Section V - Drugs Affecting Renal and Cardiovascular Function doc

With regard to transcellular Cl– flux, Cl– crosses the luminal membrane via antiport with formate and oxalate proximal tubule, symport with Na+/K+ thick ascending limb, symport with Na+

Section V Drugs Affecting Renal and Cardiovascular Function

Chapter 29 Diuretics

Overview

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 The objective of this chapter is to provide the reader with unifying concepts as to how the kidney operates and how diuretics modify renal function The chapter begins with a description of renal anatomy and physiology, as this

information is prerequisite to a discussion of diuretic pharmacology Categories of diuretics are introduced and then described with regard to chemistry, mechanism of action, site of action, effects

on urinary composition, and effects on renal hemodynamics Near the end of the chapter, diuretic pharmacology is integrated with a discussion of mechanisms of edema formation and the role of diuretics in clinical medicine Therapeutic applications of diuretics are expanded upon in Chapters 33: Antihypertensive Agents and the Drug Therapy of Hypertension (hypertension) and 34:

Pharmacological Treatment of Heart Failure (heart failure)

Renal Anatomy and Physiology

elements of the renal cortex Efferent arterioles of juxtamedullary glomeruli descend into the medulla and divide to form the descending vasa recta, which supply blood to the capillaries of the

medulla Blood returning from the medulla via the ascending vasa recta drains directly into the

arcuate veins, and blood from the peritubular capillaries of the cortex enters the interlobular veins, which, in turn, connect with the arcuate veins Arcuate veins drain into interlobar veins, which in

turn drain into segmental veins, and blood leaves the kidney via the main renal vein.

The basic urine-forming unit of the kidney is the nephron, which consists of a filtering apparatus, the glomerulus, connected to a long tubular portion that reabsorbs and conditions the glomerular ultrafiltrate Each human kidney is composed of approximately 1 million nephrons The

nomenclature for segments of the tubular portion of the nephron has become increasingly complex

as renal physiologists have subdivided the nephron into shorter and shorter named segments These subdivisions initially were based on the axial location of the segments but increasingly have been based on the morphology of the epithelial cells lining the various nephron segments Figure 29–1 illustrates the currently accepted subdivision of the nephron into 14 subsegments Commonly encountered names that refer to these subsegments and to combinations of subsegments are

 Figure 29–1 Anatomy and Nomenclature of the Nephron. 

Glomerular Filtration

In the glomerular capillaries, a portion of the plasma water is forced through a filter that has three basic components: the fenestrated capillary endothelial cells, a basement membrane lying just beneath the endothelial cells, and the filtration slit diaphragms formed by the epithelial cells that cover the basement membrane on its urinary space side Solutes of small size flow with filtered water (solvent drag) into the urinary (Bowman's) space, whereas formed elements and

macromolecules are retained by the filtration barrier For each nephron unit, the rate of filtration (single-nephron glomerular filtration rate, SNGFR) is a function of the hydrostatic pressure in the

glomerular capillaries (PGC), the hydrostatic pressure in Bowman's space (which can be equated

with pressure in the proximal tubule, PT), the mean colloid osmotic pressure in the glomerular capillaries ( GC), the colloid osmotic pressure in the proximal tubule ( T), and the ultrafiltration

coefficient (Kf), according to the equation:

SNGFR = Kf[(PGC– ( GC– T)]  (29–1)

If PGC–PT is defined as the transcapillary hydraulic pressure difference ( P), and if T is negligible (as it usually is since little protein is filtered), then:

SNGFR = Kf( P– GC)  (29–2)

This latter equation succinctly expresses the three major determinants of SNGFR However, each of

these three determinants can be influenced by a number of other variables Kf is determined by the physicochemical properties of the filtering membrane and by the surface area available for

filtration P is determined primarily by the arterial blood pressure and by the proportion of the

arterial pressure that is transmitted to the glomerular capillaries This is governed by the relative resistances of preglomerular and postglomerular vessels GC is determined by two variables, i.e.,

the concentration of protein in the arterial blood entering the glomerulus and the single-nephron

blood flow (QA) QA influences GC because, as blood transverses the glomerular capillary bed, filtration concentrates proteins in the capillaries, causing GC to rise with distance along the

glomerular bed When QA is high, this effect is reduced; however, when QA is low, GC may

increase to the point that GC= P and filtration ceases (a condition known as filtration equilibrium;

seeDeen et al. , 1972 )

Overview of Nephron Function

Approximately 120 ml of ultrafiltrate is formed each minute, yet only 1 ml/min of urine is

produced Therefore, greater than 99% of the glomerular ultrafiltrate is reabsorbed at a staggering energy cost The kidneys consume 7% of total-body oxygen intake despite the fact that the kidneys make up only 0.5% of body weight The kidney is designed to filter large quantities of plasma, reabsorb those substances that the body must conserve, and leave behind and/or secrete substances that must be eliminated

The proximal tubule is contiguous with Bowman's capsule and takes a tortuous path until finally forming a straight portion that dives into the renal medulla The proximal tubule has been

subdivided into S1, S2, and S3 segments based on the morphology of the epithelial cells lining the tubule Normally, approximately 65% of filtered Na+ is reabsorbed in the proximal tubule, and sincethis part of the tubule is highly permeable to water, reabsorption is essentially isotonic

Between the outer and inner strips of the outer medulla, the tubule abruptly changes morphology to

become the descending thin limb (DTL), which penetrates the inner medulla, makes a hairpin turn, and then forms the ascending thin limb (ATL) At the juncture between the inner and outer medulla,the tubule once again changes morphology and becomes the thick ascending limb, which is made up

of three segments: a medullary portion (MTAL), a cortical portion (CTAL), and a postmacular segment Together, the proximal straight tubule, DTL, ATL, MTAL, CTAL, and postmacular

segment are known as the loop of Henle The DTL is highly permeable to water, yet its permeability

to NaCl and urea is low In contrast, the ATL is permeable to NaCl and urea but is impermeable to water The thick ascending limb actively reabsorbs NaCl but is impermeable to water and urea Approximately 25% of filtered Na+ is reabsorbed in the loop of Henle, mostly in the thick ascendinglimb, which has a large reabsorptive capacity

The thick ascending limb passes between the afferent and efferent arterioles and makes contact with

the afferent arteriole via a cluster of specialized columnar epithelial cells known as the macula

densa The macula densa is strategically located to sense concentrations of NaCl leaving the loop of

Henle If the concentration of NaCl is too high, the macula densa sends a chemical signal (perhaps adenosine) to the afferent arteriole of the same nephron, causing it to constrict This in turn causes a

reduction in PGC and QA and decreases SNGFR This homeostatic mechanism, known as

tubuloglomerular feedback (TGF), serves to protect the organism from salt and volume wasting

Besides causing a TGF response, the macula densa also regulates renin release from the adjacent juxtaglomerular cells in the wall of the afferent arteriole

Approximately 0.2 mm past the macula densa, the tubule changes morphology once again to

become the distal convoluted tubule (DCT) The postmacular segment of the thick ascending limb

and the distal convoluted tubule often are referred to as the early distal tubule Like the thick

ascending limb, the DCT actively transports NaCl and is impermeable to water Since these

characteristics impart the ability to produce a dilute urine, the thick ascending limb and the DCT are

collectively called the diluting segment of the nephron, and the tubular fluid in the DCT is

hypotonic regardless of hydration status However, unlike the thick ascending limb, the DCT does

not contribute to the countercurrent-induced hypertonicity of the medullary interstitium (see below).

The collecting duct system (connecting tubule + initial collecting tubule + cortical collecting duct + outer and inner medullary collecting duct) is an area of fine control of ultrafiltrate composition and volume It is here that final adjustments in electrolyte composition are made, a process modulated

by the adrenal steroid, aldosterone In addition, permeability of this part of the nephron to water is modulated by antidiuretic hormone (ADH; seeChapter 30: Vasopressin and Other Agents Affecting the Renal Conservation of Water)

The more distal portions of the collecting duct pass through the renal medulla, where the interstitial fluid is markedly hypertonic In the absence of ADH, the collecting duct system is impermeable to water, and a dilute urine is excreted However, in the presence of ADH, the collecting duct system

is permeable to water, so that water is reabsorbed The movement of water out of the tubule is driven by the steep concentration gradient that exists between the tubular fluid and the medullary interstitium

The hypertonicity of the medullary interstitium plays a vital role in the ability of mammals and birds to concentrate urine and is therefore a key adaptation necessary for living in a terrestrial

environment This is accomplished via a combination of the unique topography of the loop of Henle

and the specialized permeability features of the loop's subsegments Although the precise

mechanism giving rise to the medullary hypertonicity has remained elusive, the passive

countercurrent multiplier hypothesis of Kokko and Rector (1972) is an intuitively attractive model

that is qualitatively accurate (seeSands and Kokko, 1996) According to this hypothesis, the process

begins with active transport in the thick ascending limb, which concentrates NaCl in the interstitium

of the outer medulla Since this segment of the nephron is impermeable to water, active transport in the ascending limb dilutes the tubular fluid As the dilute fluid passes into the collecting duct system, water is extracted if and only if ADH is present Since the cortical and outer medullary collecting ducts have a low permeability to urea, urea is concentrated in the tubular fluid The inner medullary collecting duct, however, is permeable to urea, so that urea diffuses into the inner

medulla where it is trapped by countercurrent exchange in the vasa recta Since the DTL is

impermeable to salt and urea, the high urea concentration in the inner medulla extracts water from the DTL and concentrates NaCl in the tubular fluid of the DTL As the tubular fluid enters the ATL,NaCl diffuses out of the salt-permeable ATL, thus contributing to the hypertonicity of the

medullary interstitium

General Mechanism of Renal Epithelial Transport

Figure 29–2 illustrates seven mechanisms by which solute crosses renal epithelial cell membranes

If bulk water flow occurs across a membrane, solute molecules will be transferred by convection

across the membrane, a process known as solvent drag Solutes with sufficient lipid solubility may

also dissolve in the membrane and diffuse across the membrane down their electrochemical

gradients (simple diffusion) Many solutes, however, have limited lipid solubility, and transport must rely on integral proteins embedded in the cell membrane In some cases, the integral protein merely provides a conductive pathway (pore) through which the solute may diffuse passively

(channel-mediated diffusion) In other cases, the solute may bind to the integral protein and, due to

a conformational change in the protein, be transferred across the cell membrane down an

electrochemical gradient (carrier-mediated or facilitated diffusion, also called uniport) However,

this process will not result in net movement of solute against an electrochemical gradient If solute must be moved "uphill" against an electrochemical gradient, then either primary active transport or secondary active transport is required With primary active transport, ATP hydrolysis is coupled directly to conformational changes in the integral protein, thus providing the necessary free energy

(ATP-mediated transport) Often, ATP-mediated transport is used to create an electrochemical

gradient for a given solute, and the free energy of that solute gradient is then released to drive the

"uphill" transport of other solutes This process requires symport (cotransport of solute species in the same direction) or antiport (countertransport of solute species in opposite directions) and is known as secondary active transport.

Figure 29–2 Seven Basic Mechanisms for Transmembrane Transport of Solutes. 1, convective flow in which dissolved solutes are "dragged" by bulk water flow; 2, simple diffusion of lipophilic solute across membrane; 3, diffusion

of solute through pore; 4, transport of solute by carrier protein down electrochemical gradient; 5, transport of solute by carrier protein against electrochemical gradient with ATP hydrolysis providing driving force; 6 and 7, cotransport and countertransport, respectively, of solutes with one solute traveling "uphill" against an electrochemical gradient and the other solute traveling down an electrochemical gradient

The kinds of transport achieved in a particular nephron segment depend mainly on which

transporters are present and whether they are embedded in the luminal or basolateral membrane A general model of renal tubular transport is shown in Figure 29–3 and can be summarized as follows:

Figure 29–3 Generic Mechanism of Renal Epithelial Cell Transport (See Text forDetails). S, symporter; A, antiporter; CH, ion channel; WP, water pore; U,

uniporter; ATPase, Na+,K+–ATPase (sodium pump); X and Y, transported solutes;

P, membrane-permeable (reabsorbable) solutes; I, membrane-impermeable

(nonreabsorbable) solutes; PD, potential difference across indicated membrane orcell

Na+,K+–ATPase (sodium pump) in the basolateral membrane hydrolyzes ATP, which results in the transport of Na+ into the intercellular and interstitial spaces and the movement of K+ into the cell Although other ATPases exist in selected renal epithelial cells and participate in the

transport of specific solutes (e.g., Ca2+–ATPase and H+–ATPase), the bulk of all transport in the kidney is due to the abundant supply of Na+,K+–ATPase in the basolateral membranes of the renal epithelial cells

Na+ may diffuse across the luminal membrane via Na+ channels into the epithelial cell down the electrochemical gradient for Na+ that is established by the basolateral Na+,K+–ATPases In addition, the free energy available in the electrochemical gradient for Na+ is tapped by integral proteins in the luminal membrane, resulting in cotransport of various solutes against their

electrochemical gradients by symporters (e.g., Na+–glucose, Na+–Pi, Na+–amino acid) This process results in movement of Na+ and cotransported solutes out of the tubular lumen into the

cell Also, antiporters (e.g., Na+–H+) countertransport Na+ out of and some solutes into the tubular lumen

  3. 

