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Mediation of calcium oxalate crystal growth on human kidney epithelial cells with different degrees of injury
Materials Science and Engineering C 32 (2012) 840–847
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Mediation of calcium oxalate crystal growth on human kidney epithelial cells with different degrees of injury Shen Zhang a, c, Ze-Xuan Su b,⁎, Xiu-Qiong Yao c, Hua Peng c, Sui-Ping Deng c, Jian-Ming Ouyang c,⁎⁎ a b c
Graduate School of Southern Medical University, Guangzhou 510515, China The First Affiliated Hospital, Jinan University, Guangzhou 510632, China Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632, China
a r t i c l e
i n f o
Article history: Received 22 January 2011 Received in revised form 16 November 2011 Accepted 30 January 2012 Available online 4 February 2012 Keywords: Calcium oxalate Renal epithelial cell Urolithiasis Abnormal biomineralization
a b s t r a c t The current study examined the role of injured human kidney tubular epithelial cell (HKC) in the mediation of formation of calcium oxalate (CaOxa) crystals by means of scanning electronic microscopy and X-ray diffraction. HKC was injured using different concentrations of H2O2. Cell injury resulted in a significant decrease in cell viability and superoxide dismutase (SOD) concentration and an increase in the level of malondialdehyde (MDA) and expression of osteopontin (OPN). Injured cells not only promote nucleation and aggregation of CaOxa crystals, but also induce the formation of calcium oxalate monohydrate (COM) crystals that strongly adhere to cells. These results imply that injured HKCs promote stone formation by providing more nucleating sites for crystals, promoting the aggregation of crystals, and inducing the formation of COM crystals. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Kidney stone affliction is prevalent worldwide. However, despite the large number of studies approaching the problem from different angles, the mechanism for the formation of stones has yet to be clearly understood. Kidney stones primarily comprise calcium oxalate (CaOxa) crystals. Renal tubular cell injury is one of the major risk factors for renal stone formation [1] because it not only provides effective nucleating sites for nascent microcrystals, but also enhances potential sites for crystal attachment to cells. Various factors, such as oxalate [2], calcium oxalate (CaOxa) crystals [2,3], hydrogen peroxide (H2O2) [4], and free radicals like superoxide anion (O2•−), peroxynitrite (ONOO•), hydroxyl radicals (OH •), and peroxyl radical (ROO •) [4], were thought to be involved in renal tubular cell injury. Lipid peroxidation is one of the important factors involved in renal tubular cell injury. Urinary excretion of oxalate and deposition of CaOxa crystals in the renal tubules damage renal epithelial cells, and this injury is mediated by lipid peroxidation reaction through the generation of oxygen free radicals [2,4–6]. Free radical scavengers, such as catalase and superoxide dismutase (SOD), could provide significant protection against renal tubular cell injury induced by oxalate and CaOxa crystals [7,8].
⁎ Corresponding author. ⁎⁎ Corresponding author. Tel./fax: + 86 20 85223353. E-mail addresses: [email protected] (Z.-X. Su), [email protected] (J.-M. Ouyang). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.01.035
H2O2 has been implicated in the pathogenesis of a variety of renal diseases and has been commonly used as a mediator in studies on the mechanisms of renal tubular cell injury. Zhuang et al. [9] reported that cell viability of primary cultures of rabbit renal proximal tubular cells exposed to 1 mmol/L H2O2 did not change within the initial 3 h but decreased to 63 ± 4% at 4 h, 55 ± 8% at 5 h, and 52 ± 3% at 6 h. Cultured epithelial cell lines have been used to examine the mechanisms in the renal epithelium leading to urolithiasis despite clear differences among various cultured epithelial cells. At present, cultured epithelial cells fall into several categories based on their origin. For example, cell cultures are derived from mice [10,11], dogs [12], monkeys [11], pigs [13], and human beings [14]. HKC used in this paper is an optimized differentiated human renal proximal tubular epithelial cell, and has the unique structure and function of human renal tubular epithelial cell. HKC can express the marker enzymes of human renal proximal tubule cell, such as alkaline phosphatase (AP) and γ-glutamyl transpeptidase (γ-GT), and cytokeratin (a marker of epithelial cell differentiation). Thus, HKC is a very valuable cell model in vitro to study the mechanisms of renal toxicity and renal disease. Current studies on crystal-cell interactions in vitro focus on two aspects. The first aspect is to investigate the injurious effect of oxalates, CaOxa crystals, and free radicals on cells. The second aspect is to investigate the process of crystal attachment to normal or injurious renal tubular cells. So far, only a limited number of reports on the direct nucleation and growth of CaOxa crystals on both normal and injured cells are available. And also there are few studies about the effect of different injury degrees of renal tubular cell on the crystallization of calcium oxalate or calcium oxalate crystal adhesion. Hence,
S. Zhang et al. / Materials Science and Engineering C 32 (2012) 840–847
the current paper aims to establish an in vitro model of H2O2-induced oxidative injury on HKC cells, and then studies the differences of injury cells in the surface potential and structure. This in vitro model will be used to investigate the difference of nucleation and growth of CaOxa crystals on HKCs with different degree of injury. The formation mechanism of kidney stones at the molecular and cellular levels is expected to be revealed. 2. Materials and methods 2.1. Reagents and equipments HKC, a line of renal proximal tubular epithelial cells of human origin, was obtained from the Shanghai Changzheng Hospital (Shanghai, China, provided by Prof. Mei Chang-Lin). Other materials include: cell proliferation assay kit (CCK-8, Dojindo Laboratories, Kumamoto, Japan); 1:1 Dulbecco's Modified Essential Medium: Nutrient Mixture F-12 (DMEM/F12) (Gibco, USA); newborn calf serum (Gibco, America); malondialdehyde (MDA) kit; and superoxide dismutase (SOD) kit (Jiancheng Institute of Biotechnology of Nanjing, China). Calcium chloride, potassium oxalate, and other conventional reagents were all analytically pure. Stock solutions of potassium oxalate (20 mmol/L) and calcium chloride (20 mmol/L) were prepared in D-Hanks balanced salt solution (D-Hanks) with pH adjusted to 7.4 and used within 3 d. A microplate reader (Safire2, Tecan, Switzerland) was used with a measuring wavelength of 450 nm and a reference wavelength of 655 nm. Samples for XL-30-type environmental scanning electron microscope (ESEM, Philips) were embedded with gold, and a measuring voltage of 20 kV was used. Laser confocal microscope (LSM510 META DUO SCAN, ZEISS, Germany) was utilized. X-ray diffraction (XRD) results were recorded on a Bruker D8 Focus X-ray Diffractometer (Bruker, Germany) using Cu-Kα radiation. Inverted fluorescence microscope (IX51) (Olympus Corporation, Japan) was also used. Inductive Coupled Plasma Emission Spectrometer (ICP) (USA, PE Optima 2000DV) was used to measure the concentrations of CaOxa after co-culture with cells. Nanoparticle size Zeta potential analyzer (Zetasizer Nano-ZS, Malvern, England) was used to measure the Zeta potential of the cell surfaces. Flow cytometry (FACS Aria, Becton Dickinson Biosciences, USA). 2.2. Cell culture The cells were cultured in a DMEM-F12 supplemented containing 10% newborn calf serum, 100 U/ml penicillin-100 μg/ml streptomycin antibiotics (Gibco) with pH 7.4 at 37 °C in a 5% carbon dioxide air atmosphere. Cell culture media were replaced every two days as needed. 2.3. Cell injury and analysis of cell viability Cell viability of HKC cells was evaluated colorimetrically using CCK-8. After the cells were trypsinized, 100 μl of cell suspension (1 × 10 4 cells/ml) was plated per well in 96-well plates (Corning Costar, Badhoevedorp, The Netherlands) and cultured in DMEM/F12 containing 10% newborn calf serum for 24 h. The medium was then aspirated and replaced with a serum-free DMEM/F12 medium. Cells were kept in the serum-free medium for 12 h to achieve quiescence [15]. Subsequent experiments were conducted using a serum-free DMEM/F12 medium. Cells were exposed to a culture medium containing 0.1, 0.3, 0.5, 1.0, and 2.0 mmol/L H2O2 for 1 h. In the control group, the medium did not contain H2O2. At the indicated time points, the medium was aspirated, cells were washed twice with D-Hanks, and freshly cultured medium was added. Then, 10 μl of CCK-8 solution was added to each well. After 4 h of incubation, absorbance at 450 nm of each
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well was measured using the microplate reader. Cell viability was calculated with Aexp/Acon × 100%, where Aexp is the absorbance of cells exposed to reagents, and Acon is the absorbance of control cells. 2.4. Detection of apoptosis and necrosis by flow cytometric analysis After the cells were trypsinized, 2 ml of cell suspension (2 × 10 5 cells/ml) was plated per well in 6-well plates. The later cell culture and H2O2 treatment were performed using a method similar to that of the analysis of cell viability above. At the indicated time points, the medium was aspirated, cells were washed twice with D-Hanks, and added 0.25% trysin digestion solution; subsequently, DMEM-F12 culture medium containing 10% newborn calf serum was added to terminate the digestion. The cells were blown well to form the single cell suspension and centrifuged for 5 min at 1500 rpm. After the supernatant had been aspirated the cells were washed once with PBS (phosphate-buffered saline) solution and centrifuged again. Then the cells were suspended in 200 μL PBS. The single cell suspension was dyed with Annexin V-FITC and PI for flow cytometric analysis, where the number of test cells was 10,000. Then the percentage of apoptotic cells, necrotic cells and viable cells were obtained [16]. 2.5. Measurements of MDA and SOD levels after cell injury 1) Measurement of MDA levels: Lipid peroxidation was assessed through MDA levels. After the establishment of quiescence and treatment of cells with a culture medium containing 0.1, 0.3, 0.5, 1.0, and 2.0 mmol/L of H2O2 for 1 h, the medium was aspirated at the indicated time points to be added to the MDA detection kit. Absorbance was measured at 532 nm. 2) Measurement of SOD levels: Cell culture and H2O2 treatment were performed using a method similar to that of MDA measurement. At the indicated time points, the medium was aspirated and added to the SOD detection kit. Absorbance was measured at 550 nm. 2.6. Observation of osteopontin (OPN) expression of injured cells Procedures for cell culture and H2O2 treatment were similar to those discussed earlier. At the indicated time points, cells were washed with PBS, and then fixed in 4% formaldehyde/0.1% glutaraldehyde for 10 min, washed, and blocked with normal goat serum for OPN staining. Cells were subsequently incubated with a rabbit antihuman OPN antibody (1:150, sc-20788, Santa Cruz) at 37 °C for 1 h, followed by incubation with an FITC-labeled goat anti-rabbit IgG (1:100, sc-2012, Santa Cruz) in a dark box at 37 °C for 0.5 h. The cells were then washed in the same buffer, mounted using an anti-fade mounting medium (sc-24941, Santa Cruz) containing DAPI to stain cell nuclei, and analyzed using confocal laser scanning microscopy to visualize cell nuclei (blue) and OPN (green). Negative controls used pre-immune rabbit serum as a substitute primary label of the OPN antibody. 2.7. SEM, XRD analysis and Zeta potential measurement Cells were cultured and treated with H2O2. At the indicated time points, the metastable CaOxa solutions were added to the cells, followed by incubation at 37 °C for 6 h. The final concentration of the metastable CaOxa solution was c(Ca 2+) = c(Oxa 2 −) = 0.50 mmol/L. After the removal of the culture medium, cells with crystals were rinsed twice with D-Hanks solution and fixed in a 2.5% glutaraldehyde solution for 2 h. The cells were washed thrice in a 0.05 mmol/L cacodylate buffer, postfixed with 1% OsO4 for 2 h, and again washed thrice in cacodylate buffer. Then, samples were dehydrated using a graded series of ethanol solutions and then critically point-dried with CO2. The results of XRD were directly recorded. After gold sputtering, the cells were examined using ESEM at 20 kV. Measurement of
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Zeta potential on cell surface was carried out according to reference [17]. 2.8. Measurement of concentrations of CaOxa after co-culture with cells The cell suspension was drawn out and inoculated in 6-well plates with cover slips on the bottom, achieving a cell concentration of 2 × 10 5 cells/ml, with each well containing 2 ml. The cover slips were basically covered with cells after incubation for 24 h. The suspension was then replaced by the serum-free culture media for cell incubation for 12 h, thereby synchronizing the cells. Then, the cells on the plate were divided into two groups: Group A, the control group, in which only the serum-free medium was added, and Group B, the injured group, in which the serum-free medium containing different concentrations of H2O2 was added to injured cells for 1 h. The culture media were then aspirated, and the cells were rinsed twice with D-Hanks. The cells in the two groups were exposed to serum-free culture media of the metastable CaOxa solution with c(Ca 2+) = c(Oxa 2 −) = 0.50 mmol/L for 6 h in an incubator. All the cover slips in the 6-well plates were taken out when the incubation time was up, and the cells on cover slips were then rinsed twice with PBS to remove all non-associated crystals. The cover slips were placed in 25 ml beakers. Then, 10 ml HNO3 and 0.5 ml HClO4 were added, and the samples were digested on an electric stove until a clear solution formed. The samples were continuously heated until the HClO4 was boiling, smoking, and nearly dried. The afterheat of the electric stove was used to dry the samples, which were then allowed to cool down at room temperature. Then, 3.0 ml water was added to the beaker to dissolve the residue. The concentration of Ca 2+ in the solution was measured using the ICP method. The number of associated CaOxa crystals was calculated from the concentrations of Ca 2+, and the results were usually expressed in micrograms per square centimeters.
same exposure time of 1 h. Concentrations of H2O2 above 1 mmol/L do not significantly affect HKC viability. Therefore, the exposure of HKCs to 0.5 mmol/L H2O2 for 1 h was selected to induce oxidative injury in cells. Under such a condition, cells become significantly injured without excessive apoptosis. Investigating the accompanying changes in molecular structure and the effect of such changes on nucleation, growth, and aggregation of CaOxa crystals is important. Fig. 2 shows the SEM images of HKC cells exposed to various concentrations of H2O2 for 1 h. HKCs in the control group exhibited many structural characteristics of epithelia, that is, they grew very well with tight connections. In contrast, cells exposed to H2O2 showed obvious morphological changes, including cell shrinkage and microvilli reduction (Fig. 1b and c). H2O2 is a major risk factor for renal proximal tubular cells. H2O2induced oxidative injury of cells could result in decreased adhesion
2.9. Statistical analysis Statistical analyses were performed using the SPSS 13.0 software package. Data were expressed as mean ± SD (x ± s). Multiple group comparisons were performed using ANOVA, followed by the Tukey post hoc test. P-value b 0.05 was considered statistically significant. 3. Results and discussion 3.1. Effect of H2O2 concentration on oxidative injury to HKC cells As shown in Fig. 1, cell viability decreased significantly with increasing concentrations of H2O2 from 0.1 to 1 mmol/L using the
Cell viability / %
100
80
60
40 0.0
0.1
0.3
0.5
1.0
2.0
Concentration of H2O2 / mmol/L Fig. 1. Inhibitory effect of H2O2 on cell viability of HKC. Exposure time: 1 h.
Fig. 2. SEM of HKC before and after injury with various concentrations of H2O2 for 1 h. (a) 0; (b) 0.3; and (c) 0.5 mmol/L. The bar: 2 μm.
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There are two types of cell death. One is apoptosis, a programmed cell death and an initiative to death. The other is necrosis, an uncontrolled death caused by the external conditions. In this study, we found that H2O2-injured cells exhibited obvious morphological changes, including cell shrinkage and microvilli reduction, but the membrane of cells still exist (Fig. 2b and c). These changes are similar to apopotosis in physiological conditions. Table 1 showed the effects of H2O2 on cellular apoptosis and necrosis. H2O2 was injurious, and induced apopotosis or necrosis of HKCs. Both apoptosis rate and necrosis rate increased with the elevated concentration of H2O2. After 1 h exposure to 0.3 mmol/L H2O2, 4.60% of the cells were apoptotic compared with 2.80% in controls. The apoptotic cells reached the highest of percentage of 9.58% when the concentration of H2O2 increased to 2.0 mmol/L. The necrosis rate was 1.31%, 1.40%, 1.60%, 5.42%, and 9.23% as HKCs were exposed to H2O2 at concentrations of 0, 0.3, 0.5, 1.0, and 2.0 mmol/L, respectively. The necrosis rate didn't increase obviously until H2O2 concentration was increased to 1.0 mmol/L. The percentage of viable cells decreased with increasing concentration of H2O2 in contrast with apoptosis rate and necrosis rate.
SOD activity / U/ml
3.2. Effect of H2O2 concentration on cellular apoptosis and necrosis
a
120
100
80
0.0
0.5
0.3
1.0
2.0
c (H2O2) / mmol/L
b MDA content / nmol/ml
between cells and basement membrane components, such as laminine and fibronectin. Moreover, H2O2 affects the capacity of renal tubule cells to attach to the collagen IV matrix, which results in actin cytoskeleton alterations [18]. All these changes contribute to cell detachment and reduce cell viability.
843
1.5
1.2
0.9
0.6
0.3 0.0
3.3. Changes in SOD and MDA levels after cell injury
0.1
0.3
0.5
1.0
2.0
Concentration of H2O2 / mmol/L 3.3.1. Decreased SOD content Fig. 3a shows the changes in the SOD level of the medium after the exposure of HKCs to various concentrations of H2O2 for 1 h. The results indicate that H2O2 results in a reduction in SOD level. After 1 h of exposure to H2O2, SOD decreased significantly from 109.6 U/ml (control) to 99.3 and 81.1 U/ml (P b 0.05) when the concentration of H2O2 was increased from 0 to 0.5 and 2.0 mmol/L, respectively (P b 0.05). Cell membrane injury is mediated by the lipid peroxidation reaction through the generation of free radicals induced by H2O2 [4], which could be responsible for the role of reactive oxygen species (ROS) in necrotic and apoptotic cell death [19]. Renal epithelial cells exposed to H2O2 have significantly higher ROS production, increased formation of MDA as a marker of lipid peroxidation, increased release of lactate dehydrogenase (LDH) as a marker for cell injury, and loss of cell viability. As an antioxidant enzyme, SOD could terminate radical chain reactions by removing free radical intermediates [11]. Free radicals are toxic to an organism, so SOD is an important antioxidant defense for nearly all cells exposed to free radicals. SOD prevents damage from oxidation attributable to free radicals, thus protecting cells against oxidative stress. Decreased SOD levels indicate that an organism has a reduced capability to prevent free radical toxicity, thus reflecting an increase in the degree of damage in the organism [13].
Fig. 3. Changes in SOD level (a) and MDA concentration (b) of HKC after exposure to different concentrations of H2O2 for 1 h.
3.3.2. Increased MDA content Fig. 3b shows the effects of H2O2 concentration on the MDA content of HKCs. MDA level increases with increasing concentration of H2O2 when exposure time is kept constant for 1 h. MDA level increased from 0.52 to 1.03 and 1.57 nmol/ml when H2O2 concentration was increased from 0 to 0.5 and 2.0 mmol/L, respectively. This finding shows that the injury to the cell membrane was aggravated. The production of MDA can be used as a biomarker for the level of oxidative stress in an organism. Changes in MDA content can reflect the degree of lipid peroxidation, and indirectly, cell injury [20]. The enzyme system generates free radicals, which most often affect polyunsaturated fatty acids because of the multiple double bonds in their carbon chains. Free radicals induce lipid peroxidation in cell membranes, resulting in cell damage and in the production of lipid peroxides [8], such as aldehydes, ketones, hydroxyl groups, carbonyl groups, and hydroperoxides. Fig. 3a and b shows that H2O2 not only increases MDA levels, but also decreases SOD levels. H2O2 was shown to induce lipid peroxidation in the cell and simultaneously weakens the capability of the cell to scavenge for free radicals. 3.4. Osteopontin (OPN) expression after cell injury
Table 1 Effect of H2O2 concentration on cellular apoptosis and necrosis. c(H2O2)/mmol/L
0
0.3
0.5
1.0
2.0
Necrotic cells/% Apoptotic cells/% Viable cells/%
1.31 2.80 95.89
1.40 4.60 94.00
1.60 5.27 93.13
5.42 7.43 87.15
9.23 9.58 81.19
The structures on the cell surface change after cell injury. For example, OPN become expressed on the cell surface [21]. Fig. 4 shows the fluorescence images of the OPN expression of HKCs after oxidative injury. Limited green fluorescence was found on the normal cell surface (Fig. 4a), indicating that normal cells did not express or weakly expressed OPN. After exposure to 0.3 mmol/L H2O2 for 1 h, green
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fluorescence was observed on the cell surface (Fig. 4b), indicating that OPN was expressed on the cell surface after injury. When the concentration of H2O2 was increased to 0.5 and 1 mmol/L, stronger green fluorescences were seen on the cell surface (Fig. 4c and d), showing that OPN expression strengthened after severe injury. These findings imply that OPN expression increases as the degree of cell injury increases. OPN is a highly negatively charged, extracellular matrix glycoprotein with a molecular weight of approximately 44 kDa and is rich in serine, aspartic acid, and glutaminic acid, making it an acidic protein. The molecular chain of OPN also has functionally important sites related to the adhesion of mineral ions. Acidic groups and their charge density on the OPN chains are important factors affecting the binding of OPN to mineral ions. If OPN expression on cells increases, the formation and adhesion of CaOxa crystals on cells will be promoted. It can be seen from Figs. 4 & 5 that cellular surface structure is closely related to the formation of calcium oxalate crystals, and OPN was also spacely related to crystals. When the expression amount of OPN molecules on the surface of injury cells increases, that is, the green fluorescence intensity becomes stronger (Fig. 4), the number of CaOxa crystals induced by the injured cells increases (Fig. 5, Table 2), furthermore,the degree of crystals' aggregation and the proportion of COM crystals increased too. 3.5. Induction of CaOxa crystal nucleation and growth by injured HKCs Fig. 5 shows the SEM images of CaOxa crystals induced by normal HKC and injured cells exposed to different concentrations of H2O2 for 1 h. Compared with the control group (Fig. 5a), crystals induced by injured cells have several differences.
3.5.1. Crystal number A small number of crystals were induced by normal cells (Fig. 5a). This number increased significantly on injured cells, showing that normal cells have a weak capability to induce the formation of CaOxa crystals, whereas injured cells obviously facilitate the nucleation of crystals. The number of CaOxa that adhered to the cell surface was measured using ICP, and the data are listed in Table 2. The number of CaOxa crystals induced by the control group was significantly less than that induced by the injured groups. After the HKC was injured by H2O2 with different concentrations, the number of the CaOxa crystals induced increased with the increase of c(H2O2) when the concentration of c(H2O2) was within the range of 0.1–1.0 mmol/L. However, the number of crystals that adhered to the cell surface changed insignificantly when c(H2O2) > 1.0 mmol/L. These finding may be attributable to the fact that when c(H2O2) b 1.0 mmol/L, the degree of injury of HKC increased with the increase of c(H2O2), resulting in the growth of induced CaOxa crystals on the cell surface, as well as the continuous increase of active sites to which the crystals subsequently adhered. Therefore, the number of crystals that adhered on the cell surface increased gradually. However, when c(H2O2) > 1.0 mmol/L, the apoptosis rate of cells increased further, and a proportion of the cells had even died (Table 1). These dead cells fell from the culture flask and floated on the culture solution without and no longer had any cellular physiological function. In addition, because we have repeatedly washed the cells during the experiment, most of these dead cells were removed. Therefore, it is impossible for these dead cells to induce the formation of CaOxa crystals. Thus, the number of the adhered crystals did not increase.
Fig. 4. Confocal laser scanning microscopy images of OPN expression of HKC cells exposed to different concentrations of H2O2 for 1 h: (a) 0; (b) 0.3; (c) 0.5; and (d) 1.0 mmol/L. OPN (green). The bar: 20 μm.
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Fig. 5. SEM images of HKC cells exposed to different concentrations of H2O2 for 1 h: (a) 0; (b) 0.3; (c) 0.5; and (d) 1.0 mmol/L. Bar: 10 μm. c(CaOxa) = 0.5 mmol/L; crytallization time: 6 h, temperature: 37 ± 0.1 °C. Bar: 10 μm.
3.5.2. Crystal size No significant difference was found between crystal sizes (P > 0.05) with increasing extent of injury in HKC cells. Crystal size remained nearly constant at 5.0 ± 0.6 μm, suggesting that injured HKC could not promote the growth of CaOxa crystals. These results imply that injured HKC promote stone formation by providing more nucleating sites for crystals, not by promoting crystal growth. 3.5.3. Degree of crystals aggregation Control cells induced scattered calcium oxalate dihydrate (COD) crystals with typical pyramidal morphology on the cell surface (Fig. 5a). In contrast, some of the crystals induced by injured cells were aggregated, and the degree of aggregation increased with the degree of
injury, indicating that injured cells promote crystal aggregation. Aggregated crystals not only significantly damage the cells, but also have stronger adhesion to cells. Thus, crystal aggregation increases the risk for the formation of urinary stones on injured cells. 3.5.4. Crystal phase COD crystals with a regular morphology were predominant in the crystals induced by control cells (Fig. 5a). In contrast, injured cells induced the formation of COM crystals. The proportion of COM crystals to the total number of crystals increased gradually with the degree of cell injury. This increase had a concomitant gradual decrease
3000 d=6.17 200*
2400
Intensity / a.u
The effect of cellular calcium on CaOxa crystallization in necrotic cells is also minimal. In normal cells, the concentration of free Ca 2+ in cell membrane is about 0.05–0.15 μmol/L. However, when cells were exposed to oxidative stress, the external Ca 2+ in cells and organelles will passively flow into the cytoplasm, making the free calcium concentration rise to 1–10 μmol/L, which led to a certain degree of physiological responses such as cell necrosis and inflammation. But this concentration of Ca 2+ ions is relatively low compared with the concentration of free Ca 2+ ions in urine (1.21±0.64 mmol/L). That is, the calcium concentration in necrotic cells is too low to cause a significant increase in urinary calcium.
1800
1200
d=2.97 202
10
Table 2 Number of CaOxa crystals after co-culture with cells measured using ICP.
d=1.99 303
600
20
30
40
50
2 theta / degree
c(H2O2)/mmol/L
0
0.1
0.3
0.5
1.0
1.5
2.0
Crystals number/μg/cm2
31.9
44.6
47.1
50.1
54.5
56.3
55.4
Fig. 6. XRD pattern of CaOxa crystals induced by injured cells. c(CaOxa) = 0.5 mmol/L; c(H2O2) = 0.5 mmol/L; exposure time: 1 h; crystallization time: 6 h. The crystal faces with asterisk show COD, and those without asterisk show COM.
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in the percentage of COD crystals. When cells were exposed to 0, 0.3, 0.5, and 1 mmol/L H2O2 for 1 h, the percentage of COM crystals was approximately 0, 5%, 14%, and 19%, respectively. Fig. 6 shows the XRD pattern of the CaOxa crystals induced by injured cells exposed to 0.5 mmol/L H2O2 for 1 h. The pattern reveals that the corresponding diffraction peaks assigned to COM and COD crystals appeared simultaneously. The corresponding interplanar distance located at 6.17 Å can be assigned to the (200) plane of COD, whereas the corresponding interplanar distance located at 2.97 Å and 1.99 Å can be assigned to the ( 202) and ( 303) planes of COM [22]. The (101), (202) and (303) planes of COM crystals are exposed to more calcium ions, so they have more positive charges, whereas COD crystals have a relatively low surface charge [23]. The promotion of the nucleation and formation of CaOxa crystals by injured cells was correlated to crystal-binding molecules expressed on the cell surface. Normal cells without oxidative injury have intact surface resistance to crystal growth and binding. This is because that there is only neutral phospholipids existing outside of the normal cell membrane, the negatively charged phospholipids [such as phosphatidylserine (PS)] is located inside the cell membrane. Hence, normal cells have a weak capability to attract positively charged Ca2+ [24] and to induce the nucleation and adhesion of CaOxa crystals [25]. However, when HKCs were injured by H2O2, the microstructures of the cell surface and the cellular polar were altered. For example, the PS molecules were translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. And also some negatively charged molecules such as OPN (Fig. 4), hyaluronic acid, and collagen protein were expressed on the surface of injured cells. The increase in the expression of negatively charged molecules on the surface of injured cells can attract more calcium ions and CaOxa crystals through electronic interaction [26]. It is an important reason why injury cells have greater ability to induce crystallization of calcium oxalate crystals than normal cells. In addition, the adhesion between injured cells and COM crystals is stronger than that between injured cells and COD crystals [27], thus increasing the risk for the formation of urinary stones. Our observation confirmed that OPN molecules were expressed on the surfaces of injured HKC cells (Fig. 4). The high-density negatively charged regions in OPN molecular chain can attract and gather calcium ions [28], thereby inducing the formation of calcium oxalate crystals [29]. Animal experiments also showed that OPN has a crucial role in the morphological conversion of CaOxa crystals retained in renal tubules to matrix-involving kidney stones, possibly leading to clinical urolithiasis [30]. By using wildtype mice (WT, with OPN) and OPN knockout mice (KO, deleting OPN) as animal models and using glyoxylate to induce renal tubular injury, the crystal deposition in the kidneys of the two kinds of genotypes mice were comparatively investigated. The results showed that the number of the deposited crystals in KO was significantly fewer than in WT. Morphological observation by polarized light optical microphotography and SEM showed large flower-shaped crystals growing in renal tubules in WT and small and uniform crystals in KO. XRD results showed these crystals components in both genotypes were calcium oxalate monohydrate. 3.6. Zeta potential changes on cell surface during the induction process of crystal growth The Zeta potential of the cell surface can show the charge state of a cell membrane, which further reflects the degree of mutual exclusion Table 3 Zeta potential of HKC cells before and after crystallization/mV. c(H2O2)/mM
0
0.1
0.3
0.5
1
1.5
HKC cells HKC + crystals
− 25.2 − 26.9
− 29.2 − 29.1
− 31.9 − 27.2
− 34.3 − 25.8
− 35.2 − 24.7
− 36.1 − 25.1
or attraction between the cell surface and foreign particles. Therefore, the capability of cells to induce crystal growth and the degree of crystal adhesion to cells can be determined by measuring the Zeta potential of the cell surface during the process of crystal growth and adhesion. Table 3 shows the change in Zeta potential change after the cells induced CaOxa crystal growth for 6 h in different states, from which the following could be concluded: 1) The Zeta potential of the control group was −25.2 mV, and that of the injured group was more negative and was less than −29.2 mV. This finding can be attributed not only to the valgus of phosphatidylserine, but also to the expression of the molecules with a negative charge such as OPN (Fig. 4). A large number of negatively charged phospholipids and proteins were expressed on the cell surface and its surroundings, thereby increasing the density of negative charges on cells, thus making the Zeta potential negative. 2) The degree of damage to the cells increased with the increase of c(H2O2), consequently increasing the concentration of negatively charged molecules expressed on the cell surface. Therefore, the Zeta potential further became negative. 3) However, after the CaOxa crystal growth was induced for 6 h, the Zeta potential of the control group cells decreased, whereas the Zeta potential of the injured cells increased. This finding resulted from the capacity of the injured cells to induce crystal growth and adhesion, which was stronger than that of the control group cells. The severely injured cell surface is covered with more crystals, thus increasing the negatively charged molecules on the cell surface and decreasing the negative charge density. Therefore, the Zeta potential increased. 4. Conclusion H2O2 could significantly injure HKCs and decrease cell viability in a dose-dependent manner at concentrations of 0.1–1 mmol/L at an exposure time of 1 h. After cell injury, MDA content and OPN expression increase, whereas SOD level decreases, resulting in an increased number of CaOxa crystals and increased crystal aggregation induced by injured cells. Moreover, injured cells promote the formation of COM crystals, thus increasing the risk for the formation of urinary stones. The in vitro model of HKCs with oxidative injury established in the current study may provide insight into the pathogenesis of nephrolithiasis at the cellular level. Acknowledgments This work was supported by the National Natural Science Foundation of China (20971057), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education of China. References [1] B.A. Vervaet, A. Verhulst, S.E. Dauwe, M.E. De Broe, P.C. D'Haese, Kidney Int. 75 (2009) 41. [2] S.H. Chen, X.F. Gao, Y.H. Sun, C.L. Xu, L.H. Wang, T. Zhou, Urol. Res. 38 (2010) 7. [3] M.S.J. Schepers, E.S. van Ballegooijen, C.H. Bangma, C.F. Verkoelen, Kidney Int. 68 (2005) 1543. [4] C. Escobar, K.J. Byer, H. Khaskheli, S.R. Khan, J. Urol. 180 (2008) 379. [5] K. Sarica, A. Erbagci, F. Yagci, K. Bakir, S. Erturhan, R. Ucak, Urol. Res. 32 (2004) 271. [6] M.S.J. Schepers, E.S. van Ballegooijen, C.H. Bangma, C.F. Verkoelen, Urol. Res. 33 (2005) 321. [7] T. Rashed, M. Menon, S. Thamilselvan, Am. J. Nephrol. 24 (2004) 557. [8] S. Thamilselvan, S.R. Khan, M. Menon, Urol. Res. 31 (2003) 3. [9] S. Zhuang, Y. Yan, R.A. Daubert, J. Han, R.G. Schnellmann, Am. J. Physiol. Ren. Physiol. 292 (2007) F440. [10] C. Escobar, K.J. Byer, S.R. Khan, BJU Int. 100 (2007) 891. [11] M.W. Bigelow, J.H. Wiessner, J.G. Kleinman, N.S. Mandel, J. Urol. 160 (1998) 1528. [12] V. Kumar, G. Farell, S. Deganello, J.C. Lieske, J. Am. Soc. Nephrol. 14 (2003) 289. [13] Y.I. Rabinovich, M. Esayanur, S. Daosukho, K.J. Byer, H.E. El-Shall, S.R. Khan, J. Colloid Interface Sci. 300 (2006) 131.
S. Zhang et al. / Materials Science and Engineering C 32 (2012) 840–847 [14] B.C. Jeong, C. Kwak, K.S. Cho, B.S. Kim, S.K. Hong, J.I. Kim, C. Lee, H.H. Kim, Urol. Res. 33 (2005) 87. [15] R. Hamamoto, Y. Furukawa, M. Morita, Y. Iimura, F.P. Silva, M.H. Li, R. Yagyu, Y. Nakamura, Nat. Cell Biol. 6 (2004) 731. [16] I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutelingsperger, J. Immunol. Methods 184 (1995) 39. [17] J.M. Ouyang, X.Q. Yao, J. Tan, F.X. Wang, J. Biol. Inorg. Chem. 16 (2011) 405. [18] D. Sáenz-Moralesa, M.M. Escribesea, K. Stamatakisc, M. García-Martosa, L. Alegrea, E. Condea, D. Pérez-Salac, F. Mampasoa, M.L. García-Bermejoa, Exp. Cell Res. 312 (2006) 3711. [19] S.M. Baek, C.H. Kwon, J.H. Kim, J.S. Woo, J.S. Jung, Y.K. Kim, J. Lab. Clin. Med. 142 (2003) 178. [20] C.K. Veena, A. Josephine, S.P. Preetha, P. Varalashmi, R. Sundarapandayan, Life Sci. 79 (2006) 1789. [21] L.C. Cao, J. Jonassen, T.W. Honeyman, C. Scheid, Am. J. Nephrol. 21 (2001) 69.
