(Hypertension. 1996;28:297-303.)
© 1996 American Heart Association, Inc.
Articles |
Neural Nitric Oxide Synthase in the Renal Medulla and Blood Pressure Regulation
the Department of Physiology, Medical College of Wisconsin, Milwaukee.
Correspondence to David L. Mattson, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.
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Abstract |
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We studied the effect of selective inhibition of the neural isoform of nitric oxide synthase in the rat renal medulla in conscious Sprague-Dawley rats. Continuous renal medullary interstitial infusion of an antisense oligonucleotide complementary to the initiation region of the mRNA for neural nitric oxide synthase increased blood pressure 14±1 mm Hg in rats maintained on a high sodium intake. Medullary interstitial infusion of saline vehicle or a scrambled oligonucleotide probe failed to alter blood pressure in separate groups of high salt control rats. Renal medullary interstitial infusion of the antisense oligonucleotide significantly decreased the level of neural nitric oxide synthase in the renal medulla by 53±8% and decreased total renal medullary nitric oxide synthase activity by 28±8%. No alterations were detected in the levels of inducible nitric oxide synthase or ß-actin in the antisense oligonucleotide–infused rats. To confirm the antisense oligonucleotide data, we administered a mechanistically different inhibitor of neural nitric oxide synthase, 7-nitroindazole, to an additional group of rats maintained on a high salt diet. Direct renal medullary interstitial infusion of this selective enzyme inhibitor significantly increased mean arterial pressure (15±6 mm Hg) and decreased total renal medullary nitric oxide synthase activity by 37±12% in rats on a high sodium diet. The present experiments demonstrate a role for the neural isoform of nitric oxide synthase in the long-term control of blood pressure in the presence of a high salt diet.
Key Words: kidney medulla • nitric oxide • blood pressure • sodium, dietary
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Introduction |
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A large amount of data indicates that NO is important in the control of renal function and the long-term regulation of BP. Data from many studies indicate that the renal medulla may be an important site of action of NO in the control of sodium and water homeostasis. In vivo experiments have demonstrated that nonpressor doses of the NOS inhibitor L-NAME depress the pressure-natriuretic relationship in anesthetized dogs independent of alterations in whole-kidney hemodynamics,1 indicating that renal tubular or medullary vascular actions may be an important effect of NO. Studies in conscious rats showed that chronic systemic administration of L-NAME selectively decreased renal medullary blood flow and led to the development of hypertension.2 In further studies, selective stimulation or inhibition of renal medullary NO led to increases or decreases in renal medullary blood flow, with parallel changes in sodium and water excretion.3 4 Finally, NO has been demonstrated to alter sodium transport in cultured collecting duct cells.5 These data demonstrate that NO is a potent controller of renal medullary vascular function and possibly of late tubular function.
In vitro studies to describe the anatomic distribution of the NOS isoforms have shown that renal medullary tissue has a greater capacity to synthesize NO than the renal cortex6 7 and that neural (nNOS), inducible (iNOS), and endothelial (eNOS) NOS isoforms are all present in the rat renal medulla.8 9 10 11 12 We recently reported that the absolute levels of eNOS, iNOS, and nNOS in the renal inner medulla are increased when normal Sprague-Dawley rats are placed on a high sodium diet.13 Since the BP response to NOS inhibitors is sodium dependent,13 14 the upregulation of NOS in the renal medulla may be important in the long-term adaptation to elevations in dietary sodium intake. Given the presence of large amounts of NOS activity in the renal medulla and modulation of the NOS isoforms with changes in sodium intake, these data suggest an important role of the endogenous renal medullary NO system in the long-term control of sodium homeostasis and arterial pressure.
The presence of the different NOS isoforms and the variety of effects attributed to NOS in the medulla have made it difficult to determine the role of the individual NOS isoforms in the control of renal function and BP. In the present experiments, we selectively inhibited renal medullary nNOS with two mechanistically different agents and determined the effects on BP in conscious rats.
