Part One DsbA-L Interacts With VDAC1 in Mitochondrionmediated Tubular Cell Apoptosis And Contributes To The Progression Of Acute Kidney Disease

Summary

1. Background

we demonstrated that disulfide-bond A oxidoreductase-like protein (DsbA-L) was involved in the progression of renal fibrosis. However, the precise function of DsbA-L in acute kidney injury (AKI), and the mechanisms involved, have yet to be elucidated.

2. Methods

We illustrate the DsbA-L interacted with VDAC1 by co-IP (co-immunoprecipitation) in vitro and Viv and found the interaction parts of them by mutation experiment. The above findings were verified by co-localization o them. In addition, we constructed the two models of PT-DsbA-L and VDAC1 KO mice to verify the function of DsbAL and VDAC1 in models of VAN, CL, P, and I/R-induced AKI.

3. Findings

The PT-DsbA-L-KO mice showed amelioration of I/R, VAN-, and CLP-induced AKI progression via the downregulation of VDAC1. Finally, we confirmed these changes in signal molecules by examining HK-2 cells and kidney biopsies taken from patients with ischemic or acute interstitial nephritis (AIN)-induced AKI. Mechanistically, DsbA-L interacted with amino acids 9-13 and 22-27 of VDAC1 in the mitochondria of BUMPT cells to induce renal cell apoptosis and mitochondrial injury.

4. Interpretation

This work suggested that DsbA-L, located in the proximal tubular cells, drives the progression of AKI, by directly upregulating the levels of VDAC1.Running Title: The Role of DsbA-L in AKI.

5. Funding

National Natural Science Foundation of China, a grant from Key Project of Hunan provincial science and technology innovation, Department of Science and Technology of Hunan Province Project of International Cooperation and Exchanges, Changsha Science and Technology Bureau project, Natural Science Foundation of Hunan Province, Fundamental Research Funds for the Central Universities of Central South University, Hunan Provincial Innovation Foundation For Postgraduate China Hunan Provincial Science and Technology Department.

Keywords

DsbA-L; VDAC_I; Bax; Cyt-c; AKI

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Introduction

Acute kidney injury (AKI), a devastating clinical complication with high rates of morbidity and mortality, affects millions of patients across the entire world(1). AKI is usually induced by sepsis, nephrotoxic agents, and ischemia-reperfusion injury (IRI)(2). As yet, the molecular mechanisms underlying AKI have yet to be fully elucidated. Consequently, there are no effective treatment strategies available for AKI.

Tubular cell injury has been recognized for decades as an important driver of AKI.3 As research has progressed, an increasing number of researchers have identified that mitochondrial damage plays a key role in tubular cell injury and that this initial damage is caused by the accumulation of ROS, the production of cytokines, and cellular death (including both necrosis and apoptosis); collectively, these processes all contribute to AKI.4-6 Although a growing body of research has focused on the mechanisms underlying mitochondrial damage in AKI,7- 12 the precise mechanism responsible for mitochondrial injury has yet to be identified.

Disulfide-bond A oxidoreductase-like protein (DsbAL) was first identified as a mitochondria protein in rat liver cells.13 Previous studies have demonstrated that DsbA-L prevented diet or diabetic nephropathy (DN)- induced obesity, inflammation, insulin resistance, or kidney injury.14-18 Interestingly, our recent study found that DsbA-L mediated unilateral ureteral obstruction (UUO)-induced renal fibrosis. However, the role and mechanism of tubular DsbA-L in AKI are still completely unknown.

In the present study, the proximal tubular deletion of DsbA-L (PT-DsbA-L-KO) in a mouse model resulted in the notable attenuation of I/R, along with vancomycin (VAN)- and cecal ligation and puncture (CLP)-induced AKI. Consistently, DsbA-L also mediated I/R, VAN-, and LPS-induced apoptosis in mouse renal proximal tubular epithelial (BUMPT) cells. Interestingly, we found that DsbA-L interacted with the 9-13 and 22
27 regions of the voltage-dependent anion channel 1 (VDAC1), a key member of the VDAC family of proteins19
21; these effects were observed in both in vivo and in vitro mitochondrial samples and human AKI samples. Furthermore, we demonstrated that PTVDAC1-KO also ameliorated renal cell apoptosis in both in vitro and in vivo models of AKI. Collectively, we demonstrated that DsbA-L interacted with VDAC1 in mitochondrion-mediated tubular cell apoptosis and therefore caused the progression of AKI.

