HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 287/1/R58    most recent 00713.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (14) Citing Articles via Google Scholar Google Scholar Articles by Dos Santos, E. A. Articles by Roman, R. J. Search for Related Content PubMed PubMed Citation Articles by Dos Santos, E. A. Articles by Roman, R. J.

THIRST AND VOLUME, ELECTROLYTE HOMEOSTASIS

Inhibition of the formation of EETs and 20-HETE with 1-aminobenzotriazole attenuates pressure natriuresis

Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Submitted 15 December 2003 ; accepted in final form 11 March 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
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 P_i·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; sodium-potassium-adenosinetriphosphatase; kidney; proximal tubule; renal hemodynamics

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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
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 (FE_Li). 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 -glutamyl transpeptidase (-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 P_i concentration in the supernatant was determined by using a colorimetric assay (catalog no. 670-C, Sigma Chemical). The values were expressed as micromoles P_i 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 P_i 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.

Western blot. 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 -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.

Immunohistochemistry. 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 x40 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.

Statistics. 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.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
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%.

View larger version (19K): [in this window] [in a new window]   Fig. 1. A: effects of daily injections of 1-aminobenzotriazole (ABT; 50 mg· kg–1·day–1 ip) on the urinary excretion of 20-hydroxyeicosatetraenoic acid (20-HETE) in rats. Values are means ± SE from n = 6 rats. 20-HETE levels were measured with a fluorescent high-performance liquid chromatography (HPLC) assay as previously described (37). B: effects of chronic treatment of rats with ABT (50 mg·kg–1·day–1 ip) (n = 18) or vehicle (n = 15) for 5 days on the cytochrome P-450 (CYP)-dependent metabolism of [14C]arachidonic acid (AA) to 20-HETE and epoxyeicosatrienoic acids (EETs) + 12,20-dihydroxyeicosatetraenoic acids (DiHETEs) by renal cortical microsomes in vitro. *P < 0.05 from the corresponding value in the kidneys perfused at a control level of renal perfusion pressure (RPP) within a treatment group. P < 0.05 from the corresponding value measured in rats treated with vehicle.

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 FE_Li 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 FE_Li 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.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Effects of chronic treatment of rats with ABT (50 mg·kg–1·day–1 ip) (n = 8) or vehicle (n = 15) for 5 days on the diuretic and natriuretic responses of urine flow (A), Na+ excretion (B), and glomerular filtration rate (GFR; C) to an elevation in RPP. Values are means ± SE. *P < 0.05 from the corresponding value in the kidneys perfused at a control level of RPP within a treatment group. P < 0.05 from the corresponding value measured in rats treated with vehicle.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Effects of chronic treatment of rats with ABT (50 mg·kg–1·day–1 ip) (n = 8) or vehicle (n = 15) for 5 days on the fractional excretion of sodium (FENa; A) and lithium (FELi; B) after an elevation in RPP. *P < 0.05 from the corresponding value in the kidneys perfused at a control level of RPP within a treatment group. P < 0.05 from the corresponding value measured in rats treated with vehicle.

View larger version (73K): [in this window] [in a new window]   Fig. 4. Comparison of the expression of the -subunit of Na+-K+-ATPase (top), a basolateral membrane (BLM) marker, and the apical membrane markers -glutamyl transpeptidase (-GT; bottom) and sodium/hydrogen exchanger (NHE)-3 (middle) in renal cortical BLM vs. homogenates.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Effects of chronic treatment of rats with ABT (50 mg·kg–1·day–1 ip) or vehicle for 5 days on the changes in Na+-K+-ATPase (vehicle, n = 13; ABT, n = 12; A) and K+-dependent p-nitrophenyl phosphatase (K-pNPPase; vehicle, n = 12; ABT, n = 24; B) activities in renal cortical BLM produced by elevations in RPP. *P < 0.05 from the corresponding value in the kidneys perfused at a control level of RPP within a treatment group. P < 0.05 from the corresponding value measured in rats treated with vehicle.

View larger version (92K): [in this window] [in a new window]   Fig. 6. Effect of chronic treatment of rats with the CYP inhibitor ABT (50 mg·kg–1·day–1 ip) or vehicle for 5 days on the distribution of immunoreactive NHE-3 and -GT proteins in renal cortical membrane fractions. Each panel presents a representative Western blot of 15 sequential membrane fractions prepared from the left (control RPP) and right (high RPP) kidneys of a rat treated with vehicle or ABT. The 2-ml fractions (fractions 1–15) were sequentially collected from the bottom of a 30-ml Percoll gradient, with 1 representing the heaviest fraction (density 1.152) and 15 representing the lightest fraction (density 1.042). Ten micrograms of protein were loaded in each lane on the gels.

