Original Article

Diagnostic and Risk Factors for Complement Defects in

Hypertensive Emergency and Thrombotic Microangiopathy
Sjoerd A.M.E.G. Timmermans, Alexis Wérion, Jan G.M.C. Damoiseaux, Johann Morelle,
Chris P. Reutelingsperger, Pieter van Paassen

Abstract—Hypertensive emergency can cause thrombotic microangiopathy (TMA) in the kidneys with high rates of end-
stage renal disease (ESRD) and vice versa. The conundrum of hypertension as the cause of TMA or consequence of
TMA on the background of defects in complement regulation remains difficult. Patients with hypertensive emergency
and TMA on kidney biopsy were tested for ex vivo C5b9 formation on the endothelium and rare variants in complement
genes to identify complement-mediated TMA. We identified factors associated with defects in complement regulation
and poor renal outcomes. Massive ex vivo C5b9 formation was found on resting endothelial cells in 18 (69%) out
of 26 cases at the presentation, including the 9 patients who carried at least one rare genetic variant. Thirteen (72%,
N=18) and 3 (38%, N=8) patients with massive and normal ex vivo complement activation, respectively, progressed
to ESRD (P=0.03). In contrast to BP control, inhibition of C5 activation prevented ESRD to occur in 5 (83%, N=6)
patients with massive ex vivo complement activation. TMA-related graft failure occurred in 7 (47%, N=15) donor kidneys
and was linked to genetic variants. The assessment of both ex vivo C5b9 formation and screening for rare variants in
complement genes may categorize patients with hypertensive emergency and TMA into different groups with potential
therapeutic and prognostic implications. We propose an algorithm to recognize patients at the highest risk for defects
in complement regulation.  (Hypertension. 2020;75:00-00. DOI: 10.1161/HYPERTENSIONAHA.119.13714.)
• Online Data Supplement
Key Words: atypical hemolytic uremic syndrome ◼ biopsy ◼ complement ◼ endothelium
◼ thrombotic microangiopathies
Downloaded from http://ahajournals.org by on December 26, 2019

M ild-to-moderate hypertension may cause a slow decline

in renal function, while acute end-stage renal disease
(ESRD) can occur in patients presenting with hypertensive
emergency. ESRD is attributed to hypertension in up to 25%
adult cases in Europe and the United States1 with, unfortunately,
no confirmative histological proof, assuming that the kidneys
are the victim rather than culprit of disease. Parenchymal renal
disease, including thrombotic microangiopathy (TMA), can
therefore be missed.2 TMA represents tissue responses to en-
dothelial damage caused by distinct mechanisms, such as me-
chanical stress to the endothelium in hypertensive emergency3
and defects in complement regulation in primary atypical he-
molytic uremic syndrome.4,5
We recently demonstrated that identical defects in com-
plement regulation can be linked to ESRD in patients present-
ing with hypertensive emergency and TMA on kidney biopsy,
resembling primary atypical hemolytic uremic syndrome.6 The
defects in complement regulation are often caused by rare vari-
ants in genes encoding proteins that either regulate or activate
complement and autoantibodies that inhibit complement reg-
ulation, lowering the threshold for unrestrained C5 activation
and TMA to occur.7 The generalized endothelial damage in
primary atypical HUS can induce thrombosis, consumptive
thrombocytopenia, and mechanical hemolysis, contributing to
target organ damage, mostly affecting the kidneys. In clinical
practice, it remains challenging to identify primary atypical
HUS among patients with hypertensive emergency because
the vast majority of patients do not present with profound he-
matologic abnormalities6,8; we therefore prefer the term com-
plement-mediated TMA (C-TMA). The correct identification
of C-TMA, however, is critical given the potential therapeutic
benefit of complement inhibition.9–11 Diagnostic methods
are not specific or need to be validated.12 Kidney Disease:
Improving Global Outcomes therefore questioned whether a
functional ex vivo complement test, clinical manifestations,
and pathological features on kidney biopsy can aid the differ-
ential diagnosis in patients with hypertensive emergency.13
The primary objective of the current study was to identify
specific factors for C-TMA in patients with hypertensive emer-
gency. We therefore screened patients with hypertensive emer-
gency and TMA on kidney biopsy for massive ex vivo C5b9
formation, a specific test to detect unrestrained C5 activation

