Kidney stone matrix proteins ameliorate calcium oxalate monohydrate induced apoptotic injury to renal epithelial cells

    Kidney stone matrix proteins ameliorate calcium oxalate monohydrate induced apoptotic injury to renal epithelial cells Shifa Narula, Simran Tandon, Shrawan Kumar Singh, Chanderdeep Tandon PII: DOI: Reference:

S0024-3205(16)30497-0 doi: 10.1016/j.lfs.2016.08.026 LFS 15003

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

20 July 2016 17 August 2016 26 August 2016

Please cite this article as: Narula Shifa, Tandon Simran, Singh Shrawan Kumar, Tandon Chanderdeep, Kidney stone matrix proteins ameliorate calcium oxalate monohydrate induced apoptotic injury to renal epithelial cells, Life Sciences (2016), doi: 10.1016/j.lfs.2016.08.026

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ACCEPTED MANUSCRIPT Kidney stone matrix proteins ameliorate calcium oxalate monohydrate induced apoptotic injury to renal epithelial cells

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Shifa Narula1, Simran Tandon2, Shrawan Kumar Singh3 and Chanderdeep Tandon1*

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Amity Institute of Biotechnology (AIB), Amity University Uttar Pradesh, Noida, Uttar Pradesh, India

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Amity Institute of Molecular Medicine and Stem Cell Research (AIMMSCR), Amity University Uttar

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Pradesh, Noida, Uttar Pradesh, India

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Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, Punjab, India

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*Corresponding author

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Correspondence: Dr. Chanderdeep Tandon,

Professor & Director, Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India.

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Pin Code- 201313

Phone- +91- 9871003672

E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Aims: Kidney stone formation is a highly prevalent disease, affecting 8-10% of the human

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population worldwide. Proteins are the major constituents of human kidney stone’s organic

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matrix and considered to play critical role in the pathogenesis of disease but their mechanism of

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modulation still needs to be explicated. Therefore, in this study we investigated the effect of human kidney stone matrix proteins on the calcium oxalate monohydrate (COM) mediated

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cellular injury.

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Main methods: The renal epithelial cells (MDCK) were exposed to 200 µg/ml COM crystals to induce injury. The effect of proteins isolated from human kidney stone was studied on COM

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injured cells. The alterations in cell-crystal interactions were examined by phase contrast,

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polarizing, fluorescence and scanning electron microscopy. Moreover, its effect on the extent of COM induced cell injury, was quantified by flow cytometric analysis.

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Key findings: Our study indicated the antilithiatic potential of human kidney stone proteins on COM injured MDCK cells. Flow cytometric analysis and fluorescence imaging ascertained that matrix proteins decreased the extent of apoptotic injury caused by COM crystals on MDCK cells. Moreover, the electron microscopic studies of MDCK cells revealed that matrix proteins caused significant dissolution of COM crystals, indicating cytoprotection against the impact of calcium oxalate injury. Significance: The present study gives insights into the mechanism implied by urinary proteins to restrain the pathogenesis of kidney stone disease. This will provide a better understanding of the formation of kidney stones which can be useful for the proper management of the disease. Keywords: Nephrolithiasis, Calcium oxalate monohydrate (COM), Kidney stones, Matrix proteins, Modulators. 2

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1. Introduction Nephrolithiasis, the deposition of stones in kidney is one of the most painful diseases affecting

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mankind since ages. The earliest historical evidence dates back to 4800 BC when a stone was

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found in the pelvic region of a mummified Egyptian [1]. Due to its multifactorial etiology and

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high rate of recurrence, this disease poses a major health burden to society. Contingent upon the various factors including socio-economic conditions, climate, lifestyle and diet, the overall

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probability of stone formation varies markedly in various parts of the world: 1-5% Asia, 5-9% Europe, 13-15% USA, 20% Saudi Arabia and with 50% rate of reoccurrence in 10 years [2, 3].

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Kidney stones are majorly composed of calcium and oxalate (70-80%), some are calcium

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phosphate (10%) or mixture of both (40-50%) [4,5]. In addition to this inorganic composition,

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kidney stones also consist of organic matrix accounting for 2-5% of the total stone weight [6, 7]. Proteins constitute a major portion (64%) of the organic matrix of human calcium oxalate (CaOx) renal stones and are considered to play an important role in the modulation of kidney stone formation [8]. The disruption in the equilibrium between the inhibitor and promoter proteins causes de-regulation of the bio-mineralization process in the kidney, thereby leading to the formation of kidney stones. The role played by these human kidney stone matrix proteins in the course of crystallization process and stone formation is still not clearly understood. In addition, it has been reported that certain proteins can have dual activity; they can either promote or inhibit a process depending upon the urinary conditions at the time of crystallization or retention [9, 10]. Till date no permanent cure for kidney stone disease in recurrent stone formers is available, and this can majorly be attributed to the fragmentary knowledge about the molecular 3

ACCEPTED MANUSCRIPT events taking place at the time of stone formation. Therefore, it is necessary to identify biomolecules that might be of etiological importance and also get an insight into the mechanism

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of stone formation at the molecular level. In addition, various studies have emphasized on the

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significance of the interaction between CaOx crystals and renal epithelial cells in kidney stone formation. Proteins being predominant modulators in stone formation could regulate this cell-

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crystal interaction to some extent [11, 12]. Although, several proteins have been identified from

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the renal stone matrix, function of only a few proteins is reported [6, 10, 13, 14]. Deep insights into the kidney stone’s molecular make-up and its role in pathogenesis are the prerequisites for

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the proper management of the disease. Thus, in the present study we evaluated the effect of human kidney stone matrix proteins on calcium oxalate monohydrate (COM) injured renal

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2. Materials and methods

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epithelial cells and their impact on cell-crystal interactions.

2.1. Protein extraction from human kidney stones The ethical clearance for the present study was obtained from Institutional Ethical Committee of Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. Surgically removed human kidney stones were Fourier transform infrared spectroscopy (FTIR) analyzed for calcium oxalate content, the stones with calcium and oxalate as their major component were collected from the Department of Urology, PGIMER, Chandigarh. The selected calcium oxalate stones were washed in 0.15 M NaCl solution with gentle stirring for 48 h to remove adhered blood and tissue at 4 oC. For extraction of matrix proteins, the stones were pulverized to fine powder and extracted with a solution containing 0.05 M EGTA, 1 mM PMSF and 1% β-mercaptoethanol for 4 days at 4 oC. The collected whole extract was then dialyzed 4

ACCEPTED MANUSCRIPT further to remove salts by using 3kDa cut-off ultra-centrifugation Amicon filter units [13]. The

2.2. Aggregation assay of calcium oxalate (CaOx) crystals

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protein concentration was determined through Bradford’s method [15].

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Solutions of calcium chloride and sodium oxalate were prepared at the final concentration of 5

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mM and 0.5 mM respectively, in a buffer containing 10 mM sodium acetate and 200 mM NaCl at pH 5.7. Both solutions were filtered through a 0.22 μm filter (Millipore). Crystallization was

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initiated by adding 1.5 ml of sodium oxalate solution to the 1.5 ml of calcium chloride solution.

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Calcium chloride solution (1.5 ml) was mixed with 1.5 ml of sodium oxalate & 100 µl desalted extract of renal matrix proteins at different concentrations of 10 μg/ml, 25 μg/ml, 50 μg/ml, 100

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μg/ml and 200 μg/ml and the temperature was maintained at 37 °C. The final solutions were

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stirred at 37 °C for 60 min and the absorbance was monitored after every minute at 620 nm. The percentage inhibition of CaOx nucleation followed by its aggregation was calculated as [1-

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(Tsi/Tsc)] X 100, where Tsc was the turbidity slope of the control and Tsi the turbidity slope in the presence of renal calculi proteins [16, 17]. 2.3. Cell Culture

The canine kidney epithelial cell line MDCK was procured from the National Centre of Cell Sciences (NCCS) Pune, India. The cells were cultured and maintained as monolayers in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 1% penicillin G (100 units/ml)–streptomycin (10,000 μg/ml) and 10% fetal bovine serum (FBS), at 37 °C and 5% CO2 in humidified incubator (Eppendorf, New Brunswick- Galaxy 170S) [13]. 2.4. Preparation of COM Crystals Calcium oxalate monohydrate (COM) crystals were prepared by mixing 10 mM calcium chloride with 1 mM sodium oxalate to make final concentrations of 5 mM and 0.5 mM, respectively, in 5

ACCEPTED MANUSCRIPT Tris buffer containing 90 mM NaCl (pH 7.4) [18]. The mixture was incubated at 25 °C overnight and COM crystals were harvested by centrifugation at 3000 rpm for 10 min. Supernatant was

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discarded, followed by re-suspension of the crystal pellet in methanol. This suspension was

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centrifuged again at 3000 rpm for 10 min, methanol was discarded and the crystal pellet was dried to fine powder at 37 °C overnight. COM crystals were then sterilized by UV irradiation for

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30 min. The morphology of COM crystals was confirmed by observing under phase contrast

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microscope. The COM crystals were then added to DMEM to achieve the final concentration of

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200 μg/ml. 2.5. Cell viability assay

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Cell viability study was conducted using MTT cell viability assay [19]. The MDCK cells were

DMEM

(Sigma-Aldrich)

supplemented

with

10%

FBS

(Gibco)

and

1%

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in

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seeded in the microwells of 96-well tissue culture plates at the seeding density of 104 cells/well

penicillinG/streptomycin (Gibco). The plates were incubated in a humidified incubator maintained at 37 °C and 5% CO2. At semi-confluent level the medium was removed from wells and 200 μl serum free media having different concentrations of proteins with and without COM crystals were added into each well. After the incubation period of 24 h, Thiazolyl Blue Tetrazolium Bromide- MTT dye (Sigma-Aldrich) at the working concentration of 0.5 mg/ml was added into each well and the plate was incubated for further 4 h. Following this, 200 μl of Dimethyl sulfoxide (DMSO) was added to dissolve the formazan precipitate. The developed color was read at a test wavelength of 570 nm and reference wavelength of 630 nm in a microplate reader (Bio-Rad 680 microplate reader). The cell viability was determined by the following formula:

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ACCEPTED MANUSCRIPT % cell viability =

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2.6. Cell-Crystal Interaction Study

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To assess cell -crystal interaction, 105 MDCK cells were seeded onto coverslips placed in individual wells of a 6 well plates, containing DMEM medium supplemented with 10% fetal

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bovine serum (FBS), 1% penicillin G/streptomycin [20]. The cultured cells were maintained in a

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humidified incubator at 37° C with 5% CO2 for 24 h. At semi-confluent level the media of the cells was replaced by serum free media for control group, 200 µg/ml COM crystal containing

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media for COM injury group, and 200 µg/ml COM crystal + 50 µg/ml of desalted matrix protein extract for test group and the cells were incubated for 24 h and the cells images were acquired by

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using various microscopy techniques:

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2.6.1. Imaging by phase contrast microscopy Following treatment for a period of 24 h, the medium was removed and the cells were washed with PBS twice and then observed under phase contrast and polarizing microscope (Olympus CH2i, Olympus Co. Ltd. Tokyo, Japan) at 20X magnification. 2.6.2. Fluorescence microscopy (Hoechst Staining) After the incubation of 24 h, the medium was removed and the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature. After washing twice with 1X PBS, cells were stained with 5 µg/ml of Hoechst 33258 dye (Sigma-Aldrich) for 10 min at room temperature in dark. Finally, the cells were washed twice with 1X PBS and the stained nuclei were observed under DAPI fluorescence filter of Olympus microscope at 20X magnification.

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ACCEPTED MANUSCRIPT 2.6.3. Scanning Electron Microscopy (SEM) To visualise the cells subjected to various treatments by SEM, the medium was removed and the

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cells were fixed with 4% paraformaldehyde and 1% gluteraldehyde for 30 min at room

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temperature. The coverslips were then washed twice with PBS, dehydrated and air-dried overnight. The dried coverslips were then mounted on aluminum stubs and coated with gold

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particles. The crystal morphology was then examined under a scanning electron microscope

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(ZEISS EVO HD, Germany) at 2000X magnification. 2.7. Flow cytometric analysis

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2.7.1. FITC Annexin V PI assay

Apoptosis in COM crystal injured cells was detected by Annexin V: FITC Apoptosis Detection

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Kit I from BD Pharmigen as per the manufacturer’s instructions. MDCK cells were cultured in 60mm dishes in complete DMEM and maintained in a humidified incubator at 37° C with 5%

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CO2. At semi-confluent level the media of the cells was replaced by serum free media for control group, 200 µg/ml COM crystal containing media for COM injury group, and 200 µg/ml COM crystals + 50 µg/ml of desalted matrix protein extract for test group and cells were incubated for 24 h. The positive control group was treated with 10 μM of CCCP- Carbonyl cyanide 3-chlorophenylhydrazone (Sigma-Aldrich), which induces apoptosis as it leads to depolarization of the mitochondrial membrane. The cells were washed twice in 1X PBS and resuspended in 1X Binding buffer at the concentration of 106cells/ml, 100 μl of this suspension was transferred to an eppendorf and stained with 5 μl of FITC labelled Annexin V and 5 μl Propidium Iodide (PI) for 15 min in dark at room temperature. Following this, 400 μl of 1X Binding Buffer was added to each tube and cells were analyzed by flow cytometry (Accuri C6, BD Biosciences).

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ACCEPTED MANUSCRIPT 2.7.2. Active Caspase-3 Apoptosis assay MDCK cells at semi confluent level were treated with serum free DMEM for control group, 200

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µg/ml COM crystal containing media for COM injury group, and 200 µg/ml COM crystal + 50

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µg/ml of desalted stone matrix protein extract for test group and incubated for 24 h in a humidified incubator at 37° C with 5% CO2. The cells were washed twice in 1X PBS and

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resuspended in 0.5 ml of BD cytofix/cytoperm solution and incubated for 20 min as per Active

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Caspase-3 kit’s instructions (BD Horizon). The cell pellet was washed twice with 1X BD Perm/Wash buffer. The suspension was then incubated with FITC rabbit active Caspase-3

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antibody for 30 min at room temperature and analyzed by BD flow cytometer. 2.8. Statistical analysis

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All data were shown as the mean ± SEM. Data were expressed as mean values of three independent experiments (each in triplicate). The statistical analysis was done by using one-way

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ANOVA (p < 0.05) to estimate the differences between values and tested using Graph Pad prism software version 6.0. 3. Results

3.1. Crystallization assays of calcium oxalate (CaOx) crystals The protein concentrations of whole EGTA extract was 174±6.2 µg/ml and that of dialyzed extract was 93.7±4.4 µg/ml. The activity of human renal calculi extract on nucleation and growth of calcium oxalate crystals has already been reported earlier (data not shown); additionally, we studied aggregation of CaOx crystals. The whole extract showed both inhibitory and promotory activities against nucleation and aggregation of CaOx crystals, whereas the desalted renal calculi protein extract showed inhibitory effect against both CaOx crystallization assays (Figure. 1).

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ACCEPTED MANUSCRIPT 3.2. Cell viability assay The desalted fraction of renal calculi protein extract exhibited significant activity towards

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calcium oxalate nucleation, aggregation and growth assay system and hence was selected for

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bioactivity studies on renal epithelial cell line. Activity of desalted renal calculi protein extract

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was analyzed to measure the effect of these proteins on COM injured MDCK cells through MTT assay. The COM crystals induced injury to the MDCK cells which led to the significant decrease

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in cell viability from 100% to 39.55 ± 1.79 % in a span of 24 h incubation. The protein extract

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alone had no cytotoxic effect on MDCK cells, as the cell viability was 93.3 ± 2.8 % similar to that of untreated cells. Moreover, the protein extract showed protective effect on COM injured

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cells in a dose dependent manner. The concentrations of 2 µg/ml and 5 µg/ml showed no

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significant effect. However, concentrations of 10 µg/ml, 25 µg/ml and 50 µg/ml showed a significant increase in viability of COM injured cells from 39.55% to 58.48 ± 2.2 % (Figure. 2).

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3.3. Cell-Crystal Interaction

The MDCK cells were microscopically studied for cell-crystal interactions under phase contrast microscope (Figure. 3) and scanning electron microscope (Figure. 4) to get the overview of injury caused by COM crystals and cyto-protective effect of kidney stone matrix proteins. The cellular morphology of untreated and renal calculi protein extract (50µg/ml) treated cells was found to be healthy, indicating no adverse effect of renal calculi extract on the cells (Figure. 3d & 3e). The morphology of MDCK cells upon treatment with COM crystals (200 µg/ml) for 24 h led to prominent injury and marked distortion in the cellular morphology. The crystals with very sharp edges were observed. Representative phase contrast (Figure. 3g) and polarizing (Figure. 3h) images revealed the COM crystal-aggregates adhering to the renal epithelial cells in the COM treated group. These crystals which possess sharp edges have a propensity to adhere 10

ACCEPTED MANUSCRIPT tightly to the cells and can subsequently lead to injury and cell death. However, the COM crystals induced insult to renal epithelial cells was significantly undermined by human kidney

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stone matrix protein extract. The extract impaired COM crystal morphology by dissolving its

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sharp edges and making it blunt (Figure. 3j & 3k).

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Hoechst is a permeable dye that stains DNA and gives blue fluorescence. The stained DNA is intact in live cells but in case of injured cells the DNA is fragmented. The cultured

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MDCK cells were stained with Hoechst dye and microscopically studied for cellular injury and

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apoptosis under fluorescence microscope. The cells treated with protein extract alone showed healthy cellular morphology (Figure 3f) similar to that of untreated cells (Figure 3c), possessing

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evenly stained, intact and rounded nucleus. The nuclear fragmentation was observed in the form

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of scattered stained patches as the consequence of cellular injury caused by COM crystal treatment; deformed nucleus was visible in the form of distorted and uneven chromatin (inset-

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Figure. 3i). The protective effect of renal calculi protein extract was observed on COM treated MDCK cells; the extent of injury was less as compared to that of only COM treated cells, as the cellular nucleus was more intact (Figure. 3l). Similarly, this protective effect of human kidney stone protein extract was also observed in Scanning Electron Microscopic (SEM) analysis of COM injured MDCK cells. The crystals with sharp edges were observed on treatment of cells with calcium oxalate monohydrate crystals alone (Figure. 4A). However, the protein extract modulated the crystal morphology, thereby minimizing the oxalate shock to the cells. The COM crystals were significantly reduced in size and architecturally altered to blunt and dissolved edges on treatment along with renal calculi protein extract (Figure. 4B). 3.4. Flow cytometry analysis: 11

ACCEPTED MANUSCRIPT The MDCK cells were treated and analyzed by flow cytometry by two different cell death assays: FITC-labeled Annexin V-PI and Active Caspase-3 assay. Both assays differentiated

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viable cells from injured/dead cells.

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In FITC Annexin V and PI staining, the Annexin V stains apoptotic cells and PI stains the

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necrotic cells, while live healthy cells would not take any of the stains as they have intact membrane. Majority of cell death by virtue of COM crystals exposure to MDCK cells was due to

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apoptosis. The percentage of viable cells as the consequence of COM crystal injury decreased

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significantly from 95.9 ± 2.5 % in control to 37.8 ± 2.43 % in COM (200 µg/ml) treated group. However, the cytotoxic effect of COM crystals was significantly modulated in the presence of

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stone matrix proteins (50 µg/ml) as the viable cells increased from 37.8% to 57.9 ± 3.06 %. On

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treatment with CCCP (10 µM) which was used as a positive control for apoptosis, only 34.0 ± 1.02 % viable cells were detected (Figure. 5). The percentage of early apoptotic, late

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apoptotic/necrotic cells and eventually total cell death, significantly decreased in COM injured cells treated along with kidney stone matrix proteins as compared to only COM treated cells (Figure. 5A).

