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

Abstract

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.

Vancomycin‐Induced Kidney Injury: Animal Models of Toxicodynamics, Mechanisms of Injury, Human Translation, and Potential Strategies for Prevention

“…Vancomycin is actively transported across the basolateral membrane into proximal tubule cells via the organic cation transporter system 55, 56 (Figure 3). The visualization of large amounts of vancomycin at the brush border also points to either ongoing secretion or reabsorption of the drug at the apical side (i.e., from the urine) 57, 58 . In support, recent preclinical data have demonstrated vancomycin transport across the brush border membrane by apical endocytosis via dehydropeptidase 59 and megalin 60 and vancomycin‐mediated inhibition of the expression and function of P‐glycoprotein, an efflux transporter 61 .…”

“…Endosomes fuse with lysosomes, which leads to accumulation of vancomycin in lysosomes. In 1992, vancomycin accumulation in the lysosomes of proximal tubule cells was first visualized using immunoelectron microscopy 58 ; and later confirmed by another group 57 . This accumulation of vancomycin in lysosomes may trigger a detrimental autophagy process.…”

Intravenous Vancomycin Dosing in the Elderly: A Focus on Clinical Issues and Practical Application

“…In these patients, loading doses of 15–25 mg/kg should be based on actual body weight [71]. Because vancomycin accumulates in the tissues [58, 72], consideration should be given to empirically reducing the maintenance dose, particularly in patients who are critically ill [9, 29, 68, 70]. Additionally, even though vancomycin is 50% bound to serum albumin [10], clinically dosing vancomycin according to serum albumin concentrations is not performed.…”

High‐Performance Liquid Chromatography Method for Rich Pharmacokinetic Sampling Schemes in Translational Rat Toxicity Models With Vancomycin

“…Complicating the understanding, clinical dosing results in homogenous exposure profiles, and patient comorbidities impact the ability to identify the true toxicodynamic relationship. Thus, there is a renewed interest in controlled laboratory experiments (e.g., animal models) to best discern mechanistic links between pharmacokinetic (PK) exposures and toxicodynamic outcomes . Additionally, there are limited data on toxicity of vancomycin in utero , and these analyses were performed before sensitive biomarkers of vancomycin toxicity were available for study.…”

“…Thus, there is a renewed interest in controlled laboratory experiments (e.g., animal models) to best discern mechanistic links between pharmacokinetic (PK) exposures and toxicodynamic outcomes. [8][9][10][11][12] Additionally, there are limited data on toxicity of vancomycin in utero, 13 and these analyses were performed before sensitive biomarkers of vancomycin toxicity were available for study.…”