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Methods |
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Experiments were performed on male Sprague-Dawley rats (300 to 350 g) obtained from Sasco (Madison, Wis). The rats were housed in the Animal Resource Center at the Medical College of Wisconsin with normal rat chow and tap water provided ad libitum. All animal procedures were approved by the Medical College of Wisconsin Animal Care Committee, and the rats were closely monitored to ensure that none experienced undue stress or discomfort.
Surgical Preparation
Rats were anesthetized with an intramuscular injection of ketamine (100 mg/kg) and acepromazine (2 mg/kg), and the right kidney was removed. The nephrectomy was carried out so that the remaining kidney was the sole determinant of renal function. A second surgery was performed 7 to 10 days after the nephrectomy to instrument the rats with chronic indwelling catheters. Catheters were placed in the abdominal aorta below the left renal artery via the femoral artery and in the vena cava via the femoral vein as described previously.2 15 They were then tunneled subcutaneously and exteriorized at the back of the neck in a piece of stainless steel spring. The spring was attached to a swivel device that allowed the rat to move freely in its cage while being continuously infused. The rats received a postoperative injection of penicillin (40 000 U IM) to prevent infection.
Interstitial Catheter Construction and Implantation
During surgery, a flank incision was made over the left kidney and a chronic renal medullary interstitial catheter was implanted. As we have previously described,3 4 15 16 a 7-mm piece of polyethylene tubing (PE-10, Clay Adams) was stretched over hot air to give a tip diameter of approximately 100 µm. The other end of the tube was heat-fused to a 5-cm piece of PE-50 tubing that was connected to a 40-cm piece of Tygon microbore tubing (1.52 mm OD, 0.51 mm ID, Norton Performance Plastics). The heat-pulled end of the catheter was implanted in the kidney by insertion directly into the kidney tissue through a small hole made in the renal capsule with a 26-gauge needle. The catheter tip was placed at or near the border of the inner and outer medullas by insertion directly through the cortex from the outer edge of the kidney. The catheter was anchored in place on the kidney surface with cyanoacrylate adhesive and a small piece of abdominal fat. The catheter was exteriorized, and sterile saline was continuously infused through a 0.2-µm syringe filter at 0.5 mL/h throughout the experiment. Placement of the interstitial catheter tip was confirmed at the end of the experiment by careful visual inspection. Rats with incorrectly placed catheters or substantial renal damage were discarded from the study.
Protocol 1: Influence on BP of Renal Medullary Interstitial Infusion of an Antisense Oligonucleotide for nNOS
Ten days after unilateral nephrectomy, surgery was performed for implantation of femoral arterial and venous catheters and a renal medullary interstitial catheter. Rats were allowed 6 to 8 days to recover, during which saline was continuously infused into the interstitial catheter at 0.5 mL/h and the rats were provided with normal rat chow and 1% NaCl drinking water (except for group 4, which received normal tap water).
After the postsurgical recovery period, daily BP measurements were made during a 3- to 4-hour period with solid-state pressure transducers (Cobe Laboratories) and a general purpose amplifier. The output of the amplifiers was directed into an analog-to-digital converter (Significat) and an Apollo DN3500 computer. The data were collected at 100 Hz, and minute averages of mean arterial pressure were calculated on-line.
