P0873GLA-RICH PROTEIN (GRP) AS AN EARLY AND NOVEL MARKER ASSOCIATED WITH VASCULAR CALCIFICATION AND CKD-MINERAL AND BONE DISORDER (MBD) IN DIABETIC PATIENTS WITH CKD: A PILOT COHORT STUDY
Authors: Viegas Carla , Silva Ana , Macedo Ana , Mendes Filipa , Guilherme Patrícia , Tavares Nelson , Dias Carolina , Rato Fátima , Santos Nélio , Faísca Marília , De Almeida Edgar A F , Neves Pedro , Simes Dina 
Published: June 2020

Abstract

Background and Aims
Cardiovascular disease (CVD) is the most life-threatening complication in chronic kidney disease (CKD) patients. In addition to traditional risk factors, most patients with CKD display abnormal mineral metabolism with underlying hormonal dysregulation, defined as chronic kidney disease-mineral and bone disorder (CKD-MBD). CKD-MBD involves changes in mineral ion homeostasis, bone quality and turnover, cardiovascular and soft tissue calcifications, which highly contribute for cardiovascular complications. Vascular calcification (VC) is associated with significant morbidity and mortality and a strong predictor of cardiovascular risk in CKD patients. Early preventive measures, including new diagnostic/prognostic tools, are required to reduce the development and progression of VC, left ventricular hypertrophy and arterial stiffness, which are crucial for the prevention of CVD outcomes in CKD patients. Gla-rich protein (GRP) is a vitamin K-dependent protein with a dual capacity to function as an inhibitor of pathological calcification and anti-inflammatory agent in the cardiovascular system, whose clinical utility is unknown. Our aim with this study was to evaluate the potential of GRP as a new marker for CKD-MBD and vascular calcification, in type 2 diabetic patients with chronic kidney disease (CKD) stages 2-4.

Method
In an observational prospective study including all eligible type 2 diabetic patients with CKD stages 2-4 (n=80) followed in outpatient nephrology consultation from 2010 to 2017, we explored correlations between levels of GRP in serum with mineral metabolism and inflammation markers, CKD developmental stage, vascular calcification and pulse pressure (PP). Vascular calcification score (VCS) was evaluated using the plain x-ray of the hands and pelvis (Adragão score), and increased cardiovascular risk was considered for VCS≥3. Measurements of GRP in serum were performed using a recently developed sandwich ELISA assay. Descriptive statistics, ANOVA and post hoc analysis with Scheffe test were used for analysis. Forward stepwise logistic regression (likelihood ratio) analysis was applied to identify predictive factors for VCS and PP, and ROC curves were used to assess the sensitivity and specificity of GRP in relation to these exposure factors.

Results
Spearman’s correlation analysis revealed the strong positive correlation between levels of serum GRP and eGFR (r=0.863, p&lt;0.0001) and α klotho (r=0.647, p&lt;0.0001), while a negative correlation with phosphate (P) (r=-0.715, &lt;0.0001), FGF23 (r=-0.676, &lt;0.0001), VCS (r=-0.822, p&lt;0.0001), PP (r=-0.533, p&lt;0.0001), calcium x phosphate (CaxP) (r=-0.302, p=0.006) and IL-6 (r=-0.349, p=0.002). Serum GRP levels were found to progressively decreased from stage 2 to stage 4 CKD. Multivariate analysis identified low levels of eGFR and GRP, and high levels of FGF-23 as independent risk factors for both the VCS and PP. The area under the ROC curves for GRP was 0.865±0.046, 95% CI (0.776-0.955), p&lt;0.0001 for VCS and 0.782±0.054, 95% CI (0.677-0.887), p&lt;0.0001 for PP.

Conclusion
Reduced levels of GRP were associated with higher levels of vascular calcification promoters such as P, FGF-23 and CaxP, and with lower levels of the VC inhibitor α-Klotho, indicating a correlation between GRP and the dysregulation of phosphate metabolism characteristic of CKD-MBD. In addition, this pilot cohort study indicates that GRP levels might be a significant clinical predictor of vascular calcifications in diabetic patients with CKD.

Funding
This research was funded by the Portuguese Society of Nephrology (SPN) through project funding 2016, by the Portuguese national funds from FCT - Foundation for Science and Technology through the transitional provision DL57/2016/CP1361/CT0006 and project UID/Multi/04326/2019.

Acknowledgments
To SPN by the attribution of the Jacinto Simões award (2018) financed by Fresenius medical care.