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NDT Advance Access published online on May 5, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn235
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Parietal epithelia cells in the urine as a marker of disease activity in glomerular diseases

Johannes Achenbach1, Michael Mengel2, Irini Tossidou1, Imke Peters1, Joon-Keun Park1, Marion Haubitz1, Jochen H. Ehrich3, Hermann Haller1 and Mario Schiffer1

1 Division of Nephrology, Department of Medicine, Hannover Medical School, Carl Neuberg Str.1, 30625 Hannover, Germany 2 Department of Pathology, Hannover Medical School, Carl Neuberg Str.1, 30625 Hannover, Germany 3 Division of Pediatric Nephrology, Hannover Medical School, Carl Neuberg Str.1, 30625 Hannover, Germany

Correspondence and offprint requests to: Mario Schiffer, Division of Nephrology, Department of Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel: +49-05115324708; Fax:  +49-0511552366; E-mail: schiffer.mario{at}mh-hannover.de



   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Discussion
 References
 
Background. The detection of viable podocytes in the urine of patients with proteinuric diseases has been described as a non-invasive method to monitor disease activity. Most of the published studies use podocalyxin (PDX) as a podocyte specific marker.

Methods. We examined the excretion of viable PDX-positive cells in a random set of spot urine from patients with biopsy-proven focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MGN) or membranoproliferative glomerulonephritis (MPGN) and characterized the excreted cells for podocyte and parietal epithelia markers as well as for proliferation activity.

Results. We found that untreated patients with active disease excrete high numbers of PDX-positive cells in their urine. In contrast to that we were not able to detect significant amounts of PDX-positive cells in the urine of patients with active minimal change disease (MCD) and patients with FSGS or MGN in full remission. When we further characterized the cells we rarely detected expression of podocyte specific markers in the PDX-positive cells, but at least 50% of the PDX-positive cells were double positive for cytokeratin (CK8–18). Immunohistochemistry of the corresponding renal biopsies showed that 100% of podocytes and parietal cells stained positive for PDX. Semiquantitative analysis revealed that 45% of parietal cells were positive for CK8–18 and 100% of proximal tubular cells. No cells of the glomerular epithelial layer stained positive for CK8–18.

Conclusions. PDX-positive cells are lost in the urine in disease states that require podocyte regeneration and are a useful non-invasive marker for glomerular disease activity. These cells are possibly derived from the parietal epithelial layer.

Keywords: FSGS; parietal epithelial cells; podocytes; urine



   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Discussion
 References
 
Damage and loss of podocytes and denudation of the glomerular basement membrane followed by proteinuria are the initial steps in the development of glomerulosclerosis [1]. The denuded basement membrane adheres to the Bowman's capsule and the continued glomerular filtration results in misfiltration into the interstitium and sets the start point for inflammatory and profibrotic responses [2]. Detachment of viable podocytes and loss into the urine has been described previously in rodent models as well as human glomerular disease [3–8]. Since podocytes are considered unable to proliferate after birth, their regenerative potential is limited. Therefore excretion of dead or viable podocytes into the urine could be a useful non-invasive marker for glomerular disease activity. Feasibility of this approach has been shown by us and other groups in various human disease states [7,9,10]. So far, little attention has been given to the parietal epithelia cells (PECs). Podocytes and PECs are both derived from the metanephric mesenchyme and they share a common phenotype until the S-shaped body stage [11]. At that stage they both express WT-1. In the capillary loop stage, podocytes lose their cytokeratin expression and begin to express vimentin, whereas PECs continue their cytokeratin expression and lose their WT-1 expression [12]. Their common embryonic origin and sharing of protein markers complicate their clear-cut identification in glomerular disease stages.

