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Original Article
Kidney International (2000) 58, 43–50; doi:10.1046/j.1523-1755.2000.00139.x
Hypoxia and interleukin-1 stimulate vascular endothelial growth factor production in human proximal tubular cells
Baha El Awad, Burkhard Kreft, Eva-Maria Wolber, Thomas Hellwig-Bürgel, Eric Metzen, Joachim Fandrey and Wolfgang Jelkmann
Institute of Physiology and Department of Internal Medicine I, Medical University of Lübeck, Lübeck, Germany
Correspondence: Wolfgang Jelkmann, M.D., Institute of Physiology, Medical University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany. E-mail: Jelkmann@physio.mu-luebeck.de
Received 14 September 1999; Revised 6 January 2000; Accepted 27 January 2000.
Top of pageAbstract
Hypoxia and interleukin-1
Background
Vascular endothelial growth factor (VEGF) promotes angiogenesis and inflammatory reactions. VEGF mRNA is detectable in the proximal tubules of inflamed kidneys but not in normals. In other organs VEGF gene expression is induced by hypoxia and cytokines such as interleukin 1 (IL-1). To identify the cellular mechanisms in control of tubular VEGF production, we studied effects of hypoxia and IL-1
Methods
PTECs were grown in monolayers from human kidneys. Hypoxia was induced by incubation at 3% O2. VEGF mRNA was quantitated by competitive polymerase chain reaction following reverse transcription. VEGF was measured by enzyme-linked immunoassay. HIF-1
Results
Significant amounts of VEGF mRNA and VEGF protein were measured in PTEC extracts and culture media, respectively. Stimulation of VEGF synthesis at low O2 tension and following IL-1
Conclusions
PTECs in culture produce VEGF. One mechanism of induction appears to be increased DNA binding of HIF-1 to hypoxia-responsive elements in the VEGF gene promoter. In inflammatory diseases of the kidney, tubular cell-derived VEGF may contribute to microvascular leakage and monocyte extravasation.
Keywords:
renal tubules, inflammation, cytokines, microvascular leakage, tumor necrosis factor-
Recent evidence suggests that renal tubules are not only targets, but are also active participants in immune reactions. Tubular cell-derived cytokines are thought to augment inflammatory processes in the kidney. Human proximal tubular cells in culture have been shown to produce tumor necrosis factor-
In situ hybridization and immunohistochemical studies indicate that tubular cells may also express the gene for vascular endothelial growth factor (VEGF) in cases of preglomerular or glomerular vascular inflammatory disease4. VEGF is a pluripotent cytokine. In addition to its mitogenicity for endothelial cells, VEGF stimulates nitric oxide-dependent dilation of blood vessels, increases the permeability of capillaries, and induces the expression of urokinase-type and tissue-type plasminogen activators, as well as of the interstitial metalloproteinase collagenase, and it is a chemoattractant for leukocytes5.
The mechanisms in control of the alleged formation of VEGF by tubular cells have not been elucidated. In other tissues, VEGF production can be stimulated by hypoxic stress, distinct cytokines, and tumor-promoting phorbol esters5. Hypoxia induces transcription of the VEGF gene and stabilization of VEGF mRNA6,7. There is a 28 bp hypoxia response element (HRE) located approximately 1 kb upstream of the transcription initiation site of the human VEGF gene8. Transcriptional activation is mediated by binding of the trans-acting dimeric protein hypoxia-inducible factor-1 (HIF-1
The aims of the present study were to investigate: (1) the capacity of primary human proximal tubular cell cultures to express VEGF mRNA and VEGF protein, (2) the influence of hypoxia, IL-1
METHODS
Tubular cell cultures
Human proximal tubular epithelial cell lines (PTECs) were cultured according to the method of Detrisac et al13 as described previously14. In brief, portions of renal cortex not involved by disease were obtained from kidneys removed by surgery for renal cell carcinoma. Primary PTEC cultures were grown on a matrix of fetal calf serum (FCS; Biochrom KG Seromed, Berlin, Germany). PTECs for maintenance and experiments were cultured in serum-free Dulbecco's modified Eagle's medium (DMEM)/HAM's F-12 medium in a 1:1 ratio (Biochrom KG Seromed). The medium was supplemented with insulin (5
Site-directed VEGF production was studied in confluent PTEC layers on polycarbonate inserts16 for separate collection of basolateral and apical medium following 4- or 24-hour incubation periods. The effects of hypoxia on VEGF mRNA levels and VEGF production were studied in nonconfluent cultures in tissue flasks (25 cm2 with 5 mL medium) placed in a pO2-controlled incubator (Heraeus, Hanau, Germany) with 20% O2 or 3% O2, 5% CO2, and balance N2 in a humidified atmosphere at 37°C for different periods of up to three days. To study the effects of proinflammatory cytokines on VEGF production, the cells were subcultured in 24-well dishes (2 cm2 with 1 mL medium). Cytokines used were recombinant human IL-1
Murine hepatoma cultures
Since the existence of HIF-1 and its capacity to bind to DNA has not been demonstrated thus far in tubular cells, control experiments were carried out with the murine hepatoma cell lines Hepa1 and Hepa1C4 (kindly provided by Dr. O. Hankinson, Los Angeles, CA, USA). Hepa1 cells exhibit HIF-1 DNA-binding activity on hypoxia exposure12, while Hepa1C4 is a Hepa1 subline that does not express a functional HIF-1
Assay of VEGF
The concentration of VEGF in the culture media was measured by commercial enzyme-linked immunoassay (ELISA; Quantikine; R&D Systems, Wiesbaden, Germany). The immobilized antibody was monoclonal, while the second horseradish peroxidase-coupled antibody was polyclonal. In a previous study, we determined the intra-assay and interassay coefficients of variance to be 5 and 7.5%, respectively, and the lower detection limit to be 9 pg/mL17. Secreted VEGF was related to the amount of total cellular protein, which was measured in lysates of washed cultures by means of a protein microdetermination kit based on the biuret/phenol reagent method (Sigma).
RNA extraction and VEGF mRNA quantitation
Vascular endothelial growth factor mRNA was quantitated by competitive polymerase chain reaction following reverse transcription (RT-PCR). Total RNA was isolated from washed cultures by the acid guanidinium thiocyanate-phenol-chloroform extraction method18. One microgram of total RNA was reverse transcribed into first-strand cDNA with oligo-dT15 primer (M-MLV reverse transcriptase; GIBCO). Qualitative RT-PCRs were carried out for GAPDH and
Table 1 - Characterization of polymerase chain reaction (PCR) primers.
Nuclear extract preparation for study of HIF-1 activation
Hypoxia-inducible factor-1
Western blot analysis
For determination of immunoreactive HIF-1
Gel shift assay of HIF-1 DNA binding
[
Sequences were TfHBSww (sense), 5'-TTCCTGCACGTACACACAAAGCGCACGTATTTC-3', and TfHBSww (antisense), 5'-GAAATACGTGCGCTTTGTGTGTACGTGCAGGAA-3'.
After 5' end labeling of the sense strand unincorporated [
Binding reactions were set up in a volume of 30
Statistics
Data are presented as the mean and standard deviation (
RESULTS
Proximal tubular epithelial cells were cultured in polycarbonate inserts for study of site-directed VEGF production. Measurements of immunoreactive VEGF carried out after 4- or 24-hour incubation periods revealed that there was no statistical difference between the amounts of VEGF secreted to the basolateral versus the apical site of unstimulated cultures Table 2. In vivo, this pattern of nonpolarized secretion would correspond to the occurrence of VEGF in both interstitial and tubular fluid. The following studies were carried out with monolayers grown on the bottom surface of common culture vessels.
Table 2 - Basolateral versus apical production of vascular endothelial growth factor (VEGF) by proximal tubular epithelial cells (PTEC) in cell culture inserts.
The rate of the secretion of VEGF was significantly increased when the cells were maintained under hypoxic conditions for up to 72 hours Figure 1. Measurements of VEGF mRNA by competitive RT-PCR were not sensitive enough to demonstrate a significant difference when normoxic and hypoxic cultures were compared (2560
Figure 1.
Vascular endothelial growth factor (VEGF) concentrations in proximal tubule epithelial cell (PTEC) culture media during normoxic (
Figure 2 shows that the addition of PMA or IL-1
Figure 2.
