Abstract In vivo tubular perfusion experiments were performed in normotensive Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats maintained from birth on a low salt (0.4% NaCl) diet to examine the role of 20-HETE in elevating loop Cl− transport in SS/Jr rats. Chloride reabsorption in the loop of Henle was significantly greater in SS/Jr than in SR/Jr rats (77±2% versus 57±3% of the perfused Cl− load). When the renal metabolism of arachidonic acid by P450 was blocked by the addition of 17-octadecynoic acid (10 μmol/L) to the perfusate, loop Cl− transport increased in SR/Jr rats to 70±2% of the delivered Cl− load, but it had no effect in SS/Jr rats. Conversely, addition of 20-HETE (10 μmol/L) to the perfusate lowered loop Cl− transport in S rats to 60±2% of perfused Cl− load, but it had no effect in SR/Jr rats. Addition of another endogenously formed HETE to the perfusate, 15-HETE (20 μmol/L), had no effect on Cl− reabsorption in the loop of Henle of SS/Jr rats. These findings indicate that endogenously produced P450 metabolites of arachidonic acid regulate Cl− transport in the loop of Henle of the rat in vivo and support the view that a diminished production of 20-HETE in the outer medulla of SS/Jr rats contributes to the elevation in loop Cl− transport and the resetting of the pressure-natriuresis relation in these animals.
- cytochrome P450
- cytochrome P450, inhibitors
- hydroeicosatetraenoic acid
- loop of Henle
- hypertension, renal
Renal transplantation studies indicate that some form of renal dysfunction underlies the development of hypertension in human1 and genetic rat2 3 4 models; however, the factors responsible for altering kidney function in hypertension have not been identified. Previous studies indicated that the pressure-natriuresis relation is reset toward higher pressures before the development of hypertension in Dahl salt-sensitive (SS/Jr) rats5 6 7 and that this is associated with an elevation in Cl− reabsorption in the loop of Henle.7 8 9 More recently, we found that the production of a P450 metabolite of AA, 20-HETE, is reduced in the outer medulla of the kidney of SS/Jr rats.10 Since 20-HETE is an endogenously produced inhibitor of the Na+, K+, 2 Cl− transporter in rabbit thick ascending loop of Henle cells,11 12 13 a deficiency in the renal production of 20-HETE might contribute to the elevation in loop Cl− transport and the development of salt-sensitive hypertension in SS/Jr rats.10 This hypothesis is further supported by our recent observations that induction of the renal formation of 20-HETE with clofibrate prevents the development of hypertension in SS/Jr rats14 and that the P4504A2 genotype cosegregates with hypertension in an F2 population of rats derived from a cross of SS/Jr and Lewis rats.15 However, it remains to be established whether endogenously produced P450 metabolites of AA influence Cl− transport in the loop of Henle of the rat in vivo and whether this system contributes to the elevation in loop Cl− transport previously reported in SS/Jr rats.5 6 7 The purpose of the present study was to compare the effects of 20-HETE and blockade of the endogenous production of this compound with 17-ODYA on loop Cl− reabsorption in SS/Jr and SR/Jr rats using in vivo microperfusion of the loop of Henle.
Experiments were performed on 9-week-old, male, inbred SS/Jr and SR/Jr rats weighing between 200 and 250 g. The SR/Jr rats were purchased from Harlan Laboratories, Madison, Wis, and the SS/Jr rats were obtained from a breeding colony that we have maintained by brother-sister mating since 1991, which is before any evidence of genetic contamination of the commercial colony.16 Moreover, eight of the rats used in this study were genotyped by polymerase chain reaction for 40 markers scattered throughout the genome, and no evidence of genetic contamination was detected.
The rats were fed a low salt diet (0.4% NaCl by weight) from the time they were weaned at 3 weeks of age, and they were housed in an animal care facility at the Medical College of Wisconsin approved by the American Association for the Accreditation of Laboratory Animal Care. All protocols involving animals received careful review and prior approval by the Animal Care Committee of the Medical College of Wisconsin.
