This study examined the effects of chronic blockade of the renal formation of epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid with 1-aminobenzotriazole (ABT; 50 mg·kg−1· day−1 ip for 5 days) on pressure natriuresis and the inhibitory effects of elevations in renal perfusion pressure (RPP) on Na+-K+-ATPase activity and the distribution of the sodium/hydrogen exchanger (NHE)-3 in the proximal tubule of rats. In control rats (n = 15), sodium excretion rose from 2.3 ± 0.4 to 19.4 ± 1.8 μeq·min−1·g kidney weight−1 when RPP was increased from 114 ± 1 to 156 ± 2 mmHg. Fractional excretion of lithium rose from 28 ± 3 to 43 ± 3% of the filtered load. Chronic treatment of the rats with ABT for 5 days (n = 8) blunted the natriuretic response to elevations in RPP by 75% and attenuated the increase in fractional excretion of lithium by 45%. In vehicle-treated rats, renal Na+-K+-ATPase activity fell from 31 ± 5 to 19 ± 2 μmol Pi·mg protein−1·h−1 and NHE-3 protein was internalized from the brush border of the proximal tubule after an elevation in RPP. In contrast, Na+-K+-ATPase activity and the distribution of NHE-3 protein remained unaltered in rats treated with ABT. These results suggest that cytochrome P-450 metabolites of arachidonic acid contribute to pressure natriuresis by inhibiting Na+-K+-ATPase activity and promoting internalization of NHE-3 protein from the brush border of the proximal tubule.
- 20-hydroxyeicosatetraenoic acid
- epoxyeicosatrienoic acids
- sodium/hydrogen exchanger-3
- proximal tubule
- renal hemodynamics
the concept that the kidney plays an important role in the long-term control of arterial pressure is based on the phenomenon of pressure natriuresis (19, 21). Despite intensive investigation, many aspects of the mechanism of pressure natriuresis remain unknown. Previous studies have indicated that pressure natriuresis is associated with elevations in renal medullary blood flow (13, 59–61) and renal interstitial hydrostatic pressure (RIHP) (15, 19, 31). Na+ transport in the proximal tubule (22, 32, 33, 57, 76) and the loop of Henle (34, 57) decreases after elevations in renal perfusion pressure (RPP). Increases in RPP have been proposed to inhibit Na+ transport in the proximal tubule by increasing backflux of Na+ through the paracellular pathway (14, 19, 36). However, this mechanism seems unlikely because of the lack of an electrochemical gradient for backdiffusion of Na+ in the proximal tubule, and the existing Cl− gradient favors reabsorption rather than backleak. The work of Magyar et al. (37, 38) and others (72, 74, 75, 76, 78, 79), indicating that elevations in RPP are associated with a fall in Na+-K+-ATPase activity and internalization of the sodium/hydrogen exchanger (NHE)-3 from the brush border of the proximal tubule, has led to the suggestion that some signal-transduction pathway probably couples elevations in RPP to inhibition of the active transport of Na+ in the proximal tubule. However, the mechanisms by which elevations in RPP and RIHP inhibit Na+ reabsorption are unknown and remain one of the key unanswered questions in hypertension research.
Previous studies have indicated that inhibitors of cyclooxygenase attenuate the reduction in proximal tubular Na+ reabsorption after elevations in RPP (8, 23, 32, 53). However, cyclooxygenase enzymes are not highly expressed in the proximal tubule (5), and there is no evidence that elevations in RPP stimulate the synthesis or release of prostaglandins in this portion of the nephron. Moreover, cyclooxygenase inhibitors reduce medullary blood flow and RIHP (17, 62). Thus they may simply modulate pressure natriuresis by altering renal medullary vascular resistance and the transmission of the pressure signal into the renal interstitium (62).
Others have suggested that nitric oxide (NO) mediates pressure natriuresis by inhibiting Na+ transport in distal nephron segments (41–44). This hypothesis is supported by the observations that elevations in RPP increase intrarenal levels of NO (40, 42), and inhibitors of NO synthase blunt pressure natriuresis (44). However, other investigators have reported that inhibitors of NO synthase do not block pressure natriuresis but rather shift the entire relationship toward higher pressures (13, 20). The blunting of the pressure-natriuresis response after inhibition of NO synthesis is associated with a fall in renal medullary blood flow (13) and RIHP (20, 43). These results suggest that NO may serve as a modulator rather than a mediator of pressure natriuresis that alters renal medullary vascular resistance and the transmission of pressure into the renal interstitium.
Leong et al. (35) recently suggested that a rapid fall in the intrarenal levels of angiotensin (ANG) II may contribute to the pressure-natriuresis response. They reported that clamping renal ANG II levels by intrarenal infusion blunted the fall in Na+-K+-ATPase activity, the redistribution of NHE-3 protein, and the natriuretic response produced by elevations in RPP.
Yet another mechanism has been suggested by the findings of Zhang et al. (80). They found that CoCl2 attenuates the inhibitory effects of elevations in RPP on proximal tubular reabsorption. CoCl2 induces heme oxygenase that inhibits the activity of a variety of heme-containing proteins, including the cytochrome P-450 enzymes that produce epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) (6). EETs and 20-HETE are produced in the proximal tubule (28, 50) and thick ascending loop of Henle (TALH) (9, 11, 28). They inhibit Na+ transport in these nephron segments (18, 29, 52, 54, 55, 64, 65, 81). Given the similarities between the effects of EETs and 20-HETE on Na+ transport and those produced by elevations in RPP, these observations suggest that EETs and/or 20-HETE may serve as mediators of pressure natriuresis if elevations in RPP and/or RIHP stimulate the formation and/or release of these compounds in the kidney. This hypothesis is also consistent with a large body of evidence indicating that the renal formation of EETs and 20-HETE is altered in hypertension (1, 50, 51, 68, 69) and that chronic induction (1, 71) or blockade (1, 25, 67, 69) of this pathway alters blood pressure in both normotensive and hypertensive animals.
Recently, 1-aminobenzotriazole (ABT) has been reported to inhibit the renal formation of EETs and 20-HETE in rats in vivo (25, 69), so the role of this pathway in pressure natriuresis can now be explored. Thus the present study examined the effects of inhibition of the renal formation of EETs and 20-HETE with ABT on pressure natriuresis and the inhibitory effects of elevations in RPP on Na+-K+-ATPase activity and the distribution of NHE-3 protein in the proximal tubule of rats.
MATERIALS AND METHODS
Experiments were performed on male Sprague-Dawley rats weighing between 300 and 350 g purchased from Taconic Farms (Germantown, NY). The rats were housed in the Animal Care Facility at the Medical College of Wisconsin that is approved by the American Association for the Accreditation of Laboratory Animal Care. The rats had free access to food and water throughout the study except that they were fasted the night before a clearance experiment. All protocols were approved by the Animal Care Committee of the Medical College of Wisconsin.
Effects of ABT on the urinary excretion of 20-HETE.
Experiments were first performed to determine the time course of the effects of ABT (AG Scientific, San Diego, CA) on the urinary excretion of 20-HETE. Rats were housed in stainless-steel metabolic cages, and overnight urine samples were collected on a control day and on days 1–3, 5, 7, and 10 after the rats were given daily intraperitoneal injections of ABT (50 mg/kg) (n = 6) or vehicle (0.9% NaCl solution) (n = 6). The urine samples were collected into glass bottles packed in dry ice. The concentration of 20-HETE in the samples was determined with a fluorescent high-performance liquid chromatography method as previously described (39).
Protocol 1: effects of chronic blockade of cytochrome P-450 metabolism of arachidonic acid on pressure natriuresis.
