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Variable Expression of Neural Cell Adhesion Molecule Isoforms in Renal Tissue: Possible Role in Incipient Renal Fibrosis.

Marković-Lipkovski J, Životić M, Müller CA, Tampe B, Ćirović S, Vještica J, Tomanović N, Zeisberg M, Müller GA - PLoS ONE (2015)

Bottom Line: Applying qRT-PCR on pure NCAM+ cells population, obtained by laser capture microdissection, significant mRNA over-expression of NCAM140kD isoform was found in NCAM+ cells within incipient IRF (p = 0.004), while NCAM120kD and NCAM180kD isoforms were not changed significantly (p = 0.750; p = 0.704; respectively).Simultaneously, qRT-PCR also showed significant αSMA (p = 0.014) and SLUG (p = 0.004) mRNAs up-regulation within the NCAM+ cells of incipient IRF, as well as highly decreased matrix metalloproteinases (MMP) -2 and -9 mRNAs (p = 0.028; p = 0.036; respectively).Fibroblast growth factor receptor 1 (FGFR1) and α5β1 integrin were extensively expressed on NCAM+ cells within the incipient IRF areas, whereas human epididymis protein-4 (HE4) was found to be present in few NCAM+ cells of both normal and interstitium with incipient fibrosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, Medical Faculty, University of Belgrade, Belgrade, Serbia.

ABSTRACT
Rare neural cell adhesion molecule (NCAM) positive cells have been previously described within the normal human adult kidney interstitium, speculating that they could increase in the interstitium with incipient interstitial renal fibrosis (IRF). In the present study, among 93 biopsy samples of various kidney diseases, NCAM+ interstitial cells were detected in 62.4% cases. An increased number of NCAM+ cells was significantly observed only in incipient IRF compared to normal renal tissues and advanced IRF stages (p<0.001), independently of underlying diseases (p = 0.657). All three major NCAM isoforms' RT-PCR bands were visible either in normal or in kidneys with incipient IRF, albeit their mRNA expression levels measured by qRT-PCR were different. Applying qRT-PCR on pure NCAM+ cells population, obtained by laser capture microdissection, significant mRNA over-expression of NCAM140kD isoform was found in NCAM+ cells within incipient IRF (p = 0.004), while NCAM120kD and NCAM180kD isoforms were not changed significantly (p = 0.750; p = 0.704; respectively). Simultaneously, qRT-PCR also showed significant αSMA (p = 0.014) and SLUG (p = 0.004) mRNAs up-regulation within the NCAM+ cells of incipient IRF, as well as highly decreased matrix metalloproteinases (MMP) -2 and -9 mRNAs (p = 0.028; p = 0.036; respectively). However, using double immunofluorescence MMP-9 could still be detectable on the protein level in rare NCAM+ cells within the incipient IRF. Further characterization of NCAM+ cells by double immunofluorescent labeling revealed their association with molecules involved in fibrosis. Fibroblast growth factor receptor 1 (FGFR1) and α5β1 integrin were extensively expressed on NCAM+ cells within the incipient IRF areas, whereas human epididymis protein-4 (HE4) was found to be present in few NCAM+ cells of both normal and interstitium with incipient fibrosis. Heterogeneity of NCAM+ interstitial cells in normal and incipient IRF, concerning molecules related to fibrosis and variable expression of NCAM isoforms, could suggest diverse role of NCAM+ cells in homeostasis and in regulation of renal fibrosis in diseased kidneys.

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Related in: MedlinePlus

Isolation of NCAM positive renal interstitial cells by laser capture microdissection (LCM) and changes in relative mRNA NCAM isofroms expression levels in incipient renal fibrosis.(A) Slide performed on cryostat section and stained by NCAM, clone Eric-1, with widespread NCAM expression, prior laser capture microdissection (arrow indicates the first selected NCAM positive cell for further LCM, while arrowhead shows second selected area). (B) Slide with rare NCAM cells within normal interstitium prior LCM. (C), (D) and (E) the same slides as Fig (A) and (B) after LCM procedure. (F) Relative expression levels of NCAM mRNAs isoforms, determined by quantitative real-time PCR (qRT-PCR), in NCAM+ cells captured by LCM from normal and from renal tissue with incipient IRF, data are presented with mean values and standard error bars; due to high variability of variables, exclusively in diseased kidneys, nonparametric Mann Whitney U test was applied to assess the difference in mRNA levels between controls and diseased kidneys; there were 6 samples (2 cases in triplicates) of control cases and 42 (14 cases in triplicates) samples of cases with incipient renal fibrosis.
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pone.0137028.g004: Isolation of NCAM positive renal interstitial cells by laser capture microdissection (LCM) and changes in relative mRNA NCAM isofroms expression levels in incipient renal fibrosis.(A) Slide performed on cryostat section and stained by NCAM, clone Eric-1, with widespread NCAM expression, prior laser capture microdissection (arrow indicates the first selected NCAM positive cell for further LCM, while arrowhead shows second selected area). (B) Slide with rare NCAM cells within normal interstitium prior LCM. (C), (D) and (E) the same slides as Fig (A) and (B) after LCM procedure. (F) Relative expression levels of NCAM mRNAs isoforms, determined by quantitative real-time PCR (qRT-PCR), in NCAM+ cells captured by LCM from normal and from renal tissue with incipient IRF, data are presented with mean values and standard error bars; due to high variability of variables, exclusively in diseased kidneys, nonparametric Mann Whitney U test was applied to assess the difference in mRNA levels between controls and diseased kidneys; there were 6 samples (2 cases in triplicates) of control cases and 42 (14 cases in triplicates) samples of cases with incipient renal fibrosis.

