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Objective: During long-term peritoneal dialysis (PD) the loss of ultrafiltration (UF) capacity determines the system survival of PD. For an adequate UF in chronic PD transcapillary UF, but also transcellular, free water transport is essential. Free water transport is considered to be associated to the expression and function of Aquaporin 1 (AQP1), a water channel protein, which has been found in peritoneal biopsy studies. With this regard, it was hypothesized that the generation of AGEs (advanced glycosylated end products) during chronic PD treatment affects the AQP1 mediated water transport by non enzymatic glycosylation. In addition to existing immunohistochemical studies in peritoneal biopsies, we now focused on the detection of free soluble AQP1 in the peritoneal effluent therefore establishing a new AQP1 sandwich ELISA. Methods: Since there are two different monoclonal AQP1 antibodies available it is possible to build up an AQP1 sandwich ELISA. Because of the fact that AQP1 is an integral protein and thus very hydrophobic it was impossible to generate a standard with purified AQP1. Therefore we enriched soluble AQP1 from the peritoneal dialysis effluent and determined the concentration via dot-blot analysis with purified AQP1. Enriched soluble AQP1 turned out to have a molecular weight of 95 kDa and therefore might be a fusion by a glycosylation process of albumin (68 kDa) and AQP1 (28 kDa). The sensitivity of the ELISA was about 1.8 ng/mL. Three samples of known concentration were tested twenty-four times on one plate to assess intra-assay precision. The CV (%) was 6.0, 5.7 and 5.8 for 8.19 ± 0.49 ng/mL, 61.8 ± 3.5 ng/mL and 132.5 ± 5.8 ng/mL, respectively. Effluent AQP1 after a 4 hrs dwell time (3.86% glucose) was measured (n = 30) in chronic PD patients (CAPD 11, CCPD 19), stable on PD. Mean PD treatment time was 13.9 ± 17.5 months. AQP1 was correlated to peritoneal transport and clinical data. Transport data were obtained by an extended peritoneal transport analysis using i.p. dextran as a volume marker. D/P and MTAC (mass transfer area coefficient) for creatinine as well as transcapillary UF, free water transport, effective ultrafiltration and lymphatic absorption were calculated. Results: The measured AQP1 concentration (4.43 ± 2.94 ng/mL; range 0-16.1 ng/mL) was corrected for the different effluent volumes. Mean AQP1 amount was 11.22 ± 7.05 mg per effluent (range 0-36.3 mg). The d/p for creatinine was 71.2 ± 10.3%, MTAC for creatinine was 11.2 ± 4.6 mL/min, transcapillary ultrafiltration was 1,108 ± 379 mL, effective ultrafiltration was 576 ± 241mL, lymphatic absorption was 174 ± 314 mL and free water transport was 149 ± 80 mL. No statistically significant correlation was found to any clinical (PD treatment time, age, CAPD/APD, diabetes/non-diabetes) or to the presented transport data. However, a tendency to an inverse relationship was seen for AQP1 concentrations and PD treatment time as well as free water transport (PD < 12 months 11.66 ± 8.64 mg vers. PD > 12 months 8.42 ± 4.02 mg; free water <140 ml 12.32 ± 9.09mg vers. > 140 ml 8.62 ± 4.76 mg). Conclusions: By composing a new sandwich ELISA for AQP1 we were able to measure soluble AQP1 in the effluent of chronic PD patients. In this pilot study effluent AQP1 concentrations did not correlate to small solute transport or parameters of peritoneal water transport especially to calculated free water transport although a tendency to an inverse relationship was seen. Correlating AQP1 to clinical data, effluent AQP1 concentrations tended to be lower in long term PD patients, but again statistical significance was missed due to the high standard deviation. [ABSTRACT FROM AUTHOR]

Objective: In peritoneal dialysis (PD) the usage of automated peritoneal dialysis (APD) has steadily increased, due to higher dialysis efficiency and quality of life. However, because of the repetitive exchanges of fresh and unphysiological dialysate in APD, we hypothesised in how far the known negative effects of low pH, glucose degradation products (GDP) and advanced glycosylation end products (AGE) on peritoneal cells might be even enhanced in APD- compared to the CAPD-mode. A recently established double chamber cell culture system, adequately mimicking the in vivo equilibration pattern and guaranteeing a sufficient lifetime of the investigated mesothelial cells, was used for a realistic comparison of CAPD and APD in vitro. Methods: Mesothelial cells (MC) (n = 10) were isolated from omental biopsies via trypsin/EDTA digestion and cultured. MC of the second passage were seeded on a microporous membrane (Anopore, Nunc, Germany; ∅25 mm, pore size 0.2µm) of a transwell insert. In a double chamber setting MC were continuously fed by the growth medium from the lower chamber. For the comparison of the PD modes, the test fluid was added to the upper chamber and with an 8 hrs lasting regimen of repetitive exchanges we compared a CAPD (4 hrs dwell time) with a CCPD setting (1 hr dwell time) -- as a typical APD regime -- using a lactate buffered PD solution (Dianeal 1.36%, Baxter). Pure medium exchanges served as a control. After a recovery phase, cell viability was assessed with a MTT-assay and cell function via constitutive and stimulated IL-6 release in the supernatant. Indicating potential induction of fibrosis TGF-1β generation and as a parameter of mesothelial cell integrity CA125 release were measured. Results were referred to the cell protein mass. Results: Both CAPD- (p < 0.05) and CCPD-mode (p < 0.01) resulted in a significantly lower mitochondrial activity (CAPD: 0.66 ± 0.22, CCPD: 0.57 ± 0.18, control: 0.80 ± 0.27 absorption/µg) and stimulated IL-6 release (CAPD: 2.08 ± 0.46, CCPD: 2.20 ± 1.20, control: 3.61 ± 1.47 pg/ml/µg; p < 0.001) compared to control. TGF-1β and CA125 release did not differ between the PD-modes and control. CAPD- and CCPD-mode itself did not differ with regard to MTT-assay, IL-6 release, TGF-1β and CA125 generation. Only constitutive IL-6 release was significantly lower in the CCPD-mode compared to the control (p < 0.05), whereas CAPD-mode and control did not differ (CAPD: 2.58 ± 1.63, CCPD: 1.96 ± 0.73, control: 2.71 ± 1.39 pg/ml/µg). Conclusions: The double chamber cell culture system with MC seeded on a microporous membrane of a transwell insert for the first time allowed an in vitro comparison of different PD modes, due to an adequate imitation of the in vivo equilibration system. As tested in this in vitro model, there is no evidence that APD due to the larger volumes of solution and more frequent contact times with fresh and unphysiological dialysate has a special and additive negative impact on biocompatibility parameters indicative for peritoneal host defence, mesothelial cell integrity and peritoneal fibrosis. [ABSTRACT FROM AUTHOR]