Abstract We previously reported that chronic systemic treatment of rats with a nitric oxide synthase inhibitor leads to a selective decrease in renal medullary blood flow, retention of sodium, and the development of hypertension. In the present studies, we used protein blotting techniques to determine the whole tissue distribution and relative quantitation of the different nitric oxide synthase isoforms in the renal cortex and medulla of Sprague-Dawley rats maintained on a low (0.4% NaCl) or high (4.0% NaCl) dietary salt intake. Neural, endothelial, and inducible nitric oxide synthase were readily detectable in homogenized renal inner and outer medullas. Only endothelial nitric oxide synthase was detectable in the renal cortex. Densitometric comparison of Western blots from equal amounts of total inner medullary tissue protein indicated that endothelial, inducible, and neural nitric oxide synthase were increased by 145%, 49%, and 119%, respectively, in rats maintained on a high NaCl diet compared with rats on a low NaCl diet. No significant differences in nitric oxide synthase levels were detected in the outer medulla, renal cortex, or aorta of rats maintained on low and high NaCl diets. In separate studies, continuous intravenous infusion of NG-nitro-l-arginine methyl ester (8.6 mg/kg per day) for 11 days in chronically instrumented rats increased mean arterial pressure 32±3 mm Hg in rats on a high NaCl diet (n=5) but only increased pressure 17±3 mm Hg in rats on a low NaCl diet (n=6). These data indicate that increased levels of renal medullary nitric oxide synthase may be important in the chronic adaptation to increased sodium intake.
A large number of studies have demonstrated the influence of acute and chronic NOS inhibition on renal function and BP control. Acute NOS inhibition shifted (or blunted) the pressure natriuretic-diuretic relationship to higher levels of perfusion pressure,1 2 3 and chronic administration of NOS inhibitors caused hypertension4 5 6 7 8 9 10 in rats and dogs. These chronic effects may be mediated by alterations in renal vascular and/or tubular function. Experiments from our laboratory have demonstrated that acute stimulation or inhibition of NO in the renal medulla increases or decreases renal medullary blood flow, with parallel changes in sodium and water excretion.11 12 More recently, we have reported that chronic infusion of L-NAME directly into the renal medullary interstitial space causes a selective decrease in renal medullary blood flow, retention of sodium and water, and the development of hypertension.13 These studies indicate that NO in the medulla plays a major role in the control of vasa recta blood flow, sodium and water excretion, and BP.
Despite the large amount of functional data that demonstrate the effects of NOS blockade on renal function and BP, very little is known about the regulation of NOS during changes in NaCl intake. Urinary excretion of NO2−/NO3−, an indirect index of endogenous NO, indicates increased NOS activity when rats are placed on a high NaCl diet.4 14 These observations are supported by functional data that demonstrate increased sensitivity of the renal vasculature to NOS inhibition in rats on a high NaCl intake.14 15 These studies provide evidence supporting a role for NOS in the chronic renal adaptation to a high salt diet. Little is known, however, about the absolute level of the different NOS isoforms in rats on a high salt diet or the specific roles the individual isoforms may play in the control of electrolyte balance after changes in NaCl intake.
In the present experiments, we characterized the levels of eNOS, iNOS, and nNOS in rats maintained on a low or high NaCl diet. We performed additional experiments to determine the influence of systemic inhibition of NOS with L-NAME on BP in rats during low or high salt.
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 low (0.4% NaCl) or high (4.0% NaCl) salt food and water provided ad libitum. All animal procedures were approved by the Medical College of Wisconsin Animal Care Committee.
Protocol 1: Influence of Low or High Dietary NaCl Intake on Relative Levels of eNOS, iNOS, and nNOS
Rats were maintained on low (n=6) or high (n=6) NaCl rat chow for 3 weeks. The rats were euthanatized with an overdose of intraperitoneal sodium pentobarbital, and pieces of renal cortex, renal outer medulla, renal inner medulla, and aorta were rapidly removed and frozen on dry ice. The tissue was stored at −80°C until extraction. In the extraction procedure, pieces of whole tissue were homogenized with a Potter-Elvehjem tissue grinder at 3000 rpm in a solution containing 250 mmol/L sucrose, 1 mmol/L EDTA, 0.1 mmol/L phenylmethylsulfonyl fluoride, and 5 mmol/L potassium phosphate, pH 7.7. All chemicals were purchased from Sigma Chemical Co. Large tissue debris and nuclear fragments were removed by two low-speed centrifuge spins (3000g, 4°C, 5 minutes; and 10 000g, 4°C, 5 minutes). The protein concentration of the tissue homogenate was determined by the method of Bradford with albumin as a standard. When necessary (in the case of the aortic and renal cortical tissues), the membrane-bound proteins were further isolated by spinning the whole-protein slurry at 101 000g for 60 minutes and resuspending the pellet for protein blotting of eNOS.
Protein samples were electrophoretically size-separated with a discontinuous system consisting of a 7.5% polyacrylamide resolving gel and 5% polyacrylamide stacking gel. High-range molecular weight markers (approximately 40 to 200 kD) were loaded into one lane as a size standard. Equivalent amounts of total protein from the same tissue from rats on a low or high salt diet were added to adjacent lanes, and the samples were run at 200 V for 45 to 60 minutes on an 8×10 cm electrophoresis gel (Bio-Rad).
After separation, the proteins were electrophoretically transferred to a nitrocellulose membrane at 100 V for 1 hour. These membranes were washed in Tris-buffered saline, blocked with 5% nonfat dried milk in Tris-buffered saline (NFM/TBS) for 2 hours, and incubated with a 1:1000 dilution of monoclonal mouse anti-nNOS (to detect both nNOS and iNOS) or polyclonal rabbit anti-eNOS (Transduction Laboratories) in 2% NFM/TBS overnight at 4°C. The membranes were then incubated with a horseradish peroxidase–labeled goat anti-mouse IgG (1:1000) or goat anti-rabbit IgG in 2% NFM/TBS for 2 hours. The bound antibody was detected by chemiluminescence (ECL, Amersham) on x-ray film. A monoclonal mouse antibody raised against the structural protein β-actin 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 β-mercaptoethanol at 50°C.
Protocol 2: Influence of Low or High Dietary NaCl Intake on L-NAME Hypertension
Rats maintained on a low (n=6) or high (n=5) NaCl diet were anesthetized with ketamine (100 mg/kg IM) and acepromazine (2 mg/kg IM), and chronic arterial and venous catheters were implanted as described previously.8 13 The catheters were placed in the abdominal aorta or vena cava via the femoral artery and vein and tunneled subcutaneously to the back of the neck. The catheters were then exteriorized in a piece of stainless steel spring that was anchored into the strap muscles with a stitch and attached to a swivel at the top of the cage. This arrangement allowed the rat to move freely about its cage while being continuously infused with saline (4.0 mL/d IV). The rats received a postoperative injection of penicillin (40 000 U IM) to prevent infection.
Beginning 1 week after surgery, daily BP determinations were made during a 2-hour period. After stable recordings of control BP, the rats received a continuous infusion of L-NAME (8.6 mg/kg IV per day in saline) for 11 days. BP determinations were made once daily on the 1st and 11th days after the initiation of the L-NAME infusion. After the 11th day of L-NAME, the infusion was returned to saline, and BP was again determined on the 3rd postcontrol day.
Data are expressed as mean±SE. An unpaired t test was used to examine the densitometric data for differences between individual isoforms in rats on low and high NaCl diets. The differences in BP values were determined with a two-way ANOVA with repeated measures in one dimension and Duncan’s post hoc test. A probability level of less than .05 was considered significant.
Protocol 1: Influence of Low or High Dietary NaCl Intake on Relative Levels of eNOS, iNOS, and nNOS
We first performed experiments to determine the viability of protein blotting for NOS isoforms in whole renal tissue (Fig 1⇓). A polyacrylamide gel was loaded with 100 μg protein from the renal cortex, outer medulla, and inner medulla; standards for eNOS (human umbilical vein endothelial cell lysate), nNOS (rat cerebellum), and iNOS (induced macrophage) were also added. The membrane was first incubated with a monoclonal anti-nNOS antibody (Fig 1⇓, top), and then it was stripped and rehybridized with a polyclonal anti-eNOS antibody (Fig 1⇓, bottom). The top panel of Fig 1⇓ shows that the anti-nNOS antibody hybridized with a protein of approximately 160 kD in the cerebellum (nNOS) and also with a 140-kD band (iNOS) in macrophages. There was no detectable signal for nNOS or iNOS in the renal cortical homogenate, although distinct bands for both nNOS and iNOS were observed in the outer and inner medullas. The bottom panel illustrates that the polyclonal anti-eNOS antibody binds to a 150-kD protein in endothelial cells (eNOS), with corresponding bands in the outer and inner medullas but no detectable band in the renal cortex.
