Background
Treatment-resistant nephrotic syndrome is a challenging clinical problem. Several patients with nephrotic syndrome are resistant to current therapies such as corticosteroid, calcineurin inhibitors, and mycophenolic acid, and they are at a higher risk of developing end stage renal disease [
1]. Among patients with treatment-resistant nephrotic syndrome, focal segmental glomerulosclerosis is the most common pathological change. Podocyte apoptosis play a key role in the development of focal segmental glomerulosclerosis. However, the mechanism underlying podocyte apoptosis has not been fully clarified. Different mechanisms could mediate podocyte apoptosis, such as cytosolic Ca
2+ overload [
2], mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and oxidative stress [
3].
Ca
2+ functions as a second messenger and plays a central role mediating apoptosis. Ca
2+ homeostasis is mainly regulated by membrane Ca
2+ channels and intracellular Ca
2+ stores; ER, mitochondria, lysosomes, endosomes and Golgi apparatus are the main Ca
2+ stores. Among the Ca
2+ stores, the transfer of Ca
2+ from the ER to mitochondria via close contacts between the two organelles named, ER-mitochondria coupling, plays a critical role in maintaining intracellular Ca
2+ homeostasis. The coupling occurs via mitochondria-associated membranes (MAMs), which account for ~ 20% of the mitochondrial surface. A single yeast cell contains around 100 couplings. Under normal physiological conditions, the cytosolic Ca
2+ concentration is ~ 100 nM, the ER Ca
2+ concentration is ~ 1000 μM, and the mitochondrial Ca
2+ concentration is ~ 1000 nM [
4]. Ca
2+ transfer from the ER to the mitochondria is required for mitochondrial ATP production and maintaining cell survival, but excessive Ca
2+ entrance into the mitochondria can lead to mitochondrial Ca
2+ overload and trigger opening of the mitochondrial permeability transition pore, release of pro-apoptotic factors such as cytochrome c, caspase activation, and apoptosis. This pathway of mitochondrial Ca
2+-dependent cell death is crucial in a plethora of cell types [
4‐
8].
The molecular basis of Ca
2+ transfer from the ER to mitochondria is the inositol 1,4,5 triphosphate receptor (IP
3R)/glucose-regulated protein 75 (Grp75)/voltage dependent anion channel 1 (VDAC1) complex, which is located at sites of ER-mitochondrial coupling. IP
3R is the Ca
2+ release channel located at the ER membrane, VDAC1 is the channel located at the outer mitochondrial membrane, and Grp75 is a bridging protein that interacts physically with IP
3R and VDAC1. The Ca
2+ released from the ER forms Ca
2+ hot spots between the ER and the mitochondria, enters the mitochondrial intermembrane space, and then finally enters the mitochondrial matrix via the mitochondrial calcium uniporter (MCU) [
5]. Based on the route of Ca
2+ transfer, we call IP
3R-Grp75-VDAC1-MCU the intracellular calcium regulation axis.
Little is known about the roles of the IP3R-Grp75-VDAC1-MCU axis in the mechanism of podocyte apoptosis. In the current study, the roles of the IP3R-Grp75-VDAC1-MCU calcium regulation axis in podocyte apoptosis were investigated in cultured mouse podocytes exposed to Adriamycin (ADR) and angiotensin II (Ang II) in vitro. Further studies on proteinuria regulation and the protective effects of calcium regulation axis antagonists on podocytes were performed in an ADR nephropathy rat model.
Methods
Induction of mouse podocyte apoptosis in vitro
Mouse podocytes (Endlich mouse podocytes, a generous gift from Prof. Hong Hui Wang from Hunan University, China) were cultured as reported previously [
9]. Briefly, 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 for 7 days. Then, they were transferred to culture media lacking IFN-γ at 37°C to differentiate for 10–14 days. To induce apoptosis, the podocytes were treated with 0.5 μg/ml ADR (D1515, Sigma, Santa Clara, California, USA) or 1 μM Ang II (A9525, Sigma) for 24 h.
