The glomerulus – a view from the inside – the endothelial cell

The International Journal of Biochemistry & Cell Biology 42 (2010) 1388–1397

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Cells in focus

The glomerulus – a view from the inside – the endothelial cell Agnes B. Fogoa,b,∗ , Valentina Konb a b

Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN, USA Division of Pediatric Nephrology, Vanderbilt University School of Medicine, Nashville, TN, USA

a r t i c l e

i n f o

Article history: Received 1 February 2010 Received in revised form 9 May 2010 Accepted 30 May 2010 Available online 9 June 2010 Keywords: Glomerulus Endothelial cell Development Fibrosis Podocyte

a b s t r a c t The glomerular endothelial cells are unique both in location and anatomy compared to most other endothelial cells throughout the body. The absence of a diaphragm with retention of a basement membrane and fenestrations enable these uniquely situated cells to have a key role in filtration performed by the kidney. Interaction with other glomerular cells such as the podocytes and mesangial cells, as well as with circulating and infiltrating inflammatory cells, contribute to the final impact of the glomerular endothelial cells on maintenance of body fluid homeostasis and modulation of disease. Thus, endothelial cells contribute to hemodynamic function, reactive oxygen stress, regulate the balance between pro-thrombotic and anti-thrombotic forces, and importantly, contribute to fibrosis, the key injury of progressive chronic kidney disease. Repair of endothelial cell damage and restoration of segmentally sclerosed glomeruli are key areas considered for intervention in chronic kidney disease. This review will focus on the structure, function, and interplay of endothelial cells with other glomerular cells and systemic factors, their impact on renal disease and give rationale for possible intervention that may forestall progressive injuries. © 2010 Elsevier Ltd. All rights reserved.

Cell facts • Glomerular endothelial cells are a component of the normal glomerular permeability barrier. • Glomerular endothelial cells are fenestrated and are covered by a glycocalyx. • Glomerular endothelial cells modulate interactions with and adhesion and infiltration of circulating cells. • Glomerular endothelial cells produce factors that modulate intraglomerular hemodynamics and hemostasis. • Glomerular endothelial cells interact with both podocytes and mesangial cells, and are modulated by key angiogenic factors derived from these cells.

glomerulus, the endothelium shares a basement membrane with the podocytes and interacts closely with this cell (Hirschberg et al., 2008). Podocyte-derived growth factors are key for maintenance of endothelial cell viability and function, and conversely, endothelial cells affect both podocytes and mesangial cells. Glomerular endothelial cells play key roles in modulation of factors regulating vascular tone and glomerular filtration, reactive oxygen species, pro-thrombotic and anti-thrombotic factors and fibrosis (Navar, 2009; Jarad and Miner, 2009). In this review, we will discuss the development and normal structure of this cell, its interplay with the other resident and infiltrating cells of the glomeruls (Fig. 1), and the complex interactions that govern the impact of the endothelial cell on glomerular functions and disease. 2. Normal development and structure 2.1. Phenotypic diversity of endothelium

1. Introduction The systemic endothelium is closely related to the pericytes with an intervening common basement membrane. In parallel, in the

∗ Corresponding author at: Department of Pathology, C-3310 Medical Center North, Vanderbilt University Medical Center, Nashville, TN 37232-2561, USA. Tel.: +1 615 3223114. E-mail address: [email protected] (A.B. Fogo). 1357-2725/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2010.05.015

Endothelium has three major phenotypes throughout the body (Segal et al., 2006). Endothelium may be continuous, i.e. lacking fenestrations, or with fenestration, or discontinuous, lacking both fenestrations and basal lamina. Basal lamina and glycocalyx are present both in the systemic capillaries and in the glomerulus, contrasting the discontinuous capillary beds in the hepatic, splenic and bone marrow sinusoidal systems. Fenestrations are present in several types of endothelium, and vary in size and presence or absence of diaphragms, as reviewed by Satchell and Braet (Satchell

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Fig. 1. Schema of interaction of glomerular endothelial cells (turquoise) with podocytes (grey) and mesangium (yellow). Podocytes produce angiogenic factors, including vascular endothelial-derived factor (VEGF-A), and angiopoietin-1 (Ang1) (see curved arrows), that reach the glomerular endothelium and are necessary for endothelial cell survival. Mesangial cells produce angiopoietin-2 (straight arrow, Ang 2) that reaches endothelial cells and modulates endothelial cell function, and may counteract proangiogenic effects of Ang-1. Endothelial cells themselves secrete numerous vasoactive factors (see right arrow, blue asterisk), such as endothelin-1, renin angiotensin system factors, nitric oxide synthase, prostacyclin; modulate hemostasis by balance of e.g. plasmin/plasminogen activator system and its inhibitors, and upregulate adhesion molecules in response to injury. The balance of these modulators and responses within the glomerulus determines the response to injury both locally and systemically.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

and Braet, 2009; Ott et al., 1993). Sinusoidal capillary beds do not have diaphragms in their fenestrae and do not express plasmalemmal vesicle-associated protein-1 (PV-1), and fenestrae are of much larger diameter. Diaphragms may contribute to modulation of flux across fenestrae. In endocrine tissue, the gastrointestinal tract and surrounding the tubules in the kidney, fenestrated endothelium is present with diameter of fenestrae 60–70 nm. These fenestrations are distinct from those seen in the glomerulus in that the diaphragms contain a type II transmembrane glycoprotein (PV-1), lacking in the mature fenestrated glomerular endothelium (Satchell and Braet, 2009). 2.2. Formation of glomerular capillaries The glomerular endothelium develops largely by vasculogenesis, i.e. from angioblasts present in situ within the metanephric mesenchyme (Ballermann, 2005, 2007). Endothelial progenitor cells present in the circulation are derived from bone marrow, and can also differentiate and give rise to endothelial cells (Lin et al., 2000). Angioblasts bear vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Flk-1), and subsequently develop into the glomerular vessels (Robert et al., 1996). Under the influence of VEGF-A, transforming growth factor-␤ (TGF-␤1) and neuropilin, angioblasts migrate during development. Neuropilin acts as a receptor for semaphorin III and binds VEGF-A (Robert et al., 2000). Angioblasts then aggregate into cord-like structures without a distinct lumen during the comma- and S-shaped stages of development. Lumen formation is modulated by TGF-␤ induced apoptosis (Fierlbeck et al., 2003). The formation of the precapillary cords is regulated by Eph-like (ELK) receptors, in response to its ligand (Daniel et al., 1996). 2.3. Formation of fenestrations At the earliest stage of angioblast migration, the cells are cuboidal and lack fenestrations. During the capillary loop stage, angioblasts proliferate and attenuate and form fenestrations. Endothelial cells derived from circulating endothelial progenitor cells also contribute to the capillary endothelium after injury, as

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they can undergo angiogenesis when co-incubated with podocytes or exogenous VEGF (Foster et al., 2006, 2008; Kamba et al., 2006). Importantly, podocytes are also present at this developmental stage, although foot processes are not yet well-formed (Ichimura et al., 2008; Quaggin and Kreidberg, 2008). The developing glomerular basement membrane separates these immature podocytes from the developing capillary lumens. Formation of the fenestrae is then mediated by VEGF-A (Breier et al., 1992; Eremina et al., 2003). VEGF-A is a major angiogenic factor, with several variants arising due to alternative mRNA splicing (Tischer et al., 1989; Kuo et al., 2008). The specific subtypes of VEGF involved in formation of glomerular endothelial fenestrations remains to be directly proven. Based on the isotypes expressed by podocytes and receptors expressed on glomerular endothelial cells, signaling pathways by VEGF-A, VEGF165 -B and VEGF-C are likely the key mediators necessary for normal glomerular endothelial cell development (Foster et al., 2006, 2008; Kamba et al., 2006). Angiopoietins also modulate mature endothelial cell growth and survival and permeability (Satchell et al., 2004; Woolf, 2010). Angiopoietin-1 protein is detected in podocytes and binds to the glomerular endothelium, whereas in normal conditions, angiopoietin-2 is not detected in podocytes in mice, but is present in mature podocytes in rats (Campean et al., 2008). Angiopoietin 2 and Tie-1 downregulate Tie-2 signaling. Absence of Tie-1, the receptor for angiopoietin, normally expressed on endothelial cells, results in abnormal glomerular microvasculature (Woolf et al., 2009). Complex interactions of additional various factors in glomerular development are demonstrated in studies of knockout mice. Thus, disruption of platelet-derived growth factor-B (PDGFB), normally expressed in the mesangial cell, results in a failure of migration of mesangial cells and ballooned capillaries, with preserved endothelium, resulting in perinatal death. The phenotype is similar with endothelial cell-specific deletion or when the PDGF-B receptor is deleted. PDGF-C is expressed in glomerular endothelial cells in the mouse, and may be angiogenic (Eitner et al., 2003; Floege et al., 2008; Li et al., 2005). The formation of fenestrae in response to VEGF involves fusion of intracellular vesicles, which contain caveolin-1 (Vasile et al., 1999). The relationship of fenestrae to caveolae has been controversial, with some suggesting that fused caveolae give rise to the endothelial cell fenestrae under the influence of VEGF. However, the absence of PV-1 and caveolin from the fenestrae of the glomerular endothelial cell argue against this hypothesis (Ballermann, 2005). Caveolin-1 −/− mice have fully differentiated glomerular endothelium including fenestrae, further providing direct evidence of distinct pathways of formation of caveolae and fenestrae (Sorensson et al., 2002). 2.4. Formation of diaphragms During the S-shaped stage of development, the glomerular endothelium is continuous. As capillary loops and fenestrations are formed in the developing glomeruli, diaphragms are present in caveolae. As foot processes develop and podocytes mature, the developing glomerular endothelial cells lose many of their diaphragms (Ichimura et al., 2008; Quaggin and Kreidberg, 2008). Observations of glomerular endothelial cell diaphragms in mature glomeruli have been variable, which may, in part, be related to the intricacies of studying these structures with standard electron microscopic techniques. With special care in processing and fixation, some normal mature rat glomeruli have been definitively shown to contain diaphragms in the fenestrations (Ichimura et al., 2008). These fenestrations appear to be arranged in clusters that are separated by ridges of cytoplasm, and occupy about 20–50% of the glomerular capillary surface area (Bulger et al., 1983). This arrangement results in alignment of fenestrations in the part of

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the cell opposite the podocyte foot processes, which is expected to facilitate filtration (Vasmant et al., 1984; Satchell and Braet, 2009). Interestingly, during injury and recovery after mesangial cell injury induced by anti-Thy-1 antibody, an increased number of glomerular endothelial cell fenestrae show diaphragms (Ichimura et al., 2008). 2.5. The glycocalyx The fenestrae are not merely empty holes open to the circulation. The luminal aspect of the endothelium has a complex surface layer, the endothelial cell surface layer, consisting of the glycocalyx and the endothelial cell coat. Importantly, standard electron microscopy does not allow visualization of this endothelial cell surface layer. Varying preparations have yielded widely divergent results of its thickness, ranging from 50 to 300 nm. Assessment of this structure in vivo has suggested that the total thickness of the endothelial cell surface layer may be up to 500 nm (Henry and Duling, 1999), which is comparable to the normal adult GBM thickness in humans of approximately 325–375 nm. The glycocalyx is not uniform in its composition. The ratio of heparan sulfates and hyaluronic acid to sialoproteins was higher in the glycocalyx within the fenestrae compared to the interfenestral regions, which may modulate filtration across fenestrae (Avasthi and Koshy, 1988; Bankston and Milici, 1983). The glycocalyx is also postulated to influence permeability. Using modeling, it has been estimated that as much as half of the overall hydraulic resistance of the capillary wall is contributed to by the glycocalyx (Drumond and Deen, 1994). Alterations of the glycocalyx may therefore also contribute to increased permeability to albumin as seen in disease states (Satchell and Tooke, 2008). Disruption of components of the endothelial cell surface layer directly leads to proteinuria (Jeansson and Haraldsson, 2006). Studies of primary cultured human glomerular endothelial cells with an endothelial cell surface layer further indicate that this component of the capillary wall is important for function (Bjornson et al., 2005). Using this system, removal of a large portion of this endothelial surface layer with neuroaminidase in this complex cultured glomerular endothelial cell system increased albumin flux and decreased electrical resistance; the latter is a surrogate marker for small molecule flux (Singh et al., 2007). When only heparan sulfate proteoglycans were removed, only albumin flux was altered (Singh et al., 2007). Elegant in vivo studies in adriamycin nephropathy, a model of nephrotic syndrome induced by this podocyte selective toxin, showed an apparent marked decrease in thickness of the endothelial cell surface layer in the nephrotic mice compared to control (Haraldsson and Jeansson, 2009). The glycocalyx is specifically modulated by angiopoietin. Ex vivo treatment of glomeruli with angiopoietin-1 resulted in increased glycosaminoglycan content of the endothelial glycocalyx and protected against removal of endothelial glycocalyx by pronase (Salmon et al., 2009b). Conversely, mice with overexpression of angiopoietin-1 did not develop increased permeability and were also resistant to increased permeability during inflammation (Thurston et al., 1999). Based on the prompt response, the effects of angiopoietin on the glycocalyx may in part be due to reassembly of the glycocalyx with reattachment to the plasma membrane after injury (Baffert et al., 2006; Gamble et al., 2000). 2.6. Impact of structure on function In the last decade, the podocyte has been center stage in studies of proteinuria and altered filtration. Podocyte foot process effacement and loss of slit diaphragms are key injuries leading to proteinuria. However, alterations of the glomerular endothelial cell fenestrae and diaphragms and its glycocalyx (see above)

also affect glomerular function and permeability. The rapid modulation of fenestrae in response to various stimuli indicate that these structures can by dynamically regulated and may influence filtration and function of the glomerular endothelial cell in physiological and pathophysiological settings. The regulation of the fenestrae and the filtration slits between foot processes are essential components of the capillary wall that allow filtration of water and small solutes. For example, decreased size and loss of fenestrations have been documented in glomeruli in preeclampsia, a lesion termed “endotheliosis”, which is rapidly reversible after delivery in most patients (see below). The induction of increased diaphragms in response to injury suggests that these structures also modulate endothelial cell function and possibly filtration in pathophysiologic settings. 3. The glomerular endothelial cell – a source of key regulatory molecules 3.1. Local accumulation versus synthesis of modulators Glomerular endothelial cells produce numerous substances that modulate glomerular function. Although serial analysis of gene expression (SAGE) has been applied to whole glomeruli, it has been difficult to dissect the unique contribution of the glomerular endothelial cell to the transcripts and proteins derived from whole glomeruli (Nystrom et al., 2009). Comparisons of glomerular endothelial cells to other cells are underway by various molecular studies including serial analysis of gene expression (SAGE), in attempts to identify further unique aspects governing their development and function (Vaughan and Quaggin, 2008). Immunohistochemical studies to examine glomerular-specific location of various components have been undertaken. Such studies must be interpreted with caution, as they do not differentiate between locally produced and locally accumulated molecules. 3.2. Vasoactive substances Vasoactive systems such as endothelin-1, nitric oxide (NO), prostacyclin and all components of the renin angiotensin system (RAS) are expressed in glomerular endothelial cells (Fig. 1) (Collino et al., 2008; Nestoridi et al., 2008; Wang et al., 2000). Endothelin-1 is a powerful vasoconstrictor that interacts with ETA and ETB receptors, both expressed locally in the glomerulus (Herman et al., 1998). Experimental and human studies show that, within the kidney, the ETB receptor predominates (ETA:ETB ratio of 1:2.3) (Karet et al., 1993; Nambi et al., 1992; Wendel et al., 2006). Within the glomerulus, mesangial cells and podocytes express both receptors, while endothelial cells express only the ETB receptor. Notably, whereas the ETA receptor leads to vasoconstriction, the ETB receptor modulates both constriction and, though stimulation of nitric oxide and prostacyclin, can effect vasodilation (Hyslop and de Nucci, 1992). The vasodilator prostacyclin is also locally expressed by the glomerular endothelium. Angiotensin II (Ang II), including its breakdown product Ang (1–7), is generated in the endothelium, although specific information on relative contribution of each component of the RAS in the glomerular versus systemic endothelium is lacking (Santos et al., 1992; Dilauro and Burns, 2009). Glomerular endothelial cell staining for angiotensinogen, ACE and angiotensin II was very weak in renal biopsies in patients without specific disease (Takamatsu et al., 2008). Whether these RAS components are synthesized locally, or accumulate at the glomerular endothelial cell, has not been directly proven in vivo in human tissues. Ang (1–7) can be generated directly from Ang I or by degradation of Ang II, a process mediated by protease enzymes including prolyl oligopeptidase, neprilysin or thimet oligopeptidase, mostly within

