Activation of Ca2+-sensing receptor as a protective pathway to reduce Cadmium-induced cytotoxicity in renal proximal tubular cells

Cadmium (Cd), as an extremely toxic metal could accumulate in kidney and induce renal injury. Previous studies have proved that Cd impact on renal cell proliferation, autophagy and apoptosis, but the detoxification drugs and the functional mechanism are still in study. In this study, we used mouse renal tubular epithelial cells (mRTECs) to clarify Cd-induced toxicity and signaling pathways. Moreover, we proposed to elucidate the prevent effect of activation of Ca2+ sensing receptor (CaSR) by Calcimimetic (R-467) on Cd-induced cytotoxicity and underlying mechanisms. Cd induced intracellular Ca2+ elevation through phospholipase C-inositol 1, 4, 5-trisphosphate (PLC) followed stimulating p38 mitogen-activated protein kinases (MAPK) activation and suppressing extracellular signal-regulated kinase (ERK) activation, which leaded to increase apoptotic cell death and inhibit cell proliferation. Cd induced p38 activation also contribute to autophagic flux inhibition that aggravated Cd induced apoptosis. R-467 reinstated Cd-induced elevation of intracellular Ca2+ and apoptosis, and it also increased cell proliferation and restored autophagic flux by switching p38 to ERK pathway. The identification of the activation of CaSR-mediated protective pathway in renal cells sheds light on a possible cellular protective mechanism against Cd-induced kidney injury.


Detection of expressions of CaSR in renal cells and R-467 prevented Cd-induced renal cells death and cytotoxicity.
To detect the expressions of CaSR in renal cells (i.e. mRTEC and HK-2 cells), western blotting (Fig. 1a) and immunofluorescence (Fig. 1b) were performed and the results showed higher expression of CaSR in mRTECs than HK-2 cells. The results of MTT assay (Fig. 1c) and LDH cytotoxicity assay (Fig. 1d) showed Cd induced cytotoxicity of renal cell mRTEC in a dose dependent manner from 1 μM, and LD50 (lethal dose, 50%) was around 5 μM that was used in the following experiments. However, Cd induced cytotoxicity were significantly prevented when co-treated with calcimimetic R-467, but not with S-467 in mRTEC cells (Fig. 1e,f). And consistent results were also detected in HK-2 cells (Fig. S1a-d). As a positive allosteric modulator of CaSR, calcimimetic R-467 (R-enantiomer) could enhance the sensitivity of activation of CaSR, but S-467 (S-enantiomer) is less activator. In hence, co-treatment of R-467 but not S-467 prevented Cd-induced cytotoxicity (Fig. 1e,f and Fig. S1c,d) and suggested that the protective effect was specific and effectively mediated by CaSR. It is noted that Cd could not affect the expressions of CaSR in mRTEC and HK-2 cells (Fig. S1e). Considering higher expression of CaSR in mRTEC cells, we chose mRTEC as cell model in the following experiments.

R-467 increased PLC activity and reinstated Cd-induced intracellular Ca 2+ level elevation.
To confirm whether Cd induced elevation of intracellular Ca 2+ level from Ca 2+ store in ER through PLC-IP 3 pathway in mRTEC cells, we measured PLC activity and intracellular Ca 2+ levels with or without the inhibitors, including U73122 (inhibitor of PLC), 2-APB (inhibitor of IP 3 R), and BAPTA (intracellular Ca 2+ chelator). Cd induced activation of PLC activity (Fig. 2a) and Cd induced intracellular Ca 2+ level elevation in mRTEC cells was suppressed by the inhibitors, U73122 and 2-APB, BAPTA, and R-467 (Fig. 2b). The results were consistent with previous study that Cd induced elevation of intracellular Ca 2+ level from Ca 2+ store in ER through PLC-IP 3 pathway. R-467 increased instead of decreased Cd-induced activation of PLC activity (Fig. 2a) but it could effectively restore intracellular Ca 2+ level (Fig. 2b), the mechanism was further studied in the following sections. In addition, Cd induced intracellular Ca 2+ elevation did not depend on CaSR, since it could not be blocked by CaSR antagonist NPS 2390 (Fig. 2b). Although block of PLC-IP 3 pathway and chelation of intracellular Ca 2+ inhibit Cd induced apoptosis (Fig. 2c), it did not prevent Cd to reduce cell viability (Fig. 2d), suggesting intracellular Ca 2+ signaling was important for cell and might more complex mechanisms were involved. R-467 reversed Cd-altered p38 and ERK signaling pathways. To further clarify the mechanism of R-467 reinstated Cd-induced intracellular Ca 2+ level, we studied the activation of MAPK pathway. The results showed Cd induced activations of p38 and JNK while suppressed ERK1/2 MAPK signaling pathways (Fig. 3a). Effects of calcimimetics R-467 and S-467 on the activations of MAPK signaling pathway suggested that R-467 prevented Cd-induced activations of p38, but not JNK1/2 (Fig. 3a). In addition, Cd-suppressed activation of ERK1/2 was restored back by R-467 (Fig. 3a). Application of the intracellular Ca 2+ chelator BAPTA indicated that Cd-induced elevation of intracellular Ca 2+ level resulted in activation of p38, but not JNK1/2 (Fig. 3b). Interestingly, Cd suppressed activation of ERK1/2 was also restored back in the presence of BAPTA (Fig. 3b). It suggested that R-467 reinstated Cd-induced intracellular Ca 2+ level to reverse Cd-altered p38 and ERK1/2 pathways. PLC inhibitor, U73122 eliminated the reverse effect of R-467 on Cd-suppressed activation of ERK, but did not alter R-467 reduced p38 activation (Fig. 3c). These results indicated that PLC activation was indispensable in reactivation of Cd-suppressed activation of ERK pathway. Cd-altered p38 and ERK signaling induced cytotoxicity. To investigate the roles of MAPK signaling pathway (i.e. p38 MAPK, JNK and ERK) in Cd-induced cell death, the specific inhibitors of these pathways, i.e. SB202190 (p38 inhibitor), SP600125 (JNK inhibitor) and PD98059 (ERK inhibitor) were applied. Inhibition of p38 and JNK reduced the expressions of cleaved caspase-3 (Fig. 4a) and decreased Cd induced cytotoxicity (Fig. 4b). It indicated that p38 and JNK pathways mediated Cd-induced apoptosis. In addition, ERK inhibitor, PD98059 did not alter R-467 reduced expression of cleaved caspase-3 in presence of Cd (Fig. 4a), which indicated R-467 could still prevent Cd-induced apoptosis when ERK activation was inhibited. However, PD98059 aggravated Cd-induced cytotoxicity in presence of Cd and R-467 (Fig. 4b), suggesting R-467-induced ERK activation played other roles in mRTEC cells. After treated with 0-20 μM Cd for 24 h, cell viability of mRTEC was evaluated by MTT assay. The condition mediums were collected for LDH cytotoxicity assay. Results are presented as mean ± SD (n = 4). (e,f) Effects of calcimimetics on Cd induced cytotoxicity in mRTEC. After treated with Cd (5 μM), R-467 (1 μM), S-467 (1 μM), Cd (5 μM) + R-467 (1 μM), or Cd (5 μM) + S-467 (1 μM) for 24 h, cell viability of mRTEC was evaluated by MTT assay. The condition mediums were collected for LDH cytotoxicity assay. Results are presented as mean ± SD (n = 4). *Statistical significance between control and treatments, or Cd treatment and co-treatment of Cd + R-467, *P < 0.05, using Student's t-test.  