Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

Pablo Ortiz

Abstract

NaCl absorption by the Thick Ascending Limb (TAL) is mediated by the apical Na+/K+/2Cl- co-transporter, NKCC2. Increased NKCC2 activity and apical trafficking are associated to salt sensitive hypertension in rodents and humans. NKCC2 endocytosis is important for maintaining surface NKCC2 such that blocking NKCC2 endocytosis increased NKCC2 surface abundance and NKCC2-mediated NaCl reabsorption. Despite its importance, NKCC2 endocytosis has been poorly studied and a part of the reason may be attributed to the lack of availability of methods with good time resolution. Hence, we developed a method to image apical NKCC2 to monitor its endocytosis in real-time by Total Internal Reflection Fluorescence (TIRF) microscopy. NKCC2 endocytosis is regulated by protein-protein interactions but only one protein has been characterized and shown to regulate NKCC2 endocytosis by interacting with a 71 amino acid region in the carboxyl terminus of NKCC2 (C2-NKCC2). To identify new TAL proteins that may mediate NKCC2 endocytosis, we performed a proteomics-based screening of TAL proteins that interacted with glutathione-S-transferase-C2-NKCC2 as bait, and identified Alström syndrome 1 (ALMS1) as an interacting partner. ALMS1 has been linked to human hypertension and renal function in genome-wide association studies, QTL in rats with salt sensitivity and mutations in the ALMS1 gene in humans causes obesity, cardiomyopathy, type-2 diabetes, hypertension and kidney disease. The role of ALMS1 in regulating blood pressure or renal Na+ handling is unknown. GST pull down with C-terminus of ALMS1 (C-ALMS1) pulled down full length NKCC2 along with several proteins known to be involved in endocytosis of other proteins. Thus, we hypothesized that ALMS1 is involved in NKCC2 endocytosis in the TAL, NaCl reabsorption and blood pressure regulation and generated ALMS1 knockout rat model in collaboration with Medical College of Wisconsin. We found that in (6-12 week old) ALMS1 KO rats, NKCC2 endocytosis was decreased and this correlated with higher NKCC2 surface abundance in TALs. ALMS1 KO rats also exhibited higher urine concentrating capacity, higher bumetanide sensitivity, a decreased ability to eliminate a volume/salt load, hypertension and salt sensitivity in these rats. Altogether, this phenotype is consistent with increased TAL function. Thus, ALMS1 interacts with NKCC2 in the TAL, mediates its endocytosis to regulate NKCC2 surface expression and activity in the TAL and thereby maintains normal blood pressure. In addition, ALMS1 KO rats develop metabolic syndrome as they age (16-18 week old) and females exhibit more severe form of metabolic dysfunction compared to their male counterparts. The mechanism causing salt-sensitive hypertension in ALMS1 KO rats may be in part due to higher TAL NaCl reabsorption and higher NKCC2 activity. However, it is possible that insulin resistance and obesity that develops in these ALMS1 KO rats may play a role in salt sensitivity. Thus, ALMS1 is not only important for renal function but may also be involved in the regulation of glucose homeostasis and metabolism in rats.

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