Simon C. Satchell

What is the mechanism of microalbuminuria in diabetes: a role for the glomerular endothelium?

Microalbuminuria is an important risk factor for cardiovascular disease and progressive renal impairment. This holds true in the general population and particularly in those with diabetes, in whom it is common and marks out those likely to develop macrovascular disease and progressive renal impairment. Understanding the pathophysiological mechanisms through which microalbuminuria occurs holds the key to designing therapies to arrest its development and prevent these later manifestations.Microalbuminuria arises from the increased passage of albumin through the glomerular filtration barrier. This requires ultrastructural changes rather than alterations in glomerular pressure or filtration rate alone. Compromise of selective glomerular permeability can be confirmed in early diabetic nephropathy but does not correlate well with reported glomerular structural changes. The loss of systemic endothelial glycocalyx—a protein-rich surface layer on the endothelium—in diabetes suggests that damage to this layer represents this missing link. The epidemiology of microalbuminuria reveals a close association with systemic endothelial dysfunction and with vascular disease, also implicating glomerular endothelial dysfunction in microalbuminuria.Our understanding of the metabolic and hormonal sequelae of hyperglycaemia is increasing, and we consider these in the context of damage to the glomerular filtration barrier. Reactive oxygen species, inflammatory cytokines and growth factors are key players in this respect. Taken together with the above observations and the presence of generalised endothelial dysfunction, these considerations lead to the conclusion that glomerular endothelial dysfunction, and in particular damage to its glycocalyx, represents the most likely initiating step in diabetic microalbuminuria.

Glomerular endothelial cell fenestrations: an integral component of the glomerular filtration barrier

Glomerular endothelial cell (GEnC) fenestrations are analogous to podocyte filtration slits, but their important contribution to the glomerular filtration barrier has not received corresponding attention. GEnC fenestrations are transcytoplasmic holes, specialized for their unique role as a prerequisite for filtration across the glomerular capillary wall. Glomerular filtration rate is dependent on the fractional area of the fenestrations and, through the glycocalyx they contain, GEnC fenestrations are important in restriction of protein passage. Hence, dysregulation of GEnC fenestrations may be associated with both renal failure and proteinuria, and the pathophysiological importance of GEnC fenestrations is well characterized in conditions such as preeclampsia. Recent evidence suggests a wider significance in repair of glomerular injury and in common, yet serious, conditions, including diabetic nephropathy. Study of endothelial cell fenestrations is challenging because of limited availability of suitable in vitro models and by the requirement for electron microscopy to image these sub-100-nm structures. However, extensive evidence, from glomerular development in rodents to in vitro studies in human GEnC, points to vascular endothelial growth factor (VEGF) as a key inducer of fenestrations. In systemic endothelial fenestrations, the intracellular pathways through which VEGF acts to induce fenestrations include a key role for the fenestral diaphragm protein plasmalemmal vesicle-associated protein-1 (PV-1). The role of PV-1 in GEnC is less clear, not least because of controversy over existence of GEnC fenestral diaphragms. In this article, the structure-function relationships of GEnC fenestrations will be evaluated in depth, their role in health and disease explored, and the outlook for future study and therapeutic implications of these peculiar structures will be approached.

Glomerular Endothelial Glycocalyx Constitutes a Barrier to Protein Permeability

Glycocalyx, composed of glycoproteins including proteoglycans, coats the luminal surface of the glomerular capillaries. Human heparanase degrades heparan sulphate glycosaminoglycans and is up-regulated in proteinuric states. In this study, we analyze the structure of the human glomerular endothelial cell glycocalyx in vitro and examine its functional relevance, especially after treatment with human heparanase. Electron microscopy of conditionally immortalized glomerular endothelial cells revealed a 200-nm thick glycocalyx over the plasma membrane, which was also demonstrated by confocal microscopy. Neuraminidase treatment removed the majority of glycocalyx, reduced trans-endothelial electrical resistance by 59%, and increased albumin flux by 207%. Heparinase III and human heparanase specifically cleaved heparan sulphate: this caused no change in trans-endothelial electrical resistance, but increased the albumin passage across the monolayers by 40% and 39%, respectively. Therefore, we have characterized the glomerular endothelial cell glycocalyx and have shown that it contributes to the barrier to flux of albumin across the cell layer. These results suggest an important role for this glycocalyx in the restriction of glomerular protein passage in vivo and suggest ways in which human heparanase levels may be linked to proteinuria in clinical disease.

