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CARDIOVASCULAR
Departments of Obstetrics and Gynecology (G.P.) and Medicine (C.P., S.D., M.I.-I., H.V.V., J.B.P.), Section of Nephrology, Tulane University School of Medicine, New Orleans, Louisiana
Received March 29, 2006; accepted May 18, 2006.
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; Pridjian and Puschett, 2002a). Yet, it is still a disease treated by symptom-directed therapy, usually magnesium sulfate and hydralazine or labetolol. Specific therapy directed to the underlying cause has not been developed.
The emerging concept regarding the etiology of preeclampsia is that it is probably multifactorial (Page, 1972; Ness and Roberts, 1996; Pridjian, 1999; Pridjian and Puschett, 2002b). Currently, there is no one satisfactory treatment regimen, which is directed at the underlying cause (Pridjian and Puschett, 2002b), although certain treatments have been proposed in certain preeclamptic subtypes (Saisto et al., 2004). We believe that successful definitive treatment of preeclampsia will be developed aimed at different subtypes.
Women with preeclampsia are volume-expanded, as are all pregnant women (Rovinsky and Jaffin, 1965; Scott, 1972). However, the added fluid resides in the interstitial, rather than intravascular, extracellular compartment (Gallery, 1999). In the past, diuretics were avoided in preeclampsia so as not to cause further volume contraction of the intravascular compartment and potential compromise of the maternalfetal circulation. However, volume contraction and decreased perfusion of the placenta with diuretics have never been proven.
We have postulated that at least some forms of preeclampsia are related to excessive expansion of the extracellular fluid (ECF) volume (Ianosi-Irimie et al., 2005; Vu et al., 2005). We hypothesize that women who develop this type of preeclampsia have an acquired or congenital defect in sodium transport, which prevents them from excreting the excess sodium. In most women, this defect would not become manifest until the patient is challenged with the 40 to 50% expansion of ECF volume that accompanies normal pregnancy (Gallery, 1999). The expansion process may actually result in the elaboration of one of more circulating factors (VanWijk et al., 2000; Hayman et al., 2001; Vu et al., 2005), which have both natriuretic and vasoconstrictive properties (Graves and Williams, 1984; Morris et al., 1988; Hilton et al., 1996; Lopatin et al., 1999). The authors have developed an animal model of preeclampsia (Ianosi-Irimie et al., 2005), which has many of the phenotypic characteristics of the human disease (Outland et al., 2005). It consists of two manipulations: 1) replacement of the drinking water of the pregnant rat with saline and 2) administration of the miner-alocorticoid desoxycorticosterone acetate (DOCA) to ensure that the excess sodium is retained. Under these circumstances, the animals develop hypertension, proteinuria, and intrauterine growth restriction (IUGR) (Vu et al., 2005).
Metolazone is a diuretic/antihypertensive agent that has been on the market for a number of years. It is a quinethazone derivative, a congener of the thiazide drugs (Puschett, 1972). It is considered safe in human pregnancy; specifically rated class B by the Food and Drug Administration use in pregnancy rating scale (Physician's Desk Reference, 2000). In low doses, the drug has antihypertensive activity with minimal diuresis and natriuresis (J. Puschett, unpublished observations). We reasoned that, if this were the case, it might be effective in treating our "preeclamptic" rats without compromising ECF, particularly intravascular volume.
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Materials and Methods |
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Blood Pressure and Sodium Excretion Measurements. Systolic blood pressure (BP) was measured using a tail-cuff method as described previously (Ianosi-Irimie et al., 2005), and 24-h urine collections were collected/measured daily. Sodium excretion was measured by flame photometry (IL 943; Instrumentation Laboratory Co., Lexington, KY).
Once animals in the PDS and PDSM groups became hypertensive (at approximately days 6–8), metolazone treatment commenced. The metolazone dose was adjusted daily according to the BP and to the last 24-h sodium (Na+) excretion measurement to ensure that metolazone was administered in non-natriuretic doses. For example, if the Na+ concentration in the 24-h urine obtained just before dosing was either greater than the Na+ concentration obtained the day before or greater than the sodium concentration of the untreated PDS group, the dose of metolazone administered would be decreased by 5 to 8%. If the BP was elevated but no evidence of natriuresis, the metolazone dose would be increased by 5 to 8%. The dose was thus individualized but ranged from 35 to 80 µg/kg body weight during the course of the experiment.
Protein, Creatinine, and Nitrite/Nitrate Assays. At days 19 to 20, a 24-h urine was collected for protein, creatinine, and NO determinations. On the 20th day of pregnancy, animals were humanely euthanized, and blood samples were taken. Pups and placentas were separated, and any pup malformations were noted.
Urinary protein was measured using the pyrogallol red method with a total protein kit (Sigma-Aldrich, St. Louis, MO). Blood and urine creatinine levels were measured using the picric acid method with a Beckman creatinine analyzer (Beckman Coulter, Fullerton, CA). Nitrite/nitrate (NOx) measurements in sera and in 24-h urine collections were performed using sulfanilamide and N-(-naphthyl)-ethylenediamine with a NO colorimetric assay (Roche Diagnostics, Indianapolis, IN). Hematocrit was measured using an Autocrit Ultra 3 centrifuge (BD Diagnostics, Sparks, MD).
Immunoblotting Analyses. Kidneys were excised, weighed, dissected, and washed in ice-cold saline buffered with 10 mM Tris-HEPES before removal of the cortex and medulla. Kidney slices (cortex and medulla) were homogenized in 50 mM mannitol buffered with 20 mM Tris-HEPES in washed sea sand (Fisher, Pittsburgh, PA). Cell extracts were obtained after centrifugation (1000g; 10 min), and protein was measured with a BCA assay kit (Pierce Chemical, Rockford, IL) with bovine albumin as the standard. Protein samples for immunoblotting analysis (10–15 µg of protein/sample) were prepared in Novex LDS sample buffer (Invitrogen, Carlsbad, CA), separated on 7% Tris-acetate NuPAGE gels (Invitrogen) (Laemmli, 1970), and transferred to a 0.2-µm nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were blocked (phosphate-buffered saline, 0.5% Tween 20, and 5% milk) for 1 h at room temperature; briefly rinsed (phosphate-buffered saline and 0.5% Tween 20); and stained with monoclonal anti-nitric-oxide synthase, endothelial (eNOS) or neuronal (nNOS), antibody (BD Transduction Laboratories, Lexington, KY). Membranes were then washed and incubated with a horseradish peroxidase-conjugated goat anti-mouse antibody (GE Healthcare, Piscataway, NJ) for 1 h. The chemiluminescent detection was performed by using ECL Western Blotting detection reagents (GE Healthcare), and autoradiographs were digitized by QuantiScan (Biosoft, Ferguson, MO). The results were normalized for 
-actin (monoclonal anti--actin antibody, clone AC-15; Sigma-Aldrich).
Statistical Analysis. Values are presented as mean ± S.E.M. Statistical comparison analyses were performed using covariant analysis for multiple determinations. A p value of less than 0.05 was considered significant.
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Twenty-four-hour Na+ excretion values (millimoles of Na+/24 h) in the control and normal pregnant animals were similar throughout the experiment (C, 3.0 ± 0.1; NP, 4.2 ± 0.3; day 19; p > 0.05) (Fig. 3). The animals in the PDS and PDSM groups had significantly higher but similar 24-h urinary Na+ excretion values (15.9 ± 2.2 and 16.1 ± 3.1, respectively; day 19; p > 0.05). The day 19, urinary Na+ mean values were consistent with sodium excretion rates obtained throughout the experiment once the animals were rendered hypertensive.
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Blood NO as estimated by NOx measurements (micromoles per liter) was significantly higher in the NP (42.4 ± 2.1; p < 0.001), PDS (34.3 ± 2.9; p < 0.01), and PDSM (31.7 ± 1.3; p < 0.05) groups compared with the nonpregnant C group (25.6 ± 2.4) (Fig. 5A). PDS animals had a significantly lower blood NOx compared with NP (p < 0.01). PDSM animals had a similar lowering compared with NP (p < 0.001). There was no difference noted between the PDS and PDSM groups (p > 0.05).
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There was no difference in urinary NO excretion (expressed in nanomolar NOx/milligram creatinine) between the C and NP groups (71.9 ± 11.8 versus 48.3 ± 10.5), but there was an increase in both PDS (151.1 ± 25.9) and PDSM (102.7 ± 19.1) groups versus NP (p < 0.001 and p < 0.05, respectively) (Fig. 5B). An increase in the urinary NOx versus controls of PDS animals was also present (p < 0.01). There was no difference in levels between PDS and PDSM groups (p > 0.05). A determination of eNOS and nNOS in Western blots from kidney cortex and medulla demonstrated no differences among the NP, PDS, and PDSM groups (data not shown).
Pup number in the PDS group was significantly decreased compared with the NP rats (13.9 ± 0.4 versus 11.2 ± 0.9; p < 0.05). However, despite a trend toward an improvement in the PDSM rats (13.4 ± 0.9), this value did not reach statistical significance (p > 0.05). With regard to malformations, none was noted in the NP group. The number of developmental malformations in the PDS animals was increased compared with NP (Fig. 6, A and B). These consisted of grossly immature and growth-retarded forms, occasional limb hypoplasia, and evidence of intrauterine death. The metolazone treatment resulted in a trend toward fewer growth malformations, but this value did not reach statistical significance.
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Hematocrit values were as follows: C, 0.52 ± 0.01; NP, 0.45 ± 0.02; PDS, 0.41 ± 0.02; and PDSM, 0.41 ± 0.08. The mean value for the C, nonpregnant animals was not statistically significantly different from that for NP (p > 0.05), but it was different from PDS (p < 0.05) and PDSM (p < 0.05) groups. However, the latter three treatments did not differ from each other.
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). Given the effectiveness of metolazone in the therapy of essential hypertension, even when given in non-natriuretic and nondiuretic doses, we elected to examine its use in our model of preeclampsia. We found that the drug was effective in lowering BP to normal pregnant levels without either a natriuresis or the disruption of fluid balance as determined by similar weights in the treated versus untreated animals (Fig. 2). We did not see resolution of proteinuria, and despite a trend toward improvement in IUGR, the values for pup number and average number of malformations did not reach statistical significance (Fig. 5A). However, it may be that the drug was given too late in the pathogenetic process to alter the abnormal glomerular permeability to protein and the vascular abnormalities leading to IUGR. We speculate that if we had instituted metolazone therapy before hypertension developed, we might have noted beneficial effects on these parameters.
The current therapy of uncomplicated mild-to-moderate preeclampsia does not include the use of antihypertensive agents. Once BP rises to levels seen in severe preeclampsia, hydralazine or labetalol is administered to control BP while delivery is planned. Avoidance of treatment of mild-to-moderate preeclamptic hypertension was practiced in the past, because despite improvement in BP, the disease process was thought to continue. Definitive proof that antihypertensive agents used in mild-to-moderate preeclampsia as a temporizing measure are harmful has never been well documented.
A general consensus (Lindheimer and Katz, 1973) that diuretics should not be used for treatment or prevention of preeclamptic hypertension and edema exists (Weseley and Douglas, 1962; Krause et al., 1966; Gray, 1968; Pitkin et al., 1972; Christianson and Page, 1976) despite some early reports of successful outcomes with these agents (Cuadros and Tatum, 1964; Finnerty and Bepko, 1966). This concept has resulted from the following observations. 1) Preeclamptic women already have a contracted intravascular volume despite expansion of the interstitial ECF space (Brown et al., 1989). 2) A rat model of pregnancy-induced hypertension, developed in the mid-1970s (Abitbol et al., 1976) and 1980s (Losonczy and Mucha, 1989) by reducing uterine perfusion pressure, has been used more recently as a paradigm for the late events of preeclampsia by Granger and coworkers. In this model, hypoperfusion of the maternal-fetal unit led to increased renal vascular resistance and hypertension (Alexander et al., 2001). Therefore, any additional decrement in ECF volume due to the use of diuretics might be expected to further reduce blood flow to the uterus and exacerbate the pathophysiology of the preeclampsia.
