Background
Vascular calcification (VC) is prevalent and well described in large- and medium-sized vessels of patients with chronic kidney disease (CKD), especially in those with end-stage kidney disease (ESKD) on dialysis [
1,
2]. Patients with CKD are at higher risk of VC compared to the general population, irrespective of age and co-morbidity burden [
3]. Medial calcification is particularly prevalent in the CKD population and is associated with arterial stiffening and higher cardiovascular morbidity and mortality [
4,
5]. Medial arterial calcification has been identified in multiple anatomical locations in patients with ESKD, including normal breast tissue from mastectomy samples [
6], as well as subcutaneous tissue of lower limb amputations [
7]. In both locations, VC has been identified in vessels in the presence of other patient risk factors for calcification, including active malignancy or peripheral vascular disease.
Conversely, the presence of VC of small vessels in the skin and subcutaneous tissue has only been systemically described in ESKD patients with calciphylaxis [
8], although recent studies suggest even this may not be a specific finding and VC can be present in patients with peripheral vascular disease without the presence of calciphylaxis [
7]. Calciphylaxis is considered an accelerated template of widespread VC. It is characterized by severe vessel ischaemia accompanied by vessel thrombosis and surrounding panniculitis [
9], ultimately resulting in painful skin lesions which typically break down to non-healing ulcers that are prone to infection, with accompanying high mortality [
10,
11]. However, calciphylaxis is a rare condition and only occurs in a very small minority of patients with ESKD with approximately 3.5 cases per 1000 patient years in those receiving chronic haemodialysis therapy [
12] To date, there is little information about the prevalence of calcification in skin and subcutaneous tissue in patients with advanced CKD and ESKD outside the setting of calciphylaxis, and if present, whether it is related to disordered bone and mineral metabolism.
The aim of the present study was to characterize VC changes in the skin and subcutaneous tissue of patients with CKD compared to a control group. We also aimed to evaluate associations between skin calcification and biochemical markers of deranged bone and mineral metabolism and identify any other histological features present that are typically described in calciphylaxis.
Methods
Patients
Between May 2017 and March 2019 patients undergoing a planned surgical procedure performed by the Nephrology Surgical Team at The Royal Melbourne Hospital were approached to participate in this single-centre cross-sectional study. Patients enrolled underwent elective surgical procedures including parathyroidectomy, hernia repair and arteriovenous fistula (AVF) formation. A fasting blood sample was collected at the time of surgical procedure. Patients over the age of 18 and able to provide informed consent were included in the study. There were no exclusion criteria.
Ethics approval and consent to participate
The study was approved by the Melbourne Health Human Research Ethics Committee (#HREC 2017.023) and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained for this study.
Histology
A full-thickness incisional skin sample was collected by the operating surgeon from the surgical site. Tissue samples were fixed in neutral buffered formalin and processed to paraffin wax for histology. Serial paraffin embedded skin sections were stained with haematoxylin and eosin (H&E) and von Kossa. Cross-sections of skin and subcutaneous tissue were evaluated in their entirety.
Counting of vessels was performed on slides stained with H&E due to ease of identifying vessels. Each cross section contained epidermal, dermal and subcutaneous tissue, with tissue blocks re-cut if they did not contain all three areas. Vessel size was estimated using a graticule. Typically, arterioles ≤50 μm in diameter have either absent or partially complete internal elastic lamina (IEL) [
13,
14]
. As a result, differentiating terminal arterioles from terminal venules is very challenging, therefore all structures with a lumen were defined as vessels. Samples with greater than 10 vessels per cross section were considered to have adequate vessel number. H&E sections were examined for evidence of intimal hyperplasia, intimal thrombosis, pannicultis or hyperplasia.
Identification of calcification was performed on slides stained with von Kossa. In brief, sections were dewaxed and rehydrated before being immersed in a solution of freshly prepared 5% Aqueous Silver Nitrate. Tissue sections were placed at close range under a bench lamp and exposed to a strong light source for 30 min. They were then washed in distilled water. To remove the unreacted silver, slides were covered with 5% Sodium Thiosulphate for 2 minutes. This was rinsed in distilled water before being counter stained with Working Eosin solution for 2 minutes. For each case, the following features were graded as either present or absent: calcification of any sized vessels, presence of extra-vascular calcification, IEL calcification. Results were expressed as a % of all vessels in the section. Histologic sections of all cases were counted independently by two investigators (IR and BW) with results presented as an average of the two observers. Malignant necrotic breast tissue was used as a positive control for the von Kossa stain.
