• Jump to main content
• Jump to navigation
• nature.com homepage
• Publications A-Z index
• Browse by subject
• ISN

• My account
• Submit manuscript
• Register
• Subscribe

Login

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.

Abstract

Hypoxia and interleukin-1 stimulate vascular endothelial growth factor production in human proximal tubular cells.

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 in VEGF mRNA levels, VEGF secretion, and activity of the hypoxia-inducible dimeric transcription factor 1 (HIF-1/) in human proximal tubular epithelial cells (PTECs) in primary culture.

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 was demonstrated by Western blot analysis and HIF-1 DNA binding by gel shift assay.

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 treatment was detectable at the protein level only. Nuclear HIF-1 protein levels and HIF-1 binding to DNA were also increased under these conditions.

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- (TNF-), and chemokines, such as RANTES (regulated upon activation, normal T-cell expressed and secreted), interleukin-8 (IL-8), and monocyte chemoattractant protein-1, on stimulation with proinflammatory cytokines or to bacterial toxins^1,2,3.

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 mRNA^6,7. 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/) to this element⁹. The HIF-1 subunit is the pO₂-sensitive partner¹⁰, as it is unstable in normoxia because of a pO₂-dependent degradation domain that targets it for ubiquitination¹¹. A recent study from our laboratory has shown that IL-1 and TNF- increase HIF-1 DNA binding in normoxic and hypoxic human hepatoma cells in culture¹². Previously, IL-1 and TNF- have been reported to stimulate VEGF production in a tissue-specific way⁵.

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, and TNF- in this process, and (3) the accompanying changes in the level of HIF-1 protein and HIF-1 DNA binding activity.

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 g/mL), transferrin (5 g/mL), selenium (5 ng/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL; all from Sigma, Deisenhofen, Germany). PTECs were characterized by binding of monoclonal antibodies directed against the epithelial membrane antigen and adenosine deaminase binding proteins¹⁵ and by electron microscopy¹⁶. Passages 2 through 4 were used for study.

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 (provided by Ciba-Geigy, Basel, Switzerland), TNF- (6.6 10⁶ U/mg by L 929 bioassay; provided by BASF/Knoll, Ludwigshafen, Germany), and IL-6 (Sigma). IL-1 was also tested for its effect on VEGF mRNA levels. Phorbol 12-myristate 13-acetate (PMA; Sigma) was used as a positive control agent for stimulation of VEGF production.

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 subunit. Hepa1 and Hepa1C4 cells were maintained in -minimal essential medium (GIBCO BRL Life Technologies, Eggenstein, Germany) supplemented with 10% FCS (Sigma).

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 -actin mRNAs to test for the integrity of cDNA. RT-PCR was performed in 1 reaction buffer (supplied by the manufacturer) with 1.5 mmol/L MgCl₂, 200 mol/L of each dNTP, 0.4 mol/L of each 5' and 3' primer, 1 L of cDNA template, and 0.75 U of Taq DNA polymerase (GIBCO) in a total reaction volume of 50 L. The primer sequences and PCR conditions are listed in Table 1. The PCR products were analyzed by agarose gel electrophoresis. The competitive PCR contained 10 L of competitor DNA for the VEGF₁₆₅ isoform and 10 L of cDNA. The VEGF competitor was constructed as described recently¹⁹. For each cDNA sample, we performed a set of four to six reactions with different amounts of competitor. Its concentration ranged from 2.5 to 0.08 pg/L in 1:2 dilutions. PCR products generated from the cDNA and the competitor template were distinguishable by size (341 vs. 269 bp) when run on an 3% (wt/vol) agarose gel stained with ethidium bromide. VEGF mRNA concentrations per g of total RNA were calculated after determination of the equilibrium between PCR products from cDNA and competitor DNA, as described by Fandrey and Bunn²⁰.

Table 1 - Characterization of polymerase chain reaction (PCR) primers.

Nuclear extract preparation for study of HIF-1 activation

Hypoxia-inducible factor-1 protein levels and HIF-1 DNA binding were determined in nuclear extracts of PTECs incubated with or without IL-1 (300 pg/mL) in a normoxic (20% O₂) or hypoxic (3% O₂) atmosphere. For these short-term experiments, PTECs were cultured in conventional Petri dishes of 55 mm diameter or in special dishes with a gas-permeable fluoroethylene-propylene copolymer Teflon membrane as the bottom support (25 m thickness; Petri-PERM®; Heraeus), which allows one to rapidly adjust the pericellular pO₂ to the pO₂ in the gas atmosphere²¹. Hepa1 and Hepa1C4 cells were grown in conventional Petri dishes of 100 mm diameter. Nuclear extracts were prepared according to Semenza and Wang with minor modifications²². Cells were washed with ice-cold phosphate-buffered saline (PBS), harvested in 6 mL PBS and centrifuged at 400 g for five minutes at 4°C. The cell pellets were washed with 2 mL ice-cold buffer A (10 mmol/L Tris, pH 7.8, 1.5 mmol/L MgCl₂, 10 mmol/L KCl), resuspended in 1 mL buffer A, and left on ice for 10 minutes. Subsequently, cells were lyzed by Dounce homogenization. Lysis was controlled with trypan blue. Nuclei were pelleted at 3500 g for five minutes at 4°C and resuspended in 150 L ice-cold buffer C (420 mmol/L KCl, 20 mmol/L Tris, pH 7.8, 1.5 mmol/L MgCl₂, 20% glycerol) and incubated for 30 minutes on ice with occasional flicking of the tubes. Just before use, buffers A and C were supplemented with 2 g/mL aprotinin, 10 g/mL leupeptin, 20 g/mL pepstatin, 1 mmol/L Na₃VO₄, 0.5 mmol/L benzamidine, 2 mmol/L levamisole, 10 mmol/L -glycerophosphate, 0.5 mmol/L dithiothreitol (DTT) and 0.4 mmol/L phenylmethylsulfonyl fluoride (PMSF). Nuclei were centrifuged at 12,000 g for 30 minutes at 4°C. The supernatant was dialyzed against 1 L of buffer D [100 mmol/L KCl, 20 mmol/L Tris, pH 7.8, 2 mmol/L ethylenediaminetetraacetic acid (EDTA), 20% glycerol] overnight at 4°C. After dialysis, nuclear extracts were collected by a short centrifugation at 4°C. Aliquots were frozen in liquid nitrogen and stored at -80°C. Protein concentrations were determined by the Bradford method using bovine serum albumin as standard.

Western blot analysis

For determination of immunoreactive HIF-1 protein, cellular or nuclear extracts were subjected to Western blot analysis as described¹². Samples were run on sodium dodecyl sulfate/7.5% polyacrylamide gels and transferred electrophoretically (Trans-Blot SD; BioRad, München, Germany) to nitrocellulose membranes (Amersham, Braunschweig, Germany). Equal loading and transfer efficiency were verified by staining with 2% Ponceau S. Membranes were blocked overnight with PBS/5% fat-free skim milk and then incubated for two hours at room temperature with a 1:500 diluted monoclonal mouse antibody against human HIF-1 (Transduction Laboratories, Heidelberg, Germany). For detection, a horseradish peroxidase-linked antimouse IgG antibody (1:2000, 1 h at room temperature; Santa Cruz, Heidelberg, Germany) and enhanced chemiluminescence substrate (Amersham) were used.

Gel shift assay of HIF-1 DNA binding

[–³²P] adenosine 5'-triphosphate (ATP) was obtained from New England Nuclear (Köln, Germany). Oligonucleotides for gel shift assays were synthesized by MWG (Ebersberg, Germany). The sequences containing the HIF-1 binding sites were derived from the human transferrin gene (TfHBSww). Rolfs et al established this experimental model, which is well suited for study of HIF-1 DNA binding, because two HIF-1 binding sites are present in the TfHBSww enhancer²³.

Sequences were TfHBSww (sense), 5'-TTCCTGCACGTACACACAAAGCGCACGTATTTC-3', and TfHBSww (antisense), 5'-GAAATACGTGCGCTTTGTGTGTACGTGCAGGAA-3'.

After 5' end labeling of the sense strand unincorporated [-³²P], ATP was removed with a Sephadex G50 (Pharmacia, Uppsala, Sweden) column. The annealing reaction was performed in the presence of a twofold molar excess of unlabeled antisense oligonucleotides and a final MgCl₂ concentration of 1 mmol/L.

Binding reactions were set up in a volume of 30 L. Nuclear extracts (3 g of protein from PTEC or 5 g of protein from murine hepatoma cells) were preincubated on ice for 30 minutes in a buffer with final concentrations of 50 mmol/L KCl, 10 mmol/L Tris, pH 7.7, 5 mmol/L DTT, 1 mmol/L EDTA, 1 mmol/L MgCl₂, 5% glycerol, 0.03% NP40, and 600 ng salmon testes DNA. After the addition of the ³²P-labeled oligonucleotide, the reactions were incubated at 4°C overnight. Samples were resolved by electrophoresis on native 5% polyacrylamide gels (polyacrylamide/bisacrylamide, 3:0.8) at room temperature. Gels were dried and analyzed directly by autoradiography.

Statistics

Data are presented as the mean and standard deviation (SD). The paired t-test was used to compare two groups of matched cultures from the same renal tissue donor. The Tukey–Kramer test was used for multiple comparisons. A statistically significant difference was assumed if the two-tailed P value was <0.05.

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 1520 amol/g total RNA vs. 4960 4160 amol/g total RNA after 24 h of incubation; mean SD of cultures from 6 different renal tissue donors, P > 0.05, paired t-test).

Figure 1.

Vascular endothelial growth factor (VEGF) concentrations in proximal tubule epithelial cell (PTEC) culture media during normoxic (, 20% O₂) and hypoxic (, 3% O₂) incubation for up to 72 hours. Data are mean SD of cultures from 5 separate renal tissue donors; *P < 0.05 compared with normoxic incubation, paired t-test.

Figure 2 shows that the addition of PMA or IL-1 increased the rate of the production of immunoreactive VEGF by PTECs. No such effect was seen, when the cells were treated with TNF- or IL-6. Measurements of VEGF mRNA revealed no significant increase in cultures treated with IL-1 (300 pg/mL) for four hours (4720 2960 amol/g total RNA vs. 3920 720 amol/g total RNA in control cultures; mean SD of cultures from 4 different renal tissue donors).

Figure 2.

Effects of the addition of phorbol 12-myristate 13-acetate (PMA; 100 nmol/L), interleukin 1- (IL-1; 300 pg/mL), tumor necrosis factor- (TNF-; 10 ng/mL), and IL-6 (20 ng/mL) on the 24-hour rates of the production of immunoreactive VEGF in PTEC. Data are mean SD of cultures from 5 separate renal tissue donors; *P < 0.001 compared to untreated control cultures, Tukey–Kramer test.

Western blot analysis showed that the amount of HIF-1 was increased in nuclear extracts from PTECs on treatment with IL-1 or exposure to hypoxia for four hours Figure 3.

Figure 3.

Western blot for hypoxia inducible factor-1 (HIF-1) in whole cell extracts from PTECs incubated under normoxic (20%O₂) or hypoxic (3% O₂) conditions without or with IL-1 (300 pg/mL) for four hours. A similar increase of HIF-1 protein on IL-1 treatment was seen on Western blots of nuclear extracts.

