Adaptive Response of HK-2 Cells to High Levels of COM Crystals Highlights CaSR as an Enhancer of Crystal Adhesion

Background: Calcium oxalate monohydrate (COM) is an aetiologic factor for urolithiasis. However, how Human Kidney-2 (HK-2) cells respond to a high COM has not yet been completely elucidated. Materials and methods: A gel-based proteomics approach was applied to investigate COM-induced cellular proteomic changes. The COM-induced upregulation of calcium-sensing receptor (CaSR) in HK-2 cells was studied. Surface phospholipids (PS), which play a role in urolithiasis formation by mediating adhesion of HK-2 cells, were labelled in the inner or outer leaet of the plasma membrane of HK-2 cells with uorescent nitrobenzoxadiazole (NBD) to form NBD-PS to detect transmembrane movements of PS. After labelling, HK-2 cells were exposed to COM in the presence of the CaSR-specic agonist gadolinium chloride (GdCl 3 ) or the CaSR-specic antagonist NPS2390. Inward and outward transmembrane movements of PS were tracked with a uorescence quenching assay. Surface-expressed PS was detected by an annexin V binding assay. Changes in aminophospholipid translocase (APLT), oxidative stress (OS), levels of apoptosis-related proteins in HK-2 cells and crystal adhesion were also assessed. Results: COM increased CaSR and surface-expressed PS levels, decreased APLT activity, impaired inward transport of PS, and enhanced outward transport of PS. However, pretreatment with GdCl 3 further effectively inhibited the inward movement of PS and APLT activity and increased surface-expressed PS levels compared with COM treatment alone. In contrast, NPS2390 promoted the inward movement of PS and APLT activity and decreased surface-expressed PS levels compared with COM treatment alone. COM increased OS, apoptosis of HK-2 cells and crystal adhesion onto cells, and this increase was further enhanced by GdCl 3 pretreatment but attenuated by NPS2390 treatment. Conclusions: These results After reaction, harvested and labelled with V-FITC in Annexin V binding buffer (BD We used a FACSAria™ ow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 530 nm to evaluate the annexin V uorescence of the cells after 15 min of incubation in the dark. The percentage of annexin V-positive cells was used to measure PS exposure. COM stimulation signicantly increased the expression of Bax, cleaved caspase-3, pro-caspase-8, cleaved caspase-8, caspase-9, cleaved caspase-9 and cytochrome C proteins and reduced Bcl-2 expression. Compared with the COM group, this effect was reversed by NPS2390 but enhanced by GdCl 3 . Therefore, COM stimulation affects the caspase cascade, and CaSR induces renal HK-2 apoptosis via the caspase-independent pathway.


Background
Urolithiasis is mainly formed by calcium oxalate (CaOx) and is often associated with potential metabolic disorders such as hypercalciuria, hyperoxaluria and hypocitraturia [1]. The formation of CaOx stones usually begins with nucleation, growth and aggregation of crystals. In addition, adhesion of CaOx crystals to renal tubular epithelial cells is critical.
Studies of the genesis of kidney stones based on intracellular CaOx levels have found that CaOx monohydrate (COM) crystals and/or oxalates can adhere directly to living tubular epithelial cells under appropriate conditions [2,3]. Because of the nature of the crystal binding sites on the top surfaces of cells, the plasma membrane promotes their adhesion [4], but the mechanism remains unclear. The COM-induced presence of high levels of surface phospholipid (PS) in the extracellular lea ets of the renal epithelial cell membrane may contribute to the retention of COM crystals on the cell surface [5,6]. However, to the best of our knowledge, the mechanism by which COM promotes the collapse of renal epithelial membrane phospholipid asymmetry is unclear.
In all mammalian cell types studied thus far, the 2 lea ets of the plasma membrane bilayer have an asymmetrical phospholipid distribution. PSs, aminophospholipids and most phosphatidylethanolamines are concentrated in the cytoplasmic (inner) lea et, while choline phospholipids, phosphatidylcholines and sphingomyelins are predominantly located in the exoplasmic (outer) lea et [7]. This asymmetrical distribution of phospholipids is established and maintained by membrane proteins, which transport phospholipids between the cytoplasmic and exoplasmic lea ets.
Three types of phospholipid transporters have been identi ed to play roles in the regulation of membrane lipid sidedness. First, ATPdependent aminophospholipid translocase (APLT), also known as ippase, mediates the localization of PS and phosphatidylethanolamine in the inner lea et by rapidly transporting these substances from the outer to the inner lea et of the plasma membrane against their respective concentration gradients. Second, the ATP-dependent phospholipid oppase slowly moves phospholipids from the inner to the outer lea et. In addition to these 2 ATP-requiring transporters, the ATP-independent, Ca 2+dependent scramblase randomly transports all phospholipids bidirectionally. Phospholipid asymmetry in biological membranes is maintained by the activity of the rst 2 transporters and by scramblase inactivation [8,9].
However, during the formation of kidney stones, multiple molecules and active proteins participate in complex interactions between COM crystals and renal epithelial cells. Most previous studies have focused only on one or two genes or proteins with varying results, so understanding of the changes in protein expression that occur after oxalate and/or COM exposure and how they participate in stone formation is limited. In addition to the proteins that have been studied, we hypothesize that other candidate proteins may also be involved in regulating the asymmetric distribution of phospholipids and the formation of renal stones in renal epithelial cells.
In our study, we investigated global protein changes in HK-2 cells exposed to a high-COM environment using a gel-based proteomics approach followed by protein analysis. A total of 11 proteins were detected, and 10 proteins, including calcium-sensing receptor (CaSR), were identi ed as differentially expressed proteins. It has been reported that CaSR increases CaOx crystal adhesion [10], and extracellular PS may promote the adhesion of crystals on the cell surface of renal epithelial cells. Therefore, we selected CaSR for further study and hypothesized that this protein alters the distribution of PS by affecting the activity of APLT, resulting in adhesion of COM on renal epithelial cells. Since the phospholipid transporters catalyse unidirectional or bidirectional transport of lipids from one membrane lea et to the other, we monitored the inward and outward movement of PS. The exposure of phosphatidylserine (PS) on the outer plasma membrane is a unique feature of apoptotic cells. Together with other "eat me" signals, phosphatidylserine exposure enables the recognition and phagocytosis of dying cells (efferocytosis), helping to explain the immunologically silent nature of apoptosis [11,12].Since apoptosis is a common mechanism underlying the pathophysiology of urolithiasis, we also evaluated it in our study.
CaOx crystals lead to the activation of NADPH oxidase (NOX), production of ROS and increased expression of molecules such as osteopontin and monocyte chemoattractant protein-1 [13,14]. Because the injurious effects of COM on renal cells are caused by ROS and oxidative stress (OS) production [15,16], we also evaluated whether ROS and OS are regulated by CaSR. GdCl 3 (a speci c CaSR activator) and NPS2390 (a speci c CaSR antagonist) were used to evaluate the involvement of CaSR in adhesion of COM on renal epithelial cells and to explore the possible underlying mechanism.

