Physiology and Pathophysiology of Diuretic Action

The term diuretic derives from the Greek diouretikos , meaning “to promote urine.” Although infusion of saline or ingestion of water would therefore qualify as being diuretic, the term diuretic usually connotes a drug that can reduce the extracellular fluid (ECF) volume by increasing urinary solute or water excretion. The term aquaretic has sometimes been applied to drugs that increase excretion of solute free water, distinguishing them from traditional diuretics, which increase solute and water together. The clinical picture of ECF volume expansion leading to edema or “dropsy” (from the Latin, hydrops ) has been recognized since the earliest days of recorded history. Ancient Egyptians referred to “flooding of the heart,” and the Hippocratic Corpus later suggested specific remedies for dropsical patients, although their results are not noted. In 1553, Paracelsus recorded the first truly effective form of therapy for dropsy, inorganic mercury (Calomel). Inorganic mercury remained the mainstay of diuretic treatment until the beginning of this century.

Introduction

In 1919, the ability of organic mercurial antisyphilitics to effect diuresis was discovered by Vogl, then a medical student. This observation led to the development of effective organic mercurial diuretics, drugs that were used commonly until the 1960s. In 1937, the antimicrobial, sulfanilamide, was found to cause metabolic acidosis in patients. Carbonic anhydrase had been discovered in 1932; it was know that sulfanilamide inhibited this enzyme. Pitts demonstrated that sulfanilamide inhibited Na bicarbonate reabsorption in dogs, and Schwartz showed that sulfanilamide could induce diuresis in patients with congestive heart failure who were resistant to organic mercurial diuretics. Soon, more potent sulfonamide-based carbonic anhydrase inhibitors were developed, but these drugs suffered from side-effects and limited potency. Nevertheless, a group at Sharp & Dohme Inc. was stimulated by these developments to explore the possibility that modification of sulfonamide-based drugs could lead to drugs that enhanced Na chloride rather than Na bicarbonate excretion. The result of this program was the synthesis of chlorothiazide and its marketing in 1957. This drug ushered in the modern era of diuretic therapy, and revolutionized the clinical treatment of edema.

The search for more potent classes of diuretics continued, based on the structure of chlorothiazide and sulonamyl derivatives. This led to the development of ethacrynic acid and furosemide in the United States and Germany, respectively. The safety and efficacy of these drugs led them to replace the organic mercurials as drugs of first choice for severe and resistant edema. Spironolactone, marketed in 1961, was developed after the properties and structure of aldosterone had been established, and steroidal analogs of aldosterone were found to have aldosterone-blocking activity. Triamterene was initially synthesized as a folic acid antagonist, but was found to have diuretic and K-sparing activity.

The availability of safe, effective, and relatively inexpensive diuretic drugs has made it possible to treat edematous disorders and hypertension effectively. Driven by clinical need, however, the development of effective diuretic drugs generated specific ligands that interact with Na and Cl transport proteins in the kidney. In the 1990s, these ligands were used to identify and clone the Na and Cl transport proteins that mediate the bulk of renal Na and Cl reabsorption. The diuretic-sensitive transport proteins that have been cloned include the sodium hydrogen exchanger (NHE) family of proteins, the bumetanide-sensitive Na-K-2Cl co-transporters, the thiazide-sensitive Na-Cl co-transporter, and the epithelial Na channel. The information derived from molecular cloning has also permitted identification of inherited human diseases that are caused by mutations in these transport proteins. The phenotypes of several of these disorders resemble the manifestations of chronic diuretic administration. The recognition, for example, that Gitelman’s syndrome results from mutation of the thiazide-sensitive Na-Cl co-transporter, has spurred interest in determining how blockade or dysfunction of this transport protein leads to magnesium-wasting. Thus, the development of clinically useful diuretics permitted identification, and later cloning, of specific ion transport pathways. The molecular cloning then helped to define mechanisms of diuretic action and diuretic side-effects, permitting the development of specific antibodies and probes, and of animals in which diuretic-sensitive transport pathways have been “knocked-out.” Many primarily historical references included in prior editions of this book have been omitted here. The interested reader is referred to prior editions for more complete references and details.

Diuretic-Sensitive Salt Transport

In a normal human kidney, approximately 23 moles of NaCl are filtered in 150 liters of fluid each day. Approximately 6–10 grams of salt (102–170 mEq NaCl) are consumed each day by individuals on a typical Western diet. To maintain balance, renal NaCl excretion must be approximately 92–160 mmol/day (the difference owing to nonrenal losses). Such calculations imply that 99.2% of the filtered NaCl load is reabsorbed by kidney tubules each day (the fractional sodium excretion, FE _Na , is 0.8%). Sodium, chloride, and water reabsorption along the nephron is driven by the metabolic energy in ATP. The ouabain-sensitive Na/K-ATPase is expressed at the basolateral cell membrane of nearly all Na transporting epithelial cells along the nephron. This pump maintains large ion gradients across the plasma membrane, with the intracellular Na concentration maintained low and the intracellular K concentration maintained high. Because the pump is electrogenic, and because it is associated with a K channel, renal epithelial cells have a voltage across the plasma membrane oriented with the inside negative relative to the outside.

