EVALUATION OF THE ANTI-HYPERTENSIVE EFFICACY OF TEST FORMULATIONS IN RAT MODEL OF TWO KIDNEY-ONE CLIP (2K1C)-INDUCED RENOVASCULAR HYPERTENSION

 EVALUATION OF THE ANTI-HYPERTENSIVE EFFICACY OF HERBAL FORMULATIONS IN RAT MODEL OF TWO KIDNEY-ONE CLIP (2K1C)-INDUCED RENOVASCULAR HYPERTENSION

1.0  TEST SYSTEM DETAILS:

Species                   : Rattus norvegicus (Rats)

Strain                     : Sprague Dawley or Wistar

Age                        : 6-7 weeks

Sex                        : Male

No. of animals        : 10/Group

Total animals         : 70

 

2.0  TEST ARTICLES DETAILS

Allium sativum, and Cinnamomum zeylanicum-based herbal formulation.

3.0  VEHICLE DETAILS

The test articles will be formulated by utilizing 0.5% methylcellulose as the vehicle.

4.0   ALLOCATION OF GROUPS:

Group No.	Group Description	Disease Induction procedure	Treatment administered	Dose Volume and Route
G1	Sham-Control	Exposure and manipulation of the left kidney and renal artery	0.5% MC, p.o., b.i.d.	5 ml/kg, p.o.
G2	Disease Control	Application of 0.25 mm silver clip on the renal artery of the left kidney	0.5% MC, p.o., b.i.d.
G3	Reference Control		Telmisartan-10 mg/kg, p.o., q.d. (morning) + 0.5% MC (evening)
G4	Treated with low dose		LD: 5-15 mg/kg, b.i.d.
G5	Treated with intermediate dose 1		ID-1: 15-50 mg/kg, b.i.d.
G6	Treated with intermediate dose 2		ID-2: 50-150 mg/kg,   b.i.d.
G7	Treated with high dose		HD: 150-500 mg/kg,  b.i.d.

Abbreviations: MC: Methylcellulose, 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 7 groups consisting of 10 animals each.

·         Animals of Group G1 will be designated as sham-control and administered 0.5% MC, p.o., b.i.d. Treatment will be initiated after establishment of hypertension.

·         Disease control animals (assigned to group G2) will receive 0.5% MC, p.o., b.i.d. Treatment will be initiated after establishment of hypertension.

·         Animals of group G3 will be treated with reference drug Telmisartan at the dose of 10 mg/kg, p.o., q.d.

·         Animals of group G4-G7 will be treated with HF, at different incremental dose levels as outlined in Section 4.0 of Annexure-I, twice daily. Treatment will be initiated after the establishment of hypertension.

·                 Rats will be anesthetized with thiopentone (50 mg/kg) administered intraperitoneally and will be placed on heating pads. The body temperature of the rats will be recorded using a digital rectal thermometer. After confirming that the rat is in the surgical plane of anesthesia, confirmed by the absence of a toe-pinching reflex, an incision will be made on the left flank of the animal, and the left kidney will be exposed.  The renal artery, renal vein, and renal nerve will be carefully isolated by blunt-tipped vascular scissors and forceps. A vascular clip (0.25 mm silver clip) will be applied to the left renal artery and a nylon suture will be applied to the clip and muscular tissue to prevent clip dislodgement. The kidney will be placed back into the abdominal cavity in its original position and the abdominal muscle and skin layers will be closed separately with absorbable catgut sutures. This procedure will be performed on rats allocated to groups G2-G7. Animals assigned to group G1 will have its kidney exposed and subjected to the manipulation of the renal artery alone.

·         Systolic arterial pressures will be measured by a non-invasive blood pressure instrument using the tail-cuff method by placing the animal in a restrainer, at 1 week, 2 weeks, 3 weeks, and 4 weeks post-surgery to determine the development of hypertension.

·         Animals of G2-G7 will be randomized after four weeks on the basis of systolic blood pressure.

·         Compound treatment will be initiated 4 weeks post-surgery and will be continued for 4 weeks.

·         On day 29, post-compound administration rats will be sacrificed under an overdose of thiopentone anesthesia. After suitable anaesthesia but before the animal dies, blood will be collected from the retro-orbital plexus for the estimation of renin and angiotensin levels in the plasma. Immediately after the animal dies, the kidney, heart, and aorta of the animal will be excised, weighed, and examined for gross pathological changes. Subsequently, they will be divided into two halves. One portion will be transferred to 10% neutral buffered formalin for histopathology and the other will be snap-frozen in liquid nitrogen and stored at -80°C for biochemical/molecular investigations.

 

6.0  PARAMETERS TO BE EVALUATED:

·         Body weight: Twice a week

·         Systolic blood pressure: Once a week for eight weeks post-arterial clipping procedure.

·         Serum levels of renin, angiotensin, and angiotensin-converting enzyme activity.

·         Kidney injury markers: KIM-1, NAG, Cystatin C in serum.

·         Heart injury markers: Lactate dehydrogenase, Troponin C in serum

·         Histological analysis of the Heart, Kidney, and Aorta.

 

7.0  REFERENCE(S):

1.  Chaykovsk, L. et al. Effects of telmisartan and linagliptin when used in combination on blood pressure and oxidative stress in rats with 2-kidney-1-clip hypertension. 31, 2290–2299 (2013).

2.  Kaur, S. & Muthuraman, A. Therapeutic evaluation of rutin in two-kidney one-clip model of renovascular hypertension in rat. Life Sci. (2016) doi:10.1016/j.lfs.2016.02.080.

3. Huang LL, Pan C, Yu TT, Guo K, Wang XH, Zhang JY, Wang HZ, Gao S. Benefical therapeutic effect of Chinese Herbal Xinji'erkang formula on hypertension-induced renal injury in the 2-kidney-1-clip hypertensive rats. Afr J Tradit Complement Altern Med. 2014 Aug 23;11(5):16-27. doi: 10.4314/ajtcam.v11i5.3.

                                                                  END OF THE DOCUMENT

You may like to read these links: 

1. List of All SOPs and Documents for the Microbiology Laboratory

2. List of All SOPs and Documents for In-vitro Laboratory

3. List of All SOPs and Documents for the In-vivo Laboratory.

4. List of All SOPs and Documents for Clinical Research

5. List of Instruments and Equipment Used In Laboratory Animal Facility

6. Animal  Facility Design - Small Laboratory Animals (Rat, Mice, Rabbit, Guinea Pig)

7. List of Chemicals Needed for In-vivo Laboratory

8. List of Physiological Data for Small Laboratory Animals 

9. Leading Lab-Diet Suppliers in India and Beyond

10. List of All SOPs and Documents for Animal House Facility