The prognosis and risk factors of baseline high peritoneal transporters on patients with peritoneal dialysis

Abstract The relationship between baseline high peritoneal solute transport rate (PSTR) and the prognosis of peritoneal dialysis (PD) patients remains unclear. The present study combined clinical data and basic experiments to investigate the impact of baseline PSTR and the underlying molecular mechanisms. A total of 204 incident CAPD patients from four PD centres in Shanghai between 1 January 2014 and 30 September 2020 were grouped based on a peritoneal equilibration test after the first month of dialysis. Analysed with multivariate Cox and logistic regression models, baseline high PSTR was a significant risk factor for technique failure (AHR 5.70; 95% CI 1.581 to 20.548 p = 0.008). Baseline hyperuricemia was an independent predictor of mortality (AHR 1.006 95%CI 1.003 to 1.008, p < 0.001) and baseline high PSTR (AOR 1.007; 95%CI 1.003 to 1.012; p = 0.020). Since uric acid was closely related to high PSTR and adverse prognosis, the in vitro experiments were performed to explore the underlying mechanisms of which uric acid affected peritoneum. We found hyperuricemia induced epithelial‐to‐mesenchymal transition (EMT) of cultured human peritoneal mesothelial cells by activating TGF‐β1/Smad3 signalling pathway and nuclear transcription factors. Conclusively, high baseline PSTR induced by hyperuricaemia through EMT was an important reason of poor outcomes in CAPD patients.


| INTRODUC TI ON
Peritoneal dialysis (PD) is now globally used as an effective replacement therapy for patients with end-stage renal disease (ESRD).
Ultrafiltration failure and cardiovascular events are still the main reasons for the mortality and withdrawal of long-term PD patients. 1 The success of PD depends on the integrity of the peritoneal structure and function. It has been demonstrated that the peritoneum may exhibit submesothelial thickening and vasculopathy at the time of PD catheter insertion in patients with ESRD. 2 Previous study had also confirmed that intraperitoneal and systemic inflammation increases during the first year of PD therapy and inflammation may partly be responsible for the development of a high peritoneal solute transport rate (PSTR). 3 The most common peritoneal functional alteration is impaired ultrafiltration and decreased dialysis efficiency caused by fast PSTR. 4 Ultrafiltration failure is the main limitation of long-term PD treatment. Peritoneal transport function refers to the permeability of the peritoneum to transport small solutes. PSTR is measured by dialysate-to-plasma (D/P) ratios of low-molecularweight solutes through the peritoneal equilibration test (PET). A high PSTR leads to rapid re-absorption of glucose and increasing in protein loss, which followed by fluid overload and malnutrition. 5 Since the transport function of peritoneal membrane varies widely among individuals, it is more accurate to study based on different types of fast transporters. There may exist two distinct types of high transporters, the early inherent phenotypes and the late acquired type. [6][7][8] The late acquired type, a consequence of continuous exposure to bioincompatible PD solutions, has no longer been considered predictors of poor outcomes with the application of automated peritoneal dialysis (APD) and the icodextrin-based PD solution. 5,[9][10][11] While among those PD patients with early inherent phenotypes of baseline high PSTR, several previous studies demonstrated that they had more technical failure and higher mortality rate. [12][13][14] However, there are also studies showed the opposite results. [15][16][17][18] The discrepancy may due to differences in the study population, regional diversity, sample size, primary disease of chronic kidney disease (CKD) or other chronic illness burden is hard to homogenize. According to these existing research results, we still do not fully understand whether a high baseline PSTR was associated with poor prognosis.
The potential causes of high PSTR might be increased peritoneal capillary perfusion and vascular numbers, both of which may be inherent or acquired. During long-term PD, peritoneal membrane is continuously exposed to high dextrose concentration dialysate, coexisted with the other inflammatory stimuli such as peritonitis, and the peritoneal membrane may develop many structural abnormalities, including angiogenesis and submesothelial fibrosis. 19,20 While persistent intraperitoneal inflammation may ultimately lead to fast PSTR and peritoneal fibrosis (PF). 2,[21][22][23] Another study considered that inflammation, along with comorbidity and low serum albumin, was independent predictors of higher baseline peritoneal permeability. 24 Moreover, Pletinck A. et al. reviewed factors including haemoglobin A1c level, salt intaken and genetic polymorphisms which have important effects on the peritoneal membrane and can result in variability of peritoneal function. 25 Therefore, there is no consistent view of the cause of the increased peritoneal permeability.
In terms of molecular mechanism, epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells has been proved to be associated with high peritoneal transport 26 which occurred even early in CAPD and will be developed during long-term exposure to high glucose dialysate, mechanical denudation, profibrotic factors such as TGFβ and inflammatory cytokines. 27 When EMT occurs, the mesothelial cells adopt a more fibrogenic characteristic and acquire a proliferative, migratory and invasive phenotype. 28 As a result, a large amount of neovascularization and accumulation of extracellular matrix accelerate tissue fibrosis, alter peritoneal transport status and lead to ultrafiltration failure. 27,29 Recently, Mizuiri et al. evaluated the association between PSTR and the expression of effluent markers related to EMT and they found effluent hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) levels were significantly higher in the patients with high transport rate. 26 Moreover, a review newly published by our team systematically summarizes the angiogenic effect of VEGF. VEGF is associated with high peritoneal transport rate while impaired mesothelial cells are the major sources of VEGF in the peritoneum. 30 Since the evaluation of peritoneal function of patients is after the start of peritoneal dialysis, there are still a large number of patients with initial high PSTR who undergo PD and their prognosis is not clear. This study was undertaken to evaluate the subsequent overall survival and technique survival of the PD patients grouped by their baseline peritoneal transport status, analyse independent predictive risk factors of high baseline peritoneal transport status and explore its potential pathogenesis through in vitro experiments.

