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J Am Soc Nephrol 15:269-275, 2004
© 2004 American Society of Nephrology

BASIC SCIENCE

Acute and Chronic Regulation of Thick Ascending Limb Endothelial Nitric Oxide Synthase by Statins

Marisela Varela and Jeffrey L. Garvin

Henry Ford Health System, Detroit, Michigan

Correspondence to Dr. Jeffrey L. Garvin, Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, 2799 W. Grand Boulevard, Detroit, MI 48202. Phone: 313-916-2048; Fax: 313-916-1479; E-mail: jgarvin1{at}hfhs.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Statins, 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, acutely increase endothelial nitric oxide synthase (eNOS) activity and chronically increase eNOS expression in endothelial cells. NO decreases transport in thick ascending limbs (TAL). We hypothesized that statins inhibit TAL transport by acutely activating eNOS, thereby increasing NO production and chronically enhancing eNOS expression. Oxygen consumption (QO2) by TAL suspensions from Sprague-Dawley rats was used as a measure of active NaCl reabsorption. Na/K ATPase activity was assessed by measuring ATP hydrolysis in the presence and absence of ouabain. eNOS expression was measured by Western blot. A total of 50 µM pravastatin decreased QO2 by 18.6 ± 3.4% (P < 0.01). In the presence of 500 µM furosemide and 200 µM amiloride, transport blockers, QO2 remained the same after pravastatin was added. Na/K ATPase activity was not different from controls and TAL treated with 50 µM pravastatin (0.33 ± 0.07 versus 0.29 ± 0.04 nmol Pi/µg protein/min, where Pi is inorganic phosphate). Nystatin stimulated QO2 to 178 ± 13.7 in pravastatin-treated TAL and 195 ± 11.5 in furosemide-treated TAL. The inhibitory effect of pravastatin on QO2 was blocked by L-nitroarginine methyl ester, an NOS inhibitor. In addition, pravastatin increased NO production as measured by the fluorescent dye DAF-2A. Pravastatin at a dose of 10 mg/kg per d had no effect on eNOS protein at 1 d (24.1 ± 2.7 versus 25.5 ± 1.1 arbitrary units [AU]) or 7 d (24.1 ± 2.7 versus 20.9 ± 1.3 AU). Similarly, at 1 d, 50 mg/kg per d had no effect on expression (24.1 ± 2.7 versus 21.2 ± 3.6 AU). At 7 d, this dose decreased eNOS protein from 24.1 ± 2.7 to 11.8 ± 4.4 AU. It is concluded that pravastatin acutely decreases NaCl entry into the TAL by releasing NO. Pravastatin does not chronically increase eNOS expression in TAL.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Statins are a group of drugs that inhibit the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, the rate-limiting step of cholesterol synthesis in the liver. This class of drugs has been shown to acutely increase nitric oxide (NO) synthesis (1–5) and enhance endothelial NO synthase (eNOS) expression by endothelial cells (1, 3, 5–9). NO is produced by many cells in the kidney (10), and NO increases urinary volume and Na excretion without changes in GFR or renal blood flow, i.e., by increasing fractional Na excretion (11–13). Similar to NO, statins have been shown to increase urinary volume and Na excretion by increasing fractional Na excretion (14). These data indicate that statins, like NO, inhibit reabsorption of salt along the nephron.

The thick ascending limb (TAL) of the loop of Henle plays a critical role in regulating urinary volume and Na excretion, and it also expresses eNOS (15–17). NO produced by eNOS acts as an autacoid to inhibit Na reabsorption (18, 19) by reducing Na entry into the cell via the Na/K/2Cl co-transporter (20) and Na/H exchanger (21) in the TAL. NO production in this segment is regulated by the same factors as in endothelial cells (22, 23 ). Thus, statins may acutely inhibit TAL transport by enhancing NO production and may chronically regulate eNOS expression in this segment. However, the effect of statins on TAL Na transport and the role of NO in this process have not been studied to our knowledge. We hypothesized that statins increase eNOS expression and NO production by the TAL and thereby decrease Na reabsorption.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) were fed a rodent diet that contains 1.1% K and 0.2% Na. Food and water were provided ad libitum. In chronic studies, pravastatin was given by gavage once a day. Rats were anesthetized with ketamine (100 mg/kg body wt intraperitoneally) and xylazine (20 mg/kg body wt intraperitoneally).

