Novel insights into osteogenesis and matrix remodelling associated ...

Nephrol Dial Transplant (2013) 28: 856–868doi: 10.1093/ndt/gfs466Advance Access publication 6 December 2012

Novel insights into osteogenesis and matrix remodellingassociated with calcific uraemic arteriolopathy

Rafael Kramann1,2*,

Vincent M. Brandenburg3,*,

Leon J. Schurgers4,

Markus Ketteler5,

Saskia Westphal2,

Isabelle Leisten2,

Manfred Bovi2,

Willi Jahnen-Dechent6,

Ruth Knüchel2,

Jürgen Floege1

and Rebekka K. Schneider2

1Division of Nephrology and Clinical Immunology, RWTH Aachen

University, Aachen, Germany,2Institute of Pathology, RWTH Aachen University, Aachen,

Germany,3Department of Cardiology, RWTH Aachen University, Aachen,

Germany,4Department of Biochemistry, Cardiovascular Research Institute

Maastricht, Maastricht University, Maastricht, The Netherlands,5Department of Medicine III, Division of Nephrology, Klinikum

Coburg, Coburg, Germany and6Institute for Biomedical Engineering, Biointerface Laboratory,

Aachen, Germany

Correspondence and offprint requests to: RafaelKramann; E-mail: [email protected]

*R.K. and V.M.B. are contributed equally to this work.

Keywords: bone morphogenic protein 2, calciphylaxis, matrixGla protein, matrix remodelling, sclerostin

ABSTRACT

Background. Calcific uraemic arteriolopathy (CUA) or calci-phylaxis is a rare, life-threatening disease predominantly oc-curring in patients with end-stage renal disease. Itspathogenesis has been suggested to include ectopic osteogen-esis in soft tissue and the vasculature associated with extra-cellular matrix (ECM) remodelling.Methods. To gain further insights into the pathogenesis ofCUA, we performed systematic analyses of skin specimensobtained from seven CUA patients including histology, im-munohistochemistry, electron microscopy, electron dispersiveX-ray analysis (EDX) and quantitative real-time RT-PCR.Skin specimens of (i) seven patients without chronic kidneydisease and without CUA and (ii) seven dialysis patientswithout CUA served as controls.Results. In the CUA skin lesions, we observed a significantupregulation of bone morphogenic protein 2 (BMP-2), itstarget gene Runx2 and its indirect antagonist sclerostin. Fur-thermore, we detected an increased expression of inactive un-carboxylated matrix Gla protein (Glu-MGP). The upregulation

of osteogenesis-associated markers was accompanied by an in-creased expression of osteopontin, fibronectin, laminin andcollagen I indicating an extensive remodelling of the subcu-taneous ECM. EDX analysis revealed calcium/phosphateaccumulations in the subcutis of all CUA patients with amolar ratio of 1.68 ± 0.06 matching that of hydroxyapatitemineral. Widespread media calcification in cutaneous arterioleswas associated with destruction of the endothelial layer andpartial exfoliation of the endothelial cells (ECs). CD31 immu-nostaining revealed aggregates of ECs contributing to intra-luminal obstruction and consecutive malperfusion resulting inthe clinical picture of ulcerative necrosis in all seven patients.Conclusions. Our data indicate that CUA is an active osteo-genic process including the upregulation of BMP-2 signalling,hydroxyapatite deposition and extensive matrix remodellingof the subcutis.

INTRODUCTION

Calcific uraemic arteriolopathy (CUA) or calciphylaxis is arare, life-threatening disease predominantly affecting patients

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with end-stage renal disease (ESRD) or chronic kidneydisease (CKD). It is characterized by media calcification ofsmall- and medium-sized cutaneous vessels, soft-tissue calci-fication and clinically by progressive painful skin indurationswith ulcerations [1, 2]. The term calciphylaxis was introducedby Hans Selye in the early 1960s [3] based on a rodent modelof systemic and local soft-tissue calcifications. Selye postu-lated a two-step hypothesis with sensitization factors (i.e. hy-perparathyreoidism, vitamin D or a diet high in calcium andphosphorus) followed by challenging factors (i.e. localtrauma, or the application of iron salt, polymycin, eggalbumin or glucocorticoids). Subsequently, a similar syn-drome with peripheral ischaemic tissue necrosis and vascularcalcification was reported in a woman with acute renal failureand this was also named calciphylaxis [4]. However, theexperimental model of Selye was free of vascular calcificationand, therefore, the human disease is more appropriately de-scribed by the term CUA [5]. The prevalence of CUA isnowadays estimated to range from less than 1–4% amongESRD patients [1, 6, 7].

The pathogenesis of CUA remains incompletely under-stood. Multiple clinical risk factors such as female gender,diabetes, obesity, warfarin use, hyperphosphataemia, hyper-parathyroidism, the use of calcium-containing phosphatebinders and particularly the presence of severe CKD havebeen identified [8, 9].

