Subcellular localization of tobramycin and vancomycin given alone and in combination in proximal tubular cells, determined by immunogold labeling

Beauchamp, Denis; Gourde, Pierrette; Simard, Marie; Bergeron, Michel G.

doi:10.1128/aac.36.10.2204

Cited by 35 publications

(32 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The double fluorescence staining demonstrated that the two drugs of VM and GM, an aminoglycoside antibiotic, can colocalize in the cytoplasmic granules in the proximal tubule cells. Also, GM has been reported to localize in lysosomes in the proximal tubule cells by the immunogold method and autoradiography (1,49), as has VM by the immunogold method (2). All these results strongly suggest that the cytoplasmic granules most probably represent lysosomes.…”

Section: Discussionsupporting

confidence: 58%

“…Although VM-associated nephrotoxicity has been studied in humans and animals, its exact mechanism remains to be elucidated (29). Using immunoelectron microscopy and commercial antisera, Beauchamp et al (2) have reported that VM given at doses of 50 mg/kg/day accumulates in the lysosomes of proximal tubule cells after 10 days of treatment. The study was limited to studies of the proximal tubules.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Immunocytochemistry for Vancomycin Using a Monoclonal Antibody That Reveals Accumulation of the Drug in Rat Kidney and Liver

Fujiwara

Yoshizaki

Shin

et al. 2012

Antimicrob Agents Chemother

View full text Add to dashboard Cite

bWe prepared monoclonal antibodies against N-(␥-maleimidobutyryloxy)succinimide-conjugated vancomycin (VM). The monoclonal antibody was specific for conjugated or free VM. The monoclonal antibody enabled us to develop an immunocytochemical method for detecting the uptake of VM in the rat kidney and liver. Three hours after a single intravenous (i.v.) injection of VM at the therapeutic dose, the immunocytochemistry revealed that VM accumulated in large amounts in both the S1 and S2 segments and in much smaller amounts in the S3 segment of the proximal tubules as well as in the distal tubules and collecting ducts. The drug was detected in the cytoplasm, cytoplasmic irregular granules, nuclei, and microvilli of the proximal tubule cells. The distal tubules and collecting ducts contained scattered swollen cells in which both the nuclei and cytoplasm were heavily immunostained. Twenty-four hours after injection, most of the swollen cells returned back to normal size and had somewhat decreased immunostaining. Also, significant amounts of VM remained accumulated for as long as 8 days postadministration. In the liver, similar drug accumulation was observed in the Kupffer cells and the endothelial cells of the hepatic sinusoids but not in the hepatocytes, suggesting that vancomycin cannot be eliminated via the liver. Immunoelectron microscopic studies demonstrated that in the collecting ducts, uptake of VM occurred exclusively in the lysosomes and cytoplasm of the principal cells and scarcely in the intercalated cells. Furthermore, double fluorescence staining using rats simultaneously administered with VM and gentamicin strongly suggests that both drugs colocalized in lysosomes in the proximal tubule cells of kidneys.

show abstract

Section: Discussionsupporting

confidence: 58%

Section: Discussionmentioning

confidence: 99%

Immunocytochemistry for Vancomycin Using a Monoclonal Antibody That Reveals Accumulation of the Drug in Rat Kidney and Liver

Fujiwara

Yoshizaki

Shin

et al. 2012

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…Using immunoelectron microscopy and commercial antisera, Beauchamp et al (1,2,3,4) have previously localized sites of uptake of GM, tobramycin, daptomycin, and vancomycin in the rat kidney. These pioneering studies were, however, limited to studies of the proximal tubules.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, subcellular fractionation (27), micropuncture techniques (24), and autoradiography using radioactively labeled aminoglycosides (6,26) have been used to study the mechanisms of nephrotoxity. Additionally, immunogold labeling methods have been used to study the subcellular distribution of aminoglycosides in proximal tubules of the nephron (1,2,3,4,20). However, these studies have been limited to ultrastructural analysis of proximal tubule cells.…”

mentioning

confidence: 99%

Light-Microscopic Immunocytochemistry for Gentamicin and Its Use for Studying Uptake of the Drug in Kidney

