Effects of Toll-like receptor antagonist 4,5-dihydro-3-phenyl-5-isoxasole acetic acid on the progression of kidney disease in mice on a high-fat diet

Abstract

Background: Obesity-related metabolic disorders are closely associated with inflammation induced by innate immunity. Toll-like receptors (TLRs) play a pivotal role in the innate immune system by activating proinflammatory signaling pathways. GIT27 (4,5-dihydro-3-phenyl-5-isoxasole acetic acid) is an active immunomodulatory agent that primarily targets macrophages and inhibits secretion of tumor necrosis factor α [as well as interleukin (IL)-1β, IL-10, and interferon γ]. However, the effect of TLR antagonist on kidney diseases has rarely been reported. We investigated whether the TLR antagonist GIT27 has beneficial effects on the progression of kidney disease in obese mice on a high-fat diet (HFD).

Methods: Six-week-old male C57BL/6 mice were divided into three groups: mice fed with normal chow diet (N=4); mice fed with a HFD (60% of total calories from fat, 5.5% from soybean oil, and 54.5% from lard, N=4); and GIT27-treated mice fed with a HFD (N=7).

Results: Glucose intolerance, oxidative stress, and lipid abnormalities in HFD mice were improved by GIT27 treatment. In addition, GIT27 treatment decreased the urinary excretion of albumin and protein in obesity-related kidney disease, urinary oxidative stress markers, and inflammatory cytokine levels. This treatment inhibited the expression of proinflammatory cytokines in the kidneys and adipose tissue, and improved extracellular matrix expansion and tubulointerstitial fibrosis in obesity-related kidney disease.

Conclusion: TLR inhibition by administering GIT27 improved metabolic parameters. GIT27 ameliorates abnormalities of lipid metabolism and may have renoprotective effects on obesity-related kidney disease through its anti-inflammatory properties.

Keywords: Kidney disease; Metabolic syndrome; Obesity; Toll-like receptors.

Figure 1

Effectsof GIT27 on glucose tolerance test. After 4 weeks, plasma glucose levels increased significantly in HFD mice compared with those in ND mice (P<0.05). GIT27 treatment improved glucose tolerance (P<0.05). * P<0.05, HFD versus HFD+GIT27. ^†P<0.05, ND versus HFD. GIT27, 4,5-dihydro-3-phenyl-5-isoxasole acetic acid; HFD, high-fat chow diet; ND, normal chow diet.

Figure 2

Effects of GIT27 on plasma lipid profiles. Lipid abnormalities improved significantly in GIT27-treated HFD mice (P<0.05). * P<0.05, ND versus HFD. ^†P<0.05, HFD versus HFD+GIT27. GIT27, 4,5-dihydro-3-phenyl-5-isoxasole acetic acid; HDL, high-density lipoprotein; HFD, high-fat chow diet; LDL, low-density lipoprotein; ND, normal chow diet.

Figure 3

Effects of GIT27 on tissue lipid content, lipid peroxidation, and oxidative stress. Compared to the kidneys and liver, the level of (A) cholesterol and (B) triglyceride were higher in the adipose tissue. (C) Lipid peroxide levels in the kidneys, adipose tissue, and liver were markedly higher in HFD mice and decreased significantly in GIT27-treated HFD mice. (D) Urinary 8-isoprostane was increased markedly in HFD mice and decreased by GIT27 treatment. * P<0.05, ND versus HFD. ^†P<0.05, ND versus HFD. Cr, creatinine; GIT27, 4,5-dihydro-3-phenyl-5-isoxasole acetic acid; HFD, high-fat chow diet; ND, normal chow diet.

Figure 4

Effects of GIT27 on proteinuria. Urinary protein excretion was significantly higher in HFD mice than in ND mice. (A) This was improved significantly after GIT27 treatment. (B) After 12 weeks of experiment, urinary albumin excretion was improved markedly in GIT27-treated HFD mice compared with that in HFD mice. * P<0.05, ND versus HFD. ^†P<0.05, HFD versus HFD+GIT27. ^‡P<0.05, ND versus HFD+GIT27. Cr, creatinine; GIT27, 4,5-dihydro-3-phenyl-5-isoxasole acetic acid; HFD, high-fat chow diet; ND, normal chow diet.