Received July 16, 2019; first decision August 9, 2019; revision accepted November 13, 2019.
From the Department of Nephrology and Clinical Immunology (S.A.M.E.G.T., P.v.P.), Central Diagnostic Laboratory, Maastricht University Medical
Center (J.G.M.C.D.), and Department of Biochemistry (S.A.M.E.G.T., C.P.R., P.v.P.), Cardiovascular Research Institute Maastricht, the Netherlands;
Division of Nephrology, Cliniques universitaires Saint-Luc (A.W., J.M.), and Institut de Recherche Expérimentale et Clinique, UCLouvain, Brussels,
Belgium (J.M.).
The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/HYPERTENSIONAHA.119.13714.
Correspondence to Pieter van Paassen, PO Box 5800, 6202 AZ, Maastricht, The Netherlands. Email p.vanpaassen@maastrichtuniversity.nl
© 2019 American Heart Association, Inc.
Hypertension is available at https://www.ahajournals.org/journal/hyp DOI: 10.1161/HYPERTENSIONAHA.119.13714

1
 2  Hypertension  February 2020
on the endothelium.14 Also, patients were tested for rare vari-
ants in complement genes linked to complement dysregulation.
Clinical manifestations and pathological features on kidney
biopsies were studied to distinguish C-TMA from hypertensive
emergency as the sole cause of TMA.13 We propose a clinical
algorithm that may guide the use of complement inhibition.
Materials and Methods
The data that support the findings of this study are available from the
corresponding author on reasonable request.
Patient Population
Patients with hypertensive emergency and TMA with typical path-
ological features of severe hypertension (ie, mucoid intimal edema)
on kidney biopsy were recruited from the Limburg Renal Registry,
Maastricht, the Netherlands,15 and the Cliniques universitaires Saint-
Luc, Brussels, Belgium, respectively. Miscellaneous causes of TMA
other than hypertensive emergency were ruled out16; the enzymatic
activity of a disintegrin and metalloproteinase with thrombospon-
din type 1 motif, member 13, that is, von Willebrand protease, was
assessed by using the FRETS-VWF73 assay in selected cases.17
Hypertensive emergency was defined as a systolic and diastolic
BP of 180/120 mm Hg and evidence of impending or progressive
extrarenal target organ damage secondary to hypertension.18 Patients
were screened for neurological and cardiac disease as considered ap-
propriate. Neurological disease was defined as acute onset of severe
headache, seizures, coma, cerebral infarction, or bleeding on imag-
ing; cardiac disease was defined as ventricular dysfunction or acute
ischemia on ECG and ultrasound.
At the time of presentation and during follow-up, serum samples
were obtained, processed, and immediately stored at −80°C to pre-
vent in vitro complement activation.19 The study was approved by
the appropriate ethics committees and is in accordance with the
Downloaded from http://ahajournals.org by on December 26, 2019
Declaration of Helsinki.
Kidney Tissue Specimens
Kidney tissue sections were processed as described.20 The sections
were scored as glomerular TMA or isolated intimal edema, the latter
of which reflects mucoid intimal edema and clear absence of acute
glomerular lesions (eg, thrombosis, endothelial cell swelling, and
mesangiolysis). Tubular atrophy and interstitial fibrosis were scored
as mild (<25%), moderate (25%–50%), or severe (>50%).
Also, sections from snap-frozen kidney specimens were stained
with rabbit anti-human C5b9 pAb (1:100; Calbiochem, San Diego,
CA) followed by Alexa488 labeled goat anti-rabbit Ab (1:100; Life
Technologies, Carlsbad, CA).
Complement Work-Up
Ex vivo C5b9 formation on microvascular endothelial cells of dermal
origin (ATCC, Manassas, VA) was assessed to identify patients with
defects in complement regulation as described.12 Briefly, endothelial
cells were plated on glass culture slides and used when >80% con-
fluent, incubated with serum diluted in test medium for 3 hours at
37°C, fixed in 3% formaldehyde, and blocked with 2% BSA for 1
hour. In selected experiments, endothelial cells were preincubated
with 10 µmol/L ADP for 10 minutes to mimic a perturbed endothe-
lium.14 Rabbit anti-human C5b9 pAb (Calbiochem) and Alexa488 la-
beled goat anti-rabbit Ab (Life Technologies) were used. The results
were compared with pooled normal human serum run in parallel.
Also, patients were screened for rare variants, that is, variants
with a minor allele frequency <1%, and single nucleotide polymor-
phisms in coding regions of CFH, CFI, CD46, CFB, C3, CFHR1-
5, THBD, and DGKE using DNA sequencing.7 The classification of
variants was based on international standards.21 Pathogenic variants
were defined as those with functional studies supporting a defect in
complement regulation, including null variants in genes linked to
complement regulation and variants that cluster in patients with a-
typical HUS as demonstrated by Osborne et al.22 Likely pathogenic
variants were defined as those with functional studies supporting a
defect in complement regulation that have been located in a muta-
tional hotspot and critical functional domain. In addition, rare vari-
ants not fulfilling these criteria have been classified as uncertain
significance. The CFH-CFHR1-5 genomic region was analyzed for
rearrangements by multiplex ligation probe amplification.12 Factor H
autoantibodies were assessed by ELISA in selected cases.23
C-TMA was defined as massive ex vivo C5b9 formation on rest-
ing endothelial cells at the time of presentation and/or the presence of
(likely) pathogenic variants in complement genes.
Statistical Analysis
Continuous variables were presented as mean (±SD) or median (in-
terquartile range) as appropriate. Differences in continuous and cate-
gorical variables were checked using the unpaired t or Mann-Whitney
U test and the χ2 or Fisher exact test, respectively. The ex vivo forma-
tion of C5b9 on the endothelium was compared with normal human
serum run in parallel by the paired sample t test or Wilcoxon signed-
rank test as appropriate. Survival was assessed using the Kaplan-
Meier method and log-rank test.
Results
Patient Population
Twenty-six patients (European, n=22; African, n=4) with
hypertensive emergency and TMA on kidney biopsy
were included (Table 1; Figure S1 in the online-only Data
Supplement). Patients invariably presented with severe kidney
failure (median serum creatinine, 723 µmol/L; interquartile
range, 423–1071) and proteinuria, 17 (65%) of whom initially
required dialysis. Kidney biopsies were needed to detect the
TMA in 19 (73%) cases because profound systemic hemol-
ysis was not present. Of note, prominent intimal fibrosis of
the arteries reflected preexisting uncontrolled hypertension.
The enzymatic activity of von Willebrand protease appeared
normal, that is, >10% in the 17 patients who have been tested
for; the other patients presented with platelet counts of over
100 000 per microliters, making thrombotic thrombocytope-
nic purpura highly unlikely.24 Drug use, infection, autoim-
mune disease, and pregnancy as causes of TMA were ruled
out. Familial disease was not noted. Extrarenal manifestations
included severe hypertensive retinopathy (ie, grade III and/or
IV; n/N=22/25, 88%), cardiac disease (n/N=18/22, 82%), and
neurological disease (n/N=6/26, 23%).
Kidney Biopsy Findings
Mucoid intimal edema was invariably present either with glo-
merular TMA (n=13) or not (n=13), that is, those with iso-
lated intimal edema. Mucoid intimal edema, however, was
not found in C-TMA without hypertensive emergency (data
not shown). The typical features on kidney biopsy have been
depicted in Figure 1. On average, 16 (±7) glomeruli were pre-
sent. Most tissue specimens classified as glomerular TMA
showed acute lesions, that is, glomerular thrombosis, endothe-
lial cell swelling, endocapillary hypercellularity, and mesan-
giolysis; 2 (15%) out of 13 specimens classified as glomerular
TMA showed chronic rather than acute lesions, that is, mem-
branoproliferative glomerulonephritis. The glomeruli from
those classified as isolated intimal edema showed wrinkling
of the glomerular basement membrane either with ischemic
collapse of the capillary tuft or not; one of these specimens
(analyzed, n/N=6/13) showed abnormalities linked to TMA on
 Timmermans et al   Hypertensive Emergency and Complement   3