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.

View larger version (28K): [in this window] [in a new window]   Fig. 7. A: representative Western blot comparing the expression of NHE-3 protein in apical membrane fractions prepared from left and right kidneys exposed to a control and high renal perfusion of a single rat treated with vehicle (left) and another rat treated with ABT (right). In these experiments, the apical membrane fractions prepared from the kidneys exposed to a control and elevated RPP from individual rats were prepared and run in the same gel to allow for a more direct comparison of the effects of an elevation in RPP on the expression of NHE-3 protein in the apical membrane. B: summary of the results obtained from rats treated with ABT and vehicle. *P < 0.05 from the corresponding value in the kidneys perfused at a control level of RPP within a treatment group.

View larger version (61K): [in this window] [in a new window]   Fig. 8. Immunohistochemical localization of NHE-3 protein in the proximal tubule of kidneys exposed to a control or high level of RPP in rats chronically treated with ABT (50 mg·kg–1·day–1 ip) or vehicle for 5 days. The sections were immunostained for NHE-3 (green) using a NHE-3 primary antibody and FITC secondary antibody. The cytoplasm of the cells were counterstained with a 0.004% Evans blue, which is visible using a Texas red fluorescence filter set. Areas of colocalization of NHE-3 and the cytoplasm are indicated in yellow.

View larger version (13K): [in this window] [in a new window]   Fig. 9. Percentage of tubular area stained in yellow, indicating areas in which NHE-3 protein (green) is colocalized with the cytoplasm (stained in red) in the proximal tubule of kidneys exposed to a control or high level of RPP for 15 min in rats chronically treated with ABT (50 mg·kg–1·day–1 ip) or vehicle for 5 days. Numbers in parenthesis indicate the number of proximal tubules scored in sections prepared from the control and high-pressure kidneys of 4 rats treated with vehicle and 3 rats treated with ABT. *P < 0.05 from corresponding value in the kidney perfused at the control RPP. P < 0.05 from the corresponding value measured in rats treated with vehicle.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

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 -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).

Perspectives

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.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-29546 and HL-36279.