Real-time reverse transcriptase polymerase chain reaction (RT-PCR)
Total RNA was isolated from a subgroup (13 out of 43) of patient samples stored in RNAlater solution (Invitrogen, Carlsbad, CA, USA) using the RNEeasy Fibrous Mini Kit (QIAGEN, Hilden, Germany) in accordance with manufacturer’s instructions. To minimise sampling error, triplicate 30 mg portions of tissue from each sample were processed. Quantification of RNA was performed using the Qubit 4 Fluorometer and Quant-IT RNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction with 1 μg used as a template for cDNA synthesis using the iScript RT supermix kit (BioRad, Hercules, CA, USA). Quantitative real-time PCR (qRT-PCR) was performed in triplicate in a CFX96 cycler (Bio-Rad) using equal volumes of cDNA (2 μL) and the SsoAdvanced™ Universal SYBR Green Supermix (Bio-Rad) and validated PCR Prime assay primer pairs (Bio-Rad): TNAP (Unique Assay ID:qHsaCID0010031); Runx2 (Unique Assay ID: qHsaCID006726); and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (Unique Assay ID: qHsaCED0038674). PCR conditions were set according to the manufacturer’s instructions. Melt curve analysis was performed to verify the purity and specificity of the amplicons. Threshold cycles were calculated using CFX Manager Software (Bio-Rad). The mRNA level of target genes was normalised to the house keeping gene Gapdh, and expressed relative to an appropriate control using the 2^(−ΔΔCt) method. Results are presented as the mean of all three samples.
Statistical analysis
A sample size estimation for this cross-sectional study was 50 patients based on feasibility with the number of patients undergoing elective surgery at our institution over the recruitment period as well as a larger enough cohort to allow for heterogeneity of skin sample sites and different stages of CKD. All data were summarised, and results reported as mean (standard deviation [SD]) or median (inter-quartile range [IQR]) for continuous data and number (percentage, %) for categorical variables. Comparison of the values of continuous variables between groups was made using an unpaired t test or Wilcoxon signed-rank test as appropriate. Chi-squared and Fisher’s exact tests were used to investigate associations between various categorical variables. Two-tailed P values of < 0.05 were considered to be significant. All statistical analyses were performed using SPSS version 21.0 for Macintosh (SPPS, Chicago, IL). Graphics were created with GraphPad Prism 8 for Macintosh (la Jolla, CA, USA).
Discussion
To our knowledge, this is the first study to show evidence of medial VC in subcutaneous and dermal vessels in multiple anatomical locations in patients with advanced CKD and ESKD undergoing elective surgery, without peripheral vascular disease or calciphylaxis. Calcification was present in 38% of samples from patients with CKD/ESKD, predominantly in the subcutaneous tissue and involving the vessel media as well as the IEL. Perieccrine calcification was identified in 64% of samples with VC, present mainly in patients with ESKD on dialysis. These microvascular calcifications were not related to biochemical markers of mineral metabolism or changes in pro-calcific gene transcripts.
Von Kossa staining is routinely used to identify tissue calcification [
2]. Using this stain, two distinct types of VC were identified in our skin samples, medial calcification and calcification of the IEL. The medial calcification identified in patients with CKD/ESKD was mostly granular but becoming confluent in some of the affected vessels. Calcification identified in the two control patients was more diffuse and milder in nature, which may represent a less advanced stage of VC progression or perhaps a different pathophysiological process. There was no evidence of intimal calcification in our study and there remains conjecture as to whether medial VC and calcification of the IEL represent progression of the same disease process. Multiple studies have shown that arterial medial calcification typically starts along the IEL and in the absence of inflammation [
16,
17]. In contrast, arterial intimal calcification is characterized by focal mineral deposition in highly inflamed and necrotic atherosclerotic lesions.
The high prevalence of perieccrine calcification in patients with VC is a novel finding in our study. There is little literature regarding this histological entity and its clinical significance. Mochel et al. [
8] initially described perieccrine calcification in a retrospective cohort of patients with calciphylaxis, suggesting that this histological feature was highly specific to calciphylaxis - the diagnosis of calciphylaxis being made based on this histological feature alone in 4 out of 56 cases. It was also identified in a case report of calciphylaxis [
18]. In a recent case series of patients with calciphylaxis (13), perieccrine calcification was not present, however this may have been due to lack of specific staining for calcification. The authors argued that given the subtleness of this histological feature, it may be possible it is routinely missed with standard H&E staining. In further case series, perieccrine calcification was identified in two biopsies in patients with low to moderate risk of calciphylaxis, and was not significantly prevalent in biopsies of patients with high clinical suspicion of calciphylaxis [
7]. The clinical significance of perieccrine calcification remains unknown and it may simply reflect the severity or progression of co-existent VC.
Calcification of skin tissue is considered to be a precursor to the development of calciphylaxis and is a common finding in skin biopsies in patients with this pathology [
8]. The specificity of this finding was recently questioned by Ellis and O’Neill in a study of 38 skin biopsies taken for high clinical suspicion for calciphylaxis and compared with lower limb amputation skin samples in patients with ESKD [
7]. All previously documented histopathological features commonly associated with calciphylaxis, including medial calcification, intimal thrombosis and extravascular calcification, were prevalent in both high-risk skin lesions and amputation samples. Only the combination of thrombosis and medial calcification was unique to the high-risk skin biopsy group. Our study confirms that medial vascular calcification and perieccrine calcification can be present in “healthy skin” taken from three different anatomical locations, and that these features indeed are not specific to calciphylaxis. We could not identify any evidence of vessel thrombosis, likely due to the low incidence of peripheral vascular disease in our CKD/ESKD cohort. This histological finding may be more prevalent in patients with significant peripheral vascular disease rather than specific to CKD alone.
There were no significant demographic or biochemical differences in patients with CKD/ESKD with and without VC. PTH in patients with VC was similar to those without, but highly variable perhaps reflecting the variability and fluctuations in PTH, as the samples used here only capture one time point. We also identified no significant differences in serum calcium, phosphate or ALP between the two groups. However, the apparent lack of discriminating factors between the two groups may in part reflect the small sample size. It is recognised that medial VC is accelerated in patients with CKD [
19]. Schlieper et al identified microcalcifications in the media of iliac arteries in uraemic patients [
20] without the presence of intimal calcification or major atherosclerotic plaques. Consistent with our results, the presence of calcification was not associated with abnormalities in serum calcium, phosphate or magnesium, although PTH was not evaluated. Despite these similarities, it is uncertain to what extent VC in skin and subcutaneous tissue reflects calcification in other vascular beds and whether it is related to more systemic pro-calcific processes affecting the larger central arteries.
RUNX2, a transcription factor, is a master regulator of bone formation and is essential for bone formation and bone remodelling. Some animal studies have shown that RUNX2 expression is required for osteogenic phenotypic change in smooth muscle cells [
21]. This transcription factor is also reportedly upregulated in human vascular fragments of large vascular territories where calcification is present [
22]. However, evidence of osteogenesis is not a consistent finding across all studies of human VC. We did not see upregulation of
RUNX2 or
TNAP in the subset of patients tested with histological evidence of calcification. Although osteogenic transformation of vascular smooth muscle cells is one proposed hypothesis for development of medial arterial calcification, other mechanisms have been implicated including elastin degradation, smooth muscle apoptosis and more passive biochemical processes [
23]. Lack of osteogenic gene upregulation in our study may also reflect temporal aspects of calcification [
6,
24] or a different underlying mechanism as a culprit. Human studies involving examination of arterial calcification in breast tissue in patients with CKD demonstrated a universal absence of staining for RUNX2 and osteocalcin in early arterial calcification [
6]. A previous study identifying upregulation of osteogenic gene expression in skin was in the setting of calciphylaxis with macroscopic evidence of calcification [
15]. This is not reflective of the subtle microscopic calcification identified in our participant cohort that is likely to occur in response to distinct triggers and mechanisms. On the other hand, this may be as a result of technical factors relating to the small sample size, or the sporadic nature of calcification identified on histological examination. Finally, given only a small amount of tissue was processed for PCR, small areas of active calcification may have been missed.
This study was limited by a small sample size and its cross-sectional design with a single skin incisional sample at one time point, without the ability to prospectively follow up VC progression, resolution or patient outcomes. It was not possible to obtain skin samples from control patients to match all anatomical locations and a future case-controlled study may better evaluate the impact of anatomical site on the presence of calcification. Assessment of VC burden at more central sites was also not undertaken. This study had many strengths, however, including a wide distribution of skin anatomical locations sampled as well large incisional biopsy sampling, which allowed for good interpretation of skin architecture and large number of vessels (mean of 40 vessels per slide). Also, patients had no underlying skin pathology to confound results of the study including presence of skin malignancy which is commonly associated with VC.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.