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J Clin Pathol: first published as 10.1136/jcp.34.11.1267 on 1 November 1981. Downloaded from J Clin Pathol 1981;34:1267-1275

Modern and the

AF LANT From the Department of Therapeutics, Westminster Medical School, London SWIP 2AP

It is particularly appropriate to discuss the pharma- THE DISTRIBUTION OF GLOMERULAR cology and mechanisms of action of drugs FILTRATION BETWEEN OUTER CORTICAL in a symposium on diseases of the kidney, since AND JUXTAMEDULLARY diuretics were the first group of drugs to be devel- The functional unit of each kidney comprises oped for the control and manipulation of selected approximately one million nephrons which lack aspects of renal function. Despite a relatively short homogeneity in their structure. About 80% of history of less than three decades since the discovery them are in the outer cortex, have short loops of of the first orally acting diuretic, , Henle, and have relatively low reabsorptive capacity modern diuretic therapy continues to enjoy an ever- for . The remaining 20% are juxtamedullary, expanding clinical demand: worldwide expenditure possessing long loops of Henle, and are largely has been estimated at around US $800 million responsible for creating the hyperosmotic inter- per annum during the last five years. Though stitium in the medulla which meditates the process originally introduced for the treatment of oedema- of concentration. Redistribution of blood flow tous conditions it is interesting that application of from outer cortical to juxtamedullary nephrons diuretics to the treatment of has out- can contribute to abnormal sodium retention, stripped their use in oedema. This has happened whilst predominance of the effect of outer cortical notwithstanding the parallel discoveries of other nephrons may lead to saluresis. A reduction in blood copyright. major antihypertensive agents such as the beta- flow to the outer part of the cortex has been found adrenoceptor blocking drugs and powerful vaso- to occur in some sodium-retaining states. Conversely, dilators. a drug which could shift blood flow from juxta- The advent of novel, orally effective diuretics medullary to outer cortical nephrons would reduce following on the prototype , chloro- sodium reabsorption and result in an effective , has had a major influence in stimulating diuretic response. progess in the basic sciences relating to nephrology or transport in body tissues in general.

HAEMODYNAMIC AND PHYSICAL FACTORS3 http://jcp.bmj.com/ Advances in fundamental knowledge have in turn Changes in physical forces within the peritubular given the impetus to further discoveries of new and, capillaries have been shown to be important de- in some cases, unique substances possessing diuretic terminants of renal sodium reabsorption, especially activity. Byand large these compounds have attained a in the . Thus, for example, renal remarkable degree of sophistication while, at the arteriolar dilatation may, by increasing the hydro- same time, remaining relatively safe during long static pressure in the vasa recta, decrease net tubular term treatment of patients. The widespread use and, reabsorption of sodium with resulting natriuresis, at times, the regrettable abuse of any group of on October 1, 2021 by guest. Protected drugs whose primary effect is directed toward while the reverse occurs with any increase of plasma interference with the renal handling of oncotic pressure. It seems probable that changes must inevitably generate secondary disturbances in in the oncotic or hydrostatic pressures within the body homeostasis which have particular relevance peritubular blood vessels achieve their effects on to the chemical pathologist. To understand these, net tubular reabsorption of sodium by influencing demands an understanding of how diuretics work the resistance of the intercellular channels or "shunt" in the context of modern views of the functional paths through which sodium ions pass to reach the organisation of the kidney. peritubular capillaries.

Renal regulation of sodium excretionl 2 HORMONAL FACTORS A number of hormonal mechanisms operate singly Four main mechanisms are believed to be involved or together in encouraging sodium retention by the in the control of sodium by the kidney. kidney. 1267 J Clin Pathol: first published as 10.1136/jcp.34.11.1267 on 1 November 1981. Downloaded from

1268 Lant

Renin-angiotensin-aldosterone4 5 FEEDBACK CONTROL SYSTEMS INVOLVING The role of this humoral system in renal sodium THE MACULA DENSA9 regulation is considerable. Yet, because of the slug- A fourth intrarenal regulating system which has gish characteristics of the system, it is unlikely been proposed is that of a servo-mechanism operat- that these hormones, especially aldosterone, are ing between the macula densa cells of the ascending involved in fine "moment to moment" modulation. limb of Henle's loop and the glomerulus of the Aldosterone influences epithelial transport of sodium same . This system functions by the release through activation of DNA-dependent RNA syn- of renin and angiotensin It locally in response to thesis, and the response to it follows a significant the sodium concentration in the tubular fluid lag period which corresponds to the time needed for impinging on the macula densa, the feedback the induction of new protein synthesis; this may loop being completed by appropriate alterations in amount to an hour or more in experimental systems. both GFR and proximal tubular reabsorption. The Yet it is clear that changes in sodium excretion detailed mechanisms involved in such an auto- in vivo may be induced abruptly by various ma- regulatory system which couples distal salt delivery noeuvres. Moreover, whenexcessive amounts ofaldos- to the filtration rate in individual nephrons remain terone or other mineralocorticoids are given experi- incompletely understood. mentally sodium retention occurs, but is transient. The "escape" from the action of aldosterone cannot Organisation of tubular function'° 11 be accounted for by changes in glomerular filtration rate or renal blood flow, and it is one of the pieces Diuretic drugs have actions on ion-transporting of evidence supporting the presence of another tissues as diverse as amphibian skin, intestinal humoral substance stimulating natriuresis-called epithelium, red and white blood cells and cornea. Third Factor or natriuretic hormone. The primary target for the action of these drugs however is the kidney, where they promote the Natriuretic hormone6 excretion of water and certain electrolytes such as

sodium and chloride by interfering with tubularcopyright. A growing body of evidence supports the existence reasorptive mechanisms. Because these reabsorptive of a natriuretic humoral agent which promotes mechanisms vary according to the degree of sophisti- the renal elimination of sodium, by inhibiting sodium cation of different portions of the epithelium lining reabsorption in the proximal tubule. It may also the tubule, a brief survey of the organisation of work in harmony with the renin-angiotensin- is to understanding aldosterone system to provide fine control of tubular functions relevant sodium excretion by an action exerted mainly on diuretic action (Fig. 1). the collecting ducts. The chemical characterisation of the natriuretic hormone remains incomplete GLOMERULUS AND PROXIMAL TUBULE

In normal man each day the renal glomeruli producehttp://jcp.bmj.com/ and its role in the sodium-retaining states is also approximately 180 litres of filtrate, and urine is uncertain. finally produced by the progressive reabsorption of 99% of this ultrafiltrate at various stages along Prostaglandins and kinins7 8 the nephron. About two-thirds of the glomerular Many experimental studies have demonstrated filtrate is reabsorbed iso-osmotically in the proximal the ability of renal tissues to generate prostaglandins tubule as a result of the active reabsorption of and kinins. Whereas renin, angiotensin and aldos- and sodium bicarbonate from the terone, like prostacyclin, are transported primarily tubular lumen into the peritubular fluid. The on October 1, 2021 by guest. Protected in the vascular compartment, kallikrein-kinin and mechanisms involved in transcellular ion movement prostaglandins of the E series are associated with are complex and involve a variety of energy- the renal interstitium and tubular lumen. One or dependent ion pumps as well as transfer paths or more prostaglandins, primarily PGE2, is probably channels in between the loose-fitting cells of the responsible for mediating the increase in medullary proximal tubule. The resistance to these intercellular blood flow that occurs in response to various stimuli shunts of ions is influenced considerably by changes including surgical trauma, salt loading, and the in the oncotic and hydrostatic pressures within the action of "loop" diuretics. The generation of peritubular capillaries. kinins in the distal tubules and collecting ducts results in the release of prostaglandins which in ASCENDING LIMB OF HENLE'S LOOP turn inhibit the local effects of antidiuretic hormone (MEDULLARY DILUTING SEGMENT)1213 (ADH) and thereby contribute to the tubular There are two morphologically distinct kinds of excretion of solute-free water. nephron, namely the outer cortical nephrons, J Clin Pathol: first published as 10.1136/jcp.34.11.1267 on 1 November 1981. Downloaded from

Modern diuretics and the kidney 1269 which are the more plentiful and have short loops of tubular microperfusion have shown that the of Henle, and the juxtamedullary nephrons which transepithelial voltage in this part of the nephron have long loops plunging down into the inner parts is positive within the lumen and that chloride ion of the medulla. The suggestion that loops of Henle is absorbed against an electrochemical gradient. might act as a countercurrent multiplier system Ion substitution studies have lent support to the was first proposed in 1942 by Kuhn and Ryffel,14 view that active chloride transport is the pri- but only received widespread attention in 1951 mary event and that sodium moves secondarily after Wirz, et a115 showed a striking osmotic gradient in this part of the nephron. Electrogenic sodium in the renal interstitium increasing from cortex reabsorption from the tubular fluid may coexist to papilla. In the ascending limb of the loop of with a separate neutral NaCI path (see later). Henle two anatomically distinct portions can be Hypertonicity of the inner medulla is the result distinguished, both of which are concerned with of combined functions of the hairpin structure of urinary dilution, namely a medullary portion the long loops of Henle acting as counter current lined by cuboidal cells and a cortical portion lined multiplier systems and of their associated vasa recta by flattened cells. The tubular epithelium of both acting as counter current exchange systems. It portions is insensitive to ADH and is thus relatively has been proposed that recycling of between the impermeable to water but the ascending limb does two limbs of the vasa recta and between the loop possess the ability to reabsorb salt actively. Because, of Henle and the collecting duct contributes to the in mammals, the diluting segments of the loop of hypertonicity of the inner medulla on which concen- Henle do not reach the surface of the kidney, tration of the urine depends. In the presence of their functional characteristics have had to be ADH16 the collecting ducts, which traverse the deduced from micropuncture of distal convoluted hypertonic medullary interstitium on their way to tubules. Recent experiments employing techniques the renal pelvis, become permeable to water PROXIMAL DISTAL

SiteIV copyright.

-____ NQv

Aldosterone A

NcCI http://jcp.bmj.com/ II3TCH20NaCl

ASCENDING LIMB on October 1, 2021 by guest. Protected (water impermeable)

Fig. I A diagram of the nephron showing the four tubular sites where diuretics act to block sodium chloride reabsorption. Urinary dilution occurs as a result ofsodium chloride reabsorption at two water-impermeable sites, the ascending limb ofHenle's loop (site MI; medullary diluting site) and the early part of the distal tubule (site II; cortical diluting site). During water diuresis, total urine volume can be divided into two moieties, the volume of urine required to excrete urinary solutes at plasma tonicity-that is, the osmolal clearance Cosm, and the volume of solute-free water generated at diluting sites II and III-that is, the solute-free water clearance CH,O. When fluid intake is restricted and hypertonic urine is formed, CH2O becomes negative and is referred to as TCH2o. TCH2O reflects the passive reabsorption into the hypertonic medulla ofsolute-free water from the collecting ducts, stimulated by A DH (see footnote on page 1270). J Clin Pathol: first published as 10.1136/jcp.34.11.1267 on 1 November 1981. Downloaded from

1270 Lant Progressive extraction of solute-free water from the luminal and peritubular membranes. Distal tubular collecting ducts into the hypertonic medulla renders secretion is largely passive and follows the residual tubular urine hypertonic. a net electrochemical gradient generated by the active uptake of sodium, and probably also of CORTICAL DILUTING SEGMENT potassium, from the luminal border of the cell. As the diluting segment of the The luminal membrane possesses an active secretory ascends out of the medulla to reach the cortex, pump for hydrogen ions which is fed by the intra- continued reabsorption of sodium without water cellular hydrogen ion pool. This pool is replenished further dilutes the tubular fluid and enables a dilute by several processes working in parallel, including urine to be excreted in the absence of ADH. The catalysed and uncatalysed hydration of C02 within sodium that is reabsorbed in the cortex does not the cell, backdiffusion of CO2 from the lumen contribute to medullary hypertonicity, so that, and uptake of hydrogen ions from the peritubular whereas a reduction of sodium transport in this space. Any diuretic acting proximal to the aldoster- cortical segment will be reflected in a reduction of one-sensitive ion-exchange sites causes an increased solute-free water clearance CH2o,* it will not reduce delivery of sodium to the distal tubule and thereby solute-free water reabsorption TCH2O,* by the col- leads to an increase in urinary loss of potassium. lecting duct which depends on the hypertonicity Thelining epitheliumofthelater part of the distal of the medulla. tubule gradually merges with that of the cortical part of the collecting duct where ADH-responsive- AND ness becomes a feature. The latter mechanism COLLECTING DUCT involves a stereospecific receptor at the basal or Sodium reabsorption in this part of the nephron blood side of the tubular cell, a modulator which is also an active process and may be accompanied receives positive or negative signals-for example, by chloride reabsorption or alternatively coupled PGE1, to be passed on to the catalytic step, phospho- to potassium and hydrogen ion secretion. The diesterase, which converts cyclic AMP to 5'AMP.

