1 Departments of Internal Medicine and 2 Anatomy and Cell Biology III, Ruperto Carola University Heidelberg, Heidelberg, Germany
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Abstract |
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Methods. To address this hypothesis, we first investigated (by RT-PCR) the expression of GDNF mRNA and the mRNAs of the GDNF receptors Ret and GFR-1 in (i) adult human renal cortex and medulla and (ii) in HMC in culture. The results were compared to the expression of these molecules in different developmental stages of the rat kidney. We found that both GDNF and its receptors were expressed in human adult kidney and HMC. Since this finding implicates a role for GDNF beyond nephrogenesis, i.e. in renal physiology/pathophysiology, we investigated the effect of GDNF on HMC growth, i.e. (i) cellular protein synthesis as an index of hypertrophy ([3H]methionine incorporation), (ii) DNA synthesis ([3H]thymidine incorporation) and cell proliferation (cell numbers) as indices of hyperplasia, and (iii) extracellular matrix synthesis, i.e. collagenous and non-collagenous extracellular proteins ([3H]proline incorporation into the collagenase-sensitive and -insensitive fraction). HMC cultures were used as a surrogate model for the development of glomerulosclerosis.
Results. GDNF induced a biphasic growth stimulatory effect in HMC with stimulation at the lowest concentration used (2 ng/ml) but had no effect at higher concentrations (20 and 50 ng/ml). In contrast, cellular protein synthesis and extracellular matrix synthesis were significantly and dose-dependently increased by GDNF.
Conclusions. These results suggest that GDNF, similar to other members of the TGF-ß superfamily, might play a role as a growth factor for mesangial cells and might thus be a player in the pathogenesis of glomerulosclerosis.
Keywords: glial cell line-derived neurotrophic factor; glomerulosclerosis; kidney; mesangial cell proliferation
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Introduction |
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Of interest, Suter-Crazzolara and Unsicker [10] reported that the kidney is the organ with the highest expression of GDNF mRNA in the newborn rat. This points to an important role of GDNF in nephrogenesis. Indeed, agenesis of the kidney has been noted in mice lacking GDNF [1113]. Mice with a disrupted Ret gene reveal a strikingly similar phenotype [14]. Mutations in the Ret coding sequence are the cause of a number of diseases (such as distinct forms of thyroid carcinomas and multiple endocrine neoplasias as well as Hirschsprung's disease (for review see [15]). The role of GDNF in these diseases is still enigmatic. Besides GDNF, another molecule of the TGF-ß superfamily, which is also highly expressed in the kidney [16], has also been shown to play an important role in nephrogenesis, i.e. bone-morphogenetic protein-7 (BMP-7). As a result of failure of differentiation of metanephric mesenchymal cells, glomeruli are virtually absent in mice with targeted deletions of BMP-7 [17,18]. Whether members of the TGF-ß superfamily such as BMP-7 or GDNF are of importance in renal physiology and pathophysiology beyond development remains unclear at present. The latter is, however, implicated by the recent finding of increased plasma GDNF levels in patients with chronic renal failure [19].
TGF-ß1 has been suggested to play an important role in the pathogenesis of excess matrix deposition in several human and experimental glomerular diseases [2023]. Furthermore several growth factors that are important during ontogenesis largely disappear in the adult kidney and are re-expressed in kidney diseases. Therefore we hypothesized that GDNF may play a role in the genesis of glomerular diseases as well. We investigated (i) whether GDNF mRNA and the mRNA of the two GDNF receptors Ret and GFR-1 are expressed in apparently normal adult human kidney (medulla/cortex) and in adult human mesangial cell (HMC) cultures as a surrogate model for the development of glomerulosclerosis. These findings were compared to different developmental stages in the rat. We then investigated (ii) whether GDNF affected HMC growth, i.e. hyperplasia, hypertrophy, and extracellular matrix synthesis.
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Materials and methods |
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Isolation of glomeruli and adult human mesangial cell (HMC) culture
Isolation of glomeruli and adult human mesangial cell cultures were performed according to standard techniques as described previously [26]. Briefly, HMC were obtained from outgrowth cultures of collagenase-treated glomeruli of macroscopically normal cortex from tumour-nephrectomy specimens. Standard growth medium was RPMI 1640 supplemented with 20% decomplemented FCS, human insulin (0.88 µmol/l), human transferrin (62 nmol/l), 1% BME vitamin solution, penicillin (50 IU/ml), and streptomycin (34 µmol/l). At near confluence trypsinEDTA 10x diluted 1 : 10 with PBS was used to detach cells for subculture. Cells were replated at a ratio of 1 : 4.
Considerable care was taken to verify the phenotype of mesangial cells, as in our previous studies [26,27]. HMC were identified by morphological (phase-contrast and electron microscopy) and immunohistochemical (positive staining for -actin, for organized smooth-muscle myosin and for vimentin) studies. Contamination of mesangial cell cultures with persisting macrophages was excluded by negative staining for macrophage-1 antigen. Epithelial and endothelial cell growth was excluded by negative staining for cytokeratin and factor VIII respectively (for details see [26]).
Mesangial cells between the third and seventh subculture were used for experiments after they had been screened negatively for mycoplasma.
RT-PCR analysis
DNA manipulation was carried out according to Sambrook et al. [28]. Expression levels of GDNF, Ret and GFR-1 were studied with a RT-PCR approach. For this purpose, rat (HanoverWistar) kidneys were taken from pre- and postnatal animals. Where possible, kidneys were dissected carefully into cortex and medulla under binocular microscopes. For analysis of the human kidney, macroscopically normal cortex and medulla from tumour-nephrectomy specimens were obtained. Total RNA was isolated with Trizol according to the manufacturer's guidelines and 1 µg was reverse transcribed in a volume of 20 µl with 50 ng random hexamer and MMLV-RT according to the manufacturer's guidelines. cDNA was amplified by PCR with the primer pairs (3' and 5') indicated in Figure 1
. Primers were selected so that they can amplify human as well as rat cDNAs. Since mRNA encoding the S6 ribosomal protein is very abundant, this cDNA was diluted 100-fold prior to amplification. All other cDNAs were amplified with undiluted cDNA. PCR was carried out with 2 µ1 cDNA, 2 µmol/l primers (Figure 1
), 100 µmol/l dNTPs, 1xTaq polymerase buffer, and 1.5 mmol/l MgCl2 in a final volume of 39.5 µ1. DNA was denatured at 93°C for 5 min, after which the temperature was lowered to 72°C. Taq polymerase (2.5 U) was added and cDNA was amplified for 24 cycles. We have chosen a relatively low number of cycles to ensure that the amount of cDNA is the rate-limiting factor for the PCR, allowing a semi-quantitative analysis well before a plateau of maximum product is reached [29]. Cycling conditions were: 30 s at 93°C, 30 s at the annealing temperature (Ta) and 30 s at 72°C. All cDNAs were amplified by touchdown RT-PCR: the initial Ta of 72°C was lowered by 0.4°C each cycle. PCR products (10 µl) were separated by agarose gel electrophoresis, visualized with ethidium bromide, and blotted to Porablot NY plus membranes according to the manufacturer's guidelines. By hybridization with an internal oligonucleotide (Figure 1
), we were able to distinguish possible PCR artefacts from specific signals and to increase the accuracy of our semi-quantitative analysis. For this, oligonucleotides were radiolabelled with polynucleotide kinase and used for Southern hybridization according to Sambrook et al. [28]. Membranes were processed and analysed with a Molecular Dynamics PhosphoImager. At least two independent tissue samples per developmental stage were analysed by RT-PCR, of which one is shown in Figure 2
.
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[3H]thymidine incorporation and mesangial cell counts
Measurement of cell proliferation was assessed by [3H]thymidine incorporation (as an index of DNA synthesis) and cell counts. For the measurement of [3H]thymidine incorporation HMC were plated with standard growth medium at a density of 104 cells/cm2 in 96-well culture plates. To render cells quiescent, the medium was changed after 48 h to standard growth medium with a reduced content (2%) of FCS (=2% FCS). Previous studies had shown that the viability of HMC is impaired in serum-free medium with reduction of cell number over time (data not shown, see also [30]). After 48 h in 2% FCS, HMC were incubated under one of the following conditions: (i) 2% FCS with various nominal concentrations (2, 20, 50 ng/ml) of GDNF, or solvent. Experiments were performed with 10-fold replicates per group. Media were changed after 24 h. HMC were incubated with 2 µCi/ml [3H]thymidinc 10 h prior to harvesting. Cells were washed twice with PBS and trypsinized; then harvesting was performed with a semiautomatic cell harvester (Skatron, Lier, Norway). Radioactivity of the samples was measured in a liquid scintillation counter (LS 1801, Beckmann Instruments). [3H]thymidine incorporation was measured after 36 h.
Growth curves were established by repeated cell counts. Therefore HMC were subcultivated at a density of 104 cells/cm2 into 24-well culture plates. After 24 h in standard growth medium, the medium was changed to 2% FCS. After 48 h in 2% FCS, viable cells per well (assessed by trypan blue stain) were counted with counting chambers (time 0). Then the medium was changed to 2% FCS and GDNF was added, as described above. Countings of eight replicates per group were performed after 24, 48, and 72 h. Results are given as cell number/cm2. Media were changed every 24 h.
