#2909 Molecular mechanisms of early vascular damage in children with chronic renal failure

Abstract

Abstract Background and Aims The risk of developing cardiovascular disease is exceedingly high in patients with chronic kidney disease (CKD), therapeutic options are limited. In order to develop specific therapies, a comprehensive molecular understanding of the underlying pathomechanisms is essential. Method Transcriptome and proteome analyses followed by pathway analyses (GO, GSEA) were performed from microdissected omental arterioles of 6 children with normal renal function and 8 children with CKD5 (median age 9.2 years), respective microproteomics were performed from laser microdissected endothelial layers. Key findings were validated independently by immunohistochemistry. Human endothelial umbilical vein cells (HUVEC) were exposed to plasma from children with normal renal function and children with CKD5. Tight junction (TJ) abundance was quantified in Transwells, ionic conductance by transendothelial resistance measurements and chromatin topology by Single Molecule Localization Microscopy (SMLM) followed by cluster/persistent homology analyses. The latter was compared to ion radiation induced chromatin re-organisation. Results Arteriolar lumen narrowing was already pronounced in children with CKD5 (lumen/vessel ratio; 0.58 vs. 0.76 in healthy individuals, p < 0.001), arteriolar intima and media thickness significantly increased. Biofunctions significantly regulated in CKD compared to controls at both transcriptome and proteome levels were immune response, chromatin organisation, actin cytoskeleton and TJ organization (FDR<0.25, p < 0.05). In independent cohorts of children with CKD5 (n = 20), the subendothelial space was infiltrated by CD68+ macrophages and endothelial histone2A abundance was reduced compared to children with normal renal function (n = 16). Repair foci at the places of DNA double strand breaks (DSB) visualized by yH2A.X were similar in both groups. HUVEC exposed to plasma from CKD5 children showed marked relaxation of H3K9 labeled heterochromatin compared to HUVEC exposed to plasma from children with normal renal function, and persistent homology analysis revealed CKD specific chromatin pattern. The CKD induced chromatin re-organisation differed from ion radiation induced DSB repair. Polarized endothelial cells exposed to plasma from CKD5 children had increased ion transport compared to cells exposed to plasma from healthy donors. Endothelial cell membrane abundance of the sealing TJ protein claudin-5 and of zonula occlundens-1, which links TJ to the actin cytoskeleton, was reduced by uremic plasma, TNFα and carbonyl stress. Co-incubation with histidine dipeptides and AlaGln ameliorated the loss of endothelial barrier function. Conclusion CKD5 leads to complex molecular pathomechanisms and functional alterations particularly including the immune response, chromatin re-organization and endothelial barrier dysfunction already in young children. Dipeptides represent potential therapeutic agents to protect the endothelial barrier function.