1 INSERM Unité 367, Institut du Fer à Moulin, Paris, France,
2 Centre de Recherche, Hôpital du Sacré-Cur, Université de Montréal, Montréal, Canada and
3 Groupe Hospitalier BichatClaude Bernard, Service de Diabétologie, Endocrinologie, Métabolisme, Paris, France
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Abstract |
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Methods. UAE was measured in normal Wistar rats and healthy humans, or in subjects with various forms of diabetes insipidus (DI), before and after acute or chronic infusion of the vasopressin V2 receptor agonist dDAVP®. Chronic dDAVP administration was also performed in normal Wistar rats previously submitted to either chronic angiotensin-converting enzyme inhibition (ACEI) or chronic blockade of AT1 receptors (ARB).
Results. In rats, acute or chronic dDAVP infusion increased UAE significantly and reversibly (4-fold and 6-fold, respectively). In healthy subjects, acute infusion of dDAVP tripled UAE (P<0.01) but did not change creatinine and ß2-microglobulin excretion, thus suggesting that the rise in UAE was due to an increased glomerular leakage of albumin. dDAVP also increased UAE in patients with central DI and in patients with hereditary nephrogenic DI bearing AQP2 mutations. However, UAE was not increased in patients with hereditary nephrogenic DI bearing mutations of the V2 receptor. In rats, ACEI and ARB blunted the dDAVP-induced rise in UAE by 70% (P<0.05) and 50% (NS), respectively.
Conclusions. The present studies reveal for the first time that vasopressin induces a marked increase in UAE in healthy rats and humans. This albuminuric effect seems to result from increased glomerular leakage, requires functional vasopressin V2 receptors, and is, at least in part, mediated by the reninangiotensin system. These results bring additional support for an involvement of vasopressin in the albuminuria observed in pathological states such as diabetes mellitus or hypertension.
Keywords: ACE inhibition; angiotensin II receptor blockade; blood pressure; diabetes insipidus; diabetes mellitus; microalbuminuria
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Introduction |
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Diabetic nephropathy is a frequent complication of DM. Early changes in renal function during DM include hyperfiltration, renal enlargement and microalbuminuria. A marked increase in VP secretion and VP plasma level is well documented in DM [6]. In a previous study, we showed that VP participates in the early manifestations of diabetic nephropathy [7]. Experimental type I DM was induced in Brattleboro rats, unable to secrete VP because of a deletion in the VP gene. Glomerular hyperfiltration, renal hypertrophy, and the progressive rise in albuminuria were absent or much less intense in these rats than in their VP-replete LongEvans controls [7].
Taken together, these findings strongly suggest that VP plays a role in the induction of albuminuria preceding and accompanying progressive renal damage due either to severe reduction in renal mass or to diabetic nephropathy. The mechanism by which VP can induce this effect is, however, unclear. Whether the elevation in UAE is due to a glomerular leakage of albumin or to a defect in its tubular reabsorption is not known. V2 receptors are most probably involved (i) because renal V1a but not V2 receptors have been shown to be downregulated in DM and (ii) because dDAVP, which induces hyperfiltration in normal rats [4], is a V2 receptor agonist with low affinity for V1a receptors [8]. However, V2 receptors or their mRNA have not been observed in glomeruli or proximal tubules [9]. This suggests that VP effects on albuminuria and hyperfiltration are indirect, as already discussed for VP-induced hyperfiltration [4,10]. They could result from associated changes in blood pressure because proteinuria and blood pressure were observed to vary in parallel when the VPwater intake axis was altered in rats with chronic renal failure [1,3]. A contribution of the reninangiotensin system, activated in response to changes in the tubulo-glomerular feedback was also suspected to contribute to the haemodynamic effects of VP [4,10].
