Biomarker for determining onset of urolithiasis and use thereof

Abstract

Provided is a novel biomarker for determining the onset of urolithiasis. This biomarker for determining the onset of urolithiasis is characterized by comprising at least one among calcium phosphate crystals and amorphous calcium phosphate in urine.

Description

Biomarkers for determining the onset of urolithiasis and their use

[0001] The present disclosure relates to biomarkers for determining the development of urolithiasis and their use.

[0002] Urolithiasis is a disease in which stones form in the urinary tract, and the stones that form are called urinary stones. It is known that the majority of urinary stones are calcium oxalate stones. For this reason, the onset of urinary stones has been determined based on the daily excretion of inorganic components such as oxalic acid calculated from a 24-hour urine collection test.

[0003] Saeed R. Khan et al, Kidney stones, Nat Rev Dis Primers, (2016) 25:2:16008.IA Karampas and CG Kontoyannis, Characterization of calcium phosphates mixtures, Vibrational Spectroscopy, 64 (2013) 126-133.LigaBerzina-Cimdina and Natalija Borodajenko, Research of Calcium Phosphates UsingFourier Transform Infrared Spectroscopy, Infrared Spectroscopy - MaterialsScience, Engineering and Technology, (2012) 123-148.

[0004] However, in recent years, it has been reported that organic components such as proteins play a promoting or inhibiting role in the growth process of urinary stones (Non-Patent Document 1). Therefore, conventional methods that evaluate only inorganic components cannot grasp the formation of urinary stones in a comprehensive environment that includes organic components, and therefore cannot accurately determine the onset of urinary stones.

[0005] Therefore, an object of the present disclosure is to provide a new biomarker for determining the onset of urolithiasis, which can accurately determine the onset of urolithiasis.

[0006] To achieve the above object, the biomarker for determining the onset of urolithiasis of the present disclosure includes at least one of calcium phosphate crystals and amorphous calcium phosphate in urine.

[0007] The method for determining the onset of urolithiasis of the present disclosure includes a detection step of detecting a biomarker for determining the onset of urolithiasis of the present disclosure in the urine of a subject, and a determination step of determining the onset of urolithiasis in the subject based on the detection result of the biomarker.

[0008] The screening method for candidate substances for inhibiting urolithiasis of the present disclosure includes a selection step of selecting, from test substances, a formation inhibitor that inhibits the formation of at least one of calcium phosphate crystals and amorphous calcium phosphate as a candidate substance for inhibiting urolithiasis.

[0009] According to the present disclosure, the onset of urolithiasis can be determined with high accuracy.

[0010] FIG. 1 is a photograph showing an image of calcium oxalate insolubilized matter formed under temperature conditions of 20°C, 10°C, and 4°C observed with an optical microscope in Example 1 of the present disclosure. FIG. 2 is a table showing basic data of patients with urinary tract stones in Example 1 of the present disclosure. FIG. 3 is a photograph showing the results of detection of calcium phosphate insolubilized matter using an optical microscope in Example 1 of the present disclosure. FIG. 4 is a graph showing the results of detection of calcium phosphate insolubilized matter by Raman spectroscopy in Example 1 of the present disclosure. FIG. 5 is a table showing the results of a statistical test between two groups, one in which no insolubilized matter was observed and one in which calcium oxalate insolubilized matter was observed, in Example 2 of the present disclosure. FIG. 6 is a table showing the results of a statistical test between two groups, one in which no insolubilized matter was observed and one in which calcium phosphate insolubilized matter was observed, in Example 2 of the present disclosure. FIG. 7 is a table showing the results of a statistical test between two groups, one in which calcium oxalate insolubilized matter was observed and one in which calcium phosphate insolubilized matter was observed, in Example 2 of the present disclosure. Figure 8 is a photograph showing an image of a thin section of a calcium oxalate stone observed with a polarizing microscope under open Nicols conditions in Example 3 of the present disclosure. Figure 9 is a photograph showing the results of mapping four elements in Area A on a thin section of a calcium oxalate stone by fluorescent X-ray analysis in Example 3 of the present disclosure. Figure 10 is a photograph showing an image of Area A on a thin section of a calcium oxalate stone observed with a polarizing microscope under crossed Nicols conditions in Example 3 of the present disclosure. Figure 11 is a graph showing the results of detection of calcium phosphate phases in Area A on a thin section of a calcium oxalate stone by laser Raman microscopy in Example 3 of the present disclosure. Figure 12 is a photograph showing an image of immunofluorescent staining of Area A on a thin section of a calcium oxalate stone observed with a fluorescence microscope in Example 3 of the present disclosure. Figure 13 is a photograph showing the results of mapping four elements in Area B on a thin section of a calcium oxalate stone by fluorescent X-ray analysis in Example 3 of the present disclosure. FIG. 14 is a photograph showing an image of Area B on a thin section of a calcium oxalate stone observed with a polarizing microscope under crossed Nicols conditions in Example 3 of the present disclosure.FIG. 15 is a graph showing the results of detecting calcium phosphate phases in Area B on a thin section of a calcium oxalate stone using a laser Raman microscope in Example 3 of the present disclosure. FIG. 16 is a photograph showing an image of immunofluorescent staining of Area B on a thin section of a calcium oxalate stone observed using a fluorescence microscope, and a photograph showing an image of insolubilized calcium phosphate in a urine sample observed using an optical microscope in Example 3 of the present disclosure. FIG. 17 is a table showing basic data of a group without urolithiasis recurrence and a group with urolithiasis recurrence in Example 4 of the present disclosure. FIG. 18 is a table showing the results of a statistical test between two groups, a group without urolithiasis recurrence and a group with urolithiasis recurrence in Example 4 of the present disclosure. FIG. 19 is a table showing the results of a logistic regression analysis in Example 4 of the present disclosure. FIG. 20 is a table showing basic data of subjects in Example 5 of the present disclosure. FIG. 21 is a table showing the results of a statistical test on the results of a 24-hour urine collection test in Example 5 of the present disclosure. FIG. 22 is a graph showing the results of a statistical test on the results of a 24-hour urine collection test in Example 5 of the present disclosure. FIG. 23 is a photograph showing the results of optical microscopy detection of each insolubilized substance in a cooled urine sample in Example 5 of the present disclosure. FIG. 24 is a table showing the results of a statistical test between each group on the number of subjects who detected each insolubilized substance in a cooled urine sample in Example 5 of the present disclosure. FIG. 25 is a graph showing the results of a statistical test between each group on the number of subjects who detected insolubilized substances in Brushite in a cooled urine sample in Example 5 of the present disclosure. FIG. 26 is a table showing the results of a logistic regression analysis in Example 5 of the present disclosure. FIG. 27 is a graph showing the correlation between SI. COD and SI. Brushite in subjects who detected insolubilized substances in a cooled urine sample in Example 5 of the present disclosure. Figure 28 is a photograph comparing the results of detecting insoluble Brushite particles by light microscopy in cooled urine samples from three groups of patients with early recurrence of urolithiasis, patients with late recurrence of urolithiasis, and patients without recurrence of urolithiasis in Example 5 of the present disclosure. Figure 29 is a graph showing the correlation between SI.COD and SI.Brushite for each group of patients with urolithiasis in Example 5 of the present disclosure.FIG. 30 is a graph showing the results of detection of insolubilized calcium phosphate by Raman spectroscopy in Example 5 of the present disclosure.

[0011] <Definition> In this specification, "urolithiasis" refers to a disease in which stones are formed in the urinary tract. Stones formed in the urinary tract are, for example, urinary stones (hereinafter also referred to as stones). The urinary tract includes, for example, the kidney, ureter, bladder, and urethra. Examples of the kidney include the right kidney, the left kidney, and both kidneys. Examples of the ureter include the right ureter, the left ureter, and both ureters. Urolithiasis is diagnosed, for example, by confirming the presence of urinary stones by an imaging test. Examples of the imaging test include simple CT scans, intravenous urography (IVU), simple X-ray tests, ultrasound tests, etc. Examples of the simple X-ray tests include simple nephroureteral and bladder radiography.

[0012] As described above, the urinary tract stone is not particularly limited and is, for example, a stone formed in the urinary tract. Examples of the components of the urinary tract stone include cystine stones, uric acid stones, magnesium ammonium phosphate stones, calcium oxalate stones, calcium phosphate stones, and mixed stones thereof. Examples of the components of the urinary tract stone may be calcium oxalate stones, calcium phosphate stones, or mixed stones of calcium oxalate stones and calcium phosphate stones. The components of the urinary tract stone can be identified, for example, by stone analysis. The stone analysis is performed, for example, by infrared spectroscopy. Examples of the site where the urinary tract stone is present include the urinary tract, such as the kidney, ureter, bladder, and urethra. Examples of the kidney include the right kidney, left kidney, and both kidneys. Examples of the ureter include the right ureter, left ureter, and both ureters. Examples of the site where the urinary tract stone is present include both kidneys or both ureters. The site where the urinary stone is present can be identified by, for example, the imaging test.

[0013] As used herein, "calcium phosphate" refers to a general term for inorganic salts containing calcium ions and phosphate ions. Examples of calcium phosphate include monocalcium phosphate (dihydrogen calcium phosphate), dicalcium phosphate (also known as hydrogen calcium phosphate or monohydrogen calcium phosphate), tricalcium phosphate (tricalcium phosphate), dicalcium phosphate (calcium pyrophosphate), hydroxylated calcium phosphate (also known as hydroxyapatite or hydroxyapatite), carbonate apatite (also known as carbonate-containing hydroxyapatite or carbonate apatite), tetracalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate. Examples of tricalcium phosphate (tricalcium phosphate) include α-tricalcium phosphate and β-tricalcium phosphate. When the calcium phosphate is dicalcium phosphate (calcium pyrophosphate), the phosphate ion is a diphosphate ion. The amorphous calcium phosphate is also referred to as amorphous calcium phosphate. The calcium phosphate may be, for example, a hydrate or an anhydrate of the calcium phosphate. The calcium phosphate may be, for example, one that is mainly contained in the urinary tract stone or a urine sample from a patient with urolithiasis, and examples thereof include hydroxyapatite, calcium hydrogen phosphate, carbonate apatite, and tricalcium phosphate.

[0014] As used herein, "crystal" refers to a solid in which atoms, molecules, or ions are periodically arranged in three dimensions. The periodicity of the crystal has, for example, long-range order. Crystals are classified, for example, into covalent crystals, ionic crystals, metallic crystals, molecular crystals, etc. based on the type of chemical bond. Examples of the crystal include single crystals and polycrystals. The crystal includes all forms formed during the crystal formation process, such as crystal nuclei. As used herein, the crystal may include, for example, quasicrystals. The quasicrystal may be referred to as, for example, a crystal lacking translational symmetry. The crystal can be formed, for example, by cooling, evaporation, etc. The crystal is also referred to as, for example, a crystallized product, an insolubilized product, a precipitated product, a crystallized product, a separated product, etc. The formation of the crystal is also referred to as, for example, crystallization, insolubilization, precipitation, precipitation, crystallization, separation, etc. The crystal formation step is also referred to as, for example, a crystallization step, an insolubilization step, a precipitation step, a precipitation process, a crystallization step, a separation step, etc. The crystal formation treatment is also referred to as, for example, a crystallization treatment, an insolubilization treatment, a precipitation treatment, a precipitation process, a crystallization treatment, a separation treatment, etc.

