The Role of Megalin in Aminoglycoside-Associated Toxicity in the Kidney

Date

2021-05

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Abstract

Multi-drug resistant (MDR) bacterial infections remain a prevalent problem due to limited effective and safe antibiotic options. While aminoglycosides have been effective in treating these life-threatening MDR bacterial infections, clinical treatment with these drugs has been limited due to their high nephrotoxic potential. As a result, aminoglycosides have been conservatively used in only the most desperate cases. In recent years, as the development of new antibiotics remains limited, there is a renewed interest in aminoglycosides and addressing their associated nephrotoxicity. Subsequent studies show that amikacin accumulates in the kidney during the filtration process and triggers a cascade of cellular events resulting in cell death, acute kidney injury and in some cases, chronic kidney failure. Additional studies show that amikacin primarily accumulates in the renal proximal tubules, densely located in the medulla, and suggest that megalin, a saturable endocytic membrane transporter, may be largely responsible for aminoglycoside accumulation. Despite the overwhelming evidence for a significant role of megalin in aminoglycoside accumulation, the complete mechanism of uptake has not been fully elucidated, nor have any appropriate interventions been successfully identified. Therefore, this project aims to further investigate the role of megalin in amikacin uptake and to identify potential interventions to improve patient outcomes following aminoglycoside treatment. To do this, a robust quantification method was developed to measure amikacin concentrations in different biological matrices, cellular uptake studies were used to gather insights of the potential role of megalin in amikacin uptake, and animal studies were used to evaluate potential pharmacotherapeutic interventions that may reduce the occurrence of amikacin-associated nephrotoxicity. For the quantification assay, a liquid chromatography – mass spectrometry (LCMS) assay was developed to circumvent common limitations associated with conventional assays. The assay was optimized with high pH mobile phases achieved with ammonium hydroxide. This method was then used to quantify amikacin in all subsequent experiments after appropriate sample preparation methods were developed for the different biological matrices including cell lysate, serum and kidney homogenate. Cellular uptake studies using wildtype and megalin-knockout cells were performed to try to clarify the quantitative role of megalin in the uptake of amikacin into the kidneys. These studies were designed to characterize amikacin uptake differences in megalin-knockdown or megalin-knockout cells and wildtype cells. These studies also provided a framework for in vitro pharmacotherapeutic interference studies whereby wildtype cells were assayed for amikacin uptake with and without pharmacotherapeutic interference. Finally, a clinically relevant animal model was used to evaluate various potential pharmacotherapeutic interventions that may mitigate aminoglycoside-associated nephrotoxicity. Clinical relevance was established by finding a dose range in the animal model that was equivalent to total drug exposure to that found in clinical data. Several pharmacotherapeutic interventions using known megalin ligands were evaluated in a series of time to nephrotoxicity studies and analyzed by Kaplan-Meier analysis. An additional proprietary drug demonstrated the potential to mitigate amikacin-associated nephrotoxicity. Additional research is necessary to continue to fully understand the mechanism of aminoglycoside uptake. This research will be an important step in the global antimicrobial crisis to provide more effective and safe therapeutic options.

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Keywords

Amikacin, LCMS, rat model, pharmacokinetics

Citation

Portions of this document appear in: Chan, Katrina, Weiqun Wang, Kimberly R. Ledesma, Taijun Yin, and Vincent H. Tam. "A robust LC–MS/MS method for amikacin: application to cellular uptake and pharmacokinetic studies." Bioanalysis 12, no. 7 (2020): 445-454.; Tam, Vincent H., Daniel N. Cohen, Kimberly R. Ledesma, Bobby Guillory, Katrina Chan, and Kevin W. Garey. "Local tissue response to subcutaneous administration of ceftriaxone in an animal model." Antimicrobial agents and chemotherapy 64, no. 3 (2019): e02090-19.