Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in patients with reduced kidney function . Nontraditional risk factors such as calcification of the vascular media play a major role in increasing CVD risk. Vascular calcification is an active process involving a phenotypic change of vascular smooth muscle cells (VSMCs) into bone lineage cells that are capable of laying down calcium phosphate minerals. This process is markedly accelerated in CKD patients.
Experimental approaches in rodents involving surgical ablation of the kidney have been shown to generate a representative model of mild to moderate CKD. On the contrary, administration of dietary adenine has been shown to generate a more severe model of CKD in rats . Excess dietary adenine saturates the normal adenine salvage pathway (adenine phosphoribosyltransferase pathway) and instead oxidizates to 2,8 dihydroxyadenine by xanthine oxidase. 2,8-dihydroxyadenine is then excreted by the kidneys; however, due to its low solubility, it forms precipitates and crystals in the tubules causing tubular injury, inflammation, obstruction and fibrosis. The phenotype in this model appears to be consistent with the complications of the chronic kidney disease-mineral bone disorder (CKD-MBD) observed in humans with CKD. That is, animals with adeninine-induced CKD develop hyperphosphatemia, secondary hyperparathyroidism, renal osteodystrophy and vascular calcification [3–7]. FGF-23 is a phosphaturic hormone that increases early in CKD to maintain normophosphatemia in the face of declining kidney function. There is a growing body of literature linking FGF-23 with cardiac hypertrophy and mortality in patients with CKD [8,9]; however, this relationship has not been studied in adenine-induced CKD.
The majority of published studies have reported approximately 50% incidence of calcification following the administration of 0.75% dietary adenine to rats for 4–6 weeks [4,5,7]. By lowering the dietary protein content to 2.5%, Price et al.  were able to show elevated levels of aortic calcium in 100% of CKD animals at the 4-week time point. However, the major confounding factor with this dietary regimen is that animals lose 30–50% of their initial body weight during the adenine intervention [11,12].
To date, it has not been studied in the adenine model of CKD whether the development of vascular calcification and its progression reflects the cardiovascular complications and changes within the circulation that are observed in humans with CKD. Therefore, we were interested in characterizing the changes in vascular compliance, blood pressure and cardiac hypertrophy at three time points in a modified adenine model of CKD [13,14]. Furthermore, it is unknown whether the presence of uremia or the duration of CKD impacts differentially upon calcification of major vessels (i.e. carotids, thoracic aorta, abdominal aorta, iliac and renal arteries).
The objectives of this study were three-fold: first, to modify the conventional 0.75% adenine to 0.25% adenine to minimize the risk of severe weight loss; second, to determine the impact of duration of adenine feeding (5, 8 and 11 weeks) on calcification of the major arteries (carotid, thoracic aorta, abdominal aorta, iliac, renal), the blood pressure profile over time and the haemodynamic consequences [pulse wave velocity (PWV), left ventricular hypertrophy); and third, to determine the impact of duration of adenine feeding (5, 8 and 11 weeks) on parameters of the CKD-MBD [phosphate, calcium and fibroblast growth factor-23 (FGF-23)] and to examine the association between these biochemical measures and the cardiovascular end-points.
MATERIALS AND METHODS
Male Sprague-Dawley rats (Charles River, St. Constant, Quebec, Canada) weighing 375–400 g at the start of the experiment were individually housed in standard polypropylene cages and maintained on a 12-h light--dark cycle (lights on at 0700 h). Animals were allowed to acclimatize for at least 1 week prior to experimentation and were provided with Purina Rat Chow (test diet) and water ad libitum. All procedures were in accordance with the guidelines of the Canadian Council on Animal Care, handling and termination.
Rats were maintained on a specially formulated and nutritionally balanced diet (Harlan, Teklad, Madison, Wisconsin, USA). This specially formulated diet contains 0.25% adenine, 1% phosphate, 1% calcium, 0.2 ppm vitamin K, 1 IU/g vitamin D and 6% protein. Diets that contain 6% protein have been demonstrated to have no significant adverse impact on growth . The reduced adenine concentration of 0.25% was selected in order to increase palatability and prevent weight loss. Body weights and food intake were monitored on a daily basis. In a few instances, animals were supplemented with a few pellets of normal chow if their weight loss reached at least 10%. Ten rats received normal rat chow (the control group), whereas the remaining animals received the 0.25% adenine diet for 5 weeks (n = 6), 8 weeks (n = 7) and 11 weeks (n = 9). These time points were chosen to allow us to characterize a temporal relationship of adenine feeding, CKD and vascular calcification development. At each time point, blood pressure and PWV were determined under anaesthesia (ketamine 100 mg/kg and xylazine 25 mg/kg) after which blood was collected from the inferior vena cava and spun (at 4°C, 4000g, 20 min). The heart was then excised, and the right ventricle was carefully separated from the left ventricle and the septum; left ventricle index (LVI) and right ventricle index (RVI) were determined as the ratio of left or right ventricle weight to body weight. Vessels were collected and carefully cleaned of extraneous connective tissue. A 5-mm section of the thoracic aorta, greater than 1 cm inferior to the arch, was kept for histology, and the remaining aorta was snap frozen in liquid nitrogen and kept at −80°C for further analysis.
Serum creatinine, urea, calcium and phosphorous levels were determined using a Roche Modular (Hytachi) in the Clinical Chemistry Core Laboratory (Department of Pathology and Molecular Medicine, Kingston General Hospital, Kingston, Ontario, Canada). Serum FGF-23 levels were measured using an FGF-23 enzyme-linked immunosorbent assay kit, according to the manufacturer's instructions (Kainos Laboratories, Inc., Tokyo, Japan).
Aortic calcium and phosphorous content
Frozen iliac, renal, abdominal aorta, thoracic aorta and carotid vessels were thawed, weighed and homogenized in 0.6 N hydrochloric acid for 24 h at 4°C. Samples were spun, and calcium content was determined colorimetrically using the O-cresolphthalein complexone method (Sigma, St Louis, Missouri, USA). O-cresolphthalein colour reagent forms a purple complex with the calcium in the samples. The absorbance for this complex was measured for both standards and tissue homogenates at 540 nm (SynergyHT Microplate Reader; Bio-Tek Instruments Inc, Winooski, Vermont, USA). Phosphate content was determined using the malachite green method. The malachite green reagent was prepared as previously described . Upon addition of ammonium molybdate, a green complex is formed between malachite green, molybdate and free phosphate. The absorbance for this complex was measured for both standards and tissue homogenates at 650 nm.
von Kossa method for visualizing vascular calcification
Five-millimetre sections of thoracic aorta were fixed in 10x neutral phosphate-buffered saline with 4% paraformaldehyde overnight. Sections were then embedded in paraffin blocks in the upright position to ensure that each aortic section could produce an average of five to six cross-sections (minimum three). Sections (3–4 μm) were stained for calcification using the von Kossa method. Briefly, sections were first deparaffinized, rehydrated in distilled water, treated with 1% silver nitrate and exposed to ultraviolet light for 20 min.
Then, sections were placed in 5% sodium thiosulfate for 2 min and counterstained with nuclear Fast Red for 5 min. Areas of calcification appeared as dark brown regions in the medial wall of the aorta.
Six rats in the 11-week group were implanted with a radiotelemetric pressure transducer (model TA11PA-C40; Data Sciences Inc., St Paul, Minnesota, USA) under isoflurane anaesthesia as described previously  and allowed to recover and acclimatize for 2 weeks prior to dietary adenine treatment. SBP, DBP, pulse pressure (PP), heart rate (HR) and activity were determined from data collected every 4 min (30 s, 150 Hz) by a digital radio signal received by units under each cage (model RA1010, RA1020, or RPC-1; Data Sciences Inc.) and transferred by a consolidation matrix (BCM100, Data Sciences Inc.) to the data acquisition system (Dataquest LabPRO or Dataquest ART, Data Sciences Inc.). Given that rats are nocturnal, daily pressure averages were pooled from data collected from 8 p.m. to 5 a.m. to reduce the impact of confounding factors that might increase the animals’ stress such as daily cage changes and typical animal handling during work hours.
