Journal of Pharmacological and Toxicological Methods 71 (2015) 147–154

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Original article

Megalin expression in human term and preterm placental villous tissues: Effect of gestational age and sample processing and storage time Amal A. Akour a, Phillip Gerk b, Mary Jayne Kennedy a,⁎ a b

Department of Pharmacotherapy and Outcomes Science, School of Pharmacy, Virginia Commonwealth University, Richmond, VA, United States Department of Pharmaceutics, School of Pharmacy, Virginia Commonwealth University, Richmond, VA, United States

a r t i c l e

i n f o

Article history: Received 29 July 2014 Accepted 3 October 2014 Available online 7 October 2014 Keywords: Megalin Placenta Receptor-mediated endocytosis Preterm mRNA stability

a b s t r a c t Introduction: The aims of this study were to characterize megalin expression in human term and preterm placental villous tissues and to assess the impact of gestational age and sample storage on receptor expression. Methods: Placental tissue samples were collected from pregnant women undergoing term and preterm Cesarean deliveries. Placental villous tissues were used to quantify megalin protein and mRNA expression by western blotting and quantitative polymerase chain reaction (q-PCR), respectively. Stability of megalin expression was also evaluated under various processing and storage conditions. Results: Megalin mRNA was detected in term and preterm placental villous tissues. Expression in early preterm samples was 6-fold higher than in late preterm and term samples. Refrigeration of processed term samples at 4 °C for up to 18 h had a slight impact on megalin mRNA expression with stored samples exhibiting mRNA levels approximately 1.5-fold lower than those frozen immediately after processing. A greater decrease in mRNA expression (up to 33-fold) was observed when processed samples were snap-frozen immediately and thawed at 4 °C. Processing of samples prior to refrigeration also appeared to improve mRNA stability with significantly higher expression levels noted in processed vs. unprocessed samples at all points for up to 48 h. Discussion: These data suggest that expression of megalin mRNA in term placental villous tissue is relatively stable for up to 18 h when samples are processed immediately and refrigerated at 4 °C prior to freezing. Processing prior to storage also appears to improve mRNA stability. This paper demonstrates the practical feasibility of analyzing stored tissue samples, thus, it will help with placental mRNA analysis. Additionally, megalin expression appears to vary inversely with gestational age with the greatest expression noted in the most premature samples. Age-dependent differences in placental megalin may therefore influence fetal exposure. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Intra-amniotic infections (IAIs) are common complications of labor and delivery occurring in up to 10% of all pregnancies and 25% of preterm births (Armer & Duff, 1991; Gibbs, Dinsmoor, Newton, & Ramamurthy, 1988; Soper, Mayhall, & Dalton, 1989). If inadequately treated, these infections can lead to significant morbidity in both the mother and the fetus. Serious, life-threatening complications may also occur in fetuses and infants born to mothers with IAI. It is estimated Abbreviations: AGs, aminoglycosides; ANOVA, analysis of variance; DPBS, Dulbecco's phosphate buffered saline; HDL-C, high lipoprotein cholesterol; HepG2, human hepatocellular carcinoma cells; HK-2, human kidney cells type 2; IAI, intra-amniotic infection; IRB, Institutional Review Board; LRP, low-density lipoprotein receptor-related protein; ML, mouse liver; MW, molecular weight; NTC, non-template control samples; PBS, phosphate buffered saline; PTMs, posttranslational modifications; PVT, placental villous tissue; q-PCR, quantitative polymerase chain reaction; RK, rat kidney; RT, reverse transcriptase enzyme; VCU, Virginia Commonwealth University. ⁎ Corresponding author at: Department of Pharmacotherapy and Outcomes Science, School of Pharmacy, Virginia Commonwealth University, 410 N. 12th Street, PO Box 980533, Richmond, VA 23298-0533, United States. Tel.: +1 804 628 3316. E-mail address: [email protected] (M.J. Kennedy).

