Niclosamide ethanolamine protects kidney in adriamycin nephropathy by regulating mitochondrial redox balance

Abstract

Chronic kidney disease (CKD) is commonly characterized by proteinuria and leads to progressive glomerulosclerosis and tubulointerstitial fibrosis. Accumulating evidence implicates mitochondrial dysfunction including reactive oxygen species (ROS) overproduction in the pathogenesis of CKD. Mitochondrial function and ROS production are regulated by mitochondrial uncoupling. Niclosamide ethanolamine salt (NEN) is a mild mitochondrial uncoupler, which reduces urinary albumin excretion in mice with diabetic kidney disease. However, its role in nondiabetic kidney disease has not been investigated. Here we show that NEN exerts renoprotective effects in adriamycin induced nondiabetic kidney disease. It reduces urinary protein excretion, restores podocyte function, ameliorates renal pathological injury, and decreases the excretion of the urinary tubular injury biomarkers NGAL and Kim-1. Specifically, NEN uncouples isolated kidney mitochondria, and dose-dependently decreases the renal production and urinary excretion of H₂O₂. Moreover, NEN increases catalase and PGC-1α expression, which might accelerate H₂O₂ scavenging. The results of this study provide the first evidence that NEN protects kidney in nondiabetic kidney disease by regulating redox balance.

Keywords: Niclosamide ethanolamine salt; adriamycin nephropathy; mitochondria; redox balance.

Figure 1

NEN reduces urinary protein, FPW, and loss of podocytes. Quantification and statistical analyses of urinary protein excretion (n = 6/group) (A), FPW (n = 3/group) (B), and the number of podocytes (n = 6/group) (C) in control mice, those with AN, and those with AN treated for 2 weeks with NEN. **P < 0.01 and ***P < 0.001 vs. control; ##P < 0.01 and ###P < 0.001 vs. AN. (D) Representative EM images of the podocyte processes in each group. The podocytes in the AN group exhibited diffuse fusion of foot processes, which was attenuated by NEN treatment. Scale bar, 500 nm. (E) Representative images of WT-1 immunostaining in the three groups. Scale bar, 20 µm.

Figure 2

Effects of NEN on renal pathology. Quantification of glomerulosclerosis (A), tubular injury (B), interstitial fibrosis (C), and the number of nuclei/glomerulus (D) in control mice, those with AN, and those with AN treated for 2 weeks with NEN (n = 6/group). ***P < 0.001 vs. control; ##P < 0.01 and ###P < 0.001 vs. AN. Representative images of PAS staining for glomeruli (scale bar, 20 µm) (E) and for tubules (scale bar, 50 µm) (F). (G) Representative images of Masson’s trichrome staining for renal tubulointerstitium. Scale bar, 50 µm.

Figure 3

NEN reduces urinary NGAL and Kim-1 excretion. Quantification of urinary NGAL (A) and Kim-1 (B) excretion in control mice, those with AN, and those with AN treated for 2 weeks with NEN (n = 6/group). ***P < 0.001 vs. control; ##P < 0.01 and ###P < 0.001 vs. AN.

Figure 4

NEN uncouples mitochondria isolated from kidneys, dose-dependently decreases renal mitochondrial H₂O₂ production, and reduces urinary H₂O₂ excretion. (A) Urinary H₂O₂ excretion in control mice, those with AN, and those with AN treated for 2 weeks with NEN (n = 6/group). **P < 0.01 vs. control; ###P < 0.001 vs. AN. (B) H₂O₂ release rates in isolated kidney mitochondria (n = 4/group). ***P < 0.001 vs. Mito (mitochondria) group; ##P < 0.01 and ###P < 0.001 vs. DMSO. Uncoupling in isolated kidney mitochondria (measured as oxygen consumption) from normal control mice in the absence (C) or presence (D) of oligomycin.

Figure 5

Effects of NEN on catalase and SOD2 expression in renal cortex. (A) Immunohistochemical staining of catalase (A) and SOD2 (B) in glomeruli (scale bars, 20 µm) and tubules (scale bars, 50 µm) in the renal cortices from control mice, those with AN, and those with AN treated for 2 weeks with NEN. (C) Representative Western blots for catalase and SOD2 expression. Quantification of catalase (D and F) and SOD2 (E and G) protein and mRNA levels, respectively, normalized to β-actin (n = 4/group). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control; #P < 0.05 vs. AN.

Figure 6

Effects of NEN on PGC-1α expression and mtDNA copy number. (A) Representative Western blots of PGC-1α in the renal cortices from control mice, those with AN, and those with AN treated for 2 weeks with NEN. Quantification of PGC-1α levels (B) and mitochondrial DNA copy number (determined by real-time quantitative PCR for cytochrome b and ND1) (C) normalized to β-actin (n = 6/group). *P < 0.05 vs. control; ##P < 0.01 vs. AN.