Arginine vasopressin modulates expression of neuronal NOS in rat renal medulla

Pierre-Yves Martin, Mathieu Bianchi, Frank Roger, Laurent Niksic, and Eric Féraille

Division of Nephrology, Hôpital Cantonal Universitaire, CH-1211 Geneva 14, Switzerland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Arginine vasopressin (AVP) plays a central role in water balance. In principal cells of the collecting duct system, AVP controls the expression of several genes, including aquaporin-2. Because nitric oxide (NO) participates in the regulation of water reabsorption by the collecting duct system, we analyzed the effect of AVP on the expression of NO synthase (NOS) isoforms in the kidney. Rats were either water restricted or water loaded to modify the circulating AVP levels, and expressions of NOS isoforms were assessed by Western blot analysis. In water-restricted rats, endothelial NOS (eNOS) expression increased in the outer medulla, and neuronal NOS (nNOS) expression rose in both the outer medulla and the papilla. Conversely, water loading induced a decrease in expression of nNOS in the outer medulla and papilla but did not alter eNOS expression. Oral administration of the specific V2-receptor antagonist SR-121463B decreased nNOS expression in the outer medulla and papilla but did not alter eNOS expression levels. Finally, the very low nNOS expression levels observed in AVP-deficient Brattleboro rats was restored by AVP infusion for 1 wk. Thus AVP specifically increases nNOS expression levels in the renal outer medulla and papilla. Because nNOS is specifically expressed in principal cells of the collecting duct system, the stimulation of nNOS expression by AVP may participate in the control of water reabsorption.

principal cells; aquaporin-2; collecting duct; nitric oxide synthase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE MAINTENANCE OF CONSTANT body fluid osmolality is achieved by complex homeostatic mechanisms in which the kidney plays a central role through the control of water balance. In higher vertebrates, water balance can be achieved mainly because the kidney has the ability to excrete either diluted or concentrated urine. Imbalance between water intake and water excretion due to impaired kidney function leads to life-threatening disorders such as hyponatremia or hypernatremia.

Once filtered by the glomerulus, water is reabsorbed by the kidney tubule. Quantitatively, the most important segments are the proximal tubule and the thin descending limb of Henle. In these segments, transcellular water reabsorption is achieved through aquaporin-1 (AQP1) water channels constitutively expressed in both apical and basolateral membrane domains (4, 21, 30). In addition, water reabsorption occurs along the paracellular route in proximal tubules (30). Regulated water reabsorption takes place in the collecting duct system, i.e., the connecting tubule and the collecting duct (14, 16). The principal cells of the collecting duct system express different water channels in their apical and basolateral membrane domains. AQP3 and AQP4 are constitutively located in the basolateral membrane domain (8, 16, 33), whereas AQP2 shuttles between intracellular stores and the apical membrane domain (16, 19).

Arginine vasopressin (AVP) is the major hormone that controls water reabsorption by the collecting duct system. Indeed, the collecting duct system is virtually impermeable to water in the absence of AVP and becomes highly permeable to water in the presence of AVP (10, 14, 16). This effect has been demonstrated in vivo (17) and by in vitro microperfusion of isolated collecting ducts (10). In principal cells, AVP acts through V2 receptors coupled to the stimulation of adenylyl cyclase (11, 16, 25). The subsequent increase in cellular cAMP concentration activates protein kinase A and induces AQP2 translocation from intracellular stores to the apical plasma membrane (15). Besides short-term stimulatory effects on water permeability (10), sodium reabsorption (9), and potassium secretion (3), AVP also controls the expression of a specific subset of genes in principal cells of the collecting duct (28). For instance, increasing and decreasing circulating AVP levels by water restriction or water loading in rats alters AQP2 expression levels (34). In addition, infusion of AVP into Brattleboro rats increases AQP2 expression (5). On the other hand, in vitro AVP treatment of a rat cortical collecting duct cell line increased the expression of the Na-K-ATPase alpha -subunit and the epithelial sodium channel beta - and gamma -subunits (6, 7).

The three nitric oxide synthase (NOS) isoforms, inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS), have been found to be expressed in the kidney (1, 18). It appears that the constitutive NOS isoforms nNOS and eNOS are responsible for modulating the physiological function of renal cells, whereas iNOS is involved in pathophysiological events (20, 26). Recent studies indicate that NO mimics the effect of AVP on AQP2 translocation by activating protein kinase G through increased intracellular cGMP levels (2). Together with the expression of nNOS and the absence of eNOS in collecting duct principal cells (36), these results suggest that endogenous NO from nNOS may participate in the regulation of water reabsorption by the collecting duct. In addition, AVP has been shown to induce transcription of several proteins that may play a role in physiological responses (28).

The purpose of this study was to investigate the effect of AVP on renal NOS activity and protein expression in rodents.


    MATERIAL AND METHODS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and study design. Male Wistar (Service de Zootechnie, Centre Medical Universitaire, Geneva, Switzerland), Brattleboro, and Long-Evans rats (Harlan, Indianapolis, IN) weighing between 150 and 300 g were used for experiments. Wistar rats were randomly assigned to either control or experimental groups, were pair-fed, and had unrestricted access to water. Two days before the start of the experimental protocol, rats were housed in metabolic cages with powdered chow. Experimental group 1 consisted of water-restricted rats that were allowed to drink 10 ml of water for 3 days and 5 ml for 2 days and were deprived of water for the remaining 24 h. Experimental group 2 consisted of water-loaded rats fed for 6 days a mixture of hydrated agar and powdered standard laboratory rat chow containing 3.33 g water/g of dry food; each animal was provided with 18 g dry food/day. Experimental group 3 consisted of rats receiving a daily intragastric administration of 1 mg/kg body weight of the specific V2-receptor antagonist SR-121463B (a kind gift from Dr. Serradeil-Le Gal, Sanofi Research, Toulouse, France) for 7 days (31); rats were killed 12 h after the last administration of SR-121463B. Measurements of water and food intake, body weight, and urine volume were undertaken at days 0 and 7. Urine specimens were collected under oil, and samples taken at days 0 and 7 were frozen at -80°C for later analysis.

Brattleboro and Long-Evans (control) rats were kept under the same conditions as the other groups. One group of Brattleboro rats was infused for 1 wk with AVP (5 µg/day) delivered by an intraperitoneal osmotic minipump (Alzet). An intraperitoneal osmotic minipump delivering solvent alone (1 µl/h of NaCl 0.9%) was implanted in both control Long-Evans and nontreated Brattleboro rats. Control Long-Evans and Brattleboro rats were then placed in individual metabolic cages for 7 days with free access to water and powdered chow, and physiological parameters were recorded as described above.

Each urine sample was centrifuged to remove any solid matter before analysis. Urine osmolality was determined by freezing point depression, and urine sodium levels were measured by flame photometry. Sodium balance was calculated as the ratio of sodium intake to urinary sodium. The protocols were approved by the Ethical Commission of the Faculty of Medicine of the University of Geneva.

Isolation of tissue. After anesthesia with pentobarbital sodium (50 mg/kg body wt), the two kidneys were infused with incubation solution containing (mM) 120 NaCl, 5 RbCl, 4 NaHCO3, 1 CaCl2, 1 MgSO4, 0.2 NaH2PO4, 0.15 Na2HPO4, 5 glucose, 10 lactate, 1 pyruvate, 4 essential and nonessential amino acids, and 20 HEPES, as well as 0.1% (wt/vol) bovine serum albumin and vitamins (1× MEM; Life Technologies), pH 7.45. Afterward, the cortex, inner stripe of the outer medulla, and papilla were separated, frozen into liquid nitrogen, and stored at -80°C until used for Western blotting or NOS activity measurement (see below).

Western blotting. Frozen tissues were pulverized and glass homogenized in 1 ml of homogenization buffer containing (in mM) 20 Tris · HCl (pH 7.4), 2 EDTA, 2 EGTA, 1 phenylmethylsulfonyl fluoride, 1 of 4-(2-aminoethyl)-benzenesulfonyl fluoride, 30 NaF, and 30 Na pyrophosphate, as well as 25 µg/ml leupeptin, 25 µg/ml aprotinin, 0.1% (wt/vol) SDS, and 1% (vol/vol) Triton X-100. After protein concentration determination (BCA assay, Pierce), equal amounts of protein (50-100 µg) were mixed with an equal volume of Laemmli 2× buffer and analyzed by SDS-PAGE. After electrophoresis on 7% polyacrylamide gels, proteins were electrotransferred on polyvinylidine difluoride membranes (Immobilon-P, Millipore, Bedford, MA) and incubated overnight at 4°C with primary antibody diluted in Tris-buffered saline (TBS) Nonidet P-40 (NP-40) medium (TBS-NP-40: 150 mM NaCl, 50 mM Tris, 0.2% NP-40, pH 7.4) with 5% (wt/vol) nonfat dry milk. After washing in TBS-NP-40, membranes were incubated with a secondary antibody coupled to horseradish peroxidase (HRP) diluted in TBS-NP-40 with 5% (wt/vol) nonfat dry milk. The antigen-antibody complexes were detected by chemiluminescence with the Super Signal Substrate method (Pierce, Rockford, IL) according to the manufacturer's instructions. For detection of nNOS, we used rabbit polyclonal antibodies (Biomol) diluted 1:1,000. For detection of eNOS, we used a mouse monoclonal antibody (Transduction Laboratories, Lexington, KY) diluted 1:1,000. For detection of iNOS, we used a mouse monoclonal antibody (Transduction Laboratories) diluted 1:1,000 or a rabbit polyclonal antibody (Biomol). With both antibodies, iNOS protein expression was not detectable in the cortex, outer medulla, or inner medulla (data not shown). For detection of AQP2, we used previously characterized rabbit polyclonal antibodies (38). Rabbit polyclonal antibodies were detected by HRP-conjugated anti-rabbit Ig diluted 1:20,000 (Transduction Laboratories), and mouse monoclonal antibodies were detected by HRP-conjugated anti-mouse Ig diluted 1:20,000 (Transduction Laboratories). Results were quantified by laser densitometry (Molecular Dynamics, Sunnyvale, CA) using Image Quant software. Data were expressed as percentage of the paired control value.

NOS activity. The NOS activity measurement assay was performed on the basis of the biochemical conversion of L-arginine to L-citrulline and NO (37). Renal cortex, outer medulla, and papilla from control and experimental rats were homogenized in a buffer containing 25 mM Tris · HCl, pH 7.4, 1 mM EDTA, and 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride. Thereafter, 50-100 µg protein contained in 10 µl of homogenization buffer were mixed with 40 µl of reaction buffer containing 50 mM Tris · HCl, pH 7.4, 6 µM BH4, 2 µM FAD, 1 mM reduced NADP, 0.1 µM calmodulin, 0.6 mM CaCl2, 100 µM L-arginine, and trace amounts (0.1 µCi) of L-[3H]arginine (Amersham, Little Chaffont, UK). Samples were incubated for 2 h at 37°C, and the reaction was stopped by the addition of 1 ml of 50 mM HEPES, pH 5.5, and 5 mM EDTA. Samples were then incubated for 10 min at 4°C with equilibrated AG 50W-X8 (Na+ form) to bind the unreacted L-arginine. Citrulline was eluted by centrifugation, and radioactivity was measured by liquid scintillation counting. In each experiment, a blank value, consisting of the radioactivity measured in samples incubated in the absence of tissue homogenate, has been determined and subtracted from each experimental value. For each experimental series, all samples were freshly homogenized, and measurement of NOS activity was performed the same day with the same batches of buffers. The NOS activity was measured in duplicate and expressed as femtomoles L-citrulline per milligram protein per minute. Time course experiments have shown that NOS activity was linear for at least 2 h, using 100 µg protein from either renal cortex or medulla (data not shown). Dose-dependence experiments have shown that NOS activity measured after 2 h incubation was linear from 25 to 200 µg protein (data not shown).

Statistics. Statistical analyses of NOS isoforms, Na-K-ATPase alpha -subunit, and AQP2 immunoreactivity were done using the Mann-Whitney U-test. Statistical analysis for NOS activity was by unpaired Student's t-test; P < 0.05 was considered significant. Results are expressed as means ± SE from several independent animals.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of water restriction on NOS activity and expression levels of NOS isoforms. To increase endogenous AVP circulating levels, rats were water restricted. As shown in Table 1, urinary volume was dramatically decreased and urinary osmolality rose close to 5,000 mosmol/kgH2O in water-restricted rats. In addition to these alterations in water handling, it is noteworthy to mention that water-restricted rats also retained significant amounts of sodium, as reflected by the 60% excretion of the amounts of ingested sodium as shown in Table 2. This sodium retention is most likely mediated by an adaptive response to hypovolemia consecutive to severe water restriction (32). Accordingly, water-restricted rats weighed less than control rats despite similar amounts of food intake, reflecting the negative water balance. The renal effect of AVP was indirectly assessed by the increase in expression of AQP2 in the inner stripe of the outer medulla from water-restricted rats (Fig. 1A).

                              
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Table 1.   Physiological parameters of water-restricted, water-loaded, SR-121463B-treated, and Brattleboro rats compared with their respective controls


                              
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Table 2.   Sodium balance parameters of water-restricted, water-loaded, SR-121463B-treated, and Brattleboro rats compared with their respective controls



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Fig. 1.   Aquaporin-2 (AQP2) expression in control and water-restricted (A), control and water-loaded (B), control and V2-receptor antagonist SR-121463B-treated (C), and Long-Evans (Control) and arginine vasopressin (AVP)-infused Brattleboro rats (D), as well as their respective controls. After completion of experimental protocols, tissues were lysed, and equal amounts of protein from the inner stripe of the outer medulla of control and experimental rats were separated by 10% SDS-PAGE. After electrotransfer, AQP2 was detected by Western blotting. Representative Western blots showing results from 3 control and 3 experimental rats are shown.

The effect of increased AVP circulating levels on renal NOS activity and NOS expression was assessed in the cortex, inner stripe of the outer medulla, and papilla from control and water-restricted rats. As indicated in Table 3, NOS activity increased to 219 ± 8% of the control value in the outer medulla of water-restricted rats but remained unchanged in the cortex and papilla. Figure 2 shows that eNOS expression levels increased to 245 ± 14% of the control value (P < 0.05) in the inner stripe of the outer medulla but remained unchanged in the cortex and papilla from water-restricted rats (Fig. 2). On the other hand, nNOS expression rose to 710 ± 34 (P < 0.05) and 159 ± 9% (P < 0.05) of the control value in the outer medulla and papilla of water-restricted rats, respectively (Fig. 3). It is worth mentioning that nNOS expression levels in the cortex of both control and water-restricted rats were below the minimal detection levels of our Western blot assay.

                              
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Table 3.   Nitric oxide synthase activity measured in cortex, outer medulla, and papilla of water-restricted, water-loaded, anti-V2-treated, and Brattleboro rats compared with their respective controls



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Fig. 2.   Effect of water restriction on endothelial nitric oxide synthase (eNOS) expression levels. Equal amounts of protein from the cortex, inner stripe of the outer medulla, and papilla of control (C) and water-restricted rats (WR) were separated by 7% SDS-PAGE. After electrotransfer, eNOS was detected by Western blotting. A: Western blot showing results from 5 control and 5 water-restricted rats. B: densitometric quantification of theWestern blot shown in A. Values are means ± SE expressed as the percentage of the optical density value of control animals in each experiment. * P < 0.05 vs. control.



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Fig. 3.   Effect of water restriction on neuronal NOS (nNOS) expression levels. Equal amounts of protein from the inner stripe of the outer medulla and papilla of control and water-restricted rats were separated by 7% SDS-PAGE. After electrotransfer, nNOS was detected by Western blotting. A: Western blot showing results from 5 control and 5 water-restricted rats. B: densitometric quantification of the Western blot shown in A. Values are means ± SE expressed as the percentage of the optical density value of control animals in each experiment. * P < 0.05 vs. control.

Effects of water loading on NOS activity and expression of NOS isoforms. A water-loading protocol by ingestion of hydrated agar was applied to decrease the endogenous circulating levels of AVP. Table 1 shows that increasing the water intake of rats increased urinary volume fivefold and decreased urinary osmolality to <400 mosm/kgH2O. Sodium balance and growth were not altered by the water-loading protocol (Tables 1 and 2). Inhibition of AVP secretion was confirmed by the decrease in AQP2 expression levels in the inner stripe of the outer medulla from water-loaded rats (Fig. 1B).

Water loading did not alter NOS activity in the cortex, inner stripe of the outer medulla, and papilla (Table 3). Similarly, renal expression of eNOS was not altered in water-loaded rats (Fig. 4). Figure 5 shows that water loading decreased nNOS expression to 54 ± 23% of the control value (not significant) in papilla but did not alter nNOS expression in the outer medulla (91 ± 37% of the control value).


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Fig. 4.   Effect of water loading on eNOS expression levels. Equal amounts of protein from the cortex, inner stripe of the outer medulla, and papilla of control and water-restricted rats were separated by 7% SDS-PAGE. After electrotransfer, eNOS was detected by Western blotting. A: Western blot showing results from 5 control and 5 water-loaded rats (WL). B: densitometric quantification of the Western blot shown in A. Values are means ± SE expressed as the percentage of the optical density value of control animals in each experiment.



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Fig. 5.   Effect of water loading on the expression levels of nNOS. Equal amounts of protein from the inner stripe of the outer medulla and papilla of control and water-restricted rats were separated by 7% SDS-PAGE. After electrotransfer, nNOS was detected by Western blotting. A: Western blot showing results from 5 control and 5 water-loaded rats. B: densitometric quantification of the Western blot shown in A. Values are means ± SE expressed as the percentage of the optical density value of control animals in each experiment.

Effects of V2-receptor blockade on NOS activity and expression of NOS isoforms. To determine whether the effect of endogenous AVP was mediated through the V2 receptor, we assessed the effect of SR-121463B, a specific V2-receptor antagonist (31). Table 1 shows that SR-121463B increased urinary volume ninefold and decreased urinary osmolality to 145 mosm/kgH2O. As already demonstrated (31), administration of SR-121463B did not change the sodium balance and growth of the animals (Tables 1 and 2). The inhibition of V2-receptor-mediated effects of AVP was illustrated by the decrease of AQP2 expression levels in the inner stripe of the outer medulla from SR-121463B-treated rats (Fig. 1C).

SR-121463B did not alter NOS activity (Table 3) or eNOS expression (Fig. 6) in the cortex, inner stripe of the outer medulla, and papilla. In contrast, Fig. 7 shows that SR-121463B decreased nNOS expression to 19 ± 24 (not significant) and 42 ± 24% (P < 0.05) of the control value in the outer medulla and papilla, respectively.


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Fig. 6.   Effect of the V2-receptor antagonist SR-121463B on eNOS expression levels. Equal amounts of protein from the cortex, inner stripe of the outer medulla, and papilla of control and water-restricted rats were separated by 7% SDS-PAGE. After electrotransfer, eNOS was detected by Western blotting. A: Western blot showing results from 5 control and 5 SR-121463B-treated rats (SR) is shown. B: densitometric quantification of the Western blot shown in A. Values are means ± SE expressed as the percentage of the optical density value of control animals in each experiment.



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Fig. 7.   Effect of SR-121463B on nNOS expression levels. Equal amounts of protein from the inner stripe of the outer medulla and papilla of control and water-restricted rats were separated by 7% SDS-PAGE. After electrotransfer, nNOS was detected by Western blotting. A: Western blot showing results from 5 control and 5 SR-121463B-treated rats. B: densitometric quantification of the Western blot shown in A. Values are means ± SE expressed as the percentage of the optical density value of control animals in each experiment. * P < 0.05 vs. control.

Comparison of NOS activity and expression of NOS isoforms in Long-Evans and Brattleboro rats. To further assess the effect of AVP on renal NOS isoforms, we compared NOS activity and expression in Brattleboro rats and in control Long-Evans rats, their normal strain counterpart, and we assessed the effect of AVP infusion in Brattleboro rats. Table 1 shows that Brattleboro rats had a higher urinary output (14-fold) and lower urinary osmolality compared with Long-Evans rats but no difference in sodium balance and growth (Tables 1 and 2). The absence of endogenous AVP in Brattleboro rats was illustrated by very low AQP2 expression levels in the inner stripe of the outer medulla (Fig. 1D). AVP infusion into Brattleboro rats markedly decreased urinary output and increased urinary osmolality but did not alter sodium balance and growth (Tables 1 and 2). The effect of AVP infusion on water excretion was associated with an increase in expression levels of AQP2 in the inner stripe of the outer medulla (Fig. 1D).