Isolation of TAL
Suspensions of medullary TAL were prepared according to the protocol described by Ortiz et al. (20). Briefly, kidneys were perfused retrograde at 6 ml/min for 6.6 min via the aorta with a physiologic solution (see Solutions) containing 0.1% collagenase (Sigma, St. Louis, MO) and 2.5 units/ml heparin. The inner stripe of the outer medulla was cut from coronal slices of the kidneys, minced, and incubated at 37°C for 30 min in 0.1% collagenase. The tissue was pelleted by centrifugation at 114 x g, resuspended in cold physiologic solution, and stirred on ice for 30 min to release the tubules. The suspension was filtered through 250-µm nylon mesh and centrifuged at 114 x g. The tubules were washed, pelleted again, and finally resuspended in cold physiologic solution.

Solutions
The physiologic solution contained (in mM) 114 NaCl, 4 KCl, 25 NaHCO3, 2.5 NaH2PO4, 1.2 MgSO4, 2 Ca lactate, 5.5 glucose, 6 alanine, and 1 Na3 citrate. Osmolality was 290 ± 3 mOsmol/kg H2O as measured by freezing-point depression. The physiologic solution was gassed with 95% O2/5% CO2. Solution A contained (in mM) 1 Na2EGTA, 5 MgCl2, 100 imidazole, 6 Na2ATP, 45 NaOH, and 5 KOH (pH 7.0). Solution B contained (in mM) 1 Na2EGTA, 5 MgCl2, 100 imidazole, 6 Na2ATP, 50 NaOH, and 2 ouabain (pH 7.0).

Determination of Oxygen Consumption
TAL were resuspended in 0.1 ml of cold physiologic solution. The medullary TAL suspension was warmed to 37°C and equilibrated with 95% O2/5% CO2. It was then added to a closed chamber maintained at 37°C, and oxygen consumption was recorded continuously with a Clark electrode. Once a constant slope was achieved, control oxygen consumption was measured. After the control period, drugs were added to the chamber and oxygen consumption was calculated again after a new constant slope was achieved. All experiments were completed within 15 min. An aliquot of the suspension was used to determine protein concentration using Coomassie protein assay reagent (Pierce, Rockford, IL) (20).

NO Production
A medullary TAL was dissected and transferred to a temperature-controlled chamber. The tubule was held in place with pipets, but the ends were left open to the bath. Only tubules with an open lumen were used. Cells were loaded for 40 min at 37°C with 2 µM DAF-2DA in HEPES-buffered solution (in mM: 130 NaCl, 4 KCl, 2.5 NaH2PO4, 1.2 MgSO4, 5.5 glucose, 6 alanine, 2 Ca lactate, 1 Na3 citrate, 10 HEPES [pH 7.4]; osmolality 290 ± 3 mOsm plus 100 µM L-arginine). Cells were then washed in dye-free solution for 10 min and for 5 min in dye-free and L-arginine–free solution. Intracellular DAF was excited using 488 nm light of a scanning confocal microscope (Noran Instruments, Middleton, WI). Emitted fluorescence was measured at wavelengths >500 nm. A basal level of fluorescence was established for 1 min, and then 50 µM pravastatin was added to the bath. Fluorescence was measured continuously for an additional 10 min.

Na/K ATPase Assay
TAL were resuspended in 200 µl of physiologic solution and divided into two Eppendorf tubes each containing 250 µl of physiologic solution (warmed at 37°C and gassed with 95% O2/5% CO2). After incubation at 37°C for 20 min in the presence or absence of 50 µM pravastatin, TAL were rinsed three times with 500 µl of 150 mM N-methyl D-glucamine chloride. Control and treated TAL were resuspended in 400 µl of N-methyl D-glucamine chloride and divided into two 1.5-ml Eppendorf tubes. They were pelleted, resuspended in 50 µl of distilled H2O, and subjected to three freeze–thaw cycles on dry ice. Then 360 µl of either solution A or B prewarmed to 37°C was added to each tube. After 10 min, 350 µl of 5% TCA was added and tubes were centrifuged at 16,000 x g for 2 min. Aliquots of 100 µl were used to measure inorganic phosphate (Pi). For dissolving proteins, 200 µl of 0.05 M NaOH containing 0.04% SDS was added to the tubes. Aliquots of 25 µl were used to determine protein concentration using Coomassie protein assay reagent (Pierce). Pi was determined by a method similar to that of Fiske and Subbarrow (24).

