Volume 66, Issue 6, Pages (December 2004)

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Volume 66, Issue 6, Pages 2300-2307 (December 2004) Overexpression of HSP-72 confers cytoprotection in experimental peritoneal dialysis Bettina Bidmon, Michaela Endemann, Klaus Arbeiter, Dagmar Ruffingshofer, Heinz Regele, Kurt Herkner, Oliver Eickelberg, Christoph Aufricht Kidney International Volume 66, Issue 6, Pages 2300-2307 (December 2004) DOI: 10.1111/j.1523-1755.2004.66040.x Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 1 Scheme of the cytoprotection assay. Human mesothelial cell cultures at confluence either underwent sham media changes (no pretreatment) or nonlethal stress by PDF or heat pretreatment (PT). The next day, nonpretreated and PT cells were either subjected to sham media changes or lethal PDF exposure (LT). Mesothelial cell cultures from the resulting 4 treatment groups (Con, LT, PT+LT, PT) were compared at appropriate recovery times for HSP expression or cellular viability, as described in the Methods section. Kidney International 2004 66, 2300-2307DOI: (10.1111/j.1523-1755.2004.66040.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 2 Cytoprotection in cultured human mesothelial cells pretreated by nonlethal PDF exposure. Immunofluorescence staining for HSP-72 is shown in the upper panel: nonpretreated cultures (right upper panel) and cultures pretreated by PDF for 60 minutes (PT) and allowed to recover for 24 hours (left upper panel). Fluorescence staining for propidium iodide (PI) after lethal treatment (lower panel) is shown in cells exposed to sham treatment (Con) or to usually lethal PDF exposure (LT, PT, PT+LT). Nonlethal PDF pretreatment resulted in marked HSP-72 induction and protection of cellular viability (PT+LT). Data are representative for 3 independent experiments. Kidney International 2004 66, 2300-2307DOI: (10.1111/j.1523-1755.2004.66040.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 3 Quantification of cytoprotection conferred by two different conditioning protocols in human mesothelial cells. After lethal PDF exposure, LDH release was compared between nonpretreated cultures and cultures pretreated (PT) either with PDF (upper panel) or heat shock (lower panel). Compared with nonpretreated cells (LT), LDH release was significantly reduced in cells with prior nonlethal pretreatment (PT+LT). There was no effect on LDH release by the pretreatment alone (PT). Cytoprotective effects were similar with both stressful stimuli. Data are derived from 9 to 12 independent experiments. Kidney International 2004 66, 2300-2307DOI: (10.1111/j.1523-1755.2004.66040.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 4 Transfection with HSP-72 or bare vector. HSP-72 expression after transfection with control (A), bare vector (B), or HSP-72 (C) shown by immunofluorescent staining and on protein level. HSC staining is shown for loading control. Kidney International 2004 66, 2300-2307DOI: (10.1111/j.1523-1755.2004.66040.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 5 Quantification of cytoprotection after transfection with HSP-72 or bare vector. Viability assessed by Vital Dye exclusion after either exposure to normal culture media or lethal PDF exposure for 120 minutes compared between HSP-72–transfected cells and bare vector. Viability is significantly preserved in cultures pretreated by HSP-72 transfection (N = 24). HSP-72 transfection resulted in a marked increase of HSP-72 expression at the time of PDF exposure compared to bare vector transfection. Kidney International 2004 66, 2300-2307DOI: (10.1111/j.1523-1755.2004.66040.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 6 Double staining of HSP-72 transfected cells. Immunofluorescentstaining for HSP-72 (green) and for ApopTag (red) in primary human mesothelial cells. Overexpression of HSP-72 after transfection and PDF exposure was particularly prevalent in vital cells (ApopTag negative), whereas ApopTag staining was exclusively found in HSP-72 negative cells, thus directly confirming HSP-72–mediated cytoprotection at a cellular basis. Data are representative for 3 independent experiments. Kidney International 2004 66, 2300-2307DOI: (10.1111/j.1523-1755.2004.66040.x) Copyright © 2004 International Society of Nephrology Terms and Conditions