A diuretic is defined as a chemical that increases the rate of urine formation. The primary action of most diuretics is the direct inhibition of Na+ transport at one or more of the four major anatomical sites along the nephron where Na+ reabsorption takes place. Because the Na+ transport systems at each of these locations are unique, there is a different set of relatively rigid structural features that a diuretic must possess to inhibit Na+ reabsorption at each site. Of additional importance are the secondary (or indirect) events that are triggered as a result of the diuretic's primary action.
The nature and magnitude of many of the observed secondary effects depend on the locus of action of the diuretic and the response of nephron sites "downstream" to an enhanced delivery of fluid Na+ or other solutes. Collectively the primary and secondary effects induced by a diuretic determine its electrolyte excretion pattern. A diuretic usually possesses some combination of natriuretic, chloruretic, saluretie, kalruretie, bicarbonaturetic, or calciuretic properties, depending on whether it enhances the renal excretion of Na + ,Cl − , Na+ / Cl− ,K + , HCO3− or Ca 2+ , respectively
Anatomy and physiology of the nephron: The functional unit of the kidney is the nephrons with its accompanying glomerulus.There are approximately a million nephrons in each kidney. The blood (or more appropriately, the plasma), from which all urine is formed, is brought to each nephron within the glomerular capillary network. Many plasma components are filtered into Bowman's space. During the process of urine formation, the resulting glomerular filtrate flows through the convoluted and straight portions of the proximal tubule, descending limb of Henle's loop, thin and thick portions of the ascending limb of Henle's loop, area of the macula densa cells, distal convoluted tubule (also referred to as the early distal tubule),
connecting tubule (also referred to as the late distal tubule), and the cortical and medullae)' collecting tubules. Each of these nephron segments consists of ultra structurally and functionally unique cell types. The physiological role of the glomerulus and each nephron segment is discussed below as it relates to the handling of important solutes and water in normally hydrated (normovolemic) and dehydrated (hypovolemic) persons and in patients afflicted with various edematous disorders (c .g. congestive heart failure, cirrhosis of the liver with ascites. and the nephrotic syndrome)
Figure 18—2: Juxtaglomerular apparatus (JGA). Urine is formed from the filtration of plasma through the glomerular capillary loops into Bowman's space. The JGA is of paramount importance for the operation of the tububoglomerular feedback mechanism, which allows a nephron to regulate the glomerular filtration rate of its own glomerulus.
Sites of sodium reabsorpion: There are four major anatomical sites along the nephron that are responsible for the bulk of Na+ reabsorption: Site 1. The convoluted and straight portions of the proximal tubule; Site 2, the thick ascending limb of Henle's loop ; Site 3, the distal convoluted tubule; and Site 4, the connecting tubule and the cortical collecting tubule.
SITE 1: The convoluted and straight portions of the proximal tubule are responsible for the reabsorption of • About 65℅ of the filtered loads of Na+, Cl−, Ca2+, and water • 80 to 90℅ of the filtered loads of HCO3−, phosphate and urate • Essentially 100% of the filtered loads of glucose, amino acids, and low-M proteins Thus, under normal circumstances, the proximal tubule has a tremendous reabsorptive capacity. There are primarily two driving forces for this high reabsorptive activity. First, because the plasma in the peritubular capillaries has a lower hydraulic pressure and a higher oncotic pressure than the luminal fluid or the
plasma delivered to the glomerulus (because of the removal of water but not protein from plasma during glomerular filtration); there is a net movement of the luminal fluid contents in a reabsorptive direction. Second, the Na+ / K+-ATPase, strategically located on the antiluminal membrane (sometimes referred to as the basolateral peritubular, or contra luminal membrane) of the proximal tubule cells, catalyzes the counter transport of intracellular Na+ into the interstitium and extra cellular K+ into the proximal tubule cells. The stoichiometry for this counter transport is 3 Na+ : 2 K +. This activity creates a deficit of intracellular Na+, a surfeit of intracellular K+, and a voltage oriented negatively inside proximal tubule cells.
In response to the action of the Na+ /K+ - ATPase, Na+ in the luminal fluid moves down the concentration gradient into proximal tubule cells by a combination of at least three distinct processes (labeled A, B. and C in Fig). The first mechanism of Na+ reabsorption at site 1 involves carbonic anhydrase (CA), which is located in the cytoplasm and on the brush border of proximal tubule cells (Fig). The H+ generated as the result of the action of intracellular CA, is exchanged (i.e., countertransported) for the filtered Na+ in the luminal fluid. The Na+ that enters proximal tubule cells during the exchange for H+ is then pumped into the interstitium by the Na+ /K+ ATPase in the antiluminal membrane.
