Abundance of megalin and Dab2 is reduced in syncytiotrophoblast during placental malaria, which may contribute to low birth weight

Abstract

Placental malaria caused by Plasmodium falciparum contributes to ~200,000 child deaths annually, mainly due to low birth weight (LBW). Parasitized erythrocyte sequestration and consequent inflammation in the placenta are common attributes of placental malaria. The precise molecular details of placental changes leading to LBW are still poorly understood. We hypothesized that placental malaria may disturb maternofetal exchange of vitamins, lipids, and hormones mediated by the multi-ligand (n ~ 50) scavenging/signaling receptor megalin, which is abundantly expressed in placenta but was not previously analyzed in pregnancy outcomes. We studied abundance of megalin and its intracellular adaptor protein Dab2 by immunofluorescence microscopy in placental biopsies from Ugandan women with (n = 8) and without (n = 20) active placental malaria. We found that: (a) abundances of both megalin (p = 0.01) and Dab2 (p = 0.006) were significantly reduced in brush border of syncytiotrophoblast of infected placentas; (b) amounts of megalin and Dab2 were strongly correlated (Spearman’s r = 0.53, p = 0.003); (c) abundances of megalin and Dab2 (p = 0.046) were reduced in infected placentas from women with LBW deliveries. This study provides first evidence that placental malaria infection is associated with reduced abundance of megalin transport/signaling system and indicate that these changes may contribute to the pathology of LBW.

Full text

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Scientific RepoRts | 6:24508 | DOI: 10.1038/srep24508
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Abundance of megalin and Dab2
is reduced in syncytiotrophoblast
during placental malaria, which
may contribute to low birth weight
Jared Lybbert1, Justin Gullingsrud2, Olga Chesnokov1, Eleanor Turyakira3, Mehul Dhorda4,5,
Philippe J. Guerin4,5, Patrice Piola5, Atis Muehlenbachs6 & Andrew V. Oleinikov1,2
Placental malaria caused by Plasmodium falciparum contributes to ~200,000 child deaths annually,
mainly due to low birth weight (LBW). Parasitized erythrocyte sequestration and consequent
inammation in the placenta are common attributes of placental malaria. The precise molecular details
of placental changes leading to LBW are still poorly understood. We hypothesized that placental
malaria may disturb maternofetal exchange of vitamins, lipids, and hormones mediated by the multi-
ligand (n ~ 50) scavenging/signaling receptor megalin, which is abundantly expressed in placenta
but was not previously analyzed in pregnancy outcomes. We studied abundance of megalin and its
intracellular adaptor protein Dab2 by immunouorescence microscopy in placental biopsies from
Ugandan women with (n = 8) and without (n = 20) active placental malaria. We found that:
(a) abundances of both megalin (p = 0.01) and Dab2 (p = 0.006) were signicantly reduced in brush
border of syncytiotrophoblast of infected placentas; (b) amounts of megalin and Dab2 were strongly
correlated (Spearman’s r = 0.53, p = 0.003); (c) abundances of megalin and Dab2 (p = 0.046) were
reduced in infected placentas from women with LBW deliveries. This study provides rst evidence that
placental malaria infection is associated with reduced abundance of megalin transport/signaling system
and indicate that these changes may contribute to the pathology of LBW.
During P. falciparum infection, pregnant women can suer from placental malaria (PM), where parasitized eryth-
rocytes (PE) sequester in the placenta1, which can lead to inammatory response. PM contributes to about 200,000
neonatal and 10,000 maternal deaths annually in malaria endemic regions2. Infant mortality in PM is largely due
to low birth weight (< 2.5 kg), in addition to stillbirth, and abortion3. Sequestration of PE in the placenta occurs
at the boundary surface of the syncytiotrophoblast4 – the interface between maternal blood and fetal vasculature
where maternofetal exchange takes place5. Anatomically, fetal blood vessels branch in the placenta into villi that
are covered by a single layer of multinucleated cell called the syncytiotrophoblast, which is created by fusion
of underlying cytotrophoblast cells. e apical surface of the syncytiotrophoblast has microvilli (brush-border)
expanding its surface to ~12.5 m2 for extensive molecular exchange5. Sequestration of PE on this surface can
lead to macrophage inltration into the intervillous space, local inammation, and pathological changes in the
syncytiotrophoblast4,6. ese processes, in turn, may lead to reduced maternofetal exchange and reduced fetal
growth during gestation, and nally to LBW and other poor outcomes including premature birth, pre-eclampsia,
and small for gestational age babies7–9. Recent insights into mechanisms of the placental pathological processes
during PM suggest involvement of various pathways, including angiogenesis10,11, insulin-like growth factor
(IGF-1) axis12, and, potentially, mammalian target for rapamycin (mTOR) (all extensively reviewed in refs 6,13).
Nevertheless, the molecular details of these processes, as well as the involvement and role of other proteins and
pathways, are still poorly understood.
1Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL, USA. 2Seattle Biomedical
Research Institute, Seattle, WA, USA. 3Mbarara University of Science and Technology, Mbarara, Uganda. 4Centre
for Tropical Medicine and Global health, Nueld Department of Clinical Medicine, University of Oxford, Oxford,
UK. 5Epicentre, Mbarara, Uganda. 6University of Washington, Seattle, WA, USA. Correspondence and requests for
materials should be addressed to A.V.O. (email: aoleinikov@health.fau.edu)
Received: 11 December 2015
Accepted: 30 March 2016
Published: 13 April 2016
OPEN

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In this respect the glycoprotein megalin (also called gp330, gp600, and LRP2) is a particularly interesting can-
didate. It is a large (~600 kDa) multi-ligand single-spanning trans-membrane endocytic receptor with substantial
physiological functions. It belongs to an ancient14 low density lipoprotein receptor family. e importance of this
receptor has been demonstrated in a large number of experiments across multiple organs15, though its role in the
placenta is not yet well characterized. It has been shown that megalin expresses in placenta and is localized on the
surface of the syncytiotrophoblast16,17. Levels of megalin expression in the placenta are third highest following
its expression by thyroid and kidney proximal tubular cells18. Many functions of megalin have been studied in
kidney and in early embryonic development15,19. In mice, megalin expresses at very early stages of embryonic
development20. Knockout of the megalin gene in mice leads to perinatal death (only 2% newborn mice survive)
and severe pathologies in various organs, most notably in brain morphology, as well as in the kidney and lung20,21.
Also, megalin-decient mouse fetuses at mid-gestation were signicantly smaller than wild-type20. It was hypoth-
esized that developmental deciency in these megalin knockout animals might be explained, at least in part, by
a vitamin and lipoprotein deciency due to defective/insucient transport of these molecules (see below) to the
fetus through megalin endocytosis in the yolk sac and placenta19,20. In humans, placental cytotrophoblast and
syncytiotrophoblast megalin starts to express at least as early as 7–8 weeks gestation22 and expresses through
term16,17,22,23. Substantial expression of megalin in the syncytiotrophoblast brush border16,17 indicates its impor-
tance for placenta function. In humans, mutation preventing megalin expression are extremely rare and only
about a dozen surviving patients with Donnai-Barrow syndrome have been previously described24, with pathol-
ogies similar to those observed in megalin-knockout mice.
In epithelial cells, megalin is transferred with its cargo to the base of microvilli and is internalized through
coated-pit endocytosis. We have demonstrated earlier that megalin interacts with a phosphotyrosine-interacting
domain of the adaptor protein Dab225,26, which binds to the internalization motif 27 in the megalin cytoplasmic
domain. Dab2 is expressed in various tissues including syncytiotrophoblast and in trophoblast cells28,29. Its mRNA
expression in placenta is highest among all tissues18. Based on previous studies of others on Dab2 functions28,30,31,
we hypothesized that megalin and Dab2 might be involved in both signal transduction as well as in endocyto-
sis through their interactions25. is hypothesis was experimentally conrmed in multiple studies15,19,32,33. As
Dab2 is important for cell growth suppression28,30, it may also have an important role in placental growth and
development.
Megalin interacts with an enormous suite of about 50 extracellular ligands which belong to several unrelated
groups of proteins15. ese ligands include nutrients, hormones and their carrier proteins, vitamin-binding and sig-
naling molecules, morphogens, and extracellular matrix proteins including serum proteases and their inhibitors.
Internalized molecules are either transcytosed or released in endosomes or lysosomes (vitamins, cholesterol, etc.)
and then can be utilized within the cell or excreted on the cell surface by various transporters. Clearly, such
diverse functions might have signicant impact on the maternofetal exchange of nutrients and signals, as well as
aect the homeostasis of various important molecules. is, in turn, may lead to placental pathology resulting in
poor outcome for fetal development, including LBW, if the megalin system is somehow disturbed.
A few interesting observations suggest that the megalin system might be aected in PM. When the megalin
gene is knocked out, the number of microvilli and the amount of endocytosis are signicantly reduced in kid-
ney21. ese changes are reminiscent of loss of syncytiotrophoblast microvilli reported during PM4 and strongly
support the idea that megalin expression/distribution in PM might be disturbed. In addition, inammatory pro-
cesses, similar to that observed during Heymann nephritis, can lead to shedding of the megalin exodomain34.
To get insights into the megalin system during PM, we determined the abundance of megalin and its intracel-
lular adaptor protein Dab2 in formalin-xed paran-embedded placental samples obtained from women living
in malaria endemic areas of Uganda, with and without PM, who had normal and low birth weights of newborn
babies (Table1 and Supplementary Table 1).
Results
Patient characteristics. Samples were selected based on availability of samples from previous study35,36.
Summary clinical information is presented in Table1, and data from individual samples are presented in
Supplementary Table S1. Groups of Ugandan mothers without (PE−) and with (PE + ) placental infection
at delivery were similar in respect to parity, hemoglobin levels, estimated gestational age, and birthweight of
newborns.
Mothers without
placental infection (PE−)
Mothers with placental
infection (PE + ) P valuea
Number of women 20 8
Parity median [interquartile range]; n 1 [1.25]; 16 1 [1]; 6 0.88
Hemoglobin level [g/dL] mean (SD); n 11.4 (2.3); 16 10.5 (2.1); 5 0.37
Estimated gestational age [weeks] mean (SD); n 39 (2); 15 38 (4); 5 0.88
Birthweight [g] mean (SD); n 2916 (513); 16 2942 (476); 5 0.89
Low birth weight n (%) 4/16 (25%) 2/6 (33%)
Stillbirth n (%)b0/16 (0%) 1/6 (17%)
Table 1. Clinical characteristics of 28 Ugandan pregnant women at delivery. aMann-Whitney test was used
to calculate all p values. bis sample was included in categorical analysis as low birth weight.

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Expression of megalin and Dab2 in human syncytiotrophoblast in term placenta.
Immunouorescence studies conrmed substantial expression of megalin in the syncytiotrophoblast of human
term placenta as previously reported17,22. However, this expression was not uniform throughout the syncyti-
otrophoblast; parts of the syncytiotrophoblast brush border may express very little of megalin (and Dab2), if any,
as was noted previously for megalin in placenta22. erefore, quantitative comparison of protein abundances in
various samples is challenging. To overcome this diculty the following steps were taken: (1) protein abundances
were measured in the parts of syncytiotrophoblast brush border (BB) with the highest signal as reecting the
ability of the tissue to express these proteins and (2) repeated blind experiments were performed using dier-
ent antibody preparations. ree non-overlapping areas encompassing the syncytiotrophoblast BB from each
placental section were selected for the highest density of signal, measured, and averaged, as described in the
Methods. Supplementary Figure S1 demonstrates that measurements obtained in independent blind experiments,
using two independent preparations of anti-megalin C-terminal peptide (in cytoplasmic tail) antibodies and
two preparations of anti-Dab2 antibodies raised against dierent regions, strongly correlate, suggesting that our
measurements accurately reect the levels of abundance of these proteins. As anti-megalin prep 2 (more recent
preparation) antibody and anti-Dab2 commercial antibody had higher signal according to linear regression anal-
ysis (Supplementary Figure S1), they were used for all analyses described below.