[0015] As used herein, "amorphous" refers to a solid in which atoms, molecules, or ions are not periodically arranged in three dimensions. The periodicity of the amorphous state has, for example, short-range order but not long-range order. The amorphous state is also referred to as, for example, non-crystalline. The amorphous state includes, for example, any substance formed during the amorphous formation process. The amorphous state can be formed by, for example, cooling, evaporation, etc. The amorphous state is also referred to as, for example, an amorphized substance, an insolubilized substance, a precipitated substance, a precipitated substance, a crystallized substance, a separated substance, etc. The formation of the amorphous state is also referred to as, for example, amorphization, insolubilization, precipitation, precipitation, crystallization, separation, etc. The amorphous formation process is also referred to as, for example, an amorphization process, an insolubilization process, a precipitation process, a precipitation process, a crystallization process, a separation process, etc. The amorphous forming treatment is also called, for example, an amorphization treatment, an insolubilization treatment, a precipitation treatment, a sedimentation treatment, a crystallization treatment, a separation treatment, or the like.

[0016] Hereinafter, embodiments and examples of the present disclosure will be described with reference to the drawings. The present disclosure is not limited to the following embodiments and examples. Furthermore, unless otherwise specified, the descriptions of the embodiments can be mutually incorporated, and the configurations of the embodiments can be combined unless otherwise specified.

[0017] <Biomarker for Determining the Onset of Urolithiasis> The biomarker for determining the onset of urolithiasis of the present disclosure (hereinafter also referred to as the biomarker of the present disclosure) is not particularly limited, and is, for example, a biomarker including at least one of calcium phosphate crystals and amorphous calcium phosphate in urine. The biomarker of the present disclosure is characterized in that at least one of calcium phosphate crystals and amorphous calcium phosphate is used as a biomarker for determining the onset of urolithiasis, and other configurations and conditions are not particularly limited. Using the biomarker of the present disclosure, for example, by detecting the biomarker of the present disclosure in the urine of a subject, the onset of urolithiasis in the subject can be determined. The biomarker of the present disclosure can be used in the method for determining the onset of urolithiasis of the present disclosure and the method for screening candidate substances for inhibiting urolithiasis of the present disclosure, which will be described later.

[0018] As a result of extensive research, the present inventors have found that at least one of calcium phosphate crystals and amorphous calcium phosphate in urine is associated with the onset of urolithiasis, leading to the establishment of the present invention. Therefore, by detecting the biomarkers disclosed herein, the onset of urolithiasis in a subject can be determined. Furthermore, screening using the biomarkers disclosed herein can also be used to obtain candidate substances for inhibiting urolithiasis.

[0019] The biomarker of the present disclosure may be, for example, a biomarker for determining the recurrence of urolithiasis. The recurrence of urolithiasis refers to, for example, the re-formation of a urolith (new nucleation, growth, aggregation, and solidification) in a patient in whom a urolith was not confirmed by imaging or other tests after treatment for urolithiasis. Therefore, the recurrence of urolithiasis does not include, for example, the re-growth of a urolith in a patient in whom a residual urolith was confirmed by imaging or other tests after treatment for urolithiasis. The recurrence of urolithiasis may be, for example, early recurrence of urolithiasis or late recurrence of urolithiasis. The early recurrence of urolithiasis refers, for example, to the re-formation of a urolith within one year, two years, three years, four years, or five years from the time when a urolith was not confirmed by imaging or other tests after treatment for urolithiasis. Late recurrence of urolithiasis refers to, for example, re-formation of a urinary stone more than 5, 6, 7, 8, 9, or 10 years after the time when a urinary stone was not detected by imaging or other tests after treatment for urolithiasis. The site at which the urinary stone re-forms may be, for example, the same site as the site at which the urinary stone was present before treatment for urolithiasis, or a site different from the site at which the urinary stone was present before treatment for urolithiasis. Examples of treatments include spontaneous stone passage, treatments promoting spontaneous stone passage, and lithotripsy. Spontaneous stone passage refers to the natural excretion of a urinary stone without the treatments promoting spontaneous stone passage or lithotripsy. Examples of treatments promoting spontaneous stone passage include, for example, drinking large amounts of fluid, physical activity, and oral medication. Examples of the treatment by lithotripsy include extracorporeal shock wave lithotripsy, endoscopic lithotripsy, percutaneous lithotripsy, open surgery, etc. The imaging test can be carried out using the above-mentioned explanation, for example.

[0020] In the present disclosure, the origin of at least one of the calcium phosphate crystals and amorphous calcium phosphate is not particularly limited and can be appropriately determined, for example, depending on the type of subject. Examples of the origin include humans and non-human animals other than humans. Examples of the non-human animals other than humans include mice, rats, dogs, cats, monkeys, rabbits, sheep, and horses. In the present disclosure, it is preferable that at least one of the calcium phosphate crystals and amorphous calcium phosphate is derived from humans, for example.

[0021] The biomarkers of the present disclosure may be used alone or in combination with biomarkers for determining the onset of urolithiasis other than the biomarkers of the present disclosure (hereinafter also referred to as other biomarkers). The other biomarkers can be detected, for example, by urinalysis, blood tests, stone analysis, imaging tests, etc. Examples of the urinalysis include a 24-hour urine collection test, a qualitative and semi-quantitative test for general substances in urine, and a urinary sediment test. Examples of the other biomarkers detected by the 24-hour urine collection test include sodium, potassium, chloride, calcium, inorganic phosphorus, magnesium, creatinine, uric acid, urea nitrogen, oxalic acid, citric acid, and amino acids. Examples of the other biomarkers detected by the qualitative and semi-quantitative test for general substances in urine include pH, occult blood reaction, white blood cell count, nitrite, and turbidity. Examples of the other biomarkers detected by the urinary sediment test include red blood cell count, white blood cell count, salts, and crystals. Examples of the other biomarkers detected by the blood test include kidney function test items (creatinine, urea nitrogen, etc.), uric acid, calcium, inorganic phosphorus, CRP, etc. Examples of the other biomarkers detected by the stone analysis include cystine stones, uric acid stones, magnesium ammonium phosphate stones, calcium oxalate stones, calcium phosphate stones, or mixed stones thereof, etc. The imaging test can be, for example, as described above.

[0022] Examples of the type of urine include early morning urine, random urine, and pooled urine. Early morning urine is, for example, the first urine immediately after waking up, and is also called first early morning urine. Random urine is, for example, urine collected at any time, and is also called spot urine. Pooled urine is, for example, urine obtained by collecting all urine excreted within a certain period of time, and is exemplified by 24-hour pooled urine. The collection conditions for the pooled urine may be, for example, acidic pooled urine or normal pooled urine. The acidic pooled urine is, for example, pooled urine containing an acidic substance. Examples of the acidic substance include hydrochloric acid. The normality of the hydrochloric acid may be, for example, 1 to 6N. The normality of the hydrochloric acid is, for example, 6N. The normal pooled urine is, for example, pooled urine that does not contain an acidic substance. The pooled urine may contain, for example, a stabilizer, a preservative, or the like. The urine may be collected by, for example, natural excretion, discharge through a urinary catheter, or discharge through bladder puncture. Furthermore, the amount of urine collected may be, for example, whole urine or partial urine. The whole urine is, for example, the entire amount of urine excreted. The partial urine is, for example, a portion of the urine excreted, and examples thereof include first urine, midstream urine, and late urine. The first urine is, for example, urine collected immediately after the start of urination. The late urine is, for example, urine collected near the end of urination. The midstream urine is, for example, urine collected while avoiding the first urine and late urine. The urine may be, for example, aliquots of urine divided into small portions from a collection container.

[0023] The biomarkers of the present disclosure can be used as markers for the onset of urolithiasis, and specifically, for example, are preferably used as markers for the recurrence of urolithiasis.

[0024] The biomarkers of the present disclosure can be detected, for example, by microscopy, automated analysis, Raman spectroscopy, infrared spectroscopy, terahertz spectroscopy, and the like, as described below.

[0025] The biomarkers of the present disclosure can be used in the determination methods of the present disclosure, the inhibitors and inhibitory methods of the present disclosure, and the screening methods of the present disclosure, which will be described later.

[0026] <Method for Determining the Onset of Urolithiasis> The method for determining the onset of urolithiasis of the present disclosure (hereinafter also referred to as the determination method of the present disclosure) is not particularly limited, and may, for example, include a detection step of detecting the biomarker of the present disclosure in the urine of a subject, and a determination step of determining the onset of urolithiasis in the subject based on the detection result of the biomarker. The determination method of the present disclosure is characterized in that the detection step detects the biomarker of the present disclosure in the urine of the subject, and other steps and conditions are not particularly limited. The determination method of the present disclosure can also be referred to as, for example, an evaluation method, a test method, etc. According to the present disclosure, the onset of urolithiasis can be determined. The determination method of the present disclosure can use the description of the biomarker of the present disclosure.

[0027] The assessment method of the present disclosure can assess, for example, the possibility of developing urolithiasis, the presence or absence of urolithiasis (whether or not a urinary stone has formed), and the prognosis. As described above, the target urolithiasis is preferably recurrent urolithiasis. Furthermore, the target urolithiasis may be a first-time urolithiasis or a urolithiasis that has recurred twice or more times.

[0028] The assessment method of the present disclosure may be, for example, a method for assessing the recurrence of urolithiasis in the subject in the assessment step. According to the assessment method of the present disclosure, for example, the possibility of recurrence of urolithiasis, the presence or absence of recurrence of urolithiasis (whether or not a urinary stone has formed), and the prognosis state can be evaluated. The recurrence of urolithiasis can be, for example, as described above.

[0029] In the determination method of the present disclosure, the subject may be, for example, a human, a non-human animal other than a human, etc. Examples of the non-human animal other than a human include a mouse, a rat, a dog, a cat, a monkey, a rabbit, a sheep, a horse, etc.

[0030] In the determination method of the present disclosure, examples of the type of urine include early morning urine, random urine, and pooled urine. Early morning urine is, for example, the first urine immediately after waking up, also referred to as first early morning urine. Random urine is, for example, urine collected at any time, also referred to as spot urine. Pooled urine is, for example, urine obtained by collecting all urine excreted within a certain period of time, such as 24-hour pooled urine. The collection conditions for the pooled urine may be, for example, acidic pooled urine or normal pooled urine. The acidic pooled urine is, for example, pooled urine containing an acidic substance. Examples of the acidic substance include hydrochloric acid. The normality of the hydrochloric acid may be, for example, 1 to 6N. The normality of the hydrochloric acid is, for example, 6N. The normal pooled urine is, for example, pooled urine that does not contain an acidic substance. The pooled urine may contain, for example, a stabilizer or a preservative. The urine may be collected by, for example, natural excretion, discharge via a urinary catheter, or discharge via bladder puncture. Furthermore, the amount of urine collected may be, for example, whole urine or partial urine. The whole urine is, for example, the entire amount of urine excreted. The partial urine is, for example, a portion of the urine excreted, and examples thereof include first urine, midstream urine, and late urine. The first urine is, for example, urine collected immediately after the start of urination. The late urine is, for example, urine collected near the end of urination. The midstream urine is, for example, urine collected while avoiding the first urine and late urine. The urine may be, for example, aliquots of urine divided into small portions from a collection container.

