(T1430-09-50) Amelioration of Drug-Induced Nephrotoxicity Using Ganglioside Liposomes

Purpose: The commonly prescribed drugs such as antibiotics, NSAIDs, diuretics, antihypertensives, and anticancer drugs are often associated with nephrotoxicity as one of their side effects. These nephrotoxic drugs cause acute kidney injury (AKI). Majority of the mechanism of nephrotoxicity is via induction of reactive oxygen species (ROS) downstream which accumulate in the cells and activate various signaling pathways leading to renal tubular cell death. Ganglioside GM 1 nanoliposomes (NLGM1) have previously been shown to be effective in fighting oxidative stress via the Nrf2-Keap1 signaling pathway. In this research we aim to investigate the utility of NLGM1 in overcoming nephrotoxicity and hence decreasing kidney injury.

Methods: In a detailed in vitro investigation, assays such as cell viability assay and ELISA assay for the sensitive biomarker of kidney injury viz. Kidney Injury marker-1 (KIM-1) and Heme Oxygenase (HO) were carried out on HK-2 cells from the proximal tubule. In a preliminary in vivo experiment, male Sprague Dawley rats were administered vancomycin hydrochloride or NLGM1 150 mg/kg/day for 3 days using an IV infusion in the left jugular vein catheter. Urine was collected after the first and the last day of IV infusions using metabolic cages. Vancomycin hydrochloride was quantified in rat urine and kidney by LC-MS/MS. Urinary KIM-1 analysis was done by using an ELISA kit.

Results: Dose-dependent HK-2 cell killing was observed with the highest killing and least viability at 100 mM and no killing (highest viability) at 0 mM in all drugs tested. Cell killing was found to be reversed to more than 90% viability in presence of NLGM1 in all concentrations tested for all drugs (p < 0.05). Supplementing this finding the KIM-1 was found to be significantly low (p < 0.05) in presence of NLGM1 with nephrotoxic drugs compared to nephrotoxic drugs alone. HO concentrations in presence of NLGM1 with nephrotoxic drugs were significanly higher (p < 0.005) compared to nephrotoxic drugs alone. Vancomycin hydrochloride concentrations in urine were higher in the vancomycin hydrochloride co-administered with NLGM1 group. Whereas in the kidney, vancomycin hydrochloride concentrations and KIM-1 concentration were lower in the vancomycin hydrochloride co-administered with NLGM1 group (p < 0.05, ANOVA and/or t test). Vancomycin hydrochloride co-administered with NLGM1 resulted in lower kidney injury as noted by a decrease in KIM-1. Utility of NLGM1 liposomes in reversing the nephrotoxic drug induced cell killing and reduction in KIM-1 levels was established in vitro and in vivo assays we performed.

Conclusion: The cell viability assay was found to be an efficient, accurate, and precise method for measuring cell toxicity caused by drugs at various concentrations in HK-2 cells. Utility of NLGM1 liposomes in reversing the nephrotoxic drug induced cell killing and reduction in KIM-1 levels and higher HO-1 levels was established in the various in vitro assays we performed. In the future, we intend to execute in vivo assays, e.g., KIM-1 ELISA, histopathology etc., in rats to establish the efficacy of NLGM1 liposomes further.

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Acknowledgements: The authors would like to acknowledge the College of Pharmacy-Glendale intramural grant awarded to Dr Medha D Joshi in 2022-2023 to pursue this research.All parts of this work, comprising housing, experimentation, and animal disposal, were performed in general agreement with the “Guide for the Care and Use of Laboratory Animals: Eighth Edition” (National Academies Press, Washington, DC, USA, 2011) in Pharmacology Discovery Services Taiwan, Ltd. (PDST), (Taipei, Taiwan), an AAALAC-accredited laboratory animal facility. In addition, the animal care and use protocol was reviewed and approved by the IACUC (number PK001–08242021) at Pharmacology Discovery Services Taiwan, Ltd. (PDST), (Taipei, Taiwan).

Cell viability of HK cells (1x105 cells/mL) after treatment with A. Vancomycin Hydrochloride OR Vancomycin Hydrochloride +NLGM1 liposomes B. Tobramycin OR Tobramycin +NLGM1 liposomes C. Gentamycin OR Gentamycin +NLGM1 liposomes D. Polymyxin B OR Polymyxin +NLGM1 liposomes B. Cisplatin OR + Cisplatin NLGM1 liposomes F. Cadmium Chloride OR Cadmium Chloride +NLGM1 liposomes G. Ciprofloxacin OR Ciprofloxacin +NLGM1 liposomes H. Amphotericin B OR Amphotericin B +NLGM1 liposomes on a half volume black 96 well plate for 48 hours. At the end of 48 hours cell supernatant was replaced with media and AlamarBlue. Incubated for 2 hours and read on plate reader. Readings were normalized with appropriate controls, n=8. Statistically significant difference was identified using unpaired t test (***p < 0.001, ****p < 0.0001)

Figure 2. KIM-1 levels of HK cells supernatants (1x105 cells/mL) after treatment with Vancomycin Hydrochloride in PBS OR Vancomycin Hydrochloride +NLGM1 liposomes on 12 well plate for 48 hours. At the end of 48 hours cell supernatant was collected and replaced with fresh media. Supernatant was treated with Quantikine ELISA Kit (R&D systems). Readings were normalized with appropriate controls, n=3. Statistically significant difference was identified using unpaired t test (*p < 0.05. **p < 0.01, ***p < 0.0001)

Figure 2.Ho-1 levels of HK cells supernatants (1x107 cells/mL) after treatment with Vancomycin Hydrochloride in PBS OR Vancomycin Hydrochloride +NLGM1 liposomes on 6 well plate for 20 hours. At the end of 20 hours cell supernatant was collected. Supernatant was treated with Human Heme Oxygenase 1 ELISA Kit (Abcam). Readings were normalized with appropriate controls, n=3. Statistically significant difference was identified using unpaired t test (*p < 0.05. **p < 0.01, ***p < 0.0001)