Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Regulatory Biology

Department

Biological, Geological, and Environmental Sciences

First Advisor

Renick, Andrew

Subject Headings

Biology; Biomedical Research; Biophysics; Cellular Biology

Abstract

Kidney cyst expansion, stagnant fluid accumulation, and insufficient vascular supply can result in localized chronic ischemia-hypoxia in kidney cysts, as well as in normal renal epithelia adjacent to a cyst. We hypothesize that in normal epithelia near a cyst, the stabilization of Hypoxia Inducible Factor 1a (HIF1a), a major regulator of cellular response to hypoxia, can cause altered paracellular and transcellular transport, transforming a normal absorptive phenotype to a secretory and paracellularly leaky phenotype, leading to cyst expansion. Using 100 µmol/L cobalt chloride (CoCl2), HIF1a was stabilized in cellular nucleus of a mouse cortical collecting duct cell line (mCCD 1296 (d)), which resulted in an increased level of erythropoietin, an effector and reporter molecule of HIF1a. The mCCD monolayers have a high transepithelial resistance (TER) value (¿ 3000 O-cm2) and around 95% amiloride-sensitive voltage value. Equivalent current was calculated to compare active ion transport. Our results showed that TER values decreased significantly after 48 and 72 hours of HIF-stabilization. The decrease of TER value was consistent with the increase in the permeability of 70 kDa FITC-dextran molecules, supporting the hypothesis that HIF-stabilization altered paracellular transport. Stabilization of HIF caused a significant decrease in the protein level of zonula occludin 1 (ZO1), which controls paracellular transport through tight junctions. Decrease in the ZO1 protein level was consistent with the decreased TER value and the increased paracellular permeability. Similarly, HIF-stabilization was found to increase paracellular permeability in Mardin-Darby Canine Kidney (MDCK) epithelial monolayers. In mCCD monolayers, HIF-stabilization for 48 hours caused loss of active sodium ion (Na+) transport, and very interestingly, 72 hours of HIF-stabilization caused a switch in the direction of net active ion transport. HIF-stabilization caused a significant decrease of protein level of sodium-potassium-ATPase (Na+/ K+-ATPase) a1 subunit, the catalytic subunit of the enzyme responsible for active Na+ transport, consistent with the loss of active transport of Na+. Our results indicate that HIF-stabilization can transform a normal absorptive epithelium to a paracellularly leaky and cyst-like secretory epithelium by reversing net Na+ transport and increasing monolayer permeability due to alterations of tight junctions, and thereby HIF-stabilization may contribute to cyst expansion.

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