Decreased vascular responsiveness produced by angiotensin-converting enzyme inhibitors in the rat isolated kidney.

The effects of three angiotensin converting enzyme (ACE) inhibitors - captopril, MK-421 diacid, and teprotide - on renal vascular responses to graded (50, 100, 200 ng) injections of norepinephrine (NE) were examined in rat isolated perfused kidneys, having a mean basal perfusion pressure of 78 +/- 10 mm Hg. The minimum dose of captopril (0.05 microgram/ml, low dose) that abolished the vasoconstrictor responses to 100 and 200 ng angiotensin I did not affect NE-induced renal vasoconstriction, whereas a dose 100 times greater (high-dose captopril, 5 micrograms/ml) reduced the vasoconstrictor action of NE. MK-421 diacid also at high dose (1 microgram/ml), caused similar reduction in renal vasoconstrictor responses to NE. In contrast, a high dose of teprotide (50 micrograms/ml) did not affect renal vascular responsiveness to NE. The threshold dose of NE that released prostaglandins, measured by bioassay, was 50 ng. Indomethacin (1 microgram/ml) prevented NE-induced release of prostaglandins but did not affect the ability of captopril to attenuate NE-induced vasoconstriction. We conclude that captopril and MK-421 diacid decreases vascular reactivity in the rat isolated kidney by a mechanism independent of ACE inhibition and unrelated to a prostaglandin-dependent vascular mechanism. Moreover, the presence of mercapto function in the ACE inhibitor is not essential since captopril, which has a sulfhydryl group, and MK-421 diacid, which lacks this group, have similar effects on renal vascular responsiveness.

Aspirin enhances the antihypertensive effect of captopril in spontaneously hypertensive rats.

Activation of renal or vascular prostaglandin mechanisms (or both) has been proposed to contribute to the antihypertensive action of captopril. In conscious spontaneously hypertensive rats (SHR) studied in the established phase of hypertension, the blood pressure-lowering effect of captopril, 30 mg/kg/12 hr p.o. given for 7 days, was greatly enhanced by the addition of aspirin, 200 mg/kg/day s.c. Systolic blood pressure decreased from 185 +/- 6 and 182 +/- 4 to 135 +/- 3 mm Hg in rats treated, respectively, with captopril and aspirin or captopril alone, and was unaltered by either vehicle or aspirin alone. Water intake was inconsistently affected by captopril but was increased (p less than 0.01) by aspirin and was even higher after captopril-aspirin treatment (p less than 0.01). Urine volume was elevated in all 3 drug-treated groups, increasing threefold after captopril-aspirin treatment. Excretion of sodium and potassium was unchanged by any treatment regimen. In the vehicle group, prostaglandin F2 alpha excretion, measured by radioimmunoassay, ranged between 65 and 93 ng/8 hr and was twofold to fourfold higher than that of prostaglandin E2. Prostaglandin F2 alpha was unaffected during captopril treatment, whereas prostaglandin E2 excretion decreased to 12 +/- 2 ng/8 hr (p less than 0.01) by Day 7. Long-term aspirin treatment, either with or without captopril, did not cause sustained inhibition of renal prostaglandin excretion, although a transient effect occurred within the first four hours of administration. These results indicate 1) aspirin potentiates the blood pressure-lowering effect of captopril in SHR, an effect that is associated with a threefold increase in urine flow.(ABSTRACT TRUNCATED AT 250 WORDS)

6-keto PGE1: a possible metabolite of prostacyclin having platelet antiaggregatory effects.

Hepatic metabolism of prostacyclin (PGI2) results in the formation of several biologically inactive lipids and one stable product that has the same chromatographic and biological properties as authentic 6-keto PGE1. Both prostaglandins, 6-keto PGE1 and PGI2, have comparable potency in their antiaggregatory and disaggregatory effects on platelets. They contract the superfused rat stomach strip but differ in their effects on the bovine coronary artery, which is contracted by 6-keto PGE1 but relaxed by PGI2. Further, 6-keto PGE1 is considerably more stable than PGI2. Thus, 6-keto PGE1 could account for some of the prolonged effects occasionally seen with PGI2.

