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Human Molecular Genetics Advance Access originally published online on November 21, 2005
Human Molecular Genetics 2006 15(1):11-21; doi:10.1093/hmg/ddi421
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Loss of polycystin-1 or polycystin-2 results in dysregulated apolipoprotein expression in murine tissues via alterations in nuclear hormone receptors

Erica Allen1, Klaus B. Piontek1, Elizabeth Garrett-Mayer2, Miguel Garcia-Gonzalez1, Kerry Lee Gorelick1 and Gregory G. Germino1,*

1Department of Medicine, Division of Nephrology 2Department of Oncology, Division of Biostatistics, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross Building 9S., Baltimore, MD 21205, USA

* To whom correspondence should be addressed. Tel: +1 4106140089; Fax: +1 4106145129; Email: ggermino{at}jhmi.edu

Received September 26, 2005; Accepted November 7, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations of PKD1 and PKD2. Murine gene targeting studies have shown that these genes play an essential role in development, with homozygous inactivation resulting in embryonic lethality. Recently, Pkd1–/– lethality has been linked to placental insufficiency. In this study, the placenta was used as a model to identify factors involved in these developmental abnormalities. Microarray analysis of Pkd1–/– placentae showed upregulation of a set of apolipoprotein-related genes. These changes were validated and were found to be associated with increased quantities of apolipoproteins in the amniotic fluid. Increased apolipoprotein gene expression was also observed in Pkd2–/–placentae and in cystic kidneys of Pkd1cond/–; Meox2cre/+ mice. Using chromatin immunoprecipitation assays, we determined that the activity of HNF-4{alpha}, a major regulator of apolipoprotein gene expression, was also increased in these organs. These findings suggest a potential role for dysregulation of nuclear hormone receptors in the pathogenesis of ADPKD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic disorders, affecting approximately one in 1000 individuals. The disease is characterized by the progressive formation of kidney cysts, leading to renal failure in 50% of patients by their mid-50s, as well as a number of extra-renal manifestations (1Go). These include the formation of hepatic and pancreatic cysts, hypertension, left ventricular hypertrophy, cardiac valvular abnormalities and an increased prevalence of intracranial aneurysms (1Go–3Go).

ADPKD results from mutation of either of two genes: PKD1, which accounts for ~85% of cases, and PKD2, accounting for most others (4Go). Polycystin-1 (PC1), the protein product of PKD1, is a large transmembrane protein thought to be involved in cell–cell and cell–matrix interactions and may also function as a flow sensor on the primary cilia of renal epithelial (4Go–6Go). Polycystin-2 (PC2), the protein product of PKD2, is considered to be the founding member of the TrpP family of TRP (transient receptor potential) channel proteins and to function as a non-specific cation channel with highest permeability for calcium (4Go,7Go–12Go). PC2 interacts with PC1 through their respective C-terminal coiled-coil domains, and PC1 is thought to modulate PC2's channel activity (11Go,13Go,14Go). The proteins are believed to form a receptor channel complex whose activity can be disrupted by mutation of either gene. This property is thought to explain the indistinguishable clinical presentations that result from mutation of PKD1 and PKD2.

A number of mouse lines with mutations of murine Pkd1 and Pkd2 have been described (15Go). Pkd1+/–and Pkd2+/–mice appear healthy with few, if any, kidney cysts but develop cystic livers of variable severity later in life. Homozygous Pkd1 and Pkd2 mutants have much more severe phenotypes. Pkd1 mutants typically die prior to 16.5 d.p.c. and may develop renal and pancreatic cysts, skeletal defects, cardiac abnormalities and vascular abnormalities including edema and hemorrhages (16Go–18Go). Pkd2 mutants also die in utero and share many of the abnormalities described for Pkd1–/– mice, though cardiac abnormalities may be more common, more severe and associated with lateralization defects (19Go,20Go). Collectively, these studies have demonstrated an essential developmental role for both genes.

Recently, we have determined that abnormalities of the placental labyrinth layer are the likely cause of Pkd1–/– fetal death (K. Piontek et al., manuscript in preparation). Pathologic changes are first noted at 11.5 d.p.c. and become more severe at later timepoints. By 14.5 d.p.c., the Pkd1–/– labyrinth layer is smaller than that of Pkd1+/–and Pkd1+/+ embryos and has decreased fetal arteriole and capillary networks that are disorganized with reduced branching. We found that tetraploid aggregation methods corrected the placental abnormalities, whereas conditional inactivation of Pkd1 in epiblast-derived tissues using cre-recombinase regulated by the Meox2 promoter (Meox2Cre/+) spared the placenta and allowed almost 50% of Pkd1 mutants to survive to birth (21Go).

Placental development shares many features with other developing organ systems. Given the critical role of Pkd1 in the placenta, we reasoned that its study could provide insight into the function of Pkd1 in other organs as well. In the present study, we compared the transcriptional profile of Pkd1 mutant and normal tissues and unexpectedly found evidence of dysregulated nuclear hormone receptor activity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
RNA expression studies of Pkd1–/– placentae
To better understand the placental abnormalities resulting from inactivation of Pkd1, we compared the expression profiles of placental tissue of littermate pairs at the latest viable timepoints (N=3 at 15.5 d.p.c. and N=4 at 14.5 d.p.c.). We used cDNA glass slide microarrays that were spotted with the NIA 15K gene set (22Go). Data were normalized by Lowess normalization and fit to a linear mixed model to determine reproducibly altered genes. T-ratios for genes were determined (higher |t-ratio|=lower P-value), as well as fold change in gene expression. Volcano plots showing |t-ratio| on the y-axis and fold change on the x-axis graphically summarize the data (Supplementary Material, Fig. S1). The cut-off for fold-change was set at >1.5 and significance was set at a |t-ratio|>3. Using these cut-offs, we identified 42 and 58 genes upregulated at 14.5 and 15.5 d.p.c., respectively, and 14 and 40 genes downregulated in the Pkd1–/–placentae (Supplementary Material, Figs S2 and S3).

One striking and unexpected finding was that a set of apolipoprotein and apolipoprotein-related genes had significantly increased expression in the Pkd1–/– versus control specimens at both 14.5 and 15.5 d.p.c. (Table 1). The difference between mutants and controls reached statistical significance for Apolipoprotein A-IV (ApoA-IV), microsomal triglyceride transfer protein (Mtp), and Apolipoprotein C-II (Apo-CII) at both timepoints. Apolipoprotein A-I (ApoA-I) and low density lipoprotein-related receptor 2 (Lrp2) were also expressed at higher levels in mutant tissues at 14.5 and 15.5 d.p.c. but the difference was not significant at the earlier stage. Given that Lrp2 serves as a receptor for Apolipoprotein B (ApoB) (among other proteins) and Mtp's primary function is in the translocation of, and the transfer of lipids to, ApoB, we re-examined our data looking for changes in ApoB expression (23Go,24Go). Although the level of expression was variable, examination of individual chips revealed that ApoB was often one of the most upregulated genes in Pkd1–/– placentae (>10-fold higher levels in some samples) and differences in ApoB expression at 15.5 d.p.c. approached significance (t-ratio=2.47).


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  		Table 1. A set of lipoprotein-related genes upregulated in 129/BS 14.5 and 15.5 d.p.c. Pkd1–/– placentae
 
We confirmed the results for three of the most highly upregulated genes (ApoA-I, ApoA-IV and ApoB) in the placentae of five littermate pairs by northern blot. Two of the pairs had previously been used for the microarray studies. Consistent with the previous findings, ApoA-IV and ApoA-I were significantly upregulated in the Pkd1–/– samples. ApoB levels also were consistently higher in the Pkd1–/– placentae but the values ranged greatly, creating too large a standard error to support a finding of statistical significance (Fig. 1). These results correlated very well with the microarray data.



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  		Figure 1. Northern blot results for ApoB, ApoA-I and Apo-IV. Northern blots of total RNA prepared from placentae of 15.5 d.p.c. Pkd1+/+ and Pkd1–/– (129/BS) littermates were probed with ApoB, ApoA-I and ApoA-IV, imaged using a PhosphorImager and analyzed using ImageQuant software. Results were normalized to those of ß–actin. Representative blots of apolipoprotein results, as well as averages, are shown. The averages are expressed as the log 2 ratio of the value for the mutant sample over the value of its littermate control (Log2(KO/WT)). ‘n’ indicates the number of pairs of specimens examined with each probe. *P<0.05 (Paired Student's t-test). Fifteen micrograms of total RNA was loaded in each lane. Average Log2(KO/WT) values: ApoA-IV=2.4±1.7; ApoB=4.9±3.3; ApoA-I=3.7±1.8.

 
We used quantitative real-time PCR (qPCR) to validate the microarray results for the 14.5 d.p.c. samples and to determine whether a similar pattern also occurred at earlier timepoints when tissues are smaller and histopathologic abnormalities are less dramatic. In the first set of studies, we compared expression of ApoA-I, ApoA-IV and ApoB in the placentae of four littermate pairs of 129Sv/BS Pkd1–/–and Pkd1+/+ 14.5 d.p.c. mice, one of which had previously been used for the microarray studies (Fig. 2A). Each of the genes was found significantly upregulated in the affected tissue (P<0.05).



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  		Figure 2. qPCR of ApoB, ApoA-I and ApoA-IV at 14.5 and 12.5 d.p.c. (A) The average Log2(KO/WT) values (left) and representative real-time graphs (right) are shown for each of the genes for littermate pairs of Pkd1–/–(red) and Pkd1+/+ (black) 14.5 d.p.c. placental specimens (129/BS). ‘n’ indicates the number of pairs of specimens examined with each probe. *P<0.05 (Paired Student's t-test). Apo gene expression levels were normalized to those of ß-actin. For the real-time graphs, the amount of PCR product is expressed in relative fluorescence units (RFU, y-axis legend) and indicates the fold increase of fluorescent signal over background. The orange horizontal line indicates the threshold level used for each reaction. Average Log2(KO/WT) values: ApoA-IV=3.3±1.7; ApoB=3.3±1.8; ApoA-I=4.0±1.9. (B) Same as in (A), except samples were derived from 12.5 d.p.c. placentae (129Sv). *P<0.05 (Paired Student's t-test). Average Log2(KO/WT) values: ApoA-IV=3.4±2.7; ApoB=4.2±2.9; ApoA-I=2.9±2.3.

 
One notable aspect of our results was that the gene expression ratios varied considerably, regardless of modality that was used. We postulated that subtle differences in gestational age between pregnancies, variability in the severity of the phenotype and genetic modifiers at other loci in this mixed strain model might account for this property. To minimize strain differences as a confounding variable and to confirm that the findings were strain independent, we compared expression levels of the apolipoprotein genes in tissues of inbred 129Sv Pkd1–/–and Pkd1+/+ mice. Because the phenotype is more severe in this inbred line, we were able to obtain only one 14.5 d.p.c. pair of samples for study. The pattern of upregulation was similar to that obtained for the other 14.5 d.p.c. samples. ApoA-I, ApoA-IV and ApoB were upregulated 28-, 24- and 58-fold, respectively, when assayed by qPCR and 4-, 11- and 24-fold when measured by microarrays. ApoA-IV and ApoB expression levels were also increased by 14- and 33-fold, respectively, by northern blot analysis (data not shown).

Lastly, we compared the level of expression of the three genes in placental samples from five 12.5 d.p.c. 129Sv littermate pairs (Fig. 2B). The pattern was similar to that found for the 14.5 d.p.c. samples. Each of the genes was significantly upregulated in the Pkd1–/–specimens when compared with littermate controls.

Pkd1–/–mice have greater quantities of apolipoproteins in their amniotic fluid
We next examined whether the increased apolipoprotein mRNA expression observed in Pkd1–/– extra-embryonic tissues was associated with an increase in protein levels. Using ApoA-I and ApoB as representative examples for the set, we found no observable increase in either protein's level in tissue lysates of placentae or whole 14.5 d.p.c. embryos (data not shown). Total protein amounts also did not differ significantly between genotypes. In contrast, we found tremendous differences in both the volume and total protein content of amniotic fluid samples that correlated with genotype. At 12.5 d.p.c., 75% of Pkd1–/– animals had polyhydramnios when compared with littermates of other genotypes, and this increased to 100% by 14.5 and 15.5 d.p.c. At every timepoint, Pkd1–/– embryos had more total protein in their amniotic fluid (Fig. 3A). We also found that band intensities for ApoA-I and ApoB were always equal or higher in amniotic samples of Pkd1–/– mice than that in their normal counterparts when equal quantities of total amniotic protein were compared. Given the much higher amounts of total protein present in the Pkd1 mutant specimens, these results show that Pkd1–/– specimens have an average of 1.7- and 7–9-fold more ApoA-I and ApoB at 12.5 and 14.5/15.5 d.p.c. timepoints, respectively (Fig. 3B).



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  		Figure 3. Pkd1–/–mice have greater quantities of apolipoproteins in their amniotic fluid. (A) Averages of total volumes (top) and total protein levels (bottom) in amniotic fluid samples from 129Sv 12.5 d.p.c. (10 WT, 13 HT, 11 KO), 129/BS 14.5 d.p.c. (9 WT, 19 HT, 9 KO) and 129/BS 15.5 d.p.c. (3 WT, 3 HT, 4 KO) specimens. *P<0.05 for KO versus WT; **P<0.01 for KO versus HT and WT; P<0.05 for HT versus WT (Student's t-test). Average volumes in µl: 12.5d: WT 33.9±19.2, HT 34.1±16.3, KO 49.9±24.1; 14.5d: WT 40.9±29.3, HT 72.1±50.6, 194.4±81; 15.5d: WT 47±34.7, HT 53.7±41.8, KO 181.8±82.8. Average microgram of protein: 12.5d: WT 27±16.7, HT 30.1±25.6, KO 46.1±25.1; 14.5d: WT 38.7±19.8, HT 83.9±52.1, 363.9±230.2; 15.5d: WT 81.7±57.1, HT 124.2±83, KO 555.8±164.4. (B) Representative western blots of ApoB and ApoA-I in amniotic fluid at various gestational ages. Equal amounts of total protein were loaded for each sample of a set. The total amount of protein in each sample is also indicated.

 
Pkd2–/– mice have placental abnormalities similar to those found in Pkd1–/– mice
Given that inactivation of Pkd1 and Pkd2 both result in fetal demise, we queried whether the placental abnormalities observed in Pkd1–/– specimens were also present in Pkd2–/– samples. We compared sections of Pkd2–/–and Pkd2+/+ 14.5 d.p.c. placentae stained with hematoxylin–eosin (H&E) and with antibodies that recognize laminin, a component of the basement membrane of fetal vessels (25Go). The results were similar to what we had previously seen in the Pkd1–/– specimens. The labyrinth layer had fewer fetal vessels, which formed disorganized networks with reduced branching (Fig. 4A). The severity of the changes was variable but the findings were present in the placentae of all Pkd2–/–mice examined.



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  		Figure 4. Pkd2–/–mouse placentae have histological and transcriptional alterations similar to those of Pkd1–/–specimens. (A) Representative sections from the labyrinth layer of 14.5 d.p.c. Pkd2+/+ (WT) and Pkd2–/–(KO) placentas stained with H&E and {alpha}-laminin. (B) Average Log2(KO/WT) values and representative real-time graphs are shown for Apo genes for littermate pairs of Pkd2–/–(red) and Pkd2+/+ (black) 12.5 d.p.c. placental specimens. Average Log2(KO/WT) values: ApoA-IV=3.4±2.2; ApoB=3.3±2; ApoA-I=4.3±2.7. ‘n’ indicates the number of pairs of specimens examined with each probe. Apo gene expression levels were normalized to those of ß-actin. *P<0.05 (Paired Student's t-test). (C) Pkd2–/–mice have greater quantities of apolipoproteins in their amniotic fluid. Top: Averages of total protein levels (top) and total volume (bottom) in amniotic fluid samples from 12.5 d.p.c (4WT, 8 HT, 6 KO) specimens. *P<0.05; **P<0.001 compared with HT and WT (Student's t-test). Average volumes in µl: WT 23.3±19.9; HT 31.3±11.6; KO 86.7±7.9. Average microgram of protein: WT 18.8±17.7; HT 22±9.3; 52.6±17.7. Bottom: Representative western blots of ApoB and ApoA-I in amniotic fluid of 12.5 d.p.c. samples. Equal amounts of total protein were loaded for each. The total amount of protein in each sample is also indicated.

 
We then tested whether the Pkd2–/– placentae had changes in apolipoprotein gene expression (ApoA-I, ApoA-IV and ApoB) similar to those found in Pkd1–/– samples. We compared expression levels in four littermate pairs of 12.5 d.p.c. Pkd2–/–mice by qPCR (Fig. 4B). As predicted, each of the three genes was expressed at higher levels in the mutant than in the normal specimens. The difference in ApoB and ApoA-I levels was significant (P<0.05); ApoA-IV levels did not reach significance (P=0.059).

Next, we examined whether the 12.5 d.p.c. Pkd2–/– mice also had an increased quantity of apolipoproteins in their amniotic fluid. Loading equal amounts of total protein for each specimen, we found that ApoA-I band intensities were approximately equal regardless of genotype, whereas ApoB band intensities were increased in the Pkd2–/– samples (Fig. 4C). We also found that all of the Pkd2–/– specimens had polyhydramnios with total amniotic fluid volumes averaging 3.7-fold higher than their normal littermates. In addition, total protein levels in the amniotic fluid of Pkd2–/– samples averaged 2.8-fold higher than that in wild-type littermates. These results suggest that both of the apolipoproteins are increased in the amniotic fluid of the Pkd2 mutant animals.

Pkd1–/cond; Meox2Cre/+ neonatal cystic kidneys have increased expression of ApoA-I, Apo-IV and ApoB
The labyrinth layer of the placenta is formed by a re-iterative process of chorioallantoic branching and thus is thought to be a useful model system for studying branching morphogenesis. Kidney development shares many features with this system, depending on reciprocal interactions between an inductive epithelium (the ureteric bud) and a responsive mesenchyme that undergoes a mesenchymal-to-epithelial transition. Re-iterative branching is also required to produce the large number of nephrons found in the fully developed organ. Given that the cystic kidneys of Pkd1–/– mutants also result from an abnormal developmental process, we queried whether their cystic kidneys had the same transcriptional alterations found in their placentae. Because relatively few Pkd1–/– mice survive to later stages of pregnancy (15.5 d.p.c.) when renal abnormalities are first apparent, we compared expression levels of ApoB, ApoA-I and ApoA-IV in kidneys of Pkd1–/cond; Meox2Cre/+ newborn mice by qPCR. These mice die within hours of birth with cystic kidneys (manuscript in preparation). We studied three Pkd1–/cond; Meox2Cre/+ neonates from two pregnancies as well as seven littermate controls (two Pkd1–/cond, three Pkd1–/+, one Pkd1+/cond; Meox2Cre/+ and one Pkd1+/cond). We found that each of the genes was always expressed at higher levels in the cystic kidneys when compared with the normal controls, with the difference reaching significance for ApoB and ApoA-I (P<0.01 and <0.05, respectively) but not for ApoA-IV (P=0.076) (Fig. 5). In contrast, expression levels were not elevated in the liver of Pkd1–/cond; Meox2Cre/+ mice, which have no obvious mutant phenotype at this life stage (data not shown).



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  		Figure 5. ApoA-I, ApoA-IV and ApoB are upregulated in the kidneys of Pkd1–/cond; Meox2-Cre neonatal mice. (A) Quantitative PCR results for kidney samples of mutant (Pkd1–/cond; Meox2-Cre) versus all other genotypes that are associated with normal phenotype. Het1 and Het2 represent all normal genotypes in pregnancy 1 and 2, respectively. Hom1 and Hom2 represent all Pkd1–/cond; Meox2-Cre mice in pregnancy 1 and 2, respectively. ‘o’ represents fitted value and lines represent 95% confidence intervals. For those lines extending to the limits of the upper edge of the plot, the upper limit of the confidence interval extends beyond (data not shown). ApoB and ApoA-I showed significant increase (P<0.01 and <0.05, respectively), ApoA-IV was close to significant (P=0.076). (B) Representative qPCR traces. (Red, Pkd1–/cond; Meox2-Cre; black, heterozygous littermate.)

 
Nuclear hormone receptors are altered in mutant Pkd1 and Pkd2 tissues
Members of the nuclear hormone receptor family of transcription factors play a central role in the transcriptional regulation of many apolipoprotein and apolipoprotein-related genes. HNF-4{alpha}, a member of this family, has been identified as an activator of transcription of all three dysregulated apolipoproteins (26Go).

We initially screened for a potential increase in HNF-4{alpha} activity by performing qPCR on a subset of genes regulated by HNF-4{alpha} and unrelated to the apolipoprotein pathway (27Go). None of these genes was present on the NIA 15K microarrays. We assayed five Pkd1–/– and Pkd1+/+ placenta pairs and three Pkd2–/– and Pkd2+/+ placenta pairs at 12.5 d.p.c. for transferrin, transthyretin and cellular retinol-binding protein mRNA levels. We found that each of the genes was significantly upregulated in the Pkd1–/– and Pkd2–/– placentae (P<0.05) (Fig. 6A). These data suggest that HNF-4{alpha} activity might be dysregulated in mutant animals.



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  		Figure 6. Nuclear hormone receptor activity is altered in tissues of Pkd1–/– and Pkd2–/– mutants. (A) Quantitative PCR was conducted on 12.5 d.p.c. placentae of Pkd1–/– and Pkd2–/– mutants and littermate controls for transferrin, transthyretin and cellular retinol-binding protein. The Log2(KO/WT) average value for each gene is shown in the top panels. The data for the Pkd1–/– mutants and controls are presented on the left and those for Pkd2–/– and its controls are on the right. Expression of each gene was significantly higher in both the Pkd1–/– and Pkd2–/– specimens than in their respective controls (*P<0.05). ‘n’ refers to the number of pairs. ß-actin was used for normalization. Average Log2(KO/WT) values for Pkd1–/–: Transferrin=3.7±2.4, Transthyretin=4.2±2.6, cRBP=2.9±2.3. Average Log2(KO/WT) values for Pkd2–/–: Transferrin=1.1±0.2, Transthyretin=1.7±0.6, cRBP=1.3±0.6. Representative real-time graphs of HNF-4{alpha} regulated genes and ß-actin (red, Pkd1–/–; black, Pkd1+/+), as well as the averages, are shown. (B) ChIP of HNF-4{alpha} binding to the ApoB promoter was carried out in mouse placental and kidney tissue. PCR amplification of a 188 bp fragment from –171 to +17 of the mouse ApoB promoter, which includes the HNF-4{alpha} binding site, was carried out after HNF-4{alpha} IP. Adult liver was used as a positive control, diluted 5-fold more than placental and kidney samples. Protein G beads were used alone as a non-immunoprecipitation control. ‘Input’ lane represents 10% of pre-IP starting material. The results depicted are representative of 10 sets of experiments using mutant and control placental specimens (four Pkd1 and six Pkd2) (top) and two independent experiments using normal neonatal and neonatal cystic kidneys (Pkd1–/cond; Meox2-Cre) (bottom). Nuclear extracts of normal and cystic kidneys from two newborn mice of the same genotype were pooled for each experiment. A very low signal was frequently observed in the negative control lanes of both mutant and control specimens in a non-specific fashion.

