Seminar Final for p 16.1.2010 New

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Diuretics

DIURETICS

Introduction:

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, a classification scheme based on mechanism of action is used in this

chapter.

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

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H2PO4 -), and uric acid. In addition, diuretics may alter renal hemodynamics

indirectly.

Objectives(1) :

1) Understand the anatomy and physiology of kidney as it relates to the

mechanism of action and therapeutic uses of diuretic drugs.

2) Know the prototypes of the major diuretic classes.

3) Understand the mechanisms of action and major side effects of the prototype

diuretic drugs.

4) Understand the therapeutic uses of diuretic drugs.

5) To study the evaluation methods of diuretic activity.

Definition and History:

• Diuretics are the agents which increase the rate of urine formation.

• Diuretics are effective for the treatment of oedema have been available since

16th century.

• Mercurous chloride was known by Paracelsus to be diuretic.

• In 1930, Swartz discovered the antimicrobial sulfanilamide could be used to

treat oedema in patients with congestive heart failure due to an increase in

renal excretion of Na+ .

• Most modern diuretics were developed when side effects of antibacterial drugs

were noted, which included changes in urine composition and output.

• Except for spironolactone,diuretics were developed empirically, without

knowledge of specific transport pathways in the nephron.

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• Diuretics are the most commonly prescribed drugs in the USA and can be

quite efficacious but they can also have an extremely wide range of adverse

effects.

• The primary therapeutic goal of diuretic use is to reduce oedema by reducing

the ECF volume.

• For this to occur, NaCl output must exceed NaCl intake.

• Diuretics primarily prevent Na+ entry into the tubule cell.

• Once diuretic enters the tubular fluid, the nephron site at which it acts

determines its effect. In addition, the site of action also determines which

electrolytes, other than Na+ will be affected.

• All diuretics except spironolactone exert their effects from the luminal side of

the nephron.

• It is necessary for diuretics to get into the tubule fluid in order to be effective.

Anatomy of Kidney(10)

Location

The paired Kidneys are reddish, kidney bean shaped organ located just above

the waist between the peritoneum &the posterior wall of abdomen . Because their

position is posterior to the peritoneum of the abdominal cavity , they are said to be

retroperitoneal The kidneys are located between the levels of the last thoracic & third

lumbar vertebrae , a position where they are partially protected by the 11 th & the 12th

pairs of ribs The right kidney is slightly lower than the left .

External anatomy -

Size :- 10-12 cm long , 5-7 cm wide , 3cm thick .

Mass :- 135-150 gm

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1) Renal hilus - Near the center of concave boarder is a deep vertical fissure

2) Layers of kidney –

Innermost - Renal capsule

Middle - Adipose capsule

Outer - Renal fascia

Internal anatomy -

1) Renal cortex - Superficial , smooth textured reddish area

2) Renal medulla - Deep, reddish brown inner region.

3) Renal pyramids - Medulla consists of 8-18 cone shaped structure.

4) Renal papilla - The base of each pyramid faces the renal cortex , its apex

7called renal papilla .

5) Nephron - Functional units within parenchyma .

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6) Minor & major calyces – The papillary ducts drain into cup – like structures.

7) Renal pelvis – Form major calyces , urine drains into a single large cavity

called renal pelvis .

8) Renal sinus – Hilus expands into a cavity within kidney called renal sinus

BLOOD & NERVE SUPPLY TO KIDNEY (10)

Kidney constitutes 0.5% of total body mass, they receive 20-25 % of the resting

cardiac output , via left and right renal arteries ,

1) Interlobar arteries- At the base of renal pyramids interlobar arteries arch

between the renal medulla, & cavity .It is also called actuate arteries.

2) Afferent arteriole – carry renal blood towards kidney

3) Efferent arteriole - carry blood out of glomerulus

4) Vasa recta - Extending from efferent arteriole which are long ,loop shaped

capillaries .

5) Peritubular capillaries – Branches of efferent arterioles

6) Interlobar veins - Peritubular capillaries reunite to from peritubular venules &

then interlobar veins which receives the blood from vasa recta Then the blood

drains though renal pyramids .

7) Renal vein - Blood leaves the kidney through a single renal vein that exits at

renal hilus & carries venous blood to inferior vena cava.

Parts of Nephron (10) –

The nephron is a tube, closed at one end , open at the other . It consists of –

1) Bowman’s capsule- Located at open end forming a double walled chamber .

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Diuretics

2) Glomerulus - A capillary network within Bowman’s capsule. Blood leaving

the glomerulus passes into a second capillary network .

3) Proximal convoluted tubule – coiled & lined with cells carpeted withmicrovilli & stuffed with mitochondria .

4) Loop of Henle – It makes a hairpin turn & returns to Distal convoluted tubule.

5) Distal convoluted tubule – which is also lightly coiled & surrounded by

capillaries .

6) Collecting tubule - It leads to pelvis of kidney periodically, on to the outside

world.

7) Juxtaglomerular apparatus - In each nephron, the final part of ascending limb

of the loop of Henle makes contact with afferent arteriole serving that renal

corpuscle. Because the columnar tubule cells in this region are crowded

together, they are known as macula densa. Alongside the macula densa the

wall of afferent arteriole contains modified smooth muscle fibers called

juxtaglomerular cells together with the macula densa they constitutes

Juxtaglomerular apparatus.

Physiology of Urine formation(4&10) :

The fluid that enters the capsular space is called glomerular filtrate on an

average, the daily volume of glomerular filtrate in adults is 150 lit in females and 180

lit. In males more than 99% of glomerular filtrate returns to the blood stream via

tubular reabsorption & only 1-2 lit are excreted as urine .

