Interaction between proximal tubular epithelial cells and infiltrating monocytes/T cells in the proteinuric state  K.N. Lai, J.C.K. Leung, L.Y.Y. Chan, H. Guo, S.C.W. Tang  Kidney International  Volume 71, Issue 6, Pages 526-538 (March 2007) DOI: 10.1038/sj.ki.5002091 Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 1 Polarized release of CCL2, CCL5, sICAM-1, and IL-6 by PTEC. (a) Increased chemokine release by PTEC was observed only when HSA was added to the apical chamber of the Transwell. (b) No change in chemokine release by PTEC was observed when HSA was added to the basolateral chamber of the Transwell. (c) Increased chemokine production by PTEC was observed only when transferrin was added to the apical chamber of the Transwell. (d) No change in chemokine release by PTEC was observed when transferrin was added to the basolateral chamber of the Transwell Results are means±s.d. obtained from five separate experiments. **P<0.01 and *P<0.05 versus values with control medium from corresponding apical or basolateral compartment. Kidney International 2007 71, 526-538DOI: (10.1038/sj.ki.5002091) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 2 Setup for co-culturing PTEC and monocytes/T-cells to study the crosstalk between monocytes/T cells and PTEC preincubated with HSA. (a) Two culture systems were used to delineate the contribution of soluble factors and cell-to-cell contact in the crosstalk between monocytes/T cells and PTEC preincubated with HSA. In system I, PTEC and monocytes/T cells (5 × 104 each) were physically separated in different chambers with monocytes/T cells seeded into the basolateral chamber) by 0.4-μm pore size semi-permeable membranes (which effectively inhibited cell movement) while sharing the same medium. In system II, PTEC (5 × 104 in 300μl medium) were first seeded onto the bottom surface of inverted insert (onto a 3-μm pore size semi-permeable membrane) for 4h to allow cell anchorage. The culture insert was then flipped over and the same numbers of T cells or monocytes were seeded onto the other surface of the chamber. (b) In system I, T cells or monocytes (arrow; stained green by fluorescein isothiocyanate-anti-CD45) are much bigger as compared with the 0.4-μm pore. (c) In system II, T cells or monocytes (arrowhead; stained green by fluorescein isothiocyanate-anti-CD45) can contact with cultured PTEC through the 3-μm pore (white circle). Transverse section shows the transmigration of the T cells/monocytes through the pore. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole diacetate (blue). Original magnification × 400. Kidney International 2007 71, 526-538DOI: (10.1038/sj.ki.5002091) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 3 Release of mediators PTEC co-cultured with monocytes/T cells with no cell contact (System I). Release of (a) CCL2, (b) CCL5, (c) sICAM-1, and (d) IL-6 by PTEC co-cultured with monocytes/T cells with no cell contact (System I). PTEC were exposed to 5mg/ml HSA on the apical surface. Results are means±s.d. obtained from five separate experiments. **P<0.01 and *P<0.05 versus values with conditioned medium (PTEC with HSA only) from corresponding apical or basolateral compartment. Kidney International 2007 71, 526-538DOI: (10.1038/sj.ki.5002091) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 4 Release of mediators by PTEC co-cultured with monocytes/T cells with cell contact (System II). Release of (a) CCL2, (b) CCL5, (c) sICAM-1, and (d) IL-6 by PTEC co-cultured with monocytes/T cells with cell contact (System II). PTEC were exposed to 5mg/ml HSA on the apical surface. Results are means±s.d. obtained from five separate experiments. **P<0.01 and *P<0.05 versus values with conditioned medium (PTEC with HSA only) from corresponding apical or basolateral compartment. Kidney International 2007 71, 526-538DOI: (10.1038/sj.ki.5002091) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 5 Effect of HSA-PTEC conditioned medium on chemotactic capacity of monocytes/T cells. Expression of chemokine receptors CCR1 (plain bar), CCR2 (dotted bar), and CCR5 (striped bar) in different subset of T cells (a) (CD3+, (b) CD4+, or (c) CD8+ T cells) as well as (d) monocytes harvested from the upper chamber following exposure to HSA-PTEC conditioned medium in the lower chamber of the Transwell. Results are means±s.d. obtained from five separate experiments and are expressed as percentage of T-cell subset/monocyte expressing specific chemokine receptors. **P<0.01 and *P<0.05 versus baseline values in the corresponding subset. NS signifies statistically not significant. Kidney International 2007 71, 526-538DOI: (10.1038/sj.ki.5002091) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 6 Chemotaxis of monocytes/T cells: effects of neutralizing anti-CCL2 or anti-CCL5 antibodies. Monocytes or T cells seeded onto the upper chamber of the Transwell insert were cultured for 2h with HSA-PTEC conditioned media (apical: light bar or basolateral medium: dark bar) added to the lower chamber of the Transwell. The number of (a) CD3+T cells, (b) CD4+ T cells, (c) CD8+ T cells or (d) monocytes migrated to the lower chamber of the Transwell was counted. Results are means±s.d. obtained from five experiments and are expressed as percentage of total number of input T-cell subset/monocyte. **P<0.01 and *P<0.05 versus baseline values in the corresponding subset. NS signifies statistically not significant. Kidney International 2007 71, 526-538DOI: (10.1038/sj.ki.5002091) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 7 Effect of HSA-PTEC conditioned medium on the expression of chemokine receptors by monocytes/T cells. CD3+T cells (a), CD4+T cells (b), CD8+T cells (c) or monocytes (d) were cultured overnight with medium alone (white bar) or conditioned media (gray bar: apical medium; black bar: basolateral medium) prepared from PTEC following overnight apical exposure to 5mg/ml HSA. Chemokine receptor expression was then determined by flow cytometry. Results are means±s.d. obtained from five separate experiments and are expressed as percentage of T-cell subset/monocyte expressing specific chemokine receptors. **P<0.01 and *P<0.05 versus control medium or apical medium. NS signifies statistically not significant. Kidney International 2007 71, 526-538DOI: (10.1038/sj.ki.5002091) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 8 The effect of different neutralizing antibodies or PDTC on the release of mediators by PTEC co-cultured with monocytes/T cells. Release of (a) CCL2, (b) CCL5, (c) sICAM-1, and (d) IL-6 by PTEC co-cultured with monocytes/T cells: effect of different neutralizing Abs or PDTC. Results are means±s.d. obtained from five separate experiments. **P<0.01 and *P<0.05 versus values with HSA-PTEC conditioned medium (with CD3+ T cells or monocytes) from corresponding apical or basolateral compartment. ##P<0.01 and #P<0.05 versus values with HSA-PTEC conditioned medium (without CD3+ T cells or monocytes) from corresponding apical or basolateral compartment. Kidney International 2007 71, 526-538DOI: (10.1038/sj.ki.5002091) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 9 Schema of interaction between T cells/monocytes and PTEC in pathogenesis of tubulointerstitial injury in proteinuric glomerulopathies. Phase I: albuminuria due either to glomerular overflow or reduced tubular reabsorption interacting with PTEC triggers the generation of reactive oxygen species and release of pro-inflammatory cytokines. Phase II: these events lead to the apical release of pro-inflammatory IL-6 and sICAM-1 as well as basolateral release of chemokines (CCL2, CCL5, and CXCL8) and sICAM-1. Phase III: subsequently, mononuclear cells are recruited from microcirculation to the interstitial tissue via chemotaxis. Phase IV: chemokines released by albumin-activated PTEC will differentially upregulate the expression of chemokine receptors on infiltrating mononuclear cells. Phase V: the T cells and monocytes stimulate the PTEC via soluble factors including TNF-α and IL-1β. These events further amplify and perpetuate the release of IL-6, sICAM-1, CCL2, and CCL5 resulting in inflammatory injury in the renal interstitium. Denotes inhibition and ⊕ denotes enhancement. Kidney International 2007 71, 526-538DOI: (10.1038/sj.ki.5002091) Copyright © 2007 International Society of Nephrology Terms and Conditions