To further understand the function of R-467 altered MAPK pathway, we detected expression and localization of Ki-67, which is used as a marker to indicate cell proliferation [51][52][53] . As shown in Fig. 4c, in control, most of the Ki-67 staining was completely superimposition with nucleus, and some of them localized to the periphery of the nucleus those are in proliferation. Cd suppressed expression of Ki-67 and restricted their localization in the nuclei (Fig. 4c), suggesting Cd inhibited cell proliferation. Co-treatment of Cd and R-467 not only significantly increased expression of Ki-67, but also improved translocalization of Ki-67 out of the nucleus (Fig. 4c), suggesting more cell proliferation. However, co-treatment of Cd and R-467-increased expression and translocalization of Ki-67 were suppressed by ERK inhibitor, PD98059 (Fig. 4c). It suggested co-treatment of Cd and R-467 induced expression of Ki-67 through ERK activation, which was necessary for translocalization of Ki-67 but was suppressed by Cd. In addition, enhanced p38 activation restricted translocalization of Ki-67 and SB202190 pre-treatment (p38 inhibitor) increased Ki-67 expression and stimulated most of the Ki-67 translocated to the periphery or out of the nuclei in presence of Cd (Fig. 4c), suggesting cell proliferation. Interestingly, BAPTA treatment increased Ki-67 expression but restricted localization of Ki-67 in the nuclei (Fig. 4c), suggested intracellular Ca 2+ was necessary for translocalization of Ki-67 and cell proliferation. In hence, pretreatment of BAPTA could not increase cell proliferation, although enhanced ERK activation and suppressed p38 activation in presence of Cd (Fig. 3b). Taken together, these results indicated that increased ERK activation and suppressed p38 activation but without dysregulation of intracellular Ca 2+ homeostasis could stimulate cell proliferation when exposed to Cd, which were coincidently satisfied when co-treatment with R-467.

R-467 reduced Cd-evoked ROS generation, autophagic flux inhibition and apoptosis.
To understand the role of Cd-increase intracellular Ca 2+ , ROS generation in mRTECs was determined. Cd significantly evoked generation of ROS in mRTEC cells (Fig. S2a), which could be abolished by inhibitors, U73122 and 2-APB, and intracellular Ca 2+ chelator, BAPTA (Fig. 5a). This suggested Cd increased ROS generation by elevation of intracellular Ca 2+ level and depended on PLC-IP 3 pathway. To determine the roles of ROS generated by Cd exposure in Cd induced mRTEC cell death, TCP (ROS scavenger) and CQ (autophagy inhibitor) were applied. In Fig. S2b and Fig. S2c, it showed these two reagents reduced Cd-induced ROS generation but aggravated Cd-induced cell death. The rate of autophagic flux can be approximated by the amount of LC3-II with degradation of p62, whereas both of them are elevated when autophagic flux was impaired 6 . Cd increased the expressions of LC3-II and p62, which was aggravated by CQ (Fig. 5b). Addition of CQ could increase Cd-induced expression of cleaved caspase-3 (apoptosis marker) (Fig. 5b) and aggravate Cd induced cytotoxicity (Fig. S2b). These results indicated that Cd impaired autophagic flux, and increased apoptotic cell death. The pretreatment of TCP alleviated the Cd-induced accumulation of LC3-II and p62 (Fig. 5b), suggesting ROS could stimulate cell autophagy. However, scavenging of ROS by TCP aggravated instead of preventing Cd-induced cytotoxicity as show in Fig. S2b, suggesting that the cytotoxicity was not attributed to ROS generation and the induced autophagy signal under the exposure of Cd.
Co-treatment of R-467 eliminated Cd-induced generation of ROS (Fig. 5a), and R-467 effectively reducing Cd-increased of p62 accumulation and cleaved caspase-3 expression (Fig. 5c). This indicated that R-467 restored Cd-impaired autophagic flux and decreased Cd induced cell apoptosis, but which was not dependent on ROS generation. On the other hand, inhibition of p38 reduced the expressions of p62 but inhibition of JNK increased Cd-increased expression of p62 ( Fig. 5d), indicating p38 pathway mediated Cd-impaired autophagic flux, whereas inhibition of JNK aggravated Cd-impaired autophagic flux. ERK inhibitor, PD98059 alone or in presence of Cd enhanced expression of p62 (Fig. S2d), and it also reversed co-treatment of R-467-reduced expression of p62 (Fig. 5d). Together with the effects of Cd and R-467 on activation of MAPK (Fig. 3a), the results indicated that R-467 restored autophagic flux by reversing Cd-altered p38 MAPK and ERK activations.

