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NDT Advance Access published online on November 21, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn611
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Expression of gremlin, a bone morphogenetic protein antagonist,is associated with vascular calcification in uraemia

Aquiles Jara1, Cecilia Chacón1, María Eugenia Burgos2, Alejandra Droguett2, Andrés Valdivieso1, Mireya Ortiz1, Pablo Troncoso1 and Sergio Mezzano2

1 Department of Nephrology, School of Medicine, Pontificia Universidad Católica de Chile, Santiago 2 Division of Nephrology, School of Medicine, Universidad Austral de Chile, Valdivia, Chile

Correspondence and offprint requests to: Aquiles Jara, Department of Nephrology, School of Medicine, Universidad Católica de Chile, Lira 85, Santiago, Chile. Tel: +5623543229; Fax: +5626397377; E-mail: ajara{at}med.puc.cl



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Vascular calcification has been widely recognized as a significant contributor to cardiovascular risk in patients with chronic kidney disease. Recent evidence suggests that BMP-7 decreases the vascular calcification observed in uraemic rats, while BMP-2 could also be participating in this process. Gremlin, a bone morphogenetic protein antagonist, has been detected in rat aortic vascular smooth muscle cells (VSMCs), and since the role of the VSMCs into vascular calcification in uraemia is considered critical in this process, we hypothesized that gremlin could be participating in its pathogenesis. With this aim, we studied its expression in aorta from uraemic rats with calcitriol-induced vascular calcification and in 16-vessel biopsies of uraemic patients undergoing kidney transplantation.

Methods. Gremlin was detected by in situ hybridization (ISH) and immunohistochemistry (IMH). BMP-7, BMP-2 and BMP-2 receptor (BMPR2) were detected by IMH. Vascular calcification was assessed by the von Kossa staining method. Sham-operated and 5/6 nephrectomized rats (NFX) (1.2%P) were treated with vehicle or calcitriol (80 ng/kg, intraperitoneally every other day). Rats were killed after 4 weeks of treatment, and abdominal aorta was dissected for assessment of gremlin expression and vascular calcification. Epigastric arteries were obtained from dialysis patients during kidney transplantation procedure. Arteries from kidney donors were also studied.

Results. NFX rats developed a mild vascular calcification, whereas NFX-calcitriol rats developed a severe vascular and tissue calcification. A marked overexpression of gremlin was observed in the vascular media of aorta from NFX-calcitriol rats as compared with NFX and sham-calcitriol groups (4.8 ± 1.3 versus 0.59 ± 0.17 versus 0.19 ± 0.07 percentage/mm2, P < 0.01), and correlated with the BMP-2 and BMPR2 expression. Sham rats showed minimal or null gremlin expression. BMP-7 was not found in sham or calcified arteries. In human studies, we observed strong expression of gremlin mRNA and protein in the media layer of vessels from uraemic patients as compared with those from normal humans (staining score 3.72 ± 0.95 versus 0.91 ± 0.08 percentage/mm2, P < 0.05).

Conclusion. We observed a marked gremlin overexpression in the media layer of vessels in uraemic rats and patients in association with vascular calcification and BMP-2 expression. We postulate that gremlin may play a role in the vascular calcification process in uraemia, and its interaction with BMP-7 or BMP-2 remains to be elucidated.

Keywords: BMP-2; BMP-7 antagonist; CKD; gremlin; vascular calcification



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Vascular calcification is a common and a widely recognized complication of chronic kidney disease (CKD), which increases the risk for cardiovascular morbidity and mortality [1–5]. The precise mechanism of vascular calcification in CKD still remains to be elucidated, but associated risk factors have been recognized, and they include age, hypertension, time on dialysis and abnormalities in calcium (Ca) and phosphorus (P) metabolism, resulting in a raised serum Ca–P product (Ca x P) and secondary hyperparathyroidism (HPT). The elevations in Ca, P and Ca x P product have been associated with an increased risk for vascular calcification [6,7].

