About Authors:
*Nilesh Sovasia, Prof.Sanjeev Thacker, Arshad Hala
Seth G.L.Bihani S.D.College Of Technical Education,
Institute Of Pharmaceutical Science & Drug Research,
Sri Ganganagar,Rajasthan,India
*nilesh.sovasia@yahoo.com
ABSTRACT
Diuretic agents are very useful for several critical conditions like hypertension, heart failure, renal failure, nephrotic syndrome, and cirrhosis.The various methods for screening of Diuretic agents provides useful tool to evaluate the safety and effectiveness of the drugs.It is also useful for determining the dose lavel of particular class of diuretic agents.
REFERENCE ID: PHARMATUTOR-ART-1472
INTRODUCTION:
Diuretics increase the rate of urine flow and sodium excretion and are used to adjust the volume and/or composition of body fluids in a variety of clinical situations, including hypertension, heart failure, renal failure, nephrotic syndrome, and cirrhosis.
By definition, diuretics are drugs that increase the rate of urine flow; however, clinically useful diuretics also increase the rate of excretion of Na+ (natriuresis) and of an accompanying anion, usually Cl-. NaCl in the body is the major determinant of extracellular fluid volume, and most clinical applications of diuretics are directed toward reducing extracellular fluid volume by decreasing total-body NaCl content. A sustained imbalance between dietary Na+ intake and Na+ loss is incompatible with life. A sustained positive Na+ balance would result in volume overload with pulmonary edema, and a sustained negative Na+ balance would result in volume depletion and cardiovascular collapse. Although continued administration of a diuretic causes a sustained net deficit in total-body Na+, the time course of natriuresis is finite because renal compensatory mechanisms bring Na+ excretion in line with Na+ intake, a phenomenon known as diuretic braking. These compensatory, or braking, mechanisms include activation of the sympathetic nervous system, activation of the renin-angiotensin-aldosterone axis, decreased arterial blood pressure (which reduces pressure natriuresis), hypertrophy of renal epithelial cells, increased expression of renal epithelial transporters, and perhaps alterations in natriuretic hormones such as atrial natriuretic peptide Historically, the classification of diuretics was based on a mosaic of ideas such as site of action (loop diuretics), efficacy (high-ceiling diuretics), chemical structure (thiazide diuretics), similarity of action with other diuretics (thiazidelike diuretics), effects on potassium excretion (potassium-sparing diuretics), etc. However, since the mechanism of action of each of the major classes of diuretics is now well understood.
Diuretics not only alter the excretion of Na+ but also may modify renal handling of other cations (e.g., K+, H+, Ca2+, and Mg2+), anions (e.g., Cl-, HCO3 -, and H2PO4 -), and uric acid. In addition, diuretics may alter renal hemodynamics indirectly.1
BIOLOGICAL EVALUATION OF DIURETIC AGENTS:
1.Diuretic and Saluretic Activity
1.1 In vitro Methods
1.1.1 Carbonic Anhydrase Inhibition in vitro:
PURPOSE AND RATIONALE
Acetazolamide (Diamox®) was one of the first synthetic non-mercurial diuretics. The mode of action was found to be inhibition of carbonic anhydrase. Carbonic anhydrase is a zinc-containing enzyme that catalyzes the reversible hydration (or hydroxylation) of CO2 to form H2CO3 which dissociates non-enzymatically into HCO3 - and H+.Although many methods to measure carbonic anhydrase activity have been developed, the micro method described by Maren (1960) is relatively simple, sensitive and reliable. The enzyme source are red cells, a rich source of the same isoenzymes found in the eye.1,4
PROCEDURE
Materials and solutions:
· Phenol red indicator solution
12.5 mg phenol red/liter 2.6 mM NaHCO3, pH 8.3 + 218 mM Na2CO3
· 1 M sodium carbonate/bicarbonate buffer, pH 9.8
· Enzyme: Carbonic anhydrase from dog blood; Blood is collected into a heparinized tube and diluted 1:100 with deionized water.
· Equipment
– Reaction vessel
– Monostat bench mounted flowmeter
– 30% CO2 – M&G Gases, Branchburg, NJ, USA
Assay
CO2 flow rate is adjusted to 30 (45) ml/min. The following solutions are added to the reaction vessel:
400 μl phenol red indicator solution
100 μl enzyme
200 μl H2O or appropriate drug concentration after
3 min for equilibration:
100 μl carbonate/bicarbonate buffer is added.
The following parameters are determined in duplicate samples:
Tu = (uncatalyzed time ) = time for the color change to occur in the absence of enzyme.
Te = (catalyzed time) = time for the color change to occur in the presence of the enzyme.
Tu – Te = enzyme rate
Ti = enzyme rate in the presence of various concentrations of inhibitor
EVALUATION
Percent inhibition of carbonic anhydrase is calculated according to the following formula:
% Inhibition = 1- (Tu - Te) - (Ti - Te)
---------------------------
Tu - Te
Standard data:
• Compound IC50 [M]
• Acetazolamide 9.0 × 10–9
• Chlorothiazide 9.0 × 10–7
CRITICAL ASSESSMENT OF THE METHOD
Determination of carbonic anhydrase inhibition is of value to characterize the activity spectrum of sulfonamide diuretics.
MODIFICATIONS OF THE METHOD
Landolfi et al. (1997) reported a modified procedure for the measurement of carbonic anhydrase activity. The measure of carbonic anhydrase activity is based on the rate of CO2 hydration by the enzyme. Such transformation was monitored by a procedure which consists in the measure of time necessary for the pH of an appropriate buffer to decrease from 8 to 7.5 in the presence of a constant CO2 flow: such time period is dosedependently reduced by the addition of the enzyme and further modified in the presence of carbonic anhydrase inhibitory compounds.1,4
1.1.2 Patch Clamp Technique In Kidney Cells
PURPOSE AND RATIONALE
In the different parts of the kidney (proximal tubules, distal tubules, collecting ducts) fluid is reabsorbed and substances may be transported either from the tubule lumen to the blood side (reabsorption) or vice versa (secretion). Besides active transport and coupled transport systems, ion channels play an important role in the function of kidney cells. The various modes of the patch clamp technique (cell-attached, cell-excised, whole-cell mode)allow the investigation of ion channels.
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PROCEDURE
The patch clamp technique can be applied to cultured kidney cells (Merot et al. 1988), to freshly isolated kidney cells (Hoyer and Gögelein 1991) or to cells of isolated perfused kidney tubules (Gögelein and Greger 1984). The latter method shall be described in more detail. Segments of late superficial proximal tubules of rabbit kidney are dissected and perfused from one end with a perfusion system (Burg et al. 1966; Greger and Hampel 1981). The non-cannulated end of the tubule is freely accessible to a patch pipette. Under optical control (differential interference contrast optics with 400× magnification) the 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 suction of the patch electrode, gigaseals form instantaneously and single potassium or sodium channels can be recorded in the cell-attached or inside- out cell-excised mode. In order to obtain exposed lateral cell membranes suitable to the application of the patch clamp method, pieces of the tubule are torn off by means of a glass pipette (diameter about 40 μm). As to facilitate the tearing off, the tubules are incubated for about 5 min in 0.5 g/l collagenase (Sigma, C 2139) at room temperature.
After tearing off part of the cannulated tubule, clean lateral cell membranes are exposed at the non-cannulated end. The patch pipette can be moved to the lateral cell membrane and gigaseals can be obtained. It was possible, to investigate potassium channels and nonselective cation channels in these membranes. As cells are still part of an epithelial layer and, therefore, are intracellularly coupled, the whole-cell technique is not appropriate in this preparation. On the other hand, cotransport systems can only be investigated by the whole-cell method because the transport rate of a single event is much too small to be resolved in a similar manner as single ion channel events. Consequently, cells of rabbit proximal tubules are isolated as described in detail elsewhere.After cervical dislocation the kidneys are rapidly excised and placed in ice-cold solution [mmol/l]: 150 K-cyclamate, 10 HEPES, 1 CaCl2, 1 MgCl2, pH 7.4. The following steps are performed on ice: After decapsulation, superficial cortical slices of about 0.5 mm thickness are dissected and minced with a scalpel. The tissue is homogenized in a Dounce homogenizer by three strokes with a loose-fitting pestle. The homogenate is then poured through graded sieves (250, 75 and 40 μm) to obtain a population of single cells. Since the predominant tubule section of the cortex of the rabbit kidney is the pars convoluta of the proximal tubule, it can be concluded that the majority of the isolated cells in the cell suspension are of proximal tubule origin. By light microscopy cells are identified by long microvilli distributed over the entire cell surface and can easily be discriminated from remaining erythrocytes, cell detritus and tubular fragments. By application of the whole-cell mode of the patch clamp technique to freshly isolated cells of convoluted proximal tubules, the sodium-alanine cotransport system could be investigated in detail.