Western Blot
The kidneys were flushed via retrograde perfusion of the aorta with 20 ml of homogenization buffer containing 20 mM HEPES (pH 7.5), 2 mM EDTA, 0.3 M sucrose, and 100 units of heparin, plus a cocktail of protease inhibitors containing 5 µg/ml antipain, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 4 mM benzamidine, 5 µg/ml chymostatin, 5 µg/ml pepstatin A, and 0.105 M pf-block (Sigma). The inner stripe of the outer medulla was excised from coronal slices of the left kidney, homogenized in 0.5 ml of homogenization buffer plus protease inhibitors and centrifuged for 30 min at 16,000 x g and 4°C. An aliquot of the supernatant was used to determine protein concentration. A total of 25 µg of protein was used per lane. The protein aliquots were mixed with 7 µl of 6x loading buffer containing 0.5 M TrisCl (pH 6.8), 10% SDS, 25% {beta}-mercaptoethanol, 50% glycerol, and 0.05% bromophenol blue. Homogenization buffer without heparin was added to adjust the final volume to 40 µl. All samples were heated in boiling water for 5 min and loaded onto an 8% polyacrylamide gel, which was run at 120 V for approximately 1 h. Proteins were then electrophoretically transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) overnight at 21 V with cooling. Nonspecific binding was blocked by incubating the membrane for 1 h at room temperature in Tris-buffered saline-Tween-20 (TBS-T) with 5% nonfat dry milk. TBS-T contained 20 mM Tris (pH 7.5), 137 mM NaCl, and 0.1% Tween-20. For detection of eNOS, the membrane was incubated for 1 h at room temperature in TBS-T with 5% nonfat dry milk and an anti-eNOS monoclonal antibody (BD Transduction Laboratories, San Diego, CA) at 1:1000 dilution. The membrane was washed with TBS-T one time for 15 min and two times for 5 min, then incubated for 1 h at room temperature in TBS-T with 5% nonfat dry milk plus anti-mouse IgG conjugated to horseradish peroxidase (Amersham, Arlington Heights, IL) diluted 1:1000, and finally washed again. Protein was detected using enhanced chemiluminescence reagents (Amersham Biosciences, UK). The films were scanned using a densitometer equipped with the Molecular Analyst program (Bio-Rad, Hercules, CA).

Statistical Analyses
Results are expressed as mean ± SEM. Oxygen consumption was evaluated by paired t test. Western blots were analyzed using ANOVA for repeated measures followed by post hoc testing with paired t test, correcting for multiple testing.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We first measured the effect of pravastatin on oxygen consumption by TAL suspensions. Oxygen consumption by TAL suspensions from male Sprague-Dawley rats was used as a measure of active NaCl reabsorption. Figure 1 shows the effect of 50 µM pravastatin on oxygen consumption. During the control period, oxygen consumption was 116.0 ± 10.3 nmol/mg protein per min. After adding pravastatin, oxygen consumption decreased to 94.3 ± 8.71 nmol/mg protein per min, an inhibition of 18.6 ± 3.4% (P < 0.01; n = 10)



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  		Figure 1. Effect of 50 µM pravastatin on oxygen consumption by thick ascending limbs (TAL).

 
Oxygen consumption is a measure of all processes that use ATP, not just active transport. To investigate whether pravastatin acts by blocking active transport, we tested whether it could still decrease oxygen consumption when Na entry via the Na/K/2Cl co-transporter and Na/H exchanger was inhibited by 500 µM furosemide and 200 µM amiloride, respectively (Figure 2). In the presence of furosemide and amiloride, oxygen consumption was 51.3 ± 6.4 nmol/mg protein per min. After 50 µM pravastatin was added, it was 50.4 ± 6.8 nmol/mg protein per min, which is not significantly different.



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  		Figure 2. Effect of 50 µM pravastatin on oxygen consumption by TAL in the presence of 500 µM furosemide and 200 µM amiloride.

