Influence of hepatocyte growth factor, epidermal growth factor, and mycophenolic acid on endothelin-1 synthesis in human endothelial cells

Cornelia Haug1,, Alexandra Schmid-Kotsas1, Theresia Linder1, Max G. Bachem1, Adolf Gruenert1 and Eva Rozdzinski2

1 Institute of Clinical Chemistry and 2 Department of Medical Microbiology, University Hospital, Ulm, Germany



   Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Endothelin-1 (ET-1) is a potent vasoconstrictive peptide which plays an important pathophysiological role in ischaemic renal failure and drug-induced renal injury such as cyclosporin A (CsA)- and tacrolimus-associated nephrotoxicity. In contrast, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) seem to accelerate renal regeneration after ischaemic and drug-induced renal injury. This study aimed to investigate the influence of HGF and EGF on ET-1 synthesis in cultured human umbilical vein endothelial cells (HUVEC) and renal artery endothelial cells (RAEC). In addition, we have investigated whether mycophenolic acid (MPA), a new immunosuppressive drug, which in contrast to CsA and tacrolimus lacks nephrotoxic side effects, modulates ET-1 synthesis in endothelial cells.

Methods. ET-1 release was measured with a specific enzyme-linked immunosorbent assay. ET-1 mRNA expression was investigated by reverse transcription polymerase chain reaction.

Results. HGF and EGF (0.001-10 nM) exerted a significant concentration-dependent inhibitory effect on ET-1 release by HUVEC and RAEC (minimum 56.1±4.3% of control, n=6, mean±SE). The suppressive effect of HGF and EGF on ET-1 synthesis was dose-dependently antagonized by the tyrosine kinase inhibitors tyrphostin AG1478, lavendustin A and methyl 2,5-dihydroxycinnamate. Incubation of HUVEC and RAEC with MPA (2.5, 10, 25, and 50 µg/ml) for 3–5 h induced a significant reduction of ET-1 mRNA expression. After 48 h incubation with MPA (1–50 µg/ml) a significant decrease of ET-1 release and DNA content per culture well was observed, whereas ET-1 release referred to the DNA content in the corresponding culture well did not differ significantly from controls.

Conclusions. The present findings demonstrate that HGF and EGF reduce ET-1 synthesis in endothelial cells via their receptor tyrosine kinase activity and suggest that the renoprotective effects of HGF and EGF might be linked to their inhibitory action on ET-1 synthesis. This study also provides evidence that, in contrast to CsA and tacrolimus, MPA does not stimulate ET-1 synthesis. This might explain the clinical observation that renal function often improves when CsA or tacrolimus is replaced by mycophenolate mofetil.

Keywords: endothelin-1; endothelial cells; epidermal growth factor; hepatocyte growth factor; mycophenolic acid; tyrosine kinase inhibitors



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endothelin is a potent vasoconstrictive peptide, originally isolated from the supernatant of cultured aortic endothelial cells. Until now, three isoforms, endothelin-1, -2 and -3, each consisting of 21 amino acid residues, have been identified [1]. Endothelial cells represent the major site of endothelin-1 (ET-1) production, however, ET-1 synthesis has also been localized to other cell types including renal mesangial cells, proximal tubule cells, and collecting duct cells [2,3]. Endothelin acts predominantly by paracrine and autocrine mechanisms via binding to specific G-protein-coupled receptors (ETA and ETB). ET-1 seems to play an essential pathophysiological role in ischaemic renal failure and drug-induced renal injury [46].

Hepatocyte growth factor (HGF), a heparin-binding glycoprotein composed of a large {alpha}-subunit and a small ß-subunit, was originally isolated from the sera of partially hepatectomized rats [7]. HGF is synthesized in various cells including endothelial cells and mesangial cells [8] and exerts mitogenic, morphogenic, and motogenic effects on tubular epithelial cells by stimulating the tyrosine kinase activity of its specific receptor c-met [911].

Epidermal growth factor (EGF) is a 53 amino acid polypeptide belonging to the EGF family of growth factors, which bind to the cell surface EGF receptor, a 170 kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity [12]. EGF binding to the receptor stimulates tyrosine kinase activity and autophosphorylation of the EGF receptor and evokes phosphorylation of a variety of cellular substrates [12,13]. Like HGF, EGF is a potent mitogen for proximal tubular cells [14,15]. Enhanced levels of mature EGF have been observed in ischaemic renal injury and administration of exogenous EGF has been shown to improve cell regeneration in ischaemic nephropathy [16,17].

