The role of lipid metabolism imbalance in copper-induced PANoptosis in broiler kidney

Abstract

Copper (Cu) is widely used in both agriculture and industry and may pose toxic risks to animals and public safety if overused. In order to gain a more profound insight into the nephrotoxic effects of Cu, a detailed analysis was performed of its impact on renal PANoptosis, with particular attention being paid to the possible involvement of lipid metabolism disorders in the kidney. In this study, one-day-old chicks were fed diets with varying Cu levels (11, 110, 220, and 330 mg/kg) over a period of 49 days. Our findings indicated that excessive Cu exposure led to vacuolar degeneration, fibrosis and mitochondrial damage in the kidney. Moreover, the assay results demonstrated that elevated Cu levels led to disturbances in lipid synthesis and catabolism, as well as the activation of lipophagy in broiler kidneys. Concurrently, genetic and protein analysis demonstrated that excess Cu triggered pyroptosis (IL-18, NLRP3, GSDMD, Caspase-1), necrosis (MLKL, Caspase-7, Caspase-8) and apoptosis (Bcl-2, Cleaved-Caspase-9/Caspase-9, Cleaved-Caspase-9/Caspase-9), ultimately resulting in PANoptosis in the chicken kidney. Furthermore, the bioinformatics analysis indicated a correlation between lipid metabolism and PANoptosis-related markers. The aforementioned results indicate that Cu-induced disruption to lipid metabolism may contribute to the process of PANoptosis in broiler kidneys.

Keywords: Broiler; Copper; Kidney; Lipid metabolism; PANoptosis.

Fig. 1

Effects of Cu exposure on histological morphology in kidney. (A) Chicken body weight at 7 weeks. (B) Chicken kidney coefficient. (C) Cu content in chicken serum. (D) Cu content in chicken kidney. (E) Fe content in chicken kidney. (F) Zn content in chicken kidney. (G) Crea content in chicken serum. (H) BUN content in chicken serum. (I) HE staining results. (J) Results of Massoon staining. (K) The ultrastructural observation. The yellow arrow denotes the location of damaged mitochondria. “N” means cell nucleus. “Mito” means mitochondria. All date were expressed as mean ± SEM; n=6 for each group. “*” indicates statistically significant with the control group (*P<0.05, **P<0.01, ***P<0.001). The same as follows.

Fig. 2

Effects of Cu exposure on lipid metabolism and lipophagy in kidney. (A-B) mRNA expression level of lipid metabolism and lipophagy. (C) Heat map. (D) Western blot result. (E-K) Quantitative analysis of protein expressions related to lipid metabolism and lipophagy.

Fig. 3

Effects of Cu exposure on pyroptosis in chicken kidney. (A) mRNA expression of mitochondrial dynamics protein. (B) Heat map. (C) Western blot result. (D-I) Results of quantitative Western blot analysis of pyroptosis-associated proteins.

Fig. 4

Immunohistochemical and immunofluorescence analyses in chicken kidney. (A-B) Immunohistochemistry staining analyzes the expression of IL-1 and NLRP3. (C) Immunofluorescence staining was used to analyze the expression of Caspase-1.

Fig. 5

Effects of Cu exposure on apoptosis in chicken kidney. (A) Relative mRNA expression of apoptosis genes in kidney. (B) Heat map. (C) Western blot result of apoptosis-associated proteins. (D-F) Results of quantitative Western blot analysis of apoptosis-associated proteins. (G) Immunofluorescence staining was used to analyze the expression of Caspase-3 in chicken kidney. (H) Fluorescence intensity analysis.

Fig. 6

Effects of Cu exposure on necrosis in chicken kidney. (A-C) Relative mRNA expression of necrosis genes in kidney. (D) Western blot result of necrosis-associated proteins. (E-H) Results of quantitative Western blot analysis of necrosis-associated proteins. (I-K) Immunofluorescence staining was employed for the analysis of RIPK3, MLKL, and Caspase-8 expression in chicken kidney.

Fig. 7

Bioinformatics correlation analysis. (A) The PCA analysis. (A) The correlation and chord plot analyses. (A) The heat map analyses.