	ACKNOWLEDGMENTS

 
The authors thank Neil Wenberg and Glenn Slocum for excellent technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. J. Roman, Medical College of Wisconsin, Dept. of Physiology, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: rroman{at}mcw.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Alonso-Galicia M, Frohlich B, and Roman RJ. Induction of P4504A activity improves pressure-natriuresis in Dahl S rats. Hypertension 3: 232–236, 1998.
2. Alonso-Galicia M, Maier KG, Greene AS, Cowley AW Jr, and Roman RJ. Role of 20-hydroxyeicosatetraenoic acid in the renal and vasoconstrictor actions of angiotensin II. Am J Physiol Regul Integr Comp Physiol 283: R60–R68, 2002.[Abstract/Free Full Text]
3. Amlal H, LeGoff C, Vernimmen C, Soleimani M, Paillard M, and Bichara M. Ang II controls Na⁺-K⁺-(NH₄)-2Cl^– cotransport via 20-HETE and PKC in the thick ascending limb. Am J Physiol Cell Physiol 274: C1047–C1056, 1998.[Abstract/Free Full Text]
4. Aperia A, Eklöf AC, Holtbäck U, Nowicki S, Sundelöf M, and Greengard P. The renal dopamine system. Adv Pharmacol 42: 870–873, 1998.[Medline]
5. Bonvalet JP, Pradelles P, and Farman N. Segmental synthesis and actions of prostaglandins along the nephron. Am J Physiol Renal Fluid Electrolyte Physiol 253: F377–F387, 1987.[Abstract/Free Full Text]
6. Botros FT, Laniado-Schwartzman M, and Abraham NG. Regulation of cyclooxygenase- and cytochrome P450-derived eicosanoids by heme oxygenase in the rat kidney. Hypertension 39: 639–644, 2002.[Abstract/Free Full Text]
7. Brand-Schieber E, Falck JF, and Schwartzman ML. Selective inhibition of arachidonic acid epoxidation in vivo. J Physiol Pharmacol 51: 655–672, 2000.[ISI][Medline]
8. Carmines PK, Bell PD, Work J, and Navar LG. Prostaglandins in the sodium excretory response to altered renal arterial pressure in dogs. Am J Physiol Renal Fluid Electrolyte Physiol 248: F8–F14, 1985.[Abstract/Free Full Text]
9. Carroll MA, Sala A, Dunn CE, McGiff JC, and Murphy RC. Structural identification of cytochrome P-450-dependent arachidonate metabolites formed by rabbit medullary thick ascending limb cells. J Biol Chem 266: 12306–12312, 1991.[Abstract/Free Full Text]
10. Croft KD, McGiff JC, Sanchez-Mendoza A, and Carroll MA. Angiotensin II releases 20-HETE from rat renal microvessels. Am J Physiol Renal Physiol 279: F544–F551, 2000.[Abstract/Free Full Text]
11. Escalante B, Erlij D, Falck JR, and McGiff JC. Effect of cytochrome P-450 arachidonate metabolites on ion transport in rabbit kidney loop of Henle. Science 251: 799–802, 1991.[Abstract/Free Full Text]
12. Felder CC, McKelvey AM, Gitler MS, Eisner GM, and Jose PA. Dopamine receptor subtypes in renal brush border and basolateral membranes. Kidney Int 36: 183–93, 1989.[ISI][Medline]
13. Fenoy FJ, Ferrer P, Carbonell L, and Garcia-Salom M. Role of NO on papillary blood flow and pressure-natriuresis. Hypertension 25: 408–414, 1995.[Abstract/Free Full Text]
14. Garcia NH, Ramsey CR, and Knox FG. Understanding the role of paracellular transport in the proximal tubule. News Physiol Sci 13: 38–43, 1998.[Abstract/Free Full Text]
15. Garcia-Estan J and Roman RJ. Role of renal interstitial pressure in the pressure diuresis response. Am J Physiol Renal Fluid Electrolyte Physiol 256: F63–F70, 1989.[Abstract/Free Full Text]
16. Garrahan PJ, Pouchan MI, and Rega AF. Potassium activated phosphatase from human red blood cells. The mechanism of potassium activation. J Physiol 202: 305–327, 1969.[Abstract/Free Full Text]
17. Gonzalez-Campoy JM, Long C, Roberts D, Berndt TJ, Romero JC, and Knox FG. Renal interstitial hydrostatic pressure and PGE₂ in pressure natriuresis. Am J Physiol Renal Fluid Electrolyte Physiol 260: F643–F649, 1991.[Abstract/Free Full Text]
18. Good DW, George T, and Wang DH. Angiotensin II inhibits HCO₃^– absorption via a cytochrome P-450-dependent pathway in MTAL. Am J Physiol Renal Physiol 276: F726–F736, 1999.[Abstract/Free Full Text]
19. Granger JP. Pressure natriuresis: role of renal interstitial hydrostatic pressure. Hypertension 19: I9–I17, 1992.[ISI][Medline]
20. Guarasci GR and Kline RL. Pressure natriuresis following acute and chronic inhibition of nitric oxide synthesis in rats. Am J Physiol Regul Integr Comp Physiol 270: R469–R478, 1996.[Abstract/Free Full Text]
21. Guyton AC, Coleman TG, Cowley AW Jr, Scheel KW, Manning RD Jr, and Norman RA Jr. Arterial pressure regulation. Overriding dominance of the kidneys in long-term regulation and in hypertension. Am J Med 52: 584–594, 1972.[CrossRef][ISI][Medline]
22. Haas JA, Granger JP, and Knox FG. Effect of renal perfusion pressure on sodium reabsorption from proximal tubules of superficial and deep nephrons. Am J Physiol Renal Fluid Electrolyte Physiol 250: F425–F429, 1986.[Abstract/Free Full Text]
23. Haas JA and Knox FG. Effect of meclofenamate or ketoconazole on the natriuretic response to increased pressure. J Lab Clin Med 128: 202–207, 1996.[CrossRef][ISI][Medline]
24. Hercule HC and Oyekan AO. Role of NO and cytochrome P-450-derived eicosanoids in ET-1-induced changes in intrarenal hemodynamics in rats. Am J Physiol Regul Integr Comp Physiol 279: R2132–R2141, 2000.[Abstract/Free Full Text]
25. Hoagland KM, Flasch AK, and Roman RJ. Inhibitors of 20-HETE formation promote salt-sensitive hypertension in rats. Hypertension 42: 669–673, 2003.[Abstract/Free Full Text]
26. Hoagland KM, Maier KG, and Roman RJ. Contribution of 20-HETE to the antihypertensive effects of Tempol in Dahl salt-sensitive rats. Hypertension 41: 697–702, 2003.[Abstract/Free Full Text]
27. Imig JD, Zhao X, Capdevila JH, Morisseau C, and Hammock BD. Soluble epoxide hydrolase inhibition lowers arterial blood pressure in angiotensin II hypertension. Hypertension 39: 690–694, 2002.[Abstract/Free Full Text]
28. Ito O, Alonso-Galicia M, Hopp KA, and Roman RJ. Localization of cytochrome P-450 4A isoforms along the rat nephron. Am J Physiol Renal Physiol 274: F395–F404, 1998.[Abstract/Free Full Text]
29. Ito O and Roman RJ. Role of 20-HETE in elevating chloride transport in the thick ascending limb of Dahl SS/Jr rats. Hypertension 33: 419–423, 1999.[Abstract/Free Full Text]
30. Kaide JI, Zhang F, Wei Y, Jiang H, Yu C, Wang WH, Balazy M, Abraham NG, and Nasjletti A. Carbon monoxide of vascular origin attenuate the sensitivity of renal arterial vessels to vasoconstrictors. J Clin Invest 107: 1163–1171, 2001.[ISI][Medline]
31. Khraibi AA and Knox FG. Effect of renal decapsulation on renal interstitial hydrostatic pressure and natriuresis. Am J Physiol Regul Integr Comp Physiol 257: R44–R48, 1989.[Abstract/Free Full Text]
32. Kinoshita Y and Knox FG. Role of prostaglandins in proximal tubule sodium reabsorption: response to elevated renal interstitial hydrostatic pressure. Circ Res 64: 1013–1018, 1989.[Abstract/Free Full Text]
33. Kinoshita Y and Knox FG. Response of superficial proximal convoluted tubule to decreased and increased renal perfusion pressure. In vivo microperfusion study in rats. Circ Res 66: 1184–1189, 1990.[Abstract/Free Full Text]
34. Kunau RT and Lameire NH. The effect of an acute increase in renal perfusion pressure on sodium transport in the rat kidney. Circ Res 39: 689–695, 1976.[Abstract/Free Full Text]
35. Leong PK, Yang LE, Holstein-Rathlou NH, and McDonough AA. Angiotensin II clamp prevents the second step in renal apical NHE3 internalization during acute hypertension. Am J Physiol Renal Physiol 283: F1142–F1150, 2002.[Abstract/Free Full Text]
36. Liang M, Ramsey CR, and Knox FG. The paracellular permeability of opossum kidney cells, a proximal tubule cell line. Kidney Int 56: 2304–2308, 1999.[CrossRef][ISI][Medline]
37. Magyar CE, Zhang Y, Holstein-Rathlou NH, and McDonough AA. Proximal tubule Na transporter responses are the same during acute and chronic hypertension. Am J Physiol Renal Physiol 279: F358–F369, 2000.[Abstract/Free Full Text]
38. Magyar CE, Zhang Y, Holstein-Rathlou NH, and McDonough AA. Downstream shift in sodium pump activity along the nephron during acute hypertension. J Am Soc Nephrol 12: 2231–2240, 2001.[Abstract/Free Full Text]
