Effect of ethacrynic acid on the thick ascending limb of Henle's loop

Effect of ethacrynic acid on the thick ascending limb of Henle's loop

Kidney International, Vol. 4 (1973), p. 301—308 Effect of ethacrynic acid on the thick ascending limb of Henle's loop MAURICE BURG and NORDICA GREEN ...

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Kidney International, Vol. 4 (1973), p. 301—308

Effect of ethacrynic acid on the thick ascending limb of Henle's loop MAURICE BURG and NORDICA GREEN Laboratory of Kidney and Electrolyte Metabolism, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland

it inhibits NaCI reabsorption at this site is uncertain. The thick ascending limb can be studied directly by means of in vitro perfusion of tubule fragments dissected from rabbit kidneys [7, 8]. Since the rabbit is one of the species in which the drug is effective [1], a direct study of its action in this preparation was of interest. In previous studies it was found that NaCI reabsorption from the thick ascending limb involves active transport of chloride and is associated with a transepithelial electrical potential difference (PD) oriented positive in the lumen [7, 8]. The active Cl transport is inhibited by the presence of ouabain in the bath [7, 8] and by furosemide [9] and mersalyl [10] in the tubule lumen. The purpose of the present studies was to investigate directly the action of ethacrynic acid in this tubule segment.

Effect of ethacrynic acid on the thick ascending limb of Henle's

loop. Cortical thick ascending limbs of Henle's loop were dissected from rabbit kidneys and perfused in vitro. The diuretic ethacrynic acid (10—i M) in the tubule lumen caused a reversible decrease in the electrical PD. Ethacrynic-cysteine complex, a major urinary excretion product of ethacrynic acid, also caused a reversible decrease in the PD as well as in net chloride trans-

port, but at a much lower concentration (3 x lO M) in the lumen. Ethacrynic-cysteine in the bath (1O M) had no effect on the PD. Ethacrynic-cysteine had little effect on Na and Cl conductances, measured electrically. On the basis of these results

we suggest that the ethacrynic-cysteine complex is the major active form of the drug in vivo and that it acts in the lumen of the thick ascending limb of Henle's loop to inhibit active Cl transport and thereby diminish NaC1 reabsorption. Effet de l'acide éthacrynique sur Ia partie large de Ia branche ascendante de l'anse de Henle. Des branches ascendantes larges corticales d'anses de Henle ont été disséquées a partis de veins

de lapins et perfusées in vitro, L'acide éthacrynique (lOs M) Methods

dans Ia lumière tubulaire determine une inhibition reversible de

Ia difference de potentiel electrique. Le complexe ethacrynique-

cystCine, un produit d'excrCtion urinaire majeur de l'acide éthacrynique, determine aussi une diminution reversible de la difference de potentiel et du transport net de chlore, mais a une concentration luminale moindre (3 x lO M). Le complexe éthacrynique-cysteine dans Ic bain n'a pas d'effet sur Ia différence de potential. Le complexe a peu d'effets sur les conductances Na et Cl mesurées electriquement. Sur Ia base de ces résultats, nous suggerons que Ic complexe ethacrynique-cysteine est le forme active majeure de Ia drogue in vivo et qu'il agit dans la lumiCre de Ia partie large de Ia branche ascendante de l'anse de Henle en inhibant Ic transport actif du chlore et, de cc fait, en diminuant Ia reabsorption du NaCI.

The method employed in these studies has previously been described in detail [7, 11] and is summarized below with additions and modifications. Cortical segments of thick ascending limbs of Henle's loop were dissected from rabbit kidney and were perfused at 37°C using concentric glass pipets. The tubules were dissected at room temperature in the saline buffer that was used in the bath during perfusion. It contains the following (in millimoles): NaCI, 115; KCI, 5;

Ethacrynic acid is one of the most potent diuretics known [1—5]. Its principal site of action within the kidney has been

identified by clearance techniques as the ascending limb of Henle's loop [2—4, 6]; however, the mechanism by which

NaHCO3, 25; Na acetate, 10; CaCI2, 1; MgSO4, 1.2; NaH2PO4, 1.2; glucose, 5; plus 5% v/v rabbit serum. The perfusate contained NaCI, 150; K2HPO4, 2.5; CaCl2, 1.0; and MgSO4, 1.2, adjusted to pH 7.4 (unless otherwise noted) with HCI.

