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Screening models for
diuretic agents
Presented by: Ms. Hiran Suthar
M.Pharm Sem 1 department of pharmacology
LMCP
11/11/17
Diuretics
• 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.
Types of diuretics
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
Evaluation models
1. In vitro:
isolated tubule preparation
carbonic anhydrase inhibition
patch clamp technique
2. In vivo :
Lipschitz test
saluretic activity in rats
stop flow technique
clearance method
micropuncture technique
Isolated tubule preparation
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
OIL
Suction
Perfusion Pipette
Perfusion Fluid
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 tubule lumen
 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 collecting pipette
 To approximate the in vivo situation, an isotonic rabbit serum is
perfused while the tubule is immersed in a bath of rabbit serum
EVALUTION :
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
CARBONICANHYDRASE INHIBITION
PRINCIPLE :
Carbonic anhydrase is Zn containing enzyme.
H2O + CO2
H2CO3
H+ + HCO3
_
Carbonic anhydrase
PROCEDURE:
Here the reaction vessel is used
CO2 flow rate is adjusted to 30-45 ml/min.
Reaction vessel
Phenol Red
CA enzyme
(from Dog blood)
Water/ Water +
Drug
HCO3
- buffer
 Following parameters are determined.
Tu = Time for color change in absence of enzyme
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 :
PATCH CLAMPTECHNIQUE
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
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 gigaseal
Over 1 μm
Rseal≥10 GΩ
Gigaseal
Amplifier
Cell-attached, cell-excised,whole-cellmode of this techniqueallow
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
Glass pipette
Cell
attached
Electrode
Whole-cell mode :
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
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.
Electrode
Glass pipette
Cell
attached
Inside-out
patch
Discard
cell
Suction
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
Glass pipette
Cell
attached
Electrode Outside-out
patch
Discard
cell
Suction
EVALUTION :
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
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
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….
EVALUATION :
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
SALURETICACTIVITYIN RATS
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
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
EVALUATION:
For Saluretic activity :
Na+ + Cl- excretion calculated
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
STOPFLOWTECHNIQUE
PRINCIPLE :
Useful in localization of transport process along the length of nephron
During clamping of ureter, GFR is grossly reduced
 Contact time for tubular fluid in respective nephron segment increases &
conc. of constituents of tubular fluid approximate the static head situation
 After releasing clamp, rapid passage of tubular fluid modify composition of
fluid only slightly
 Urine is sampled sequentially
PROCEDURE :
This method can be Performed in different animals during
anesthesia
Ureter of animal is clamped allowing static column of urine to
remain in contact with tubular segments for longer than usual
time period
Clamp released & urine is sampled sequentially
Substances examined are administered along with inulin
before the application of occlusion
EVALUTION :
Concentration of inulin and substance under study is measured
 Fractional excretion of substance & inulin are plotted against
cumulative urine volume
CLEARANCE METHOD
PRINCIPLE :
 Method for evaluation of renal function & provide information
about site of action of diuretics
Where,
CH2O : Clearance of solute free water during water diuresis
TCH2O : Reabsorption of solute free water during water restriction
Drug acting on CH2O & TCH2O
PCT Increase Both CH2O & TCH2O
LOH Impaire both CH2O & TCH2O
DCT Reduce CH2O But Not TCH2O
PROCEDURE :
Test may be performed in species from which urine and
plasma can be readily collected.
Low level changes in concentration may be more readily
reflect in rat than in dog
(1) water diuresis :
Rat :
Administration of oral dose of water by gavage representing 5%
of the rat body weight
Placed in Metabolic cage
Urine samples (every 30 mins. For 2 hrs.)
Dog :
Oral administration 50ml water /kg. body weight
Maintained by continuous infusion into jugular vein of 2.5 %
glucose soln.+ 0.58 % NaCL soln. at 0.5 ml/min/kg.body
weight
Urine sample by urethral catheter
On the day of experiment 20 mU/kg vasopressin injected i.v.
followed by infusion of 50 mU/kg vasopressin
To obtain constant urine flow 5% NaCl solution infused up
to i.v. dose of test compound
Withdrawing water 48 hr before experiment &
0.5 U/kg vasopressin in oil injected i.m. before 24 hr
Urine & blood sample collected
2) Hydropenia:
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 hyppurate is used
CH2O & TCH2O
Free water clearance ( cH2o ):
 Amount of the urine excreted in excess that needed to clear
salt
CH2O = V – Cosm
Free water reabsorption ( Tch2o ) :
In the presence of ADH urine is concentrated at that time
V < Cosm.
TCH2O = Cosm – V
Osmolar clearance (cosm) :
 Volume of urine containing the solute at the osmolal conc.
equal to that of plasma (Posm)
Cosm = V(Uosm / Posm)
where,
V : Urine flow (ml/min.)
Uosm : Urine osmolarity (mosm/kg.body weight)
Posm : Plasma osmolarity (mosm/kg.body weight)
MICROPUNCTURETECHNIQUES
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
Animals :
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
 According to the site where micropuncture is going to be
performed, appropriate animal model is chosen.
Anesthesia :
 Thiobarbital and Pentobarbital is injected via i.p. route
Surgical preparation :
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.
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.
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 37
o
C by heating coil
Tubule blockage :
 Done by castor oil stained with sudan black and filtered. It doesn’t
penetrate into Tubular epithelial cells
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
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
DIFFERENT
MICROPUNCTURE
TECHNIQUES
PROCEDURE :
 After identification of segment micropipette is advanced
at 10º-30º angle
 An oil drop is introduced
 Collection is done by gentle suction in such a way that
oil block remains in its place
 Reference point for proximal tubule : Glomerulus
for distal tubule : Macula densa
FREE FLOW MICROPUNCTURE
 Absolute reabsorptive rate ( C ) is calculated by.
C = ( VO – VC ) / L
Where VO = Single nephron filtration rate.
VC = Collection rate (nl/min)
L = Length of tubule
EVALUATION :
Conti…..
Measurement of Single nephron filtration rate :
GFR (ml/min) = V (ml/min) · ( U / P ) In
SNGFR (nl/min) = Vtf (nl/min) · ( tf / P ) In
Where,
GFR = Filtration rate for kidney or inulin clearance
SNGFR = Filtration rate for single nephron
( tf / P ) In = Conc. of inulin in tubular fluid / Conc. of inulin in plasma
(tf / P)In is an index of volume reabsorption at the point of
micropuncture
Vtf = Flow rate of tubule fluid
It is obtained by a timed quantitative collection of the tubule fluid.
• Similar to free flow tech. but limited to collecting system
• An oil filled catheter is fixed to a heatable platinum loop
• Catheter is advanced into a collecting duct at a tip of
papilla
• Pressure in the catheter is lowered and urine collection is
started
MICROCATHETERISATION
MICROINJECTIONAND MICROINFUSION
MICROINJECTION :
Small fluid volume containing 3H and 14C labeled inulin and
labeled tracer of the substance is injected into the segments of
superficial nephron
After introduction of pipette, 30 nl of tracer containing solution
is injected over a period of 15 - 30 sec.
Urine is collected at every 30 sec and tracer is estimated by
scintillation counting.
EVALUTION :
Urinary recovery of the substance and inulin are compared which
allows the estimation of fractional reabsorption of given substance.
In this method pipette is mounted on a micro infusion pump.
With help of this pump micro infusion is made at a very low flow
rate for several minutes.
After completion of micro infusion period, the pipette is left in
the place so that fluid cannot escape from the hole. It prevents loss
of tracer from hole.
During micro infusion and for 20 additional minutes, urine is
collected at every 2 min.
EVALUTION :
Cumulative recovery of the substance and inulin are compared
which allows the estimation of fractional reabsorption of given
substance.
MICROINFUSION :
MICROPURFUSION
It involves perfusion of tubule segment. The decrease or
increase in the concentration of substances under study
indicates influx or out flux
Water reabsorption is monitored through labeled inulin.
An oil filled pipette is injected
into proximal tubule
The micro perfusion pipette is
introduced in one of the
successive loop and the collecting
pipette is inserted in the last
accessible loop
MICROPURFUSION OF PROXIMAL CONVOLUTED TUBULE :
The proximal and distal oil
block is inserted
Micro perfusion is initiated
after insertion of oil blocks
Samples are collected
Conti…..
Insertion of pipette filled with
Lissamine green
An oil filled pipette is inserted into
next loop
Oil block is introduced with
pipette
Perfusion pipette is introduced
To keep the oil block at its place,
tubular fluid proximal to the oil
block is collected by pipette
containing Lissamine green collects.
MICROPERFUSION OF LOOP OF HENLE :
 Oil filled pipette is
withdrawn and is reinserted
in distal tubule. Distal oil
block is injected
 Sampling is done by
using collecting pipette
Conti….
