Metabolism, protein binding and renal clearance of microbiota derived p-cresol in patients with chronic kidney disease

Ruben Poesen1, MD, Pieter Evenepoel1, MD PhD, Henriette de Loor1, MSc, Dirk Kuypers1, MD PhD, Patrick Augustijns2, PharmD PhD, Björn Meijers1, MD PhD

1 Department of Microbiology and Immunology, Division of Nephrology, University Hospitals

Leuven, B-3000 Leuven, Belgium2 Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and

Disposition, University of Leuven, B-3000 Leuven, Belgium

Word count abstract: 298

Word count main body: 3255

Tables: 4

Figures: 4

Running title: Metabolism of p-cresol in CKD

Keywords: microbiota, p-cresol, chronic renal insufficiency

Address for correspondence: Björn Meijers, MD, PhD

Division of Internal Medicine, Department of Nephrology

University Hospitals Leuven

Herestraat 49

B-3000 Leuven, Belgium

Tel. +32 16 344580

Fax. +32 16 344599

E-mail: bjorn.meijers@uzleuven.be

1

Abstract

Background and objectives: Colonic microbial metabolism substantially contributes to uremic

retention solutes in CKD. p-Cresyl sulfate is the main representative of this group of solutes,

relating to adverse outcomes. Besides sulfate conjugation, p-cresol is subjected to

endogenous glucuronide conjugation. Whether the balance between sulfate and glucuronide

conjugation is relevant in CKD is unexplored.

Design, setting, participants, and measurements: We prospectively followed 488 patients

with CKD stage 1–5 (enrollment between November 2005 and September 2006, follow-up

until December 2010). Serum and urine levels of p-cresyl sulfate and p-cresyl glucuronide

were measured using liquid chromatography–mass spectrometry. Total amount of microbial

p-cresol was calculated by sum of serum p-cresyl sulfate and p-cresyl glucuronide. Outcome

analysis was performed for mortality and cardiovascular disease.

Results: Serum p-cresyl sulfate was median 193.0-fold (IQR 121.1–296.6) higher than serum

p-cresyl glucuronide with a significant correlation between eGFR and proportion of serum p-

cresyl sulfate to glucuronide (ρ 0.23, P 0.001). There was also a significant correlation

between eGFR and proportion of 24h urinary excretion of p-cresyl sulfate to glucuronide (ρ

0.32, P<0.001). Higher serum p-cresol and lower proportion of serum p-cresyl sulfate to

glucuronide were jointly and significantly associated with mortality (HR per SD higher of 1.58

(1.10 – 2.29), P 0.01 and HR 0.65 (0.47 – 0.89), P 0.008, respectively) and cardiovascular

disease (HR 1.68 (1.27 – 2.22), P<0.001 and HR 0.55 (0.42 – 0.72), P<0.001, respectively)

after adjustment for eGFR, Framingham risk factors, mineral bone metabolism markers, C-

reactive protein and albumin.

Conclusions: p-Cresol demonstrates a preponderance to sulfate conjugation, although a

relatively diminished sulfotransferase activity can be suggested in patients with advanced

2

CKD. Along with total p-cresol burden, a relative shift from sulfate to glucuronide

conjugation is independently associated with mortality and cardiovascular disease,

warranting increased focus to the dynamic interplay between microbial and endogenous

metabolism.

3

Introduction

There has been mounting evidence that the colonic microbial metabolism contributes

substantially to uremic retention solutes accumulating in patients with chronic kidney

disease (CKD)(1,2). p-Cresyl sulfate (PCS) can be considered representative of this group of

solutes and has been associated with overall mortality, cardiovascular disease and

progression of CKD(3-7). In addition, mechanistic studies relate PCS to oxidative stress,

endothelial dysfunction, proximal tubular injury and insulin resistance(8-10).

Besides sulfate conjugation, microbial p-cresol is subjected to endogenous glucuronidation

with serum levels of p-cresyl glucuronide (PCG) being substantially lower than those of

PCS(11,12). Although it can be hypothesized that this imbalance is due to differences in

phase 2 metabolism, possibly influenced by renal dysfunction, this has not been studied to

date. Furthermore, renal handling of PCG is unknown, but may be different when compared

with PCS, which mainly depends on tubular secretion(13-16). In addition, although it is well

established that serum PCS is highly protein-bound(17,18), protein binding characteristics of

serum PCG are less clear, possibly contributing to differential renal clearance of PCS and

PCG. A better understanding of the determinants of serum PCG is needed as PCG has also

been related to adverse outcomes in patients with CKD(19). Whether a relative shift of p-

cresol from sulfation to glucuronidation, or vice versa, is relevant in CKD remains, however,

unknown.