http://dx.doi.org/10.1016/j.vascn.2014.10.001 1056-8719/© 2014 Elsevier Inc. All rights reserved.

that up to 4% of deaths in term infants and more than 10% of those in preterm infants are directly related to IA (Newton, 1993). Approximately 20–40% of early-onset newborn sepsis and pneumonia cases are also associated with IAI and occur, most likely, as a consequence of infection acquired in utero (Newton, 1993). While there is unequivocal evidence that maternal antibiotic administration during delivery significantly improves outcomes in both the fetus and the newborn, (Gibbs et al., 1988) the impact of intrapartum antibiotics on morbidity and mortality is highly dependent on attainment of therapeutic drug concentrations in the fetus. It is therefore essential that any maternally-administered antibiotics readily cross the placenta so that adequate fetal levels are achieved. Aminoglycoside (AG) antibiotics (gentamicin, tobramycin, amikacin) are frequently used during pregnancy to treat maternal infections and are recommended by the American College of Obstetrics and Gynecology (ACOG) as one of the first line antibiotic treatments for documented or suspected IAI (ACOG educational bulletin, 1998). These agents are highly active against gram-negative bacteria and, when used in combination with a β-lactam, provide excellent coverage against the most frequently isolated pathogens in early-onset newborn sepsis (E. Coli and Group B

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nature of preterm deliveries, a waiver of consent was granted by the VCU IRB for the collection of preterm placentas. The following information was collected from the subject's medical record: maternal race, placental weight and gestational age.

Streptococcus). AGs also readily cross the placenta achieving maximum fetal serum concentrations that are 21-48% of maternal peak levels within 1 to 2 h of maternal administration (Good & Johnson, 1971; Kauffman, Morris, & Azarnoff, 1975; Weinstein, Gibbs, & Gallagher, 1976; Yoshioka, Monma, & Matsuda, 1972). This level of exposure is above the minimum inhibitory concentration (MIC) for the most common pathogens in IAI and is sufficient to provide rapid, bactericidal activity in the fetus. The highest and most persistent fetal AG levels, however, are found in renal tissue where concentrations of up to 7.2 μg/g (3.5 times the recommended trough serum level) have been observed for up to 34 h after maternal administration (Bernard et al., 1977; Bernard et al., 1977). AG levels are also detectable in fetal kidney tissue within 2 h of maternal administration and continue to steadily increase despite decreasing serum levels (Bernard, Abate, et al., 1977; Bernard, Garcia-Cazares, et al., 1977). There is a direct correlation between the rate and extent of AG accumulation in renal tissue and individual susceptibility to nephrotoxicity, a common and well-characterized side effect of AG treatment (Schentag, Cerra, & Plaut, 1982). The fetus is therefore particularly vulnerable to the nephrotoxic effects of AGs given the rapid and significant renal tissue accumulation that occurs following drug exposure. Persistence of AGs in renal tissue after birth also increases the susceptibility of the newborn to injury during the early postnatal period when AGs are administered as the standard of care to prevent and/or treat infections acquired in utero. Consequently, the need to achieve therapeutic AG levels in the fetal serum must be carefully balanced with the inherent risk of developmental nephrotoxicity. It has been recently demonstrated that megalin, an endocytic receptor expressed on the apical surfaces of absorptive epithelia, is responsible for the uptake of AGs into renal proximal tubular epithelial cells, the physiologic site of AG induced renal injury (Nagai, Tanaka, Nakanishi, Murakami, & Takano, 2001). Pharmacologic blockade of the megalin receptor has been shown to limit renal accumulation of AGs and prevent nephrotoxicity in animal models (Watanabe et al., 2004). This strategy may be useful in preventing drug accumulation in the fetal kidney during intrapartum AG administration but only if placental drug transport remains unaltered by megalin receptor blockade. It is therefore important to understand the molecular mechanisms involved in placental AG transport so that targeted strategies to limit renal accumulation without compromising placental transport can be developed. Expression of the megalin receptor has been previously demonstrated in human term placenta (Larsson et al., 2003) and it is reasonable to speculate, given its role in renal AG uptake, that it is similarly involved in the placental uptake of AGs. It is not known, however, whether megalin is expressed in human preterm placenta and if its expression varies with gestational age. The objective of this study, therefore was to characterize megalin expression in human term and preterm placental villous tissues and to assess the impact of gestational age on receptor expression.

