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Patil Aishwarya Mahaveer.
M pharm (Pharmacology)
Roll no.: 06
1
CONTENTS
Definition
Mechanism of action
In-vitro methods
In-vivo methods
2
DIURETICS
Definition :
“Elimination of excess of urine (more than normal levels) is termed as
diuresis and the drugs that facilitate the process are known as Diuretics.”
The normal volume of urine in healthy person is 800 to 2000 ml per day.
Urine consist of metabolic waste materials, water and some electrolytes.
3
CLASSIFICATION
CLASS OF DRUG LOCATION EXAMPLE
Carbonic anhydrase
inhibitors
Proximal tubule Acetazolamide
Loop diuretics Loop of Henle Furosemide/Bumetanide
Thiazide diuretics Distal tubule Hydrochlorothiazide/Bendr
oflumethiazide
Potassium sparring diuretics Collecting duct Spironolactone/triamterene
/Amiloride
Osmotic Diuretics Lumen of nephron Mannitol
4
MECHANISM OF ACTION
5
IN-VITRO AND IN-VIVO METHODS
In-vivo methods :-
I. Diuretic activity in rats (LIPSCHITZ TEST)
II. Saluretic and diuretic activity in dogs
III. Saluretic activity in rats
IV. Stop flow techniques
V. Micro puncture technique in rat
In-vitro methods :-
I. Carbonic anhydrase inhibition in vitro
II. Patch clamp technique in kidney cells
III. Isolated perfused kidney
6
IN VIVO METHODS
(i) Diuretic activity in rats (Lipschitz test)
 Aim: In 1943diuretic activity in rats (Lipschitz test) purpose to determine diuretic activity of test drug by lipschitz
method by comparing water and sodium excretion in test animals.
 Requirements:
Animals : Male Wistar rats (150-200 gm)
Equipments : Metabolic cages - wire mesh at bottom and funnel for
collection of urine Stainless steel sieves, Flame photometer.
Chemicals : Test drug, normal saline, urea
 Procedure:
1. Rats divided into 4 groups of 3, each placed in metabolic cages, provided with wire mesh at bottom and funnel for
collecting urine. Stainless steel sieves placed in funnel retain feces and allow only urine for measurement.
2. The rats fed with standard diet and water ad libitum.
3. 15 hr prior to test stop food and water.
7
4. 3 animals are placed in one metabolic cage.
5. 2 groups (6 rats) used for 1 dose of test drug. Test drug is given by orally to these 6 rats.
6. To other 2 groups (6 rats)
For one group 1gm/kg of urea was administered.
To another group 5 ml of normal saline solution per 100 gm by oral route is given.
 Evaluation:
Urine excretion is recorded after 5 hr upto 24 hr. Sodium content of urine is estimated by flame photometer
depending upon response doses of test compound, adjusted for graded response, determined by ED 50.
 Results for calculation:
Urine volume excreted
x body weight
100
Urine volume excreted/100 gm body weight is worked out expressed as "lipschitz value".
Ratio = T/ U
 T-Response of test compound
 U-Response of urea treatment
If ratio is 1 and more indicates the positive diuretic effect (Calculated for 5 hr upto 24 hr).
Similar quotients can be calculated for sodium excretion. 8
(ii) Saluretic and diuretic activity in dogs
 Aim:
Dogs are most dependable animals for screening diuretic to study renal physiology and action of
diuretics. Because of renal physiology of dog is similar to human than that of rat.
Requirements:
Animals required : Beagle dogs (either sex)
Chemicals required: Urea
Equipments required: Metabolic cages, plastic catheter,
gavage, Osmometer
 Procedure:
1. Dogs are subjected to intensive training for accepting feeding through gavage and hourly
catheterization without any resistance. Then placed in metabolic cages.
2. Minimum 4 dogs used as control group receiving water only, 1 gm/kg urea p.o. or 5 mg/kg
furosemide orally is given to test group.24 hr prior to experiment, food is withdrawn, and water is
withdrawn on morning of that day urinary bladder is emptied with plastic catheter.
3. The dogs receive 20 ml/kg of water by gavage followed by hourly doses of 4 ml/kg of drinking
water. Bladder is catheterized twice in an interval of 1 hr and urine is collected for analysis of
initial values.
9
4. The test drug and standard is applied either orally or i.v. hourly catheterization is repeated for 6hr
without further water dosing, animals placed in metabolic cages over night. After 24 hr of test compound
dogs catheterized once again. Urine collected through catheter measured together.
 Evaluation:
Urine samples are analyzed for sodium, potassium and chloride. Osmolarity is measured through
osmometer. Urine volume also measured.
 Conclusion:
For assessment of activity of test compound urine volume, electrolyte concentration and osmolarity of
each animal recorded and averages for each group are calculated. Values plotted against time to allow
comparison with pretreatment values as well as with water controls and standard drugs.
10
(iii) Stop flow technique:
This is very useful in localization of transport processes along length of nephron. After clamping of ureter
glomerular filtration rate GRF is decreased.
 Aim:
To determine diuretic activity of test drug by using clamp which is inserted to ureter.
 Requirements:
Animals required : Male Wistar rats (150-200 gm)
Equipments required : Metabolic cages - wire mesh at bottom and funnel for collection of urine
Chemicals required : Insulin
 Procedure:
1. The ureter of animal undergoes intense osmatic diuresis, it is damped for several minutes allowing a relatively
static column of urine to remain in contact with various tabular segments for longer than useful period of time. This
way the operation of each segment on tubular fluid is exaggerated.
2. Then the clamp is released and urine sample sequentially. Small serial samples collected rapidly.
3. Earliest samples represent fluid which had been in contact with most distal nephron segment.
4. Substances to be examined are administered along with insulin before the application of ureteral occlusion.
5. The tubular segments downstream from proximal segments may modify the tubular fluid composition during its
degrees. For assessment in each sample the concentration of a glomerular marker such as insulin and concentration
of substance under study are measured.
11
 Evaluation:
Fractional exertion of substance and glomerular market are plotted against the cumulative urinary
volume. This method has been found useful in the evaluation of uricosuric compounds.
12
IN-VITRO METHODS
(i) Isolated tubule preparation :
 Purpose and rationale:
Measurement of change in concentration of solutes in perfusion fluid
 Procedure:
1. This technique has been used in the kidney segments of several species like rat, mouse, hamster, rabbit
etc.
2. The thin (<1 mm) tubule segments are dissected from kidney slices
3. Segment is transferred into perfusion chamber
4. To perfuse a suitable tubule, one end of the tubule is holded by micropipette.
5. A perfusion pipette is inserted into tubule lumen. The other end of the tubule is sucked into collecting
pipette.
6. The oil inside the collecting pipette prevents the evaporation
7. All the accumulated fluid is collected at periodic intervals by inserting a narrow calibrated pipette in the
collecting pipette
13
 Evaluation:
The absolute volume of reabsorption is determined from the change in the concentration of an
impermeable marker like (3H) inulin, (125I) isothalamate in the collecting fluid.
Leaks around the perfusion pipette detected from the appearance of the marker in the external
bath.
14
(ii) Patch clamp technique:
 Purpose and procedure:
This technique allows the study of single-ion channels as well as whole-cell ion channel currents.
It requires a patch electrode with a relatively large tip (>1 mm) that has a smooth surface.
 Procedure:
The patch-clamp electrode is pressed against a cell membrane
and suction is applied to pull the
cell membrane inside the electrode tip.
The suction causes the cell to form a tight,
high- resistance seal with the rim of the electrode,
usually greater than 10 giga Ohms,
which is called a giga seal.
15
Cell-attached, cell-excised, whole-cell mode of this technique allow investigation of ion channels.
Cell-attached mode:
With this mode, the patch electrode remains sealed to the
cell membrane, permitting the recording of currents
through single-ion channels from the patch of membrane
surrounded by the tip of the electrode.
Inside-out mode:
From the cell-attached configuration, the
electrode is quickly pulled out from the cell.
This leaves the patch of membrane attached to
the electrode exposing the intracellular surface
of the membrane to the external environment,
allowing pharmacological manipulations to the
intracellular side of the ion channels.
16
Outside-out mode:
After achieving cell-attached configuration, the electrode is slowly withdrawn from the cell, allowing a
membrane patch to be Excised.
Which will then reorganize on the edge of the electrode with the original interior of the cell membrane
facing inside the electrode solution.
This mode allows researchers To examine ion channel properties, studying effects of membrane non
permeable molecules on the intracellular part of the channel
17
18
CONTENTS
Definition
Mechanism of action
In-vitro methods
In-vivo methods
19
ANTIHYPERTENSIVE
WHAT IS HYPERTENSION ?
