IN-VITRO MODEL FOR TESTING DIURETICS ACTIVITY BY USING THE FOLLOWING MODELS METHODOLOGY AND ITS IMPROVMENT BY MODIFICATION.

1. MODELS FOR TESTING ACTIVITY OF DIURETICS
Presented by : MOHANLAL
15PHM2007
M. PHARM
mohanlalchoudhary1992@gmail.com
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2. INVIVO METHODS
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1. Diuretic activity in rats (LIPSCHITZ test)
2. Saluretic activity in rats
3. Diuretic and saluretic activity in dogs
4. Clearance methods
5. Micropuncture techniques in the rats

3. DIURETIC ACTIVITY IN RATS
(LIPSCHITZ TEST)
Purpose and rationale
 The test is based on water and sodium excretion in test animals
as compared to the rats treated with high dose of Urea (control).
 The “LIPSCHITZ value” = Excretion by test animals
Excretion by urea control
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5. CONTI….
Methodology
 Speces - Male Wistar rats (100–200 gm)
 Sex - male rats
 Three animals in each group both in test and control
Procedure
 3 animals per group are placed in metabolic cages provided with a wire mesh
bottom and funnel to collect the urine.
 SS sieves are placed in the funnel to retain feces but allow urine to pass.
 Rats are fed with std diet(Altromin pellets) and water.
 17-24 hrs prior to the experinment food and water is withdrawn.
 For screening,2 groups of 3 animals are used for 1 dose of test compound.
 Test compound is applied orally at a dose of 50mg/kg in 5mlwater/kg body
weight.
 2 groups of 3 animals act as control and are treated with urea
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6. PROCEDURE
 Test compound is applied orally at a dose of 50mg/kg in 5mlwater/kg body
weight.
 2 groups of 3 animals act as control and are treated with urea.
 Additionally,5ml of 0.9% Nacl solution/100g body weight are given by gavage.
 Urine excretion is recorded after 5 and 24 hrs.
 The Na content of urine is determined by Flame photometry.
 Active compounds are tested again with lower doses.
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7. EVALUATION
 Urine volume excreted /100g of body weight is calculated for each group.
 Lipschitz value= Excretion by test animals is calculated
Excretion by urea control
 Lipschitz value of more than 1 is taken as positive.
 With potent diuretics Lipschitz value of 2 or more can be found.
 Calculating this index for 5 and 24 hrs indicates duration of diuretic effect.
 Quotients can also be calculated for Na excretion.
 DRC can be established for various doses.
 Loop diuretics like furosemide are characterized by steep DRC and lipschitz value of 4.0.
 Saluretic drugs like Hydrochlorthiazide have Lipschitz value of 1.8.
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8. MODIFICATIONS
 Screening diuretic agents in rats using normal saline as hydrating fluid.
 Homozygous Brattleboro rats exhibit symptoms of Diabetes insipidus due
to failure of hypothalamic neurons to produce vassopressin,which is due to
single point deletion in vassopressin gene.
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9. SALURETIC ACTIVITY IN RATS
Purpose and Rationale
 Excretion of electrolytes and water is important for treatment in peripheral
oedema and CHF as well as in hypertension.
 However K loss has to be avoided and therefore saluretic and K sparing
diuretics were developed.
 The diuresis test in Rats was modified in such a way that in addition to
determining Na and water content,K and chloride content as well as osmolality
can be determined.
 The ratios between the electrolytes can be calculated indicating CA inhibition or
K sparing effect.
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10. METHODOLOGY
 Speces- Wistar rats (100-200g)
 Sex - Male
 Theree animals in each group both in test and in control to test the test compound
PROCEDURE
 Male Wistar rats fed with std diet (Altromin pellets) and water are used.
 15 hrs prior to the test food but not water is withdrawn.
 Test compounds are applied 50mg/kg orally in 0.5ml/100g body weight starch
suspension.
 3 animals are placed in 1 metabolic cage with a wire mesh bottom and a funnel to
collect the urine.
 2 groups of 3 animals are used for each dose of the test compound.
 Urine excretion is registered every hr upto 5 hrs.
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11.  The 5 hr urine is analysed by flame photometry Na and K and Argentometrically by
potentiometrical end pt. for chloride.
 To evaluate compounds with prolonged effect 24 hr urine is analysed.
 Furosemide(25mg/kg) or Hydrochlorthiazide(25mg/kg) is used as standard.
Cont….
EVALUATION
The sum of Na and Chloride excretion is a measure of saluretic activity.
The ratio of Na/K is calculated for natriuretic activity.Values >2 indicate natriuretic effect.Ratios>10
indicate K sparing effect.
The ratio of chloride/Na+K is known as ion quotient is calculated for CA inhibition.
MODIFICATIONS
Adrenolectomized Rats treated with Aldosterone can be used to study the Aldosterone antagonists.
