REVIEWS

Endothelium structure and function

in kidney health and disease
Noemie Jourde-​Chiche1,2*, Fadi Fakhouri3, Laetitia Dou2, Jeremy Bellien4,
Stéphane Burtey1,2, Marie Frimat5,6, Pierre-​André Jarrot2,7, Gilles Kaplanski2,7,
Moglie Le Quintrec8,9, Vincent Pernin8,9, Claire Rigothier10,11, Marion Sallée1,2,
Veronique Fremeaux-​Bacchi12,13, Dominique Guerrot 14 and Lubka T. Roumenina 13,15,16
*
Abstract | The kidney harbours different types of endothelia, each with specific structural and
functional characteristics. The glomerular endothelium, which is highly fenestrated and covered
by a rich glycocalyx, participates in the sieving properties of the glomerular filtration barrier
and in the maintenance of podocyte structure. The microvascular endothelium in peritubular
capillaries, which is also fenestrated, transports reabsorbed components and participates in
epithelial cell function. The endothelium of large and small vessels supports the renal vasculature.
These renal endothelia are protected by regulators of thrombosis, inflammation and complement,
but endothelial injury (for example, induced by toxins, antibodies, immune cells or inflammatory
cytokines) or defects in factors that provide endothelial protection (for example, regulators
of complement or angiogenesis) can lead to acute or chronic renal injury. Moreover, renal
endothelial cells can transition towards a mesenchymal phenotype, favouring renal fibrosis and
the development of chronic kidney disease. Thus, the renal endothelium is both a target and a
driver of kidney and systemic cardiovascular complications. Emerging therapeutic strategies
that target the renal endothelium may lead to improved outcomes for both rare and common
renal diseases.

Uraemic toxins
Blood compounds that are
normally eliminated in the
urine but accumulate in
patients with chronic kidney
disease or acute kidney injury
and exert deleterious effects.
*e-​mail: noemie.jourde@
ap-hm.fr; lubka.roumenina@
crc.jussieu.fr
https://doi.org/10.1038/
s41581-018-0098-z
The endothelium constitutes the inner monolayer of
cells that lines blood and lymphatic vessels. It was tradi-
tionally considered to act as a simple conduit for trans-
porting blood, as a barrier to separate blood from the
vessel wall and in the regulation of vessel permeability
and diapedesis of immune cells at sites of inflammation.
However, we now understand that the endothelium is
a dynamic organ. Different populations of endothelial
cells (ECs) exist, each with different architectures and
functions that together promote an antithrombotic
and anti-​inflammatory environment and maintain tissue
perfusion and vascular tone (Figs. 1,2).
The kidney is a highly vascularized organ, character-
ized by a remarkable diversity of EC populations1 (Fig. 3).
ECs from the microvasculature, in particular, regulate
blood flow to local tissue beds and modulate coagula-
tion, inflammation and vascular permeability. Beyond
these functions, the ECs of the glomerulus contribute
to the glomerular filtration barrier and support podo-
cyte structure, whereas ECs of the microvasculature
in kidney tubules contribute to tubular reabsorption.
These varied functions of ECs make them key driv-
ers and targets of inflammatory and thrombotic pro-
cesses that can damage the kidney (for example, in the
context of common diseases such as diabetes melli-
tus and hypertension, in rare diseases such as atypical
haemolytic uraemic syndrome (aHUS) and glomer-
ulonephritides related to systemic diseases2 and in
situations associated with acute kidney injury (AKI))3.
In addition, the accumulation of uraemic toxins that
occurs with kidney dysfunction contributes to endothe-
lial dysfunction and the cardiovascular burden of
patients with chronic kidney disease (CKD)4,5. The
transition of renal ECs towards a mesenchymal phe-
notype in a process called endothelial-​to-mesenchymal
transition (EndMT) promotes renal fibrosis and the
development of CKD. In addition, systemic endothe-
lial dysfunction and accelerated atherosclerosis result-
ing from CKD might itself accelerate decline in renal
function. Thus, endothelial damage in the context of
kidney dysfunction can drive further kidney injury and
systemic complications.
Improved understanding of the role of endothe-
lial injury in renal disease has paved the way for new
therapeutic strategies (for example, complement block-
ade in aHUS) that target the endothelium. The fur-
ther development of strategies to protect EC structure
and function has the potential to reverse coagulation and

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Reviews
Key points
• The kidney contains diverse populations of endothelial cells, including the
glomerular endothelium, microvascular endothelium in peritubular capillaries and
the endothelium of large and small vessels, and each of these populations has specific
characteristics and functions.
• Homeostasis of renal endothelial cells is crucial for the preservation of glomerular
structure and function, the preservation of an anti-​inflammatory and an
antithrombotic environment and the prevention of renal fibrosis.
• Glomerular endothelial cells, in particular, are susceptible to injury in typical and
atypical haemolytic uraemic syndrome, lupus nephritis, antineutrophil cytoplasmic
antibody vasculitides and antibody-​mediated rejection as well as in situations of
vascular endothelial growth factor (VEGF) depletion.
• Common forms of chronic kidney disease (CKD) — diabetic kidney disease and
arteriolar nephrosclerosis — are also characterized by renal endothelial dysfunction.
• Alterations in endothelial repair capacity, endothelial-​to-mesenchymal transition
and capillary rarefaction contribute to the fibrogenic processes that lead to CKD.
• Therapeutic strategies aimed at preserving and/or restoring the integrity of the
endothelial glycocalyx, reversing the procoagulant and pro-​inflammatory
phenotype of injured endothelial cells and slowing renal fibrosis hold promise for
the treatment of renal disease.
inflammatory processes in other glomerulonephritides
and to slow the progression of CKD in patients with dia-
betic kidney disease (DKD) or arteriolar nephrosclero-
sis. Here, we describe the unique features of the healthy
renal endothelium and the pathological mechanisms
leading to endothelial damage in emblematic renal
diseases. We discuss the processes and consequences of
renal EndMT and the effects of CKD and uraemic toxins
on the systemic endothelium. We also describe the role
of the renal endothelium in the initiation and progres-
sion of the two most frequent kidney diseases — DKD
and arteriolar nephrosclerosis.
Author addresses
1
Aix-​Marseille University, Centre de Nephrologie et Transplantation Renale, AP-​HM
Hopital de la Conception, Marseille, France.
2
Aix-​Marseille University, C2VN, INSERM 1263, Institut National de la Recherche
Agronomique (INRA) 1260, Faculte de Pharmacie, Marseille, France.
3
Centre de Recherche en Transplantation et Immunologie, INSERM, Université de
Nantes and Department of Nephrology, Centre Hospitalier Universitaire de Nantes,
Nantes, France.
4
Department of Pharmacology, Rouen University Hospital and INSERM, Normandy
University, Université de Rouen Normandie, Rouen, France.
5
Université de Lille, INSERM, Centre Hospitalier Universitaire de Lille, U995, Lille
Inflammation Research International Center (LIRIC), Lille, France.
6
Nephrology Department, Centre Hospitalier Universitaire de Lille, Lille, France.
7
Assistance Publique-​Hôpitaux de Marseille, Service de Médecine Interne et
d’Immunologie Clinique, Hôpital de La Conception, Marseille, France.
8
Centre Hospitalier Universitaire de Lapeyronie, Département de Néphrologie Dialyse et
Transplantation Rénale, Montpellier, France.
9
Institute for Regenerative Medicine and Biotherapy (IRMB), Montpellier, France.
10
Tissue Bioengineering, Université de Bordeaux, Bordeaux, France.
11
Service de Néphrologie Transplantation, Dialyse et Aphérèse, Centre Hospitalier
Universitaire de Bordeaux, Bordeaux, France.
12
Assistance Publique-​Hôpitaux de Paris, Service d’Immunologie Biologique, Hôpital
Européen Georges Pompidou, Paris, France.
13
INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France.
14
Normandie Université, Université de Rouen Normandie, Rouen University Hospital,
Department of Nephrology, Rouen, France.
15
Sorbonne Universités, Paris, France.
16
Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
Renal endothelia
The environment of renal ECs
The phenotype and properties of ECs vary between vas-
cular beds according to their location and microenviron-
ment. In vivo, the vascular endothelium is continuously
subjected to the oscillating mechanical shear stress of
blood flow, the extent of which depends on the type
of blood vessel and its geometry6. This shear stress is a
major determinant of EC morphology, function, gene
expression and behaviour6–8.
The kidney harbours a wide variety of ECs, includ-
ing glomerular ECs (GECs), microvascular ECs within
peritubular capillaries and ECs within larger venous and
arterial blood vessels (Fig. 3). These populations of ECs
are exposed to different environments within the kidney
(for example, different oxygen pressures and osmolalities
within the kidney). Importantly, renal ECs contribute
to different transport activities: GECs are involved in
glomerular filtration whereas ECs of peritubular capil-
laries are involved in tubular secretion and reabsorption.
Both GECs and peritubular ECs are fenestrated,
although ultrastructural differences exist between them9.
This fenestration is critical for the permselectivity of the
glomerular filtration barrier and for the efficient passage
of high volumes of fluids and the formation of urine.
GECs are lined by a particularly thick filamentous
glycocalyx that is enriched in negatively charged pro-
teoglycans (mostly heparan and chondroitin sulfate)
that form a network with glycosaminoglycans (mostly
hyaluronic acid). The glycocalyx contributes to the reg-
ulation of vascular permeability and fluidic balance and
repels blood cells away from the vascular wall10. Densely
packed hyaluronic acid anchors the glycocalyx to the glo-
merular basement membrane (GBM) and fills endothe-
lial fenestrae, preventing the passage of albumin11. The
thickness of the glycocalyx exceeds the dimensions
of cellular adhesion molecules such as intercellular
adhesion molecule 1 (ICAM1), vascular cell adhesion
protein 1 (VCAM1), P-​selectins and E-selectins, which
are expressed by ECs, thus hampering cell adhesion to
the endothelium.
ECs of peritubular capillaries have a thin stroma.
As seen with electron microscopy, EC fenestrations are
covered by a thin diaphragm, which is composed of gly-
coproteins organized in radial fibrils and modulates the
sieving properties of these ECs12,13. These ECs lie on a
basement membrane, on the opposite side of which are
pericytes. These pericytes14 drive capillary constriction
to regulate medullary and cortical blood flow; they also
regulate capillary permeability, participate in angiogene-
sis and are a source of myofibroblasts in renal fibrogene-
sis15. Peritubular microvascular ECs also provide a niche
for resident organ stem cells, which reside in close prox-
imity and benefit from the paracrine effects of the angi-
ocrine factors they release. This angiocrine signalling is
organ-​specific and has a crucial role in tissue repair and
in preventing scarring and fibrosis16.
Response of renal ECs to stress
In addition to acting as a barrier, renal ECs are a source
and target of circulating factors. They express vasoac-
tive factors (such as endothelin 1 (ET1), prostacyclin
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Leukocyte trafficking Inflammation inflammatory receptors2. Activation of ECs under sit-

