Tumor Lysis Syndrome and Aki Beyond Crystal.14

BASIC RESEARCH www.jasn.
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Tumor Lysis Syndrome and AKI: Beyond

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Crystal Mechanisms
Marine Arnaud,1 Maud Loiselle,1 Camille Vaganay,2 Ste phanie Pons ,1
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 04/12/2024
Emmanuel Letavernier, Jordane Demonchy, Sofiane Fodil,1 Manal Nouacer,1

3,4 1
re,3 Eden Arrii,1 Julien Lion,1 Nuala Mooney,1

Sandrine Placier,3 Perrine Fre
Raphael Itzykson,2,5 Chakib Djediat,6 Alexandre Puissant,2 and Lara Zafrani1,7
Due to the number of contributing authors, the affiliations are listed at the end of this article.
ABSTRACT
Background The pathophysiology of AKI during tumor lysis syndrome (TLS) is not well understood due to
the paucity of data. We aimed to decipher crystal-dependent and crystal-independent mechanisms of TLS-
induced AKI.
Methods Crystalluria, plasma cytokine levels, and extracellular histones levels were measured in two
cohorts of patients with TLS. We developed a model of TLS in syngeneic mice with acute myeloid leuke-
mia, and analyzed ultrastructural changes in kidneys and endothelial permeability using intravital confocal
microscopy. In parallel, we studied the endothelial toxicity of extracellular histones in vitro.
Results The study provides the first evidence that previously described crystal-dependent mechanisms are
insufficient to explain TLS-induced AKI. Extracellular histones that are released in huge amounts during
TLS caused profound endothelial alterations in the mouse model. The mechanisms of histone-mediated
damage implicates endothelial cell activation mediated by Toll-like receptor 4. Heparin inhibits extracellu-
lar histones and mitigates endothelial dysfunction during TLS.
Conclusion This study sheds new light on the pathophysiology of TLS-induced AKI and suggests that
extracellular histones may constitute a novel target for therapeutic intervention in TLS when endothelial
dysfunction occurs.
JASN 33: 1154–1171, 2022. doi: https://doi.org/10.1681/ASN.2021070997
Tumor lysis syndrome (TLS) is a life- sometimes mimics sepsis, presenting as sys-
threatening complication of cancer treatment temic inflammatory response syndrome with
in patients with proliferative and/or chemosen- multiple organ failures.3 TLS-induced AKI,
sitive malignancies with high tumor burden. occurring in up to 64% of patients with TLS,
TLS occurs either as a consequence of diverse strongly reduces the likelihood of complete
anticancer treatments or, less frequently, spon-
taneously. Cancers with high potential for cell
lysis include acute leukemias, high-grade lym- Received July 27, 2021. Accepted March 12, 2022.
phomas, and other rapidly proliferating Published online ahead of print. Publication date available at
tumors.1,2 Tumor cell lysis results in the release www.jasn.org.
of intracellular ions and metabolites into the See related editorial, “Crystals or His(stones): Rethinking AKI in
circulation, thus leading to hyperuricemia, Tumor Lysis Syndrome,” on pages 1055–1057.
hyperkalemia, hyperphosphatemia, and hypo- Correspondence: Prof. Lara Zafrani, Medical Intensive Care
calcemia. Clinical TLS is present when labora- Unit, Assistance Publique des H^ opitaux de Paris, INSERM UMR
tory TLS is accompanied by an increased 976, Saint Louis Hospital, 1 Avenue Claude Vellefaux, 75010
Paris, France. Email: lara.zafrani@aphp.fr
creatinine level, seizures, cardiac dysrhythmia,
or even multiple organ failure and death.1 TLS Copyright ß 2022 by the American Society of Nephrology
1154 ISSN : 1533-3450/1046-1154 JASN 33: 1154–1171, 2022

www.jasn.org BASIC RESEARCH
remission of the underlying malignancy and is associated

Significance Statement
with a higher mortality rate.4 Crystal-induced kidney
injury occurs in TLS when calcium phosphate, uric acid, The pathophysiology of AKI during tumor lysis syndrome (TLS) is
and xanthine precipitate in renal tubules. Nowadays, not fully understood. We aimed to decipher crystal-dependent
and crystal-independent mechanisms of TLS-induced AKI. Ana-
most patients prone to development of TLS receive ras- lyzing urine and blood from patients with TLS provided data on
buricase. Moreover, urine alkalinization that may increase crystal-independent mechanisms of the pathogenesis of AKI dur-
calcium-phosphate precipitation is no longer recom- ing TLS. We also explored mechanisms of TLS-induced AKI
mended. The pathologic mechanisms underlying TLS- in vitro and in vivo in a murine model of TLS (syngeneic mice
induced AKI are not well defined because of a scarcity of with acute myeloid leukemia receiving chemotherapy). We found
that extracellular histones released in huge amounts during TLS
data. Studies published so far consist mainly of individual profoundly alter the endothelium. Nonanticoagulant heparin mit-
case reports.5,6 Indeed, case reports that described renal igated AKI in this model.
calcium-phosphate crystals during TLS were published in
1979 and 1984, when alkalinization was largely used in
the management of TLS.5,6 Moreover, in these case METHODS
reports, patients had extreme phosphatemia levels (.5
mmol/L) that are rarely seen during TLS because most Patients
patients would undergo dialysis before reaching such lev- Plasma samples were obtained from two cohorts of
els. Many patients with TLS experience AKI without any patients.
evidence of hyperphosphatemia or hyperuricemia-
induced crystals, especially because rasburicase, a recom- First Cohort
binant urate oxidase, is considered a standard of care Frozen plasma samples were obtained from an existing and
treatment for patients at high risk of TLS.7 Darmon et al.4 available biobank of 736 patients with hematologic malig-
have shown, in a study focusing on high-risk patients nancies admitted to the intensive care unit (ICU). The
with TLS and AKI in the rasburicase era, that 28.3% of study was carried out in 17 ICUs participating in a research
patients with TLS experienced AKI, 53% of them requir- network, initiated in 2005, in France and Belgium.11 From
ing RRT. These results are similar to those of previous January 2010 to May 2011, consecutive patients with hema-
studies of TLS undertaken before the widespread use of tologic malignancies admitted to the ICUs for any reason
rasburicase, when allopurinol and alkalinization were were included. Exclusion criteria were complete cure of the
mainly used, with AKI occurring in up to one third of malignancy for .5 years and age ,18 years. In each center,
patients with TLS.8,9 investigators used a standardized electronic case report
Extracellular histones are increasingly recognized for form to collect the study data. Clinical data, biologic data,
their cytotoxic effects. Histones consist of five cationic and plasma were collected prospectively. A total of 94
proteins (H1, H2A, H2B, H3, and H4) in the nucleus of patients were diagnosed as having TLS at admission. Labo-
eukaryotic cells. When cell death is extensive, high levels ratory TLS and clinical TLS were defined on the basis of
of unchained extracellular histones are released that con- the daily recorded laboratory and clinical values, using the
vey cytotoxic effects.10 The vascular endothelium forms Cairo–Bishop criteria (Supplemental Table 1).12 Definition
the active interface between blood and tissues and can and staging of AKI were defined on the basis of serum cre-
thereby potentiate multiple organ failure. In this context, atinine and urine output criteria, according to Kidney Dis-
some authors have suggested that extracellular histones ease Improving Global Outcomes guidelines (Supplemental
may contribute to endothelial dysfunction during sepsis Table 2).13
or trauma.10 To date, extracellular histones have not Peripheral blood samples were collected within 24 hours
been examined in the context of the pathophysiology of of ICU admission and processed within 2 hours of sam-
TLS. pling. Specimens were centrifuged at 1500 3 g for 10
In this study, we aimed to decipher crystal-dependent minutes at 20 C, and the plasma was stored as 500-ml
and -independent mechanisms of TLS- induced AKI. To aliquots at 280 C.
this end, we studied crystalluria and the effect of crystals Urine samples were unavailable in this cohort.
on AKI in patients with TLS. We developed a model of
TLS in syngeneic mice with acute myeloid leukemia Second Cohort
(AML), analyzed ultrastructural changes in kidneys, and In parallel, a prospective reliability cohort of 55 patients
found profound endothelial alterations, leading us to inter- with TLS (hospitalized in the Department of Intensive Care
rogate the role of extracellular histones in endothelial dys- Medicine, H^ opital Saint Louis) were analyzed. All patients
function during TLS. included in this cohort presented with laboratory or clinical
JASN 33: 1154–1171, 2022 Tumor Lysis Syndrome and Kidney 1155
BASIC RESEARCH www.jasn.org
TLS on admission. Uricemia was assessed every 6 hours concentrations were determined by spectrophotometry at
during the first 72 hours of TLS, and then every 12 hours 450 nm (Molecular Devices).
until day 7. Urine and plasma samples were collected pro-
spectively at day 1, 3, 5, and 7. Extracellular Histone Measurements
Plasma from healthy blood donors were obtained in We measured the levels of extracellular histones in the
accordance with institutional regulations from the Eta- plasma of patients and mice using a Cell Death Detection
blissement Français du Sang, H^ opital Saint Louis, Paris, ELISA kit (Roche Applied Science, Penzberg, Germany).
France. Purified mixed calf thymus histones were used to generate
standard curves.
Clinical and Biologic Data

The following clinical and biologic data were recorded: Mice
demographic parameters, medical history, presenting symp-
toms and treatments. Liver enzyme levels, lactate dehydro- Animal Experiments
genase (LDH) levels, calcemia, uricemia, and phosphatemia C57BL/6 mice (8 weeks old) from Envigo were housed and
were recorded at day 1, 2, 3, and 7. Serum creatinine was studied under sterile conditions at the University Institute
recorded daily throughout the patients’ stay in the ICU and of Hematology (Hôpital Saint Louis).
at 3 months. The Sequential Organ Failure Assessment
score was computed at admission then daily throughout AML Mice
the patient’s stay in the ICU, as previously described.14 We For the in vivo model of TLS, we used a mouse model of
assessed patients’ vital status at ICU discharge, hospital dis- AML driven by retroviral expression of a single mutational
charge, and at 3 and 6 months. hit, the MLL-AF9 gene fusion, as previously described. 15,16
Expression of MLL-AF9 in normal granulocyte macrophage
progenitor cells (GMPs) is sufficient to generate an aggres-
Crystalluria
sive, transplantable myeloid leukemia in syngeneic, immuno-
Crystalluria was analyzed at day 1, 3, 5, and 7 using a phase
competent C57BL/6 mice, with a clonal leukemic stem cell
contrast microscope, with a polarized light device (Labora-
population that displays an immunophenotype similar to that
toire d’Explorations Fonctionnelles, H^ opital Tenon, Paris,
of normal GMPs (Linlow, Sca-1, c-Kit1, CD16/32high, and
France). Urine samples brought to the laboratory within 2
CD34high). We further enriched for stem cell activity by seri-
hours of voiding were kept at room temperature and were
ally transplanting the MLL-AF9–driven leukemias through
processed rapidly. Urine-specific gravity and pH were mea-
secondary, tertiary, and quaternary recipients, generating
sured. Undiluted urine was then homogenized by gentle leukemias with 100% penetrance in about 3–4 weeks. We
shaking and rotation (neither centrifuged nor filtered) and established that cytarabine, given at 100 mg/kg daily for
10 mm3 was immediately placed in a Malassez cell (CML, 5 days, in combination with doxorubicin, given at 1 mg/kg
Nemours, France) for examination by light microscopy daily for the first 3 days, was the maximum tolerated chemo-
using a polarizing microscope (Optiphot-2; Nikon, Cham- therapeutic regimen in this model. This treatment clears
pigny-sur-Marne, France). The entire cell was examined at about 60%–70% of leukemic blasts from bone marrow. TLS
2003 magnification to localize crystals and aggregates, and occurs 24 hours after the end of the chemotherapeutic regi-
then at 4003 magnification (high power field). All crystals men. In the control group, mice received 150 ml of vehicle
and aggregates were counted on the entire cell and their Hanks’ balanced salt solution (HBSS). In mice who received
size determined using the included micrometric scale. The heparin, we injected 3 mg/kg heparin intraperitoneally
results were expressed as number of crystals per millimeter (i.p.) twice a day before and during the administration of
cubed. Only one Malassez cell was examined in each chemotherapy.
instance. Examiners of crystalluria were blinded to the clin-
ical status of the patients.
Toll-Like Receptor 4 Knockout Mice
Toll-like receptor 4 (TLR4)–deficient female mice (Tlr42/2,
ELISA C57BL/6 background) and the corresponding wild-type
A multiplex assay was used to measure soluble factors in (WT) mice were provided by Prof. Shizuo Akira (Osaka
patients with TLS, including IL-6, IL-8, TNF-a, soluble University, Osaka, Japan).
intercellular adhesion molecule 1 (sICAM-1), IL-1b, IL-10,
and IFN-g (V-PLEX Proinflammatory Panel 1, Meso Scale Injection of Recombinant Histones
Discovery). C57BL/6 female mice were injected with recombinant his-
The concentrations of cytokines and chemokines, tones (25 mg/kg body wt, i.p. injection) or vehicle (0.9%
including IL-6 and IL-8, in culture supernatants were deter- saline) (single bolus). Previously published data on mice
mined using ELISA kits (Human ELISA MAX; BioLegend). using different doses of histones have shown that doses
The manufacturer’s instructions were followed, and the .50mg/kg were lethal.10 Other authors used 10–50 mg/kg
1156 JASN JASN 33: 1154–1171, 2022

according to studies and evidenced toxic effects of extracel- “continuous” if red blood cell movement was uninter-
lular histones.17–19 Therefore, we chose an intermediate rupted, “intermittent” if red blood cell movement stopped
dose of 25 mg/kg i.p. in our study. or reversed, or “no flow” if no red blood cell movement
was observed. Data were expressed as the percentage of ves-
Clinical Chemistry Analyses in Mice sels in each of the three categories.
Serum creatinine was measured using a high-performance

liquid chromatography method.20 BUN and LDH in serum Morphologic Analysis
were measured using commercially available kits and an Kidneys were rapidly excised after euthanasia. Kidneys
automatic analyzer. Data are expressed as serum BUN in were partly fixed in alcohol-formalin– acetic acid solution,
milligrams per deciliter and serum LDH concentration in dehydrated, embedded in paraffin, and further processed
international units per liter. for Masson trichrome staining or Periodic acid–Schiff
staining (5-mm sections). Histologic changes in the cortex
Intravital Microscopy Imaging and in the outer stripe of the outer medulla were assessed
Intravital microscopy imaging was performed using two by quantitative measurements of tissue damage, as previ-
different techniques: an inverted Zeiss Axiovert fluorescent ously described.22 Tubular damage was defined as tubular
microscope (Zeiss, Paris, France) and confocal microscopy. epithelial swelling, loss of brush border, vacuolar degenera-
tion, necrotic tubules, cast formation, and desquamation.
Intravital Confocal Microscopy The degree of kidney damage was estimated at 3200 mag-
To visualize kidney microvascular perfusion and permeabil- nification using five randomly selected fields for each ani-
ity in vivo, confocal microscopy was performed using an mal by the following criteria: zero, normal; one, areas of
Olympus Inverted Microscope IX83 equipped with DSD2 damage ,25%; two, damage involving 25%–50% of tubules;
system attached to an Andor Zyla camera (Andor Technol- three, damage involving 50%–75% of tubules; four, damage
ogy) and a 37 C heated chamber. Briefly, the left kidney of involving 75%–100% of tubules.
anesthetized mice was exposed by flank incision and gently
pulled to allow insertion of a slit plate at the level of the Immunohistochemical Analyses
renal artery and vein. This system allows the physical sepa- Renal fragments embedded in paraffin were cut into 3-mm
ration of the organ from the rest of the body. At that time, sections. Kidneys were immunostained with purified rat
a total volume of 200 ml of albumin-FITC (100 mg/g body anti-mouse panendothelial cell antigen (MECA-32; BD Bio-
wt; 50 mg/ml stock solution; Sigma), was intravenously sciences) (for peritubular capillary density), a purified rat
injected in the eye sinus. The animal was then flipped over anti-mouse CD54 antibody (ICAM-1; Biolegend), and a
to position the kidney on a window plate at the microscope purified rabbit anti-cleaved PARP (Asp214) antibody
stage, and image acquisition (one image every 5 seconds (c-PARP; Ozyme) (for apoptosis analysis). Results were
for 30 minutes) could start instantaneously. visualized using the Anti-Rat HRP-DAB Cell & Tissue
Masked playback analysis of videotaped images were Staining Kit (R&D Systems). Density of peritubular
performed offline by a blind examinator. Leakage of capillaries and staining intensity were determined on
albumin-FITC from peritubular capillaries into the intersti- pictures at 2003 magnification using ImageJ Fiji software,
tium (outside the vessels) was analyzed every 1 minute dur- as previously described.23
ing the first 15 minutes of each video, using a
semiquantitative score (from one to three) for each mouse. Transmission Electron Microscopy
For scanning electron microscopy, kidneys were extracted
Renal Microvascular Perfusion and fixed for 24 hours in paraformaldehyde. Sections were
Renal microvascular perfusion was analyzed in WT mice postfixed in 1% buffered osmium tetroxide for 1 hour,
injected with recombinant histones or vehicle. Intravital dehydrated in an ascending series of ethanol with 1% ura-
video microscopy was performed as described previously.21 nyl acetate included in 70% ethanol, and flat embedded in
The left kidney was exposed by a flank incision and posi- epoxy mixture (Spurr resin). Ultrathin sections were cut
tioned on a glass stage above an inverted Zeiss Axiovert using a Reichert Jung ultramicrotome (Leica, Nussloch,
200M fluorescent microscope equipped with an Axiocam Germany) and collected on copper grids. Sections were
HSm camera (Zeiss). Videos of 10 seconds (approximately stained on drops of lead citrate for 3 minutes and examined
30 frames per second) at 2003 magnification were acquired in a Hitachi H-7100 electron microscope (Hitachi, Tokyo,
from five randomly selected, nonoverlapping fields of view. Japan). Digital images were acquired using a charge-
Body temperature was maintained at 35 C–37 C with a coupled device camera (Hamamatsu AMT 540.3, Hamama-
warming lamp. Approximately 150 capillaries were ran- tsu, Japan). The number of capillary fenestrations was
domly selected and analyzed from these fields of view from semiquantitatively graded and expressed as a score between
the kidney of each animal. Capillaries were categorized as zero and four, with a score of zero representing 0%–5%,
one representing 6%–25%, two representing 26%–50%, Statistical Analyses

three representing 51%–75%, and four representing For statistical analyses, we used Prism (GraphPad software).
76%–100% of the total endothelial capillary circumference All data are expressed as mean6SEM. All metric data were
having fenestrations (i.e., a score of four being the finding tested for normal distribution using the Shapiro–Wilks W
in healthy kidneys). The areas with the endothelial nucleus test. Differences between groups were analyzed for statisti-
were omitted, and at least ten micrographs of capillaries cal significance by t test or one-way ANOVA for repeated
per mouse taken at an original magnification of 310,000 measures and subsequent Bonferroni post hoc test. Non-
were evaluated. All analyses were performed in a blinded normally distributed single measurements were compared
manner. using the Mann–Whitney U test or the Kruskal–Wallis test
with Dunn post hoc test. A P value ,0.05 was accepted as
statistically significant.
In Vitro Experiments
Cell Lines and Culture Reagents Study Approval