Table 1. The Patients’ Characteristics at the Time of Presentation

Parameter Total C-TMA No Complement Defects P Value

N 26 18 8
M/F 15/11 9/9 6/2 0.39
European, % 22–85 18–100 4, 50 <0.01
Age, y 39 (±11) 40 (±9) 40 (±8) 0.81
SBP, mm Hg 217 (±32) 210 (±29) 236 (±35) 0.08
DBP, mm Hg 130 (120–148) 120 (120–140) 146 (140–154) 0.02
LDH, U/L 731 (335–1168) 638 (303–1200) 762 (612–1128) 0.33
Platelets, ×103/µL 168 (±79) 184 (±78) 113 (±55) 0.04
Creatinine, µmol/L 723 (423–1071) 820 (579–1152) 605 (410–971) 0.19
Dialysis, % 17 14–78 3–38 0.08
Glomerular thrombosis 13 12 1 0.03
Extrarenal manifestations
 Neurological disease, n/N 6/26 5/18 1/8 0.63
 Hypertensive retinopathy, n/N 22/25 14/17 8/8 0.53
 Cardiac disease, n/N 18/22 11/14 7/8 1.00
 Systemic hemolysis, % 7–27 4–22 3–38 0.64
Complement
 Low C4, n/N 0/22 0/15 0/7
 Low C3, n/N 7/24 6/17 1/7 0.62
 Rare variant(s), % 9–35 9–50 0 0.02
Outcome
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 Renal response 10 5 5 0.19

 ESRD at 3 mo 16 13 3 0.03
 Donor kidneys 15 12 3
 Relapse 7 7 0 0.20
Systemic hemolysis was defined as microangiopathic hemolytic anemia (ie, hemoglobin <10 g/dL, lactate dehydrogenase >500 U/L,
schistocytes on peripheral blood smear) and platelets <150 000/µL. C-TMA indicates complement-mediated thrombotic microangiopathy; DBP,
diastolic blood pressure; ESRD, end-stage renal disease; F, female; LDH, lactate dehydrogenase; M, male; and SBP, systemic blood pressure.

electron microscopy, that is, widening of the glomerular base-
ment membrane with electron lucent material. Mild tubular
atrophy and interstitial fibrosis was present in 11, moderate in
8, and severe in 7 cases.
Frozen kidney specimens of 20 patients were available
for immunofluorescence microscopy. Focal granular C3c
and C5b9 deposits were found at the vascular pole and along
segments of the glomerular basement membrane in 9 (53%,
N=17) and 7 (47%, N=15) cases, respectively; colocalization
of C3c and C5b9 was found in 6 of these cases. Focal granular
IgM deposits were found along segments of the glomerular
basement membrane in 7 (44%, N=16) and sclerotic areas in
3 (19%, N=16) cases, while immune complex deposits were
not appreciated on electron microscopy (analyzed, n/N=9/10),
indicating unspecific entrapment of IgM.
Complement Work-Up
At presentation, serum samples were available to test for
ex vivo C5b9 formation from all but one patient (ie, the pa-
tient with combined variants in CFI and THBD as described
below). Massive ex vivo C5b9 formation on the resting endo-
thelium was found in 17 (68%) out of 25 cases tested for. Of
note, ex vivo C5b9 formation normalized on resting but not
on perturbed endothelial cells at the time of quiescent disease
(analyzed, n/N=7/17), underscoring the key role of a second
hit for unrestrained C5 activation to occur. The 8 samples with
normal test results at presentation also showed normal ex vivo
C5b9 on the perturbed endothelium, indicating normal com-
plement regulation.12,14 It is noteworthy that the ex vivo test’s
specificity is 97% (Table S1).
DNA samples were tested and rare variants in complement
genes were found in 9 (35%) out of 26 patients. The charac-
teristics of the variants, including C3 (n=2), CFH (n=2), CFI
(n=2), CD46 (n=1), and THBD (n=1) have been depicted in
Table 2; 5 and 1 out of 8 variants were considered pathogenic
and likely pathogenic, respectively. Two patients presented
with combined variants: CFI combined with THBD and CD46
combined with CFH. The at-risk CFH-H3 and MCPGGAAC hap-
lotypes were found in 8 (32%) and 2 (8%) cases, respectively;
the homozygous genomic deletion of CFHR1 and CFHR3
 4  Hypertension  February 2020
was identified in 2 patients, while factor H autoantibodies
were not found.
Thus, C-TMA was diagnosed in 18 (69%) out of 26
patients (Figure S2). Details about the complement work-up
are provided in Tables 2 and 3.
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Patient Characteristics and Outcome Depending on
Complement Defects
None of the 4 patients from African descent had C-TMA
(P=0.004). The diastolic BP appeared to be slightly lower,
whereas platelet counts were higher in patients with C-TMA
as compared with those with no complement defects (Table 1).
Extrarenal manifestations did not differ between both groups.
Most of the patients with C-TMA presented with glomerular
TMA on kidney biopsy (n/N=12/13 versus n/N=6/13 with iso-
lated intimal edema, P=0.03), while the prevalence of rare
variants in complement genes did not differ between both
pathological groups (n/N=6/13 versus n/N=3/13, P=0.4). No
specific staining for C-TMA was found on immunofluores-
cence microscopy (data not shown).
Patients were followed for a median of 2.6 (interquartile
range, 0.8–10.8) years. BP was controlled by intravenous ad-
ministration of antihypertensive agents. Thirteen (72%) of the
18 patients with C-TMA and 3 (38%) of the 8 patients with no
complement defects had ESRD (P=0.03) at 3 months follow-up,
while the other 5 patients in both groups had chronic kidney di-
sease. Except for one case, none of the patients with C-TMA
achieved a renal response (ie, renal recovery or >25% decrease
in serum creatinine) upon BP control alone. Five patients with
no complement defects who had chronic kidney disease at 3
months follow-up achieved a renal response after BP control;
the 3 nonresponders, however, had a serum creatinine of >700
µmol/L at presentation. From 2015 onward, the anti-C5 mAb
eculizumab was started in 7 cases (C-TMA, n=6) because of
the lack of a renal response. Five (83%) out of 6 patients with
Figure 1. Features on kidney biopsy of 2
patients with a pathogenic variant in C3 (ie,
c.418C>T). Mucoid intimal edema was present
(C; periodic acid-Shiff stain, 400×) either with
glomerular thrombotic microangiopathy (A;
Jones methenamine silver, 400×) or not (B;
Jones methenamine silver, 630×). Electron
microscopy revealed widening of the GBM with
electron lucent material (D, 1900×).
C-TMA who received eculizumab recovered and improved
renal function (Figure 2A); no response, however, was achieved
in the patient with no complement defects. Follow-up serum
samples from 6 treated patients (C-TMA, n=5), indeed, con-
firmed adequate blockade of C5 activation (Table 3).
Fifteen donor kidneys were transplanted in 7 recipients with
C-TMA (carriers of pathogenic variants in complement genes,
n=6) and 2 recipients with no complement defects (Figure 2B).
TMA manifested in 7 donor kidneys of 4 recipients with path-
ogenic variants in complement genes (ie, C3, n=3, and CD46
combined with CFH,25 n=1) but not in those with no comple-
ment defects (P=0.1). Recurrence of TMA after kidney trans-
plantation was linked to graft failure in all cases.
Discussion
Here, we demonstrate defects in complement regulation as the
key causative factor of poor renal outcomes in patients with
hypertensive emergency, TMA, and severe kidney failure.
Neither clinical manifestations, including hypertensive reti-
nopathy grade III and IV, nor pathological features on kidney
biopsy could differentiate C-TMA from hypertensive emer-
gency as the key cause of disease. Massive ex vivo C5b9 for-
mation on the endothelium, however, indicated C-TMA. Most
importantly, massive ex vivo C5b9 formation on the endothe-
lium predicted a poor response upon BP control, while com-
plement inhibition appeared effective in most of the treated
cases. Half the patients with abnormal test results carried rare
variants in complement genes, confirming the genetic predis-
position for C-TMA to develop. Thus, our findings indicate
that massive ex vivo C5b9 formation holds promise for the
recognition of C-TMA and may be suitable to guide the use
of complement inhibition in patients with hypertensive emer-
gency. Moreover, pathogenic variants in complement genes
predicted the risk for TMA and subsequent graft failure after
kidney transplantation.
 Timmermans et al   Hypertensive Emergency and Complement   5

