Most renal K⁺ is excreted through secretory K⁺ channels located in the apical membrane of principal cells in the cortical collecting tubule [10-12]. The electrochemical driving force for distal nephron K⁺ secretion (that is, increased urinary lumen negativity and high intracellular K⁺ levels) is maintained by luminal Na⁺ entry through apical Na⁺ channels and serosal K⁺ uptake through the basolateral Na^+/K+ -adenosine triphosphatase pump [10-13].

Pentamidine is an aromatic diamidine structurally similar to "potassium-sparing" diuretics such as amiloride, triamterene, and trimethoprim [5, 13, 14] (Figure 1). We therefore applied transepithelial and single-channel measurement techniques to two well-established models of cortical collecting tubule ion transport (A6 amphibian cell line and primary cultured rabbit cortical collecting tubules) to investigate the effects of pentamidine on renal tubular Na⁺ reabsorption and, therefore, K⁺ secretion.

View larger version (20K): [in this window] [in a new window]   Figure 1. Structures of pentamidine and "potassium-sparing" diuretics such as amiloride, triamterene, and trimethoprim.

Methods

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Transepithelial Measurements on A6 Distal-Nephron Cell Line Cultures

The A6 cell (American Type Culture Collection, Rockville, Maryland) subpassages 91 to 96 were grown to confluency on collagen-coated polycarbonate filters (Costar; Cambridge, Massachusetts) in the presence of 1.5 µmolars of aldosterone. Filters were mounted in a modified Ussing chamber, and macroscopic current was measured at room temperature with a DVC-1000 voltage clamp (World Precision Instruments, Sarasota, Florida) [14-16]. We measured short-circuit current under voltage-clamp conditions and added 10 µmolars of amiloride to the luminal bath at the end of each experiment to determine the amiloride-sensitive component of the short-circuit current. Bath solutions contained the following: 100 mM of NaCl, 4 mM of KCl, 2.5 mM of NaHCO₃, 1 mM of KPO₄, 1 mM of CaCl (₂), 11 mM of glucose, and 10 mM of n-2-hydroxyethylpiperazine-n-2-ethanesulfonic (HEPES) (pH, 7.4).

Patch Clamp Measurements on Primary Cultures of Rabbit Cortical Collecting Tubules

As previously described [17], renal cortices from New Zealand white rabbits (body weight, 1 to 2 kg) were collagenase digested and subjected to Percoll density centrifugation. Rabbit cortical collecting tubule fragments were separated and grown to confluency on permeable, collagen-coated Millipore-CM filters (Millipore Corp., Bedford, Massachusetts) in the presence of 1.5 µmolars of aldosterone. Unitary channel events were measured at 37 °C with a List EPC-7 Patch Clamp (Medical Systems Corp., Greenvale, New York). Data were digitized, recorded, and analyzed as previously described [10, 14, 17]. Patch pipette and extracellular bath solutions consisted of a physiologic saline solution containing the following: 140 mM of NaCl (final NaCl concentration after titration to a pH of 7.4 with NaOH), 5 mM of KCl, 1 mM of CaCl₂, 1 mM of MgCl₂, and 10 mM of HEPES (pH, 7.4).

Chemicals

Pentamidine (1,5-bis[p-Amidinophenoxyl]-pentane bis[2-hydoxyethane-sulfonate salt]) was of the highest commercial grade available (Sigma Chemical, St. Louis, Missouri). We added appropriate solvent vehicles to control baths that by themselves did not change the Na (⁺) channel activity.

Results

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The amiloride-sensitive component of the short-circuit current is a measure of the net Na⁺ transport across the renal epithelium [14, 16, 18]. When we applied various concentrations of pentamidine to the solution, bathing the luminal or "urinary" surface of A6 distal nephron cell monolayers, we observed a dose-dependent inhibition of the amiloride-sensitive, short-circuit current (five experiments) (Figure 2). This inhibition developed rapidly, and 50% blockage of the amiloride-sensitive, short-circuit current (50% inhibitory concentration) occurred with 700 µmolars of pentamidine at a pH of 7.4.

View larger version (11K): [in this window] [in a new window]   Figure 2. Effect of pentamidine on short-circuit current (Isc) in A6 distal nephron cells. The amiloride-sensitive component of the short-circuit current is expressed as a percentage of the control values. For pentamidine applied to the luminal surface, data are presented as the mean ±SE (five experiments).

In previous studies, we have characterized the amiloride-sensitive, 4-picosiemen Na⁺ channel that is responsible for physiologic, mineralocorticoid-dependent Na⁺ reabsorption in the mammalian distal nephron [17]. We therefore examined the effect of pentamidine on 4-picosiemen Na⁺ channel activity in apical, cell-attached patches on principal cells of primary cultured rabbit cortical collecting tubules. The results, summarized in Table 1, show that addition of 1.0 µmolars of pentamidine to the serosal bath or luminal bath (outside the cell-attached pipette) did not significantly affect Na⁺ channel activity within the unexposed patch membrane (no pentamidine was added to the pipette solution). In contrast, when pentamidine was placed in direct contact with the luminal surface of the patch membrane (1.0 µmolars of pentamidine in the pipette solution), Na⁺ channel activity (measured as the number of channels times the open probability) decreased to 40% of control values.