Primary human renal glomerular endothelial cells Experimental studies on mice were approved by the Euro-
(HRGECs) were purchased from Innoprot (Spain) and cul- pean ethical committee (Autorisation de Projet Utilisant
tured in Endothelial Cell Medium (ScienCell), as previously des Animaux a des Fins Scientifiques #8909).
described.24 The human dermal microvascular endothelial
cell line (HMEC-1) was cultured in supplemented MCDB-
First Cohort
131 medium (Thermo Fisher Scientific, Watham, MA), as The study and plasma collection were approved by the
previously described.24,25 For experiments using nonanti- appropriate ethics committees in France and Belgium. No
coagulant heparin, we used N-acetyl heparin sodium data allowing patient identification were collected. All
(Sigma-Aldrich). patients or relatives gave their informed consent to study
participation (Programme Hospitalier de Recherche Clini-
Stimulation Assays que AOM 08235).
Highly-purified human recombinant histones H3 (New
England BioLabs Inc., Ipswich, MA) were used for stimula- Second Cohort
tory experiments on HMEC-1s and HRGECs. For in vitro This project was approved by the appropriate ethical com-
assays, 500,000 cells per well seeded in six-well plates were mittee (H^ opitaux Universitaires Paris Nord Val de Seine
stimulated with 5–40 mg/ml of histones in cell culture Institutional Review Board number 00006477, Paris 7 Uni-
media supplemented with 1% FCS. Unstimulated cells were versity, Assistance Publique-Hôpitaux de Paris).
used as negative control. Experiments were performed in
triplicate, at a minimum.
RESULTS
Immunofluorescence Confocal Laser Microscopy
HMEC-1s were incubated with extracellular histones for 4 Characteristics of Patients with TLS
hours. A rabbit polyclonal antibody to histone H3 (1791; A total of 94 patients with hematologic malignancies were
Abcam) was used as primary antibody, and a Cy3- included in the study in the first cohort and 55 patients in
conjugated anti-rabbit IgG antibody was used as secondary the second cohort. All 149 patients were admitted to the
antibody. An anti-TLR4 primary antibody and a FITC- ICU with TLS. Of the 149 patients, 129 (86.6%) developed
labeled secondary antibody were used to visualize TLR4, AKI during ICU stay (patients with TLS-AKI). Their clini-
and 49 ,6-diamidino-2-phenylindole (Vector) was used to cal characteristics, laboratory findings, treatments, and out-
stain the nuclei. Confocal imaging was performed with a comes are shown in Table 1 and Table 2.
Zeiss LSM 510 Confocal microscope.
Urine Crystals Are Uncommon in Patients with TLS
Inhibition of TLR2 and TLR4 Analysis of crystalluria on fresh urine samples was performed
For TLR2 and TLR4 inhibition, endothelial cells (ECs) were in 55 patients with TLS at day 1, 3, 5, and 7 after ICU admis-
preincubated with anti-TLR2 antibody (50 mg/ml; Thermo sion. All patients received intravenous saline hydration. Ninety
Fisher Scientific) or anti-TLR4 antibody (50 mg/ml; percent of patients had received urate oxidase, according to
Thermo Fisher Scientific) for 1 hour before histone stimu- current guidelines, because of the high risk of developing
lation. Mouse IgG2a,k antibody (50 mg/ml; Thermo Fisher TLS.7 Only three patients had uric-acid crystalluria, two in the
Scientific) was used as an isotype control. A specific TLR4 TLS-AKI group and one in the non-AKI TLS group. Of note,
antagonist, TAK-242 (1 mM, Tocris Biotechne), was also these patients had not received urate oxidase. Only one patient
used to confirm specific inhibition of TLR4. had calcium-phosphate crystals and he did not develop AKI.
1158 JASN JASN 33: 1154–1171, 2022

Table 1. Clinical characteristics, laboratory findings, treatment, and outcome of patients at admission (first cohort)
Patients with Patients with
TLS
Characteristic TLS-AKI Non-AKI TLS P Value
(n594)
(n583) (n511)
Male sex, n (%) 65 (69.1) 58 (69.9) 7 (63.6) 0.42
Age (yr), mean6SD 58612 61614 50620 0.67

Past history of CKD, n (%) 6 (6.4) 5 (6.0) 1 (9.0) 0.69
Baseline serum creatinine levels 88.0 (80.0–97.0) 88.0 (80.0–97.0) 97.0 (80.0–106.0) 0.71
(mmol/L), median (IQR)
Past history of hypertension, n (%) 35 (37.2) 33 (39.7) 2 (18.2) 0.16
Chronic cardiac dysfunction, n (%) 11 (11.7) 11 (13.3) 0 0.19

Diabetes, n (%) 12 (12.7) 11 (13.3) 1 (9.0) 0.69
Underlying malignancy, n (%)
AML 30 (31.9) 27 (32.5) 3 (27.3) 0.73
Acute lymphoid leukemia 13 (13.8) 10 (12.0) 3 (27.3) 0.17
Lymphoma 44 (46.8) 39 (46.9) 5 (45.5) 0.92
Chronic lymphocytic leukemia 4 (4.3) 4 (4.8) 0 0.46
Myeloma 1 (1.1) 1 (1.2) 0 0.71
Nephrotoxic drugs 19 (20.2) 19 (22.9) 0 0.08
Laboratory findings at admission
Phosphates (mmol/L), median (IQR) 2.1 (1.9–2.3) 2.1 (1.9–2.3) 2.2 (2.1–2.3) 0.7
Calcium (mmol/L), median (IQR) 1.6 (1.2–2.1) 1.6 (1.3–2.2) 1.2 (0.9–1.5) 0.02a
Potassium (mmol/L), median (IQR) 4.2 (3.6–4.9) 4.3 (3.6–5.1) 4.1 (3.6–4.7) 0.70
LDH (3 normal) at admission, median 4.0 (2.8–15.5) 4.0 (3.3–8.0) 20.0 (2.0–38.0) .0.99
(IQR)
Serum creatinine (mmol/L), median (IQR) 157.5 (106.8–289.8) 170.0 (124.0–298.0) 78.0 (51.0–105.0) ,0.001a
Uric acid (mmol/L) (before rasburicase), 503.0 (155.0–788.0) 539.0 (165.8–862.0) 200.0 (61.0–383.0) 0.009a
median (IQR)
Lactatemia (mmol/L), median (IQR) 3.9 (1.8–9.2) 4.2 (1.9–9.4) 2.4 (1.6–3.1) 0.39
Diuresis (ml/24 h), median (IQR) 665 (382–1125) 750 (400–1300) 1200 (1000–2370) 0.007a
SOFA at admission, median (IQR) 6 (3–10) 6 (3–10) 3 (1–3) 0.0001a
AKI stage 1, n (%) 16 (19.3)
AKI stage 2, n (%) 11 (13.3)
AKI stage 3, n (%) 56 (67.5)
Treatments
Hypouricemic therapy, n (%) 90 (95.7) 80 (96.4) 10 (90.9) 0.39
Need for RRT, n (%) 67 (80.7) 0 ,0.001a
RRT duration (d), median (IQR) 4 (2–13)
Need for vasopressors, n (%) 33 (35.1) 33 (39.8) 0 0.009
Need for mechanical ventilation, n (%) 46 (48.9) 44 (53.0) 2 (18.2) 0.03a
ICU mortality, n (%) 34 (36.2) 33 (39.8) 1 (9.0) 0.04a
28-Day mortality, n (%) 38 (40.4) 37 (44.6) 1 (9.0) 0.02a
P values are reported for comparisons across the two patient groups. IQR, interquartile range.
a
P,0.05.
The vast majority of patients with AKI did not have positive LDH, BUN, phosphate, and creatinine levels 24 hours after
crystalluria (Table 2). the end of chemotherapy (Figure 1, B and C). In AML
mice, in vivo intravital microscopy revealed leukemic cell
infiltration of peritubular capillaries that was drastically
High-Dose Chemotherapy in AML Mice Induced TLS reduced after chemotherapy (Figure 1D). In this in vivo
and Renal EC Dysfunction In Vivo model of TLS, we first analyzed histologic lesions using
To further analyze AKI during TLS, we used a mouse Masson trichrome and Periodic acid–Schiff staining.
model of AML driven by the MLL-AF9 gene fusion (Figure Despite the high degree of AKI in TLS mice, only mild
1A).26 Treatment with a combination of an anthracycline tubular vacuolization was observed, with no significant dif-
(doxorubicin) and the antimetabolite cytarabine at the ference between leukemic mice that had versus had not
maximally tolerated dose, mimicking AML standard of care received chemotherapy (Supplemental Figure 1, A–C).
intensive chemotherapy,27 led to a sharp reduction in leu- We then looked for renal ultrastructural changes by
kemic burden over an 8-day course in the blood, spleen, transmission electronic microscopy and observed pro-
and bone marrow of treated mice, compared with vehicle found alterations of peritubular ECs in TLS mice. Indeed,
control, and the induction of biologic TLS with increased in healthy kidneys, transmission electron microscopy
Table 2. Clinical characteristics, laboratory findings, crystalluria, treatment, and outcome of TLS patients (second cohort)
Patients with Patients with
TLS
Characteristics TLS-AKI Non-AKI P Value
(n555)
(n546) TLS (n59)
Male sex, n (%) 40 (72.7) 36 (78.3) 4 (44.4) 0.09
Age (yr), mean6SD 57617 59615 44620 0.25