As an enhanced ET-1 synthesis seems to play an important pathophysiological role in acute and chronic renal injury whereas HGF and EGF have been shown to attenuate ischaemic and drug-induced renal injury [16,1822], the present study aimed to investigate the influence of HGF and EGF on ET-1 synthesis in cultured human umbilical vein endothelial cells (HUVEC) and renal artery endothelial cells (RAEC).

In addition, we have investigated the effect of mycophenolic acid (MPA) on ET-1 release and ET-1 mRNA expression in HUVEC and RAEC. Mycophenolate mofetil (MMF) is a new immunosuppressive drug that in vivo is converted to MPA [23]. MMF does not seem to exert nephrotoxic side effects, and recently published studies have demonstrated that renal function was markedly improved when the immunosuppressive therapy was switched from cyclosporin A (CsA) or tacrolimus to MMF, or when MMF was added and CsA or tacrolimus doses were reduced [2428]. As previous studies have suggested that ET-1 is an important mediator of CsA- and tacrolimus-associated nephrotoxicity, and CsA and tacrolimus have been shown to stimulate ET-1 synthesis in endothelial cells [4,29,30], our study aimed to examine the effect of MPA on ET-1 synthesis in endothelial cells.



   Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
HUVEC (passage 1) and RAEC (passage 3) were purchased from Clonetics (San Diego, CA, USA). HUVEC were cultured (37°C, 5% CO2) in gelatin-coated culture flasks in EGM-2 medium (Clonetics; 2% fetal calf serum). RAEC were propagated in EGM-2-MV medium (Clonetics; 5% fetal calf serum) in culture flasks coated with collagen type I (Sigma, Deisenhofen, Germany). Medium was changed every 2 days, and cells were subcultured after reaching confluency. For investigation of ET-1 release and ET-1 mRNA expression, HUVEC and RAEC were seeded into collagen type I-coated 24-well plates and 6-well plates (Falcon, Franklin Lakes, NJ, USA), respectively, and experiments were performed with confluent monolayers in culture medium (HUVEC: EGM-2, RAEC: EGM-2-MV) containing 1% fetal calf serum (PAA Laboratories, Linz, Austria).

For investigation of ET-1 release into the cell culture supernatant, cells (HUVEC: passage 4, RAEC: passages 5–6) were incubated for 48 h with HGF, EGF (0.001–10 nM; HGF, R&D Systems, Wiesbaden, Germany; EGF, Bachem, Heidelberg, Germany), tyrosine kinase inhibitors tyrphostin, lavendustin A and erbstatin analogue methyl 2,5-dihydroxycinnamate (0.1–10 µM; tyrphostin AG1478, Calbiochem, Bad Soden, Germany; lavendustin A, Calbiochem; erbstatin analogue: methyl 2,5-dihydroxycinnamate (MDC), Biomol, Hamburg, Germany) [11,31,32] MPA (Sigma; 1–50 µg/ml) or vehicles. In experiments performed with a combination of growth factors and tyrosine kinase inhibitors, cells were pre-incubated with tyrosine kinase inhibitors for 1 h.

To investigate the effect of growth factors and tyrosine kinase inhibitors on ET-1 mRNA expression, HUVEC (passages 3–5) were incubated for 1–6 h with HGF (1 nM), EGF (1 nM), tyrphostin AG1478 (10 µM), lavendustin A (10 µM), MDC (10 µM), or vehicle alone or co-incubated with growth factors and tyrosine kinase inhibitors (cells were pre-incubated with tyrosine kinase inhibitors for 30 min before addition of growth factors). The effect of MPA on ET-1 mRNA expression was investigated by incubating HUVEC (passage 3) for 3, 4, and 5 h and RAEC (passage 6) for 3.5 h with 2.5, 10, 25, and 50 µg/ml MPA.

Experiments for phosphotyrosine and HGF receptor western blotting were performed with HUVEC (passages 2–3) grown in gelatin-coated culture flasks. HUVEC were incubated in serum-free medium (M 199, Sigma) for 5 min (37°C) with vehicle or 1 nM HGF either with or without pre-incubation (30 min, 37°C) with tyrphostin AG1478 (1–100 µM), lavendustin A (1–10 µM), or MDC (1–10 µM).