Upon collection, placental tissues were inspected for the presence of any gross abnormalities. Any tissues with visible infarcts, calcifications, hematomas or other abnormalities were excluded from analysis. Pieces of placental tissue were snap-frozen in liquid nitrogen and stored at − 80 °C. Placental villous tissue fragments were also prepared using methods previously described by our laboratory (Vaidya, Walsh, & Gerk, 2009). Briefly, the umbilical cord was cut gently to release any blood and subsequently excised. Triangular wedges of tissue (approximately 100 g) were cut and the basal and chorionic plates removed. Tissues were then rinsed with ice-cold sterile saline and blotted with sterile gauze. Tissue wedges were then gently cut into small pieces, washed in antibiotic-supplemented Dulbecco's phosphate buffered saline (DPBS) filtered through gauze and subsequently minced into smaller pieces. After several cycles of mincing and washing, 300–400 mg of the resulting villous tissue was frozen until analysis (maximum of 4 months). Standard protocol at VCU Medical Center requires that all preterm placentas be evaluated by the Department of Pathology prior to becoming available for research purposes. Because of the inherent variation in processing/storage of preterm samples, stability is therefore a concern. Consequently, we collected, processed and stored term samples under varying conditions to determine the impact of variation in these parameters on megalin stability. To evaluate the effect of storage conditions, term placental tissues were collected and divided into two groups. In the first group, samples were processed immediately and then stored at 4 °C for 1, 2, 4, 6 and 18 h (n = 3 per time point). Samples were then frozen at −80 °C until analysis (Fig. 1-A). In the second group, tissues were snap-frozen immediately after processing and then thawed in the refrigerator for 1, 2, 4, 6 and 18 h prior to analysis (n = 3 per time point). We also evaluated the effect of sample processing pre- and postrefrigeration on megalin stability. Term placental tissue samples were collected and pieces of tissue from each sample were processed using 2 different methods. In the first group, samples were processed immediately after delivery, stored in the refrigerator for 1, 2, 4, 6, 10, 24, and 48 h (n = 3 per time point) and then frozen at −80 °C. In the second group, samples were left unprocessed at 4 °C for 1, 2, 4, 6, 10, 24, and 48 h (n = 3 per time point) and after each time point were subsequently processed and frozen at −80 °C (Fig. 1-B). mRNA expression in these samples were then compared to a snap-frozen sample of the same placental tissue.

2. Methods

2.3. Protein separation and in-gel western blotting

2.1. Study subjects

Frozen placental villous tissue samples were homogenized on ice in 1:10 tissue protein extraction buffer (t-PER®; Thermo Scientific Pierce Inc, Rockford, IL) containing 1:100 Halt® Protease inhibitor (Thermo Scientific, Rockford, IL). Homogenization was done for approximately 1 min at a speed setting of 6.5 using a Polytron PT 10–35 homogenizer with a PTA 10 TS generator (Kinematica, Lucerne, Switzerland). Protein concentrations were determined in tissue supernatants using the BCA protein assay kit (Thermo Scientific, Rockford, IL) with bovine serum albumin (BSA) as a standard. Approximately 100–200 μg of membrane protein were loaded onto a 4−12% polyacrylamide BioRad® tris−glycine denaturing gel (Biorad, Hercules, CA) and electrophoresed at 100 V for 45 min to 1 h. Separated proteins were subsequently fixed with 50% isopropyl alcohol and 12% acetic acid for 15 min at room temperature and after fixation, the gel was washed with ultra-pure water. Binding of the primary (rabbit anti-human megalin, 1:200; mouse

