How do kidney cells adapt to survive in hypertonic inner medulla?

Abstract

The hypertonic inner medulla poses challenges to the cells that inhabit this area of the nephron. We employed discovery tools including proteomics and genomics to identify proteins that subserve the adaptive response. The gamma subunit of the Na/K-ATPase is critical to the survival of cells in hypertonic conditions, as silencing it increases osmosensitvity, and overexpression increases osmotolerance. The inner medullary collecting duct (IMCD) has high transepithelial resistance (TER). Proteins responsible for tight junction integrity are upregulated in hypertonic states. Multi PDZ protein 1 (MUPP1), a PDZ scaffolding protein, targets Claudin 4 to the tight junction. The silencing of either of these proteins decreases TER and renders the epithelium leaky. The accumulation of inert osmolytes is integral to the adaptive response. The genes involved are regulated by the transcription factor Tonicity Enhancer Binding Protein. An osmoregulated nuclear protein Nup88 is critical to the retention of this transcription factor in the nucleus and to the generation of the osmolytes. In summary, IMCD cells bring forth a coordinated response to hypertoncity that is necessary for cell survival and function of these cells in anisotonic conditions.

Fig. 1

Panel A: Effect of silencing the γ subunit of Na/K-ATPase on cell survival upon exposure to sublethal hypertonic stress (550 mOsm/Kg H₂O). While empty vector (EV) controls, (squares) withstand this stress well, the silenced cells (circles) display significantly decreased survival ability. Panel B: Effect of overexpressing the γ subunit of Na/K-ATPase on cell survival in cells exposed to lethal osmotic stress (675 mOsm/Kg H₂O). The empty vector controls (squares) cannot withstand such a stress, the overexpressing cells (triangles) have high levels of survival. Adapted from reference .

Fig. 2

Effect of silencing MUPP1 Panel A and Claudin 4 Panel B on transepithelial resistance (TER) in monolayers of inner medullary collecting duct cells previously adapted to 550 mOsm/Kg H₂O, compared to empty vector (EV) control. Under both circumstances the silenced cells generated a significantly lower TER. Adapted from reference and .

Fig. 3

Panel A: Effect of silencing Nup 88 on the retention of TonEBP in the nucleus. The upper panel depicts that most of the fluorescent signal for the transcription factor is in the nucleus in the empty vector control, while the lower panel shows that the fluorescence is primarily in the cytoplasm in the silenced cells following 8 hours of exposure to hypertonicity. Panel B: Quantitative PCR for target genes of TonEBP in empty vector controls (open bars) and Nup 88 silenced cells (solid bars) following acute exposure to hypertonicity. Note the decrement in message for all target genes but not for the control alpha subunit of Na K ATPase that is not a target gene for TonEBP. Panel C: A representative Western blot for aldose reductase (AR) and Hsp 70 protein expression in empty vector (EV) and Nup 88 silenced cells. The β actin is a loading control. With permission, from reference .

Fig. 4

Coordinated upregulation of proteins to maintain cellular viability and function in IMCD cells.