In the antidiuretic state cells that inhabit the inner medulla of terrestrial animals must adapt to a hypertonic environment and to rapidly respond to changes in tonicity. This response involves the activation of a sequential network of protein kinases and ultimately the upregulation of proteins that promote the cellular uptake of the inert osmolytes. It is now well recognized that the adaptive response involves a more complex array of both genes and proteins. The primary aim of this proposal is to examine by both genomic and proteomic approaches, the repertoire of genes and proteins that are specifically upregulated by hypertonicity in inner medullary collecting duct (IMCD3) cells. These studies are designed to establish the temporal sequence for appearance and disappearance of genes over a 24 hour period and to contrast these observations to those seen in cells chronically adapted to hypertonic conditions (600-900 mOsm/kg). The differences in gene-protein expression between IMCD3 and more osmosensitive cortical collecting duct cells (M1) will be examined. Subsequent experiments, employing pharmacologic inhibition will examine the role of the modules of the MAP kinase family (ERKs, JNKs, and P38 MAP kinases) as well as PI3 kinase in the transcription of osmoregulated genes and expression of proteins. We will further focus our attention on the role of the c-JUN N-terminal kinase (JNK). Dominant negative mutants of this kinase are sensitized to hypertonicity and have impaired synthesis of some osmoprotective proteins. However, since dominant negative mutants still exhibit significant JNK activity, we will instead employ cells derived from immorto mice that are entirely deficient in JNK1 or JNK2. In addition to characterizing such cells and analyzing their genomic - proteomic response to hypertonicity, we will examine their osmosensitivity. The physiologic significance of the above observations will be assessed by analyzing the changes in gene-protein expression in rodents in various states of hydration. Furthermore, the importance of selected proteins that are found to be robustly upregulated in the above settings will be studied employing a silencing RNA technique that can render cells incapable of synthesizing selective proteins. The significance of the JNK activation will be tested in JNK1 and JNK2 knockout mice. These animals will be subjected to changing water balance states and to histologic examination of their inner medulla. The described experiments should define genes and proteins involved in osmoregulation and particularly dissect the role of JNK kinase and its isoforms in the adaptation of the mammalian inner medulla to hypertonicity.
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