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[22] J.Y. He, S.P. Deng, J.M. Ouyang, IEEE Trans. Nanobioscience 9 (2010) 156. [23] L. Tunik, F. Hueredi-Milhofer, N. Garti, Langmuir 14 (1998) 3351. [24] J.H. Wiessner, A.T. Hasegawa, L.Y. Hung, G.S. Mandel, N.S. Mandel, Kidney Int. 59 (2001) 637. [25] S. Chutipongtanate, V. Thongboonkerd, Chem. Biol. Interact. 188 (2010) 421. [26] J.C. Lieske, S. Deganello, F.G. Toback, Nephron 81 (1999) 8. [27] M.T. Moriyama, K. Miyazawa, K. Noda, M. Oka, M. Tanaka, K. Suzuki, Urol. Res. 35 (2007) 295. [28] Y.C. Chien, D.L. Masica, J.J. Gray, S. Nguyen, H. Vali, M.D. McKee, J. Biol. Chem. 284 (2009) 23491. [29] G.K. Hunter, B. Grohe, S. Jeffrey, J. O'Young, E.S. Sørensen, H.A. Goldberg, Cells Tissues Organs 189 (2009) 44. [30] A. Okada, S. Nomura, Y. Saeki, Y. Higashibata, S. Hamamoto, M. Hirose, Y. Itoh, T. Yasui, K. Tozawa, K. Kohri, J. Bone Miner. Res. 23 (2008) 1629.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Mediation of calcium oxalate crystal growth on human kidney epithelial cells with different degrees of injury Shen Zhang a, c, Ze-Xuan Su b,⁎, Xiu-Qiong Yao c, Hua Peng c, Sui-Ping Deng c, Jian-Ming Ouyang c,⁎⁎ a b c
Graduate School of Southern Medical University, Guangzhou 510515, China The First Affiliated Hospital, Jinan University, Guangzhou 510632, China Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632, China
a r t i c l e
i n f o
Article history: Received 22 January 2011 Received in revised form 16 November 2011 Accepted 30 January 2012 Available online 4 February 2012 Keywords: Calcium oxalate Renal epithelial cell Urolithiasis Abnormal biomineralization
a b s t r a c t The current study examined the role of injured human kidney tubular epithelial cell (HKC) in the mediation of formation of calcium oxalate (CaOxa) crystals by means of scanning electronic microscopy and X-ray diffraction. HKC was injured using different concentrations of H2O2. Cell injury resulted in a significant decrease in cell viability and superoxide dismutase (SOD) concentration and an increase in the level of malondialdehyde (MDA) and expression of osteopontin (OPN). Injured cells not only promote nucleation and aggregation of CaOxa crystals, but also induce the formation of calcium oxalate monohydrate (COM) crystals that strongly adhere to cells. These results imply that injured HKCs promote stone formation by providing more nucleating sites for crystals, promoting the aggregation of crystals, and inducing the formation of COM crystals. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Kidney stone affliction is prevalent worldwide. However, despite the large number of studies approaching the problem from different angles, the mechanism for the formation of stones has yet to be clearly understood. Kidney stones primarily comprise calcium oxalate (CaOxa) crystals. Renal tubular cell injury is one of the major risk factors for renal stone formation [1] because it not only provides effective nucleating sites for nascent microcrystals, but also enhances potential sites for crystal attachment to cells. Various factors, such as oxalate [2], calcium oxalate (CaOxa) crystals [2,3], hydrogen peroxide (H2O2) [4], and free radicals like superoxide anion (O2•−), peroxynitrite (ONOO•), hydroxyl radicals (OH •), and peroxyl radical (ROO •) [4], were thought to be involved in renal tubular cell injury. Lipid peroxidation is one of the important factors involved in renal tubular cell injury. Urinary excretion of oxalate and deposition of CaOxa crystals in the renal tubules damage renal epithelial cells, and this injury is mediated by lipid peroxidation reaction through the generation of oxygen free radicals [2,4–6]. Free radical scavengers, such as catalase and superoxide dismutase (SOD), could provide significant protection against renal tubular cell injury induced by oxalate and CaOxa crystals [7,8].
⁎ Corresponding author. ⁎⁎ Corresponding author. Tel./fax: + 86 20 85223353. E-mail addresses: [email protected] (Z.-X. Su), [email protected] (J.-M. Ouyang). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.01.035
H2O2 has been implicated in the pathogenesis of a variety of renal diseases and has been commonly used as a mediator in studies on the mechanisms of renal tubular cell injury. Zhuang et al. [9] reported that cell viability of primary cultures of rabbit renal proximal tubular cells exposed to 1 mmol/L H2O2 did not change within the initial 3 h but decreased to 63 ± 4% at 4 h, 55 ± 8% at 5 h, and 52 ± 3% at 6 h. Cultured epithelial cell lines have been used to examine the mechanisms in the renal epithelium leading to urolithiasis despite clear differences among various cultured epithelial cells. At present, cultured epithelial cells fall into several categories based on their origin. For example, cell cultures are derived from mice [10,11], dogs [12], monkeys [11], pigs [13], and human beings [14]. HKC used in this paper is an optimized differentiated human renal proximal tubular epithelial cell, and has the unique structure and function of human renal tubular epithelial cell. HKC can express the marker enzymes of human renal proximal tubule cell, such as alkaline phosphatase (AP) and γ-glutamyl transpeptidase (γ-GT), and cytokeratin (a marker of epithelial cell differentiation). Thus, HKC is a very valuable cell model in vitro to study the mechanisms of renal toxicity and renal disease. Current studies on crystal-cell interactions in vitro focus on two aspects. The first aspect is to investigate the injurious effect of oxalates, CaOxa crystals, and free radicals on cells. The second aspect is to investigate the process of crystal attachment to normal or injurious renal tubular cells. So far, only a limited number of reports on the direct nucleation and growth of CaOxa crystals on both normal and injured cells are available. And also there are few studies about the effect of different injury degrees of renal tubular cell on the crystallization of calcium oxalate or calcium oxalate crystal adhesion. Hence,
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the current paper aims to establish an in vitro model of H2O2-induced oxidative injury on HKC cells, and then studies the differences of injury cells in the surface potential and structure. This in vitro model will be used to investigate the difference of nucleation and growth of CaOxa crystals on HKCs with different degree of injury. The formation mechanism of kidney stones at the molecular and cellular levels is expected to be revealed. 2. Materials and methods 2.1. Reagents and equipments HKC, a line of renal proximal tubular epithelial cells of human origin, was obtained from the Shanghai Changzheng Hospital (Shanghai, China, provided by Prof. Mei Chang-Lin). Other materials include: cell proliferation assay kit (CCK-8, Dojindo Laboratories, Kumamoto, Japan); 1:1 Dulbecco's Modified Essential Medium: Nutrient Mixture F-12 (DMEM/F12) (Gibco, USA); newborn calf serum (Gibco, America); malondialdehyde (MDA) kit; and superoxide dismutase (SOD) kit (Jiancheng Institute of Biotechnology of Nanjing, China). Calcium chloride, potassium oxalate, and other conventional reagents were all analytically pure. Stock solutions of potassium oxalate (20 mmol/L) and calcium chloride (20 mmol/L) were prepared in D-Hanks balanced salt solution (D-Hanks) with pH adjusted to 7.4 and used within 3 d. A microplate reader (Safire2, Tecan, Switzerland) was used with a measuring wavelength of 450 nm and a reference wavelength of 655 nm. Samples for XL-30-type environmental scanning electron microscope (ESEM, Philips) were embedded with gold, and a measuring voltage of 20 kV was used. Laser confocal microscope (LSM510 META DUO SCAN, ZEISS, Germany) was utilized. X-ray diffraction (XRD) results were recorded on a Bruker D8 Focus X-ray Diffractometer (Bruker, Germany) using Cu-Kα radiation. Inverted fluorescence microscope (IX51) (Olympus Corporation, Japan) was also used. Inductive Coupled Plasma Emission Spectrometer (ICP) (USA, PE Optima 2000DV) was used to measure the concentrations of CaOxa after co-culture with cells. Nanoparticle size Zeta potential analyzer (Zetasizer Nano-ZS, Malvern, England) was used to measure the Zeta potential of the cell surfaces. Flow cytometry (FACS Aria, Becton Dickinson Biosciences, USA). 2.2. Cell culture The cells were cultured in a DMEM-F12 supplemented containing 10% newborn calf serum, 100 U/ml penicillin-100 μg/ml streptomycin antibiotics (Gibco) with pH 7.4 at 37 °C in a 5% carbon dioxide air atmosphere. Cell culture media were replaced every two days as needed. 2.3. Cell injury and analysis of cell viability Cell viability of HKC cells was evaluated colorimetrically using CCK-8. After the cells were trypsinized, 100 μl of cell suspension (1 × 10 4 cells/ml) was plated per well in 96-well plates (Corning Costar, Badhoevedorp, The Netherlands) and cultured in DMEM/F12 containing 10% newborn calf serum for 24 h. The medium was then aspirated and replaced with a serum-free DMEM/F12 medium. Cells were kept in the serum-free medium for 12 h to achieve quiescence [15]. Subsequent experiments were conducted using a serum-free DMEM/F12 medium. Cells were exposed to a culture medium containing 0.1, 0.3, 0.5, 1.0, and 2.0 mmol/L H2O2 for 1 h. In the control group, the medium did not contain H2O2. At the indicated time points, the medium was aspirated, cells were washed twice with D-Hanks, and freshly cultured medium was added. Then, 10 μl of CCK-8 solution was added to each well. After 4 h of incubation, absorbance at 450 nm of each
