1. ADR nephropathy rat model development and MCU inhibitor treatment
All protocols were approved by the Institutional Animal Care and Use Committee of Peking University First Hospital (number: 11400700229305). This study used a previously reported model . ADR (D1515, Sigma, Santa Clara, California, USA) was successfully used to induce a nephropathy model in Sprague-Dawley rats (Beijing Vital River Laboratory Animal Technology Co. Ltd.) as previously reported. After the injection of ADR, 24 h urinary protein excretion in the rats increased significantly after 6 weeks. We previously confirmed that the injection of RR (R2751, Sigma), an MCU inhibitor, significantly improves proteinuria induced by ADR after 6 weeks . As reported, the Sprague-Dawley rats were divided into four groups: a normal saline control group (Ctl, n = 6), an RR control group (RR, n = 6), an ADR group (ADR, n = 10), and an ADR plus RR group (ADR + RR, n = 6). All rats were sacrificed and harvested at the 6-week time point . Stored renal tissues were used for this study.
2. Western blot analysis
Western blot analysis was performed to detect target molecules in the renal cortex or cultured mouse podocytes. RIPA lysis buffer (89901, Thermo Scientific) containing protease inhibitor was used to extract total cellular proteins, and the proteins were boiled at 100℃ for 10 min After SDS-PAGE on 6%-15% gels, the proteins were transferred to Immuno-Bot PVDF membranes (Bio-Rad). The membranes were blocked with 5% BSA for 1 h and then incubated overnight at 4℃ with the primary antibodies anti-IP3R diluted 1:1000 (ab5804, Abcam), anti-Grp75 diluted 1:1000 (#3593, Cell Signalling Technology), anti-VDAC1 diluted 1:1000 (ab14734, Abcam), anti-MCU diluted 1:1000 (D2Z3B, #14997, Cell Signalling Technology) and anti-GAPDH diluted 1:3000 (C1312, Applygen). Blots were then incubated with HRP-labelled secondary antibody diluted 1:3000 (C2247, C1309, Applygen). ImageJ software was used to semi-quantitate the ratio of the target protein/GAPDH. The specific details of these methods were the same as those that we reported previously .
3. Immunohistochemical analysis showing glomerular expression of members of the IP3R-Grp75-VDAC1-MCU calcium axis
Paraffin-embedded sections (4 μM) from ADR-induced nephropathy rat’s kidneys were used. After dewaxing with xylene and hydration using a gradient series of ethanol, the renal slides underwent antigen retrieval with a sodium citrate solution (pH 6.0) in an autoclave for 20 min, and endogenous peroxidase was then blocked with 3% hydrogen peroxide for 5 min. Afterwards, the sections were sealed with normal goat serum for 30 min at 37°C and then incubated with anti-IP3R (1:1000, ab5804, Abcam), anti-Grp75 (1:1000, ab2799, Abcam), anti-VDAC1 (1:4000, ab14734, Abcam) and anti-MCU (1:200, ab121499, Abcam) antibodies overnight at 4℃. After washing with phosphate-buffered saline (PBS), the sections were incubated with HRP-conjugated anti-rabbit secondary antibody (PV-9000, ZSGB-BIO) for 20 min at room temperature. Then, the sections were sequentially stained with diaminobenzidine (DAB) and haematoxylin. Finally, the sections were sealed with resin. Fifteen images of randomly glomeruli from each rat were captured with a 40× objective under a light microscope (Olympus, Tokyo, Japan). Ratios of the glomerular area showing positive staining for the target molecule to the total glomerular area were analysed using Image-Pro Plus 6.0 software and used to define the glomerular expression of each molecule [6, 7].
4. Immunofluorescence staining showing glomerular expression of members of the IP3R-Grp75-VDAC1-MCU calcium axis
Frozen sections (4 μM) from ADR-induced nephropathy rat kidneys were used for immunofluorescence staining. Sections were fixed with pre-cooled acetone for 10 min, permeabilized with 0.5% Triton X-100 for 10 min at room temperature and blocked with 5% PBS-BSA for 60 min at room temperature to decrease non-specific protein binding. Then, the renal slides were incubated with anti-IP3R (1:100, ab5804, Abcam) and anti-synaptopodin (1:200, ab224491, Abcam), anti-Grp75 (1:100, ab2799, Abcam) and anti-podocin (1:100, ab50339, Abcam), anti-VDAC1 (1:100 ab14734, Abcam) and anti-podocin, or anti-MCU (1:200, ab121499, Abcam) and anti-synaptopodin antibodies overnight at 4℃. After washing with PBS, sections were incubated with Alexa 488-conjugated goat anti-mouse IgG and Alexa 594-conjugated goat anti-rabbit IgG (Invitrogen) for 1 h each. Nuclei were stained for 2 min with 4’6-diamidino-2-phenylindol dihydrochloride (DAPI). Images were taken with a laser scanning confocal microscope (Olympus FluoView FV1000) equipped with a 100× oil immersion objective lens .
5. Real-time quantitative- polymerase chain reaction (PCR)
Total RNA was extracted from podocytes using Trizol (Invitrogen, USA) according to the protocol. The cDNA was synthesized using TransScript First-Strand cDNA Synthesis SuperMix kit (TransGene, China) following the instructions of the kit, then the cDNA was used for real-time quantitative-PCR analysis using PowerUp SYBR Green Master Mix (Applied Biosystems, USA) and ABI Prism 7500 (Applied Biosystems, USA) according to the protocols provided by the kit . The 2-ΔΔCT method was used to calculate the relative expression of target genes to GAPDH . The primers used were as followed: GAPDH: 5’-TCCTCGTCCCGTAGACAAAATG-3’ and 5’-CGCCCAATACGGCCAAA-3’; IP3R1(Itrp1): 5’-CCTTAACAATCCACCCAAGAAATT and 5’-TGCGGAGTATCGATTCATAGGA-3’; Grp75 (Hspa9): 5’-AGAGATTATGCATCAGAAGCAATCA-3’ and 5’-TCCATAACAGCCACACAGGAGTT-3’; VDAC1: 5’-AGCTCACCTTTGATTCGTCATTC-3’ and 5’-CCCTGTCTTGATTTTAGCATTTTTTT-3’; MCU: 5’-ACCACGTACGGCCACCAA-3’ and 5’-CAGGGTCTTCACGTCGTTCA-3’.
6. Mouse podocyte culture
Mouse podocytes (Endlich mouse podocytes, a generous gift from Prof. Hong Hui Wang from Hunan University, China) were cultured as we previously reported . The podocytes were cultured at 33°C in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, and 10 U/ml recombinant mouse interferon-γ (IFN-γ) to induce proliferation and transferred to culture media lacking IFN-γ at 37°C to differentiate for 10–14 days . The podocytes were pre-treated with the MCU inhibitor RR for 60 min before the addition of 0.5 μg/ml ADR for 24 h .
7. Co-immunoprecipitation assay
Co-immunoprecipitation assay was used to detect the effect of RR on the interactions between members of the IP3R-Grp75- VDAC1-MCU complex in cultured mouse podocytes, as we previously reported . Podocyte lysates were collected and centrifuged, following which protein concentrations were determined. Co-immunoprecipitation was performed using a Thermo Scientific Pierce Co-IP Kit (26149, Thermo Fisher Scientific) according to the manufacturer’s protocols. Anti-Grp75 antibody (D13H4, #3593, Cell Signalling Technology) was used to capture proteins coupled in mitochondria/the ER, and normal rabbit IgG without antigenicity was used as a negative control. After co-immunoprecipitation, proteins pulled down by the anti-Grp75 antibody were analysed by western blotting. Lysates from both Ctl and ADR-treated podocytes without immunoprecipitation were used as positive controls (input).
8. Statistical analysis
Data are presented as the means ± SDs and were analysed using one-way AVONA and Student’s t-tests. Differences for which P<0.05 were considered statistically significant.