It involves three phases -

1) Glomerular filtration

2) Tubular reabsorption

3) Tubular secretion

1) Glomerular filtration :

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Fluid is driven from the capillaries into the tubular capsule (Bowman’s

capsule) by hydrodynamic force. It crossed three layers the capillary

epithelium, the basement membrane and the epithelial cell layer of the

capsule. These from a complex filter that excludes large molecules. Normally

all constituents in the plasma, except plasma proteins, appear in the filtrate

&the blood which passes through the efferent arteriole to the particular

capillaries has a higher concentration of plasma proteins & thus higher

osmotic pressure than normal.

Glomerular filtration Rate - (G. F. R )

The amount of filtrate formed in all the renal capsule of both kidneys each min

is glomerular filtration rate.

In adults, GFR averages 125ml/min in males & 105 ml/min in females. It GFR

is too high, needed substances may not reabsorbed & are lost in urine. It the GFR is

too low, nearly all the filtrate may be reabsorbed and certain waste products may not

be adequately excreted.

GFR is directly related to pressures that determine net filtration pressure. Any

change in net filtration pressure will affect GFR. In case of severe blood loss, mean

arterial pressure reduces & decreases glomerular blood hydrostatic pressure G.F.R. In

case of severe blood loss, mean arterial pressure reduces & decreases glomerular

blood hydrostatic pressure G.F.R. is nearly constant when mean arterial blood

pressure is anywhere in between 80-180 mm Hg .

The mechanisms that regulate GFR operate in two ways

1) By adjusting blood flow into and out of the glomerulus.

2) By altering glomerular capillary surface area available for filtration .

GFR increases when blood flow into glomerular capillaries increases.

Coordinated control of diameter of afferent & efferent arterioles regulate glomerular

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blood flow Constriction of afferent arteriole, decreases blood flow into the

glomerulus, whereas dilation of afferent areteriole increases blood flow.

Three mechanisms control GFR –

a) Renal autoregulation

b) Neural regulation

c) Hormonal regulation

(a) Renal autoregulation

The kidney themselves helps to maintain a constant renal blood Flow & GFR .

Despite normal, everyday changes in B.P (e.g. exercise) this capability is called

renal auto regulation. It consists of a feedback mechanism. As blood pressure

rises, however, the elevated blood pressure stretches the walls of afferent

arteriole. In response, smooth muscle fibers in the wall of afferent arteriolecontract, which narrows the arterioles lumen. As a result renal blood flow leaves

thus reducing GFR to its previous level & vice versa.

(b) Neural regulation of GFR

The blood vessels of kidney are supplied by sympathetic ANS fibers that

release nor-epinephrine (NE). NE causes vasoconstriction through activation of

alpha1 receptor. At rest, sympathetic stimulation is moderately low & GFR is

normal. With moderate sympathetic stimulation both afferent & efferent

arterioles constricts to the same degree. Blood flow into & out of glomerular is

restricted to same extent, which decreases GFR slightly. With greater

sympathetic stimulation, vasoconstriction of afferent arteriole predominates. As a

result, blood flow through glomerular capillaries is greatly decreased & GFR

drops. This lowering of renal blood flow has two consequences

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Diuretics

1. It reduces urine output which helps to conserve blood volume.

2. It permits greater blood flow to other body tissues.

(c) Hormonal regulation of G F R

Two hormones contribute to regulation of G F R.

1) Angiotensin-II reduces GFR

2) Atrial natriuretic peptide (ANP) increases GFR.

Angiotensin-II reduces GFR

Angiotenisin II is a potent vasoconstrictor that narrows both

afferent & efferent arterioles, reduces renal blood flow, there by GFR. Cells in the

atria of the heart secret ANP. Stretching of the atria as occurs when blood volume

incrteases, stimulates secretion of ANP. By causing relaxation of the glomerular

mesengial cells, ANP increases, Capillary surface area available for filtration . GFR

rises as the surface area increases.

2) Tubular reabsorption & 3) Tubular secretion(4) :->

In the epithelium of the tubules, the apex or luminal surface of each cell is surrounded

by a Zonula accludens , a specialized region of membrane that from a tight junction

between it & neiglbouring cells. The movement of ions & water through the cell and

between the cells through zonulae occludentes. The tightness or leakiness of the

epithelium of various portions of nephron is important factor in their function Tight

epithelium is found in dlistal portion of nephron which is the site of action of major

hormones involved in the control of NaCl & water excretion.

Proximal convoluted tubule - (PCT) :=>

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About 60-70% filtered load of Na+& water is reabsorbed in PCT. The most

imp. mechanism for Na+ entry into the cell from the filtrate is the Na+/H+ exchanger.

Glucose , amino acids are also reabsorbed along with Na+. sodium is transported out

of the cell into the interstitial &then into the blood by primary active transport

mechanism of the nephron , the Na+/ K +/ATP –ase in basolateral membrame. Cl-

absorption is largely passive, some diffuses through zonula occludens . Bicarbonate is

returned to the plasma mainly in PTC. By an indirect method involving action of

carbonic anhydrase. Remaining 30-40% of the filtrate passes onto the Henle’s loop.

The Loop of Henle :=>

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The loop consists of a descending & ascending portion. During passage

through loop of Henle 20n-30% of Na+ in the tubule is reabsorbed. This part of the

nephron plays an imp. Part in regulating the osmolality of urine, and osmotic balance

of body.

The descending limb is highly permeable to water, which moves out passively.

In juxtamedullary nephrons with long loops, there is extensive movement of water

eventually reaching the tip of the loop has high osmolality.

The ascending limb has very low permeability to water that 20-30% of filtered

Na+ is reabsorbed Both Na+ & Cl- move into the cell by a co-transport system

involving Na+/ K +/2Cl-. This process is driven by the electrochemical gradient for Na+

produced by the Na+/ K +/ATP ase in the basolateral memberance. Most of the K +

taken into the cell by co-transport system cycles back to the lumen through potassium

chancel but some K + is reabsorbed along with Mg+2& Ca2+. Loop of Henle is

sometimes referred as diluting segment because absorption of NaCl with very little

water result in marked dilution of filtrate .