It is high blood pressure. Hypertension is defined as conventionally increase in
blood pressure above 140/90 mmHg.
Normal blood pressure: 120/80mmHg.
Definition :
“ The drugs which are used in the treatment of hypertension are known as antihypertensive.”
Types of hypertension:-
1. Primary or essential hypertension –
In this the cause of rise in the blood pressure is unknown, several factors affecting are as follow:
 High salt intake
 Cigarette smoking
2. Secondary hypertension-
It is cause due to another medical conditions. Several factors affecting are as follow:
Acute or chronic renal disease, renal artery stenosis, drugs like oral contraceptives, estrogen, steroids.
20
MECHANISM OF ACTION
21
IN-VITRO AND IN-VIVO METHODS
 In-vivo models :
1. Renovascular hypertension
(i) Goldblatt method
I. Two kidney one clip (2KIC) hypertension
II. One kidney one clip (IKIC) hypertension
III. Two kidney two clip (2K2C) hypertension
(ii) Hypertension induced by external compression of renal parenchyma
I. Page hypertension
(iii) Grollman hypertension
I. Two kidney one ligature (2K1L)
II. One kidney one ligature (IKIL)
III. Coarctation of aorta
IV. Reduced renal mass
22
2. Dietary hypertension
I. Increased salt intake
3. Endocrine hypertension
I. Mineralocorticoid induced hypertension
II. Adrenal regeneration hypertension
4. Neurogenic hypertension
I. Denervation of sinoaortic baroreceptors
5. Psychogenic hypertension
6. Genetic hypertension
7. Other models
I. Obesity related hypertension
II. Hypertension induced by cholinomimetic agents
III. Angiotensin-II induced hypertension
IV. Hypertension induced by cadmium
23
In-vitro methods:
I. Langendorff (isolated heart system)
II. Endothelin receptor antagonism in porcine isolated heart
III. Alpha-2 adreno-receptors activity
IV. Beta adreno-receptors activity
24
IN VIVO METHODS
1. Renovascular hypertension
The renovascular hypertension is the most common hypertension animal model, which is
associated with the activation of renin-angiotensin-aldosterone system (RAAS). Experimentally,
renovascular hypertension is produced by constriction of the renal artery, which causes the activation
of the sympathetic nervous system and renal renin secretion. The renin secretion triggers the
peripheral RAAS and produces angiotensin-II. Additionally, angiotensin-II increases aldosterone
secretion leading to salt and water retention leading to increased blood volume and hypertension .
(i) Goldblatt method :-
Goldblatt et al (1934) induced hypertension by partial constriction of the renal artery of dog.
Thereafter many renal induced models of hypertension have been successfully established in several
experimental animals (rats, mice, rabbit, and monkey).
Experimentally, animals are anesthetized with a suitable anesthetic agent like hexobarbitone sodium 140
mg/kg body weight)-than kidney and the renal artery are visualized by lateral abdominal incision, just
below and parallel to costal margin.
The renal artery is constricted/ligated by a U-shaped silver ribbon clip (0.2 mm of internal diameter) or
silk suture (4-0). Constriction of renal artery should be more than 50%.
The animal is considered hypertensive if systolic BPTS more than 160 mm of Hg for two consecutive
days after 4 weeks of surgical ligation.
25
 There are three types of renal hypertension induced by Goldblatt's techniques.
26
I. Two Kidney One Clip (2K1C) hypertension:
The renal artery is constricted on only one side with the other artery (or kidney) left untouched which causes sustained
increase in BP due to increased Plasma Renin Activity (PRA), which in turn increases circulating angiotensin-II, a
potent vasoconstrictor. However, there is no salt and water retention because of the other normal kidney being intact.
Therefore, the resultant hypertension at this stage is renin- angiotensin dependent. After about 6 weeks, the increased
angiotensin-II releases aldosterone from adrenal cortex leading to a gradual retention of salt and water. Decreased
renin production is because of the retention of salt and water. From this stage onwards, hypertension is volume
dependent. This clearly shows that salt and water balance is critically involved in the pathogenesis of renovascular
hypertension. Increased BP and increased renin activity returns to normal by unclipping or removal of the affected
kidney.
II. One Kidney One Clip (IKIC) hypertension:
Constriction of renal artery is done on one side and the contralateral kidney is removed. BP rises within few hours. As
there is no other kidney, there is no pressure diuresis and natriuresis, so rapid salt and water retention is there. Plasma
renin activity is usually normal. Hypertension soon becomes volume dependent
III. Two Kidney Two Clip (2K2C) hypertension:
Constriction of aorta or both renal arteries is done. There is a patchy ischemic kidney tissue, which secretes renin
causing increased BP. The remaining kidney tissue retains salt and water. In fact, one of the most common causes of
renal hypertension in human beings is such a patchy ischemic kidney disease.
(ii) Dietary hypertension:-
I. Increased salt intake:
Physiologically, normal kidney has the ability to excrete easily the daily salt load without allowing a
marked rise in extracellular volume.
Excess salt intake produces hypertension in rats, which mimics human hypertension.
High salt intake hypertension has been produced in rats, rabbits and chicks by replacing drinking water
with 1-2% sodium chloride for 9-12 months
27
IN-VITRO METHODS
(i)Alpha-2 adreno-receptors activity :
Alpha-adrenergic agonists most potently displace 3H clonidine.The purpose of this assay is to assess the
interaction of hypotensive agents with central a2-receptors and determine possible clonidine-like
mechanisms of action.
 PROCEDURE
Reagents:
1. Tris buffer pH 7.7
2. [4-3H]-Clonidine hydrochloride
3. Clonidine-HCI
4. Test compounds:1 mM stock solution is made up in a suitable solvent and serially diluted, so that the
final concentrations in the assay range from 10-5 to 10-8 M.
28
Tissue Preparation
29
Stored on ice and final tissue concentration is 10 mg/ml.
Supernatant is discarded and the final pellet rehomogenized in 50 vol of buffer 1
Centrifuged at 40 000 g for 15 min
Homogenized in 50 volumes of 0.05 M Tris buffer pH 7.7
Cortical tissue is rapidly dissected
Male Wistar rats are sacrificed by decapitation
Assay
Tissue homogenates are incubated for 20 min at 25 °C with 3 nM 3H-clonidine and varying drug
concentrations
Filtered under reduced pressure on Whatman filters
Filters are washed with 3 ml volumes of 0.05 M Tris buffer pH 7.7
Transferred to scintillation vials
Specific clonidine binding is defined as the difference between total bound radioactivity and that
bound in the presence of 1 µM clonidine.
30
31
Tha
nk
you

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In-vitro and in-vivo methods of diuretics & antihypertensive final.pptx

  • 1. Patil Aishwarya Mahaveer. M pharm (Pharmacology) Roll no.: 06 1
  • 3. DIURETICS Definition : “Elimination of excess of urine (more than normal levels) is termed as diuresis and the drugs that facilitate the process are known as Diuretics.” The normal volume of urine in healthy person is 800 to 2000 ml per day. Urine consist of metabolic waste materials, water and some electrolytes. 3
  • 4. CLASSIFICATION CLASS OF DRUG LOCATION EXAMPLE Carbonic anhydrase inhibitors Proximal tubule Acetazolamide Loop diuretics Loop of Henle Furosemide/Bumetanide Thiazide diuretics Distal tubule Hydrochlorothiazide/Bendr oflumethiazide Potassium sparring diuretics Collecting duct Spironolactone/triamterene /Amiloride Osmotic Diuretics Lumen of nephron Mannitol 4
  • 6. IN-VITRO AND IN-VIVO METHODS In-vivo methods :- I. Diuretic activity in rats (LIPSCHITZ TEST) II. Saluretic and diuretic activity in dogs III. Saluretic activity in rats IV. Stop flow techniques V. Micro puncture technique in rat In-vitro methods :- I. Carbonic anhydrase inhibition in vitro II. Patch clamp technique in kidney cells III. Isolated perfused kidney 6
  • 7. IN VIVO METHODS (i) Diuretic activity in rats (Lipschitz test)  Aim: In 1943diuretic activity in rats (Lipschitz test) purpose to determine diuretic activity of test drug by lipschitz method by comparing water and sodium excretion in test animals.  Requirements: Animals : Male Wistar rats (150-200 gm) Equipments : Metabolic cages - wire mesh at bottom and funnel for collection of urine Stainless steel sieves, Flame photometer. Chemicals : Test drug, normal saline, urea  Procedure: 1. Rats divided into 4 groups of 3, each placed in metabolic cages, provided with wire mesh at bottom and funnel for collecting urine. Stainless steel sieves placed in funnel retain feces and allow only urine for measurement. 2. The rats fed with standard diet and water ad libitum. 3. 15 hr prior to test stop food and water. 7