Pregnant adult females (18 to 45 years) admitted to Virginia Commonwealth University (VCU) Medical Center for labor and delivery were eligible for enrollment. Women delivering at term (≥36 weeks) and preterm (b36 weeks) were included. Subjects were excluded if any of the following criteria were met: (Soper et al., 1989) maternal history of diabetes, pre-eclampsia, hypertension or HIV infection; (Gibbs et al., 1988) maternal history of tobacco, drug or alcohol abuse; and/or (Armer & Duff, 1991) documented or suspected placental disorders. The research protocol and informed consent were reviewed and approved by the VCU Institutional Review Board prior to study initiation. Term placentas were collected from women undergoing scheduled Cesarean section at VCU Medical Center and written informed consent was obtained prior to sample and data collection. Given the unplanned

2.2. Collection, processing and storage of placental tissue

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149

A

B

Fig. 1. A. Schematic diagram of the experimental set-up used to assess the effect of refrigerator storage time on megalin mRNA stability. B. Schematic diagram of the experimental set-up used to assess the effect of sample processing pre-and post-refrigeration on megalin mRNA stability.

anti-human β-actin, 1:2000, Sigma Aldrich, St. Louis, MO) and secondary (goat anti-mouse Alexa Fluor 680, 1:5000; goat anti-rabbit IR Dye 800, 1:2000; Li-Cor®, Lincoln, NE) antibodies was performed in 5% bovine serum albumin at 4 °C overnight and at room temperature for 1 h in the dark, respectively. The resulting fluorescent complexes were detected and the band visualized using the Odyssey® Infrared Imaging System (Li-Cor® Corporate, Lincoln, NE). Mouse kidney and liver tissue were used as positive and negative controls, respectively. 2.4. RNA isolation and assessment of mRNA expression Total RNA was isolated from fresh frozen placental villous tissue according to the Trizol® isolation protocol (Invitrogen, Carlsbad, CA). Trizol® lysates of human kidney (HK-2) and human hepatocellular carcinoma (HepG2) cells grown on 12-well Transwell® plates were used as positive and negative controls, respectively. Briefly, 1 mL of Trizol® was added to 100 mg of tissue and homogenized with a Polytron homogenizer. For phase separation, 0.2 mL of chloroform was added for each 1 mL of Trizol® and then samples were centrifuged at 10,000 ×g for 15 min. The RNA was precipitated from the aqueous phase with 0.5 mL of isopropyl alcohol and the resulting pellet was washed with 75% ethanol. After the remaining ethanol was removed, the pellet was dissolved in 30 μL of water treated with diethylpolycarbonate (DPEC).

Total RNA (2.5 μg) was digested by DNAse I and the total concentration of mRNA measured using the Nanodrop® 2000c (Thermo Scientific, Rockford, IL). Purity of the RNA preparation was assessed using the 260/280 nm ratio. Samples with ratios between 1.7 and 2.1 were subjected to reverse transcription in a 20 μL reaction mixture containing the following: reverse transcriptase enzyme (RT); 25 mM Mg(Cl)2; 5× reaction buffer; PCR nucleotide mix and nuclease-free water added to oligo(dT)15 and random primers (0.5 μg/reaction). RNA in the reverse transcription mixture was denatured at 70 °C for 5 min and then annealed at 25 °C for 5 min. First strand synthesis occurred at 42 °C for 60 min and RT was inactivated at 70 °C for 15 min. Taqman® Universal PCR master mix and 40 × Taqman® human megalin probes rs_2229263 were used for the quantification of gene expression. Megalin mRNA expression was normalized to that of 18S (reference gene) which was measured using the Taqman® gene expression assay (Hs99999901_s1). PCR reactions were performed using the Bio-Rad C1000 Thermal Cycler® (Bio-Rad®, Hercules, CA) via the Bio-Rad C1000 Manager software®. Expression of mRNA was expressed as the normalized expression ratio as described by Livak & Schmittgen (2001). This method uses the number of cycles at which the fluorescence signal is detected (Ct) to quantify gene expression. It normalizes the expression of the target gene (megalin) to that of the reference gene (8S) and a calibrator sample (arbitrary sample that is designated