• ICAM1 • MCP1 uations of stress can either be rapid and independent
• VCAM • Complement activation
• E-selectin • Oxidative stress of the transcription of new genes (a process known
as type I activation) or slower and dependent on the
Metabolism
transcription of new genes (a process known as type II
Angiogenesis
• VEGF–VEGFR • Glycolysis activation)17. For example, ECs exposed to stress stimuli
• Angiopoietin 2–TIE2 • FAO (such as cytokines, histamine, thrombin, ADP, anaphyl-
• HIF activation • Glutamine and
• CD146 asparagine metabolism atoxins of the complement system and the complement
• Glycolysis • mTOR membrane attack complex C5b-9, haem, reactive oxygen
• FAO • HIF species (ROS) and bacterial lipopolysaccharide (LPS))
• Glutamine and Endothelial
asparagine metabolism cell respond within minutes with exocytosis of intracellular
• vWF granules known as Weibel–Palade bodies, which con-
• Jagged 1–NOTCH-1 Vascular permeability
and glomerular tain components such as the prothrombotic glycoprotein
filtration vWF, P-​selectin, IL-8 and ET1. Longer-​term exposure of
Control of VSMC proliferation • Endothelial ECs to inflammatory cytokines (such as TNF, IFNγ and
• Microparticles fenestrations
• Jagged 1–NOTCH-1 • Glycocalyx IL-1), pathogen-​associated molecular patterns (PAMPs;
• miR-126 • VEGF such as LPS and Shiga toxin (Stx)) or haemolysis-​derived
• NO • CD146 products (such as haem and haemoglobin) induces sig-
nalling within ECs to increase the expression and synthe-
Haemostasis and coagulation Vascular tone sis of adhesion molecules (such as E-​selectin, VCAM1
• TM • vWF Vasodilators: Vasoconstrictors:
• Glycocalyx • TF • NO • Endothelin and ICAM1) while decreasing the production of protec-
• tPA – • TXA2 + • H2S – • TXA2 + tive molecules such as thrombomodulin18. The above-​
• TFPI • PAI1 • PGI2 • PGH2 mentioned inflammatory cytokines, as well as histamine,
• PGI2
thrombin, bradykinin, complement component C5a,
Fig. 1 | role of the endothelium in health and disease. The endothelium has a crucial vascular endothelial growth factor (VEGF) and haem,
role in the regulation of multiple processes, including angiogenesis (through the actions increase endothelial permeability and impair EC barrier
of vascular endothelial growth factor (VEGF)–VEGF receptor (VEGFR), angiopoietin function. P-​selectin released from Weibel–Palade bod-
2–TIE2 and hypoxia-​inducible factor (HIF) activation; CD146; glycolysis; fatty acid ies stimulates recruitment of leukocytes to the activated
oxidation (FAO); glutamine and asparagine metabolism; von Willebrand factor (vWF); endothelium, whereas E-​selectin promotes the rolling
and jagged 1–NOTCH1 signalling); haemostasis and coagulation (through the protective and adherence of monocytes and activated lymphocytes
role of the glycocalyx and the synthesis of procoagulant factors, such as vWF, tissue to ECs, and VCAM1 and ICAM1 facilitate the arrest of
factor (TF), thromboxane A2 (TXA2) and plasminogen activator inhibitor 1 (PAI1), and
leukocytes allowing their diapedesis. Moreover, in addi-
anticoagulant factors, such as thrombomodulin (TM), tissue plasminogen activator (tPA),
TF pathway inhibitor (TFPI) and prostaglandin I2 (PGI2)); vascular tone (through the
tion to increasing the expression of adhesion molecules,
synthesis of vasodilators such as nitric oxide (NO), hydrogen sulfide (H2S) and PGI2 and pro-​inflammatory mediators also induce shedding of
vasoconstrictors such as endothelin, TXA2 and prostaglandin H2 (PGH2) and through the the glycocalyx, resulting in increased exposure of the
control of vascular smooth muscle cell (VSMC) proliferation); vascular permeability and adhesion molecules and leukocyte recruitments.
glomerular filtration (through the functions of endothelial fenestrations, the glycocalyx, Inflammation and coagulation are closely linked
VEGF and CD146); inflammation (through the synthesis of intercellular adhesion in the endothelial response to stress. Protease-​activated
molecule 1 (ICAM1), vascular cell adhesion protein 1 (VCAM1), E-​selectin and membrane receptors 1–4 (PAR1–PAR4)19 are G protein-​coupled
cofactor protein 1 (MCP1); regulation of complement activation and oxidative stress; receptors (GPCRs) that are expressed on the surface
and the recruitment and trafficking of leukocytes); control of VSMC proliferation of ECs. They are specifically activated by coagulation
(through the functions of microparticles, jagged 1–NOTCH1 signalling, miR-126 and NO);
proteases and modulate the inflammatory and throm-
and metabolism (through the functions of glycolysis, FAO, glutamine and asparagine
metabolism as well as mTOR and HIF).
botic phenotype of ECs. For example, the activation of
PAR1 by thrombin upregulates the endothelial expres-
sion of inflammatory cytokines (such as IL-1, IL-6 and
and nitric oxide (NO)); thrombotic regulators (such as TNF) and cell adhesion molecules (such as E-​selectin,
von Willebrand factor (vWF), tissue factor (TF), plas- P-selectin, ICAM1 and VCAM1)19, promotes EC apop-
minogen activators and plasminogen activator inhibi- tosis and increases endothelial permeability19. The acti-
tor 1 (PAI1)); intercellular adhesion molecules (such vation of PAR2 by activated factor X (FXa) participates in
as platelet EC adhesion molecule (PECAM1), ICAM1, tissue remodelling, mesangial proliferation and kidney
ICAM2 and VCAM1); and inflammatory modulators, damage in animal models of ischaemia–reperfusion
including chemokines2, and thus contribute actively injury20, mesangioproliferative and crescentic glomer-
to the microenvironment. Under physiological condi- ulonephritides21,22 and renal fibrosis23. Indeed, FXa and
tions, ECs exhibit antithrombotic properties through the activation of PARs induces the expression of inflam-
activation of protein C by expressing the thrombin matory mediators, such as monocyte chemoattractant
regulator thrombomodulin, and specific proteoglycans protein 1 (MCP1; also known as CCL2), IL-1β and TNF
and by releasing tissue plasminogen activator (tPA)1. in many cell types, including glomerular macrophages,
Upon activation (for example, by inflammation, oxida- ECs and podocytes24. Thus, PARs have an important role
tive stress, injury, uraemic toxins or in the context of in the interplay between coagulation and inflammation.
infection), ECs undergo phenotypic changes, charac- The peptide vasoconstrictor ET1, which is produced
terized by the downregulation of protective regulators locally in the kidney not only by ECs but also by other
and the upregulation of pro-​adhesive molecules and cells, including podocytes, mesangial cells, parietal

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Reviews

epithelial cells and tubular cells, acts in an autocrine or development of CKD28. As mentioned above, ET1 is
paracrine manner to regulate renal haemodynamics25. released from Weibel–Palade bodies by exocytosis
For example, binding of ET1 to the ETA receptor from ECs under conditions of endothelial stress. The
increases renal vasculature resistance and reduces renin–angiotensin–aldosterone system (RAAS), which
glomerular filtration rate (GFR), whereas binding of is involved in hypertension, renal damage and CKD pro-
ET1 to the ETB receptor increases tubular water and gression29, interacts closely with the endothelin system29.
sodium reabsorption26. Activation of the endothelin Angiotensin II (ANGII), which induces vasoconstriction
system is observed in various renal diseases and pro- of glomerular efferent arterioles, increases intraglomer-
motes mitochondrial stress in ECs, leading to disrup- ular pressure and favours the development of glomeru-
tion of the podocyte cytoskeleton27 as well as mesangial losclerosis, also increases the production of ET1 in the
cell proliferation, renal inflammation, fibrosis and the kidney, the expression of ETA and the binding of ET1
to ETA, further increasing renal vasculature resistance.
a Anti-inflammatory and anticoagulant phenotype
Key features of renal ECs
Although renal ECs behave largely like other ECs
in terms of their function as a barrier, a conduit for
EC transporting blood and a source of circulating factors,
Repair
by EPCs differences exist in the composition of their microenvi-
ronment, in their proteome and in the extent of their
Shear-stress-induced Glycocalyx Repair by response to various stimuli (Supplementary Table 1).
NO synthesis mature ECs Differences in the glycocalyx composition, such as the
abundance of sialic acid and the sulfation level of hepa-
ran sulfate30, determine the pattern of interaction of ECs
with plasma factors, such as complement factor H (FH).
This regulatory factor controls the activation of comple-
ment by recognizing and binding glycosaminoglycan on
host tissue. One glycosaminoglycan-​binding region of
FH — CCP6–CCP8 — anchors FH to Bruch's membrane
b Pro-inflammatory, procoagulant and complement-activating phenotype in the eye, whereas another, CCP19–20, anchors FH to
Activation Lesion Repair GECs30. Genetic variations in the CCP6–8 and CCP19–
20 domains therefore contribute differently to the dam-
age of eyes or kidneys in diseases such as age-related
TF Adhesion molecules macular degeneration and aHUS, respectively.
C3a Compared with cultured human umbilical vein ECs
Reduced mobilization (HUVECs), which are considered the classic model of
C5a
Coagulation
Monocyte and survival of EPCs ECs, GECs produce more fibrinolytic factors at resting
state. Moreover, the fibrinolytic properties of GECs are
potentiated upon activation with TNF or LPS, whereas
ROS Release Reduced
generation of EMPs proliferation Glycocalyx HUVECs become more pro-​thrombotic31. These differ-
breakdown ences suggest that the glomerular endothelium is par-
Detachment ticularly resistant to thrombus formation, even under
of CECs
conditions of EC stress. In contrast to differences in the
expression of thrombotic factors, only weak differences
exist between GECs and HUVECs in their expression
Reduced NO availability levels of adhesion molecules, complement factors, com-
plement activation capacity and permeability at resting
state and after activation with TNF, IFNγ, IL-1 or brief
Fig. 2 | endothelium dynamics in health and disease. a | Under physiological
conditions, the endothelium handles shear-​stress-induced synthesis of nitric oxide (NO) exposure to haem18,32–34. Nevertheless, GECs have a dif-
and maintains an anticoagulant and anti-​inflammatory environment through the ferent metabolic profile than HUVECs at resting state
production of regulatory factors and through the actions of the intact protective and after exposure to Stx and/or TNF35. Exposure of
glycocalyx. Endothelial repair is achieved by the proliferation of adjacent mature GECs to Stx and TNF strongly reduced NAD metabo-
endothelial cells (ECs) or by the incorporation of endothelial progenitor cells (EPCs), lism, leading to depletion of the energy substrate acetyl
which also promote angiogenesis through the synthesis of pro-​angiogenic factors. CoA and the antioxidant glutathione. By contrast, these
b | Situations of stress or injury lead to activation of the endothelium, characterized by effects were less pronounced in HUVECs, in which cellu­
the release of reactive oxygen species (ROS) and endothelial microparticles (EMPs) and lar acetyl CoA content even increased, which may partly
activation of complement and other pro-​inflammatory mediators. Disruption of the explain the particular susceptibility of GECs to Stx-
glycocalyx exposes adhesion molecules on the endothelium surface, leading to
induced injury35. These findings are in line with a study
leukocyte recruitment and inflammation; exposure of tissue factor (TF) induces
activation of the coagulation cascade. A reduction in NO release as a consequence of showing that oxidative stress, membrane lipid peroxi-
endothelial dysfunction induces impairment of flow-​mediated vasodilation. Apoptotic dation and depletion of reduced glutathione contrib-
or damaged ECs become detached and form circulating ECs (CECs), exposing the uted to renal injury in a mouse model of Stx-​associated
extracellular matrix with its procoagulant TF to components of the coagulation cascade. HUS36. These findings suggest that Stx and TNF render
Endothelium repair by adjacent mature cells or by EPCs is also compromised. GECs particularly susceptible to oxidative stress and lead

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Endothelium in medium and large vessels Podocyte

Endothelial
cell
Endothelial cell Glycocalyx
Mesangial
cell
Smooth Glomerular
muscle cell endothelium

Peritubular capillary

Endothelial Tubular
cell epithelial
cell

Fig. 3 | Diverse populations of endothelial cells in the kidney. The kidney harbours three main populations of
endothelial cells with distinct architectures and functions. Endothelial cells within medium and large vessels are
continuous, confluent, connected by intercellular junctions and elongated in the direction of blood flow. Glomerular
endothelial cells are highly fenestrated and are covered on their luminal surface by a thick glycocalyx, which participates in
the sieving properties of the glomerular filtration barrier. Crosstalk between glomerular endothelial cells, mesangial cells
and podocytes is crucial for the maintenance of glomerular structure and function. Similar to glomerular endothelial
cells, endothelial cells of the peritubular capillaries are also fenestrated, and their fenestrae are covered by a thin
diaphragm composed of glycoproteins. This endothelial population facilitates the reabsorption and secretion of fluids
and molecules from the adjacent tubular epithelial cells.