Perfusions were carried out by gravity. Sodium and potassium concentrations were measured in the perfusion

fluids and baths using an IL flame photometer, and

Received for publication May 29, 1973; and in revised form July 11, 1973. © 1973, by the International Society of Nephrology.

osmolality was measured using the Bowman-Aminco freez-

ing point apparatus [13]. Potassium was measured in 301

302

Burg! Green

microsamples of the perfusate and collected fluid using the Aminco helium glow photometer [12]. Chloride was measured in bulk solutions using the Cotlove-Aminco chloridometer [14] and in microsamples of perfused and collected fluid using a modification [15] of the Ramsay method [16]. The transepithelial electrical PD was measured between calomel cells connected to the fluids in the bath and perfusion pipet with bridges containing 0.16 M NaCI-agar. The PD was displayed on an oscilloscope (Tektronix 561A) and a recorder (Hewlett Packard 680 M) connected via an amplifier (Bak ELSA-3 or 4). The PD was not corrected

for liquid junction potentials since those are small and cannot be readily calculated for the complex solutions used [17]. (The liquid junction potential with the standard solutions listed above is approximately + 1.5 my which would be subtracted from the recorded PD for correction.) The transepithelial electrical resistance was calculated by cable analysis using the voltage change at both ends of the tubule resulting from passage of current from the perfusion pipet to a ground connected to the bath [18]. The current (5 x 10-8 amp) was passed through the lumen of the perfusion pipet. The voltage artifact resulting from the passage of current through the perfusion pipet was nulled with a bridge circuit [18].

Ethacrynic acid was provided by Merck, Sharpe, & Dohme. It was alkalinized with NaOH in order to dissolve it. Ethacrynic-cysteine was prepared by mixing the ethacrynic acid solution with cysteine in a molar ratio of 1:1.1. The ethacrynic-cysteine was freshly prepared during each experiment since it lost activity after standing for a few hours. Except as indicated, no rabbit serum was added to

• °Ethacrynic-cysteine • Ethacrynic acid

80 60 -

4020 10_o

0 0 8 0 0 8

0 0 0

. 0 0

iO

lO—

I .

S

-1-

iO

Concentration, M Fig. 1. Effect of ethacrynic acid in the lumen on the thick ascending

limb of Henle's loop. The PD is the steady value reached one to ten minutes after adding the drug and is given as percent of the initial control value.

ducts is the cysteine adduct of ethacrynic acid [1]. When ethacrynic-cysteine was added to the perfusate in the present experiments, the PD was inhibited by a much lower concentration (Figs. 1, 2B, 4) than of ethacrynic acid, itself. Thus, 3 x 10 6 M ethacrynic-cysteine had an effect on the PD (Fig. 2B) at least as great as that of l0— M ethacrynic acid, indicating that the ethacrynic-cysteine is more

the bath containing the ethacrynic acid or ethacryniccysteine in order to avoid possible protein binding. Results are given as mean standard error (number of tubules studied). Statistical significance was estimated using the Student's 1-test. Results

The mear electrical PD across the epithelium of the cortical thick ascending limb of Henle's loop is 6.4 2.0 SD (lumen positive) when the solutions in the lumen and bath

contain equal concentrations of Na [7]. The PD results from the active transport of Cl across the epithelium, and it drives the Na transport which is largely, and possibly entirely, passive [7]. Ethacrynic acid in the perfusate caused a decrease in the

electrical PD (Figs. 1, 2A, 3) similar to the effect of furosemide [9] and the mercurial diuretic mersalyl [10]. The concentration of ethacrynic acid required is high (Fig. 1). There was no effect with 10 M ethacrynic acid in the lumen. A concentration of at least 3x 10 M was required in order to cause the PD to decrease. Beyer et al [I]

have reported that most of the ethacrynic acid that is administered to dogs appears in the urine in a chemically modified form. One of the major urinary excretion pro-

0 Minutes Fig. 2. Effect of ethacrynic acid (A) and ethacrynic-cysteine (B) in the lumen on the thick ascending limb of Henle's loop. Representative experiments are shown in order to illustrate the time course at different concentrations of the drugs.

