US20090104649A1 - Markers for preeclampsia - Google Patents

Abstract

This document provides methods and materials related to determining whether or not a pregnant mammal (e.g., a pregnant human) has preeclampsia. For example, methods and materials related to the use of urinary podocytes to determine whether or not a pregnant human has preeclampsia are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
• This application is a continuation-in-part and claims the benefit of U.S. application Ser. No. 12/137,350, filed Jun. 11, 2008, which claims the benefit of U.S. Provisional Application Ser. No. 60/943,242, filed Jun. 11, 2007. The disclosures of the prior applications are incorporated by reference in entirety.
BACKGROUND
• 1. Technical Field
• This document relates to methods and materials involved in determining whether or not a pregnant mammal has preeclampsia. For example, this document provides methods and materials related to the use of urinary podocytes and/or plasma microvesicles to determine whether or not a pregnant mammal (e.g., a pregnant human) has preeclampsia.
• 2. Background Information
• Preeclampsia is a pregnancy-specific disease that affects about 5 percent of all pregnancies and remains a leading cause of both maternal and fetal morbidity and death worldwide. It is characterized by hypertension (blood pressure, ≧140/90 mm Hg) and proteinuria (≧300 mg in a 24-hour urine sample) that occur after 20 weeks of gestation. Proteinuria in preeclampsia is associated with characteristic renal pathologic changes of glomerular endotheliosis, which is considered to be a hallmark of preeclampsia in humans.
SUMMARY
• This document relates to methods and materials involved in determining whether or not a pregnant mammal has preeclampsia. For example, this document provides methods and materials related to the use of urinary podocytes and/or plasma microvesicles to determine whether or not a pregnant mammal (e.g., a pregnant human) has preeclampsia. Identifying patients who have preeclampsia can allow such patients, who are at risk for both maternal and fetal morbidity and death, to be treated effectively. In addition, identifying patients who do not have preeclampsia can avoid unnecessary treatment and patient suffering. As described herein, the presence of urinary podocytes and/or plasma microvesicles can be used to identify pregnant humans as having preeclampsia.
• In general, one aspect of this document features a method for assessing a pregnant mammal for preeclampsia. The method comprises, or consists essentially of, determining whether or not a urine sample from the mammal contains an elevated level of urinary podocytes, wherein the presence of the elevated level indicates that the mammal has preeclampsia. The mammal can be a human. The determining step can comprise using an antibody to detect podocytes. The antibody can be an anti-podocin antibody. The antibody can be an anti-podocalyxin antibody. The antibody can be an anti-nephrin antibody. The antibody can be an anti-synaptopodin antibody. The method can comprise classifying the mammal as having preeclampsia if the elevated level is present, and classifying the mammal as not having preeclampsia if the elevated level is not present. In general, one aspect of this document features a method for assessing a pregnant mammal for preeclampsia. The method comprises, or consists essentially of, determining whether or not a plasma sample from the mammal contains an elevated level of microvesicles expressing a VEGFR-1 polypeptide, and classifying the mammal as having preeclampsia if the plasma contains the elevated level. The mammal can be a human. The determining step can comprise using flow cytometry. The determining step can comprise using an antibody to detect the microvesicles. The antibody can be an anti-VEGFR-1 antibody. The method can comprise classifying the mammal as having preeclampsia if the elevated level is present, and can comprise classifying the mammal as not having preeclampsia if the elevated level is not present.
• In another aspect, this document features a method for assessing a mammal for risk of developing a complication of preeclampsia. The method comprises, or consists essentially of, determining whether or not plasma from the mammal contains an elevated level of endothelium-derived microvesicles and classifying the mammal as being at risk for developing a complication of preeclampsia. The determining step can comprise using flow cytometry. The determining step can comprise using an antibody to detect the endothelium-derived microvesicles. The complication of preeclampsia can comprise HEELP syndrome. The method can comprise classifying the mammal as being at risk of developing a complication of preeclampsia if the elevated level is present, and can comprise classifying the mammal as not being at risk of developing a complication of preeclampsia if the elevated level is not present.
• In another aspect, this document features a method for assessing a pregnant mammal for preeclampsia. The method comprises, or consists essentially of, determining whether or not a urine sample from the mammal contains an elevated level of urinary podocytes, wherein the presence of the elevated level indicates that the mammal has preeclampsia. The mammal can be a human. The determining step can comprise using an antibody to detect podocytes. The antibody can be an anti-podocin antibody. The antibody can be an anti-podocalyxin antibody. The antibody can be an anti-nephrin antibody. The antibody can be an anti-synaptopodin antibody. The method can comprise classifying the mammal as having preeclampsia if the elevated level is present, and classifying the mammal as not having preeclampsia if the elevated level is not present.
• Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
• The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
DESCRIPTION OF THE DRAWINGS
• FIG. 1 contains photographs of urinary cells plated on collagen-coated slides, cultured for 24 hours, and stained for podocin (A), podocalyxin (B), nephrin (C), and synaptopodin (D) immunoreactivity.
• FIG. 2 contains graphs plotting ROC curves for podocyturea as determined by staining for podocin (A), podocalyxin (B), nephrin (C), and synaptopodin (D) immunoreactivity.