Placental malaria is associated with reduced abundance of megalin and Dab2. e abundance
of both megalin and Dab2 was reduced in the syncytiotrophoblast brush border in malaria-infected placentas by
indirect uorescence microscopy, with statistical signicance for both proteins (Fig.1). Data on parity is available
for 22 samples used in this analysis (Supplementary Table S1). Only including these 22 samples in the analysis
shows that median parity is not dierent (p = 0.88) for groups stratied by placental infection, while abundance
of both proteins is still reduced in the infected group (Supplementary Figure S2), demonstrating that parity is not
a confounding factor. Samples with previous (resolved) malaria infection, as dened by the presence of extra-
cellular hemozoin (Hz) in brin, are indicated in Fig.1. If samples with active and past placental infection were
combined, megalin and Dab2 continue to demonstrate a statistically signicant reduction of abundance in these
samples against normal controls (p = 0.04 and 0.005 for megalin and Dab2, respectively). Two data points for
megalin and one for Dab2 with past (resolved) placental infections were above the median protein abundance
value in the group of non-infected placenta samples (Fig.1), which may point out the possibility for restoration of
megalin and Dab2 expression in the placenta aer placental infection is cleared. Similar results (as in Fig.1) were
obtained when protein abundance was measured in the entire syncytiotrophoblast when cytoplasmic staining was
included (though uorescence values were lower).
When data were stratied by the presence of placental inammation in malaria-infected samples (n = 3 )
against samples without infection and inammation, a similar decrease in megalin and Dab2 abundance was
observed (Fig.2), with statistical signicance for Dab2.
Also, irrespective of PM status of samples, statistically signicant positive correlation of megalin and Dab2
expression in the brush border region was observed (Fig.3). A similar correlation was observed when the entire
syncytiotrophoblast was analyzed (data not shown).
Figure 1. Megalin and Dab2 abundance is reduced in brush border area of syncytiotrophoblast in
placentas with malarial infection at delivery. Abundance of megalin and Dab2 was assessed in brush border
of placentas with PE (n = 8) and without PE (n = 20) using immunouorescence assay as described in Methods.
Medians of arbitrary uorescence units (AFU) are reported (gray bars). Filled symbols represent samples with
extracellular hemozoin in brin indicative of previous (resolved) placental infections, clear symbols represent
samples without hemozoin. Protein abundance between 2 groups was compared using Mann-Whitney test.
PE – parasitized erythrocytes in the placenta at the time of delivery.

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Expression of megalin and Dab2 is reduced in placentas with malaria parasites and low birth
weight. Moderate correlation of birth weight with abundance of megalin and Dab2 in the brush border of
the syncytiotrophoblast was identied, though below the level of statistical signicance (Spearman correlation
coecients, Megalin: r = 0.35 [CI = − 0.09981 to 0.6782], p = 0.11; Dab2: r = 0.23 [CI = − 0.2242 to 0.6030,
p = 0.3). When Dab2 expression levels were stratied by median Dab2 abundance, birth weight was 350 g greater
in those with high (above median) versus low (below median) placental expression, although the dierence was
not statistically signicant (p = 0.095) (a woman with stillbirth and no detectable Dab2 expression was excluded).
Abundance of both megalin and Dab2 is substantially reduced in the brush border of malaria-infected placentas
obtained from LBW deliveries (Fig.4), with statistical signicance for Dab2 (p = 0.046). Figure5 illustrates com-
parative dierences observed between two samples from infected placenta and LBW versus uninfected placenta
and normal birth weight (NBW). Specically, the brush border of syncytiotrophoblast demonstrates the low or
absent levels of megalin and Dab2 in the LBW samples with active malaria infection.
Discussion
PM-related changes in the maternofetal interface of the placenta are associated with LBW6. Several mechanisms
contributing to LBW have been suggested, including dysregulated angiogenesis9,10,37, impaired growth hormone
production12,38, and decreased nutrient transport6. In this study a novel role for megalin system in PM pathologies
Figure 2. Megalin and Dab2 abundance is reduced in brush border area of syncytiotrophoblast in
placentas with malarial intervillous inammatory inltrates. Megalin and Dab 2 abundance in placentas
with PE and malaria-relevant inammation (n = 3) and without PE and inammation (n = 19) was measured
using immunouorescence assay. Medians of arbitrary uorescence units (AFU) are reported (gray bars).
Protein abundance between 2 groups was compared using Mann-Whitney test. PE – parasitized erythrocytes in
the placenta at the time of delivery.
Figure 3. Correlation of megalin and Dab2 abundance in brush border area of syncytiotrophoblast.
Correlation between amounts of Megalin and Dab2 was assessed using Spearman’s rank correlation coecient
(r). Megalin and Dab 2 abundance in all placentas (n = 28) was measured in arbitrary uorescence units (AFU)
using immunouorescence assay as described in Methods. Six study participants (4 with PE and hemozoin, 1
with hemozoin only, and 1 without PE or hemozoin) did not have detectable megalin and Dab2. Line indicates
linear regression, slope = 0.77 + 0.19, p = 0.0003. PE – parasitized erythrocytes in the placenta at the time of
delivery. Hemozoin in the placenta at the time of delivery is indicative of past infection.

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was explored, including PM-associated LBW. Results demonstrate that PM is associated with reduced syncyti-
otrophoblast abundance of megalin and Dab2 proteins, known to provide endocytosis and signaling pathways,
which, in turn, may play a role in fetal growth restriction and low birth weight pathology.
Specically, the study provides evidence that megalin and Dab 2 abundance is reduced in placentas with active
infections (Fig.1). Similar reduction (with statistical signicance for Dab2) was also observed in infected placen-
tas with inammation (Fig.2). As PM is oen characterized by inltration of immune cells, especially monocytes
and macrophages, into intervillous space39, these results suggest that inammation may aect megalin system
expression in syncytiotrophoblast. e small number of samples with malaria-related inammation (n = 3) limits
statistical power in our analysis.
Further, amounts of megalin and Dab2 are positively correlated in the placental brush border (Fig.3). It has
been shown earlier that Dab2, as an intracellular ligand of megalin25, co-localizes with megalin in renal proximal
tubules40. In addition, expression levels of both proteins are mutually dependent, where knockout of one of them
reduces expression of the other one40. e results presented here expand this observation to placental tissue and
indicate inter-relevance of megalin and Dab2 expression. Moreover, this underscores the importance of signi-
cant associations identied in this work, even found for one of these proteins, as reduction of its abundance may
aect the function of the entire system.
While all samples with placental infection and LBW had low or undetectable megalin and Dab2 (3/3), there
were 4/11 and 3/11 normal controls with NBW that also had low or undetectable levels of megalin and Dab2,
respectively (Fig.4). In addition, the reduction of megalin system proteins in the placenta correlated with the
reduction in birth weight irrespective of PM status, but these data were not statistically signicant. ough small
sample size denitely limited the power of our analysis, it allows to speculate that such correlation or association
might be the case not only during PM, but, potentially, in other diseases of pregnancy. Low levels of these proteins
in a proportion of malaria-negative placentas may indicate that other factors might aect their abundance. For
example, renal inammatory processes such as those that occur in Heymann nephritis can lead to shedding of the
megalin exodomain34; both megalin and its co-receptor cubilin were downregulated in gallbladder epithelium in
gallstone patients at the mRNA and protein levels41; mice fed a cholesterol rich lithogenic diet showed a reduction
in megalin mRNA expression in these cells15. Megalin expression might be responsive to treatment, for example,
rosiglitazone signicantly increased megalin mRNA expression in the gallbladder15. Similarly, multiple factors
may contribute to the expression of megalin system proteins in uninfected placentas. Also, in spite of mega-
lin importance, even megalin knock-out newborn mice can survive, though in low numbers (2%), indicating
that some redundant mechanism(s) may compensate for megalin system insuciency during fetal development
which allows for survival20,21.
Nevertheless, categorical analysis of protein abundance data stratified by LBW and placental infection
revealed statistically signicant reduction for Dab2 abundance (and very similar trend for megalin) compared to
non-infected placental samples with NBW (Fig.4). is result suggests that reduction in abundance of megalin
transport/signaling system proteins in PM may contribute to the pathology of LBW. Below is a discussion of the
potential mechanisms of pathological changes in pregnancy in relevance to the disturbance of the megalin system
in placenta, which was observed in this work during PM.
