Mechanisms of decreased tubular flow-induced nitric oxide in Dahl salt-sensitive rat thick ascending limbs

Abstract

Dahl salt-sensitive (SS) rat kidneys produce less nitric oxide (NO) than those of salt-resistant (SR) rats. Thick ascending limb (TAL) NO synthase 3 (NOS3) is a major source of renal NO, and luminal flow enhances its activity. We hypothesized that flow-induced NO is reduced in TALs from SS rats primarily due to NOS uncoupling and diminished NOS3 expression rather than scavenging. Rats were fed normal-salt (NS) or high-salt (HS) diets. We measured flow-induced NO and superoxide in perfused TALs and performed Western blots of renal outer medullas. For rats on NS, flow-induced NO was 35 ± 6 arbitrary units (AU)/min in TALs from SR rats but only 11 ± 2 AU/min in TALs from SS (P < 0.008). The superoxide scavenger tempol decreased the difference in flow-induced NO between strains by about 36% (P < 0.020). The NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) decreased flow-induced superoxide by 36 ± 8% in TALs from SS rats (P < 0.02) but had no effect in TALs from SR rats. NOS3 expression was not different between strains on NS. For rats on HS, the difference in flow-induced NO between strains was enhanced (SR rats: 44 ± 10 vs. SS: 9 ± 2 AU/min, P < 0.005). Tempol decreased the difference in flow-induced NO between strains by about 37% (P < 0.012). l-NAME did not significantly reduce flow-induced superoxide in either strain. HS increased NOS3 expression in TALs from SR rats but not in TALs from SS rats (P < 0.003). We conclude that 1) on NS, flow-induced NO is diminished in TALs from SS rats mainly due to NOS3 uncoupling such that it produces superoxide and 2) on HS, the difference is enhanced due to failure of TALs from SS rats to increase NOS3 expression.

NEW & NOTEWORTHY The Dahl rat has been used extensively to study the causes and effects of salt-sensitive hypertension. Our study suggests that more complex processes other than simple scavenging of nitric oxide (NO) by superoxide lead to less NO production in thick ascending limbs of the Dahl salt-sensitive rat. The predominant mechanism involved depends on dietary salt. Impaired flow-induced NO production in thick ascending limbs most likely contributes to the Na⁺ retention associated with salt-sensitive hypertension.

INTRODUCTION

Nitric oxide (NO) is an important regulator of kidney function (1–8). It inhibits salt reabsorption by several nephron segments, including the thick ascending limb (TAL) (2, 7–10). NO production may be diminished by lack of substrate, changes in NO synthase (NOS) expression, or uncoupling so that NO is no longer produced. When NOS is uncoupled, superoxide (${\text{O}}_2^{-}$) is generated rather than NO. NO bioavailability can be reduced by reactions with other reactive oxygen species (ROS) such as ${\text{O}}_2^{-}$, a process frequently referred to as NO scavenging. It is often difficult in experimental settings to distinguish between changes in NO production and NO bioavailability. Reductions in renal NO production and/or bioavailability have been implicated in the development of hypertension in many animal models (11–16), including the Dahl salt-sensitive (SS) rat (1, 17–21). The causes for this are not fully understood.

Thick ascending limbs comprise most of the mass of the outer medulla and are a major source of renal NO. NO is produced primarily by NO synthase 3 (NOS3) (9, 22) in this segment even though all three NOS isoforms are expressed (23–27). Acute increases in luminal flow stimulate NO production (28–31) and ${\text{O}}_2^{-}$ synthesis (32) by thick ascending limbs from Sprague-Dawley rats. However, flow-stimulated NO attenuates flow-induced ${\text{O}}_2^{-}$ (32). The blockade of flow-induced ${\text{O}}_2^{-}$ production by flow-induced NO likely evolved as a mechanism that enhances Na⁺ excretion and minimizes NO scavenging by ${\text{O}}_2^{-}$ in this rat strain during periods of high urinary flows.

Similar to luminal flow, angiotensin II (ANG II) enhances NO production/bioavailability in thick ascending limbs from Sprague-Dawley rats (33, 34). It also increases ${\text{O}}_2^{-}$ production (35–37). However, unlike flow-induced, ANG II-induced NO does not blunt ANG II-stimulated ${\text{O}}_2^{-}$ (38); rather, ANG II causes partial NOS uncoupling. Thus, NOS becomes a source of ${\text{O}}_2^{-}$ (38) in addition to NO.

On normal salt, medullary ROS are greater (39, 40) and NO is lower (19, 21, 41) in SS rats than in Dahl salt-resistant (SR) rats. Thick ascending limb cells in renal medulla strips of SS rats produce more ROS than those from normotensive controls (39). This has led to the assumption that bioavailable NO is reduced in thick ascending limbs of SS rats primarily due to scavenging by other ROS. This assumption is supported by data showing that ANG II-induced NO bioavailability in tubules of SS rats is blunted due, at least in part, to scavenging by ${\text{O}}_2^{-}$ (20). However, reduced NO bioavailability has also been shown to be due to NOS uncoupled in the medulla of SS rats (39). Although luminal flow is a critical regulator of NO and ROS production by thick ascending limbs, the effects of flow on NO production/bioavailability in thick ascending limbs of SS and SR rats have not been directly studied.

A high-salt diet increases the expression of NOS3 in thick ascending limbs likely via both increases in interstitial osmolality (42) and luminal flow (11, 43). This increase in expression augments the capacity of thick ascending limbs to produce NO (44). However, the effect of dietary salt on flow-induced NO production/bioavailability and NOS3 expression in thick ascending limbs of SS and SR rats has not been investigated.

We hypothesized that 1) when on a normal-salt diet, flow-induced NO production is decreased in thick ascending limbs of SS rats compared with SR rats primarily due to NOS uncoupling and reduced NOS3 expression rather than simple scavenging by ROS and 2) this difference is exaggerated by a high-salt diet due to increased NOS3 expression in tubules of SR rats but not those of SS rats.

MATERIALS AND METHODS

Chemicals and Solutions

The NO-sensitive dye 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA) was purchased from Life Technologies (Eugene, OR). Unless otherwise noted, all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). The physiological saline solution used to perfuse and bathe thick ascending limbs contained (in mM) 130 NaCl, 2.5 NaH₂PO₄, 4 KCl, 1.2 MgSO₄, 6 l-alanine, 1 Na₃citrate, 5.5 glucose, 2 Ca(lactate)₂, and 10 HEPES (pH 7.4 at 37°C) and was adjusted to 290 ± 3 mOsmol/kgH₂O as measured by vapor pressure osmometry.

Animals and Diets

All animal protocols were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Four- to six-wk-old male SS and SR rats (Envigo, Indianapolis, IN) were fed an artificial purified diet containing 0.6% NaCl (normal-salt diet, TestDiet No. 5876) or 4% NaCl (high-salt diet, TestDiet No. 9GDV) for 7–9 days. Rats were anesthetized with ketamine and xylazine (100 and 20 mg/kg body wt, respectively) for terminal surgeries.

Blood Pressure

Systolic blood pressure was measured via noninvasive tail-cuff plethysmography (CODA High Throughpoint System, Kent Scientific, Torrington, CT). After 3 days of training, baseline normal-salt diet blood pressure was measured just before changing to a high-salt diet. Blood pressure data for a high-salt diet were measured the day before or on the day of the experiment (6–8 days on a high-salt diet).

Isolation and Perfusion of Thick Ascending Limbs

After animals weighing between 120 and 150 g were anesthetized, the abdominal cavity was opened and the left kidney was bathed in ice-cold saline and removed. Kidney slices were placed in cold physiological saline solution, and medullary thick ascending limbs were dissected from the outer medulla under a stereomicroscope at 4–10°C. Tubules measuring 0.5–1.0 mm were transferred to a temperature-regulated chamber (37 ± 1°C) and perfused using concentric glass pipets as previously described (45).

Flow-Induced NO Production

NO was measured in isolated thick ascending limbs similar to what we have previously done (30, 31). Tubules were loaded with 2 μM DAF FM-DA in physiological saline solution for 15 min and then washed in dye-free physiological saline solution for 20 min. The dye was excited at 488 nm with a xenon arc lamp, while the emitted fluorescence was collected with a ×40 immersion oil objective mounted on an inverted microscope (Nikon Eclipse TE300) equipped with a 505-nm dichroic mirror and a 520- to 560-nm barrier filter. Emitted fluorescence was digitally imaged with a CoolSnap HQ digital camera (Photometrics, Tucson, AZ), and data were recorded with MetaFluor version 6.2r6 imaging software (Molecular Devices, Downingtown, PA). NO production was measured in the absence of luminal flow once every 30 s for 5 min during the first period. The luminal flow was then increased to 20 nL/min, and NO was measured for 15 min. The change in emitted fluorescence over time was taken as NO bioavailability and expressed as arbitrary units (AU) per minute. We assumed that the increase in NO bioavailability was primarily due to increased production rather than reduced degradation. To account for differences in dye loading, data were divided by the initial fluorescence reading. The difference in NO availability between period 1 (no flow) and period 2 (flow) was used to calculate flow-induced NO bioavailability. Experiments were performed in the presence of 0.4 mM l-arginine in the bath to support endogenous NO production.

Flow-Induced ${\text{O}}_2^{-}$ Production

${\text{O}}_2^{-}$ was measured in isolated thick ascending limbs similar to what we have previously done (32). Since thick ascending limbs produce NO and ${\text{O}}_2^{-}$ in response to increases in flow, for the N-nitro-l-arginine methyl ester (l-NAME) experiments, we measured ${\text{O}}_2^{-}$ in the absence of l-arginine to avoid the possible confounding effects of flow-induced NO. Tubules were perfused at 15 nL/min and loaded with 5 μM dihydroethidium (Invitrogen, Eugene, OR) in physiological saline solution for 15 min and then washed in dye-free physiological saline solution for 30 min. Fifteen minutes before the end of the wash, the flow was stopped. Oxyethidium and dihydroethidium were excited using 488- and 365-nm light, respectively. Using a ×40 oil immersion objective on a Nikon Diaphot inverted microscope, fluorescence emitted between 520 and 600 nm (oxyethidium) and 400 and 450 nm (dihydroethidium) was imaged digitally with an image intensifier connected to a charge-coupled device camera and recorded once every 5 s with MetaFluor imaging software. After 1 min of measurements in the absence of flow, the luminal flow was increased to 15 nL/min, and measurements were acquired for 3 min and then another 3 min in the presence of the NOS inhibitor l-NAME (5 mM, Cayman Chemical, Ann Arbor, MI). Regression analysis of the fluorescence ratio measurements was performed to obtain rates for each period. Differences in rates measured in the absence and presence of flow were taken to represent flow-stimulated ${\text{O}}_2^{-}$ production. Time controls were also performed.

Western Blot Analysis for NOS3

Western blot analysis was conducted in fresh samples as previously described (46). In brief, the samples were obtained by homogenizing fresh outer medullary tissue in cold lysis buffer (CelLytic M, C2978, Sigma) plus 1:100 protease inhibitor cocktail (P8340, Sigma) using a glass homogenizer. Lysed samples were rested for at least 5 min on ice, and the debris was removed by centrifugation (5,600 g, 5 min, 4°C). Protein content was determined by a colorimetric assay. Equal amounts of protein (6–8 µg) were loaded and run on 7.5% polyacrylamide gels (Bio-Rad, Hercules, CA). After overnight transfer of proteins to PVDF membranes (Bio-Rad), the membranes were rinsed with Tris-buffered saline with Tween 20 (TBS-T) containing 20 mM Tris (pH 7.6), 137 mM NaCl, and 0.1% Tween 20. After being blocked with 5% nonfat milk in TBS-T, the membranes were incubated with NOS3 antibody (No. 610296, BD Transduction, San Jose, CA) diluted 1:2,000 in blocking buffer for 2 h. This antibody has been previously validated in NOS3 knockout mice (22). After washes with TBS-T, the membranes were incubated for 1 h with 1:2,500 anti-mouse IgG-horseradish peroxidase (HRP; NA931, GE Healthcare Life Sciences, Pittsburg, PA) and washed again. The membranes were then incubated with HRP substrate, and the films were exposed for not less than 1 min. Finally, as a loading control, the membranes were washed, stripped, and reblotted for GAPDH (ab9385, Abcam, Cambridge, MA) using 1:15,000 dilution in 5% BSA in TBS-T for 2 h. All incubations were performed at room temperature. All films were scanned using an EPSON Expression 1680 scanner with EPSON-Scan software (positive film, 16-bit greyscale, 600 dpi). The densitometry of the bands was measured using ImageJ 1.47p software (https://imagej.nih.gov/ij/). The average peak area of all experimental lanes in each membrane was calculated, and the peak areas of individual lanes were expressed as a fraction of this value. Results were expressed as NOS3-to-GAPDH ratios. Samples from SR and SS rats from the same diet group were processed in pairs and loaded on the same gel, so each gel had its own control.

When Western blot analysis was performed to investigate the effect of salt, normal- and high-salt samples from the same strain were processed in pairs and loaded on the same gel. For these samples, a 1:1,000 dilution of NOS3 antibody for 1 h was used. The IgG-HRP secondary antibody was diluted 1:1,500. After the membranes had been stripped, vinculin was used as a loading control (47). It was detected using a 1:30,000 dilution of monoclonal anti-vinculin antibody (V9131, Sigma-Aldrich) for 1 h and 1:30,000 anti-mouse IgG-HRP. Results were expressed as NOS3-to-vinculin ratios.

