Volume 67, Issue 2, Pages 602-612 (February 2005) Activation of tubular epithelial cells by mesangial-derived TNF-α: Glomerulotubular communication in IgA nephropathy  Loretta Y.Y. Chan, Joseph C.K. Leung, Anita W.L. Tsang, Sydney C.W. Tang, K.A.R. Neng Lai  Kidney International  Volume 67, Issue 2, Pages 602-612 (February 2005) DOI: 10.1111/j.1523-1755.2005.67116.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 Flow cytometry assay of IgA binding to PTEC and HMC. (A) Binding of IgA from IgAN patients (N = 22) to PTEC and HMC is increased when compared with healthy control subjects (N = 15). (B) Representative histogram showing a lower binding of IgA to PTEC than to HMC when same amount of IgA (50 μg/mL) and the equal number of cells (1 × 106 cells) are used for the experiments. Kidney International 2005 67, 602-612DOI: (10.1111/j.1523-1755.2005.67116.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 mRNA expression of IgA receptors by reverse transcription-polymerase chain reaction (RT-PCR). PTEC do not express mRNA of known IgA receptors, including FcαR, pIgR, and Fcα/μR, except TfR. As for ASGPR, only the H1 chain of the ASGPR is expressed in PTEC. Kidney International 2005 67, 602-612DOI: (10.1111/j.1523-1755.2005.67116.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 Inhibition binding assay. Inhibition of competitor proteins on the binding of (A) FITC-mIgA or (B) FITC-pIgA to PTEC. The binding of FITC-mIgA or FITC-pIgA (solid bars) to PTEC is not blocked by preincubating PTEC with different competitor proteins (dotted bars). Open bar represents the background MFI using isotype antibody as control. Preincubation of PTEC with mIgA and pIgA results in reduction of FITC-mIgA binding by 75% and 86.5%, respectively. Preincubation of PTEC with mIgA and pIgA results in reduction of FITC-pIgA binding by 42% and 90%, respectively. The results represent the mean ± standard deviation from 5 individual experiments. Kidney International 2005 67, 602-612DOI: (10.1111/j.1523-1755.2005.67116.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 Protein synthesis of TNF-α, MIF, and sICAM-1 in PTEC incubated with IgA. There is no difference in protein synthesis of (A) TNF-α, (B) MIF, and (C) sICAM1 when PTEC are incubated with total IgA prepared from patients with IgA nephropathy (IgAN), minimal change nephropathy (MCNS), or Henoch-Schönlein purpura (HSP) as compared with IgA from healthy control subjects (Ctl) or incubation with culture medium alone (Medium Ctl). Kidney International 2005 67, 602-612DOI: (10.1111/j.1523-1755.2005.67116.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 Cell proliferation assay of PTEC incubated with IgA-HMC spent medium. Increased proliferation of PTEC is observed when cultured with IgA-HMC spent medium prepared from IgAN patients when compared with healthy control subjects (Ctl). PTEC incubated with IgA-HMC spent medium from patients with minimal change nephropathy (MCNS) or with Henoch-Schönlein purpura (HSP) demonstrate no increased proliferation when compared to IgA isolated from healthy control subjects. #P < 0.001 vs. IgAN. Kidney International 2005 67, 602-612DOI: (10.1111/j.1523-1755.2005.67116.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 6 Expression of TNF-α, MIF, and ICAM-1 in PTEC incubated with IgA-HMC spent medium. The gene expression of (A) TNF-α, (B) MIF, and (C) ICAM-1 is up-regulated in PTEC incubated with IgA-HMC spent medium prepared from IgAN patients when compared to that from healthy control subjects (Ctl). Gene expression of TNF-α or MIF is not increased in PTEC incubated with IgA-HMC spent medium prepared from patients with minimal change nephropathy (MCNS) or with Henoch-Schönlein purpura (HSP) when compared with that of healthy control subjects. The protein synthesis of (D) TNF-α, (E) MIF, and (F) sICAM-1 is up-regulated in PTEC incubated with IgA-HMC spent medium prepared from IgAN patients when compared to that from healthy control subjects (Ctl). Protein synthesis of TNF-α, MIF, or sICAM-1 is not increased in PTEC incubated with IgA-HMC spent medium prepared from patients with minimal change nephropathy (MCNS) or with Henoch-Schönlein purpura (HSP) when compared with that of healthy control subjects. #P < 0.01 vs. IgAN and *P < 0.01 vs. Ctl. Kidney International 2005 67, 602-612DOI: (10.1111/j.1523-1755.2005.67116.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 7 Synthesis of TNF-α in HMC incubated with IgA. TNF-α is up-regulated when HMC are incubated with IgA prepared from IgAN patients as compared to healthy control subjects (Ctl). TNF-α synthesis is not increased in HMC incubated with IgA prepared from patients with minimal change nephropathy (MCNS) or with Henoch-Schönlein purpura (HSP) when compared with that of the Ctl. #P < 0.001. Kidney International 2005 67, 602-612DOI: (10.1111/j.1523-1755.2005.67116.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 8 Synthesis of MIF and sICAM-1 by PTEC incubated with IgA-HMC spent medium in the presence of a neutralizing anti-TNF-α antibody. A neutralizing anti-TNF-α at a concentration of 0.1 μg/mL (shaded bar) down-regulates (A) MIF and (B) sICAM-1 synthesis in PTEC incubated with IgA-HMC spent medium from patients with IgAN as compared with IgA-HMC spent medium from control subjects. Kidney International 2005 67, 602-612DOI: (10.1111/j.1523-1755.2005.67116.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 9 Synthesis of MIF and sICAM-1 by PTEC incubated with IgA-HMC spent medium in the presence of neutralizing antibodies to TNF-α, IL-1β, TGF-β, or PDGF. Only the neutralizing anti-TNF-α (shaded bar) down-regulates (A) MIF and (B) sICAM-1 synthesis in PTEC incubated with IgA-HMC spent medium (black bar) from patients with IgAN as compared with IgA-HMC spent medium from control subjects (white bar). Synthesis of MIF or sICAM-1 is not reduced by neutralizing antibodies to IL-1β, TGF-β, or PDGF. Kidney International 2005 67, 602-612DOI: (10.1111/j.1523-1755.2005.67116.x) Copyright © 2005 International Society of Nephrology Terms and Conditions