Angiopoietin 1 and Vascular Endothelial Growth Factor Modulate Human Glomerular Endothelial Cell Barrier Properties

Normal glomerular filtration depends on the combined properties of the three layers of glomerular capillary wall: glomerular endothelial cells (GEnC), basement membrane, and podocytes. Podocytes produce endothelial factors, including angiopoietin 1 (ang1), and vascular endothelial growth factor (VEGF), whereas GEnC express their respective receptors Tie2 and VEGFR2 in vivo. As ang1 acts to maintain the endothelium in other vascular beds, regulating some actions of VEGF, these observations suggest a mechanism whereby podocytes may direct the unique properties of the glomerular endothelium. This interaction was investigated by studies on the barrier properties of human GEnC in vitro. GEnC were examined for expression of endothelium-specific markers by immunofluorescence and Western blotting and for typical responses to TNF-alpha by a cell-based immunoassay. Expression of angiopoietin and VEGF receptors was examined similarly. Barrier properties of GEnC monolayers cultured on porous supports were investigated by measurement of transendothelial electrical resistance (TEER) and passage of labeled albumin. Responses to a cAMP analogue and thrombin were examined before those to ang1 and VEGF. Results confirmed the endothelial origin of GEnC and their expression of Tie2 and VEGFR2. GEnC formed monolayers with a mean TEER of 30 to 40 Omega/cm(2). The cAMP analogue and thrombin increased and decreased TEER by 34.4 and 14.8 Omega/cm(2), respectively, with corresponding effects on protein passage. Ang1 increased TEER by 11.4 Omega/cm(2) and reduced protein passage by 45.2%, whereas VEGF reduced TEER by 12.5 Omega/cm(2) but had no effect on protein passage. Both ang1 and VEGF modulate GEnC barrier properties, consistent with potential in vivo roles; ang1 stabilizing the endothelium and resisting angiogenesis while VEGF induces the high permeability to water, characteristic of the glomerular endothelium.

Endothelial glycocalyx dysfunction in disease: albuminuria and increased microvascular permeability

Appreciation of the glomerular microcirculation as a specialized microcirculatory bed, rather than as an entirely separate entity, affords important insights into both glomerular and systemic microvascular pathophysiology. In this review we compare regulation of permeability in systemic and glomerular microcirculations, focusing particularly on the role of the endothelial glycocalyx, and consider the implications for disease processes. The luminal surface of vascular endothelium throughout the body is covered with endothelial glycocalyx, comprising surface‐anchored proteoglycans, supplemented with adsorbed soluble proteoglycans, glycosaminoglycans and plasma constituents. In both continuous and fenestrated microvessels, this endothelial glycocalyx provides resistance to the transcapillary escape of water and macromolecules, acting as an integral component of the multilayered barrier provided by the walls of these microvessels (ie acting in concert with clefts or fenestrae across endothelial cell layers, basement membranes and pericytes). Dysfunction of any of these capillary wall components, including the endothelial glycocalyx, can disrupt normal microvascular permeability. Because of its ubiquitous nature, damage to the endothelial glycocalyx alters the permeability of multiple capillary beds: in the glomerulus this is clinically apparent as albuminuria. Generalized damage to the endothelial glycocalyx can therefore manifest as both albuminuria and increased systemic microvascular permeability. This triad of altered endothelial glycocalyx, albuminuria and increased systemic microvascular permeability occurs in a number of important diseases, such as diabetes, with accumulating evidence for a similar phenomenon in ischaemia–reperfusion injury and infectious disease. The detection of albuminuria therefore has implications for the function of the microcirculation as a whole. The importance of the endothelial glycocalyx for other aspects of vascular function/dysfunction, such as mechanotransduction, leukocyte–endothelial interactions and the development of atherosclerosis, indicate that alterations in the endothelial glycocalyx may also be playing a role in the dysfunction of other organs observed in these disease states. Copyright

Hyperfunctional C3 convertase leads to complement deposition on endothelial cells and contributes to atypical hemolytic uremic syndrome

Complement is a major innate immune defense against pathogens, tightly regulated to prevent host tissue damage. Atypical hemolytic uremic syndrome (aHUS) is characterized by endothelial damage leading to renal failure and is highly associated with abnormal alternative pathway regulation. We characterized the functional consequences of 2 aHUS-associated mutations (D(254)G and K(325)N) in factor B, a key participant in the alternative C3 convertase. Mutant proteins formed high-affinity C3-binding site, leading to a hyperfunctional C3 convertase, resistant to decay by factor H. This led to enhanced complement deposition on the surface of alternative pathway activator cells. In contrast to native factor B, the 2 mutants bound to inactivated C3 and induced formation of functional C3-convertase on iC3b-coated surface. We demonstrated for the first time that factor B mutations lead to enhanced C3-fragment deposition on quiescent and adherent human glomerular cells (GEnCs) and human umbilical vein endothelial cells (HUVECs), together with the formation of sC5b-9 complexes. These results could explain the occurrence of the disease, since excessive complement deposition on endothelial cells is a central event in the pathogenesis of aHUS. Therefore, risk factors for aHUS are not only mutations leading to loss of regulation, but also mutations, resulting in hyperactive C3 convertase.

