Orally active, species-independent novel A3 adenosine receptor antagonist protects against kidney injury in db/db mice

Figures

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  Fig. 1. db/db mice presented higher expression of renal A3AR than db/m mice. Mice were sacrificed at 20 weeks old, and kidney tissues were collected to analyze the expression levels of A1AR, A2aAR, A2bAR, and A3AR. (a) Protein and (b) mRNA expression levels of all AR subtypes are shown. Data are presented as the mean ± SE of 7–10 mice/group. **p < 0.01 vs. db/m mice

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  Fig. 2. LJ -2698 improved kidney morphology in db/db mice. LJ-2698 was administered to db/db mice at doses of 1.5, 5, or 10 mg/kg. CMC was administered to control db/db and db/m mice. After 12 weeks of treatment, mice were sacrificed, and the right kidney was fixed and sectioned. (a) Sections were stained with PAS reagent. The scale bar indicates 10 μm; the original magnification was ×630. (b) Glomerular volume and (c) FMA were then analyzed using Image-Pro Plus 4.5.1. Data are presented as the mean ± SE of 7–10 mice/group. *p < 0.05 vs. db/m and †p < 0.05 vs. db/db mice

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  Fig. 3. LJ-2698 exhibited renoprotective effects in a dose-dependent manner in db/db mice. LJ-2698 was administered to db/db mice at doses of 1.5, 5, or 10 mg/kg. CMC was administered to control db/db and db/m mice. After 12 weeks of treatment, (a) urinary albumin was measured. mRNA expression levels of (b) nephrin, (c) NGAL, (d) TGF-β, (e) collagen IV, (f) fibronectin, (g) MCP-1, and (h) ICAM-1 were analyzed by qRT-PCR. (i) The urinary LPO concentration was measured by the thiobarbituric acid method. Data are presented as the mean ± SE of 7–10 mice/group. *p < 0.05 vs. db/m and †p < 0.05 vs. db/db mice

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  Fig. 4. LJ-2698 prevented kidney injury in db/db mice. LJ-2698 (10 mg/kg) was administered in db/db mice and age-matched control db/m mice for 12 weeks. Losartan (1.5 mg/kg) was administered in diabetic mice to compare with the effect of LJ-2698. After 12 weeks of treatment, urine and plasma were collected for analysis of (a) urinary albumin, (b) plasma cystatin C, and (c) urinary KIM-1. Paraffin-embedded kidney sections were stained with (d) PAS staining and (e) nephrin antibody. The scale bar indicates 10 μm, original magnification was ×630. Quantitative analysis of (f) glomerular volume, (g) FMA, and (h) glomerular nephrin area are depicted. Data are presented as the mean ± SE of 7–10 mice/group. *p < 0.05 vs. db/m and †p < 0.05 vs. db/db mice

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  Fig. 5. LJ-2698 inhibited kidney fibrosis in db/db mice. mRNA expression levels of (a) type IV collagen, (b) PAI-1, and (c) TGF-β were determined by qRT-PCR. Representative photomicrographs of (d) picrosirius red-stained kidney sections are displayed. The scale bar indicates 50 μm; the original magnification was ×100. Quantitative analysis of (e) picrosirius red in kidney cortex is depicted. CM control mice; DM db/db diabetic mice. Data are presented as the mean ± SE of 7–10 mice/group. *p < 0.05 vs. db/m, †p < 0.05 vs. db/db, and #p < 0.05 vs. db/db + LJ-2698

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  Fig. 6. LJ-2698 ameliorated kidney inflammation in db/db mice. After 12 weeks of LJ-2698 administration to db/db mice, mRNA expression of inflammatory cytokines in the kidney tissue was measured. (a) MCP-1 and (b) TNF-α mRNA expression was analyzed by qRT-PCR. Paraffin-embedded kidney sections were stained with (c) F4/80 antibody. The scale bar indicates 20 μm; the original magnification was ×200. Quantitative analysis of (d) F4/80 positive stained area is depicted. CM control mice; DM db/db diabetic mice. Data are presented as the mean ± SE of 7–10 mice/group. *p < 0.05 vs. db/m and †p < 0.05 vs. db/db mice

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  Fig. 7. LJ-2698 prevented kidney oxidative stress in db/db mice. Paraffin-embedded kidney sections were stained with (a) 8-oxo-dG antibody. The scale bar indicates 20 μm; the original magnification was 200 × . Quantitative analysis of (b) 8-oxo-dG in kidney cortex is depicted. The LPO concentration was detected in both (c) kidney tissues and (d) urine samples following 12 weeks of drug treatment. CM, control mice; DM, db/db diabetic mice, Data are presented as the mean ± SE of 7–10 mice/group. *p < 0.05 vs db/m and †p < 0.05 vs db/db mice

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  Fig. 8. LJ-2698 inhibited renal lipid accumulation in db/db mice. (a) Renal lipids were detected by ORO staining and (b) quantified using Image-Pro Plus 4.5 software. The scale bar indicates 20 μm; the original magnification was 200 × . (c, d) Protein expression of PGC1α was detected by immunoblotting. Transcription factors interacting with PGC1α were observed, including (e) PPARα protein and (f) NRF1 mRNA. Mitochondrial transcripts were indicated by (g) TFAM mRNA. Oxidative phosphorylation was represented by (h,i) protein expression of Cox4i1 and (j) Cytb mRNA. (k) CPT1α and (l) MCAD mRNAs were analyzed to examine fatty acid β-oxidation. Data are presented as the mean ± SE of 7–10 mice/group. CM, control mice; DM, db/db diabetic mice, *p < 0.05 vs db/m, †p < 0.05 vs db/db, and #p < 0.05 vs db/db + LJ-2698