A metabonomic and proteomic analysis of changes in IMCD3 cells chronically adapted to hypertonicity.

BACKGROUND: The genomic response to adaptation of IMCD3 cells to hypertonicity results in both upregulation and downregulation of a variety of genes. METHOD: The present study was undertaken to assess the metabonomic and proteomic response of IMCD3 cells that have been chronically adapted to hypertonicity (600 and 900 mosm/kg H(2)O) as compared to cells under isotonic conditions. RESULTS: Adaptation of IMCD3 cells to hypertonic conditions resulted in a change of a wide range of organic osmolytes, including sorbitol (+8,291%), betaine (+1,099%), myo-inositol (+669%), taurine (+113%) and glycerophosphorylcholine (+61%). Evaluation of the polyol pathway for sorbitol production revealed a reduction in sorbitol dehydrogenase and an increase in aldose reductase mRNA in adapted cells. Proteome analysis revealed increased expression of six glycolytic proteins, including malic enzyme and pyruvate carboxylase, indicating the activation of the pyruvate shunt and changes in glucose metabolism. This study showed that the observed reduction in cell replication could possibly reflect a redirection of cellular energy from cell growth and replication to maintenance of intracellular ion levels in chronically adapted cells. CONCLUSION: The combined metabonomic and proteomic analysis was shown to be a very helpful tool for the analysis of the effects caused by chronic adaptation to hypertonicity. It made it possible to better evaluate the importance of certain changes that occur in the process of adaptation.

The tight junction protein, MUPP1, is up-regulated by hypertonicity and is important in the osmotic stress response in kidney cells.

Antibody array proteomics was used to detect differentially expressed proteins in inner medullary collecting duct 3 (IMCD3) cells grown under isotonic and chronic hypertonic conditions. Of 512 potential proteins, >90% were unchanged in expression. Noteworthy was the up-regulation of several tight junction-related proteins, including MUPP1 (multi-PDZ protein-1), ZO1 (zonula occludens 1), and Af6. The most robustly up-regulated protein under hypertonic conditions was MUPP1 (7.2x, P < 0.001). Changes in expression for MUPP1 were verified by quantitative PCR for message and Western blot for protein. In mouse kidney tissues, MUPP1 expression was substantial in the papilla and was absent in the cortex. Furthermore, MUPP1 expression increased 253% (P < 0.01) in the papilla upon 36 h of thirsting. Localization of MUPP1 protein expression was confirmed by immunocytochemical analysis demonstrating only minor staining under isotonic conditions and the substantial presence in chronically adapted cells at the basolateral membrane. Message and protein half-life in IMCD3 cells were 26.2 and 17.8 h, respectively. Osmotic initiators of MUPP1 expression included NaCl, sucrose, mannitol, sodium acetate, and choline chloride but not urea. Stable IMCD3 clones silenced for MUPP1 expression used the pSM2-MUPP1 vector. In cell viability experiments, clones silenced for MUPP1 demonstrated only a minor loss in cell survival under acute sublethal osmotic stress compared with empty vector control cells. In contrast, a 24% loss (P < 0.02) in transepithelial resistance for monolayers of MUPP1-silenced cells was determined as compared with controls. These results suggest that MUPP1 specifically, and potentially tight junction complexes in general, are important in the renal osmoadaptive response.

Expression of the calcium-binding protein S100A4 is markedly up-regulated by osmotic stress and is involved in the renal osmoadaptive response.

Proteomic analysis of Inner Medullary Collecting Duct (IMCD3) cells adapted to increasing levels of tonicity (300, 600, and 900 mosmol/kg H(2)O) by two-dimensional difference gel electrophoresis and mass spectrometry revealed several proteins as yet unknown to be up-regulated in response to hypertonic stress. Of these proteins, one of the most robustly up-regulated (22-fold) was S100A4. The identity of the protein was verified by high pressure liquid chromatography-mass spectrometry. Western blot analysis confirmed increased expression with increased tonicity, both acute and chronic. S100A4 protein expression was further confirmed by immunocytochemical analysis. Cells grown in isotonic conditions showed complete absence of immunostaining, whereas chronically adapted IMCD3 cells had uniform cytoplasmic localization. The protein is also regulated in vivo as in mouse kidney tissues S100A4 expression was many -fold greater in the papilla as compared with the cortex and increased further in the papilla upon 36 h of thirsting. Increased expression of S100A4 was also observed in the medulla and papilla, but not the cortex of a human kidney. Data from Affymetrix gene chip analysis and quantitative PCR also revealed increased S100A4 message in IMCD3 cells adapted to hypertonicity. The initial expression of message increased at 8-10 h following exposure to acute sublethal hypertonic stress (550 mosmol/kg H(2)O). Protein and message half-life in IMCD3 cells were 85.5 and 6.8 h, respectively. Increasing medium tonicity with NaCl, sucrose, mannitol, and choline chloride stimulated S100A4 expression, whereas urea did not. Silencing of S100A4 expression using a stable siRNA vector (pSM2; Open Biosystems) resulted in a 48-h delay in adaptation of IMCD3 cells under sublethal osmotic stress, suggesting S100A4 is involved in the osmoadaptive response. In summary, we describe the heretofore unrecognized up-regulation of a small calcium-binding protein, both in vitro and in vivo, whose absence profoundly delays osmoadaptation and slows cellular growth under hypertonic conditions.

The gamma subunit of Na/K-ATPase: an exceptional, small transmembrane protein.

The gamma subunit has been characterized as a fine-tuning modulator of the Na/K-ATPase expressed in kidney tissues. This small single transmembrane domain protein interacts with the alpha subunit of Na/K-ATPase to increase affinity for ATP and decrease affinity for Na allowing medullary cells to continue pump activity under reduced cellular ATP levels. The gamma subunit is undetectable in kidney cell cultures grown under isotonic conditions and expression is induced with acute or chronic exposure to hypertonicity. The gamma subunit demonstrates remarkable regulatory complexity including induction by chloride ions rather than sodium, the differential expression of at least 2 isoforms, involvement of separate MAP kinase signaling pathways for transcription (JNK) and translation (PI3K) as well as cell type regulation of expression. Mutation in the transmembrane domain of the gamma subunit has been implicated in cases of primary hypomagnesemia. Alternative roles have been established for the gamma subunit in embryonic development and quite possibly additional functions in cell signaling as yet unrecognized may be possible.