The H+ secreted (i.e., transported uphill or against its gradient) into the luminal fluid reacts there with the filtered HCO3− to generate carbonic acid. The carbonic acid decomposes, both spontaneously and with the aid of the brush border-bound CA, to carbon dioxide and water. The carbon dioxide diffuses into the proximal tubule cells and is converted back into HCO3−, which subsequently passes from the proximal tubule cells, across the antiluminal membrane, and into the interstitium by way of a Na +/ HCO3− symporter in the antiluminal membrane. CA is very plentiful in the convoluted portion of the human proximal tubule but is nonexistent in the straight portion.
Thus, the processes just described occur primarily in the convoluted portion of the proximal tubule and account for the reabsorption of about 20 to 25℅ of the filtered load of Na+ (or about one third of the filtered load of Na+ that is reabsorbed at site 1, and about 80 to 90% of the filtered load of HCO3−, The second mechanism by which Na+ moves out of the luminal fluid at site 1 involves its cotransport into proximal tubule cells along with glucose, amino acids, or phosphate. The latter three solutes enter proximal tubule cells against their concentration gradients. The reabsorption of the Na+ that enters proximal tubular cells by these processes is completed when it is subsequently pumped into the interstitium by the antiluminal membrane-bound Na+ /H+ ATPase and then passes into the adjacent peritubular capillaries.
<ul><li>Figure 18—3: Site 1: The Na+ transport systems responsible for the reabsorption of Na+ and associated solutes in the proximal tubule. </li></ul><ul><li>Transcellular reabsorption of Na which is controlled by carbonic anhydrase (CA). Acetazolamide and other CA inhibitors block Na+ reabsorption by this route. </li></ul><ul><li>B. Transcellular reabsorption of Na+ coupled to glucose, amino acids, and phosphate </li></ul><ul><li>C. Paracellular transport of Na+/ Cl). No commercially available agents inhibit Na+ reabsorption by routes B or C. Na+/K+ -ATPase is indicated by filled circles on the antiluminal membrane. </li></ul>
FIgure 18—4: Site 2: The Na transport systems responsible for the reabsorption of Na+ and associated solutes in the water impermeable cortical and medullary portions of the thick ascending limb of Henle's loop. The collective actions of the antiluminal membrane— bound Na+ /K+ -ATPase and the luminal membrane—bound 1 Na+ /1 K+ /2 Cl- cotransport system account for transcellular reabsorption of Na+ /CI-, in a Na+ /Cl- ratio of 3:6. and the generation of a lumen-positive potential that drives the reabsorption of Na+ and other cations via the paracellular pathway (dashed line). Diuretic agents that block Na+ reabsorption in the thick ascending limb by inhibition of the luminal membrane—bound 1 Na+/1 K+ / 2Cl- cotransport system include furosemide, bumetanide, torsemide, ethacrynic acid and a number of miscellaneous agents.
SITE 3: Following its sojourn past the macula densa cells, the luminal fluid comes into contact with the third major site for the reabsorption of Na+, the relatively short, water-impermeab1e, distal convoluted tubule. Again, the major driving force for Na+ reabsorption from the luminal fluid at site 3 involves the deficit of intracellular Na+ produced by the action of the antiluminal membrane-bound Na+ / K+ ATPase. In this instance, the luminal membrane-bound Na+/ Cl- cotransport system moves luminal fluid Na+ downhill and luminal fluid Cl- uphill into distal convoluted tubule cells.
The reabsorption of Na+ is completed when the antiluminal membrane -hound Na+ /K+ ATPase actively pumps it into the interstitium with subsequent passage into the surrounding vasculature: intracellular Cl- enters the interstitium through channels in the antiluminal membrane. Approximately 5 to 8% of the filtered load of Na+ is reabsorbed at site 3.
Figure 18—5, Site 3: The Na+ transport systems responsible for the reabsorption of Na+ and Cl- in the water-impermeable distal convoluted tubule. Inhibitors of the luminal membrane— bound Na+/Cl- cotransport system include the thiazide and thiazide-like diuretics.
SITE 4: The connecting tubule (i.e., late distal tubule) and the cortical collecting tubule house the fourth and final major site for the reabsorption of Na+ from the luminal fluid. This portion of the nephron is composed of two distinct cell types, the principal cells and the intercalated cells. The principal cells are important for Na+ reabsorption and K+ secretion, whereas the intercalated cells (subtype AI are important for the generation and secretion of H+ the intercalated cells possess only small quantities of the Na+ / K+ ATPase on their antiluminal membranes, but they contain abundant quantities of intracellular CA, which catalyzes the formation of carbonic acid from CO2 and water.
The carbonic acid ionizes, yielding H+ and HCO3−. The H+ is then pumped actively into the luminal fluid by the luminal membrane-bound H+ -ATPase. The driving force for the reabsorption of Na+ in the principal cells is once again the deficit of intracellular Na+ created by the Na+ /K+ ATPase on the antiluminal membrane, which counter transports 3 Na+ uphill from the principal cells into the interstitium and 2 K+ uphill from the interstitium into the principal cells. In response to the deficit of Na+ in the principal cells, the Na+ in the luminal fluid moves downhill into the principal cells through Na+ channels in the luminal membrane and is subsequently pumped actively into the interstitium by the antiluminal membrane-hound Na+ / K+ -ATPase. These events create a lumen-negative transepithelial voltage.
In response to this voltage difference, some combination of the following three processes occurs • Cl- moves paraccllularly from the lumen into the Interstitium • K+ in the principal cells moves downhill into the luminal fluid through K+ channels in the luminal membrane • H+ generated in the intercalated cells moves into the luminal fluid by way of the H+ ATPase Because the latter two processes predominate, one may view the activities at site 4 as an exchange of luminal fluid Na+ for principal cell K+ and intercalated cell H+.
The exchange of luminal fluid Na+ for intracellular H+ or K+ normally is associated with the reabsorption of only 2 to 3% of the filtered load of Na+ and the distal location of this exchange system dictates the final acidity and K+ content of the urine. The amount of Na+ reabsorbed at site 4 and, therefore, the amount of H+ and K+ present in the final urine are modulated by • Plasma and renal levels of mineralocorticoids like aldosterone the higher the levels of circulating aldosterone. The greater the Na+ reabsorptiom and K+ and H+ excretion • Luminal fluid flow rate and the percentage of the filtered load of Na+ presented to the exchange sites-the greater the flow and the load of Na+, the greater the amount of exchange • Acid-base status of the individual-acidosis favors exchange of Na+ and H+ . Whereas alkalosis favors exchange of Na+ and K+
The classes of diuretics that inhibit the reabsorption of Na+ at sites 1, 2.or 3 (i.e., sites proximal to site 4) ultimately increase, to varying degrees, the luminal fluid flow rate and the percentage of the filtered load of Na+ delivered to site 4. Thus, many diuretics acutely enhance the urinary loss of K+ and may he associated with the induction of hypokalctnia (i.e. abnormally low levels of K+ in the circulating blood)
FIgure 18—6, Site 4: The Na+ transport systems responsible for the reabsorption of Na+ in the connecting and corlicat collecting tubules. Na reabsorption and K+ secretion take over in the principal cells; H+ formation and secretion occur in intercalated cells. Spironolactone inhibits Na+ competitively antagonizing the effects of aldosterone on principal cells. Triamterene and amiloride "plug" the Na+ channels in the luminal membrane of the principal cells, then: preventing Na+ reabsorption and K+ and H+ secretion. Those while producing a modest natriuresis, these drugs prevent the loss of K+ and are commonly referred to as K -sparing diuretic
Site 1 Diuretic: Carbonic Anhydrase Inhibitor: Although the available CA inhibitors are used infrequently as diuretics, they not only played an important role in the development of other major classes of diuretics that are currently in widespread use but also aided in our understanding of basic renal physiology. Shortly after its introduction for the treatment of bacterial infections, sulfanilamide was observed to produce a mild diuresis characterized by the presence of urinary Na+ and a substantial amount of HCO3−. It was subsequently shown that it induced this effect through inhibition of renal CA. However, it was a relatively weak inhibitor of renal CA, and the dose needed to exert adequate diuresis was associated with severe adverse effects.
To improve on the CA-inhibitory property of sulfanilamide, many sulfamoyl containing compounds (SO2.NH2,) were synthesized and screened for their diuretic cavity in vivo and their ability to inhibit CA in vitro. Two pups of CA inhibitors emerged:simple heterocyclic sulfonamides and into meta-disulfamoylbenzene derivatives.
STRUCTURE-ACTIVITY RELATIONSHIPS 1. SAR studies involving the simple heterocyclic sulfonamides yielded the prototypic CA inhibitor, acetazolamide. 2. The sulfamoyl group is essential for in vitro CA inhibitory activity and for diuresis production in vivo. 3. The sulfamoyl nitrogen atom must remain unsubstituted to retain both in vivo and in vitro activities. This feature explains why all antibacterial sulfonamides except sulfanilamide are capable of inhibiting CA or exerting diuresis. 4. In contrast, substitution of a methyl group on one of acetazolamide’s ring nitrogen yields methazolamide, a product that retains CA inhibitor activity. 5. The moiety to which the sulfamoyl group is attached must possess aromatic character.
6. In addition, within a given series of heterocyclic sulfonamides, the derivatives with the highest lipid/water partition coefficients and the lowest pKa values have the greatest CA inhibitory and diuretic activities. The SAR studies involving the meta- disulfamoylbenzenes revealed that the parent 1, 3 disulfamoylbenzene lacked diuretic activity, but key substitutions led to compounds with diuretic activity. The first commercially available analogue, dichlorphenamide, is similar to acctazolamide in its CA-inhibitory activity, but it is also a chloruretic agent. Subsequently, chloraminophenamide when given by the intravenous route was shown to possess less CA-inhibitory activity but more chloruretic activity. Poor diuretic activity following the oral administration of chloraminophenamide precluded its marketing.
SITE AND MECHANISM OF ACTION: CA is located both intracellularly (type II CA) and in the luminal brush border membrane (type IV CA) of proximal convoluted tubule cells. Both of these site locations arc major targets of the CA inhibitors. This group of diuretics also inhibits intracellular CA in the intercalate cells of the connecting and cortical collecting tubules (i.e. site 4).During the first 4 to 7 days of continuous therapy with CA inhibitor, several noteworthy events occur that lead to an increase in Na+ and HCO3−, excretion: (a) inhibition of the intracellular CA in proximal tubule cells decreases the available H+ normally exchanged for luminal fluid Na+, thus decreasing proximal tubule reabsorption of Na+: and
(b) inhibition of CA on the luminal brush border membrane of proximal tubule cells causes a decrease in the production of carbon dioxide within the luminal fluid and a decrease in the proximal tubule uptake of carbon dioxide. The net result is a decrease in the reabsorption of HCO3−. One might assume that a massive diuresis would follow inhibition of the portion of proximal tubule Na+: reabsorption under the control of CA (i.e., one third of the 65% of the filtered load of Na+: normally reabsorbed from the proximal luminal fluid, or about 22c% of the filtered load of Na+).
However, Na+ reabsorption sites downstream (especially site 2) compensate for such an action by reabsorbing much of the additional Na+ presented to them. Some of the luminal fluid HCO3− is reabsorbed downstream by a non-CA-mediated system. Thus, the actions of the CA inhibitors ultimately result in the urinary loss of only 2 to5% of the filtered load of Na+ and up to 30% of the filtered load of HCO3−.Secondarily, the CA inhibitors enhance the urinary excretion of a substantial amount of K+ .The urinary loss of K+ increases because the proximal tubule actions of CA inhibitors present a greater percentage of the filtered load of Na+ to site 4, increase the flow rate of luminal fluid through the distal convoluted tubule and collecting tubule, and decrease the availability of intracellular H+ at site
4.All three changes favor enhanced exchange of luminal fluid Na+ for intracellular K+ at site 4. The urinary concentration of Cl- actually decreases after the administration of CA inhibitors. Hence, CA inhibitors are primarily natriuretic bicarbonaturetic, and kaliuretic agents. Toward the end of the first week of continuous therapy with a CA inhibitor, resistance develops to its diuretic effect .This is primarily due to two factors. First, there is marked reduction in the filtered load of, HCO3− because CA inhibitors Produce both a 20% reduction in the GFR via the tuhuloglomerular feedback mechanism, and a reduction in the plasma concentration of HCO3−. When there is less HCO3− present in the luminal fluid. There is less HCO3− reabsorption to inhibit. Second, the metabolic acidosis created by these diuretics pr video a sufficient amount non-CA-generated intracellular H+ to exchange from the luminal fluid Na+. The Na+ reabsorption at site I progressively return to a near-normal rate, and diuresis wanes.
Use: The major use of the CA inhibitors is in the treatment of glaucoma. CA is a functionally important enzyme in the eye where it plays a key role in the formation of aqueous humor. Inhibition of this ocular enzyme reduces the rate of formation of the aqueous humor, thereby reducing the intraocular pressure associated with glaucoma. Interestingly a reduction in the intraocular pressure usually persists at a time when resistance has developed to the renal effects of the CA inhibitors. CA inhibitors have been used prophylactically to counteract acute mountain sickness, to act as adjuvant for the treatment of epilepsy, and to create alkaline urine in an attempt to hasten the renal excretion of certain noxious weak acids or to maintain the urinary solubility of certain poorly water soluble, endogenous weak acids.