Site of action of diuretic drugs

Transcription

1 Kidney International, VOL II (l977)p. 1-8 EDITORIAL REVIEW Site of action of diuretic drugs Diuretic drugs continue to attract the interest of renal physiologists not only for their intrinsic tubular effects but equally importantly for the insight that such studies provide into normal and abnormal mechanisms of renal function. Much new information has accumulated in recent years from experiments at a single nephron level particularly as a result of work on the isolated kidney tubule initiated by Burg and his collaborators [1, 2]. This makes it appropriate at the present time to review current knowledge about the site of action of commonly used diuretics with particular attention to the results of micropuncture studies. We plan to consider the available evidence in each segment of the nephron for the following groups of drugs: the potent loop diuretics furosemide, ethacrynic acid and mercurials; the benzothiadiazides, or simply the thiazide group of diuretics; the carbonic anhydrase inhibitors; and the potassium sparing diuretics. Before considering the specific action of diuretic drugs on the transport of water and electrolytes in the renal tubule, brief attention should be given to their effects on glomerular filtration since such effects may influence to a major extent the magnitude of the diuresis. The use of all diuretic drugs is often associated with a decrease in filtration rate for reasons that are not entirely clear. An important factor appears to relate to secondary changes in extracellular fluid (ECF) volume. Burke, Robinson, and Clapp [3] have shown that the decrease in glomerular filtration rate (GFR) that occurs following an administration of furosemide can be prevented if the diuretic induced volume losses are adequately replaced. The same laboratory [4], however, has found that another potent loop diuretic, ethacrynic acid, still occasionally causes a fall in GFR despite concurrent replacement of fluid losses. In view of the action of these drugs on peripheral venous compliance and venous return [5 7], which appears to be independent of their renal effects, it is not surprising that changes in renal hemodynamics and filtration rate may occur despite prevention of ECF volume depletion, mediated perhaps through subtle changes in Received for publication April 22, 1976: and in revised form August 27, , by the International Society of Nephrology. cardiac output. It is likely that the mechanism whereby ECF volume losses lead to decreases in GFR is related to a reduction in glomerular capillary plasma flow [8], although there have been no direct studies to establish this point. Other diuretics, such as thiazides, mercurials, and carbonic anhydrase inhibitors, invariably cause a decrease in GFR whether diuretic losses are replaced or not, clearly indicating other mechanisms rather than changes in ECF volume in the control of filtration rate. Recent evidence presented by Wright and Schnermann [9, 10] supports the notion that these effects may in part be mediated by an intrarenal feedback mechanism which couples distal salt delivery to filtration rate in individual nephrons. Proximal convoluted tubule. The various hemodynamic and possibly intrarenal effects of diuretics have made detection of primary transport effects within the proximal convoluted tubule very difficult to establish with certainty. The only drugs at the present time for which the evidence strongly supports a major inhibitory action on proximal salt and water reabsorption are the carbonic anhydrase inhibitors and the thiazide diuretics. The carbonic anhydrase inhibitor acetazolamide has been extensively studied using a variety of techniques. Virtually all groups using this drug have demonstrated inhibitory effects within the proximal tubule. The magnitude alone of the urinary bicarbonate losses that follow acetazolamide administration attests to a reduction in proximal bicarbonate reabsorption and this has been subsequently confirmed in free flow micropuncture studies. The aspect of much more recent interest relates to the effects of carbonic anhydrase inhibition on sodium and chloride reabsorption and whether or how such effects can be attributed to the reduction of sodium and bicarbonate reabsorption. Free flow micropuncture studies have repeatedly shown that following carbonic anhydrase inhibition there is a rise in tubular fluid to plasma (TF/P) bicarbonate ratios [11 17]. This has also been reflected by a corresponding fall in TF/P chloride ratios towards unity [14, 17 19]. Whereas earlier work employing the quinhydrone electrode has reported TF/P bicarbonate values in excess of unity after acetazolamide administration, TF/P chloride values do not fall below one, suggesting that the calculated I

2 2 Seely and Dirks bicarbonate results using this electrode were spuriously high. The reduction in bicarbonate reabsorption after acetazolamide administration has been attributed to a reduction in active hydrogen ion secretion across the luminal cell membrane, which is in line with current concepts of bicarbonate reabsorption [20]. Malnic et al [21]'have studied the effects of acetazolamide on the rate of intratubular acidification of split drops of known buffer composition. Their results suggest that acetazolamide inhibits the active step of hydrogen ion secretion as well as reducing passive permeability of the epithelium to bicarbonate. Several free flow studies have demonstrated that in addition to the effects on bicarbonate transport the fractional reabsorption of sodium, chloride and water was also reduced after carbonic anhydrase inhibition [17, 22, 23]. Kunau [17] has reported that another potent carbonic anhydrase inhibitor, benzolamide, significantly reduced proximal fractional reabsorption of chloride by 29%, sodium by 34%, and bicarbonate by 55%. Earlier reported studies, in which volume losses were not replaced, had given equivocal results [14, 24, 25]. Several groups have also shown a reduction in volume reabsorption measured by the split oil droplet technique [26, 27]. Grantham [28] has reported a reduction in absorption from both convoluted and straight portions of the proximal tubule using a modified in vitro technique to study the isolated rabbit tubule. The studies of Radtke et al [26], which have shown a prolongation of the split droplet half time in the absence of bicarbonate using the organic buffer glycodiazine, were interpreted to mean that acetazolamide may also inhibit a noncarbonic anhydrase mediated hydrogen ion secretory transport system. The mechanism whereby sodium and chloride transport is inhibited secondary to the inhibition of bicarbonate reabsorption is still unsettled at the present time. Chloride transport appears to be passively driven down a favourable electrochemical gradient created by the preferential reabsorption of sodium bicarbonate. The rise in luminal chloride concentration generates a small diffusion potential, lumen positive in the later portions of the proximal convoluted tubule [18, 29, 30]. It is possible that this could also facilitate the passive reabsorption of sodium in this segment in view of the high permeability to sodium and low electrical resistance of this portion of the nephron [31 33]. Interference with preferential bicarbonate reabsorption by acetazolamide, which has been shown to result in lower IF/P chloride ratios in the late proximal tubule, leads therefore to a smaller positive potential [18, 29, 34] and thus might account for the inhibition of sodium and chloride reabsorption that occurs. This scheme, though attractive, lacks firm experimental support. Furthermore, direct testing of this hypothesis in the in vitro perfused rabbit proximal tubule failed to show any consistent relationship between fluid absorption and the transepithelial potential difference [35].An alternative hypothesis recently proposed by Frömter [36] and by Schafer, Patlak, and Andreoli [37] appears more likely. This attributes the coupling of solute and water transport to the fact that the transported bicarbonate species has a higher reflection coefficient than chloride. The positive potential difference is a consequence of the transepithelial chloride gradient under in vivo conditions, but is not of itself a driving force for solute reabsorption. Certain ones of the thiazide diuretics which have been studied by micropuncture exhibit many of the same effects as acetazolamide on the proximal tubule, although their net effects on the whole kidney show some important differences, The early micropuncture study of Dirks, Cirksena, and Berliner [24] failed to show inhibition of fractional reabsorption in the proximal tubule after hydrochlorothiazide administration, probably as a result of a failure to prevent diuretic induced ECF volume losses and consequent large falls in GFR. A subsequent study from the same laboratory [38] has shown a small depression in proximal fractional reabsorption (10 to 15%) after chlorothiazide administration in the dog under both hydropenic and mild saline loaded conditions. When volume losses were replaced, Fernandez and Puschett [39] have also shown that both chlorothiazide and a related drug, metolazone, inhibit fractional sodium and water reabsorption in the proximal tubule of hydropenic parathyroidectomized dogs, provided that the GFR did not drop by more than 26%. Several groups have shown that chlorothiazide leads to a depression of split drop reabsorption [27, 28, 40]. In recent studies of Kunau, Weller, and Webb [41], fractional chloride reabsorption was reduced by chlorothiazide to the same extent as by the carbonic anhydrase inhibitor benzolamide. End proximal TF/P chloride values were similarly reduced by both drugs. These similarities suggest that the proximal effect of chlorothiazide may be attributable to inhibition of carbonic anhydrase. However, metolazone, which is not a carbonic anhydrase inhibitor, resulted in a similar depression of proximal fractional reabsorption. Unfortunately, no direct studies of bicarbonate transport in the proximal tubule are yet available with either drug. Nevertheless, it is clear that the major differences in the whole kidney effects of thiazides and acetazolamide must be due to their action beyond the proximal tubule. The evidence for a proximal tubular effect of drugs