We then used the antibodies described above to examine the levels of the different NOS isoforms in 100 μg total protein from the outer and inner medullas of rats on a low and high NaCl intake. A representative blot is shown in Fig 2⇓. The top panel demonstrates that both nNOS and iNOS levels are increased in the inner medulla of rats on a high salt diet compared with rats on a low salt diet. The middle panel indicates that eNOS levels are also increased in the inner medulla of rats on a high NaCl diet. The bottom panel is the same membrane probed with a monoclonal antibody raised against the structural protein β-actin; the equal density of binding in all the lanes indicates that equal amounts of inner and outer medulla protein were loaded into each lane. Densitometric analysis of protein blots from inner medulla of six rats on a low and six rats on a high NaCl diet indicated that eNOS, iNOS, and nNOS were significantly increased by 145%, 49%, and 119%, respectively, compared with rats on a low NaCl diet (Fig 3⇓). There was no difference in the density of binding of β-actin in the inner medulla protein between the rats maintained on a low and high NaCl diet. Although iNOS and nNOS levels tended to increase in the outer medulla of rats on a high NaCl diet, they did not reach statistical significance compared with levels in rats on a low NaCl diet.
The relative quantity of eNOS in the renal cortex and aorta was examined in the 101 000g fraction of the renal cortex and aorta of rats on a low and high NaCl intake. A representative blot for eNOS and β-actin in the 101 000g pellet from the whole renal cortex of low and high salt rats is shown in Fig 4⇓; there was no difference in the density of the eNOS or β-actin band in the renal cortex of low and high salt rats. There was also no difference in eNOS or β-actin in the aorta between rats maintained on a low and high NaCl diet (data not shown).
Protocol 2: Influence of Low or High Dietary NaCl Intake on L-NAME Hypertension
As shown in Fig 5⇓, two groups of rats maintained on low and high salt diets had similar levels of control BP. After 1 day of L-NAME infusion (8.6 mg/kg IV per day), BP was significantly elevated in each group of rats. After 11 days of continuous L-NAME infusion, mean arterial pressure was increased 32±3 mm Hg in rats on a high NaCl diet, an increase significantly different from the 17±3 mm Hg increase in BP in the rats maintained on a low NaCl diet. By the 3rd postcontrol day, BP had returned to levels not significantly different from control.
The present data confirm the presence of eNOS, iNOS, and nNOS in the renal medulla of normal Sprague-Dawley rats. On a whole tissue basis, the renal medulla contains more NOS than the renal cortex. In addition, the relative levels of renal medullary eNOS, iNOS, and nNOS are increased when rats are placed on a high NaCl diet. Finally, we observed that chronic systemic administration of the NOS inhibitor L-NAME had a greater effect on BP in rats maintained on a high NaCl diet than in rats on a low NaCl diet. These data indicate that NOS may play a major role in the regulation of sodium and water homeostasis during increases in dietary salt intake.
Greater amounts of NOS protein in the medulla are consistent with previous reports which demonstrated that the renal medulla of the rat16 and dog17 kidney has a greater capacity to produce NO than the renal cortex. The greater levels of the NOS isoforms in the medulla compared with the renal cortex may help explain the preferential effects of chronic systemic L-NAME infusion on renal medullary function. Chronic intravenous infusion of L-NAME in the same dose as used in the present study in normotensive rats leads to a decrease of renal medullary blood flow, no change in renal cortical blood flow, retention of sodium and water, and the development of hypertension.8 The elevated levels of NOS in the medulla may render renal tubular and vascular function especially sensitive to chronic NOS inhibition. It is important, however, to note that the renal cortical vasculature is also highly sensitive to NOS inhibition,12 14 15 and although NOS is in lower amounts in the renal cortex on a total tissue basis, the precise distribution of the different NOS isoforms will probably dictate the physiological effects of NO and the pharmacological response to NOS inhibition.
