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Hepatocyte growth factor gene therapy enhances infiltration of macrophages and may induce kidney repair in db/db mice as a model of diabetes.

Flaquer M, Franquesa M, Vidal A, Bolaños N, Torras J, Lloberas N, Herrero-Fresneda I, Grinyó JM, Cruzado JM - Diabetologia (2012)

Bottom Line: The aim of this study was to test whether bone-marrow-derived cells are also involved in this HGF-induced reparative process.We have created chimeric db/db mice as a model of diabetes that produce enhanced green fluorescent protein (EGFP) in bone marrow cells.These cells are mainly monocyte-derived macrophages, which may contribute to the renal tissue repair and regeneration consistently observed in our model.

View Article: PubMed Central - PubMed

Affiliation: Nephrology Laboratory, Departament de Ciències Clíniques, IDIBELL, University of Barcelona, Bellvitge Hospital, Barcelona, Spain.

ABSTRACT

Aims/hypothesis: We previously demonstrated hepatocyte growth factor (HGF) gene therapy was able to induce regression of glomerulosclerosis in diabetic nephropathy through local reparative mechanisms. The aim of this study was to test whether bone-marrow-derived cells are also involved in this HGF-induced reparative process.

Methods: We have created chimeric db/db mice as a model of diabetes that produce enhanced green fluorescent protein (EGFP) in bone marrow cells. We performed treatment with HGF gene therapy either alone or in combination with granulocyte-colony stimulating factor, in order to induce mobilisation of haematopoietic stem cells in these diabetic and chimeric animals.

Results: We find HGF gene therapy enhances renal expression of stromal-cell-derived factor-1 and is subsequently associated with an increased number of bone-marrow-derived cells getting into the injured kidneys. These cells are mainly monocyte-derived macrophages, which may contribute to the renal tissue repair and regeneration consistently observed in our model. Finally, HGF gene therapy is associated with the presence of a small number of Bowman's capsule parietal epithelial cells producing EGFP, suggesting they are fused with bone-marrow-derived cells and are contributing to podocyte repopulation.

Conclusions/interpretation: Altogether, our findings provide new evidence about the therapeutic role of HGF and open new opportunities for inducing renal regeneration in diabetic nephropathy.

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Related in: MedlinePlus

Evaluation of glomerular macrophages expressing M2 markers. a Macrophages are stained with F4/80 (in red) and nuclear M2 marker galectin-3 (green), or transmembrane M2 marker mannose receptor (green). Co-staining of both macrophages and M2 markers shows as yellow (arrows) (×630). b Number of macrophages per glomeruli. HGF gene therapy increased the presence of glomerular macrophages (*p = 0.05, db/db–BMT vs db/db–HGF), the majority of them (60.0%) expressing M2 markers. Each bar represents the mean number of macrophages F4/80+ per glomeruli; the black shading in the bars indicates the percentage of the total macrophages that are M2 macrophages
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Fig7: Evaluation of glomerular macrophages expressing M2 markers. a Macrophages are stained with F4/80 (in red) and nuclear M2 marker galectin-3 (green), or transmembrane M2 marker mannose receptor (green). Co-staining of both macrophages and M2 markers shows as yellow (arrows) (×630). b Number of macrophages per glomeruli. HGF gene therapy increased the presence of glomerular macrophages (*p = 0.05, db/db–BMT vs db/db–HGF), the majority of them (60.0%) expressing M2 markers. Each bar represents the mean number of macrophages F4/80+ per glomeruli; the black shading in the bars indicates the percentage of the total macrophages that are M2 macrophages

Mentions: In order to test whether HGF modifies the inflammatory response we measured serum levels of some Th1, Th2 and pro-inflammatory cytokines in HGF-treated groups (Table 2). HGF alone and in combination with G-CSF significantly reduced IL-6 and MCP-1. There was also a trend to reduce Th1 cytokines (TNF-α, IFN-γ, IL-12p70) and to enhance some Th2 cytokines (IL-4, IL-10), although these relationships did not reach statistical significance. We evaluated the expression of M2 markers in glomerular macrophages by confocal microscopy (Fig. 7). In agreement with the results shown in Fig. 5, HGF, G-CSF and the combination of both were associated with an increased number of glomerular macrophages. Nevertheless, the percentage of macrophages expressing the M2 markers galectin-3 and mannose receptor was nearly double in animals treated with HGF vs those receiving G-CSF alone (Fig. 7b). In diabetic non-treated mice, glomerular macrophages were seldom encountered, although a high proportion of macrophages expressed M2 markers.Table 2


Hepatocyte growth factor gene therapy enhances infiltration of macrophages and may induce kidney repair in db/db mice as a model of diabetes.

