Kidney-Targeted Epoxyeicosatrienoic Acid Analog, EET-F01, Reduces Inflammation, Oxidative Stress, and Cisplatin-Induced Nephrotoxicity

Abstract

Although epoxyeicosatrienoic acid (EET) analogs have performed well in several acute and chronic kidney disease models, targeted delivery of EET analogs to the kidney can be reasonably expected to reduce the level of drug needed to achieve a therapeutic effect and obviate possible side effects. For EET analog kidney-targeted delivery, we conjugated a stable EET analog to folic acid via a PEG-diamine linker. Next, we compared the kidney targeted EET analog, EET-F01, to a well-studied EET analog, EET-A. EET-A or EET-F01 was infused i.v. and plasma and kidney tissue collected. EET-A was detected in the plasma but was undetectable in the kidney. On the other hand, EET-F01 was detected in the plasma and kidney. Experiments were conducted to compare the efficacy of EET-F01 and EET-A for decreasing cisplatin nephrotoxicity. Cisplatin was administered to WKY rats treated with vehicle, EET-A (10 mg/kg i.p.) or EET-F01 (20 mg/kg or 2 mg/kg i.p.). Cisplatin increased kidney injury markers, viz., blood urea nitrogen (BUN), N-acetyl-β-(D)-glucosaminidase (NAG), kidney injury molecule-1 (KIM-1), and thiobarbituric acid reactive substances (TBARS). EET-F01 was as effective as EET-A in decreasing BUN, NAG, KIM-1, TBARS, and renal histological injury caused by cisplatin. Despite its almost 2×-greater molecular weight compared with EET-A, EET-F01 was comparably effective in decreasing renal injury at a 10-fold w/w lower dose. EET-F01 decreased cisplatin nephrotoxicity by reducing oxidative stress and inflammation. These data demonstrate that EET-F01 targets the kidney, allows for a lower effective dose, and combats cisplatin nephrotoxicity. In conclusion, we have developed a kidney targeted EET analog, EET-F01, that demonstrates excellent potential as a therapeutic for kidney diseases.

Keywords: chemotherapy; epoxyeicosatrienoic acid; kidney-targeted; nephrotoxicity; novel therapy.

Figure 1

Comparison of plasma and kidney EET-F01 and EET-A following i.v. administration for 6 h. Top panel: Chemical structures for EET-A and EET-F01. Left panel: Plasma concentration of EET-F01 and EET-A. Right panel: Kidney levels of EET-F01 and EET-A. Data expressed as mean ± SEM, n = 3/group.

Figure 2

Kidney injury induced by cisplatin is reduced by EET-F01 and EET-A treatments. Left panel: blood urea nitrogen (BUN), Middle panel: urinary N-acetyl-β-(D)-glucosaminidase (NAG), Right panel: kidney injury molecule-1 (KIM-1) in cisplatin administered rats treated with vehicle, EET-F01, or EET-A. Bottom panel: Representative photomicrographs of Periodic acid-Schiff (PAS) Staining depicting vacuolation and desquamation of the renal epithelial cells along with severe intra-tubular proteinaceous cast tubular cast formation in the renal sections of different experimental groups. * p < 0.05 vs. control–vehicle group; † p < 0.05 vs. cisplatin–vehicle group. Data expressed as mean ± SEM, n = 6/group.

Figure 3

Renal inflammation in cisplatin nephrotoxicity is reduced by EET-F01 and EET-A treatments. RT-PCR analysis for mRNA expressions of IL-6 (left panel) and TNFα (right panel). * p < 0.05 vs. control–vehicle group; † p < 0.05 vs. cisplatin–vehicle group. Data expressed as mean ± SEM, n = 6/group.

Figure 4

Renal oxidative stress in cisplatin nephrotoxicity is reduced by EET-F01 and EET-A treatments. RT-PCR analysis for mRNA expressions of (a) p47phox, (b) p67phox, (c) gp91phox, (d) NOX4. * p < 0.05 vs. control–vehicle group; † p < 0.05 vs. cisplatin–vehicle group. Data expressed as mean ± SEM, n = 6/group.

Figure 5

Kidney thiobarbituric acid reactive substances (TBARS) in cisplatin nephrotoxicity is reduced by EET-F01 and EET-A treatments. * p < 0.05 vs. control–vehicle group; † p < 0.05 vs. cisplatin–vehicle group. Data expressed as mean ± SEM, n = 6/group.

Figure 6

Tumor growth of a human breast cancer cell line, MBA-MB-231, in athymic mice is not altered by EET-A treatment. Left panel: tumor volume, Right panel: tumor weight in MBA-MB-231 tumor bearing mice treated with vehicle (control) or EET-A. Bottom panel: Representative photomicrographs of Periodic acid-Schiff (PAS) Staining depicting similar tumor hyperplasia and proliferation in human breast tumors. Data expressed as mean ± SEM, n = 5 mice and n = 10 tumors/group.

Figure 7

Synthesis of EET-F01.