Active Caspase-3 flow cytometric assay was also performed to ascertain the cytoprotective potential of kidney stone matrix proteins. The level of active and inactive Caspase-3 enzyme was detected by Caspase-3 antibody. Ideally, the active Caspase-3 is well expressed in cells undergoing death through apoptosis and in case of healthy and proliferating cells the inactive isoform of Caspase-3 enzyme is expressed. Figure. 6 showed COM treated MDCK cells had only 34.6 ± 1.8 % of inactive Caspase population and remaining population \ 66 ± 2.1 % of active Caspase-3 enzyme. The kidney stone protein extract treatment on COM injured cells resulted in significant elevation in the level of inactive enzyme 62.1 ± 1.78 % with a 12

ACCEPTED MANUSCRIPT concomitant decrease in the active Caspase-3 level, owing to the protective effect of the extract to the renal epithelial cells against oxalate insult. CCCP (10 µM) induced apoptosis in cells

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decreased the level of inactive Caspase population to 24.5 ± 0.9 % and the active Caspase

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containing population was considerably increased to 75.7 ± 1.2 %.

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4. Discussion

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Nephrolithiasis is a chronic kidney disease and its incidence has been constantly rising for the past several decades. Its multifactorial etiology, being the major obstacle for the permanent cure

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of this disease, makes it even more unpredictable. The calcium oxalate induced cytotoxicity could lead to several renal pathologies, including kidney stone disease which is one of the

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serious health burdens. Proteins being major component of kidney stone organic matrix are

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considered to play a regulatory function in cell-crytsal interactions and lithogenesis inside the

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kidney [21]. Under physiological conditions, urine is generally supersaturated with respect to salts and very small crystals of CaOx are normally excreted out in urine. There are certain organic and inorganic molecules in the urine that play key role on their own or in combination, in the inhibition of crystal growth and retention within the kidney tissue. However, in pathological state the inhibitory activities of these biomolecules, especially proteins get masked by some stimulatory factors which facilitate CaOx crystal growth and retention thereby leading to the stone formation [22]. Earlier, our group identified and reported nine kidney stone matrix proteins with their activity on crystallization process [13, 23, 24]. Although, several studies have identified and characterized few proteins from kidney stones but their modulatory effect and mechanism of action are not completely understood. Therefore, the purpose of our study was to evaluate the potential of human kidney stone matrix proteins on the modulation of kidney stone formation on an in vitro renal epithelial cell line model. 13

ACCEPTED MANUSCRIPT In our study, we found that the proteins isolated from the matrix of human kidney stones have antilithiatic effect, showing approx. 20% of protective effect on COM injured MDCK cells. The

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calcium and oxalate crystallization can result in the formation two hydrated forms of crystals;

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CaOx monohydrate (COM) and CaOx dihydrate (COD) crystals. Although, both types of CaOx crystals can nucleate and adhere to renal tubular epithelial cells, several studies have indicated

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that COM being thermodynamically more stable and least soluble form, has more potent

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adhesive ability and can induce more toxic effects to renal tubular epithelial cells [25]. Therefore, calcium oxalate monohydrate crystals being more potent injury inducer were prepared

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and used for the study. The microscopic studies suggested the dissolution of COM crystals and its aggregates in the presence of stone matrix proteins, which was subsequently followed by a

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significant decrease in the extent of injured cells. This observation was in sync with in vitro calcium oxalate crystallization assay, in which the human kidney stone protein extract was found

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to be inhibitory against CaOx nucleation and aggregation. Similarly, in the scanning electron micrograph images, dissolution of the COM crystal edges and surface due to the presence of stone matrix proteins was evident. This alteration in the morphology of rigid structure like COM crystal can be attributed to the presence of crystal binding sites on these proteins, as a mode of interaction with cells and crystals. Similarly, many studies have emphasized on the crucial role of calcium binding, oxalate binding and calcium oxalate crystal binding activities of certain proteins in nephrolithiasis [26-31]. These studies have reported the significance of crystal coating by the proteins by virtue of the active binding sites on the latter, thereby inhibiting adhesion of the crystal to the epithelial cells. Lieske et al, have reported the adsorption of proteins on the crystal faces can inhibit crystal aggregation and adhesion [29].

