Cells of the thick ascending limb of Henle's loop (TALH) in the human kidney are exposed to extreme hyperosmotic stress during urine formation. They are actively involved in transporting salts (mostly NaCl) from the renal tube into the extracellular space of the medulla. However, because the ascending limb of the loop is impermeable to water, the accumulation of ions in the cells creates extreme hyperosmotic conditions. Such cellular conditions are known to trigger an increase in sorbitol due to the upregulation of aldose reductase, an enzyme that directs glucose towards sorbitol. In addition, several heat-shock proteins have also been found previously to be upregulated under osmotic stress in cells of the TALH. Knowing that extreme osmotic conditions can trigger such physiological changes, it would be intriguing to take a bird's-eye view of these cells to see other changes in protein expression that characterize their response to one of the most, if not the most, severe stresses that human cells can be exposed to. Hassan Dihazi and colleagues from the Georg-August University of Göttingen in Germany used a proteomic approach to obtain this kind of global view to gain new insights and make some very interesting discoveries.

The authors used two-dimensional gel electrophoresis to investigate the differences in protein expression in an epithelial cell line from the outer medulla of a rabbit, which has been shown to be a suitable model for the function of TALH cells. They exposed cells to normal (300 mosmol kg^–1) and hyperosmotic (600 mosmol kg^–1) stress conditions. After comparing expression levels, they isolated protein spots from the gels, digested them with trypsin and analyzed the mass of the resulting peptide fragments. The distribution of the masses of these fragments that one can obtain with mass spectrometry provides a so-called `mass fingerprint'. Comparing the mass fingerprints with the genome, the team found 25 proteins to be overexpressed and 15 downregulated in the cells when exposed to hyperosmotic stress. What did they learn?

Importantly, the 15-fold upregulation they found in aldolase reductase levels confirmed an expected finding and validated the approach taken. In support of the importance of sorbitol accumulation, the authors also observed the upregulation of lactate and malate dehydrogenase, enzymes that participate in gluconeogenesis and provide increased levels of glucose, which is needed for the synthesis of sorbitol.

Hyperosmotic stress causes cell shrinking and therefore requires changes in the expression of cytoskeletal proteins. Vimentin and tropomyosin, two proteins that are associated with the cytoskeleton, changed expression levels, which confirmed the importance of cell-volume modifications during hyperosmotic stress.

The authors also compared the response of their control cell line with cells that were selected after exhibiting increased resistance to higher osmotic conditions (600 mosmol kg^–1), under which they were still able to grow. The resistant cells showed higher expression levels of several heat-shock proteins, such as α-crystalline, Hsp70 and Hsp90, which have several functions including the maintenance of protein structure during stress. Surprisingly, other stress proteins, such as glucose-regulated proteins that are located in the endoplasmic reticulum and are involved in Ca²⁺-binding, were downregulated, which the authors suggest may augment Ca²⁺ homeostasis, which plays an important yet still rather unknown role during osmotic stress.

The diversity of cellular responses that such a proteomic approach reveals is unsettling, reminding us how little we actually know about how the cells in the human kidney respond when they are exposed to one of the most severe stressors that human cells experience. In many ways, this bird's-eye view has raised more questions than it answered.