Figure 1.
Human cytokine array analysis of multiple cytokines secreted by mesangial cells after cIgA1 treatment.
A: The right upper image is a human cytokine array coordinates displaying the location of detected cytokines. Symbol + means the positive reference spot, while symbol – means the negative control. Other nine images were array results of 1ml culture medium of mesangial cells treated with 100μg/ml cIgA1 from 4 IgAN patients (IgAN-1,2,3,4), 4 health controls (HC-1,2,3,4) and without cIgA1 (blank control) for 48 hours. Images were from one minute exposures to X-ray film. B: Grouped bar chart showed multiple cytokines secreted by mesangial cells after cIgA1 treatment. Levels of CCL1, sTREM-1, C5a, IL-8, IL-6, MCP-1 and CXCL1 showed a higher tendency in mesangial medium in IgAN group than in HC group. Mann-Whitney U test was performed to compare IgAN group and HC group.
Figure 2.
Increased secretion of CXCL1 and TGF-β1 by human mesangial cells treated with cIgA1 from IgAN patients.
Compared with from healthy controls (HC), cIgA1 from IgAN patients (IgAN) significantly up-regulated mesangial cells CXCL1 (A) and TGF-β1 secretion (B) (CXCL1: 718.4 ± 532.1 pg/ml vs. 311.6 ± 191.8 pg/ml, P=0.002; TGF-β1: 1535.5±255.5 pg/ml vs. 1304.9±363.8 pg/ml, P=0.031).
Figure 3.
Elevated urinary levels of CXCL1 and TGF-β1 in IgAN patients.
Patients with IgAN (IgAN) showed higher urinary levels of CXCL1 (A) and TGF-β1 (B) than that in healthy controls (HC) (CXCL1: 43.85±142.49 pg/mg Vs 1.38±4.09 pg/mg, p<0.01; TGF-β1: 107.56±283.95 pg/mg Vs 43.83±33.85 pg/mg, p=0.026). Individual symbols represent individual person (104 IgAN patients and 74 healthy controls).
Figure 4.
Upregulation of CXCR2 expression in podocytes cultured with conditioned medium of mesangial cells exposed to cIgA1 from IgAN patients (IgAN-HMCM) and rhTGF-β1.
The mRNA (A) and protein (B) expression of CXCR2 were significantly increased in podocytes cultured with conditioned medium of mesangial cells exposed to cIgA1 from IgAN patients (IgAN-HMCM) than from healthy controls (HC-HMCM). Similar findings were observed in podocytes cultured with 2 ng/ml TGF-β1 (C), but not under rhCXCL1.
Figure 5.
Increased podocytes death and reduced podocytes adhesion cultured with conditioned medium of mesangial cells exposed to cIgA1 from IgAN patients (IgAN-HMCM) and rhTGF-β1 together with rhCXCL1.
A: The podocytes released LDH were significantly increased when podocytes were cultured with conditioned medium of mesangial cells exposed to cIgA1 from IgAN patients (IgAN-HMCM) compared to from healthy controls (HC-HMCM). B: Similar increased LDH could also be observed when podocytes were treated with 2ng/ml CXCL1 and 2ng/ml TGF-β1 together (symbol * represented p<0.05 compared with control). The increased podocytes released LDH, induced by IgAN-HMCM or rhTGF-β1 (2ng/ml) together with rhCXCL1 (2ng/ml) could be partially rescued by simultaneous incubation with 0.5ug/ml blocking antibody against CXCR2 (symbol # represented p<0.05 compared with without CXCR2 blocking antibody). C: The number of adherent podocytes were significantly decreased when podocytes were cultured with IgAN-HMCM compared to HC-HMCM (5790±377 cells vs. 9272±736 cells, P=0.002). D: Similar decreased podocyte adhesion could also be observed when podocytes were treated with 2ng/ml CXCL1 and 2ng/ml TGF-β1 together (7327±326 cells vs. 10468±499 cells, P=0.001) (symbol * represented p<0.05 compared with control). The decreased podocyte adhesion, induced by IgAN-HMCM or rhTGF-β1 (2ng/ml) together with rhCXCL1 (2ng/ml) could be partially rescued by simultaneous incubation with 0.5ug/ml blocking antibody against CXCR2 (symbol # represented p<0.05 compared to without CXCR2 blocking antibody).
Figure 6.
Immunofluorescence staining of CXCL1 and podocalyxin in urine exfoliated podocytes in IgAN patients.
Urine exfoliated nucleated cells were stained with DAPI (blue). Expression of CXCL1 (green) staining was observed in urine exfoliated podocalyxin (red) staining positive cells. Images in left panel were showed in low magnitude (200 ×) and images in right panel were showed in high magnitude (400 ×).