Some macromolecules 2 D map of proteins from human renal stone matrix and evaluation of their effect on oxalate induced renal tubular epithelial cell injury

ARTICLE INFO _________________________________________________________ ___________________ Vol. 39 (1): 128-136, January February, 2013 doi: 10.1590/S1677-5538.IBJU.2013.01.16 IBJU | exPloring the Matrix Proteins in huMan renal stone Matrix 129 in these systems are responsible for initiating mineralisation, defining its physical limits and dictating its cessation, but others provide an architectural framework upon which the inorganic salts are laid down. Less clearly defined, however, are the roles played by macromolecules in the formation of human uroliths, a process possessing all the hallmarks of uncontrolled mineralisation (5,6). Many proteins occur in stone, but their role in urolithiasis remains unknown. Calculi contains some proteins normally present in urine, in addition to others arising from injury inflicted by the stones themselves, making it impossible to discriminate between those that bind to the stone as it grows, but play no role in its development (7); the inhibition is generally understood to arise mainly from the non-dialyzable molecules of urine, particularly acid glycoproteins, and acidic glycoproteins and glycosaminoglycans (8,9). Some inhibitor molecules have been identified, including Tamm-Horsefall Protein, uropontin (10,11), calgranulin (12), bikunin (13), and prothrombin F1 fragment (14). Thus, in order to understand the mechanism of stone genesis, it is essential to determine the characteristics of molecules constituting the urinary stone matrix. In the present study, we analysed a 2D map (2dimensional polyacrylamide gel electrophoresis) of human renal stone matrix proteins by MALDI-TOF (Matrix-assisted laser desorption/ionization-Time of Flight) to throw light on the matrix proteins and also study their effect on oxalate injured renal epithelial cells. MATERIALS AND METHODS Human Renal stones collection Stones surgically removed by Percutaneous nephrolithotomy (PNL) from the kidney stone patients were obtained from the Department of Urology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. Stones were preserved at 4o C before study. Stones were of non-infectious nature and were collected from those patients who were more than 25 years of age and were suffering from no other abnormality. After FTIR (Fourier transform infrared spectroscopy) analysis, the stones with calcium and oxalate as their major components were selected for present study. Thirty stones with calcium and oxalate, as the major components were used for further studies. Thirty stone samples were randomly pooled into 5 groups, each group containing 6 stone samples. Protein extraction from Human Renal stones Proteins were isolated from the matrix of kidney stones containing calcium oxalate (CaOx) as the major constituent using EGTA as a demineralising agent. Stones were washed in 0.15 M sodium chloride (NaCl) and were then dried and pulverized with a mortar and pestle. For extraction of the organic matrix of powdered stone, each gram of stone was suspended in 10 mL of 0.05 M EGTA (ethylene glycol tetraacetic acid), 1 mM PMSF (phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride) and 1% β-mercaptoethanol. The extraction was carried out for 4 days at 4o C with constant stirring. The suspension was centrifuged for 30 minutes at 10,000 g and at 4o C. The supernatant of EGTA extract was filtered through Amicon ultra centrifugal filter device (Catalog UFC 800324) with a molecular weight cut off 3kDa at 4o C and concentrated to a known volume. The excess of the 3kDa fractions were stored at -20o C for further studies (15). Protein determination Total protein concentration was determined by Lowry’s method using BSA as a standard (16). Assay to measure inhibitory activity of protein w.r.t Calcium Oxalate (CaOx) crystal nucleation The method used was similar to that described by Hennequin et al. with some minor modifications (17). Solutions of calcium chloride (CaCl2) and sodium oxalate (Na2C2O4) were prepared at the final concentration of 3 mmoL/L and 0.5 mmoL/L, respectively, in a buffer containing Tris 0.05 moL/L and NaCl 0.15 moL/L at pH 6.5. Both solutions were filtered through a 0.22 μm filter; 1.5 mL of CaCl2 solution was mixed with different concentrations of extracted proteins. Crystallization was started by adding 1.5 mL of Na2C2O4 solution. The final solution was stirred at 37o C repeatedly after an interval of 60 sec for 8 min. The IBJU | exPloring the Matrix Proteins in huMan renal stone Matrix 130 absorbance of the solution was monitored at 620 nm after every 60 sec. The percentage inhibition produced by the protein extract was calculated as [1-(Tsi/Tsc)] X 100, where Tsc was the turbidity slope of the control and Tsi the turbidity slope in the presence of the inhibitor. Assay to measure activity of protein w.r.t. CaOx crystal growth Activity against CaOx crystal growth was measured using the seeded, solution-depletion assay, according to the method described by Nakagawa et al. (18). Cell Culture Madin-Darby Canine kidney (MDCK) cells were obtained from National Centre of Cell Sciences (NCCS, Pune). The cells were maintained as monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM) with 2.0 mM L-glutamine adjusted to contain 3.7 g/L sodium bicarbonate, 4.5 g/L glucose. Media was supplemented with 1% Penicillin (100 units/mL)-Streptomycin (10,000 μg/mL) and 10% fetal bovine serum. Cells were cultured in 25 cm2 tissue-culture treated flasks at 37o C and 5% CO2 in humidified chambers (19). Oxalate-induced Cell Injury MDCK cells were incubated in DMEM containing 1 mM sodium oxalate in the presence of different concentrations of protein samples for 48 hours (20). Cell injury was assessed by measuring the cell viability through MTT and LDH (Lactate dehydrogenase) Assay. Preparation of the protein samples For cell culture studies, the proteins was dialysed through Millipore Amicon Ultra Centrifugal Filters, 3kDa and desalted by ReadyPrep 2-D Cleanup Kit (catalog 163-2130) and it was reconstituted in 0.22 μm filtered distilled water using Millipore Millex GV Filter Unit 0.22 μm (Catalog SLGU033RS). MTT Assay Cell viability studies via MTT test were conducted by the method described by Fulya Karamustafa et al. with slight modifications (21). MDCK cells were suspended in DMEM with serum and plated into the microwells of 96-well tissue culture plates. Plates were incubated for 24 h at 37o C in a humidified incubator containing 5% CO2. Then the medium was removed from wells. 200 μL DMEM (without serum) containing different concentrations of proteins with and without sodium oxalate were added into the wells. After 48 hours, the medium was removed. Each well was treated with 100 μL medium and 13 μL MTT solution, and incubated for a further 3 hours. Then, plates were emptied and 100 μL isopropanol was added to dissolve the formazan precipitate. The developed colour was read at a wavelength of 570 nm with spectrophotometer. LDH Leakage Assay MDCK cells were suspended in DMEM with serum and plated into the microwells of 96-well tissue culture plates. Plates were incubated for 24 h at 37o C in a humidified incubator containing 5% CO2. Then the medium was removed from wells. 200 μL DMEM (without serum) containing different concentrations of proteins with and without sodium oxalate were added into the wells for 48 hours. LDH leakage assay was performed by the LDH Cytotoxicity Assay Kit (Cayman 10008882) according to the manufacturer’s instructions (22). Statistical analysis Data were expressed as mean values of three independent experiments (each in triplicate) and analyzed by the analysis of variance (p < 0.05) to estimate the differences between values. 2-D Gel Electrophoresis The samples were desalted using ReadyPrep 2-D Cleanup Kit and dissolved in 125 μL of sample rehydration buffer containing 8 M urea, 2% w/v CHAPS, 50 mM DTT, 0.2% w/v Ampholytes and 0.0002% bromophenol blue. IEF was first carried out using Bio-Rad IPG strip (pH 3-9; 7 cm) in Bio-Rad protean IEF cell according to manufacturer’s instructions, followed by equilibration for 15 minutes each in equilibration buffer I (6 M Urea, 2% SDS, 0.375 M Tris HCl (pH 8.8), 20% Glycerol, 130mM DTT) and equilibraIBJU | exPloring the Matrix Proteins in huMan renal stone Matrix 131 tion buffer II (6 M Urea, 2% SDS, 0.375 M Tris HCl (pH 8.8), 20% Glycerol, 135mM Iodoacetamide). Equilibrated IPG strips were loaded onto a 10% polyacrylamide gel sealed with overlay agarose, and electrophoresed at a constant voltage of 100 V. The gel was stained by silver staining and analysed using Biorad PD Quest Advanced 2D Analysis Software. The spots of interest were manually excised from the gel and were destained using destainer provided in the ProteoSilverTM Plus Silver Stain Kit (PROTSIL2, Sigma-Aldrich Co.) followed by in-gel digestion using Trypsin profile IGD kit (PPO100, Sigma-Aldrich Co.). The proteins were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) MS followed by MASCOT database search. Tryptic in-gel digestion of purified protein Single band detected after molecular-sieve chromatography was excised from the gel and was destained with destainer provided in the ProteoSilverTM Plus Silver Stain Kit (PROTSIL2, Sigma-Aldrich Co.). Trypsin profile IGD kit (PPO100, Sigma-Aldrich Co.) was used for in-gel digestion of purified protein. Destained gel piece was dried for approximately 15 to 30 min. Trypsin solubilised in 1 mmoL/L HCl and mixed with 40 mmoL/L ammonium bicarbonate and 9% acetonitrile was added to the destained gel piece. Gel piece was fully covered by the addition of 40 mmoL/L ammonium bicarbonate and 9% acetonitrile (pH 8.2) solution and was incubated for 5 hours at 37o C. After the incubation, liquid was removed from the gel piece and transferred to a new labeled Eppendorf tubes and was preserved for mass spectroscopic analysis (23). Peptide mass fingerprinting by MALDI-TOF-MS Peptides were e