Induction of molecular chaperones by hyperosmotic stress in mouse inner medullary collecting duct cells.

The extreme hyperosmotic conditions that exist in the renal inner medulla enable the urinary concentrating mechanism to function. In this study, we evaluated whether stress-related molecular chaperones are induced in response to hyperosmotic stress in mouse inner medullary collecting duct (mIMCD3) cells. Exposure of cells to medium supplemented with 100 mM NaCl for 4 or 24 h resulted in an increase in heat shock protein-72 (HSP-72) (inducible form) by Western blot. Immunocytochemistry confirmed the increase of HSP-72 and showed that hyperosmotic stress resulted in a localization of HSP-72 predominantly to the nucleoplasm that surrounds the nucleoli and to the cytoplasm, a subcellular distribution pattern different from that seen with heat shock. Using a denatured protein (casein)-affinity column with ATP elution, we identified a number of putative molecular chaperones (46, 60, 78, and 200 kDa) that are upregulated in response to 4 h of hyperosmotic NaCl treatment. Microsequencing identified one of these proteins to be the mitochondrial chaperone mtHSP-70, a member of HSP-70 family, and another to be similar to beta-actin. We also found high levels of HSP-72 in cells chronically adapted to hypertonicity, indicating that chaperones are still required to maintain certain cellular functions even after nonperturbing organic osmolytes are known to accumulate. These results suggest an important role for molecular chaperones in the adaptation of renal medullary epithelial cells to the hyperosmotic conditions that exist in the inner medulla in vivo.

Molecular cloning and chromosome localization of a putative basolateral Na(+)-K(+)-2Cl- cotransporter from mouse inner medullary collecting duct (mIMCD-3) cells.

Electroneutral Na(+)-K(+)-2Cl- cotransporters represent one of the major routes for Cl- movement in epithelia. A secretory form of the cotransporter has been described in the basolateral membrane of a variety of epithelia from fish to mammals. We isolated a putative bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter cDNA, BSC2, from mIMCD-3 cells. Northern analysis indicates that in contrast to BSC1, the recently cloned renal-specific apical isoform of the cotransporter, BSC2 is expressed in secretory epithelia and thus appears to represent the basolateral isoform. Furthermore, BSC2 is also expressed in non-polarized cells, such as red cells and myocytes. Sequence comparison and chromosome localization demonstrate that BSC2 and BSC1 are different genes that diverged before the evolution of vertebrates.

An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse.

The terminal inner medullary collecting duct (IMCD) plays an important role in determining the final urinary composition. Currently, there is no continuous cell line derived from this nephron segment. We have developed a cell line derived from the terminal IMCD of mice transgenic for the early region of simian virus SV40 (large T antigen). This cell line, mIMCD-3, retains many differentiated characteristics of this nephron segment including high transepithelial resistance (1,368 +/- 172 omega.cm2), inhibition of apical-to-basal sodium flux by amiloride (41 +/- 7%) and by atrial natriuretic peptide (ANP) (40 +/- 9%), the presence of the amiloride-sensitive sodium channel as determined by Western blot analysis, and accumulation of the major organic osmolytes in response to hypertonic stress. Significantly, mIMCD-3 cells adapted readily and were able to grow in hypertonic medium supplemented with NaCl and urea up to 910 mosmol/kgH2O. These extreme osmotic conditions exist in the renal medulla in vivo but are known to be lethal to most other cells. This cell line should be highly useful for the study of the cellular adaptation to osmotic stress and the cell biology and transport physiology of this nephron segment.

Expression of GLUT-2 cDNA in human B lymphocytes: analysis of glucose transport using flow cytometry.

The molecular characterization of transport proteins is often limited by transient functional expression or the need for a simple method to select functional cDNA clones. We used a mammalian expression system to obtain long-term expression of GLUT-2, an isoform of glucose permease. Rat GLUT-2 cDNA was ligated into an EBV vector (pLPP) and transfected into B lymphocytes which lack GLUT-2. Northern and Western analyses confirmed expression of GLUT-2 protein in membranes of transfected cells. Two functional assays using flow cytometry were developed to distinguish GLUT-2 transfectants from control/pLPP transfectants. Uptake of NBD-glucosamine, a fluorescent analogue of glucose, was increased in GLUT-2 transfectants. In addition, when exposed to hypertonic glucose medium, GLUT-2 transfectants and control/pLPP transfectants exhibited a difference in forward-angle light scatter (FALS), an index of cell volume, indicating a difference in glucose permeability. Independent measurements of glucose uptake (isotopic) and cell volume (video microscopy) confirmed the flow cytometry observations. This expression system used in combination with flow cytometry is useful for studying the functional properties of glucose and other solute transporters.