[0031] The determination method of the present disclosure may include, for example, an insolubilization step in which the subject's urine is subjected to an insolubilization treatment to form the biomarker of the present disclosure. The insolubilization step is not particularly limited, and for example, a known insolubilization method can be used. The insolubilization step can also be referred to as, for example, a crystallization process, an amorphization process, a precipitation process, a precipitation process, a crystallization process, a separation process, etc. The insolubilization step can also be referred to as, for example, a crystallization process, an amorphization process, a precipitation process, a precipitation process, a crystallization process, a separation process, etc. The insolubilization process can be performed, for example, by adding an inorganic component, a poor solvent, a seed crystal, a pH adjuster, an ionic strength adjuster, etc. The insolubilization process can also be performed, for example, by supersaturation, as described below. The addition of the inorganic component can be, for example, by adding a solution containing sodium oxalate and calcium chloride to the subject's urine. The inorganic component may be added, for example, by adding a solution containing calcium phosphate, a solution containing inorganic phosphorus, or a solution containing calcium to the urine of the subject. The rate of addition of the inorganic component may be, for example, slow or fast. The rate of addition of the poor solvent may be, for example, slow or fast. The addition of the pH adjuster may be, for example, by adding an alkaline agent to the urine of the subject. Examples of the alkaline agent include sodium hydroxide and calcium hydroxide.

[0032] The determination method of the present disclosure may be, for example, a determination method in which the insolubilization step is supersaturation. Supersaturation, for example, refers to a state in which a solute is contained in a solution at a concentration equal to or greater than its solubility. In the case of the present disclosure, supersaturation, for example, refers to a state in which calcium phosphate is contained in the urine of the subject at a concentration equal to or greater than its solubility. The solubility is, for example, the limit amount of solute that can be dissolved in a certain amount of solvent. Supersaturation may, for example, refer to a state in which a solute is contained in a solution at a concentration equal to or greater than its supersolubility. Supersaturation, for example, refers to changing a solution to the supersaturated state. In the case of the present disclosure, supersaturation, for example, refers to changing the urine of the subject to the supersaturated state. In the case of the present disclosure, supersaturation can also be referred to as, for example, supersaturation of calcium phosphate in the urine of the subject. The supersaturation may be performed in parallel with an insolubilization treatment, for example, by adding the inorganic component, the antisolvent, the seed crystal, the pH adjuster, or the ionic strength adjuster, or the like, or the treatment may be performed before or after the supersaturation. The supersaturation can be performed, for example, by cooling the urine of the subject or evaporating the urine of the subject. Examples of the evaporation include evaporation by heating and evaporation by reducing pressure. Since the patient with urolithiasis contains, for example, calcium phosphate in the urine, it is presumed that the biomarker of the present disclosure is formed by the generation of crystalline nuclei of the biomarker of the present disclosure due to the supersaturation. Furthermore, since the patient with urolithiasis contains, for example, crystalline nuclei of the biomarker of the present disclosure in the urine, it is presumed that the biomarker of the present disclosure is formed by the growth of the crystalline nuclei due to the supersaturation. However, the mechanism of formation of the biomarker of the present disclosure by the supersaturation is not limited to, for example, each of the above presumptions. According to the supersaturation, for example, the biomarker of the present disclosure can be formed by supersaturating calcium phosphate in the urine of the subject. The above-mentioned explanation can be applied to the calcium phosphate, and it includes any product formed in the process of forming the biomarker of the present disclosure.

[0033] The supersaturation may be, for example, cooling. It is presumed that the supersaturation caused by cooling involves, for example, changing calcium phosphate in the urine of the subject to a supersaturated state, further generating crystalline nuclei of the biomarker of the present disclosure, and growing the crystalline nuclei to form the biomarker of the present disclosure. It is also presumed that the supersaturation caused by cooling involves, for example, growing crystalline nuclei of the biomarker of the present disclosure contained in the urine of the subject, thereby forming the biomarker of the present disclosure. However, the supersaturation caused by cooling is not limited to, for example, the above presumptions. Examples of devices used for cooling include refrigerators, refrigerators, refrigerator-freezers, low-temperature incubators, constant-temperature low-temperature chambers, and refrigerator-warmer chambers. The device used for cooling may be, for example, for home use or for pharmaceutical use. The cooling temperature may be, for example, 0 to 20°C, 2 to 18°C, 4 to 16°C, 6 to 14°C, or 8 to 12°C. The cooling temperature may be, for example, 0 to 10°C, 2 to 8°C, or 4 to 6°C. The cooling temperature is preferably, for example, 0 to 4°C. The cooling temperature may be, for example, constant or may differ between the initial, middle, and later stages of cooling. The cooling rate may be, for example, constant or may differ between the initial, middle, and later stages of cooling. The cooling time is not particularly limited, and examples include 24 hours or more, 36 hours or more, and 48 hours or more. During cooling, the urine may be left to stand or stirred, for example. The stirring can be performed using, for example, a stirrer. The stirring rate may be, for example, constant or may differ between the initial, middle, and later stages of cooling. The supersaturation by cooling may be carried out in parallel with an insolubilization treatment by, for example, adding the inorganic component, the poor solvent, the seed crystals, the pH adjuster, the ionic strength adjuster, or the like, or may be carried out before or after the cooling.

[0034] The determination method of the present disclosure may be a determination method in which, for example, if the biomarker of the present disclosure is detected in the urine of the subject in the detection step, the subject is determined to have urolithiasis in the determination step. The detection of the biomarker of the present disclosure in the detection step may be, for example, an analysis of the presence or absence of the biomarker of the present disclosure in the urine (qualitative analysis), or an analysis of the amount of the biomarker of the present disclosure (quantitative analysis).

[0035] The detection target in the detection step is the biomarker of the present disclosure, and specifically includes at least one of crystalline calcium phosphate and amorphous calcium phosphate.

[0036] In the detection step, the biomarkers of the present disclosure may be used alone or in combination with a biomarker for determining the onset of urolithiasis other than the biomarkers of the present disclosure (the other biomarkers). For example, the above-mentioned explanations can be used for the other biomarkers.

[0037] In the detection step, the method for detecting the biomarker of the present disclosure is not particularly limited, and known methods can be used. Specific examples of the method for detecting the biomarker of the present disclosure include microscopy, automated analysis, Raman spectroscopy, infrared spectroscopy, X-ray diffraction, and terahertz spectroscopy.

[0038] The microscopic examination can be performed using, for example, an optical microscope, a polarizing microscope, an electron microscope, or the like. It is known that, in the microscopic examination using the optical microscope, the biomarker of the present disclosure can be observed, for example, as chrysanthemum-shaped or plate-shaped, colorless to grayish-white crystals. Furthermore, it is known that the biomarker of the present disclosure can be differentiated from other formed components based on its solubility in acid, since it dissolves and disappears in, for example, acetic acid, hydrochloric acid, or the like. Therefore, the biomarker of the present disclosure can be differentiated from other formed components based on, for example, the shape, color, size, solubility in acid, or the like of the biomarker of the present disclosure. Furthermore, the biomarker of the present disclosure can be qualitatively analyzed for the presence or absence of the biomarker of the present disclosure, or quantitatively analyzed for the amount of the biomarker of the present disclosure, based on the results of the differentiation. In this case, the amount of the biomarker of the present disclosure may be, for example, the number, size, or the like of the biomarker of the present disclosure.

[0039] The automated analysis method can be performed, for example, by an image processing method, a cytometry method, or the like. The automated analysis method can also be referred to, for example, as a urinary formed element analysis method. The image processing method is, for example, a method that automates the microscopic examination method, and can qualitatively analyze the presence or absence of the biomarker of the present disclosure or quantitatively analyze the amount of the biomarker of the present disclosure by analyzing images captured by a CCD camera or the like. Furthermore, the cytometry method can qualitatively analyze the presence or absence of the biomarker of the present disclosure or quantitatively analyze the amount of the biomarker of the present disclosure by analyzing scattered light in response to laser light irradiation, for example.

[0040] In the case where at least one of crystalline calcium hydrogen phosphate and amorphous calcium hydrogen phosphate contained in the biomarker of the present disclosure is detected by the Raman spectroscopy, the biomarker of the present disclosure has a peak at, for example, 990 cm －１ Near and 875-880 cm －１ It is known that the biomarker of the present disclosure exhibits a characteristic peak at the Raman shift of 990 cm －１Near and 875-880 cm －１ The biomarker of the present disclosure can be distinguished from other contained components based on a characteristic peak in the Raman shift of the biomarker of the present disclosure. Furthermore, for example, based on the result of the distinction, the presence or absence of the biomarker of the present disclosure can be qualitatively analyzed, or the amount of the biomarker of the present disclosure can be quantitatively analyzed. In this case, the amount of the biomarker of the present disclosure can be determined, for example, by analyzing the amount of the biomarker of the present disclosure at 990 cm －１ Near and 875-880 cm －１ The concentration may be calculated by applying the Raman scattering intensity of a characteristic peak at a Raman shift of 0.01 to a calibration model created from a standard sample of known amount. Furthermore, Non-Patent Document 2 discloses that at least one of hydroxyapatite crystalline and amorphous hydroxyapatite, at least one of carbonate apatite crystalline and amorphous carbonate apatite, and at least one of tricalcium phosphate crystalline and amorphous tricalcium phosphate contained in the biomarkers of the present disclosure exhibits a characteristic peak at a specific Raman shift in the Raman spectroscopy. Therefore, each of the biomarkers of the present disclosure can be differentiated from other contained components, for example, based on the characteristic peak at the Raman shift described in Non-Patent Document 2. Furthermore, each of the biomarkers of the present disclosure can be qualitatively analyzed for the presence or absence of each of the biomarkers of the present disclosure, or quantitatively analyzed for the amount of each of the biomarkers of the present disclosure, based on the results of the differentiation. It should be noted that the description in Non-Patent Document 2 is merely an example, and the Raman shift of the biomarker of the present disclosure in the Raman spectroscopy can be, for example, a known one, and is not limited in any way to the description in Non-Patent Document 2.

[0041] Examples of the infrared spectroscopy include dispersive infrared spectroscopy and Fourier transform infrared spectroscopy (FTIR). Non-Patent Documents 2 and 3 disclose that at least one of calcium hydrogen phosphate crystals and amorphous calcium hydrogen phosphate, at least one of hydroxyapatite crystals and amorphous hydroxyapatite, at least one of carbonate apatite crystals and amorphous carbonate apatite, and at least one of tricalcium phosphate crystals and amorphous tricalcium phosphate, contained in the biomarkers of the present disclosure, exhibits a characteristic peak at a specific wavenumber in the FTIR method. Therefore, the biomarkers of the present disclosure can be differentiated from other components, for example, based on the characteristic peaks at the wavenumbers described in Non-Patent Documents 2 and 3. Furthermore, the biomarkers of the present disclosure can be used, for example, to qualitatively analyze the presence or absence of the biomarkers of the present disclosure or quantitatively analyze the amount of the biomarkers of the present disclosure based on the results of the differentiation. The descriptions in Non-Patent Documents 2 and 3 are merely examples, and the wavenumbers of the biomarkers of the present disclosure in the infrared spectroscopy may be, for example, publicly known ones, and are not limited in any way to the descriptions in Non-Patent Documents 2 and 3.

[0042] Examples of the X-ray diffraction method include single crystal X-ray diffraction and powder X-ray diffraction (PXRD). Non-Patent Document 2 discloses that at least one of calcium hydrogen phosphate crystals and amorphous calcium hydrogen phosphate, at least one of hydroxyapatite crystals and amorphous hydroxyapatite, at least one of carbonate apatite crystals and amorphous carbonate apatite, and at least one of tricalcium phosphate crystals and amorphous tricalcium phosphate, contained in the biomarker of the present disclosure, exhibits a characteristic peak at a specific diffraction angle 2θ in the PXRD method. Therefore, the biomarker of the present disclosure can be differentiated from other contained components, for example, based on the characteristic peak at the diffraction angle 2θ described in Non-Patent Document 2. Furthermore, the biomarker of the present disclosure can be used, for example, to qualitatively analyze the presence or absence of the biomarker of the present disclosure or quantitatively analyze the amount of the biomarker of the present disclosure based on the results of the differentiation. The description in Non-Patent Document 2 is merely an example, and the diffraction angle 2θ of the biomarker of the present disclosure in the X-ray diffraction method may be, for example, a known value, and is not limited in any way to the description in Non-Patent Document 2.

[0043] Examples of the terahertz spectroscopy include Fourier transform infrared spectroscopy (FTIR) and terahertz time-domain spectroscopy (THz-TDS). In the terahertz spectroscopy, the biomarker of the present disclosure exhibits, for example, a characteristic peak at a specific terahertz frequency. Therefore, the biomarker of the present disclosure can be differentiated from other contained components based on the characteristic peak at the specific terahertz frequency, for example, by utilizing the specific terahertz frequency that is known for the biomarker of the present disclosure. Furthermore, the biomarker of the present disclosure can be used, for example, to qualitatively analyze the presence or absence of the biomarker of the present disclosure or to quantitatively analyze the amount of the biomarker of the present disclosure based on the results of the differentiation.

[0044] The determination method of the present disclosure may, for example, detect the presence or absence of the biomarker of the present disclosure in the urine of the subject in the detection step, and determine that the subject has urolithiasis if the presence or absence of the biomarker of the present disclosure is determined to be present in the determination step. Alternatively, the determination of the presence or absence of the biomarker of the present disclosure in the determination step may be, for example, determining that the biomarker of the present disclosure is present if the biomarker of the present disclosure is detected in the detection step. Alternatively, the determination of the presence or absence of the biomarker of the present disclosure in the determination step may be, for example, determining that the biomarker of the present disclosure is absent if the biomarker of the present disclosure is not detected in the detection step.

[0045] The assessment method of the present disclosure may include a step of assessing the onset of urolithiasis in the subject by comparing the amount of the biomarker of the present disclosure in the subject's urine with a reference value. The reference value is not particularly limited, and examples thereof include the amount of the biomarker of the present disclosure in a healthy subject, a patient with urolithiasis, a patient with non-recurrent urolithiasis, or a patient with recurrent urolithiasis. In the case of prognosis assessment, the reference value may be, for example, the amount of the biomarker of the present disclosure in the same subject after treatment (e.g., immediately after treatment). The healthy subject is, for example, a subject who has never previously developed urolithiasis. The patient with urolithiasis is, for example, a first-time patient who has developed urolithiasis. The non-recurrent urolithiasis patient is, for example, a patient who has not recurred urolithiasis within five years or a patient who has not recurred urolithiasis within ten years after treatment for urolithiasis. It is known that nearly half of urolithiasis cases recur within five years, and more than half within ten years. Therefore, the non-recurrent urolithiasis patients can be referred to as, for example, patients who have been in remission from urolithiasis or patients who have been completely cured of urolithiasis. The recurrent urolithiasis patients are, for example, patients who have recurred from urolithiasis after treatment for urolithiasis. The onset and recurrence of urolithiasis can be diagnosed, for example, by imaging tests. The imaging tests can be, for example, as described above.

[0046] The reference value can be obtained, for example, using urine collected from a healthy subject, a patient with non-recurrent urolithiasis, a patient with urolithiasis, and / or a patient with recurrent urolithiasis (hereinafter also referred to as reference urine). Furthermore, in the case of prognosis evaluation, for example, reference urine collected from the same subject after treatment may be used in addition to or instead of the reference urine. The reference value may be measured, for example, simultaneously with the test urine of the subject, or may be measured in advance. The latter case is preferable because, for example, it is not necessary to obtain a reference value each time the test urine of the subject is measured. It is preferable that the test urine of the subject and the reference urine are collected under the same conditions, and the biomarkers of the present disclosure are detected under the same conditions.

[0047] In the determination step, the method for evaluating the onset of urolithiasis in a subject is not particularly limited and can be appropriately determined depending on the type of the reference value. Specifically, the subject can be evaluated as being at risk or at high risk for developing urolithiasis if the amount of the biomarker of the present disclosure in the test urine of the subject is significantly higher than the amount of the biomarker of the present disclosure in the reference urine of the healthy subject or the patient without recurrence of urolithiasis, if the amount of the biomarker of the present disclosure in the reference urine of the patient with urolithiasis or the patient with recurrence of urolithiasis is the same as (if there is no significant difference with) the amount of the biomarker of the present disclosure in the reference urine of the patient with urolithiasis or the patient with recurrence of urolithiasis, and / or if the amount of the biomarker of the present disclosure in the reference urine of the patient with urolithiasis or the patient with recurrence of urolithiasis is significantly higher than the amount of the biomarker of the present disclosure in the reference urine of the patient with urolithiasis or the patient with recurrence of urolithiasis. Furthermore, if the amount of the biomarker of the present disclosure in the test urine of the subject is the same as the amount of the biomarker of the present disclosure in the reference urine of the healthy subject or the patient with non-recurrent urolithiasis (if there is no significant difference), if it is significantly lower than the amount of the biomarker of the present disclosure in the reference urine of the healthy subject or the patient with non-recurrent urolithiasis, and / or if it is significantly lower than the amount of the biomarker of the present disclosure in the reference urine of the patient with urolithiasis or the patient with recurrent urolithiasis, the subject can be evaluated as having no or a low risk of developing urolithiasis.

[0048] When evaluating the prognosis in the determination step, for example, evaluation may be performed in the same manner as described above, or the amount of a biomarker of the present disclosure in a reference urine sample of the same subject after treatment may be used as a reference value for evaluation. Specifically, if the amount of a biomarker of the present disclosure in the subject's test urine is significantly higher than the reference value, the subject can be evaluated as being at risk of recurrence or worsening after the treatment. Furthermore, if the amount of a biomarker of the present disclosure in the subject's test urine is the same as the reference value (if there is no significant difference) and / or significantly lower than the reference value, the subject can be evaluated as being at no risk or at a low risk of recurrence after the treatment.

[0049] In the present disclosure, for example, urine may be collected from the same subject over time, and the amounts of the biomarkers of the present disclosure in the urine may be compared. This allows, for example, a determination that the likelihood of onset has increased if the amount increases over time, and a determination that the likelihood of onset has decreased or that the disease has been cured if the amount decreases over time.

[0050] The assessment method of the present disclosure may further include, for example, a step of administering a therapeutic drug for urolithiasis to a subject who has been assessed in the assessment step as being at risk or at high risk of developing urolithiasis or to a subject who has been assessed as being at risk of recurrence or worsening after treatment (administration step). In this case, the assessment method of the present disclosure can also be referred to as a method for assessing and treating urolithiasis.

[0051] The therapeutic agent for urolithiasis is not particularly limited, and examples thereof include the inhibitor of the present disclosure (a pharmaceutical containing a formation inhibitor described below), Quercus salicina extract, or a combination thereof. The administration conditions of the therapeutic agent for urolithiasis (administration target, dosage, administration form, administration method, etc.) are not particularly limited, and can be determined appropriately depending on the type of the therapeutic agent.

[0052] The determination method of the present disclosure can accurately determine, for example, the onset of urolithiasis. Furthermore, the determination method of the present disclosure can accurately determine, for example, the recurrence of urolithiasis. Therefore, the determination method of the present disclosure can determine, for example, the possibility of onset of urolithiasis, whether or not urolithiasis has occurred, evaluation of the prognosis of the onset of urolithiasis, the possibility of recurrence of urolithiasis, whether or not urolithiasis has recurred, evaluation of the prognosis of the recurrence of urolithiasis, etc.

[0053] <Urolithiasis Inhibitor and Urolithiasis Inhibitor> The urolithiasis inhibitor of the present disclosure (hereinafter also referred to as the inhibitor of the present disclosure) includes a formation inhibitor that inhibits the formation of at least one of calcium phosphate crystals and amorphous calcium phosphate. Furthermore, the urolithiasis inhibition method of the present disclosure (hereinafter also referred to as the inhibition method of the present disclosure) includes an administration step of administering the inhibitor of the present disclosure to a subject. The inhibitor and inhibition method of the present disclosure are characterized by inhibiting the formation of at least one of calcium phosphate crystals and amorphous calcium phosphate, and other configurations and conditions are not particularly limited. The inhibitor and inhibition method of the present disclosure can inhibit the onset of urolithiasis. For the inhibitor and inhibition method of the present disclosure, for example, the descriptions of the biomarkers and the assessment methods of the present disclosure can be used.

[0054] Examples of the formation inhibitor include substances that adjust urine pH and substances that chelate inorganic components. Examples of the substance that chelates inorganic components include sodium citrate. Examples of the formation inhibitor include diuretics and uric acid production inhibitors. Examples of the diuretics include thiazide diuretics. Examples of the uric acid production inhibitors include allopurinol and febuxostat. The formation inhibitor can be used as an ingredient in, for example, foods, beverages, food additives, drink additives, supplements, pharmaceuticals, etc. Furthermore, the formation inhibitor can be used as, for example, foods, beverages, food additives, drink additives, supplements, pharmaceuticals, etc.

[0055] Examples of the inhibitory substance include foods containing the formation inhibitor, beverages containing the formation inhibitor, food additives containing the formation inhibitor, beverage additives containing the formation inhibitor, supplements containing the formation inhibitor, and pharmaceuticals containing the formation inhibitor.

[0056] When the inhibitor of the present disclosure is a food containing the formation inhibitor, the food is not particularly limited and includes, for example, processed grain foods, processed vegetable foods, processed fruit foods, processed meat foods, processed seafood foods, dairy products, confectionery, etc. Examples of the confectionery include candy, taffy, gummy candy, lemonade, snacks, baked goods, steamed goods, etc.

[0057] When the inhibitor of the present disclosure is a pharmaceutical containing the formation inhibitor, the administration form of the pharmaceutical is not particularly limited. Furthermore, the dosage form of the pharmaceutical is not particularly limited and can be determined appropriately depending on the administration form, for example. Furthermore, the amount of the formation inhibitor in the pharmaceutical is not particularly limited. Furthermore, the administration conditions of the formation inhibitor in the pharmaceutical are not particularly limited.

[0058] <Screening Method for Candidate Substances for Inhibiting Urolithiasis> The screening method for candidate substances for inhibiting urolithiasis of the present disclosure (hereinafter also referred to as the screening method of the present disclosure) is not particularly limited, and is, for example, a screening method including a selection step of selecting, from test substances, a formation inhibitor that inhibits the formation of at least one of calcium phosphate crystals and amorphous form as a candidate substance for inhibiting urolithiasis. The screening method of the present disclosure is characterized in that, in the selection step, the candidate inhibitor is selected based on the biomarker of the present disclosure, and other steps and conditions are not particularly limited. For example, the screening method of the present disclosure can refer to the descriptions of the biomarker of the present disclosure, the determination method of the present disclosure, and the inhibitor and inhibition method of the present disclosure.

[0059] Examples of the formation inhibitor include a substance that adjusts urine pH, a substance that chelates inorganic components, etc. Examples of the substance that chelates inorganic components include sodium citrate, etc. The formation inhibitor may be, for example, a formation inhibitor for food, a formation inhibitor for beverages, a formation inhibitor for food additives, a formation inhibitor for beverages, a formation inhibitor for supplements, a formation inhibitor for pharmaceuticals, etc.

[0060] The screening method of the present disclosure may be a screening method that includes, for example, an insolubilization step in which the test substance is subjected to an insolubilization treatment in the presence of the test substance to form at least one of calcium phosphate crystals and amorphous calcium phosphate, and a detection step in which the calcium phosphate crystals and amorphous calcium phosphate are detected, and in the selection step, the candidate inhibitor is selected from the test substance based on the detection result of at least one of the calcium phosphate crystals and amorphous calcium phosphate. The insolubilization treatment is not particularly limited, and the above-mentioned explanation can be used, for example. Furthermore, the biomarker detection method of the present disclosure is not particularly limited, and the above-mentioned explanation can be used, for example.

[0061] The screening method of the present disclosure may be, for example, a screening method in which the presence or absence of at least one of calcium phosphate crystals and amorphous calcium phosphate is detected in the detection step, and the test substance determined to be absent from at least one of calcium phosphate crystals and amorphous calcium phosphate in the selection step is selected as the candidate inhibitor. The insolubilization treatment is not particularly limited, and the above-mentioned explanation can be used, for example. Furthermore, the biomarker detection method of the present disclosure is not particularly limited, and the above-mentioned explanation can be used, for example.

[0062] The screening method of the present disclosure may be, for example, a screening method in which the detection step detects the amount of at least one of calcium phosphate crystals and amorphous calcium phosphate, and the selection step selects, as the candidate inhibitor, a test substance in which the amount of at least one of calcium phosphate crystals and amorphous calcium phosphate is lower than that of a control in which the test substance is not present. The insolubilization treatment is not particularly limited, and the above-mentioned explanation can be used, for example. Furthermore, the biomarker detection method of the present disclosure is not particularly limited, and the above-mentioned explanation can be used, for example.

[0063] In the screening method of the present disclosure, for example, the insolubilization step may be performed by supersaturation. The above-mentioned explanation can be applied to the insolubilization treatment by supersaturation, for example.

[0064] In the screening method of the present disclosure, for example, the supersaturation may be achieved by cooling in the insolubilization step. The above-mentioned explanation can be applied to the supersaturation by cooling, for example.

[0065] The test substance is not particularly limited, and may be, for example, at least one selected from the group consisting of low molecular weight compounds, peptides, and proteins.

[0066] <Use of Formation Inhibitor> The present disclosure relates to a formation inhibitor that inhibits the formation of at least one of calcium phosphate crystals and amorphous calcium phosphate, or use thereof, for use in the treatment of urolithiasis. The present disclosure relates to a formation inhibitor that inhibits the formation of at least one of calcium phosphate crystals and amorphous calcium phosphate, or use thereof, for use in the manufacture of a therapeutic agent for urolithiasis.

[0067] <Use of at least one of calcium phosphate crystals and amorphous calcium phosphate in urine> The present disclosure relates to the use of at least one of calcium phosphate crystals and amorphous calcium phosphate in urine for determining the onset of urolithiasis. The present disclosure relates to the use of at least one of calcium phosphate crystals and amorphous calcium phosphate in urine for determining the recurrence of urolithiasis. The present disclosure relates to the use of at least one of calcium phosphate crystals and amorphous calcium phosphate in urine for determining the early recurrence of urolithiasis.

[0068] Next, examples of the present disclosure will be described. However, the present disclosure is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise specified.

[0069] Example 1 It was confirmed that at least one of calcium phosphate crystals and amorphous calcium phosphate can be formed by cooling a urine sample.

[0070] (1) Effect of Temperature Conditions on Insolubilized Matter Formation Urine samples were collected from patients with urolithiasis who had calcium oxalate stones. The collected urine samples were cooled at 20°C, 10°C, and 4°C for at least 24 hours in a stationary state. The cooled urine samples were placed on glass slides, and a cover glass was placed directly on top of the urine samples to prepare specimens. The specimens were examined under an optical microscope at a magnification of 200x without staining. As shown in Figure 1, the lower the cooling temperature, the larger and more numerous the calcium oxalate crystals were. Therefore, the cooling temperature was set to 4°C for the following examples.

[0071] (2) Basic Data of Urolithiasis Patients Forty-seven patients with urolithiasis, as shown in Figure 2, were recruited as subjects in Example 1. Among these subjects, 14 patients with recurrent urolithiasis were those in whom the absence of residual urinary stones was confirmed by imaging tests (ultrasound, plain X-ray, or plain CT) after spontaneous stone passage, stone passage with treatment, or stone fragmentation with treatment, and in whom urolithiasis recurred. Furthermore, these subjects were urolithiasis patients who had previously excluded patients whose urinary stones were determined by stone analysis to be cystine stones, uric acid stones, or magnesium ammonium phosphate stones. That is, the subjects were urolithiasis patients whose urinary stones were determined by stone analysis to be calcium oxalate stones, calcium phosphate stones, or mixed calcium oxalate and calcium phosphate stones.

[0072] (3) Observation by Optical Microscope: Urine samples collected from the subjects during a 24-hour urine collection test were centrifuged at 2500 rpm for 10 minutes, and the supernatant was recovered. The supernatant was filtered through a 0.45 μm pore size filter to remove impurities and obtain a filtrate. The filtrate was cooled at 4°C for 24 hours or more. The cooled filtrate was placed on a glass slide, and a cover glass was placed directly on top of the filtrate to prepare a specimen. The specimen was examined under an optical microscope under unstained, high-magnification (400x) conditions to confirm the presence or absence of insolubilized matter in the cooled filtrate. The type of insolubilized matter in the cooled filtrate was morphologically classified based on its shape, color, size, etc.

[0073] As shown in Figure 3, it was confirmed that various insolubilized substances appeared in the filtrate by cooling the filtrate at 4°C for 24 hours or more. As a result of morphological classification of the insolubilized substances, insolubilized substance A was classified as calcium oxalate crystals because it was a regular octahedral, colorless crystal. Insolubilized substances B and C were classified as calcium phosphate crystals because they were chrysanthemum-shaped or plate-shaped, colorless to off-white crystals. Insolubilized substance D was classified as uric acid crystals because it was a diamond-shaped, yellowish-brown crystal.

[0074] (4) Raman Spectroscopic Analysis The cooled filtrate was placed on a slide glass with a silicone sheet, and a quartz cover glass was placed directly on top of the filtrate to prepare a specimen. The specimen was microscopically examined using a Raman spectrometer, and the insolubilized matter in the cooled filtrate was irradiated with a laser. The type of insolubilized matter in the cooled filtrate was classified based on the peak of the Raman shift obtained by spectroscopically analyzing the Raman scattered light. The laser irradiation was performed under the following conditions: excitation wavelength 532 nm; exposure time 10 seconds; number of accumulations 6; and laser output 5 to 10 mW.

[0075] As shown in FIG. 4, the Raman scattering light of the insolubilized material was analyzed by Raman spectroscopy. As a result, the insolubilized material A exhibited a peak intensity of 910 cm －１ Nearby and nearby 1479 cm －１The insolubilized materials B and C contained calcium oxalate dihydrate, as a Raman shift peak was confirmed around 990 cm. －１ Nearby and 878 cm －１ Since a peak was confirmed in the Raman shift around 623 cm, the insolubilized material of D contained calcium hydrogen phosphate (calcium hydrogen phosphate dihydrate (Brushite)). －１ Near 1004 cm －１ Near 1431 cm －１ and around 1500 cm －１ A peak was confirmed in the Raman shift around this range, indicating that the solution contained uric acid.

[0076] From the results of Example 1 (3) and (4), the 47 patients with urinary tract stones were classified into 26 patients (55%) in whom calcium oxalate crystals were found, 6 patients (13%) in whom calcium phosphate crystals were found, 2 patients (4%) in whom uric acid crystals were found, and 13 patients (28%) in whom no crystals were found.

[0077] From the above results, it was found that the insolubilization treatment of cooling a urine sample can form at least one of calcium phosphate crystals and amorphous calcium phosphate. It was also found that the insolubilization treatment of cooling a urine sample can form at least one of calcium oxalate crystals and amorphous calcium oxalate. It was also found that the insolubilization treatment of cooling a urine sample can form at least one of uric acid crystals and amorphous uric acid. Therefore, it was inferred that the insolubilization treatment of cooling a urine sample can form at least one of inorganic component crystals and amorphous inorganic component.

[0078] [Example 2] The relationship between the presence or absence of insolubilized matter and the recurrence of urolithiasis was investigated, and it was confirmed that when at least one of calcium phosphate crystals and amorphous calcium phosphate was detected, there was a high possibility of recurrence of urolithiasis.

[0079] Forty-five urolithiasis patients were recruited as subjects in Example 2, excluding two subjects in whom insolubilized uric acid was found from the subjects in Example 1. The subjects in Example 2 were divided into three groups: 13 subjects in whom insolubilized uric acid was found, 26 subjects in whom insolubilized calcium oxalate was found, and 6 subjects in whom insolubilized calcium phosphate was found. The relationship between the presence or absence of insolubilized urinary stones and the recurrence of urolithiasis was examined for these three groups.

[0080] As shown in Figure 5, there was no statistically significant difference in the number of patients with recurrent urinary tract stones (recurrence) between the group in which calcium oxalate insolubilized matter was not detected (no crystals) and the group in which calcium oxalate insolubilized matter was detected (calcium oxalate crystals). Furthermore, the BMI value was statistically significantly higher in the group in which calcium oxalate insolubilized matter was detected than in the group in which calcium oxalate insolubilized matter was not detected. In contrast, there was no statistically significant difference in the other evaluation items between the group in which calcium oxalate insolubilized matter was not detected and the group in which calcium oxalate insolubilized matter was detected.

[0081] As shown in Figure 6, the number of patients with recurrent urinary tract stones (recurrence) was statistically significantly higher in the group with calcium phosphate insolubilized particles (calcium phosphate crystals) than in the group without insolubilized particles (no crystals). Furthermore, the BMI value was statistically significantly higher in the group with calcium phosphate insolubilized particles than in the group without insolubilized particles. In contrast, there were no statistically significant differences in other evaluation items between the group without insolubilized particles and the group with calcium phosphate insolubilized particles.

[0082] As shown in Figure 7, the number of patients with recurrent urinary tract stones (with recurrence) was statistically significantly higher in the group with calcium phosphate insolubilized substances (calcium phosphate crystals) than in the group with calcium oxalate insolubilized substances (calcium oxalate crystals).In contrast, there were no statistically significant differences in other evaluation items between the group with calcium oxalate insolubilized substances and the group with calcium phosphate insolubilized substances.

[0083] These results indicate that the detection of calcium phosphate crystals and / or amorphous calcium phosphate indicates a high possibility of urinary stone recurrence. In contrast, the detection of calcium oxalate crystals and / or amorphous calcium oxalate indicates that the presence or absence of urinary stone recurrence cannot be determined.

[0084] Example 3 The structure and composition of calcium oxalate stones collected from patients with urolithiasis were analyzed, and it was confirmed that either crystalline or amorphous calcium phosphate serves as the initiation site for stone growth.

[0085] A calcium oxalate stone was collected from the kidney of a 60-year-old male patient with urolithiasis. The calcium oxalate stone was primarily composed of calcium oxalate dihydrate (COD) crystals. The calcium oxalate stone was immersed in a calcium oxalate solution to induce a solution-mediated phase transition. The solution-mediated phase transition transformed the inner and outermost layers of the calcium oxalate stone into calcium oxalate monohydrate (COM) crystals. The solution-mediated phase transition-induced calcium oxalate stone was then pretreated, and thin sections with thicknesses of 20 to 30 μm were prepared from the pretreated calcium oxalate stone using a resin embedding method.

[0086] Fig. 8 shows an image of the thin section observed under an open Nicol condition using a polarizing microscope (OPTIPHOT2-POL, manufactured by Nikon Corporation). In element mapping on the thin section by XRF analysis (described later), co-localization of phosphorus and zinc elements was confirmed in Area A and Area B in Fig. 8, and an investigation was carried out.

[0087] <Area A> (1) XRF Analysis Device The thin sections were analyzed by X-ray fluorescence (XRF). Specifically, the locations of four elements (Ca, P, Zn, and Si) on the thin sections were identified using an XRF analysis device (XGT9000, manufactured by HORIBA). In all measurements, the X-ray tube voltage was set to 30 kV and the X-ray tube current was set to 1,000 μA.

[0088] As shown in Figure 9, the four elements on the thin section of Area A were analyzed using an XRF analyzer, and it was confirmed that calcium element was distributed throughout the stone. In contrast, phosphorus element was confirmed to be concentrated and localized in a specific region, as shown by the dashed circle. Furthermore, in the region where phosphorus element was localized, zinc element was also confirmed to be concentrated and localized, as shown by the dashed circle. Therefore, it was found that phosphorus element and zinc element were co-localized in a specific region in Area A.

[0089] (2) Observation by Polarizing Microscope Under Crossed Nicols Conditions When Area A in Figure 8 was observed under crossed Nicols conditions using a polarizing microscope, a layered structure (indicated by circles 2 and 3) and a dark, light-opaque region (indicated by circles 4 and 5, and the dashed boundary line) were observed, as shown in Figure 10. It is known that when observed under crossed Nicols conditions using a polarizing microscope, aggregates of microcrystals scatter light at their boundaries, resulting in the appearance of a dark region. Therefore, the dark region was considered to be a structure in which some kind of microcrystals were aggregated. Furthermore, when the elemental distribution in the dark region was confirmed using the elemental mapping results using the XRF analyzer described in (1), it was found that the dark region coincided with a region in which phosphorus and zinc elements were concentrated and co-localized.

[0090] (3) Laser Raman Microscope In order to identify the type of each layer on the thin section in more detail, the thin section was observed using a laser Raman microscope (RAMANTouch, manufactured by Nanophoton). Specifically, the COD phase, COM phase, and calcium phosphate phase in the thin section were identified by detecting specific peaks associated with chemical bonds specific to each layer using the laser Raman microscope. The measurement conditions for the laser Raman microscope were as follows: laser wavelength 787 nm; laser output 1.50±0.25 mW; diffraction grating groove count 600 gr / mm; measurement wavenumber range 600 to 1600 cm －１ .

[0091] The points indicated by circles 1 to 5 on the thin section shown in Figure 10 were analyzed by a laser Raman microscope. As a result, as shown in Figure 11, peaks characteristic of the COM phase were detected at the points indicated by circles 1 to 3 (regions of the layered structure in Area A). In contrast, peaks characteristic of calcium phosphate phases, particularly hydroxyapatite, were detected at the dark areas indicated by circles 4 and 5. This revealed that the dark areas contained aggregates of calcium phosphate microcrystals.

[0092] (4) Immunofluorescence Staining To visualize the distribution of proteins in the thin sections, immunofluorescence staining was performed on the thin sections, and the stained thin sections were observed using a fluorescence microscope (A1R, Nikon Corporation). The immunostaining targeted a protein called osteopontin (OPN). OPN is known as a calcium-binding protein and has been reported to suppress or promote urinary stone formation.

[0093] As shown in Figure 12, immunofluorescent staining of the thin sections revealed that the striped structure of OPN was more clearly visualized in the outer layer than in the inner layer. Furthermore, the structure of the aggregates of calcium phosphate microcrystals contained in the dark regions could not be confirmed on the micrometer scale, suggesting that their size was on the nanometer order.

[0094] From these results, it was found that the dark areas on the thin sections contained aggregates of calcium phosphate microcrystals co-localized with zinc element. It is known that in urolithiasis, crystal nuclei called Randall's plaque are formed in the interstitium of the renal papilla, and the Randall's plaque serves as a scaffold for stone formation, allowing the crystals to grow. Previous studies have reported that Randall's plaque is an aggregate of calcium phosphate microcrystals on the nanometer order, and that zinc is co-localized. Therefore, it was speculated that the dark areas were Randall's plaque.

[0095] In addition, in Figure 10, the COM phase, which is a layered structure region in Area A, extends outward from the dark region. Previous research has shown that Randall's plaque serves as a scaffold for stone formation. Therefore, it was speculated that the results confirmed that Randall's plaque serves as a starting point for the nucleation of COM crystals.

[0096] <Area B> (1) XRF Analysis Area B on the thin section was analyzed by XRF in the same manner as Area A. As shown in FIG. 13 , the four elements on the thin section of Area B were analyzed using an XRF analyzer. As a result, it was confirmed that calcium element was distributed throughout the stone. In contrast, phosphorus element was found to be concentrated and localized in a specific region, as indicated by the circle drawn with a dashed line. Furthermore, in the region where phosphorus element was localized, zinc element was also found to be concentrated and localized, as indicated by the circle drawn with a dashed line. Therefore, it was found that phosphorus element and zinc element were co-localized in a specific region in Area B.

[0097] (2) Observation by Polarizing Microscope Under Crossed Nicols Conditions When Area B in Fig. 8 was observed by a polarizing microscope under crossed Nicols conditions, a layered structure region (indicated by circles 1 and 2) and a slightly light-transmitting dark region (indicated by a circle drawn with a dashed line) were observed, as shown in Fig. 14. Furthermore, when the elemental distribution in the slightly light-transmitting dark region was confirmed by the elemental mapping results in the XRF analyzer described in (1), it was found that the slightly light-transmitting dark region coincided with a region where phosphorus and zinc elements were concentrated and co-localized.

[0098] (3) Laser Raman Microscopy Area B on the thin section was observed using a laser Raman microscope in the same manner as Area A. Points circled 1 and 2 on the thin section shown in Figure 14 (the layered structure region) and the slightly light-transmitting dark region were analyzed using a laser Raman microscope. As a result, as shown in Figure 15, peaks characteristic of the COM phase were detected at points circled 1 and 2 (the layered structure region). However, a Raman spectrum could not be obtained from the slightly light-transmitting dark region due to the influence of background fluorescence. Therefore, it was speculated that the slightly light-transmitting dark region contained some kind of autofluorescent protein.

[0099] (4) Immunofluorescent Staining: Immunofluorescent staining was performed on Area B on the thin section using the same method as for Area A to confirm whether the slightly light-transmitting dark region contained the protein OPN. As shown in Figure 16(a), immunofluorescent staining of the thin section revealed that OPN-containing platelet crystal aggregates were observed in the slightly light-transmitting dark region. Furthermore, the region where the OPN-containing platelet crystal aggregates were observed coincided with the region where phosphorus and zinc elements were colocalized in the XRF analysis. Furthermore, as shown in Figure 16(a), the OPN-containing platelet crystal aggregates were very similar in morphology to the calcium phosphate crystals shown in Figure 16(b) (observed in a urine sample using an optical microscope), and their size was on the order of micrometers. Therefore, although the slightly light-transmitting dark region was a calcium phosphate phase colocalized with zinc, its size indicated that it was not Randall's plaque.

[0100] These results revealed that the slightly light-transmitting dark regions on the thin sections contained micrometer-sized aggregates of calcium phosphate crystals, not Randall's plaque. Furthermore, in Figure 13, the COM phase, which is the layered structure region, extends outward from the slightly light-transmitting dark regions. Therefore, it was speculated that either calcium phosphate crystals or amorphous calcium phosphate, even if not Randall's plaque, served as the starting point for nucleation of COM crystals.

[0101] Furthermore, the micrometer-order calcium phosphate crystal aggregates contained the protein OPN, suggesting that the formation of either calcium phosphate crystals or amorphous calcium phosphate is influenced by organic components such as proteins contained in urine.

[0102] [Example 4] The relationship between the presence or absence of recurrence of urolithiasis and insoluble matter was investigated, and it was confirmed that in patients with recurrence of urolithiasis, there is a high possibility that at least one of calcium phosphate crystals and amorphous calcium phosphate is detected.

[0103] (1) Study of the Relationship Between the Presence or Absence of Urolithiasis Recurrence and Insolubilized Matter Forty-seven urolithiasis patients (the same urolithiasis patients as in Example 1) shown in FIG. 17 were used as subjects in Example 4. The subjects in Example 4 were divided into two groups: 33 non-recurrent urolithiasis patients (no recurrence) and 14 recurrent urolithiasis patients (recurrence). The recurrent urolithiasis patients had urolithiasis located in both kidneys or both ureters statistically significantly more frequently than the non-recurrent urolithiasis patients. The relationship between the presence or absence of urolithiasis recurrence and insolubilized matter was studied for these two groups.

[0104] As shown in "No Crystal Formation vs. CaP Crystals" in Figure 18, the number of patients with recurrent urolithiasis was statistically significantly higher in subjects with calcium phosphate insolubilization (CaP crystals) than in subjects without insolubilization (no crystal formation). Also, as shown in "CaOx Crystals vs. CaP Crystals" in Figure 18, the number of patients with recurrent urolithiasis was statistically significantly higher in subjects with calcium phosphate insolubilization (CaP crystals) than in subjects with calcium oxalate insolubilization (CaOx crystals). In contrast, as shown in "Crystal Formation" in Figure 18, there was no statistically significant difference between the group of non-recurrent urolithiasis patients and the group of recurrent urolithiasis patients in the number of subjects with calcium oxalate insolubilization, calcium phosphate insolubilization, or uric acid insolubilization (crystal formation). Furthermore, as shown in Figure 18 "No crystal formation vs. CaOx crystals," there was no statistically significant difference in the number of patients with recurrent urinary tract stones between subjects in whom no insolubilized matter was observed (no crystal formation) and subjects in whom insolubilized matter of calcium oxalate was observed (CaOx crystals).

[0105] Furthermore, as shown in Figure 18, the values ​​of the evaluation items related to renal function (Ca, inorganic phosphorus, Mg, Cre, urea nitrogen) were statistically significantly higher in the group of patients with recurrent urolithiasis than in the group of patients with non-recurrent urolithiasis.

[0106] The above results indicate that in patients with recurrent urolithiasis, at least one of calcium phosphate crystals and amorphous calcium phosphate is likely to be detected, whereas in patients with recurrent urolithiasis, at least one of calcium oxalate crystals and amorphous calcium oxalate is not likely to be detected.

[0107] (2) Evaluation of Insolubilized Calcium Phosphate as a Biomarker for Determining the Recurrence of Urolithiasis As in Example 4(1), 47 subjects from Example 1 were recruited as subjects in Example 4(2). The subjects in Example 4(2) consisted of 33 non-recurrent urolithiasis patients and 14 recurrent urolithiasis patients. The data of the subjects from Example 4(2) was analyzed using logistic regression analysis to identify risk markers for urolithiasis recurrence. In the logistic regression analysis, the presence or absence of urolithiasis recurrence was used as the dependent variable, and the presence or absence of urinary stones in both kidneys or both ureters, the results of a 24-hour urine collection test (Ca, Mg, Cre, urea nitrogen), and the presence or absence of insolubilized matter (presence or absence of insolubilized calcium phosphate) were used as explanatory variables.

