Am J Physiol Renal Physiol 314: F454–F461, 2018.

First published November 22, 2017; doi:10.1152/ajprenal.00376.2017.

REVIEW

Advances in our understanding of the pathogenesis of hemolytic uremic

syndromes
E. E. Bowen and R. J. Coward
Academic Renal Unit, School of Clinical Sciences, University of Bristol, Bristol, United Kingdom
Submitted 27 July 2017; accepted in final form 16 November 2017

Bowen EE, Coward RJ. Advances in our understanding of the pathogenesis of

hemolytic uremic syndromes. Am J Physiol Renal Physiol 314: F454 –F461, 2018.
First published November 22, 2017; doi:10.1152/ajprenal.00376.2017.—Hemo-
lytic uremic syndrome (HUS) is major global health care issue as it is the leading
cause of acute kidney injury in children. It is a triad of acute kidney injury,
microangiopathic hemolytic anemia, and thrombocytopenia. In recent years, major
advances in our understanding of complement-driven inherited rare forms of HUS
have been achieved. However, in children 90% of cases of HUS are associated with
a Shiga toxin-producing enteric pathogen. The precise pathological mechanisms in
this setting are yet to be elucidated. The purpose of this review is to discuss
advances in our understanding of the pathophysiology underlying HUS and identify
the key questions yet to be answered by the scientific community.
complement; hemolytic uremic syndrome; thrombotic microangiopathy

INTRODUCTION This review will focus on the current understanding of the

Hemolytic uremic syndrome (HUS) is the leading cause of
acute kidney injury in children. It is a triad of acute kidney
injury, microangiopathic hemolytic anemia, and thrombocyto-
penia; first described in 1955 by Gasser et al. (as noted in Ref.
44). Over 90% of childhood HUS cases are associated with
infection (19). Shiga toxin-producing Escherichia coli found in
contaminated food and water supplies is typically the infective
trigger (STEC HUS), although HUS has also been reported
following exposure to Shigella, Campylobacter, and Strepto-
coccus pneumoniae (37). In the United States, Shiga toxin
associated HUS affects 0.5–2.1 people per 100,000 population
per year. Most of these cases are reported in children under 5
yr of age (19).
The remaining noninfective HUS cases are termed “atypi-
cal.” Atypical HUS is rare, with an estimated incidence in the
United States of two cases per million population per year (17).
However, unlike Shiga toxin HUS, the majority of adult HUS
cases are atypical. These include familial HUS due to genetic
abnormalities in complement regulation and other secondary
triggers such as pregnancy, drugs, malignancy, connective
tissue disorders, and transplantation (Fig. 1) (14). Over the last
decade there has been a renewed appreciation and considerable
interest in the role of the complement system in renal disease,
which has been magnified by the effectiveness of eculizumab
(a monoclonal humanized antibody against C5) in the treat-
ment of atypical HUS (17, 24).
pathogenesis of atypical HUS and STEC HUS and try to
identify some of the key questions yet to be answered by the
scientific community.
CLASSIFICATION OF HUS
HUS is categorized histopathologically as a thrombotic
microangiopathy (TMA). This term was first introduced in
1952 by Symmers (as noted in Ref. 44) to describe capillary
wall thickening, swelling, and detachment of the endothelial
cell from the basement membrane, accumulation of debris in
the subendothelial space, and intraluminal platelet thrombosis,
culminating in partial or complete obstruction of the vessel.
When this process occurs in the microvasculature of the kidney
(as is the case in HUS), renal impairment is seen due to organ
ischemia. The microangiopathic hemolytic anemia that ensues
is a consequence of erythrocyte shear, resulting in red cell
fragmentation. Platelet activation and entrapment in micro-
thrombi as well as trafficking to the reticuloendothelial system
lead to thrombocytopenia, local thrombosis, and organ ischemia
(19, 35). Thus TMA represents a final common pathway in a
number of disease processes that among others include HUS and
thrombotic thrombocytopenic purpura (TTP). (Fig. 1). The clini-
cal sequelae observed is dependent on the vascular bed and organ
affected (19). Both HUS and TTP are a consequence of endothe-
lial cell injury but are now accepted to be different diseases with
distinct underlying mechanisms (21).
ATYPICAL HUS DUE TO COMPLEMENT DYSREGULATION

Address for reprint requests and other correspondence: R. J. M. Coward, It is now established that most familial cases of atypical
Bristol Renal, School of Clinical Sciences, Whitson St., Bristol BS1 3NY, UK HUS are caused by hyperactivation of the alternative comple-
(e-mail: richard.coward@bristol.ac.uk). ment pathway as a result of loss-of-function mutations in
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 UNDERSTANDING THE PATHOGENESIS OF HUS F455

Classification of TMA

Microangiopathic anemia, Microangiopathic anemia, Fig. 1. Classification of thrombotic microan-

↓PLTs, AKI. ↓PLTs, AKI, fever and giopathy (TMA). It is now widely accepted
neurological features with that classification of TMA according to etiol-
ADAMTS13 <10%. ogy (rather than clinical features) is a more
useful guide to prognosis and treatment. TMA
Infection represents a final common pathway in many
Transplantation
- STEC, Shigella, Campylobacter
- Solid organ
- Streptococcus Pneumoniae
disease processes (19). PLTs, platelets; HUS,
- Hemopoietic TTP
hemolytic uremic syndrome; AKI, acute kid-
Acquired Genetic
ney injury; CNI, calcineurin inhibitors; SLE,
HUS - autoantibodies systemic lupus erythematosus; APLS, anti-
Complement disorders phospholipid syndrome; STEC, Shiga toxin-
Pregnancy
- Genetic (Atypical HUS) producing Escherichia coli; TTP, thrombotic
- HELLP
- Acquired
thrombocytopenic purpura; HELLP, hemoly-
sis, elevated liver enzymes, low platelet count
syndrome.
Drugs Systemic Disease
DGKε - HIV
- Oral contraceptives
- Chemotherapy - Malignancy
- CNIs - Connective tissue diseases (SLE, APLS)

complement inhibitory regulatory factors or gain of function
mutations in C3 or factor B (14). Autoantibodies against
regulatory complement factors have also been described in
atypical HUS (27). It was the discovery by Warwicker et al. in
1998 (as noted in Ref. 33) that a mutation in factor H led to the
development of atypical HUS, which prompted the search and
subsequent discovery of over 120 mutations in complement
regulatory genes responsible for the disease.
Complement system. The complement pathway forms part of
the innate immune response and consists of 60 proteins that
play a vital role in the host defense against pathogens and in the
maintenance of tissue homeostasis (1, 33). These proteins can
be plasmatic (fluid) or membrane-bound receptors (solid) (43).
Three pathways leading to activation of the complement cas-
cade have been described: classic, lectin, and alternative. All
culminate in the formation of the membrane attack complex
(MAC), leading to the insertion of pores in the target cell
membrane, resulting in osmotic lysis and cell death (2) (Fig. 2).
The classic pathway is activated by binding of C1q to anti-
body-antigen complexes. The lectin pathway is triggered by
binding of mannose binding lectin (MBL) to mannose-contain-
ing carbohydrates on microbial surfaces that activates MBL-
associated serine protease 1 and 2 (MASP 1 and MASP 2) (2)
(33). Both the classic and lectin pathways lead to the genera-
tion of the same C3 convertase C4b2a on target cell surfaces,
resulting in proteolytic cleavage of C3 into C3a and C3b (33).
The alternative pathway differs from the other two in that it
is continually active at low levels in the plasma. Spontaneous
hydrolysis of the thioester bond in C3 induces a conformational
change in the protein, which facilitates binding of complement
factor B. Once bound to C3, factor B is cleaved by factor D to
Ba and Bb. This generates the alternative pathway C3 conver-
tase C3Bb, resulting in proteolytic cleavage of C3 into C3a and
C3b (see Fig. 2) (2) (33, 43). Association of C3b with C3
convertases (generated by any of the 3 pathways) leads to
formation of C5 convertases, which cleave factor C5 into C5a
and C5b, initiating the assembly of the MAC on target cell
surfaces (33). This terminal pathway in the complement cas-
cade also acts to alert the host defenses by the release of
chemoattractant and inflammatory mediators; C3a and C5a are