Therefore, we explored the behavior of PCS and PCG in patients with CKD not yet on dialysis,

focusing on potential differences in phase 2 metabolism, protein binding and renal clearance

4

between both p-cresol derivatives. In addition, the relative contribution of sulfation and

glucuronidation of p-cresol to adverse outcomes was examined.

Material and Methods

Study population

This is an ancillary analysis of the Leuven Mild-to-Moderate CKD study (NCT00441623)(5).

Prevalent CKD patients, followed at the nephrology outpatient clinic of University Hospitals

Leuven, 18 year of age or older and able to provide consent, were eligible for inclusion.

Patients were screened between November 2005 and September 2006. The study was

performed according to the Declaration of Helsinki and approved by the ethics committee of

University Hospitals Leuven. Informed consent was obtained from all patients.

Biochemical measurements

Serum levels of PCS and PCG were quantified using a dedicated ultra-performance liquid

chromatography–tandem mass spectrometry (UPLC–MS/MS) method (Supplemental

Material). To gain further insights in the behavior of PCS and PCG, we also measured free

solute levels, 24h urinary excretion and renal clearance (total and free) in subgroup of

patients with availability of 24h urinary collection. Completeness was assessed using 24h

urinary creatinine excretion and collections were considered complete when creatinine

excretion was within 2 standard deviations (SD) of the mean creatinine excretion for the

geographical region of this study (International Study of Salt and Blood Pressure (INTERSALT)

study)(20). Free solute levels were measured after ultrafiltration of serum using Centrifree

UF Device (EMD Millipore, Billerica, MA) (molecular weight cut-off of 30kDa). Free solute

fraction was defined as ratio of free to total solute level. Assuming steady state conditions

5

and negligible non-renal clearance, 24h urinary excretion of PCS and PCG can be considered

an estimate of endogenous sulfate and glucuronide conjugation of p-cresol. In addition,

combined 24h urinary excretion of PCS and PCG equals total intestinal uptake of precursor p-

cresol.

Equilibrium dialysis

To explore protein binding characteristics of PCG, we performed equilibrium dialysis using

HTDialysis 96b system (cell volume 150µl, HTDialysis, Gales Ferry, CT) (molecular weight cut-

off of 12-14kDa). Experimental solutions were prepared by spiking PCG at different

concentrations (2, 5, 10, 25, 50, 75, 100, 200, 400, 800 and 1200µM) in an albumin solution

(target 40g/l dissolved in phosphate buffered saline (PBS)), healthy serum, uremic serum or

healthy serum with addition of PCS (target 200µM). PCG, fatty acid free human serum

albumin and PBS were purchased from Sigma-Aldrich (St. Louis, MO). Pooled healthy serum

was obtained from 8 healthy study participants, while pooled uremic serum was derived

from 8 hemodialysis patients taken immediately prior to the midweek hemodialysis session.

PCS was synthesized according to Feigenbaum and Neuberg(21). To determine binding

equilibrium, experimental solutions were dialyzed against an equal volume of PBS with

temperature kept constant at 37°C and for a duration of 4 hours. All experiments were

performed in octet with pooling of 4 chamber volumes of each experimental condition for

quantification. Free fractions of PCG were defined as ratio between solute concentrations in

PBS solution and experimental solution.

Outcome analysis

6

After inclusion, patients were prospectively followed at the nephrology outpatient clinic at 3-

to 6-month intervals. In the original study, follow-up was available until December 1, 2008.

For this study, follow-up was extended until December 31, 2010. Endpoint evaluation has

been described previously(5) and consisted of overall mortality and first cardiovascular

event (Supplemental Material).

Statistics

Data are expressed as mean (SD) for normally distributed variables or median (interquartile

range (IQR)) for non-normally distributed variables. Correlations were calculated by

Spearman’s rank correlation coefficients. Differences were tested using Wilcoxon rank-sum,

Kruskal-Wallis or chi-square test as indicated. Linear regression analysis was performed for

renal clearance (total and free) of PCS and PCG, free fraction of PCS and PCG, and proportion

of 24h urinary excretion of PCS to PCG. Time to first event analysis was performed for both

serum total p-cresol (sum of serum total PCS and PCG) and proportion of serum total PCS to

PCG using Cox proportional hazards analysis. For multivariate analysis, we used a double

backward elimination approach, with inclusion of all variables at P<0.20 for secondary

backward elimination at P<0.05. To test the proportionality assumption, each model was

tested against log(time). For overall mortality, data were censored at start of renal

replacement therapy, loss to follow-up or the end of study observation period. With respect

to cardiovascular disease, additional censoring was performed for non-cardiovascular death.

All statistical analyses were performed using SAS (version 9.3, the SAS institute, Cary, NC).

Results

Study population

7

Study population consisted of 499 patients with CKD stage 1-5 and has been described

previously(5). Of these, total serum levels of PCS and PCG were measured in 488 patients for

outcome analysis. In addition, 24h urinary collection was available in 203 patients, in which

we also measured free serum levels, 24h urinary excretion and renal clearance (total and

free) of PCS and PCG. Besides a small age difference, we observed no significant differences

in baseline characteristics between both groups (Table 1).

Serum level of PCS versus PCG

Total serum levels of PCS amounted to median 49.7µM (IQR 21.0–104.1), being

approximately 193.0-fold (IQR 121.1–296.6) higher than those of PCG. There was a

statistically significant correlation between serum total PCS and PCG (ρ 0.88, P<0.001), and

both serum total PCS and PCG were significantly correlated with eGFR (ρ -0.67, P<0.001 for

PCS, ρ -0.65, P<0.001 for PCG) (Figure 1A-B). In addition, we observed a significant

relationship between proportion of serum total PCS to PCG and eGFR (ρ 0.23, P 0.001)

(Figure 1E), with relatively more serum total PCG in patients with advanced CKD, especially

when eGFR is below 30 ml/min/1.73m² (Supplemental Figure 1A).

Free serum levels of PCS (median 1.8µM, IQR 0.6–4.1) were also 10.1-fold (IQR 6.4–15.5)

higher than those of PCG. In addition, free serum levels of PCS and PCG were significantly

correlated with each other (ρ 0.93, P<0.001), with their corresponding total serum levels (ρ

0.97, P<0.001 for PCS, ρ 0.97, P<0.001 for PCG) and with eGFR (ρ -0.69, P<0.001 for PCS, ρ -

0.65, P<0.001 for PCG) (Figure 1C-D). Also, the proportion of serum free PCS to PCG was

significantly lower in patients with lower eGFR (ρ 0.20, P 0.005) (Figure 1F) (Supplemental

Figure 1B).

8

24h urinary excretion of PCS versus PCG

To estimate the degree of sulfate versus glucuronide conjugation, we calculated 24h urinary

excretion of PCS and PCG. 24h urinary excretion of PCS amounted to median 510.7µM (IQR

271.0–77.5), being 12.7-fold (IQR 8.1–18.6) higher than 24h urinary excretion of PCG, both

correlating with each other (ρ 0.78, P<0.001). A significant correlation was noted between

24h urinary excretion and total serum levels of both compounds (ρ 0.68, P<0.001 for PCS, ρ

0.69, P<0.001 for PCG). There was also a significant correlation between eGFR and 24h

urinary excretion of PCG (ρ -0.25, P<0.001) and a borderline significant correlation between

eGFR and 24h urinary excretion of PCS (ρ -0.13, P 0.07) (Figure 2A-B). In addition, we

observed a significant relationship between proportion of 24h urinary excretion of PCS to

PCG and eGFR (ρ 0.32, P<0.001) with relatively more 24h urinary excretion of PCG in patients

with advanced CKD. To estimate total intestinal uptake of precursor p-cresol, 24h urinary

excretion of PCS and PCG was combined, demonstrating a significant correlation with eGFR

(ρ -0.15, P 0.04). In linear regression analysis, eGFR (β 0.43 per ml/min/1.73m² (Ln), P 0.03),

but not 24h urinary excretion of p-cresol (β 0.03 per µmol (Ln), P 0.74) was associated with

proportion of 24h urinary excretion of PCS to PCG. There was a significant correlation

between 24h urinary excretion of urea and 24h urinary excretion of PCS (ρ 0.32, P<0.001),

PCG (ρ 0.20, P 0.006) and p-cresol (ρ 0.31, P<0.001), while there was no correlation between

24h urinary excretion of urea and proportion of 24h urinary excretion of PCS to PCG (ρ 0.11,

P 0.13).

