Caspase-Dependent and Caspase-Independent Pathways Are Involved in Cadmium-Induced Apoptosis in Primary Rat Proximal Tubular Cell Culture

We designed this study to investigate whether cadmium induces caspase-independent apoptosis and to investigate the relationship between the caspase-dependent and caspase-independent apoptotic pathways. Cadmium (1.25–2.5 μM) induced oxidative stress in rat proximal tubular (rPT) cells, as seen in the reactive oxygen species levels; N-acetylcysteine prevented this. Cyclosporin A (CsA) prevented mitochondrial permeability transition pore opening and apoptosis; there was mitochondrial ultrastructural disruption, mitochondrial cytochrome c (cyt c) translocation to the cytoplasm, and subsequent caspase-9 and caspase-3 activation. Z-VAD-FMK prevented caspase-3 activation and apoptosis and decreased BNIP-3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) expression levels and apoptosis-inducing factor/endonuclease G (AIF/Endo G) translocation. Simultaneously, cadmium induced prominent BNIP-3 expression in the mitochondria and cytoplasmic AIF/Endo G translocation to the nucleus. BNIP-3 silencing significantly prevented AIF and Endo G translocation and decreased the apoptosis rate, cyt c release, and caspase-9 and caspase-3 activation. These results suggest that BNIP-3 is involved in the caspase-independent apoptotic pathway and is located upstream of AIF/Endo G; both the caspase-dependent and caspase-independent pathways are involved in cadmium-induced rPT cell apoptosis and act synergistically.


Introduction
Cadmium is gaining attention as a known occupational hazard and environmental pollutant that can cause a series of biochemical and physiological dysfunctions in humans. The exposure routes have principally been contact with batteries, paints, fertilizers, and automobiles. As with other complex organic pollutants, microorganisms cannot degrade cadmium. Cadmium accumulates in the ecosystem and enters the food chain through contaminated water and soil and individually on a 12 h light/dark cycle with unlimited standard rat food and double distilled water (DDW). All experimental procedures were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council and were approved by the Animal Care and Use Committee of Yangzhou University (Approval ID: SYXK (Su) 2007-0005). All surgeries operations were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize any suffering experienced by the animals used in this study.

Cell culture and cadmium exposure conditions
The rPT cells were obtained from the kidneys of Sprague-Dawley rats (from the Comparative Medicine Centre of Yangzhou University) with body weights between 180 g and 200 g. Intraperitoneal injection of sodium pentobarbital (2%, 0.31 ml/100 g) to anesthetize rats. Breaking the neck to death 5 min latter when the rats were in a deep coma. The rats were transferred to super-clean worktable after 75% alcohol soak for 2 minutes. Then, opened the abdominal cavity and removed kidneys of rats under aseptic conditions. rPT cell isolation, identification, and culture were performed as previously described [24]. Primary cells and subcultures were cultured in DMEM/F12 supplemented with 15% FBS, 0.25 g/L glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37˚C in 95% air and 5% CO 2 . rPT cell identity was confirmed by alkaline phosphatase antibody staining against specific proximal tubular antigens. The purity of the isolated primary rPT cells was >95%; the cells were subcultured using trypsin-EDTA digestion. Cells cultured for 12 h had the highest viability (according to the growth curve, data not shown). Based on the doses in a previous study [25], cells were treated with 1.25, 2.5, or 5.0 μM cadmium; the cadmium acetate stock solution was dissolved in sterile ultrapure water.

DAPI staining
Apoptotic morphological changes in the nuclei were detected by staining with DAPI (4',6-diamidino-2-phenylindole). rPT cells (2 × 10 5 cells per well) were seeded onto sterile cover slips in 24-well plates. After 12-h treatment with 0, 1.25, 2.5, or 5.0 μM cadmium, the medium was removed. The cells were washed with ice-cold phosphate-buffered saline (PBS), fixed with paraformaldehyde (4% w/v) for 10 min at room temperature, and incubated with DAPI staining solution (50 mM in PBS) for 10 min in the dark. After washing in PBS three times, the cells were viewed under a Leica inverted fluorescence microscope (Wetzlar, GER) at an excitation wavelength of 352 nm. To assess the extent of cadmium-induced apoptosis, 200 cells per experiment were randomly selected and the apoptotic cells therein were counted; each experiment was performed in triplicate.