Classical Pathway C1q C2 Fig. 2. The complement cascade. Three path-

C4 ways have been described leading to activa-
tion of the complement pathway; classical,
Y C3 convertase lectin and alternative. All culminate with the
(C4b2a)
formation of the membrane attack complex
Lectin Pathway MASP 1 MASP 2
(MAC) leading to the insertion of pores into
C3 C5 MAC the target cell membrane resulting in osmotic
MBL lysis and cell death. The alternative pathway
differs from the other two in that it is contin-
ually active at low levels in the plasma and is
C3 convertase C3a C5a
(C3Bb)
activated by spontaneous hydrolysis of a thio-
ester bond in C3. To protect host cells from
non-specific destruction complement activa-
C3 tion is regulated by a number of plasma
Factor B Ba CFH, CFI
C3 (CFH, CFI) and membrane-bound factors (2,
Bb
Alternative Pathway C3 33). MBL, mannose-binding lectin; MASP,
Factor D MBL-associated serine protease; CFH, com-
Spontaneous
plement factor H; CFI, complement factor I.
Hydrolysis
Amplification Loop

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F456 UNDERSTANDING THE PATHOGENESIS OF HUS
potent anaphylatoxins that recruit phagocytes and induce en-
dothelial cell activation (46).
The alternative pathway acts as a positive feedback loop,
amplifying complement activity. C3b on target cell surfaces
generated by the classic or lectin pathways acts as a site of
C3Bb formation, and the activation of alternative pathway
itself leads to deposits of C3b on cell surfaces, further poten-
tiating this loop. As such, the alternative pathway may account
for up to 80% terminal pathway activity (2). Hence, the
complement cascade has great potential for nonspecific de-
struction, which host cells must be protected from (2).
Complement regulation. It is essential that complement
activation is regulated to prevent excessive cell injury and
inflammation (2, 27). Host cells are protected by surface-bound
and soluble plasma complement regulatory proteins. Of partic-
ular importance are the plasma proteins factor H and factor I,
which negatively regulate the C3b amplification loop (Fig. 2).
Although found in the plasma both can act in the fluid phase
and on cellular surfaces. Their role on cell surfaces and
specialized biomembranes (such as the glomerular basement
membrane in the kidney), which lack membrane-bound com-
plement regulators, is vital, as they are the only defense against
complement attack and thrombus formation (27, 46). This may
explain why the kidney is so susceptible to injury in atypical
HUS, where impaired regulation of the alternative complement
pathway in the glomerulus due to mutation in complement
regulators or autoantibodies against them results in loss-of-host
protection (12).
Notable membrane-bound complement factors include
CD46 [membrane cofactor protein (MCP)], CD55 [decay ac-
tivating factor (DAF)], CD59, and CR1. By definition, these
surface-bound complement regulatory proteins are restricted in
activity by their cellular expression and distribution, the ex-
ception being CR1, which is expressed by neutrophils, lym-
phocytes, and erythrocytes, which circulate throughout the
body rather than being tissue bound (27, 34).
GENETICS OF ATYPICAL HUS
Atypical HUS is a rare condition associated with significant
morbidity and mortality, historically leading to end-stage renal
disease in approximately one-half of patients affected (27).
Inheritance may be sporadic, autosomal recessive, or auto-
somal dominant with incomplete penetrance (34). This low-
level genetic penetrance highlights the importance of genetic
modifiers and environmental triggers in the development of the
disease (3). Interestingly, identification of the specific gene
polymorphisms resulting in abnormalities of the complement
cascade provides powerful prognostic information allowing
patients to be counselled about likelihood of posttransplanta-
tion recurrence (5, 14). Mutations in Complement factor H are
associated with the worst outcomes, whereas those with MCP
mutations do far better. This is not surprising given that MCP
is a transmembrane protein that is highly expressed in the
kidney; hence, kidney transplantation corrects the MCP defect
and restores MCP solid phase activity. This is in contrast to
mutations in circulating (fluid phase) complement factors H
and I, which are predominantly of hepatic origin (33).
What is intriguing about complement regulatory proteins
(whether soluble or membrane bound) is that they have