Discussion
The expression of CaSR in renal proximal tubules had been report in rat [47][48][49] . The renal expression of CaSR plays important roles in calciotropic signals responsible for Ca 2+ homeostasis 54,55 . Altered CaSR expression and disruption of Ca 2+ homeostasis had been shown to be associated with renal insufficiency 56,57 . Previous studies suggested that Cd disrupted intracellular Ca 2+ homeostasis through reducing the influx of extracellular Ca 2+23,24 , or increasing Ca 2+ release from intracellular Ca 2+ store 22,25 and resulting in cell apoptosis of renal tubular cells 21,22 . In renal distal epithelial A6 cells, Cd disrupted intracellular Ca 2+ homeostasis through a divalent cation receptor (CSR) mediated PLC-IP 3 pathway 27 . Moreover, CSR was different from CaSR and CaSR agonist neomycin could diminish the effect of Cd on intracellular Ca 2+27 . However, the underlying mechanism and function of activation of CaSR on Cd-induced disruption of intracellular Ca 2+ homeostasis and Cd-regulated pathways were still unclear. Interestingly, our previous study in gill cells of Japanese eels indicated that activation of CaSR and it mediated PLC-ERK pathway could as a protective pathway to reduce Ca 2+ -induced cytotoxicity 50 . It naturally raised a question that whether there is crosstalk between CaSR and CSR for competition of PLC pathway. In present study, we investigated whether activation of CaSR affected Cd-induced cell death of renal proximal tubular cells. Calcimimetics as allosteric modulator of CaSR is currently being tested for the treatment of primary hyperparathyroidism, and CaSR-based therapeutics will likely be applicable to other disorders in which CaSRs are under-or overactive 58 . In this study, the R-enantiomer (R-467) was used, which is classified as a type II calcimimetic and functions as a positive allosteric modulator of CaSR to amplify receptor sensitivity to Ca 2+ or other full agonists 59 .
(10 μM) for 30 min, followed Cd treatment (5 μM) for 24 h, cell viability was evaluated by MTT assay. *Statistical significance between control and treatments, or Cd treatment and in presence of inhibitors, *P < 0.05, using Student's t-test and one-way ANOVA followed by Duncan's multiple range tests.
The S-enantiomer S-467 is known to be less active 60 . Activation of CaSR by R-467 effectively protected renal cells from Cd-induced cytotoxicity and reducing Cd-evoked disruption of intracellular Ca 2+ homeostasis, followed ROS generation, autophagic flux inhibition and apoptosis. The present study demonstrated that activation of CaSR could restore of the intracellular Ca 2+ homeostasis and related physiological disorders.
