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Obstetrics & Gynecology:
doi: 10.1097/01.AOG.0000207698.74104.4f
Original Research

Fractional Excretion of Angiogenic Factors in Women With Severe Preeclampsia

Buhimschi, Catalin S. MD; Magloire, Lissa MD1; Funai, Edmund MD1; Norwitz, Errol R. MD1; Kuczynski, Edward PhD1; Martin, Ryan MD1; Richman, Susan MD1; Guller, Seth PhD1; Lockwood, Charles J. MD1; Buhimschi, Irina A. MD1

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Author Information

From the 1 Department of Obstetrics, Gynecology and Reproductive Science, Yale University, New Haven, Connecticut.

Corresponding author: Dr. Catalin Buhimschi, Yale University, Department of Obstetrics, Gynecology and Reproductive Science, 333 Cedar Street, LLCI 804 New Haven, CT 06520; e-mail: catalin.buhimschi@yale.edu.

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Abstract

OBJECTIVE: We estimated the fractional excretions of soluble fms-like tyrosine kinase 1 (sFlt-1), vascular endothelial growth factor, and placental growth factor of severely preeclamptic women at the time of disease clinical manifestation.

METHODS: Levels of free sFlt-1, vascular endothelial growth factor, and placental growth factor were measured by immunoassay from time-matched serum-urine samples from 64 women in the following groups: nonpregnant reproductive aged (n = 9), healthy pregnant controls (n = 13), mildly preeclamptic women (n = 15), and women with severe preeclampsia (n = 27). Urinary concentrations of angiogenic factors were normalized to creatinine and fractional excretions calculated. Correlations were estimated between fractional excretions of angiogenic factors, albuminuria, nonspecific proteinuria and urine protein-to-creatinine ratio.

RESULTS: Severely preeclamptic women had more than double urinary vascular endothelial growth factor (P = .01) and fractional excretion of vascular endothelial growth factor compared with mildly preeclamptic women (P = .007) or pregnant controls (P < .001). Serum, urine and fractional excretion levels of sFlt-1 were much higher among severely preeclamptic women compared with all the other pregnant groups (P < .001). Conversely, severely preeclamptic women had lower serum placental growth factor levels compared with healthy pregnant women (P < .05) and mildly preeclamptic groups (P < .05). Severely preeclamptic women had increased fractional excretions of placental growth factor, albumin, proteinuria, and random urine total protein/creatinine ratio. Among severely preeclamptic women there was no correlation between proteinuria and fractional excretion of vascular endothelial growth factor (r = 0.30, P = .127) or sFlt-1 (r = 0.35, P = .07). There was a significant correlation between fractional excretion for placental growth factor, random urine total protein/creatinine ratio (r = 0.60, P = .002), and nonspecific proteinuria (r = 0.50, P = .01).

CONCLUSION: Severe preeclampsia is characterized by increased fractional excretion of angiogenic factors and especially of vascular endothelial growth factor, likely reflecting 2 separate phenomena that may have additive effects: “endogenous” renal production and glomerular “leakage.”

LEVEL OF EVIDENCE: II-2

Preeclampsia remains a leading cause of pregnancy-associated maternal and perinatal morbidity and mortality worldwide.1,2 Despite extensive research efforts, the etiology of this multisystemic disorder specific to human pregnancy remains unknown. Theories of its cause include abnormal implantation and development of the placenta, oxidative stress, impaired endothelial prostanoid and nitric oxide homeostasis, genetic polymorphisms, abnormal circulating autoantibodies, and an abnormal maternal systemic inflammatory response.3–7 Most recently, there has been increased attention focused on alterations in serum levels of circulating proangiogenic factors and their inhibitors.8

Previous reports have shown that maternal serum concentrations of soluble fms-like tyrosine kinase 1 (sFlt-1), vascular endothelial growth factor, and placental growth factor are altered in preeclamptic women.9 Alterations appear to precede the onset of clinically identifiable disease by approximately 5 weeks.9 Evidence from several quarters supports the view that defective placentation leads to placental ischemia followed by systemic release of cytotoxic products that damage maternal vascular endothelium. Thus, it is plausible that derangements in angiogenesis may also exert indirect effects on the general maternal vasculature including that of the kidney. Moreover, widespread kidney endothelial cell injury is evidenced by one of the most characteristic morphologic lesion of preeclampsia, glomerular endotheliosis.10–14

We and others recently demonstrated that profound alteration in excretion of urinary angiogenic factors occur at the time of clinical manifestation of severe preeclampsia.15,16 Glomerular damage often occurs in women with severe preeclampsia and may account for the increased release of these factors in the urine. Alternatively, the increased urinary excretion of angiogenic factors in severe preeclampsia may reflect increased placental or systemic vascular synthesis and thus higher serum concentrations. In support of this theory, measured concentrations of the urinary angiogenic factors sFlt-1 and placental growth factor but not of vascular endothelial growth factor were consistently reported to be below maternal serum levels.15,16 Therefore, our previous findings seem to beg the question: “What is the origin of the urinary angiogenic factors in severe preeclampsia?” We sought to answer this question by investigating, in parallel, the concentration of these angiogenic factors within maternal serum and urine and by calculating the fractional excretion indicators for sFlt-1, vascular endothelial growth factor, and placental growth factor in healthy nonpregnant, pregnant, and preeclamptic women.

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MATERIALS AND METHODS

We studied paired collections of serum and urine samples from 64 women admitted at Yale New Haven Hospital between February 2004 and January 2005. Samples were collected under protocols approved by the Human Investigation Committee of Yale University. All participants provided informed consent before enrollment and all women solicited for enrollment agreed to participate. The urine, but not serum immunoassay, results of 14 women were previously reported.15 Gestational age was established based on menstrual date and/or ultrasonographic examination before 20 weeks gestation. Subjects were recruited from women evaluated or admitted to labor and birth unit the antepartum high and low risk units. Our subjects were solicited for enrollment prospectively based on the availability of one of the investigators (C.S.B.). None of the enrolled women were excluded from the final analysis.

We enrolled patients in the following groups: severe preeclampsia (n = 27), mildly preeclamptic hypertensive and proteinuric women who did not meet criteria for severe preeclampsia (n = 15), healthy pregnant control women (n = 13), and healthy nonpregnant women of reproductive age (n = 9). Mild preeclampsia was defined according to established criteria, as a diastolic blood pressure of at least 140/90 mm Hg and urinary excretion of at least 0.3 g protein/24-hour urine specimens (or at least 1+ or greater on dipstick testing), each on 2 occasions 4–6 hours apart.17 Severe preeclampsia was defined as HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count), blood pressure above 160/110 mm Hg on at least 2 occasions 6 hours apart, more than 5 g in a 24-hour urinary protein excretion, and or persistent +3 proteinuria on dipstick testing.17 Other elements of the severe preeclampsia definition included in utero growth restriction below the 10th percentile, persistent neurologic symptoms (headache, visual disturbances), epigastric pain, oliguria (less than 500 mL/24 hours), serum creatinine above 1.0 mg/dL, elevated liver enzymes (greater than 2 times normal), and thrombocytopenia (< 100,000 cells/μL).17 Chronic hypertension was defined as a sustained elevation in blood pressure above 140/90 mm Hg before pregnancy or before 20 completed weeks gestation. Proteinuria was defined as more than 300 mg protein in a 24-hour period of urine collection.17