• FIG. 3 contains graphs plotting sFlt-1 (A; pg/mL), soluble endoglin (B; ng/mL), serum PIGF (C; pg/mL), and urine PIGF (D; adjusted for Mg creatinine) for normotensive and preeclamptic pregnancies as a function of age. For normotensive pregnancies, open circles indicate individual values with a trend regression line. The dashed portion represents extrapolation. For preeclamptic pregnancies, values are stratified by intervals of gestational age in which data distributions are summarized with box plots indicating median values and inter-quartile ranges.
• FIG. 4 contains graphs plotting ROC curves for sFlt-1 (A), soluble endoglin (B), serum PIGF (C), and urine PIGF (D).
• FIG. 5 contains representative scatter plots obtained by FACSCanto™ flow cytometry showing control gates of buffer with fluorescein-conjugated antibodies and calibration beads (size and True Count Beads™) in the absence of sample (A), gates derived from adding a sample containing microvesicles to the buffer with fluorescein conjugated antibodies and calibration beads (B), and representative quadrants derived from the microvesicle gates shown in panels A and B, respectively, with counts separated by antibody binding, andquadrant3 representing microvesicles (C and D).
DETAILED DESCRIPTION
• This document provides methods and materials related to determining whether or not a pregnant mammal (e.g., a pregnant human) has preeclampsia. For example, this document provides methods and materials related to the use of urinary podocytes and/or plasma microvesicles to determine whether or not a pregnant human has preeclampsia. As described herein, if the level of urinary podocytes is elevated in a pregnant mammal, then the mammal can be classified as having preeclampsia. If the level of urinary podocytes is not elevated in a pregnant mammal, then the mammal can be classified as not having preeclampsia.
• This document also provides methods and materials for using microvesicles to determine whether or not a pregnant mammal has preeclampsia. For example, a marker for preeclampsia can be an elevated level of microvesicles positive for a vascular endothelial growth factor/vascular permeability factor receptor-1 (VEGFR-1) polypeptide in a sample taken from a pregnant mammal. As disclosed herein, if the level microvesicles expressing a VEGFR-1 polypeptide in a sample from a pregnant mammal is elevated, then the mammal can be classified as having preeclampsia. If the level microvesicles expressing a VEGFR-1 polypeptide is not elevated, then the mammal can be classified as not having preeclampsia.
• Microvesicles, which circulate in the peripheral blood, are a heterogeneous population of spheres (vary in size from about 0.1 to 1.5 μm) formed from intact phospholipid rich membranes. Typically, a microvesicle contains at least half of the surface polypeptides, receptors, and lipids of their cells of origin. Microvesicles are differentiated from microparticles, the latter of which can refer to chemical particles or aggregates such as those formed from plasma lipoprotein or other chemicals. Micro-vesicles are smaller than platelets, which are typically between 2 and 2.5 μm in diameter, and are generated during cell activation and apoptosis induced by oxidative damage, inflammatory cytokines and chemokines, thrombin, bacterial lipopolysaccharide, shear stress, and hypoxia.
• Any appropriate type of sample can be used to evaluate the level of microvesicles in a mammal including, without limitation, serum, blood, and plasma. In addition, any method can be used to obtain a sample. For example, a blood sample can be obtained by peripheral venipuncture. Once obtained, a sample can be manipulated prior to measuring the level of microvesicles. For example, a blood sample can be centrifuged to separate serum and plasma, and the separated serum and plasma can be liquid frozen for future analysis. Once obtained, the sample can be analyzed by flow cytometry based on size or using antibodies to determine the total number of microvesicles, the level of microvesicles of a particular cellular origin, or the level VEGFR-1 expressing microvescicles, present within a sample.
• This document also provides methods and materials related to the use of microvesicles to determine whether or not a pregnant mammal is at risk for developing complications associated with preeclampsia (e.g., HEELP syndrome). As described herein, if the level of endothelium-derived microvesicles is elevated in a sample taken from a pregnant mammal, then the pregnant mammal can be classified as being at risk for developing a complication of preeclampsia. If the level of endothelium-derived microvesicles is not elevated in a sample taken from a pregnant mammal, then the pregnant mammal can be classified as not being at risk for developing a complication of preeclampsia.
• The methods and materials provided herein can be used to assess any pregnant mammal for preeclampsia, or a complication of preeclampsia. For example, a human, cat, dog, or horse can be assessed for preeclampsia. In some cases, a human pregnant for 18 to 36 weeks (e.g., between 18 and 35 weeks, between 20 and 35 weeks, or between 20 and 30 weeks) can be assessed.
• Any appropriate method can be used to determine the level of podocytes, or any fragment of podocytes, in a mammal's urine. For example, cell staining techniques that include using antibodies that bind to podocytes or polypeptides expressed by podocytes can be used. Examples of such antibodies include, without limitation, antibodies that have the ability to bind podocin, podocalyxin, nephrin, synaptopodin, Neph1, GLEPP1, WT1, CD2AP, actin, actinin, cadherin, catenin, integrin, vinculin, talin, paxillin, and ZO-1.
• Any appropriate method can be used to determine the presence of microvesicles in a sample obtained from a pregnant mammal. In some cases, the methods and materials provided herein can be used to detect microvesicles generated in vivo from many cell types. For example, a FACSCanto™ (New fourth or fifth generation) machine with high sensitivity and six colors detectors can be used to detect microvesicles. In some cases, blood can be prepared for analysis as follows. The sample can be centrifuged (e.g., 3000 g for 15 minutes). The resulting supernatant from this spin can be removed and re-spun (e.g., using the same speed and duration). The absence of platelets from the supernatant of the second spin can be validated by Coulter counter. This platelet free plasma can be centrifuged (e.g., 20,000 g for 30 minutes), and the pellet can be washed (e.g., washed once with HEPES/Hanks buffer) and centrifuged (e.g., 20,000 g for 30 minutes) to prepare washed microvesicles. The supernatant can be discarded, and the pellet reconstituted with buffer (e.g., HEPES/Hanks buffer). These microvesicles can be stained to identify their cell of origin (all cells, not only platelets). Higher than 95% of microvesicles that circulate in the blood of healthy people can originate from platelets. Both buffers and antibodies can be filtered through 0.2 μm filters to remove contaminants before staining the isolated microvesicles. The signal to noise ratio can be high (e.g., 15-30,000 events:200-500 events). Scanning and transmission electron microscopy and Cyto viva can be used to verify the presence of microvesicles.
• Any appropriate method can be used to determine the level of microvesicles, present within a sample, that express, for example, a marker such as a VEGFR-1 polypeptide. For example, standards such as those described herein (e.g., anti-VEGFR-1 antibodies) can be used to identify and quantify microvesicles expressing of a VEGFR-1 polypeptide present within a sample. In some cases, the expression of a VEGFR-1 polypeptide on particular microvesicles (e.g., platelet-derived microvesicles, procoagulant microvesicles, or endothelium-derived microvesicles) can be determined using antibodies as markers associated with particular cell types. In some cases, antibodies against the markers set forth in Table 1 can be used to identify and quantify microvesicles of a particular cell origin that express VEGFR-1 polypeptides using flow cytometry techniques. For example, anti-CD62e can be used to identify endothelium-derived microvesicles present within a sample.
• Any appropriate method can be used to determine the cellular origin of microvesicles present within a sample. For example, standards such as those described herein (e.g., beads of known size or cellular membrane antibodies) can be used to identify and quantify microvesicles present within a sample. In some cases, the level of particular microvesicles (e.g., platelet-derived microvesicles, procoagulant microvesicles, or endothelium-derived microvesicles) can be determined using antibodies to detect markers associated with particular cell types. For example, antibodies against the markers set forth in Table 1 can be used to identify and quantify particular microvesicles using flow cytometry techniques. In some cases, anti-CD62e can be used to identify endothelium-derived microvesicles present within a sample.
• The term “elevated level” as used herein with respect to the level of urinary podocytes is any level that is above a median urinary podocytes level in urine from a random population of pregnant mammals (e.g., a random population of 10, 20, 30, 40, 50, 100, or 500 pregnant mammals) lacking preeclampsia. In some cases, an elevated level of urinary podocytes can be a detectable level of podocin-positive cells within a urine sample. The presence or absence of such a detectable level of podocin-positive cells can be determined using an anti-podocin antibody.
• The term “elevated level” as used herein with respect to the level microvesicles expressing a VEGFR-1 polypeptide is any level that is greater than a control level of microvesicles expressing a VEGFR-1 polypeptide associated with a sample of microvesicles from normal, healthy mammals lacking signs or symptoms of preeclampsia. In some cases, an elevated level of VEGFR-1 expressing microvesicles can be a detectable level. In some cases, an elevated level of VEGFR-1 expressing microvesicles can be any level that is greater than a reference level for an elevated level of VEGFR-1 expressing microvesicles. For example, a reference level of microvesicles expressing a VEGFR-1 polypeptide can be the average level of microvesicles expressing a VEGFR-1 polypeptide that is present in samples obtained from a random sampling of 50 healthy pregnant mammals matched for age. It will be appreciated that levels from comparable samples are used when determining whether or not a particular level is an elevated level.
• An elevated level of VEGFR-1 expressing microvesicles can be any level provided that the level is greater than a corresponding reference level of VEGFR-1 expressing microvesicles. For example, an elevated level of VEGFR-1 expressing microvesicles can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more times greater than the reference level VEGFR-1 expressing microvesicles. In addition, a reference level can be any amount. For example, a reference level for VEGFR-1 expressing microvesicles can be zero. In this case, any level of microvesicles expressing a VEGFR-1 polypeptide greater than zero would be an elevated level.
• The term “elevated level” as used herein with respect to the level of endothelium-derived microvesicles is any level that is greater than a control endothelium-derived microvesicle level associated with mammals lacking signs or symptoms of preeclampsia, or a complication of preeclampsia. In some cases, an elevated level of endothelium-derived microvesicles can be a detectable level. In some cases, an elevated level of endothelium-derived microvesicles can be any level that is greater than a reference level for endothelium-derived microvesicles.
• The term “reference level” as used herein with respect to an endothelium-derived microvesicle level is the level of endothelium-derived microvesicles typically found in healthy mammals, for example, mammals free of signs and symptoms of preeclampsia, or complications of preeclampsia. For example, a reference level of endothelium-derived microvesicles can be the average level of endothelium-derived microvesicles that is present in samples obtained from a random sampling of 50 healthy pregnant mammals matched for age. It will be appreciated that levels from comparable samples are used when determining whether or not a particular level is an elevated level.
• An elevated level of endothelium-derived microvesicles can be any level provided that the level is greater than a corresponding reference level for endothelium-derived microvesicles. For example, an elevated level of endothelium-derived microvesicles can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more times greater than the reference level for endothelium-derived microvesicles. In addition, a reference level can be any amount. For example, a reference level for endothelium-derived microvesicles can be zero. In this case, any level of endothelium-derived microvesicles greater than zero would be an elevated level.