Preparation of COM crystals
We prepared crystals as described previously [17]. COM crystals formed immediately as we mixed equal volumes of 10 mM CaCl 2 and 10 mM sodium oxalate at approximately 26°C and after approximately 3 days at 4°C. Deionized water was used to wash the COM, which was then dried at 62°C. Fourier transform infrared spectroscopy was used to con rm that the crystals were COM. A stock solution was created with 5 mg/ml COM in sterile phosphate-buffered saline (PBS). The COM was evenly distributed in a monolayer (67 μg of crystals/cm 2 of cells) and settled on the cells under the force of gravity. PBS was added to ensure that the volume was the same for each group.
Protein extraction, two-dimensional gel electrophoresis (2-DE) and staining After cultivating HK-2 cells with or without COM (67 μg of crystals/cm 2 of cells) for 24 h, we harvested the cells into tubes containing 0.5 M EDTA in PBS and incubated at 4°C for 30 min before removing the adherent crystals. Then, the cells were washed with PBS to remove EDTA, resuspended in lysis buffer, and incubated at 4°C for 30 min. The suspensions were then centrifuged to remove insoluble debris and particulate matter.
A total of 200 mg of protein derived from each sample was mixed with a rehydration buffer (8 M urea, 2% CHAPS, 18 mM DTT, 0.5% IPG buffer, trace bromophenol blue) to obtain a nal volume of 200 ml per sample. The samples were then rehydrated onto individual Immobiline DryStrips (7 cm long, nonlinear pH gradient of 3-10; GE Healthcare, Uppsala, Sweden) for 16 h at room temperature.
Isoelectric focusing (IEF) was performed at 500 V for 1 h, 1,000 V for 1 h, and 8,000 V for 10 h to reach a total of approximately 60-80 kVh. After IEF, the strips were placed in an equilibration buffer (130 mM DTT, 112 mM Tris-base, 6 M urea, 30% glycerol, 4% SDS, and 0.002% bromophenol blue) for 20 min and then incubated with a similar buffer (in which 130 mM DTT was replaced with 135 mM iodoacetamide) for 20 min. The proteins on the equilibrated strips were further separated on 12.5% polyacrylamide gels at 120 V for approximately 1.5 h. The proteins were detected by a modi ed silver-staining method compatible with MS analysis. Image analysis and protein identi cation by ESI-MS/MS We analysed differences in protein levels among samples with Image Master software. Following spot detection, a matched set including all three batches of gels was built. A reference gel was selected from the control gels, and unmatched spots were added to the reference gel. Normalization was based on the total spot density.
We excised differentially expressed protein spots from the preparative gels, destained them and digested them overnight. For protein identi cation, a Finnigan LTQ mass spectrometer coupled with a Surveyor HPLC system (ThermoQuest) was used to analyse the extracted peptides. Brie y, we separated the protein digests with Microcore RP columns (C18 0.15 mm*150 mm; ThermoHypersil, San Jose, CA, USA). Solvent A was 0.1% v/v formic acid, and solvent B was 0.1% v/v formic acid in 100% v/v acetonitrile (can). The gradient was held at 2% solvent B for 20 min and increased linearly to 98% solvent B in 1.5 h. The peptides were eluted from C18 microcapillary columns (120 μl per min) and electrosprayed directly into an LCQ-Deca mass spectrometer (with a spray voltage of 3.0 kV and a capillary temperature of 180°C). The full scan ranged from M/Z 400 to 2,000. Protein identi cation using the MS/MS raw data was performed with SEQUEST software based on the Swiss-Prot database. Both y ions and b ions were included in the database search. The results of protein identi cation were ltered by the DelCn (≥0.1) and Xcorr (1+≥1.9, 2+≥2.2, 3+≥3.75) values. The NCBI and ExPASy protein databases were used to determine the main functions of all the identi ed proteins.

Annexin V binding analysis
A FITC-labelled annexin V staining assay was used to measure the PS exposure of HK-2 cells. Cells were seeded in 6-well plates, grown to approximately 80% con uence (67 μg of crystals/cm 2 of cells) and treated with COM crystals with or without the CaSR activator GdCl 3 (300 μM) for 30 min or the CaSR inhibitor NPS2390 (10 μM) for 60 min. After the reaction, the cells were harvested and labelled with annexin V-FITC in Annexin V binding buffer (BD Pharmingen™). We used a FACSAria™ ow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 530 nm to evaluate the annexin V uorescence of the cells after 15 min of incubation in the dark. The percentage of annexin V-positive cells was used to measure PS exposure.