The present study was designed to evaluate whether VP has an influence on albumin excretion through a V2-receptor-dependent pathway in rats and humans with normal kidney function, and whether the reninangiotensin system and/or associated changes in blood pressure mediate these effects. First, changes in albumin excretion in response to acute or chronic administration of dDAVP were evaluated in normal rats. Second, albumin excretion was measured after short-term infusion of dDAVP in healthy humans and in subjects with hereditary diabetes insipidus due to different aetiologies. Third, the possible contribution of the reninangiotensin system to dDAVP-induced albuminuria was evaluated in rats by studying the effect of dDAVP after chronic blockade of this system by converting-enzyme inhibition or by selective antagonism of angiotensin II AT1 receptors.
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Subjects and methods |
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Studies in rats
Adult male Wistar rats [body weight (BW) 230240 g], purchased from Iffa Credo (L'Arbresle, France), were housed individually in metabolic cages during the whole experiments with free access to tap water and powdered food (A03, UAR, Epinay/Orge, France) except when otherwise mentioned. They were allowed at least 4 days of adaptation before starting the experiments.
Experimental protocols
Acute infusion of dDAVP (Experiment A)
Eight rats were studied during 3 successive days. On the first 2 days, at 8:00 a.m., rats received an intraperitoneal (i.p.) injection of either 500 µl isotonic saline (basal day) or 500 ng dDAVP in 500 µl isotonic saline (experimental day). No injection was given on the third, recovery, day. On each day, urine was collected from 8:00 a.m. to 2:00 p.m. (6 h following the injection), and from 2:00 p.m. to 8:00 a.m. of the following day (18 subsequent hours). Urine flow rate (V), urine osmolality (Uosm) and urinary albumin concentration (Ualb) were measured (see below).
Chronic infusion of dDAVP (Experiment B)
In experiment B, seven rats were studied during 3 successive weeks (basal, experimental, and recovery weeks). During the experimental week, rats were infused with dDAVP, 8.33 ng/h (200 ng/day), delivered at a rate of 1 µl/h through osmotic minipumps (model 2001, Alza Corporation, Palo Alto, USA) implanted i.p. under brief anaesthesia (ketamine, 6.25 mg/100 g bodyweight (BW) plus xylazine, 0.5 mg/100 g BW). This dDAVP treatment is similar to that used in our previous study demonstrating the influence of vasopressin on GFR [4]. This infusion increased urinary concentrating activity within a physiological range (taking into account that dDAVP is 5-fold more potent than VP because of its longer half-life). The pumps were removed under anaesthesia at the end of the experimental week. During the last 2 days of each week, urine was collected and daily urine volume, BW, and water and food intakes were measured as well as V, Uosm and Ualb. Immediately after completion of urine collections, a blood sample (400 µl) was drawn from a jugular vein in a heparinized syringe under ketamine/xylazine anaesthesia, for measurement of plasma solute concentrations.
Chronic infusion of dDAVP after chronic blockade of the reninangiotensin system (Experiments C and D)
Experiment C followed exactly the same protocol as experiment B except that two groups of rats were studied in parallel, one being submitted to chronic angiotensin-converting enzyme inhibition (ACEI) and the other used as control. Sixteen rats were placed in metabolic cages. In order to limit interindividual variations in food intake (and thus in osmolar excretion) as well as drug spilling during the treatment period, all rats were offered a limited amount of food per day (25 g), corresponding roughly to the previously measured average food intake of rats of this BW. After 4 days of adaptation, renal function was studied for 2 consecutive days and rats were divided into two equal groups of equivalent BW, urinary concentrating activity and UAE. One of the groups was then given Coversyl (tert-butylamine salt of perindopril, Servier, Orléans, France) at a dose of 1 mg/100 g BW per day, mixed in the powdered food. Ten days after initiation of the treatment, the same 3-week protocol as that described for Experiment B was performed. In addition, systolic blood pressure (SBP) was measured by the tail-cuff method (BP recorder 8005, Apelex, Massy, France) 3 days per week, starting during the first week of ACEI treatment. Rats were placed for 20 min under a specially adapted, thermostatic heating element before measurements. Results of the last 2 days of each week were averaged for each rat.