Previous studies had show that Cd disrupted intracellular Ca 2+ homeostasis, resulting in cell apoptosis in a variety of cells 9,[16][17][18][19] , including renal tubular cells 21,22 . One source of disruption of cadmium on intracellular Ca 2+ homeostasis was through increasing Ca 2+ release from ER store mediated by PLC-IP 3 pathway 18 . Our data confirmed this notion by pretreatment of U73122 and 2-APB, inhibitors of PLC and IP 3 receptor respectively, inhibited Cd-induced elevation of intracellular Ca 2+ levels in mRTEC. However, inhibition of PLC and IP 3 receptor by U73122 and 2-APB, and chelation of intracellular Ca 2+ by BAPTA could not inhibited Cd-induced cytotoxicity of mRTEC. It indicated that there should be other pathways mediated Cd-induced cytotoxicity and the underlying mechanism is more complex than our speculation. On the other hand, our results suggested that activation of , or pre-treated with BAPTA (10 μM) for 30 min, followed Cd treatment (5 μM) for 24 h, total proteins were extracted for western blotting analysis. Activations of and p38 MAPK and JNK represented by in vitro phosphorylations of p38 MAPK and JNK and showed significantly increase in response to Cd treatment. R-467 suppressed Cd-induced activation of p38 MAPK, but not JNK. Activation of ERK was decreased by Cd treatment, but it was increased when co-treated with R-467. BAPTA suppressed Cd-induced activation of p38 MAPK, but not JNK, and BAPTA increased Cd-suppressed activation of ERK. Total p38 MAPK, JNK and ERK1/2 served as loading control. (c) Inhibition of PLC on activation of MAPK pathway. Cells pretreated with U73122 (1 μM) for 30 min, followed by Cd treatment (5μM) or co-treated with R-467 (1 μM) for 24 h, total proteins were extracted for western blotting analysis of expression of phosphorylation of ERK, p38 and JNK. Total ERK1/2, p38 and JNK served as loading control. *Statistical significance between control and treatments or Cd treatment and in presence of inhibitors or calcimimetics, *P < 0.05, using Student's t-test and one-way ANOVA followed by Duncan's multiple range tests. CaSR by R-467 reduced Cd-evoked disruption of intracellular Ca 2+ homeostasis in mRTECs, which was similar with the effect of CaSR agonist neomycin on Cd-increased intracellular Ca 2+ in renal distal epithelial A6 cells 27 . The co-treatment of Cd + R-467 increased even higher PLC activity than Cd, but they displayed reverse effects on intracellular Ca 2+ level. However, what elements decide the downstream effectors and fate of PLC pathway needs further study.
Furthermore, previous study suggested Cd-induced intracellular Ca 2+ elevation resulted in induction of ROS, triggering cell apoptosis 19,61 . Pretreatment with BAPTA attenuated Cd-induced ROS in the neuronal cells 19,61 . In this study, our results confirmed that Cd induced elevation of intracellular Ca 2+ level resulted in ROS generation in mRTEC. Notably, previous studies also had indicated that Cd induced autophagy through ROS-activated pathways 22,62 . Cd recruited Ca 2+ and ROS to act as 2nd messengers to control key Ca 2+ -and redox-sensitive molecular switches dictating cell function and fate. Severe ROS/Ca 2+ signals activate cell death, whereas low localized Ca 2+ and ROS levels promote cellular adaptation and survival 63 . In addition, ROS promoted autophagic flux serves as a cell survival mechanism to protect cell death [64][65][66] . The data in present study showed Cd induced generation of ROS and intracellular ROS in mRTEC cells stimulated autophagy, but Cd impaired autophagic flux (accumulation of p62), which contributed to apoptotic cell death. It suggested Cd induced ROS generation and autophagy signal as passive responses of mRTEC cells to promote adaptation and cell survival in Cd exposure. Autophagy is considered as an adaptive and protective mean against Cd-induced damage and dysfunction. Hence, inhibition of autophagic flux, such as by CQ, aggravated Cd-induced apoptosis. More importantly, activation of CaSR by R-467 could eliminate Cd-induced generation of ROS, but restore Cd-impaired autophagic flux (reduced expression of p62) and reduce Cd-induced apoptosis (reduced expression of cleaved caspase-3).