The pentamidine-induced inhibition of Na⁺ channel activity appeared to be primarily caused by a decrease in open probability (percentage of time an individual channel is open) rather than the number of channels per patch (membrane channel density) (Table 1). We confirmed this impression in four cell-attached patches that contained only one Na⁺ channel and therefore permitted us to directly calculate open probability (Figure 3). In these latter experiments, we could also show reversibility of the inhibitory effects of luminal pentamidine on Na⁺ channel kinetics.

View larger version (21K): [in this window] [in a new window]   Figure 3. Effect of intrapipette pentamidine on Na+ channel activity in rabbit cortical collecting tubule primary cultures.Top. Open probability was measured from apical, cell-attached patches containing only one Na+ channel. Open probability was calculated from 3 minutes of continuous recording at the resting principal cell membrane potential. Lines connect data from the same patch experiment. Bottom. Actual single-channel traces from the patch represented by open circles in the top panel. Downward channel transitions represent inward current (pette to cell) or Na+ reabsorption at resting membrane potential. Original corner frequency was done at 1 KHz, sampling at 2 KHz, and additional software filtering at 200 Hz. C = zero current level (closed state); O = open state; pA = picoamperes.

Discussion

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The electrochemical driving force for K⁺ secretion in the cortical collecting tubule is maintained by luminal Na⁺ entry through apical Na (⁺) channels and by serosal K⁺ uptake through the basolateral Na^+/K+ -adenosine triphosphatase pump [10-12]. We have previously shown that the mechanism of action for "potassium-sparing" diuretics, such as amiloride, trimethoprim, and triamterene, is direct blockade of these apical Na⁺ channels [13, 14, 17, 18]. Therefore, unlike most diuretics, which promote both natriuresis and kaliuresis, these latter drugs actually inhibit kaliuresis [13, 16].

In this study, we showed that luminal exposure to pentamidine inhibits Na⁺ reabsorption in both mammalian and amphibian distal nephron cells. Luminal pentamidine inhibited both amiloride-sensitive, macroscopic short-circuit currents and individual Na⁺ channels at concentrations greater than 50 µmolars and 1.0 µmolars, respectively. Differences in the apparent inhibitory constant of pentamidine for the Na⁺ channel in rabbit principal cells and in amphibian A6 cells may reflect either subtle variabilities within the structure of Na⁺ channels expressed in mammalian and amphibian renal cells or the different techniques used to examine Na⁺ transport [14, 19].

Little information is available on the pharmacokinetics of pentamidine in humans [5]. Pentamidine has a serum half-life of 5 to 6 hours after one parenteral dose, but the half-life increases to a mean of 52 ±89 hours after multiple doses [20]. Only 15% to 20% of a single intramuscular dose is excreted daily in the urine, yielding urinary concentrations of 20 to 25 µg/mL (1 µg/mL micromolars) [5]. In patients with impaired renal function, parenteral pentamidine is excreted in the urine at a rate of 1.85 to 7.13 mg/d [20]. Urinary concentrations after daily administration of aerosolized pentamidine range from 1.3 to 778 ng/mg of creatinine per milliliter [21]. Therefore, most of the administered pentamidine becomes protein- and tissue-bound, with an estimated volume of distribution of 3 L/kg body weight and the greatest accumulation occurring in the kidney [5]. Urinary levels are detectable for months after therapy is discontinued. In patients with AIDS, autopsy studies have found detectable levels of pentamidine in renal tissue for as long as 1 year after the last dose [1]. Tissue depot release probably accounts for the wide range of reported urinary concentrations and the observation that pentamidine-induced hyperkalemia can persist for days after the drug is withdrawn [2].

In conclusion, pentamidine therapy for treating HIV-infected patients with P. carinii pneumonia can be associated with life-threatening hyperkalemia. We have shown that pentamidine, at concentrations found clinically in the urine, directly and reversibly blocks apical Na⁺ channels in a manner similar to potassium-sparing diuretics. The result is a decrease in the electrochemical driving force for both K⁺ and H⁺ secretion in the cortical collecting tubule. It is therefore not surprising that hyperkalemia and hyperchloremic metabolic acidosis are observed in patients treated with pentamidine, amiloride, triamterene, or trimethoprim [6, 7, 9, 14, 22]. These renal tubular effects provide a mechanism for pentamidine-induced hyperkalemia in patients without severe renal failure, tubulointerstitial damage, adrenocortical insufficiency, or hyporeninemic hypoaldosteronism.

Portions of this work were presented at the American Society of Nephrology Annual Meeting in November 1993.