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.
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