us to speculate that pro-​oxidative haemolysis-​derived context of tissue ischaemia45, complement activation

products, such as haemoglobin, haem and erythrocyte
microvesicles37,38, are particularly noxious for kidney
microvessels and will amplify the cell damage, comple-
ment activation and thrombosis in the context of renal
thrombotic microangiopathy (TMA)2,39,40.
Endothelial-​to-mesenchymal transition
ECs exhibit remarkable plasticity during both embry-
ogenesis and in adulthood 41. Maintenance of the
endothelial phenotype is a physiologically active process,
requiring energy and signalling input in ECs41. Some
forms of endothelial injury (for example, in the setting
of AKI resulting from sepsis or ischaemia–reperfusion
injury) induce ECs to undergo EndMT, leading to vascu-
lar rarefaction, organ fibrosis and dysfunction41. EndMT
involves a partial or complete phenotypic switch of ECs
to a mesenchymal cell fate. In this process, which can
be driven by activation of TGFβ, AKT and/or mTOR
pathways42, ECs lose markers typical of the endothelial
lineage (for example, VE-​cadherin and CD31), their
polarization and primary cilia43 and acquire myofi-
broblastic characteristics, including the expression
of α-smooth muscle actin (αSMA) and N-cadherin41.
EndMT has a central role in cardiac development
during embryogenesis44. After birth, EndMT occurs
in several pathologic conditions (for example, in the
and inflammatory cytokine stimulation).
Renal EndMT is a specific feature of endothelial
dysfunction and can contribute to renal microvas-
cular rarefaction 45, fibrosis and the progression of
CKD46. This process has been investigated in experi-
mental models of renal injury, including ischaemia–
reperfusion injury, unilateral ureteral obstruction
(UUO)46 and streptozotocin-​induced DKD42. Use of
endothelial-​lineage tracing demonstrated the presence
of αSMA-​positive myofibroblasts of endothelial ori-
gin in the interstitium of fibrotic kidneys from mice
with streptozotocin-​induced DKD42, suggesting that
EndMT contributes to renal fibrosis. A small number of
myofibroblasts of endothelial origin were also observed
in the afferent and efferent glomerular arterioles of
these mice, suggesting that EndMT may participate
in glomerulosclerosis.
As mentioned above, excessive TGFβ signalling
probably contributes to the activation of EndMT.
TGFβ-knockout mice are non-viable; however, mice with
heterozygous endothelium-specific deletion of the
TGFβ receptor are viable and display enhanced angio-
genic potential under basal conditions47. These mice are
also protected from tubulo-​interstitial fibrosis and renal
microvasculature rarefaction, with preserved renal blood
flow and low levels of tissue hypoxia after induction of

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Flow-​mediated dilation
Noninvasive, ultrasonography-​
based test of endothelial
function that measures
brachial arterial diameter in
response to a substantial
increase in arterial flow after
the release of brachial
constriction with a cuff. Flow-​
mediated dilation reflects the
ability of endothelial cells to
secrete nitric oxide (NO) and
induce arterial vasodilation in
response to shear stress.
Alterations in flow-​mediated
dilation represent an early
marker of endothelial
dysfunction and can be
reversed with endothelial
recovery.
folic-​acid-associated nephropathy or UUO47, implicating
a role for TGFβ signalling and EndMT in the regulation
of angiogenic and fibrotic responses to kidney injury.
In humans, EndMT contributes to renal damage
in sepsis-​induced AKI48,49. In kidney transplant recip-
ients, EndMT can result from ischaemia–reperfusion
injury and from antibody-​mediated rejection (ABMR).
Markers of EndMT are expressed in peritubular cap-
illaries of patients with ABMR50 and are predictive of
long-term graft dysfunction and proteinuria.
The circulating endothelial compartment
The circulating endothelial compartment consists of cir-
culating ECs (CECs), endothelial-​derived microvesicles
or microparticles (EMPs) and endothelial progenitor
cells (EPCs). The balance between endothelial injury,
as reflected by the presence of CECs and EMPs, and
the capacity for endothelial repair, as reflected by the
recruitment of functional EPCs, is crucial for maintain-
ing endothelial homeostasis51. Although it is currently
not possible to determine the organ of origin of CECs
and EMPs, these cells and cell-​derived packages are
of interest as potential biomarkers of acute or chronic
endothelial dysfunction in renal diseases (Table 1). CECs
are mature ECs that have been shed from the injured
vessel wall (for example, after myocardial infarction
or arterial angioplasty)52. EMPs are submicrometric
vesicles that are shed from stressed or damaged ECs53
upon remodelling of the plasma membrane, with exter-
nalization of phosphatidylserine53. Numbers of CECs
and EMPs, which are most commonly measured in the
blood by CD146-based immunomagnetic separation54
and flow cytometry53, are increased in diseases involv-
ing EC activation and/or injury52,53 and typically corre-
late with disease severity and clinical outcome52. Patients
with systemic lupus erythematosus (SLE) have increased
numbers of CECs, especially those with lupus nephri-
tis and renal TMA, reflecting microvascular injury55.
Numbers of CECs are also increased in patients on hae-
modialysis, especially those with active cardiovascular
disease4 in whom it predicts the occurrence of future
cardiovascular events56, suggesting that the number of
CECs reflects endothelial damage and is a biomarker
of cardiovascular complications. Levels of EMPs are also
elevated in patients with CKD57,58 and correlate with
clinical markers of vascular damage58.
Circulating EMPs and CECs are not only markers
of vascular damage but also mediate endothelial dys-
function59. Apoptotic and/or necrotic CECs can interact
with healthy endothelium and induce pro-​inflammatory
signals60. Both apoptotic and necrotic CECs are bound
and engulfed by healthy ECs, and incubation of cultured
ECs with apoptotic CECs results in increased expression
of chemokines and increased binding of leukocytes to
ECs60. In patients with systemic diseases, such as vasculitis
or SLE, EMPs can contribute to a pro-​inflammatory envi-
ronment by transporting inflammatory mediators from
the site of injury into the circulation61. In patients with
antineutrophil cytoplasmic antibody (ANCA)-associated
vasculitides (AAV), numbers of EMPs in plasma corre-
late with disease activity62. EMPs express procoagula-
tion factors, such as phosphatidylserine and TF, on their
membrane surface, thereby facilitating thrombus forma-
tion53. EMPs derived from patients with CKD have higher
levels of exposed phosphatidylserine than those obtained
from control individuals63, and exposure of cultured ECs
to uraemic toxins promotes the release of EMPs with
TF-​dependent procoagulant activity64. High numbers
of EMPs are also observed in the blood of patients with
antiphospholipid antibodies (aPLs)65, but only plasma
from patients with a history of thrombosis stimulates
cultured HUVECs to release procoagulant EMPs66, high-
lighting the fact that both the number and the properties
of EMPs are of interest as biomarkers in this setting.
EMP activation is probably closely linked to comple-
ment activation. The deposition of complement on cell
surfaces and the formation of the membrane attack com-
plex C5b−9 can induce the release of EMPs67. Moreover,
treatment of mice with ciclosporin induces activation
of the alternative pathway of complement within the
kidneys, leading to glomerular deposition of C3 (ref.68)
and the release of EMPs carrying C3 deposited on their
surface. These C3-carrying EMPs interact poorly with
the regulatory FH, resulting in further activation of the
alternative pathway68. This process might amplify intra-
renal endothelial damage and favour the development
of TMA. The involvement of EMPs in this amplifying
loop of endothelial injury might be of interest in the
context of haemolytic uraemic syndrome (HUS) and in
organ transplantation.
Endothelial lesions can also result from a defect in
repair capacity51. EPCs were first described in 1997
(ref.69) as a population of cells from the haematopoietic
lineage that incorporated into sites of angiogenesis. They
were subsequently shown to originate either from the
bone marrow or from tissue-​specific resident progenitor
cells16 and to promote angiogenesis and microvascular
endothelial repair through the production of angiocrine
factors such as VEGFA, FGF2 and angiopoietins16,51.
Studies in animal models of endothelial kidney injury,
such as experimental glomerulonephritis or CKD
resulting from 5/6 nephrectomy, indicate that endothe-
lial regeneration occurs predominantly via EPCs of
renal origin, with ECs of extrarenal origin contributing
0–10% of ECs following regeneration70–72. Intravenous
infusion of autologous cultured EPCs obtained from
bone marrow to mice following 5/6 nephrectomy atten-
uated damage to the remaining renal tissue, probably
through the release of paracrine factors such as VEGF73.
Homing of these EPCs to glomerular and peritubular
capillaries, as well as to liver and spleen, was observed
after 4 weeks73. In larger vessels, resident EPCs from the
vessel wall are most likely the main drivers of vascular
repair, as demonstrated by the minimal contribution of
bone-​marrow-derived EPCs to endothelial replacement
in a model of transplant atherosclerosis13,74.
Evaluating EC dysfunction and damage
Several approaches are used to evaluate endothelial func-
tion in the clinic (Table 1). These include functional tests
(mainly measurement of flow-​mediated dilation); evalu-
ation of markers indicative of a pro-​inflammatory or
prothrombotic endothelium phenotype; dysregulation
of NO metabolism or disruption of the glycocalyx4,75;
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Table 1 | Noninvasive approaches to evaluate endothelial function in humans

Type of exploration Markers Consequence of endothelial dysfunction
Functional tests
FMD NA Reduced
FMD with administration of an NO NA Unaltered
donor
Forearm blood flow NA Decreased
Cutaneous circulation NA Decreased
Plasmatic markers
Adhesion molecules sVCAM1 Increased
sICAM1 Increased
Soluble E-​selectin Increased
Pro-​inflammatory markers Inflammatory cytokines Increased
Oxidized LDL Increased
Endocan Increased
Thrombotic mediators Thrombomodulin Increased
von Willebrand factor Increased
Tissue factor Increased
Markers of NO dysregulation NO metabolites Modified
Asymmetric dimethylarginine Increased
Symmetric dimethylarginine Increased
Glycocalyx disruption Heparan sulfate Increased
Circulating endothelial compartment
Markers of activation or lesion Circulating endothelial cells Increased
Endothelial microparticles Increased
Markers of repair capacity Endothelial progenitor cells Decreased numbers in the blood and
altered functions in vitro
Circulating angiogenic cells Decreased numbers in the blood and
altered functions in vitro
FMD, flow-​mediated vasodilation; NA , not applicable; NO, nitric oxide; sICAM1, soluble intercellular adhesion molecule; sVCAM1,
soluble vascular cell adhesion protein 1.

Tubulo-​reticular inclusions
Cytoplasmic clusters of
tubule-like structures arising
from the membrane of the
endoplasmic reticulum,
thought to result from
activation of type I interferons
in endothelial cells and
lymphocytes.
Intravital multiphoton
microscopy
(IVM). A form of microscopy
that enables the observation of
biological processes in living
animals (owing to its low
energy and phototoxicity,
which allow the prolonged
exposure of tissues) with high
resolution owing to the deep
penetration of tissues by
photons.
and evaluation of circulating endothelial compartments,
indicative of an imbalance between endothelial damage
and repair76. All of the available markers that are used
to evaluate endothelial function in humans reflect sys-
temic endothelial dysfunction rather than organ-​specific
endothelial dysfunction.
Histological examination of renal biopsy samples is
required to assess endothelial damage specifically in the
kidney and typically demonstrates capillary or small-​
vessel thrombosis with fibrin deposition. Renal TMA
is also evidenced by the finding of swollen and turgid
ECs, obstructing blood flow in the glomerular capil-
lary or small arteries and inducing their detachment
from the GBM or vascular wall, with the formation of
‘double contours’ (whereby the GBM seems duplicated
on light microscopy, reflecting the synthesis of GBM-​
like material, which is possibly initiated to fill a void
between non-​adherent GECs and the GBM) and clear
subendothelial spaces (that is, empty spaces between the
GECs and GBM on light microscopy, reflecting a par-
tial detachment of GECs from the GBM)77. Glomerular
fibrinoid necrosis is also encountered in biopsy samples
from patients with renal vasculitides or lupus nephritis,
with endocapillary and/or extracapillary proliferation
of glomerular cells, with or without immune complex
deposition. Peritubular capillary rarefaction, vascular
fibrosis and interstitial fibrosis (resulting in part from
EndMT) can also be seen in renal biopsy samples.
Ultrastructurally, endothelial damage can be observed
in glomeruli, peritubular capillaries and the vasa
recta, evidenced by reduced fenestrations, increased
endothelial vacuoles, thickened basement membranes
and fibrin-​like intraluminal deposits78,79. In virus-​
associated nephropathies and lupus nephritis, typical
tubuloreticular inclusions can be observed in GECs80.
Intravital multiphoton microscopy (IVM) has also been
used81–83 to study renal blood flow, leukocyte rolling and
recruitment, cell death and cell shedding and vascular
permeability in the glomerular and peritubular vascula-
ture of the living kidney in rodents. IVM enables inves-
tigation of endothelial and microvascular alterations
in the living animal, showing, for example, the reduced
peritubular capillary blood flow in sepsis-​induced AKI
or after ischaemia–reperfusion injury or increased vas-
cular permeability of ischaemic areas of renal tissue.
At the cellular level, IVM has been used to observe how

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Reviews
alterations in the endothelial glycocalyx might favour
albuminuria and increase vascular permeability in
living rats84.
Future strategies to study EC populations
Our understanding of EC diversity among different vas-
cular beds is limited. Microarray studies of cultured ECs
have demonstrated differences in the gene expression
profiles of ECs isolated from different vascular beds8.
However, the use of classic 2D culture conditions inevi-
tably modifies the behaviour and gene expression profile
of ECs, including renal ECs, limiting the utility of this
system. The construction of 3D renal vessels to enable
exposure of ECs to physiological mechanical stresses and
surrounding cells, such as pericytes and podocytes, is a
key goal for the study of the endothelium in renal dis-
eases15. Although progress has been made in the develop-
ment of vascular scaffolds85–87, further studies are needed
to more closely mimic the in vivo environment88. Of note,
engraftment of renal organoids below the kidney capsule
of living rats89 resulted in the formation of vascularized
glomeruli with differentiated capillary walls and basic
physiologic functions89, providing a model with which
to study renal EC function. The development of ex vivo
perfused kidneys-​on-a-​chip that recapitulate compo-
nents of the glomerular barrier, such as GECs, the GBM
and podocytes, and enable glomerular fluid shear stress
conditions would also advance studies of EC function.
Diseases with endothelial damage
As mentioned earlier, the renal endothelium can be
activated or injured by a variety of factors, including
toxins, hyperglycaemia, complement activation frag-
ments, autoantibodies or alloantibodies, immune cells
and cytokines. Defects in mechanisms of endothelium
protection (for example, through dysregulation of com-
plement regulatory factors such as FH as occurs in aHUS
or through an imbalance between pro-​angiogenic and
anti-​angiogenic factors as occurs in pre-​eclampsia) can
also induce glomerular endothelial damage. Injury of
peritubular capillary ECs in settings such as ischaemia–
reperfusion can also induce AKI and vascular rarefac-
tion, which can lead to renal fibrosis and subsequent
CKD in part owing to EndMT. Some of these processes
are specific for particular kidney diseases (such as aHUS)
whereas others contribute more broadly to the mecha-
nisms of renal dysfunction seen in common complex
diseases, such as hypertension-​attributed nephropathy
and DKD. Irrespective of its aetiology, CKD is associ-
ated with an accumulation of uraemic toxins, which
contribute to systemic endothelial dysfunction and car-
diovascular disease. The below discussion describes the
mechanisms of renal endothelial damage in renal dis-
eases and explains how CKD can, in turn, exacerbate
systemic endothelial dysfunction.
Direct endothelial injury or activation
The most emblematic renal diseases resulting from
direct endothelial injury are typical HUS, lupus nephri-
tis and antiphospholipid syndrome (APS) nephrop-
athy, ABMR in renal transplant recipients, AAV and
sepsis-induced AKI.
Typical Shiga toxin-​associated haemolytic uraemic
syndrome. TMA is characterized by vascular damage,
with the formation of fibrin and platelet thrombi90.
TMA translates clinically into a triad of peripheral
thrombocytopenia, mechanical haemolytic anaemia
and acute or chronic organ damage predominantly in
kidney and brain. The most common type of kidney-​
predominant TMA is typical HUS caused by an infec-
tion with Stx-​secreting bacteria, including some strains
of Escherichia coli (Fig. 4a). Animal studies of typical
HUS have been hampered by the finding that conven-
tional mice infected with enterohemorrhagic E. coli do
not develop key features of the disease, including renal
lesions. One study showed that induction of the full
renal TMA phenotype required co-​injection of Stx and
LPS into mice91. Another study engineered Citrobacter
rodentium to secrete Stx, which reproduced the human
phenotype in mice92. Mechanistic studies have shown
that Stx causes glomerular injury through binding of
its B-​subunit of globotriaosylceramide (GB3)93. This
receptor is expressed at particularly high levels by
GECs (~50-fold higher than in HUVECs), explaining
the susceptibility of the glomerular endothelium to this
toxin94. Once bound to its receptor, the Stx undergoes
endocytosis and inhibits protein synthesis within the
EC by cleaving an adenine base from the 28S ribosomal
RNA (rRNA) of the 60S ribosomal subunit95. Moreover,
Stx modulates microvascular EC signalling pathways
and ribosomal activity, inducing endothelial permea-
bility and cytotoxic changes in ECs by stimulating the
CXCR4–CXCR7–SDF1 pathway96. Following infection,
Stx is also transported in the bloodstream bound to
platelets, neutrophils, monocytes and red blood cells;
these cells, together with ECs, release Stx-​containing
complement-​coated microvesicles that enable GB3-
independent endocytosis of Stx into GECs and peritu-
bular capillary ECs and through basement membranes
into podocytes and tubular cells67. In GECs, Stx induces
activation of complement, mobilization of the Weibel–
Palade bodies and cell surface expression of the adhe-
sion molecule P-​selectin97, which provides a platform
for assembly of the alternative pathway C3 convertase
(C3bBb) and further cleavage of C3 into C3a and C3b97.
The release of C3a further potentiates the expression of
P-​selectin and stimulates tPA-​dependent thrombomod-
ulin shedding and loss of thromboresistance, leading to
a prothrombotic state and thrombus formation. Stx also
binds to the ultra-​large multimers of the glycoprotein
vWF, reducing their cleavage by ADAMTS13 and fur-
ther stimulating thrombosis98. Acceleration of the TMA
process through these pathways eventually leads to AKI.
Lupus nephritis and APS nephropathy. The pathogene-
sis of SLE is multifactorial, involving a loss of tolerance
to self-​antigens and the production of a wide range of
autoantibodies, with formation of immune complexes
and culminating in the damage of tissues, such as the
kidney in lupus nephritis99. Autoantibodies that react
specifically with the EC surface (anti-​EC antibodies
(AECAs)) have been found in patients with SLE100,101,
in whom their presence is associated with markers of
endothelial damage102, and with the occurrence and
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pathological activity of lupus nephritis101,103,104. The
specific antigenic targets of these AECAs are yet to be
identified; however, their binding to native or modified
endothelial antigens might promote a pro-​inflammatory
and procoagulant EC phenotype, stimulating oxidative
stress and EC apoptosis100 and promoting the develop-
ment of lupus nephritis, renal vasculopathy and accel-
erated systemic atherosclerosis105 (Fig. 4b). Apoptosis
of cultured HUVECs is increased following exposure
to purified immunoglobulin G (IgG) from patients
with lupus nephritis101. Moreover, anti-​complement C3
autoantibodies from patients with lupus nephritis stim-
ulated complement activation in cultured HUVECs,
contributing indirectly to endothelial injury106.
SLE is also associated with an imbalance between
endothelial lesion and repair. Patients with SLE —
particularly patients with lupus nephritis and renal TMA
— have increased numbers of CECs55 and decreased
numbers and functions of EPCs and circulating angio-
genic cells (CACs)107, which are susceptible to apoptosis,
synthesize few pro-​angiogenic molecules, incorporate
poorly into vascular structures and differentiate poorly
into mature ECs107. The functional defects in EPCs
and CACs in patients with SLE are mediated by type I
interferons107, which repress the pro-​angiogenic factors
IL-1β and VEGF and activate the inflammasome, and
the pro-inflammatory cytokine IL-18 (ref.108).
In association with SLE or in primary APS, aPLs
can also contribute to renal endothelial damage. APS
is defined as the coexistence of thrombotic or obstetric
events in the presence of persisting specific aPLs. The
renal manifestations of APS are heterogeneous109, com-
prising both large renal vessel manifestations (throm-
bosis, stenosis or infarction) and microvascular lesions
(TMA or fibrous intimal hyperplasia), which can induce
progressive or acute renal dysfunction — a condition
known as APS nephropathy110. The diversity of renal
manifestations implies that aPLs exert various, poten-
tially overlapping effects on the vasculature, including
promotion of coagulation or impairment of fibrinolysis
by binding proteins of the coagulation pathway; direct
cytotoxic effects on EC membrane phospholipids; and
the recruitment of pro-​inflammatory, pro-​oxidative
and prothrombotic factors through increased inter-
actions of the circulating β2-glycoprotein 1b (β2GPI)
with different receptors, such as Toll-​like receptor 2
(TLR2), TLR4, annexin A2 and the α-​chain of platelet
glycoprotein Ib (GPIbα) on ECs or blood cells111 (Fig. 4b).
Most studies of the endothelial effects of aPLs have
used either monoclonal murine antibodies, human
affinity-​purified antibodies (mostly anti-​glycoprotein 1
(GP1) antibodies) or patient-​derived IgG antibodies112.
Sequence homology between human and mouse
GP1 enables use of patient-​derived aPLs in murine
models113,114. Injection of human aPLs into mice does
not induce direct thrombosis in the absence of under-
lying endothelial dysfunction and/or precipitating
thrombotic events, but it does induce a dose-​dependent
increase in thrombus size once thrombosis is initiated,
which is indicative of a ‘two-​hit’ concept115,116. Thrombus
formation is decreased in mice deficient in complement
components C3 (ref.117) or C5 (ref.118), and glomerular
Nature Reviews | Nephrology
Reviews
TMA lesions are reduced in mice lacking the C5a
receptor (C5aR)119, highlighting the role of complement
activation in APS and APS nephropathy. Moreover, the
monoclonal anti-​C5 antibody eculizumab has been
successfully used in patients with post-​transplantation
aPL-​induced TMA or refractory catastrophic APS110,120.
Activation of the proliferation and survival AKT–mTOR
pathway by aPLs is also involved in APS nephropa-
thy, especially in the chronic vasculopathy character-
ized by EC proliferation and intimal hyperplasia110,115.
Heterogeneous vascular lesions involving endothelial
activation or damage are therefore involved in both the
systemic and renal manifestations of SLE and APS.
Antibody-​mediated rejection in kidney transplantation.
The renal endothelium is particularly prone to injury in
kidney transplant recipients as ECs from the transplant,
which bear the HLA antigens of the donor, are directly
exposed to the recipient’s immune system. The main
cause of graft loss in kidney transplant recipients is
ABMR121, defined by the presence of donor-​specific
antibodies (DSAs) directed against donor HLA alloan-
tigens, microvascular inflammation (glomerulitis and
peritubular capillaritis) and variable C4d deposition
in peritubular capillaries121. Of kidney transplant recipi-
ents, 24% develop de novo DSAs within 10 years of trans-
plantation, increasing the risk of impaired long-​term
graft survival122,123, whereas preformed DSAs are associ-
ated with a high risk of ABMR and early graft failure124.
DSAs cause endothelial damage through direct activation
of ECs, through activation of the complement system and
through recruitment of inflammatory cells such as natu-
ral killer (NK) cells via their crystallizable fragment (Fc)
receptors, inducing antibody-​dependent cell cytotoxicity
(ADCC)125 (Fig. 4c). In vitro studies show that anti-​HLA
antibodies bind to ECs within the transplant, leading to
activation of the mTOR pathway, EC proliferation and
intimal hyperplasia with subsequent ischaemia126,127.
The Fc regions of IgM and IgG DSAs bind C1q, acti-
vating the classical complement pathway and resulting
in endothelial injury. Moreover, ischaemia–reperfusion
injury that can occur with kidney transplantation pro-
motes local activation of complement via lectin and alter-
native pathways, independent of DSAs128,129. Deposition
of sublytic levels of C5b-9 causes EC activation, with
recruitment and activation of T cells contributing to
transplant vasculopathy 130, although complement-​
induced lysis of ECs is hampered by the high consti-
tutive expression of protective complement regulators
such as membrane cofactor protein (MCP; also known as
CD46), CD55 (also known as decay accelerating factor,
DAF) or CD59. Inflammation triggered by the anaphyl-
atoxins C3a and C5a can also contribute to ABMR131.
In allograft biopsy samples, the presence of glomerulitis
and peritubular capillaritis indicates ongoing interaction
of antibodies with the endothelium130.
Complement-​independent pathways, in particu-
lar the NK-​cell-mediated ADCC, are also involved in
ABMR132. NK cells interact with antibodies via their
FcγR and can induce rapid apoptosis or lysis of target
ECs through the release of perforin and granzyme and
the production of IFNγ and other pro-​inflammatory
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a Typical haemolytic uraemic syndrome C3a

Platelet
aggregation
Stx-containing Soluble TM
microvesicles C3b
TM
Stx C3
convertase Factor B
vWF
tPA