Electrophoretic mobility shift assays showed that hypoxia led to increased DNA binding of the HIF-1 complex Figure 4. Moreover, incubation with IL-1 was as effective as hypoxia in activating the HIF complex even under normoxic conditions. The effect was stronger when cells were studied in gas-permeable Petri-PERM® culture dishes. To demonstrate the effectiveness of the hypoxic incubation, nuclear extracts of mouse hepatoma cells were tested for HIF-1 DNA binding. Only Hepa1 cells, which contained both HIF-1 subunits in an active form, showed hypoxic inducibility, whereas Hepa1C4 cells lacked a functional HIF-1 subunit. Specificity of HIF-1 binding was confirmed by adding unlabeled TfHBSww-oligonucleotide to the binding reaction. This experiment was not performed with nuclear extracts from PTECs because of the limited availability of the renal cells.

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 (300 pg/mL) for four hours. In the competition experiment (lane H, Hepa1 + competitor), a 150-fold molar excess of unlabeled oligonucleotide was added to the binding reaction. Abbreviations are: i, inducible DNA-binding activity; c, constitutive DNA-binding activity, n.s., nonspecific DNA-binding activity.

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 podocytes^24,25. While renal VEGF seems to be essential for the capillarization of the metanephros^26,27, 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 immunohistochemistry^4,25. 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 hypoxia^6,7,8,30. 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 fibroblasts^34,35,36, and tumor cells^37,38. The action of TNF- in VEGF gene expression is more tissue restricted. However, this cytokine is effective in human keratinocytes³⁵ and some tumor cell lines^37,39.

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 was added to the cells. TNF- did not stimulate the secretion of VEGF. In addition, we failed to demonstrate significant differences in VEGF mRNA levels, when cultures grown at 20% O₂ were compared with those grown at 3% O₂ or treated with IL-1. This failure was probably due to the moderate effects of low O₂ concentration and IL-1, which increased the amount of secreted VEGF by 20 and 30%, respectively. Whereas the ELISA for immunoreactive VEGF could still detect this stimulation, the effect was likely below the resolution threshold of the RT-PCR assay for VEGF mRNA. Several transcription factors have been implicated in the control of the VEGF gene, including HIF-1⁹, AP-1^30,37, and SP-1³⁷. Interest has been focused on HIF-1 because this transcription factor complex is the key activator of O₂-controlled genes^40,41,42. Originally, HIF-1 was thought to function solely to mediate responses to O₂ and glucose deprivation. However, we have recently observed that IL-1 and TNF- increase HIF-1 DNA binding in the human hepatoma cell lines, HepG2 and Hep3B, under normoxic and hypoxic conditions. HIF-1 protein accumulation was seen only on IL-1 treatment of the hepatoma cells, while TNF- was ineffective in this regard¹². This present study extends these findings, as the addition of IL-1 to cultured primary human proximal tubular cells resulted in increased HIF-1 immunoreactivity and HIF-1 DNA binding. Thus, the transcription factor HIF-1 may not only play an important role in O₂ homeostasis but also in IL-1–mediated inflammatory processes.

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 treatment. The secretion of VEGF into the renal interstitium will result in increased microvascular permeability and monocyte extravasation. The extent to which renal proximal tubular cells contribute to this processes remains to be clarified, because VEGF will also be produced by other resident renal cells and by leukocytes, when these are challenged by hypoxia and inflammation.

