Dialysate hyaluronan concentration predicts survival but not peritoneal sclerosis in continuous ambulatory peritoneal dialysis

Dialysate Hyaluronan Concentration Predicts Survival But Not Peritoneal Sclerosis in Continuous Ambulatory Peritoneal Dialysis Cheuk-Chun Szeto, MBChB, MRCP(UK), Teresa Yuk-Hwa Wong, MBChB, MRCP(UK), Ka-Bik Lai, MPhil, Christopher Wai-Kei Lam, PhD, FACB, Kar-Neng Lai, DSc, FRCP, and Philip Kam-Tao Li, FRCP, FACP ● Hyaluronan is an important component of extracellular matrix and plays a critical role in early phases of wound healing. Peritoneal mesothelium is a major site of hyaluronan production. Serum hyaluronan concentration has been shown to predict survival in maintenance hemodialysis patients. We hypothesize that mesothelial production of hyaluronan during the stable phase of continuous ambulatory peritoneal dialysis (CAPD) predicts the risk of peritoneal adhesion and mortality. We studied peritoneal dialysate effluent (PDE) hyaluronan levels from 116 stable CAPD patients. They were then followed-up for 3 years. During the follow-up period, there were 196 episodes of peritonitis in 78 patients. Tenckhoff catheter was removed in 31 episodes (15.8%). Tenckhoff catheter was reinserted successfully in 12 cases, and CAPD was resumed. Peritoneal adhesion developed in 16 cases. Three patients died before Tenckhoff catheter reinsertion was attempted. There was no difference in stable-phase PDE hyaluronan levels between patients who developed peritoneal adhesion and those who did not (159 ⴞ 63 versus 227 ⴞ 194 ␮g/L, P ⴝ 0.27). Thirty-three patients died during the study period. Patients who died had significantly higher PDE hyaluronan concentration than survivors (272 ⴞ 194 versus 170 ⴞ 105 ␮g/L, P < 0.01). Univariate analysis showed that increased PDE hyaluronan level was associated with a shorter patient survival (P < 0.001). There was no association between PDE hyaluronan level and serum albumin, protein nitrogen appearance, and percentage of lean body mass. Multivariate analysis confirmed that PDE hyaluronan level, serum albumin, and diabetic state were independent predictors of survival. We conclude that PDE hyaluronan level during stable phase of CAPD does not predict the risk of postperitonitis adhesion. However, it is a strong independent predictor of survival in CAPD patients. © 2000 by the National Kidney Foundation, Inc. INDEX WORDS: Hyaluronan; survival; peritoneal dialysis (PD).

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YALURONAN, previously known as hyaluronic acid, is a major component of extracellular matrices.1 Serum hyaluronan concentration is elevated in patients receiving hemodialysis2 as well as in continuous ambulatory peritoneal dialysis (CAPD).3 Hyaluronan production is increased in uremic patients, probably because of altered connective tissue metabolism.4 Serum hyaluronan concentration has been implicated as an independent predictor of survival in chronic hemodialysis patients.3 Previous experiments showed that peritoneal dialysate effluent (PDE) hyaluronan concentration is higher than concurrent serum level in CAPD patients.5,6 In fact, PDE hyaluronan largely represents local synthesis by peritoneal mesothelial cells.6-10 However, there are few prospective data on the longterm significance of PDE hyaluronan. We hypothesize that PDE hyaluronan concentration is a predictor of survival in CAPD patients. Hyaluronan plays a critical role in the early stages of wound healing.11,12 It has been implicated in the process of peritoneal sclerosis.7,8,13 In stable CAPD patients, PDE hyaluronan levels correlate with peritoneal permeability.6,9 During

peritonitis, a rapid upsurge of PDE hyaluronan level is observed.6 Peritonitis induces mesothelial cell hyaluronan production, mainly by the action of interleukin-1␤ (IL-1␤).14 Our second hypothesis is hyaluronan production by mesothelium during a stable period of peritoneal dialysis predicts the risk of peritoneal adhesion when peritonitis develops. PATIENTS AND METHODS In 1995, we performed a cross-sectional study of 116 randomly selected CAPD patients.6 Patients who had peritonitis within 2 months before the study period were excluded.

From the Departments of Medicine & Therapeutics and Chemical Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, China. Received December 20, 1999; accepted in revised form April 21, 2000. Supported in part by the CUHK research account 6900570 and CUHK Direct Research Grant project code 2040756. Address reprint requests to Cheuk-Chun Szeto, MBChB, MRCP(UK), Department of Medicine & Therapeutics, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China. E-mail: [email protected] © 2000 by the National Kidney Foundation, Inc. 0272-6386/00/3603-0019$3.00/0 doi:10.1053/ajkd.2000.16201

American Journal of Kidney Diseases, Vol 36, No 3 (September), 2000: pp 609-614

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PDE hyaluronan production and peritoneal transport kinetics were studied at that time.6 In the current study, we examine the 3-year outcome of these patients, with emphasis on patient survival and the risk of peritoneal adhesion.

Determination of Hyaluronan Concentration in PDE This has been described previously.6 Briefly, overnight PDE samples (glucose 1.36% for 8 hours) were collected and the volume measured. The total volume of well-mixed PDE samples were centrifuged at 514g for 10 minutes at room temperature. Cell-free PDE samples were collected and stored at ⫺70°C until analysis. Plasma was drawn from 34 randomly selected patients at the time of PDE collection for determination of PDE hyaluronan concentration. Hyaluronan was measured by a radioimmunoassay with a detection limit of 10 ␮g/L (Pharmacia, Uppsala, Sweden). All samples were assayed at the same time to avoid interbatch variation. Intra-assay coefficient of variation was less than 8%.

Peritoneal Membrane Transport Characteristics We used the fast peritoneal equilibration test (PET) as described by Twardowski.6,15 Creatinine and glucose concentrations in PDE (glucose 1.36%) at 0 and 4 hours were measured by the hexokinase and alkaline-picrate methods (Hitachi 911 analyzer; Boehringer Mannheim, Mannheim, Germany), respectively. Creatinine concentration in dialysate was corrected for glucose interference according to a formula provided by our laboratory.16 Dialysate-to-plasma ratios of creatinine (D/P) at 4 hours was calculated.

Clinical Follow-Up Baseline data, including age, sex, underlying renal disease, CAPD regimen, duration on dialysis, hepatitis B status, and presence of diabetes mellitus (DM), were obtained. Clearance studies were performed at least yearly by 24-hour dialysate and urine collections. The method of clearance study was reported previously.17,18 Protein nitrogen appearance (PNA) and percentage of lean body mass (%LBM) were determined by standard methods.19,20 Serum albumin level was measured by bromcresol purple method. All patients were followed for 36 months. The dialysis regimen was that prescribed by the individual patient’s nephrologist. Changes of dialysis regimen were made for clinical indications and were not affected by the study. Seven patients received a renal transplant during the 3-year study period. Individual patient survival was censored at the time of death, transplantation, or completion of the study.

Peritonitis and Peritoneal Adhesion During the follow-up period, peritonitis was defined by conventional criteria.21,22 Peritonitis episodes were treated with a standard antibiotics protocol of our center at that time. Initial antibiotics for peritonitis were generally vancomycin plus either imipenem-cilastatin or a third-generation cephalosporin. Antibiotic regimens for individual patients were modified when culture results were available. Tenckhoff catheters were removed and patients were put on temporary

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hemodialysis when peritonitis failed to resolve with antibiotics, or when there was fungal peritonitis. Tenckhoff catheter reinsertion was attempted in all cases. “Success” cases were defined as those patients of whom CAPD could be resumed. According to the practice described in our previous study,18 patients were only switched to long-term hemodialysis when attempts of Tenckhoff catheter re-insertion failed because of peritoneal adhesion, or when there was ultrafiltration failure due to peritoneal sclerosis. These were defined as “Failure” cases.

Statistics Statistical analysis was performed by SYSTAT 7.0 for Windows software (SPSS Inc., Chicago). Results were expressed as mean ⫾ SD unless otherwise stated. PDE hyaluronan concentrations during the stable phase of CAPD of the “Failure” group were compared with that of the “Success” group by Kruskal-Wallis test because the data were significantly skewed. The Cox proportional hazards model was used for statistical analysis of patient and technique survival.23 All clinical and laboratory parameters described were included for univariate analysis. For time-dependent indices (including serum albumin, PNA, %LBM, and Kt/V), average values during the study period was used for analysis. Kaplan-Meier analysis was used to explore their effect on survival. Age, duration of dialysis before the study, diabetic status, PDE hyaluronan concentration, albumin, and Kt/V were identified by univariate analysis. Regression with backward stepwise elimination was then applied to determine independent predictors. All probabilities were two-tailed.

RESULTS

A total of 116 patients were studied for 3,792 patient-months. There were 54 males and 62 females. The mean age was 53.0 ⫾ 11.9 years. The median duration of CAPD before enrollment was 18.5 months (range, 3 to 97 months). The average Kt/V of our patients during the study period was 1.82 ⫾ 0.45. Seventy-three patients had no previous peritonitis; 17 had more than 4 episodes of peritonitis; and the remaining 26 had 1 to 3 episodes. The incidence of peritonitis in our patients before enrollment was one episode per 16.3 patient-months. Eighteen patients (15.5%) were hepatitis B surface antigen (HbsAg) positive. The PDE hyaluronan levels of HbsAg-positive patients were not significantly different from those of HbsAgnegative patients (156 ⫾ 71 versus 207 ⫾ 151 ␮g/L, P ⫽ 38). Peritoneal Adhesion There were 196 episodes of peritonitis in 78 patients during the study period. The peritonitis

PERITONEAL HYALURONAN PREDICTS SURVIVAL

Fig 1.

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Clinical outcome of the 118 study cases.

rate during the study period was one episode per 19.3 patient-month. Their clinical courses were summarized in Fig 1. Six patients had fulminant peritonitis and died before Tenckhoff catheter removal was possible. Tenckhoff catheter was removed in 31 episodes (15.8%). In 14 patients, attempts of Tenckhoff catheter re-insertion failed because of peritoneal adhesion. In 2 other patients, ultrafiltration failure developed despite successful reinsertion of Tenckhoff catheter. These 16 patients were the “Failure” group. In another 12 patients, Tenckhoff catheter re-insertion was successful (the “Success” group). Patients of the “Failure” group had slightly lower PDE hyaluronan concentration during the stable phase of CAPD compared with the “Success” group (Fig 2). However, the result was not statistically significant (159 ⫾ 63 versus 227 ⫾ 194 ␮g/L, P ⫽ 0.27). Patient Survival Thirty-three patients died during the 3-year study period. The causes of death were cardiovascular disease (13 cases), peritonitis (9 cases), nonperitonitis infection (3 cases), malignancy (3 cases), liver failure (3 cases), and unknown (2 cases). All patients that died of liver failure had cirrhosis related to chronic hepatitis B infection.

Patient who died during the study period had significantly higher PDE hyaluronan concentration than survivors (272 ⫾ 194 versus 170 ⫾ 105

Fig 2. Comparison of PDE hyaluronan concentrations between 16 patients who developed postperitonitis adhesion (“Fail”) and 14 patients who successfully resumed CAPD after severe peritonitis and Tenckhoff catheter removal (“Success”) by box-and-line plot. The box indicates median, 25th, and 75th percentiles. The lines indicate 5th and 95th percentiles. KruskalWallis test, P ⴝ 0.27.

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Fig 3. Survival by KaplanMeier plot of patients with PDE hyaluronan (HA) concentrations of <150 ␮g/L and >150 ␮g/L (59 patients each). Log-rank P < 0.001.

␮g/L, P ⬍ 0.01). Survival by Kaplan-Meier plot, dividing patients into those with “low” (⬍150 ␮g/L) and “high” (⬎150 ␮g/L) hyaluronan concentrations showed a marked difference in survival (Fig 3). Actuarial patient survival was 87.0% and 63.5% for low and high hyaluronan groups, respectively (P ⬍ 0.001). There was no difference in peritonitis rates of “low” and “high” hyaluronan groups (20.0 versus 18.6 patientmonth per episode; P ⫽ 0.55). The results of Cox proportional hazard model for actuarial patient survival are summarized in Table 1. By multivariate analysis, independent factors for patient survival were PDE hyaluronan concentration, presence of diabetes, and serum albumin level. Age and total weekly Kt/V had a trend to be associated with patient survival, but the result did not reach statistical significance. The causes of death for the two groups of patients were summarized in Table 2. There was a generalized increase in mortality from cardiovascular disease, peritonitis, and liver failure. Table 1.

Because the number of observations was small, subgroup statistic analysis was not performed. DISCUSSION

We observed a strong association between PDE hyaluronan level during stable phase of peritoneal dialysis and all-cause mortality in CAPD patients. The association persists after correction for age, diabetic state, serum albumin, and total Kt/V, all major predictors of patient survival.24 We did not observe significant effects of age and total Kt/V on patient survival because of the small sample size, although there were trends of such effects. Our findings are consistent with that reported by Woodrow et al,25 who showed that serum hyaluronan levels were predictive of survival in hemodialysis patients. Previous studies have suggested that the peritoneum is a major site of hyaluronan production.5-7,10,13 In 34 randomly selected CAPD patients, hyaluronan concentration in plasma was approximately 85% of that in PDE.6

Cox Proportional Hazards Model of Patient Survival

Variable

Relative Risk

95% CI

P

Diabetes PDE hyaluronan level (1 150 ␮g/L) Serum albumin (1 1 g/L) Age (1 10 years) Kt/V (1 0.1 unit/week)

3.49 1.66 0.87 1.55 0.96

2.08-5.87 1.23-2.02 0.82-0.93 0.91-2.25 0.91-1.02

⬍0.02 ⬍0.05 ⬍0.05 NS (P ⫽ 0.09) NS (P ⫽ 0.07)

Abbreviation: PDE, peritoneal dialysis effluent. See text for details.

