Identification of acute kidney injury after ischemia-reperfusion by using cysteine-sensitive fluorescent probe, and application thereof

Abstract

The present invention relates to: a fluorescent probe compound containing a 4-nitro-7-perfluorophenoxybenzo[c][1,2,5]oxadiazole structure for cysteine detection; a composition comprising same for diagnosing delayed graft function due to ischemia-reperfusion injury; and a method for providing information for diagnosing delayed graft function due to ischemia-reperfusion injury using the composition, wherein the compound of the present invention can be easily and conveniently synthesized and can exhibit values of intense fluorescence in urine and kidneys of patients with delayed graft function, which may occur after transplantation of a kidney from a brain-dead person, and thus can be usefully employed for diagnosing the onset of delayed graft function due to ischemia-reperfusion injury by detecting cysteine in cells and animal- and human-derived samples, particularly in urine or kidney tissues.

Description

Detection of acute kidney injury after ischemia-reperfusion using a cysteine-sensitive fluorescent probe and its application

[0001] The present invention relates to a composition for diagnosing delayed transplant function due to ischemia-reperfusion injury, comprising a cysteine-sensitive fluorescent probe compound, and a method for providing information for diagnosing delayed transplant function due to ischemia-reperfusion injury using the same.

[0002] One of the ideal treatments for patients with end-stage organ failure is organ transplantation. Brain-dead patients are typically the primary donors for organ transplantation. Typically, during transplantation, a healthy kidney from a brain-dead patient is exposed to ischemia-reperfusion. Ischemia is a process in which tissue is damaged by vascular blockage and narrowing. This process is unavoidable when transplanting from a brain-dead patient to a recipient, and rapid reperfusion is essential to minimize ischemic damage. However, even with rapid reperfusion, many transplant patients experience acute kidney injury (AKI) and delayed graft function (DGF), leading to poor outcomes. This is called ischemia-reperfusion injury (I / R injury) and is caused by endothelial dysfunction and renal tissue damage during the ischemia-reperfusion process. The primary damage may be due to increased reactive oxygen species (ROS) caused by the inflow of oxygen into the blood during reperfusion after ischemia. ROS are various types of oxygen with increased reactivity, including hydrogen peroxide, superoxide, hydroperoxyl, and hydroxyl radicals. ROS are primarily generated in aerobic organisms that consume oxygen and are known to cause various diseases, including cancer. To resist ROS, cells import cystine from the outside and increase the amount of cysteine ​​internally through enzymatic reactions. Furthermore, cells overproduce the amino acid cysteine ​​within themselves. Cysteine ​​is a sulfur-containing amino acid that is oxidized under high ROS levels, thereby suppressing ROS levels. Based on this background, it is predicted that cysteine ​​overproduction, driven by high ROS levels, occurs in kidneys of donors undergoing ischemia-reperfusion.Additionally, this may demonstrate the potential of cysteine ​​as a biomarker for acute renal injury and transplant renal dysfunction induced by ischemia-reperfusion.

[0003] Diagnostic methods for ischemia-reperfusion-induced renal injury used in clinical settings rely on assessments of renal function. These methods primarily involve measuring serum creatinine concentrations, cystatin C, and urine output. However, because these methods are not histologically dependent on the degree of renal damage, they cannot be used to specifically predict ischemia-reperfusion injury. Furthermore, most diagnostic procedures are complex, time-consuming and costly, relying on the physician's empirical judgment. Therefore, developing new diagnostic methods that reduce time and cost while facilitating objective physician judgment is an essential challenge.

[0004] Among human organs, the kidney plays a crucial role in regulating endogenous hydrogen sulfide production. Hydrogen sulfide is produced as an antioxidant in tissue IRI through cystathionine-γ-lyase (CSE). Furthermore, increased CSE mRNA expression has been reported immediately after ischemia and reperfusion, suggesting an increased cysteine ​​production in IRI. Cysteine ​​is an essential substrate for glutathione and plays a crucial role in the antioxidant defense system. Because it participates in various antioxidant mechanisms, it is recognized as a biomarker for disease. However, despite the importance of thiol groups, existing methods for detecting them in vivo present several obstacles. Known methods for measuring thiol groups include mass spectrometry, HPLC, voltammetry, and capillary electrophoresis. However, these methods are complex, time-consuming, and expensive. Furthermore, real-time in vivo observation is impossible.