References

1. Yard, BA, Daha, MR, Kooymans-Couthino, M, Bruijn, JA, Paape, MA, Schrama, E, van Es, LA, van der Woude, FJ: IL- stimulated TNF- production by cultured human proximal tubular epithelial cells. Kidney Int 1992 42: 383–389,  | PubMed | ISI | ChemPort |
2. Deckers, JGM, van der Woude, FJ, van der Kooij, SW, Daha, MR: Synergistic effect of IL-1, IFN-, and TNF- in RANTES production by human renal tubular epithelial cells in vitro. J Am Soc Nephrol 1998 9: 194–202,  | PubMed | ISI | ChemPort |
3. Hughes, AK, Stricklett, PK, Kohan, DE: Shiga toxin-1 regulation of cytokine production by human proximal tubule cells. Kidney Int 1998 54: 1093–1106,  | Article | PubMed | ISI | ChemPort |
4. Gröne, H-J, Simon, M, Gröne, EF: Expression of vascular endothelial growth factor in renal vascular disease and renal allografts. J Pathol 1995 177: 259–267,  | PubMed | ISI | ChemPort |
5. Ferrara, N, Davis-Smyth, T: The biology of vascular endothelial growth factor. Endocr Rev 1997 18: 4–25,  | Article | PubMed | ISI | ChemPort |
6. White, FC, Carroll, SM, Kamps, MP: VEGF mRNA is reversibly stabilized by hypoxia and persistently stabilized in VEGF-overexpressing human tumor cell lines. Growth Factors 1995 12: 289–301,  | PubMed | ISI | ChemPort |
7. Levy, AP, Levy, NS, Goldberg, MA: Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem 1996 271: 2746–2753,  | Article | PubMed | ISI | ChemPort |
8. Liu, Y, Cox, SR, Morita, T, Kourembanas, S: Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Circ Res 1995 77: 638–643,  | PubMed | ISI | ChemPort |
9. Forsythe, JA, Jiang, B-H, Iyer, NV, Agani, F, Leung, SW, Koos, RD, Semenza, GL: Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 1996 16: 4604–4613,  | PubMed | ISI | ChemPort |
10. Huang, E, Arany, Z, Livingston, DM, Bunn, HF: Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its subunit. J Biol Chem 1996 271: 32253–32259,  | Article | PubMed | ISI | ChemPort |
11. Salceda, S, Caro, J: Hypoxia-inducible factor 1 (HIF-1) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. J Biol Chem 1997 272: 22642–22647,  | Article | PubMed | ISI | ChemPort |
12. Hellwig-Bürgel, T, Rutkowski, K, Metzen, E, Fandrey, J, Jelkmann, W: Interleukin-1 and tumor necrosis factor- stimulate DNA-binding of hypoxia-inducible factor-1. Blood 1999 94: 1561–1567,  | PubMed | ISI | ChemPort |
13. Detrisac, CJ, Sens, MA, Garvin, AJ, Spicer, SS, Sens, DA: Tissue culture of human kidney epithelial cells of proximal tubule origin. Kidney Int 1984 25: 383–390,  | PubMed | ISI | ChemPort |
14. Kreft, B, Brzoska, S, Doehn, C, Daha, MR, van der Woude, FJ, Sack, K: 1,25-Dihydroxycholecalciferol enhances the expression of MHC class II antigens and intercellular adhesion molecule-1 by human renal tubular epithelial cells. J Urol 1996 155: 1448–1453,  | PubMed | ISI | ChemPort |
15. Leeker, A, Kreft, B, Sandmann, J, Bates, J, Wasenauer, G, Müller, H, Sack, K, Kumar, S: Tamm-Horsfall protein inhibits binding of S- and P-fimbriated Escherichia coli to human renal tubular epithelial cells. Exp Nephrol 1997 5: 38–46,  | PubMed | ISI | ChemPort |
16. Krüger, S, Kliniker, M, Krüger, S, Brandt, E, Kreft, B: Interleukin-8 secretion of cortical tubular epithelial cells is directed to the basolateral environment and is not enhanced by apical exposure to Escherichia coli. Infect Immun 2000 68: 328–334,  | PubMed | ISI | ChemPort |
17. Heits, F, Katschinski, DM, Wiedemann, GJ, Weiss, C, Jelkmann, W: Serum vascular endothelial growth factor (VEGF), a prognostic indicator in sarcoma and carcinoma patients. Int J Oncol 1997 10: 333–337,  | ISI | ChemPort |
18. Chomczynski, P, Sacchi, N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987 162: 156–159,  | Article | PubMed | ISI | ChemPort |
19. Gassmann, M, Fandrey, J, Bichet, S, Wartenberg, M, Marti, HH, Bauer, C, Wenger, RH, Acker, H: Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells. Proc Natl Acad Sci USA 1996 93: 2867–2872,  | Article | PubMed | ChemPort |
20. Fandrey, J, Bunn, HF: In vivo and in vitro regulation of erythropoietin mRNA: Measurement by competitive polymerase chain reaction. Blood 1993 81: 617–623,  | PubMed | ISI | ChemPort |
21. Wolff, M, Fandrey, J, Jelkmann, W: Microelectrode measurements of pericellular pO2 in erythropoietin-producing human hepatoma cell cultures. Am J Physiol 1993 265: C1266–C1270,  | PubMed | ISI | ChemPort |