 
Although the data described above indicate that pravastatin inhibits Na transport, they do not indicate whether entry or exit is affected. To study this issue, we investigated the effect of pravastatin on Na/K ATPase by measuring ATPase activity directly. Na/K ATPase activity was taken as the difference between total and ouabain-insensitive activity. Control total ATPase activity was 0.80 ± 0.08 nmol Pi/µg protein per min (n = 6), and ouabain-insensitive ATPase activity was 0.48 ± 0.04 nmol Pi/µg protein per min (n = 6). Consequently, Na/K ATPase activity was 0.33 ± 0.07 nmol Pi/µg protein per min in control TAL (n = 6; Figure 3). In pravastatin-treated TAL, total ATPase activity was 0.75 ± 0.05 nmol Pi/µg protein per min (n = 6) and ouabain-insensitive ATPase activity was 0.46 ± 0.05 nmol Pi/µg protein per min (n = 6). Consequently, Na/K ATPase activity was 0.29 ± 0.04 nmol Pi/µg protein per min (n = 6) in pravastatin-treated TAL (P > 0.6 versus control).



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  		Figure 3. Effect of 50 µM pravastatin on ATPase activity in TAL.

 
That pravastatin had no effect on oxygen consumption in the presence of furosemide and amiloride could be due to pravastatin’s inhibiting either Na transport or ATP generation. When Na transport is inhibited, the demand for ATP generation is less than when Na transport processes are intact. To ensure that pravastatin does not inhibit Na transport by directly inhibiting the pump or dramatically reducing ATP production and levels, we investigated whether pravastatin reduces nystatin-stimulated oxygen consumption. As shown in Figure 4, 50 µM pravastatin decreased TAL oxygen consumption by 13 ± 1.8% (P < 0.01 versus control; n = 6). After 300 U/ml nystatin was added, oxygen consumption increased to 178 ± 13.7% (P < 0.01 versus control; n = 6). Similarly, after the Na/K/2Cl co-transporter was inhibited with furosemide, nystatin increased TAL oxygen consumption to 195 ± 11.5% (P < 0.01 versus control; n = 6), not significantly different from the stimulation caused by nystatin in the presence of pravastatin.



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  		Figure 4. Effect of 300 U/ml nystatin on oxygen consumption by 500 µM furosemide- and 50 µM pravastatin-treated TAL.

 
NO from eNOS inhibits NaCl reabsorption in the TAL (18, 19), and statins activate NOS in endothelial cells (1, 3, 5–8). Consequently, we investigated whether NO mediates the inhibitory effect of pravastatin on TAL transport by testing whether L-nitroarginine methyl ester (L-NAME), a NOS inhibitor, could block its effect on TAL transport. In the presence of 1 mM L-NAME, oxygen consumption was 136.5 ± 23.2 nmol/mg protein per min. After 50 µM pravastatin was added, it was 131.0 ± 24.0 nmol/mg protein per min (n = 5), not significantly different (Figure 5). In the presence of L-NAME, QO2 did not change with time.



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  		Figure 5. Effect of 50 µM pravastatin on oxygen consumption by TAL when NOS is inhibited by 1 mM L-nitroarginine methyl ester.

 
Because the effects of pravastatin were inhibited by L-NAME, we measured its effect on NO production directly using the fluorescent dye DAF-2DA. During the control period, fluorescence intensity was 64.3 ± 2.7 arbitrary units (AU). After tubules were treated with 50 µM pravastatin, fluorescence increased by 5.4 ± 0.7%, indicating an increase in NO production.

Chronic statin treatment increases eNOS expression in endothelial cells (1, 3, 5–9). To study the chronic effects of pravastatin on eNOS expression in the TAL, we measured eNOS protein abundance in medullary TAL from rats that were treated with varying doses of pravastatin: 0, 10, or 50 mg/kg per d. Pravastatin at a dose of 10 mg/kg per d had no significant effect on eNOS protein abundance at 1 d (24.1 ± 2.7 versus 25.5 ± 1.1 AU; n = 8). This dose also had no significant effect on eNOS expression at 7 d (24.1 ± 2.7 versus 20.9 ± 1.3 AU; n = 6). Similarly, a much higher dose of 50 mg/kg per d caused no significant change in eNOS abundance after 1 d (24.1 ± 2.7 versus 21.2 ± 3.6 AU; n = 7). However, after 7 d of treatment, this dose of pravastatin decreased eNOS protein abundance from 24.1 ± 2.7 to 11.8 ± 4.4 AU (49%; P < 0.015; n = 4; Figure 6).



