nejmoa1911481.pdf

The new engl and jour nal of medicine
n engl j med 382;5 nejm.org January 30, 2020
416
From the Division of Cardiology, Depart-
ment of Medicine, University of Michi-
gan, Ann Arbor (S.S.H.); the Divisions
of Renal Medicine (D.E.L., S. Sharma,
S.S.W.) and Pulmonary and Critical Care
Medicine (R.M.B.), Brigham and Wom-
en’s Hospital, the Section of Nephrology,
Department of Medicine, Boston Univer-
sity School of Medicine (S.S.W.), and the
Divisions of Nephrology (S. Sever) and
Cardiology (A.C., N.E.I., J.L.J.), Massachu-
setts General Hospital — all in Boston;
Emory Clinical Cardiovascular Research
Institute, Emory University School of
Medicine, Atlanta (A.S.T., M.R., A.A.Q.);
the Department of Medicine, Rush Uni-
versity Medical Center, Chicago (X.W.,
R.R.D., M.M.A., C.W., J.R.); the Section of
Nephrology, Department of Medicine,
Baylor College of Medicine, Houston
(D.S.-H., J.S.-C.P., M.W.H.); and the Vet-
erans Affairs Pittsburgh Healthcare Sys-
tem and the University of Pittsburgh
School of Medicine, Pittsburgh (S.D.W.).
Address reprint requests to Dr. Hayek
at the University of Michigan–Internal
Medicine, Frankel Cardiovascular Center,
1500 E. Medical Center Dr., Ann Arbor,
MI 48109-1382, or at shayek@med.umich.
edu; or to Dr. Reiser at Rush University,
1717 W. Congress Pkwy., Chicago, IL
60612, or at jochen_reiser@rush.edu.
Drs. Hayek, Leaf, and Samman Tahhan
contributed equally to this article.
N Engl J Med 2020;382:416-26.
DOI: 10.1056/NEJMoa1911481
Copyright © 2020 Massachusetts Medical Society.
BACKGROUND
Acute kidney injury is common, with a major effect on morbidity and health care utiliza-
tion. Soluble urokinase plasminogen activator receptor (suPAR) is a signaling glycopro-
tein thought to be involved in the pathogenesis of kidney disease. We investigated
whether a high level of suPAR predisposed patients to acute kidney injury in multiple
clinical contexts, and we used experimental models to identify mechanisms by which
suPAR acts and to assess it as a therapeutic target.
METHODS
We measured plasma levels of suPAR preprocedurally in patients who underwent coronary
angiography and patients who underwent cardiac surgery and at the time of admission to
the intensive care unit in critically ill patients. We assessed the risk of acute kidney injury
at 7 days as the primary outcome and acute kidney injury or death at 90 days as a second-
ary outcome, according to quartile of suPAR level. In experimental studies, we used a
monoclonal antibody to urokinase plasminogen activator receptor (uPAR) as a therapeutic
strategy to attenuate acute kidney injury in transgenic mice receiving contrast material.
We also assessed cellular bioenergetics and generation of reactive oxygen species in
human kidney proximal tubular (HK-2) cells that were exposed to recombinant suPAR.
RESULTS
The suPAR level was assessed in 3827 patients who were undergoing coronary angiogra-
phy, 250 who were undergoing cardiac surgery, and 692 who were critically ill. Acute
kidney injury developed in 318 patients (8%) who had undergone coronary angiography.
The highest suPAR quartile (vs. the lowest) had an adjusted odds ratio of 2.66 (95% con-
fidence interval [CI], 1.77 to 3.99) for acute kidney injury and 2.29 (95% CI, 1.71 to 3.06)
for acute kidney injury or death at 90 days. Findings were similar in the surgical and
critically ill cohorts. The suPAR-overexpressing mice that were given contrast material
had greater functional and histologic evidence of acute kidney injury than wild-type mice.
The suPAR-treated HK-2 cells showed heightened energetic demand and mitochondrial
superoxide generation. Pretreatment with a uPAR monoclonal antibody attenuated kidney
injury in suPAR-overexpressing mice and normalized bioenergetic changes in HK-2 cells.
CONCLUSIONS
High suPAR levels were associated with acute kidney injury in various clinical and
experimental contexts. (Funded by the National Institutes of Health and others.)
ABSTR ACT
Soluble Urokinase Receptor and Acute
Kidney Injury
Salim S. Hayek, M.D., David E. Leaf, M.D., Ayman Samman Tahhan, M.D.,
Mohamad Raad, M.D., Shreyak Sharma, M.B., B.S.,
Sushrut S. Waikar, M.D., M.P.H., Sanja Sever, Ph.D., Alex Camacho, Ph.D.,
Xuexiang Wang, M.D., Ph.D., Ranadheer R. Dande, M.D.,
Nasrien E. Ibrahim, M.D., Rebecca M. Baron, M.D., Mehmet M. Altintas, Ph.D.,
Changli Wei, M.D., Ph.D., David Sheikh‑Hamad, M.D., Jenny S.‑C. Pan, M.D.,
Michael W. Holliday, Jr., M.D., Ph.D., James L. Januzzi, M.D.,
Steven D. Weisbord, M.D., Arshed A. Quyyumi, M.D.,
and Jochen Reiser, M.D., Ph.D.
Original Article
The New England Journal of Medicine
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Copyright © 2020 Massachusetts Medical Society. All rights reserved.

n engl j med 382;5 nejm.org January 30, 2020 417
Soluble Urokinase Receptor and Acute Kidney Injury
T
he incidence of acute kidney in-
jury is increasing globally. Acute kidney
injury occurs in 2 to 5% of hospitalized
adults and has a major effect on morbidity and
health care utilization.1-4
The largest burden of
acute kidney injury occurs in critically ill pa-
tients and in persons with cardiovascular dis-
ease, who are at increased risk for both acute
kidney injury and chronic kidney disease owing
to their older age and multiple coexisting condi-
tions, as well as their greater likelihood of un-
dergoing procedures that may directly affect the
kidneys, such coronary angiography or cardiac
surgery.4-6
Despite recent gains in our under-
standing of the causes and underlying mecha-
nisms of acute kidney injury, few therapeutic or
preventive options exist.7
Thus, uncovering new
therapeutic targets for the prevention of acute
kidney injury is of importance.
Inflammation and oxidative stress are central
components of the pathogenesis of acute kidney
injury, implicating multiple subtypes of immune
cells.8,9
Evidence of a pathway linking the bone
marrow to kidney injury has emerged, involving
soluble urokinase plasminogen activator receptor
(suPAR)7,10-17
— the circulating form of a glycosyl-
phosphatidylinositol–anchored three-domain
membrane protein. This receptor is normally
expressed at very low levels on a variety of cells,
including endothelial cells, podocytes, and, with
induced expression, immunologically active cells
such as monocytes and lymphocytes.11,16,18
Levels
of suPAR are strongly predictive of progressive
decline in kidney function.17,19-23
Long-term ex-
posure to elevated suPAR levels directly affects
the kidneys by means of pathologic activation of
αvβ3 integrin expressed in podocytes, resulting
in proteinuria.7,12,16,24
Whether suPAR has an ef-
fect on kidney tubular cells — the cells most
affected in acute kidney injury — is unclear.
We investigated whether a high level of suPAR
was associated with acute kidney injury in pa-
tients undergoing coronary angiography and
sought to replicate the findings in two other
clinical contexts in which patients are at high
risk for acute kidney injury: cardiac surgery and
critical illness. We then used experimental mod-
els to determine whether the overexpression of
suPAR led to worsening of renal function and
assessed the potential for prevention of acute
kidney injury by means of pharmacologic inhibi-
tion of suPAR.
Methods
Acute Kidney Injury and suPAR
We evaluated the association between suPAR
levels and postprocedural acute kidney injury in
two prospective cohorts of patients undergoing
coronary angiography for suspected coronary
artery disease: the Emory Cardiovascular Biobank
(EmCAB) and the Catheter Sampled Blood Archive
in Cardiovascular Diseases (CASABLANCA). To
determine whether suPAR was associated with
acute kidney injury unrelated to the use of con-
trast material we sought to replicate our find-
ings in patients at high risk for acute kidney
injury who were undergoing cardiac surgery and
in critically ill patients who had been admitted
to the intensive care unit (ICU).25-27
Coronary Angiography Cohorts
EmCAB and CASABLANCA are prospective ob-
servational cohorts consisting of adult patients
(≥18 years of age) undergoing coronary angiog-
raphy for suspected ischemic heart disease.25,26
EmCAB enrolled patients at three Emory Health-
care sites in Atlanta between 2003 and 2015, and
CASABLANCA enrolled patients at Massachusetts
General Hospital in Boston between 2008 and
2011. EmCAB excluded patients with congenital
heart disease, severe anemia, a recent blood
transfusion, myocarditis, or a history of active
inflammatory disease or cancer. The only exclu-
sion criterion in CASABLANCA was an unwill-
ingness to participate.
Patients without end-stage kidney disease who
had a serum creatinine–based measurement of
kidney function at baseline and at least one mea-
surement obtained within 7 days after angiogra-
phy were included in this analysis. We measured
suPAR in blood samples that were obtained at
the time of the procedure before the injection
of contrast material. Both studies were approved
by the institutional review board at the respective
institutions.
The authors had full access to the data and
take responsibility for the completeness and ac-
curacy of the data and for the integrity of the
analysis. All the participants provided written
informed consent at the time of enrollment.
Details of the cardiac surgery cohort and the
ICU cohort are provided in Section I in the
Supplementary Appendix, available with the full
text of this article at NEJM.org.