evolved from a common structural domain characterized by
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several large genomic repeat regions: the short consensus
repeat (8). This facilitates nonhomologous recombination that
results in chromosomal rearrangements in these regions that
produce diverse outcomes. This has allowed evolution of
differential specificities for binding ligands (in this case the
various complement proteins) but has also resulted in deletions
and duplications of chromosomal fragments that lead to mu-
tated complement regulatory proteins and predisposition to
HUS (49).
Factor H is known to be key in discrimination between host
cells and invading microbes. Indeed in a recent review by
Jokiranta (12), mutations in factor H are responsible for up to
28% of atypical HUS cases with factor I mutations seen in only
8% of patients. Autoantibodies against the C terminus of factor
H or factor I can also result in acquired complement dysregu-
lation, accounting for up to 10% of cases (3). In approximately
one-third of cases of atypical HUS, the underlying mutation is
unknown (12).
When factor H binds to cell surface C3b, it prevents activa-
tion and spares the cell from destruction (12). Hence, factor H
is central in discriminating self and non-self and regulation of
the alternative complement pathway. This is via simultaneous
binding of factor H to C3b and cell surface polyanions (sialic
acid, glycosaminoglycans, and phospholipids). Until recently,
the glycosaminoglycans heparin and heparin sulfate were con-
sidered to be the most important polyanions involved in self-
protection of host cell surfaces. However, Hy Arinen et al. (7)
demonstrated that mutations in factor H specifically disrupt
binding to sialic acid. They also showed that removal of sialic
acid from glomerular endothelial cells reduced factor H bind-
ing and resulted in complement attack (29). Furthermore, sialic
acids are under dynamic regulation and as such cell surface
composition varies at different ages and conditions, which may
contribute to the incomplete genetic penetrance observed in
atypical HUS associated with factor H mutations. Fascinat-
ingly, pathogens such as Borrelia burgdorferi, streptococci,
and Gram-negative bacteria have evolved to escape comple-
ment attack by binding to factor H, thereby mimicking host cell
surfaces (29).
The membrane-bound regulators MCP, DAF, and CR1 limit
complement activation at the C3 step. CD59 inhibits polymer-
ization of C9 and prevents MAC formation. They are largely
species selective and protect host cell surfaces against comple-
ment from homologous species (29). Mutations in these cell
surface regulators account for 10 –15% of atypical HUS cases
(12). A lack of DAF and CD59 (due to an acquired defect in
anchorage to cell membrane phospholipids) in erythrocytes
results in the disease paroxysmal nocturnal hematuria (29).
Red blood cells are rendered susceptible to complement attack,
and patients develop a tendency to thrombosis. Together,
paroxysmal nocturnal hematuria and the glomerular thrombotic
microangiopathy seen in atypical HUS suggest that the coag-
ulation system and complement pathways are closely intercon-
nected and that thrombosis itself may act to trigger further
complement activation (12, 29).
MCP is expressed by all nucleated cells (i.e., it is absent
from erythrocytes). CR1 is present on all human blood cells
(except platelets and most T cells) where it acts as a cofactor
for the cleavage of C4b and C3b by factor I and deactivates the
alternative complement pathway through direct binding to
C3b. CR1 also binds to C4b accelerating the decay of classical
 UNDERSTANDING THE PATHOGENESIS OF HUS
and MBL convertases (9). In red blood cells, CR1 acts as an
immune adherence receptor binding antigens coated in C3b/
C4b and transporting them for clearance in the reticuloendo-
thelial system (9). Of particular interest, CR1 is also expressed
on selected epithelial cells: the podocyte in the kidney and the
retinal pigment cells in the eye. Its role in these cells remains
a mystery. Java et al. (9) have recently shown that CR1 is a
potent inhibitor of the alternative pathway and binds immune
complexes directly that remain on the surface of the epithelial
cell. This observation has led to the hypothesis that podocytes
are fundamental in modulating the innate immune response
through CR1 binding of immune complexes trapped within the
glomerulus. However, difficulties with any further study in this
area have been that conditionally immortalized podocytes in
vitro lack CR1 expression and that mouse erythrocytes lack
CR1 (8).
ATYPICAL HUS INDEPENDENT OF COMPLEMENT
ACTIVATION
The discovery in 2013 by Lemaire et al. (as noted in Ref. 42)
that recessive mutations in DGKε cause atypical HUS in a
significant proportion of children in the first year of life has led
to an alternative pathophysiological mechanism being pro-
posed for the condition. DGKε is the first gene to be associated
with familial HUS that has not been shown to have a direct role
in alternative pathway complement activation (4, 25). Diacyl-
glycerol kinases (DGKs) are intracellular lipid kinases that
phosphorylate diacylglycerol (DAG) to phosphatidic acid
(PA), leading to termination of DAG signaling (4). The DGKε
isoform preferentially phosphorylates arachidonic acid con-
taining DAG (AADAG) to PA thereby terminating AADAG
signaling (4) (Fig. 3).
AADAG is a key intracellular signaling molecule that acti-
vates protein kinase C (PKC). AADAG-dependent PKC sig-
naling promotes thrombin-induced platelet activation. In endo-
thelial cells, it has been shown to increase production of
prothrombotic factors such as von Willebrand factor and tissue
factor; as well as downregulating VEGF receptor VEGFR2
TRPC6
PLC
PIP PIP2 AADAG PKC
ATP
DGKε
ADP
cPLA2
PA
AA
COX-1
COX-2
Prostaglandins
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F457
signaling, which has been linked to TMAs previously (25, 42).
Bruneau et al. (4) have demonstrated in endothelial cells that
knockdown of DGKε results in a proinflammatory and pro-
thrombotic state via over activation of p38 and p44/42 MAP
kinases. In podocytes, DAGs have been shown to modify slit
diaphragm function and through PKC-dependent pathways
downregulate expression of VEGFR2 (25). Hence, the hypoth-
esis is that loss-of-function mutations in DGKε results in
sustained AADAG signaling and a subsequent prothrombotic
state. This finding has important implications for the manage-
ment of HUS, which to date have focused on complement
blockade.
VASCULAR ENDOTHELIAL GROWTH FACTOR AND HUS
In the kidney, podocytes are the major source of vascular
endothelial growth factor (VEGF-A), which is essential for
maintaining the nearby glomerular endothelium. The clinical
observation that cancer patients treated with the VEGF inhib-
itor bevacizumab develop a glomerular TMA has led to studies
in transgenic mouse models (6). Interestingly, Eremina et al.
(6) have shown that disruption of VEGF signaling in mature,
fully developed podocyte-specific VEGFA knockout mice re-
sults in a glomerular TMA with a phenotype that recapitulates
the glomerular injury seen in HUS. Moreover, recent work
published by Keir et al. (20) has provided evidence that local
VEGF availability modulates complement regulation in the
microvasculature of the kidney (and retina) via VEGFR2/PKC-
␣/CREB signaling.
In further support of the importance of the glomerular
microenvironment and local VEGF signaling; Jin et al. (10)
have shown that sFLT1 (a soluble VEGF receptor) is produced
by podocytes. sFLT1 is known to bind VEGF thereby acting as
a decoy receptor to inhibit proangiogenic activity via
VEGFR2. High circulating levels of sFLT1 have been linked to
preeclampsia, another glomerular disease associated with en-
dotheliosis and complement activation (10, 20). Elimination of
podocyte sFLT1 production led to podocyte actin cytoskeleton
rearrangement with damage to the glomerular filtration barrier
Fig. 3. Effect of AADAG signaling. DGKε is an
Platelet activation intracellular kinase that preferentially phosphor-
ylates AADAG to PA thereby terminating
AADAG signaling. Loss-of-function mutations
vWF in DGKε lead to unregulated AADAG signaling
and PKC dependent platelet activation and in-
TF creased production of prothrombotic factors
(25, 42). PIP, phosphatidylinositol; PIP2, phos-
phatidylinositol-4,5-bisphosphate; PLC, phos-
VEGFR2
pholipase C; AADAG, arachidonic acid diacyl-
glycerol; ATP, adenosine triphosphate; ADP,
adenosine diphosphate; DGKε, diacylglycerol
kinase ε; PA, phosphatidic acid; TRCP6, tran-
sient receptor potential cation channel 6; PKC,
protein kinase C; vWF, von Willebrand factor;
TF, tissue factor; VEGFR2, vascular endothelial
growth factor receptor 2; cPLA2, cytosolic
phospholipase A2; AA, arachidonic acid; COX,
cyclooxygenase enzyme.
Thromboxane
F458 UNDERSTANDING THE PATHOGENESIS OF HUS

