Abstract 08: Functional Characterisation Of Uromodulin Promoter Risk Variants Associated With Blood Pressure

Abstract

Using a genome wide association study (GWAS), we identified a locus on the promoter of the human uromodulin (UMOD) gene associating with primary hypertension. In this study we aimed to identify genotype-differential protein binding partners which influence UMOD expression and to examine pathways linking UMOD expression to blood pressure variability in humans.We examined a variant on this locus, by incubating 50mer oligonucleotides featuring alternate alleles (C/G) with HEK-293 nuclear protein extract. Subsequently, we performed an electrophoretic mobility shift assay (EMSA) and purified protein complexes to analyse by Tandem-mass-spectroscopy (MS2). Filtering for peptide matches >20, we interpreted these data by Ingenuity Pathway Analysis (IPA). From a bank of 98 human renal biopsy samples, we assessed UMOD expression by qRT-PCR. RNA-seq was performed using the NextSeq-500 system on a subset of high and low UMOD expressors (n=3/ group). Differentially expressed (false-discovery-rate (FDR) <0.05) genes were identified, with strong correlations in these data validated by qRT-PCR. Implicated pathways were interpreted using both IPA and gene set enrichment analysis (GSEA).The C allele at our risk variant displayed greater binding affinity versus the G allele; 0.77 (0.13, 1.40, 95%CI). In contrast no difference in binding affinity was observed between the T and C alleles of our control variant, 0.08 ( -1.22, 1.39, 95%CI). Mass spectroscopy of EMSA complexes identified PARP1 binding at this target. By IPA we identified a complex between PARP1 and HNF1β as driving expression of UMOD. RNA-sequencing identified positive correlation in expression of UMOD and the Na-K-Cl cotransporter (NKCC2) (FDR=2.58e-9, log2-fold-change [L2FC]= 3.3), the renal outer medullary potassium channel (ROMK) (FDR=3.69e-2, L2FC= 2.79) and the epithelial sodium channel (ENaC) (FDR=1.83e-2, L2FC= 1.25). We validated this coexpression between UMOD and NKCC2 by qRT-PCR and show a significant correlation (p=2.2e-16, R2= 0.844, n=84).Together these data suggest our risk variant contributes to differences in UMOD expression via a PARP1:HNF1β transcriptional complex and these differences associate with blood pressure through sodium homeostasis in the human kidney.