Figure 4. Megalin and Dab2 abundance is reduced in brush border area of syncytiotrophoblast in
placentas with malarial infection and low birth weight. Megalin and Dab 2 abundance in placentas with PE
and low birth weight (LBW) (n = 3) and without PE or past infection and normal birth weight (NBW) (n = 11)
was measured using immunouorescence assay. Medians of arbitrary uorescence units (AFU) are reported
(gray bars). Protein abundance between 2 groups was compared using Mann-Whitney test. PE – parasitized
erythrocytes in the placenta at the time of delivery.

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Maternal cholesterol is essential for fetal growth42. It has been shown that fetal growth restriction is associ-
ated with alterations in placental lipoprotein receptors (LDL, LRP1) and maternal lipoprotein composition43.
Megalin is involved in transport of the same lipoparticles, plus it interacts and transports additional types of
Apo-lipoproteins and might substantially contribute to cholesterol transport, necessary for fetal growth.
Megalin also plays an important role in vitamin homeostasis by re-absorbing various vitamins in complexes
with their carrier proteins in kidneys19,44. Because vitamins for the developing fetus can only be supplied from
maternal blood, megalin may play a similar function in placenta supplying retinol22, cobalamins45, folate, and
vitamin B1245, as well as vitamin D, which is important for regulation of calcium homeostasis46. Moreover, de-
ciency of vitamin D in pregnancy may increase the risk of preterm delivery and fetal growth restriction47.
Further, megalin co-expresses on the syncytiotrophoblast surface with several receptors specialized for interac-
tions with hormones/growth factors (that are also megalin ligands) including insulin-like growth factor-1 (IGF-1),
parathyroid hormone and epidermal growth factor5, which play important roles in the regulation of fetal growth
throughout pregnancy48. yroid hormones (TH) are involved in placenta villous development and free TH (T4)
levels are associated with birth weight49. In maternal blood, T4 is almost completely bound to transthyretin, a
ligand for megalin50 that is secreted to maternal circulation and re-absorbed by trophoblasts51, and therefore may
maintain T4 homeostasis. As mentioned above, PM was associated with disturbance of the IGF axis, with signi-
cant changes in IGF-1 but not IGF-2, and no detectable changes in mRNA levels for IGF-1 receptor12. Since IGF-1
is also a ligand for megalin and modulation of the megalin system may aect levels of IGF-152, megalin reduction
may contribute to the eects of PM on IGF-1 level changes12.
How does PM aect the megalin system? One potential mechanism might be that sequestration of PE through
surface-expressed PfEMP1 parasite adhesins may cross-link the corresponding host adhesion receptor, chon-
droitin sulfate A1, on the syncytiotrophoblast surface. is clustering of the surface receptors may directly aect
multiple membrane processes including megalin-mediated endocytic and/or signaling pathways, as was the case
in other studies on the eects of cross-linking of other cell surface receptors53. Inltrating macrophages during
PM may directly damage the syncytiotrophoblast surface, linking placental inammation and megalin system
abundance (Fig.2) and/or function.
Figure 5. Expression of megalin and Dab2 in placentas from mothers with low birth weight babies is
reduced. Megalin (A,B) and Dab2 (C,D) expression in syncytiotrophoblast of placentas with malaria infection
and low birth weight (A,C) and without malaria infection and normal weight (B,D) was measured using
immunouorescence assay. Protein expression is shown in green, DAPI nuclear stain in blue. Pairs of pictures
(A–D) were taken and processed under identical conditions. Bright cells inside of fetal vessels are auto-
uorescing erythrocytes (e). Brush border (BB) is indicated by arrows. LBW – low birth weight. NBW – normal
birth weight.

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TGFβ negatively regulates endocytosis of albumin through megalin and cubilin54. During placental malaria
TGF-beta concentration in placenta is signicantly increased55. Hence, systemic regulation of placental megalin
expression in malaria is possible and may lead to reduced endocytosis in infected placentas.
is study is limited by the number of samples investigated. A logical continuation would be a similar study
with larger sample size and samples obtained from various geographical areas, taking into account a larger set
of variables, including timing of malaria infection during pregnancy and nutritional status. Further, eects of
PE adhesion to cytotrophoblast primary cells or cell lines, like BeWo cells, on megalin system functioning in the
absence and presence of monocytes/macrophages may test our hypothesis in vitro. ese studies may contribute
to development of therapy alleviating pathological eects of megalin system dysfunction, for example by aecting
transcription of relevant genes, and/or by supplementation of nutrients and ligands.
