Experimental Pharmacology:l 34

Experimen
EXPERIMENT No.4
STUDY OF DIURETIC ACTIVITY.OFDRUGS USINGRATSIMICE
Purpose:
At the endof practical class, the students shall be able to
1. Know about the diuretics and anti-diuretics.
2. Knowthe mechanism and uses of diuretics.
3. Know the different animal models used for screening of diuretiCs.
Terminology:
Diuretics: These are the agents which increase the flow of urine. Elimination
excess urine is termed as diuresis and the drugs that facilitate this process are calle
as diuretics.
Anti-diuretics: These are drugs that reduce urine volume, particularly used i
diabetes insipidus.
Description:
Diuretics are amongthe most widely prescribed drugs. These are the compounds, which
increase the flow of urine. A number of proven measures and drugs capable of producing
diuresis, in congestive heart failure and other clinical conditions associated with oedema
have been available for many years. Application of diuretics for the management d
hypertension has surpassed their use in oedema. Availability of diuretics has also had a major
impact on the understaning of renal physiology. Diuretics increase the excretion of Na" and
water. They decrease the reabsorption of Na and C[ from the glomerular filtrate, increased
water loss being secondary to the increased excretion of NaCl (natriuresis). This can be
achieved by adirect action on the cells of the nephron and indirectly by modifying the
content of the filtrate. The folowing drugs are used às diuretics:
1. High efficacy diuretics or Loop diuretics (acts on loop of Henle by inhibit Na*-K*-2C
cotransport): Furosemide, Bumetanide, Torasemide.
2. Medium efficacy diuretics (acts on distal tubule by inhibit Na*-cr symport)
(a) Benzothiadiazines (thiazides): Hydrochlorothiazide, Benzthiazide, Hydro
flumethiazide, Bendroflumethiazide.
(b) Thiazide like (related heterocyclics): Chlorthalidone, Metolazone, Xipamide,
Indapamide, Clopamide.
3. Weak or adjunctive diuretics
(a) Carbonic anhydrase inhibitors (acts on proximal tube): Acetazolamide
(b) Potassium sparing diuretics (acts on collecting duct):
() Aldosterone antagonist: Spironolactone.
i) Inhibitors of renal epithelial Na channel: Triamterene, Amiloride.
i Osmotíc diuretics (acts on lumen of nephron): Mannitol, Isosorbide, Glycerol.
 Experimental Pharmacology-I 35
Experiments
A method using rats for estimation of
antidiuretic potency was described by Burn in
1931. This method or a modification of it has been used for
subsequent workers. Investigators who used dog as an diuretic assays by most of the
diuretics varied in the technical details much more than experimental animal for assay
of
those who used
wd in-Vitro and n-VIvo sCreening methods are used for studying the rats. The following
different drugs. diuretic activity of
In-vitro methods:
Tsolated tubule preparation: This method measures the change in
concentration
solutes in perfusion fluid. The thin tubule segments (< 1 mm) are dissected from .of
kidney slices
af several species lIke rat, mouse, hamster, rabbit etc. and transferred into perfusion
chamber.The absolute volume of reabsorption is determined.
(b) Carbonic anhydrase inhibition: Inhibition of carbonic anhydrase enzyme is
measured.
(c) Patch clamp technique: This method measures the current across the ion
channel.This technique allows the study of single-ion channel 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.
In-vivo methods:
(a) Diuretic activity in rats (Lipschitz test): Measurement of urine volume and sodium
excretion in wistar albino rats.
(b) Saluretic activity in rats: Measurement of electrolytes excretion in wistar albino rats.
() Diuretic and saluretic activity in dogs.
(d) Stop flow technique: Ureter of animal is clamped allowing static column of urine to
remain in contact with tubular segments for longer than usual time period and then
collection of urine. This method can be performed in different anaesthetized animals.
(e) Clearance Method: Measurement of water and electrolyte excretion, glomerular
filtration rate (GFR), renal plasma flow etc.
) Micro-puncture Techniques: Measure the changes in tubular fluid re-absorptive rates
or dog) is
and electrolyte concentration. Appropriate animal model (rat, hamster
chosen, according to the site where micro-puncture is to be performed.
Objective: To study the effect of furosemide on the output of urine in rats.
Principle:screening of diuretics for clinical utility is a matter of practical importance. Normal
urine formation in rat like small rodents is very less. Hence, to produce measurable quantity
of urine the rats are hydrated with sufficient quantity of saline. The urine output is increased
after administration of diuretics like urea and furosemide. Increase in volume of urine is
output in drug
trdeeatteerdminanied mals vwith the control animals.
with the help of measuring cylinder and compared the urine
Experimental Pharmacology-II 36
Experiment
The urine volume in rats can be easily measured by placing the rat in a metabolic cage
The metabolic cage is designed to allow measurement of fluid intake and urine formation. l
provides a perfect separation of faeces and urine through the special design of the funne
and of the separation cone. Metabolic cage contains wire mesh at bottom, a funnel to collec
urine, and astainless-stillsieve placed into the funnel toretain faeces and to allow the urine
to pass into the measuring cylinder, which can be used for numerouS qualitative and
quantitative determinations (Fig 4.1).

Fig. 4.1: Metabolic cage for collecting the urine

Requirements:
Animals: Wistar albino rats (150-200 gof either sex).
Drug: Furosemide.
Equipment: Metabolic cages.
Others: Syringe, needle, graduated measuring cylinder and beaker.
Procedure:
1. Randomly divide the rats of either sex with a body
weight between 150-200 g into
two groups (n =6) i.e. control and test.
2. Weigh and number the animals of each group. Keep the
3. Prepare the drug solution by adding 5 mg of animals overnight fasting.
4. Administer normal saline (25 ml/kg) to the
furosemide in 25 ml saline water.
control group rats, and furosemide
solution(25 ml/kg) to the test group rats, orally with the help of oral feeding
Over a period of 10min in divided doses.
needle
5. After administration, place the rats in
different metabolic cages and collect the urine
free from faeces in measuring cylinder.
'6 Note the time after collection of first drop of urine of
each animal in the measuring
cvlinder and also record the total volume of urine collected from each animal in
different time interval up to 5 hours (Table 4.1).
7. Calculate the mean volume of urine collected from different group of animals in
different time intervals and compare the urine output between control and drug
treated groups. Differences among test and control group mean values should be
significantly different, by using statistical methods.
Experimental Pharmacology-II
37
o Experiments
Also calculate the
percentage increase in
group rats as per the following formula. urine volume in test group over control
Urine volume in the test
% increase = group-Urine volume in the control group x 100
Urine volume in the control
group
Observation:
Table 4.1: Effect of furosemide on the
urine output in rats
Body Time (min) Volume of urine collected at %
Sr. after first different time intervals (h) increase
No. weight
Group
9) drop of urine 05 n urine
collected r S hrS. hrs volume
Control 1
(Saline) 2

3
4

5
6

Mean t SEM
Test 1
(Furosemide 2
solution)
3

6
Mean t SEM

Inference:
Furosemide like high efficacy diuretics increases the urine formation by inhibiting the
Na'k*2CIr CO-transporter at ascending part of loop of Henle. After administration of
furosemi de, urine output may significantly increase in test group animals as compared to
control group animals.
Note:
and osmolarity of
Diuresis test in rat is designed to determine Na", K*, Ci, water content
urine. All the Samples of urine are analysed by flame photometry for sodium, potassium and
chloride Content. may be also expressed in Lipschitz value and
Results
Saluretic activity value.
Experimental Pharmacology-I 38

1) Lipschitz test: Lipschitz value for both urine and electrolyte (Na ) excretion
Experimen
calculated as below.
Urine output in test animal
Lipschitz value = Urine output in control animal
Lipschitz value 1 indicates positive effect, and Lipschitz value 2 indicates pote
diuretic activity. P

Lipschitz values for Hydrochlorothiazide 1.8 and for loop diuretics z4

2) Saluretic activity in rats: The folowing parameters may be obtained by calculating t
different ionic contents in the urine.
(a) Estimation of saluretic activity: The sum of sodium and chloride exXcretion
calculated as parameter for saluretic activity.
Saluretic activity = Na + Cl excretion
is calculate
(b) Estimation of natriuretic activity: The ratio of sodium and potassium
for natriuretic activity.
Na
Natriuretic activity = K*
Values greater than 2.0 indicate a favourable natriuretic effect. Ratio greater than 10
indicates a potassium sparing effect.
(c) Estimation of carbonic anhydrase (CA) inhibition: The ratio of CI/Na + K)
calculated to estimate carbonic anhydrase inhibition.
CIr
CA inhibition = Na + K*
Precautions:
1. Handle the animals with care.
2. Clean the metabolic cage properly so that it provides uncontaminated, reliab{
samples for accurate metabolic monitoring and to minimize animal stress.
3. Acclimatize the animals prior to testing in the apparatus.
4. Maintain the laboratory conditions properly.
Questions for the students:
1. Define and classify diuretics.
2. Discuss the mechanism of action of different diuretic agents.
3. Discuss the normal physiology of urine formation.
4. Discuss the various methods used for screening of diuretics.
5. Discuss about Lipschitz test and Salureticactivity test.