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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.

No MeSH data available.


Related in: MedlinePlus

Presence of NCAM and its isoforms in normal and fibrotic kidneys.(A) RT-PCR: three NCAM isoforms in different renal samples, fibrosis was present in 3 cases, lanes IV, VIII and X. (B) RT-PCR: presence of all NCAM isoforms in FSGS. (C) RT-PCR: presence of all NCAM isoforms in MPGN. (D) Same case as Fig (B): increased NCAM expression in areas with slight fibrosis on cryostat section, immunoperoxidase, clone Eric-1, x200. (E) Same tissue as Fig (C): NCAM positivity in peritubular incipient interstitial fibrosis shown on cryostat section, immunoperoxidase, clone Eric-1, x400.
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pone.0137028.g003: Presence of NCAM and its isoforms in normal and fibrotic kidneys.(A) RT-PCR: three NCAM isoforms in different renal samples, fibrosis was present in 3 cases, lanes IV, VIII and X. (B) RT-PCR: presence of all NCAM isoforms in FSGS. (C) RT-PCR: presence of all NCAM isoforms in MPGN. (D) Same case as Fig (B): increased NCAM expression in areas with slight fibrosis on cryostat section, immunoperoxidase, clone Eric-1, x200. (E) Same tissue as Fig (C): NCAM positivity in peritubular incipient interstitial fibrosis shown on cryostat section, immunoperoxidase, clone Eric-1, x400.

Mentions: Considering that the three major NCAM isoforms could not be differentiated by immunohistochemical staining, we further proceeded RT-PCR analyzes on normal and fibrotic tissue lysates in order to detect presence of specific isoforms. The mRNA expression of all NCAM isoforms was detected in eight control renal tissues and five cases with incipient IRF (Fig 3A: lanes IV, VIII and X; Fig 3B and 3C). All three major isoforms were found to be expressed both in normal and kidneys with incipient IRF. The same cases were also stained for NCAM, using cryostat sections, and two cases are presented on Fig 3: FSGS with focal incipient fibrosis (Fig 3D) and results of RT-PCR (Fig 3B), and MPGN with incipient fibrosis (Fig 3E) and results of RT-PCR (Fig 3C). We need to underline that NCAM staining was more intensively visible on these cryostat sections (Fig 3D and 3E) than on slides from paraffin-embedded tissues (Fig 1) that were used in our study for the assessments of NCAM positivity in various kidney diseases with variable degree of IRF (Fig 1).


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)

Presence of NCAM and its isoforms in normal and fibrotic kidneys.(A) RT-PCR: three NCAM isoforms in different renal samples, fibrosis was present in 3 cases, lanes IV, VIII and X. (B) RT-PCR: presence of all NCAM isoforms in FSGS. (C) RT-PCR: presence of all NCAM isoforms in MPGN. (D) Same case as Fig (B): increased NCAM expression in areas with slight fibrosis on cryostat section, immunoperoxidase, clone Eric-1, x200. (E) Same tissue as Fig (C): NCAM positivity in peritubular incipient interstitial fibrosis shown on cryostat section, immunoperoxidase, clone Eric-1, x400.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556687&req=5

pone.0137028.g003: Presence of NCAM and its isoforms in normal and fibrotic kidneys.(A) RT-PCR: three NCAM isoforms in different renal samples, fibrosis was present in 3 cases, lanes IV, VIII and X. (B) RT-PCR: presence of all NCAM isoforms in FSGS. (C) RT-PCR: presence of all NCAM isoforms in MPGN. (D) Same case as Fig (B): increased NCAM expression in areas with slight fibrosis on cryostat section, immunoperoxidase, clone Eric-1, x200. (E) Same tissue as Fig (C): NCAM positivity in peritubular incipient interstitial fibrosis shown on cryostat section, immunoperoxidase, clone Eric-1, x400.
Mentions: Considering that the three major NCAM isoforms could not be differentiated by immunohistochemical staining, we further proceeded RT-PCR analyzes on normal and fibrotic tissue lysates in order to detect presence of specific isoforms. The mRNA expression of all NCAM isoforms was detected in eight control renal tissues and five cases with incipient IRF (Fig 3A: lanes IV, VIII and X; Fig 3B and 3C). All three major isoforms were found to be expressed both in normal and kidneys with incipient IRF. The same cases were also stained for NCAM, using cryostat sections, and two cases are presented on Fig 3: FSGS with focal incipient fibrosis (Fig 3D) and results of RT-PCR (Fig 3B), and MPGN with incipient fibrosis (Fig 3E) and results of RT-PCR (Fig 3C). We need to underline that NCAM staining was more intensively visible on these cryostat sections (Fig 3D and 3E) than on slides from paraffin-embedded tissues (Fig 1) that were used in our study for the assessments of NCAM positivity in various kidney diseases with variable degree of IRF (Fig 1).

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