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Am J Physiol Cell Physiol 294: C543-C554, 2008. First published December 5, 2007; doi:10.1152/ajpcell.00307.2007
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EXTRACELLULAR MATRIX, CELL INTERACTIONS

Influence of microvascular endothelial cells on transcriptional regulation of proximal tubular epithelial cells

Sonia Aydin,1 Sara Signorelli,1 Thomas Lechleitner,1 Michael Joannidis,2 Clara Pleban,3 Paul Perco,3 Walter Pfaller,1 and Paul Jennings1

1Division of Physiology, Department of Physiology and Medical Physics and 2Clinical Division of General Internal Medicine, Clinical Department of Internal Medicine, Innsbruck Medical University, Innsbruck; and 3Institute for Theoretical Chemistry, University of Vienna, Vienna, Austria

Submitted 19 July 2007 ; accepted in final form 4 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the renal cortex the peritubular capillary network and the proximal tubular epithelium cooperate in solute and water reabsorption, secretion, and inflammation. However, the mechanisms by which these two cell types coordinate such diverse functions remain to be characterized. Here we investigated the influence of microvascular endothelial cells on proximal tubule cells, using a filter-based, noncontact, close-proximity coculture of the human microvascular endothelial cell line HMEC-1 and the human proximal tubular epithelial cell line HK-2. With the use of DNA microarrays the transcriptomes of HK-2 cells cultured in mono- and coculture were compared. HK-2 cells in coculture exhibited a differential expression of 99 genes involved in pathways such as extracellular matrix (e.g., lysyl oxidase), cell-cell communication (e.g., IL-6 and IL-1β), and transport (e.g., GLUT3 and lipocalin 2). HK-2 cells also exhibited an enhanced paracellular gating function in coculture, which was dependent on HMEC-1-derived extracellular matrix. We identified a number of HMEC-1-enriched genes that are potential regulators of epithelial cell function such as extracellular matrix proteins (e.g., collagen I, III, IV, and V, laminin-{alpha} IV) and cytokines/growth factors (e.g., hepatocyte growth factor, endothelin-1, VEGF-C). This study demonstrates a complex network of communication between microvascular endothelial cells and proximal tubular epithelial cells that ultimately affects proximal tubular cell function. This coculture model and the data described will be important in the further elucidation of microvascular endothelial and proximal tubular epithelial cross talk mechanisms.

human microvascular endothelial cells; human proximal tubular cells; transepithelial electrical resistance; gene expression; cell culture

IN THE RENAL CORTEX the peritubular capillary network is in close proximity to the proximal tubular epithelium, where the majority of nutrient exchange processes occur (39). It is here that solutes and water, reabsorbed from the proximal tubular lumen, are taken up by the vasculature and reenter the circulation and also substances are presented to the proximal tubule for secretion. In addition, the recruitment of circulating immune cells to sites of tubular damage is promoted by the microvasculature (7). Nephrogenesis and microvessel assembly are also tightly coupled in the developing kidney (1). Thus, from the very origin of renal development to mature kidney formation, the fates of the microvasculature and proximal tubular epithelium are intertwined. They remain in close functional contact under physiological and pathophysiological conditions.

Over the last decades in vitro cell culture models have delivered a great deal of information to all major fields of biological research. However, cell type interactions are for the most part neglected. Since there is a myriad of ways in which cells communicate to each other, it would be expected that culturing two different cell types in coculture would alter certain functional pathways. There is evidence to support the hypothesis that epithelial cells can be influenced by neighboring cells. Linas and Repine (27) demonstrated that bovine aortic macrovascular cells influence sodium transport in proximal tubular epithelial cells. In another study it was demonstrated that enteric glia cells could positively influence the barrier function of both Caco-2 and Madin-Darby canine kidney (MDCK) cells (36). However, there is little information in the literature pertaining to the effect of microvascular endothelial cells on renal tubular epithelial function.

Our aim was to model the influence of the endothelial cells of the peritubular capillary network on the epithelial cells of the proximal tubular epithelium. To this end, we cultured HMEC-1 (a human dermal microvasculature cell line) together with HK-2 (a human proximal tubular epithelial cell line) cells in a noncontact, close-proximity coculture system. We recently described (4) a microvascular endothelial-proximal tubular epithelial coculture system for the study of leukocyte transmigration. For transmigration studies it is necessary to use filters with pore sizes >1 µm; thus there is a potential for direct contact. Indeed, on morphological examination we could demonstrate cell protrusions through the pores allowing direct cell contact. Additionally, in the transmigration system it was necessary to seed the epithelial cells first. The model described in the present study is different in that the endothelial cells were seeded first and aluminum oxide filters with a pore size of 0.2 µm were used, preventing direct contact of the cell monolayers but maintaining the correct polarization of interaction.

