Cells of the renal medulla are exposed to extraordinarily high concentrations of the potent denaturant, urea, as a consequence of the renal concentrating mechanism. The molecular mechanisms through which renal medullary cells adapt to and tolerate this harsh environment have important implications for the understanding of water and urea physiology, and for the understanding of and potential enhancement of cellular protection from metabolic stress in diverse pathophysiological contexts. The applicant has obtained abundant evidence that, in marked contrast to the other principal medullary solute, NaC1, urea activates a novel receptor tyrosine kinase pathway in renal epithelial cells. Two candidate urea sensing molecules exhibiting prompt, solute- and tissue-specific, urea-inducible autophosphorylation or tyrosine phosphorylation have been partially purified by the applicant through co-immunoprecipitation with urea-responsive signaling molecules, and through lectin affinity precipitation, respectively. The overall objective of the project is to understand the molecular mechanism and physiological consequences of the activation of urea-inducible kinase signaling pathways in renal medullary cells in vitro and in vivo, and to identify these and other potentially novel urea sensing and effector molecules. The ability of known SH2 domain-containing receptor tyrosine kinase effector molecules to mediate elements of the renal epithelial cell adaptive response to urea stress (Aim 1) will be determined through inhibition of effector pathways with pharmacological agents and through transient and stable transfection with inducible dominant-negative expression constructs. The two candidate urea sensors will be partially biochemically purified (and other potential candidates identified, if necessary) using lysates prepared from 32Pi metabolically labeled control- and urea-treated cells, through chromatographic and batch affinity strategies in conjunction with 2-D gel electrophoresis (Aim II). Lastly, the candidate urea sensing proteins will be identified through tandem mass spectrometry, or, if novel, cloned through peptide microsequencing followed by cDNA library screening (Aim III). Thereafter, their physiological regulation at the transcriptional, translational, and post-translational levels will be determined in renal medullary cells in culture and in rodent models associated with abnormal water and urea metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK052494-04
Application #
6329412
Study Section
Pathology A Study Section (PTHA)
Program Officer
Scherbenske, M James
Project Start
1997-12-20
Project End
2002-06-30
Budget Start
2000-12-01
Budget End
2002-06-30
Support Year
4
Fiscal Year
2001
Total Cost
$241,699
Indirect Cost
Name
Oregon Health and Science University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Komers, Radko; Lindsley, Jessie N; Oyama, Terry T et al. (2007) Renal p38 MAP kinase activity in experimental diabetes. Lab Invest 87:548-58
Xu, Hongshi; Tian, Wei; Lindsley, Jessie N et al. (2005) EphA2: expression in the renal medulla and regulation by hypertonicity and urea stress in vitro and in vivo. Am J Physiol Renal Physiol 288:F855-66
Tian, Wei; Salanova, Michele; Xu, Hongshi et al. (2004) Renal expression of osmotically responsive cation channel TRPV4 is restricted to water-impermeant nephron segments. Am J Physiol Renal Physiol 287:F17-24
Xu, Hongshi; Zhao, Hongyu; Tian, Wei et al. (2003) Regulation of a transient receptor potential (TRP) channel by tyrosine phosphorylation. SRC family kinase-dependent tyrosine phosphorylation of TRPV4 on TYR-253 mediates its response to hypotonic stress. J Biol Chem 278:11520-7
Komers, Radko; Tian, Wei; Lindsley, Jessie N et al. (2002) Effects of cyclooxygenase-2 (COX-2) inhibition on plasma and renal renin in diabetes. J Lab Clin Med 140:351-7
Zhao, Hongyu; Tian, Wei; Cohen, David M (2002) Rottlerin inhibits tonicity-dependent expression and action of TonEBP in a PKCdelta-independent fashion. Am J Physiol Renal Physiol 282:F710-7
Tian, Wei; Cohen, David M (2002) Urea stress is more akin to EGF exposure than to hypertonic stress in renal medullary cells. Am J Physiol Renal Physiol 283:F388-98
Tian, W; Cohen, D M (2001) Urea inhibits hypertonicity-inducible TonEBP expression and action. Am J Physiol Renal Physiol 280:F904-12
Yang, X Y; Zhao, H; Zhang, Z et al. (2001) Urea signaling to ERK phosphorylation in renal medullary cells requires extracellular calcium but not calcium entry. Am J Physiol Renal Physiol 280:F162-71
Tian, W; Bonkovsky, H L; Shibahara, S et al. (2001) Urea and hypertonicity increase expression of heme oxygenase-1 in murine renal medullary cells. Am J Physiol Renal Physiol 281:F983-91

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