Screening method for preventive or therapeutic drug for urolithiasis, established cell line therefor, and preventive or therapeutic drug for urolithiasis

Abstract

Disclosed is a novel in-vitro screening method capable of efficiently carrying out primary screening for a prophylactic or therapeutic drug for urinary calculosis. Also disclosed is a novel established cell line that can be used for the screening. The method for screening a prophylactic or therapeutic drug for urinary calculosis includes culturing, in the presence of an analyte, of cells derived from proximal tubule, the cells capable of forming calcareous deposit in in-vitro culture. The suppression of calcareous deposit formation indicates a high probability that the analyte has the effect of preventing or suppressing the urinary calculosis.

Description

[Technical Field]

[0001] The present invention relates to an in vitro method for screening for preventive or therapeutic agents for urolithiasis, an established cell line therefor, and a preventive or therapeutic agent for urolithiasis. [Background technology]

[0002] Urolithiasis is a common disease in which stones composed of calcium oxalate (sometimes abbreviated as "CaOx" herein) or calcium phosphate (sometimes abbreviated as "CaP" herein) obstruct the urinary tract, causing severe pain during an attack. Currently, urolithiasis is primarily treated by non-invasive surgical treatments such as extracorporeal shock wave lithotripsy (ESWL), transurethral ureterolithotripsy (TUL), and percutaneous nephrolithotripsy (Non-Patent Document 1).

[0003] Although urolithiasis can be treated by these surgical treatments, there is a problem of a high recurrence rate (approximately 50% within 5 years) (Non-Patent Document 2). Methods for preventing the recurrence of urolithiasis include increased fluid intake, dietary therapy, and exercise therapy, but no prophylactic drugs are used (Non-Patent Document 3). As is clear from the recommendations for dietary therapy and exercise therapy, urolithiasis is a disease of metabolic syndrome and is a risk factor for hypertension (Non-Patent Document 4), myocardial infarction (Non-Patent Document 5), and chronic renal failure (Non-Patent Document 6). In particular, the association between obesity, diabetes, and urolithiasis is considered almost certain (Non-Patent Document 7).

[0004] Therefore, in order to prevent the progression of these serious chronic diseases, the search for and solution of methods for preventing the recurrence of urolithiasis and for treatment are also important medical and economical urgent issues to be resolved. However, it is not easy to continue the recommended dietary therapy and intake of large amounts of fluid every day for many years, and there is a strong desire to provide an effective preventive drug.

[0005] One screening method for urolithiasis preventive drugs is a cell-free screening method based on the supersaturation theory. Specifically, a model system for calcium oxalate crystal formation by combining calcium chloride and oxalic acid in an artificial urine composition is known as an in vitro experimental system (without involving cells) based on the physicochemical "supersaturation theory." Based on the theory that the initial stages of urolithiasis based on the supersaturation theory theoretically proceed chronologically from nucleation to crystal growth, followed by crystal aggregation and finally to cell adhesion, the results of studies on numerous different compounds have been systematically summarized by step (Non-Patent Document 8). However, none of the numerous candidate compounds identified by the supersaturation theory have yet been used to prevent urolithiasis.

[0006] On the other hand, known in vitro screening systems using cells include the NRK-52E cell line derived from rat proximal tubules, the MDCK cell line derived from dog distal tubules, the Vero cell line derived from African green monkey kidney proximal tubules, and the HK-2 cell line derived from human fetal proximal tubules.

[0007] For example, it has been reported that 14 types of herbal medicines have an inhibitory effect in an evaluation system of cell damage induced by the adhesion of completely synthetic calcium oxalate crystals to canine distal tubule cells (MDCK strain) (Non-patent document 9).

[0008] Furthermore, among the above cell lines, MDCK cells have the remarkable property of spontaneous calcification. Specifically, it has been shown that when MDCK cells are cultured for a long period of 30 days in a standard DMEM medium containing 10% FBS, microcalculi composed of calcium phosphate are deposited and formed on the basement membrane side of the cells (between the basement membrane and the culture substrate) in two-dimensional planar culture (Non-Patent Documents 10 and 11).

[0009] It is known that alendronate, a drug used to treat osteoporosis, has an inhibitory effect on the formation of minute CaP crystals that form in three-dimensional soft agar cultures of MDCK cells in this spontaneous calcification system (Non-Patent Document 12). Based on the results of this in vitro MDCK spontaneous calcification system, alendronate has been evaluated in clinical trials in patients with postmenopausal osteoporosis as a substance that may be effective in preventing not only osteoporosis but also urinary tract stones (Non-Patent Document 13). [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Kenjiro Gun, ed., All about urinary tract stones, Igaku-Shoin, Tokyo, 2008 [Non-patent document 2] Saigal, C.S., et al., Kidney Int., 68, 1808-1814, 2005 [Non-patent document 3] Khan, SR et al., 2016, Nature Rev. Dis. Primers, 2, 16008 [Non-patent document 4] Madore, F. et al., Am. J. Hypertens., 11, 46-53, 1998 [Non-Patent Document 5] Rule AD et al., J. Am. Soc. Nephrol., 21, 1641-1644, 2010 [Non-patent document 6] Rule, AD et al., Clin. J Am. Soc. Nephrol., 4, 804-811, 2009 [Non-Patent Document 7] Khan, SR, Urol. Res., 40, 95-112, 2012 [Non-patent document 8] Aggarwal, KP et al., Biomed. Res. International, 2013, Article 20, 292953 [Non-Patent Document 9] Nishihata, M. et al., Int. J. Urol., 20, 1032-1036, 2013 [Non-Patent Document 10] Kageyama, S. et al., Int. J. Urol., 3, 23-26, 1996, [Non-Patent Document 11] Naito, Y. et al., Urol. Res., 25, 59-65, 1997 [Non-Patent Document 12] Senzaki, H. et al., Urol. Res., 32, 223-228, 2004 [Non-Patent Document 13] Yasui, T et al., CLINICAL CALCIUM, 21, 1511-1515, 2011 Summary of the Invention [Problem to be solved by the invention]

[0011] A screening method using in vitro culture of MDCK cells (hereinafter referred to as "MDCK screening" for convenience) is effective for discovering preventive drugs for urolithiasis, and as mentioned above, several compounds effective for preventing urolithiasis have been found so far. However, the preventive effect of these compounds for urolithiasis is not necessarily satisfactory, and therefore, none of them has been put to practical use.

[0012] To screen for compounds that have practical and excellent urinary stone prevention effects, it is necessary to test a very large number of compounds. Naturally, the preventive effect must be confirmed through animal experiments and subsequent clinical trials, but animal experiments are time-consuming and expensive, so it is unrealistic to conduct animal experiments on all compounds. A more realistic approach is to find promising candidate compounds through primary in vitro screening, such as MDCK screening, and then subject only the promising compounds found to animal testing.

[0013] MDCK screening is an excellent method because it naturally calcifies even when cultured in a normal culture medium without the need for any special external stimuli, and some promising compounds have been discovered using this method. However, it is difficult to say that this is a highly efficient (high-throughput (HTS)) screening method, as it requires microscopic observation and quantification of MDCK cell spheroids cultured in soft agar. In order to subject more compounds to primary screening than before, it is desirable to provide a new HTS screening method.

[0014] Therefore, an object of the present invention is to provide a novel in vitro screening method that enables primary screening of preventive agents for urolithiasis more efficiently than MDCK screening. Another object of the present invention is to provide a novel established cell line that can be used for such screening. A further object of the present invention is to provide a novel preventive or therapeutic agent for urolithiasis discovered by the screening method of the present invention. [Means for solving the problem]

[0015] As a result of extensive research, the present inventors have succeeded in establishing a novel cell line from the proximal tubules of dogs with renal cancer that spontaneously forms calcified foci when cultured in vitro. They have also found that this novel established cell line forms more calcified foci than the above-mentioned MDCK cells, and that the inhibition of calcification can be easily evaluated not only by colorimetric quantification of staining results but also by visual observation. They have conceived that an in vitro screening method using this established cell line in the same way as MDCK cells can enable highly efficient screening of preventive drugs for urolithiasis, and have completed the present invention. Furthermore, by actually conducting screening using the screening method of the present invention, they have found that lactoferrin (sometimes abbreviated as "LF" in this specification) is effective in preventing or treating urolithiasis.

[0016] That is, the present invention provides the following. (1) A method for screening for a preventive or therapeutic agent for urolithiasis, comprising culturing cells derived from proximal tubules, which are capable of forming calcified foci when cultured in vitro, in the presence of a test substance, wherein inhibition of the formation of calcified foci indicates a high possibility that the test substance has a preventive or therapeutic effect on urolithiasis. A method for screening for a preventive or therapeutic agent for urolithiasis, wherein the cells are an established cell line derived from the proximal tubules of an animal suffering from kidney cancer, and the established cell line is a CJ cell deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE P-03184. (2) The cells are cultured using (i) Dulbecco's MEM (High Glucose) or α-MEM containing fetal bovine serum, or (ii) Dulbecco's MEM (Low Glucose), Dulbecco's MEM / Ham-F12, or Eagle's MEM as a medium. (1) How to post. (3) The cells are cultured in a serum-free, growth factor-containing, synthetic medium with known components. Or (2) How to post. (4) The method according to (3), wherein the cell growth factor is at least one selected from the group consisting of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). (5) The cells are cultured using a glyoxylic acid-containing medium as a medium. (4) 10. The method according to any one of claims 1 to 9. (6) The degree of calcification of the cultured cells is determined by Alizarin Red S staining and / or Yasue staining. (5) 10. The method according to any one of claims 1 to 9. (7) The culture is performed by juxtaposition or comparative culture with osteoblast and / or odontogenic cell lines, (1) to (6) 10. The method according to any one of claims 1 to 9. (8) An established cell line derived from the proximal tubules of animals with renal cancer, which can form calcified foci when cultured in vitro. The established cell line is a CJ cell deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE P-03184. . [Effects of the Invention]

[0017] The present invention provides a novel screening method and a novel established cell line useful for the primary screening of drugs for preventing or treating urolithiasis. The screening method of the present invention uses cells that form calcified foci upon in vitro culture, similar to MDCK screening. Therefore, the inhibitory effect of a test substance on urolithiasis formation can be evaluated by assessing the degree of inhibition of the formation of calcified foci (hereinafter sometimes referred to as "calcification").

[0018] In the screening method of the present invention, the calcification level of the cells used is greater than that of MDCK cells, and the degree of calcification inhibition can be determined by visual observation as well as colorimetric quantification after staining without using a microscope, so that the screening efficiency is high and a large number of compounds can be screened in a short time. Therefore, the possibility of discovering a preventive or therapeutic drug for urolithiasis that exerts excellent pharmacological effects is increased. Furthermore, the screening method of the present invention has completely novelly discovered that lactoferrin has excellent preventive and therapeutic effects for urolithiasis. [Brief explanation of the drawings]

[0019] [Figure 1]This figure explains the key points of the present invention and the clear differences (from the perspective of calcification and decalcification) between similar prior art examples. It shows the background of the field and the key points of the present invention relative to prior art. The left side of the figure, centered on lactoferrin, shows the bone formation-promoting effect of lactoferrin on calcium metabolism in bone, a normotopic calcified tissue, along with abundant literature information. Meanwhile, calcium metabolism in stones, an ectopic calcified tissue, shown in the right frame of the figure, was extremely scarce and virtually unknown until the new CJ cells of the present invention were obtained. The arrows indicate that calcium deposition and shedding in bone tissue and stone tissue are completely opposite directions, respectively, using only lactoferrin as a single substance.

[0020] In bone tissue, calcification is carried out by mesenchymal osteoblasts, and decalcification is carried out by osteoclasts derived from the hematopoietic system, but the calcification and decalcification functions of the CJ cells of the present invention are both carried out by glandular cells derived from renal tubules, which are clearly different from the cells responsible in bone tissue. In order to clarify the significance of the present invention, it is essential to clarify the positions of the cells that exhibit similar functions involved, and it will be easier to consider the matter if we give provisional names to cells that are similar in properties but by no means identical.

[0021] Therefore, in the present invention, CJ cells that have reached the logarithmic growth phase or dome formation but have not yet begun calcification under a microscope are considered to be glandular epithelial cells, epithelial-like cells, or glandular cells. On the other hand, CJ cells that have begun calcification are provisionally named "stoneblasts," as they are cells that produce stones. Furthermore, CJ cells that produce CaOx crystals through glyoxylic acid induction are also stoneblasts, but at the stage where the CaOx crystals have subsequently progressed to dissolution and disintegration, they are provisionally named "stoneclasts," as they are phagocytes derived from CJ cells that have acquired the ability to phagocytose CaOx crystals.

[0022] [Figure 2]Figures 2(a) and 2(b) show the morphology of CJ cells established in the present invention under a phase-contrast microscope, images of natural calcified foci, and images of calcified foci visually observed with the naked eye. Figure 2(a) shows the morphology of CJ cells adherently grown and spread on a standard dish for adherent cells. Figure 2(b) shows the morphology of CJ cells adherently grown on a dish whose surface has been treated with collagen. Figure 2(c) shows calcified foci scattered on the bottom of a Corning flask for culturing adherent cells (C25 flask). Figure 2(d) shows calcified foci formed extensively by CJ cells (photographed at 40x magnification). Figure 2(e) shows calcified foci in the same field of view as Figure 1(d) (photographed at 100x magnification). Figure 2(f) shows calcified foci in the same field of view as Figure 1(d) (photographed at 200x magnification).

[0023] [Figure 3] Figure 3(a) shows a comparison of CJ cells and MDCK cells double-stained with Alizarin Red S (sometimes abbreviated as "AR" herein) and Light Green (sometimes abbreviated as "LG" herein). Figure 3(a) shows a double-stained image of CJ cells cultured in a C25 flask; calcified foci are stained red and cells are stained green. Figure 3(b) shows a double-stained image of CJ cells cultured in a collagen-treated dish; calcified foci are stained red and cells are stained green. Figure 3(c) shows a double-stained image of MDCK cells cultured in a C25 flask. No red-stained calcified foci were detected, and only green-stained cells were present. Figure 3(d) shows a double-stained image after 30 days of incubation of culture medium alone in a C25 flask. No red-stained deposits or precipitates were detected in the green-stained cells.

[0024] [Figure 4] These are images of alkaline phosphatase (sometimes abbreviated as "ALPase" herein) staining in CJ cells and MDCK cells. Figure 4(a) is an image of CJ cells stained with ALPase (x100). Figure 4(b) is an image of MDCK cells stained with ALPase (x100).

[0025] [Figure 5-1]Figure 5(a) shows a comparison of the spontaneous calcification abilities of CJ cells and MDCK cells. CJ cells and MDCK cells were used as test cells. Cell concentrations were varied on the first day of the experiment (day 0). On day 8, all wells were washed, fixed with 95% EtOH, and then stained with AR (left half) and crystal violet (CV) (right half). Figure 5(b) shows the results of extraction and quantification of CV dye in the wells shown in Figure 4(a). The cell number increased over time depending on the number of days of culture (only three CJ cell concentrations are plotted in the figure). Figure 5(c) shows the results of extraction and quantification of AR dye in the wells shown in Figure 4(a). This figure also shows the progression of calcification over time for three CJ cell concentrations (corresponding to Figure 4(b)) seeded on day 0. The progression of calcification was observed depending on the initial cell number seeded. Figure 5(d) shows the quantification of the increase in cell number over time for three different MDCK cell concentrations seeded on Day 0. The increase in cell number over time was quickly detected and quantified by the amount of dye, depending on the number of cells seeded initially. Figure 5(e) shows the quantification of the progression of calcification over time for three different MDCK cell concentrations seeded on Day 0. No tendency for calcification was observed in the extract after AR staining, regardless of the number of cells seeded initially. [Figure 5-2] Figure 5(f) shows a comparison of the time-dependent growth curves of CJ cells and MDCK cells measured using the commercially available cell counting reagent, Cell Counting Kit-8 (Dojin Chemical Research Institute). CJ cells and MDCK cells proliferated over time with approximately the same doubling time, which is consistent with the results of the growth curves obtained using the CV staining method.

[0026] [Figure 6]This figure shows the effect of fetal bovine serum (FBS) on the spontaneous calcification of CJ cells and MDCK cells. CJ-AR: Values ​​obtained by quantitating the calcified portions of CJ cells on the day of assessment after AR staining and extraction with 5% formic acid. MDCK-AR: Values ​​obtained by quantitating the calcified portions of MDCK cells on the day of assessment after AR staining and extraction with 5% formic acid. CJ-CV: Values ​​obtained by quantitating the CJ cells adhering to the bottom of the wells on the day of assessment after CV staining. MDCK-CV: Values ​​obtained by quantitating the MDCK cells adhering to the bottom of the wells on the day of assessment after CV staining. Results are shown as mean ± SD (n=6).

[0027] [Figure 7] Figure 7(a) illustrates the effect of DMSO on the spontaneous calcification of CJ cells. Figure 7(a) shows the time course of the effect of DMSO at a final concentration of 0.1% (v / v) on the calcification of CJ cells (initial cell number: 6 x 103 cells / well). Results are shown as mean ± SD. Figure 7(b) shows the time course of the effect of DMSO at a final concentration of 0.1% (v / v) on the calcification of CJ cells (initial cell number: 1.2 x 104 cells / well). Results are shown as mean ± SD. Figure 7(c) shows the time course of the effect of DMSO at a final concentration of 0.1% (v / v) on the calcification of CJ cells (initial cell number: 2.5 x 104 cells / well). Results are shown as mean ± SD. Figure 7(d) shows the effect of adding DMSO at a final concentration of 0.1% (v / v) on the calcification of CJ cells over time (initial cell number: 5 x 104 cells / well). The results are shown as mean ± SD.

[0028] [Figure 8]Figure 8(a) shows the validation of the spontaneous calcification ability of CJ cells in a calcium phosphate stone formation system (plate staining images). Figure 8(a) shows an image of an air-dried plate after fixation and AR staining on day 8 of validation of calcification evaluation using a 96-well multiwell plate. Figure 8(b) shows an image of an air-dried plate after fixation and AR staining on day 10 of validation of calcification evaluation using a 96-well multiwell plate. Figure 8(c) shows an image of an air-dried plate after fixation and AR staining on day 12 of validation of calcification evaluation using a 96-well multiwell plate. The top table in Figure 8(d) is a heat map of the measured values ​​(Ca content) obtained by AR staining, extraction, and OD 450 nm measurement using the plate fixed on day 10 in Figure 7(b). The table at the bottom is a heat map of the measured values ​​(reflecting the number of cells adhered to the bottom of the well) obtained by discarding the extract and washing the plate with water, followed by post-staining with CV, extraction, and OD 595 nm measurement on the same plate.

[0029] [Figure 9] Figure 9 shows a summary of validation parameters for spontaneous calcification of CJ cells. An experiment similar to that in Example 7 was performed twice. Figure 9 summarizes the results of each experiment, along with the types and significance of validation parameters, and also lists the desired pass criteria for a high-throughput screening system.

[0030] [Figure 10]This figure shows the clear influence of different media on the spontaneous calcification of CJ cells. Figure 10(a) shows the visual and qualitative results of the degree of calcification of CJ cells in six commercially available cell culture media using 24-well plates, as determined by AR staining and Von Kossa staining (bottom two rows), and the adhesion and proliferation status of CJ cells using CV staining and Giemsa staining (top two rows). CJ cells were plated at 2 x 105 cells / ml / well on day 0, washed with PBS(-) on day 10, fixed with 95% ethanol (sometimes abbreviated as "EtOH" herein), air-dried, and then stained. Figure 10(b) shows the quantitative results of the degree of calcification of CJ cells in five commercially available cell culture media using AR staining and extraction, and the adhesion and proliferation status of CJ cells using CV staining and extraction (n = 6) using 96-well plates. Test cells were plated at 4 x 104 cells / 200 μl / well (day 0), washed with PBS(-) on day 10, fixed with 95% EtOH, air-dried, stained, extracted, and then colorimetrically quantified. The results are shown as mean ± SD.

[0031] [Figure 11] This figure shows that, of the three oxalate precursors, only glyoxylic acid (hereinafter sometimes abbreviated as "GA") can induce CaOx crystal formation. Figure 11(a) shows a phase-contrast image of CJ cells (10 PDLs) treated with only the vehicle HBSS(-) on day 2, taken at 200x magnification on day 9. Figure 11(b) shows a phase-contrast image of CJ cells (10 PDLs) treated with 10 mM ethylene glycol on day 2, taken at 200x magnification on day 9. Figure 11(c) shows a phase-contrast image of CJ cells (10 PDLs) treated with 10 mM sodium glycolate on day 2, taken at 200x magnification on day 9. Figure 11(d) shows a phase-contrast image taken at 200x magnification on day 9 after adding 10 mM glyoxylic acid monohydrate to CJ cells (109 PDLs) on day 2. The formation of calcium oxalate dihydrate crystals with a typical shape is observed.

[0032] [Figure 12]Figure 12(a) shows an image of calcium oxalate crystal dihydrate aggregates formed by CJ cells upon addition of GA to a final concentration of 10 mM. Figure 12(a) was photographed at 100x magnification under a phase-contrast microscope on the sixth day after GA addition, after washing with PBS(-) and fixation with 95% EtOH. The image shows spherical, pseudopodia-mediated aggregates of multiple crystals. Figure 12(b) is a 200x magnification image of the same field of view as Figure 11(a). Figure 12(c) is a 400x magnification image of the same field of view as Figure 11(a). Figure 12(d) is a 100x magnification image of crystal aggregates from a different field of view than Figure 11(a). Numerous calcium oxalate crystal aggregates are clearly visible, centered around fibrous structures presumably derived from FBS. Figure 12(e) is a 200x magnification image of the same field of view as Figure 11(d). Figure 12(f) is an image taken at 400x magnification with the same field of view as Figure 11(d).

[0033] [Figure 13] This figure shows a model of a mixed CaOx and CaP crystal stone produced by CJ cells after shifting from a glyoxylic acid (GA)-containing medium to a GA-free medium. Figure 13(a) is a 200x magnification image taken with a phase-contrast microscope. A group of CaP crystals is easily visible in the center, with numerous CaOx crystals to the left. Figure 13(b) shows the coexistence of CaOx and CaP crystals in a different field of view from Figure 12(a). CaOx crystals being swallowed by the CaP crystal mass can be just barely distinguished at the 2 o'clock and 6 o'clock positions of the central CaP crystal mass. Figure 13(c) shows the beginning of CaOx disintegration in a different field of view. Figure 13(d) shows the disintegration of CaOx in a different field of view, with particularly noticeable disintegration of CaOx crystals in the center.

[0034] [Figure 14]Figure 14(a) shows the formation and disintegration of CaOx· dihydrate crystals induced by glyoxylic acid (phase-contrast images in EtOH fixative). Figure 14(a) shows CaOx crystals formed 6 days after the addition of GA at a final concentration of 10 mM (8 days after seeding CJ cells). The image was taken at 400x magnification in 95% EtOH fixative after removing the culture medium and washing with PBS(-). The crystal surface has lost its luster. Figure 14(b) shows an image of a CaOx crystal in 95% EtOH fixative from a different field of view in the same well as Figure 13(a). Small spherical cells can be seen covering the ridges of the crystal at the 5 o'clock and 11 o'clock positions. Figure 14(c) shows an image of a CaOx crystal in 95% EtOH fixative in a different well of the plate used in this example. Linear grooves extending from 10 o'clock to 6 o'clock are visible on the crystal surface, and small spherical cells are also present nearby. Figure 14(d) shows an image of a CaOx crystal in 95% EtOH fixative in another well of the plate. Missing areas are visible at the 8 and 9 o'clock edges of the crystal, and two cells, identified by small spherical nuclei, are visible adhering to the crystal surface at the 1 and 2 o'clock positions. Figure 14(e) shows a photograph of the intracellular vacuole or vacuole stained with Oil Red O after treatment with 10 mM GA. The intracellular sac-like structures do not stain at all with Oil Red O, indicating that they do not contain lipids. Figure 14(f) shows a Giemsa stained image of the same specimen as in Figure 13(e), which did not stain with Oil Red O. The frames of various sizes of structures within the intracellular space are stained as a meshwork.

[0035] [Figure 15]This figure shows the biological degradation of CaOx crystals by "lithoclasts (tentative name)" induced from CJ cells and the change in the form of Ca within the lithoclasts. Figure 15(a) is an AR-stained image of a CJ cell line containing calcium oxalate dihydrate crystals formed by the addition of GA. Only a portion of the upper surface of the crystal is stained red with a clearly defined irregular border. This image clearly depicts the appearance of the "lithoclasts" tentatively defined in this invention, which are presumed to be semi-professional phagocyte-like cells transformed from CJ cells. Photographed at 200x magnification. Figure 15(b) is a different view of Figure 14(a) photographed at 400x magnification. A red-stained amoeba-like object (cell body) covers and bridges the two central regular octahedral crystals. Because calcium oxalate is unstainable by AR, the intense red staining of the "lithoclasts" (tentative name in this study) strongly suggests that the Ca ions derived from the ingested CaOx were converted back into calcium phosphate or carbonate within the cell cytoplasm. Figure 15(c) shows an image of the same specimen after the AR staining shown in Figures 14(a) and 14(b) in this study, but with additional Yasue staining, including pretreatment with 5% acetic acid (sometimes abbreviated as "AcOH" in this specification), which selectively dissolves and removes CaP. In the center of the tetrahedral crystal, the undigested CaOx (unstainable by AR) that was not absorbed by the "lithoclasts" (tentative name in this study) appears black and prominent due to Yasue staining, which stains only CaOx. This image was taken at 200x magnification. Figure 15(d) shows the same image as Figure 14(c), taken at 400x magnification, of a crystal in a different well.

[0036] [Figure 16]These figures show that CaOx crystal formation and decay also occur in cloned CJ cell lines. Figure 16(a) shows an image of spontaneous calcification with strong AR staining in the CJ-5H5 clone, which was isolated and expanded from the parent CJ cell line. Photographed at 200x magnification. Figure 16(b) shows an image of spontaneous calcification with strong AR staining in the CJ-6G4 clone, which was isolated and expanded from the parent CJ cell line. Photographed at 200x magnification. Figure 16(c) shows an image of the CJ-7E12 clone, which was isolated and expanded from the parent CJ cell line. Photographed at 200x magnification, it shows a partially calcified, AR-stained area intermixed with the majority of the AR-unstained cell sheet. Figure 16(d) shows an unstained, phase-contrast image clearly showing the formation and decay of CaOx in the CJ-5H5 clone, which was isolated and expanded from the parent CJ cell line after the addition of GA to a final concentration of 10 mM. Photographed at 400x magnification. Figure 16(e) is an unstained, phase-contrast image (photographed at 400x magnification) clearly showing the CaOx formed and its decay in the CJ-6G4 clone, which was isolated and grown from the parent CJ cell line, after the addition of GA to a final concentration of 10 mM. Figure 16(f) is an unstained, phase-contrast image (photographed at 400x magnification) of the CJ-7E12 clone, which was isolated and grown from the parent CJ cell line, after the addition of GA to a final concentration of 10 mM. CaOx crystals precipitated as early as 48 hours after the addition of GA, and their decay also progressed more rapidly than in the other two clones.

[0037] [Figure 17]This figure shows the time difference between the onset of CaOx crystal formation and decay in different CJ cell clones. Figure 17(a) is a phase-contrast image taken two days after adding 10 mM GA to CJ parent cells. Figure 17(b) is a phase-contrast image taken three days after adding 10 mM GA to CJ parent cells. Figure 17(c) is a phase-contrast image taken four days after adding 10 mM GA to CJ parent cells. The appearance of CaOx crystals is observed on day four. Figure 17(d) is a phase-contrast image taken eight days after adding 10 mM GA to CJ parent cells. Although spherical cells adhere to the surface of the CaOx crystals on day eight, the surface remains smooth. Figure 17(e) is a phase-contrast image taken two days after adding 10 mM GA to CJ-3C1 clone cells. Figure 17(f) is a phase-contrast image taken three days after adding 10 mM GA to CJ-3C1 clone cells. The appearance of CaOx crystals can be observed on day 3, one day earlier than in the parent CJ cells. Figure 17(g) shows a phase-contrast image taken on day 4 after adding GA to CJ-3C1 clone cells at a final concentration of 10 mM. Spherical cell adhesion to the CaOx crystal surface and various sizes of coarse debris resulting from cell fragments or disintegration of disintegrated CaOx crystals are clearly visible. Figure 17(h) shows a phase-contrast image taken on day 8 after adding GA to CJ-3C1 clone cells at a final concentration of 10 mM. The image clearly shows the further disintegration and dissolution of the CaOx crystals. The 15 μm-sized, dark-brown, rectangular objects with irregular edges in the field of view are the digested residue of the octahedral CaOx crystals. An increase in the number of various sizes of coarse debris resulting from cell fragments or disintegration of disintegrated CaOx crystals is also clearly visible, clearly distinguishing this from the image taken on day 8 of the parent CJ cells (Figure 16(d)).

[0038] [Figure 18]These figures show the anti-lithiasis effects of bisphosphonates and resveratrol on spontaneous calcification of CJ cells. Figure 18(a) shows the dose-dependent anti-lithiasis effect of risedronate on spontaneous calcification of CJ cells. At a final concentration of 10-5M, approximately 60% of the anti-lithiasis effect was observed compared to the control, but at 10-fold higher concentrations (10-4M), the cell count significantly decreased. Each measurement point is shown as the mean value of n=4. Figure 18(b) shows the dose-dependent anti-lithiasis effect of alendronate on spontaneous calcification of CJ cells. At a final concentration of 10-5M, approximately 40% of the anti-lithiasis effect was observed compared to the control, but at 10-fold higher concentrations (10-4M), the cell count significantly decreased. Each measurement point is shown as the mean value of n=4. Figure 18(c) shows the dose-dependent anti-lithiasis effect of pamidronate on spontaneous calcification of CJ cells. At a final concentration of 10-5M, an approximately 45% anti-calcification effect was obtained compared to the control, but at 10 times that concentration (10-4M), the cell count significantly decreased. Each measurement point is shown as the average of n=4. Figure 18(d) shows the dose-dependent anti-calcification effect of resveratrol on the spontaneous calcification of CJ cells. At a final concentration of 50μM, an approximately 20% anti-calcification effect was obtained compared to the control, and at 100μM, a 70% anti-calcification effect was obtained, but the cell count also decreased by 35% compared to the control. Each measurement point is shown as the average of n=4.

[0039] [Figure 19] These figures show the anti-calcification effect of interferon-γ as a false positive compound. Figure 19(a) shows that human interferon-α (HuIFN-α) has no effect on spontaneous calcification of CJ cells. Each measurement point is shown as the average of n=4. Figure 19(b) shows that canine interferon-γ (Canine IFN-γ) inhibits spontaneous calcification of CJ cells. Each measurement point is shown as the average of n=4.

[0040] [Figure 20]This figure shows the dose-dependent anti-calculus effect of lactoferrin (LF) on the spontaneous calcification of CJ cells. Figure 20 shows the anti-calculus effect of bovine lactoferrin (sometimes abbreviated as "bLF" herein) in relation to cell count. Compared to the control, 3 μM, which did not show any decrease in cell count, reduced stone mass by 20%, 6 μM reduced stone mass by approximately 50%, and 12 μM reduced stone mass by approximately 65%. At 25 μM, which inhibited cell proliferation by 20%, a reduction of stone mass of more than 90% was observed.

[0041] [Figure 21] This figure shows the antilithiasis effect of lactoferrin on both calcium phosphate and calcium oxalate stones. Figure 21(a) is a photograph showing the antilithiasis effect of bovine lactoferrin by AR staining under CaP stone-forming conditions (spontaneous calcification conditions, top four rows of wells) and CaOx stone-forming conditions (GA-induced conditions, bottom four rows of wells) with CJ cells seeded on a 96-well plate. Figure 21(b) quantitatively shows the dose-dependent antilithiasis effect of bLF under the CaP stone-forming conditions (top four rows of wells in the 96-well plate photograph) in Figure 20(a), where the AR staining intensity at each bLF concentration is relative to the AR staining intensity in the bLF-free well group, which is set at 100% (each point in the figure is the average value of four cases). Figure 21(c) quantitatively shows the dose-dependence of the anti-calculus effect of bLF addition under the CaOx stone formation conditions (the bottom four rows of the 96-well plate photograph) in Figure 20(a) (each point in the figure is the average value of four cases).

[0042] As already shown in Figure 14, this figure detects the "lithotripsy activity" of a kind of "lithotripsy cells" as an "indirect" assessment of the amount of CaP and / or calcium carbonate that have become AR-stainable within the cytoplasm of CJ cells that have transformed from epithelial-like cells to phagocyte-like cells after phagocytosis of CaOx crystals (AR unstainable). In other words, this figure reflects the inhibition of CaOx formation itself, the promotion of CaOx degradation after formation, or the dose-dependent effect of bLF on both effects.

[0043] In particular, as can be seen in Figure 23 below, even when bLF is added at a final concentration of 25 μM or higher, a certain amount of CaOx·dihydrate crystals are initially formed. Specifically, as can be seen from (f) of the same figure, the addition of bLF dose-dependently removes the internal CaOx that would be stained black with Yause staining, leaving only the crystalline structure framework (a type of ghost structure), resulting in a "crushed residual crystalline structure." The bLF enhancement of lithoclast activity, which processes these once-formed CaOx crystals, naturally reflects the "therapeutic effect" of CaOx stones.

[0044] [Figure 22] The anti-lithiasis effect of lactoferrin (LF) on CaOx formation in CJ cells was semi-quantitatively shown by Alizarin Red S (AR) staining. Figure 22(a) shows an AR stained image without LF (Control). Figure 22(b) shows an AR stained image with LF added at a final concentration of 0.4 μM. Figure 22(c) shows an AR stained image with LF added at a final concentration of 6 μM. Figure 22(d) shows an AR stained image with LF added at a final concentration of 50 μM.

[0045] [Figure 23] Figure 23 shows the anti-lithiasis effect of lactoferrin (LF) on CaOx formation in CJ cells, semi-quantitatively shown by Yasue staining. Figure 23(a) shows the control without LF addition; Figure 23(b) shows the effect of LF addition at a final concentration of 0.4 μM; Figure 23(c) shows the effect of LF addition at a final concentration of 3.0 μM; Figure 23(d) shows the effect of LF addition at a final concentration of 6.0 μM; Figure 23(e) shows the effect of LF addition at a final concentration of 12 μM; Figure 23(f) shows the effect of LF addition at a final concentration of 25 μM; Figure 23(g) shows the effect of LF addition at a final concentration of 50 μM; and Figure 23(h) shows the effect of LF addition at a final concentration of 100 μM.

[0046] [Figure 24]This figure shows that transferrin, which has the same iron ion binding capacity, has no anti-calculus effect. Figure 24(a) shows the dose-dependence of the anti-calculus effect of bovine transferrin in a CaP formation system using CJ cells. No anti-calculus effect was observed. Figure 24(b) shows the dose-dependence of the anti-calculus effect of bovine transferrin in a CaOx formation system using CJ cells. No anti-calculus effect was observed.

[0047] [Figure 25]This figure shows the formation of stones within the lumen of spheroid cultures of CJ cells. Figure 25(a) shows CJ cells that reached confluence in an adhesive surface-treated flask. They were detached with 0.25% trypsin-1 mM EDTA, dispersed well with a pipette, and plated onto a non-adhesive surface flask. This cell suspension was then seeded onto a non-adhesive surface flask for subculture, resulting in a phase-contrast image 8 hours after subculture. Spheroid formation was observed early, and stone deposition was observed on the cell surface or lumen of some of the spheroids. The formed spheroid dispersion was divided into two equal parts and plated onto a new non-adhesive surface flask and another onto an adhesive surface flask, and culture was continued. Figure 25(b) shows a phase-contrast image of the CJ spheroid dispersion divided into two equal parts in Figure 25(a) two days after culture in a non-adhesive surface flask. The distribution of stones within and on the surface of the spheroids is uneven. The fusion and connection of multiple spherical spheroids is also evident. Figure 25(c) is a phase-contrast image of the CJ spheroid dispersion divided into two equal parts in Figure 25(a) after two days of culture in a flask with an adhesive surface. After transplantation into an adhesive flask, epithelial-like CJ cells began to spread from the spheroids attached to the bottom toward the periphery. Figure 25(d) is a phase-contrast image of the CJ spheroid dispersion divided into two equal parts in Figure 25(a) after nine days of culture in a flask with a non-adhesive surface. Stone clusters are clearly visible in most spheroids under phase contrast. Figure 25(e) is a phase-contrast image of the CJ spheroid dispersion divided into two equal parts in Figure 25(a) after two days of culture in a flask with an adhesive surface. Epithelial-like CJ cells have remarkably spread widely from the spheroids attached to the flask bottom toward the periphery. The spherical stones are also fixed to the flask bottom and remain immobile. DETAILED DESCRIPTION OF THE INVENTION

[0048] The cells used in the screening method of the present invention are proximal tubule-derived cells that can spontaneously calcify when cultured in vitro. Since the cells are advantageously capable of being subcultured in vitro, they are preferably immortalized established cell lines prepared from kidneys derived from humans or animals suffering from renal cancer.

[0049] As a preferred example of such an immortalized cell line derived from the proximal tubules of an animal suffering from renal cancer, as specifically described in the Examples below, the present inventors established a cell line consisting mainly of proximal tubule cells from excised kidney tissue of a dog suffering from renal cancer, which can be subcultured in vitro and which spontaneously calcifies when cultured in vitro. These cells were designated CJ( C anine J apan cells.

[0050] CJ cells have been deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation under accession number NITE P-03184. CJ cells were established by the method specifically described in the Examples below, have various properties specifically described in the Examples below, and can be subcultured in vitro. Furthermore, as specifically described in the Examples below, CJ cells have the property of varying the degree of calcification depending on the medium used for in vitro culture. It is possible to use a medium that causes little or no calcification (low to no calcification medium), a medium that causes moderate calcification (moderate calcification medium), or a medium that causes high calcification (high calcification medium) (described below).

[0051] In the screening method of the present invention, the cells are cultured in the presence of a test substance. Naturally, the culture is carried out under culture conditions that allow the cells to grow. In order to use the ability of the test substance to inhibit cell calcification as an index, it is preferable that little or no calcification has occurred at the start of culture.

[0052] Since urinary stones consist of CaOx, CaP, or a mixture of these, little or no calcification occurs when cultured in a medium containing low concentrations of calcium (e.g., 0.4 mM in RPIM-1640 medium), and screening is therefore preferred using cells subcultured in such a medium. Of course, calcification of CJ cells begins after the cells reach confluence, so there is no problem with subculturing them in, for example, the above-mentioned high-calcification medium or moderate-calcification medium before they reach confluence.

[0053] On the other hand, the culture for screening is carried out in a medium containing a higher Ca concentration (e.g., 1.8 mM in the following medium) than RPMI-1640, although glucose concentration is also a factor. Examples of such a medium include (i) Dulbecco's MEM (High Glucose) and α-MEM containing fetal bovine serum, and (ii) Dulbecco's MEM (Low Glucose) and Eagle's MEM.

