EVALUATION OF DIURETICS

GUIDED BY, KIRTI V. PATEL

PREPARED BY JINKAL SHARMA M .PHARM SEM-2 PHARMACOLOGY DEPARTMENT

Introduction Evaluation Techniques In Vitro Methods In Vivo Methods References

INTRODUCTION
Diuretics means
a. Drugs that increase the urine output by an action on kidney b. Also called as natriuretic agents or saluretic agents

Diuretic effect can be achieved by.

a. Direct action on the cells of nephron OR b. Indirectly modifying the content of the filtrate

Classification of diuretics
A.Diuretics acting directly on the cells of the nephron: 1.Loop diuretics: Furosemide Torasemide Bumetanide Piratanide Indacrinone Ethacrynic acid 2.Thiazide diuretics: Cyclopenthiazide Bendrofluazide Hydrochlorthiazide Chlorthalidone Benzthaizide Metalazone

3.K+-Sparing diuretics: Aldosterone antagonists: Spironolactone Eplerenone 4. Na+ reabsorption channel blocker: Triamterene, Amiloride

B.Diuretics that act indirectly by modifying the content of the filtrate: 4.Carbonic anhydrase (CA) inhibitors: Oral administration: Acetazolamide
Methazolamide Dichlorphenamide

Eye drops:

Dorzolamide Brinzolamide Mannitol Isosorbide Sucrose Dextrose Glycerol

5.Osmotic diuretics:

TYPE Carbonic Anhydrase inhibitor Loop diuretic

SITE OF ACTION Proximal Tubule Loop of Henle

MECHANISM OF ACTION Inhibition of carbonic anhydrase Block Na+/K+/2CLsymport Block Na+/CL- symport Block Na+ channel

Thiazide diuretic Distal tubule Potassium sparing Collecting duct Lumen of Osmotic diuretic Creating osmotic draw nephron

In vitro methods
1. 2. 3. 4.

Carbonic anhydrase inhibition Patch clamp technique in kidney cells Perfusion of isolated kidney tubules Isolated perfused kidney

Invivo methods
1. 2. 3. 4. 5. 6.

Diuretic activity in rats (LIPSCHITZ test) Saluretic activity in rats Diuretic and saluretic activity in dogs Clearance methods Stop flow technique Micropuncture techniques in rats

1.

2. 3. 4. 5. 6.

Physiological salt solution: Tyrode, buffer, Krebs solution. Temperature : 37 1 C Basal Tension on lever :500mg-1g Sensitivity :50-500 mV Aeration :Air Contact Time :30sec

1. 2. 3. 4. 5.

Acclimatization: It means adaptation to new environment. It is generally for 15 days. Temperature: 22-28C. Humidity: 35-55 % RH. Light and Dark cycle: 12/12 hours. Food: Rat- Chow diet.

PURPOSE AND RATIONALE

Test is based on H2O and Na+ excretion in test animals and compared to rats treated with high dose of urea. This test is standard,very useful and simple. Lipschitz value is between excretion by test animals and excretion by urea control. Urea is given to increase the urine output as in rats the urine output per day is very less (1-2 ml/rat/day)

PROCEDURE ANIMAL: Rat STRAIN : Wistar SEX : Male WEIGHT : 100-200 g Male Wistar rats (100200 g) are used. Three animals/ group are placed in metabolic cages provided with a wire mesh bottom and a funnel to collect the urine. Stainless-steel sieves are placed in the funnel to retain feces and to allow the urine to pass. The rats are fed with standard diet (Altromin pellets) and water ad libitum.

Fifteen hours prior to the experiment food and water are withdrawn. Three animals are placed in one metabolic cage. For screening procedures 2 groups of 3 animals (6 rats)are used for 1 dose of the test compound. The test compound is applied orally at a dose of 50 mg/kg in 5.0 ml water/kg body weight. Two groups of 3 animals receive orally 1 g/kg urea.

Additionally, 5 ml of 0.9% NaCl solution per 100 g body weight are given by gavage. Urine excretion is recorded after 5 hr and after 24 h. The sodium content of the urine is determined by flame photometry. Active compounds are tested again with lower doses.

EVALUATION

a) LIPSCHITZ value: Urine output of [ test treated / urea treated ] animals b) LIPCSHITZ value: Electrolyte output of [ test treated / urea treated ] animals c) LIPSCHITZ values of 5 hrs and 24 hrs are to be compared

LIPCSCHITZ value: >1 : diuretic activity : >2: potent diuretic value LIPSCHITZ value for loop diuretics: around 1.8 for thiazide diuretics: around 4.0

Calculating this index for 24 hr excretion period as well as for 5 hr indicates duration of diuretic effect. Similar quotients can be calculated for Na+ excretion. DRC established using various doses. For loop diuretics = a steep DRC. CRITICAL ASSESSMENT OF METHOD

Test has been proven to be a standard method and a vary useful tool for screening of potential diuretics.

PERPOSE AND RESIONAL: Clearance method represent indirect method for evaluation of renal function & provide information on site of action of diuretics & other pharmacological agents within nephron

A drug that act solely on proximal convoluted tubule by causing the delivery of increased amount of filtrate to loop of henle & distal convolution, would augment the clearance of solute free water (CH2O) during water diuresis & reabsorption of solute free water (TH2O) during water restriction .

In contrast, drug that inhibit sodium reabsorption in Henles loop would impair both CH2O & TH2O. On the other hand drug that act in distal tubule would reduce CH2O but not TCH2O.

PROCEDURE:

Clearance experiment are performed either in conscious or anesthesised beagle dog under condition of water diuresis and hydropenia. Water diuresis is induced by oral administration of 50 ml of water/kg body weight and maintained by continuous infusion into jugular vein of 2.5% glucose solution and 0.58 % NaCl solution at 0.5 ml/min/ kg body weight.

When water diuresis is well established the glucose infusion is discontinued and control urine samples are collected by urethral catheter. Blood samples are obtained in the middle of each clearance period. After the control period, compounds to be tested are administered and further clearance test are performed. Hydropenia is induced by withdrawing the drinking water 48 h before experiment.

On the day before experiment 0.5 U/ kg body weight vasopresinin oil is injected intramuscularly. On the day of experiment 20 mU/kg vasopressin is injected i.v., followed by infusion of 50 mU/kg per hour vasopressin. To accomplish constant urine flow 5%NaCl solution is infused at 1 ml/ min/kg body wt.up to i.v. administration of compound to be tested, followed by i.v. Infusion of 0.9%NaCl solution at a rate equal to the urine flow. GFR and RPF are measured by clearance of insulin and p-amino hippurate , respectively. Therefore appropriate infusion of insulin and Pamino hipppurate are initiated.

EVALUATION:

o o o o o o o

The following parameters are may be determined: Water and electrolyte excretion GFR RPF CH2O TH2O Plasma renin activity Results of test compound are compared with control and standard drug treated animals.

MODIFICATION OF METHOD: 1. Ronnhedt et al. (1996) described a simple method to perform serial renal clearance studies without urine collection in rats. This was applied to non-radiolabelled para-aminohippurate sodium and iothalamate sodium which were used respectively to estimate renal blood flow and GFR. 2. Gabel et al. (1996) described fast and accurate assay for measuring GFR and effective renal blood flow in conscious rats. An enzymatic method was developed for the determination of insulin and colorimetric method was developed for determination of p-aminohippurate in the plasma and urine of rats.

PURPOSE AND RATIONALE

This procedure is of considerable value in the localization of transport processes along the length of the nephron. During clamping of the ureter, glomerular filtration is grossly reduced. The contact time for the tubular fluid in the respective nephron segments increases, and the concentration of the constituents of tubular fluid should approximate the static-head situation.

After release of the clamp, the rapid passage of the tubular fluid should modify the composition of the fluid only slightly. The first samples should correspond to the distal nephron segment, the latest to glomerular fluid. However,

with introduction of the micropuncture technique, the stop-flow method appears less attractive.

PROCEDURE ANIMAL: Rat STRAIN : Wistar SEX : male/female WEIGHT: 100-200 g

This method can be performed in different animals during anesthesia. The ureter of an animal undergoing intense osmotic diuresis is clamped for several minutes allowing a relatively static column of urine to remain in contact with the various tubular segments for longer than the usual periods of time. Thus, the operation of each segment on the tubular fluid is exaggerated. Then the clamp is released, and the urine is sampled sequentially.

Small serial samples are collected rapidly, the earliest sample representing fluid which had been in contact with the most distal nephron segment. Substances examined are administered along with inulin before the application of uretral occlusion. However, tubular segments downstream from the proximal segments may modify the tubular fluid during its egress.

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.

PURPOSE AND RATIONALE

Excretion of electrolytes is as important as the excretion of water for treatment of peripheral edema and ascites in congestive heart failure and hypertension treatment. Potassium loss has to be avoided. The diuresis test in rats was modified in such a way that potassium and chloride as well as osmolality are determined.