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as a control). The resulting ratio is multiplied by 100% to show percentage. 2.5. Sample size calculation and statistical analysis There are currently no data regarding the differential expression of placental megalin mRNA in preterm and term tissues. The number of samples needed to discern the impact of ontogeny of megalin mRNA expression was therefore estimated from ontogenic data of other transporters that are expressed on the apical (maternal) side of trophoblasts [multi-drug resistance P-glycoprotein (P-gp) (Mathias et al., 2005; Sun et al., 2006), multi-drug resistance related protein 2 (MRP2) (Meyer zu Schwabedissen et al., 2005) and breast cancer related protein (BCRP)] (Meyer zu Schwabedissen et al., 2006). Using these estimates of transporter expression and the demonstrated effect sizes, an average sample size of six subjects per group (range 3–12) is required to detect a significant difference in megalin mRNA expression between term and preterm samples. Sample size calculation was performed using nQuery advisor 7.0® (Statistical Solutions, Boston, MA), (α = 0.05, β = 0.8). The significance of differences observed between data means for megalin mRNA expression was assessed via one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for multiple comparisons. The effects of processing and storage time on megalin mRNA expression were evaluated using two-way ANOVA. Pearson correlation was used to assess the relationship between gestational age (weeks) and megalin mRNA expression. All statistical analyses were performed using GraphPad® Prism 5 (GraphPad Software, Inc., La Jolla, CA) and the level of significance for all analyses was α = 0.05. Unless otherwise indicated, data are presented as mean ± SD. 3. Results 3.1. Sample characteristics A total of ten term and five preterm placentas were collected. All samples were free of gross abnormalities and no visible infarcts, calcifications, hematomas or other abnormalities were noticed. Mothers delivering at term were illness-free and had no history of smoking or alcoholism. Sample characteristics are presented in Table 1. Given that preterm samples were obtained from the Department of Pathology (per institutional standard protocol), placental weight and maternal race information was not available for these samples. 3.2. Megalin protein expression Initial immunoblot analysis showed a specific protein band at a molecular weight greater than 225 kDa in 2 term placental villous tissue samples (gestational ages of 42 and 39 weeks) and in the positive control (rat kidney) (Fig. 2-A, left blot). The corresponding band was Table 1 Summary of placental sample characteristics. Weeks of gestation (n)

Placental weight (g)

Race (n)

Time from delivery to sample processing

42 (3)

695.3 (58.58)

Fresh frozen

39 (6)

760.2 (87.57)

38 (1) 35 (1) 34 (1) 33 (1) 31 (1) 26 (1)

716.2

White: 1 African American: 2 White: 2 Hispanic: 2 African American: 1 Middle Eastern: 1 African American