GB3 P-selectin C3aR

Endocytic
vesicle GEC
• Inhibition of
protein synthesis Weibel–Palade body
• Cytotoxic changes

b Lupus nephritis APS nephropathy

Podocyte EC Direct Complement
β2GPI activation
membrane
toxicity

Cellular
Stabilization receptor

EC activation
EC activation
Recruitment of and proliferation
inflammatory cells
Immune
complexes Anti-C3b Antiphospholipid
EPC and CAC
dysfunction antibody antibody
Subepithelial Subendothelial Endothelial Anti-endothelial
deposits deposits antigen antibody

c Antibody-mediated rejection
Classical complement C3a
pathway activation C5a
NK
cell
C1q C3 convertase Thrombus formation
DSA
Donor HLA C4d

NK cytotoxic Membrane attack

enzymes complex (C5b-9)
ADCC Complement-dependent lesion

d ANCA-associated vasculitis
MPO or PR3

Priming ANCA
antibody ROS

Neutrophil
Neutrophil
serine Leukocyte
proteases Integrin recruitment
Complement
alternative
NET Adhesion
pathway activation
molecule

NETosis
EC activation
EC lysis

Tissue damage and Neutrophil

fibrinoid necrosis diapedesis

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◀ Fig. 4 | renal diseases associated with endothelial injury in kidney diseases.
a | In typical haemolytic uraemic syndrome, Shiga toxin (Stx) enters glomerular endothelial
cells (GECs) by binding the B-​subunit of globotriaosylceramide (GB3), which induces
Stx endocytosis, leading to inhibition of protein synthesis and cytotoxic damage. The
release of Stx-​containing complement-​coated microvesicles enables entry of Stx into
other cell populations. Stx induces complement activation, mobilization of Weibel–Palade
bodies, P-​selectin expression and secretion of von Willebrand factor (vWF) multimers,
stimulating platelet aggregation; P-​selectin stimulates C3 convertase activity and tissue
plasminogen activator (tPA)-dependent shedding of thrombomodulin to generate
soluble thrombomodulin (TM), further promoting thrombosis. Released C3a will bind to
its receptor, C3aR, and will further stimulate the GECs to mobilize the Weibel–Palade
bodies, perpetuating the process. b | Lupus nephritis is characterized by the glomerular
deposition of circulating immune complexes. GECs can also be activated by
anti-endothelial antibodies; moreover, anti-​complement C3 autoantibodies stimulate
complement activation and recruitment of inflammatory cells, exacerbating glomerular
damage. Lupus nephritis is also characterized by an imbalance between endothelial
damage and repair, with decreased numbers and functions of circulating angiogenic
cells (CACs) and endothelial progenitor cells (EPCs). Antiphospholipid antibodies
associated with systemic lupus erythematosus or in primary antiphospholipid syndrome
(APS) have direct cytotoxic effects on endothelial cell (EC) membrane phospholipids; they
can bind and stabilize β2-glycoprotein 1b (β2GPI) and induce complement activation;
their interaction with cellular receptors (for example, Toll-​like receptor 2 (TLR2), TLR4,
annexin A2 and α-​chain of platelet glycoprotein Ib (GPIbα)) can induce EC activation and
proliferation. c | In kidney transplant recipients with antibody-​mediated rejection,
donor-specific antibodies (DSAs) directed against donor HLAs can directly activate ECs.
DSAs can also trigger complement activation by binding C1q, activating the classical
complement pathway and inducing thrombus formation. Inflammatory cells, such as
natural killer (NK) cells, can be recruited via their crystallizable fragment (Fc) receptor,
leading to the generation of NK cytotoxic enzymes and antibody-​dependent cell
cytotoxicity (ADCC). d | In antineutrophil cytoplasmic antibody (ANCA)-associated
vasculitis, activation of neutrophils by ANCAs follows neutrophil cell priming and the
migration of proteinase 3 (PR3) or myeloperoxidase (MPO) to the cell surface. Neutrophil
activation induces the formation of neutrophil extracellular traps (NETs), and stimulates
oxidative burst, with the production of reactive oxygen species (ROS), which together
cause EC damage and fibrinoid necrosis. Binding of neutrophils to adhesion molecules
expressed by activated ECs promotes neutrophil recruitment and diapedesis,
contributing further to tissue damage.
chemokines that contribute to recruitment of cells of
the adaptive immune system and EC activation133. High
levels of transcripts corresponding to NK cells were
found in kidney and heart allograft biopsy samples from
patients with DSAs and ABMR134,135, and the contribu-
tion of NK cells to chronic allograft vasculopathy was
confirmed in a murine model of heart transplantation132.
In ex vivo samples from kidney transplant recipients,
ADCC reactivity of NK cells against allogeneic target
cells was predictive of a decline in transplant function136.
Moreover, in vitro cytotoxicity of patient-​derived DSAs
was predictive of ABMR136.
Oxidative burst In addition to DSAs, non-​HLA preformed autoan-
Also known as respiratory tibodies directed against cryptic epitopes exposed after
burst, oxidative burst is the tissue damage (such as ischaemia–reperfusion injury,
rapid release of reactive
oxygen species (ROS) from
vascular injury and/or allograft rejection) might par-
phagocytes (such as ticipate in renal transplant dysfunction137. For example,
neutrophils or macrophages) to LG3, a fragment of the proteoglycan perlecan normally
degrade internalized particles present in the vasculature, is a neoantigen that is released
or pathogens within the
by injured or apoptotic ECs. Patients with early vascular
phagosome. Primed
neutrophils can also rejection have high levels of anti-​LG3 antibodies, and the
degranulate and undergo an pre-​existence of anti-​LG3 antibodies in patients awaiting
oxidative burst in the absence kidney transplantation is associated with an increased
of pathogens, as in risk of vascular rejection137.
antineutrophil cytoplasmic
antibody (ANCA)-associated
Finally, ECs from the transplant not only are a passive
vasculitis, leading to target of alloimmunization but also participate in this
surrounding tissue damage. process138. Upon exposure to hypoxia in the context of
Nature Reviews | Nephrology
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ischaemia–reperfusion injury, ECs increase their expres-
sion of HLA-​DR, which facilitates the presentation of
antigens to T cells and is itself an important target for
allogeneic T cells139,140. Under inflammatory conditions,
ECs of the graft also modify the local allogeneic response,
promoting either an IL-6-dependent T helper 17 (TH17)
cell pro-​inflammatory response or a contact-​CD54-
dependent regulatory T cell response, depending on the
cytokine environment, thus contributing to or regulating
the rejection process141.
ANCA-​ a ssociated vasculitides. Renal involvement
in AAV — a group of small-​vessel vasculitides with a
broad spectrum of clinical manifestations — is char-
acterized by a necrotizing crescentic glomerulone-
phritis with absent or very few immune deposits142.
ANCA autoantibodies and neutrophils contribute to
the injury of ECs and small-​vessel walls by stimulating
oxidative burst, the release of proteolytic enzymes and
the formation of neutrophil extracellular traps (NETs)143
(Fig. 4d). Activation of neutrophils by ANCAs requires
cell priming, which is mediated by pro-​inflammatory
cytokines, and induces migration of the enzymes pro-
teinase 3 (PR3) or myeloperoxidase (MPO), which are
contained in neutrophil azurophilic granules142, to the
neutrophil membrane144,145. Binding of ANCAs to MPO
or PR3 on the neutrophil membrane then promotes
activation of neutrophil β2 integrin, which mediates
adhesion of leukocytes to the endothelium and favours
microvascular permeability and neutrophil diapedesis.
In vitro, ANCA-​stimulated neutrophils activate ECs in
a process mediated by NF-κB, which favours neutrophil
adhesion. Use of targeted small interfering RNA (siRNA)
to block NF-​κB specifically in the endothelium attenu-
ated the development of crescentic glomerulonephritis
in a mouse model of AAV, demonstrating an important
role for EC in the induction of AAV-​associated glo-
merulonephritis146. ANCAs also induce the release of
enzymes contained in neutrophil toxic granules, such as
neutrophil serine proteases147, which induce EC injury
and small-​vessel fibrinoid necrosis148. In addition, neu-
trophil activation induces the release of ROS, mediated
by NADPH oxidase 1 (NOX2). Surprisingly, one study
showed that transplantation with NOX2-deficient bone
marrow induced a more severe phenotype in a mouse
model of AAV than transplantation with wild-​type bone
marrow149. The more severe phenotype was associated
with increased renal concentrations of IL-1β, suggest-
ing that NOX2 interferes with inflammasome-​induced
IL-1β processing and ANCA-​induced inflammation149.
Activated neutrophils also exert pathogenic effects
through the excessive generation of NETs143. ANCA-​
induced NETs containing MPO and PR3 have been
found in the kidneys of patients with AAV-​induced glo-
merulonephritis and might induce EC death through
histone-dependent cytotoxicity — a mechanism that is
thought to trigger adaptive immunity by exposing extra-
cellular autoantigens to antigen-​presenting cells146,150. The
alternative pathway of complement also contributes to
AAV pathogenesis. In a mouse model of anti-​MPO vas-
culitis, deficiency of C5 or factor B protected mice from
disease151. Activation of complement and the generation
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Neutrophil extracellular
traps
(NETs). NETs are composed of
DNA in association with
histones and granular proteins
such as neutrophil elastase and
myeloperoxidase (MPO). They
are released by neutrophils in a
process called NETosis, which
allows pathogens to be
captured in a bactericidal net
and destroyed. NETs can also
cause tissue damage, such as
in ANCA-​associated vasculitis,
or increase the exposure of
autoantigens to the immune
system, such as in lupus.
Cell priming
Cell priming of neutrophils is
the transition from a steady
state with limited antimicrobial
activity to an activated state,
allowing the rapid enhancement
of phagocytic activity and
oxidative burst upon a second
stimulation. Neutrophils can be
primed by microbial products,
chemo-​attractants and
inflammatory cytokines.
Azurophilic granules
Azurophilic granules of
neutrophils are
intracytoplasmic vesicles
containing peptides with
antimicrobial activity, such as
MPO, proteinase 3 (PR3), α-​
defensins, elastase, cathepsin G
and bactericidal/permeability
increasing protein (BPI).
Histone-​dependent
cytotoxicity
Pro-​inflammatory and
cytotoxic signalling induced by
extracellular histones. Histones
and DNA normally reside in
the nucleus and form
nucleosomes. Extracellular
histones are damage-​
associated molecular patterns
(DAMPs), which elicit pro-​
inflammatory signalling
through Toll-​like receptors
(TLRs) and promote cell death.
Opsonization
Tagging of a pathogen, a cell or
an apoptotic body, with
opsonins, which increases their
interaction with phagocytes
and natural killer (NK) cells.
These opsonins can be
antibodies (immunoglobulin G
(IgG) or IgE) or complement
fractions (C3b or C4b).
of C5a might result from interaction with infectious trig-
gers such as bacterial infections, neutrophil membranes,
plasma circulating microparticles from different cell types
or NETs, inducing a pro-​inflammatory amplification
loop involving NETs and the complement cascade152.
Sepsis-​induced acute kidney injury. Sepsis is caused by
an overwhelming host response to infection and results
in life-​threatening dysfunction of organs, including the
kidneys. AKI develops in approximately 50% of patients
with sepsis who are admitted to the intensive care unit
(ICU) and is associated with 30–50% mortality in this
setting153. Systemic hypotension and ischaemia–reper-
fusion injury are not the only culprits in sepsis-​induced
AKI, with studies over the past decade highlighting a
central role of endothelial dysfunction153. ECs are both
targets and facilitators of kidney damage in sepsis1. ECs
are exposed to bacterial components (such as LPS), pro-​
inflammatory cytokines (including TNF and IL-1) and
immune cells, promoting their activation or apopto-
sis. Together with pericytes, ECs also contribute to the
increased renal vascular resistance and reduced renal
blood flow typical of sepsis-​induced AKI by promot-
ing vasoconstriction and tissue oedema as a result of
fluid leakage and by facilitating leukocyte adhesion and
microthrombus formation1. Multiple factors (includ-
ing increased generation of thrombin resulting from
the heightened exposure of TF as a consequence of EC
apoptosis, dysfunction of antithrombin and the throm-
bomodulin–protein C anticoagulant system observed in
sepsis and impaired fibrinolysis induced by increased
PAI1 expression) result in the endothelial dysfunction
that contributes to coagulopathy in sepsis153. Levels
of soluble thrombomodulin, which reflect the extent of
thrombomodulin detachment from ECs, independently
predicted development of AKI in a large cohort of
patients with sepsis admitted to the ICU153.
Defective endothelium protection
The renal endothelium is vulnerable, and in addition
to direct injury, it can be damaged through the loss of
protective factors, such as regulators of the complement
alternative pathway as occurs in aHUS, through an
imbalance between pro-​angiogenic and anti-​angiogenic
factors as occurs in pre-​eclampsia or with use of VEGF
blockade therapies in oncology.
Atypical haemolytic uraemic syndrome. Complement
is a component of the innate immune defence system
and is critical for protecting the host against path-
ogens154. The alternative pathway of complement is
constitutively active at low levels as a sentinel against
pathogens; however, owing to its powerful lytic capac-
ity, dysregulation or overactivation of this pathway can
trigger host tissue damage155. A healthy endothelium is
protected against complement activity by the presence
of multiple regulatory molecules, such as FH, factor I,
MCP, CD55 and CD59. aHUS is associated with dys-
regulation of the alternative pathway, resulting in mas-
sive EC opsonization, release of the anaphylatoxins C3a
and C5a and formation of sublytic membrane attack
complex C5b-9 on GECs, culminating in endothelial
damage2,90,154. Genetic abnormalities or the presence
of autoantibodies that affect the quantity or function of
FH are the most common causes of alternative pathway
dysregulation156–158, followed by mutations in MCP159
and factor I160. Overactivation of the alternative path-
way due to mutations in the C3 convertase components
C3 (refs161,162) and factor B163,164 are also associated with
aHUS (Fig. 5a). Genetic abnormalities independent of
complement have also been described, notably in dia-
cylglycerol kinase-​ε (DGKE)165 — a gene involved in
the protein kinase C (PKC) signalling pathway — or in the
gene that encodes thrombomodulin166. DGKE deficiency
induces a permanent procoagulant phenotype in ECs167,
whereas decreased expression of thrombomodulin pro-
motes thrombi formation and inhibits the inactivation
of C3a, C5a and C3b166.
Despite differences in the complement system
between mice and humans, mouse models of aHUS
have been developed168. Mice with complete deficiency
in FH develop spontaneous C3 glomerulopathy but not
aHUS169 owing to a lack of complement haemolytic acti­
vity as a result of increased C3 and C5 consumption170.
However, mice with hepatocyte-​s pecific FH defi-
ciency, which exhibit plasma FH levels of 20% of nor-
mal, develop severe renal TMA, although this effect is
not spontaneous and requires concurrent induction of
serum nephrotoxic nephritis171. Transgenic mice with
a truncated FH that is functionally equivalent to that
of FH mutants in humans with aHUS have also been
developed172. The presence of a point mutation in FH
that impairs interaction of FH with host cells without
reducing plasma FH levels also induces renal TMA and
systemic thrombosis in mice173. These models are val-
uable for the study of aHUS disease mechanisms and
therapeutic strategies. Indeed, blockade of C5 (ref.171)
or properdin, which stabilizes the alternative pathway
C3 convertase174, showed therapeutic efficacy in animal
models of aHUS. The reason why the kidney micro-
vasculature is particularly susceptible to complement-​
mediated damage is unclear, but a new study showing
that renin can cleave C3 suggests a possible explanation
for this renal tropism175. Of note, the penetrance of aHUS
among carriers of mutations in any of the susceptibi­
lity genes is incomplete176, and a triggering event (such
as infection or pregnancy) and additional genetic pre-
disposing factors (such as risk haplotypes in FH and
MCP) are needed to induce the disease32,177. Treatment
of cultured ECs that had been activated with cytokines,
haem or ADP with serum from patients with aHUS
induced greater complement deposition than treatment
with serum from healthy donors32,39,178,179. Haemolysis,
which accompanies early disease stages, can exacerbate
aHUS owing to the complement-​activating capacity of
released haem38–40.
Pre-​eclampsia and anti-​VEGF therapy. VEGF, con-
trolling the proliferation, differentiation, migration and
permeability of ECs, is the most potent regulator of angi-
ogenesis180. Several isoforms of VEGF (predominantly
VEGF121, VEGF165 and VEGF189) bind to three trans-
membrane VEGF receptors (VEGFRs): VEGFR1 (also
known as FLT1), VEGFR2 (also known as KDR) and
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 Reviews