 
We then directly assayed HNF-4{alpha} mRNA and protein levels in 12.5 d.p.c. placentae to determine whether there was an upregulation in expression but observed no substantial change (data not shown). However, post-translational modifications have been suggested to play an important role in the regulation of HNF-4{alpha}-binding activity (28Go,29Go). We therefore performed chromatin immunoprecipitation (ChIP) assays using the well-defined murine ApoB promoter to directly assay in vivo activation of HNF-4{alpha} (30Go). HNF-4{alpha}-binding activity was evaluated in six sets of mutant versus control Pkd1 specimens (two kidneys and four placentae) as well as six sets of mutant versus control Pkd2 placental specimens. Given the small size of the newborn kidneys, we pooled nuclear extracts isolated from both kidneys of two mice of the same genotype for a single assay. We immunoprecipitated DNA/protein cross-linked complexes using anti-HNF-4{alpha} antibody and then amplified the consensus binding site in the ApoB promoter by PCR. HNF-4{alpha} was found bound to the ApoB promoter in all 10 mutant placentae and in both pools of cystic kidneys, whereas binding was absent (n=8 placentae, n=2 pools of normal kidneys) or reduced (n=1 placenta) in control specimens (Fig. 6B). This demonstrates a dysregulation of HNF-4{alpha} activity in Pkd1 and Pkd2 mutant tissues, consistent with its potential role as a positive regulator of apolipoprotein gene expression in our samples.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
In the current study, we used microarray analyses of placental tissue as a screen to identify molecules whose expression is dysregulated upon inactivation of Pkd1. Unexpectedly, we found upregulation of a group of genes related to apolipoprotein synthesis and transport. Multiple independent methods validated our results and showed that the transcriptional changes were associated with increased protein levels in amniotic fluid. We found that Pkd2–/– mice have similar placental abnormalities and showed that changes in apolipoprotein gene expression were not limited to placental tissue, finding similar changes in the cystic kidneys of Pkd1–/cond; Meox2Cre/+ neonatal mice. Lastly, we determined that a set HNF-4{alpha}-regulated genes, unrelated to the apolipoprotein pathway, was also transcriptionally upregulated and that binding of the transcription factor HNF-4{alpha} to the ApoB promoter was increased in mutant Pkd1and Pkd2 placentae and cystic Pkd1 kidneys. These studies suggest a possible role for dysregulated nuclear hormone signaling in the pathogenesis of ADPKD.

It is presently unknown whether the increased HNF-4{alpha} activity is a direct or indirect consequence of loss of polycystin expression. It is possible that loss of polycystin expression may disrupt normal placental or vascular development that results in secondary alterations in nuclear hormone receptor activity. Alternatively, polycystins may play a more direct role in the regulation of HNF-4{alpha} activity. It has been demonstrated in an injury response model that HNF-4{alpha} activity is decreased by phosphorylation in a JAK2-dependent manner (29Go). As polycystins have been shown to activate JAK2, it is possible that loss of either polycystin results in decreased JAK2 activity, reduced HNF-4{alpha} phosphorylation and an increase in HNF-4{alpha} activity (31Go). If this mechanism holds true in non-injured cell models, it could suggest a direct link between polycystins and HNF-4{alpha} activation.

Regardless of whether the alterations in HNF-4{alpha} activity and upregulation of apolipoproteins are a direct or indirect consequence of loss of polycystins, these abnormalities could have important pathologic consequences in the Pkd1 and Pkd2 null mice. Recent evidence showing that apolipoproteins are required for proper Hedgehog and Wingless signaling during Drosophila development suggests one mechanism by which apolipoproteins may exert their effect (32Go). We speculate that induction of apolipoprotein expression by HNF-4{alpha} in cells where apolipoprotein genes are normally minimally expressed may have adverse consequences on cell signaling and contribute to the observed pathology.

The fact that HNF-4{alpha} is an essential developmental factor makes it reasonable to predict that its dysregulation may disrupt the normal developmental program by other means as well (33Go). Although there is currently no murine model of HNF-4{alpha} over-expression reported in the literature, the COUP-TFII null mouse might provide clues as to what might result (34Go). COUP-TFII is a transcriptional repressor that binds to the same consensus promoter sequence as HNF-4{alpha}. In the absence of COUP-TFII, HNF-4{alpha} activity likely increases because of reduced promoter competition. Conversely, increased HNF-4{alpha} activity may compete with COUP-TFII, reducing the activity of the latter. It is thus interesting to note that COUP-TFII–/– mutants develop edema, hemorrhages and cardiac abnormalities that may be similar to those observed in Pkd1 and Pkd2 null mice. Additionally, just a 50% reduction of COUP-TFII levels in COUP-TFII+/– mice results in perinatal death in 2/3 of mice, indicating that even modest changes in its activity can have very detrimental effects (34Go).

The cross-talk between nuclear hormone receptor pathways also may help to explain the beneficial effects of a PPAR-{gamma} agonist on Pkd1 mutant mice as previously reported by Muto et al. They found that treatment of pregnant Pkd1+/– mutant mice with pioglitazone rescued embryonic lethality, ameliorated the severity of the cystic kidney disease and corrected abnormalities in the heart (35Go). We speculate that pioglitazone may have also led to increased levels of COUP-TFII in the tissues of Pkd1 mutant mice as had been previously reported in normal mouse hearts after treatment with troglitazone, another PPAR-{gamma} agonist (36Go). By increasing the levels of COUP-TFII, the authors may have additionally counteracted some of the detrimental effects of increased HNF-4{alpha} activity.

There are several other noteworthy aspects of our findings. We found that the placentae of Pkd2–/– embryos had histopathologic and transcriptional abnormalities similar to those found in Pkd1–/– specimens. These results are not surprising in light of previous studies that described PC2 expression and function in human placental trophoblasts (9Go). Our data suggest that the PC1/2 complex cooperatively regulates branching morphogenesis of the placental labyrinth layer and inactivation of either partner compromises this process. Our findings also provide an alternative explanation for the fetal demise of Pkd2–/– mice. Wu et al. (19Go) reported a range of cardiac abnormalities in Pkd2–/– mice and attributed their in utero death to this problem. We note that many of the cardiac defects are very mild and unlikely to compromise cardiac function sufficiently to result in pre-natal death. Future studies utilizing conditional Pkd2 alleles or tetraploid rescue will be required to establish this with certainty.

We also note that our report lends support to recent studies that have challenged the conventional model of ApoB regulation. Older studies suggest that ApoB is constitutively transcribed and then regulated post-translationally (37Go). However, it has recently been shown that changes in ApoB gene expression may also play a role in the regulation of ApoB levels. For example, in vitro manipulations that increase ApoB mRNA levels in cultured cells result in increased amounts of ApoB secretion (38Go,39Go). Likewise, transgenic over-expression of human APOB in mice and mutations of the ApoB gene promoter have been shown to result in increased plasma ApoB levels (40Go,41Go). In the current work, we describe the first in vivo example where dysregulation of an upstream factor (Pkd1 or Pkd2) results in upregulation of ApoB at the transcriptional level and show that this correlates with increased amounts of protein. These data add further weight to the newly emerging concept that ApoB is complexly regulated at both transcriptional and post-translational levels.

In conclusion, we have identified a pattern of similar transcriptional changes in the placentae and cystic kidneys of mice with inactivating mutations of ADPKD genes. These findings suggest that there are transcriptional pathways regulated by the PC1/2 complex that are likely to be common to the two organs and support the use of the placenta as a developmental model to study PC1/2 function. The data also suggest that HNF-4{alpha} activity is altered, directly or indirectly, by changes in polycystin expression and that its dysregulation plays a role in disease progression. Given the numerous functions of this class of transcription factors and the complexity of cross-talk between them, we speculate that its dysregulation may be important in other organs as well. Finally, these results have potential therapeutic implications as they provide additional evidence in support of therapies that alter nuclear hormone signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Animals/sample isolation
The Pkd1ß-gal-null (referred to as Pkd1–/– in this report), Pkd1cond and Pkd2– alleles have been previously described (19Go,42Go). The Pkd1ß-gal-null allele is maintained in both a mixed (129Sv/Black Swiss, [129/BS]) and an inbred strain (129Sv), and the strains used for each study are identified in the text. Extra-embryonic samples were isolated from timed pregnancies, with the presence of the vaginal plug defined as 0.5 d.p.c. Fetal mouse tails were used to prepare DNA for genotyping. RNA was isolated using Trizol (Invitrogen). RNA samples used for microarray studies were further purified using RNeasy columns or Oligotex-dt mRNA kits (Qiagen). All studies were performed using approved protocols in pathogen-free facilities that are accredited by the AAALAC and meet federal (NIH) guidelines for the humane and appropriate care of laboratory animals.

Microarrays
Microarray experiments were done on NIA 15K glass cDNA mouse slides using Cy3/Cy5 dual labeling of 5 µg of total RNA or 1 µg of mRNA with Invitrogen's Superscript labeling kit (22Go). Dye-swap experiments were performed to control for potential dye bias. Mutant and wild-type specimens from the same pregnancy were paired for the analyses.

Statistical analysis of microarry results
Lowess normalization was performed using the marrayNorm library of Bioconductor to adjust gene expression log ratios for spatial artifacts. A covariate matrix was set up, including dye (i.e. wild-type labeled with Cy3 or Cy5), timepoint, sample pairs (total of seven). A linear mixed model was used to analyze the data. Instead of averaging over replicates, each chip was included in the analysis. The mixed model accounts for correlation of replicates. The model fit was as follows:

where yijk is the log ratio of knock-out expression to wild-type expression for sample pair i, gene j, replicate k; timei is an indicator of whether sample pair i is from 14.5 days and dyeik is an indicator of whether wild-type was labeled with Cy3 for sample i, replicate k. The random effect is bij, which is a random intercept for sample pair i for gene j, and eijk is the random error term, which is normally distributed with mean 0 and constant variance. The parameters of interest are ß0j: the estimated log ratio comparing knock-out with wild-type at 15.5 days in gene j, adjusting for dye effects; ß0j+ß1j: the estimated log ratio comparing knock-out with wild-type at 14.5 days in gene j, adjusting for dye effects. For each parameter of interest, we determined its statistical significance using a z-score.