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Materials and methods

1. Ethics statement

The study was approved by the Review Board of the Second Xiangya Hospital, People’s Republic of China (NO. 2018065) and this study recruited 18 patients. All participants before inclusion in the study were recruited with written informed consent. All animal experiments complied with the guiding principles approved by the Animal Care Ethics Committee of Second Xiangya Hospital, People’s Republic of China (NO. 2020310).

2. Antibodies and reagents

Anti-COXIV (ab33985, RRID: AB_879754), VDAC1 (ab14734, RRID: AB_443084), and PGC-1a (ab191838, RRID: AB_2721267) antibodies were obtained from Abcam (Cambridge Science Park, Cambridge, UK). Anti-Caspase3 (9662, RRID: AB_331439) and cleavedcapase3 (9664, RRID: AB_2070042) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Bax (50599-2-Ig, RRID: AB_2061561), Cyt-c (10993-1-AP, RRID: AB_2090467), NRF1 (12482- 1-AP, RRID: AB_2282876), GAPDH (60004-1-Ig, RRID: AB_2107436), and b-tubulin (10094-1-AP, RRID: AB_2210695) antibodies were obtained from Proteintech (Rosemont, IL, USA). The anti-DsbA-L antibody was provided by Dr. Feng Liu(14). All secondary antibodies (MitoTracker Green FM and MitoTracker Red CMXRos) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The calcium ionophore was purchased from Sigma-Aldrich (Shanghai, China). Antimycin A (>95% pure, ab141904) and a Mitochondria/Cytosol Fractionation Kit (ab65320) were purchased from Abcam (Cambridge Science Park, Cambridge, UK). The target sequence for mouse DsbAL has been described previously.22

3. The creation of AKI models by ischemic reperfusion, CLP, and VAN

C57BL/6J male mice (RRID: MGI:5657312) aged 8-10 weeks were purchased from Hunan SJA Laboratory Animal Co., Ltd. Mice exhibiting proximal tubule-specific DsbA-L or VDAC1 deletion were produced by crossing DsbA-L (flox/flox) mice (provided by Dr. Feng Liu) or VDAC1 (flox/flox) mice (obtained from Shanghai model organisms) with PEPCK-Cre mice (provided by Dr. Volker Haase (the University of Pennsylvania, Philadelphia, PA) as described previously.22,23 Male mice (8-10 weeks of age) were subjected to ischemia, CLP, and VAN nephrotoxic AKI, as described previously.23
27 For ischemic AKI, the bilateral renal artery was continuously clipped for 28 min followed by reperfusion for 24 h or 48 h. The body temperature of the mice was maintained at approximately 37°C. For CLP-induced AKI, the cecum was tightly ligated at a position 1.5 cm from the tip. This was followed by a puncture for 18 h. For VAN injury, mice were intraperitoneally injected with a single dose of VAN at a dose of 600 mg/kg for 7 consecutive days, as described previously.28,29 In addition, the C57BL/6 mice were injected with DsbA-L or VDAC1 plasmids, or VDAC1 siRNA (at a dose of 15 mg/kg) via the tail vein twice a week22; saline was used as a control injection. For each study, we analyzed the samples together to avoid bias, based on the previous studies, and used the PASS software to determine the sample size(n=6). We excluded the sample that died or failed the model establishment before the endpoint. The experimental mice were grouped by random-numbers table. Investigators were not blinded during the experiment but were blinded during the allocation, sample collection, and data analysis. All animal experiments complied with the guiding principles approved by the Animal Care Ethics Committee of Second Xiangya Hospital, People’s Republic of China. Mice were housed in a 12-h light/ dark environment with free access to a standard rodent diet and water.

