Development of a 3D in vitro Model of the Kidney Cortical Collecting Duct

Abstract

The cortical collecting duct (CCD) of the mammalian kidney plays a critical role in the regulation of body volume, sodium pH and osmolarity and is composed of two distinct cells types, principal cells (PCs) and intercalated cells (ICs). Each cell type is detectable in the kidney by the localization of specific transport proteins such as Aqp2 and ENaC in principal cells and V‐ATPase B1 in intercalated cells. The inter‐relationship between PCs and ICs is complex and yet to be fully elucidated. The ratio of the number of PCs to ICs (typically 70:30 in healthy mice) has been shown to be influenced by multiple factors involved in Notch signalling [1]. A transitional cell expressing both PC and IC markers, was also recently described. mCCDcl1 cells have been widely used as a mouse principal cell line on the basis of their physiological characteristics. However, previous studies have shown plasticity of mCCDcl1 cells, a direct lineage relationship between principal and intercalated cells, and evidence consistent with mCCDcl1 cells being precursor cells [2]. These characteristics make mCCDcl1 cells a useful tool in the study of collecting duct physiology and cell differentiation. However, classic (2D) cell culture techniques have limitations for the development of an optimal in vitro model of the collecting duct.In this study, a 3D model of collecting duct was developed using polyHIPEs, an emulsion‐based polymer materials offering a highly interconnected micro‐porosity network [3]. 3D‐printed polyHIPEs structures were designed and integrated in a PDMS‐free fluidic device and offered a neutral support structure for mCCDcl1 cells, allowing cell attachment and polarization. Cells were cultured on the 3D scaffold for up to 6 weeks, showing an improved lifespan compared to classic cell culture, and displayed a continuous monolayer. Cell thickness was determined using confocal microscopy and increased from 7.5 ± 1.1 μm in 2D culture to 20.2 ± 2.5 μm in 3D culture. Immunocytochemistry of sodium channels (α‐ENaC) showed a highly polarized monolayer and increased expression in 3D cultures with its fluorescence mean intensity up to 31.64 ± 2.69 %, from 23.74 ± 2.19 % on a 2D culture. ENaC expression was confirmed by q‐PCR, which showed a 2.82 ± 0.15 fold increase in cells on the 3D scaffold, and V‐ATPase B1 expression increased by a factor of 23.77 ± 8.20. Amiloride sensitive sodium transport was observed using a fluorescent sodium‐binding dye and demonstrates that 3D polyHIPEs scaffolds may enable quantitative visualisation of ion transport in cultured collecting duct cells.The 3D model developed here is a useful tool for elucidating the mechanisms involved in collecting duct plasticity, to study drug toxicity, and for studying the cellular mechanisms underlying genetic diseases such as the syndrome of apparent mineralocorticoid excess (SAME).Support or Funding InformationBHF (British Heart Foundation)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.