NOVEL ADP-RIBOSYL CYCLASE AND INHIBITOR THEREOF

Abstract

The present disclosure relates to a pharmaceutical composition containing an inhibitor against the expression or activation of a novel ADP-ribosyl cyclase or a naturally occurring variant thereof as an active ingredient for preventing or treating an ADP-ribosyl cyclase-mediated disease. In addition, the present disclosure relates to a composition for diagnosis of an ADP-ribosyl cyclase-mediated disease, the composition containing an agent for measuring a gene expression level or protein level of the ADP-ribosyl cyclase or a naturally occurring variant thereof. The composition of the present disclosure has the effect of inhibiting calcium increase in kidney cells, which is attributed to angiotensin II-induced ADP-ribosyl cyclase expression or activation, and as such can be advantageously used as a therapeutic agent for an ADP-ribosyl cyclase-mediated disease, particularly a renal disease.

Claims

1. An ADP-ribosyl cyclase (ADPRC) comprising an amino acid sequence of SEQ ID NO 1 or a naturally occurring variant thereof.

2. The ADP-ribosyl cyclase or naturally occurring variant thereof according to claim 1, wherein the naturally occurring variant of ADP-ribosyl cyclase is a naturally occurring variant selected from a group consisting of an interspecies variant, a species homolog, an isoform, an allelic variant, a conformational variant, a splice variant and a point mutation variant.

3. The ADP-ribosyl cyclase or naturally occurring variant thereof according to claim 1, wherein the naturally occurring variant of ADP-ribosyl cyclase originates from an organism selected from a group consisting of mammals, birds, reptiles, amphibians and fish.

4. The ADP-ribosyl cyclase or naturally occurring variant thereof according to claim 1, wherein the naturally occurring variant of ADP-ribosyl cyclase is an ADP-ribosyl cyclase comprising an amino acid sequence selected from a group consisting of SEQ ID NOS 2-21.

5. The ADP-ribosyl cyclase or naturally occurring variant thereof according to claim 1, wherein the ADP-ribosyl cyclase or variant thereof converts NAD.sup.+ to cyclic ADP-ribose (cADPR).

6. A nucleic acid molecule encoding the ADP-ribosyl cyclase or naturally occurring variant thereof according to claim 1.

7. A vector comprising the nucleic acid molecule according to claim 6.

8. A host cell comprising the vector according to claim 7.

9. A method for converting NAD+ into cyclic ADP-ribose (cADPR), comprising the step of treating NAD+ to produce the ADP-ribosyl cyclase or naturally occurring variant thereof according to claim 1, a nucleic acid molecule encoding the ADP-cyclase or naturally occurring variant thereon, or a vector comprising the nucleic acid molecule.

10. A method for preventing or treating an ADP-ribosyl cyclase-mediated disease, comprising administering an inhibitor against the expression or activation of an ADP-ribosyl cyclase comprising an amino acid sequence of SEQ ID NO 1 or a naturally occurring variant thereof as an active ingredient to a subject.

11. The method according to claim 10, wherein the inhibitor against the expression of an ADP-ribosyl cyclase or a naturally occurring variant thereof is selected from a group consisting of an antisense oligonucleotide, a siRNA, a shRNA, a miRNA, a ribozyme, a DNAzyme and a PNA (protein nucleic acid).

12. The method according to claim 11, wherein the siRNA comprises a nucleotide sequence of SEQ ID NO 22.

13. The method according to claim 10, wherein the inhibitor against the activation of the ADP-ribosyl cyclase or naturally occurring variant thereof is selected from a group consisting of a compound, a peptide, a peptide mimetic, an aptamer and an antibody.

14. The method according to claim 13, wherein the compound is selected from a group consisting of 4,4′-dihydroxyazobenzene, 2-(1,3-benzoxazol-2-ylamino)-1-methylquinazolin-4(1H)-one and dicaffeoylquinic acid.

15. The method according to claim 10, wherein the ADP-ribosyl cyclase-mediated disease is a renal disease.

16. The method according to claim 15, wherein the renal disease is renal failure, nephropathy, nephritis, renal fibrosis or nephrosclerosis.

17. The method according to claim 16, wherein the renal failure is chronic renal failure, acute renal failure or mild renal failure before dialysis.

18. The method according to claim 16, wherein the nephropathy is nephropathy syndrome, lipoid nephropathy, diabetic nephropathy, immunoglobulin A (IgA) nephropathy, analgesic nephropathy or hypertensive nephropathy.