3 Diuretic site of action 3 known to exert major inhibitory effects on the loop of Henle, i.e. ethacrynic acid, furosemide and the mercurials, is much more conflicting. In part, the reason for this appears to be related to their hemodynamic effects. Dirks, Cirksena and Berliner [24] have shown that the use of all three drugs led to large decreases in GFR and increases in proximal fractional reabsorption. This was attributed to the volume depletion that ensued, since diuretic losses were not replaced. Moreover, this seemed likely in view of their previous demonstration that acute volume expansion led to a reduction in proximal fractional reabsorption [42]. Furthermore, when losses were replaced during ethacrynic acid administration, fractional reabsorption was then found to be unchanged. Several studies have subsequently confirmed that acute volume depletion enhances fractional and absolute proximal reabsorption [43, 44]. A number of other variables may also interfere with or obscure any direct effects of diuretic drugs on the proximal tubule. Recent studies of Burke and collaborators [3, 4] have emphasized the role of changes in whole kidney filtration rate on proximal reabsorption following administration of furosemide and ethacrynic acid. In the case of furosemide, alterations in GFR appeared to be related to ECF volume depletion since replacement of losses prevented a decrease in GFR. In this instance proximal fraction reabsorption was observed to decrease [3]. When losses were not replaced, GFR fell and fractional reabsorption was found to increase. Similar increases in fractional reabsorption could be demonstrated when GFR was reduced by means of partial renal artery constriction despite volume replacement. In comparable experiments performed with ethacrynic acid, the same laboratory reported that changes in fractional reabsorption could still be correlated with changes in GFR despite apparently adequate volume replacement [4]. Animals showing a severe reduction in GFR (45 to 64%) showed a significant rise in fractional reabsorption, whereas fractional reabsorption fell only in those animals in which there was a minimal or no fall in GFR. All animals, however, showed significant increases in absolute and fractional sodium excretion whether proximal fractional reabsorption had increased or decreased. Brenner et al (45) have drawn attention to a possible artifact introduced by retrograde collection of tubular fluid under diuretic conditions when intratubular pressures are elevated. He showed in the rat that, provided large oil blocks were placed distal to the puncture site and thus prevented retrograde flow, absolute reabsorption in the proximal tubule decreased after furosemide administration. When these precautions were not taken, a significant degree of contamination Table 1. Major site of diuretic action Proximal tubule Ascending limb - Distal tubule and collecting duct - - Carbonic anhydrase inhibitors Thiazides Furosemide Ethacrynic acid Mercurials Thiazides Spironolactone Amiloride occurred which was reflected in falsely high values for both single nephron GFR and fractional reabsorption. It is possible that this may account for the failure of many reported free flow studies to show inhibition of proximal reabsorption with furosemide despite attempts at volume replacement. It must also be recognized, however, that the attempt to fully replace fluid losses after furosemide administration carries with it a danger of overcorrection and some degree of volume expansion because of the magnitude of the diuresis. In contrast to the somewhat conflicting results of furosemide under free-flow conditions, almost all experiments designed to test the effects of this drug on the intrinsic reabsorptive rate of the proximal tubule have indicated an inhibitory action. Studies in at least seven laboratories [25, 26, 40, 43, 45-47] have demonstrated inhibition by the shrinking droplet technique either after systemic or local administration of the drug in the peritubular circulation or split droplet. The in vivo microperfusion studies of Morgan et al [48] have demonstrated both a reduction in net sodium transport as well as an elevation of the steady state sodium concentration, The steady state chloride ratios have also been found to be significantly lower after furosemide [49], suggesting that steady state bicarbonate concentrations should also be higher. Radtke et at [26] and Malnic and Giebisch [20] have also shown a reduction in the rate of proximal hydrogen ion secretion after furosemide administration, although to a somewhat lesser extent than after acetazolamide administration, These results are in keeping with an inhibition of carbonic anhydrase which has been observed with this drug. In contrast to the foregoing studies performed under in vivo conditions, recent experiments of Burg et al [50] have failed to show any inhibition of either net sodium transport or generation of a negative potential difference in the isolated rabbit proximal tubule in luminal concentrations of up to 104M. Peritubular concentrations of 103M also failed to alter the electrical resistance or potential difference of the epithelium. Grantham [28], however, has reported inhibitory effects of furosemide when using a modified method of studying fluid absorption from the isolated rabbit

4 4 Seely and Dirks proximal tubule. With this method, one end of the tubule is closed on itself and the rate of absorption studied by the rate at which fluid leaves the cannulating pipette. It is possible that the composition of the luminal fluid may resemble more closely that found under in vivo conditions when using the latter method, rather than when short segments are perfused in vitro, since there may be more time for changes in perfusate composition to occur. If such changes are important in determining the net rate of salt and water transport, this might then account for the differences observed. It is also possible that in such experiments secretion of the drug into the l.umen may osmotically restrict fluid movement apart from any intrinsic or pharmacological effects. These conflicting results make it difficult to draw any firm conclusion about the extent of proximal inhibition after furosemide administration. Some of the data cited do suggest that salt and water absorption may be inhibited secondary to inhibition of carbonic anhydrase and that such effects may not be evident with the in vitro technique used to study the rabbit proximal tubule. It is clear, however, that the proximal effects may be easily overcome since, unless extracellular fluid volume is carefully sustained during the diuresis, fractional reabsorption actually increases. It is likely that this occurs in the clinical conditions for which this drug is used. Nevertheless, as Clapp and his colleagues have emphasized [3, 4], it is likely that an inhibitory action in the proximal tubule may reduce the degree to which fractional reabsorption is increased by any given degree of volume contraction and, in this sense, may enhance the extent of diuresis by ensuring a higher degree of delivery out of the proximal tubule than would otherwise occur. Ethacrynic acid, which closely resembles furosemide in its overall effects on the human kidney, has been far less studied by micropuncture techniques, in large part because it is relatively inactive in the rat compared to furosemide. Nevertheless, the studies that are available indicate a close similarity of proximal effects. Both Deetjen [51] and Clapp, Nottebohm, and Robinson [4] have demonstrated that ethacrynic acid leads to a reduction in proximal fractional reabsorption provided volume losses are replaced and that filtration rate remains constant. Split-drop studies in the rat [27] failed to show any significant inhibitory effects. Mercurial diuretics are almost of historical interest only, at the present time having largely been replaced by the more potent oral agents now available. Experiments in the dog have shown that when volume losses are not replaced, GFR falls and fractional reabsorption in the proximal tubule rises [24]. A subsequent experiment from the same laboratory has shown no further change in fractional reabsorption when a mercurial diuretic was superimposed on a modest saline diuresis [52]. These few results suggest that mercurials do not exert any significant effects on proximal tubular function. The clearcut effects of furosemide, ethacrynic acid, and mercurials in the loop of Henle (see following), in contrast to the proximal tubular studies, give added support to the notion that different transport systems are involved in these two segments. Loop of Henle. Studies of the effects of diuretics within the loop of Henle have generated far less controversy than their action in the proximal tubule, reflecting the large degree of agreement between the results of different laboratories. In this segment, in particular, studies of isolated perfused tubules have provided new insight into mechanisms of transport and of diuretic action. Clearance studies based on measures of urinary concentration and dilution have previously pointed to the loop of Henle as an important site of action for many of the more potent diuretics. This has been subsequently supported by in vivo micropuncture evidence of the composition of tubular fluid as it emerged from the ascending limb into the distal convoluted tubule. Clapp and Robinson [53], for instance, have been able to show an increase in the tubular fluid osmolality of early distal fluid samples after administration of a number of diuretic drugs. Other groups subsequently have demonstrated significant increases in distal sodium and chloride concentrations in a variety of species