Na+ exits the basolateral membrane into the intercellular and interstitial spaces via the Na+ pump

or via symporters or antiporters in the basolateral membrane.

  4. 

The action of Na+-linked symporters in the luminal membrane causes the concentration of substrates for these symporters to rise in the epithelial cell These electrochemical gradients thenpermit simple diffusion or mediated transport (symporters, antiporters, uniporters, and channels)

of solutes into the intercellular and interstitial spaces

  5. 

Accumulation of Na+ and other solutes in the intercellular space creates a small osmotic

pressure differential across the epithelial cell In water-permeable epithelium, water moves into the intercellular spaces driven by the osmotic pressure differential Water moves through

aqueous pores in both the luminal and the basolateral cell membranes as well as through the tight junctions (paracellular pathway) Bulk water flow carries some solutes into the

intercellular space by solvent drag

  6. 

Movement of water into the intercellular space concentrates other solutes in the tubular fluid, resulting in an electrochemical gradient for these substances across the epithelium Membrane-permeable solutes then move down their electrochemical gradients into the intercellular space

via both the transcellular (simple diffusion, symporters, antiporters, uniporters, and channels)

and paracellular pathways Membrane-impermeable solutes remain in the tubular lumen and are excreted in the urine with an obligatory amount of water

  7. 

As water and solutes accumulate in the intercellular space, the hydrostatic pressure increases, thus providing a driving force for bulk water flow Bulk water flow carries solute (solute

convection) out of the intercellular space into the interstitial space and, finally, into the

peritubular capillaries The movement of fluid into the peritubular capillaries is governed by the same Starling forces that determine transcapillary fluid movement for any capillary bed

Mechanism of Organic Acid and Organic Base Secretion

The kidney is a major organ involved in the elimination of organic chemicals from the body

Organic molecules may enter the renal tubules by glomerular filtration of molecules not bound to plasma proteins or may be actively secreted directly into the tubules The proximal tubule has a highly efficient transport system for organic acids and an equally efficient but separate transport system for organic bases Current models for these secretory systems are illustrated in Figure 29–4 Both systems are powered by the sodium pump in the basolateral membrane, involve secondary andtertiary active transport, and utilize a poorly characterized facilitated-diffusion step The antiporter that exchanges -ketoglutarate for organic acids has been cloned from several species, including human beings (Lu et al. , 1999 ) The optimal substrate for transport by the organic acid secretory mechanism is a molecule with a negative or partial negative charge, separated by 6 to 7 Å from a second negative charge, and a hydrophobic region 8 to 10 Å long However, much flexibility exists

around this optimal motif, and the system transports a large variety of organic acids The organic base secretory mechanism is even less discriminating and may involve a family of related transport mechanisms This system(s) transports many drugs containing an amine nitrogen positively charged

at physiological pH

Figure 29–4 Mechanisms of Organic Acid (A) and Organic Base (B) Secretion inthe Proximal Tubule. The numbers 1, 2, and 3 refer to primary, secondary, and tertiary active transport A–, organic acid (anion); C+, organic base (cation);

KG2–, -ketoglutarate, but also other dicarboxylates

Renal Handling of Specific Anions and Cations

Reabsorption of Cl– generally follows reabsorption of Na+ In segments of the tubule with

low-resistance tight junctions (i.e., "leaky" epithelium), such as the proximal tubule and thick ascending

limb, Cl– movement can occur paracellularly With regard to transcellular Cl– flux, Cl– crosses the

luminal membrane via antiport with formate and oxalate (proximal tubule), symport with Na+/K+

(thick ascending limb), symport with Na+ (DCT), and antiport with HCO3 (collecting duct system)

Cl– crosses the basolateral membrane via symport with K+ (proximal tubule and thick ascending limb), antiport with Na+/HCO3 (proximal tubule), and Cl– channels (thick ascending limb, DCT, collecting duct system)

Eighty to ninety percent of filtered K+ is reabsorbed in the proximal tubule (diffusion and solvent

drag) and thick ascending limb (diffusion), largely via the paracellular pathway In contrast, the

DCT and collecting duct system secrete variable amounts of K+via a conductive (channel-mediated)

pathway Modulation of the rate of K+ secretion in the collecting duct system, particularly by aldosterone, allows urinary excretion of K+ to be matched with dietary intake The transepithelial

potential difference (VT), lumen-positive in the thick ascending limb and lumen-negative in the collecting duct system, provides an important driving force for K+ reabsorption and secretion, respectively

Most of the filtered Ca2+ (approximately 70%) is reabsorbed by the proximal tubule by passive

diffusion, probably via a paracellular route Another 25% of filtered Ca2+ is reabsorbed by the thick

ascending limb, mostly via a paracellular route driven by the lumen-positive VT, although a

component of active Ca2+ reabsorption also may exist The remaining Ca2+ is reabsorbed in the

distal convoluted tubule and the connecting tubule via a transcellular pathway that is modulated by parathyroid hormone (PTH; seeChapter 62: Agents Affecting Calcification and Bone Turnover:

Calcium, Phosphate, Parathyroid Hormone, Vitamin D, Calcitonin, and Other Compounds) PTH appears to increase Ca2+ channels in the luminal membrane, thereby facilitating the passive

movement of Ca2+ into the epithelial cell Ca2+ is extruded across the basolateral membrane by a

Ca2+–ATPase and via Na+–Ca2+ antiport

Inorganic phosphate (Pi) is largely reabsorbed (80% of filtered load) by the proximal tubule A Na+–

Pi symporter uses the free energy of the Na+ electrochemical gradient to effect secondary active transport of Pi into the cell The Na+–Pi symporter is inhibited by PTH Pi exits the basolateral membrane down its electrochemical gradient by a poorly understood transport system

Only 20% to 25% of Mg2+ is reabsorbed in the proximal tubule, and only 5% is reabsorbed by the DCT and collecting duct system The bulk of Mg2+ is reabsorbed in the thick ascending limb via a paracellular pathway driven by the lumen-positive VT However, transcellular movement of Mg2+

also may occur with basolateral exit via Na+–Mg2+ antiport or via a Mg2+–ATPase

The renal tubules play an extremely important role in the reabsorption of HCO3 and secretion of protons (tubular acidification) and thus participate critically in the maintenance of acid–base

balance A description of these processes is presented in the section on carbonic anhydrase

inhibitors

Principles of Diuretic Action

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 as 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 (thiazide-like diuretics), effects on potassium excretion sparing diuretics), etc However, since the mechanism of action of each of the major classes of diuretics is now reasonably well understood, a classification scheme based on mechanism of action

(potassium-is now possible and (potassium-is used in th(potassium-is 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 H2PO4), and uric acid In addition, diuretics may indirectly alter renal hemodynamics Table 29–1 gives a comparison of the general effects of the major classes of diuretics

Inhibitors of Carbonic Anhydrase

Acetazolamide (DIAMOX) is the prototype of a class of agents that have limited usefulness as diuretics but have played a major role in the development of fundamental concepts of renal

physiology and pharmacology

Chemistry

When sulfanilamide was introduced as a chemotherapeutic agent, metabolic acidosis was

recognized as a side effect This observation led to in vitro and in vivo studies demonstrating that

sulfanilamide is an inhibitor of carbonic anhydrase Subsequently, an enormous number of

sulfonamides were synthesized and tested for the ability to inhibit carbonic anhydrase; of these compounds, acetazolamide has been most extensively studied Table 29–2 lists the chemical

structures of the three carbonic anhydrase inhibitors currently available in the United States—

acetazolamide, dichlorphenamide (DARANIDE), and methazolamide (NEPTAZANE) The common molecular motif of available carbonic anhydrase inhibitors is an unsubstituted sulfonamide moiety.Mechanism and Site of Action

Proximal tubular epithelial cells are richly endowed with the zinc metalloenzyme carbonic

anhydrase, which is found in the luminal and basolateral membranes (type IV carbonic anhydrase,

an enzyme tethered to the membrane by a glycosylphosphatidylinositol linkage) as well as in the cytoplasm (type II carbonic anhydrase) Davenport and Wilhelmi (1941) were the first to discover this enzyme in the mammalian kidney, and subsequent studies revealed the key role played by

carbonic anhydrase in NaHCO3 reabsorption and acid secretion (seeMaren, 1967 and 1980).

In the proximal tubule, the free energy in the Na+ gradient established by the basolateral Na+ pump

is used by a Na+–H+ antiporter (also referred to as a Na+–H+ exchanger or NHE) in the luminal membrane to transport H+ into the tubular lumen in exchange for Na+ (Figure 29–5) In the lumen,

H+ reacts with filtered HCO3 to form H2CO3, which rapidly decomposes to CO2 and water in the presence of carbonic anhydrase in the brush border Normally the reaction between CO2 and water occurs slowly, but carbonic anhydrase reversibly accelerates this reaction several thousandfold CO2

is lipophilic and rapidly diffuses across the luminal membrane into the epithelial cell where it reactswith water to form H2CO3, a reaction catalyzed by cytoplasmic carbonic anhydrase (The actual reaction catalyzed by carbonic anhydrase is OH–+ CO2 HCO3 ; however, H2O OH–+ H+ and HCO3 + H+ H2CO3, so that the net reaction is H2O + CO2 H2CO3.) Continued operation of the

Na+–H+ antiporter maintains a low proton concentration in the cell, so that H2CO3 spontaneously ionizes to form H+ and HCO3, creating an electrochemical gradient for HCO3 across the

basolateral membrane The electrochemical gradient for HCO3 is used by a Na+–HCO3 symporter (also called Na+–HCO3 cotransporter or NBC) in the basolateral membrane to transport NaHCO3

into the interstitial space The net effect of this process is transport of NaHCO3 from the tubular lumen to the interstitial space followed by movement of water (isotonic reabsorption) Removal of water concentrates Cl– in the tubular lumen, and consequently Cl– diffuses down its concentration

gradient into the interstitium via the paracellular pathway.

Figure 29–5 NaHCO3 Reabsorption in Proximal Tubule and Mechanism of Diuretic Action of Carbonic Anhydrase (CA) Inhibitors. A, antiporter; S, symporter; CH, ion channel (The actual reaction catalyzed by carbonic anhydrase is OH–+ CO2 HCO3 ; however, H2O OH–+ H+, and HCO3+ H+

H2CO3, so that the net reaction is H2O + CO2 H2CO3.) Numbers in parentheses indicate stoichiometry

Carbonic anhydrase inhibitors potently inhibit (IC50 for acetazolamide is 10 nM) both the

membrane-bound and cytoplasmic forms of carbonic anhydrase, resulting in nearly complete abolition of NaHCO3 reabsorption in the proximal tubule (Cogan et al. , 1979 ) Studies with a high-molecular-weight carbonic anhydrase inhibitor that only inhibits luminal enzyme because of limitedcellular permeability indicate that inhibition of both the membrane-bound and cytoplasmic pools of carbonic anhydrase contributes to the diuretic activity of carbonic anhydrase inhibitors (Maren et

al.

, 1997) Because of the large excess of carbonic anhydrase in proximal tubules, a high percentage

of enzyme activity must be inhibited before an effect on electrolyte excretion is observed Although the proximal tubule is the major site of action of carbonic anhydrase inhibitors, carbonic anhydrase also is involved in secretion of titratable acid in the collecting duct system (a process that involves aproton pump); therefore the collecting duct system is a secondary site of action for this class of drugs

Effects on Urinary Excretion

Inhibition of carbonic anhydrase is associated with a rapid rise in urinary HCO3 excretion to approximately 35% of filtered load This, along with inhibition of titratable acid and ammonia secretion in the collecting duct system, results in an increase in urinary pH to approximately 8 and development of a metabolic acidosis However, even with a high degree of inhibition of carbonic anhydrase, 65% of HCO3 is rescued from excretion by poorly understood mechanisms that may involve carbonic anhydrase–independent HCO3 reabsorption at downstream sites Inhibition of the transport mechanism described in the preceding section results in increased delivery of Na+ and Cl–

to the loop of Henle, which has a large reabsorptive capacity and captures most of the Cl– and a portion of the Na+ Thus, only a small increase in Cl– excretion occurs, HCO3 being the major anionexcreted along with the cations Na+ and K+ The fractional excretion of Na+ may be as much as 5%, and the fractional excretion of K+ can be as much as 70% The increased excretion of K+ is

secondary to increased delivery of Na+ to the distal nephron The mechanism by which increased distal delivery of Na+ enhances K+ excretion is described in the section on inhibitors of sodium channels Carbonic anhydrase inhibitors also increase phosphate excretion (mechanism unknown), but have little or no effect on the excretion of Ca2+ or Mg2+ The effects of carbonic anhydrase inhibitors on renal excretion are self-limiting, probably because, as metabolic acidosis develops, thefiltered load of HCO3 decreases to the point that the uncatalyzed reaction between CO2 and water issufficient to achieve HCO3 reabsorption

Effects on Renal Hemodynamics

By inhibiting proximal reabsorption, carbonic anhydrase inhibitors increase delivery of solutes to the macula densa This triggers tubuloglomerular feedback (TGF), which increases afferent

arteriolar resistance and reduces renal blood flow (RBF) and glomerular filtration rate (GFR) (Persson and Wright, 1982)

Other Actions

Carbonic anhydrase is present in a number of extrarenal tissues including the eye, gastric mucosa, pancreas, central nervous system (CNS), and red blood cells (RBCs) Carbonic anhydrase in the ciliary processes of the eye mediates the formation of large amounts of HCO3 in aqueous humor For this reason, inhibition of carbonic anhydrase decreases the rate of formation of aqueous humor and consequently reduces intraocular pressure Acetazolamide frequently causes paresthesias and somnolence, suggesting an action of carbonic anhydrase inhibitors in the CNS The efficacy of acetazolamide in epilepsy is in part due to the production of metabolic acidosis; however, direct

actions of acetazolamide in the CNS also contribute to its anticonvulsant action Due to interferencewith carbonic anhydrase activity in RBCs, carbonic anhydrase inhibitors increase CO2 levels in peripheral tissues and decrease CO2 levels in expired gas Large doses of carbonic anhydrase inhibitors reduce gastric acid secretion, but this has no therapeutic applications.

Absorption and Elimination

The oral bioavailability, plasma half-life, and route of elimination of the three currently available carbonic anhydrase inhibitors are listed in Table 29–2 Carbonic anhydrase inhibitors are avidly bound by carbonic anhydrase and, accordingly, tissues rich in this enzyme will have higher

concentrations of carbonic anhydrase inhibitors following systemic administration

Toxicity, Adverse Effects, Contraindications, Drug Interactions

Serious toxic reactions to carbonic anhydrase inhibitors are infrequent; however, these drugs are sulfonamide derivatives and, like other sulfonamides, may cause bone-marrow depression, skin toxicity, and sulfonamide-like renal lesions and may cause allergic reactions in patients

hypersensitive to sulfonamides With large doses, many patients exhibit drowsiness and

paresthesias Most adverse effects, contraindications, and drug interactions are secondary to urinary alkalinization or metabolic acidosis, including: (1) diversion of ammonia of renal origin from urine into the systemic circulation, a process that may induce hepatic encephalopathy (the drugs are contraindicated in patients with hepatic cirrhosis); (2) calculus formation and ureteral colic due to precipitation of calcium phosphate salts in an alkaline urine; (3) worsening of metabolic or

respiratory acidosis (the drugs are contraindicated in patients with hyperchloremic acidosis or severe chronic obstructive pulmonary disease); (4) interference with the urinary tract antiseptic methenamine; and (5) reduction of the urinary excretion rate of weak organic bases

Therapeutic Uses

Although acetazolamide is used for treatment of edema, the efficacy of carbonic anhydrase

inhibitors as single agents is low, and carbonic anhydrase inhibitors are not widely employed in thisregard However, studies by Knauf and Mutschler (1997) indicate that the combination of

acetazolamide with diuretics that block Na+ reabsorption at more distal sites in the nephron causes amarked natriuretic response in patients with low basal fractional excretion of Na+ (<0.2%) who are resistant to diuretic monotherapy Even so, the long-term usefulness of carbonic anhydrase

inhibitors often is compromised by development of metabolic acidosis

The major indication for carbonic anhydrase inhibitors is open-angle glaucoma Carbonic anhydraseinhibitors also may be employed for secondary glaucoma and preoperatively in acute angle-closure

glaucoma to lower ocular pressure before surgery (seeChapter 66: Ocular Pharmacology)

Acetazolamide also is used for the treatment of epilepsy (seeChapter 21: Drugs Effective in the Therapy of the Epilepsies) The rapid development of tolerance, however, may limit the usefulness

of carbonic anhydrase inhibitors for epilepsy Acetazolamide may provide symptomatic relief in patients with acute mountain sickness; however, it is more appropriate to give acetazolamide as a prophylactic measure (Coote, 1991) Acetazolamide also is useful in patients with familial periodic paralysis (Links et al. , 1988 ) The mechanism for the beneficial effects of acetazolamide in

mountain sickness and familial periodic paralysis is not clear, but it may be related to the induction

of a metabolic acidosis Finally, carbonic anhydrase inhibitors can be useful for correcting a

metabolic alkalosis, especially an alkalosis caused by diuretic-induced increases in H+ excretion

Osmotic Diuretics

Osmotic diuretics are agents that are freely filtered at the glomerulus, undergo limited reabsorption

by the renal tubule, and are relatively inert pharmacologically Osmotic diuretics are administered

in large enough doses to increase significantly the osmolality of plasma and tubular fluid Table 29–

3 gives the molecular structures of the four currently available osmotic diuretics—glycerin ,

isosorbide, mannitol , and urea.

Mechanism and Site of Action

For many years it was thought that osmotic diuretics act primarily in the proximal tubule (Wesson and Anslow, 1948) By acting as nonreabsorbable solutes, it was reasoned that osmotic diuretics limit the osmosis of water into the interstitial space and thereby reduce luminal Na+ concentration tothe point that net Na+ reabsorption ceases Indeed, early micropuncture studies supported this concept (Windhager et al. , 1959 ) However, subsequent studies suggest that this mechanism, while operative, may be of only secondary importance For instance, mannitol only slightly increases the delivery of Na+ and moderately increases the delivery of water out of the proximal tubule (Seely and Dirks, 1969), and urea does not alter proximal tubular reabsorption in rats at the time of a large osmotic diuresis (Kauker et al. , 1970 ) Mannitol, on the other hand, markedly increases the delivery

of Na+ and water out of the loop of Henle (Seely and Dirks, 1969), suggesting that the major site of action is the loop of Henle

By extracting water from intracellular compartments, osmotic diuretics expand the extracellular fluid volume, decrease blood viscosity, and inhibit renin release These effects increase RBF, and the increase in renal medullary blood flow removes NaCl and urea from the renal medulla, thus reducing medullary tonicity Also, under some circumstances, prostaglandins may contribute to the renal vasodilation and medullary washout induced by osmotic diuretics (Johnston et al. , 1981 ) A reduction in medullary tonicity causes a decrease in the extraction of water from the DTL, which in turn limits the concentration of NaCl in the tubular fluid entering the ATL This latter effect

diminishes the passive reabsorption of NaCl in the ATL In addition, the marked ability of osmotic diuretics to inhibit reabsorption of Mg2+, a cation that is mainly reabsorbed in the thick ascending limb, suggests that osmotic diuretics also interfere with transport processes in the thick ascending limb The mechanism of this effect is unknown

In summary, osmotic diuretics act both in the proximal tubule and the loop of Henle, with the latter being the primary site of action Also, osmotic diuretics probably act by an osmotic effect in the tubules and by reducing medullary tonicity

Effects on Urinary Excretion

Osmotic diuretics increase the urinary excretion of nearly all electrolytes, including Na+, K+, Ca2+,

Mg2+, Cl–, HCO3, and phosphate

Effects on Renal Hemodynamics

As indicated in the preceding section, osmotic diuretics increase RBF by a variety of mechanisms

Osmotic diuretics dilate the afferent arteriole, which increases PGC, and dilute the plasma, which decreases GC These effects would increase GFR were it not for the fact that osmotic diuretics also

increase PT In general, superficial SNGFR is increased but total GFR is little changed

Absorption and Elimination

The oral bioavailability, plasma half-life, and route of elimination of the four currently available osmotic diuretics are listed in Table 29–3 Glycerin and isosorbide can be given orally, whereas mannitol and urea must be administered intravenously

Toxicity, Adverse Effects, Contraindications, Drug Interactions

Osmotic diuretics are distributed in the extracellular fluid and contribute to the extracellular

osmolality Thus, water is extracted from intracellular compartments, and the extracellular fluid volume becomes expanded In patients with heart failure or pulmonary congestion, this may cause frank pulmonary edema Extraction of water also causes hyponatremia, which may explain commonadverse effects, including headache, nausea, and vomiting On the other hand, loss of water in excess of electrolytes can cause hypernatremia and dehydration In general, osmotic diuretics are contraindicated in patients who are anuric due to severe renal disease or who are unresponsive to test doses of the drugs Urea may cause thrombosis or pain if extravasation occurs, and it should not

be administered to patients with impaired liver function because of the risk of elevation of blood ammonia levels Both mannitol and urea are contraindicated in patients with active cranial bleeding.Glycerin is metabolized and can cause hyperglycemia

Therapeutic Uses

A rapid decrease in GFR, i.e., acute renal failure (ARF), is a serious medical condition that occurs

in 5% of hospitalized patients and is associated with a significant mortality rate ARF can be caused

by diverse conditions both extrinsic (prerenal and postrenal failure) and intrinsic to the kidney

Acute tubular necrosis (ATN), i.e., damage to tubular epithelial cells, accounts for the majority of

cases of intrinsic ARF In animal models, mannitol is effective in attenuating the reduction in GFR associated with ATN when administered before the ischemic insult or offending nephrotoxin The renal protection afforded by mannitol may be due to removal of obstructing tubular casts, dilution

of nephrotoxic substances in the tubular fluid, and/or reduction of swelling of tubular elements via

osmotic extraction of water Although prophylactic mannitol is effective in animal models of ATN, the clinical efficacy of mannitol is less well established Most published clinical studies have been

uncontrolled, and controlled studies have not shown a benefit over hydration per se (seeKellum,

1998) In patients with mild-to-moderate renal insufficiency, hydration with 0.45% sodium chloride

is as good as or better than either mannitol or furosemide in protection against decreases in GFR

induced by radiocontrast agents (Soloman et al., 1994) Studies of prophylactic mannitol indicate

effectiveness in jaundiced patients undergoing surgery (Dawson, 1965) However, in vascular and open heart surgery, prophylactic mannitol maintains urine flow but not GFR In established ATN, mannitol will increase urine volume in some patients, and those patients converted from oliguric to nonoliguric ATN appear to recover more rapidly and require less dialysis compared with patients who do not respond to mannitol (Levinsky and Bernard, 1988) However, it is not clear whether these benefits are due to the diuretic or whether "responders" have lesser degrees of renal damage from the outset compared with "nonresponders." Repeated administration of mannitol to

nonresponders is not recommended, and nowadays loop diuretics are more frequently used to convert oliguric to nonoliguric ATN

Another use for mannitol and urea is in the treatment of dialysis disequilibrium syndrome Too rapid a removal of solutes from the extracellular fluid by hemodialysis or peritoneal dialysis results

in a reduction in the osmolality of the extracellular fluid Consequently, water moves from the extracellular compartment into the intracellular compartment, causing hypotension and CNS

symptoms (headache, nausea, muscle cramps, restlessness, CNS depression, and convulsions) Osmotic diuretics increase the osmolality of the extracellular fluid compartment and thereby shift water back into the extracellular compartment.

By increasing the osmotic pressure of the plasma, osmotic diuretics extract water from the eye and brain All four osmotic diuretics are used to control intraocular pressure during acute attacks of glaucoma and for short-term reductions in intraocular pressure, both preoperatively and

postoperatively, in patients who require ocular surgery Also, mannitol and urea are used to reduce cerebral edema and brain mass before and after neurosurgery

Inhibitors of Na +–K+–2 Cl– Symport (Loop Diuretics; High-Ceiling Diuretics)

Inhibitors of Na+–K+–2Cl– symport are a group of diuretics that have in common an ability to block the Na+–K+–2Cl– symporter in the thick ascending limb of the loop of Henle; hence these diuretics

also are referred to as loop diuretics Although the proximal tubule reabsorbs approximately 65% of

the filtered Na+, diuretics acting only in the proximal tubule have limited efficacy because the thick ascending limb has a great reabsorptive capacity and reabsorbs most of the rejectate from the proximal tubule Diuretics acting predominantly at sites past the thick ascending limb also have limited efficacy, because only a small percentage of the filtered Na+ load reaches these more distal sites In contrast, inhibitors of Na+–K+–2Cl– symport are highly efficacious, and for this reason they

often are called high-ceiling diuretics The efficacy of inhibitors of Na+–K+–2Cl– symport in the thick ascending limb of the loop of Henle is due to a combination of two factors: (1) Approximately25% of the filtered Na+ load normally is reabsorbed by the thick ascending limb; and (2) nephron segments past the thick ascending limb do not possess the reabsorptive capacity to rescue the flood

of rejectate exiting the thick ascending limb

Chemistry

Inhibitors of Na+–K+–2Cl– symport are a chemically diverse group of drugs (seeTable 29–4)

Furosemide, bumetanide, azosemide, piretanide, and tripamide all contain a sulfonamide moiety, whereas ethacrynic acid is a phenoxyacetic acid derivative Muzolimine has neither of these

structural features, and torsemide is a sulfonylurea Only furosemide ( LASIX ), bumetanide ( BUMEX

), ethacrynic acid (EDECRIN), and torsemide ( DEMADEX ) are available currently in the United

States

Mechanism and Site of Action

Inhibitors of Na+–K+–2Cl– symport act primarily in the thick ascending limb Micropuncture of the DCT demonstrates that loop diuretics increase the delivery of solutes out of the loop of Henle (Dirks and Seely, 1970) Also, in situ microperfusion of the loop of Henle (Morgan et al. , 1970 ) and

in vitro microperfusion of the CTAL (Burg et al. , 1973 ) indicate inhibition of transport by low concentrations of furosemide in the perfusate Some inhibitors of Na+–K+–2Cl– symport may have additional effects in the proximal tubule; however, the significance of these effects is unclear

It was initially thought that Cl– was transported by a primary active electrogenic transporter in the luminal membrane independent of Na+ Discovery of furosemide-sensitive Na+–K+–2Cl– symport in other tissues caused Greger (1981) to investigate more carefully the Na+ dependence of Cl– transport

in the isolated perfused rabbit CTAL By scrupulously removing Na+ from the luminal perfusate, Greger demonstrated the dependence of Cl– transport on Na+ It is now well accepted that, in the thick ascending limb, flux of Na+, K+, and Cl– from the lumen into the epithelial cell is mediated by

a Na+–K+–2Cl– symporter (seeFigure 29–6) This symporter captures the free energy in the Na+

electrochemical gradient established by the basolateral Na+ pump and provides for "uphill" transport

of K+ and Cl– into the cell K+ channels in the luminal membrane (called ROMK) provide a

conductive pathway for the apical recycling of this cation (Ho et al. , 1993 ; Kohda et al. , 1998 ), and basolateral Cl– channels (called CLCN) provide a basolateral exit mechanism for Cl– The luminal membranes of epithelial cells in the thick ascending limb have conductive pathways (channels) onlyfor K+; therefore the apical membrane voltage is determined by the equilibrium potential for K+

(EK) In contrast, the basolateral membrane has channels for both K+ and Cl–, so that the basolateral

membrane voltage is less than EK; i.e., conductance for Cl– depolarizes the basolateral membrane Depolarization of the basolateral membrane results in a transepithelial potential difference of approximately 10 mV, with the lumen positive with respect to the interstitial space This lumen-positive potential difference repels cations (Na+, Ca2+, and Mg2+) and thereby provides an important driving force for the paracellular flux of these cations into the interstitial space

Figure 29–6 NaCl Reabsorption in Thick Ascending Limb and Mechanism of Diuretic Action of Na+ –K + –2Cl– Symport Inhibitors  S, symporter; CH, ion channel Numbers in parentheses indicate stoichiometry Designated voltages are the potential differences across the indicated membrane or cell

As the name implies, inhibitors of Na+–K+–2Cl– symport bind to the Na+–K+–2Cl– symporter in the thick ascending limb (Koenig et al. , 1983 ) and block its function, bringing salt transport in this segment of the nephron to a virtual standstill (Burg et al. , 1973 ) The molecular mechanism by which this class of drugs blocks the Na+–K+–2Cl– symporter is unknown, but evidence suggests that these drugs attach to the Cl––binding site (Hannafin et al. , 1983 ) located in the symporter's

transmembrane domain (Isenring and Forbush, 1997) Inhibitors of Na+–K+–2Cl– symport also inhibit Ca2+ and Mg2+ reabsorption in the thick ascending limb by abolishing the transepithelial potential difference that is the dominant driving force for reabsorption of these cations

Na+–K+–2Cl– symporters are an important family of transport molecules found in many secretory and absorbing epithelia The rectal gland of the dogfish shark is a particularly rich source of the protein, and a cDNA encoding a Na+–K+–2Cl– symporter was isolated from a cDNA library

obtained from the dogfish shark rectal gland by screening with antibodies to the shark symporter (Xu et al. , 1994 ) Molecular cloning revealed a deduced amino acid sequence of 1191 residues containing 12 putative membrane-spanning domains flanked by long N and C termini in the

cytoplasm Expression of this protein resulted in Na+–K+–2Cl– symport that was sensitive to

bumetanide The shark rectal gland Na+–K+–2Cl– symporter cDNA subsequently was used to screen

a human colonic cDNA library, and this provided Na+–K+–2Cl– symporter cDNA probes from this tissue These latter probes were used to screen rabbit renal cortical and renal medullary libraries, which allowed cloning of the rabbit renal Na+–K+–2Cl– symporter (Payne and Forbush, 1994) This symporter is 1099 amino acids in length, is 61% identical to the dogfish shark secretory Na+–K+–2Cl– symporter, has 12 predicted transmembrane helices, and contains large N- and C-terminal cytoplasmic regions Subsequent studies demonstrated that Na+–K+–2Cl– symporters are of two

varieties (seeKaplan et al. , 1996 ) The "absorptive" symporter (called ENCC2, NKCC2, or BSC1)

is expressed only in the kidney, is localized to the apical membrane of the thick ascending limb, and

is regulated by cyclic AMP (Obermüller et al. , 1996 ; Kaplan et al. , 1996 ; Nielsen et al. , 1998 ; Plata

et al , 1999) At least six different isoforms of the absorptive symporter are generated by alternative mRNA splicing (Mount et al. , 1999 ) The "secretory" symporter (called ENCC3, NKCC1, or BSC2) is a "housekeeping" protein that is widely expressed and, in epithelial cells, is localized to the basolateral membrane A model of Na+–K+–2Cl– symport has been proposed based on ordered binding of ions to the symporter (Lytle et al. , 1998 ) Mutations in the genes coding for the

absorptive Na+–K+–2Cl– symporter, the apical K+ channel, or the basolateral Cl– channel give rise to

Bartter's syndrome (inherited hypokalemic alkalosis with salt wasting and hypotension) (seeSimon

and Lifton, 1998)

Effects on Urinary Excretion

Due to blockade of the Na+–K+–2Cl– symporter, loop diuretics cause a profound increase in the urinary excretion of Na+ and Cl– (i.e., up to 25% of the filtered load of Na+) Abolition of the

transepithelial potential difference also results in marked increases in the excretion of Ca2+ and

Mg2+ Some (e.g., furosemide), but not all (e.g., bumetanide and piretanide), sulfonamide-based

loop diuretics have weak carbonic anhydrase–inhibiting activity Those drugs with carbonic

anhydrase–inhibiting activity increase the urinary excretion of HCO3 and phosphate The

mechanism by which inhibition of carbonic anhydrase increases phosphate excretion is not known All inhibitors of Na+–K+–2Cl– symport increase the urinary excretion of K+ and titratable acid This effect is due in part to increased delivery of Na+ to the distal tubule The mechanism by which increased distal delivery of Na+ enhances excretion of K+ and H+ is discussed in the section on inhibitors of Na+ channels Acutely, loop diuretics increase the excretion of uric acid, whereas chronic administration of these drugs results in reduced excretion of uric acid The chronic effects

of loop diuretics on uric acid excretion may be due to enhanced transport in the proximal tubule secondary to volume depletion, leading to increased uric acid reabsorption, or to competition between the diuretic and uric acid for the organic acid secretory mechanism in the proximal tubule, leading to reduced uric acid secretion

By blocking active NaCl reabsorption in the thick ascending limb, inhibitors of Na+–K+–2Cl–

symport interfere with a critical step in the mechanism that produces a hypertonic medullary

interstitium Therefore, loop diuretics block the kidney's ability to concentrate urine during

hydropenia Also, since the thick ascending limb is part of the diluting segment, inhibitors of Na+–

K+–2Cl– symport markedly impair the kidney's ability to excrete a dilute urine during water diuresis.Effects on Renal Hemodynamics

If volume depletion is prevented by replacing fluid losses, inhibitors of Na+–K+–2Cl– symport generally increase total RBF and redistribute RBF to the midcortex (Stein et al. , 1972 ) However, the effects on RBF are variable The mechanism of the increase in RBF is not known, but

prostaglandins have been implicated (Williamson et al. , 1974 ) In fact, nonsteroidal

antiinflammatory drugs (NSAIDs) attenuate the diuretic response to loop diuretics, most likely by preventing prostaglandin-mediated increases in RBF (Brater, 1985) Loop diuretics block TGF by inhibiting salt transport into the macula densa, so that the macula densa no longer can "sense" NaCl concentrations in the tubular fluid Therefore, unlike carbonic anhydrase inhibitors, loop diuretics

do not decrease GFR by activating TGF Loop diuretics are powerful stimulators of renin release This effect is due to interference with NaCl transport by the macula densa and, if volume depletion occurs, to reflex activation of the sympathetic nervous system and to stimulation of the intrarenal baroreceptor mechanism Prostaglandins, particularly prostacyclin, may play an important role in mediating the renin release response to loop diuretics (Oates et al. , 1979 )

Other Actions

Loop diuretics may cause direct vascular effects (seeDormans et al. , 1996 ) Loop diuretics,

particularly furosemide, acutely increase systemic venous capacitance and thereby decrease left ventricular filling pressure This effect, which may be mediated by prostaglandins and requires intact kidneys (Johnston et al. , 1983 ), benefits patients with pulmonary edema even before diuresis ensues Furosemide and ethacrynic acid can inhibit Na+, K+–ATPase, glycolysis, mitochondrial respiration, the microsomal Ca2+ pump, adenylyl cyclase, phosphodiesterase, and prostaglandin

dehydrogenase; however, these effects do not have therapeutic implications In vitro, high doses of

inhibitors of Na+–K+–2Cl– symport can inhibit electrolyte transport in many tissues Only in the inner ear, where alterations in the electrolyte composition of endolymph may contribute to drug-induced ototoxicity, is this effect clinically important

Absorption and Elimination

The oral bioavailability, plasma half-life, and route of elimination of the four inhibitors of Na+–K+–2Cl– symport available in the United States are listed in Table 29–4 Because furosemide,

bumetanide, ethacrynic acid, and torsemide are extensively bound to plasma proteins, delivery of these drugs to the tubules by filtration is limited However, they are efficiently secreted by the organic acid transport system in the proximal tubule and thereby gain access to their binding sites

on the Na+–K+–2Cl– symport in the luminal membrane of the thick ascending limb Probenecid shifts the plasma concentration–response curve to furosemide to the right by competitively

inhibiting furosemide secretion by the organic acid transport system (Brater, 1983) The most recentloop diuretic to receive FDA approval is torsemide, which has a longer half-life than the other loop diuretics available in the United States (Brater, 1991: seeKnauf and Mutschler, 1997)

Toxicity, Adverse Effects, Contraindications, Drug Interactions

Adverse effects unrelated to the diuretic efficacy are rare, and most adverse effects are due to abnormalities of fluid and electrolyte balance Overzealous use of loop diuretics can cause serious depletion of total body Na+ This may be manifest as hyponatremia and/or extracellular fluid

volume depletion associated with hypotension, reduced GFR, circulatory collapse, thromboembolic

episodes, and, in patients with liver disease, hepatic encephalopathy Increased delivery of Na+ to the distal tubule, particularly when combined with activation of the renin–angiotensin system, leads

to increased urinary excretion of K+ and H+, causing a hypochloremic alkalosis If dietary K+ intake

is not sufficient, hypokalemia may develop, and this may induce cardiac arrhythmias, particularly inpatients taking cardiac glycosides Increased Mg2+ and Ca2+ excretion may result in

hypomagnesemia (a risk factor for cardiac arrhythmias) and hypocalcemia (rarely leading to

tetany)

Loop diuretics can cause ototoxicity that manifests itself as tinnitus, hearing impairment, deafness, vertigo, and a sense of fullness in the ears Hearing impairment and deafness are usually, but not always, reversible Ototoxicity occurs most frequently with rapid intravenous administration and least frequently with oral administration Ethacrynic acid appears to induce ototoxicity more often than do other loop diuretics Loop diuretics also can cause hyperuricemia (rarely leading to gout) and hyperglycemia (rarely precipitating diabetes mellitus) and can increase plasma levels of low-density lipoprotein (LDL) cholesterol and triglycerides, while decreasing plasma levels of high-density lipoprotein (HDL) cholesterol Other adverse effects include skin rashes, photosensitivity, paresthesias, bone marrow depression, and gastrointestinal disturbances

Contraindications to the use of loop diuretics include severe Na+ and volume depletion,

hypersensitivity to sulfonamides (for sulfonamide-based loop diuretics), and anuria unresponsive to

a trial dose of loop diuretic

Drug interactions may occur when loop diuretics are coadministered with: (1) aminoglycosides (synergism of ototoxicity caused by both drugs); (2) anticoagulants (increased anticoagulant

activity); (3) digitalis glycosides (increased digitalis-induced arrhythmias); (4) lithium (increased plasma levels of lithium); (5) propranolol (increased plasma levels of propranolol); (6)

sulfonylureas (hyperglycemia); (7) cisplatin (increased risk of diuretic-induced ototoxicity); (8) NSAIDs (blunted diuretic response; salicylate toxity when given with high doses of salicylates); (9) probenecid (blunted diuretic response); (10) thiazide diuretics (synergism of diuretic activity of bothdrugs leading to profound diuresis); and (11) amphotericin B (increased potential for nephrotoxicityand toxicity and intensification of electrolyte imbalance)

Therapeutic Uses

A major use of loop diuretics is in the treatment of acute pulmonary edema A rapid increase in venous capacitance in conjunction with a brisk natriuresis reduces left ventricular filling pressures and thereby rapidly relieves pulmonary edema Loop diuretics also are widely used for the

treatment of chronic congestive heart failure when diminution of extracellular fluid volume is

desirable to minimize venous and pulmonary congestion (seeChapter 34: Pharmacological

Treatment of Heart Failure) Diuretics are widely used for the treatment of hypertension

(seeChapter 33: Antihypertensive Agents and the Drug Therapy of Hypertension), and controlled

clinical trials demonstrating reduced morbidity and mortality have been conducted with Na+–Cl–

symport (thiazides and thiazide-like diuretics), but not Na+–K+–2Cl– symport, inhibitors

Nonetheless, Na+–K+–2Cl– symport inhibitors appear to lower blood pressure as effectively as Na+–

Cl– symport inhibitors while causing smaller perturbations in the lipid profile (van der Heijden et

al.

, 1998) The edema of nephrotic syndrome often is refractory to other classes of diuretics, and loop diuretics often are the only drugs capable of reducing the massive edema associated with this renal disease Loop diuretics also are employed in the treatment of edema and ascites of liver cirrhosis; however, care must be taken not to induce encephalopathy or hepatorenal syndrome In patients with a drug overdose, loop diuretics can be used to induce a forced diuresis to facilitate

more rapid renal elimination of the offending drug Loop diuretics—combined with isotonic saline administration to prevent volume depletion—are used to treat hypercalcemia Loop diuretics

interfere with the kidney's ability to produce a concentrated urine Consequently, loop diuretics combined with hypertonic saline are useful for the treatment of life-threatening hyponatremia Loopdiuretics also are used to treat edema associated with chronic renal insufficiency However, animal

studies have demonstrated that loop diuretics increase PGC by activating the renin–angiotensin system, an effect that could accelerate renal injury (Lane et al. , 1998 ) Most patients with ARF receive a trial dose of a loop diuretic in an attempt to convert oliguric ARF to nonoliguric ARF However, there is no evidence that loop diuretics prevent ATN or improve outcome in patients with

ARF (seeKellum, 1998).

Inhibitors of Na+– Cl– Symport (Thiazide and Thiazide-Like Diuretics)

The benzothiadiazides were synthesized in an effort to enhance the potency of inhibitors of carbonicanhydrase However, unlike carbonic anhydrase inhibitors, which primarily increase NaHCO3

excretion, benzothiadiazides were found predominantly to increase NaCl excretion (Beyer, 1958),

an effect shown to be independent of carbonic anhydrase inhibition Chlorothiazide was the first serious challenge to the mercurial diuretics, a now-obsolete class of organometallic compounds that dominated diuretic therapy for more than thirty years

Chemistry

Inhibitors of Na+–Cl– symport are sulfonamides (seeTable 29–5) and many are analogs of

1,2,4-benzothiadiazine-1,1-dioxide Because the original inhibitors of Na+–Cl– symport were

benzothiadiazine derivatives, this class of diuretics became known as thiazide diuretics

Subsequently, drugs that are pharmacologically similar to thiazide diuretics but are not thiazides

were developed and are called thiazide-like diuretics The term thiazide diuretics often is used to

refer to all members of the class of inhibitors of Na+–Cl– symport, and this usage is employed in the present chapter

Mechanism and Site of Action

Some studies using split-droplet and stationary-microperfusion techniques have described

reductions in proximal tubule reabsorption by thiazide diuretics; however, free-flow micropuncture studies have not consistently demonstrated increased solute delivery out of the proximal tubule following administration of thiazides In contrast, micropuncture (Kunau et al. , 1975 ) and in situ microperfusion studies (Costanzo and Windhager, 1978) clearly indicate that thiazide diuretics inhibit NaCl transport in the DCT Furthermore, the renal cortex has a high-affinity receptor for thiazide diuretics (Beaumont et al. , 1988 ), and binding of thiazides localizes to the DCT (Beaumont

et al , 1989) It is now well accepted that the primary site of action of thiazide diuretics is the DCT, whereas the proximal tubule may represent a secondary site of action

Figure 29–7 illustrates the current model of electrolyte transport in the DCT As with other nephron segments, transport is powered by a Na+ pump in the basolateral membrane The free energy in the electrochemical gradient for Na+ is harnessed by a Na+–Cl– symporter in the luminal membrane, which moves Cl– into the epithelial cell against its electrochemical gradient Cl– then passively exits

the basolateral membrane via a Cl– channel Thiazide diuretics inhibit the Na+–Cl– symporter, perhaps by competing for the Cl– binding site (Beaumont et al. , 1988 )

Figure 29–7 NaCl Reabsorption in Distal Convoluted Tubule and Mechanism of Diuretic Action of Na+ –Cl – Symport Inhibitors  S, symporter; CH, ion channel Numbers in parentheses indicate stoichiometry

Using a functional expression strategy (Cl–-dependent Na+ uptake in Xenopus oocytes), Gamba et

al (1993) isolated a cDNA clone from the urinary bladder of the winter flounder that codes for a

Na+–Cl– symporter This Na+–Cl– symporter is inhibited by a number of thiazide diuretics (but not

by furosemide, acetazolamide, or an amiloride derivative), has 12 putative membrane-spanning domains, and its sequence is 47% identical to the cloned dogfish shark rectal gland Na+–K+–2Cl–

symporter Subsequently, Gamba et al (1994) cloned the rat and Mastroianni et al (1996) cloned

the human Na+–Cl– symporter The Na+–Cl– symporter (called ENCC1 or TSC) is expressed

predominantly in the kidney (Chang et al. , 1996 ) and is localized to the apical membrane of DCT epithelial cells (Bachmann et al. , 1995 ; Obermüller et al. , 1995 ; Plotkin et al. , 1996 ) Expression of the Na+–Cl– symporter is regulated by aldosterone (Velázquez et al. , 1996 ; Kim et al. , 1998 ;

Bostonjoglo et al. , 1998 ) Mutations in the Na+–Cl– symporter cause a form of inherited

hypokalemic alkalosis called Gitelman's syndrome (seeSimon and Lifton, 1998).

Effects on Urinary Excretion

As would be expected from their mechanism of action, inhibitors of Na+–Cl– symport increase Na+

and Cl– excretion However, thiazides are only moderately efficacious (i.e., maximum excretion of

filtered load of Na+ is only 5%), since approximately 90% of the filtered Na+ load is reabsorbed before reaching the DCT Some thiazide diuretics also are weak inhibitors of carbonic anhydrase, aneffect that increases HCO3 and phosphate excretion and probably accounts for the weak proximal tubular effects of some thiazide diuretics Like inhibitors of Na+–K+–2Cl– symport, inhibitors of

Na+–Cl– symport increase the excretion of K+ and titratable acid due to increased delivery of Na+ to the distal tubule Acute administration of thiazides increases the excretion of uric acid However, uric acid excretion is reduced following chronic administration by the same mechanisms discussed for loop diuretics The acute effects of inhibitors of Na+–Cl– symport on Ca2+ excretion are variable; when administered chronically, thiazide diuretics decrease Ca2+ excretion The mechanism is unknown but may involve increased proximal reabsorption due to volume depletion as well as direct effects of thiazides to increase Ca2+ reabsorption in the DCT Thiazide diuretics may cause a mild magnesuria by a poorly understood mechanism, and there is increasing awareness that long-

term use of thiazide diuretics may cause magnesium deficiency, particularly in the elderly (Martin and Milligan, 1987) Since inhibitors of Na+–Cl– symport inhibit transport in the cortical diluting segment, thiazide diuretics attenuate the ability of the kidney to excrete a dilute urine during water diuresis However, since the DCT is not involved in the mechanism that generates a hypertonic medullary interstitium, thiazide diuretics do not alter the kidney's ability to concentrate urine duringhydropenia.

Effects on Renal Hemodynamics

In general, inhibitors of Na+–Cl– symport do not affect RBF and only variably reduce GFR due to increases in intratubular pressure Since thiazides act at a point past the macula densa, they have little or no influence on TGF

Other Actions

Thiazide diuretics may inhibit phosphodiesterase, mitochondrial oxygen consumption, and renal uptake of fatty acids; however, these effects are not of clinical significance

Absorption and Elimination

The relative potency, oral bioavailability, plasma half-life, and route of elimination of inhibitors of

Na+–Cl– symport currently used in the United States are listed in Table 29–5 Of special note is the wide range of half-lives for this class of drugs Sulfonamides are organic acids and therefore are secreted into the proximal tubule by the organic acid secretory pathway Since thiazides must gain access to the tubular lumen to inhibit the Na+–Cl– symporter, drugs such as probenecid can attenuatethe diuretic response to thiazides by competing for transport into the proximal tubule However, plasma protein binding varies considerably among thiazide diuretics, and this parameter determines the contribution that filtration makes to tubular delivery of a specific thiazide

Toxicity, Adverse Effects, Contraindications, Drug Interactions

Thiazide diuretics rarely cause CNS (vertigo, headache, paresthesias, xanthopsia, weakness), gastrointestinal (anorexia, nausea, vomiting, cramping, diarrhea, constipation, cholecystitis,

pancreatitis), hematological (blood dyscrasias), and dermatological (photosensitivity, skin rashes)

disorders The incidence of sexual dysfunction (i.e., erection problems) is greater with Na+–Cl–

symport inhibitors than with several other antihypertensive agents ( -adrenergic receptor

antagonists, calcium channel blockers, angiotensin converting enzyme inhibitors, 1-receptor antagonists) (Grimm et al. , 1997 ), but usually is tolerable However, like loop diuretics, most serious adverse effects of thiazides are related to abnormalities of fluid and electrolyte balance These adverse effects include extracellular volume depletion, hypotension, hypokalemia,

hyponatremia, hypochloremia, metabolic alkalosis, hypomagnesemia, hypercalcemia, and

hyperuricemia Thiazide diuretics have caused fatal or near fatal hyponatremia, and some patients are at recurrent risk of hyponatremia when rechallenged with thiazides

Thiazide diuretics also decrease glucose tolerance, and latent diabetes mellitus may be unmasked during therapy The mechanism of the reduced glucose tolerance is not completely understood but appears to involve reduced insulin secretion and alterations in glucose metabolism Hyperglycemia may be related in some way to K+ depletion, in that hyperglycemia is reduced when K+ is given along with the diuretic (Tannen, 1985) Thiazide diuretics also may increase plasma levels of LDL cholesterol, total cholesterol, and total triglycerides Thiazide diuretics are contraindicated in

individuals who are hypersensitive to sulfonamides.

With regard to drug interactions, thiazide diuretics may diminish the effects of anticoagulants, uricosuric agents used to treat gout, sulfonylureas, and insulin and may increase the effects of anesthetics, diazoxide, digitalis glycosides, lithium, loop diuretics, and vitamin D The effectiveness

of thiazide diuretics may be reduced by NSAIDs, bile acid sequestrants (reduced absorption of thiazides), and methenamines (alkalinization of urine may decrease effectiveness of thiazides) Amphotericin B and corticosteroids increase the risk of hypokalemia induced by thiazide diuretics

A potentially lethal drug interaction warranting special emphasis is that involving thiazide diuretics with quinidine (Roden, 1993) Prolongation of the QT-interval by quinidine can lead to the

development of polymorphic ventricular tachycardia (torsades de pointes) due to triggered activity originating from early afterdepolarizations (seeChapter 35: Antiarrhythmic Drugs) Although usually self-limiting, torsades de pointes may deteriorate into fatal ventricular fibrillation

Hypokalemia increases the risk of quinidine-induced torsades de pointes, and thiazide diuretics

cause hypokalemia It is likely, therefore, that thiazide diuretic–induced K+ depletion accounts for

many cases of quinidine-induced torsades de pointes.

drugs (seeChapter 33: Antihypertensive Agents and the Drug Therapy of Hypertension) In this

regard, thiazide diuretics are inexpensive, as efficacious as other classes of antihypertensive agents, and well tolerated Thiazides can be administered once daily, do not require dose titration, and have few contraindications Moreover, thiazides have additive or synergistic effects when combined withother classes of antihypertensive agents Although thiazides may marginally increase the risk of

sudden death (seeHoes and Grobbee, 1996) and renal cell carcinoma (Grossman et al. , 1999 ), in general these agents are safe and reduce cardiovascular morbidity and mortality in hypertensive patients Because the adverse effects of thiazides increase progressively in severity at doses higher than maximally effective antihypertensive doses, only low doses should be prescribed for

hypertension (see Ramsey, 1999) Thiazide diuretics, which reduce urinary excretion of Ca2+, sometimes are employed to treat calcium nephrolithiasis and may be useful for the treatment of

osteoporosis (seeChapter 62: Agents Affecting Calcification and Bone Turnover: Calcium,

Phosphate, Parathyroid Hormone, Vitamin D, Calcitonin, and Other Compounds) Thiazide

diuretics are also the mainstay for treatment of nephrogenic diabetes insipidus, reducing urine volume by up to 50% The mechanism of this paradoxical effect remains unknown (Grønbeck et al. , 1998) Since other halides are excreted by renal processes similar to those for Cl–, thiazide diuretics may be useful for the management of Br– intoxication

Inhibitors of Renal Epithelial Na+ Channels (K+-Sparing Diuretics)

Triamterene (DYRENIUM, MAXZIDE ) and amiloride (MIDAMOR) are the only two drugs of this class

in clinical use Both drugs cause small increases in NaCl excretion and usually are employed for

their antikaluretic actions to offset the effects of other diuretics that increase K+ excretion

Consequently, triamterene and amiloride, along with spironolactone (see next section), often are

classified as potassium (K+)-sparing diuretics.

Chemistry

Amiloride is a pyrazinoylguanidine derivative, and triamterene is a pteridine (Table 29–6) Both drugs are organic bases and are transported by the organic base secretory mechanism in the

proximal tubule

Mechanism and Site of Action

Available data suggest that triamterene and amiloride have similar mechanisms of action Of the two, amiloride has been studied much more extensively, so its mechanism of action is known with ahigher degree of certainty As illustrated in Figure 29–8, principal cells in the late distal tubule and collecting duct have in their luminal membranes a Na+ channel that provides a conductive pathway for the entry of Na+ into the cell down the electrochemical gradient created by the basolateral Na+

pump The higher permeability of the luminal membrane for Na+ depolarizes the luminal

membrane, but not the basolateral membrane, creating a lumen-negative transepithelial potential difference This transepithelial voltage provides an important driving force for the secretion of K+

into the lumen via K+ channels (ROMK) in the luminal membrane Carbonic anhydrase inhibitors, loop diuretics, and thiazide diuretics increase the delivery of Na+ to the late distal tubule and

collecting duct, a situation that is often associated with increased K+ and H+ excretion It is likely that the elevation in luminal Na+ concentration in the distal nephron induced by such diuretics

augments depolarization of the luminal membrane and thereby enhances the lumen-negative VT, which facilitates K+ excretion In addition to principal cells, the collecting duct also contains type A intercalated cells that mediate the secretion of H+ into the tubular lumen Tubular acidification is driven by a luminal H+–ATPase (proton pump), and this pump is aided by the lumen-negative transepithelial voltage However, increased distal delivery of Na+ is not the only mechanism by which diuretics increase K+ and H+ excretion Activation of the renin–angiotensin–aldosterone axis

by diuretics also contributes to diuretic-induced K+ and H+ excretion by a mechanism explained in the section on mineralocorticoid antagonists

Figure 29–8 Na+ Reabsorption in Late Distal Tubule and Collecting Duct and Mechanism of Diuretic Action of Epithelial Na+ -Channel Inhibitors  Cl–

reabsorption (not shown) occurs both paracellularly and transcellularly, and

precise mechanism of Cl– transport appears to be species specific A, antiporter;

CH, ion channel; CA, carbonic anhydrase Numbers in parentheses indicate stoichiometry Designated voltages are the potential differences across the indicated membrane or cell

Considerable evidence indicates that amiloride blocks Na+ channels in the luminal membrane of principal cells in the late distal tubule and collecting duct This evidence includes data from

epithelia of nonrenal origin (amphibian skin and toad bladder) (Garty and Palmer, 1997) as well as

a number of electrophysiological studies in isolated mammalian collecting ducts (O'Neil and Boulpaep, 1979) Amiloride produces half-maximal inhibition at concentrations <1 mM and, depending on the study, amiloride may interact with Na+ in the channel either competitively or noncompetitively It is important, however, to bear in mind that renal epithelial Na+ channels inhibited by this class of diuretics are not the same as voltage-gated Na+ channels found in many cell types (for instance, neurons and myocytes)

Molecular cloning studies have revealed that the amiloride-sensitive Na+ channel (called ENaC) consists of three subunits ( , , ) (Canessa et al. , 1994 ) Although the subunit is sufficient for channel activity, maximal Na+ permeability is induced when all three subunits are coexpressed in the same cell, suggesting a minimal oligomeric structure in which one copy of each subunit is

associated in a heterotrimeric protein Studies in Xenopus oocytes expressing ENaC suggest that

triamterene and amiloride bind to ENaC by similar mechanisms (Busch et al. , 1996 ) Liddle's syndrome (pseudohyperaldosteronism) is an autosomal dominant form of low-renin, volume-expanded hypertension that is due to mutations in the or subunits, leading to increased basal activity of ENaC (Ismailov et al. , 1999 )

Effects on Urinary Excretion

Since the late distal tubule and collecting duct have a limited capacity to reabsorb solutes, blockade

of Na+ channels in this part of the nephron results in only a mild increase in the excretion rates of

Na+ and Cl– (approximately 2% of filtered load) Blockade of Na+ channels hyperpolarizes the luminal membrane, reducing the lumen-negative transepithelial voltage Since the lumen-negative potential difference normally opposes cation reabsorption and facilitates cation secretion,

attenuation of the lumen-negative voltage decreases the excretion rates of K+, H+, Ca2+, and Mg2+ Volume contraction may increase reabsorption of uric acid in the proximal tubule; hence, chronic administration of amiloride and triamterene may decrease uric acid excretion

Effects on Renal Hemodynamics

Amiloride and triamterene have little or no effect on renal hemodynamics and do not alter TGF.Other Actions

Amiloride, at concentrations higher than needed to elicit therapeutic effects, also blocks the Na+–H+

and Na+–Ca2+ antiporters and inhibits the Na+ pump

Absorption and Elimination

The relative potency, oral bioavailability, plasma half-life, and route of elimination for amiloride and triamterene are listed in Table 29–6 Amiloride is eliminated predominantly by urinary

excretion of intact drug Triamterene is extensively metabolized to an active metabolite,

4-hydroxytriamterene sulfate, and this metabolite is excreted in the urine The pharmacological activity of 4-hydroxytriamterene sulfate is comparable to that of the parent drug Therefore, the toxicity of triamterene may be enhanced in both hepatic disease (decreased metabolism of

triamterene) and renal failure (decreased urinary excretion of active metabolite)

Toxicity, Adverse Effects, Contraindications, Drug Interactions

The most dangerous adverse effect of Na+-channel inhibitors is hyperkalemia, which can be threatening Consequently, amiloride and triamterene are contraindicated in patients with

life-hyperkalemia as well as in patients at increased risk of developing life-hyperkalemia (e.g., patients with

renal failure, patients receiving other K+-sparing diuretics, patients taking angiotensin converting enzyme inhibitors, or patients taking K+ supplements) Even NSAIDs can increase the likelihood of hyperkalemia in patients receiving Na+-channel inhibitors Cirrhotic patients are prone to

megaloblastosis because of folic acid deficiency, and triamterene, a weak folic acid antagonist, mayincrease the likelihood of this adverse event Triamterene also can reduce glucose tolerance and induce photosensitization and has been associated with interstitial nephritis and renal stones Both drugs can cause CNS, gastrointestinal, musculoskeletal, dermatological, and hematological adverse effects The most common adverse effects of amiloride are nausea, vomiting, diarrhea, and

headache; those of triamterene are nausea, vomiting, leg cramps, and dizziness

Therapeutic Uses

Because of the mild natriuresis induced by Na+-channel inhibitors, these drugs seldom are used as

sole agents in the treatment of edema or hypertension Rather, their major utility is in combination

with other diuretics Coadministration of a Na+-channel inhibitor augments the diuretic and

antihypertensive response to thiazide or loop diuretics More importantly, the ability of Na+-channelinhibitors to reduce K+ excretion tends to offset the kaliuretic effects of thiazide and loop diuretics; consequently, the combination of a Na+-channel inhibitor with a thiazide or loop diuretic tends to result in normal values of plasma K+ (Hollenberg and Mickiewicz, 1989) Liddle's syndrome can be

treated effectively with Na+-channel inhibitors Aerosolized amiloride has been shown to improve mucociliary clearance in patients with cystic fibrosis (Zahaykevich, 1991) By inhibiting Na+

absorption from the surface of airway epithelial cells, amiloride augments hydration of respiratory secretions and thereby improves mucociliary clearance Amiloride also is useful for lithium-inducednephrogenic diabetes insipidus because it blocks Li+ transport into the cells of the collecting

tubules

Antagonists of Mineralocorticoid Receptors (Aldosterone Antagonists; K+-Sparing Diuretics)Mineralocorticoids cause retention of salt and water and increase the excretion of K+ and H+ by

binding to specific mineralocorticoid receptors Kagawa et al (1957) observed that some

spirolactones block the effects of mineralocorticoids; this finding led to the synthesis of specific antagonists for the mineralocorticoid receptor (MR) Spironolactone (ALDACTONE), a 17-

spirolactone, is the only member of this class available in the United States (Table 29–7)

Mechanism of Action

Epithelial cells in the late distal tubule and collecting duct contain cytoplasmic MRs that have a high affinity for aldosterone This receptor is a member of the superfamily of receptors for steroid

hormones, thyroid hormones, vitamin D, and retinoids (seeChapter 2: Pharmacodynamics:

Mechanisms of Drug Action and the Relationship Between Drug Concentration and Effect)

Aldosterone enters the epithelial cell from the basolateral membrane and binds to MRs; the MR–aldosterone complex translocates to the nucleus, where it binds to specific sequences of DNA (hormone-responsive elements) and thereby regulates the expression of multiple gene products called aldosterone-induced proteins (AIPs) Figure 29–9 illustrates some of the proposed effects of AIPs, including: activation of "silent" Na+ channels and "silent" Na+ pumps that preexist in the cell membrane; alterations in the cycling of Na+ channels and Na+ pumps between the cytosol and cell membrane so that more channels and pumps are located in the membrane; increased expression of

Na+ channels and Na+ pumps; changes in permeability of the tight junctions; and increased activity

of enzymes in the mitochondria that are involved in ATP production The precise mechanisms by which AIPs alter transport are incompletely understood However, the net effect of AIPs is to increase Na+ conductance of the luminal membrane and sodium pump activity of the basolateral membrane Consequently, transepithelial NaCl transport is enhanced and the lumen-negative transepithelial voltage is increased The latter effect increases the driving force for secretion of K+

and H+ into the tubular lumen

Figure 29–9 Effects of Aldosterone on Late Distal Tubule and Collecting Duct and Diuretic Mechanism of Aldosterone Antagonists. AIP, aldosterone-induced proteins; ALDO, aldosterone; MR, mineralocorticoid receptor; CH, ion channel;

1, activation of membrane-bound Na+ channels; 2, redistribution of Na+ channels

from cytosol to membrane; 3, de novo synthesis of Na+ channels; 4, activation of membrane-bound Na+,K+–ATPase; 5, redistribution of Na+,K+–ATPase from

cytosol to membrane; 6, de novo synthesis of Na+,K+–ATPase; 7, changes in permeability of tight junctions; 8, increased mitochondrial production of ATP

Drugs such as spironolactone competitively inhibit the binding of aldosterone to the MR (Marver et

al.

, 1974) Unlike the MR–aldosterone complex, the MR–spironolactone complex is not able to induce the synthesis of AIPs Since spironolactone and other drugs in this class block the biological

effects of aldosterone, these agents also are referred to as aldosterone antagonists.

Effects on Urinary Excretion

The effects of spironolactone on urinary excretion are very similar to those induced by renal

epithelial Na+-channel inhibitors However, unlike that of the Na+-channel inhibitors, the clinical efficacy of spironolactone is a function of endogenous levels of aldosterone The higher the levels

of endogenous aldosterone, the greater the effects of spironolactone on urinary excretion

Effects on Renal Hemodynamics

Spironolactone has little or no effect on renal hemodynamics and does not alter TGF

Other Actions

High concentrations of spironolactone have been reported to interfere with steroid biosynthesis by inhibiting 11 - and 18-, 21-, and 17 -hydroxylase These effects have limited clinical relevance

(seeChapter 60: Adrenocorticotropic Hormone; Adrenocortical Steroids and Their Synthetic

Analogs; Inhibitors of the Synthesis and Actions of Adrenocortical Hormones)

Absorption and Elimination

Spironolactone is partially absorbed (approximately 65%), is extensively metabolized (even during its first passage through the liver), undergoes enterohepatic recirculation, is highly protein-bound, and has a short half-life (approximately 1.6 hours) However, an active metabolite of

spironolactone, canrenone, has a half-life of approximately 16.5 hours, which prolongs the

biological effects of spironolactone Although not available in the United States, canrenone and the

K+ salt of canrenoate also are in clinical use Canrenoate is not active per se but is converted to

canrenone in the body MR antagonists are the only diuretics that do not require access to the tubular lumen to induce a diuresis

Toxicity, Adverse Effects, Contraindications, Drug Interactions

As with other K+-sparing diuretics, spironolactone may cause life-threatening hyperkalemia

Therefore, spironolactone is contraindicated in patients with hyperkalemia and in patients at

increased risk of developing hyperkalemia, either because of disease or because of administration ofother medications Spironolactone also can induce metabolic acidosis in cirrhotic patients

Salicylates may reduce the tubular secretion of canrenone and decrease the diuretic efficacy of spironolactone, and spironolactone may alter the clearance of digitalis glycosides Due to its steroid structure, spironolactone may cause gynecomastia, impotence, decreased libido, hirsutism,

deepening of the voice, and menstrual irregularities Spironolactone also may induce diarrhea, gastritis, gastric bleeding, and peptic ulcers (the drug is contraindicated in patients with peptic ulcers) CNS adverse effects include drowsiness, lethargy, ataxia, confusion, and headache

Spironolactone may cause skin rashes and, rarely, blood dyscrasias Breast cancer has occurred in patients taking spironolactone chronically (cause and effect not established), and high doses of spironolactone have been associated with malignant tumors in rats Whether or not therapeutic doses of spironolactone can induce malignancies remains an open question

diuretic of choice in patients with hepatic cirrhosis Pitt et al (1999) have reported that

spironolactone, when added to standard therapy, substantially reduces morbidity and mortality in

patients with New York Heart Association (NYHA) class III and class IV heart failure (seeChapter

34: Pharmacological Treatment of Heart Failure)

Mechanisms of Edema Formation and the Role of Diuretics in Clinical Medicine

Mechanism of Edema Formation

A complex set of interrelationships (Figure 29–10) exists among the cardiovascular system, the kidneys, the CNS (Na+ appetite, thirst regulation), and the tissue capillary beds [distribution of extracellular fluid volume (ECFV)], so that perturbations at one of these sites can affect all of the remaining sites A primary law of the kidney is that Na+ excretion is a steep function of mean arterial blood pressure (MABP) such that small increases in MABP cause marked increases in Na+

excretion (Guyton, 1991) Over any given time interval, the net change in total body Na+ (either positive or negative) is simply the dietary Na+ intake minus the urinary excretion rate minus other

losses (e.g., sweating, fecal losses, vomiting) When a net positive Na+ balance occurs, the

concentration of Na+ in the ECF will increase, stimulating water intake (thirst) and reducing urinary

water output (via ADH release) Opposite changes occur during a net negative Na+ balance

Changes in water intake and output adjust ECFV concentration toward normal, thereby expanding

or contracting total ECFV Total ECFV is distributed among many body compartments; however, since the volume of extracellular fluid on the arterial side of the circulation pressurizes the arterial tree, it is this fraction of ECFV that determines MABP, and it is this fraction of ECFV that is

"sensed" by the cardiovascular system and kidneys Since MABP is a major determinant of Na+

output, a closed loop is established (Figure 29–10) This loop cycles until net Na+ accumulation is

zero; i.e., in the long run, Na+ intake must equal Na+ loss

Figure 29–10 Interrelationships among Renal Function, Na+ Intake, Water Homeostasis, Distribution of Extracellular Fluid Volume (ECFV), and Mean Arterial Blood Pressure (MABP). Pathophysiological mechanisms of edema formation: 1, rightward shift of renal–pressure natriuresis curve; 2, excessive dietary Na+ intake; 3, increased distribution of ECFV to peritoneal cavity (e.g.,

liver cirrhosis with increased hepatic sinusoidal hydrostatic pressure) leading to

ascites formation; 4, increased distribution of ECFV to lungs (e.g., left heart

failure with increased pulmonary capillary hydrostatic pressure) leading to

pulmonary edema; 5, increased distribution of ECFV to venous circulation (e.g.,

right heart failure) leading to venous congestion; 6, peripheral edema caused by altered Starling forces causing increased distribution of ECFV to interstitial space

(e.g., diminished plasma proteins in nephrotic syndrome, severe burns, liver

disease)

The above discussion implies that three fundamental types of perturbations contribute to venous congestion and/or edema formation: (1) A shift to the right in the renal pressure–natriuresis

relationship (e.g., chronic renal failure) causes reduced Na+ excretion for any level of MABP If all other factors remain constant, this would increase total body Na+, ECFV, and MABP The

additional ECFV would be distributed throughout various body compartments, according to the state of cardiac function and prevailing Starling forces, and would predispose toward venous

congestion and/or edema Even so, in the absence of any other predisposing factors for venous congestion and/or edema, a rightward shift in the renal pressure–natriuresis curve generally causes hypertension with only a slight (usually immeasurable) increase in ECFV As elucidated by Guyton and coworkers (Guyton, 1991), ECFV expansion triggers the following series of events: expanded

ECFV augmented cardiac output enhanced vascular tone (i.e., total body autoregulation)

increased total peripheral resistance elevated MABP pressure natriuresis reduction of ECFV and cardiac output toward normal Most likely, a sustained rightward shift in the renal pressure–natriuresis curve is a necessary and sufficient condition for long-term hypertension but is only a predisposing factor for venous congestion and/or edema (2) An increase in dietary Na+ intake

would have the same effects as a rightward shift in the renal pressure–natriuresis relationship (i.e.,

increased MABP and predisposition to venous congestion/edema) However, changes in salt intake may have minimal or large effects depending on the shape of the patient's renal pressure–natriuresiscurve (3) Any pathophysiological alterations in the forces that govern the distribution of ECFV among the various body compartments would cause abnormal amounts of ECFV to be trapped at the site of altered forces This would deplete the "sensed" ECFV, which would be restored back to normal by the mechanisms described in the preceding paragraph ECFV may be trapped at several sites by different mechanisms For instance, cirrhosis of the liver increases lymph in the space of

Disse, leading to spillover via the glissonian wall into the peritoneal cavity (ascites) Left heart

failure, both acute and chronic, increases hydrostatic pressure in the lung capillaries, leading to pulmonary edema Chronic right heart failure redistributes ECFV from the arterial to the venous circulation, resulting in venous, hepatic, and splenic congestion and peripheral tissue edema

Decreased levels of plasma protein, particularly albumin (e.g., in nephrotic syndrome, severe burns,hepatic disease), increase the distribution of ECFV into the interstitial spaces, causing generalized peripheral edema Peripheral edema also may be "idiopathic" due to unknown alterations in the Starling forces at the capillary bed

The Role of Diuretics in Clinical Medicine

Another implication of the mechanisms illustrated in Figure 29–10 is that three fundamental

strategies exist for mobilizing edema fluid—correct the underlying disease, restrict Na+ intake, or administer diuretics The most desirable course of action would be to correct the primary disease; however, this is often impossible For instance, the increased hepatic sinusoidal pressure in cirrhosis

of the liver and the urinary loss of protein in nephrotic syndrome are due to structural alterations in the portal circulation and glomeruli, respectively, which may not be remediable Restriction of Na+

intake is the favored nonpharmacological approach for the treatment of edema and hypertension andshould usually be attempted; however, compliance is a major obstacle

Diuretics, therefore, remain the cornerstone for the treatment of edema or volume overload,

particularly that due to congestive heart failure, ascites, chronic renal failure, and nephrotic

syndrome With regard to heart failure, diuretics reduce pulmonary edema and venous congestion, and it may be possible to manage mild cardiac failure with diuretics alone However, most patients ultimately will require additional therapy with digitalis and/or angiotensin converting enzyme inhibitors (Chapter 34: Pharmacological Treatment of Heart Failure) Periodic administration of diuretics to cirrhotic patients with ascites may eliminate the necessity for or reduce the interval between paracenteses, adding to patient comfort and sparing protein reserves that are lost by

paracenteses Although diuretics can reduce the edema associated with chronic renal failure,

increased doses of the more powerful loop diuretics may be required In the nephrotic syndrome, the response to diuretics often is disappointing

Whether a patient should receive diuretics and, if so, what therapeutic regimen should be used (i.e.,

type of diuretic, route of administration, and speed of mobilization of edema fluid) depends on the clinical situation Massive pulmonary edema in patients with acute left heart failure is a medical emergency requiring rapid, aggressive therapy including intravenous administration of a loop diuretic In this setting, use of oral diuretics or diuretics with lesser efficacy is inappropriate On the other hand, mild pulmonary and venous congestion associated with chronic heart failure is best treated with an oral loop diuretic, the dosage of which should be titrated carefully to maximize the benefit-to-risk ratio In many situations, edema will not pose an immediate health risk Even so, uncomfortable, oppressive, and/or disfiguring edema can greatly reduce quality of life, and the decision to treat will be based in part on quality-of-life issues In such cases, only partial removal ofedema fluid should be attempted, and the fluid should be mobilized slowly using a diuretic regimen that accomplishes the task with minimal perturbation of normal physiology Brater (1998) has provided an algorithm for diuretic therapy (specific recommendations for drug, dose, route, and drug combinations) in patients with edema caused by renal, hepatic, or cardiac disorders

In many clinical situations, edema is not caused by an abnormal intake of Na+ or by an altered renal handling of Na+ Rather, edema is the result of altered Starling forces at the capillary beds, i.e., a

"Starling trap." Use of diuretics in these clinical settings represents a judicious compromise betweenthe edematous state and the hypovolemic state In such conditions, reducing total ECFV with

diuretics will decrease edema but also will cause depletion of "sensed" ECFV, possibly leading to hypotension, malaise, and asthenia.

Diuretic resistance refers to edema that is or has become refractory to a given diuretic If diuretic

resistance develops against a less efficacious diuretic, a more efficacious diuretic should be

substituted, e.g., a loop diuretic for a thiazide However, resistance to loop diuretics is not

uncommon and can be due to several causes (Brater, 1985) NSAIDs block prostaglandin-mediated increases in RBF, resulting in resistance to loop diuretics In chronic renal failure, a reduction in RBF decreases the delivery 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 protein binds diuretics and thereby limits the response 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 (Knauf and Mutschler, 1997)

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

Use of combination therapy to sequentially block more than one site in the nephron may result

in a synergistic interaction between two diuretics For instance, a combination of a loop diuretic with a K+-sparing or a thiazide diuretic may improve therapeutic response; however, nothing is gained by the administration of two drugs of the same type Thiazide diuretics with significant

proximal tubular effects, e.g., metolazone, are particularly well suited for sequential blockade

when coadministered with a loop diuretic

  5. 

Scheduling of diuretic administration shortly before food intake will provide effective

concentrations of diuretic in the tubular lumen when the salt load is highest

Prospectus

All currently available diuretics perturb K+ homeostasis However, studies in animals have

established that blockade of adenosine A1 receptors induces a brisk natriuresis without significantly increasing urinary K+ excretion (Kuan et al. , 1993 ) Two clinical studies with FK453, a highly selective A1-receptor antagonist, confirm that blockade of A1 receptors induces natriuresis in humanbeings with minimal effects on K+ excretion (Balakrishnan et al. , 1993 ; van Buren et al. , 1993 ) Thenatriuretic mechanism of this novel class of diuretics has been partially elucidated (Takeda et al. , 1993) Endogenous adenosine acts on A1 receptors in the proximal tubule to inhibit adenylyl

cyclase As cyclic AMP inhibits basolateral Na+–HCO3 symport activity, a reduced level of cyclic AMP increases Na+–HCO3 symport activity in the basolateral membrane Blockade of A1 receptors prevents the inhibition of adenylyl cyclase by endogenous adenosine, increases epithelial cyclic AMP levels, and consequently decreases Na+–HCO3 symport activity in the basolateral membrane

of the proximal tubule Because A1 receptors are involved in TGF, A1-receptor antagonists uncoupleincreased distal delivery of Na+ from activation of TGF (Wilcox et al. , 1999 ) Other mechanisms, including an effect in the collecting tubules, contribute to the natriuretic response to A1-receptor antagonists; however, it is not known why this class of diuretics has little effect on K+ excretion An

A1-receptor antagonist is under clinical development for the treatment of edema due to heart failure.Recently, the water channels of the proximal tubule (aquaporin 1) and of the collecting duct

(aquaporins 2, 3, and 4) were cloned and their functional characteristics examined (Agre et al. , 1993; Fushimi et al. , 1993 ; seeYamamoto and Sasaki, 1998) The cloning of these proteins

represents an important step in our understanding of water homeostasis Currently, there are no specific inhibitors of aquaporins; however, these proteins are important targets for the development

of novel diuretics Inhibition of water channels in the proximal tubule would greatly diminish the flux of water across proximal tubular epithelial cells, which would reduce luminal Na+

concentration to the point where Na+ reabsorption would cease Therefore, inhibitors of proximal tubular aquaporin 1 may be useful natriuretic diuretics On the other hand, inhibitors of collecting duct aquaporins 2, 3, and 4 would prevent water reabsorption in the collecting duct and therefore

would be highly efficacious "aquaretic" diuretics, i.e., diuretics with a predominant effect on water,

rather than on Na+, excretion Since aquaporin 2 is regulated by the vasopressin V2 receptor, anotherpotential class of aquaretic diuretics would be nonpeptide, orally active V2-receptor antagonists Exciting progress has been made, and this subject is developed in Chapter 30: Vasopressin and Other Agents Affecting the Renal Conservation of Water Finally, apical K+ channels (ROMK) are another potential molecular target for the development of K+-sparing diuretics with high efficacy

Chapter 30 Vasopressin and Other Agents Affecting the Renal Conservation of Water

Overview

Precise regulation of body fluid osmolality is essential It is controlled by a finely tuned, intricate homeostatic mechanism that operates by adjusting both the rate of water intake and the rate of

solute-free water excretion by the kidneys—i.e., water balance Abnormalities in this homeostatic

system can be caused by genetic diseases, acquired diseases, or drugs and may result in serious and potentially life-threatening deviations in plasma osmolality The goals of this chapter are to describethe physiological mechanisms that regulate plasma osmolality, to discuss the diseases that perturb those mechanisms, and to examine pharmacological approaches for treating disorders of water balance

Arginine vasopressin (the antidiuretic hormone in human beings) is the main hormone involved in regulation of body fluid osmolality Many diseases of water homeostasis and many pharmacologicalstrategies for correcting such disorders pertain to vasopressin Accordingly, vasopressin is the majorfocus of this chapter and is discussed with regard to: (1) chemistry (including the chemistry of vasopressin agonists and antagonists); (2) physiology (including anatomical considerations; the synthesis, transport, and storage of vasopressin and the regulation of vasopressin secretion); (3) basic pharmacology (including vasopressin receptors and their signal transduction pathways, renal actions of vasopressin, pharmacological modification of the antidiuretic response to vasopressin, and nonrenal actions of vasopressin); (4) diseases affecting the vasopressin system (diabetes

insipidus, syndrome of inappropriate secretion of antidiuretic hormone, and other water-retaining states); and (5) clinical pharmacology of vasopressin peptides (therapeutic uses, pharmacokinetics, toxicities, adverse effects, contraindications, and drug interactions) A small number of other drugs

can be used to treat abnormalities of water balance; a discussion of these agents is integrated into the section on diseases affecting the vasopressin system.

Introduction to Vasopressin

Immunoreactive vasopressin has been observed in neurons from organisms belonging to the first

animal phylum with a nervous system (e.g., Hydra attenuata), and vasopressin-like peptides have

been isolated and characterized from both mammalian and nonmammalian vertebrates as well as from invertebrates (Table 30–1) Genes encoding vasopressin-like peptides probably evolved more than 700 million years ago

With the emergence of life on land, vasopressin became the mediator of a remarkable regulatory system for the conservation of water The hormone is released by the posterior pituitary whenever water deprivation causes an increased plasma osmolality or whenever the cardiovascular system is challenged by hypovolemia and/or hypotension In amphibians, the target organs for vasopressin areskin and the urinary bladder, whereas in other vertebrates, including human beings, the site of action is primarily the renal collecting duct In each of these target tissues, vasopressin acts by increasing the permeability of the cell membrane to water, thus permitting water to move passively down an osmotic gradient across skin, bladder, or collecting duct into the extracellular

Chemistry of Vasopressin Receptor Agonists and Antagonists

Chemistry of Vasopressin Receptor Agonists

du Vigneaud and coworkers (1954) determined the structures of vasopressin and oxytocin and accomplished the complete synthesis of each A number of vasopressin-like peptides occur

naturally (Table 30–1) All are nonapeptides; contain cysteine residues in positions 1 and 6; have anintramolecular disulfide bridge between the two cysteine residues (essential for agonist activity); have additional conserved amino acids in positions 5, 7, and 9 (asparagine, proline, and glycine, respectively); contain a basic amino acid in position 8; and are amidated on the carboxyl terminus

In all mammals except swine, the neurohypophyseal peptide is 8-arginine vasopressin, and the

terms vasopressin, arginine vasopressin (AVP), and antidiuretic hormone (ADH) are used

interchangeably The chemical structure of oxytocin is closely related to that of vasopressin, i.e.,

oxytocin is [Ile3, Leu8]AVP Oxytocin binds to specific oxytocin receptors on myoepithelial cells in the mammary gland and on smooth muscle cells in the uterus, causing milk ejection and uterine contraction, respectively Inasmuch as vasopressin and oxytocin are structurally similar, it is not surprising that vasopressin and oxytocin agonists and antagonists can bind to each other's receptors Therefore, most of the available peptide vasopressin agonists and antagonists have some affinity foroxytocin receptors; at high doses, they may block or mimic the effects of oxytocin (Manning and

Sawyer, 1989).

With the advent of solid-phase peptide synthesis, many vasopressin analogs were synthesized with the goal of increasing duration of action and selectivity for vasopressin receptor subtypes (V1versus

V2 vasopressin receptors, which mediate pressor responses and antidiuretic responses, respectively)

In 1967, Zaoral and coworkers announced the synthesis of desmopressin: 1-deamino-8-D-arginine vasopressin (DDAVP) (Table 30–1) Deamination at position 1 increases duration of action and increases antidiuretic activity without increasing vasopressor activity Substitution of D-arginine for L-arginine greatly reduces vasopressor activity without reducing antidiuretic activity Thus, the antidiuretic-to-vasopressor ratio for desmopressin is approximately 3000-fold greater than that for vasopressin, and desmopressin now is the preferred drug for the treatment of central diabetes insipidus (Robinson, 1976) Substitution of valine for glutamine in position 4 further increases the antidiuretic selectivity, and the antidiuretic-to-vasopressor ratio for deamino[Val4, D-Arg8] AVP (Table 30–1) is approximately 11,000-fold greater than that for vasopressin Increasing V1

selectivity has proved more difficult than increasing V2 selectivity (Thibonnier, 1990) However, a limited number of agonists have been developed with modest selectivity for V1 receptors (seeTable

been described (seeTable 30–1).

Chemistry of Vasopressin Receptor Antagonists

The impetus for the development of specific vasopressin receptor antagonists is the belief that such drugs may be useful in a number of clinical settings Selective V1a antagonists may be beneficial

when total peripheral resistance is increased (e.g., congestive heart failure and hypertension);

selective V2 antagonists could be useful whenever reabsorption of solute-free water is excessive

(e.g., the syndrome of inappropriate secretion of antidiuretic hormone and hyponatremia associated

with a reduced effective blood volume) Combined V1a/V2 receptor antagonists might be beneficial

in diseases associated with a combination of increased peripheral resistance and dilutional

hyponatremia (e.g., congestive heart failure).

Shortly after the synthesis of vasopressin, du Vigneaud and coworkers began designing antagonists

of vasopressin's pharmacological effects Since that time, numerous vasopressin receptor

antagonists have been synthesized (Manning et al. , 1993 ; László et al. , 1991 ) Highly selective V1

and V2 peptide antagonists that are structural analogs of vasopressin have been synthesized

(seeTable 30–2 for examples), including both cyclic and linear peptides [1( mercapto ,

-cyclopentamethyleneproprionic acid),2-O-methyltyrosine] Arginine vasopressin, also known as

d(CH2)5[Tyr(Me)2]AVP, has a greater affinity for V1a receptors than for either V1b or V2 receptors; this antagonist has been widely employed in physiological and pharmacological studies (Manning and Sawyer, 1989) Although [1-deaminopenicillamine, 2-O-methyltyrosine] arginine vasopressin, also called dP[Tyr(Me)2]AVP, is a potent V1b receptor antagonist with little affinity for the V2

receptor, it also blocks V1a receptors No truly selective V1b receptor antagonist currently is

available Peptide antagonists currently available have limited oral activity, and the potency of peptide V2 antagonists is species-dependent Also, with prolonged infusion, peptide V2 antagonists

appear to express significant agonist activity (Kinter et al. , 1993 ) However, in the early 1990s a nonpeptide, orally active V1-selective antagonist (OPC-21268) (Yamamura et al., 1991) and a

nonpeptide, orally active V1a-/V2-selective antagonist (OPC-31260) (Yamamura et al. , 1992 ) were synthesized Neither compound had partial agonist activity Other highly potent nonpeptide

vasopressin receptor antagonists, such as SR 49059, SR 121463A, VPA-985, and YM 087

(Mayinger and Hensen, 1999) have been synthesized (seeTable 30–2)

Anatomy

The antidiuretic mechanism in mammals involves two anatomical components: a CNS component for the synthesis, transport, storage, and release of vasopressin, and a renal collecting duct system composed of epithelial cells that respond to vasopressin by increasing their permeability to water The relevant anatomy of the renal collecting duct system is described in Chapter 29: Diuretics The

CNS component of the antidiuretic mechanism is called the hypothalamiconeurohypophyseal

system and consists of neurosecretory neurons with perikarya located predominantly in two specifichypothalamic nuclei, the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) The long axons of neurons in the SON and PVN traverse the supraopticohypophyseal tract to terminate

in the median eminence and pars nervosa of the posterior pituitary

Synthesis

Vasopressin and oxytocin are synthesized in the perikarya of magnocellular neurons in the SON andPVN; the two hormones are synthesized predominantly in separate neurons Vasopressin synthesis appears to be regulated solely at the transcriptional level (Robinson and Fitzsimmons, 1993), and the molecular mechanism of vasopressin synthesis has been elucidated in considerable detail

(Archer, 1993) In human beings, a 168-amino-acid preprohormone (Figure 30–1) is synthesized, and a signal peptide (residues -23 to -1) assures incorporation of the nascent polypeptide into ribosomes During synthesis, the signal peptide is removed to form the vasopressin prohormone, and vesicle-mediated translocations maneuver the prohormone through the rough endoplasmic

reticulum and cis-, medial-, and trans-Golgi compartments, so that the prohormone emerges

incorporated into large (0.1- to 0.3-micron) membrane-enclosed granules The prohormone consists

of three domains: vasopressin (residues 1 to 9), vasopressin (VP)–neurophysin (residues 13 to 105),and VP–glycopeptide (residues 107 to 145) The vasopressin domain is linked to the VP–

neurophysin domain through a glycine–lysine–arginine processing signal, and the VP–neurophysin domain is linked to the VP–glycopeptide domain by an arginine-processing signal In the secretory granules, an endopeptidase, exopeptidase, monooxygenase, and lyase act sequentially on the

prohormone to produce vasopressin, VP–neurophysin (sometimes referred to as neurophysin II or MSEL–neurophysin), and VP–glycopeptide (sometimes called copeptin) The synthesis and

transport of vasopressin are dependent on the conformation of the preprohormone In particular, VP–neurophysin binds vasopressin and is critical to the correct processing, transport, and storage ofvasopressin (Breslow, 1993) Genetic mutations in either the signal peptide or VP–neurophysin giverise to central diabetes insipidus (Raymond, 1994)

Figure 30–1 Processing of the 168–Amino Acid Human 8-Arginine Vasopressin (AVP) Preprohormone to AVP, Vasopressin (VP)–Neurophysin, and VP–

Glycopeptide.