  • 8. 4. 3 animals are placed in one metabolic cage. 5. 2 groups (6 rats) used for 1 dose of test drug. Test drug is given by orally to these 6 rats. 6. To other 2 groups (6 rats) For one group 1gm/kg of urea was administered. To another group 5 ml of normal saline solution per 100 gm by oral route is given.  Evaluation: Urine excretion is recorded after 5 hr upto 24 hr. Sodium content of urine is estimated by flame photometer depending upon response doses of test compound, adjusted for graded response, determined by ED 50.  Results for calculation: Urine volume excreted x body weight 100 Urine volume excreted/100 gm body weight is worked out expressed as "lipschitz value". Ratio = T/ U  T-Response of test compound  U-Response of urea treatment If ratio is 1 and more indicates the positive diuretic effect (Calculated for 5 hr upto 24 hr). Similar quotients can be calculated for sodium excretion. 8
  • 9. (ii) Saluretic and diuretic activity in dogs  Aim: Dogs are most dependable animals for screening diuretic to study renal physiology and action of diuretics. Because of renal physiology of dog is similar to human than that of rat. Requirements: Animals required : Beagle dogs (either sex) Chemicals required: Urea Equipments required: Metabolic cages, plastic catheter, gavage, Osmometer  Procedure: 1. Dogs are subjected to intensive training for accepting feeding through gavage and hourly catheterization without any resistance. Then placed in metabolic cages. 2. Minimum 4 dogs used as control group receiving water only, 1 gm/kg urea p.o. or 5 mg/kg furosemide orally is given to test group.24 hr prior to experiment, food is withdrawn, and water is withdrawn on morning of that day urinary bladder is emptied with plastic catheter. 3. The dogs receive 20 ml/kg of water by gavage followed by hourly doses of 4 ml/kg of drinking water. Bladder is catheterized twice in an interval of 1 hr and urine is collected for analysis of initial values. 9
  • 10. 4. The test drug and standard is applied either orally or i.v. hourly catheterization is repeated for 6hr without further water dosing, animals placed in metabolic cages over night. After 24 hr of test compound dogs catheterized once again. Urine collected through catheter measured together.  Evaluation: Urine samples are analyzed for sodium, potassium and chloride. Osmolarity is measured through osmometer. Urine volume also measured.  Conclusion: For assessment of activity of test compound urine volume, electrolyte concentration and osmolarity of each animal recorded and averages for each group are calculated. Values plotted against time to allow comparison with pretreatment values as well as with water controls and standard drugs. 10
  • 11. (iii) Stop flow technique: This is very useful in localization of transport processes along length of nephron. After clamping of ureter glomerular filtration rate GRF is decreased.  Aim: To determine diuretic activity of test drug by using clamp which is inserted to ureter.  Requirements: Animals required : Male Wistar rats (150-200 gm) Equipments required : Metabolic cages - wire mesh at bottom and funnel for collection of urine Chemicals required : Insulin  Procedure: 1. The ureter of animal undergoes intense osmatic diuresis, it is damped for several minutes allowing a relatively static column of urine to remain in contact with various tabular segments for longer than useful period of time. This way the operation of each segment on tubular fluid is exaggerated. 2. Then the clamp is released and urine sample sequentially. Small serial samples collected rapidly. 3. Earliest samples represent fluid which had been in contact with most distal nephron segment. 4. Substances to be examined are administered along with insulin before the application of ureteral occlusion. 5. The tubular segments downstream from proximal segments may modify the tubular fluid composition during its degrees. For assessment in each sample the concentration of a glomerular marker such as insulin and concentration of substance under study are measured. 11
  • 12.  Evaluation: Fractional exertion of substance and glomerular market are plotted against the cumulative urinary volume. This method has been found useful in the evaluation of uricosuric compounds. 12
  • 13. IN-VITRO METHODS (i) Isolated tubule preparation :  Purpose and rationale: Measurement of change in concentration of solutes in perfusion fluid  Procedure: 1. This technique has been used in the kidney segments of several species like rat, mouse, hamster, rabbit etc. 2. The thin (<1 mm) tubule segments are dissected from kidney slices 3. Segment is transferred into perfusion chamber 4. To perfuse a suitable tubule, one end of the tubule is holded by micropipette. 5. A perfusion pipette is inserted into tubule lumen. The other end of the tubule is sucked into collecting pipette. 6. The oil inside the collecting pipette prevents the evaporation 7. All the accumulated fluid is collected at periodic intervals by inserting a narrow calibrated pipette in the collecting pipette 13
