Diuretics - Renal Anatomy and Physiology

Renal Anatomy

The main renal artery branches close to the renal hilum into segmental arteries, which, in turn, subdivide to form interlobar arteries that pierce the renal parenchyma. The interlobar arteries curve at the border of the renal medulla and cortex to form arc-like vessels known as arcuate arteries. Arcuate arteries give rise to perpendicular branches, called interlobular arteries, which enter the renal cortex and supply blood to the afferent arterioles. A single afferent arteriole penetrates the glomerulus of each nephron and branches extensively to form the glomerular capillary nexus. These branches coalesce to form the efferent arteriole. Efferent arterioles of superficial glomeruli ascend toward the kidney surface before splitting into peritubular capillaries that service the tubular 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 291 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 included.

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 (P_GC), the hydrostatic pressure in Bowman's space (which can be equated with pressure in the proximal tubule, P_T), the mean colloid osmotic pressure in the glomerular capillaries (_GC), the colloid osmotic pressure in the proximal tubule (_T), and the ultrafiltration coefficient (K_f), according to the equation:

SNGFR = K_f[(P_GC (_{GC T}

If P_GCP_T 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 = K_f(P _GC

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. K_f 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 (Q_A). Q_A 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 Q_A is high, this effect is reduced; however, when Q_A 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 since this 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 ascending limb, 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 P_GC and Q_A 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 292 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.

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 293 and can be summarized as follows:

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 294. Both systems are powered by the sodium pump in the basolateral membrane, involve secondary and tertiary 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 from a second negative charge, and a hydrophobic region 8 to 10 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.

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 (V_T), 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 V_T, 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 (P_i) is largely reabsorbed (80% of filtered load) by the proximal tubule. A Na⁺P_i symporter uses the free energy of the Na⁺ electrochemical gradient to effect secondary active transport of P_i into the cell. The Na⁺P_i symporter is inhibited by PTH. P_i 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 V_T. 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 acidbase balance. A description of these processes is presented in the section on carbonic anhydrase inhibitors.

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 292 lists the chemical structures of the three carbonic anhydrase inhibitors currently available in the United Statesacetazolamide, 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 295). 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 reacts with 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.

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 limited cellular 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 a proton 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 anhydraseindependent 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 anion excreted 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, the filtered load of HCO₃ decreases to the point that the uncatalyzed reaction between CO₂ and water is sufficient 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 interference with 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 292. 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 this regard. 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 a marked 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 anhydrase inhibitors 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.

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) Approximately 25% 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 294). 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 296). 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) only for K⁺; therefore the apical membrane voltage is determined by the equilibrium potential for K⁺ (E_K). In contrast, the basolateral membrane has channels for both K⁺ and Cl, so that the basolateral membrane voltage is less than E_K; 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.

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 Clbinding 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 (Obermller 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 anhydraseinhibiting activity. Those drugs with carbonic anhydraseinhibiting 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 294. 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 concentrationresponse curve to furosemide to the right by competitively inhibiting furosemide secretion by the organic acid transport system (Brater, 1983). The most recent loop 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 reninangiotensin 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 in patients 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 both drugs leading to profound diuresis); and (11) amphotericin B (increased potential for nephrotoxicity and 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 diureticscombined with isotonic saline administration to prevent volume depletionare 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. Loop diuretics also are used to treat edema associated with chronic renal insufficiency. However, animal studies have demonstrated that loop diuretics increase P_GC by activating the reninangiotensin 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).

The benzothiadiazides were synthesized in an effort to enhance the potency of inhibitors of carbonic anhydrase. 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 295) 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 297 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).

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; Obermller et al., 1995; Plotkin et al., 1996). Expression of the Na⁺Cl symporter is regulated by aldosterone (Velzquez 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, an effect 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 during hydropenia.

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 295. 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 attenuate the 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, -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 diureticinduced K⁺ depletion accounts for many cases of quinidine-induced torsades de pointes.

Therapeutic Uses

Thiazide diuretics are used for treatment of the edema associated with heart (congestive heart failure), liver (hepatic cirrhosis), and renal (nephrotic syndrome, chronic renal failure, acute glomerulonephritis) disease. With the exceptions of metolazone and indapamide, most thiazide diuretics are ineffective when GFR is <30 to 40 ml/min.

Thiazide diuretics decrease blood pressure in hypertensive patients by increasing the slope of the renal pressurenatriuresis relationship (Saito and Kimura, 1996), and thiazide diuretics are widely used for the treatment of hypertension, either alone or in combination with other antihypertensive 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 with other 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 (Grnbeck 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.

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 296). 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 a higher degree of certainty. As illustrated in Figure 298, 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 V_T, 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 reninangiotensinaldosterone axis by diuretics also contributes to diuretic-induced K⁺ and H⁺ excretion by a mechanism explained in the section on mineralocorticoid antagonists.

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 296. 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 life-threatening. Consequently, amiloride and triamterene are contraindicated in patients with hyperkalemia as well as in patients at increased risk of developing 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, may increase 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⁺-channel inhibitors 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-induced nephrogenic diabetes insipidus because it blocks Li⁺ transport into the cells of the collecting tubules.

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 297).

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 MRaldosterone 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 299 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.

Drugs such as spironolactone competitively inhibit the binding of aldosterone to the MR (Marver et al., 1974). Unlike the MRaldosterone complex, the MRspironolactone 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 of other 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.

Therapeutic Uses

As with other K⁺-sparing diuretics, spironolactone often is coadministered with thiazide or loop diuretics in the treatment of edema and hypertension. Such combinations result in increased mobilization of edema fluid while causing lesser perturbations of K⁺ homeostasis. Spironolactone is particularly useful in the treatment of primary hyperaldosteronism (adrenal adenomas or bilateral adrenal hyperplasia) and of refractory edema associated with secondary aldosteronism (cardiac failure, hepatic cirrhosis, nephrotic syndrome, severe ascites). Spironolactone is considered the 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).

Mechanism of Edema Formation

A complex set of interrelationships (Figure 2910) 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 2910). This loop cycles until net Na⁺ accumulation is zero; i.e., in the long run, Na⁺ intake must equal Na⁺ loss.

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 pressurenatriuresis 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 pressurenatriuresis 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 pressurenatriuresis 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 pressurenatriuresis 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 pressurenatriuresis curve. (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 2910 is that three fundamental strategies exist for mobilizing edema fluidcorrect 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 and should 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 of edema 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 between the 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: