INTRODUCTION

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ABUNDANT EXPRESSION of endothelin (ET)-1 peptide and mRNA for its precursor, preproendothelin-1 (prepro-ET-1), have been found in the vascular endothelium of the renal vascular bed, including glomerular capillaries, arterioles, and peritubular capillaries (15, 24). Subsequent studies have demonstrated that ET-1 is present in high concentrations in the inner medulla of the kidney (24), where ET-1 receptors are also located (5), and that ET-1 secretion is not limited to vascular endothelium, because primary epithelial cell cultures derived from the renal tubule secrete abundant amounts of ET-1 (10). ET-1 secretion in the renal medulla is highly compartmentalized in tubules with the following secretion hierarchy: inner medullary collecting ducts (IMCD) > medullary thick ascending limb > cortical collecting ducts > proximal tubule (11, 29).

The physiological activities of ET-1 on renal sodium and water handling still remain to be clarified (11). Convincing evidence exists that ET-1 may inhibit arginine vasopressin (AVP)-stimulated water reabsorption in the collecting ducts. Several studies have demonstrated that ET-1 binding to the ET_B receptor subtype reduces Na⁺-K⁺-ATPase activity in the IMCD (34) and inhibits vasopressin-stimulated cAMP accumulation and water transport on isolated cortical collecting ducts from rats (23, 30). These and other studies (11, 29) indicate that ET-1 might regulate water reabsorption independently of its effects on Na⁺ reabsorption or renal hemodynamics.

Studies investigating the activity of ET-1 on sodium reabsorption have given conflicting or unconvincing results. ET-1 inhibited Na⁺-K⁺-ATPase activity in a suspension of rabbit IMCD (34), suggesting a possible natriuretic effect. Administration of anti-ET-1 antibodies to rats on a low-sodium diet increased urinary salt and water (32). Experiments performed by infusing ET-1 in isolated rat kidney (22) or in vivo gave conflicting results, because either a sodium-retentive (8) or a natriuretic (13, 31) effect was observed. However, the natriuresis observed after ET-1 infusion might result from release of the atrial natriuretic peptide (ANP) induced by ET-1 infusion (19). Moreover, the natriuretic effect might be secondary to hemodynamic changes such as the increase in arterial blood pressure (pressure natriuresis) (31). On the other hand, the sodium retention reported after ET-1 infusion both in rabbits (8) and in humans (25) might be caused by intrarenal blood flow redistribution even if the total renal blood flow was not decreased (25).

Thus the pharmacological approach to investigation of physiological function as performed by infusing ET-1 may be confounding because the active agents can provoke additional known or unknown effects. Studies performed in humans without ET-1 infusion but reproducing some physiological situations have shown that urinary ET-1 is inversely related to changes in blood volume. Acute volume expansion caused, in addition to the notable increase in urinary volume, a marked decrease in urinary ET-1 excretion (21). Conversely, a sustained enhancement of urinary ET-1 excretion with a considerable decrease in urinary volume and sodium excretion occurred during long-lasting standing posture (17). These findings suggest that renal ET-1 may be involved in the mechanisms of control of diuresis and natriuresis.

To investigate this hypothesis we studied whether different sodium intakes affect synthesis of renal ET-1. Because experimental studies (27) and investigations performed in humans (17) suggest that angiotensin-converting enzyme (ACE) inhibition may influence renal ET-1 excretion, we studied renal ET-1 excretion during a low-sodium diet in the presence of inhibition of ANG II formation.

	METHODS

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Subjects Investigated

Experimental Protocols

In another separate set of experiments, after one wash-out week on a normal-sodium diet, the subjects were treated with ramipril (5 mg/day) for 7 days and then underwent a 7-day low-sodium diet period while they continued ramipril treatment.

Throughout the experimental periods the subjects had the same basic diet containing a constant amount of protein (1.3 g/kg), calories (126 kJ/kg), calcium (800 mg/24 h), and potassium (80 meq/day). Urinary and plasma samples for the determination of ET-1, sodium, lithium, osmolality, creatinine, and plasma renin activity (PRA) were taken daily during the last 3 days before the different diet regimens started and every day throughout the study periods.

Sampling Procedure and Radioimmunoassays

Blood samples for PRA were collected in ice-cold tubes containing EDTA (0.084 ml tripotassic EDTA in 7 ml blood; final concentration 0.34 mol/l) and immediately centrifuged, then stored at 20°C. PRA measurement was performed by measuring the quantity of ANG I generated in vitro by radioimmunoassay with a commercial kit (Sorin Biomedica) and was expressed as nanograms of ANG I per milliliter of plasma per hour of incubation.

Analytical Methods and Calculations

Free water clearance (C_H2O) was calculated as where U_osm (mosmol/kg H₂O) and P_osm (mosmol/kg H₂O) are the simultaneous urinary and plasma osmolalities, respectively, and V (ml/min) is the simultaneous rate of urinary volume flow. Negative values of C_H2O occur when the urine is hypertonic to the plasma.

Osmolar clearance, sodium clearance (C_Na), lithium clearance (C_Li), and C_Cr were calculated using standard formulas. C_Li is a measure of fluid delivery from the proximal tubule to the loop of Henle. The ratio of sodium to lithium clearances (C_Na/C_Li) is thus a measure of that fraction of the sodium delivered to the distal tubule which is finally excreted, and C_Li  C_Na is a measure of the absolute rate of sodium reabsorption from the distal tubule (3). Finally, we calculated the cumulative sodium balance during the last 3 days of sodium diet or ACE treatment period.

In Situ Hybridization

Kidney specimens were cut from the pole opposite the tumor. Kidney portions containing cortex and medulla from the six kidneys were fixed in 4% paraformaldehyde in 0.12 M phosphate buffer (pH 7.2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and prepro-ET-1 probes were transformed from Escherichia coli with separate plasmid vectors harboring the cDNA for GAPDH (pHcGAP, ATCC no. 57090) and prepro-ET-1 (ET1c, ATCC no. 65698). Recombinant plasmids were purified from 5 ml each of E. coli growth using the FlexyPrep kit (Pharmacia). After digestion we separated the cDNA fragments from the plasmid by electrophoresis on a 1% agarose gel and then purified by cutting the gel slice of the corresponding molecular insert size (1.2 kb for both ET-1 and GAPDH) using the BandPrep Kit Sephaglas (Pharmacia). Streptavidin-biotinylated horseradish peroxidase complex in buffered sodium chloride was used as the detection reagent (28). Specimens were stained with 3-amino-9-ethyl carbazole (Sigma) for 10 min at 37°C and counterstained with Mayer's hematoxylin. To ensure the specificity of the in situ hybridization signals, negative controls were performed by testing the sections with hybridization mixture 1) without the probe and 2) after incubation with RNAase A (1 Kunitz unit/l) for 1 h at 37°C. Positive controls were obtained for each sample using a cDNA probe for the constitutively expressed GAPDH to ensure that mRNA in kidney specimens was intact. In situ hybridization staining was performed at the same time for all specimens and at least twice on serial sections in each specimen. The presence of mRNA signals was assessed by light microscopy.

Statistical Analysis

	RESULTS

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Effects of Different Sodium Intakes

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Urinary endothelin (ET)-1 excretion (A) and plasma ET-1 concentration (B) in healthy subjects at baseline (day 2 to day 0) and during three 7-day sodium diet regimens (day 1 to day 7), indicated by bars. , Low-sodium diet, 20 meq/day; , normal-sodium diet, 108 meq/day; , high-sodium diet, 300 meq/day. * P < 0.05 vs. baseline (day 0).

The values of the different analytical parameters obtained at the end of the 7-day normal-sodium diet are reported in Table 1.

Urinary ET-1 excretion at the end of the high-sodium diet period had significantly decreased (28%, P < 0.05), whereas ET-1 plasma concentration had on average increased by 103% (P < 0.05) (Table 1). The decrease in urinary ET-1 excretion became significant from day 2 (22 %, P < 0.05) and reached a nadir at day 3 (34%, P < 0.05), and then values plateaued (Fig. 1, Table 1). PRA decreased from 1.50 ± 0.21 to 0.28 ± 0.10 ng ANG I · ml¹ · h¹ (P < 0.05). GFR did not change, whereas urinary flow rate and C_Na increased and C_H2O decreased (Table 1).

The low-sodium diet was associated with an increased daily ET-1 urinary excretion (from 2.58 ± 0.62 to 4.28 ± 1.34 pg/min, +66%, P < 0.05). Plasma ET-1 concentration did not change significantly (Fig. 1, Table 1). PRA increased after 2 days of diet (P < 0.05), and it was 4.26 ± 0.37 ng ANG I · ml¹ · h¹ (P < 0.05) at the end of the low-sodium diet. During the low-sodium diet period, GFR did not significantly change, whereas the urinary flow rate decreased from 54 ± 16 to 46 ± 15 ml/h at the end of the diet period (P < 0.05). Similarly, there was a marked reduction in sodium excretion (from 117 ± 5 to 21 ± 3 meq/24 h) and C_Na (from 0.57 ± 0.02 to 0.10 ± 0.01 ml/min, P < 0.05).

At the end of the low-sodium diet, C_Li was reduced by 51% (P < 0.05). At the same time, the fraction of sodium delivered to the distal tubule that is finally excreted, expressed by the ratio C_Na/C_Li, decreased by 62% (P < 0.05) (Table 1). C_H2O significantly increased on the second day (from 0.88 ± 0.30 to 0.38 ± 0.40 ml/min, P < 0.05) and rose further up to the seventh day (0.17 ± 0.13 ml/min, P < 0.05) (Table 1).

Urinary ET-1 excretion changes from a normal- to a low-sodium diet were negatively related to contemporary changes in sodium excretion (r = 0.97, P < 0.05) and were positively correlated to C_H2O (r = 0.97, P < 0.05) (Fig. 2).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Relationships of urinary ET-1 with sodium excretion (A) and free water clearance (CH2O; B) during low-sodium diet without () and with () angiotensin-converting enzyme (ACE) inhibition. Values are expressed per unit of glomerular filtration rate (GFR).

In situ hybridization. In situ hybridization studies performed in kidneys from patients on a normal-sodium diet showed that the prepro-ET-1 mRNA signal was present in the smooth muscle cells and endothelial cells of arcuate and interlobular arteries, whereas almost no signal was observed within the glomeruli (Fig. 3). The hybridization signal was observed in the peritubular capillary network in the cortical region (Fig. 3), in vasa recta, and in the loose connective tissue of the inner medulla. In the cortical region the epithelial cells did not show the hybridization signal in either the proximal or the distal tubule (Fig. 3). A weak, spotted hybridization signal was observed in the epithelial cells of the medullary collecting tubule (Fig. 3).

View larger version (110K): [in this window] [in a new window]   Fig. 3.   In situ hybridization in kidney specimens from patients on normal-sodium diet. A: no signal for preproendothelin (prepro-ET-1) mRNA was observed within glomeruli and in epithelial cells; positive prepro-ET-1 mRNA hybridization signal in peritubular capillary network (×200). B: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in epithelial cells of inner medulla (×1,000). C and D: weak, spotted prepro-ET-1 mRNA hybridization signal was observed in epithelial cells of medullary collecting tubule in longitudinal (C) and transverse (D) sections (×1,000).

View larger version (117K): [in this window] [in a new window]   Fig. 4.   In situ hybridization for prepro-ET-1 mRNA in kidney specimens from patients on low-sodium diet. A: increased hybridization signal in peritubular capillary network; no signal is detectable in epithelial cells of the proximal as well as of the distal tubule (×200). B: in situ hybridization of RNAase-treated specimen (×1,000). C and D: hybridization signal of prepro-ET-1 mRNA expression in epithelial cells of collecting tubules in longitudinal (C) and transverse (D) sections (×1,000).

Effects of ACE Inhibition

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View larger version (34K): [in this window] [in a new window]   Fig. 5.   Effects of ACE inhibition (day 8 to day 14) in patients on low-sodium diet (day 1 to day 14) on rate of ET-1 urinary excretion (A), urinary sodium excretion (B), and cumulative sodium balance of last 3 days of each period (C), and CH2O (D). * P < 0.05 vs. baseline (day 0).

Sodium excretion showed consensual modifications to those of ET-1 excretion (Fig. 5B). On the first day of ramipril administration there was a marked and transient increase in C_Na (from 0.10 ± 0.01 to 0.23 ± 0.04 ml/min, P < 0.05), which, however, showed a tendency to return to the pretreatment values (0.13 ± 0.02 ml/min, P < 0.05 vs. both pretreatment values and vs. the first day of ramipril administration). The cumulative sodium balance of the last 3 days of ramipril treatment, when sodium and ET-1 excretion remained steady, was 16 ± 12 meq in comparison to 2 ± 5 meq (P < 0.05), observed during the low-sodium diet before ramipril treatment (Fig. 5C).

