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Low biocompatibility of peritoneal dialysis fluid (PDF) injures mesothelial cells and activates their stress response. In this study, we investigated the role of heat shock proteins (HSP), the main cytoprotective effectors of the stress response, in cytoskeletal stabilization of mesothelial cells in experimental peritoneal dialysis. In cultured human mesothelial cells, cytoskeletal integrity was assessed by detergent extractability of marker proteins following in vitro PDF exposure. Effects of HSP on stabilization of ezrin were evaluated by a conditioning protocol (PDF pretreatment) and repair assay, based on coincubation of cytoskeletal protein fractions with recombinant HSP-72 or HSP-72 antibodies. In the rat model, detachment of mesothelial cells from their peritoneal monolayer during in vivo PDF exposure was assessed with and without overexpression of HSP-72 (by heat conditioning). In vitro, cytoskeletal disruption on sublethal PDF exposure was demonstrated by significantly altered detergent extractability of ezrin and ZO-1. Restoration was associated with significant induction and cytoskeletal redistribution of HSP during recovery. Both the conditioning protocol and in vitro repair assay provided evidence for HSP-72-mediated cytoskeletal stabilization. In the rat model, overexpression of HSP-72 following heat conditioning resulted in significantly reduced detachment of mesothelial cells on in vivo exposure to PDF. Our results establish an essential role of HSP in repair and cytoprotection of cytoskeletal integrity in mesothelial cells following acute in vitro and in vivo exposure to PDF. Repeated exposure to PDF, as is the rule in the clinical setting, may not only cause repeat injury to mesothelial cells but rather represents a kind of inadvertent conditioning treatment.

Background: Peritoneal dialysis is complicated by mesothelial cell injury due to low biocompatibility of peritoneal dialysis fluid (PDF). We have previously demonstrated that heat shock protein (HSP)-72 is potently up-regulated in response to PDF exposure of mesothelial cells in in vitro and in vivo models of peritoneal dialysis. The aim of this study was to evaluate potential cytoprotective effects of overexpression of HSP-72., Methods: Cytoprotection was assessed by comparing cellular viability between pretreated versus nonpretreated human mesothelial cells (Met 5a; ATCC, Manassas, VA, USA, and primary cell cultures) subjected to extended, usually lethal PDF exposure times (120 min, CAPD2; Fresenius, Bad Homburg, Germany). Pretreatment was performed with exposure to PDF (60 min, CAPD2; Fresenius) or heat (15 min, 41.5 degrees C), and by transient transfection with HSP-72., Results: When mesothelial cells were pretreated by nonlethal exposure to PDF or heat, HSP-72 was markedly up-regulated (>5-fold, P < 0.01). Pretreated human mesothelial cells were significantly protected against subsequent "lethal" exposures to PDF, as assessed by dye exclusion (>50% reduction, P < 0.05) and lactate dehydrogenase (LDH) release (>30% reduction, P < 0.05). Comparable cytoprotection (50% reduction by dye exclusion) was indicated by overexpression of HSP-72 in cultered human mesothelial cells (>5-fold) after transient transfection with HSP-72. This cytoprotection was confirmed at a cellular basis by double staining techniques with HSP-72 and ApopTag (apoptosis detection kit)., Conclusion: Our study therefore shows that the mesothelial stress response confers cytoprotection in experimental peritoneal dialysis, mediated by the induction of HSP-72, and that the stimulus of the pretreatment does not have to be identical to the subsequent injury. These data offer the basis for an attractive novel therapeutic approach against PDF toxicity.

In experimental transplant models, upregulation of renal heat shock proteins (HSP) represents a new therapeutic tool to improve allograft survival. In this clinical study, we hypothesized that HSP-72 expression in pretransplant donor kidney biopsies predicts posttransplant outcome. Expression of HSP-72 in pretransplant biopsies was assessed by immunohistochemistry and in situ hybridization in 82 consecutive renal transplantations and clinical data were collected prospectively during the first six months posttransplant. Renal tubular expression of HSP-72 was low and not influenced by donor-, graft-, or procedure-related risk factors. Neither strength nor pattern of pretransplant HSP-72 staining discriminated allografts with complicated (40%) posttransplant courses from those without complications (60%). The low HSP-72 expression in pretransplant donor kidney biopsies failed to predict delayed graft function or acute rejection. These findings suggest that constitutive HSP-72 gene expression at the time of engraftment does not play a role in graft protection.

