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Abstract
Abstract Plasma concentration of endothelin-1, a potent vasoconstrictor produced by the vascular endothelium, has been observed to be significantly increased in a number of pathophysiological states, including preeclampsia. In the present study we have evaluated the effects of elevated plasma endothelin-1 in pregnant sheep by continuous exogenous endothelin-1 administration. Nine pregnant ewes (110±5 days’ gestation) were instrumented for measurements of maternal mean arterial pressure, renal blood flow, and uterine blood flow. After recovery, endothelin-1 was infused intravenously for 4 hours at a dose that was adjusted to raise mean arterial pressure by ≈20 mm Hg by the end of the first hour (range 5 to 20 ng/kg per minute). Mean arterial pressure, renal blood flow, uterine blood flow, urinary protein excretion, hematocrit, and plasma endothelin-1 concentration were measured hourly, and renal and uterine vascular resistances were calculated. Endothelin-1 produced significant increases (% change from baseline at t=4 hours) in mean arterial pressure (45±8%), renal vascular resistance (353±66%), and uterine vascular resistance (59±21%). Endothelin-1 also increased microvascular permeability both systemically and within the kidney, as suggested by marked increases in hematocrit (0.27±0.01 to 0.32±0.01) and urinary protein concentration (0.95±0.1 to 7.9±3.2 mg/mL per mg creatinine). There was a highly significant correlation (P<.0001) between plasma endothelin-1 and mean arterial pressure, renal vascular resistance, uterine vascular resistance, hematocrit, and urinary protein content in all sheep studied. In addition, plasma endothelin-1 corresponded well with the time course of the changes in cardiovascular parameters and urinary protein excretion observed. These results provide evidence to suggest that elevation of circulating endothelin-1 in pregnant sheep can produce cardiovascular and hemodynamic changes that in many ways resemble the human disease preeclampsia. This supports the hypothesis that endothelial cell damage and/or dysfunction that is associated with increased production of endothelin-1 could directly contribute to the progression of preeclampsia.
Since the discovery of endothelin, a potent vasoactive peptide released by the endothelial lining of the vasculature,1 2 there has been an accumulation of evidence demonstrating that circulating endothelin-1 (ET-1) is significantly elevated in many pathological conditions.3 4 5 Increased plasma ET-1 has also been observed in situations in which effective circulating volume is decreased, such as heart failure,6 hepatorenal syndrome,7 and renal failure.8 9 Preeclampsia, a serious complication of pregnancy that occurs in an estimated 5% to 10% of pregnant women, also is associated with increased plasma ET-1.5 10 11 12 13 14
Clinically, preeclampsia is typically characterized by a syndrome of hypertension, proteinuria, and edema; however, common manifestations also include reduced uterine blood flow, hemoconcentration, abnormal platelet function, and renal pathology.15 16 17 Although it is unclear what may cause plasma ET-1 levels to rise in preeclampsia, there is experimental evidence to suggest that maternal endothelial cell dysfunction may be involved in the development of the syndrome.5 11 18 19 Such pathogenesis could conceivably give rise to a number of vasoactive endothelium-derived factors, including ET-1, that in turn could serve to mediate the clinical symptoms of preeclampsia.
The idea that ET-1 might participate in the development and/or maintenance of preeclampsia is in keeping with some of the many known effects of ET-1. In addition to its systemic pressor actions,20 this peptide has been shown to be a powerful vasoconstrictor in both the renal21 and uterine22 23 vascular beds. Thus, abnormally elevated ET-1 levels would be expected to increase systemic, renal, and uterine vascular resistances. In addition, ET-1 has been shown to directly alter renal protein handling24 25 and has been implicated in several experimental models of progressive proteinuric renal disease.26 27 28 29 Based on the above evidence, the present studies sought to determine whether elevation of circulating ET-1 in pregnant ewes, via continuous systemic infusion of ET-1, would produce changes in systemic, renal, and uterine vascular resistances, urinary protein excretion, and intravascular volume, consistent with the clinical manifestations of human preeclampsia.
Methods
Animals
Nine pregnant ewes (110 to 115 days’ gestation) of mixed breed were purchased from Tom Morris, Inc. Animals were housed in individual portable stainless steel cages in a temperature- and light-controlled, American Association for Accreditation of Laboratory Animal Care–accredited facility with free access to food and water.