following thiazide, furosemide, and mercurial administration. The non reabsorbed fraction of filtered sodium has been shown to be increased with the latter two despite the lack of any change in proximal fractional reabsorption, providing strong evidence for an inhibition of salt reabsorption within the 1oop itself. Direct analysis of the effects of diuretics within loop structures has had to await the development of techniques for perfusion of isolated segments of the loop in an in vitro system. Work in two laboratories with this method has revealed evidence for an active chloride transport system in the thick ascending limb of the loop of Henle [54, 55]. This segment has been shown to generate a small potential difference, lumen positive, when an identical solution is placed on the two sides of the epithelium. The potential is abolished in the absence of chloride and is increased when sodium is removed from the bathing solution. This segment is relatively impermeable to water and in the presence of slow or stop flow conditions can generate hypotonic fluid within the lumen. In this situation the potential becomes more positive due to the impo-

5 Diuretic site of action 5 sition of a dilution potential for sodiuni chloride across the epithelium in view of the fact that the permeability to sodium exceeds that of chloride. In contrast to the thick ascending limb, both the descending limb and thin ascending limbs of Henle's loop have not convincingly been shown to be capable of active transport. The mechanism of osmotic equilibration of fluid within the descending limb of Henle's loop and the role of the thin ascending limb in the generation of inner medullary hypertonicity remain controversial, but in any case are secondary to the action of the thick ascending limb which provides the ultimate source of energy for urinary concentration and in part for urinary dilution. It is here that all the potent diuretics appear to have their major site of action. Burg and his collaborators have shown that furosemide [50], ethacrynic acid [56] and the mercurial diuretic, mersalyl [57], are all capable of inhibiting both the electrical potential and net solute transport when applied in low doses to the luminal bathing medium. It is of interest that these drugs appear to be far less potent or inactive when presented to the peritubular side of the epithelium. The effect of mersalyl could be reversibly inhibited by p chloromercuribenzoate, whereas the effect of mercuric chloride could not, suggesting that the action of the mercurial group of diuretics is not simply dependent on the mercuric ion alone. Burg was also able to show that the ethacrynic cysteine adduct, in which form the drug is partly excreted, is active at a far lower concentration than the native compound. In view of the preferential action of these drugs from the luminal side of the membrane, it is relevant that furosemide is secreted into the lumen of the proximal tubule and may thus exert an inhibitory action on the ascending limb when only small doses of the drug are administered, Interference with the secretion of furosemide by the organic acid pathway may account in part for the reason that larger doses of the drug need to be administered for a diuretic effect in uremic patients. With the exception of amiloride, which is inactive in the ascending loop [58], there is no direct information on the action of other diuretic drugs on the ascending limb studied in the in vitro system. Clearance studies suggest that acetazolamide has little or no inhibitory effects within the loop [59]. A recent study [41] has demonstrated that net chloride reabsorption within the loop was increased after administration of the carbonic anhydrase inhibitor benzolamide, since fractional chloride delivery rose from 54% to 68% at the end of the accessible proximal tubule in contrast to a very small change from 4.5 to 6.3% in fractional delivery to the early distal tubule. This gives further evidence for the lack of an inhibitory action of this group of drugs on ascending limb chloride transport. The action of thiazides in the loop of Henle has been less clear cut. The results of experiments which demonstrated no effect on free water reabsorption, although free water clearance was inhibited, led to the concept of a "cortical diluting segment" which was inhibited by these drugs [59]. The exact location of this site within the nephron, however, was not defined and could conceivably involve the outer medullary ascending limb of Henle's loop, distal convoluted tubule or collecting duct system since inability to generate or maintain hypotonic tubular fluid within any one of these segments could account for the impairment in free water clearance. Free flow micropuncture experiments in the rat [41] have demonstrated that early distal chloride delivery after thiazide administration is increased to a very small extent, compared to the marked increase in end proximal delivery which is very similar to the results seen after carbonic anhydrase