J Korean Med Sci.  2016 Sep;31(9):1403-1412. 10.3346/jkms.2016.31.9.1403.

Renoprotective Effects of a Highly Selective A3 Adenosine Receptor Antagonist in a Mouse Model of Adriamycin-induced Nephropathy

• Min HS
• Cha JJ
• Kim K
• Kim JE
• Ghee JY
• Kim H
• Lee JE
• Han JY
• Jeong LS
• Cha DR
• Kang YS

• KMID: 2468272
• DOI: http://doi.org/10.3346/jkms.2016.31.9.1403

Abstract

The concentration of adenosine in the normal kidney increases markedly during renal hypoxia, ischemia, and inflammation. A recent study reported that an A3 adenosine receptor (A3AR) antagonist attenuated the progression of renal fibrosis. The adriamycin (ADX)-induced nephropathy model induces podocyte injury, which results in severe proteinuria and progressive glomerulosclerosis. In this study, we investigated the preventive effect of a highly selective A3AR antagonist (LJ1888) in ADX-induced nephropathy. Three groups of six-week-old Balb/c mice were treated with ADX (11 mg/kg) for four weeks and LJ1888 (10 mg/kg) for two weeks as following: 1) control; 2) ADX; and 3) ADX + LJ1888. ADX treatment decreased body weight without a change in water and food intake, but this was ameliorated by LJ1888 treatment. Interestingly, LJ1888 lowered plasma creatinine level, proteinuria, and albuminuria, which had increased during ADX treatment. Furthermore, LJ1888 inhibited urinary nephrin excretion as a podocyte injury marker, and urine 8-isoprostane and kidney lipid peroxide concentration, which are markers of oxidative stress, increased after injection of ADX. ADX also induced the activation of proinflammatory and profibrotic molecules such as TGF-β1, MCP-1, PAI-1, type IV collagen, NF-κB, NOX4, TLR4, TNFα, IL-1β, and IFN-γ, but they were remarkably suppressed after LJ1888 treatment. In conclusion, our results suggest that LJ1888 has a renoprotective effect in ADX-induced nephropathy, which might be associated with podocyte injury through oxidative stress. Therefore, LJ1888, a selective A3AR antagonist, could be considered as a potential therapeutic agent in renal glomerular diseases which include podocyte injury and proteinuria.

Keyword

Adriamycin; Nephropathy; Adenosine Receptor Antagonist; Mouse Model

MeSH Terms

Actins/metabolism
Adenosine/pharmacology/*therapeutic use
Adenosine A3 Receptor Antagonists/pharmacology/*therapeutic use
Albuminuria/prevention & control
Animals
Body Weight/drug effects
Creatinine/blood
Dinoprost/analogs & derivatives/urine
Disease Models, Animal
Doxorubicin/toxicity
Immunohistochemistry
Kidney/pathology
Kidney Diseases/chemically induced/*drug therapy
Lipid Peroxidation/drug effects
Male
Membrane Proteins/urine
Mice
Mice, Inbred BALB C
NF-kappa B/genetics/metabolism
Oxidative Stress/drug effects
Plasminogen Activator Inhibitor 1/genetics/metabolism
Proteinuria/prevention & control
Transforming Growth Factor beta1/genetics/metabolism
Actins
Adenosine A3 Receptor Antagonists
Membrane Proteins
NF-kappa B
Plasminogen Activator Inhibitor 1
Doxorubicin
Creatinine
Dinoprost
Adenosine

Figure

• 

  Fig. 1 Effects of LJ1888 on urinary excretion of protein and albumin in experimental ADX-induced nephropathy. (A) Twenty-four hour urinary protein excretion level. (B) Urinary excretion of albumin level. Urinary protein, albumin, and nephrin levels were corrected for urine creatinine levels. Values are expressed as (means ± SEM). ADX, adriamycin. *P < 0.01; †P < 0.05 vs. control; ‡P < 0.001 vs. ADX.

• 

  Fig. 2 Effects of LJ1888 on oxidative stress in ADX-induced nephropathy. (A) Twenty-four hour urinary level of 8-isoprostane. Urinary 8-isoprostane levels were corrected for urine creatinine levels. (B) LPO concentration in the kidney tissue. LPO concentration levels were corrected for kidney weight. (C) Urinary excretion of nephrin. Values are expressed as (means ± SEM). ADX, adriamycin; LPO, lipoperoxidase. *P < 0.01 vs. control; †P < 0.001; ‡P < 0.01 vs. ADX.

• 

  Fig. 3 Effects of LJ1888 on inflammation and fibrosis in ADX-induced nephropathy. (A and B) Effects of LJ1888 on the expression of inflammatory and fibrotic cytokines. (C) Representative western blots of NF-κB, TGF-β1, NOX4, and TLR4 in the kidney and quantitative analysis scoring of western blot. Values are expressed as (means ± SEM). ADX, adriamycin; Col-4, type IV collagen; PAI-1, plasminogen activator inhibitor-1; MCP-1, macrophage/monocyte chemoattractant peptide-1; TGF-β1, transforming growth factor-β1; NOX-4, NADPH oxidase 4; TLR-4,Toll-like receptor 4; A3AR, A3 adenosine receptor; TNFα, tumor necrosis α; IL-1β, interleukine-1β; IFN-γ, interferon-γ; NF-κB, nuclear factor-κB. *P < 0.05; †P < 0.01 vs. control; ‡P < 0.05; §P < 0.001; ∥P < 0.01 vs. ADX.

• 

  Fig. 4 Representative renal histological and immunohistochemical staining in experimental models at the time of sacrifice. Representative sections show that the histological changes and immunohistochemical staining for PAS, F4/80, Masson’s trichrome and α-SMA, nephrin, type I collagen, TGF-β1, and type IV collagen are increased in ADX-induced nephropathy and decreased after treatment with LJ1888. Original magnification X400. ADX, adriamycin; PAS, periodic acid-Schiff; α-SMA, α-smooth muscle actin; TGF-β1, transforming growth factor-β1.

• 

  Fig. 5 Quantitation of immunohistochemical staining. (A) Glomerulosclerosis index, (B) F4/80 positive cell staining score, (C) Tubulointerstitial fibrosis index, (D) Quantitation of immunohistochemical staining of nephrin, type I Collagen, TGF-β1 and Type 4 collagen. Values are expressed as (means ± SEM). ADX, adriamycin. *P < 0.01 vs. control; †P < 0.001; ‡P < 0.01 vs. ADX.

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