The rats were anesthetized with ketamine (30 mg/kg IM) and thiobutylbarbitol (30 mg/kg IP) and placed on a thermostatically controlled warming table to maintain body temperature at 37°C. Cannulas were placed in the right external jugular vein for intravenous infusions and in the right femoral artery for measurement of arterial pressure. An abdominal incision was made to expose the left kidney for micropuncture, and both ureters were cannulated for the collection of urine. After surgery, the rats were given an intravenous bolus of 1 to 2 mL 0.9% NaCl solution containing 6% albumin to replace surgical fluid losses and to return hematocrit to 45%. Thereafter, the rats received an intravenous infusion of 3% bovine serum albumin in a 0.9% NaCl solution at a rate of 20 μL · min−1 · 100 g body wt−1 throughout the experiment.
Loop Microperfusion Study
These experiments were performed on 17 SS/Jr and 16 SR/Jr rats surgically prepared as described above. After surgery and a 60-minute equilibration period, late proximal convoluted tubules along with their associated early distal tubule were identified by injection of a small bolus of a 5% solution of lissamine green into the tubule. Selected tubules were then blocked with bone wax by use of a glass micropipette and a hydraulic microdrive. A pipette connected to a nanoliter pump (model A1400, World Precision Instruments) was inserted downstream of the wax block to perfuse the loop of Henle with either a control or an experimental perfusate at a rate of 20 nL/min. The control perfusate contained (in mmol/L) NaCl 145, KCl 5, MgSO4 1, CaCl2 1, and urea 5, 100 μCi/mL [3H]inulin, and vehicle (0.02% ethanol). Experimental perfusates contained a suicide-substrate inhibitor of the renal metabolism of AA by P450, 17-ODYA (10 μmol/L),17 18 20-HETE (10 μmol/L), or another HETE formed in the outer medulla of the kidney, 15-HETE (20 μmol/L).10 After the tubule was perfused for 15 minutes with either a control or an experimental perfusate, a collection pipette was placed in the distal tubule and a timed tubular fluid collection was obtained for measurement of sample volume, tubular fluid inulin, and chloride concentrations. The concentration of 17-ODYA used in these experiments was based on our previous findings that 17-ODYA blocks the formation of 20-HETE in renal microsomes and microvessels at concentrations >1 μmol/L.17 18 In perfused tubule studies, however, we found that a higher concentration of 17-ODYA (10 μmol/L) had to be added to tubular fluid to block tubuloglomerular feedback responses that are dependent on the endogenous formation of 20-HETE.19 The reason for this is at present unknown, but we suspect that it may be because 17-ODYA is highly lipid soluble and a large portion of the infused compound is resorbed before it can diffuse into and achieve an effective inhibitory concentration (approximately 1 μmol/L) in thick ascending limb cells. Similarly, 20-HETE is also extensively resorbed when added to tubular fluid,19 and it also has to be added in a relatively high concentration to tubular perfusates to raise intracellular levels of 20-HETE and elicit functional responses in thick ascending limb cells in vivo. In this regard, the concentration of 20-HETE used in the present study was based on our previous finding that 10 μmol/L 20-HETE is required to restore tubuloglomerular feedback responses when added to the fluid perfusing the loop of Henle after inhibition of this response with 17-ODYA.19
Additional studies were performed in 8 SS/Jr rats and 10 SR/Jr rats to compare baseline renal function under the present experimental conditions. These rats were surgically prepared as described above except that [3H]inulin (1 μCi/mL) was added to the intravenous infusion solution for measurement of GFR, and an electromagnetic flow probe (2 mm) was placed around the left renal artery for measurement of RBF. After surgery and a 1-hour equilibration period, urine flow, hematocrit, RBF, GFR, mean arterial pressure, and the urinary excretion of sodium, potassium, and chloride were measured during two 30-minute clearance periods.
The volume of the tubular fluid samples was determined by measurement of sample length in 1-μL capillary tubes. The concentration of [3H]inulin in tubular fluid and plasma samples was determined by use of a liquid scintillation spectrophotometer. Tubular fluid chloride concentration was measured with a microtitrator (model F-25, World Precision Instruments). The percentage of the perfused load of chloride resorbed in the loop of Henle was calculated as described previously.20
In the clearance studies, urine flow was determined gravimetrically. Sodium and potassium concentrations of urine and plasma samples were measured with a flame photometer, and chloride concentration was measured columetrically. GFR was calculated as the product of urine flow and the ratio of urine to plasma inulin concentration. Urinary excretion data, RBF, and GFR were all factored per gram of kidney weight.