A random urine sample (5–10 mL) was collected by standard use of sterile containers. At the time of enrollment all severe preeclampsia women had a Foley catheter placed to allow for accurate monitoring of urinary output. In the absence of a Foley catheter urine samples were collected using other techniques (bladder catheterization or “clean catch” method). Samples obtained from mildly preeclamptic women, healthy pregnant controls, and healthy nonpregnant controls were also collected under sterile conditions (Foley, bladder catheterization, or “clean catch” technique). Seventy percent of severely preeclamptic women were enrolled before initiation of magnesium sulfate seizure prophylaxis. A sample of blood was collected by venipuncture at the time of urine collection and allowed to clot. Samples were collected at the time of admission, before labor induction or Cesarean delivery. Serum and urine samples were spun at 3,000g at 4°C for 20 minutes, and the supernatant was aliquoted and immediately stored at −80°C until sFlt-1, vascular endothelial growth factor, and placental growth factor levels were measured using specific immunoassays.

Enzyme-linked immunosorbent assays for human unbound vascular endothelial growth factor, sFlt-1, and placental growth factor were performed according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). Briefly, serum and urine samples were assayed in duplicate in a 96-well plate precoated with a capture antibody directed against free vascular endothelial growth factor, sFlt-1, or placental growth factor. Incubation protocols were performed followed by washings and reading at 450 nm in accordance with the procedure summary. The minimal detectable concentrations in the assays for vascular endothelial growth factor, sFlt-1, and placental growth factor were 2, 5, and 7 pg/mL, respectively. The interassay and intraassay coefficients of variation varied from 3% to 10%. Plates were read at 450 nm with 570-nm wavelength correction using a VERSAmax microplate reader with Softmax Pro 3.1.1 software (Molecular Devices, Sunnyvale, CA). This software reports a positive value if the optical density of the sample wells is above that of the zero standard (Blank wells). If the optical density of a sample well is below that of the zero standard, a negative value is reported and automatically converted to zero by the computer. Serum vascular endothelial growth factor was the only analyte where we had instances of undetectable levels (values lower than zero standard) in any of the assays.

For albumin immunoassays microtiter plates (Immuno MaxiSorp, Nalge Nunc, Rochester, NY) were coated with capture antibody (10 μg/mL goat anti–human albumin antibody (Bethyl Laboratories). The plates were washed, blocked and incubated with urine (1:1,000 dilution or 1:100 for nonpregnant patients) or serum samples (diluted 1:150,000) or human albumin calibrants (Bethyl Laboratories) in a range from 6.25 to 400 ng/mL. Detection was accomplished using a goat anti–human albumin antibody conjugated to horseradish peroxidase (1:150,000 dilution, Bethyl Laboratories) and 3,3′,5,5′-tetramethylbenzidine (Vector Laboratories, Burlingame, CA) as substrate. The color reaction was stopped with 2 M sulfuric acid and plates were read at 450 nm with 650-nm wavelength correction. The intraassay coefficient of variation was less than 5%. The sensitivity of the assay was 1 ng/mL.

Creatinine levels in serum and urine were measured in the same aliquot used for immunoassays using a colorimetric assay (Stanbio Laboratory, Boerne, TX) against standard curves derived from known concentrations. Total protein levels were also measured using a bicinchoninic acid/cupric sulfate reagent (BCA kit, Pierce, Rockford, IL). Urinary levels of angiogenic factors, protein or albumin were normalized to urinary creatinine concentrations.

The urine random total protein/creatinine ratio has been long advocated as a strong predictor of a 24-hour urine total protein excretion, and was used for our correlation analysis.18

We subjected all data sets to normality testing using the Kolmogorov-Smirnov method and report our data as either mean and 95% confidence interval (CI) or standard error of the mean (for normally distributed data) or as median with range (for nonnormally distributed data). The statistical analysis for the fractional excretion of angiogenic factors was completed following logarithmic transformation of the data. Pairwise multiple comparison procedures were performed using one-way analysis of variance (ANOVA), Student-Newman-Keuls, or Kruskal-Wallis analysis of variance on ranks followed by Dunn's tests as appropriate. Proportions were compared with the Fisher exact test. A Spearman's product moment correlation was used to measure colinearity between the selected independent variables as well as other relevant correlations between dependent and independent variables. The fractional excretion of a substance represents the proportion of the substance excreted in the urine compared with that filtered by the glomeruli. It is generally reported relative to creatinine clearance because creatinine is neither resorbed nor significantly secreted and thus any effects of urine concentration/dilution are cancelled out. For each angiogenic factor a fractional excretion indicator was calculated using the following formula: [Ua] × [Sc] / [Sa] × [Uc], with [Ua] and [Sa] representing the urinary and serum concentration of the angiogenic factor, respectively, and [Uc] and [Sc] representing urinary and serum creatinine concentration. Similar calculations were performed for albumin and total proteins. A P value of less than .05 indicated statistical significance.

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RESULTS

The clinical characteristics of our subjects are presented in Table 1. The mildly preeclamptic women were significantly older compared with severely preeclamptic women (Student-Newman-Keuls, P = .04) and healthy pregnant controls (P = .04) (Table 1). Severely preeclamptic women developed hypertensive proteinuria at an earlier gestational age (P = .03) and delivered babies with significantly lower birth weights (P = .02) compared with the mildly preeclamptic women. Due to severity of disease and fetal malposition (breech, transverse), 74% of the women with severe and 60% of the women with mild preeclampsia were delivered by cesarean.

Table 1
Table 1
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Hypertensive women in both the severe and mild preeclampsia groups had significantly higher blood pressures compared with pregnant controls (mean arterial pressure [95% CI]: severe preeclampsia, 124 [120–128.5]; mild preeclampsia, 114 [110.5–118]; healthy pregnant control women, 80 [74–85] mm Hg; Kruskal-Wallis ANOVA P < .001). Our clinical diagnosis was supported by clinical laboratory changes (Table 2).

Table 2
Table 2
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We present the results of our analyses for serum and urinary protein and creatinine concentrations and immunoassays for vascular endothelial growth factor, sFlt-1, placental growth factor, and albumin in Table 3. Severely preeclamptic women had significantly lower serum (Kruskal-Wallis ANOVA, P = .017) but not urinary (P = .495) protein concentrations compared with mildly preeclamptic women. Severely preeclamptic but not mildly preeclamptic women had significantly increased urine random total protein-to-creatinine ratios compared with the other study groups (Kruskal-Wallis ANOVA, P = .007).

Table 3
Table 3
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Serum albumin levels did not differ significantly among groups (Table 3). However, we demonstrated that there was a significant difference in albuminuria among groups as assayed in a random urine sample (P < .001). There was no significant correlation between proteinuria and albuminuria for mild preeclampsia (r = 0.477, P = .080) or severe preeclampsia groups (r = 0.143, P = .472) Serum creatinine concentrations of women with severe preeclampsia were significantly higher compared with mild preeclampsia (one-way ANOVA, P = .04) and healthy pregnant control women (P = .005).

There were significant differences in the serum and urine concentrations among groups for vascular endothelial growth factor, sFlt-1, and placental growth factor. Pregnancy was characterized by decreased serum level of vascular endothelial growth factor (healthy nonpregnant controls versus healthy pregnant controls, P < .001) (Table 3). Severely preeclamptic women had significantly lower serum concentration of free vascular endothelial growth factor compared with healthy pregnant controls (Kruskal-Wallis, ANOVA, P < .05) but not mildly preeclamptic women (P > .05) (Fig. 1A). There was no difference in the urinary concentrations of vascular endothelial growth factor among healthy nonpregnant controls, healthy pregnant controls, and mildly preeclamptic women (One-Way ANOVA, P = .371). In contrast, severely preeclamptic women had significantly higher urine levels of vascular endothelial growth factor compared with mildly preeclamptic women (P = .01) (Fig. 1B). There was no significant correlation between albuminuria and urinary levels of vascular endothelial growth factor for the severe preeclampsia group (r = 0.083, P = .860). There was a significant correlation between proteinuria and urinary concentration of vascular endothelial growth factor in the mild preeclampsia (r = 0.713, P = .003) but not in the severe preeclampsia group (r = −0.021, P = .918). There was no correlation between urinary vascular endothelial growth factor and proteinuria when all preeclamptic subjects were analyzed as a group (r = 0.164, P = .30). These findings suggest that severe preeclampsia alters profoundly the serum and urinary concentration of this angiogenic factor but independently of proteinuria.