Surgical Procedures
Animals were sedated with sodium pentobarbital (15 mg/kg IV) and anesthesia was maintained by ventilation with a mixture of 2% to 3% isoflurane and oxygen. Under sterile conditions, the right kidney was exposed by a small retroperitoneal flank incision, and a transit-time flow probe (Transonic Systems) was positioned on the renal artery for subsequent monitoring of unilateral renal blood flow. After a 1-week recovery period, ewes were again anesthetized and chronic indwelling polyvinyl catheters were implanted in the maternal and fetal femoral arteries and veins and advanced to the level of the distal aorta and vena cava, respectively. Through a 15-cm lower abdominal incision, transit-time flow probes were placed bilaterally on the maternal middle uterine arteries for measurement of uterine blood flow. In three of the pregnant ewes, a flow probe was also placed on the fetal common umbilical artery via a fetal flank incision in order to monitor umbilical blood flow. All catheters and flow probe cables were exteriorized through the respective incision site, passed subcutaneously to the ewe’s flank, placed in a cloth pouch, and secured to the ewe’s side. A 1-week recovery period was allowed after the final surgery before study.
Antibiotics (3 mL IM) (penicillin G procaine, Jeffers Co) were administered on the day of and 3 days after surgery. Maternal catheters were flushed daily with sterile saline and filled with sodium heparin (1000 USP units/mL) (Elkins-Sinn Inc) to maintain patency; fetal catheters were flushed daily with bacteriostatic-free sterile saline and filled with sodium heparin (500 USP units/mL). Fetal arterial blood samples were collected anaerobically into heparinized syringes daily, and fetal blood gas values (Pao2, Paco2, and pH) were determined with a blood gas analyzer (model 288, Ciba-Corning Diagnostics) to verify fetal well-being. All surgical and experimental procedures were performed in accordance with the Institutional Animal Care and Use Committee guidelines of the University of Cincinnati.
Experimental Protocol
After surgical recovery (1 week), baseline measurements were obtained over a 2-hour control period in conscious ewes. A continuous intravenous infusion of ET-1 (American Peptide Co) was then begun at a dose of 5 ng/kg per minute (infusion rate=0.1 mL/min). ET-1 infusion was maintained for a 4-hour period with the dose adjusted as described below. Maternal mean arterial pressure (MAP), heart rate, renal blood flow, total uterine blood flow, and umbilical blood flow were recorded continuously throughout the control and experimental periods. Urine samples were collected by clean-catch at the end of the control period and at the end of hours 1, 2, 3, and 4 of ET-1 infusion, and stored for determination of urinary protein and creatinine excretion. Maternal arterial blood samples were collected at these same time points, hematocrit was recorded, and plasma was frozen at −20°C for later measurement of plasma ET-1.
The metabolic clearance rate of endothelin-1 may be increased during pregnancy in sheep,30 and also appears to vary considerably among individual animals (authors’ unpublished observations); thus, the same dose of ET-1 infused into different animals did not necessarily increase circulating ET-1 to the same degree. To produce more consistent changes in plasma ET-1 concentration, the dose of endothelin-1 infused was adjusted during each experiment to achieve an increase in MAP of ≈20 mm Hg and a decrease in renal blood flow of not more than 40% by the end of the first hour of infusion. These parameters were used as indices of circulating ET-1 since they were found to correlate most strongly with plasma ET-1 concentration. The ET-1 infusion rate necessary to achieve the above criteria ranged from 5 to 20 ng/kg per minute.
Analytical Methods
Systemic, renal, and uterine vascular resistances were calculated as MAP divided by the respective blood flow rate. In order to measure urinary protein concentration, urine samples were extracted with 72% trichloroacetic acid and 0.15% sodium deoxycholate to precipitate proteins. Protein pellets were resuspended in 5% SDS. Urinary protein concentration was then determined by a modification of the Lowry method (BioRad DC Protein Assay). Creatinine concentration for each urine sample was measured using a standard alkaline picrate colorimetric assay, and protein concentration was expressed per unit of creatinine to normalize for changes in glomerular filtration rate.