Urine analysis is a fundamental examination for differential diagnosis of glomerular disease states [13]. The presence of gross proteinuria, red blood cells or acanthocytes and casts indicates the presence of glomerular injury. However, it would be helpful to have a reliable test for the presence of specific cells of the glomerular origin, i.e. podocytes and other glomerular cells, since they would be a direct readout of ongoing tissue damage. The technical approaches in the literature vary from urine cytospin preparations to cultivation of viable cells. We experienced technical difficulties with immunofluorescence stainings of whole cytospin preparations [5,8]. Since the urine sediment can be filled with red blood cells or casts that are fixed and stained after regular cytospin procedures, their presence can lead to a strong background signal. When we switched to overnight cultures we obtained much better results. The aim of our study was to establish reliable staining procedure for cells of glomerular origin. We wanted to validate our staining system on a random set of untreated and treated patients with glomerular disease and healthy controls to test whether detection of glomerular cells is a useful non-invasive marker in the differential diagnosis of glomerular disease. Finally we wanted to further characterize the heritage of the glomerular cells to see if excretion of different cell populations is characteristic for a disease entity.



   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Discussion
 References
 
Sample processing/cell culture
Spot urine samples (10–100 ml) of patients as well as healthy controls were collected and processed within 3 h. Prior to processing, all samples were stored at room temperature. Processing steps were carried out as follows: after centrifugation at 1200 rpm for 8 min, the supernatant was removed and the pellet was resuspended in a sterile HDF solution (137 mM NaCl, 5 mM KCl, 5.5 mM glucose, 4 mM NaHCO3 and 0.2% EDTA). After additional centrifugation at 1200 rpm for 8 min, the pellet was resuspended in a DMEM/F-12 medium containing 10% FCS supplemented with antibiotics [0.5 U/l penicillin, 0.5 mg/dl streptomycin, 1 mg/ml kanamycin (GIBCO/ Invitrogen, Karlsruhe, Germany)] and seeded in a 24-well dish containing collagen I coated cover slides. For coating, glass slides were covered with coating solution [20 mM sodium acetate, collagen type I, rat tail (4.5 mg/ml) (BD Bioscience, Erembodegem, Belgium]. Samples were incubated at 37°C with 5% CO2 overnight. The following day, slides were gently rinsed and washed with PBS removing excess media, non-adherent cells and cell detritus. Samples were then fixed at –20°C for 10 min using ice-cold methanol and stored at 4°C in PBS.

Immunocytochemistry
After fixation slides were processed following a standard protocol for indirect immunofluorescence labelling. Incubation with primary antibodies was carried out in a humidity chamber overnight at 4°C. The following day slides were incubated with secondary antibodies in a humidity chamber for 1 h at room temperature. Finally, slides were mounted on glass slides using VectaShield with DAPI (Vector laboratories, Burlingame, CA, USA). Primary antibodies used were goat anti-human podocalyxin (PDX) (R&D Systems, Minneapolis, MN, USA), mouse anti-human PDX (gift from Dontscho Kerjaschki, Medical University of Vienna), mouse anti-human CK8–18 (Neomarker, CA, USA), rabbit anti-WT-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human podocin (Alpha Diagnostic International, San Antonio, TX, USA) and mouse anti-synaptopodin (Progen Biotechnik, Heidelberg, Germany). Secondary antibodies of donkey anti-mouse, FITC labelled, and donkey anti-goat, Cy3 labelled (Jackson ImmunoResearch, Suffolk, UK), were used.

Quantification
The total number of cells was obtained by the number of DAPI-positive nuclei. PDX-positive cells with a DAPI-positive nucleus and CK8–18 double stainings were quantified separately to obtain the number of double positive cells and cells solely positive for PDX or CK8–18. All quantifications were carried out by two independent investigators blinded to the study. The average cell count was determined on two slides. The results were normalized to the sample volume (ml) and the urine creatinine content (mg/ml) of the sample. As a result we obtained the number of positive cells per mg urine creatinine. The percentage of PDX-positive cells was determined as well as the percentage of cells positive for CK8–18 and PDX of the entire cell number detected and the PDX positive fraction.

Classification of disease activity
In cooperation with the university's department of clinical chemistry albumin and creatinine content of each sample was measured to obtain the urine albumine–creatinine ratio (UAC) (mg/g). The UAC ratio of spot urine samples strongly correlates with the 24-h urine protein excretion. Activity of disease was defined by the UAC ratio as follows. UAC >3000 was defined as the fully active form of the diseases, a UAC of 300–3000 and 50% of proteinuria decrease after treatment was defined as partial remission and a UAC of <300 was defined as complete remission. For the FSGS cases, no differentiation was made between primary and secondary FSGS.



