Gentamicin traffics rapidly and directly to the Golgi complex in LLC-PK1 cells

Ruben M. Sandoval, Kenneth Dunn, Bruce Molitoris

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

To study the intracellular mechanisms of aminoglycoside toxicity, we used a 1:1 fluorescent conjugate of Texas Red and gentamicin (TRG) to quantify early uptake dynamics in renal epithelial (LLC-PK1) cells. Utilizing a protocol that quenches TRG fluorescence from lysosomes, the bulk of intracellular accumulation, we determined a portion rapidly trafficked directly to the Golgi complex when identified by a FITC-conjugated lectin from Lens culinaris agglutinin (LCA). A kinetic study over 120 min on cells showing total and quenched TRG fluorescence was then carried out, and the fluorescence intensity from the images was quantified. Trafficking of TRG to the Golgi complex occurred within 15 min and accounted for ~20% of total cellular accumulation in the kinetic study. Colocalization studies using compartment-specific markers, 6-[N-(7-nitro-benz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl sphingosine (C6-NBD ceramide) and LCA, for the TGN trans-Golgi network, and the cis/medial-Golgi compartments, respectively, determined colocalization occurred with both Golgi compartments. These data support the existence of a pathway that directly and rapidly shuttles a portion of internalized gentamicin to the Golgi complex. We believe this pathway may be responsible for the early negative effects seen on protein synthesis in renal proximal epithelia after aminoglycoside administration.

Original languageEnglish
JournalAmerican Journal of Physiology - Renal Physiology
Volume279
Issue number5 48-5
StatePublished - 2000

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LLC-PK1 Cells
Golgi Apparatus
Gentamicins
Fluorescence
Aminoglycosides
trans-Golgi Network
Kidney
Sphingosine
Fluorescein-5-isothiocyanate
Lysosomes
Lectins
Epithelium
Texas red
Proteins

Keywords

  • Aminoglycosides
  • Lysosomal-fluorescence quenching
  • Nephrotoxicity

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)

Cite this

Gentamicin traffics rapidly and directly to the Golgi complex in LLC-PK1 cells. / Sandoval, Ruben M.; Dunn, Kenneth; Molitoris, Bruce.

In: American Journal of Physiology - Renal Physiology, Vol. 279, No. 5 48-5, 2000.

Research output: Contribution to journalArticle

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