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Physiol. Genomics 28: 193-202, 2007. First published September 19, 2006; doi:10.1152/physiolgenomics.00147.2006
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Received 10 July 2006; accepted in final form 15 September 2006.
Physiological Genomics 28:193-202 (2007)
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Microarray interrogation of human metanephric mesenchymal cells highlights potentially important molecules in vivo

Karen L. Price 1,*, David A. Long 1,*, Nipurna Jina 2, Helen Liapis 3, Mike Hubank 2, Adrian S. Woolf 1 and Paul J. D. Winyard 1

1 Nephro-Urology, University College London Institute of Child Health, London, United Kingdom
2 Molecular Haematology Units, University College London Institute of Child Health, London, United Kingdom
3 Department of Pathology & Immunology, Washington University Medical School, St. Louis, Missouri


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Many molecules have been implicated in kidney development, often based on experimental animal studies with organ cultures and cell lines. There are very few studies, however, that have directly addressed equivalent living human embryonic tissues. We generated renal mesenchymal cell lines from normal human metanephroi and used a microarray strategy to define changes in gene expression after stimulation with growth factors which enhance nephrogenesis in rodents. Changes were observed in 1) genes modulating diverse general cellular processes, such as matrix metalloproteinase 1 and stanniocalcin 1; 2) genes previously implicated in organogenesis e.g., sprouty 4 and midline 1; and 3) genes involved in blood vessel growth, including angiopoietin 1 and 4. Expression of these same genes was subsequently confirmed in vivo. Our novel data have identified several previously unhighlighted genes that may be implicated in differentiation programs within early human nephrogenesis.

leukemia inhibitory factor; mesenchyme; nephrogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE HUMAN METANEPHROS INITIATES at 5 wk gestation and comprises ureteric bud epithelium and mesenchyme. Mutual inductions between these two lineages lead to normal kidney development (41). Additionally, mesenchyme gives rise to stromal-interstitial tissues and contributes to the vasculature. Defects in the interactions between the mesenchyme and ureteric bud give rise to congenital anomalies such as dysplastic kidneys that have either no functioning nephrons or significant nephron deficits with incomplete differentiation and metaplasia (42).

The identification of human mutations linked to renal malformations have elucidated some information about key genes involved in nephrogenesis, but most detailed functional data derive from mutant or transgenic mice and murine organ culture experiments. Further studies have utilized rodent renal precursor cells (3): for example, transforming growth factor (TGF)-ß superfamily members and fibroblast growth factors (FGF) modulate ureteric bud growth (9), whereas a growth factor cocktail including leukemia inhibitory factor (LIF) promotes differentiation of renal mesenchyme (4, 27).

There are very few studies, however, that have directly addressed equivalent living human embryonic tissues. Burrow and Wilson (8) isolated cells from 12- to 14-wk gestation metanephroi, demonstrating that an (undefined) factor secreted by the G401 renal tumor line enhanced proliferation. Drummond et al. (14) generated several immortalized cell lines, with mesenchymal or epithelial phenotypes, from first trimester metanephroi. Dekel et al. (12) used metanephroi, isolated just a few weeks after inception of the organ, demonstrating continued differentiation after xenotransplantation.

We previously generated renal mesenchymal cell lines from first trimester human metanephroi, which expressed nephrogenic molecules including Wilms' tumor 1 (WT1) and glial cell line-derived neurotrophic factor (GDNF) (28). Herein, we used a microarray-based strategy to define changes in gene expression after stimulation with growth factors considered to enhance nephrogenesis in rodent studies (4). After stimulation, changes were observed in genes that have hitherto not been implicated in human kidney development: expression of these same genes was subsequently confirmed in vivo. Our novel data define several genes that may be important in early stages of human nephrogenesis.


   METHODS AND MATERIALS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All reagents were obtained from Sigma Chemical (Poole, UK) unless otherwise stated. Experiments were all approved by the Institute's Ethical Committee.

