Renal Ca2+ reabsorption plays an important role in total body Ca2+ homeostasis. The distal tubule (distal convoluted tubule (DCT), connecting tubule (CNT), and cortical collecting duct (CCD)) reabsorbs only about 10% of the filtered load of Ca2+, but is the major regulatory site for Ca2+ reabsorption in the kidney under the control of parathyroid hormone (PTH). Recently, Bindels et. al. suggested that l,25-dihydroxyvitamin D3 also regulated transepithelial Ca2+ flux. In the distal tubule it is generally accepted that Ca2+ enters the cell across the apical membrane passively down a large electrochemical gradient via calcium channels, but must then exit actively across the basolateral membrane. The exit step can occur through at least two different transport pathways, the Na+/Ca2+ exchanger, and the Ca2+-ATPase. Studies by Shimizu et. al. in isolated perfused tubules (CNT-rabbit) and Bindels et. al. in primary cultures (CNT and CCD- rabbit) suggest that the Na+/Ca2+ exchanger plays a major role in the basolateral exit of Ca2+ in this nephron segment. PTH appears to regulate both the apical entry of Ca2+ and its basolateral exit Gesek et al. in an immortalized mouse cell line from medullary thick ascending limb and DCT, showed that PTH initially stimulated the opening of a chloride channel that hyperpolarized the cell membrane. After a short delay, this was then followed by the opening of a dihydropyridine- sensitive Ca2+ channel that was activated by hyperpolarization. That will directly affects Na+/Ca2+ exchange activity in kidney was reported in basolateral membrane vesicles by several groups. The effect was the result of a change in the Vmax for Na+ gradient-dependent Ca2+ uptake with no change in Km, and was not mimicked by dibutyryl cAMP. Recently, we reported the molecular cloning of a renal Na+/Ca2+ exchanger from rabbit kidney cortex and its immunolocalization using both monoclonal and polyclonal antibodies directed against an intracellular epitope. We showed that the Na+/Ca2+ exchanger is highly homologous to the canine cardiac sarcolemmal Na+/Ca2+ exchanger (95% amino acid identity), and is primarily expressed in the distal nephron (CNT) of both rabbit and rat in this proposal we will use these cDNAs and antibodies to examine the molecular mechanisms responsible for regulation of the renal Na+/Ca2+ exchanger in a primary culture of CNT isolated by indirect immunodissection. These studies will require an antibody directed against an extracellular epitope (for indirect immunodissection). Therefore, a monoclonal antibody will first be generated to an extracellular epitope of the exchanger, and employed to isolate the primary culture. The mechanisms of regulation of the exchanger in response to the acute (1-3 hrs.) and chronic (72 hrs.) exposure to PTH and 1,25-dihydroxyvitamin D3 will then be examined. These studies will provide important novel insights into the molecular mechanisms responsible for the regulation of the renal Na+/Ca2+ exchanger.
| Shugrue, C A; Obermuller, N; Bachmann, S et al. (1999) Molecular cloning of NHE3 from LLC-PK1 cells and localization in pig kidney. J Am Soc Nephrol 10:1649-57 |
| White, K E; Gesek, F A; Reilly, R F et al. (1998) NCX1 Na/Ca exchanger inhibition by antisense oligonucleotides in mouse distal convoluted tubule cells. Kidney Int 54:897-906 |
