vascular calcification is an active process similar to the mineralization that occurs in bone [ 1 , 2 ] . vascular smooth muscle cells undergo phenotypic differentiation into osteoblast - like or chondroblast - like cells and they synthesize calcification regulating proteins and matrix components typically found in bone and in cartilage [ 3 , 4 ] . calcification of the arterial media , observed even in small vessels ( mnckeberg 's calcification ) , is common in uremic patients and seems to be less associated with inflammation as compared to intimal mineralization typical for atherosclerosis [ 5 , 6 ] . vascular mineralization advances with age and is intensified in diabetes , dyslipidemia , chronic kidney disease , and hypertension . in newly treated hemodialysis and peritoneal dialysis patients , diabetes , dialysis duration , and the previous presence of aortic arch calcification ( aac ) hyperinsulinemia and insulin resistance ( clinical signs of type 2 diabetes ) are positively correlated with the arterial calcification [ 8 , 9 ] . arterial medial calcifications often occur also in diabetic individuals as a component of the diabetic macroangiopathy . in the animal model , insulin resistance induced in rats by fructose feeding resulted in increased aortic calcium deposition , elevated calcium - phosphate index , and local hyperplastic changes in the aortic media . blood vessels obtained from end stage renal disease ( esrd ) patients were often studied by various histological techniques to assess vascular calcification . they were collected during renal transplantation ( epigastric or iliac arteries ) or by peripheral arterial biopsy ( radial artery ) [ 3 , 11 , 12 ] . in the present study we used small samples ( otherwise routinely discarded ) of radial arterial walls obtained during creation of arteriovenous fistula for hemodialysis access . the study was aimed at investigating the relationship between selected clinical and biochemical parameters , with special emphasis on diabetes markers , and the level of histologically assessed radial artery calcification in end stage renal disease patients . the study population consisted of 59 patients ( 38 males , 21 female ; mean age at the beginning of the study 61  16  yrs ) , including 36 on maintenance hemodialysis ( hd ) and 23 on predialysis ( stage 5 of ckd ) . the study was approved by the bioethics committee of the jagiellonian university and all patients signed an informed consent for their participation . the data on mortality was collected over a period of three years . during this period , the mortality data , including the causes of death , was based on the patients ' records . in all patients , selected biochemical parameters were assessed : serum concentrations of total cholesterol , hdl - cholesterol , ldl - cholesterol , triglycerides ( tg ) , serum creatinine , peripheral blood counts , albumin , glucose , intact parathyroid hormone ( ipth ) , total calcium ( ca ) and phosphate ( pi ) , high sensitive c - reactive protein ( hscrp ) , asymmetric dimethylarginine ( adma ) , osteopontin ( opn ) , osteoprotegerin ( opg ) , and osteocalcin ( oc ) . homeostasis model of assessment - insulin resistance ( homa - ir ) was calculated by application of the international formula : fasting insulin ( iu / ml )  fasting glucose ( mmol / l)/22.5 . routine biochemical tests were carried out using automatic biochemical analyzers : hitachi 917 ( hitachi , japan ) and modular p ( roche diagnostics , mannheim , germany ) . concentrations of hscrp were measured using immunonephelometric method on nephelometer bn ii ( siemens healthcare diagnostics , germany ) . opn , oc , opg , and adma were determined using elisa microplate immunoassays and elx808 automatic reader ( bio - tek instruments , inc . , the following kits of reagents were applied : opn ( r&d systems ) , oc ( metra , germany ) , opg ( quidel , biovendor , czech republic ) , and adma ( immundiagnostik , germany ) . the mean arterial pressure ( map ) was calculated according to the formula : map = dbp + 1/3(sbp - dbp ) , where sbp is systolic blood pressure and dbp is diastolic blood pressure . the intima - media thickness of the common carotid artery trunk ( cca - imt ) was assessed by ultrasonography ( b presentation , acuson 128 xp/10 apparatus equipped with a linear head at 5/7  mhz ) . the measurements were performed bilaterally at 0.5  cm and 2  cm below the division of the common carotid artery during diastolic phase of the heart cycle . the results were expressed as the arithmetic means of the values obtained for the left and right arteries . 