Vascular inflammation and media calcification are already present in early stages of chronic kidney disease

    Vascular inflammation and media calcification are already present in early stages of chronic kidney disease Kerstin Benz, Ildiko Varga, Daniel Neureiter, Valentina Campean, Christoph Daniel, Christian Heim, Albrecht Reimann, Michael Weyand, Karl F. Hilgers, Kerstin Amann PII: DOI: Reference:

S1054-8807(16)30140-5 doi: 10.1016/j.carpath.2017.01.004 CVP 6968

To appear in:

Cardiovascular Pathology

Received date: Revised date: Accepted date:

29 September 2016 16 January 2017 17 January 2017

Please cite this article as: Benz Kerstin, Varga Ildiko, Neureiter Daniel, Campean Valentina, Daniel Christoph, Heim Christian, Reimann Albrecht, Weyand Michael, Hilgers Karl F., Amann Kerstin, Vascular inflammation and media calcification are already present in early stages of chronic kidney disease, Cardiovascular Pathology (2017), doi: 10.1016/j.carpath.2017.01.004

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ACCEPTED MANUSCRIPT Vascular inflammation and media calcification are already present in early

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stages of chronic kidney disease

Kerstin Benz1,2*, Ildiko Varga1*, Daniel Neureiter3, Valentina Campean1, Christoph

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Daniel1, Christian Heim4, Albrecht Reimann4, Michael Weyand4, Karl F. Hilgers5, Kerstin Amann1

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Departments of 1Nephropathology, 2Pediatrics, 4Cardiac Surgery, 5Nephrology and Hypertension, University of Erlangen-Nürnberg, Erlangen, Germany, 3Pathology, Salzburger Landeskliniken, Salzburg, Austria

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Short Title: Vascular alterations in early and advanced CKD

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Corresponding Author: Kerstin Amann, M.D. Department of Nephropathology

University of Erlangen-Nürnberg Krankenhausstr. 8-10, 91054 Erlangen Germany Phone:

0049-9131-8522291

Fax:

0049-9131-8522601

Email: [email protected]

* both authors contributed equally

ACCEPTED MANUSCRIPT Abstract: Background: While patients with CKD have a high prevalence of classical coronary

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risk factors, there is increasing evidence that atherosclerosis is different in renal

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compared to non-renal patients. Therefore, the present study compares changes in different vessels obtained at cardiac surgery between patients with early and

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advanced CKD and non-renal control patients.

Methods and Results: Fifty patients undergoing cardiac bypass surgery were

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divided into 3 groups: (i) 24 control patients with creatinine<1.3 mg/dl, (ii) 14 patients with early CKD (creatinine 1.3-2.0 mg/dl), (iii) 12 patients with advanced CKD (creatinine>2.0 mg/dl). Aorta, A. mammaria interna and V. saphena were analysed using morphometry, Kossa stain for vascular calcification and immunohistochemistry

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for markers of inflammation and proosteogenic differentiation of vascular smooth

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muscel cells (VSMC). Thereby, aortic wall thickness, calcification score of aortic intima and of V. saphena were significantly higher in advanced CKD patients than in control

patients

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non-renal

whereas

significant

vascular

inflammation

and

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proosteogenic dedifferentiation of VSMC and calcification of the aortic media were already present in early CKD. Interestingly, marked calcification of the V. saphena magna was seen in advanced CKD. Of note, CaxP product correlated well with markers of inflammation, but not with calcification itself. Conclusions: Early stages of CKD are already associated with local upregulation of proinflammatory and proosteogenic molecules in the vascular wall and calcification of the aortic media. These findings point to the importance of local microinflammation in CKD and may shed new light on the potentially overestimated role of the CaxP product for vessel calcification. Keywords: vascular calcification, intima, media, chronic kidney disease, uremia

ACCEPTED MANUSCRIPT 1 Introduction: The prevalence of chronic kidney disease (CKD) is increasing worldwide. In patients

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with CKD cardiovascular morbidity and mortality are major clinical problems. The

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majority of this patient population will die from cardiovascular events as the risk for cardiovascular disease is 10-30-fold higher compared to individuals with normal renal

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function1. Of interest, even mild renal insufficiency is an independent predictor of cardiovascular disease or death2 and many patients with CKD die from

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cardiovascular events prior to progressing to end-stage renal disease3. While CKD patients certainly have a high prevalence of classical cardiovascular risk factors e.g. hyperlipidemia, hypertension, increased oxidative stress and diabetes, there is increasing evidence that atherosclerosis, i.e. the evolution and composition of

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plaques is different in renal compared to non-renal patients. In animal models and

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patients with CKD specific structural changes of the heart, i.e. left ventricular hypertrophy (LVH) with interstitial myocardial fibrosis, capillary deficit and

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microarteriopathy4,5, and of extracardiac arteries and veins6 could be identified

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accounting at least in part for this high cardiovascular death rate. Patients with coronary heart disease and end stage CKD show more advanced and more heavily calcified coronary plaques compared to age and gender matched non-renal control patients7. Moreover, it is of special importance that even in children with CKD, but lower exposure to classical cardiovascular risk factors, increased intima-media thickness of the common carotid artery was found 8. Vascular thickening was most pronounced in dialysis patients8, but was also seen in patients with only mild to moderate renal failure. Morphologically, in some animal models of atherosclerosis and calcification as well as in most patients, intima and media calcification can be distinguished and this may be of importance as the clinical consequences differ6. While intima calcification is

ACCEPTED MANUSCRIPT eventually associated with increased plaque formation and presumably higher plaque vulnerability, media calcification primarily causes arterial stiffness, increased pulse

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pressure, and finally LVH6. As one potential pathomechanism of increased

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calcification in CKD the switch of vascular smooth muscle cells (VSMC) from a contractile to a more secretory and proosteogenic phenotype has been described 6.

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There is a definite lack of in-vivo human data concerning these morphological characteristics of various vascular beds in CKD patients with different stages of renal

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dysfunction. In order to identify the prevalence and time course of typical vascular alterations seen in these patients and to get an idea about the underlying pathogenetic mechanisms, the present study compares changes in different vessels obtained at cardiac bypass surgery of patients with early (early CKD) and late stages

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of renal dysfunction (advanced CKD), and non-renal control patients.

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It is of particular importance that the present in-vivo study did not address the difference between CKD patients and presumably healthy normal non-renal patients,

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but aims to determine the specific characteristics of changes in patients with coronary

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heart disease and thus comparable cardiovascular risk profile. Of note, all study patients had a manifest severe coronary heart disease necessitating cardiac bypass surgery and we compared the differences in inflammation, calcification and vessel thickness in standardised samples of the aorta, the A. mammaria int. and the V. saphena magna in the 3 subgroups of patients, i.e. without known kidney disease, patients with earlier stages of CKD and patients with advanced stages of CKD.

ACCEPTED MANUSCRIPT 2 Materials and methods: 2.1 Patient selection:

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Fifty consecutive patients (31 male, 19 female) with a median age of 67 years (range

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49-81 years) undergoing cardiac bypass surgery were divided into 3 groups depending on patient history and pre-operative serum creatinine levels (tab. 1): (i)

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control group: 24 patients with creatinine <1.3 mg/dl (age: 67.4 ± 7.63 years; 11 male, 13 female). (ii) early CKD: 14 patients with creatinine of 1.3-2.0 mg/dl (age:

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69.9 ± 8.19 years; 10 male, 4 female). (iii) advanced CKD: 12 patients with creatinine >2.0 mg/dl or dialysis treatment (age: 65.6 ± 7.85 years; 10 male, 2 female). Basal patient characteristics including age, body weight and body mass index (BMI) as well as cardiovascular risk profile, e.g. hypertension, nicotine abuse, diabetes,

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dyslipidemia, dialysis treatment and former cardiac bypass surgery were obtained

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from hospital records. Before surgery the following laboratory measurements were documented: inorganic phosphate [mg/dl], calcium [mmol/l], cholesterol [mg/dl],

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triglycerides [mg/dl] and creatinine [mg/dl]. The study protocol was approved by the

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local Ethics Committee (University of Erlangen-Nürnberg, Germany, No. 2684). 2.2 Tissue preparation: At cardiac bypass surgery, samples from the aorta (cannulation biopsy), the A. mammaria interna (int.), and the V. saphena magna were taken from comparable sites using a standardised protocol. For morphological investigation, the specimens were fixed instantly after removal with formaldehyde (4%), embedded in paraffin, sectioned (1 µm) and stained using haematoxylin-eosin (H&E), Elastica-van-Gieson (EvG, for fibrous tissue) and van Kossa technique (for tissue calcification). 2.2.1 Morphometric evaluation: Morphometric analysis of aorta, A. mammaria int. and V. saphena magna was performed by planimetry using a semi-automatic image analysing system

ACCEPTED MANUSCRIPT (AnalysisPRO; SIS, Münster, Germany) as described10-12. Parameters of interest were wall thickness of intima and media (mean of 10 measurements per vessel),

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outer and inner (lumen) diameter. The vessel diameters were measured at the two

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smallest opposing sites of the vascular wall since this is the site which is least affected by artefacts, i.e. the sectioning angle. Wall thickness was calculated as

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(outer diameter-inner diameter)/2. Of note, in aortic biopsies only intima and media thickness could be measured.

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2.2.2 Calcification analysis:

For tissue calcification van Kossa stain was performed and all vessels including the small ones in the periadventitial tissue were analysed semiquantitatively according to a scoring system from 0-4 (0:no calcification, 4:highest calcification; see Suppl. Fig.

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1)12. In the aorta and the A. mammaria int. calcification was separately analysed for

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intima and media while in the V. saphena magna where intima and media could not be clearly dissociated from each other only one score per vessel was used.

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2.2.3 Immunohistochemical analysis:

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Immunohistochemistry for parameters of inflammation was performed on paraffin sections of aorta, A. mammaria int. and V. saphena magna using the following antibodies: rabbit polyclonal antibody against human CRP (1:100, Sigma Aldrich, Munich, Germany), mouse monoclonal antibody against human CD40 (1:400, AbD Serotec, Düsseldorf, Germany), rabbit polyclonal antibody against human CD154 (1:300, Santa Cruz Biotechnology Inc, CA) as described before7. Sections were examined using light microscopy (magnification x40) and a semiquantitative scoring system (0-4): 0:no staining, 1:mild staining, 2:moderate staining, 3:strong staining, 4 very strong staining11(Suppl. Fig. 1). Separate analysis for vessel media and intima staining was done for the aorta and the A. mammaria int.; all analyses were performed by one observer in a blinded manner 7. In order to adress the important

ACCEPTED MANUSCRIPT issue of osteogenic conversion of VSMC we performed immunohistological investigations using the following antibodies and compared the expression levels on

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a qualitative basis: Alpha-smooth muscle actin (1:100, DAKO, Germany), Vimentin

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(1:200, DAKO, Germany), Galectin-3 (1:100, Diagnostic Biosystems, USA), a proinflammatory molecule involved in vascular osteogenesis in atherosclerosis and

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special AT-rich sequence-binding protein 2 (SATB2, 1:200, Abcam, USA ) which is known to induced Runx2 overproduction and VSMC calcification.

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We had also used antibodies against CD3 (1:100, Zytomed, Germany) and CD20 (1:500, DAKO, Germany) for T- and B-cell detection but since we found only very few cells on inspection we did not formally analyse these stainings.

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2.3 Statistics:

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Statistical analysis was performed with SPSS 18.0 (SPSS Inc., Chicago, IL, USA). First, continuous data were tested for normal distribution using the Kolmogorov-

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Smirnov goodness-of-fit test. Spearman's rank correlation (rho). was used for the

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correlation analysis. ²-test, Mann-Whitney U test or Student’s t test was used to compare data of nominal, ordinal or interval level. Additionally, Kruskal-Wallis H test and the univariate ANOVA (analysis of variance; using Least significant difference test and the Bonferroni’s post-hoc test to adjust for multiple comparisons) was used to test for differences between groups of tissue samples for data with ordinal and interval level, respectively. Finally, a multiple logistic regression analysis with backward stepwise method (Wald) was applied to identify significant predictors between controls and early as well as advanced CKD, respectively. Additionally, we have included covariates (like gender and smoking as well as others co-comorbidities i.e. diabetes, hypertension and hyperlipidemia) in the logistic regression models for

ACCEPTED MANUSCRIPT adjusting for baseline predictors. These statistical approaches did not affect any of

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the predictors.Statistical significance was determined with a level P<0.05.

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3 Results:

3.1 Basic characteristics and laboratory findings (table 1):

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The mean age of the 3 groups (control, early and advanced CKD) was not significantly different. Gender distribution revealed a higher number of males in both

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CKD groups compared to controls, but this difference was not statistically significant. There were also no significant differences between the 3 groups with respect to the incidence of classical risk factors such as hypertension, diabetes, dyslipidemia and nicotine abuse. As expected due to the classification criteria serum creatinine was

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significantly different between the 3 groups (p<0.0001) with highest values in the

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advanced CKD group. Serum phosphate was significantly (p<0.05) higher in advanced CKD compared to early CKD and controls whereas serum calcium was not

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significantly different between the 3 groups. The calcium-phosphate product (CaxP)

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was significantly (p<0.05) higher in advanced CKD than in early CKD and controls. Serum cholesterol values did not differ significantly whereas triglycerides were significantly higher in advanced CKD than in controls and early CKD patients.

3.2 Morphometric analysis of intima and media thickness in the different vascular beds (table 2, fig.1A-C, fig.2A-C, fig.3A-C): -

Aortic intima thickness was increased in advanced, but not in early CKD

Patients with advanced CKD had significantly (p=0.010) higher aortic intima thickness than controls and early CKD patients. In contrast, in the A. mammaria int. intima as well as media thickness was not significantly different between the 3

ACCEPTED MANUSCRIPT groups. In parallel, wall thickness of V. saphena magna did also not show significant

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3.3 Analysis of vascular calcification (figs. 1-3, table 3):

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differences between the 3 groups.

Aortic media calcification is already present in early CKD with unaltered CaxP

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and is progressive in advanced CKD with increased CaxP

In the aorta, intima calcification score was significantly increased in advanced CKD

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patients and slightly higher in early CKD patients compared to controls. Of interest, calcification of the aortic media (fig.1A-G) was already significantly higher in patients with early CKD compared to controls. In the A. mammaria int. (fig.2A-G) intima and media calcification was slightly more pronounced in both CKD groups than in

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controls; due to the high standard variation, however, the differences were not

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statistically significant. In contrast, in the V. saphena magna (fig.3A-D) calcification score was significantly higher in advanced CKD compared to controls and early CKD.

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Of particular note, in some patients with advanced CKD extremely heavy

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circumferential venous calcification was seen (fig.3C and fig. 4W-Y).

3.4 Analysis of vascular inflammation (figs.1-3, table 4): -

In-situ markers of vascular inflammation were already elevated in the aorta and the A. mammaria int. in early CKD

- 3.4.1 CRP: The aortic intima (fig.1H) showed significantly higher in-situ CRP protein expression in advanced CKD and a slightly higher expression in early CKD compared to controls. In contrast, in the aortic media (fig.1I) CRP score was significantly higher in both CKD groups compared to controls. In contrast, CRP expression in the intima and media of the A. mammaria int. and in the V. saphena magna were not significantly different between the 3 analysed groups.

ACCEPTED MANUSCRIPT - 3.4.2 CD40: Significantly higher CD40 scores of the aortic intima were seen in advanced CKD compared to both other groups. In the aortic media CD40 expression

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was significantly increased in early and advanced CKD patients compared to

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controls. In the intima of the A. mammaria int. (fig.2H) CD40 expression score was also significantly higher in both early and advanced CKD compared to controls

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whereas in the media the difference was only significant for advanced CKD versus control patients with the value in early CKD being in between. In the V. saphena

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magna (fig.3E) CD40 expression score was significantly (p<0.001) higher in advanced CKD compared to controls, but was not significantly higher than in early CKD. -

3.4.3 CD154: In the aortic intima the CD154 score was significantly higher in

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early and advanced CKD compared to controls. In contrast, CD154 expression in the

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aortic media was significantly higher in advanced CKD compared to controls slightly higher than in early CKD. Intima and media of the A. mammaria int. showed

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significantly (p<0.001) higher CD154 scores in both CKD groups compared to

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controls with a significantly higher intima score in advanced CKD than in early CKD. In the V. saphena magna significantly higher CD154 expression in advanced CKD compared to early CKD and control patients was seen. Of note, on inspection we did not see many T- and B-cells in the different vascular specimens.

3.5 Analysis of osteogenic conversion of vascular cells in the A. mammaria int. and the V. saphena (figs.4-5): On qualitative inspection expression of the contractile marker smooth muscle actin did not differ in the A. mammaria int. between the various groups (fig. 4A-C). In contrast, remodelling of the V. saphena was associated with changes in the

ACCEPTED MANUSCRIPT distribution of VSMCs (fig. 4D-F) and particularly invasion of the neointima by such actin positive cells (fig. 4F). In early and late CKD expression of the mesenchymale

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marker vimentin increased slightly in the A. mammaria int (fig. 4 G-I), whereas it was

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more or less the same in the saphenous vein (fig. 4 J-L). Of note, the proinflammatory molecule galectin-3 was not expressed in the vessel wall of control

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patients but expression in VSMCs increased in the A. mammaria int. and the V. saphena already in early CKD and was even more intense in late CKD (fig. 5 A-F).

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SATB2 expression in the A. mammaria int. was very similar (fig. 5 G-I) but was completely absent in the V. saphena. Representative examples of immunhistological staining for vimentin, smooth muscle actin and galectin-3 in calcificated veins are shown at various magnifications and (fig. 5 J-L).

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Thus, in summary the above qualitative data in patients with early and late CKD are

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in line with the idea of early an osteogenic switch of VSMC that might be caused or is

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at least associated with vascular inflammation.

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3.6 Multiple logistic regression analysis for independent explanatory predictors between the dependant categorial subgroups of (i) controls versus advanced CKD, (ii) controls versus early CKD as well as (iii) early versus advanced CKD: (i) Multiple logistic regression analysis revealed the thickness of the aortic intima (p=0.068, Odds Ratio (OR) 1.034, Confidence Interval (CI) [0.997;1.071]) in combination with the expression of the inflammatory marker CRP inside the aortic intima (p=0.022, OR 47.645, CI [1.753;1294.91]) as independent significant discriminators between controls and advanced CKD (2=15.0, p=0.001). (ii) Additionally, statistical analysis revealed a highly significant logistic regression model (2=16.5, p=0.001) between controls and early CKD, whereby the identified independent predictors CD40 inside the A. mammaria int. (p= 0.060 , OR 8.34, CI

ACCEPTED MANUSCRIPT [0.915;75.969] as well as media thickness of the A. mammaria int. (p= 0.077, OR 1.04, CI [0.996; 1.086]) and CRP inside the aortic media (p= 0.058, OR 19.38, CI

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[0.904;415.522]) partially reached significance level.

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(iii) Finally, statistical analysis only yielded CRP score in the aortic intima (p= 0.115, OR 4.842, CI [0.550;42.589]) as an independent predictor between early and late

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CKD based on a non-significant multiple logistic regression model (2=2.4, p=0.116). Additionally, we included covariates (like gender and cocomorbidities) to adjust for

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baseline predictors. These statistical approaches did not affect any of the predictors.

3.7 Correlation analysis (see table 5)

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Serum creatinine significantly (p<0.05) correlated with markers of vascular inflammation, i.e. CD40 and CD154 score of the intima and media of the A.

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mammaria int. and the aorta as well as intima and media CRP expression of the aorta. Interestingly, low, but not high serum Ca levels correlated significantly

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(p=0.032) with the degree of intima calcification in the A. mammaria int. whereas

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Phosphate values and CaxP product positively correlated (p<0.05) with CD154scores of the aorta, but not with calcification itself. Analysis of subgroups revealed an association of low serum Ca levels with the degree of intima calcification in the A. mammaria interna (p=0.006) for the control group. For the early CKD group a significant linkage of the serum triglyceride level with the intima CRP score of the A. mammaria int. (p=0.048) was seen. For the advanced CKD group an association of the serum cholesterol level with CD40 score of the aortic intima and serum Ca with the calcification score of the aortic media (p<0.05) was found. Compared to the cardiovascular risk factors not unexpectedly, the presence of diabetes mellitus correlated (p<0.05) with media calcification, CD40/CD154 scores of

ACCEPTED MANUSCRIPT the A. mammaria int. and aortic intima calcification, whereas the presence of hypertension correlated (p<0.05) with CRP and CD154 scores of the aortic intima.

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Smoking correlated (p<0.05) with CRP score in the aortic intima. Interestingly, no

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correlations could be found for dyslipidemia.

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4 Discussion:

In the present study morphological alterations, vascular calcification and in-situ

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expression of inflammatory markers were analysed at 3 different vascular sites of patients with early and advanced CKD in comparison to non-renal control patients undergoing cardiac bypass surgery. First and most importantly, we could show that significant inflammation of all vessels and calcification of the aortic media was

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already present in early CKD, i.e. a situation where no major derangement of CaxP is

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present. Second, we found calcification of the aortic intima and most interestingly also of the V. saphena magna significantly more pronounced in advanced CKD than

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in controls with the CaxP product correlating well with markers of inflammation, but

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not with calcification itself. Third, we could show significantly higher thickness of the aortic intima in advanced, but not early CKD compared to controls whereas the intima and media of the A. mammaria int. was not significantly different in both CKD groups compared to control patients indicating different vascular remodelling processes at various sites of the vascular bed. Cardiovascular morbidity and mortality is much higher in CKD patients compared to non-renal control patients and other cardiovascular risk groups, i.e. patients with diabetes mellitus1,13. Of interest, the Framingham Heart Study and subanalysis observed an association even between mild renal insufficiency and increased rate of cardiovascular events14,15. This finding was recently confirmed and extended in the HOPE study2 where in parallel to further impairment of renal function an increase in

ACCEPTED MANUSCRIPT cardiovascular events was seen in patients with and without diabetes mellitus. Go et al16 observed an independent, graded association between a reduced estimated

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GFR and the risk of death and cardiovascular events in a large community-based

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population. It is well known that serum creatinine is an independent risk factor for cardiovascular morbidity and mortality per se17 and this may explain why many

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patients with CKD will die from cardiovascular events prior to progressing to endstage renal disease3. As CKD is often associated with so-called classical or

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traditional risk factors, it is difficult to differentiate the role of kidney function per se from that of associated risk factors. However, in a multivariate analysis it could be demonstrated that cardiovascular events are only partly explained by associated risk factors with renal disease18. In our study the classical risk factors hypertension,

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diabetes, nicotine abuse and hyperlipidemia did not show significant differences

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between control, early and advanced CKD patients, but there was a strong tendency to a higher rate of diabetes in the advanced CKD group. The sex distribution

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revealed a higher number of males in both CKD groups compared to controls. Unless

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this difference was not significant, the different gender distribution is explained by the well-known higher prevalence of CKD in males and we had to accept this for our study as one potential confounder. Of note, the female subgroup of patients was in a postmenopausal age when known sex differences are no longer seen in kidney diseases19. One important aspect of the present study was the patient selection since we only included patients undergoing cardiac bypass surgery. Thus, all study patients had a high cardiovascular risk profile per se as they all suffered from relevant coronary heart disease making bypass surgery necessary. Therefore, the study did not address the difference between patients with CKD and presumably healthy nonrenal patients, but aims to determine the specific characteristics of vascular changes in coronary heart disease patients with early and advanced CKD as compared to

ACCEPTED MANUSCRIPT those without relevant renal dysfunction but comparable classical cardiovascular risk profile.

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Using detailed morphological analyses we found marked aortic intima thickening in

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advanced CKD compared to early CKD and controls whereas the intima and media of the A. mammaria int. was not significantly thicker in both CKD groups compared to

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control patients. Obviously, vascular reaction patterns in CKD markedly differ from one arterial site to the other. Wall thickening, intimal hyperplasia, atrophy of vessel

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wall musculature, vessel calcification and vascular inflammation were already described in CKD patients20,21. Previous studies on arteriolar wall thickness in CKD using ultrasonography of the carotid artery found that even minor deterioration in renal function was independently associated with increased intima media thickness

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(IMT) in middle-aged male population and in postmenopausal women 18,22. Of note,

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no correlation between the severity of kidney disease and carotid IMT was found 22. Patients with different stages of CKD show significant higher IMT as well as

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increased plaque formation in the coronary arteries and the A. carotis communis 10,22.

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Altered vascular remodeling with marked changes in intima thickness, lumen diameter and composition of the vascular wall was also seen in an experimental model of renal failure, i.e. the subtotally nephrectomized rat (SNX)11,24. In this model of moderate stable renal insufficiency an early increase in media thickness of mesenteric and intramyocardial arteries due to permissive roles of ET-1, PTH and P was documented25-27. In hemodialysis patients P, but not Ca was associated with increased IMT28. When IMT and carotid plaque formation were studied in CKD patients on peritoneal dialysis Ca was associated with plaque formation, but not with IMT29. In patients with end-stage renal disease (ESRD) only plaque formation was found to be associated with cardiovascular events30. In dialysis patients IMT of the V. brachialis at the AV-fistula arm showed a continuous increase with the duration of

ACCEPTED MANUSCRIPT dialysis31. Hypertrophy of venous media and intima was shown to be due to hyperplasia of vascular smooth muscle cells (VSMCs) and an increase of

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extracellular fibrous tissue32. This is in line with experimental data in the SNX model

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where significant thickening of the aorta was also found to be associated with hyperplasia of VSMCs and increased extracellular matrix deposition 33.

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Vascular calcification, which in patients is mostly assessed by CT, is associated with increased mortality in renal and non-renal patients34. In dialysis patients more

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pronounced media, but not intima calcification could be detected directly before renal transplantation8,35. In parallel, increased media calcification of smaller arteries, increased vascular stiffness and reduced elasticity module was found in CKD patients36. In these patients, an association of high P levels and particularly

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increased CaxP product with cardiovascular morbidity and mortality was shown in

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large clinical studies37,38 . In addition, in-vitro studies39,40 provided insights into the mechanisms of how P stimulates the phenotypic switch of VSMCs from contracting

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matrix producing cells to osteoblastic calcifying cells thus favouring vascular

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calcification. Of note, our data on expression of galectin-3 and STAB2 in patients with early and late CKD are in line with this idea of an early osteogenic switch of VSMC that might be caused or is at least associated with vascular inflammation. We have to acknowledge, however, that the van Kossa stain to detect histological microcalcification has certainly some limitations in terms of sensitivity although we used this stain routinely and thus have ample experience with it. Of particular interest, however, is our finding that not only arterial but also venous calcification was markedly increased in advanced CKD. Venous alterations in CKD have been investigated only occasionally; in parallel to arterial changes increased venous wall thickness and marked alterations of the venous structure had also been documented31,32. Despite the fact that the finding of such venous alterations could

ACCEPTED MANUSCRIPT probably reflect and support the theory that renal dysfunction may result in a proinflammatory environment that may be reflected throughout the circulation the issue

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of venous changes in CKD has been very much neglected. More importantly, marked

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venous calcification as in our study has not been described so far albeit this may be extremely relevant when it comes to shunt surgery, renal transplantation or venous

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bypass grafting. Our finding that aortic media calcification was already significantly more pronounced in early CKD patients compared to controls points to alternative

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pathogenetic pathways since in these patients P and CaxP product were not yet significantly altered. This important finding is paralleled by the fact that media calcification was associated with in-situ upregulation of pro-inflammatory molecules in early CKD, but not with increased serum levels of P, Ca or CaxP. Alternative factors

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that could be involved in early calcification in mild CKD are increased oxidative stress

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and microinflammation that both are seen in early renal dysfunction and are known to be involved in the development of atherosclerosis42. An interesting in vitro study43

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showed that only endothelial microparticles from endothelial cells stimulated by TNFα

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were able to induce calcification in VSMC thereby indicating a special initial role for inflammation in the process of vascular calcification. In particular CRP and other acute phase proteins were already significantly elevated in early stages of CKD; CRP values, however, were about 10 times higher in dialysis patients 17,42,44. In coronary arteries lesions of CKD patients more pronounced macrophage infiltration and higher CRP-deposition were seen whereas fetuin A deposition was lower than in control patients12. As a pro-inflammatory and pro-atherogenic system the CD40-CD154 receptor ligand system is associated with initiation and progression of atherosclerotic plaques. Increased CD40 expression of media myocytes and plaque-associated endothelial cells, T lymphocytes, macrophages and fibroblasts was seen in advanced CKD compared to control patients7. In-situ expression of CD40, CRP and CD154 was

ACCEPTED MANUSCRIPT increased in calcified compared to non-calcified areas7. These cytokines as well as TNFα are also substantially involved in pathologic soft tissue calcification and

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particularly vascular calcification6,45. Thus, a direct action on VSMC seems

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conceivable inducing a switch into a VSMC phenotype which favours or thereby allows calcification46,47.

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In the present study we found only very few T- and B-cells in the aorta, the A. mammaria interna and the V. saphena. This is in line with data from the previous

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study in more advanced atherosclerotic lesions7 were we found no inflammatory cells at all in 6/57 specimens with no difference between uremic and control patients. In this study scores for lymphocytes and macrophages tended to be higher in calcified versus non-calcified plaques and CD40-CD154 generally correlated with CD3

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positive T-cell and macrophages but not with B-cells (again without any difference

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between the groups).

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In conclusion, in our study early CKD was already associated with local upregulation

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of proinflammatory and proosteogenic molecules in the vascular wall and calcification of the aortic media compared to non-renal patients with comparable coronary heart disease. These findings confirm earlier findings of inflammation in calcific cardiac valves in various patient cohorts48,49 as well as in experimental renal failure50 and underscore the importance of microinflammation as a non-classical cardiovascular risk factor in CKD maybe sheding new light on the pathogenesis of early vascular calcification in patients with CKD. Additionally, we found significantly more calcification of the aortic intima and V. saphena magna as well as increased thickness of the aortic intima in advanced CKD than in control patients whereas the intima and media thickness of the A. mammaria int. were comparable in all groups,

ACCEPTED MANUSCRIPT indicating

different

and

potentially

pathogenetically

heterogeneous

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remodelling processes at various sites of the vascular bed.

vascular

ACCEPTED MANUSCRIPT Acknowledgements: The authors thank M. Klewer, S. Söllner, M. Reutelshöfer, K. Schmitt and S. Tauber

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for excellent assistance. The study was supported by the Interdisciplinary Centre for

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Clinical Research (IZKF, projects A11,A31,J4) at the University Hospital of Erlangen-

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The authors declare no conflicts of interest.

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Nürnberg and the Deutsche Forschungsgemeinschaft (AM93/12-1 and HI510/9-1).

ACCEPTED MANUSCRIPT References: 1. Sarnak MJ, Levey AS, Schoolwerth AC, Coresh J, Culleton B, Hamm LL et al. Kidney disease as a risk factor for development of cardiovascular disease: a

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statement from the American Heart Association Councils on Kidney in

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25. Nabokov AV, Amann K, Wessels S, Münter K, Wagner J, Ritz E. Endothelin

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27. Amann K, Törnig J, Kugel B, Gross ML, Tyralla K, El-Shakmak A et al.

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28. Ishimura E, Taniwaki H, Tabata T et al. Cross-sectional association of serum phosphate with carotid intima-medial thickness in hemodialysis patients. Am J

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Kidney Dis 2005; 45: 859-65. 29. Wang AY, Ho SS, Liu EK et al. Differential associations of traditional and nontraditional risk factors with carotid intima-media thickening and plaque in

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35. Moe SM, O'Neill KD, Duan D, Ahmed S, Chen NX, Leapman SB et al. Medial

matrix proteins. Kidney Int. 2002;61:638-47.

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36. London GM, Guerin AP, Marchais SJ, Metivier F, Pannier B, Adda H. Arterial

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media calcification in end-stage renal disease : impact on all-cause and cardiovascular mortality, Nephrol Dial Transplant 2003;18 : 1731 – 1740.

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disease, Circ Res 2004; 95: 560 – 567. 40. Giachelli CM. The emerging role of phosphate in vascular calcification. Kidney

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Int. 2009;75: 890-7.

41. Feinfeld DA, Batista R, Mir R, Babich D. Changes in venous histology in chronic hemodialysis patients, Am J Kidney Dis 1999; 34: 702 – 705

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42. Drüeke TB, Massy ZA. Atherosclerosis in CKD: differences from the general population. Nat Rev Nephrol. 2010;6:723-35. 43. Buendía P, Montes de Oca A, Madueño JA, Merino A, Martín-Malo A, Aljama P et al. Endothelial microparticles mediate inflammation-induced vascular calcification. FASEB J. 2015; 29(1):173-81. 44. Bergström J, Lindholm B, Lacson E Jr, Owen W Jr, Lowrie EG, Glassock RJ et al. What are the causes and consequences of the chronic inflammatory state in chronic dialysis patients? Semin Dial 2000; 13: 163 – 175. 45. Zimmermann J, Herrlinger S, Pruy A, Metzger T, Wanner C. Inflammation enhances cardiovascular risk and mortality in hemodialysis patients.Kidney Int. 1999; 55:648-58. 46. Moe SM, Chen NX. Inflammation and vascular calcification. Blood Purif 2005; 23: 64-71

ACCEPTED MANUSCRIPT 47. Hattori Y, Matsumara M, Kasai K. Vascular smooth muscle cell activation by Creactive protein. Cardiovasc Res 2003; 58; 186-195 48. Sádaba JR1, Martínez-Martínez E1, Arrieta V1, Álvarez V1, Fernández-Celis A1,

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Ibarrola J1, Melero A1, Rossignol P2, Cachofeiro V3, López-Andrés N4,2.Role for

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Galectin-3 in Calcific Aortic Valve Stenosis. J Am Heart Assoc. 2016;5(11) pii: e004360.

49. Mosch J, Gleissner CA, Body S, Aikawa E. Histopathological assessment of

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calcification and inflammation of calcific aortic valves from patients with and without diabetes mellitus. Histol Histopathol. 2016 Jun 29:11797. [Epub ahead

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of print]

50. Shuvy M1, Abedat S2, Mustafa M2, Duvdevan N2, Meir K3, Beeri R2, Lotan C2. Cellular Changes during Renal Failure-Induced Inflammatory Aortic Valve

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Disease. PLoS One. 2015 Jun 12;10(6):e0129725.

ACCEPTED MANUSCRIPT Legends to the figures: Fig. 1:

Aorta: Representative histology (A-C,Kossa stain,x20), CD40 score

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(D-Fx10), media calcification score (G), intima (H) and media (I) CRP

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expression score.

Please note that aortic intima thickness (C) was significantly higher in advanced CKD

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than in early CKD (B) and controls (A). In contrast, aortic media calcification (A-C, G) and CD40 expression (D-F) were already significantly higher (p<0.05 and 0.001) in

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early CKD than in controls. In-situ expression of CRP in the aortic intima (H) was significantly higher in advanced CKD than in controls whereas in the aortic media expression (I) was already significantly (p<0.005) increased in early CKD and this

A. mammaria interna: Representative histology (A-C,HE,x5), media

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Fig. 2:

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increase was also shown in advanced CKD. *:p<0.05, **:p<0.005

calcification (D-G,Kossa,x20) and CD40 expression score (H).

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Intima and media thickness of the A. mammaria int. were not significantly different

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between the 3 groups (A-C). Media calcification was markedly increased in early and advanced CKD compared to control patients (D-G). The difference, however, just failed to reach statistical significance. Expression score of CD40 (H) was significantly higher in early and advanced CKD compared to controls. *:p<0.05

Fig. 3:

V. saphena magna: Representative histology (A-C,Kossa,x20), vessel calcification (D) and CD40 score (E)

Media calcification (A-D) and CD40 expression (E) of the V. saphena magna were significantly higher in advanced CKD than in early CKD and control patients. *:p<0.05, **:p<0.005

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Analysis

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wall

structure

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expression

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alpha smooth muscle actin and vimentin as early markers for

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mammaria int. and the V. saphena magna.

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dedifferentiation of vascular smooth muscle cells (VSMC) in the A.

A-F: Expression of alpha-smooth muscle actin did not differ in the A. mammaria int.

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(A-C) in the 3 patient groups. In contrast, remodelling of the V. saphena magna (D-F) was associated with changes in the distribution of alpha-actin positive VSMCs and

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particularly invasion of the neointima by such actin positive cells was seen (F). G-L: Expression of the mesenchymal marker vimentin slightly increased in the A. mammaria int. (G-I) in early and late CKD compared to control patients. In contrast,

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in the V. saphena magna (J-L) there was no difference.

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Fig. 5: Analysis of vessel wall structure and expression of Galectin-3 and SATB2 as early markers for osteogenic changes of vascular smooth

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muscle cells (VSMC) in the A. mammaria int and the V. saphena

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magna.

A-F: Expression of the proinflammatory molecule galectin-3 was not seen in control patients at all, but its expression increased in the A. mammaria int. (A-C) and the V. saphena (D-F) already in early CKD and was even more intense in late CKD (E-F). G-I: Expression of the proosteogenic molecule SATB2 was not seen in control patients (G) in the A. mammaria int., but its expression increased in early (H) and particularly late CKD (I). J-L:

Representative

magnifications.

examples

of

marked

venous

calcification

at

various

ACCEPTED MANUSCRIPT Basic characteristics and laboratory findings

early CKD patients (n=14)

67.4±7.6

69.9±8.2

65.6±7.9

13/11

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age [years]

advanced CKD patients (n=12)

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Non-renal control patients (n=24)

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Table 1:

2/10

diabetes (%)

3/20 (15)

2/11 (18.1)

5/10 (50)

hypertension (%)

16/20 (80)

10/12 (83.3)

9/10 (90)

nicotine abuse (%)

8/18 (44.4)

3/9 (33.3)

6/8 (75)

hyperlipidemia (%)

13/24 (54.1)

9/14 (64.2)

