stm.sciencemag.org/cgi/content/full/12/543/eaay7591/DC1

Supplementary Materials for

Nuclear receptor PXR targets AKR1B7 to protect mitochondrial metabolism and

renal function in AKI

Xiaowen Yu, Man Xu, Xia Meng, Shumin Li, Qianqi Liu, Mi Bai, Ran You, Songming Huang, Li Yang, Yue Zhang*, Zhanjun Jia*, Aihua Zhang*

*Corresponding author. Email: zhaihua@njmu.edu.cn (A.Z.); jiazj72@hotmail.com (Z.J.);

zyflora2006@hotmail.com (Y.Z.)

Published 13 May 2020, Sci. Transl. Med. 12, eaay7591 (2020) DOI: 10.1126/scitranslmed.aay7591

The PDF file includes:

Materials and Methods Fig. S1. PXR deficiency aggravated cisplatin-induced tubular inflammation and mitochondrial damage. Fig. S2. MnTBAP therapy attenuated cisplatin-induced AKI in wild-type and PXR–/– rats. Fig. S3. PCN treatment ameliorated cisplatin-induced apoptosis, inflammation, and the dysregulation of mitochondrial genes in vitro. Fig. S4. PXR overexpression attenuated cisplatin-induced apoptosis and mitochondrial dysfunction in vitro. Fig. S5. PCN treatment or PXR overexpression attenuated cisplatin-induced apoptotic response in three-dimensional cultured HK2 cells. Fig. S6. Expressions of other nuclear receptors remained unchanged in PXR–/– rats. Fig. S7. AKR1B7 overexpression prevented cisplatin-induced mitochondrial injury in vitro. Fig. S8. PXR deficiency changed mitochondrial metabolic pathways. Fig. S9. PXR/AKR1B7 activation attenuated cisplatin-induced lipid accumulation and improved mitochondrial fatty acid β-oxidation. Fig. S10. The protective effect of PXR on mitochondrial oxidative capacity and fatty acid β-oxidation was diminished by silencing AKR1B7. Fig. S11. PXR deficiency aggravated I/R-induced mitochondrial damage. Table S1. Clinical data of patients with AKI. References (47–54)

Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/12/543/eaay7591/DC1)

Data file S1 (Microsoft Excel format). Primary data. Data file S2 (Microsoft Excel format). Proteomics analysis of all differential proteins between wild-type and PXR–/– groups. Data file S3 (Microsoft Excel format). Untargeted lipidomics analysis of all differential lipids between wild-type and PXR–/– groups. Data file S4 (Microsoft Excel format). Untargeted metabolomics analysis of all differential metabolites between wild-type and PXR–/– groups. Data file S5 (Microsoft Excel format). RNA-seq analysis of all differential transcriptomes between NC and AKR1B7-overexpressed RTECs. Data file S6 (Microsoft Excel format). Primers used for PCR amplification.

Materials and Methods

Analysis of SCr and BUN in blood

The concentrations of serum creatinine (SCr) and blood urea nitrogen (BUN) were

evaluated using a serum biochemical autoanalyzer (Hitachi 7600 modular chemistry

analyzer).

ELISA assay of IL-6 and TNF-α

The circulating, kidney, and cell culture medium IL-6 and TNF-α were measured by

ELISA kits (Elabscience Biotech) in accordance with the manufacturer's instructions.

Histological analysis

Kidneys from animals were fixed in 4% paraformaldehyde (PFA) for 24 h at room

temperature and embedded in paraffin. Sections (3 μm) were stained with PAS and analyzed

by a pathologist in a blinded manner. A minimum of 10 fields on each kidney slide were

examined and scored for pathological injury. A pathological assessment score from 0 to 4 was

given: 0, normal histology; 1, mild injury—5% to 25% of tubules showed pathological

damage; 2, moderate injury—25% to 50% of tubules showed pathological damage; 3, severe

injury—50% to 75% showed pathological damage; and 4, almost all tubules in the field of

view were damaged. The average histological score for each sample was calculated (41).

Neutral lipids in frozen kidney sections were detected by oil red O staining. Images were

acquired with an Olympus BX51 microscope (Olympus).

TUNEL assay

In situ cell death was measured by a TdT–mediated dUTP nick-end labeling (TUNEL)

in situ cell death detection kit (Roche Diagnostics) according to the manufacturer's

instructions (47). Detection of the apoptotic cells showing green fluorescence was performed

by Carl Zeiss LSM 5 PASCAL laser scanning confocal microscopy. The numbers of apoptotic

cells were shown as percentages of TUNEL-positive cells out of total cells.

