Modulation of angiotensin II-mediated signalling by heparan sulphate glycosaminoglycans
Hannes Köppel1,
Benito A. Yard1,
Michael Christ2,
Martin Wehling2 and
Fokko J. van der Woude1
1V. Medizinische Klinik and 2Institüt für Klinische Pharmakologie, Klinikum Mannheim, University of Heidelberg, Mannheim, Germany
Correspondence and offprint requests to: Dr Hannes Köppel, V. Medizinische Universitätsklinik, Klinikum Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68135 Mannheim, Germany. Email: KoeppelH{at}verw.ma.uni-heidelberg.de
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Abstract
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Background. Heparin and angiotensin-converting enzyme inhibitors can be used as a therapeutic option in diabetic nephropathy (DN). Although the mode of action is poorly understood, both agents may retard the progression of DN. Previously, we demonstrated that angiotensin II (Ang II) has an inhibitory effect on the production of heparan sulphate proteoglycan (HSPG) in mesangial cells (MCs). We have now studied the influence of heparin on the Ang II-induced intracellular Ca2+ release and activation of nuclear factor kappa B (NF-
B).
Methods. Human MCs were isolated from renal cortex and cultivated to measure Ca2+ influx and NF-
B activation.
Results. Stimulation of MCs with 100 nM Ang II resulted in a rapid increase in the intracellular Ca2+ concentration ([Ca2+]i), followed by a decline to baseline level. The addition of heparin resulted in an oscillatory pattern of Ca2+ influxes upon Ang II stimulation. Whereas the rapid increase in [Ca2+]i was most likely due to release from intracellular stores, oscillations in [Ca2+]i were dependent on the presence of extracellular Ca2+. Heparin alone did not induce Ca2+ influx. Both the initial increase and the subsequent oscillations in [Ca2+]i could be blocked by losartan. In MCs with chemically or enzymatically altered membrane-associated heparan sulphate glycosaminoglycan (HS-GAG), Ang II stimulation resulted in [Ca2+]i oscillations. Interestingly, in these cells, the addition of heparin or GAG completely prevented [Ca2+]i oscillations. Heparin inhibited NF-
B activation in Ang II-stimulated MCs that expressed either normal or chemically altered GAG.
Conclusions. These findings suggest that alterations in HS-GAG chemistry or metabolism under pathological conditions, such as DN, may have direct functional consequences for the local effect of Ang II.
Keywords: angiotensin II; Ca2+ signalling; heparan sulphate glycosaminoglycan; heparin; mesangial cells; NF-
B
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Introduction
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Diabetic nephropathy (DN) is characterized by mesangial matrix expansion, thickening of the glomerular basement membrane (GBM) and a concomitant loss of heparan sulphate proteoglycan (HSPG) in the mesangial matrix and the GBM. Hypertension is one of the main features of DN and is present in most, if not all, patients. During the last decade it has become evident that elevated blood pressure is the major determinant in the deterioration of renal function. The prognosis of DN is greatly improved by adequate treatment of hypertension using angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor antagonists.
In addition, evidence that heparin or heparan sulphate may have renoprotective effects in streptozotocin-induced diabetes in rats [1] and patients with overt DN [2] is accumulating. The studies of Gambaro et al. [1] and Oturai et al. [3] demonstrated that long-term administration of low-molecular-weight heparin and dermatan sulphate in diabetic rats prevented GBM thickening, glomerular anionic charge reduction and albuminuria. Treatment with sulphated glycosaminoglycans in patients with DN resulted in a reduction in urinary albumin excretion [2].
It has been thought for a long time that the beneficial effects of ACE inhibition on the development and progression of DN are primarily related to the haemodynamic properties of these compounds. However, both in vitro and in vivo data support the notion that angiotensin II (Ang II) does affect intrinsic properties of the glomerular filtration barrier, independent of its haemodynamic properties [4]. It has been demonstrated that the production of transforming growth factor ß (TGF-ß) in mesangial cells (MCs) is induced by Ang II [5], thereby indirectly stimulating the production of extracellular matrix (ECM) components. The inhibition of Ang II production may thus ameliorate renal disease by reducing renal TGF-ß production. Moreover, Ang II is able to inhibit the production of HSPG in MCs [5]. As heparan sulphate can inhibit MC proliferation [6], the loss of HSPG may then lead to an increased proliferation of MCs. Ang II may also directly stimulate MC proliferation [7]. Therefore, the beneficial effects of ACE inhibition or heparin on the development of DN may also be related to the inhibition of MC proliferation and the inhibition of TGF-ß production.
Little is known about the mechanisms by which heparin influences gene expression. However, several studies have provided evidence that heparan sulphate or heparin may interact with specific transcription factors [8] or interfere with signalling events in a variety of human cell lines. In addition, the action of a variety of growth factors (e.g. fibroblast growth factor and vascular endothelial growth factor) is known to be regulated by binding to heparan sulphate on the cell surface or the ECM, which promotes or restricts interactions with their signal transducing receptors.
Inasmuch as Ang II is able to influence the production of HSPG, nothing is known regarding the influence of HSPG on Ang II signalling. Therefore, we studied the effects of heparin on Ang II signalling in MCs. To this end, the intracellular Ca2+ concentration [Ca2+]i and activation of the Ca2+-dependent nuclear factor kappa B (NF-
B) in Ang II-stimulated MCs were determined.
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Materials and methods
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Cell culture
MCs were cultured from normal renal tissue acquired from multiple sources, including from allografts unsuitable for transplantation and from grossly normal surgical nephrectomy specimens. MCs were isolated using standard methods described previously [9]. In brief, MCs were subcultured from hillocks, usually appearing 3 weeks after outgrowth of the glomeruli, in 24-well plates (Greiner, Frickenhausen, Germany) in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum (Gibco, Karlsruhe, Germany). Single wells were trypsinized and seeded into a T25-culture flask on the basis of cell morphology (multilayer, spindle shaped) and the absence of epithelial cells with cobblestone morphology. Depending on the cell density, cultures were divided and transferred by trypsinization into new T25-culture flasks. MCs were characterized after the third passage by a uniform staining with fluorescein isothiocyanatephalloidin for actin, a positive staining with monoclonal antibodies against vimentin and the absence of staining using monoclonal antibodies against von Willebrand factor, cytokeratin and desmin. A purity of >95% was obtained after the third passage. Prior to each experiment, MCs were growth arrested by serum starvation for 24 h. For reasons of purity and senescence, only MCs in passages 48 were used in the experimental settings. MCs, grown on fibronectin-coated cover slips, were stimulated with Ang II in a concentration of 100 nmol/l in the absence or presence of 1050 µg/ml heparin (Heparin Braun) or heparan sulphate glycosaminoglycan (HS-GAG). In some experiments the cells were cultured for 3 days in the presence of 25 mmol/l D-glucose or 25 mmol/l chlorate (NaClO3) in sulphate-free medium before being stimulated with Ang II and/or heparin as described above. Heparinase and heparitinase treatment was performed by culturing MCs overnight in the presence of 10 U of heparinase and 1 U/ml of heparitinase, respectively.
Measurement of [Ca2+]i
Cells were grown on round, fibronectin-coated glass cover slips for at least 2 days prior to each experiment. The cover slips were then washed twice in physiological saline solution (PSS) buffer (135 mmol/l NaCl, 5 mmol/l KCl, 1.8 mmol/l CaCl2, 0.8 mmol/l MgCl2, 10 mmol/l HEPES and 5.5 mmol/l glucose, pH 7.4) and then the MCs were loaded with 5 µmol/l Fura-2 AM in PSS buffer containing 10% Pluronic for 60 min at 37°C. Then, the cells were washed twice with PSS buffer to remove residual dye. The cover slips were placed in a thermostatically controlled chamber (37°C) containing 450 µl of PSS buffer. Ang II (100 nmol/l) was added alone or together with heparin (1050 µg/ml) in a volume of 50 µl. A Zeiss Axiovert 35 (Zeiss, Hanau, Germany) inverted fluorescence microscope, equipped with a fluor 40/1.30 oil immersion objective and a charge-coupled device imaging camera (General Scanning, Planegg, Germany), was employed to detect fluorescence changes. Dual wavelength excitation at 340 and 380 nm was performed by an imaging system (Till Photonics, Planegg, Germany). The time increment was 3 s at an integration time of 200 ms for the 340 nm and 150 ms for the 380 nm excitation wavelength. Autofluorescence was determined in each experiment by the addition of 5 µmol/l ionomycin and 5 mmol/l MnCl2 to quench intracellular dye. Calibration was performed according the method of Grynkiewicz [10] by use of the ratio for Fura-2 acid in solution at zero Ca2+ (10 mmol/l EGTA) and the ratio at 1.8 mmol/l Ca2+.
Typically, a region of 815 cells was monitored. All administered drugs were tested for autofluorescence, which was insignificant for these conditions of excitation and emission. All readings were checked for baseline stability for at least 90 s. The 340/380 nm ratio was analysed on serial images in regions of interest. Each experiment was performed at least five times with similar results.
Electrophoretic mobility shift assay
Nuclear proteins were isolated by lyses of 106 MCs in 200 µl of buffer A (10 mmol/l HEPESKOH, pH 7.9, at 4°C, 1.5 mmol/l MgCl2, 10 mmol/l KCl, 0.5 mmol/l DTT and 0.2 mmol/l PMSF) for 10 min on ice. The supernatant was discarded and the pellet was resuspended in 50 µl of cold buffer C (10 mmol/l HEPESKOH, pH 7.9, 25% glycerol, 420 mmol/l NaCl, 1.5 mmol/l MgCl2, 0.2 mmol/l EDTA, 0.5 mmol/l DTT and 0.2 mmol/l PMSF), incubated on ice for 20 min and centrifuged at high speed for 2 min at 4°C. The supernatant was quick frozen in liquid nitrogen and stored at 80°C until use. Protein concentrations were determined by Pierce reagent (Pierce, Rockford, IL). Briefly, NF-
B consensus oligonucleotides were labelled to a specific activity of >5 x 107 c.p.m./µg DNA using T4-polynucleotide kinase (Promega, Mannheim, Germany). Binding of NF-
B was performed in 10 mmol/l HEPES, pH 7.5, 0.5 mmol/l EDTA, 100 mmol/l KCl, 2 mmol/l DTT, 2% glycerol, 4% Ficoll 400, 0,25% NP-40, 1 mg/ml bovine serum albumin (DNase free) and 0.1 µg/µl poly dI/dC in a total volume of 20 µl. Labelled oligonucleotide (1 ng) was added to 10 µg of nuclear extract and incubated at room temperature for 20 min in the appropriate binding buffer. ProteinDNA complexes were separated from the free DNA probe by electrophoreses through 5% native polyacrylamide gels containing 2.5% glycerol and 0.5x TBE. The gels were run at room temperature with 30 mA for 2.5 h. Gels were dried on Whatmann D-81 paper (Schleicher und Schüll, Dassel, Germany) and exposed for 1248 h to Amersham Hyperfilms at 80°C.
Statistical analysis
The significance of changes in Ca2+ responses between MCs stimulated with Ang II and those stimulated with Ang II plus heparin was assessed by application of the
2 test. P-values of <0.05 were considered to be significant. Results have been expressed as Mean ± SD.