A large drop of stained castor oil is
injected into the proximal tubule
through one barrel of the double
barreled pipette
A drop of saline injected through the
other barrel of the pipette so the drop
of castor oil is splitted
As saline drop is reabsorbed by
tubule epithelium, drops of castor oil
approach each other and saline drop
shrinkage occurs. The rate of
shrinkage of the saline drop is
measured by sequence photography
SHRINKING DROPLET METHOD
EVALUTION :
The time for reabsorption of one half of the volume of the
saline drop (t1/2) and tubule radius (r) is measured.
 Absolute reabsorptive rate (C) is determined by
following equation.
C = ( 0.693 π r 2 / t1/2 ) ·10-3
STATIONARYMICROPERFUSION
PRINCIPLE :
It includes the collection and analysis of the injected fluid
droplet
Fluid reabsorption is retarded or prevented by addition of
solute which cannot be reabsorbed
Alteration in the concentration of substance under study is
used as an indicator of tubule transport
 An oil filled pipette , Pipette with
test fluid , Additional oil filled
pipette are introduced in proximal
tubule
 An oil column is inserted from
first pipette
Conti…..
Splitting of oil column is done by
injection of test fluid from the
second pipette. Fluid droplet is
exposed to tubule epithelium
Further oil is introduced from
pipette to push fluid downstream.
Collection of fluid is done using
third oil pipette
Conti…..
 Peritubular capillary puncture is best performed at
vasa recta in rat or in large capillaries of dog
ADVANTAGES :
 To determine protein conc. in afferent arteriole
 To determine PH
and CO2 conc. in kidney papilla
LIMITATIONS :
 Handling of small samples is difficult
 Damage of blood cells causes considerable errors
MICROPUNCTURE OFPERITUBULAR
CAPILLARIES
PERITUBULAR MICROINJECTION :
 Similar to intratubular microinjection
 Test substance is injected into vasa recta capillaries and
radioactive labeled substance is monitored in urine
LIMITATION : Short contact with tubular epithelium
PERITUBULAR MICROPERFUSION :
 Pipette is inserted into superficial capillaries
ADVANTAGE : Damage to cell is less likely
LIMITATIONS :
 High pressure is required to push the blood
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
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
INVIVO METHODS
References
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) Powerpoint presentation acknowledgement : Mr. vijay patel LMCP
THANK YOU

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Diuretics screening models

  • 1. Screening models for diuretic agents Presented by: Ms. Hiran Suthar M.Pharm Sem 1 department of pharmacology LMCP 11/11/17
  • 2. Diuretics • 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.
  • 3. Types of diuretics 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
  • 4. Evaluation models 1. In vitro: isolated tubule preparation carbonic anhydrase inhibition patch clamp technique 2. In vivo : Lipschitz test saluretic activity in rats stop flow technique clearance method micropuncture technique
  • 5. Isolated tubule preparation 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
  • 6. OIL Suction Perfusion Pipette Perfusion Fluid 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 tubule lumen  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 collecting pipette  To approximate the in vivo situation, an isotonic rabbit serum is perfused while the tubule is immersed in a bath of rabbit serum
  • 7. EVALUTION : 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
  • 8. CARBONICANHYDRASE INHIBITION PRINCIPLE : Carbonic anhydrase is Zn containing enzyme. H2O + CO2 H2CO3 H+ + HCO3 _ Carbonic anhydrase
  • 9. PROCEDURE: Here the reaction vessel is used CO2 flow rate is adjusted to 30-45 ml/min. Reaction vessel Phenol Red CA enzyme (from Dog blood) Water/ Water + Drug HCO3 - buffer
  • 10.  Following parameters are determined. Tu = Time for color change in absence of enzyme 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 :
  • 11. PATCH CLAMPTECHNIQUE 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
  • 12. 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 gigaseal Over 1 μm Rseal≥10 GΩ Gigaseal Amplifier
  • 13. Cell-attached, cell-excised,whole-cellmode of this techniqueallow 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 Glass pipette Cell attached Electrode
  • 14. Whole-cell mode : 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
  • 15. 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. Electrode Glass pipette Cell attached Inside-out patch Discard cell Suction
  • 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 Glass pipette Cell attached Electrode Outside-out patch Discard cell Suction
  • 17. EVALUTION : 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
  • 18. 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
  • 19. 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….