In experiment D, the possible influence of angiotensin II was blocked by chronic administration of the AT1 receptor antagonist losartan (MSD, Wilmington, USA) added to the drinking water so as to provide a daily dose of 3 mg/100 g BW (the concentration of losartan in the drinking water was increased during the dDAVP week in order to compensate for the reduction in fluid intake induced by this hormone). The losartan treatment was started 8 days before the beginning of urine collection. The same 3-week protocol as that described for experiments B and C was then performed (blood pressure was not measured in experiment D).
Studies in human subjects
Subjects
In previous studies at the Hôpital du Sacré-Cur (Montreal), healthy subjects and patients with hereditary diabetes insipidus (DI) of different aetiologies had been submitted to a dDAVP infusion to evaluate the influence of this drug on coagulation factors and plasma cyclic adenosine monophosphate (cAMP). The following protocol had been used (as reported in [11]). dDAVP, 0.3 µg/kg BW, was infused i.v. over 20 min and urine was collected by spontaneous voiding every 30 min for 60 min before and 180 min after the beginning of dDAVP infusion. Mean arterial blood pressure was measured every 30 min. Blood samples were taken every 10 min for the first 30 min after initiation of dDAVP infusion and every 30 min thereafter, for measurement of plasma renin activity. Urinary flow rate and osmolality, and urinary creatinine concentration were measured, and representative aliquots of urine were frozen at -70°C.
In the present study, we reconsidered six healthy subjects and 15 DI patients for whom frozen urine samples were still available. In urine of healthy subjects (3 male, 3 female, 31±4 years, 67±6 kg), we measured urinary albumin and ß2-microglobulin concentrations. We also report plasma renin activity (PRA), urinary flow rate and osmolality, and creatinine excretion (not reported in the initial paper), and mean arterial blood pressure (MAP) [11]. Albumin concentration was also evaluated in urine of four of these healthy subjects collected with the same timing during a sham test involving infusion of isotonic saline instead of dDAVP.
Among the patients with DI, two had central DI (CDI) and 13 had nephrogenic DI (NDI) due to mutations of either the vasopressin V2 receptor gene (NDI-VPR2, n=10) or the aquaporin 2 gene (NDI-AQP2, n=3). The mutations involved are shown in Table 2. CDI patients stopped their dDAVP for 24 h and NDI patients stopped their hydrochlorothiazide medication for 3 days. Urinary albumin concentration was measured in urine collected from these DI patients before and after dDAVP infusion, with the same timing as in healthy subjects.
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Results are expressed as means±SEM. All urine data in rat studies of experiments B, C and D are means of the two successive 24-h urine collections for each rat. Data were analysed by ANOVA with repeated measures followed by Fisher post-hoc test. In healthy humans, the influence of dDAVP was evaluated by ANOVA with repeated measures followed by Dunnett post-hoc test, comparing values observed at each time after the beginning of dDAVP infusion to those obtained during the basal period (mean of the first two 30-min urine collections). For patients with DI, only descriptive statistics are provided because the number of subjects was too small in two of the groups to allow valid statistical comparisons.
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Results |
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The chronic effects of dDAVP are shown in Table 1 (experiment B). Plasma sodium concentration fell by 3 mmol/l during the dDAVP week (from 143±0 to 140±1 mmol/l, P<0.01) and returned to control values during recovery. In the whole, the antidiuretic effects were of similar magnitude to those observed after acute dDAVP administration. The albuminuric effect, however, was more intense (5.8-fold increase, P<0.01).
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Figure 2 shows the results observed in patients with diverse forms of DI. The two patients with central DI exhibited the expected antidiuretic response to dDAVP infusion. A very intense rise in UAE was also observed, largely exceeding that seen in healthy subjects. From 90 to 150 min after the dDAVP infusion, UAE reached a plateau at a level 12- and 31-fold above values recorded in the basal period. In patients with NDI, no antidiuretic response was observed (as could be expected). However, there was a distinct rise in UAE in those with AQP2 mutations but no or only a very modest rise in nine of the 10 patients with mutations of the V2 receptor (Table 2
). Note that four of the 13 patients with NDI exhibited unusually high basal UAE,
510 times higher than that in healthy subjects and other DI patients (Figure 2
and Table 2
).