Transmission electron microscopy

Kidney samples (approximately 2 mm3) were immersed in 2.5% glutaraldehyde /0.1 M

phosphate buffer (PB; pH 7.4) for 2 h. Next, samples were postfixed in 1% OsO4 /0.1 M PB

for 2 h. Tissues were dehydrated in ethanol and embedded in epoxy resin (Epok 812; Epok).

Ultrathin sections (60 nm) were cut with an ultramicrotome (Leica Ultracut UC7; Leica

Microsystems) and mounted on a copper grid. Sections were stained in uranyl acetate for 30

min and lead citrate for 4 min at room temperature. Grids were viewed with a transmission

electron microscope (JEOL JEM-1010).

Immunohistochemistry (IHC) of human and animal kidney samples

IHC was performed as previously described (47). Briefly, paraffin-embedded human and

animal kidney sections (4 μm) were deparaffinized. Slides were boiled in improved Citrate

Antigen Retrieval Solution (Beyotime, P0083) for 1 min and cooled on the bench top for 20

min. After a 15 min incubation in 3% hydrogen peroxide, sections were blocked with 10%

normal goat serum for 60 min at 37°C and then incubated with rabbit monoclonal primary

antibodies against PXR (Abcam, 1:100), CD68 (Abcam, 1:200), and Cytochrome c (CST,

1:100) overnight at 4°C. After washing with TBST buffer three times, sections were

incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody for 60 min.

The localization of peroxidase conjugates was determined using a DAB kit (ZLI-9018, zsbio).

Slides were examined under a microscope, and the signals were analyzed using Image-Pro

Plus software analysis tools.

Dihydroethidium (DHE) staining of kidney tissues.

Cryosections from frozen kidney tissues (5 μm) were prepared with a Leica CM1900

cryostat (Leica). The sections were stained with DHE solution (2 μM) (S0063, Beyotime) for

30 minutes in the dark at 37°C, then washed 3 times with PBS. Finally, LSM710 laser

confocal microscope (Zeiss) was used to photograph them under 543 nm excitation light.

Thiobarbituric acidreactive substances (TBARS) assay

The measurement of kidney tissue thiobarbituric acidreactive substances (TBARS) was

based on the formation of malondialdehyde by using a commercial TBARS assay kit

(Cayman Chemical) following the manufacturer’s instructions. MDA value was normalized

to the kidney protein content determined by a BCA kit (Beyotime). The amount of lipid

peroxidation was expressed as μg/g protein.

Cell culture and treatments

Mouse renal tubular epithelial cells (RTECs) were obtained from the American Type

Culture Collection (ATCC). Cells were cultured in DMEM/F-12 medium [Wisent

supplemented with 10% fetal bovine serum (GIBCO), penicillin (100 U/mL) and

streptomycin (100 μg/mL)] and maintained at 37 °C in 5% CO2 in a humidified incubator.

Cells were grown to 80% confluence and pretreated with PCN for 2 h. Then, cisplatin (5

μg/mL) was added to the serum-free medium to stimulate RTECs for 24 h. In another

experiment, cells were grown to 60-70% confluence and transfected with siPXR or

siAKR1B7. Then the cells were pretreated with PCN or PXR plasmids, followed by cisplatin

administration. Sequences of siRNAs are as follows: siPXR: GGAGGAAGATGGAGGTCTT;

siAKR1B7: GGAATGCGTTATTACCCAA. In HK2 cell (a human renal epithelial cell line

obtained from ATCC) study, an in vitro 3D culture model was developed using non-adherent

Nunclon Sphera 6-well plate (Thermo Scientific), which allows cells to grow in suspension

with virtually no attachment. When HK2 cells were grown for three days in non-adherent

Nunclon Sphera 6-well plate, they were pretreated with PCN for 2 h or overexpression of

PXR for 24 h with a lentiviral approach. Then, cisplatin (10 μg/mL) was added to the

serum-free medium to stimulate HK2 cells for 24 h.

Annexin V/PI double staining

After treatment, cells were washed three times with PBS, trypsinized, centrifuged (1500

rpm at room temperature) for 5 min, adjusted to 5 × 104 cells/mL, and double-stained with

annexin V-FITC and PI (Annexin V-FITC Apoptosis Detection Kit, BD Biosciences)

according to the manufacturer's instructions. After incubation for 15 min at room temperature

in the dark, the fluorescence intensity was measured using a flow cytometer (BD

Biosciences).