Table 2. Complement Work-Up in 26 Patients With Hypertensive Emergency and Thrombotic Microangiopathy on Kidney Biopsy

CFH- ΔCFHR1-
Patient no. Variant(s) Protein MAF In Vitro Defect H3/MCPGGAAC CFHR3 FHAA Resting ADP-Activated
M99918 None N/N N ND 121% 121%
M09018 C3 c.463A>C* K155Q 0.2–0.4 GOF Y/Y N ND 326%† 403%‡
M06018 None N/Y Y None 297%‡ ND
M09717 None N/N Y None 223%† 190%†
M99917 CFI c.148C>G* P50A <0.02 LOF Y/N Y ND ND ND
THBD c.1433C>T§ T478I <0.01 Unknown
M01217 None Y/N Y None 160% 117%
M10216 None N/N Y None 353%† 205%†
M04516 None N/N N None 198%‡ 204%‖
M01416 None N/N N None 245%‡ 340%‡
M01016 None Y/N N None 224%‖ ND
M02715 C3 c.481C>T* R161W <0.01 GOF N/N Y¶ None 373%‡ 246%†
M01715 CFI c.452A>G* N151S <0.01 LOF N/N N ND 395%‡ 252%‖
M04010 CFH c.2558G>A* C853Y 0 LOF Y/N N ND 339%† 253%†
M03307 C3 c.481C>T* R161W <0.01 GOF Y/N N ND 463%‡ 404%‡
M04306 CD46 ΔD271/S272 0 LOF Y/N Y None 284%‡ 272%‡
c.811_816delGACAT*
CFH c.2850G>T§ Q950H 0.1–0.4 LOF(?)
M00105 C3 c.481C>T* R161W <0.01 GOF N/N N None 310%‡ 325%†
M03802 None N/N N None 140% 92%
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M05595 None N/N N ND ND 95%