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  		Figure 6. Effect of 50 mg/kg pravastatin on eNOS expression by TAL after 7 d of treatment.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We believe this is the first study to evaluate a specific nephron segment as a target of action for statins. We found that pravastatin acutely decreases NaCl transport in the TAL by inhibiting Na entry. Pravastatin does not directly affect Na/K ATPase or significantly reduce ATP generation. We also found that inhibition of TAL transport by pravastatin is mediated by NO. Our results show that chronic pravastatin does not increase eNOS expression.

Because NO inhibits NaCl reabsorption in the TAL and the effects of statins are mediated by NO in endothelial cells, the TAL is a likely target for statins. However, little is known about their effects on TAL transport. To study the effect of statins on TAL transport, we first measured the effect of pravastatin on oxygen consumption in TAL suspensions. Pravastatin decreased oxygen consumption by the TAL by approximately 15%. This decrease could be related to inhibition of active Na transport, other processes that use ATP, or ATP production in the mitochondria. To study how pravastatin reduces oxygen consumption, we tested whether (1) its effects were blocked by Na transport inhibitors, (2) it altered Na/K ATPase activity, and (3) it reduced ATP production.

In the TAL, Na transport can be described as a two-step process in which Na first enters the cell via either the Na/K/2 Cl co-transporter or the Na/H exchanger and then exits via Na/K ATPase in the basolateral membrane. Na/K ATPase pumps Na out of the cell, maintaining the electrochemical gradient for Na, which provides the driving force for NaCl entry (25, 26). We found that pravastatin had no effect on oxygen consumption in the presence of furosemide and amiloride, which block the entry of Na into the cell via the Na/K/2 Cl co-transporter and the Na/H exchanger, respectively. It is important to note that oxygen consumption was lower in the presence of furosemide and amiloride than in their absence because active Na transport, which accounts for approximately 50% of oxygen consumption in this segment, is blocked. We conclude from these experiments that pravastatin reduces oxygen consumption by inhibiting Na transport. However, they do not indicate whether entry or exit of Na is reduced. To examine this issue, we measured ATPase activity directly. Total and ouabain-insensitive ATPase activities were not significantly altered by pravastatin. Thus, Na/K ATPase activity, measured as the difference between total and ouabain-insensitive ATPase activity, was not significantly different. These data indicate that pravastatin affects Na entry rather than Na/K ATPase activity.

A 15% reduction in oxygen consumption amounts to a 30% reduction in Na transport, because approximately 50% of oxygen consumption is related to Na absorption in the TAL. The Na/K/2Cl co-transporter accounts for approximately 70 to 80% of Na entry in this segment, and the Na/H exchanger accounts for the remainder (27). Given that pravastatin reduces Na transport by 30%, it inhibits Na/H exchange completely and Na/K/2Cl not at all, only inhibits the co-transporter, or inhibits both. It seems unlikely that pravastatin would completely inhibit Na/H exchange; therefore, the co-transporter is probably affected as well.

Although the experiments discussed above show that pravastatin inhibits Na transport, they do not rule out the possibility that it reduces ATP production. When Na transport is inhibited by furosemide and amiloride, the demand for ATP is reduced by 50%, which could mask an effect of pravastatin on ATP generation. To investigate whether pravastatin affects ATP production, we measured the effect of nystatin on TAL oxygen consumption in the presence of 144.5 mM extracellular Na. We found that nystatin increased oxygen consumption by the same amount in pravastatin- and furosemide-treated tubules. Nystatin causes the Na/K ATPase to function at maximum rate because it increases intracellular Na to saturating concentrations. Because 50% of ATP is used by the pump in TAL, this dramatically increases ATP hydrolysis. The resultant increase in ADP levels causes a "quasi" state 3 respiration rate in the mitochondria. If ATP generating capacity were significantly reduced by pravastatin, then the increased demand for ATP caused by nystatin could not be met and oxygen consumption would not increase to the same extent as in control tubules. These data indicate that ATP production is not rate-limiting for the Na/K ATPase after pravastatin treatment and that pravastatin does not significantly alter ATP production. However, they do not address whether maximum ATP production rate or maximum transport of electrons through the electron transport chain is altered one way or the other.