PROCEDURE ANIMAL: Rat STRAIN: Wistar SEX : Male Weight : 100-200 g

Male Wistar rats weighing 100200 g fed with standard diet (Altromin pellets) and water ad libitum are used. Fifteen hours prior to the test, food but not water is withdrawn. Test compounds are applied in a dose of 50 mg/kg orally in 0.5 ml/100 g body weight starch suspension. Three animals are placed in one metabolic cage provided with a wire mesh bottom and a funnel to collect the urine. Two groups of 3 animals are used for each dose of a test drug.

Urine excretion is registered every hour up to 5 h. The 5-h urine is analyzed by flame photometry for sodium and potassium and argentometrically by potentiometrical end point titration for chloride. To evaluate compounds with prolonged effects the 24 h urine is collected and analyzed. Furosemide (25 mg/kg p.o.), Hydrochlorothiazide (25 mg/kg p.o.), triamterene(50 mg/kg p.o.), or amiloride (50 mg/kg p.o.) are used as standards.

EVALUATION
1)The sum of Na+ and Cl excretion is calculated as parameter for saluretic activity. 2) The ratio Na+/K+ is calculated for natriuretic activity. Values greater than 2.0 indicate a favorable natriuretic effect. Ratios greater than 10.0 indicate a potassium-sparing effect. 3) The ratio cl-/Na+ + K+ (ion quotient) is calculated to estimate carbonic anhydrase inhibition

4)Carbonic anhydrase inhibition can be excluded at ratios between 1.0 and 0.8. MODIFICATION OF THE METHOD

adrenalectomized rats treated with DOCA or aldosterone can be utilized to test aldosterone antagonists.

Principle Dogs have been extensively used to study renal physiology and the action of diuretics. 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.

PROCEDURE ANIMAL: Beagle dogs SEX : male/female WEIGHT: 20-25 kg

a) Beagle dogs trained for gavage feeding and hourly catheterization, kept only on water b) Administration of drinking water 20 ml/kg and 4 ml/kg hourly, on the day of experiment(twice collection of urine for initial values measurements) c) Test drug (50mg/kg) and std. drug [Urea (1 g/kg) / Furosemide (5 mg/kg)] : {p.o/i.v.} d) Readings taken at an interval of hour for 6 hrs and final reading at 24 hrs e) Measurements of Na+, K+, Cl- and Osmolarity

EVALUATION
a) Saluretic activity and Osmolarity b) Natriuretic activity c) CA index

ISOLATED TUBULE PREPARATION

PRINCIPLE: Isolated kidney is a good tool for studying proximal tubule, but of limited value for distal tubule function. The kidney can be perfused in situ and isolated in vitro. The isolated kidney can be perfused by a pump using blood or plasma-like solutions. One specific problem of the blood-perfused dog kidney in vitro is its instability. After only 1 h of perfusion, glomerular filtration and renal blood flow decline markedly. It was reported that in situ-perfused isolated dog kidney seems to be more stable..

 In isolated perfused rat kidney plasma-like solutions are used for perfusion. This system by inclusion of a dialyzing unit, provides optimal conditions for maintaining a constant electrolyte composition of the perfusate. However, function of distal tubule is also grossly impaired in this rat model. The isolated kidney does not acidify tubular fluid, and the concentrating ability is reduced.

PROCEDURE: Kidneys are obtained from anaesthetized male rats with a body weight of 300 to 400 g. The donor animals are fasted overnight prior to surgery, but have free access to water. After the abdominal cavity is exposed by a ventricular incision, the right ureter is cannulated with PE-50 polyethylene tubing and heparin is injected into the vena cava (500 U/kg body weight). The venous cannula is introduced into the vena cava below the right renal vein. The right kidney is freed from the perirenal fat, not disrupting the renal capsule.

The renal artery is cannulated via the superior mesenteric artery without interruption of flow. Thereafter, the kidney is continuously perfused with a perfusion solution fed from the gravity system situated 130 cm above the cannula. Ligatures around the renal artery and vena cava above the renal pedicle are tied. The kidney is then removed from the animal and placed in a Plexiglas chamber. A perfusion pressure of 8090 mm Hg in the renal artery is maintained by adjusting the speed of the perfusion Pump.

EVALUATION: After the equilibration period, clearance periods of 20 min are used. Urine samples are collected and perfusate is obtained at midpoint of the clearance period for the evaluation of overall kidney function. For determination of glomerular filtration rate (GFR) and fluid transport, 3H-labelled polyethylene glycol is added to a modified Krebs-Henseleit bicarbonate buffer. Electrolytes are determined in urine by standard flame photometry. Fractional excretions of water, electrolytes and test compounds are calculated.

MODIFICATION OF METHOD: Tarako et al.evaluated oxygen supply and energy state in the isolated perfused rat kidney. Metabolic activities of the isolated perfused rat kidney were described by Nishiitsutsuji-Uwo et al.

CARBONIC ANHYDRASE INHIBITION

PRINCIPLE : Inhibitors block CA that results into blockage of Na+ recovery by inhibiting H+ excretion. PROCEDURE In reaction vessels following solution are added. Phenol red + enzyme (from dog blood) + H2O / Drug After 3 mins CO3-2/HCO3- buffer. CO2 flow 30-45 ml/min

EVALUATION % Inhibition = 1 Tu T i Tu Te
Following parameters are determined. Tu = Time for colour change in absence of enzyme Te = Time for colour change in presence of enzyme Tu Te = Enzyme rate Ti = Enzyme rate in presence of inhibitor

X 100

APPLICATION
Useful to characterize the activity spectrum of sulfonamide diuretics.

PATCH CLAMP TECHNIQUE

PRINCIPLE : Ion channels play an important role in the function of kidney cells. Patch clamp technique allow the investigation of ion channels. It can be applied to cultured cells or isolated cells. Conc. response curve of ion channel inhibitors can be obtained by this method. a) Patch (formation of seal) + Clamp (setting desired voltage) b) Modes of Patch-clamp technique: Cellattached, cell-excised and whole-cell mode

PROCEDURE : ANIMAL: NZ white rabbits SEX : male/female WEIGHT : 2-3 kg

NZ white rabbits are used. segments of late superficial proximal tubules of rabbits kidney are dissected and perfused from one end with a perfusion system. The non cannulated end of the tubule is freely accessible to a patch pipette. Under optical control (differential interference contrast optics with 400x magnification) into patch pipette can be moved through the open end into the tubule lumen and is brought in contact with the brush border membrane

After slight incision of the patch electrode gigaseals form immediately and single pottasium or sodium channels can be recoreded in cell attached or inside out cell excised mode.

EVALUATION

In isolated perfused renal tubules CRC curves of drugs which inhibits ion channel can be obtain with this technique. the apparent Km values for sodium and Lalanine can be recorded.

PRINCIPLE: Isolated kidney is a good tool for studying proximal tubule, but of limited value for distal tubule function. The kidney can be perfused in situ isolated in vitro. The isolated kidney can be perfused by a pump using blood or plasma like solution. After only 1 hr of perfusion, glomerular filtration and renal blood flow decline markly. The isolated kidney does not acidify tubular fluid, and the concentrating ability is reduced.

PROCEDURE: ANIMAL : Rat STRAIN : Wistar SEX : Male Weight : 300-400 g. Kidney are obtained from anesthetized male rats with a body wt of 300 to 400 g.

The donor animals are fasted overnight prior to surgery, but have free access to water. After the abdominal cavity is exposed by a ventricular incision, the right ureter is cannulated with PE-50 polyethylene tubing heparin is injected into the vena cava(500 U/kg body wt). The venous cannula is introduced into vena cava below the right renal vein. The renal artery is cannulated via the superior mesentric artery without interruption of flow.

Thereafter, the kidney is continuously perfused with a perfusion solution fed from the gravity system situated 130 cm above the cannula. The kidney is then removed from the animal and placed in a Plexiglas chamber. A perfusion pressure of 80-90 mm Hg in the renal artery is maintained by adjusting the speed of the perfusion pump.

EVALUATION: After the equilibrium period, clearance periods of 20 min are used. Urine samples are collected and perfusate is obtained at midpoint of the clearance period for the evaluation of overall kidney function.

For determination of glomerular filtration rate (GFR) and fluid transport, 3H-labelled polyethylene glycol is added to a modified Krebs-Henseleit bicarbonate buffer. Electrolytes are determined in urine by standard flame photometry. Fractional excretion of water, electrolytes and test compounds are calculated.

Drug Discovery and Evaluation Pharmacological Assays,Co-Editors:Wolfgang H.Vogel,Bernward A. Schlkens,Jrgen Sandow,Wolfgang F. Vogel,Second Edition Pg No. 323 to 328