[0054] Here, "high glucose" refers to a glucose concentration in the range of 15 mM to 40 mM, and "low glucose" refers to a glucose concentration in the range of 1 mM to 10 mM. Culture in medium (i) results in a strong degree of calcification, while culture in medium (ii) results in a moderate degree of calcification. Therefore, these can be used appropriately depending on the purpose of screening. For example, if one wishes to collect a wide range of candidates, the above-mentioned medium (ii), which results in a moderate degree of calcification, can be used, whereas if one wishes to narrow down to only those with a significant inhibitory effect, the above-mentioned medium (i) can be used. It is also possible to first perform screening using medium (ii), and then further narrow down the candidates obtained by screening using medium (i).

[0055] In addition to the media mentioned above, various commercially available artificial culture media can be used for subcultivating CJ cells and screening these cells and their clones (for stone-suppressing and stone-promoting compounds), including Ham's MEM medium supplemented with fetal bovine serum (FBS), Ham's MEM (Kainghn's Modification), D-MEM / Ham's F-12 medium, McCoy's 5A medium, Medium-199 medium, Fisher's medium, IMDM medium, Williams' medium E, MCDB medium, and Leibovitz's L-15 medium.

[0056] These media are commercially available and well-known, and are merely examples. Naturally, other existing media or media newly designed based on the present invention can also be used depending on the purpose. That is, as shown in Example 10, all media can be automatically classified into "highly mineralized media," "moderately mineralized media," or "low to no mineralized media" based on the quantitative parameter of spontaneous calcification of CJ cells.

[0057] Culturing is preferably carried out at a temperature suitable for the growth of the cells used, and for CJ cells, a temperature of 35° C. to 39° C. The culturing time is not particularly limited as long as it is long enough to cause sufficient calcification in the absence of the test substance, but is usually about 3 to 30 days, and preferably about 6 to 12 days.

[0058] The concentration of the test substance (final concentration; hereinafter, the same applies to the concentration in the culture medium) may be any concentration that does not inhibit cell growth, and a concentration that allows accurate examination of the ability to inhibit calcification can be appropriately selected. If this concentration is unknown or difficult to predict before the test, screening can be performed at multiple concentrations. The test substance may be present from the start of culture, or it may be added after a certain period of time (e.g., 1 to 2 days) has passed since the start of culture. If the test substance is added later, calcification will have already progressed to a certain extent, so the effect of dissolving already formed calcified lesions can also be examined.

[0059] A test substance that has the effect of dissolving calcified foci once formed has not only a preventive effect on urolithiasis but also a therapeutic effect (lactoferrin, which will be described later, falls into this category), so the screening method of the present invention also makes it possible to screen for therapeutic drugs for urolithiasis. Furthermore, culture can be carried out in 96-well or 364-well microplates, as in conventional cell culture.

[0060] Furthermore, differences in calcification ability in these media can be utilized to screen for substances that inhibit stone formation as medicines or supplements, or compounds that may promote stone formation, to preemptively exclude them from food additives or drug candidates. For example, for screening for anti-stone substances useful as preventive or therapeutic agents, a medium that induces severe to moderate stone formation is preferable, while for detecting substances that may promote stone formation, a medium that induces no to low stone formation is preferable. However, the degree of stone formation is not necessarily a limiting factor. For example, as shown in Figure 19(b) of Example 18, even in DMEM medium, which induces severe stone formation, not only can interferon-γ exert its anti-stone effect at high concentrations but also exert its opposite effect at low concentrations, i.e., promote stone formation.

[0061] It is also possible to culture cells in a serum-free, completely defined medium containing growth factors and whose components are known. The use of such a serum-free, completely defined medium can eliminate the effects of various components contained in serum. Examples of growth factors include, but are not limited to, at least one selected from the group consisting of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). The concentration of these growth factors in the medium can be appropriately selected within a range that maintains cell growth, and is typically approximately 0.1 ng / ml to 200 ng / ml for each growth factor.

[0062] Furthermore, based on the fact that the degree of stone formation differs depending on the type of medium used to culture CJ cells in the present invention, substances that promote or inhibit stone formation can be identified by the presence or absence and differences in concentration of known compounds that make up the medium. This can be done by sequentially adding or subtracting each substance that makes up the medium and comparing them. Alternatively, the components to be investigated can be divided into groups and compared. DMEM (High Glucose) medium and RPMI-1640 medium are preferably used as comparative media, but are not limited to these two types.

[0063] In any case, this research method is only possible if CJ cells that express the parameter calcification, which can be determined by simple colorimetric quantification, are obtained. Of course, the measurement of the parameter calcification is not limited to colorimetry, and any known method for measuring calcium can be used. Furthermore, the resulting calculi may be CaP, CaOx, or a mixture of CaP and CaOx.

[0064] In the case of CJ cells, the calcification foci formed by the above culture are CaP, but most urinary stones (approximately 85%) are CaOx. To investigate the inhibition of CaOx formation or dissolution, the medium can be supplemented with glyoxylic acid, a precursor to oxalic acid. A mixed CaOx and CaP culture can be obtained by periodically switching between glyoxylic acid (GA)-containing and GA-free medium. There are no restrictions on the order or frequency of addition of GA-containing and GA-free medium; if CaP or CaOx crystals are found under microscopic observation, the medium can be switched to one that induces the other Ca crystal species. The concentration of glyoxylic acid added to the medium can be adjusted as needed; however, the final concentration is typically selected within the range of approximately 8 mM to 20 mM for parent CJ cell lines and approximately 2 mM to 10 mM for CJ clone cell lines.

[0065] Furthermore, when evaluating CJ cells that form stones as ectopic calcification, osteoblasts and / or dental cells can be cultured side-by-side in wells of the same culture plate to compare with calcification in bone tissue, the site of normal calcification, or comparative screening tests can be performed in independent plates. Such side-by-side or comparative cultures make it possible to select test substances that suppress only urinary stone formation without inhibiting normal calcification processes such as bone formation and tooth formation.

[0066] In other words, by selecting a test substance that suppresses the calcification of stone-forming cells such as CJ cells but has no effect on or does not suppress normal calcification (normtopic calcification) such as in osteoblasts and odontocytes, it will be possible to efficiently select a test substance that suppresses only the formation of urinary stones as ectopic calcification.

[0067] After culturing, the degree of calcification of the cells is examined. This can be done, for example, by staining the calcium deposits with AR staining or Yasue staining, and examining the degree of staining by visual inspection or absorbance measurement. Specific staining methods are described in the Examples below. Since the calcium deposits are stained by staining, the greater the degree of calcification, the stronger the staining. Therefore, a weaker staining indicates a stronger calcification-inhibiting effect of the test substance, and thus a stronger preventive or therapeutic effect on urinary tract stones.

[0068] Furthermore, by combining the glyoxylic acid (GA)-added CJ cell culture system with alizarin red S (AR) staining and dye extraction techniques, it is possible to quantitatively evaluate the amount of CaOx-derived Ca de novo produced in stone-clad cells using a simple colorimetric method, without color interference from background CaP formation. In other words, it is possible to quantitatively evaluate the "stone-clad activity" of stone-clad cells. Of course, it is also possible to analyze the cell number and staining intensity by directly capturing images of stone-clad cells stained red on CaOx crystals using an image analyzer, without dye extraction.

[0069] Furthermore, after quantifying the AR staining of the stoneclasts on the CaOx crystals, the same specimen can be extracted with 5% AcOH and thoroughly washed to remove the CaP from the AR-stained stoneclasts. Yasue staining can then be performed to observe the remaining CaOx crystals and quantitatively evaluate them using image analysis.

[0070] Of course, after the 5% AcOH extraction and washing step, it is also possible to extract Ca from the remaining CaOx by a stronger acid treatment, such as 2% hydrochloric acid, without going through the Yasue staining step, and then perform colorimetric quantification.

[0071] Since the screening method of the present invention is used as a primary screening prior to animal testing, the level of effectiveness of compounds to be subjected to animal testing can be determined arbitrarily. For example, for candidate antilithiasis compounds, the molar concentrations of the IC50 values ​​of the main effect can be easily compared using a dose-response curve, and compounds in the top 0.1% can be subjected to animal testing. Of course, in addition to the main effect, the IC50 values ​​of "cytotoxicity" as a side effect can also be determined using CV staining, Giemsa staining, etc., as described below. Therefore, the ratio of the IC50 value of the main effect to the IC50 value of the side effect can also be useful information for narrowing down the candidates for animal testing. Furthermore, in this narrowing down process, the presence or absence of "selectivity," i.e., promoting or at least having no effect on bone mineralization (normative mineralization) and inhibiting calcification in stones (ectopic mineralization), can also provide valuable information when initiating animal testing.

[0072] As specifically described in the Examples below, lactoferrin (LF) was identified as a substance with excellent calcification inhibitory activity by the screening method of the present invention. Furthermore, LF not only inhibits calcification but also dissolves calcified deposits once they have formed, making it effective in both the prevention and treatment of urinary stones.

[0073] LF may be of any origin as long as it is from a mammal, and commercially available bovine lactoferrin (bLF) is preferred. LF can also be produced by genetic engineering techniques, and LF produced by such genetic engineering techniques may be used. Furthermore, while natural LF is preferred, lactoferrin (LF) of the present invention also encompasses those containing an amino acid sequence in which one or more amino acids have been substituted, deleted, and / or inserted, or in which one or more amino acids have been added, that are effective for the prevention or treatment of urolithiasis. Furthermore, lactoferrin (LF) of the present invention also encompasses polypeptides containing an amino acid sequence that shares 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more sequence identity with the amino acid sequence of natural LF, and that are effective for the prevention or treatment of urolithiasis.

[0074] When LF is used as a drug for the prevention or treatment of urolithiasis, it can be administered by various routes, such as oral administration, injection (intravenous, subcutaneous, intramuscular, etc.), transurethral, ​​vaginal, enteral, nasal, transmucosal, pulmonary, etc. Among these, oral administration is preferred because it is simple and can be easily administered by patients themselves. Recent research results using high-resolution equipment, such as micro-XCT and EDX (energy dispersive X-ray), have shown that the site of calcification initiation is not the conventional idea that it is the loop of Henle, but rather that it traces the tubular pathway back to the proximal tubule. The role of the lymphatic system in the theory of calcification (fixed particle) on the mesenchymal side of the tubule has also been reevaluated (Wiener, SV et al., Connective Tissue Research, 58, 102-110, 2018). As a route via the body's vascular system, methods such as lymphatic-directed drug-delivery systems can be used to increase the distribution of lactoferrin to the lymphatic system in addition to the vascular system through which blood flows.

[0075] The dosage of LF can be appropriately determined depending on the type and severity of the disease or symptom to be treated or prevented, the condition of the subject, the dosage form, etc. The daily dosage for an adult is usually 0.1 g to 50 g / day, preferably 0.5 g to 20 g / day, and more preferably 2 g to 10 g / day.

[0076] While LFs may be used alone for pharmaceutical purposes, they may also contain pharmaceutically acceptable carriers and pharmaceutical additives. Examples include most carriers suitable for parenteral administration, including water, physiological saline, Ringer's solution, Hank's solution, glucose solution, lactose, dextrose, ethanol, glycerol, albumin, and other solutions. These compositions may optionally contain stabilizers, antioxidants, antibacterial agents, preservatives, buffers, surfactants, and other auxiliary additives. The actual additives are selected from the above or in combination depending on the dosage form, depending on the condition of the urinary stone patient targeted by the present invention, for the purpose of preventing recurrence, inhibiting the growth of stones already formed, or dissolving stones, but are not limited to these.

[0077] Furthermore, the preventive or therapeutic drug for urolithiasis of the present invention, which contains LF as an active ingredient, can be used in a solid state or in a solution state. When it is a solid, it may be used as is, or it may be further pulverized into a powder or molded into tablets or the like. When it is a liquid, it can be administered orally as is, or it can be administered by spray administration as an aerosol using a spray inhaler (nebulizer). Furthermore, a formulation further pulverized can be administered by inhalation administration using a powder inhaler.

[0078] Furthermore, it is naturally preferable to use LF as a so-called "enteric-coated drug" by formulating it into micelles, granules, tablets, or capsules, which will prevent the degradation of LF, a polypeptide, in the stomach and allow it to be efficiently dissolved and absorbed in the intestine.

[0079] Furthermore, LF is known to have anticancer, immune-enhancing, bactericidal, and antiviral effects, and supplements and health foods containing LF are commercially available. The prophylactic or therapeutic agent for urolithiasis of the present invention may be in the form of such supplements or health foods.

[0080] Furthermore, the CJ cells of the present invention can be used as a suitable research material for elucidating the mechanisms of kidney stone formation and elimination.

[0081] For example, this method can be used to efficiently study changes in gene expression that occur at the onset of early "calcified kidney." For this purpose, the use of commercially available tools such as Affymetrix's Canine GeneChip is a preferred specific approach. Specific comparison and narrowing down methods include: (1) comparing the presence or absence of stone formation in CJ cells grown in DMEM (high glucose) and RPMI-1640 media; (2) comparing the strength of stone formation in CJ cells grown in DMEM media differing only in glucose concentration, i.e., DMEM (high glucose) and DMEM (low glucose) media; and (3) comparing the presence or absence of calcification ability between the CJ cells (proximal tubules) of the present invention and known MDCK cells (distal tubules) derived from the same animal species, dogs, cultured in the same medium.

[0082] These three approaches allow us to focus on "stone formation" within calcification, enabling us to compare and examine the associated changes in gene expression and patterns of increase or decrease in related protein synthesis. In particular, the use of CJ cells provides strong evidence that narrowing down to the truly essential gene expression components of nephrocalcinosis corresponds to the "presence or absence, increase or decrease, of calcification as a clear final event." Needless to say, the easy detection and quantification of this clear final event is the basis for the role that many osteoblastic cell lines, including the MC3T3-E1 cell line, have played in basic and applied research on bone metabolism and the development of preventive and therapeutic drugs. CJ cells can occupy a similar position in basic and development research on kidney stones.

[0083] Regarding the process of bone tissue and kidney stone calcification, although it is a matter of gene expression level, there are two theories: one is that a similar process is at work in stone calcification, centered on Runx2, a master gene involved in bone formation, which is the result of research on bone metabolism, and the other is that the stone calcification process proceeds through a mechanism completely separate from that of bone. The reason for the argument regarding bone metabolism-related genes is that an increase or decrease in the expression of this master gene is supported by the phenomenon of "increases or decreases in calcification at a visible level."

[0084] CJ cells are kidney stone-forming cells, and they are the ones that achieve this "visible level of calcification." In in vitro anti-calcification tests using CJ cells, the effects of lactoferrin and resveratrol were inhibitory to calcification. Conversely, these substances are known to promote calcification in in vitro bone formation tests using osteoblasts. Therefore, both substances acted in completely opposite directions in the phenomenon of calcium deposition in in vitro cell tests.

[0085] In other words, the final event of calcification in "osteoblasts" derived from bone tissue and "lithoblasts" derived from kidney tissue is likely to indicate that the mechanisms of calcification in both tissues are different, and the LF of the present invention can be used as a preventive and therapeutic agent for stones without adversely affecting bone, which is a normotopic calcifying tissue.

[0086] This is because resveratrol has been reported to promote bone formation and also inhibit kidney stones in animals, while LF has also been reported to promote bone formation in many animals.

[0087] Therefore, as a further step (4), by comparing the expression of genes and their products between "lithoblasts (CJ cells and their cloned cells)" and "osteoblasts (whether primary culture or immortalized cells)" as defined in this invention, it may be possible to find new pathways to discover target tissue-selective antilithiasis compounds and bone formation-promoting compounds that differ from LF.

[0088] In the examples of the present invention, CJ cells, which produce stones as an ectopic calcification reaction due to calcium deposition, are glandular epithelial cells that, when cultured, express "polarity," dividing into a cell membrane facing the open lumen and a cell membrane attached to the matrix. The morphology of these epithelial cells is clearly different from the fibroblast-like morphology of osteoblasts, which promote calcification in bone tissue, a process known as entopic calcification. Furthermore, unlike osteoblasts, the CJ cells of the present invention also differ in their ability to produce CaOx crystals in addition to CaP crystals.

[0089] Furthermore, the inventors discovered that phagocytes that engulf and dissolve poorly soluble CaOx crystals are induced and formed from these CJ cells. Needless to say, it is the well-known osteoclasts that perform a similar demineralization role in bone, a normotopic mineralized tissue. While osteoclasts are derived from monocytes and macrophages that differentiate from hematopoietic stem cells, CJ cells that are induced to differentiate by GA and demineralize CaOx are renal glandular epithelial cells, and therefore their differentiation pathway is completely different from that of osteoclasts.

[0090] That is, in the present invention, CJ cells that have reached the logarithmic growth phase or dome formation but have not yet begun to form calcification under a microscope are called "glandular epithelial cells," "epithelial-like cells," or "glandular cells," but CJ cells that have begun to form calcification are provisionally given the name "stoneblast," meaning that they are cells that produce stones.

[0091] On the other hand, the CJ cells that produce CaOx crystals through GA induction are also stoneclasts, but at the stage where CaOx crystals have progressed to dissolution and disintegration, they are tentatively named "stoneclasts," meaning that they are phagocytes derived from CJ cells that have acquired the ability to phagocytose CaOx crystals. In research into the relationship between stoneclasts vs. osteoblasts and between stoneclasts vs. osteoclasts, CJ cells can also be used as a research material for comparative studies at the genetic level.

[0092] The present invention will be specifically described below based on examples, although the present invention is not limited to the following examples.

[0093] Example 1: Establishment of immortalized canine proximal tubule cells (CJ line) 1. Tissue origin of the CJ cells established in this invention An intraperitoneal tumor that developed in a 3-year-old Chihuahua (female, spayed) served as the starting material for the preparation of the CJ cell line of the present invention. Preoperative CT and MRI scans revealed a tumor on the left side of the abdominal cavity measuring 12 x 9.1 x 8.3 cm, with a calcified lesion showing a CT value of 10-50 (some calcifications with a CT value of 100-200). Since the left kidney could not be identified, it was determined to be tumorous in the left kidney, and the tumor was surgically removed. A portion of the removed tumor was subjected to pathological examination, cell isolation, and in vitro culture and passage. Pathological examination revealed that the tumor was a renal adenocarcinoma, with the original renal tissue structure having been lost and almost the entire area replaced by neoplastic proliferation of atypical epithelial cells accompanied by widespread necrosis and interstitial proliferation. The proliferating atypical cells were arranged in a tubular to papillary pattern, with individual cells possessing weakly eosinophilic cuboidal to columnar cytoplasm and atypical nuclei of varying sizes, and the necrotic areas were accompanied by significant calcification.

[0094] 2. Enzymatic Digestion of Excised Tissue and Cell Separation A portion of the excised tumor mass (approximately 5 x 5 x 5 mm) was minced in calcium- and magnesium-containing Hank's solution (hereinafter sometimes abbreviated as "HBSS(+)"). The minced pieces were then transferred to a digestion solution containing 0.1% (w / v) collagenase (Type I, Sigma) and 0.01% (w / v) DNase I (Sigma) dissolved in HBSS(+) at a final concentration of 0.1% (w / v). The total volume of the solution was 50 ml and stirred at 37°C for 1 hour. The resulting cell dispersion was filtered through a 100 μm stainless steel mesh to remove digestion residue, and the filtrate was centrifuged at 1,200 rpm at 4°C for 5 minutes to recover the precipitated cell fraction.