[0005] The field of fluorescent molecular probes is a rapidly developing field, utilized in numerous studies due to its convenient visual observation of various molecules, molecular activities, proteins, and protein activity within the body. In particular, they demonstrate outstanding utility in detecting metabolites in the body with high sensitivity and specificity. Furthermore, their importance is expected to increase in the future due to their ability to easily observe metabolites and proteins in real time. Among the fluorescent molecular probes developed to date, the development of a probe that selectively detects cysteine, a thiol group, has been challenging. Furthermore, its structure is highly complex. The difficulty in selectively detecting cysteine ​​is primarily due to its structural similarity to homocysteine ​​(homocysteine ​​carbon number minus cysteine ​​carbon number = 1, a difference of one carbon). To distinguish cysteine ​​from homocysteine, a structure with high electrophilicity and a low steric density, making it susceptible to nucleophilic attack, is essential. In addition, fluorescent molecular probes have been developed using principles such as intramolecular charge transfer (ICT), Förster resonance energy transfer (FRET), photoinduced proton transfer (PET), and excited state intramolecular proton transfer (ESIPT) to induce fluorescence. Typically, fluorescent molecular probes for detecting biometabolites are designed as fluorescence-on, fluorescence-off, and fluorescence-changing systems. Among these, fluorescent molecular probes of the fluorescence-on system are characterized by having higher resolution and sensitivity with lower error potential in vivo than the fluorescence-off system. In addition, the fluorescence-on system can help stable imaging in real-time imaging in vivo with higher resolution than the fluorescence-changing system.For these reasons, the development of cysteine-sensing fluorescent probes with a fluorescence-activating system remains a very challenging task despite its importance, and its development is an important task for diagnosing and further overcoming various diseases.

[0006] Accordingly, the inventors of the present invention developed a fluorescent probe using a simple and mass-synthetic method to detect cysteine ​​in urine and predict DGF caused by IRI from organ transplantation from brain-dead individuals, and completed the present invention by verifying this in cells, animals, and human-derived materials (urine).

[0007] The purpose of the present invention is to provide a composition and a diagnostic method for predicting delayed renal function (DGF) due to ischemia-reperfusion injury (IRI) through detection of cysteine ​​in urine or renal tissue using a cysteine-sensitive fluorescent probe compound that can be manufactured by a simple, mass-producible synthetic method.

[0008] To achieve the above purpose,

[0009] The present invention provides a composition for diagnosing kidney damage or renal function decline, comprising a compound represented by the following chemical formula 1, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof:

[0010] [Chemical Formula 1]

[0011]

[0012] In addition, the present invention provides a method for providing information for diagnosing kidney damage or renal function decline, comprising a step of treating a sample with a compound represented by the above chemical formula 1, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.

[0013] In one embodiment, the renal damage or renal function decline may be acute kidney injury (AKI) or delayed graft function (DGF), and preferably may be due to ischemia-reperfusion injury (IRI).

[0014] In one embodiment, the compound may be capable of predicting the onset of graft dysfunction due to ischemia-reperfusion injury after organ transplantation from a brain-dead donor.

[0015] In one embodiment, the compound may emit fluorescence upon contact with cysteine.

[0016] In one embodiment, the compound may selectively react with cysteine ​​in urine or kidney tissue and not react with other biomolecules.

[0017] In one embodiment, the compound may exhibit fluorescence after a SMILE rearrangement, whereby the thiol group (-SH) of cysteine ​​in urine or kidney tissue attacks a carbon atom in the molecule of the compound, breaking the ether linkage and binding to cysteine.