418
Measurement of Kidney Function
and Definition of Acute Kidney Injury
Measurements of serum creatinine at enrollment
and all subsequent values obtained during the
index hospitalization were obtained from elec-
tronic medical records. The estimated glomeru-
lar filtration rate (eGFR) was calculated with the
use of the Chronic Kidney Disease Epidemiology
Collaboration equation.28
Acute kidney injury was
defined according to the Kidney Disease: Im-
proving Global Outcomes (KDIGO) Work Group
criteria as an absolute increase in the creatinine
level of at least 0.3 mg per deciliter (30 μmol per
liter) within the first 48 hours after the proce-
dure or ICU admission, a relative increase of at
least 50% in the creatinine level within the first
7 days after the procedure or ICU admission, or
use of dialysis.29
The creatinine measurement
obtained immediately before the procedure or
on admission to the ICU was used as the base-
line value for all analyses.
Sample Collection and Measurement of suPAR
Blood samples were obtained as described, and
plasma was stored at −80°C in EDTA-coated
tubes. Experienced technicians who were un-
aware of the clinical data measured suPAR in
plasma using a commercially available enzyme-
linked immunosorbent assay (ViroGates). The
lower limit of detection was 100 pg per milli
liter. The interassay coefficient of variation, which
was determined with the use of blinded replicate
samples obtained from study patients, was 10.9%.
We and others have found that suPAR levels are
stable in stored plasma and serum samples and
that levels are reproducible in samples that have
been stored for more than 5 years at −80°C de-
spite exposure to multiple freeze–thaw cycles.14,30
Animal Model of Acute Kidney Injury
We used C57BL/6J wild-type mice and trans-
genic mice overexpressing suPAR in an animal
model of contrast-induced nephropathy to study
the effect of suPAR on kidney function.12,17
The
median suPAR level in the suPAR-transgenic
mice at 10 weeks of age was 210 ng per milliliter
(interquartile range, 160 to 256). We injected
iohexol intraperitoneally in suPAR-transgenic
mice (20 mice, 9 of which were male) and in
wild-type controls (16 mice, 8 of which were
male) following published protocols.31
The mice
were also randomly assigned to receive an intra-
peritoneal injection of either urokinase plasmino-
gen activator receptor (uPAR)–blocking mono-
clonal antibody or the same concentration of
IgG isotype. Serum creatinine and kidney histo-
logic tests were used to assess the severity of
acute kidney injury. Experimental details are
provided in the Supplementary Appendix.14,16,32,33
The study was approved by the Institutional Ani-
mal Care and Use Committee of Rush University
in Chicago.
Effect of SuPAR on Kidney Tubular Cell
Bioenergetics
We quantified the generation of reactive oxygen
species and cellular bioenergetics of human kid-
ney proximal tubular (HK-2) cells that were ex-
posed to recombinant suPAR at a concentration
of 10 ng per milliliter, with or without anti-uPAR
antibody, using the MitoSOX (Invitrogen) and
Seahorse Extracellular Flux Analyzer (Agilent),
respectively. Details of the experiments are pro-
vided in the Supplementary Appendix.34
Statistical Analysis
Continuous variables are presented as means
(±SD) or as medians (with interquartile ranges)
for normally and nonnormally distributed data,
respectively. Categorical variables are presented
as percentages. To compare patients across
quartiles of suPAR level, we used analysis of
variance or the Kruskal–Wallis test for continu-
ous variables and chi-square tests for categorical
variables.
We used logistic regression to characterize the
association between suPAR levels and acute kid-
ney injury at 7 days as the primary outcome and
acute kidney injury or death at 90 days as a sec-
ondary outcome. We assessed suPAR levels both
as a continuous variable (natural log–transformed)
and as quartiles, with the lowest quartile serving
as the reference group. We adjusted for covari-
ates using three models. In all the cohorts,
model 1 was unadjusted. In the coronary angi-
ography cohort, model 2 was adjusted for age,
sex, race, smoking history, diabetes mellitus,
congestive heart failure, hypertension, and cohort
(EmCAB or CASABLANCA); model 3 incorpo-
rated the aforementioned variables in addition
to acute myocardial infarction, revascularization,
volume of contrast material, and baseline eGFR.
In the cardiac surgery cohort, model 2 was ad-
justed for age, sex, race, diabetes mellitus,

congestive heart failure, and preoperative eGFR;
model 3 incorporated the aforementioned vari-
ables in addition to urgent procedure and cardio-
pulmonary-bypass time of more than 120 min-
utes. In the ICU cohort of critically ill patients,
model 2 was adjusted for age, sex, race, baseline
eGFR, diabetes mellitus, congestive heart failure,
chronic lung disease, and chronic liver disease;
model 3 was further adjusted for vasopressors
received during the first 24 hours of ICU admis-
sion, mechanical ventilation during the first 24
hours of ICU admission, and the hemoglobin
level and white-cell count at ICU admission.
To investigate the possibility of effect modifi-
cation attributed to differences in baseline char-
acteristics, we computed odds ratios for the as-
sociation between suPAR levels and acute kidney
injury in relevant subgroups and performed tests
of interaction. Finally, we calculated the area
under the curve (AUC) to assess the incremental
value of adding suPAR to the Simplified Integer
Risk Score for Calculating the Risk of Acute
Kidney Injury, a validated clinical score derived
from the National Cardiovascular Data Registry
(NCDR) and used to predict the risk of contrast-
induced nephropathy; the score includes age,
preprocedural eGFR, history of stroke, history of
heart failure, history of percutaneous coronary
intervention, acute coronary syndrome on pre-
sentation, diabetes, chronic lung disease, hyper-
tension, cardiac arrest, anemia, heart failure at
presentation, balloon-pump use, and cardio-
genic shock.35
For the experiments in animals, we used a
two-way analysis of variance and post hoc tests
(least significant differences) to compare creati-
nine levels and kidney injury scores between
suPAR-transgenic mice and wild-type mice and
between the mice that received IgG isotype and
those that received uPAR monoclonal antibody.
Two-tailed P values of 0.05 or less were consid-
ered to indicate statistical significance. All the
analyses were performed with the use of SPSS
software, version 24 (IBM).
Results
Baseline Characteristics of Patients
and Determinants of Acute Kidney Injury
The study included 3827 patients undergoing
coronary angiography: 2752 from the EmCAB
cohort, and 1075 from the CASABLANCA co-
hort. Summary statistics for each of these co-
horts are reported in Table S1 in the Supplemen-
tary Appendix.
Postprocedural acute kidney injury developed
in 318 patients (8%). The mean increase in the
creatinine level was 0.44±0.54 mg per deciliter
(39±50 μmol per liter) among patients with
acute kidney injury, as compared with 0.01±0.22
mg per deciliter (1±20 μmol per liter) among
those without acute kidney injury. The majority
of cases of acute kidney injury within 7 days
after angiography were mild (98% of the cases
were of KDIGO stage 1), with 28 patients having
KDIGO stage 2 acute kidney injury and 3 pa-
tients having KDIGO stage 3 acute kidney injury.
Patients with acute kidney injury after coronary
angiography were more likely than those with-
out acute kidney injury to be older, to have dia-
betes mellitus, to have a history of heart failure,
to have a higher suPAR level, to have a lower
baseline eGFR, to have received a lower volume
of contrast material, and to have undergone per-
cutaneous coronary intervention at the time of
angiography (Table 1). In multivariable analysis,
only diabetes mellitus, history of heart failure,
lower eGFR, and higher suPAR levels were inde-
pendently associated with acute kidney injury.
Association of suPAR and Acute Kidney Injury
The characteristics of the patients, stratified ac-
cording to quartiles of suPAR level, are shown in
Table S2. After coronary angiography, the inci-
dence of acute kidney injury was 14% in the
highest suPAR quartile (≥4184 pg per milliliter)
and 4% in the lowest quartile (<2475 pg per milli
liter), which yielded an unadjusted odds of acute
kidney injury that was 3.8 times as high in the
highest quartile as in the lowest quartile (Fig. 1A).
The association between suPAR level and post-
procedural acute kidney injury persisted despite
adjustment for clinical characteristics (model 2),
including the volume of contrast material and
baseline kidney function (model 3; adjusted
odds ratio, 2.66; 95% confidence interval [CI],
1.77 to 3.99). The results were consistent when
we examined the suPAR level as a continuous
variable (per natural log) (adjusted odds ratio,
2.10; 95% CI, 1.54 to 2.87). The suPAR level was
also strongly associated with the combined out-
come of acute kidney injury or death from any
cause at 90 days (adjusted odds ratio, 2.29; 95%
CI, 1.71 to 3.06) (Table S3). In subgroup and

420
sensitivity analyses, the odds ratios (per natural
log of suPAR) for acute kidney injury remained
consistent across relevant subgroups, including
each cohort separately (Fig. 1B).
Last, we examined the incremental value of
adding suPAR to the NCDR Simplified Integer
Risk Score in predicting the risk of acute kidney
injury after coronary angiography.35
The AUC for
the NCDR risk score was 0.579 (95% CI, 0.560 to
0.597). The addition of suPAR to the NCDR score
modestly improved the AUC to 0.628 (95% CI,
0.610 to 0.647), with a change in the AUC of
0.050 (95% CI, 0.013 to 0.087).
Cardiac Surgery and ICU Cohorts
The demographic and clinical characteristics of
the patients in the surgical and ICU cohorts are
listed in Tables S4 through S7. Among 250 pa-
tients who underwent cardiac surgery, the inci-
dence of acute kidney injury was 40% in the
highest suPAR quartile (≥5100 pg per milliliter)
and 16% in the lowest quartile (<2860 pg per
Figure 1 (facing page). Risk of Acute Kidney Injury
after Coronary Angiography.
Panel A shows the odds ratios and 95% confidence
intervals (CIs; I bars) for acute kidney injury according
to quartiles of soluble urokinase plasminogen activator
receptor (suPAR) level before the procedure. Model 1
was unadjusted; model 2 was adjusted for age, sex,
race, smoking history, diabetes mellitus, congestive
heart failure, hypertension, and cohort (EmCAB [Emory
Cardiovascular Biobank] or CASABLANCA [Catheter
Sampled Blood Archive in Cardiovascular Diseases]);
and model 3 incorporated the aforementioned variables
in addition to acute myocardial infarction, revasculariza-
tion, volume of contrast material, and baseline kidney
function (estimated glomerular filtration rate). Quartile
1 was the reference group (1.00) in all models, with a
suPAR level of less than 2475 pg per milliliter. The suPAR
levels in quartiles 2, 3, and 4 were 2475 to 3198 pg per
milliliter, 3199 to 4183 pg per milliliter, and 4184 pg per
milliliter or more, respectively. Panel B shows the odds
ratios for acute kidney injury per 1 unit natural log of
suPAR according to subgroup in the unadjusted analy-
sis (model 1). Stage 3 chronic kidney disease was de-
fined as an estimated glomerular filtration rate of less
than 60 ml per minute per 1.73 m2
of body-surface area.
Characteristic
No Acute Kidney Injury
(N = 3509)
Acute Kidney Injury
(N = 318) P Value
Age — yr 66±12 68±12 <0.001
Male sex — no. (%) 2413 (69) 224 (70) 0.54
Black race — no. (%)† 467 (13) 37 (12) 0.40
Body-mass index‡ 29±6 30±7 0.26
Smoking — no. (%) 2314 (66) 196 (62) 0.12
Type 2 diabetes mellitus — no. (%) 1206 (34) 139 (44) 0.001
Hypertension — no. (%) 2783 (79) 261 (82) 0.24
History of myocardial infarction — no. (%) 959 (27) 87 (27) 0.99
History of heart failure — no. (%) 1147 (33) 135 (42) <0.001
Estimated glomerular filtration rate§
Mean — ml/min/1.73 m2
of body-surface area 71±22 62±22 <0.001
<60 ml/min/1.73 m2
— no. (%) 1098 (31) 157 (49) <0.001
Median suPAR level (IQR) — pg/ml 3162 (2451–4115) 3937 (2935–5070) <0.001
Percutaneous coronary intervention — no. (%) 1905 (54) 143 (45) 0.001
Acute myocardial infarction — no. (%) 449 (13) 47 (15) 0.10
Median volume of contrast material (IQR) — ml 157 (95–230) 136 (77–210) 0.002
*
Plus–minus values are means ±SD. IQR denotes interquartile range, and suPAR soluble urokinase plasminogen activator receptor.
†
Race was reported by the patient.
‡
The body-mass index is the weight in kilograms divided by the square of the height in meters.
§
The estimated glomerular filtration rate was calculated with the use of the Chronic Kidney Disease Epidemiology Collaboration equation.
Table 1. Demographic and Clinical Characteristics of Patients Who Underwent Coronary Angiography, Stratified According to Incidence
of Postprocedural Acute Kidney Injury.*

milliliter). Among 692 critically ill patients ad-
mitted to the ICU, the incidence of acute kidney
injury was 53% in the highest suPAR quartile
(≥9440 pg per milliliter) and 15% in the lowest
quartile (5150 pg per milliliter).
Among patients in the surgical cohort, acute
kidney injury developed postoperatively in 67
(27%); of those, 14 (6%) had severe (stage 2 or 3)
acute kidney injury, and 8 (3%) underwent dialy-
sis. In both the surgical and ICU cohorts, the
P0.001
P0.001
P0.001
P=0.003
P0.001
P=0.007
P0.001
P=0.02
P=0.003
B Acute Kidney Injury According to Subgroup
A Odds Ratio for Acute Kidney Injury after Angiogram
Odds
Ratio
(95%
CI)
6
4
5
3
2
1
0
Model 2 Model 3
Model 1
Quartile 1
Quartile 2
Quartile 3
Quartile 4
Reference
1.85 (1.23–2.78)
2.27 (1.53–3.36)
3.79 (2.61–5.51)
Reference
1.73 (1.15–2.61)
2.04 (1.36–3.06)
3.14 (2.13–4.64)
Reference
1.67 (1.11–2.53)
1.88 (1.25–2.83)
2.66 (1.77–3.99)
suPAR Quartiles (N=3827)
1 2 3 4
All patients
Age
65 yr
≥65 yr
Sex
Male
Female
Chronic kidney disease, stage 3
Yes
No
Congestive heart failure
Yes
No
Diabetes mellitus
Yes
No
Acute myocardial infarction
Yes
No
Revascularization
Yes
No
Volume of contrast material
≤155 ml
155 ml
Cohort
EmCAB
CASABLANCA
No. of Patients Odds Ratio (95% CI)
Subgroup
2.95 (2.24–3.88)
2.97 (1.89–4.65)
2.66 (1.86–3.80)
2.67 (1.93–3.69)
4.24 (2.50–7.21)
2.54 (1.65–3.90)
2.27 (1.51–3.42)
2.49 (1.64–3.80)
3.07 (2.13–4.43)
3.15 (2.08–4.76)
2.59 (1.78–3.75)
3.79 (1.81–7.94)
2.85 (2.12–3.82)
4.03 (2.65–6.12)
2.16 (1.50–3.12)
2.40 (1.69–3.42)
3.78 (2.45–5.85)
2.68 (1.90–3.78)
2.60 (1.56–4.33)
3827
1705
2122
2637
1190
1255
2572
1282
2545
1345
2482
496
3331
2048
1779
1823
1904
2752
1075
0.5 1.0 8.0
2.0 4.0

422
risk of acute kidney injury increased steadily with
increasing suPAR quartiles, with an increase of
3.5 to 4 times in the risk of acute kidney injury
in the highest suPAR quartile as compared with
the lowest suPAR quartile. The association be-
tween suPAR and acute kidney injury was only
minimally attenuated in multivariable analyses
and did not differ between subgroups. (Details
are provided in Figs. S1 through S3.)
SuPAR Overexpression and Worsening Acute
Kidney Injury in Experimental Models
Before the injection of iohexol, baseline kidney
function and histologic findings were similar in
the wild-type mice and the suPAR-transgenic
mice at 10 weeks of age (Fig. 2A, 2D, and 2G),
despite higher suPAR levels in the transgenic
mice than in the wild-type mice (210±56 ng per
milliliter vs. 2±1 ng per milliliter). At 24 hours
after the injection of contrast material, both
wild-type mice and suPAR-transgenic mice had
an increase in the serum creatinine level. How-
ever, suPAR-transgenic mice had significantly
higher creatinine levels and more severe histo-
pathological features of acute kidney injury than
their wild-type counterparts that had received
the IgG isotype (Fig. 2E and 2H). One mouse
in the suPAR-transgenic group that received
IgG died unexpectedly, so data are shown for
19 mice.
Attenuation of Acute Kidney Injury
with Anti-uPAR Antibody
Mice that were pretreated with a uPAR monoclo-
nal antibody had lower creatinine levels at 24
hours than their counterparts that received the
IgG isotype (Fig. 2G). When comparing the renal
histopathological findings in the wild-type mice
and suPAR-transgenic mice, we found that both
groups had largely normal histologic features at
baseline (Fig. 2A and 2D). At 48 hours after the
administration of iohexol, all the mice had his-
tologic features typical of contrast-induced acute
kidney injury, including tubular vacuolization,
tubular necrosis, and casts (Fig. 2B, 2C, 2E,
and 2F). The suPAR-transgenic mice that were
pretreated with a uPAR monoclonal antibody
had milder histopathological features of acute
kidney injury and lower kidney injury scores than
the mice that received the IgG isotype (Fig. 2E
and 2F).
Effect of suPAR on Bioenergetic Profile and
Oxidative Stress of HK-2 Cells and Podocytes
HK-2 cells that were exposed to suPAR had sig-
nificantly higher energetic demand under base-
line conditions, with increased mitochondrial
basal respiration and ATP production, and high-
er maximum rate of respiration and spare re-
spiratory capacity than cells exposed to media
alone. The suPAR-treated cells also had a higher
rate of nonmitochondrial oxygen consumption,
indicating an active involvement of other cellular
oxygen–consuming reactions in addition to that
catalyzed by the mitochondrial cytochrome c oxi-
dase. The oxygen-consuming rates that were at-
Figure 2 (facing page). Acute Kidney Injury in Wild-Type
and Transgenic Mice before and after Treatment
with Anti-uPAR Monoclonal Antibody.
Panels A through F show representative kidney his
tologic findings, on high-power view, with the use of
periodic acid–Schiff stain in samples obtained from
wild-type mice and suPAR-transgenic mice at baseline
(Panels A and D) and 48 hours after the administration
of iohexol (Panels B, C, E, and F) stratified according
to treatment (IgG isotype, in Panels B and E; or uro
kinase plasminogen activator receptor [uPAR] mono-
clonal antibody, in Panels C and F). Wild-type mice
and suPAR-transgenic mice had largely normal kidney
morphologic features at baseline. At 48 hours after

iohexol administration, tubular vacuolization could
be seen in all wild-type and suPAR-transgenic mice

(arrows). The suPAR-transgenic mice that received IgG
(Panel E) had more severe renal injuries than mice in
any other studied groups. The suPAR-transgenic mice
that received the uPAR monoclonal antibody (Panel F)
had significantly less severe tubular vacuolization than
their counterparts that received the IgG isotype (Panel E).
Panel G shows serum creatinine levels measured before
and after the administration of contrast material. To
convert the values for creatinine to micromoles per liter,
multiply by 88.4. As compared with baseline, the serum
creatinine level at 24 hours after iohexol injection was
increased in all examined groups. The suPAR-transgenic
mice that received the IgG isotype had much higher
creatinine levels than mice in any other groups. There
was no significant between-group difference at base-
line. Panel H shows a semiquantitative scoring system
that accounts for glomerular and tubular changes asso-
ciated with acute kidney injury; kidney-injury scores
range from 0 to 12, with higher scores indicating more
severe kidney injury. (One mouse in the suPAR-trans-
genic group that received IgG died unexpectedly, so
data are shown for 19 mice.) The analyses in Panels G
and H were conducted with two-way analysis of vari-
ance. In Panels G and H, bars represent means, and
I bars ±1 SD; circles or squares indicate values in indi-
vidual mice.

tributed to proton leak across the mitochondrial
membrane did not differ between suPAR-exposed
and nonexposed cells, which indicated that
mitochondria were not damaged by suPAR and
that the mitochondrial integrity was maintained
after suPAR treatment. Superoxide generation
was increased by a factor of 2 in the presence
of suPAR, an effect that was completely abro-
gated by co-exposure to uPAR antibody. These
effects were attenuated when uPAR antibody
was coadministered with suPAR. These ef-
fects were not seen in podocytes that were
exposed to suPAR. (Details are provided in Figs.
S4 and S5.)
D suPAR-Transgenic
A Wild-Type
100 µm 100 µm 100 µm
100 µm 100 µm 100 µm
B Wild-Type with Iohexol+IgG C Wild-Type with Iohexol+uPAR
Antibody
E suPAR-Transgenic with Iohexol+IgG F suPAR-Transgenic with Iohexol+
uPAR Antibody
G Serum Creatinine Level H Kidney Injury Score
Value
(mg/dl)
8
6
7
5
4
2
1
3
0
W
i
l
d
-
T
y
p
e
w
i
t
h
I
g
G
W
i
l
d
-
T
y
p
e
w
i
t
h
u
P
A
R
A
n
t
i
b
o
d
y
s
u
P
A
R
-
T
r
a
n
s
g
e
n
i
c
w
i
t
h
I
g
G
s
u
P
A
R
-
T
r
a
n
s
g
e
n
i
c
w
i
t
h
u
P
A
R
A
n
t
i
b
o
d
y
Baseline 24 Hr
Score
8
6
7
5
4
2
1
3
0
I
g
G
u
P
A
R
A
n
t
i
b
o
d
y
Wild-Type suPAR-Transgenic
P0.001
P0.001
P0.001
P=0.006

424
Discussion
This study showed that suPAR was associated
with subsequent acute kidney injury in several
cohorts (4769 patients who were exposed to intra-
arterial contrast material for coronary angiogra-
phy, who underwent cardiac surgery, or who
were critically ill). Concurrently, we obtained
experimental evidence that suPAR may be directly
involved in the pathogenesis of acute kidney in-
jury by sensitizing kidney proximal tubules to
injury through modulation of cellular bioener-
getics and increased oxidative stress. Inhibiting
suPAR with the use of a monoclonal antibody
attenuated the effect of iohexol on kidney func-
tion in mice overexpressing suPAR and abrogated
bioenergetic changes in HK-2 cells exposed to
suPAR.
There has been little progress in the overall
risk stratification, prevention, and treatment of
acute kidney injury. Therapies such as intrave-
nous saline hydration, acetylcysteine, and sodium
bicarbonate have had little success.27,36
Biomark-
ers that are currently under study, such as cystatin
C, neutrophil gelatinase–associated lipocalin,
and kidney injury molecule 1, are early markers
of acute kidney injury whose levels increase only
after renal injury has occurred.37,38
We found that
preprocedural suPAR levels were predictive of
acute kidney injury in both low-risk and high-
risk cohorts and across subgroups, independent
of relevant clinical characteristics, including base-
line kidney function. In addition, suPAR mod-
estly improved risk discrimination when this
variable was added to the NCDR Simplified Inte-
ger Risk Score for acute kidney injury. These
findings are in line with one previous smaller
study involving 107 patients who underwent car-
diac surgery.39
Improved assessment of the pre-
procedural risk of acute kidney injury would al-
low for more informed decision making and
would help to identify a subgroup of patients
who would benefit from an intervention to
minimize procedural acute kidney injury, poten-
tially in the form of anti-suPAR therapies.
The wide spectrum of clinical contexts in
which suPAR levels are associated with incident
acute kidney injury suggests that the underlying
mechanism is not dependent on the type of in-
citing event. On the basis of our animal models,
we speculate that there may be a synergistic ef-
fect between suPAR — which acts as a meta-
bolic sensitizer and increases the workload of
tubular cells — and various injuries such as
ischemia, cytotoxic effects, and oxidative stress
(e.g., induced by cardiac surgery or the use of
iodinated radioactive contrast material).40,41
In
response to high suPAR levels, proximal tubular
cells, but not podocytes, showed an increase in
mitochondrial respiration. Extramitochondrial ox-
ygen consumption was increased in both types
of cells by suPAR, but to a greater degree in tu-
bular cells than in podocytes — a finding that
suggests activation of extramitochondrial enzy-
matic oxidation.