and massive proteinuria. This work illustrates the complexity
of glomerular cell cross talk and the autocrine function of
sFLT1 in the regulation of podocyte behavior (10).
SHIGA TOXIN-ASSOCIATED HUS
The commonest cause of HUS is infection, which accounts
for 90% of cases in childhood (19). The pathophysiology
underlying STEC HUS is yet to be determined and as such
there are currently no specific treatments for the disease other
than supportive care (38). Acute mortality outcomes for STEC
HUS have improved from 30 to 5% with the introduction of
early dialysis. However, of those patients that survive the
initial insult up to 30% develop proteinuria and 18% progress
to chronic renal failure. Furthermore, some patients develop
extra-renal sequelae such as diabetes mellitus, colonic stric-
tures, and devastating neurological disease (13). A better un-
derstanding of the pathophysiology underlying STEC HUS is
essential if targeted therapy for this condition is to be devel-
oped.
Shiga toxins are exotoxins produced by E. coli, as well as by
other bacteria including Shigella dysenteriae and Campylobac-
ter jejuni (11). They consist of a single 30-kDa enzymatic A
subunit in noncovalent association with five identical B sub-
units of 7 kDa each (37, 52). The genes encoding Shiga toxin
are found within lambdoid bacteriophages present in all patho-
genic STEC. Toxin secretion occurs by phage-mediated bac-
terial lysis (11). It is the B subunit of Shiga toxin that binds to
the glycosphingolipid receptor Gb3. It has been shown that
cells lacking Gb3 expression are resistant to the toxic effects of
Shiga toxin, meaning the pattern of expression of Gb3 in
different cell types is a reliable predictor of Shiga toxin site of
action (41).
Following binding to Gb3, Shiga toxin is endocytosed and
transported in a retrograde manner from the endosome to the
trans-Golgi network. From here it is transported to the endo-
plasmic reticulum (ER), where using the ER-associated degra-
dation machinery it is able to translocate into host cell cyto-
plasm (53) (Fig. 4). Shiga toxin acts in three main ways: first,
it inactivates ribosomes by enzymatically removing an adenine
residue from 28S ribosomal RNA. This triggers the ribotoxic
stress response leading to MAPK signaling and activation of
cytokines and chemokines that result in proinflammatory and
proapoptotic pathways (37) (52). Second, the unfolded protein
response may be triggered by Shiga toxin unfolding within the
ER. Prolonged signaling via the unfolded protein response will
induce apoptosis in cells (52). Finally, the binding of the B
subunit itself can initiate a cytoplasmic transduction cascade
that is distinct from the ribotoxic stress response but that also
culminate in a prothrombotic, proinflammatory cellular envi-
ronment (1a, 37). Attempts made to block Shiga toxin binding
with synthetic Shiga toxin binders such as STARFISH or
Synsorb-Pk or to inhibit intracellular transport of the toxin with