To further understand the molecular mechanisms of Cd-induced cytotoxicity and the protective roles of action of R-467, we detected the role of MAPK pathway. Previous studies had indicated Cd regulated activation of MAPK pathway (i.e. JNK, p38 and ERK) and induced cell apoptosis 35,36,67,68 . The JNK pathway usually mediates stress response and apoptosis [69][70][71] , and p38 activation was indicated to be involved in suppressing autophagy but promoting apoptosis [72][73][74][75] . Conversely, ERK is activated to induce cell proliferation 76 or promote cellular adaptation and survival 63 . Our results showed Cd disrupt autophagic flux by p38 activation, which was consent with previous studies. In addition, the results indicated Cd induced apoptotic cell death through activation of p38 MAPK and JNK pathways, and Cd inhibited cell proliferation through reducing activation of ERK. It agreed with the effects of Cd on activation of MAPK in the human non-small cell lung carcinoma cell line, CL3 36 , human osteosarcoma cell line, MG63 77 , and human renal proximal tubular cells, HK-2 12 . In rat pheochromocytoma (PC12), human neuroblastoma (SH-SY5Y) cell lines and primary murine neurons, intracellular Ca 2+ signaling mediated Cd-induced neuronal apoptosis via induction of activation of MAPK 61 . In mesangial cells, Cd-stimulated Ca 2+ release from the endoplasmic reticulum induced activation of ERK, which leaded to predominantly autophagic cell death and a minor level of apoptotic cell death and resulted in nephrotoxicity 78 . In present study, the results suggested that in mRTEC cells, Cd-induced activation of p38 MAPK depended on intracellular Ca 2+ signaling, while Cd-induced activation of JNK was intracellular Ca 2+ independent manner. Pretreatment with BAPTA attenuated Cd-reduced activation of ERK. It indicated that different MAPK families mediated Cd-induced intracellular Ca 2+ signaling in different cell types. Our previous study had suggested CaSR mediated PLC-ERK pathway to regulate calcium transport in gill cells 50 . Here, the results indicated that activation of CaSR by R-467 induced activation of PLC-ERK pathway while reduced activation of p38 MAPK, which was similar with effects of BAPTA on activation of p38 MAPK and ERK. It was notable that although both R-467 and BAPTA could restore intracellular Ca 2+ level, p38 and ERK activations, R-467 instead of BAPTA prevent Cd-induced cytotoxicity and proliferation inhibition in mRTEC cells, suggesting R-467 might play important role in restoring back of intracellular Ca 2+ to ER store or induce other pathways to stimulate cell proliferation that need further studies. Through reducing activation of p38 MAPK and increasing activation of ERK pathway, R-467 restored autophagic flux and reduced apoptotic cell death and increased cell proliferation. The results here indicated that activation of CaSR by R-467 could switch Cd-activated PLC-Ca 2+ -p38 MAPK to activate PLC-ERK pathway. However, it cannot rule out other possible mechanisms, such as R-467 might also stimulate other receptors to prevent Cd-induced cytotoxicity or alter the binding affinity of Cd onto its receptor CSR, or there might be interaction or cross-talking between the receptors (CSR and CaSR) and their other downstream effectors which were altered by R-467, and how can R-467 switch Cd-activated PLC-Ca 2+ -p38 MAPK to activate PLC-ERK pathway and what key elements decide the fate of PLC activation are still unclear. Therefore, the clarification of other pathways and deep mechanisms behind the protective effect of R-467 need further studies.
In summary, the results of this study demonstrated that Cd induced Ca 2+ release from ER store through PLC-IP 3 pathway in mRTEC cells, and elevation of intracellular Ca 2+ level leaded to ROS generation. Cd-induced elevation of intracellular Ca 2+ also leaded to p38 activation, which impaired autophagic flux, stimulated apoptosis and suppressed cell proliferation. In addition, Cd induced apoptosis through JNK activation, and inhibited cell proliferation through suppression of ERK activation. Activation of CaSR by R-467 reinstated intracellular Ca 2+ level and ROS generation, restored Cd-impaired autophagic flux and reduced Cd-induced apoptosis, and increased cell proliferation by competing with Cd for PLC to switch Cd activated p38 MAPK to R-467 activated PLC-ERK pathway (Fig. 6). The identification of the activation of CaSR-mediated protective pathway in mRTEC cells sheds light on a possible cellular protective mechanism against Cd-induced kidney injury. In some experiments, cells were either untreated or pre-treated with kinase inhibitors for 30 min, i.e. U73122 (1 μM), 2-APB (50 μM), BAPTA/AM (10 μM), PD98059 (10 μM), SB202190 (10 μM), SP600125 (10 μM), TCP (100 μM) and CQ (20 μM). After 24 hours, the culture mediums were collected for cytotoxicity analysis, and the cell viability was measured by MTT assay. In some experiments, cell lysates were collected for western blotting analysis.