Fresh frozen

Fresh frozen 1h 5h 3h 7h 5h

very weak or absent in mouse liver (negative control). Halving the amount of total protein loaded led to decreased intensity of the corresponding band in a third sample (gestational age of 39 weeks) (Fig. 2-A, right blot). Additional Western blot analysis of a fourth term (42 weeks) placental villous tissue sample showed a specific protein band at a molecular weight greater than 460 kDa where megalin is known to be expressed; parallel bands were present in both positive controls (rat and mouse kidney) and absent in the negative control (mouse liver) (Fig. 2-B and C). Collectively, these results are consistent with previous investigations, which reported expression of the megalin protein based on detection of a band with a molecular weight greater than 200 kDa (Nagai et al., 2001) (Schmitz et al., 2002)(Gburek et al., 2002; Takano et al., 2002) (Yates, C.I., & Nakorchevsky, 2009). Given its large molecular size (N 460 kDa), and its selective expression in kidney vs. liver (as previously reported), it is likely that this high-molecular weight band is megalin. Using Western blot analysis, however, we were unable to confirm that this high molecular weight band is indeed the megalin protein. Given that the double color detection of megalin and actin could not be achieved simultaneously on the same gel, different methods also had to be used to detect these 2 proteins (in-gel for megalin and conventional for actin) eliminating the possibility of accurately quantitating megalin expression via immunoblot analysis. We therefore also evaluated megalin mRNA expression via quantitative PCR (q-PCR). 3.3. Megalin mRNA expression q-PCR analysis demonstrated expression of megalin in both term and preterm placenta villous tissue samples. A representative chart of mRNA expression in a term sample compared to the positive and negative controls is presented in Fig. 3. Expression of megalin mRNA in the positive control (HK-2 cells) was significantly higher than that in term placental villous tissue and in the negative control (HepG2 cells) (p b 0.05). In contrast, megalin mRNA expression was significantly higher in the term placental villous tissue than the negative control (p b 0.05). 3.4. Effect of processing and storage on mRNA stability Processed placental villous tissues from six term subjects were stored in the refrigerator at 4 °C for up to 18 h before freezing and megalin mRNA expression was subsequently compared to that of a snap-frozen sample. One-way ANOVA followed by Tukey's post-hoc test showed that there is a significant difference in relative megalin mRNA expression in snap-frozen samples vs. samples stored at 4 °C for 1, 2, 4, 6 and 18 h prior to freezing (F = 5.53, df = 16, p b 0.05; Fig. 4-A). Expression in the snap frozen sample was approximately 1.5-fold higher (30% difference) than that in the other samples. After the first hour (H1) there was no significant difference in mRNA expression among samples from the remaining time points. In contrast, when processed samples were snap-frozen at the same time and subsequently thawed at 4 °C for 1, 2, 4, 6 and 18 h, megalin mRNA expression was significantly lower in the thawed samples compared to that in the snap frozen samples (p b 0.05) (Fig. 4-B). Tukey's post-hoc test showed that mRNA expression in the snap-frozen sample was 4.5, 3.4, 6.25, 6.25 and 33.3-fold higher that that in samples thawed for 1, 2, 4, 6, and 18 h, respectively. Two-way ANOVA to evaluate the effect of sample processing pre- and post-storage at 4 °C for up to 48 h on mRNA expression demonstrated a significant interaction between storage time at 4 °C and mRNA expression. Megalin mRNA expression was significantly higher at each time point in samples that were processed prior to storage at 4 °C (Group A) than in samples that were refrigerated prior to processing (Group B) (F = 6.52, p b 0.05) (Fig. 4-C).

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A

B

C

Fig. 2. Immunoblots of megalin in different tissues. A. Immunoblots of term placental villous tissues 1, 2 and 3 (PVT1, PVT2 and PVT3), rat kidney (RK), and mouse liver (ML), and the molecular weight marker (MW). B. Immunoblots of term placental villous tissue (PVT), mouse kidney (MK), and rat kidney (RK). E: Empty lane. C. Immunoblots of mouse kidney (MK), placental villous tissue (PVT), and mouse liver (ML) with a high molecular weight marker (MW).

* * †

One-way ANOVA analysis of megalin mRNA expression in each group showed that when samples were processed shortly after collection, there was no significant difference in mRNA expression between snap frozen samples and that in samples frozen after 1, 2, 4, 6, 10, and 18 h of refrigeration (Group A). In contrast, expression was significantly decreased in samples that were left unfrozen for 24 (mean difference of 48, 8%, 95% CI 7.44–90.10) and 48 h (mean difference 54%, 95% CI 17.01– 90.16). For samples that were refrigerated prior to processing (Group B), there was a significant decline in megalin mRNA expression, 40% of which occurred within the first hour of storage (F = 86.04, df = 24, p b 0.05). 3.5. Effect of gestational age on megalin mRNA expression

Fig. 3. Megalin mRNA expression in human kidney cells (HK-2), term placental villous tissue (PVT) and HepG2 cells. Expression is expressed as normalized ratio multiplied by 100% (data represent mean ± SD of n = 3). *p b 0.05 for the statistical comparison of mRNA expression between HK-2 cells vs. PVT and HepG2; †P b 0.05 for the statistical comparison of mRNA expression of PVT vs. HepG2.