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the proximal tubule. Angiotensin converting enzyme 2 (ACE2) is a homologue of ACE that degrades Ang II to Ang (1–7) and also converts Ang I to angiotensin (1–9). Angiotensin (1–9) can also give rise to Ang (1–7) by actions of ACE. ACE2 localizes to the proximal tubule with lesser amounts in other nephron segments. Thus, the formation of Ang (1–7) and its related effects are largely downstream to the glomerular endothelial cell. However, ACE2 expression has been observed in the renal endothelium and in podocytes, suggesting a possible local production of Ang (1–7) within the glomerulus as well (Ye et al., 2006; Lely et al., 2004; Velez et al., 2007). Interesting evidence supports that Ang (1–7) may be counterregulatory and increase renal blood flow and inhibit Ang II responses (Dilauro and Burns, 2009). Glomerular endothelial cells also modulate the production of nitric oxide (NO). Both endothelial and inducible nitric oxide synthase (iNOS, eNOS) are expressed in cultured human glomerular endothelial cells, and their expression is influenced by VEGF interacting with the VEGF R-2 receptor (Pala et al., 2005). Thus, local activity of these keys vasoactive substances may be differentially regulated compared to the systemic circulation. 3.3. Modulators of inflammation/cytokines/chemokines Glomerular endothelial cells normally express numerous molecules that govern interaction with circulating elements, including adherence and ultimately infiltration by circulating leukocytes, and also govern local hemostasis, including platelet endothelial cell adhesion molecule-1, intercellular adhesion molecule-1 and -2 (ICAM-1, ICAM-2), VCAM-1, von Willebrand factor and plasminogen activators (PA) and plasminogen activator inhibitor-1 (PAI-1) (Nagao et al., 2007; Satchell et al., 2006). These molecules are typically expressed at low basal levels but increase (Akis and Madaio, 2004) in response to injury, promoting cell adhesion and release of both pro-thrombotic and anti-thrombotic factors. The net result depends on the balance ensuing of these modulators. The uniqueness of glomerular endothelial cell expression of inflammatory modulators versus other renal endothelial cells has been demonstrated. Thus, endothelial cells in different renal compartments showed differential chemokine expression profiles. The chemokine IP-10/CXCL10 acts via CXCLR3 on activated T cells, and MCP-1/CCL2 which acts via CCR2 on monocytes. In response to renal artery perfusion with an anti-endothelial antibody, both were strongly upregulated, but predominantly in endothelial cells in the tubulointerstitial area, co-localizing with infiltrating cells. Of note, IP-10/CXCL10 was not expressed by glomerular endothelial cells after this injury, whereas MCP-1/CCL2 mRNA was expressed both in the glomerulus and the tubulointerstitial endothelial cells (Panzer et al., 2006). These responses would thus favor interstitial rather than glomerular T cell infiltration. In contrast, monocyte accumulation was promoted by increased chemokine expression both in the glomerulus and the interstitium after injury. In contrast, monocyte accumulation is promoted by increased chemokine expression, including CXCR3, both in the glomerulus and the interstitium after injury (Menke et al., 2008; Panzer et al., 2007). Experiments with knockout mice for various ligands for CXCR3 showed that deletion of Mig/CXCL9 or CXCR3, but not deletion of CXCL10, resulted in decreased glomerular and interstitial T cell and macrophage infiltration and decreased glomerular IgG deposits in immune-mediated nephritis. Thus, Mig/CXCL9, but not IP-10/CXCL10, is the important ligand for glomerular T cell accumulation. Further, both CCL2 and CXCL9 were upregulated in glomeruli after injury, although distinction of endothelial versus other cell origin was not determined (Menke et al., 2008; Panzer et al., 2007). These studies demonstrate the finely regulated and heterogeneous expression of chemokines that directs leukocyte trafficking and tissue infiltration and ultimately renal injury. Further study will be

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necessary to specifically determine whether the specific modulators expressed in glomerular endothelium that may thus serve as therapeutic targets. Expression of angiopoietins also contributes to glomerular cell infiltration (Fig. 1). Angiopoietin-2 often acts as an inhibitor of endogenous angiopoietin-1, binding to Tie-2 but blocking its activation. Thus, contrary to angiopoietin-1, angiopoietin-2 enhances leukocyte adhesion to endothelial cells (Kuo et al., 2008). In contrast, angiopoetin-1 strengthens endothelial adhesion in microvessels injured by inflammation, and promotes maintenance of the glycocalyx and protects against increased permeability after injury (Baffert et al., 2006; Gamble et al., 2000; He, 2009). 4. Endothelial cell-podocyte cross-talk How then may factors released from the podocyte reach the endothelium? As elegantly shown in knockout studies, deletion of VEGF specifically from the podocyte has profound effects on the endothelium. This may be contributed to by movement of substances counter to the net direction of filtration (Fig. 1). One mechanism is by the so-called eddy effect, whereby non-linear flow results in areas of turbulence and countercurrent flow. In addition, recent elegant three-dimensional reconstruction of the glomerular tuft by electron microscopy has shown a distinct sub-podocyte space (Neal et al., 2005). This area is bound by the podocyte cell body and thin plate-like extensions from the cell body and glomerular filtration barrier underneath. Thus, filtrate that exits through the basement membrane and the foot processes enters into these cave-like domains, with only small areas available for direct egress into the urinary space and the proximal tubule outlet. The sub-podocyte spaces may cover around 60% of the entire filtering surface area of the capillary walls. These three-dimensional reconstructions show that the sub-podocyte space is highly restrictive to flow, with estimates that hydraulic resistance within the sub-podocyte space is 2.5 times more than that of the underlying barrier. Changes in the dimension of the exit sites from the sub-podocyte space could thus be key in changing the net pressure within the sub-podocyte space (Salmon et al., 2007). Thus, this sub-podocyte space may also serve to enhance movement of molecules counter to the net filtration direction, allowing VEGF and other podocyte-derived molecules to reach the endothelium (Salmon et al., 2009a). 5. Glomerular endothelial cells in disease 5.1. Direct versus indirect glomerular endothelial cell injury Glomerular endothelial cells are altered in a wide range of disease states, often in parallel to systemic endothelial injury, in e.g. diabetes, or more specifically, in hemolytic uremic syndrome (HUS). Increased susceptibilties of the unique glomerular endothelium may underlie some of these mechanisms. It is also possible that there is increased local generation of injurious factors, or a combination of both possibilities. Indirect glomerular endothelial injury is observed when podocytes are severely injured, resulting in loss of key angiogenic factors necessary for maintenance of glomerular endothelial cell survival and function. In preeclampsia, for instance, loss of functional podocyte VEGF is a key contributor to pathogenesis of the characteristic severe glomerular endothelial injury, so-called “endotheliosis” (see below). In diabetes, the generalized direct endothelial cell injury is contributed to by an array of factors including high glucose, reactive oxygen species (ROS), advanced glycation end products (AGE), and increased RAS activation. In the kidneys with diabetic nephropathy, podocytes are also depleted (Wolf et al., 2005), resulting in a combination of direct and indirect noxious stimuli contributing to endothelial cell injury.