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ACCEPTED MANUSCRIPT The flow cytometry and fluorescence microscopy analysis revealed that the mode of cell death in COM injured renal epithelial cells was through apoptosis. This observation was in

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accordance to the findings of Bigelow et al, who had also reported the mode of calcium oxalate

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induced cell injury to be apoptosis in renal cells [32]. Khan et al, have suggested that during apoptotic cell death, phosphatidylserine gets exposed on the outer cell surface. These exposed

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negatively charged headgroups of phosphatidylserine, attract calcium and thereby can promote

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crystal adhesion and binding to the renal cells [33]. The human kidney stone matrix proteins controlled the extent of COM induced apoptotic toxicity in the renal epithelial cells. In summary,

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the stone matrix proteins markedly altered COM crystal morphology; a consequence of proteincrystal interactions. This also reflects the possible defense mechanism used by these matrix

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proteins, when present in the active form in urinary conditions, against the calcium oxalate crystallization. Further, we need to assess the molecular interactions between the individual

5. Conclusion

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kidney stone matrix proteins and calcium oxalate crystal in silico.

The result of the present study highlights the crucial role played by proteins extracted from the human kidney stone matrix in nephrolithiasis. The interaction of these stone matrix proteins with COM crystals, leads to the significant alteration in the crystal morphology and subsequent attenuation of cellular injury; A putative mechanism operating in kidney by active modulators to overcome and minimize the impact of calcium oxalate crystal induced insult on renal epithelial cells can hence be put forward from this study. Conflict of interest All authors have declared no conflict of interest exists.

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ACCEPTED MANUSCRIPT Acknowledgements This study was supported by grant from the Indian Council of Medical Research (52/10/2011-

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BMS), New Delhi, India and was performed at the Amity Institute of Biotechnology, Amity

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University, Noida (UP), India.

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of calcium oxalate crystal with human phosphate cytidylyltransferase 1: a novel inhibitor purified from human renal stone matrix. J Biomed Eng., 4 (9) (2011), pp. 591–598.

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Intricate Mechanism of Urolithiasis. Plos One., 8(7) (2013), pe. 69916. 25. Thongboonkerd V, Chutipongtanate S, Semangoen T, Malasit P. Urinary trefoil factor 1 is a novel potent inhibitor of calcium oxalate crystal growth and aggregation. J. Urol.,

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27. Kalaiselvi P, Selvam R. Effect of experimental hyperoxaluria on renal calcium oxalate monohydrate binding proteins in the rat. Br. J. Urol. Int., 87 (2001), p. 110. 28. Verkoelen CF, Romijn JC, DeBruijn WC, Boeve ER, Cao CL, Schroder FH. Association of calcium oxalate monohydrate crystals with MDCK cells. Kidney Int., 48 (1995), pp. 129-38.

29. Verkoelen CF, van der Boom BG, Kok DJ, Romijn JC. Sialic acid and crystal binding. Kidney Int., 57 (2000), pp. 1072–1082. 30. Kramer G, Steiner GE, Prinz-Kashani M, Bursa B, Marberger M. Cell-surface matrix proteins and sialic acids in cell-crystal adhesion; the effect of crystal binding on the viability of human CAKI-1 renal epithelial cells. Braz J Urol Int., 91 (2003), pp. 554– 559. 31. Lieske JC, Leonard R, Toback FG. Adhesion of calcium oxalate monohydrate crystals to renal epithelial cells is inhibited by specific anions. Am J Physiol., 268 (1995), pp. 604– 612.

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ACCEPTED MANUSCRIPT 32. Bigelow MW, Wiessner JH, Kleinman JG et al. Surface exposure of phosphatidylserine increases calcium oxalate crystal attachment to IMCD cells. Am J Physiol., 272 (1997), pp. F55–F62.

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Figure 1. Percentage inhibitory activity of human kidney stone matrix protein extract against CaOx crystal aggregation. This percentage inhibition data of different concentrations of kidney stone matrix protein extract was obtained by comparing it to inhibitory activity of control experiment in triplicates. Data are mean ± SD of three independent observations (*p < 0.05, **p < 0.005, ***p < 0.0005).

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Figure 2. MTT cell viability assay: Effect of human kidney stone matrix proteins on the COM injured MDCK cells. Data are mean ± SD of three independent observations. * p < 0.05, ****p < 0.0001 versus untreated control; and ns- non significant, #p < 0.05, ##p < 0.005, ###p < 0.0005 versus calcium oxalate monohydrate treated control.

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Figure 3. Effect of kidney stone matrix protein extract on calcium oxalate monohydrate induced renal injury in MDCK cells, visualized under phase contrast (Lane 1), polarizing (Lane 2) and fluorescence microscope (Lane 3). The MDCK cells were treated with COM crystals and kidney stone matrix proteins, post incubation (24 h) these cells were fixed and stained with Hoechst 33258 dye. The Hoechst stained cells when seen under DAPI filter appear blue in color, inset images are the zoomed in images at 20X magnification to show the changes in morphology. Group A. Control (Untreated MDCK cells); Group B. Kidney stone matrix protein extract (50 ug/ml) treated MDCK cells; Group C. COM (200 ug/ml) treated MDCK cells; Group D. COM (200 ug/ml) + kidney stone matrix protein extract (50 ug/ml) treated MDCK cells. (Original Magnification- 20X) 21

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Figure 4. Scanning electron microscopic images of COM crystal adhered on the monolayer of MDCK cells; A. Only COM (200 ug/ml) treated; sharp edged COM crystal and cells with ruptured morphology as shown by arrows, B. COM (200 ug/ml) + stone matrix proteins (50 ug/ml) treated; shows blurred and dissolved borders (illustrated with arrows) of COM crystals.

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Figure 5. Representative Flow cytometric analysis images of cell death of MDCK cells treated with COM crystals (200 μg/ml) and kidney stone matrix proteins (50 μg/ml). Apoptosis and necrosis of these cells were evaluated using FITC labeled Annexin V and PI staining. The viable cells are the percentage of population in the left lower quadrant (live cells) and the percentage of cell death was determined by a summation of the number of cells in the right lower quadrant (early apoptotic cells), right upper quadrant (late apoptotic cells and/or necrotic cells) and left upper quadrant (necrotic cells), and compared to total cell count in all four quadrants. A. Unstained Control, B. Stained Control, C. Apoptosis Control- 10 uM CCCP, D. COM (200 ug/ml), E. COM (200 ug/ml) + Protein (50 ug/ml), F. Only Protein (50 ug/ml) treated.

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Figure 5A. Graphical representation of cell death caused by exposure of COM crystals to MDCK cells, detected by Annexin V/ PI flow cytometric analysis for apoptosis and necrosis. Data are mean ± SD of three independent observations. ****p < 0.0001 versus untreated control; and #p < 0.05, ##p < 0.005 versus calcium oxalate monohydrate treated control.

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Figure 6. Flow cytometric analysis of cell death through apoptosis in MDCK cells incubated with COM crystals (200 μg/mL) and kidney stone matrix proteins (50 μg/ml). Cell death by the virtue of apoptosis was evaluated using Active Caspase-3 flow cytometric apoptosis assay. The M1 population (left plot) is the percentage of cells with inactive Caspase-3 and they are the viable cells; M2 population (right plot) is the percentage of cells having active Caspase-3 enzyme, showing apoptotic or dead cells. A. Untreated MDCK cells, B. Only protein (50 ug/ml), C. 10 uM CCCP treated. D. COM crystals (200 ug/ml), E. COM (200 ug/ml) + Protein (50 ug/ml)

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