Characterisation of thiazide-sensitive pathways of sodium reabsorption in the kidney
Publication Date
July 24, 2020
Creator
Abstract
With increasing individuals developing hypertension and with no known cause, it is becoming ever more vital to research how our blood pressure is regulated throughout the body. The kidney is often a forgotten regulatory organ for blood pressure, however, it’s biochemical role for increasing and decreasing pressure within the circulatory system is essential to maintain a healthy balance. This thesis aims to further characterise the functions of thiazide-sensitive sodium-chloride cotransporter located within the distal nephron of the kidney using urinary and cell culture exosomes. The co-transporter plays a key role in regulating blood pressure for genetic conditions like Gordon Syndrome and Gitelman Syndrome, providing the possibility to regulate blood pressure in essential hypertension.
As a source for protein and nucleic acids, the use of urinary exosomes is a novel non-invasive procedure. Urinary exosomes were extracted from urine and cell culture media allowing the collection of genetic and proteomic information from the kidney tubule cells. Specific biomarkers have been identified, in order to ensure the study of the area of interest for example the thiazide-sensitive sodium-chloride co-transporter (NCC) or ALG-2-interacting protein X (ALIX).
Validation protein work in chapter 3 was performed to determine the optimal conditions for exosomal fraction storage and quantify the exosomal fraction from the ultrafiltration extraction method, using NCC and phosphorylated NCC. It was identified that ALIX should be used as an exosomal marker over Aquaqporin-2 (AQP2). These validation methods were applied to the rest of the thesis and the communication experiments presented in Chapter 3. These communication experiments aimed to show exosomes as a communication vessel by transferring exosomal NCC into Proximal Kidney Tubule Cells (PKTC). Despite initial findings showing that exosomes can transfer NCC to PKTC, it was concluded further experiments were needed to confirm if NCC had transferred successfully and was physiologically functional.
Using ultra-filtration methods demonstrated in Chapter 3, exosomes were extracted from donated normonatraemic, non-thiazide patient urine to demonstrate the influence dietary electrolytes have on the expression and activation of NCC. In Chapter 4, 24hr electrolyte excretion was used as an alternative to electrolyte intake, which were then correlated to NCC and other relatable proteins. Decreasing levels of expression were seen in phosphorylated NCC when correlated with serum potassium producing a negative correlation, but the opposite was observed when serum potassium was compared to NCC3 or NCC1/2. In addition to serum potassium, 24hr urinary potassium did not correlate with NCC or phosphorylated NCC. In addition to potassium, other electrolytes e.g. chloride were correlated with NCC. It was identified that serum chloride negatively correlated with NCC3, but not with phosphorylated NCC or NCC1/2.
It has long been reported that exosomes hold Ribonucleic acid (RNA), which may be used as a form of communication between cells. However, extracting viable messenger RNA (mRNA) has proven difficult due to its instability outside of the exosomal environment. The studies presented in this thesis attempt various methods to extract the best quality and quantity of mRNA. The top four methods for extracting mRNA were followed up with Next Generation RNA Sequencing (performed by Oxford Genomics) in attempt to detect exosomal RNAs. Although the extracted exosomal RNA was poor quality, RNA sequencing was sensitive enough to report protein coding genes. Some of the RNAs detected by RNA sequencing were validated using Quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR). The combination of RNA sequencing and qRT-PCR identified WNK Lysine Deficient Protein Kinase 1 (WNK1) and Cullin-3 (CUL3) from the WNK-SPAK/OSR1-NCC pathway.
RNA sequencing studies were continued to compare RNA expression between Acute and Convalescent Thiazide-Induced Hyponatremia (TIH) patients and control groups using the Seramir RNA extraction from Chapter 5’s validation experiments. Additional RNase inhibitor was used to improve RNA quality. No known RNAs associated with TIH were identified despite using the hypothesis free method and the candidate approach method. It may be that despite attempts to reduce degradation, the exosomal RNA suffered due to this factor, hence why no associated genes were identified. However, glucose-related proteins, GYS2 and SLC5A2, were reported as differentially expressed between acute and convalescent TIH samples, suggesting that glucose channels may play a role in the development of TIH. Further pathway analysis, as well as genomic and proteomic analysis would help identify with these glucose-related proteins affect the pathophysiology of TIH patients.
Overall, the studies presented in this thesis provide further evidence towards thiazide-sensitive sodium-chloride cotransporter’s relationship towards dietary electrolytes within a healthy human body, whilst also showing that exosomes can be used to characterise protein expression of the thiazide-sensitive sodium-chloride cotransporter and its regulatory proteins. The work carried out in this thesis has commented on the advantages and disadvantages of working with urinary exosomes, but it has also contributed to the growing knowledge of how these extracellular vesicles can be used to study the intracellular functions of cells without invasive biopsies.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Medicine
eprints ID
59378
UoN Repository URI
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Thesis - revised from viva.2.pdf
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