• TABLE 1
  Markers for identifying the source of microvesicles.

  Cell-derived
  microvesicles	Markers
  Procoagulant microvesicles	Annexin-V or thrombin
  	generation assay
  Leukocytes-derived microvesicles	CD45, CD11b
  Granulocytes-derived microvesicles	CD33, CD15, CD11b; CD177
  NK cells-derived microvesicles	CD56
  Monocytes-derived microvesicles	CD14
  T-lymphocyte-derived microvesicles	CD3 and CD134
  B-lymphocyte-derived microvesicles	CD19 or CD20
  Platelet-derived microvesicles	CD41 or CD61 or CD42a
  Endothelium-derived microvesicles	CD62e, CD106, CD146
  Erythrocyte-derived microvesicles	Glycophorin A

• An antibody can be, without limitation, a polyclonal, monoclonal, human, humanized, chimeric, or single-chain antibody, or an antibody fragment having binding activity, such as a Fab fragment, F(ab′) fragment, Fd fragment, fragment produced by a Fab expression library, fragment comprising a VL or VH domain, or epitope binding fragment of any of the above. An antibody can be of any type (e.g., IgG, IgM, IgD, IgA or IgY), class (e.g., IgG1, IgG4, or IgA2), or subclass. In addition, an antibody can be from any animal including birds and mammals. For example, an antibody can be a human, rabbit, sheep, or goat antibody. An antibody can be naturally occurring, recombinant, or synthetic. Antibodies can be generated and purified using any suitable methods known in the art. For example, monoclonal antibodies can be prepared using hybridoma, recombinant, or phage display technology, or a combination of such techniques. In some cases, antibody fragments can be produced synthetically or recombinantly from a gene encoding the partial antibody sequence. An anti-podocin antibody can bind to podocin polypeptides at an affinity of at least 10⁴mol⁻¹(e.g., at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹²mol⁻¹).
• Once the level of urinary podocytes, endothelium-derived microvesicles, or all microvesicles expressing a VEGFR-1 polypeptide in a sample from a mammal is determined, then the level can be compared to a median level or a cutoff level and used to evaluate the mammal for preeclampsia, or risk of developing a complication of preeclampsia. A level of urinary podocytes, VEGFR-1 expressing microvesicles, or endothelium-derived microvesicles that is higher than the median level of urinary podocytes, VEGFR-1 expressing microvesicles, or endothelium-derived microvesicles in samples taken from a population of mammals of the same species having no preeclampsia (or a cutoff level) can indicate that the mammal has preeclampsia, or at risk for developing a complication of preeclampsia. A level of urinary podocytes, VEGFR-1 expressing microvesicles, or endothelium-derived microvesicles that is lower than the median level of urinary podocytes, VEGFR-1 expressing microvesicles, or endothelium-derived microvesicles in samples taken from a population of mammals of the same species having no preeclampsia (or a cutoff level) can indicate that the mammal does not have preeclampsia, or is not a risk of developing a complication of preeclampsia. A cutoff level can be set to any level provided that the values greater than that level correlate with an increased level of urinary podocytes, VEGFR-1 expressing microvesicles, or endothelium-derived microvesicles indicative of a mammal having preeclampsia. For example, a cutoff level can be equal to, or greater than 1 cell/mg creatinine.
• In some cases, a pregnant mammal can be classified as having preeclampsia if it is determined that the podocyte level in a urine sample from the mammal is greater than the podocyte level in a urine sample obtained previously from that mammal. In some cases, a pregnant mammal can be classified as having preeclampsia if it is determined that the level of VEGFR-1 expressing microvesicles in a sample is greater than the level of VEGFR-1 expressing microvesicles in a sample obtained previously from that mammal. In some cases, a pregnant mammal can be classified as being at risk for developing a complication of preeclampsia if it is determined that the endothelium-derived microvesicles level in a sample from the mammal is greater than the endothelium-derived microvesicles level in a urine sample obtained previously from that mammal.
• A mammal that has been or is being treated for preeclampsia can be monitored using the methods and materials provided herein. For example, the level of podocytes, VEGFR-1 expressing microvesicles, or endothelium-derived microvesicles in a sample from a mammal being treated for preeclampsia can be assessed to determine whether or not the mammal is responding to the treatment.
• This document also provides methods and materials to assist medical or research professionals in determining whether or not a pregnant mammal has preeclampsia. Medical professionals can be, for example, doctors, nurses, medical laboratory technologists, and pharmacists. Research professionals can be, for example, principle investigators, research technicians, postdoctoral trainees, and graduate students. A professional can be assisted by (1) determining the level of urinary podocytes in a urine sample, the level of endothelium-derived microvesicles, or the level of VEGFR-1 expressing microvesicles, and (2) communicating information about the level to that professional.
• Any appropriate method can be used to communicate information to another person (e.g., a professional). For example, information can be given directly or indirectly to a professional. In addition, any type of communication can be used to communicate the information. For example, mail, e-mail, telephone, and face-to-face interactions can be used. The information also can be communicated to a professional by making that information electronically available to the professional. For example, the information can be communicated to a professional by placing the information on a computer database such that the professional can access the information. In addition, the information can be communicated to a hospital, clinic, or research facility serving as an agent for the professional.
• The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLESExample 1Urinary Podocyte Excretion as a Marker for Preeclampsia
• An approved study was conducted with the consent of all included women. A diagnosis of preeclampsia was made in the presence of (a) hypertension after 20 weeks of gestation, which was defined as a blood pressure of ≧140/90 mm Hg, (b) proteinuria, which was defined as ≧300 mg of protein in a 24-hour urine specimen, and/or 1+(30 mg/L) dipstick urinalysis in the absence of urinary tract infection and/or a predicted 24-hour urine protein of ≧300 mg on a random urine collection, and (c) resolution of hypertension and proteinuria by 12 weeks after delivery. Women with severe forms of preeclampsia such as eclampsia and HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome, the diagnosis of which was confirmed on the basis of previously published criteria (Jones,Hematopathol. Mol. Hematol.,11: 147-71 (1998)), also were included. Healthy, normotensive pregnant women without hypertension and proteinuria served as control subjects. An additional control group consisted of women with hypertension and proteinuria.
• A cross-sectional study was conducted, and blood and urine samples were collected close to and typically ≦24 hours before delivery. In total, 67 women were recruited. Preeclampsia was present in 33 of the patients, and HELLP was diagnosed in 11 patients; 23 normotensive pregnancies served as control subjects (Table 1). Blood samples were obtained in all 67 women, and urine samples for podocyturia were collected in a subset of 31 pregnant women (15 cases and 16 control subjects).

