Date of Award

2020

Document Type

Dissertation

Degree Name

Philosophy (Ph.D)

Department

Pharmaceutical Sciences

First Advisor

Sue Ford

Second Advisor

Louis Trombetta

Third Advisor

Francis Schanne

Abstract

In vitro methods can be cost effective and facilitate large scale drug screening in the pharmaceutical industry. Renal proximal tubular cells have been a prime target for drug induced toxicity. LLC-PK1 cells possess many transport functions of the proximal tubule epithelia, however, its energy metabolism differs from the highly oxidative tubule metabolism in vivo. This may impact its reliability in predicting toxicity. Modulating the growth media composition has been shown to shift cultured liver and muscle cells from high glycolytic activity to increased oxidative metabolism, but few studies have been done on renal cells. This study is designed to test the impact of growth media modulation on mitochondrial respiration and glycolytic activity of LLC-PK1 cells via measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XFp Analyzer (Agilent Technologies). Cells were grown to confluence in SFFD (50:50 DMEM-Ham’s nutrient mix F-12 with 15 mM HEPES) culture medium containing 3% FBS and 5mM glucose or 5mM glucose supplemented with 5mM acetoacetate. Extracellular flux (XF) analysis showed that the basal respiration, maximal respiration, spare respiratory capacity and ATP-linked respiration were significantly increased, indicating an increase in mitochondrial function. The increase in mitochondrial function was accompanied by an increased expression in biomarkers   of mitochondrial biogenesis, PGC-1α and TFAM. There was also an increase in mitochondrial COX IV expression. Additionally, XF analysis also showed a significant decrease in glycolytic capacity and reserve for the acetoacetate group. LC50 values for mitochondrial toxicants, clotrimazole, diclofenac and ciprofibrate were assessed via a modified trypan blue staining assay. Cells grown in media complemented with acetoacetate displayed significantly lower LC50 values when treated with clotrimazole and diclofenac. There was a marked increase in uncoupled respiration in the presence of diclofenac, while clotrimazole and ciprofibrate significantly decreased respiration in the acetoacetate group. In conclusion, acetoacetate complemented media can alter the cellular metabolism of the LLC-PK1 cell line and increase its sensitization to toxicants, thus making this a better model for toxicity drug screening.

Available for download on Thursday, September 16, 2021

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Biochemistry Commons

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