The combination of the low intracellular Na concentration and the plasma membrane voltage generates a large electrochemical gradient favoring Na entry from lumen or interstitium. Specific diuretic-sensitive Na transport pathways are expressed at the apical surface of cells along the nephron, permitting vectorial transport of Na from lumen to blood (see Figure 40.1 ). Along the proximal tubule, where approximately two-thirds of filtered Na is reabsorbed, a major component of Na reabsorption is exchanged for H ⁺ via an isoform of the Na/H exchanger (NHE3) at the apical membrane. Along the thick ascending limb, where approximately 20–25% of filtered Na is reabsorbed, an isoform of the Na-K-2Cl co-transporter (NKCC2) is expressed at the apical membrane. Along the distal convoluted tubule (DCT), where approximately 5% of filtered Na is reabsorbed, the thiazide-sensitive Na-Cl co-transporter (NCC) is expressed. The DCT comprises two subsegments, the DCT1 (or “early DCT”) and the DCT2 (or “late DCT”). Along the DCT1, the NCC is the predominant Na transporter. Along the connecting tubule and cortical collecting duct, where approximately 3% of filtered Na is reabsorbed, isoforms of the amiloride-sensitive epithelial Na channel are expressed (ENaC). DCT2 cells express both NCC and ENaC. Together, these apical Na transport pathways along the nephron form the molecular targets for diuretic action.

Figure 40.1, Sites of diuretic action along the nephron.

This chapter will discuss the physiological and pharmacological bases for diuretic action in the kidney. Although some aspects of clinical diuretic usage will be discussed, we have emphasized physiological principles and mechanisms of action. Several recent texts provide detailed discussions of diuretic treatment of clinical conditions. Extensive discussions of diuretic pharmacokinetics are also available. The influence of renal disease on diuretic drug usage is discussed in the following chapter of this volume.

One classification of diuretic drugs is based on the primary nephron site of action. Such a scheme emphasizes that drugs of more than one chemical class can affect the same ion transport mechanism. Although most diuretic drugs have actions on more than one nephron segment, most owe their clinical effects primarily to their ability to inhibit Na transport by one particular nephron segment. An exception is the osmotic diuretics. Although these drugs were initially believed to inhibit solute and water flux primarily along the proximal tubule, subsequent studies have revealed effects in multiple segments. Other diuretics, however, will be classified according to their primary site of action.

Osmotic Diuretics

Osmotic diuretics are substances that are freely filtered at the glomerulus, but are poorly reabsorbed (see Figure 40.2 ). The pharmacological activity of drugs in this group depends entirely on the osmotic pressure exerted by the drug molecules in solution, and not on interaction with specific transport proteins or enzymes. Mannitol is the prototypical osmotic diuretic. Its diuretic effect is not due to interactions with receptors or renal transporters, but rather it is due to more complex mechanisms that involve osmotic effects on tubule epithelium and reduction of the medullary interstitial osmolality. Because the relationship between the magnitude of effect and concentration of osmotic diuretic in solution is linear, all agents used clinically are small molecules. Other agents considered in this class include urea, sorbitol, and glycerol.

Urinary Electrolyte Excretion

Although osmotic agents do not act directly on transport pathways, the rate of transport of ions is affected. Following the infusion of mannitol, the excretion of sodium, potassium, calcium, magnesium, bicarbonate, and chloride is increased (see Table 40.1 ). The fractional reabsorption rates for sodium and water are reduced substantially following the infusion of mannitol. Reabsorption of magnesium and calcium are also reduced in the proximal tubule and loop of Henle, and phosphate reabsorption is inhibited slightly along the proximal tubule. In addition to increasing electrolyte excretion, mannitol infusion increases cortical and medullary blood flow, and has a variable effect on GFR. The most pronounced effect observed with mannitol is a brisk diuresis and natriuresis.

Table 40.1

Effects of Diuretics on Electrolyte Excretion

	Na	Cl	K	Pi	Ca	Mg
Osmotic diuretics	↑(10–25%)	↑(15–30%)	↑(6%)	↑(5–10%)	↑(10–20%)	↑(>20%)
Carbonic anhydrase inhibitors	↑(6%)	↑(4%)	↑(60%)	↑(>20%)	↑ or ⇔ (<5%)	↑(<5%)
Loop diuretics	↑(30%)	↑(40%)	↑(60–100%)	↑(>20%)	↑(>20%)	↑(>20%)
DCT diuretics	↑(6–11%)	↑(10%)	↑(200%)	↑(>20%)	↓	↑(5–10%)
Na channel blockers	↑(3–5%)	↑(6%)	↓(8%)	⇔	⇔	↓
Spironolactone	↑(3%)	↑(6%)	↓	⇔	⇔	↓

Figures indicate approximate maximal fractional excretions of ions following acute diuretic administration in maximally effective doses. ↑ indicates that the drug increases excretion; ↓ indicates that the drug decreases excretion; ⇔ indicates that the drug has little or no direct effect on excretion. During chronic treatment, effects often wane (Na excretion), may increase (K excretion during DCT diuretic treatment) or may reverse as with uric acid (not shown). For references that support this table, please see .

Mechanism of Action

The mechanisms by which mannitol produces a diuresis are thought to be secondary to: (1) an increase in osmotic pressure in the proximal tubule fluid and loop of Henle, thereby retarding the passive reabsorption of water; and (2) an increase in renal blood flow and washout of the medullary tonicity.

Mannitol is freely filtered at the glomerulus, and its presence in the tubule fluid minimizes passive water reabsorption primarily by the proximal tubule and by the thin limbs of the loop of Henle. Normally, within the proximal tubule, sodium reabsorption creates an osmotic gradient for water reabsorption. When an osmotic diuretic is administered, however, the osmotic force of the nonreabsorbable solute in the lumen opposes the osmotic force produced by sodium reabsorption. Isoosmolality of the tubule fluid is preserved, because molecules of mannitol replace sodium ions reabsorbed. Sodium reabsorption eventually stops, however, because the luminal sodium concentration is reduced to a point where a limiting gradient is reached. Surprisingly, mincropuncture experiments showed that mannitol has a greater effect on inhibiting Na and water reabsorption in the loop of Henle than in the proximal tubule. Within the loop of Henle the site of action of mannitol appears to be restricted to the thin descending limb, resulting in a decrease in reabsorption of Na and water. In the thick ascending limb, reabsorption of Na will continue in proportion to its delivery to this segment. The sum of net transport in the thin and thick limbs will determine the net effect of mannitol in the loop of Henle. Further downstream in the collecting duct, mannitol also reduces sodium and water reabsorption.

Renal Hemodynamics

During the administration of mannitol, its molecules diffuse from the bloodstream into the interstitial space. In the interstitial space, the increased osmotic pressure draws water from the cells to increase ECF volume. This effect increases total renal plasma flow. Cortical blood flow and medullary blood flow are both increased following mannitol infusion. Single nephron GFR, on the other hand, increases in the cortex and decreases in the medulla, this action on the medulla washes out the medullary osmotic gradient by reducing papillary sodium and urea content. The mechanisms that contribute to the increase in renal blood flow include a decrease in hematocrit and blood viscosity, and the release of vasoactive agents. Experimental studies indicate that the osmotic effect of mannitol to increase water movement from intracellular to extracellular space leads to a decrease in hematocrit and in blood viscosity. This fact contributes to a decrease in renal vascular resistance and increase in renal blood flow. Both prostacyclin (PGI ₂ ) synthesis and atrial natriuretic peptide could mediate the effect of mannitol on renal blood flow. The vasodilatory effect of mannitol is reduced when the recipient is pretreated with indomethacin or meclofenamate, suggesting that PGI ₂ is involved in the vasodilatory effect.

The effect of mannitol on GFR has been variable, but most studies indicate that the overall effect of mannitol is to increase GFR. Whereas mannitol increased cortical and medullary blood flow, it increased cortical but decreased medullary single nephron GFR. The mechanisms by which mannitol reduces the GFR of deep nephrons are not known, but it has been postulated that mannitol reduces efferent arteriolar pressure. Micropuncture studies examining the determinants of GFR in superficial nephrons have demonstrated that the increase in single nephron GFR is owing to an increase in single nephron plasma flow, and a decrease in oncotic pressure. Alterations in renal hemodynamics contribute to the diuresis observed following administration of mannitol. An increase in medullary blood flow rate reduces medullary tonicity primarily by decreasing papillary sodium and urea content, and increasing urine flow rate.

Pharmacokinetics

Mannitol is not readily absorbed from the intestine; therefore it is routinely administered intravenously. Following infusion, mannitol distributes in ECF with a volume of distribution of approximately 16 liters; its excretion is almost entirely by glomerular filtration. Of the filtered load, less than 10% is reabsorbed by the renal tubule, and a similar quantity is metabolized, probably in the liver. With normal GFR, plasma half-life is approximately 2.2 hours.

Clinical Use

Mannitol is often used prophylactically to help prevent acute kidney injury (AKI) in the setting of rhabdomyolysis, although some controlled studies have not confirmed benefit. It was previously used for prophylaxis from contrast-induced nephropathy, but appears of no benefit here, and is of potential harm. In the past, it was also used to treat established AKI, but its use here has also fallen from favor, as convincing evidence of benefit has been lacking.

Mannitol is used for short-term reduction of intraocular pressure. By increasing the osmotic pressure, mannitol reduces the volume of aqueous humor and the intraocular pressure by extracting water. Mannitol also decreases cerebral edema and the increase in intracranial pressure associated with trauma, tumors, and neurosurgical procedures, where its benefits are most clearly established. Mannitol is used perioperatively in patients undergoing cardiopulmonary bypass surgery. The beneficial effects may be related to its osmotic activity reducing intravenous fluid requirement, and its ability to act as a free radical antioxidant. Mannitol and other osmotic agents have been used in the treatment of dialysis disequilibrium syndrome. This syndrome is characterized acute symptoms immediately following hemodialysis. Most significant symptoms are attributable to disorders of the central nervous system, such as headache, nausea, blurred vision, confusion, seizure, coma, and death. Rapid removal of small solutes such as urea during dialysis of patients who are markedly azotemic is associated with the development of an osmotic gradient for water movement into brain cells producing cerebral edema and neurologic dysfunction. Dialysis disequilibrium syndrome can be minimized by slow solute removal and raising plasma osmolality with saline or mannitol.

Adverse Effects

In patients with reduced cardiac output, an increase in extracellular volume induced by mannitol infusion may lead to pulmonary edema. Intravenous administration of mannitol increases cardiac output and pulmonary capillary wedge pressures. Acute and prolonged administration of mannitol leads to different electrolyte disturbances. Acute overzealous use or the accumulation of mannitol leads to dilutional metabolic acidosis and hyponatremia. Accumulation of mannitol also produces hyperkalemia, as a result of an increase in plasma osmolality. An increase in plasma osmolality increases potassium movement from intracellular to extracellular fluid from bulk solute flow, and increases the electrochemical gradient for potassium secretion. Prolonged administration of mannitol can lead to urinary losses of sodium and potassium, leading to volume-depletion, hypernatremia (as urinary loss of sodium is invariably less than water), and hypokalemia. Marked accumulation of mannitol in patients can lead to reversible AKI that appears to be due to vasoconstriction and tubular vacuolization. Mannitol-induced AKI usually occurs when large cumulative doses of ~295 g are given to patients with previously compromised renal function.

Proximal Tubule Diuretics

(Carbonic Anhydrase Inhibitors)

Through the development of carbonic anhydrase inhibitors, important compounds were discovered that have utility as therapeutic agents, and as research tools. Carbonic anhydrase inhibitors (CAI) have a limited therapeutic role as diuretic agents, because of weak natriuretic properties. They are used primarily to reduce intraocular pressure in glaucoma, and to enhance bicarbonate excretion in metabolic alkalosis. CAIs have been useful in the development of other diuretic agents, such as thiazide and loop diuretics, and have been instrumental in elucidating transport function in proximal and distal nephron segments. Structures of carbonic anhydrase inhibitors are shown in Figure 40.3 .

Urinary Electrolyte Excretion

Through their effects on carbonic anhydrase in the proximal tubule, CAIs increase bicarbonate excretion by 25–30% (see Table 40.1 ). Chronic CAI administration, however, causes only a modest natriuresis, despite the magnitude of carbonic anhydrase-dependent proximal Na reabsorption. Several factors account for this. First, carbonic anhydrase is required for reabsorption of HCO ₃ ⁻ , whereas about two thirds of the proximal Na ⁺ reabsorption is accompanied by Cl ⁻ . Second, some proximal HCO ₃ ⁻ reabsorption persists even after apparently full inhibition of carbonic anhydrase. Third, some of the HCO ₃ ⁻ that is delivered from the proximal tubule can be reabsorbed at more distal sites. Fourth, the metabolic acidosis that develops limits the filtered load to HCO ₃ ⁻ , and thereby curtails the natriuresis. Fifth, the increased delivery of filtered Na ⁺ to the macula densa elicits a tubuloglomerular feedback (TGF)-induced reduction in the GFR. Micropuncture studies of mice with deletion of the proximal Na ⁺ /H ⁺ exchanger, NHE3, also show that inhibition of proximal Na reabsorption is largely balanced by reduced GFR, suggesting that the reduction of GFR with CAI use contributes importantly to limiting their natriuretic potency.

The effecs of CAI on calcium excretion are complex. Proximal tubule calcium and phosphate reabsorption are inhibited by acetazolamide, partly because sodium and calcium reabsorption are closely linked within this segment. Yet fractional calcium excretion is often unchanged or reduced, because distal calcium reabsorption is stimulated and because luminal bicarbonate promotes calcium reabsorption. Over the longer-term, however, CAI can increase urinary calcium excretion and predispose to nephrocalcinosis and kidney stone formation. In contrast, phosphate appears to escape distal reabsorption following acetazolamide administration, resulting in an increase in fractional excretion of phosphate by ~3%. Although proximal tubule magnesium transport is inhibited by CAI, fractional excretion is either unchanged or is increased as a result of variable distal reabsorption.

Acetazolamide increases potassium excretion. Although a direct effect of acetazolamide has not been established, it is likely that several indirect effects could contribute to the observed kaliuresis. Carbonic anhydrase inhibition could block proximal tubule potassium reabsorption and increase delivery to the distal tubule, but the reported effects of carbonic anhydrase inhibition on proximal tubule transport have been conflicting. Whereas CAI decreases proximal tubule sodium, bicarbonate, and water absorption during both free flow micropuncture and microperfusion, the effects of CAI on proximal tubule potassium transport have been less consistent. In free flow micropuncture studies, carbonic anhydrase inhibition did not affect proximal tubule potassium reabsorption, whereas net potassium transport was reduced during proximal perfusion in vivo . The effect of acetazolamide on the proximal tubule ion transport does, however, facilitate an increase in tubular fluid flow rate and delivery to the distal nephron of sodium bicarbonate. This effect is thought to increase the concentration of nonreabsorbable anions, creating an increase in lumen-negative voltage and an increase in flow rate, factors known to increase potassium secretion by the distal tubule.

Most diuretics have some CAI action. This contributes to the weak inhibition of proximal reabsorption by furosemide and chlorothiazide, and to the relaxation of vascular smooth muscle cells by high-dose furosemide Goldfarb diuretics.

Mechanism of Action

In the kidney, CAI likely acts via three distinct, but related mechanisms (see Figure 40.4 ). First, they inhibit the hydration of CO ₂ within cells, thereby reducing the generation of substrate for H ⁺ and HCO ₃ ⁻ transporters; second, they reduce the dehydration of carbonic acid to CO ₂ and H ₂ O in the luminal compartment, thereby inhibiting continued H ⁺ secretion; finally, intracellular CA appears to associate with several membrane transport proteins, and may affect their activity more directly. These actions take place along the nephron, but actions along the proximal tubule and collecting duct are especially important. The biochemical, morphological, and functional properties of carbonic anhydrase have been reviewed previously. Carbonic anhydrase (CA), a metalloenzyme containing one zinc atom per molecule, is important in sodium bicarbonate reabsorption and hydrogen ion secretion by renal epithelial cells.

Figure 40.4, Localization of CA isoforms along the nephron.

CA is expressed by many tissues, including erythrocytes, kidney, gut, ciliary body, choroid plexus, and glial cells. Although at least 15 isoforms of CA have been identified, two play predominant roles in renal acid–base homeostasis: CAII and CAIV. CAII is widely expressed, comprising the enzyme expressed by red blood cells and a variety of secretory and absorptive epithelia. In the kidney, CAII is a cytoplasmic protein expressed, comprising 95% of renal CA. It is present in proximal tubule cells and intercalated cells of the aldosterone-sensitive distal nephron (ASDN) (see Figure 40.4 ). In rodents, carbonic anhydrase XIV is expressed at the luminal border of the cells of the proximal tubule, thick ascending limb (TAL) of the loop of Henle, and α-intercalated cells of the ASDN, but its role in other species is not as clear.

Carbonic anhydrase (CA) catalyzes the reversible hydration of CO ₂ according to the reaction:

H^{+} + {HCO}_{2}^{-} \leftrightarrow H_{2} {CO}_{2} \leftrightarrow {CO}_{2} + H^{+}

$H^{+} + {HCO}_{2}^{-} \leftrightarrow H_{2} {CO}_{2} \leftrightarrow {CO}_{2} + H^{+}$

CO ₂ gas dissolves in water and is in equilibrium with the acid H ₂ CO ₃ . The Henderson–Hasselbalch equation relates pH, HCO ₃ ⁻ concentration, and partial pressure of CO ₂ gas in physiologic solutions:

pH = 6.1 + \log_{10} \frac{({HCO}_{3}^{-})}{0.03 \times {pCO}_{2}}

$pH = 6.1 + \log_{10} \frac{({HCO}_{3}^{-})}{0.03 \times {pCO}_{2}}$

The uncatalyzed hydration of CO ₂ is relatively slow, whereas the turnover number for CAII is in the order of 10 ⁶ s ⁻¹ .

Type IV carbonic anhydrase is bound to renal cortical membranes, comprising up to 5% of the overall activity in kidney, and is sensitive to sulfonamides. Type IV carbonic anhydrase, expressed on basolateral and luminal plasma membranes of proximal tubule cells and luminal membrane of intercalated cells, catalyzes the dehydration of intraluminal carbonic acid generated from secreted protons.

Evidence for the physiological importance of carbonic anhydrase is apparent, as a deficiency of CAII leads to a renal acidification defect resulting in renal tubular acidosis. Furthermore, metabolic acidosis leads to an adaptive increase in both CAII and CAIV mRNA expression in kidney, suggesting the importance of both carbonic anhydrase isoforms in this disorder.