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In African Americans with hypertension-associated chronic kidney disease, prevailing evidence has pointed to an underlying primary microvascular defect rather than direct hemodynamic injury causing the sclerosis (Fogo et al., 1997). Recent data point to a remarkable increase in prevalence of a polymorphism for nonmuscle myosin heavy chain 9 (MYH9) in African Americans, especially in association with hypertension and chronic kidney disease and with HIA-associated collapsing glomerulopathy, but interestingly not in association with diabetic nephropathy in this ethnic group (Freedman et al., 2009). As the polymorphism is also present in persons without disease, a two-hit model of genetic susceptibility has been proposed, with additional genetic and/or environmental factors contributing to the ultimate disease phenotype of excessive global glomerulosclerosis and chronic kidney disease, with consequential hypertension. This MYH9 molecule is expressed in platelets and in podocytes. Rare autosomal dominant diseases with nonsense or missense mutations of MYH9, such as May–Hegglin, Sebastian, Fechtner, and Epstein syndromes are characterized by platelet abnormalities with very rare associated kidney disease (Bostrom and Freedman, 2010). The mechanisms underlying the rarity of renal disease in these patients, and the common association of intronic polymorphisms of MYH9 with chronic kidney disease are unknown. One may thus speculate that both direct endothelial cell injury, contributed to by abnormal platelet–endothelial interaction, and indirect injury, contributed to by podocyte abnormalities, may be involved. 6. Markers and consequences of glomerular endothelial cell dysfunction Markers of endothelial dysfunction include elevated serum levels of von Willebrand factor (vWF). Increased generation of ROS, which disrupts the glycocalyx, caused marked proteinuria without changes in the remaining capillary wall (Henry and Duling, 2000; Yoshioka et al., 1991). Direct effects of ROS on the glomerular endothelium include decreased production of heparan sulfate proteoglycans and interference with nitrous oxide by increased availability and activation of NF␬B (Kashihara et al., 1992; Vink and Duling, 1996). Increased ROS may be contributed to both by resident glomerular cells, including the endothelium, as well as by circulating inflammatory cells. Further, increased ROS may promote more adhesion and infiltration of circulating cells, thus furthering a cycle of increased local injury. Any initial injury of the endothelium results in upregulation of adhesion molecules such as ICAM-1 and VCAM-1 (Nagao et al., 2007), furthering adhesion of inflammatory cells and promoting their migration into the glomerulus proper. Conversely, when the antioxidant enzyme superoxide dismutase, which degrades ROS, is genetically overexpressed, the effects of increased glucose to prevent eNOS activity are stopped (Du et al., 2001). 7. Endothelial cells and fibrosis/sclerosis versus repair 7.1. Glomerular endothelial cells and sclerosis Endothelial cells respond to injury by elaboration of increased pro-thrombotic substances, including plasminogen activator inhibitor-1 (PAI-1). PAI-1 is the inhibitor of tissue type and urokinase type plasminogen activators (tPA, uPA). tPA and uPA activate inert plasminogen to plasmin. Plasmin promotes not only lysis of fibrin, but also lysis of matrix proteins that accumulate in sclerosis. Increased PAI-1 is linked not only to thrombosis but to fibrosis. We have further shown that regression of glomerulosclerosis by high dose ARB is associated with decreased PAI-1 (Eddy and Fogo, 2006; Ma et al., 2005). Our proteomic studies of glomeruli in various

stages of sclerosis identified thymosin-␤4 (T␤4) as a key molecule upregulated in various cells, including glomerular endothelial cells, during development of scarring. Cultured glomerular endothelial cells respond to angiotensin II with increased PAI-1, a profibrotic response that was completely prevented by knockdown of T␤4 (Xu et al., 2005). T␤4 is a G-actin sequestering protein with widespread actions in angiogenesis, and would healing. It is degraded by prolyl oligopeptidase into the tetrapeptide AcSDKP. AcSDKP is degraded by ACE, and is increased with treatment with ACEI. Increased AcSDKP has been linked to the anti-fibrotic effects of ACEI (Lin et al., 2008). Whether the balance of T␤4 and AcSDKP from glomerular endothelium or other cells is pivotal for determining response to injury has not yet been determined. The possibility of transition of various cell types contributing to repair versus injury is an area of intense investigation. Epithelial–mesenchymal transition has been postulated to give rise to interstitial fibrosis, as has endothelial–myofibroblast transition (Kalluri and Neilson, 2003; Li et al., 2009). Other studies have shed doubt on the importance of such transition for fibrosis, and have rather focused on pericyte–mesenchymal transition (Humphreys et al., 2010; Cook, 2010). Whether similar transitions can happen in the glomerulus, and the role of cross-talk between podocytes and endothelial cells in mediating such fibrotic responses, remain unknown. Recent data show a fibroblast-like marker expressed in podocytes in diabetic nephropathy, namely fibroblast specific protein-1 (FSP-1) (Yamaguchi et al., 2009). We have also seen a stage specific increase of FSP-1 expressed in podocytes in diabetic nephropathy and lupus nephritis, with increases more dominant at early stages of diabetic injury and in proliferative types of lupus nephritis (unpublished data, Rossini et al., 2005). Whether this change in podocyte phenotype also alters glomerular endothelial cell behavior is not yet known. In the single-dose anti-Thy-1.1 model mesangial injury induced by anti-mesangial cell antibody, there is mesangiolysis, capillary injury and microaneurysms, followed by exuberant mesangial proliferation, and eventual repair. Treatment with exogenous VEGF during the acute phase promotes repair (Shimizu et al., 2004; Miyamoto et al., 2004). Repair is mediated by successive branching of capillary loops and endothelial cell proliferation, so-called intussusceptive capillary growth (Notoya et al., 2003). This capillary growth is also contributed to by bone marrow derived-cells (Rookmaaker et al., 2003). Capillary growth also occurs early in diabetic nephropathy. The glomerular enlargement seen early in experimental models of diabetic nephropathy has been investigated in detail with stereological assessment at the electron microscopic level. Glomerular enlargement was contributed to both by increased length and surface area of glomerular capillaries. Whether this increased total capillary number is contributed to by angiogenesis or vasculogenesis or both remains to be determined (Guo et al., 2005). However, in the chronic sclerosing stage of kidney disease, there is evidence for altered vascular homeostasis, with decreased VEGF and increased circulating endothelial cells. These findings implicate a defect in local angiogenesis, resulting in stimulation of other potential sources of endothelial cells (Futrakul et al., 2008). The mechanisms and potential for repair vary at different stages of injury. Others and we have examined the potential for glomerular capillary repair and regression of established glomerulosclerosis in chronic kidney injury models. We have observed that sclerosed glomeruli may undergo remodeling with increased open capillaries and increased capillary branches and complexity of branching in response to high dose angiotensin receptor blocker (ARB), documented by three-dimensional confocal imaging and graph theory analysis of branching (Ma et al., 2005; Fogo, 2006). Studies by the group of Ritz have further shown that capillaries are increased when regression of sclerosis in a model of secondary FSGS is

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induced with ARB (Adamczak et al., 2004). Our mathematical modeling supports that although regression may occur, it is limited to glomeruli with less than half of the tuft sclerosed. In glomeruli with sclerosis involving most loops, progressive sclerosis ensued despite intervention with high dose ARB (Ikoma et al., 1991). In vitro, we showed that high dose ARB could also influence injured podocytes exposed to puromycin aminonucleoside, restoring podocyte ability to produce VEGF and angiopoietin-1 (Liang et al., 2006). Thus, glomerular endothelial cells exposed to supernatant from injured podocytes failed to grow, whereas those exposed to supernatant from injured podocytes treated with ARB had enhanced growth and branching. These ARB effects were linked to increased phosphorylation of AKT, Erk and p38 MAP kinases. Thus, angiotensin blockade may influence glomerulosclerosis and ameliorate injury and even restore glomerular capillaries by enhancing podocyte production of key endothelial cell survival factors (Liang et al., 2006). Whether additional interventions to decrease both direct and indirect ongoing endothelial cell injury, promote mesenchymal–endothelial transition rather than the converse, and increase angiogenesis and vasculogenesis can promote further repair, is unknown. 8. Specific disease states and glomerular endothelial injury We will review selected diseases with evidence for direct and/or indirect glomerular endothelial injury contributing to disease pathogenesis. 8.1. Diabetic nephropathy In human diabetic nephropathy, endothelial cell dysfunction precedes microalbuminuria. When microalbuminuria develops, fenestration loss is also documented (Toyoda et al., 2007). In early diabetic injury, there is increased glomerular endothelial cell proliferation, and an increase in capillary surface area and microaneurysm formation (Hohenstein et al., 2006; Ichinose et al., 2005; Nyengaard and Rasch, 1993; Yamamoto et al., 2004). Microaneurysm formation is due to mesangial cell injury and mesangiolysis, key early lesions seen during development of diabetic nephropathy. Inhibition of angiogenesis prevented the increased glomerular size otherwise characteristic of early diabetic nephropathy. Subtle podocyte injury due to hyperglycemia and other injuries associated with diabetes resulting in increased VEGF are thought to underlie these early angiogenic changes (Wang et al., 2006; Hohenstein et al., 2006). In animal models of diabetic nephropathy, glomerular angiopoietin-2 expression was also increased, and may also be a target to prevent early pathologic angiogenesis (Yamamoto et al., 2004; Rizkalla et al., 2005). With subsequent progression of injury, podocytes then are lost, which likely contributes to loss of glomerular endothelial cells and sclerosis observed at later states of diabetic nephropathy (Wang et al., 2006; Hohenstein et al., 2006). Restoration of this imbalance of angiogenic factors with treatment with bone morphogenic-protein 7 indeed protected against injury in an experimental model of the diabetic nephropathy (Kuo et al., 2008; Tischer et al., 1989). Advanced glycation end products (AGEs) are increased and implicated in pathogenesis of diabetic nephropathy (D’Agati et al., 2009). The receptor for AGE (RAGE) is expressed in glomerular endothelial cells and in podocytes. When cultured human glomerular endothelial cells were exposed to AGEs, VEGFR2 was upregulated, with decreased growth and decreased expression of heparan sulfate proteoglycans (Pala et al., 2005). Thus, increased AGE can mediate increased permeability and albuminuria both directly and by indirect upregulation of podocyte VEGF (D’Agati et al., 2009).

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In response to pathogenic stimuli such as high glucose and angiotensin, relevant to diabetic nephropathy, glomerular endothelial cells increase synthesis of prostacyclin (Jaimes et al., 2010). This may be relevant to early states of diabetic injury, where hyperfiltration occurs. Cultured glomerular endothelial cells respond to increased glucose and oxidative stress induced by free fatty acids and Ang II by dysfunctional expression of eNOS and increased reactive oxygen species (ROS), induced in part from NADPH oxidase. The combined injury of high glucose and angiotensin also upregulated cyclooxygenase-2 (COX-2) and prostacyclin. In contrast, free fatty acid only increased prostacyclin but did not upregulate COX-2 (Jaimes et al., 2010). Of note, deletion of eNOS results in worsening of injury in the db/db model of diabetic nephropathy (Zhao et al., 2006). Glomerular endothelial cells also can generate extracellular succinate, which can interact with the kidney specific G protein-coupled metabolic receptor GP391. Within the juxtaglomerular apparatus, complex interactions of multiple cell types likely contribute to local succinate accumulation in disease states such as diabetes (Toma et al., 2008). Taken together, these findings show local cell-specific differential responses to the generalized direct and indirect injury in diabetes. 8.2. Obesity The possible contribution of adipokines to microvascular endothelial dysfunction is particularly relevant in obese patients, with or without type 2 diabetes. Adipokines, such as leptin and adiponectin, are produced by adipose tissue. Obesity is also associated with a proinflammatory state with increased TNF-␣ and IL-6, increased leptin and downregulated adiponectin (Yang and Smith, 2007). Microalbuminuria might be contributed to by concerted interactions of TNF-␣, IL-6 and leptin, which induces vascular permeability and stimulates angiogenesis together with VEGF (Cao et al., 2001). The decreased adiponectin seen with obesity might also contribute to microalbuminuria as it normally decreases endothelial cell activation and inflammation. 8.3. Preeclampsia/eclampsia The glomerular endothelial cell is central in the injury of preeclampsia/eclampsia (Kanasaki and Kalluri, 2009). Morphologically, the glomeruli appear bloodless due to swollen endothelium with loss of fenestrae, a lesion called endotheliosis. Patients with preeclampsia have increased levels of the soluble receptor for VEGF, sFlt-1, which thus acts as an inhibitor of VEGF actions, likely due to a primary injury in the placenta with failure of proper maturation of spiral arterioles. Serum from preeclampsia patients increased secretion of endothelin from endothelial cells (Collino et al., 2008). Blockade of VEGF also induced endothelin release, both of which may contribute to hypertension (Collino et al., 2008). sFlt-1 levels and glomerular structure return to normal shortly after delivery. Experimental studies point to a key pathogenic role of abnormal VEGF in preeclampsia. In rats, increased sFlt-1 resulted in lesions typical of endotheliosis. Direct evidence for a key role of VEGF-A in glomerular endothelial development and in preeclampsia comes from elegant studies of knockout mice. Deletion of VEGF-A specifically in the podocytes led to a lack of endothelial cell migration and differentiation. Heterozygous knockout mice hypomorphic for VEGF had lack of complete differentiation, with failure of glomerular capillary endothelium to flatten or form fenestrae, a lesion similar to endotheliosis (Eremina et al., 2003; Maynard et al., 2003). Soluble endoglin receptor is also increased in preeclampsia. This receptor is present in endothelial cells and binds with TGF-␤ and is required for activation of ALK 1 signaling in endothelial cells. Knockout mice deficient in endoglin die due to a defect in vasculo-