a Atypical haemolytic uraemic syndrome and organization of the actin cytoskeleton. Podocyte-​
C3a Spontaneous Alternative specific deletion of sFLT1 leads to massive proteinuria
FI hydrolysis pathway
C3b or trigger regulators in mice, highlighting the role of sFLT1 in preserving the
integrity of the glomerular filtration barrier184. sFLT1
Alternative
pathway auto- released by podocytes might also contribute to the regula-
amplification tion of VEGF function by binding VEGF and inducing its
FH loop
antibody C5a uptake by podocytes184. Furthermore, sFLT1 acts on peri­
Factor B
cytes to modulate the differentiation and proliferation
FH of ECs; selective deletion of FLT1 in vascular pericytes
MCP peripheral to the kidney leads to increased vascularization
C3 convertase and excessive neoangiogenesis within the kidney184.
GEC C5b-9 The importance of this balance between VEGF and
sFLT1 within glomeruli is highlighted by two conditions:
pre-​eclampsia and following the use of anti-​VEGF ther-
b VEGF inhibition apies (Fig. 5b). Pre-​eclampsia is a complication of preg-
nancy characterized by hypertension and proteinuria
Podocyte
occurring after 20 weeks of gestation. It is associated
with a dramatic increase in the release of sFLT1 and sol-
uble endoglin by placental cells, leading to an imbalance
between anti-​angiogenic factors (sFLT1 and endoglin)
Placental and pro-​angiogenic factors (VEGF and placental growth
sFLT1
VEGF factor)185,186. The relative deficiency in VEGF damages
the glomerular filtration barrier, leading to effacement
Anti-VEGF of podocyte foot processes, swelling and detachment of
VEGFR2 antibody ECs from the GBM and ultimately the development
of TMA. Similar renal lesions can be seen in patients who
receive anti-​VEGF drugs for the treatment of cancers183.
Normal crosstalk between Pre-eclampsia Anti-VEGF Up to one-​third of patients receiving anti-​VEGF therapy
EC and podocytes (increased sFLT1) therapy develop hypertension, 20–60% of patients receiving anti-​
Fig. 5 | renal damage associated with defective endothelial protection. a | Atypical VEGF therapy develop proteinuria and a few patients will
haemolytic uraemic syndrome is characterized by deficient regulation of the develop TMA187. Importantly, podocyte-​specific deple-
complement alternate pathway due to quantitative or functional deficiency or tion of VEGF induces the same clinical and pathological
autoimmune inhibition of factor H (FH), factor I (FI) or membrane cofactor protein (MCP), phenotype in mice183. Improved understanding of the bal-
leading to uncontrolled alternative pathway activation with formation of the membrane ance between VEGF and anti-​angiogenic factors might
attack complex C5b-9 and endothelial cell (EC) injury. b | Vascular endothelial growth facilitate the development of approaches to manage severe
factor (VEGF) synthesized by podocytes exerts protective effects on glomerular ECs diseases, as exemplified by the use of apheresis to remove
(GECs) by binding VEGF receptors (VEGFRs). VEGF availability is compromised in circulating sFLT1 in women with pre-​eclampsia188, and
pre-eclampsia, in which increased placental secretion of the soluble VEGFR sFLT1 acts as shed new light on the pathophysiology of other vascular
a decoy to reduce the availability of VEGF to signal through VEGFR2 on GECs. Similarly,
renal diseases, such as AAV189.
use of anti-​VEGF therapies also reduces levels of available VEGF, leading to podocyte
and endothelial damage.
Diabetic kidney disease. DKD is the leading cause of
end-​stage renal disease (ESRD) in high-​income coun-
neuropilin 1 (ref.180). The pro-​angiogenic effect of VEGF tries190,191. In patients with type 1 diabetes mellitus
is mainly mediated through VEGFR2; mice lacking this (T1DM) or type 2 diabetes mellitus (T2DM), DKD is
receptor die in utero owing to an absence of endothelial characterized by glomerular hypertrophy and hyper-
and haematopoietic cells181. Two major splice variants filtration, typically associated with albuminuria, and
of VEGFR1 exist: the full-​length transmembrane recep- progressive CKD. Pathological examination shows
tor and a secreted, soluble protein called soluble FLT1 GBM thickening, mesangial expansion and podocyte
(sFLT1), which lacks the transmembrane and intra- detachment; the development of glomerulosclerosis192,
cellular domains. Full-​length VEGFR1 and sFLT1 are tubular atrophy and interstitial fibrosis 193; and EC
decoy receptors and reduce the availability of VEGF for loss and capillary rarefaction in glomeruli and the
signalling through VEGFR2 (ref.180); mice deficient in tubulo-interstitium194 (Fig. 6a).
VEGFR1 die owing to excess EC growth182. Hyperglycaemia induces apoptosis of GECs, leading
The renal GBM provides one of the best examples of to a progressive reduction in the surface of fenestrated
the importance of VEGF in EC and pericyte physiology. endothelia195–197. This process is triggered by local acti-
Podocytes are the main source of VEGF in the glomer- vation of the RAAS194,198, ROS-​induced activation of
ulus183. Secreted VEGF is delivered to GECs across the caspase 3 (ref.199) and reduced activation of protein C
GBM, against the flow of urine filtrate, through ill-​defined by thrombomodulin200. Under physiological conditions,
mechanisms that probably involve diffusion183, where it activated protein C prevents glucose-​induced apopto-
binds to VEGFR2. Podocytes also produce sFLT1, which in sis of GECs and podocytes by binding to endothelial
an autocrine fashion binds to glycosphingolipid GM3 protein C receptor (EPCR) and activating the serine/
in lipid rafts to promote podocyte adhesion to the GBM threonine-​protein kinase, PAR1, leading to a decrease

Nature Reviews | Nephrology

Reviews

a Diabetic kidney disease

Loss of fenestrae

Glycocalyx disruption
Podocyte foot
process detachment
High glucose
ROS

AGEs

Heparanase

EC

Glycocalyx

Podocyte
↓eNOS
• Reduced NO
• Increased apoptosis
• Increased permeability RAGE

b Arteriolar nephrosclerosis
Bowman
capsule Ischaemic collapse
of capillary loops
(hypoperfusion)
• Reduced NO, prostaglandin I
and epoxyeicosatrienoic acids Smooth
• Increased ET1, ANGII and muscle cell
thromboxane A2

Vessel lumen Mesangial

narrowing cell

Arteriolar Myointimal thickening, SMC

hyalinosis hypertrophy and proliferation

c Endothelial dysfunction in CKD

EC

Reduced repair
Release of
procoagulant factors
Uraemic Reduced
Inflammation
toxins EPC survival
EMPs
TF

AhR activation EC detachment

Reduced NO availability Glycocalyx breakdown

Fig. 6 | endothelial dysfunction in common diseases. a | Multiple pathways lead to endothelial dysfunction in diabetic
kidney disease. Hyperglycaemia, binding of advanced glycation end products (AGEs) to their receptor RAGE and oxidative
stress promote glycocalyx disruption through increased production of heparanase, reduced synthesis of heparan sulfate,
apoptosis of glomerular endothelial cells (GECs) and reduced nitric oxide (NO) synthesis. Progressive loss of GEC
fenestrae and detachment of podocyte foot processes increases glomerular permeability. b | Arteriolar nephrosclerosis
(sometimes called hypertension-​associated nephropathy) is characterized by a narrowing of renal arterioles, with smooth
muscle cell (SMC) hypertrophy and proliferation, and arterial hyalinosis, with downstream ischaemic collapse of
glomerular capillary loops. Renal endothelial dysfunction leads to a decrease in endothelium-​derived vasodilators (such
as NO, prostaglandin I and epoxyeicosatrienoic acids) and an increase in vasoconstrictive mediators (such as endothelin 1
(ET1), angiotensin II (ANGII) and thromboxane A2), which contribute to increased vasoconstriction, promoting hypoxia,
inflammation and fibrosis. c | Chronic kidney disease (CKD) itself can cause endothelial dysfunction. Uraemic toxins
induce endothelial oxidative stress and a procoagulant phenotype, with increased expression of tissue factor (TF), and
release of procoagulant endothelial microparticles (EMPs). CKD also leads to reduced NO bioavailability, breakdown of
the glycocalyx and reduced endothelial repair by mature endothelial cells (ECs) and endothelial progenitor cells (EPCs).
AhR, aryl hydrocarbon receptor; eNOS, endothelial NO synthase; ROS, reactive oxygen species.