[0108] As shown in "No crystal formation vs. CaP crystals" in Figure 19, the presence or absence of calcium phosphate insolubilized products was a factor that statistically significantly affected the recurrence of urolithiasis when comparing subjects who did not have insolubilized products (no crystal formation) with subjects who had calcium phosphate insolubilized products (CaP crystals). Also, as shown in "CaOx crystals vs. CaP crystals" in Figure 19, the presence or absence of calcium phosphate insolubilized products was a factor that statistically significantly affected the recurrence of urolithiasis when comparing subjects who had calcium oxalate insolubilized products (CaOx crystals) with subjects who had calcium phosphate insolubilized products (CaP crystals). In contrast, the values ​​or presence or absence of other evaluation items did not have a statistically significant effect on the recurrence of urolithiasis.

[0109] From the above results, it was found that at least one of calcium phosphate crystals and amorphous calcium phosphate can be used as a biomarker for determining the recurrence of urolithiasis. It was also found that at least one of calcium phosphate crystals and amorphous calcium phosphate can be used as a method for determining the recurrence of urolithiasis in a subject based on the detection results of at least one of calcium phosphate crystals and amorphous calcium phosphate. It is known that patients with recurrent urolithiasis are likely to develop the same type of urolithiasis as at the initial onset. Therefore, based on the results of the above examples, it was inferred that at least one of calcium phosphate crystals and amorphous calcium phosphate can be used as a biomarker for determining the onset of urolithiasis, or that at least one of calcium phosphate crystals and amorphous calcium phosphate can be used as a method for determining the onset of urolithiasis in a subject based on the detection results of at least one of calcium phosphate crystals and amorphous calcium phosphate.

[0110] Example 5: The relationship between the timing of urolithiasis recurrence and insoluble matter was investigated, and it was confirmed that in patients with early recurrence of urolithiasis, at least one of calcium phosphate crystals and amorphous calcium phosphate is likely to be detected. In particular, it was confirmed that in patients with early recurrence of urolithiasis, calcium hydrogen phosphate dihydrate (Brushite) crystals are likely to be detected.

[0111] (1) Basic Data As shown in Figure 20, 135 patients with urolithiasis and 15 healthy subjects were recruited as subjects in Example 5. Of the 135 patients with urolithiasis, 30 (22.2%) were early recurrent patients who experienced a recurrence of urolithiasis within 5 years, 27 (20.0%) were late recurrent patients who experienced a recurrence of urolithiasis beyond 5 years, and 78 (57.8%) were non-recurrent patients who did not experience a recurrence of urolithiasis. As shown in Figure 20, the subjects in Example 5 were classified into four groups: the early recurrent patient group (early recurrent patients), the late recurrent patient group (late recurrent patients), the non-recurrent patient group (non-recurrent patients), and the healthy subject group (healthy subjects). Patients with recurrent urolithiasis (i.e., the early recurrent patients and the late recurrent patients) were patients in whom new urinary stone formation was confirmed by plain CT scan or plain X-ray examination after spontaneous stone passage or stone passage or stone fragmentation due to treatment. The recurrent urolithiasis patients were patients who were confirmed to have recurrent urolithiasis due to a residual urolith. Furthermore, the recurrent urolithiasis patients were patients who were confirmed to have cystine stones, uric acid stones, or magnesium ammonium phosphate stones by stone analysis. The recurrent urolithiasis patients were patients whose urolithiasis stones were confirmed to be calcium oxalate stones or calcium phosphate stones by stone analysis. The recurrent urolithiasis patients were patients who were confirmed to have renal tubular acidosis or hyperparathyroidism (i.e., patients who are prone to specific urolithiasis conditions).

[0112] The results of comparing the basic data for the four groups are shown in Figure 20. The basic data included age, BMI, sex, presence or absence of diabetes, presence or absence of hyperlipidemia, presence or absence of hypertension, presence or absence of oral citric acid use, presence or absence of a history of surgery related to urolithiasis, and presence or absence of staghorn stones. As shown in Figure 20, there was no statistically significant difference in the basic data among the four groups.

[0113] (2) 24-Hour Urine Collection Test A 24-hour urine collection test was conducted on the four groups. Specifically, a 24-hour urine collection was collected from the subjects using a 24-hour urine proportional collector, Urinmate (SB Kawasumi Co., Ltd.), and various components, such as electrolytes, in the 24-hour urine were then analyzed using an analytical device. The supersaturation indices (SIs) of calcium oxalate (CaOx) and calcium phosphate (CaP) were calculated as SI. CaOx and SI. CaP using geochemical calculation software PHREEQC. In this example, the CaOx and CaP with the lowest solubility at human body temperature were used to calculate the supersaturation indices. Specifically, the SI. CaOx was calculated using the supersaturation index of calcium oxalate monohydrate (COM), and the SI. For CaP, the supersaturation index of hydroxyapatite (HAP) was used.

[0114] The results of the 24-hour urine collection test are shown in Figures 21 and 22. In Figure 21, the results of the 24-hour urine collection test include sodium (Na), potassium (K), chloride (Cl), magnesium (Mg), inorganic phosphorus, calcium (Ca), creatinine (Cr), uric acid, urea nitrogen, oxalic acid, urine volume, pH, SI. CaOx, and SI. CaP. In Figure 22A, the Ca is shown as 24-hour urine Ca. In Figure 22B, the oxalic acid is shown as 24-hour urine oxalic acid. In Figure 22C, the SI. CaOx is shown as 24-hour urine SI. CaOx. In Figure 22D, the SI. CaP is shown as 24-hour urine SI. CaP. As shown in Figures 21 and 22, the SI. CaOx values ​​in each group of urolithiasis patients were significantly higher than those in the control group. The SI.CaOx was statistically significantly higher than that of the healthy control group. In contrast, as shown in Figures 21 and 22, there was no statistically significant difference in SI.CaOx among the three groups of urolithiasis patients (i.e., the early recurrence group, the late recurrence group, and the non-recurrence group). Also, as shown in Figures 21 and 22, there were no statistically significant differences in other results of the 24-hour urine collection test among the four groups.

[0115] These results demonstrate that SI.CaOx, a 24-hour urine collection test, can differentiate between healthy subjects and urolithiasis patients. However, the results of the 24-hour urine collection test do not necessarily differentiate between the recurrence of urolithiasis and the timing of recurrence of urolithiasis among urolithiasis patients.

[0116] (3) Insolubilized Matter in the Chilled Urine Sample The urine sample collected from the subject in the 24-hour urine collection test was treated in the same manner as in Example 1(3), except that it was centrifuged at 3,000 rpm for 10 minutes, to obtain a filtrate after cooling (i.e., a chilled urine sample). The types of insolubilized matter in the filtrate after cooling were classified by observation using an optical microscope and analysis by Raman spectroscopy.

[0117] As shown in Fig. 23, six types of insolubilized products A-F, each exhibiting various shapes, were identified in the cooled filtrate by observation using an optical microscope. Furthermore, as shown in Fig. 30, Raman spectroscopy analysis of the insolubilized products identified using an optical microscope confirmed that, of the insolubilized products, insolubilized product A, exhibiting a regular octahedral shape, is a calcium oxalate dihydrate (COD) crystal, insolubilized products B and C, exhibiting a chrysanthemum or plate-like shape, are calcium hydrogen phosphate dihydrate (Brushite) crystal, insolubilized product D, exhibiting a bulky shape, is a magnesium ammonium phosphate crystal, insolubilized product E, exhibiting a diamond shape, is a uric acid crystal, and insolubilized product F is a carbonate apatite crystal.

[0118] (4) Study of the Relationship Between the Time of Recurrence of Urolithiasis and Insoluble Matter The relationship between the time of recurrence of urolithiasis and insoluble matter in cooled urine samples was studied for the four groups. Subjects who detected magnesium ammonium phosphate crystals or uric acid crystals were excluded from this study. As shown in Figures 24 and 25 , the number of subjects who detected insoluble matter in Brushite was statistically significantly higher in the early recurrence group than in the other groups. Specifically, the percentages of subjects who detected insoluble matter in Brushite were 57.6% in early recurrence patients, 11.5% in late recurrence patients, 13.3% in non-recurrence patients, and 6.7% in healthy controls. In contrast, the percentage of subjects who detected insoluble matter in COD exceeded 60% in each group. The percentage of subjects with carbonate apatite insolubilization varied between 10% and 30% among the four groups, but as shown in Figure 24, there was no statistically significant difference in the number of subjects with COD or carbonate apatite insolubilization among the four groups.

[0119] These results indicate that calcium phosphate crystals and / or amorphous calcium phosphate are highly likely to be detected in chilled urine samples from patients with early recurrence of urolithiasis, whereas calcium oxalate crystals and / or amorphous calcium oxalate are universally found in chilled urine samples, regardless of the timing of recurrence of urolithiasis, the presence or absence of urolithiasis, or the presence or absence of urolithiasis.

[0120] (5) Evaluation of calcium phosphate insolubilized substances as a biomarker for determining early recurrence of urolithiasis. The relationship between the timing of urolithiasis recurrence and insolubilized substances was further examined using logistic regression analysis for the four groups. In the logistic regression analysis, the presence or absence of urolithiasis recurrence was used as the dependent variable, and the results of 24-hour urine collection tests (Ca, oxalic acid, SI. CaOx, SI. CaP) and the presence or absence of insolubilized substances (COD, carbonate apatite, brushite) were used as explanatory variables. As shown in Figure 26, the presence or absence of insolubilized brushite was a factor that statistically significantly influenced the presence or absence of urolithiasis recurrence in the early recurrence patient group compared with the other groups (i.e., the late recurrence patient group, the non-recurrence patient group, and the healthy control group). In addition, SI. CaOx and SI. CaP was a factor that statistically significantly influenced the recurrence of urolithiasis in the early recurrence patient group compared with the healthy control group. In contrast, the other explanatory variables did not have a statistically significant influence on the recurrence of urolithiasis.

[0121] From the above results, it was found that at least one of calcium phosphate crystals and amorphous calcium phosphate can be used as a biomarker for determining the early recurrence of urolithiasis. It was also found that at least one of calcium phosphate crystals and amorphous calcium phosphate can be used as a determination method for determining the early recurrence of urolithiasis in a subject based on the detection results of at least one of calcium phosphate crystals and amorphous calcium phosphate.

[0122] (6) Correlation between SI.COD and SI.Brushite in subjects who observed insoluble matter in cooled urine samples The supersaturation index (SI) of calcium oxalate dihydrate (COD) and monobasic calcium phosphate dihydrate (Brushite) was calculated as SI.COD and SI.Brushite using geochemical calculation software PHREEQC. In subjects who observed insoluble matter of Brushite or insoluble matter of COD in cooled urine samples, the correlation between SI.COD and SI.Brushite was examined for each type of insoluble matter. In Figure 27A, subjects who observed insoluble matter of Brushite are indicated by a circle symbol, and subjects who did not observed insoluble matter of Brushite are indicated by a triangle symbol. In addition, in Figure 27B, subjects who observed insoluble COD are indicated by circles, and subjects who did not observe insoluble COD are indicated by triangles. As shown in Figure 27A, subjects who observed insoluble COD in Brushite observed a correlation between SI.COD and SI.Brushite. Also, as shown in Figure 27B, subjects who observed insoluble COD also observed a correlation between SI.COD and SI.Brushite.

