Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Jul;56(7):1661-9.
doi: 10.1007/s00125-013-2907-z. Epub 2013 Apr 19.

Fractalkine and its receptor mediate extracellular matrix accumulation in diabetic nephropathy in mice

K H Song  1 , J Park, J H Park, R Natarajan, H Ha
Affiliations

Fractalkine and its receptor mediate extracellular matrix accumulation in diabetic nephropathy in mice

K H Song et al. Diabetologia. 2013 Jul.

Abstract

Aims/hypothesis: Fractalkine (FKN) is a unique chemokine that works as a chemoattractant and an adhesion molecule. Previous studies have demonstrated that FKN plays a role in ischaemic and protein-overload renal injury via its cognate receptor chemokine (C-X3-C motif) receptor 1 (CX3CR1). However, involvement of the FKN/CX3CR1 system in diabetic nephropathy remains unclear. We examined the role of FKN/CX3CR1 in diabetic mice and mouse mesangial cells (MMCs).

Methods: Streptozotocin (50 mg kg(-1) day(-1)) was intraperitoneally administered for 5 days to male Cx3cr1-knockout (KO) mice and wild-type (WT) mice. MMCs transfected with Fkn (also known as Cx3cl1) or Cx3cr1 siRNA, respectively, were used to elucidate the role of FKN/CX3CR1 in extracellular matrix (ECM) synthesis.

Results: At 12 weeks, diabetic Cx3cr1 KO mice showed no significant changes in plasma glucose, but markers of renal inflammation, fibrosis and ECM, such as the fractional mesangial area, fibronectin and collagen, were significantly lower in diabetic Cx3cr1 KO mice compared with diabetic WT mice. High glucose, oleic acid and TGF-β1 stimulated FKN and CX3CR1 expression, together with the expression of ECM proteins in MMCs, but the effects were significantly attenuated by Fkn or Cx3cr1 siRNA. More importantly, FKN itself increased mesangial ECM through CX3CR1 and subsequent activation of reactive oxygen species and mitogen-activated protein kinases. A neutralising TGF-β antibody inhibited FKN/CX3CR1 in MMCs treated with diabetic stimuli and decreased FKN-induced ECM accumulation.