Once the rats reached a stable BP level for 2 to 3 days, the experimental treatment was begun. Group 1 (n=9) received a continuous 4-day infusion into the medullary interstitial space with an antisense oligonucleotide probe (7.5 nmol/h) complementary to the initiation region (bases 335-351, 5'-CATGGTATCTGTGTCCT-3') of the mRNA for nNOS.17 This probe was synthesized in the Protein–Nucleic Acid Facility of the Medical College of Wisconsin with five phosphothioate bonds on the 5' and 3' ends and phosphodiester bonds in the remainder. Group 2 (n=6) was continuously infused for 4 days with saline as a vehicle time control. Group 3 (n=8) was continuously infused for 4 days into the renal medullary interstitial space with a scrambled oligonucleotide probe (7.5 nmol/h delivered in saline at 0.5 mL/h). This scrambled probe (5'-CGTCTCTGTTTGATACG-3') was composed of the same constituent bases but in random order to control for nonspecific effects of oligonucleotide infusion. Group 4 (n=5) was continuously infused for 4 days with the antisense oligonucleotide to nNOS (5'-CATGGTATCTGTGTCCT-3', 7.5 nmol/h delivered in saline at 0.5 mL/h), but these rats were maintained on normal rat chow and tap water. An additional group of uninephrectomized rats (group 5, n=6) was studied for determination of the reversibility of the antisense inhibition. These rats were infused for 5 days with the antisense oligonucleotide probe into the renal medullary interstitial space (7.5 nmol/h in saline at 0.5 mL/h). After the fifth day of oligonucleotide treatment, the renal medullary interstitial infusion was returned to saline for 5 postcontrol days. Daily measurements of BP and body weight were made throughout the protocol in this rat group.
Protocol 2: Influence of Renal Medullary Interstitial Infusion of Antisense Oligonucleotide on Levels of nNOS and Total NOS Activity
A separate group of uninephrectomized rats was infused for 4 days with the antisense oligonucleotide (7.5 nmol/h), the scrambled probe (7.5 nmol/h), or saline into the medullary interstitial space. No difference was detected between the saline and scrambled probe infusion, so these data were later pooled into a control group. The rats were maintained on normal rat chow with 1% NaCl drinking water. After the fourth day of infusion, the rats were euthanized with an overdose of sodium pentobarbital, and the renal medulla and cerebellum were rapidly removed and frozen on dry ice. The tissue was stored at -80°C until protein extraction. In the extraction procedure, pieces of whole tissue were homogenized with a tissue grinder (Potter-Elvehjem) at 3000 rpm in a solution containing (mmol/L) sucrose 250, EDTA 1, phenylmethylsulfonyl fluoride 0.1, and potassium phosphate 5, pH 7.7. All chemicals were purchased from Sigma Chemical Co unless otherwise noted. The homogenate was centrifuged at low speed (16 000g, 4°C, 20 minutes), and the protein concentration of the supernatant tissue homogenate was determined with a Coomassie blue protein assay (Pierce Chemical Co) with albumin as a standard. The individual NOS isoform distribution and total NOS activity of the tissue were determined with the protein blotting and NOS enzyme assay protocols described below. We attempted but were unable to measure enzyme activity and the level of the individual NOS isoforms in the renal cortex and aorta of these rats. The level of total NOS activity in these tissues is below the detection level of our present enzyme assay. Also, as we have previously shown,13 nNOS is not detectable in the whole renal cortex with our whole-tissue protein blotting techniques despite its presence in the macula densa.18 19
Western Blotting Protocols
Protein samples were electrophoretically size-separated by a discontinuous system consisting of a 7.5% polyacrylamide resolving gel and 5% polyacrylamide stacking gel. Molecular weight markers (range, approximately 40 to 200 kD) were loaded into one lane as a size standard. Equivalent amounts of total protein from antisense-treated or control rats were added to adjacent lanes, and the samples were run at 200 V for 45 to 60 minutes on an 8x10-cm electrophoresis cell (Bio-Rad).
After separation, the proteins were electrophoretically transferred to a nitrocellulose membrane at 100 V for 1 hour. The membranes were washed in Tris-buffered saline (TBS), blocked with 5% nonfat dried milk in TBS (NFM/TBS) for 2 hours, and incubated with a 1:1000 dilution of monoclonal mouse anti-nNOS (Transduction Laboratories) in 2% NFM/TBS overnight at 4°C. We previously demonstrated that this antibody binds to both rat nNOS and iNOS.13 The membranes were then incubated with a horseradish peroxidase–labeled goat anti-mouse IgG (1:1000) in 2% NFM/TBS for 2 hours. The bound antibody was detected by chemiluminescence (Amersham) on x-ray film. Binding of a monoclonal antibody against the structural protein ß-actin (Sigma) was used as a loading control. Membranes were stripped between incubations with different antibodies in a Tris-buffered solution containing 2% sodium dodecyl sulfate and 100 mmol/L ß-mercaptoethanol at 50°C. Densitometry was performed with a Phosphoimager Personal Densitometer (Molecular Dynamics).
Tissue NOS Activity
The NOS enzyme assay was based on previously described methods.20 21 The total tissue homogenate was incubated with 2 mmol/L CaCl2, 1 mmol/L NADPH, 25 µmol/L FAD, 1.25 µg/mL calmodulin, 10 µmol/L tetrahydrobiopterin, and [3H]arginine (approximately 300 000 cpm; specific activity, 68 Ci/mmol) in 20 mmol/L HEPES buffer, pH 7.2, at 37°C for 5 minutes. The arginine and converted citrulline were separated by isocratic reversed-phase high-performance liquid chromatography with a Supelco LC-18-DB column (mobile phase: 11.5% methanol, 11.5% acetonitrile, 1% tetrahydrofuran, 0.1 mol/L KH2PO4, pH 5.9). The amount of converted citrulline and the total counts were measured by radiochemical detection (Packard).
Protocol 3: Renal Medullary Interstitial Infusion of 7-NI
To inhibit renal medullary nNOS with a mechanistically different approach, 7-NI (BIOMOL Research Laboratories), an inhibitor specific to nNOS when administered in vivo,22 23 24 25 26 was infused into the medullary interstitial space of a separate rat group maintained on normal rat chow and 1% NaCl drinking water (n=7). Rats were prepared as described above except that 7-NI was continuously infused directly into the renal medullary interstitial space at 100 µg/h for 2 days, 200 µg/h for an additional 2 days, and finally at a dose of 400 µg/h for the final 4 days. The 7-NI was added to hot (approximately 80°C) 0.5% NaCO3 in saline and rigorously stirred to dissolve in solution. Some precipitation was noted in the syringe filters at the highest concentration of 7-NI even though the drug went into solution in hot 0.5% NaCO3. A separate rat group was infused with 0.5% NaCO3 in saline and maintained on the high salt diet as a vehicle time control (n=5).
Statistical Methods
Data are expressed as mean±SE. Within-group changes in BP and body weight were evaluated with a one-way ANOVA for repeated measures and Duncan's post hoc test. Between-group comparisons of BP values were made with one-way ANOVA for unpaired data with Duncan's post hoc test. Comparison of densitometry and enzyme activity data between control and treated groups was done with an unpaired two-tailed t test. A probability level of less than .05 was considered significant.
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Results |
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Protocol 1: Influence on BP of Renal Medullary Interstitial Infusion of an Antisense Oligonucleotide for nNOS
As shown in Fig 1
, mean arterial pressure in groups 1 and 2 was not significantly different during the control period. Renal medullary interstitial infusion of the antisense oligonucleotide significantly increased BP 14±1 mm Hg in the antisense oligonucleotide–infused rats but had no significant effect on BP in the vehicle-infused rats. The mean changes in BP from control after 4 days of infusion in the different rat groups are summarized in Fig 2. Control levels of arterial pressure averaged 101±3, 97±1, 105±2, and 104±4 mm Hg in groups 1 through 4, respectively. Although renal medullary interstitial infusion of the antisense oligonucleotide for nNOS significantly increased BP in the rats maintained on a high sodium diet, neither infusion of saline nor infusion of the scrambled oligonucleotide probe altered mean arterial pressure from control levels. Infusion of the antisense oligonucleotide in rats maintained on a normal sodium diet also failed to alter BP.
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Mean arterial pressure data from group 5 are summarized in Fig 3. Renal medullary interstitial infusion of the antisense oligonucleotide for nNOS significantly increased BP from a mean control value of 97±3 to 112±2 mm Hg by the fifth day of infusion. Mean arterial pressure decreased to a level not significantly different from control by the fifth postcontrol day. The average body weight of these rats progressively and significantly increased from a control level of 377±5 g on the final control day to 392±6 g on the final day of antisense infusion. During the postcontrol period, body weight did not return to control but did not significantly increase from the final experimental period value, averaging 397±5 g on the final postcontrol day.
