University of Groningen Towards personalized cardiovascular risk management in renal transplant recipients de Vries, Laura Victorine

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Towards personalized cardiovascular risk management in renal transplant recipients

de Vries, Laura Victorine

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de Vries, L. V. (2018). Towards personalized cardiovascular risk management in renal transplant recipients.

Rijksuniversiteit Groningen.

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Chapter 4

TWENTY-FOUR HOUR URINARY CORTISOL

EXCRETION AND THE METABOLIC SYNDROME

IN PREDNISOLONE-TREATED RENAL

TRANSPLANT RECIPIENTS

Laura V. de Vries Wilhelmina H.A. de Jong

Daan J. Touw Stefan P. Berger Gerjan Navis Ido P. Kema Stephan J.L. Bakker Steroids. 2017; 127: 31-39.

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ABSTRACT

Chronic prednisolone treatment in renal transplant recipients (RTR) causes metabolic abnormalities, which cluster in the metabolic syndrome (MS). It also suppresses the hypothalamic-pituitary-adrenal (HPA) axis. We investigated whether HPA axis sup-pression, as measured by 24h urinary cortisol excretion, is associated with presence of the MS and its individual components, in outpatient RTR with a functioning graft for >1 year. Urinary cortisol was measured in 24h urine, using LC-MS/MS (LOQ 0.30 nmol/L). We included 563 RTR (age 51 ± 12 years; 54% male) at median 6.0 [IQR, 2.6-11.5] years post-transplantation. MS was present in 439/563 RTR (78%). Median 24h urinary cortisol excretion was 2.0 [IQR, 0.9-5.1] nmol/24h. Twenty-four hour urinary cortisol excretion was independently associated with MS presence (OR=0.80 [95% CI, 0.66-0.98], P=0.02). It was also independently associated with bodyweight (st.β=-0.11, P=0.007), waist circumference (st.β=-0.10, P=0.01), BMI (st.β=-0.14, P=0.001), fasting triglycerides (st.β=-0.15, P=0.001), diabetes (st.β=-0.12, P=0.005), and number of anti-hypertensives used (st.β=-0.13, P=0.003). Suppressed HPA axis activity, as reflected by decreased 24h urinary cortisol excretion, is associated with higher prevalence of MS and its individual components (i.e. central obesity, dyslipidemia, diabetes, hyper-tension) in prednisolone-treated RTR. Assessment of 24h urinary cortisol excretion by LC-MS/MS may be a tool to monitor metabolic side-effects of prednisolone in RTR.

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4 INTRODUCTION

Cardiovascular disease is the leading cause of mortality after kidney transplantation, with annual risk of cardiovascular incidents and mortality being 10 to 50-fold higher in renal transplant recipients (RTR) than in the general population.1,2_{In recent years,} many conventional risk factors, such as central obesity, dyslipidemia, hypertension, and diabetes mellitus, have been identified to increase post-transplantation car-diovascular risk. Clustering of these risk factors in the metabolic syndrome occurs frequently in RTR.3-5_{In addition, several transplantation-related risk factors have} been identified, such as decreased kidney function and use of immunosuppressive drugs.2,6

Corticosteroids are known to cause a wide range of metabolic abnormalities, includ-ing weight gain, lipid derangement, new-onset diabetes mellitus after transplantation (NODAT), and hypertension.7,8_{Therefore, there has been a great effort to get rid of} corticosteroids as part of maintenance immunosuppressive regimens after kidney transplantation.7-9_{Nevertheless, it has recently been concluded that corticosteroids} have to remain part of the immunosuppressive regimen in order to maintain low acute rejection rates and optimal long-term graft survival.10,11 _{Unfortunately, corticosteroid} dosing regimens remain empiric to date, usually with fixed doses independent of either body size and/or steroid sensitivity.12

The most often used corticosteroids in RTR are prednisone and prednisolone. Long-term treatment with these drugs is known to suppress the hypothalamic-pi-tuitary-adrenal (HPA) axis, leading to reduced endogenous cortisol production.13,14 Twenty-four hour urinary cortisol excretion is often used to measure HPA axis activity in patients with increased or normal endogenous cortisol production.15-17_Twenty-four hour urinary cortisol excretion may, therefore, also be used to measure HPA axis activity in patients with suppressed endogenous cortisol production, such as pred-nisolone-treated RTR.

Measurement of urinary cortisol in prednisolone-treated patients remains a challenge to date, because of structural similarity of both compounds. Historically used immune-based assays were susceptible to cross-reactivity of exogenous steroids, leading to gross overestimation of urinary cortisol concentrations.18,19

Using a highly sensitive and validated liquid chromatography-tandem mass spec-trometry (LC-MS/MS) method, allows for accurate separation of urinary cortisol from prednisolone and for studying HPA axis activity in prednisolone-treated RTR. Using

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such a method, we aimed to investigate whether the degree of HPA axis suppression, as measured by 24h urinary cortisol excretion, reflects the pharmacological effects of prednisolone, and thus is associated with the metabolic syndrome and its individual components, including central obesity, dyslipidemia, hypertension, and NODAT.

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4 MATERIALS AND METHODS

Research design and subjects

In this cross-sectional study, we invited all adult stable RTR who visited our outpatient transplant clinic between August 2001 and July 2003 and had a functioning graft for at least 1 year. A total of 606 of 847 eligible RTR (72%) signed written informed con-sent. The group that did not sign informed consent was comparable with the group that signed consent. Samples for determination of urinary cortisol were available in 565 RTR (93%). Additional details of this study have been published previously.3,20-22 The Institutional Review Board approved the study protocol (METc 2001/039), which adhered to the Declaration of Helsinki.

Patient characteristics

The Groningen Renal Transplant Database contains information on all kidney trans-plantations performed at our center since 1968. Relevant transplantation-related characteristics were extracted from this database. Current medication was taken from the medical record. Standard immunosuppression regimen was as described previ-ously.20_{In short, it consisted of 5-10 mg prednisolone daily in combination with either} cyclosporine or tacrolimus, and/or either azathioprine or mycophenolate mofetil. Body mass index (BMI), waist circumference, and blood pressure (BP) were measured as described previously.20

Study procedures

At the study visit, fasting blood samples and 24h urine collections of the preceding day were taken. To ensure adequate 24h urine collection, RTR were carefully instructed to start 24h urine collection with emptying of the bladder, collect all subsequent urine through the next 24 hours, and include the next morning’s specimen on the day of their visit. Blood and urine samples were stored at -80°C until assessment of biochem-ical measures for this study. Creatinine in plasma and urine were measured using a modified version of the Jaffé method (MEGA AU 510; Merck Diagnostics). Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epide-miology Collaboration (CKD-EPI) equation.23_{Glucose, insulin, hemoglobin A1c (HbA}

1c),

total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides, were measured as described previously.3,20-22

Measurement of urinary cortisol

Urinary cortisol was measured by on-line solid phase extraction (SPE) coupled with liquid chromatography and tandem mass spectrometry (XLC-MS/MS). 250 μL of urine was mixed directly in an autosampler vial with an internal standard solution

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(corti-sol-d4, Cambridge Isotope Laboratories (Andover, MA) and diluted with water to reach a final volume of 1 mL. Fifty µL of the sample was injected into the XLC-MS/MS system. Sample clean-up was performed by on-line SPE, using a Spark-Holland Symbiosis® system (Spark Holland, Emmen, the Netherlands), as described previously.24_{We used} HySphere C18 HD SPE cartridges (Spark Holland, Emmen) for sample extraction and performed LC by use of a Phenomenex Luna Phenyl-Hexyl column (particle size 3 µm, 2.0 mm internal diameter by 150 mm; Waters). AJ0-4350 Security Guard Cartridges (4 x 2.0 internal diameter) were used as guards for the LC column. Detection was performed with a Quattro Premier®_{tandem mass spectrometer operated in positive} electrospray ionization mode (Waters, Milford, MA). Cortisol and its deuterated internal standard were protonated to produce ions at the form [M+H]+_{, with m/z 363 and m/z} 367, respectively. Upon collision-induced dissociation with Argon gas, these precursor ions produced characteristic product ions of m/z 121 for both cortisol and the deuter-ated internal standard. A multiple reaction monitoring mode was developed for the specific m/z transitions 363→121 and 367→121 (internal standard). For the aim of this study, in which high concentrations of prednisolone were present in urine, it was nec-essary to achieve complete chromatographic separation of cortisol and prednisolone. However, initial ionization lead to the same mass transitions for both cortisol and pred-nisolone. To accomplish complete separation of these molecules, an Agilent Zorbax SB-Phenyl column (particle size 1.8 μm, 2.1 mm internal diameter by 100 mm; Agilent Technologies, Santa Clara, CA) was added.25_{To minimize the effects of inter-assay} vari-ability on the data, all urine samples were analyzed in one lot. Intra- and inter-assay variation coefficients were 2.4% and 7.8% for the lower range and 1.4% and 3.8% for the higher range, respectively. Lower limit of quantitation was 0.30 nmol/L (assay linear range: 0.30 to 1419 nmol/L). Cortisol concentrations were stable when stored up to 7 days at 10 °C or 4 °C. At room temperature, it was stable up to 24 hours. No changes in measured concentrations were observed in urine that had been subjected to 1, 2, or 3 freeze-thaw cycles. Twenty-four hour urinary cortisol excretion was calculated by multiplication of urinary cortisol concentration with urinary volume (in liters); urinary cortisol-to-creatinine ratio was calculated by dividing urinary cortisol concentration (in nmol) by urinary creatinine concentration (in mmol); body surface area (BSA)-corrected 24h urinary cortisol excretion was calculated by dividing 24h urinary cortisol excretion by BSA, which was calculated according to the formula of Dubois and Dubois (BSA = (Weight0.425_{x Height}0.725_{) x 0.007184).}26

Definitions

The metabolic syndrome was defined according to the Third Adult Treatment Panel of the U.S. National Cholesterol Education Plan (NCEP) de finition.5_{This definition requires} patients to have at least three of the five following criteria: (1) central obesity (waist

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circumference ≥102 cm for men and ≥88 cm for women); (2) serum triglycerides of

≥1.7 mmol/L, or specific treatment; (3) serum high-density lipoprotein (HDL) choles-terol <1.03 mmol/L for males and <1.29 mmol/L for females, or specific treatment; (4) systolic BP ≥130 and/or diastolic BP ≥85 mmHg, or treatment of previously diagnosed hypertension; (5) fasting plasma glucose ≥6.1 mmol/L, or previously diagnosed type 2 diabetes mellitus. For secondary analyses we used the International Diabetes Feder-ation (IDF) definition.5_{The main difference between the NCEP definition and the IDF} definition is that the former does not obligatory require central obesity to be present for diagnosis of the metabolic syndrome, whereas it is an obligatory requirement for the latter. Thus, the IDF definition requires patients to have central obesity and at least two of the four remaining criteria. In addition, the IDF defines central obesity as waist circumference ≥90 cm for men and ≥80 cm for women, and increased fasting plasma glucose as ≥5.6 mmol/L. Criteria for triglycerides, HDL cholesterol and blood pressure are the same for both definitions. New-onset diabetes mellitus after transplantation (NODAT) was defined as: clinical symptoms of diabetes mellitus in combination with casual plasma glucose ≥ 11.0 mmol/L or fasting plasma glucose ≥ 7.0 mmol/L.27

Statistical analysis

Data were analyzed with SPSS statistics version 22.0 for Windows (SPSS Inc.) and GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA). Normally dis-tributed data are presented as mean ± standard deviation, non-normally disdis-tributed data as median [interquartile range (IQR)], and nominal data as number (percentage). A two-sided P-value < 0.05 was considered to indicate statistical significance. Vari-able distribution was tested with histograms and probability plots. Skewed data were normalized by logarithmic transformation in all analyses. Initial explorative analyses included all 565 RTR in which data on urinary cortisol were available. In these analy-ses it appeared that two RTR who were not treated with prednisolone were extreme outliers with regard to their urinary cortisol. Therefore, further data presentation and analyses were limited to the 563 patients that were treated with prednisolone. Differences in 24h urinary cortisol excretion, urinary cortisol-to-creatinine ratio, and BSA-corrected 24h urinary cortisol excretion between 7.5 mg and 10 mg prednisolone groups were tested using a Wilcoxon signed rank test.

First, univariable regression analyses were used to explore associations of patient- and transplantation-related characteristics with 24h urinary cortisol excretion, urinary cortisol-to-creatinine ratio, and BSA-corrected 24h urinary cortisol excretion. Logistic regression was applied for dichotomized variables and linear regression for contin-uous variables. Second, multivariable regression analyses were used to determine which components of the metabolic syndrome and which metabolic parameters were

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independently associated with 24h urinary cortisol excretion, urinary cortisol-to-cre-atinine ratio, and BSA-corrected 24h urinary cortisol excretion. In both multivariable logistic and linear regression analyses, crude associations were adjusted for age and sex (model 1), and creatinine clearance (model 2), because they are known to influence urinary cortisol concentrations, and use of calcineurin inhibitors and transplant vintage (model 3), because these parameters were associated with urinary cortisol in initial explorative analyses. All primary analyses were performed using the NCEP criteria for the metabolic syndrome. Secondary analyses were performed using the IDF criteria. Because the vast majority of RTR in our cohort (87%) were treated with antihyperten-sive drugs, increased BP was present in 98% of RTR according to the NECP definition, and only 2% of the population served as control, resulting in low power for finding associations. Therefore, we performed secondary analyses, in which we changed the original NCEP criterion for increased BP to an adapted criterion, defining increased BP as systolic BP ≥130 and/or diastolic BP ≥85 mmHg and use of ≥2 antihypertensive drugs.

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4 RESULTS

Study population

A total of 565 RTR (54% male, mean age 51 ± 12 years) were included at a median time of 6.0 [IQR, 2.6-11.5] years after transplantation. They had stable kidney function with a mean creatinine clearance of 62 ± 22 mL/min and median urinary protein excretion of 0.2 [IQR, 0.0-0.5] g/24h. Of the 565 RTR included, 563 RTR (99.6%) used prednisolone as maintenance immunosuppressive therapy. Median daily prednisolone dose was 10.0 [IQR, 7.5-10.0] mg/day. More specifically, nine RTR (2%) used 5 mg prednisolone per day, five RTR (1%) used 6.25 mg/day, 140 RTR (25%) used 7.5 mg/day, 30 RTR (5%) used 8.75 mg/day, and 379 RTR (67%) used 10 mg/day. Other patient- and transplant-related characteristics are shown in Table 1.