Pathologically, two distinct patterns of vascular calcification have been identified in the uraemic patient. The intimal calcification, in association with atherosclerosis, has been associated with lipid-laden macrophages and intimal hyperplasia contributing to plaque formation and rupture. The medial calcification, which causes vascular stiffening, occurs in the media of the vessel in conjunction with a phenotypic transformation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells (Mönckeberg's calcification) [8,9]. It has been demonstrated that VSMCs isolated from human or animal arteries were capable of mineralizing in vitro in a similar manner to osteoblast when they are exposed to high phosphorous content in the medium [7,10–13]. The presence of proteins such as alkaline phosphatase, osteopontin, bone sialoprotein and type I collagen in arteries from dialysis patients suggests an osteogenic process in vascular calcification in CKD patients, and that VSMCs phenotypically behave like osteoblasts [14].

Hruska et al. have established the concept that CKD is a state of bone morphogenetic protein-7 (BMP-7) deficiency [15–17], and that BMP-7 would play a role in maintaining VSMC differentiation and preventing transdifferentiation of VSMCs into an osteoblast phenotype. When uraemic rats were treated with BMP-7, the calcification in these animals was prevented [15]. The authors believed that BMP-7's effects are mediated via influences on cellular differentiation programs, with a positive influence on VSMCs differentiation, and that transdifferentiation of these cells is the critical first step in the mechanism of vascular calcification. In addition, it has been demonstrated that BMP-2 may induce osteogenic differentiation of VSMCs through induction of transcription factors Cbfa1, osterix and the msh homeobox homologue MSX-2 [18,19]. BMP-2 could also contribute to calcification by inhibitory effects on the Matrix Gla protein (MGP) [20].

Gremlin, as BMP antagonist, has been reported to influence diverse processes in growth, differentiation and development, in many cases by heterodimerization with BMP-2, -4 and -7, thereby inhibiting the ability of these ligands to bind to their receptors. Moreover, a role for gremlin in the epithelial mesenchymal transition (EMT) and tubule interstitial fibrosis has been proposed, both as BMP-7 antagonist and directly as a downstream mediator of TGB-β in that process [21–24].

Recently, it has been demonstrated that gremlin is constitutively expressed in VSMCs of rats and is regulated by several growth factors [25]. Gremlin overexpression increases VSMCs proliferation and migration, and its expression is markedly increased following vascular injury [25]. The purpose of this study is to examine the presence of gremlin in different layers of vascular tissue from uraemic rats with secondary hyperparathyroidism with calcitriol-induced vascular calcification and in uraemic patients undergoing renal transplantation.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Studies in aorta from uraemic rats
Male Sprague-Dowley rats weighing 250 g were used. CKD was induced by 5/6 nephrectomy (NFX). Briefly, NFX in a two-step procedure reduced the original functional renal mass by five-sixths. In the first step, rats were anaesthetized using xylazine (5 mg/kg, intraperitoneally) and ketamine (80 mg/kg, intraperitoneally), an incision was made on the left mediolateral surface of the abdomen and the left kidney was exposed. The left renal artery was visualized, and two of the three branches were ligated tightly. The rats were placed back into its individual home cages. After 1 week of recovery, the rat was reanaesthetized with the same previous procedure, and a similar incision was made on the right mediolateral surface of the abdomen. The right kidney was exposed and unencapsulated, the renal pedicle was clamped and ligated, and the kidney was removed. Rats were allowed to recover in their home cages. Sham-operated rats underwent the same procedures without renal manipulations [26].

Experimental design
One week after the second surgery, rats were randomly assigned into five experimental groups: (1) sham-operated with normal diet (0.6%Ca–0.6%P), used as controls (group sham, n = 10), (2) sham-operated with high phosphorous diet (0.6%Ca–1.2%P) (sham-HPD, n = 10), (3) sham-operated + HPD + calcitriol 80 ng/kg (Calcijex®, Lab. Abbott, Santiago, Chile) intraperitoneally every other day (sham-calcitriol, n = 10), (4) 5/6 nephrectomy + HPD (NFX + HPD, n = 9) and (5) 5/6 nephrectomy + HPD + calcitriol (NFX-calcitriol, n = 12). We used this last group as a model of vascular calcification as it was previously demonstrated [27]. The high mortality in 5/6 nephrectomized rats precludes maintaining them for more than 4 weeks in this group. After 4 weeks, all rats were killed by aortic puncture and exsanguinated 24 h after the last dose of calcitriol. After the rats were killed, the abdominal aorta, the heart and the remnant kidney were dissected.