Data are presented as mean±SEM. The significance of differences within and between groups was evaluated by two-way ANOVA factored for rat strain and treatment followed by a Duncan’s multiple range test as previously suggested by Raman et al21 to deal with the potential problem of skewing of the data due to an unequal number of tubules sampled per rat. A value of P<.05 by two-tailed test was considered significant.
Basal Renal Function
A comparison of baseline renal function in SS/Jr and SR/Jr rats is presented in the Table⇓. Mean arterial blood pressures were not different in 9-week-old SS/Jr and SR/Jr rats maintained on a low salt (0.4% NaCl) diet. RBF and GFR were about 25% lower in SS/Jr rats than in SR/Jr rats. Urine flow, sodium, potassium, and chloride excretion were all significantly lower in SS/Jr rats than in SR/Jr rats, and this was associated with lower fractional excretions of Na+ and Cl− in the SS/Jr rats compared with the values observed in SR/Jr rats.
Loop Microperfusion Studies
The effects of inhibiting the endogenous P450 metabolism of AA with 17-ODYA on loop Cl− reabsorption are presented in Fig 1⇓. Basal reabsorption of water was similar in SS/Jr and SR/Jr rats when the loop of Henle was perfused with a control solution containing vehicle alone; however, loop Cl− reabsorption was significantly greater in SS/Jr than in SR/Jr rats. The percentage of the perfused Cl− load reabsorbed in the loop of Henle averaged 77±2% in SS/Jr rats (n=27 nephrons in 17 rats) and only 57±3% of the perfused Cl− load in SR/Jr rats (n=29 nephrons in 16 rats). Addition of 17-ODYA (10 μmol/L) to the perfusate had no effect on water reabsorption in the loop of Henle in either group (Fig 1⇓, top). Loop Cl− reabsorption, however, increased to 70±2% (n=19 nephrons in 9 rats) after 17-ODYA in SR/Jr rats, but it had no significant effect on Cl− reabsorption in SS/Jr rats (n=13 nephrons in 7 rats) (Fig 1⇓, bottom).
The effects of 20-HETE on water and Cl− reabsorption in SS/Jr and SR/Jr rats are presented in Fig 2⇓. Addition of 20-HETE to the fluid perfusing the loop of Henle had no effect on water reabsorption in either group (Fig 2⇓, top). However, 20-HETE (10 μmol/L) significantly reduced the percentage of the perfused load of Cl− reabsorbed in the loop of Henle from 77±2% to 60±2% in SS/Jr rats, but it had no effect on loop Cl− reabsorption in SR/Jr rats (Fig 2⇓, bottom). In 3 additional SS/Jr rats, the effects of adding another endogenously formed HETE to the perfusate on loop Cl− transport were examined in 8 nephrons. In these experiments, the addition of 15-HETE (20 μmol/L) to the perfusate had no effect on water or chloride reabsorption in the loop of Henle. In tubules perfused with 15-HETE, the percentage of water load reabsorbed in the loop of Henle averaged 62±2% and the percentage of the perfused Cl− reabsorbed averaged 82±2% (n=8 nephrons in 3 rats). These values are not different from control values observed in tubules perfused with vehicle alone in SS/Jr rats.
The present study examined the role of 20-HETE on the regulation of Cl− transport in the loop of Henle of SS/Jr and SR/Jr rats and the possible contribution of an abnormality in the renal production of this compound to the elevation of loop Cl− transport previously documented in SS/Jr rats.7 8 9 The loop microperfusion results again confirm previous reports that Cl− reabsorption in the loop of Henle is markedly elevated in SS/Jr rats compared with SR/Jr rats.7 8 9 The clearance data are also consistent with this view. Urine flow and sodium and chloride excretion were markedly reduced in 9-week-old normotensive SS/Jr rats maintained on a low salt diet compared with the corresponding values obtained in SR/Jr rats. The retention of water and electrolytes in SS/Jr rats was largely due to elevations in tubular reabsorption, because fractional excretions of sodium and chloride were 50% lower in SS/Jr than in SR/Jr rats. However, consistent with our previous findings in SS/Jr rats,6 RBF and GFR were significantly reduced in SS/Jr rats. Thus, changes in renal hemodynamics and the filtered load of sodium and chloride probably contribute to the inability of SS/Jr rats to excrete water and electrolytes as well as SR/Jr rats at equivalent levels of arterial pressure.