Analysis of podocyte apoptosis by flow cytometry
Cells were washed twice with cold PBS and then resuspended in 1× binding buffer (556,547, BD Biosciences, Franklin Lake, New Jersey, USA) at a concentration of 1 × 10
6 cells/ml. Then, a 100 μl cell suspension was transferred to a 5-ml culture tube. The podocytes were stained with 5 μl FITC-conjugated annexin V and 5 μl propidium iodide (PI, 556547, BD Biosciences) for 15 min at room temperature in the dark and analyzed by flow cytometry (Flow Cytometer, FACSCanto II, BD Biosciences) within 1 h to analyze apoptosis [
10]. When doing flow cytometry, naked cells without staining, cells stained with FITC-annexin V only and cells stained with PI only were used for adjustment of the detecting parameters of flow cytometry in advance. The cells which were annexin high and PI low were counted as apoptotic cells. The apoptotic rate of podocytes among different groups were compared.
Analysis of mitochondrial Ca2+-related apoptosis
Podocytes were cultured in 96 well plates at 1 × 10
5 cells/well for 24 h, treated with different drugs or transfection and used for mitochondrial Ca
2+ detection. Mitochondrial Ca
2+ was labeled with Rhod-2 AM (Invitrogen, Karlsruhe, Germany) [
11]. The working solution was dispensed from a 1 mM DMSO stock and diluted to 5 μM in PBS. Podocytes were incubated with 5 μM Rhod-2 AM at 4°C for 30 min, washed, and detected using Synergy 4 Multi-Detection Microplate Reader (Synergy 4, BioTek, Vermont, USA). Rhod-2 AM was detected using excitation at 552 nm and emission at 581 nm. Before labeling of Rhod-2 AM, the background fluorescence intensity of cells in each well was measured as F0. After labeling of Rhod-2 AM, the fluorescence intensity of cells in each well was measured as F1. The value of F1-F0 was used to reflect mitochondrial Ca
2+ level of cells in each well.
Western blotting was used to detect active caspase-3 [
10]. Rabbit polyclonal anti-active caspase-3 (ab2302, Abcam, Cambridge, UK) and mouse monoclonal anti-GAPDH (RM2002, Beijing Ray Antibody Biotech) were used. Total cellular proteins were extracted using non-denatured RIPA lysis buffer containing protease inhibitor and then quantified using a Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, Massachusetts, USA). After SDS-PAGE electrophoresis on 6–15% gels, the proteins were transferred to nitrocellulose membranes using the Mini Trans Blot Cell (Bio-Rad). The membranes were blocked with 5% BSA/PBS-T for 60 min and then incubated overnight at 4°C with primary antibodies. After three washes with PBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Applygen Technologies Inc., Beijing, China) for 1 h at room temperature and the signal was developed using an ECL chemiluminescence detection kit (Millipore, Massachusetts, USA). The ratio of active caspase-3/GAPDH was semi-quantitated using ImageJ software.
Western blot analysis of expression of IP3R, Grp75, VDAC1, and MCU
For western blot analysis, rabbit polyclonal anti-IP3R (ab5804, Abcam), rabbit monoclonal anti-Grp75 (D13H4, #3593, Cell Signaling Technology), mouse monoclonal anti-VDAC1 (ab14734, Abcam), rabbit monoclonal anti-MCU (D2Z3B, #14997, Cell Signaling Technology), and mouse monoclonal anti-GAPDH antibodies were used. Western blotting and semi-quantitation were performed as the method described above.
Co-immunoprecipitation (co-IP) analysis of the IP3R-Grp75-VDAC1 complex
Podocytes lysates (generating ~ 1.5 mg total protein) were collected using ice-cold IP lysis buffer (26,149, ThermoFisher Scientific
). The lysates were transferred to a micro centrifuge tube and centrifuged at ~ 13,000×
g for 10 min to pellet the cell debris. Then, the supernatant was transferred to a new tube for protein concentration determination and further analysis. Co-IP was performed using a Thermo Scientific Pierce Co-IP Kit
(26,149, ThermoFisher Scientific) according to the manufacturer’s protocols. Anti-Grp75 antibody was used as the bait antibody to capture mitochondria-ER coupling proteins. Rabbit monoclonal anti-Grp75 antibody (D13H4, #3593, Cell Signaling Technology) was first immobilized using AminoLink Plus Coupling Resin
(26,149, ThermoFisher Scientific). Then, the resin was washed and incubated with lysate overnight. After incubation, the resin was washed again and proteins were eluted using Elution Buffer
(26,149, ThermoFisher Scientific). Normal rabbit IgG without antigenicity provided with the kit was used as a negative control to detect nonspecific binding. The control was treated in the same way as the Co-IP samples, including incubation with the Grp75 antibody. After Co-IP, the proteins pulled down by anti-Grp75 antibodies were analyzed by western blotting [
12,
13]. Lysates from both Ctl and ADR- or Ang-II treated podocytes without immunoprecipitation were used as a positive control (input).
IP3R-Grp75-VDAC1-MCU axis agonists
D-myo-inositol 1,4,5-triphosphate tripotassium salt (IP3, 74,148, Sigma) was used at a concentration of 10 μM diluted in ultra-pure water to stimulate IP3R in cultured mouse podocytes for 24 h. Spermine (S3256, Sigma) was used at a concentration of 20 μM, diluted in ultra-pure water, to stimulate MCU in cultured mouse podocytes for 2 h.
IP3R-Grp75-VDAC1-MCU axis antagonists
The IP
3R inhibitor Xestospongin C (XeC, ×2628, 10 μM, Sigma) [
14] and the MCU inhibitor Ru360 (557,440, 10 μM, Merck, Kenilworth, New Jersey, USA) [
15] were used to block ER calcium release and mitochondrial Ca
2+ uptake, respectively. Podocytes were pre-treated with the above inhibitors for 60 min before treatment with ADR or Ang II, respectively. Specific siRNA targeting the bridging protein Grp75 and a non-targeted negative control siRNA were synthesized by Invitrogen. Podocytes were plated in six-well plates and treated with 100 pmol/well siRNA duplexes using 10 μl RNAiMAX reagent (Invitrogen) according to the manufacturer’s protocol. After 8–12 h, the media were changed according to the status of cell growth at 40–50% confluence. The podocytes were collected for further experiments 24 h after transfection.
ADR nephropathy rat model and MCU inhibitor treatment
All protocols were approved by the Institutional Animal Care and Use Committee of Peking University First Hospital (Number: 11400700229305). Ruthenium red (RR, R2751, Sigma) was used as a specific inhibitor of MCU. Thirty-two male Sprague Dawley rats weighing 80–100 g were randomly divided into four groups: normal saline control (Ctl, n = 6), RR control (RR, n = 6), ADR group (ADR, n = 10), and ADR plus RR (ADR + RR, n = 10). The rats were fed a standard diet, and water was given ad libitum; they were maintained using alternating 12-h cycles of light and dark. After acclimatization for 48 h, the rats in ADR and ADR + RR group received a tail vein injection of 0.8 mg/100 g bodyweight ADR in sterile water (the 0 time point). Immediately after ADR injection, rats in the ADR + RR group were administered RR at a dose of 2.5 mg/kg/d by intraperitoneal injection for 14 days. The rats in Ctl and RR groups received normal saline and RR injections for 14 days, respectively. All rats were sacrificed at the 6-week time point and the kidneys were harvested. Twenty-four hours urine was collected from each rat at 0, 2 weeks, 4 weeks, and 6 weeks using metabolic cages.
Effects of RR on proteinuria and podocyte in ADR nephropathy
Urinary proteins were analyzed using the Pyrogallol red-molyb-date dye-binding method and a Hitachi-7150 Automatic biochemical analyzer (Hitachi, Tokyo, Japan). Ultrathin sections of renal cortex were made for electron microscopy using a method reported previously [
10]. Averaged 24 electron microscopic photographs of glomeruli were taken randomly from each rat. Podocyte foot process width was analyzed as reported previously [
16] using Olympus Scandium SEM imaging software. Total glomerular basement membrane length was measured. Total number of foot processes in each glomerular basement membrane was counted. Total glomerular basement membrane length divided by number of foot process was used as mean foot process width.
Statistical analysis
SPSS20.0 statistical analysis software was used for all analyses. All data are presented as means ± SD. Unpaired Student’s t-tests were used to compare differences between two groups. One-way ANOVA was used to compare differences among more than two groups. P < 0.05 was used to define statistical significance.
Discussion
Podocytes are core target cells for proteinuria control [
1]. The mechanism underlying podocyte apoptosis has been investigated in different pathways [
3]. Previous studies revealed that disturbance of cytosolic Ca
2+ homeostasis, which is regulated by membrane Ca
2+ channels, such as TRPC6 and TRPC5, is a key mediator of podocyte apoptosis or injury [
2]. Recently, mounting evidence has demonstrated that dysregulation of Ca
2+ transfer from the ER to mitochondria by the IP
3R-Grp75-VDAC1-MCU axis, located at sites of ER-mitochondria coupling, will lead to mitochondrial Ca
2+ overload, opening of mitochondrial permeability transition pore, the release of pro-apoptotic factors such as cytochrome c, caspase activation, and apoptosis in many different cells and diseases including neurodegeneration, metabolic diseases, and cancer [
4‐
8]. However, it is unclear whether this pathway also mediates podocyte apoptosis.