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Results
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Influence of heparin or HS-GAG on Ang II-induced Ca2+ signalling
The addition of 100 nmol/l Ang II to MCs immediately resulted in a rapid increase of [Ca2+]i, followed by a gradual decline to baseline level (Figure 1A). Decreasing concentrations of Ang II resulted in a decline in peak [Ca2+]i and a slower return to baseline (data not shown). As higher concentrations of Ang II (>100 nmol/l) did not differ from 100 nmol/l with respect to the increase in [Ca2+]i, 100 nmol/l Ang II was used in all further experiments. When MCs were stimulated with a mixture of 100 nmol/l Ang II and 50 µg/ml heparin or HS-GAG (data not shown), the initial increase in [Ca2+]i was followed by oscillations (Figure 1B). Although 10 µg/ml heparin also induced [Ca2+]i oscillations in MCs, when stimulated with Ang II (data not shown), 50 µg/ml heparin was more effective in this respect and therefore used in all experiments. Oscillations in [Ca2+]i were specific for heparin, as dextran sulphate was not able to influence Ang II-induced Ca2+ signalling (Figure 1C). Similar negative findings for chondroitin sulphate were observed (data not shown). Statistical analysis of four independent experiments, in which a total number of 83 cells were analysed (37 stimulated with Ang II alone and 46 stimulated with Ang II plus heparin), revealed a highly significant correlation (P < 0.01) between Ang II stimulation in the presence of heparin and the occurrence of [Ca2+]i oscillations. There was not a significant difference in the initial increase in [Ca2+]i from baseline to peak between Ang II and Ang II plus heparin (113 ± 92
340/380 and 143 ± 63
340/380 nm, respectively) nor in the time to peak (14.4 ± 7.9 and 14.1 ± 10.6 s, respectively). Heparin was required during Ang II stimulation to exert a modulatory effect, as heparin preincubation and subsequent removal prior to Ang II stimulation did not change the Ang II response (data not shown). Both the initial increase and oscillations in [Ca2+]i were exclusively mediated via the Ang II type 1 receptor (AT1R), as 1 nmol/l losartan (Figure 2A and B), but not PD 123319 (Figure 2C), completely blocked Ang II-induced increases in [Ca2+]i. Both the initial increase and subsequent oscillations in [Ca2+]i were significantly inhibited by 1 µmol/l thapsigargin (Figure 2D), and oscillations in [Ca2+]i could be selectively blocked by the addition of 5 µmol/l nifedipin (Figure 2E) or by stimulating in Ca2+-free PSS buffer (Figure 2F). Thus, these data suggest that the initial increase in [Ca2+]i was due to the release of Ca2+ from the endoplasmic reticulum and that the oscillations in [Ca2+]i were dependent on the presence of extracellular Ca2+.

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Fig. 1. Cultured human MCs were stimulated with 100 nmol/l angiotensin II (Ang II) alone (A) or with 50 µg of heparin (B) or dextran sulphate (C) at the indicated times (arrows). The response of a single representative cell is depicted and is expressed as 340/380 nm ratio of Fura-2 AM as described in Materials and methods. In each graph, the response of two cells is depicted. At least four experiments with similar results were performed.
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Fig. 2. Cultured human MCs were stimulated with 100 nmol/l angiotensin II (Ang II) at the indicated times (arrows). When losartan (dotted arrow) was added prior to Ang II stimulation, no increase in [Ca2+]i was observed (A). Oscillations induced by heparin and Ang II stimulation were stopped directly after the addition of losartan (B). The addition of PD 123319 (arrowhead) did not affect the Ca2+ signal (C). To investigate the involvement of extracellular Ca2+ or the involvement of intracellular Ca2+ stores, cells were also incubated for 10 min with 1 µmol/l thapsigargin (D) or 5 µmol/l nifedipin (E) prior to Ang II stimulation (arrows) or they were stimulated in Ca2+-free PSS buffer (F). The response of a single representative cell (n = 8) is depicted and expressed as 340/380 nm ratio of Fura-2 AM as described in Materials and methods. A total number of three experiments with similar results were performed.
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Altered cell-associated HS-GAG expression changes Ang II-induced Ca2+ signalling
Since the expression of HSPG is decreased in DN, we investigated whether an altered HSPG expression in MCs could change Ang II-induced Ca2+ signalling. To this end, the expression of cell-associated HSPG was modulated in three ways. First, MCs were cultured for 3 days in the presence of 25 mmol/l D-glucose, which inhibits both the production of HSPG and its N-linked sulphation [11]. Secondly, MCs were cultured in 25 mmol/l chlorate for 3 days to inhibit sulphation of HS-GAG [12]. Thirdly, MCs were cultured for 24 h in the presence of 1 U/ml heparitinase to remove cell-associated HS-GAG. Whereas in MCs cultured in medium alone a single peak in [Ca2+]i was detected (Figure 3A), oscillations in [Ca2+]i were observed upon Ang II stimulation in MCs that were cultured in the presence of D-glucose (Figure 3B), chlorate (Figure 3C) or heparitinase (Figure 3D). When MCs were cultured in medium alone, oscillations were induced by the combination of Ang II and heparin (Figure 3E). Ang II and heparin were added after culturing the cells in the presence of D-glucose (Figure 3F), chlorate (Figure 3G) or heparitinase (Figure 3H). Ang II-induced oscillations in MCs that were cultured in D-glucose or chlorate were completely prevented by heparin (Figure 3F and G), whereas heparin was without effect when the cells were cultured in the presence of heparitinase (Figure 3H).

