Effects of Azithromycin on kidney cells in culture treated with and without 2,8-Dihydroxyadenine crystals

Abstract

Adenine phosphoribosyl transferase (APRT) deficiency is a rare metabolic disorder caused by a mutation in the APRT gene that causes increased accumulation of adenine, which is broken down by xanthine oxireductasedase (XOR) to 2,8-dihydroxyadenine (2,8-DHA). Continuous excretion of 2,8-DHA leads to the formation of crystalline deposits in the renal tubules, which can cause both inflammation and fibrosis. Azithromycin (Azm) is a broad-spectrum macrolide antibiotic and has been shown to have both anti-inflammatory and epithelial barrier enhancing effects on the respiratory epithelium. Studies have also shown that Azm prevents epithelial-mesenchymal transition (EMT) in the lung epithelium.The purpose of the study was to investigate the effects of Azm on kidney cells and see if Azm treatment could prevent further damage on the epithelium caused by 2,8-DHA. The renal cell lines HK-2 and MDCK were treated with and without Azm and exposed to 2,8-DHA. HK-2 cells were also treated with TGF-β to induce EMT in cells. The renal cells were treated in both 2D and 3D cultures, along with a liquid-liquid interface (LLI) culture. The phenotypic changes of the cells were assessed by live cell imaging, cell viability assay, RT-qPCR, western blotting and fluorescence staining, as well as measurement of the trans-epithelial electrical resistance (TEER). The expression of genes and proteins involved in EMT as well as genes involved in the inflammatory response was specifically examined.Based on my results, Azm alone seemed to alter the morphology of renal cells. In 2D culture, HK-2 cells were seen to become more elongated with Azm, and in 3D culture, cell-cell adhesion appeared to increase. Azm treatment, had no significant effect on gene expression in the cells. Azm treatment did not appear to be able to prevent or reduce the formation of EMT in HK-2 cells. The results indicated that Azm treatment could not affect the epithelial barrier and molecular changes after 2,8-DHA exposure in MDCK cells. However, this study was conducted as a pilot, so more detailed studies are needed to determine whether Azm treatment could have a positive effect on renal epithelial cells after 2,8-DHA exposure.