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The Distal Tubule :=>

In early distal tubule , active NaCl transport , coupled c impermeability of

zonula outudens continues to dilute the tubular fluid and osmolality falls At this point , K +& H+ are added to the filtrate . The transport is driven by Na+/ K + pump in

the basolateral membrame , Na+ entering the cell from the lumen accompanied by Cl

by means of an electroneutral Na+/Cl carrier . Thiazide diuretics act by inhibiting this

carrier,

The collecting tubule & collecting duct :=>

Several distal tubules empty into each collection tubule & the collection tubules join

to from collecting ducts. Collecting tubule has two diff. cell types –

1) Principal cells reabsorb Na+& secrete K +

2) Intercalated cells – involves in H+ secretion.

In this portion ions & water regulation is influenced by hormones. Absorption of

Nacl is controlled by a mineralocorticoid – Aldosteron & absorption of water is

controlled by Antidiaretic Hormone (ADH/vassopressin) Aldosteron enhances Na+

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reabsorption & promots K + excretion. Aldosterone secretion is indirectly controlled

by Juxtaglomerular apparatus.

CLASSIFICATION (1 & C)

From Knauf & Mutschler Klin. Wochenschr. 1991 69:239-250

70%

20%

5%

4.5%

0.5%Volume 1.5 L/day

Urine Na 100 mEq/L

Na Excretion 155 mEq/day

100%GFR 180 L/day

Plasma Na 145 mEq/L

Filtered Load 26,100 mEq/day

CA Inhibitors

Proximal tubule

Loop Diuretics

Loop of Henle

Thiazides

Distal tubule

Antikaliuretic

s

Collecting

duct

ThickAscendin

g

Limb

©

Drugs acting at-

1. PCT

Carbonic anhydrase inhibitors-

e.g. Acetazolamide, dichlorphenamide, methazolamide.

Osmotic diuretics-

eg. Mannitol, glycerine, isosorbide, Urea etc.

2. Loop

Na-K-2Cl Co-transport inhibitors e.g. Furosemide, ethacrynic acid,

Bumetahide, torsemide.

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Diuretics

3. DCT

Na-Cl co-transport inhibitors-

e.g. Chlorthiazide, hydrochlorthiazide, Indapamide, metolazone.

4. Collecting Duct

Potassium sparing diuretics-

Renal epithelial Na channel inhibitors-e.g. Amiloride, triamterine.

Aldosterone antagonists-

e.g. Spironolactone, epelerinone, canrenone.

CARBONIC ANHYDRASE INHIBITORS ( 1 & 6 )

They are structural related to sulphonamides. Diuretic action is a side effect of

sulphanilamide ,first discovered antibiotic .Acetazolamide is prototype of this class

of drug .

Major site of action is proximal convoluted tubule. Intracellularly CO 2 and

H2O are converted to H2CO3 (carbonic acid ) which requires activity of carbonic

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anhydrase ,it further dissociates into H+ and HCO3- These HCO3

- ions go to basolateral

membrane and H+ ion are exchanged with Na+ in the lumen . The filtered bicarbonate

ions are associated with H+ ions , which form H2CO3 which immediately

dissociates in CO2 and H2O ( requiring carbonic anhydrase activity ) which is

reabsorbed . This action is like a circuit, so bicarbonate ions are reabsorbed for every

H+ secretion .Carbonic anhydrase inhibitors act in this stage and there is loss of

NaHCO3 .

b)Effects on Electrolytes and Renal Haemodynamics

Metabolic acidosis results in HC03-loss. There is an increase in Na+ and K -

excretion. However, the natriuresis is modest due to the large Na+ capacity of the

ascending loop of Henle and the distal tubule. By inhibiting proximal tubule

reabsorption, the increase in solute delivery to the distal tubule increases afferent

arteriolar resistance thus decreasing renal blood flow and GFR (tubuloglomerular

feedback regulation).

b)Toxicity and Adverse Effects

Metabolic acidosis, sedation and paresthesia Also, because of the structuralsimilarity to sulfonamides, carbonic anhydrase inhibitors can cause bone morrow

depression and allergic reactions.

b) Therapeutic Uses

Carbonic anhydrase inhibitors are not used for their diuretic properties. Rather

these agents are used to reduce intraocular pressure in the treatment of glaucoma. This

is because these agents inhibit intraocular carbonic anhydrase and thus the formation

of aqueous humor. Carbonic anhydrase inhibitors are also used to treat epilepsy and

motion sickness.

THIAZIDE DIURETICS ( 1 & 6 )

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These were originally synthesized as carbonic anhydrase inhibitors. While

some thiazides have carbonic anhydrase inhibitory activity, the major site of action is

in the distal tubule. Hydrochlorothiazide is the prototype for this class of drug.

Chlorthalidone, indapamide and metolazone are long acting diuretics. These drugs do

not have the thiazide structure but are referred to as thiazide-like. The clinically

available thiazides and thiazide-like agents have the same mechanism of action. They

differ only in pharmacokinetic properties such as the plasma half-life.

a. Mechanism of Action

Thiazide diuretics are secreted into the tubular fluid by proximal tubulecells. These agents act in the distal convoluted tubule and block a Na+, Cr

symporter that is associated with the luminal membrane. This transport system

moves both Na+ and cr into the cell using the free energy produced by the Na+,

K +, ATPase. The Na+ is pumped out of the epithelial cell via this transport system

in the basolateral membrane. The Cl- exits the cell via a Cl channel. Because

thiazides are related in structure to carbonic anhydrase inhibitors, some of these

agents have weak carbonic anhydrase activity.

b. Effects on Electrolytes and Renal Hemodynamics

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1. Na+, Cl excretion is enhanced. However, the effect on Na+ is small

because most of the Na + has already been absorbed prior to reaching the

distal tubule.

2. K + excretion is enhanced due to the increase in Na + delivered to the

distal tubule.