[0095] Erythrocytes present in the precipitated cells were removed by the following hemolysis procedure: 1 ml of room temperature PBS(-) was added to the precipitate, and after thorough dispersion, the precipitate was transferred to a centrifuge tube. 2.3 ml of sterile distilled water was added and the erythrocytes were disrupted by hypotonic treatment for approximately 15 seconds. 1 ml of 3% (w / v) sterile NaCl solution was immediately added to restore isotonicity, and the total volume was increased to 14 ml with PBS(-). The mixture was then centrifuged at 1,200 rpm for 5 minutes at 4°C to obtain the precipitate. This precipitate was then dispersed again in cold PBS(-) and the same centrifugal washing procedure was repeated.

[0096] The finally obtained cell fraction was dispersed in DMEM medium (High Glucose) containing a final concentration of 10% (v / v) FBS (BioWest), 100 units / ml penicillin, and 100 μg / ml streptomycin (hereinafter, unless otherwise specified, this DMEM culture medium supplemented with 10% FBS and antibiotics will be abbreviated as DMEM medium in this specification). The cells obtained by enzyme treatment were dispersed in DMEM medium and then plated on multiple 25 cm plates. 2 The cells were plated onto a culture flask (C25 flask, Corning) and a 9 cm diameter collagen-coated culture dish (Celltite C-1, MS-0390K, Sumitomo Bakelite), and culture was initiated at 37°C in a 5% carbon dioxide incubator.

[0097] 3.Culture results CJ cells proliferated at approximately the same doubling time in both standard adherent cell culture flasks and collagen-treated cell culture dishes, but their morphology was significantly different. Specifically, in standard cell culture flasks without collagen treatment, typical cobblestone-like colonies of epithelial cells were formed (Figure 2(a)), whereas in collagen-treated dishes, short spindle-shaped cells were scattered on the bottom (Figure 2(b)). This morphological difference did not persist even after repeated passages; Figure 2(a) shows images of cultures at 48 PDLs, and Figure 2(b) shows images of cultures at 50 PDLs.

[0098] Specifically, when CJ cells reach confluence in a normal culture flask, they first form domes (or blisters), which are characteristic of glandular cells. However, if they are left alone without subculture, island-like precipitates that are easily visible to the naked eye appear everywhere (Fig. 2(c)). Figures 2(d), 2(e), and 2(f) show the results of experiments using 25 cm 2 The image was taken at 40x, 100x, and 200x magnifications under a phase-contrast microscope in the same field of view, focusing on an area of ​​visible island-like precipitates in the flask (Fig. 2(c)). Precipitates consisting of clumps of various sizes are visible throughout the field of view, with a small area in the center where a monolayer of CJ cells is observed.

[0099] Example 2: Comparison of AR and LG double staining of established CJ cells and MDCK cells as a control 1. Purpose of the experiment Next, AR staining was performed to confirm that the precipitates produced by CJ cells observed in Example 1 were Ca. LG staining was also performed to differentiate the CJ cell layer, which is detected as a monolayer in addition to the precipitates. In addition to spontaneously calcifying CJ cells (57 PDLs), Madin-Darby Canine Kidney (MDCK) cells, canine distal tubule cells that have already been reported to spontaneously calcify, were also used as a control. To confirm that the observed precipitates were not the result of a physicochemical "supersaturation phenomenon" caused by the culture medium itself, but rather a biological phenomenon that only occurs in the presence of CJ cells—so-called biomineralization—a culture experiment using only cell-free DMEM medium was also performed as a control.

[0100] 2. Experimental Method CJ and MDCK cells were grown at 1 x 10 in DMEM medium. 5 The CJ cell suspension was prepared at a concentration of 25 cm 2 5 ml of the MDCK cell suspension was seeded in two types of culture vessels: a culture flask (C25 flask, Corning) and a collagen-coated culture dish (Celltite C-1, MS-0060K, Sumitomo Bakelite) with a diameter of 6 cm.2 The cells were seeded only in a culture flask (C25 flask, Corning). Cell-free DMEM medium alone was also seeded at 25 cm 2 After dispensing into culture flasks (C25 flasks, Corning), incubation of all culture vessels was initiated at 37°C under 5% carbon dioxide and 95% air (day 0). Medium was changed every 3 days, and double staining with AR and LG was performed 30 days later.

[0101] Double staining with two dyes was performed as follows. On day 30, the medium was removed, the cells were washed twice with PBS(-), and then 95% EtOH was added and fixed at room temperature for 30 minutes. The fixative was discarded, and the cells were air-dried using a hair dryer. 5 ml of 1% (w / v) AR aqueous solution (pH 6.4) was added per culture vessel and stained at room temperature for 15 minutes. Next, the dye was discarded, and excess dye was removed by rinsing with water. 1% (w / v) LG aqueous solution was added, and staining was continued for 1 minute at room temperature. The LG solution was immediately discarded and the cells were quickly rinsed with water to remove excess dye. Finally, 5 ml of distilled water was added to the culture vessel, and microscopic examination and photography were performed in this state.

[0102] 3. Results and Discussion The results are shown in Figure 2. CJ cells formed precipitates of various sizes on both a regular flask (Figure 3(a)) and a collagen-coated dish (Figure 3(b)), and these precipitates were strongly stained with AR, which stains Ca red. Areas without precipitates or protuberances were stained green with LG dye and consisted only of cells.

[0103] That is, when there is sufficient space between cells before confluence, there was a significant difference in the morphology of CJ cells depending on whether or not the surface of the culture vessel was collagen-coated, but it was also found that calcification began in the same way as the cell density increased. In fact, although not shown in the photographs, at low cell density, CJ cells on the collagen coat exhibited a short spindle shape similar to the cell morphology of mesenchymal cells, but upon reaching confluence, they changed into "pavement-like" epithelial-like cells, demonstrating a "mesenchymal to epithelial" transformation.

[0104] On the other hand, in the MDCK cells used as a control in this example, the precipitate stained red by AR was completely undetectable with the naked eye, and even under a microscope, the presence of the cells could only be confirmed by LG staining (Fig. 3(c)). Such extremely weak AR staining results could be speculated to be the reason why the screening of "anti-calcium compounds" using MDCK cells had to be performed under a microscope using a special culture medium, soft agar.

[0105] Visual inspection under a microscope is time-consuming and laborious, and searching for hundreds of thousands of candidate compounds would be impossible due to time and cost constraints. Conversely, the calcification of CJ cells, which was strongly stained with AR, clearly demonstrated the possibility of easily transferring this method to HTS using multi-well plates (e.g., 96-well plates), since dye extraction and colorimetric quantification at OD 405 nm are available.

[0106] In addition, in flasks in which only cell-free DMEM medium was replaced every three days, no precipitate-like structures appeared under phase-contrast microscope observation at each time point of medium replacement, and as shown in Figure 3(d), naturally, no structures stained with either dye were observed when assessed on day 30. Furthermore, it has been repeatedly confirmed in preliminary screening that no precipitates appear even when incubation is continued for 6 to 14 days in this cell-free DMEM medium without any medium replacement, as shown in the blank well row at the far right of the 96-well plate in Figure 21(a).

[0107] In other words, the spontaneous calcification produced by CJ cells is not the result of a physical or chemical reaction caused by supersaturation of Ca in the culture medium, but is a biological calcification involving living cells (bio-mineralization). Furthermore, although a wide variety of cells have been cultured in DMEM culture medium containing FBS, there have been no reports of spontaneous calcification except for MDCK cells, and therefore, at present, this is a phenomenon specific to CJ cells.

[0108] Example 3: Comparison of alkaline phosphatase (ALPase) staining in established CJ cells and control MDCK cells 1. Purpose of the experiment CJ cells, which were successfully isolated and cultured from canine renal adenocarcinoma, take on an epithelial-like morphology, and when they reach confluence on the surface of the culture vessel, they form a dome and express a so-called "polarity" in which the apical side (luminal side) and basal side (substrate side) are distinguished in the cell arrangement, which is essential for the movement of solution and solutes.

[0109] Polarity is also expressed in the canine MDCK cell line used as a control in Example 2, and is a common property. Glandular cells in the kidney primarily comprise the proximal and distal tubules. ALPase staining was performed to determine the origin of CJ cells from which tubule. Although the kidney is composed of many cell types, only the proximal tubule exhibits high ALPase activity (e.g., Miyazawa, K. et al., Jpn. J. Vet. Sci., 47, 895-900, 1985; Ghung, S. D. et al., J. Cell Biol., 95, 118-126, 1982). Counterstaining was performed on MDCK cells derived from the distal tubules as a control.

[0110] 2. Experimental Method CJ cells (54 PDLs) and MDCK cells were cultured at 1 x 10 in DMEM medium. 5 After adjusting the cell concentration to 1000 cells / ml, the cells were seeded into 35 mm culture dishes (MS-10350, Sumitomo Bakelite) at 2 ml per dish and cultured at 37°C under 5% carbon dioxide and 95% air. Observed daily under a phase-contrast microscope, the medium was removed upon reaching confluence. Both cells were rinsed twice with 3 ml per dish of saline (0.9% NaCl). Next, 3 ml per dish of ice-cold fixative (methanol:formalin stock:glacial acetic acid = 9:1:0.01) was added for 5 seconds, followed by immediate rinsing with distilled water and air-drying. Next, 2 ml of the following substrate solution was added per dish and incubated at room temperature for 10 minutes.

[0111] After confirming the degree of color development due to the enzymatic reaction with the naked eye and microscopic examination, excess substrate solution was removed by decantation, and the enzymatic reaction was stopped by rinsing the Petri dishes under running water. Finally, 2 ml of distilled water was added to each Petri dish for microscopic examination and photography. The substrate solution was prepared freshly by dissolving 10 mg of sodium naphthol AS-BI phosphate in 20 ml of 50 mM 2-amino-2-methyl-1,3-propanediol buffer (pH 9.8), followed by the addition of 10 mg of Fast Blue RR salt, and then immediately filtering the filtrate through No. 2 filter paper.

[0112] 3. Results and Discussion As shown in Figure 4(a), CJ cells expressed extremely high ALPase activity. On the other hand, MDCK cells derived from distal tubule tissue expressed ALPase activity, but it was extremely weak (Figure 4(b)). This indicates that the obtained CJ cell line is primarily composed of cells derived from proximal tubule tissue.

[0113] Example 4: Qualitative and quantitative comparison of spontaneous mineralization in established CJ and control MDCK cells in multi-well plates (96 wells)

[0114] 1. Purpose of the experiment Next, the inventors investigated whether it would be possible to easily screen candidate anti-calculus drugs by combining CJ cells in a multi-well plate (Multiplate 96F, MS-8096F5, Sumitomo Bakelite) with a simple colorimetric method. In this study, the cell concentration required to detect sufficient calcification approximately one week after seeding CJ cells on the plate was also investigated. MDCK cells were also used as control cells in this study.

[0115] 2. Experimental Method Dilute CJ and MDCK cells in DMEM medium to 5 x 10 5Starting with the highest concentration of 100 cells / ml, serial 2-fold dilutions were made with DMEM medium to create a cell concentration array, which were plated at 100 μl / well in the array shown in Figure 5. CJ cells were plated in the top four rows of a 96-well plate, and MDCK cells in the bottom four rows. The left and right half of the wells were assigned to AR staining for Ca quantification and CV staining, reflecting cell number. Thus, each 96-well plate was used for one cell type and one cell concentration, with n=4 experiments. After plating, the medium was not replaced with fresh medium until the evaluation day. On day 0, cells were plated in the same way on five plates, and quantification was performed at five time points: day 0, day 2, day 4, day 6, and day 8. The day 0 evaluation score was obtained by microscopic examination 6 hours after plating to confirm that the cells had adhered and spread to the incubator bottom, at which point they were fixed and stained.

[0116] At each time point, the culture medium in the wells was discarded, any remaining liquid on the plate was wiped off by tapping on a paper towel (Kimwipe), and 200 μl of PBS(-) was dispensed into each well to rinse. After this PBS(-) washing procedure was repeated, 100 μl of 95% EtOH was dispensed into each well at room temperature for 10 minutes, and the wells were then air-dried using a hair dryer. Prior to the following staining procedures, plate seals were attached to the left and right halves of the plate to cover the wells intended for staining with one dye until the staining procedure with the other dye was completed, preventing the intrusion of dye solution and washing solution.

[0117] For AR staining, 100 μl of 1% dye solution was dispensed per well and stained at room temperature for 30 minutes. The staining solution was then discarded, and the excess dye was washed by immersing the plate in gently running water and decanting the wells. This process was repeated 3-4 times, and the washing solution was finally wiped away and the plate was air-dried at room temperature. For CV staining, 100 μl of 0.1% crystal violet solution was dispensed per well and stained at room temperature for 30 minutes. After staining, excess dye was removed, and the plate was washed and air-dried in the same manner as for AR staining. AR extraction from the wells was performed by dispensing 100 μl of 5% (v / v) formic acid per well and shaking the plate for 30 seconds using a plate mixer (TUPLE MIXER TWIN 3-28, IWAKI). CV extraction was performed by dispensing 100 μl of extraction solution (70% EtOH:ethylene glycol:citric acid = 50:50:1) into wells and shaking for 30 seconds using a plate mixer (TUPLE MIXER TWIN 3-28, IWAKI). The extracted pigments were measured using a Microplate Reader (Benchmark, BIO-RAD) at OD 450 nm for AR and OD 590 nm for CV.

[0118] Cell counts were also measured using a commercially available kit reagent (Cell Counting Kit-8, Dojin Chemical Research Institute). Specifically, on day 0, CJ cells (145 PDLs) or MDCK cells were dispersed in DMEM medium in a 96-well plate and counted at 1 x 10 4 Following the kit instructions, 10 μl of color reagent was added to each well and incubated at 37°C for 1 hour. The absorbance was then measured at OD 450 nm (Figure 5(e)).

[0119] 3. Results and Discussion Figure 5(a) shows the actual colorimetric test plate 8 days after the start of the test. For example, the number of cells per well that had proliferated by day 8 can be visually determined by the blue color tone of the CV of the well in the right half of the photograph. At the cell concentration seeded on day 0, there was a maximum difference of just under 40-fold, but after day 8, the blue color tone had reached almost the same level, and no difference was observed between CJ cells and MDCK cells. On the other hand, a clear difference emerged in the level of calcification. In CJ cells, calcified foci were clearly stained red, and even at lower cell concentrations (1.5 x 10 3 The red color of wells seeded with 100 cells / well was weaker than that of wells seeded with a higher cell density. A time lag was also observed between the time the cells reached confluence in the culture vessel and the start of calcification, suggesting that some metabolic change or progression of a process was responsible for the time lag in the onset of calcification.

[0120] On the other hand, the detection of calcium deposits by AR staining was extremely weak in the MDCK cells used as a control, even when cultured in a 96-well plate. Therefore, even though both cells spontaneously calcify in a normal culture medium, there is a large difference in ability between the CJ cells (derived from proximal tubules) of the present invention and the known MDCK cells (derived from distal tubules), demonstrating the superiority of CJ cells for HTS development.

[0121] In Figures 5(b) to 5(e), the X axis represents the number of days in culture, and the Y axis represents the changes in the amount of calcification and cell mass of CJ cells and MDCK cells (Mean ± SD, n = 4). As shown in Figures 5(b) and 5(c), 5 x 10 4 cells / well and 6 x 10 3 Although the CJ cells were seeded on day 0 at a rate of almost 10-fold higher than the 1.5 x 10 cells / well, both CJ cells reached confluence on day 6 and reached almost the same amount of calcium deposition two days later on day 8. 3 When cells were plated at a smaller number than the number of cells / well, the number of CJ cells was 5 x 10 on day 8. 4 cells / well group and 6 x 10 3The level of calcification was insufficient, although it was comparable to that of the cells / well group.

[0122] In the case of MDCK cells (Fig. 5(d) and Fig. 5(e)), it was difficult to measure the amount of calcium deposition by AR staining using a 96-well plate and colorimetric methods. In fact, the measured values ​​were equivalent to the blank values ​​(background values ​​obtained by injecting only DMEM medium without adding cells into the wells and culturing). The growth kinetics of both cells, measured using the Cell Counting Kit-8 (Dojin Chemical Research Institute), a commercially available cell counting reagent, were interesting. As shown in Fig. 5(f), CJ cells and MDCK cells proliferated at almost the same rate until day 10.

[0123] On the other hand, after day 10, the number of MDCK cells did not increase, but the number of CJ cells continued to increase. Since no medium changes were performed between the time the cells were seeded on day 0 and the final measurement day, day 14, the decrease in MDCK cell number is thought to reflect a deterioration of the culture environment, such as the depletion of nutrients and the accumulation of metabolites. This result suggests that CJ cells have extremely robust properties that allow them to withstand harsh environments, and that at calcified sites, CJ cells are present on top of the calcified foci (in other words, calcified deposits are present between the basement membrane of the CJ cells and the culture dish).

[0124] This is because, unlike CV staining (Figure 5(a)), the cell counting method using the Cell Counting Kit-8 (Figure 5(f)) is based on measuring intracellular dehydrogenase activity, not on measuring the amount of accumulated protein, a non-cellular component secreted and accumulated in the extracellular matrix. Furthermore, unlike MDCK cells, CJ cells, as seen in non-calcified areas, reach confluence without layering, and more than 50% of the well bottom area is covered with calcified foci. Despite this, the fact that the number of CJ cells is greater than that of MDCK cells can be explained by considering that the site of calcification foci formation is between the cell basement membrane and the dish matrix, and the following literature report seems to support this interpretation.

[0125] Kageyama et al., who discovered spontaneous calcification in MDCK cells, showed by electron microscopy that in monolayer cultures, calcified crystals of MDCK cells were formed and detected between the cells and the substrate of the culture dish, i.e., on the stromal side of polarized glandular cells (Kageyama, S. et al., Int. J. Urol., 3, 23-26, 1996). Similarly, Naito et al. reported that microcalcified stones formed in MDCK cells cultured on a plate, in a 3D collagen gel, or in a 3D agar medium were detected only on the basolateral side of the cells, not the apical side, by electron microscopy (Naito, Y. et al., Urol. Res., 25, 59-65, 1997). Furthermore, it has been reported that microliths are distributed on the basement membrane side of the follicles in tumors formed by subcutaneous transplantation of MDCK cells into nude mice (Sakakura, T. et al., Urol. Res., 27, 200-205, 1999).

[0126] The problem with these pioneering studies using MDCK cells was the small amount of microstones formed. This meant that all steps, from the culture method and sample processing to the quantification step of counting under a microscope, were extremely time-consuming and unsuitable for screening a large number of candidate compounds. Despite these circumstances, a search for anti-stone substances using a small number of substances was undertaken, and alendronate was reported as one candidate (Senzaki, H. et al., Urol. Re., 32, 223-228, 2004).

[0127] However, unfortunately, there have been no subsequent reports of the search for anti-stone substances using a similar 3D culture system using MDCK cells. In any case, for the purpose of this example, when using CJ cells for screening in a 96-well plate, 5 x 10 4 ~6 x 10 cells / well 3Almost the same results were obtained within the range of 1.5 x 10 cells / well, and above all, it was found that quantitative evaluation using multi-well plates is possible. However, if the culture and evaluation time is extended, the minimum cell concentration in this example, 1.5 x 10 cells / well, can be obtained. 3 Since calcification proceeds sufficiently even when cells are plated per well, the present invention is not limited to this cell concentration range.

[0128] Example 5: CJ vs MDCK, effect of fetal bovine serum (FB) on spontaneous calcification 1. Purpose of the experiment Generally, cell culture requires the addition of 5% to 10% FBS, which contains cell growth factors, to the basic commercially available culture medium. The FBS requirement for spontaneous calcification of CJ cells was examined by varying the concentration of FBS added to commercially available DMEM medium. MDCK cells were used as a control cell, and comparisons were made from two aspects: calcification and cell proliferation.

[0129] 2. Experimental Method Commercially available DMEM culture medium was supplemented with 100 units / ml penicillin and 100 μg / ml streptomycin to a final concentration of 100 units / ml, and various concentrations of FBS were added to this to achieve final concentrations of 0, 0.5, 1.0, 2, 4, 6, 8, and 10% (v / v) to prepare test culture media. CJ cells (86 PDLs) and MDCK cells passaged in 10% FBS-containing DMEM medium were washed with PBS(-) according to the standard method, then detached from the flask bottom using 0.25% trypsin-1mM EDTA treatment. The detached cells were thoroughly dispersed in 10% FBS-containing DMEM medium, centrifuged at 1,200 rpm for 5 minutes at 4°C, and the supernatant was removed. PBS(-) was added to the precipitated fraction, dispersed well, and centrifuged under the same conditions.

[0130] After repeating this centrifugation and washing procedure, the final precipitated cell fraction was dispersed in FBS-free DMEM, the number of viable cells was counted, and the cells were divided into equal portions and diluted to 1x10 in the previously prepared DMEM medium containing various concentrations of FBS. 5 Each cell suspension was diluted to 2 x 10 cells / ml in a 96-well plate. 4The cells were dispensed at 200 μl per well, and incubation was initiated at 37°C in a 5% CO incubator (day 0). Evaluation was performed on day 8. After removing the medium by decantation, 200 μl of PBS(-) was dispensed per well to wash the wells, which were then removed by decantation. This PBS(-) washing procedure was repeated twice, after which 200 μl of 95% EtOH was added per well and the plate was left to stand at room temperature for 15 minutes to delipidate and fix the cells. After removing the ethanol, excess EtOH was removed with a hairdryer, and the plate was air-dried. After air-drying, staining of each well with AR and CV, followed by extraction and quantification of each dye, was performed as described in Example 4.

[0131] 3. Results and Discussion On the other hand, no progression of calcification was observed in MDCK cells at any FBS concentration, as shown by the open histogram in Figure 6. As qualitatively shown in Example 2, MDCK cells have been reported to spontaneously calcify, but it was difficult to quantitatively evaluate them using the Alizarin Red staining method even in this example using a multi-well plate.

[0132] Example 6: Examination of DMSO concentration on proliferation and spontaneous calcification of CJ cells 1. Purpose of the experiment Compound libraries contain over hundreds of thousands of drug candidate compounds, ranging from hydrophilic (polar) compounds that are easily soluble in water to lipophilic (non-polar) compounds that are insoluble in water but soluble in organic solvents. When adding test substances to live cell screening systems, dimethyl sulfoxide (DMSO) is widely used as a solvent that dissolves a wide range of both hydrophilic and lipophilic compounds and has low cytotoxicity. Typically, a final DMSO concentration of 0.1% (v / v) is used, so we compared the effects of DMSO on the calcification phenomenon of CJ cells in the present invention with and without DMSO.

[0133] 2. Experimental Method CJ cells were cultured at 5 x 10 in DMEM medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin. 5The cells were diluted to 1000 cells / ml and seeded into three 96-well plates on day 0 as follows. The three plates were fixed and stained on days 1, 7, and 14 after the start of culture, respectively, to assess calcification. The test group was arranged in the 96-well plate as follows: rows 1 to 4 were wells for culturing CJ cells alone, and rows 5 to 8 were wells for culturing CJ cells with DMSO at a final concentration of 0.1%. However, columns 1 and 12 (a total of 16 wells) were used as blanks, with 100 μl of DMEM medium dispensed into each well.

[0134] Next, 100 μl / well of DMEM medium was dispensed into all wells except for the empty wells in rows 1 and 5 and the 16 blank wells, and 200 μl / well of the above starting test cell concentration solution was dispensed into the empty wells in rows 1 and 5 (n=10 each). Of this 200 μl, 100 μl was transferred to the wells in the next row below (i.e., row 2 and row 6, respectively), and the cell solution was diluted 2-fold by pipetting three times to thoroughly disperse the solution. 100 μl of this solution was then transferred to the next row below (i.e., row 3 and row 7, respectively), and similar 2-fold serial dilutions were performed sequentially. The final 100 μl of the solution in the wells in rows 4 and 8 was discarded. The plates after seeding the cells were transferred to a 5% CO2 incubator, and culture was initiated at 37°C. One plate was removed at a time (day 1, day 7, and day 14), and washing, fixation, and drying were performed on the same day. Note that no medium change was performed from the start of culture (day 0) to the day each culture plate was collected.

[0135] The procedures for washing and fixing cells from plates collected over time were as follows: After microscopic observation of the cells in the plate, the culture medium was decanted, and 150 μl / well of HBSS(+) was added to wash the wells. This wash solution was then decanted and discarded, and 150 μl / well of HBSS(+) was added again to wash the wells twice. Next, 100 μl / well of 10% neutral interference formalin was dispensed and left at room temperature for 60 minutes to fix the cells. The fixative was then decanted and discarded, and the plate was immersed in a box under weak running water for washing. Next, 100 μl / well of 99.5% EtOH was added, and the wells were degreased for 10 minutes at room temperature. The EtOH was then discarded, and the inner surfaces of the wells were air-dried using a hair dryer. The plate was then stored at room temperature until the following staining and extraction date.

[0136] After day 14, when plate collection and fixation were completed over time, all plates were stained, washed, dried, extracted, and quantified on the same day. Cell-produced calcium precipitates were removed by dispensing 100 μl of 1% (w / v) AR aqueous solution (pH 6.4) into wells and leaving the plate at room temperature for 30 minutes. The staining solution was then decanted off, and the plate was immersed in a box under weak running water and decanted repeatedly. The plate was gently tapped on a stack of Kimwipes to remove as much moisture as possible and allowed to air-dry at room temperature. AR was extracted by adding 100 μl of 5% (v / v) formic acid into wells and leaving the plate at room temperature for 30 minutes. The absorbance was measured at OD 450 nm (BIO-RAD, Microplate Reader, Benchmark).

[0137] 3. Results and Discussion We examined calcium phosphate stone (CaP) deposition in the first and second weeks of culture with and without a medium change, using a wide range of cell concentrations, in the presence or absence of DMSO at a final concentration of 0.1%, but no effect on calcification was observed. Therefore, we confirmed that DMSO can be used without problems as a dissolving solution for test substances, and that the versatility of the usual primary screening method is not affected by any culture period between days 7 and 14.

[0138] Example 7: Validation of CJ cell calcification evaluation in a calcium phosphate stone (CaP) formation system 1. Purpose of the experiment Our studies to date have greatly increased the possibility of using CJ cells as cells for HTS in the search for anti-calcification compounds. However, because biomineralization requires the presence of CJ cells, rather than physicochemical calcium stone formation, if the test substance has a cytocidal effect due to toxicity to CJ cells, the amount of calcium stones formed will inevitably be reduced, leading to erroneous interpretation of the results. Therefore, in addition to measuring the amount of calcification, it is also necessary to measure the amount of cells in the well.

[0139] Initially, the inventors conducted experiments using separate plates for AR staining and CV staining. However, if both staining, extraction, and quantification could be performed on the same plate, the labor, time, and cost would be halved, which would be particularly advantageous when screening a large number of candidate "antilithic compounds." Preliminary studies by the inventors demonstrated that the objectives could also be fully achieved by performing additional CV staining in the same well after AR staining, extraction, and measurement, followed by extraction and measurement. The results of the validation test in Example 7 are shown as an example.

[0140] 2. Experimental Method Specifically, the validation test was performed as follows: Columns 1 and 12 of three 96-well plates were left blank (200 μl / well of DMEM medium containing 0.1% DMSO). All remaining wells were filled with 1 x 10 CJ cells diluted in DMEM medium containing 0.1% DMSO. 4 Cells were seeded at 200 μl per well (day 0), and culture was initiated in 5% carbon dioxide, 95% air, and 37°C. On days 8, 10, and 12, each plate was removed from the incubator, and calcification and cell mass were quantified sequentially in the same 96-well plate. Note that the medium was not changed during the culture.

[0141] On the day of analysis, the culture medium was discarded and the plates were washed with 200 μl / well of PBS(-). Then, 200 μl / well of 95% EtOH was added and left at room temperature for 30 minutes to fix the cells. After discarding the fixative, the wells were dried with hot air from a hair dryer and left at room temperature until all three plates were ready. When the air-dried plates for Days 8, 10, and 12 were ready, 100 μl / well of 1% AR solution was added and left at room temperature for 30 minutes. Excess dye was removed by rinsing the plates in running water. After air drying, 100 μl / well of 5% formic acid was added and the OD was measured at 450 nm.

[0142] After the calcium quantification, the AR extract was decanted, and the 96-well plate was immersed in running water to rinse the wells and then air-dried at room temperature. Next, 100 μl of 0.05% (w / v) CV solution dissolved in 10% formalin water was dispensed into the wells of the same air-dried plate at room temperature. After 30 minutes of staining, excess dye solution was removed by immersion in running water. After air-drying, 100 μl of extraction solution (70% EtOH:ethylene glycol:citric acid = 50:50:1) was added at 100 μl per well, and absorbance was measured at OD 595 nm. In this experiment, the cells were fixed with EtOH due to prior Ca removal treatment during CV staining, but a staining solution containing 10% formalin was used due to concerns about cell detachment. However, subsequent studies demonstrated that the presence of formalin was not necessary.

[0143] 3. Results and Discussion Figures 8(a) to 8(c) show photographs of plates stained with AR and air-dried on the 8th, 10th, and 12th days after evaluation. While the blank wells in columns 1 and 12, which contained only medium, were completely transparent, the remaining wells were strongly stained red with the AR dye, and the signal was extremely strong compared to the background. Furthermore, there was little difference in the degree of staining between evaluation days. Although not shown in the photographs, the signal intensity of the CV staining, which was additionally performed after AR staining, extraction, quantification, and washing, was sufficiently strong. The results are quantified in the heat map in Figure 8(d).

[0144] The upper table in Figure 8(d) shows the amount of calcium measured by AR staining, and the lower table shows the cell count measured by CV staining. For example, in the day-10 determination plate, the mean ± standard deviation of the 0% control for the calcium quantification system was 0.048 ± 0.005, with a coefficient of variation of 10%, and the mean ± standard deviation of the 100% control was 2.468 ± 0.182, with a coefficient variance of 7%. In the cell count quantification system, the mean ± standard deviation of the 0% control was 0.100 ± 0.006, with a coefficient of variation of 5%, and the mean ± standard deviation of the 100% control was 2.030 ± 0.084, with a coefficient of variation of 4%, demonstrating that this is an excellent evaluation system.

[0145] Example 8: Summary of validation of spontaneous calcification reaction of CJ cells (results of two experiments) 1. Purpose of the experiment Institutions that hold hundreds of thousands of candidate pharmaceutical compounds (for example, the compound bank managed by the University of Tokyo's Drug Discovery Initiative) require potential information on the screening system possessed by the recipient before supplying candidates (http: / / www.ddi.u-tokyo.ac.jp / ). To determine whether the screening system for novel anti-calculus compounds using CJ cells that we established as part of the present invention complies with these standards, we calculated the measured values ​​in Example 8, a follow-up test similar to Example 7, and determined parameter values.

[0146] 2. Results and Discussion As summarized in Figure 9, in two independent tests, the pass criteria were met for all items. In particular, the Z'-factor, which is considered the most important indicator of assay system accuracy, exceeded the standard value with a margin of error. The results also provided extremely useful information, showing that even if there was a four-day difference in the assessment date from cell seeding, even if there was a two-day difference before and after, the assessment results would not be affected at all. Furthermore, the CJ cells of the present invention have an extremely advantageous property for efficient screening in that they not only conserve compound but also reduce labor, time, and cost.

[0147] Example 9: Differences in culture media on spontaneous calcification of CJ cells 1. Purpose of the experiment CJ cells spontaneously calcify without the need for any special external stimuli, using only commercially available culture medium supplemented with FBS, which is commonly used in cell cultures. Furthermore, the amount of calcium stones produced is so large that it has never been known before. The mechanism of spontaneous calcification is currently unknown, and many calcium stones observed clinically are of unknown origin. While different from clinical cases and the in vitro CJ cell line of the present invention, even if there are superficial similarities, the existence of some common mechanism seems to be apparent.

[0148] Therefore, we considered whether there were any other characteristics common to clinical calcium stone formation. In the course of this research, the inventors first focused on the concept that metabolic syndrome is a contributing factor to the development of urinary stones, which has been widely advocated in recent years based on epidemiological analysis. In fact, there have been numerous reports on the relationship between urinary stones and myocardial infarction, hypertension, diabetes, and chronic renal failure.

[0149] In clinical practice, these chronic diseases can be diagnosed based on the presence or absence or amount of substances in body fluids such as blood. In other words, known and unknown pathogenic components exist in each patient's body as additional elements to the basic components that make up body fluids in living organisms, which correspond to the basic components that make up cell culture medium.

[0150] On the other hand, if urinary tract stones are also considered a form of metabolic syndrome, we wondered whether differences in the constituents of "artificial" cell culture media, which correspond to bodily fluid components, might also lead to differences in in vitro calcification in CJ cells. Currently, a wide variety of cell culture media are commercially available, with a wide variety of constituent types and contents. These culture media have been empirically improved and developed through a process of trial and error, focusing primarily on cell survival and proliferation as parameters.

[0151] Therefore, in most cases, when inducing the expression of some function other than proliferation, the chemical substance is added externally to these commercially available basic media. In addition to survival and proliferation, CJ cells also express a parameter called spontaneous calcification, and we considered the possibility that some component of the DMEM culture medium may be involved in this.

[0152] In other words, if differences in the body fluid environment in the body affect metabolic syndrome, differences in cell culture media, which correspond to differences in the body fluid environment for in vitro cultured cells, may affect the expression of parameters (calcification) even if there is no difference in proliferation ability. Therefore, the inventors cultured CJ cells in several commercially available culture media with different constituents and contents, and compared both cell proliferation and calcification.

[0153] 2. Experimental Method In this example, the following six commercially available cell culture media were used: DMEM (High Glucose), DMEM (Low Glucose), DMEM / Ham's F-12, RPMI-1640, and α-MEM were obtained from Wako Pure Chemical Industries. Eagle's MEM was obtained from Nissui Pharmaceutical. CJ cells (92 PDLs) that had been repeatedly passaged in DMEM (High Glucose) medium were used in this example.

[0154] According to the law, 75 cm 2 CJ cells grown in flasks were washed with PBS(-) and then detached by heating in 0.25% trypsin-1 mM EDTA-4Na solution. The cells were then dispersed in DMEM medium and counted. The cell suspension was dispensed into six 15 ml centrifuge tubes and centrifuged at 1200 rpm at 4°C for 5 minutes to remove the supernatant. 10 ml of cold PBS(-) was added to each of the precipitated cell fractions at the bottom of the tubes, and the cells were dispersed using a 5 ml pipette. The cells were then centrifuged at 1200 rpm at 4°C for 5 minutes to obtain a washed cell pellet with PBS(-). This cell pellet was then washed again by the same centrifugation with PBS(-) to obtain the precipitated cell fraction.

[0155] After removing the supernatant and washing solution, 10 ml of the six types of culture medium prepared in advance, supplemented with 10% (v / v) FBS (same manufacturer, same lot), 100 units / ml penicillin, and 100 μg / ml streptomycin, was added to each 15 ml centrifuge tube. Disperse well using a 5 ml pipette, then transfer the cells to a 50 ml centrifuge tube at 2 x 10 5 The cells were diluted to 100 cells / ml and dispensed into a 24-well multi-well plate at 1 ml / well to initiate culture (day 0). On day 10, the supernatant was removed, the plate was washed with PBS(-), and 2 ml / well of 95% EtOH was dispensed to fix the cells at room temperature for 30 minutes, after which the plate was air-dried.

[0156] Prior to culturing, cells were dispensed onto plates in 4 wells for each culture medium, as shown in Figure 10(a), and stained after fixation with EtOH. During each staining and washing, plate seals were attached to the other wells to prevent liquid contamination. Quantitative testing using a 96-well plate was performed using 2 x 10 cells in each medium as described above. 5 Prepare a cell suspension of 4 x 10 cells / ml. 4 The cells were plated at 200 μl / well (day 0), washed with BS(-) on day 10, fixed with 95% EtOH, air-dried, stained, extracted, and quantified.

[0157] 3. Results and Discussion The results were very interesting. As shown in Figure 10(a), qualitative differences were readily apparent and visible to the naked eye. In other words, there was almost no difference in the survival and proliferation of CJ cells due to the differences in culture medium. This result was shown to be equivalent not only by CV staining but also by Giemsa staining.

[0158] On the other hand, significant differences in calcification were observed depending on the culture medium. Specifically, based on the degree of AR staining, CJ cells showed high calcification in DMEM (high glucose) and α-MEM media, whereas they hardly formed any calcified foci in RPMI-1640 media. In DMEM (low glucose), DMEM / Ham F-12, and Eagle's MEM, they showed intermediate calcification, falling between the two.

[0159] As shown in the figure, the staining results using AR closely matched the staining intensity obtained with Von Kossa staining, which is said to stain calcium more sensitively. Similar results were obtained in quantitative evaluations using 96-well plates containing five commercially available media. The amount of cells stained with CV in each well did not differ depending on the type of culture medium used. However, the amount of calcium deposits stained, extracted, and quantified using AR was high in DMEM (high glucose) and α-MEM media, extremely low in RPMI-1640 media, and intermediate in DMEM (low glucose) and Eagle's MEM media.

[0160] This result is truly surprising. In culture research using various cells, we have all experienced large differences between the lots of FBS used. We have simply believed that it was conventional wisdom to pre-test and select serum lots without investigating unknown components in the serum. Commercially available culture media are completely artificial, with known components and contents other than serum. However, because the same lot of serum from the same manufacturer was dispensed on the same day for each experiment, the significant differences in calcification ability cannot be attributed to unknown components in the serum. The cause must be the presence or absence and / or differences in content of components in the completely artificial medium, or a combination of these factors.

[0161] Of particular interest are the differences between DMEM medium (high glucose) and DMEM medium (low glucose). Both media are commercially available from the same manufacturer, and the only difference in their components is the glucose concentration. Specifically, the glucose content in DMEM medium (high glucose) is 4.5 g / L, while that in DMEM medium (low glucose) is 1.0 g / L, a 4.5-fold difference. As mentioned above, diabetes has also been reported to be related to urinary stone formation. Therefore, DMEM medium (high glucose), as if reflecting high blood glucose levels, may promote calcium crystal formation, the starting point for urinary stone formation, in CJ cells. Therefore, CJ cells may be a useful in vitro model for investigating the pathogenesis of diabetes and urinary stone formation.

[0162] However, the glucose content of α-MEM, which showed a high promotion of mineralization in both media, was low at 1.0 g / L. Furthermore, the glucose content of RPMI-1640, which showed almost no mineralization, was 2.0 g / L, twice that of α-MEM, and the glucose content of DMEM / Han F-12, which showed only moderate to moderate mineralization, was 3.15 g / L, 3.15 times that of α-MEM.

[0163] Therefore, calcification is not affected solely by differences in glucose concentration, but other medium components and their combination with glucose metabolism may be related to calcification and its inhibition (for example, in the case of RPMI-1640).In fact, from speculative research based on the results of Example 9, when considering chronic diseases other than diabetes that are thought to be related to urinary tract stones, such as myocardial infarction, hypertension, obesity, and chronic renal failure, as well as metabolic syndrome, it is natural to assume that physiologically active factors other than glucose concentration are also involved.

[0164] From the perspective of achieving scientific and long-term goals, the fact that the majority of commercially available culture media are completely synthetic and comprise highly pure compounds is undoubtedly a major advantage. In other words, by using CJ cells and various culture media with known components and contents, and taking an additive or subtractive scientific approach based on the degree of calcification as an indicator, it will inevitably be possible to investigate and identify the factors that promote and inhibit calcification in known culture medium components.

[0165] This fundamental approach will lead to the identification of factors related to urolithiasis, which is associated with metabolic syndrome and chronic diseases, and the development of drugs targeting these factors. From the perspective of achieving practical, short-term goals, on the other hand, it is an applied approach to evaluate compounds by combining the CJ cells of the present invention with various media. For example, the combination of CJ cells with DMEM medium enables highly efficient screening of "anti-lithiasis compounds" for the purpose of discovering and developing drugs to prevent or treat urolithiasis. The combination of CJ cells with RPMI-1640 medium, for example, enables highly efficient screening of "lithogenic compounds" for the purpose of confirming the safety of drug candidate compounds, foods, and food additives.

[0166] Example 10: Addition of three oxalate precursors to CJ cell culture system 1. Purpose of the experiment Next, we focused on the type of calcium stones commonly seen in clinical practice. In clinical patients, CaOx-only stones and mixed CaOx-CaP stones were found at roughly equal rates, accounting for 90% of all calcium stones. Meanwhile, CaP-only stones accounted for a surprisingly low 10%. As mentioned above, even in the CaOx-rich growing masses seen in Randall's plaques, CaP crystals form the nucleus at the center of the stone, and as a result, pure CaP stones may be rare. If CaP crystal nucleation precedes the process of stone recurrence, spontaneous calcification by CJ cells is limited to CaP. It would be meaningful to utilize this cellular characteristic to inhibit CaP crystal formation. Furthermore, if we could discover that CJ cells also have the ability to form CaOx crystals, their value as a model for exploring anti-calculus substances would be even greater.

[0167] Because there are no suitable in vitro cell culture models for stone formation, CaOx stone formation models have mainly been studied in animal models. A typical model involves administering three oxalate precursors, namely ethylene glycol, glycolic acid, and glyoxylic acid, to animals, based on the metabolism of oxalate in the body. Interestingly, even though they are rodents, rats can produce CaOx stones with any of the three oxalate precursors, whereas in mice, only glyoxylic acid can induce CaOx crystals in the kidney.

[0168] Therefore, the present inventors added these three types of oxalic acid precursors to a CJ cell culture system to examine whether CaOx crystals or CaOx stones similar to those in animal stone models could be produced.

[0169] 2. Experimental Method 2 x 10 cells in DMEM medium 5 CJ cells (10 PDLs) adjusted to 10 cells / ml were plated at 2 ml / well in a multi-well plate (12 wells, Sumitomo Bakelite) on day 0. Culture was initiated at 37°C in a 5% CO2 incubator. On day 2, three oxalate precursors were added to the culture medium to a final concentration of 10 mM. The oxalate precursors, ethylene glycol (Lot ECP4599), sodium glycolate (Lot LKQ2021), and glyoxylic acid monohydrate (Lot PDF4037), were all obtained from Wako Pure Chemical Industries. These reagents were dissolved in HBSS(-). The pH of the first two solutions was neutral, but glyoxylic acid monohydrate has a pH of 2.4. Therefore, the pH was adjusted to 6.8 with dropwise addition of NaOH before use. All solutions were stored at 4°C until use. HBSS(-) alone was added to the control group. Each treatment group was run in triplicate. On day 9 (7 days after the addition of the oxalic acid precursor), counting from the start of culture on day 0, the cells were observed under a phase contrast microscope and photographed.

[0170] 3. Results and Discussion In preliminary studies, we sought to determine the appropriate concentration range for the precursor to be added. This was based on the fact that the timing of the onset of spontaneous calcification in CJ cells tended to be more time-dependent than the initial cell number seeded. In other words, cells seeded on day 0 appeared as CaP around day 4. Unlike physicochemical crystal precipitation, the existence of this time lag suggests the true nature of biomineralization, which involves cellular metabolism. In particular, in this example, intracellular enzymatic conversion from the precursor to oxalic acid, which can ultimately bind to Ca ions, is required. Therefore, we considered that CJ cells must survive for at least 4 days after precursor addition. As a result, a final concentration of 10 mM was selected, and the results of this example are presented.

[0171] As shown in Figures 11(a)-11(d), only glyoxylic acid (GA) (Figure 11(d)) induced the formation of CaOx crystals. As can be seen in the phase-contrast micrograph in Figure 11(d), the cells are still readily visible on the well bottom, spreading and tightly adhering to each other, suggesting cell survival. On the other hand, 10 mM ethylene glycol and sodium glycolate are not toxic, and although the image of cells adhering to the well bottom is comparable to that observed with GA, they did not produce any CaOx, at least not as CaOx crystals observable under a microscope. This result is consistent with the mouse model (Okada, A. et al., Urol. Res., 35, 89-99, 2007) but inconsistent with the rat model (Fan, J. et al., J. Am. Soc. Nephrol., 14, S376-S380, 1999) in in vivo stone formation animal models.

[0172] Specifically, Okada et al. reported that when various oxalate precursors were administered intraperitoneally to mice, only GA was able to produce calcium oxalate crystals in the kidney, while ethylene glycol and sodium glycolate were ineffective. On the other hand, Fan et al. showed that in rats, two other oxalate precursors besides GA could also produce stones. CJ cells are renal proximal tubule cells derived from dogs, but they are likely to be an in vitro model closer to mice than rats.

[0173] That is, the completely new in vitro discovery of crystal formation and crystal dissolution / disintegration, and the fact that their time course, i.e., kinetics, are very similar, will be shown in the following examples. In any event, the initial goal of this example was to answer the working hypothesis of whether CJ cells can form calcium oxalate stones, which account for the majority of calcium stone patients, and the result was a clear yes, further enhancing the value of CJ cells as an in vitro model of urinary stones.

[0174] Example 11: Aggregation image of calcium oxalate (CaOx) crystals formed by adding glyoxylic acid 1. Purpose of the experiment According to a recent review (Alelign, T and Petros, B., 2018, Advance in Urology vol. 2018, Article ID 3068365), the timeline of stone formation is said to progress as follows: oxidative stress → cell damage and cell membrane destruction → crystal nucleation → crystal growth → crystal aggregation → cell-crystal interaction → crystal retention and adhesion to cells → stone formation. In our previous studies, for example, we were able to observe the appearance and growth of CaOx crystals, but were unable to observe the subsequent process of crystal aggregation.

[0175] Although the actual trigger for the crystal aggregation process in the body is unknown, it is speculated that matrix produced by the cells or dead cell fragments may act as glue. If the formed CaOx crystals were able to move freely within the culture system, they could eventually form crystal aggregates through collisions, associations, and adhesion with the movement of the culture medium. However, the CaOx octahedral crystals observed in the culture dish were firmly attached to the surface of the CJ cells or the surface of the dish substrate, and were not even able to be detached or moved by shaking or pipetting.

[0176] As discussed in Example 4, CaP stones formed by spontaneous calcification are sandwiched between the basement membrane of CJ cells and the culture vessel. These results suggest that a similar phenomenon occurs during CaOx crystal formation, and in fact, staining experiments have been obtained that strongly support this hypothesis (see the Example below). The two major theories of stone formation are the fixed particle theory and the free particle theory. As the names suggest, in the former, particles grow in the interstitial tissue beneath epithelial cells while attached to the tissue (immobile), while in the latter, particles grow detached from the tissue (mobile) and primarily in the lumen of the renal tubules. If CJ cells retain the CaOx crystals they themselves are involved in producing in an immobile state, then this cell culture system can be said to be close to the fixed particle theory, and we hypothesized that clues to aggregation may be hidden there, so we conducted extensive microscopic observations.

[0177] 2. Experimental Method Columns 1 and 12 of a 96-well plate were left blank (200 μl / well of DMEM medium only), and all remaining wells were filled with 2 x 10 CJ cells (135 PDLs) diluted in DMEM medium. 4 Cells were plated at 200 μl per well (day 0) and cultured at 37°C in 5% carbon dioxide and 95% air. On day 2, GA was added to a final concentration of 10 mM. On day 8, the culture medium was discarded and the cells were washed with 200 μl per well of PBS(-). 200 μl of 95% EtOH was added per well and the cells were left at room temperature for 30 minutes to fix the cells. Images of the cells in this fixative were observed and photographed under a phase-contrast microscope.

[0178] 3. Results and Discussion Observation of each well of the 96-well plate revealed that the majority of CaOx crystals were isolated and scattered, but two interesting crystal aggregation images were obtained. One of the images, shown in Figures 12(a)–12(c), is a cell-mediated aggregation image. The other, shown in Figures 12(d)–12(f), is a crystal aggregation image mediated by fibrous foreign matter contaminated in the culture system. The fibrous foreign matter in Figures 12(d)–12(f) is a fibrous material often observed in culture systems. It is thought to be derived from fetal bovine serum (FBS) added to the culture system, and the inventors have speculated that it is likely fibrin. Low-magnification (100x) images show numerous CaOx crystals closely attached to the fibrous material, while no isolated CaOx crystals were found in the surrounding area. It is intriguing whether these foreign filaments entangled the surrounding crystals as the liquid moved, or whether crystal precipitation itself initiated on the fibrous material.

[0179] On the other hand, in Figures 12(a) to 12(c), what appear to be cells appear to extend pseudopodia-like structures between the crystals, entangling and grabbing the crystals. This morphology is the complete opposite of the pattern of crystals gathering and agglomerating around dead cell fragments. The cells themselves are alive, and appear to be grabbing and grabbing the crystals in the manner described above, resulting in the appearance of aggregation. Contrary to accepted theory, the following examples reveal that, at least in the case of CJ cells, living cells, particularly cells that have not previously been recognized, play a major role in the fate of the CaOx crystals formed.

[0180] In any event, it has become clear that the crystal aggregation process, which is one step in stone formation, can also be actively intervened in the CJ cell culture system of the present invention, i.e., screening for substances that promote or inhibit aggregation is possible. Specifically, whether dead cell fragments serve as "nuclei for aggregation" can be confirmed by adding separately prepared dead cell fragments, and if the serum-derived filaments are fibrin, known fibrin can be added. Furthermore, the present inventors naturally recognize that if a crystal aggregation promoter is found in any form or amount, it can then be used as a crystal aggregation system, enabling screening for crystal aggregation inhibitors.

[0181] Example 12: Preparation of mixed CaOx and CaP stones by exchanging the medium with glyoxylic acid (±) 1. Purpose of the experiment Our previous experiments have revealed that CJ cells, when cultured in a standard medium, primarily form CaP crystals and their aggregated precipitates, whereas the addition of GA reverses this process by forming CaOx single crystals and their aggregates. Meanwhile, in clinical settings, in addition to CaP and CaOx-based single stones, some patients develop mixed stones (sometimes abbreviated as CaP-CaOx mixed stones herein), with a reported ratio of 10:50:40. Given that 90% of urinary stones are calcium stones, and 40% of these are mixed stones, generating mixed stones using CJ cells would provide a highly convenient in vitro model, further enhancing the value of the CJ cells of the present invention as a research tool. After extensive research, we discovered that the addition and removal of GA can easily induce the formation of CaP-CaOx mixed stones in CJ cells in an in vitro culture system, thereby achieving our goal.

[0182] 2. Experimental Method On Day 0, 2 x 10 5 CJ cells diluted to 4 x 10 cells / ml were plated in a 12-well plate. 5 Cells were seeded at 2 ml / well and cultured at 37°C in a carbon dioxide incubator. On day 2, GA was added to final concentrations of 0 mM (control), 0.1 mM, 1 mM, or 10 mM (n=3 per group), and culture continued. On day 6, the medium was replaced with DMEM containing GA at each concentration, and culture continued until day 13. After confirming sufficient CaOx crystal formation under a microscope on day 13, the culture medium was replaced with DMEM without GA (GA-free DMEM medium). On day 17, the medium was replaced again with GA-free DMEM, and on day 19, microscopic observation and photography were performed.

[0183] 3. Results and Discussion Figures 13(a) and 13(b) show typical mixed CaP and CaOx stones observed on Day 19. In the center of the image, a cluster of CaP crystals of varying sizes can be seen incorporating nearby CaOx crystals to form a mixed stone. Therefore, as expected, a CaP-CaOx mixed stone formed on the bottom of the petri dish, albeit in a two-dimensional plane, rather than the three-dimensional shape seen in an actual living body.

[0184] However, when we found what appeared to be remnants of CaOx crystals slightly below the center of the photograph in Figure 13(d), we were forced to change our interpretation and interpret the roughening of the plain CaOx crystal surface as not being the surface deposition of small CaP particles, but as the crystals themselves dissolving or collapsing. The validity of this interpretation was supported by completely new and powerful supporting evidence in the following examples.

[0185] In any case, we can conclude that our initial goal was achieved. In other words, we have demonstrated that CJ cells are multipotent cells capable of not only forming CaP crystals and CaOx crystals individually, but also producing mixed stones of both, demonstrating their applicability as an in vitro model system consistent with clinical cases. Below, we further investigated the biological breakdown / dissolution phenomenon of crystals (bio-degradation), which begins simultaneously with CaOx crystal formation and is clearly associated with cells.

[0186] Example 13: Relatively early onset of calcium oxalate crystal dissolution and breakdown (bio-degradation) 1. Purpose of the experiment In Example 12, we found that long-term treatment (19 days) with GA (±) resulted in the loss of luster on the crystal surface and the dissolution and collapse of the crystal structure itself. This is an important discovery that leads to the mysterious disappearance of calcium oxalate crystals in vivo, which was observed and reported by Okada, A. et al. 10 years ago in an animal model, as described below. In particular, the discovery that similar crystal collapse and dissolution occurs at the in vitro cell culture level is one of the most important points in this invention.

[0187] 2. Experimental Method (1) Parts (a) to (d) CJ cells (135 PDLs) were diluted in DMEM medium and plated at 2 x 10 4 Cells were plated at 200 μl per well (day 0). On day 2, GA was added to a final concentration of 10 mM, and culture was initiated at 37°C in a 5% CO2 incubator. On day 8, the culture supernatant was removed, and 200 μl per well of 0.9% NaCl (physiological saline) was dispensed to wash the cells. Then, 200 μl per well of 95% EtOH was dispensed to fix the cells. Images of the cells in this fixative were observed and photographed under a phase-contrast microscope.

[0188] (2) Parts (e) and (f) To confirm whether the structures in the cytoplasm of cells formed by GA addition were lipid droplets, fat staining with oil red O dye was performed. CJ cells (97 PDLs) were diluted in DMEM medium and placed in 5 x 10 wells of a 96-well plate. 4 On day 0, cells were plated at 200 μl per well. On day 1, after confirming by microscopic examination that the cells in the wells had reached sufficient confluence, GA was added to a final concentration of 10 mM, and culturing was initiated at 37°C in a 5% CO2 incubator.

[0189] On day 8, the culture supernatant was removed and the cells were washed twice with 300 μl / well of 0.9% NaCl (physiological saline). Then, 150 μl / well of 10% neutral hornorhinol solution was added to fix the cells. After removing the formalin, the plate was immersed in gently running water to wash the wells (by pouring water into the wells and decanting it), and 100 μl / well of 60% isopropanol was dispensed, and the fixed cells were infiltrated with alcohol for 1 minute.

[0190] After removing the alcohol, 100 μl of Oil Red O staining solution was dispensed into each well and stained at 37°C for 15 minutes. The Oil Red O staining solution was prepared immediately before use (Oil Red O was dissolved in 99% isopropanol at 0.3% (w / v) to form a stock solution. Four volumes of distilled water were added to six volumes of this stock solution, and the solution was stirred and filtered through No. 2 filter paper before use). After removing the Oil Red O, 200 μl / well of 60% isopropanol was added, incubated for two minutes, and then replaced with 200 μl / well of distilled water. Microscopic examination and photography were then performed.

[0191] 3. Results and Discussion As shown in Figure 14(a), the flat surfaces of the octahedral crystals characteristic of calcium oxalate dihydrate (COD) lose their luster and become rough. At the 10, 2, and 7 o'clock positions, there are cells that appear to cover the top surface of the crystal or penetrate the bottom surface. Furthermore, the CJ cells that adhere to and extend from the bottom surface have undergone significant morphological changes, with numerous vacuoles or vacuole-like structures observed within the cytoplasm. In Figure 14(b), a different field of view, the crystal surface also becomes rough, and cells can be seen invading and expanding onto the top or bottom surface of the crystal from the 12, 2, and 5 o'clock positions.

[0192] Although not shown in the photograph, the positive control cells for Oil Red O staining were the mouse fibroblast 3T3L1 cell line (JCRB9014 3T3L1) obtained from the JCRB Bioresource Bank of the National Institutes of Medical Basics, Health and Nutrition. The intracellular structures obtained by replacing 3T3L1 cells with a differentiation-inducing medium (containing insulin, isobutylmethylxanthine, and dexamethasone) stained intensely with Oil Red O after the same treatment, demonstrating that there were no problems with the fat staining process.

[0193] Among stones, calcium oxalate stones are extremely insoluble, and unlike some other stones, drug dissolution therapy has traditionally been thought to be nearly impossible. While CaP has low solubility, it can be dissolved in 5% hydrochloric acid. However, taking advantage of the fact that CaOx is insoluble even in 5% hydrochloric acid, Yasue staining has emerged as a CaOx-specific staining method (Yasue, T., Acta Histochem. Cytochem., 2, 83-95, 1969).

[0194] Interestingly, when this stubborn CaOx crystal is artificially formed in vivo, it has been reported that the CaOx crystals disappear, and moreover, this disappearance occurs in a short period of time, in mouse and rat stone models. Okada et al. reported that when GA was administered intraperitoneally at 80 mg / kg for 15 consecutive days to C57BL / 6N male mice, CaOx crystals appeared and increased in the kidneys for the first 6 days after the start of administration. However, despite continued daily GA administration, the number of CaOx crystals in the kidneys steadily decreased, reaching undetectable levels by the 12th day (Okada, A. et al., JBMR, 24(5), 908-924, 2009).

[0195] Meanwhile, in the same year, Vervaet also gave male Wistar rats free access to water containing 0.75% (v / v) ethylene glycol for four days, and followed their progress over time by pathological analysis of the kidneys. He reported that the amount of CaOx crystals in the kidneys increased two and five days after cessation of EG intake, but decreased significantly after 10 days, and was completely undetectable after 25 days (Vervaet, BA et al., Kidney Intern., 75, 41-51, 2009).

[0196] Of the two animal model experiments, the results in the mouse model seem to have important implications. The fact that CaOx crystals disappear even with continued administration of GA, an oxalate precursor, suggests that even patients with hyperoxaluric calculi may have hope for stone dissolution. Furthermore, recent geobiological techniques, combining stone sectioning techniques and optical techniques (brightfield, polarized light, confocal, and autofluorescence), have demonstrated that CaOx stones undergo repeated crystallization and dissolution during their formation and growth in the kidney (Sivaguru, M. et al., Scientific Reports (2018) 8: 13731). Khan has already expressed hope for the possibility of crystal dissolution therapy, asking, "Even if we could suppress supersaturation of CaOx, what approaches would be possible for existing CaP deposits? For example, would it be possible for CaP to be naturally removed by the action of macrophages, etc.?" (Khan, SR et al., Nat. Rev. Dis. Primers 2016, 2: 16008).

[0197] However, a major obstacle to this hope is the lack of an in vitro experimental system capable of evaluating a large number of substances from the perspective of "stone dissolution." Whether searching for substances that inhibit the stone formation process or those that promote stone disappearance, screening systems using animals are not realistic in terms of the time required, cost, and number of samples that can be processed. In fact, despite important discoveries being made in both mouse and rat stone models, it is not surprising that even now, 10 years after their discovery, there have been no reports of compounds that have shown promise in these models.