Plasma ET-1 concentration was measured by enzymatic immunoassay using kits purchased from Cayman Chemical. Plasma samples were assayed without prior extraction, which was possible since the ET-1 infusion was sufficient to place plasma ET-1 concentrations in the detectable range of the assay. Baseline values were generally at or near the lower sensitivity limit of the assay. The assay has 100% cross-reactivity with ET-2 and ET-3; thus, baseline values may be somewhat overestimated since the measured ET-1 may actually reflect any circulating ET-2 and ET-3 as well. However, since the endothelin infused was pure ET-1, the elevation of plasma ET measured by the assay during the infusion period can be attributed largely (if not solely) to ET-1. There was negligible (<0.01%) cross-reactivity with Big-ET. Coefficients of variation were ≤15% (interassay) and ≤5% (intra-assay), and dilutions of plasma samples yielded linear measurements.
Statistical Analysis
Results are presented as the group mean±SEM. Multiple comparisons were conducted by two-way ANOVA for repeated measures. Mean differences were determined by the Student-Newman-Keuls test. The .05 level of probability was used as the criterion of significance.
Results
In pregnant sheep, baseline levels of plasma ET-1 were generally less than 40 pg/mL but were considerably elevated in one sheep (355 pg/mL). This was reflected as a high level of variability about the mean for baseline plasma ET-1 (86±54 pg/mL) as seen in Fig 1⇓. Plasma ET-1 rose markedly during the course of exogenous infusion and appeared to plateau at approximately 250 pg/mL (Fig 1⇓). This elevation in circulating ET-1 produced a marked increase in MAP by the end of hour 1 in all animals, and MAP rose to a maximum of approximately 45% above baseline (from 76±3 to 110±7 mm Hg) by 4 hours of infusion (Fig 2⇓). When MAP was evaluated as a function of plasma ET-1 for all time points, a strong correlation was observed (r=.91; P<.001).
Hourly measurement of plasma ET-1 concentration during continuous ET-1 infusion in pregnant ewes. **P<.01; ***P<.001 vs control period.
Hourly measurement of MAP (expressed as percent of baseline) during continuous ET-1 infusion in pregnant ewes. ***P<.001 vs control period.
As shown in Fig 3a⇓, ET-1 infusion caused significant constriction within the uterine vasculature as reflected by an increase in mean UVR to 64% above baseline by the end of hour 2 that was maintained for the rest of the infusion period. The uterine vasculature of one sheep in particular was highly responsive, with UVR increasing 300%; this explains the large standard error associated with the mean values. Although the magnitude of the changes in UVR varied somewhat among individual animals, the rise in UVR was strongly correlated with the rise in plasma ET-1 concentration over the course of the infusion period (r=0.94; P<.001). Despite the pronounced increase in UVR, uterine blood flow was not significantly altered due to the concomitant increase in MAP of similar magnitude. Furthermore, while it has been shown that ET-1 is a potent constrictor of the umbilical vasculature,31 32 no changes in umbilical blood flow were observed in the present study (data not shown); thus, it does not appear that ET-1 crossed the placenta during the course of its infusion in pregnant sheep.
Hourly measurement (expressed as percent of baseline) of uterine and renal vascular resistances during continuous ET-1 infusion in pregnant ewes: a, UVR; b, RVR; and c, comparison of both renal and uterine vascular resistances using the same scale on y axis. *P<.05; **P<.01; ***P<.001 vs control period.
The renal vasculature was found to be extremely sensitive to the vasoconstrictor action of ET-1. RVR reached 353±66% above baseline by 4 hours of ET-1 infusion (Fig 3b⇑). The degree of RVR at each time point was found to correlate well with observed concentrations of plasma ET-1 (r=.89; P<.01). This increase in RVR was associated with a substantial fall in renal blood flow (526±45 mL/min at baseline to 201±34 mL/min by end of hour 4; data not shown). Thus, in pregnant sheep the renal vasculature was found to be much more sensitive to ET-1 than the uterine vasculature, as is shown by the direct comparison of vascular resistance in these beds (Fig 3c⇑). In addition to its effects on the renal vasculature, ET-1 was also found to directly affect renal protein handling. As shown in Fig 4⇓, urinary protein excretion increased markedly during the course of ET-1 infusion, although there was some variability between the responses of individual animals. One sheep did not develop proteinuria at all, but one became severely proteinuric (30 mg/mL per mg/mL creatinine) by the end of hour 2; interestingly, this animal also showed greater vascular responses as well. In most animals, the appearance of significant proteinuria was somewhat delayed as compared with the changes in other parameters. It should be noted that urinary protein concentration is expressed as a ratio of the corresponding urinary creatinine concentration, thus normalizing for changes in glomerular filtration rate which likely occurred in conjunction with the reduced renal blood flow.
Hourly measurement of urinary protein excretion during continuous ET-1 infusion in pregnant ewes. *P<.05 vs control period.
In addition to its effects on renal permeability, ET-1 also appeared to have a direct effect on the permeability of the systemic microvasculature. Hematocrit rose dramatically by 19% in response to ET-1 infusion, from a baseline of 0.27±0.01 to 0.32±0.01 by hour 4 (Fig 5⇓). The increase in hematocrit correlated well with the rise in plasma ET-1 that occurred during the infusion (r=.87; P<.01). This hemoconcentration is indicative of a significant decrease in intravascular volume in these animals.
Hourly measurement of hematocrit during continuous ET-1 infusion in pregnant ewes. ***P<.001 vs control period.
Discussion
The present studies were conducted to evaluate the effects of chronic elevation of plasma ET-1, via continuous intravenous ET-1 infusion, on systemic, renal, and uterine hemodynamics in pregnant sheep. As plasma levels of ET-1 rose over a 4-hour period, we observed the onset of hypertension, renal and uterine vasoconstriction, hemoconcentration, and, finally, proteinuria. These changes resemble the clinical manifestations of human preeclampsia, a disease that affects approximately 10% of all pregnant women and that is a leading cause of perinatal morbidity and mortality.
While there are a number of plausible theories regarding the etiology of preeclampsia, the event(s) that initiate the onset of this disease remain unclear. Available evidence suggests that these unknown initiating factor or factors trigger a process that ultimately leads to endothelial cell damage and/or dysfunction, which may be intimately involved in the pathogenesis of preeclampsia.5 17 18 19 This hypothesis is supported by evidence that plasma concentrations of endothelial cell markers (such as cellular fibronectin, tissue plasminogen activator, plasminogen activator inhibitor-1, and von Willebrand factor) are significantly higher in preeclamptic women than in normal pregnant women.33 34 Furthermore, a variety of endothelium-derived vasoactive substances appear to be altered in preeclampsia, including decreased capacity for nitric oxide production, elevation of the thromboxane/prostacyclin ratio, and elevation of plasma ET-1 concentration.5 10 11 12 13 14
While it is likely that the overall balance of endothelial factors (ie, vasoconstrictors versus vasodilators) is of critical importance, the idea that abnormally elevated ET-1 may in part contribute to the progression of preeclampsia is intriguing for a number of reasons. Endothelin-1 has been shown to be an extremely potent and long-lasting vasoconstrictor of the uterine22 23 and umbilical31 32 circulations. Heightened production of ET-1 could also have an important influence on renal function, since renal vessels are particularly responsive to the vasoconstricting effects of ET-1.21 ET-1 has also been shown to inhibit renin secretion,35 which is in keeping with reports that plasma renin activity is decreased in preeclamptic women.36
In addition to its well-known effects on vascular smooth muscle, ET-1 has been found in other studies to have a profound effect on microvascular permeability, both systemically and within the kidney. ET-1 administration in rats has been reported to cause significant proteinuria characterized by an increase in albumin excretion and by the appearance of proteins with molecular weights of 20 000 to 280 000.25 This may be the result of ET-1’s action on the renal glomerular mesangial cells,37 38 the contractile cells that govern the size of the renal filtration barrier. In the present investigation, it is unclear whether the proteinuria observed in response to ET-1 infusion was in fact due to a direct effect within the glomerulus or whether it was an indirect result of the increase in renal perfusion pressure. Also, the fact that the appearance of proteinuria was delayed as compared with changes in other parameters suggests that there may be a critical threshold and/or duration for elevation of plasma ET-1 in order to cause the development of proteinuria. Further studies are planned to better understand the mechanism of this response.
Systemically, ET-1 has also been shown to dramatically enhance microvascular permeability and protein extravasation in conscious rats.39 This is consistent with the present findings in sheep that hematocrit rose markedly in response to ET-1 infusion, indicating a significant reduction of intravascular volume. The loss of plasma protein that occurs via urinary excretion and systemic vascular leakage likely has important systemic consequences. Reduced colloid osmotic pressure may contribute to the shift of extracellular fluid volume from intravascular to interstitial compartments, as is observed in preeclampsia patients.40 In preeclampsia, such a decrease in plasma volume theoretically could perpetuate the need for elevated MAP to maintain uteroplacental perfusion; the redistribution of extracellular fluid volume may also increase the risk of pulmonary edema.41
A survey of a number of clinical studies that report significant elevations of plasma ET-1 in preeclamptic women reveals that plasma ET-1 can increase from ≈1.0 to 6.0 pg/mL in normal pregnancy to averages of ≈8.0 to 16.0 pg/mL in preeclampsia.5 10 11 13 In the present study, baseline plasma ET-1 in pregnant sheep was found to be ≈89 pg/mL and was increased to ≈300 pg/mL after 4 hours of ET-1 infusion, by which time significant cardiovascular and hemodynamic changes were observed. While it is not clear why pregnant sheep appear to have high baseline levels of circulating ET-1 compared with humans, it is not surprising that peak plasma ET-1 concentrations measured in humans are substantially lower than the ovine values obtained in the present study. Since it is intended as a local rather than a circulating regulatory factor, ET-1 produced by endothelial cells is primarily released toward the vascular smooth muscle rather than into the vessel lumen. This suggests that when plasma ET-1 increases endogenously, as is observed in preeclampsia, this actually reflects a greatly magnified amount of ET-1 being produced by the endothelium and consequently seen by the vascular smooth muscle. To achieve similarly high concentrations of ET-1 at the level of the target cell (vascular smooth muscle) via exogenous ET-1 administration, it is thus necessary to elevate plasma ET-1 to a much greater degree than would occur physiologically.
Finally, it should be noted that the effects of continuous ET-1 infusion reported herein do not appear to be specific for pregnancy, as similar responses were observed when experiments were conducted in nonpregnant sheep (authors’ unpublished observations). This is not surprising since studies by other investigators (discussed above) that corroborate the present findings were conducted largely in nonpregnant animals. However, the overall hypothesis that suggests a role for ET-1 in preeclampsia maintains that an event that is unique to pregnancy and likely occurs very early acts as an initiating factor in a cascade of events that ultimately results in widespread endothelial dysfunction, and thereby elevated ET-1 production. If this hypothesis is true, then an artificial elevation of ET-1 (by exogenous infusion) would in essence serve to mimic some of the diagnostic criteria of preeclampsia while “bypassing” the earlier pathogenic events. Because it is these early pathogenic events, and not the actions of ET-1, that presumably are unique to pregnancy, it becomes irrelevant whether ET-1 infusion produces similar effects in nonpregnant animals.
In summary, the present studies demonstrated that elevation of plasma ET-1, produced by continuous systemic ET-1 infusion, resulted in increased systemic, uterine, and renal vascular resistances as well as alterations in microvascular permeability in pregnant sheep. While these data are not evidence that preeclampsia is caused by elevations in ET-1 per se, the present study does support the overall hypothesis that early pathogenic events that are unique to pregnancy may ultimately result in widespread endothelial dysfunction and that increased production of ET-1 associated with this dysfunction (particularly within the kidney) could contribute to some of the clinical manifestations of preeclampsia.
Selected Abbreviations and Acronyms
| ET-1, ET-2, ET-3 | = | endothelin-1, -2, and -3, respectively |
| MAP | = | mean arterial pressure |
| RVR | = | renal vascular resistance |
| UVR | = | uterine vascular resistance |
Acknowledgments
This research work was supported by grants from the American Heart Association (Ohio Affiliate), the Kidney Foundation of Greater Cincinnati, and the National Institutes of Health (HL-51051 and HL-49901). The authors wish to gratefully acknowledge Angella Friedman for her diligent assistance with the daily maintenance of animals used in this study.
- Received April 2, 1997.
- Revision received May 2, 1997.
- Accepted July 9, 1997.
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- Effects of Continuous Infusion of Endothelin-1 in Pregnant Sheep
