INTRODUCTION

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NITRIC OXIDE (NO) regulates glomerular hemodynamics and renal blood flow and induces natriuresis (11). Within the kidney, the capacity to synthesize NO has been demonstrated in both tubular epithelium and vascular endothelium. Recently, the renal medulla has been shown to produce the highest levels of NO in the kidney (26). In this regard, there are at least three isoforms of nitric oxide synthase (NOS) that could contribute to medullary NO production: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). In the medulla, abundant nNOS mRNA has been demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) in inner medullary collecting ducts (IMCD), with lesser expression in the vasa recta and the inner medullary thin limb (22). Bachmann et al. (3), however, demonstrated immunoreactivity for nNOS in the macula densa and afferent arteriole but did not detect nNOS in IMCD. With regard to iNOS mRNA, studies have demonstrated its presence in IMCD and inner medullary interstitial cells (1). Histochemical analysis of renal tissue, however, has not detected basal iNOS protein expression in the IMCD (2). In contrast, eNOS mRNA and protein are distributed in endothelial cells throughout the renal vasculature, although expression of this isoform in renal tubules is not consistently detected (20, 25).

In rats, chronic infusion of an inhibitor of NOS directly into the renal medullary interstitium results in a selective decrease in medullary blood flow, associated with salt and water retention and development of hypertension (14). Inhibition of nNOS expression by intramedullary antisense oligonucleotide administration increases blood pressure in rats maintained on a high-salt diet, suggesting that the ability of the kidneys to excrete sodium may be impaired in the absence of medullary nNOS activity (12). Furthermore, during high dietary salt intake, the serum concentration and urinary excretion of the NO decomposition products NO₂ and NO₃ increase (18), suggesting that enhanced NO production represents an adaptive mechanism that induces vasodilatation and natriuresis, thereby contributing to the maintenance of normal blood pressure.

The potential importance of nNOS-derived NO in the inner medulla led us to examine whether this isoform is expressed in tubular cells of the rat IMCD and to test the hypothesis that high dietary NaCl increases nNOS expression in this segment. Our results demonstrate that nNOS protein is readily detected in IMCD by immunofluorescence and that high dietary NaCl differentially regulates nNOS mRNA and protein expression in the IMCD and renal cortex. Whereas high dietary NaCl inhibits nNOS mRNA and protein expression in the cortex, it increases nNOS protein expression without affecting mRNA levels in the IMCD. In contrast, high dietary NaCl has no effect on iNOS mRNA expression in the IMCD.

	MATERIALS AND METHODS

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Animals. Male Sprague-Dawley rats (200-250 g) were fed a standard (0.3% NaCl, control) or high-salt (4.0% NaCl) diet (Harlan Teklad, Madison, WI) for either 3 days or 3 wk, with unlimited access to water. The diets were equivalent in contents of all other electrolytes. For 24 h prior to death, the rats were in metabolic cages for urine collection. After death, the kidneys were immediately removed for dissection of cortices and for isolation of IMCD. Kidneys from two rats were pooled for each IMCD preparation.

IMCD preparation. Rat IMCDs were isolated using a modification of the method of Grenier and Smith (8), essentially as described (10). Briefly, renal papillary tissue was rapidly minced and suspended in Krebs buffer containing (in mM) 118 NaCl, 14 glucose, 25 NaHCO₃, 4.7 KCl, 2.5 CaCl₂, 1.8 MgSO₄, and 1.8 KH₂PO₄; 0.1% collagenase A (type I; Boehringer-Mannheim, Laval, Quebec, Canada); and 0.01% deoxyribonuclease (Boehringer-Mannheim). The minced tissue was incubated for 120 min at 37°C in 5% CO₂-95% air with gentle agitation. After collagenase digestion, the tubule suspension was pelleted by centrifugation at 100 g for 30 s at room temperature and washed three times with Krebs buffer. The pellet was then reconstituted in a hypotonic solution consisting of Krebs buffer and sterile water (1:2 respectively, vol/vol), gently mixed for 4 min, and centrifuged at 100 g for 30 s. This procedure has been shown to disrupt all cells, except those of the IMCD (8). The tubules were washed three times with Krebs buffer and then used immediately for either RNA or protein isolation. Cell viability was assessed by trypan blue exclusion, which demonstrated that at least 95% of IMCD cells were viable.

Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from cortex and suspensions of IMCD using a commercially available kit (RNeasy; Qiagen, Chatsworth, CA). Prior to RT-PCR, residual genomic DNA was digested by incubating RNA with amplification grade DNase I (Life Technologies, Burlington, ON, Canada) for 15 min at room temperature. DNase I was inactivated by adding ethylenediaminetetraacetic acid (final concentration, 1.6 mM) followed by incubation at 65°C for 15 min. RNA quality was determined by running samples on 1% agarose-formaldehyde gels stained with ethidium bromide, and RNA concentration and purity were determined by optical density measurement at 260 and 280 nm. RNA samples were uniformly of high quality by these standards.

Serial dilutions of total RNA (6, 30, 150, and 750 ng) were prepared in RNase-free water and reverse transcribed in a buffer containing 5 mM MgCl₂, 1× PCR buffer (50 mM KCl, 10 mM Tris · HCl, pH 8.3), 1 mM each of deoxynucleoside triphosphates, 1 µCi [-³²P]dCTP (sp act, 3,000 Ci/mmol; Amersham, Mississauga, ON), 20 U RNase inhibitor, 50 U Moloney murine leukemia virus reverse transcriptase, and 2.5 µM random hexamers (GeneAmp RNA PCR kit; Perkin-Elmer, Norwalk, CT) in a total volume of 20 µl. After reverse transcription, reactions were brought up to 100 µl with 1× PCR buffer supplemented with (final concentrations) 2 mM MgCl₂, 2.5 U AmpliTaq DNA polymerase (Perkin-Elmer), and 1 µM of either nNOS- or iNOS-specific oligonucleotide primers. After initial denaturation at 94°C for 3 min, 33 cycles of amplification [94°C for 30 s, 68°C (nNOS) or 60°C (iNOS) for 30 s, and 72°C for 30 s] were performed, followed by final extension at 72°C for 10 min, in a Perkin-Elmer GeneAmp PCR system 2400 apparatus. The PCR products were separated on ethidium bromide-stained 2% agarose gels, the gel bands were excised, and the incorporated radioactivity was determined by liquid scintillation counting.

To determine relative differences in NOS mRNA abundance in rats on 0.3% and 4.0% NaCl diets, we used the titration analysis method as described (17). This is a noncompetitive, semiquantitative method that utilizes the linear relationship between the logarithm of the initial amount of target mRNA and the logarithm of the amount of amplification product generated, providing that the PCR reactions have not reached plateau phase. To ensure that all data were collected before PCR reactions reached this plateau, we measured ³²P incorporation into PCR products as a function of cycle number for both nNOS and iNOS mRNA amplifications. In both cases, PCR product amplification was exponential for at least 40 cycles. Accordingly, we used 33 cycles of amplification for all further experiments. To minimize tube-to-tube variation in PCR efficiency, we used a master mix of reaction components in all experiments. Under these conditions, the logarithm of incorporated radioactivity varied linearly with the logarithm of initial RNA amount (not shown). To control for variations in RNA isolation and for the efficiency of the reverse transcription reaction, -actin mRNA was amplified at the time of nNOS and iNOS mRNA amplifications. Total RNA (150 ng) was reverse transcribed and amplified as described above, in the presence of specific primers for -actin (20 nM). As a control for any possible genomic DNA contamination, a reaction lacking the reverse transcriptase enzyme was included in all experiments.

The primer sequences for nNOS, iNOS, and -actin products are reported in Table 1. The primers for nNOS and iNOS were chosen from the rat brain (5) and murine macrophage (28) cDNA sequences, respectively. The specificity of the primers was verified through GenBank. The -actin primers were selected according to the human cDNA sequence (9).