[0198] However, this difficult problem can be easily overcome by using the CJ cells discovered by the present inventors. This point also brings us back to the origin of the problem to be solved by this invention. Below, we will present specific substances that are candidates for the prevention and treatment of calcium stones, as well as examples in which their effects can be quantitatively evaluated. However, before that, we first demonstrated that the crystals observed to appear with GA addition were CaOx, not only by their characteristic morphology but also by Yasue staining, which specifically stains calcium oxalate. Furthermore, during this demonstration experiment, we made another interesting discovery, which opened the door to a simple quantitative evaluation of the crystal dissolution phenomenon.

[0199] Example 14: Biodegradation of CaOx crystals by lithoclasts (tentative name) induced from CJ cells and changes in the form of Ca within the lithoclasts' cytoplasm 1. Purpose of the experiment Alizarin red and silver nitrate are known as staining methods for histologically detecting calcium, but Yasue used 14 other reagents in addition to these, and examined the staining properties and intensity of three calcium compound specimens, calcium phosphate (CaP), calcium carbonate (CaC), and calcium oxalate (CaOx), each embedded in celloidin, as well as kidneys from oxaluric rats with CaOx stones, renal tissue from patients with Alport syndrome, and transplanted kidneys, as comparison materials.

[0200] As a result, we developed a highly specific CaOx staining method that stains only CaOx black to dark brown, overcoming the drawbacks of Von Kossa staining and Pizolato staining, even though both methods use the same silver nitrate (Yasue T., Hitochem. Cytochem., 2, 83-95, 1969).

[0201] Yasue discovered and reported an excellent method for differentially staining calcium precipitates, CaP, CaOx, and CaC, through model experiments. The staining method is characterized by the use of rubeanic acid in addition to silver nitrate. The key to Yasue's CaOx-specific staining is the removal of contaminating CaP and CaC by acid treatment of the specimen. In other words, the former two dissolve in 5% acetic acid (AcOH), but the latter, CaOx, does not. Conversely, the former two stain with alizarin red dye, but the latter, CaOx, does not stain at all.

[0202] Therefore, the inventors qualitatively examined whether the forming, disintegrating, or dissolving octahedral crystals would stain black or brown with Yasue staining. However, just to be sure, Alizarin Red staining was also performed before Yasue staining.

[0203] 2. Experimental Method CJ cell dispersion (2 x 10 5 Cells / ml) were seeded at 2 ml / well in a 12-well plate, and after 48 hours, GA was added to a final concentration of 10 mM. After a further 7 days of culture (day 9), the cells were fixed with 95% ethanol (EtOH) and stained with 1% AR solution for 30 minutes (Figures 14(a) and 14(b)). The cells were then immersed in distilled water and observed and photographed according to the standard method. Next, additional staining was performed using the same plate using the Yasue method. First, 5% (w / v) AcOH (2 ml / well) was dispensed into each well of the AR-stained plate, and a shaking extraction treatment was performed at room temperature for 30 minutes (Shaking Mixer SHM2002, LMS).

[0204] This acid extraction process removes CaP and CaC, even if they are present. Yasue staining was then performed. The wells were lightly rinsed with distilled water, and 50 μl of 5% (w / v) silver nitrate solution was added per well and allowed to stand at room temperature for 15 minutes. After removing the silver nitrate, the wells were washed with gently running water. Saturated rubeanic acid solution (50 μl per well) was then dispensed and incubated at room temperature for 1 minute, after which the rubeanic acid was removed by decantation. The wells were then rinsed with 50% EtOH (100 μl per well), and finally, the wells were filled with distilled water (200 μl per well) for phase contrast observation and photography.

[0205] 3. Results and Discussion Phase contrast observation revealed no granular structures resembling CaP other than the regular octahedral crystals of CaOx. However, as a precaution, AR staining was performed, revealing an unexpected crystal image, as shown in Figures 15(a) and 15(b). In other words, almost all of the CaOx crystals scattered across the field of view were partially stained deep red. The red-stained circular to irregularly circular shapes clearly revealed the appearance of phagocytes covering the crystals. Cell images of this magnitude would have been difficult to detect using phase contrast observation.

[0206] In cases where the crystals were close to each other (Figure 15(b)), they were observed as red-stained amoeba-shaped structures that straddled and covered the two crystals. Furthermore, the parts of the octahedral crystals that were not covered by the amoeba-shaped phagocytes (lithoclasts) were unstained by AR, and the intact CaOx in these areas was indeed unstained by AR dye, as reported by Yasue.

[0207] As mentioned above, it is difficult to recognize and identify these phagocytes present on the crystals using phase contrast observation (e.g., Figure 13), and in some cases they can barely be observed as spherical cells approaching the crystals in 95% EtOH (Figure 14). However, surprisingly, when AR staining was performed (Figure 15), the boundary of the phagocytes' crystal coverage could be clearly observed.

[0208] The mechanism of AR staining is probably the release of free ionized Ca (CaOx) from the CaOx crystal during the phagocytosis process by phagocytes. 2+ ) is dissociated by acid treatment, and this Ca 2+ This is thought to be because the calcium recombined with phosphate or carbonate in the stone-clad cells and changed into the AR-positive forms of calcium phosphate and calcium carbonate.

[0209] On the other hand, as shown in Figures 15(c) and 15(d), Yasue staining clearly reveals black staining in the crystals, particularly in the center of the crystals, suggesting that this is the CaOx portion that has not yet been phagocytosed. Furthermore, it is clearly evident that the crystal disintegration proceeds gradually from the periphery toward the center. In both photographs, the peripheral areas of small crystals that do not stain black with Yasue staining can be observed under phase contrast, preserving the general shape of the crystal. This is interesting because it is assumed to represent the remains of matrix components (probably primarily protein components) other than Ca that were not dissolved in 5% AcOH.

[0210] CJ cells are proximal tubule cells (glandular cells) that exhibit a renal epithelial-like morphology. The crystal disintegration and dissolution observed in the CaOx formation system of CJ cells makes it difficult to imagine that these cells are contaminated macrophages that survived for as long as 100 PDLs after cell isolation. Rather, they are more likely to resemble so-called semi-professional phagocytes (Ichimura, T. et al., J. Biol. Chem., 273, 4135-4143, 1998; Bonventre, J. V., J. Am. Soc. Nephrol., 14, S55-S61, 2003), which are formed by transformation of proximal tubule cells themselves, which process debris in damaged proximal tubule tissue. Of course, current data alone cannot rule out the possibility that terminally differentiated glandular cells may dedifferentiate to hematopoietic stem cells, which then redifferentiate into macrophages.

[0211] Example 15: CaP formation, CaOx formation, and disintegration of CaOx crystals in three CJ clone cell lines. 1. Purpose of the experiment Next, the inventors used CJ cells that had reached 90 PDLs as a parent line and cloned the cells by limiting dilution, comparing their calcification ability in CaP and CaOx systems with their decalcification ability in CaOx systems in an attempt to obtain clues. As shown in Example 15 below, a portion of the results was obtained. Essentially, many cloned cells grown from single cells also yielded positive results in the CaP system (spontaneous calcification when cultured in DMEM medium alone) and the CaOx system (CaOx crystal formation and collapse / dissolution with the external addition of GA). This is described in detail in Example 15 below.

[0212] 2. Experimental Method First, we performed cell cloning by limiting dilution. Specifically, we used CJ cells that had reached 90 PDLs as the parent line. These were serially diluted with DMEM medium to prepare a cell suspension containing 10 cells / ml. This suspension was then seeded into seven 96-well multiwell plates at 1 cell / 100 μl / well and cultured at 37°C in a carbon dioxide incubator. Because the CJ cells proved robust during the handling process, we scanned the wells with a phase-contrast microscope the day after the start of culture to check for the presence or absence of cells. We recorded the distribution of cells (0, 1, or 2 or more / well) and their location within the well.

[0213] On the 7th day of culture, microscopic examination confirmed the formation of single colonies at the location of the cells. Then, the culture was continued while examining the cells under a microscope. From the wells that reached confluence, the medium was removed, washed with PBS(-), and the cells were detached by trypsin treatment. 2 The flask and culture area were gradually expanded to create cryopreservation ampoules. During this cryopreservation process, a portion of each clone's cells was seeded onto two 48-well plates (day 0). One of the 48-well plates was cultured in DMEM medium alone (with three medium changes every 3 to 5 days). On day 16, the cells were fixed with EtOH and their spontaneous mineralization ability was qualitatively examined by AR staining according to the method described in Example 2. GA was added to the other 48-well plate at a final concentration of 10 mM on day 2, and the plate was observed and photographed under a phase-contrast microscope on day 6.

[0214] 3. Results and Discussion Of the 672 wells in seven 96-well plates, 420 wells showed no cell adhesion or proliferation. Microscopic examination confirmed one cell adhesion on day 1 and detected proliferation as a single cell colony on day 7 in 164 wells. Two cells / well, three cells / well, and four or more cells / well were detected on day 0 in 53 wells, six wells, and three wells, respectively. The remaining 26 wells showed cell adhesion on day 1 but no cells on day 7. Of the 164 clones that successfully expanded from single cells, 135 clones with good proliferation were obtained and cryopreserved.

[0215] These 135 clones were subjected to CaP and CaOx crystal formation tests. Except for two clones, 133 clones exhibited nearly identical CaP crystal formation potential. While there were differences among clones in terms of the timing, abundance, and crystal size of CaOx crystal formation, octahedral crystal formation was observed in 58 clones, or roughly half of the 133 clones. Furthermore, four clones formed crystals of a different morphology, although their identification was not possible. These results clearly suggest that the formation and collapse of CaOx crystals, particularly evident upon the addition of 10 mM GA, is not the result of separate cells with different functions present in the parent CJ cells, but rather that the same cells (at least 58 clones) may be involved in both processes. Representative examples are shown in Figures 16(a)–16(f).

[0216] Figures 16(a) to 16(c) show the results of the CaP formation system, i.e., spontaneous calcification, in DMEM medium. Both clone CJ-5H5 and clone CJ-6G4 cells showed high spontaneous calcification potential as measured by the production of CaP crystals, and most other clone lines were similar. On the other hand, clone CJ-7E12 cells had extremely low CaP production potential, and there was one other clone with a similar potential (CJ-1D6). Interestingly, these two clones showed areas stained with AR, i.e., partial calcification foci, in various parts of the field of view.

[0217] On the other hand, the results of adding GA to these cloned cells at a final concentration of 10 mM are shown in Figures 16(d) to 16(f), respectively. In Examples 10 to 14, the formation of regular octahedral crystals of CaOx dihydrate (COD) was observed, similar to that shown in the CJ parent cell line, and the crystal collapse and dissolution patterns observed in the parent cell line were also clearly observed. In particular, time-dependent observations revealed that the formation and decomposition of CaOx crystals occurred quickly in the cloned CJ-7E12 cells.

[0218] Example 16: Time lag between CaOx crystal formation and decay in CJ parent cells and CJ-3C1 clone 1. Purpose of the experiment In Example 15, the formation and decay of calcium oxalate (CaOx) crystals in the CJ-7E12 strain was faster than in the other two clone strains (CJ-5H5, CJ-6G4). We investigated whether there were any clones that exhibited similar early formation and decay of CaOx.

[0219] 2. Experimental Method Test cells dispersed in DMEM medium were cultured at 5 x 10 on day 0. 4 Cells were plated at 100 μl / well, and 24 hours later, on day 1, 100 μl / well of DMEM medium containing 20 mM glyoxylic acid was added (final volume: 200 μl / well, final glyoxylic acid concentration: 10 mM). The CJ parental cell line was placed in the top four rows of the 96-well plate, and the CJ-3C1 clone cell line was placed in the bottom four rows. Observations were performed daily using a phase-contrast microscope, and photographs were taken and recorded.

[0220] 3. Results and Discussion As a result of the study, the addition of GA to a final concentration of 10 mM also showed a similar response, namely, early formation and early decay of CaOx, in the CJ-3C1 clone. Figure 17 shows phase-contrast microscopic images in comparison with the parent CJ cell line. Furthermore, although not shown in the figure, unlike the CJ-7E12 clone, the CJ-3C1 clone, without the addition of GA, showed accelerated formation of calcium phosphate (CaP) crystals that stained extensively with AR, similar to the CJ-5H5 and CJ-6G4 clones.

[0221] This suggests that although the CJ-3C1 and CJ-7E12 lines both showed early formation and decay of CaOx, they may not have been derived from the same clone, but rather from different clones.At present, it is unclear why the CJ-7E12 and CJ-3C1 lines exhibited CaOx crystal formation and decay approximately one day earlier than the parent CJ cell line or the other two clones (CJ-5H5 and CJ-6G4).

[0222] In any event, the various novel findings presented in the above examples led the inventors to the following invention. Specifically, this invention is positioned as a research tool patent that can lead to useful discoveries and inventions by appropriately using the CJ parent cell line, as well as the clone lines that make up this parent line. Needless to say, the value of a research tool patent depends on whether it can actually produce a compound that is scientifically and practically useful.

[0223] Example 17: Effects of bisphosphonates and resveratrol on CJ cell proliferation and stone formation 1. Purpose of the experiment Therefore, the present inventors first used a bisphosphonate derivative, which has been reported to have anti-calculus effects on microliths formed in vitro in 3D cultures of MDCK cells and to be expected to have anti-calculus effects in vivo in postmenopausal osteoporosis patients at risk of stone formation (Senzaki, H. et al., Urol. Res., 32, 223-228, 2004; Yasui, T. et al., CLINICAL CALCIUM, 21, 1511-1515, 2011), to evaluate whether a similar effect would be observed in the spontaneous CaP crystal formation system of the CJ cells of the present invention.

[0224] Three bisphosphonate derivatives were used: risedronate, alendronate, and pamidronate. Similarly, although the antilithiasis effect of resveratrol, a polyphenol, in an EG-induced rat stone disease model has been reported (Hong, S. H. et al., Pharmacological Reports, 65, 970-979, 2013), there are no reports of resveratrol inhibiting calcification in the in vitro "MDCK screening" referred to herein. Therefore, we also examined whether resveratrol inhibits calcification using the CJ cells of the present invention.

[0225] 2. Experimental Method CJ cells (93 PDLs) were cultured in DMEM medium for 10 5 Dilute cells / ml to 10 in a 96-well plate. 4 The cells were plated at 100 μl / well and culture was initiated at 37°C in a 5% CO2 incubator (day 0). The first row of the plate was used as a blank, to which only the culture medium was added. Bisphosphonate 3 derivative (Tokyo Chemical Industry Co., Ltd.) was dissolved in distilled water and diluted to 100 μl. -2 After preparing the stock solution of M, dilute it further in DMEM medium to 2x10 -4 M~2x10 -12 Ten-fold serial dilutions between M were prepared.

[0226] Resveratrol (synthetic) powder was dissolved in 99.5% EtOH to prepare stock solutions, which were then stored in a freezer until use. On Day 2, two-step dilutions of these stock solutions were made with DMEM medium on a transfer plate, and the diluted solutions were added at 100 μl / well to continue culturing. After microscopic examination on Day 10, the same procedures as in Example 7 were performed: medium removal, PBS(-) washing, EtOH fixation, AR staining, washing, drying, pigment extraction, and absorbance measurement. Cell counts were also quantified in the same wells as in Example 7, using CV staining (except using a formalin-free 0.05% (w / v) CV aqueous solution) followed by washing, pigment extraction, and absorbance measurement.

[0227] 3. Results and Discussion As shown in Figures 18(a) to 18(c), all bisphosphonates showed similar dose-response curves. That is, as shown by the black circles, the anti-stone effect of all bisphosphonates was 10 -6 M~10 -4 However, a dose-dependent change was observed between 10 -4 At the 10 M concentration, as shown by the open bars, stone formation was inhibited due to the manifestation of cytotoxicity. At the 10 M concentration, no cytotoxicity was observed and an inhibitory effect on calcification was observed. -5 The effect of MDCK cells on the 3D culture system was limited to the M concentration range. There are some differences when comparing the results above with those reported by Senzaki et al. (Senzaki, H. et al., Urol. Res., 32, 223-228, 2004), which evaluated the effect of MDCK cells on the 3D culture system.

[0228] They found that alendronate had a 10% inhibitory effect on calcification. -9 M and 10 -7 However, in this example, as shown in Figure 18(b), no anti-calcification effect was observed in the corresponding concentration range, and -5 In M, approximately 40% inhibition of calcification was finally observed, with an effective concentration difference of more than 100 times. They also reported that incadronate did not have the anti-stone effect of alendronate.

[0229] On the other hand, in this example, incadronate (Fig. 18(d)) was slightly weaker than alendronate (Fig. 18(b)), but 10 -5 An anti-stone effect was observed at the M level. Of course, even though they are both canine-derived kidney cells, the CJ cells of the present invention are primarily derived from proximal tubules, while MDCK cells are derived from distal tubules, and they share the common characteristic of spontaneous calcification in vitro without induction by externally added factors. However, the CJ cells of the present invention are cultured in two-dimensional plates, while Senzaki et al.'s method is three-dimensional, and there are differences in the concentration range of the bisphosphonates examined, so the present inventors are fully aware that a simple comparison cannot be made.

[0230] On the other hand, as shown in Figure 18(d), resveratrol showed a stone-forming inhibitory effect of approximately 20% at a final concentration of 50 μM, and at the highest concentration of 100 μM, the anti-stone effect reached 70% at a condition where cytotoxicity of approximately 35% appeared, although not as great as that of bisphosphonates. From the above, it was revealed that two candidate compounds, bisphosphonates and resveratrol, which are expected to have anti-stone effects in human patients or animal models, also react with the CJ cell evaluation system of the present invention.

[0231] Example 18: Antilithiasis effect of interferon-γ as a false positive hit compound 1. Purpose of the experiment In addition to bisphosphonates, interferon (hereinafter abbreviated as IFN, as necessary) is a compound involved in regulating calcium metabolism in bone tissue, a normotopic mineralized tissue. The present inventors previously found that interferon has a potent inhibitory effect on osteoclast formation in vitro (Takayanagi, H. et al., Nature, 416, 744-749, 2002).

[0232] In particular, IFN-γ not only inhibited osteoclast activity, which is involved in bone dissolution (i.e., demineralization), but also inhibited calcification in mouse MC3T3-E1 osteoblasts (Nobutaka Ida et al., WO97 / 04799). Therefore, we focused on the effect of IFN on stone formation due to ectopic calcification and investigated this using the CJ cells of the present invention. Note that IFN is species-specific, and canine IFN-γ was used for canine-derived CJ cells. However, since IFN-α has weak species specificity, human IFN-α was also used in the experiments.

[0233] 2. Experimental Method CJ cells (100 PDLs) were cultured in DMEM medium for 10 min. 5 Dilute cells / ml to 10 in a 96-well plate. 4 The cells were plated at 100 μl / well and culture was initiated at 37°C in a 5% CO2 incubator (day 0). The first column of the plate was used as a blank, to which only the culture medium was added. Canine IFN-γ (Interdog®), 3 x 10 5 (units / vial, Toray) was dissolved in 15 ml of DMEM medium in a vial and 2 x 10 4 A solution of human IFN-α (Sumiferon®), 3 x 10 units / ml was prepared. 6 units / vial, Sumitomo Pharmaceuticals) was dissolved in DMEM medium and diluted with distilled water to 2 x 10 4 After preparing a unit / ml solution, dilute it further with DMEM medium to 2x10 -4 M~2x10 -12 Ten-fold serial dilutions between M and M were prepared. On Day 2, these diluted IFN solutions were added at 100 μl / well, and the culture was continued. On Day 7, after microscopic examination, the medium was removed, washed with PBS(-), fixed with EtOH, stained with AR, washed, dried, extracted with dye, and measured for absorbance, in the same manner as in Example 7. The cell number was also quantified in the same manner as in Example 7, by staining with crystal violet in the same well after Ca quantification, followed by washing, extracted with dye, and measured for absorbance.