303

Ethacrynic acid 10

12

9

10

8

S

7

+ 6 4

2

4 3

Control Fig. 4. Effect of ethacrynic-cysteine (10 M) in the lumen on the thick ascending limb of Henle's loop. The PD with the drug is the

2

steady value reached three to ten minutes after it was added. The after control is the maximum value reached ten to thirty minutes after the drug was removed. The bar equals 2 SEM. Control

Ethacrynic acid Control

Fig. 3. Effect of ethacrynic acid (0.3 to 2 X lO M) in the lumen

on the thick ascending limb of Henle's loop. The PD with the drug is the steady value reached three to ten minutes after it was added. The after control is the maximum value reached 10 to 30 minutes after drug was removed. The bar equals 2 SEM.

than 100 times as potent.' Net chloride transport was also

blocked by ethacrynic-cysteine (Table 1) which together with the decrease in PD is most consistent with inhibition by the drug of active chloride transport.2 The diuretic action of ethacrynic acid in vivo is notably rapid both in its onset and in its reversibility. Consistent with this, both ethacrynic acid and ethacrynic-cysteine

was no reversal of the effect after the drug was removed. In other preliminary experiments (not shown) concentrations of ethacrynic acid lower than iO- M in the bath had no definite effect on the PD. When the ethacrynic acid (10 M) was dissolved in rabbit serum in the bath, there was no effect on the PD (Table 2). (As a control, rabbit serum, by itself in the bath, has little effect in transport [7]). These results are similar to those previously observed when

mersalyl was placed in the bath in high concentrations. Mersalyl in the bath caused the PD to decrease irreversibly, Table 1. Effect of ethacrynic-cysteine in the lumen on the thick ascending limb of Henle's loop a

required only one to ten minutes to decrease the PD

Control

maximally (Fig. 2) depending on the concentration used,

(Cl)

and the effect reversed substantially within 10 to 20 minutes

after the drug was removed (Figs. 2—4). The time course in vitro is similar to that of the mercurial diuretic mersalyl [10]. Furosemide in vitro is even more rapid in its action and reversibility [9]. When ethacrynic acid was removed

from the lumen, the net chloride transport (Table I), as well as the PD, returned toward the control value. Ethacrynic acid (10 M) also caused the PD to decrease when placed in the bath, provided the bath contained the saline buffer without added protein (Fig. 5 and Table 2). The decrease was not as rapid as when the drug was placed in the lumen, however (compare Figs. 2 and 5), and there As a control, cysteine alone (10 and iO— M) in the lumen had no effect on the PD [10].

2 In two preliminary experiments net chloride transport was also blocked by iO— M ethacrynic acid in the lumen.

Mean SEM

12.0 5.2 10.8 11.4 9.9

Ethacryniccysteine

(E), 3x l0

1.6

After control

(C2)

1.4

5.5

0.8

3.2

3.5

7.2

3.9 2.4

7.0 0.8

5.7

0.9

a As judged from chloride transport rate in pEq cm' scc1. Each value is the mean of three collections from an individual tubule taken over 15 to 25 minutes. The mean paired differ-

/

C1+C2\

P< 0.01. The mean

was —5.4 0.8 SEM, ence E— 2 ) tubule length was 2.2 mm and the mean perfusion rate was 1.9 nI min1. The mean concentrations of chloride were as follows: bath, 119 mM; perfusate, 150 mM; and control collected perfusion fluid, 83 m. Net chloride transport was calculated as previously described [101 from the concentrations of chloride in the perfused and collected fluid, assuming no net fluid transport.

