Abstract
BackgroundBicarbonate-based peritoneal dialysis (PD) fluids enhance the migratory capacity and damage-repair ability of human peritoneal mesothelial cells by upregulating AQP1. However, little is known about the underlying molecular mechanisms.ResultsHere we used HEK-293T cells to investigate the effect of pH on AQP1 gene transcription levels. We found that AQP1 mRNA levels increases with pH. Transfection of HEK-293T cells with luciferase reporter vectors containing different regions of the AQP1 promoter identified an upstream region in the AQP1 gene between − 2200 and – 2300 bp as an enhancer required for pH-mediated regulation of AQP1 expression. Site-directed mutagenesis of this specific promoter region revealed a critical region between − 2257 and − 2251 bp, and gene knock-down experiments and ChIP assays suggested that the Spi-B transcription factor SPIB is involved in pH-mediated regulation of AQP1 expression.ConclusionsWe identified an upstream region in the AQP1 gene and the transcription factor SPIB that are critically involved in pH-mediated regulation of AQP1 expression. These findings provide the basis for further studies on the pH- and buffer-dependent effects of PD fluids on peritoneal membrane integrity and function.
Highlights
Bicarbonate-based peritoneal dialysis (PD) fluids enhance the migratory capacity and damage-repair ability of human peritoneal mesothelial cells by upregulating aquaporin 1 (AQP1)
Effect of pH on AQP1 expression To investigate the regulatory effects of pH on AQP1 gene expression, HEK-293T cells were first used as a research tool to examine whether AQP1 mRNA levels were upregulated at certain pH levels
Quantitative PCR demonstrated a significant upregulation of AQP1 expression with pH, with two-fold higher mRNA levels at pH 8 than those at pH 6 (P = 0.0009, one way analysis of variance (ANOVA), Fig. 1a)
Summary
Bicarbonate-based peritoneal dialysis (PD) fluids enhance the migratory capacity and damage-repair ability of human peritoneal mesothelial cells by upregulating AQP1. Novel PD solutions that separate glucose from the buffer at a very low pH contain fewer glucose degradation products (GDPs) and have a neutral to physiological pH after mixing. Several studies suggest that these solutions can reduce peritoneal cell damage and preserve the peritoneal membrane host defence and transport functions. The solutions contain lactate, bicarbonate or a mixture of both buffers. A prospective paediatric trial that compared a pure bicarbonate buffer to a purely lactate-based low GDP fluid demonstrated better preservation of ultrafiltration capacity over 10 months [10]. The underlying mechanisms remained elusive, and the respective impacts of the solutions on cellular pH are unknown. The ultrafiltration capacity of the peritoneum is closely related to peritoneal aquaporin 1 (AQP1) expression. AQP1 is expressed in peritoneal capillaries, post-capillary venules and peritoneal mesothelial
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