Conclusions/interpretation: These results demonstrate that FKN/CX3CR1 may play an important role in diabetic renal injury through upregulation of ECM synthesis and could therefore be a therapeutic target for preventing diabetic nephropathy.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Inhibiting CX3CR1 results in decreased glomerular volume and FMA in mouse kidneys. (a) Glomerular volume and (b) FMA. (c–f) PAS-stained mouse kidney sections (scale bar, 20 µm): (c) WT non-diabetic mice; (d) Cx3cr1 KO non-diabetic mice; (e) WT diabetic mice; (f) Cx3cr1 KO diabetic mice. Data are mean±SE or images representative of PAS staining in 8–12 mice. *p<0.05 vs control Cx3cr1 WT mice; †p<0.05 vs diabetic WT mice. Black bars, control; white bars, diabetes. +/+, WT; −/−, Cx3cr1 KO
Fig. 2
Fig. 2
Renal fibrosis was reduced in diabetic Cx3cr1 KO mice. Kidney tissue sections from each mouse were stained with Masson’s trichrome (a–d, scale bar, 50 µm; e–h, scale bar, 20 µm) and Picrosirius Red (i–l, scale bar, 50 µm; m–p, scale bar, 20 µm) and quantified using Image J software (20 images per animal) (q–r). Data are mean±SE or representative stained images of 8–12 mice. *p<0.05 vs control Cx3cr1 WT mice; †p<0.05 vs diabetic WT mice. Black bars, control; white bars, diabetes. +/+, WT; −/−, Cx3cr1 KO
Fig. 3
Fig. 3
ECM markers were downregulated in diabetic Cx3cr1 KO mice. mRNA expression of (a) Tgf-β1, (b) Fn, (c) Col1α1 and (d) Col1α2 was determined by real-time PCR. (e–h) Protein production was measured by ELISA or western blot. Data are mean±SE or western blots representative of those obtained with 8–12 mice. *p<0.05 vs control Cx3cr1 WT mice; †p<0.05 vs diabetic WT mice. Black bars, control; white bars, diabetes. +/+, WT; −/−, Cx3cr1 KO
Fig. 4
Fig. 4
Macrophage infiltration in glomerular regions was decreased in diabetic Cx3cr1 KO mice. The recruitment of macrophages was examined by immunohistochemical staining with anti-F4/80 (1:200) in renal tissues (a–d; scale bar, 20 µm): (a) WT non-diabetic mice; (b) Cx3cr1 KO non-diabetic mice; (c) WT diabetic mice; and (d) Cx3cr1 KO diabetic mice. The areas staining positive were quantified using Image J software (20 images per animal) (e). The mRNA expression of (f) F4/80 and (g) Mcp-1 was determined by real-time PCR. Data are shown as mean±SE or images showing immunohistochemical staining representative of that obtained in 8–12 mice. *p<0.05 vs control Cx3cr1 WT mice; †p<0.05 vs diabetic WT mice. Black bars, control; white bars, diabetes. +/+, WT; −/−, Cx3cr1 KO
Fig. 5
Fig. 5
FKN directly induced ECM synthesis through CX3CR1 in MMCs. The mRNA expression of (a) Tgf-β1 (b) Fn and (c) Col4α1 was determined by real-time PCR. In Cx3cr1 siRNA-transfected MMCs, mRNA expression induced by exposure to FKN, 50 mg/ml, of Tgf-β1 (d), Fn (e) and Col4α1 (f) was measured by real-time PCR. Data are mean±SE of four experiments. *p<0.05 vs FKN at 0 ng/ml or negative siRNA; †p<0.05 vs conditions of FKN and negative siRNA; siScr, negative siRNA; siCx3cr1, Cx3cr1 siRNA
Fig. 6
Fig. 6
Fkn/Cx3cr1 siRNA inhibited diabetes-induced ECM synthesis in MMCs. Tgf-β1 (a,d), Fn (b,e), and Col4α1 (c,f) mRNA expression was assessed by real-time PCR in MMCs exposed to 50 nmol/l Fkn or Cx3cr1 siRNA under diabetic conditions that included HG, OA and TGF-β1. Data are mean±SE of four experiments. *p<0.05 vs negative siRNA without other treatments; †p<0.05 vs negative siRNA with HG (30 mmol/l d-glucose), OA (100 µmol/l OA) or TGF-β1 (10 ng/ml). siFkn, Fkn siRNA; siCx3cr1, Cx3cr1 siRNA; TGF, TGF-β1
Fig. 7
Fig. 7
Inhibition of diabetic-stimuli-induced FKN/CX3CR1 and FKN-induced ECM secretion in MMCs treated with TGF-β-neutralising antibody (FKN at 50 ng/ml). Fkn (a) and Cx3cr1 (b) mRNA levels were measured in MMCs exposed to HG (30 mmol/l d-glucose) and OA (100 µmol/l OA) with or without a TGF-β-neutralising antibody. ECM markers such as Fn (c) and Col4α1 (d) mRNA expression were assessed in MMCs incubated with a TGF-β-neutralising antibody before stimulation with FKN. Data are mean±SE of four experiments. *p<0.05 vs control; †p<0.05 vs HG, OA or FKN. Black bars, treated in 5 µg/ml IgG; white bars, treated in 5 µg/ml TGF-β-neutralising antibody. Con, control
Fig. 8
Fig. 8
Inhibition of FKN-induced ECM protein levels in MMCs treated with inhibitors of ROS, ERK or p38 MAPK. FKN-induced mRNA expression in the ECM was determined in MMCs pretreated with antioxidant (NAC, 5 mmol/l), PD98059 (50 µmol/l) and p38i (10 µmol/l). FKN was used at 50 ng/ml. Data are mean±SE of four experiments. *p<0.05 vs control; †p<0.05 vs FKN vehicle. Con, control; PD, PD98059; vehicle, FKN
Fig. 9
Fig. 9
Suggested model for FKN/CX3CR1 in the regulation of diabetic nephropathy. HG, 30 mmol/l d-glucose
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Tervaert TW, Mooyaart AL, Amann K, et al. Pathologic classification of diabetic nephropathy. J Am Soc Nephrol. 2010;21:556–563. - PubMed
    1. Whiting DR, Guariguata L, Weil C, Shaw J. IDF diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030. Diabetes Res Clin Pract. 2011;94:311–321. - PubMed
    1. Belobradkova J, Filipensky B, Roztocil A, et al. The effect of self-monitoring on perinatal outcome in insulin therapy of diabetic women during pregnancy. Vnitr Lek. 1992;38:1077–1081. - PubMed
    1. Gilbert RE, Cooper ME. The tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury? Kidney Int. 1999;56:1627–1637. - PubMed
    1. Sassy-Prigent C, Heudes D, Mandet C, et al. Early glomerular macrophage recruitment in streptozotocin-induced diabetic rats. Diabetes. 2000;49:466–475. - PubMed

Publication types