,
    3. Deen WM
    : Resolved: Normal glomeruli filter nephrotic levels of albumin. J Am Soc Nephrol 19: 427–432, 2008pmid:18216311
    OpenUrlAbstract/FREE Full Text
  70. ↵
    1. Birn H,
    2. Christensen EI
    : Renal albumin absorption in physiology and pathology. Kidney Int 69: 440–449, 2006pmid:16514429
    OpenUrlCrossRefPubMed
  71. ↵
    1. Birn H,
    2. Fyfe JC,
    3. Jacobsen C,
    4. Mounier F,
    5. Verroust PJ,
    6. Orskov H,
    7. Willnow TE,
    8. Moestrup SK,
    9. Christensen EI
    : Cubilin is an albumin binding protein important for renal tubular albumin reabsorption. J Clin Invest 105: 1353–1361, 2000pmid:10811843
    OpenUrlCrossRefPubMed
  72. ↵
    1. Gold EW
    : The quantitative spectrophotometric estimation of total sulfated glycosaminoglycan levels. Formation of soluble alcian blue complexes. Biochim Biophys Acta 673: 408–415, 1981pmid:6784773
    OpenUrlPubMed
  73. ↵
    1. Pagtalunan ME,
    2. Rasch R,
    3. Rennke HG,
    4. Meyer TW
    : Morphometric analysis of effects of angiotensin II on glomerular structure in rats. Am J Physiol 268: F82–F88, 1995pmid:7840251
    OpenUrlPubMed
  74. ↵
    Weibel ER: Stereological Methods: Practical Methods for Biological Morphometry, London, Academic Press, 1979
  75. ↵
    1. Michel CC,
    2. Mason JC,
    3. Curry FE,
    4. Tooke JE,
    5. Hunter PJ
    : A development of the Landis technique for measuring the filtration coefficient of individual capillaries in the frog mesentery. Q J Exp Physiol Cogn Med Sci 59: 283–309, 1974pmid:4548540
    OpenUrlPubMed
  76. ↵
    1. Bates DO
    : The chronic effect of vascular endothelial growth factor on individually perfused frog mesenteric microvessels. J Physiol 513: 225–233, 1998pmid:9782172
    OpenUrlCrossRefPubMed
  77. ↵
    1. Kajimura M,
    2. Wiig H,
    3. Reed RK,
    4. Michel CC
    : Interstitial fluid pressure surrounding rat mesenteric venules during changes in fluid filtration. Exp Physiol 86: 33–38, 2001pmid:11429617
    OpenUrlCrossRefPubMed
  78. ↵
    1. Michel CC,
    2. Phillips ME
    : Steady-state fluid filtration at different capillary pressures in perfused frog mesenteric capillaries. J Physiol 388: 421–435, 1987pmid:3498833
    OpenUrlCrossRefPubMed
  79. ↵
    1. Savin VJ,
    2. Terreros DA
    : Filtration in single isolated mammalian glomeruli. Kidney Int 20: 188–197, 1981pmid:7289403
    OpenUrlCrossRefPubMed
  80. ↵
    1. Salmon AH,
    2. Neal CR,
    3. Bates DO,
    4. Harper SJ
    : Vascular endothelial growth factor increases the ultrafiltration coefficient in isolated intact Wistar rat glomeruli. J Physiol 570: 141–156, 2006pmid:16239266
    OpenUrlCrossRefPubMed
  81. ↵
    1. Avasthi PS,
    2. Koshy V
    : The anionic matrix at the rat glomerular endothelial surface. Anat Rec 220: 258–266, 1988pmid:2966599
    OpenUrlCrossRefPubMed
  82. ↵
    1. Wijnhoven TJ,
    2. Lensen JF,
    3. Wismans RG,
    4. Lamrani M,
    5. Monnens LA,
    6. Wevers RA,
    7. Rops AL,
    8. van der Vlag J,
    9. Berden JH,
    10. van den Heuvel LP,
    11. van Kuppevelt TH
    : In vivo degradation of heparan sulfates in the glomerular basement membrane does not result in proteinuria. J Am Soc Nephrol 18: 823–832, 2007pmid:17251387
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

View Selected Citations (0)
Print
Download PDF
Email Article
Citation Tools
Request Permissions
Loss of the Endothelial Glycocalyx Links Albuminuria and Vascular Dysfunction
Andrew H.J. Salmon, Joanne K. Ferguson, James L. Burford, Haykanush Gevorgyan, Daisuke Nakano, Steven J. Harper, David O. Bates, Janos Peti-Peterdi
JASN Aug 2012, 23 (8) 1339-1350; DOI: 10.1681/ASN.2012010017
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section