Mentions: Since tissue lysates could contain other NCAM expressing cells, not only the interstitial spindle shaped NCAM+ cells which were within the focus of our study, we decided to perform laser capture microdissection (LCM) which allowed us to separate and collect pure cell populations of the relevant NCAM+ cells out of tissue samples. Isolated pure NCAM+ cell populations were the most suitable starting material for downstream quantitative real-time PCR (qRT-PCR). Fig 4A and 4B represent renal tissues stained with anti-NCAM antibody prior to LCM procedure, and illustrate widespread NCAM expression in incipient IRF (Fig 4A) and scarce NCAM positivity in normal renal interstitium (Fig 4B). Fig 4C–4E illustrate tissues after LCM procedure. Statistically significant changes in the relative mRNA expression levels of NCAM isoforms have been revealed after applying qRT-PCR in the pure NCAM+ cell population. NCAM+ cells captured from incipient IRF significantly up-regulated NCAM140kD isoform compared to NCAM+ cells in normal kidneys, p = 0.004 (Fig 4F). Nevertheless, mRNA expression levels of NCAM120kD and NCAM180kD isoforms were not changed significantly in comparison to normal kidneys (p = 0.750; p = 0.704; respectively).


Variable Expression of Neural Cell Adhesion Molecule Isoforms in Renal Tissue: Possible Role in Incipient Renal Fibrosis.

Marković-Lipkovski J, Životić M, Müller CA, Tampe B, Ćirović S, Vještica J, Tomanović N, Zeisberg M, Müller GA - PLoS ONE (2015)

Isolation of NCAM positive renal interstitial cells by laser capture microdissection (LCM) and changes in relative mRNA NCAM isofroms expression levels in incipient renal fibrosis.(A) Slide performed on cryostat section and stained by NCAM, clone Eric-1, with widespread NCAM expression, prior laser capture microdissection (arrow indicates the first selected NCAM positive cell for further LCM, while arrowhead shows second selected area). (B) Slide with rare NCAM cells within normal interstitium prior LCM. (C), (D) and (E) the same slides as Fig (A) and (B) after LCM procedure. (F) Relative expression levels of NCAM mRNAs isoforms, determined by quantitative real-time PCR (qRT-PCR), in NCAM+ cells captured by LCM from normal and from renal tissue with incipient IRF, data are presented with mean values and standard error bars; due to high variability of variables, exclusively in diseased kidneys, nonparametric Mann Whitney U test was applied to assess the difference in mRNA levels between controls and diseased kidneys; there were 6 samples (2 cases in triplicates) of control cases and 42 (14 cases in triplicates) samples of cases with incipient renal fibrosis.
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Related In: Results  -  Collection