Urinary cortisol excretion

There was considerable inter-individual variation in 24h urinary cortisol excretion, urinary cortisol-to-creatinine ratio, and BSA-corrected 24h urinary cortisol excre-tion in RTR treated with the same prednisolone dose (Figure 1). For the whole study population, median 24h urinary cortisol excretion was 2.0 [IQR, 0.94-5.1] nmol/24h, median cortisol-to-creatinine ratio was 0.19 [IQR, 0.09-0.42] nmol/mmol, and median BSA-corrected 24h urinary cortisol excretion was 1.06 [IQR, 0.49-2.65] nmol/24h/m2_, respectively. Twenty-four hour urinary cortisol excretions in patients who were not treated with prednisolone were 76.8 and 154.7 nmol/24h, cortisol-to-creatinine ratios were 5.1 and 18.9 nmol/mmol, and BSA-corrected 24h urinary cortisol excretions were 39.4 and 85.6 nmol/24h/m2_{, respectively (Figure 1). Since we aimed to study urinary} cor-tisol in prednisolone-treated RTR, further data presentation and analyses are restricted to the 563 RTR who were treated with prednisolone. Median 24h urinary cortisol excre-tion was lower in RTR who were treated with 10 mg prednisolone than in RTR who were treated with 7.5 mg prednisolone (1.8 [IQR, 0.9-4.9] vs. 2.4 [IQR, 1.2-5.3] nmol/24h; Figure 1), albeit borderline significantly (P=0.06). Median cortisol-to-creatinine ratio was significantly lower (0.16 [IQR, 0.08-0.38] vs. 0.23 [IQR, 0.11-0.52] nmol/mmol; P=0.01; Figure 1). Median BSA-corrected 24h urinary cortisol excretion was also significantly lower (0.95 [IQR, 0.48-2.5] vs. 1.31 [0.61-2.99] nmol/24h/m2_{; P=0.03; Figure 1).}

Associations with the metabolic syndrome and its components

Metabolic syndrome was present in 439 RTR (78%) according to the NCEP definition. Prevalences of the individual components of the metabolic syndrome are presented in Figure 2. Multivariable logistic regression analyses showed that presence of the metabolic syndrome, central obesity, and increased fasting glucose, were associated with 24h urinary cortisol excretion, independent of age, sex, creatinine clearance,

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cyc-losporine use and transplant vintage (Table 2). In similar logistic regression analyses, presence of the metabolic syndrome, central obesity, decreased HDL cholesterol, and increased fasting glucose were independently associated with urinary cortisol-to-cre-atinine ratio and BSA-corrected 24h urinary cortisol excretion (Table 2).

Figure 1. Distribution of [A] 24h urinary cortisol excretion (nmol/24h), [B] urinary corti-sol-to-creatinine ratio (nmol/mmol), and [C] BSA-corrected 24h urinary cortisol excretion according to daily prednisolone dose (mg/24h) for the total study population (n=565).

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Figure 2. Prevalence of the metabolic syndrome (MS) and its individual components in prednisolone-treated renal transplant recipients (n=563), according to the Third Adult Treatment Panel of the U.S. National Cholesterol Education Program (NCEP) definition. Individual components include: [1] central obesity (CenOb), [2] increased triglycerides or specific treatment (↑TG), [3] decreased HDL cholesterol or specific treatment (↓HDLc), [4]

increased blood pressure or antihypertensive treatment (↑BP), and [5] increased fasting blood glucose or diagnosis of diabetes mellitus (↑FBG).

Associations with metabolic parameters

Multivariable regression analyses showed that bodyweight (standardized (st.) β=-0.11, P=0.007), waist circumference (st.β=-0.10, P=0.01), BMI (st.β=-0.14, P=0.001), fasting triglycerides (st.β=-0.15, P=0.001), the number of antihypertensive drugs used (st.β=-0.13, P=0.003), use of more than two antihypertensive drugs (β=-0.27, P=0.001), fasting insulin (st.β=-0.10, P=0.03), HbA1c (st.β=-0.09, P=0.04), and presence of NODAT (β=-0.31, P=0.006) were inversely and independently associated with 24h urinary cortisol excretion, while SBP and DBP were not independently associated with 24h urinary cortisol excretion. (Table 3). In similar multivari-able regression analyses, bodyweight (st.β=-0.18, P<0.001), waist circumference (st.β=-0.14, P=0.001), BMI (st.β=-0.18, P<0.001), fasting triglycerides (st.β=-0.15, P<0.001), the number of antihypertensive drugs used (st.β=-0.16, P<0.001), use of more than two antihypertensive drugs (β=-0.31, P<0.001), fasting insulin (st.β=-0.10, P=0.02), HbA1c (st.β=-0.10, P=0.02), and presence of NODAT (β=-0.30, P=0.01) were

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inversely and independently associated with urinary cortisol-to-creatinine ratio (Table 4). There was no significant association of daily prednisolone dose with any of the metabolic parameters (Suppl. Table 1).

Suppl. Figure 1. Prevalence of the metabolic syndrome (MS) and its individual components in prednisolone-treated renal transplant recipients (n=563), according to the International Diabetes Federation (IDF) definition. Individual components include: [1] central obesity (CenOb), [2] increased triglycerides or specific treatment (↑TG), [3] decreased HDL cho-lesterol or specific treatment (↓HDLc), [4] increased blood pressure or antihypertensive treatment (↑BP), and [5] increased fasting blood glucose or diagnosis of diabetes mellitus (↑FBG).

Secondary analyses

Metabolic syndrome was present in 391 RTR (69%) according to the IDF definition. Prevalence of the individual components of the metabolic syndrome are presented in Suppl. Figure 1. Using the IDF definition, multivariable logistic regression analyses showed that presence of the metabolic syndrome and central obesity were borderline significantly associated, and increased fasting glucose was significantly associated with 24h urinary cortisol excretion (Suppl. Table 2). Results for urinary cortisol-to-creatinine ratio and BSA-corrected 24h urinary cortisol excretion were similar, but slightly stron-ger (Suppl. Table 2).

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According to the NCEP criteria, increased BP was present in as much as 551 (98%) out of

563 RTR, which does not allow for meaningful analyses, because of the small number of RTR without increased BP. To overcome this limitation, we applied an adapted, slightly less strict criterion for increased BP, which resulted in 533 RTR (94%) having increased BP. Using the original NCEP BP criterion, increased BP was borderline significantly associated with 24h urinary cortisol excretion, urinary cortisol-to-creatinine ratio, and BSA-corrected 24h urinary cortisol excretion, whereas it was significantly associated with these parameters using the adapted criterion (Suppl. Table 3). Associations of the metabolic syndrome with 24h urinary cortisol excretion, urinary cortisol-to-creatinine ratio, and BSA-corrected 24h urinary cortisol excretion also became stronger using the adapted criterion (Suppl. Table 3).

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Table 1. Patient and transplant-related characteristics and results of univariable linear regression analyses with 24h urinary cortisol excretion, urinary

cortisol-to-creatinine ratio, and BSA-corrected 24h urinary cortisol excretion as dependent variables. Variable (n=563)

Distribution Associations Distribution MIN MAX 24h urinary cortisol excretion

cortisol/creatinine ratio in urine

24h urinary

cortisol excretion / BSA

Stand. β P value Stand. β P value Stand. β P value

Recipient demographics Age (yrs)

51.3 ± 12.0 21 80 0.11 0.008 0.17 < 0.001 0.11 0.009 Male sex, n (%) 301 (54) - 0.03 0.5 - 0.16 < 0.001 - 0.07 0.08 Weight (kg) 76.9 ± 13.7 35.0 128.5 - 0.05 0.15 - 0.19 < 0.001 - 0.14 0.001 Waist (cm) 97.0 ± 13.8 61.5 142.0 - 0.05 0.2 - 0.11 0.01 - 0.11 0.01 BMI (kg/m 2) 26.1 ± 4.3 14.6 42.7 - 0.07 0.10 - 0.11 0.008 - 0.11 0.007

Blood pressure Systolic BP (mmHg)

153 ± 23 108 235 - 0.04 0.3 - 0.03 0.5 - 0.05 0.3 Diastolic BP (mmHg) 90 ± 10 61 122 - 0.06 0.15 - 0.08 0.05 - 0.07 0.10 Number of AHD ( n) 1.9 ± 1.2 0 5 - 0.15 < 0.001 - 0.17 < 0.001 - 0.17 < 0.001 No AHD, n (%) 74 (13) 1 AHD, n (%) 135 (24) 2 or more AHD, n (%) 354 (63)

Lipids Total cholesterol (mmol/L)

5.6 ± 1.1 2.3 15.2 - 0.07 0.08 - 0.04 0.3 - 0.06 0.12 HDL cholesterol (mmol/L) 1.1 ± 0.3 0.4 2.7 0.05 0.2 0.11 0.009 0.08 0.05 LDL cholesterol (mmol/L) 3.6 ± 1.0 0.7 12.0 0.01 0.9 0.01 0.8 0.01 0.8 Triglycerides (mmol/L) 1.9 [1.4-2.6] 0.4 12.3 - 0.17 < 0.001 - 0.15 < 0.001 - 0.18 < 0.001 Use of statins, n (%) 281 (50) - 0.03 0.6 - 0.01 0.8 - 0.03 0.5 Diabetes FBG (mmol/L) 4.9 ± 1.4 2.9 16.6 - 0.03 0.4 - 0.02 0.6 - 0.04 0.4

Fasting insulin (µU/mL)

11.2 [7.9-11.6] 2.0 66.8 - 0.06 0.18 - 0.08 0.06 - 0.08 0.07 HbA1c (%) 6.5 ± 1.1 3.9 11.4 - 0.10 0.02 - 0.09 0.04 - 0.11 0.009 NODAT, n (%) 97 (17) - 0.08 0.07 - 0.05 0.2 - 0.08 0.06

Use of antidiabetic drugs, n (%)

72 (13) - 0.09 0.04 - 0.07 0.10 - 0.09 0.02

Kidney function Serum creatinine (µmol/L)

134 [111-165] 63 510 - 0.18 < 0.001 - 0.23 < 0.001 - 0.20 < 0.001 eGFR (mL/min*1.73) 47 ± 16 10 108 0.17 < 0.001 0.17 < 0.001 0.18 < 0.001

Creatinine clearance (mL/min)

62 ± 23 8 166 0.24 < 0.001 0.07 0.08 0.21 < 0.001

Urinary protein excretion (g/24h)

0.2 [0.0-0.5] 0.0 6.8 0.01 0.8 0.01 0.9 0.01 0.9

Transplantation Transplant vintage (yrs)

5.9 [2.6-11.4] 1.0 31.6 0.03 0.5 0.07 0.07 0.04 0.3 Living donor, n (%) 76 (14) 0.02 0.4 0.01 0.7 0.06 0.2 No. of transplantations, n ( % ) - 0.04 0.4 - 0.03 0.5 - 0.03 0.5 1 503 (89) ≥ 2 60 (11) Acute rejection, n (%) No acute rejection 314 (56) - 0.04 0.4 - 0.02 0.7 - 0.03 0.6

High dose corticosteroids

172 (30) 0.05 0.2 0.06 0.17 0.05 0.2 Anti-lymphocyte antibodies 77 (14) - 0.01 0.8 - 0.05 0.2 - 0.02 0.6

Previous dialysis duration (mo)

27 [14-49] 0 398 - 0.03 0.5 - 0.04 0.3 - 0.04 0.4 HLA mismatches (n) 1.7 ± 1.4 0 6 0.04 0.3 0.01 0.8 0.02 0.6

Immunosuppression Prednisolone dose (mg/24h)

10.0 [7.5-10.0] 5.0 10.0 - 0.05 0.25 - 0.08 0.07 - 0.07 0.11 Calcineurin inhibitor, n (%) 441 (78) - 0.18 < 0.001 - 0.19 < 0.001 - 0.22 < 0.001 Proliferation inhibitor, n (%) 420 (75) 0.03 0.4 0.04 0.3 0.04 0.03 Nominal data are presented as absolute number (percentage), normally distributed data as mean ± standard deviation, and non-normally distributed data as median [interquartile range]. For continuous variables minimum and maximum values are also displayed. For linear regression analyses, skewed data were normalized by log -arithmic transformation. Abbreviations: AHD, antihypertensive drugs; BMI, body mass index; BP, blood pressure; BSA, body surface area; eGFR, estimated glomerular filtration rate; FBG, fasting blood glucose; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; HLA, human leukocyte antigen; LDL, low-density lipoprotein; NODAT,

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Table 1. Patient and transplant-related characteristics and results of univariable linear regression analyses with 24h urinary cortisol excretion, urinary

cortisol-to-creatinine ratio, and BSA-corrected 24h urinary cortisol excretion as dependent variables. Variable (n=563)

Distribution Associations Distribution MIN MAX 24h urinary cortisol excretion

cortisol/creatinine ratio in urine

24h urinary

cortisol excretion / BSA

Stand. β P value Stand. β P value Stand. β P value

Recipient demographics Age (yrs)

51.3 ± 12.0 21 80 0.11 0.008 0.17 < 0.001 0.11 0.009 Male sex, n (%) 301 (54) - 0.03 0.5 - 0.16 < 0.001 - 0.07 0.08 Weight (kg) 76.9 ± 13.7 35.0 128.5 - 0.05 0.15 - 0.19 < 0.001 - 0.14 0.001 Waist (cm) 97.0 ± 13.8 61.5 142.0 - 0.05 0.2 - 0.11 0.01 - 0.11 0.01 BMI (kg/m 2) 26.1 ± 4.3 14.6 42.7 - 0.07 0.10 - 0.11 0.008 - 0.11 0.007

153 ± 23 108 235 - 0.04 0.3 - 0.03 0.5 - 0.05 0.3 Diastolic BP (mmHg) 90 ± 10 61 122 - 0.06 0.15 - 0.08 0.05 - 0.07 0.10 Number of AHD ( n) 1.9 ± 1.2 0 5 - 0.15 < 0.001 - 0.17 < 0.001 - 0.17 < 0.001 No AHD, n (%) 74 (13) 1 AHD, n (%) 135 (24) 2 or more AHD, n (%) 354 (63)

5.6 ± 1.1 2.3 15.2 - 0.07 0.08 - 0.04 0.3 - 0.06 0.12 HDL cholesterol (mmol/L) 1.1 ± 0.3 0.4 2.7 0.05 0.2 0.11 0.009 0.08 0.05 LDL cholesterol (mmol/L) 3.6 ± 1.0 0.7 12.0 0.01 0.9 0.01 0.8 0.01 0.8 Triglycerides (mmol/L) 1.9 [1.4-2.6] 0.4 12.3 - 0.17 < 0.001 - 0.15 < 0.001 - 0.18 < 0.001 Use of statins, n (%) 281 (50) - 0.03 0.6 - 0.01 0.8 - 0.03 0.5 Diabetes FBG (mmol/L) 4.9 ± 1.4 2.9 16.6 - 0.03 0.4 - 0.02 0.6 - 0.04 0.4

11.2 [7.9-11.6] 2.0 66.8 - 0.06 0.18 - 0.08 0.06 - 0.08 0.07 HbA1c (%) 6.5 ± 1.1 3.9 11.4 - 0.10 0.02 - 0.09 0.04 - 0.11 0.009 NODAT, n (%) 97 (17) - 0.08 0.07 - 0.05 0.2 - 0.08 0.06

Use of antidiabetic drugs, n (%)

72 (13) - 0.09 0.04 - 0.07 0.10 - 0.09 0.02

Kidney function Serum creatinine (µmol/L)

134 [111-165] 63 510 - 0.18 < 0.001 - 0.23 < 0.001 - 0.20 < 0.001 eGFR (mL/min*1.73) 47 ± 16 10 108 0.17 < 0.001 0.17 < 0.001 0.18 < 0.001

Creatinine clearance (mL/min)

62 ± 23 8 166 0.24 < 0.001 0.07 0.08 0.21 < 0.001

Urinary protein excretion (g/24h)