Past history of CKD, n (%) 1 (1.8) 0 (0.0) 1 (11.1) 0.16
Baseline serum creatinine levels (mmol/L), median (IQR) 75.5 (61.0–90.8) 76.5 (62.5–90.3) 67.5 (58.3–94.5) 0.71
Past history of hypertension, n (%) 15 (27.3) 14 (30.4) 1 (11.1) 0.42
Chronic cardiac dysfunction, n (%) 2 (3.6) 2 (4.3) 0 (0.0) .0.99
Diabetes, n (%) 8 (14.5) 8 (17.4) 0 (0.0) 0.33

Underlying malignancy, n (%)
AML 10 (18.2) 9 (19.6) 1 (11.1) .0.99
Acute lymphoid leukemia 7 (12.7) 5 (10.9) 2 (22.2) 0.59
Lymphoma 35 (63.6) 30 (65.2) 5 (55.5) 0.71
Chronic lymphocytic leukemia 1 (1.8) 1 (2.3) 0 (0) .0.99
Others 2 (3.6) 1 (2.3) 1 (11.1) 0.30
Nephrotoxic drugs 25 (45.5) 21 (45.7) 4 (44.4) .0.99
Laboratory findings at admission
Phosphates (mmol/L), median (IQR) 4.4 (4.1–5.2) 4.6 (4.1–5.2) 4.2 (3.7–4.6) 0.25
Calcium (mmol/L), median (IQR) 2.2 (2.0–2.3) 2.2 (2.0–2.3) 2.1 (1.9–2.3) 0.34
Potassium (mmol/L), median (IQR) 4.4 (4.1–5.2) 4.6 (4.1–5.2) 4.2 (3.7–4.6) 0.25
LDH at admission (UI/L), median (IQR) 2764 (1431–6750) 2764 (1431–6750) 2883 (1191–7344) 0.95
Serum creatinine (mmol/L), median (IQR) 124.0 (89.0–190.5) 124.0 (94.5–195.0) 83.5 (64.5–138.0) 0.04a
Uric acid (mmol/L) (before rasburicase), median (IQR) 497.0 (226.0–615.0) 520.0 (270.5–696.5) 404.0 (220.9–511.3) 0.27
AKI stage 1, n (%) 15 (32.6)
AKI stage 2, n (%) 6 (13.0)
AKI stage 3, n (%) 25 (54.4)
Delay between ICU admission and AKI (d), median (IQR) 1 (0–1)
Positive crystalluria, n (%)b 7 (12.7) 5 (10.8) 2 (22.2) 0.46
Weddellite crystals (calcium oxalate dihydrate) 0 0 0 .0.99
Whewellite crystals (calcium oxalate monohydrate) 2 (3.6) 2 0 .0.99
Calcium-phosphate crystals 1 (1.8) 0 1 0.16
Uric-acid crystals 3 (5.5) 2 1 0.42
Struvite (magnesium ammonium phosphate hexahydrate) 1 (1.8) 1 0 .0.99
Urine pH (day 1), median (IQR) 6.2 (5.7–6.5) 6.2 (5.3–6.5) 6.2 (5.9–6.3) 0.94
Calcium urinary excretion, median (IQR) 0.1 (0.0–0.5) 0.13 (0.0–0.5) 0.1 (0.0–0.5) 0.76
Calcium-creatinine urinary ratio, mmol/mmol (day 1)
Phosphate urinary excretion (Ph/creat), mmol/mmol 5.6 (3.2–7.2) 4.9 (3.1–7.1) 7.1 (3.4–7.9) 0.39
(day 1) median (IQR)
Uric acid excretion (Uric/creat), mmol/mmol (day 1) 0.5 (0.1–0.9) 0.4 (0.1–1.0) 0.5 (0.0–0.7) 0.49
median (IQR)
Uric acid supersaturation index (day 1), median (IQR) 1.5 (0.1–4.3) 1.3 (0.1–3.5) 1.4 (0.1–2.6) 0.94
Calcium phosphate supersaturation index (day 1), 0.21 (0.10–0.45) 0.20 (0.08–0.47) 0.28 (0.18–0.61) 0.36
median (IQR)
Treatments
Rasburicase use, n (%) 46 (83.6) 38 (82.6) 8 (88.9) .0.99
Delay between ICU admission and first rasburicase 0 (0–0) 0 (0–0) 0 (0–0) 0.82
administration (d), median (IQR)
Allopurinol use, n (%) 9 (16.4) 8 (17.4) 1 (11.1) .0.99
Need for RRT, n (%) 25 (45.5) 25 (54.4) 0 0.03a
RRT duration (d), median (IQR) 2 (1–4) 2 (1–4)
Need for vasopressors, n (%) 9 (16.4) 8 (17.4) 1 (11.1) .0.99
Need for mechanical ventilation, n (%) 11 (20) 10 (21.7) 1 (11.1) 0.67
ICU mortality, n (%) 9 (16.4) 7 (15.2) 2 (22.2) 0.63
28-Day mortality, n (%) 20 (36.4) 16 (34.8) 4 (44.4) 0.71
P values are reported for comparisons across the two patient groups. IQR, interquartile range; Ca, calcium; creat, creatinine; Ph, phosphate; Uric, uric acid.
a
P,0.05.
b
Crystalluria is positive if at least one urine sample (day 1, 3, or 5) is positive. Crystalluria was negative at day 7 in all patients (49 patients had crystalluria analysis at
day 7).
1160 JASN JASN 33: 1154–1171, 2022

A MLL-AF9
B Disease burden in Bone Marrow Disease burden in Spleen Weight of spleen
translocation p<0.0001 p=0.0019 p=0.0020
80 20 200
MLL-AF9+ cells in
Bone Marrow (%)
MLL-AF9+ cells
in Spleen (%)
60 15
Weight (mg)
150
C57BL/6 GMP
actin dsRed
40 10 100
7–10 days
20 5 50
3 days 2 days 1 day

Cyt+Doxo Cyt
0 0 0
le
py
py
le
py
cl
ic
AML Tumor Lysis Syndrome
c
Chemotherapy
ra
ra
ra
hi
hi
h
he
he
Ve
Ve
he
Ve
ot
ot
ot
m
m
C
he
he
he
C
C
40 * * * *
Serum creatinine (mg/dl)
0.8
30
BUN (mg/dl)
0.6
D AML mice + vehicle
20 0.4
10 0.2
0 0.0
le
le
py
S
S
py
TL
ic
ic
TL
ra
ra
h
he
ve
ve
he
ot
ot
+
em
em
L
L
AM
AM
ch
ch
+
+
T
T
W
W
Serum phosphorus (mmol/l)
2.5
* 1000 ** *
800
AML mice + chemotherapy (TLS mice)
2.0
LDH (UI/l)
1.5 600
1.0 400
0.5 200
0.0 0
e
py
le
py
S
S
cl
ic
TL
TL
ra
ra
hi
h
he
he
ve
ve
ot
ot
+
+
em
em
L
L
AM
AM
ch
ch
+
+
T
T
W
Figure 1. Biochemical and microscopic analysis of a mouse model of AML mice show significant TLS after administration of
chemotherapy. (A) Model of TLS in AML mice. (B) Disease (percent MLL-AF91 blasts) in the bone marrow and spleen of MLL-AF9 mice
after treatment with vehicle or with chemotherapy (24 hours after treatment). n53 per group. (C) BUN, LDH, serum creatinine, and
phosphate were measured in AML mice treated with vehicle (n56) or chemotherapy (cytarabine 1 doxorubicin; n56) and WT mice
treated with chemotherapy (n56) 24 hours after treatment. (D) In vivo renal intravital microscopy in MLL-AF9 mice that received vehicle
or chemotherapy. MLL-AF9 leukemic blasts are DsRED positive and visible in renal peritubular capillaries. After chemotherapy, blast cell
infiltration is no longer visible. All data are presented as mean6SEM *P,0.05, **P,0.01 versus vehicle, Mann–Whitney test.
showed continuous fenestrations of the endothelium. (Figure 2C, Supplemental Videos 1 and 2). Chemotherapy in
Interestingly, TLS mice had profound alterations of the WT mice did not decrease MECA-32 staining or alter capil-
peritubular endothelium: the capillary shape was more lary permeability (Figure 2, B and C, Supplemental Figure 2,
irregular with swollen endothelium and lost fenestrations B and C, Supplemental Video 3). There was no difference in
(Figure 2A). To confirm these alterations were not simply terms of ICAM-1 staining in the TLS group when compared
due to the toxicity of the chemotherapeutic drugs, we also with AML mice treated with vehicle and WT mice
analyzed the endothelium in WT mice (without leukemia) (Supplemental Figure 3). Moreover, we did not observe sig-
after chemotherapy. We did not detect any significant dif- nificant apoptosis in vivo in TLS mice, as assessed by renal
ference between the peritubular endothelium in these c-PARP staining (Supplemental Figure 4).
mice and that of WT mice (Supplemental Figure 2A).
Moreover, we did not find any crystal deposits in TLS Circulating Histones Are Elevated during TLS and
mice (as has been observed using transmission electron Correlate with Renal Outcome
microscopy in other experimental models28) nor evidence Because renal microvascular endothelial dysfunction
of tubular injury. appeared to be central to the pathophysiology of TLS-
We confirmed renal endothelial alterations by immunohis- induced AKI, we then explored the mechanisms of endo-
tochemical analysis. Indeed, renal MECA-32 staining was thelial dysfunction. Extracellular histones have previously
reduced in AML mice treated with chemotherapy compared been reported to induce endothelial cytotoxicity. The
with AML mice treated with vehicle (Figure 2B). Vascular intracellular content is released after cell lysis in TLS,
permeability was assessed by confocal intravital microscopy. therefore, we hypothesized that extracellular histones may
Increased vascular permeability was observed in TLS mice, also be released during TLS. Indeed, assays of patient
detected by the leakage of albumin-FITC into the interstitium plasma demonstrated significantly elevated histones levels
A AML mice + vehicle