Association between the natriuretic action of angiotensin-(1-7) and selective stimulation of renal prostaglandin I2 release.

We previously reported that angiotensin-(1-7) [Ang-(1-7)], a heptapeptide derived from the metabolism of either Ang I or Ang II, was biologically active in the rat isolated kidney, producing a marked diuresis and natriuresis that could be dissociated from the modest increase in glomerular filtration rate. The natriuretic response was accompanied by an increase in sodium concentration and concomitant decrease in urinary potassium concentration. Ang-(1-7) has also been shown to stimulate arachidonic acid release from isolated proximal tubules and elicit prostaglandin release from a number of tissues. Therefore, in the present study we tested the hypothesis that prostaglandins participate in the renal actions of Ang-(1-7). Rat isolated kidneys were perfused at 37 degrees C with gassed (95% O2/5% CO2) Krebs-Henseleit buffer containing oncotic agents and amino acids for six 10-minute clearance periods at a constant pressure of 90 mm Hg. Ang-(1-7) was infused at a rate that achieved a final concentration of 3 pmol/mL in the presence and absence of 10 mumol/L indomethacin. Prostaglandin E2 (PGE2) and PGI2 released into ureteral and venous effluents were measured by enzyme-linked immunoassay. During Ang-(1-7) infusion there was a selective increase in 6-keto-PGF1 alpha, an index of PGI2, appearing in both urine and perfusate; PGE2 levels were unchanged. Inhibition of stimulated 6-keto-PGF1 alpha release with indomethacin halved the fourfold increase in urine flow and sevenfold increase in sodium excretion rate without altering the increase in urinary sodium concentration produced by Ang-(1-7).(ABSTRACT TRUNCATED AT 250 WORDS)

Chloride anion concentration as a determinant of renal vascular responsiveness to vasoconstrictor agents.

1. The role of chloride concentration in modulating vasoconstrictor responses of the rat isolated kidney, perfused with Krebs-Henseleit solution, to angiotensin II (AII), arginine vasopressin (AVP) and phenylephrine (PE) was investigated. 2. Reduction of perfusate chloride from a high (117 mM) to low (87 mM) concentration, by substitution of sodium chloride with a mixture of sodium salts of propionate, acetate and methanesulphonate, reduced responsiveness to all three vasoconstrictors, the change for AII being most pronounced. 3. For AII, reduced vasoactivity with low chloride was evident both in terms of the threshold dose and on the linear part of the dose-response curve but not for the maximum response. This attenuating effect of low chloride on the vasoconstrictor response to AII was reversed when perfusion with high chloride was reinstituted. Continuous perfusion with high chloride progressively increased the vasoconstrictor effect of low doses of AII for successive dose-response curves. 4. In addition to reducing responses on the linear part of the dose-response curve for both AVP and PE, low chloride also reduced the maximum vasoconstrictor response to PE, whereas the threshold dose for the two agonists was unchanged. In contrast to the enhanced pressor response to AII, during continuous perfusion with high chloride, tachyphylaxis occurred with AVP and PE. 5. The ability of chloride to modify renal responsiveness to vasoconstrictor agents may contribute to the increase in renal vascular resistance and decrease in glomerular filtration rate (GFR) which occurs during infusion of hyperchloremic solutions into the renal artery and explain the need for chloride as the anion accompanying sodium in salt-sensitive hypertensive models.

Natriuretic action of angiotensin(1-7).

Evidence that angiotensin(1-7) (Ang(1-7)) is biologically active and can be synthesized by the kidney prompted us to examine its actions in the rat, isolated kidney. Ang(1-7) had three major effects producing, (1) a substantial natriuresis and diuresis, (2) an increase in urinary sodium concentration associated with a fall in potassium concentration and (3) an increase in glomerular filtration rate without affecting renal vascular resistance. Thus, Ang(1-7) may participate in the renal effects of the renin-angiotensin system.

Urinary epidermal growth factor is excreted from the rat isolated perfused kidney in the absence of plasma.

Large amounts of epidermal growth factor (EGF) are excreted in urine and the majority of this urinary EGF appears to be of renal origin. EGF is synthesized in the kidneys as a membrane-bound 160 kDa precursor, in the thick ascending limb of Henle and in the early part of the distal convoluted tubule. Very little is known about how EGF is released from cell membranes into urine but proteolytic cleavage of the membrane-bound EGF precursor seems likely. The purpose of this study was to examine whether plasma constituents are necessary for urinary excretion of EGF. In the rat isolated kidney perfused at a pressure of 90 mmHg with a modified Krebs-Henseleit buffer containing oncotic agents, the quantity of EGF excreted into the ureteral effluent was 67% of the amount excreted by the rat kidney in vivo. The EGF excreted by the isolated kidney behaved like urinary EGF upon gel filtration. Administration of the proteinase inhibitor aprotinin reduced urinary EGF excretion from the rat isolated perfused kidney by approximately 50%. In conclusion, the rat isolated perfused kidney excreted significant amounts of urinary EGF without having access to plasma, and EGF excretion was reduced by aprotinin. This is further evidence suggesting an intrarenal source of urinary EGF and suggests that the EGF precursor in the rat kidney is processed by enzyme(s) of renal origin.

Hypotensive and renovascular actions of 6-keto-prostaglandin E1, a metabolite of prostacyclin.

Either intra-aortic or intravenous injections of a stable prostaglandin metabolite, 6-keto-prostaglandin E1 (6-keto-PGE1), caused similar dose-dependent falls in blood pressure and reductions in renovascular resistance in the anesthetized rat. The threshold dose was 0.3 microgram/kg. A maximum hypotensive effect occured at 10 micrograms/kg, but renal blood flow was further reduced by a dose of 30 microgram/kg. 6-keto-PGE1, like prostacyclin, could be a circulating hormone.

Captopril decreases vascular reactivity independently of changes in converting enzyme activity and prostaglandin release in the rat isolated kidney.

The relationship of the vascular effect of captopril to angiotensin converting enzyme activity and prostaglandin-dependent mechanism was studied in rat isolated kidneys, perfused with Krebs-Henseleit at 20 ml/min per 2 kidneys, with basal perfusion pressures of 78 +/- 1 mm Hg (Mean +/- S.E.M.). Two doses of captopril were used; both low (0.05 microgram/ml) and high doses (5 microgram/ml) inhibited maximally the vasoconstrictor responses to 100 and 200 ng of angiotensin I. Captopril at the low dose did not affect the renal vasoconstrictor responses to norepinephrine (NE) (25-400 ng), whereas high-dose reduced the vasoconstriction to all doses of NE. Treatment with captopril tended to diminish dose-related release of prostaglandins in response to NE. Indomethacin (1 microgram/ml) prevented NE-induced release of bioassayable and radioimmunoassayable prostaglandins but did not affect the ability of captopril to reduce NE-induced vasoconstriction. High-dose captopril also decreased the vascular reactivity to angiotensin II (5 ng) and lysine vasopressin (10 mU); however, the renal vasoconstriction caused by PGE2 (80 ng) was unaffected by captopril. We conclude that high-dose captopril decreased vascular reactivity by a mechanism independent of converting enzyme inhibition and unrelated to a prostaglandin-dependent vascular mechanism.

The contribution of cytochrome P450-dependent arachidonate metabolites to integrated renal function.

Arachidonic acid can be metabolized to diverse products which differ widely in their biological activities. These metabolites affect basic biological mechanisms such as vascular reactivity and transport function in critical nephron segments. Metabolism of arachidonic acid is discretely localized to specific cells and differs within segments of the nephron; for example, cells of the medullary ascending limb of Henle's loop have a considerable capacity to generate cytochrome P450-dependent arachidonate metabolites but have negligible cyclooxygenase activity. Arachidonic acid metabolites participate in fluid and electrolyte homeostasis, and in the regulation of tissue blood flow, and act as modulators of vasoactive hormones, and, thereby, make important contributions to integrated renal function.

Prostaglandin release by the chick embryo heart is increased by 2,3,7,8-tetrachlorodibenzo-p-dioxin and by other cytochrome P-448 inducers.