PWV was assessed using the foot-to-foot method, as previously described . Specifically, the method used determines the time for an aortic pulse to travel from the carotid artery to the iliac bifurcation. Two catheters were inserted at the superior (i.e. carotid) and inferior (i.e. femoral) ends of the aorta, and were used to measure blood pressure simultaneously. Blood pressure was recorded as a pulsatile waveform at a frequency of 1000 Hz. The distance from the tip of the carotid catheter to the iliac catheter was measured. PWV was calculated using the following formula: PWV = propagation distance / propagation time (m/s) at a pressure between 80 and 90 mmHg. At least 10 normal and consecutive waveforms were individually analysed and averaged. Only three out of the seven animals at 8 weeks survived the full PWV procedure. However, SBP, DBP, as well as PP from all animals were determined from the carotid catheter, and calculated using Chart version 5 software (ADInstruments, Colorado Springs, Colorado, USA). The six animals with radiotelemetry also had their PWV measured using this method.
A stepwise linear regression analysis was performed on the following parameters: creatinine, urea, phosphorous, calcium, FGF-23, tissue calcium and phosphorus, heart weight and PP. All data from control and CKD rats were presented as means ± SD. Differences between groups were analysed using a one-way analysis of variance (ANOVA) followed by a Newman--Keuls posthoc test. A P value less than 0.05 was considered statistically significant. Statistical analyses were performed on GraphPad Prism version 5 (GraphPad Software, Inc., San Diego, California, USA).
Effects of 0.25% dietary adenine on chronic kidney disease progression
Animals on the adenine diet (0.25%) lost 2.3 ± 2.4, 12.2 ± 1.6 and 9.4 ± 2.6% of their initial weight after 5, 8, and 11 weeks, respectively (Table 1). All animals developed CKD after 5 weeks of dietary exposure to 0.25% adenine. Serum creatinine was significantly elevated (>4.5-fold) at 5 weeks of CKD over control and remained elevated at 8 (5.5-fold) and 11 weeks (5-fold) of CKD (Table 1). Serum urea was also significantly elevated at 8 weeks of CKD (4.4-fold) and remained elevated at 11 weeks of CKD (4.8-fold). Although serum phosphate was increased two-fold over control animals at week 5 and week 8 of CKD, this was not sustained at 11 weeks (Table 1). FGF-23 was significantly elevated at all three time-points (13-fold, 122-fold and 63-fold increase at 5, 8 and 11 weeks, respectively). In contrast, serum calcium did not differ between controls or adenine-fed rats at any duration of CKD (Table 1).
Effects of 0.25% dietary adenine on vascular calcification
Calcification was determined by tissue calcium and phosphate content as well as by von Kossa staining. A vessel was considered calcified if it contained levels of calcium that were greater than 3 SD above control vessel values. As demonstrated in the histological sections (Fig. 1a--d), calcification was localized to the media of the artery. The relationship between tissue calcium and tissue phosphate content was strongly correlated (r 2 = 0.97, P < 0.05), and the calcium:phosphate ratio was 1.5 ± 0.1.
In the 5-week group (n = 6), two animals had elevated levels of calcium in the iliac artery, but only one of these animals also had elevated levels of calcium in their other vessels (thoracic aorta, carotid, abdominal aorta and renal artery) (Fig. 2a). In the 8-week (n = 7) and 11-week groups (n = 9), a majority of animals had elevated levels of calcium in all vessels; however, at both time points, the thoracic aorta and carotid artery were the least likely to be calcified compared with the more distal vascular beds (iliac, renal, abdominal aorta) (Fig. 2b and c). That is, all animals with thoracic and carotid calcification (i.e. 57 and 78% of the animals at 8 and 11 weeks, respectively) also had abdominal, renal and iliac artery calcification.
