EVALUATION OF NEPHROPROTECTIVE ACTIVITY OF AYURVEDIC FORMULATIONS IN RAT MODEL OF ADENINE-INDUCED CHRONIC KIDNEY DISEASE

EVALUATION OF NEPHROPROTECTIVE ACTIVITY OF AYURVEDIC FORMULATIONS IN RAT MODEL OF ADENINE-INDUCED CHRONIC KIDNEY DISEASE

1.0 TEST SYSTEM DETAILS:

Species : Rattus norvegicus (Rats)

Strain : Albino Wistar

Age : 7-8 weeks

Sex : Male

No. of animals : 6 /Group

Total animals : 30

2.0 TEST ARTICLES DETAILS

Test Formulation – Herbal Extract

3.0 VEHICLE DETAILS

The test articles will be formulated using 0.5% sodium carboxymethylcellulose (Na-CMC) as the vehicle.

4.0 ALLOCATION OF GROUPS:

Groups	Treatment	Dose*	Chronic Kidney Disease Study
G1	Normal Control	Normal Diet, 0.5% Na-CMC, p.o., b.i.d.	6
G2	Disease Control	Adenine-2.5 mg/kg Feed	6
G3	Herbal Extract – LD	Adenine-2.5 mg/kg Feed, Treatment with X/3 mg/kg in 0.5% Na-CMC, p.o., b.i.d.	6
G4	Herbal Extract –MD	Adenine-2.5 mg/kg Feed, Treatment with X mg/kg in 0.5% Na-CMC, p.o., b.i.d.	6
G5	Herbal Ext ract –HD	Adenine-2.5 mg/kg Feed, Treatment with 3X mg/kg in 0.5% Na-CMC, p.o., b.i.d.	6

Abbreviations: Na-CMC: Sodium carboxymethylcellulose, p.o.-per os. q.d.: quaque die; bid: bis in die.

5.0 METHOD:

· Healthy animals will be selected for the study, randomized based on body weight, and will be assigned to 5 groups consisting of 6 animals each.

· Animals of the Group G1 will be designated as normal-control and administered 0.5% Na-CMC, p.o., b.i.d. and provided with the normal chow diet

· Disease control animals (assigned to group G2) will receive 0.5% Na-CMC, p.o., b.i.d. and and provided with the normal chow diet mixed with 0.25% adenine.

· Animals of group G3-G5 will be treated with Herbal extract their fix dose level of p.o., b.i.d. as mentioned in above table. And provided with the normal chow diet mixed with 0.25% adenine.

· Normal control group (G1) will be provided with normal standard diet, whereas animals allocated to groups G2 – G5 will be administered normal diet mixed with 0.25% adenine from day 1 to day 30.

· All animals will be individually housed in metabolic cages for a 24- hour urine collection from W0-W4. Every week, urine sample will be collected for the estimation of relevant biochemical parameters.

· Subsequently, blood samples will be collected from the retro- orbital plexus under transient isoflurane anesthesia using aseptic techniques. The collection blood will be allowed to clot, and serum will be separated by centrifugation for the assessment of biochemical parameters.

· Subsequently, on the 29^th day after 24 hours of last of test sample administration, animals will be sacrificed under an overdose of thiopentone anaesthesia. Immediately after the animals dies, the kidney will be weighed and the left kidney will be fixed in 10% neutral buffered formalin for histopathological whereas the right kidney will be stored at- 20C for the ensuring biochemical evaluations (Balkrishna et al. 2026; Jin et al. 2009; Kim et al. 2025; Li et al. 2021; Su et al. 2023; Vinuela- Berni et al. 2025; Yang et al. 2024).

6.0 PARAMETERS TO BE EVALUATED:

· Body weight (weekly).

· Food and Water intake (weekly).

· Urine output (24 h metabolic) 29th day.

· Relative Kidney weight.

· Serum biochemistry Parameters: Blood Urea Nitrogen, Creatinine.

· Urinary biochemistry Parameters: Creatinine and Albumin.

· Inflammatory markers: TNF-α

· Ex- Vivo biochemistry Parameters: MDA, SOD, CAT, GR, TAC.

· Histological analysis of kidneys (Hematoxylin & Eosin- Stained).

7.0 REFERENCE(S):

7.1 Balkrishna, Acharya, Monali Joshi, Sunil Shukla, Meenu Tomer, Sandeep Sinha, and Anurag Varshney. 2026. “Renogrit Protects against Folic Acid-Induced Kidney Damage in Rat Model of Acute Renal Failure by Regulating Serum Creatinine and Urea Levels.” Fitoterapia 190. doi: 10.1016/j.fitote.2026.107158.

7.2 Jin, Q. R., W. S. Shim, M. K. Choi, G. Y. Tian, I. S. Song, S. G. Yang, D. D. Kim, S. J. Chung, and C. K. Shim. 2009. “Decreased Urinary Secretion of Belotecan in Folic Acid-Induced Acute Renal Failure Rats Due to Down-Regulation of Oat1 and Bcrp.” Xenobiotica 39(10): 711–21. doi:10.1080/00498250903026458.

7.3 Kim, Jong Min, Yiseul Kim, Hyun Jin Na, Haeng Jeon Hur, Sang Hee Lee, and Mi Jeong Sung. “Magnolia Kobus DC. Alleviates Adenine-Induced Chronic Kidney Disease by Regulating Ferroptosis in C57BL/6 Mice.” Frontiers in Pharmacology 16(April): 1–11. doi:10.3389/fphar.2025.1548660.

7.4 Li, Xue, Yu Zou, Yuan Yuan Fu, Jia Xing, Kai Yue Wang, Peng Zhi Wan, Mo Wang, and Xiao Yue Zhai. 2021. “Ibudilast Attenuates Folic Acid–Induced Acute Kidney Injury by Blocking Pyroptosis Through TLR4-Mediated NF-ΚB and MAPK Signaling Pathways.” Frontiers in Pharmacology 12(May): 1–17. doi:10.3389/fphar.2021.650283.

7.5 Su, Lianjiu, Jiahao Zhang, Hernando Gomez, John A. Kellum, and Zhiyong Peng. 2023.“Mitochondria ROS and Mitophagy in Acute Kidney Injury.” Autophagy 19(2): 401–14.doi: 10.1080/15548627.2022.2084862.Viñuela-Berni, Verónica, María Antonieta Carbajo-Mata, Rebeca Corona, and Teresa Morales.

7.6 2025. “Gavage versus Dietary Adenine Administration Is More Effective for the Female Mouse Model of Chronic Kidney Disease.” Scientific Reports 15(1): 1–11. doi:10.1038/s41598-025-06143-1.

7.7 Yang, Qiao, Songya Su, Nan Luo, and Gang Cao. 2024. “Adenine-Induced Animal Model of Chronic Kidney Disease: Current Applications and Future Perspectives.” Renal Failure 46(1). doi:10.1080/0886022X.2024.2336128.

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