Paracellular transport across the proximal tubule, which is regulated by a complex interaction of tight junction proteins (10), is a major route of solute and water reabsorption. Thus we first investigated the effect of coculture on established barrier function parameters. Also, with the use of microarray analysis the effect of coculture on HK-2 transcription was investigated. Finally, the transcriptomes of HK-2 and HMEC-1 cells were compared in an attempt to identify potential endothelium-borne regulatory mechanisms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals All chemicals were purchased from Sigma (Vienna, Austria) unless otherwise mentioned.

Cell Culture HK-2 cells (human proximal epithelial cell) were purchased from American Type Culture Collection (ATCC, no. CRL-2190). Cells were routinely cultured on 10-cm culture dishes from Sarstedt (Manassas, VA). Culture medium was a 1 to 1 mixture of DMEM and Ham's F-12 medium (11966-025 and 21765-029, respectively, GIBCO-Invitrogen, Lofer, Austria) containing 5 mM glucose supplemented with 10 ng/ml human recombinant epidermal growth factor (EGF), 36 ng/ml hydrocortisone, 5 µg/ml bovine insulin, 5 µg/ml human transferrin, 5 ng/ml sodium selenite, 2 mM Glutamax (GIBCO), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were fed three times weekly and subcultivated by trypsinization when near confluence.

The immortalized human dermal microvascular endothelial cell line HMEC-1, originally described by Ades et al. (2), was maintained in MCDB-131 medium supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany), 2 mM Glutamax, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.2 µg/ml hydrocortisone, and 10 ng/ml EGF. Cells were fed three times weekly and subcultivated by trypsinization when near confluence.

Human primary proximal tubular cells (HPT) and glomerular microvascular endothelial cells (GMEC) were obtained from the healthy region of nephrectomized tissue as previously described (18). The protocol was reviewed and approved by the Ethical Commission of Innsbruck Medical University, and informed consent was obtained. Briefly, renal cortex was minced into small pieces and digested with 1.5 mg/ml collagenase type II for 35 min at 37°C. Digested cortex was centrifuged in 42% isosmotic Percoll solution at 25,000 g for 30 min at 4°C. Fraction 1 (top fraction) was sieved through a 180-µm mesh, and the filtrate containing glomeruli was further digested in 1.5 mg/ml collagenase type II for 10 min at 37°C. Digested glomeruli were washed and plated on human fibronectin (2 µg/cm2)-coated cell culture dishes. Cell culture medium for microvascular growth was MCDB-131 with 20% (vol/vol) FCS, 50 µg/ml endothelial cell growth supplement (Eubio, Vienna, Austria), 30 U/ml heparin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM Glutamax. Cell culture medium was changed after 7 days and thereafter every third day until the mixed population of glomerular cells covered 80–85% of the culture dish. GMEC were isolated from the mixed glomerular culture by CD31 [platelet endothelial cell adhesion molecule (PECAM)] magnetic bead sorting. GMEC stained positive for von Willebrand factor and PECAM. Fraction 2 (lower fraction), containing predominantly proximal tubular fragments, was washed and seeded at ~5 x 105 fragments onto human fibronectin-coated 10-cm diameter culture dishes. Cell culture medium was DMEM-Ham's F-12 without L-valine or glucose and supplemented with 10% (vol/vol) FCS, 26 mM sodium bicarbonate, 0.8 mM D-valine, 2 mM L-proline, 10 mM sodium pyruvate, 2 mM Glutamax, 100 U/ml penicillin, and 100 µg/ml streptomycin. Medium was changed on the first day and twice weekly thereafter.

Experimental Design Coculture setup common procedure. Two systems were used for coculture setups: 1) aluminum oxide tissue culture inserts (Nunc, Wiesbaden, Germany) and 2) self-made filter holders. The holders consisted of two polysulfone parts, a top male thread and a bottom female thread. Loose aluminum oxide filters (Anodisc, Whatman International, Maidstone, UK) were placed into the female compartment, followed by a 2.5-cm outer diameter, 1.8-cm inner diameter, 0.2-cm thick silicone O-ring. The male part was then screw mounted, ensuring a tight seal. The unit was autoclaved before cell seeding. HMEC-1 cells were seeded at ~4 x 105 cells/ml on inverted filter inserts or holders and allowed to attach for ~5 h and reverted. HMEC-1 medium was applied to both compartments. After HMEC-1 confluence was reached (2–3 days, as examined by microscopic analysis) HK-2 cells were seeded at ~1.5 x 105 cells/ml into the upper compartment (Fig. 1). After this time HK-2 medium was used for both compartments. For epithelial and endothelial monocultures the same procedure was conducted, with the exception that partner cells were not added. Where it was necessary to separate the partner cells, two aluminum oxide filters were inserted into the filter holders and mono- and cocultures were set up as described above. At day 3 filters were removed from filter holders and separated, ensuring no cross-contamination of partner cells.


Figure 1
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  		Fig. 1. Schema of epithelial-endothelial mono- and coculture systems. Proximal tubular epithelial cells (HK-2) and microvascular endothelial cells (HMEC-1) were cultured on aluminum oxide filters with 0.2-µm pores and 50-µm thickness. Cells were either cultured in monoculture (A and B) or in coculture with a single filter (C) or a double filter (D). The double-filter system allows end point parameters to be investigated in each cell line once the filters are separated without cross-contamination.

 
HMEC-1 conditioned medium was obtained by washing confluent HMEC-1 cells in 10-cm dishes with PBS and applying fresh hormonally defined DMEM-F-12 for 3–4 days. Supernatant was centrifuged to remove contaminating cells. Conditioned medium was mixed 1:1 with fresh hormonally defined DMEM-F-12 before use. HMEC-1 extracellular matrix (ECM) conditioned filters were obtained by culturing HMEC-1 cells on filters as described for HMEC-1 monocultures. After 3–4 days of culture, filters were washed in PBS and allowed to air dry for 1 h. As aluminum oxide filters become opaque when dry, complete drying was readily observed visually. Filters were washed several times in PBS and then in DMEM-F-12. Microscopic analysis confirmed the loss of viable cells.

Electrophysiological studies. At day 3 of coculture in filter holders, transepithelial electrical resistance (TEER) of cell monolayers and cocultures was measured with the Endohm and EVOM systems from World Precision Instruments (Berlin, Germany). TEER of blank filters was subtracted. Experimental TEER values were expressed as ohms times centimeter squared. In a subset of experiments TEER was measured from separated filters that were previously in coculture.

Paracellular permeability: inulin leakage. At day 3 of coculture, in tissue culture inserts, 500 µl of medium containing 50 µg/ml fluorescein isothiocyanate (FITC)-conjugated inulin was applied to the top compartment and 300 µl of medium was applied to the bottom compartment. Cultures were incubated at 37°C in a 5% CO2-95% air humidified incubator for 24 h. These conditions ensure enough hydrostatic pressure gradient to allow close to 100% inulin leakage in blank filter inserts. Samples were drawn from both compartments and measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm with a TECAN GENios microtiter plate reader.

RNA extraction. RNA was extracted with an RNA high pure isolation kit from Roche (Roche Diagnostics, Applied Science, Vienna, Austria). For both DNA arrays and quantitative real-time polymerase chain reaction (qRT-PCR), lysates pooled from two filters were used per column and the samples were eluted with 25 µl of RNase-free water. RNA was quantified by the Ribogreen assay (Molecular Probes, Invitrogen, Lofer, Austria).

Affymetrix DNA arrays and analysis. Hybridization target preparations were performed according to protocols recommended by Affymetrix. Briefly, 5 µg of total RNA was reverse transcribed into cDNA with an oligo(dT)-T7 promotor primer and transformed into double-stranded cDNA by Escherichia coliDNA polymerase with the Affymetrix one-cycle cDNA synthesis kit. After purification of double-stranded cDNA with the Affymetrix GeneChip Sample Cleanup Module, biotin-labeled cRNA was produced by T7 polymerase (Affymetrix IVT Labeling kit). After Agilent-based quantification and integrity control, 20 µg of cRNA was fragmented by alkaline treatment (Affymetrix GeneChip Sample Cleanup Module) and 15 µg of fragmented cRNA was added to the hybridization cocktail (300 µl final volume). The arrays were washed and stained according to the recommended Fluidics Station protocol (EukGE-WS2 version 5_450). Fluorescence signal intensities from each feature on the microarrays were determined with the Affymetrix GeneChip Scanner 3000 and GCOS software (version 1.2) according to the manufacturer's recommendations. CEL files were uploaded onto the CARMA web tool (https://carmaweb.genome.tugraz.at/carma/) and condensed with the Mas5 algorithm (scaled to 100). Data were deposited to ArrayExpress (http://www.ebi.ac.uk/arrayexpress/; accession no. E-MEXP-1178). For monoculture vs. coculture comparison the following procedure was used to determine statistically significant differentially expressed genes. With Microsoft Excel 2007 the following filters were applied (in this order): above a value of 15; having <33% absent values per treatment except when all values are absent in one treatment and present in the other treatment; a twofold differential expression; and finally a P value ≤0.05 by Student's unpaired t-test. Since there was a very large difference in gene expression of HMEC-1 monoculture compared with HK-2 monoculture, more stringent filters were applied for the HMEC-1/HK-2 comparison. Genes upregulated over fourfold with a P value ≤0.001 (Student's t-test) were selected. Values that were Absent in >66% of HK-2 and HMEC-1 data were removed.

Quantitative real-time PCR. RNA was transcribed to cDNA with Escherichia coli reverse transcriptase, oligo(dT), and random primers (Qiagen). For determination of gene expression levels commercially available predesigned primer sets were used (Applied Biosystems). 18S RNA (Applied Biosystems) served as an endogenous control. PCR reagent mixes for designed primers contained 900 nM sense and antisense primer, 200 nM probe, and a TaqMan Mix (Applied Biosystems). Thermal cycling profile started with 2 min at 50°C (RNAse inhibitor activation) and 10 min at 95°C to activate polymerase. Repeating cycles were performed 40 times at 95°C for 15 s, followed by 60°C for 1 min. Samples were run in duplicate, and the gene expression levels were calculated with the comparative threshold cycle (Ct) method known as the 2–{Delta}{Delta}Ct method and converted to fold change over monoculture. Experiments were carried out with the ABI PRISM 7700 and evaluated with the SDS 1.9.1 software package.

Cytokine secretion assays. Mono- and cocultures in filter inserts were provided with 500 µl of HK-2 medium in both compartments. At day 3 supernatants were collected and interleukin (IL)-6 concentrations were measured by enzyme immunoassay (EIA) (DuoSet, R&D Systems, Minneapolis, MN), according to the manufacturer's instructions. Hepatocyte growth factor (HGF) was measured by EIA (DuoSet, R&D Systems) in 3-day supernatants from HK-2 and HMEC-1 confluent monolayers cultured in 10-cm dishes. Fresh cell culture medium was used as a control. One hundred microliters of 20 µM Amplex Red Ultra (Molecular Probes)-5 mM H2O2 in PBS was used for horseradish peroxidase (HRP) detection. Fluorescence was measured at an excitation wavelength of 540 nm and an emission wavelength of 595 nm.

H2O2 production. Mono- and cocultures in tissue culture carriers were incubated for 24 h in HK-2 medium containing 500 µl/compartment of 160 µM Amplex Red Ultra (Molecular Probes)-1/200 streptavidin HRP (R&D Systems). Fluorescence was measured at an excitation wavelength of 540 nm and an emission wavelength of 595 nm.

ATP assay. At day 3, epithelial and endothelial filters were separated and 0.5% trichloroacetic acid cell extracts were prepared. ATP was measured with a commercially available luciferin luciferase assay from Molecular Probes.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of Endothelial Cells on Epithelial Cell Barrier Function Paracellular permeability is an important function of epithelial cells, especially in "leaky" epithelia such as the proximal tubule (10). TEER is a measure of the electrical resistance of a cell monolayer and thus predominantly reflects the paracellular pathway. Paracellular tightness is controlled by the proteins assembled in the tight junction. Inulin transfer from the apical to the basolateral compartment, under conditions of a hydrostatic pressure gradient, also predominantly measures the paracellular pathway. Inulin leakage is inversely proportional to TEER. Monocultures of HMEC-1 cells presented a very low TEER, while monocultures of HK-2 exhibited a modest TEER (Fig. 2A). However, when both cells were cultured in coculture there was an increase in TEER 1.92-fold above the sum of the HMEC-1 and HK-2 monocultures (Fig. 2A). Monocultures of HMEC-1 cells also presented a weak barrier to FITC-inulin, while monocultures of HK-2 cells exhibited a modest barrier to FITC-inulin (Fig. 2B). The coculture exhibited an enhanced barrier to FITC-inulin 1.33-fold higher than the sum of the HK-2 and HMEC-1 monocultures (Fig. 2B). Similar results were obtained with primary GMEC and HPT cells (Fig. 2, C and D). Since HMEC-1 cells exhibit a weak barrier function, one would expect that the increase in coculture barrier function arises because of alterations in HK-2 barrier formation. To investigate this possibility we cultured HK-2 cells and HMEC-1 cells in a two-filter coculture system (Fig. 1D). The filters were separated at day 3 of coculture, and TEER was measured in HMEC-1 and HK-2 monolayers. As expected, only HK-2 cell monolayers that had been cultured with HMEC-1 cells exhibited a significant increase in TEER (Fig. 2E).


Figure 2
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  		Fig. 2. Barrier function of epithelial and endothelial monocultures and cocultures. A: transepithelial electrical resistance (TEER) of monoculture and coculture (co-c) of HK-2 and HMEC-1 cell lines. B: 24-h fluorescein isothiocyanate (FITC)-inulin leakage of mono- and cocultures of HK-2 and HMEC-1 cell lines. At day 3 of coculture FITC-conjugated inulin was loaded into the top compartment and incubated for 24 h. Blank, filter inserts without cells. C: TEER of mono- and cocultures of human primary proximal tubular cells (HPT) and human primary glomerular microvascular endothelial cells (GMEC). D: 24-h FITC-inulin leakage of mono- and cocultures. E: TEER of separated filters. Mono- and cocultures were set up in a 2-filter system. After 3–4 days of coculture TEER was measured for individual monolayers after filter separation. cc-s, Coculture separated. F: effect of sequence of coculture construction on TEER. Two types of cocultures were set up by altering the sequence of cell seeding: HMEC-1 cells seeded first (co-HMEC 1st) or HMEC-1 cells seeded second (co-HMEC 2nd). Each value represents mean + SE of 3–5 independent experiments each performed with 3–6 replicates. Statistical significance between groups was determined by 1-way ANOVA with Tukey's multiple comparison test. *P < 0.05, **P < 0.001, ***P < 0.0001.

 
To determine whether the order of seeding has an impact on TEER we constructed the coculture system by seeding the HMEC-1 cells first and then the HK-2 cells (as normal) or by seeding HK-2 cells first and then the HMEC-1 cells. Only when the HMEC-1 cells were seeded first was there a statistically significant increase in TEER (Fig. 2F).

To determine whether endothelial ECM or endothelium-derived soluble factors were responsible for an increase in HK-2 TEER, HK-2 cells were cultured in HMEC-1 conditioned medium and on filters where HMEC-1 cells had been cultured and then air dried to remove cells but maintain ECM. HK-2 cells cultured in conditioned medium did not exhibit a significantly enhanced TEER (Fig. 3). HK-2 cells cultured on HMEC-1 ECM conditioned filters did, however, demonstrate an enhanced TEER equal to that of cocultures. Culturing HK-2 cells on laminin-coated filters had a similar but weaker effect. In addition, inhibition of the enzymatic activity of lysyl oxidase (LOX) by incubation of cocultures with the LOX inhibitor 3-aminopropionitrile (34) had no effect on TEER.


Figure 3
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  		Fig. 3. Effect of HMEC-1 conditioned medium and HMEC-1 extracellular matrix (ECM) on HK-2 TEER. HK-2 cells were cultured on aluminum oxide filters under various conditions, and TEER was measured after 3 days. HMEC-1 cond med, HK-2 monoculture incubated in a 1:1 mix of HMEC-1 conditioned medium; coculture, HMEC-1/HK-2 coculture; co-c + LOX inhib, HMEC-1/HK-2 coculture + 100 µM lysyl oxidase (LOX) inhibitor 3-aminopropionitrile; HMEC-1 ECM, HK-2 cells seeded onto HMEC-1 ECM conditioned filters (HMEC-1 cells cultured on underside of aluminum oxide filters, air dried, and washed in PBS); laminin, HK-2 cells cultured onto 1 µg/cm2 Engelbreth-Holm-Swarm tumor laminin-coated filters. Each value represents mean + SE TEER of 8–15 cultures from 2 independent experiments. Statistical significance between groups was determined by 1-way ANOVA with Tukey's multiple comparison test. *P < 0.05, **P < 0.001, ***P < 0.0001 compared with HK-2 monoculture control (dashed line).

 
Alterations in Cocultured HK-2 Cells by Gene Expression Profile Transcriptomic profiling of HK-2 cells cultured in monoculture compared with HK-2 cells cultured in coculture revealed 114 differentially expressed transcripts (99 unique genes) with 84 differentially expressed genes of known function (55 upregulated and 29 downregulated). The genes are listed in functional categories in Table 1.


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  		Table 1. Differential gene expression in HK-2 cells cultured with HMEC-1 cells in coculture compared with HK-2 cells cultured in monoculture

 
Identification of Genes Enriched in HMEC-1 Cells A selection of genes that play a role in extracellular processes were selected from 756 genes that were over fourfold enriched in HMEC-1 monocultures compared with HK-2 monocultures. In addition, a number of endothelium-specific genes, which should be highly elevated in endothelial cells, were included as a validating control. Data are presented in Table 2 in raw form as average spot intensity (after Mas5 condensation and scaling to 100). Present and Absent calls are shown. The actual numerical value of the fold increase is not very meaningful here, especially when HK-2 data sets are very low. However, the data set does show genes that are highly enriched in HMEC-1 cells compared with HK-2 cells. The presence of genes such as PECAM-1 and von Willebrand factor that are known to be expressed in endothelial cells and not expressed in epithelial cells (30, 40) demonstrates that such an analysis is appropriate and meaningful.


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  		Table 2. Selected genes that are over fourfold enriched in HMEC-1 cells compared with HK-2 cells

 
Quantitative Real-Time PCR mRNA levels of selected genes were determined from HK-2 cells cultured under mono- and coculture conditions by qRT-PCR (Fig. 4A). Expression levels are represented as HK-2 coculture over HK-2 monoculture. These experiments confirm, by an independent method, the regulation of several genes identified by microarray analysis. Additionally, it was investigated whether endothelium-derived ECM or endothelium-derived soluble factors were responsible for these changes (Fig. 4B). HMEC-1 conditioned medium alone was sufficient to induce LCN2, while HMEC-1 ECM alone was sufficient to induce IL-1β, LOX, CALCR, and KISS1R. No significant alterations in HIG2 or CA12 were observed in these two conditions individually.


Figure 4
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  		Fig. 4. Quantitative PCR analysis of selected gene products differentially expressed in HK-2 cells under HMEC-1 coculture (A) and in the presence of HMEC-1 conditioned medium or HMEC-1 ECM (B). A: HK-2 cells were cultured under mono- and coculture conditions as described in text. Quantitative real-time PCR was carried out, and mRNA levels for selected gene products were quantified. Data represent mean + SE HK-2 coculture expression level as fold change over HK-2 monoculture. B: HK-2 cells were cultured in monoculture, in monoculture with HMEC-1 conditioned medium, and in monoculture with HMEC-1-derived ECM. Values are expressed as fold change over HK-2 monoculture controls. Statistical significance was assessed in comparison to monoculture controls with an unpaired 2-tailed student's t-test. *P < 0.05, **P < 0.001, and ***P < 0.0001. Dashed line represents control.

 
Further Characterization Since IL-6, one of the major regulators of inflammation, was decreased in HK-2 coculture microarrays (Table 1), we investigated the secretion of IL-6 protein from mono- and cocultures. HK-2 cells secreted 33.6-fold higher IL-6 into the supernatant medium than HMEC-1 cells (Fig. 5A). In addition, there was a 3.2-fold decrease of IL-6 secretion in the coculture compared with HK-2 monoculture (Fig. 5A). This result confirms the observed decrease in IL-6 mRNA observed by DNA microarray analysis. A decrease in IL-6 secretion may point to an overall reduction in inflammatory processes. Since IL-6 has been shown to be induced by H2O2 exposure (8, 29), we also investigated the H2O2 environment of mono- and coculture systems. HK-2 and HMEC-1 cells produced similar amounts of total H2O2 (Fig. 5B). However, when the cells were in coculture there was a modest decrease in H2O2 production (HK-2 monoculture vs. coculture; P = 0.055 by Student's t-test) when one would expect an additive effect from the cell types.


Figure 5
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  		Fig. 5. Further characterization. Twenty-four-hour IL-6 (A) and H2O2 (B) were measured in mono- and coculture systems. For comparative purposes the algebraic sum of the total HK-2 monoculture and the total HMEC-1 monoculture extracellular H2O2 or IL-6 is shown. Measurements were conducted in apical and basolateral compartments and are presented as the total amount. H2O2 data are shown as arbitrary relative fluorescent units (RFU). C: cellular ATP was determined in HK-2 mono- and coculture lysates (P = 0.393 by Student's t-test). D: hepatocyte growth factor (HGF) was measured in 3-day supernatants from HK-2 and HMEC-1 grown in 10-cm plastic dishes. No detectable HGF was measured in HK-2 supernatants. All data represent means + SE from at least 3 experiments. **P < 0.001, ***P < 0.0001 compared with HK-2 monoculture by 1-way ANOVA with Tukey's multiple comparison test.

 
A reduction in intracellular ATP levels is associated with cell stress and has been shown to predispose cells to H2O2 toxicity (29). Our results show that cellular ATP in HK-2 cells was unaffected by culture conditions (P = 0.3938; Fig. 5C).

As a proof of concept for HMEC-1-upregulated genes (presented in Table 2), we measured the secretion of HGF protein in HK-2 and HMEC-1 supernatants. HMEC-1 cells produced modest amounts of HGF, whereas HGF production from HK-2 cells was not detected (Fig. 5D).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The aim of this study was to investigate the influence of microvascular endothelial cells on proximal tubular cell gene expression and functional phenotype with a noncontact coculture system. To this end, we cocultured microvascular endothelial cells with renal proximal tubular epithelial cells on opposite sides of aluminum oxide filters, allowing no direct contact between the monolayers. Microarray analysis demonstrated an altered regulation of 99 genes in HK-2 cells when cocultured with HMEC-1 cells. To establish which endothelium-derived factors might be important in epithelial regulation we compared HK-2 and HMEC-1 transcription profiles and identified a number of potential endothelium-derived ECM factors and endothelium-derived soluble factors. Endothelium-derived ECM and endothelial conditioned medium exhibited specific effects on epithelial cells.