Fujiwara

Shin

Matsunaga

et al. 2009

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Gentamicin (GM) is a widely used antibiotic but shows renal toxicity. We produced a serum against GM (anti-GM) conjugated to bovine serum albumin with N-(gamma-maleimidobutyryloxy)succinimide. The antiserum was monospecific for GM and did not cross-react with the analog streptomycin, tobramycin, kanamycin, or amikacin. The antiserum also detected glutaraldehyde-fixed GM, and this enabled us to develop an immunocytochemical method for detecting the uptake of GM in rat kidney. Twelve hours after a single intravenous administration of GM, immunocytochemistry revealed that GM accumulated in the S1, S2, and S3 segments of the proximal tubules, as well as in the distal tubules and collecting ducts. By 12 h after injection, the drug was detected in cytoplasmic granules of the proximal tubule cells. However, early (1 h) after injection, drug accumulation was detected in the microvilli of these cells. The distal tubules and collecting ducts contained scattered swollen cells, reminiscent of necrotic cells, in which both the nuclei and the cytoplasm reacted strongly with GM. No staining occurred in the kidneys of saline-injected control rats. These results agree with previous studies showing that GM is endocytosed in the proximal tubules and accumulates in lysosomes. Additionally, our results show that GM also accumulates in the distal tubules and collecting ducts. This was achieved by systematically varying the pretreatment conditions-an approach necessary for detecting GM in different subcellular compartments. This approach should be useful for accurately detecting the uptake and toxicity of the antibiotic in different tissues.Gentamicin (GM) is widely used as a bactericidal agent for the treatment of severe infections with gram-negative bacteria, such as Pseudomonas aeruginosa. Its antibacterial activity is due to an irreversible inhibition of bacterial protein synthesis. However, clinical use of aminoglycosides, such as GM, is limited by their ototoxicity and nephrotoxicity. The mechanisms behind the nephrotoxity have been investigated extensively, but how these drugs induce cellular malfunction and necrosis remains to be elucidated. Accurate localization of GM in cells and tissues can be expected to contribute to this effort. Previously, subcellular fractionation (27), micropuncture techniques (24), and autoradiography using radioactively labeled aminoglycosides (6, 26) have been used to study the mechanisms of nephrotoxity. Additionally, immunogold labeling methods have been used to study the subcellular distribution of aminoglycosides in proximal tubules of the nephron (1,2, 3,4,20). However, these studies have been limited to ultrastructural analysis of proximal tubule cells. Recently, we have successfully developed immunocytochemical (ICC) procedures for detecting the cellular uptake of other water-diffusible small-molecule drugs, such as daunomycin (DM) (14,15,22,23). These procedures make use of glutaraldehyde (GA)-fixed tissues, which undergo a series of pretreatments to unmask sites of accumulation of the drug...

show abstract

“…Few studies have dealt with glycopeptides. Vancomycin shows a slow uptake and modest accumulation in macrophages (up to eightfold in 24 hours [65]) and is supposed to accumulate in lysosomes (at least in proximal tubular cells of the kidney after in vivo administration [66]). Conversely, teicoplanin, a more lipophilic compound, shows a more extensive and faster accumulation (40-to 60-fold [64,67]) but its localization is not known.…”

Section: Other Antibioticsmentioning

confidence: 99%

Intracellular pharmacodynamics of antibiotics

Carryn

Chanteux

Seral

et al. 2003

Infectious Disease Clinics of North America

175

151

View full text Add to dashboard Cite

Subcellular localization of tobramycin and vancomycin given alone and in combination in proximal tubular cells, determined by immunogold labeling

Cited by 35 publications

References 24 publications

Immunocytochemistry for Vancomycin Using a Monoclonal Antibody That Reveals Accumulation of the Drug in Rat Kidney and Liver

Immunocytochemistry for Vancomycin Using a Monoclonal Antibody That Reveals Accumulation of the Drug in Rat Kidney and Liver

Light-Microscopic Immunocytochemistry for Gentamicin and Its Use for Studying Uptake of the Drug in Kidney

Intracellular pharmacodynamics of antibiotics

Contact Info

Product

Resources

About