0.2 [0.0-0.5] 0.0 6.8 0.01 0.8 0.01 0.9 0.01 0.9

Transplantation Transplant vintage (yrs)

5.9 [2.6-11.4] 1.0 31.6 0.03 0.5 0.07 0.07 0.04 0.3 Living donor, n (%) 76 (14) 0.02 0.4 0.01 0.7 0.06 0.2 No. of transplantations, n ( % ) - 0.04 0.4 - 0.03 0.5 - 0.03 0.5 1 503 (89) ≥ 2 60 (11) Acute rejection, n (%) No acute rejection 314 (56) - 0.04 0.4 - 0.02 0.7 - 0.03 0.6

High dose corticosteroids

172 (30) 0.05 0.2 0.06 0.17 0.05 0.2 Anti-lymphocyte antibodies 77 (14) - 0.01 0.8 - 0.05 0.2 - 0.02 0.6

Previous dialysis duration (mo)

27 [14-49] 0 398 - 0.03 0.5 - 0.04 0.3 - 0.04 0.4 HLA mismatches (n) 1.7 ± 1.4 0 6 0.04 0.3 0.01 0.8 0.02 0.6

Immunosuppression Prednisolone dose (mg/24h)

10.0 [7.5-10.0] 5.0 10.0 - 0.05 0.25 - 0.08 0.07 - 0.07 0.11 Calcineurin inhibitor, n (%) 441 (78) - 0.18 < 0.001 - 0.19 < 0.001 - 0.22 < 0.001 Proliferation inhibitor, n (%) 420 (75) 0.03 0.4 0.04 0.3 0.04 0.03 Nominal data are presented as absolute number (percentage), normally distributed data as mean ± standard deviation, and non-normally distributed data as median [interquartile range]. For continuous variables minimum and maximum values are also displayed. For linear regression analyses, skewed data were normalized by log -arithmic transformation. Abbreviations: AHD, antihypertensive drugs; BMI, body mass index; BP, blood pressure; BSA, body surface area; eGFR, estimated glomerular filtration rate; FBG, fasting blood glucose; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; HLA, human leukocyte antigen; LDL, low-density lipoprotein; NODAT,

(17)

Table 2 . Results of multivariable logistic regression analyses, showing associations of 24h urinary cortisol excretion, urinary cortisol-to-creatinine

ratio, and BSA-corrected 24h urinary cortisol excretion with the metabolic syndrome and its individual components, using NCEP criteria.

Model 1 Model 2 Model 3 OR [95% CI] P-value OR [95% CI] P-value OR [95% CI] P-value

24h urinary cortisol excretion Metabolic syndrome (yes)

0.78 [0.65-0.94] 0.008 0.79 [0.65-0.95] 0.02 0.80 [0.66-0.98] 0.02

Central obesity (yes)

0.85 [0.72-1.00] 0.05 0.81 [0.68-0.96] 0.02 0.83 [0.70-0.99] 0.04 Triglycerides ↑ (yes) 0.81 [0.68-0.97] 0.02 0.85 [0.71-1.02] 0.08 0.86 [0.72-1.04] 0.13 HDL cholesterol ↓ (yes) 0.83 [0.69-1.01] 0.06 0.82 [0.67-1.00] 0.05 0.84 [0.68-1.03] 0.09 Blood pressure ↑ (yes) 0.63 [0.39-1.02] 0.06 0.64 [0.39-1.06] 0.08 0.69 [0.41-1.12] 0.12 FBG ↑ or DM2 (yes) 0.78 [0.63-0.96] 0.02 0.77 [0.62-0.95] 0.01 0.78 [0.63-0.98] 0.03

Urinary cortisol-to-creatinine ratio Metabolic syndrome (yes)

0.74 [0.62-0.90] 0.003 0.76 [0.63-0.92] 0.004 0.77 [0.63-0.93] 0.008

0.80 [0.68-0.95] 0.009 0.79 [0.67-0.93] 0.005 0.81 [0.68-0.96] 0.01 Triglycerides ↑ (yes) 0.82 [0.69-0.98] 0.03 0.84 [0.70-1.01] 0.06 0.86 [0.71-1.03] 0.10 HDL cholesterol ↓ (yes) 0.77 [0.64-0.94] 0.009 0.77 [0.63-0.93] 0.008 0.79 [0.64-0.96] 0.02 Blood pressure ↑ (yes) 0.60 [0.38-0.96] 0.03 0.60 [0.37-0.97] 0.04 0.64 [0.39-1.05] 0.08 FBG ↑ or DM2 (yes) 0.79 [0.64-0.97] 0.02 0.78 [0.64-0.97] 0.02 0.80 [0.65-0.99] 0.04

24h urinary cortisol excretion / BSA Metabolic syndrome (yes)

0.75 [0.62-0.90] 0.002 0.76 [0.63-0.92] 0.005 0.77 [0.63-0.94] 0.009

0.78 [0.66-0.92] 0.003 0.75 [0.63-0.89] 0.001 0.76 [0.64-0.91] 0.002 Triglycerides ↑ (yes) 0.81 [0.67-0.96] 0.02 0.84 [0.70-1.01] 0.06 0.85 [0.71-1.03] 0.09 HDL cholesterol ↓ (yes) 0.81 [0.67-0.98] 0.03 0.80 [0.66-0.97] 0.02 0.82 [0.67-0.99] 0.04 Blood pressure ↑ (yes) 0.61 [0.38-0.99] 0.04 0.63 [0.38-1.02] 0.06 0.67 [0.40-1.11] 0.12 FBG ↑ or DM2 (yes) 0.77 [0.62-0.94] 0.01 0.76 [0.61-0.94] 0.01 0.77 [0.62-0.96] 0.02 Data are presented as odds ratios (OR) plus 95% confidence intervals (95% CI) and corresponding P-values. Presence (yes/no) of the metabolic syndrome and its individual components were entered in the logistic regression as dependent variables; 24h urinary cortisol excretion, urinary cortisol-to-creatinine ratio, and BSA-corrected 24h urinary cortisol excretion as independent variables. Abbreviations: BSA, body surface area; DM2, diabetes mellitus type 2; FBG, fasting blood glucose; HDL, high-density

lipoprotein; NCEP, National Cholesterol Education Program;

↑ , increased; ↓ , decreased. Model 1: age- and sex-adjusted associations; Model 2: as model 1 + adjustment

(18)

4

Table 3

. Results of multivariable regression analyses, showing associations of 24h urinary cortisol excretion with metabolic parameters.

Model 1 Model 2 Model 3 Stand. β P-value Stand. β P-value Stand. β P-value Obesity Weight (kg) -0.06 0.12 -0.12 0.002 -0.11 0.007 Waist circumference (cm) -0.08 0.04 -0.11 0.007 -0.10 0.01 BMI (kg/m 2) -0.09 0.02 -0.15 < 0.001 -0.14 0.001

-0.08 0.06 -0.05 0.2 -0.05 0.3 HDL cholesterol (mmol/L) 0.04 0.3 0.01 0.8 0.01 0.8 LDL cholesterol (mmol/L) 0.01 0.9 0.03 0.5 0.04 0.4 Triglycerides (mmol/L) -0.19 < 0.001 -0.15 0.001 -0.15 0.001

-0.08 0.06 -0.04 0.4 -0.01 0.9 Diastolic BP (mmHg) -0.05 0.2 -0.03 0.4 -0.01 0.9 No. of AHD (n) -0.15 < 0.001 -0.14 0.001 -0.13 0.003 ≥ 2 AHD (yes) -0.29 < 0.001 -0.28 0.001 -0.27 0.001 Diabetes FBG (mmol/L) -0.05 0.2 -0.05 0.2 -0.04 0.4

-0.05 0.2 - 0.09 0.04 -0.10 0.03 HbA1c (%) -0.14 0.001 -0.10 0.01 -0.09 0.04 NODAT (yes) -0.31 0.007 -0.34 0.004 -0.33 0.006 Continuous variables are presented as standardized β coefficients (stand. β) with corresponding P-values from linear regression analyses; dichotomous variables (≥2 antihypertensive drugs (yes/no), NODAT (yes/no)) are presented as non-standardized β coefficients and P-values from logistic regression analyses. Metabolic parameters were entered in the linear regression as dependent variables; 24h urinary cortisol excretion as independent variable. Skewed data were normalized by logarithmic trans -formation in all analyses. Abbreviations: AHD, antihypertensive drugs; BMI, body mass index; BP, blood pressure; FBG, fasting blood glucose; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NODAT, new-onset diabetes mellitus after transplantation. Model 1: age- and sex-adjusted associations; Model

(19)

Table 4

. Results of multivariable regression analyses, showing associations of urinary cortisol-to-creatinine ratio with metabolic parameters.

Model 1 Model 2 Model 3 Stand. β P-value Stand. β P-value Stand. β P-value Obesity Weight (kg) -0.16 < 0.001 -0.19 < 0.001 -0.18 < 0.001 Waist circumference (cm) -0.13 0.001 -0.15 < 0.001 -0.14 0.001 BMI (kg/m 2) -0.17 < 0.001 -0.19 < 0.001 -0.18 < 0.001

-0.06 0.17 -0.05 0.3 -0.04 0.3 HDL cholesterol (mmol/L) 0.06 0.17 0.05 0.3 0.05 0.3 LDL cholesterol (mmol/L) 0.02 0.7 0.03 0.5 0.03 0.5 Triglycerides (mmol/L) -0.18 < 0.001 -0.16 < 0.001 -0.15 < 0.001

-0.08 0.05 -0.06 0.13 -0.02 0.6 Diastolic BP (mmHg) -0.07 0.12 -0.06 0.18 -0.02 0.7 No. of AHD (n) -0.18 < 0.001 -0.17 < 0.001 -0.16 < 0.001 ≥ 2 AHD (yes) -0.32 < 0.001 -0.31 < 0.001 -0.31 < 0.001 Diabetes FBG (mmol/L) -0.05 0.3 -0.05 0.3 -0.03 0.5

-0.08 0.06 -0.10 0.02 -0.10 0.02 HbA1c (%) -0.14 0.001 -0.12 0.004 -0.10 0.02 NODAT (yes) -0.30 0.009 -0.31 0.007 -0.30 0.01 Continuous variables are presented as standardized β coefficients (stand. β) with corresponding P-values from linear regression analyses; dichotomous variables (≥2 antihypertensive drugs (yes/no), NODAT (yes/no)) are presented as non-standar dized β coefficients and P-values from logistic regression analyses. Metabolic parameters were entered in the linear regression as dependent variables; urinary cortisol-to-creatinine ratio as independent variable. Skewed data were normalized by logarithmic transformation in all analyses. Abbreviations: AHD, antihypertensive drugs; BMI, body mass index; BP, blood pressure; FBG, fasting blood glucose; HbA1c, glycated hemo -gl ob in ; H D L, hi gh -d en si ty li po pro te in ; LD L, low -d en si ty li po pro te in ; N O D AT, ne w -o ns et d iab et es me lli tu s af te r t ran sp lan tat io n. M od el 1 : ag an d se x-ad ju st ed ass oci at io ns ;

(20)

4

Suppl. Table 1

. Results of multivariable regression analyses, showing associations of daily prednisolone dose with metabolic parameters.

Model 1 Model 2 Model 3 Stand. β P-value Stand. β P-value Stand. β P-value

Daily prednisolone dose Obesity Weight (kg)

0.03 0.5 0.05 0.2 0.03 0.5 Waist circumference (cm) 0.01 0.7 0.02 0.6 0.02 0.7 BMI (kg/m 2) 0.01 0.8 0.03 0.5 0.03 0.5

-0.02 0.7 -0.03 0.5 0.02 0.7 HDL cholesterol (mmol/L) 0.02 0.6 0.03 0.5 0.06 0.2 LDL cholesterol (mmol/L) 0.01 0.8 0.01 0.9 0.05 0.3 Triglycerides (mmol/L) 0.01 0.9 -0.01 0.8 -0.01 0.9

0.03 0.5 0.03 0.5 0.02 0.7 Diastolic BP (mmHg) 0.06 0.15 0.06 0.2 0.05 0.3 No. of AHD (n) 0.03 0.5 0.02 0.7 0.04 0.4 ≥ 2 AHD (yes) -0.01 0.9 -0.01 0.9 0.01 0.8 Diabetes FBG (mmol/L) 0.01 0.9 0.01 0.9 0.01 0.9

0.07 0.09 0.09 0.05 0.06 0.16 HbA1c (%) 0.03 0.5 0.02 0.7 0.01 0.9 NODAT (yes) -0.04 0.4 -0.04 0.4 -0.02 0.7 Continuous variables are presented as standardized β coefficients (stand. β) with corresponding P-values from linear regression analyses; dichotomous variables (≥2 anti -hypertensive drugs (yes/no), NODAT (yes/no)) are presented as non-standardized β coefficients and P-values from logistic regression analyses. Metabolic parameter s wer e

entered in the linear regression as dependent

variables; daily prednisolone

dose as independent

variable. Skewed

data were normalized by logarithmic transformation in

all analyses. Abbreviations: AHD, antihypertensive drugs; BMI, body mass index ; BSA, body surface area; BP, blood pressure; FBG, fasting blood glucose; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NODAT, new-onset diabetes mellitus after transplantation. Model 1: age- and sex-adju sted asso

(21)

Suppl. Table 2 . Results of multivariable logistic regression analyses, showing associations of 24h urinary cortisol excretion, urinary cortisol-to-cre

-atinine ratio, and BSA-corrected 24h urinary cortisol excretion with the metabolic syndrome and its individual components, using the IDF criteria.

0.87 [0.73-1.03] 0.11 0.82 [0.69-0.98] 0.03 0.85 [0.71-1.02] 0.08

0.90 [0.75-1.09] 0.3 0.82 [0.67-1.00] 0.05 0.84 [0.68-1.02] 0.09 Triglycerides ↑ (yes) 0.81 [0.68-0.97] 0.02 0.85 [0.71-1.02] 0.08 0.86 [0.72-1.04] 0.13 HDL cholesterol ↓ (yes) 0.83 [0.69-1.01] 0.06 0.82 [0.67-1.00] 0.05 0.84 [0.68-1.03] 0.09 Blood pressure ↑ (yes) 0.63 [0.39-1.02] 0.06 0.64 [0.39-1.06] 0.08 0.69 [0.41-1.12] 0.12 FBG ↑ or DM2 (yes) 0.78 [0.64-0.95] 0.01 0.76 [0.62-0.93] 0.009 0.78 [0.63-0.96] 0.02

0.79 [0.66-0.94] 0.008 0.77 [0.65-0.92] 0.004 0.79 [0.66-0.95] 0.01

0.81 [0.68-0.96] 0.01 0.76 [0.64-0.91] 0.003 0.79 [0.65-0.94] 0.009

0.82 [0.68-0.99] 0.04 0.75 [0.61-0.91] 0.004 0.77 [0.63-0.94] 0.009 Triglycerides ↑ (yes) 0.81 [0.67-0.96] 0.02 0.84 [0.70-1.01] 0.06 0.85 [0.71-1.03] 0.09 HDL cholesterol ↓ (yes) 0.81 [0.67-0.98] 0.03 0.80 [0.66-0.97] 0.02 0.81 [0.67-0.99] 0.04 Blood pressure ↑ (yes) 0.61 [0.38-0.99] 0.04 0.62 [0.38-1.02] 0.06 0.67 [0.40-1.11] 0.12 FBG ↑ or DM2 (yes) 0.77 [0.63-0.94] 0.01 0.75 [0.61-0.92] 0.006 0.77 [0.63-0.95] 0.01 Data are presented as odds ratios (OR) plus 95% confidence intervals (95% CI) and corresponding P-values. Presence (yes/no) of the metabolic syndrome and its individual components were entered in the logistic regression as dependent variables; 24h urinary cortisol excretion, urinary cortisol-to-creatinine ratio, and BSA-corrected 24h urinary cortisol excretion as independen t variables. Abbreviations: BSA, body surface area, DM2, diabetes mellitus type 2; FBG, fasting blood glucose; HDL, high-density lipoprotein; IDF, International Diabetes Federation; ↑ , increased; ↓ , decreased. Model 1: age- and sex-adjusted associations; Model 2: as model 1 + adjustment for creat

(22)

4

Suppl. Table 2 . Results of multivariable logistic regression analyses, showing associations of 24h urinary cortisol excretion, urinary cortisol-to-cre

-atinine ratio, and BSA-corrected 24h urinary cortisol excretion with the metabolic syndrome and its individual components, using the IDF criteria.

0.87 [0.73-1.03] 0.11 0.82 [0.69-0.98] 0.03 0.85 [0.71-1.02] 0.08

0.90 [0.75-1.09] 0.3 0.82 [0.67-1.00] 0.05 0.84 [0.68-1.02] 0.09 Triglycerides ↑ (yes) 0.81 [0.68-0.97] 0.02 0.85 [0.71-1.02] 0.08 0.86 [0.72-1.04] 0.13 HDL cholesterol ↓ (yes) 0.83 [0.69-1.01] 0.06 0.82 [0.67-1.00] 0.05 0.84 [0.68-1.03] 0.09 Blood pressure ↑ (yes) 0.63 [0.39-1.02] 0.06 0.64 [0.39-1.06] 0.08 0.69 [0.41-1.12] 0.12 FBG ↑ or DM2 (yes) 0.78 [0.64-0.95] 0.01 0.76 [0.62-0.93] 0.009 0.78 [0.63-0.96] 0.02

0.79 [0.66-0.94] 0.008 0.77 [0.65-0.92] 0.004 0.79 [0.66-0.95] 0.01

0.81 [0.68-0.96] 0.01 0.76 [0.64-0.91] 0.003 0.79 [0.65-0.94] 0.009

0.82 [0.68-0.99] 0.04 0.75 [0.61-0.91] 0.004 0.77 [0.63-0.94] 0.009 Triglycerides ↑ (yes) 0.81 [0.67-0.96] 0.02 0.84 [0.70-1.01] 0.06 0.85 [0.71-1.03] 0.09 HDL cholesterol ↓ (yes) 0.81 [0.67-0.98] 0.03 0.80 [0.66-0.97] 0.02 0.81 [0.67-0.99] 0.04 Blood pressure ↑ (yes) 0.61 [0.38-0.99] 0.04 0.62 [0.38-1.02] 0.06 0.67 [0.40-1.11] 0.12 FBG ↑ or DM2 (yes) 0.77 [0.63-0.94] 0.01 0.75 [0.61-0.92] 0.006 0.77 [0.63-0.95] 0.01 Data are presented as odds ratios (OR) plus 95% confidence intervals (95% CI) and corresponding P-values. Presence (yes/no) of the metabolic syndrome and its individual components were entered in the logistic regression as dependent variables; 24h urinary cortisol excretion, urinary cortisol-to-creatinine ratio, and BSA-corrected 24h urinary cortisol excretion as independen t variables. Abbreviations: BSA, body surface area, DM2, diabetes mellitus type 2; FBG, fasting blood glucose; HDL, high-density lipoprotein; IDF, International Diabetes Federation; ↑ , increased; ↓ , decreased. Model 1: age- and sex-adjusted associations; Model 2: as model 1 + adjustment for creat

-inine clearance; Model 3: as model 2 + adjustment for cyclosporine use and transplant vintage. Suppl.

Table 3

. Results of multivariable logistic regression analysis,

showing

associations of 24h

urinary cortisol excretion, urinary cortisol-to-creati

-nine

ratio, and BSA-corrected 24h

urinary cortisol excretion with the metabolic syndrome and its

individual

component “increased blood

pressure”. Secondary analyses compared the following BP criteria: [1] SBP ≥130 and/or DBP ≥85 mmHg and/or use of antihypertensive drugs (original NCEP criterion) and [2]

SBP ≥130 and/or DBP ≥85 mmHg and/or use of ≥2 antihypertensive drugs (adapted criterion).

24h urinary cortisol excretion Increased blood pressure (yes) Original NCEP BP criterion (≥1 AHD)

0.63 [0.39-1.02] 0.06 0.64 [0.39-1.06] 0.08 0.69 [0.41-1.12] 0.12

Adapted BP criterion (≥2 AHD)

0.56 [0.41-0.76] < 0.001 0.57 [0.41-0.79] 0.001 0.64 [0.45-0.90] 0.01

Metabolic syndrome (yes) Original NCEP BP criterion (≥1 AHD)

0.78 [0.65-0.94] 0.008 0.79 [0.65-0.95] 0.02 0.80 [0.66-0.98] 0.02

0.74 [0.62-0.89] 0.001 0.75 [0.62-0.91] 0.004 0.78 [0.64-0.95] 0.01

Urinary cortisol-to-creatinine ratio Increased blood pressure (yes) Original NCEP BP criterion (≥1 AHD)

0.60 [0.38-0.96] 0.03 0.60 [0.37-0.97] 0.04 0.64 [0.39-1.05] 0.08

0.55 [0.40-0.75] < 0.001 0.56 [0.41-0.76] < 0.001 0.62 [0.44-0.86] 0.005

0.74 [0.62-0.90] 0.003 0.76 [0.63-0.92] 0.004 0.77 [0.63-0.93] 0.008

0.72 [0.59-0.86] < 0.001 0.73 [0.60-0.88] 0.001 0.75 [0.62-0.91] 0.003

24h urinary cortisol excretion / BSA Increased blood pressure (yes) Original NCEP BP criterion (≥1 AHD)

0.61 [0.38-0.99] 0.04 0.63 [0.38-1.02] 0.06 0.67 [0.40-1.11] 0.12

0.55 [0.40-0.75] < 0.001 0.56 [0.41-0.77] < 0.001 0.63 [0.45-0.88] 0.007

0.75 [0.62-0.90] 0.002 0.76 [0.63-0.92] 0.005 0.77 [0.63-0.94] 0.009

0.71 [0.59-0.85] < 0.001 0.73 [0.60-0.87] 0.001 0.75 [0.61-0.91] 0.003 Data are presented as odds ratios (OR) plus 95% confidence intervals (95% CI) and corresponding P-values. Presence (yes/no) of the metabolic syndrome and increased blood pressure were entered in the logistic regression as dependent variables; 24h urinary cortisol excretion, urinary cortisol-to-creatinine ratio, and BSA-corrected 24h urin ary cortisol excreti on , as in dep en den t variab les. Ab breviation s: AH D, an tih yp erten sive dru gs; BP, blood p ressu re; BS A, bod y su rf ace area; DBP, diastolic blood p ressu re; NCEP, National Cholesterol Education Plan; SBP, systolic blood pressure. Model 1: age- and sex-adjusted associations; Model 2: as model 1 + adjustment for creatinine

(23)

DISCUSSION

In the current study, we found that there is considerable variation in 24h urinary cor-tisol excretion in RTR who are chronically treated with prednisolone. In addition, we found that a higher degree of HPA axis suppression by prednisolone, as reflected by decreased 24h urinary cortisol excretion, is associated with higher prevalence of the metabolic syndrome and its individual components. When we used urinary corti-sol-to-creatinine ratio or BSA-corrected 24h urinary cortisol excretion as measures for HPA axis activity, associations were similar, but generally stronger.

