Table 1.
Primer sequences used for qRT-PCR procedures.
Fig 1.
NCAM positive interstitial staining among various kidney diseases and their relationship to severity of renal interstitial fibrosis.
(A) Frequency of interstitial NCAM positivity among various kidney diseases. (B) Number of detected NCAM+ cells per field of view on ×400 magnification in controls and in diseased kidneys with regard to severity of interstitial renal fibrosis (IRF); p values after applying Mann-Whiteny U test. (C-D) FSGS with slight interstitial fibrosis (IRF-1) without tubular atrophy exhibiting an increased diffuse NCAM interstitial positivity detected on slides from paraffin-embedded tissue. (C) PAS, x400. (D) Immunoperoxidase staining, NCAM clone 123C3.D5, x400. (E-G) Lupus nephritis with NCAM positive interstitial cells detected focally around tubuli in the area with slight IRF (IRF-1). (E) PAS, x400. (F) Massone trichrome staining, x400. (G) Immunoperoxidase staining, NCAM clone 123C3.D5, x400.
Table 2.
Distribution of diagnosis and neural cell adhesion molecule (NCAM) interstitial positivity (including the number of NCAM positive interstitial cells presented with mean ±SD) observed among stages of interstitial renal fibrosis (IRF).
Fig 2.
Double immunofluorescent labeling of NCAM with erythropoietin (EPO) and granzyme B.
(A-C) NCAM positive interstitial cells did not express EPO. Merge of NCAM (clone Eric-1) and EPO, cryostat sections, double immunofluorescent labeling, x400. (D) Diffuse NCAM (clone EP257Y) staining in peritubular incipient interstitial fibrosis of FSGS case, without any granzyme B positivity, cryostat section, double immunofluorescene, x400. (E) Overlapping of NCAM (clone EP257Y) and granzyme B in a single cell within the whole biopsy core of the case illustrated in previous picture, cryostat section, double immunofluorescence, x400. (F) Mononuclear interstitial inflammatory infiltrate of lupus nephritis, arrow indicates two NCAM+ cells without overlapping with granzyme B, cryostat section, double immunofluorescence, x400.
Fig 3.
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.
Fig 4.
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.
Fig 5.
Expression of FGFR1, HE4 and α5β1 integrin on NCAM positive cells in normal renal interstitium.
(A) FGFR1 expression on renal interstitial cells. (B) HE4 expression on renal interstitial cells. (C) Integrin α5β1 expression on renal interstitial cells. (A-C) cryostat sections, immunoperoxidase, x400; (D) FGFR1 expression. (E) HE4 expression. (F) Integrin α5β1 expression. (G-I) NCAM expression. (J) Coexpression FGFR1 and NCAM on the same interstitial cells. (K) Coexpression HE4 and NCAM on the same interstitial cell. (L) coexpression of α5β1 and NCAM on the same interstitial cells. (D-L) cryostat section, immunofluorescent labeling, x600; staining techniques are described in detail under Material and Methods.
Fig 6.
Expression of FGFR1, HE4 and α5β1 integrin on NCAM positive cells in incipient renal fibrosis.
(A) Double immunofluorescent labeling of NCAM and FGFR1; merge of these two markers clearly shows that all NCAM+ cells coexpressed FGFR1 (white arrows); diffuse NCAM expression on interstitial cells; strong FGFR1 expression on bold vessels (white stars) and diffuse expression on interstitial cells; x200. (B) Double immunofluorescent labeling of NCAM and HE4; merge of NCAM and HE4 revealed single cells coexpressing both markers (white arrow); x400. (C) Double immunofluorescence labeling of NCAM and α5β1; merge of these two markers clearly shows co-expression of NCAM and α5β1 on renal interstitial cells in area of incipient fibrosis (white arrows); x600. (D) Double immunofluorescence labeling of NCAM and αSMA; merge of these two markers showed no overlapping of NCAM and αSMA on renal interstitial cells in area of incipient fibrosis, although areas of NCAM+ and SMA+ interstitial cells are close to each other; x100. Staining techniques are described in detail under Material and Methods.
Fig 7.
Differences in relative mRNA expression levels of molecules relevant for renal fibrosis among NCAM positive cells from normal and renal interstitium with incipient fibrosis, obtained by laser capture microdissection.
(A) Relative mRNA expression levels of various molecules relevant for renal fibrosis; data are presented with mean values and standard error bars; *- indicate statistically significant difference, p<0.05; graph is made of mean values in order to unify variable presentations, although only αSMA, SLUG and ALK3 followed normal distribution; thus, some variables differed extremely among cases and consequently these data did not display normal distribution; due to influence of these extreme values on the mean value presented in the graph, bars are high but without statistical significance (such as BMP7); Student’s t test was used for variables with normal distribution both in control and kidneys with fibrosis: αSMA, SLUG and ALK3; due to high variability of other variables, exclusively in diseased kidneys, we applied nonparametric Mann Whitney U test 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. (B) NCAM positivity in peritubular incipient interstitial fibrosis shown on cryostat section, immunofluorescene, clone EP257Y, x400. (C) MMP-9 positivity in peritubular incipient interstitial fibrosis shown on cryostat section, immunofluorescence, clone 6-6B, x400. (D) arrows indicate the overlapping of NCAM and MMP-9 in interstitial cells.
Table 3.
Relative mRNA levels of molecules related to renal fibrosis in NCAM positive renal interstitial cells laser captured from normal and renal interstitium with incipient fibrosis.