Excessive volume expansion as an important etiologic factor in the pathogenesis of preeclampsia may not be a universal phenomenon in the human syndrome. In our rat model of preeclampsia, we have identified a circulating inhibitor of sodium-potassium ATPase, the excretion of which is elevated before hypertension develops (Vu et al., 2005). This substance, marinobufagenin, could serve as a predictor of the later development of preeclampsia if the data obtained in rats is applicable to the human subject. Thus, therapy with metolazone might be attempted only in those patients in whom marinobufagenin excretion is elevated.
Past studies of the treatment of preeclamptic hypertension with diuretics have been problematic. The literature is replete with anecdotal reports. There have been no double-blinded, randomized, controlled investigations with adequate numbers to achieve valid conclusions. Confounding the results of many of these reports is the fact that patients with preeclampsia, superimposed upon preexisting hypertension from various etiologies, were included. Finally, the doses of the diuretics used to treat the hypertension and edema of preeclampsia were natriuretic, and by today's standards rather large. For example, chlorothiazide was given in doses of 500 to 1000 mg/day, equivalent to a dose of hydrochlorothiazide of 50 to 100 mg/day. These dosages are no longer used even in the therapy of essential hypertension. Thus, any potentially beneficial effect of this class of drugs on the disease process was most probably vitiated by the introduction of ECF volume contraction. Only in recent years has it been recognized that small doses of these drugs are effective in the therapy of essential hypertension (Puschett, 1999).
IUGR is a common concomitant of preeclampsia (Eskenazi et al., 1993; Obegard et al., 2000; Xiao et al., 2003). In the animal model described herein (Ianosi-Irimie et al., 2005), IUGR occurred. Furthermore, although the values did not reach statistical significance, the data suggest that IUGR might be either prevented or mitigated in this rat model of preeclampsia by the administration of metolazone (Fig. 5).
It is not possible, given our results, to implicate NO as an important pathogenetic factor in these studies. Furthermore, because there were no differences, either in the blood or urine NO levels between the PDS and PDSM rats, it is clear that the mechanism by which metolazone lowered BP did not involve alterations in NO. These conclusions are verified by the fact that there were no changes either in eNOS or nNOS. We suspect that the increased excretion of NO in the expanded animals was the result of an increment in Na+ excretion (Shultz and Tolins, 1993) and that the augmented clearance of this substance by the kidney may have led to a decline in the blood levels that were observed.
The mechanism by which metolazone reduced BP is currently unknown. However, because volume contraction was not involved, it must have represented a vasodilatory effect. Because a NO mechanism is not supported by our data, further studies will be required to determine the nature of this effect.
In conclusion, metolazone has proven effective in ameliorating the hypertension in this rat model of preeclampsia. There was a trend toward a reduction in pup malformations and an improvement in pup number with metolazone that did not reach statistical significance. Perhaps provision of the drug earlier in pregnancy would result in an improvement in these parameters. The latter studies are currently planned.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ECF, extracellular fluid; DOCA, desoxycorticosterone acetate; IUGR, intrauterine growth restriction; NP, normal pregnant; PDS, pregnant + DOCA + saline; PDSM, pregnant + DOCA + saline + metolazone; BP, blood pressure; NOx, nitrite/nitrate; eNOS, endothelial nitric-oxide synthase; nNOS, neuronal NOS; C, control.
Address correspondence to: Dr. Gabriella Pridjian, Department of Obstetrics and Gynecology, SL-11, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112-2699. E-mail: gabriella.pridjian{at}tulane.edu
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