Statistical Analysis

Data were analyzed using GraphPad Prism V6.07 (GraphPad, La Jolla, CA). Results are expressed as means ± SE of each group. Each n value represents the number of rats used per group. Means were compared using unpaired two-tailed Student’s t tests. All P values of <0.05 were considered significant. For analysis of ${\text{O}}_2^{-}$ experiments, repeated-measures two-way ANOVA with matched stacked values was used, and Bonferroni’s method was used to correct for multiplicity in post hoc two-tailed paired t tests.

RESULTS

We first measured blood pressure in male SR and SS rats fed normal salt. Systolic blood pressure for SR rats was 110 ± 4 mmHg (n = 7), whereas for SS rats it was 120 ± 2 mmHg (n = 6). Next, we studied the effect of flow on NO production by thick ascending limbs from SR and SS rats on a normal-salt diet (Fig. 1). We found that flow-stimulated NO bioavailability by thick ascending limbs from SR rats was 35 ± 6 AU/min (n = 8), whereas in tubules from SS rats, it was only 11 ± 2 AU/min (n = 6, P < 0.008). Basal NO bioavailabilities in the absence of flow were not significantly different. Thus, the rate for SS rats was only ∼30% of that for SR rats.

Figure 1.

Flow-induced nitric oxide (NO) production by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed normal salt. Data were analyzed by an unpaired t test (n = 8 SR rats and 6 SS rats, P < 0.008). AU, arbitrary units.

To study whether acute scavenging of NO by ${\text{O}}_2^{-}$ accounts for any difference in NO bioavailability between tubules from SS and SR rats, we repeated the flow-induced NO experiments in the presence of 100 µM tempol to eliminate ${\text{O}}_2^{-}$. We found that tempol increased flow-induced NO bioavailability in SS rats from 11 ± 2 to 19 ± 2 AU/min (n = 6 for control and n = 9 for tempol, P < 0.020; Fig. 2). In contrast, tempol had no significant effect on NO bioavailability in tubules from SR rats on normal salt. Thus, scavenging of NO by ${\text{O}}_2^{-}$ accounted for ∼36% of the difference in flow-induced NO bioavailability between thick ascending limbs of SS and SR rats.

Figure 2.

Effect of the ${\text{O}}_2^{-}$ scavenger tempol on flow-induced nitric oxide (NO) production by thick ascending limbs from Dahl salt-sensitive (SS) rats fed normal salt. Data on SS rats from Fig. 1 are repeated here for comparison with tempol-treated tubules. Data were analyzed by an unpaired t test (n = 6 for untreated tubules and n = 9 for tempol-treated tubules, each tubule came from a different rat, P < 0.020). AU, arbitrary units.

Since NO bioavailability was lower in tubules from SS rats, we investigated whether NOS activity was uncoupled from NO production. To explore whether NOS in thick ascending limbs was uncoupled, we measured flow-induced ${\text{O}}_2^{-}$ production in the absence and presence of the NOS inhibitor l-NAME. Two-way ANOVA showed that the effect of l-NAME was significant (P < 0.028). The post hoc analysis of data is shown in Fig. 3. The addition of l-NAME significantly reduced flow-induced ${\text{O}}_2^{-}$ production by thick ascending limbs of SS rats from 373 ± 56 to 249 ± 58 AU/min, a decrease of 36 ± 8% (n = 6, P < 0.02), whereas it had no effect in tubules from SR rats (control: 263 ± 64 AU/min vs. l-NAME: 230 ± 17 AU/min, n = 5). Time controls in the absence of l-NAME for the entire experiment were also performed.

Figure 3.

Effect of the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester (l-NAME) on flow-induced ${\text{O}}_2^{-}$ production by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed normal salt. Data analyzed by a post hoc paired t test after two-way ANOVA are shown (n = 5 SR rats, no significance; n = 6 SS rats, P < 0.02). AU, arbitrary units; Con, control period.

In addition to acute scavenging and uncoupling, lower NO bioavailability by thick ascending limbs from SS rats could be due to blunted production as a result of reduced NOS3 expression. To test this, we measured NOS3 expression in tubules from SS and SR rats on a normal-salt diet. Figure 4A shows a representative Western blot from two different animals from each strain. Figure 4B shows the mean data. NOS3 expression by SR rat tissue was 1.09 ± 0.08 (n = 5), whereas it was 0.91 ± 0.08 for SS rat tissue (n = 5), not significantly different.

Figure 4.

Nitric oxide synthase isoform 3 (NOS3) expression by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed normal salt. A: representative blot. B: mean data of NOS3 expression normalized to the loading control GAPDH. Data were analyzed by an unpaired t test (n = 5 rats for each strain).

