A zebrafish model of infection-associated acute kidney injury

Abstract

Sepsis-associated acute kidney injury (S-AKI) independently predicts mortality among critically ill patients. The role of innate immunity in this process is unclear, and there is an unmet need for S-AKI models to delineate the pathophysiological response. Mammals and zebrafish ( Danio rerio) share a conserved nephron structure and homologous innate immune systems, making the latter suitable for S-AKI research. We introduced Edwardsiella tarda to the zebrafish. Systemic E. tarda bacteremia resulted in sustained bacterial infection and dose-dependent mortality. A systemic immune reaction was characterized by increased mRNA expressions of il1b, tnfa, tgfb1a, and cxcl8-l1 ( P < 0.0001, P < 0.001, P < 0.001, and P < 0.01, respectively). Increase of host stress response genes ccnd1 and tp53 was observed at 24 h postinjection ( P < 0.0001 and P < 0.05, respectively). Moderate E. tarda infection induced zebrafish mortality of over 50% in larvae and 20% in adults, accompanied by pericardial edema in larvae and renal dysfunction in both larval and adult zebrafish. Expression of AKI markers insulin-like growth factor-binding protein-7 (IGFBP7), tissue inhibitor of metalloproteinases 2 (TIMP-2), and kidney injury molecule-1 (KIM-1) was found to be significantly increased in the septic animals at the transcription level ( P < 0.01, P < 0.05, and P < 0.05) and in nephric tubules compared with noninfected animals. In conclusion, we established a zebrafish model of S-AKI induced by E. tarda injection, with both larval and adult zebrafish showing nephron injury in the setting of infection.

Keywords: acute kidney injury; innate immunity; zebrafish infection.

Fig. 1.

Evidence of systemic infection in zebrafish. A: larvae were injected in the duct of Cuvier at 3 days postfertilization. B: bacterial loads of zebrafish larvae over time. The scatterplot represents means ± SE. Numbers of colony-forming units (CFU) were calculated using homogenates of 5 larvae per sample, n = 6 samples per group. Significant differences were detected between controls and septic larvae at 24, 48, and 72 h postinfection. C: Kaplan-Meier curve of 7 days postinjection revealed dose-dependent mortality of both larval and adult zebrafish in response to Edwardsiella tarda injections. Sample size for larval groups: control, n = 281; 100 CFU, n = 118; 300 CFU, n = 330; 600 CFU, n = 312; 1,000 CFU, n = 202; for adult groups: control, n = 10; 6 × 10⁶ CFU, n = 12; 6 × 10⁷ CFU, n = 12. Compared with control animals, 100-CFU injections to larvae and 6 × 10⁶-CFU injections to adults had no significant impact on mortality, whereas larvae injected with ≥300 CFU and adults injected with 6 × 10⁷ CFU E. tarda exhibited significantly increased mortality. D: fold changes in mRNA expressions (presented as means ± SE) of inflammatory cytokines and stress response genes were estimated using samples containing homogenates of 50 larvae per sample, n = 4 samples per group. Significant differences were found between control and infected zebrafish larvae injected with 300 CFU E. tarda 1 day postinjection. C, control; ccnd1, cyclin D1; cxcl8-l1, zebrafish homolog of mammalian C-X-C motif chemokine ligand 8; dpi, days postinjection; hpi, hours postinjection; il1β, interleukin-1β; INF, infected; tgfb1a, transforming growth factor-β1a; tnfα, tumor necrosis factor-α; tp53, tumor protein p53. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.

Manifestations of nephron dysfunction during systemic infection in zebrafish. A: representative images of septic larvae showing larval edema in the pericardial area (red arrows) at 2 days after 300-CFU Edwardsiella tarda injection. B: after injection with 300 CFU E. tarda for 2 days, septic vs. control larvae were injected with FITC-dextran (40 kDa) and subjected to fluorescence intensity measurements in the pericardium under the stereomicroscope. Dextran excretions at 24 h post-dextran injection were compared with intensities at 3 h after the injections in the same animal. Representative images of fluorescence accumulation around the zebrafish pericardium (red arrows pointing to yellow triangular area) at the two time points are shown. C: bar graph shows quantified fluorescence intensities (means ± SE) at the two time points; n = 5 larvae per group. The scatterplot (presented as means ± SE) displays nephron clearance index (FITC intensity_3h – FITC intensity_24h) normalized by the larval heart rate. D: impaired endocytosis of dextran by the adult zebrafish nephron tubule depending on severity of infection. Representative images show lower nephron fluorescence accumulations in the infected animal nephrons compared with control, with the least seen in the severely infected animal. High-magnification images show endocytosis-defective cells in the infected animal nephrons (yellow arrows). The corresponding quantified fluorescence intensities are shown in the bar graph (means ± SE). Statistical analysis suggests significant differences among groups. n = 3 animals per group. C, control; HM, high magnification; hpi, hours postinjection; INF, infected; LM, low magnification; Mod, moderate; Sv, severe. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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.