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genesis (Lebrin et al., 2005). Lack of proper endoglin signaling may thus also contribute to endothelial cell injury in preeclampsia. 8.4. Thrombotic microangiopathy/hemolytic uremic syndrome A range of diseases is characterized by fibrin thrombi within the microvasculature, a lesion called thrombotic microangiopathy (TMA) (Tsai, 2006). The clinical spectrum ranges from classic diarrhea (D)-associated hemolytic uremic syndrome (HUS) to thrombotic thrombocytopenic purpura (TTP). In classic D-positive HUS, verotoxin is produced by pathogenic E. coli that produce verotoxin/shiga toxin. Exposure to combined TNF-␣ and shiga toxin, with or without additional exogenous Ang II, upregulated tissue factor activity from cultured human glomerular endothelial cells. This expression was mediated via the Ang II type 1 receptor (AT1). Importantly, both mRNA and activity of the AT1 receptor were implicated, suggesting that local RAS activation could be important in the development of thrombotic microangiopathy (Nestoridi et al., 2008). The increased susceptibility of children of D-positive HUS has been proposed to be due to increased glomerular expression of globotriaosylceramide (Gb3). This receptor binds shiga toxin and is highly expressed on glomerular versus systemic endothelium. However, detailed examination of this receptor in renal biopsies in children versus adults failed to find differences in expression, thus suggesting other factors may be implicated in the differential susceptibilities (Ergonul et al., 2003). In some patients without diarrhea, so-called atypical HUS, hereditary or acquired deficiency in various complement regulatory molecules has been described. Imbalance in complement regulation locally then is implicated in local destruction of glomerular endothelial cells (Noris and Remuzzi, 2009). Inherited deficiencies of the von Willebrand factorcleaving protease ADAMTS-13 or acquired auto-antibodies to this protease have been found in many, but not all, patients with classical TTP (Tsai, 2006; Moake, 2004). TMA is also associated with various drugs including calcineurin inhibitor, HIV infection, and following solid organ or bone marrow transplant. Further support for the key role of normal podocyte-endothelial cell crosstalk in maintaining the glomerulus is seen by the recent reports of thrombotic microangiopathy occurring in a small number of patients treated with neutralizing VEGF antibody for malignancy (Eremina et al., 2007, 2008; Ranieri et al., 2006). VEGF is regulated by splicing factors, and splice site selection can switch from the anti-angiogenic VEGF(xxx) b isoforms to the pro-angiogenic VEGF(xxx) isoforms, increased expression of the proangiogenic VEGF164 results in glomerular dysfunction (Eremina et al., 2003). In contrast, increased VEGF165b does not result in overt glomerular disease, although altered glomerular endothelial fenestrations are evident by electron microscopy, and these mice do show decreased glomerular permeability (Qiu et al., 2009). These complexities of VEGF regulation may underlie the varying renal effects observed with various experimental maneuvers with administration of antibodies or exogenous VEGF. 8.5. Immune complex/crescentic glomerulonephritides Direct injury to the glomerular endothelium is also observed in ANCA-associated disease and in immune complex disease with subendothelial deposits where activation of complement and membrane attack complex occurs in the subendothelial space. Glomerular endothelial cell staining for angiotensinogen, ACE and Ang II was also increased in human IgA nephropathy (Takamatsu et al., 2008), supporting a possible pathogenic role for heightened RAS activity in the pathogenesis. TNF-␣ is increased in a variety of inflammatory conditions, and increases endothelial cell adhesion molecule expression and expression of IL-6. TNF-␣ disrupts

the glycocalyx and also directly increases endothelial permeability (Henry and Duling, 2000; Friedl et al., 2002). Activation of TNF receptor 2 may be critical for the injurious cascade of recruitment of leukocytes, proteinuria and complement activation that characterizes these inflammatory glomerulonephritides (Vielhauer et al., 2005; Segerer et al., 2006; Panzer et al., 2006), and thus could be a target for intervention. The balance of angiopoietin-1 versus angiopoietin-2 is altered in several inflammatory disease states, with decreased Ang-1 and increased Ang-2 in anti-GBM nephritis in the mouse, associated with sclerosis and loss of endothelium and capillary loops (Yuan et al., 2002). In the rat anti-Thy-1.1 model of mesangial proliferative glomerulonephritis both Ang-1 and Ang-2 were upregulated (Campean et al., 2008). Angiopoietin-2 glomerular expression is increased in immune-mediated glomerulonephritis, and may thus alter glomerular endothelial function (Kuo et al., 2008; Tischer et al., 1989). Interestingly, increased eNOS resulted in upregulation of both angiopoietin-1 and VEGF in a model of angiogenesis in the mesenteric artery (Benest et al., 2008). Whether similar interaction of eNOS, angiopoietins and VEGF occur within the glomerular endothelium has not been shown. Glomerular endothelial cells also respond to viral double stranded type DNA by increased production of various cytokines, chemokines, and interferon, with resulting increased leukocyte adhesion to endothelial cells, increased permeability and decreased endothelial proliferation. These effects were mediated independently of Toll-like receptors. Glomerular endothelial cells only express Toll-like receptor-3, which binds double-stranded RNAs. The effects of viral double-stranded DNA were not affected by inhibiting Toll-like receptors that act through MyD88-dependent pathways. These results indicate that glomerular endothelial cells can trigger local anti-viral defense mechanisms, promoting a glomerular inflammatory and albuminuric response to viral injury, independent of classic Toll-like receptors (Hagele et al., 2009). 8.6. Transplant glomerulopathy Transplant glomerulopathy is a lesion of chronic endothelial injury in the transplant, tightly linked to preceding humoral rejection. Morphologically, it is characterized by splitting of the glomerular basement membrane by light microscopy, often with accompanying segmental sclerosis, and increased lamina rara interna and interposed cells by electron microscopy, with accompanying positive C4d in peritubular capillaries. C4d is a breakdown product of C4 and binds covalently to tissues at sites of complement activation. Its presence in peritubular capillaries is a marker of humoral rejection, and correlates well with the presence of antidonor antibodies. Of note, positive C4d may not be present at the time of histologic diagnosis, but may precede the chronic stage of injury (Fotheringham et al., 2009). Multilamellation of peritubular capillary basal lamina is observed in chronic rejection, presumed to result from previous acute rejection. Glomerular C4d staining by frozen section immunofluorescence is present even in normal kidneys, and is not specifically correlated with humoral rejection. However, although peritubular capillary C4d localization is the site that sensitively and specifically correlates with anti-donor antibodies, the glomerular capillaries are also injured by humoral rejection. 9. Future prospects The glomerular endothelial cell is both a target in injury and a contributor to perpetuation of injury. Maintenance of glomerular endothelium and restoration of capillaries is key to preventing chronic kidney disease or restoring functionality after injury. Understanding of the key complex balance of various angiogenic

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factors and interaction of the glomerular endothelium with the podocyte and other glomerular infiltrating and resident cells will be key for developing further therapeutic interventions.

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