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pone.0137028.g004: Isolation of NCAM positive renal interstitial cells by laser capture microdissection (LCM) and changes in relative mRNA NCAM isofroms expression levels in incipient renal fibrosis.(A) Slide performed on cryostat section and stained by NCAM, clone Eric-1, with widespread NCAM expression, prior laser capture microdissection (arrow indicates the first selected NCAM positive cell for further LCM, while arrowhead shows second selected area). (B) Slide with rare NCAM cells within normal interstitium prior LCM. (C), (D) and (E) the same slides as Fig (A) and (B) after LCM procedure. (F) Relative expression levels of NCAM mRNAs isoforms, determined by quantitative real-time PCR (qRT-PCR), in NCAM+ cells captured by LCM from normal and from renal tissue with incipient IRF, data are presented with mean values and standard error bars; due to high variability of variables, exclusively in diseased kidneys, nonparametric Mann Whitney U test was applied to assess the difference in mRNA levels between controls and diseased kidneys; there were 6 samples (2 cases in triplicates) of control cases and 42 (14 cases in triplicates) samples of cases with incipient renal fibrosis.
Mentions: Since tissue lysates could contain other NCAM expressing cells, not only the interstitial spindle shaped NCAM+ cells which were within the focus of our study, we decided to perform laser capture microdissection (LCM) which allowed us to separate and collect pure cell populations of the relevant NCAM+ cells out of tissue samples. Isolated pure NCAM+ cell populations were the most suitable starting material for downstream quantitative real-time PCR (qRT-PCR). Fig 4A and 4B represent renal tissues stained with anti-NCAM antibody prior to LCM procedure, and illustrate widespread NCAM expression in incipient IRF (Fig 4A) and scarce NCAM positivity in normal renal interstitium (Fig 4B). Fig 4C–4E illustrate tissues after LCM procedure. Statistically significant changes in the relative mRNA expression levels of NCAM isoforms have been revealed after applying qRT-PCR in the pure NCAM+ cell population. NCAM+ cells captured from incipient IRF significantly up-regulated NCAM140kD isoform compared to NCAM+ cells in normal kidneys, p = 0.004 (Fig 4F). Nevertheless, mRNA expression levels of NCAM120kD and NCAM180kD isoforms were not changed significantly in comparison to normal kidneys (p = 0.750; p = 0.704; respectively).

Bottom Line: Applying qRT-PCR on pure NCAM+ cells population, obtained by laser capture microdissection, significant mRNA over-expression of NCAM140kD isoform was found in NCAM+ cells within incipient IRF (p = 0.004), while NCAM120kD and NCAM180kD isoforms were not changed significantly (p = 0.750; p = 0.704; respectively).Simultaneously, qRT-PCR also showed significant αSMA (p = 0.014) and SLUG (p = 0.004) mRNAs up-regulation within the NCAM+ cells of incipient IRF, as well as highly decreased matrix metalloproteinases (MMP) -2 and -9 mRNAs (p = 0.028; p = 0.036; respectively).Fibroblast growth factor receptor 1 (FGFR1) and α5β1 integrin were extensively expressed on NCAM+ cells within the incipient IRF areas, whereas human epididymis protein-4 (HE4) was found to be present in few NCAM+ cells of both normal and interstitium with incipient fibrosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, Medical Faculty, University of Belgrade, Belgrade, Serbia.

ABSTRACT
Rare neural cell adhesion molecule (NCAM) positive cells have been previously described within the normal human adult kidney interstitium, speculating that they could increase in the interstitium with incipient interstitial renal fibrosis (IRF). In the present study, among 93 biopsy samples of various kidney diseases, NCAM+ interstitial cells were detected in 62.4% cases. An increased number of NCAM+ cells was significantly observed only in incipient IRF compared to normal renal tissues and advanced IRF stages (p<0.001), independently of underlying diseases (p = 0.657). All three major NCAM isoforms' RT-PCR bands were visible either in normal or in kidneys with incipient IRF, albeit their mRNA expression levels measured by qRT-PCR were different. Applying qRT-PCR on pure NCAM+ cells population, obtained by laser capture microdissection, significant mRNA over-expression of NCAM140kD isoform was found in NCAM+ cells within incipient IRF (p = 0.004), while NCAM120kD and NCAM180kD isoforms were not changed significantly (p = 0.750; p = 0.704; respectively). Simultaneously, qRT-PCR also showed significant αSMA (p = 0.014) and SLUG (p = 0.004) mRNAs up-regulation within the NCAM+ cells of incipient IRF, as well as highly decreased matrix metalloproteinases (MMP) -2 and -9 mRNAs (p = 0.028; p = 0.036; respectively). However, using double immunofluorescence MMP-9 could still be detectable on the protein level in rare NCAM+ cells within the incipient IRF. Further characterization of NCAM+ cells by double immunofluorescent labeling revealed their association with molecules involved in fibrosis. Fibroblast growth factor receptor 1 (FGFR1) and α5β1 integrin were extensively expressed on NCAM+ cells within the incipient IRF areas, whereas human epididymis protein-4 (HE4) was found to be present in few NCAM+ cells of both normal and interstitium with incipient fibrosis. Heterogeneity of NCAM+ interstitial cells in normal and incipient IRF, concerning molecules related to fibrosis and variable expression of NCAM isoforms, could suggest diverse role of NCAM+ cells in homeostasis and in regulation of renal fibrosis in diseased kidneys.

No MeSH data available.


Related in: MedlinePlus