• TABLE 1
  Patient characteristics.

  	Normal	Preeclampsia	HELLP	Preeclampsia +
  Variable	(n = 23)	(n = 33)	(n = 11)	HELLP (n = 44)
  Maternal age (y)	28.7 ± 5.4	26.2 ± 5.1	33.0 ± 6.0	27.9 ± 6.1
  Gestational age	39.2 ± 2.2	34.3 ± 3.8*	33.5 ± 5.6*	34.1 ± 4.2*
  (wk)
  Primiparous (%)	47.8	81.8	9.1	63.6
  Systolic blood	110.5 ± 9.5	159 ± 19.8*	162.6 ± 23*	159.9 ± 20.3*
  pressure (mm Hg)
  Diastolic blood	66.9 ± 9.8	97.8 ± 9.4*	98.3 ± 10.6*	97.9 ± 9.6*
  pressure (mm Hg)
  Proteinuria (g/24	247 ± 294	2693 ± 3164*	4373 ± 5962*	3113 ± 4032*
  hr)
  Platelet count	242,000 ± 35,519	232,333 ± 66,787	100,273 ± 42,245*	199,318 ± 84,146
  Data are given as mean ± SD.
  *P < .05, compared with normal group.

Serum Studies
• Blood samples for the determination of sFlt-1, free PIGF, and soluble endoglin levels were drawn within 24 hours before delivery. Serum creatinine level, liver function tests, and platelet counts were performed according to standardized laboratory procedures. Serum levels of sFlt-1, soluble endoglin, and free PIGF were measured with Quantikine ELISA (enzyme-linked immunosorbent assay) kits (R&D Systems, Minneapolis, Minn.).
Urine Chemistry
• Concurrent with serum collection, clean-catch urine specimens (50-100 mL) were obtained. Urine albumin, total protein, and creatinine concentrations were measured by standard methods on a Hitachi 911 Chemistry Analyzer (Roche Diagnostics, Indianapolis, Ind.). Urinary PIGF determinations were performed with the PIGF ELISA (enzyme linked immunosorbent assay) kit (R&D Systems).
Podocyturia
• Random urine samples (25-50 mL each) were centrifuged for 8 minutes at 700 g at room temperature. The pellets were rinsed twice with human diploid fibroblast (HDF) solution. Next, the pellets were resuspended in Dulbecco's modified eagle's medium (DMEM) F-12 medium with 10% fetal bovine serum that was supplemented with antibiotics for the prevention of bacterial contamination. One-milliliter aliquots were plated in four-chamber, collagen-coated tissue culture slides, which was followed by overnight incubation at 37° C. in 5% CO₂. The next day, the media were removed, followed by two phosphate-buffered saline solution washes. Slides were fixed with 1 mL of ice cold methanol for 10 minutes at −20° C. Each of the four slide chambers was incubated with one of four different antibodies to podocyte proteins: podocalyxin (dilution, 1:40), podocin (dilution, 1:200), nephrin (dilution, 1:100), and synaptopodin (undiluted). After being washed with phosphate-buffered saline solution, a secondary fluorescein isothiocyanate-labeled antibody was added at a dilution of 1:40 for 30 minutes. The sediment was counterstained with Hoechst nuclear stain to facilitate differentiation of whole cells from cell fragments. Coverslips were mounted with Vectashield (Vector Labs, Burlington, Calif.), and the slides were viewed with a fluorescence microscope (Leica, Germany). Nucleated, positive-staining cells were considered to be podocytes. A renal pathologist, who was blinded to the clinical diagnosis and laboratory findings, evaluated each sample to determine the number of cells that were present and the percentage of cells that were stained for podocyte markers. Podocyturia was expressed as a ratio of the number of podocytes to the creatinine content of the respective urine sample, which was performed for each of the four podocyte markers.
Statistical Methods
• Descriptive statistics are reported for quantitative traits as means and SDs or as medians and interquartile ranges and for categoric traits as percentages. The operating characteristics of podocyte and angiogenic markers of preeclampsia were assessed by consideration of the trait either as a categoric measure (i.e., absence/presence) and estimation of its sensitivity and specificity or by the consideration of it as a quantitative measure and the generation of the receiver-operating characteristic (ROC) curve. The areas under the curve were estimated with confidence intervals and contrasted among markers with a method described elsewhere (DeLong et al.,Biometrics,44:837-45 (1988)). All statistical tests were carried out at the two-sided 0.05 significance level.
Podocyturia
• In the women with urinary measures of podocyturia (i.e., 15 cases and 16 control subjects), those women with preeclampsia or HELLP had podocin-positive cells in the urine (FIG. 1A), whereas none of the normotensive control subjects had any podocin-positive cells. Thus, the sensitivity and specificity of podocyturia, as determined by the podocin-positive cells, for the diagnosis of preeclampsia were both 100%. A positive correlation between the degree of proteinuria and podocyturia, as determined by podocin staining, was present (P=0.04).
• Compared with podocin, measurements of podocyturia that were based on podocalyxin, nephrin, and synaptopodin stains (FIGS. 1B, C, and D, respectively) had both lower sensitivity and specificity (Table 2). For sFLT-1, endoglin, and PIGF, the sensitivity and specificity were calculated twice, with two different cutoffs to define a positive test. The cutoffs for podocyturia (podocin, nephrin, podocalyxin, and synaptopodin) were expressed as cells per milligram of creatinine (Table 2). The ROC curves for all four podocyte markers were generated (FIG. 2), and their respective areas under the curve were compared as a measure of diagnostic accuracy.