Measurement of MDA, LDH, and SOD levels
The levels of MDA, SOD, and LDH were measured using a commercial kit according to the manufacturer's instructions.
HK-2 cell labelling with nitrobenzoxadiazole (NBD)-labelled PS (NBD-PS) PS transmembrane bidirectional movement was observed with NBD-PS (Avanti® Polar Lipids), a uorescent analogue of phosphatidylserine. NBD-PS dissolved in chloroform was dried under nitrogen and solubilized in absolute ethanol (6 nmol for 2× 10 7 cells). We labelled 2 groups of cells. In one group, called group A, we inserted NBD-PS into the outer plasma membrane [18,19]; in the other group, group B, we inserted NBD-PS into the inner plasma membrane. We prevented internal movement in group A by cooling the cells to 2°C. Then, an ethanolic solution of NBD-PS was added to a suitable volume of cell suspension (approximately 2 × 10 7 cells per ml) in mPBS, and the mixture was vortexed. Non-inserted NBD-PS was removed by washing the cells with ice-cold mPBS after incubation on ice for 15 min. The group B cell suspensions were incubated with NBD-PS in mPBS at 37°C for 1 h with 5 mM diisopropyl uorophosphate (Sigma-Aldrich) to prevent NBD-PS degradation. Redistribution of NBD-PS to the cytoplasmic membrane lea et was mediated by APLT, which mediates the localization of PS and phosphatidylethanolamine to the inner lea et of the plasma membrane by rapidly transporting these substances from the outer to the inner lea et. Any NBD-PS remaining in the outer lea et was removed by washing the cells 3 times with mPBS containing BSA (1% w/v).

Western blot analysis
We prepared total proteins according to the kit manufacturer's instructions. A Bradford protein assay was performed to determine the supernatant protein concentration with BSA as the reference standard. We blotted all of the proteins (20 μg) onto a nitrocellulose membrane in transfer buffer at 100 V for 1 h in a water-cooled apparatus after electrophoresis in Tris-glycine electrophoresis buffer with standard 10% SDS-PAGE. The membrane was blocked in TBS-T buffer with 5% skimmed milk at 37°C for 1 h and then incubated overnight at 4°C with anti-CaSR antibodies (1:2,500), APLT antibodies (1:1,500), and antibodies against SOD, NOX, Bcl-2, Bax, procaspase-9, caspase-9, pro-caspase-8, cleaved-caspase-8, cleaved-caspase-3 and cytochrome C. TBS-T was used to wash the membrane 3 times. The membrane was then incubated with alkaline phosphatase-conjugated anti-IgG antibodies diluted 1:1,000 in TBS-T for 1 h at room temperature. Western Blue Stabilized Substrate for Alkaline Phosphatase was used to detect antibody-antigen complexes. A Bio-Rad ChemiDoc TM EQ densitometer and Bio-Rad Quantity One software were used to evaluate the protein band densities.
Crystal adhesion assay.
One of the methods used before was performed to carry out crystal adhesion experiments [20]. The cells were randomly divided into 4 groups after culture for 24 hours: (1) control group: cells were cultured in DMEM at 37°C; (2) COM group: cells were incubated with crystals (67 µg/cm2 of cells); (3) COM+GdCl3: GdCl3 (300 µM) was added to growth medium for half an hour, and then crystals were added; (4) COM+NPS2390 group: After NPS2390 (10 µM) was added to growth medium for 1 hour, the crystals were also added. After cells were treated with crystals for 6, 12, 24, and 48 hours, the unbound crystals were removed and collected. They were counted in 5 randomized high-power elds (HPFs) under a phase contrast microscope. This experiment was carried out in triplicate.

Statistical analysis
Quantitative data are presented as the mean ± SEM. All statistical analyses were performed using SPSS software version 13.0. ANOVA was used to analyse differences among multiple groups, and t tests were used to compare 2 groups. A 2-sided p<0.05 was considered to indicate statistical signi cance.

Proteomic analysis
Approximately 900 protein spots were visualized in each 2-D gel by spot matching. Quantitative intensity analysis and statistical analysis showed that the expression levels of only 11 protein spots (10 de ned proteins) in the high-COM exposure group were signi cantly changed by more than 1.5 times (including 9 upregulated proteins and 1 downregulated protein) (Fig 1). These changed protein spots were detected using ESI-MS/MS analysis. Table 1 summarizes the identi ed peptide numbers, protein scores, degrees of change (multiples) and other relevant information for all altered proteins (Table 1).

PS exposure
Since the uorescent molecule Annexin V-FITC binds to PS when it appears on the cell surface, it can be used to detect the redistribution of PS. HK-2 cells were exposed to COM crystals (67 µg of crystals/cm 2 of cells) for 24 h, and PS exposure was assessed using ow cytometry. The percentage of Annexin V-FITC-positive cells was greater in the COM group than in the control group (p < 0.05). This nding indicates that PS exposure on the HK-2 cell membrane surface increased under the action of COM. GdCl 3 further enhanced PS exposure, while NPS-2390 decreased it (p < 0.05), which suggests that CaSR helps to enhance PS exposure (Fig 2).
Measurement of LDH, SOD, and MDA levels The LDH and MDA levels in the COM group were signi cantly higher than those in the control group, but the SOD levels were signi cantly lower (p < 0.05). The levels of LDH and MDA were elevated under GdCl 3 treatment, while those of SOD were reduced (p <0.01). However, NPS2390 reduced the concentrations of LDH and MDA and increased those of SOD (p < 0.05) (Fig 3).

NBD-PS inward movement
Fluorescent NBD-PS was used to label outer membrane lea ets to verify whether COM crystals affected the inward transport of PS from the outer membrane lea et to the inner lea et, and its internalization was monitored over time ( Fig 4A). Approximately 90% of NBD-PS was internalized by HK-2 cells within 24 h (Fig 4B). However, under COM treatment (67 µg of crystals/cm 2 of cells), the percentage of internalized NBD-PS decreased to 48% (Fig 4B), which was lower than the control rate (p < 0.05). The inward transport rate of PS increased after the addition of the CaSR inhibitor NPS2390 to COM-treated cells (p < 0.05). Approximately 71% of NBD-PS was internalized, similar to the percentage observed in the control group. In contrast, the addition of the CaSR activator GdCl 3 to COMtreated cells signi cantly inhibited the inward transport rate of PS (p < 0.05) (Fig 4B).
We also preincubated cells with the APLT inhibitor N-ethylmaleimide (NEM, Sigma-Aldrich) (5 mM) for 15 min and tested the inward movement of NBD-PS. After NEM treatment, the intrinsic kinetics became similar regardless of the presence or absence of other agents and decreased compared to the control values (p <0.05, Fig 4B). These ndings indicate that the inward movement of PS in HK-2 cells is mediated by APLT activity and that CaSR can modulate APLT activity in COM lesions.

NBD-PS outward movement
We also used NBD-PS to label cells intracellularly, and we determined the degree of movement of NBD-PS to the outer lea et to investigate whether the outward transport of PS was affected by COM. The incubation medium contained BSA (1% w/v), which can quickly extract NBD-PS expressed on the cell surface, thereby eliminating interference with APLT-mediated inward transport. In control cells, only approximately 20% of NBD-PS migrated to the outer lea ets within 24 h (Fig 5B), which may have been due to normal oppase activity. However, COM crystal treatment (67 μg of crystals/cm 2 of cells) increased the PS outward transfer rate to 70%, which was signi cantly greater than the rate in control cells (p < 0.05). However, the addition of the CaSR activator GdCl 3 or the CaSR inhibitor NPS2390 did not signi cantly affect this abnormal process, suggesting that the mechanism by which COM induces PS outward transport may not involve CaSR (Fig 5B).