Impact of 0.25% dietary adenine on blood pressure
Blood pressure was analysed by radiotelemetry in conscious animals exposed to 11 weeks of 0.25% dietary adenine. PP measured over a 24-h time period was first significantly elevated at 8 weeks (42.9 ± 4.2--51.0 ± 4.7 mmHg, P < 0.05) and then progressively increased through to 11 weeks (59.0 ± 4.7mmHg) (Fig. 3). Changes in PP were largely driven by a significant drop in DBP rather than by changes in SBP (Fig. 4a). DBP was significantly lower at weeks 9, 10 and 11 than all other weeks (−7.9 ± 4.4, −15.9 ± 3.8 and −11.1 ± 2.4 mmHg, respectively, P < 0.05). Although there was no change in mean SBP at weeks 9–11 compared with earlier weeks, SBP demonstrated significantly greater variability from week 9 to week 11 than all other weeks (Fig. 4b). Variability in DBP was only significantly greater at week 11 (Fig. 4a). There was a general downward trend in HR, HR variability and activity with age. Specifically, HR fell between 4 and 11 weeks (from 380.1 ± 3.6 to 323.2 ± 4.0 bpm, P < 0.001) in telemetry animals in a manner consistent with the reduction in activity (from 4.3 ± 0.4 to 1.2 ± 0.1 counts/min) [↓HR was significantly correlated with ↓activity, r 2 = 0.77]. HR variability fell between 4 and 8 weeks (from 36.8 ± 1.5 to 27.7 ± 2.0, P < 0.0001), but did not change further in the last 3 weeks (24.8 ± 1.3 at 11 weeks).
Impact of 0.25% dietary adenine on haemodynamic consequences
Blood pressure was analysed in all rats under anaesthesia via carotid catheters at 5, 8 and 11-week time points. Anaesthetized blood pressure recording demonstrated elevated SBP in the 8 and 11-week groups, and decreased DBP in the 11-week group (Fig. 5a and b). Although PP was elevated in the 8-week group, it was only significantly elevated above control in the 11-week group (Fig. 5c). Blood pressure had an impact on PWV measurements. That is, in animals without calcification (regardless of age or CKD status), there was a positive linear relationship between blood pressure [DBP, SBP and mean arterial pressure (MAP)] and PWV. In contrast, in animals with calcification, this relationship was lacking (i.e. less than one-third of the slope of control animals). Nonetheless, PWV was found to be increased only in CKD animals with thoracic calcification at 5 (n = 1), 8 (n = 3) and 11 (n = 7) weeks (Fig. 6).
The severity of CKD (determined by serum creatinine level) predicted the LVI (r 2 = 0.38, P = 0.0001), but not the RVI (r 2 = 0.08). LVI was also linked with the duration of adenine feeding and was elevated the most at 11 weeks at which point all animals had vascular calcification (Fig. 7). A stepwise linear regression of LVI with haemodynamic variables (i.e. PP, DBP, SBP, MAP and PWV), tissue calcium and phosphate, and serum calcium, phosphate, and log (FGF-23) revealed that LVI was highly correlated with increased FGF-23 and PP (Fig. 8a and b). All other variables either demonstrated a low or insignificant correlation with LVI.
The present study establishes that lowering the dietary adenine concentration to 0.25% produces stable CKD at 5, 8 and 11 weeks but without severe weight loss; extending the duration of adenine feeding from 5 to 11 weeks increases the propensity for vascular calcification and that distal vessels appear more susceptible to vascular calcification; medial calcification in this model has a unique haemodynamic profile, which includes increased PP, lower DBP and increased variability in SBP; and abnormalities in phosphorus homeostasis, reflected by elevated levels of FGF-23, are associated with left ventricular hypertrophy (LVH).