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Fig. 3. Angiotensin II (Ang II)-induced Ca2+ signalling in MCs with altered HS-GAG expression. Cells were cultured for 72 h either in normal culture medium (A and E) or in medium supplemented with 25 mmol/l -glucose (B and F) or 25 mmol/l chlorate (C and G). The cells were also treated for 24 h with 1 U/ml of heparitinase (D and H). Thereafter, the cells were stimulated with 100 nmol/l Ang II in the absence (AD) or presence of 50 µg of heparin (EH). The response of a single representative cell is depicted (n = 10) and is expressed as 340/380 nm ratio of Fura-2 AM as described in Materials and methods. For each condition, at least five experiments were performed with similar results.
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Inhibition of NF-
B by heparin
To investigate whether heparin was able to inhibit the nuclear translocation of NF-
B in Ang II-stimulated MCs, the cells were stimulated with Ang II in the absence or presence of 50 µg/ml heparin. In nuclear extracts of MCs, three specific bands with apparent binding activity for NF-
B consensus oligonucleotides were detected, as demonstrated by the disappearance of these bands when the extracts were preincubated with cold competitor oligonucleotides. The upper two bands were increased after Ang II stimulation in MCs that were either cultured under normal conditions or cultured in the presence of chlorate. Both bands consisted of NF-
B p65, as the addition of anti-p65 antibody to the nuclear extract markedly decreased these bands and resulted in a super shift. Heparin completely prevented the increase in Ang II-mediated NF-
B activation, as both p65-containing bands did not increase when MCs were stimulated with Ang II in the presence of heparin. No differences were found between normal MCs and those that were cultured in the presence of chlorate (Figure 4) or those that were cultured in the presence of 25 mmol/l D-glucose (data not shown).
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Discussion
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The results presented here demonstrate that heparin or HS-GAG changes Ang II-induced Ca2+ signalling by inducing [Ca2+]i oscillations. As oscillations could be blocked by the addition of nifedipin, which might also affect the refilling of intracellular Ca2+ stores and did not occure in Ca2+-free PSS buffer, our data demonstrate that these oscillations were at least dependent on the presence of extracellular Ca2+. The requirement of extracellular Ca2+ for such oscillations has also been reported by Harootunian et al. [13] using cultured fibroblasts. Engagement of the AT1R was required for Ca2+ signalling, as losartan completely prevented an increase in [Ca2+]i and heparin alone did not change it.
In DN, the expression of HSPG is altered in both the GBM and mesangial matrix. Therefore, we investigated whether quantitative or qualitative alterations in HSPG expression could influence Ang II-induced Ca2+ signalling. We and others have previously demonstrated that glucose and chlorate alter cell-associated HS-GAG expression in MCs [11] and fibroblasts [12]. Ang II-induced [Ca2+]i oscillations in MCs that were cultured in the presence of 25 mmol/l D-glucose or cultured in the presence of chlorate, suggesting that cell-bound HS-GAG may have a functional relevance in Ang II-induced Ca2+ signalling. In contrast to MCs with unaltered HS-GAG expression, the addition of heparin to these cells completely normalized Ca2+ signalling. These observations may be explained by the requirement of endogenous HS-GAG for normal AT1R activation. If this interaction does not occur, the receptor cannot be properly activated by Ang II, resulting in a change in Ca2+ response. The interaction of HS-GAG with the AT1R may be disturbed by exogenous HS-GAG and desulphation of, or reduction in, endogenous HS-GAG. In the latter situation, the interaction with exogenous HS-GAG may restore a normal Ca2+ signalling. Thus, in our view, oscillations always occur when the interaction of HS-GAG with the AT1R is disrupted. A discrete balance between membrane-associated HS-GAG and HS-GAG in the cell surrounding may therefore determine the pattern of Ca2+ response after Ang II stimulation. In contrast to MCs cultured in the presence of 25 mmol/l D-glucose or 25 mmol/l chlorate, heparin was not able to restore a normal Ca2+ response in heparitinase-treated MCs. It should be mentioned, however, that heparitinase treatment does not completely liberate the core protein from its HS-GAG side chains, but only acts on the N-acetylated or N-sulphated glucosamineglucoronic linkage that has a sulphate group at the 6-position of the glucosamine moiety [14]. Heparitinase treatment would thus result in the generation of HS-GAG chains, varying in length, that are attached to the core protein. We speculate that these HS-GAG stumps may prevent the interaction of exogenous HS-GAG with the AT1R, but may not be sufficient to enable a normal Ca2+ response after Ang II stimulation.
There is evidence for the requirement of cell-bound HS-GAG in the activation of several receptors, including the basic fibroblast growth factor receptor [15]. These receptors cannot be activated in cell lines deficient in HS-GAG unless exogenous heparin is added, suggesting an absolute requirement for HS-GAG in receptor activation. The AT1R differs in this respect, as HS-GAG does not prevent or promote AT1R activation, but merely changes the Ca2+ response that is evoked upon receptor engagement.
Several patterns of Ang II-induced increases in [Ca2+]i have been observed in vascular smooth muscle cells, including a transient rise [16], an initial peak followed by a low sustained plateau or an initial peak accompanied by discrete oscillations [17]. The differences in Ca2+ response within one cell type using similar concentrations of Ang II may be explained by differences in HS-GAG expression among the vascular smooth muscle cells used in various laboratories. Our study clearly suggests that cell-bound HS-GAG plays an important role that may determine the pattern of Ca2+ response displayed upon Ang II stimulation in MCs, although this may be different in vascular smooth muscle cells.
The AT1R is known to undergo agonist induced desensitization. Desensitization of the AT1R occurs independently of receptor internalization [18] and requires the activation of G protein-coupled receptor kinase (GRK). Although GRKs are heparin-sensitive kinases, our data do not support a role for receptor desensitization in [Ca2+]i oscillations. First, to date, receptor desensitization could only be blocked by intracellular injection of heparin. Labelled heparin is taken up by cells and degraded with a half-time of several hours [19]. Thus, this process would be too slow to account for desensitization, as desensitization occurs within minutes. Secondly, if receptor desensitization was blocked by exogenous heparin, this would occur in MCs with both normal and altered HS-GAG expression. Therefore, oscillations in [Ca2+]i should occur independently of the HS-GAG expressed in MCs, which was clearly not found in this study. Thirdly, oscillations also occur in heparitinase-treated MCs in the absence of heparin. Fourthly, heparin did not influence Ca2+ signalling when MCs were stimulated with endothelin 1 (data not shown), which also signals through GRK.
The antimitogenic effects of heparin in MCs may be explained by the inhibition of Ca2+/calmodulin-dependent kinase II activation, the inhibition of c-fos induction or the inhibition of mitogen-activated protein kinase activation [8]. Moreover, oscillations in [Ca2+]i are known to affect the activation of the calmodulin-dependent kinase II and subsequently influence gene expression [20]. In the present study, we have demonstrated that Ang II-mediated NF-
B activation is also inhibited by heparin in MCs cultured in normal medium or medium supplemented with chlorate or glucose. Thus, oscillations per se did not influence NF-
B activation, nor was the inhibitory effect of heparin on NF-
B activation mediated by [Ca2+]i oscillations.
In conclusion, we have demonstrated that AT1R-mediated Ca2+ signalling and NF-
B activation is modulated by HS-GAG. The addition of exogenous heparin to MCs with decreased or desulphated HS-GAG content can restore a normal Ca2+ signalling. These findings suggest that alterations in HS-GAG chemistry or metabolism under pathological conditions, such as DN, may have direct functional consequences for the local effect of Ang II.
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Acknowledgments
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This study was supported by a grant from the Deutsche Forschungsgemeinschaft DFG WO 686/2-1.
Conflict of interest statement. None declared.