   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Discussion
 References
 
Viable podocalyxin positive cells exhibit primary and secondary processes
When we began to establish our staining system we used patient cases with nephrotic range proteinuria. For each sample 50 ml of fresh spot urine was processed within 3 h after the sample was obtained. Until processing the urine was kept at room temperature. Since the quality of cytospin samples did not convince us in most of the cases, we decided to use overnight cultures and introduce a series of washing steps that would clear casts, cellular detritus and red blood cells. After overnight culture at 37°C in a culture medium, the sediments were washed with PBS and then fixed with methanol (see the Materials and methods section). Therefore we had already introduced a selection step at this early point, concentrating only on viable cells that attached to cover slides, whereas dead or apoptotic cells would be lost with the washing steps. Then we decided first to stain for PDX expression since most published work uses this plasma membrane protein as a podocyte marker, and published work suggests this marker as well preserved in most glomerular diseases [14]. After that, we counterstained with DAPI and quantified the number of DAPI-positive nuclei as the total cell number. In a second counting step we quantified the PDX-positive cells with a DAPI-positive nucleus. Interestingly ~40–50% of the cells on the slides were DAPI positive but PDX negative. These cells were considered ‘of other origin’ and served as an internal negative control for specificity of PDX-staining on each slide. The cells that stained positive for PDX either exhibited a fan-shaped morphology (Figure 1A, panel a) with a few primary processes, or showed primary and secondary foot processes (Figure 1A, panel b), but the majority of cells had a rounded shape without primary or secondary processes (Figure 1A, panel c). These different cellular morphologies could be present side by side, which might already indicate a different degree of differentiation or the presence of different cell populations.


Figure 1
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  		Fig. 1 (A) Morphology of podocalyxin positive cells cultured from urine sediments of nephrotic patients. Three different shapes of podocalyxin positive cells were observed after overnight culture. Cells had either long thin cytoplasmic processes (white arrowheads, panel a), or cells with primary and secondary processes (panel b) or exhibited a rounded cobblestone shaped pattern with lamellopodia (white arrowheads, panel c). All forms can be visible in parallel in a single patient sediment. (B) Number of podocalyxin positive cells (black bars) and albumin/creatinine ratio (white bars) from the sediment and spot urine of healthy controls (n = 10), patients with active FSGS (n = 11), FSGS in partial remission (n = 19), FSGS in full remission (n = 6), active MCD (n = 6), MCD in partial remission (n = 6), MCD in full remission (n = 7), active MGN (n = 12), MGN in partial remission (n = 10), MGN in full remission (n = 3), active MPGN (n = 5) and MPGN in partial remission (n = 2). **P < 0.0001 FSGS active versus partial remission; *P = 0.0017 MGN active versus partial remission; {dagger}P = 0.04 MPGN active versus partial remission. (C) Correlation of podocalyxin positive cells with the total number of cells quantified by DAPI in cell containing samples pooled (All) (r2 = 0.875), patients with FSGS (r2 = 0.8992), MGN (r2 = 0.9958) and MPGN (r2 = 0.5269).

 
The number of podocalyxin positive cells correlates with disease activity in patients with active FSGS, MGN and MPGN
Next we examined a random set of spot-urine samples of patients with focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), membranous nephropathy (MGN) and membranoproliferative glomerulonephritis (MPGN) (see Table 1 for clinical profile of patients). The number of PDX-positive cells was quantified by an observer blinded to the patients’ diagnosis. The number of cells was normalized to the total volume of sample and set relative to the urine creatinine measured in the supernatant. We found that in the urine of patients with active FSGS (UAC ratio 9654) on average 64.28 PDX-positive cells per mg urine creatinine were detectable (Table 1). In patients with FSGS in partial remission (UAC ratio 1117.8) we found only 0.3 PDX-positive cells per mg creatinine. FSGS patients after successful therapy in full remission (UAC ratio 123.2) excreted no PDX-positive cells in their urine. Strikingly, patients with active MCD (UAC ratio 13101.3) did not excrete PDX-positive cells in their urine. We also examined other nephrotic patients with membranous and membranoproliferative glomerulonephritis and we were surprised to find a high excretion of PDX-positive cells in both diseases in the active state. We detected on average 46.3 PDX-positive cells per mg urine creatinine in the urine of patients with active MGN (UAC ratio 6481) and 91.5 PDX-positive cells in the urine of patients with active MPGN (UAC ratio 5190.2). Similar to our observations in FSGS patients we found lower numbers in patients in partial remission and no excretion of cells if patients were in full remission (Figure 1B). When we correlated the number of PDX-positive cells with the total number of cells, we detected a highly significant correlation when we pooled all data from cell containing samples (r2 = 0.875). In addition, when we subdivided in the different disease groups the number of PDX-positive cells correlated with the number of DAPI positive cells (FSGS: r2 = 0.8992 and MGN: r2 = 0.9958). Only for MPGN the r2 of 0.5269 cannot be considered significant, which might be related to the low case number. These data indicate that the total amount of cells simply quantified by DAPI staining could be used as a read-out of disease activity as well.


