Hypertension Produced by Reductions in Uterine Perfusion in the Pregnant Rat
Role of Tumor Necrosis Factor-α
Inflammatory cytokines such as tumor necrosis factor (TNF)-α are elevated in preeclamptic women and are thought to be an important link between placental ischemia and endothelial dysfunction. The purpose of this study was to determine the role of TNF in mediating hypertension in response to chronic reductions in uterine perfusion (RUPPs) in pregnant rats. Arterial pressure was significantly higher in RUPP rats (138±1 mm Hg) than in pregnant rats (107±1 mm Hg). Serum TNF-α levels in the RUPP rats were 17±4 pg/mL compared with 8±1 pg/mL in normal pregnant rats. To determine the long-term effects of a 2- to 3-fold elevation in plasma TNF-α on renal and systemic hemodynamics in pregnant rats, we infused TNF-α for 5 days at a rate of 50 ng/d during days 14 to 19 of gestation in pregnant rats. Serum levels were 7±2 pg/mL in the control pregnant rats and 14±2 pg/mL in the TNF-α–treated pregnant rats. Mean arterial pressure was higher in the TNF-α–treated pregnant rats (123±3 mm Hg) compared with pregnant controls (96±3 mm Hg) at day 19 of gestation. TNF-α increased renal vascular resistance in pregnant rats by 182%. Renal plasma flow was 5.4±1.2 mL/min in the TNF-α–treated group and 9.2±1.6 mL/min in the control group. Glomerular filtration rate was 1.7±0.4 mL/min in the TNF-α–treated group and 2.6±0.4 mL/min in the control group. In summary, these data suggest that TNF-α may play an important role in mediating the increased arterial pressure in response to chronic RUPPs in pregnant rats.
Although reductions in blood flow to the uteroplacental unit are known to result in cardiovascular and renal abnormalities consistent with the pathophysiologic characteristics of human preeclampsia, the physiologic mechanisms linking placental ischemia with abnormalities in the maternal circulation are unclear.1,2 Several lines of evidence support the hypothesis that the ischemic placenta contributes to endothelial cell activation/dysfunction of the maternal circulation by enhancing the synthesis of cytokines such as tumor necrosis factor (TNF)-α.1–4 Inflammatory cytokines such as TNF-α have been shown to induce structural as well as functional alterations in endothelial cells.1,5–10 TNF-α also enhances the formation of a number of endothelial cell substances, such as endothelin; reduces acetylcholine-induced vasodilatation; and destabilizes the mRNA of endothelial nitric oxide synthase (eNOS).5–9 Also supporting a potential role for TNF-α in preeclampsia are findings that plasma levels of TNF-α are significantly elevated in women with preeclampsia by ≈2-fold.1,11–13 Furthermore, interleukin-6, which is activated by TNF-α, has also been reported to be elevated in preeclamptic women.14
We recently reported that chronic reductions in uterine perfusion pressure (RUPPs) in pregnant rats resulted in significant elevations in mean arterial pressure (MAP) and reductions in renal plasma flow (RPF) and glomerular filtration rate (GFR).15 Although serum TNF-α levels are elevated in women with preeclampsia, it is unclear whether the hypertension associated with chronic RUPPs in pregnant rats is associated with elevated plasma levels of TNF-α. Therefore, the purpose of this study was to determine the role of TNF-α in mediating the cardiovascular and renal changes observed during chronic RUPPs in pregnant rats. To achieve this goal, we first determined whether the hypertension associated with chronic RUPPs in pregnant rats was associated with elevated serum levels of TNF-α. The second objective was to determine the arterial pressure and renal hemodynamic effects of infusing TNF-α into chronically instrumented pregnant rats at a rate that mimics plasma levels observed in RUPP rats.
All studies were performed in timed pregnant Sprague-Dawley rats purchased from Harlan Sprague Dawley Inc (Indianapolis, Ind). Animals were housed in a temperature-controlled room (23°C) with a 12:12-hour light/dark cycle. All experimental procedures executed in this study were in accordance with National Institutes of Health guidelines for use and care of animals, and the institutional Animal Care and Use Committee at the University of Mississippi Medical Center approved all protocols.
