ERA

No preview available

Analytics

Share

Permanent link (DOI): https://doi.org/10.7939/R3BK16X75

Download

Export to: EndNote  |  Zotero  |  Mendeley

Communities

This file is in the following communities:

Graduate Studies and Research, Faculty of

Collections

This file is in the following collections:

Theses and Dissertations

Renal proximal tubular reabsorption of sodium, water and calcium are interconnected Open Access

Descriptions

Other title
Subject/Keyword
RENAL PROXIMAL TUBULE
RENAL SODIUM ABSORPTION
RENAL CALCIUM ABSORPTION
RENAL WATER ABSORPTION
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Krishnan, Devishree
Supervisor and department
Alexander, Todd and Departments of Physiology / Pediatrics
Examining committee member and department
Casey, Joseph and Departments of Physiology / Biochemistry
Fliegel, Larry and Department of Biochemistry
Leslie, Elaine and Department of Physiology
Kapus, Andras and Department of Biochemistry
Department
Department of Physiology
Specialization

Date accepted
2016-09-21T13:22:11Z
Graduation date
2016-06:Fall 2016
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Intravascular volume is maintained by complex interplay between organ systems with a main role for renal sodium, and water handling. The glomerulus filters a large quantity of water and salt daily with the majority of sodium, water and calcium being reabsorbed from the proximal tubule (PT). The renal reabsorption of sodium, water and calcium are interconnected. Apical influx of sodium from the PT occurs via NHE3 in exchange for a cytosolic proton. Cytosolic sodium is excreted back into the blood via either sodium potassium ATPase or a sodium dependent bicarbonate transporter. The rate-limiting step for NHE3 activity is the presence of a cytosolic proton. This is generated by cytosolic carbonic anhydrase II (CAII), an enzyme mediating the catalysis of CO2 and H2O to form HCO3- and H+. CO2 and H2O enter the PT epithelial cell through the water channel aquaporin-1 (AQP1). Osmotically driven water flux across the PT also drives calcium reabsorption from this segment, a process linked by NHE3. CAII interacts with many membrane transporters including NHE1, AE1, MCT1 and NBCe1. We identified potential CAII binding sites in both NHE3 and AQP1. A primary hypothesis of this thesis is that CAII physically and functionally interacts with both NHE3 and AQP1. CAII and NHE3 were closely associated in a renal proximal tubular cell culture model as revealed by a proximity ligation assay. Direct physical interaction was confirmed in solid-phase binding assays with immobilized CAII and C-terminal NHE3 glutathione-S-transferase fusion constructs. To assess the effect of CAII on NHE3 function, we expressed NHE3 in a proximal tubule cell line and measured NHE3 activity. NHE3-expressing cells had a significantly greater rate of intracellular pH recovery than controls. Inhibition of endogenous CAII activity with acetazolamide significantly decreased NHE3 activity, indicating that CAII activates NHE3. To ascertain whether CAII binding per se activates NHE3, we expressed NHE3 with wild-type CAII, a catalytically inactive CAII mutant (CAII-V143Y), or a mutant unable to bind other transporters (CAII-HEX). NHE3 activity increased upon wild-type CAII coexpression, but not in the presence of the CAII-V143Y or HEX mutant. These studies support an association between CAII and NHE3 that increases the transporter's activity. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII increased water flux relative to AQP1 expression alone. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring an acidic cluster of amino acids in the cytosolic tail of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Renal cortical membrane vesicles isolated from CAII-deficient mice has reduced water flux, which is measured by stopped-flow light scattering. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins. Evaluation of urinary calcium excretion from humans and mice fed altered sodium containing diets revealed that urinary calcium excretion is proportionate to sodium intake. We hypothesized that angiotensin II mediated regulation of NHE3 activity is central to alterations in urinary calcium excretion in response to altered salt intake. To assess this possibility we performed mRNA and protein expression studies on sodium transporters and tight junction proteins along the nephron involved in sodium and calcium reabsorption. We did not find a significant decrease in the expression of these transporters when animals were fed high salt diets. This diet did however decrease renal renin mRNA expression. Previous studies found that the angiotensin-I receptor (AT1R) and NHE3 are expressed in the proximal tubule. Using a cell culture model we demonstrate that angiotensin II increases NHE3 membrane localization and enhances its activity. Thus we postulate that increased sodium intake decreases renin production, and AngII levels that in turn translocate NHE3 from the brush border to an endomembrane compartment. This would decrease both sodium reabsorption from the proximal tubule and consequently increase calcium excretion in the urine. Together these studies highlight an interconnection between sodium, water and calcium reabsorption from the renal proximal tubule.
Language
English
DOI
doi:10.7939/R3BK16X75
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
Citation for previous publication

File Details

Date Uploaded
Date Modified
2016-09-21T19:22:12.069+00:00
Audit Status
Audits have not yet been run on this file.
Characterization
File format: zip (ZIP Format)
Mime type: application/zip
File size: 7681645
Last modified: 2016:11:16 14:38:06-07:00
Filename: uuid:7ab93312-26d9-4ec2-bb7e-52cc6b573408.zip
Original checksum: 58375f872f0e6fd675cd782ac95b8a5f
Activity of users you follow
User Activity Date