22. Semenza, GL, Wang, GL: A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol 1992 12: 5447–5454,  | PubMed | ISI | ChemPort |
23. Rolfs, A, Kvietikova, I, Gassmann, M, Wenger, RH: Oxygen-regulated transferrin expression is mediated by hypoxia-inducible factor-1. J Biol Chem 1997 272: 20055–20062,  | Article | PubMed | ISI | ChemPort |
24. Berse, B, Brown, LF, Van De Water, L, Dvorak, HF, Senger, DR: Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors. Mol Biol Cell 1992 3: 211–220,  | PubMed | ISI | ChemPort |
25. Brown, LF, Berse, B, Tognazzi, K, Manseau, EJ, Van De Water, L, Senger, DR, Dvorak, HF, Rosen, S: Vascular permeability factor mRNA and protein expression in human kidney. Kidney Int 1992 42: 1457–1461,  | PubMed | ISI | ChemPort |
26. Kitamoto, Y, Tokunaga, H, Tomita, K: Vascular endothelial growth factor is an essential molecule for mouse kidney development: Glomerulogenesis and nephrogenesis. J Clin Invest 1997 99: 2351–2357,  | PubMed | ISI | ChemPort |
27. Simon, M, Gröne, H-J, Jöhren, O, Kullmer, J, Plate, KH, Risau, W, Fuchs, E: Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney. Am J Physiol 1995 268: F240–F250,  | PubMed | ISI | ChemPort |
28. Klanke, B, Simon, M, Röckl, W, Weich, HA, Stolte, H, Gröne, H-J: Effects of vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) on haemodynamics and permselectivity of the isolated perfused rat kidney. Nephrol Dial Transplant 1998 13: 875–885,  | PubMed | ISI | ChemPort |
29. Webb, NJA, Watson, CJ, Roberts, ISD, Bottomley, MJ, Jones, CA, Lewis, MA, Postlethwaite, RJ, Brenchley, PEC: Circulating vascular endothelial growth factor is not increased during relapses of steroid-sensitive nephrotic syndrome. Kidney Int 1999 55: 1063–1071,  | Article | PubMed | ISI | ChemPort |
30. Goldberg, MA, Schneider, TJ: Similarities between the oxygen-sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin. J Biol Chem 1994 269: 4355–4359,  | PubMed | ISI | ChemPort |
31. Marti, HH, Risau, W: Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors. Proc Natl Acad Sci USA 1998 95: 15809–15814,  | Article | PubMed | ChemPort |
32. Sandner, P, Gess, B, Wolf, K, Kurtz, A: Divergent regulation of vascular endothelial growth factor and of erythropoietin gene expression in vivo. Eur J Physiol 1996 431: 905–912,  | ChemPort |
33. Li, J, Perrella, MA, Tsai, J-C, Yet, S-F, Hsieh, C-M, Yoshizumi, M, Patterson, C, Endege, WO, Zhou, F, Lee, M-E: Induction of vascular endothelial growth factor gene expression by interleukin-1 in rat aortic smooth muscle cells. J Biol Chem 1995 270: 308–312,  | Article | PubMed | ISI | ChemPort |
34. Ben-Av, P, Crofford, LJ, Wilder Rl Hla, T: Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: A potential mechanism for inflammatory angiogenesis. FEBS Lett 1995 372: 83–87,  | Article | PubMed | ChemPort |
35. Jackson, JR, Minton, JAL, Ho, ML, Wei, N, Winkler, JD: Expression of vascular endothelial growth factor in synovial fibroblasts is induced by hypoxia and interleukin 1. J Rheumatol 1997 24: 1253–1259,  | PubMed | ISI | ChemPort |
36. Frank, S, Hübner, G, Breier, G, Longaker, MT, Greenhalgh, DG, Werner, S: Regulation of vascular endothelial growth factor expression in cultured keratinocytes. J Biol Chem 1995 270: 12607–12613,  | Article | PubMed | ISI | ChemPort |
37. Ryuto, M, Ono, M, Izumi, H, Yoshida, S, Weich, HA, Kohno, K, Kuwano, M: Induction of vascular endothelial growth factor by tumor necrosis factor in human glioma cells. J Biol Chem 1996 271: 28220–28228,  | Article | PubMed | ISI | ChemPort |
38. Akagi, Y, Lui, W, Xie, K, Zebrowski, B, Shaheen, RM, Ellis, LM: Regulation of vascular endothelial growth factor expression in human colon cancer by interleukin-1. Br J Cancer 1999 80: 1506–1511,  | Article | PubMed | ISI | ChemPort |
39. Cohen, T, Nahari, D, Cerem, LW, Neufeld, G, Levi, B-Z: Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem 1996 271: 736–741,  | Article | PubMed | ISI | ChemPort |
40. Wang, GL, Semenza, GL: Oxygen sensing and response to hypoxia by mammalian cells. Redox Rep 1996 2: 89–96,  | ISI | ChemPort |
41. Gassmann, M, Wenger, RH: HIF-1, a mediator of the molecular response to hypoxia. News Physiol Sci 1997 12: 214–218,  | ISI | ChemPort |
42. Gleadle, JM, Ratcliffe, PJ: Hypoxia and the regulation of gene expression. Mol Med Today 1998 4: 122–129,  | PubMed | ISI | ChemPort |