Assessment of vascular calcification
Samples of abdominal aorta, the heart and the remnant kidney were fixed in 4% buffered formalin and subsequently sectioned and stained for mineralization by the von Kossa method as has been previously reported [27].

Studies in arteries from ESRD patients
All patients over the age of 18 years undergoing a renal transplantation at Hospital Clínico Pontificia Universidad Católica de Chile were eligible for the study. The samples were obtained after obtaining patients’ consent. This project was approved by the Ethics Committee for Research of School of Medicine, Universidad Católica de Chile. During the transplant procedure, the proximal portion of the epigastric artery, which had previously undergone ligation according surgical protocol, was removed and placed in a saline buffer. The vessel was dissected free of fat and fixed in 4% paraformaldehyde. Demographical and medical information was obtained from patient's records. Duration of dialysis, type of dialysis, causes of ESRD, presence or absence of diabetes mellitus, story of coronary or peripheral vascular disease, use of vitamin D sterols, antihypertensive drugs, average serum calcium, serum phosphorous and intact PTH from last 6 months were considered. As control, vessels were obtained from kidney donors for transplantation using similar procedure.

Assessment of gremlin, BMP-2, BMP-7 and BMPR2 expression by immunohistochemistry (IMH)
This technique for gremlin has been recently reported [22]. For light microscopy, vessel tissues were fixed in 4% buffered formalin, dehydrated and embedded in paraffin by conventional techniques. Sections were stained with haematoxylin and eosin (HE), and von Kossa for mineral demonstration. Paraffin-embedded biopsy specimens were used for detection of gremlin and BMP-2, BMP-7 and BMPR2.

The following primary antibodies were employed: rabbit polyclonal anti-gremlin (ABGENT, AP6133a, San Diego, CA, USA); monoclonal anti-BMP-2 (ABCAM, Cambridge, UK); goat polyclonal anti-BMP-7 (Santa Cruz Biotechnology, CA, USA); rabbit polyclonal anti-BMPR2 (ABGENT). Briefly, 5-µm-thick formalin-fixed vessel sections from humans and rats were deparaffinized through xylene, alcohol and distilled water. Endogenous peroxidase was blocked by 3% H2O2 for 15 min, and then the sections were treated in a microwave oven in a solution of 0.1 mM citrate buffer, pH 6.0, for 10 min. After blocking, the sections were incubated overnight at 4°C with the specific primary antibody. The sections were then incubated with the correspondent biotinylated secondary antibodies for 30 min at 22°C. After three rinses in Tris saline phosphate (TPS), they were incubated with streptavidin peroxidase (Dako, Carpinteria, CA, USA) 1/1000 for 30 min. Colour was developed with a substrate (Dako) and then counterstained with haematoxylin, dehydrated and mounted with Canadian balsam (Polysciences, Inc., Warrington, PA, USA). The specificity was checked by omission of primary antibodies and use of non-immune sera.

We also used R.T.U. Vectastain Kit®(Vector, Burlingame, CA, USA) for confirmation of gremlin localization.

Assessment of the pattern of gremlin expression by in situ hybridization (ISH)
This technique has been recently reported [23]. ISH was performed using biotin-labelled human gremlin probes: 478 antisense 5'-TGAAAGGAACCTTCCTCCTTCC3', 2416 antisense 5'-ATGGGAGAGCACTGGATCAAAA-3' and 3553 antisense 5'-CAGGCACTGACTCAGGAAGACA-3' (Invitrogen, Carlsbad, CA, USA). For gremlin analysis, pretreatment with an endogenous biotin blocking system (Dako Co., Carpinteria, CA, USA) was performed prior to proteinase K digestion. The sections were incubated with a pre-hybridization solution (Dako, mRNA ISH Solution) for 60 min at 37°C and with the antisense probe overnight at 37°C. The slides for gremlin were washed with 2 x SSC and 1 x SSC for 10 min at room temperature and then with 0.5 x SCC for 20 min at 37°C.

Detection was performed with avidin–alkaline phosphatase conjugate (Dako Co., Carpinteria, CA, USA) for 30 min at room temperature, washed 5 min with 1 x TBS and using NBT-BCIP as the enzyme substrate for 120 min at 37°C (R&D Systems, Minneapolis, MN, USA). Tissues were then dehydrated in ethanol series and mounted in Canadian balsam (Polysciences Inc., Warrington, Pennsylvania, USA).

The specificity of the reaction was confirmed (a) by demonstrating the disappearance of the hybridization signal when RNAse (100 µg/ml) (Sigma Chemicals Co., St Louis, MO, USA) was added in 0.05 M Tris after the digestion with proteinase K; (b) by the use of a sense probe (R&D Systems, Minneapolis, MN, USA); (c) with a negative control (Plasmid DNA) (Dako Co., Carpinteria, CA, USA) and (d) without probe.

For gremlin ISH slides, Dako nuclear fast red was used for 10 min.

IMH quantification
In human and rat vessels, the surface area and density labelled was evaluated by quantitative image analysis using a KS 300 imaging system 3.0 (Zeiss, München-Hallbergmoos, Germany).

For each sample, the mean staining area was obtained by analysis of 10 different fields (40x). Quantification was done twice, independently, and interassay variations were not significant. The staining score is expressed as percentage/mm2.

Biochemical measurements
Creatinine, phosphate and total calcium were measured by spectrophotometry (Sigma Diagnostic, St Louis, MO, USA). PTH was quantified according to the vendor's instructions using the rat-specific IRMA assay (Nichols Institute, San Juan de Capistrano, CA, USA)

Statistical analysis
The statistical analysis was performed with the GraphPad Instat, GraphPad Software, San Diego, CA, USA. Results are expressed as mean ± SE. Pearson correlation was used to correlate the gremlin mRNA and protein expression. Spearman correlation was used to correlate the gremlin protein expression and the score of aortic calcification. A Mann–Whitney U-test was used to compare the expression of gremlin, BMP-2, BMPR2 and calcification between the different groups, and a Kruskal–Wallis test to compare all the rat groups.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Experimental study
All animals were killed at Day 28. The mean serum creatinine concentration in sham rats (0.6%P), sham-HPD (1.2%P) and sham-calcitriol were 0.36 ± 0.02, 0.35 ± 0.03 and 0.33 ± 0.02 mg/dl, respectively (P = n.s.). As expected, 5/6 nephrectomized rats had significantly higher creatinine levels (0.90 ± 0.13 mg/dl, P < 0.01). Moreover, treatment with calcitriol in 5/6 NFX rats resulted in a significant increase in the serum creatinine concentration (1.17 ± 0.12 mg/dl, P < 0.05) in relation to the 5/6 NFX rats without calcitriol. All 5/6 NFX rats developed marked secondary hyperparathyroidism; however, the differences in PTH, serum phosphate and serum calcium were not significant between both NFX groups (PTH = 849 ± 141 versus 679 ± 185 pg/ml; serum P = 15.9 ± 3.08 versus 10.5 ± 1.37 mg/dl; serum Ca 7.85 ± 0.55 versus 8.19 ± 0.60 mg/dl). However, the increase on the Ca x P product was significantly higher in the NFX-calcitriol group (111 ± 10.1 versus 76.3 ± 5.4 mg2/dl2, P < 0.05) (Table 1).


