Supported in part by grants from the Ministry of Science and Technology (PB98-0700-C02-01/02 and SAF 2002-04356-C01/03), Comunidad Autónoma de Madrid (CAM 08.6/0038.2000 1/ 2) and the Spanish Society of Nephrology.
Both renin-angiotensin system and PTHrP are overexpressed after folic acid (FA)-induced acute renal failure (ARF). Recently, we demonstrated that PTHrP was upregulated in the renal parenchyma following AngII infusion. In the present in vitro study, we evaluated the putative role of AngII on the mechanism of PTHrP overexpresion induced by FA in renal tubular cells.
Results: We found that a nephrotoxic dose of FA (10 mM) stimulates PTHrP gene and protein expression (2-fold over control) in two renal tubuloepithelial cell lines (NRK 52E and MCT). The AT1 receptor antagonist losartan, at 10 µM, abolished this effect of FA in these cells. Inmunofluorescence analysis by using a confocal microscope showed a rapid (within 10-15 min) translocation of AngII from the cytoplasm to the nucleus of NRK 52E cells after FA treatment; an effect which was inhibited by losartan. Using Western blot, we observed an early activation of phospho-ERK ¾ after stimulation with either FA or AngII (100 nM) in both cell lines. Since the promoter region of the PTHrP gene has binding sites for cAMP-responsive elements (CREB), we assessed the putative activation of CREB (by EMSA) in these cells after stimulation with FA or AngII. Both agonists were found to induce an activation of CREB within 5-10 min in these cells. This was abolished after pretreatment with the MAPK inhibitor PD098059 or losartan, both at 10 µM. Furthermore, cells which had been transfected with a dominant-negative mutant vector preventing CREB activation, failed to show any PTHrP gene response following either FA or AngII treatments.
Conclusion: These findings support the notion that AngII, acting through AT1-induced CREB activation, has a paramount role on the PTHrP upregulation in FA-induced ARF.
