Gentamicin causes endocytosis of Na/Pi cotransporter protein (NaPi-2).
BACKGROUND
Renal toxicity is a major side-effect of aminoglycoside antibiotics and is characterized by an early impairment in proximal tubular function. In a previous study, we have shown that gentamicin administration to the rat causes an early impairment in sodium gradient-dependent phosphate (Na/Pi) cotransport activity. The purpose of our current study was to determine 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 injections of 30 mg/kg body weight gentamicin or the vehicle.


RESULTS
Gentamicin caused a progressive decrease in superficial cortical apical 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 SC-BBM Na/Pi cotransporter protein abundance, a 50% decrease after one day of treatment, a 63% decrease after two days of treatment, and an 83% decrease after three days treatment with gentamicin. In contrast, gentamicin treatment had no effect on Na/Pi cotransport activity or Na/Pi cotransporter protein abundance in BBM isolated from the juxtamedullary cortex (JMC-BBM). Immunofluorescence 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. Colocalization studies showed that in gentamicin-treated rats, Na/Pi protein was colocalized in the early endosomes and especially in the lysosomes. Northern blot analysis of cortical RNA interestingly showed no reduction in Na/Pi cotransporter mRNA abundance even after three days of gentamicin treatment.


CONCLUSIONS
We conclude that gentamicin inhibits Na/Pi cotransport activity by causing a decrease in the expression of the type II Na/Pi cotransport protein at the level of the proximal tubular apical BBM and that inhibition of Na/Pi cotransport activity is most likely mediated by post-transcriptional mechanisms.
University of Zaragoza, Zaragoza, Spain.
V
V
Sorribas
N
N
Halaihel
K
K
Puttaparthi
T
T
Rogers
R E
RE
Cronin
A I
AI
Alcalde
J
J
Aramayona
M
M
Sarasa
H
H
Wang
P
P
Wilson
H
H
Zajicek
M
M
Levi
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005839:Gentamicins / Q000494:pharmacology*; D066298:In Vitro Techniques; D007672:Kidney Cortex / Q000187:drug effects / Q000378:metabolism*; D007679:Kidney Medulla / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters
D000900:Anti-Bacterial Agents; D002352:Carrier Proteins; D005839:Gentamicins; D012333:RNA, Messenger; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters