Metabolic acidosis is a pronounced disturbance in acid-base balance that is caused by genetic or acquired defects in metabolism, in renal handling of bicarbonate, and in the excretion of titratable acid. Chronic acidosis causes mental retardation in children and osteomalacia, nephrocalcinosis and urolithiasis in adults. During metabolic acidosis, a compensatory increase in renal glutamine catabolism occurs in the proximal convoluted tubule. The resulting increases in ammonium and bicarbonate ion synthesis facilitate the excretion of acid and partially restore acid-base balance. This essential adaptive response is sustained by pronounced changes in gene expression that occur primarily through post-transcriptional regulation. A paradigm for this response is the sustained increase in glutaminase (GA) that occurs through selective stabilization of GA mRNA and is mediated by two 8-nt AU-sequences that function as a pH-response element (pHRE). The rapid turnover of GA mRNA that occurs during normal acid base-balance is preceded by a rapid deadenylation, while the pH-responsive stabilization is associated with a decreased rate and extent of deadenylation. HuR and p40-AUF1 bind to the pHRE with high affinity and specificity. Preliminary experiments establish that onset of acidosis activates an ER-stress signaling pathway in rat renal proximal tubules and promotes the formation of RNA stress granules and the release of HuR from the nucleus in cultured kidney cells. Therefore, during normal acid-base balance, a pHRE binding protein may facilitate the recruitment of a deadenylase and the subsequent exonucleolytic decay of GA mRNA. During metabolic acidosis, association with stress granules may promote remodeling of the GA mRNA and facilitate its association with stabilizing factors such as HuR or p40AUF1. To test these novel hypotheses, the following specific aims are proposed: to identify the ER-stress signaling pathway that is activated in response to onset of acidosis and to utilize dominant negative and siRNA constructs to establish its role in stabilization of GA mRNA;to characterize the potential roles of HuR and the cytosolic isoforms of AUF1 by performing over expression and siRNA knockdown experiments and by utilizing protein/RNA pull-down, immunofluorescence, and mass spectroscopic analysis to establish their temporal association with GA mRNA, cellular localization, and sites of phosphorylation;and to employ a novel protocol to rapidly purify and identify pHRE/protein complexes that are formed in intact cells. The resulting data will define the mechanism by which the proximal tubule cell senses slight changes in pH and mediates the remodeling of its proteome in response to acidosis. It will also significantly enhance understanding of the fundamental mechanisms that regulate changes in mRNA stability and mediate an essential physiological response of the kidney.

Public Health Relevance

Metabolic acidosis is a common clinical condition that contributes to osteomalacia, nephrocalcinosis and urolithiasis in adults and causes mental retardation in children. During end stage renal disease, the development of metabolic acidosis correlates with increased peripheral insulin resistance and is an additional morbidity risk factor. To compensate for the onset of acidosis requires an appropriate increase in renal acid excretion and bicarbonate production. This essential response is initiated by an adaptive increase in renal catabolism of glutamine that occurs solely within the proximal convoluted tubule and is sustained, in large part, by the cell-specific and pronounced increase in the level of glutaminase mRNA and protein. The proposed experiments will greatly enhance knowledge of the mechanism by which the kidney senses changes in acid-base balance and transmits this information to promote selective stabilization of glutaminase mRNA. Recent proteomic analysis indicates that expression of multiple genes in the proximal tubule during acidosis is regulated through the same mechanism of mRNA stabilization. This system is also an important paradigm for understanding the mechanism by which the physiological regulation of mRNA turnover contributes to an essential adaptive response. In addition, the resulting data will contribute fundamental knowledge of the mechanism and regulation of mRNA turnover, both in this system and in general. This knowledge will constitute the basis for biochemical and pharmacological studies that may improve the treatment of patients who present with chronic forms of metabolic acidosis.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Cellular and Molecular Biology of the Kidney Study Section (CMBK)
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Rasooly, Rebekah S
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Colorado State University-Fort Collins
Schools of Arts and Sciences
Fort Collins
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