The levels of the mitochondrial glutaminase and glutamate dehydrogenase and of the cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) are increased within the rat renal proximal convoluted tubule in response to metabolic acidosis. This adaptation is necessary to sustain increased renal ammoniagenesis and gluconeogenesis during a compensated chronic acidosis. The increased enzyme activities result from increased synthesis due to an increased level of the respective mRNAs. The induction of PEPCK is regulated at the level of transcription. The mechanism which initiates and coordinates the associated changes in renal gene expression are unknown. The entire rat PEPCK gene has been isolated and sequenced. Many of the PEPCK promoter elements and the associated transcription factors that participate in the regulation of the liver PEPCK gene have been identified and characterized. Furthermore, LLC-PKF+ cells, an established gluconeogenic line of renal proximal tubular epithelial cells, exhibit adaptive changes in PEPCK mRNA levels in response to growth in acidic medium that closely mimic those observed in vivo. Thus, this system is extremely well suited to characterize the mechanism by which specific cells within the kidney sense changes in pH and/or HCO3 concentration and transduce this information to alter gene expression. This mechanism may also regulate the coordinate adaptations in the interorgan metabolism of glutamine that are required to sustain increased renal ammoniagenesis.
The specific aims of the proposed research are to determine the mechanism of the pH-response in PEPCK gene expression that occurs in LLC-PK-F+ cells, to map the cis-regulatory elements that participate in this response, to utilize transgenic mice to determine if the identified regulatory elements are essential for the in vivo response to acidosis, to develop assays to identify and quantitate the associated trans-acting factors, and to clone the participating transacting factors. The results of the proposed studies should provide insight into potential pharmacologic approaches that may stimulate ammoniagenesis in various clinical conditions which cause a metabolic acidosis.
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|Andratsch, Manfred; Feifel, Elisabeth; Taylor, Lynn et al. (2007) TGF-beta signaling and its effect on glutaminase expression in LLC-PK1-FBPase+ cells. Am J Physiol Renal Physiol 293:F846-53|
|O'Hayre, Morgan; Taylor, Lynn; Andratsch, Manfred et al. (2006) Effects of constitutively active and dominant negative MAPK kinase (MKK) 3 and MKK6 on the pH-responsive increase in phosphoenolpyruvate carboxykinase mRNA. J Biol Chem 281:2982-8|
|Dhakras, Purabi S; Hajarnis, Sachin; Taylor, Lynn et al. (2006) cAMP-dependent stabilization of phosphoenolpyruvate carboxykinase mRNA in LLC-PK1-F+ kidney cells. Am J Physiol Renal Physiol 290:F313-8|
|Hajarnis, Sachin; Schroeder, Jill M; Curthoys, Norman P (2005) 3'-Untranslated region of phosphoenolpyruvate carboxykinase mRNA contains multiple instability elements that bind AUF1. J Biol Chem 280:28272-80|
|Feifel, Elisabeth; Obexer, Petra; Andratsch, Manfred et al. (2002) p38 MAPK mediates acid-induced transcription of PEPCK in LLC-PK(1)-FBPase(+) cells. Am J Physiol Renal Physiol 283:F678-88|
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