PRECLINICAL SCREENING MODELS In vitro methods • Patch clamp technique in kidney cells • Perfusion of isolated kidney tubules • Isolated perfused kidney In vivo methods • Diuretic activity in rats (LIPSCHITZ test) • Saluretic activity in rats • Diuretic and saluretic activity in dogs • Clearance methods • Micro puncture techniques in the rat • Stop-flow technique

1. PRECLINICAL SCREENING
MODEL OF DIURETICS AND
SALURETIC ACTIVITY
1
NAME :- YADAV ABHAYKUMAR ATMAPRASAD
FIRST YEAR MASTER OF PHARMACY
(DEPARTMENT OF PHARMACOLOGY)
GUIDE : DR (MRS) .VANITA KANASE

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3. DIURETICS
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• Elimination of excess urine (more than normal levels) is
termed as diuresis and the drugs that facilitate this process
are called diuretics
• Urine consists of metabolic waste materials, water and some
electrolytes.
• Saluretics are agents that facilitate the removal of salt or
especially sodium ion.

4. TYPES OF
DIURETICS
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CLASS OF DRUG LOCATION EXAMPLE
Carbonic anhydrase
inhibitors
Proximal tubule Acetazolamide
Loop diuretics Loop of Henle Furosemide/bumetamid
e
Thiazide diuretics Distal tubule Hydrochlorthiazide/bend
roflumethiazide
Potassium sparring
diuretics
Collecting duct Spironolactone/triampte
rene/amiloride
Osmotic diuretics Lumen of nephron Mannitol

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6. EVALUATIONMODELS
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INVITRO METHODS
1. CARBONIC ANHYDRASE INHIBITION
2. PATCH CLAMP TECHNIQUE IN KIDNEY CELLS
3. PERFUSION OF ISOLATED KIDNEY TUBULES
INVIVO METHODS
1. DIURETIC ACTIVITY IN RATS (LIPSCHITZ TEST)
2. SALURETIC ACTIVITY IN RATS
3. DIURETIC AND SALURETIC ACTIVITY IN DOGS
4. MICROPUNCTURE TECHNIQUES IN RATS
5. STOP FLOW TECHNIQUE

7. INVITRO
METHODS
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8. ISOLATED TUBULE PREPARATION
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PRINCIPLE :
Measurement of change in concentration of solutes in
perfusion fluid
PROCEDURE :
This technique has been used in the kidney segments of
several species like rat, mouse, hamster, rabbit etc.
The thin (<1 mm ) tubule segments are dissected from kidney
slices
Segment is transferred into perfusion chamber

9. OIL Perfusion Pipette
Perfusion Fluid
Suction
Accumulated
Fluid
Narrow Pipette
For Sampling
 To perfuse a suitable tubule, one end of the tubule is holded by
micropipette
 A perfusion pipette is inserted into tubulelumen
 The other end of the tubule is sucked into collecting pipette
 The oil inside the collecting pipette prevents the evaporation
 All the accumulated fluid is collected at periodic intervals by
inserting a narrow calibrated pipette in the collectingpipette
 To approximate the in vivo situation, an isotonic rabbit serum is
perfused while the tubule is immersed in a bath of rabbit serum
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10. EVALUTION:
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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 is detected from the
appearance of the marker in the external bath

11. CARBONIC ANHYDRASE INHIBITION
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H+
_
+ HCO3
PRINCIPLE :
Carbonic anhydrase is Zn containing enzyme.
H2O + CO2
H2CO3

12. PROCEDURE:
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Here the reaction vessel is used
CO2 flow rate is adjusted to 30-45ml/min.
Reaction vessel
Phenol Red
CA enzyme
(from Dog blood)
Water/ Water +
Drug 3HCO - buffer

13.  Following parameters are determined.
Tu = Time for color change in absence ofenzyme
Te= Time for color change in presence of enzyme
Tu – Te= Enzyme rate
Ti = Enzyme rate in presence of inhibitor
% Inhibition = 1 –
(Tu
_Te) - (Ti -Te)
Tu – Te
X 100
EVALUATION:
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14. PATCH CLAMP TECHNIQUE
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PRINCIPLE :
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