EVALUATION
In isolated perfused renal tubules, concentration response curves of drugs which inhibit ion channels can be obtained with the patch clamp technique. In isolated cells of the proximal tubule, the whole-cell mode of the patch clamp technique enables the investigation of the sodium-alanine cotransport system. The apparent Km values for sodium and L-alanine can be recorded.
MODIFICATIONS OF THE METHOD
Schlatter (1993) recorded membrane voltages of macula densa cells with the fast or slow whole-cell patch-clamp method. The effects of diuretics and the conductance properties of these cells were examined.1,5
1.1.3 Perfusion Of Isolated Kidney Tubules
PURPOSE AND RATIONALE
The various tubule segments: proximal tubule (PT, S1–S3); descending thin limb of the loop of Henle (DTL); ascending thin limb of the loop of Henle (ATL); thick ascending limb of the loop of Henle (TAL); distal convoluted tubule (DCT); connecting tubule (CNT); cortical collecting duct (CCD); medullary collecting duct (MCD); papillary collecting duct (PCD) have different functional properties. The in vitro perfusion of isolated tubule segments is the method of choice if one has to identify the site and the mechanism of action of a pharmacological agent which has been shown to act on kidney function in clearance and micropuncture studies.
PROCEDURE
After its invention by Burg et al. (1966) this technique has been used successfully in the kidney tubule segments of several species: man; rabbit; rat; mouse; hamster; snake; birds etc. The tubule segments are dissected from thin kidney slices (<1 mm thickness). Usually dissection can be done using sharpened forceps or needles without the addition of proteases (collagenase). The segment is identified by its anatomical location and by its appearance. A 20–50× lens is used for dissection. Dark field illumination is helpful for the identification of the segment under study. PT, TAL, DCT, CNT, CCD, MCD can all be dissected quite easily. The dissection of DTL, ATL and PCD is much more difficult because these segments are damaged easily by the mechanical dissection. Dissection is usually performed at 4 °C in a Ringer type solution. The dissected segment is transferred into the perfusion chamber by a transfer pipette. The perfusion chamber is mounted in the stage of an inverted microscope (20–400×). The chamber is usually kept at 37 °C, and the bath perfusate is also preheated to this temperature. The bath perfusate will depend on the tubule segment under study. In most instances it will contain HCO3 – and will be bubbled with CO2. The metabolic substrate will be acetate for PT and D-glucose for TAL, CCD etc. The actual perfusion is performed with two sets of concentric glass pipettes, one set at the perfusion end; and one at the collection end of the segment. These pipettes are manufactured with special glass forges. The most refined one has been designed by Hampel and Greger. The glass tube is rotated at approximately 1 rps, is moved in perpendicular direction by a remote control, and the heating filament is moved in xy direction also by a remote control. The shaping of the glass is observed continuously by a lens (5–50×). The pulling force is provided by weights fixed on the lower end of the glass tube. The pipettes are cut either by a diamond or by the pulling force of a small glass bead, fixed on the edge of a vertical platinum filament and melted sidewise on the pipette. When the heating current of the platinum filament is switched off the filament retracts and brakes the pipette a the desired site. Greger and Hampel (1981) have modified the original perfusion system of Burg and co-workers. Their device is optimized inasmuch as it guarantees concentric alignment of the various pipettes. The forward and backward movement is controlled by small electric motors. At the perfusion side they use 4 concentric pipettes. The outermost one contains sylgard and is driven over the perfused end of the tubule in order to seal this end. The tubule is held by a holding pipette with appropriate dimensions. The tubule is sucked into this pipette up to the constriction. Then the perfusion pipette with a tip diameter smaller than the inner diameter of the perfused segment is advanced into the segment held by the holding pipette. The perfusion pipette is put under hydrostatic pressure of a few to 3 Contribution by R. Greger (first edition) and M. Bleich. 100 cm to achieve a perfusion rate of 1–20 nl/min. Usually the collapsed tubule lumen opens when the perfusion pipette is advanced. The pipette is advanced in the lumen until it reaches an area of the segment where it appears intact by inspection (200–400×). Within its lumen the perfusion pipette contains yet another pipette, the fluid exchange pipette. With this pipette the composition of the perfusate can be replaced very rapidly.The collection end of the tubule segment is sucked into a holding pipette. A sylgard pipette is advanced to seal the collection site. The holding pipette at the collection site will contain mineral oil in flux measurements. Then a collection pipette is advanced through the oil to quantitatively collect the perfusate delivered by the tubule.
The measured parameters can be as follows:
Flux measurements The collection rate (Vc, nl/min) can be measured by the constant bore collection pipette by timed collections. Radioactive tracers can be added to the lumen or bath fluid. For instance, radioactively labelled inulin can be added to the perfusate (Inp) and can be used to measure volume absorption (ΔV = perfusion rate (Vi – Vc).
Unidirectional fluxes, bath to lumen and lumen to bath, for any given substance can be quantified, and permeabilities (Px) can be determined:
Px = (Vi - Vc) L-1 [In ( xp Inc xc-1 Inp ) + 1 ]
where L is the length of the segment; xp and xc are the concentrations of x in the perfusate and in the collected fluid; and Inc is the inulin concentration in the collected fluid. Net fluxes of x can be determined as the difference of the unidirectional fluxes or by the chemical determination of Δx (perfusate – collected fluid). This requires very sensitive methods. Electron probe analysis of Na+, K+, Ca2+, Mg2+ etc. has been used to determine the net transport of these ions in various tubule segments.Flux studies are usually performed at low luminal perfusion rates of a few nl/min. Substances under study can be added to the luminal and bath perfusate, and paired data can be obtained under control and experimental conditions.
Transepithelial electrical measurements. The perfusion pipette can be connected to the high impedance input of an electrometer. The voltage is referenced to the grounded bath. The connections are usually made with agar bridges (80 g agar in 1 l Ringer’s solution), and appropriate corrections for liquid junction voltages must be applied. With identical solutions in the bath and in the lumen and with high luminal perfusion rates (> 10 nl/min), any transepithelial voltage (Vte) must be caused by active transport = active transport potential. Hence, the effectiveness of putative inhibitors of active transport can also be examined by the measurement of Vte. According to Ohm’s law the determination of the flux of ions also requires the measurement of transepithelial resistance. Greger (1981) has introduced a method which utilizes a dual channel perfusion pipette, made of Q-shaped glass. One channel is used for perfusion and the other for current (Ite) injection. The current is defined by a resistor chosen such that the deflection in Vte generated by this pulse is in the order of 10–20 mV. Transepithelial resistance (Rte) can now be calculated from ΔVte and Ite. The ratio of Vte and Rte is called equivalent short circuit current. It is directly proportional to active transport. The measurement of Vte and Rte is much more efficient than flux studies for pharmacological screening, provided that the process under study produces a transepithelial voltage. Several substances can be examined in one single tubule in strictly paired fashion . The time resolution of the measurements is on the order of 1 s, whereas that of flux studies is several minutes at best.