  • 14.  Evaluation: The absolute volume of reabsorption is determined from the change in the concentration of an impermeable marker like (3H) inulin, (125I) isothalamate in the collecting fluid. Leaks around the perfusion pipette detected from the appearance of the marker in the external bath. 14
  • 15. (ii) Patch clamp technique:  Purpose and procedure: This technique allows the study of single-ion channels as well as whole-cell ion channel currents. It requires a patch electrode with a relatively large tip (>1 mm) that has a smooth surface.  Procedure: The patch-clamp electrode is pressed against a cell membrane and suction is applied to pull the cell membrane inside the electrode tip. The suction causes the cell to form a tight, high- resistance seal with the rim of the electrode, usually greater than 10 giga Ohms, which is called a giga seal. 15
  • 16. Cell-attached, cell-excised, whole-cell mode of this technique allow investigation of ion channels. Cell-attached mode: With this mode, the patch electrode remains sealed to the cell membrane, permitting the recording of currents through single-ion channels from the patch of membrane surrounded by the tip of the electrode. Inside-out mode: From the cell-attached configuration, the electrode is quickly pulled out from the cell. This leaves the patch of membrane attached to the electrode exposing the intracellular surface of the membrane to the external environment, allowing pharmacological manipulations to the intracellular side of the ion channels. 16
  • 17. Outside-out mode: After achieving cell-attached configuration, the electrode is slowly withdrawn from the cell, allowing a membrane patch to be Excised. Which will then reorganize on the edge of the electrode with the original interior of the cell membrane facing inside the electrode solution. This mode allows researchers To examine ion channel properties, studying effects of membrane non permeable molecules on the intracellular part of the channel 17
  • 18. 18
  • 20. ANTIHYPERTENSIVE WHAT IS HYPERTENSION ? It is high blood pressure. Hypertension is defined as conventionally increase in blood pressure above 140/90 mmHg. Normal blood pressure: 120/80mmHg. Definition : “ The drugs which are used in the treatment of hypertension are known as antihypertensive.” Types of hypertension:- 1. Primary or essential hypertension – In this the cause of rise in the blood pressure is unknown, several factors affecting are as follow:  High salt intake  Cigarette smoking 2. Secondary hypertension- It is cause due to another medical conditions. Several factors affecting are as follow: Acute or chronic renal disease, renal artery stenosis, drugs like oral contraceptives, estrogen, steroids. 20
  • 22. IN-VITRO AND IN-VIVO METHODS  In-vivo models : 1. Renovascular hypertension (i) Goldblatt method I. Two kidney one clip (2KIC) hypertension II. One kidney one clip (IKIC) hypertension III. Two kidney two clip (2K2C) hypertension (ii) Hypertension induced by external compression of renal parenchyma I. Page hypertension (iii) Grollman hypertension I. Two kidney one ligature (2K1L) II. One kidney one ligature (IKIL) III. Coarctation of aorta IV. Reduced renal mass 22
  • 23. 2. Dietary hypertension I. Increased salt intake 3. Endocrine hypertension I. Mineralocorticoid induced hypertension II. Adrenal regeneration hypertension 4. Neurogenic hypertension I. Denervation of sinoaortic baroreceptors 5. Psychogenic hypertension 6. Genetic hypertension 7. Other models I. Obesity related hypertension II. Hypertension induced by cholinomimetic agents III. Angiotensin-II induced hypertension IV. Hypertension induced by cadmium 23
  • 24. In-vitro methods: I. Langendorff (isolated heart system) II. Endothelin receptor antagonism in porcine isolated heart III. Alpha-2 adreno-receptors activity IV. Beta adreno-receptors activity 24
  • 25. IN VIVO METHODS 1. Renovascular hypertension The renovascular hypertension is the most common hypertension animal model, which is associated with the activation of renin-angiotensin-aldosterone system (RAAS). Experimentally, renovascular hypertension is produced by constriction of the renal artery, which causes the activation of the sympathetic nervous system and renal renin secretion. The renin secretion triggers the peripheral RAAS and produces angiotensin-II. Additionally, angiotensin-II increases aldosterone secretion leading to salt and water retention leading to increased blood volume and hypertension . (i) Goldblatt method :- Goldblatt et al (1934) induced hypertension by partial constriction of the renal artery of dog. Thereafter many renal induced models of hypertension have been successfully established in several experimental animals (rats, mice, rabbit, and monkey). Experimentally, animals are anesthetized with a suitable anesthetic agent like hexobarbitone sodium 140 mg/kg body weight)-than kidney and the renal artery are visualized by lateral abdominal incision, just below and parallel to costal margin. The renal artery is constricted/ligated by a U-shaped silver ribbon clip (0.2 mm of internal diameter) or silk suture (4-0). Constriction of renal artery should be more than 50%. The animal is considered hypertensive if systolic BPTS more than 160 mm of Hg for two consecutive days after 4 weeks of surgical ligation. 25
  • 26.  There are three types of renal hypertension induced by Goldblatt's techniques. 26 I. Two Kidney One Clip (2K1C) hypertension: The renal artery is constricted on only one side with the other artery (or kidney) left untouched which causes sustained increase in BP due to increased Plasma Renin Activity (PRA), which in turn increases circulating angiotensin-II, a potent vasoconstrictor. However, there is no salt and water retention because of the other normal kidney being intact. Therefore, the resultant hypertension at this stage is renin- angiotensin dependent. After about 6 weeks, the increased angiotensin-II releases aldosterone from adrenal cortex leading to a gradual retention of salt and water. Decreased renin production is because of the retention of salt and water. From this stage onwards, hypertension is volume dependent. This clearly shows that salt and water balance is critically involved in the pathogenesis of renovascular hypertension. Increased BP and increased renin activity returns to normal by unclipping or removal of the affected kidney. II. One Kidney One Clip (IKIC) hypertension: Constriction of renal artery is done on one side and the contralateral kidney is removed. BP rises within few hours. As there is no other kidney, there is no pressure diuresis and natriuresis, so rapid salt and water retention is there. Plasma renin activity is usually normal. Hypertension soon becomes volume dependent III. Two Kidney Two Clip (2K2C) hypertension: Constriction of aorta or both renal arteries is done. There is a patchy ischemic kidney tissue, which secretes renin causing increased BP. The remaining kidney tissue retains salt and water. In fact, one of the most common causes of renal hypertension in human beings is such a patchy ischemic kidney disease.
  • 27. (ii) Dietary hypertension:- I. Increased salt intake: Physiologically, normal kidney has the ability to excrete easily the daily salt load without allowing a marked rise in extracellular volume. Excess salt intake produces hypertension in rats, which mimics human hypertension. High salt intake hypertension has been produced in rats, rabbits and chicks by replacing drinking water with 1-2% sodium chloride for 9-12 months 27
  • 28. IN-VITRO METHODS (i)Alpha-2 adreno-receptors activity : Alpha-adrenergic agonists most potently displace 3H clonidine.The purpose of this assay is to assess the interaction of hypotensive agents with central a2-receptors and determine possible clonidine-like mechanisms of action.  PROCEDURE Reagents: 1. Tris buffer pH 7.7 2. [4-3H]-Clonidine hydrochloride 3. Clonidine-HCI 4. Test compounds:1 mM stock solution is made up in a suitable solvent and serially diluted, so that the final concentrations in the assay range from 10-5 to 10-8 M. 28
  • 29. Tissue Preparation 29 Stored on ice and final tissue concentration is 10 mg/ml. Supernatant is discarded and the final pellet rehomogenized in 50 vol of buffer 1 Centrifuged at 40 000 g for 15 min Homogenized in 50 volumes of 0.05 M Tris buffer pH 7.7 Cortical tissue is rapidly dissected Male Wistar rats are sacrificed by decapitation
  • 30. Assay Tissue homogenates are incubated for 20 min at 25 °C with 3 nM 3H-clonidine and varying drug concentrations Filtered under reduced pressure on Whatman filters Filters are washed with 3 ml volumes of 0.05 M Tris buffer pH 7.7 Transferred to scintillation vials Specific clonidine binding is defined as the difference between total bound radioactivity and that bound in the presence of 1 µM clonidine. 30