Shiga toxin A subunit

Shiga toxin B subunit

Plasma Membrane Gb3 receptor

Endocytosis

Trans-Golgi
Network
Endoplasmic Reticulum

Apoptosis

ATF6
PERK
IRE

Unfolded Protein Response Ribotoxic Stress Response

Ribosome

MAPK

Pro-inflammatory Responses

Fig. 4. Intracellular responses to Shiga toxin binding. The B subunit of Shiga toxin binds to the glycosphingolipid Gb3 in the target cell membrane. Following
binding Shiga toxin is endocytosed and transported in a retrograde manner to the trans-Golgi network. From here it is transported to the endoplasmic reticulum
(ER) where it triggers 3 responses; the ribotoxic stress response, proinflammatory cytokine release and the unfolded protein response (37) (52). ATF6, activating
transcription factor 6; PERK, protein kinase R (PKR)-like endoplasmic reticulum kinase; IRE, iron-response element; MAPK, mitogen-activated protein kinase.

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 UNDERSTANDING THE PATHOGENESIS OF HUS
molecules such as Exo1 and Golgicide A have to date been
ineffective (11).
The exact mechanisms underlying the trafficking of Shiga
toxin from the gut to the kidney (and other target organs such
as the brain) are yet to be elucidated. Interestingly, there is
evidence in human intestinal cells that an alternative pathway
for toxin translocation exists, which is independent of the Gb3
receptor and results in IL-8 secretion without cytotoxicity (36).
Shiga toxin also stimulates the release of proinflammatory
cytokines from macrophages, which are also refractory to
toxicity due to low-level expression of Gb3 (36). At present,
the most widely accepted hypothesis is that neutrophils are
responsible for binding and transportation of Shiga toxin to
susceptible tissues (37).
Gb3 expression and the effect of Shiga toxin in eukaryotic
cells. As already alluded to, there is an association between the
amount of Gb3 expressed on the cell membrane and its sensi-
tivity to Shiga toxin. For example, endothelial cell lines de-
rived from large vessels such as the human umbilical vein
(which express low levels of Gb3) are relatively resistant to
Shiga toxin in comparison to glomerular endothelial cells (36).
Moreover, cells lacking the Gb3 receptor can be rendered
sensitive through artificially incorporating Gb3 into their mem-
brane (36). However, the amount of Gb3 extractable from a
tissue does not directly correlate to its sensitivity to Shiga
toxin. Increasing evidence suggests that the membrane mi-
croenvironment consisting of cholesterol, other glycolipids and
fatty acids also plays a role in Gb3 receptor binding. It is,
therefore, vital to determine how reactive each cell type is
when challenged with Shiga toxin not just the amount of Gb3
expressed (37). Furthermore, the Gb3 fatty acid chain length
has also been shown to affect how sensitive a cell is to Shiga
toxin (36). Therefore, caution is advised when interpreting in
vitro studies to acknowledge the differences in the cell mem-
brane fatty acid profiles in culture vs. their in vivo counterparts,
which likely affect Gb3 receptor binding to Shiga toxin (39).
All human glomerular cells (mesangial cells, podocytes, and
endothelial cells) express Gb3. However, mouse glomerular
cells lack Gb3 (19). Human podocytes and glomerular endo-
thelial cells are particularly sensitive to the cytotoxicity of
Shiga toxin. Interestingly, human podocytes exposed to the
toxin express 60% less VEGFA compared with untreated
control cells (19). In light of the finding from Eremina et al. (6)
that podocyte-specific VEGFA knockout mice develop a glo-
merular TMA, it is conceivable that the podocyte may prove to
be more important in the pathogenesis of STEC HUS than first
thought.
Human and most other animals also express Gb3 on their
tubular cells and collecting duct epithelium (37). Conse-
quently, STEC HUS results in primary renal tubular injury and
mesangial cell lysis, resulting in the release of proinflammatory
chemokines (IL-1 and TNF-␣) that potentiate the renal cyto-
toxicity of Shiga toxin (30). Van Setten et al. (47) have
demonstrated that these proinflammatory cytokines increase
Gb3 synthesis in glomerular endothelial cells. The expression
of such an abundance of biologically reactive Gb3 in the
human glomerulus and the upregulation of functional Gb3
expression in response to inflammation in glomerular endothe-
lial cells may provide one explanation as to why the kidney is
the predominant target in STEC HUS. Others have suggested
that the rich vascular supply of the kidney and filtration rate of
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F459
blood may increase exposure of the renal microvasculature to
Shiga toxin and predispose to renal injury (37).
Animal models of STEC HUS. The development of rodent
models of STEC HUS that recapitulate the glomerular TMA
lesions seen in humans is yet to be achieved. Mice challenged
with Shiga toxin alone (orally or intraperitoneally) develop
lethal tubular disease without histological evidence of glomer-
ular TMA, presumably due to the absence of Gb3 expression in
their glomeruli (19). Any vascular changes seen are driven by
the combined action of Shiga toxin and lipopolysaccharide (18,
37). In an effort to address this discrepancy several other
animal models have been used to study STEC HUS in the
rabbit, pig, and baboon.
Rabbits develop acute renal failure following STEC chal-
lenge, but this is a variable and inconsistent model. Porcine
models have not been described in detail but, similar to murine
models, tubular lesions predominate. Primate studies most
closely recapitulate the disease process observed in human
kidneys but only following intravenous infusion with Shiga
toxin (22). Enteric bacterial infection with Shiga toxin-produc-
ing E.coli in primates does not lead to HUS, and bacterial
translocation from the gut is not seen baboons (23, 51). This is
in contrast to humans where STEC induce attaching and
effacing lesions and inflammatory cytokine production in in-
testinal epithelial cells (26). As such, non-human primate
models are of use in the validation of observations in mouse
studies but are expensive and impractical given the need for
intravenous toxin administration. Furthermore, the lack of
intestinal injury observed in baboons is a limitation of this
model and suggests species-specific host factors are likely to be
very important in STEC (23). It is clear that animal models that
more closely recapitulate human HUS are needed.
EVIDENCE OF COMPLEMENT ACTIVATION IN STEC HUS
In the last decade, evidence for the role for complement
activation in Shiga toxin-associated HUS has increased. Some
patients with STEC HUS have been reported to have low levels
of circulating C3 and evidence of increased levels of C3
convertases and factor B (33). However, retrospective analyses
of the benefit of complement blockade with eculizumab in
patients during STEC HUS outbreaks have been difficult to
meaningfully interpret (28).
A recent publication by Ozaki et al. (38) has described a
novel mouse model in human MBL-expressing mice (that lack
murine MBL). Mice were given intraperitoneal Shiga toxin in
combination with or without a monoclonal antibody to human
MBL. Fascinatingly, the antibody attenuated Shiga toxin-in-
duced renal injury (38). Conventionally, atypical HUS has
been accepted to be a product of dysregulated alternative
pathway activation. This paper suggests that STEC HUS may
initially trigger the MBL pathway, which then leads to alter-
native pathway activation and amplification. There is increas-
ing evidence that components of the MBL pathway may be
able to activate the alternative pathway directly via a C2 bypass
mechanism (32). Indeed, given the high-affinity binding of
MBL to the lipopolysaccharide membrane of E. coli, it is
feasible that the initial innate immune response to STEC
infection in the gut may be predominantly driven by the MBL
pathway (48). Furthermore, MASPs activated in the MBL
immune response (specifically MASP2) cleave prothrombin to
F460 UNDERSTANDING THE PATHOGENESIS OF HUS
thrombin, resulting in a procoaguable state, which may con-
tribute to the glomerular TMA seen in HUS (16). This inter-
play between the MBL and alternative complement pathways
may even explain the low genetic penetrance of atypical HUS
where an environmental trigger is usually required alongside a
genetic predisposition (17). However, at odds with the impor-
tance of the MBL complement pathway in STEC HUS is the
finding that children with MBL deficiency are not predisposed
to the condition (40).
Evidently, our understanding of the precise pathophysiolog-
ical mechanisms underlying STEC HUS is far from complete.
Additionally, we do not understand why children and the
elderly are more prone to develop STEC HUS. A popular
theory had been that children express more renal Gb3 than
adults, but studies have shown the reverse (37). It may be the
case that the Gb3 expressed in pediatric kidneys is more
biologically active due to the membrane microenvironment
(37). Another more recent concept in complement biology has
been the theory of trained immunity, whereby the innate
immune response is capable of immunological memory (31).
This may explain why the elderly and immunosuppressed have
a higher incidence of STEC HUS.
CONCLUSIONS
Complement pathway hyperactivation is emerging as a po-
tential common mechanism underlying the pathogenesis of
glomerular TMA. Identification of the genetic mutations in
complement regulatory proteins, C3 convertase components,
and anti-CFH antibodies in atypical HUS has led to the advent
of a new era of pharmacological complement modulation (33).
Indeed, the success of eculizumab in the management of
atypical HUS has transformed the management of these pa-
tients and resulted in reduced morbidity and mortality (17).
However, with the discovery of recessive loss-of-function
mutations in DGKε an alternative mechanism for atypical HUS
independent of complement activation has been demonstrated,
suggesting other non-complement-driven pathways may also
be important (42).
In STEC HUS, delineating the role of complement, eluci-
dating which precise signaling pathways are involved, and
defining whether there is a cellular target of Shiga toxin in the
glomerulus will be important. This will be helped by develop-
ing better animal models that more accurately recapitulate
human Shiga toxin HUS (28). The challenge for the future lies
in identifying the molecular and pathophysiological mecha-
nisms responsible for STEC HUS and subsequent development
of specific treatment strategies for this devastating disease.
GRANTS
This review article was supported by Kidney Research UK Clinical Re-
search PhD Fellowship Grant TF_007_20151127 (to E. E. Bowen) and
Medical Research Council Senior Research Fellowship Grant MR/K010492/1
(to R. J. Coward).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
E.E.B. prepared figures; E.E.B. drafted manuscript; E.E.B. and R.J.C.
edited and revised manuscript; E.E.B. and R.J.C. approved final version of
manuscript.
AJP-Renal Physiol • doi:10.1152/ajprenal.00376.2017 • www.ajprenal.org
Downloaded from www.physiology.org/journal/ajprenal (195.022.251.172) on January 20, 2020.
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