304

Burg! Green

across the epithelium, resulting from the imposed chemical concentration gradient (sodium conductance exceeds chlo-

8

ride conductance) [7]. All other things being equal, the magnitude of the diffusion potential depends on the ratio of sodium to chloride conductance. In the experiments in Table 3, tubules were perfused with solutions containing

7 6

both a normal (150 mM) and a low (50 mM) NaCl concentration, The mean difference in PD with the two solutions was approximately the same whether ethacrynic-cysteine was present (24.0 my difference) or absent (23.5 my differ-

5

E

ence), indicating little change in the ratio of sodium to chloride permeabilities with the drug. Ethacrynic-cysteine

(l0 M) in the lumen caused a small increase in the

3

electrical resistance. The mean electrical resistance in 5 tubules was 4280 ohm cm after the drug in the lumen had caused the PD to decrease (E), compared to 3290 in

2

the preceding control periods (Cl) and 3790 10 to 20 min-

utes after removing the drug (C 2). The difference

1

I 0

10

30 4050 60

20 Minutes

Hg. 5. Effect of ethacrynic acid (1O— M) in the bath on the thick ascending limb of Henle's loop. The bath contained no added protein.

except in the presence of serum which prevented the mersalyl effect [19]. Ethacrynic cysteine (10—s) in the bath

had no effect on the PD even in the absence of added protein (Table 2). This result is similar to that with furosemide which also has little effect in the bath [9]. In order to test whether the effect of ethacrynic-cysteine in the lumen of the thick ascending limb of Henle's loop is accompanied by any major change in sodium or chloride permeability, the NaCI diffusion potential and electrical resistance were measured. When the thick ascending limb is perfused with a solution containing a lower NaCI concentration than in the bath, the electrical PD increases. This is principally a consequence of a diffusion potential

E—

Cl+C2\ was 740 270 SEM ohm cm (P = 0.05). Since 2

)

the electrical resistance increased slightly without any change in the diffusion PD for NaCI, it is likely that there was a small decrease in the conductance of the epithelium to both of the major ions present (Cl and Na). A similar

result was previously found with furosemide [9]. The changes in sodium and chloride conductance induced by both drugs are relatively minor, however, and the major effect of the drugs evidently is to inhibit the active chloride transport. Ethacrynic acid and furosemide both cause an increase in the urinary excretion of potassium as well as of sodium [1—3, 6]. It is conceivable that inhibition by the drugs of

potassium reabsorption in the thick ascending limb of Henle's loop might contribute to the kaliuresis. The results

of studies of potassium transport in this tubule segment indirectly support the possibility. Potassium transport was Table 3. Effect of ethacrynic cysteine and NaCI concentration in the lumen on the thick ascending limb of Henle's loopa Pert usate

Table 2. Effect of ethacrynic acid in the bath on the thick

Perfusate

ascending limb of Henle's loopa

150

50

150

50

0

0

105b

iO

8.3 8.7 7.3 7.2 8.4

30.5 32.8

3.0

25.2

1.1

28.0

31.0

1.6

32.1

30.8

0.5 0.9

22.0 25.8 26.0

31.5

1.4

[NaCI], mEq litert [Ethacrynic-cysteine], M

Control Experi-

After Tubules,

mental control N

PD,mV Tubule 1

Ethacrynic acid, 1O— M,saline buffer

Ethacrynic acid,

IO M, rabbit serum

6,5

1.6

1.0

3

4

7.8

7.4

7.2

3

8.1

7.8

7.8

3

5

Mean

Ethacrynic-cysteine,

1O M, saline buffer

2 3

a As measured by electrical potential difference (PD) in millivolts. The PD was measured for 10 minutes in the presence of the drug (Experimental) and for 10 to 20 minutes in the subsequent period (After control).

Difference

8.0 23.5

25.4 24.0

a Each value of the PD is the mean of two readings taken one minute apart. The perfusate with 50 mEq liteC' of NaCI was otherwise identical to that containing 150 mEq liter'. b Except for tubule 1 in which 3 x l06 M was used.