C_Li and C_Na/C_Li increased during the first 24 h of ramipril administration (Table 1), whereas C_H2O decreased. After the first day of ACE inhibition all these functional indexes returned to the pretreatment values, whereas C_H2O remained significantly reduced in comparison to pretreatment values (Table 1; Fig. 5D). Thus the partial recovery of ET-1 excretion despite ACE inhibition was associated with consensual changes in C_H2O (from 0.52 ± 0.11 to 0.29 ± 0.16 ml/min, P < 0.05; Fig. 5D).

When ACE inhibition was started before the low-sodium diet no significant differences in ET-1 excretion, urinary volume, sodium excretion, or C_H2O were found in comparison to ACE inhibition started during the low-sodium diet (data not shown). The very good correlations found between ET-1 excretion and both sodium excretion and C_H2O during the low-sodium diet were also maintained when the low-sodium diet was started under ACE inhibition (r = 0.99, P < 0.05 and r = 0.98, P < 0.05, respectively), although they had slightly shifted to the left (Fig. 2).

	DISCUSSION

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The present findings indicate that a low sodium intake is associated with an increase in prepro-ET-1 mRNA expression in endothelial cells of the peritubular capillary network and, especially, in epithelial cells of the medullary collecting tubules. These changes of mRNA expression for prepro-ET-1 were associated with increased urinary ET-1 excretion. Conversely, the high-sodium diet was associated with a decrease in urinary ET-1 excretion.

Our results demonstrate for the first time that renal ET-1 synthesis in humans is affected by changes in sodium intake. The modifications in renal prepro-ET-1 mRNA expression were associated with consensual variations in urinary ET-1 excretion, thus indicating that urinary ET-1 excretion is the main expression of renal ET-1 formation. Previous studies showed that infusion of 30 ml/kg of a 5% glucose solution over 1 h caused a marked increase in plasma ET-1 concentration associated with an almost complete disappearance of urinary ET-1 (21). In the present study the increase in urinary ET-1 excretion without any changes in plasma ET-1 during low sodium intake, and, conversely, the increased plasma ET-1 concentration with a contemporary decrease in ET-1 excretion after a high-sodium diet, suggest that plasma ET-1 and renal ET-1 are two independent compartments.

The expression of mRNA for prepro-ET-1 during the low-sodium diet increased in the endothelial cells of the peritubular capillary network and in the epithelial cells of the medullary collecting tubules. Although the contribution of this ET-1 arising from endothelial cells to the total amount of urinary ET-1 cannot be ruled out, it is probably negligible, because studies performed in rats showed that only trace amounts of injected ¹²⁵I-labeled ET-1 passed in the urine (from 0.27 to 0.30% of the total infused ¹²⁵I-labeled ET-1) (1); as a consequence, the urinary ET-1 almost entirely derives from the renal tubule.

The increased renal ET-1 formation was associated with an enhancement in sodium retention. The increased prepro-ET-1 mRNA expression in endothelial cells of the postglomerular vascular capillary network circumventing the proximal and distal tubules probably contributes to the mild decrease in renal blood flow that occurs during low sodium intake (9). The decrease in renal blood flow mainly occurs in the renal medulla, where vasoconstrictor ET_A receptors are highly expressed on interstitial cells and pericytes of the vasa recta (6). As a consequence of the decreased blood flow, the medullary osmolarity increases; this in turn produces an augmented sodium reabsorption (9). This sequence of events is suggested by the decreased fraction of sodium delivered to the distal tubule that is finally excreted (C_Na/C_Li). This distal sodium reabsorption was largely independent of ANG II activity, because ramipril administration only mildly reduced distal sodium reabsorption (Table 1). Thus these findings suggest a sodium-retentive activity of ET-1 in vivo. Several experimental findings obtained in vitro seem to speak against a sodium-retentive activity of ET-1. In a suspension of isolated IMCD cells, ET-1 has been demonstrated to inhibit Na⁺-K⁺-ATPase activity (34) and to inhibit Na⁺ channels in the luminal membrane by a Ca⁺-dependent process in the cortical collecting duct cells (14), thus resulting in a decrease of sodium entry from the lumen of epithelial cells. However, the effects of ET-1 in vivo appear to be more complex. Clavell et al. (6), using selective ET_A and ET_B antagonists, elegantly showed in dogs that the natriuretic effect of ET-1 at the level of epithelial cells becomes evident only when ET_A vasoconstrictor receptors are selectively blocked. When ET-1 was infused in humans at low doses (1 ng · kg¹ · min¹), it caused sodium retention accompanied by a fall in the fractional excretion of lithium (25). Thus, in vivo, the sodium-retentive activity of ET-1 seems to prevail over the natriuretic effect found in in vitro studies. The increased sodium retention resulting from the increased medullary osmolality found in the present investigation may be considered as an indirect effect of ET-1, but the net result is a reduced sodium excretion.

An increased C_H2O was the other important functional modification associated with the increase in ET-1 urinary excretion. In addition to the peritubular capillaries, a marked increase in mRNA expression for ET-1 was also observed at the epithelial cells of inner medullary collecting tubules and ducts during low sodium intake. AVP secretion is the only condition affecting C_H2O, and thus a decrease in this secretion might be responsible for the increase in C_H2O observed in subjects during the low-sodium diet. We did not measure AVP plasma concentration, but it does not increase in low sodium intake (4). Several studies have shown that ET-1 negatively modulates the effects of AVP on the distal tubule by inhibiting both AVP-stimulated cAMP accumulation (12) and water permeability in the rat terminal IMCD (20, 23, 29). Therefore, the increased C_H2O seems to be attributable to the increased ET-1 formation by the epithelial cells of the collecting tubules and ducts.

The factors responsible for the increased ET-1 renal formation remain to be clarified. Several studies have shown that ANG II increases mRNA expression for prepro-ET-1 in isolated cells (27). Therefore, the increased mRNA expression for prepro-ET-1 in the endothelial cells of the peritubular capillary network found during low sodium intake might be caused by the increased ANG II formation. In the present investigation, changes in ET-1 urinary excretion paralleled renin-angiotensin system activity. The further PRA increase after ramipril administration in patients on a low-sodium diet is an index of the inhibition of ACE and then of a reduced ANG II formation. However, ET-1 excretion was only partially reduced by ACE inhibition, thus indicating that renal ET-1-forming activity was largely independent of the renin-angiotensin system. Moreover, the increased kinin activity that occurs during ACE inhibition (2) might directly inhibit ET-1 renal production, because previous studies showed that bradykinin inhibited ET-1 production from cultured endothelial cells (18).

An increase in renal medulla osmolality was found to stimulate mRNA expression for prepro-ET-1 in rat and rabbit epithelial tubular cells (16, 33). Moreover, reduced renal perfusion in rats and rabbits considerably enhanced ET-1 synthesis by the epithelial cells of the medullary tubules (7). During low sodium intake, blood flow decreases and medullary osmolality increases (9). Thus the increased medullary osmolality rather than PRA appears to be the main factor causing the increased mRNA expression for prepro-ET-1.

Plasma ET-1 levels increased during high sodium intake in contrast to the decrease in urinary ET-1 excretion. The augmented plasma ET-1 levels are probably caused by the volume expansion that occurs in this condition (26), because experimental acute volume expansion (21) or spontaneous chronic volume expansion as observed in congestive heart failure (27) were both found to be associated with increased plasma ET-1 levels.

In conclusion, the present results indicate that renal ET-1 is influenced by sodium intake. More particularly, a low-sodium diet is associated with increased mRNA expression for prepro-ET-1 both in the endothelial cells of the peritubular capillaries and in the epithelial cells of the medullary collecting tubules. The increase in ET-1 synthesis is associated with an increase in both sodium retention and C_H2O. The enhanced mRNA expression for prepro-ET-1 in the endothelial cells of the peritubular capillaries is indirectly responsible for the increased sodium reabsorption, which is mediated by the enhanced medullary osmolality, whereas the increased synthesis of ET-1 by the epithelial cells of the medullary collecting ducts may explain the increase in C_H2O observed during a low-sodium diet.