Influence of previous blockade of the reninangiotensin system on dDAVP-induced albuminuria in rats
To evaluate the possible contribution of the reninangiotensin system in the effects of dDAVP on albumin excretion, dDAVP was infused chronically in rats previously submitted to chronic ACEI or blockade of AT1 receptors (Figures 3 and 4,
respectively). Treatment with perindopril markedly lowered SBP in the basal week (-26 mmHg, P<0.001). SBP was not measured in rats receiving losartan, but it was shown to fall by 30 mmHg in Wistar rats treated in the same way in a previous experiment performed in the same laboratory [12]. Thus, it may be assumed that SBP fell similarly in ACEI- and ARB-treated rats of the present experiment. No change in SBP occurred during the week of dDAVP infusion, but a modest fall was observed after discontinuation of this infusion (P<0.01) (Figure 3
).
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In experiment D, chronic blockade of AT1 receptors did not alter basal urinary concentration or the antidiuretic response to dDAVP (data not shown). Uosm and V during the 3 weeks in the two groups were very similar to those in experiment C (data not shown). UAE rose by 2.70±0.77 mg/day during dDAVP infusion in the control group (x5.8 compared with basal), and by only 1.24±0.30 mg/day in the group receiving the ARB (x3.0 compared with basal) (Figure 4). The difference in the dDAVP-induced rise in UAE between the two groups did not reach statistical significance. Thus, both ACEI and ARB reduced the dDAVP-mediated albuminuria but this effect was weaker with the latter.
Plasma urea increased significantly during dDAVP infusion in all groups (Table 3). This increase was due to a more intense urea reabsorption known to occur in the collecting duct at low urine flow rates and to the vasopressin-dependent diffusion of urea from the terminal collecting ducts into the medullary interstitium. Part of this urea fails to be recycled within the kidney and thus returns to the general circulation [10]. Note that rats with depressed reninangiotensin system (due to ACEI or to AT1 blockade) had 813% higher basal plasma urea concentration (Purea) than control rats, and that rats with ACEI exhibited a much more intense rise in Purea under dDAVP than the three other groups (Table 3
), suggesting an interaction between the reninangiotensin system (and especially angiotensin-converting enzyme) and dDAVP on urea reabsorption. Purea returned to basal values after dDAVP discontinuation in all groups.
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Discussion |
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Albumin excretion is usually kept to a very low level, due to the relatively low permeability of the glomerular filtration barrier to albumin, and to efficient reabsorptive mechanisms in the proximal tubule. An increase in UAE is interpreted as a sign of early renal damage in several diseases including DM and hypertension. The present results suggest that VP could contribute to albuminuria in these pathological situations involving a rise in its plasma level.
The fact that ß2-microglobulin excretion did not rise in parallel with that of albumin in healthy humans suggests that the dDAVP-induced rise in UAE originated from an increased leakage of albumin through the glomerular filtration barrier rather than from a reduction in its tubular reabsorption. The rise in UAE was transient. This may be due, at least in part, to the fact that dDAVP concentration in plasma fell with time after cessation of the 20-min infusion. In contrast, the antidiuretic effects lasted for several hours, most probably because the kidney's architecture slows the dissipation of the gradient of solutes accumulated in the renal medulla. These different time courses suggest that dDAVP-induced albuminuria does not result from the physicochemical changes occurring in the kidney in relation to the process of urine concentration. The dDAVP-induced rise in UAE in normal rats was greater after a prolonged treatment than after a single injection. This suggests that dDAVP-dependent albuminuria may progressively worsen with prolonged exposure to the hormone.
In healthy humans, a marked fall in MAP and rise in PRA was observed in the first 30 min after dDAVP infusion, as already reported by Bichet and colleagues [11,13]. The fall in blood pressure may be due to extra-renal V2 receptor-mediated vasodilatation [14] and may induce a rise in renin release [11,13,15] which contributes to the prompt restoration of blood pressure. The rise in PRA observed here was also short-lived but preceded the rise in albumin excretion. However, it may be assumed that the change in UAE was concomitant with that in PRA but was observed later due to the dead space in the urinary tract. The change in glomerular permeability to albumin was thus most probably synchronous with the presence of exogenous dDAVP in the blood and to the increased activity of the reninangiotensin system. The precise mechanism by which these hormonal systems influence glomerular permselectivity cannot be inferred from the present study.
dDAVP infusion markedly increased UAE in all patients with DI who had functional V2 receptors, whether they were able to concentrate urine in response to vasopressin (CDI) or not (NDI-AQP2). In contrast, dDAVP failed to increase (or increased only very modestly) UAE in patients with mutated, non functional V2 receptors. These results thus clearly establish that functional V2 receptors are required for the manifestation of dDAVP-induced albuminuria. Interestingly, these patients also exhibited no change in blood pressure, PRA, and heart rate in response to dDAVP, as reported earlier [11,13].
The reason why basal UAE was markedly higher in four of the 13 NDI patients is unknown. These patients had normal creatinine clearance and no sign of renal insufficiency, except patient no. 7 (BER Mi) who had a creatinine clearance of 40 ml/min. It may be worth mentioning that Brattleboro rats with hereditary CDI also tend to have higher basal UAE and much greater individual variability in UAE than LongEvans controls with normal vasopressin secretion [7]. Of note also, the dDAVP-induced rise in UAE tended to be more intense and was more prolonged in DI patients (with functional V2 receptors) than in healthy subjects (compare Figures 2 and 3
). The possibility that sustained production of dilute urine may favour glomerular and/or tubular leakage of albumin needs to be evaluated in further studies.
Possible mechanism responsible for the albuminuric effect of dDAVP
The influence of dDAVP on glomerular leakage is probably indirect because no V2 receptors or V2-receptor mRNA have been noted in the glomerulus [9]. More probably this influence depends on a sequence of events initiated by the action of the drug on renal tubular V2 receptors. However, an antidiuretic response is not a prerequisite for this albuminuric effect, because it was also observed in NDI-AQP2 patients who were unable to concentrate urine after dDAVP administration. Vasodilatory effects mediated by extra-renal V2 receptors [14] may also be involved. The present experiments do not provide any information on these intermediate steps. However, several observations suggest that an activation of the reninangiotensin system could participate in this mechanism. Angiotensin II is known to increase the glomerular filtration of albumin or proteins in vivo or in the isolated kidney and to enhance glomerular albumin permeability in isolated glomeruli in vitro [16]. A marked rise in PRA was observed in healthy subjects just before the rise in UAE in the present study, and patients with NDI-VPR2, who failed to exhibit a rise in UAE, also showed no change in PRA.
Activation of the intrarenal reninangiotensin system may induce glomerular hypertension and thus favour albumin leakage, even in the absence of systemic hypertension [17]. In a previous study on the influence of VP or dDAVP on GFR, an inhibition of the activity of the tubulo-glomerular feedback (TGF) was suspected. This inhibition should result from a decrease in the sodium concentration of the tubular fluid at the macula densa, due to the more intense vasopressin-dependent intrarenal urea recycling that brings more urea in the lumen of loops of Henle [6,10]. The involvement of the intrarenal reninangiotensin system in the TGF-dependent regulation of GFR is well established. The present study suggests that V2-mediated actions of VP, in addition to increasing GFR and kidney weight [4,5], also enhance the glomerular leakage of albumin through a mechanism involving the reninangiotensin system. Accordingly, VP probably activates the previously described vicious circle that leads from hyperfiltration and/or glomerular hypertension to progressive deterioration of renal function [18].