Measurement of MPTP activity
MPTP opening in rPT cells was detected using calcein-AM and CoCl 2 loading, resulting in mitochondrial localization of calcein fluorescence [26]; these reagents were used to monitor the MPTP activity. rPT cells (2 × 10 5 cells per well) were seeded onto sterile cover slips in 24-well plates, loaded for 30 min at 37˚C with 2 μM calcein-AM, followed by 1-h incubation with 2 mM CoCl 2 after 12-h incubation with 2.5 μM cadmium, and then washed twice with PBS. The cover slips were fixed with paraformaldehyde (4% w/v) for 10 min at room temperature and then imaged under a laser scanning confocal microscope (LSM 710; Zeiss, Jena, Germany). The change in fluorescence intensity was measured with excitation at 488 nm and emission at 525 nm.

Flow cytometry analysis
All subsequent assays were carried out on a Beckman Coulter fluorescence-activated cell sorter (CyAn ADP 7; Brea, CA, USA). rPT cells were seeded in 6-well plates and treated with 0, 1.25, 2.5, or 5.0 μM cadmium for 12 h when the cell fusion rate was 60-70%. Subsequently, the adherent cells were collected with the Accutase cell detachment solution by 5-min centrifugation at 1500 rpm. Each treatment group yielded at least 1.5 × 10 6 cells, which were washed twice with PBS and incubated with fluorescent dyes for the flow cytometric analysis.

Detection of apoptosis
Apoptotic cells were evaluated using annexin V-FITC/PI staining. The total apoptotic proportion is presented as the sum of early and late apoptotic cells, which was determined as the percentage of annexin V + /PIand annexin V + /PI + cells, respectively. After 12-h staining, the harvested cells were labeled with annexin V-FITC and PI according to the manufacturer's protocol. FITC and PI fluorescence was characterized using an FL-1 filter (530 nm) and FL-2 filter (585 nm), respectively; 10,000 events were acquired.

ROS measurement
Intracellular ROS were determined using flow cytometry and DCFH-DA staining. DCFH-DA can be cleaved to form non-fluorescent dichlorofluorescein (DCFH) in the cells and is oxidized to fluorescent dichlorofluorescein (DCF) by ROS. Cells (1.5 × 10 6 ) were incubated with 100 μM DCFH-DA at 37˚C for 30 min, washed twice with PBS, and the fluorescence intensity (FL-1, 530 nm) of 10,000 cells was measured using a flow cytometer.

Cell fraction preparation
After 12-h treatment with 0, 1.25, 2.5, or 5.0 μM cadmium, cells were harvested by Accutase™ Cell Detachment Solution and washed twice with PBS. To obtain the mitochondrial and cytosolic protein extracts, the harvested cells were subfractionized in homogenization buffer. The mitochondrial and cytosolic fractions were isolated with the method described by Jayanthi et al. [27]. The pellet and supernatant contained the mitochondrial fraction and cytosolic fraction, respectively.

Immunofluorescence assays
rPT cells (2 × 10 5 cells per well) were seeded onto sterile cover slips in 24-well plates and treated with 0, 1.25, 2.5, or 5.0 μM cadmium for 12 h; there were three replicates per group. Next, the cells were washed twice with PBS, and fixed on the coverslips with 4% paraformaldehyde. Then, the monolayer was permeated with 0.5% Triton X-100 and the cells were blocked with 5% bovine serum albumin (BSA). The cells were incubated with anti-AIF antibody (1:100) overnight at 4˚C, washed with PBS, stained with Alexa Fluor 488-labeled goat anti-rabbit immunoglobulin G (IgG) (H+L) (1:500) for 1 h at room temperature, and the nuclei were stained with DAPI (5 μg/mL) in the dark. A Leica inverted phase contrast microscope (Wetzlar, GER) was used to examine and analyze the transfer rate of AIF into the nuclei.

Statistical Analysis
Data from the present study are presented as mean±SD from at least three independent experiments with different batches of cells, and each one was performed in duplicate or triplicate. Statistical comparisons were made using one-way analysis of variance (ANOVA) (Scheffe's F test) after ascertaining the homogeneity of variance between the treatments. All statistical data were analyzed using SPSS 19.0 (SPSS, Chicago, IL, USA). The critical value for statistical significance was P<0.05.

Cadmium induces mitochondrial oxidative stress and dysfunction in rPT cells
We detected intracellular superoxide accumulation in cadmium-treated rPT cells. The cells were stained with DCFH-DA, a superoxide-specific dye, after 12-h cadmium treatment ( Fig 1A). Cadmium significantly increased the intracellular superoxide levels, which co-treatment with 100μM N-acetylcysteine (NAC) eliminated ( Fig 1B). Next, we examined the changes in MPTP opening and mitochondrial morphology following cadmium treatment. Fig  1C and 1D shows that reduced mitochondrial calcein fluorescence represented MPTP opening and it was dose-dependent during cadmium exposure. The cadmium-induced changes in mitochondrial morphology were assessed by transmission electron microscopy. The morphological changes were typical of mitochondrial damage, i.e., swelling, rupture of the outer membrane, and distorted cristae (disruption or loss); the severity of the damage increased with the cadmium dose (Fig 1E). Fig 2A and 2B show that co-incubation with CsA (an MPTP inhibitor) significantly reversed cadmium-mediated MPTP opening. The mitochondrial calcein fluorescence drastically increased from 35.7% (2.5 μM cadmium alone) to 83.6% (cadmium+CsA). However, CsA alone had no effect on MPTP opening. Annexin V/PI staining was used to determine the apoptotic cells after 12-h cadmium exposure. Fig 2C and 2D show that 2.5 μM cadmium significantly enhanced the number of apoptotic cells (early and late), being 2.84-fold that of the control. CsA co-treatment significantly prevented apoptosis in the treated cells, being 1.37-fold that of the control.

Effect of CsA on MPTP and apoptosis
3.3. Cyt c release and caspase-9 and caspase-3 activation as a measure of the caspase-dependent apoptotic pathway Fig 3A show that immunoblotting indicated significant mitochondrial cyt c release to the cytoplasm after 12-h cadmium exposure. In addition, quantification (Fig 3B) demonstrated that cadmium induced cyt c release dose-dependently. Cyt c released into the cytoplasm activates caspase-9. Cadmium increased cleaved caspase-9 protein expression dose-dependently; Cleaved caspase-3 is an execution protein in apoptosis; cadmium also increased its expression dose-dependently (Fig 3C and 3D). These results confirm that the caspase-dependent pathway is involved in cadmium-induced apoptosis in rPT cells.

BNIP-3 is involved in the caspase-independent apoptotic pathway and induces mitochondrial AIF and Endo G nuclear translocation
Immunoblotting demonstrated that BNIP-3 protein levels were increased, as was mitochondrial AIF and Endo G translocation to the nucleus, dose-dependently after 12-h cadmium exposure (Fig 4A-4F). Fig 4G shows that AIF staining exhibited a granular pattern in the cytosol of the control group and was restricted mainly to the nucleus, as indicated by colocalization with DAPI labeling after 12-h cadmium exposure. Immunoblotting showed that mitochondrial AIF and Endo G translocation to the nucleus was decreased after BNIP-3 knockdown (Fig 4H-4M). The results confirm that BNIP-3 is involved in the caspase-independent apoptotic pathway and causes AIF and Endo G nuclear translocation.

