NR4A1 silencing alleviates high-glucose-stimulated HK-2 cells pyroptosis and fibrosis via hindering NLRP3 activation and PI3K/AKT pathway

Figure 1 Metabolic indices and renal pathological alterations in diabetic kidney disease rats. A: Comparison of blood glucose, urinary microalbumin, blood creatinine, body weight, and renal index of rats; B: Representative images of histopathological changes in the rat kidneys. Hematoxylin and eosin: Lobular structure (blue arrow), vacuolated cytoplasm (black arrow), inflammatory cell infiltration in the periphery (orange arrow); Periodic acid-Schiff: Hyperplasia of the mesangial matrix (black arrow), basement membrane thickening (orange arrow). Scale bar = 20 μm, n = 9. ^aP < 0.05 vs control. CON: Control; DKD: Diabetic kidney disease; KI: Kidney index.

Figure 2 Nuclear receptor subfamily 4 group A member 1 expression was upregulated in kidney tissue of rats with diabetic kidney disease. A: Representative western blotting bands and integrated density study of nuclear receptor subfamily 4 group A member 1 (NR4A1) in rat kidney tissues; B: Quantitative real-time PCR of NR4A1 mRNA expression in rat kidney tissues, n = 3. ^aP < 0.05 vs control. CON: Control; DKD: Diabetic kidney disease; NR4A1: Nuclear receptor subfamily 4 group A member 1.

Figure 3 Markers of NOD-like receptor protein 3-mediated pyroptosis were increased in rats with diabetic kidney disease. A: Representative western blotting bands and integrated density analysis of NOD-like receptor protein 3, cleaved-caspase-1, and gasdermin D N-terminal structural domain in rat kidney tissues; B: Quantitative real-time PCR of interleukin (IL)-1β mRNA expression in rat kidney tissues and concentration of IL-1β in rat serum, n = 3. ^aP < 0.05 vs control. CON: Control; DKD: Diabetic kidney disease; GSDMD-N: Gasdermin D N-terminal structural domain; IL-1β: Interleukin-1β; NLRP3: NOD-like receptor protein 3.

Figure 4 Markers of PI3K/AKT signaling pathway-mediated fibrosis were upregulated in rats with diabetic kidney disease. A: Representative western blotting and integrated density study of AKT, p-AKT, PI3K, and p-PI3K in rat kidney tissues; B: Representative western blotting and integrated density study of TGF-β1 and collagen III in rat kidney tissue, n = 3. ^aP < 0.05 vs control. AKT: Protein kinase B; CON: Control; DKD: Diabetic kidney disease; PI3K: Phosphoinositide 3-kinase; TGF-β1: Transforming growth factor β1.

Figure 5 Silencing of NR4A1 attenuated NOD-like receptor protein 3-mediated pyroptosis in high glucose-stimulated HK-2 cells. A and B: Quantitative real-time PCR of NR4A1 mRNA expression and siRNA transfection efficiency in HK-2 cells; C and D: Illustrative western blotting and integrated density study of NOD-like receptor protein 3, cleaved-caspase-1, and gasdermin D N-terminal structural domain in HK-2 cells; E: Illustrative immunocytochemistry images of interleukin-1β in HK-2 cells. Scale bar = 20 μm, n = 3. ^bP < 0.05 vs NC, ^cP < 0.05 vs high glucose + negative control. GSDMD-N: Gasdermin D N-terminal structural domain; NC: Normal control; HG: High glucose; NLRP3: NOD-like receptor protein 3; NR4A1: Nuclear receptor subfamily 4 group A member 1; si-NR4A1: SiRNA targeting nuclear receptor subfamily 4 group A member 1.

Figure 6 Silencing of nuclear receptor subfamily 4 group A member 1 attenuated phosphoinositide 3-kinase/protein kinase B pathway-mediated fibrosis in high glucose-activated HK-2 cells. A: Illustrative Western blotting bands and integrated density analysis of protein kinase B (AKT), p-AKT, phosphoinositide 3-kinase (PI3K), and p-PI3K in HK-2 cells; B: Representative western blotting and integrated density analysis of transforming growth factor-β1 in HK-2 cells; C: Illustrative immunocytochemistry images of collagen III in HK-2 cells. Scale bar = 20 μm, n = 3. ^bP < 0.05 vs normal control, ^cP < 0.05 vs high glucose + negative control. HG: High glucose; NC: Normal control; NR4A1: Nuclear receptor subfamily 4 group A member 1; AKT: Protein kinase B; PI3K: Phosphoinositide 3-kinase; si-NR4A1: SiRNA targeting nuclear receptor subfamily 4 group A member 1; TGF-β1: Transforming growth factor-β1.

Figure 7 Diagram of possible mechanisms by which silencing of NR4A1 ameliorates diabetic kidney disease in vitro. HG: High glucose; AKT: Protein kinase B; PI3K: Phosphoinositide 3-kinase; si-NR4A1: SiRNA targeting nuclear receptor subfamily 4 group A member 1; TGF-β1: Transforming growth factor-β1; NLRP3: NOD-like receptor protein 3; GSDMD: Gasdermin D; IL-1β: Interleukin-1β.