M05486 C3 c.481C>T* R161W <0.01 GOF Y/N N ND 336%‡ 283%‡
M06880 None Y/N N ND 306%‡ 255%†
B47 None N/N N None 99% 121%
B30 None N/N N None 162% 125%
B17 None N/N N None 87% 117%
B04 None N/N N None 394%† ND
B03 None N/N Y¶ None 159% 85%
B02 None N/N N None 197%‖ ND
FHAA indicates factor H autoantibodies; MAF, minor allele frequency according to Exome Variant Server (EVS) and Genome Aggregation Database (gnomAD); N, not
found; ND, not determined; and Y, found.
*Pathogenic variant.
†P value <0.01.
‡P value <0.001.
§Variant of uncertain significance.
‖P value <0.05.
¶ΔCFHR1-CFHR3 came in homozygosity.

Patients with hypertensive emergency may present with
acute kidney failure and TMA in over 50% and up to 25% of
cases, respectively.26 TMA may be under-recognized as most
patients with hypertensive emergency and TMA on kidney
biopsy lack systemic hemolysis. Thus, a kidney biopsy is
imperative to detect the TMA; obviously, control of BP and
other clinical routines is mandatory to lower the risk of bleed-
ing.27 Patients with TMA should be screened for severe von
Willebrand protease deficiency and other causes.16 The conun-
drum of hypertensive emergency as a cause or consequence of
TMA, however, remains difficult.13
DNA testing can confirm the genetic predisposition for
C-TMA in half the patients13 but does not reflect the dynamic pro-
cess of complement activation. Levels of circulating complement
proteins and markers of complement activation also lack sensitivity
and specificity as complement in C-TMA is activated on the endo-
thelium (ie, the solid phase) rather than the fluid phase.12,14 Ex vivo
C5b9 formation reflects solid phase but not fluid phase comple-
ment activation.12,14 Here, we demonstrated that massive ex vivo
C5b9 formation reflects unrestrained C5 activation on the endothe-
lium regardless of the genetic predisposition. Ex vivo C5b9 forma-
tion, indeed, normalized on resting but not on perturbed endothelial
 6  Hypertension  February 2020
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Figure 2. The clinical outcome before and after kidney transplantation. A,
The cumulative incidence of the composite renal end point, that is, renal
response and recovery, was higher in patients with complement-mediated
thrombotic microangiopathy (TMA) who had been treated with eculizumab
vs those who had not been treated; the composite renal end point
occurred after a median of 1.8 mo (P=0.001). B, TMA and subsequent graft
failure was common in kidney donor recipients with pathogenic variants;
TMA recurred after a median of 5.0 y (P=0.05).
cells at the time of quiescent disease. The clinical course of patients
with massive ex vivo C5b9 formation was poor,4,5 with early ESRD
occurring in most patients. BP control was ineffective in all but
one case, while eculizumab blocked ex vivo C5b9 formation and
improved the renal function among patients classified as C-TMA.
In contrast, BP control was effective in over half the patients with
normal ex vivo test results at presentation, resembling the natural
course of so-called malignant nephrosclerosis.28 Thus, our observa-
tions indicate that massive ex vivo C5b9 formation differentiates
C-TMA from TMA caused by hypertensive emergency alone.12
C-TMA was associated with a poor prognosis, indicating
C5 as a therapeutic target for the treatment of TMA in patients
with hypertensive emergency. Eculizumab’s dosing schedule
and treatment duration, however, remain to be established. In
line with Galbusera et al,29 a prolonged interdose interval of
3 to 4 weeks retained normal ex vivo C5b9 formation on the
perturbed endothelium despite a residual activity of the clas-
sical pathway above the recommended goal of <10% during
complement inhibition.13 None of the reported patients to date
developed a relapse of disease, suggesting that trough levels
below the recommended target concentration30 can prevent un-
restrained C5 activation, potentiating lower costs of treatment.
Knowledge of the genetic predisposition is instrumental to
make informed decisions regarding transplantation in poten-
tial recipients with TMA in their native kidneys.13 Patients with
(likely) pathogenic variants in CFH, CFB, C3, and those with
combined mutations, are at high risk of disease recurrence.31
In our cohort, TMA recurrence occurred in high-risk recipients
who carried pathogenic variants in complement genes,13 under-
scoring the importance of genetic testing before kidney trans-
plantation. Eculizumab can prevent TMA to manifest and its
sequelae.32 Living kidney donation and adequate BP control may
postpone the initiation of eculizumab33 and, in particular, be-
cause TMA developed as a late complication after kidney trans-
plantation. However, one should keep in mind that the allograft’s
capacity to recover is limited as compared with native kidneys.34
Of note, in our experience, several patients with C-TMA in
their native kidneys have been misdiagnosed as hypertensive
ESRD. Neither clinical manifestations nor pathological features
on kidney biopsy appeared specific for C-TMA in patients with
hypertensive emergency. Thus, impending hypertensive organ
damage outside the kidneys, including severe retinopathy, should
not be used to exclude atypical HUS as such manifestations are
common in patients with atypical HUS who present with hyper-
tensive emergency.12,35 Larsen et al36 recently suggested that the
presence of isolated intimal edema on kidney biopsy can exclude
C-TMA as none of their patients carried rare variants in comple-
ment genes, contrasting our data. Further conclusions from their
study, however, were limited by incomplete clinical data. Most of
Larsen et al37 patients are from African descent, a population with
a high burden of hypertensive emergency and ESRD as compared
with those from European descent. No complement defects were
identified in our patients from African descent and all showed an
excellent response on BP control, pointing toward hypertension
as the sole cause of disease in this particular group of patients.
Larsen et al’s cohort might represent a mixture of distinct causes,
some of which may be related to well-known secondary etiologies
not linked to complement.38,39 Potential recipients classified as hy-
pertensive ESRD with proof or a high suspicion of TMA should
therefore be screened for rare variants in complement genes.
Based on our studies,6,12 we propose an algorithm for the eval-
uation of TMA in patients presenting with hypertensive emer-
gency (Figure 3). Patients with hypertensive emergency and severe
kidney failure who lack systemic hemolysis should be biopsied to
assess whether TMA is present or not. Patients should be screened
for other causes of TMA and, if no cause can be identified, for ex
vivo C5b9 formation. Abnormal results are indicative of a poor re-
sponse to BP control and thus, C-TMA. Furthermore, patients and,
in particular, potential kidney donor recipients, should be screened
for rare variants in complement genes to adopt suitable measures
before kidney transplantation. Prospective studies, however, are
needed to test the hypothesis that complement inhibition, either
with eculizumab or other therapies under development, will im-
prove the outcome of patients with massive ex vivo C5b9 forma-
tion. The predictive role of pathological features, such as chronic
vascular and tubulointerstitial damage,40,41 and clinical phenotype
about the response to treatment should also be addressed.
In conclusion, these observations show that assessment of
both ex vivo C5b9 formation and screening for rare variants in
 Timmermans et al   Hypertensive Emergency and Complement   7