Renal clearance of PCS versus PCG

9

Total renal clearance of PCS amounted to median 6.6ml/min (IQR 3.6–12.0), which was

correlated with (ρ 0.70, P<0.001), but substantially lower than total renal clearance of PCG

(median 98.9ml/min, IQR 40.6–212.4). Correlation between eGFR and total clearance was

nominally higher for PCS (ρ 0.81, P<0.001) than for PCG (ρ 0.55, P<0.001) (Figure 3A-B).

Furthermore, there was a correlation between eGFR and fractional excretion, albeit more

pronounced for total PCS (median 16.6%, IQR 13.2–22.3) (ρ 0.29, P<0.001) than for total PCG

(median 259.5%, IQR 161.1–385.1) (ρ 0.16, P 0.02). On the other hand, we observed no

relationship between proportion of total clearance of PCS to PCG and eGFR (ρ -0.01, P 0.90).

When focusing on free solute renal clearance, clearance of PCS (median 190.0ml/min, IQR

94.2–374.6) was correlated with (ρ 0.72, P<0.001), but higher than clearance of PCG (median

136.5ml/min, IQR 57.5–295.9) (Figure 3C-D). Again, correlation between eGFR and free

clearance was nominally higher for PCS (ρ 0.81, P<0.001) than for PCG (ρ 0.58, P<0.001), and

correlation between eGFR and fractional excretion was stronger for free PCS (median

463.2%, IQR 353.3–625.8) (ρ 0.52, P<0.001) than for free PCG (median 371.5%, IQR 232.3–

567.3) (ρ 0.25, P<0.001). There was no relationship between eGFR and proportion of free

clearance of PCS to PCG (ρ 0.08, P 0.27).

In linear regression analysis, eGFR and serum albumin were significantly associated with

total and free renal clearance of PCS with higher serum albumin relating to lower total

clearance, but also to higher free clearance. For PCG, eGFR, but not serum albumin was a

determinant of both total and free clearance (Table 2).

Protein binding of PCS versus PCG

10

Median free fraction of PCS was 3.5% (IQR 2.9–4.2), while median free fraction of PCG was

72.8% (IQR 62.2–80.0). Free fraction of PCS and PCG correlated with each other (ρ 0.50,

P<0.001) and with eGFR (ρ -0.35, P<0.001 for PCS, ρ -0.33, P<0.001 for PCG) and albumin (ρ -

0.45, P<0.001 for PCS, ρ -0.26, P<0.001 for PCG). In linear regression analysis, both lower

eGFR and lower serum albumin were associated with higher free fraction of PCS and PCG

(Table 3).

To extend clinical data, we performed ex vivo equilibrium dialysis (Figure 4). Mean free

fraction of PCG was 76.9% (SD 2.6) in albumin in PBS solution, being slightly higher than

mean free fraction in healthy serum (74.5% (SD 3.3), P 0.008). When comparing healthy

versus uremic serum, a significantly higher mean free fraction of PCG was noted in uremic

serum (88.1% (SD 3.7), P<0.001). There was no difference in free fraction of PCG between

healthy serum and healthy serum with addition of PCS (74.3% (SD 3.7), P 0.88). In addition,

there was no correlation between total spiked concentrations of PCG and free fraction of

PCS in both healthy (ρ -0.02, P 0.92) and uremic serum (ρ -0.23, P 0.28) with mean free

fraction of PCS being higher in uremic than in healthy serum (7.2% (SD 0.71) vs. 3.7% (SD

0.71), P<0.001).

Outcome analysis

We investigated the relationship between serum total p-cresol (sum of serum total PCS and

PCG), proportion of serum total PCS to PCG and adverse outcomes (Table 4). During follow-

up, we noted 51 deaths and 75 cardiovascular events. Higher serum total p-cresol and lower

proportion of serum total PCS to PCG were jointly associated with mortality, even after

adjustment for eGFR, Framingham risk factors, mineral bone metabolism markers, albumin

11

and C-reactive protein (HR per higher of 1.58 (95% confidence interval 1.10 – 2.29), P 0.01

for p-cresol, HR 0.65 (0.47 – 0.89), P 0.008 for proportion of PCS to PCG). In addition, higher

serum total p-cresol and lower proportion of serum total PCS to PCG were independent

predictors for cardiovascular events (HR 1.68 (1.27 – 2.22), P<0.001 for p-cresol, HR 0.55

(0.42 – 0.72), P<0.001 for proportion of PCS to PCG). Further analyses also demonstrated a

significant independent relationship between higher proportion of serum total PCG to p-

cresol, along with higher serum total p-cresol, and both overall mortality and cardiovascular

disease (Supplemental Table 1). Finally, higher serum total PCG itself was a significant and

independent predictor of adverse outcomes (Supplemental Table 2).

Discussion

In this study, we explored the differential behavior of PCS and PCG in patients with CKD not

yet on dialysis. The key findings are as follows: (i) total and free serum levels of PCS are

substantially higher than those of PCG; (ii) p-cresol is predominantly subjected to sulfation

although the contribution of glucuronidation was greater among patients with lower eGFR;

(iii) renal clearance of PCS and PCG depend on tubular secretion with serum levels of

albumin also contributing to renal clearance of PCS; (iv) protein binding of serum PCS is

substantially higher as compared to PCG and protein binding of both p-cresol derivatives is

diminished in patients with advanced CKD and lower serum albumin; (v) a relative shift from

sulfation to glucuronidation, along with higher serum total p-cresol, associates with

mortality and cardiovascular disease.

PCS originates from colonic microbial fermentation of tyrosine to p-cresol with subsequent

endogenous sulfate conjugation(2,22). PCG is another p-cresol derivative, albeit with

12

substantially lower total serum levels when compared with PCS(11,12). As the differential

behavior of PCS and PCG has been largely unexplored, we measured serum levels and 24h

urinary excretion of both p-cresol derivatives in our Leuven Mild-to-Moderate CKD cohort.

In agreement with previous studies(11,12), total serum levels of PCG were considerably

lower than those of PCS in patients across the whole range of eGFR. In addition, when

focusing on free serum solute levels, the balance between PCS and PCG, albeit less

pronounced, was still in favor of PCS. As these findings can be explained by differences in

both phase 2 metabolism and renal clearance, we compared 24h urinary excretion and renal

clearance between both solutes. Assuming steady state conditions and negligible non-renal

clearance, 24h urinary excretion of PCS and PCG can be considered an estimate of

endogenous sulfate and glucuronide conjugation of p-cresol. As we noted a substantially

higher 24h urinary excretion of PCS, it can be suggested that phase 2 metabolism of p-cresol

demonstrates preponderance to sulfate conjugation. Furthermore, we observed a lower

proportion of both serum and 24h urinary excretion of PCS to PCG in patients with lower

eGFR, which is indicative of a relatively diminished sulfotransferase activity in patients with

advanced CKD. These findings confirm and extend a previous observation of a decreased

serum total PCS to PCG ratio in patients on maintenance hemodialysis, in which a different

dialytic clearance should be taken into account(23). In agreement, glucuronide conjugation

of paracetamol was also more pronounced as compared to sulfate conjugation when

administered to patients with CKD, again pointing to a decrease in sulfotransferase function,

especially for sulfotransferase 1A1(24). A relative shift from sulfate to glucuronide

conjugation may, however, be detrimental as we demonstrated that lower proportion of

serum total PCS to PCG, along with higher serum total p-cresol burden, was an independent

13

predictor of mortality and cardiovascular disease. Serum PCG has already been related to

worse survival in CKD, although direct comparison to PCS was not performed(19).

Mechanistic studies of PCG are also rather scarce as compared to PCS. In this regard, it has

been demonstrated that PCG per se has no effect on leucocyte oxidative burst activity,

whereas it may induce a synergistic activating effect in the presence of PCS(12). In addition,

the effect of PCG on proximal tubular cells is equivocal(25,26). Further research is required

to elucidate the relevance of a decrease in sulfotransferase activity in patients with

advanced CKD with respect to phase 2 metabolism of p-cresol and, possibly, also to

endogenous and drug metabolism in general.

