Skip Navigation


NDT Advance Access originally published online on March 2, 2007
Nephrology Dialysis Transplantation 2007 22(6):1558-1566; doi:10.1093/ndt/gfm006
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
22/6/1558    most recent
gfm006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Markovic-Lipkovski, J.
Right arrow Articles by Müller, G. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Markovic-Lipkovski, J.
Right arrow Articles by Müller, G. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Neural cell adhesion molecule expression on renal interstitial cells

Jasmina Markovic-Lipkovski1, Claudia A. Müller2, Gerd Klein2, Thomas Flad2, Tatjana Klatt2, Sabine Blaschke3, Johannes T. Wessels3 and Gerhard A. Müller3

1Institute of Pathology, School of Medicine, University of Belgrade, Serbia, 2Section for Transplantation Immunology and Immunohematology, ZMF, University Medical Clinic, Tübingen and 3Department of Nephrology and Rheumatology, Center of Internal Medicine, Georg-August University, Göttingen, Germany

Correspondence and offprint requests to: Prof. Jasmina Markovic-Lipkovski, MD, PhD, Institute of Pathology, University of Belgrade, 11000 Belgrade, Dr Subotica 1, Serbia. Email: acal{at}matf.bg.ac.yu



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. At early stages of kidney development, the neural cell adhesion molecule (NCAM) is highly expressed on cells of the metanephrogenic mesenchyme. During maturation of the fetal kidney, NCAM gradually disappears. So far, it has been widely accepted that NCAM in the adult kidney is only expressed by nerves, and not by other cell types.

Methods. NCAM expression was analysed in human adult healthy and diseased kidneys by immunohistochemistry and western blot analysis. NCAM+ renal interstitial cells were further characterized by double immunofluorescent staining using antibodies against neurofilaments, {alpha} smooth muscle actin, vimentin, {alpha}5ß1 integrin, CD68, CD11c, HLA-DR and the potential progenitor cell markers CD34, CD117, CD133, CD24, nestin and cadherin-11.

Results. In adult human kidneys, NCAM expression is restricted to rare interstitial cells with dendritic morphology, which are neurofilament-negative and predominantly localized on the corticomedullary junction. They are also negative for fibroblast cell markers, but co-express the haematopoietic stem cell markers CD34 and CD133. The number of NCAM+ interstitial cells increased in the initial phases of interstitial fibrosis. Western blot analysis of renal tissues with incipient interstitial fibrosis tissues showed the expression of the 140 kDa NCAM isoform.

Conclusions. These data indicate that a rare subpopulation of NCAM+ interstitial cells could represent renal progenitors, and that NCAM+ interstitial cells can participate in the initial phase of interstitial fibrosis.

Keywords: neural cell adhesion molecule; renal interstitial cells; renal progenitor cells



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
The neural cell adhesion molecule (NCAM), CD56, comprises a family of cell surface sialoglycoproteins of the immunoglobulin superfamily. There are three major NCAM isoforms: NCAM 180 and NCAM 140 are transmembrane proteins with cytoplasmatic domains, whereas NCAM 120 is covalently attached to the plasma membrane via a glycerophospholipid anchorage [1]. NCAM is involved in calcium-independent homotypic and heterotypic intercellular adhesion by homophilic binding to NCAM on other cells [2] and it is mainly involved in neuron–neuron and nerve–muscle interactions. In addition, NCAM represents a specific marker of neuroectodermal/neuroendocrine differentiation [3]. It is also present on essentially all resting and activated natural killer (NK) cells and some NK-like T cells [4].

During mammalian development, NCAM expression seems to be less restricted. It plays an important role in organogenesis not only of the brain, the muscle, the peripheral neural, neuroectodermal and neuroendocrine tissues, but also of several other organs including the kidney. During embryogenesis NCAM is expressed in the uninduced metanephrogenic mesenchyme at the early stage of kidney development [5]. It is still present on the condensing mesenchyme and the comma- and S-shaped bodies during early tubular development. Later, in mice, during tubular epithelial cell differentiation NCAM gradually disappears and remains on the surrounding mesenchyme up to the perinatal period [6]. However, to date, expression of NCAM has not been detected in healthy adult kidneys, except for nerves and interstitial nerve fibres.

In the present study, the NCAM expression in human renal normal tissues and renal tissues with interstitial fibrosis was investigated. We observed by means of immunohistochemistry for the first time that some rare interstitial cells of normal adult kidneys do express NCAM. Moreover, we also recognized that NCAM+ interstitial cells are increased in areas of incipient interstitial fibrosis. Therefore, the major goals of our present study were to further characterize the NCAM+ cells in the adult human kidney and to try to analyse NCAM+ cells during induction of interstitial fibrosis.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Acknowledgements
 References
 
Renal tissues
Adult human renal tissues were obtained from five healthy organ donors that were not transplanted because of vascular abnormalities, and from seven ‘normal’ tissues adjacent to kidney tumours. Ten renal needle biopsies from patients with different diagnoses (three IgA nephropathy, two focal segmental glomerulosclerosis, one membranous glomerulonephritis, one rapidly progressive glomerulonephritis, one diabetic nephropathy and one amyloidosis), were also analysed. One part of each specimen was processed routinely for light-microscopical evaluation. The other part of the kidney samples was put into cell culture medium (RPMI 1640, Gibco, Eggenstein) immediately after removal, snap frozen and stored in liquid nitrogen. Five micrometre thick frozen sections cut from each tissue were fixed in acetone for 10 min, air-dried at room temperature for 1 h and then used for immunostaining.

In addition, paraffin-embedded tissues of five diseased kidney biopsies (two focal segmental glomerulosclerosis, two IgA nephropathy and one membranous glomerulonephritis) and of human fetal renal tissues at different gestation weeks (from 14 to 27) obtained from medically indicated abortions were immunohistochemically investigated.

Antibodies
For analyses of NCAM expression the monoclonal antibody (mAb) clone ERIC-1 [7] that detects the 120, 140 and 180 kDa isoforms of human NCAM (CD56, Ancell, Bayport) was applied. This antibody was also used for double immunofluorescence staining of NCAM+ cells and immunoblotting. For double immunofluorescence labelling of tissues, a panel of directly labelled monoclonal antibodies and several polyclonal antibodies was applied in addition to ERIC-1. Nerves were differentiated by a rabbit polyclonal antiserum against neurofilament (SAD2186, NatuTec, Frankfurt) and by a rabbit polyclonal anti-S100 antiserum (Ab-2, Calbiochem, Darmstadt). For the detection of renal interstitial fibroblasts/myofibroblasts, anti-vimentin mAb (clone V9, Sigma, Deisenhofen), polyclonal rabbit anti-{alpha}5 integrin antibody (Biotrend, Köln) and anti-{alpha} smooth muscle actin ({alpha}SMA) mAb (clone 1A4, Sigma) were used. Polyclonal rabbit antibody to CD68 (Santa Cruz Biotechnology, Heidelberg) and dendritic cell markers anti-CD11c (clone HL3, BD Pharmingen, Sandiego) and anti HLA-DR (L243, Santa Cruz Biotechnology) were applied also. The progenitor/stem cell markers on NCAM+ interstitial cells were analysed with CD34 mAb (clone AC136, Miltenyi, Bergisch Gladbach), CD133 mAb (clone AC 133, Miltenyi), CD24 mAb (clone 1B5, ImmunoTools, Friesoythe), a mAb against c-kit (CD117, clone 104D2, NatuTec), a rabbit anti-cadherin-11 antiserum (Acris Antibodies, Hiddenhausen) and a rabbit polyclonal anti-nestin (H-85, Santa Cruz Biotechnology).

Immunoperoxidase staining
In the present study immunoperoxidase staining was used on cryostat as well as on paraffin sections in order to correlate immunolabelling with specific antibody with precise histomorphological details.

For investigation of NCAM expression on renal tissues the EnVisionTM staining method (DAKO, Hamburg) was performed on cryosections, followed by counterstaining with hemalaun (Merck, Darmstadt). All experiments included sections stained with undiluted supernatant of the mAb W6/32.HL (anti-HLA-ABC heavy chain) as positive control, and of the mAb W6/32.HK (inactive variant of W6/32.HL) [8] as negative control to exclude non-specific staining. Controls were also performed by omitting the first antibody. Paraffin sections of fetal kidneys and some biopsy specimens were treated by microwave for 3 x 3 min at 400 W in citrate buffer (pH 6.0) after deparaffinization and dehydration and stained by the EnVisionTM method. The slides were evaluated using the Zeiss Axiophot light microscope (Zeiss, Jena).

Immunofluorescence labelling of tissues
For the detection of NCAM+ cells in renal tissue, indirect immunofluorescent staining was also applied on frozen renal tissues. The tissue sections were incubated for 45 min with the mAb ERIC-1 (diluted 1:200) followed by Cy3TM-conjugated goat anti-mouse antibody diluted 1:700 (Dianova, Hamburg) as a second antibody. Controls were performed in all experiments by omitting the first antibody and applying mAb W6/32HK as the first antibody [9]. False positive staining was eliminated by thorough comparison with control slides. Negative staining was easily estimated, since the kidney slides contain structures which are normally stained with corresponding antibodies, e.g. NCAM—nerves, CD34—endothelium, CD133—some tubular epithelium, {alpha}SMA—arterioles, etc.

For further immunomorphological characterization of renal NCAM+ cells, double immunofluorescence staining was performed. After fixation, the slides were first incubated for 1 h at room temperature with the mAb ERIC-1. Subsequently a Cy3TM-conjugated goat anti-mouse antibody diluted 1:700 (Dianova) or a Cy2TM-conjugated goat anti-mouse antibody diluted 1:100 (Rockland, Gilbersville) was applied for 30 min depending on the immunofluorescent label of the second primary antibody available. The second labelling consisted of either a directly conjugated monoclonal antibody or a polyclonal reagent as follows: CD34-FITC (1:100); CD133-PE (1:15); CD24-FITC (1:5); CD45-FITC (1:10); vimentin-Cy3 (1:100); {alpha}SMA-FITC (1:1000); CD11c-FITC (1:100); HLA-DR-FITC (1:200); polyclonal rabbit anti-neurofilament (1:600); anti-S100 (1:100); anti-{alpha}5 integrin (1:10); anti-CD68 (1:100); anti-cadherin-11 (1:100) and anti-nestin (1:50) antibodies. Binding of the polyclonal antibodies was visualized by Cy3TM-labelled goat anti-rabbit IgG (d 1:1000, Dianova). The cell nuclei were identified by counterstaining with 1 µg/ml 4',6-diamidino-2-phenylindolyl-dihydrochloride (DAPI). The staining was visualized by epifluorescence microscopy (Zeiss). Digital pictures from every fluorescence channel were taken and superimposed for the specific antibody staining as well as for each negative control labelling using the software AnalySIS from Soft Imaging Systems (Leinfelden–Echterdingen) as previously published [9].

Immunoblotting
Protein extracts from normal kidney, kidney biopsies with incipient interstitial fibrosis, as well as renal cell carcinoma (RCC) tissue and cell lines as control, were obtained by incubating the cells or tissue specimen for 30 min on ice in extraction buffer containing 1% Triton-X100, 1% NP40, 1 mM CaCl2, 1 mM MgCl2, 150 mM NaCl and 50 mM Tris–HCl pH 7.6. Proteins were separated on 10% polyacrylamide gels and transferred to nitrocellulose filters. Non-specific protein binding sites were blocked with 2% blocking reagent (Roche, Mannheim). The filters were probed for 1 h with primary antibodies against NCAM and subsequently washed for 15 min with PBS. Bound antibodies were detected by alkaline phosphatase-conjugated goat anti-mouse (DAKO), followed by colorimetric reaction with the FastTM BCIP/NBT system (Sigma).



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Acknowledgements
 References
 
NCAM expression in fetal and normal adult kidneys
Immunoperoxidase staining of paraffin-embedded tissue of human fetal kidneys at the early stage of gestation revealed strong NCAM expression on cells of the non-induced and induced mesenchyme, as well as on epithelial cells of the comma and S-shaped bodies (Figure 1A). Later on during fetal kidney development, NCAM expression gradually disappeared from epithelial cells, but persisted on some cellular islands of medullary interstitium (Figure 1B).


Figure 1
View larger version (96K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. NCAM expression in human fetal and adult kidneys. Fetal paraffin-embedded tissues (A, B) and frozen sections of adult kidneys (C–E) were labelled by immunoperoxidase staining, visualized with DAB and counterstained with hemalaun (A–E). (A) In the nephrogenic zone of the embryonic kidney, NCAM expression can be found on almost all interstitial cells (arrowheads), but also on the epithelial S-shaped bodies (arrows). (B) Deeper in the cortex of the embryonic kidney, NCAM expression is restricted to small areas of interstitial cells (arrowheads). (C–E) These three micrographs show the morphology of NCAM+ interstitial cells in adult kidneys. (F–H) Immunofluorescence staining with Cy3-labelled antibody also revealed NCAM+ interstitial cells. (I, J) Double immunofluorescence labelling with antibodies against neurofilaments (Cy3 staining, shown in red) and against NCAM (FITC staining, shown in green) revealed (J) a colocalization of both antigens on nerve fibres (yellow–orange staining, arrow), but no co-localization could be observed on interstitial NCAM+ cells (green, arrowheads). (I) NCAM+ interstitial cell did not show any neurofilament positive staining.

 
Twelve adult human kidneys were investigated for NCAM expression on cryosections by indirect immunoperoxidase staining as well as by indirect immunofluorescent labelling. None of the analysed specimens revealed a staining on glomeruli or on tubuli with the antibody against NCAM. All kidneys showed strong staining signals of nerves and of interstitial nerve fibres. However, on higher magnification by light as well as by fluorescence microscopic investigation, we were able to identify single NCAM+ interstitial cells which were clearly identified by hemalaun or DAPI-stained nuclei as cellular constituents and not as nerves (Figure 1C–E, F–H). These cells had fusiform (Figure 1C and D) or dendritic morphology (Figure 1E), with elongated nuclei (Figure 1G and H) and usually displayed cytoplasmatic protrusions (Figure 1C and F). They were very rare and predominantly located in the interstitium at the corticomedullary junction. The number of these rare cells varied from 0 up to 10 cells per tissue slide and also differed from kidney to kidney as well as from section to section of the same kidney.

Phenotyping of NCAM+ interstitial renal cells
In order to exclude nerves as the exclusively NCAM+ structures in adult kidneys, we performed double immunofluorescent labelling with antibodies against NCAM and neurofilament, including DAPI. Renal interstitium contains single NCAM+ cells which are labelled only with the antibody against NCAM (Figure 1J), in addition to nerves which are labelled with both antibodies (Figure 1I).

The first aim of our study was to identify expression of some dendritic cell markers such as CD11c and HLA-DR on NCAM+ cells. There was no overlapping between these markers and NCAM+ cells.

For further immunomorphological characterization of NCAM+ interstitial cells, the renal tissue was labelled by double immunofluorescent staining with antibodies against marker molecules of hematopoietic stem cells (CD133, CD34, CD117), with antibodies against some potential renal progenitor markers (CD24, cadherin-11 or nestin) or with antibodies that could define renal interstitial fibroblasts/myofibroblasts (vimentin, {alpha}5ß1 integrin, {alpha}SMA).

Strong CD133-PE labelling was found on some tubular epithelial cells (Figure 2B). Interestingly, we could also observe that some, but not all NCAM+ interstitial cells were also positive for CD133, according to yellow–orange stained cells observed in the merged pictures (Figure 2A–C). Using the horizontal intensity profile measurement function of AnalySIS software, we obtained strong peak signals for both the green and the red channel at the level of the measured NCAM+ cell, clearly indicating that this cell co-expressed both antigens (Figure 2A). In the double staining shown in Figure 2B, a NCAM+ nerve could be clearly differentiated from a NCAM+ interstitial cell which co-expresses CD133. An additional subpopulation of NCAM+ renal interstitial cells was also observed, which did not express CD133 simultaneously (data not shown).


Figure 2
View larger version (128K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. NCAM+ CD133+ interstitial cells. Frozen sections of adult kidneys (A–C) were double-stained with fluorescent-labelled antibodies against NCAM (Cy2 staining, shown in green) and CD133 (PE staining, shown in red) including nuclear labelling (DAPI staining, shown in blue). Overlapping signals of both antibodies could be observed as yellow–orange areas. (A) The micrograph shows the merged picture of a staining for NCAM, CD133 and DAPI. Intensities of the single antibody staining were evaluated using the software AnalySIS-Doku (intensity measurement function) at the level of the horizontal line and displayed as the inserted intensity curves for Cy2 and PE (arrow). (B, C) Rare NCAM+ CD133+ cells could be detected in the interstitium (arrowheads). (B) A nerve is present in the vicinity of a blood vessel, as shown by the green labelling with NCAM antibodies (arrow) in contrast to yellow–orange stained interstitial cells (arrowheads) which co-express NCAM and CD133. (C) Expression of CD133 can also be detected on renal tubular cells (stars).

 
We further analysed the expression of CD34 on NCAM+ interstitial cells. In adult kidneys, antibodies against CD34 labelled almost all endothelial cells, as expected (Figure 3F). In addition to endothelial cells, some rare renal interstitial cells co-expressed NCAM and CD34, as can be seen in Figure 3A. Using AnalySIS, the common peak for the green and the red fluorescence channel was obtained at the level of the measured NCAM+ renal interstitial cell, confirming that this cell expressed CD34 antigen, too (Figure 3A).