The identity of the nNOS PCR product was confirmed by restriction enzyme digestion with either Bgl II or Sca I, which generated the expected products (210 and 389 bp and 101 and 498 bp, respectively). To confirm the fidelity of iNOS RT-PCR, the rat IMCD iNOS PCR product was subcloned, and DNA derived from a single bacterial colony was partially sequenced (92 bp, corresponding to bp 1194-1286; Ref. 28), using the Taq Cyclist DNA sequencing kit (Stratagene, La Jolla, CA). This product demonstrated 98% nucleotide identity to the murine macrophage iNOS cDNA (28) and 100% nucleotide identity to rat vascular smooth muscle iNOS cDNA (7) in the sequenced region.

Western blotting. IMCDs from 0.3% and 4.0% NaCl-fed rats were homogenized with a cell disrupter in boiling lysis buffer [1% sodium dodecyl sulfate (SDS), 10 mM Tris · HCl, pH 7.4]. The lysate was then boiled for 10 min, followed by centrifugation at 12,000 g for 2 min to remove insoluble debris. Protein concentrations in the supernatant were determined by the Bradford method (Bio-Rad, Montreal, PQ), using bovine serum albumin (BSA; Sigma, St.Louis, MO) as standard. Tubule lysates (20, 40, or 60 µg) were separated on 7.5% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad). The membranes were blocked with 10% skimmed milk in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 (TBS-T) and 0.01% sodium azide for 3 h at room temperature. The membranes were then incubated for 18 h at 4°C with 1:1,000 dilution of mouse monoclonal antibody to nNOS or 1:400 dilution of mouse monoclonal antibody to iNOS (Transduction Laboratories, Lexington, KY). The mouse monoclonal antibody to nNOS was raised against a human peptide fragment of nNOS and reacts against both human and rat nNOS at 160 kDa. By Western blot analysis, we determined that this antibody did not cross-react with endothelial cell (eNOS) protein lysates (Transduction Laboratories) but did detect a 130-kDa band, clearly discernible from nNOS, from cytokine-stimulated macrophages (iNOS), as reported by the manufacturer. In some experiments, two separate rabbit polyclonal antibodies to iNOS were used (Transduction Laboratories; Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were then incubated with 1:2,000 dilution of either anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Amersham). Primary antibodies were diluted in TBS-T supplemented with 5% skimmed milk and 0.01% sodium azide, whereas the secondary antibodies were diluted in TBS-T supplemented with 2% milk. Proteins were detected by enhanced chemiluminescence (ECL, Amersham) on Hyperfilm (Amersham), according to the manufacturer's instructions. Prestained standards were used as molecular weight markers (Bio-Rad), and rat pituitary and macrophage lysates (4 µg, Transduction Laboratories) were used as positive controls for nNOS and iNOS, respectively. To control for protein loading, all membranes were stripped and probed with a monoclonal anti--actin antibody (mouse ascites fluid, Sigma). Signals on Western blots were quantified by densitometry and corrected for the -actin signal, using an image analysis software program (NIH Image 1.47).

Immunofluorescence studies. IMCD segments were microdissected from rat kidney medulla, as described (16), dried onto microscope slides for 1 h at room temperature, and then fixed in a 1:1 (vol/vol) acetone-methanol mixture for 15 min at room temperature. After fixation, the tubules were permeabilized with 0.3% Triton X-100 in phosphate-buffered saline (PBS, pH 7.4) for 30 min at room temperature. Slides were then incubated in blocking buffer consisting of PBS supplemented with 1% BSA for 30 min. A 1:200 dilution of polyclonal nNOS antibody (Santa Cruz Biotechnology) was added to the slides and incubated for 18 h at 4°C. In separate experiments, this antibody did not cross-react with protein lysates (Transduction Laboratories) from cytokine-stimulated macrophages (iNOS) or endothelial cells (eNOS) on Western blots (not shown), confirming the manufacturer's description regarding its specificity. In contrast, the antibody detected the expected nNOS protein band of 160 kDa in the pituitary and IMCD lysates. IMCDs incubated in the presence of primary antibody that had been previously neutralized with the immunizing antigen were used as negative controls (Santa Cruz Biotechnology). The slides were then washed three times for 5 min each in PBS and incubated with donkey anti-rabbit secondary antibody (1:20 dilution, Amersham) conjugated to fluorescein isothiocyanate (FITC) for 60 min at 37°C. All antibodies were diluted in PBS supplemented with 0.3% Triton X-100. After three washes with PBS, the slides were covered with an anti-fade compound (PBS containing 0.1 mM phenylaminediamine and 10% glycerol) and viewed with a Zeiss Axioplan fluorescent microscope. Photographs were taken with Kodak Tri-X black and white film.

