Diabetes İnsipidus’lu Hastaların Aqp2 Geninde Tanımlanan Mutasyonların Fonksiyonel Analizleri

Abstract

ABSTRACT FUNCTIONAL ANALYSIS OF MUTATIONS FOUND IN AQP2 GENE OF PATIENTS WITH DIABETES INSIPIDUS TUĞÇE KARADUMAN Master of Philosophy, Department of Biology Supervisor: Prof. Dr. Hatice MERGEN June 2018, 111 pages

The water homeostasis in the body is provided by balancing water loss and intake. Arginine vasopressin (AVP, ADH, antidiuretic hormone) is a hormone synthesized in the hypothalamus region of the brain and stored in the posterior part of the pituitary gland to be secreted when stimulated. In Diabetes Insipidus (DI) patients, production and activity of this hormone is reduced. This hormone regulates a vital function by increasing the reabsorption of water in the kidney collecting channel. Once the AVP hormone is secreted, AVP bounds to the arginine vasopressin receptor 2 (AVPR2) which is a transmembrane protein localized on the basolateral membrane of polarized epithelial cells of kidney nephron. AVPR2 is a G protein bound receptor (GPCR). Thus, G protein becomes active and activates adenylate cyclase. As a result, produced cAMP stimulates protein kinase A. Protein kinase A phosphorylates the aquaporin (AQP2) proteins which are present in the cell to enable localization of these proteins in apical membrane in tetramers. Absorption of water from the urine to the kidney cells is ensured through these channels. When the water reabsorption stimulus is over, AQP2 is removed from the membrane by endocytosis. Any pathology that may occur within this control system causes impairment of water homeostasis of the body and generation of DI.

AQP2 is a homotetrameric water channel responsible for the reabsorption of water in the kidney's main collecting duct cells. Mutations in AQP2 gene induce nephrogenic DI (NDI), a pathogenic condition in maintaining body water homeostasis, causing excess volume of urine to be produced. There are several hypotheses that have been proven by clinical and experimental observations in the literature regarding the role of these mutations in the pathogenesis of NDI. It has been proven that AQP2 gene mutations generally lead to misfolding of mutant protein or misleading of this protein; It has also been reported that nonfunctional water channels indicate correct membrane targeting. The purpose of this study is to perform functional analysis of A45T, R85X and A147T mutations identified in the AQP2 gene in NDI patients. It is aimed to contribute to the treatment approaches for the development of pharmacological agents which can be more effective in the treatment of the disease through the evaluation of the results obtained from the experimental studies in correlation with the clinical information of the patients. For the purposes of this study; expression vector carrying the wild-type coding AQP2 sequence was provided and appropriate expression vectors containing mutant AQP2 gene sequences were prepared using the site-directed mutagenesis method. Following this process, stable transfection of wild-type and mutant vectors into MDCK cells was performed. Deglycosylation experiments were conducted to investigate the intracellular maturation process of mutant proteins by expression of the vectors in MDCK cells. Cycloheximide analysis was also performed to determine the half-lives of the proteins and the results of both the deglycosylation experiments and the cyclohexamide experiments were evaluated by immunoblot analysis. Immunocytochemical analyses were performed to determine the intracellular traffic of mutant AQP2 proteins. Within the framework of functional analysis studies performed on the Xenopus laevis oocyte expression system of AQP2 gene mutations, oocyte expression vectors containing wild type and mutation were injected into oocyte cells and total membrane and plasma membranes were isolated in order to determine the expression levels of AQP2 protein in oocytes; wild type protein and mutant proteins membrane ratios were compared through immunoblot analysis. To determine the effects of mutant AQP2 proteins on water uptake mechanism, oocyte cells were tested for water permeability. As a result, within the framework of this thesis, all mutant AQP2 proteins showed alteration of function compared to the wild type AQP2.

Keywords: Diabetes insipidus, AQP2, functional analysis