**
6
**
Fenestrations score (0–4)

4
2
AML mice + chemotherapy (TLS mice)
)
py
T
py
ic
W
ra
ra
ot e
he
em ic
he
L
ch S m
AM
ot
em
L+ TL
ch
+
T
M
W
(A
B AML mice + vehicle C AML mice + vehicle
**
**
Peritubular capillary permeability score

**
MECA-32 (mean staining intensity)
25 3
* **
20 **
2
15
10
1
AML mice + chemotherapy (TLS) 5 AML mice + chemotherapy (TLS)
0
0
T
py
)
py
cl
W
le
)
py
ra
py
T
hi
ra
ic
W
ra
he
ve
ra
h
he
he
ve
he
ot
ot
+
em
ot
ot
+
em
e
em
ic
em
L
ch
AM
ch
ch
ch
+
L+
AM
L+
T
+
M
W
M
(A
(A
e
ic
e
m
ic
m
S
TL
S
TL
Figure 2. Peritubular endothelium is significantly altered in TLS leukemic mice. (A) Peritubular capillaries undergo ultrastructural
alterations in TLS mice. Details of peritubular capillaries in transmission electron are shown for MLL-AF9 mice and TLS mice. Capillar-
ies from MLL-AF9 kidneys had a flat endothelium with regular contours and multiple fenestrations (arrows). During TLS, the capillary
shape became more irregular and the endothelium became swollen and lost fenestrations (asterisk). Leukemic blasts infiltrating the
peritubular capillaries are seen in MLL-AF9 mice. A semiquantitative analysis showed that, compared with WT, MLL-AF9, and
WT1chemotherapy kidneys, the capillary area with fenestrations was significantly decreased in TLS mice (n56 per group). Scales
bars, 6 mm in left, 1 mm in middle-left, 500 nm in middle-right, 200 nm in right panel. (B) Kidneys from WT mice treated with vehicle,
WT mice receiving chemotherapy, and AML mice receiving chemotherapy (TLS mice) or vehicle were immunostained with a panen-
dothelial cell antigen antibody (MECA-32). Decreased peritubular capillary staining was observed in TLS (peritubular capillary den-
sity, as quantified by computer image analysis of MECA-32 immunostaining). n56 per group. Original magnification, 2003. (C) Peri-
tubular capillary permeability in WT mice treated with vehicle, WT mice receiving chemotherapy, TLS mice, or AML mice. Analysis of
interstitial leakage of albumin-FITC was performed using confocal videomicroscopy. Representative still images from the captured
videos at 10 minutes in AML mice and TLS mice are presented in (C). Increased peritubular capillary permeability was observed after
TLS. n55–6 per group. *P,0.05, **P,0.01, Mann–Whitney test.
in patients with TLS (median [interquartile range (IQR)], the mouse model of TLS (AML mice treated with chemother-
4.43 [1.34–7.64] mg/ml) compared with samples from apy), extracellular histones increased significantly 24 hours
patients with acute myeloid/lymphoid leukemia without after high-dose chemotherapy administration (median levels
TLS (median [IQR], 0.75 [0.03–2.46] mg/ml; P,0.001) or of 0.17 mg/ml in vehicle-treated mice versus 2.47 mg/ml in
from patients with AKI due to other causes (0.58 chemotherapy-treated mice; P,0.002; Figure 3E).
[0.46–1.08] mg/ml; P,0.001) (Figure 3A).
Moreover, levels of circulating histones significantly
correlated with AKI severity and mortality (Figure 3, B and C). Extracellular Histones Increase Renal Microvascular
In patients who required RRT, levels of circulating histones Dysfunction In Vivo
were significantly decreased at the end of RRT (single session To establish the pathogenic role of extracellular histones on
of 6 hours of hemodialysis): median (IQR) of 3.89 endothelial dysfunction in the context of TLS, we per-
(2.23–11.93) mg/ml at the initiation of RRT versus 1.89 formed transmission electronic microscopy analysis in kid-
(1.02–4.72) mg/ml at the end of RRT (P,0.02; Figure 3D). In neys of WT mice injected with 25 mg/kg of recombinant
1162 JASN JASN 33: 1154–1171, 2022

***
A *** B C
*
Extracellular histones (Pg/ml)

15 *** 15 15 **
10 10 10
5 5 5
0 0 0
rs
rs
I
I1
I2
I3
l
S
tro
AK
vo
ivo
TL
TL
TL
AK
AK
AK
on
vi
rv
o
o
no
r
N
C
In
Su
su
L
AK
AM
AKI stage
on
N
D E
15 * 5 **
* 4
10
3
2
5
1
0 0
le
on
e
RT
RT
ic
ic
si
m
h
R
rR
is
ve
S
m
te
TL
r
+
ad
fo
af
e
be
ic
at
s
ne
m
s
s
ne
ne
to
L
AM
to
is
to
H
is
is
H
H
Figure 3. Circulating histones are elevated in patient plasma during TLS and in a mouse model of TLS. (A) Plasma histone con-
centrations were determined in four independent cohorts of patients. n584 patients with TLS, n58 controls, n514 patients with AKI
without TLS, n517 patients with acute leukemia without TLS (Kruskal–Wallis test with Dunn post-test). (B) Plasma histone concentra-
tions are shown according to AKI stage in patients with TLS-AKI (Kruskal–Wallis test with Dunn post-test). (C) Plasma histone concen-
trations in survivors and nonsurvivors at day 28 (paired t test). (D) Kinetic of plasma histones concentrations in patients who required
RRT (hemodialysis; n524) before and after a single session of 6 hours of RRT (Kruskal–Wallis test with Dunn post-test). (E) Extracellu-
lar histone concentrations in the AML mouse model with or without TLS. n56 per group (Mann–Whitney test). All data are presented
as mean6SEM. *P,0.05, **P,0.01, ***P,0.001.
histones. Median (IQR) plasma histones levels were 3.34 capillaries to the interstitial compartment in confocal micros-
(2.56–9.3) mg/ml at 1 hour and 21.19 (20.32–21.85) mg/ml copy (Figure 4C, Supplemental Videos 4–6). Capillary perfu-
at 24 hours (Supplemental Figure 5). Twenty-four hours sion was then analyzed. We categorized the cortical
after injection, we observed similar ultrastructural changes distribution of peritubular capillary perfusion as continuous,
compared with those observed in TLS mice (Figure 4A). intermittent, or no flow, as previously described.29 After 4
After histone exposure, MECA-32 staining was also hours, mice injected with vehicle only had a high percentage
decreased (Figure 4B). of continuously perfused renal cortical capillaries (81%65%),
We then used intravital confocal microscopy to study and only a small percentage of capillaries with no flow
whether histone exposure induced renal microvascular dys- (4%61.8%) (Figure 4C). In contrast, 4 hours after histone
function. Leukocyte recruitment and renal microvascular per- administration, the percentage of capillaries with continuous
meability were evaluated by intravital microscopy after perfusion was reduced to 57%66.2% (P,0.008 versus vehi-
intravenous administration of 25 mg/kg of histones in C57BL/ cle), and the percentage of capillaries with no perfusion was
6 mice. We did not find any difference in leukocyte recruit- increased to 21%64.6% (P,0.02 versus vehicle) (Figure 4C,
ment in mice treated with histones compared with control Supplemental Video 7). After histone injection, renal function
mice (Supplemental Figure 6). However, histone exposure sig- was altered with increased BUN and serum creatinine 24
nificantly increased leakage of FITC-albumin from peritubular hours after injection (Figure 4D).
A WT mice + vehicle B WT mice + vehicle
6 **
(mean staining intensity)

25

*
MECA-32 staining
20
4 15
10
2 5
WT mice + recombinant histones WT mice + histones
0
le
es
0
ic
on
h
ve
st
le
es
hi
ic
+
on
h
+
T
ve
st
T
hi
W
+
+
T
W
T
W
D 80
***
C
BUN (mg/dl)
WT mice + vehicle 60
100
WT + vehicle 3 ** 40
WT + histones
% capillary perfusion
80
Peritubular capillary
20
permeability score
60
** 2 0
le
es
ic
on
h
ve
st
40
hi
+
1
+
T
*
T
WT mice + histones
W
20