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Protocol 2: Influence of Renal Medullary Interstitial Infusion of Antisense Oligonucleotide on Levels of nNOS and Total NOS Activity
Fig 4 shows a representative Western blot of total medullary protein obtained from control and antisense-infused rats. The first four lanes were loaded with 100 µg of total renal medullary protein from control rats, and the last four lanes were loaded with 100 µg total medullary protein from antisense-infused rats. The middle lane contains the molecular weight markers with the 199-kD size standard. The membrane was probed with an anti-nNOS antibody (top), which we have previously shown to bind to both nNOS and iNOS,13 and an antibody against the structural protein ß-actin (bottom). The intensity of the nNOS bands was reduced in the antisense-treated rats, whereas the iNOS and ß-actin bands were not altered. Densitometric analysis of total medullary protein homogenates from seven antisense-treated and seven control rats indicated that nNOS was significantly reduced by 53% in the antisense-treated rats, whereas the intensities of iNOS and ß-actin were not altered (Fig 5).
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Supporting the Western blot results are the NOS assay data, which demonstrated that total renal medullary NOS activity was reduced by 30%, whereas cerebellar NOS activity was unaltered by renal medullary interstitial infusion of the antisense oligonucleotide. Total counts converted to citrulline in the antisense-treated group (n=6) averaged 20 088±2230 cpm, whereas the total counts converted to citrulline in the scrambled probe control group (n=5) averaged 28 592±1950 cpm. The total counts converted to citrulline by the cerebellar tissue averaged 241 437±2075 and 242 259±3099 cpm in the treated and control groups, respectively.
Protocol 3: Influence of Renal Medullary Interstitial Infusion of 7-NI on Mean Arterial Pressure and NOS Activity
The effects of renal medullary interstitial infusion of 7-NI, a NOS inhibitor reportedly selective for nNOS whenadministered in vivo,22 23 24 25 26 are illustrated in Fig 6. The baseline level of arterial pressure averaged 108±3 mm Hg in the control period. Arterial pressure was not significantly altered during the renal medullary interstitial infusion of 7-NI at rates of either 100 or 200 µg/d. Mean arterial pressure was significantly elevated from control by the second day of drug infusion at a rate of 400 µg/d, with a total increase in BP of 15±6 mm Hg by the final day of infusion. Renal medullary interstitial infusion of vehicle (0.5% NaCO3 in 0.9% NaCl) did not alter BP from the mean control value (106±4 mm Hg) after 8 days of renal medullary interstitial infusion of vehicle. Body weight progressively and significantly increased in both groups during the experimental period. The body weight of rats infused with 7-NI averaged 409±21 g in the control period and was significantly increased to 429±24 g by the final day of 7-NI infusion. Similarly, the body weight of the vehicle control rats significantly increased from 422±27 to 458±16 g over the same time period.
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Comparison of the total NOS activity from homogenates of whole renal medullary tissue demonstrated that 7-NI infusion significantly decreased NOS activity by 37%. Total NOS activity in the cerebellum of the 7-NI–treated rats was not altered compared with that in the vehicle-treated rats, indicating a lack of recirculation of the enzyme inhibitor. The calculated total counts converted to citrulline by the total renal medullary homogenate of the control rats (n=6) averaged 21 862±2146 cpm, and the total citrulline counts in the 7-NI–treated group (n=6) averaged 13 842±2565 cpm. The total counts converted to citrulline by the cerebellar tissue averaged 264 946±4120 and 272 418±1046 cpm in the control and treated groups, respectively.
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Discussion |
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A large amount of data describing the effects of NOS inhibition on renal function and BP control has been reported.27 28 29 30 The bulk of this work, however, has been performed with arginine analogue inhibitors of NOS, which are not specific for the individual isoforms. In the present study, selective inhibition of renal medullary nNOS with an antisense oligonucleotide or the specific enzyme inhibitor 7-NI led to a progressive increase in arterial pressure. The increase in BP was sodium dependent and reversible. Selective nNOS blockade was confirmed by protein blotting, which demonstrated decreased levels of nNOS in the antisense oligonucleotide–infused rats compared with control rats. Two other gene products found in the renal medulla, iNOS and ß-actin, were unaltered by antisense treatment. In addition, infusion of either the antisense oligonucleotide or 7-NI into the renal medullary interstitial space led to a decrease in total renal medullary NOS activity. Inhibition of nNOS in the renal medulla led to a sustained increase in mean arterial pressure in normal rats maintained on a high sodium diet.
Renal Medullary NOS
Although renal medullary nNOS was inhibited in the present study, it cannot be determined where in the medulla this isoform exerts its actions. A large number of studies have identified the three NOS isoforms throughout the rat kidney. Immunohistochemistry, in situ hybridization, and reverse transcriptase coupled to the polymerase chain reaction (RT-PCR) of microdissected renal vessels and tubules have demonstrated the presence of nNOS mRNA or protein in the macula densa, inner and outer medullary collecting ducts, glomerulus, vasa recta, arcuate artery, and renal nerves in perivascular connective tissue as well as nerves adjacent to the pelvic epithelium.9 11 18 19 eNOS and iNOS are also present in the kidney. RT-PCR studies have also shown eNOS in the glomeruli, preglomerular vasculature, proximal tubules, thick ascending limbs, and collecting ducts.12 Two separate isoforms of iNOS have been identified in renal vascular and tubular segments. The vascular smooth muscle isoform of iNOS has been detected in arcuate and interlobular arteries, whereas macrophage NOS was identified in the glomeruli, proximal tubules, thick ascending limbs, and collecting ducts.8 10 These studies demonstrate that not only nNOS but also eNOS and iNOS are distributed throughout the rat kidney. The site at which renal medullary nNOS was inhibited in the present study could be in almost any structure in the renal medulla.
The present study demonstrates that inhibition of renal medullary nNOS leads to an elevation of BP in rats on a high sodium diet but does not address the mechanism of this hypertensive effect. We previously reported that systemic and renal medullary interstitial infusion of L-NAME leads to a selective reduction in renal medullary blood flow, retention of sodium and water, and increased BP.2 15 These observations could be due to the inhibition of any or all of the NOS isoforms. Since the neural isoform has been identified in both the vasa recta blood vessels and the collecting ducts11 and NO has been implicated in the control of both vasa recta blood flow2 15 and collecting duct transport,5 direct vascular and tubular influences are possible.
Antisense Oligonucleotide Inhibition of Renal Medullary nNOS
The antisense oligonucleotide used in the present experiment is a short, single-stranded piece of DNA complementary to the initiation region of nNOS mRNA. The exact mechanism of action of antisense oligonucleotides is not thoroughly understood, but it has been proposed that the oligonucleotides are internalized into cells by endocytosis and rapidly localized in nuclear structures where translation by the targeted mRNA is inhibited.31 32 33 The previous use of antisense oligonucleotide probes in vivo has been limited because of the difficulty in delivery. Single-stranded DNA is highly susceptible to extracellular degradation by exonucleases and endonucleases, and the oligonucleotides are hydrophilic, which reduces the amount of oligonucleotide available to enter the cell. Nevertheless, this antisense oligonucleotide approach has been successfully used to block the production of both cell-cycle regulatory proteins in the smooth muscle of injured rat carotid arteries34 and angiotensinogen and the angiotensin type 1 receptor in rat brain.35
We modified the oligonucleotide used in the present experiments with phosphothioate bonds on each end in an attempt to increase the stability by decreasing degradation.32 Administration of this antisense oligonucleotide, which significantly increased BP, decreased the level of nNOS protein without changing iNOS or ß-actin, two other gene products found in the renal medulla. These results support the view that the antisense oligonucleotide had specific effects on nNOS. The antisense oligonucleotide also significantly decreased total tissue NOS activity in the medulla without changing the cerebellar NOS activity in these same rats. The lack of effects on cerebellar NOS activity suggests minimal systemic spillover, although we did not examine the influence of systemic infusion of the oligonucleotide (or 7-NI) on cerebellar and renal medullary NOS activities in this study. Neither the medullary interstitial infusion of saline nor the scrambled oligonucleotide probe altered BP. The scrambled oligonucleotide consisted of the same bases but in randomized order to account for any nonspecific chemical effects of oligonucleotide infusion. These experiments support a selective influence of the antisense oligonucleotide to decrease the level of renal medullary nNOS and increase BP in chronically treated rats.