Effect of Chronic RUPPs on Arterial Pressure and Serum Levels of TNF-α
Experiments were performed in the following groups of rats: pregnant controls (n=24) and RUPP pregnant rats (n=26). All pregnant rats undergoing surgical procedures were anesthetized with 2% isoflurane (W. A. Butler Co) delivered by an anesthesia apparatus (Vaporizer for Forane Anesthetic, Ohio Medical Products). Pregnant rats entering the RUPP group underwent a clipping procedure at day 14 of gestation. After a midline incision was made, the lower abdominal aorta was isolated and a silver clip (0.230-mm i.d.) was placed around the aorta above the iliac bifurcation. Branches of both the right and left ovarian arteries were also clipped with a silver clip (0.10-mm i.d.) as previously described.15 Rats were also surgically instrumented with a carotid catheter for subsequent arterial pressure measurement on day 19. At day 19 of gestation, arterial pressure was recorded and blood samples were taken.
Effect of TNF-α on Arterial Pressure and Renal Hemodynamics in Conscious, Chronically Instrumented Pregnant Rats
Experiments were performed in the following groups of rats: pregnant controls (n=5) and TNF-α–treated pregnant rats (50 ng/d, n=7). Recombinant, purified, rat TNF-α (BioSource International) was infused for 5 days during days 14 to 19 of gestation in pregnant rats. During isoflurane anesthesia, rats at day 14 of pregnancy were surgically instrumented with catheters (PE50 tubing) in the femoral vein and carotid artery for blood sampling and blood pressure monitoring.15 TNF-α–treated pregnant rats were also instrumented with jugular vein catheters connected to miniosmotic infusion pumps. A midline lower abdominal incision was made, and the bladder was cannulated with flare-tipped PE90 tubing for urine collection. All catheters were tunneled to the back of the neck and exteriorized.
Renal hemodynamics and arterial pressures were determined in control pregnant rats and pregnant rats treated with TNF-α at day 19 of gestation. The femoral vein catheter was connected to an infusion pump that delivered isotonic saline containing [I125]iodothalamate (Isotex Diagnostics; 0.05 mCi · kg−1 · min−1) and [I131]iodohippurate (Syncor International Corp; 0.1 mCi · kg−1 · min−1) at a fixed rate of 3 mL/h. Arterial pressure was monitored with a pressure transducer connected to an arterial pressure–recording device. After a 1-hour equilibration period, two 20-minute clearances were obtained in each rat. Urine volume was determined gravimetrically. GFR and RPF were calculated from concentrations of [I125] and [I131], respectively, in plasma and urine.16
TNF-α ELISA Assay
Serum TNF-α was measured with a rat parameter TNF-α ELISA purchased from R&D Systems. Precision assays are 5% for intraassay and 10% for interassay. The sensitivity of the assay was 5.0 pg/mL.
All data are expressed as mean±SEM. Comparisons of control rats with experimental rats were analyzed by factorial ANOVA followed by Scheffe test. A value of P<0.05 was considered statistically significant.
MAP and Serum TNF-α Levels in Response to RUPPs in Pregnant Rats
MAP was significantly higher (P<0.05) in RUPP rats (138±1 mm Hg) than in pregnant rats (107±1 mm Hg). Associated with the elevation in arterial pressure in RUPP rats were increases in serum levels of TNF-α (Figure 1). Serum TNF-α levels in the RUPP rats were 17±4 pg/mL compared with 8±1 pg/mL in normal pregnant rats (P=0.0584).
Serum TNF-α Levels and Arterial Pressures in Control and TNF-α–Treated Pregnant Rats
Figure 2 illustrates serum levels of TNF-α in control pregnant rats and in pregnant rats infused with TNF-α for 5 days at a rate of 50 ng/d. Plasma TNF-α concentration was 7±2 pg/mL in control pregnant rats. Pregnant rats infused with TNF-α at a rate of 50 ng/d had a 2-fold elevation (P<0.05) in plasma levels of TNF-α (14±2 pg/mL, P<0.05), which is comparable to the serum levels observed in RUPP rats. Figure 2 also illustrates MAPs in control pregnant rats and in pregnant rats infused with TNF-α for 5 days at a rate of 50 ng/d. MAP averaged 96±3 mm Hg in control pregnant rats. Arterial pressure in pregnant rats infused with TNF-α at a rate of 50 ng/d averaged 123±3 mm Hg, which was ≈28% above control rats, P<0.05.