Urinary nitrate/nitrite content. Urinary nitrate/nitrite content was determined by the nitrate reductase/Griess reaction assay as described (18). First, nitrate present in the urine samples was reduced to nitrite in the presence of nicotinamide adenine dinucleotide phosphate (150 µg/ml), flavin adenine dinucleotide (3.0 µg/ml), and 0.4 U nitrate reductase (Boehringer-Mannheim) for 30 min at room temperature. Nitrite was then measured by combining reduced urine aliquots with a 1:1 mixture (vol/vol) of 1% sulfanilamide (in 30% acetic acid) and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride (in 60% acetic acid). Absorbances were read on an ELISA reader at 550 nm. Nitrate and nitrite content was calculated using nitrate and nitrite standard curves.

Urinary sodium excretion. Urinary sodium concentration was determined by flame photometry (model IL-943; Instrumentation Laboratory, Milan, Italy), kindly performed by the laboratory of Dr. D. Z. Levine (University of Ottawa).

Statistics. Results are presented as means ± SE. Data were analyzed by the unpaired Student t-test. A value of P < 0.05 was considered significant.

	RESULTS

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Effect of high dietary NaCl on nNOS mRNA and protein expression in IMCD and kidney cortex. Relatively high levels of nNOS mRNA are found in the IMCD (22), yet histochemical methods have been unable to detect nNOS protein in this segment (3). As shown in Fig. 1A, using a polyclonal antibody to nNOS, cells within freshly microdissected IMCDs stained strongly for nNOS, with a cytoplasmic pattern. The specificity of the response was verified by staining IMCD with nNOS antibody, which had been previously neutralized with immunizing antigen, which resulted in complete loss of immunofluorescence (Fig. 1B). A monoclonal nNOS antibody (Transduction Laboratories) also strongly stained microdissected IMCD (not shown).

View larger version (92K): [in this window] [in a new window]   Fig. 1.   Immunolocalization of neuronal nitric oxide synthase (nNOS) in inner medullary collecting duct (IMCD). Shown are fluorescence micrographs of microdissected IMCD segments labeled with a polyclonal nNOS antibody (A) or with nNOS polyclonal antibody neutralized by preincubation with immunizing antigen (B). Similar results were obtained in 2 additional experiments.

The effect of high dietary NaCl on the expression of nNOS mRNA and protein in IMCD was determined, using RT-PCR and Western blotting, respectively. In rats fed a 4.0% NaCl diet for 3 days or 3 wk, urinary sodium excretion significantly increased (Table 2, P < 0.001 vs. 0.3% NaCl diet). This was associated with significant increases in urinary NO₂/NO₃ excretion (Fig. 2: 3 days of 0.3% NaCl, 2.82 ± 0.31, vs. 4.0% NaCl, 3.79 ± 0.43 µmol · 100 g body wt¹ · 24 h¹, P < 0.05, n = 12; 3 wk of 0.3% NaCl, 1.21 ± 0.26, vs. 4.0% NaCl, 1.92 ± 0.08 µmol · 100 g body wt¹ · 24 h¹, P < 0.01, n = 7). Three days of 4.0% NaCl diet had no effect on nNOS mRNA expression in IMCD (Fig. 3) [0.3% NaCl, 19.84 ± 1.57 × 10³, vs. 4.0% NaCl, 20.44 ± 3.14 × 10³ cpm, P = not significant (NS), n = 3] but significantly increased nNOS protein expression in IMCD (Fig. 4) (0.3% NaCl, 0.51 ± 0.12, vs. 4.0% NaCl, 0.92 ± 0.14 arbitrary units, P < 0.05, n = 5).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effect of high-NaCl diet on urinary excretion of NO2 + NO3. A: 24-h urinary content of NO2 + NO3 after 3 days of 4.0% NaCl intake. B: 24-h urinary content of NO2 + NO3 after 3 wk of 4.0% NaCl intake. Results are means ± SE. A: * P < 0.05, n = 12. B: * P < 0.01, n = 7.