PERITONEAL HYALURONAN PREDICTS SURVIVAL Table 2.

Cause of Death by Patient Group

Cause of Death

Cardiovascular Peritonitis Nonperitonitis infection Malignancy Liver failure Unknown Total

HA ⬍150 ␮g/L

HA ⱖ150 ␮g/L

4 2 2 2 0 1 11

9 7 1 1 3 1 22

Mortalities from cardiovascular disease, peritonitis, and liver failure were all increased in the group with high PDE hyaluronan level (Table 2). The reasons remain speculative. A high PDE hyaluronan level may indicate underlying cirrhosis.26 However, only 3 patients in the present study died of liver failure. One possible explanation is the association of hyaluronan and malnutrition. A weak inverse correlation between serum hyaluronan and albumin levels has been described.25 However, we found no association between PDE hyaluronan and serum albumin level in our study. Neither was PDE hyaluronan level associated with other nutritional markers such as PNA and %LBM (details not shown). Furthermore, the association of mortality persists after correction for serum albumin level by multivariate analysis. We do not think malnutrition is the complete explanation of the observed difference. Conversely, hyaluronan is a marker of systemic inflammation. Serum hyaluronan concentration correlates with disease activity in a number of chronic inflammatory diseases.27-29 In hemodialysis patients, serum hyaluronan level correlates with other inflammatory markers.30,31 Evidence suggests that C-reactive protein, another marker of inflammation, is a strong predictor of survival in dialysis patients.32 Since peritoneum is an important site of hyaluronan synthesis in CAPD patients,7-10 PDE hyaluronan may represent a direct estimate of their inflammatory state and therefore reliable predictor of patient survival. In the current report, the PDE hyaluronan level was measured only at the beginning of the study period because of practical convenience. However, a single measurement might be biased. Yamagata et al recently described a gradual rise of PDE hyaluronan level in a longitudinal study

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of 7 CAPD patients.9 However, the observed fluctuation within individual patient was relatively small. We found no correlation between PDE hyaluronan level and duration of dialysis.6 Furthermore, duration of dialysis at the time of PDE hyaluronan assay did not appear in the survival model with multivariate analysis. We did not find that PDE hyaluronan level during stable phase of CAPD predicted postperitonitis adhesion. However, several groups have suggested the important role of this mediator. Impaired mesothelial hyaluronan production has been implicated as possible reasons of adhesion after peritonitis and laparotomy.33 Hyaluronatebased bioresorbable membrane has been found effective in reducing postoperative abdominal adhesions.34 It is possible that upregulation of mesothelial hyaluronan production during early period of peritonitis, rather than the baseline production, is determinative in peritoneal healing. Hyaluronan is mainly involved in the early phases of wound healing.11,12 A rapid upsurge of peritoneal dialysate hyaluronan level has been observed during peritonitis.6 In summary, we show that PDE hyaluronan level is a strong independent predictor of allcause mortality in CAPD patients. However, PDE hyaluronan level during a stable phase of CAPD does not predict the risk of peritoneal adhesion after peritonitis. Further study on peritoneal hyaluronan production during early phase of peritonitis is needed to determine the role of hyaluronan on peritoneal adhesion. REFERENCES 1. Mason RM: Recent advances in the biochemistry of hyaluronan. Prog Clin Biol Res 54:87-112, 1981 2. Turney JH, Davison AM, Forbes MA, Cooper EH: Hyaluronic acid in end-stage renal failure treated by haemodialysis: Clinical correlates and implications. Nephrol Dial Transplant 6:566-570, 1991 3. Lipkin GW, Forbes MA, Cooper EH, Turney JH: Hyaluronic acid metabolism and its clinical significance in patients treated by continous ambulatory peritoneal dialysis. Nephrol Dial Transplant 8:357-360, 1993 4. Hallgren R, Engstrom-Laurent A, Nisbeth U: Circulating hyaluronate: A potential marker of altered metabolism of the connective tissue in uremia. Nephron 46:150-154, 1987 5. Lai KN, Szeto CC, Lam CW, Lai KB, Wong TY, Leung JC: Increased ascitic level of hyaluronan in liver cirrhosis. J Clin Lab Med 131:354-359, 1998 6. Lai KN, Szeto CC, Lai KB, Lam CW, Chan DT, Leung JC: Increased production of hyaluronan by peritoneal cells

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