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.

Main navigation

ISN resources

• Nephrology Gateway
• Chinese edition
• Spanish edition
• Portuguese edition
• Contact us
• Society news

NPG resources

• KI Japanese home page
• Nature Reviews Nephrology
• Nature Reviews Urology

NPG Journals

by Subject Area

• Chemistry

  • Chemistry
  • Drug discovery
  • Biotechnology
  • Materials
  • Methods & Protocols

• Clinical Practice & Research

  • Cancer
  • Cardiovascular medicine
  • Dentistry
  • Endocrinology
  • Gastroenterology & Hepatology
  • Methods & Protocols
  • Pathology & Pathobiology
  • Urology

• Earth & Environment

  • Earth sciences
  • Evolution & Ecology

• Life sciences

  • Biotechnology
  • Cancer
  • Development
  • Drug discovery
  • Evolution & Ecology
  • Genetics
  • Immunology
  • Medical research
  • Methods & Protocols
  • Microbiology
  • Molecular cell biology
  • Neuroscience
  • Pharmacology
  • Systems biology

• Physical sciences

  • Physics
  • Materials

by A - Z Index

Extra navigation

ARTICLE NAVIGATION - FULL TEXT

• Table of contents
• Download PDF
• Send to a friend
• Rights and permissions
• Order Commercial Reprints
• CrossRef lists 29 articles citing this article
• Scopus lists 84 articles citing this article
• Save this link
• Abstract
• METHODS
• RESULTS
• DISCUSSION
• References
• Acknowledgments
• Figures and Tables

• Export citation
• Export references

• Papers by El Awad

Top

• Kidney International
• ISSN: 0085-2538
• EISSN: 1523-1755
• © 2010 International Society of Nephrology

• About NPG
• Contact NPG
• RSS web feeds
• Help

• Privacy policy
• Legal notice
• Accessibility statement
• Terms

• Nature News
• Nature jobs
• Nature Asia
• Nature Education

• partner of AGORA, HINARI, OARE, INASP, CrossRef and COUNTER