Expression of the G-CSF receptor (G-CSFR) in kidneys.

<p>(<b>A</b>) RT-PCR analysis of G-CSFR mRNA expression in kidney tissue. Hypothalamus tissue was used as a positive control, together with a no-template negative control. (<b>B</b>) G-CSFR immunostained via antibody (green, a) and DAPI (blue, b) in glomeruli of a kidney section (magnification x400). G-CSF receptor, G-CSFR; positive control, P; negative control, N.</p

Experimental design.

<p>Experiment 1: a rat model of diabetic nephropathy (male OLETF rats), Experiment 2: a rat model of diabetic nephropathy with bone marrow transplantation (BMT) (donors: male OLETF rats, recipients: female OLETF rats).</p

FISH imaging and immunostained ED-1 in glomeruli of BMT female rats after treatment.

<p>(<b>A</b>) Stained with hematoxylin and eosin (HE) (magnification x400). (<b>B</b>) Higher magnification views of the boxed regions in (A), stained with FISH using a Cy3-labeled Y-chromosome (red, white arrow) and DAPI-labeled nucleus (blue) (magnification x400). (<b>C</b>) Macrophages immunostained with ED-1 antibody (black arrow). Kidney of the LETO rat (a), the saline-treated OLETF rat (b), and the G-CSF-treated OLETF rat (c). (<b>D</b>) Quantitative analysis of Y-chromosome-positive cells in glomeruli. (<b>E</b>) Quantitative analysis of ED-1-positive cells in glomeruli. Fluorescence <i>in situ</i> hybridization, FISH; 4′–6-Diamidino-2-phenylindole, DAPI; Bone marrow transplantation, BMT. All data are expressed as mean±SD. *<i>P</i><0.05 vs. LETO rats. ^†<i>P</i><0.05 vs. untreated OLETF rats (n = 3).</p

Histological changes in the kidney after treatment.

<p>(<b>A</b>) Stained with periodic acid-Shiff (PAS) (magnification x400). (<b>B</b>) Electron micrograph of a glomerulus (magnification x20.000). Kidney of the LETO rat (a), the saline-treated OLETF rat (b), and the G-CSF-treated OLETF rat (c). (<b>C</b>) Quantitative analysis of images of PAS-stained kidney sections. (<b>D</b>) Quantitative analysis of images of GBM thickness via electron micrographs. (<b>E</b>) Quantitative analysis of foot process width via electron micrographs. All data are expressed as mean±SE. *<i>P</i><0.05 vs. LETO rats. ^†<i>P</i><0.05 vs. untreated OLETF rats (n = 4).</p

Sequences of primers.

<p>Sequences of primers.</p

Levels of metabolic parameters before treatment with G-CSF or saline.

<p>Long-Evans Tokushima Otsuka rats, LETO; Otsuka Long-Evans Tokushima Fatty rats, OLETF; total cholesterol, TC; triglyceride, TG; urine albumin creatinine ratio, UACR. All data are expressed as mean±SD. *<i>P</i><0.05 vs. LETO rat (LETO, <i>n</i> = 4; OLETF, <i>n</i> = 8).</p

Changes of body weight, serum glucose, and UACR prior to treatment.

<p>Body weight (<b>A</b>), plasma glucose (<b>B</b>), and urine albumin creatinine ratio (UACR) (<b>C</b>) in rats. White circles: OLETF rats (n = 12) black circles: LETO rats (n = 7). All data are expressed as mean±SD. *<i>P</i><0.05 vs. LETO rats.</p

Anti-Obesity Effects of Granulocyte-Colony Stimulating Factor in Otsuka-Long-Evans-Tokushima Fatty Rats

