1. Volume 61, Issue 5, Pages 1655-1665 (May 2002)
Transferrin up-regulates chemokine synthesis by human proximal tubular epithelial cells: Implication on mechanism of tubuloglomerular communication in glomerulopathic proteinura 
Sydney Tang, Joseph C.K. Leung, Anita W.L. Tsang, Hui Yao Lan, Tak Mao Chan, Kar Neng Lai 
Kidney International 
Volume 61, Issue 5, Pages (May 2002)
DOI: /j x
Copyright © 2002 International Society of Nephrology Terms and Conditions

2. Figure 1
Proliferative response of mesangial cells (MC). Subconfluent MC on 24-well plates were subjected to proliferation assay as determined by their incorporative capacity of tritiated thymidine after different experimental conditions overnight: (A) Growth-arrested MC grown in serum-free medium as negative control. (B) MC incubated in medium containing 10% fetal calf serum (FCS) as a positive control. (C) MC incubated in medium containing 5 mg/mL of transferrin. (D) MC incubated with supernatant from growth-arrested proximal tubule epithelial cells (PTEC) grown in serum free medium for a further 6 to 12 hours, which served as control for column E. (E) MC grown in “conditioned” medium (defined in the Methods section). (F) MC grown in PTEC supernatants obtained directly after 6 hours of transferrin stimulation. All experiments were performed in quadruplicate, and results are expressed as mean ± SD.
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

3. Figure 2
Expression of platelet-derived growth factor (PDGF) by MC. Constitutive PDGF expression (Control) by MC was up-regulated after overnight incubation with “conditioned” medium (E) compared with unstimulated cells or cells incubated with PTEC supernatants obtained directly after 6 hours of transferrin stimulation (F). All experiments were performed in triplicate. Right hand panel showed the typical appearances of gels from three representative experiments.
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

4. Figure 3
PDGF secretion by PTEC (▪) and MC (□). The concentration of PDGF protein in culture supernatants was assayed by ELISA in quiescent PTEC (Control), “conditioned” PTEC (defined in the Methods section) (E), and PTEC incubated in the presence of 5 mg/mL transferrin for 6 hours (F). These supernatants were then transferred to MC for overnight incubation to reproduce the experimental conditions (D, E and F, respectively) detailed in the legend to Figure 1, and then assayed for PDGF protein. *P < 0.01, #P < compared with the corresponding control (Control). Experiments were performed in triplicate, and the results are expressed as mean ± SD.
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

5. Figure 4
Effect of PDGF blockade on MC proliferation. Proliferative responses in subconfluent MC were quantified by thymidine uptake after incubation with supernatant from growth arrested PTEC grown in serum free medium for a further 6 to 12 hours (Control), “conditioned” medium (see text for definition) (E), or with PTEC supernatants obtained directly after 6 hours of transferrin stimulation (F), in the presence () or absence (□) of neutralizing anti-PDGF antibody. Experiments were performed in triplicate, and the results are expressed as mean ± SD.
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

6. Figure 5
Time response of interleukin-8 (IL-8; ▪), migration inhibitory factor (MIF; □), monocyte chemoattractant protein-1 (MCP-1;) and RANTES (normal T-cell expressed and secreted, regulated upon activation;) gene expression in PTEC after transferrin challenge. Confluent growth-arrested PTECs were incubated with serum- and transferrin-free medium (STFM) or medium containing 5 mg/mL transferrin for 3 to 48 hours. cDNA was extracted from cells and subjected to PCR amplification. The intensity of the amplicon scanned from agarose gels stained with ethidium bromide was compared to that of α-actin as the internal control. All experiments were performed in triplicate from three kidney preparations. Results are expressed as mean ± SD. *P < 0.05, **P < 0.01, #P < compared with cells grown in STFM for an equivalent duration.
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

7. Figure 6
Dose response of IL-8 (▪), MIF (□), MCP-1 (), and RANTES () gene expression in PTEC after transferrin challenge. Confluent growth-arrested PTECs were incubated with STFM or medium containing 1.25 to 20 mg/mL of transferrin. Duration of incubation was adjusted in accordance with the maximal time response observed for each chemokine: 12 hours for IL-8, 3 hours for MCP-1, and 24 hours for MIF and RANTES. Results were obtained from triplicate experiments of three different kidney preparations, and are shown as mean ± SD. *P < 0.01, **P < 0.005, #P < compared with cells grown in STFM for an equivalent duration.
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

8. Figure 7
Effect of transferrin on chemokine secretion by PTECs. Confluent growth-arrested cells were cultured in serum-free medium containing escalating doses of transferrin. After distinct time points, culture supernatants were harvested for enzyme immunoassay for the four chemokines, while cell number was counted. (A) Kinetics of chemokine biosynthesis after overnight incubation with 5 mg/mL transferrin. (B) Dose effect of transferrin stimulation on chemokine secretion. Results were obtained from triplicate experiments. Data are expressed as mean ± SD. Symbols are: (▪) IL-8; (□) MIF; () MCP-1; () RANTES; *P < 0.02, **P < 0.005, §P = 0.001, and #P < compared with unstimulated, growth-arrested cells grown in STFM for equivalent duration.
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions