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Low pre-transplant adiponectin multimers are associated with adverse allograft outcomes in kidney transplant recipients a 3-year prospective study
Regulatory Peptides 178 (2012) 11–15
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Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
Low pre-transplant adiponectin multimers are associated with adverse allograft outcomes in kidney transplant recipients a 3-year prospective study Marcel Roos a,⁎, 1, Marcus Baumann a, 2, Dan Liu a, 2, Falko M. Heinemann b, Monika Lindemann b, Peter A. Horn b, Tobias Türk c, 2, Jens Lutz a, 2, Uwe Heemann a, 2, Oliver Witzke c, 1, 3, Maximilian von Eynatten a, 1, 3 a b c
Dept. of Nephrology, Technische Universität München, Munich, Germany Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany Dept. of Nephrology, University Hospital Essen, Essen, Germany
a r t i c l e
i n f o
Article history: Received 14 December 2011 Accepted 19 June 2012 Available online 27 June 2012 Keywords: Adiponectin Endothelial dysfunction Kidney transplantation Renal allograft failure
a b s t r a c t Background: In kidney transplant recipients endothelial dysfunction is almost a universal risk factor for allograft failure. Adiponectin, an adipocyte derived hormone, has endothelial-protective properties and the highmolecular weight (HMW) multimer is the major active form, exerting anti-inflammatory and anti-apoptotic effects on endothelial cells. This study evaluated, whether pre-transplant total and HMW multimer adiponectin levels are associated with markers of endothelial dysfunction and arteriosclerosis and predict long-term graft survival in patients after kidney transplantation. Methods: In 206 renal transplant recipients pre-transplant total and HMW adiponectin levels were measured in serum by ELISA and Western blot, respectively. During the 36 months active follow up (median [interquartile range] 1249 [1020; 1445] days) 13 patients died (94% patient survival) and renal allograft failure was reported in 18 patients (91% graft survival). Results: Pre-transplant total and HMW adiponectin levels were significantly associated with lipid and glucose parameters at baseline. After 3 years follow-up pre-transplant total and HMW adiponectin levels were significantly inversely associated with the incidence of allograft failure (adiponectin: r = − 0.216; p = 0.002: HMW: r = −0.218; p = 0.002). In multivariable adjusted Cox proportional hazard regression models patients in the lowest total and HMW adiponectin quartile had a significantly increased risk for allograft failure within 3 years post-transplantation: odds ratio [95%CI]: total adiponectin: 4.25 [1.27–14.24; p = 0.019], and HMW multimers: 3.35 [1.04–10.76; p = 0.042], respectively. Conclusion: Low pre-transplant levels of total and HMW adiponectin reflect a pro-atherogenic endothelial milieu and independently predict an increased risk of allograft failure in kidney-transplant recipients. Measurement of adiponectin levels may identify patients at risk for adverse allograft outcomes after kidney transplantation. © 2012 Published by Elsevier B.V.
1. Introduction Long term survival of renal allografts is the major goal of clinical kidney transplantation [1]. Immunological risk factors, such as age, race, HLA matching, and acute rejection episodes are well established risk factors for impaired allograft survival. In addition, traditional vascular pro-atherogenic risk factors, such as arterial hypertension, ⁎ Corresponding author at: Department of Nephrology, Klinikum rechts der Isar, Ismaninger Str. 22, 81675 München, Germany. Tel.: +49 89 4140 2231; fax: +49 89 4140 4878. E-mail address: [email protected] (M. Roos). 1 Designed the research, performed the study, analyzed the data, and wrote the paper. 2 Designed the research and collected data, FH, ML, PH performed the research, collected the data, and wrote the paper. 3 These authors contributed equally to this work. 0167-0115/$ – see front matter © 2012 Published by Elsevier B.V. doi:10.1016/j.regpep.2012.06.001
hyperlipidemia, and metabolic syndromes including impaired glucose tolerance add to the risk of renal allograft failure and poor posttransplant patient survival [2,3]. More recently the introduction of novel, additional biomarkers further improved risk prediction strategies after kidney transplantation [4–6]. Thus, pre-transplant risk evaluation by immunologic and non-immunologic risk assessment has gained substantial clinical importance. However, despite all recent progresses, reliable identification of patients at high risk for adverse renal allograft outcomes is still vague and novel, and innovative riskidentifying strategies are still an unmet medical need; these should be amended in order to improve pre-transplant risk evaluation and subsequent preventive efforts. Adiponectin, the most abundant adipokine in humans, circulates in different multimer complexes with distinct molecular weights [7,8]. The high-molecular weight (HMW) form of adiponectin has been shown to be the predominant active form of this hormone, exerting
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M. Roos et al. / Regulatory Peptides 178 (2012) 11–15
anti-inflammatory, anti-diabetic and endothelial protective properties [9]. In clinical studies low HMW levels were associated with systemic inflammation, atherogenic dyslipidemia, impaired insulin sensitivity, risk for type 2 diabetes, endothelial dysfunction, arteriosclerosis and eventually coronary artery disease (CAD) [10–13]. The link between low adiponectin and both, endothelial and metabolic dysfunction was previously confirmed in patients undergoing renal transplantation. In these patients, low adiponectin levels were associated with increased markers of endothelial dysfunction, suggesting elevated adiponectin to act as a counter regulatory defense system in consequence of an accelerated endothelial inflammatory milieu [14]. Moreover, in a prospective follow-up study low pre-transplant adiponectin levels were associated with an increased risk for the development of new onset diabetes mellitus after transplantation (NODAT) [15]. The aim of this study was to evaluate whether pre-transplant adiponectin and HMW multimer levels are associated with markers of endothelial dysfunction and arteriosclerosis in 206 patients undergoing renal transplantation. In addition, the predictive value for total adiponectin and HMW multimers on long-term renal allograft survival was investigated during a 36 month follow-up period. 2. Material and methods 2.1. Patients 206 renal transplant recipients were enrolled in this study and active follow-up was conducted for 36 months (median [interquartile range] 1249 [1020–1445] days). The study design and methods have been reported previously [5]. Briefly, all patients were transplanted with kidney (n = 194) or kidney/pancreas (n = 12) allografts in a consecutive series from May 2001 to June 2004 in the University Hospital of Essen, Germany, of whom 174 (84%) were transplanted with a kidney from a deceased donor and 32 (16%) patients received an allograft from a living donor. Pediatric transplantations and combined kidney/ liver transplantations were excluded. Primary kidney diseases of the patients studied were glomerulonephritis (n = 75), cystic kidney disease (n = 29), diabetes mellitus types 1 and 2 (n = 20), interstitial nephritis (n = 20), renal vascular disease (n = 13), and other diseases (n = 49). Sixty-three patients were treated with cyclosporine (CsA) (100–150 ng/ml whole blood through level, tested by FPIA Abbott TDx monoclonal assay; Abbott Laboratories Limited, Wiesbaden, Germany) and 136 patients with tacrolimus (4–10 ng/ml whole blood through level, tested by Abbott Imx tacrolimus assay). A total of 109 received MMF in combination with tacrolimus and 56 patients in combination with CsA. Additionally, all but four patients received prednisone prior to transplantation; antithymocyte globulin was given to eight patients, combined with tacrolimus or MMF. Loss of renal allograft function and renal rejection episodes were histologically diagnosed in all patients concerned. In total, 13 patients (6%) died within 3 years after transplantation. Patients who died with a functioning graft (n = 9) were excluded from the survival analysis. Cytomegalovirus (CMV) infections within the first 4 months posttransplantation were detected by immunohistological pp65 assay and occurred in 26 patients (13%). HLA typing of patients and donors was performed by serology or polymerase chain reaction using sequence specific primers (PCR-SSP) for the HLA-A, -B, ‐DR, and ‐DQ loci as previously described [5]. 2.2. Clinical chemistry Adiponectin levels were measured with ELISA according to the manufacturer's instructions (BioVendor, Brno, Czek Republic). Both, the intra- and inter‐assay CVs were b5.0%. HMW adiponectin multimer levels were quantified by Western blot analysis, as previously described [8]. In brief, serum proteins were separated by 10% SDS-PAGE under
nonreducing and nonheating conditions, and transferred to nitrocellulose membranes. Membranes were blocked with TBS-Tween20 containing 5% skim milk and incubated with a goat antihuman adiponectin polyclonal antibody (1:500, R&D Systems, Wiesbaden, Germany). After being washed, membranes were incubated with horseradish peroxidise–conjugated donkey antigoat antibody (1:4000, Santa Cruz Biotechnology, Santa Cruz, California, USA). Bands were visualized by the use of lumi-light Western blotting substrate (Roche Diagnostics, Mannheim, Germany), and the image was acquired with a Kodak IS440CF Imaging Station. Densitometry analysis was performed with Adobe Photoshop software. Relative distributions of adiponectin multimers were calculated by dividing the band density by the total density. Percentages of multimers were multiplied by the total adiponectin concentrations obtained by ELISA to calculate the absolute values. 2.3. Statistics Bivariate correlations between pre-transplant adiponectin levels and parameters of interest were calculated by the Spearman's correlation coefficient and the Mann–Whitney U test was applied to compare two independent groups. Cox proportional multivariable analyses were performed to analyze the independent association between adiponectin, HMW and other covariates with the risk of allograft failure. For analyses pre-transplant adiponectin and HMW levels were divided into quartiles (adiponectin: 1st: b 16.0 μg/ml, 2nd: ≥16.0–37.5 μg/ml, 3rd: ≥37.6–65.2 μg/ml and 4th: >65.2 μg/ml. HMW 1st: b7.6 μg/ml, 2nd ≥7.6–19.1 μg/ml, 3rd: ≥19.2–35.4 μg/ml and 4th: >35.4 μg/ml). Further independent parameters were included in the analyses as previously described [5]: recipient sex, previous transplantations, percent PRA (positive vs. negative), donor type (cadaveric vs. living), kidney or kidney/pancreas transplantation, number of HLA-A, -B, -DR mismatches (0, 1, 2 vs. 3, 4, 5, 6), CMV infections, and allograft rejection. Statistical analyses were performed with SPSS software version 15.0 (SPSS Inc., Chicago, IL) and a p-value of b0.05 was considered as statistically significant. 3. Results Patient demographics and clinical characteristics at baseline are presented in Table 1. A total of 206 patients were included in this study and active follow up was conducted for 36 months (median [interquartile range] 1249 [1020–1445] days). The follow-up data revealed a 94% patient survival rate (13 patients died). Nine patients died with a functioning graft and were therefore excluded from the survival analysis. In the remaining group of 197 patients, 18 patients (9%) experienced a graft failure within 2 years after transplantation. Pre-transplant adiponectin and HMW levels were significantly lower in patients with incident graft failure as compared to patients with functional graft after long-term follow-up (adiponectin: 38.1 ± 41.0 μg/ml vs. 14.6 ± 33.5 μg/ml; p = 0.005, HWM: 19.6 ± 22.6 μg/ml vs. 7.2 ± 17.8 μg/ml p = 0.007, Fig. 1A and B). In addition, donor age was significantly higher in patients experiencing graft failure during the follow-up as compared to non-failure individuals (52.3 ± 21.6 vs. 46.3 ± 16.6 years; p = 0.013). No significant difference between patients with and without graft failure was seen for recipient age, cold ischemic time, and HLA-mismatches from cadaveric donors. In order to evaluate pre-transplant associations between adiponectin levels and atherogenic risk factors, we evaluated Spearman correlation coefficients between pre-transplant total adiponectin and HMW levels and various surrogate parameters for endothelial dysfunction and arteriosclerosis (presented in Table 2). Both adiponectin measures were significantly associated with atherogenic markers of lipid and glucose metabolism. Total adiponectin and HMW levels significantly correlated with HDL-cholesterol (both r> 0.45; both p b 0.05), and fasting plasma glucose levels (both r b −0.21; both p b 0.05).