Compared with control Long-Evans rats, Brattleboro rats exhibit a significantly lower NOS activity in the outer medulla (46 ± 15% of the control value, P < 0.05) and papilla (42 ± 15% of the control value, P < 0.05). Outer medullary and papillary NOS activity of Brattleboro rats returned to the levels of control rats after 1 wk of AVP infusion. In contrast, cortical NOS activity was similar in control and Brattleboro rats (Table 3).

As shown in Fig. 8, eNOS expression was similar in the cortex, inner stripe of the outer medulla, and papilla of Brattleboro and control Long-Evans rats. In contrast (Fig. 9), compared with Long-Evans rats, Brattleboro rats displayed low levels of nNOS expression in the outer medulla (17 ± 31% of the control value, P < 0.05) and papilla (34 ± 27% of the control value, P < 0.05). AVP infusion significantly increased Brattleboro rat NOS expression to 54 ± 24 (P < 0.05) and 99 ± 20% (P < 0.05) of the control value in the outer medulla and papilla, respectively.


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Fig. 8.   Expression levels of eNOS in Long-Evans (Control; C), untreated (B), and AVP-infused Brattleboro rats (B+A). Equal amounts of protein from the cortex, inner stripe of the outer medulla, and papilla of Long-Evans and untreated or AVP-infused Brattleboro rats were separated by 7% SDS-PAGE. After electrotransfer, eNOS was detected by Western blotting. A: Western blot showing results from 5 control Long-Evans rats, 5 untreated, and 5 AVP-infused Brattleboro rats. B: densitometric quantification of the Western blot shown in A. Values are means ± SE expressed as the percentage of the optical density value of control animals in each experiment.



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Fig. 9.   Expression levels of nNOS in Long-Evans and Brattleboro rats. Equal amounts of protein from the inner stripe of the outer medulla and papilla of Long-Evans and untreated or AVP-infused Brattleboro rats were separated by 7% SDS-PAGE. After electrotransfer, nNOS was detected by Western blotting. A: Western blot showing results from 5 control Long-Evans rats, 5 untreated Brattleboro, and 5 AVP-infused Brattleboro rats is shown. B: densitometric quantification of the Western blot shown in A. Values are means ± SE expressed as the percentage of the optical density value of control animals in each experiment. * P < 0.05 vs. control; &P < 0.05 vs. Brattleboro.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our study has investigated the relationship between renal expression of NOS isoforms and AVP. The results demonstrate that AVP specifically modulates the expression of renal nNOS through V2 receptors but does not influence the expression of renal eNOS. In addition, our results confirm the renal expression pattern of eNOS and nNOS with a predominant expression of eNOS throughout the kidney, whereas nNOS is mainly restricted to the renal medulla and papilla.

The present study shows that nNOS, specifically expressed in the renal outer medulla and papilla, follows the same pattern of expression as AQP2, the AVP-regulated water channel, in response to water deprivation or water loading. Our observations are consistent with previous results obtained by Shin et al. (32), who showed an increase in nNOS mRNA levels in the outer medulla and papilla of water-deprived rats. We are adding more information by demonstrating that protein expression of nNOS is significantly altered by water deprivation or loading. Moreover, administration of the specific V2-receptor antagonist SR-121463B (31) decreased expression of nNOS, strengthening the link with AVP through its action on the basolateral V2 receptor of principal cells. Finally, Brattleboro rats, which are genetically AVP deficient, had very low renal nNOS expression levels that significantly increased after chronic AVP infusion. Altogether, these observations demonstrate that in vivo AVP stimulates nNOS protein expression through V2 receptors. However, our results cannot discriminate between the direct or indirect effects of AVP. This is the limitation of in vivo studies, as already mentioned with the results suggesting a long-term control of AQP2 (4a, 5, 34) and epithelial sodium channel subunits (7) expression by AVP. Nevertheless, one could conceive that AVP increases expression of nNOS through paracrine and/or autocrine factors or by decreasing the action of an endogenous protein inhibitor of nNOS (12). Importantly, the variations of nNOS protein expression are independent of alterations in food intake, growth, or sodium balance because, with the exception of water-restricted rats, these parameters were similar in both control and experimental groups of rats. In contrast, eNOS expression does not appear to be under AVP control. Indeed, neither decreasing endogenous circulating levels of AVP by water loading nor treatment with a V2-receptor antagonist altered renal eNOS expression. Furthermore, Brattleboro rats exhibited similar eNOS expression levels to control rats despite the complete absence of circulating AVP, and AVP infusion did not significantly alter eNOS expression levels. Thus the increase in eNOS expression restricted to the outer medulla of water-restricted rats, confirming previous observations (32), was most likely secondary to dehydration and subsequent hypovolemia, which was illustrated by oliguria, weight loss, and sodium retention in these rats. This interpretation is consistent with increased plasma and kidney angiotensin II levels in water-deprived rats (32).