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Heparanase
An enzyme that degrades
polymeric heparan sulfate
molecules into shorter-​
chain-length oligosaccharides.
The action of heparanase
disrupts the endothelial
glycocalyx.
The polyol pathway
Pathway that converts hexose
sugars such as glucose into
sugar alcohols (polyols). In this
metabolic pathway, glucose is
reduced to sorbitol (by the
aldose reductase), which is
then converted to fructose (by
sorbitol dehydrogenase). The
polyol pathway is activated in
hyperglycaemia, owing to the
saturation of physiological
glucose metabolism, and leads
to a cellular oxidative stress.
Sorbitol
Sugar alcohol derived from
glucose through the action of
aldose reductase. Sorbitol is a
component of the polyol
pathway.
eNOS uncoupling
Occurs when endothelial NO
synthase (eNOS) is not coupled
with its substrate (mainly
l-arginine) or cofactors. eNOS
uncoupling results in the
production of the pro-​oxidative
superoxide anion (O2−) instead
of NO, which characterizes
endothelial dysfunction.
Prostanoids
Physiologically active lipid
compounds that are
metabolites of the fatty acid
arachidonic acid. Prostanoids
comprise prostaglandins
(which have vasodilator and
anti-​aggregant properties) and
thromboxanes (which are
potent vasoconstrictors that
also activate platelet
aggregation).
Incretins
Glucose-​lowering hormones
secreted by the stomach
during a meal. Incretins
increase the release of insulin
from pancreatic β-​cells, slow
gastric emptying and inhibit
glucagon release from
pancreatic α-​cells. The two
main incretins are glucagon-​
like peptide 1 (GLP1) and
gastric inhibitory peptide (GIP),
which are both rapidly
inactivated by the enzyme
dipeptidyl peptidase 4 (DPP4).
Nature Reviews | Nephrology
in the mitochondrial apoptosis pathway in glucose-​
stressed cells200. Activated protein-​C-mediated inhi-
bition of apoptosis within glomeruli protects against
hyperglycaemia-​induced renal dysfunction and there-
fore identifies the crucial role of endothelial function in
the pathogenesis of diabetic nephropathy. Moreover,
in Pima Indians with T2DM, the reduction in GEC
fenestrae correlated with the level of albuminuria and
loss of GFR197. Hyperglycaemia also induces alterations
in the glycocalyx. For example, a reduction in heparan
sulfate synthesis201 contributes to increased albumin
passage201. In rats with streptozotocin-​induced DKD,
proteinuria increased concurrently with decreasing
heparan sulfate content202. In addition to reduced hep-
arin sulfate synthesis, heparan sulfate degradation is
promoted by the increased synthesis of heparanase by
GECs and podocytes203,204. The increase in heparanase
synthesis by podocytes occurs as a result of increased
ROS and ANGII205 and the activation of NF-​κB by
advanced glycation end products (AGEs)206. In Zucker
fatty rats, oxidative stress increased glomerular filtration
barrier permeability to macromolecules by increasing
expression of heparanase207. Heparanase might also
contribute to the inflammatory processes that promote
tubulo-interstitial fibrosis in DKD207,208.
Activation of PKC by diacylglycerol might also medi-
ate cell signalling and endothelial hyperpermeability in
DKD209. Inhibition of PKC reduces hyperglycaemia-​
induced hyperpermeability of GECs209, whereas inhi-
bition of the PKC isoform, PKCβ, reduces albumin
excretion as well as glomerular and tubulo-​interstitial
injury in diabetic rats210.
As mentioned above, podocytes synthesize VEGF9,194,
which acts on VEGFRs expressed by GECs to regu-
late GEC function211. Reduced VEGF levels have been
reported in patients and rodents with diabetes, possibly
as a consequence of reduced podocyte number; how-
ever, increased VEGF levels have also been reported
in association with diabetes194,211,212. Hyperglycaemia
might also alter the responsiveness of GECs to VEGF211.
A study of biopsy samples from patients with diabetes
showed increased binding of VEGF to its receptor in the
endothelium of mildly injured glomeruli but decreased
binding in severely injured glomeruli212. Thus, an initial
upregulation of VEGF signalling in response to endothe-
lial stress could contribute to increased glomerular per-
meability in early stages of DKD, whereas subsequent
downregulation of VEGF might underlie the ineffec-
tive capillary repair in advanced stages of disease212,213.
VEGF inhibition promotes endothelial injury and accel-
erates the progression of glomerular lesions in mice
with diabetes213.
In contrast to adipose or muscle cells, ECs do not
require insulin to uptake glucose194, with different EC
populations regulating glucose uptake through differ-
ent mechanisms214,215. Although information specific to
GECs are lacking, the finding that microvascular reti-
nal ECs are not able to limit glucose uptake, whereas
ECs in the brain and heart can limit glucose uptake214,
might in part explain why some populations, such as
retinal ECs and GECs, are particularly susceptible to
a high-​glucose environment. Under hyperglycaemic
Reviews
conditions, saturation of physiological glucose
metabolism pathways in ECs leads to the activation of
deleterious pathways (such as the polyol pathway and for-
mation of sorbitol), which produces more oxidative stress
and EC damage216. Inhibition of the polyol pathway
attenuates proteinuria, decreases GBM thickening in
diabetic rats and reduces glomerular hyperfiltration
in humans213.
GECs might have a reciprocal role in supporting
podocyte function through the production of protective
factors such as endothelial NO synthase (eNOS). Diabetic
mice with eNOS deficiency have accelerated DKD and
severe podocyte injury198. One way in which chronic
hyperglycaemia affects eNOS expression is through the
formation of AGEs — a group of proteins and/or lipids
that are glycated following exposure to sugars213,217. The
AGE receptor, RAGE, is upregulated in ECs by hyper-
glycaemia213, and binding of AGEs to RAGE increases
ROS synthesis in ECs through activation of NAD(P)H
oxidase (NOX2)218,219. The production of ROS by GECs
is increased in hyperglycaemic states via PKC-​dependent
activation of NOX2220,221. AGEs also reduce NO bio-
availability by decreasing eNOS expression and inacti-
vating NO219. In addition, hyperglycaemia reduces NO
bioavailability221 by inducing the expression of ADMA
(asymmetric dimethylarginine), an endogenous inhibi-
tor of eNOS213, and through eNOS uncoupling195, leading
to the formation of superoxide anion (O2−) instead of
NO. ROS also increases expression of pro-​inflammatory
COX2 (refs221,222) and subsequent production of COX-​
derived vasoconstrictor prostanoids221–223, which might
promote glomerular efferent arteriole constriction and
glomerular capillary pressure. ROS production in late
stages of disease might promote renal fibrosis through
upregulation of the profibrotic molecules TGFβ1 and
ANGII224,225. Mesangial expansion and the accumulation
of extracellular matrix (ECM) might also be favoured
by the endothelial synthesis of platelet-​derived growth
factor (PDGF)226, the expression of which is induced by
high glucose213. Blockade of PDGF receptor signalling
inhibits the proliferation of pericytes and their differen-
tiation into myofibroblasts, attenuating the development
of kidney fibrosis213,227. Genetic deletion of a specific
PDGF (PDGFD) attenuated renal fibrosis after UUO or
ischaemia–reperfusion injury226.
The fibrotic process of DKD also involves EndMT194,
which is promoted by hyperglycaemia, TGFβ, ANGII,
AGEs35 and the enzyme dipeptidyl peptidase 4 (DPP4)228,
which degrades incretins. Inhibition of DPP4 with lina-
gliptin reduces kidney fibrosis associated with EndMT
in diabetic mice229.
Of note, endothelial injury and loss in DKD is not
compensated by increased repair. In fact, the number
of EPCs is reduced in patients with T1DM or T2DM194
and is associated with progression of renal disease230.
Moreover, the angiogenic properties of EPCs from
patients with diabetes are reduced, with impaired
proliferation and ability to incorporate into vascular
structures194, reduced eNOS expression, decreased NO
bioavailability and increased pro-​oxidative properties231.
Of interest, new agents that have shown promise in
patients with DKD — glucagon-​like peptide 1 receptor
Reviews
Autoregulation
Autoregulation of renal blood is
a homeostatic mechanism,
relying both on myogenic
response of afferent arterioles
(vasoconstriction in case of
transmural pressure elevation)
and tubulo-​glomerular
feedback (depending on the
sensing of sodium chloride
delivery to the macula densa).
Autoregulation protects the
glomerular capillaries from
elevations in arterial pressure
and allows the kidney to
maintain a fairly constant
blood flow and glomerular
filtration rate.
Indolic compounds
Chemical compounds
comprising an aromatic bicyclic
structure resembling that of
indole (C8H7N). Indolic uraemic
compounds result from
tryptophan metabolism by the
gut and include indoxyl sulfate,
indole-3 acetic acid and
indoxyl-​β-d-​glucuronide.
(GLP1R) agonists and sodium–glucose cotransporter 2 of local vascular homeostasis; increase vasoconstric-
(SGLT2) inhibitors — might in part act by improving tion; and, in turn, promote hypoxia, inflammation and
endothelial function232. fibrosis in the artery and surrounding interstitium.
Beyond the kidney, endothelial dysfunction is
Arteriolar nephrosclerosis implicated in the macrovascular complications of
Epidemiological studies reveal a strong association hypertension, including coronary artery disease, heart
between the presence and severity of arterial hyper- microvascular dysfunction, stroke or conduit arterial
tension and the occurrence of ESRD233. In this con- dysfunction and stiffness. This dysfunction in part
text, endothelial dysfunction is suspected to contribute results from the reduced release of endothelium-​derived
substantially to the pathophysiology of hypertension-​ vasodilatory mediators241,251. Structural (myointimal
induced kidney injury. The typical presentation of this thickening) and functional (vasoconstriction) con-
so-​called arteriolar nephrosclerosis234 involves nonspe- sequences of endothelial dysfunction consequently
cific criteria, including a slow decline in GFR, absent increase exposure of the renal vasculature to alterations
or low-​range proteinuria, normal urine sediment and in blood flow and impaired autoregulation, increasing
the absence of an alternative identifiable cause of kid- the risk of glomerular ischaemia and tissue hypoxia.
ney disease in a patient with long-​standing hypertension. Ultimately, vascular rarefaction at the peritubular capil-
Histology typically demonstrates a narrowing of renal lary level, as a result of endothelial damage and EndMT,
arterioles with smooth muscle cell hypertrophy and an drives tubulo-​interstitial fibrosis and the development
accumulation of ECM in the vascular wall. Additional of CKD252–254.
lesions, dependent on the pathogenic factors and the
chronicity of the disease, include ischaemic glomeruli Systemic endothelial dysfunction associated with CKD.
and tubulo-​interstitial fibrosis (Fig. 6b). Of note, CKD in As described above, renal endothelial injury can result
the context of hypertension is not always causally related, in the development of renal dysfunction. However, CKD
as has been discussed elsewhere235–237. itself is a cause of profound systemic endothelial dys-
Despite this caveat, multiple studies have shown function, which contributes to the cardiovascular burden
that patients with essential hypertension have impaired of this disease255. Endothelial dysfunction, characterized
systemic and renal endothelial function238–241, although by elevated soluble biomarkers such as soluble adhesion
whether renal endothelial dysfunction is a cause molecules or EMPs (Table 1) and altered flow-​mediated
or a consequence of hypertension remains unclear. vasodilation256,257, is observed at every stage of CKD4,258,
Pharmacological inhibition of NO with L-​NAME including children with CKD259,260. Patients with CKD
induces endothelial dysfunction, severe hypertension also show evidence of an imbalance between EC injury
and renal vascular lesions similar to those observed (reflected by the number of EMPs and CECs) and
in patients with malignant hypertension242,243, and repair (reflected by the number of EPCs and CACs)4,261.
fawn-​hooded hypertensive rats, which have impaired The accumulation of uraemic toxins that occurs as
autoregulation of renal blood flow, develop progressive a consequence of impaired renal clearance contributes
kidney failure with age. In this inbred model of spon- to CKD-​associated endothelial dysfunction4; endothe-
taneous renal disease, endothelial dysfunction in the lial dysfunction worsens with progression of CKD and
renal artery precedes the development of kidney dam- improves with resolution of uraemia following kid-
age244 and involves direct exposure of the renal arteri- ney transplantation257. The endothelium is constantly
ole endothelium to increased pressure. Renal ECs also exposed to uraemic toxins that accumulate in the blood
integrate signalling from circulating vasoactive and pro-​ of patients with CKD262 (Fig. 6c). The increase in uraemic
inflammatory mediators. Specifically, studies from the toxin concentrations occurs early in CKD and is observed
past couple of years have demonstrated a central role in living kidney donors despite maintaining an estimated
for components of the immune system, particularly GFR >60 ml/min (ref.263). More than 100 uraemic solutes
TH17 and γδ T cells, in endothelial dysfunction and have been described, which can be classified into three
target organ damage in ANGII-​induced hypertension. categories: small molecules, medium-​sized molecules
These deleterious effects are dependent on the arte- and protein-​bound solutes262. Among protein-​bound
rial pro-​inflammatory milieu induced by T cell pro-​ toxins, some indolic compounds that result from trypto-
inflammatory cytokines, especially IL-17A and IFNγ. phan metabolism by gut microbiota have documented
The attributable renal damage may similarly relate to endothelial toxicity264: they inhibit endothelial prolifer-
immune-​mediated dysfunction of renal ECs, although ation and wound repair; decrease NO production; and
this remains to be established245,246. Oxidative stress promote EC senescence, EMP formation, leukocyte
also contributes to renal damage in hypertension247. adhesion, glycocalyx disruption and EPC apoptosis265.
Experimental studies have demonstrated that cytoplas- The indolic uraemic toxins indoxyl sulfate and indole-3
mic and mitochondrial dysfunction can be induced acetic acid (IAA), for example, promote endothelial oxi-
by ANGII, leading to increased ROS and contributing dative stress266 and induce a pro-​inflammatory267 and
to endothelial dysfunction and atherosclerosis248–250. procoagulant268 phenotype in different types of ECs.
Renal endothelial dysfunction leads to a decrease in Indoxyl sulfate and IAA are ligands for the transcription
endothelium-​derived vasodilators (such as NO, prosta- factor aryl hydrocarbon receptor (AhR), which induces
glandin I and epoxyeicosatrienoic acids) and an increase endothelial synthesis of TF and COX2, in addition to
in vasoconstrictive mediators (such as ET1, ANGII and increasing expression of the cytochromes CYP1A1 and
thromboxane A2), which contribute to the disruption CYP1B1 for the metabolism of exogenous pollutants64,267.
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Dioxin
Also known as 2,3,7,8-tetra-
chlorodibenzodioxin (TCDD),
dioxin and dioxin-​like
compounds are chemical
pollutants of the environment,
resulting from industrial
practices (mostly incineration
processes) and from forest fires
and volcanic eruptions. Dioxin
is the exogenous ligand of the
transcription factor aryl
hydrocarbon receptor (AhR).
Exposure to dioxins, mostly
through ingestion of
contaminated food, is
mutagenic and increases the
risks of cardiovascular
diseases.
Nature Reviews | Nephrology
Chronic exposure to environmental pollutants such as
dioxin, an exogenous ligand of AhR, causes endothelial
dysfunction and cardiovascular disease similar to that
seen in CKD269. Chronic exposure of mice to indoxyl
sulfate following uninephrectomy270, or UUO269, induces
endothelial dysfunction. In patients with CKD, serum
levels of indoxyl sulfate and IAA are associated with car-
diovascular events and mortality267,271, and reduction in
indoxyl sulfate concentration with use of the oral sorbent
AST-120 improves markers of endothelial dysfunction in
mice272 and in humans273.
Therapies that target the endothelium
Understanding of the physiological roles of the endothe-
lium as well as the different mechanisms of EC dysfunc-
tion have led to the development of targeted strategies
to improve endothelium health.
Protection of the glycocalyx
As described earlier, the glycocalyx covers GECs and acts
as a filtration barrier at fenestrations. Factors that cause
injury to GECs (for instance, in the setting of DKD) also
induce the sulfation and deacetylation of heparan sulfate
and activate heparanase in podocytes, resulting in gly-
cocalyx injury. Mice lacking heparanase are protected
from streptozotocin-​induced DKD274, and treatment of
diabetic mice with anti-​heparanase antibodies reduces
albuminuria and kidney injury274. However, two clinical
trials that aimed to replenish the glycocalyx through the
oral ingestion of sulodexide (a mixture of proteoglycans)
failed to demonstrate a renoprotective effect on GFR
decline or proteinuria275,276.
The potent vasoconstrictor, ET1, is produced by ECs
and other renal cell populations in response to stress.
Endothelin-​receptor antagonists (ERAs) have shown
beneficial renal effects in animal models of CKD and
anti-​proteinuric effects in patients with DKD and non-​
diabetic CKD, even in the presence of optimal RAAS
blockade 28,277. However, treatment of hypertensive
patients with ERAs is associated with an increased risk
of fluid retention and congestive heart failure owing to
a dose-​dependent reduction in the fractional excretion
of sodium, possibly through inhibitory effects on the
ETB receptor28. Nevertheless, the attenuation of albu-
minuria achieved by the ERA atrasentan in a murine
model of diabetic nephropathy277 was associated with
restoration of glomerular endothelial glycocalyx cov-
erage through downregulation of heparanase and was
associated with a shift in the balance of macrophages
from a pro-​inflammatory phenotype towards a more
regulatory phenotype. Future studies, such as the ongo-
ing SONAR trial in patients with DKD278, will determine
whether ERAs can safely slow the progression of renal
dysfunction in patients with DKD.
Anti-​inflammatory strategies
RAAS blockade has been the cornerstone of nephro-
protection in CKD for many years. The benefit of
RAAS blockade has been considered until now as
a class effect, resulting from the haemodynamic,
anti-​inflammatory and anti-​f ibrotic properties of
angiotensin-​converting-enzyme (ACE) inhibitors and
Reviews
ANGII receptor blockers (ARBs); however, a new study
challenges this view. This study demonstrated the ability
of renin to cleave C3 into C3b and C3a, as would a C3
convertase. This cleavage, and consequent complement
activation, was specifically blocked by the renin inhibitor
aliskiren175. Administration of aliskiren to three patients
with dense deposit disease (a C3 glomerulopathy asso-
ciated with dysregulation of the alternative pathway of
complement) led to decreased systemic and renal com-
plement activation. In a separate study, aliskiren and the
ACE inhibitor enalaprilat were successful in controlling
malignant hypertension in two paediatric patients with
aHUS279. Although these studies indicate that RAAS
inhibitors might exert their beneficial effects in part
by inhibiting the deleterious effects of complement on
the endothelium, further studies are warranted to eval-
uate the effect of this treatment on complement and
TMA injury279.
Blockade of the complement terminal pathway by
eculizumab, a monoclonal anti-​C5 antibody, has dramat-
ically improved outcomes for adults and children with
aHUS280,281, including recurrent aHUS after transplanta-
tion282. The role of eculizumab in the treatment of other
complement-​mediated renal diseases, such as typical
HUS and other forms of renal TMA, remains to be deter-
mined283–286. As mentioned earlier, the anaphylatoxin
C5a and its receptor contribute to the pathogenesis of
AAV. Avacopan, an orally administered C5aR antagonist,
has shown promising results in AAV as a replacement for
high-​dose corticosteroids287. In addition, experimental
targeting of pro-​inflammatory cytokines such as IL-1β or
pro-​inflammatory signalling pathways, such as SMAD–
TGFβ or NF-​κB signalling pathways, reduces the inflam-
matory consequences of renal endothelial dysfunction
in murine models of crescentic glomerulonephritis and
DKD and in human ECs in vitro146,288,289.
Inhibition of coagulation
FXa is an important component of the coagulation
cascade. FXa is responsible for thrombin generation,
but it also exerts direct effects on a variety of cells via
its receptors, PAR1 and PAR2 (ref.290). As mentioned
earlier, the PARs participate in the interplay between
coagulation and inflammation. The mesangial pro-
liferation, glomerular inflammation and fibrin depo-
sition observed in animal models in response to FXa
signalling through PARs are reversed by FXa inhibi-
tion21,291,292. In diabetic db/db mice, inhibition of FXa
attenuates glomerular hypertrophy, ECM accumula-
tion and proteinuria and limits angiogenesis293. In an
experimental model of kidney transplantation-​induced
ischaemia–reperfusion injury, administration of the
anti-​F Xa anticoagulation therapy fondaparinux to
the transplanted kidney, before warm ischaemia and
during cold ischaemia, improved renal function post-​
transplantation, with reduced indices of fibrosis com-
pared with organs treated with heparin294. Targeting
FXa might therefore be promising for preventing the
fibrotic evolution of glomerulonephritides and early
DKD293,294. By contrast, despite promising results in
pilot studies, administration of recombinant activated
protein C failed to improve outcomes in patients with
Reviews

