Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616355
Title: Characterisation of Uromodulin as a candidate gene for human essential hypertension
Author: Graham, Lesley A.
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2013
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Abstract:
Essential hypertension is a highly hereditable trait of complex aetiology, where multiple environmental and life style factors contribute to blood pressure variation. Family health studies of blood pressure suggest that heritability accounts for 30 – 50 % of variation. Consequently the study of genetic architecture has proven useful to detect a small number of genes, loci, and single nucleotide polymorphisms (SNPs) that have appreciable effects on blood pressure. Genetic linkage and association methods have long provided the foundation of gene identification in humans. Although linkage studies have proven to be highly successful in identifying genes of monogenic (or Mendelian) disorders, this analysis has minimal or limited power to detect gene of complex traits and disease. Furthermore, candidate gene approaches have not yet reported any reproducible associations with hypertension. Accordingly, gene identification efforts have become increasingly reliant on association approaches. A recent genome wide association study (GWAS) identified a locus upstream of the Uromodulin (UMOD) gene transcriptional start site, which was associated with hypertension. This group used an extreme case - control design in a discovery sample of 1,621 hypertension cases and 1,699 hypercontrols, representing the top 2% and bottom 20% of the BP distribution. The minor G allele of rs13333226 when adjusted for estimated glomerular filtration rate (eGFR) was associated with a 7 % lower risk of developing hypertension. UMOD encodes the protein uromodulin which is interchangeably known as Tamm Horsfall protein (THP). It is a kidney specific protein and is exclusively synthesised at the level of the thick ascending limb of the loop of Henle (TAL) and is the most abundant protein in human urine. The biological role of uromodulin still remains unclear; however other UMOD variants have been associated with chronic kidney disease. Due to UMODs exclusive expression at the kidney it may have a role in regulating blood pressure via sodium homeostasis mechanisms. As hypertension is characterised by a disturbance of renal function that subsequently leads to an augmented Na+ reabsorption, the present study aimed to follow up the GWAS signal to assess whether altered 18 UMOD expression and/or function impacts on sodium homeostasis and influence blood pressure phenotypes. Promoter activity assays here demonstrate that the index SNP (rs13333226) is not a functional variant causing altered transcription, however the minor G allele of rs13333226 is associated with reduced promoter activity. These findings are consistent with the original GWAS study that this allele is associated with lower risk of hypertension. In this study we reported a SNP in LD with rs13333226 within the 2 Kb promoter region (rs4997081) that may be a causal variant altering transcriptional activity of UMOD. Furthermore, with computational and experimental evidence we show that binding of rs4997081 to TFAP2A in a genotype dependent manner leads to transcriptional changes of UMOD which were associated with altered sodium reabsorption via downstream signalling of Tumor necrosis factor alpha (TNF-α). Cardiovascular characterisation of UMOD knockout mice (KO) revealed significantly lower systolic blood pressure (SBP) in comparison to the wild type (WT) counterparts. The reported novel blood pressure phenotypes in the KO mice were not sensitive to change by salt loading (2% NaCl) over a six week period. KO mice displayed increased concentrations of sodium in the urine upon salt loading, to greater levels than the WT mice (± 2% NaCl). Urinary electrolyte analysis corrected to creatinine levels revealed augmented sodium loss in the KO mice during the high salt diet. Chronic renal function curves demonstrate that the reduced SBP is attained by increased natriuresis via augmented GFR in the KO mice. Histological examination illustrated cellular swelling and papillary oedema in the KO mice before and after salt loading which may be triggered by the pro-inflammatory cytokines TNF-α and Interleukin 1 (IL-1) according to metabolomic analysis. These inflammatory signals may affect Na+ homeostasis at the TAL in the KO mice by reducing NKCC2 expression. Expression analysis studied in outer medulla tissue illustrated down regulation of the major NaCl transporters in the absence of UMOD which were further attenuated upon salt loading conditions possibly by increased levels of TNF-α at the TAL. KO mice displayed increased levels of urinary TNF-α in addition to augmented mRNA abundance in the outer medulla tissue. In addition, immunohistochemical analysis revealed reduced NKCC2 staining with increased TNF-α staining in renal tissue of the KO mice during normal and high salt diets. These results were 19 confirmed in vitro and suggest UMOD acts as a negative regulator of TNF-α production by the TAL to maintain NaCl/volume homeostasis. We have confirmed with a small pilot study using human renal tissue samples from normotensive and hypertensive individuals, that in times of altered UMOD expression there are changes in NKCC2 and NHE3 expression levels, but not TNF- α. More interestingly we have demonstrated that UMOD, NKCC2, and NHE3 expression levels are altered in a genotype dependant manner, in that the minor G allele of rs13333226 appears to be associated with blood pressure via altered sodium homeostasis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.616355  DOI: Not available
Keywords: Q Science (General) ; QP Physiology
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