39. Maier KG, Henderson L, Narayanan J, Alonso-Galicia M, Falck JR, and Roman RJ. Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid. Am J Physiol Heart Circ Physiol 279: H863–H871, 2000.[Abstract/Free Full Text]
40. Majid DS, Godfrey M, Grisham MB, and Navar LG. Relation between pressure natriuresis and urinary excretion of nitrate/nitrite in anesthetized dogs. Hypertension 25: 860–865, 1995.[Abstract/Free Full Text]
41. Majid DS and Navar LG. Nitric oxide in the mediation of pressure-natriuresis. Clin Exp Pharmacol Physiol 24: 595–599, 1997.[ISI][Medline]
42. Majid DS, Omoro SA, Chin SY, and Navar LG. Intrarenal nitric oxide activity and pressure natriuresis in anesthetized dogs. Hypertension 32: 266–271, 1998.[Abstract/Free Full Text]
43. Majid DS, Said KE, Omoro SA, and Navar LG. Nitric oxide dependency of arterial pressure-induced changes in renal interstititial hydrostatic pressure in dogs. Circ Res 88: 347–351, 2001.[Abstract/Free Full Text]
44. Majid DS, Williams A, and Navar LG. Inhibition of nitric oxide synthesis attenuate pressure-induced natriuretic responses in anesthetized dogs. Am J Physiol Renal Fluid Electrolyte Physiol 264: F79–F87, 1993.[Abstract/Free Full Text]
45. Miyata N, Taniguchi K, Seki T, Ishimoto T, Sato-Wantanabe M, Yasuda Y, Doi M, Kametani S, Tamishima Y, Ueki T, Sato M, and Kameo K. HET0016, a potent and selective inhibitor of 20-HETE synthesizing enzyme. Br J Pharmacol 133: 325–329, 2001.[CrossRef][ISI][Medline]
46. Oyekan A, Balazy M, and McGiff JC. Renal oxygenases: differential contribution to vasoconstriction induced by ET-1 and ANG II. Am J Physiol Regul Integr Comp Physiol 273: R293–R300, 1997.[Abstract/Free Full Text]
47. Oyekan AO, McAward K, Conetta J, Rosenfeld L, and McGiff JC. Endothelin-1 and CYP450 arachidonate metabolites interact to promote tissue injury in DOCA-salt hypertension. Am J Physiol Regul Integr Comp Physiol 276: R766–R775, 1999.[Abstract/Free Full Text]
48. Oyekan AO and McGiff JC. Cytochrome P-450 derived eicosanoids participate in the renal functional effecs of ET-1 in the anesthetized rat. Am J Physiol Regul Integr Comp Physiol 274: R52–R61, 1998.[Abstract/Free Full Text]
49. Nowicki S, Chen SL, Aizman O, Cheng XJ, Li D, Nowicki C, Nairn A, Greengard P, and Aperia A. 20-Hydroxyeicosatetraenoic acid (20 HETE) activates protein kinase C. Role in regulation of rat renal Na⁺,K⁺-ATPase. J Clin Invest 99: 1224–1230, 1997.[ISI][Medline]
50. Omata K, Abraham NG, and Schwartzman ML. Renal cytochrome P-450 arachidonic acid metabolism: localization and hormonal regulation in SHR. Am J Physiol Renal Fluid Electrolyte Physiol 262: F591–F599, 1992.[Abstract/Free Full Text]
51. Omata K, Tsutsumi E, Sheu HL, Utsumi Y, and Abe K. Effect of aging on renal cytochrome P450-dependent arachidonic acid metabolism in Dahl rats. J Lipid Mediators 6: 369–373, 1993.[ISI][Medline]
52. Ominato M, Satoh T, and Katz AI. Regulation of Na-K-ATPase activity in the proximal tubule: role of the protein kinase C pathway and of eicosanoids. J Membr Biol 152: 235–243, 1996.[CrossRef][ISI][Medline]
53. Pawlowska D, Haas JA, Granger JP, Romero JC, and Knox FG. Prostaglandin blockade blunts the natriuresis of elevated renal interstitial hydrostatic pressure. Am J Physiol Renal Fluid Electrolyte Physiol 254: F507–F511, 1988.[Abstract/Free Full Text]
54. Quigley R, Baum M, Reddy KM, Griener JC, and Falck JR. Effects of 20-HETE and 19(S)-HETE on rabbit proximal straight tubule volume transport. Am J Physiol Renal Physiol 278: F949–F953, 2000.[Abstract/Free Full Text]
55. Ribeiro CM, Dubay GR, Falck JR, and Mandel LJ. Parathyroid hormone inhibits Na⁺-K⁺-ATPase through a cytochrome P-450 pathway. Am J Physiol Renal Fluid Electrolyte Physiol 266: F497–F505, 1994.[Abstract/Free Full Text]
56. Roman RJ and Cowley AW Jr. Characterization of a new model for the study of pressure-natriuresis in the rat. Am J Physiol Renal Fluid Electrolyte Physiol 248: F190–F198, 1985.[Abstract/Free Full Text]
