Targeting mPGES-2 to protect against acute kidney injury via inhibition of ferroptosis dependent on p53

Acute kidney injury (AKI) is a clinical syndrome with high morbidity and mortality but no specific therapy. Microsomal prostaglandin E synthase-2 (mPGES-2) is a PGE2 synthase but can metabolize PGH2 to malondialdehyde by forming a complex with heme. However, the role and mechanism of action of mPGES-2 in AKI remain unclear. To examine the role of mPGES-2, both global and tubule-specific mPGES-2-deficient mice were treated with cisplatin to induce AKI. mPGES-2 knockdown or overexpressing HK-2 cells were exposed to cisplatin to cause acute renal tubular cell injury. The mPGES-2 inhibitor SZ0232 was used to test the translational potential of targeting mPGES-2 in treating AKI. Additionally, mice were subjected to unilateral renal ischemia/reperfusion to further validate the effect of mPGES-2 on AKI. Interestingly, both genetic and pharmacological blockage of mPGES-2 led to decreased renal dysfunction and morphological damage induced by cisplatin and unilateral renal ischemia/reperfusion. Mechanistic exploration indicated that mPGES-2 deficiency inhibited ferroptosis via the heme-dependent regulation of the p53/SLC7A11/GPX4 axis. The present study indicates that mPGES-2 blockage may be a promising therapeutic strategy for AKI.

Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, Jiangsu, P. R.China Email: jiazj72@hotmail.com
Cell viability was detected by Variokan LUX multimode microplate reader (VL0000D0) and calculated according to the instruction.

Generation of mPGES-2-overexpressing or -knockdown HK-2 cells
Human PTGES2 cloned into the expression vector pCDH-CMV2-puro was designed and provided by Jiangsu Laisen Biotechnology Co., Ltd.The shRNA sequence (5'-CAGCGCCCTCAAGACCTACC-3') targeting PTGES2 was designed and cloned into the lentiviral eukaryotic expression vector pLKO.1-puro(Shanghai GenePharma Co. Ltd.).Briefly, lentiviral particles were generated in human HEK293T cells, and cells were then transiently transfected with plasmids containing PTGES2 cDNA or shRNA sequences with viral packaging plasmids psPAX2 and pMD2.G.After 48 h, the viral supernatant was harvested and filtered.Subsequently, HK-2 cells were infected with the virus for 48 h, and positive cells were selected using puromycin dihydrochloride (ab141453, Abcam).

Detection of intracellular reactive oxygen species production
Briefly, cells were seeded on poly L-lysine-coated glass coverslips in 12-well plates, followed by treatment with cisplatin.After stimulation, cells were washed twice with PBS and incubated in the presence of 10 μM 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA, S0033S, Beyotime) in serum-free DMEM for 30 min at 37°C.DCFH-DA was de-esterified intracellularly and converted into highly fluorescent 2′-7-dichlorofluorescein (DCF) upon oxidation by cellular esterases.Intracellular levels of reactive oxygen species (ROS) were reflected by DCF fluorescence intensity (excitation wavelength, 485 nm; emission wavelength, 530 nm).Images were captured using an OLYMPUS-BX43F microscope and quantified using Image-Pro Plus 6.0 blindly.

Mitochondrial JC-1 staining
A fluorescent, lipophilic, and cationic probe, JC-1 (C2006, Beyotime), reflecting mitochondrial membrane potential (∆Ψm), was used according to the manufacturer's instructions.Briefly, HK-2 cells were seeded in 12-well plates and stimulated with cisplatin.After that, the cells were then incubated with the JC-1 staining solution for 30 min at 37°C.Fluorescence was detected using a Fluostar Optima microplate reader (BMG Technologies).For detecting the monomeric form of JC-1, the excitation and emission wavelengths were 490 and 535 nm, respectively.Wavelengths of 525 nm (green) and 590 nm (red) were used to detect aggregation of JC-1.The ratio of red to green fluorescence represents the ∆Ψm of HK-2 cells.

BODIPY™ 581/591 C11 detection
Briefly, cells were treated with cisplatin for 24 h and then incubated with BODIPY 581/591 C11 (D3861, Thermo Fisher) at 2 μM and Mito Tracker Red CMXRos (C1035, Beyotime) at 100 nM for 30 min.After incubation, cells were prepared for image analysis.Confocal images were captured using a Leica STELLARIS 5 microscope with Leica Application Suite X (LAS X) imaging software.The excitation and emission band of oxidized type is pass of 460 -495 and 510 -550, respectively.
But the excitation and emission band of reduced type is pass of 565 -581 and 585 -591, respectively.We used the ratio of oxidized form to reduced form to present lipid peroxidation.For image quantification, analyses were performed using Image-Pro Plus 6.0 and analyzed blindly.

Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) analysis
Kidney tissues embedded in Tissue-Tek Optimal Cutting Temperature (OCT) compound were prepared for TUNEL analysis using the In Situ Cell Death Detection Kit (Meilunbio, MA0223-1) according to the manufacturer's instructions.Cells with positive nuclear staining and DNA breakage were identified and captured by fluorescence microscopy.TUNEL-positive cells were quantified using Image-Pro Plus 6.0 software and defined as (number of dead cells/total number of nucleated cells × 100).

Quantitative reverse transcription-polymerase chain reaction
RNA was isolated from kidney samples using TRIzol reagent (15596026, Invitrogen, Carlsbad, CA, USA) and was reverse transcribed to cDNA using the Superscript III First-Strand Synthesis System (RR037A, Takara Bio).Oligonucleotides were designed using Primer3 software (available at http://frodo.wi.mit.edu/primer3/).TB Green Premix (RR820A, Takara Bio) was used for qRT-PCR amplification.The qRT-PCR data was acquired by Roche LightCycler 480 II/96 with Light Cycler 480 SW 1.5 software.The sequence of primers used are listed in Supplementary Table 1.

RNA-seq
Kidney cortex tissues were collected from mPGES-2 WT and KO mice treated with cisplatin.RNA extraction and expression profiling were performed and analyzed by Hangzhou Cred Technology Co. (Hangzhou, China).The RNA-seq data was deposited in NCBI with accessible number of PRJNA838832.

Western blotting
Tissues or cells were homogenized in RIPA lysis buffer containing a protease inhibitor cocktail (04693159001, Roche) and quantified using a BCA Protein Assay Kit (23227, Thermo Fisher Scientific) according to the manufacturer's protocols.