Urea overdose causes pathological injury and activates necroptosis in lung of Jianzhou Da’er Goat (Capra hircus)

Abstract

Urea, as a feed additive, causes poisoning in excessive dosage. In this study, we explored the mechanism of lung damage in urea poisoning on Jianzhou Da'er goat by adding different levels of urea. We used pathological histological analysis and Real-time fluorescence quantitative PCR to investigate the extent of lung damage. 9 Jianzhou Da’er goats of similar age and body condition were selected and equally randomized into three groups: 0 % urea group with 0 urea diet, 5 % urea group with 5 % urea diet and 10 % urea group with 10 % urea diet. Equal feeding was used 3 times daily (8:00 a.m., 2:00 p.m., 8:00 p.m.) in 7-d pre-feeding stage and 21-d formal experiment. After the experiment, we found the lungs of 5 % urea group and 10 % urea group both had collagen fibrosis proliferation and hemosiderin production. The nuclei of alveolar epithelial cells in the 5 % urea group were constricted and the mitochondria swelled; the alveolar epithelial cells in the 10 % urea group were necrotic and disintegrated. The expressions of RIP3 (Receptor interacting protein kinase 3) and TNF-α (tumor necrosis factor-α) mRNA in the lung of the 5 % urea group and 10 % urea group were increased significantly (P < 0.01) while the mRNA expressions of cIAP2 (cellular inhibitor of apoptosis 2) and TRADD (TNFR1-associated death domain) in the 10 % urea group were significantly decreased (P < 0.01); the mRNA expression of NEMO (NF-κB essential modulator) and Caspase-8 (cysteine aspartate protease-8) in lung of the 5 % urea group and 10 % urea group were significantly decreased (P < 0.01) and the mRNA expression of HMBG-1 (High mobility group box-1) and HSP70(Heat Shock Protein 70) were significantly up-regulated (P < 0.01). The expression of RIP3 protein in 5 % urea group and 10 % urea group were increased significantly (P < 0.01). In summary, evidence of lung damage from urea feeding includes disruption of alveolar structure, lysis of organelles, and a significant rise in genes associated with necroptosis. We expect to provide scientific value for the study of necroptosis and lung injury caused by urea toxicity.