Role of Endothelium-Derived ECM Renal epithelial cells exhibited an enhanced barrier function when cocultured with microvascular endothelial cells. Since TEER and FITC-inulin flux measure predominantly the paracellular pathway that is controlled by tight junction proteins, this observation can be seen as an enhancement of the epithelial paracellular gating function. Apart from an increase in LOX transcription, which was previously shown to be a regulator of barrier function (35), we did not find any obvious relationship to gene regulation and enhanced barrier function. (However, LOX inhibition with 3-aminopropionitrile had no effect on TEER.) Thus it is unlikely that the elevated paracellular tightness is due to an altered transcription of tight junction proteins. Importantly, the enhancement in barrier function was dependent on the order of coculture construction: the effect was seen only when HMEC-1 cells were seeded first. In line with this finding HMEC-1 conditioned medium had no effect on HK-2 TEER. This would appear to point to endothelium-specific production of ECM proteins, which influence the HK-2 cell fate at attachment. Indeed, HMEC-1 cells exhibited transcriptional activity of a wide variety of ECM-associated genes that were present only in low levels in HK-2 cells, including ADAMS6, punctin, punctin-2, biglycan, collagen (type I-{alpha}1, III-{alpha}1, IV-{alpha}6, V-{alpha}1, VI-{alpha}3, fibrillin 2, laminin-{alpha}4, MMP 1, -2, and -19, and TIMP 3). Thus in monoculture (or in coculture when seeded first) the HK-2 cells experience a naked aluminum oxide filter and in coculture (when seeded second) the filter will have a complex deposit of endothelium-derived ECM material distinctly different from what the HK-2 cells produce. The role of endothelium-derived ECM in epithelial barrier function was conclusively proven in experiments where HK-2 cells were cultured on filters preconditioned with HMEC-1 ECM. Endothelium-derived ECM enhanced HK-2 TEER equal to that of cocultures and was responsible for an induction of HK-2 LOX, CALCR, and KISS1R mRNA. Endothelium-derived ECM may confer a higher paracellular tightness through interaction with the cytoskeleton (11).

A number of genes that are known to interact with the ECM resulting in signal transduction were regulated in HK-2 cocultures. Eph receptor A3, SHANK-2, and LOX were upregulated in coculture. Eph receptor A3 is a member of the largest subfamily of receptor tyrosine kinases (RTKs). Eph receptors and their ephrin ligands are important mediators of cell-cell communications regulating cell attachment, shape, and mobility (17). SHANK-2 is a scaffold protein that directs polarized activities in apical and basolateral membranes and was recently demonstrated to be a regulator of the Na+/H+ exchanger NHE3 (15). LOX is a protein involved in the maturation of collagen and elastin matrix proteins, dictating their stability against metalloproteinases (20, 33). Matrilin 3, cysteine-rich 61 (CYR61 or CCN1), and connective tissue growth factor (CTGF or CCN2) were downregulated in coculture. Matrilin 3 is a subfamily of ECM proteins and is normally absent in noncartilaginous tissue (41). Interestingly, two members of the proangiogenic CCN family were downregulated in coculture, CCN1 and CCN2 (22, 32). Intuitively, it makes sense that proangiogenic factors would no longer be necessary in a situation where there is sufficient microvascular interaction. An increase in the G protein-coupled receptors, the calcitonin receptor (CALCR), and the KISS1 receptor in coculture may also be indicative of increased interaction with the extracellular environment and subsequent signal transduction.

Role of Endothelium-Derived Soluble Factors HMEC-1 cells exhibited transcriptional activation of a number of secreted growth factors or cytokines that were highly enriched compared with HK-2 cells, including HGF, bone morphogenetic protein (BMP)6, gremlin-1, adrenomedullin, endothelin-1, FGF 5, IL-17, insulin-like growth factor binding proteins 2, 4, and 5, parathyroid hormone-like hormone, placental growth factor, secreted frizzled-related protein 1, serglycin, stanniocalcin 1, and VEGF-C. Since endothelial conditioned medium did not enhance epithelial TEER it is unlikely that any of these factors play a role in epithelial barrier function. Nonetheless it would be expected that these factors either individually or in combination (with the possible exception of adrenomedullin) would have an effect on other aspects of epithelial regulation. [The receptor for adrenomedullin, calcitonin-like receptor, is known to be expressed predominantly in endothelial cells, where activation alters vascular tone (14). We also demonstrate that calcitonin-like receptor is highly enriched in HMEC-1 cells (Table 2).]