inhibition. If chloride is the only actively transported anion species in the ascending loop, this would strongly suggest that this process is not inhibited by thiazides. Distal TF/P sodium concentration ratios are found to be increased to a small extent after thiazides [38], which could result from the increased delivery of sodium and bicarbonate from the proximal segment to the distal tubule. Distal convoluted tubule and collecting duct. Studies of the distal portion of the nephron, distal convoluted tubule, collecting tubule, and ducts have been relatively hampered more by greater methodological problems than the more proximal portions. The distal convoluted tubule is shorter and less easily accessible to the surface for in vivo free flow studies and few in vitro studies have been performed on this segment. The cortical collecting tubules have been studied by perfusion techniques but are not generally as accessible for micropuncture as are the medullary collecting ducts which have also been studied by microcatheterization techniques. One of the major problems of studies using free flow micropuncture techniques has been the fact that changes in proximal or loop function may have resulted in different rates of volume and solute delivery to the distal tubule. Unless the response of this segment to such changes in load produced by other means is known, it is impossible to detect changes in reabsorption due to diuretic effects from those due to alterations in load, apart from any other hemodynamic or other effects of the diuretic on the kidney. Many authors have surmised that an elevation of the intratubular sodium concentration in the distal tubule implies an inhibition of sodium reabsorption both in the loop and

6 6 Seely and Dirks distal tubule; however, perfusion studies make clear that high rates of delivery to the loop alone may result in high intratubular concentrations of sodium and chloride. In a free flow study of the rat distal tubule, Duarte, Chomety, and Giebisch [60] found that furosemide almost completely abolished the sodium gradient along the distal tubule; however, the fraction of the filtered sodium reabsorbed along the distal tubule appeared to be 20 to 30%, which is two to three times greater than the normal amounts reabsorbed in this portion. It seems highly likely that such apparent increases in absolute sodium reabsorption in this portion result from the higher delivery and the low gradient against which sodium must be absorbed. Morgan et al [48] have studied the effects of furosemide on distal tubular function by microperfusion of the loop of Henle and sequential collections from two sites in the same distal nephron. They attempted to control for differences in the loop by providing higher perfusion rates in the control state to achieve comparable flow rates in the early distal tubule. They were unable to show any significant differences in reabsorptive rates in control or furosemide treated animals; however, the scatter in the results was large and the concentrations of sodium and potassium were not equal in the two situations. Work with carbonic anhydrase inhibitors has confirmed that distal hydrogen ion secretion is reduced [20] and that free-flow distal sodium concentrations are elevated along with concurrent increases in fractional sodium reabsorption [60, 61]. Wiederholt, Sullivan and Giebisch [611 concluded that the increased load provided additional sodium to undersaturated distal reabsorptive sites. Recent work of Kunau, Weller, and Webb [41] presents strong evidence that thiazide diuretics inhibit the process of sodium and chloride reabsorption in the distal tubule. The carbonic anhydrase inhibitor, benzolamide, in contrast, had no inhibitory effects on distal chloride reabsorption. This effect appears to account for the major natriuretic and chloruretic effect of thiazide diuretics and probably accounts for the depression of transport in the "cortical diluting segment" identified by clearance studies. In contrast to the relative paucity of data regarding changes ii sodium reabsorption as a result of diuretics in the distal tubule, there is abundant evidence to suggest that major changes in potassium transport occur in this segment. Duarte, Chometry and Giebisch [60] found in their study that whereas early distal tubular potassium concentrations appeared to rise following furosemide, late distal TF/P potassium concentration ratios were equal or lower than controls, yet fractional secretion and urinary fractional excretion were considerably increased owing to the high urine flow. They concluded that the effects of furosemide on potassium transport could be attributed entirely to changes in flow rate along the distal tubule. The effects of carbonic anhydrase inhibitors on the distal transport of potassium has been studied extensively by Giebisch and his collaborators. Studies of both amphibian and mammalian kidneys have demonstrated that these agents lead to augmented potassium secretion [22, 60]. Wiederholt, Sullivan, and