Western blot analysis
CaSR was expressed in each group (Fig 6A). CaSR expression was clearly upregulated in the COM group ( Fig 6A) compared with the control group. The expression of CaSR in the COM + GdCl 3 group was greater than that in the COM group (p < 0.05) (Fig 6B).
Furthermore, CaSR expression was lower in the COM + NPS2390 group than in the COM group (p < 0.05) (Fig 6B). After COM treatment, APLT and SOD expression were downregulated, and NOX expression was upregulated (p < 0.05).
Upregulation of APLT and SOD expression and downregulation of NOX were observed in the COM + NPS2390 group compared with the COM group (p <0.05). APLT and SOD expression were lower in the COM + GdCl 3 group than in the COM group. NOX expression, by contrast, was higher (p <0.05) (Fig 6B).
We screened and examined the levels of caspase-dependent and caspase-independent apoptosis-related proteins in cells with or without NPS2390 and GdCl 3 . As shown in Fig 6D, COM stimulation signi cantly increased the expression of Bax, cleaved caspase-3, pro-caspase-8, cleaved caspase-8, caspase-9, cleaved caspase-9 and cytochrome C proteins and reduced Bcl-2 expression. Compared with the COM group, this effect was reversed by NPS2390 but enhanced by GdCl 3 . Therefore, COM stimulation affects the caspase cascade, and CaSR induces renal HK-2 apoptosis via the caspase-independent pathway.

Crystal adherence to cells in groups
The results showed that compared to the COM group, the CaSR activator signi cantly decreased the number of unbound crystals, which also means that the CaSR activator increased the number of adherent crystals (Figure 7) (p < 0.05). In contrast, the CaSR inhibitor resulted in a reduction in adherent crystals (p< 0.05). Crystal adherence to cells exposed to varying times (6, 12, 24, or 48 hours) was also determined in the four groups. The response was time-dependent ( Figure 7). Figure 7 indicates that crystal adherence to HK-2 cells was the highest following 48 hours.