Furthermore, we studied renal clearance of both p-cresol derivatives. Total clearance of PCS

was lower when compared with PCG, while free clearance of PCS exceeded free clearance of

PCG. As free renal clearances of PCS and PCG were higher than eGFR or creatinine

clearances, active tubular secretion can be expected for both solutes. Furthermore,

fractional excretion was lower in patients with advanced CKD, thus also pointing to

saturation of tubular transport for PCS and PCG. Although mechanisms underlying tubular

secretion of PCS are increasingly being unraveled(13,14,27), less is known about renal

handling of PCG. Recent data point to potential involvement of breast cancer resistance

protein (BCRP) and multidrug resistance-associated protein 4 (MRP4) for tubular transport of

PCG, while secretion of PCS may depend on MRP4, but not on BCRP(23). Differences in free

renal clearance of PCS and PCG may also be derived from their binding characteristics as it

has been suggested that protein binding enhances clearance by providing a readily

accessible reservoir for efficient removal of the solute throughout its passage within the

native kidney(15). Interestingly, even in the range present in our cohort, lower serum

14

albumin was a significant determinant of lower free renal clearance of PCS, while also being

associated with higher total renal clearance.

Finally, we investigated protein binding characteristics of PCG, demonstrating rather low

protein binding as compared to PCS. Although serum albumin was the major protein

responsible for PCG binding, as also observed for PCS(17), there was no competitive binding

between PCG and PCS, thus possibly pointing to another albumin binding site for PCG than

for PCS (Sudlow site II)(28). In addition, protein binding of PCG was significantly diminished

in patients with advanced CKD, as well as in experimental uremic conditions. Diminished

protein binding along with renal function decline has already been noted for PCS(17), but

also for various relevant drugs(29), and may relate to hypoalbuminemia, chemical

modifications or conformational changes of albumin, and competitive binding with

increasing levels of uremic retention solutes(30).

There are limitations to our study. First, the study design precludes causal inferences.

Second, as measurements of proteinuria were only available in a subgroup of patients,

adjustment for proteinuria was not possible in outcome analysis. Finally, phase 2

metabolism of p-cresol was estimated by 24h urinary excretion of PCS and PCG, assuming

negligible non-renal clearance. Preliminary own data in healthy volunteers demonstrate only

minor biliary excretion of both PCS and PCG.

In conclusion, p-cresol demonstrates preponderance to sulfate conjugation, although

relatively diminished sulfotransferase activity can be observed in patients with advanced

CKD. Along with total p-cresol burden, a relative shift from sulfate to glucuronide

15

conjugation is independently associated with mortality and cardiovascular disease. The

pathophysiological relevance of these findings requires further investigation.

Disclosures

None

Acknowledgements

RP is the recipient of a Ph.D. fellowship of the Research Foundation - Flanders (FWO) (grant

11E9813N). Part of the research has been funded by the Research Foundation - Flanders

(FWO) (grant G077514N). Technical assistance by T. Coopmans and M. Dekens is highly

appreciated.

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Tables

Table 1 – Study population

Variable Subgroup (n = 203) Full group (n = 488) PAge (yr) 60 (47 – 72) 64 (50 – 74) 0.02Gender: male/female (%) 120/83 (59.1/40.9) 270/218 (55.3/44.7) 0.37Albumin (g/dl) 4.51 (4.20 – 4.68) 4.48 (4.24 – 4.69) 0.81Creatinine (mg/dl) 1.81 (1.29 – 2.50) 1.78 (1.26 – 2.43) 0.45eGFR (ml/min per 1.73 m²) 34 (23 – 56) 35 (23 – 56) 0.87Creatinine clearance (ml/min) 40 (27 – 61) - -24h proteinuria (g) 0.31 (0.11 – 1.13) - -Serum total PCS (µM) 49.7 (21.0 – 104.1) 53.2 (21.6 – 106.5) 0.24Serum free PCS (µM) 1.8 (0.6 – 4.1) - -Serum total PCG (µM) 0.22 (0.08 – 0.60) 0.23 (0.08 – 0.59) 0.85Serum free PCG (µM) 0.13 (0.05 – 0.50) - -

Data are expressed as median (IQR) or proportion.eGFR, estimated glomerular filtration rate; PCS, p-cresyl sulfate; PCG, p-cresyl glucuronide