In addition to hormones and neuromediators (9), renal sodium and water handling is also controlled by local factors such as NO, which plays an increasingly recognized role (18). Active sodium transport has been shown to be regulated through an increase in NO and cGMP (22, 35). Our study suggests that nNOS may be the major NOS isoform involved in the control of sodium and water handling by NO. Quantitatively, however, eNOS seems to be responsible for most renal NO production (13), but principal cells of the collecting duct system and inner medullary collecting duct cells express nNOS (18, 36). A strong body of evidence suggests that nNOS plays an important role in this renal segment in which fine regulation of sodium and water balance occurs. Indeed, high-salt intake increases renal nNOS expression (29). In addition, NO exerts AVP-like effects in principal cells of the collecting duct system through indirect stimulation of the epithelial sodium channel by apical membrane hyperpolarization (22) and direct stimulation of phosphorylation and apical membrane insertion of AQP2 through activation of protein kinase G by cGMP (2). Therefore, the increase in expression of nNOS, leading to increased NO production, may participate in the effects of AVP on water (16) and sodium transport (27) in the collecting duct system. Besides direct effects on transporters, NO can also modulate renal water and sodium handling through its hemodynamic effects. Indeed, NO regulates medullary blood flow and administration of NG-nitro-L-arginine methyl ester into the medullary interstitial space, decreasing sodium and water excretion without modifying glomerular filtration rate or blood pressure (23).

Our results confirm the distribution pattern of renal eNOS and nNOS protein expression (1, 18). In agreement with most authors, eNOS protein expression is detected throughout the kidney, and nNOS protein expression is predominantly located in the medulla and papilla. In the renal cortex, nNOS expression is restricted to macula densa cells (1) and cortical collecting duct principal cells (36) and is below the detection threshold of Western blot analysis in crude cortex homogenates. With the exception of Brattleboro rats, NOS activity measured at Vmax was parallel to eNOS expression levels. The absence of AVP-dependent modulation of NOS activity as well as the parallel increase in NOS activity and eNOS protein expression in the outer medulla of water-restricted rats suggests that eNOS generated the majority of the renal NO, thus occulting changes in nNOS-generated NO production that are quantitatively less important in Wistar rats. However, compared with Long-Evans rats, NOS activity is significantly lower in Brattleboro rats and varies in parallel with nNOS expression levels. This finding may suggest that the contribution of nNOS to overall NOS activity is larger in this specific rat strain. Finally, we did not find any protein expression of iNOS isoform, which does not exclude that a low level of iNOS protein was present, because iNOS mRNA has been demonstrated in the cortex and inner and outer medulla (18, 20). However, such a low level may not have a role in this setting.

In conclusion, our results indicate that AVP specifically increases the expression of nNOS in the renal medulla and papilla. This effect may participate in long-term control of water and sodium reabsorption by AVP in the collecting duct system.


    ACKNOWLEDGEMENTS

We thank Dr. Serradeil-Le Gal for the gift of SR-121463B.


    FOOTNOTES

This work was supported in part by Swiss National Foundation for Science Grant 31-56504 and the Sir Jules Thorn Charitable Overseas Trust Shaan (P.-Y. Martin).

Address for reprint requests and other correspondence: P.-Y. Martin, Div. of Nephrology, Hôpital Cantonal Universitaire, 24 rue Micheli du Crest, 1211 Geneva 14, Switzerland (E-mail: Pierre-Yves.Martin{at}hcuge.ch).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

March 19, 2002;10.1152/ajprenal.00309.2001

Received 3 October 2001; accepted in final form 11 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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