Exposure of chick embryos in ovo to cytochrome P-448 inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3,4,3',4'-tetrachlorobiphenyl, 3,4,5,3',4',5'-hexachlorobiphenyl and beta-napthoflavone, increased cardiac prostaglandins in vitro. The dose response relationships were biphasic with prostaglandin release increasing at the low doses and returning to basal levels at higher doses. Phenobarbital was ineffective. Increased cardiac prostaglandin release was detected at doses that induced hepatic 7-ethoxyresorufin deethylase (7-ER) but which were below the threshold for cardiac induction. The fall in prostaglandin release coincided with induction of cardiac 7-ER and therefore may be attributable to increased prostaglandin metabolism. These studies show that the P-450 system may interact with the arachidonic acid metabolizing system to increase PG release and that this effect may be part of the pleiotypic response to Ah-receptor activation.

Isomers of 12-hydroxy-5,8,10,14-eicosatetraenoic acid reduce renin activity and increase water and electrolyte excretion.

A metabolite of arachidonic acid, 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), which can be formed either in the 12-S or 12-R configuration, has a diversity of biological actions and is generated by a number of tissues including the renal glomerulus and the vasculature. As the two isomers have been shown to differ in their effects on epithelial transport mechanisms and vascular responsiveness, we studied their direct effects on the rat isolated kidney, perfused for four consecutive 15-min clearance periods at a pressure of 90 mm Hg with a modified Krebs' buffer containing oncotic agents. At a dose of 20 nmol, both 12(S)- and 12(R)-HETE doubled urine volume (P less than .05) and sodium and potassium excretion rate in the first, postinjection clearance period. The effects of 12(R)-HETE were sustained during all three post-treatment clearance periods, whereas those of 12(S)-HETE were short-lived, excretion rates being similar to control values by the second post-treatment clearance period. At a higher dose of 40 nmol, 12(R)-HETE significantly reduced the usual rate of decline in glomerular filtration rate, characteristic of the rat isolated kidney, and caused an even greater initial increase in urine volume and sodium excretion rate than that achieved with 20 nmol. Renin concentration in the venous effluent was reduced immediately by 12(R)- and 12(S)-HETE (P less than .01), to approximately half of the control value. Again the response to the (R)-isomer was more prolonged. Thus, a 12-HETE of glomerular origin may alter renal function through direct and indirect tubular and hemodynamic effects.

Novel arachidonate metabolites generated by cytochrome P450-dependent mono-oxygenases.

Cytochrome P450-dependent arachidonic acid (AA) metabolism by medullary thick ascending limb of the loop of Henle (mTALH) cells, corneal epithelium and other transporting epithelia, such as those of the intestines, generate metabolites which affect Na(+)-K(+)-ATPase activity, vasomotion and, thereby, organ function. Further, these novel AA metabolites contribute to the control of blood pressure in the SHR through participation in the local control of blood flow and in the regulation of extracellular fluid volume.

Endothelin-3 effects on renal function and prostanoid release in the rat isolated kidney.

The direct effects of rat endothelin (ET-3) on renal function and prostanoid levels were examined in the isolated, oncotically perfused kidney of the rat. ET-3 at 0.75 and 2.0 ng/ml produced sustained increases in perfusion pressure of 46 and 83 mm Hg, respectively, as compared with control kidneys. Glomerular filtration rate was significantly higher than control after additions of ET-3 as was the absolute and fractional excretion of water and electrolytes. ET-3 increased perfusate 6-keto-prostaglandin (PG)F1 alpha (breakdown product of PGI2) levels and stimulated greater urinary excretion of PGE2 and PGF2 alpha than 6-keto-PGF1 alpha. ET-3 did not affect urinary excretion or perfusate levels of thromboxane B2. Time-dependent increases in renin release were suppressed by ET-3. Indomethacin (10 microM) prevented ET-3-induced increases in urinary and perfusate prostanoids; however, renal vasoconstrictor and excretory responses were the same in the presence or absence of indomethacin. These results indicate that ET-3 acts directly on the perfused rat kidney to increase the release of prostanoids from the vascular and urinary compartments. A modulatory influence of prostaglandins on the acute renal hemodynamic and excretory effects of ET-3 was not observed under these conditions.