Fig. 1
Fig. 1
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Pregnancy was associated with increased serum levels of sFlt-1 (Kruskal-Wallis ANOVA, healthy nonpregnant controls versus healthy pregnant controls, P < .001) (Fig. 2A). At the time of clinical diagnosis, both mild preeclampsia and severe preeclampsia groups had significantly elevated mean serum levels of sFlt-1 compared with healthy pregnant controls (P < .05). Severely preeclamptic women distinguished themselves from mildly preeclamptic women in that their serum sFlt-1 concentrations were 47% higher (severe preeclampsia versus mild preeclampsia, P < .05). Urinary concentrations of sFlt-1 were not influenced by pregnancy per se (healthy nonpregnant controls versus healthy pregnant controls, P > .05) (Fig. 3B). However, mildly preeclamptic subjects had significantly higher urinary levels of sFlt-1 compared with healthy pregnant controls (Kruskal-Wallis ANOVA, P = .007). Urinary concentrations of sFlt-1 increased with the degree of disease severity (severe preeclampsia versus mild preeclampsia, P < .05). There was no significant correlation between albuminuria and urinary levels of sFlt-1 within the severely preeclamptic group (r = 0.324, P = .873).There was no significant correlation between proteinuria and urinary concentration of sFlt-1 for either mild preeclampsia (r = 0.137, P = .628) or severe preeclampsia groups (r = 0.336, P = .087). In summary, preeclamptic women have significantly elevated serum and urinary concentrations of sFlt-1. This derangement varies with the severity of preeclamptic disease.

Fig. 2
Fig. 2
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Fig. 3
Fig. 3
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Serum and urine concentration of placental growth factor were significantly elevated during normal pregnancy (Kruskal-Wallis ANOVA, healthy pregnant controls versus healthy nonpregnant controls, P < .05) (Figs. 3A and 3B). Moreover, serum placental growth factor levels in women with severe preeclampsia or mild preeclampsia were significantly lower (approximately 20%) compared with healthy pregnant controls (severe preeclampsia versus healthy pregnant control women, P < .05; mild preeclampsia versus healthy pregnant controls, P < .05). Women with severe preeclampsia had significantly lower serum levels of placental growth factor compared with mildly preeclamptic women (severe preeclampsia versus mild preeclampsia, P < .05) (Fig. 4A). Likewise, this ratio was maintained concerning urinary concentrations of placental growth factor (severe preeclampsia versus healthy pregnant controls, P = .004). There was no significant difference in the urinary concentration of placental growth factor among preeclamptic women (severe preeclampsia versus mild preeclampsia, P = .733). There was no significant correlation between albuminuria and urinary levels of placental growth factor for the severe preeclampsia group (r = –0.091, P = .653). There was no correlation between urinary placental growth factor and proteinuria when all preeclamptic subjects (mild and severe) were analyzed as a group (r = –0.04, P = .770). There was an inverse and significant correlation between proteinuria and urinary placental growth factor concentration in severe preeclampsia (r = –0.6, P = .002). In summary, severely preeclamptic women had significantly lower serum placental growth factor levels compared with mildly preeclamptic and healthy pregnant controls. The urinary levels of placental growth factor were also lower in preeclamptic women and did not vary with the severity of hypertensive disease.

