screening procedures

1. SCREENING DIURETIC
DRUGS
Dr Saurav Misra
PGIMS Rohtak

2. INTRODUCTION
DIURETICS :
Increases urine production
Increases rate of urine flow and sodium excretion are used to adjust the volume
and/or composition of body fluids in a variety of clinical situations
Elimination of excessurine (more than normal levels)is termed asdiuresis
Saluretics are agentsthat facilitate the removal of salt or especially sodiumion.

5. In-vivo models
• Diuretic activity in rats (LIPSCHITZ test)
• Saluretic activity in rats
• Diuretic and saluretic activity in dogs
• Clearance methods
• Micropuncture techniques in rats
• Stop flow technique

6. Metabolic cage
Metabolic cages provide a perfect separation of feces and urine through the
special design of the funnel and of the separation cone.
Cages are available for single mouse (with single chamber feeder), for grouped
mice (with double chamber feeder).
Provide uncontaminated, reliable samples for accurate metabolic monitoring
Designed to minimize animal stress:

7. LIPSCHITZTEST
PURPOSE AND RATIONALE:
Based on water & Na+ excretion in test animal (rats) & compared to rats treated
with high high dose of urea.
The Lipschitz -value is the quotient between excretion by test animals and
excretion by the urea control.

8. PROCEDURE:
Male Wistar rats weighing 100-200g are used
3 animals per group are placed in Metabolic cages provided with wire mesh bottom
and a funnel to collect the urine.
Rats are fed with std diet & water.
15 hr. before the experiment, food & water are withdrawn.

9. The test compound is given orally at a dose of 50 mg/kg in 5 ml water/kg body
weight.
Two groups of 3 animals receive orally 1g/kg urea and 5 ml of 0.9% NaCL
solution per 100g body weight are given by gavage.
Urine excretion is recorded after 5 and after 24 hours.
The sodium content of the urine is determined by flame photometry.

10. EVALUATION :
❖ Urine volume excreted per 100 g body weight is calculated for each group.
❖ Results are expressed as LIPSCHITZ value ( the ratio of T/U)
LIPSCHITZ value = T (response of test compound)
U (response of urea treatment )
Lipschitz value ≥ 1 indicates positive effect
Lipschitz value ≥ 2 potent diuretic activity
➢For studying prolonged effect, 24 hr urine sample collected & analyzed

11. SALURETICACTIVITYIN RATS
PURPOSE AND RATIONALE :
➢ Diuresis test in rats was designed to determine sodium, potassium, chloride and water
content & osmolarity of urine
➢ Ratio between electrolytes can be calculated, indicating carbonic anhydrase inhibition or
K+ sparing effect

12. PROCEDURE:
➢ Male Wistar Rats weighing 100-200 g are fed with std diet & water
➢ 15 hrs prior to experimentfood is withdrawn but not water.
➢ Test compounds is given in a dose of 50 mg/kg orally in 0.5 ml/100 g body weight starch
suspension.
➢ 3 animals placed in one metabolic cage
➢ 2 groups, each of 3 rats used for test & std drug
➢ Urine excretion measured every hr up to 5 hr
➢ 5 hr urine is analysed by flame photometry for sodium, potassium and chloride.

13. EVALUATION
The sum of sodium and chloride excretion is calculated as parameter for
saluretic activity.
The ratio of sodium/potassium is calculated for natriuretic activity.
Values greater than 2.0 indicate a favourable natriuretic effect. Ratio greater
than 10.0 indicate a potassium sparing effect.
The ratio of Cl-
Na+ + K+
Is calculated to estimate carbonic anhydrase inhibition.

14. Diuretic and Saluretic activity in
dogs
PURPOSE AND RATIONALE:
Dogs have been extensively used to study renal physiology and the action of
diuretics.
Renal physiology of dogs is claimed to be closer to man than that of rats.
Using catheters, interval collections of urine can be made with more reliability
than in rats.

15. PROCEDURE
Beagle dogs of either sex have to undergo intensive training to be accustomed to accept gavage
feeding and hourly catheterisation without any resistance.
24 hrs prior to the experiment food but not water is withheld.
On the morning of experiment, the urinary bladder is emptied with a plastic catheter.
The dogs are placed in metabolic cages.
The dogs receive 20 ml/kg body weight water by gavage, followed by hourly doses of 4 ml/kg body
weight drinking water.

16. Bladder is catheterised twice in an interval of 1 hr and the urine collected for analysis of
initial values.
Then test compound and standard are given orally or i.v. Hourly catheterization is repeated
over the next 6 hrs.
Without further water dosage the animals are placed in metabolic cages overnight.
24 hours after dosage of the test compound, the dogs are catheterised once more and this
urine together with the urine collected overnight in the metabolic cage registered.
All the samples of urine are analysed by flame photometry for sodium, potassium and
chloride content.

17. 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 applied.

18. CLEARANCE METHOD
PRINCIPLE :
➢ Method for evaluation of renal function & provide information about site of action of
diuretics.
➢ A drug that acts solely in the proximal convoluted tubule, 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.
➢ The drugs that inhibits sodium reabsorption in Henle`s loop would impair both CH2O and
TCH2O
➢ Drugs that act only in distal tubule would reduce CH2O but not TCH2O

19. PROCEDURE:
Test may be performed in species from which urine and plasma can be readily
collected.
Clearance experiments are performed either in conscious or anaesthetized beagle
dogs under conditions of water diuresis and hydropenia.
1. Water diuresis:
Oral administration 50ml water /kg. body weight
Maintained by continuous infusion into jugular vein of 2.5 % glucose soln and
0.58 % NaCl soln. at 0.5 ml/min/kg.body weight
Control urine sample taken by urethral catheter

20. 2. Hydropenia:
Withdrawing water 48 hr before experiment
Day before the experiment 0.5 U/kg vasopressin is injected i.m.
On the day of experiment 20 mU/kg vasopressin injected i.v. followed by
infusion of 50 mU/kg per hour vasopressin
To obtain constant urine flow 5% NaCl solution is infused at 1 ml/min per kg body weight
upto i.v administration of test compound.
Urine & blood sample collected

21. EVALUTION:
❖ Following parameters are determined & results of test drug compared with std drug
➢ Water and electrolyte excretion
➢ Glomerular filtration rate : Inulin is used
➢ Renal plasma flow : Para amino hippurate is used
➢ CH2O &TCH2O
❑ Free water clearance ( cH2o ):
➢Volume of water that must be removed from urine in order to render urine isosmotic
with plasma
➢CH2O = V – Cosm

22. ❑ Free water reabsorption ( TCH2O ) :
➢Volume of free water reabsorbed per unit time
➢In the presence of ADH urine is concentrated at thattime V< Cosm.
TCH2O = Cosm – V
❑ Osmolar clearance (cosm) :
➢Theoretical volume of plasma per unit time that the kidney has to filter in order to extract
all the solutes out of that volume of plasma.
Cosm = V(Uosm / Posm)
where, V= Urine flow (ml/min.)
Uosm = Urine osmolarity (mosm/kg.body weight)
Posm. =. Plasma osmolarity (mosm/kg.body weight)

23. MICROPUNCTURETECHNIQUES
PRINCIPLE :
➢ Direct investigation of the effects of diuretics on single nephron function.
➢ The observed changes in tubular fluid reabsorptive rates and electrolyte
concentrations can be used to assess 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.

24. Animals
According to the site where micro puncture is going to be performed,
appropriate animal model is chosen.
Anesthesia: Thiobarbital and Pentobarbital is injected via i.p route
Location Animals to be used
Bowman`s space Rat
Loop of Henle Rat
Proximal tubule Dog and Rat
Distal tubule Dog and Rat
Collecting duct Rat and Hamsters

25. Procedure
Rats fasted for 12-18 hrs but have free access to water & anaesthetized
Rats are tracheotomized and carotid artery and jugular vein cannulation is done for
B.P recording and for infusion of compounds.
Left kidney is exposed and embedded in a small plastic vessel with cotton wool and
bathed with paraffin oil at 37o C.
Ureter is cannulated and rectal temperature is monitored

26. Bolus injection of 75 µCi inulin 3H in 0.7% NaCl solution is given
After 45 mins. the control puncture of tubules is performed
Direct collection of tubular fluid samples from proximal and distal tubules is
carried out by glass capillary (8-10 µm external diameter)
Control period followed by the test period & after equilibration of 30 min with test
compound micropuncture is performed and tubular fluid collected

27. Evaluation
The following parameters are determined: Inulin clearance (GFR) single
nephron GFR, fractional delivery of water, sodium and potassium in proximal
and distal tubules and in urine.
All data are expressed as mean±SEM
Comparison of the effects of compounds to be tested with controls is performed
by ANOVA.

28. STOP FLOW TECHNIQUE
PURPOSE AND RATIONALE
Useful in the localization of transport processes along the length of the nephron
During clamping of the ureter, glomerular filtration is decreased
Contact time for the tubular fluid in the respective nephron segments increased
After release of the clamp, the rapid passage of the tubular fluid modify the composition
of the fluid slightly
The first samples correspond to the distal nephron segment and the last to the glomerular
fluid

29. PROCEDURE
Method can be performed in different animals under anaesthesia.
Ureter of animal undergoing intense osmotic diuresis is clamped for several minutes allowing
a relatively static column of urine to remain in contact with various tubular segments for
longer than usual periods of time.
Then clamp is released
Small serial samples are collected rapidly, the earliest sample representing fluid which had
been in contact with most distal nephron segment.
Substances examined are administered along with inulin before the application of ureteral
obstruction

30. Evaluation
In each sample, the concentration of a glomerular marker such as inulin and the
concentration of the substance under study are measured
Fractional excretion of the substance and the glomerular marker are plotted
versus the cumulative urinary volume

31. In-vitro models
• Carbonic anhydrase inhibition
• Patch clamp technique in kidney cells
• Perfusion of isolated kidney tubules
• Isolated perfused kidney technique

32. REFERENCES
• Vogel HG. Drug discovery and evaluation: Pharmacological assays. 2nd ed.
● Goodman & Gilman’s the pharmacological basis of therapeutics 12th edition.
● K D Tripathi`s Essential of medical pharmacology 7th edition.

33. THANKYOU