Experiments were performed on rats chronically treated with daily intraperitoneal injections of vehicle (n = 15) or ABT (n = 8) for 5 days at a dose of 50 mg/kg to block the renal formation of 20-HETE and EETs. On the day of the acute experiment, the rats were anesthetized with an intraperitoneal injection of thiobutabarbitol (100 mg/kg) and prepared for the study of pressure natriuresis as previously described (56). Catheters were inserted in the right femoral vein for intravenous infusions, in the carotid and femoral arteries for monitoring of blood pressure above and below the renal arteries, and in both ureters for the collection of urine. An adjustable clamp was placed on the aorta between the left and right renal arteries so that RPP to the left kidney could be controlled. The rats received an intravenous infusion of a 0.9% NaCl solution containing 2% albumin at a rate of 100 μl/min throughout the experiment. Vasopressin (52 pg/min), aldosterone (20 ng/min), norepinephrine (100 ng/min), and hydrocortisol (20 μg/min) were included in the infusion solution to fix the circulating levels of these hormones as previously described (56). [3H]inulin (2 μCi/ml) and LiCl (20 mM) were added to the infusion solution for measurement of glomerular filtration rate and the fractional excretion of lithium (FELi). After surgery and a 1-h equilibration period, RPP was acutely increased to 150 mmHg by tying off the celiac and mesenteric arteries. The right kidney was exposed to the elevated pressure while RPP to the left kidney was maintained at the control level by tightening the clamp on the aorta between the renal arteries. After a 15-min equilibration period, urine and plasma samples were collected from both kidneys during a 30-min clearance period. At the end of each experiment, a 500-μl sample of blood was collected for measurement of plasma Na+, K+, and Li+ concentrations and plasma renin activity. Both kidneys were removed and rapidly frozen in liquid nitrogen for later measurement of the renal metabolism of arachidonic acid (AA).
Renal metabolism of AA.
Microsomes were prepared from the renal cortex by differential centrifugation as previously described (2, 25, 26). The metabolism of AA to EETs and 20-HETE was determined by incubating the microsomes (500 μg of protein) with [14C]AA (0.1 μCi, 42 μM) for 30 min at 37°C. The products were extracted with ethyl acetate and separated by a C18-reverse-phase high-performance liquid chromatography column and monitored with a radioactive flow detector, as previously described (1, 25, 26). Values are expressed as picomoles formed per minute per milligram of protein.
Protocol 2: role of cytochrome P-450 metabolites of AA in mediating the effects of RPP on Na+-K+-ATPase activity.
Experiments were performed to determine the effects of chronic blockade of the renal formation of EETs and 20-HETE on the changes in renal Na+-K+-ATPase activity associated with elevations in RPP. These experiments were performed on rats that were chronically treated with vehicle (n = 13) or ABT (n = 12). The rats were surgically prepared as described above. After a 30-min equilibration period, RPP was increased to 150 mmHg by tying off the celiac and mesenteric arteries. The right kidney was exposed to the elevated RPP, while RPP to the left kidney was maintained at the control level by adjusting a clamp on the aorta between the renal arteries. After 15 min, both kidneys were removed, and renal cortical basolateral membranes (BLMs) were prepared for measurement of Na+-K+-ATPase activity.
The BLMs were prepared using a Percoll gradient as previously described (12). Briefly, the renal cortex was homogenized in 20 ml of a sucrose buffer containing 0.25 M sucrose, 10 mM Tris·HCl, 2.3 mM Tris-base and 7.5 μl/ml of a protease inhibitor cocktail (cat no. P-8340, Sigma Chemical, St. Louis, MO), pH 7.6. The homogenate was centrifuged at 2,500 g for 15 min to remove tissue chunks and intact cells. The supernatant was collected and centrifuged at 24,000 g for 20 min. The fluffy layer that was suspended above the pellet and contains both apical and BLMs was collected and diluted with 30 ml of sucrose buffer. Percoll (9%) was added to this solution, and the membranes were centrifuged at 30,000 g for 35 min. The pellet containing the enriched BLMs was collected and resuspended in 30 ml of a 100 mM NaCl buffer (pH 7.2), containing 100 mM mannitol, 5 mM HEPES-Tris, 5 mM Tris·HCl, and 5 mM Tris-base. BLMs were repelleted at 34,000 g for 30 min. The pellet was suspended in 0.5 ml of sucrose buffer containing 2.4 mM deoxycholic acid. BLMs were then frozen in liquid nitrogen and stored at −80°C until Na+-K+-ATPase activity was measured. The purity of the preparations was assessed by measuring the enrichment of Na+-K+-ATPase and K+-dependent p-nitrophenyl phosphatase (K+-pNPPase) activity. We also compared the expression of the basolateral enzyme marker Na+-K+-ATPase and the brush border marker γ-glutamyl transpeptidase (γ-GT) in BLMs vs. the levels expressed in renal homogenates.
Measurement of Na+-K+-ATPase activity.
Renal cortical BLMs or homogenates (50 μg of protein) were incubated in 1 ml of solution containing (in mM) 100 NaCl, 20 KCl, 3 MgCl2, 30 imidazole, 1 EDTA, and 5 ATP (pH 7.2) at 37°C for 30 min in the presence and absence of 1 mM ouabain. The reactions were stopped by adding 5% trichloroacetic acid, and the proteins were removed by centrifugation. The free Pi concentration in the supernatant was determined by using a colorimetric assay (catalog no. 670-C, Sigma Chemical). The values were expressed as micromoles Pi released per milligram of protein per hour.
Because ATP can be hydrolyzed by enzymes other than Na+-K+-ATPase, K+-pNPPase activity was also measured. The substrate for this assay, p-nitrophenyl phosphate, has been reported to be more specific for Na+-K+-ATPase than ATP (16). BLMs or cortical homogenates (10 μg of protein) were incubated in 0.6 ml of a 50 mM Tris·HCl buffer, pH 7.8, containing 10 mM MgSO4, 5 mM EDTA, and 90 mM KCl, in the presence or absence of 1 mM ouabain. The reactions were initiated by adding 0.5 mM p-nitrophenyl phosphate. After a 30-min incubation, the reactions were stopped with 0.1 M NaOH. The concentration of p-nitrophenol released in the media was determined by measuring the absorbance at 410 nm. K+-pNPPase activities were expressed as micromole Pi released per milligram of protein per hour.
Protocol 3: effect of ABT and RPP on the distribution of NHE-3 protein in renal cortical membrane fractions.
Additional experiments were performed to determine the effects of chronic blockade of the renal formation of 20-HETE and EETs on the distribution of NHE-3 protein in renal cortical membranes separated on a Percoll density gradient. These experiments were performed on rats chronically treated with vehicle (n = 4) or ABT (n = 4) for 5 days. The kidneys were exposed to either a control (110 mmHg) or an elevated (150 mmHg) RPP for 15 min as described above. The left (control) and right (high pressure) kidneys of four vehicle and four ABT-treated rats were collected, and homogenates were prepared separately. Renal cortical membranes were isolated from these homogenates by differential centrifugation as described above.
Density gradient separation of the cortical membranes.
The renal cortical membranes prepared from the left (control) and right (high pressure) kidneys of four rats treated with vehicle and four rats that received ABT were independently fractioned on Percoll density gradients. The membranes were resuspended in 5 ml of a sucrose buffer and layered on top of 25 ml of a 3–37% Percoll density gradient (density, 1.042–1.152 g/ml) formed in a sucrose buffer. The samples were centrifuged at 30,000 g for 30 min. Fifteen 2-ml fractions were collected from the bottom of the tube. The fractions were diluted with 1 ml of the sucrose buffer, and the membranes were pelleted by centrifugation at 100,000 g for 12 h. The pellets were resuspended in 0.25 ml of the sucrose buffer, and the protein concentration of the fractions was measured using the Bradford method.
The distribution of NHE-3 in the membrane fractions was determined by a Western blot technique prepared from the left and right kidneys of vehicle- and ABT-treated rats. Ten micrograms of protein from the 15 membrane fractions prepared from each kidney were run on 16 different 7.5% polyacrylamide gels. The proteins were separated by electrophoresis at 100 V for 1 h and then transferred to a nitrocellulose membrane. The membranes were blocked overnight at 4°C in a Tris-buffered saline buffer plus Tween-20 (TBST-20 buffer) containing 10 mM Tris·HCl, 150 mM NaCl, 0.08% Tween 20, and 10% nonfat dry milk. The blots were incubated for 1 h with primary antibodies raised against the 131 amino acids on the COOH terminus of the NHE-3 protein (catalog no. MAB3136, Chemicon, Temecula, CA) and γ-GT at dilutions of 1:4,000 and 1:16,000, respectively. The membranes were then washed with TBST-20 buffer and incubated with a 1:8,000 dilution of a horseradish peroxidase-coupled (Santa Cruz Biotech, Santa Cruz, CA) secondary antibody for 1 h. The immunoblots were coated with a chemiluminescence substrate (SuperSignal West Dura, Pierce Chemical, Rockford, IL) exposed to X-ray film, and the films were developed.