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  		Table 1 Biochemical data, weight of heart and remnant kidney, and gremlin staining score from sham-operated and 5/6 nephrectomized rats (NFX), treated or not treated with calcitriol

 
Vascular calcification, established by the von Kossa staining method as previously described, was studied in aortic tissues. As shown in Figure 1, we observed a marked media layer calcification in NFX-calcitriol groups (Figure 1D), whereas it was not observed in sham groups (Figure 1A, B). Furthermore, we did not find aortic calcification in NFX rats, except the ones with greater serum creatinine (Figure 1C).


Figure 1
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  		Fig. 1 Von Kossa-stained sections of the aorta in sham-operated rats (A), in sham-operated rats treated with calcitriol (80 ng/kg on alternate days) (B), in 5/6 nephrectomized rats treated with vehicle (C) or 5/6 nephrectomized rats treated with calcitriol for 4 weeks (D).

 
Gremlin expression was studied by IMH and ISH, and we found a strong expression of gremlin in the vascular media of NFX-calcitriol rats in the same area of vascular calcification (Figure 2B, C), as compared with NFX and sham-calcitriol groups (4.8 ± 1.3 versus 0.59 ± 0.17 versus 0.19 ± 0.07 percentage/mm2, P < 0.05). On the other hand, sham rats without calcitriol showed minimal or null gremlin expression (Figure 2A, Table 1). As illustrated in Figure 3, there was a strong correlation between aortic calcification and gremlin protein expression (r = 0.85, P < 0.01) (Figure 3). Furthermore, in NFX-calcitriol rats, positive von Kossa staining was also detected in muscle fibres of ventricles (Figure 4B) as well as in myocardial arteries (Figure 4C). In those calcified arteries, we also found positive staining for gremlin (Figure 4D). Mineral deposition was also observed in the basement membrane of renal tubules from kidney tissue remnant.


Figure 2
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  		Fig. 2 Immunohistochemistry (IMH) for gremlin in aorta from normal and uraemic rats. There is no expression of gremlin protein in vascular media of sham-operated rats (A). There is a strong gremlin protein expression in the media of aorta in 5/6 nephrectomized rats treated with calcitriol (80 ng/kg on alternate days) for 4 weeks (B, C). Black arrow shows mineral deposition in the media vascular of aorta of 5/6 nephrectomized rats treated with calcitriol (C).

 

Figure 3
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  		Fig. 3 Spearman's correlation between aortic calcification, measured with the von Kossa staining method, and gremlin protein expression from 5/6 nephrectomized rats and sham calcitriol-treated rats.

 

Figure 4
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  		Fig. 4 Von Kossa-stained sections of the heart from sham-operated rats (A) or 5/6 nephrectomized rats treated with calcitriol (80 ng/kg on alternate days) for 4 weeks (B, C, D). Positive staining is observed in muscle fibres of myocardium (B), and the myocardial arteries (C). Positive gremlin staining observed on the same calcified myocardial arteries (D).

 
As gremlin antagonizes BMPs, we also studied the expression of BMP-2, BMP-7 and their receptors by IMH in this animal model. BMP-7 was not expressed in normal or calcified arteries as is shown in Figure 5B. However, BMP-2 and their receptor BMPR2 while weakly expressed in the intimal layer of sham animals (Figure 5C, E) were strongly expressed in the intima and media layer of NFX-calcitriol animals, in association with areas of calcification (Figure 5 D, F). The percentage of staining for BMP-2 and BMPR2 were significantly increased in NFX-calcitriol animals versus sham-calcitriol animals: BMP-2 8.97 ± 0.92 versus 3.58 ± 1.03 (P = 0.029), BMPR2 11.7 ± 3.3 versus 2.37 ± 0.72 (P = 0.028).


Figure 5
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  		Fig. 5 Immunohistochemistry (IMH) staining for BMP-7, BMP-2 and BMPR2 from sham-operated rats (A, C, E) or 5/6 nephrectomized rats treated with calcitriol for 4 weeks (B, D, F). BMP-7 was normally expressed in the luminal side of kidney distal tubules (A); however, it is not expressed in arteries from sham or 5/6 nephrectomized rats treated with calcitriol (B). BMP-2 weakly expressed in the intimal layer of sham animals (C) was strongly expressed at the media of NFX-calcitriol rats, in association with areas of calcification (D). The receptor BMPR2 weakly expressed at the intimal layer of sham animals (E) was strongly expressed in the media of NFX-calcitriol rats, in association with areas of calcification (F).

