Kidney function evaluation and safety evaluation test using a rat model of adenine-induced kidney damage using UMO concentrated solution
- KIWITA
- Jan 18
- 9 min read
Test Report
Kidney function evaluation and safety evaluation test using a rat model of adenine-induced kidney damage using UMO concentrated solution
(Test number: 21775)
APA Corporation
Testing contractor : EP Mediate Co., Ltd.
April 30, 2021
Terminology used in this research report
Mean | Average Value |
S.E.M（standard error of the mean） | Standard Error |
P.O.（per os） | Oral route |
Test Overview
1.1 Test title
Renal function and safety evaluation test of UMO concentrated solution using a rat model of adenine-induced nephropathy
1.2 Test number
21775
1.3 Test objective
A comparative study was conducted on the effects of UMOUmo concentrated solution on adenine-induced nephropathy by oral administration for 28 days. A safety evaluation of the test substance was also conducted.
1.4 Testing contractor
APA Corporation
4-7 Shinmei-cho, Anjo City, Aichi Prefecture, 446-0019
1.5 Testing contractor
EP Mediate Co., Ltd.
9F Tsuruya Building, 2-23 Shimomiyabi-cho, Shinjuku-ku, Tokyo, 162-0822
Testing contractor: Kunihiko Wasaki, Yuki Shiraishi
(TEL 03-5657-4983, FAX 03-5657-4984)
1.6 Testing facility
ITEC Lab Co., Ltd.
52-1 Fukue, Kaizu-cho, Kaizu City, Gifu Prefecture, 503-0628
(TEL 0584-47-2712, FAX 0584-48-0022)
1.7 Testing schedule
Testing start date : March 1, 2021
Animal arrival date : March 1, 2021
Quarantine and acclimatization end date : March 8, 2021
Operation start date : March 8, 2021
Operation end date : April 5, 2021
Final report (draft) creation date : April 22, 2021
Final report creation date (test end date) : April 30, 2021
1.8 Test director and test staff
<Test director>
Masaki Matsuura
<Responsible for animal rearing>
Masaki Matsuura, Makiko Idota, Kazuhiro Kawai
<Quarantine manager>
Fusako Mitsunaga
<Responsible for test substance preparation and administration>
Masaki Matsuura, Makiko Idota, Kazuhiro Kawai
<Responsible for general condition observation>
Masaki Matsuura, Makiko Idota, Kazuhiro Kawai
<Responsible for weight measurement>
Makiko Idota
<Responsible for urine and blood collection>
Masaki Matsuura, Makiko Idota
<Responsible for urine and blood testing>
Haruki Tanaka (DIMS Inc.) Institute of Medical Science)
<Data Processing>
Masaki Matsuura, Makiko Idota
1.9 Animal Ethics
Animal experiments will be conducted in accordance with the "Guidelines for Proper Conduct of Animal Experiments" (June 1, 2006, Science Council of Japan), the "Law Concerning Welfare and Management of Animals" (Law No. 105 of 1973, amended August 30, 2011: Law No. 105), and the "Standards for the Care and Storage of Experimental Animals and the Relief of Pain" (Ministry of the Environment Notification No. 88, April 28, 2006), based on the "Guidelines for Animal Experiments at ITEC Lab Co., Ltd." (December 1, 2011). This study was reviewed and approved by the ITEC Lab Co., Ltd. Animal Experiment Ethics Committee based on the above guidelines.
1.10 Biosafety
Animal experiments are conducted in accordance with the "Laboratory Biosafety Manual" by the World Health Organization (WHO) and the "Guidelines for Biosafety at ITEC Lab Co., Ltd." (December 1, 2011), which are based on the "Law Concerning the Conservation of Biological Diversity through Restrictions on the Use of Genetically Modified Organisms, etc. (Cartagena Law)." This study was reviewed and approved by the ITEC Lab Co., Ltd. Biosafety Committee in accordance with the above guidelines.