activity of these distal tubular exchange mechanisms The cyclic nucleotide diffuses through the cyto-copyright. is controlled to a large extent by aldosterone, whose plasm to reach the effector site-the luminal plasma modulating action involves the synthesis of mineralo- membrane. corticoid-induced proteins capable of stimulating the sodium pump located at the peritubular border Sites of action of diuretic drugs in the nephron17 18 19 of distal tubular cells. Permeability to sodium at the luminal border of these cells and those of the Many techniques have been employed to localise cortical collecting ducts may also be increased. the sites of action of diuretics in the nephron. The trans-tubular electrical potential difference These include various in vitro approaches such as in the distal tubule is negative within the lumen and stop-flow, free-flow micropuncture, autoradiographyhttp://jcp.bmj.com/ largely generated by the active reabsorption of and single nephron microperfusion. The use of sodium. It amounts to about -10 mV in the early different species has made it difficult to interpret part ofthe tubule and reaches -45 mV more distally; the results of such experiments and evidence from it is increased further by the presence of poorly in vitro investigation may not be directly applica- reabsorbable anions within the tubule. ble to man. Understandably in vitro methods have In common with most other cells distal tubular been much more limited in scope but have neverthe- cells are rich in potassium, and a chemical gradient less provided valuable evidence on the possible exists for diffusion out of the cells across both the sites of action in the human nephron. on October 1, 2021 by guest. Protected If it is recalled that of total sodium reabsorption *The free water clearance, CH,O, is defined as the volume of in the nephron, approximately 65% takes place in urine excreted in excess of that required to excrete the solute the proximal tubule, 25% in the loop of Henle, iso-osmotically with plasma. If the urinary solute concentra- 8-9% in the distal tubule and the remaining 1-2% tion and volume are Uosm and V respectively, the total in the collecting duct, the maximal natriuretic solute content = Uosm x V, and the volume required to excrete this iso-osmotically is response to a diuretic can give a clue to its site of x V action (Table). Similarly the pattern of excretion Uosm = the osmolal clearance Cosm. of anions and cations evoked by diuretics can be POSM related to the known .CH. =O V- Cosm functional characteristics If there is net reabsorption of solute-free water, CH,O is of specific portions of the tubule. negative; it is therefore expressed as Probably the most informative in vivo technique TcH,o = -CH,o = Cosm - V has been the study of diuretic effects on the mech- (Eds) J Clin Pathol: first published as 10.1136/jcp.34.11.1267 on 1 November 1981. Downloaded from Modern diuretics and the kidney 1271 Table Classification ofdiuretics reabsorption, a metabolic acidosis is engendered which has been found to limit the diuretic effect. Group Predominant site of action* There are other reasons why direct effects of diuretic drugs on the proximal tubule may be obscured or High efficacy diuretics ( > 15%) Organomercurials Medullary diluting even annulled. When proximal tubular reabsorption Ethacrynic acid segment of sodium chloride is blocked, compensatory Frusemide () (site 11) Humetanide increases in reabsorption further down the nephron may follow so that little additional saluresis occurs in the final urine. The reserve reabsorptive capacity Medium efficacy diuretics (5-10%) can Chlorothiazide and Thia7ide Family Cortical diluting of the diluting segments is considerable and segment overshadow effects occurring in more proximal (site III) parts of the nephron. By contrast, a diuretic having Phthalimidines Chlorthalidone, a primary effect on the medullary diluting segment may lead to the excretion of a substantial amount of excess salt. The main reason for this is the limited Ilenzenesulphonamides reabsorptive capacity for salt of the distal tubule Chlorobenzamides and collecting duct. The latter explains why diuretics acting primarily in the distal tubule such Salicylamides as the potassium-sparing compounds, evoke only at to Weak or adjunct diuretics (< 5 %) a modest saluretic effect amounting, most, Xanthines Glomerular only 5 % of the filtered load of sodium (see Table). Aminophylline arteriolar dilatation Carbonic anhydrase inhibitors Proximal tubule Subcellular mechanisms of diuretic drug action'9 20 (site I) Osmotic agents Proximal tubule (site I) With the exception ofcarbonic anhydrase inhibitors, Potassium-sparing compounds the biochemical basis for the action of diuretics (a) Aldosterone antagonists Distal There is no doubt that the copyright. and canrenoate Na+/K+H+ is still largely unknown. (b) Pteridines and pyrazine- Exchange availability of diuretically active substances has carboxamides- and (site IV) stimulated considerable interest in the physiology and biochemistry of nephron function and has Numbers in parentheses are the maximal fractional excretion of permitted the characterisation of intricacies of sodium (sodium excretion expressed as a percentage of the sodium been un- filtered). tubular function which had hitherto *In certain instances, experimental evidence has been adduced for recognised. Such studies have also emphasised, secondary sites of diuretic action additional to the major locus within however, the unique anatomical and functional the nephron-for example, frusemide and piretanide may also exert relations within the kidney which make it such a some effects upon the proximal site I, as may the thiazide-like com- http://jcp.bmj.com/ pound, metolazone. difficult organ to investigate experimentally. Because tubular epithelium shares certain trans- anisms for urinary concentration and dilution. The port characteristics with other cellular systems, results of such investigations have permitted in- simpler tissues have been utilised to study the ferences to be drawn on the inhibitory effects of mechanisms of diuretic action with a view to various diuretics on electrolyte transport within explaining the latter in biochemical terms. This is the medullary and cortical diluting segments of the the reason why such an assortment of non-renal loop of Henle. Four main tubular sites can be tissues as-for example, amphibian skin, urinary on October 1, 2021 by guest. Protected identified as of importance in the action of diuretics bladder, blastocyst and corneal membranes have in the kidney (Fig. 1). been used. Whilst undoubted parallels can be drawn In view of the substantial reabsorptive capacity from the findings in such diverse tissues, it is of the proximal tubule, it might at first sight appear important that direct extrapolation to the function to be useful to produce diuretics whose main locus of the intact human kidney be made with caution. of action lay proximally. However, those agents What may be demonstrable in isolated tissues in which do seem to work in the proximal tubule one species may be drastically altered by the many are either too weak to be effective alone-for extra- and intrarenal mechanisms known to be example, inhibitors of carbonic anhydrase, or operative in man. Despite these criticisms, the use inconvenient, because of the need for intravenous of diuretics as "membrane probes" has yielded administration-forexample, mannitol. Furthermore, fascinating insights into some of the mechanisms a major disadvantage of carbonic anhydrase block- whereby solutes are transferred across cell mem- ade is that by inhibiting proximal bicarbonate branes. Numerous questions about the nature of J Clin Pathol: first published as 10.1136/jcp.34.11.1267 on 1 November 1981. Downloaded from

1272 the "cell receptors" for diuretic drugs remain CH-CH unanswered and await further exploration. 1 NH-(CH CLCCH2-CNH-, C CH CH3 STRUCTURE :ACTIVITY RELATIONS21 22 ~~0~~~ The history of the evolution of diuretic drugs reveals H2N 02S COOH an interesting sequence based on chance observa- H2NB02 d COOH tions of unwanted effects. Although the diuretic Frusemide effects of mercury compounds were known to Fig. 3 Comparison of two sulphamoyl loop diuretics, Paracelsus in the sixteenth century, it was Vogl frusemide and bumetanide. Both are 5-sulphamoyl- who, as a medical student in Vienna in 1921, carboxylic acids, but the change from the 4-chloro observed diuresis in a patient being treated with an substituent in frusemide to a phenoxy group, as in organo-mercurial for syphilis and paved the way bumetanide, results in a substantial increase in diuretic for the development of mercurial diuretics. By the potency on a weight for weight basis. early 1940s, it was known that various phenyl- and heterocyclic- substituted sulphonamides in- hibited carbonic anhydrase and caused a natriuresis especially straightforward in the case of the sul- with the passage of alkaline urine but at the expense phamoyl radical (SO2-NH2). The converse is the of inducing a metabolic acidosis. A major milestone case. Without doubt, the target enzyme for drugs in the development of the sulphonamide diuretics like acetazolamide or dichlorphenamide is carbonic was the discovery of chlorothiazide in 1957. Subse- anhydrase, but precisely how the natriuretic effects quent molecular manipulation of the hydrogenated of carbonic anhydrase inhibition are achieved is benzothiadiazine structure yielded many different not clear. The evidence for involvement of a bi- thiazide and thiazide-like agents with progressively carbonate-activated ATPase coupled to sodium dwindling capacity to inhibit carbonic anhydrase transport remains uncertain. Although all thiazide (Fig. 2). Then, by opening the thiadiazine ring, and and thiazide-like diuretics possess a free sulphamoyl group, the hydrogenated derivatives which followed chlorothiazide have negligible carbonic anhydrasecopyright. HAcC HN sS.O/S02N inhibitory capacities yet are potent saluretic agents. 0 II To date, no consistent biochemical mechanism of N-N action has been worked out for this large group of Acetazolamide diuretics. When high ceiling compounds such as Rs }6#fCH-R3NH,3 frusemide and ethacrynic acid are compared in terms of their inhibitory effects on respiration and H2N-02S 2 glycolysis in renal tissue it is again disappointing Thiazide group to find that natriuretic activity does not necessarily Fig. 2 Structural relation between a classical carbonic correlate with metabolic effects. For example, the http://jcp.bmj.com/ anhydrase inhibitor, such as acetazolamide, and the] concentration of diuretic needed to inhibit glycolysis benzothiadiazine (thiazide) group ofdiuretics. The in vitro may be higher than that which could be three main positions for substitution in the thiazide achieved in vivo within the renal tubular cells. molecule are depicted as R2, R3 and R6, the latter a or invariably consisting of halogen (eg, Cl) RENAL ION PUMPS AND DIURETIC ACTION1018 trifluoromethyl (eg, CF3) radical. An impressive body of evidence now exists to show that the Na, K-ATPase of most cells represents on October 1, 2021 by guest. Protected exploring derivatives of anthranilic acid, frusemide or is an integral part of the sodium pump. The emerged in the early 1960s (Fig. 3). The discovery enzyme is found in high concentrations in the kidney of ethacrynic acid, on the other hand, followed a and especially in the ascending limb of the loop of quite separate avenue of research, in that it rep- Henle. Not surprisingly, Na, K-ATPase has been resented an orally effective "high ceiling" diuretic* considered a potential candidate as the cell "re- which bound to sulphydryl groups in renal tissue ceptor" for diuretics, especially high ceiling "loop" but did not contain any mercury. compounds. Yet a number of important observations It might, at first sight, appear that the biochemical conflict with such a view. Although loop diuretics mechanisms of actions of diuretics would be inhibit glycoside-sensitive Na, K-ATPase, they also inhibit that part of sodium transport that persists *The term "high ceiling" is applied to diuretics which act even in the presence of full doses of the cardiac on the loop of Henle because the dose/response curves continue to rise, even with very large doses, unlike the glycoside, ouabain. The diuretic effects of "loop" esponse to , which reaches a plateau. compounds has been ascribed to blockade of active J Clin Pathol: first published as 10.1136/jcp.34.11.1267 on 1 November 1981. Downloaded from