15. PROCEDURE:
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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 gigaseal
Over 1 μm
Gigaseal
Amplifier

16. CELL-ATTACHED,WHOLE-CELL MODE OF THIS
TECHNIQUE ALLOW
INVESTIGATION OF ION CHANNELS
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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
Glass pipette
Cell
attached
Electrode

17. WHOLE-CELL
MODE :
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From the initial cell-attached configuration, additional suction
is applied to rupture the cell membrane, thus providing access to
the intracellular space of the cell.
The soluble contents of the cell will slowly be replaced by the
contents of the electrode
Whole-cell mode records currents through all channels from the
entire cell membrane at once
Electrode
Glass pipette
Cell
attached
Whole-cell
configurationSuction

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19. EVALUTION:
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Concentration response curve of the drugs which inhibit ion
channels can be obtained
Single ion channel events studied by cell-attached &
cell-excised technique
Co transport system only studied by whole cell patch clamp
technique as transport rate of single event is too small to detect

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21. INVIVO
METHODS
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22. PRINCIPLE : Based on water & Na+ excretion in test animal &
compared to rats treated with std. drug
PROCEDURE:
Male Wistar rats weighing 100-200 g are used & placed
in Metabolic cages
 Metabolic cages :
 Wire mesh at bottom
 Funnel to collect urine
Stainless-still sieves are placed into the funnel
to retain feces and to allow the urine to pass
 Rats are fed with std diet & water
15 hr. before the experiment, food & water are withdrawn
LIPSCHITZ TEST
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25. Urine excretion recorded up to 5 hr & 24 hr.
Na+ content of urine estimated by flame photometer & Urine
vol. excreted calculated for each group
Test
2 group(6 rats)
Std
2 group(6 rats)
 Animals are divided as treated with test and standard drug
CONTI
….
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26. EVALUATION :
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Results expressed in LIPSCHITZ value for both urine
excretion & for electrolyte
LIPSCHITZ value = Urine output in test animal
Urine output in std. drug treated animal
Lipschitz value ≥ 1 indicates positive effect
Lipschitz value ≥ 2 potent diuretic activity
For studying prolonged effect, 24 hr urine sample collected &
analyzed
 For Saluretic drugs
Hydrochlorothiazide = 1.8
For loop diuretics ≥ 4

27. SALURETICACTIVITYIN RATS
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PRINCIPLE :
Excretion of electrolytes is important for the treatment of
peripheral edema, CHF, hypertension
so, need to develop diuretic with saluretic & K+ sparing
effect
Diuresis test in rats is designed to determine Na+ ,K+ ,Cl-
, water content & osmolarity of urine
Ratio b/w electrolytes can be calculated indicating
carbonic anhydrase inhibition or K+ sparing effect

28. PROCEDURE:
Male Wistar Rats weighing 100-200 g are fed with std diet &
water
 15 hrs prior to experiment food is withdrawn but not water
 3 animals placed in one metabolic cage
Urine excretion measured every hr up to 5 hr & collected urine is
analyzed for Na+ ,K+ & Cl-
 2 groups, each of 3 rats used for test & std drug
Furosemide, HCT, triamterene & amiloride are used as
standards
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29. EVALUATION:
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For Saluretic activity :
Na+ + Cl- excretioncalculated
For Natriuretic activity : Na+ is calculated
K+
Natriuretc effects > 2
Potassium sparing effect > 10
For estimating CA inhibition :
Cl- is calculated
Na+ +K+
Inhibition can be excluded at ratio between 1 to 0.8 with
decreasing ratio slight to strong carbonic anhydrase inhibition
can be assumed

30. 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.
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31. 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.
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32.  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.
 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.
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33. 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. 33

34. MICROPUNCTURE TECHNIQUES
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PRINCIPLE :
Micropuncture technique allows localization of tubular
site of action
Measure the Changes in tubular fluid reabsorptive rates
and electrolyte concentration and there by infer
mechanism of action

35. ANIMALS:
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According to the site where micropuncture is going to be
performed, appropriate animal model is chosen.
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
Anesthesia :
 Thiobarbital and Pentobarbital is injected via i.p. route

36. SURGICAL
PREPARATION :
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Rat fasted for 12-18 hrs & anaesthetized by pentobarbital
Tracheotomy is performed
Jugular vein cannulation for infusion
Femoral artery is catheterized for obtaining blood
One ureter & bladder is catheterized to collect urine
Bolus injection of inulin 3H given, followed by 0.85% NaCl
solution.
Left kidney exposed by flank incision in which 3 cm cut made at left
subcostal margin & cavity filled with oil at 37°C.

37. After 45 mins. the control puncture of tubules is performed
Micropipette is used to collect tubular fluid sample
Control period followed by the test period & after equilibration of
30 min with test compound micropuncture is performed and
tubular fluid collected
Microscope :
 A stereo microscope and light source are required
with magnification range (10X100).
Zoom optic – most convenient for changing
magnification.
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38. Micropipette : Diameter from 6 nm to 12 nm
Micro perfusion Pump : Delivery rates – 1 to 40 nl/min
Temp control : Perfusing oil is kept at 37o
C by heating coil
oesn’t
Tubule blockage :
Done by castor oil stained with sudan black and filtered. It d
penetrate into Tubular epithelial cells
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39. ADVANTAGE:
Allow localization & analysis of renal transport in vivo
DISADVANTAGE:
Require anesthesia & extensive surgery
Exposure of nephron may alter its function
Increase in luminal pressure lead to decrease in glomerular
function
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40. 1. Free flow micro puncture
2. Micro catheterisation
3. Microinjection and micro infusion
4. Micro perfusion
5. Shrinking droplet method
6. Stationary micro perfusion
7. Peritubular micro perfusion and microinjection
MICROPUNCTURE
TECHNIQUES
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41. IN VITRO METHODS
Isolated tubule preparation : Measurement of change in
concentration of solutes in perfusion fluid
Carbonic anhydrase inhibition : Inhibition of carbonic
anhydrase enzyme
Patch clamp technique : Measurement of the current
across the ion channel
SUMMARY
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42. Lipschitz Test : Measurement of urine volume and sodium
excretion
Saluretic activity in rats : Measurement of electrolytes
excretion
Stop flow technique : Allow urine to remain in nephron for
long period and then collection of urine
Clearance Method : Measurement of Water and electrolyte
excretion, GFR, Renal plasma flow, CH2O &TCH2O
Micropuncture Techniques :Measure the Changes in tubular
fluid reabsorptive rates and electrolyte concentration
IN VIVOMETHODS
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43. RESEARCH PAPERS OF DIURETIC ACTIVIES
 Evaluation of Diuretic Activity of Alcoholic Extract of
Roots of Cissampelos Pareira in Albino Rats
 Diuretic Activity of Leaves of Garcinia Cambogia in
Rats
 Diuretic activity of different extracts of Biophytum
sensitivum (Linn.) DC
 Diuretic Activity of the Aqueous Extract Leaves of Ficus
glumosa Del. (Moraceae) in Rats
 Evaluation of the diuretic activity of the aqueous and
80% methanol extracts of Ajuga remota Benth
(Lamiaceae) leaves in mice
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44. REFERENCES
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1) Drug discovery and evaluation: pharmacological assays 2nd edition, by Gerhard
vogel.
2) RANG and DALE’S Pharmacology Sixth Edition by H.P.RANG, M.M. DALE, J.M.
RITTER, R.J. FLOWER
3) https://brilliantnurse.com/nclex-diuretics/
4) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4080013/
5) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3267310/
6) www.ayujournal.org/article.asp?issn=0974-
8520;year=2015;volume=36;issue=3;spage=356;epage=358;aulast=Ch
andavarkar
7) Powerpoint presentation acknowledgement : DR.(MRS) VANITAKANASE, OCP

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46. THANKYOU
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