Effect of standard and neutral-pH peritoneal dialysis solutions upon fibroblasts proliferation

Correspondência para: Bartira Ercilia Pinheiro da Costa. Pontifícia Universidade Católica do Rio Grande do Sul. Av. Ipiranga, no 6690, Hospital São Lucas da PUCRS, 2o andar Laboratório de Nefrologia, Instituto de Pesquisas Biomédicas. Porto Alegre, RS, Brazil. CEP: 90610-000. E-mail: bart@pucrs.br Tel: (51) 3367-700. Fax: (51) 3367-700. Peritosteril® and Balance® bags were provided by Fresenius Medical Care. Effect of standard and neutral-pH peritoneal dialysis solutions upon fibroblasts proliferation

Introduction: Continuous exposition of the peritoneal membrane to conventional dialysis solutions is an important risk factor for inducing structural and functional alterations.Objective: To compare in vitro mouse fibroblast NIH-3T3 cell viability after exposition to a neutral pH dialysis solution in comparison to cells exposed to a standard solution.Methods: Experimental study to compare the effects of a conventional standard or a neutral-pH, low-glucose degradation products peritoneal dialysis solution on the viability of exposed fibroblasts in cell culture.Both solutions were tested in all the commercially available glucose concentrations.Cell viability was evaluated with tetrazolium salt colorimetric assay.Results: Fibroblast viability was significantly superior in the neutral pH solution in comparison to control, in all three glucose concentrations (Optical density in nm-means ± SD: 1.5% 0.295 ± 0.047 vs. 0.372 ± 0.042, p < 0.001; 2.3% 0.270 ± 0.036 vs. 0.337 ± 0.051, p < 0.001; 4.25% 0.284 ± 0.037 vs. 0.332 ± 0.032, p < 0.001; control vs. neutral pH respectively, Student t Test).There was no significant difference in cell viability between the three concentrations of glucose when standard solution was used (ANOVA p = 0.218), although cell viability was higher after exposition to neutral pH peritoneal dialysis fluid at 1.5% in comparison to 2.3 and 4.25% glucose concentrations (ANOVA p = 0.008: Bonferroni 1.5% vs. 2.3% p = 0.033, 1.5% vs. 4.25% p = 0.014, 2.3% vs. 4.25% p = 1.00).Conclusion: Cell viability was better in neutral pH dialysis solution, especially in the lower glucose concentration.A more physiological pH and lower glucose degradation products may be responsible for such results.

IntRoductIon
Long-term peritoneal dialysis (PD) as therapy for end-stage renal disease (ESRD) patients is only feasible if adequate peritoneal membrane structure and function are preserved.Standard PD solutions are acid (pH = 5.5), lactate-buffered, with high glucose concentrations (75-220 mmol/L), hyperosmolar (334-486 mOsm/L) and contain high concentration of glucose degradation products (GDP) -known to affect peritoneal function. 1Lasting PD therapy with an intact peritoneal membrane has been, as yet, an unreached goal and the main drive to develop biocompatible PD solutions.Peritoneal membrane continuous exposition to acid solutions seems to be an important risk factor for structural and functional changes.Loss of mesothelial cells, small vessels changes, thickened and fibrotic sub-mesothelial compact zone have been observed at peritoneal biopsies. 2Functional changes have included ultrafiltration loss, small solutes clearance decline and, eventually, technique failure. 3sing biocompatible dialysis solutions is paramount in accomplishing long-term membrane survival. 4A neutral-pH, poor GDP dialysis solution, presented in a double-chambered bag has been developed in anticipation of improved biocompatibility. 3 Previous clinical and experimental studies have suggested its beneficial effects. 3,5,6The aim of the current study was to compare mouse fibroblast NIH-3T3 -a well characterized cell lineage -viability after exposition to either the standard acid or the neutral-pH dialysis solution.

methods
Mouse fibroblast NIH-3T3 cell cultures were placed in media containing conventional (Control) PD solution or a neutral-pH PD fluid.All the commercially available glucose (1.5, 2.3 and 4.25%) concentrations were tested.