Giebisch [611 suggested that these effects resulted from enhanced uptake of potassium at the peritubular membrane and an increase in the intracellular potassium transport pool. The action of the mildly natriuretic potassium sparing agents have received little study by micropuncture. The effect of aldosterone within the distal convoluted tubule and collecting duct has been well established [62, 63]. In view of the evidence that spironolactone is a competitive antagonist of aldosterone and other mineralocorticoids, it is reasonable to expect any future micropuncture studies under in vivo conditions or in isolated tubules to support such a distal action. Amiloride and triamterene, compounds which are physiologic though not specific antogonists to aldosterone, also result in modest natriuresis with reduced potassium excretion. During free flow and microperfusion studies of the loop of Henle, amiloride was not found to alter early distal tubular concentrations of sodium or potassium [60] nor has any effect been observed with this drug in studies of the isolated ascending limb. However, as expected from clearance studies, amiloride has marked effects on the distal convoluted tubule and collecting duct. Duarte, Chomety, and Giebisch [60] observed a tendency for distal sodium concentration ratios to rise along the distal convoluted tubule. This was accompanied by a small increase (2%) in fractional sodium excretion. Conversely, the normal increase in potassium concentration along the distal tubule was blunted, indicating inhibition of distal potassium secretion and hence leading to a reduction in urinary potassium excretion. The mean distal transtubular potential difference fell from 46 to 26 mv. They considered that amiloride directly reduced luminal sodium permeability, a finding consistent with studies of this drug on anuran membranes. The recent work of Stoner, Burg, and Orloff on the perfused rabbit cortical collecting tubule [64] demonstrated that amiloride rapidly reversed the negative transtubular electrical potential leading to a small positive potential difference. Sodium reabsorption and potassium secretion were also sharply decreased. Further addition of acetazolamide abolished the positive potential difference, suggesting the presence of a current of acid-

7 Diuretic site of action 7 ification. The small natriuretic effect compared to the antikaliuretic effect was attributed to the meagre sodium load normally presented to the collecting tubule but could also reflect the possibility that sodium reabsorption is unimpaired by the drug beyond the cortical collecting tubule. Meng [65] has recently confirmed, using the split oil droplet technique, that intraluminal application of amiloride (up to lo-6m) strongly inhibits isotonic fluid absorption in the distal convoluted tubule of the rat kidney. Proximal effects were also seen but only at very much higher concentrations. In conclusion, the availability of many different diuretic drugs that can selectively inhibit one or more of a variety of transport processes within the nephron provides a wide range of options that can be rationally used in the treatment of edema states and nonedematous disorders. Caution must be exercised before the results at a segmental level of the nephron can be extrapolated to whole kidney in vivo performance. For instance, hemodynamic effects, changes in ECF volume, intratubular pressure, tubular fluid flow rate, or electrolyte composition, etc., may all exert effects on solute transport independently of any direct effect of the drug in a particular nephron segment. Many questions remain unanswered. The effects of certain diuretics upon proximal tubular transport are still unsettled and the results of in vivo experiments have not been reconciled with in vitro studies in the isolated perfused tubule. The mechanism whereby carbonic anhydrase inhibitors affect sodium and chloride transport is still unclear as is the extent to which such effects can account for the proximal action of thiazide diuretics. The answers to these and many other issues that have been raised by such studies require much further work and will undoubtedly contribute to a better understanding of the physiology of the kidney. JOHN F. SEELY JOHN H. DIRKS Montreal Reprint requests to Dr. John F. Seety, Renal and Electrolyte Division, Department of Medicine, Royal Victoria Hospital and McGill University, Montreal, P.Q., Canada. References 1. BURG MB, GRANTHAM J, ABRAMOV M, ORLOFF J: Preparation and study of fragments of single rabbit nephrons. Am J Physio/210: , BURG MB, ORLOFF J: Perfusion of isolated renal tubules, in section 8 of Handbook of Physiology, edited by ORL OFF J, BERLINER RW, Washington, D.C., Am. Physiol. Soc , pp BURKE Ti, ROBINSON RR, CLAPPJR: Determinants of the effect of furosemide on the proximal tubule. Kidney mt 1:12 20, CLAPP JR, NOTTEBOI-IM GA, ROBINSON RR: Proximal site of action of ethacrynic acid: Importance of infiltration rate. 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