Characterisation of Uromodulin as a candidate gene for human essential hypertension

Graham, Lesley A. (2013) Characterisation of Uromodulin as a candidate gene for human essential hypertension. PhD thesis, University of Glasgow.

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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
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
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
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.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > Q Science (General)
Q Science > QP Physiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Life Sciences
Supervisor's Name: McBride, Dr. Martin W.
Date of Award: 2013
Depositing User: mrs lesley graham
Unique ID: glathesis:2013-5215
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 07 Jul 2014 14:00
Last Modified: 07 Jul 2014 14:01

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