Osmotic diuretics are used successfully to alleviate acute tubular necrosis (ATN) produced by chemotherapeutic agents and aminoglycoside antibiotics. The beneficial action of these agents likely involves rapid elimination of the nephrotoxic agents from the kidney by promoting diuresis. Adenosine A(1) receptor (A(1)AR) subtype present on renal proximal tubular epithelial and cortical collecting duct cells mediates the antidiuretic and cytoprotective actions of adenosine. These receptors are induced by activation of nuclear factor (NF)-kappaB, a transcription factor reported to mediate hyperosmotic stress-induced cytoprotection in renal medullary cells. In this study, we tested the hypothesis that induction of the A(1)AR in renal proximal tubular cells by NF-kappaB contributes to the cytoprotection afforded by osmotic diuretics. Exposure of porcine renal proximal tubular epithelial (LLC-PK1) cells to mannitol or NaCl produced a significant increase in A(1)AR. This increase was preceded by adenosine release and NF-kappaB activation. Expression of an IkappaB-alpha mutant, which acts as a superrepressor of NF-kappaB, abrogated the increase in A(1)AR. Cells exposed to mannitol demonstrated increased reactive oxygen species (ROS) generation, which was attenuated by inhibiting xanthine oxidase with allopurinol. Allopurinol attenuated both the increase in A(1)AR expression and NF-kappaB activation produced by osmotic diuretics, indicating a role of adenosine metabolites in these processes. Treatment of LLC-PK1 cells with cisplatin ( 8 muM) resulted in apoptosis, which was attenuated by mannitol but exacerbated by selective A(1)AR blockade. Administration of mannitol to mice increases A(1)AR expression and activation of NF-kappaB in renal cortical sections. Taken together, these data provide novel mechanisms of nephroprotection by osmotic diuretics, involving both activation and induction of the A(1)AR, the latter mediated through activation of a xanthine oxidase pathway leading to ROS generation and promoting activation of NF-kappaB.