Osteopontin levels in the drained dialysate reflect the peritoneal solute transport rate

Background and objectives: Long-term peritoneal dialysis (PD) is accompanied by low-grade intraperitoneal inflammation, may eventually lead to peritoneal membrane injury with high solute transport rate and ultrafiltration failure. Osteopontin(OPN) is highly expressed with the pro-inflammatory cytokines stimulation in many cell types, and evolves in the process of tissue fibrosis. This study aimed to investigate the potential of OPN as a new indicator of peritoneal injury. Methods: We analyzed a total of 125 PD patients with end-stage renal disease, including 16 patients with continuous ambulatory PD(CAPD)-related peritonitis and 109 patients without peritonitis in a single renal center. The OPN levels in the overnight peritoneal effluents or in serum were analyzed using ELISA. In HMrSV5 cells, The OPN and fibronectin(FN) protein expression were identified using western blot analysis. Results: The OPN levels in overnight drained dialysate were significantly correlated with D/P Cr (P < 0.0001, R =0.54) and D/D0 glucose (P < 0.0001 R=-0.39). Logistical regression analysis showed that the OPN levels in peritoneal effluents was an independent predictive factor for the increased peritoneal solute transport rate (PSTR) (p < 0.001). The area under the receiver operating characteristic (ROC) curve of the OPN-PSTR model for identifying PSTR was 0.88, with 95% confidence interval (CI):0.81-0.95. The OPN was more abundant in peritoneal effluents of the CAPD-related peritonitis group compared with the patients without peritonitis (18.64±13.04 vs. 2.23±1.63 ng/ml, p<0.001). In the in vitro experiment, lipopolysaccharides(LPS) increased the OPN expression in HMrSV5 cells, whereas downregulation of OPN suppressed FN induction with transforming growth factor-β1(TGF-β1)stimulation. Conclusions: The OPN in effluents in patients with CAPD-related peritonitis. Peritoneal mesothelial cells displayed a high expression of OPN under inflammatory stimuli and OPN was likely to be implicated in the progression of peritoneal fibrosis. Thus, OPN may be a useful indicator of peritoneal injury in patients with PD.


Introduction
Peritoneal dialysis (PD) is a vital replacement therapy for patients with end-stage kidney disease. Approximately 11% of the dialysis population uses PD worldwide. During PD, the peritoneal membrane (PM) naturally removes waste products and excess fluid from the blood and transports them to the dialysis solution. However, long-term exposure to hyperglycemia, hyperosmotic, and acidic dialysis solutions often cause low-grade chronic inflammation and PM injury. The PM presents as progressive fibrosis, angiogenesis, and vasculopathy, which lead to increased solute transport and ultrafiltration failure. Among these conditions, progressing peritoneum fibrosis plays a significant role in peritoneal transport dysfunction, which is characterized by the accumulation of myofibroblasts in the peritoneum with fibronectin (FN) and collagen expression [1][2][3][4]. The peritoneal equilibration test (PET) is widely employed to assess PM transport function [5].
Taken together, we suggest that OPN may be secreted from mesothelial cells, macrophages, or fibroblasts in the PM through long-term and low-grade chronic inflammation stimulation during PD treatment. OPN is also likely to be involved in the development and progression of peritoneal fibrosis, which eventually affects the solute transport and ultrafiltration. We investigated whether OPN may be a new indicator of PM injury characterized by high solute transport via analyzing the association between the OPN concentration in drained dialysate and peritoneal solute transport rate (PSTR) obtained with PET.

Patients
From February 2018 to December 2018, 231 PD patients with end-stage renal disease at the PD unit of the First Hospital Affiliated of Soochow University were followed-up regularly. Sixteen patients who suffered from CAPD-related peritonitis were analyzed.
Among the remaining 215 patients, we selected the patients who were less than 75 years old and being treated with a CAPD prescription (four times exchanges per day). Other exclusion criteria included acute inflammatory processes, diagnosis peritonitis or abdominal trauma within the past six months prior to the study, active autoimmune diseases, and tumors, with incomplete clinical characteristics. Eventually, 109 CAPD patients without peritonitis were analyzed. Table 1

ELISA Osteopontin
All participants in the experiment were asked to perform a dialysis exchange according to the usual overnight dialysis regimen prior to PD center visit. The overnight effluent was fully drained next morning in the PD center. We collected a 10ml sample from each patient, which were stored at-80℃ immediately. OPN levels were quantified in peritoneal

Statistical analysis
Clinical data were presented as means ± Standard deviation (SD). Baseline characteristics of the study population were compared using one-way analysis of variance (ANOVA) for continuous variables and χ2 testing for categorical variables. Relationships between clinical variable and D/P Cr levels were analyzed with Spearman's correlation coefficient test. Independent factors affecting PSTR were analyzed by Logistic regression analysis, the model with prespecified adjustments for: gender, age, duration, blood albumin, Serum phosphate. In addition, to identify the possible predictors of increased PSTR, a logistic regression model was constructed for the probability of increased PSTR, and the equation as follows: probability = exp(c)/[1+exp(c)], where c is 1.23×OPN levels in peritoneal effluents-0.13×blood albumin+0.944. The optimal cut-off point was identified based on the maximum Youden index (sensitivity + specificity-1). In vitro data were presented as mean±S. E. Comparison between groups was made using one-way analysis of variance, followed by the Mann-Whitney U test. Western blot analysis was completed by scanning and analyzing the intensity of hybridization signals by using NIH ImageJ software package. P-value < 0.05 (two-tailed) was considered statistically significant. These statistical

Patient characteristics
A total of 109 CAPD patients without peritonitis were enrolled in this study. Among these patients, 55.3% were males with a mean age of 49.14 ±13.25 years and a median PD duration of 37.32 ± 35.01 months. Table 1 summarized the clinical characteristics of the study.

Correlation between OPN and peritoneal transport characteristics
The PSTR determined using PET was correlated with the OPN concentrations in overnight peritoneal effluents. Significant correlations were found between the OPN levels and D/P Cr (p < 0.0001, R = 0.54), D/D0 glucose (P < 0.0001 R = -0.39) through Spearman's correlation coefficient test (Figure 1). In the blood samples, no firm correlation was observed between the OPN levels and PET results ( Table 2). The patients were divided into either low and low-average transport (L/A) group, or high and high average transport (H/A) group based on the PET results. The OPN concentrations in peritoneal effluents were considerably higher in the H/A group than in the L/A group (3.05±1.94 vs. 1.25 ±1.03 ng/ml, P < 0.0001). The H/A group was more likely to be older (p = 0.007), with lower serum albumin (p<0.0001), and higher serum phosphate levels (p = 0.004) ( Table 3).
Logistic regression analysis showed that OPN in effluents was an independent predictive factor for the PSTR after adjusting for age, PD duration, gender, serum albumin, and serum phosphate (Table 4). To future examine the diagnostic accuracy of the OPN measured for identifying increased PSTR, the OPN-PSTR model was constructed, using two variables (OPN levels in the effluents and serum albumin). As described in Figure 2

Discussion
During long-term PD, the PM undergoes functional and structural alterations, eventually leading to PD drop-out and increased mortality [2,3,13]. PET is the most significant method for evaluating the transfer rate of solute and water across the peritoneal barrier, and includes three parameters: D/P Cr, D/D0 glucose, and 4h ultrafiltration volume.

Rajnish Mehrotra et al. demonstrated that D/P Cr is a robust predictor of all-cause
mortality and hospitalization through a study that enrolled a large and diverse cohort of patients undergoing PD in both unadjusted and adjusted models [5,14,15]. However, PET is an invasive method that requires blood sampling. In addition, patients need to be hospitalized. It will be of great interest to seek relevant biomarkers in peritoneal effluents, which can represent the transfer rate of the solute. The effluent concentrations of interleukin6(IL6), cancer antigen 125(CA125), vascular endothelial growth factor(VEGF), plasminogen activator inhibitor-1(PAI1), matrix metalloproteinase 2(MMP2), matrix metalloproteinase3(MMP-3), bone morphogenetic protein-7(BMP-7), and tissue inhibitor of metalloproteinases1(TIMP-1) levels in the dialysate obtained using PET were correlated with the D/P Cr ratios [14,[16][17][18]. The present study showed significant correlations exist between the OPN levels in the dialysate and D/P Cr, D/D0 glucose. The OPN concentrations in peritoneal effluents were remarkably higher in the H/A group than in the L/A group. OPN was an independent predictor factor for increased PSTR, which was evaluated using logistic regression analysis. In addition, by combining the concentrations of serum albumin and OPN in drained dialysate, our novel model demonstrated good performance in identifying increased PSTR.
Increasing evidence shows that chronic inflammation might be an initial factor for PM injury. After prolonged PD treatment, both MCs and peritoneal macrophages produce a wide array of inflammatory cytokines, such as IL-1 , tumor necrosis factor-(TNF-), and IL-6. Moreover, pro-fibrotic cytokines such as MMP-2, TGF-β, and CTGF, are also secreted, which may eventually lead to PM damage and functional abnormalities [3,13,19]. An In addition, OPN increased the recruitment of macrophages and T cells, which are implicated in the pathogenesis of acute and chronic inflammatory diseases, such as rheumatoid arthritis and bacterial infections [20]. Additionally, OPN can transform fibroblasts into active myofibroblast phenotype, and evolve in the EMT programs in several different cell lines, which results in fibrotic phenotypes [7,8] [21]. The partial EMT of mesothelial cells is implicated in peritoneal fibrosis and TGFβ1, which is a potent cytokine, that stimulates EMT and induces peritoneal fibrosis [22]. Thus, we further  Table3. Data are presented as means ± standard deviation (SD) or n (%);

Table4
Association between D/P Cr and OPN levels in the peritoneal effluents.    The levels of OPN in the peritoneal effluents from patients without or with infectious peritonitis. ***P < 0.001.