Discussion
COM is critical in the formation of CaOx stones and impairs tubular epithelial cells [21,22]. However, the understanding of the mechanism by which renal tubular cells respond to COM is still limited. Therefore, we performed a gel-based proteomics study to determine the protein changes that occur in renal tubular cells due to high COM exposure ( Fig. 1 and Table 1). The induction of 10 proteins by CaOx crystals was detected by 2-DE of HK-2 cells. These altered proteins are involved in several cellular functions.
One of the differentially expressed proteins was CaSR. There is increasing evidence that CaSR may be involved in the formation of kidney stones [23][24][25]. In our previous study, we found that activation of CaSR by COM may lead to crystal retention and renal insu ciency [10]. However, the exact mechanism by which CaSR mediates crystal adhesion is not clear. Studies have shown that the appearance of extracellular PS in renal epithelial cells may promote the adhesion of COM crystals on the cell surface [26]. Therefore, we speculate that the appearance of extracellular PS in renal epithelial cells may be promoted by CaSR.
In our study, we observed increased CaSR expression, PS exposure, apoptosis, ROS production and crystal adhesion in HK-2 cells after treatment with COM. Moreover, the CaSR inhibitor NPS2390 reduced ROS production, PS exposure, apoptosis and crystal adhesion, while the CaSR activator GdCl 3 signi cantly increased ROS production, PS exposure, apoptosis and crystal adhesion. To the best of our knowledge, we are the rst to demonstrate that COM-induced PS exposure and apoptosis are mediated by CaSR. In addition, we observed enhanced outward movement of NBD-PS after COM treatment. However, changes in CaSR activity did not signi cantly affect the outward movement of NBD-PS, suggesting that the PS outward movement caused by the interaction of COM with membrane lipids is not mediated by CaSR.
In addition to increased outward movement, we observed impaired inward movement of NBD-PS in COM-treated HK-2 cells, which may be due to reduced APLT activity. This nding indicated that the cells were unable to rapidly transport the externalized PS back to the inner lea ets of their plasma membranes. The inhibition of APLT activity mediated by COM was accompanied by increased cellular CaSR production and was attenuated by treatment with the CaSR inhibitor NPS2390 (or enhanced by treatment with the CaSR activator GdCl 3 ), implying that the COM-mediated decrease in APLT activity was dependent upon CaSR intermediates.
Injury to HK-2 cells, such as necrosis and apoptosis, is a common mechanism underlying the pathophysiology of urolithiasis [27]. HK-2 cell injury and apoptosis promote crystallization by providing substrates for heterogeneous crystal nucleation [28]. Cell degradation following HK-2 cell injury provides numerous membrane vesicles, which are good nucleators of crystals, promotes nucleation at low supersaturation, and enhances interactions between cells and crystals [29]. However, the detailed mechanisms by which COM promotes the apoptosis and necrosis of HK-2 cells are unknown. Many pro-apoptotic and anti-apoptotic proteins, such as Bcl-2, Bcl-xL, Bax and Bak, participate in the apoptosis process. Studies have narrowed the range of downstream factors to the caspase family. Ultimately, we selected caspase-8 and caspase-9 (the activated initiators of the apoptotic pathway) and caspase-3 (a key apoptotic protein).
Western blotting was used to analyse the levels of these members of the caspase family, and we found that COM-induced apoptosis depends on caspase-3, -8 and − 9 via activation of CaSR.
Studies have shown that oxalate-induced increased ROS generation impairs APLT activity and that decreased APLT activity has a role in hyperoxaluria-promoted CaOx stones by facilitating phosphatidylserine redistribution in kidney epithelial cells [30].
CaSR-mediated loss of APLT activity may be a key cause of COM-induced PS exposure in renal epithelial cells. In addition, disturbance of the physiochemical milieu may lead to ROS generation and OS development. ROS begin a signalling cascade culminating in the generation of macromolecules that suppress crystal nucleation, growth and aggregation. In the case of transitory disorder, either no crystal will take shape or the formed crystals will remain tiny and well dispersed and will be expelled as particles, causing crystalluria.
Persistent disorders, such as hyperoxaluria, hyper-COM and hypocitraturia, are associated with imbalances between oxidative and antioxidative forces. ROS-induced cell damage leads to cell death and the formation of membrane-bound vesicles that support crystal adhesion [31,32]. In our study, high COM exposure led to OS development mediated by CaSR. PS exposure, apoptosis and increased OS may eventually lead to crystal retention and kidney damage. We have thus provided a new theoretical basis for the mechanism of CaOx stone formation (Fig. 8).

Conclusions
Our study presents novel, direct evidence that COM-induced increases in CaSR increase OS production and impair APLT activity. This impairment of APLT activity results in PS externalization on the renal epithelial cell membrane and apoptosis of renal epithelial cells.
Since the negatively charged PS on the cell surface may serve as an anionic molecule to mediate COM crystal adhesion, the sequence of events suggested in this study might explain the mechanism of COM-induced CaOx stone disease. Decreasing CaSR activity can attenuate the aforementioned abnormalities, which partly demonstrates the mechanism underlying the protective effects of CaSR

Availability of data and materials
The data used to support the ndings of this study are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
The present study was supported by the National Natural Science Foundation  Figure 1 2-DE maps of proteins from COM-treated HK-2 cells and control HK-2 cells. Con uent HK-2 cells were exposed to COM crystals (67 μg of crystals/cm2 of cells) for 24 h. HK-2 cells without COM crystals were used as controls. At the end of the experimental period, the cells were washed and harvested. The cell suspensions were subsequently sonicated for protein extraction. The gels were stained with silver diamine.     The data are expressed as the mean ± SEM. #p<0.05 vs the COM group.