19. (canceled)

20. A non-human animal model wherein a hetero-type gene of the ADP-ribosyl cyclase (ADPRC) or the naturally occurring variant thereof according to claim 1 is deleted.

21. A method for identifying an ADP-ribosyl cyclase-mediated disease, comprising: (a) a step of inducing a specific disease in the animal model according to claim 20 and a wild-type animal model; and (b) a step of identifying the difference between the animal models.

22. A method for providing information for diagnosis of an ADP-ribosyl cyclase-mediated disease, comprising: 1) a step of measuring the expression or activation level of an ADP-ribosyl cyclase according to claim 1 in a sample isolated from a subject; and 2) a step of determining a risk of the ADP-ribosyl cyclase-mediated disease of the subject by comparing the expression or activation level of the ADP-ribosyl cyclase or naturally occurring variant thereof in the step 1) with a normal control group.

23. A composition for diagnosis of an ADP-ribosyl cyclase-mediated disease, comprising an agent for measuring the gene expression level or protein level of an ADP-ribosyl cyclase according to claim 1.

24. A kit for diagnosis of an ADP-ribosyl cyclase-mediated disease, comprising an agent for measuring the gene expression level or protein level of an ADP-ribosyl cyclase according to claim 1.

25. A method for screening a substance for preventing or treating an ADP-ribosyl cyclase-mediated disease, comprising: 1) a step of treating a cell expressing an ADP-ribosyl cyclase according to claim 1 with a test substance; 2) a step of measuring the gene expression level or protein level of the ADP-ribosyl cyclase as a result of treating with the test substance; and 3) a step of screening the test substance as a substance for preventing or treating an ADP-ribosyl cyclase-mediated disease if the gene expression level or protein level is decreased as compared to a control group not treated with the test substance.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0105] FIG. 1 shows a result of measuring the NAD glycohydrolase (NADase) activity of new ADPRC in HEK293 cells and MES13 cells.

[0106] FIG. 2 shows a result of evaluating the cADPR synthesis capability of purified FLAG-ADPRC.

[0107] FIG. 3 shows a result of measuring the intracellular cADPR production by new ADPRC when MES13 cells are stimulated with angiotensin II.

[0108] FIG. 4 shows a result of comparing the interspecies sequence homology of ADP-ribosyl cyclase of the present disclosure (human (96%), rat (96%), dog (90%), pig (88%), rabbit (89%), sheep (90%), chicken (70%), cattle (78%), chimpanzee (93%), horse (91%), frog (63%), goat (90%), turkey (62%), guinea pig (91%), Echinococcus granulosus (27%), Schistosoma haematobium (22%), Trichinella spiralis (22%), Drosophila (19%) and zebrafish (56%) as compared to mouse).

[0109] FIG. 5 shows a result of measuring the effect of new inhibitors inhibiting the NAD glycohydrolase (NADase) activity of new ADPRC.

[0110] FIG. 6 shows a result of measuring the effect of dicaffeolylquinic acid (DCQA) on the blood glucose (FIG. 6A), kidney-to-body weight ratio (FIG. 6B), creatinine clearance rate (FIG. 6C) and urine albumin level (FIG. 6D) of a diabetic renal disease mouse model.

[0111] FIG. 7 shows a result of measuring the effect of DCQA on the ADPRC activity (FIG. 7A) and cADPR concentration (FIG. 7B) in the kidney tissue of a diabetic renal disease mouse model.

[0112] FIG. 8 shows a result of measuring the effect of DCQA on the change in expression of TGF-β1, fibronectin and collagen IV in the kidney tissue of a diabetic renal disease mouse model.

[0113] FIG. 9 shows a result of observing the histopathological change in the kidney tissue of a diabetic renal disease mouse model by hematoxylin and eosin (H&E) staining.

[0114] FIG. 10 shows a result of measuring the blood glucose (FIG. 10A), kidney-to-body weight ratio (FIG. 10B) and creatinine clearance rate (FIG. 10C) in normal mouse and a diabetic renal disease model of ADPRC hetero (ADPRC (+/−)) mouse.

[0115] FIG. 11 shows a result of measuring the effect of DCQA on the blood pressure (FIG. 11A) and creatinine clearance rate (FIG. 11B) in normal mouse and a hypertension mouse model.

BEST MODE

[0116] Hereinafter, the present disclosure is described in more detail through examples. These examples are only for illustrating the present disclosure more specifically and it will be obvious to those having ordinary knowledge in the art that the scope of the present disclosure is not limited by the examples.

Examples

Example 1. NAD Glycohydrolase (NADase) Activity of New ADPRC

[0117] In order to investigate the ADP-ribosyl cyclase (ADPRC) activity of SEQ ID NO 1, a FLAG-ADPRC plasmid was prepared by ligating a cDNA sequence encoding ADPRC into a FLAG-CMV-2 vector and the overexpression of new ADPRC was induced by treating HEK293 cells or MES13 cells using a transfection reagent. The overexpressed new ADPRC was lysed with a lysis buffer. 45 μL of the lysed sample was treated with 5 μL of 2 mM ε-NAD (nicotinamide 1,N6-ethenoadenine dinucleotide) and incubated at 37° C. for 1 hour. Then, the enzyme-substrate reaction was stopped by treating with 50 μL of 10% trichloroacetic acid. After centrifuging for 10 minutes and adding 80 μL of the supernatant to 720 μL of 0.1 M sodium phosphate buffer, absorbance was measured at 297 nm (excitation) and 410 nm (emission) with a fluorescence spectrometer. The result is shown in FIG. 1.

Example 2. Evaluation of cADPR Synthesis Capability of New ADPRC

[0118] The cADPR synthesis capability of ADPRC was evaluated by the method reported by Graeff R et al. [Graeff R, Lee H C. Biochem. J. 361: 379-384, 2002].

[0119] Specifically, the FLAG-ADPRC plasmid was overexpressed in HEK293 cells using a transfection reagent and then lysed with a lysis buffer. The overexpressed FLAG-ADPRC was purified from the lysed sample using a FLAG-agarose column. The purified sample was treated with 100 μM β-NAD and incubated at 37° C. for 1 hour. Then, after extracting cADPR by treating with trichloroacetic acid to a final concentration of 0.6 M, 0.1 mL of the extract or 0.1 mL of a standard cADPR solution was reacted at room temperature for 30 minutes after adding 50 μL of a mixture solution of ADPR cyclase (0.3 μg/mL), nicotinamide (30 mM) and sodium phosphate (100 mM).

[0120] After adding ethanol (2%), alcohol dehydrogenase (100 μg/mL), resazurin (20 μM), diaphorase (10 μg/mL), FMN (10 μM), nicotinamide (10 mM), bovine serum albumin (BSA, 0.1 mg/mL) and sodium phosphate (100 mM) to the mixture solution, reaction was conducted for 2-4 hours. Then, absorbance was measured between 544 nm and 590 nm using a fluorescence spectrophotometer. The result is shown in FIG. 2.

Example 3. Change in Intracellular cADPR Concentration in MES13 Cells by ADPRC

[0121] The change in intracellular cADPR concentration by ADPRC was investigated by the method reported by Graeff R et al. [Graeff R, Lee H C. Biochem. J. 361: 379-384, 2002].

[0122] Specifically, the expression of the new ADPRC in MES13 cells was inhibited by using a small interfering RNA (SEQ ID NO 22) as a transfection reagent. After treating the ADPRC expression-inhibited cells with 150 nM angiotensin II for 60 seconds and then extracting cADPR with 0.6 M trichloroacetic acid, 0.1 mL of the extract or 0.1 mL of a standard cADPR solution was reacted at room temperature for 30 minutes after adding 50 μL of a mixture solution of ADPR cyclase (0.3 μg/mL), nicotinamide (30 mM) and sodium phosphate (100 mM).

[0123] After adding ethanol (2%), alcohol dehydrogenase (100 μg/mL), resazurin (20 μM), diaphorase (10 μg/mL), FMN (10 μM), nicotinamide (10 mM), bovine serum albumin (BSA, 0.1 mg/mL) and sodium phosphate (100 mM) to the mixture solution, reaction was conducted for 2-4 hours. Then, absorbance was measured between 544 nm and 590 nm using a fluorescence spectrophotometer. The result is shown in FIG. 3.

Example 4. Effect of New Inhibitor Inhibiting NAD Glycohydrolase (NADase) Activity of New ADPRC

[0124] In order to find an inhibitor which inhibits the activation of new ADP-ribosyl cyclase (ADPRC) of SEQ ID NO 1, 5 μL of each of 4,4′-dihydroxyazobenzene (4-DHAB, TCI (Japan)), 2,2′-dihydroxyazobenzene (2-DAB, Sigma-Aldrich (USA)), 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one (Quercetin, Sigma-Aldrich (USA)) and San4825 (synthesized by Kannt A et al. [Kannt A, Sicka K, Kroll K, Kadereit D, Gogelein H. Naunyn. Schmiedebergs. Arch. Pharmacol. 385: 717-727, 2012], China) and 2-(1,3-benzoxazol-2-ylamino)-1-methylquinazoline-4(1H)-one (2-BMQ) and 1,4-dicaffeoylquinic acid (1,4-DCQA, Biopurify (China), hereinafter DCQA), which were newly found through screening, was reacted with 40 μL of new ADPRC in iced water for 15 minutes. Then, reaction was conducted at 37° C. for 1 hour after treating with 5 μL of 2 mM ε-NAD (nicotinamide 1,N6-ethenoadenine dinucleotide). Then, the enzyme-substrate reaction was stopped by treating with 50 μL of 10% trichloroacetic acid. After centrifuging for 10 minutes and adding 80 μL of the supernatant to 720 μL of 0.1 M sodium phosphate buffer, absorbance was measured at 297 nm (excitation) and 410 nm (emission) using a fluorescence spectrometer. The result is shown in FIG. 5. As shown in FIG. 5, the NAD glycohydrolase (NADase) activity was inhibited by 42.40% for 4-DHAB, 36.49% for 2-BMQ and even 79.64% for DCQA as compared to the control group.

Example 5. Effect of DCQA on Blood Glucose, Kidney-to-Body Weight Ratio, Creatinine Clearance Rate and Urine Albumin Level in Renal Disease Mouse Model

[0125] The blood glucose, kidney-to-body weight ratio, creatinine clearance rate and urine albumin level in a renal disease mouse model were measured by the method reported by Kim S Y et al. [Kim S Y, Park K H, Gul R, Jang K Y, Kim U H. Am. J. Physiol. Renal Physiol. 296: F291-F297, 2009]

[0126] Specifically, 0.2 mL of 50 mM citrate buffer (pH 4.8) (control group and DCQA group) or 200 μL of 5 mg/mL streptozotocin (STZ, Sigma-Aldrich (USA)) dissolved in 50 mM citrate buffer (STZ group and STZ+DCQA group) was intraperitoneally injected to C57BL/6J mice. Blood glucose was measured from day 2 after the STZ injection and the mice whose blood glucose level was 300 mg/dL were divided into an STZ group and an STZ+DCQA group. To the mice that showed increased blood glucose level, DCQA which showed the highest inhibitory effect against novel ADPRC in Example 5 was intraperitoneally administered every day (100 μL) for 6 weeks after dissolving in dimethyl sulfoxide to 9 mg/mL and diluting with saline to 9 μg/mL. On the last day, the mouse was put in a metabolic cage and urine was collected for 24 hours.

[0127] Then, the body weight and blood glucose of the mouse were measured (FIG. 6A). In addition, in order to investigate the possibility as a therapeutic agent for a renal disease, the mouse was sacrificed and serum and kidney were taken. After measuring kidney-to-body weight ratio (FIG. 6B), some of the kidney was fixed in 10% formalin for fluorescent staining and H&E staining, and the remainder was subjected to ADPR cyclase activity and cADPR concentration measurement. Creatinine clearance rate was calculated by measuring the creatinine levels in urine and serum using a creatinine assay kit (Bioassay Systems, USA) (FIG. 6C), and urine albumin level was measured using an albumin assay kit (Bioassay Systems, USA) (FIG. 6D). The result is shown in FIGS. 6A to 6D.

[0128] As can be seen from FIGS. 6A to 6D, although the STZ+DCQA group did not show decreased in blood glucose as compared to the control group (STZ) (FIG. 6A), the possibility as a candidate for a therapeutic agent for a renal disease such as chronic renal failure or diabetic nephropathy was confirmed from the significant decrease in kidney to body weight (FIG. 6B), significant increase in creatinine clearance rate (FIG. 6C) and significant decrease in the urine albumin level (FIG. 6D).