57. Roman RJ. Pressure-diuresis in volume-expanded rats: tubular reabsorption in superficial and deep nephrons. Hypertension 12: 177–183, 1988.[Abstract/Free Full Text]
58. Roman RJ. P450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev 82: 131–185, 2002.[Abstract/Free Full Text]
59. Roman RJ, Cowley AW Jr, Garcia-Estan J, and Lombard JH. Pressure-diuresis in volume-expanded rats: cortical and medullary hemodynamics. Hypertension 12: 168–176, 1988.[Abstract/Free Full Text]
60. Roman RJ and Kaldunski ML. Renal cortical and papillary blood flow in spontaneously hypertensive rats. Hypertension 11: 657–663, 1988.[Abstract/Free Full Text]
61. Roman RJ and Kaldunski ML. Pressure-natriuresis and cortical and papillary blood flow in inbred Dahl rats. Am J Physiol Regul Integr Comp Physiol 261: R595–R602, 1991.[Abstract/Free Full Text]
62. Roman RJ and Lianos E. Influence of prostaglandins on papillary blood flow and pressure-natriuretic response. Hypertension 15: 29–35, 1990.[Abstract/Free Full Text]
63. Roman RJ, Ma YH, Frohlich B, and Markham B. Clofibrate prevents the development of hypertension in Dahl salt-sensitive rats. Hypertension 21: 985–988, 1993.[Abstract/Free Full Text]
64. Romero MF, Madhun ZT, Hopfer U, and Douglas JC. An epoxygenase metabolite of arachidonic acid 5,6 epoxyeicosatrienoic acid mediates angiotensin-induced natriuresis in proximal tubular epithelium. Adv Prostaglandin Thromboxane Leukot Res 21: 205–208, 1991.
65. Sanchez-Mendoza A, Lopez-Sanchez P, Vazquez-Cruz B, Rios A, Martinez-Ayala S, and Escalante B. Angiotensin II modulates ion transport in rat proximal tubules through CYP metabolites. Biochem Biophys Res Commun 272: 423–430, 2000.[CrossRef][ISI][Medline]
66. Schwartzman M, Ferreri NR, Carroll MA, Songu-Mize E, and McGiff JC. Renal cytochrome P-450-related arachidonate metabolite inhibits (Na⁺-K⁺)-ATPase. Nature 314: 620–622, 1985.[CrossRef][Medline]
67. Stec DE, Mattson DL, and Roman RJ. Inhibition of renal outer medullary 20-HETE production produces hypertension in Lewis rats. Hypertension 29: 315–319, 1997.[Abstract/Free Full Text]
68. Stec DE, Trolliet MR, Krieger JE, Jacob HJ, and Roman RJ. Renal cytochrome P4504A activity and salt sensitivity in spontaneously hypertensive rats. Hypertension 27: 1329–1336, 1996.[Abstract/Free Full Text]
69. Su P, Kaushal KM, and Kroetz DL. Inhibition of renal arachidonic acid omega-hydroxylase activity with ABT reduces blood pressure in the SHR. Am J Physiol Regul Integr Comp Physiol 275: R426–R438, 1998.[Abstract/Free Full Text]
70. Thorup C, Jones CL, Gross SS, Moore LC, and Goligorsky MS. Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS. Am J Physiol Renal Physiol 277: F882–F889, 1999.[Abstract/Free Full Text]
71. Wilson TW, Alonso-Galicia M, and Roman RJ. Effects of lipid-lowering agents in the Dahl salt-sensitive rat. Hypertension 31: 225–231, 1998.[Abstract/Free Full Text]
72. Yang L, Leong PK, Chen JO, Patel N, Hamm-Alvarez SF, and McDonough AA. Acute hypertension provokes internalization of proximal tubule NHE3 without inhibition of transport activity. Am J Physiol Renal Physiol 282: F730–F740, 2002.[Abstract/Free Full Text]
73. Yang LE, Leong PK, Ye S, Campese VM, and McDonough AA. Responses of proximal tubule sodium transporters to acute injury-induced hypertension. Am J Physiol Renal Physiol 284: F313–F322, 2003.[Abstract/Free Full Text]
74. Yang LE, Zhong H, Leong PK, Perianayagam A, Campese VM, and McDonough AA. Chronic renal injury-induced hypertension alters renal NHE3 distribution and abundance. Am J Physiol Renal Physiol 284: F1056–F1065, 2003.[Abstract/Free Full Text]
75. Yip KP, Tse CM, McDonough AA, and Marsh DJ. Redistribution of Na⁺/H⁺ exchanger isoform NHE3 in proximal tubules induced by acute and chronic hypertension. Am J Physiol Renal Physiol 275: F565–F575, 1998.[Abstract/Free Full Text]
76. Yip KP, Wagner AJ, and Marsh DJ. Detection of api