Renal Hemodynamics in Control and TNF-α–Treated Pregnant Rats
The differences in renal hemodynamics observed in control pregnant rats and in pregnant rats infused with TNF-α for 5 days at a rate of 50 ng/d are shown in Figure 3. Renal vascular resistance was significantly increased in pregnant rats infused with TNF-α at a dose of 50 ng/d (21.6±5.7 mm Hg · mL−1 · min−1, P<0.05) compared with control pregnant rats (7.7±1.0 mm Hg · mL−1 · min−1). RPF and GFR were decreased in pregnant rats infused with TNF-α at a dose of 50 ng/d compared with control rats. RPF decreased from 9.2±1.6 mL/min in control pregnant rats to 5.4±1.2 mL/min (P<0.05) in pregnant rats infused with TNF-α. GFR decreased from an average of 2.6±0.4 mL/min in control pregnant rats to 1.7±0.4 mL/min (P<0.05) in pregnant rats infused with TNF-α.
We previously reported that chronic RUPPs in pregnant rats resulted in significant elevations in arterial pressure and reductions in RPF and GFR.15 We report in this study that the elevation in arterial pressure in RUPP rats is also associated with significant increases in serum levels of TNF-α. Moreover, we found that a chronic 2-fold elevation in serum levels of TNF-α, comparable to levels observed in RUPP rats, results in significant hypertension in pregnant rats. The increase in arterial pressure is associated with increases in renal vascular resistance and reductions in RPF and GFR. These results indicate that TNF-α may play a role in mediating the hypertension and reduction in renal hemodynamics observed in RUPPs in pregnant rats.
Most, but not all, investigators have reported significant elevations in plasma levels of TNF-α in women with preeclampsia.1 On average, plasma levels of TNF-α are increased by ≈2- to 3-fold in women with preeclampsia.1 The source of the TNF-α in preeclampsia or in response to placental ischemia is uncertain. Previous in vitro studies have implicated placental cytotrophoblast and/or Hofbauer cells as potential sites of cytokine synthesis in preeclampsia.1 However, a recent study by Benyo et al11 suggested that sources other than the placenta contribute to the elevated concentrations of TNF-α found in the circulation of preeclamptic women. Those investigators found no significant differences in TNF-α mRNA or protein levels between normal-term and preeclamptic placentas. Although peripheral and uterine venous levels of TNF-α were elevated in preeclamptic women, there were no significant differences in the concentration of TNF-α between peripheral and uterine venous plasma samples. Thus, identification of the source of the TNF-α in preeclampsia remains an important area of investigation.
In this study, we infused TNF-α into pregnant rats to mimic levels of serum TNF-α observed in RUPP rats. Rat TNF-α was infused at a rate of 50 ng/d for 5 days, and plasma levels of rat TNF-α were measured by ELISA. The 50 ng/d dose increased plasma levels of TNF-α 2-fold, which is comparable to the increase observed in RUPP rats and in women with preeclampsia.1 The 2-fold elevation of TNF-α resulted in significant increases in arterial pressure (>25%) compared with untreated pregnant rats at day 19 of gestation. The increase in arterial pressure in the TNF-α–treated rats was associated with a 30% to 40% reduction in RPF and GFR. The decrease in renal hemodynamics in response to TNF-α is similar to the decrease in renal hemodynamics we previously reported to occur in RUPP rats.
Enhanced susceptibility to the renal and cardiovascular effects of lipopolysaccharide (LPS) during pregnancy has also been reported. Faas and colleagues17 previously demonstrated that a single low-dose infusion of LPS elevated blood pressure and albumin excretion in pregnant rats. Although it is presumed that the LPS effects were cytokine mediated, it cannot be certain, because TNF-α was not measured in that study. A more recent study by Faas et al,18 however, demonstrated that pretreatment with corticosterone inhibited the low-dose endotoxin-induced glomerular inflammatory reaction in pregnant rats.
Although TNF-α is known to mediate hypotension during sepsis via activation of inducible NOS, the mechanisms whereby modest increases in plasma TNF-α elevate arterial pressure during pregnancy are not known. TNF-α is known to activate a variety of vasoconstrictors, such as endothelin and platelet-derived growth factor from cultured endothelial cells.1,5–7 TNF-α has also been reported to reduce endothelium-dependent vasodilation.8,9 Additionally, a modest elevation of TNF-α has been shown to destabilize the mRNA of endothelial NOS.16 We previously reported that protein expressions of both neuronal and inducible NOS were significantly decreased in the medulla of pregnant rats infused with TNF-α.19 Moreover, we have preliminary data that indicate that TNF-α enhances the expression of pre-proendothelin in pregnant rats.20 Although all of these effects of TNF-α on endothelial cell function can potentially lead to enhanced vasoconstriction and hypertension, the quantitative importance of each of these actions in mediating the in vivo effects of TNF-α during pregnancy are unknown and remain an important area of investigation.
Preeclampsia, which affects 5% to 10% of all pregnancies in the United States, is a multisystemic disorder of pregnancy that is associated with hypertension and endothelial dysfunction. Despite being 1 of the leading causes of maternal and perinatal morbidity and mortality, the pathophysiologic mechanisms underlying hypertension during preeclampsia are unknown. Increases in circulating factors such as inflammatory cytokines, including TNF-α, may serve as an important mediator of maternal endothelial activation and/or dysfunction in preeclampsia. In this study, we report that the elevation in arterial pressure in RUPP rats is also associated with significant increases in serum levels of TNF-α. Moreover, we found that a chronic 2-fold elevation in serum levels of TNF-α, comparable to levels observed in RUPP rats, results in significant hypertension in pregnant rats. The increase in arterial pressure is associated with increases in renal vascular resistance and reductions in RPF and GFR. These results indicate that TNF-α may play a role in mediating the hypertension and reduction in renal hemodynamics observed in RUPPs in pregnant rats.
Although we have shown that TNF may play an important role in mediating the elevation in arterial pressure in response to RUPPs, the importance of TNF and other cytokines in mediating the cardiovascular and renal alterations during preeclampsia in humans remain unclear. Furthermore, it is unknown whether drugs that inhibit the actions of inflammatory cytokines may be of benefit to women at high risk of developing preeclampsia. These important questions will not be answered until well-controlled clinical studies, using specific inhibitors of cytokines, are performed in women with preeclampsia.
This work was supported by NIH grants HL38499and HL51971.
This paper was sent to Gerald F. DiBoni, associate editor, for review by expert referees, editorial decision, and final disposition.
- Received May 5, 2005.
- Revision received May 25, 2005.
- Accepted June 15, 2005.
Hajjar KA, Hajjar DP, Silverstein RL, Nachman RL. Tumor necrosis factor-mediated release of platelet-derived growth factor from cultured endothelia cells. J Exp Med. 1987; 166: 235–245.
van Hinsbergh VW, Kooistra T, van den Berg EA, Princen HM, Fiers W, Emeis JJ. Tumor necrosis factor increases the production of plasminogen activator inhibitor in human endothelial cells in vitro and rats in vivo. Blood. 1988; 72: 1467–1473.
Benyo DF, Smarason A, Redman CW, Sims C, Conrad KP. Expression of inflammatory cytokines in placentas from women with preeclampsia. J Clin Invest. 2001; 86: 2505–2512.
Alexander BT, Kassab SE, Miller MT, Abram SR, Reckelhoff JF, Bennett WA, Granger JP. Reduced uterine perfusion pressure during pregnancy in the rat is associated with increases in arterial pressure and changes in renal nitric oxide. Hypertension. 2001; 37: 1191–1195.
Yoshizumi M, Perrella MA, Burnett JC, Lee ME. Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. Circ Res. 1993; 73: 205–209.
LaMarca BB, Gadonski G, Cockrell K, Sullivan E, Granger JP. Endothelin type A receptor blockade attenuates TNF-α-induced hypertension in pregnant rats. FASEB J. 2004; 475. Abstract.