Flaquer M, Franquesa M, Vidal A, Bolaños N, Torras J, Lloberas N, Herrero-Fresneda I, Grinyó JM, Cruzado JM - Diabetologia (2012)

Evaluation of glomerular macrophages expressing M2 markers. a Macrophages are stained with F4/80 (in red) and nuclear M2 marker galectin-3 (green), or transmembrane M2 marker mannose receptor (green). Co-staining of both macrophages and M2 markers shows as yellow (arrows) (×630). b Number of macrophages per glomeruli. HGF gene therapy increased the presence of glomerular macrophages (*p = 0.05, db/db–BMT vs db/db–HGF), the majority of them (60.0%) expressing M2 markers. Each bar represents the mean number of macrophages F4/80+ per glomeruli; the black shading in the bars indicates the percentage of the total macrophages that are M2 macrophages
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Related In: Results  -  Collection

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Fig7: Evaluation of glomerular macrophages expressing M2 markers. a Macrophages are stained with F4/80 (in red) and nuclear M2 marker galectin-3 (green), or transmembrane M2 marker mannose receptor (green). Co-staining of both macrophages and M2 markers shows as yellow (arrows) (×630). b Number of macrophages per glomeruli. HGF gene therapy increased the presence of glomerular macrophages (*p = 0.05, db/db–BMT vs db/db–HGF), the majority of them (60.0%) expressing M2 markers. Each bar represents the mean number of macrophages F4/80+ per glomeruli; the black shading in the bars indicates the percentage of the total macrophages that are M2 macrophages
Mentions: In order to test whether HGF modifies the inflammatory response we measured serum levels of some Th1, Th2 and pro-inflammatory cytokines in HGF-treated groups (Table 2). HGF alone and in combination with G-CSF significantly reduced IL-6 and MCP-1. There was also a trend to reduce Th1 cytokines (TNF-α, IFN-γ, IL-12p70) and to enhance some Th2 cytokines (IL-4, IL-10), although these relationships did not reach statistical significance. We evaluated the expression of M2 markers in glomerular macrophages by confocal microscopy (Fig. 7). In agreement with the results shown in Fig. 5, HGF, G-CSF and the combination of both were associated with an increased number of glomerular macrophages. Nevertheless, the percentage of macrophages expressing the M2 markers galectin-3 and mannose receptor was nearly double in animals treated with HGF vs those receiving G-CSF alone (Fig. 7b). In diabetic non-treated mice, glomerular macrophages were seldom encountered, although a high proportion of macrophages expressed M2 markers.Table 2

Bottom Line: The aim of this study was to test whether bone-marrow-derived cells are also involved in this HGF-induced reparative process.We have created chimeric db/db mice as a model of diabetes that produce enhanced green fluorescent protein (EGFP) in bone marrow cells.These cells are mainly monocyte-derived macrophages, which may contribute to the renal tissue repair and regeneration consistently observed in our model.

View Article: PubMed Central - PubMed

Affiliation: Nephrology Laboratory, Departament de Ciències Clíniques, IDIBELL, University of Barcelona, Bellvitge Hospital, Barcelona, Spain.

ABSTRACT

Aims/hypothesis: We previously demonstrated hepatocyte growth factor (HGF) gene therapy was able to induce regression of glomerulosclerosis in diabetic nephropathy through local reparative mechanisms. The aim of this study was to test whether bone-marrow-derived cells are also involved in this HGF-induced reparative process.

Methods: We have created chimeric db/db mice as a model of diabetes that produce enhanced green fluorescent protein (EGFP) in bone marrow cells. We performed treatment with HGF gene therapy either alone or in combination with granulocyte-colony stimulating factor, in order to induce mobilisation of haematopoietic stem cells in these diabetic and chimeric animals.

Results: We find HGF gene therapy enhances renal expression of stromal-cell-derived factor-1 and is subsequently associated with an increased number of bone-marrow-derived cells getting into the injured kidneys. These cells are mainly monocyte-derived macrophages, which may contribute to the renal tissue repair and regeneration consistently observed in our model. Finally, HGF gene therapy is associated with the presence of a small number of Bowman's capsule parietal epithelial cells producing EGFP, suggesting they are fused with bone-marrow-derived cells and are contributing to podocyte repopulation.

Conclusions/interpretation: Altogether, our findings provide new evidence about the therapeutic role of HGF and open new opportunities for inducing renal regeneration in diabetic nephropathy.

Show MeSH
Related in: MedlinePlus