Aristolochic acid I and ochratoxin A differentially regulate VEGF expression in porcine kidney epithelial cells--the involvement of SP-1 and HIFs transcription factors

Abstract

Aristolochic acid I (AAI) and ochratoxin A (OTA) cause chronic kidney diseases. Recently, the contribution of hypoxic injuries and angiogenic disturbances to nephropathies has been suggested, but underlying mechanisms have not been fully clarified yet. In porcine kidney epithelial cell line, LLC-PK1 cells, treatment with non-toxic doses of AAI increased whereas with OTA decreased production of vascular endothelial growth factor (VEGF), the angiogenic factor with well-defined functions in kidney. Moreover, the activity of transcription factors regulating VEGF expression was differentially affected by examined compounds. Activity of hypoxia inducible factors (HIFs) and SP-1 was increased by AAI but diminished by OTA. Interestingly, AP-1 activity was inhibited while NFκB was not influenced by both toxins. Mithramycin A, a SP-1 inhibitor, as well as chetomin, an inhibitor of HIFs, reversed AAI-induced up-regulation of VEGF synthesis, indicating the importance of SP-1 and HIFs in this effect. Additionally, adenoviral overexpression of HIF-2α but not HIF-1α prevented OTA-diminished VEGF production suggesting the protective effect of this isoform towards the consequences exerted by OTA. These observations provide new insight into complex impact of AAI and OTA on angiogenic gene regulation. Additionally, it adds to our understanding of hypoxia influence on nephropathies pathology.

Fig. 1

AAI and OTA affect LLC-PK1 cell viability and proliferation. Cells were cultured in the presence of increasing concentrations of AAI and OTA for 24 h. Cell viability, assessed by measurement of LDH release showed that 10 μM AAI (A) and 25 μM OTA (B) are the highest non-toxic concentrations. After 24 h stimulation with non-toxic concentration of both toxins analysis of BrdU incorporation pointed out inhibition of cell proliferation after delivery of 5 and 10 μM AAI as well as after 25 μM OTA (C). Mean of at least 3 experiments performed in duplicates; *p < 0.05 vs control

Fig. 2

AAI elevates whereas OTA diminishes VEGF production in LLC-PK1 cells. LLC-PK1 cells were transfected with reporter plasmid containing luciferase encoding sequence under the control of VEGF promoter and then stimulated with toxins. The elevation of VEGF promoter activity 24 h after AAI and diminishment after OTA delivery was observed (A). Analysis of mRNA level by real-time PCR (B) and protein production by ELISA (C) indicated the enhancement and down-regulation of VEGF expression after AAI and OTA adding, respectively. Mean of 3 experiments performed in duplicates; *p < 0.05 vs control.

Fig. 3

AAI and OTA differentially regulate the activity of HRE and SP-1 but both attenuate AP-1 activity. 24 h after transfection with HRE-luc (A), SP-1-luc (B) and AP-1-SEAP (C) reporter plasmids LLC-PK1 cells were stimulated with AAI and OTA for next 24 h. Analysis demonstrated AAI-elevated and OTA-diminished HRE activity (A). SP-1 activity was enhanced after AAI and down-regulated after OTA (B) adding. Both AAI and OTA diminished AP-1 activity (C). Immunocytochemical staining showed that the stabilization of HIF-1α (D) and HIF-2α (E) was elevated after AAI (middle column) but decreased after OTA (right column) delivery. HIF-2α expression was increased by AAI and diminished by OTA as shown by Western blot (F). ROS generation was increased after OTA but not after AAI stimulation (G). Mean of 3 (A, B, G) and 2–3 (C) experiments performed in duplicates, D, E-representative immunofluorescent staining, F–representative western blot; *p < 0.05 vs control.

Fig. 4

Induction of VEGF by AAI goes through SP-1 and HIFs activation. LLC-PK1 cells were transfected with reporter plasmid containing luciferase encoding sequence under the control of VEGF promoter. 24 h after transfection cells were prestimulated with 1 μM mithramycin A (A–C) or 60 nM chetomin (D) for 30 min and then AAI was added for 24 h. The elevation of VEGF promoter activity (A), mRNA level (B) as well as VEGF protein level (C) evoked by AAI was reversed by inhibition of SP-1 activity by mithramycin A. Moreover, inhibition of HIF activity by chetomin caused the reversal effect on AAI-induced VEGF protein level (D). Mean of 3 (A, B, C) or 2 (D) experiments performed in duplicates; *p < 0.05 vs control; ^#p < 0.05 vs AAI-treated cells.

Fig. 5

Hypoxia and HIF-2α attenuate the diminishment of VEGF production evoked by OTA. LLC-PK1 cells were cultured under normoxic and hypoxic conditions for 24 h and stimulated by 25 μM OTA. OTA attenuated induction of VEGF expression evoked by hypoxia on mRNA level determined by real-time PCR (A) as well as on the protein level assessed by ELISA (B). Overexpression of HIF-2α but not HIF-1α abolished the diminishment of VEGF expression evoked by OTA on mRNA (C) and protein (D) level. Mean from 2 (A), 3 (B), 3–5 (C) or 3–4 (D) experiments performed in duplicates, *p < 0.05 vs. control (A, B); vs. relevant AdGFP control (C, D), ^#p < 0.05 vs. OTA (A, B); vs. AdGFP + OTA (C, D).