Figure 5

Anti-inflammatory and antifibrotic effects of GIT27. (A) Levels of TNFα and IL-2 in urine were increased in HFD mice and decreased in GIT27-treated HFD mice. In particular, the level of IL-2 was improved significantly in GIT27-treated HDF mice compared with that in HFD mice. (B) The gene expression of proinflammatory cytokines such as MCP-1, PAI-1, and TNFα in the adipose tissue tended to increase in HFD mice and decrease in GIT27-treated HFD mice. (C and D) The gene expression of proinflammatory and profibrotic cytokines in kidney tissue tended to increase in HFD mice and decrease in GIT27-treated HFD mice and that of type IV collagen, PAI-1, MCP-1, IL-1b, and SREBP1 were decreased significantly in GIT27-treated HFD mice compared with that in HFD mice. * P<0.05, HFD versus HFD+GIT27. ^†P <0.05, HFD versus HFD+GIT27. ^‡P<0.05, ND versus HFD. ABCA, adenosine triphosphate-binding cassette transporter A; AMPK, AMP-dependent kinase; Col(IV), type lV collagen alpha; Cr, creatinine; FAS, fatty acid synthase; GIT27, 4,5-dihydro-3-phenyl-5-isoxasole acetic acid; HFD, high-fat chow diet; HMG coA, 3-hydroxy-3-methylglutaryl-coenzyme A; IL, interleukine; INF-γ, interferon gamma; MCP, monocyte chemoattractant protein; ND, normal chow diet; PAI, plasminogen activator inhibitor; PPARγ, peroxisome proliferator activator receptor gamma; SREBP, sterol-regulatory element-binding protein; TGF, transforming growth factor; TNF, tumor necrosis factor.

Figure 6

Renal pathology and immunohistochemistry. (A) Representative sections show that the histological changes and immunohistochemical staining for PAS, type IV collagen, PAI-1, F4/80, and TGFβ1 were increased in HFD mice and decreased in GIT27-treated HFD mice (original magnification 400× for PAS, type IV collagen, F4/80 staining, and TGF-β1 staining; and 1000× for PAI-1 staining). (B) Bar graphs represent quantification of glomerulosclerosis and the immunostaining scores in the kidney tissue. Data are shown as the mean±SEM. * P <0.01 ND versus HFD. ^†P<0.05 HFD versus HFD+GIT27. ^‡P<0.05 ND versus HFD. GIT27, 4,5-dihydro-3-phenyl-5-isoxasole acetic acid; GSI, glomerulosclerosis index; HFD high-fat chow diet; ND, normal chow diet; PAI-1, plasminogen activator inhibitor-1; PAS, periodic acid-Schiff; SEM, standard error of the mean; TGF, transforming growth factor.

Figure 7

TLR4/NFκB pathway in animal experiments. Protein synthesis for TLR4, NFκB, IκBβ, and NOX4 did not show a significant change in renal cortical tissues among ND, HFD, and GIT27-treated HFD mice. GIT27, 4,5-dihydro-3-phenyl-5-isoxasole acetic acid; HFD, high-fat chow diet; IκB, inhibitory kappa B; ND, normal chow diet; NFκB, nuclear factor kappa B; NOX, nicotinamide adenine dinucleotide phosphate oxidase; TLR, Toll-like receptor.

Figure 8

TLR4/NFκB pathway in cultured podocytes. (A, B) FFA induced NFκB activation and IκBβ inhibition in podocytes, which were suppressed by GIT27. TLR4, TRAF6, NOX4, p-AMPK, and SREBP1 protein expressions tended to increase after FFA treatment, an effect that was reversed by GIT27 treatment. However, these changes were not significant on densitometric analysis of Western blot results. Data are shown as mean±SEM. * P < 0.01 NG versus NG+FFA and HG versus HG+FFA. ^†P<0.05 NG+FFA versus NG+FFA+GIT27 and HG+FFA versus HG+FFA+GIT27. AMPK, AMP-dependent kinase; FFA, free fatty acid; GIT27, 4,5-dihydro-3-phenyl-5-isoxasole acetic acid; HG, high glucose; IκB, inhibitory kappa B; NFκB, nuclear factor kappa B; NG, normal glucose; NOX, nicotinamide adenine dinucleotide phosphate oxidase; SREBP1, sterol regulatory element binding transcription protein1; TLR, Toll-like receptor; TRAF, TNF receptor associated factor.

Figure 9

TLR4/NFκB pathway in adipocytes. In adipocytes, no changes were observed in the protein expression of SREBP1, TLR4, NFκB, IκBβ, NOX4, and p-AMPK. AMPK, AMP-dependent kinase; FFA, free fatty acid; GIT27,4,5-dihydro-3-phenyl-5-isoxasole acetic acid; HG, high glucose; IκB, inhibitory kappa B; NFκB, nuclear factor kappa B; NG, normal glucose; NOX, nicotinamide adenine dinucleotide phosphate oxidase; SREBP1, sterol regulatory element binding transcription protein1; TLR, Toll-like receptor.