Carbonic anhydrase, which is associated with the brush border, prevents H ⁺ from accumulating in tubule fluid, and secondarily permits the continued secretion of H ⁺ (see Figure 40.5 ). Carbon dioxide rapidly diffuses from the lumen into the cell across the apical membrane. Within the cell, H is secreted into the tubule lumen via Na/H exchange, and perhaps other pathways such as H-ATPase. Following H secretion, OH ⁻ formed combines with CO ₂ , forming HCO ₃ , which exits the basolateral membrane via Na(HCO ₃ ) ₃ co-transport. Thus, in the early proximal tubule, the net effect of the process described results in the isosmotic reabsorption of NaHCO ₃ . The lumen chloride concentration increases, because water continues to be reabsorbed producing a lumen-positive potential. These axial changes provide an electrochemical gradient for transport of chloride via paracellular and transcellular pathways. The latter pathway for chloride likely involves a chloride–base exchanger operating in parallel with a Na/H proton exchanger. The dual operation of these parallel exchangers results in net transepithelial NaCl absorption.

Figure 40.5, Mechanisms of carbonic anhydrase inhibitors in the proximal tubule.

The participation of a membrane-bound component of carbonic anhydrase was first suggested by Rector, Carter, and Seldin. The observation that carbonic anhydrase inhibitors produced an acid disequilibrium pH in the proximal tubule suggested that luminal fluid was normally in contact with carbonic anhydrase. Disequilibrium pH refers to the difference between the pH of tubule fluid in situ (in this case during infusion of carbonic anhydrase inhibitors), and the pH achieved after the tubule fluid is allowed to reach chemical equilibrium at known pCO ₂ . Thus, when carbonic anhydrase is present, the pH measured in situ should be the same as the pH measured at equilibrium (in other words, CA should make the HCO ₃ dissociate into CO ₂ and H ₂ O very rapidly). When carbonic anhydrase is inhibited by the administration of CAI, the dissociation of HCO ₃ to OH and CO ₂ is slow, allowing H to accumulate in the lumen, and reducing pH.

The demonstration of an acid disequilibrium pH provided physiological evidence in support of previous histochemical findings that a fraction of enzymatic activity was present in the tubule lumen. Although the cytoplasmic carbonic anhydrase constitutes the majority of enzyme activity in kidney, it is believed that the membrane bound carbonic anhydrase plays a significant role in bicarbonate reabsorption by the proximal tubule. Studies addressing this question have employed CAIs that differ in their ability to penetrate proximal tubule cell membranes. Benzolamide is charged at normal pH and is relatively impermeant, whereas acetazolamide enters the cell relatively easily. Both intravenous and intratubular administration of benzolamide resulted in an acid disequilibrium pH, indicating that luminal carbonic anhydrase inhibition contributes to bicarbonate absorption. Furthermore, proximal tubular perfusion of benzolamide resulted in 90% inhibition of bicarbonate reabsorption. Despite near equal efficacy in inhibiting proximal tubule bicarbonate reabsorption, benzolamide lowered tubular fluid pH, whereas acetazolamide increased tubular fluid pH. These results suggest that the site of action of benzolamide is at the luminal membrane, whereas the site of action of acetazolamide is within the cell. Inhibition of luminal carbonic anhydrase causes lumen pH to decrease, because of the continued secretion of hydrogen ions and its accumulation in the tubular lumen. In contrast, acetazolamide does not produce an acid disequilibrium pH. The conclusion that tubular fluid was in direct contact with membrane carbonic anhydrase was substantiated by the use of dextran-bound carbonic anhydrase inhibitor. In proximal tubules perfused in vivo , Lucci et al. determined that dextran-bound inhibitors, which inhibit only luminal carbonic anhydrase, decreased proximal tubule bicarbonate absorption by approximately 80%, and reduced lumen pH. Although these studies establish the importance of luminal carbonic anhydrase, they also support a role for intracellular carbonic anhydrase, and as acetazolamide is the only CAI used for its renal properties, suggesting that cytoplasmic CA is the predominant drug target in humans.

Following administration of carbonic anhydrase inhibitors, proximal tubule bicarbonate reabsorption is inhibited variably between 35 and >85%. As suggested from the sites of expression along the nephron (see Figure 40.4 ), however, additional sites of action of carbonic anhydrase inhibitors include proximal straight tubule or loop of Henle, distal tubule, collecting tubule, and papillary collecting duct.

As noted, CAII may participate more directly in facilitating net bicarbonate and, perhaps, NaCl reabsorption. CAII has been shown to associate with several proximal tranpsporters, including the basolateral bicarbonate exit transport system, kNBC1, the apical Cl/base exchanger, SCL26A6, and the basolateral Na/H exchanger, NHE1, by binding to their respective carboxyl-terminal tails, associations that may be physiologically relevant. Thus, CAIs may act both directly on transporters and via inhibition of substrate production.

In α-intercalated cells (see Figure 40.5 ) within the DCT2, connecting tubule, and collecting duct, CA facilitates acid secretion that is mediated by a vacuolar H adenosinetriphosphatase (H-ATPase) H-K-ATPase, and the blood group protein RhCG at the apical membrane; RhCG is now known to be an important contributor to renal ammonia secretion. The anion exchanger (AE3) at the basolateral cell membrane participates importantly in bicarbonate reabsorption. As for the proximal tubule, both membrane-associated and cytosolic forms of CA likely contribute to distal acidification. Individuals with CAII deficiency display both proximal and distal acidification defects, confirming a role for this enzyme in the distal nephron. Yet, luminal administration of acetazolamide produced an acid disequilibrium pH in the outer medullary collecting duct, suggesting contribution a of luminal carbonic anhydrase as well. In subsequent studies, a membrane-impermeant carbonic anhydrase inhibitor reduced bicarbonate absorption, thus confirming the presence of membrane-bound carbonic anhydrase in the outer medullary collecting duct. The K _i for inhibition of bicarbonate absorption was 5 µM, consistent with the inhibition of Type IV carbonic anhydrase.

Like the proximal tubule, where CAII association with solute transporters may contribute to their regulation and activity, CAII has been shown to associate with CAII at the basolateral membrane, and inhibition of CA nearly completely blocked the bicarbonate transport induced by transfection of cells with AE1.

Effects of CAI on renal calcium transporters have also been examined. In mice, acetazolamide increased urinary pH and urinary calcium excretion, in association with a reduced TRPV5 abundance. Similar changes were induced by loading with NH ₄ Cl, but not by treatment with NaHCO ₃ , suggesting that they were the result of systemic acidosis, and not high urine pH. The importance of TRPV5 in these processes was confirmed by showing that acetazolamide did not alter urinary calcium excretion in TRPV5 knockout mice. These studies suggest that the hypercalciuria and tendency to stone formation induced by CAI is the result of systemic acidosis.

Renal Hemodynamics

Inhibition of carbonic anhydrase produces an acute decrease in GFR by activating TGF. Systemic infusion of acetazolamide resulted in a 30% decrease in GFR. Distally measured single nephron glomerular filtration rate (SNGFR) was reduced by 23% during acetazolamide infusion, whereas proximally measured SNGFR was not affected. These results indicated that acetazolamide blocked activated TGF, which in turn reduced GFR. Similar results were observed following infusion of benzolamide. Sar-ala8-angiotensin I, an angiotensin II antagonist, prevented the decrease in SGNFR, suggesting the involvement of local angiotensin II in response to benzolamide.

Pharmacokinetics

Acetazolamide is well-absorbed from the gastrointestinal (GI) tract. More than 90% of the drug is plasma protein-bound. The highest concentrations are found in tissues that contain large amounts of carbonic anhydrase (e.g., renal cortex, red blood cells). Renal effects are noticeable within 30 minutes, and are usually maximal at 2 hours. Acetazolamide is not metabolized, but is excreted rapidly by glomerular filtration and proximal tubular secretion. The half-life is approximately 5 hours, and renal excretion is essentially complete in 24 hours. In comparison, methazolamide is absorbed more slowly from the GI tract, and its duration of action is long, with a half-life of approximately 14 hours.

Adverse Effects

Generally, carbonic anhydrase inhibitors are well-tolerated, with infrequent serious adverse effects. Side-effects of carbonic anhydrase inhibitors may arise from the continued excretion of electrolytes. Significant hypokalemia and metabolic acidosis may develop. In elderly patients with glaucoma treated with acetazolamide (250 mg to 1000 mg/day), metabolic acidosis was a frequent finding in comparison to a control group. Acetazolamide is also associated with nephrocalcinosis and nephrolithiasis, due to its effects on urine pH facilitating stone formation. Premature infants treated with furosemide and acetazolamide are particularly susceptible to nephrocalcinosis, presumably due to the combined effect of an alkaline urine and hypercalciuria. Other adverse effects include drowsiness, fatigue, CNS depression, and parathesias. Bone marrow suppression has been reported.

Clinical Use

The popularity of carbonic anhydrase inhibitors as diuretics has waned, principally because more effective agents are available, but in specific settings these drugs remain useful. In general, tolerance to the natriuretic effects of CAI develops rapidly, and renders them relatively ineffective in treatment of edema. Daily use produces systemic acidemia from an increase in urinary excretion of bicarbonate. Nevertheless, acetazolamide can be administered for short-term therapy, usually in combination with other diuretics to patients who are resistant or who do not respond adequately to other agents. The rationale for using a combination of diuretic agents is based on the summation of their effect at different sites along the nephron.

The major indication for the use of acetazolamide as a diuretic agent is in the treatment of patients with metabolic alkalosis that is accompanied by edematous states or chronic obstructive lung disease. In patients with cirrhosis, congestive heart failure or nephrotic syndrome, aggressive diuresis with loop diuretics promotes intravascular chloride and volume-depletion, secondary hyperaldosteronism, and renal insufficiency, conditions that promote metabolic alkalosis. Administration of sodium chloride to correct the metabolic alkalosis may exacerbate edema. Acetazolamide can improve metabolic alkalosis by decreasing proximal tubule bicarbonate reabsorption and distal proton secretion, thereby increasing the fractional excretion of bicarbonate. An increase in urinary pH (>7.0) indicates enhanced bicarbonaturia. However, it should be noted that potassium-depletion should be corrected prior to acetazolamide use, as acetazolamide will increase potassium excretion. The time-course of acetazolamide effect is rapid. In critically ill patients on ventilators, following the correction of fluid and electrolyte disturbances, intravenous acetazolamide produced an initial effect within 2 hours, and a maximum effect in 15 hours.

Acetazolamide was used to treat chronic open-angle glaucoma, but its popularity has been limited by side-effects and limited efficacy. The high bicarbonate concentration in aqueous humor is carbonic anhydrase-dependent, and oral carbonic anhydrase inhibition can be used to reduce aqueous humor formation. Dorzolamide is a topical CAI that is in clinical use to treat glaucoma.

Acute mountain sickness usually occurs in sojourners who ascend to heights greater than 2500–3000 feet. Symptoms occur within the 12–72 hours, and are characterized by a symptom complex consisting of headache, nausea, dizziness, and breathlessness. Carbonic anhydrase inhibitors improve symptoms and arterial oxygenation.

The administration of acetazolamide has been used in the treatment of familial hypokalemic periodic paralysis, a disorder characterized by intermittent episodes of muscle weakness and flaccid paralysis. Its efficacy may be related to a decrease in influx of potassium as a result of a decrease in plasma insulin and glucose or to metabolic acidosis. Carbonic anhydrase inhibitors can also be used as an adjunct treatment of epilepsy, pseudotumor cerebri, and central sleep apnea.

By increasing urinary pH, acetazolamide has been used effectively in certain clinical conditions. Acetazolamide is used to treat cystine and uric acid stones by increasing their solubility in urine. Acetazolamide, in combination with sodium bicarbonate infusion, is also used to treat salicylate toxicity. Salicylates are weak acids (pK _a 3.0), therefore their ionic and nonionic forms exist in equilibrium. They are excreted primarily by the kidney through secretion via the organic anion transport pathway in the proximal tubule. Acetazolamide and sodium bicarbonate infusions increase urinary pH, thereby favoring formation of a nondiffusible nonionic form of salicylate, thus increasing excretion of salicylates.

Loop Diuretics

The loop diuretics inhibit sodium and chloride transport along the loop of Henle. Although these drugs also impair ion transport by proximal and distal tubules under some conditions, these effects probably contribute little to their action clinically. Although the predominant natriuretic effects result from blockade of NKCC2, the apical transporter of the TAL, other effects may result from blockade of NKCC1, the more widely-expressed form. The affinity of loop diuretics for the two classes of transporter appears to be similar; differential selectivity for the renal isoform during topical use is likely the result of the concentration of secreted drug within the tubule lumen, leading to a higher concentration adjacent to TAL cells. The role of NKCC1 inhibition in pharmacological actions of loop diuretics is discussed below. The loop diuretics available in the United States include furosemide, bumetanide, torsemide, and ethacrynic acid (see Figure 40.6 ). Organic mercurial diuretics also inhibit ion transport along the loop of Henle, but these drugs are of historical interest, as they are no longer available for clinical use.

Urinary Electrolyte and Water Excretion

Loop diuretics increase the excretion of water, Na, K, Cl, phosphate, magnesium, and calcium (see Table 40.1 ). The dose–response relationship between loop diuretic and urinary Na and Cl excretion is sigmoidal (see Figure 40.7 ). The steep dose–response relationship in the therapeutic range has led many to refer to these as “threshold” drugs. Loop diuretics have the highest natriuretic and chloriuretic potency of any class of diuretics; they can increase Na and Cl excretion to more than 25% of the filtered load. Following oral water-loading, the administration of a loop diuretic decreases free water clearance ( C _H2O ) and increases osmolar clearance, although the urine always remains dilute. This effect contrasts with that of osmotic diuretics, in which increases in osmolar clearance are associated with increase in C _H2O . During water deprivation, loop diuretics impair the reabsorption of solute free water ( $T_{H 2 O}^{C}$ $T_{H 2 O}^{C}$ ). Loop diuretics may induce a “negative” $T_{H 2 O}^{C}$ $T_{H 2 O}^{C}$ , even during water deprivation. During maximal loop diuretic action, the urinary Na concentration is usually between 75–100 mM. Because urinary K concentrations during furosemide-induced natriuresis remain low, the clearance of electrolyte free water ( C _H2O ) is increased when loop diuretics are administered during conditions of water diuresis or water deprivation.

Figure 40.7, Dose–response curve for loop diuretics.

Mechanisms of Action

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