DiAlysis solutions
Both solutions used glucose as osmotic agent.The control solution was Peritosteril ® (Fresenius Medical Care, Jaguariúna, Brazil) and the neutral-pH solution was Balance ® (Fresenius Medical Care, Bad Homburg, Germany) provided in a dual-chambered bag.The composition of both dialysis solutions is presented in Table 1.The 1.5% glucose Balance ® bag contains an alkaline chamber with sodium lactate (sodium: 75 mmol/L; lactate: 70 mmol/L) and a second acid fluid chamber with electrolytes and glucose (sodium: 193 mmol/L; calcium: 3.5 mmol/L; magnesium: 1 mmol/L; chloride: 203 mmol/L; glucose: 166.5 mmol/L).The Balance ® solution is ready for use by opening the seal between the two chambers and mixing their contents, resulting in a fluid with pH in the range of 6.8 to 7.4.Differently, Peritosteril ® has a pH of 5.5.Both Peritosteril ® and Balance ® bags were provided by Fresenius Medical Care.

Results
Cells viability after exposition to standard (Control) and neutral-pH solutions with different glucose concentrations is depicted in Figure 1.Cells viability was significantly greater on the neutral pH than on the acid solution, at all tested glucose concentrations, measured by optical density (in nm): 0.295 ± 0.047 vs. 0.372 ± 0.042, p < 0.001 for 1.5% glucose control and neutral pH, respectively; 0.270 ± 0.036 vs. 0.337 ± 0.051, p < 0.001 for 2.3% control and neutral pH, respectively; 0.284 ± 0.037 vs. 0.332 ± 0.032, p < 0.001 for 4.25% control and neutral pH solution, respectively.No significant differences in cell viability among the three control solution glucose concentrations were observed (repeated-measures ANOVA: p = 0.218).However, cells viability on neutral-pH solution was greater after exposition to 1.5 than to 2.

dIscussIon
The current experiments demonstrated that cells viability is greater when fibroblasts are exposed, in vitro, to a neutral pH than to an standard PD solution.Besides, greater viability of cells exposed to 1.5%, in comparison to 2.3 and 4.25% glucose on the neutral pH solution was evidenced.The significance of such findings is not yet clear, but it is conceivable that a neutral pH and less glucose degradation products are better for cell viability and that such effect may be amplified at lower glucose concentrations.Improved cell viability with the neutral pH solution does not mean that it is nontoxic or does not induce fibrosis.Fibroblasts were used as an experimental model [7][8][9] and evaluating the effects of such exposition in other cell models, or in experimental animals, would be desired. 10,11Use of the neutral pH dialysis solution, in vivo, resulted in improved peritoneal ultrafiltration and peritoneal membrane integrity markers. 3,12It also improved mesothelial cells proliferation and viability. 13 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide-based (MTT) colorimetric assay was used to evaluate cell viability.Its use allows measuring citotoxicity, cell proliferation and cell activation.The assay is based on viable cell mitochondrial dehydrogenases to transform MTT into a water-insoluble blue formazan.The assay method is not time consuming, cheap and allows for using multiwall scanning spectrophotometry. 14ibroblasts have also been used in a previous study, which found no difference in cell proliferation with incubation in either icodextrin-containing or standard glucose dialysis solution. 15The study was carried out on pH-adjusted solutions mixed with culture medium, a major distinction from the present work, where test solutions pH difference was a major characteristic being evaluated.Interestingly, heat sterilized 4.25% glucose solution caused a significant reduction in in vitro cell growth, compared with filter-sterilized solution, supposedly associated with increased GDP. 15 Occurrence of GDP and low pH seem to be important factors related with biocompatibility of PD solutions.GDP have been implicated in the formation of advanced glycation end products (AGEs) occurring during the heat sterilization process.The presence of AGEs has been associated with reduce mesothelial cell growth in vitro. 16It has been previously shown that in vitro cells exposed to lower glucose concentrations (1.5 or 2.27%) grow better than at higher (3.86%). 17uch results are in agreement with findings of this study, suggesting that GDP are highly inhibitory of in vitro cell growth (17) and could be related with clinical consequences. 18,19The neutral pH peritoneal dialysis solution was designed as a two-chamber bag, one containing the alkaline component with lactate and the other with electrolytes and glucose.Such approach allows for a higher pH and sterilization with reduced formation of GDP.The two-compartments fluids are mixed immediately before infusion, by rupturing a seal between the chambers.
In conclusion, fibroblasts viability was higher on neutral-pH dialysis solution, especially with lower glucose concentrations.It is possible that a more physiological pH and lower GDP are associated with such results.

Figure 1 .
Figure 1.Fibroblast viability after cells were exposed to acid and neutral pH solutions at different glucose concentrations.