3. Uric acid excretion is reduced.

4. Ca2+ excretion is decreased via a poorly understood mechanism.

5. The excretion of Mg2+ is enhanced.

6. Thiazides have little effect on renal blood flow or total glomerular

filtration rate.

c. Side Effects, Drug Interactions and Toxicity

The likelihood of side effects with the thiazides increases with the plasma

concentration of the drug. These drugs were introduced in the 1950s and earlyclinical trials were carried out with high drug concentrations (200 mg/day)

designed to produce significant diuresis. As a consequence many side effects were

reported. However, more recently, clinical trials have showed that low doses of

thiazide diuretics (12-25 mg/day) are actually more effective than higher doses in

reducing cardiovascular events. The reported side effects are provided below. In

many instances the likelihood of observing a particular side effect and the severity

is dependent on the dose of diuretic. Potential side effects of diuretic drugs

include;

1. Electrolyte abnormalities include volume depletion, hypokalemia,

hyponatremia, hypochloremia, hypercalcemia, hyperuricemia and

hypomagnesemia.

2. A decrease in glucose tolerance and reduces the efficacy of

hypoglycemic drugs.

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3. Increases plasma levels of LDL cholesterol, and triglycerides. While

the effects on glucose levels, cholesterol and triglycerides have been

observed, it is not clear that these result in an increase of risk for diuretic

usage.

4. Sexual dysfunction (impotence)

5. The hypokalemia resulting from thiazides increases the likelihood of

ventricular rhythm disturbances. This is a potentially serious consequence,

6. A variety of drug-drug interactions have also been reported, such as a

decrease in the efficacy of a variety of drugs including anticoagulants and

uricosorics (drugs used to treat gout). Thiazides increase the risk of toxicity

(i.e. increase the likelihood of rhythm disturbances) of cardiac glycosides,

quinidine and antiarrhythmic drugs.

7. Nonsteroidal anti-inflammatory agents reduce the efficacy oftrnazide

diuretics. Thiazides can increase the blood levels of lithium.

d. Clinical Uses

Thiazides can be used to treat edema associated with a variety of

pathophysiologic conditions including congestive heart failure, cirrhosis, renal

insufficiency and the nephrotic syndrome. However, their major use is in the

therapy of hypertension. They can be used alone or in combination with

numerous other antihypertensives.

Initially, thiazides reduce plasma volume which contributes to the

hypotensive effect. . Long term use is associated with a decrease in peripheral

vascular resistance. The effects on vascular resistance are due to direct effects on

vascular smooth muscle. However, the mechanisms underlying these effects are

not well understood.

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LOOP OR HIGH CELING DIURETICS ( 1 & 6 )

Furosemide

Ethacrynic Acid

Bumetanide

Torsemide

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a) Mechanism of Action

Like thiazides these agents must be secreted into the tubular fluid by

proximal tubule cells. In the thick ascending loop Na+ and Cl- reabsorption is

accomplished by a Na+, K +,Cl- symporter. The thick ascending limb has a high

reabsorptive capacity and is responsible for reabsorbing 25% of the filtered load

of Na+. The loop diuretics act by blocking this symporter. Because of the large

absorptive capacity and the amount of Na+ delivered to the ascending limb, loop

diuretics have a profound diuretic action. In addition, more distal nephron

segments do not have the reabsorptive capacity to compensate for this increased

load. The osmotic gradient for water reabsorption is also reduced resulting in an

increase in the amount of water excreted.

b) Effects on Electrolytes and Renal Blood Flow

Loop diuretics cause a significant increase in Na+,K + and Cl- excretion. Ca2+

and Mg2+ excretion are also enhanced. Loop diuretics block the Na+, K +, Cl-

symporter in the macula densa. As a result the tubuloglomerular feedback

mechanism is blocked & because of this loop diuretics maintain renal blood flow.

c) Side Effects, Drug Interactions and Toxicity

1. Many side effects occur as a result of abnormalities in fluid or electrolytes.

This would include volume depletion, hyponatremia, hypokalemia,

hypochloremia, hypocalcemia and hypomagnesemia.

2. Ototoxicity, especially with ethacrynic acid

3. Hypotension

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4. Metabolic effects-hyperuricemia, hyperglycemia, increase triglyceride and

cholesterol levels, increase LDL cholesterol and decrease HDL

cholesterol.

5. As with thiazides the hypokalemia produced by high ceiling diuretics can

induce arrhythmias.

6. Potential drug interactions could occur with certain antiarrhythmic drugs

and cardiac glycosides can increase the likelihood of ventricular

disturbances. This is a potentially serious interaction. Co-administration of

drugs, like aminoglycoside antibiotics or the anticancer drug cisplatin that

are also can produce ototoxicity. Nonsteroidal antiinflammatory drugs

blunt the actions of loop diuretics. Loop diuretics cart increase the blood

levels of certain drugs such as lithium.

Agents, such as probenecid, that block secretion into the into the distal tubule

will decrease the response to high ceiling diuretics.

d) Clinical Uses

The vigorous diuresis produced by loop diuretics makes these especially

useful for rapid reduction of edematous fluid. Indications include renal

insufficiency, ascites, nephrotic syndrome, pulmonary edema and congestive

heart failure. While not drug of first choice in the treatment of hypertension, loop

diuretics can also be used to treat hypertension. The increase in Ca2+ excretion

caused by these agents makes them useful in the treatment of hypercalcemia.

POTASSIUM SPARING DIURETICS ( 1 & 6 )

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Hypokalemia is a problem associated with the use of thiazide or loop diuretics.