Table 3. Ex Vivo C5b9 Formation During Follow-Up in Patients Treated With Eculizumab or Not

Ex Vivo C5b9, % of the

Control
Interval of Platelets, SCr, ADP-
Patient no. Variant(s) Treatment, w Hb, mmol/L LDH, U/L 103/μL μmol/L C4, g/L C3, g/L CPFA, % Resting Activated
TMA, active disease
M03307* C3† N/a 6.1 383 210 426 0.27 0.90 65 366‡ 307‡
TMA, quiescent disease
M04516 None N/a§ 7.3 85 ND ESRD ND ND ND 136 ND
M01416 None N/a§ 7.2 192 301 187 ND ND 121 74 251‖
M02715 C3† N/a 7.0 239 333 ESRD 0.34 1.14 114 160 231‖
M01715 CFI† N/a§ 6.6 142 399 278 0.33 1.41 >125 141 256¶
M04010* CFH† N/a 6.8 197 174 205 0.20 0.47 107 163 293¶
M03307* C3† N/a 6.1 181 232 220 0.28 0.97 77 87 170¶
M00105* C3† N/a 7.5 ND 198 98 ND ND ND 116 248‡
TMA, eculizumab treatment
M06018 None 3 7.7 134 236 399 0.22 1.10 3 ND 14¶
M99917 CFI, † 2 8.9 277 211 227 ND 1.09 2 ND 3‡
THBD#
3 8.6 165 210 190 ND ND 21 25¶ 45‖
M01217 None 1 4.9 241 309 ESRD ND ND 4 0 0
M04516 None 2 5.5 143 492 ESRD 0.34 1.36 6 0 0
M01416 None 2 6.3 154 298 234 0.28 1.12 2 17‡ 14¶
M01715 CFI† 2 5.5 179 291 315 ND ND 0 0¶ 24¶
Downloaded from http://ahajournals.org by on December 26, 2019

4 7.4 141 393 288 0.29 1.16 102 160 229‡

M04010* CFH† 4 6.8 138 216 132 0.21 0.56 0 9‡ 29¶
M03307* C3† 2 6.7 264 216 437 ND ND 2 ND 12‡
Reference values for C4, C3, and CPFA are 0.11–0.35, 0.75–1.35 g/L, and >75%, respectively. ADP indicates adenosine diphosphate; CPFA, classical pathway
functional activity; ESRD, end-stage renal disease; LDH, lactate dehydrogenase; ND, not determined; and TMA, thrombotic microangiopathy.
*Kidney donor recipient.
†Pathogenic variant.
‡P<0.001.
§Eculizumab has been discontinued for over 3 mo.
‖P<0.05.
#Variant of uncertain significance.
¶P<0.01.

complement genes may categorize the TMA in patients with
hypertensive emergency, into different groups with potential
therapeutic and prognostic implications. Furthermore, ex vivo
C5b9 formation may guide the dosage of treatment.
Perspectives
Hypertensive emergency can cause TMA via mechanical stress
to the endothelium. This cohort study revealed defects in com-
plement regulation as the key causative factor of poor renal
outcomes in patients with hypertensive emergency, TMA, and
severe kidney failure, indicating C-TMA and not mechanical
stress as the key cause of disease. In clinical practice, however,
most patients with C-TMA remain unidentified assuming that
the kidneys are the victim rather than culprit of disease. Patients
with hypertensive emergency and TMA, either on kidney bi-
opsy or blood smear, and severe kidney failure should therefore
be screened for C-TMA. Neither clinical manifestations, such
as hypertensive retinopathy grade III and IV, nor pathological
features can differentiate C-TMA from mechanical stress. We
therefore propose to screen patients for ex vivo complement ac-
tivation on the endothelium and rare variants in complement
genes. Massive ex vivo complement activation indicates a poor
response to BP control, while complement inhibition appeared
promising. Future prospective trials should focus on the use of
complement inhibition in patients with abnormal test results.
Rare variants in complement genes predict the risk of TMA
after kidney transplantation. Thus, kidney donor recipients car-
rying pathogenic variants warrant close and careful monitoring.
Acknowledgments
We gratefully thank the nephrologists affiliated with the Limburg Renal
Registry and the members of the multidisciplinary TMA/HUS team at the
 8  Hypertension  February 2020
Cliniques universitaires Saint-Luc and the UCLouvain Kidney Disease
Network for the recruitment and excellent care of patients. Furthermore,
we appreciate Nele Bijnens, Erik Geelkens, Henk van Rie, and Ruud
Theunissen (Maastricht University Medical Center) for the excellent
technical assistance and Sébastien Druart and Yvette Cnops for the
management of the biobank (Cliniques universitaires Saint-Luc). This
work was supported in part by funding from the Fondation Saint-Luc
(J. Morelle), the National Fund for Scientific Research (J. Morelle), the
Fonds de Recherche des Cliniques universitaires Saint-Luc (J. Morelle),
and the Association pour l’Information et la Recherche sur les Maladies
Rénales Génétiques (J. Morelle). S.A.M.E.G. Timmermans and P. van
Paassen participated in research idea and study design; S.A.M.E.G.
Timmermans, A. Wérion, and J. Morelle participated in data acquisition.
S.A.M.E.G. Timmermans participated in statistical analysis. J.G.M.C.
Downloaded from http://ahajournals.org by on December 26, 2019
Damoiseaux, C.P. Reutelingsperger, and P. van Paassen participated in
supervision and mentorship . Each author contributed important intel-
lectual content during manuscript drafting or revision and accepts ac-
countability for the overall work.
Sources of Funding
None.
Disclosures
We declare that the results presented in this paper have not been
published previously in whole or part, except in abstract format. P.
van Paassen declares that he has received an unrestricted educational
grant from Alexion Pharmaceuticals, Inc, which has not had any in-
fluence on the results or interpretation in this article. The other au-
thors report no conflicts.
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mediated TMA as the cause of disease.
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patients with hypertensive emergency, TMA, and severe kidney failure.
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• Rare variants in complement genes, classified as pathogenic, predict the
long-term prognosis, that is, risk of TMA after kidney transplantation.
Summary
Patients with hypertensive emergency, TMA, and severe kidney
failure should be screened for ex vivo C5b9 formation on the endo-
thelium and rare variants in complement genes to better categorize
the TMA, having impact on treatment and prognosis.