Multiplex Optical Urinalysis for Early Detection of Drug-Induced Kidney Injury

Article abstract of DOI:10.1021/acs.analchem.0c00989

Drug-induced kidney injury (DIKI) is a significant contributor of both acute and chronic kidney injury and remains a major concern in drug development and clinical care. However, current clinical diagnostic methods often fail to accurately and timely detect nephrotoxicity. This study reports the development of activatable molecular urinary reporters (MURs) that are able to specifically detect urinary biomarkers including γ-glutamyl transferase (GGT), alanine aminopeptidase (AAP), and N-acetyl-β-d-glucosaminidase (NAG). By virtue of their discrete absorption and emission properties, the mixture of MURs can serve as a cocktail sensor for multiplex optical urinalysis in the mouse models of drug-induced acute kidney injury (AKI) and chronic kidney disease (CKD). The MURs cocktail not only detects nephrotoxicity earlier than the tested clinical diagnostic methods in drug-induced AKI and CKD mice models, but also possesses a higher diagnostic accuracy. Therefore, MURs hold great promise for detection of kidney function in both preclinical drug screening and clinical settings.

Full text of DOI:10.1021/acs.analchem.0c00989

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Article
Multiplex Optical Urinalysis for Early Detection of Drug-induced Kidney Injury
Penghui Cheng, Qingqing Miao, Jiaguo Huang, Jingchao Li, and Kanyi Pu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.0c00989 • Publication Date (Web): 02 Apr 2020
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Page 1 of 8
Analytical Chemistry
Multiplex Optical Urinalysis for Early Detection of Drug-induced Kidney
Injury
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Penghui Cheng, Qingqing Miao, Jiaguo Huang, Jingchao Li and Kanyi Pu*
School of Chemical and Biomedical Engineering, Nanyang Technological University, 637457 Singapore. Email:
kypu@ntu.edu.sg
ABSTRACT: Drug-induced kidney injury (DIKI) is a significant contributor of both acute and chronic kidney injury, and
remains a major concern in drug development and clinical care. However, current clinical diagnostic methods often fail to
accurately and timely detect nephrotoxicity. This study reports the development of activatable molecular urinary reporters
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(
MURs) that are able to specifically detect urinary biomarkers including γ-glutamyl transferase (GGT), alanine
aminopeptidase (AAP) and N-acetyl-β-D-glucosaminidase (NAG). By virtue of their discrete absorption and emission
properties, the mixture of MURs can serve as a cocktail sensor for multiplex optical urinalysis in the mouse models of drug-
induced acute kidney injury (AKI) and chronic kidney disease (CKD). The MURs cocktail not only detects nephrotoxicity
earlier than the tested clinical diagnostic methods in drug-induced AKI and CKD mice models, but also possesses a higher
diagnostic accuracy. Therefore, MURs hold great promise for detection of kidney function in both pre-clinical drug screening
and clinical settings.
Kidney is a vital organ in the body that plays important
roles in regulating body fluid homeostasis and blood
filtration as well as maintaining electrolyte balance. As it is
the primary site for metabolism and excretion of drugs,
imaging contrast agents and environmental hazards, it is
vulnerable to the exposure of these toxicants. In particular,
drug-induced kidney injury (DIKI) is
medical applications.13-16 With a high signal-to-background
ratio, fluorescent probes often have high sensitivity and
strong ability for early detection of diseases such as
1
7-20
21,22
23-25
cancer,
skin diseases,
acute liver injury
and
2
6
diabetes. Additionally, multiplex fluorescence imaging
provides a unique approach to simultaneously study
multiple interrelated biomarkers at the same time,
1
a
frequent
27,28
complication in intensive care unit (ICU) and has a high risk
of morbidity and mortality. Moreover, DIKI remains a
potentially improving diagnostic accuracy.
However,
2
activatable fluorescent probes have been less exploited for
2
9-31
significant contributor to acute kidney injury (AKI) and
underlines 19-25% of all cases in critically ill patients.3
Therefore, development of sensitive detection methods for
DIKI is of great importance for pharmaceutical companies
to screen nephrotoxic drugs in drug development process,
and for clinicians to closely monitor patient safety in clinical
care.
detection of DIKI,
imaging.
not to mention multiplex fluorescence
Current clinical diagnosis often relies on measurement of
serum creatinine (sCr) and blood urea nitrogen (BUN),
which are factors reflecting changes in glomerular filtration
rate (GFR). However, sCr/BUN methods not only fail to
sensitively detect kidney injury because substantial
histological injuries could occur before measurable GFR
change, but also lack in accuracy because patients’ age and
muscle mass can cause fluctuations in the biomarker levels.
In contrast, urinalysis using the biomarkers such as
clusterin, Cystatin-C and β2-microglobulin has recently
shown to be more sensitive and reliable than clinical
4
5
6
7
diagnostic methods for detection of DIKI in animal studies.
However, the detection methods for these urinary
biomarkers are limited to commercial immunoassays and
matrix-assisted laser desorption ionization time of flight
mass
spectroscopy
(MALDI-TOF-MS).
Commercial
immunoassays generally suffer from low sensitivity and
tedious measurement procedures; MALDI-TOF-MS is a
highly specific method in biomarker detection, but its
application has been limited by number of reference MS
spectra in the database.
8
Scheme 1. Design and urinalysis mechanism of MURs. (a)
Multiplex optical urinalysis mechanism of MURs. (b) Molecular
structures and sensing mechanisms of MURs towards urinary
biomarkers.
9
Activatable fluorescent probes that specifically emit
signals in response to the biomarker of interest have
become indispensable tools in fundamental biology,
1
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and
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Analytical Chemistry
Page 2 of 8
In this study, we report the development of three
Synthesis of compound 2. Pure compound 1 (100 mg, 0.25
mmol) was dissolved in anhydrous tetrahydrofuran (THF)
in ice bath under N atmosphere, and PBr (37 μL, 0.38
2 3
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molecular urinary reporters (MURs) for multiplex optical
urinalysis in early detection of DIKI. Because elevated
urinary excretion of proximal tubule brush border enzymes
(γ-glutamyl transferase: GGT; alanine aminopeptidase:
AAP) and lysosomal enzyme (N-acetyl-D-glucosamine:
NAG) has been found at the early stage of both
mmol) in anhydrous THF was added via a syringe. The
reaction mixture was stirred for 2 h before the solvent was
concentrated under reduced pressure. The resulting
mixture was diluted with ethyl acetate and washed with
saturated sodium bicarbonate solution for three times to
remove residual acid. The organic layer was dried was
anhydrous sodium sulfate and concentrated under reduced
pressure. The crude product was then taken for next step
synthesis without further purification.
3
2
33
glomerulonephritis and acute tubular necrosis, they are
chosen as the DIKI biomarkers for the design of MURs.
Activatable molecular fluorophores responsive towards
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4-36
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38,39
GGT,
AAP, and NAG
have been previously reported,
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the molecules are designed by caging the fluorophore
optically-tunable atoms (typically oxygen or nitrogen) with
an enzyme cleavable moiety. However, they were not
applied for multiplex imaging. MURs1-3 are devised to emit
blue, orange and near-infrared (NIR) fluorescence signals
upon activation by GGT, AAP, and NAG, respectively. Due to
the minimal spectral overlap, MURs1-3 can form a cocktail
sensor for multiplex optical urinalysis of DIKI.
Synthesis of compound 3. Resorufin (10 mg, 0.05 mmol)
and grinded potassium carbonate (25 mg, 0.2 mmol) were
vigorously stirred in anhydrous dimethylformamide (DMF)
2
under N atmosphere for 30 minutes, before compound 2
(
95 mg, 0.2 mmol) dissolved in anhydrous DMF was added
via a syringe. The mixture was continuously stirred for 5 h,
and then pure compound 3 (29 mg, 95%) could be obtained
after HPLC purification. H NMR (300 MHz, DMSO-d6) δ:
1
Experimental Section
Materials
7.89 (d, J = 6 Hz, 2H), 7.63-7.79 (m, 6H), 7.53 (d, J = 12Hz,
1H), 7.42 (m, 3H), 7.32 (m, 2H), 7.18 (d, J = 3 Hz, 1H), 7.12
(
4
m, 1H), 6.78 (m, 1H), 6.27 (d, J = 2.1 Hz, 1H), 5.22 (s, 2H),
.24 (m, 4H), 1.30 (d, J = 7 Hz, 3H). MS (ESI): m/z = 611.75
All chemicals were purchased from Sigma-Aldrich unless
otherwise stated. Commercially available reagents were
used without further purification unless otherwise noted.
Fmoc-Ala-OH was purchased from Sangon Biotech. Alanine
aminopeptidase (AAP), fibroblast activation protein-alpha
(FAP-), furin, matrix metalloproteinase-2 (MMP2), and
dipeptidyl peptidase-4 (DPP4) were purchased from R&D
Systems.
+
[M + H] .
Synthesis of MUR2. Pure compound 3 (20 mg, 0.03 mmol)
was stirred in 5% piperidine/DMF for 10 minutes at rt,
before 10-times diluted acetic acid was added dropwise
until pH was adjusted to 7. Pure MUR2 (10 mg, 90%) could
1
be obtained after HPLC purification. H NMR (300 MHz,
3
CD CN) δ: 7.64 (d, J = 9 Hz, 1H), 7.53 (d, J = 6Hz, 2H), 7.40 (s,
Instrumentation
1
1
H), 7.38 (m, 2H), 6.97 (m, 1H), 6.90 (d, J = 3Hz, 1H),6.67 (m,
UV/Vis spectra were measured on a Shimadzu UV-2450
spectrophotometer. Fluorescence measurements were
performed on a Fluorolog 3-TCSPC spectrofluorometer
(Horiba Jobin Yvon). HPLC analyses were done on an
Agilent 1260 system equipped with a G1311B pump, UV
detector and an Agilent Zorbax SB-C18 RP (9.4 × 250 mm)
H), 6.15 (d, J = 3Hz, 1H), 5.12 (s, 2H), 4.05 (m, 1H), 1.49 (d,
+
J = 6Hz, 3H). MS (ESI): m/z = 389.79 [M + H] .
Synthesis of CyOH. Synthesis was carried out according to
4
0 1
literature report. H NMR (300 MHz, CDCl
3
) δ: 8.18 (d, J =
1
1
3
5 Hz, 1H), 7.70 (m, 1H), 7.35 (s, 1H), 7.31 (s, 1H), 7.22 (s,
H), 7.10 (t, 1H), 6.89 (t, 2H), 5.67 (d, J = 12 Hz, 1H), 2.65 (m,
H), 1.91 (t, 2H), 1.69 (s, 6H), 1.25 (s, 4H), 0.86 (m, 3H). MS
3
column, with methanol (0.1% CF COOH) and water (0.1%
CF COOH) as the eluent. Fluorescence images of living cells
3
+
(
ESI): m/z = 386.45 [M + H] .
Synthesis of Compound
pentaacetate (780 mg, 2 mmol) was dissolved and stirred in
mL anhydrous DCM in ice bath under N atmosphere, and
and immunofluorescent imaging of kidney slices were
acquired on Laser Scanning Microscope LSM800 (Zeiss).
4.
β-D-galactosamine
1
Proton-nuclear magnetic resonance ( H NMR) spectra were
6
2
recorded on a Bruker Advance II 300 MHz NMR.
Electrospray ionization-mass spectrometry (ESI-MS)
spectra were acquired on a Thermo Finnigan Polaris Q
quadrupole ion trap mass spectrometer (ThermoFisher
Corporation) equipped with a standard ESI source.
HBr (33 wt% in acetic acid, 4.7 mL, 2 mmol) was added via
a syringe. The mixture was continuously stirred in ice bath
for 6 h. The residue mixture was then washed with ice-cold
saturated sodium bicarbonate solution for three times to
remove residue acid. The organic layer was dried with
anhydrous sodium sulfate and concentrated under reduced
pressure. The crude product was stored at -20 C and
directly taken for next step synthesis without further
Syntheses
Synthesis of Compound 1. Fmoc-Ala-OH (310 mg, 1
mmol), p-aminobenzyl alcohol (250 mg, 2 mmol) and N-
Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (300 mg, 2
mmol) were dissolved and mixed in anhydrous
+
purification. MS (ESI): m/z = 409.71 [M + H] .