The kidneys of additional rats that were chronically treated with vehicle (n = 4) or ABT (n = 3) were exposed to a control (110 mmHg) or elevated (150 mmHg) RPP for 15 min. They were then collected and immersed in a solution containing 2% paraformaldehyde, 75 mM lysine, and 10 mM Na-periodate, pH 7.4 (72). After an overnight fixation, 5-μm-thick cryosections were prepared. The sections were incubated with a primary antibody raised against NHE-3 (catalog no. MAB3136, Chemicon, Temecula, CA) at a dilution of 1:50. The sections were washed and incubated with a FITC-conjugated goat-anti-mouse secondary antibody (Santa Cruz Biotechnology) at a dilution of 1:1,000. The slides were washed with TBS and counterstained with 0.004% Evans blue to stain the cytoplasm and reduce autofluorescence. The sections were viewed with a Nikon fluorescence microscope fitted with a 5-MHz camera (MicroMax, Princeton Instruments, Monmouth Junction, NJ) using a ×40 objective. Overlays of the distribution of the FITC-stained NHE-3 protein (green) with the native images of the cells (red) were created with video imaging software (MetaMorph, Universal Imaging, Downington, PA) to visualize areas of colocalization. The percentage of tubular area that appeared yellow, indicating colocalization of NHE-3 protein in the cytoplasm, was traced and measured in 20–30 proximal tubules per slide.
Means ± SE are presented. The significance of differences in mean values obtained from the kidneys exposed to the control and high level of RPP within the same animal was determined by a paired t-test. Between-group comparisons of corresponding values studied at the same level of RPP were determined by an unpaired t-test. A P < 0.05 was considered to be significant.
Effects of ABT on the renal metabolism of AA and 20-HETE excretion.
The effects of ABT on the urinary excretion of 20-HETE are presented in Fig. 1A. The urinary excretion of 20-HETE first decreased significantly on the third day of treatment in rats given ABT (50 mg·kg−1·day−1 ip). It fell by 60% of control after 5 days of treatment with ABT. Over the next 5 days, the urinary excretion of 20-HETE fell by an additional 20%.
The effects of chronic treatment of the rats with ABT for 5 days on the metabolism of AA by renal cortical microsomes are presented in Fig. 1B. Chronic treatment of rats with ABT reduced the formation of 20-HETE and EETs in renal cortical microsomes by 80 and 60%, respectively.
Protocol 1: pressure natriuresis.
The effect of ABT on the pressure-natriuresis response is presented in Figs. 2 and 3. In control rats, urine flow and sodium excretion increased ninefold as RPP was increased from 114 to 156 mmHg (Fig. 2). Fractional excretion of sodium increased fivefold, and FELi rose from 28 to 42% of the filtered load (Fig. 3). Chronic treatment of the rats with ABT lowered baseline blood pressure by ∼5 mmHg. However, the levels of RPP that the kidneys were exposed to in vehicle- and ABT-treated rats were not significantly different. Chronic treatment of rats with ABT blunted the diuretic and natriuretic responses to elevations in RPP by 75% and reduced the increase in the FELi by 45% (Figs. 2 and 3). There was a small but significant increase in glomerular filtration rate in both ABT- and vehicle-treated rats after RPP was elevated. Baseline plasma renin activities were similar in vehicle- and ABT-treated rats and averaged 5.72 ± 0.42 and 5.91 ± 0.23 ng ANG I·ml−1·h−1, respectively.
Protocol 2: effects of RPP and ABT on Na+-K+-ATPase activity in renal BLM preparations.
Na+-K+-ATPase activity was sixfold higher in BLM preparations than in renal cortical homogenates (27.7 ± 3.4 vs. 4.96 ± 0.6 μmol Pi·mg protein−1·h−1). Similarly, K+-pNPPase activity was 11-fold higher in the BLM preparations than in renal homogenates (11.2 ± 1.3 vs. 1.0 ± 0.1 μmol Pi·mg protein−1·h−1). The expression of the α-subunit of Na+-K+-ATPase (BLM marker) was enriched in BLMs (Fig. 4). In contrast, the expression of the brush-border markers NHE-3 and γ-GT protein was not detectable in BLMs compared with the high level of expression seen in renal cortical homogenates
The effects of chronic treatment of rats with ABT on Na+-K+-ATPase activity in the BLMs isolated from kidneys exposed to a control and high RPP are presented in Fig. 5. In vehicle-treated rats, an elevation in RPP from 110 to 150 mmHg for 15 min reduced ouabain-sensitive Na+-K+-ATPase and K+-pNPPase activities by 40%. Chronic treatment of rats with ABT prevented the fall in Na+-K+-ATPase and K+-pNPPase activities in BLMs isolated from kidney exposed to high pressure.