Prednisolone has remained an integral part of transplant immunosuppression for more than 50 years, despite the fact that its use is often accompanied with signifi-cant side effects.7,8_{These side effects include profound induction of components of} the metabolic syndrome, and they are for many RTR and their transplant physicians reason to try to avoid or stop prednisolone treatment. Indeed, the proportion of RTR discharged from hospital after transplantation with a steroid avoidance regimen has steadily been rising in the past, but has now plateaued at a level of about 35% of all transplantations.28_{Furthermore, it appears that, with an initial steroid avoidance rate} of 35%, steroid treatment is added to immunosuppressive regiments within one year after transplantation in approximately 20-25% of cases with initial steroid avoidance.28 Hence, in the United States, at one year after transplantation, prednisolone is still part of the long-term maintenance regimen in 70-75% of RTR.28_{In our stable renal} transplant population, only 2 out of 565 RTR were not on maintenance prednisolone treatment.

We hypothesized that 24h urinary cortisol excretion could be a measure of the degree of suppression of the HPA axis by prednisolone, and thus reflect the pharmacological effects of prednisolone. Indeed, we found that RTR with the lowest amount of endog-enous cortisol production, and thus the highest degree of HPA axis suppression, had the highest prevalence of the metabolic syndrome. In addition, they suffered most severely from metabolic abnormalities; they had higher bodyweight, higher waist circumference, higher BMI, higher fasting triglycerides, and a higher prevalence of NODAT. We did not find an association of systolic or diastolic blood pressure with 24h urinary cortisol excretion. This is likely due to adequate pharmacological treatment of hypertension in RTR, as the vast majority of RTR (87%) in our study used antihyper-tensive medication. Therefore, the number of antihyperantihyper-tensive drugs by which RTR are treated, probably better reflects the tendency for high blood pressure than blood pressure itself. In line, we found a strong association of the number of antihypertensive drugs used with 24h urinary cortisol excretion.

(24)

4

We did not find an association of daily prednisolone dose with any of the metabolic

parameters. This suggests that daily dose of prednisolone does not reliably reflect its pharmacological effects in individual patients, that variation in dose was too low to allow for finding associations, or a combination of both. Indeed, the majority of patients in our cohort were treated with either 7.5 mg or 10 mg of prednisolone, resulting in little variation in this parameter, while 24h urinary cortisol excretion varied greatly between individuals, with also variation between RTR who were treated with a similar daily prednisolone dose.

We used LC-MS/MS for the measurement of urinary cortisol concentrations. For the analysis of urinary cortisol, mass spectrometry (MS) methods are known to offer better specificity and accuracy than immunoassay-based methods.18,19 _{But even with more} recent sensitive methods using liquid chromatography (LC) coupled with mass spec-trometry (MS), it remains a challenge to accurately separate cortisol from prednisolone and to measure the very low cortisol concentrations caused by prednisolone treatment, because cross-reactivity of prednisolone with cortisol remains.25,29,30_{This is especially} a problem in patient populations with a suppressed HPA axis, such as ours, in whom urinary cortisol concentrations are relatively low and prednisolone concentrations relatively high. We achieved complete chromatographic separation of prednisolone and cortisol, using a Zorbax SB-Phenyl column, which was previously used by Ionita et al to separate cortisol and prednisolone in plasma.25_{Another advantage of our} method is that it allowed us to measure urinary cortisol concentrations as low as 0.30 nmol/L. We could only find two recent studies using LC-MS/MS, which could measure urinary cortisol concentrations below 1 nmol/L.31,32_{In these studies, which were aimed} at developing new LC-MS/MS methods, lower limits of quantitation were 0.14 nmol/L31 and 0.44 nmol/L,32_{respectively.}

Twenty-four hour urinary cortisol excretion is often used to measure HPA axis activity, both in subjects with normal17,33_{and increased HPA axis function.}15,16_{It has the} advan-tage of being unaffected by the circadian rhythm of cortisol and by varying plasma protein binding capacities.17,34_{In addition, it is relatively easy to collect in a large study} population.17,33_{Despite of its use in other populations, 24h urinary cortisol excretion} has, to our knowledge, not been studied in subjects with a suppressed HPA axis, such as RTR. In general, studies trying to monitor HPA axis activity in prednisolone-treated RTR are scarce. We could find only two small studies, which measured serum cortisol concentration to evaluate prednisolone pharmacodynamics.35,36 _{Both studies found} serum cortisol to be significantly lower in patients who experienced prednisolone side effects compared to patients who did not.35,36_{This is in line with the results we found} for urinary cortisol excretion.

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Post-transplantation metabolic syndrome is known to occur frequently. However, its reported prevalence varies in literature. In our study, metabolic syndrome prevalence was 78% according to the NCEP definition, and 69% according to the IDF definition. Courivaud et al reported a prevalence of 32% in 337 RTR, who were one year post-trans-plantation.37_{In addition, Porrini et al reported a prevalence of 23% in 230 RTR at one} year, which increased to 38% at 18-months post-transplantation.38_{Metabolic syndrome} prevalence in these studies was more or less similar to that of the (European) general population.5,39_{Compared to these studies, which both used the NCEP definition, like} we did, metabolic syndrome in our study was twice as prevalent. However, the study of Porrini et al also showed that metabolic syndrome prevalence after kidney trans-plantation may increase over time.38_{Since median time after transplantation in our} study was 6 years, this could contribute to higher prevalence in our study. Although prednisolone dosages in these studies were similar to those in our study, longer expo-sure to pharmacological effects of prednisolone may have contributed to increased metabolic syndrome prevalence.

The present study has several limitations. First, given its cross-sectional nature, it is hard to disentangle causal relationships. Although we used multivariable logistic and linear regression models and adjusted for several potential confounding variables, including parameters of kidney function, the possibility of residual confounding cannot be excluded. Second, urinary cortisol excretion was measured at a single time point only and therefore we could not take potential changes over time into account. How-ever, previous studies found that intra-individual prednisolone pharmacokinetics are relatively constant40_{and 24h urinary cortisol values are relatively stable over time.}17 The main strength of this study is that it is, to our knowledge, the first study to mea-sure 24h urinary cortisol excretion in a large cohort of prednisolone-treated RTR. In addition, next to analyzing 24h urinary cortisol excretion, we also analyzed urinary cortisol-to-creatinine ratio and BSA-corrected 24h urinary cortisol excretion, which showed similar results, adding to the robustness of our findings. Other strengths of our study include the well-characterized patient population and the very sensitive method used to measure urinary cortisol.

In conclusion, using a highly sensitive and validated LC-MS/MS method, we show for the first time that some HPA axis activity, as measured by 24h urinary cortisol excre-tion, is present in the majority of RTR treated with prednisolone. In addiexcre-tion, we show that there is considerable inter-individual variation in the degree of HPA axis suppres-sion. Moreover, lower 24h urinary cortisol excretion is associated with the metabolic syndrome and its individual components, including central obesity, dyslipidemia, impaired glucose metabolism, and increased blood pressure. Accurate assessment of

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24h urinary cortisol excretion by a mass spectrometry based method may be a tool

to monitor pharmacological effects of prednisolone and to reduce the burden of its metabolic side-effects in RTR.

ACKNOWLEDGEMENTS

The authors kindly thank Claude van der Ley and Bettine Haandrikman for their valu-able technical assistance.

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