Determination of mitochondrial ROS production and mitochondrial membrane potential

(MMP)

Cell mitochondrial reactive oxygen species (ROS) production was measured using

MitoSOXRed mitochondrial superoxide indicator (Life Technologies) (41). Briefly, 50 μg of

MitoSOX mitochondrial superoxide indicator was dissolved in 13 μL of dimethylsulfoxide

(DMSO) to make a 5 mM MitoSOX reagent stock solution. Then, the reagent stock solution

was diluted using buffer to make a 5 μM MitoSOX reagent working solution. Then, 1.0 mL

of 5 μM MitoSOX reagent working solution was applied to cover cells adherent to coverslips

and incubated for 10 min at 37°C in the dark. After incubation, the accumulation of

mitochondrial superoxide anions in RTECs was analyzed by flow cytometry. The MMP,

△Ψm, of cells was determined using a mitochondrial membrane potential assay kit

(Beyotime Biotechnology) with JC-1, and the quantification of JC-1 fluorescence was

performed by flow cytometry as described previously (41).

Determination of ATP content

The RTEC ATP content was determined with an Enhanced ATP Assay Kit (Beyotime)

according to the manufacturer's instructions, and the results are shown in arbitrary units.

Plasmid construction and luciferase reporter assay

The mutant and wild-type pGL3 promoter-luciferase constructs were generated by Obio

Technology Co., Ltd., and the mutation site of AKR1B7 was based on a previous study (20).

HEK293T cells were plated in a 24-well plate and cultured in Dulbecco's Modified Eagle

Medium: Nutrient Mixture F-12 (DMEM/F-12) containing 10% FBS. Transient transfections

were performed as described (20). Briefly, the pGL3-AKR1B7-wt and -mutant luciferase

reporter constructs were cotransfected into HEK293T cells with plasmids expressing PXR,

followed by treatment with either vehicle or PCN. Twenty-four hours after transfection, cells

were assayed using a Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase

activity was normalized to the corresponding Renilla luciferase activity.

Establishment of the PXR- and AKR1B7-overexpressing cell line

Lentiviruses carrying the PXR and AKR1B7 genes were constructed by Obio

Technology Co., Ltd.. RTECs were seeded in a 12-well plate and infected with lentivirus

according to protocols recommended by the manufacturer (48). After 24 h, the medium was

replaced with complete medium. To obtain a stable PXR- and AKR1B7-overexpressing cell

line, lentivirus-infected cells were selected by incubation with 2 μg/mL puromycin. The

expression of PXR and AKR1B7 in RTECs stably infected with lentivirus was assessed by

Western blotting. To detect changes in mitophagy, pDsRed2-mito and mt-mKeima-cox8

lentivirus vectors were constructed by Obio Technology Co., Ltd. for transfection of RTECs.

RNA fluorescence in situ hybridization (RNA FISH)

Cy3-labeled Akr1b7 probes were obtained from RiboBio. RNA FISH was performed

using a fluorescence in situ hybridization kit (RiboBio) following the manufacturer's

instructions (49).

Western blotting

Frozen kidney cortex tissue (100 mg) was ground in liquid nitrogen. Cells were seeded

in 6-well plates. Two hundred microliters of lysis buffer supplemented with 1 × protease

inhibitor cocktail (Roche Diagnostics, 04693132001) was added to the ground tissue or cells

and incubated on ice for 30 min. The protein concentration was measured using the Bradford

method, and 50 μg of total protein was used for Western blotting analysis following standard

methods with primary antibodies against BAX (CST, 1:1000), BCL-2 (CST, 1:1000),

caspase-3 (CST, 1:1000), cleaved caspase-3 (CST, 1:500), PXR (Abcam, 1:1000),

Cytochrome c (CST, 1:1000), LC3B (Sigma-Aldrich, 1:1000), P62/SQSTM1 (Abcam,

1:1000), FLAG (Sigma-Aldrich, 1:1000), and GAPDH (CST, 1:1000), followed by the

addition of HRP-labeled secondary antibodies (CST, 1:2500). GAPDH was used as an

internal control for all Western blotting analyses. Densitometry was analyzed with ImageJ

software (NIH).