For the first time, this study investigated the roles of the IP
3R-Grp75-VDAC1-MCU calcium regulation axis during podocyte apoptosis. First, we explored whether this calcium regulation axis affects mitochondrial Ca
2+ and apoptosis in podocytes by using IP
3 to specifically stimulate Ca
2+ release from IP
3R located at the ER membrane and Spermine to specifically activate the mitochondria calcium uniporter. As expected, our results revealed that both agonists significantly increased mitochondrial Ca
2+ levels and apoptosis rate in mouse podocytes. Second, to assess whether the IP
3R-Grp75-VDAC1-MCU calcium regulation axis plays a role in podocyte apoptosis, we used the common apoptosis-inducing drugs ADR and Ang II. Western blotting identified significantly increased levels of IP
3R, Grp75, VDAC1, and MCU during ADR- or Ang II-induced podocyte apoptosis. Next, Co-IP experiments revealed that the interaction among the IP
3R, Grp75, and VDAC1 complex was enhanced, which suggests that these drugs may increase ER-mitochondria coupling during podocyte apoptosis. As previously described [
5], ER-mitochondria coupling is the site through which Ca
2+ transfer from the ER to mitochondria occurs; therefore, more coupling might lead to more Ca
2+ transfer to the mitochondrial matrix. Indeed, significantly increased mitochondrial Ca
2+ levels accompanied by increased active caspase-3 levels were found during ADR- and Ang II-induced mouse podocyte apoptosis. Together, these data revealed that the increased expression of IP
3R, Grp75, VDAC1 and MCU, enhanced interaction of IP
3R-Grp75-VDAC1, increased mitochondrial Ca
2+ and active caspase-3 were observed during ADR- and Ang II-induced mouse podocyte apoptosis.
To demonstrate the possible causative effects of an enhanced IP
3R-Grp75-VDAC1-MCU axis on mitochondrial Ca
2+ overload and apoptosis in mouse podocytes, three different antagonists were used to inhibit different proteins in the axis. XeC is a specific IP
3R inhibitor that decreases Ca
2+ release from the ER. Si-Grp75 was used to knockdown Grp75 and hence decouple the IP
3R-Grp75-VDAC1 axis [
17]. RU360 is a specific MCU inhibitor that was used to block Ca
2+ entry into the mitochondrial matrix. The current results clearly demonstrated that these three antagonists all prevented ADR- and Ang II-induced mitochondrial Ca
2+ overload, increased active caspase-3 levels, and mouse podocyte apoptosis. These results confirmed that an enhanced IP
3R-Grp75-VDAC1-MCU axis mediates mouse podocyte apoptosis by facilitating Ca
2+ transfer from the ER to mitochondrial and mitochondrial Ca
2+ overload. Therefore, antagonists of the IP
3R-Grp75-VDAC1-MCU axis could prevent ADR- and Ang II-induced mitochondrial Ca
2+ overload and apoptosis in mouse podocytes.
Little is known about the roles of this Ca
2+ regulation axis in podocyte apoptosis. During palmitic acid-induced podocyte apoptosis, Yuan et al. [
18] found that MCU was upregulated, which was accompanied by an increase in mitochondrial Ca
2+ and cytochrome c levels. They also showed that inhibiting MCU prevented mitochondrial Ca
2+ uptake and the mouse podocyte apoptosis induced by palmitic acid. To our knowledge, no studies have investigated the roles of Grp 75 and VDAC1 in podocyte apoptosis. Gong et al. [
19] compared kidney proteins between diabetic and non-diabetic rats using method of proteomic analysis and found upregulation of VDAC1 in kidney proteins from diabetic rats. Further investigations into the mechanisms regulating the IP
3R-Grp75-VDAC1-MCU calcium axis in podocytes are needed.
Finally, an ADR-induced nephropathy model was used to assess whether inhibiting the transfer of Ca
2+ to mitochondrial matrix can protect against proteinuria. Since MCU is the final channel that regulates the entry of Ca
2+ into the mitochondrial matrix, the MCU-specific inhibitor RR was used to treat ADR nephropathy rats. The dose of RR was selected based on a previous report in a subarachnoid hemorrhage rat model [
20]. Interestingly, significantly decreased proteinuria accompanied by significantly improved podocyte foot process effacement was observed in the rats treated with ADR plus RR.