The effect of gestational age on megalin mRNA expression was evaluated by comparing the relative megalin mRNA expression (normalized to 18S) in term (n = 10) and preterm (n = 5) placental villous tissue samples. A significant correlation between gestational age and megalin mRNA expression was observed (r2 = 0.74, p b 0.05) (Fig. 5-A). Furthermore, when preterm samples were divided into two clinically relevant groups [early preterm (b32 weeks; n = 2) and moderate preterm (32 to 35 weeks, n = 3)], one-way ANOVA analysis demonstrated that megalin mRNA expression was significantly greater in

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A

B

*

C

Fig. 4. A. Effect of storage time at 4 °C on megalin mRNA expression (Group A: refrigerated then processed). B. Effect of thawing time at 4 °C on megalin mRNA expression (Group B: refrigerated then processed). C. Effect of processing pre- and post-storage at 4 °C on megalin mRNA expression. Expression is expressed as normalized ratio multiplied by 100% (n = 3 per time point; *p b 0.05).

early pre-term samples compared to those from moderate preterm and term deliveries (F = 172, df = 14, p b 0.05). Tukey's multiple comparison test showed a mean difference of 495% and 526% in mRNA expression between early pre-term and late preterm and term; respectively. Tissues from early preterm samples demonstrated megalin expression levels that were approximately 6-fold higher than that in late preterm and term tissues (Fig. 5-B).

4. Discussion and conclusion In this study, we demonstrated that megalin is expressed in human term and preterm placental villous tissues and that megalin mRNA expression is inversely correlated with gestational age. Placental expression of the megalin protein was highly suggested by the results of our immunoblot analysis and was subsequently confirmed by q-PCR

A

*

*

Fig. 5. A. Relationship between gestational age (weeks) and megalin mRNA expression. B. Effect of gestational age on megalin mRNA expression. Data represent mean ± SD for 3 replicates of a single sample at each gestational age except 40 weeks. At 40 weeks, data represent the mean ± SD of all term samples (n = 10).