Measurement of ET-1 release
ET-1 was measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Biotrend, Köln, Germany; cross reactivity: ET-1 100%, ET-2 3.3%, ET-3 <0.1%, Big ET-1 <0.1%, Big ET-3 <0.1%). Standards or samples (100 µl) were incubated overnight in coated microtitre plates. After washing, labelled anti-ET-1 antibody was added and incubated for 30 min at 37°C. After another washing step, substrate solution was added, followed by an incubation period of 30 min at room temperature. Stop solution was added, and optical density was read at 450 nm after blanking against the substrate blank. Intra-assay coefficients of variation (n=24) were 2.9% (9.7 pg/ml) and 2.1% (56.2 pg/ml), inter-assay coefficients of variation (n=8) were 2.9% (10.0 pg/ml) and 3.3% (55.4 pg/ml), respectively. No immunoreactive ET-1 was detected in cell culture medium, which had not been incubated with the cells. ET-1 concentrations in the cell culture supernatants were referred to the DNA content in the corresponding culture wells.

Measurement of DNA
DNA content of cells was measured as described previously [33] by fluorescent DNA staining with bisbenzimide (Sigma) using calf thymus DNA as a standard. Fluorescence (excitation 350 nm, emission 450 nm) was measured with a Victor 1420 Multilabel Counter (Wallac, Turku, Finland).

RNA isolation and RT–PCR of ET-1 mRNA
ET-1 mRNA expression was investigated by reverse transcription polymerase chain reaction (RT–PCR). Total RNA was extracted with a High Pure RNA Isolation Kit, which includes DNA digestion (Roche Diagnostics, Mannheim, Germany). The concentration and purity of RNA was determined by measuring the absorbance at 260 and 280 nm, and 1 µg of total RNA was reverse transcribed into cDNA with 0.02 U/ml reverse transcriptase (Superscript, Gibco, Germany) at 42°C for 50 min. For RT–PCR the following oligonucleotide primers (0.5 µM) were used: preproET-1 [34] from nucleotide +415 to +662 (a 248 bp fragment, Accession number Y00749), sense primer 5'-TGCTCGTCCCTGATGGATA-3', antisense primer 5'-TTCTCCATAATGTCTTCAGCC-3' (exon2–exon4); ß-actin [35] from nucleotide +144 to +683 (a 540 bp fragment, Accession number M10277), sense primer 5'-GTGGGGCGCCCCAGGCCCA-3', antisense primer 5'-CTCCTTAATGTCACGCACGATTTC-3' (exon2 to exon4). DNA amplification was performed with 60 ng cDNA using the LightCycler technology (Idaho Technology, Salt Lake City, UT, USA; LightCycler–DNA Master SYBR Green I; Roche Diagnostics). Reactions were cycled 34–40 times (95°C denaturation: 1 s; annealing: ET-1 52°C, 5 s, ß-actin 58°C, 7 s; extension 72°C: ET-1 12 s, ß-actin 22 s; slopes were 20°C/s). Fluorescence was measured at the end of the extension phase. To confirm the specificity of the amplified products, melting curves were performed at the end of the amplification by cooling the samples at 20°C/s to 62 (ET-1) or 68°C (ß-actin) and then increasing the temperature to 95°C at 0.1°C/s with fluorescence measurement every 0.1°C. For evaluation of the amplification efficiency, PCR products were purified with the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and sequenced (Sequiserve, Vaterstetten, Germany). Concentrations were determined by measuring the absorbance at 260 nm, and samples were used as standard in a 10-fold serial dilution. PCR products were quantified using the LightCycler software. ET-1 mRNA was referred to ß-actin mRNA in the corresponding samples. No detectable PCR products were present in water controls and in controls, amplified without prior reverse transcription. For visualization, PCR products were applied to 1% agarose gel in 0.5x Tris–borate and stained with GelStar (Biozym, Hessisch Oldendorf, Germany).

Western blotting
For phosphotyrosine and HGF receptor western blotting HUVEC were incubated with vehicle or HGF either with or without pre-incubation with tyrosine kinase inhibitors. Cells were washed with phosphate-buffered saline, and cellular proteins were solubilized in ice-cold RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM activated sodium orthovanadate, 20 mM benzamidine, 10 µM zinc chloride, 0.5 mM phenylmethylsulfonylfluoride; 15 min, 4°C). Samples were centrifuged (13000 g, 8 min, 4°C), supernatants were diluted with Lämmli buffer (reducing conditions), sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed, and separated proteins were electroblotted to polyvinylidene difluoride membranes (PVDF Western Blotting Membranes, Roche, Germany). After blocking, samples were incubated for 2 h at room temperature with anti-phosphotyrosine antibody (PT04, Oncogene Research Products, Cambridge, MA, USA; 1:100) or antibody against c-met (Santa Cruz Biotechnology, Heidelberg, Germany; 1:400), followed by incubation with biotinylated secondary antibodies (phosphotyrosine: rabbit anti-mouse, Dako, Hamburg, Germany; c-met: swine anti-rabbit, Dako; 1:3000) and peroxidase-conjugated streptavidin (Dako; 1:3000). Western blots were visualized by chemiluminescence detection (Lumi-LightPLUS, Roche).

Statistical analysis
Results represent means±SE. Differences between ET-1 synthesis were evaluated by one-way analysis of variance, followed by the Newman–Keuls test (comparison of more than two groups) or the two sample t-test, respectively.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
HGF and EGF induced a significant concentration-dependent reduction of ET-1 release by HUVEC and RAEC (Figure 1Go). The suppressive effect of HGF and EGF on ET-1 release by HUVEC was concentration-dependently antagonized by the tyrosine kinase inhibitors tyrphostin AG1478, lavendustin A, and MDC (Figure 2Go). Incubation with tyrphostin AG1478 and MDC (0.1–10 µM) alone did not result in relevant changes of ET-1 release, incubation with lavendustin A induced a slight, dose-dependent increase of ET-1 release (lavendustin A 0.1–10 µM: 96.6±1.5%, 101.4±3.4%, 105.9±1.3%, 122.2±9.7%, not significant, control 100±2.0%, n=3). Similarly, the inhibitory effect of 1 nM HGF or EGF on ET-1 release by RAEC (74.0±4.9% and 80.1±5.7% of controls, respectively; P<0.01 vs controls) was also antagonized by tyrphostin AG1478 (1 µM; 97.5±3.6%, 100.4±3.3%), lavendustin A (10 µM; 109.1±1.3%, 107.6±4.1%) and MDC (1 µM; 97.1±2.3%, 101.3±5.4%; n=3). Tyrphostin AG1478 and MDC alone did not alter ET-1 release by RAEC significantly, and lavendustin A induced a slight, however not significant, increase of ET-1 release (maximum 116.2±1.9%).



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Fig. 1. Effect of (A) HGF and (B) EGF on ET-1 release by cultured HUVEC and RAEC. Cells were incubated for 48 h with HGF or EGF, and ET-1 concentrations in the supernatants were measured with a specific ELISA. ET-1 concentrations were referred to the DNA content in the corresponding culture wells. Results represent means±SE and are expressed as relative ET-1 release compared to controls; n=6; *P<0.05; **P<0.01; ***P<0.001 vs controls.

 


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Fig. 2. Antagonizing effect of tyrosine kinase inhibitors (TKI) tyrphostin AG1478, lavendustin A and erbstatin analogue MDC on the inhibitory effect of 1 nM HGF (A) or EGF (B) on ET-1 release by HUVEC. Cells were incubated for 48 h with HGF or EGF alone or in combination with tyrosine kinase inhibitors, and ET-1 concentrations were measured by a specific ELISA. ET-1 concentrations were referred to the DNA content in the corresponding culture wells. Results represent means±SE and are expressed as relative ET-1 release compared with controls; n=3; **P<0.01 vs controls.

 
ET-1 mRNA expression in HUVEC was significantly decreased after incubation with HGF and EGF (maximum decrease after 3 h incubation). The HGF- and EGF-induced decrease of ET-1 mRNA expression was antagonized by tyrphostin AG1478, lavendustin, and MDC (Figure 3Go); tyrphostin, lavendustin, and MDC (10 µM) alone induced no relevant changes of ET-1 mRNA expression.



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Fig. 3. ET-1 mRNA expression in HUVEC after incubation (3 h) with 1 nM HGF or EGF alone or co-incubation with tyrphostin AG1478 (Tyr., 10 µM), lavendustin A (Lav., 10 µM), or erbstatin analogue MDC (10 µM). ET-1 mRNA expression was evaluated by RT–PCR using the LightCycler technology and was referred to ß-actin mRNA in the corresponding samples. Results represent means±SE and are given as relative ET-1 mRNA expression compared to controls; n=3; **P<0.01 vs control. Inset: Lane 1, base pair ladder; lane 2, ET-1 mRNA, RT–PCR product visualized by application to 1% agarose gel, predicted size 248 bp.

 
Phosphotyrosine western blotting confirmed the inhibitory action of tyrphostin AG1478, lavendustin A, and MDC on HGF receptor tyrosine kinase activity (Figure 4Go).



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Fig. 4. Effect of tyrphostin AG1478, lavendustin A, and MDC on HGF receptor (c-met) tyrosine kinase activity, detected by anti-phosphotyrosine western blotting (lanes 1–10). Cells were incubated for 5 min with vehicle or with 1 nM HGF either with or without pre-incubation with tyrphostin, lavendustin A or MDC. Proteins were run under reducing conditions. Lane 1, molecular weight marker; lane 2, vehicle; lane 3, HGF; lane 4, HGF+tyrphostin 1 µM; lane 5, HGF+tyrphostin 10 µM; lane 6, HGF+tyrphostin 100 µM; lane 7, HGF; lane 8, HGF+lavendustin A 10 µM; lane 9, HGF; lane 10, HGF+MDC 10 µM. To confirm the identity with c-met tyrosine kinase activity, Western blotting was also performed with a c-met antibody; lane 11: molecular weight marker, lane 12: c-met protein expression in cells incubated with vehicle. Results from one representative experiment (n=3).

 
Incubation of HUVEC and RAEC with the immunosuppressive substance MPA induced a significant reduction of ET-1 release and DNA content per well, whereas ET-1 release referred to the DNA content in the corresponding culture well did not differ significantly from controls (Figure 5Go). In both cell types, ET-1 mRNA was significantly decreased after incubation with MPA (Figure 6Go). ß-Actin mRNA was only slightly reduced in HUVEC and was not altered significantly in RAEC.



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Fig. 5. Effect of MPA on ET-1 release referred to the DNA content in the corresponding culture well, ET-1 release per culture well and DNA content per well in HUVEC and RAEC. Cells were incubated for 48 h with MPA, and ET-1 concentrations in the supernatants were measured with a specific ELISA. Results represent means±SE; n=8; ***P<0.001 vs controls.

 


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Fig. 6. Effect of MPA on ET-1 mRNA expression in cultured HUVEC and RAEC. Cells were incubated for 3–5 (HUVEC) or 3.5 h (RAEC) with MPA. ET-1 mRNA expression was evaluated by RT–PCR using the LightCycler technology and was referred to ß-actin mRNA in the corresponding samples. Results represent means±SE and are given as relative ET-1 mRNA expression compared with controls; HUVEC: means from 3–5 h, n=7; RAEC: n=3; *P<0.05, **P<0.01, ***P<0.001 vs control.

 



   Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study demonstrates an inhibitory effect of HGF and EGF on ET-1 synthesis in HUVEC and RAEC. Accumulating evidence suggests that an enhanced synthesis of the potent vasoconstrictive peptide ET-1 is an important mediator of acute ischaemic renal failure as well as drug-induced renal injury [46]. In a recently published study, a substantial rise of ET-1 mRNA expression has been observed after renal ischaemia, and immunohistochemical analysis showed a marked increase of ET-1 peptide expression in the endothelium of cortical peritubular capillaries [36]. In addition, nephrotoxic drugs such as CsA and tacrolimus have been shown to stimulate endothelin synthesis in endothelial cells [29,30], and application of ETA receptor antagonists attenuated post-ischaemic renal injury and CsA-induced renal hypoperfusion [4,6].

In contrast to the adverse effects of ET-1 in ischaemic and drug-induced renal injury, HGF and EGF have been demonstrated to accelerate recovery from ischaemic renal injury [16,21], and HGF has been shown to exert a preventive effect on CsA- and tacrolimus-associated nephrotoxicity [18,22].

In previous experiments, we have observed an inhibitory effect of HGF and EGF on the basal as well as the CsA- and tacrolimus-stimulated ET-1 release in a rabbit proximal tubule cell line [37,38]. In the present study, performed with human endothelial cells, the suppressive action of HGF and EGF on ET-1 synthesis was concentration-dependently antagonized by the tyrosine kinase inhibitors tyrphostin AG1478, lavendustin A, and erbstatin analogue MDC. These substances are known to be potent inhibitors of EGF receptor tyrosine kinase activity [3941]. Phosphotyrosine western blotting was performed to confirm that the HGF-induced tyrosine phosphorylation of c-met was antagonized by tyrphostin AG1478, lavendustin A, and methyl 2,5-dihydroxycinnamate. Thus, the present data provide evidence that HGF and EGF inhibit ET-1 synthesis via their receptor tyrosine kinase activity. These results suggest that the renoprotective actions of HGF and EGF are not only due to their mitogenic and morphogenic effects on renal epithelial cells but might also be linked to their inhibitory action on ET-1 synthesis.

In addition to the inhibitory effect of HGF and EGF on ET-1 synthesis, we also have demonstrated that the new immunosuppressive drug, MPA, applied at concentrations in the range of clinically observed blood levels [28,42], significantly inhibits ET-1 mRNA expression in HUVEC and RAEC. Incubation of HUVEC and RAEC with MPA for 48 h induced a significant decrease of ET-1 release and a parallel decrease of DNA, whereas ET-1 release referred to the DNA content in the corresponding culture well did not differ significantly from controls. This discrepancy between ET-1 mRNA expression and ET-1 release might be explained by the different incubation periods (3–5 h for detection of changes in mRNA expression and 48 h for collection of cell culture supernatants—previous data from our group have shown that steady state levels of ET-1 concentrations in cell culture supernatants are reached after at least 36 h incubation) as well as by a different behaviour of ß-actin and DNA, to which ET-1 mRNA expression and ET-1 release were referred, respectively. The immunosuppressive action of MPA is mediated by inhibition of inosine monophosphate dehydrogenase, a key enzyme in de novo DNA synthesis. Lymphocytes are more dependent on de novo DNA synthesis than other cell types; however, an inhibitory effect of MPA on DNA synthesis in cultured endothelial cells has also been observed in a previous study [43]. The effect of MPA on ET-1 and DNA synthesis in cultured endothelial cells might differ from the in vivo situation, and from in vitro studies it might be difficult to understand the effect of long-term MPA application on in vivo ET-1 synthesis. Fitting with the data in endothelial cells, we also have observed an inhibitory effect of MPA on ET-1 release by cultured rabbit proximal tubule cells (reduction of ET-1 release, referred to the corresponding cell number, to about 85% of controls—data not shown). Thus, in summary our in vitro results suggest that MPA induces a slight to moderate inhibition of ET-1 synthesis or at least does in contrast to CsA and tacrolimus not stimulate ET-1 synthesis. This finding might explain the clinical experience, confirmed by several studies, that MPA in contrast to CsA and tacrolimus does not exert nephrotoxic side effects [23] and that switching immunosuppressive therapy from these substances to MPA or addition of MPA and reduction of CsA or tacrolimus is often accompanied by an improvement of renal function [2428].

In summary, this study demonstrates that HGF and EGF inhibit ET-1 synthesis in human endothelial cells via their receptor tyrosine kinase activity. These data suggest that the renoprotective effects of HGF and EGF might be linked to their inhibitory action on ET-1 synthesis and might encourage further studies to evaluate the therapeutic potential of HGF and EGF in ischaemic and drug-induced renal injury. The present study also provides evidence that MPA, in contrast to CsA and tacrolimus, does not stimulate ET-1 synthesis. This might explain the clinical observation of an improved renal function when CsA or tacrolimus is replaced by MMF.



   Acknowledgments
 
We wish to thank Gisela Sailer, Martina Adam-Jäger and Ingrid Kränzle for expert technical assistance.



   Notes
 
Correspondence and offprint requests to: Dr Cornelia Haug, Institute of Clinical Chemistry, University Hospital Ulm, Robert-Koch-Strasse 8, D-89070 Ulm, Germany. Email: cornelia.haug{at}medizin.uni\|[hyphen]\|ulm.de Back



   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 10. 1.01
Revision received 11. 7.01.