[0018] In one embodiment, the compound reacts with cysteine ​​in urine or kidney tissue to cause a fluorescence-on phenomenon, and the fluorescence-on phenomenon may occur in a group of patients with DGF by IRI compared to a normal group.

[0019] In one embodiment, if an increase in fluorescence emission or fluorescence intensity is confirmed in a sample after treatment with the compound compared to a sample before treatment, it may be determined that kidney damage or renal function decline has occurred.

[0020] In one embodiment, the sample may be a biological sample, preferably urine or kidney tissue.

[0021] The 4-nitro-7-perfluorophenoxybenzo[c][1,2,5]oxadiazole compound (compound 1) according to the present invention can be easily prepared, and the compound 1 can be used to image cysteine ​​in urine and kidney. In particular, the compound 1 according to the present invention can be used to perform imaging analysis of urine and kidneys in an IRI animal model at the cellular level, and further, to analyze urine from brain-dead donors, thereby enabling early prediction and diagnosis of DGF due to IRI.

[0022] The composition and diagnostic method for DGF by IRI using compound 1 of the present invention have been verified in cells, animals, and human-derived materials (urine and kidney tissue), and thus, the results are highly reliable, have sufficient clinical value, and are readily commercializable. Therefore, the prospects for future commercialization of the technology are clear. In addition, since compound 1 of the present invention focuses on metabolites that are difficult to access in the field of molecular diagnosis, it is endowed with high technological innovation in that it can quickly detect metabolites that are difficult to detect using existing molecular diagnosis methods, such as polymerase chain reaction (PCR) and Western blot.

[0023] Figure 1 shows the results of a spectrum analysis of the fluorescence change of compound 1 when cysteine ​​was added to normal rat urine. The fluorescence spectrum was acquired at an excitation wavelength of 488 nm.

[0024] Figure 2 shows the results of measuring the fluorescence change of compound 1 in normal rat kidneys treated with cysteine ​​and cysteine ​​inhibitors using a fluorescence tissue imaging system (a) and confirming the light spot efficiency value (b).

[0025] Figure 3 shows the results of fluorescence imaging of compound 1 at the HK-1 cell level under hypoxic conditions confirmed by confocal microscopy (a) and the results of quantifying fluorescence intensity (b).

[0026] Figure 4 shows the results of measuring (a) and confirming the light spot efficiency value (b) using a fluorescence tissue imaging system in a kidney from an ischemia-reperfusion animal model treated with compound 1, and the results confirmed using a two-photon excitation fluorescence microscope (c).

[0027] Figure 5 shows the results of measuring the fluorescence intensity of urine from an ischemia-reperfusion animal model and urine from a patient after organ transplantation from a brain-dead donor treated with compound 1.

[0028] Hereinafter, the present invention will be described in detail.

[0029] The present inventors developed a cysteine-sensitive fluorescent probe compound by a simple synthetic method that can be synthesized in large quantities, and attempted to detect cysteine ​​in urine to predict DGF by IRI caused by organ transplantation from brain-dead patients. As a result, they confirmed that a high fluorescent-on signal was observed in the urine of a group of DGF patients, thereby confirming the possibility of cysteine ​​in urine as a new biomarker of DGF and the applicability of the present fluorescent probe compound as a reference index of DGF by IRI, and completed the present invention by verifying this in cells, animals, and human-derived materials (urine) (see Examples 1 to 6).

[0030] In one aspect, the present invention relates to a composition for diagnosing kidney damage or renal function decline, comprising a compound represented by the following chemical formula 1, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof:

[0031]

[0032] In an embodiment of the present invention, the compound represented by the chemical formula 1 was identified as 4-nitro-7-perfluorophenoxybenzo[c][1,2,5]oxadiazole, and is referred to as 'compound 1' in this specification (see Example 1).

[0033] In one embodiment, the renal damage or renal function decline may be acute kidney injury (AKI) or delayed graft function (DGF), and preferably may be due to ischemia-reperfusion injury (IRI).

[0034] In one embodiment, the compound may be capable of predicting the onset of graft dysfunction due to ischemia-reperfusion injury after organ transplantation from a brain-dead donor.

[0035] In one embodiment, the compound may emit fluorescence upon contact with cysteine.

[0036] In one embodiment, the compound may selectively react with cysteine ​​in urine or kidney tissue and not react with other biomolecules. For example, the compound may emit a higher intensity fluorescence signal when reacted with a sample containing cysteine ​​than when reacted with a sample containing at least one selected from the group consisting of homocysteine, glutathione, and hydrogen sulfide. The above biomolecules may be at least one selected from the group consisting of homocysteine, glutathione, hydrogen sulfide, aspartic acid, glutamine, lysine, arginine, adenosine triphosphate, sucrose, ammonium, HSA (human serum sulbumin), PbCl2, MgCl2, FeCl3, Hg(NO3)2, CdCl2, ZnCl2, NiCl2, CoCl2, NaCl, AuCl3, AgCl, CuCl2, and KCl, but are not limited thereto.

[0037] In one embodiment, the compound may exhibit fluorescence after a SMILE rearrangement, whereby the thiol group (-SH) of cysteine ​​in urine or kidney tissue attacks a carbon atom in the molecule of the compound, breaking the ether linkage and binding to cysteine.

[0038] In one embodiment, the compound reacts with cysteine ​​in urine or kidney tissue to cause a fluorescence-on phenomenon, and the fluorescence-on phenomenon may occur in a group of patients with DGF by IRI compared to a normal group.

[0039] The above "fluorescence turn-on" characteristic of the present invention means a characteristic in which the detection target substance does not fluoresce in the absence of the detection target substance and cannot be identified with the naked eye, but reacts with the detection target substance and fluoresces when the detection target substance is present.

[0040] In the case of a "fluorescence-on" reference system such as compound 1 according to an example of the present invention, when the target substance is not present in a living tissue, tissue isolated from a living body, or other sample, no fluorescence is present, or only weak fluorescence that cannot be identified with the naked eye is present, but when the target substance is present, clear fluorescence (at least 10-fold increase in fluorescence intensity) occurs, so that it is possible to clearly visually determine whether the target substance is detected in a living tissue, tissue isolated from a living body, or other sample.

[0041] The fluorescence on-state exhibited by the compound of the present invention may mean that it does not fluoresce before contact with cysteine, but fluoresces upon contact with cysteine. Accordingly, the compound may be used as a cysteine-sensitive fluorescent probe and may be used for the diagnosis of DGF by IRI through the detection of cysteine ​​in urine or kidney tissue.

[0042] In the present invention, the term "probe," also known as "sensor," is defined as something capable of detecting or imaging an in vivo or ex vivo target. It is generally used interchangeably with terms such as imaging agent, contrast agent, and radiopharmaceutical.

[0043] The compound represented by the chemical formula 1 of the present invention does not show fluorescence because it contains a nitrogen dioxide (NO2) group that withdraws electrons and a pentafluorophenol that withdraws electrons at the para (-para) position. However, when an amine group (NH2) of cysteine ​​that combines with cysteine ​​and donates electrons at the para position is introduced, a fluorescence-on phenomenon is observed around 550 nm, and when an aromatic ring compound that is deficient in electrons is present, the fluorescence-on phenomenon may occur only in cysteine ​​without interference from thiols such as homocysteine ​​and glutathione.

[0044] In one embodiment of the present invention, the compound 1 reacts with cysteine ​​in urine or kidney tissue to cause a fluorescence-on phenomenon (Examples 2 to 4), and it was confirmed that the fluorescence-on phenomenon occurred in the DGF patient group due to IRI compared to the normal group, thereby confirming that cysteine ​​may be present in greater amounts in the urine or kidney tissue from ischemia-reperfusion patients than in the normal group (Examples 5 to 6). Through these results, it was confirmed that the compound 1 has the potential to enable the early diagnosis of DGF due to IRI through the detection of cysteine ​​fluorescence in urine or kidney tissue.

[0045] The term "diagnosis" in the present invention refers to confirming the presence or characteristics of a pathological condition, the progression of a disease, the therapeutic effect of a disease during treatment, etc. For the purposes of the present invention, the diagnosis refers to, but is not limited to, diagnosing renal damage or renal function decline, preferably DGF due to IRI, by detecting cysteine ​​in urine or renal tissue.

[0046] According to one embodiment of the present invention, when the compound is administered intravenously, cysteine ​​can be detected in the urine and kidney of the IRI animal model as a fluorescence-on phenomenon occurs in the urine and kidney after 4 hours. However, the administration method and circulation time are not specific time points and are not limited to the above conditions.

[0047] In one embodiment, the composition of the present invention may further comprise one or more selected from a solvent, an acid, a base, and a buffer solution in addition to the compound, and may be prepared by adding the compound described above to a solvent, a buffer solution, or a mixture thereof, and then adding an acid and / or a base thereto. In addition, the composition may additionally comprise other additives that can be used in the art. The contents of the solvent, acid, base, and buffer solution contained in the composition may be appropriately adjusted depending on the required performance. The solvent may include water, THF, methanol, ethanol, HI aqueous solution, N,N-dimethylformamide, or a combination thereof. Alternatively, the composition may be mixed with a biological sample.

[0048] The above biological sample may include cells, tissues, body fluids (saliva, etc.), blood, spinal fluid, cerebrospinal fluid, serum, plasma, urine or feces, etc. isolated from humans, animals (rodents, mammals, etc.), and preferably may be urine, kidney cells or kidney tissue, but is not limited thereto, and any biological sample that can be used in the relevant technical field may be used.

[0049] In another embodiment of the present invention, the compound of the present invention is capable of detecting cysteine ​​in urine and kidney tissue, and has high utility as a material for early diagnosis and prediction of diseases in which cysteine ​​accumulates in large amounts in urine or kidney tissue.

[0050] In addition, the present invention provides a method for providing information for diagnosing kidney damage or renal function decline, comprising a step of treating a sample with a compound represented by the above chemical formula 1, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.

[0051] In the method for providing information for diagnosis according to the present invention, the description of the compound, renal damage, renal dysfunction and diagnosis is as described above.

[0052] In one embodiment, the sample may be a biological sample, preferably urine or kidney tissue.

[0053] In one embodiment, the method may include a step of treating a sample with a compound represented by the above chemical formula 1, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof (step 1); a step of irradiating the sample treated in step 1 with an excitation light source (step 2); and a step of measuring a change in emitted fluorescence (step 3).

[0054] In the method for providing information for diagnosis according to the present invention, step 3 includes a method for confirming that the fluorescence intensity of the sample to be measured has increased compared to the fluorescence intensity before the compound is treated.

[0055] More specifically, the step 3 may include a step of confirming that changes in absorption and fluorescence emission spectra occur as the compound represented by the chemical formula 1 according to the present invention selectively reacts with cysteine ​​in the sample. Preferably, the fluorescence-on and fluorescence intensity may increase in the presence of cysteine ​​in the sample, and if an increase in fluorescence emission and fluorescence intensity is confirmed after treating the sample with the compound according to the present invention compared to the sample before treatment, it may be determined that renal damage or renal function decline, preferably, delayed transplant kidney function due to ischemia-reperfusion injury, has occurred.

[0056] The excitation light source of step 3 above may be a light source having an excitation wavelength of 450 to 500 nm, preferably 480 to 490 nm, and 488 nm may be most preferable.

[0057] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0058] Example 1. Synthesis and structural analysis of a compound for detecting cysteine ​​in urine.

[0059] The present inventors designed the molecular structure of 4-chloro-7-nitrobenzofurazan to facilitate nucleophilic attack by introducing pentafluorophenol, an electron-deficient aromatic ring compound, in order to develop a fluorescent probe compound of the present invention for rapid measurement of cysteine ​​in urine, an in vivo product, and synthesized the compound 1 according to the following reaction formula:

[0060]

[0061] (1) Synthesis of 4-nitro-7-perfluorophenoxybenzo[c][1,2,5]oxadiazole

[0062] Specifically, the synthetic starting materials, 4-chloro-7-nitrobenzofurazan (20 mg, 0.1 mol, TCI, A55921G) and pentafluorophenol (55.2 mg, 0.3 mol, TCI, 771-61-9), were dissolved in 3 mL of N,N-dimethylformamide (DMF) deoxygenated with nitrogen and stirred at 25°C (rotations per minute: 300 rpm). Then, 30 μL of N,N-diisopropylethylamine (DIPEA) and 20.7 mg of potassium carbonate (K2CO3) were added to the stirred solution, and the temperature was raised to 60°C in a silicone oil container. When the temperature reached 60℃, it was stirred for 10 minutes, then the temperature was lowered again to 25℃, and 30 mL of water was added to terminate the reaction. To extract the organic layer, ethyl acetate (EtOAc) and water were mixed in a 1:1 ratio using a separatory funnel, and only the organic layer could be obtained through this process. Afterwards, the organic layer was dried over anhydrous sodium sulfate (Na2SO4, 2 g) and concentrated using an evaporator at 37℃ and 20 to 500 mmHg. The dark brown solid compound thus obtained was separated using column chromatography (diameter 6 cm, height 15 cm) using silica gel (silica gel, Merck-silica gel 60, 230-400 mesh) (eluent: Et OAc / Hexane = 10 / 90) to obtain a dark yellow solid compound (compound 1, 12.5 mg, 35.9%).

[0063] Nuclear Magnetic Resonance (NMR) analysis was performed to confirm the successful synthesis. The analysis confirmed the successful synthesis of Compound 1. The NMR analysis results are as follows:

[0064] 1 H-NMR (CDCl3, 500 MHz); δ 8.52(d, 1H, J=8.0 Hz), 6.85(d, 1H, J=8.0 Hz). 19 F-NMR (CDCl3containing hexafluoro-benzene, 471 MHz); δ -155.9(s, 2F), -158.1(d, 1F), -162.8(d, 2F).

[0065] Through this, the compound 1 was identified as '4-nitro-7-perfluorophenoxybenzo[c][1,2,5]oxadiazole'.

[0066] Example 2. Confirmation of fluorescence emission characteristics of compound 1 in normal rat urine.

[0067] In order to confirm the fluorescence emission characteristics of compound 1 prepared in Example 1 above, the fluorescence emission graph was measured after reaction with cysteine ​​in normal rat urine.

[0068] Specifically, compound 1 was dissolved in DMSO solution at 10 mM and used, and the amount of DMSO in the final solvent conditions was controlled to be the same in each container (1% or less). To confirm the reaction of compound 1 and cysteine ​​in urine, urine (198 μL) of rats that had not been pretreated was treated with cysteine ​​at various concentrations (3.125 - 200 μM), then treated with compound 1 (100 μM), and the fluorescence spectrum was measured after stirring for 40 minutes in a shaker at 37°C (excitation wavelength: 488 nm). A fluorescence measurement device (Spectrofluorophotometer; SHIMADZU CORP. RF-6000, Japan) was used to analyze the emission spectrum, and the glass cuvette used for fluorescence measurement was a standard quartz cell with a 1 cm thick side (Internal volume = 0.1 cm, Hellma Analytics, Jena, Germany).

[0069] Figure 1(a) shows the results obtained after treating normal rat urine with compound 1 (100 μM), and then treating normal rat urine with cysteine ​​(100 μM), followed by treatment with compound 1 (100 μM), stirring in a shaker at 37°C for 40 minutes, and excitation at 488 nm. According to Figure 1(b), it was confirmed that the fluorescence change when treating normal rat urine with cysteine ​​was dependent on the concentration of cysteine. In addition, the fluorescence emission spectrum in Figure 1(a) showed the highest fluorescence value at 550 nm or more and 600 nm or less.

