Peritoneal dialysis

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Introduction and Aims: Increased expression of the C-C chemokine monocyte chemoattractant protein-1 (MCP-1) in mesothelial cells in response to high glucose concentrations and/or high osmolality plays a crucial role in the development of peritoneal fibrosis during continuous ambulatory peritoneal dialysis (CAPD). Recent studies suggest that in kidney cells osmolality-induced MCP-1 upregulation is mediated by the osmosensitive transcription factor nuclear factor of activated T cells 5 (NFAT5). We addressed the question of whether activation of NFAT5 by hyperosmolality, as present in PD fluids, contributes to MCP-1 expression in the mesothelial cell line Met5A. Methods: Met5A cells were incubated in isoosmotic (300 mosm/kg H2O) or hyperosmotic (400 mosm/kg H2O) medium. The medium osmolality was elevated by the addition of glucose. To distinguish between glucose-specific effects and osmolality-induced effects, mannitol or NaCl was used as osmotic controls. Expression of MCP-1 and NFAT5 was measured by RT-PCR and by ELISA or immunoblot assay. Cellular NFAT5 activity was tested by reporter gene assays. To evaluate the role of NFAT5 in osmolality-induced MCP-1 expression, NFAT5 was knocked down using a siRNA approach. Activation of the transcription factor NF-kB under hyperosmotic conditions was measured by reporter gene assay and by the use of phospho-specific antibodies. Results: Hyperosmolality, induced by addition of glucose, NaCl or mannitol to the growth medium, increased NFAT5 activity and stimulated MCP-1 expression in Met5A cells. NFAT5 activity was enhanced by multiple mechanisms: first, by increased NFAT5 expression; second, by stimulation of the c-terminal transactivation domain; and third, by enhanced translocation from the cytoplasm to the nucleus. siRNA-mediated knockdown of NFAT5 decreased osmolality-induced MCP-1 upregulation substantually. Hyperosmolality also induced activation of nuclear factor-kB (NF-kB). Accordingly, pharmacological inhibition of NF-kB significantly decrased osmolality-induced MCP-1 expression. Conclusions: Taken together, the present study indicates that the transcription factor NFAT5 is activated in response to high osmolalities in mesothelial cells, and that this activation contributes to increased expression of MCP-1, probably in collaboration with NF-kB. This mechanism may play an important role in the development of peritoneal fibrosis during CAPD.

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