Protocol 3: effect of ABT and RPP on the distribution of NHE-3 protein in renal cortical membrane fractions.
The effects of ABT on the distribution of NHE-3 protein in renal cortical membranes fractioned on a Percoll density gradient are presented in Fig. 6. Each panel presents a representative Western blot of the distribution of immunoreactive NHE-3 and γ-GT protein expressed in density gradient membrane fractions prepared from the left (control RPP) and the right (high RPP) kidneys of a rat chronically treated with vehicle or ABT for 5 days. In the kidney of the vehicle-treated rat exposed to a control level of RPP, intact NHE-3 protein (molecular weight of 83) was detected in the apical membrane fractions (fractions 7–11) enriched with the brush border marker γ-GT. A small amount of a second immunoreactive band that migrates at a lower molecular weight (i.e., 57 kDa) was also observed. No intact NHE-3 protein was detected in the heavier membrane fractions (fractions 1–6) containing lysosomes and endosomes. However, the smaller molecular weight NHE-3 immunoreactive band was highly expressed in these fractions. We believe this lower band is a degradation product of NHE-3.
In vehicle-treated rats, exposure of the kidney to a high RPP for 15 min greatly reduced the amount of intact NHE-3 protein found in the apical membrane fractions (fractions 7–11) (Fig. 6). In addition, the expression of the lower molecular weight NHE-3 immunoreactive band in the heavy membrane fractions (fractions 1–6) tended to increase (Fig. 6). Chronic treatment of rats with ABT had no effect on the distribution of NHE-3 protein across the membrane fractions obtained from kidneys exposed to a control level of RPP. However, treatment of rats with ABT markedly attenuated the fall in the expression of intact NHE-3 protein in the apical membrane fractions in kidneys exposed to an elevated RPP (Fig. 6). ABT also reduced the increase in the expression of the lower molecular weight NHE-3 immunoreactive band typically seen in heavy membrane fractions (fractions 1–6) after RPP is elevated.
Because it difficult to compare changes in the expression of proteins run on different gels, the apical membrane fractions (fractions 7–11) prepared from the control and high-pressure kidneys of ABT- and vehicle-treated rats were rerun on the same gels to allow for a direct comparison of the effects of elevations in RPP on the expression of NHE-3 protein. A representative Western blot is presented in Fig. 7A, and the summary data obtained from rats treated with vehicle and ABT are presented in Fig. 7B. The expression of NHE-3 protein in the apical membrane fractions fell significantly after RPP was elevated in the vehicle-treated rats. In contrast, the expression of NHE-3 protein was not significantly altered after an elevation in RPP in rats treated with ABT.
The effects of ABT on the distribution of NHE-3 protein in the proximal tubule after an elevation in RPP are presented in Fig. 8. In the kidneys of vehicle-treated rats exposed to a normal RPP, most of the NHE-3 protein (green) was associated with the brush border. After an elevation in RPP, NHE-3 protein was internalized as indicated by the increase in the intensity of the yellow staining (colocalization with red-stained cytoplasm) and there was a decrease in the amount of green staining seen in the apical border of the proximal tubules. The effects of elevations in RPP to promote internalization of NHE-3 protein in the proximal tubule were attenuated in rats treated with ABT.
A comparison of the percentage of tubular area exhibiting colocalization of NHE-3 protein with the cytoplasm (yellow area) is presented in Fig. 9. In vehicle-treated rats, the percentage of tubular area exhibiting colocalization increased significantly after an acute (15 min) elevation in RPP. The amount of NHE-3 protein that was internalized also increased in rats treated with ABT, but the magnitude of the increase was attenuated compared with that seen in vehicle-treated animals.
The present study examined the influence of cytochrome P-450 metabolites of AA on pressure natriuresis and on the inhibitory effects of elevations in RPP on Na+-K+-ATPase activity and distribution of NHE-3 protein in the proximal tubules of rats. Our results confirm previous findings (35, 37, 38, 72–75, 76, 78–80) that acute elevations in RPP inhibit renal Na+-K+-ATPase activity and promote the internalization of NHE-3 protein from the brush border of proximal tubules. The new finding of the present study is that chronic treatment of rats for 5 days with a dose of ABT that blocks the synthesis of 20-HETE and EETs in renal microsomes and reduces the urinary excretion of 20-HETE by >60% blunted the pressure-natriuretic response by 75%. This was associated with a significant fall in the FELi, indicating that ABT attenuates the inhibitory effects of elevations in RPP on the reabsorption of sodium and water in the proximal tubule. Chronic treatment of rats with ABT also prevented the fall in renal Na+-K+-ATPase activity in renal BLM and the redistribution of NHE-3 protein from the brush border to the cytoplasm of the proximal tubule after an elevation in RPP. These results are consistent with previous findings indicating that 20-HETE inhibits Na+-K+-ATPase activity both in the kidney (49, 66) and proximal tubule (52) as well as Na+ transport in proximal tubules of the rabbit perfused in vitro (54). Our results also fit with previous observations that 20-HETE serves as a second messenger that mediates the inhibitory effects of ANG II (65), parathyroid hormone (52, 55), and dopamine (4, 49) on Na+-K+-ATPase activity and Na+ transport in the proximal tubule. Overall, the present results suggest that elevations in RPP, perhaps acting through elevations in RIHP, stimulate the synthesis and/or release of cytochrome P-450 metabolites of AA (EETs or 20-HETE) in the proximal tubule and that one or both of these compounds may decrease Na+ reabsorption by inhibiting Na+-K+-ATPase activity and promoting the internalization of NHE-3 protein from the brush border of these cells.
Although the present study emphasizes the potential role of cytochrome P-450 metabolites of AA in mediating changes in proximal tubular reabsorption after elevations in RPP, it must be emphasized that 20-HETE is produced in the TALH and regulates Na+ transport in this nephron segment. Pressure natriuresis is also associated with inhibition of Na+ reabsorption in the TALH (57). Thus it remains possible that elevations in the synthesis and production of 20-HETE may also contribute to pressure natriuresis by inhibiting Na+ transport in the TALH.
The hypothesis that 20-HETE or EETs might couple elevations in RPP and/or RIHP to inhibition of Na+ transport in the proximal tubule is consistent with the results of previous studies, which indicated that treatment of rats with CoCl2 blunts pressure natriuresis and the internalization of NHE-3 protein from the brush border of the proximal tubule (80). These authors suggested that the heme oxygenase inducer CoCl2 may act by depleting the cellular stores of heme and reducing the formation of cytochrome P-450 metabolites of AA. However, it was not established that treatment of rats with CoCl2 actually reduced the renal formation of EETs or 20-HETE in this previous study (80). Moreover, CoCl2 increases the formation of carbon monoxide that has important effects on Na+ transport and renal hemodynamics (6, 30). Carbon monoxide also inhibits NO synthase (70) and alters the activity of other heme-containing enzymes that may influence sodium transport (6). Thus it remains to be determined whether the effects of CoCl2 on pressure natriuresis is due to inhibition of the renal formation of EETs and/or 20-HETE or one of its many other effects.
Recent studies have suggested that ANG II stimulates the renal formation of EETs (64) and 20-HETE (1, 3, 24, 46) and that 20-HETE mediates some of the inhibitory actions of ANG II (3, 11, 65) and endothelin (24, 46, 47) on tubular transport of Na+. These results may provide a possible link between the synthesis of EETs and 20-HETE and the recent observation (35) that clamping the renal levels of ANG II with captopril and an intrarenal infusion of ANG II blunts pressure natriuresis. We would propose that, under these conditions, the release of 20-HETE and EETs may already be stimulated by the infusion of ANG II, and the levels of these metabolites cannot increase further after an elevation in RPP. This may help explain why elevated intrarenal levels of ANG II blunt pressure natriuresis.
The mechanism by which 20-HETE and/or EETs modulate pressure natriuresis remains uncertain, but the results of the present study suggest that they may act by inhibiting Na+-K+-ATPase and by promoting the internalization of NHE-3 protein from the brush border of the proximal tubule. This conclusion is based on our finding that chronic treatment of rats with ABT blocked the fall in Na+-K+-ATPase activity and internalization of NHE-3 protein from the brush border of proximal tubules after elevations in RPP, as determined by immunohistochemisty. It is also supported by the results of the density gradient experiments, which indicated that the amount of NHE-3 protein associated with apical membrane fractions is reduced after an elevation in RPP in vehicle-treated rats and that this effect is blocked in the rats treated with ABT. Another interesting finding is that the expression of a low molecular weight protein that cross reacts with the NHE-3 antibody increased after elevations in RPP in vehicle-treated rats and that this protein did not increase as much in the kidneys of rats treated with ABT. These findings suggest that NHE-3 may be cleaved to a smaller protein after it is internalized into endosomes and that ABT reduces the formation of this product by preventing the movement of NHE-3 out of the brush border.