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  		Table 1 Clinical comparison of patients with the average and range of age, serum creatinine (S-creatinine), urinary albumine–creatinine ratio (UAC-ratio) and number of podocalyxin (PDX)-positive cells stained in an overnight culture normalized for urinary creatinine (U-creatinine) in the corresponding spot urine sample

 
Podocalyxin positive cells exhibit markers of cell proliferation and are double positive for parietal cell markers
From the published work of others one would imply that the PDX-positive cells are podocytes. However, when we double stained for podocyte specific markers like WT-1, synaptopodin, nephrin or GLEPP-1 we were unable to detect specific staining with various antibodies and under varying conditions. Only podocin staining was of comparable quality in some samples but not in all (data not shown). As a positive control we used cultured podocytes to exclude technical issues as explanation for negative staining. Since this variety of podocyte specific markers did not confirm that the PDX-positive cells are podocytes, we decided to characterize the cultured cells in more detail. In some samples we found PDX-positive cells that were double positive for the proliferation marker Ki-67 (Figure 2A, panels a–d). However, in the examined samples we could not detect any regularity of cell proliferation with regards to the patients’ diagnosis. Proliferating Ki-67 positive cell populations could be detected in all disease groups. Interestingly, we detected many cells that were double positive for cytokeratin (CK8–18) (Figure 2A, panels e–h) and the parietal cell marker PGP9.5 (Figure 2A, panels i–l). Since we found PDX and CK8–18 positive cells in all samples, we performed double stainings and quantified the sediments of five patients with active FSGS (Figure 2B) and five patients with active membranous glomerulonephritis (Figure 2C) for PDX and CK8–18. In both diseases 50–60% of attached cells stained positive for PDX. Approximately 30% of the cells in FSGS and 40% in MGN stained positive for CK8–18. Interestingly, almost all CK8–18 positive cells in both disease groups were double positive for PDX. Only a minority of the cells was positive for CK8–18 only. These data indicate that a high percentage of PDX-positive cells that we detected in the urine samples were not of visceral epithelial origin.


Figure 2
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  		Fig. 2 Podocalyxin positive cells exhibit markers of proliferation and are double positive for parietal cell markers. (A) Cultured sediments of an FSGS patient were stained for podocalyxin (panels b, f, j) double labelled for Ki-67 (panels a–d), CK8–18 (panels e–h) and PGP 9.5 (panels i–l). White open arrows depict nuclear Ki-67 and podocalyxin double positive cells (panels a–d), the green arrow depicts CK8–18 single positive cell (panel e, merged in h) and the red arrow depicts podocalyxin only positive cell (panel f, merged in h). (B) Quantification of podocalyxin and CK8–18 positive cells in urine sediments of five patients with active FSGS. About 60% of the cultured cells are positive for podocalyxin. Thirty percent are positive for CK8–18. Almost all of the CK8–18 positive cells are positive for podocalyxin. About 30% of cells were podocalyxin only positive; <1% were CK8–18 only positive. (C) Quantification of podocalyxin and CK8–18 positive cells in urine sediments of five patients with active membranous glomerulonephritis. On average 50% of the cultured cells are positive for podocalyxin. Fourty-five percent are positive for CK8–18. Almost all of the CK8–18 positive cells are positive for podocalyxin. About 15% of cells were podocalyxin only positive, <10% were CK8–18 only positive.

 
Podocalyxin is expressed in parietal cells in kidney biopsies
Since we wanted to clarify the origin of the double positive cells in our urine cultures, we stained the corresponding biopsies of patients for PDX and CK8–18 (Figure 3). When we examined patients with FSGS (panels a–c), MGN (panels d–f) and MPGN (panels g–i) we found that in all examined patients PDX stained positive in all cells of the visceral side of the glomerulus, as well as all cells on the parietal side (Figure 3, panels b, e and h). As expected, all tubular epithelial cells express CK8–18. But we also detected positive CK8–18 staining in up to 40% of the PECs. We detected positive staining in cells close to the urinary pole as well as in cells distant from the urinary pole that appeared flat and parietal cell-like. In some cases we even saw CK8–18 positivity around the whole circumference of Bowmann's capsule (data not shown).


Figure 3
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  		Fig. 3 PAS, podocalyxin and CK8–18 staining in corresponding kidney biopsies from a patient with focal-segmental glomerulosclerosis (FSGS) (a–c), membranous glomerulonephritis (MGN) (d–f) and membranoproliferative glomerulonephritis (MPGN) (g–i). (a) FSGS with segmental attached sclerosed capillary loops (PAS stain, 400x magnification). (d) Membranous glomerulonephritis with granular to linear IgG deposits (insert) in the thickened glomerular basement membranes (PAS stain and immunohistochemistry, 400x magnification). (g) Membranoproliferative glomerulonephritis with lobular accentuation of the glomerular tuft and C3 deposits (insert) in peripheral glomerular basement membranes (PAS) stain and immunohistochemistry, 400x magnification). (panels b, e and h) Immunohistochemistry for podocalyxin with expression in visceral podocytes and parietal cells (400x magnification). (panels c, f and i) Immunohistochemistry for cytokeratin 8/18 with expression in some parietal cells (400x magnification).

 


   Discussion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
References
 
Previous work by us and others has demonstrated that podocyte damage, apoptosis and podocyte loss are critical steps in the development of glomerulosclerosis [15–18]. The reason for podocyte damage can be a genetic defect leading to misexpression or loss of podocyte specific proteins, activation of TGF–β and/or a changed expression of extracellular or intracellular signalling modifiers [16–19]. As a result podocytes may dedifferentiate, detach and/or undergo apoptosis. As a logical consequence, the detached cells should be detectable in the urine. Many groups have tried to quantify excreted podocytes with various technical approaches in human disease. Hara et al. report excretion of podocytes detected with a PDX immunofluorescence technique on cytospins [5–7,14]. They report higher levels of PDX-positive cells in FSGS compared to MCD and MGN as well as in IgA and Henoch Schönlein Purpura. Vogelmann et al. describe excretion of viable PDX-positive cells in patients with active FSGS and active lupus nephritis [8]. Their results are similar but all related to the specificity of PDX as a podocyte specific marker. We can partially confirm their results; however, with our method we cannot detect markers of mature differentiated podocytes (e.g. synaptopodin, WT-1 or nephrin) in the majority of the PDX-positive cells. Instead we detected by double immunofluorescence PGP9.5 a marker of PECs and CK8–18, a cytokeratin, that is regularly not expressed on mature podocytes. When we stained the corresponding biopsies of patients for these markers, we found that the majority of the detected cells originate from the parietal side of the glomerulus rather than the visceral epithelium. Nevertheless we find a strong relationship between the number of excreted PDX-positive cells and disease activity in patients with FSGS, MGN and MPGN. Strikingly, we never detected PDX-positive cells in the urine of patients with active MCD. These results would indicate that PDX-positive cells might be a good non-invasive marker for disease activity in FSGS, MGN and MPGN and could be used for monitoring of treatment success in these patients. This is especially of interest since detachment of the cells might be a more specific marker of ongoing glomerular damage than proteinuria, as proteinuria can also be the expression of residual scarring processes rather than active disease. A similar finding was described by Yu and co-workers in three different rodent models of glomerular disease [3]. They demonstrated persistent proteinuria despite remission of podocyturia. Since the rate of PDX-positive cell excretion reflects disease activity, but not the disease type, monitoring of these cells cannot replace a biopsy; however, it might be a very helpful tool for the differentiation of MCD and FSGS. Excretion of these cells certainly reflects better on both organs and all glomeruli, whereas a biopsy might miss early stages of a focal disease. Interestingly, we can obtain similar results if we just quantify the total amount of cells that adhere in our urine cultures overnight, since the total amount of intact cells correlates highly with the amount of PDX-positive cells. PDX was initially described as a podocyte-associated membrane protein. Later it was also detected on endothelial cells [20–23]. Our results suggest that PECs express PDX as well. We used two different antibodies: a polyclonal antibody (R&D Systems) and a monoclonal antibody (gift from Dontscho Kerjaschki). Both antibodies resulted in positive staining of PECs on kidney biopsies in a concentration dependent effect: after serial dilutions with both antibodies, we can only detect PDX staining on podocytes whereas endothelial cells and PECs are negative (data not shown). Therefore PDX staining of PECs can be easily missed when the research focus is on podocytes. Interestingly Bariety et al. describe ‘parietal podocytes’ in normal kidney samples [24]. They found cells expressing podocyte specific proteins lining Bowman's capsule and demonstrated PDX staining along the circumference of Bowman's capsule. The other cell population we detected is the PDX-positive cells in the urine that do not co-express cytokeratin. These cells cannot be attributed to a specific cell type. They might originate from either the visceral or the parietal side, and thus these cells might have been podocytes. PDX staining seems to persist in various disease states, whereas other markers have been described to get lost with dedifferentiation [14,25]. Podocytes might undergo phenotypic changes before, during or after detachment from the glomerular basement membrane [26–28]. This might be the same for other glomerular cells that are shed into the urine.

Therefore the identification of glomerular cells detected in the urine sediment will always carry the risk of describing artificially expressed marker proteins. In the present study we present evidence that PDX-positive cells are excreted in active glomerular diseases. We demonstrate that a significant amount of cells originate from the parietal epithelial cell layer, indicating that in active glomerular disease parietal epithelial cells might react with proliferation and shedding. The number of cells reflects the disease activity; however, it is not a tool to differentiate between various glomerular diseases. Nevertheless, in paediatric patients these cells might help with decision making of taking a biopsy since we could not detect the cells in active MCD. Further studies are on the way to validate our data in other glomerular diseases and in prospective studies.

Since PECs are found in high amounts in the urine in active disease states, this could be interpreted as reaction secondary to ongoing tissue damage. Therefore our data provide evidence for a significant crosstalk of glomerular cells in active disease. The question arises why PECs shed in the urine in high amounts and why this correlates with disease activity. There is recent evidence that PECs can transdifferentiate to podocytes and repopulate the glomerulus in animal models (personnel communication Marcus Moeller, Aachen). In addition there is evidence that a subset of PECs coexpresses stem cell markers CD24 and CD133 as well as the stem cell specific transcription factors Oct-4 and BmI-1 [29]. It is an intriguing hypothesis that the PDX-positive cells that we are detecting in the urine are not a read-out of ongoing cell damage, but instead are a read-out of ongoing regeneration. However a more detailed characterization of the urinary cells is necessary to prove that they truly originate from the parietal epithelial cell layer. With our overnight cultures we might have selected out this specific subfraction of cells, whereas the detached and dying podocytes are not detected using our method. We are in the process of analyzing expression of stem cell markers in our cell population and characterizing the transdifferentiation potential of the PDX-positive cells isolated from patient urine in vitro. Further analysis and understanding of this ‘parietal epithelial shedding’ in the urine might help to understand disease progression in glomerular disease.



   Acknowledgments
 
We would like to thank Heike Lührs for excellent technical assistance. This work was supported by grant support from the German Research Council (Emmy Noether Fellowship Grant Schi 587/2) to M.S.

Conflict of interest statement. None declared. The results presented in this paper have not been published previously in whole or part, except in abstract format.



   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received for publication: 6. 1.08
Accepted in revised form: 7. 4.08


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