7/12 (58.3)

0.92±0.15

1.57±0.20 *

5.24±2.27 *,#

3.82±0.81

3.67±0.73

4.65±0.99 *,#

calcium [Ca, mmol/l]

2.30±0.14

2.25±0.15

2.18±0.28

Ca x P [mg2/dl2]

35.26±7.76

33.27±7.33

40.25±8.53 #

cholesterol [mg/dl]

194.0±34.2

203.6±60.0

194.3±57.9

triglycerides [mg/dl]

165.7±57.1

156.0±64.1

252.9±143.5 *,#

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creatinine [mg/dl]

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laboratory findings

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cardiovascular risk factors1

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phosphate [P, mg/dl]

4/10

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gender [female/male]

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: excluding cases with definitive unknown risk factor

* versus control (p<0.05) # versus early CKD (p<0.05)

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Wall thickness of different vessels (µm)

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20.9±17.7

165.5±41.0

190.0±63.3

199.4±80.5

466±165

410±279

503±173

A.mammaria Intima thickness interna Media thickness

14.3±11.05

Wall thickness

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* versus control (p<0.05)

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# versus early CKD (p<0.05)

96.1±53.9*

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89.1±63.7

V. saphena magna

advanced CKD patients (n=12)

158±80.4*

Intima thickness

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Aorta

early CKD patients (n=14)

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Non-renal control patients (n=24)

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Median [interquartile range]

A. mammaria interna

3.17±0.72* 3.0 [2]

2.73±1.00 3.0 [2]

1.30±0.66 1.0 [1]

1.83±0.58 2.0 [1]

1.82±1.1 1.0 [2]

2.16±0.97 2.0 [2]

2.67±0.65 2.5 [1]

2.55±0.82 3.0 [1]

1.00±0.74 1.0 [1]

1.36±0.63 2.0 [1]

2.10±0.88* 2.0 [1]

1.04±0.64 1.0 [0]

Media

2.48±0.51 3.0 [1]

Intima

Media

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V. saphena magna

* versus control (p<0.05)

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# versus early CKD (p<0.05)

advanced CKD patients (n=12)

1.67±1.12* 2.0 [2]

Intima

1.27±0.79 1.0 [0]

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Aorta

early CKD patients (n=14)

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Non-renal control patients (n=24)

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Mean±standard deviation

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Table 3:

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Median [interquartile range]

Aorta

Intima

CRP CD40

Media

CRP

CD40

CRP

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A.mammaria Intima interna

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CD154

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CD40

Media

CD154

CRP CD40 CD154

V. saphena magna

CRP CD40 CD154

3.70±0.48 4.0 [1] 2.70±1.34*# 3.0 [2] 3.20±0.32* 4.0 [0]

1.78±0.67 2.0 [1] 2.35±0.71 2.0 [1] 2.76±0.44 3.0 [1]

2.80±0.79 3.0 [1] 3.20±0.42* 3.0 [0] 3.25±0.75 3.0 [1]

2.90±0.99 3.0 [2] 3.60±0.52* 4.0 [1] 3.80±0.42* 4.0 [1]

1.89±0.76 1.0 [2] 1.11±0.90 1.0 [2] 2.11±0.76 2.0 [0]

1.91±0.83 2.0 [2] 2.09±1.38* 3.0 [2] 2.91±0.70** 3.0 [1]

2.33±1.23* 2.0 [2] 2.00±1.12* 2.0 [2] 3.55±0.52** 4.0 [1]

2.67±0.97 2.0 [1] 2.67±0.97 3.0 [1] 2.44±0.86 2.0 [1]

2.64±1.03 2.0 [2] 3.09±0.54 3.0 [0] 3.36±0.50 3.0 [1]

2.67±1.00 2.0 [1] 3.56±0.53* 4.0 [1] 3.73±0.47** 4.0 [0]

2.70±0.98 3.0 [2] 2.41±0.80 2.0 [1] 2.90±0.70 2.0 [1]

2.92±0.99 3.0 [2] 2.86±0.66 3.0 [1] 3.17±0.58 3.0 [2]

2.84±0.80 3.0 [1] 3.45±0.83** 3.75 [2] 3.89±0.33*# 4.0 [1]

* versus control (p<0.05), ** versus control (p<0.001) # versus early CKD (p<0.05)

advanced CKD patients (n=12)

3.20±0.63 3.0 [1] 2.00±0.94 2.0 [2] 3.58±0.51* 4.0 [1]

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CD154

2.74±0.62 3.0 [1] 1.83±1.03 2.0 [1] 2.43±0.68 3.0 [1]

early CKD patients (n=14)

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Non-renal control patients (n=24)

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Mean±standard deviation

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Table 4:

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Spearman's rank

Arteria mammaria interna

Aorta

significance Media

(0.331/0.040) CD154-Score

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(0.675/0.000)

Calcium [mg/dl]

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Phosphate [mg/dl]

Media

CRP-Score

CRP-Score

(0.408/0.007)

(0.324/0.036)

CD40-Score

CD40-Score

CD40-Score

(0.349/0.029)

(0.449/0.003)

(0.722/0.000)

CD154-Score

CD154-Score

CD154-Score

(0.647/0.000)

(0.502/0.001)

(0.569/0.000)

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Creatinine [mg/dl]

CD40-Score

Intima

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Intima

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correlation (rho)/

Thickness (0.315/0.037) CRP-Score (0.383/0.015) CD154-Score (0.376/0.014)

Kossa-Score (0.327/0.030)

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Ca x P [mg²/dl²]

Triglyceride [mg/dl]

CD154-Score (0.348/0.024)

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