In summary, it is likely megalin is involved in fetal growth via a complex network of regulatory activities par-
ticipating in endocrine, paracrine, and autocrine signaling by interacting with and internalizing various ligands
of maternal and fetal origin. We propose that dysregulation of these networks in the placenta aects development
of the fetus. To our knowledge, this is the rst report linking the abundance of placental megalin system proteins
with the birth weight of newborn babies and associating PM infection with changes in abundance of megalin
system proteins.
Methods
Ethics Statement. Written informed consent was provided and specimens were collected under approval
by the Uganda National Committee for Science and Technology (#HS207). e use of coded specimens was
approved and deemed not human subjects research by the University of Washington Human Subjects Division
(#35425) and by the Florida Atlantic University Institutional Review Board (#801504). All experiments were
performed in accordance with the relevant guidelines and regulations.
Placental samples. Specimens were from a cohort of a nested randomized control trial of
artemether-lumefantrine versus quinine to treat malaria in pregnancy conducted in 2006–2009 in Mbarara,
Uganda, as previously described35. Briey, the randomized controlled trial inclusion criteria included viable preg-
nancy at gestational age of 13 weeks or later and positive malaria blood smear by microscopy; exclusion criteria
included P. falciparum parasitemia greater than 250 × 103/L, severe anemia (hemoglobin < 7 g/dL), or severe or
complicated malaria needing parenteral treatment. As previously described56, placental biopsies were xed and
stored in neutral buered formalin, routinely processed and scored for the presence or absence of parasitized
erythrocytes (PE + or PE− ) and hemozoin in brin57, which were semi-quantitatively scored together with the
presence of intervillous inammatory inltrates58. Placentas positive for parasitized erythrocytes (PE) by his-
tology were referred as malaria-infected. To select samples for this study (n = 28) all available samples that had
parasites identied by histology (n = 8) were matched to sequential controls that had no identiable placental
malaria-related changes on histopathology (n = 17), maintaining similarities in parity (Supplementary Figure S2).
These control samples included available placental specimens from the larger cohort of women, who were
screened negative for malaria by blood smear at weekly visits and not included in the randomized controlled
trial36 (samples 23–28 in Supplementary Table S1). In addition, a subset of women with a relatively large amount
of hemozoin in brin but no parasites (heavy past infections) were also available and were included (n = 3).
For the immunofluorescence assays, sections of 6 m were prepared from paraffin embedded blocks of
formalin-xed placentas obtained from these women. Relevant clinical parameters of placenta samples are
described in Supplementary Table S1.
Antibodies. Primary antibodies used were monospecic puried rabbit antibodies, raised against mega-
lin C-terminal 17-mer peptide (2 independently puried preparations) and Dab2 phosphotyrosine interaction
domain, which were described and characterized previously25, and commercial anti-Dab2 antibody H-110
(# sc-13982, Santa Cruz Biotechnology, CA). Secondary antibodies used were Alexa Fluor 594-labeled polyclonal
Donkey anti-Rabbit IgG (#711-585-152, Jackson Immunoresearch, West Grove, PA).
Measurement of megalin and Dab2 abundance by indirect fluorescence microscopy. The
paran embedded tissue samples were rehydrated by a series (3 min each) of graded xylene/ethanol washes
(2 times with 100% xylene, once with 1:1 xylene/ethanol, once sequentially with 100%, 95%, 70% and 50% eth-
anol in deionized water), followed by a nal wash in phosphate buered saline (PBS) for 5 min. e rehydrated
slides were then heated in Tris-EDTA buer (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween-20, pH 9.0)
and microwaved to boiling at full power and then maintained at 50% power for 8 minutes and cooled in PBS59.
Sections were then pre-incubated with PBS, 1% BSA and 0.25% Triton-X for 30 minutes, incubated with primary
antibody (Dab2 commercial 1:100; Dab2-PID 1:100; Megalin C-terminal 17-mer peptide 1:50) in the presence
of 2.5% non-immune donkey serum (#017-000-121, Jackson Immunoresearch, West Grove, PA, USA), either
for one hour at room temperature or overnight at 4 °C in a humidied chamber and washed with PBS for 5 min-
utes 3 times. Slides were then incubated with secondary antibody in the presence of non-immune donkey IgG
(# 017-000-002, 1:100, Jackson Immunoresearch, West Grove, PA, USA) for one hour at room temperature and
counterstained with DAPI (5 g/mL, Sigma) for 3 minutes. Aer three washes with PBS for 5 minutes, slides were
processed with mounting medium (Vectashield H-1000, Vector Laboratories, Burlingame, CA, USA) and cov-
erslip applied. Negative controls were processed without primary antibody or with antibodies puried from the
pre-immunized animal serum on the same column as monospecic antibodies. Both methods produced similar
background levels (data not shown). For the staining, to provide maximally identical conditions, each placental
section was divided in sub-sections processed with all primary antibodies, and then incubated with secondary
antibody simultaneously. As a positive control for the procedures used, we stained fresh-frozen mouse kidney

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Scientific RepoRts | 6:24508 | DOI: 10.1038/srep24508
6 m sections as described above and obtained strong staining of the proximal tubule cells with anti-megalin and
anti-Dab 2 antibodies, as described previously40,60 (data not shown).
Slides were imaged using a confocal microscope (Zeiss LSM 700 equipped with Plan-Apochromat 63x/1.40 Oil
DIC M27 objective) and processed using Zen 2012 Blue soware (Zeiss, Jena, Germany). Images were captured
using identical conditions to allow for quantication of signal strength. Collected images of syncytiotrophoblast
were selected for the brush border only (~1 m from the surface to inside of the cell) or the entire syncytiotropho-
blast (up to ~10 m in depth and excluding nuclei). e process of selection is illustrated in Supplementary Figure S3:
selected region A indicates brush border area, and region A+B indicates entire syncytiotrophoblast. In each
placental section at least 3 non-overlapping areas encompassing syncytiotrophoblast 50 m–100 m along the
surface were selected for the highest density of signal (in arbitrary uorescence units, AFU) using visual appear-
ance and density-measuring feature of the Zen 2012 Blue soware, measured as described above, and averaged
for each placental sample. If staining was similar throughout the section, 3 random areas were selected. Negative
control AFU were then subtracted from the experimental AFU to obtain nal AFU for each sample used in the
analysis. All slides were processed by the same individual, blindly.
Statistical analyses. Statistical analyses were performed using GraphPad Prism 6 (La Jolla, California, US)
statistical soware using non-parametric statistics: Mann-Whitney tests (two-tail) for group dierences and
Spearman tests for analyses of correlations. P value of < 0.05 was considered signicant. Qualitative level of sig-
nicance indicated by stars: *p < 0.05, **p < 0.01, ***p < 0.001.
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Acknowledgements
is work was supported by the National Institutes of Health (grants 5R01HD058005 and 1R21AI105506 to
A.V.O.), by the Florida Atlantic University Graduate Research & Inquiry Program (GRIP grant to J.L.), and by
the Florida Atlantic University (Start-up fund to A.V.O.). e original study which produced the initial placental
sample collection was supported by Médecins Sans Frontières and the European Commission. We are grateful to
the entire research team at the Epicentre, Mbarara, Uganda and all the study participants and their families who
made this study possible.
Author Contributions
A.V.O. conceived and designed the experiments. J.L., J.G., O.C. and A.M. performed the experiments. J.L., J.G.,
and A.V.O. analyzed the data. E.T., M.D., P.J.G., P.P. and A.M. contributed reagents/materials. A.M. and A.V.O.
wrote the manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Lybbert, J. et al. Abundance of megalin and Dab2 is reduced in syncytiotrophoblast
during placental malaria, which may contribute to low birth weight. Sci. Rep. 6, 24508; doi: 10.1038/srep24508
(2016).

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Scientific RepoRts | 6:24508 | DOI: 10.1038/srep24508
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