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References
|
---|
- Gambaro G, Venturini AP, Noonan DM et al. Treatment with glycosaminoglycan formulation ameliorates experimental diabetic nephropathy. Kidney Int 1994; 46: 797806[ISI][Medline]
- Tamsma TJ, van der Woude FJ, Lemkes HH. Effect of sulphated glycosaminoglycans on albuminuria in patients with overt diabetic (type 1) nephropathy. Nephrol Dial Transplant 1996; 11: 182185[Abstract]
- Oturai PS, Rasch R, Hasselager E et al. Effects of heparin and aminoguanidine on glomerular basement membrane thickening in diabetic rats. APMIS 1996; 104: 259264[ISI][Medline]
- Gansevoort RT, de Zeeuw D, de Jong PE. Dissociation between the course of the hemodynamic and antiproteinuric effects of angiotensin I converting enzyme inhibition. Kidney Int 1993; 44: 579584[ISI][Medline]
- Van Det NF, Tamsma JT, van den Born J et al. Differential effects of angitensin II and transforming growth factor-ß on the production of heparan sulfate proteoglycan by mesangial cells in vitro. J Am Soc Nephrol 1996; 7: 10151023[Abstract]
- Groggel GC, Marinides GN, Hovingh P, Hammond E, Linker A. Inhibition of rat mesangial cell growth by heparan sulfate. Am J Physiol 1990; 258: F259F265[ISI][Medline]
- Ray PE, Aguilera G, Kopp JB, Horikoshi S, Klotman PE. Angiotensin II receptor mediated proliferation of cultured human fetal mesangial cells. Kidney Int 1991; 40: 764771[ISI][Medline]
- Busch SJ, Martin GA, Barnhat RL, Mano M, Cardin AD, Jackson RL. Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription. J Cell Biol 1992; 116: 3142[Abstract]
- Muller EW, Kim Y, Michael AF, Vernier RL, van der Hem GK, van der Woude FJ. Explantation of mesangial cell hillocks: a method for obtaining human mesangial cells in culture. Int J Exp Path 1992; 73: 920[ISI][Medline]
- Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 34403450[Abstract]
- Van Det NF, van den Born J, Tamsma J et al. Effects of high glucose on the production of heparan sulfate proteoglycan by mesangial and glomerular visceral epithelial cells. Kidney Int 1996; 49: 10791089[ISI][Medline]
- Keller KM, Brauer PR, Keller JM. Modulation of sulfate structure by growth of cells in the presence of chlorate. Biochemistry 1989; 28: 81008107[ISI][Medline]
- Harootunian AT, Kao JPY, Paranjape S, Tsien RY. Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3. Science 1991; 251: 7577[ISI][Medline]
- Nader HB, Porcionatto MA, Teraiol IL et al. Purification and substrate specificity of heparitinase I and heparitinase II from flavobacterium heparinum. Analysis of the heparin and heparan sulfate degradation products by 13C NMR. J Biol Chem 1990; 256: 1680716813
- Aviezer D, Levy E, Safran M et al. Differential structural requirements of heparin and heparan sulfate proteoglycans that promote binding of basic fibroblast growth factor to its receptor. J Biol Chem 1994; 269: 114121[Abstract/Free Full Text]
- Carriu C, Andre P, Schott C, Michel M, Stoclet JC. ANG II receptor expression and function during phenotypic modulation of rat aortic smooth muscle cells. Am J Physiol 1994; 266: H631H636[ISI][Medline]
- Johnson EM, Theler JM, Capponi AM, Valloton MB. Characterization of oscillations in cytosolic free Ca2+ concentration and measurement of cytosolic changes evoked by angiotensin II and vasopressin in individual rat aortic smooth muscle cells. J Biol Chem 1991; 266: 1261812626[Abstract/Free Full Text]
- Abdellatif MM, Neubauer FC, Lederer WJ, Rogers TB. Angiotensin induced desensitization of the phosphoinositide pathway in cardiac cells occurs at the level of the receptor. Circ Res 1991; 69: 800809[Abstract]
- Letourneur D, Caleb B, Castellot JJ Jr. Heparin binding, internalization, and metabolism in vascular smooth mucle cells: I. Upregulation of heparin binding correlates with antiproliferative activity. J Cell Physiol 1995; 165: 676686[ISI][Medline]
- Dolmetsch RE, Xu K, Lewis RS. Calcium oscillations increase the efficiency and specificity of gene expression. Nature 1998; 392: 933936[CrossRef][ISI][Medline]
Received for publication: 15.12.02
Accepted in revised form: 23. 5.03