Synthesis of compound 5. Pure CyOH (20 mg, 0.054 mmol)
and grinded potassium carbonate (28 mg, 0.2 mmol) was
mixed and stirred in anhydrous DCM under N atmosphere
2
dichloromethane (DCM) and stirred overnight under N
2
atmosphere. The organic solvent was concentrated under
reduced pressure. After HPLC purification, pure compound
1
for 30 minutes before compound 4 (86 mg, 0.2 mmol)
dissolved in anhydrous DCM was added via a syringe. The
residue mixture was stirred overnight and concentrated
under reduced pressure. The crude product was directly
used for next step synthesis without further purification. MS
1 could be obtained (330 mg, 0.8 mmol). H NMR (300 MHz,
CDCl
J = 9 Hz, 2H), 7.39 (t, 2H), 7.30 (d, J = 9 Hz, 4H), 4.65 (s, 2H),
.46 (d, J = 6 Hz, 2H), 4.36 (s, 1H), 4.21 (t, 1H), 1.46 (d, J = 6
3
) δ: 7.76 (d, J = 6 Hz, 2H), 7.57 (d, J = 6 Hz, 2H), 7.47 (d,
4
+
Hz, 3H). MS (ESI): m/z = 417.24 [M + H] .
+
(
ESI): m/z = 713.56 [M + H] .
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Analytical Chemistry
Synthesis of MUR3. Crude compound 5 (38 mg, 0.054
experimental group and control group, respectively. The
assays were performed in five sets for each concentration.
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4
5
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mmol) and MeONa (25 wt% in methanol [MeOH], 550 μL, 2
mmol) were dissolved and stirred in MeOH in ice bath. The
reaction was continuously monitored by thin-layer
chromatography, and 10-times diluted acetic acid was
added dropwise until pH was adjusted to 7. The residue
mixture was concentrated under reduced pressure and
Mice cisplatin-induced acute kidney injury model
development. All animal experiments were performed in
accordance with the Guidelines for Care and Use of
Laboratory Animals of the Nanyang Technological
University-Institutional Animal Care and Use Committee
(NTU-IACUC) and approved by the Institutional Animal
Care and Use Committee (IACUC) for Animal Experiment,
Singapore. The choice of drug dosage and injection methods
could be found in literature reports. Cisplatin (1 mg/mL in
purified with column chromatography to yield pure MUR3
1
(
20 mg, 64%). H NMR (300 MHz, MeOD) δ: 8.78 (d, J = 15
Hz, 1H), 7.67 (d, J = 8 Hz, 1H), 7.47 (m, 3H), 7.33 (s, 1H), 7.13
s, 1H), 7.00 (d, J = 9 Hz, 1H), 6.55 (J = 15 Hz, 1H), 5.21 (m,
(
1
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H), 3.96 (m, 4H), 3.73 (m, 1H), 3.59 (m, 2H), 3.42 (m, 2H),
.75 (d, J = 15 Hz, 4H), 1.96 (m, 6H), 1.81 (s, 3H), 1.28 (s,
H). MS (ESI): m/z = 587.30 [M + H] .
0
.9% NaCl solution) was injected once intraperitoneally
into Ncr mice (18-20 g, drug dosage 20 mg/kg bw). Age-
matched control Ncr mice were injected with an equal
volume of isotonic saline. All mice were housed in a
temperature controlled (22 °C) room with 12 h dark light
cycles (0700 h on and 1900 h off). All mice were weighed
twice daily. Mice were sacrificed at different drug post-
treatment time points (t = 12, 24, 48 and 72 h). An overnight
urine collection of spontaneously voided urine was made
from each animal in metabolic cages on the night before
sacrifice. Blood samples for serum creatinine (sCr) and
Blood Urea Nitrogen (BUN) were obtained from central tail
artery in non-anesthetized mice. Obtained blood samples
were stored in ice and centrifuged at 3000 rpm for 10
minutes to separate serum.
+
Enzyme kinetic studies. Various concentrations of MUR1
10, 20, 40, 80, 160, 320, 640, 800 μM) were incubated with
(
GGT (5 µg) at 37 °C for 5 min in a 100 µL system of PBS
buffer (1×, pH = 7.4) with 0.02% Tween@20. Similarly,
various concentrations of MUR2 (5, 10, 20, 40, 80, 160, 240,
320 μM) were incubated with AAP (0.1 μg) at 37 °C for 5 min
in a 100 µL system of Tris buffer (50 mM, pH = 7.4). Lastly,
various concentrations of MUR3 (10, 25, 50, 100, 150, 300,
6
00 μM) were incubated with NAG (10 mU) at 37 °C for 5
min in a 100 µL system of PBS buffer (1×, pH = 7.4). After
incubation, the mixture was injected into HPLC (eluent:
methanol/water) for quantification analyses. The initial
reaction velocity (µmol/s) was calculated, plotted against
MURs1-3 concentration, and fitted to a Michaelis-Menten
curve. The kinetic parameters were calculated using
Mice doxorubicin-induced chronic kidney injury model
development. The choice of drug dosage and injection
methods could be found in literature reports. Doxorubicin
M
Michaelis-Menten equation: V = Vmax*[S] (K + [S]), where V
is initial velocity, and [S] is substrate concentration.
(
1 mg/mL in 0.9% NaCl solution) was intravenously
injected into BALB/c mice (18-20 g, 10 mg/kg bw). Age-
matched control BALB/c mice were injected with an equal
volume of isotonic saline. All mice were housed in a
temperature controlled (22 °C) room with 12 h dark light
cycles (0700 h on and 1900 h off). All mice were weighed
twice daily. Mice were sacrificed at different drug post-
treatment time points (t = 5, 8, 10, 14, 21, 28 days). An
overnight urine collection of spontaneously voided urine
was made from each animal in metabolic cages on the night
before sacrifice. Blood samples for serum albumin and
creatinine (Cr) were obtained from central tail artery in
non-anesthetized mice. Obtained blood samples were
stored in ice and centrifuged at 3000 rpm for 10 minutes to
separate serum.
Cell culture. HK-2 and NDF cells were cultured in
Dulbecco’s Modified Eagle Media (DMEM) medium
supplemented with heat-inactivated fetal bovine serum
(
10%) and antibiotics penicillin/streptavidin (1%) in a
humidified environment containing 5% CO and 95% air at
2
37 °C. Cell media was replaced every two to three days.
Cell fluorescence imaging. For cell fluorescence imaging,
5
HK-2 and NDF cells (10 cells per well in 1 mL) were seeded
in the dishes (dia. 35mm) and incubated overnight to reach
80% confluency, respectively. The cells were incubated
with 30 μM MURs cocktail (containing 10 μM of each MUR)
for 30 minutes. The medium was removed, and the cells
were washed with PBS for three times. Then the cells were
fixed with 4% polyformaldehyde solution. Fluorescence
images of fixed cells were acquired on a Laser Scanning
Microscope LSM800 (Zeiss). The excitation and emission
wavelength for cell imaging were 405/410-470nm for
MUR1, 561/580-630nm for MUR2 and 640/655-710nm for
MUR3. ImageJ software was utilized to remove signal
background and quantify cellular fluorescence intensity.
Statistics analysis. Statistical comparisons between the
two groups were determined by Student's t-test (1-tailed,
unpaired). For all tests, p < 0.05 was considered as
statistically significant. For analysis of diagnostic
performance of MURs, ROC curves were drawn and the
AUCs were calculated and compared according to Hanley &
41
McNeil. The combination of MURs is calculated by a linear
relationship of MURs1-3 derived from logistic regression:
MURs = -2.16 + 0.32 × [MUR1] + 0.37 × [MUR2] + 0.17 ×
Cytotoxicity assay. HK-2 and NDF cells were seeded in 96-
4
well plates (10 cells per well) for 24 h, and then different
[
MUR3], [MUR] indicates normalized fluorescence
concentrations of MUR cocktail (5, 10, 20, 30 and 40 μM,
containing equimolar amount of each MUR) were added to
the cell culture medium. Cells were incubated with or
without (control) MUR cocktail, followed by addition of MTS
assay (Promega Cat. no. G3581, 100 mL, 0.1 mg/mL) for 4 h.
The absorbance of MTS at 490 nm was measured by using a
microplate reader. The cytotoxic effects (VR) of MUR
cocktail were assessed using the following equation: VR =
enhancement for the three MURs, respectively. All
statistical calculations were performed using GraphPad
Prism v.6 (GraphPad Software Inc., CA, USA).
