|Reinhardt, Timothy - Tim|
Submitted to: American Society for Bone and Mineral Research
Publication Type: Abstract Only
Publication Acceptance Date: 6/15/2008
Publication Date: 9/12/2008
Citation: Forster, M.A., Masilamani, S., Reinhardt, T.A., Beckman, M.J. 2008. Metabolic alkalosis transition in renal proximal tubule cells facilitates an increase in CYP27B1, while blunting responsiveness to PTH [abstract]. Journal of Bone and Mineral Research. 23(abstracts supplement):S422. Interpretive Summary:
Technical Abstract: Parathyroid hormone (PTH) is the central activator of renal proximal 1-alpha-hydroxylase (CYP27B1), the enzyme responsible for synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Past studies have documented a disruption of CYP27B1 activity in chronic metabolic acidosis in vivo, while simulated acidosis in cultured renal cells failed to demonstrate impairment of 1,25(OH)2D3 synthesis. To determine the factors involved in regulating responsiveness of CYP27B1 to PTH in renal proximal tubule cells, we developed a dietary protocol to study the transition from metabolic acidosis (diet low in potassium) to metabolic alkalosis (diet high in potassium-bicarbonate) in young adult rats, n = 9. Gene expression was determined in renal proximal tubules isolated from each group using Affymetrix GeneChip® microarray, n = 3. The high potassium bicarbonate diet was found to induce metabolic alkalosis (> 7.5) by day 10 as tested by urine pH. The rats being fed the control diet were maintained at a slightly acidic urine pH (< 7.4) throughout the study. Blood concentrations of phosphate, calcium and 25-OH-D3 were replete under the conditions effected by both acidic and basic diets, however, blood 1,25(OH)2D3 concentrations were significantly increased by the switch to the high potassium-bicarbonate alkaline diet. This was matched by an increase in gene expression of 1a-hydroxylase (CYP27B1), 9kDa-CaBP and NR4A2, and a decrease in VDR following alkalosis in renal proximal tubules. The orphan nuclear receptor family 4A, member 2 (NR4A2, also known as HZF-2, NURR1) was recently identified as a positive regulator of CYP27B1 transcription in renal proximal tubule epithelial cells. In the present study, intermediate components of PTH cell signaling such as adenylate cyclase and CREB were expressed at significantly higher levels in animals fed the acidic control diet, while NR4A2 was closely associated with the regulation pattern of CYP27B1 in metabolic alkalosis. These results indicate that the important link between PTH responsiveness and renal CYP27B1 gene expression is predisposed by levels of cAMP/PKA/CREB pathway intermediates, but CYP27B1 expression requires NR4A2. In conclusion, chronic acidosis suppressed CYP27B1 gene expression in proximal tubules but maintained enhanced responsiveness to PTH. In contrast, metabolic alkalosis displayed blunted PTH responsiveness.