DOMINANT-NEGATIVE C-JUN NH2-TERMINAL KINASE-2 SENSITIZES RENAL INNER MEDULLARY COLLECTING DUCT CELLS TO HYPERTONICITY-INDUCED LETHALITY INDEPENDENT OF ORGANIC OSMOLYTE TRANSPORT

The c-Jun NH2-terminal protein kinases (JNKs), as well as the extracel lular signal-regulated protein kinases (ERKs) and p38 mitogen-activate d protein kinase, are activated in renal cells in response to extracel lular hypertonicity, To determine whether activation of JNKs by hypert onicity is isoform-specific, renal inner medullary collecting duct cel ls were stably transfected with cDNA's encoding hemagglutinin (HA)-tag ged JNK1 and JNK2 isoforms, and the expressed kinases were immunopreci pitated with an anti-HA. antibody, Whereas both recombinant kinases we re equivalently expressed, only immunoprecipitates from the HA-JNK2 ce lls displayed hypertonicity-inducible JNK activity, Furthermore, expre ssion of dominant-negative JNK2 (HA-JNK2-APF) in stable clones inhibit ed hypertonicity-induced JNK activation by 40-70%, whereas expression of dominant-negative JNK1 (HA-JNK1-APF) had no significant inhibitory effect, Independent HA-JNK2-APF (but not HA-JNK1-APF) clones displayed greatly reduced viability relative to neomycin controls after 16 h of exposure to 600 mosM/kg hypertonic medium with percent survival of 20 .5 +/- 2.7 and 31.5 +/- 7.3 for two independent HA-JNK2-APF clones com pared with 80.1 +/- 1.0 for neomycin controls (p < 0.001, n = 5, mean +/- S.E.). However, neither JNK mutant blocked either regulatory volum e increase or hypertonicity-induced enhancement of uptake of inositol, an organic osmolyte putatively involved in long term adaptation to hy pertonicity, These results define JNK2 as the primary hypertonicity-ac tivated JNK isoform in IMCD-3 cells and demonstrate its central import ance in cellular survival in a hypertonic environment by a mechanism i ndependent of acute regulatory volume increase as well as regulation o f organic osmolyte uptake.