Intracellular electrical measurements. Greger and Schlatter have developed a method for the use of impalement techniques in the isolated perfused tubule. Very fine tip microelectrodes (Ø < 100 nm) are used to impale the tubule cell across the basolateral membrane. The actual impalement is performed by a piezo stepper which accelerates the microelectrode to high speed, which makes it possible to penetrate the rigid basal membrane. The simultaneous measurement of Vte, Rte, and basolateral membrane voltage (Vbl) allows for a complete analysis of voltages and resistances. Ion selective microelectrodes can also be used in impalement studies, and the cytosolic ion activities for e.g. Na+, K+, Cl- can also be determined . These methods are all rather difficult to perform. They are of high relevance for the understanding of the function of a given tubule segment and for the detailed description of the mechanism of action of a drug, which, in preceding studies has been shown to act in a given tubule segment.
Fluorescent dyes in the isolated perfused tubule. Several fluorescent dyes for the monitoring of Na+, K+, Cl-, Ca2+, pH have become available during the past few years. These dyes can be used in the in vitro perfused tubule. The inverted microscope is equipped with an appropriate illumination and filter wheel for excitation. The emission is measured by photon counting or by a video camera. When compared with impalement methods, these techniques are probably easier for routine use.
EVALUATION
For each of the above protocols paired measurementsof one or several given parameters of tubule transportare obtained under control conditions and in the presenceof a substance under study. Also concentrationresponse curves can be obtained in one single preparation. Intracellular measurements areusually required to define the mechanism of action. Especially the electrical and opticalmeasurements have a very high reproducibility. Forscreening usually 3 preparations are sufficient. Approximately10 preparations are required for concentrationresponse curves.1,6
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1.1.4 Isolated Perfused Kidney
Purpose And Rationale
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 80–90 mm Hg in the renal artery is maintained by adjusting the speed of the perfusion pump. For more details see references.
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.
MODIFICATIONS OF THE METHOD
Tarako et al. (1991) 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. (1967). Cox et al. (1990) used the isolated perfused rat kidney as a tool in the investigation of renal handling and effects of nonsteroidal anti-inflammatory drugs.1,3
1.2 IN VIVO METHODS
1.2.1 Diuretic Activity In Rats (LIPSCHITZ Test)
Purpose And Rationale
A method for testing diuretic activity in rats has been described by Lipschitz et al. (1943). The test is based on water and sodium excretion in test animals and compared to rats treated with a high dose of urea. The “Lipschitz-value” is the quotient between excretion by test animals and excretion by the urea control.
Procedure
Male Wistar rats weighing 100–200 g are used. Threeanimals per group are placed in metabolic cages providedwith a wire mesh bottom and a funnel to collectthe urine. Stainless-steel sieves are placed in the funnelto retain feces and to allow the urine to pass. Therats are fed with standard diet (Altromin® pellets) andwater ad libitum. Fifteen hours prior to the experimentfood and water are withdrawn. Three animals areplaced in one metabolic cage. For screening procedures two groups of three animals are used for one dose ofthe test compound. The test compound is applied orallyat 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 gbody weight are given by gavage. Urine excretion isrecorded after 5 and after 24 h. The sodium content ofthe urine is determined by flame photometry. Activecompounds are tested again with lower doses.
Evaluation
Urine volume excreted per 100 g body weight is calculated for each group. Results are expressed as the “Lipschitz-value”, i.e., the ratio T/U, in which T is the response of the test compound, and U, that of urea treatment. Indices of 1.0 and more are regarded as a positive effect. With potent diuretics, Lipschitz values of 2.0 and more can be found. Calculating this index for the 24 h excretion period as well as for 5 h indicates the duration of the diuretic effect. Similar to urine volume, quotients can be calculated for sodium excretion. Dose response curves can be established using various doses. Loop diuretics are characterized by a steep dose-response curve. Saluretic drugs, like hydrochlorothiazide, show Lipschitz values around 1.8, whereas loop diuretics (or high ceiling diuretics) like furosemide, bumetanide or piretanide reach values of 4.0 and more.
Critical Assessment Of The Method
The Lipschitz test has been proven to be a standard method and a very useful tool for screening of potential diuretics.
Modifications Of The Method
The method has been modified in various ways by severalauthors. Cummings et al. (1960) recommended asequential procedure with criteria for acceptance orrejection of test drugs. Kau et al. (1984) recommendeda method for screening diuretic agents in the rat usingnormal saline (4% body weight) as hydrating fluid.
Homozygous Brattleboro rats exhibit symptoms of diabetes insipidus (Valtin et al. 1965). The condition is due to the failure of hypothalamic neurons to produce vasopressin, which is due to a single base point deletion in the vasopressin gene (Schmale and Richter 1984). The abnormal quinine drinking aversion in the Brattleboro rat with diabetes insipidus is reversed by a vasopressin agonist (Laycock et al. 1994).
These animals can be used to study vasopressin agonism and antagonism and the aquaretic effects of synthetic drugs.
Klatt et al. (1975) described a method of collecting urine excreted by cats. On the basis of urine funnel used in rats, an appropriate larger metabolism cage made out of transparent, rigid polyvinyl chloride was used. The cage was improved by a built-in sieve cone which assured good separation of urine and feces. A device to measure and record the time and amount of voided urine was attached. Urine was collected in a vessel with a hose connection from the bottom to a pressure sensor. An attached overflow tube could be occluded. The initial pressure of the sensor was fed into a linear recorder. Before the test, the recorder was calibrated with a sufficient amount of distilled water to adjust the number of division intervals for direct measurement of voided urine in milliliters. This allowed calculation of the time point of voiding from the chart speed.1,2
1.2.2 Saluretic Activity In Rats
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 as well as for treatment of hypertension. Potassium loss has to be avoided. As a consequence, saluretic drugs and potassium-sparing diuretics were developed. The diuresis test in rats was modified in such a way that potassium and chloride as well as osmolality are determined in addition to water and sodium. Ratios between electrolytes can be calculated indicating carbonic anhydrase inhibition or a potassium sparing effect.
Procedure
Male Wistar rats weighing 100–200 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 (Chloride-Titrator Aminco) 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
• The sum of Na+ and Cl- excretion is calculated as parameter for saluretic activity.
• 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.
• The ratio
CI-
---------------
Na+ + K+
(ion quotient) is calculated to estimate carbonic anhydrase inhibition.
• Carbonic anhydrase inhibition can be excluded at ratios between 1.0 and 0.8. With decreasing ratios slight to strong carbonic anhydrase inhibition can be assumed.
Modifications Of The Method
Adrenalectomized rats treated with DOCA or aldosterone can be utilized to test aldosterone antagonists. Spironolactone has no effect in the absence of a mineralocorticoid, but reverses in a dose-related manner the effect of DOCA on the Na+/K+ ratio in the urine.1,3,5
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1.2.3 Diuretic And Saluretic Activity In Dogs
Purpose And Rationale
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
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 urine 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. 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 (Chloride Titrator Aminco) for chloride content. Furthermore, osmolality is measured with an Osmometer.
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.
1.2.4 Clearance Methods
Purpose And Rationale
Investigations of clearance represent indirect methods for the evaluation of renal function and provide information on the site of action of diuretics and other pharmacological agents within the nephron. The discovery of the countercurrent multiplier system as the mechanism responsible for the concentration and dilution of the urine has been the prerequisite for the identification of the site of action of diuretic drugs. A drug that acts solely in the proximal convoluted tubule, by causing the delivery of the increased amounts of filtrate to the loop of Henle and the distal convolution, would augment the clearance of solute-free water (CH2O) during water diuresis and the reabsorption of solute-free water (TCH2O) during water restriction. In contrast, drugs that inhibit sodium reabsorption in Henle’s loop would impair both CH2O and TCH2O.On the other hand, drugs that act only in the distal tubule would reduce CH2O but not TCH2O.
Procedure
Clearance experiments are performed either in conscious or anaesthetized beagle dogs under conditions of water diuresis and hydropenia. The status of water diuresis and hydropenia may be accomplished as described by Suki et al. (1965). Water diuresis is induced by oral administration of 50 ml of water per kg body weight and maintained by continuos infusion into jugular vein of 2.5% glucose solution and 0.58% NaCl solution at 0.5 ml/min per 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 tests are performed. Hydropenia is induced by withdrawing the drinking water 48 h before experiment. On the day before the experiment 0.5 U/kg body weight of vasopressin in oil is injected intramuscularly. On the day of the 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 per kg body weight up to i.v. administration of a compound to be tested, followed by i.v. infusion of 0.9% NaCl solution at a rate equal to the urine flow. Glomerular filtration rate (GFR) and renal plasma flow (RPF) are measured by the clearance of inulin and para-aminohippurate, respectively. Therefore, appropriate infusion of inulin (bolus of 0.08 g/kg followed by infusion of 1.5 mg/kg per min) and para-aminohippurate (bolus 0.04 g/kg followed by infusion of 0.3 mg/kg per min) are initiated. Inulin and para-aminohippurate are measured according to Walser et al. (1955) and Smith and al (1945), respectively.
Evaluation
The following parameters may be determined: water and electrolyte excretion, GFR, RPF, CH2O, TCH2O and plasma renin activity. Results of test compound are compared statistically with control and standard drug treated animals.
Modifications Of The Method
Rönnhedh et al. (1996) described a simple method to perform serial renal clearance studies without urine collection in rats. This was applied to non-radiolabeled para-aminohippurate sodium and iothalamate sodium which were used respectively to estimate renal blood flow and glomerular filtration rate. Gabel et al. (1996) described fast and accurate assays for measuring glomerular filtration rate and effective renal blood flow in conscious rats. An enzymatic method was developed for the determination of inulin and a colorimetric method was developed for determination of p-aminohippurate in the plasma and urine of rats. Hropot et al. (1985) described clearance methods in monkeys. Chimpanzees weighing 30.7 ±10.6 kg were anesthetized with 1 mg/kg Sernylan i.m. Food was withdrawn 24 h prior to the experiment and the animals received only tap water ad libitum. In the morning before the experiment, the urinary bladder of the animals was emptied by catheterization. The urine was discarded. To determine the glomerular filtration rate (inulin clearance), a bolus injection of 50 mg/kg inulin i.v. was given and followed by a continuous infusion of 3 ml/min inulin dissolved in Ringer lactate solution. After an equilibrium of 60 min, urine and 4 Contribution by M. Hropot. blood samples were collected for two control clear327 ance periods of 30 min each. The control periods were followed by intravenous administration of the test preparation in a dose of 20 mg/kg. Thereafter, urine and blood samples were collected during 6 clearance periods. The following parameters were determined: urine excretion, inulin clearance and urate clearance [ml/kg/min], fractional excretion of urate and plasma urate [mmol/l]. Tanaka et al. (1990) evaluated uricosuric and diuretic properties of diuretic agents using clearance studies in urate-loaded dogs and urate-loaded rabbits.1,3,5
CONCLUSION
The various methods for screening of Diuretic agents provides useful tool to evaluate the safety and effectiveness of the drugs.It is also useful for determining the dose lavel of particular class of diuretic agents.
REFERENCES
1. H.Gerhard Vogel,Drug Discovery And Evaluation Pharmacological Assay Second Edition Published by Springer P.No.317-348
2. Tarako T, Nakata K, Kawakami T, Miyazaki Y, Muakami M, Seo Y, Suzuki E (1991) Validation of a toxicity testing model by evaluating oxygen supply and energy state in the isolated perfused rat kidney. J Pharmacol Meth 25:195–204
3. Klatt P, Muschaweck R, Bossaller W, Magerkurth KO, Vanderbeeke O (1997) Method of collecting urine and comparative investigation of quantities excreted by cats and dogs after furosemide. Am J Vet Res 36:919–923
4. Muschaweck R, Hajdu P (1964) Die saludiuretische Wirksamkeit der Chlor-N-(2-furylmethyl)-5-sulfamyl-anthranilsäure. Arzneim Forsch 14:44–47
5. Nyunt-Wai V, Laycock JF (1990) The pressor response to vasopressin is not attenuated by hypertonic NaCl in the anaesthetized Brattleboro rat. J Physiol 430:35P
6. Schmale H, Ivell M, Breindl D, Darmer D, Richter D (1984) The mutant vasopressin gene from diabetes insipidus (Brattleboro) rats is transcribed but the message is not efficiently translated. EMBO J 3:3289–3293
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