The relative importance of EETs vs. 20-HETE in mediating the inhibitory effects of elevations in RPP and Na+ transport in the proximal tubule could not be addressed from the results of the present study because we found that ABT was equally effective in blocking the formation of both compounds. Recently, several more selective inhibitors of the formation of EETs (PPOH, PPOMS) (7) and 20-HETE (DDBB, DDMS, HET0016, HET0225) (45, 48) have been identified. There is also evidence that PPOH selectively reduces the renal synthesis of EETs in rats in vivo (7) and that DDBB lowers the excretion of 20-HETE in rats (48). However, we have found that these compounds are very difficult to use in that they are not very soluble and avidly bind to plasma proteins. Moreover, we have not been able to demonstrate that these drugs selectively block the renal formation of EETs and 20-HETE after in vivo administration to rats. Thus the issue of whether inhibition of the formation of EETs or 20-HETE mediates the effects of ABT on pressure natriuresis will have to remain unresolved until additional work is done to identify better ways to selectively inhibit the renal formation of EETs and/or 20-HETE in vivo.
In theory, either 20-HETE or EETs may contribute to the modulation of the pressure-natriuretic response. 20-HETE has long been known to inhibit Na+-K+-ATPase activity (66). It activates protein kinase C, which phosphorylates a serine-23 residue in the α-subunit of Na+-K+-ATPase (4, 49). 20-HETE has also been shown to inhibit Na+ transport in isolated perfused rabbit proximal tubule (54) and in the TALH of rats both in vivo and in vitro (29, 81). Inhibitors of the formation of 20-HETE attenuate the inhibitory effects of PTH (52, 55), dopamine (52), and ANG II (65) on Na+-K+-ATPase activity and Na+ transport in the proximal tubule and in proximal tubule-derived cell lines. 20-HETE also inhibits Na+ transport in cultured proximal tubule cells (52, 55) and in the TALH (11, 18). Similarly, EETs have been reported to inhibit Na+/H+ exchange in the proximal tubule (64) and to mediate the inhibitory effects of ANG II on proximal tubular Na+ reabsorption. Thus it is possible that elevations in RPP might activate phospholipase A2 and stimulate the formation and/or release of 20-HETE and EETs in the proximal tubule by increasing RIHP. 20-HETE then may contribute to pressure natriuresis by inhibiting both Na+-K+-ATPase activity and Na+/H+ exchange, whereas EETs would be expected to act by inhibiting the Na+/H+ exchanger alone.
The view that 20-HETE and/or EETs contribute to pressure natriuresis is consistent with the large body of evidence indicating that the formation of these compounds is altered in genetic and experimental animal models of hypertension (1, 26, 29, 47, 50, 51, 63, 67–69, 71). It also fits with previous findings that inhibitors of the renal formation of 20-HETE and/or EETs promote the development of salt-sensitive hypertension in normotensive strains of rats (25, 67), that increasing the renal formation of 20-HETE with clofibrate or tempol lowers blood pressure in Dahl S rats (26, 63, 71), and that increasing renal levels of EETs with soluble epoxide hydrolase inhibitors lowers blood pressure in spontaneously hypertensive (77) and ANG II-hypertensive rats (27).
The present results indicate that EETs and/or 20-HETE contribute to pressure natriuresis by inhibiting Na+-K+-ATPase activity and by promoting the movement of NHE-3 protein from the brush border to the cytoplasm of the proximal tubule. These results are consistent with the view that EETs and 20-HETE participate in the long-term control of arterial pressure and that a deficiency in renal formation of EETs and/or 20-HETE promotes the development of some forms of salt-sensitive hypertension in humans and experimental animals.
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-29546 and HL-36279.
The authors thank Neil Wenberg and Glenn Slocum for excellent technical assistance.
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