Cell culture.
The normal human metanephric cell line was generated from a 10-wk gestation fetus, as previously described (28). The kidneys were diced and placed in Dulbecco's modified eagle medium/Hams F12 (1:1) mix (Invitrogen, Paisley, UK) supplemented with 5% fetal calf serum (FCS; Invitrogen), epidermal growth factor (10 ng/ml), hydrocortisone (36 ng/ml) 3,3',5-Triiodo-L-thyronine sodium salt (4 pg/ml), insulin-transferrin-selenium liquid medium supplement (100x), penicillin G (1,000 U/l), streptomycin (1 mg/ml), and amphotericin (25 mg/ml). Cells were cultured at 37°C in a 5% CO2 incubator, medium was changed twice weekly, and confluent cells were passaged 1:3 or 1:5. To assess potential differentiation, cells were grown to 70% confluence, serum-starved for 24 h, and then treated with either serum-free medium alone (control group, n = 3) or serum-free medium with LIF (20 ng/ml), FGF2 (50 ng/ml), and TGF-{alpha} (10 ng/ml) (treated group; n = 3). We elected 1) to propagate cells on tissue culture plastic, as the most basic substrate; 2) to use defined, serum-free media, to avoid confounding, unknown influences in serum; 3) to use concentrations of LIF, FGF2, and TGF-{alpha} based on studies in which rat mesenchymes were stimulated to differentiate (4); and 4) to stimulate cells with LIF, FGF2, and TGF-{alpha} for 72 h because murine mesenchyme shows evidence of differentiation after this period (4).

Microarray analysis.
We used 5 µg of RNA isolated from control and treated cells with Tri-reagent for microarray analysis and assessed it on the Bioanalyser 2100 (Agilent Technologies, Palo Alto, CA). cDNA and subsequent cRNA synthesis was then performed as described (17). Purified cRNA transcripts were fragmented and hybridized to human U133 plus 2.0 GeneChips according to Affymetrix standard protocols (http://www.affymetrix.com) (n = 3 for control and treated cells at 3 days). Signal values were calculated using MAS 5.0, scaled to 100 and normalized to the median prior to analysis with GeneSpring 7.2 software (Agilent Technologies). Genes were excluded if the signal strength did not significantly exceed background values and if expression did not reach a threshold value for reliable detection [based on the relaxed Affymetrix MAS 5.0 probability of detection; P ≤ 0.1 (31)], in all three of either the control or treated chips. Finally, genes were excluded if the level of expression between control and treated cells did not vary by >1.8-fold. The resulting gene lists were statistically analyzed by the Wilcoxon-Mann-Whitney test, with a P value cut-off at 0.05, and applying the Benjamini and Hochberg false discovery multiple testing correction. The data presented in table form show the mean relative values for genes on three chips. Functional categories for genes were assigned using gene ontology terms from the Gene Ontology Consortium (www.geneontology.com).

RT-PCR.
RT-PCR was performed to characterize human metanephric cells and later to confirm the presence of selected genes altered in the microarray in extracts from whole metanephroi between 9 and 12 wk gestation. The primers used are listed in Supplementary Tables 1 and 2 (the online version of this article contains supplemental material). cDNA was prepared from 1 µg of total RNA according to the provider's instructions (Bio-Rad Laboratories, Hemel Hempstead, UK). A 25-µl final volume reaction was set up containing 1 µl cDNA product, 1.5 mM MgCl2, 200 µl dNTP, iTaq buffer (200 mM Tris·HCl pH 8.4, 500 mM KCl), 100 nM of both forward and reverse primers, and 1.25 units of iTaq DNA polymerase (Bio-Rad Laboratories). PCR amplifications were performed on a DNA Engine Dyad (MJ Research, Waltham, MA). Negative controls of reactions without cDNA template were included. The housekeeping gene, ß-actin, was used as a loading control. In the case of matrix metalloproteinase 1 (MMP1) and plasminogen activator (urokinase), an additional set of commercially available primers was also used (Tebu-Bio, Peterborough, UK). Validation of alterations in expression of selective genes from the microarray was sought by quantitative real-time RT-PCR. RNA was prepared from control and treated cells of three independent experiments, and all measurements were then performed in duplicate. Quantitative real-time RT-PCR was performed on a PTC-200 DNA Engine Opticon System (MJ Research) for all of the primers listed in Supplementary Table 2. Each reaction contained 1 µl of cDNA product, 25 µl of SYBR Green Supermix (100 mM KCl, 40 mM Tris·HCl, pH 8.4, 0.4 mM dNTP, 50 units/ml iTaq DNA polymerase, 6 mM MgCl2, SYBR Green I, 20 nM fluorescein) (Bio-Rad Laboratories), and 100 nM of forward and reverse primers. For each primer, a standard curve was generated, and ß-actin primers were used as a housekeeping gene to allow quantification. Alterations in gene expression in treated cells were expressed relative to the mean intensity in the control cells, which was given a standardized value of 1. Data were statistically analyzed by the Mann-Whitney U-test, with a P value of <0.05 considered significant. Negative controls consisting of reactions without cDNA template were included.


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  		Table 1. Upregulated genes of >1.8-fold (83 in total)

 

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  		Table 2. Downregulated genes of >1.8 fold-change (36 in total)

 
Immunohistochemistry.
Five normal human metanephroi (9–10 wk gestation) were fixed in 4% paraformaldehyde, dehydrated, wax-embedded, and sectioned at 5 µm. Sections were dewaxed and rehydrated, and antigens were unmasked by treatment with proteinase K (20 µg/ml), protease (20 µg/ml), or microwaved in citric acid (2.1 g/l, pH 6.0), depending on the antibody used. Slides were treated with 3% hydrogen peroxide in distilled water to quench endogenous peroxidase. Nonspecific binding was blocked by incubation with 10% FCS, 0.2% bovine serum albumin, and 0.1% Tween 20 in phosphate-buffered saline for 30 min before overnight incubation at 4°C with goat anti-human LIF (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-human LIFR (1:500, Santa Cruz Biotechnology), rabbit anti-human gp130 (1:200, Santa Cruz Biotechnology), rabbit anti-human MMP1 (1:200; Chemicon, Temecula, CA), rabbit anti-human sprouty-4 (SPRY4; 1:100, Abcam, Cambridge, UK), rabbit anti-human midline-1 (MID1, 1:50; Orbigen, San Diego, CA), and goat anti-human stanniocalcin-1 (STC1, 1:50; Santa Cruz Biotechnology). As negative controls, primary antibodies were preincubated with excess immunizing peptides (10:1 wt/wt) or they were omitted. Washed slides were exposed to secondary antibodies, which were detected using an EnVision kit (DAKO, High Wycombe, UK) followed by diaminobenzidine and finally counterstained with hematoxylin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of mesenchymal cells.
As assessed by RT-PCR (Fig. 1), the unstimulated cell line expressed the mesenchymal markers WT1 and GDNF: They did not express E-cadherin, an epithelial marker, nor WNT11 and RET, ureteric bud markers. We noted that these cells, cultured in serum-free media, did not express PAX2, although our earlier studies demonstrated PAX2 expression when cultured in serum-containing media (28).


Figure 1
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  		Fig. 1. RT-PCR characterization of the metanephric mesenchymal cell line. Unstimulated cells (Cells) expressed Wilms' tumor 1 (WT1), glial cell line-derived neurotrophic factor (GDNF), leukemia growth factor (LIF), leukemia growth factor receptor (LIFR), and gp130 but not PAX2, E-cadherin, RET, or WNT11. All genes were expressed in a 12-wk gestation metanephros (12 wk). Negative controls are also shown.

 
Expression of LIF in human renal development.
Using RT-PCR, we found LIF and its two receptors (LIFR and gp130) to be expressed by the unstimulated renal mesenchymal cell line, and all three transcripts were detected in whole human metanephroi (Fig. 1). In tissue sections of first trimester metanephroi (10 wk gestation), LIF immunolocalized in the metanephric capsule and ureteric bud branches (Fig. 2A), LIFR was detected in nephrogenic mesenchyme and also stromal cells between tubules (Fig. 2B), and gp130 signal was detected in subsets of cells in nephrogenic mesenchyme, stroma, and ureteric bud branches (Fig. 2C).


