#3257 EFFECTS OF CKD ASSOCIATED GENETIC VARIATION ON TRANSCRIPTOMIC REGULATION OF RENAL CELLS
Authors: van Dijk Christian , Chrifi Ihsan , Verhaar Marianne , Duncker Dirk , Cheng Caroline 
Published: June 2023

Background and Aims

Chronic kidney disease (CKD) is an universal health problem characterized by a decline of renal function. This progressive disease leads to kidney failure and CKD patients have a high risk of cardiovascular diseases, including endothelial dysfunction in the heart. Genetic variability in the general population has a strong impact on the risk of CKD development. Genome wide association studies (GWAS) already identified more than 250 single nucleotide polymorphisms (SNPs) associated with renal function. These SNPs are studied in relation with their location in the genome in protein coding regions, however, genetic variability in the genome could also affect the function of DNA regulatory elements (DREs). These non-protein coding regions regulate transcriptional expression of genes by transcription factor binding. These elements are ubiquitously present in the genome and can regulate promotor activity from a long distance (&gt;100 kb from the start site) via 3D chromatin folding. DRE activity is cell type dependent and also changes in different conditions (e.g. disease). Therefore, genetic variability e.g. SNPs located in DREs and their regulation of gene transcription could play a key role in the development and progression of CKD. Previous studies identified more than 30 DREs localized SNPs in renal cells which could be important in CKD development. We focus on SNP rs881858, a CKD associated SNP region without a protein coding sequence. Moreover, vascular endothelial growth factor (VEGF) is identified as target gene of this SNP which could therefore impact renal cell-endothelial cell interaction. Six possible active DREs were identified in this rs881858 DNA region. We hypothesize that the CKD associated SNP rs881858 induce DRE mediated transcriptional changes that contribute to the development and progression of CKD. We aim to study the effect of the DREs in DNA region rs881858 on transcriptional gene regulation in renal cells.

Method

All variations of the genetic variability in 6 identified DREs in DNA region rs881858 were introduced by site-directed mutagenesis. These variations including the reference variant, were cloned into a luciferase reporter plasmid with a minimal promoter and subsequently transfected in human embryonal kidney (HEK) cells. Luciferase activity was measured using spectrophotometry and represent DRE regulation of transcription. A decline in luciferase activity of one of the variants in a DRE imply a reduced activity of that specific variant compared to the reference variant. To assess the impact of the DREs in the genome, a CRISPR/Cas9 construct was made to knockout each individual DRE on the risk locus rs881858.

Results

We considered a DRE functional if (almost) all variants within this DRE demonstrate a change in luciferase activity. Based on this, we identified 3 DRE DNA regions in which SNPs gave a decline in luciferase activity. CRISPR/Cas9 mediated knockout of DRE in the HEK cells was validated using PCR and qPCR analysis of SNP rs881858 associated genes is planned to confirm the DRE mediated transcriptomic regulation of these genes.

Conclusion

These primary results shows that CKD associated SNP rs881858 has a negative impact on DRE function compared to the reference variant. Next we will study the effect of DRE mediated gene transcription in the DRE knockout lines using qPCR. Furthermore, CRISPR/Cas9 mediated DRE-knockout will be performed in an iPSC derived renal organoid model. Since this in vitro model consists of both renal and vascular cell types, the interaction between these cell types can be evaluated in a background of epigenetic variability. This study provides new knowledge in the effect of genetic variant in the development of CKD. Results could lead to identification of novel pathways and therefore personalized strategies for intervention. This method is a stepping stone for studying other known genetic variants in other diseases, including renal and cardiovascular diseases.