• TABLE 2
  Test characteristics for markers of preeclampsia.

  Pretest probability for preeclampsia

  5%

  25%

  			Positive	Negative	Positive	Negative
  	Sensitivity	Specificity	predictive	predictive	predictive	predictive

  Test	Cutoff	(%)	(%)	value (%)	value (%)	value (%)	value (%)

  sFLT-1*	7463	pg/mL	83	58	9.4	98.5	39.7	91.1
  	9795	pg/mL	71	68	10.5	97.8	42.5	87.6
  Endoglin*	21.3	ng/mL	94	58	10.5	99.5	42.7	96.7
  	24.6	ng/mL	86	63	10.9	98.8	43.7	93.1
  Serum P1GF†	84.92	pg/mL	74	58	8.5	97.7	37.0	87.0
  	102.7	pg/mL	86	47	7.9	98.5	35.1	91.0
  Urine P1GF†	1.22	pg/mL‡	79	50	7.7	97.8	34.5	87.7
  	2.18	pg/mL†	86	38	7.5	98.4	33.9	90.4
  Podocin*	0.85	cells†	100	100	100.0	100.0	100.0	100.0
  Nephrin*	0.75	cells†	93	75	16.4	99.5	55.4	97.0
  Podocalyxin*	0.83	cells†	93	75	16.4	99.5	55.4	97.0
  Synaptopodin*	1.11	cells†	93	81	20.5	99.5	62.0	97.2
  *A positive test is defined as having a value higher than the cutoff.
  †A positive test has a lower value than the cutoff.
  ‡Expressed per milligram of creatinine in the respective urine samples

• The analysis indicated that podocin had a greater diagnostic accuracy than did podocalyxin (P=0.04) or nephrin (P=0.05) and possibly better than did synaptopodin (P=0.08); diagnostic accuracy of the other three markers (podocalyxin, nephrin, and synaptopodin) did not differ. For the cases, the rate of podocyte excretion, which was expressed as median cell number per milligram of creatinine, was 3.7 for podocin and synaptopodin, 5.0 for podocalyxin, and 3.3 for nephrin. For the control subjects, the rate of podocyte excretion for synaptopodin was 0.6, and 0 for podocalyxin, podocin, and nephrin. An additional control group consisted of women with gestational hypertension (n=6), essential hypertension (n=2), and preexisting proteinuria (n=3), who did not have clinical signs and symptoms of superimposed preeclampsia. None of these 11 women demonstrated podocyturia, as determined by podocin staining.
Angiogenic Markers of Preeclampsia
• Serum sFlt-1 levels were significantly higher in women with preeclampsia or HELLP than in normotensive pregnant control subjects (17,326±12,124 pg/mL vs. 8,160±5,186 pg/mL; P<0.001; Table 3). SerumsFlt-1 levels did not differ significantly between patients with preeclampsia and HELLP(P=0.11). Patients with preeclampsia and HELLP displayed higher sFlt-1 levels than normal patients, if they delivered early in pregnancy (FIG. 3A). This difference became less apparent closer to full term delivery.

• TABLE 3
  Normal and preeclamptic levels of sFLT-1, endoglin, and P1GF.

  	Normal	Preeclampsia	HELLP	Preeclampsia +
  Variable	(n = 23)	(n = 33)	(n = 11)	HELLP (n = 44)
  sFLT-1 (pg/mL)	8160 ± 5186	18,231 ± 11,216*	14,711 ± 14,876*	17,326 ± 12,124*
  Endoglin (ng/mL)	27.2 ± 23.9	56.5 ± 31.7*	52.1 ± 32.7*	55.4 ± 31.6*
  Serum P1GF (pg/mL)	173 ± 175	66.2 ± 44.2*	59.8 ± 48.5*	64.6 ± 44.7*
  Urine P1GF (pg/mL	2.94 ± 3.56	1.17 ± 1.54	†	†
  per mg creatinine)
  Data are given as mean ± SD.
  *P < 0.05, compared with normal group.
  † None of the 11 patients with HELLP had urine samples.

• Serum soluble endoglin levels were significantly higher in women with preeclampsia or HELLP than in normotensive pregnant control subjects (55.4±31.6 ng/mL vs. 27.2±23.9 ng/mL; P<0.001). Serum soluble endoglin levels did not differ significantly between patients with preeclampsia and HELLP(P=0.69). The difference between normal and preeclamptic pregnancies was greater with an earlier delivery and became less apparent in those patients who were delivered at full term (FIG. 3B).
• Serum-free PIGF levels were lower in women with preeclampsia or HELLP than in normotensive pregnant control subjects (64.6±44.7 pg/mL vs. 173±174.8 pg/mL; P=0.0005). Serum-free PIGF levels did not differ significantly between patients with preeclampsia and HELLP(P=0.36). In those patients delivering at an earlier gestational age, free PIGF levels were lower in patients with preeclampsia and HELLP vs. control subjects, but this difference became less apparent as pregnancies were carried towards full term (FIG. 3C).
• There was a statistically insignificant trend towards lower urine PIGF levels in women with preeclampsia or HELLP, compared with normotensive pregnant control subjects (1.17±1.54 pg/mL/mg vs.2.94±3.56 pg/mL/mg creatinine; P=0.11; Table 3). Urine PIGF levels in women with preeclampsia were not different than in normal women, regardless of gestational age at delivery (FIG. 3D).
Angiogenic Factors as Diagnostic Tests for Preeclampsia: Comparison to Podocyturia
• ROC curves were generated for sFlt-1, soluble endoglin, and both serum and urine PIGF (FIG. 4). The positive predictive value and the negative predictive value for podocyturia, as determined by the four podocyte-specific markers and the angiogenic factors that were evaluated (Table 2), were calculated. Because the value of a diagnostic test can depend on the pretest probability of disease, the diagnostic accuracy of each test was estimated for two different pretest probabilities: 5%, which reflects the pretest probability for preeclampsia in the general population, and 25%, which is a commonly cited percentage risk in women with preexisting hypertension. The negative predictive value did not differ between the podocyturia and angiogenic factor tests in patients with a low (5%) pretest probability. However, in patients with a pretest probability of 25%, the negative predictive value was higher with podocyturia. The positive predictive value was higher with podocyturia, compared with angiogenic factors tests in both the low and high pretest probability groups.
• The results provided herein demonstrate that podocyturia (i.e., urinary excretion of podocytes) is present in patients with preeclampsia at the time of delivery. These cells retain the ability to attach to tissue culture plates in vitro, which indicates that they are viable. Urinary shedding of podocytes may contribute to proteinuria in preeclampsia, because these cells have a very limited regenerative capacity. Therefore, podocyturia may indicate podocyte loss from the glomerulus which may lead to a disruption of the glomerular filtration barrier and consequent proteinuria.
• Podocyturia is present at the time of the clinical diagnosis of preeclampsia, and the number of podocytes can correlate with the degree of proteinuria. Urinary podocyte excretion, which was quantified by four podocyte-specific markers (namely, podocalyxin, podocin, nephrin, and synaptopodin), is a sensitive marker of renal damage and proteinuria in preeclampsia. Among these four markers, podocin exhibited a high sensitivity and specificity of 100% each. In addition, a positive correlation between the degree of proteinuria and podocyturia was found, which suggests a possible common/shared underlying pathogenic mechanism. Women with normotensive pregnancies and women with either hypertension or proteinuria, but in the absence of the clinical syndrome of preeclampsia, did not have podocyturia. Therefore, podocyturia does not appear to be merely a result of hypertensive kidney damage or a marker of proteinuria.
• There are several possible explanations for podocin being a better marker for podocyturia in preeclampsia than podocalyxin, nephrin, or synaptopodin. Staining for podocalyxin, nephrin, and synaptopodin may be less specific, because these proteins, unlike podocin, are expressed in cells other than podocytes. These cells might be present in the urine particularly around the time of delivery, when urine samples can be contaminated with decidual, amniotic, and red and white blood cells. Moreover, staining for podocin may be more sensitive than staining for other podocyte proteins. Glomerular expression of nephrin and synaptopodin, but not podocin, was found to be decreased in kidney sections from women with preeclampsia. Consequently, podocytes that are shed in the urine may have lower expressions of nephrin and synaptopodin than podocin, which makes the latter a more sensitive marker of podocyte presence in the urine. It is particularly intriguing to postulate that podocyturia, as a marker of subclinical renal damage, may be detected before overt proteinuria and the full clinical picture of preeclampsia develops.
• The results provided herein demonstrate that differences in sFlt-1, PIGF, and soluble endoglin levels between normotensive and preeclamptic pregnancies were greatest in those women who delivered earliest. Early delivery was a marker of severe disease that resulted in termination of pregnancy. These differences become less apparent as pregnancies are carried toward full term. There is a significant overlap in free PIGF and sFlt-1 values between mild forms of preeclampsia and normotensive pregnancies closer to full term, which could lead potentially to both false-positive and false-negative screening test results. In addition, no difference in urinary PIGF was observed between the cases (n=15) and control subjects (n=16), and no significant difference in circulating endoglin levels was observed between the cases of preeclampsia (n=33) and HELLP (n=11).
• In summary, the results provided herein indicated that podocyturia is a marker of renal damage and proteinuria in preeclampsia.
Example 2Detecting Levels of Microvesicles Expressing VEGFR-1
• Blood and urine samples were collected from 8 preeclamptic women and 15 normal pregnant women just prior to delivery (IRB # 166-00). The patient characteristics are provided in Table 4.

