The classification of peritoneal transport characteristics is primarily measured 4–8 weeks after PD start by PET. This proteomic study focused on developing new and better biomarkers to distinguish the transport characteristics directly before PD start. This research identified and verified two proteins, ORM2 and CRP, as biomarkers to predict the baseline peritoneal transport characteristics.
ORM, also known as AGP (alpha-1-acid glycoprotein), mainly including ORM1 and ORM2, belongs to acute-phase protein family and is heavily glycosylated (45%). ORM is almost synthesized in the liver, and it can also be produced by endothelial cells, then released into the circulation. Increased ORM expression has been associated with acute infection, chronic diseases, and different kinds of cancers[15, 16]. Previous research showed that serum ORM was higher in ESRD patients before PD start compared with healthy controls in small series, but they do not classify different peritoneal transport characteristics. In this study, the result of western blot showed that ORM2 in LA&L group was higher than H, HA and NC group, clearly confirmed our proteomic data. Therefore, LA&L group could be distinguished from H, HA group by increased serum ORM2 level.
We also validated that elevated serum ORM2 was associated with its increased expression in peritoneum, suggesting ORM2 was directly involved in peritoneal transport. Peritoneum has size barriers and charge barriers, which affected the ability of a molecule to permeate the peritoneum[18, 19]. ORM is the negatively charged plasma protein, due to the high level of sialic acid content at the terminal of glycan chains[13, 20]. The isoelectric point (pI) value of ORM is 2.7–3.2, while the pI value of CRP is 7.9-9.0[21, 22]. ORM has been reported to increase the polyanionic charge selectivity, inhibit the trans-vascular leakage of negatively charged substances like proteins or albumin, and protect vascular permeability in endothelial barrier and blood-brain barrier by increasing tight junction protein (occludin and zonula occludens-1) expression[24, 25]. It also can suppress the renal filtration of albumin as the major components of the glomerular endothelial cell coat. In our study, we found that peritoneal total proteins and albumin loss were reduced after giving exogenous ORM2 to mice. Due to the negative charge effect of ORM2 as known, we thought that local high expression of ORM or giving exogenous ORM may add more negative charge to the surface of cell, thus reduce the loss of negatively charged proteins or albumin into the PDE (Fig. 7). Therefore, it may explain why the patients in LA&L group with higher serum ORM2 had higher serum albumin than H group. Previous study also showed that higher baseline peritoneal transport characteristics were associated with lower serum albumin. Peritoneal albumin loss was one of the causes of hypoalbuminemia in PD patients, which was also related to malnutrition, increased plasma viscosity, carotid atherosclerotic plaques and cardiovascular events[29, 30]. These results indicated that supplying exogenous ORM in PD patients is likely to have a protective effect by preventing hypoalbuminemia caused by high peritoneal transport.
Creatinine and urea are both small molecule toxins which can be partly eliminated by PD therapy. D/P urea is correlated positively with D/P creatinine. The 2 h PET result in mice showed that there was no significant difference of D/P urea between PET and PET + ORM groups. It suggested that giving 30 mg/kg ORM to mice as an appropriate concentration did not influence the elimination of small molecule toxins.
Higher peritoneal transport characteristics was reportedly associated with intraperitoneal inflammation, which was largely independent of systemic inflammation[31, 32]. ORM has both pro-inflammatory and anti-inflammatory roles in different diseases[25, 33, 34]. Whether ORM can stimulate HPMCs and PVECs to express pro-inflammatory or anti-inflammatory cytokines remains unknown. Thus, we measured the mRNA level of cytokines in mice peritoneum to exclude the influence of local inflammation. PET with or without ORM neither increased the expression of IL-6, TNF-α and IL-10 in peritoneum (Additional file 2: Figure S1), suggesting that the mice did not have peritonitis, and ORM was safe and effective in reducing proteins loss without causing an excessive pro-inflammatory response.
Increased serum CRP is associated with inflammation, peritoneal fibrosis, cardiovascular morbidity and overall mortality[35, 36]. CRP-transgenic mice had higher D/P creatinine ratio than that of WT mice. A cross-sectional study showed that CRP in H and HA group was higher than LA and L group, and CRP has been verified to be able to distinguish different transport status in long dialysis vintage PD patients. We found that H group also had higher serum CRP concentration compared with LA&L group or NC group, which means CRP may have the capacity to discriminate the H transport characteristic from LA&L transport characteristic in incident PD patients, too. Additionally, a collection of biomarkers, ORM2 and CRP, can outperform a single biomarker to discriminate different peritoneal transport characteristics.
The limitations of this study were that the sample size was small; the function of ORM was based on experiment in non-uremic animals; the result could only predict the baseline peritoneal transport characteristics. Peritoneal transport characteristics change over time, and the serum ORM2 and CRP may be influenced by dialysis vintage. Future studies should focus on the quantitative investigation of ORM2 in larger PD patient cohorts to provide robust validation, and the mechanism associated with ORM and peritoneal transport characteristics by assessing the charge on the human peritoneum.