M. Roos et al. / Regulatory Peptides 178 (2012) 11–15
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A
Table 1 Patient characteristics and demographics. Parameter
Total
Failures
Non-failures
Patients (n; %) Age (years, mean ± SD) Male (n; %) Female (n; %) Adiponectin (μg/ml, mean ± SD) HMW-adiponectin (μg/ml, mean ± SD) Cold ischemic time (hours, mean ± SD) Donor age (years, mean ± SD) Kidney transplantation (n; %) Kidney and pancreas transplantation (n; %) First transplantation (n; %) Retransplantation (n; %) Transplantation from living donor (n; %) Transplantation from deceased donor (n; %) HLA-MM cadaveric (mean ± SD) HLA-MM living (mean ± SD) PRA b 5% (n; %) PRA ≥ 5% (n; %) Immunosuppression CsA + MMF (n; %) CsA (n; %) Tacrolimus + MMF (n; %) Tacrolimus (n; %) Other (n; %) Patients death [3 years posttransplant] (n; %)
206 (100) 47.8±13.7 130 (63.1) 76 (36.9) 37.2±41.2 18.0±22.3 15.2 ± 8.3 47.5 ± 16.6 194 (94.2) 12 (5.8)
18 (8.7) 48.8±15.0 12 (5.8) 6 (2.9) 14.6±33.5 7.2 ± 17.8 18.8 ± 9.0 52.3 ± 21.6 18 (8.7) –
186 (91.3) 47.7 ± 13.6 118 (57.3) 70 (34.0) 38.1±41.0⁎⁎ 19.6±22.6⁎⁎ 15.1 ± 8.0 46.3 ± 16.6⁎ 176 (83.5) 12 (5.8)
164 (79.6) 42 (20.4) 32 (15.5) 174 (84.5)
8 (3.9) 10 (4.9) – 18 (8.7)
156 (75.7) 32 (15.6) 32 (15.5) 156 (75.8)
2.71 ± 1.78 3.50 ± 1.10 186 (90.3) 20 (9.7)
2.94 ± 1.47 – 15 (7.3) 3 (1.4)
2.64 ± 1.81 3.50 ± 1.10 171 (83.0) 17 (8.3)
56 (27.2) 7 (3.4) 109 (52.9) 27 (13.1) 7 (3.4) 13 (6.3)
4 1 6 5 2 4
52 (25.3) 6 (2.9) 103 (50.0) 22 (10.7) 5 (2.5) 9 (4.4)
(1.9) (0.5) (2.9) (2.4) (0.9) (1.9)
Failure: graft failure within 3 years posttransplant. ⁎ p b 0.05. ⁎⁎ p b 0.01 between failures and non‐failures (by Mann–Whitney U‐test).
In the next step, we performed multivariable Cox proportional regression analyses to investigate the independent association between pre-transplant adiponectin and HMW multimer levels and renal allograft outcome after the 36 month follow-up. Analyses were adjusted for traditional risk factors for renal allograft loss (including recipient sex, previous transplantations, percent PRA, donor type, kidney or kidney/pancreas transplantation, number of HLA-A, ‐B, ‐DR mismatches, CMV infections, and allograft rejection). Patients in the lowest total and HMW adiponectin quartile showed an independent and a significantly increased risk for renal allograft failure as compared with patients in the highest total and HMW quartile: odds ratio [95%CI]: total adiponectin: 4.25 [1.27–14.24; p = 0.019], and HMW multimers: 3.35 [1.04–10.76; p =0.042], respectively (Fig. 2A and B). 4. Discussion This study demonstrates that pre-transplant adiponectin and HMW multimer levels are significantly associated with markers of endothelial dysfunction and arteriosclerosis in patients undergoing renal transplantation. Moreover, total adiponectin and HMW multimers significantly predict long-term graft survival in renal transplant recipients. Our findings of an inverse association between adiponectin and HMW multimer levels and renal allograft survival may at least in part be explained by adiponectins' protective effects on endothelial cells [16] and vascular inflammation [17,18]. In general, renal allograft loss is the consequence of cumulative damages from a series of time dependent stressors and factors [3] and there is considerable evidence, that endothelial dysfunction and arteriosclerosis are crucial risk factors for allograft failure [19]. In clinical studies endothelial dysfunction has been shown to be highly prevalent in patients with chronic kidney disease (CKD) and kidney transplant recipients [19,20]. Novel risk assessment strategies including innovative biomarkers are clearly needed, in order to evaluate high-risk potentials for adverse renal allograft outcomes. This approach may support early identification of renal transplant recipients
p=0.005
Total Adiponectin [µg/ml]
Graft failure n= 18
No graft failure n = 188
B p=0.007
HMW Adiponectin [µg/ml]
Graft failure n= 18
No Graft failure n = 188
Fig. 1. A: Pre-transplant total adiponectin levels in renal recipients with and without allograft failure during the 36 month follow-up. B: pre-transplant HMW multimer levels in renal recipients with and without allograft failure during the 36 month follow-up.
in which intensified preventive strategies and clinical treatment options for arteriosclerosis and endothelial dysfunction are warranted. Over the last decade adiponectin has emerged as an important vasculoprotective molecule with insulin-sensitizing, anti-inflammatory, and anti-atherogenic properties, thereby improving endothelial function [19,21,22]. In this context, a recent study by Malyszko et al. reported a significant association between circulating adiponectin levels and markers of endothelial dysfunction in patients after renal transplantation with concomitant coronary artery disease (CAD). The authors speculate that increased levels of adiponectin may serve as a counter-regulative defense mechanism in consequence of an accelerated endothelial inflammatory milieu [14]. Our study significantly adds to the proposed link between adiponectin and endothelial dysfunction by expanding the knowledge toward patients in the pre-transplant state. We analyzed plasma adiponectin and HMW multimer levels before patients were transplanted, thereby providing the potential to identify patients with vascular dysfunction prior to renal transplantation. Furthermore, our results demonstrate that lower pre-transplant adiponectin and HMW levels independently predict a subsequent increased risk of long-term renal allograft failure. Hence, low pre-transplant adiponectin and HMW levels may not only reflect impaired endothelial function but also serve as an independent risk
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M. Roos et al. / Regulatory Peptides 178 (2012) 11–15
Table 2 Spearman correlation coefficients between pre-transplant total adiponectin and HMW multimer levels and cardiovascular risk factors.