An alternative approach to validate the antisense experiments was to perform the same experiment with a mechanistically different inhibitor of nNOS. Renal medullary interstitial infusion of the enzyme inhibitor 7-NI, which has been reported to selectively block nNOS when administered in vivo,22 23 24 25 26 led to an increase in arterial pressure of 15 mm Hg. The hypertensive effects of 7-NI were accompanied by a 37% decrease in total renal medullary NOS activity. In addition, the renal medullary interstitial 7-NI infusion did not alter cerebellar NOS activity, indicating minimal systemic spillover of the 7-NI from the infused kidney. The functional and biochemical data from the rats chronically treated with 7-NI indicate that inhibition of renal medullary nNOS leads to increased BP. These data also support the antisense oligonucleotide data since these two compounds, which have different mechanisms of action, caused the same functional effects in chronically treated rats maintained on a high sodium diet.
Although the present data document inhibition with the antisense oligonucleotide or 7-NI in the renal medulla without peripheral effects on cerebellar NOS activity, we were unable to measure the influence of these inhibitors on renal cortical NOS activity. The techniques we used in this study are not sensitive enough to measure nNOS or NOS activity in the renal cortex. Since nNOS has been demonstrated in the macula densa18 19 and L-NAME administration has potent effects on the tubuloglomerular feedback response,19 36 it is possible that the effects observed in the present experiment involve alterations in renal cortical hemodynamics or even tubular responses due to alterations in tubuloglomerular feedback. However, this possibility seems unlikely because renal medullary interstitial infusion of L-NAME into acutely or chronically instrumented rats had no influence on renal cortical blood flow or glomerular filtration rate.4 16 Nevertheless, it is possible that the hypertensive effects of medullary interstitial infusion of the antisense against nNOS and 7-NI are due to effects on the renal cortex. An additional concern is the use of the uninephrectomized rat. Although this preparation is necessary for determination of the long-term effects of renal medullary interstitial infusion, it is possible that the remnant kidney exhibits increased NOS activity to compensate for its increased excretory requirements. Although this remains to be determined, it could lead to an exaggerated functional response to renal medullary nNOS inhibition.
Conclusions
The results of the present experiments may help explain the importance of renal medullary NOS during elevated NaCl intake in the rat. We previously reported that L-NAME infusion directly into the medullary interstitial space led to a selective decrease in renal medullary blood flow, retention of sodium and water, and the development of hypertension.15 Further studies demonstrated that the relative levels of eNOS, iNOS, and nNOS were all increased in rats on a high NaCl diet compared with levels in rats on a low NaCl diet13 and that the chronic hypertension associated with systemic L-NAME infusion is sodium sensitive.13 14 Each of these studies indicated that renal medullary NOS is important in the control of sodium balance and BP. The present study clearly demonstrates that nNOS is important in the long-term regulation of arterial pressure, although the mechanism of this hypertension remains to be determined.
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Selected Abbreviations and Acronyms |
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-nitro-L-arginine methyl ester
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Acknowledgments |
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This work was partially supported by National Heart, Lung, and Blood Institute grant HL-29587 and American Heart Association, Wisconsin Affiliate, grant 95-GS-76.
Received February 12, 1996; first decision March 6, 1996; accepted April 3, 1996.
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