[0234] 3. Results and Discussion Human IFN-α was tested at concentrations up to 100,000 units / ml, but no effect on either cytotoxicity or calcification parameters was observed (Figure 19(a)). In contrast, canine IFN-γ showed almost no cytotoxicity up to a maximum concentration of 10,000 units / ml, but showed an interesting biphasic response to calcification. This was characterized by promotion of calcification at low concentrations and inhibition at high concentrations. The "anti-calcification effect" at high concentrations, in particular, was almost negligible with cytotoxicity, making it a "hit compound" in the cell-based assay using CJ cells of the present invention. Fortunately, there is a wealth of clinical information, as well as information on the physiological and pharmacological effects of IFN. Therefore, we understand that concentrations of 5,000 units / ml to 10,000 units / ml are local concentrations that are impossible to achieve in clinical practice, and we therefore avoid making a misjudgment.

[0235] Rather, the problem lies in the low concentration range in this experimental data where a tendency for increased "calcification" was observed, and these may be blood and tissue concentrations that are clinically relevant. If this is the case, the hit compound will be a stone-promoting drug rather than an anti-stone drug, and even if it is eventually eliminated in secondary and tertiary screening, it is truly an example of a "false-positive compound." This is also an example demonstrating that the CJ cell line of the present invention is not only a detection system for "stone-promoting compounds," but is also capable of detecting negative compounds that promote the formation of various stones, including urinary stones.

[0236] Example 19: Inhibitory effect of lactoferrin (LF) on spontaneous calcification of CJ cells 1. Purpose of the experiment Both bisphosphonates and interferons have been approved as drugs, and there is a wealth of research and data on their effects on bone metabolism, the tissue responsible for normal mineralization. Lactoferrin (LF) is a compound that affects bone metabolism and is close to a drug, and there have been numerous reports of its promotion of bone formation in vitro and in vivo, making it a candidate for the treatment of osteoporosis.

[0237] However, the only reported effect of LF on stone formation as ectopic calcification was in an in vitro cell-free experimental system, where the physicochemical interaction between CaOx crystals and the test protein promoted the formation of CaOx crystals, and the conclusion was that LF, which is a component of stones, acts to promote crystal growth (Farmanesh, S. et al., J. Am. Chem. Soc., 2014, Sep. 10: 136(36)12648-12657). Therefore, we investigated whether promotion of stone formation would also occur in our in vitro calcified stone model using the CJ cell system.

[0238] 2. Experimental Method First, we performed an experiment using CJ cells in a spontaneous calcification model where CaP was formed without any medium exchange. CJ cells (100 PDLs) were cultured in DMEM medium for 10 5 Dilute cells / ml to 10 in a 96-well plate. 4 The cells were plated at 100 μl / well and culture was initiated at 37° C. in a 5% carbon dioxide incubator (day 0). The first column of the plate was used as a blank, to which only the culture medium was added.

[0239] Bovine lactoferrin (bLF, biochemical grade, derived from milk, Lot WDK 2023, Wako Pure Chemical Industries, Ltd.) was dissolved and diluted with DMEM medium to a concentration of 2 x 10 -4 On Day 2, 2 x 10 cells were cultured on a transfer plate. -4 Starting with M solution, LF solutions were prepared by serially diluting them two-fold, and 100 μl / well of the diluted LF solutions were added to a 96-well plate containing CJ cells plated two days earlier. The cells were then cultured for 24 hours. After microscopic examination on Day 7, the cells were subjected to the same procedures as in Example 7: medium removal, washing with PBS(-), fixation with EtOH, AR staining, washing, drying, dye extraction, and absorbance measurement. Cell counts were also quantified in the same manner as in Example 7, using CV staining in the same wells as in Ca quantification, followed by washing, dye extraction, and absorbance measurement.

[0240] 2. Results and Discussion The results obtained were completely opposite to what was expected. As shown in Figure 20, LF inhibited CaP formation in a dose-dependent manner, with an IC50 value of approximately 6 μM, as can be seen from the figure. At this concentration, no cell detachment was observed, and at an LF concentration of 25 μM, which showed more than 90% stone formation inhibition, approximately 20% cell reduction was observed.

[0241] The reason why we did not state 20% cytotoxicity here is that observation under a phase-contrast microscope showed that the glandular epithelial CJ ​​cells detached from the dish surface and changed into cells that were attached but had a spindle-like morphology. It is currently unclear whether the cells detached and became rounded, or whether they detached because they had become rounded. In any case, it is important to note that these floating cells were removed during the washing procedure performed during AR staining on the day of evaluation, and the result of the decrease in CV staining intensity was reflected in the cell count.

[0242] In any case, it was clearly demonstrated that lactoferrin does not promote stone formation as is conventionally believed (Farmanesh, S. et al., J. Am. Chem. Soc., 2014, Sep.10: 136(6), 12648-12657, Rimer, ID. et al., Urolithiasis, 5(1) 57-74, 2017), but rather inhibits it in a CaP-dominated spontaneous calcification model.

[0243] Incidentally, in the CaP evaluation system using the CJ cell line, the anti-stone effect, which is the main effect of LF, was approximately 10 times superior in molar ratio to the IC50 value of the aforementioned resveratrol, and conversely, the dose at which LF manifested its side effect, cytotoxicity, was lower than that of resveratrol (see Figure 18(d)).

[0244] As shown in Figure 1, there have been many reports of LF's bone formation promoting effect (normtopic calcification) in vitro and at the animal experiment level, while its stone formation inhibitory effect (ectopic calcification) showed a completely opposite Ca metabolism. When the discovery of resveratrol's anti-stone effect in CJ cells and publicly known reported examples are taken into consideration, a similar opposite Ca metabolism relationship is established.

[0245] For example, in vitro, resveratrol promotes bone differentiation from canine bone marrow mesenchymal stem cells, accompanied by mineralization (Zhao, Xiao-E, et al., Cell Reprogram 2018, 20(6), 371-381), and inhibits osteoclast formation from RAW 264.7 cells, precursors of osteoclasts (He, Xu, et al., Biochem. Biophys. Res. Commun., 2010, 401(3), 356-362). In addition to these in vitro experimental reports, there are also abundant evidences that resveratrol promotes bone formation in animal experiments (Murgia, D., et al., Biomolecules, 2019, 9,94).

[0246] On the other hand, there are few reports on renal stones, which are ectopic calcification, in in vitro or animal experimental systems, with the only report being an ethylene glycol-induced rat stone model (Hong, SH et al., Pharmacological Reports, 65, 7099, 13). They are the only ones to demonstrate the anti-stone (anti-calcification) effect of resveratrol at this in vivo level. The anti-calcification effect of resveratrol in the CJ cell line of the present invention provides some support for the anti-stone mechanism in Hong et al.'s in vivo data.

[0247] Looking at this factual situation from the other side, it is clear that the strong anti-stone effect of LF on CJ cells provides a theoretical basis for the reasonable assumption that the anti-stone effect of LF can be expected even at the animal experiment level.

[0248] Example 20: Determination of the anticalculus activity of lactoferrin in CaP and CaOx systems 1. Purpose of the experiment The experiments in Examples 14 to 16 revealed that the addition of GA specifically promoted the formation of CaOx crystals. However, it is difficult for laboratories without the necessary equipment to perform mass screening of anti-calculus compounds using image analysis of Yasue-stained images in this CaOx-forming system and quantify the results. Meanwhile, preliminary studies revealed that the dye eluted with 5% acetic acid from the discovered "lithoclasts" stained red with AR exhibited a reasonable absorbance at 450 nm. Therefore, we also used LF to examine whether it was possible to quantitatively evaluate anti-CaOx calculus activity from the perspective of "lithoclast activity."

[0249] 2. Experimental Method A comparative experiment on the anti-lithiasis effect of LF was conducted using CJ cells in the CaP and CaOx forming systems. As shown in the photograph of the 96-well plate in Figure 21(a), the experiment was conducted with the CaP forming system in the top four rows and the CaOx forming system in the bottom four rows. First, on Day 0, CJ cells (141 PDLs) were cultured in DMEM medium for 10 5 Dilute cells / ml to 10 in a 96-well plate. 4 Cells were plated at 100 μl / well and culture was initiated at 37°C in a 5% CO2 incubator. The first row of the plate was used as a blank, to which only culture medium was added. The bLF used was the sterilized 200 μM stock solution (stored at 4°C) used in Example 19.

[0250] On Day 2, 2 x 10 cells were transferred to a transfer plate. -4 Starting with the M solution, LF solutions were serially diluted two-fold to prepare 2-fold serial dilutions, and 100 μl / well of the LF diluted solutions were added to the 96-well plate containing CJ cells plated two days earlier, followed by the addition of the LF diluted solutions. The plate was then returned to the incubator.

[0251] On day 3, the plate was removed, and 20 μl of HBSS alone was added to each well of the CaP formation system in the top four rows, while 20 μl / well of HBSS (pH 6.4) containing 100 mM GA was added to each well of the bottom four rows, and the culture was continued.

[0252] After microscopic examination on Day 7, the same procedures as in Example 7 were followed: removal of medium, washing with PBS(-), fixation with EtOH, AR staining, washing, drying, dye extraction, and absorbance measurement. For the CaOx formation system, AR staining and Yasue staining after quantification were also performed in the same 96-well plate. Morphological observation and photography of cells and crystals stained with each dye were performed under the condition that 200 μl of distilled water was added per well after washing.

[0253] 3. Results and Discussion First, this experiment also reproduced the clear dose-dependent anti-lithiasis effect of LF in the CaP formation system, with an IC50 value of approximately 5 μM, which was almost the same as the 6 μM value in the previous experiment (Figure 21(b)). In addition to observing the number and size of CaOx crystals, the objective of this experiment was to successfully quantify the "lithoclast activity," and the anti-lithiasis effect of added LF was also measured.

[0254] In this case, although the absolute value of the OD450 nm reading was considerably lower than that in the CaP system, when the control value without LF was set to 100%, a clear dose-response curve was obtained, as shown in Figure 21(c). It may be more appropriate to express the "lithoclast activity" in the CaOx-forming system as EC50 (Effective Concentration of 50%) rather than IC50 (Inhibiting Concentration of 50%) in the CaP-forming system.

[0255] However, the measured value here is merely the remaining rate of AR pigment in the stone cell, and not the concentration of Ca in the stone cell. 2+ The amount of calcium and calcium phosphate released into the stone-clad cells and the subsequent outflow of calcium from the stone-clad cells are not included. In any case, LF clearly exerts a dose-dependent effect on the formation and / or activity of stone-clad cells, and the 50% reading from this figure is approximately 3 μM.

[0256] If the same inhibitory effect of LF on osteoclast formation in bone tissue were to occur in this stone model, inhibiting stone-breaking activity, the Ca within the CaOx crystals should be preserved, and the higher the LF concentration, the more the CaOx crystals should stain black with Yasue stain. However, in reality, as shown in Figure 23, the exact opposite was true, with the CaOx crystals at the high LF concentration side completely missing the CaOx that should have been stained with Yasue stain, leaving only crystal frames (ghosts) (Figures 23(f) to 23(h)).

[0257] From this figure, including the difference in CaOx crystal size, it is clear that LF not only inhibits the formation of CaOx crystals themselves, but also induces the appearance of "lithoclasts" and enhances lithoclast activity, although this is semi-quantitative. In other words, it is clear that LF inhibits osteoclast function, which is involved in decalcification in bone tissue, which is the site of normal calcification, but conversely promotes the function of the "lithoclasts" of the present invention, which are involved in decalcification in stones, which are the site of ectopic calcification.

[0258] Example 21: Transferrin has no anti-calculus effect in either the CaP-forming system or the CaOx-forming system. 1. Purpose of the experiment LF belongs to the transferrin family and shares the common property of binding iron ions. If iron ion metabolism is involved in the anti-calcification effect of LF revealed in this invention, it is possible that transferrin may also have a similar effect, and this was investigated. The anti-calcification effect of transferrin was examined using two evaluation systems: a spontaneous calcification CaP formation system and a glyoxylic acid-induced CaOx formation system.

[0259] 2. Experimental Method Experiments were performed on the CaP formation system in the top four wells of a 96-well plate, and on the CaOx formation system in the bottom four wells. CJ cells (100 PDLs) were cultured in DMEM medium for 10 min. 5 Dilute cells / ml to 10 in a 96-well plate. 4 The cells were plated at 100 μl / well and culture was initiated at 37° C. in a 5% carbon dioxide incubator (day 0). The first column of the plate was used as a blank, to which only the culture medium was added.

[0260] Human transferrin (T-1147, Sigma) was dissolved and diluted in DMEM medium to 2 x 10 -4 A 200 μM solution was prepared and then sterile filtered for use in the experiment. The CaP formation test was performed on day 2, with 2 x 10 -4 Starting with solution M, two-fold serial dilutions of transferrin solutions were made, and 100 μl / well of these solutions were added to the cultured CJ cell plates. Microscopic examination was performed on day 7, and on the same day, the same procedures as in Example 7 were carried out: removal of the medium, washing with PBS(-), fixation with EtOH, AR staining, washing, drying, pigment extraction, and absorbance measurement.

[0261] On the other hand, for the test using the CaOx formation system, 20 μl / well of 100 mM GA was added to the transfer plate on day 3, 24 hours after the addition of the transferrin solution on day 2, and microscopic examination, fixation, AR staining, extraction, and quantification were performed on day 7.

[0262] 3. Results and Discussion Transferrin had no antilithiatic effect in either the CaP system (Figure 24(a)) or the CaOx system. In the CaOx-forming system, AR staining showed no change, and microscopic observations showed no change in the size or number of the CaOx crystals formed by transferrin.

[0263] Transferrin was also investigated in the physicochemical experimental system for CaOx crystal formation by Farmanesh et al., and the conclusion was that transferrin is an inhibitor of CaOx crystal growth (Farmanesh, S. et al., J. Am. Chm. Soc., 136(1), 367-37). Therefore, following LF, the results for transferrin are inconsistent, and the reason for this is likely to be the difference in the presence or absence of living cells in the experimental systems used.

[0264] Vogel, HJ stated that although both LF and transferrin have the ability to bind iron ions, transferrin plays an important role in transporting iron to various parts of the body via the bloodstream, while LF does not appear to play such a role, raising the question of what the role of LF is (Vogel, HJ Biochem. Cell Biol., 90, 233-244, 2013).

[0265] Transferrin is also thought to exert various physiological effects through its iron transport ability, similar to that of LF, but the possibility that the mechanism of the anti-stone effect of LF involves iron ion metabolism has been virtually ruled out. In other words, the anti-stone effect of LF is due to a different biological activity specific to LF that is not dependent on iron metabolism.

[0266] Example 22: Intraluminal stone formation in spheroid cultures of CJ cells 1. Purpose of the experiment In recent years, it has become clear that cell function differs significantly between two-dimensional planar cultures formed in flasks for adherent cell culture and three-dimensional cell mass (spheroid) states. While spontaneous calcification progresses in two-dimensional cultures of the CJ cells of the present invention, we investigated what happens in three-dimensional cultures.

[0267] 2. Experimental Method First, CJ cells were dispersed in DMEM medium and cultured in a conventional adherent culture flask (Cat No. 690-170, 25 cm 2The cells were seeded in suspension culture flasks (Cat. No. 690-190, Greiner Bio-One) and confirmed to undergo passage and spontaneous calcification similarly to Corning flasks. After the flasks reached confluence or showed slight calcification, the medium was removed from the flasks, washed with PBS(-), and a 0.01% (w / v) solution of pronase (Actinase E®, 1,000,000 tyrosine units / g) in PBS(-) was added. The cells were incubated at 37°C for 30 minutes. To the floating cells, 8 ml of DMEM medium was added and the cells were thoroughly dispersed by pipetting. The entire cell suspension was seeded in suspension culture flasks (Cat. No. 690-190, Greiner Bio-One) and cultured statically in a CO2 incubator for 12 hours. After the initiation of spheroid formation was confirmed under a microscope, half of the suspension culture flask was left in place and the other half was seeded into a new adhesion flask. For both, 2 ml of fresh DMEM medium was added, and static culture was continued in a carbon dioxide incubator, followed by morphological observation and photography over time.

[0268] 3. Results and Discussion CJ cells were well dispersed after 30 minutes of treatment with 0.01% pronase. These dispersed cells were also transferred to a suspension culture flask and cultured for 12 hours, resulting in the formation of cell clusters consisting of multiple cells, as shown in Figure 25(a). Because the doubling time for CJ cells is approximately 24 hours, we determined that the cell clusters were not formed by division of a single cell, but rather by aggregation of cells that had been separated in the liquid. The fact that these cell clusters were so-called spheroids is clear from the shape of the cells in the culture flask on day 2 (Figure 25(b)) and day 9 (Figure 25(d)).

[0269] It was also found that CaP stones formed in the lumen of the spheroids. Furthermore, it was also found that the percentage of spheroids containing CaP stones in the lumen increased with the passage of culture time. This indicates that even in a three-dimensional state, calcium deposition progresses over time due to calcium ion movement from the apical to the basal side of the cell membrane.

[0270] This CaP deposition in the lumen of CJ cell (proximal tubule) spheroids is consistent with results observed in MDCK cells, which are distal tubule cells (e.g., Naito, Y. et al., Urol. Res., 25, 59-65, 1997). On the other hand, when spheroids were reseeded in adherent culture flasks, more than half of them adhered to the flask bottom by day 2, and CJ cells began to proliferate and spread toward the periphery (Figure 25(c)). By day 9, the spreading area had further increased (Figure 25(e)). When these CJ spheroids were re-expanded in two dimensions, the CaP spherical masses that had already formed in the lumen appeared to adhere to the cells, and although not shown in the figure, they were confirmed to stain red with AR. [Accession number]

[0271] NITE P-03184

Claims

1. A screening method for a preventive or therapeutic agent for urolithiasis, comprising culturing cells derived from the proximal tubules, which are capable of forming calcified foci when cultured in vitro, in the presence of a test substance, wherein inhibition of the formation of calcified foci indicates a high possibility that the test substance has a preventive or therapeutic effect on urolithiasis, wherein the cells are an established cell line derived from the proximal tubules of an animal suffering from kidney cancer, and the established cell line is CJ cells deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE P-03184.

2. The method according to claim 1, wherein the cells are cultured using (i) Dulbecco's MEM (High Glucose) or α-MEM containing fetal bovine serum, or (ii) Dulbecco's MEM (Low Glucose), Dulbecco's MEM / Ham-F12, or Eagle's MEM as a medium.

3. 3. The method according to claim 1, wherein the cells are cultured in a serum-free, growth factor-containing, synthetic medium with known components.

4. 4. The method according to claim 3, wherein the cell growth factor is at least one selected from the group consisting of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF).

5. The method according to any one of claims 1 to 4, wherein the cells are cultured using a medium containing glyoxylic acid.

6. The method according to any one of claims 1 to 5, wherein the degree of calcification of the cultured cells is determined by Alizarin Red S staining and / or Yasue staining.

7. The method according to any one of claims 1 to 6, wherein the culturing is carried out by juxtaposition or comparative culturing with an osteoblast and / or an odontoblast cell line.

8. An established cell line derived from the proximal tubules of an animal suffering from kidney cancer, which is capable of forming calcification foci when cultured in vitro, said established cell line being a CJ cell deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE P-03184.

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