Total adiponectin HMW adiponectin Body mass index (BMI) LDL-cholesterol HDL-cholesterol Triglycerides High-sensitive C-reactive protein Fasting plasma glucose (FPG)
HMW
BMI
LDL-cholesterol
HDL-cholesterol
Triglycerides
hsCRP
FPG
r = 0.984 p b 0.001 /
r = −0.084 p = 0.24 r = −0.091 p = 0.20 /
r = 0.399 p = 0.07 r = 0.424 p = 0.06 r = 0.116 p = 0.63 /
r = 0.457 p = 0.028 r = 0.519 p = 0.011 r = 0.017 p = 0.94 r = 0.413 p = 0.08 /
r = −0.138 p = 0.38 r = −0.142 p = 0.36 r = 0.462 p = 0.002 r = 0.206 p = 0.37 r = −0.271 p = 0.21 /
r = −0.154 p = 0.08 r = −0.143 p = 0.10 r = 0.165 p = 0.57 r = 0.074 p = 0.76 r = −0.294 p = 0.18 r = 0.021 p = 0.89 /
r = −0.219 p = 0.028 r = −0.224 p = 0.025 r = 0.080 p = 0.42 r = 0.183 p = 0.48 r = 0.240 p = 0.32 r = −0.046 p = 0.79 r = 0.069 p = 0.49 /
r = −0.910 p = 0.20 r = 0.424 p = 0.06 r = 0.519 p = 0.011 r = −0.142 p = 0.36 r = −0.143 p = 0.10 r = −0.224 p = 0.025
r = 0.116 p = 0.63 r = 0.017 p = 0.94 r = 0.462 p = 0.002 r = 0.165 p = 0.57 r = 0.080 p = 0.42
r = 0.413 p = 0.08 r = 0.206 p = 0.37 r = 0.074 p = 0.76 r = 0.183 p = 0.48
r = −0.271 p = 0.21 r = −0.294 p = 0.18 r = 0.240 p = 0.32
r = 0.021 p = 0.89 r = −0.046 p = 0.79
r = 0.069 p = 0.49
r = correlation coefficient, p b 0.05 indicates a statistically significant difference between the values in the table.
factor for a progressive deterioration of microcirculatory capacity potentially leading to premature graft failure. The proposed effect of adiponectin on vascular graft function is derived from previous findings linking this highly abundant circulatory adipokine to anti-inflammation and vascular protection. Inflammation of the systemic microcirculation is widely appreciated to underlie post-transplant kidney organ damage [23]. Adiponectin exerts antiinflammatory effects on the vascular wall by suppressing the expression of pro-inflammatory adhesion molecules and by inhibiting endothelial NF-κB signaling [24,25]. Adiponectin reverses the deleterious endothelial effects of TNF-α and other cytokines which trigger an
Odds ratio for allograft failure
A 15
10
5
0 < 16.0
≥ 16.0 - 37.5 ≥ 37.6 - 65.2
> 65.2
Total adiponectin [μg/mL]
inflammatory signaling cascade and enhance leukocytes–endothelial interactions [19,26]. In turn, it has been demonstrated that adiponectin deficient mice show an accelerated endothelial expression of the vascular cell adhesion molecule-1 (VCAM-1). This molecule is known to play a crucial role in leukocyte–endothelial cell–cell interactions, including leukocyte trafficking, adhesion and transmigration [27]. Recent studies have convincingly shown that the HMW mulitmeric isoform specifically confers the vascular-protective activities of this adipokine. Thus, only HMW dose-dependently suppressed apoptosis and caspase-3 activity in human umbilical vein endothelial cells (HUVECs) [28]. This suggests, that decreased circulating total and HMW adiponectin levels in patients undergoing renal transplantation fail to sufficiently counter-act a microenvironment of chronic inflammation and monocyte recruitment. This condition subsequently promotes secretion of pro-inflammatory cytokines, leading to mononuclear cell infiltration and long-term allograft dysfunction and eventually failure. In conclusion, our results demonstrate that pre-transplant total and HMW adiponectin levels are significantly associated with markers of endothelial dysfunction and arteriosclerosis and independently predict long-term allograft outcome in kidney-transplant recipients. Adiponectin and HMW multimers may play a causal role in adverse allograft outcomes after kidney transplantation by affecting microvascular inflammation and endothelial dysfunction. Total adiponectin levels and HMW multimers are known to positively respond to pharmacological interventions [29,30] and randomized controlled trials are needed to explore the importance of hypoadiponectinemia as a modifiable risk factor for long-term renal allograft failure.
Odds ratio for allograft failure
B References
15
10
5
0 < 7.6
≥ 7.6 - 19.1 ≥ 19.2 - 35.4
> 35.4
HMW adiponectin [μg/mL] Fig. 2. Odds ratios for renal allograft failure during the 36 month follow-up in the first, second, and third quartiles compared with the fourth quartile of the total adiponectin (A), and high-molecular weight adiponectin (HMW) multimers (B). All models were adjusted for recipient sex, previous transplantations, percent PRA, donor type, kidney or kidney/pancreas transplantation, number of HLA-A, ‐B, ‐DR mismatches, CMV infections, and renal allograft rejection as covariables. Vertical bars indicate 95% CI.
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Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
Low pre-transplant adiponectin multimers are associated with adverse allograft outcomes in kidney transplant recipients a 3-year prospective study Marcel Roos a,⁎, 1, Marcus Baumann a, 2, Dan Liu a, 2, Falko M. Heinemann b, Monika Lindemann b, Peter A. Horn b, Tobias Türk c, 2, Jens Lutz a, 2, Uwe Heemann a, 2, Oliver Witzke c, 1, 3, Maximilian von Eynatten a, 1, 3 a b c
Dept. of Nephrology, Technische Universität München, Munich, Germany Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany Dept. of Nephrology, University Hospital Essen, Essen, Germany
a r t i c l e
i n f o
Article history: Received 14 December 2011 Accepted 19 June 2012 Available online 27 June 2012 Keywords: Adiponectin Endothelial dysfunction Kidney transplantation Renal allograft failure
a b s t r a c t Background: In kidney transplant recipients endothelial dysfunction is almost a universal risk factor for allograft failure. Adiponectin, an adipocyte derived hormone, has endothelial-protective properties and the highmolecular weight (HMW) multimer is the major active form, exerting anti-inflammatory and anti-apoptotic effects on endothelial cells. This study evaluated, whether pre-transplant total and HMW multimer adiponectin levels are associated with markers of endothelial dysfunction and arteriosclerosis and predict long-term graft survival in patients after kidney transplantation. Methods: In 206 renal transplant recipients pre-transplant total and HMW adiponectin levels were measured in serum by ELISA and Western blot, respectively. During the 36 months active follow up (median [interquartile range] 1249 [1020; 1445] days) 13 patients died (94% patient survival) and renal allograft failure was reported in 18 patients (91% graft survival). Results: Pre-transplant total and HMW adiponectin levels were significantly associated with lipid and glucose parameters at baseline. After 3 years follow-up pre-transplant total and HMW adiponectin levels were significantly inversely associated with the incidence of allograft failure (adiponectin: r = − 0.216; p = 0.002: HMW: r = −0.218; p = 0.002). In multivariable adjusted Cox proportional hazard regression models patients in the lowest total and HMW adiponectin quartile had a significantly increased risk for allograft failure within 3 years post-transplantation: odds ratio [95%CI]: total adiponectin: 4.25 [1.27–14.24; p = 0.019], and HMW multimers: 3.35 [1.04–10.76; p = 0.042], respectively. Conclusion: Low pre-transplant levels of total and HMW adiponectin reflect a pro-atherogenic endothelial milieu and independently predict an increased risk of allograft failure in kidney-transplant recipients. Measurement of adiponectin levels may identify patients at risk for adverse allograft outcomes after kidney transplantation. © 2012 Published by Elsevier B.V.
1. Introduction Long term survival of renal allografts is the major goal of clinical kidney transplantation [1]. Immunological risk factors, such as age, race, HLA matching, and acute rejection episodes are well established risk factors for impaired allograft survival. In addition, traditional vascular pro-atherogenic risk factors, such as arterial hypertension, ⁎ Corresponding author at: Department of Nephrology, Klinikum rechts der Isar, Ismaninger Str. 22, 81675 München, Germany. Tel.: +49 89 4140 2231; fax: +49 89 4140 4878. E-mail address: [email protected] (M. Roos). 1 Designed the research, performed the study, analyzed the data, and wrote the paper. 2 Designed the research and collected data, FH, ML, PH performed the research, collected the data, and wrote the paper. 3 These authors contributed equally to this work. 0167-0115/$ – see front matter © 2012 Published by Elsevier B.V. doi:10.1016/j.regpep.2012.06.001
hyperlipidemia, and metabolic syndromes including impaired glucose tolerance add to the risk of renal allograft failure and poor posttransplant patient survival [2,3]. More recently the introduction of novel, additional biomarkers further improved risk prediction strategies after kidney transplantation [4–6]. Thus, pre-transplant risk evaluation by immunologic and non-immunologic risk assessment has gained substantial clinical importance. However, despite all recent progresses, reliable identification of patients at high risk for adverse renal allograft outcomes is still vague and novel, and innovative riskidentifying strategies are still an unmet medical need; these should be amended in order to improve pre-transplant risk evaluation and subsequent preventive efforts. Adiponectin, the most abundant adipokine in humans, circulates in different multimer complexes with distinct molecular weights [7,8]. The high-molecular weight (HMW) form of adiponectin has been shown to be the predominant active form of this hormone, exerting
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M. Roos et al. / Regulatory Peptides 178 (2012) 11–15
anti-inflammatory, anti-diabetic and endothelial protective properties [9]. In clinical studies low HMW levels were associated with systemic inflammation, atherogenic dyslipidemia, impaired insulin sensitivity, risk for type 2 diabetes, endothelial dysfunction, arteriosclerosis and eventually coronary artery disease (CAD) [10–13]. The link between low adiponectin and both, endothelial and metabolic dysfunction was previously confirmed in patients undergoing renal transplantation. In these patients, low adiponectin levels were associated with increased markers of endothelial dysfunction, suggesting elevated adiponectin to act as a counter regulatory defense system in consequence of an accelerated endothelial inflammatory milieu [14]. Moreover, in a prospective follow-up study low pre-transplant adiponectin levels were associated with an increased risk for the development of new onset diabetes mellitus after transplantation (NODAT) [15]. The aim of this study was to evaluate whether pre-transplant adiponectin and HMW multimer levels are associated with markers of endothelial dysfunction and arteriosclerosis in 206 patients undergoing renal transplantation. In addition, the predictive value for total adiponectin and HMW multimers on long-term renal allograft survival was investigated during a 36 month follow-up period. 