To examine the role of endogenous P450 metabolites of AA in the regulation of loop Cl− transport, the loop of Henle of SS/Jr and SR/Jr was perfused with a solution containing the mechanism-based inhibitor P450 ω-hydroxylase inhibitor 17-ODYA at a concentration greater than that previously reported to inhibit the formation of 20-HETE in renal cortical microsomes17 and renal microvessels.18 This same concentration has also been reported to produce a gradual block of tubuloglomerular feedback responses over a 15-minute period when added to solutions perfusing the loop of Henle of the rat in vivo.19 Addition of 17-ODYA to the perfusate increased Cl− reabsorption in the loop of Henle of SR/Jr rats, whereas the addition of 20-HETE (10 μmol/L) to the perfusate inhibited Cl− transport in the loop of Henle of SS/Jr rats. The effects of 20-HETE on loop Cl− transport in SS/Jr rats appear to be specific to this compound, since addition of an even higher concentration of a closely related fatty acid produced in the outer medulla of rats, 15-HETE, had no effect on loop Cl− transport. Overall, these observations provide the first in vivo evidence that endogenously formed P450 metabolites of AA, in particular 20-HETE, participate in the regulation of loop Cl− reabsorption. In this regard, the present results are consistent with previous in vitro studies by Escalante et al11 and Carroll et al12 in the rabbit and Omata et al13 in the rat indicating that 20-HETE is the primary metabolite of AA produced by the thick ascending limb and that this metabolite is a potent inhibitor of Na+, K+, 2 Cl− transport in these cells.11 However, we have reported that 17-ODYA also inhibits the formation of P450-derived metabolites of AA other than 20-HETE, such as 19-, 18-, and 16-HETE and 11-, 12-, 14-, and 15-EETs and diHETEs.17 Thus, to the extent that other P450 metabolites of AA are produced in the thick ascending loop of Henle, we cannot exclude the possibility that inhibition of the endogenous formation of one or more of these other compounds might also contribute to the elevation in loop Cl− transport produced by 17-ODYA in Dahl SR/Jr rats.
Another important aspect of the present study was to determine whether the previously reported abnormality in the production of 20-HETE in the outer medulla of the kidney of SS/Jr rats10 contributes to the elevation in loop Cl− transport seen in these rats.7 8 9 This was done by comparing the effects of a P450 inhibitor on loop Cl− transport in SS/Jr and SR/Jr rats. The present results indicating that addition of 17-ODYA to the perfusate markedly enhanced Cl− reabsorption in SR/Jr rats to the same level as that seen in SS/Jr rats but that it had no effect on Cl− transport in SS/Jr rats are consistent with this possibility. Further evidence that a deficiency in the endogenous production of 20-HETE may contribute to the elevated Cl− reabsorption in the loop of Henle of SS/Jr rats was obtained in the studies in which exogenous 20-HETE was added to the perfusate. In these studies, 20-HETE inhibited Cl− reabsorption in the loop of Henle of SS/Jr rats to the same level as that seen in SR/Jr rats, but it had no effect in SR/Jr rats, in which the production of 20-HETE in the outer medulla was higher than that seen in SS/Jr rats.10
The present results implicating a role for P450 metabolites of AA in altering loop chloride transport and sodium excretion in no way imply that this is the only mechanism that alters renal function in SS/Jr. Indeed, hypertension in SS/Jr is thought to be a polygenic disease, and there is considerable evidence that a number of different genetic loci that impact on renal function cosegregate with hypertension in this strain. In this regard, markers linked to renin,22 11β-hydroxylase (a steroid hydroxylase),23 guanyl cyclase,24 inducible nitric oxide synthetase,25 the Sa gene26 (which maps to a region near the amiloride-sensitive sodium channel), and endothelin-2 synthase27 all cosegregate with arterial pressure in F2 crosses of SS/Jr and normotensive strains. Strain differences in the renal production of nitric oxide,28 prostaglandins,29 kallikrein,30 and EETs31 and abnormalities in the tubular effects of deoxycorticosterone and vasopressin32 have also been identified and suggested to play a role in altering renal function in Dahl salt-sensitive rats. However, it should be noted that except for inducible nitric oxide synthase, there is no genetic evidence linking any of these factors with the development of hypertension in SS/Jr, nor is there any evidence suggesting that these factors play a major role in the regulation of Cl− transport in the loop of Henle.