  • 20. EVALUATION : 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
  • 21. SALURETICACTIVITYIN RATS 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
  • 22. 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
  • 23. EVALUATION: For Saluretic activity : Na+ + Cl- excretion calculated 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
  • 24. STOPFLOWTECHNIQUE PRINCIPLE : Useful in localization of transport process along the length of nephron During clamping of ureter, GFR is grossly reduced  Contact time for tubular fluid in respective nephron segment increases & conc. of constituents of tubular fluid approximate the static head situation  After releasing clamp, rapid passage of tubular fluid modify composition of fluid only slightly  Urine is sampled sequentially
  • 25. PROCEDURE : This method can be Performed in different animals during anesthesia Ureter of animal is clamped allowing static column of urine to remain in contact with tubular segments for longer than usual time period Clamp released & urine is sampled sequentially Substances examined are administered along with inulin before the application of occlusion EVALUTION : Concentration of inulin and substance under study is measured  Fractional excretion of substance & inulin are plotted against cumulative urine volume
  • 26. CLEARANCE METHOD PRINCIPLE :  Method for evaluation of renal function & provide information about site of action of diuretics Where, CH2O : Clearance of solute free water during water diuresis TCH2O : Reabsorption of solute free water during water restriction Drug acting on CH2O & TCH2O PCT Increase Both CH2O & TCH2O LOH Impaire both CH2O & TCH2O DCT Reduce CH2O But Not TCH2O
  • 27. PROCEDURE : Test may be performed in species from which urine and plasma can be readily collected. Low level changes in concentration may be more readily reflect in rat than in dog (1) water diuresis : Rat : Administration of oral dose of water by gavage representing 5% of the rat body weight Placed in Metabolic cage Urine samples (every 30 mins. For 2 hrs.)
  • 28. Dog : Oral administration 50ml water /kg. body weight Maintained by continuous infusion into jugular vein of 2.5 % glucose soln.+ 0.58 % NaCL soln. at 0.5 ml/min/kg.body weight Urine sample by urethral catheter
  • 29. On the day of experiment 20 mU/kg vasopressin injected i.v. followed by infusion of 50 mU/kg vasopressin To obtain constant urine flow 5% NaCl solution infused up to i.v. dose of test compound Withdrawing water 48 hr before experiment & 0.5 U/kg vasopressin in oil injected i.m. before 24 hr Urine & blood sample collected 2) Hydropenia:
  • 30. 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 hyppurate is used CH2O & TCH2O Free water clearance ( cH2o ):  Amount of the urine excreted in excess that needed to clear salt CH2O = V – Cosm
  • 31. Free water reabsorption ( Tch2o ) : In the presence of ADH urine is concentrated at that time V < Cosm. TCH2O = Cosm – V Osmolar clearance (cosm) :  Volume of urine containing the solute at the osmolal conc. equal to that of plasma (Posm) Cosm = V(Uosm / Posm) where, V : Urine flow (ml/min.) Uosm : Urine osmolarity (mosm/kg.body weight) Posm : Plasma osmolarity (mosm/kg.body weight)
  • 32. MICROPUNCTURETECHNIQUES 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
  • 33. Animals : 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  According to the site where micropuncture is going to be performed, appropriate animal model is chosen. Anesthesia :  Thiobarbital and Pentobarbital is injected via i.p. route
  • 34. Surgical preparation : 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.
  • 35. 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.
  • 36. 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 37 o C by heating coil Tubule blockage :  Done by castor oil stained with sudan black and filtered. It doesn’t penetrate into Tubular epithelial cells
  • 37. 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
  • 38. 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 DIFFERENT MICROPUNCTURE TECHNIQUES
  • 39. PROCEDURE :  After identification of segment micropipette is advanced at 10º-30º angle  An oil drop is introduced  Collection is done by gentle suction in such a way that oil block remains in its place  Reference point for proximal tubule : Glomerulus for distal tubule : Macula densa FREE FLOW MICROPUNCTURE
  • 40.  Absolute reabsorptive rate ( C ) is calculated by. C = ( VO – VC ) / L Where VO = Single nephron filtration rate. VC = Collection rate (nl/min) L = Length of tubule EVALUATION :
  • 41. Conti….. Measurement of Single nephron filtration rate : GFR (ml/min) = V (ml/min) · ( U / P ) In SNGFR (nl/min) = Vtf (nl/min) · ( tf / P ) In Where, GFR = Filtration rate for kidney or inulin clearance SNGFR = Filtration rate for single nephron ( tf / P ) In = Conc. of inulin in tubular fluid / Conc. of inulin in plasma (tf / P)In is an index of volume reabsorption at the point of micropuncture Vtf = Flow rate of tubule fluid It is obtained by a timed quantitative collection of the tubule fluid.
  • 42. • Similar to free flow tech. but limited to collecting system • An oil filled catheter is fixed to a heatable platinum loop • Catheter is advanced into a collecting duct at a tip of papilla • Pressure in the catheter is lowered and urine collection is started MICROCATHETERISATION
  • 43. MICROINJECTIONAND MICROINFUSION MICROINJECTION : Small fluid volume containing 3H and 14C labeled inulin and labeled tracer of the substance is injected into the segments of superficial nephron After introduction of pipette, 30 nl of tracer containing solution is injected over a period of 15 - 30 sec. Urine is collected at every 30 sec and tracer is estimated by scintillation counting. EVALUTION : Urinary recovery of the substance and inulin are compared which allows the estimation of fractional reabsorption of given substance.
  • 44. In this method pipette is mounted on a micro infusion pump. With help of this pump micro infusion is made at a very low flow rate for several minutes. After completion of micro infusion period, the pipette is left in the place so that fluid cannot escape from the hole. It prevents loss of tracer from hole. During micro infusion and for 20 additional minutes, urine is collected at every 2 min. EVALUTION : Cumulative recovery of the substance and inulin are compared which allows the estimation of fractional reabsorption of given substance. MICROINFUSION :
  • 45. MICROPURFUSION It involves perfusion of tubule segment. The decrease or increase in the concentration of substances under study indicates influx or out flux Water reabsorption is monitored through labeled inulin.
  • 46. An oil filled pipette is injected into proximal tubule The micro perfusion pipette is introduced in one of the successive loop and the collecting pipette is inserted in the last accessible loop MICROPURFUSION OF PROXIMAL CONVOLUTED TUBULE :
  • 47. The proximal and distal oil block is inserted Micro perfusion is initiated after insertion of oil blocks Samples are collected Conti…..
  • 48. Insertion of pipette filled with Lissamine green An oil filled pipette is inserted into next loop Oil block is introduced with pipette Perfusion pipette is introduced To keep the oil block at its place, tubular fluid proximal to the oil block is collected by pipette containing Lissamine green collects. MICROPERFUSION OF LOOP OF HENLE :
  • 49.  Oil filled pipette is withdrawn and is reinserted in distal tubule. Distal oil block is injected  Sampling is done by using collecting pipette Conti….
  • 50. A large drop of stained castor oil is injected into the proximal tubule through one barrel of the double barreled pipette A drop of saline injected through the other barrel of the pipette so the drop of castor oil is splitted As saline drop is reabsorbed by tubule epithelium, drops of castor oil approach each other and saline drop shrinkage occurs. The rate of shrinkage of the saline drop is measured by sequence photography SHRINKING DROPLET METHOD
  • 51. EVALUTION : The time for reabsorption of one half of the volume of the saline drop (t1/2) and tubule radius (r) is measured.  Absolute reabsorptive rate (C) is determined by following equation. C = ( 0.693 π r 2 / t1/2 ) ·10-3
  • 52. STATIONARYMICROPERFUSION PRINCIPLE : It includes the collection and analysis of the injected fluid droplet Fluid reabsorption is retarded or prevented by addition of solute which cannot be reabsorbed Alteration in the concentration of substance under study is used as an indicator of tubule transport
  • 53.  An oil filled pipette , Pipette with test fluid , Additional oil filled pipette are introduced in proximal tubule  An oil column is inserted from first pipette Conti…..
  • 54. Splitting of oil column is done by injection of test fluid from the second pipette. Fluid droplet is exposed to tubule epithelium Further oil is introduced from pipette to push fluid downstream. Collection of fluid is done using third oil pipette Conti…..
  • 55.  Peritubular capillary puncture is best performed at vasa recta in rat or in large capillaries of dog ADVANTAGES :  To determine protein conc. in afferent arteriole  To determine PH and CO2 conc. in kidney papilla LIMITATIONS :  Handling of small samples is difficult  Damage of blood cells causes considerable errors MICROPUNCTURE OFPERITUBULAR CAPILLARIES
  • 56. PERITUBULAR MICROINJECTION :  Similar to intratubular microinjection  Test substance is injected into vasa recta capillaries and radioactive labeled substance is monitored in urine LIMITATION : Short contact with tubular epithelium
  • 57. PERITUBULAR MICROPERFUSION :  Pipette is inserted into superficial capillaries ADVANTAGE : Damage to cell is less likely LIMITATIONS :  High pressure is required to push the blood
  • 58. 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
  • 59. 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 INVIVO METHODS
  • 60. References 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) Powerpoint presentation acknowledgement : Mr. vijay patel LMCP