The development of a successful bioartificial kidney faces some challenges such as overgrowth and dedifferentiation of epithelial cells. It is known that some renal epithelial cells are exposed to hypertonic environment in nature and it modulates the synthesis of extracellular matrix (ECM) proteins, therefore we hypothesize that hypertonicity may influence the performance of epithelial cells.
Cells were treated with regular (300 mOsm) and hypertonic media (400 and 500 mOsm) and were investigated by MTS (for mitochondrial activity), Q-PCR, siRNA technology and microscopy.
We found that hypertonic media suppressed the mitochondrial activity, suggesting the suppression of overgrowth of cells. Morphological evaluation revealed that hypertonic media maintained an intact epithelial monolayer, while isotonic medium treatment resulted in a disrupted layer of cells, where some cells looked like mesenchymal cells. Hypertonic media treatment improved most of the investigated epithelial markers (e.g. E-cadherin, EPCAM and ZO-1) and renal transporters (e.g. OATP4C1 and MRP4), while it suppressed mesenchymal markers (e.g. SM22 alpha and vimentin). mRNA expression of ECM genes (e.g. COL4A1, LAMA1, LAMA5 and FN) was not influenced, suggesting that hypertonic effects were not due to modulation of ECM genes. This prompted us to explore other underlying molecular mechanisms. Tonicity-responsive enhancer binding protein (TonEBP) is the key transcription factor that protect the cells from hypertonic deleterious effects. Consequently, we silenced the mRNA expression of TonEBP and it appeared that it was not only responsible for the induction of epithelial markers, but also for the prevention of inducing mesenchymal markers.
Our results are promising for bioartificial kidney, since they suggest that hypertonicity inhibits (via TonEBP) the overgrowth of the cells and maintains an intact differentiated epithelial monolayer, which are some of the major obstacles that counteract the development of bioartificial kidney. In the future we will investigate the effect of hypertonicity on certain epithelial functions and we will further study the signaling pathway of TonEBP.