[0070] Example 3. Confirmation of Compound 1 in imaging of normal rat kidney tissue

[0071] The present inventors measured the results using a tissue fluorescence imaging system to confirm whether compound 1 could be imaged after cysteine ​​binding in the kidney of a normal rat, and the results are shown in Fig. 2.

[0072] Specifically, to determine whether compound 1 binds to cysteine ​​in the kidneys of normal rats, N-ethylmaleimide (NEM) (500 μM), a cysteine ​​inhibitor, was used in the kidneys of normal rats. In addition, cysteine ​​was additionally added to increase the concentration of cysteine. To introduce cysteine ​​and NEM into the kidneys, the kidneys were placed in a solution containing both cysteine ​​and NEM to induce osmosis.

[0073] Figure 2(a) shows the result of imaging a normal rat kidney that went through the above pretreatment process with compound 1. According to Figure 2(a), the group pretreated with cysteine ​​showed high fluorescence intensity, and the group sequentially treated with NEM and cysteine ​​showed low fluorescence intensity, which was confirmed through imaging, indicating that compound 1 can react with cysteine ​​in the kidney. The result is shown in Figure 2(b) through photon efficiency analysis, and the statistical method for confirming the significant result is as follows; The error bar represents mean ±SD (n=3) with one-way ANOVA, Turkey's multiple comparisons tests; ** p < 0.01.

[0074] Example 4. Fluorescence response test of compound 1 in renal tissue under hypoxic conditions.

[0075] The present inventors attempted to confirm at the cellular level whether compound 1 could image increased cysteine ​​under hypoxic conditions (1.0% O2), and the results are shown in Fig. 3.

[0076] Figure 3(a) shows the results of confocal microscopy of fluorescence changes observed when human kidney tissue cells (HK-2) were subjected to hypoxic conditions for 1 hour (1 h) and 5 hours (5 h). At this time, compound 1 was treated at 5 μM for 15 minutes at 37°C to observe the fluorescence changes. As a result, the 5-hour hypoxic condition showed higher fluorescence intensity and imaging results than the 1-hour hypoxic condition. The results for fluorescence intensity are shown in Figure 3(b). The statistical methods for confirming the significant results are as follows; The error bar represents mean ± SEM with one-way ANOVA, Turkey's multiple comparisons tests; **p < 0.001 (A vs B), ***p < 0.001 (C vs D). The dot plots represent jitter points.

[0077] Example 5. Fluorescence response test of compound 1 in renal tissue of an ischemia-reperfusion animal model

[0078] To determine whether compound 1 can image renal tissue in an ischemia-reperfusion rodent model, the inventors performed fluorescence tissue imaging system analysis and two-photon excitation fluorescence microscopy analysis.

[0079] Prior to this experiment, mice (C57BL / 6, purchased from Orient Bio) were 7-9 weeks old and randomly divided into three groups: a normal group (control group, n=9), a 20-minute ischemia-reperfusion group (n=16), and a 45-minute ischemia-reperfusion group (n=16). In addition, the ischemia-reperfusion model in mice was established as follows: After inhaled anesthesia, a mid-abdominal laparotomy was performed to dissect the left renal vascular pedicle and occlude it with an atraumatic vascular clamp for the time specified for each experimental group. During the occlusion, the abdominal incision site was closed to minimize body temperature and water loss. After the clamp was removed, reperfusion of the left kidney was visually confirmed, and the right kidney was excised. The excised abdomen was sutured using 4-0 Vicryl suture.

[0080] Fluorescence tissue imaging system analysis and two-photon excitation fluorescence microscopy analysis were performed on kidney tissues obtained from normal rats and ischemia-reperfusion rats, respectively, using the above method, and the results are shown in Fig. 4.