Figure 3
View larger version (95K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. NCAM+ CD34+ interstitial cells. Frozen sections of adult kidneys were double-stained with fluorescent-labelled antibodies against NCAM (Cy3 staining, shown in red), CD34 (FITC staining, shown in green) and a nuclear labelling (DAPI staining, shown in blue). Co-localization of both antibodies can be visualized as yellow–orange stained areas. (A) Intensities of the individual antibody staining were evaluated by the software AnalySIS (intensity measurement function) at the level of the horizontal line and displayed as the inserted intensity curves (arrow). The arrowhead marks one interstitial CD34+ cell which does not express NCAM. (B–D) Simultaneous expression of NCAM (B), CD34 (C) and the merged picture shown with the DAPI staining (D). (E, F) Rare NCAM+ CD34+ cells can be detected in the interstitium (arrowheads). The antibody against CD34 also labels endothelial cells (F; star).

 
As further potential progenitor markers, CD117 and CD24 were also analysed. There was no cross-reactivity with NCAM+ cells, since both antigens were completely absent from renal tissue. Although cadherin-11 was expressed by mesangial and certain interstitial cells, no overlay was detected with cells positive for NCAM. Nestin revealed strong positivity on some cells in glomeruli without any positivity in the renal interstitium, and therefore no overlapping was present. We were also not able to observe any co-expression of NCAM with antibodies against {alpha}SMA, vimentin, {alpha}5ß1 or CD68 on renal interstitial cells.

By immunoblotting we tried to verify the presence of NCAM in normal renal parenchyma. However, due to the low abundance of NCAM in adult kidneys, no signals for NCAM could be detected in lysates from normal renal tissue (Figure 4, lane 1).


Figure 4
View larger version (49K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Detection of NCAM by immunoblotting of renal tissues. Protein extracts of renal tissues (lanes 1–4) normal kidney (lane 1), IgA nephropathy (the case also shown in Figure 5B) (lane 2), membranous glomerulonephritis (lane 3), renal cell carcinoma (lane 4) and renal carcinoma cell line A-498 (lane 5). The anti-NCAM antibody detected a specific band of 140 kDa in extracts of the renal carcinoma tissue and cell lines, as well as in kidneys in the initial phase of fibrosis.

 
NCAM in kidneys with incipient fibrosis
Immunohistochemical analysis of NCAM expression in 15 renal biopsy specimens with mild interstitial fibrosis due to different diseases was performed. In comparison with normal renal tissues, usually a focal (Figure 5A–C), but sometimes also a diffuse (Figure 5D) increase of NCAM expression on interstitial cells was observed in 13 cases with incipient interstitial fibrosis. However, in two biopsies with advanced interstitial fibrosis, one due to amyloidosis and the other due to diabetes, no increase of NCAM+ interstitial cells was detected. Although the majority of the analysed cases with prominent NCAM+ interstitial cells also showed an increase of {alpha}5ß1 integrin and {alpha}SMA in the interstitium, no overlapping expression pattern between these two molecules and NCAM was observed by double immunofluorescence labelling (data not shown). However, we did not observe an increased expression of CD133 or CD34 in the areas of the interstitium, where an increase of NCAM+ cells could be observed.


Figure 5
View larger version (111K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. NCAM expression on kidneys with incipient interstitial fibrosis. Focal increase of NCAM+ interstitial cells in (A–C): (A) case of IgA nephropathy; this tissue was also used for immunoblotting as shown in Figure 4, lane 2 (immunofluorescence staining with Cy3-labelled antibodies); (B) membranous glomerulonephritis (immunoperoxidase staining on cryostat section); (C) focal segmental glomerulosclerosis (immunoperoxidase staining on paraffin section). Diffuse increase of NCAM+ interstitial cells in (D): rapidly progressive glomerulonephritis (immunoperoxidase staining).

 
By immunoblotting, anti-NCAM antibodies detected a specific band of 140 kDa (Figure 4, lane 2) in the renal biopsy specimens (the case shown in Figure 5D) of an IgA nephropathy with mild interstitial fibrosis. Thus, the presence of NCAM could be confirmed by immunoblotting in the case where an increase of NCAM tissue staining was observed by immunofluorescence microscopy in small areas of interstitial fibrosis.



   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Acknowledgements
 References
 
It has been widely accepted that NCAM molecules are only expressed on nerves and nerve fibres, but not on parenchymal cells, of adult healthy human kidneys [6]. In the present study, however, precise immunomorphological analysis by light and fluorescent microscopy revealed additional conspicuous NCAM+ interstitial cells with characteristic dendritic morphology in normal kidneys. Using double immunofluorescence labelling with antibodies against neurofilaments and other markers for neural tissue, it was possible to clearly exclude nerves as exclusive NCAM+ structures in renal tissue. Rare renal interstitial cells expressing NCAM with cytoplasmatic extensions and elongated nuclei, which also stained positively for markers of early hematopoietic progenitor cells, were predominantly found at the corticomedullary junction. Although conspicuous and individually localized with long protrusions, these cells could easily be overlooked or misinterpreted as interstitial nerve fibres. In the present study, we were able to notify by immunoblotting and immunomorphology an increase of NCAM+ renal interstitial cells in kidneys with incipient interstitial fibrosis.

A small subpopulation of renal interstitial cells does express NCAM. It could be speculated that these cells are remnants of the embryonic metanephric mesenchyme. In fetal kidneys, the metanephric mesenchyme from which all renal cells with the exception of collecting ducts originate, strongly expresses NCAM molecules [5]. Later on in embryonic kidney development, during conversion of mesenchymal cells into polarized epithelia, NCAM gradually disappears from developing tubuli, but remains to be present on surrounding stromal cells up to the perinatal period. In postnatal renal development, NCAM is almost completely lost from the stroma [6]. Only some solitary conspicuous cells continue to express this molecule in the renal interstitium as shown in the present study. It has been published by Oliver et al. [10] that the metanephric mesenchyme contains embryonic renal stem cells that could develop into all kidney cell types including endothelial, epithelial and stromal cells. These embryonic renal stem cells express NCAM, and it could be speculated that undifferentiated mesenchymal renal cells, which express NCAM and might represent renal progenitors, still persist in the adult human renal interstitium.

Since the NCAM+ renal cells have clear dendritic morphology, the first issue of our study was addressed to DC markers such as CD11c and HLA-DR. Although there was a certain amount of HLA-DR-positive cells in the interstitium, especially with incipient fibrosis, there were no coexpressions of these markers.

Further immunophenotyping of NCAM+ cells revealed that these cells were negative for fibroblast cell markers such as vimentin {alpha}5ß1 integrin, and progenitor cell markers CD117, as well as CD24, cadherin-11 and nestin which have been proposed to characterize murine renal progenitor cells [11,12]. However, in the intact renal interstitium, NCAM+ renal cells could co-express CD34 or CD133 antigens. CD34 is known to be present on most endothelial cells [13], but also on some cells belonging to the interstitium. However, not all interstitial cells that express CD34 share NCAM expression as shown in this study. In the healthy renal interstitium, NCAM+ and CD34-negative cells and vice versa CD34+ and NCAM-negative cells exist, including cells expressing both antigens. CD133 has been reported to be expressed on certain tubular epithelial cells in adult kidneys [14]. In addition, we found expression of CD133 on some NCAM+ interstitial cells. All these NCAM+ interstitial cells were variably present in different normal kidneys. Although a variety of exciting reports have confirmed that bone marrow-derived cells could be involved in the maintenance and repair of almost all compartments of the kidney [15–19], the existence of adult kidney progenitor cells is not excluded. There is evidence for the presence of resident stem cells in the niche of papilla in adult mice [20]. Interestingly, these cells could acquire a dendritic appearance in a cell culture showing similar morphological characteristics as the NCAM+ cells we detected in human renal tissue. Also in other organs, NCAM has been shown to be expressed by cells which seem to belong to the local organ-specific progenitor/stem cell pool such as human liver oval cells [21] or craniofacial muscle-derived cells [22]. Resident stem/progenitor cells of different human adult organs are known to express haematopoietic stem cell markers such as CD34, CD117 and CD133 [21–24]. Thus, it might be possible that the NCAM/CD34 or NCAM/CD133 molecule combinations might indicate the local progenitor cell type of the human kidney [25]. Recently, CD133+ renal progenitor cells with limited self-renewal capacity and capability for differentiation into epithelial and endothelial cells have been isolated from the adult human kidney [26].

Under normal circumstances, NCAM+ interstitial cells are very rare, but during certain pathological conditions their number could increase. We particularly noticed an increase of NCAM+ interstitial cells at the early phase of interstitial fibrosis due to different forms of glomerulonephritis. In addition, in experimental acute renal failure investigated by Abate et al. [27], the interstitium adjacent to the NCAM+ recovering tubular epithelium also showed a transitional significant increase of NCAM+ cells. As in early kidney development, an increased NCAM expression could be an indication of different remodelling processes during kidney diseases. NCAM+ interstitial cells might participate in the phases of kidney repair and also in interstitial fibrosis as an undesired result of the healing processes. Renal interstitial cells, expressing {alpha}SMA and {alpha}5ß1 integrin as a receptor for fibronectin which might regulate cell–matrix interactions, have been supposed to be involved in the development of interstitial fibrosis [28,29]. Although there was clear evidence for an increased number of {alpha}SMA and {alpha}5ß1 integrin-positive renal interstitial cells in the corresponding area of interstitium with increased NCAM expression, no overlap between NCAM and the expression of these molecules was detected. An increase of CD34-positive renal interstitial cells in areas of fibrosis due to glomerulonephritis, including those investigated in the present study, has also been reported [30]. Unfortunately, we were not able to detect any increase of CD34- or CD133-labelled cells in the area with mild fibrosis where interstitial accumulation of NCAM+ cells was observed. That could indicate a dynamic pool of renal interstitial cells in normal and diseased kidneys.

Our present results clearly show that a small subpopulation of kidney interstitial cells expresses NCAM and these cells might be renal progenitors.



   Acknowledgements
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
References
 
J.M.-L. is grateful to Alexander von Humboldt Foundation (Bonn, Germany) for financial support. Part of this work was presented in abstract form at the 19th European Congress of Pathology, and received the first award for free paper presentation. This study was financially supported by the grant no. 1990 of the Ministry of Science of the Republic of Serbia. We thank Inge Steiert for technical assistance and Jon Tolson for critical comments on the manuscript.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Goridis C, Brunet JF. NCAM: structural diversity, function and regulation of expression. Semin Cell Biol (1992) 3:189–197.[Medline]
  2. Rutishauser U, Acheson A, Hall AK, Mann DM, Sunshine J. The neural cell adhesion molecule (NCAM) as a regulator of cell–cell interactions. Science (1988) 240:53–57.[Abstract/Free Full Text]
  3. Al-Khafaji B, Noffsinger AE, Miller MA, DeVoe G, Stemmermann GN, Fenoglio-Preiser C. Immunohistologic analysis of gastrointestinal and pulmonary carcinoid tumors. Hum Pathol (1998) 29:992–999.[CrossRef][Web of Science][Medline]
  4. Warren HS, Kinnear BF, Skipsey LJ. Human natural killer (NK) cells: requirements for cell proliferation and expansion of phenotypically novel subpopulations. Immunol Cell Biol (1993) 71:87–97.
  5. Klein G, Langegger M, Goridis C, Ekblom P. Neural cell adhesion molecules during embryonic induction and development of the kidney. Development (1988) 102:749–761.[Abstract/Free Full Text]
  6. Nouwen EJ, Dauwe S, van der Biest I, De Broe ME. Stage- and segment-specific expression of cell-adhesion molecules N-CAM, A-CAM, and L-CAM in the kidney. Kidney Int (1993) 44:147–158.[Web of Science][Medline]
  7. Bourne SP, Patel K, Walsh F, Popham CJ, Coakham HB, Kemshead JT. A monoclonal antibody (ERIC-1), raised against retinoblastoma, that recognises the neural cell adhesion molecule (NCAM) expressed on brain and tumours arising from the neuroectoderm. J Neurooncol (1991) 10:111–119.[CrossRef][Medline]
  8. Barnstable CJ, Bodmer WF, Brown G, et al. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell (1978) 14:9–20.[CrossRef][Web of Science][Medline]
  9. Müller CA, Markovic-Lipkovski J, Klatt T, et al. Human {alpha}-defensins HNPs-1, -2 and -3 in renal cell carcinoma. Influences on tumor cell proliferation. Am J Pathol (2002) 160:1311–1324.[Abstract/Free Full Text]
  10. Oliver JA, Barasch J, Yang J, Herylinger D, Al-Awqati Q. Metanephric mesenchyme contains embryonic renal stem cells. Am J Physiol Renal Physiol (2002) 283:F799–F809.[Abstract/Free Full Text]
  11. Challen GA, Martinez G, Davis MJ, et al. Identifying the molecular phenotype of renal progenitor cells. J Am Soc Nephrol (2004) 15:2344–2357.[Abstract/Free Full Text]
  12. Chen J, Boyle S, Zhao M, et al. Differential expression of the intermediate filament protein Nestin during renal development and its localization in adult podocytes. J Am Soc Nephrol (2006) 17:1283–1291.[Abstract/Free Full Text]
  13. Lin G, Finger E, Gutierrez-Ramos JC. Expression of CD34 in endothelial cells, hematopoietic progenitors and nervous cells in fetal and adult mouse tissues. Eur J Immunol (1995) 25:1508–1516.[Web of Science][Medline]
  14. Corbeil D, Roper K, Hellwig A, et al. The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions. J Biol Chem (2000) 275:5512–5520.[Abstract/Free Full Text]
  15. Poulsom R, Forbes SJ, Hodivala-Dilke K, et al. Bone marrow contributes to renal parenchymal turnover and regeneration. J Pathol (2001) 195:229–235.[CrossRef][Web of Science][Medline]
  16. Imasawa T, Utsunomiya Y, Kawamura T, et al. The potential of bone marrow-derived cells to differentiate to glomerular mesangial cells. J Am Soc Nephrol (2001) 12:1401–1409.[Abstract/Free Full Text]
  17. Kale S, Karihaloo A, Clark PR, Kashgarian M, Krause DS, Cantley LG. Bone marrow stem cells contribute to repair of the ischemically injured renal tubule. J Clin Invest (2003) 112:42–49.[CrossRef][Web of Science][Medline]
  18. Gupta S, Verfaillie C, Chmielewski D, Kim Y, Rosenberg ME. A role for extrarenal cells in the regeneration following acute renal failure. Kidney Int (2002) 62:1285–1290.[CrossRef][Web of Science][Medline]
  19. Cornacchia F, Fornoni A, Plati AR, et al. Glomerulosclerosis is transmitted by bone marrow-derived mesangial cell progenitors. J Clin Invest (2001) 108:1649–1656.[CrossRef][Web of Science][Medline]
  20. Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q. The renal papilla is a niche for adult kidney stem cells. J Clin Invest (2004) 114:795–804.[CrossRef][Web of Science][Medline]
  21. Strain AJ, Crosby HA, Nijjar S, Kelly DA, Hubscher SG. Human liver-derived stem cells. Semin Liver Dis (2003) 23:373–384.[CrossRef][Web of Science][Medline]
  22. Sinanan AC, Hunt NP, Lewis MP. Human adult craniofacial muscle-derived cells: neural-cell adhesion-molecule (NCAM; CD56)-expressing cells appear to contain multipotential stem cells. Biotechnol Appl Biochem (2004) 40:25–34.[CrossRef][Web of Science][Medline]
  23. Crosby HA, Kelly DA, Strain AJ. Human hepatic stem-like cells isolated using c-kit or CD34 can differentiated into biliary epithelium. Gastroenterology (2001) 120:534–544.[CrossRef][Web of Science][Medline]
  24. Miraglia S, Godfrey W, Yin AH, et al. A novel five-transmembrane hematopoietic stem cell antigen: isolation, characterization, and molecular cloning. Blood (1997) 90:5013–5021.[Abstract/Free Full Text]
  25. Al-Awqati Q, Oliver JA. Stem cells in the kidney. Kidney Int (2002) 61:387–395.[CrossRef][Web of Science][Medline]
  26. Busolatti B, Bruno S, Grange C, et al. Isolation of renal progenitor cells from adult human kidney. Am J Pathol (2005) 166:545–555.[Abstract/Free Full Text]
  27. Abbate M, Brown D, Bonventre JV. Expression of NCAM recapitulates tubulogenic development in kidneys recovering from acute ischemia. AJP – Renal Physiol (1999) 277:F454–F463.
  28. Hewitson TD, Becker GJ. Interstitial myofibroblasts in IgA glomerulonephritis. Am J Nephrol (1995) 15:111–117.[Web of Science][Medline]
  29. Hillis GS, Roy-Chaudhury P, Duthie LA, et al. Expression of ß1 integrins in IgA nephropathy. Nephrol Dial Transplant (1997) 12:1137–1142.[Abstract/Free Full Text]
  30. Okon K, Szumera A, Kuzniewski M. Are CD34+ cells found in renal interstitial fibrosis? Am J Nephrol (2003) 23:409–414.[CrossRef][Web of Science][Medline]
Received for publication: 31. 8.06
Accepted in revised form: 4. 1.07


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
22/6/1558    most recent
gfm006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Markovic-Lipkovski, J.
Right arrow Articles by Müller, G. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Markovic-Lipkovski, J.
Right arrow Articles by Müller, G. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?