Fig. 4
Fig. 4
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Calculations of fractional excretions for each angiogenic factor suggested that healthy pregnant women excrete significantly more vascular endothelial growth factor compared with healthy nonpregnant controls (one-way ANOVA, P < .001) (Fig. 4). Mild preeclampsia did not impact on fractional excretion of vascular endothelial growth factor compared with healthy pregnant controls (P = .346). However, severe preeclampsia increased fractional excretion of vascular endothelial growth factor significantly compared with both the mild preeclampsia (P = .007) or healthy pregnant control groups (P < .001). Pregnancy was further characterized by a marked decrease in the fractional excretion of sFlt-1 (healthy pregnant controls versus healthy nonpregnant controls, P < .001). Mild preeclampsia was not associated with a significant change in the fractional excretion of sFlt-1 (healthy pregnant controls versus mild preeclampsia, P = .43), whereas severe preeclampsia reversed pregnancy-induced changes to cause an increased fractional excretion of sFlt-1 compared with the healthy pregnant control group (P < .001) and mild preeclampsia group (P < .001) (Fig. 1).

Fractional excretion of placental growth factor followed a model similar to the one of sFlt-1. Healthy pregnant control women had decreased excretion fraction of placental growth factor compared with healthy nonpregnant controls (healthy pregnant control women versus healthy nonpregnant controls, P < .001). Similarly, mild preeclampsia does not further impact on the excretion fraction of placental growth factor (healthy pregnant control women versus mild preeclampsia, P = .125), whereas this effect was partially reversed in women with severe preeclampsia (severe preeclampsia versus mild preeclampsia, P < .001).

Pregnancy was not characterized by an increase in the fractional excretion of albumin in healthy controls (healthy nonpregnant controls versus healthy pregnant controls, P = .385). However, there was a significant increase in the fractional excretion of albumin in the severe preeclampsia group compared with the mild preeclampsia and healthy pregnant control groups (one-way ANOVA, P < .001).

Our analysis demonstrated a highly significant increase in the fractional excretion of total proteins in severely preeclamptic compared with mildly preeclamptic women at the time of the clinical manifestation of the disease (severe preeclampsia versus mild preeclampsia, P < .001). Instead, there was no significant change of the fractional excretion of total proteins in association with healthy pregnancy (healthy nonpregnant controls versus healthy pregnant controls, P > .05) or mild preeclampsia (healthy pregnant controls versus mild preeclampsia, P > .05).

We further analyzed the fractional excretion of each of the identified angiogenic factors in relationship to albuminuria, nonspecific proteinuria that reflects with high probability impairment of the glomerular filtration capacity of the kidney.19–21 Correlations were estimated between fractional excretion of angiogenic factors and urine random total protein-to-creatinine ratio for each of the study groups. There was no significant correlation between the fractional excretion of albumin and that of any of the angiogenic factors, with the exception of women with mild preeclampsia and only for the sFlt-1 (r = 0.639, P = .01) and placental growth factor (r = 0.687, P = .004).

There was no correlation between proteinuria and fractional excretion of any of the examined angiogenic factors in healthy nonpregnant controls (vascular endothelial growth factor [r = 0.33, P = .382], sFlt-1 [r= 0.40, P = .296], placental growth factor [r = 0.59, P = .09]). We identified a significant correlation between proteinuria and fractional excretion of sFlt-1 in healthy pregnant controls (sFlt-1 [r = 0.59, P = .03]). The fractional excretion of placental growth factor and sFlt-1 did not correlate with proteinuria in healthy pregnant controls (P > .05). However, there was no correlation between proteinuria and fractional excretion of any of the angiogenic factors in mildly preeclamptic women: vascular endothelial growth factor (r = 0.42, P = .118), sFlt-1 (r = 0.23, P = .414), or placental growth factor (r = 0.34, P = .214). In severe preeclampsia there was no correlation between proteinuria and fractional excretion of vascular endothelial growth factor (r = 0.30, P = .127) or sFlt-1 (r = 0.35, P = .07). In contrast, there was a significant correlation between proteinuria and fractional excretion of placental growth factor (r = 0.50, P = .01). Furthermore, in women with severe preeclampsia there was a significant correlation between the urinary random total protein/creatinine ratio, and fractional excretion of placental growth factor (r = 0.60, P = .002) or sFlt-1 (r = 0.50, P = .007).

In summary, women with severe preeclampsia were observed to have higher excretion of vascular endothelial growth factor than normal controls. The magnitude of such increase does not correlate with the degree of proteinuria as reflected by the fractional excretion of total proteins, albuminuria, or the urine random total protein-to-creatinine ratio.

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DISCUSSION

We demonstrate in the present investigation that the fractional excretions of vascular endothelial growth factor, sFlt-1, and placental growth factor vary significantly with pregnancy and with the severity of the hypertensive disorder. We observed that serum vascular endothelial growth factor and placental growth factor levels are significantly lower in severely preeclamptic women compared with healthy pregnant controls whereas sFlt-1 is significantly higher. Unlike sFlt-1 and placental growth factor, urinary vascular endothelial growth factor concentrations remain significantly higher than their serum level in all study groups. However, even when accounting for the degree of proteinuria as a reflection of impaired glomerular integrity, we found that severe preeclampsia is associated with increased fractional excretion of vascular endothelial growth factor and sFlt-1. In contrast, placental growth factor (a much lower-molecular-weight protein) previously proposed as a marker for preeclampsia was correlated with proteinuria and the urine random total proteinuria/creatinine ratio. Severe preeclampsia distinguishes itself as a divergent hypertensive clinical entity based on our finding that the serum and urinary levels for all angiogenic factors vary with the severity of hypertensive disease. Yet, the change in serum and urine concentrations does not necessarily occur in parallel.

The factors responsible for the development of preeclampsia remain unknown. Recently, a number of published reports have focused the attention of clinical investigators because alteration in levels of circulating angiogenic factors may play a causal role in the pathogenesis of preeclampsia.8,9 Still, there are limitations and several unanswered questions regarding the developing story of circulating proangiogenic factors and their inhibitors, including whether they are a cause or a consequence of the preeclamptic syndrome. Compelling evidence supports the view that the blueprint for the development of preeclampsia most probably lays in the initial phase of implantation and placental development.22

Pregnancy involves coordinated formation of new blood vessels via a process known as angiogenesis. Physiologically, the vascular growth factors vascular endothelial growth factor and placental growth factor, together with the oxygen diffusive placental tissue conductance, may promote remodeling of the maternofetal interface.14,23 Remodeling of the uterine arteries and vascular growth during placentation is critical,24 and it is thought that dysregulation in the expression of key angiogenic factors contributes to a number of obstetric complications including preeclampsia.25,26 Interestingly, immunohistochemical reactivity of angiogenic factors in placental tissue was observable both in controls and hypertensive cases.26 This raises again the issue of whether the increased serum concentrations of sFlt-1 reported by others and in our current study precipitate preeclampsia or are simply the consequence of placental hypoxia, which in turn releases angiogenic factors to the maternal circulation.9,15

The significance of angiogenic factors vascular endothelial growth factor, sFlt-1, and placental growth factor in regulation of human kidney glomerular vascular physiology also warrants increased attention. Human kidneys express mRNA for vascular endothelial growth factor and its receptors vascular endothelial growth factor R-1 (Flt-1) and vascular endothelial growth factor R-2 (Flk-1/KDR), predominantly in glomerular podocytes, distal tubules, and collecting ducts.27–31 This is particularly relevant to our study because excess sFlt-1 plays an important role in promoting glomerular endothelial hypertrophy, apoptosis, cell detachment from the glomerular basement membrane and thus hypertension, and proteinuria.8 In our previous and current study, we demonstrated that severe preeclampsia is associated with an increased urinary output of sFlt-1 and vascular endothelial growth factor but a decreased output of placental growth factor at the time of clinical manifestation.15