Proteomics sample pretreatment and analysis.

Frozen kidney cortex tissues (400 mg) from PXR+/+ and PXR-/- rats were harvested and

ground as previously described (50). The total protein concentration was measured using the

Bradford method. For protein digestion, 2 μg trypsin was added and incubated overnight at

37°C. Then, an equal volume of 0.1% formic acid was added for acidization, and peptides

were purified on a Strata-X C18 column. The dried peptide power was redissolved in 20 μL

of 0.5 M Triethylammonium Bicarbonate (TEAB) for peptide labeling.

iTRAQ labeling and fractionation were performed according to a previous method (51).

Briefly, samples were labeled with an iTRAQ Reagent-8 plex Multiplex Kit (AB Sciex U.K.

Limited). Next, the labeled samples were fractionated using a high-performance liquid

chromatography (HPLC) system (Thermo DINOEX Ultimate 3000 BioRS) with a Durashell

C18 column, and 12 fractions were collected for further analysis. Liquid

chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS)/MS

analysis was conducted with an AB Sciex nanoLC-MS/MS (Triple TOF 5600 plus) system.

Samples were analyzed using a 90-min gradient from 2–30% (buffer A: 0.1% (v/v) formic

acid, 5% (v/v) acetonitrile; buffer B: 0.1% (v/v) formic acid, 95% (v/v) acetonitrile). MS1

spectra were collected over the range of 350–1500 m/z for 250 ms. The 20 most intense

precursors with charge states of 2–5 were selected for fragmentation. MS2 spectra were

collected over the range 50–2000 m/z for 100 ms. Precursor ions were excluded from

reselection for 15 s. ProteinPilot Software v4.5 was used to analyze the original data. For

protein identification, the Paragon algorithm, which was integrated into ProteinPilot, was

used for searching against the UniProt Rattus norvegicus database (7974 items). Proteins with

at least one unique peptide and an unused value of greater than 1.3 were collected for further

analysis. For normalized protein abundance ratios measured using iTRAQ, we established the

threshold to identify significant changes as an upregulated ≥1.5-fold or downregulated

≤0.67-fold and a P-value of <0.05. All differentially expressed proteins are listed in data file

S2.

Untargeted Lipidomics Analysis

1. Sample preparation

Sample preparation was performed in an ice bath to avoid the degradation of metabolites.

Biological replicates were essential for data analysis (5 replicates were used) (52). Lipids

were extracted using the FOLCH method (53): 1) Homogenize tissue samples with tissue

lysis buffer; 2) Place 200 μL of the samples in a tube, add 300 μL of water, and vortex for 1

min; 3) Add 1.5 mL chloroform:MeOH (2:1, v/v) and vortex for 1 min. Centrifuge for 15 min

at 3,000 rpm to separate the different phases and move the lower phase to a new tube for

evaporation; and 4) Reconstitute the dried extract with 100 μL of isopropyl alcohol:MeOH

(1:1, v/v) after centrifugation, and submit 2 μL of the supernatant for LC-MS analysis.

2. UPLC-QTOF-MS/MS analysis

UPLC-QTOF-MS/MS analysis was performed on an Agilent 1290 Infinity LC system

equipped with an Agilent 6538 Accurate Mass Quadrupole Time-of-Flight mass spectrometer

(Agilent). The LC system was comprised of an Agilent ZORBAX RRHD Eclipse Plus C18

column (100×3 mm, 1.8 µm) with Phenomenex Security Guard ULTRA. The mobile phase

consisted of solvent A (0.1% HCOOH+10 mM HCOONH4+40% H2O +60% ACN) and

solvent B (0.1% HCOOH+10 mM HCOONH4 +10% ACN+90% isopropyl alcohol) with a

gradient elution (0−4 min, 95% A; 4−25 min, 95-5% A). The flow rate of the mobile phase

was 0.5 mL·min-1. Mass spectrometry was performed on a time-of-flight mass spectrometer

(QTOF-MS; Agilent Technologies) using a Dual Agilent Jet Stream (AJS) ESI source. The

scanning mass-to-charge (m/z) range was 50 to 1500 m/z, with a scan rate of 1.00

spectra·sec-1. The capillary voltages were set to 4000 V and 3500 V (for positive and negative

mode, respectively), and the fragmentor was set to 175 V. The pressure of the nebulizer was

set to 35 psi, the gas temperature was set to 325°C, and the continuous gas flow was set to 5

L·min-1. The instrument mode was set to extended dynamic range. QC samples were used to

demonstrate the stability of the LC-MS system. The QC samples were run in positive and

negative mode at regular intervals throughout the entire sequence.

3. Identification of lipids

Mass spectrometry identifications of the lipids (Beijing Biotech-Pack Scientific) was

performed. Three procedures were used to identify metabolites: (1) The mass-to-charge ratios

were used to search online databases such as the Human Metabolome Database (HMDB)

(http://www.hmdb.ca/), METLIN (http://metlin.scripps.edu/), and LIPID MAPS

(http://www.lipidmaps.org/); (2) The MS/MS spectra were compared with MS/MS data from

the above databases to verify the structures of the putative metabolites; and (3) the structures

were predicted from the MS/MS spectra based on specific cleavage rules for lipids. All

differentially expressed lipids are listed in data file S3.

Untargeted Metabolomics Analysis

1. Sample preparation

Kidney tissue (100 mg) was harvested, washed immediately with physiological saline,

and stored at -80°C for the following metabolomics study. Samples were homogenized in 250

μL of ice-cold acetonitrile. Samples were placed on ice for 20 minutes and centrifuged at

14,000 × g for 5 minutes. The supernatant from tissue samples was diluted in 80%

acetonitrile. The extract was resuspended before analysis.

2. UPLC/Q-TOFMS Conditions

Metabolites were separated using a Waters ACQUITY UPLC HSS T3 C18 column (100

mm×2:1 mm, 1.8 μm) using a Waters ACQUITY UPLC I-Class system (Waters). The mobile

phase was composed of solvent A (0.1% formic acid-water) and solvent B (0.1% formic

acid-acetonitrile) with a gradient elution (0−1 min, 95% A; 1−6 min, 95−70% A; 6−20 min,

70−5% A). The flow rate of the mobile phase was 0.5 mL·min-1. The column temperature

was maintained at 45°C, and the sample manager temperature was set at 4°C. Mass

spectrometry was performed using a Waters XevoTM G2S-QTof/MS. Metabolites were

measured separately in positive and negative electrospray ionization (ESI) modes. At the

beginning of the sequence, we ran four quality control (QC) samples from mixed samples to

avoid small changes in both the chromatographic retention time and signal intensity. The QC

sample was also injected at regular intervals throughout the analytical run (54).

3. Metabolite Identification

Metabolite concentrations that were considered markedly different between wild-type

and PXR-/- rats in multivariate and univariate analyses were searched in the HMDB

(www.hmdb.ca), METLIN (www.metlin.scripps.edu), and the Mass Bank (www.massbank.jp)

database. Fragmentation patterns for each metabolite were compared to putative database

compound fragmentation patterns using MassFragment. All differentially expressed

metabolites are listed in data file S4.

RNA sequence analysis

For RNA sequencing (RNA-seq), RNA samples were collected from NC and

AKR1B7-overexpressing RTECs. RNA isolation, library construction, and sequencing were

performed by BGI on a BGISEQ-500 RNA-seq platform (Beijing Genomic Institution,

www.genomics.org.cn, BGI). The GRCm38.p5 reference genome of mouse was used to map

with clean tags. For gene expression analysis, the significance of the differential expression

of genes among different groups was analyzed and defined by the bioinformatics service of

BGI according to the combination of the absolute value of log2-Ratio ≥ 1 and FDR ≤ 0.001.

All differentially expressed metabolites are listed in data file S5.

RNA isolation and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from kidney cortexes and cells using TRIzol (Invitrogen)

following the manufacturer’s instructions (39). cDNA was synthesized with the PrimeScript

Reverse Transcriptase System for reverse transcription PCR (TaKaRa Biotechnology).

qRT-PCR was performed in duplicate with the SYBR Green Master Mix (Vazyme) on a

QuantStudio 3 Real-time PCR System (Applied Biosystems). The expression of each gene

was normalized to that of the internal control gene Gapdh. The primers used for PCR

amplification are listed in data file S6.

Mitochondrial DNA copy number

Total DNA was extracted from cells using a DNeasy Tissue Kit (69506, QIAGEN

Sciences) following the manufacturer’s instructions. The primers listed in data file S6 were

designed using Primer 5 software and synthesized by Invitrogen. qRT-PCR was performed to

determine the mtDNA copy number. Relative mtDNA copy numbers were normalized to 18s

RNA and calculated from the threshold cycle numbers using the delta-delta method.