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Table 2 – Linear regression analysis of renal clearance of p-cresyl sulfate and p-cresyl

glucuronide

p-cresyl sulfateVariable β P

Total renal clearance (ml/min) (Ln)

eGFR (ml/min/1.73m²) (Ln) 1.07 < 0.001Serum albumin (g/dl) - 0.18 0.03

Model R² 0.65

Free renal clearance(ml/min) (Ln)

eGFR (ml/min/1.73m²) (Ln) 1.22 < 0.001Serum albumin (g/dl) 0.19 0.05

Model R² 0.65p-cresyl glucuronide

Total renal clearance (ml/min) (Ln)

eGFR (ml/min/1.73m²) (Ln) 0.91 < 0.001Serum albumin (g/dl) - 0.07 0.81

Model R² 0.09

Free renal clearance (ml/min) (Ln)

eGFR (ml/min/1.73m²) (Ln) 1.01 < 0.001Serum albumin (g/dl) 0.05 0.85

Model R² 0.12eGFR, estimated glomerular filtration rate; Ln, natural logarithmic transformation

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Table 3 – Linear regression analysis of free fraction of p-cresyl sulfate and p-cresyl glucuronide

Variable β PFree fraction ofp-cresyl sulfate (%)

eGFR (ml/min/1.73m²) (Ln) - 0.15 < 0.001Serum albumin (g/dl) - 0.36 < 0.001

Model R² 0.25

Free fraction of p-cresyl glucuronide (%)

eGFR (ml/min/1.73m²) (Ln) - 6.78 < 0.001Serum albumin (g/dl) - 7.31 0.007

Model R² 0.11eGFR, estimated glomerular filtration rate; Ln, natural logarithmic transformation

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Table 4 – Cox proportional hazards analysis of overall mortality and cardiovascular disease for serum total p-cresol and proportion of serum total p-cresyl sulfate to p-cresyl glucuronide

Variable Hazard ratio per SD higher

(95 % confidence interval)

P

MO

RTAL

ITY

Model 1: p-cresol (Ln)

p-cresyl sulfate to glucuronide (Ln)

1.90 (1.37 – 2.61)

0.59 (0.45 – 0.78)

< 0.001

< 0.001

Model 2: p-cresol (Ln)

p-cresyl sulfate to glucuronide (Ln)

1.58 (1.10 – 2.29)

0.65 (0.47 – 0.89)

0.01

0.008

CARD

IOVA

SCU

LAR

DISE

ASE

Model 1: p-cresol (Ln)

p-cresyl sulfate to glucuronide (Ln)

2.10 (1.61 – 2.75)

0.53 (0.41 – 0.69)

< 0.001

< 0.001

Model 2: p-cresol (Ln)

p-cresyl sulfate to glucuronide (Ln)

1.68 (1.27 – 2.22)

0.55 (0.42 – 0.72)

< 0.001

< 0.001

Model 1 included serum total p-cresol and proportion of serum total p-cresyl sulfate to p-cresyl glucuronide. Model 2 included additional adjustment for eGFR (Ln), age, gender, systolic blood pressure, current smoker, diabetes mellitus, cholesterol, calcium, phosphate, parathyroid hormone (Ln), c-reactive protein (Ln), albumin. Serum total p-cresol is the sum of serum total p-cresyl sulfate and p-cresyl glucuronide.eGFR, estimated glomerular filtration rate; Ln, natural logarithmic transformation

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Legends to figures

Figure 1 – Serum levels of p-cresyl sulfate and p-cresyl glucuronide versus eGFR

Correlation between eGFR and serum (A) total p-cresyl sulfate, (B) total p-cresyl glucuronide,

(C) free p-cresyl sulfate, (D) free p-cresyl glucuronide, (E) proportion of total p-cresyl sulfate

to p-cresyl glucuronide and (F) proportion of free p-cresyl sulfate to p-cresyl glucuronide

Figure 2 – 24h urinary excretion of p-cresyl sulfate and p-cresyl glucuronide versus eGFR

Correlation between eGFR and 24h urinary excretion of (A) p-cresyl sulfate and (B) p-cresyl

glucuronide

Figure 3 – Renal clearance of p-cresyl sulfate and p-cresyl glucuronide versus eGFR

Correlation between eGFR and renal clearance of (A) total p-cresyl sulfate, (B) total p-cresyl

glucuronide, (C) free p-cresyl sulfate and (D) free p-cresyl glucuronide

Figure 4 – Protein binding characteristics of p-cresyl glucuronide

Free fraction of p-cresyl glucuronide in (A) albumin in PBS solution versus healthy serum and

(B) healthy serum versus uremic serum and healthy serum with addition of p-cresyl sulfate

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