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4. Cell culture and an ischemia-reperfusion model

BUMPT cells obtained from Dr. Wilfred Lieberthal (Boston University School of Medicine) or HK-2 cells obtained from Shanghai Zhongqiaoxinzhou Biotech were cultured with DMEM (Gibco, 11965092) added 10% FBS (Gibco,10100147) and 1% Penicillin-Streptomycin(Gibco, 15140163) at 37°C in 5% CO2. For the ischemic cell model, BUMPTs cells were treated with 10mM antimycin A (an inhibitor of mitochondrial complex III, Abcam, ab141904) and 1.5mM calcium ionophore (Sigma, A23187) in Hanks’ Balanced Salt Solution (Hyclone, SH30030.02) for 2 h.30 We then replaced HBSS with DMEM medium for 0, 2, and 4 h for the reperfusion stage. Cell apoptosis was detected by morphology and immunoblotting for apoptotic indicators, including cleaved-caspase3, Bax, and Cyt-c. Several plasmids were created and transfected into cells by lipofection: DsbA-L, HA-VDAC1-1-25, HA-VDAC1-26-366, HA-VDAC1-full length, HA- VDAC1-D9-13,22-27, HAVDAC1-D39-45,55-57, and HA- VDAC1-D9-13,22-27 D39-45,55-57, and siRNA for DsbA-L and VDAC1. Six to eight hours after transfection, the culture medium was replaced with DMEM. The sequences of DsbA-L siRNA and VDAC1 siRNA for mice were 50 - GCAUGGAGCAACCAGAGAUTT -30 and 50 - CCAGAGCAACTTCGCAGTT -30, respectively; the sequences for the scrambled NC siRNA were 50 -UUCUCCGAACGUGUCACGUTT-30. The sequences of DsbA-L siRNA and VDAC1 siRNA for humans were 50 - UCAUUUGCCAUGUAUAGUCCU-30 and 50 -UAUUAAGCCAAAUCCAUAGCC -30, respectively; the sequences for the scrambled NC siRNA were 50 -AACCACUCAACUUUUUCC CAA -30. The model of IR was then induced when the cell density reached 90%.

5. Flow cytometry

The flow cytometry was operated according to the instruction of the FITC Annexin V Apoptosis Detection Kit (BD, 556547). BUMPT or HK-2 Cells with different treatments were collected using trypsin without EDTA and washed three times with PBS followed by the FITC for 15min and PI for 5min at room temperature, and finally examined by the flow cytometry.

6. AKI patients and sample collection

The protocol was approved by the Review Board of the Second Xiangya Hospital, People’s Republic of China. This study recruited 18 patients, including 12 males and 6 females. The recruitment principle was based on kidney biopsy. Patient information is supplemented in Table 1. The kidney biopsy specimens were obtained from patients living with minimal change diseases (MCD) (n=6) and ischemic or acute interstitial nephritis (AIN)-induced AKI (n=6). We declare that all study complies with all relevant ethical regulations for research with human participants and was carried out in compliance with the Declaration of Helsinki principles and that the study is compliant with the guidance of the Ministry of Science and Technology for the Review and Approval of Human Genetic Resources. The inclusion and exclusion criteria of MCD were described in our previous study.23 For IR: Inclusion criteria:1 Biopsy for ischemia-reperfusion patients,2 Age less than 75 years old; Exclusion criteria1: exclude patients with tumors found in postoperative medical examinations,2 exclude patients with a history of diabetes, gout, hypertension, urinary tuberculosis or infection respectively. For AIN: Inclusion criteria1: Clinically diagnosed as acute interstitial nephritis2 Biopsy confirmed AIN patients,3 Age less than 75 years old; Exclusion criteria1: Exclude patients with abnormal blood creatinine before admission,2 exclude patients with a history of diabetes, gout, hypertension, urinary tuberculosis or infection. These specimens were then used for staining of HE, TUNEL, and immunohistochemistry as well as immunoprecipitation (IP) and immunoblotting.

7. Immunoprecipitation

The cytoplasm and mitochondria from BUMPT cells and kidney tissue from mice and AKI patients were separated using the Mitochondria/Cytosol Fractionation Kit (Abcam, ab65320); the kit was used by the manufacturer’s instructions. The antibodies (DsbA-L, VDAC1, HA, or IgG) were bound onto the magnetic beads. Next, we added the mitochondria/cytoplasm lysate and incubated it for three hours. Next, the mixture was eluted and investigated for the expression of associated markers by immunoblotting.

8. Relative quantitative PCR (qPCR)

RNA was extracted from BUMPT cells or kidney tissue by the Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA was then reverse-transcripted into first-strand cDNA using the Prime Script RT Reagent kit and gDNA Eraser (TaKaRa, RR037A), as previously described.31‘ 34 Next, we used the cDNA as a template with TB green (TaKaRa, RR820A) and a Light cycler 96 (Roche) with the following primer pairs: DsbA-L: 5
- AAATATGGGGCCTT TGGGCT-3‘ (forward) and 5‘- TAGCAACTCCAAGCGGTCA G-3‘ (reverse); and GAPDH: 5‘-GGTCTCCTCTGACTTCACA-3‘(forward) and 5‘-GTGAGGGTC TCTCTCTTCCT-3‘(reverse); PGC- 1a: 5‘-ATGTGTCGCCTTCTTGCTCTTCC-3 (forward) and 5‘-CTCCCGCTTCTCGTGCTCTTTG-3‘(reverse); NrF1:5’-TCTGCTGTGGCTGATGGAGA GG-3‘(forward) and NrF1:5’-GATGCTTGCGTCGTCTGGATGG-3 (reverse);

9. BUN and creatinine detection

The detection of BUN and creatinine were performed according to the protocol of BUN (Urea Nitrogen Content Assay Kit Beijing Boxbio Science & Technology Co, Ltd.) and Creatinine Assay kit(Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

10. HE staining, TUNEL staining, immunochemistry, immunofluorescence, and immunoblotting

Renal tissue was embedded in paraffin and then cut into sections for various types of staining, as described previously.26,35 Histology was assessed by hematoxylin and eosin staining. The criteria used to score renal tubular injury were described previously.23 TUNEL staining was used to evaluate apoptosis in the renal cells. The proportion (%) of TUNEL-positive cells in 10-20 microscopic fields per tissue section was used as a quantitative indicator of apoptosis.26 For immunochemistry staining, tissue sections were incubated overnight at 4° C with a specific primary antibody (DsbA-L 1:200 or VDAC1 1:100). The following morning, the sections were incubated with a secondary antibody for 30min at 37°C and then reacted with DAB for 5-10 min. For immunofluorescence staining, the sections were incubated with a specific primary antibody (DsbA-L 1:200, VDAC1 1:100) overnight at 4°C followed by a secondary fluorescent antibody for 1 h at 37°C in the dark. DAPI was then added for 3-5 min and the sections were observed by fluorescent microscopy. Mitochondrial staining was performed by a standard protocol. Protein lysates from BUMPT cells or kidneys were harvested and then centrifuged to collect the supernatant containing the proteins. The supernatant was subjected to SDS-PAGE and then transferred to a PVDF membrane. The membranes were then incubated with a primary antibody overnight at 4°C followed by a secondary antibody for 1 h at room temperature. The concentration of antiCOXIV, VDAC1, PGC-1a, Bax, Cyt-c, NRF1, GAPDH, and b-tubulin is 1:1000. The concentration of anti-Caspase3 and cleaved-capase3 is 1:2000.

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11. Statistics

All data were presented as means § SD. Two-tailed Student t-tests were used for two group comparisons. Oneway ANOVA followed by Tukey’s post hoc analysis was used for multiple comparisons. The Kruskal
Walls test was used for the data with non-normal distribution. The Graph Pad software 8.0 was used to analyze the data and P<0.05 was considered statistically significant.

12. Role of funding source

The funders were not involved in the study design, data collection, analysis, interpretation, or writing of the manuscript.

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Xiaozhou Li,a,b,1 Jian Pan,a,b,1 Huiling Li,c Guangdi Li,g Bohao Liu,a,b Xianming Tang,a,b Xiangfeng Liu,f Zhibiao He,a,b Zhenyu Peng,a,b Hongliang Zhang,a,b Luxiang Wang,a,b Yijian Li,d Xudong Xiang,a,b Xiangping Chai,a,b Yunchang Yuan,e Peilin Zheng,h and Dongshan Zhang a,b *

a Department of Emergency Medicine, People’s Republic of China

b Emergency Medicine and Difficult Diseases Institute, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, People’s Republic of China

c Department of Ophthalmology, People’s Republic of China

d Department of Urinary Surgery, People’s Republic of China

e Department of Chest Surgery, People’s Republic of China

f Department of General Surgery, Second Xiangya Hospital, People’s Republic of China

g Department of Public Health, Central South University, Changsha, Hunan, People’s Republic of China

h Department of Endocrinology, Shenzhen People's Hospital, The Second Clinical Medical College of Jinan University, The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen, People’s Republic of China

1 Xiaozhou Li and Jian Pan contributed equally to this study