Modern diuretics and the kidney 1273 24l chloride reabsorption in the medullary diluting Amiloride 12jug segment, an action shared with organomercurials. wash with Ringer I The driving force for active chloride transport in (x2) renal tissue has not been fully defined nor has the 20 relation of Na, K-ATPase or other Mg-dependent ATPases to chloride as opposed to sodium transport. 0. Percentage A cyclic AMP-activated Na, K cotransport system change in -20 has been described in avian erythrocytes which is short circuit inhibited by loop compounds, whilst a chloride current -40* self-exchange system in human erythrocytes has also been found to be inhibited by these diuretics. -60 The relation between these transport systems in human red cells and the renal tubule remains to be -80 clarified.23 24 -luu It is of considerable interest to note that about 0 10 20 30 40 50 60 7fl 80 thirty years ago in the era when organomercurial Time ( min) diuretics reigned supreme, it was proposed that Fig. 4 Effects of 12 and 24 pzg Amiloride (8 and these compounds worked primarily by inhibiting 16 x 10-7M) added to the Ringer solution bathing the chloride transport. This conclusion was encompassed external surface of isolatedfrog skin. The immediate fall in short circuit current is readily reversed by washing in the view that "the relationship to plasma chloride the skin surface with fresh Ringer solution (based on is the significant one, and that if mercury specifically Baba et a128). blocks one ion absorptive mechanism, that mech- anism is the one for chloride absorption. Increased sodium excretion following mercurial diuretics is aldosterone made it likely that diuretic effects are thus a more or less passive consequence of increased mediated by competitive binding to the mineralo-

chloride elimination."25 In retrospect, clearly the corticoid-sensitive binding protein in cells located copyright. major role of chloride transport in the action of within the distal convoluted and cortical collecting diuretics has been overshadowed over the years. tubules. Triamterene and amiloride act by blocking Yet, it remains likely that the "uphill" reabsorptive the passive entry of sodium into transporting cells in transport of chloride in the loop of Henle may rep- these same regions of the nephron. The evidence resent a transport process tied to the Na, K-ATPase. for this has been obtained for example in experi- This means that translocation of chloride across ments where diuretics have been added to the outside membranes occurs by utilising the energy of the surface offrog skin (Fig. 4) or the epithelial surface of sodium gradient. This would still imply a degree of the apex of the toad bladder. Triamterene and amilo-

primacy in the operation of the electrogenic sodium ride are unique among diuretics in blocking sodium http://jcp.bmj.com/ pump.26 Vanadate occurs naturally in cells throughout entry mechanisms in a rapid and easily reversible the body and has been found to inhibit the sodium manner. Both compounds have been extensively pump reversibly from the cytoplasmic side of the employed as membrane probes to investigate cell membrane, in contrast with cardiac glyco- sodium-selective channels in a variety of transport- sides such as ouabain which bind to the exterior ing epithelia. of cells.27 Infusion of vanadate can induce saluresis by inhibition of tubular reabsorption and it has NEW GENERATION DIURETICS3031 been proposed that vanadate may have a modulating The lack of any easily definable relation between on October 1, 2021 by guest. Protected role on the performance of renal Na, K-ATPase in chemical structure and mechanism of action has normal as well as pathological states. The relation been emphasised clearly in the sulphonamide if any, between pump modification by vanadate series of diuretics. At least three different modes of and the cellular actions of diuretics remains obscure. operation occur: classical carbonic anhydrase inhibition, benzothiadiazine action and high ceiling POTASSIUM-SPARING DIURETICS AND action. It is often questioned whether there is any PASSIVE SODIUM-ENTRY CHANNELS2829 merit in further work involving molecular manipula- The use of non-renal tissues in the study of diuretic tion within a drug family, when apparently all the drug action has paid off most handsomely in the useful variations in substitution have been carried case of the potassium-sparing drugs, where most out. Experience teaches us that the unpredictable work on the mechanisms has been carried out on can always emerge even from such mundane isolated amphibian epithelia. In the case of spiro- exercises. Thus, for example, the unexpected diuretic nolactone, the close chemical similarity with profile of frusemide emerged from molecular play J Clin Pathol: first published as 10.1136/jcp.34.11.1267 on 1 November 1981. Downloaded from

1274 Lant 05,

C2H,--C C O-CH,-COOH 0 -6 CHP E Ethocryr ic cicid 0 04 'Thr

Ln o2 C O-CH COOH CHOO-CH - 16H, Indocrinone (MK 196i 031 0 fTi 'ynrfen) 2 4 6 8 24 Collection time(h) Fig. 5 Structural relation between ethacrs'nic acid 201 and two polyvalent phenoxyacetic acid derivatives, tienilic acid and , both capable of causing r simultaneous saluresis and uricosuria. Indacrinone exi.st.s as a racemic mixture, isomerism occurlring at the 2 position of the indanone rting. with the well-explored benzothiadiazine series. Subsequent high ceiling developments which have followed include bumetanide and piretanide. Mole- cular manipulation of the ethacrynic acid molecule has led to the emergence of a new class of phenoxy- -1 1-2 2-4 4-6 6-8 8-12 12- 24 acetic acid derivatives possessing an unusual Collection periods (h) copyright. combination of saluretic and uricosuric properties. Fig. 6 Changes in handling of luic acid in iiorinal ,,,ali The first of these so-called polyvalent diuretics aifter a single close of iacemic indacrinone. nig. was tienilic acid (tricrynafen) which unfortunately The top half of the figure shows the plasma urcate ran into major toxicological problems early in its concentrations on control da -)anid dr}ug daX clinical application to antihypertensive treatment. A significant fall occur.s at 1, 2 and 4 h after Development of human hepatotoxicity led to the indacrinone conipared to control. This parallel.s a Significant increase in fractional Ienal clearanic withdrawal of tienilic acid from clinical use by its measured as Eurate Cturate/Cre(itwinei 1X/00.x At tim7e in A further phenoxyacetic manufacturers 1980. ofpeak uiricosuria (2-4h) Es a increased frotn 0.55 + 0.09 acid derivative, indacrinone lacks the thienyl (control) to 2.92 + 0.30 (p < 0.001) http://jcp.bmj.com/ feature of stereo- substituent and has the unusual L control day; dr-ug day isomerism, each of its constituent enantiomers Results are expr-essed as Iiieani values + SEM (ti ') possessing different pharmacological profiles in (based on Brooks et a132). relation to saluresis and uricosuria.32 Availability of this new generation of compounds transtubular fluxes and also postsecretory reabsorp- has offered a means of investigating in greater tion. The latter process is of particular importance depth the mechanisms of uric acid handling by the in accounting for renal urate retention associated on October 1, 2021 by guest. Protected kidney and how these are affected by diuretics. with chronic thiazide administration and occurs Current concepts of renal urate handling support secondarily to diuretic-induced ECF volume con- the view that uric acid may undergo bidirectional traction. transfer via one single transport system located at various sites along the nephron.33 Such a mechanism References is analogous to the forwards and backwards move- ment of sodium and potassium ions which has been ' Levinsky NG. Some aspects of the regulation of sodiuln. excretion. In: Fisher JW, ed. Renal pharmacologi. demonstrated to occur through the conventional London: Butterworths, 1971:85-98. sodium pump.34 Disturbance in the relative balance 2 Blythe WB. Regulation of sodium excretioni. 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J Clin Pharmacol 1977: 21 Axelrod DR, Pitts RF. The relationship of plasma pH 17 :673-80. and anion pattern to mercurial diuresis. J Clin Invest 8 McGiff JC, Wong P Y-K. Prostaglandins and renal 1952;31 :171-9. function: implications for the activity of diuretic 26 Epstein FH, Silva, P, Kormanik G. Role of Na-K-ATPase agents. In: Cragoe EJ, ed. Diuretic agents. Washington: in chloride cell function. Am J Physiol 1980;238:R246-50. American Chemical Society, 1978:1-11. 27 Grantham JJ. The renal sodium pump and vanadate. 9 Thurau K, Mason J. The intrarenal function of the juxta- Am J Physiol 1980;239:F97-106. glomerular apparatus. In: Thurau K, ed. MTP Inter- 28 Baba WI, Lant AF, Smith AJ, Townshend MM, Wilson national Review of Science Vol 6, Kidney and urinary GM. Pharmacological effects in animals and normal tract physiology. London: Butterworths, 1974:357-89. human subjects of the diuretic, amiloride hydrochloride 10 Sachs G. Ion pumps in the renal tubule. Am J Physiol (MK 870). 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13 Valtin H. Structural and functional heterogeneity of 31 Lemieux G, Steele TU, eds. Symposium on antihyperten- copyright. mammalian nephrons. Am J Physiol 1977;233:F491-501 sive uricosuric diuretics. A new class of renally active 14 Kuhn W, Ryffel K. Herstellung konzentrierter Losungen compounds with antihypertensive, diuretic, uricosuric ans verdunnten durch blosse Membranewirkung: properties. Nephron 1979;23 Suppl 1. ein Modellversuch zur Funktion der Niere. Z Physiol 32 Brooks BA, Blair EM, Finch R, Lant AF. Studies on the Chem 1942 ;276:145-78. mechanism and characteristics of action of a uricosuric 15 Wirz H, Hargitay B, Kuhn W. Lokalisation des konzen- diuretic, indacrinone (MK-196). Br J Clin Pharmacol trierungsprozesses in der Niere durch direkte kryoscopie. 1980;10:249-58. Helv Physiol Pharmacol Acta 1951 ;9:196-207. 33 Weiner IM. Urate transport in the nephron. Am J Physiol 16 Bisset GW, Jones NF. Antidiuretic hormone. In: Jones NF, 1979;237:F 85-92. ed. Recent advances in renal disease. London: Churchill 34 Lant AF, Priestland RN, Whittam R. The coupling of

Livingstone, 1975:350-416. downhill ion movements associated with reversal of http://jcp.bmj.com/ 17 Lant AF, Wilson GM. Diuretics. In: Black DAK, ed. the sodium pump in human red cells. J Physlol (Lond) Renal disease 2nd ed. Oxford: Blackwell Scientific 1970;207:291-301. Publications, 1972:594-637. 18 Burg MB. Tubular chloride transport and the mode of action of some diuretics. Kidney Int 1976;9:189-97. 19 Lant AF. Relief of oedema and the action of diuretics. Requests for reprints to: Prof AF Lant, Department of In: Vere DW, ed. Topics in therapeutics. London: Pitman Therapeutics, Page Street Wing, Westminster Hospital, Publishing, 1978:150-66. London SWIP 2AP, England. on October 1, 2021 by guest. Protected