A/A
0
× 100%, where A and A
0
are the absorbance of the
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Analytical Chemistry
Page 4 of 8
Results and Discussion
Syntheses of MURs
catalytic efficiencies of GGT, AAP and NAG towards MURs1-
3 were respectively calculated to be 31.8, 0.24 and 0.0198
μM s , and linear relationships existed between
fluorescence intensity and the concentration of biomarkers
(Figure S1 and Table S2).
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-1 -1
The sensing mechanism of MURs1-3 are shown in Scheme
. MUR1 is L-Glutamic acid γ-(7-amido-4-methylcoumarin),
1
which is a specific GGT-activatable probe and emits blue
fluorescence signals after enzymatic cleavage. MUR2 was
derived from an orange-fluorescent dye, resorufin, by
caging its optically tunable hydroxyl group with a self-
immolative linker connected to AAP-cleavable alanine
amino acid (Ala). Upon AAP cleavage at the terminal alanyl
group, the linker is released by 1,6-immolation, leading to
free resorufin unit with enhanced electron-donating ability
from the oxygen atom. Similarly, MUR3 was synthesized
from a NIR fluorescent hemicyanine dye (CyOH), with its
hydroxyl group caged by a NAG-cleavable responsive group
(N-acetyl-β-D-glucosaminide). In the presence of NAG, the
terminal glycosidic bond is cleaved, forming free CyOH unit
with enhanced electron-donating ability from the oxygen
atom. Therefore, MURs2-3 are able to turn on their orange
and NIR fluorescence signals in the presence of AAP and
NAG, respectively.
0
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MURs2-3 were synthesized through
a synergistic
approach (Scheme S1). The amino acid alanine was first
coupled to p-aminobenzyl alcohol with subsequent 1º
bromination to yield a highly active compound 2.
Compound 2 was then conjugated to the hydroxyl group on
resorufin followed by Fmoc deprotection on Alanyl amino
group to afford MUR2. As for MUR3, its precursor CyOH was
obtained by reacting IR775-Cl with resorcinol via a retro-
Knoevenagel reaction. For its biomarker responsive
compartment, a highly reactive compound 4 was afforded
by brominating N-acetyl-β-D-glucosamine tetraacetate at
its anomeric center, it was then reacted with the hydroxyl
group of CyOH to afford compound 5, and compound 5 was
deprotected of acetate groups to yield MUR3. The structures
of these compounds were confirmed by NMR and mass
spectra (Supporting Information).
Figure 1. Evaluation of MURs sensing capabilities. (a-c) UV-vis
absorption and fluorescence spectra of MURs1-3 (20 M) in the
absence or presence of their respective biomarkers (0.8 g/mL
GGT, 0.8 g/mL AAP and 60 mU/mL NAG) in PBS (10mM, pH =
7
.4 with 0.02% Tween@20), Tris (50 mM, pH = 7.4) and PBS
(
10mM, pH = 7.4), respectively. (d-f) HPLC traces of MURs1-3
in the absence or presence of their respective biomarkers, and
traces of pure AMC (7-amino-4-methylcoumarin), resorufin
and CyOH. (g-i) Fluorescence signal changes of MUR1 (440
nm), MUR2 (590 nm) and MUR3 (710 nm) relative to the blank
in the presence of different enzymes. Error bars are standard
deviation from three separate measurements. Fibroblast
activation protein-alpha (FAP훼), matrix metalloproteinase-2
In vitro detection
(
MMP2), dipeptidyl peptidase-4 (DPP4).
To investigate detection sensitivity and specificity of
MURs, their UV-vis absorption and fluorescence spectra
were measured in the absence or presence of biomarkers
MURs cocktail multiplex imaging
To test the capability of MURs for multiplex imaging,
MUR1-3 were mixed at an optimized equimolar ratio (MURs
cocktail) and the fluorescence spectra was measured in the
absence or presence of all three biomarkers by selective
excitation at their respective absorption wavelengths (380,
550, and 670 nm). Upon excitation at 380 nm, only
fluorescence signal of MUR1 at 440nm was detected, but
signals of MURs2-3 were negligible. This was attributed to
the absence of fluorescence resonance energy transfer
(
Figure 1 and Table S1). Note that all MURs were barely
fluorescent at their intrinsic states. After incubation of
MUR1 with GGT, its absorption maximum shifted from 325
to 345 nm, and fluorescence signals at 440 nm enhanced 67-
times (Figure 1a). Similarly, upon treatment of MUR2 with
AAP, its absorption maximum shifted from 480 to 570 nm,
which is the characteristic peak for released resorufin
(
Figure 1b). At this time point, MUR2 showed 60-fold
fluorescence enhancement. Moreover, when MUR3 was
incubated with NAG, its absorption maximum shifted from
(
FRET) among MURs, because individual MUR molecules
were free and away from each other in solutions and their
spectra had minimal overlap. Similar spectral behaviours
were upon excitation at 550 or 670 nm, only showing the
signal of MUR2 or MUR3. Thus, the MURs cocktail could
realize simultaneous detection of these biomarkers with
minimal signal interference (Figure 2a).
