1. Volume 82, Issue 5, Pages 548-560 (September 2012)
Activated innate immunity and the involvement of CX3CR1–fractalkine in promoting hematuria in patients with IgA nephropathy 
Sharon N. Cox, Fabio Sallustio, Grazia Serino, Antonia Loverre, Francesco Pesce, Margherita Gigante, Gianluigi Zaza, Patrizia F. Stifanelli, Nicola Ancona, Francesco P. Schena 
Kidney International 
Volume 82, Issue 5, Pages (September 2012)
DOI: /ki
Copyright © 2012 International Society of Nephrology Terms and Conditions

2. Figure 1
Functional analysis of the top selected genes identified by microarray during the gross hematuria episode. The network was algorithmically constructed by the ingenuity pathway analysis (IPA) software on the basis of the functional and biological connectivity of genes. The network is graphically represented as nodes (genes) and edges (the biological relationship between genes). Red and green shaded nodes represent upregulated and downregulated genes, respectively; others (empty nodes) are those that IPA automatically includes because they are biologically linked to our genes based on the evidence in the literature. The analysis of this top-ranked network (score 48, 54 associated genes, P<0.0001) revealed an important role of the immunoproteasome pathway, the central role of STAT1, and the presence of a gene characterizing cytotoxic effector lymphocytes CX3CR1.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

3. Figure 2
Gene expression levels evaluated by real-time (RT)-PCR in peripheral blood mononuclear cells from 12 IgA nephropathy (IgAN) patients during the gross hematuria (GH) episode and during the remission phase characterized by permanent microscopic hematuria (MH). (a) CX3CR1- and (b) PSMB9- normalized gene expression levels were significantly higher during the GH phase of the disease compared with the same subject in the MH phase (*P=0.0001, **P=0.006, respectively). (c) FADD-normalized gene expression level was significantly lower during the GH phase of the disease compared with the same subject during the MH phase (***P=0.01). P-values obtained by paired t test.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

4. Figure 3
CX3CR1 and STAT1 protein expression on isolated peripheral blood mononuclear cells (PBMCs) of IgA nephropathy (IgAN) patients at two different time points: during the gross hematuria (GH) episode and during the remission phase characterized by permanent microscopic hematuria (MH). (a) Surface marker expression of CX3CR1 as measured by flow cytometry on PBMCs was significantly higher during the GH (gray line) compared with the same subject during the MH (black line). Histograms are compared with isotype control MoAB (black solid filled histogram). (b) Surface marker expression of CX3CR1 as measured by flow cytometry on PBMCs during the GH compared with the same subject in MH (*P=0.001). (c) In accordance with results obtained by flow cytometry, CX3CR1 protein levels assessed by western blotting showed significantly higher levels during the GH compared with the same subject in the MH (**P=0.001). (d) STAT1 protein levels assessed by western blotting were significantly higher during the GH compared with the same subject in the MH (***P=0.002). (e) Representative western blotting experiment for CX3CR1 and STAT1 on two IgAN patients during the GH and the same subjects in the MH. P-values obtained by paired t test. FITC, fluorescein isothiocyanate.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

5. Figure 4
Enhancement of CX3CR1 expression on the surface of cytotoxic leukocyte subsets during the gross hematuria episode. (a) CX3CR1 was evaluated on natural killer (NK) cells (CD16+CD56+CD3), natural killer T (NKT) cells (CD16+CD56+CD3+), and on CD4+, CD8+, and γδ-T cells (CD4−CD8−CD3+) gated on CD3+ T cells from isolated peripheral blood mononuclear cells (PBMCs), at two different time points: during the gross hematuria (GH) episode and during the remission phase characterized by permanent microscopic hematuria (MH). Quadrants and histograms were based on cell staining with isotype control monoclonal antibodies. Results are depicted for one IgA nephropathy (IgAN) patient during the GH compared with the same subject in MH, but they are representative of five IgAN patients. (b) The histograms represent the enhancement of CX3CR1 protein levels during the GH episode compared with MH expressed as mean±s.e.m. (n=5, *P<0.05, paired t test).
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

6. Figure 5
CX3CR1 and STAT1 protein expression levels in peripheral blood mononuclear cells (PBMCs) of 12 IgA nephropathy (IgAN) patients and 10 healthy blood donors (HBD) in response to antigenic stimuli. (a, b) CX3CR1 and STAT1 expression increased in a dose-dependent manner only in IgAN patients and not in HBDs, reaching a statistically significant fold increase with the dose of 1μg/ml (n=3). (c, d) CX3CR1 and STAT1 protein levels, assessed by western blotting, showed, respectively, a 1.8- and 1.6-fold increase in IgAN patients compared with HBDs after lipopolysaccharide (LPS) stimulation (1μg/ml). (e) Representative western blotting experiment for CX3CR1 and STAT1 of an IgAN patient and HBD. The histograms represent the mean±s.e.m. of CX3CR1 protein levels (*P<0.01, **P<0.05).
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

7. Figure 6
Glomerular immunolocalization and urinary level of fractalkine (FKN). (a) Strong deposition of FKN was found in the glomerulus of a patient with a history of recurrent gross hematuria (rGH) episodes after upper respiratory tract infections. (b) Magnified image of a showing diffused staining that corresponds to the secreted form of FKN (b, red arrow) and a more linear staining that corresponds to the membrane-bound form (b, black arrow, × 100 objective magnification). (c) Weak glomerular FKN expression was found in IgA nephropathy (IgAN) patients who had permanent microscopic hematuria (pMH) and (d) in normal biopsy specimens. (e) FKN-specific staining in the glomerular area was higher in the rGH group compared with pMH (P<0.0001) and controls (P=0.002); no statistical difference was found when we compared pMH and controls. Quantification of the extent of immunoperoxidase staining was carried out as described in the Materials and Methods and Supplementary Methods online section. (f) Urinary level of fractalkine was higher in IgAN patients with rGH patients compared with pMH patients (P=0.005), IgAN patients without microscopic hematuria (w/o H, P=0.04), membranous nephropathy patients with microscopic hematuria (MN, P=0.001), and membranoproliferative glomerulonephritis patients with microscopic hematuria (MPGN, P=0.04). Values are expressed as mean±s.e.m.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

8. Figure 7
Glomerular expression of fractalkine (FKN) in IgA nephropathy (IgAN) patients characterized by recurrent gross hematuria episodes. Immunofluorescent labeling of FKN in biopsy specimens, with histological diagnosis of IgA nephropathy. We found a perfect colocalization of FKN within glomerular endothelial cells, as shown by (a–f) dual labeling with anti-CD31 and within podocytes as shown by (g–l) dual labeling with anti-synaptopodin.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

9. Figure 8
Potential pathogenic mechanism in IgA nephropathy (IgAN) patients characterized by recurrent episodes of gross hematuria (rGH). Antigenic stimuli enhance CX3CR1 expression on circulating blood cytotoxic effector cells including natural killer (NK) cells, CD8+ T cells, and γδ-T cells, which contain cytoplasmic granules of perforin and granzyme. IgAN patients who have permanent microscopic hematuria (pMH) are characterized by low glomerular fractalkine (FKN) expression. On the other hand, IgAN patients characterized by rGH during the follow-up have an enhanced FKN expression by both podocytes and endothelial cells (ECs) and an enhanced urinary level of this chemokine. FKN captures CX3CR1+ cells under both static and flow conditions. The CX3CR1–FKN interaction promotes transendothelial migration of cytotoxic effector cells, leading to a break in continuity of the glomerular capillary wall. This mechanism may explain the massive presence of red blood cells in the urine during the GH. GBM, glomerular basement membrane.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions