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Collecting Duct Is a Site of Sodium Retention in PAN Nephrosis: A Rationale for Amiloride Therapy

GEORGES DESCHÊNES^*,, MONIKA WITTNER, ANTONIO DI STEFANO, SYLVIE JOUNIER and ALAIN DOUCET

^* Service de Néphrologie Pédiatrique, Hôpital Armand-Trousseau Assistance Publique-Hôpitaux de Paris, Paris, France.
Laboratoire de Biologie Intégrée des Cellules Rénales, CNRS URA 1859, Service de Biologie Cellulaire, Commissariat à l'Energie Atomique, Saclay, France.

Correspondence to Dr. Alain Doucet, URA 1859, Bâtiment 520, CE Saclay, 91191 Gif sur Yvette, France. Phone: 33-1-69-08-97-63; Fax: 33-1-69-08-35-70; E-mail: doucet{at}dsvidf.cea.fr

Abstract

Abstract. Micropuncture studies of the distal nephron and measurements of Na,K-ATPase activity in microdissected collecting tubules have suggested that renal retention of sodium in puromycin aminonucleoside (PAN) nephrotic rats originates in the collecting duct. The present study demonstrated this hypothesis by in vitro microperfusion and showed that amiloride was able to restore sodium balance. Indeed, isolated perfused cortical collecting ducts from PAN-treated rats exhibited an abnormally high transepithelial sodium reabsorption that was abolished by amiloride, and in vivo administration of amiloride fully prevented decreased urinary sodium excretion and positive sodium balance in nephrotic rats. As expected from the aldosterone independence of Na⁺ retention in PAN nephrotic rats, blockade of aldosterone receptor by potassium canrenoate did not alter urinary Na⁺ excretion, Na⁺ balance, or ascites formation in PAN nephrotic rats.

Nephrotic syndrome is defined by massive proteinuria and is clinically characterized by edema and ascites secondary to renal sodium retention. In vivo micropuncture studies in the unilateral model of puromycin aminonucleoside (PAN) nephrosis have suggested that renal retention of sodium originates in the collecting duct (1). Whereas tubular sodium load at the end of the accessible distal convoluted tubule was similar in PAN and control kidneys of the same rat, the final urine sodium excretion was threefold lower in the PAN ureter compared with the control ureter (1). Recently, it was found that sodium retention in PAN nephrosis is correlated in time with increased Na,K-ATPase activity in the cortical collecting duct (CCD) (2). These findings suggest that the CCD is the renal site of sodium retention in nephrotic syndrome.

The aim of this study was to demonstrate directly the role of CCD in PAN nephrosis (1) by evaluating sodium reabsorption in in vitro microperfused CCD isolated from normal and PAN nephrotic rats and (2) by evaluating the potency of the collecting duct diuretics amiloride (sodium channel blocker) and potassium canrenoate (aldosterone-receptor antagonist) to prevent sodium retention and ascites in PAN nephrotic rats.

Materials and Methods

Animals
PAN nephrosis was induced in Sprague-Dawley male rats that weighed 140 to 160 g by a single intraperitoneal injection of PAN (Sigma, L'Isle d'Abeau Chesnes, France; 15 mg/ml in 0.9% NaCl, 1 ml/100 g body wt). Control rats received 1 ml/100 g body wt of the vehicle. Twenty ml of amiloride solution (150 mg/L [pH 7.0]) or potassium canrenoate solution (750 mg/L [pH 7.0]) or tap water was given overnight as drinking solution to three groups of PAN-treated and of vehicle-injected rats. Amiloride and potassium canrenoate treatments were initiated after the peak of sodium excretion observed at day 1 after PAN injection (Figure 1) to prevent massive salt loss. Rats had free access to tap water during the daytime period. Diuretic-treated PAN nephrotic rats had free access to food throughout the study, and their daily food intake was measured. Because of nonsignificant differences in food intake between the two groups of diuretic-treated PAN nephrotic rats, the mean daily food intake in these two groups was used as the index for a single group of pair-fed, diuretic-untreated, PAN nephrotic rats.

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Effect of amiloride and potassium canrenoate on urinary sodium excretion (A) and sodium balance (B) in puromycin aminonucleoside (PAN) nephrotic rats. Amiloride or potassium canrenoate were given in the drinking solution between days 2 and 6 to PAN-treated rats. Pair-fed control PAN nephrotic rats had free access to water. Results are means ± SD of 8 amiloride-treated, 6 potassium canrenoate—treated, and 11 control PAN nephrotic rats. Statistically significant differences (ANOVA Fisher test: *, P < 0.05; ***, P < 0.001) were observed between amiloride-treated and pair-fed PAN nephrotic rats at days 2 to 6, whereas no statistically significant differences were observed between potassium canrenoate—treated and pair-fed PAN nephrotic rats. (A) , amiloride; , K+ canrenoate; , vehicle. (B) [UNK], amiloride; , K+ canrenoate; , vehicle.

Animals were housed in individual metabolic cages with free access to food for 2 d before the onset of the experiments throughout the study. Urine was collected daily under mineral oil from 1 d before PAN or vehicle injection (day 0) to 6 d. Urine volume and food intake were measured each day to evaluate net urinary sodium balance. Blood samples were obtained by retro-ocular puncture on days 1 and 3 and by aortic puncture on day 6. Plasma and urine sodium, protein, creatinine, and plasma albumin concentrations were measured with an automatic analyzer (Hitachi 911, Boehringer-Mannheim, Germany). Ascites volume was measured after the rats were killed at day 6 by moistening and weighing an absorbent paper.

In Vitro Microperfusion
Experiments were carried out on medullary thick ascending limbs (MTAL), and CCD were microdissected from normal rats and PAN nephrotic rats at day 6 and perfused in vitro as previously described (3). In CCD, both transepithelial voltage (V_T) and transepithelial net sodium flux (J_Na) were determined, whereas in MTAL, only V_T was measured. Briefly, after microdissection, nephron segments were mounted on a set of concentric micropipettes and perfused at rates of 2.50 ± 0.26 nl/min (perfusate composition: 150 mM NaCl, 1.7 mM K₂HPO₄, 0.2 mM KH₂PO₄, 1 mM MgCl₂, 1 mM CaCl₂ [pH 7.4]) while the bath flow rate was 15 to 20 ml/min (bath composition: 150 mM NaCl, 1.7 mM K₂HPO₄, 0.2 mM KH₂PO₄, 1 mM MgCl₂, 1 mM CaCl₂, 5 mM glucose [pH 7.4]). In CCD, volume reabsorption was monitored by addition of [methoxy-³H]-inulin to the perfusate. Each experiment included an equilibration period followed by a first control series of three 10-min collections. After addition of furosemide (10 ^-4 M) or amiloride (10^-6 M) to the perfusate, a second period of equilibration was followed by a second experimental series of three 10-min collections. After withdrawal of the diuretic and a third equilibration period, a final recovery series of three 10-min collections were performed. Intratubular fluid was collected using siliconized micropipettes filled with water-saturated paraffin oil. A separate pipette was used for each experimental condition (with and without amiloride). The concentrations of sodium in the perfused solutions and the collected tubular fluids were measured by flame microspectrophotometry. J_Na (in pmol/mm^-1 per min^-1) was calculated for each collection period as previously reported (3). For each animal, a single tubule was studied and results were expressed as the mean of the three collections of a given series. V_T was measured throughout all experiments via NaCl-agar bridges and calomel half cells via the grounded bath. For each tubule, V_T values were the mean for a given experimental condition.

Results and Discussion

As previously described (4), V_T and J_Na were not statistically different from zero in CCD from normal rats (Table 1). Conversely, CCD from PAN nephrotic rats repeatedly displayed high J_Na and lumen-negative V_T. Luminal addition of amiloride (10^-6 M) fully abolished V_T and J_Na in CCD from PAN nephrotic rats, whereas it had no significant effect in control rats. Amiloride effect was fully reversible, as attested by V_T recovery after withdrawal of the drug. Altogether, these results demonstrate that the CCD is a site of increased sodium reabsorption and indicate that the apical step of sodium reabsorption in CCD from PAN rats is mediated by amiloride-sensitive sodium channels. This suggests their upregulation along with the previously reported stimulation of Na,K-ATPase activity (1, 5, 6).

As expected from an in vivo study (1), MTAL from normal and PAN nephrotic rats displayed similar sodium reabsorption capacity, as evaluated by the lumen-positive V_T (Table 1). At a concentration of 10^-4 M, sufficient for maximum inhibition of sodium transport in normal rat MTAL (7), furosemide also maximally inhibited V_T in MTAL from PAN nephrotic rats (Table 1). Thus, the lower diuretic efficiency of furosemide reported in nephrotic animals is not due to an intrinsic tissue resistance to the drug (8). Although the apparent resistance to furosemide was attributed initially to its binding to albumin within the tubular fluid (9), this has been ruled out recently in nephrotic patients (10). Rather, furosemide resistance in nephrotic syndrome might be due mainly to the increased potency of CCD to reabsorb an overload of sodium.