2. Material and methods 2.1. Patients 206 renal transplant recipients were enrolled in this study and active follow-up was conducted for 36 months (median [interquartile range] 1249 [1020–1445] days). The study design and methods have been reported previously [5]. Briefly, all patients were transplanted with kidney (n = 194) or kidney/pancreas (n = 12) allografts in a consecutive series from May 2001 to June 2004 in the University Hospital of Essen, Germany, of whom 174 (84%) were transplanted with a kidney from a deceased donor and 32 (16%) patients received an allograft from a living donor. Pediatric transplantations and combined kidney/ liver transplantations were excluded. Primary kidney diseases of the patients studied were glomerulonephritis (n = 75), cystic kidney disease (n = 29), diabetes mellitus types 1 and 2 (n = 20), interstitial nephritis (n = 20), renal vascular disease (n = 13), and other diseases (n = 49). Sixty-three patients were treated with cyclosporine (CsA) (100–150 ng/ml whole blood through level, tested by FPIA Abbott TDx monoclonal assay; Abbott Laboratories Limited, Wiesbaden, Germany) and 136 patients with tacrolimus (4–10 ng/ml whole blood through level, tested by Abbott Imx tacrolimus assay). A total of 109 received MMF in combination with tacrolimus and 56 patients in combination with CsA. Additionally, all but four patients received prednisone prior to transplantation; antithymocyte globulin was given to eight patients, combined with tacrolimus or MMF. Loss of renal allograft function and renal rejection episodes were histologically diagnosed in all patients concerned. In total, 13 patients (6%) died within 3 years after transplantation. Patients who died with a functioning graft (n = 9) were excluded from the survival analysis. Cytomegalovirus (CMV) infections within the first 4 months posttransplantation were detected by immunohistological pp65 assay and occurred in 26 patients (13%). HLA typing of patients and donors was performed by serology or polymerase chain reaction using sequence specific primers (PCR-SSP) for the HLA-A, -B, ‐DR, and ‐DQ loci as previously described [5]. 2.2. Clinical chemistry Adiponectin levels were measured with ELISA according to the manufacturer's instructions (BioVendor, Brno, Czek Republic). Both, the intra- and inter‐assay CVs were b5.0%. HMW adiponectin multimer levels were quantified by Western blot analysis, as previously described [8]. In brief, serum proteins were separated by 10% SDS-PAGE under
nonreducing and nonheating conditions, and transferred to nitrocellulose membranes. Membranes were blocked with TBS-Tween20 containing 5% skim milk and incubated with a goat antihuman adiponectin polyclonal antibody (1:500, R&D Systems, Wiesbaden, Germany). After being washed, membranes were incubated with horseradish peroxidise–conjugated donkey antigoat antibody (1:4000, Santa Cruz Biotechnology, Santa Cruz, California, USA). Bands were visualized by the use of lumi-light Western blotting substrate (Roche Diagnostics, Mannheim, Germany), and the image was acquired with a Kodak IS440CF Imaging Station. Densitometry analysis was performed with Adobe Photoshop software. Relative distributions of adiponectin multimers were calculated by dividing the band density by the total density. Percentages of multimers were multiplied by the total adiponectin concentrations obtained by ELISA to calculate the absolute values. 2.3. Statistics Bivariate correlations between pre-transplant adiponectin levels and parameters of interest were calculated by the Spearman's correlation coefficient and the Mann–Whitney U test was applied to compare two independent groups. Cox proportional multivariable analyses were performed to analyze the independent association between adiponectin, HMW and other covariates with the risk of allograft failure. For analyses pre-transplant adiponectin and HMW levels were divided into quartiles (adiponectin: 1st: b 16.0 μg/ml, 2nd: ≥16.0–37.5 μg/ml, 3rd: ≥37.6–65.2 μg/ml and 4th: >65.2 μg/ml. HMW 1st: b7.6 μg/ml, 2nd ≥7.6–19.1 μg/ml, 3rd: ≥19.2–35.4 μg/ml and 4th: >35.4 μg/ml). Further independent parameters were included in the analyses as previously described [5]: recipient sex, previous transplantations, percent PRA (positive vs. negative), donor type (cadaveric vs. living), kidney or kidney/pancreas transplantation, number of HLA-A, -B, -DR mismatches (0, 1, 2 vs. 3, 4, 5, 6), CMV infections, and allograft rejection. Statistical analyses were performed with SPSS software version 15.0 (SPSS Inc., Chicago, IL) and a p-value of b0.05 was considered as statistically significant. 3. Results Patient demographics and clinical characteristics at baseline are presented in Table 1. A total of 206 patients were included in this study and active follow up was conducted for 36 months (median [interquartile range] 1249 [1020–1445] days). The follow-up data revealed a 94% patient survival rate (13 patients died). Nine patients died with a functioning graft and were therefore excluded from the survival analysis. In the remaining group of 197 patients, 18 patients (9%) experienced a graft failure within 2 years after transplantation. Pre-transplant adiponectin and HMW levels were significantly lower in patients with incident graft failure as compared to patients with functional graft after long-term follow-up (adiponectin: 38.1 ± 41.0 μg/ml vs. 14.6 ± 33.5 μg/ml; p = 0.005, HWM: 19.6 ± 22.6 μg/ml vs. 7.2 ± 17.8 μg/ml p = 0.007, Fig. 1A and B). In addition, donor age was significantly higher in patients experiencing graft failure during the follow-up as compared to non-failure individuals (52.3 ± 21.6 vs. 46.3 ± 16.6 years; p = 0.013). No significant difference between patients with and without graft failure was seen for recipient age, cold ischemic time, and HLA-mismatches from cadaveric donors. In order to evaluate pre-transplant associations between adiponectin levels and atherogenic risk factors, we evaluated Spearman correlation coefficients between pre-transplant total adiponectin and HMW levels and various surrogate parameters for endothelial dysfunction and arteriosclerosis (presented in Table 2). Both adiponectin measures were significantly associated with atherogenic markers of lipid and glucose metabolism. Total adiponectin and HMW levels significantly correlated with HDL-cholesterol (both r> 0.45; both p b 0.05), and fasting plasma glucose levels (both r b −0.21; both p b 0.05).
M. Roos et al. / Regulatory Peptides 178 (2012) 11–15
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A
Table 1 Patient characteristics and demographics. Parameter
Total
Failures
Non-failures
Patients (n; %) Age (years, mean ± SD) Male (n; %) Female (n; %) Adiponectin (μg/ml, mean ± SD) HMW-adiponectin (μg/ml, mean ± SD) Cold ischemic time (hours, mean ± SD) Donor age (years, mean ± SD) Kidney transplantation (n; %) Kidney and pancreas transplantation (n; %) First transplantation (n; %) Retransplantation (n; %) Transplantation from living donor (n; %) Transplantation from deceased donor (n; %) HLA-MM cadaveric (mean ± SD) HLA-MM living (mean ± SD) PRA b 5% (n; %) PRA ≥ 5% (n; %) Immunosuppression CsA + MMF (n; %) CsA (n; %) Tacrolimus + MMF (n; %) Tacrolimus (n; %) Other (n; %) Patients death [3 years posttransplant] (n; %)
206 (100) 47.8±13.7 130 (63.1) 76 (36.9) 37.2±41.2 18.0±22.3 15.2 ± 8.3 47.5 ± 16.6 194 (94.2) 12 (5.8)
18 (8.7) 48.8±15.0 12 (5.8) 6 (2.9) 14.6±33.5 7.2 ± 17.8 18.8 ± 9.0 52.3 ± 21.6 18 (8.7) –
186 (91.3) 47.7 ± 13.6 118 (57.3) 70 (34.0) 38.1±41.0⁎⁎ 19.6±22.6⁎⁎ 15.1 ± 8.0 46.3 ± 16.6⁎ 176 (83.5) 12 (5.8)
164 (79.6) 42 (20.4) 32 (15.5) 174 (84.5)
8 (3.9) 10 (4.9) – 18 (8.7)
156 (75.7) 32 (15.6) 32 (15.5) 156 (75.8)
2.71 ± 1.78 3.50 ± 1.10 186 (90.3) 20 (9.7)
2.94 ± 1.47 – 15 (7.3) 3 (1.4)
2.64 ± 1.81 3.50 ± 1.10 171 (83.0) 17 (8.3)
56 (27.2) 7 (3.4) 109 (52.9) 27 (13.1) 7 (3.4) 13 (6.3)
4 1 6 5 2 4
52 (25.3) 6 (2.9) 103 (50.0) 22 (10.7) 5 (2.5) 9 (4.4)
(1.9) (0.5) (2.9) (2.4) (0.9) (1.9)
Failure: graft failure within 3 years posttransplant. ⁎ p b 0.05. ⁎⁎ p b 0.01 between failures and non‐failures (by Mann–Whitney U‐test).
In the next step, we performed multivariable Cox proportional regression analyses to investigate the independent association between pre-transplant adiponectin and HMW multimer levels and renal allograft outcome after the 36 month follow-up. Analyses were adjusted for traditional risk factors for renal allograft loss (including recipient sex, previous transplantations, percent PRA, donor type, kidney or kidney/pancreas transplantation, number of HLA-A, ‐B, ‐DR mismatches, CMV infections, and allograft rejection). Patients in the lowest total and HMW adiponectin quartile showed an independent and a significantly increased risk for renal allograft failure as compared with patients in the highest total and HMW quartile: odds ratio [95%CI]: total adiponectin: 4.25 [1.27–14.24; p = 0.019], and HMW multimers: 3.35 [1.04–10.76; p =0.042], respectively (Fig. 2A and B). 4. Discussion This study demonstrates that pre-transplant adiponectin and HMW multimer levels are significantly associated with markers of endothelial dysfunction and arteriosclerosis in patients undergoing renal transplantation. Moreover, total adiponectin and HMW multimers significantly predict long-term graft survival in renal transplant recipients. Our findings of an inverse association between adiponectin and HMW multimer levels and renal allograft survival may at least in part be explained by adiponectins' protective effects on endothelial cells [16] and vascular inflammation [17,18]. In general, renal allograft loss is the consequence of cumulative damages from a series of time dependent stressors and factors [3] and there is considerable evidence, that endothelial dysfunction and arteriosclerosis are crucial risk factors for allograft failure [19]. In clinical studies endothelial dysfunction has been shown to be highly prevalent in patients with chronic kidney disease (CKD) and kidney transplant recipients [19,20]. Novel risk assessment strategies including innovative biomarkers are clearly needed, in order to evaluate high-risk potentials for adverse renal allograft outcomes. This approach may support early identification of renal transplant recipients
p=0.005
Total Adiponectin [µg/ml]
Graft failure n= 18
No graft failure n = 188
B p=0.007
HMW Adiponectin [µg/ml]
Graft failure n= 18
No Graft failure n = 188
Fig. 1. A: Pre-transplant total adiponectin levels in renal recipients with and without allograft failure during the 36 month follow-up. B: pre-transplant HMW multimer levels in renal recipients with and without allograft failure during the 36 month follow-up.
in which intensified preventive strategies and clinical treatment options for arteriosclerosis and endothelial dysfunction are warranted. Over the last decade adiponectin has emerged as an important vasculoprotective molecule with insulin-sensitizing, anti-inflammatory, and anti-atherogenic properties, thereby improving endothelial function [19,21,22]. In this context, a recent study by Malyszko et al. reported a significant association between circulating adiponectin levels and markers of endothelial dysfunction in patients after renal transplantation with concomitant coronary artery disease (CAD). The authors speculate that increased levels of adiponectin may serve as a counter-regulative defense mechanism in consequence of an accelerated endothelial inflammatory milieu [14]. Our study significantly adds to the proposed link between adiponectin and endothelial dysfunction by expanding the knowledge toward patients in the pre-transplant state. We analyzed plasma adiponectin and HMW multimer levels before patients were transplanted, thereby providing the potential to identify patients with vascular dysfunction prior to renal transplantation. Furthermore, our results demonstrate that lower pre-transplant adiponectin and HMW levels independently predict a subsequent increased risk of long-term renal allograft failure. Hence, low pre-transplant adiponectin and HMW levels may not only reflect impaired endothelial function but also serve as an independent risk
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Table 2 Spearman correlation coefficients between pre-transplant total adiponectin and HMW multimer levels and cardiovascular risk factors.