Failure of chronic aspirin treatment to inhibit urinary prostaglandin excretion in spontaneously hypertensive rats: comparison with indomethacin and flurbiprofen.

The inability of chronic treatment with aspirin to cause sustained inhibition of urinary prostaglandin (PG) excretion observed previously prompted us to compare the effects of 9-day treatment of spontaneously hypertensive rats with aspirin, 200 mg/kg/day s.c., flurbiprofen, 2.5 mg/kg/b.i.d. s.c. and indomethacin, 2.5 mg/kg/b.i.d. s.c. on the excretion rate of radioimmunoassayable PGE2 and PGF2 alpha. Conversion of 1-[14C]arachidonic acid and the release of PGs from endogenous substrate by the renal papilla were also examined. In vehicle-treated control rats, PGF2 alpha excretion ranged from 32.2 +/- 6.2 (mean +/- S.E.M.) to 41.6 +/- 7.3 ng/6 h, and was 2- to 4-fold higher than that of PGE2. Within 6 h of administration all three drugs reduced excretion of PGF2 alpha and PGE2 to less than 20% and 35% of control rats, respectively. Thereafter, PGF2 alpha and PGE2 excretion in aspirin-treated rats returned to values similar to the vehicle-treated group, whereas inhibition of PG excretion in indomethacin and flurbiprofen groups was sustained. Urine volume was doubled by aspirin throughout the study. In contrast, urine volume in flurbiprofen- and indomethacin-treated rats was unaffected. Paradoxically, metabolism of 1-[14C]arachidonic acid to PGs by renal papilla dissected on day 10, 2 to 4 h after the last drug dose, was reduced markedly by aspirin as was the release of immunoreactive PGs but was unaffected by flurbiprofen or indomethacin. The failure of long-term aspirin treatment to inhibit urinary PG excretion and the disparity between in vivo and ex vivo indices of PG release emphasize the need to verify their intended action by measuring PGs in biological fluids.

Metabolism of prostacyclin: formation of an active metabolite in the liver.

Hepatic metabolism of prostacyclin (PGI2) results in the formation of several biological inactive lipids and one product that has the chromatographic and biological properties of 6-keto-PGE1; the latter, unlike prostacyclin, is stable. Further, authentic 6-keto-PGE1, like PGI2, escapes pulmonary degradation and is a potent inhibitor of platelet aggregation. It could be generated from either prostacyclin or its inactive hydrolysis product 6-keto-PGF1 alpha via the 9-hydroxyprostaglandin dehydrogenase pathway, which has been identified in the liver and kidney. The prolonged biological activity of PGI2, which is difficult to explain in view of its inherent instability, may derive from transformation of PGI2 to 6-keto-PGE1. These studies raise the question: What, if any, of the effects of prostacyclin on platelets and the circulation are dependent on its conversion to 6-keto-PGE1?

Role of chloride in the variable response of the kidney to cyclooxygenase inhibition.

The role of prostanoids in renal function remains unclear, as inhibitors of cyclooxygenase (COX) have contrasting effects. We postulated that these inconsistencies were related to differential effects of the prevailing chloride concentration on COX-dependent mechanisms. In oncotically perfused rat kidneys, in the presence of either high (117 mM) or low (87 mM) chloride with sodium held constant, low chloride resulted in a higher glomerular filtration rate (GFR) than with high chloride, i.e., 1.2 +/- 0.2 and 0.5 +/- 0.1 ml/min, respectively, for the last clearance period. Water and electrolyte excretion and levels of immunoassayable prostaglandins were higher with low chloride. Indomethacin (10 microM) had opposite effects on renal function depending on the chloride levels, although prostaglandin release was inhibited similarly. For example, indomethacin substantially reduced the elevated urine flow and sodium excretion in the low-chloride group, which, by the last period, were reduced from 111 +/- 32 to 37 +/- 3 microliters/min and 8.3 +/- 2.9 to 3.1 +/- 0.6 mu eq/min, respectively, whereas the lower urine flow and sodium excretion in the high-chloride group increased from 32 +/- 8 to 109 +/- 15 microliters/min and 2.5 +/- 0.8 to 7.1 +/- 1.6 mu eq/min, respectively. In summary, inhibition of COX has differential effects depending on the prevailing chloride concentration, or conversely, high and low chloride have contrasting effects on renal function, which are reversed by COX inhibition. We suggest that prohypertensive and antihypertensive COX-dependent mechanisms are linked to chloride; the latter is an integral component in the development of salt-sensitive hypertension.

Analysis of eicosanoid mediation of the renal functional effects of hyperchloremia.

Depression of GFR and antinatriuresis in response to high chloride has been linked to a cyclooxygenase (COX)-dependent mechanism involving thromboxane A2 (TxA2) and prostaglandin endoperoxide (PGH2), because inhibition of COX prevented the fall in GFR and antinatriuresis produced by hyperchloremia. However, hyperchloremia did not increase, but unexpectedly decreased, renal prostaglandin and TxA2 efflux (Yin et al., 1995). To resolve questions regarding the role of eicosanoids in mediating the renal functional effects of high chloride (117 mM), by stimulating either TxA2 synthesis or TxA2/PGH2 receptors, we compared the ability of indomethacin to block high-chloride effects in the rat isolated kidney with that of BMS 180291 and SQ 29548, antagonists of the TxA2/PGH2 receptor. These antagonists differ in terms of their selectivity and their capacity to inhibit isoforms of the TxA2/PGH2 receptor. Indomethacin and SQ 29548 had identical actions, preventing the decrease of GFR and antinatriuresis evoked by hyperchloremia, e.g., sodium excretion rate in the SQ 29548 and indomethacin groups increased to 7.2 +/- 1.3 and 7.1 +/- 1.2 microEq/min, respectively, compared with 2.6 +/- 0.7 microEq/min in the control group. In contrast, neither BMS 180291 nor the TxA2 synthase inhibitors, OKY 046 and CGS 13080, modified the negative effects of high chloride on GFR or sodium excretion. These results argue against either TxA2 or PGH2 acting as mediator of the effects of high chloride on renal function and suggest a product of COX activity such as a 20-HETE analog of prostaglandin endoperoxide. Evidence to support this proposal was obtained: 1) Hyperchloremia increased 20-HETE release from the rat kidney by 2-fold when compared with low-chloride conditions of renal perfusion. 2) The renal vasoconstrictor action of 20-HETE was shown to be dependent on COX activity and to be antagonized by blockade of the TxA2/PGH2 receptor.

Renovascular actions of angiotensin II in the isolated kidney of the rat: relationship to lipoxygenases.

Several actions of angiotensin II have been linked to metabolism of arachidonic acid by lipoxygenases. To evaluate the importance of this interaction intrarenally we tested the effect of three different lipoxygenase inhibitors, BW755c (50 microM), a dual lipoxygenase-cyclooxygenase inhibitor, MK447 (200 microM), a nonselective lipoxygenase inhibitor which can stimulate cyclooxygenase, and baicalein (1 microM), a highly selective 12-lipoxygenase inhibitor, on angiotensin II-evoked hemodynamic changes in the rat isolated kidney, perfused with oncotic agents. Kidneys were pretreated with indomethacin (10 microM) to exclude participation of cyclooxygenase-dependent arachidonate products. Renal perfusion pressure was kept constant at 90 mm Hg by continuous adjustments in perfusate flow rate. Inhibition of cyclooxygenase alone produced a transient potentiation of the vasoconstrictor response to angiotensin II without altering GFR. On the other hand, the lipoxygenase inhibitors attenuated the angiotensin II-induced increase in renal vascular resistance by approximately 50% and promoted an increase in GFR above that of kidneys infused with angiotensin II in the presence of only indomethacin. Base-line values were essentially unchanged by lipoxygenase inhibition. Furthermore, the vasoconstrictor response to the thromboxane/endoperoxide agonist U46619 was unaffected. We conclude that products of the lipoxygenase pathway, arising within the kidney, contribute to the renal hemodynamic effects of angiotensin II.