Gentamicin causes endocytosis of Na/Pi cotransporter protein (NaPi-2)

Background. Renal toxicity is a major side-effect of aminoglycoside antibio tics and is characterized by an early impairment in proximal tubular functi on. In a previous study, we have shown that gentamicin administration to th e rat causes an early impairment in sodium gradient-dependent phosphate (Na /Pi) cotransport activity. The purpose of our current study was to determin e the molecular mechanisms of the impairment in Na/Pi cotransport activity, specifically the role of the proximal tubular type II Na/Pi cotransporter. Methods. Rats were treated for one, two, and three days with two daily inje ctions of 30 mg/kg body weight gentamicin or the vehicle. Results. Gentamicin caused a progressive decrease in superficial cortical a pical brush-border membrane (SC-BBM) Na/Pi cotransporter activity (856 +/- 93 in control vs. 545 +/- 87 pmol/mg BBM protein in 3-day gentamicin, P < 0 .01). Western blot analysis showed a parallel and progressive decrease in S C-BBM Na/Pi cotransporter protein abundance, a 50% decrease after one day o f treatment, a 63% decrease after two days of treatment, and an 83% decreas e after three days treatment with gentamicin. In contrast, gentamicin treat ment had no effect on Na/Pi cotransport activity or Na/Pi cotransporter pro tein abundance in BBM isolated from the juxtamedullary cortex (JMC-BBM). Im munofluorescence microscopy showed a major decrease in the expression of Na /Pi cotransporter protein in the apical membrane of the proximal convoluted tubule, with progressive intracellular accumulation of Na/Pi protein. Colo calization studies showed that in gentamicin-treated rats, Na/Pi protein wa s colocalized in the early endosomes and especially in the lysosomes. North ern blot analysis of cortical RNA interestingly showed no reduction in Na/P i cotransporter mRNA abundance even after three days of gentamicin treatmen t. Conclusion. We conclude that gentamicin inhibits Na/Pi cotransport activity by causing a decrease in the expression of the type II Na/Pi cotransport p rotein at the level of the proximal tubular apical BBM and that inhibition of Na/Pi cotransport activity is most likely mediated by post-transcription al mechanisms.