Before the injection of contrast material, we
found no histopathological or biochemical mea-
sures of renal dysfunction in 10-week-old mice
overexpressing suPAR. After the injection of con-
trast material, the suPAR-transgenic mice had
significantly more severe acute kidney injury than
the wild-type mice. The effect of the administra-
tion of contrast material on kidney injury in
suPAR-transgenic mice was attenuated with a
monoclonal antibody to uPAR, which suggests
that chronically elevated suPAR levels sensitized
the kidney to acute injury (although suPAR con-
centrations were 50 times as high as median
levels in humans) and that this sensitizing effect
of suPAR could be reversed pharmacologically.
These conclusions are in line with a recent re-
port showing that targeting the urokinase recep-
tor in a rat model of diabetic kidney disease re-
sulted in improvement in kidney function.42
The
mechanisms of suPAR in kidney dysfunction
have focused on its source of production and its
role in binding and activating podocyte αvβ3
integrins.15,17
Other reports have suggested that
suPAR also affects proximal tubules and drives
kidney fibrosis in an integrin-dependent man-
ner.14,16,17,43
Although it will be helpful for studies
to be expanded into other models of acute kidney
injury, it is plausible that prolonged suPAR expo-
sure affects podocytes and tubular cells by means
of different mechanisms, a hypothesis in line
with the different bioenergetic profiles seen in
HK-2 cells and podocytes in response to suPAR.
Our study has several strengths. We found
consistent results across several well-character-
ized cohorts in three clinical contexts and a to-
tal of 4769 participants, allowing for subgroup
analyses and adjustment for confounders. The

accompanying experimental findings have com-
pelling translational and therapeutic implica-
tions. Our study also has several important
limitations. One is the retrospective nature of
the cohort studies and risk of selection bias.
However, the collection of blood samples and
the end points were prespecified in all the co-
horts included in the study, and the incidence of
acute kidney injury in these cohorts was consis-
tent with rates reported in the NCDR registry.35
We could not compare suPAR to other biomark-
ers such as kidney injury molecule 1 and neutro-
phil gelatinase–associated lipocalin in the pres-
ent study, because these were not systematically
measured in all cohorts.
In conclusion, high suPAR levels were associ-
ated with incident acute kidney injury in several
patient cohorts. The experimental models used
here suggest that suPAR may be a pathogenic fac-
tor in acute kidney injury.
Supported by grants (1R61HL138657-02, 1P30DK111024-
03S1,5R01HL095479-08,3RF1AG051633-01S2,5R01AG042127-06,
2P01HL086773-08, U54AG062334-01, 1R01HL141205-01,
5P01HL101398-02, 1P20HL113451-01, 5P01HL086773-09,
1RF1AG051633-01, R01NS064162-01, R01HL89650-01, HL095479-
01, 1DP3DK094346-01, and 2P01HL086773, to Dr. Quyyumi;
R01HL089650-01 and R01DK101350, to Drs. Sever, Wei, and
Reiser; K23DK106448, to Dr. Leaf; and R01HL142093, to
Dr. Baron) from the National Institutes of Health, by a grant
(15SFCRN23910003, to Dr. Quyyumi) from the American Heart
Association, by an American Society of Nephrology Foundation
for Kidney Research Carl W. Gottschalk Research Scholar Grant
(to Dr. Leaf), by the Hutter Family Professorship (to Dr. Januzzi),
by a grant (W81XWH1810667, to Dr. Baron) from the Depart-
ment of Defense, by a Merit Award (BX002006, to Dr. Sheikh-
Hamad) and a Career Development Award (BX002912, to Dr. Pan)
from the Department of Veterans Affairs, and by the American
Society of Nephrology Foundation for Kidney Research George
B. Rathmann Research Fellowship Award, as part of the Ben J.
Lipps Research Fellowship Program (to Dr. Holliday).
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
We thank the members of the Emory Cardiovascular Biobank
team, specifically Yi-An Ko for database maintenance and statis-
tical support, Mosaab Awad and Ayman Alkhoder for serving as
coordinators, the staff of the Emory Clinical Cardiovascular Re-
search Institute, and the staff of the Atlanta Clinical and Trans-
lational Science Institute for the recruitment of participants,
compilation of data, and preparation of samples; Beata Samelko
and Jing Li for technical help with mouse tissue and enzyme-
linked immunosorbent assay for soluble urokinase plasminogen
activator receptor; the members of the Brigham and Women’s
Hospital Registry of Critical Illness (Mayra Pinilla, Sam Ash,
Paul Dieffenbach, Laura Fredenburgh, and Anthony Massaro);
and Myles Wolf (Duke University) and Monnie Wasse (Rush Uni-
versity) for critical reading of an earlier version of the manu-
script.
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