 
Human studies
Biopsies of epigastric arteries from 14 haemodialysis and 2 peritoneal-dialysis patients undergoing renal transplantation and biopsies of renal arteries from donors were included. The mean age of the patients was 37.3 ± 3.3 years, and 10 were females. Only two patients were diabetics. The time of dialysis in 14 patients before renal transplantation was 39.5 ± 5.7 months; two patients were transplanted in the predialysis stage. The gremlin expression was studied by IMH and ISH, as is illustrated in Figure 6. Gremlin was constitutively expressed in the medial layer of a normal human vessel (Figure 6A, B). Conversely, abundant gremlin protein staining was observed in the vessels from uraemic patients as compared with normal (staining score 3.72 ± 0.95 versus 0.91 ± 0.08 percentage/mm2, P < 0.05, Figure 7). As is shown in Figure 6C, D, the gremlin protein was most prominently detected in the vascular media, and intimal neo-induction was observed. Gremlin mRNA was also strongly expressed in the vascular media in uraemic patients (Figure 6F) in relation to the control artery (Figure 6E). A strong correlation between the gremlin mRNA and protein expression was observed in the samples studied (r = 0.8; P < 0.01).


Figure 6
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  		Fig. 6 Immunohistochemistry (IMH) and in situ hybridization (ISH) for gremlin in vessels from normal humans (kidney donors) and uraemic patients. There is no expression or it is weakly observed in arteries from kidney donors (A, 10x, and B, 20x). A strong gremlin expression is observed in the media vascular of the epigastric arteries of patients in chronic dialysis (C–D, 20x). Gremlin mRNA expression is markedly observed in the media layer of arteries of uraemic patient (F, 20x). Control sense negative (E, 20x)

 

Figure 7
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  		Fig. 7 Staining score of the gremlin protein by immunohistochemistry in vascular media of normal (kidney donors) and uraemic patients (patients in dialysis). *P < 0.05 versus normal.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Vascular calcification in chronic renal disease has a complex pathogenesis that is still not completely understood. The critical role of the VSMCs has been recognized, and many underlying causes of arterial calcification initiate the process by transforming VSMCs to a chondrocyte or osteoblast-like cells. Normally, mesenchymal stem cells differentiate to adypocites, osteoblasts, chondrocytes and VSMCs. In CKD through different mechanisms that include hyperphosphataemia, elevated calcium x phosphorus product, uraemic serum, oxidized lipids, decreased levels of BMP-7 and several other factors, these VSMCs can dedifferentiate or transform into osteoblast/chondrocyte-like cells by up-regulation of transcription factors such as Cbfa1/RUNX-2 and MSX2 [12,13,18,19]. These transcription factors are critical for normal bone development, and thus their up-regulation observed in VSMCs is indicative of a phenotypic switch.

This study provides the first morphological evidence that gremlin, an antagonist of BMPs, which are involved in the vascular calcification process, could be participating in this transition. Thus, these results indicate that the developmental gene gremlin re-emerges in the context of arterial calcification suggesting that gremlin might have a role in the pathogenesis of vascular calcification, probably as an antagonist of BMPs such as BMP-7 or BMP-2.

The relevance of these findings is in relation to the accelerated cardiovascular disease (CV) observed in patients with chronic renal disease, with a CV risk of 20- to 1000-fold in patients with uraemia [28].

Vascular calcification in uraemia is now recognized not only as a passive precipitation of Ca and P in the presence of excessively high extracellular concentrations but also as an orchestrated, highly regulated process that produces a medial artery calcification and that includes three key steps: (1) presence of the traditional and uraemic-related promoters of calcification, (2) decreased circulating inhibitors of calcification (fetuin-A, BMP-7, parathyroid hormone-related peptide) and local inhibitors [MGP, osteopontin, inorganic pyrophosphate (PPi), osteoprotogerin] and (3) transdiffentiation of VSMCs towards cells with osteogenic phenotype (osteoblast/chondrocyte-like cells) expressing the major bone-specific proteins, as a key event in the process of medial arterial calcification.

Supporting a key role of VSMC's dedifferentiation into osteoblast/chondrocyte-like cells in the arterial vascular calcification, Hruska et al. demonstrated that the administration of BMP-7 decreased the arterial calcification in LDL receptor null mice [15]. In the adult, BMP-7 is expressed in the distal collecting tubule and podocyte of the kidney, where it maintains differentiated phenotypes of tubular cells through autocrine and paracrine signalling [29]. BMP-7 levels are detectable in adults suggesting a hormonal role in osteoblast function. BMP-7 expression is down-regulated early in chronic renal failure [30], and the concept of CKD has been proposed as a state of BMP-7 deficiency.

On the other hand, gremlin antagonizes effects of BMPs to form heterodimerization with BMP-2, -4 and -7, thereby inhibiting the ability of these ligands to bind to their receptors. Gremlin could mediate its action via induction of epithelial to mesenchymal feedback signalling [31], as it has been suggested that gremlin has a role in the EMT of tubular cells in renal tubule interstitial fibrosis, and in the glomerular crescent formation [22–24]. It is tempting to speculate a role for gremlin in the VSMC's dedifferentiation into osteoblast/chondrocyte-like cells, also considering the recent finding that gremlin is constitutively expressed in VSMCs of rats and is regulated by several growth factors [25].

As has been previously noted, up-regulation of transcription factors such as Cbfa1/RUNX-2 and Msx2 has been proposed as a marker of phenotypic switch to osteoblast lineage of vascular cells. BMP-2–muscle segment homeobox homologue (Msx2) signalling cascade can be activated by mural oxidative stress and inflammatory cytokines, suggesting that these signals participate in the arterial calcification as it is also observed in diabetic patients and animal model of chronic renal failure and the metabolic syndrome [17]. It has been reported that a subset of myofibroblasts in the fibro fatty aortic adventitia and aortic valve interstitium, but not the tunica media, elaborated this early BMP-2–Msx2 response. Recent studies show that vascular osteogenic signals, such as paracrine Wnt signal initiated by adventitial BMP-2–Msx2 actions, are concentrically conveyed to the calcifying tunica media. Thus, BMP-2 is a key stimulus for Msx2 expression and enhances Wnt signalling. Aortic Msx2–Wnt signalling is also stimulated by BMP-2. Moreover, BMP-2 administration for 4 weeks augments arterial calcification 2-fold in LDLR–/– mice fed with high-fat diabetogenic diets and mineral accumulation again localizes to the vascular tunica media. Thus, BMP-2 can activate a vascular Wnt signalling cascade that drives osteogenic mineralization of vascular progenitors. The strong staining for BMP-2 and their receptor in areas of vascular calcification is supporting a role of BMP-2 in this process [32–34]. Therefore, BMP-2 would be actively acting in vascular calcification, and the functional role of gremlin, as a BMP-2' effect antagonist, by heterodimerization with its ligand and thereby preventing receptor binding, must be elucidated.

In conclusion, in this study, gremlin expression is strongly associated with arterial calcification. Whether this overexpression at the medial vascular layer from uraemic patients and azotemic rats with calcitriol-induced vascular calcification is contributing to enhance mineral deposition blocking the beneficial action of BMP-7 [35] or might have a salutary effect by blocking actions of BMP-2-cascade signalling of calcification remains to be established. Studies in vitro with VSMCs are in progress to further elucidate the role of gremlin in the vascular calcification observed in chronic renal failure.



   Acknowledgments
 
This study was supported by a grant (no. 1050783 and 1080083), FONDECYT, Chile.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 11. 5.08
Accepted in revised form: 7.10.08


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