Endothelial NOX5 Obliterates the Reno-Protective Effect of Nox4 Deletion by Promoting Renal Fibrosis via Activation of EMT and ROS-Sensitive Pathways in Diabetes

Abstract

Chronic hyperglycemia induces intrarenal oxidative stress due to the excessive production of reactive oxygen species (ROS), leading to a cascade of events that contribute to the development and progression of diabetic kidney disease (DKD). NOX5, a pro-oxidant NADPH oxidase isoform, has been identified as a significant contributor to renal ROS in humans. Elevated levels of renal ROS contribute to endothelial cell dysfunction and associated inflammation, causing increased endothelial permeability, which can disrupt the renal ecosystem, leading to progressive albuminuria and renal fibrosis in DKD. This study specifically examines the contribution of endothelial cell-specific human NOX5 expression in renal pathology in a transgenic mouse model of DKD. This study additionally compares NOX5 with the previously characterized NADPH oxidase, NOX4, in terms of their relative roles in DKD. Regardless of NOX4 pathway, this study found that endothelial cell-specific expression of NOX5 exacerbates renal injury, albuminuria and fibrosis. This is attributed to the activation of the endothelial mesenchymal transition (EMT) pathway via enhanced ROS formation and the modulation of redox-sensitive factors. These findings underscore the potential therapeutic significance of NOX5 inhibition in human DKD. The study proposes that inhibiting NOX5 could be a promising approach for mitigating the progression of DKD and strengthens the case for the development of NOX5-specific inhibitors as a potential therapeutic intervention.

Keywords: NOX4; NOX5; diabetic kidney disease; fibrosis; inflammation; reactive oxygen species (ROS).

Figure 1

ECNOX5 increases albuminuria and renal injury in diabetes. Assessment of albuminuria (n = 15/gp) (A,B), periodic acid-Schiff staining (C,D), relative mesangial area expansion (E,F) and glomerulosclerotic index (G,H) in Nox4 wild-type (WT) and Nox4 knockout (Nox4^−/−) mice with and without NOX5 expression after 10 weeks of STZ diabetes. Scale bar: 20 μm, in all photomicrographs (n = 7–10/gp). Data are shown as mean ± SEM. Asterisks represent p-values for comparisons of the indicated groups: * <0.05, ** <0.01 and *** <0.001.

Figure 2

EC-NOX5 enhances renal ROS formation in diabetes. Immunostaining of glomerular nitrotyrosine (A,B) and its quantification (C,D), gene expression of renal Nox2 (E,F) and eNOS (G,H) and urinary 8-isoprostane levels (L,M) in Nox4WT and Nox4^−/− mice with and without NOX5 expression after 10 weeks of STZ-induced diabetes (n = 7–10/gp). Scale bar: 20 μm, in all photomicrographs. Gene and protein expression of NRF2 in Nox4^−/− mice with and without NOX5 expression after 10 weeks of STZ-induced diabetes (I–K). Western blot showing the protein expression of renal NRF2 (61 kDa) and its quantification (J,K) (n = 3/group). β-Actin (42 kDa) serves as a housekeeping protein. Data are shown as mean ± SEM. Asterisks represent p-values for comparisons of the indicated groups: * <0.05, ** <0.01, *** <0.001 and ns, not significant.

Figure 3

EC-NOX5 upregulates markers of inflammation and ROS-sensitive factors. Gene expression of renal Mcp-1 (A,B), Tlr4 (C,D), Vegf (E,F), Egr1 (G,H), and Pkc-α (I,J) in Nox4WT and Nox4^−/− mice with and without NOX5 expression after 10 weeks of STZ-induced diabetes (n = 7–10/gp). Western blot showing the protein expression of renal PKC-α (77 kDa) and its quantification (K,L) (n = 3/group). β-Actin (42 kDa) serves as a housekeeping protein. Immunostaining for F4/80 (M) and CD68 (O) and their quantification (N,P) as well as protein level of MCP-1 (Q) by ELISA (n = 7–10/gp). Scale bar: 20 μm, in all photomicrographs. Data are shown as mean ± SEM. Asterisks represent p-values for comparisons of the indicated groups: * <0.05, ** <0.01, *** <0.001 and ns, not significant.

Figure 4

EC-NOX5 upsurges ECM accumulation and renal fibrosis. The renal gene expression of fibronectin (Fn1) (A,B) and collagen IV (Col4) (C,D) as well as immunostaining of collagen IV (E,F) and its quantification (G,H) in Nox4WT and Nox4^−/− mice with and without NOX5 expression after 10 weeks of STZ-induced diabetes. Scale bar, 20 μm in all photomicrographs (n = 7–10/gp). Data are shown as mean ± SEM. Asterisks represent p-values for comparisons of the indicated groups: * <0.05, ** <0.01 and *** <0.001.

Figure 5

EC-NOX5 promotes renal fibrosis by activating EMT-related factors. Gene expression of renal α-SMA (A,B), Vimentin (E,F), Ki67 (G,H), and Ctgf (I,J) in Nox4WT and Nox4^−/− mice with and without NOX5 expression after 10 weeks of STZ-induced diabetes (n = 7–10/gp). Immunostaining of renal α-SMA (C) and its quantification (D) (n = 6/gp). Scale bar: 20 μm, in all photomicrographs. Data are shown as mean ± SEM. Asterisks represent p-values for comparisons of the indicated groups: * <0.05 and ** <0.01.