Urinary acidification is required for normal acid-base homeostasis, and disorders of acidification are important causes or morbidity in acute and chronic kidney diseases. Distal acidification emanates from the intercalated cells, a specialized cell-population exhibiting high plasma membrane levels of a vacuolar-type H+ ATPase. Recent experiments indicate that this specialization involves an intercalated-cell specific amplification of one isoform of the H+ ATPase 56kD subunit. Vacuolar H+ ATPases in other segments of the nephron also contribute to tubular bicarbonate reabsorption, but their biochemical properties and physiologic responses differ significantly from those of intercalated cells. The long term objectives of this proposal are to examine the mechanisms for the specialized expression and function of the vacuolar H+ ATPase in renal intercalated cells and other renal epithelial cells.
The specific aims are: 1. To investigate the basis for tissue-specific amplification of the 56 kD subunit """"""""kidney"""""""" isoform in renal intercalated cells: a) Isolation of cDNA clones for the rat vacuolar H+ ATPase 56 kD subunit isoforms. b) Are the high steady state level of mRNA encoding the kidney isoform of vacuolar H+ ATPase 56 kD subunit due to high levels of transcription? c) Genomic cloning of the human """"""""kidney"""""""" isoform of vacuolar H+ ATPase 56 kD subunit. d) Determination of the transcriptional initiation site. e) Determination of the minimum promoter in the 5' flanking region of the 56 kD subunit """"""""kidney"""""""" isoform gene required for transcription. f) Use of the transgenic mouse to identify the genomic elements responsible for amplified expression of the 56 kD subunit """"""""kidney"""""""" isoform in intercalated cells. g) Examine the genomic elements controlling amplified expression of the 56 kD subunit """"""""kidney"""""""" isoform by retrovirally mediated DNA transfer of promoter-reporter gene constructs. g) Determine the extent to which mRNA stability contributes to tissue specific and amplified expression of the """"""""kidney"""""""" isoform of the vacuolar H+ ATPase 56 kD subunit. 2. Development of an immortalized intercalated cell line using 56 kD subunit """"""""kidney"""""""" isoform gene/transforming antigen constructs: a) Introduction of 56 kD subunit """"""""kidney"""""""" isoform gene/transforming antigen constructs into primary cultures of rat collecting ducts by retroviral DNA transfer. b) Preparation of transgenic mice with 56 kD subunit """"""""kidney"""""""" isoform promoter/transforming gene constructs. 3. Isolation and sequence analysis of cDNA clones for other subunits of the kidney H+ ATPase. 4. Identification of additional heterogeneity in proton pump structure and evaluation of its functional significance in renal epithelial cells. These studies will enhance our understanding of the biochemical mechanisms regulating urinary acidification.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK038848-07
Application #
3238397
Study Section
General Medicine B Study Section (GMB)
Project Start
1986-12-01
Project End
1997-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
7
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Barnes-Jewish Hospital
Department
Type
DUNS #
City
Saint Louis
State
MO
Country
United States
Zip Code
63110
Sautin, Yuri Y; Lu, Ming; Gaugler, Andrew et al. (2005) Phosphatidylinositol 3-kinase-mediated effects of glucose on vacuolar H+-ATPase assembly, translocation, and acidification of intracellular compartments in renal epithelial cells. Mol Cell Biol 25:575-89
Chen, Shih-Hua; Bubb, Michael R; Yarmola, Elena G et al. (2004) Vacuolar H+-ATPase binding to microfilaments: regulation in response to phosphatidylinositol 3-kinase activity and detailed characterization of the actin-binding site in subunit B. J Biol Chem 279:7988-98
Lu, Ming; Vergara, Sandra; Zhang, Li et al. (2002) The amino-terminal domain of the E subunit of vacuolar H(+)-ATPase (V-ATPase) interacts with the H subunit and is required for V-ATPase function. J Biol Chem 277:38409-15
Lu, M; Holliday, L S; Zhang, L et al. (2001) Interaction between aldolase and vacuolar H+-ATPase: evidence for direct coupling of glycolysis to the ATP-hydrolyzing proton pump. J Biol Chem 276:30407-13
Holliday, L S; Lu, M; Lee, B S et al. (2000) The amino-terminal domain of the B subunit of vacuolar H+-ATPase contains a filamentous actin binding site. J Biol Chem 275:32331-7
Lee, B S; Gluck, S L; Holliday, L S (1999) Interaction between vacuolar H(+)-ATPase and microfilaments during osteoclast activation. J Biol Chem 274:29164-71
Gluck, S L (1998) Acid-base. Lancet 352:474-9
Gluck, S L; Lee, B S; Wang, S P et al. (1998) Plasma membrane V-ATPases in proton-transporting cells of the mammalian kidney and osteoclast. Acta Physiol Scand Suppl 643:203-12
Holliday, L S; Dean, A D; Lin, R H et al. (1997) Low NO concentrations inhibit osteoclast formation in mouse marrow cultures by cGMP-dependent mechanism. Am J Physiol 272:F283-91
Lee, B S; Krits, I; Crane-Zelkovic, M K et al. (1997) A novel transcription factor regulates expression of the vacuolar H+-ATPase B2 subunit through AP-2 sites during monocytic differentiation. J Biol Chem 272:174-81

Showing the most recent 10 out of 39 publications