Defects in renal NO bioavailability in SS rats only cause a phenotypic change in blood pressure when animals are on a high-salt diet. Thus, we measured the effect of high salt on systolic blood pressure. Blood pressure was 125 ± 3 mmHg in SR rats (n = 7) on high salt, whereas it was 166 ± 2 mmHg in SS rats (n = 6, P < 0.0001; Fig. 5). We next studied whether the difference in flow-induced NO bioavailability in thick ascending limbs from SS and SR rats was greater when tubules were taken from animals fed a high-salt diet. Flow increased NO bioavailability by 44 ± 10 AU/min (n = 6) in thick ascending limbs from SR rats on high salt but by only 9 ± 2 AU/min in tubules from SS rats (n = 6, P < 0.005 vs. SR rats; Fig. 6). Thus, thick ascending limbs from SS rats produced less NO in response to flow than those from SR rats when rats were on a high-salt diet, and the magnitude of the difference between strains was greater by ∼50% than when rats were fed normal salt.

Figure 5.

Effect of a 6–8 days of high-salt diet on systolic blood pressure of Dahl salt-resistant (SR) and salt-sensitive (SS) rats. Data were analyzed by an unpaired t test (n = 7 SR rats and 6 SS rats, P < 0.0001).

Figure 6.

Flow-induced nitric oxide (NO) production by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed high salt. Data were analyzed by an unpaired t test (n = 6 rats for each strain, P < 0.005). AU, arbitrary units.

We next investigated whether acute scavenging of NO by ${\text{O}}_2^{-}$ is involved in blunted NO bioavailability by thick ascending limbs from SS rats on a high-salt diet. We found that tempol increased flow-induced NO bioavailability in tubules from SS rats on high salt from 9 ± 2 to 23 ± 4 AU/min (n = 6 for each group, P < 0.012; Fig. 7). In contrast, tempol had no significant effect on flow-induced NO bioavailability by thick ascending limbs from SR rats on high salt. Thus, scavenging of NO by ${\text{O}}_2^{-}$ accounted for only ∼37% of the difference in flow-induced NO production by thick ascending limbs from SS and SR rats when rats were on a high-salt diet.

Figure 7.

Effect of the ${\text{O}}_2^{-}$ scavenger tempol on flow-induced nitric oxide (NO) production by thick ascending limbs from Dahl salt-sensitive (SS) rats fed high salt. Data on SS rats from Fig. 6 are repeated here for comparison with tempol-treated tubules from SS rats. Data were analyzed by an unpaired t test (n = 6 rats for each group, P < 0.012). AU, arbitrary units.

To assess whether NOS3 uncoupling contributes to the difference in flow-induced NO between strains as it did when rats were fed normal salt, we measured the effect of l-NAME on flow-stimulated ${\text{O}}_2^{-}$ in rats fed high salt. Figure 8 shows that the addition of l-NAME had no significant effect on flow-induced ${\text{O}}_2^{-}$ production by both SS thick ascending limbs of SS rats (control: 492 ± 70 AU/min vs. l-NAME: 580 ± 83 AU/min, n = 7) and tubules of SR rats (control: 374 ± 84 AU/min vs. l-NAME: 364 ± 54 AU/min, n = 7).

Figure 8.

Effect of the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester (l-NAME) on flow-induced ${\text{O}}_2^{-}$ production by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed high salt. Data analyzed by two-way ANOVA showed no significance (n = 7 rats for each strain). AU, arbitrary units; Con, control period.

A high-salt diet enhances NOS3 expression in Sprague-Dawley rats (44), but whether this is true for tubules from SS and SR rats is unknown. Thus, we investigated whether changes in NOS3 expression contributed to the increased difference in flow-stimulated NO production between strains when on high salt. Figure 9A shows a representative Western blot for NOS3. In contrast to the data for normal salt, we found that NOS3 expression by SR rat tissue was 1.26 ± 0.08 (n = 5) but only 0.74 ± 0.08 in SS rat tissue, significantly lower (n = 5, P < 0.003; Fig. 9B).

Figure 9.

Nitric oxide synthase isoform 3 (NOS3) expression by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed high salt. A: representative blot. B: mean data of NOS3 expression normalized to the loading control GAPDH. Data were analyzed by an unpaired t test (n = 5 rats for each strain, P < 0.003).

The data shown in Fig. 9 suggest that high salt increases NOS3 expression in tubules from SR rats, but not those from SS rats. However, it may be that expression in tubules from SR rats was not altered by salt, whereas it was reduced in tubules from SS rats. To test which was the case, we performed Western blots in which samples from the same strain fed either normal or high salt were run on the same gel. One-way ANOVA showed a significant difference (P < 0.006). Figure 10 shows that for SR rats, high salt increased NOS3 expression by fourfold when normalized to the normal-salt group (n = 5 each, post hoc unpaired t test: P < 0.04). In contrast, there was no difference in NOS3 expression between dietary groups for SS rats (n = 5 each).