The increased levels of eNOS, iNOS, and nNOS in the medulla of Sprague-Dawley rats maintained on a high NaCl diet may be important in the chronic adaptation to a high salt diet. This observation is consistent with increased urinary NO2−/NO3− excretion in normotensive rats placed on a high NaCl diet,4 14 which indicates that in vivo NO activity is increased when rats are maintained on an elevated sodium intake. The salt sensitivity of the hypertensive response to chronic L-NAME in the high salt rats lends further support to the concept that NO is important in the chronic adaptation to high salt diets. Increased levels of NOS, and presumably NO, could have significant effects on renal medullary function to participate in the maintenance of sodium and water balance.
It is interesting that the present data indicate that the level of renal cortical eNOS is not altered under conditions of elevated salt intake when functional data have demonstrated increased renal cortical vasoconstriction after L-NAME in conditions in which dietary sodium is elevated.14 15 A number of possibilities could explain this apparent paradox. First, the level of NOS protein may not accurately reflect endogenous NO production. Second, the present study examined total renal cortical tissue; it is possible that renal cortical blood vessels actually show increased NOS under conditions of high sodium intake. Finally, as proposed by Deng et al,18 the endogenous level of arginine, the substrate for NOS, may be increased under conditions of elevated sodium intake, which could also lead to elevated NOS activity. Any of these possibilities could help explain the enhanced sensitivity of the renal cortical vasculature to L-NAME in high salt rats.
Although the present data describe changes in the distribution of the NOS isoforms in the different regions of the kidney when the NaCl intake of the rats is increased, the present studies do not elucidate in which tubular and/or vascular segments these changes occur. At least four NOS isoforms have been identified in the rat kidney. Immunohistochemical data indicate the nNOS is present in the macula densa,19 20 whereas microdissection coupled with reverse transcription–polymerase chain reaction (RT-PCR) has demonstrated the presence of nNOS mRNA in the inner medullary collecting duct, outer medullary collecting duct, glomerulus, vasa recta, and arcuate artery.21 Immunohistochemistry and in situ hybridization were used to identify nNOS in the thick ascending loop of Henle, the efferent arteriole, and renal nerves in perivascular connective tissue and adjacent to the pelvic epithelium.22 Further studies with RT-PCR demonstrated that eNOS mRNA is present in high levels in the glomeruli and preglomerular vasculature, with lower levels in the proximal tubules, thick ascending limbs, and collecting ducts.23 The mRNAs for two separate iNOS isoforms have also been identified in renal vascular and tubular segments of normal rats. The vascular smooth muscle isoform (vsmNOS) of iNOS has been found in arcuate and interlobular arteries, and an NOS partially homologous to macrophage NOS (macNOS) has been identified in the glomeruli, proximal tubules, thick ascending limbs, and collecting ducts.24 25 The macNOS appears to be the predominant inducible isoform in the medulla of the normal rat. These combined data obtained by microdissection coupled with RT-PCR, immunohistochemistry, and in situ hybridization techniques indicate that eNOS, iNOS, and nNOS are present in the normal rat kidney, although the functional importance of the different isoforms is unknown. Presently, the site or sites in the nephron or renal vasculature where the NOS isoforms are increased when sodium intake is altered and the site of action of NO produced by these enzymes are unknown.
As evidenced by the failure of the Western blotting technique to detect nNOS in the renal cortex when this isoform is clearly present in the macula densa cells, the technique used in the present study has a limited sensitivity and could have missed important changes in the NOS isoforms in discrete portions of the renal tubules or vasculature. The present data cannot be used to eliminate the importance or possible changes with a high salt diet of NOS enzymes located in discrete parts of the kidney or other parts of the body. In addition, a number of other factors, including the availability of substrate and cofactors and the presence or absence of stimulators or inhibitors of NO release may also be important in the determination of the endogenous levels of NO. The present study does indicate, however, that relatively large amounts of the three NOS isoforms are present in the renal medulla of the normal Sprague-Dawley rat. In addition, large changes in eNOS, iNOS, and nNOS are observed in the inner medulla when rats are placed on a high NaCl diet. These findings could indicate an important regulatory role for the NO system in the control of renal medullary function, in the control of sodium and water homeostasis, and in the long-term regulation of arterial pressure.
Selected Abbreviations and Acronyms
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|NOS||=||nitric oxide synthase|
This work was partially supported by the National Heart, Lung, and Blood Institute (grant HL-29587) and the American Heart Association, Wisconsin Affiliate (grant 95-GS-76).
Reprint requests to David L. Mattson, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.
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