Renal ischemia not only causes injury but also induces repair mechanisms, such as the cellular induction of the 72-kilodalton heat shock protein HSP-72. The aim of this study was to determine whether HSP-72 is excreted in urine after ischemic renal injury. The first urine of six pediatric allograft recipients was examined for proteinuria and urinary HSP-72 excretion. Sprague-Dawley rats were treated with renal ischemia or hyperthermia and renal cortex and urinary HSP-72 levels were determined. HSP-72 was excreted in the first urine of renal allografts. In rats, renal HSP-72 was induced both by renal ischemia or hyperthermia. However, only renal ischemia resulted in urinary excretion of HSP-72. Urinary excretion of HSP-72 indicates an increased renal stress response and loss of tubular cell integrity after clinical and experimental renal ischemia.

Background: We recently designed an in vitro system based on differential Triton-extractability of Na,K-ATPase from the cytoskeletal protein fraction isolated from rat renal cortex after renal ischemia. In the present study, we hypothesized that heat shock protein (HSP)-70, HSP-25 and HSP-90 work synergistically to stabilize the cytoskeletal anchorage of Na,K-ATPase., Methods: Cellular proteins were fractionated by differential centrifugation into cytoskeletal pellets (I-PEL) obtained early (exhibiting abnormally high Triton extractability of Na,K-ATPase) and non-cytoskeletal supernatants (R-SUP) obtained late (exhibiting high abundance of HSP) after renal ischemia. For assessment of the role of HSP-70, HSP-25 and HSP-90 upon in vitro re-compartmentalization, I-PEL was either incubated in R-SUP with/without HSP antibodies, or in buffer with/without HSPs at different titers and combinations. Effects were evaluated by changes of Triton extractability of Na,K-ATPase after co-incubation., Results: R-SUP was shown to contain significant amounts of HSP-70, HSP-25 and HSP-90. Incubation of I-PEL in R-SUP reduced Triton extractability of Na,K-ATPase. Addition of antibodies against each HSP significantly abolished these effects of R-SUP. Incubation of I-PEL with purified HSP-70, HSP-25 or HSP-90 each partly reproduced the effects of R-SUP, whereas the combination of all three HSP demonstrated a strong and more than additive effect on the cytoskeletal stabilization of Na,K-ATPase., Conclusions: The molecular mechanisms responsible for postischemic re-compartmentalization of Na,K-ATPase in rat renal cortex likely involves interactions between HSP-70, HSP-25 and HSP-90, stress proteins known to be induced in the ischemic kidney.

Background: Low biocompatibility of peritoneal dialysis fluids (PDF) contributes to mesothelial injury. We investigated whether the heat shock proteins (HSP)-27, HSP-72, and HSP-90 are differentially induced upon exposure of mesothelial cells to PDF and whether this was affected by selective modulation of the physicochemical properties of PDF., Methods: Human mesothelial cells (Met5A and primary human mesothelial cells) were exposed to acidic lactate and glucose-monomer based PDF (CAPD2 and CAPD3), to control culture media, or to a neutral lactate and glucose-monomer-based PDF with reduced levels of glucose degradation products (BALANCE). Expression of HSP-27, HSP-72, and HSP-90 and cellular distribution of HSP-72 were assessed by Western blotting and immunocytochemistry., Results: Mesothelial cells exhibited strong constitutive expression of HSP-27 and to a lesser extent HSP-72 and HSP-90. Exposure of the cells to CAPD2 and CAPD3 resulted in strong up-regulation of HSP-72. HSP-27 levels were slightly increased, but HSP-90 levels were unchanged upon exposure to CAPD2 or CAPD3. In contrast, exposure of the cells to BALANCE did not affect HSP-27 or HSP-72 expression. The acidic pH and glucose degradation products were found to be principal in mediating increased HSP-72 expression upon exposure to PDF., Conclusions: Analysis of HSP expression represents a novel tool to assess biocompatibility of PDF. Among the HSP investigated, HSP-72 is the most predictive and accurate parameter to assess mesothelial cell injury in the early phase of exposure to PDF.