Our current findings bring new perspectives, because we analyzed in parallel maternal serum and urinary levels of sFlt-1, vascular endothelial growth factor, and placental growth factor in nonpregnant, healthy pregnant, and preeclamptic women. Further, we compute the fractional excretion of each of the studied angiogenic factors, and as a result we posit a plausible mechanism responsible for the changes in both serum concentrations and urinary excretion of vascular endothelial growth factor, sFlt-1, and placental growth factor. We theorize first that podocytes and mesangial cell destruction as well as loss of glomerular basal membrane integrity (glomerular endotheliosis) in women with severe preeclampsia result from increased exposure to serum sFlt-1 and reduced exposure to placental growth factor. This would be consistent with our finding that severely preeclamptic women had increased serum and urinary levels of sFlt-1. Therefore, it make sense to accept as true that in severe preeclampsia the placenta releases more angiogenic factors including sFlt-1 into the maternal circulation to attempt restoration of a normal uteroplacental blood flow. Instead, the increased fractional excretion of sFlt-1 (a molecule of approximately 100 kd) in severely preeclamptic women may relate only in part to structurally compromised glomeruli, because there is no correlation between the increased urinary outpouring of sFlt-1 and proteinuria. Interestingly, there was a significant correlation between fractional excretion of sFlt-1 and the urinary total protein-to-creatinine ratio, demonstrating the complexity of the issue and the importance of identifying a correct answer to the question concerning the fraction of maternal serum angiogenic factors cleared by the kidney. Thus, the hypothesis that a damaged glomerular barrier is the sole explanation for the urinary presence of sFlt-1 appears to be overly simplistic from the perspective of constitutive expression of sFlt-1 in the adult kidney.31 Our findings suggest that the dramatic increase in sFlt-1 urinary concentration characteristic of severe preeclampsia may be the result of 2 discrete phenomena occurring in parallel (namely endogenous renal production and glomerular “leakage”) that may have additive effects and influence each other's level of activity and function.

We further determined that women with severe preeclampsia have decreased serum levels of free vascular endothelial growth factor, but an exceptional increase in its urinary output. Clearly, the decreased serum levels may be explained via its substantial binding to plasma proteins such as sFlt-1. As a consequence, vascular endothelial growth factor might not be detected by the highly specific enzyme-linked immunosorbent assay that detects only the free form. It follows that the dramatic increase in urinary levels and fractional excretion is more likely the result of intrinsic kidney production and less likely due to glomerular leakage.32 Our finding that the dramatic increase in urinary levels of vascular endothelial growth factor is independent of proteinuria and serum levels of vascular endothelial growth factor further supports this argument, although our findings have to be confirmed in a larger number of patients. Considering that preeclampsia is a dynamic illness, it will be interesting to determine in a serial fashion whether this lack of correlation is continuous or time dependent. However, the mechanisms through which the kidneys enhance production of vascular endothelial growth factor are currently unknown, and this requires further investigation.

The mechanisms proposed here for the regulation of sFlt-1 and vascular endothelial growth factor apparently do not apply to placental growth factor. Severely preeclamptic women had significantly lower serum levels of placental growth factor compared with mildly preeclamptic women and healthy pregnant controls, whereas the fractional excretion of placental growth factor was partially increased compared with mildly preeclamptic women. But, in contrast to sFlt-1 and vascular endothelial growth factor, there was a significant correlation between total proteinuria and fractional excretion of placental growth factor. This suggests that placental growth factor, a much smaller molecule (approximately 30 kd), likely escapes into the urine through a damaged glomerular basement membrane, rather than being produced by the affected kidney.

In conclusion, our study suggests that altered serum levels and glomerular damage may not be the sole mechanism responsible for the increased output of urinary angiogenic factors in preeclampsia. The further increase in fractional excretion of angiogenic factors and especially of vascular endothelial growth factor is likely the result of a complex interaction among a local nephrogenous response to hypoxia, derangements in tubular resorption, and/or structurally compromised glomeruli.

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