The severity of CKD was found to be robust in that all animals demonstrated a greater than 3.5-fold increase in creatinine levels after 5 weeks of adenine treatment. Furthermore, creatinine levels appeared to be relatively stable over time, such that between weeks 8 and 11, creatinine levels remained approximately five-fold elevated over control animals. The frequency of vascular calcification increased over time; that is, at the 5-week time point, 33% of rats exhibited vascular calcification, but at the 11-week time point, 100% of animals were calcified. Alterations in haemodynamic parameters were associated with the progression of CKD, as well as the onset and progression of vascular calcification. There was a progressive increase in left ventricular mass that correlated with the duration of CKD, associated biochemical changes (e.g. ↑FGF-23) and haemodynamic alterations (↑PP). Lastly, our findings suggest that there is a likely link between progression of calcification, haemodynamic changes and changes in cardiac structure and function.
Vessels distal from the heart appeared to be most susceptible to vascular calcification, as calcification in these vessels consistently preceded that in the thoracic aorta and carotid artery. All vessels were exposed to identical CKD conditions and prevailing concentrations of phosphorus. That is, the exposure of VSMCs to the calcifying risk factors associated with the CKD environment would be the same. Therefore, these data suggest that there may be regional differences in structural components of the vessel or in the local expression of key inhibitors of vascular calcification, such as matrix Gla protein (MGP), which impact upon susceptibility to vascular calcification. One possible explanation could relate to the fact that VSMCs in different arterial regions develop from different embryonic origins. Recently, Leroux-Berger et al.  in a series of in-vitro and in-vivo experiments using aortas from MGP knockout mice demonstrated that susceptibility to calcification differed between the different regions of the aorta. Regional susceptibility to vascular calcification has not been well studied in the CKD population; most studies only report coronary or thoracic calcification. However, one study  recently showed in obese patients that abdominal and iliac arteries were more frequently calcified than were thoracic aorta or carotid arteries.
Duration of CKD was linked to haemodynamic alterations within the circulation, although the greatest circulatory changes occurred in animals that also had thoracic calcification. That is, most of the circulatory changes were significant at 11 weeks (↑SBP, ↓DBP, ↑PP), reflecting the progression of calcification. Furthermore, the finding that PWV was only increased in animals with calcification of the thoracic aorta demonstrates the importance of calcification in determining vascular stiffness. These findings are consistent with cardiovascular complications reported in CKD patients. For example, CKD patients develop systolic hypertension and elevated PWV, both of which have been associated with vascular calcification [21–24].
In contrast to blood pressure assessments under anaesthesia, the use of radiotelemetry eliminates the confounding influence of anaesthetic agents on the circulation and allows for the examination of the temporal component of haemodynamic alterations in CKD rats. All of the animals implanted with radiotelemetry for 11 weeks developed vascular calcification. The initial and accelerated change in PP occurred at week 8 and was progressive through to week 11. It was unexpected that the sudden rise in PP was associated with a drop in DBP, rather than a rise in SBP. Although mean SBP did not change throughout the 11 weeks study period, variability markedly increased between 9 and 11 weeks. Blood pressure variability has been consistently associated with end-organ damage in humans [25,26], but has never been studied in the context of vascular calcification in CKD. Although most previous work has considered the effect of 24-h blood pressure variability, here we show chronic SBP variability in the awake hours (i.e. variability was not due to day/night dipping effect), which may be a better marker of LVH and vascular remodelling . The increase in SBP variability could be an indication of reduced compliance as well as stiffness. As the changes in HR and HR variability did not account for the changes in blood pressure, the distinctive pressure profile likely resulted from alterations in the mechanical properties (i.e. compliance) of the large elastic arteries due to medial wall calcification. Evidence for altered vascular properties in vascular calcification has previously been well established by Sutliff et al. .
The observed changes in arterial compliance are likely a major contributing factor to the increased left ventricular weight observed in the CKD animals. That is, the increased PWV and PP may be the physical stimulus contributing to LVH . However, in this model, the presence of LVH is not fully explained by calcification-associated abnormalities generated within the circulation, and we found an interesting link with FGF-23 levels. There is previous evidence linking FGF-23 with mortality and LVH in humans with and without CKD. It has been hypothesized that FGFs, including FGF-23, may stimulate myocardial cell growth and fibrosis via activation of FGF receptors, which are expressed in adult myocardial cells. In the present study, we found an association between FGF-23 concentrations and LVH in CKD animals regardless of their calcification status. It is plausible that markedly elevated levels of FGF-23 in CKD may nonselectively activate FGF receptors in the heart, and thereby mediate myocardial cell enlargement and fibrosis; however, further studies are needed.
Clinical studies [30–32] have shown that elevated serum phosphate levels in CKD are associated with coronary artery calcification, elevated FGF-23 and mortality. CKD rats had an elevated serum phosphate at 5 and 8 weeks, but not at 11 weeks, whereas serum FGF-23 remained elevated throughout the study. Although phosphate is believed to be a key signalling molecule in the development of vascular calcification, an isolated serum phosphate value per se in the late-stage CKD animals did not predict this event. Other experimental models have also shown minimal or no elevations in serum phosphate, yet these animals also developed vascular calcification . The observed decline in serum phosphate at 11 weeks could be due to multiple factors such as decreased food consumption related to deterioration of animal health, although increased tissue uptake of phosphate could also have occurred . FGF-23 may therefore represent a more robust marker of the duration and severity of one of the key stimuli of vascular calcification and cardiovascular health in this model .
In conclusion, the experimental approach of the present study attempted to model the development of CVD in the setting of a modified adenine CKD approach, which minimizes weight loss. Our findings reveal that there is regional susceptibility to calcification and that a distinctive haemodynamic profile appears to be linked with its development. Finally, using this modified adenine-induced model of stable CKD, we established that prolonged elevation of FGF-23 was a better sentinel of both the positive phosphate balance (which impacts calcification) and its negative impact on cardiac structure than was the transient hyperphosphatemia.
The authors acknowledge the financial support provided by the Heart & Stroke Foundation of Ontario, the Kidney Foundation of Canada and Canadian Institute of Health Research and Amgen. Authors would like to thank the contributions of David Beseau and Dr Steven Pang for their involvement with assays and histology, respectively.
The whole or part of the work presented in the article has been presented as oral or poster presentations at the following meetings: American Society of Nephrology, International Society of Hypertension, Experimental Biology, Canadian Society of Nephrology, Ontario Hypertension Society, Canadian Hypertension Society and 63rd High Blood Pressure Research Conference (American Heart Association).
Conflicts of interest
There are no conflicts of interest.
Reviewers’ Summary Evaluations Referee 1
This paper describes evolution of vascular calcification in renal failure induced by dietary adenine in rats. A calcification in distal arteries was associated with increased BP variability and a widening of the pulse pressure which correlated with LV hypertrophy. Author's suggestion that the adenine ingestion mimics human renal failure and that the findings in rats reflected hemodynamic alterations are highly speculative. Weight loss and early deaths suggest severe adenine toxicity in rats. It is not clear whether quick calcifications in animals are caused by hemodynamic factors and it is doubtful that the experimental model replicates the slow calcification in humans.
Using experimental chronic kidney disease (CKD) in rats the study demonstrated that the development of medial vascular calcification was accompanied by widening of the pulse pressure driven by the diastolic BP drop, PWV increase in calcified thoracic aorta and positive association between LVH with both FGF-23 level and pulse pressure. This integrated effect of vascular calcification appears to have good potential for explaining cardiovascular complications reported in CKD patients. However, the observed associations did not identify the stimuli contributing to the development of LVH, which may require additional measures for structural variations in the cardiovascular system that accompany the calcification process.
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Keywords:© 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins
adenine; chronic kidney disease; fibroblast growth factor-23; hyperphosphatemia; left ventricular hypertrophy; pulse pressure; systolic variability; vascular calcification