Figure 10.

Nitric oxide synthase isoform 3 (NOS3) expression by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed normal salt (NS) or high salt (HS). A: representative blots. B: mean data of NOS3 expression normalized to the loading control vinculin. Data analyzed by a post hoc unpaired t test after ANOVA are shown (n = 5 rats for each group, P < 0.04 for NS vs. HS in SR rats).

DISCUSSION

Our hypothesis was that flow-induced NO production is decreased in thick ascending limbs from SS rats primarily due to reduced NOS3 expression and NOS uncoupling rather than simple scavenging by ROS. We found that flow-stimulated NO production was reduced in thick ascending limbs from SS rats on normal salt compared with that in SR rats. NOS uncoupling appeared to account for most of the difference while less than 35% of the difference was attributable to simple scavenging. Although rats were on normal salt, there were no differences in NOS3 expression. We found that flow-stimulated NO production was reduced in thick ascending limbs from SS rats on high salt compared with that in SR rats. In contrast to the situation when rats were on normal salt, NOS uncoupling did not appear to account for most of the difference. Similar to when rats were fed normal salt, only ∼35% of the difference was attributable to simple scavenging. High salt also greatly augmented NOS3 expression in SR rats but not in SS rats. Thus, changes in expression likely accounted for most of the increased magnitude of the difference in flow-induced NO between strains when rats are fed high salt.

We measured NOS3 expression in the outer medulla tissue, and these data most likely reflect NOS3 expression in thick ascending limbs for several reasons. First, the vast majority of the outer medulla consists of thick ascending limbs (>80%). Second, NOS3 is highly expressed in thick ascending limbs in terms of the amount of NOS3 per milligram of tissue. In fact, our data suggest that thick ascending limbs express more NOS3 per unit of tissue than freshly isolated endothelial cells (48). Therefore, although there may be changes in vasculature NOS3, it most likely would not contribute significantly to the changes observed for NOS3 in outer medulla homogenates.

Because we have previously shown that flow stimulates NO production by thick ascending limbs from Sprague-Dawley rats (28), we were interested in whether responses to increases in flow were reduced in SS rats. Our finding in the present study is that thick ascending limbs from SS rats have a blunted response to increases in luminal flow, which has not been previously shown. These data support studies by our laboratory (49) and others (19, 21). NO production by thick ascending limbs due to ANG II type 2 receptor activation was blunted in tubules from SS rats (49). Renal medullary NO production is lower in SS rats than in SR rats (19, 21). Our current findings are also consistent with a report showing that ANG II treatment did not increase measurable NO in isolated thick ascending limbs from SS rats but did in thick ascending limbs from SS^BN13 rats, a normotensive rat in which chromosome 13 of the SS rat is changed to one from the Brown Norway rat (20). Additionally, ANG II-induced NO in the outer medulla was reduced in SS rats compared with Brown Norway rats; however, basal NO concentration in the two strains was similar (19). In SS rats, ANG II-induced NO was reduced in thick ascending limbs compared with a SR control strain (20).

It was thought that ${\text{O}}_2^{-}$ and NO only interact by reacting with each other to form peroxynitrite (ONOO⁻) in a process called scavenging. Previously, we showed that in Sprague-Dawley rats, ${\text{O}}_2^{-}$ did blunt NO bioavailability (6). Therefore, this could explain reductions in flow-stimulated NO in SS rats. However, under normal salt conditions, we found that the difference in NO between SS and SR rats remained even in the presence of the ${\text{O}}_2^{-}$ scavenger tempol. Therefore, scavenging plays only a minor role in the observed difference in flow-induced NO between strains.

NOS can become uncoupled to produce ${\text{O}}_2^{-}$ rather than NO. We found that when animals are fed normal salt, NOS-derived ${\text{O}}_2^{-}$ was the primary mechanism by which flow-induced NO by thick ascending limbs was reduced in SS rats. These data are consistent with our previous report showing that uncoupling of NOS occurs in this nephron segment in Sprague-Dawley rats (38). Interestingly, in that study, although ANG II stimulated both NO and ${\text{O}}_2^{-}$, ANG II-stimulated NO did not reduce ANG II-stimulated ${\text{O}}_2^{-}$, whereas in the case of acute increases in flow, the thick ascending limb is able to keep in check the effects of ${\text{O}}_2^{-}$ with NO (32).