5  2  mm in size , were collected during the first creation of arteriovenous fistula for hemodialysis access . the samples were immediately immersed in 10% phosphate - buffered formalin and fixed overnight and then rinsed in pbs and soaked in 30% sucrose . the material was snap - frozen and tissue blocks were positioned in a cryostat for cutting sections in a plane encompassing the entire thickness of the vascular wall . serial 10  m - thick cryosections were cut and thaw - mounted on poly - l - lysine coated slides . sections were stained routinely with mayer 's haematoxylin and eosin ( he ) , with von kossa method and with alizarin red . the stained sections were examined using an olympus bx-50 microscope ( olympus , tokyo , japan ) in brightfield mode and images were registered using olympus dp-71 digital ccd camera controlled by olympus analysis five software . the advancement of vascular calcification was semiquantitatively evaluated in von kossa and alizarin red - stained sections by two independent observers . the degree of mineralization was classified according to the following scale : 0 : no mineral content , 1 : a few small dispersed concretions , 2 : numerous small dispersed concretions , 3 : larger granular concretions , and 4 : large areas occupied by fused mineral deposits . the reproducibility of the morphological analysis was confirmed by bland - altman method and by calculating intraclass correlation coefficient ( icc ) which was 0.88 . the number of patients ( percentage of the group ) was reported for categorical variables and mean  standard deviation or median ( lower - upper quartile ) for continuous variables , depending on distribution . student t - test or mann - whitney test was used for simple comparisons between the groups . multiple logistic regression models were constructed using the variables that differed significantly between the groups in simple comparisons and/or predefined sets of confounders , as pointed in the results . odds ratios ( or ) for 1 unit increase with 95% confidence intervals ( 95% ci ) being reported , unless otherwise stated . all tests were two - tailed and the results were considered statistically significant at p  0.05 . statistica 10 software ( statsoft , tulsa , ok , usa ) was used for computations . routine histology ( he ) showed general morphology of the radial artery , with intimal thickening in the vast majority of the examined specimens ( figure 1(a ) ) . basophilic deposits were visible in arterial wall in cases of very intense calcification ( figure 1(b ) ) . the preliminary comparison of two staining methods used for the assessment of the mineralization degree showed that von kossa method was less sensitive ; thus we employed alizarin red staining for further analysis and for correlation of the vascular calcification level with clinical and biochemical data . morphologically , mineral deposits were found in all layers of the arterial wall but they were most frequently localized in the media ( figures 1(b)1(d ) ) . in less advanced lesions , deposits were preferentially located close to the outer and inner elastic laminae ( figure 1(e ) ) . some deposits were fine and dispersed ( figure 1(f ) ) , while others occupied larger areas and in the most advanced cases even the entire thickness of the media ( figures 1(b)1(d ) ) . only very scanty mineral deposits were occasionally seen in the vascular intima . among 59 radial artery samples examined histologically , 34 showed positive alizarin red staining indicative of the calcification process . the proportion of samples with arterial wall mineralization ( table 1 ) as well as its advancement ( figure 2(a ) ) did not differ between hd and predialysis patients ( p = 0.6 ) . in further analysis , hd patients were studied together with predialysis patients ; nevertheless all multiple regression models were adjusted for hd status . table 1 summarizes differences in clinical and biochemical parameters between the groups with and without calcifications as assessed by alizarin red staining of radial artery . vascular calcification was associated with higher age of patients , higher glucose , and diabetes . map was slightly lower in patients with calcifications and the levels of adma and opg were higher in this group . clinical criteria of the metabolic syndrome [ 14 , 15 ] were compared between patients with and without vascular calcifications in radial artery ( table 2 ) . among patients with vascular calcifications , the number of individuals with fasting glucose level above 5.6 mmol / l , that is , patients with ifg ( prediabetes )   and diabetes , was significantly higher ( p = 0.0004 ) . moreover , vascular calcifications were more severe in the group of patients with ifg and diabetes ( figure 2(b ) ) . other criteria of the metabolic syndrome did not differ between the groups with or without calcifications . the association of radial artery calcifications with ifg and diabetes was further confirmed by multiple logistic regression ( table 3 ) . three models were constructed , containing age and fasting glucose > 5.6  mmol / l as independent variables : the first one was adjusted for gender , hd status , and ca  pi product , and the second was additionally adjusted for dyslipidemia , hypertension , high bmi , and hscrp . the third model , adjusted as the first one , included other variables that were significantly associated with arterial calcification in simple comparisons , that is , map , adma , and opg . fasting glucose > 5.6  mmol / l was the only variable independently associated with the vascular calcifications in all three models . the results were similar when diabetes was substituted for increased fasting glucose level in the models : or = 17.2 ( 1.79166 ) ; p = 0.011 in model 1 ; or = 25.4 ( 1.74372 ) ; p = 0.015 in model 2 ; or = 24.6 ( 1.45419 ) ; p = 0.022 in model 3 . patients with calcifications in radial arteries presented higher cca - imt ( table 1 ) . the relation between cca - imt and histologically detected vascular calcification remained significant after adjustment for dialysis status , gender , ca  pi , hscrp , and the presence of metabolic syndrome ( or for vascular calcification 2.31 per 0.1  mm increase in cca - imt ; 95%  ci 1.184.52 ; p = 0.011 ) but not after adjustment for age . cca - imt was significantly higher in patients with ifg and diabetes ( 0.98  0.13 versus 0.88  0.15  mm ; p = 0.007 ) . sixteen patients with calcifications in the radial artery ( 47% ) died during 3 years of the followup , while in the group without calcifications the mortality was lower : 3 deaths ( 12% ) . vascular calcification was significantly associated with patients ' mortality in simple analysis ( p = 0.004 ) and after adjustment for hd status and age ( or for death 5.39 ; 95%  ci 1.2024.1 ; p = 0.024 ) . this study presents a comprehensive comparison of biochemical and clinical data with the calcification status assessed histologically in the peripheral arteries of esrd patients . biopsies of radial artery collected during the creation of vascular access for hemodialysis were used previously by other authors to study calcification . however , in that study , the surgical anastomosis was performed in an end - to - end fashion ; thus a sample encompassing the entire circumference of the artery could be excised and used for further analysis , allowing for a more reliable assessment of the calcification extent in the arterial wall . in the study mentioned above , the authors found mineral deposits in 37% of the examined radial arteries . after adopting stringent morphological criteria to include even the finest calcifications and using more sensitive alizarin red staining for calcium detection we found arterial calcification in 57% of cases . intimal calcification occurs in advanced atherosclerotic lesions and is associated with lipid accumulation and infiltration of the inflammatory cells , such as macrophages and t cells . medial arterial calcification ( mac ) displays features very similar to those of physiological calcification in bone . mac develops independently of atherosclerosis and is a commonly observed pathology in diabetes , esrd , and ageing . the present study revealed a significant association of arterial medial calcifications with impaired fasting glucose ( ifg , prediabetes ) and diabetes but not with other criteria of the metabolic syndrome including overweight . our results are in accordance with the study of lim et al .   demonstrating the relationship between anthropometric parameters , metabolic profiles , and coronary artery calcium scoring ( cacs ) . subjects with ifg or diabetes had higher cacs and more advanced coronary stenosis than normal subjects . moreover , several studies confirmed that fasting plasma glucose is a better independent determinant of the progression of coronary artery calcification than the other metabolic syndrome risk factors [ 2022 ] . the above mentioned papers presented a relationship between hyperglycemia and vascular calcification based on noninvasive imaging of blood vessels . in our study , this relationship was analyzed for the first time using histologically examined samples of peripheral arteries . our study showed that fasting hyperglycemia , mostly associated with type 2 diabetes , was the only significant predictor of vascular calcifications in esrd patients . recent evidence suggests that medial calcification in diabetes is an active , cell - mediated process , similar to that observed in patients with end stage renal disease [ 23 , 24 ] . vascular calcifications and atherosclerosis are frequent in patients with esrd and they are associated with increased cardiovascular morbidity . coronary artery calcification ( cac ) was found in 70.2% of dialysis patients and was significantly associated with cca - imt and the thickness of atherosclerotic plaques . these results indicate that both , medial calcification and atherosclerotic lesions , frequently coexist in patients with esrd and that cca - imt , increased in patients with calcifications examined in this study , may serve as a surrogate marker of vascular calcification . the mechanisms responsible for vascular calcification include inflammation and oxidative stress , as well as bone and mineral metabolism disturbances . in our study higher adma and opg levels were associated with vascular calcification . serum levels of adma in chronic kidney disease increase due to its defective inactivation and excretion . coen at al .   postulated that adma may play a role in the pathogenesis of vascular calcification in dialysis patients . increased serum opg is associated with type 2 diabetes , chronic kidney disease , and the severity of vascular calcification and coronary artery disease [ 2931 ] . it could represent a compensatory mechanism for vascular damage , also showing a protective effect against vascular calcification [ 32 , 33 ] . arterial calcification was associated with higher mortality in esrd patients . according to our knowledge , the relationship between vascular calcification assessed histologically and the long - term mortality in chronic kidney disease patients has not yet been studied . ogawa et al .   examined the effect of ct - assessed aortic arch calcification on mortality in the 401 hemodialysis patients during 4-year follow - up period and demonstrated that cardiovascular mortality was significantly higher in patients with calcification . in our study , employing a different assessment model ( radial artery and histology ) this effect of arterial calcification on mortality in esrd patients has been confirmed . small samples of radial artery obtained during creation of vascular access for hemodialysis may successfully serve as source of the material for histological assessment of vascular mineralization . in end stage renal disease patients , impaired fasting glucose ( prediabetes ) and diabetes predict vascular calcification which is significantly associated with higher mortality . these results indicate that combination of renal insufficiency and hyperglycemic conditions exerts a synergistic effect on vascular calcification and increases the risk of death .
objective . 
 the objective of the study was to assess the relationship between selected clinical and biochemical parameters of end stage renal disease ( esrd ) patients and arterial calcification . 
 materials and methods . 
 the study comprised 59 stage 5 chronic kidney disease patients ( 36 hemodialyzed and 23 predialysis ) . 
 the examined parameters included common carotid artery intima - media thickness ( cca - imt ) , bmi , incidence of diabetes and impaired fasting glucose ( ifg ) , dyslipidemia , hypertension , and 3-year mortality . 
 plasma levels asymmetric dimethylarginine ( adma ) , osteopontin ( opn ) , osteoprotegerin ( opg ) , and osteocalcin ( oc ) were also measured . 
 fragments of radial artery obtained during creation of hemodialysis access were stained for calcifications using von kossa method and alizarin red . 
 results . 
 calcification of radial artery was significantly associated with higher prevalence of ifg and diabetes ( p = 0.0004 ) and older age ( p = 0.003 ) , as well as higher opg ( p = 0.014 ) and adma concentrations ( p = 0.022 ) . 
 fasting glucose > 5.6  mmol / 
 l ( ifg and diabetes ) significantly predicted vascular calcification in multiple logistic regression . 
 the calcification was also associated with higher cca - imt ( p = 0.006 ) and mortality ( p = 0.004 ; or for death 5.39 [ 1.2024.1 ] after adjustment for dialysis status and age ) . 
 conclusion . 
 combination of renal insufficiency and hyperglycemic conditions exerts a synergistic effect on vascular calcification and increases the risk of death .