Effects of the addition of phorbol 12-myristate 13-acetate (PMA; 100 nmol/L), interleukin 1-
Western blot analysis showed that the amount of HIF-1
Figure 3.
Western blot for hypoxia inducible factor-1
Electrophoretic mobility shift assays showed that hypoxia led to increased DNA binding of the HIF-1 complex Figure 4. Moreover, incubation with IL-1
Figure 4.
Gel shift analysis with nuclear extracts from PTECs and mouse hepatoma cells (Hepa1 and Hepa1C4). The cells were incubated under normoxic (20% O2; N) or hypoxic (3% O2; H) conditions. Normoxic PTECs were challenged with IL-1
Top of page
DISCUSSION
Initial studies on the localization of the expression of the VEGF gene revealed the lung, kidneys, heart, and adrenal glands as the dominating sites in normal adult guinea pigs24. In situ hybridization and immunohistochemical studies have indicated that the main intrarenal sites of the generation of VEGF are the renal corpuscles and, here, the podocytes24,25. While renal VEGF seems to be essential for the capillarization of the metanephros26,27, its function in the kidneys of adults is incompletely understood. Experimental studies have not supported the hypothesis that high levels of circulating VEGF might cause an increase of glomerular permeability for macromolecules and, therefore, lead to proteinuria. In isolated perfused rat kidneys, the addition of VEGF to the perfusion does not enhance albumin excretion rates28. Neither does the intravenous administration of high doses of VEGF to rats result in the development of proteinuria29. As pointed out by Webb et al, however, VEGF normally approaches the endothelium from the abluminal side rather than from the luminal side29. Thus, paracrine effects of intrarenally produced VEGF cannot be excluded.
In normal human kidney, proximal tubules exhibit only faint, if any, labeling for VEGF mRNA and VEGF protein, when studied by in situ hybridization and immunohistochemistry4,25. However, in the damaged tubules of kidneys with necrotizing vasculitis and glomerulonephritis, significant staining for VEGF mRNA and VEGF protein has been demonstrated4. Two mechanisms could be responsible for this reaction. First, VEGF mRNA levels may increase on the induction of hypoxia6,7,8,30. Exposure of rodents to inspiratory hypoxia resulted in increased renal VEGF mRNA levels in one study31, but not in another32. Although impaired renal blood flow may also cause focal hypoxia, it is noteworthy that ischemic acute renal failure is not generally accompanied by significant peritubular inflammation. Second, VEGF gene expression can be induced by immunomodulatory peptides. IL-1 increases VEGF mRNA levels in rat smooth muscle cells33, human fibroblasts34,35,36, and tumor cells37,38. The action of TNF-
To the best of our knowledge, the present study is the first to show that human proximal tubular cells produce VEGF in culture. By RT-PCR and ELISA, VEGF mRNA and immunoreactive VEGF protein were demonstrated in PTECs and the culture supernatants, respectively. Studies using culture inserts indicated that the cells secreted VEGF in a nonpolarized fashion. The occurrence of VEGF on the apical site of the cultures is in line with the fact that human urine contains VEGF in measurable amounts. Interestingly, the urinary VEGF/creatinine ratio is not increased in nephrotic subjects compared with normal subjects29. Based on the present in vitro study, we speculate that urinary VEGF may partly originate from tubules. A functional role cannot be assigned to urinary VEGF at present.
The rate of VEGF secretion in PTEC cultures was increased when the O2 concentration in the incubator was reduced from 20 to 3% or when IL-1
In conclusion, the demonstration of HIF-1 activation in human proximal tubular cells supports the concept that HIF-1 is ubiquitously controlling oxygen-dependent genes in mammalian cells. Increased HIF-1 DNA binding to hypoxia-responsive elements in the VEGF gene promoter may partly account for the stimulation of VEGF production in tubular cells on exposure to hypoxic stress or IL-1
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Acknowledgments
This study was supported by the Deutsche Forschungsgemeinschaft (SFB 367-B3, SFB 367-C8). Baha El Awad is a visiting scientist from the University of Khartoum by courtesy of the Sudanese government. Thanks are due to Dr. O. Hankinson (Los Angeles, CA, USA) for the gift of murine hepatoma cells.
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