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in both term and preterm placental villous tissues, a model that has not been previously used to study megalin expression. While expression of megalin in term placenta has been reported by other investigators, quantitative analyses of megalin protein and mRNA in term placenta have not been completed to date (Casslen, Gustavsson, Angelin, & Gafvels, 1998) (Lambot et al., 2006) (Larsson et al., 2003). Megalin expression in preterm placenta has also not been previously reported. In the current investigation, we were able to quantify expression of megalin mRNA in human term and preterm placental villous tissues. Given that megalin is a large protein that undergoes post-translational modification, however, mRNA expression might not accurately reflect functional protein expression and additional quantitative studies of protein expression may be needed. This is also the first study to characterize changes in megalin mRNA expression occurring as a function of gestational age. Based on the physiologic function of megalin in the placenta, we would expect to see a correlation between gestational age and receptor expression. Megalin is involved in the placental uptake of endogenous substances that are necessary for fetal growth and development (e.g., HDL-C, vitamin B12) (Hammad, Barth, Knaak, & Argraves, 2000) (Moestrup et al., 1996). Our data, which demonstrate higher levels of megalin expression at an earlier gestational age, are consistent with the physiologic role of megalin in supplying specific nutritional substances during this period of gestation via receptor-mediate endocytosis. Our data, which demonstrate a decrease in receptor expression with increasing gestational age, are consistent with the changing nature of fetal nutritional requirements during the gestational period. The early period of fetal growth is characterized by rapid and extensive growth and, as such, a greater nutritional demand (Fall et al., 2003; Wu, Bazer, Cudd, Meininger, & Spencer, 2004). It would be therefore be expected for a receptor like megalin, which contributes to supplying the fetus with vitamins and nutrients, to have higher levels of expression in the early phases of fetal growth (b 32 weeks of gestation) and for the expression levels to decline with fetal growth rate. Interestingly, our mRNA stability data suggest that processed (i.e., cut and washed) placental villous tissue samples can be stored for up to 18 h in the refrigerator at 4 °C before freezing without a significant change in megalin mRNA expression. These data are in agreement with previous studies that have demonstrated stability of TNF-α and cyclooxygenase 2 total RNA in human placental samples stored at 4 °C for up to 48 h (Fajardy et al., 2009). This phenomenon can most likely be explained by the expression of endogenous RNase inhibitor in human placenta, a protein that inhibits the activity of ribonucleases and consequently protects mRNA from degradation. Human placental RNAse inhibitor was purified from placenta by ion-exchange and affinity chromatography in 1977 (Blackburn, Wilson, & Moore, 1977) and it was found to abolish both the angiogenic and ribonucleolytic activities of angiogenin toward 18S and 28S rRNAs (Shapiro & B.L., 1987). Currently, RNAse inhibitors that are isolated from human placenta are commercially available as such or in PCR kits to protect RNA against RNases A, B and C (RNasin® plus RNase inhibitor from Promega). Our data also demonstrate that processing placental villous samples prior to storage at 4 °C improves the stability of megalin mRNA potentially through removal of tissue RNase by tissue cutting and washing. Thawing samples appeared to have a detrimental effect on megalin mRNA expression. This result is consistent with a previously published investigation that examined the effect of thawing on the mRNA expression of the following proteins: B-cell CLL/lymphoma 2; v-fos FBJ murine osteosarcoma viral oncogene homolog; hypoxia-inducible factor 1a subunit, proliferating cell nuclear antigen; and transforming growth factor (Botling et al., 2009). In this investigation, RNA degradation started within a few minutes after thawing the tissue and then declined variably over 16 h of thawing (Botling et al., 2009). There is no explicit explanation for the effect of thawing on the RNA stability, but it could be due to loss of placental RNase inhibitor activity after freezing.

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In conclusion, our data suggest that megalin mRNA expression in processed placental villous tissue samples is relatively stable for up to 18 h when stored at 4 °C and that processing prior to refrigeration significantly improves mRNA stability. An 18-hour refrigeration window therefore appears to be acceptable for processed placental tissue samples intended for studies assessing megalin expression. Given that these stability data are likely to be both tissue and transcript specific, however, caution should be taken when extrapolating these data to tissues or proteins other than megalin. Results from this paper demonstrate the practical feasibility of analyzing stored tissue samples, thus, it will help future researchers in this field with placental mRNA analysis. Megalin expression also appears to vary inversely with gestational age with the greatest expression noted in the most premature samples. Age-dependent differences in placental megalin expression may therefore influence fetal AG exposure to maternally administered megalin substrates such as essential nutrients (e.g., vitamin B12) (Moestrup et al., 1996) and therapeutic medications (e.g., aminoglycoside antibiotics).

Acknowledgments This study was supported, in part, by a grant from the Thomas F. and Kate Miller Jeffress Trust and funds from the VCU School of Pharmacy. Amal Akour was supported by a fellowship from the University of Jordan. The authors would like to thank Dr. Scott Walsh and Ms. Sonya Washington (Department of Obstetrics and Gynecology, Virginia Commonwealth University) for their assistance with procurement of placental samples. In addition, we would like to acknowledge Dr. Matthew Beckman (VCU School of Pharmacy) for his assistance with the qPCR analyses.

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