In the case of nitric oxide synthase, there is now some physiological evidence that this system contributes to the alterations in renal function in SS/Jr. In this regard, l-arginine has been reported to prevent the development of hypertension and renal disease in SS/Jr.28 The antihypertensive effects of l-arginine are associated with an elevation in renal interstitial hydrostatic pressure33 and a shift in the pressure-natriuresis relation toward lower pressures. However, l-arginine feeding does not alter Cl− transport in the loop of Henle of SS/Jr,34 indicating that this abnormality is not dependent on the nitric oxide system. Thus, although it is clear that there are strain differences in a number of renal paracrine systems that are important in the renal handling of sodium, the relative importance of each of these systems versus 20-HETE in altering renal function and in the development of hypertension in SS/Jr will have to be sorted out in future studies.
In summary, the present results indicate that 20-HETE is an endogenously formed inhibitor of Cl− transport in the loop of Henle of the rat in vivo and that the previously reported deficiency in the production of this substance in the outer medulla of the kidney of SS/Jr rats relative to other normotensive strains (SR/Jr and Lewis Wistar rats)10 15 contributes to the elevation in loop Cl− reabsorption in SS/Jr rats. The elevation in loop Cl− transport has previously been suggested to play an important role in the resetting of the pressure-natriuresis relation and the development of hypertensive SS/Jr rats.7 8 9 Moreover, our recent observations that P4504A genotype cosegregates with hypertension in an F2 population of rats derived from SS/Jr and Lewis Wistar rats and that induction of the production of 20-HETE15 in the kidneys of SS/Jr rats with clofibrate prevents the development of hypertension14 provide further support for this hypothesis.
Selected Abbreviations and Acronyms
|GFR||=||glomerular filtration rate|
|RBF||=||renal blood flow|
This work was supported by grant HL-36279 from the National Institutes of Health.
Reprint requests to Richard J. Roman, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.
Dahl LK, Heine M, Thompson K. Genetic influence of renal homografts on the blood pressure of rats from different strains. Proc Soc Biol Med. 1972;140:852-856.
Bianchi G, Fox U, DiFrancesco GF, Bardi U, Radice M. The hypertensive role of the kidney in spontaneously hypertensive rats. Clin Sci Mol Med. 1973;45(suppl):135s-139s.
Roman RJ. Abnormal renal hemodynamics and pressure-natriuresis relationship in Dahl-salt sensitive rats. Am J Physiol. 1986;251:F57-F65.
Roman RJ, Kaldunski ML. Pressure-natriuresis and cortical and papillary blood flow in inbred Dahl rats. Am J Physiol. 1991;261:R595-R602.
Kirchner KA. Greater loop chloride uptake contributes to the blunted pressure natriuresis in Dahl S rats. J Am Soc Nephrol. 1990;1:180-186.
Kirchner KA. Increased loop chloride uptake precedes hypertension in Dahl salt-sensitive rats. Am J Physiol. 1992;262:R263-R268.
Ma Y-H, Schwartzman ML, Roman RJ. Altered renal P-450 metabolism of arachidonic acid in Dahl salt-sensitive rats. Am J Physiol. 1994;267:R579-R589.
Escalante B, Erlij D, Falck JR, McGiff JL. Effect of cytochrome P450 arachidonate metabolites on ion transport in rabbit kidney loop of Henle. Science. 1991;251:799-802.
Carroll MA, Sala A, Dunn CE, McGiff JC, Murphy RC. Structural identification of cytochrome P450-dependent arachidonate metabolites formed by rabbit medullary thick ascending limb cells. J Biol Chem. 1991;266:12306-12312.
Omata K, Abraham N, Schwartzman ML. Renal cytochrome P450 arachidonic acid metabolism: localization and hormonal regulation in SHR. Am J Physiol. 1992;262:F591-F599.
Roman RJ, Ma Y-H, Frohlich B, Markham B. Clofibrate prevents the development of hypertension in Dahl salt-sensitive rats. Hypertension. 1993;26:985-988.
Stec D, Deng AY, Rapp JP, Roman RJ. Cytochrome P4504A genotype cosegregates with hypertension in Dahl S rats. Hypertension. 1996;27:564-568.
Zou AP, Ma Y-H, Sui Z, Ortiz de Montellano PR, Clark JE, Masters BS, Roman RJ. Effects of 17-octadecynoic acid, a suicide-substrate inhibitor of cytochrome P450 fatty acid ω-hydroxylase, on renal function in rats. J Pharmacol Exp Ther. 1994;268:474-481.
Zou AP, Imig JD, Kaldunski M, Ortiz de Montellano PR, Sui Z, Roman RJ. Inhibition of renal vascular 20-HETE production impairs autoregulation of renal blood flow. Am J Physiol. 1994;266:F275-F284.
Zou AP, Imig JD, Kaldunski M, Ortiz de Montellano PR, Sui Z, Falck JR, Roman RJ. Effect of P450 ω-hydroxylase metabolites of arachidonic acid on tubuloglomerular feedback. Am J Physiol. 1994;266:F934-F941.
Roman RJ, Kauker ML. Renal effect of prostaglandin synthetase inhibition in rats: micropuncture studies. Am J Physiol. 1978;235:F111-F118.
Raman S, Mousseau G, Levine DZ. Statistical models for renal micropuncture studies. Am J Physiol. 1977;233:F349-F357.
Rapp JP, Wang SM, Dene H. A genetic polymorphism in the renin gene of Dahl rats cosegregates with blood pressure. Science. 1989;243:542-544.
Deng AY, Rapp JP. Locus for the inducible, but not a constitutive, nitric oxide synthase cosegregates with blood pressure in the Dahl salt-sensitive rat. J Clin Invest. 1995;95:2170-2177.
Harris EL, Dene H, Rapp JP. Sa gene and blood pressure cosegregation using Dahl salt-sensitive rats. Am J Physiol. 1993;6:330-334.
Deng AY, Dene H, Pravenec M, Rapp JP. Genetic mapping of two new blood pressure quantitative trait loci in the rat by genotyping endothelin system genes. J Clin Invest. 1994;93:2701-2709.
Chen PY, Sanders PW. l-Arginine abrogates salt-sensitive hypertension in Dahl Rapp rats. J Clin Invest. 1991;88:1559-1567.
Takenaka T, Suzuki H, Sakamaki Y, Itaya Y, Saruta T. Contribution of prostaglandins to pressure-natriuresis in Dahl salt-sensitive rats. Am J Hypertens. 1991;4:489-493.
Rapp JP, McPartland RP, Sustarsic DL. Anomalous response of urinary kallikrein in rats bred for their susceptibility and resistance to the hypertensive effect of salt. Circ Res. 1982;4:20-26.
Makita K, Takahasi K, Karara A, Jacobson HR, Falck JR, Capdevila JH. Experimental and/or genetically controlled alterations of the renal microsomal cytochrome P450 epoxygenase induce hypertension in rats fed a high salt diet. J Clin Invest. 1994;94:2414-2420.
Hawk CT, Schaffer JA. Effects of AVP and deoxycorticosterone on Na+ and water transport in the Dahl salt-sensitive rat CCD. Am J Physiol. 1991;260:F471-F478.
Patel AR, Granger JP, Kirchner KA. l-Arginine improves transmission of perfusion pressure to the renal interstitium in Dahl salt-sensitive rats. Am J Physiol. 1994;266:R1730-R1735.
Kirchner KA, Crosby BA, Patel AR, Granger JP. Segmental chloride transport in the Dahl-S kidney during l-arginine administration. J Am Soc Nephrol. 1995;5:1567-1572.