Cell culture studies: BUMPT cells, a mouse proximal tubular cell line, was initially obtained from Drs. William Lieberthal and John Shwartz at Boston University. Cells were cultured in high glucose DMEM supplemented with 10% FBS and antibiotics, and they were maintained at 37oC in a humidified atmosphere of 5% CO2. Various lentiviruses (purchased from Vigene Biosciences), including CHIP overexpression lentivirus (Lv-CHIP), CHIP knockdown lentivirus (shCHIP) and empty vector lentivirus (Lv-NC and shNC), were employed to transfect the cells. Puromycin (3 μg ml-1) was used to generate stably transfected cell lines following 24 hrs treatment. The cells were seeded into 96-well plates or 6-well plates containing culture medium supplemented with 2% FBS. The cells were treated with 20 μM cisplatin (Sigma-Aldrich, catalog P4394) for 16-24 hours. They were then harvested for various studies. Polyethylenimine was used for transfection of 293t cells, a highly transfectable cell line.. Cyclohexamide (50μg ml-1, MCE, catalog HY-12320) was used to treat the cells for 2-8 hours, while the treatment with 15μM of MG132 (MCE, catalog HY-13259) was carried out for 2-12 hours. For CHIP activation, 2μM YL-109 (MCE, catalog HY-18619) was used to pre-treat the BUMPT cells for 8 hours, and they were co-treated with cisplatin.
Animal experiments: All experiments were performed by following the guidelines of the Animal Ethical and Welfare Committee of the Second Xiangya hospital at Central South University (Approval number: 2022620). Male C57BL/6J mice (8-10 weeks old) were used, and they were grouped into various groups (6 mice in each group). For inducing cisplatin-induced AKI, cisplatin was intraperitoneally administered to mice at the dose of 25 mg kg-1. Two days later, kidneys and blood samples were harvested for various studies. In some studies, YL-109 (MCE, catalog HY-18619) was subcutaneously injected in the scruff of the neck (15 mg kg-1, every 2 days) three times before cisplatin injection.
For the modulation of CHIP expression in the kidneys, adenovirus (Vigene Biosciences) was used in in vivo studies. Mice were anesthetized with pentobarbital sodium, and their dorsum of the flank was shaved. The left kidney was exposed with a dorsal incision, and the renal pedicle was clamped. Following clamping, a total of 100 μl adenovirus solution (at the concentration of 1.5 - 2 Í 1012 particles ml-1) was injected in four different regions of the kidney (25 μl at each site) with a 31-gauge needle. The clamp was then removed from the renal pedicle 5 min after the injection. The adenoviral vectors used in the present studies included: CHIP overexpressing adenovirus (Ad-CHIP), CHIP knockdown adenovirus (Ad-shCHIP), and empty adenovirus vector (Ad-NC and Ad-shNC). Two days after the administration of adenoviral vectors, the mice were injected with cisplatin.
Western Blotting procedures: The protocol for immunoblotting has been described [4]. Briefly, cells or renal cortices were homogenized in RIPA buffer, and the protein concentration was measured by BCA assay (Biyotime, catalog P0012S). Equal amounts of proteins were then subjected to SDS-PAGE, and the fractionated proteins were electroblotted onto the PVDF membranes. The membranes were immersed in 5% milk solution, and then incubated with diluted primary antibody overnight at 4oC. The membranes were washed and incubated with secondary diluted antibodies at 22oC for 1 hr. Finally, the membranes were subjected to ECL chemiluminescence, and the signal was detected by exposing the membranes to X-ray film. The antibodies used in these studies included: anti–CHIP (Abcam, catalog ab134064; 1:2,000), anti–β-actin (proteintech, catalog 66009-1-Ig; 1:5,000) and anti-GAPDH (proteintech, catalog 60004-1-Ig; 1:5,000), anti-tubulin (proteintech, catalog 66031-1-Ig; 1:5,000), anti-Bax (proteintech, catalog 60267-1-Ig; 1:2,000), anti-cleaved caspase3 (CST, catalog 9664; 1:1,000), anti-Bcl2 (proteintech, catalog 26593-1-AP; 1:1,000), anti-Flag (Sigma, catalog F1804; 1:5,000), anti-HA (proteintech, catalog 51064-2-AP; 1:5,000, and Santa Cruz, catalog sc-7392; 1:5,000), anti-Nur77 (proteintech, catalog 25851-1-AP; 1:1,000), anti-ubiquitin (CST, catalog 3936; 1:1,000), anti-myc (proteintech, catalog 60003-2-Ig; 1:2000) and anti-COXIV (proteintech, catalog 11242-1-AP; 1:500).
qRT-PCR analysis: The mRNA levels were detected by qRT-PCR, as previously described [26]. Briefly, the cells or renal cortex were homogenized with RNA Trizol (Invitrogen, catalog 15-596-026), and the mRNA was isolated. After removal of the contaminating DNA, ~1 μg mRNA was used for reverse transcription with PrimeScriptTM RT reagent kit (Takara). The cDNA samples thus generated were diluted with an equal volume of water and subjected to an ABI PRISM 7900 Sequence Detector System (Applied Biosystems) with ChamQTM Universal SYBR® qPCR Master Mix (Vazyme). For the quantitative analysis in Figure 1, the relative expression of the genes was obtained using ΔCt values, and their relative expression were compared with the mean value of CHIP to reveal their differences in expression. For the quantitative analysis in Figures 4-6 were calculated with the ΔΔCt method. The primers used in our studies were given in Supplemental table 1.
Immunohistochemistry methods: 4-μm thick paraffin embedded tissue sections were used for carrying out immuno-histochemical procedures [4]. Briefly, the sections were deparaffinized, rehydrated, and rinsed with PBS. Following which, the tissue sections were subjected to antigen retrieval. After blocking of endogenous peroxidase, the sections were immersed in goat serum for 1 hr at 22oC. The tissue sections were then immersed in diluted primary CHIP antibody (Abcam, catalog ab134064; 1:200) solution and kept them overnight at 4oC. After a brief rinse with PBS, they were incubated with diluted secondary antibody solution for 1 hr at 22oC. Subsequently, the sections were treated with DAB solution, followed by staining with hematoxylin for 30 seconds. The sections were re-rinsed with PBS, dehydrated and coverslip mounted and evaluated by light microscopy.
Immunofluorescence staining methods: For in vitro studies, the cells were seeded on the coverslips prior to cisplatin treatment. They were washed, fixed with 4% paraformaldehyde, and permeabilized. For in vivo studies, ~4-μm paraffin embedded thick sections were deparaffinized, rehydrated, permeabilized, and subjected to antigen-retrieval. The sections were immersed in 5% BSA solution for 1 hr at 22oC. They were then incubated with diluted primary antibody solution overnight at 4℃. The sections were rinsed with PBS and incubated with diluted secondary antibody solution for 1-2 hs at 22oC. The sections were stained with DAPI to visualize the nuclei by fluorescence microscopy. The antibodies used in these studies were as follows: anti–CHIP (Abcam, catalog ab134064; 1:1,000), anti–Tomm20 (Abcam, catalog ab565783; 1:500), anti–Megalin (Abcam, catalog ab 184676; 1:500), anti–Nur77 (proteintech, catalog 25851-1-AP; 1:200), and anti-GFP (proteintech, catalog 66002-1-Ig; 1:200).
Morphological analysis: Cellular morphology was evaluated by light microscopy immediately after cisplatin treatment and following H & E staining. Briefly, 4-μm paraffin embedded thick sections were deparaffinized and rehydrated. They were then treated with Hematoxylin for 3 minutes and eosin for 30 seconds. The sections were dehydrated and coverslip mounted for microscopic evaluation.
Renal functional analysis: Renal functions were assessed by measuring serum creatinine and blood urea nitrogen (BUN). Briefly, the blood samples were kept at 22oC for ~20 minutes; following which they were centrifuged to collect the clear serum. Serum creatinine kit (Jiancheng, China, catalog C011-2-1) and BUN kit (Jiancheng, China, catalog C013-2-1) were used to measure the blood concentrations of creatinine and BUN, per the manufacturer’s instructions.
Preparation of mitochondrial and mitochondria-deficient cellular extracts: For the isolation of mitochondria from BUMPT cells, a special kit (Proteintech, catalog PK10016) was used, as per vendor’s instructions. Briefly, cells were washed with cold PBS solution after various treatments and then collected in a centrifuge tube. Mitochondria Isolation Solution A (~1ml for 20 million cells) was added, and the cells were homogenized at 4oC. The same amount of Mitochondrial Isolation Solution B was added in another tube. The cellular homogenate was then carefully added into 2nd tube. The resulting mixture was centrifuged at 600 g for 10 min, and the supernatant was collected. The supernatant was then centrifuged at 10,000 g for 10 min. Finally, the mitochondria at the bottom of the tube were collected and designated as mitochondrial extracts while the supernatant was designated as mitochondria-deficient extracts. The mitochondrial extracts were lysed in the Mitochondrial Lysis Buffer, and the lysate was processed for immuno-blotting procedures.