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well was measured using the microplate reader. Cell viability was calculated with Aexp/Acon × 100%, where Aexp is the absorbance of cells exposed to reagents, and Acon is the absorbance of control cells. 2.4. Detection of apoptosis and necrosis by flow cytometric analysis After the cells were trypsinized, 2 ml of cell suspension (2 × 10 5 cells/ml) was plated per well in 6-well plates. The later cell culture and H2O2 treatment were performed using a method similar to that of the analysis of cell viability above. At the indicated time points, the medium was aspirated, cells were washed twice with D-Hanks, and added 0.25% trysin digestion solution; subsequently, DMEM-F12 culture medium containing 10% newborn calf serum was added to terminate the digestion. The cells were blown well to form the single cell suspension and centrifuged for 5 min at 1500 rpm. After the supernatant had been aspirated the cells were washed once with PBS (phosphate-buffered saline) solution and centrifuged again. Then the cells were suspended in 200 μL PBS. The single cell suspension was dyed with Annexin V-FITC and PI for flow cytometric analysis, where the number of test cells was 10,000. Then the percentage of apoptotic cells, necrotic cells and viable cells were obtained [16]. 2.5. Measurements of MDA and SOD levels after cell injury 1) Measurement of MDA levels: Lipid peroxidation was assessed through MDA levels. After the establishment of quiescence and treatment of cells with a culture medium containing 0.1, 0.3, 0.5, 1.0, and 2.0 mmol/L of H2O2 for 1 h, the medium was aspirated at the indicated time points to be added to the MDA detection kit. Absorbance was measured at 532 nm. 2) Measurement of SOD levels: Cell culture and H2O2 treatment were performed using a method similar to that of MDA measurement. At the indicated time points, the medium was aspirated and added to the SOD detection kit. Absorbance was measured at 550 nm. 2.6. Observation of osteopontin (OPN) expression of injured cells Procedures for cell culture and H2O2 treatment were similar to those discussed earlier. At the indicated time points, cells were washed with PBS, and then fixed in 4% formaldehyde/0.1% glutaraldehyde for 10 min, washed, and blocked with normal goat serum for OPN staining. Cells were subsequently incubated with a rabbit antihuman OPN antibody (1:150, sc-20788, Santa Cruz) at 37 °C for 1 h, followed by incubation with an FITC-labeled goat anti-rabbit IgG (1:100, sc-2012, Santa Cruz) in a dark box at 37 °C for 0.5 h. The cells were then washed in the same buffer, mounted using an anti-fade mounting medium (sc-24941, Santa Cruz) containing DAPI to stain cell nuclei, and analyzed using confocal laser scanning microscopy to visualize cell nuclei (blue) and OPN (green). Negative controls used pre-immune rabbit serum as a substitute primary label of the OPN antibody. 2.7. SEM, XRD analysis and Zeta potential measurement Cells were cultured and treated with H2O2. At the indicated time points, the metastable CaOxa solutions were added to the cells, followed by incubation at 37 °C for 6 h. The final concentration of the metastable CaOxa solution was c(Ca 2+) = c(Oxa 2 −) = 0.50 mmol/L. After the removal of the culture medium, cells with crystals were rinsed twice with D-Hanks solution and fixed in a 2.5% glutaraldehyde solution for 2 h. The cells were washed thrice in a 0.05 mmol/L cacodylate buffer, postfixed with 1% OsO4 for 2 h, and again washed thrice in cacodylate buffer. Then, samples were dehydrated using a graded series of ethanol solutions and then critically point-dried with CO2. The results of XRD were directly recorded. After gold sputtering, the cells were examined using ESEM at 20 kV. Measurement of
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Zeta potential on cell surface was carried out according to reference [17]. 2.8. Measurement of concentrations of CaOxa after co-culture with cells The cell suspension was drawn out and inoculated in 6-well plates with cover slips on the bottom, achieving a cell concentration of 2 × 10 5 cells/ml, with each well containing 2 ml. The cover slips were basically covered with cells after incubation for 24 h. The suspension was then replaced by the serum-free culture media for cell incubation for 12 h, thereby synchronizing the cells. Then, the cells on the plate were divided into two groups: Group A, the control group, in which only the serum-free medium was added, and Group B, the injured group, in which the serum-free medium containing different concentrations of H2O2 was added to injured cells for 1 h. The culture media were then aspirated, and the cells were rinsed twice with D-Hanks. The cells in the two groups were exposed to serum-free culture media of the metastable CaOxa solution with c(Ca 2+) = c(Oxa 2 −) = 0.50 mmol/L for 6 h in an incubator. All the cover slips in the 6-well plates were taken out when the incubation time was up, and the cells on cover slips were then rinsed twice with PBS to remove all non-associated crystals. The cover slips were placed in 25 ml beakers. Then, 10 ml HNO3 and 0.5 ml HClO4 were added, and the samples were digested on an electric stove until a clear solution formed. The samples were continuously heated until the HClO4 was boiling, smoking, and nearly dried. The afterheat of the electric stove was used to dry the samples, which were then allowed to cool down at room temperature. Then, 3.0 ml water was added to the beaker to dissolve the residue. The concentration of Ca 2+ in the solution was measured using the ICP method. The number of associated CaOxa crystals was calculated from the concentrations of Ca 2+, and the results were usually expressed in micrograms per square centimeters.
same exposure time of 1 h. Concentrations of H2O2 above 1 mmol/L do not significantly affect HKC viability. Therefore, the exposure of HKCs to 0.5 mmol/L H2O2 for 1 h was selected to induce oxidative injury in cells. Under such a condition, cells become significantly injured without excessive apoptosis. Investigating the accompanying changes in molecular structure and the effect of such changes on nucleation, growth, and aggregation of CaOxa crystals is important. Fig. 2 shows the SEM images of HKC cells exposed to various concentrations of H2O2 for 1 h. HKCs in the control group exhibited many structural characteristics of epithelia, that is, they grew very well with tight connections. In contrast, cells exposed to H2O2 showed obvious morphological changes, including cell shrinkage and microvilli reduction (Fig. 1b and c). H2O2 is a major risk factor for renal proximal tubular cells. H2O2induced oxidative injury of cells could result in decreased adhesion
2.9. Statistical analysis Statistical analyses were performed using the SPSS 13.0 software package. Data were expressed as mean ± SD (x ± s). Multiple group comparisons were performed using ANOVA, followed by the Tukey post hoc test. P-value b 0.05 was considered statistically significant. 3. Results and discussion 3.1. Effect of H2O2 concentration on oxidative injury to HKC cells As shown in Fig. 1, cell viability decreased significantly with increasing concentrations of H2O2 from 0.1 to 1 mmol/L using the
Cell viability / %
100
80
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40 0.0
0.1
0.3
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Concentration of H2O2 / mmol/L Fig. 1. Inhibitory effect of H2O2 on cell viability of HKC. Exposure time: 1 h.
Fig. 2. SEM of HKC before and after injury with various concentrations of H2O2 for 1 h. (a) 0; (b) 0.3; and (c) 0.5 mmol/L. The bar: 2 μm.
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There are two types of cell death. One is apoptosis, a programmed cell death and an initiative to death. The other is necrosis, an uncontrolled death caused by the external conditions. In this study, we found that H2O2-injured cells exhibited obvious morphological changes, including cell shrinkage and microvilli reduction, but the membrane of cells still exist (Fig. 2b and c). These changes are similar to apopotosis in physiological conditions. Table 1 showed the effects of H2O2 on cellular apoptosis and necrosis. H2O2 was injurious, and induced apopotosis or necrosis of HKCs. Both apoptosis rate and necrosis rate increased with the elevated concentration of H2O2. After 1 h exposure to 0.3 mmol/L H2O2, 4.60% of the cells were apoptotic compared with 2.80% in controls. The apoptotic cells reached the highest of percentage of 9.58% when the concentration of H2O2 increased to 2.0 mmol/L. The necrosis rate was 1.31%, 1.40%, 1.60%, 5.42%, and 9.23% as HKCs were exposed to H2O2 at concentrations of 0, 0.3, 0.5, 1.0, and 2.0 mmol/L, respectively. The necrosis rate didn't increase obviously until H2O2 concentration was increased to 1.0 mmol/L. The percentage of viable cells decreased with increasing concentration of H2O2 in contrast with apoptosis rate and necrosis rate.
SOD activity / U/ml
3.2. Effect of H2O2 concentration on cellular apoptosis and necrosis
a
120
100
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0.0
0.5
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c (H2O2) / mmol/L
b MDA content / nmol/ml
between cells and basement membrane components, such as laminine and fibronectin. Moreover, H2O2 affects the capacity of renal tubule cells to attach to the collagen IV matrix, which results in actin cytoskeleton alterations [18]. All these changes contribute to cell detachment and reduce cell viability.
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3.3. Changes in SOD and MDA levels after cell injury
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Concentration of H2O2 / mmol/L 3.3.1. Decreased SOD content Fig. 3a shows the changes in the SOD level of the medium after the exposure of HKCs to various concentrations of H2O2 for 1 h. The results indicate that H2O2 results in a reduction in SOD level. After 1 h of exposure to H2O2, SOD decreased significantly from 109.6 U/ml (control) to 99.3 and 81.1 U/ml (P b 0.05) when the concentration of H2O2 was increased from 0 to 0.5 and 2.0 mmol/L, respectively (P b 0.05). Cell membrane injury is mediated by the lipid peroxidation reaction through the generation of free radicals induced by H2O2 [4], which could be responsible for the role of reactive oxygen species (ROS) in necrotic and apoptotic cell death [19]. Renal epithelial cells exposed to H2O2 have significantly higher ROS production, increased formation of MDA as a marker of lipid peroxidation, increased release of lactate dehydrogenase (LDH) as a marker for cell injury, and loss of cell viability. As an antioxidant enzyme, SOD could terminate radical chain reactions by removing free radical intermediates [11]. Free radicals are toxic to an organism, so SOD is an important antioxidant defense for nearly all cells exposed to free radicals. SOD prevents damage from oxidation attributable to free radicals, thus protecting cells against oxidative stress. Decreased SOD levels indicate that an organism has a reduced capability to prevent free radical toxicity, thus reflecting an increase in the degree of damage in the organism [13].