• TABLE 4
  Baseline patient characteristics

  		Normal
  		pregnancy	Preeclampsia
  	Variable	(n = 15)	(n = 8)
  	Age (yrs) - mean (range)	29.0 (19-40)	27.3 (21-38)
  	Gestational age* (wks) - mean	38.9 (36-41)	31.1 (27-34)
  	(range)
  	MAP (mm Hg) - mean (range)	80.5 (74-88)	108.3 (75-127)
  	*At time of enrollment

Isolation of Blood Microvesicles
• Blood was drawn through a 19 gauge needle into tubes containing anticoagulants, hirudin and soybean trypsin inhibitor (which inactivates platelets). Plasma was separated by centrifugation (3000×g for 15 minutes, twice) to obtain platelet free plasma. The absence of platelets in the plasma was validated by Coulter counter (platelet count≦1), and flow cytometry (FACS Canto™) using fluorescent beads (1 μm and 2 μm).
• The cell free, platelet-free plasma sample (0.5 μL) was centrifuged (20,000×g for 30 minutes), and the supernatant was removed. The pellet obtained was reconstituted with 0.5 mL of 20 mM HEPES/HANK'S/0.05% glucose buffer (pH 7.4) which had been filtered using 0.2 μm pore size membrane filter. The sample was washed by vortexing, and centrifuged (20,000×g for 30 minutes). After the final centrifugation, buffer was discarded, and the pellet was reconstituted with 0.5 mL fresh buffer. The sample was vortexed for 1-2 minutes to detach microvesicles from the sides of the tube and to separate microvesicles from each other.
Staining of Blood Microvesicles
• The microvesicle preparation was placed into flow cytometry tubes in 50 μL aliquots. Antibodies were added, based on the cell or receptor of interest, and incubated for 30 minutes. Antibody concentration needed for optimal staining was determined by experimentation. Anti-CD42a-PE mouse anti-human monoclonal antibodies (GPIX, BD) were used to label platelet-derived microvesicles. Anti-CD62e PE mouse anti-human monoclonal antibodies (e-selectin, BD) were used to identify endothelium-derived microvesicles. Anti-VEGFR-1 PE mouse anti-human monoclonal antibodies (R&D) were used to label VEGFR-1. Annexin V mouse anti-human monoclonal antibodies (BD) and anti-B48/B100 mouse anti-human monoclonal antibodies (AbCam) were used as markers of phospatidylserine and lipid expression. Controls were run for each sample using same isotype IgG FITC or PE stained samples.
Identification of Blood Microvesicles
• Flow cytometry (FACSCanto™) was used to detect microvesicles by size and positive antibody fluorescence. Gates to define size were set using an internal standard of 1 μm and 2 μm beads (FIG. 5). For quantification, samples were spiked with a known quantity of beads of 4.2 μm diameter (FIG. 5). All buffers and antibodies were filtered through a 0.2 μm pore size membrane filter to eliminate chemical particles and to reduce instrument noise.
Separation and Quantification of Microvesicles Based on Staining
• The absolute numbers of annexin-V microvesicles were calculated based on counts of calibration beads. The absolute count of microvesicles equaled the number of events in the microvesicle gate per number of events in the calibration bead region as multiplied by the number of beads per test (spiked known count/test volume). The same calculation was applied to quantification of microvesicles positive or negative for annexin-V or other cell specific monoclonal antibodies.
Data Analysis
• Data were analyzed using Microsoft Excel. The diagnosis of preeclampsia was made in the presence of hypertension accompanied by proteinuria, as recommended elsewhere (Am J Obstet Gynecol183(1): S1-S22 (2000)). Hypertension was defined as blood pressure of 140/90 mmHg; excretion of 300 mg of protein or more in a 24-hour urine specimen was considered diagnostic of significant proteinuria.
Results
• Preeclamptic women had median (inter-quartile range) proteinuria (mg/24 hour) of 1466 (561-2642). A portion of plasma microvesicles expressed VEGFR-1 in both preeclamptic and normal pregnant women. VEGFR-1 positive microvesicles were significantly elevated in preeclamptic as compared to normal pregnant women. VEGFR-1 positive microvesicles were 30.0 (14.3-81.0) microvesicles per liter plasma in preeclamptic women as compared to 12.0 (8.0-14.0) microvesicles per liter plasma in normal pregnant women (p=0.01 by Wilcoxon rank sum test). There was not a statistically significant difference in total microvesicles per liter plasma between the two groups: 327 (289-509) in preeclamptic women vs. 333 (232-385) in normal pregnant women (p=0.54 by Wilcoxon rank sum test).
• In summary, the results provided herein indicated that an increased level of VEGFR-1 expressing microvesicles is associated with preeclampsia in women.
OTHER EMBODIMENTS
• It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (11)

1. A method for assessing a pregnant mammal for preeclampsia, said method comprising determining whether or not a plasma sample from said mammal contains an elevated level of microvesicles expressing a VEGFR-1 polypeptide, and classifying said mammal as having preeclampsia if said plasma contains said elevated level.

2. The method ofclaim 1, wherein said mammal is a human.

3. The method ofclaim 1, wherein said determining step comprises using flow cytometry.

4. The method ofclaim 1, wherein said determining step comprises using an antibody to detect said microvesicles.

5. The method ofclaim 1, wherein said antibody is an anti-VEGFR-1 antibody.

6. The method ofclaim 1, wherein said method comprises classifying said mammal as having preeclampsia if said elevated level is present, and classifying said mammal as not having preeclampsia if said elevated level is not present.

7. A method for assessing a mammal for risk of developing a complication of preeclampsia, wherein said method comprises determining whether or not plasma from said mammal contains an elevated level of endothelium-derived microvesicles and classifying said mammal as being at risk for developing a complication of preeclampsia.

8. The method ofclaim 7, wherein said determining step comprises using flow cytometry.

9. The method ofclaim 7 wherein said determining step comprises using an antibody to detect said endothelium-derived microvesicles.

10. The method ofclaim 7, wherein said complication of preeclampsia comprises HEELP syndrome.

11. The method ofclaim 7, wherein said method comprises classifying said mammal as being at risk of developing a complication of preeclampsia if said elevated level is present, and classifying said mammal as not being at risk of developing a complication of preeclampsia if said elevated level is not present.

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