2 Test substances, media and reagents
2.1 Test substances
① Name: Concentrated Umo solution (silicon content 8000-9300ppm)
② Provided by: APA Corporation
③ Amount obtained: 450mL x 1 bottle
④ Date of acquisition: February 25, 2021
⑤ Properties or dosage form: Colorless, transparent liquid
⑥ Storage conditions: Store at room temperature
⑦ Storage location: Room temperature storage cabinet for test substances
⑧ Disposal of residual substances after use: Dispose of residual test substances.
2.2 Media
① Name: Water for injection
② Lot number: 8A93N
③ Manufacturer: Otsuka Pharmaceutical Factory, Inc.
④ Storage conditions: Room temperature (store in refrigerator after opening)
⑤ Storage location: Room temperature storage cabinet for test substances or refrigerator for storing test substances
2.3 Reagent I
① Name: Adenine sulfate
② Lot number: SKF5159
③ Manufacturer: Fujifilm Wako Pure Chemical Industries, Ltd.
④ Storage conditions: Store at room temperature
⑤ Storage location: Room temperature storage cabinet for test substances
2.4 Reagent II
① Name: 0.5w/v% methylcellulose 400 solution, sterilized (hereinafter MC)
② Lot number: KCF7015
③ Manufacturer: Fujifilm Wako Pure Chemical Industries, Ltd.
④ Storage conditions: Refrigerated storage
⑤ Storage location: Refrigerator for storing test substances
3 Test system
3.1 Animal species, lineage and sex
Rat, Slc:Wistar, SPF, male
3.2 Supply source
Japan SLC Co., Ltd. (Hamamatsu, Shizuoka Prefecture)
3.3 Age and number of animals received
① Age at arrival: 6 weeks old
② Number of animals received: 13 animals
③ Age at administration of test substance: 7 weeks old
3.4 Quarantine and acclimation method
· After arrival, a quarantine and acclimation period of 8 days was set.
· During the quarantine and acclimation period, the animals were observed for general condition once a day, and their weight was measured the day after arrival and on the day quarantine and acclimation was completed.
· Healthy animals that showed good growth in general condition and weight performance during the quarantine and acclimation period were used for the test.
3.5 Grouping method
Based on the weight on the day acclimation was completed, the animals were divided into 4 groups by a computer-assisted complete random sampling method so that the average weight of each group was equal.
3.6 Identification of the test system
① Animals were identified with oil-based ink.
② During the "acclimation period," cages were identified by cage cards that listed the test number, arrival date, age at arrival, animal species, sex, cage number, and quarantine number.
3.6.1 After grouping
Based on the weight on the final day of acclimation, animals were divided into four groups using a computer-based complete random sampling method so that the average weight of each group was equal.
3.7 Method of euthanasia
After the end of the test, animals were euthanized by exsanguination under anesthesia.
4 Animal Management
4.1 Breeding Room
Area VI, Room IV (Clean Breeding Room)
4.2 Breeding Racks and Cages
① Cage: Stainless steel hanger cage (32.0D x 20.5W x 19.5H cm)
② Number of animals per cage: 3 animals/cage, 1 animal/metabolic cage (at urine collection)
③ Absorbent paper (Tech Elite, EP Trading Co., Ltd.) was used as the dropping tray.
④ The dropping tray was replaced at least once a week.
4.3 Breeding Environment Conditions
① Temperature: 18-28°C
② Relative Humidity: 30-80%
③ Lighting Time: 12 hours (7:00-19:00)
4.4 Feed and Drinking Water
4.4.1 Feed
· Commercially available solid feed [MF; Oriental Yeast Co., Ltd. (Itabashi-ku, Tokyo)] was given ad libitum.
4.4.2 Drinking water
· Drinking water was tap water (Kaizu-cho, Kaizu-shi, Gifu Prefecture) provided ad libitum via water bottle.
5. Group composition and administration method
5.1. Group composition, test substance, administration dose, administration solution volume, and number of subjects
5.2 Method of administration of vehicle, test substance, and adenine administration solution
① Number of administrations and administration period: The vehicle and test substance were administered repeatedly once a day for 28 days.
Adenine was administered 30 minutes after administration of the vehicle and test substance. Administration was carried out between 8:00 and 12:00.
② Administration method: Oral administration was performed using a disposable syringe and a disposable oral probe.
③ Reason for selecting the administration method: This method ensures accurate administration of the correct amount.
④ Calculation of the amount of administration solution: Calculated for each individual based on the body weight on the most recent measurement date.
6 Preparation and storage of test substance and reagents
6.1 Test substance
The concentrated Umo solution administration solution was already prepared, so the original solution was used.
6.2 Adenine administration solution
① The adenine administration solution was prepared by weighing 600 mg of adenine sulfate powder using an electronic balance, suspending it in 0.5% MC solution, and measuring the volume in a graduated cylinder to make the total volume 6 mL.
② The solution was prepared twice a week, divided into small portions, and stored in a refrigerator.
6.3 Disposal of remaining test substance
The prepared solution was discarded after use.
7. Procedures
7.1. Observation of general condition and survival
During the test period, all animals were observed once a day for general condition and survival.
7.2. Body weight
Weight was measured on the day after arrival, on the day of the end of acclimation, and once a week after administration of the test substance.
7.3. Urine collection
① Urine was collected individually in metabolic cages overnight on the day before administration of the test substance, on the 14th day after administration of the test substance, and on the last day of administration of the test substance.
② After measuring the urine volume, the collected urine was centrifuged at 1700×g, 4℃, 15 min to collect the supernatant, which was then frozen and stored (500μL was used for blood biochemistry measurements, and the remainder was stored for additional analysis).
7.4. Blood and plasma collection
① Before administration of the test substance and on the 15th day after administration of the test substance, blood was collected from the jugular vein under no anesthesia using a syringe that had been passed through with heparin injection as an anticoagulant. The collected blood was centrifuged at 1700×g, 4℃, 15 min to collect plasma, which was then frozen and stored (for further analysis).
② On the day after the administration of the test substance, the animals were anesthetized by intramuscular administration of 0.8 mL/kg of ketamine (Ketalar® for intramuscular injection 500 mg, Sankyo Co., Ltd.) and 0.8 mL/kg of xylazine (Seractal® 2% injection, Bayer Co., Ltd.), and euthanized by exsanguination from the inferior vena cava.
③ Approximately 2 mL of the collected blood was collected in a blood collection tube containing EDTA-2K (for blood count test).
④ The remaining blood was collected in a blood collection tube containing sodium heparin, centrifuged at 1700×g, 4℃, 15 min to collect plasma, which was then frozen and stored (500 μL was used for blood biochemistry measurements, and the remainder was stored for further analysis).
7.5 Urine measurements
The urine samples stored in a freezer were measured by an outsourced service (DIMS Medical Science Research Institute, Inc.).
The measurement items and methods are as follows. The measurement equipment used was a Hitachi Automated Analyser 3500 (Hitachi, Ltd.).
7.6 Blood biochemistry tests (A1 and A2 groups)
① Frozen plasma was measured by an outside contractor (DIMS Medical Science Research Institute, Inc.).
② The measurement items and methods were as follows. The measurement equipment used was a Hitachi Automated Analyser 3500 (Hitachi, Ltd.).
7.7 Blood count test (A1 and A2 groups)
The collected blood was measured by an outsourced company (DIMS Medical Research Institute, Inc.).
The measurement items and methods are as follows. The measurement equipment used was a multi-parameter automated blood cell analyzer (XT-2000i, Sysmex Corporation).
Test Results
1) Body Weight
The changes in body weight of rats administered with UMO concentrate solution for 28 days are shown in Fig. 1, Table 1, and Appendix Table 1. Both the vehicle-only group and the UMO concentrate solution-only group exhibited steady weight gain throughout the observation period. However, in the adenine-administered vehicle and UMO concentrate groups, weight gain was not observed after the second week.
2) Urine Analysis (Urine Volume, Creatinine, and Inorganic Phosphate)
The urine analysis results of rats administered with UMO concentrate solution for 28 days are shown in Fig. 2, Table 2, and Appendix Table 2. Urine volume in the vehicle-only and UMO concentrate-only groups remained consistent at 2 and 4 weeks. In contrast, in the adenine-administered vehicle and UMO concentrate groups, urine volume increased approximately 5-fold in the vehicle group and 4-fold in the UMO concentrate group compared to pre-administration levels at week 2.
Urinary creatinine levels remained stable in the vehicle-only and UMO concentrate-only groups at 2 and 4 weeks. However, in the adenine-administered vehicle and UMO concentrate groups, creatinine levels decreased approximately 6-fold and 4-fold, respectively, compared to pre-administration levels at week 2.
Urinary inorganic phosphate levels showed large individual differences, particularly in the UMO concentrate-only group and the adenine + UMO concentrate group, where pre-administration values appeared relatively low. At weeks 2 and 4, clustering was observed between adenine-administered and non-administered groups. Notably, the adenine-administered group showed a significant decrease in urinary inorganic phosphate (uncorrected for creatinine). For the creatinine-corrected phosphate values (IP-corrected values), the adenine-administered group tended to show higher levels than the non-administered group at week 4. At this time, the UMO concentrate group exhibited a mild (23%) suppressive effect on the increase in IP-corrected values induced by adenine compared to the non-administered group.
3) Blood Biochemistry
The blood biochemistry results of rats administered with UMO concentrate solution for 28 days are shown in Fig. 3-1 to 3-4, Table 3-1, 3-2, and Appendix Tables 3-1 and 3-2.There were no significant differences between the vehicle-only group and the UMO concentrate-only group in the measured parameters, including AST, ALT, ALP, T-BIL, BUN, CRE, GLU, TP, ALB, A/G, LAC, IP, CA, MG, NA, K, and CL.
However, in the UMO concentrate group, the liver lipid metabolism marker γ-GTP was 23% lower, triglycerides (TG) were 32% lower, total cholesterol (T-CHO) was 14% lower, and phospholipids (PL) were 13% lower compared to the vehicle group.
4) Hematology
The hematology results of rats administered with UMO concentrate solution for 28 days are shown in Fig. 4, Table 4, and Appendix Table 4.
No significant differences were observed between the vehicle-only group and the UMO concentrate-only group in blood RBC, HCT, HGB, PLT, and WBC counts.
Summary and Discussion
This study evaluated the effect of the test substance, UMO concentrate solution, on adenine-induced kidney injury in rats. UMO concentrate solution was administered orally for 28 consecutive days, and parameters such as urine volume, urinary creatinine, and inorganic phosphate were measured. Additionally, blood biochemistry and hematology tests were conducted as part of a non-clinical safety evaluation.
Throughout the study period, there were no fatalities or abnormalities in general condition in either the vehicle or UMO concentrate groups. Based on blood biochemistry and hematology results, no toxicity was observed, suggesting that the no-observed-adverse-effect level (NOAEL) of UMO concentrate solution exceeds 6 g/kg.
Notably, four lipid metabolism markers were reduced in the UMO concentrate group, suggesting a potential effect of the UMO concentrate solution on enhancing lipid metabolism.
In the adenine-administered groups, an increase in urine volume was observed shortly after adenine administration, and body weight gain ceased in the latter half of the study period. Concurrently, urinary creatinine and inorganic phosphate levels decreased, indicating the induction of adenine-induced kidney injury. A comparison of IP-corrected values suggested a mild suppressive effect of UMO concentrate solution on IP-corrected value increases, implying a potential preventive or ameliorative effect on kidney injury.
However, the small sample size and large individual differences in this study limit the reliability of these findings. Future studies with larger sample sizes are recommended to further confirm and verify the function and effects of the test substance.
Note: This paper is translated from the following URL. The content is provided for reference on the scientific research of the raw material only. Whether APA raw materials are used or not, we hope this research will help increase understanding and awareness of body minerals.
Comments