6
00 to 690 nm, due to enzymatic cleavage at the ester bond
and generation of free CyOH unit (Figure 1c). Meanwhile,
MUR3 showed 33-fold fluorescence enhancement. High-
performance liquid chromatography (HPLC) analysis
confirmed that in the presence of their respective
biomarkers, MURs1-3 were totally converted into the free
The MURs cocktail was then used for multiplex imaging of
endogenous biomarkers in kidney proximal tubule
epithelial cells (HK-2) after confirming their low
cytotoxicity in living cells (Figure S2). Compared to normal
fluorescent dyes AMC (quantum yield, Φ
f
= 0.70), resorufin
(
Φ
f
= 0.39), and CyOH (Φ = 0.21), respectively (Figure 1d-
f
f). All MURs showed negligible fluorescence changes in the
presence of interfering enzymes (Figure 1g-i). Besides, the
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dermal fibroblasts (NDF) as control, the fluorescence
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intensities of MURs1-3 in HK-2 were ~20-times higher,
because of the high expression levels of these enzymes in
HK-2 cells (Figure 2b-c). These results confirmed the
feasibility of MURs cocktail for multiplex fluorescence
imaging of multiple biomarkers.
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Figure 3. Multiplex urinalysis for detection of DIKI. (a)
Timeline for development of cisplatin and doxorubicin–
induced kidney injury. Mice were treated with cisplatin
(
(
intraperitoneal injection, 20 mg/kg bw) or doxorubicin
intravenous injection, 10 mg/kg bw), and urine samples that
Figure 2. Multiplex fluorescence imaging in solution and renal
cells. (a) Fluorescence spectra of MURs cocktail (containing 15
μM of each MUR) in the absence or presence of all three
biomarkers–GGT, AAP and NAG in PBS (1×, pH = 7.4)
containing 5% DMSO. Arrows indicate respective excitation
wavelength for MURs1-3. (b) Fluorescence intensity
enhancement of single HK-2 relative to NDF cells in Figure 2c
were collected from drug-treated mice at indicated post-
treatment timepoints were then incubated with MURs cocktail
(containing 10 μM of each MUR) for 1 h at 37 ºC in PBS (1×, pH
= 7.4) containing 5% DMSO. Commercial assays were used to
measure Cr, BUN in serum and proteinuria in urine. (b-c)
Fluorescence enhancement of MURs1-3 after incubation with
urine samples collected from drug-treated living mice, and fold
changes in urinary and serum biomarkers at different post-
treatment times. *Statistically significant difference in
fluorescence enhancement and biomarkers fold change
between pre-treatment and indicated statistically significant
timepoints post-treatment of drugs. (n=5, mean  s.d.) (d) ROC
curves for the exclusion analysis (nephrotoxicant-treated
animals with observed renal injuries versus animals not-
treated with nephrotoxicant and without renal injuries) with
the two nephrotoxicant studies. MURs is linear combination of
MURs1-3 derived from logistic regression. (kidney injury [n =
28] vs control [n = 13]) n.s.: not significant; *p<0.05; **p<0.01;
(
n = 3, mean ± s.d.). (c) Multiplex fluorescence images of human
primary dermal fibroblasts (NDF) and kidney proximal tubule
epithelial cells (HK-2) after incubation with MURs cocktail
(
containing 10 μM of each MUR).
***p<0.001.
Multiplex Optical Urinalysis
After confirming their plasma stability (Figure S3), MURs
were then used to detect urinary biomarkers in murine
models of DIKI with two nephrotoxicants – cisplatin and
doxorubicin. Cisplatin is an antineoplastic drug with side
effects of acute kidney injury (AKI), which is characterized
by sequential physiological events: acute tubular necrosis,
42
rapid GFR decline and kidney dysfunction. Differently,
doxorubicin-induced kidney injury is a mouse model that
mimics human chronic kidney disease (CKD) caused by
4
3
focal segmental glomerulosclerosis. Specifically, healthy
mice received one-time drug treatment of cisplatin (20
mg/kg bw) or doxorubicin (10 mg/kg bw) (Figure 3a). At
different post-treatment timepoints, urine and blood
samples were collected, and mice were sacrificed to collect
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kidneys for histological analysis. The urine samples were
Page 6 of 8
and 5 days after drug challenge, which were respectively 48
h and 5 days earlier than tested clinical diagnostic methods.
Furthermore, an ROC exclusion analysis highlighted that
MURs had better diagnostic accuracy than traditional
diagnostic methods. However, in this study, MURs are only
tested in drug-induced kidney injury models. To further
validate the specificity of MURs, future studies should be
conducted to include urine samples from patients with
some common diseases like diabetes, cardiovascular
disease, and other urinary disease like bladder cancer. Thus,
this study exemplified a pioneering multiplex molecular
detection method with a great potential for real-time
monitoring of kidney function in preclinical drug screening
and clinical diagnosis.
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incubated with MURs cocktail before multiplex
fluorescence analysis, and serum biomarkers were tested
using commercial assays. In the context of CKD, proteinuria
was also tested as a clinical urinary biomarker reflecting
4
4
glomerulosclerosis.
In the cisplatin-induced AKI model, the first statistically
significant fluorescence enhancement of MURs1-3 was
observed at 24 (1.52-fold), 48 (4.05-fold) and 24 h (3.02-
fold), respectively (Figure 3b and Figure S4). In contrast, the
statistically significant increase of serum sCr and BUN was
observed at a later timepoint, 72 h (2.81 & 2.15-fold) after
cisplatin treatment, when histological injuries like loss of
brush border and renal cell debris could be readily
observed (Figure S5). In the doxorubicin-induced CKD
model, the first statistically significant fluorescence
enhancement of MURs1-3 was observed at 5 (1.74-fold), 5
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ASSOCIATED CONTENT
Supporting Information
(
2.06-fold) and 8 days (1.37-fold). Proteinuria and serum
The Supporting Information is available free of charge on the
ACS Publications website.
creatinine had statistically significant increase at later
timepoints, 10 (2.17-fold) and 21 days (2.45-fold).
Histological injuries were observed from 14 days onwards
(Figure S6). Meanwhile, significant mice weight loss for
cisplatin and doxorubicin- induced kidney injury models
were observed at 48 h and 8 days, respectively (Figure S7).
Thereafter, MURs1-3 detected cisplatin and doxorubicin-
induced kidney injury at 24 h and 5 days post-treatment,
which are respectively 48 h and 5 days earlier than
traditional diagnostic methods including sCr, BUN and
proteinuria. The differences in statistically significant
timepoints among MURs1-3 could be probably attributed to
the biomarkers’ individual regulation pathways in renal
cells after drug challenge.
Supporting Information. Supporting Figures and Tables (PDF).
AUTHOR INFORMATION
Corresponding Author
Kanyi Pu – School of Chemical and Biomedical Engineering,
Nanyang Technological University, 637457 Singapore. Email:
kypu@ntu.edu.sg
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
In order to quantitatively study the diagnostic
performance (sensitivity and specificity), receiver
operating characteristic (ROC) exclusion analysis was
conducted by comparing the diagnosis results of MURs
between two groups – healthy mice without histology
injuries and drug-treated mice with obvious histology
injuries. Besides, the area under curve (AUC) has statistical
meaning of diagnostic accuracy and enables comparison
between MURs and traditional diagnostic methods. MURs1-
K.P. thanks Nanyang Technological University (Start-Up grant:
M4081627), Singapore Ministry of Education Academic
Research Fund Tier 1 (2017-T1-002-134, RG147/17; 2019-T1-
002-045, RG125/19), and Academic Research Fund Tier 2
(
MOE2018-T2-2-042) for the financial support.
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