PEPCK mRNA localization in proximal tubule and gene regulation during metabolic acidosis.
To identify the nephron segments expressing PEPCK in control and acidotic conditions, PEPCK mRNA was localized in rat kidney using the technique of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected S1 S2, and S3 segments of the rat proximal tubule. In controls, the number of tubules expressing PEPCK mRNA was greatest in the S3 segment, moderate in the S2 segment, and least in the S1 segment of the proximal tubule. After NH4Cl feeding, strong signals for PEPCK mRNA were detected in all three proximal tubule segments. In situ hybridization demonstrated expression of PEPCK mRNA only in the medullary rays in controls. After NH4Cl, PEPCK mRNA was expressed throughout the cortex, confirming the RT-PCR results. These data demonstrate the ability of the rat kidney cortex to modulate the expression of PEPCK mRNA during metabolic acidosis by recruitment of additional cells in the proximal nephrons. Studies with cultured LLC-PK1-F+ cells indicated that increased PEPCK gene transcription at acid pH required a cis-acting element (enhancer) in the more distal 5' flanking region of the promoter.
Drewnowsk
K D
KD
Department of Medicine, Virginia Commonwealth University, Richmond 23298-0160, USA.
Craig
M R
MR
Digiovanni
S R
SR
McCarty
J M
JM
Moorman
A F M
AF
Lamers
W H
WH
Schoolwerth
A C
AC
D000138:Acidosis / Q000201:enzymology*; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D053938:DNA Fragmentation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D017403:In Situ Hybridization; D007687:Kidney Tubules, Proximal / Q000201:enzymology* / Q000648:ultrastructure; D018374:LLC-PK1 Cells; D008297:Male; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000096:biosynthesis* / Q000235:genetics; D012313:RNA; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014162:Transfection
C034477:RNA primers; D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D019764:Phosphoenolpyruvate Carboxykinase (ATP)