Northern blot analysis
Total RNA (15 µg) was size fractionated and transferred to GeneScreen Plus Hybridization Transfer Membranes (Perkin Elmer) using standard methods. Hybridizations were performed at 68°C using QuickHyb buffer (Stratagene). Probes for ApoB (base pairs 12097–11223), ApoA-I (base pairs 166884), ApoA-IV (base pairs 43–934) and ß-actin (base pairs 212–1437) were prepared from cloned cDNA. Probes were labeled with [{alpha}-32P]dCTP using the Random Primed Labeling Kit (Roche). Probed membranes were exposed to a PhosphorImager screen overnight, and hybridization signals were quantified using ImageQuant software.

Real-time PCR analysis
cDNA was synthesized using Invitrogen's Superscript 1st Strand System for RT-PCR from 5 µg of total RNA using random hexamers. Quantitative PCR of apolipoproteins was done using SYBR green PCR master mix (Applied Biosystems). Cloned cDNA or amplified cDNA products were used to construct standard curves, and RT-negative samples were used to control for DNA contamination. Samples were run in triplicate and quantitation was done according to Applied Biosystem's User Bulletin no. 2. Annealing temperatures and primer sequences were as follows: ApoB, 62.5°C: 5'-gcaaagccctctgtgtccttggag and 5'-gcctgactcgtggaagaagttggtg; ApoA-I, 55.8°C: 5'-cagattgggtgagacaggagatg and 5'-attcgtccaggtagggctgc; ApoA-IV, 58°C: 5'-gagcaaggtgaagggcaacac and 5'-tccacagtgcgtcggaactc; ß-actin, 58.7°C: 5'-gcagttggttggagcaaacatc and 5'-ttgggagggtgagggacttc.

PCR conditions were 95°C for 10 min, followed by 35 cycles of 95°C for 30 s, annealing temperature for 30 s and 72°C for 30 s, followed by a melt curve. Values were normalized to those of ß-actin.

Transthyretin (Mm00443267_m1), cRBP (Mm00436300_m1) and Transferrin (Mm00446708_m1) were assayed using Applied Biosystem Taqman assays according to manufacturer's protocol.

Western blot analysis
Protein concentrations were measured using the Bradford Assay. Samples were run on either 4% (ApoB) or 10% acrylamide (ApoA-I) gels, transferred to Immobilon-P membrane (Millipore) and then probed with {alpha}-ApoB (US Biological A2299-55) or {alpha}-ApoA-I (Rockland no. 600-101-196) and the ECL chemiluminescence system (Amersham).

Immunohistochemistry
Placentae were fixed in 4% paraformaldehyde, embedded and then cut into 5 µm sections. H&E staining was done by standard protocols. Laminin staining was done as previously described using {alpha}-laminin (L9393, Sigma) at a final concentration of 1:400 and the InnoGenex IHC DAB kit (25Go).

Statistical analysis of kidney and liver samples
‘Expression’ values were standardized within each litter by dividing by the average heterozygous expression value for the litter. Owing to skewness of standardized values, all expression values were transformed by subtracting the reciprocal of the square root. To determine whether the expression in the homozygous and heterozygous expression values differed, a multiple linear regression was performed including fixed effects for heterozygous versus homozygous and for litter. Model assumptions were checked using residual plots. Statistical significance of homozygosity was determined on the basis of P-value of the homozygosity coefficient in the regression model. Fitted model results were transformed back to the original expression scale for plotting and interpretation.

Chromatin immunoprecipitation assay
Assays were carried out using the protocol from Hu et al. (43Go), with minor modifications. Specifically, Protein G agarose was used instead of Protein A agarose. Prior to use, the ssDNA/Protein G beads were incubated for 1 h at 4°C with 100 µg Hek293 cell protein lysate and 1.5 µg Hek293 DNA in order to block non-specific binding to the beads. After the 1 h incubation, beads were washed once and resuspended in ChIP dilution buffer. HNF-4{alpha} antibody was purchased from Santa Cruz (sc-6556). Primers were designed to amplify a region from –171 to +17 of the ApoB promoter, which contains the HNF-4{alpha} binding site: forward: 5'-AGGTGGCGGCGTTTGAA-3'; reverse: 5'-CAGGCACTGATGAGAC-3'. PCR cycles were as follows: 94°C for 4 min, 40 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s, followed by 7 min at 72°C.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank the Germino laboratory, the JHU NIDDK PKD Center of Excellence, Dr Irene Kim of the JHU NIDDK Gene Expression Core and Dr Norman Rosenblum (Toronto) for helpful discussions and Dr Stefan Somlo (Yale University) for his generous gift of Pkd2– mice. This work was supported by the NIH (G.G.G.: [DK48006, DK 57325 and U24DK58757]) and the PKRF (K.B.P.: no. 99024). G.G.G. is the Irving Blum Scholar of JHU. All microarray data have been uploaded to the GEO database and have the following accession nos: GSM51337–GSM51353.

Conflicts of Interest statement. None to declare.


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 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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