Example 6. Effect of DCQA on ADPRC Activity and cADPR Concentration in Kidney Tissue of Renal Disease Mouse Model

[0129] The ADPRC activity and cADPR concentration in the kidney tissue of a renal disease mouse model were measured by the method reported by Kim S Y et al. [Kim S Y, Park K H, Gul R, Jang K Y, Kim U H. Am. J. Physiol. Renal Physiol. 296: F291-F297, 2009]

[0130] Specifically, 0.2 mL of 50 mM citrate buffer (pH 4.8) (control group and DCQA group) or 200 μL of 5 mg/mL streptozotocin (STZ, Sigma-Aldrich (USA)) dissolved in 50 mM citrate buffer (STZ group and STZ+DCQA group) was intraperitoneally injected to C57BL/6J mice. Blood glucose was measured from day 2 after the STZ injection and the mice whose blood glucose level was 300 mg/dL were divided into an STZ group and an STZ+DCQA group. To the mice that showed increased blood glucose level, DCQA which showed the highest inhibitory effect against novel ADPRC in Example 5 was intraperitoneally administered every day (100 μL) for 6 weeks after dissolving in dimethyl sulfoxide to 9 mg/mL and diluting with saline to 9 μg/mL. In order to investigate the possibility as a therapeutic agent for a renal disease, the mouse was sacrificed and serum and kidney were taken.

[0131] After lysing some of the kidney tissue with a lysis buffer, 45 μL of the lysed sample was treated with 5 μL of 2 mM NGD (nicotinamide guanine dinucleotide) and reacted at 37° C. for 1 hour. Then, the enzyme-substrate reaction was stopped by treating with 50 μL of 10% trichloroacetic acid. After centrifuging for 10 minutes and adding 80 μL of the supernatant to 720 μL of a 0.1 M sodium phosphate buffer, absorbance was measured at 297 nm (excitation) and 410 nm (emission) with a fluorescence spectrometer (Hitachi, Japan).

[0132] In addition, after extracting cADPR by treating some of the kidney tissue taken from each group with 0.2 mL of 0.6 M trichloroacetic acid, 0.1 mL of the extract or 0.1 mL of a standard cADPR solution was reacted at room temperature for 30 minutes after adding 50 μL of a mixture solution of ADPR cyclase (0.3 μg/mL), nicotinamide (30 mM) and sodium phosphate (100 mM). The mixture solution was reacted for 2-4 hours after adding ethanol (2%), alcohol dehydrogenase (100 μg/mL), resazurin (20 μM), diaphorase (10 μg/mL), FMN (10 μM), nicotinamide (10 mM), bovine serum albumin (BSA, 0.1 mg/mL) and sodium phosphate (100 mM). Then, absorbance was measured between 544 nm and 590 nm using a fluorescence spectrophotometer. The result is shown in FIG. 7.

[0133] As shown in FIG. 7A, the DCQA of the present disclosure decreased the ADP-ribosyl cyclase activity increased by STZ to the level of the control group. And, as shown in FIG. 7B, the DCQA of the present disclosure decreased the cADPR concentration increased by STZ to the level of the control group. Therefore, it was confirmed that DCQA has the ability to decrease ADPRC activity and cADPR concentration, which are increased due to failure or loss of kidney function associated with a renal disease.

Example 7. Effect of 1,4-DCQA on Change in Expression of TGF-β1, Fibronectin and Collagen IV in Kidney Tissue of Renal Disease Mouse Model

[0134] The change in the expression of TGF-β1, fibronectin and collagen IV in the kidney tissue of a renal disease mouse model was measured by the method reported by Kim S Y et al. [Kim S Y, Park K H, Gul R, Jang K Y, Kim U H. Am. J. Physiol. Renal Physiol. 296: F291-F297, 2009].