View larger version (24K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effect of 3 days of 4.0% NaCl diet on expression of nNOS mRNA in IMCD. A: representative ethidium bromide-stained agarose gel of IMCD nNOS RT-PCR products (599 bp) from 0.3% (lanes 1-4) and 4.0% (lanes 5-8) NaCl-fed rats. Dilutions of total RNA were (from left) 750, 150, 30, and 6 ng. -Actin PCR products (350 bp) from IMCD of 0.3% and 4.0% NaCl-fed rats are shown in lanes 9 and 10, respectively. B: radioactivity incorporated into nNOS mRNA amplification products. Results are means ± SE for 30 ng of initial total RNA (n = 3).

View larger version (45K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   Fig. 4.   High-NaCl diet increases nNOS protein expression in IMCD at 3 days. A: representative Western blot of nNOS protein expression in IMCD of rats fed a 4.0% NaCl diet for 3 days. Lanes 1 and 3: proteins from 0.3% NaCl-loaded rats, 40 and 20 µg of total protein, respectively. Lanes 2 and 4: proteins from 4.0% NaCl-loaded rats, 40 and 20 µg of total protein, respectively. Corresponding bands for -actin are depicted, demonstrating equal loading. B: densitometric quantification of Western blot signals for nNOS protein in IMCD. Results are means ± SE (* P < 0.05, n = 5).

After 3 wk of 4.0% NaCl intake, both nNOS mRNA (Fig. 5) (0.3% NaCl, 11.02 ± 1.05 × 10³, vs. 4.0% NaCl, 13.71 ± 3.02 × 10³ cpm, P = NS, n = 3) and nNOS protein (Fig. 6: 0.3% NaCl, 1.03 ± 0.16, vs. 4.0% NaCl, 1.13 ± 0.12 arbitrary units; P = NS, n = 3) in the IMCD did not differ from control values, indicating that the salt-induced nNOS protein upregulation is transient.

View larger version (25K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effect of 3-wk 4.0% NaCl diet on expression of nNOS mRNA in IMCD. A: representative ethidium bromide-stained agarose gel of IMCD nNOS RT-PCR products (599 bp) from 0.3% (lanes 1-4) and 4.0% (lanes 5-8) NaCl-fed rats. Dilutions of total RNA were (from left) 750, 150, 30, and 6 ng. -Actin PCR products (350 bp) from 0.3% and 4.0% NaCl-fed rats are shown in lanes 9 and 10, respectively. B: radioactivity incorporated into nNOS mRNA amplification products. Results are means ± SE for 30 ng of initial total RNA (n = 3).

View larger version (22K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effect of 3-wk 4.0% NaCl diet on nNOS protein expression in IMCD. A: representative Western blot of nNOS protein expression in IMCD of rats fed 4.0% NaCl diet for 3 wk. Lane 1: rat pituitary lysate (positive control). Lanes 2 and 4: proteins from 0.3% NaCl-fed rats, 20 and 40 µg total protein, respectively. Lanes 3 and 5: proteins from 4.0% NaCl-fed rats, 20 and 40 µg total protein, respectively. B: densitometric quantification of Western blot signals for nNOS protein in IMCD. Results are means ± SE (n = 3).

Previous studies have demonstrated downregulation of renal cortical nNOS mRNA by high dietary salt (4, 20). In agreement with these studies and in contrast to our results in the IMCD, kidney cortical nNOS mRNA significantly decreased after 3 days of 4.0% NaCl intake (Fig. 7) (0.3% NaCl, 30.88 ± 4.36 × 10³, vs. 4.0% NaCl, 16.4 ± 4.70 × 10³ cpm; P < 0.05, n = 5). This was associated with a significant decrease in kidney cortical nNOS protein expression, determined by Western blot analysis (Fig. 8) (0.3% NaCl diet, 1.88 ± 0.14, vs. 4.0% NaCl, 0.92 ± 0.20 arbitrary units; P < 0.05, n = 5).

View larger version (24K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   Fig. 7.   High-NaCl diet decreases nNOS mRNA expression in kidney cortex at 3 days. A: representative ethidium bromide-stained agarose gel of kidney cortex nNOS RT-PCR products (599 bp) from 0.3% (lanes 1 and 2) and 4.0% (lanes 3 and 4) NaCl-fed rats. Dilutions of total RNA were (from left) 150 and 30 ng. -Actin PCR products (350 bp) from 0.3 and 4.0% NaCl-fed rats are shown in lanes 5 and 6, respectively. B: radioactivity incorporated into nNOS mRNA amplification products. Results are means ± SE for 150 ng initial total RNA. * P < 0.05, n = 5.

View larger version (27K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   Fig. 8.   High-NaCl diet decreases nNOS protein expression in kidney cortex at 3 days. A: representative Western blot of nNOS protein expression in kidney cortex of rats fed 4.0% NaCl diet for 3 days. Lane 1: 0.3% NaCl-fed rats (40 µg total protein); lane 2: 4.0% NaCl-fed rats (40 µg total protein). B: densitometric quantification of Western blot signals for nNOS protein from kidney cortex. Results are means ± SE. * P < 0.05, n = 5.

In all experiments, to control for possible variations in RNA isolation and reverse transcription reactions, -actin mRNA was PCR amplified along with the nNOS mRNA amplifications. There were no differences in radioactivity incorporated into -actin PCR products among samples derived from 0.3 or 4.0% NaCl-fed rats (not shown). Similarly, there were no differences in -actin protein abundance among samples derived from 0.3 or 4.0% NaCl-fed rats by Western blot analysis.

Effect of high dietary NaCl on iNOS mRNA expression in IMCD. Basal expression of iNOS mRNA has been reported in the IMCD (15). High dietary NaCl intake had no effect on IMCD iNOS mRNA expression after either 3 days (Fig. 9) (0.3% NaCl, 3.43 ± 0.14 × 10³, vs. 4.0% NaCl, 3.14 ± 0.52 × 10³ cpm; P = NS, n = 5) or 3 wk (Fig. 10) (0.3% NaCl, 3.90 ± 0.32 × 10³, vs. 4.0% NaCl, 4.44 ± 0.74 × 10³ cpm; P = NS, n = 3).

View larger version (30K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   Fig. 9.   Effect of 3-day 4.0% NaCl diet on expression of iNOS mRNA in IMCD. A: representative ethidium bromide-stained agarose gel of IMCD iNOS RT-PCR products (705 bp) from 0.3% (lanes 1-4) and 4.0% (lanes 5-8) NaCl-fed rats. Dilutions of total RNA were (from left) 750, 150, 30, and 6 ng. -Actin PCR products (350 bp) from IMCD of 0.3% and 4.0% NaCl-fed rats are shown in lanes 9 and 10, respectively. B: radioactivity incorporated into IMCD iNOS mRNA PCR products. Results are means ± SE for 6 ng initial total RNA (n = 5).

View larger version (26K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   Fig. 10.   Effect of 3-wk 4.0% NaCl diet on expression of iNOS mRNA in IMCD. A: representative ethidium bromide-stained agarose gel of IMCD iNOS RT-PCR products (705 bp) from 0.3% (lanes 1-4) and 4.0% (lanes 5-8) NaCl-fed rats. Dilutions of total RNA were (from left) 750, 150, 30, and 6 ng. -Actin PCR products (350 bp) from IMCD of 0.3% and 4.0% NaCl-fed rats are shown in lanes 9 and 10, respectively. B: radioactivity incorporated into IMCD iNOS PCR products. Results are means ± SE for 6 ng of initial total RNA (n = 3).

Western blots, using a mouse monoclonal antibody to iNOS (Transduction Laboratories) and with loading of up to 100 µg protein per lane, failed to detect the expected 130-kDa iNOS protein in lysates from IMCD in either 0.3 or 4.0% NaCl-fed rats. Two other polyclonal antibodies to iNOS also did not detect the iNOS protein on Western blots.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The present studies determined the effect of high dietary salt on nNOS expression in the IMCD. Our data clearly demonstrate that nNOS mRNA and protein are expressed in the IMCD. In addition, we show that high dietary salt intake differentially regulates nNOS mRNA and protein expression in the IMCD and renal cortex but has no effect on expression of iNOS mRNA in the IMCD.

Localization of nNOS. Utilizing RT-PCR, Terada et al. (22) detected the highest levels of intrarenal nNOS mRNA in the IMCD. The results of the present study, in which we readily detected nNOS mRNA in normal rat IMCD by RT-PCR, are consistent with this study and with the data of Singh et al. (20). Previous immunolocalization studies, however, have not detected nNOS protein expression in the IMCD, although an abundance of nNOS is present in the macula densa (3). In contrast, in the present studies, immunofluorescence revealed intense cytoplasmic staining for nNOS in cells of freshly microdissected IMCD, and this staining was specific for nNOS, since preincubation of the polyclonal nNOS antibody with immunizing antigen resulted in complete elimination of immunofluorescence. Furthermore, Western blot analysis of proteins extracted from IMCD revealed a single band of ~160 kDa that comigrated with nNOS from a rat pituitary lysate, used as a positive control. Taken together, therefore, our data clearly demonstrate presence of nNOS mRNA and protein in IMCD. The explanation for the lack of detection of nNOS protein in IMCD by Bachmann et al. (3) is unclear, although we speculate that differences in either tissue processing methods, antibodies, or immunodetection methods may account for the varying results.

Regulation of nNOS by dietary salt in IMCD and cortex. Inhibition of renal medullary nNOS activity increases mean arterial pressure in rats maintained on high-salt diets, suggesting that this NOS isoform may be involved in the long-term regulation of blood pressure (12). In the present study, nNOS protein expression in the IMCD significantly increased after 3 days of high dietary NaCl, but nNOS mRNA levels remained unchanged. This suggests either increased translation of nNOS mRNA into protein or enhanced protein stability due to posttranslational modification. The regulation of nNOS gene transcription and translation, however, is incompletely understood. In this regard, nNOS mRNAs with distinct 5'-untranslated regions that are encoded by separate promoters have been described in human cerebellum (27). These mRNAs likely encode identical nNOS proteins but could differ in stability, processing, or translation efficiency. Accordingly, it is possible that high dietary salt could induce a conversion to formation of nNOS mRNA transcripts with increased efficiency of translation, via as yet undefined signaling mechanisms. Alternatively, the possibility exists that the semi-quantitative RT-PCR method utilized in the present study was not sensitive enough to detect small increases in mRNA expression that could have accounted for the increased expression of nNOS at 3 days. Our results, however, are in agreement with those of Singh et al. (20), who did not detect an effect of high-salt diet for 7 days on nNOS mRNA expression in the rat IMCD by competitive RT-PCR.

In rats on high-NaCl diet for 3 wk, IMCD nNOS mRNA and protein expression did not significantly change from control values, suggesting that the increase in nNOS protein observed at 3 days is transient. Although information regarding factors regulating nNOS expression is limited (reviewed in Ref. 11), our data suggest that the molecular signals for stimulation of nNOS protein expression are either no longer present or are counteracted by other factors after 3 wk of high-salt diet. In rat total inner medullary tissue, Mattson and Higgins (13) reported that protein expression of eNOS, nNOS, and iNOS increased significantly after 3 wk of high dietary salt intake. We cannot readily provide an explanation for these differences from our present data, although it is conceivable that high dietary salt might increase nNOS expression in other inner medullary structures, such as within renal nerves, vasa recta, or inner medullary thin limbs (3, 22). It must be noted, furthermore, that the Mattson and Higgins (13) study utilized a single monoclonal antibody to detect both nNOS and iNOS by Western blot analysis, and, in contrast to our results, they did not detect nNOS in renal cortical homogenates.