The transforming growth factor (TGF)-β superfamily ligand BMP6 and the BMP antagonist gremlin-1 would likely have opposing roles in the TGF-β/SMAD pathway. HGF in particular has been shown to be an antagonist of this pathway (6, 28). In our coculture model we observed an increase in SMAD9 and a decrease in TGF-β2 mRNA. HGF also induces the mRNA expression and production of lipocalin 2 (LCN2 or Ngal) in renal epithelial cells (13). Here we show that both under coculture conditions and with HMEC-1 conditioned medium there is an upregulation of LCN2 mRNA in HK-2 cells, while HMEC-1 ECM had no effect on HK-2 LCN2 expression. LCN2 binds matrix metalloproteinase (MMP)9, preventing its degradation while maintaining enzymatic activity (42). Thus HGF through the induction of LCN2 and the stabilization of MMP9 can enhance ECM degradation. However, this may be offset in our coculture model by the endothelium-derived ECM induction of epithelial LOX.

Genes that have been demonstrated to be upregulated by hypoxia were also upregulated in coculture, namely, NDRG1 (37), HIG2 (9), BNIP3 (12), TXNIP (24), HGTD-P (25), REDD1 (19), PDK-1 (21), PGK1 (26), and SLC2A3 (26). Although it cannot be ruled out that the HK-2 cells under coculture, because of the extra monolayer of oxygen-consuming cells, experience a hypoxic level of pericellular oxygen tension, we have several lines of evidence to argue that this is not the case here. 1) Intracellular ATP concentrations were not different between mono- and cocultured HK-2 cells. 2) There is growing evidence that hypoxia induces reactive oxygen species, including H2O2 (reviewed in Ref. 5), while we demonstrate a decrease in H2O2 production in coculture. 3) In a model of ischemia-perfusion injury exposure of HK-2 cells to hypoxic conditions caused a collapse of barrier function, whereas in our coculture model we observed the opposite effect (26). 4) A number of hypoxia-upregulated genes {IL-6 (3), AREG [hypoxia-inducible factor (HIF)-1 independent] (31), CYR61 (23), and CTGF (16)} were downregulated in HK-2 cocultures. HIF-1 is the main transcriptional regulator of the hypoxia response. The HIF-1{alpha} subunit is ubiquitinated and degraded under normoxia, but under hypoxia it remains stable and translocates to the nucleus, where it regulates transcription. Spinella et al. (38) demonstrated that endothelin-1 can stabilize HIF-1{alpha} under normoxic conditions, leading to a full HIF response. Thus it is tempting to speculate that HMEC-1-derived endothelin-1 (11.6 times higher transcription in HMEC-1 cells than HK-2 cells) is responsible for some of the observed hypoxia-like transcriptional effects.

Additionally, genes involved in catalytic activity, cell communication, transport, cytoskeleton, proliferation, and protein trafficking were altered by coculture. Genes known to be involved in acid-base balance (CA12), phosphatidylcholine biosynthesis (PCYT1B), glycolytic pathway (PGK1, ENO2, PDK1), amino acid transport (SLC36A1, SLC16A10), Na+/myo-inositol transport (SLC5A3), water transport (AQP9), and glucose transport (GLUT3) were upregulated, while genes involved in sulfate transport (SLC26A2) and proliferation (CCND1) were downregulated. IL-1β was upregulated, and IL-6 (message and protein) was downregulated. How these epithelial genes may be regulated by endothelial cells cannot as yet be clarified.

This study provides a detailed characterization of an in vitro model of endothelial to epithelial signal transduction using transcriptomic profiling and demonstrates a highly complex network of interactions. We have identified a number of potential endothelium-derived factors including ECM proteins and soluble growth factors that are most likely involved in the regulation of the renal epithelium. This model and the data described will be important in the further elucidation of the microvascular endothelial and proximal tubular epithelial cross talk mechanisms.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Austrian Federal Ministry of Education Science and Culture (GZ 70.078/2-Pr/4/2002), the EU 6th Framework project "PREDICTOMICS" (LSHB-CT-2004-504761), and the EU Framework project "CARCINOGENOMICS" (LSHB-CT-2006-037712).


   ACKNOWLEDGMENTS
 
We thank Martin Streicher for his ingenuity in the design and production of the filter holders.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Jennings, Div. of Physiology, Dept. of Physiology and Medical Physics, Fritz Pregl Strasse 3, Innsbruck Medical Univ., Innsbruck, Austria A6020 (e-mail: paul.jennings{at}i-med.ac.at)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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