[0135] Specifically, 0.2 mL of 50 mM citrate buffer (pH 4.8) (control group and DCQA group) or 200 μL of 5 mg/mL streptozotocin (STZ, Sigma-Aldrich (USA)) dissolved in 50 mM citrate buffer (STZ group and STZ+DCQA group) was intraperitoneally injected to C57BL/6J mice. Blood glucose was measured from day 2 after the STZ injection and the mice whose blood glucose level was 300 mg/dL were divided into an STZ group and an STZ+DCQA group. To the mice that showed increased blood glucose level, DCQA which showed the highest inhibitory effect against novel ADPRC in Example 5 was intraperitoneally administered every day (100 μL) for 6 weeks after dissolving in dimethyl sulfoxide to 9 mg/mL and diluting with saline to 9 μg/mL. In order to investigate the possibility as a therapeutic agent for a renal disease, the mouse was sacrificed and serum and kidney were taken. Some of the extracted kidney was fixed in 10% formalin. The kidney tissue fixed in 10% formalin was cut into kidney tissue sections on a slide glass using a cryostat microtome. The kidney tissue sections were washed with a TTBS (Tris-buffered saline (TBS) with 0.1% Tween 20) buffer and then incubated with a TTBS buffer containing 1% bovine serum albumin (BSA) for 1 hour. The tissues were reacted with primary antibodies (TGF-β1 (Santa Cruz, USA), fibronectin (Santa Cruz, USA) and collagen IV (Abcam, UK)) diluted in a TTBS buffer containing 1% bovine serum albumin (BSA) to 1:200 at 4° C. for 12 hours or longer. After washing the tissues that reacted with the primary antibodies 3 times with a TTBS buffer, they were reacted with FITC-labeled secondary antibodies diluted in a TTBS buffer to 1:200 in the dark at room temperature for 1 hour. Then, after washing 3 times with a TTBS buffer, a cover glass was attached using a mounting solution. The stained kidney tissue was observed using a fluorescence microscope (Carl Zeiss, Germany) for observing green fluorescence. The result is shown in FIG. 8.

[0136] As shown in FIG. 8, the DCQA of the present disclosure decreased the expression level of TGF-β1, fibronectin and collagen IV increased by STZ to the level of the control group. Therefore, it was confirmed that DCQA has the ability to decrease the expression level of TGF-β1, fibronectin and collagen IV, which are increased due to failure or loss of kidney function associated with a renal disease.

Example 8. Investigation of Histopathological Change in Kidney Tissue of Renal Disease Mouse Model Through Hematoxylin and Eosin (H&E) Staining

[0137] The histopathological change in the kidney tissue of a renal disease mouse model was measured by the method reported by Shu B et al. [Shu B, Feng Y, Gui Y, Lu Q, Wei W, Xue X, Sun X, He W, Yang J, Dai C. Cell. Signal. 42:249-258, 2018]

[0138] Specifically, 0.2 mL of 50 mM citrate buffer (pH 4.8) (control group and DCQA group) or 200 μL of 5 mg/mL streptozotocin (STZ, Sigma-Aldrich (USA)) dissolved in 50 mM citrate buffer (STZ group and STZ+DCQA group) was intraperitoneally injected to C57BL/6J mice. Blood glucose was measured from day 2 after the STZ injection and the mice whose blood glucose level was 300 mg/dL were divided into an STZ group and an STZ+DCQA group. To the mice that showed increased blood glucose level, DCQA which showed the highest inhibitory effect against novel ADPRC in Example 5 was intraperitoneally administered every day (100 μL) for 6 weeks after dissolving in dimethyl sulfoxide to 9 mg/mL and diluting with saline to 9 μg/mL. In order to investigate the possibility as a therapeutic agent for a renal disease, the mouse was sacrificed and serum and kidney were taken. Some of the extracted kidney was fixed in 10% formalin. The kidney tissue fixed in 10% formalin was cut into kidney tissue sections on a slide glass using a cryostat microtome. The kidney tissue sections were washed with running water for 5 minutes and then stained with hematoxylin for 5 minutes. After the staining, the tissue was washed with running water for 5 minutes. Then, the tissue was immersed in 157 mM hydrochloric acid 2 times and taken out quickly. Then, the tissue was immersed once in 0.25% ammonia water and taken out quickly. After washing again with running water for 5 minutes and staining with eosin for about 30 seconds, the tissue was reacted with 70% ethanol for 30 seconds, with 80% ethanol for 30 seconds, with 90% ethanol for 30 seconds, with 100% ethanol for 30 seconds, again with 100% ethanol for 30 seconds, and then again with 100% ethanol for 30 seconds. Finally, after reacting with xylene for 5 minutes and again with xylene for 5 minutes or longer, a cover glass was attached using a mounting solution. The stained kidney tissue was observed with an optical microscope (Lieca, Germany). The result is shown in FIG. 9.

[0139] As shown in FIG. 9, the DCQA of the present disclosure recovered the formation of glomerulus hypertrophy, infiltration of inflammatory cells and formation of transitional epithelial cells increased by STZ to a level similar to that of the control group. Therefore, it was confirmed that DCQA has the ability to recover the histopathological changes of the kidney caused by failure or loss of kidney function associated with a renal disease.

Example 9. Comparison of Blood Glucose, Kidney-to-Body Weight Ratio and Creatinine Clearance Rate Between Normal Mouse and ADPRC Hetero (ADPRC (+/−)) Mouse Renal Disease Model

[0140] The blood glucose, kidney-to-body weight ratio and creatinine clearance rate of a renal disease mouse model were measured by the method reported by Kim S Y et al. [Kim S Y, Park K H, Gul R, Jang K Y, Kim U H. Am. J. Physiol. Renal Physiol. 296: F291-F297, 2009]

[0141] Specifically, 200 μL of streptozotocin (STZ) dissolved in a 50 mM citrate buffer (pH 4.8) to 5 mg/mL was intraperitoneally injected to 12951/SvImJ mouse (wild type, WT) and ADPRC hetero knockout (ADPRC(+/−)) mouse acquired from The Jackson Laboratory (USA). Blood glucose was measured from day 2 after the injection and the mice whose blood glucose level was 300 mg/dL were used. 6 weeks later, the mouse was put in a metabolic cage and urine was collected for 24 hours.

[0142] Then, the body weight and blood glucose of the mouse were measured (FIG. 10A). The mouse was sacrificed and serum and kidney were taken. Then, kidney-to-body weight ratio was measured using the kidney (FIG. 10B). Creatinine clearance rate was calculated by measuring the creatinine levels in urine and serum using a creatinine assay kit (Bioassay Systems, USA) (FIG. 10C). The result is shown in FIGS. 10A to 10C.

[0143] As shown in FIGS. 10A to 10C, although blood glucose was not decreased in the (ADPRC(+/−) group as compared to the control group (FIG. 10A), significant decrease in kidney to body weight was observed in the ADPRC(+/−)+STZ group of the present disclosure as compared to the WT+STZ group (FIG. 10B). In addition, the decrease in creatinine clearance rate caused by STZ was not observed in the ADPRC(+/−)+STZ group (FIG. 10C), which confirms the importance of the new ADPRC in a renal disease such as chronic renal failure or diabetic nephropathy.

Example 10. Effect of DCQA on Blood Pressure and Creatinine Clearance Rate in Normal Mouse and Hypertension Mouse Model

[0144] The blood pressure and creatinine clearance rate in a hypertension mouse model were measured by the method reported by Allagnat et al. [Allagnat F, Haefliger J A, Lambelet M, Longchamp A, Berard X, Mazzolai L, Corpataux J M, Deglise S. Eur. J. Vasc. Endovasc. Surg. 51: 733-742, 2016] and Kim S Y et al. [Kim S Y, Park K H, Gul R, Jang K Y, Kim U H. Am. J. Physiol. Renal Physiol. 296: F291-F297, 2009]

[0145] Specifically, 0.2 mL of 8 mg of L-NAME (Nw-nitro-L-arginine-methyl-ester, Sigma-Aldrich, USA) dissolved in 1 mL of saline was orally administered to C57BL/6J mouse every day. On days 7 and 14 of the oral administration, blood pressure was measured at the tail of the mouse. Then, DCQA which showed the highest inhibitory effect against novel ADPRC in Example 5 was intraperitoneally administered every day (100 μL) for 7 days after dissolving in dimethyl sulfoxide to 9 mg/mL and diluting with saline to 9 μg/mL and L-NAME was administered orally. On day 6 after the DCQA treatment, the mouse was put in a metabolic cage and urine was collected for 24 hours.

[0146] After measuring the blood pressure of the mouse (FIG. 11A), the mouse was sacrificed and serum was obtained.

[0147] Creatinine clearance rate was calculated by measuring the creatinine levels in urine and serum using a creatinine assay kit (Bioassay Systems, USA) (FIG. 11B). The result is shown in FIGS. 11A and 11B.

[0148] As shown in FIG. 11A, DCQA had no effect of lowering blood pressure in the hypertension model. However, the possibility of DCQA as a therapeutic agent for a renal disease such as hypertensive nephropathy was confirmed through the increase in creatinine clearance rate by DCQA (FIG. 11B).

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[0162] Although the specific exemplary embodiments of the present disclosure have been described in detail, it will be obvious to those having ordinary knowledge in the art that they are merely preferred exemplary embodiments and the scope of the present disclosure is not limited by them. It is to be understood that the substantial scope of the present disclosure is defined by the appended claims and their equivalents.