| Study design and participants
The present analysis was a multicentre retrospective cohort study that included all of the incident PD patients, aged between 18 and performed CAPD 3 to 5 times a day, and daily dialysis dose ranged from 6000 to 10000ml. All the participants underwent a PET after the first month of PD treatment. The exclusion criteria were as follows: patients who underwent PD treatment for less than 6 months, aged less than 18 years or over than 75 years, patients who suffered from peritonitis within the first 3 months, patients who had malignant tumour, liver cirrhosis, active tuberculosis, history of acute myocardial infarction and major surgical trauma within 3 months before starting PD, patients who initiated PD in other PD centres and previously accepted haemodialysis (HD) or kidney transplantation. These enrolled patients were followed until cessation of PD, death or on 30 September 2020.

| Demographic and Clinical Data
The baseline data collected consist of information about the demographic details, clinic and biochemical tests and a limited range of comorbidities including diabetes mellitus (DM), hypertension and cardiovascular disease (CVD). CVD included previous and present history of congestive heart failure, ischaemic heart disease or cerebrovascular disease. A fasting venous blood sample was collected before the morning exchange. Blood biochemical examination was analysed by standard techniques. Corrected serum calcium (cSCa) was correction with the following formula: cSCa = serum Ca + 0.8*(4.0-serum albumin [ALB]) (if serum ALB < 4g/dl).

| Peritoneal equilibration test
Baseline PET was performed 1 month after initial of dialysis.
According to Twardowski, 31 a standard 4-h dwell period was used, using a 2.5% dextrose PD solution for a 2 L volume exchange after 2.5% dextrose PD solution dwelling overnight (8 ~ 12 h

| Outcomes
The endpoint of the study was the patient status (dead or alive) or technical failure like transferring to HD at termination of the followup period (30 September 2020).
Reactive oxygen species (ROS) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Peritoneal dialysis fluid (2.5%) was purchased from Baxter Healthcare (Guangzhou, China). In order to confirm the additive effect of uric acid and high glucose (HG) in HPMCs, uric acid (800 µM) and 2.5% HG peritoneal dialysis fluid were used singly or in combination for 36 h before cell harvesting. All of the in vitro experiments were repeated for at least three times.

| CCK-8 proliferation assay
The CCK-8 proliferation kit was used according to the manufacturer's instructions. HPMCs were starved for 24 h with DMEM/F12 containing 0.5% FBS and then exposed to uric acid in different doses

| Wound-healing assay
HPMCs were seeded into a 6-well plate and allowed to reach 90% confluence. A scratch wound was created on the cell surface using a micropipette tip. Then, cells were washed with PBS in three times and incubated in serum-free DMEM/F12 with uric acid (800 µM).

| Reactive oxygen species assay
ROS level was examined with a ROS assay kit that sets DCFH-DA as the probe. HPMCs were grown in 6-well plates and were incubated in serum-free media containing DCFH-DA (10 μM) in the presence of groups for 60 min at 37°C and 5% CO2 in the dark. After washing with PBS for three times, the positive cells of DCFH-DA were measured using immunofluorescence photography.

| Immunoblot analysis
Cell lysates were collected from each group. Immunoblot analysis was conducted as described previously. 32 The densitometry analysis of immunoblot results was conducted by using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

| Immunofluorescence staining
Immunofluorescence staining was carried out according to the procedure described in our previous study. 33 HPMCs from different treatment groups were immobilized and incubated with primary antibodies against α-SMA or E-cadherin, and then Texas Red-or FITClabelled secondary antibodies (Invitrogen).

| Statistical analysis
Results are expressed as mean ± SD for continuous data and as frequencies (n) and percentages (%) for categorical data. Data distribution normality was evaluated by the Kolmogorov-Smirnov test.
A comparison among the different peritoneal transport types was performed by analysis of variance (ANOVA, parametric distribution) or the Kruskal-Wallis test (non-parametric distribution). The Kaplan-Meier survival curves were drawn for each event of interest (patient survival and technique survival), and the log-rank test was used to compare curves. Univariate and multivariate Cox proportional hazards models were used to search significant risk factors associated with study outcomes. Data were censored at the time of renal transplantation; 30 September 2020 or transfer to haemodialysis for the overall survival analyses, whereas the death-censored technique analyses were censored at the time of renal transplantation; death; or 30 September 2020. Multivariate logistic regression modelling was used for the analysis of risk factors associated with baseline high PSTR. A two-tailed p value <0.05 was considered statistically significant. In multivariate model, the adjustment variables included peritoneal transport category; age; gender; smoking status; BMI category; weekly residual renal Kt/V; the presence or absence of hypertension; CVD and diabetes, the covariate of laboratory parameters included ALB; C-reactive protein (CRP); cSCa; cardiac troponin (cTnT); haemoglobin (Hb); glycosylated haemoglobin (HbA1c); phosphate (P); parathyroid hormone (PTH); serum creatinine (Scr); total cholesterol (TC); triglyceride (TG); UA; and 4-h D/P Cr.
All the in vitro experiments were conducted at least three times.
Data depicted in graphs represent the means ± SEM for each group.
Intergroup comparison was made using one-way analysis of variance. Multiple means were compared using Tukey's test. The differences between two groups were determined by Student's t test.
Statistical significant difference between mean values was marked in each graph. p < 0.05 was considered significant.
The statistical analyses were conducted by using IBM SPSS Statistics 20.0 (version X; IBM).  Table 1.

| Outcomes of CAPD patients
The total average survival time of the study patients was

| Predictors of Baseline Peritoneal Transport Status
In order to explore the possible reasons for the baseline high PSTR, we  Figure 3). Otherwise, peritoneal permeability was not associated with the other clinical characteristics which were shown in Table 5.

| Uric acid induces EMT of cultured human peritoneal mesothelial cells in a dosedependent manner
The present results noted us that baseline uric acid levels were obviously associated with both all-cause death and high PSTR, Further results indicated that uric acid had the additive effect of EMT with 2.5% HG peritoneal dialysis fluid. Expression levels of α-SMA, collagen I and vimentin were higher in combined use of UA and HG than separate use (Supplemental Figure S1). Moreover, 800 μM uric acid could increase ROS production according to the DCFH-DA immunofluorescence (Supplemental Figure S2), suggesting that UA contributed to oxidative stress in HPMCs.

| Uric acid induces EMT of cultured human peritoneal mesothelial cells in a timedependent manner
Moreover, we also explored the level of EMT marker expression in the HPMCs stimulated with uric acid at 800 μM in different periods of time. As shown in Figure 5

| Uric acid facilitates the proliferation and migration of peritoneal mesothelial cells
To investigate whether uric acid is also involved in proliferation of

| The uric acid concentration in dialysate
Finally, in order to clarify the difference between uric acid concentration in PD solution and serum, forty-three PD effluent samples were collected after 4 h dwelling in the abdominal cavity when performed the first PET test. We measured the concentration of uric acid in PD effluent and found that the concentration of uric acid was lower in PD effluent compared with that in serum (p < 0.001). Compared with patients in low average group, the concentration of uric acid in PD effluent was higher in high transporters (Supplemental Figure S3).

| DISCUSS ION
This study retrospectively evaluated a cohort of patients that had their initial peritoneal membrane permeability analysed as categorical variable (H, HA, LA and L transport status). In our research, transport classes were significantly associated with deathcensored technique survival. Compared with LA group, baseline H transporters have a more than 5 times higher risk of technical failure (p = 0.008). SUA was significantly associated with baseline high PSTR (p = 0.020) and independently predicted mortality (p < 0.001). more studies had confirmed that the high transport of peritoneal solutes is one of the risk factors for poor prognosis. 37 Our findings were broadly consistent with those of previous studies. The suggested mechanism of adverse outcomes was mainly concentrated on peritoneal ultrafiltration failure and fluid overload do to rapid glucose absorption followed by reduction in osmotic gradient and abundant loss of protein in the dialysate leading to malnutrition. [38][39][40] It was apparently to see in the present study that high transporter   44 In the publication from the ANZDATA Registry, poor prognosis was associated with fast transporters only on CAPD but not on APD. 13 Another larger study of 4128 patients showed that APD leads to better survival of fast transporters. 45 On the other hand, with the utilization of biocompatible neutral pH, low GDP dialysate, peritoneal ultrafiltration may be increased and the function of peritoneum can also be preserved. 11,46 For patients who fail to achieve ultrafiltration goals with CAPD, APD implementation in combination with icodextrin dialysate could prolong technique survival. 47 In our present study, even after adjustment for nutrition and inflammation factors such as serum albumin, CRP, concentrations of creatinine, phosphorus and body mass index, hyperuricemia was still an independent predictor of all-cause mortality. To our knowledge, UA is the metabolic end product of nucleic acid purine which is mainly eliminated via renal excretion. Hyperuricaemia is very common in CKD patients due to the decreased ability of the kidneys to remove uric acid. In individuals at high risk for cardiovascular disease, despite uric acid has been proven to be a potent radical scavenger and antioxidant, 48

ACK N OWLED G EM ENT
We acknowledge and appreciate our colleagues for their valuable efforts and comments on this paper.

CO N FLI C T O F I NTE R E S T
No conflicts of interest, financial or otherwise, are declared by the author(s).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.