The simplest explanation for decreased NO production by thick ascending limbs from SS rats compared with SR rats would be decreased NOS3 expression. However, under normal salt intake conditions, we found that lower flow-induced NO in SS versus SR rats was not due to differences in NOS3 protein expression. Findings for SS rats on low salt are consistent with another study that measured NOS3 protein expression in various tissues, including the kidney, in SS and SR rats (50). Quantitative immunohistochemistry also found no difference between 3-wk-old SS and SR rats (51). However, other reports that measured NOS3 mRNA and/or protein abundance are contradictory. Reduced NOS3 and NOS1 mRNA levels in the renal medulla were found in SS rats compared with SR rats (41) and Brown Norway rats (21) on normal salt. Additionally, NOS3 protein levels as detected by Western blot analysis (19) and immunohistochemistry (51) were lower in the outer medulla of SS rats compared with SR rats. Finally, immunohistochemical analysis detected less outer medulla NOS3 in 9-wk-old SS rats compared with SR rats (50). It is difficult to reconcile all of these discrepancies, but differences in age, strains of SS rats used, and the strain used as the control for comparison most likely are involved.

A high-salt diet increases luminal flow through thick ascending limbs. The thick ascending limb, which regulates Na⁺ transport via NO, plays an important role in preventing hypertension. Therefore, we next investigated if flow-induced NO is reduced in SS rats under high-salt conditions. Increased flow resulted in lower NO in tubules of SS rats than those of SR rats, and the difference between strains was greater than what we saw for normal salt.

We then elucidated the mechanisms involved in this difference between strains. Similar to the case with normal salt, we found that scavenging played only a minor role. Next, we tested whether ${\text{O}}_2^{-}$ derived from uncoupled NOS could account for reduced NO in SS rats as it did with normal salt. We observed that the magnitude of flow-induced ${\text{O}}_2^{-}$ for thick ascending limbs from SS rats is higher than that for thick ascending limbs from SR rats, and, as one might expect, the magnitude for each strain is higher for animals fed high salt than those fed normal salt. Surprisingly, l-NAME did not significantly affect flow-induced ${\text{O}}_2^{-}$ in tubules from SS rats as it did in tubules from animals on normal salt. There are several possible explanations for these data. First, high salt increases oxidative stress by stimulating NADPH oxidase (NOX) activity, thereby decreasing the NADPH-to-FAD ratio. Reducing the absolute NADPH concentration and the relative amount compared with FAD would be expected to blunt the NOS-derived ${\text{O}}_2^{-}$ normally observed in the absence of elevated oxidative stress. This is consistent with studies showing that redox ratios (NADH/FAD) of kidney tissue from SS rats fed low- and high-salt diets were significantly lower than those of salt-resistant SS p67^phox-null or SS NOX4^−/− null rats (52, 53). These data indicate that reduced levels of mitochondrial electron transport chain metabolic activity contribute to the enhanced oxidative stress in SS rats. Alternatively, the method used to investigate NOS uncoupling may be important. Classically, l-NAME-inhibitable ${\text{O}}_2^{-}$ production has been used to demonstrate NOS uncoupling (54, 55). However, NOS contains both a reductase domain and an oxidase domain. l-NAME only blocks the ${\text{O}}_2^{-}$ generated from the oxidase domain but not the reductase domain. In contrast, S-glutathionylation causes NOS to produce ${\text{O}}_2^{-}$ from the reductase domain (56). This type of uncoupling has been demonstrated in endothelial cells in preeclampsia placentas (57, 58), acute lung injury (59), and ANG II-induced ${\text{O}}_2^{-}$ (55). Thus, NOS may still be producing ${\text{O}}_2^{-}$ due to uncoupling in tubules from SS rats on a high-salt diet, but it would not be inhibited by l-NAME. Finally, levels of the NOS cofactor tetrahydrobiopterin could also play a role (60).

We found that NOS uncoupling plays a major role in reduced flow-induced NO in SS rats fed normal salt, whereas it has no significant effect when SS rats are on high salt. This is consistent with the findings that thick ascending limb ${\text{O}}_2^{-}$ is higher in SS rats than in SR rats. Cellular stretch, a mechanical component of flow, induced more ${\text{O}}_2^{-}$ in thick ascending limbs in SS rats than in SR rats (61) on normal salt. Similarly, in medullary strips containing thick ascending limbs, ANG II increased ${\text{O}}_2^{-}$ production to a greater extent in prehypertensive SS rats than SR rats (20). However, our data conflict with a previous study of outer medullary tissue; in that study, NOS uncoupling was due to differences in tetrahydrobiopterin during high salt intake for 6 wk (54). l-NAME decreased ${\text{O}}_2^{-}$ in hypertensive SS rats but not in prehypertensive SS rats. Uncoupling did not occur in the normotensive SS^BN13 strain. Part of the explanation for these disparate results could be that different strains of Dahl SS and SR rats were used. Additionally, we studied specifically flow-induced NO in isolated tubules rather than basal NO and ${\text{O}}_2^{-}$ levels in outer medullary tissue. Furthermore, these contradictory data may simply be due to the time course of salt administration. Perhaps early, acute effects of high salt (1 wk) are due to one mechanism (a failure by SS rats to increase NOS3), whereas the chronic assault of high salt for 6 wk causes uncoupling to occur in SS rats (54).