Measurement of cell growth and cytotoxicity assay.
After treated for 24 h, the culture mediums were collected to measure the activity of lactate dehydrogenase (LDH) using the LDH Cytotoxicity Assay Kit (Cayman) according to the manufacturer's instructions. The absorbance at 490 nm was detected using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, USA). Cell growth was measured by the MTT (3-(4,5-dimethyl-2-thiaz olyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay. In brief, cells were incubated in 100 μL MTT solution (0.5 mg/ml in RPMI 1640 medium) in 96-well plate for 4-h before the end of incubation. The supernatant was then discarded, and 100 μL DMSO was added to dissolve the colored product (formazan). The absorbance was measured at 540 nm (690 nm as reference) using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, USA).
PC-PLC assay. The mRTEC cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris of pH 7.4, 150 mM NaCl, 1% NP40) at 1, 2, 5 min post-treatment with CdCl 2 (5 μM), or 2 min post-treatment with R-467 (1 μM), and R-467 (1 μM) + CdCl 2 (5 μM). After centrifugation at 13 000 g for 15 min at 4 °C, the supernatant was collected and the total protein concentration was determined by the Bradford method (Bio-Rad). The protein lysates (15 μg/sample) were analyzed for PC-PLC activity using an Amplex Red PC-PLC assay kit according to the manufacturer's instruction (Molecular Probes, Eugene, OR, USA). The reactions were incubated in darkness at 37 °C for 1 h. The fluorescence was measured with a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, USA) with excitation at 530 nm and emission at 590 nm.

Measurement of intracellular calcium.
To visualize the effect of Cd on intracellular Ca 2+ levels in renal cells, mRTEC cells were seeded at a density of 2 × 10 4 cells/well in 24-well plates. Next day, the cells were loaded with 1 μM Fluo-3/AM in the RMPI 1640 medium (phenol red free) for 30 min at 37 °C in the dark. After dye loading, the cells were washed twice with the medium. Then, the cells were pre-treated with inhibitors for 30 min, i.e. U73122 (1 μM), 2-APB (50 μM), and BAPTA/AM (10 μM), then added CdCl 2 (5 μM) or CdCl 2 (5 μM) + R467 (1 μM) for 1 hour. Finally, calcium imaging was observed by Olympus IX73 microscopy (Japan).
Immunofluorescence. To detect the expression of Ki-67, the mRTEC cells were treated with CdCl 2 (5 μM) with or without R-467 (1 μM), or S-467 (1 μM) for 24 h, or pre-treated with PD98059 (10 μM), SB202190 (10 μM) for 30 min. The cells were fixed for 30 min in 4% Formaldehyde (FA, Sigma-Aldrich). Then, the cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 20 min. After blocked with 3% normal goat serum, the cells were incubated with mouse anti-Ki-67 (1:100, Cell Signaling Technology) antibody overnight at 4 °C, followed by 1 h incubation with Alexa Fluor 488 goat anti-mouse IgG (1:200, Molecular Probes, Invitrogen). After washing twice with PBS, cell nucleus was stained by the DAPI (Invitrogen) for several minutes. The cells were washed in PBS for 10 min and the cells were mounted, then examined by Olympus IX73 microscopy (Japan). To detect the expression of CaSR (anti-CaSR, 1:40, Thermo fisher Scientific) on the membranes of HK-2 and mRTEC cells, the process was performed without the permeabilization step.
Statistical analysis. Drug treatments were performed in triplicate in each experiment and every experiment was repeated at least three times. All data are represented as means ± s.e.m. Statistical significance was assessed with Student's t-test or one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests. Groups were considered significantly different if P < 0.05.