Total adiponectin HMW adiponectin Body mass index (BMI) LDL-cholesterol HDL-cholesterol Triglycerides High-sensitive C-reactive protein Fasting plasma glucose (FPG)
HMW
BMI
LDL-cholesterol
HDL-cholesterol
Triglycerides
hsCRP
FPG
r = 0.984 p b 0.001 /
r = −0.084 p = 0.24 r = −0.091 p = 0.20 /
r = 0.399 p = 0.07 r = 0.424 p = 0.06 r = 0.116 p = 0.63 /
r = 0.457 p = 0.028 r = 0.519 p = 0.011 r = 0.017 p = 0.94 r = 0.413 p = 0.08 /
r = −0.138 p = 0.38 r = −0.142 p = 0.36 r = 0.462 p = 0.002 r = 0.206 p = 0.37 r = −0.271 p = 0.21 /
r = −0.154 p = 0.08 r = −0.143 p = 0.10 r = 0.165 p = 0.57 r = 0.074 p = 0.76 r = −0.294 p = 0.18 r = 0.021 p = 0.89 /
r = −0.219 p = 0.028 r = −0.224 p = 0.025 r = 0.080 p = 0.42 r = 0.183 p = 0.48 r = 0.240 p = 0.32 r = −0.046 p = 0.79 r = 0.069 p = 0.49 /
r = −0.910 p = 0.20 r = 0.424 p = 0.06 r = 0.519 p = 0.011 r = −0.142 p = 0.36 r = −0.143 p = 0.10 r = −0.224 p = 0.025
r = 0.116 p = 0.63 r = 0.017 p = 0.94 r = 0.462 p = 0.002 r = 0.165 p = 0.57 r = 0.080 p = 0.42
r = 0.413 p = 0.08 r = 0.206 p = 0.37 r = 0.074 p = 0.76 r = 0.183 p = 0.48
r = −0.271 p = 0.21 r = −0.294 p = 0.18 r = 0.240 p = 0.32
r = 0.021 p = 0.89 r = −0.046 p = 0.79
r = 0.069 p = 0.49
r = correlation coefficient, p b 0.05 indicates a statistically significant difference between the values in the table.
factor for a progressive deterioration of microcirculatory capacity potentially leading to premature graft failure. The proposed effect of adiponectin on vascular graft function is derived from previous findings linking this highly abundant circulatory adipokine to anti-inflammation and vascular protection. Inflammation of the systemic microcirculation is widely appreciated to underlie post-transplant kidney organ damage [23]. Adiponectin exerts antiinflammatory effects on the vascular wall by suppressing the expression of pro-inflammatory adhesion molecules and by inhibiting endothelial NF-κB signaling [24,25]. Adiponectin reverses the deleterious endothelial effects of TNF-α and other cytokines which trigger an
Odds ratio for allograft failure
A 15
10
5
0 < 16.0
≥ 16.0 - 37.5 ≥ 37.6 - 65.2
> 65.2
Total adiponectin [μg/mL]
inflammatory signaling cascade and enhance leukocytes–endothelial interactions [19,26]. In turn, it has been demonstrated that adiponectin deficient mice show an accelerated endothelial expression of the vascular cell adhesion molecule-1 (VCAM-1). This molecule is known to play a crucial role in leukocyte–endothelial cell–cell interactions, including leukocyte trafficking, adhesion and transmigration [27]. Recent studies have convincingly shown that the HMW mulitmeric isoform specifically confers the vascular-protective activities of this adipokine. Thus, only HMW dose-dependently suppressed apoptosis and caspase-3 activity in human umbilical vein endothelial cells (HUVECs) [28]. This suggests, that decreased circulating total and HMW adiponectin levels in patients undergoing renal transplantation fail to sufficiently counter-act a microenvironment of chronic inflammation and monocyte recruitment. This condition subsequently promotes secretion of pro-inflammatory cytokines, leading to mononuclear cell infiltration and long-term allograft dysfunction and eventually failure. In conclusion, our results demonstrate that pre-transplant total and HMW adiponectin levels are significantly associated with markers of endothelial dysfunction and arteriosclerosis and independently predict long-term allograft outcome in kidney-transplant recipients. Adiponectin and HMW multimers may play a causal role in adverse allograft outcomes after kidney transplantation by affecting microvascular inflammation and endothelial dysfunction. Total adiponectin levels and HMW multimers are known to positively respond to pharmacological interventions [29,30] and randomized controlled trials are needed to explore the importance of hypoadiponectinemia as a modifiable risk factor for long-term renal allograft failure.
Odds ratio for allograft failure
B References
15
10
5
0 < 7.6
≥ 7.6 - 19.1 ≥ 19.2 - 35.4
> 35.4
HMW adiponectin [μg/mL] Fig. 2. Odds ratios for renal allograft failure during the 36 month follow-up in the first, second, and third quartiles compared with the fourth quartile of the total adiponectin (A), and high-molecular weight adiponectin (HMW) multimers (B). All models were adjusted for recipient sex, previous transplantations, percent PRA, donor type, kidney or kidney/pancreas transplantation, number of HLA-A, ‐B, ‐DR mismatches, CMV infections, and renal allograft rejection as covariables. Vertical bars indicate 95% CI.
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