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12. DIURETIC AND SALURETIC ACTIVITY IN DOGS
 Renal physiology of the dog is claimed to be closer
to man than that of rats.
 Oral absorbability of diuretic substances can
appropriately be studied in dogs.
 Using catheters, interval collections of urine can be
made with more reliability than in rats.
 Simultaneously, blood samples can be withdrawn to
study pharmacokinetics.
PURPOSE AND RATIONALE
Methodology
Speces – Dogs
Sex - male or female
Four animals are used in each group for testing of
comp.
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13. PROCEDURE
 Beagle dogs of either sex have to undergo intensive training to be accustomed to accept
gavage feeding and hourly catheterization without any resistance.
 The dogs are placed in metabolic cages.
 At least 4 dogs are used as controls receiving water only, as standard controls 1 g/kg
urea p.o. or 5 mg/kg furosemide p.o. or the test drug group.
 Twenty-four hours prior to the experiment food but not water is withheld.
 On the morning of the experiment, the urinary bladder is emptied with a plastic
catheter.
 The dogs receive 20 ml/kg body weight water by gavage, followed by hourly doses of 4
ml/kg body weight drinking water.
 The bladder is catheterized twice in an interval of 1 h and the urine collected for
analysis of initial values.
 Then, the test compound or the standard is applied either orally or intravenously.
 Hourly catheterization is repeated over the next 6 h.
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14.  Without further water dosage the animals are placed in metabolic cages overnight.
 Twenty-four hours after dosage of the test compound, the dogs are catheterized once more and
this urine together with the urine collected over night in the metabolic cage registered.
 All urine samples are analyzed by flame photometry for sodium and potassium and by
argentometry for chloride content.
 Furthermore, osmolality is measured with an Osmometer.
EVALUATION
Urine volume, electrolyte concentrations and osmolality are averaged for each group.
The values are plotted against time to allow comparison with pretreatment values as well as with water
controls and standards.
The non-parametric U-test is used for statistical analysis.
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15. CLEARANCE METHOD
Purpose and Rationale
 A drug that acts solely in the PCT by causing the delivery of the increased amounts of filtrate to the loop
of Henle and the distal convolution, would augment the clearance of solute-free water (CH2O) during
water diuresis and the reabsorption of solute-free water (TCH2O) during water restriction.
 In contrast, drugs that inhibit sodium reabsorption in Henle’s loop would impair both CH2O and TCH2O.
 On the other hand, drugs that act only in the distal tubule would reduce CH2O but not TCH2O.
PROCEDURE
 Clearance experiments are performed either in conscious or anaesthetized beagle dogs under conditions
of water diuresis and hydropenia.
 Water diuresis is induced by oral administration of 50 ml of water per kg body weight and maintained by
continuos infusion into jugular vein of 2.5% glucose solution and 0.58% NaCl solution at 0.5 ml/min per
kg body weight.
 When water diuresis is well established, the glucose infusion is discontinued and control urine samples
are collected by urethral catheter.
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16. CONT….
 Blood samples are obtained in the middle of each clearance period. After the control
period,compounds to be tested are administered and further clearance tests are
performed.
 Hydropenia is induced by withdrawing the drinking water 48 h before experiment.
 On the day before the experiment 0.5 U/kg body weight of vasopressin in oil is injected
intramuscularly.
 On the day of the experiment 20 mU/kg vasopressin is injected i.v., followed by
infusion of 50 mU/kg per hour vasopressin.
 To accomplish constant urine flow 5% NaCl solution is infused at 1 ml/min per kg body
weight up to i.v. administration of a compound to be tested, followed by i.v. infusion of
0.9% NaCl solution at a rate equal to the urine flow.
 Glomerular filtration rate (GFR) and renal plasma flow (RPF) are measured by the
clearance of inulin and para-aminohippurate, respectively.
 Therefore, appropriate infusion of inulin and para-aminohippurate are initiated.
 Inulin and para-aminohippurate are measured.
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17. EVALUATION
 The following parameters may be determined: water and electrolyte
excretion, GFR, RPF, CH2O, TCH2O and plasma renin activity.
 Results of test compound are compared statistically with control and
standard drug treated animals.
Modification
1. A simple method to serial renal clearance studies without urine collection in
rats.
2. This was applied to non-radiolabeled para-aminohippurate sodium and
iothalamate sodium which were used respectively to estimate renal blood
flow and glomerular filtration rate.
3. An enzymatic method was developed for the determination of inulin and a
colorimetric method was developed for determination of p-aminohippurate in
the plasma and urine of rats.
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18. 3. clearance methods described in monkeys ( Hropot et al.)
 Chimpanzees weighing 30.7 ±10.6 kg were anesthetized with 1 mg/kg Sernylan i.m.
injection.
 Food was withdrawn 24 h prior to the experiment and the animals received only tap
water.
 In the morning before the experiment, the urinary bladder of the animals was emptied
by catheterization. The urine was discarded.
 To determine the glomerular filtration rate (inulin clearance), a bolus injection of 50
mg/kg inulin i.v. was given and followed by a continuous infusion of 3 ml/min inulin
dissolved in Ringer lactate solution. After an equilibrium of 60 min, urine and blood
samples were collected for two control clearance for 30 mins each.
 The control periods were followed by intravenous administration of the test
preparation in a dose of 20 mg/kg.
 Thereafter, urine and blood samples were collected during 6 clearance periods.
 The following parameters were determined: urine excretion, inulin clearance and
urate clearance, fractional excretion of urate and plasma urate .
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19. MICROPUNCTURE TECHNIQUES IN THE RATS
Purpose and Rationale
 Micropuncture techniques have been applied to the direct
investigation of the effect of diuretics on single nephron function.
 The observed changes in tubular fluid reabsorptive rates and
electrolyte concentrations can be used to asses the mechanism of
action.
 The rat is the model of choice since proximal and distal tubules as
well as collecting ducts are accessible for micropuncture.
Methodology
Speces -Rats ( 250 gm)
Sex - male or female
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20. PROCEDURE
 Rats with a body weight of 250g are anaesthetized with intraperitoneal injection of Thiopentone.
 The animals are fasted for 16 h before the beginning of the experiment, but have free access to
tap water.
 After anesthesia the animals are placed on a thermostatically heated table.
 Thereafter rats are tracheotomized and carotid artery and jugular vein are cannulated for blood
pressure recording, blood sampling, and for infusion of compounds, respectively.
 The left kidney is carefully exposed by a flank incision, embedded in a small plastic vessel with
cotton wool, and bathed with paraffin oil at 37 °C.
 The ureter is cannulated and rectal temperature monitored continuously.
 A bolus injection of inulin 3H in NaCl solution is given, followed by 0.85% NaCl solution at a
rate of 2.5 ml/min per 100 g body weight.
 The control puncture of tubules is performed 45 min after beginning of the intravenous infusion.
 The direct collection of tubular fluid samples from proximal and distal tubules is carried out
with glass capillaries of 8 to10 μm external diameter using a micromanipulator and microscopic
observation.
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21.  Distal tubules are identified by intravenous injection of lissamine green.
 The control period is followed by the test period.
 After an equilibration period of 30 min with the compound to be tested, micropuncture is
performed again and tubular fluid is collected.
 The uretral urine is collected and blood sampling is performed in the middle of each clearance
period.
1. The following parameters may be determined: inulin clearance (GFR), single nephron
GFR, fractional delivery of water, sodium and potassium in proximal and distal tubules and
in urine.
2. All data are expressed as mean values ±SEM.
3. Comparison of the effects of compounds to be tested with controls is performed by one
way analysis of variance and by Student’s t-test for paired and unpaired data.
Evaluation
Cont….
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22. STOP FLOW TECHNIQUE
Purpose and Rationale
 This procedure is of considerable value in the localization of transport processes
along the length of the nephron.
 During clamping of the ureter, glomerular filtration is grossly reduced.
 The contact time for the tubular fluid in the respective nephron segments
increases, and the concentration of the constituents of tubular fluid should
approximate the static-head situation.
 After release of the clamp, the rapid passage of the tubular fluid should modify
the composition of the fluid only slightly.
 The first samples should correspond to the distal nephron segment, the latest to
glomerular fluid.
 However, with introduction of the micropuncture technique, the stop-flow
method appears less attractive.
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23. PROCEDURE
 This test can be performed on different animals.
 The ureter of an animal undergoing intense osmotic diuresis is clamped for several minutes
allowing a relatively static column of urine to remain in contact with the various tubular
segments for longer than the usual periods of time.
 Thus, the operation of each segment on the tubular fluid is exaggerated.
 Then the clamp is released, and the urine is sampled sequentially.
 Small serial samples are collected rapidly, the earliest sample representing fluid which had been
in contact with the most distal nephron segment.
 Substances examined are administered along with inulin before the application of uretral
occlusion.
 However, tubular segments downstream from the proximal segments may modify the tubular
fluid during its egress.
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24. EVALUATION
 Each sample the concentration of a glomerular marker, such as inulin, and the
concentration of the under study are measured.
 Fractional excretion of the substance and the glomerular marker are plotted
versus the cumulative urinary volume.
Modifications of the method
1. stop-flow studies on tubular transport of uric acid in rats treated with
pyrazinoic acid, an inhibitor of tubular urate secretion.
2. Tanaka et al. used stop-flow experiments to test uricosuric and diuretic
activities of new compounds in dogs.
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25. REFERENCES
 H. Gerhard Vogel “Drug Discovery and Evaluation” ,Pharmacological Assays , springer
publication London , 2nd edition ,2002, pg (316-331).
 http://www.pharmatutor.org/articles/screening-diuretic-agents-overview
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