Supplementary Material for: Unravelling Pathophysiology of Crystalline Nephropathy in Ceftriaxone-Associated Acute Kidney Injury: A Cellular Proteomic Approach
datasetposted on 16.01.2018, 15:58 by Chatchen S., Pongsakul N., Srisomsap C., Chiangjong W., Hongeng S., Svasti J., Chutipongtanate S.
Background: Previous studies showed that ceftriaxone can cause acute kidney injury (AKI) in the pediatric population. This study proposed a cellular model of crystalline nephropathy in ceftriaxone-associated AKI and explored the related pathophysiology by using a proteomic approach. Methods: Ceftriaxone was crystallized with calcium in artificial urine. Madin-Darby Canine Kidney (MDCK) cells, a model of distal renal tubular cell, were cultured in the absence (untreated control) or presence of ceftriaxone crystals for 48-h (n = 5 each). MDCK cells were harvested and subsequently analyzed by proteomic analysis. Protein bioinformatics (i.e., STRING and Reactome) was used to predict functional alterations, and subsequently validated by Western blotting and cellular studies. p < 0.05 was considered statistically significant. Results: Phase-contrast microscopy showed increased intracellular vesiculation and cell enlargement as a result of ceftriaxone crystal exposure. Proteome analysis revealed a total of 20 altered proteins (14 increased, 5 decreased and 1 absent) in ceftriaxone crystal-treated MDCK cells as compared to untreated cells (p < 0.05). Protein bioinformatics and validation studies supported heat stress response mediated by heat shock protein 70 (Hsp70) and downregulation of annexin A1 as the proposed pathophysiology of crystalline nephropathy in ceftriaxone-associated AKI, in which impaired proliferation and wound healing of crystal-induced distal tubular cells were outcomes. Conclusions: This study, for the first time, used the in vitro model of crystalline nephropathy to investigate the underlying pathophysiology of ceftriaxone-associated AKI, which should be investigated in vivo for potential clinical benefits in the future.