[3H]methionine and [3H]proline incorporation
Intracellular protein synthesis of HMC was measured by [3H]methionine incorporation. Pretreatment of cells and procedures were as described for the [3H]thymidine assay. Eight hours prior to harvesting 1 µCi/ml [3H]methionine was added to the cells. [3H]methionine incorporation was measured after 24 h.
Extracellular matrix synthesis was assessed by [3H]proline incorporation into the collagenase-sensitive and -insensitive fraction, i.e. collagenous and non-collagenous extracellular protein synthesis. Pretreatment of cells and procedures were as described for the cell counts. Extracellular matrix synthesis was measured after 24 h in eightfold replicates per group. Mesangial cells were incubated with 5 µCi/ml [3H]proline and 50 µg/ml ascorbic acid 24 h prior to precipitation of cell and media proteins with an equal volume of 12% ice-cold trichloroacetic acid (TCA). Cells were scraped from the plates and transferred together with the supernatant into 2-ml polypropylene tubes. The precipitated material was sedimented at 4°C and 1000 g for 10 min. The resulting pellet was washed three times with 6% TCA and then solubilized in 2 ml 0.2 N NaOH. Aliquots from each sample were subjected to liquid scintillation counting. The remainder was adjusted to contain 100 mmol/l NaCl, 50 mmol/l HEPES, and 3 mmol/l CaCl2. pH 7.4. Collagenase (100 U/ml) was then added, followed by incubation for 16 h at room temperature. After collagenase digestion the proteins were again TCA precipitated as described above, washed three times with ice-cold TCA, solubilized in 0.2 N NaOH and subjected to liquid scintillation counting. Collagenase-sensitive [3H]proline incorporation was defined as the difference between TCA precipitable counts before and after collagenase digestion. For all experiments, cell numbers were determined in replicate wells. [3H]methionine and [3H]proline incorporation were corrected for cell number.
Evaluation of potential cytotoxicity of GDNF
Potential cytotoxic effects of GDNF were assessed by using the trypan blue exclusion method (cell counts) and by measuring the release of LDH (Hitachi A705 Automatic Analyzer) into the supernatant.
Statistical analysis
Data are presented as mean±SD. Data were analysed using the one-way analysis of variance (ANOVA) ( post-hoc comparisons between groups with the Bonferroni test). P<0.05 was defined as the level of statistical significance.
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Results |
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GDNF specific RT-PCR may result in two PCR-products (s- and l-GDNF [10]), since in some cells and tissues a smaller mRNA-form (648 in contrast to 720 bases) can be detected. GDNF signals (Figure 3) could be observed in the total RNA preparations of all developmental stages investigated. Highest expression levels are found at embryonic day 18 (E18), after which the signal intensities are reduced. In adult rat kidney only weak PCR signals can be observed. The expression of GDNF mRNA is comparable in cortex and medulla. Strikingly, the abundance of the smaller GDNF mRNA variant increases (as compared to the larger form) during development.
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Our data suggest a role for GDNF mostly in perinatal stages. However, the presence of the Ret receptor in the adult medulla indicates that this receptor may fulfil activities in the adult kidney as well.
Expression of GDNF and its receptors in adult human kidney and HMC
We next addressed the question of whether the mRNAs for GDNF and its receptors are also detected in the human kidney. For this purpose, cortex and medulla of adult human kidney and HMC were investigated. GDNF mRNA can be observed in HMC (Figure 2, lanes 9 and 10), but here only the smaller GDNF mRNA form is present. As can be seen in Figure 3
, this smaller mRNA form can also be detected in human renal cortex and medulla, but at a much lower level. The GDNF receptor mRNAs for Ret and GFR
-1 can be found in the medulla, but in contrast to GFR
-1, signals for Ret are very low in the cortex. Taken together, expression levels of all three mRNAs in human adult kidney are much lower than those found in the developing rat kidney, when compared to the PCR signals for S6 mRNA. Importantly, our data show that GDNF may have effects on HMC, since these cells express the mRNAs for both GDNF receptors.
Cytotoxicity studies
A potential cytotoxic effect of GDNF on HMC could be ruled out. No increase of trypan blue-positive cells and LDH release into the supernatant were noted with GDNF concentrations up to the maximum concentration used in the cytotoxicity study, i.e. 500 ng/ml (data not shown).
Effect of GDNF on DNA synthesis and cell numbers of HMC
GDNF had a biphasic effect on DNA synthesis and cell proliferation, as assessed by [3H]thymidine incorporation and cell numbers respectively (Figures 4 and 5
).
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Effect of GDNF on cellular protein synthesis of HMC
GDNF significantly and dose-dependently stimulated cellular protein synthesis of HMC, as assessed by [3H]methionine incorporation (Figure 6).
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Effect of GDNF on collagen synthesis of HMC
GDNF significantly and dose-dependently stimulated extracellular matrix synthesis of HMC, as assessed by [3H]proline incorporation. This is true both for collagenous and non-collagenous extracellular proteins (Figures 7 and 8
).
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Discussion |
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Our results document that GDNF and its receptors Ret and GFR-1 are expressed during nephrogenesis in the rat, mainly perinatally. Signals can also be detected in the rat adult organ. In the rat adult medulla, the mRNA for Ret can be detected at high levels, whereas relatively little GDNF mRNA can be observed. This may suggest the presence of an additional ligand for Ret in the adult. Indeed, it was shown that neurturin, a TGF-ß-superfamily member closely related to GDNF, also employs Ret for signalling [32]. The rat adult kidney, however, expresses high levels of GFR
-1, a receptor that is essential and exclusive for GDNF signalling [33]. This observation indicates that in the adult rat kidney, GDNF may function as an important signalling molecule in physiological and pathophysiological states.
To date, there is only little information about the distribution of GDNF mRNA and the mRNAs of the two GDNF receptors Ret and GFR-1 in human tissues. This is the first study to document the expression of these molecules in human adult kidney and HMC. GDNF and GFR
-1 mRNAs are found in both renal cortex and medulla. In contrast, Ret mRNA expression is relatively low in renal cortex. All three molecules are expressed in HMC. This is an important finding, since it suggests that GDNF may have effects on these cells.
Several animal and clinical studies indicate that TGF-ßs play an important role in inflammatory and fibrotic diseases, including renal fibrosis [34]. Our study documents that GDNFsimilar to TGF-ß [35]increases the production of extracellular matrix proteins in HMC. Furthermore, GDNF also induced hypertrophy and hyperplasia (hyperplasia only at the lowest concentration used) of HMC under our culture conditions. The mitotic effect of GDNF is, however, only modest as compared to other growth factors like platelet-derived growth factor, angiotensin II, or endothelin-1 [26,36]. When GDNF was added to HMC cultured in serum-free medium, survival of HMC was improved: the cell numbers remained equal over a period of 120 h, whereas the number decreased by 25% in serum-free control cultures. The use of growth medium containing 5 and 10% of FCS did not result in a more pronounced effect of GDNF on the parameters investigated. No co-stimulatory effect was seen with different concentrations of platelet-derived growth factor, angiotensin II, and endothelin-1 (data not shown). To our knowledge, a mitotic effect of GDNF on renal cells has previously only been shown in primary cultures of ureteric bud cells [37].
Whether the GDNF related peptide TGF-ß can induce hyperplasia in mesangial cells is controversial. The majority of authors found a potent antiproliferative effect of TGF-ß in mesangial cells in vitro [3840], but some reported a stimulatory effect [41]. The reason for this discrepancy is probably due to different culture conditions and the use of different concentrations of TGF-ß. The hypertrophic effect of TGF-ß on mesangial cells is, however, undoubted [34,38,42]. Our finding of a more pronounced effect of GDNF on mesangial cell hypertrophy as compared to the modest mitotic effect is somehow similar to the findings reported concerning TGF-ß.
Since the actions of TGF-ß on HMC can be reproduced in vivo by transfection with the TGF-ß gene [43] or in TGF-ß transgenic mice [44], it is tempting to speculate that GDNF might play a role as a growth factor in experimental and human kidney diseases as well. The specific physiological and pathophysiological roles of the TGF-ß superfamily in vivo, however, remain unclear. Furthermore, GDNF and neurturin are the only members of the TGF-ß superfamily that employ a tyrosine kinase receptor. The pathways of TGF-ß and GDNF signalling might therefore differ. This may explain why the effects of GDNF on HMC are not identical to those of TGF-ß. As far as the actions of TGF-ßs are concerned, the cellular response is strongly contextual and appears to be an integrated function of cell type, its state of differentiation and its environmental context, particularly regarding the nature of extracellular matrix and the activities of other cytokines [42].
The present study, however, documents for the first time that GDNF is a growth factor for HMC and may thus be a player in the pathogenesis of glomerulosclerosis. The clinical relevance of our in vitro finding will have to be investigated in renal damage models and biopsy specimens of patients with renal diseases, including in situ hybridization studies. There is no doubt that further studies to clarify the role of GDNF as a potential growth factor in renal disease progression will be rewarding.
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Acknowledgments |
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Notes |
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References |
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