Vascular calcification is now considered an active cell-mediated process similar to bone morphogenesis. Neverthe-less, both origins of the mineralizing cell population and in-itiating stimuli remain controversial. One possible stimulus isbone morphogenic protein 2 (BMP-2). It belongs to thetransforming growth factor-β superfamily and acts bybinding to its specific type II receptor followed by the acti-vation of the type I receptor [10]. BMP-2 function can be in-hibited by matrix Gla protein (MGP) [11], but only by theactive Gla-MGP [12]. MGP activation is vitamin K dependentwhich needs to be activated via gamma-carboxylation [13].Vitamin K antagonists such as warfarin inhibit the recyclingof vitamin K, thereby limiting the activation of MGP. [14].Vitamin K antagonist usage appears to be an independentrisk factor for the development of CUA [15]. If BMP-2 is notinhibited by MGP, the BMP-2 downstream signalling cascadeleads to an increased expression of the key osteogenic tran-scription factor Runx2 [16]. Runx2 controls the expression ofmatrix proteins of osteoblastic differentiation such as osteo-pontin and collagen I [17]. Sclerostin, the glycoproteinproduct of the SOST gene, is expressed by mature osteocytesand also antagonizes the effect of BMP-2 indirectly via inhi-bition of the Wnt/β-catenin signalling pathway [18, 19].

CUA can be regarded as a particular subtype of vascularcalcification. It has been hypothesized that CUA is, similar tovascular calcification in large arteries, an active process withosteoblast-like cells contributing to calcification [20]. Thus, incalcified skin lesions of CUA patients, increased expression ofosteopontin, suggesting an osteogenic process, was observed[20]. Besides osteogenesis, an altered extracellular matrix(ECM) with increased expression of matrix proteins such ascollagen I, osteopontin and fibronectin is important in

calcification processes [21, 22]. Clinically, hardening of thesoft tissue with a dense, plate-like aspect is a frequent earlyfinding in CUA patients, which proceeds later to ulcerations.And similarly, once soft-tissue ulcerations have developed,these lesions are often surrounded by areas of tissue indura-tion typically palpable as leather-like rigidifications of skinareas in association with calcified and necrotic lesions [1].

Our study aimed to gain further insights into mechanisticaspects of CUA development with a special focus on the in-terplay between altered ECM remodelling, osteogenesis,ectopic calcification and vascular obstruction finally leadingto tissue ulcerations in patients with CUA. We performed asystematic, detailed analysis of calcified skin specimens inseven CUA patients by histology, immunohistochemistry,electron microscopy, electron dispersive X-ray analysis (EDX)and quantitative real-time RT-PCR (Qt-RT-PCR). We par-ticularly investigated markers involved in osteogenesis suchas BMP2, Runx2, sclerostin and key proteins of the ECMsuch as collagen I, IV, fibronectin, osteopontin, MGP andlaminin.

MATERIALS AND METHODS

CUA Patients

Based on data collected in our calciphylaxis registry initiat-ive (International Collaborative Calciphylaxis Network), weretrospectively identified all patients who were treated for cal-ciphylaxis in the University Hospital Aachen since 2006 andfrom whom tissue samples were available (n = 7; two males/five females, age 50 ± 19 years, range 21–77). We examinedskin specimens obtained from consented clinical autopsies(n = 2) and operative resections (n = 5) of these seven CUApatients. The diagnosis of CUA of all patients was based onthe clinical picture and confirmed by histological analysis ofone or more skin sections. We used two different patientgroups as controls: (i) patients without CUA and withoutCKD (non-CKD controls) and (ii) dialysis patients withoutCUA (dialysis controls). (i) As non-CKD controls, wematched skin specimens of seven non-CKD, non-CUApatients for age, gender, the presence or the absence ofobesity and diabetes (n = 2 men, age 49 ± 18 years, range 25–76; diabetes in n = 4, obesity in n = 4). Specimens from non-CKD controls were obtained during consented clinical autop-sies (n = 2), and by surgical skin resections in n = 5. Thecause of death in all autopsy patients was septicaemia. Inautopsy cases, the skin samples were always resected within8 h after death, and significant signs of autolysis were ex-cluded. Due to our experience, early sampling of tissues ofdeceased subjects does not reduce the reliability of immuno-histochemical or EDX analysis and is also routinely appliedfor clinical autopsies. Only two autopsy cases were includedinto the PCR analysis (one in the CUA group and one in thenon-CKD control group. (ii) As dialysis controls, we includedskin samples obtained from dialysis patients without CUA(three males/four females, age 54 ± 10, range 45–71, diabetesin n = 4, obesity in n = 3, time on dialysis 4.2 ± 3.4 years).Specimens of dialysis control patients were obtained from

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two lower leg amputations and from five surgical resectionsof healthy skin after skin cancer (melanoma). All patientsand controls were of Caucasian ethnicity. Data about themedical history and medications were obtained by retrospec-tive chart review and obtained from the above-mentioned cal-ciphylaxis registry (www.calciphylaxie.de). The study wasapproved by the ethical committee of the RWTH AachenUniversity Hospital and carried out according to the prin-ciples of the Declaration of Helsinki.

