Diuretics screening models

Diuretics screening models, Screening of drugs increasing Urination

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/ bumetamide Thiazide diuretics Distal tubule Hydrochlorthiazide / bendroflumethiazide Potassium sparring diuretics Collecting duct Spironolactone/ triampterene / amiloride Osmotic diuretics Lumen of nephron Mannitol

4. Evaluation models In vitro: isolated tubule preparation carbonic anhydrase inhibition patch clamp technique 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. 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. CARBONIC ANHYDRASE INHIBITION PRINCIPLE : Carbonic anhydrase is Zn containing enzyme. H 2 O + CO 2 H 2 CO 3 H + + HCO 3 _ Carbonic anhydrase

9. PROCEDURE: Here the reaction vessel is used CO 2 flow rate is adjusted to 30-45 ml/min. Reaction vessel Phenol Red CA enzyme (from Dog blood) Water/ Water + Drug HCO 3 - buffer

10. Following parameters are determined. T u = Time for color change in absence of enzyme T e = Time for color change in presence of enzyme T u – T e = Enzyme rate T i = Enzyme rate in presence of inhibitor % Inhibition = 1 – (T u _ T e ) - (T i -T e ) T u – T e X 100 EVALUATION :

11. PATCH CLAMP TECHNIQUE 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 μ m Rseal≥10 G Ω Gigaseal Amplifier

13. Cell-attached, cell-excised, whole-cell mode of this technique allow 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 configuration Suction

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 Pull 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 Pull 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. SALURETIC ACTIVITY IN 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. STOP FLOW TECHNIQUE 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, C H2O : Clearance of solute free water during water diuresis T CH2O : Reabsorption of solute free water during water restriction Drug acting on C H2O & T CH2O PCT Increase Both C H2O & T CH2O L OH Impaire both C H2O & T CH2O DCT Reduce C H2O But Not T CH2O

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 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 C H2O & T CH2O Free water clearance ( c H2o ): Amount of the urine excreted in excess that needed to clear salt C H 2 O = V – C osm

31. Free water reabsorption ( T ch2o ) : In the presence of ADH urine is concentrated at that time V < C osm. TC H 2 O = C osm – V Osmolar clearance (c osm ) : Volume of urine containing the solute at the osmolal conc. equal to that of plasma (P osm ) C osm = V(U osm / P osm ) where, V : Urine flow (ml/min.) U osm : Urine osmolarity (mosm/kg.body weight) P osm : Plasma osmolarity (mosm/kg.body weight)

32. MICROPUNCTURE TECHNIQUES 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 3 H 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 = ( V O – V C ) / L Where V O = Single nephron filtration rate. V C = 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) = V tf (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 V tf = 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. MICROINJECTION AND MICROINFUSION MICROINJECTION : Small fluid volume containing 3 H and 14 C 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 (t 1/2 ) and tubule radius (r) is measured. Absolute reabsorptive rate (C) is determined by following equation. C = ( 0.693 π r 2 / t 1/2 ) ·10 -3

52. STATIONARY MICROPERFUSION 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 P H and CO 2 conc. in kidney papilla LIMITATIONS : Handling of small samples is difficult Damage of blood cells causes considerable errors MICROPUNCTURE OF PERITUBULAR 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, C H2O & T CH2O Micropuncture Techniques :Measure the Changes in tubular fluid reabsorptive rates and electrolyte concentration IN VIVO METHODS

60. References Drug discovery and evaluation: pharmacological assays 2 nd edition, by Gerhard vogel . RANG and DALE’S Pharmacology Sixth Edition by H.P. RANG, M.M. DALE, J.M. RITTER, R.J. FLOWER Powerpoint presentation acknowledgement : Mr. vijay patel LMCP

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