A prevalent C3 mutation in aHUS patients causes a direct C3 convertase gain of function

Atypical hemolytic uremic syndrome (aHUS) is a rare renal thrombotic microangiopathy commonly associated with rare genetic variants in complement system genes, unique to each patient/family. Here, we report 14 sporadic aHUS patients carrying the same mutation, R139W, in the complement C3 gene. The clinical presentation was with a rapid progression to end-stage renal disease (6 of 14) and an unusually high frequency of cardiac (8 of 14) and/or neurologic (5 of 14) events. Although resting glomerular endothelial cells (GEnCs) remained unaffected by R139W-C3 sera, the incubation of those sera with GEnC preactivated with pro-inflammatory stimuli led to increased C3 deposition, C5a release, and procoagulant tissue-factor expression. This functional consequence of R139W-C3 resulted from the formation of a hyperactive C3 convertase. Mutant C3 showed an increased affinity for factor B and a reduced binding to membrane cofactor protein (MCP; CD46), but a normal regulation by factor H (FH). In addition, the frequency of at-risk FH and MCP haplotypes was significantly higher in the R139W-aHUS patients, compared with normal donors or to healthy carriers. These genetic background differences could explain the R139W-aHUS incomplete penetrance. These results demonstrate that this C3 mutation, especially when associated with an at-risk FH and/or MCP haplotypes, becomes pathogenic following an inflammatory endothelium-damaging event.

The molecular and functional phenotype of glomerular podocytes reveals key features of contractile smooth muscle cells

The glomerular podocyte is a highly specialized cell, with the ability to ultrafilter blood and support glomerular capillary pressures. However, little is known about either the genetic programs leading to this functionality or the final phenotype. We approached this question utilizing a human conditionally immortalized cell line, which differentiates from a proliferating epithelial phenotype to a differentiated form. We profiled gene expression during several time points during differentiation and grouped the regulated genes into major functional categories. A novel category of genes that was upregulated during differentiation was of smooth muscle-related molecules. We further examined the smooth muscle phenotype and showed that podocytes consistently express the differentiated smooth muscle markers smoothelin and calponin and the specific transcription factor myocardin, both in vitro and in vivo. The contractile contribution of the podocyte to the glomerular capillary is controversial. We demonstrated using two novel techniques that podocytes contract vigorously in vitro when differentiated and in real time were able to demonstrate that angiotensin II treatment decreases monolayer resistance, morphologically correlating with enhanced contractility. We conclude that the mature podocyte in vitro possesses functional apparatus of contractile smooth muscle cells, with potential implications for its in vivo ability to regulate glomerular dynamic and permeability characteristics.

Hemopexin Induces Nephrin-Dependent Reorganization of the Actin Cytoskeleton in Podocytes

Hemopexin is an abundant plasma protein that effectively scavenges heme. When infused into rats, hemopexin induces reversible proteinuria, and activated hemopexin is increased in children with minimal change nephrotic syndrome. These observations suggest a role for hemopexin in glomerular disease; in this study, the effects of active hemopexin on human podocytes and glomerular endothelial cells, the two cell types that compose the glomerular filtration barrier, were investigated. Within 30 min of treatment with hemopexin, actin reorganized from stress fibers to cytoplasmic aggregates and membrane ruffles in wild-type podocytes. This did not occur in nephrin-deficient podocytes unless they were transfected with nephrin-expressing plasmids. Furthermore, hemopexin did not affect actin organization in cells that do not express nephrin, specifically human glomerular endothelial cells, fibroblasts, and HEK293 cells. The effects of hemopexin on wild-type podocytes reversed within 4 h and were inhibited by preincubation with human plasma. Treatment with hemopexin activated protein kinase B in both wild-type and nephrin-deficient podocytes but activated RhoA only in wild-type cells. In addition, hemopexin led to a selective increase in the passage of albumin across monolayers of glomerular endothelial cells and to a reduction in glycocalyx. In summary, active hemopexin causes nephrin-dependent remodeling of podocytes and affects permeability of the glomerular filtration barrier by degrading the glycocalyx.

Reactive oxygen species modulate the barrier function of the human glomerular endothelial glycocalyx.

Reactive oxygen species (ROS) play a key role in the pathogenesis of proteinuria in glomerular diseases like diabetic nephropathy. Glomerular endothelial cell (GEnC) glycocalyx covers the luminal aspect of the glomerular capillary wall and makes an important contribution to the glomerular barrier. ROS are known to depolymerise glycosaminoglycan (GAG) chains of proteoglycans, which are crucial for the barrier function of GEnC glycocalyx. The aim of this study is to investigate the direct effects of ROS on the structure and function of GEnC glycocalyx using conditionally immortalised human GEnC. ROS were generated by exogenous hydrogen peroxide. Biosynthesis and cleavage of GAG chains was analyzed by radiolabelling (S35 and 3H-glucosamine). GAG chains were quantified on GEnC surface and in the cell supernatant using liquid chromatography and immunofluorescence techniques. Barrier properties were estimated by measuring trans-endothelial passage of albumin. ROS caused a significant loss of WGA lectin and heparan sulphate staining from the surface of GEnC. This lead to an increase in trans-endothelial albumin passage. The latter could be inhibited by catalase and superoxide dismutase. The effect of ROS on GEnC was not mediated via the GAG biosynthetic pathway. Quantification of radiolabelled GAG fractions in the supernatant confirmed that ROS directly caused shedding of HS GAG. This finding is clinically relevant and suggests a mechanism by which ROS may cause proteinuria in clinical conditions associated with high oxidative stress.