Renal Epithelial Function Can Be Altered by Hypoxia Inducible Mechanisms
Authors: Nag Subhra S. , Resnick Andrew H. 
Published: November 2023

Polycystic Kidney Disease (PKD) is characterized by fluid filled cysts which interfere with renal function and can lead to kidney failure. In cystic epithelia, hypoxia inducible factor 1α (HIF1α) and HIF2α were reported to be elevated [1]. Cyst expansion, stagnant fluid accumulation, and insufficient vascular supply may 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 HIF1α, a major regulator of cellular response to hypoxia, can cause altered paracellular and transcellular epithelial transport. Thus, increased level of HIF1α can be a triggering factor in the transformation of normal renal epithelia from an absorptive to a secretory phenotype, leading to cyst expansion.

Cobalt chloride (CoCl2) was used at a concentration of 100 μM to stabilize HIF1α in the cellular nucleus. Using western blots, we confirmed that increased HIF1α levels in the nuclear extracts by CoCl2 also increased cellular erythropoietin (EPO) levels. Application of a HIF1α inhibitor, methyl 3‐[[2‐[4‐(2‐adamantyl)phenoxy]acetyl]amino]‐4‐hydroxybenzoate was found to suppress the increased level of EPO expression by CoCl2. Transcellular and paracellular transport studies were performed in a mouse cortical collecting duct cell line (mCCD 1296 (d)) which has a high monolayer transepithelial resistance (TER) value around 3000 Ω‐cm2 and a detectable 95% amiloride sensitive voltage value. A few paracellular transport studies were also carried out in Mardin‐Darby Canine Kidney (MDCK) epithelial cell line which has relatively low monolayer TER value around 400 Ω‐cm2 with no detectable voltage value. Short circuit current (Isc) was calculated using Ohm's law (I = V/R) to compare active ion transport. TER and voltage values were recorded using an Endohm chamber. Higher TER values in mCCD cells can reflect reduced paracellullar permeability which was verified by permeability assay using FITC‐conjugated dextran molecules (3 and 70 kDa). MTT assay was used in order to make sure CoCl2 treated and untreated groups maintained same level of cellular viability. Cells exposed to CoCl2 showed altered active ion transport in mCCD cells. Treatment with CoCl2 caused increased 70 kDa FITC‐dextran paracellular permeability both in mCCD and MDCK monolayers. In MDCK cell line, we performed a paracellular permeability experiment in which we used the HIF1α inhibitor along with CoCl2. The HIF1α inhibitor was found to block the effect of CoCl2 treatment on FITC‐dextran permeability to a certain extent. Thus, CoCl2 can alter epithelial transport most likely due to hypoxia inducible mechanisms. Currently, we are trying to strengthen the connection between HIF1α levels and renal epithelial function using HIF1α inhibitors in active and passive transport experiments in the presence of CoCl2 or another HIFα stabilizer, the iron chelator desferrioxamine.

Support or Funding Information

The project is supported by National Institute of Diabetes and Digestive and Kidney Diseases 1R15DK092716 to AR and Cleveland State University Dissertation Research Award to SN.