If the collecting duct is a site of increased sodium reabsorption in PAN nephrotic rats, then inhibition of this process should prevent sodium retention and ascites formation. Thus, we evaluated the effect of the sodium channel blocker amiloride and of the aldosterone-receptor antagonist potassium canrenoate on urine sodium excretion in PAN nephrotic rats. In the absence of amiloride treatment, urinary sodium excretion varied with time as reported previously in PAN nephrotic rats (2). After increasing at day 1, urinary sodium excretion (in mmol Na⁺/mmol creatinine) decreased from 25 to 30 to approximately 10 at days 2 to 4 and down to 3 at days 5 to 6 (Figure 1A). Accordingly, urinary Na⁺ balance was slightly negative at day 1 and became highly positive from day 2 to day 6 (Figure 1B). Oral administration of amiloride, at a high dose of 18.2 ± 3.7 mg/kg per d to circumvent impaired drug bioavailability, fully prevented Na⁺ retention. Urine Na⁺ excretion at days 2 to 6 was not statistically different from control values (day 0) and was significantly higher than in diuretic-untreated, pair-fed PAN nephrotic rats (Figure 1A). Accordingly, amiloride equilibrated urinary Na⁺ balance within 1 d of treatment (Figure 1B), and, consequently, amiloride-treated PAN nephrotic rats exhibited a significantly lower body weight than pair-fed PAN nephrotic rats (Table 2). The normalization by amiloride of urinary Na⁺ excretion and Na⁺ balance in PAN nephrotic rats rules out any involvement in Na⁺ retention of tubular segments upstream of the collecting duct. In PAN-untreated rats, amiloride (17.5 mg/kg per d) had a transient effect on Na⁺ handling: urinary Na⁺ excretion increased (from 32.4 ± 4.0 to 50.4 ± 6.5 mmol Na⁺/mmol creatinine; n = 6; P < 0.0001) at day 1 but not during the following 3 d.

Conversely, potassium canrenoate given to PAN nephrotic rats at an oral dose of 100 mg/kg per d had no effects on Na⁺ excretion, Na⁺ balance (Figure 1, A and B), or body weight (Table 2). This result is consistent with most studies that deny a role for aldosterone in the Na⁺ retention of PAN nephrotic rats: (1) adrenalectomy does not prevent PAN-induced ascites and renal Na⁺ retention in rats (5, 11); (2) plasma volume is not reduced in PAN nephrotic rats (12); (3) captopril fails to induce natriuresis in PAN nephrotic rats (13); and (4) in the unilateral model of PAN nephrosis, Na⁺ retention occurs in the proteinuric kidney but not in the unaffected kidney (1). In addition, in three independent threapeutic trials, spironolactone or potassium canrenoate had no to mild natriuretic effect (1- to 1.5-fold stimulation) in nephrotic patients (14,15,16). Potassium canrenoate administration (100 mg/kg per d) to PAN-untreated rats led to a transient increase in Na⁺ excretion (from 25.5 ± 1.9 to 33.4 ± 4.0 mmol Na⁺/mmol creatinine; n = 6; P < 0.001) at day 1 but not during the following 5 d.

Irrespective of amiloride or potassium canrenoate administration, proteinuria appeared at day 4 after PAN administration and reached similar levels at day 6 in amiloride-treated (9.1 ± 2.0 g/mmol creatinine), potassium canrenoate—treated (9.4 ± 1.3 g/mmol creatinine), and pair-fed PAN nephrotic rats (7.9 ± 4.2 g/mmol creatinine; not significant). Accordingly, plasma total protein and albumin fell to similar levels in the three groups at day 6 (Table 2). Plasma creatinine remained stable throughout the study in the three groups of rats, except for a 60% increase in amiloride-treated PAN nephrotic rats at day 6. This likely was due to the superimposition of a low plasma protein level with a cumulative negative sodium balance. In contrast with proteinuria but as expected from changes in Na⁺ balance, ascites was present at day 6 in pair-fed PAN nephrotic rats (4.9 g ± 3.0; n = 6) and in potassium canrenoate—treated PAN nephrotic rats (6.6 ± 3.5; n = 6; not significant) but not in amiloride-treated PAN nephrotic rats (n = 5).

In conclusion, (1) in vitro perfused CCD isolated from Na⁺-retaining PAN nephrotic rats exhibited an abnormally high transepithelial Na⁺ reabsorption that was completely inhibited by amiloride; (2) in vivo administration of amiloride normalized urinary Na⁺ excretion and Na⁺ balance in nephrotic rats, suggesting that the effect of amiloride should be evaluated in human nephrotic syndrome; (3) normalization of renal Na⁺ balance by amiloride did not modify albuminuria; and (4) as expected from the aldosterone independence of Na⁺ retention in PAN nephrotic rats, blockade of aldosterone receptor did not influence the pattern of urinary Na⁺ excretion, Na⁺ balance, or ascites formation in nephrotic rats.

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