Ethacrynic acid

studied in 31 experiments in 19 tubules (the results of studies of sodium transport in most of these tubules was reported previously [7]. Potassium concentration in the bath and perfusate was 5 mEq liter'. In 14 experiments the tubules were perfused with the standard solution con-

taining 150 mEq liter' of NaCI. The mean PD was +6 my. There was a statistically insignificant increase in potassium concentration in the collected fluid of 0.59

0.28 SEM mEq liter' (P >0.05). In 17 experiments the tubules were perfused with solution containing 50 to 75 mEq liter1 of NaC1, but otherwise identical to the standard solution. The mean PD was +19 my. There was a mean decrease in potassium concentration in the collected

fluid of 0.76 0.26 mEq liteC1 (P <0.01). Therefore, there was net potassium reabsorption, but only when the NaCI concentration in the perfusate was low, as it presumably is in the late portions of the ascending limb in viva. The existence of net potassium reabsorption in the ascending Henle's loop was previously deduced from micropuncture experiments in which the potassium concentration in the early distal tubule was found to be low [20, 21]. It is possible that the potassium reabsorption is passive since the electrical gradient is favorable. Fractional potassium reabsorption, however, is much less than sodium reabsorption studied in the same tubules. In the previous experiments [7] with equal sodium concentration in lumen and bath, sodium concentration decreased by approximately 35%, whereas, as indicated above, potassium concentration did not decrease at all under those conditions. Either potassium permeability is less than sodium permeability or there is an opposing active secretion of potassium (which is also suggested by the equivocal increase in potassium concentration under these conditions). Ethacrynic acid and

305

transport across the renal tubule epithelium and the discovery of what was presumed to be a potent inhibitor of sodium transport was of obvious interest. The effect of ethacrynic acid on the intact kidney was studied by conventional clearance and micropuncture techniques, and in addition, the drug was tested in numerous other tissue preparations in order to define its mechanism of action. In human red blood cells that were already maximally inhibited by ouabain, ethacrynic acid caused a further decrease in sodium efflux [26—30] and potassium influx [29].

The drug also caused a decrease in sodium efflux and/or an increase in cell sodium content in muscle cells [31—34], frog

oocytes [35], and intestinal epithelial cells [36]. Further, ethycrynic acid caused an increase in renal cell water and NaC1 content and a decrease of cell potassium content in kidney slices and suspensions of renal tubules [37—41]. There were changes in metabolism, however, that could not be completely explained on the basis of inhibition of cation transport [34, 36], and it was suggested that some of the effects of ethacrynic acid were secondary to inhibition of cell metabolism rather than a primary effect of the drug on a cation transport system [34, 40—42]. Ethacrynic acid

was found to inhibit respiration and glycolysis both in intact cells and in tissue homogenates from nonrenal [34, 43—46] and renal tissue [1, 47—51]. The inhibition of

renal metabolism required a concentration of ethacrynic acid that exceeded that found in vivo, however, and the effect was not regarded as related to the diuresis [48, 52].

Ethacrynic acid was also found to inhibit Na- and Kactivated adenosine triphosphatase (Na, K-ATPase) [1, 34,

48, 53—63]. The effect of ethacrynic acid on the renal ATPase was not specific, however, as was that of ouabain.

furosemide both cause the PD to decrease in the thick

Whereas ouabain inhibited Na- and K-activated, but not Mg-activated ATPase activity, ethacrynic acid inhibited

ascending limb of Henle's loop and they prevented the fall in NaCI concentration in the lumen. The combined result

both [34,56, 57, 62]. It is evident that ethacrynic acid has a number of differ-

could be to eliminate net potassium reabsorption in this segment and thus add to the kaliuresis. Calcium is also extensively reabsorbed in Henle's loop [22]. Calcium excretion is greatly increased by these diuretics [23, 24] and part of the mechanism of this increase might be analogous to that proposed above for potassium.

ent biological actions. We believe, however, that the

Discussion

Ethacrynic acid is one of a class of aryloxyacetic acid derivatives that were synthesized in order to produce more effective diuretic agents than the organic mercurials which were the most potent diuretics then known [1]. Since the mercurials were believed to act by inhibiting sulfhydrylcatalyzed systems, the aryloxyacetic acid derivatives were chosen for their selective reactivity with functionally important sulfhydryl groups [25]. Ethacrynic acid proved to be an extremely potent diuretic [1—5], even more effective than the organic mercurials [5]. At the time it was believed

that diuretics in general acted to inhibit active sodium

diuretic effect may be distinct from all of these and that, whereas the other effects may involve reactions with tissue sulfhydryl groups, the diuretic effect may not. These conclusions are principally based on the findings in the present