0 0.8
0 **
le
es
ic
us
ow
t
0.6
on
en
h
uo
ve
Fl
st
itt
tin
hi
rm
+
N
0.4
on
+
T
te
T
C
In
W
0.2
0.0
es
le
ic
on
h
ve
st
hi
+
+
T
W
T
W
Figure 4. Recombinant histones induce endothelial dysfunction in vivo. (A) Peritubular capillaries undergo ultrastructural altera-
tions in mice injected with recombinant histones (25 mg/kg). Twenty-four hours after histone injection, the endothelium became
swollen and lost fenestrations (asterisk). A semiquantitative analysis showed that, compared with WT mice, the capillary area with
fenestrations was significantly decreased. n56 per group. Scale bars, 600 nm in left panel, 400 nm in right panel. (B) Kidneys from
WT mice receiving vehicle and histones were immunostained with anti-mouse panendothelial cell antigen antibody (MECA-32).
Decreased peritubular capillary staining was observed 24 hours after recombinant histone injection. Peritubular capillary density was
quantified by computer image analysis of MECA-32 immunostaining. n55 per group. Original magnification, 2003. (C) Representa-
tive still images from the captured videos of peritubular capillaries in WT mice injected with vehicle or recombinant histones at 25
mg/kg. Analysis of interstitial leakage of albumin-FITC and capillary perfusion were performed using confocal videomicroscopy.
Increased peritubular capillary permeability was observed 24 hours after histone exposure. Capillary perfusion also decreased in
some areas of the kidney after histone exposure. n56 per group. (D) BUN and serum creatinine were measured in WT mice treated
with vehicle (n57) or recombinant histones (n57) 24 hours after treatment. *P,0.05, **P,0.01, ***P,0.001, compared with WT
mice, Mann–Whitney test.
Extracellular Histones Activate ECs In Vitro and Figure 7). Thus, high levels of extracellular histones can
Mediate Cellular Damage decrease the viability of microvascular ECs in vitro.
Extracellular histones induce EC activation with increased
expression of CD54 (ICAM-1) and CD106 (vascular cell Histone Effect on Endothelial Phenotype Involved
adhesion molecule-1) and increased secretion of IL-6 and TLR4 but Not TLR2
IL-8 by ECs (Figure 5, A and B). Extracellular histones did On the basis of previous data showing extracellular histones
not change CD62E (E-selectin) expression. At high concen- have agonistic effects on TLRs (TLR2 and TLR4),30 we then
trations (40 mg/ml), histones induced EC damage, as asked if these receptors were involved in EC activation after
revealed by the production of reactive oxygen species in exposure to histones. Neutralizing antibodies specific for
live cells (Figure 5C). Moreover, the proportion of annexin either TLR2 or TLR4 were preincubated with ECs before
V–positive, 7AAD-negative cells was increased in histone- histone stimulation in vitro. Anti-TLR4, but not anti-TLR2,
stimulated ECs compared with control cells (Supplemental strongly reduced histone-induced upregulation of IL-6 by
1164 JASN JASN 33: 1154–1171, 2022

Control
A Positively stained cells (%)
50 CD54 CD106 CD62E CD54 CD106 CD62E 100
HMEC
500
* * 80 HMEC + H3 20 Pg/mL
40 400 HMEC + H3 40 Pg/mL
60
30 300
MFI
20 40
200 *
10 100 20
0 0 0
3 4 5
l
3
l
C 3
l
3
0 10 10 10
l
3
l
3
l
3
tro
tro
tro
tro
tro
tro
H
H
on
on
on
on
on
on
CD54+
C
C
300 * *
*** 400 400
B 800
*
*** 300 300
IL-6 (pg/ml)
IL-8 (pg/ml)
IL-8 (pg/ml)
600 200
IL-6 (pg/ml)
200 200
400
100
200 100 100
0 0 0 0
4h
8h
h
l
4h
8h
tro
tro
/m
/m
/m
/m
tro
tro
/m
/m
/m
/m
24
24
Pg
Pg
Pg
on
g
Pg
Pg
Pg
3
on
on
on
5P
5P
3
H
H
10
20
40
10
20
40
C
C
3
3
H
3
H
H
C 100
Mean fluorescence intensity
HMEC
2500
HMEC + H3 20Pg/mL
80 HMEC + H3 40Pg/mL 2000
Positive control
60 1500
1000
40
500
20
0
0
EC
L
/m
/m
M
3 4 5
Pg
Pg
0 10 10 10
H
20
40
ROS+
3
3
H
H
+
+
EC
EC
M
M
H
Figure 5. Endothelial cells are activated by extracellular histones in vitro. (A) CD54, CD106, and CD62E expression were assessed
in ECs activated with recombinant histones. Mean fluorescence intensity (MFI) and percentage of CD541, CD1061, and
CD62E1positive ECs nonactivated or activated with 20 mg/ml histones over 24 hours. Representative plots for CD54 expression are
gated on microvascular ECs (HMECs). Mean6SEM is shown from four to six independent experiments. Mann–Whitney test. (B) IL-6 and
IL-8 levels in the supernatant of HMEC treated with histones at the indicated concentrations (5, 10, 20, 40 mg/ml) and different time
points at 20 mg/ml. Mean6SEM is shown from four to ten independent experiments. Kruskal–Wallis test with Dunn post-test. (C) Effect
of histones on reactive oxygen species (ROS) production. HMECs were treated with histones at the indicated concentrations. Production
of ROS by live cells was analyzed by flow cytometry using the CellROXTM Green Flow Cytometry Assay Kit. Tert-butyl hydroperoxide
(THBP) solution, an inducer of ROS, was used as positive control. Kruskal–Wallis test with Dunn post-test. *P,0.05, ***P,0.001.
ECs and their expression of CD54 (Figure 6, A and B). significant increase of IL-6, IL-8, and sICAM-1 in patients
Moreover, confocal microscopy examination of ECs with TLS, as compared with other groups (Figure 7A). The
revealed colocalization of TLR4 and extracellular histones decrease of plasma IL-6 from ICU admission to day 7 paral-
on the EC surface (Supplemental Figure 6C). In vivo, leled the decrease of extracellular histone concentrations
TLR4-knockout mice injected with recombinant histones (Figure 7, B and C).
were protected against renal endothelial dysfunction, as
assessed by intravital microscopy and MECA-32 staining
Nonanticoagulant Heparin Alleviates Histone-Induced
(Figure 6D, Supplemental Videos 8 and 9).
Endothelial Cytotoxicity In Vitro and In Vivo
Heparin has a high negative charge density and a strong
Patients with TLS Have Increased Endothelial affinity for histones. Indeed, because histones are positively
Dysfunction Markers charged, heparin has been shown to neutralize extracellular
Having observed the above modifications of human ECs histones.31 Heparin significantly decreased plasma histone
in vitro, we then assessed plasma concentration of inflamma- levels in WT mice injected with recombinant histones
tory markers in patients with TLS using a multiplex electro- (Supplemental Figure 5). We thus investigated the effects of
chemiluminescent assay of seven cytokines/chemokines (IL-6, nonanticoagulant heparin on ECs in vitro and in vivo dur-
IL-8, IL-10, TNF-a, IL-1b, IFN-g, and sICAM-1). We found a ing TLS. Administration of heparin decreased the activation
A B * *
D TLR4 KO mice + vehicle TLR4 KO mice + histones
1.5 * 800 * * 60
normalized IL-6
600
production
CD54 MFI
CD54 %
1.0 40
400
0.5 20
200
0.0 0 0
4
L
4
tro
tro
H
R
/m
/m
/m
/m
R
L
TL
on
-T
TL
on
l+
l+
µg
µg
µg
µg
i
t i-
C
t
t i-
tro
tro
an
20
20
20
an
an
2
on
on
e
3+
3+
3
3
yp
C
H
H
ot
2+
4+
4+
l+
l+
is
R
tro
G
tro
an
TL
TL
lg
on
on
t i-
t i-
C
an
an
R
TL
t i-
an
C H3 TLR-4 Overlay
25 0.8
20
permeability score
MECA-32 staining
0.6
15
0.4
10
0.2
5
0 0.0
le
le
es
es
ic
ic
on
on
h
h
ve
ve
st
st
hi
hi
+
+
+
+
KO
KO
KO
KO
4
4
R
R
4
4
R
TL
R
TL
TL
TL
Figure 6. TLR4, but not TLR2, was implicated in histone-mediated activation of ECs. (A) HMECs were preincubated with mouse
isotype control IgG (Iso-IgG, 50 mg/ml), mouse anti-human TLR2 (anti-TLR2, 50 mg/ml), and/or anti-human TLR4 (anti-TLR4,
50 mg/ml) for 60 minutes, and then stimulated with 20 mg/ml histones (H3) for 24 hours. The secretion of IL-6 was then determined
by ELISA. Data are presented as mean6SEM from four different experiments. (B) HMECs were preincubated with anti-TLR4 for
60 minutes (TAK-242, 1 mM) and stimulated with 20 mg/ml histones (H3) for 24 hours. Mean fluorescence intensity (MFI) and percent-
age of CD54-positive ECs after histones 6TAK-242 exposure. Data are presented as mean6SEM from six different experiments. (C)
TLR4 and extracellular H3 colocalize in ECs. Confocal images showed the localization of histones H3 (red) and TLR4 (green) in ECs.
49 ,6-Diamidino-2-phenylindole (blue) was used to stain the nuclei. (D) MECA-32 staining by immunohistochemistry and peritubular
capillary permeability (intravital confocal microscopy) in TLR4 knockout (TLR4 KO) mice injected with vehicle or histones (24 hours
after injection of 25 mg/kg of recombinant histones). No difference was observed between the two groups (Mann–Whitney test).
n54 per group. *P,0.05, Mann–Whitney test.
of ECs after histone exposure (Figure 8A). In the mouse induced AKI, whereby (1) endothelial dysfunction partici-
model of TLS, administration of heparin at 3 mg/kg twice a pates in TLS-induced AKI; (2) plasma levels of extracellu-
day, on the day before and during chemotherapy adminis- lar histones are hugely increased during TLS, and this is
tration, was able to prevent renal endothelial dysfunction, associated with the occurrence and severity of AKI; (3)
as assessed by electron microscopy, MECA-32 immu- extracellular histones induce renal endothelial dysfunction
nostaining, and intravital microscopy (Figure 8, B–D, in vitro and in vivo; and (4) nonanticoagulant heparin can
Supplemental Videos 10–12). Although nonanticoagulant neutralize extracellular histones and prevent endothelial
heparin improved kidney function of WT mice injected damage.
with histones (Figure 8E), it was not able to significantly The evidence for calcium-phosphate crystals during TLS
improve renal function in TLS mice (Figure 8E). relies on scarce data from relatively old case reports.5,6
Although we cannot exclude that crystal deposits may have
been missed by electron microscopy in our experimental
DISCUSSION model, uric-acid and calcium-phosphate crystals were
uncommon, despite the high prevalence of AKI in patients
In this study coupling a multicenter cohort of patients with TLS. This led us to question the role of additional
with TLS with an in vivo model of chemotherapy-induced crystal-independent mechanisms in the pathophysiology of
TLS and in vitro studies, we demonstrate, for the first TLS-induced AKI. Indeed, crystalluria is a sensitive method
time, that uric-acid and calcium-phosphate crystals are to predict the occurrence of crystal-induced nephropathy.
rare during TLS-induced AKI in the rasburicase era and Although calcium-phosphate crystal–induced nephropathy
establish a new model for the pathophysiology of TLS- and uric-acid nephropathy are always preceded by
1166 JASN JASN 33: 1154–1171, 2022