Figure 2
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  		Fig. 2. Immunohistochemistry of the LIF axis in 1st-trimester metanephroi. Sections of 10-wk-old human fetal kidneys were counterstained with hematoxylin: positive immunohistochemical signal is brown. A: LIF detected in capsule and ureteric bud branches. B: LIFR detected in nephrogenic mesenchyme and stromal cells. C: gp130 detected in subsets of cells in nephrogenic mesenchyme, stroma, and ureteric bud branches. D: negative control. Bar is 50 µm.

 
Mesenchymal cells cultured with LIF, FGF2, and TGF-{alpha}.
Parallel cultures were performed and analyzed in triplicate in basal medium alone or media supplemented with LIF, FGF2, and TGF-{alpha}. At the start of the observation period, cells at ~70% confluence had a typical "mesenchymal" morphology with irregular, elongated outlines (Fig. 3, A and B). All cultures became confluent over the next 3 days, and there were no gross changes in cell morphology in either treated or control groups, although those in basal medium alone tended to form randomly aligned elongated cells (Fig. 3C), whereas those exposed to LIF, FGF2, and TGF-{alpha} appeared to take up a more aligned conformation (Fig. 3D). To assess changes in gene expression in LIF/FGF2/TGF-{alpha}-treated vs. control cells, we undertook microarray analysis 72 h after stimulation (n = 3 for each group; Tables 1 and 2). Eighty-three genes showed statistically significant increases of >1.8 fold in treated cells, while 36 genes were downregulated using the same criteria. Changes were observed for: 1) genes modulating diverse cellular processes, particularly MMP1 and STC1, which were the two most highly upregulated genes at 78- and 20- to 25-fold respectively; 2) genes previously implicated in organogenesis e.g., muscle-segment homeobox (msh) homeobox homolog 1 (MSX1; upregulated x5.6), nestin (upregulated x4.6), SPRY4 (upregulated x4.0), and MID1 (downregulated x3.3); and 3) genes involved in blood vessel growth e.g., angiopoietin-like factor 4 (ANGPTL4, upregulated x3.3), angiopoietin 1 (ANGPT1, downregulated x2.3), and vascular cell adhesion molecule 1 (VCAM1, downregulated x3.2). The full data set has been submitted to the ArrayExpress database at the European Bioinformatics Institute. Real-time quantitative RT-PCR was performed on 12 selected genes of biological interest, seven of which were upregulated and the remainder downregulated (Fig. 4). This technique confirmed directions and magnitudes of change suggested by the microarray analysis.


Figure 3
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  		Fig. 3. Human renal mesenchymal cells in culture. Parallel cultures at start of observation period (0 h; A and B) and after three days (72 h; C and D), in basal media (cont; A and C) or exposed to the growth factor cocktail of LIF, fibroblast growth factor (FGF)2, and transforming growth factor (TGF)-{alpha} (LIF; B and D). Note that cells become confluent over the 3 days and that those exposed to the growth factor cocktail appear more aligned. Bar is 50 µm in A and B, and 20 µm in C and D.

 

Figure 4
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  		Fig. 4. Quantitative real-time PCR on selected genes. Left-hand panels in A–C show fluorescence in treated and control samples for stanniocalcin (STC)1, midline (MID)1, and ß-actin, whereas the corresponding right-hand panels show the melting curve for each of the primer sets. D: 7 upregulated genes; E: 5 downregulated genes.