Supplementary Figures

Fig. S1

Fig. S1. PXR deficiency aggravated cisplatin-induced tubular inflammation and

mitochondrial damage. (A) Construction of Nr1i2 gene knockout rats by TALEN

(Transcription Activator-Like Effector Nuclease). (B) Representative images of

immunohistochemical staining of CD68 in the kidneys of CP-treated WT and PXR-/- rats

(n=6; scale bars=20 μm). (C) qRT-PCR analysis for mitochondrial genes in the kidneys of

CP-treated WT and PXR-/- rats (n=6). (D) qRT-PCR analysis for Pgc-1α, Tfam, Sod2, and

Nrf2 mRNA expressions (n=6). Data are expressed as means ± SD. Statistically significant

differences were determined by two-way ANOVA. # P < 0.05, ## P < 0.01, ### P < 0.001, *

P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. S2

Fig. S2. MnTBAP therapy attenuated cisplatin-induced AKI in wild-type and PXR–/–

rats. (A) SCr and BUN concentrations were measured in CP-treated WT and PXR-/- rats with

or without MnTBAP administration. (B) qRT-PCR analysis for renal Kim-1 and Ngal mRNA

expressions. (C) ELISA assay for the protein concentrations of IL-6 and TNF-α in kidney

tissues. (D) qRT-PCR analysis for renal Il-1β, Il-6, and Tnf-α mRNA expressions. (E)

Representative images for PAS (scale bars=20 μm) in the kidneys (left). Tubular injury scores

in rats were analyzed (right). Data are expressed as mean ± SD of 6 random fields taken from

each kidney. (F) Representative images for DHE staining (scale bars=20 μm) in the kidneys.

(G) TBARS assay was used to detect the lipid peroxidation in the kidneys. Data are expressed

as means ± SD (n=6).Statistically significant differences were determined by two-way

ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig. S3

Fig. S3. PCN treatment ameliorated cisplatin-induced apoptosis, inflammation, and the

dysregulation of mitochondrial genes in vitro. (A) qRT-PCR analysis for Bax and Bcl-2

mRNA expressions in primary mouse kidney tubular cells. (B) qRT-PCR analysis for

mitochondrial genes in CP-induced RTEC injury with PCN administration. (C) RTECs were

transfected with PXR siRNA followed by PCN treatment. qRT-PCR analysis for Il-1β, Il-6,

and Tnf-α mRNA expressions in RTECs. (D) RTECs were transfected with PXR siRNA

followed by PCN treatment. ELISA was used to detect the concentrations of IL-6 and TNF-α

in cell culture medium. Data are expressed as means ± SD (n=3). Statistically significant

differences were determined by one-way ANOVA and two-way ANOVA, # P < 0.05, ##

P < 0.01, ### P < 0.001, #### P < 0.0001, * P < 0.05, ** P < 0.01, **** P < 0.001.

Fig. S4.

Fig. S4. PXR overexpression attenuated cisplatin-induced apoptosis and mitochondrial

dysfunction in vitro. (A) Representative images of FACS analysis after Annexin V and PI

staining. RTECs overexpressed PXR and were then incubated with cisplatin (5 μg/mL) for

24 h. The percentages of apoptotic cells were quantified by FACS (n=3). (B) Western blotting

showing BAX, BCL-2, cleaved caspase-3, and CYT-C in CP-induced RTEC injury with or

without PXR overexpression. (C) qRT-PCR analyses for Il-1β, Il-6, and Tnf-α mRNA

expressions (n=3). (D) FACS analysis for the accumulation of reactive oxygen species in the

mitochondria and mitochondrial membrane potential using the MitoSOX Red Mitochondrial

Superoxide Indicator and JC-1 fluorescent probe in RTECs, respectively. qRT-PCR analyses

for the mtDNA copy number in RTECs. 18s was used as an internal control (n=3). (E)

Cellular ATP content was detected by the luciferase assay (n=3). (F) Seahorse 96xf was used

to detect basal oxygen consumption rate (n=8). (G) Seahorse 96xf was used to detect spare

respiratory capacity (n=8). (H) qRT-PCR analysis for Pgc-1α, Tfam, Sod2, and Nrf2 mRNA

expressions (n=3). (I) qRT-PCR analysis for p62, Atg3, Atg5, and Atg7 mRNA expressions

(n=3). (J) qRT-PCR analysis for Pink1, Parkin, Mfn1, Mfn2, Opa1, Fundc1, and Nix mRNA

expressions (n=3). (K) qRT-PCR analysis for mitochondrial genes in PXR-overexpressed

RTECs. 18s was used as an internal control (n=3). (L) RTECs were transfected with the

pDsRed2-mito plasmids and red fluorescence was detected (n=3; scale bars=10 μm). (M)

RTECs stably expressing mt-mKeima-cox8 were used to detect green and red fluorescence

(n=3; scale bars=10 μm). (N) PXR-overexpressing RTECs were pretreated with Bafilomycin

A1 (Baf-A1), and then incubated with cisplatin (5 μg/mL) for 24 h. The percentage of

apoptotic cells was determined by FACS (n=3). Data are expressed as means ± SD.

Statistically significant differences were determined by one-way ANOVA and two-way

ANOVA. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001, * P < 0.05, ** P < 0.01, ***

P < 0.001, **** P < 0.0001.

Fig. S5

Fig. S5. PCN treatment or PXR overexpression attenuated cisplatin-induced apoptotic

response in three-dimensional cultured HK2 cells. (A) Representative morphology images

of PCN-treated HK2 cells cultured in non-adherent Nunclon Sphera 6-well plates (scale

bars=100 μm). (B) qRT-PCR analysis for Bax and Bcl-2 mRNA expressions in PCN-treated

HK2 cells cultured in non-adherent Nunclon Sphera 6-well plates. (C) Representative

morphology images of PXR-overexpressing HK2 cells cultured in non-adherent Nunclon

Sphera 6-well plates (scale bars=100 μm). (D) qRT-PCR analysis for Bax and Bcl-2 mRNA

expressions in PXR-overexpressing HK2 cells cultured in non-adherent Nunclon Sphera

6-well plates. Data are expressed as means ± SD (n=3). Statistically significant differences

were determined by two-way ANOVA, ### P < 0.001, *** P < 0.001.

Fig. S6

Fig. S6. Expressions of other nuclear receptors remained unchanged in PXR–/– rats. (A)

Quantification of detectable nuclear receptors in the kidneys from WT and PXR-/- rats based

on iTRAQ-based quantitative proteomic analysis. (B) qRT-PCR analysis for the mRNA

expressions of renal nuclear receptors in the kidneys of PXR-/- and WT rats (n=7). Data are

expressed as means ± SD. Statistically significant differences were determined by student’s t

test.

Fig. S7

Fig. S7. AKR1B7 overexpression prevented cisplatin-induced mitochondrial injury in

vitro. (A) qRT-PCR analysis for Akr1b7 in RTECs exposed to CP (dose- and time-dependent).

(B) qRT-PCR analyses for mitochondrial genes in AKR1B7-overexpressed RTECs. 18s was

used as an internal control. (C) In situ hybridization of AKR1B7 with a Cy3-labeled probe

(red) and Mito-Tracker (green) in RTECs (scale bars= 10 μm). (D) qRT-PCR analysis for the

expression of Akr1b7 between mitochondria and cytoplasm in AKR1B7-overexpressing

RTECs. (E) RNA-seq analysis of mitochondria-related genes in NC and

AKR1B7-overexpressing RTECs. (F) AKR1B7-overexpressing RTECs were pretreated with

BafilomycinA1 (Baf-A1), and then incubated with CP (5 μg/mL) for 24 h. The mRNA

expressions of Bax and Bcl-2 were detected. Data are expressed as means ± SD (n=3).

Statistically significant differences were determined by student’s t test, one-way ANOVA, and

two-way ANOVA, # P < 0.05, ## P < 0.01, ### P < 0.001, * P < 0.05, ** P < 0.01, ***

P < 0.001, **** P < 0.0001.

.

Fig. S8

Fig. S8. PXR deficiency changed mitochondrial metabolic pathways. (A) The

iTRAQ-based quantitative proteomic analysis revealed that the metabolic pathway in kidneys

was mostly affected by PXR deficiency. (B) The differential mitochondria-related protein

expressions in the kidneys from WT and PXR-/- rats detected by iTRAQ-based quantitative

proteomic analysis.

Fig. S9

Fig. S9. PXR/AKR1B7 activation attenuated cisplatin-induced lipid accumulation and

improved mitochondrial fatty acid β-oxidation. (A) OPLS-DA analysis based on

non-targeted metabolomics was conducted in the kidneys from WT and PXR-/- rats (n=5). (B)

Hierarchical cluster analysis was used to characterize the metabolites that differed between

WT and PXR-/- rats (n=5). (C) OPLS-DA analysis based on liposomes was conducted in the

kidneys from WT and PXR-/- rats (n=5). (D) Hierarchical cluster analysis was used to

characterize the lipids that differed between WT and PXR-/- rats (n=5). (E) qRT-PCR analyses

for renal Cpt-1α, Mcad, Hmgcs2, Pgc-1α, and Acox1 mRNA expressions in the kidneys of

WT and PXR-/- rats (n=6). (F) qRT-PCR analyses for renal Cpt-1α, Mcad, and Acox1 mRNA

expressions in the kidneys from WT and PXR-/- rats challenged with CP (n=6). (G)

Representative images of Oil Red O staining in the kidneys from WT and PXR-/- rats

challenged with CP (n=6; scale bars=20 μm). (H) Representative images of Oil Red O

staining in the kidneys from PCN-treated mice challenged with CP (n=6; scale bars=20 μm).

(I) Representative images of Oil Red O staining in the kidneys from PXR-overexpressing

mice challenged with CP (n=6; scale bars=20 μm). (J) qRT-PCR analysis for renal Cpt-1α,

Mcad, Hmgcs2, and Acox1 mRNA expressions in the kidneys from PCN-treated mice

challenged with CP (n=6). (K) qRT-PCR analysis for renal Cpt-1α, Mcad, Hmgcs2, and

Acox1 mRNA expressions in the kidneys from PXR-overexpressing mice challenged with CP

(n=6). Data are expressed as means ± SD. Statistically significant differences were

determined by student’s t-test and two-way ANOVA, # P < 0.05, ## P < 0.01, ### P < 0.001, *

P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. S10

Fig. S10. The protective effect of PXR on mitochondrial oxidative capacity and fatty

acid β-oxidation was diminished by silencing AKR1B7. (A) RTECs were transfected with

AKR1B7 siRNA followed by PCN treatment. Seahorse 96xf was used to detect basal oxygen

consumption rate (n=8). (B) Seahorse 96xf was used to detect spare respiratory capacity

(n=8). (C) RTECs were transfected with AKR1B7 siRNA followed by PCN treatment.

qRT-PCR analysis for Cpt-1α, Mcad, Hmgcs2, and Acox1 mRNA expressions (n=3). (D)

PXR-overexpressing RTECs were transfected with AKR1B7 siRNA, and then incubated with

CP (5 μg/mL) for 24 h. Seahorse 96xf was used to detect basal oxygen consumption rate

(n=8). (E) Seahorse 96xf was used to detect spare respiratory capacity (n=8). (F)

PXR-overexpressing RTECs were transfected with AKR1B7 siRNA, and then incubated with

CP (5 μg/mL) for 24 h. qRT-PCR analysis for Cpt-1α, Mcad, Hmgcs2, and Acox1 mRNA

expressions (n=3). Data are expressed as means ± SD. Statistically significant differences

were determined by two-way ANOVA, # P < 0.05, ## P < 0.01, ### P < 0.001, ####

P < 0.0001, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig. S11

Fig. S11. PXR deficiency aggravated I/R-induced mitochondrial damage. qRT-PCR

analyses for mitochondrial genes in the kidneys of I/R-treated wild-type and PXR-/- rats. Data

are expressed as means ± SD (n=6). Statistically significant differences were determined by

two-way ANOVA, # P < 0.05, ## P < 0.01, * P < 0.05, ** P < 0.01, *** P < 0.001.

Table S1. Clinical data of patients with AKI.

Number SCr（μmol/L） BUN（mmol/L）

2003-381 248 15.5

2009-1579 200 29.97

2004-1396 1000 21.13

2016-3169 159 9.2

2016-3166 1002 28.3

2015-1674 432 16.4

2016-0019 230 18.9

2016-0418 447 27.1

2016-0483 173.75 11.44

2016-1032 369.6 38.89

2016-1146 682 17.51

2016-1220 124.7 6.88

2016-1550 277 14.5

2017-0546 172 13.76

2013-1731 767 17.9

2013-1746 195 5.7

2017-2947 182.2 6.18

2017-3243 1115.2 39.68

2017-3431 1149 21

2016-0031 188 6.8