<div><p>Granulocyte-colony stimulating factor (G-CSF) has molecular structures and intracellular signaling pathways that are similar to those of leptin and ciliary neurotropic factor (CNTF). It also has immune-modulatory properties. Given that leptin and CNTF play important roles in energy homeostasis and that obesity is an inflammatory condition in adipose tissue, we hypothesized that G-CSF could also play a role in energy homeostasis. We treated 12 38-week-old male Otsuka-Long-Evans-Tokushima fatty rats (OLETF, diabetic) and 12 age-matched male Long-Evans-Tokushima rats (LETO, healthy) with 200 µg/day G-CSF or saline for 5 consecutive days. Body weight reduction was greater in G-CSF-treated OLETF (G-CSF/OLETF) than saline-treated OLETF (saline/OLETF) following 8 weeks of treatment (−6.9±1.6% vs. −3.1±2.2%, <i>p</i><0.05). G-CSF treatment had no effect on body weight in LETO or on food intake in either OLETF or LETO. Body fat in G-CSF/OLETF was more reduced than in saline/OLETF (−32.2±3.1% vs. −20.8±6.2%, <i>p</i><0.05). Energy expenditure was higher in G-CSF/OLETF from 4 weeks after the treatments than in saline/OLETF. Serum levels of cholesterol, triglyceride, interleukin-6 and tumor necrosis factor-α were lower in G-CSF/OLETF than in saline/OLETF. Uncoupling protein-1 (UCP-1) expression in brown adipose tissue (BAT) was higher in G-CSF/OLETF than in saline/OLETF, but was unaffected in LETO. Immunofluorescence staining and PCR results revealed that G-CSF receptors were expressed in BAT. In vitro experiments using brown adipocyte primary culture revealed that G-CSF enhanced UCP-1 expression from mature brown adipocytes via p38 mitogen-activated protein kinase pathway. In conclusion, G-CSF treatment reduced body weight and increased energy expenditure in a diabetic model, and enhanced UCP-1 expression and decreased inflammatory cytokine levels may be associated with the effects of G-CSF treatment.</p></div

UCP-1 and G-CSFR expression in BAT.

<p>(A) UCP-1 mRNA levels are higher in the G-CSF-treated OLETF than those in the saline-treated OLETF. UCP-1 mRNA levels are unaffected by G-CSF treatment in LETO; (B) UCP-1 protein levels are higher in the G-CSF-treated OLETF than those in the saline-treated OLETF. UCP-1 protein levels are unaffected by G-CSF treatment in LETO; (C) RT-PCR results show the presence of G-CSFR in BAT; (D), (E) and (F) Immunofluorescence staining revealed that G-CSFR was presented in the islands of brown adipocytes (white arrows) (D, immunofluorescence staining for G-CSFR, 200; E, DAPI, 200; F, merged, 200). * p<0.05. - The data were presented as the mean ± S.E.M. - Measurements of UCP-1 mRNA levels were duplicated, and the mRNA levels were normalized against those of the saline-treated LETO rats. - UCP-1 protein levels were normalized against those of the saline-treated LETO rats. UCP-1, uncoupling protein-1; G-CSFR, G-CSF receptor; BAT, brown adipose tissue.</p

UCP-1 expression in primary cultured brown adipocytes by G-CSF via p38 MAPK pathway.

<p>(A) Oil red-O staining for brown adipocytes; (B) Indictors for differentiation of brown adipocytes. UCP-1 were expressed from D2 to D4 and the band density were decreased on D4; (C) mRNA expression and immunofluorescence staining of G-CSFR in primary cultured brown adipocytes. Cytoplasm was stained by G-CSFR specific antibodies (white arrow); (D) UCP-1 mRNA and protein expression in mature brown adipocytes on D4. After 1 hour from G-CSF treatment, UCP-1 expressions were significantly enhanced. (E) UCP-1 mRNA and protein expression was enhanced after G-CSF treatment and repressed after G-CSF treatment when brown adipocytes were pretreated with SB203580 (p38 MAPK inhibitor); (F) p38 MAPK phosphorylation was enhanced 30 minutes after G-CSF treatment and repressed by SB203580 pretreatment. * p<0.05 vs. UCP-1 level at 0 hour; † p<0.05 vs. UCP-1 levels at 1 hour; ‡ p<0.05 vs. the others. - The relative quantities of UCP-1 mRNA were measured 5 times and averaged. D(n), n days after differentiation; G-CSFR, Granulocyte colony-stimulating factor receptor; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; MSC; Bone marrow derived mesenchymal stem cell; UCP-1, Uncoupling protein-1; PPAR Peroxisome proliferator-activated receptor; PGC-1α, PPAR γ co-activator 1-α; SB203580; p38 Mitogen-activated protein kinase inhibitor; p38 MAPK, p38 Mitogen-activated protein kinase; p38-P, phosphorylated form of p38 MAPK; p38-T, total form of p38 MAPK.</p