Histomorphological analysis

For histological and immunohistochemical analyses, skinspecimens were fixed in 3.7% formaldehyde for 24 h. Skinspecimens were paraffin-embedded, cut with a rotating mi-crotome at 3 µm thickness (Leica) and stained according toroutine histology protocols. Immunohistochemical analysiswas performed in an autostainer (DAKO cytomation) usingprimary antibodies specific for fibronectin (mouse mono-clonal, 1:200; Sigma, Hamburg, Germany), collagen I (mousemonoclonal, 1:2000; Sigma), collagen IV (mouse monoclonal,1:250; Sigma), laminin (mouse monoclonal, 1:1000; Sigma),CD68 (mouse monoclonal, 1:400; DAKO, Hamburg,Germany), CD31 (mouse monoclonal, 1:500; DAKO), BMP-2 (rabbit polyclonal, 1:200; Abcam, Cambridge, UK), Glu-and Gla-MGP (mouse monoclonal, 1:200; Vascular Products,Maastricht, the Netherlands) and osteopontin (mouse mono-clonal, 1:3000; Santa Cruz Biotechnology, Heidelberg,Germany) as described before [23, 24]. The immunostainingfor sclerostin was performed manually. After blocking thesections for endogenous avidin/biotin (Avidin-Biotin Block-ing Kit, Vector Laboratories Burlingame, CA, USA) and per-oxidase activity (3% H2O2), slides were immediatelyincubated with a goat polyclonal anti-sclerostin antibody(1:200, R&D Systems; Minneapolis, MN, USA). Biotinylatedhorse monoclonal anti-goat antibody (Vector Laboratories)was used as a secondary antibody. Detection was realizedusing the Vectastin ABC Kit (Vector Laboratories).

Semi-quantitative immunohistochemical analysis

Protein expression and von Kossa staining were quantifiedby two blinded investigators (R.K. and R.K.S.) using a semi-quantitative scoring system (0, no expression; 1,weakexpression; 2 moderate expression; 3, strong expression; 4,very strong expression) in high-power fields (×200 magnifi-cation) as described before [25, 26]. The number of small ar-teries and arterioles with CD31+ endoluminal debris wasestimated by counting all arteries and arterioles containingendoluminal CD31+ cells in one section (magnification ×200)and dividing by all arteries and arterioles in the correspond-ing section.

Scanning electron microscopy

Tissue sections were fixed in 3% glutaraldehyde for at least24 h, rinsed with sodium phosphate buffer (0.2 M, pH 7.39,MERCK, Darmstadt, Germany) and dehydrated by incubat-ing consecutively in ascending acetone series (30, 50, 70 and90%) with a final incubation in 100% acetone for 10 min.The tissue sections were critical-point dried in liquid CO2.

Samples were analysed using an environmental scanning elec-tron microscope (ESEM XL 30 FEG, FEI, PHILIPS, Eindho-ven, the Netherlands) in a high vacuum environment. EDXanalysis was performed using an EDAX Falcon Genesis Spec-trum 5.21 energy-dispersive X-ray spectroscopy system withan ultrathin window liquid nitrogen cooled Si(Li) X-ray de-tector (EDAX Inc., Mahwah, NJ, USA).

Real-time reverse trancriptase polymerase chain reaction

Total RNA was isolated from the formalin-fixed, paraffin-embedded skin specimens using the RNeasy FFPE Kit(Qiagen, Hilden, Germany) according to the manufacturerinstructions. We achieved an isolation of RNA feasible forreal-time PCR from only four CUA patients (patient no. 1, 3,5, 6) due to the origin of RNA from formalin-fixed paraffin-embedded sections. We compared gene expression of thesefour patients with gene expression of n = 4 non-CUA non-CKD controls and n = 4 non-CUA dialysis controls. TheRNA concentration was determined by measuring absor-bance at 260 nm (Nanodrop, Thermo Scientific, Wilmington,USA). One microgram of RNA was reverse transcribed usingthe high capacity cDNA Reverse Transcriptase Kit (AppliedBiosystems, 7300 Real-Time PCR System, Foster City, CA,USA). Quantitative PCR reactions were carried out withPower SYBR Green PCR Master Mix (Applied Biosystems).For each sample, 1.2 µL of cDNA was added as a template inPCR reactions. Amplification was monitored with the ABIPrism 7300 (Applied Biosystems). The expression of genes ofinterest was normalized against the housekeeping gene gly-ceraldehyde-3-phosphate dehydrogenase in all samples. ThecDNA of the skin sections from non-CUA non-CKD controlpatients was used as a relative standard for the genes of inter-est and set as one for all genes. Relative gene expression wasanalysed with the 2− ΔΔCt method. Primers are listed in Sup-plementary data, Table S1.

Statistical analysis

Data are presented as mean ± standard deviation (SD).After testing for normal distribution, one-way analysis of var-iance with the post hoc Scheffé procedure (PCR data) orKruskall–Wallis test with post hoc Dunńs multiple compari-son (histological scoring) was used where appropriate. Stat-istical significance was defined as P < 0.05. Analyses wereperformed using PASW Statistic 18.0 (SPSS Inc., Chicago, IL,USA) and GraphPad Prism 5.0c (GraphPad Software Inc., LaJolla, CA, USA).

RESULTS

Clinical results

Clinical data of the patients are presented in Table 1. Allcalciphylaxis patients were admitted to the University Hospi-tal Aachen due to complicated CUA, and in all patients CUAwas associated with chronic or severe acute kidney dysfunc-tion: five patients were chronic haemodialysis (HD) patients,one patient suffered acute renal failure before CUA was diag-nosed and one patient had CKD stage III. Six patients were

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of Caucasian and one was of Asian ethnicity. Four patientswere receiving long-term anti-coagulation with coumadin,four patients were substituted with oral vitamin D3, fourpatients were receiving subcutaneous injections of erythro-poietin, one patient was receiving intravenous iron, sixpatients were receiving oral phosphorus binders (four seve-lamer, one aluminium hydroxide, one lanthanum carbon-ate) and one patient was receiving cinacalcet. Onehaemodialysis patient had undergone parathyroidectomy.Laboratory data of the seven CUA patients are shown inTable 2. Three deaths occurred in HD CUA patients due torefractory septic shock.

The clinical picture of CUA was deep ulcerative proximallesions in four patients, while three patients revealed distalcalciphylaxis at the lower extremities.

Distribution of calcified lesions and upregulation ofBMP-2, sclerostin and MGP

von Kossa staining revealed calcified areas in the subcu-tis of all CUA patients. Calcification was observed aroundadipocytes (Figure 1A), in the media of subcutaneous ar-terioles (Figure 1B, inset) and in connective tissue strands(Figure 1B) of the subcutis. Calcification was never ob-served in the dermis. To evaluate the hypothesis that activebone formation contributes to the process of subcutaneouscalcification in CUA, we analysed the expression of BMP-2, its receptor bone morphogenic protein receptor 2(BMPR-2), its downstream target gene Runx2 and itsantagonists sclerostin and MGP in the skin of CUApatients. Immunohistochemistry showed areas positive forBMP-2 and sclerostin adjacent to calcified areas in the sub-cutis (Figure 1C–F) in all seven CUA patients. In contrast,the subcutis of controls (non-CKD and dialysis controls)was negative for sclerostin and BMP-2 (Table 3, Sup-plementary data, Figures S1 and S2). The results of the Qt-RT-PCR of four patients confirmed this observation at themRNA level. BMP-2, sclerostin and Runx2 were signifi-cantly upregulated in the skin of CUA patients comparedwith the skin of both controls (Figure 1G). BMPR-2 wasnon-significantly upregulated in the skin of CUA patientscompared with controls. Staining for inactive (uncarboxy-lated) Glu-MGP and active (carboxylated) Gla-MGP re-vealed an expression of both forms in CUA skin lesions(Figure 1H and Supplementary data, Figure S3). However,compared with the controls only the inactive Glu-MGPwas significantly increased in the CUA skin (Table 3). Weobserved no differences in the expression of both MGPforms between coumadin-treated and non-treated CUApatients (data not shown).

CUA skin lesions exhibit extensive remodelling of thesubcutaneous ECM

As fibrotic procalcific remodelling of the ECM supportscalcification in heart valves and vessels [21, 22, 27], wehypothesized that a similar process is detectable in skin in-durations of CUA patients which finally end in calcification.Semi-quantitative analysis of the protein expression in thesubcutis revealed a significantly increased subcutaneous

expression of collagen I, fibronectin, osteopontin and lamininin calcified skin specimens of CUA patients compared withnon-CKD controls (Table 3). However, despite a trendtowards upregulation of all analysed ECM proteins, semi-quantitative histological scoring revealed a significantly in-creased expression only for osteopontin and laminin in theCUA skin lesions compared with the dialysis controls(Figure 2 and Table 3). Osteopontin exhibited a comparabledistribution pattern to von Kossa staining in CUA with astrong expression in the subcutis close to adipocytes and inconnective tissue strands (Figure 2A–B). In contrast to thecontrol skin (Figure 2D, inset and Supplementary data,Figure S4), the fine, reticular fibres surrounding adipocytes inCUA patients were broadened and strongly positive for fibro-nectin (Figure 2D).

In CUA, laminin and collagen I showed increasedexpression in association with adipocytes (Figure 2E and G),whereas collagen IV expression did not differ quantitativelyfrom control skin, although the expression pattern was irre-gular around adipocytes in the subcutis of CUA patients, interms of adiponecrosis (Figure 2F, Table 3). We comparedthe immunohistochemistry distribution pattern for ECM pro-teins to the von Kossa staining and observed an upregulationof fibronectin, collagen I and laminin even in areas that didnot show overt calcification. Qt-RT-PCR analysis supportedthe immunohistochemial findings regarding the extensiveECM remodelling process in CUA skin (Figure 2H), as osteo-pontin, fibronectin and laminin were significantlyupregulated.

Calcification of the subcutaneous adipose tissue involvescalcium hydroxyapatite formation

Scanning electron microscopy (SEM) confirmed calcifiedareas in the skin sections of all CUA patients (Figure 3). Themost extensive calcification was detected in the media of sub-cutaneous arterioles (Figure 3B) and within the subcutaneousconnective tissue (Figure 3F). Comparable with von Kossastaining, we detected calcifications around adipocytes(Figure 3E). Energy-dispersive X-ray spectroscopy (EDX)showed carbon, oxygen, phosphorus and calcium in all calci-fied regions (Figure 3D), with a molar ratio of calcium/phos-phate of 1.68 ± 0.06 matching that of calcium hydroxyapatitemineral. Sodium was detected in five, iron in three, mag-nesium in two and silicium in one skin section of CUApatients (data not shown). Control spectra from skin sectionsof non-CKD controls showed carbon and oxygen (Figure 3C),but never calcium or phosphate.

Clogging of subcutaneous vessels by endothelial cells

In CUA lesions, lumina of small cutaneous vessels wereoften obstructed by cell debris (Figures 1B and 3B, inset).This observation had previously led to a ‘2-hit model’where vascular calcification is followed by endoluminal ob-struction, thrombus formation and tissue necrosis [1, 28].Anti-CD31 staining revealed CD31+ endothelial cells (ECs)within the endoluminal debris in about 30% of the calcifiedcutaneous arteries and arterioles of calcified CUA skin sec-tions (Figure 4A and B), while the normal CD31+ ECs

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layer of the intima was often destroyed in those vessels. Incontrast, anti-CD31 staining of skin sections from non-CKD controls showed a regular CD31+ layer of intimal ECand never intraluminal cellular debris accumulation(Figure 4C). Furthermore, we observed histological signs ofthrombosis in very small cutaneous vessels (diameter15 μm) with remnants of CD31+ cells within the thrombus(Figure 4D–E).

Haemosiderophages in the subcutis

Iron has been discussed as a potential contributor to CUAdevelopment [29, 30] and, as noted above, we detected ironby EDX analysis in skin lesions of three of the seven CUApatients. In line with our EDX analysis, Prussian blue stainingwas positive in only these three skin sections. In two sections,

only small Prussian blue-positive areas were observed inassociation with small subcutaneous vessels (Figure 5A,inset), while in the skin section of one CUA patient, we ob-served large Prussian blue-positive areas in the subcutaneousconnective tissue (Figure 5A and B). SEM and EDX analysesrevealed that the iron deposits were not closely correlatedwith the calcified areas, as we observed carbonate, oxygen,sodium, phosphorus and sulphate associated with the ironspectra, but not calcium (Figure 5C and D). Rather, anti-CD68 staining confirmed CD68+ macrophages within theiron deposits (Figure 5E and F), identifying them ashaemosiderophages.

We observed no significant differences in any performedanalyses between the proximal and distal form of CUA. Thismight be due to the small patient cohort.

Table 2. Laboratory values of seven CUA patients upon admission

Patientno.

Albumin(g/L)

Calcium(mmoL/L)

Phosphorus(mmoL/L)

AP(U/L)

PTH(ng/L)

Creatinine(mg/dL)

CRPa

(mg/L)

1 17.3 2.4 0.8 196 60 4.9 230

2 33 2.1 2.7 115 896 12.2 38

3 36.8 2.7 1.4 108 787 3.1 17

4 32.5 2.2 1.2 96 276 3.3 93

5 32.8 1.4 1.0 157 4 1 62

6 10.7 2.5 0.8 276 118 4.9 84

7 41.5 2.4 1.2 105 80 0.7 5

AP, alkaline phosphatase; CRP, C-reactive protein.aNormal value <5 mg/L.

Table 1. Clinical characteristics of seven CUA patients

Patientno.

Age(year)

Sex Obesity DM Wounddistribution

Cause ofCKD/ESRD

RRTmode

Time onHD(years)

Outcomea

1 44 F Yes Yes Abdominal Diabetic HD 5 Expired

2 43 M Yes Yes Distal legs Hypertensive HD 6 Alive

3 77 F No No Distal leg Hypertensive HD 3 Alive

4 67 F No No Abdominal Hypertensive HD 5 Expired

5 21 M Yes No Abdominal ARFb NoRRT

– Alive

6 39 F Yes Yes Abdominal Diabetic HD 3 Expired

7 56 F Yes Yes Distal leg CKD IIIdiabetic

NoRRT

– Alive

RRT, renal replacement therapy; ESRD, end-stage renal disease; DM, diabetes mellitus; HD, haemodialysis; CKD, chronic kidney disease;F, female; M, male. Obesity was defined as body mass index >30.aAt discharge of hospital.bIncrease of creatinine up to 4.2 mg/dL in medical history.

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DISCUSSION

Once thought to be a passive process, vascular calcificationhas emerged as an active and tightly regulated cell-mediated

process including matrix remodelling and deposition [16].CUA can be regarded as a high-speed template for ectopic,vascular calcification processes with a rapid onset. Ourcurrent study points towards a number of yet unidentifiedissues in the pathogenesis of CUA at the tissue level: (i) we

F IGURE 1 : Up-regulation of osteogenic markers in skin lesions of CUA patients. (A and B) von Kossa positive subcutaneous adipose tissue(A), connective tissue (B) and arteriole (B, inset). (C and D) BMP-2 staining and (E and F) sclerostin staining indicated an osteogenic processin the skin of CUA patients. We observed BMP2-positive areas in the dermis (C arrows) and the subcutaneous adipose tissue (D arrows) onlyin the calcified skin and subcutaneous areas of the CUA patients. Similarly, sclerostin positive areas were observed only in the subcutaneousadipose tissue of calcified skin in CUA patients (E and F arrows). The subcutis of control subjects was completely negative for these stainings(Supplementary data, Figures S1 and S2). (G) mRNA expression of BMP-2, sclerostin (SOST) and Runx2 was significantly enhanced in theskin lesions of CUA patients compared with the skin of both control groups (non-CKD controls = expression set as 1 for all genes). (H)Uncarboxylated inactive Glu-MGP expression was increased in the subcutaneous arterioles (arrow) and in the connective tissue surroundingadipocytes of the subcutis (H, insets, arrowheads) *P < 0.05 versus non-CKD controls, #P < 0.05 versus dialysis controls, values are expressedas mean ± SD. All scale bars 200 µm, insets 100 µm.

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describe the type and distribution of soft-tissue (not just mi-crovascular) calcification in CUA specimens; (ii) we presentdata on extensive remodelling of the subcutaneous tissue, (iii)our data present evidence for an interplay of increased BMP-2, Glu-MGP and sclerostin expression to calciphylaxis and(iv) demonstrate evidence for destruction of the endothelialcell layer in subcutaneous vessel leading to vascularobstruction.

Our first major finding was that BMP-2 was upregulatedin skin lesions of CUA patients associated with a significantupregulation of the downstream key osteogenic transcriptionfactor ‘Runt-related transcription factor 2’ (Runx2, also calledcore-binding factor alpha 1, Cbfa1). BMP-2 induction in theskin may be related to inflammation or reactive oxygenspecies (ROS) [9, 31–33]. One potential source of BMP-2expression might be ECs as it is known that BMP can be up-regulated in ECs by oscillatory shear stress, inflammatory cy-tokines and ROS [34]. In one case report of CUA, expressionof BMP-4 in the periarteriolar adventitia was described [35].Both BMP-2 and BMP-4 have also been described in calcifiedareas of atherosclerotic lesions [36, 37]. BMP-4 is thought topromote endothelial proliferation and angiogenesis, whereasBMP-2 has a direct link to calcification as it promotes miner-alization [34]. The increased BMP-2 expression isaccompanied by an increased expression of MGP in its inac-tive, uncarboxylated form (Glu-MGP), which might result ina decreased capability of MGP to inhibit BMP-2 signalling.Our finding of increased expression of inactive Glu-MGP inCUA skin lesions might reflect local vitamin K deficiency.However, we did not observe any differences between

coumadin-treated and non-treated CUA patients in our smallcohort.

Runx2 induction is an essential early step responsible forpre-osteoblast formation and plays an important role in en-chondral ossification [38]. Further, Runx2 upregulates theexpression of ECM proteins critical for osteogenesis such asosteopontin and collagen I [39]. This theory of a cascade inCUA development was supported by our second majorfinding, namely a significant upregulation of several ECMproteins critical for mineralization (osteopontin, fibronectin,collagen I and laminin) in skin lesions of CUA patients.ECM remodelling is important for osteogenesis and calcifica-tion in bone [38], heart valves [27] and vessels [21, 40]. Os-teopontin expression in skin lesions of CUA was firstdescribed by Ahmed et al. [20]. These authors predominantlyobserved osteopontin-positive areas in the media of calcifiedcutaneous vessels. We additionally detected osteopontin inthe calcified connective tissue surrounding adipocytes. As os-teopontin is an important regulator of mineralization andblocks apatite crystal growth [41, 42], its increased expressionin CUA might be a feedback mechanism to control uncon-trolled growth of crystals in the CUA lesions. The most pro-nounced alteration of the ECM in the subcutis of CUApatients compared with control subjects was the strongexpression of fibronectin even in non-calcified areas. Fibro-nectin might be a critical ECM protein for the calcificationobserved in the CUA lesions as it promotes the calcificationof vascular smooth muscle cells in vitro [22]. Collagen I isanother ECM protein in the CUA lesions and the majorprotein of bone that is essential for osteogenesis and also

Table 3. Semiquantitative evaluation of protein expression and von Kossa staining in the subcutisof calcified CUA skin and control skin (from non-CKD patients and dialysis patients)

Non-CKDcontrols(n = 7)

Dialysiscontrols(n = 7)

Non-CKDcontrols versusdialysis controls(P-valuea)

CUA(n = 7)

CUA versusnon-CKDcontrols (P-valuea)

CUA versusdialysiscontrols (P-valuea)

von Kossa 0 0.07 ± 0.19 ns 3.57 ± 0.35 <0.001 <0.01

BMP-2 0 0.21 ± 0.27 ns 1.57 ± 0.19 <0.001 <0.01

Sclerostin 0 0.14 ± 0.38 ns 1.79 ± 0.64 <0.001 <0.01

Glu-MGP 0.17 ± 0.29b 0.43 ± 0.53 ns 2.5 ± 0.71 <0.05 <0.01

Gla-MGP 0.67 ± 0.29b 1.07 ± 0.67 ns 1.57 ± 0.45 ns ns

Osteopontin 0 0.21 ± 0.27 ns 3.64 ± 0.48 <0.001 <0.01

Collagen I 0.5 ± 0.5 1.14 ± 0.24 ns 1.86 ± 0.48 <0.001 ns

Collagen IV 1.79 ± 0.27 1.57 ± 0.45 ns 1.64 ± 0.38 ns ns

Fibronectin 0.86 ± 0.24 1.57 ± 0.45 ns 3.43 ± 0.45 <0.001 ns

Laminin 0.57 ± 0.53 0.92 ± 0.19 ns 1.71 ± 0.39 <0.01 <0.05aKruskall–Wallis test with post hoc Dunn’s multiple comparison test.bMGP staining in healthy controls was only performed in n = 3 skin samples.ns, non significant.

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plays an important role in atherosclerosis and vascular calcifi-cation [21]. Collagen I expression was apparently upregulatedin CUA skin specimen. Finally, we detected a significant

upregulation of laminin in CUA skin, i.e. a key regulator ofcell migration and adhesion [43]. This is in line with our pre-vious finding of increased expression of laminin in calcified

F IGURE 2 : ECM remodelling in the subcutis of CUA skin lesions. Immunohistochemistry demonstrated expression of various ECM pro-teins in CUA skin lesions in comparison with skin sections of dialysis controls (insets). (A and B) Osteopontin staining revealed positiveareas with a similar distribution pattern as von Kossa staining (in Figure 1) around adipocytes (A and B arrows) and in the connective tissuestrands of the subcutis (asterisk in A). (C and D) Fibronectin staining demonstrated strongly positive subcutaneous connective tissue withthickening of periadipocytic (arrows) and connective tissue (asterisk) fibres. (E) Laminin expression was slightly enhanced in CUA skin (F).Staining for collagen IV showed a more irregular distribution pattern in CUA subcutis (arrows) compared with skin of dialysis controls(inset). (G) Collagen I expression was increased in the subcutis of CUA patients (arrows). (H) mRNA expression of osteopontin (OPN) andfibronectin (FN) was significantly increased in the skin of CUA patients compared with both non-CKD and dialysis controls, whereas laminin(Lam) expression was only significantly increased in CUA versus non-CKD controls, collagen I (Col I) expression was non-significantly in-creased and collagen IV (Col IV) expression remained unchanged compared with skin of controls. Non-CKD controls = expression set as 1for all genes; *P < 0.05 versus non-CKD controls, #P < 0.05 versus dialysis controls; values are expressed as mean ± SD; all scale bars 200 µm,except (F) and (F, inset) 100 µm.

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vessels of dialysis patients [44]. In summary, we were able todetect a sophisticated concert of proteins in CUA lesionspointing towards a pro-calcifying ECM environment. This re-modelling of the subcutaneous ECM in CUA may also con-tribute to the clinical finding of skin induration of CUApatients, which is a frequent early finding in areas that laterprogress to ulcerative lesions.

ECM remodelling in the so-called matrix maturationphase is associated with the expression of osteopontin whichis thought to promote the deposition of mineral by regulatingthe amount and size of hydroxyapatite crystals formed [45].This is consistent with our third major finding, namely thedetection of hydroxyapatite within the calcified lesions ofCUA patients. Calcium and phosphate were described within

the calcified lesions of CUA patients before [5, 20], but weare the first to identify a molar ratio of calcium to phosphateof 1.6 matching that of hydroxapatite mineral. This findingsupports our hypothesis of active osteogenesis in CUAlesions. Hydroxyapatite is characterized by the incorporationof numerous foreign ions, which is consistent with our detec-tion of small amounts of magnesium, sodium and the rareearth element silicium in the calcified CUA sections [46, 47].However, our study cohort is too small to exclude the pres-ence of any other calcium biominerals in CUA. Interestingly,besides hydroxyapatite, the presence of whitlockite was alsoreported in uraemic vascular calcification [48, 49].

Our fourth major novel finding was a significant upregula-tion of the osteocyte marker sclerostin in skin lesions of CUA

F IGURE 3 : Hydroxyapatite accumulation in the subcutis of CUA skin lesions. SEM demonstrated calcified areas in the subcutis (B, E andF) of CUA patients, whereas no calcification was observed in the skin of non-CKD controls (A). Adipose tissue (A) and subcutaneous vesselsof non-CKD control (A, inset) did not show any calcification. In contrast, in CUA subcutis multiple calcified areas (B arrowhead, E and F)with strongly calcified subcutaneous arteries (B, inset) that partly contain endoluminal thrombosis (asterisk) were found. The calcificationpattern surrounded the adipocytes (E arrows). EDX analysis only showed carbon and oxygen of the biological matrix in non-CKD skin (C),whereas calcium and phosphate were detected in the skin of CUA patients with a molar ratio of 1.6 matching that of hydroxyapatite mineral(D). The calcified areas in the subcutis of CUA patients were partially finely granular around adipocytes (E) and partially massive in theconnective tissue (F). Scale bars 100 µm, except (F, inset) 20 µm.

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patients. Sclerostin, first identified in 2001 [50], is an inhibi-tor of canonical Wnt signalling via binding to the frizzled-LRP5/6 membrane receptor complex leading to increasedbone formation [51]. The regulation of sclerostin expressionin osteocytes is not yet fully understood [52]. Calcitriol, glu-cocorticoids, TNF-α, BMP2, 4 and 6 are able to increase thesclerostin expression [52]. Increased serum values of scleros-tin in ESRD patients compared with healthy control subjectshave been described [53]. Recently, an upregulated sclerostinexpression during vascular smooth muscle cell calcification invitro and in the calcified aorta of ectonucleotide pyropho-sphate/phosphodiesterase 1-null (Enpp1−/−) mice were ob-served [54]. Didangelos et al. [55] detected sclerostinexpression in intact human aortas obtained during aorticvalve replacement. Overall, the role of sclerostin in uraemicbone disease and uraemic vascular calcification is incomple-tely understood. However, our immunohistochemistry andPCR data point towards an important role of sclerostin alsoin CUA. At this time-point, we are unable to establish anycausality. However, regarding its role in the bone, the upregu-lation of sclerostin might be an antiregulatory process, as alast effort to prevent soft-tissue calcification.

The devastating clinical picture of the very severe form ofcalciphylaxis—the proximal ulcerative form [1]—is mainlycaused by large ulcerative areas that often trigger infectiouscomplications (septicaemia). It is likely that in CUA the

primarily fibrotic and calcifying processes ultimately result ina fatal malperfusion syndrome. Indeed, we detected detachedendoluminal CD31+ ECs and a destroyed endothelial celllayer of small- and medium-sized subcutaneous arteries incalcified CUA skin. Others previously also noted that theendothelium is ‘lifted off’ in calcified subcutaneous vessels ofCUA skin sections [20]. Furthermore, we detected signs oftotal vascular occlusion in subcutaneous terminal vessels withthrombosis and remnants of CD31+ cells. Thus, a sequencemay exist, whereby vascular calcification triggers endothelialdamage finally leading to off-sheared ECs, which may be thenidus for thrombogenesis resulting in malperfusion and ne-crosis of the skin. The subcutaneous reduced blood flowmight also promote the calcification of the subcutaneous softtissue. In the context of thrombus formation, it is also inter-esting that BMPs (BMP-2/4) induce a proinflammatory endo-thelial phenotype and cause an induction of endothelialadhesion molecules in ECs with increased monocyteadhesion capability [31, 33, 56].

Prussian blue-positive areas have previously been reportedin close spatial relation with vessels in CUA skin biopsies[29]. Iron deposition is considered a ‘challenging factor’ forCUA in analogy to what Hans Selye postulated in his animalmodels. Indeed, we detected iron via Prussian blue staining inthe subcutis of three CUA patients, and EDX analysis con-firmed the presence of iron by three typical iron peaks.

F IGURE 4 : Obstruction of subcutaneous vessels by ECs in CUA. CD31 staining demonstrated a completely destroyed endothelial cell layerwith clogging of small and medium subcutaneous arterioles (A and B arrows) by detached ECs in about 30% of the subcutanoues vessels inCUA patients in comparison with (C) regular endothelium in the subcutis of a non-CKD control (arrows CD31). We observed total endolum-inal occlusion with residual CD31+ areas (arrow D and E) within thrombotic formations in smaller subcutaneous vessels (D and E). Scale barsA–C 50 µm, D and E 10 µm.

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However, we do not consider iron as being a trigger factor forCUA. Given our observation that CD68 staining revealed anaccumulation of macrophages exactly in these ‘iron-positive’areas, the iron accumulation in the subcutis of CUA patientsmight be due to haemosiderophages. Interestingly, the onepatient in our study who received i.v. iron therapy did notexhibit iron accumulation in the skin, which is in line with ourhypothesis of secondary iron deposition due to haemorrhages.

In summary, although the present cross-sectional studydoes not allow a clear causality to be established, our datapoint towards an active, cell-mediated, BMP-2-driven osteo-genic process with extensive ECM remodelling and depo-sition of hydroxyapatite mineral in the subcutis of CUApatients. Although yet unproven, the present data confirmour clinical impression of a cascade starting with matrix re-modelling, followed by calcification, endothelial damage andthrombus formation, and finally leading to luminal obstruc-tion with tissue necrosis. Future research should focus on

early causative factors for matrix remodelling and on theidentification of driving forces for the progress of the above-postulated cascade in CUA.

SUPPLEMENTARY DATA

Supplementary data are available online at http://ndt.oxfordjournals.org.

FUNDING

This study was funded by intramural funding to R.K. and R.S. (START grants) from RWTH Aachen University. V.B. andM.K. received a project grant from AMGEN for the GermanCalciphylaxis Registry conduct. The results presented in thispaper have not been published previously in whole or part.

F IGURE 5 : Haemosiderophages in the subcutis of CUA lesions. (A and B) Positive Prussian blue staining in the subcutaneous connective tissuestrands of CUA lesions was observed in close association to vessels (asterisks). (C and D) SEM and EDX analyses revealed iron with small amountsof sulphate and phosphorus in the connective subcutaneous tissue (arrows in C). (E and F) CD68 staining demonstrated an accumulation ofmacrophages (arrows) in all ‘iron-positive’ subcutaneous areas. Comparable with iron deposition in (A and B), macrophages aggregate in theconnective tissue strands of the subcutis (E) and around the subcutaneous vessels (asterisk in F). All scale bars 200 µm, except (C, inset) 20 µm.

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http://ndt.oxfordjournals.org






ACKNOWLEDGEMENTS

We thank Norina Labude (Institute of Pathology) and EstherStuettgen (Department of Nephrology) for excellent technicalassistance in the lab.

CONFLICT OF INTEREST STATEMENT

None declared.

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Received for publication: 11.4.2012; Accepted in revised form: 22.8.2012

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