Cell death evaluation: MTT assay was used to detect cell viability. Briefly, 3,000-5,000 cells were seeded into each well of 96-well plates. After cisplatin treatment, the culture medium was removed, and 100 μl MTT mixture (cell culture medium with 1 mg ml-1 Thiazolyl Blue Tetrazolium Bromide) was added into the wells. The cells were then incubated in this mixture for 4 hrs. Afterwards, the mixture was removed and 150 μl DMSO was added into the wells to dissolve the formazan. The plates were then gently shaken for 5 min on an orbital shaker, and then readings were recorded using a microplate reader at an OD of 495 nm. The background readings were subtracted. The mean values of the relative data of untreated groups were also calculated. Finally, the values of cisplatin-treated groups were divided by the mean values of their related-untreated groups, and thus the relative cell survival rate was calculated. For studies related to apoptosis, TUNEL assay Kit (UElandy, catalog T6014L) was used to evaluate the extent of cellular DNA damage, per the manufacturer's instructions. The tissue sections were photographed with a fluorescent microscope equipped with UV illumination, and the quantitation of cellular damage was assessed.
Evaluation of intracellular ROS: Dihydroethidium (DHE) and 2′,7′-dichlorodihydrofluorescein diacetate (DCF) were used to assess the intracellular ROS in cells and kidney tissues. For DHE staining, BUMPT cells or cryosections of kidney tissues were incubated with 10 μM DHE at 37°C for 30 min and then evaluated by fluorescence microscopy. For DCF staining, BUMPT cells were incubated with 10 μM DCF for 60 min at 37°C, and the sections were then subjected to microscopic evaluation,and flow cytometry was used to evaluate the fluorescence intensity.
Evaluation of protein-protein interaction: The interaction between CHIP and Nur77 was initially predicted by String analysis. The website of String analysis is as follows: STRING: functional protein association networks (string-db.org). Co-immunoprecipitation (CO-IP) assay was used to confirm their interaction, as previously described [26]. Briefly, the cells were homogenized with CO-IP lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5% glycerol, 1.0% NP40, and 1 mM MgCl2), supplemented with protease inhibitors. The protein concentration was measured, and 500-1,000 μg protein in a total volume of 250-350 μl was used for these studies. The primary antibodies were added into the cell lysate and gently mixed using an orbital shaker at 4oC. 25 μl protein A/G beads (Santa-cruz) were added into the lysate mixtures, and they were gently shaken for another 4 hours. The beads were washed, centrifuged, and boiled with the SDS-PAGE loading buffer. The final mixtures were then subjected to SDS-PAGE analysis. The antibodies used in these studies included: anti-Flag (Sigma, catalog F1804), anti-myc (proteintech, catalog 60003-2-Ig) and anti-HA (proteintech, catalog 51064-2-AP and Santa, catalog sc-7392).
Protein-protein docking prediction: The protein sequence of CHIP (PDB, PDB sequence number: 2C2L) and Nur77 (PDB, PDB sequence number: 2QW4) were obtained from the Protein Data Bank. Subsequently, Cluspro was used to generate their interaction model [27-31], and the potential protein-protein docking sites were predicted by using LigPlus software. Finally, the biomolecular structures of the interaction were acquired from PyMOL.
Plasmid construction: CHIP and Nur77 CDS nucleotide sequence were obtained from Miaolingbio (Wuhan, China). The CDS sequence was used as the template to duplicate or truncate CHIP and Nur77 with the primers as indicated in Supplemental table 1. The PCR products generated were subcloned into PCDH-3 ÍFlag or PCDH-3ÍHA plasmid. For point mutation analysis, the primers were designed as indicated in Supplemental table 1. The full-length CHIP plasmid was used as the template, and PCR amplification was carried out. Subsequently, the PCR products were mixed and used as the template for the secondary fusion PCR. The products of fusion PCR were finally subcloned into PCDH-3 ÍFlag plasmid or PCDH-3ÍHA plasmid.
Statistics: Graphpad Prism 9.0 was used to calculate the differences. The values were presented as mean ± SD. 1-way ANOVA with Dunn’s multiple comparisons was performed.