Stabilization of Hypoxia Inducible Factor by Cobalt Chloride Can Alter Renal Epithelial Transport

Abstract

Around 600,000 people in the United States alone have polycystic kidney disease (PKD) which is characterized by the formation of fluid‐filled pouch‐like structures, called cysts. These cysts tend to expand over the years, finally leading to kidney failure. Previously, kidney cysts were reported to be hypoxic [1]. Cyst expansion, stagnant fluid accumulation, and insufficient vascular supply can result in localized chronic ischemia‐hypoxia in cysts and as well as in normal epithelia adjacent to a cyst. We hypothesize that in normal epithelia near a cyst, the stabilization of Hypoxia Inducible Factor 1α (HIF1α), a major regulator of cellular response to hypoxia, can cause altered paracellular and transcellular transport in normal renal epithelia, transforming the normal absorptive epithelia to a secretory and leaky phenotype, leading to cyst expansion.Using cobalt chloride (CoCl2), HIF1α 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 molecule of HIF1α. The mCCD monolayers have high transepithelial resistance (TER) value around 3000 Ω‐cm2 and around 95% amiloride sensitive voltage value. Equivalent current (Ieq) was calculated to compare active ion transport and FITC‐conjugated dextran molecules (3 and 70 kDa) were used to quantify transepithelial transport in terms of permeability values. MTT assay was used in order to make sure CoCl2 treated and untreated groups maintained same levels of metabolically active cells. Our results showed that TER values decreased significantly after 48 and 72 hours of HIF stabilization by CoCl2. The decrease of TER value was consistent with the increase in the permeability of large‐size 70 kDa FITC‐dextran molecules, supporting HIF stabilization actually altered paracellular transport. Western blot analysis showed HIF stabilization caused a significant decrease in the protein level of one of the major tight junction proteins, zonula occludin 1 (ZO1). Decrease in the ZO1 protein level was consistent with the decreased TER value and the increased paracellular permeability. HIF stabilization by CoCl2 for 48 hours caused almost complete loss of active sodium ion transport, and very interestingly, 72 hours of HIF stabilization caused a switch in the direction of active ion transport. Western blot analysis showed that HIF stabilization caused a decrease in the protein level of sodium‐potassium‐ATPase (Na+‐K+‐ATPase) α1 subunit, the catalytic subunit of the enzyme responsible for active sodium ion transport. Decreased level of Na+‐K+‐ATPase α1 subunit due to HIF stabilization was consistent with the loss of active sodium ion transport. Thus, HIF stabilization by CoCl2 can affect Na+‐K+‐ATPase α1 subunit and ZO1 protein levels, leading to alterations of transcellular and paracellular transport through normal kidney epithelial monolayers. Thus, our results indicate that localized ischemia‐hypoxia can transform a normal absorptive epithelia to a secretory and leaky epithelia and may contribute to cyst expansion.Support or Funding InformationThis research was supported by the Dr. John Vitullos Pilot and Bridge Funding Program at the Center for Gene Regulation in Health and Disease (GRHD), NIH DK092716 award (both to AR) and Cleveland State University Dissertation Research Award to SN.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.