Signs and symptoms of hypokalemia include muscle weakness, drowsiness (and a

variety of other CNS manifestations and cardiac rhythm disturbances). The potassium

loss can be managed with K + supplementation (K + salts or foods rich in K +) or the use

of K + sparing diuretics. There are two types of potassium sparing diuretics. They are;

1. Renal epithelial Na+ channel inhibitors - amiloride, triamterene

2. Aldosterone Antagonists - spironolactone, eplerenone

1. Na + Channel Inhibitors

Mechanism of action of amiloride and triamterene

In the distal tubule a Na channel is expressed and conducts Na down

the concentration gradient established by the Na+, K + ATPase. The elevation of

intracellular Na + depolarizes the luminal side of the cell. This relative depolarization

results in K + secretion into the tubular lumen. Diuretics that work at a site proximal to

the distal convoluted tubule increase the Na+ load and thus increase the amount of K +

that is secreted. This is why most diuretics produce hypokalemia. Amiloride and

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triamterene block the epithelial Na + channel. As a result the driving force for K+

secretion is eliminated, hence K+ secretion ceases, The diuretic effect is modest.

Toxicity and Side Effects

The most obvious side effect is an extension of the therapeutic action

of these drugs, that is hyperkalemia. These drugs are contraindicated in situations in

which hyperkalemia occurs as well as patients predisposed to hyperkalemia (renal

failure, treatment with ACE inhibitors).

2. Aldosterone antagonists

Mechanism of action -spironolactone and eplerenone

Aldosterone interacts with a cytoplasmic mineralocorticoid receptor to

enhance the expression of the Na+, K +-ATPase and the Na+ channel involved in a Na+

K + transport in the distal tubule. Spironolactone and eplerenone bind to this receptor

blocking the actions of aldosterone on gene expression.

Spironolactone: Like the Na+ channel blockers spironolactone has limited

efficacy alone but is often given with other diuretics. Spironolactone is metabolized tocanrenone which is the active drug molecule. The diuretic and natriuretic effects of

spironolactone are modest. Therefore, its agents are not usually used alone to treat

edema or hypertension. Rather, it is used with thiazides and loop diuretics in the

therapy of hypertension. This enhances the hypotensive effect of the more potent

diuretics and counteracts the K+ loss seen with these diuretics. Recent studies indicate

that aldosterone contributes to cardiac hypertrophy. The Randomized Aldactone

Evaluation Study (RALES) showed that spironolactone, when added to a standard

treatment regimen, decreased the risk of morbidity and mortality in patients with

severe congestive heart failure. Toxicities and cautions are similar to Na+ channel

inhibitors. Due to its steroid structure, spironolactone can cause antiandrogen effects

such as gynaecomastia, decreased libido and impotence in men as well as menstrual

irregularities and hair growth in women.

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Eplerenone: Is an analog of spironolactone introduced in 2003. Eplerenone

has lower affinity compared to spironolactone for the mineralocorticoid receptor.

Nonetheless, it blocks aldosterone-induced gene expression. However,eplerenone has little affinity for androgen or progesterone receptors. Therefore it is

void of the unpleasant steroid hormone-like effects (gynaecomastia, hair growth, etc).

Clinical trials have shown that eplerenone can be used effectively to treat

hypertension. In addition, eplerenone has been shown to improve outcomes in patients

with heart failure.

Osmotic Diuretics

Glycerin

Isosorbide

Mannitol

Urea

a) Mechanism of Action

Osmotic diuretics are freely filterable but not reabsorbed and prevent H20

reabsorption in the proximal tubule. Osmotic diuretics also extract H20 from systemic

body compartments. This expands extracellular fluid volume and increases renal blood

flow. This increase in blood flow removes NaCI and urea from the renal medulla. The

loss of these solutes decreases the medullary toxicity and hence the ability to generate

a concentrated urine.

b) Toxicity and Adverse Effects

Osmotic diuretics increase the excretion of all electrolytes. The increase in

extracellular fluid volume could exacerbate congestive heart failure or pulmonary

congestion.

c) Clinical Uses

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Diuretics

Osmotic diuretics maintain renal blood flow in patients with acute renal failure.

These agents can also be used to treat increases in intraocular pressure in glaucoma

as well as reduce cerebral edema.

PHARMACOKINETICS ( 5 )

The pharmacologic characteristics of all loop diuretics are similar.

Therefore, a lack of response to adequate doses of one loop diuretic militates against

the administration of another loop diuretic; instead, combinations of diuretics with

different mechanisms of action should be given.

Loop diuretics block the sodium-potassium-chloride transporter, thiazide

diuretics block the electroneutral sodium-chloride transporter, and amiloride and

triamterene block apical sodium channels. All diuretics except spironolactone reach

these luminal transport sites through the tubular fluid; all but osmotic diuretics are

actively secreted into the urine by proximal tubule cells. A high degree of protein

binding (> 95 percent) limits glomerular filtration, even in patients with

hypoalbuminemia. In effect, binding to serum proteins traps the diuretic in the

vascular space so that it can be delivered to secretory sites of proximal tubule cells.

Loop and thiazide diuretics and acetazolamide are secreted through the organic-acid

pathway, and amiloride and triamterene through the organic-base pathway

About 50 percent of a dose of furosemide is excreted in unchanged form

into the urine& the other 50 percent is conjugated to glucuronic acid in the kidneys.

Thus, in patients with renal insufficiency, the plasma half-life of furosemide is

prolonged because not only urinary excretion but also renal conjugation is

decreased. The other two loop diuretics available in the United States, bumetanide

and torsemide, are largely metabolized by the liver (50 and 80 percent, respectively)

; therefore, their half-lives are not prolonged in patients with renal insufficiency,

although renal disease impairs their delivery to the tubular fluid. In contrast, in

patients with liver disease, the plasma half-lives of these drugs are prolonged, and

more drug reaches the tubular fluid.

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Diuretics

Although the pharmacologic characteristics of ethacrynic acid have been

characterized as typical of those of loop diuretics, there are no data on its

pharmacokinetics. The drug's ototoxic potential is greater than that of other loop

diuretics, and it is therefore now given only to patients who have allergic reactions

to other loop diuretics.

The pharmacokinetics of thiazide diuretics have been studied less

extensively than those of loop diuretics. Some thiazide diuretics are metabolized

primarily by the liver (e.g., bendroflumethiazide, polythiazide, and indapamide);

others are primarily excreted in unchanged form in the urine (e.g., chlorothiazide,

chlorthalidone, hydrochlorothiazide, hydroflumethiazide, and trichlormethiazide).

There is little information about the influence of disease on the pharmacokinetics

of these drugs.

Since amiloride is excreted by the kidneys, renal disease prolongs its

plasma half life, whereas liver disease has little effect on the drug. The

pharmacokinetics of triamterene are complicated, because it is converted to an

active metabolite by the liver, and the metabolite is then secreted into the tubular

fluid. Renal disease impairs the secretion of this metabolite into the tubular fluid.The amount of metabolite that reaches the tubular fluid is also reduced in patients

with liver disease, because of diminished formation of the metabolite in the liver.

The pharmacokinetics of spironolactone are even more complex, because it is

converted to numerous active metabolites.

In addition to the routes of metabolism, the pharmacokinetic features of

diuretics that are clinically important are bioavailability and half-life. On average,

the amount of an oral dose of furosemide that is absorbed is 50 percent, but it

ranges from 10 to 100 percent. This wide range makes it difficult to predict how

much furosemide will be absorbed in an individual patient, and different doses

must be tried before the drug is judged to be ineffective. In contrast, absorption of

bumetanide and torsemide is nearly complete, ranging from 80 to 100 percent.

There is therefore probably less need for titration of these drugs when one is

switching from an intravenous to an oral dose. The variation in the absorption of

A.B.C.P. Sangli.26



Diuretics

furosemide may be clinically important; patients with heart failure treated with a

completely absorbed loop diuretic (torsemide) may require hospitalization less

often and have a better quality of life than patients treated with furosemide. The

amount increased exposure to solute causes hypertrophy of distal nephron

segments, with concomitant increases in the reabsorption of sodium. Sodium that

escapes from the loop of Henle is therefore reabsorbed at more distal sites,

decreasing overall diuresis. The result is long-term tolerance of the loop diuretic.

Thiazide diuretics block the nephron sites at which hypertrophy occurs,

accounting for the synergistic response to the combination of a thiazide and a loop

diuretic. This phenomenon reinforces the logic of using combinations of loop and

thiazide diuretics in patients who do not have adequate responses to optimal doses

of a loop diuretic.

DIURETIC RESISTANCE ( 3 )

Diuretic resistance refers to edema that is or has been become refractory to

a given diuretics.

If diuretic resistance develops against a less efficacious diuretic, a moreefficacious one can be substituted, e.g. a loop diuretic is substituted for a thiazide.

However, resistance to loop diuretic is not common and can be due to several

causes.

• NSAIDs block prostaglandin mediated increase in renal blood flow,

resulting in resistance to loop diuretic.

•

In chronic renal failure, a reduction in renal blood flow decreases thedelivery of diuretics to the kidney, and accumulation of endogenous

organic acids competes with loop diuretics for transport at the proximal

tubule. Consequently, the concentration of diuretic at the active site in

the tubular lumen is diminished.

• In nephrotic syndrome, urinary proteins bind with diuretics and

thereby limit the response.

A.B.C.P. Sangli.27



Diuretics

• In hepatic cirrhosis or heart failure~ the kidney may have a diminished

responsiveness to diuretics because of increased proximal tubular Na

reabsorption, leading to diminished delivery of Na+ to the distal

nephron segments.

Other causes

• Poor patient compliance

• Poor absorption

• Continued sodium intake

Faced with resistance to loop diuretics, the clinician has several options:

1) Bed rest

2) Increase in dose

3) Combination therapy

4) Intake of diuretic shortly before food intake

5) Continuous I.V. infusion.

Diuretic Therapy in Patients with Edema

1. Renal Insufficiency( 1 )

A loop diuretic is the diuretic of choice in patients with renal insufficiency.

Although a large dose of a thiazide will cause diuresis in patients with mild renal

insufficiency, the response in patients with a creatinine clearance of less than

about 50 ml per minute is poor.

A.B.C.P. Sangli.28



Diuretics

In patients with a creatinine clearance of 15 ml per minute, 1/5 to 1/10 as

much loop diuretic is secreted into the tubular fluid as in normal subjects. Thus, a

large dose must be given to attain an effective amount of diuretic in the tubular

fluid. The relation between the rate at which the diuretic is excreted and the

response to it is the same in patients with renal insufficiency as it is in normal

subjects. Thus, the remaining nephrons in patients with renal insufficiency retain

their responsiveness to the diuretic; the problem is getting enough drug to the site

of action

A frequent question is what is the largest single dose of a loop diuretic that

can be given to a patient with severe renal insufficiency? The maximal natriuretic

response occurs with intravenous bolus doses of 160 to 200 mg of furosemide or

the equivalent doses of bumetanide and torsemide, and nothing is gained by using

larger doses. Some patients may require these large doses several times a day. The

maximal response is the excretion of about 20 percent of filtered sodium. In a

patient with a creatinine clearance of 15 ml per minute, this means that about 25

mmol of sodium will be excreted. If the patient ingests 75 mmol of sodium per

day, then the single dose of 25 mmol to be excreted must be administered three

times per day, and sodium will be retained if the intake is higher. Single

intravenous bolus doses of 160 to 200 mg can occasionally cause transient tinnitus

but this effect can be minimized by administering the dose over a period of 20 to

30 minutes.

The bioavailability of loop diuretics is the same in patients with renal

insufficiency as it is in normal subjects. Therefore, the intravenous and oral doses

of bumetanide and torsemide are similar. For furosemide, the usual maximal oraldose is twice the intravenous dose (160 to 320 mg in patients with moderate renal

insufficiency and 320 to 400 mg in those with severe renal insufficiency).

However, the absorption of furosemide varies from one patient to another.

Occasionally, a very small fraction of the dose is absorbed, and very large oral

doses are therefore required. Before concluding that a patient has not had a

response to furosemide and contemplating the use of dialysis to control volume,

A.B.C.P. Sangli.29



Diuretics

the physician should administer larger oral doses of furosemide or a maximal oral

dose of either bumetanide or torsemide.

In patients who have poor responses to intermittent doses of a loopdiuretic, a continuous intravenous infusion can be tried. If an effective amount of

the diuretic is maintained at the site of action at all times, a small but clinically

important increase in the response may occur. There are other reasons to consider

giving a continuous infusion of a loop diuretic. It may be easier for nursing staff to

give a continuous infusion than intermittent bolus intravenous doses. In addition,

with a continuous infusion, decisions about the timing of doses of an additional

diuretic are simplified. Finally by closely monitoring urinary output, one can

unambiguously determine whether the added drug was beneficial.

Before administering a continuous infusion of a loop diuretic, the

physician should give a loading dose in order to decrease the time needed to

achieve therapeutic drug concentrations otherwise, 6 to 20 hours is required to

achieve a steady state, depending on the diuretic used. The rate of the continuous

infusion is governed by the patient's renal function. If an adequate response has

not occurred after the drug has been given for an hour, the loading dose should berepeated, and then the infusion rate can be increased.

Another strategy to enhance the response to a loop diuretic is to add an

oral thiazide diuretic. Metolazone is frequently given in the United States,

whereas otherthiazides are given elsewhere. The pharmacologic characteristics of

metolazone are similar to those of other thiazides. Some formulations of the drug

are absorbed poorly and slowly, and it has a long elimination half-life (about two

days). Thus, metolazone accumulates over a period of about 10 days. Other

thiazides have the same synergistic effects when combined with a loop diuretic.

Since the absorption of other thiazides, such as hydrochlorothiazide, is more rapid

and predictable, they may be preferable to metolazone.

Because thiazide diuretics must reach the lumen ofthe nephron to be

effective, higher doses are required in patients with renal insufficiency than in

other patients. Patients with mild-to-moderate renal insufficiency require 50 to

A.B.C.P. Sangli.30



Diuretics

100 mg of hydrochlorothiazide per day; those with more severe disease require

100 to 200 mg per day. Thiazides can be administered once or twice a day.

2. The Nephrotic Syndrome

( 1 & 6 )

It is often difficult to achieve a satisfactory diuresis in patients with the

nephrotic syndrome. In such patients, serum albumin concentrations are frequently

low, and the diffusion of diuretics into the extracellular fluid is therefore increased.

This may reduce the amount of drug delivered to renal secretory sites. If so, the

efficacy of diuretic therapy may be increased by administering a mixture of albumin

and a loop diuretic; in several patients with severe hypoalbuminemia, an infusion 000

mg of furosemide mixed with 25 g of albumin enhanced diuresis. However, in most

patients with the nephrotic syndrome (and in those with cirrhosis ), renal tubular

secretion of furosemide is normal (unless the patient also has renal insufficiency), and

combined infusions are therefore unnecessary. This conclusion may not be applicable

to patients with serum albumin concentrations of less than 2 g per deciliter. In such

patients, it may be reasonable to try combined infusions.

The diuretic response is subnormal in patients with the nephrotic syndrome,

despite an adequate rate of excretion of drug into the tubular fluid. In animals, and

presumably also in humans, diuretics become bound to albumin in tubular fluid,

resulting in a diminished amount of unbound, active drug and a decreased diuretic

response When urinary albumin concentrations exceed 4 g per liter, one half to two

thirds of the diuretic that reaches the tubular fluid is bound to albumin in the fluid.

Consequently, doses two to three times the normal dose are needed to deliver

adequate amounts of unbound, active drug to the site of action In addition, patients

with the nephrotic syndrome may have a diminished response because of a decrease

in the drug's action on cells within the loop of Henle and because of increased

proximal or distal reabsorption of sodium Doses must therefore be sufficient to

overcome urinary binding and must be administered more frequently than in other

patients, and combinations of diuretics may be necessary

3. Cirrhosis ( 1 & 6 )

A.B.C.P. Sangli.31



Diuretics

The mainstay of diuretic therapy for patients with cirrhosis who have edema is

spironolactone, because secondary hyperaldosteronism is an important cause of

sodium and water retention in such patients. Spironolactone causes only a moderate

diuresis, which is desirable because greater diuresis may compromise the

intravascular volume Even if patients need additional diuretics, spironolactone should

be continued Repeated large-volume paranthesis may be used to minimize the need

for more potent diuretics.

The initial dose of spironolactone is usually 50 mg per day. The drug and its

active metabolites have sufficiently long half-lives that once-daily administration is

adequate Its biologic half-life is such that three to four days of treatment are needed to

attain steady-state effects. The dose can be increased to as much as 400 mg per day,

although doses higher than 200 mg per day are often poorly tolerated.

If maximal doses of spironolactone do not cause an adequate diuresis, a

thiazide diuretic can be added, the dose being determined by the level of renal

function If diuresis is still inadequate, a loop diuretic can be given instead of the

thiazide while spironolactone is continued.

The pharmacokinetics and pharmacodynamics of loop diuretics have been

well characterized in patients with cirrhosis. In patients with normal renal function,

the diuretic concentration in the tubular fluid is normal. Thus, a decreased response to

a loop diuretic in a patient with cirrhosis is not due to decreased delivery of the drug

to its site of action, and there is no need to administer large doses, unless the patient

has concomitant renal dysfunction.

Responses to loop diuretics are decreased in patients with cirrhosis because

the relation between the excretion rate and the natriuretic response is shifted

downward and to the right, so that the response to a maximally effective dose is

substantially less than the normal response. The cause of this shift is unknown. The

maximal response in a patient with severe cirrhosis may be the excretion of only 25 or

30 mmol of sodium, as compared with 200 to 250 mmol in normal subjects. This

response is not increased with larger doses, but with more frequent doses, hence given

alone or with a thiazide diuretic, may be effective.

A.B.C.P. Sangli.32



Diuretics

4. Congestive Heart Failure ( 1, 5, 9 )

Patients with edema caused by mild congestive heart failure should be treated

initially with a thiazide diuretic but most will require a loop diuretic. In patients with

normal or nearly normal renal function, the delivery of loop diuretics to the tubular

fluid is normal. The rate of absorption of loop diuretics is slowed in diuretic therapy

that may be increased by administering a mixture of albumin and a loop diuretic; in

several patients with severe hypoalbuminemia, an infusion of 30 mg of furosemide

mixed with 25 g of albumin enhanced diuresis. However, in most patients with the

nephrotic syndrome (and in those with cirrhosis ), renal tubular secretion of

furosemide is normal (unless the patient also has renal insufficiency), and combined

infusions are therefore unnecessary. This conclusion may not be applicable to patients

with serum albumin concentrations of less than 2 gm per deciliter. In such patients, it

may be reasonable to try combined infusions.

The diuretic response is subnormal in patients with the nephrotic syndrome,

despite an adequate rate of excretion of drug into the tubular fluid. In animals, and

presumably also in humans, diuretics become bound to albumin in tubular fluid,

resulting in a diminished amount of unbound, active drug and a decreased diuretic

response When urinary albumin concentrations exceed 4 gm per liter, one half to two

thirds of the diuretic that reaches the tubular fluid is bound to albumin in the fluid.

Consequently, doses two to three times the normal dose are needed to deliver

adequate amounts of unbound, active drug to the site of action In addition, patients

with the nephrotic syndrome may have a diminished response because of a decrease

in the drug's action on cells within the loop of Henle and because of increased

proximal or distal reabsorption of sodium Doses must therefore be sufficient to

overcome urinary binding and must be administered more frequently than in other

patients, and combinations of diuretics may be necessary

5. Hypertension: ( 6 )

Diuretics and or beta blockers are currently recommended as the first-line drug

therapy for hypertension. Low dose diuretic therapy is safe & effective in preventing

stroke, myocardial infarction, congestive heart failure & total mortality,

A.B.C.P. Sangli.33



Diuretics

Recent data suggest that diuretics are superior to beta blocker in older adults,

Clinically, the thiazides have long been mainstay of antihypertensive medication,

since they are inexpensive, convenient to administer, & well tolerated, 1< They are

effective in reducing systolic & diastolic blood pressure.

EVALUATION : ( 7 )

Diuretic and saluretic activity

In vitro methods

Carbonic anhydrase inhibition in vitro

Patch clamp technique in kidney cells

Perfusion of isolated kidney tubules of Isolated kidney

In vivo methods

Diuretic activity in rats (LIPSCHITZ test)

Saluretic activity in rats

• Diuretic and saluretic activity in dogs

• Clearance methods

• Micropuncture techniques in rats

• Stop flow technique

I. DIURETIC ACTIVITY IN RATS (LIPSCHITZ TEST)

PURPOSE AND RATIONALEA 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

uotient between excretion by testanimals and excretion by the urea control.

PROCEDURE

A.B.C.P. Sangli.34



Diuretics

Male Wistar rats weighing 100–200 g are used. Three animals per 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 two groups of three animals are used for one 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 and after 24 h. The sodium content of

the urine is determined by flame photometry. Active compounds 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

of2.0 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, whereasloop diuretics (or high ceiling diuretics) like

furosemide,bumetanide or piretanide reach values of 4.0 and more.

II. 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

A.B.C.P. Sangli.35



Diuretics

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

metaboliccage 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 calcu lated 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 (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

A.B.C.P. Sangli.36



Diuretics

mineralocorticoid, but reverses in a dose-related manner the effect of DOCA on

the Na+/K+ ratio in the urine (Kagawa et al. 1957; Bicking et al. 1965).

III. DIURETIC AND SALURETIC ACTIVITY IN DOGS


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 I nterval 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 anOsmometer.

A.B.C.P. Sangli.37



Diuretics

EVALUATION

Urine volume, electrolyte concentrations and osmolality are averaged for each

group. The values are plotted against time to allow comparison with pretreatmentvalues as well as with water controls and standards. The non- parametric U-test is

used for statistical analysis.

IV. CLEARANCE METHODS


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

A.B.C.P. Sangli.38



Diuretics

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 infusi on

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.

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Diuretics

References

1. Goodman Gilman’s Basics of Pharmactherapy, MeGraw Hill Publication .

742-761

2. Pathology , Harsh Mohan 80

3. Lippincott’s Pharmacology , Second Edition ,223

4. Pharmacology Fifth Edition H.P. Rang , M.M. Dale, J.M. Ritter, P.K. Moor.

354-358

5. Essentials of Medical Pharmacology By K.D. Tripathi, Fourth Edition 450

6. Essentials of Pharmacotherapeutics By F. S.K. Barar.

7. Drug Discovery and Evaluation By H.G. Vogel , Springer Publication 2002

317-328

8. The New England Journal Of Medicine

9. Pharmacology & Pharmacotherapeutics , R.S. Satoskar, S.D. Bhandarkar, 16th

Edition Popular Prakashan 520

10.Principles of Anatomy And Physiology By Tortora, Grabowski. Tenth

Edition. 950-964

Web Addresses:

a. www.sciencedirect.com

b. www.pubmed.com

c. www.diuretics.com

d. www.healthcenter.com

http://www.sciencedirect.com/

http://www.pubmed.com/

http://www.diuretics.com/

http://www.healthcenter.com/

http://www.sciencedirect.com/

http://www.pubmed.com/

http://www.diuretics.com/

http://www.healthcenter.com/

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