Statins increase NO production in endothelial cells (1, 3, 5–8). TAL, similar to endothelial cells, express eNOS. The NO that it releases acts as an autacoid to inhibit transport (18, 19). Therefore, we investigated whether the effects of pravastatin on the TAL are mediated by NO. We found that pravastatin did not significantly change oxygen consumption in the presence of L-NAME. Because we found that L-NAME inhibited the effect of pravastatin, we measured its ability to stimulate NO. We found that pravastatin acutely enhances NO production by isolated TAL. These data indicate that the inhibitory effect of pravastatin on NaCl transport in the TAL is mediated by NO. The mechanism by which pravastatin activates eNOS was not studied here. However, it may involve inhibiting the activity of small G proteins such as Rho and Rac (28, 29) or acylation of eNOS itself. Full activation of eNOS requires acylation, and statins have been shown to alter the acylation of several proteins (2).

In this study, we found that NO mediates the effects of pravastatin. Previously, we reported that NO inhibits the Na/K/2Cl co-transporter (20) and the Na/H exchanger (21) rather than Na/K ATPase in the TAL. Thus, our current findings agree with previous data that NO reduces net Na transport by inhibiting Na entry rather than exit via Na/K ATPase. Given that we previously reported that NO inhibits both Na/H exchange and Na/K/2Cl co-transport, and given that the effects of pravastatin are mediated by NO, pravastatin probably reduces oxygen consumption by reducing the activity of both transporters.

NO synthesis inhibitors administered intrarenally lower urinary Na excretion (30, 31), whereas intrarenal administration of NO donors induces natriuresis (32). In in vivo studies, Na excretion increased when NO production was stimulated (13) and decreased when NOS was inhibited in the absence of renal hemodynamic changes (12). In humans, Na excretion also increased when NO synthesis was stimulated (11). Under physiologic conditions, the TAL reabsorbs approximately 25% of filtered Na and Cl (33). Because this part of the nephron is impermeable to water, absorption of salt results in a hypertonic medulla and dilute tubular fluid. Since the TAL is pivotal in creating a corticomedullary osmotic gradient and dilute tubular fluid, NaCl reabsorption in this segment is also essential for urine concentration. Our data show that pravastatin inhibits NaCl transport by the TAL and that this effect is mediated by NO. NO-induced inhibition of TAL transport will tend to produce both natriuresis and diuresis, thereby favoring a reduction in BP. Thus, part of the effect of statins on BP and natriuresis may be due to enhanced NO production by the TAL.

Because pravastatin acutely inhibits NaCl transport and this effect is mediated by NO, we investigated whether chronic pravastatin treatment increases eNOS expression. Our data showed a decrease in eNOS protein abundance when rats were treated with 50 mg/kg pravastatin for 7 d. We found no change when rats were treated for 1 d at the same dose. The decrease in eNOS protein at 7 d with high-dose pravastatin may represent a feedback mechanism in the TAL. When rats were treated with 10 mg/kg pravastatin either for 1 or 7 d, we found no significant change in eNOS protein abundance. Our results are similar to those of Hayashidani et al. (34), who showed that eNOS protein did not change after treatment with fluvastatin in myocardial tissue. Conversely, other investigators have found an increase in eNOS protein abundance after chronic statin treatment of cultured endothelial cells (3, 5–9). This may be because we studied fresh homogenized TAL, whereas the above authors studied cultured endothelial cells. In contrast, our data may indicate that chronic pravastatin acts in TAL by a different mechanism than in endothelial cells.

Because we found that pravastatin had no effect or decreased eNOS expression, we did not investigate further the regulation of expression. However, statins have been reported to both increase eNOS mRNA abundance or stability (3, 5, 6) and decrease eNOS mRNA abundance (1). These data could be related to the fact that measurements were made at different time points or that the regulation of eNOS mRNA by statins is related to many factors that will not be investigated easily.

We concluded that pravastatin acutely decreases NaCl transport in the TAL by inhibiting Na entry. Because this effect is mediated by NO, the Na/K/2 Cl co-transporter and/or the Na/H exchanger are likely affected. NO-induced inhibition of TAL transport tends to produce both natriuresis and diuresis, thereby lowering BP. Therefore, the ability of statins to enhanced NO production by the TAL will favor a reduction in BP. We also concluded that pravastatin does not chronically increase eNOS expression in TAL as it does in endothelial cells, suggesting that chronic pravastatin acts by a different mechanism in TAL.


   Acknowledgments
 
This work was supported in part by grants from the National Institute of Health to J.G. (HL 28982 and HL 70985). M.V. was supported by a fellowship from the American Heart Association (#0225424Z).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication February 21, 2003. Accepted for publication October 24, 2003.




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