The onset of metabolic acidosis causes a rapid increase in the renal catabolism of glutamine. This adaptation results in increased ammoniagenesis and gluconeogenesis, processes that facilitate the excretion of acid and generate HCO3-. The increased metabolism is sustained, in part, by increased expression of the mitochondrial glutaminase (GA) and cytosolic phosphoenolpyruvate carboxykinase (PEPCK) within the proximal convoluted segment of the nephron. The primary objectives of our research are to determine how these cells sense changes in acid-base balance and transmit this information to mediate the cell specific regulation of gene expression.

An initial proteomic analysis of this response was performed using difference gel electrophoresis and MALDI/TOF/TOF mass spectroscopy. This approach identified 17 additional proteins that are increased between 1.5- and 5.6-fold and 16 proteins that are decreased between 0.67- and 0.03-fold in rat renal proximal tubules during chronic metabolic acidosis. Many of these initial observations have been confirmed by western blot analysis and in preliminary experiments using ICAT labeling. Current studies are utilizing iTRAQ labeling techniques to confirm and extend the initial analysis. In addition, proteomic analyses are being used to detect and quantify changes in membrane proteins and the phosphoproteome during acute and chronic metabolic acidosis. Finally, bioinformatic analysis will be performed to identify the regulatory elements and potential signaling mechanisms that mediate the homeostatic adaptations and contribute to regulation of acid-base balance.

Previous studies established that induction of the mitochondrial glutaminase results primarily from a pH-responsive stabilization of its mRNA. A set of chimeric β-globin-GA genes and RNA-gel shift assays were used to establish that a direct repeat of 8-base AU-sequences within the 3'-UTR of the GA mRNA functions as the pH-response element (pHRE). AUF1, HuR and ζ-crystallin:NADPH quinone reductase were identified as proteins that bind to the pHRE with high affinity and specificity. Current efforts are directed at using siRNA knock-downs and RNA immunoprecipitation assays to characterize the function of the identified pHRE binding proteins. Immunostaining experiments and in situ hybridization analysis will be performed to determine the role of the ER-stress signaling pathway and the formation of stress granules in the selective stabilization of the GA mRNA during acidosis. Induction of the PEPCK mRNA initially results from increased transcription that is mediated through activation of the p38-MAPK pathway and phosphorylation of the ATF-2 transcription factor. Additional studies are being conducted to identify the proteins that mediate the rapid turnover of the PEPCK mRNA and to characterize the role of the p38-MAPK pathway in its selective stabilization.

Following a stroke, the release of brain GA may contribute to the sustained increase in extracellular glutamate that causes progressive neuronal atrophy. Release of glutaminase may also contribute to HIV-1 induced dementia. As a result, we have recently developed bacterial expression vectors that encode various truncated forms of the human brain glutaminase containing an N-terminal His₆-tag. We have also developed protocols to rapidly purify mg quantities of the recombinant glutaminases and are currently testing different protocols to crystallize and determine the 3-dimensional structure of the glutaminase by X-ray diffraction analysis. The resulting structural information may lead to the development of an effective GA inhibitor that is of therapeutic value in treating stroke.

3'-Nontranslated region of phosphoenolpyruvate carboxykinase mRNA contains multiple instability elements that bind AUF1. Sachin Hajarnis, Jill M. Schroeder and Norman P. Curthoys, J. Biol. Chem. 280, 28272-28280 (2005).

cAMP-dependent stabilization of phosphoenolpyruvate carboxykinase mRNA in LLC-PK₁-F^+, kidney cells. Purabi S. Dhakras, Sachin Hajarnis, Lynn Taylor and Norman P. Curthoys, Am. J. Physiol. Renal Physiol. 290, F313-F318 (2006).

Role of deadenylation and AUF1 binding in the pH-responsive stabilization of glutaminase mRNA. Jill M. Schroeder, Hend Ibrahim, Lynn Taylor and Norman P. Curthoys, Am. J. Physiol. Renal Physiol. 290, F733-F740 (2006).

Effects of constitutively active and dominant negative MKK3 and MKK6 on the pH-responsive increase in phosphoenolpyruvate carboxykinase mRNA. Morgan O'Hayre, Lynn Taylor, Manfred Andratsch, Elisabeth Feifel, Gerhard Gstraunthaler, and Norman P. Curthoys, J. Biol. Chem. 281, 2982-2988 (2006).

Proteomic Analysis of the Adaptive Response of Rat Renal Proximal Tubules to Metabolic Acidosis. Norman P. Curthoys, Lynn Taylor, Jason D. Hoffert, Mark A. Knepper, Am J Physiol Renal Physiol. 292, F140-147 (2007).

Glutamatergic or GABAergic neuron specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter. Morten Rasmussen, Lingxin Kong, Guo-ring Zhang, Meng Liu, Xiaodan Wang, Gabor Szabo, Norman P. Curthoys, and Alfred Geller, Brain Res. 1144, 19-32 (2007).

Renal ammonium ion production and excretion. Norman P. Curthoys, in The Kidney: Physiology and Pathophysiology, 4th Edition, (R.J. Alpern and S.C. Hebert, Eds.), Chapter 56, pp. 1601-1619, Elsevier, Inc., San Diego, (2007).

Glutamate production by HIV-1 infected human macrophage is blocked by the inhibition of glutaminase. Nathan Erdmann, Jianxing Zhao, Alicia Lopez, Shelley Herek, Norman Curthoys, Terry D. Hexum, Takashi Tsukamoto, Dana Ferraris, and Jialin Zheng, J. Neurosci. 102, 539-549 (2007).

Novel Mechanism of Inhibition of Rat Kidney-type Glutaminase by BPTES. Mary M. Robinson, Steven J. McBryant, Takashi Tsukamoto, Camilo Rojas, Dana V.Ferraris, Sean K. Hamilton, Jeffrey C. Hansen and Norman P. Curthoys, Biochem J. 406, 407-414 (2007).

TGF-&beta signaling and its effect on glutaminase expression in LLC-PK₁-FBPase⁺ cells. Manfred Andratsch, Elisabeth Feifel, Lynn Taylor, Morgan O´Hayre, Herbert Schramek, Norman P. Curthoys, and Gerhard Gstraunthaler, Am. J. Physiol. Renal Physiol. 293, F846-F853 (2007).

Renal response to acidosis: role of mRNA stabilization. Hend Ibrahim, Yeon J. Lee and Norman P. Curthoys, Kidney Int. 73, 11-18 (2008).