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. 2017 Jun 16;8(43):74217-74232.
doi: 10.18632/oncotarget.18540. eCollection 2017 Sep 26.

A novel pan-Nox inhibitor, APX-115, protects kidney injury in streptozotocin-induced diabetic mice: possible role of peroxisomal and mitochondrial biogenesis

Guideock Kwon  1 , Md Jamal Uddin  1 , Gayoung Lee  1 , Songling Jiang  1 , Ahreum Cho  1 , Jung Hwa Lee  1 , Sae Rom Lee  2 , Yun Soo Bae  2 , Sung Hwan Moon  3 , Soo Jin Lee  3 , Dae Ryong Cha  4 , Hunjoo Ha  1
Affiliations

A novel pan-Nox inhibitor, APX-115, protects kidney injury in streptozotocin-induced diabetic mice: possible role of peroxisomal and mitochondrial biogenesis

Guideock Kwon et al. Oncotarget. .

Abstract

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are increasingly recognized as a key factor in inflammation and extracellular matrix accumulation in diabetic kidney disease. APX-115 (3-phenyl-1-(pyridin-2-yl)-4-propyl-1-5-hydroxypyrazol HCl) is a novel orally active pan-Nox inhibitor. The objective of this study was to compare the protective effect of APX-115 with a renin-angiotensin system inhibitor (losartan), the standard treatment against kidney injury in diabetic patients, on streptozotocin (STZ)-induced diabetic kidney injury. Diabetes was induced by intraperitoneal injection of STZ at 50 mg/kg/day for 5 days in C57BL/6J mice. APX-115 (60 mg/kg/day) or losartan (1.5 mg/kg/day) was administered orally to diabetic mice for 12 weeks. APX-115 effectively prevented kidney injury such as albuminuria, glomerular hypertrophy, tubular injury, podocyte injury, fibrosis, and inflammation as well as oxidative stress in diabetic mice, similar to losartan. In addition, both APX-115 and losartan treatment effectively inhibited mitochondrial and peroxisomal dysfunction associated with lipid accumulation. Our data suggest that APX-115, a pan-Nox inhibitor, may become a novel therapeutic agent against diabetic kidney disease by maintaining peroxisomal and mitochondrial fitness.

Keywords: APX-115; diabetic kidney disease; mitochondria and peroxisome; oxidative stress; pan-Nox inhibitor.

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Conflict of interest statement

CONFLICTS OF INTEREST None of the authors except SHM and SJL declared any competing interest. SHM and SJL work at Aptabio Therapeutics Inc, Korea which develops APX-115.

Figures

Figure 1
Figure 1. Effect of APX-115 on kidney function and morphology
Diabetes was induced in mice by intraperitoneal injection of STZ (50 mg/kg). Then APX-115 (60 mg/kg/day) or losartan (1.5 mg/kg/day) was administered orally for 12 weeks to diabetic mice. After 12 weeks, urine and blood samples were collected for analysis of (A) urinary albumin excretion, (B) albumin/creatinine ratio, (C) creatinine clearance rate, and (D) plasma cystatin C. (E) Kidneys were fixed in paraffin and cut into 3 μm sections that were subsequently stained with PAS reagent. Scale bar: 10 μm; original magnification: 630×. After PAS staining, (F) glomerular volume, (G) mesangial area, and (H) tuft area were analyzed using Image-Pro Plus 4.5.1. DM, STZ-induced diabetic mice. Data are presented as means ± SE of 10–12 mice/group; *p < 0.05 vs. control, †p < 0.05 vs. DM.
Figure 2
Figure 2. Effect of APX-115 on tubular and podocyte injury in STZ-induced diabetic mice
Tubular injury markers such as (A) urinary KIM1 by ELISA and (B) KIM1 and (C) NGAL mRNA levels in kidney tissues using real-time PCR were measured. (D and E) Paraffin-embedded kidney sections were stained with anti-nephrin antibodies (1:100; Scale bar: 10 μm; original magnification: 630×). Data are presented as means ± SE of 10–12 mice/group; *p < 0.05 vs. control, †p < 0.05 vs. DM.
Figure 3
Figure 3. Effect of APX-115 on fibrosis in STZ-induced diabetic mice
The mRNA levels of fibrosis markers (A) TGFβ1 and (B) fibronectin in kidney tissues were measured using real-time PCR. Paraffin-embedded kidney sections were stained with (C and D) picrosirius red stain, (E and F) anti-αSMA (1:200), and (G and H) anti-collagen IV (1:200) antibodies. Original magnification: 200×; scale bar: 100 μm. Data are presented as means ± SE of 5 mice/group; *p < 0.05 vs. control, †p < 0.05 vs. DM.
Figure 4
Figure 4. Effect of APX-115 on inflammation in STZ-induced diabetic mice
The mRNA levels of inflammation markers (A) TNFα and (B) MCP1 in kidney tissue were measured using real-time PCR. (C and D) Paraffin-embedded kidney sections were stained anti-F4/80 antibodies (1:200; original magnification: 100×; scale bar: 100 μm). Data are presented as means ± SE of 5 mice/group; *p < 0.05 vs. control, †p < 0.05 vs. DM.
Figure 5
Figure 5. Effect of APX-115 on oxidative stress in STZ-induced diabetic mice
(A) Plasma LPO, (B) urinary LPO, (C) kidney tissue LPO, (D) Nox1, (E) Nox2, and (F) Nox4 mRNA expression levels in kidneys were measured using real-time PCR. (G and H) Frozen kidney sections were stained with DHE at 5 µM (original magnification: 400×; scale bar: 20 μm). (A–H) Data are presented as means ± SE of 10–12 mice/group; *p < 0.05 vs. control, †p < 0.05 vs. DM. (I) Mesangial cells were incubated with or without APX-115 (1 µM) for 30 min and stimulated with or without 30 mM high glucose (HG) for 24 h followed by angII for 30 min. After that cells were incubated with 10 µM DCF-DA for 10 min and the fluorescence intensity was measured with a Zeiss vision system. Data are presented as means ± SE of at least 2 independent experiments; *p < 0.05 vs. control, †p < 0.05 vs. angII or angII+HG in DMSO.
Figure 6
Figure 6. Effect of APX-115 on lipid accumulation in kidney of STZ-induced diabetic mice
(A) Frozen kidney sections were stained Oil Red O staining (original magnification: 200×; scale bar: 100 μm). (B) Quantification of (A). (C) mRNA expression of CPT1, (D and E) protein expression of ACOX1, and (F and G) mRNA expression of lipogenic markers (SREBP1c and FAS) in kidney tissues. Data are presented as means ± SE of 10–12 mice/group; *p < 0.05 vs. control, †p < 0.05 vs. DM.
Figure 7
Figure 7. Effect of APX-115 on mitochondrial biogenesis in STZ-induced diabetic mice
(A) Paraffin-embedded kidney sections were stained with anti-PGC1α antibody (1:200; original magnification: 200×; scale bar: 100 μm). (B) Quantification of PGC1α stained positive area. mRNA expression levels of NRF1 (C) and TFAM (D) in kidney tissues were measured. Data are presented as means ± SE of 10–12 mice/group; *p < 0.05 vs. control, †p < 0.05 vs. DM.
Figure 8
Figure 8. Effect of APX-115 on peroxisomal biogenesis in STZ-induced diabetic mice
(A) Protein expression levels of PMP70, catalase, and PEX5 in kidneys. (B) Quantification of PMP70, (C) quantification of catalase, and (D) quantification of PEX5. Protein densities were normalized against β-actin. (E) Paraffin-embedded kidney sections were stained with anti-PMP70 and anti-catalase antibodies (1:200; original magnification: 600×; scale bar: 20 μm). Data are presented as mean ± SE of 10–12 mice/group; *p < 0.05 vs. control, †p < 0.05 vs. DM.
Figure 9
Figure 9. Suggested scheme of therapeutic effects of APX-115 on STZ-induced diabetic kidney injury
We summarize here our overall findings schematically, with a focus on how Noxs-mediated ROS production may be a trigger for increased oxidative metabolism leading to mitochondrial and peroxisomal dysfunction associated with lipid accumulation as well as activation of different signaling pathway leading to increased inflammatory responses and profibrotic factors in DKD. In addition, ROS triggers kidney cells to secret cytokines which potentiate recruitment and activation of macrophages and then, the activated macrophages also contribute to DKD. Interestingly, pan-Nox inhibitor, APX-115 attenuates most of the ROS-mediated effects (shown as green arrows), suggesting that APX-115 may become a new therapeutic strategy against DKD.
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