#6643 MOLECULAR PATHWAYS ASSOCIATED WITH VASCULAR CALCIFICATIONS IN CHILDREN WITH CHRONIC KIDNEY DISEASE
Authors: Bernardor Julie , Bartosova Maria , Zhang Conghui , Marinovic Iva , Herzog Rebecca , Kratochwill Klaus , Farlay Delphine , Jaminon Armand , Schurgers Leon , Schmitt Claus Peter 
Published: June 2023

Abstract

Background and Aims
Patients with advanced stages of chronic disease (CKD) suffer from 30-times higher risk of cardiovascular disease (CVD) as compared to the general population, vascular calcifications are prevalent. Underlying molecular mechanisms are only partly understood. Children are devoid of life-style and aging related CVD risk factors and therefore allow for a highly sensitive and specific analyses of CKD induced pathomechanisms.

Method
Arteriolar tissues of 55 children with normal renal function and 29 children with CKD5 (median age 9.2 years) underwent digital histomorphometry, calcium deposits were assessed by Von Kossa staining and 18F-sodium fluoride positron emission tomography (18F-NaF PET). Gene set enrichment (GSEA) and Ingenuity pathway analysis were performed on multi-omics data sets obtained from micro-dissected omental arterioles from children with normal renal function and with CKD5 (n = 7/group; age 7.3 ± 3/6.8 ± 3 years). Based on literature review, we established a vascular calcification (VC) pathway library comprising 442 biological processes/molecular functions and extracted linked genes from Gene Ontology database. Key identified mechanisms were validated in independent patient cohorts (n = 32) and controls (n = 20) by quantitative immunostaining.

Results
Significant arteriolar lumen obliteration was present in CKD5 children (p = 0.001), intima and media thickness were increased (p&lt;0.0001 and p = 0.02). Von Kossa staining was negative, but 18F-NaF PET revealed microcalcifications in children with CKD5. Subendothelial arteriolar inflammatory cells (CD68+ macrophages) were present in CKD5 cohort (p&lt;0.0001 compared to non-CKD controls) and correlated with serum inorganic phosphorus (R2 = 0.18; p = 0.04). GSEA identified top enriched pathways including telomere extension by telomerase and chromatin histone methylation. IPA cross-omics showed suppression of actin cytoskeleton, tight junction signaling, and focal adhesion. VC pathway analysis identified 30/442 pathways related to actin cytoskeleton, Wnt signaling, extracellular matrix (ECM) organization, complement activation, apoptosis, endoplasmic reticulum stress and ossification regulation. Fibronectin-1 (FN1) was identified as Hubgene, involved in ECM regulation. In independent age-matched cohorts, complement factor C3d was higher in CKD5, vascular endothelial growth factor and endothelial cell number/µm endoluminal circumference were reduced. Arteriolar osteoglycin, a protein inducing ectopic bone formation, was increased and correlated with serum PTH (R2 = 0.61; p = 0.01). FN1 was less abundant in arterioles from CKD5 children and negatively correlated with serum creatinine levels (R2 = −0.62; p = 0.02).

Conclusion
Vascular disease and microcalcifications are present in young children with CKD5. We provide the first comprehensive analysis of the underlying molecular mechanisms associated with the vascular calcifications and identified specific molecular pathomechanisms, of which several represent potential therapeutic targets.