The results obtained here in rats submitted to either ACEI or ARB confirm the involvement of the reninangiotensin system in the V2 receptor-dependent rise in albuminuria. When angiotensin II actions were prevented by either approach, this rise was largely blunted. The slightly lower inhibition with losartan than with perindopril could be due either to small inter-experiment differences or to differing pharmacokinetics of the two drugs. Alternatively, it could suggest the involvement of additional factors suppressed by the latter but not by the former. Bradykinin, the half-life and thus the functional effects of which are potentiated by ACEI, may thus have played a role, through a mechanism that remains to be determined. Similarly, it is not possible to exclude a direct influence of angiotensin-converting enzyme, which is expressed in the nephron (especially along the brush border in the proximal tubule). However, even during efficient blockade of the reninangiotensin system, a residual rise in albumin excretion after dDAVP exposure was still observed. This suggests either that the reninangiotensin system was not fully blocked by the treatments (this is unlikely in view of the marked fall in blood pressure they induced), or that an additional mechanism, independent of the reninangiotensin system, participated in the effect of dDAVP.
VP has been shown to stimulate the secretion of ANP, a hormone that could also induce a rise in UAE [19]. However, it seems unlikely that the dDAVP-induced rise in UAE could be mediated by ANP in the present study because of the very different time course and intensity of the rises observed in ANP and in UAE in healthy subjects.
Expected consequences in diverse pathological situations
The results of this study suggest that VP could contribute to albuminuria in pathological situations in which VP secretion is enhanced. This is the case in DM. Patients with uncontrolled type 1 or type 2 DM exhibit elevated plasma VP concentration. VP is also elevated in rats with either streptozotocin-induced DM or with a genetic form of DM (BB rats) [6]. Moreover, basal levels of VP in type 2 diabetic patients showing albuminuria were reported to be significantly higher than in diabetics without complications [20]. Experimental data in rats strongly support the notion that endogenous VP contributes to the albuminuria of streptozotocin-induced DM. First, the usual rise in UAE was almost completely blunted when DM was induced in VP-deficient Brattleboro rats [7]. Second, chronic administration of a selective, non-peptide V2 receptor antagonist (SR 121463, Sanofi-Recherche) to Wistar rats with DM totally prevented the rise in UAE seen in untreated rats [21]. In the present experiments, only V2 effects of vasopressin were considered because the V2 agonist, dDAVP, had been shown to induce hyperfiltration in normal rats [4] and it was thus interesting to see whether the same VP agonist would also increase albuminuria. Moreover, studies in rats and humans have shown that V1a but not V2 receptors are down-regulated in diabetes mellitus [6]. However, a recent study showed that 3-week treatment with a vasopressin V1a antagonist induced a modest decrease in albuminuria in diabetic patients, thus suggesting that the V1a effects of the hormone, if still significant, could also increase albumin excretion [20].
Because VP effects on albuminuria appear to be mediated by the reninangiotensin system, it is conceivable that the protection against progression of microalbuminuria and diabetic nephropathy afforded by ACEI or ARBs could be due, at least in part, to the interruption of a V2-receptor-dependent renal action [6]. Another situation in which VP is chronically elevated is the syndrome of inappropriate secretion of antidiuretic hormone. To our knowledge, no study has evaluated UAE in such patients. Similarly, no information is available regarding UAE in patients with central DI who are chronically treated with dDAVP.
Besides DM, albuminuria also represents a risk factor for cardiovascular morbidity and mortality in hypertension. Now, an elevated VP plasma level has been documented in some groups of hypertensive patients [22,23]. High VP secretion is also observed in Sabra rats, a model of salt-sensitive hypertension [24]. Thus, it may be assumed that VP also contributes to enhanced albumin excretion associated with high blood pressure. Interestingly, smoking, a proven risk factor in hypertension and diabetic nephropathy, is also a potent stimulus for VP secretion.
In summary, the present studies demonstrate that VP, or at least its V2 moiety, in addition to its well-known effects on urinary concentrating activity, also induces an increase in urinary albumin excretion in rats and humans. This effect is probably of glomerular origin, seems to depend on the effects of the hormone on its renal and/or extra-renal V2 receptors and to involve, at least for part of it, the reninangiotensin system. More information is needed to fully elucidate the mechanism of this novel effect. Together with previously published observations, these results also suggest that VP, known to be elevated in diabetes mellitus, may contribute to the albuminuria often observed in this disease.
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Acknowledgments |
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Notes |
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References |
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