Within the renal cortex, the macula densa contains the highest levels of nNOS mRNA and protein (3, 20). In rats, salt deprivation stimulates nNOS mRNA expression in the macula densa, whereas salt loading significantly decreases mRNA expression (4, 20). Our data on nNOS mRNA expression in renal cortex, performed as a positive control for our assay system, are in agreement with these observations. In addition, we have extended these findings by demonstrating that renal cortical nNOS protein expression is significantly downregulated by high dietary salt intake. The cellular signaling mechanisms mediating the inverse relationship between salt intake and macula densa nNOS expression are unknown, but a recent study revealed that blockade of systemic AT₂ ANG II receptors significantly inhibited sodium depletion-induced increases in renal interstitial cGMP levels, suggesting that AT₂ receptors may be linked to stimulation of renal NO production (21). In addition to the possibility that differential regulation of nNOS expression between IMCD and renal cortex is due to separate cell signaling pathways, variable expression patterns may also be secondary to transcription of mRNAs from separate promoters, as discussed above (27). Furthermore, alternative splicing of nNOS mRNA yielding a novel nNOSµ protein isoform has been reported in skeletal muscle, suggesting that tissue-specific regulation of nNOS mRNA may occur (19).

Our data demonstrate an increase in urinary excretion of NO decomposition products after 3 days of high dietary salt intake, a time at which IMCD nNOS protein expression is increased but at which renal cortical nNOS is inhibited. However, urinary excretion of NO₂/NO₃ remained significantly increased after 3 wk of high salt intake, despite normalization of IMCD nNOS protein expression. In rats fed a high-salt diet for 2 wk, an increase in both serum concentration and urinary excretion of NO₂ and NO₃ has also been reported (18). Thus our data suggest a dissociation between IMCD nNOS protein expression and excretion of urinary NO products. Furthermore, although absolute levels of urinary NO₂/NO₃ were comparable in rats at 3 days and 3 wk, we observed decreased excretion at 3 wk, when correction for body weight was made. This suggests there may be age-dependent effects on urinary excretion of NO products.

The urinary excretion of NO products is complex, representing the contributions from glomerular filtration of serum NO₂ and NO₃, tubular handling, and de novo intrarenal NO synthesis. Thus the effects of enhanced nNOS protein in IMCD on intrarenal NO production cannot be estimated from our data. The gene expression and activity of eNOS have been shown to be upregulated by shear stress (24), suggesting that increased urinary levels of NO decomposition products with high salt intake could also reflect increased intrarenal activity of this isoform. In addition, the activity of nNOS protein may be affected by levels of intracellular calcium or endogenous inhibitors (reviewed in Ref. 11), independent of protein expression. Whether these mechanisms are involved in the regulation of nNOS activity in the IMCD with high salt is unknown.

Dietary NaCl and iNOS in IMCD. Our data demonstrate tonic expression of iNOS mRNA in the IMCD of normal rats, with levels not affected by high dietary salt. Abundant iNOS mRNA has previously been detected in rat IMCD by in situ hybridization (1). Restriction mapping of iNOS RT-PCR products in rat kidney revealed that the murine macrophage homologue of iNOS was the principal isoform expressed in tubular cells, including the IMCD, with lesser amounts of the rat vascular isoform of iNOS (15). The present studies were not designed to determine the relative amounts of these two mRNAs in IMCD cells. Rather, we chose PCR primers to examine total iNOS transcript levels in this segment. In this regard, it is of interest that a recent study identified four alternatively spliced iNOS mRNAs in human tissues, including kidney, with upregulation of all splice variants by cytokines (6). Accordingly, it is possible that multiple splice variants of iNOS exist in IMCD, although our data suggest that dietary salt intake does not affect total mRNA transcript levels.

In the present studies, Western blotting with three different antibodies failed to detect iNOS protein in IMCD. Recently, immunohistochemistry using antibodies to the macrophage-type iNOS in rat kidney demonstrated strong staining in outer medulla, cortical collecting ducts, and some proximal tubules, with no detection in IMCD (2). Tojo et al. (23), using a vascular smooth muscle iNOS antibody in rat kidney, did not report iNOS staining in IMCD, although there was immunolabeling in terminal afferent arteriole, distal tubule, and thick ascending limb. Our results are in agreement with these studies and suggest that iNOS mRNA is not efficiently translated into protein in IMCD under basal conditions or with high dietary salt.

In summary, the present studies demonstrate that cells of the normal rat IMCD express nNOS mRNA and protein. Protein expression of nNOS but not mRNA is upregulated by high dietary salt after 3 days. In contrast, high-salt diet decreases renal cortical mRNA and protein expression of nNOS. These data suggest that selective activation of inner medullary nNOS could be involved in the early adaptive response to high dietary salt intake.