A **
800 * 1000 100 100
***
*
800 80 80
sIC AM-1 (µg/ml)

600
TNF-D (pg/ml)
IL-10 (pg/ml)
IL-6 (pg/ml)
600 60 60
400
400 40 40
200
200 20 20
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600 6
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400 4
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300 3
200 2
100 1
0 0
on
on
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si
is
is
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Figure 7. Endothelial dysfunction markers were elevated in plasma of patients with TLS. (A) Plasma cytokine concentrations
(multiplex analysis) in four independent cohorts of patients: n57–27 patients with TLS, n54–9 controls, n53–13 patients with AKI
without TLS, and n56–8 patients with acute leukemia without TLS. Values are expressed as mean6SEM. (B) Time course of plasma
IL-6 concentrations in nine patients with TLS. (C) Time course of IL-6 and extracellular histones concentrations. The left panel shows
the kinetic of plasma IL-6 concentration in one patient with TLS from ICU admission to day 7 (D7) and the parallel decrease of extra-
cellular histones concentrations from ICU admission to D7. *P,0.05, **P,0.01, ***P,0.001, Kruskal–Wallis test with Dunn post-test.
crystalluria, the reverse is not true and crystalluria may occur In patients with TLS-induced AKI, we have found dras-
without resulting in crystal-induced nephropathy or stone tically higher levels of extracellular histones compared with
formation.32 Calcium-phosphate crystallization may result patients experiencing postrenal AKI. However, although
from high concentrations of calcium and phosphate, but the histones were significantly elevated in AKI stage 3, the
main factor increasing calcium-phosphate supersaturation in increase of histones in AKI stage 1 and 2 was not signifi-
urine is high pH. Therefore, calcium-phosphate crystals occur cant. This may be due to a lack of statistical power of these
when urinary alkalinization is performed. Because urine alka- results. Nevertheless, the association between AKI and his-
linization is no longer recommended during TLS,7 this may tone elevation does not prove a causal effect of histones on
also explain the absence of calcium-phosphate crystals in our AKI. The endothelial toxicity of extracellular histones has
study. Uric-acid nephropathies are also reported during been previously described during sepsis or trauma.10,34 We
TLS.1,33 However, the wide use of hypouricemic therapies show in this study that extracellular histones induce activa-
(90% of the patients in our cohort) has probably decreased tion of microvascular ECs. In addition, injection of recom-
the risk of urate nephropathies. Despite these elements, AKI binant histones in vivo induces renal endothelial
still develops during TLS. Our data concord with previous dysfunction with increased microvascular leakage. These
studies4 and AKI remains prevalent during TLS, despite wide results are in agreement with a previous study suggesting a
use of rasburicase, use of saline for hydration, and close mon- role for histones in renal endothelial dysfunction in a
itoring of patients with TLS in the centers involved in this model of endotoxin-induced AKI, in a TLR2/TLR4-depen-
study. Moreover, crystal-induced mechanisms do not explain dent manner.30 Histones can bind EC membranes on
sepsis-like syndromes with multiple organ failure that may be TLR4 and increase permeability. Although TLR4 was
observed during TLS.3 involved in the modifying adhesion molecule expression
A *
B AML mice + chemotherapy+heparin
400
* 5 **

300 4 *
IL-6 (pg/ml)
3
200
2
100
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WT mice + histones + heparin
25
** 3
**
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20 60
permeability score
BUN (mg/dl)
*
15 2
40
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AML mice + chemotherapy (TLS) + heparin AML mice + chemotherapy (TLS) + heparin
L
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T
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MECA-32 staining
15 0.8 ** * *
3 **

permeability score
10 0.6
2
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ic
ic
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W
T
ic
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on
m
m
L
st
S
S
AM
hi
TL
TL
+
T
W
Figure 8. Heparin mitigates the effect of extracellular histones and TLS on renal endothelial dysfunction. (A) IL-6 in the superna-
tant of ECs (HRGECs/HMECs) treated with 20 mg/ml histones and heparin at the indicated concentrations (400 and 600 mg/ml) for
24 hours. Mean6SEM is shown from four to ten independent experiments. *P,0.05, Mann–Whitney test. (B) Peritubular capillaries
were examined by transmission electron microscopy in MLL-AF9 mice and TLS mice, having received chemotherapy and heparin 3
mg/kg twice a day 24 hours before and during the administration of chemotherapy. Capillaries from these mice have a flat endothe-
lium with multiple fenestrations (arrows). A semiquantitative analysis showed that, compared with TLS mice without heparin, the cap-
illary area with fenestrations was significantly increased. n56 per group. Scale bars, 1 mm in left panel, 200 nm in right panel.
*P,0.05, **P,0.01, Mann–Whitney test. (C) Kidneys from WT mice injected with histones and heparin (left panel) and TLS mice
receiving heparin (right panel) were immunostained with panendothelial cell antigen antibody (MECA-32). Decreased peritubular
capillary staining was observed after TLS or histone exposure, but not after heparin administration. Peritubular capillary density was
quantified by computer image analysis of MECA-32 immunostaining. n54–6 per group. Original magnification, 2003. **P,0.01,
Mann–Whitney test. (D) Representative still images extracted after 10 minutes from videos of peritubular capillaries of WT mice
injected with recombinant histones and heparin (left panel) and from TLS mice injected with heparin (right panel). Analysis of intersti-
tial leakage of albumin-FITC and capillary perfusion were performed by confocal videomicroscopy. Increased peritubular capillary
permeability was observed after TLS or exposure to histones, but not after heparin administration. n55–6 per group. *P,0.05,
**P,0.01, Mann–Whitney test. (E) BUN and serum creatinine were measured in WT mice treated with vehicle (n57) or recombinant
histones with (n57) or without (n57) heparin, in AML mice treated with vehicle (n56), and TLS mice treated with (n56) or without
heparin (n56). Nonanticoagulant heparin significantly decreased creatininemia of WT mice injected with histones. *P,0.005,
**P,0.001, ***P,0.0001, Kruskal–Wallis test with Dunn post-test. Hep, heparin.
and IL-6/IL-8 secretion by ECs, we did not find evidence of some patients experienced hemodynamic failure, neurologic
TLR2 being implicated in this model. failure, and AKI, and may have required mechanical venti-
The cytokine production, including increased IL-6 lation, none of our patients with TLS experienced acute
release, observed in our patients, may participate in AKI. respiratory distress syndrome. In AML mice treated with
Indeed, emerging data show that local activation of IL-6 is vehicle and TLS mice, we did not detect any evidence of
implicated in renal endothelial dysfunction and AKI.35,36 acute lung injury (Supplemental Figure 8).
Beyond AKI, extracellular histones have been linked to Forming a dynamic interface between blood and tis-
acute lung injury in the literature.10,37 However, although sues, the vascular endothelium may have implications in
1168 JASN JASN 33: 1154–1171, 2022

