THIAZIDE TREATMENT EFFECTS ON NCC ABUNDANCE AND PHOSPHORYLATION IN 2 FHHT PATIENTS

A WU1, M WOLLEY1,2, Q WU3, R GORDON1, R FENTON3, M STOWASSER1

1Endocrine Hypertension Research Centre, University of Queensland Diamantina Institute, Greenslopes and Princess Alexandra Hospitals, Brisbane, Australia, 2Royal Brisbane and Women’s Hospital Department of Nephrology, Brisbane, Australia, 3Aarhus University Department of Biomedicine, Aarhus, Denmark

Aim: To analyse the proteome in urinary extracellular vesicles (uEVs) isolated from 2 FHHt patients before and after thiazide diuretic treatment.
Background: Mutations responsible for familial hyperkalaemic hypertension (FHHt) cause over-activation of NCC in the DCT, leading to a phenotype which is a mirror image of Gitelman syndrome and which is reversed by thiazide treatment. Recent evidence in human suggests NCC is downregulated by plasma K+.
Methods: Urine samples were collected before and after thiazide treatment in 2 male FHHt patients. uEVs were harvested by ultracentrifugation and analysed by label-free LC-MS/MS and western blotting.
Results: In both patients thiazide treatment corrected hyperkalaemia (5.6 to 4.6mmol/L in patient 1 and 6.2 to 4.8mmol/L in patient 2), and reduced aldosterone (1120 to 502pmol/L in patient 1 and 910 to 210pmol/L in patient 2) and the aldosterone-to-renin ratio (106 to 31 in patient 1 and 114 to 23 in patient 2)
In uEVs isolated from patient 1, label-free LC-MS/MS demonstrated an increase in NCC (IBAQ ratio from 0.05% to 0.63% after 55-day thiazide treatment. There were consistent increases in the relative abundance of NCC (to uEV marker ALIX, 1.49 vs 6.11) in western blotting. There were also increases in pNCC (1.49 vs 9.12) and pNCC/NCC ratio (1.21 vs 1.49). In patient 2 relative abundance of NCC decreased from 6.74 to 2.36, pNCC increased from 2.99 to 4.59, and pNCC/NCC ratio increased from 0.41 to 1.49.
Conclusions: In the two FHHt patients, thiazide treatment corrected hyperkalaemia by inhibiting NCC function. But there were varying degrees of increase in relative abundance of pNCC and pNCC/NCC ratio in uEVs, which may related to the decreased plasma K+ level.


Biography:
Aihua is a Third-year PhD student interested in the regulation of sodium chloride cotransporter (NCC) in health and disease of salt homeostasis.

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