studies that 1) the ethacrynic-cysteine complex in the tubule lumen is more than 100 times more effective than ethacrynic acid itself in inhibiting NaC1 absorption in the

thick ascending limb of Henle's loop and that 2) it is active chloride transport that is inhibited by the drug, rather than active sodium transport, as previously believed. Most of the in vitro effects of ethacrynic acid are elicited

only by a high concentration of the drug. Approximately iO M was used in most of the studies quoted above and 10 M generally had little if any effect. Further, the addition of cysteine or dithiothreitol blocked the effect of ethacrynic acid [1, 28, 47, 62, 64]. A similarly high concentration of ethacrynic acid was required to decrease the PD across the thick ascending limb of Henle's loop. In contrast to the effects in other tissues, however, premixing the ethacrynic

306

Burg / Green

acid with cysteine greatly increased its effectiveness in the

lumen of renal tubule (although only the ethacryniccysteine complex is referred to here, it is possible that other analogous complexes are formed and have a similar effect).

Cysteine should block the reactivity of ethacrynic acid with other sulfhydryl groups rather than enhance it. It is conceivable that cysteine facilitated the access of ethacrynic

acid to sulfhydryl-containing receptors in the thick ascending limb of Henle's loop, but not in other tissues. We consider this possibility unlikely, however, and question whether diuresis in vivo involves sulfhydryl inhibition, as was previously believed. In support of our view, Beyer et al [1] noted that the diuretic effect of ethacrynic acid is not readily reversed by dimercaprol (BAL) as it might be were inhibition of sulfhydryl-containing enzymes essential to its action. In fact, there is little evidence that reactivity with sulfhydryl groups is involved in the diuretic action of ethycrynic acid except for the observation that the concentration of protein-bound sulfhydryl groups in dog kidneys is reduced [65] by the drug. If ethacrynic-cysteine is the form of the drug that causes diuresis, it is possible to explain why ethacrynic acid is

relatively nontoxic when used as a diuretic despite the multiple nonspecific effects mentioned above. The nonspecific effects of ethacrynic acid (e.g., its effects on metabolism and nonrenal sodium transport) could be prevented in vivo by complexing of the drug with proteins

or cysteine in plasma. The drug could then be concentrated in proximal tubule cells and secreted into the urine by the organic acid transport system (1]. Prior to or during

this process, the ethacrynic acid could be converted to ethacrynic-cysteine. Consistent with this view, ethacrynic acid, when incubated with kidney cells in vitro, becomes complexed to cysteine [55]. This mechanism explains how

a compound as reactive as ethacrynic acid can greatly reduce NaCI reabsorption in the thick ascending limb of Henle's loop without causing nonspecific effects in the kidney and other tissues. Not only is the ethacryniccysteine complex less toxic, but lower concentrations are required than of ethacrynic acid to inhibit NaCI reabsorption in the ascending limb of Henle's loop. Thus, the concentration of ethacrynic acid (1O M) necessary to decrease the PD when placed in the lumen of the thick ascending

limb caused an irreversible inhibition of the PD when placed in the bath, but a concentration of ethacrynic cysteine greater than that effective in the lumen had no effect when placed in the bath (10 M in the bath vs. 3 x 106ri in the lumen). Since ethacrynic cysteine decreased both the PD across the thick ascending limb of Henle's loop and the net absorption of chloride without any major change in sodium or chloride conductance, the drug most likely acts by inhibiting active chloride transport. This conclusion is consistent with the observation that chloride (not HCO3) excretion is specifically increased by the drug [1—4, 6, 66].

In most tissues the principal ions that are actively trans-

ported are sodium and potassium, and the mechanism of transport is believed to involve Na, K-ATPase. As discussed previously, it is difficult to see how the inhibition of Na- and K-activated ATPase by diuretic drugs could

directly explain their inhibition of the active chloride transport system in the thick ascending limb of Henle's loop [9]. In addition, it seems unlikely that inhibition of the Na- and K-activated ATPase could be involved in the diuretic action of ethacrynic acid since inhibition of the

active chloride transport is enhanced by addition of cysteine to ethacrynic acid, whereas inhibition of ATPase is reduced by cysteine [1, 62]. Acknowledgments

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