Influence of caspase inhibitor on the caspase-independent apoptotic pathway
Cadmium (2.5 μM) and Z-VAD-FMK (20 μM, a caspase inhibitor) co-treatment decreased cleaved caspase-3 protein expression (Fig 5A and 5B). Flow cytometry revealed a significantly decreased apoptotic rate in the co-treatment group (Fig 5C and 5D). Immunoblotting showed that BNIP-3 protein levels (Fig 6A and 6B) and AIF transfer levels (Fig 6C) in the co-treatment group were significantly decreased compared with the cadmium-only group.

Influence of BNIP-3 silencing on the caspase-dependent apoptotic pathway
The earlier results confirmed that BNIP-3 is involved in the caspase-independent apoptotic pathway and induces mitochondrial AIF and Endo G translocation to the nucleus. BNIP-3 silencing blocked the caspase-independent pathway in cadmium-induced apoptosis, and clearly decreased the apoptotic rate (Fig 7A and 7B). Immunoblotting showed significantly decreased mitochondrial cyt c release to the cytoplasm after BNIP-3 knockdown (Fig 7C and  7D) and decreased cleaved caspase-9 and caspase-3 expression levels (Fig 7E and 7F).

Discussion
Using rPT cells as an in vitro model, we demonstrate that cadmium preferentially induces mitochondrial oxidative stress, dysfunction, MPT, and apoptosis. Oxidative stress promotes apoptosis in primary rPT cell cultures exposed to cadmium [5,25] and exploring the role of the mitochondrial apoptosis pathway, the caspase-dependent and caspase-independent apoptotic pathways, and the relationship between the two in rPT cell apoptosis, is necessary.
Cadmium induces rPT cell apoptosis, in which oxidative stress plays a pivotal role [25]. Our results showed that cadmium-induced preferential mitochondrial superoxide accumulation leads to mitochondrial dysfunction, which NAC prevented. ROS can directly result in MPTP opening, which facilitates MPT induction [3], the closing or opening state of MPTP, enabling tight regulation of mitochondria-mediated apoptosis [28]. Our results reveal that cadmium (a, c) Representative western blots of cyt c, cleaved caspase-9, and cleaved caspase-3. (b, d) Quantitative analysis of cyt c, cleaved caspase-9, and cleaved caspase-3 western blots; grayscale of the control was set at 1. Quantitative analysis was performed with images from three independent experiments (mean ± SD, n = 3). *P < 0.05, **P < 0.01, and ## P < 0.01 as compared to control. can lead to morphological changes typical of mitochondrial damage, including matrix swelling, outer membrane rupture, and distorted cristae. Cadmium also induced MPTP opening, triggering the release of apoptogenic proteins into the cytosol, which CsA prevented. In the cadmium and CsA co-treatment group, the rate of rPT cell apoptosis was prevented partially as compared to the cadmium-only group. Therefore, the cadmium-induced oxidative stressinduced MPTP opening, which triggers apoptogenic factor release, was halted.
The release of mitochondrial pro-apoptotic proteins such as cyt c (caspase-dependent), and AIF and Endo G (caspase-independent) strengthened the occurrence of multiple apoptotic pathways. Caspase-9 activation subsequently activates caspase-3, and requires cyt c for apoptosome formation [29,30]. We found that oxidative stress induced cyt c release and caspase-9 and caspase-3 activation, and ultimately led to apoptosis, which is in accordance with previous research on different cell types [31][32][33][34]. A similar study indicated that the use of 10 μmol/L of Cd induces cyt c release after 24 hours of Cd treatment [35]. Compared with our results, they used a lower dose and shorter time that induced cyt c release may due to primarily cultured cells was more sensitive to Cd. Intracellular zinc (Zn) depletion also induce apoptosis and shown as loss of ΔC, release of cyt c and activation of caspase-9 and caspase-3 [36]. This similar phenomenon may due to Zn from the zinc enzyme could be replaced by Cd and loss of function. Meanwhile, Zn is a part of the antioxidant defence system. Zn depletion will lead to oxidative stress which also induced by Cd.
AIF is a mitochondrial protein that translocates to the cytosol and the nucleus, mediating caspase-independent apoptosis in a number of model systems [37][38][39][40]. Endo G participates in mitochondrial DNA copying, recombination, and repair [41]; induced by oxidative stress, it translocates from the mitochondria to the nucleus [42]. Endo G released from the mitochondria interacts with AIF in the nucleus and is involved in caspase-independent apoptosis in Caenorhabditis elegans [43,44]; in neurodegenerative disease, both Endo G and AIF expression levels are decreased in the mitochondria but are increased in the nuclei [45]. AIF cannot cut DNA; it is possible that both Endo G and AIF are involved in nuclear DNA degradation [44,46]. BNIP-3 is a BH3-only pro-apoptotic member of the Bcl-2 family: it mediates cell death via different pathways, including the mitochondrial pathway [47]. Endoplasmic reticulum-targeted BNIP-3 induces cell death that the anti-apoptotic protein Bcl-2 can block. Mitochondria-targeted BNIP-3 initiates apoptosis, inducing MPT and mitochondrial membrane potential dissipation [48], and Bcl-2 expression cannot prevent it [47]. BNIP-3 and AIF cooperate to induce apoptosis and cavitation in epithelial morphogenesis [16]. Meanwhile,  silencing BNIP-3 prevents Endo G translocation and DNA degradation [49]. We found that BNIP-3 protein levels and AIF and Endo G translocation were increased during cadmiuminduced rPT cell apoptosis, while BNIP-3 silencing decreased AIF and Endo G translocation. In short, BNIP-3 is involved in the caspase-independent apoptotic pathway and it is located upstream of AIF/Endo G.
Under different stimuli, PARP-1 [poly (ADP-ribose) polymerase-1] activation triggers mitochondrial AIF release and translocation to the nucleus [50][51][52]. PARP-1 activation produces PAR in the nucleus, which is released into the cytosol and colocalizes with the mitochondria to induce AIF release [53]. The mature AIF is loosely bound on the mitochondrial outer membrane [54], from which PAR can detach it [55]. Others have reported that without caspase activity, AIF/Endo G still translocates to the nucleus under cadmium induction [6,56]. Consequently, the relationship between the caspase-dependent and caspase-independent apoptotic pathways is controversial due to the uncertainty as to whether there is a site upstream of AIF/Endo G. We show that BNIP-3 is involved in the caspase-independent apoptotic pathway during cadmium treatment and that it is located upstream of AIF/Endo G. BNIP-3 silencing inhibits mitochondrial cyt c release to the cytosol and caspase-dependent apoptosis in embryoid body differentiation [57]. BNIP-3 overexpression or recombinant BNIP-3 treatment of isolated mitochondria induce MPT and cyt c release in fibroblasts [48,58]. We obtained similar results in cadmium-induced rPT cells. Here, the cadmium and Z-VAD-FMK co-treatment group had significantly decreased BNIP-3 protein levels, and apoptosis was significantly prevented, as expected. This proves that the caspase-dependent apoptotic pathway affects the caspase-independent apoptotic pathway. Z-VAD-FMK co-treatment revealed that the two pathways play a similar role (co-promotion or co-suppression), acting synergistically in cadmium-induced rPT cell apoptosis.
The part data was also observed in programmed necrosis induced by Cd. Therefore, we detected level of intracellular ATP and expression level of HMGB1 in the cytoplasm of Cdtreated rPT cells. Mitochondrial ATP production is essential for maintaining ΔC and preventing apoptosis [59]. In this study, decreased ATP level (S1 Fig) indicated that abnormal cellular energy metabolism promoted Cd-induced apoptosis in rPT cells; and it was not detected any HMGB1 in the cytoplasm (date not shown). Thus, rPT cells experienced apoptosis rather than programmed necrosis during Cd exposure.
In summary, BNIP-3 acts as an upstream factor in the caspase-independent apoptotic pathway to induce AIF/Endo G translocation. Cadmium activates both the caspase-dependent and caspase-independent apoptotic pathways in rPT cells, and inhibiting one restrains the other. That is, the caspase-dependent and caspase-independent apoptotic pathways are complementary in cadmium-induced rPT cell apoptosis.