Fig. 3. Changes in SOD level (a) and MDA concentration (b) of HKC after exposure to different concentrations of H2O2 for 1 h.
3.3.2. Increased MDA content Fig. 3b shows the effects of H2O2 concentration on the MDA content of HKCs. MDA level increases with increasing concentration of H2O2 when exposure time is kept constant for 1 h. MDA level increased from 0.52 to 1.03 and 1.57 nmol/ml when H2O2 concentration was increased from 0 to 0.5 and 2.0 mmol/L, respectively. This finding shows that the injury to the cell membrane was aggravated. The production of MDA can be used as a biomarker for the level of oxidative stress in an organism. Changes in MDA content can reflect the degree of lipid peroxidation, and indirectly, cell injury [20]. The enzyme system generates free radicals, which most often affect polyunsaturated fatty acids because of the multiple double bonds in their carbon chains. Free radicals induce lipid peroxidation in cell membranes, resulting in cell damage and in the production of lipid peroxides [8], such as aldehydes, ketones, hydroxyl groups, carbonyl groups, and hydroperoxides. Fig. 3a and b shows that H2O2 not only increases MDA levels, but also decreases SOD levels. H2O2 was shown to induce lipid peroxidation in the cell and simultaneously weakens the capability of the cell to scavenge for free radicals. 3.4. Osteopontin (OPN) expression after cell injury
Table 1 Effect of H2O2 concentration on cellular apoptosis and necrosis. c(H2O2)/mmol/L
0
0.3
0.5
1.0
2.0
Necrotic cells/% Apoptotic cells/% Viable cells/%
1.31 2.80 95.89
1.40 4.60 94.00
1.60 5.27 93.13
5.42 7.43 87.15
9.23 9.58 81.19
The structures on the cell surface change after cell injury. For example, OPN become expressed on the cell surface [21]. Fig. 4 shows the fluorescence images of the OPN expression of HKCs after oxidative injury. Limited green fluorescence was found on the normal cell surface (Fig. 4a), indicating that normal cells did not express or weakly expressed OPN. After exposure to 0.3 mmol/L H2O2 for 1 h, green
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fluorescence was observed on the cell surface (Fig. 4b), indicating that OPN was expressed on the cell surface after injury. When the concentration of H2O2 was increased to 0.5 and 1 mmol/L, stronger green fluorescences were seen on the cell surface (Fig. 4c and d), showing that OPN expression strengthened after severe injury. These findings imply that OPN expression increases as the degree of cell injury increases. OPN is a highly negatively charged, extracellular matrix glycoprotein with a molecular weight of approximately 44 kDa and is rich in serine, aspartic acid, and glutaminic acid, making it an acidic protein. The molecular chain of OPN also has functionally important sites related to the adhesion of mineral ions. Acidic groups and their charge density on the OPN chains are important factors affecting the binding of OPN to mineral ions. If OPN expression on cells increases, the formation and adhesion of CaOxa crystals on cells will be promoted. It can be seen from Figs. 4 & 5 that cellular surface structure is closely related to the formation of calcium oxalate crystals, and OPN was also spacely related to crystals. When the expression amount of OPN molecules on the surface of injury cells increases, that is, the green fluorescence intensity becomes stronger (Fig. 4), the number of CaOxa crystals induced by the injured cells increases (Fig. 5, Table 2), furthermore,the degree of crystals' aggregation and the proportion of COM crystals increased too. 3.5. Induction of CaOxa crystal nucleation and growth by injured HKCs Fig. 5 shows the SEM images of CaOxa crystals induced by normal HKC and injured cells exposed to different concentrations of H2O2 for 1 h. Compared with the control group (Fig. 5a), crystals induced by injured cells have several differences.
3.5.1. Crystal number A small number of crystals were induced by normal cells (Fig. 5a). This number increased significantly on injured cells, showing that normal cells have a weak capability to induce the formation of CaOxa crystals, whereas injured cells obviously facilitate the nucleation of crystals. The number of CaOxa that adhered to the cell surface was measured using ICP, and the data are listed in Table 2. The number of CaOxa crystals induced by the control group was significantly less than that induced by the injured groups. After the HKC was injured by H2O2 with different concentrations, the number of the CaOxa crystals induced increased with the increase of c(H2O2) when the concentration of c(H2O2) was within the range of 0.1–1.0 mmol/L. However, the number of crystals that adhered to the cell surface changed insignificantly when c(H2O2) > 1.0 mmol/L. These finding may be attributable to the fact that when c(H2O2) b 1.0 mmol/L, the degree of injury of HKC increased with the increase of c(H2O2), resulting in the growth of induced CaOxa crystals on the cell surface, as well as the continuous increase of active sites to which the crystals subsequently adhered. Therefore, the number of crystals that adhered on the cell surface increased gradually. However, when c(H2O2) > 1.0 mmol/L, the apoptosis rate of cells increased further, and a proportion of the cells had even died (Table 1). These dead cells fell from the culture flask and floated on the culture solution without and no longer had any cellular physiological function. In addition, because we have repeatedly washed the cells during the experiment, most of these dead cells were removed. Therefore, it is impossible for these dead cells to induce the formation of CaOxa crystals. Thus, the number of the adhered crystals did not increase.
Fig. 4. Confocal laser scanning microscopy images of OPN expression of HKC cells exposed to different concentrations of H2O2 for 1 h: (a) 0; (b) 0.3; (c) 0.5; and (d) 1.0 mmol/L. OPN (green). The bar: 20 μm.
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845
Fig. 5. SEM images of HKC cells exposed to different concentrations of H2O2 for 1 h: (a) 0; (b) 0.3; (c) 0.5; and (d) 1.0 mmol/L. Bar: 10 μm. c(CaOxa) = 0.5 mmol/L; crytallization time: 6 h, temperature: 37 ± 0.1 °C. Bar: 10 μm.
3.5.2. Crystal size No significant difference was found between crystal sizes (P > 0.05) with increasing extent of injury in HKC cells. Crystal size remained nearly constant at 5.0 ± 0.6 μm, suggesting that injured HKC could not promote the growth of CaOxa crystals. These results imply that injured HKC promote stone formation by providing more nucleating sites for crystals, not by promoting crystal growth. 3.5.3. Degree of crystals aggregation Control cells induced scattered calcium oxalate dihydrate (COD) crystals with typical pyramidal morphology on the cell surface (Fig. 5a). In contrast, some of the crystals induced by injured cells were aggregated, and the degree of aggregation increased with the degree of
injury, indicating that injured cells promote crystal aggregation. Aggregated crystals not only significantly damage the cells, but also have stronger adhesion to cells. Thus, crystal aggregation increases the risk for the formation of urinary stones on injured cells. 3.5.4. Crystal phase COD crystals with a regular morphology were predominant in the crystals induced by control cells (Fig. 5a). In contrast, injured cells induced the formation of COM crystals. The proportion of COM crystals to the total number of crystals increased gradually with the degree of cell injury. This increase had a concomitant gradual decrease
3000 d=6.17 200*
2400
Intensity / a.u
The effect of cellular calcium on CaOxa crystallization in necrotic cells is also minimal. In normal cells, the concentration of free Ca 2+ in cell membrane is about 0.05–0.15 μmol/L. However, when cells were exposed to oxidative stress, the external Ca 2+ in cells and organelles will passively flow into the cytoplasm, making the free calcium concentration rise to 1–10 μmol/L, which led to a certain degree of physiological responses such as cell necrosis and inflammation. But this concentration of Ca 2+ ions is relatively low compared with the concentration of free Ca 2+ ions in urine (1.21±0.64 mmol/L). That is, the calcium concentration in necrotic cells is too low to cause a significant increase in urinary calcium.
1800
1200
d=2.97 202
10
Table 2 Number of CaOxa crystals after co-culture with cells measured using ICP.
d=1.99 303
600
20
30
40
50
2 theta / degree
c(H2O2)/mmol/L
0
0.1
0.3
0.5
1.0
1.5
2.0
Crystals number/μg/cm2
31.9
44.6
47.1
50.1
54.5
56.3
55.4
Fig. 6. XRD pattern of CaOxa crystals induced by injured cells. c(CaOxa) = 0.5 mmol/L; c(H2O2) = 0.5 mmol/L; exposure time: 1 h; crystallization time: 6 h. The crystal faces with asterisk show COD, and those without asterisk show COM.
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in the percentage of COD crystals. When cells were exposed to 0, 0.3, 0.5, and 1 mmol/L H2O2 for 1 h, the percentage of COM crystals was approximately 0, 5%, 14%, and 19%, respectively. Fig. 6 shows the XRD pattern of the CaOxa crystals induced by injured cells exposed to 0.5 mmol/L H2O2 for 1 h. The pattern reveals that the corresponding diffraction peaks assigned to COM and COD crystals appeared simultaneously. The corresponding interplanar distance located at 6.17 Å can be assigned to the (200) plane of COD, whereas the corresponding interplanar distance located at 2.97 Å and 1.99 Å can be assigned to the ( 202) and ( 303) planes of COM [22]. The (101), (202) and (303) planes of COM crystals are exposed to more calcium ions, so they have more positive charges, whereas COD crystals have a relatively low surface charge [23]. The promotion of the nucleation and formation of CaOxa crystals by injured cells was correlated to crystal-binding molecules expressed on the cell surface. Normal cells without oxidative injury have intact surface resistance to crystal growth and binding. This is because that there is only neutral phospholipids existing outside of the normal cell membrane, the negatively charged phospholipids [such as phosphatidylserine (PS)] is located inside the cell membrane. Hence, normal cells have a weak capability to attract positively charged Ca2+ [24] and to induce the nucleation and adhesion of CaOxa crystals [25]. However, when HKCs were injured by H2O2, the microstructures of the cell surface and the cellular polar were altered. For example, the PS molecules were translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. And also some negatively charged molecules such as OPN (Fig. 4), hyaluronic acid, and collagen protein were expressed on the surface of injured cells. The increase in the expression of negatively charged molecules on the surface of injured cells can attract more calcium ions and CaOxa crystals through electronic interaction [26]. It is an important reason why injury cells have greater ability to induce crystallization of calcium oxalate crystals than normal cells. In addition, the adhesion between injured cells and COM crystals is stronger than that between injured cells and COD crystals [27], thus increasing the risk for the formation of urinary stones. Our observation confirmed that OPN molecules were expressed on the surfaces of injured HKC cells (Fig. 4). The high-density negatively charged regions in OPN molecular chain can attract and gather calcium ions [28], thereby inducing the formation of calcium oxalate crystals [29]. Animal experiments also showed that OPN has a crucial role in the morphological conversion of CaOxa crystals retained in renal tubules to matrix-involving kidney stones, possibly leading to clinical urolithiasis [30]. By using wildtype mice (WT, with OPN) and OPN knockout mice (KO, deleting OPN) as animal models and using glyoxylate to induce renal tubular injury, the crystal deposition in the kidneys of the two kinds of genotypes mice were comparatively investigated. The results showed that the number of the deposited crystals in KO was significantly fewer than in WT. Morphological observation by polarized light optical microphotography and SEM showed large flower-shaped crystals growing in renal tubules in WT and small and uniform crystals in KO. XRD results showed these crystals components in both genotypes were calcium oxalate monohydrate. 3.6. Zeta potential changes on cell surface during the induction process of crystal growth The Zeta potential of the cell surface can show the charge state of a cell membrane, which further reflects the degree of mutual exclusion Table 3 Zeta potential of HKC cells before and after crystallization/mV. c(H2O2)/mM
0
0.1
0.3
0.5
1
1.5
HKC cells HKC + crystals
− 25.2 − 26.9
− 29.2 − 29.1
− 31.9 − 27.2
− 34.3 − 25.8
− 35.2 − 24.7
− 36.1 − 25.1
or attraction between the cell surface and foreign particles. Therefore, the capability of cells to induce crystal growth and the degree of crystal adhesion to cells can be determined by measuring the Zeta potential of the cell surface during the process of crystal growth and adhesion. Table 3 shows the change in Zeta potential change after the cells induced CaOxa crystal growth for 6 h in different states, from which the following could be concluded: 1) The Zeta potential of the control group was −25.2 mV, and that of the injured group was more negative and was less than −29.2 mV. This finding can be attributed not only to the valgus of phosphatidylserine, but also to the expression of the molecules with a negative charge such as OPN (Fig. 4). A large number of negatively charged phospholipids and proteins were expressed on the cell surface and its surroundings, thereby increasing the density of negative charges on cells, thus making the Zeta potential negative. 2) The degree of damage to the cells increased with the increase of c(H2O2), consequently increasing the concentration of negatively charged molecules expressed on the cell surface. Therefore, the Zeta potential further became negative. 3) However, after the CaOxa crystal growth was induced for 6 h, the Zeta potential of the control group cells decreased, whereas the Zeta potential of the injured cells increased. This finding resulted from the capacity of the injured cells to induce crystal growth and adhesion, which was stronger than that of the control group cells. The severely injured cell surface is covered with more crystals, thus increasing the negatively charged molecules on the cell surface and decreasing the negative charge density. Therefore, the Zeta potential increased. 4. Conclusion H2O2 could significantly injure HKCs and decrease cell viability in a dose-dependent manner at concentrations of 0.1–1 mmol/L at an exposure time of 1 h. After cell injury, MDA content and OPN expression increase, whereas SOD level decreases, resulting in an increased number of CaOxa crystals and increased crystal aggregation induced by injured cells. Moreover, injured cells promote the formation of COM crystals, thus increasing the risk for the formation of urinary stones. The in vitro model of HKCs with oxidative injury established in the current study may provide insight into the pathogenesis of nephrolithiasis at the cellular level. Acknowledgments This work was supported by the National Natural Science Foundation of China (20971057), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education of China. References [1] B.A. Vervaet, A. Verhulst, S.E. Dauwe, M.E. De Broe, P.C. D'Haese, Kidney Int. 75 (2009) 41. [2] S.H. Chen, X.F. Gao, Y.H. Sun, C.L. Xu, L.H. Wang, T. Zhou, Urol. Res. 38 (2010) 7. [3] M.S.J. Schepers, E.S. van Ballegooijen, C.H. Bangma, C.F. Verkoelen, Kidney Int. 68 (2005) 1543. [4] C. Escobar, K.J. Byer, H. Khaskheli, S.R. Khan, J. Urol. 180 (2008) 379. [5] K. Sarica, A. Erbagci, F. Yagci, K. Bakir, S. Erturhan, R. Ucak, Urol. Res. 32 (2004) 271. [6] M.S.J. Schepers, E.S. van Ballegooijen, C.H. Bangma, C.F. Verkoelen, Urol. Res. 33 (2005) 321. [7] T. Rashed, M. Menon, S. Thamilselvan, Am. J. Nephrol. 24 (2004) 557. [8] S. Thamilselvan, S.R. Khan, M. Menon, Urol. Res. 31 (2003) 3. [9] S. Zhuang, Y. Yan, R.A. Daubert, J. Han, R.G. Schnellmann, Am. J. Physiol. Ren. Physiol. 292 (2007) F440. [10] C. Escobar, K.J. Byer, S.R. Khan, BJU Int. 100 (2007) 891. [11] M.W. Bigelow, J.H. Wiessner, J.G. Kleinman, N.S. Mandel, J. Urol. 160 (1998) 1528. [12] V. Kumar, G. Farell, S. Deganello, J.C. Lieske, J. Am. Soc. Nephrol. 14 (2003) 289. [13] Y.I. Rabinovich, M. Esayanur, S. Daosukho, K.J. Byer, H.E. El-Shall, S.R. Khan, J. Colloid Interface Sci. 300 (2006) 131.
S. Zhang et al. / Materials Science and Engineering C 32 (2012) 840–847 [14] B.C. Jeong, C. Kwak, K.S. Cho, B.S. Kim, S.K. Hong, J.I. Kim, C. Lee, H.H. Kim, Urol. Res. 33 (2005) 87. [15] R. Hamamoto, Y. Furukawa, M. Morita, Y. Iimura, F.P. Silva, M.H. Li, R. Yagyu, Y. Nakamura, Nat. Cell Biol. 6 (2004) 731. [16] I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutelingsperger, J. Immunol. Methods 184 (1995) 39. [17] J.M. Ouyang, X.Q. Yao, J. Tan, F.X. Wang, J. Biol. Inorg. Chem. 16 (2011) 405. [18] D. Sáenz-Moralesa, M.M. Escribesea, K. Stamatakisc, M. García-Martosa, L. Alegrea, E. Condea, D. Pérez-Salac, F. Mampasoa, M.L. García-Bermejoa, Exp. Cell Res. 312 (2006) 3711. [19] S.M. Baek, C.H. Kwon, J.H. Kim, J.S. Woo, J.S. Jung, Y.K. Kim, J. Lab. Clin. Med. 142 (2003) 178. [20] C.K. Veena, A. Josephine, S.P. Preetha, P. Varalashmi, R. Sundarapandayan, Life Sci. 79 (2006) 1789. [21] L.C. Cao, J. Jonassen, T.W. Honeyman, C. Scheid, Am. J. Nephrol. 21 (2001) 69.
847
[22] J.Y. He, S.P. Deng, J.M. Ouyang, IEEE Trans. Nanobioscience 9 (2010) 156. [23] L. Tunik, F. Hueredi-Milhofer, N. Garti, Langmuir 14 (1998) 3351. [24] J.H. Wiessner, A.T. Hasegawa, L.Y. Hung, G.S. Mandel, N.S. Mandel, Kidney Int. 59 (2001) 637. [25] S. Chutipongtanate, V. Thongboonkerd, Chem. Biol. Interact. 188 (2010) 421. [26] J.C. Lieske, S. Deganello, F.G. Toback, Nephron 81 (1999) 8. [27] M.T. Moriyama, K. Miyazawa, K. Noda, M. Oka, M. Tanaka, K. Suzuki, Urol. Res. 35 (2007) 295. [28] Y.C. Chien, D.L. Masica, J.J. Gray, S. Nguyen, H. Vali, M.D. McKee, J. Biol. Chem. 284 (2009) 23491. [29] G.K. Hunter, B. Grohe, S. Jeffrey, J. O'Young, E.S. Sørensen, H.A. Goldberg, Cells Tissues Organs 189 (2009) 44. [30] A. Okada, S. Nomura, Y. Saeki, Y. Higashibata, S. Hamamoto, M. Hirose, Y. Itoh, T. Yasui, K. Tozawa, K. Kohri, J. Bone Miner. Res. 23 (2008) 1629.