[0123] (7) Comparison of the size and amount of insoluble brushite particles in patients with early recurrence of urolithiasis, patients with late recurrence of urolithiasis, and patients without recurrence of urolithiasis. The size and amount of insoluble brushite particles observed in chilled urine samples were compared using an optical microscope among patients with early recurrence of urolithiasis, patients with late recurrence of urolithiasis, and patients without recurrence of urolithiasis. Photographs in Figures 28A-D show insoluble brushite particles observed in chilled urine samples from patients with early recurrence of urolithiasis. Photographs in Figure 28E show insoluble brushite particles observed in chilled urine samples from patients with late recurrence of urolithiasis. Photographs in Figures 28F-H show insoluble brushite particles observed in chilled urine samples from patients without recurrence of urolithiasis. In Figures 28A-D, the insoluble brushite particles observed in the cooled urine samples represent all insoluble particles in the photographs. In contrast, in Figures 28E-H, the insoluble brushite particles observed in the cooled urine samples are those indicated by arrows in the photographs. Comparing Figures 28A-B and 28E-H, the amount of insoluble brushite particles observed in the cooled urine samples of patients with early recurrence of urolithiasis was significantly greater than that of patients with late recurrence of urolithiasis and patients with no recurrence of urolithiasis. From these results, it can be said that the amount of at least one of calcium phosphate crystals and amorphous calcium phosphate in patients with early recurrence of urolithiasis was approximately 100 or more per field of view under conditions of, for example, a 20x lens magnification. 28C-D and 28E-H, the size of insoluble brushite observed in cooled urine samples from patients with early recurrence of urolithiasis was significantly larger than that of patients with late recurrence of urolithiasis and patients without recurrence of urolithiasis. This result suggests that the size of at least one of calcium phosphate crystals and amorphous calcium phosphate in patients with early recurrence of urolithiasis is, for example, approximately 100 μm or more.

[0124] The correlation between SI.COD and SI.Brushite was also examined in each group of urolithiasis patients. In Figure 29, symbols A-D indicate early recurrent patients in whom insolubilized matter was observed, as shown in Figures 28A-D. In Figure 29, symbol E indicates late recurrent patients in whom insolubilized matter was observed, as shown in Figure 28E. In Figure 29, symbols F-H indicate non-recurrent patients in whom insolubilized matter was observed, as shown in Figures 28F-H. In Figure 29, early recurrent patients are indicated by circles, late recurrent patients are indicated by triangles, and non-recurrent patients are indicated by squares. As shown in Figure 29, no correlation between SI.COD and SI.Brushite was observed in early recurrent urolithiasis patients, late recurrent urolithiasis patients, or non-recurrent urolithiasis patients.

[0125] From the above results, it was found that the amount of at least one of calcium phosphate crystals and amorphous calcium phosphate was higher in patients with early recurrence of urolithiasis compared with patients with late recurrence of urolithiasis. It was also found that the size of at least one of calcium phosphate crystals and amorphous calcium phosphate was larger in patients with early recurrence of urolithiasis compared with patients with late recurrence of urolithiasis. Therefore, it was inferred that early recurrence of urolithiasis can be determined based on the size and amount of at least one of calcium phosphate crystals and amorphous calcium phosphate.

[0126] Therefore, it was speculated that the size and amount of at least one of calcium phosphate crystals and amorphous calcium phosphate could be used as a biomarker for determining the early recurrence of urolithiasis. It was also speculated that the size and amount of at least one of calcium phosphate crystals and amorphous calcium phosphate could be used as a determination method for determining the early recurrence of urolithiasis in a subject based on the detection results of the size and amount of at least one of calcium phosphate crystals and amorphous calcium phosphate.

[0127] Furthermore, it was found that the size and amount of insoluble matter in Brushite are not necessarily related to SI.COD and SI.Brushite. Therefore, it was inferred that evaluation based solely on inorganic components such as SI.COD and SI.Brushite cannot necessarily determine the early recurrence of urolithiasis. In contrast, evaluation based on insoluble matter in a cooled urine sample can grasp the formation of urolithiasis in a comprehensive environment including organic components such as proteins. Therefore, it was inferred that evaluation based on at least one of calcium phosphate crystals and amorphous calcium phosphate in a cooled urine sample can determine the early recurrence of urolithiasis.

[0128] Although the present disclosure has been described above with reference to embodiments and examples, the present disclosure is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure. Furthermore, each embodiment can be combined with other embodiments as appropriate.

[0129] This application claims priority based on Japanese Patent Application No. 2024-004939, filed January 17, 2024, and Japanese Patent Application No. 2024-129618, filed August 6, 2024, the disclosures of which are incorporated herein in their entireties.

[0130] <Appendix> Some or all of the above embodiments can be described as the following appendices, but are not limited to them. <Biomarkers> (Appendix 1) A biomarker for determining the onset of urolithiasis, comprising at least one of calcium phosphate crystals and amorphous calcium phosphate in urine. (Appendix 2) The biomarker according to Appendix 1, wherein the calcium phosphate comprises monobasic calcium phosphate. (Appendix 3) The biomarker according to Appendix 1 or 2, wherein the calcium phosphate comprises monobasic calcium phosphate dihydrate. (Appendix 4) The biomarker according to any of Appendixes 1 to 3, wherein the urine is collected urine. (Appendix 5) The biomarker according to any of Appendixes 1 to 4, wherein the urine is collected urine over 24 hours. (Appendix 6) The biomarker according to any of Appendixes 1 to 5, wherein the biomarker is a biomarker for determining the recurrence of urolithiasis. (Appendix 7) The biomarker according to any of Appendixes 1 to 6, wherein the biomarker is a biomarker for determining the early recurrence of urolithiasis. <Method of Determination> (Appendix 8) A method of determining the onset of urolithiasis, comprising: a detection step of detecting a biomarker according to any one of Appendices 1 to 5 in the urine of a subject; and a determination step of determining the onset of urolithiasis in the subject based on the detection result of the biomarker. (Appendix 9) The method of determination according to Appendix 8, comprising an insolubilization step of forming the biomarker by subjecting the urine of the subject to an insolubilization treatment. (Appendix 10) The method of determination according to Appendix 9, wherein in the insolubilization step, the insolubilization treatment is supersaturation. (Appendix 11) The method of determination according to Appendix 10, wherein the supersaturation is cooling. (Appendix 12) The method of determination according to Appendix 11, wherein the cooling temperature is 0 to 4°C. (Appendix 13) The method of determination according to Appendix 11, wherein the cooling time is 24 hours or longer. (Supplementary Note 14) The method of determining according to Supplementary Note 11, wherein the cooling temperature is 0 to 4°C and the cooling time is 24 hours or longer. (Supplementary Note 15) The method of determining according to any of Supplementary Notes 8 to 14, wherein, if the biomarker is detected in the urine of the subject in the detection step, the subject is determined to have developed urolithiasis in the determination step.(Appendix 16) The method of any one of Appendices 8 to 15, wherein the recurrence of urolithiasis in the subject is determined in the determination step. (Appendix 17) The method of any one of Appendices 8 to 16, wherein the early recurrence of urolithiasis in the subject is determined in the determination step. <Screening Method> (Appendix 18) A method of screening for a candidate substance for inhibiting urolithiasis, comprising a selection step of selecting, from test substances, a formation inhibitor that inhibits the formation of at least one of calcium phosphate crystals and amorphous calcium phosphate, as a candidate substance for inhibiting urolithiasis. (Appendix 19) The screening method of Appendices 18, wherein the calcium phosphate comprises calcium hydrogen phosphate. (Appendix 20) The screening method of Appendices 18 or 19, wherein the calcium phosphate comprises calcium hydrogen phosphate dihydrate. (Supplementary Note 21) The screening method according to any one of Supplementary Notes 18 to 20, comprising: an insolubilization step of forming at least one of calcium phosphate crystals and amorphous calcium phosphate by performing an insolubilization treatment in the presence of the test substance; and a detection step of detecting at least one of the calcium phosphate crystals and amorphous calcium phosphate, wherein the candidate inhibitor substance is selected from the test substance based on the detection result of at least one of the calcium phosphate crystals and amorphous calcium phosphate in the selection step. (Supplementary Note 22) The screening method according to Supplementary Note 21, wherein the insolubilization treatment in the insolubilization step is supersaturation. (Supplementary Note 23) The screening method according to Supplementary Note 22, wherein the supersaturation is cooling. (Supplementary Note 24) The screening method according to Supplementary Note 23, wherein the cooling temperature is 0 to 4°C. (Supplementary Note 25) The screening method according to Supplementary Note 23, wherein the cooling time is 24 hours or longer. (Appendix 26) The screening method according to appendix 23, wherein the cooling temperature is 0 to 4°C, and the cooling time is 24 hours or longer.(Appendix 27) The screening method according to any one of Appendices 18 to 26, wherein in the selection step, the test substance in which the amount of at least one of calcium phosphate crystals and amorphous calcium phosphate is lower than that of a control in which the test substance is not coexisting is selected as the candidate inhibitory substance. <Use> (Appendix 28) Use of at least one of calcium phosphate crystals and amorphous calcium phosphate in urine for determining the onset of urolithiasis. (Appendix 29) The use according to Appendices 28, wherein the calcium phosphate comprises calcium dihydrogen phosphate. (Appendix 30) The use according to Appendices 28 or 29, wherein the calcium phosphate comprises calcium dihydrogen phosphate dihydrate. (Appendix 31) The use according to any one of Appendices 28 to 30, wherein the urine is collected urine. (Appendix 32) The use according to any one of Appendices 28 to 31, wherein the urine is collected urine over 24 hours. (Appendix 33) The use according to any one of Appendices 28 to 32, wherein the onset of urolithiasis is recurrent urolithiasis. (Appendix 34) The use according to any one of Appendices 28 to 33, wherein the onset of urolithiasis is early recurrence of urolithiasis.

[0131] According to the present disclosure, for example, the onset of urolithiasis can be accurately determined, and therefore the present disclosure is applicable to various fields such as the medical field.

Claims

1. A biomarker for determining the onset of urolithiasis, comprising at least one of calcium phosphate crystals and amorphous calcium phosphate in urine.

2. The biomarker according to claim 1, wherein the biomarker is a biomarker for determining recurrence of urolithiasis.

3. A determination method for determining the onset of urolithiasis, comprising: a detection step of detecting the biomarker according to claim 1 in a subject's urine; and a determination step of determining the onset of urolithiasis in the subject based on the detection result of the biomarker.

4. The determination method according to claim 3, wherein in the determination step, recurrence of urolithiasis in the subject is determined.

5. The determination method according to claim 3, comprising a precipitation step of forming the biomarker by subjecting the subject's urine to a precipitation treatment.

6. The determination method according to claim 5, wherein in the precipitation step, the precipitation treatment is supersaturation.

7. The determination method according to claim 3, wherein when the biomarker in the subject's urine is detected in the detection step, in the determination step, the subject is determined to have developed urolithiasis.

8. A screening method for candidate substances for suppressing urolithiasis, comprising a selection step of selecting, as a candidate substance for suppressing urolithiasis, a formation inhibitor that inhibits the formation of at least one of calcium phosphate crystals and amorphous calcium phosphate from a test substance.

9. The screening method according to claim 8, comprising: a precipitation step of subjecting the test substance to a precipitation treatment in the coexistence thereof to form at least one of calcium phosphate crystals and amorphous calcium phosphate; and a detection step of detecting at least one of calcium phosphate crystals and amorphous calcium phosphate, wherein in the selection step, the candidate substance for suppression is selected from the test substance based on the detection result of at least one of calcium phosphate crystals and amorphous calcium phosphate.

10. The screening method according to claim 9, wherein in the selection step, the test substance in which the amount of at least one of calcium phosphate crystals and amorphous calcium phosphate is lower than that of a control without the coexistence of the test substance is selected as the candidate substance for suppression.

11. The screening method according to claim 9 or 10, wherein in the insolubilization step, the insolubilization treatment is supersaturation.

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