septic shock295. Likewise, the benefit of recombinant
thrombomodulin in sepsis coagulopathy remains to be
clearly demonstrated296.
Fibrogenesis as a target
Irrespective of their origin, interstitial myofibroblasts
drive fibrosis in CKD. The relative contribution of res-
ident interstitial fibroblasts, infiltrating fibrocytes and
cells derived from kidney pericytes, epithelial cells
and ECs is still a matter of debate, although cell fate stud-
ies suggest that EndMT-​derived cells represent approx-
imately 10% of interstitial myofibroblasts297. Despite the
likely limited contribution of EndMT to renal myofi-
broblasts, blocking EndMT by targeting core mediators
of this phenotypic switch, including TGFβ, TWIST1,
SNAI1 or sirtuin 3, might hold important therapeutic
implications in halting CKD progression298–301.
Conclusions
Endothelial activation, and the switch of ECs from a reg-
ulatory state to a prothrombotic and pro-​inflammatory
phenotype, is a common feature of multiple renal
diseases. Improvements in techniques to study the
endothelium, either indirectly through the assessment
of circulating biomarkers or directly through the use of
new imaging techniques to study EC structure in vivo
and/or assess EC function ex vivo, as well as develop-
ments in the bioengineering of vessels and organoids,
hold great promise for the development and evaluation
of therapeutic strategies for renal diseases associated
with endothelial injury. Improved understanding of
endothelial physiology is required to identify therapeu-
tic targets. For example, knowledge of the mechanisms
by which complement drives endothelial lesions paved
the way for the use of C5 blocking strategies in patients
with aHUS. Understanding of endothelial biology is
also needed to anticipate adverse effects of therapeutic
agents, such as the adverse consequences of VEGF inhi-
bition. Greater understanding of the interaction between
complement and coagulation pathways will also likely
lead to the development of new therapeutic strategies
for a number of diseases, including DKD and arteri-
olar nephrosclerosis, leading to improved outcomes for
patients with renal diseases.
Published online xx xx xxxx

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Acknowledgements
The authors are grateful to all members of the French Renal
Endothelial Society (FRENDS) for fruitful discussions.
FRENDS is a collaborative group of nephrologists and
researchers working on the endothelium in different renal
diseases. The research was supported by grants from the
Agence Nationale de la Recherche (ANR) JCJC–INFLACOMP
2015–2018 (ANR-15-CE15-0001; to L.T.R.); INSERM (to
L.T.R.); EU FP7 grant 2012–305608 (EURenOmics;
to V.F.-B.); Assistance Publique-​Hôpitaux de Paris (AP-​HP)-
Programme Hospitaliser de Recherche Clinique (PHRC)
AOM08198 (to V.F.-B.); Association pour l'Information et la
Recherche sur les maladies rénales Génétiques (AIRG) (to
V.F.-B.); the French Society of Nephrology (to V.F.-B.); a grant
from Appel d'Offre de Recherche Clinique (AORC) 2011-
A01347 (to G.K.); and Assistance Publique-​Hôpitaux de
Marseille (AP-​HM)-PHRC grant 2012-A00217-36 (to G.K.).
Author contributions
All authors contributed to researching data for the article,
discussing the article’s content and writing, reviewing and
editing the manuscript before submission.
Competing interests
F.F. has received consultancy fees and/or speaker honoraria
from Alexion and Roche. M.L.Q. has received consultancy
fees and/or speaker honoraria from Alexion. The other
authors declare no competing interests.
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Supplementary information
Supplementary information is available for this paper at
https://doi.org/10.1038/s41581-018-0098-z.
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