potentiating AKI and multiple organ failure. The role of increased inflammatory response in vitro and in vivo.44 In
microvascular dysfunction during TLS has not been pre- our study, we used a nonanticoagulant heparin to maintain
viously explored. We set up an experimental model of the neutralizing effect of heparin on histones, without
MLL-AF9 AML to induce TLS and to allow in vivo stud- increasing the risk of bleeding in patients with underlying
ies. Indeed, patients with AML have a high risk of TLS hematologic malignancies who are already at high risk of
because of the chemosensitivity and rapid proliferation spontaneous bleeding. Nevertheless, nonanticoagulant hep-
rate in AML. Moreover, the expression of MLL-AF9 in arin did not significantly improve renal function in TLS
normal GMPs is sufficient to generate an aggressive, mice, suggesting mechanisms other than histone-mediated
transplantable myeloid leukemia with a high tumor bur- AKI may be have been involved in our model.
den. To be sure that the chemotherapy alone did not Interestingly, we found a strong decrease of plasma
induce renal endothelial toxicity, we performed control extracellular histones after RRT in patients with TLS-AKI.
experiments by administering chemotherapy in WT Given the small size of extracellular histones (11–21 kD),
mice, and we did not observe any evidence of AKI or we believe that RRT using membranes with a molecular
renal endothelial dysfunction. The chemotherapy regimen mass cutoff around 20 kD may have removed extracellular
(doxorubicin and cytarabine) is currently used as the histones. On the other hand, early initiation of RRT in
main chemotherapeutic regimen in patients with AML. patients with high-grade hematologic malignancies may
Extremely rare case reports of acute rhabdomyolysis result in underdosing chemotherapeutic drugs and may,
have been reported with cytarabine chemotherapy,38 but therefore, decrease the chance of achieving complete remis-
there are no reports of AKI induced by doxorubicin sion. Nonanticoagulant heparin may then be of particular
chemotherapy. interest to delay the initiation of RRT. To date, there are
We confirmed in our model that TLS was associated no randomized clinical studies that have focused on the
with high circulating levels of histones and that renal endo- timing and indications of RRT in patients with TLS.
thelial dysfunction was present during AKI-induced TLS. In conclusion, this study provides the first evidence that
Our results do not exclude the involvement of other the previously described crystal-dependent mechanisms are
mechanisms involved in the pathophysiology of TLS- insufficient to explain TLS-induced AKI.
induced AKI. Indeed, other potentially toxic damage- Crystal-independent mechanisms of TLS-induced AKI
associated molecular patterns, such as high mobility group involve extracellular histones and endothelial dysfunction.
box 1, produced by dying cells are released after chemo- This study sheds new light on the pathophysiology of TLS-
therapy.39 Moreover, other cancer-associated nephrotoxic induced AKI and extracellular histones may, therefore,
insults may contribute to AKI40 and these mechanisms and
constitute a novel target for either diagnostic and/or therapeu-
extracellular histone release are not necessary mutually
tic intervention in TLS when endothelial dysfunction occurs.
exclusive mediators of AKI in TLS. Nevertheless, the drastic
increase of histones in the TLS mouse model and in
patients with TLS, their association with AKI, and their DISCLOSURES
endothelial toxicity suggest that extracellular histones are
important mediators of TLS-induced endothelial dysfunc- R. Itzykson reports consulting for Abbvie, Amgen, BMS/Celgene, Daiichi-
tion. Moreover, we found that heparin administration Sankyo, Jazz Pharma, Karyopharm, Novartis, and Stemline Therapeutics;
could prevent renal endothelial damage in the TLS model. and receiving research funding from Novartis and Janssen; none of which is
Because of their electrostatic interactions, heparin neutral- related to this work. E. Letavernier reports receiving consulting fees, and
honoraria, from Biocodex; receiving research funding from Coloplast; and
izes extracellular histones. In a murine model of septic AKI
having patents or royalties via Inserm Transfert. N. Mooney reports receiv-
using cecal ligation and puncture, Wang et al.17 showed ing research funding from CSL-Behring. L. Zafrani reports receiving a Jazz
that heparin was able to alleviate renal inflammation Pharmaceuticals grant. All remaining authors have nothing to disclose.
through the neutralization of histones. Moreover, Kawai
and colleagues41 demonstrated that heparin was able to
improve multiple organ injuries and the survival rate of FUNDING
mice injected with a fatal dose of histones by inhibiting
histone-EC binding. It is, however, possible that heparin This work was supported by a Jazz Pharmaceuticals grant, ESICM Basic
may have other roles in AKI, independent of histone neu- Science Award, and Ministere de la Sante grant PHRC AOM 08235.
tralization. For example, recent data showed that heparin
binding protein (HBP) may promote macrophage activa-
ACKNOWLEDGMENTS
tion and inflammation during sepsis-induced AKI,42,43 and
elevated plasma levels of HBP have been associated with The Groupe de Recherche Respiratoire en Reanimation Onco-Hematologique
AKI during sepsis in humans.44 Different heparin deriva- (GRRROH) members include: Elie Azoulay, Djamel Mokart, Frederic Pene,
tives were indeed able to abrogate the HBP-induced Achille Kouatchet, Julien Mayaux, François Vincent, Martine Nyunga,
Fabrice Bruneel, Pierre Perez, Anne-Pascale Meert, Dominique Benoit, Supplemental Video 8. Intravital confocal microscopy in TLR4KO mice 1
Michael Darmon, and Virginie Lemiale. vehicle.
Supplemental Video 9. Intravital confocal microscopy in TLR4KO mice 1
histones 25mg/kg.
Supplemental Video 10. Intravital confocal microscopy in AML mice 1
AUTHORS CONTRIBUTIONS
histones 1 heparin.
Supplemental Video 11. Intravital confocal microscopy in TLS mice 1
M. Arnaud¸ E. Arrii, J. Demonchy, C. Djediat, S. Fodil, P. Frere, E. Leta- histones 1 heparin.

vernier, M. Loiselle, M. Nacer, S. Placier, S. Pons, C. Vaganay, and L. Zafrani Supplemental Video 12. Intravital confocal microscopy in WT mice 1
were responsible for investigation; M. Arnaud, C. Djediat, P. Frere, R. Itzyk- histones 25mg/kg 1 heparin.
son, E. Letavernier, S. Placier, S. Pons, and L. Zafrani were responsible for
methodology; M. Arnaud, P. Frere, R. Itzykson, E. Letavernier, N. Mooney,

S. Pons, A. Puissant, and L. Zafrani were responsible for validation; M. REFERENCES
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AFFILIATIONS
1
Human Immunology and Immunopathology, Institut National de la Sant e et de la Recherche Medicale (INSERM) U 976, University of Paris Cit
e,
Paris, France
2
INSERM UMR 944, Saint Louis Hospital, University of Paris Cit
e, Paris, France
3
INSERM UMR S 1155, Sorbonne University, Paris, France
4
Multidisciplinary Functional Explorations Department, Assistance Publique des H^ opitaux de Paris, Tenon Hospital, Paris, France
5
Department of Hematology, Assistance Publique des H^ opitaux de Paris, Saint Louis Hospital, Paris, France
6
Electron Microscopy Department, UMR 7245, Museum National D’Histoire Naturelle, Paris, France
7
Medical Intensive Care Unit, Assistance Publique des H^
opitaux de Paris, Saint Louis Hospital, Paris, France

Tumor Lysis Syndrome and Aki Beyond Crystal.14

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Tumor Lysis Syndrome and Aki Beyond Crystal.14

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BASIC RESEARCH www.jasn.

Tumor Lysis Syndrome and AKI: Beyond

Emmanuel Letavernier, Jordane Demonchy, Sofiane Fodil,1 Manal Nouacer,1

re,3 Eden Arrii,1 Julien Lion,1 Nuala Mooney,1

JASN 33: 1154–1171, 2022. doi: https://doi.org/10.1681/ASN.2021070997

1154 ISSN : 1533-3450/1046-1154 JASN 33: 1154–1171, 2022

remission of the underlying malignancy and is associated

Clinical and Biologic Data

1156 JASN JASN 33: 1154–1171, 2022

Serum creatinine was measured using a high-performance

one representing 6%–25%, two representing 26%–50%, Statistical Analyses

Cell Lines and Culture Reagents Study Approval

1158 JASN JASN 33: 1154–1171, 2022

Age (yr), mean6SD 58612 61614 50620 0.67

Chronic cardiac dysfunction, n (%) 11 (11.7) 11 (13.3) 0 0.19

Age (yr), mean6SD 57617 59615 44620 0.25

Diabetes, n (%) 8 (14.5) 8 (17.4) 0 (0.0) 0.33

1160 JASN JASN 33: 1154–1171, 2022

3 days 2 days 1 day

A AML mice + vehicle

Fenestrations score (0–4)

Peritubular capillary permeability score

1162 JASN JASN 33: 1154–1171, 2022

Extracellular histones (Pg/ml)

Extracellular histones (Pg/ml)

A WT mice + vehicle B WT mice + vehicle

(mean staining intensity)

Fenestrations score (0–4)

Serum creatinine (mg/dl)

1164 JASN JASN 33: 1154–1171, 2022

(mean staining intensity)

1166 JASN JASN 33: 1154–1171, 2022

sIC AM-1 (µg/ml)

Fenestrations score (0–4)

WT mice + histones + heparin

Serum creatinine (mg/dl)

1168 JASN JASN 33: 1154–1171, 2022

M. Arnaud¸ E. Arrii, J. Demonchy, C. Djediat, S. Fodil, P. Frere, E. Leta- histones 1 heparin.

methodology; M. Arnaud, P. Frere, R. Itzykson, E. Letavernier, N. Mooney,

1170 JASN JASN 33: 1154–1171, 2022

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