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. 2018 Mar 14;315(2):F291–F299. doi: 10.1152/ajprenal.00328.2017

Fig. 3.

Fig. 3.

Expressions of kidney injury markers during systemic infection in zebrafish. A: fold changes in mRNA expressions (mean ± SE) of tubule injury marker genes in 300 CFU-infected vs. control larvae 24 h postinjection. Each sample contains 50 larvae, with n = ~3–4 samples per group. Significant differences were detected for igfbp7, timp2, and kim1 expression, as opposed to no changes of the chronic fibrosis marker gene col1a1a. B: expressions of kidney injury markers in septic animal (300 CFU) nephrons. Protein staining was colocalized with expression of enhanced green fluorescent protein (eGFP) in the transgenic zebrafish lines Tg(PT::eGFP) and Tg(cdh17::eGFP), which fluorescently label cells in the proximal and distal tubule, respectively. Whole mount and cross-section immunofluorescent staining identified the expression of kidney injury markers kidney injury molecule-1 (KIM-1), insulin-like growth factor-binding protein-7 (IGFBP7), and tissue inhibitor of metalloproteinases 2 (TIMP-2) (red) in the pronephric tubule (green) 48 h postinfection. Increased KIM-1 and IGFBP7 expressions were observed in the proximal tubule and increased KIM-1 and TIMP-2 expressions were observed in the distal tubule of septic zebrafish larvae. C: immunofluorescence staining of igfbp7−/− or timp2−/− larvae showing absent target proteins at 2 days post-Edwardsiella tarda (300 CFU) injections. As IGFBP7 and TIMP-2 protein expressions were inactivated, KIM-1 protein expression remained existent in TIMP-2−/− animals whereas absent in IGFBP7−/− animals during infection. D: pathway analysis showing the possible roles of upregulated kidney injury markers in addition to activated Toll-like receptor (TLR)-based host-pathogen cascades. Three major downstream functions were chosen, namely, migration of cells, differentiation of cells, and movement of phagocytes. Increased TIMP-2 was involved in TLR signaling, and KIM-1 [hepatitis A virus cellular receptor 1 (HAVCR1)] and IGFBP7 expressions had a combined inhibitory effect on cellular differentiation per the analysis. C, control; CCND1, cyclin D1; col1a1a, collagen, type I, α1a; CS, cross section; DAPI, fluorochrome 4′,6-diamidino-2-phenylindole; IL1B, interleukin-1β; INF, infected; Tg(cdh17::eGFP), transgenic zebrafish line that fluorescently labels cells in the pronephros whole length tubule; Tg(PT::eGFP), transgenic zebrafish line that fluorescently labels cells in the pronephros proximal tubule; TP53, tumor protein p53. *P < 0.05, **P < 0.01.