In contrast to our data with normal salt, we found that renal medullary NOS3 expression is lower in SS rats compared with SR rats during high salt intake. This is consistent with a previous report from our laboratory showing that a high-salt diet enhances thick ascending limb NO synthesis by increasing NOS3 expression and activity in Sprague-Dawley rats (44). It is important to make the distinction that the difference in NOS3 expression between thick ascending limbs of SS and SR rats on high salt is due to a failure of tubules from SS rats to increase NOS3 expression. Tubules from SR rats respond to high salt by increasing NOS3 to counterbalance the deleterious effects of ${\text{O}}_2^{-}$ exacerbated by increased dietary salt, whereas thick ascending limbs from SS rats do not. Thus, salt-induced increases in luminal flow prevent salt retention and increases in blood pressure under normal circumstances in Sprague-Dawley and SR rats.

Our data provide further evidence that the primary renal defect that results in abnormal salt reabsorption in SS rats is most likely in the thick ascending limb (17, 18, 62, 63). Our current findings support the importance of flow-induced effects on NO-regulated Na⁺ transport. In the thick ascending limb, increases in tubular flow stimulate NO production (28), and NO directly inhibits NaCl transport (2, 7) in Sprague-Dawley rats. NO inhibits Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2), which must be present at the apical surface to reabsorb Na⁺ and Cl⁻ from the tubule lumen. Apical surface NKCC2, and therefore presumably NKCC2 activity, was greater in the thick ascending limbs of SS rats (64). Others have shown in vitro and in vivo that NKCC2 activity is increased in thick ascending limbs of SS rats (65). Our current data are also consistent with our previous findings that the effect of NO on thick ascending limb transport is blunted in the SS rat (18). Increases in factors that decrease NO bioavailability can lead to enhanced Na⁺ retention. It is well known that lack of renal NO and excess renal ${\text{O}}_2^{-}$ contribute to salt-induced elevations of blood pressure in the Dahl rat model (18, 19, 21, 39, 66). We have previously reported that flow-stimulated Na⁺ reabsorption depends on ${\text{O}}_2^{-}$ (4, 67) but that flow-induced NO buffers flow-stimulated NaCl reabsorption by directly inhibiting NaCl transport (2, 7) and blunting flow-induced ${\text{O}}_2^{-}$ (32) in thick ascending limbs from Sprague-Dawley rats.

Under chronic high-salt conditions, ONOO⁻ may contribute to the enhanced difference in flow-stimulated NO between SS and SR rats. Because a high-salt diet chronically elevates flow (11, 43) and ONOO⁻ inhibits NOS3 expression in the thick ascending limb (68), it is possible that the formation of ONOO⁻ would eventually reduce NOS3 expression and NO production. This would begin a vicious cycle in which NO decreases and ${\text{O}}_2^{-}$ increases. Ultimately, instead of flow leading to an increase in urinary NaCl excretion as it would in an acute setting, in the chronic situation, elevated flow would lead to diminished NO, elevated ${\text{O}}_2^{-}$, and thus a decrease in NaCl excretion. This could contribute to increases in blood pressure and renal damage.

In summary, we found that flow-induced stimulation of NO is diminished in thick ascending limbs from SS rats compared with tubules from SR rats and that it is further reduced when animals are fed a high-salt diet. When animals are on a normal-salt diet, this difference between strains is not due to altered NOS3 expression or scavenging of NO by ${\text{O}}_2^{-}$ but rather NOS-derived ${\text{O}}_2^{-}$. A high-salt diet causes an enhanced difference in flow-induced NO between strains. In contrast to the findings with normal salt, the major reason why flow-induced NO is lower in SS rats is a failure to increase NOS3 expression.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL70985 (to J.L.G.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.L.G. conceived and designed research; N.J.H., A.G-V., F.S., and J.L.G. performed experiments; N.J.H., A.G-V., F.S., and J.L.G. analyzed data; N.J.H., A.G-V., F.S., and J.L.G. interpreted results of experiments; N.J.H., A.G-V., F.S., and J.L.G. prepared figures; N.J.H. drafted manuscript; N.J.H., F.S., and J.L.G. edited and revised manuscript; N.J.H., A.G-V., F.S., and J.L.G. approved final version of manuscript.