[0081] According to the results of Fig. 4(a), when compound 1 was treated at 100 μM in the kidney tissue of normal rats and the kidney tissue of an ischemia-reperfusion rat model and stirred for 2 hours, it was confirmed that high fluorescence intensity was observed in the kidney tissue of the ischemia-reperfusion rat model, and this was statistically significant through photon efficiency analysis (Fig. 4b). In addition, the results of two-photon excitation fluorescence microscopy analysis showed high fluorescence intensity in the ischemia-reperfusion rat model, and this was confirmed even at a depth of 60 μm (Fig. 4c).

[0082] Based on these results, it was confirmed that cysteine ​​may be present in greater amounts in renal tissue derived from an ischemia-reperfusion model than in a normal group, and it can be stated that damage caused by ischemia-reperfusion can be diagnosed using compound 1 of the present invention.

[0083] Example 6. Fluorescence response test of compound 1 in an ischemia-reperfusion animal model and in urine from patients who received organ transplants from brain-dead donors.

[0084] To determine whether an increase in cysteine ​​occurred in the urine of an ischemia-reperfusion rodent model and in the urine of patients with acute kidney injury (AKI) and delayed graft function (DGF) after receiving organ transplantation from brain-dead donors, we performed a fluorescence intensity analysis using compound 1. The results are also shown in Fig. 5.

[0085] According to the results in Fig. 5(a), when compound 1 was treated at 100 μM in the urine of normal rats and the urine of an ischemia-reperfusion rat model and stirred at 37°C for 40 minutes, a high fluorescence intensity was observed in the urine of the ischemia-reperfusion rat model, which showed a tendency to increase over the duration of ischemia. In addition, the statistical significance thereof is as follows: The error bar represents mean±SEM with one-way ANOVA, Turkey's multiple comparisons tests; *p < 0.05.

[0086] In addition, according to the results of Fig. 5(b), when compound 1 was treated at 100 μM in the urine of the normal group and the patients (recipients) who received organ transplants from brain-dead donors and stirred at 37°C for 40 minutes, it was confirmed that the urine of the patients who received organ transplants from brain-dead donors showed a higher fluorescence intensity than the urine of the normal group, and statistical significance was derived: The error bar represents mean±SEM with unpaired t-test; *p < 0.05.

[0087] Therefore, since the fluorescence-on phenomenon occurred in the DGF patient group by IRI compared to the normal group, it was confirmed that cysteine ​​may be present in the urine of the patient group, and this can be detected using compound 1 of the present invention, and through this, DGF by IRI can be diagnosed through urine.

[0088] The above description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that modifications to other specific embodiments can be made without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A composition for diagnosing renal damage or renal function decline, comprising a compound represented by the following chemical formula 1, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] 2. In paragraph 1, A composition, wherein the above renal damage or renal function decline is acute kidney injury (AKI) or delayed graft function (DGF).

3. In paragraph 1, A composition wherein the above renal damage or renal function decline is caused by ischemia-reperfusion injury (IRI).

4. In paragraph 1, A composition wherein the compound can predict the onset of delayed graft function due to ischemia-reperfusion injury after organ transplantation from a brain-dead donor.

5. In paragraph 1, A composition wherein the compound emits fluorescence upon contact with cysteine.

6. In paragraph 1, A composition wherein the compound selectively reacts with cysteine ​​in urine or kidney tissue and does not react with other biomolecules.

7. A method for providing information for diagnosing renal damage or renal function decline, comprising the step of treating a sample with a compound represented by the following chemical formula 1, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] 8. In paragraph 7, A method wherein the above renal damage or renal function decline is acute kidney injury (AKI) or delayed graft function (DGF).

9. In paragraph 7, A method wherein the above renal damage or renal function decline is caused by ischemia-reperfusion injury.

10. In paragraph 7, A method in which, if an increase in fluorescence emission or fluorescence intensity is confirmed in a sample after treatment compared to a sample before treatment with the compound, it can be determined that kidney damage or renal function decline has occurred.

11. In paragraph 7, A method wherein the above sample is a biological sample.

12. In paragraph 11, A method wherein the above sample is urine or kidney tissue.

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