 
Expression of selected target genes in whole metanephroi.
A striking feature of our results, obtained using cultured cells, was how few of the genes identified had previously been highlighted in early (i.e., first trimester) human nephrogenesis. Using RT-PCR we therefore sought, and confirmed, expression of the 12 genes alluded to above in whole metanephroi isolated at 9 and 12 wk gestation (Fig. 5). Additionally, we sought expression of selected genes using immunohistochemistry in sections from similarly staged metanephroi (9–10 wk gestation). First, for comparison, we demonstrated that WT1 immunolocalized to subsets of cells in the nephrogenic mesenchyme and was upregulated in condensed mesenchyme (Fig. 6, A and B), as previously described (39). Of the factors upregulated in treated cells, SPRY4, MMP1, and STC1 were immunolocalized in several areas including renal mesenchyme (Fig. 6, C–J). For MID1, which was downregulated in treated cells, subsets of positive cells were detected in the renal capsule and nephrogenic mesenchyme (Fig. 6, K and L).


Figure 5
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  		Fig. 5. Expression of selected target genes in whole metanephroi. RT-PCR performed in the control cell line (Cells) and in 2 human fetal kidneys at 9 wk (9w1 and 9w2) and 12 wk (12wk) of gestation. All target genes were detected at both stages in vivo, apart from matrix metalloproteinase (MMP)1, angiopoietin-like factor (ANGPTL)4, and PAPLN, which were only detected at 12 wk gestation.

 

Figure 6
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  		Fig. 6. Immunohistochemistry of human metanephroi. We used 10-wk-old human fetal kidneys for all antibodies, with the exception of STC1 where a 9-wk-old kidney was used. All sections counterstained with hematoxylin: positive immunohistochemical signal is brown. A, C, E, G, I, and K are low-power views of the metanephric cortex, whereas B, D, F, H, J, and L are enlarged frames of regions of interest. A and B: WT1 in subsets of cells in nephrogenic mesenchyme and in condensed mesenchyme. Note nonspecific background staining in ureteric bud. C and D: MMP1 in subsets of nephrogenic mesenchyme cells. E and F: sprouty (SPRY)4 in nephrogenic and condensed mesenchyme. G and H: negative control for SPRY4. I and J: STC1 immunolocalized in mesenchyme and ureteric bud branches. K and L: MID1 in the capsule and subsets of nephrogenic mesenchyme cells. Bar is 50 µm for low-powered views and 15 µm for enlarged frames of regions of interest.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Treatment of our human renal mesenchymal cells with a combination of LIF/FGF2/TGF-{alpha} induced a series of reproducible and significant changes in expression of numerous genes. Our in vitro and in vivo data have identified several previously unhighlighted genes which may be implicated in first trimester human nephrogenesis.

Barasch and colleagues demonstrated that LIF is expressed by murine ureteric bud cells, with mesenchyme expressing its receptors. In addition, administration of FGF2 and TGF-{alpha} to isolated mesenchymes prevented apoptosis but, in combination with LIF, led to differentiation with upregulation of the epithelial marker E-cadherin and formation of primitive tubules within 48 h (4). An earlier report, using whole metanephric explants, reported that LIF inhibited nephrogenesis (5); the apparent conflicts between these studies might be explained by confounding (negative) regulatory effects of LIF on the bud itself, or by differences in the concentration of added growth factor.

In the current study, we present the first report of expression of LIF and its receptors in the human metanephros. LIF was immunodetected in the ureteric bud, as in the mouse (4), but we also detected strong signal in the renal capsule. The capsule has recently been reported to contain Foxd1-positive cells and be essential for normal renal development (21). LIFR and gp130 were detected in mesenchyme and stroma of human metanephroi, with the latter protein also found in ureteric bud branches. Thus, in vivo, both human mesenchymal and bud lineages might be targets for LIF, although it is notable that gp130 can also bind several other interleukin-6 family members not sought in this study (24).

The renal mesenchymal cell line used in the current experiments also expressed both LIF receptors, providing a rationale for treatment with LIF (along with FGF2 and TGF-{alpha}). Our initial analyses showed that they expressed both WT1 and GDNF, markers of renal mesenchyme, but not E-cadherin, an epithelial marker, nor ureteric bud markers RET and WNT11. This expression profile is unlike rodent lines such as the 7.1.1 metanephric cells, which express both mesenchymal and epithelial markers (25), and is consistent with the contention that the human cell line represents undifferentiated renal mesenchyme. This impression is also supported by the disorganized morphology of the cells in monolayer culture.

Following exposure of the human cell line to LIF, FGF2, and TGF-{alpha}, 83 and 36 genes were up- or downregulated, respectively, as assessed by microarray analysis. The two factors with the highest change in expression levels following stimulation were MMP1 and STC1. Appropriate breakdown of the extracellular matrix is a critical process in development and MMP1 degrades collagens, aggrecan, proteoglycans, and tenascin (34). Little is known about MMP1 in renal development, but because there is no direct homolog in mice (2), the current insight could not have been elicited in murine studies. Intriguingly, MMP1 was not detected at 9 wk gestation by RT-PCR, but positive immunostaining was detected at 10 wk (and RT-PCR at 12 wk). It would be interesting to repeat these studies with several samples at each time point to assess whether this upregulation occurs consistently, but, unfortunately, sufficient material is not available to study. A related protease, MMP14, was also significantly upregulated (x3.2) and has been reported to be expressed in mouse metanephroi (26). STC1 is a polypeptide hormone regulating phosphate and calcium flux in diverse tissues. Previous studies in mature human kidneys demonstrated expression in principal and {alpha}-intercalated cells (11), but metanephric expression has only previously been reported in rodents (40). In the mouse metanephros, STC1 mRNA is first detected in mesenchyme but is later restricted to ureteric bud derivatives; protein is more widely distributed (13), as might be expected for a secreted protein. Based on expression patterns, it has been speculated that STC1 has a possible role in differentiation in mouse urogenital development and our results raise the same possibility in humans.

The two most downregulated genes in our cells following stimulation were RDH10 and RARRES2. Retinoid signaling is important in renal development, modulating ureteric bud division and final nephron complement (15), and lower urinary tract morphogenesis (7). Exposure of pregnant mice to all-trans retinoic acid in midgestation causes a failure of renal mesenchyme to express WT1 along with fulminant apoptosis (36). The next most downregulated factor was CITED2, a member of the CITED family of nuclear transactivators. CITED2 has been shown to modulate MMP levels in vitro in an immortalized human chondrocyte cell line, with increased CITED2 linked to a fall in MMP1 mRNA and protein levels via a TGF-ß-dependent pathway (45); here we observed a similar converse relationship as the significant fall in CITED2 occurred in conjunction with a massive increase in MMP1.

The MSX1 gene was upregulated x5.6 after 3 days. Msx genes are a family of homeobox-containing factors related to the Drosophila msh that are expressed in diverse vertebrate tissues, including neural crest, bone, and teeth. MSX1 has not previously been reported in human kidney cells, but several of its interacting partners including BMP and WNT family members are important in nephrogenesis (42). Mutations of MID1 are the cause of the X-linked Opitz G/BBB syndrome, which comprises defects of the ventral midline such as hypertelorism, cleft lip/palate, and heart malformations (33). MID1 was downregulated x3.3 on microarray and is known to be associated with microtubules (30) and catalyses degradation of protein phosphatase 2A (PP2Ac), a central cellular regulator (35).

Nestin (two transcripts upregulated x2.2 and x4.6) is an intermediate filament protein classically associated with neuroepithelial stem cells that is now recognized in multilineage progenitor cells (38). Recent reports describe mesenchymal expression at the inception of mouse metanephrogenesis with subsequent expression in podocytes (10). Numerous processes including proliferation, differentiation, migration, and survival are controlled by receptor tyrosine kinase signaling pathways, and the Spry proteins are ligand-inducible inhibitors of these systems (23). We detected significant upregulation of SPRY4 (4.0x) upon exposure to LIF/FGF2/TGF-{alpha}. Several reports have implicated SPRY4 in lung development, where it is expressed in mesenchyme and may antagonize FGF signaling. In the murine kidney, it is also expressed at the tips of the ureteric bud and adjacent mesenchyme (46). SPRY domain-containing SOCS box protein was also significantly upregulated (1.9-fold). Interestingly, SPRY1 mRNA was also upregulated 2.5-fold on microarray, although the P value for this change was just above our cut-off value of 0.05; this factor is thought to modulate GDNF/RET signaling (6), an important process in kidney development as mutations in either of these factors causes renal agenesis.

Alterations in several angiogenic genes were also observed following treatment. The developmental origins of the renal vasculature is much debated with initial suggestions that all of the vessels grew into the kidney by angiogenesis, based on transplantation experiments in the chick, refuted by the finding of vascular precursors within the early metanephros (22) and in metanephric mesenchymal cell lines (37). We detected changes in expression of several "vascular factors" after exposure to LIF/FGF2/TGF-{alpha}. ANGPTL4 was upregulated, whereas VCAM1 and ANGPT1 were decreased. ANGPTL4 is a strongly proangiogenic, hypoxia-inducible factor, and high levels have been described in renal cell carcinoma (20). ANGPT1 is a secreted growth factor that enhances endothelial cell survival and capillary morphogenesis, and expression has already been confirmed in the murine metanephros (43). VCAM1 is expressed in glomerular parietal cells, proximal tubules, and endothelia of mature kidneys (32), and angiotensin II-driven upregulation occurs in diverse renal conditions (19). These are not the only factors associated with vessel development, however, as several of the previously detailed genes may also be implicated. Examples include MMPs, which are important in degradation of the vascular basement membrane and matrix remodeling to facilitate endothelial cell migration (29), and nestin, which has been described in nascent skin blood vessels (1) and in glomerular vascular precursors (10).

Despite these highly significant changes in expression of diverse factors, we did not observe either gross morphological changes in the cells or upregulation of epithelial markers such as collagen IV, cadherin-6, or E-cadherin following treatment, which contrasts markedly with rodent studies (4, 27, 44). These discrepancies raise intriguing questions about potential differences between human and rodent kidney development, but, to inform and design future studies, we must also consider other possible reasons why these human cells failed to differentiate. Firstly, we may have used the wrong subset of cells. Although our cell lines have several renal mesenchymal characteristics alluded to above, they did not express either PAX2 or WNT4 (as assessed on microarray; data not shown) in serum-free conditions, markers of the nephron lineage in rodent studies (27, 44). Interestingly, we previously reported that the same line expressed PAX2 when propagated in serum-containing media (28), and Drummond et al. (14) showed that fetal calf serum was crucial to induce renal mesenchymal differentiation and could enhance expression of epithelium-specific genes. Secondly, the combination of growth factors used may have been insufficient to induce human differentiation. Differentiation has only been demonstrated with these factors in rat mesenchymes and is less successful in mice (4), so perhaps human cells needed a modified regimen. LIF responsiveness appears linked to upregulation of cadherin 16, at least for adult murine renal mesenchymal differentiation (16), and this factor was actually downregulated 1.7-fold by treatment here (data not shown). Clearly, other growth factor combinations could be assessed in future and potential factors worthy of consideration are retinoic acid, activin-A, and bone morphogenetic protein 7 because embryonic stem cells treated with this combination are capable of differentiating into kidney epithelia (18). Finally, specific cell substrata may be critical for differentiation. Uncoated tissue culture plastic was used in these experiments, but perhaps coating dishes with collagen I or IV, growing the cells on filters, or culture in gels would be more efficacious.

In summary, we report expression of components of the LIF-signaling pathway for the first time in developing human kidneys and investigated the potential differentiation of human renal mesenchymal cells in response to a combination of growth factors including LIF. Our observations concerning changes of human gene expression, ascertained initially in vitro and then confirmed in whole organs, uncover several novel candidates that may be important in understanding pathways of early human nephrogenesis.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Kidney Research UK (R18/1/2000), Kids Kidney Appeal, Special Trustees, Great Ormond Street Hospital, UCL Institute of Child Health Human Developmental Biology Resource.


   FOOTNOTES
 
Address for reprint requests and other correspondence: P. J. D. Winyard, Nephro-Urology Unit, UCL Institute of Child Health, 30 Guilford St., London, UK (email p.winyard{at}ich.ucl.ac.uk).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

* K. L. Price and D. A. Long contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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