Stem cell transplantation increases antioxidant effects in diabetic mice

Abstract

Intra bone marrow-bone marrow transplantation (IBM- BMT) + thymus transplantation (TT) has been shown to reduce the incidence of graft versus host disease (GVHD) and restore donor-derived T cell function. In addition, an increase in insulin sensitivity occurred in db/db mice after IBM-BMT+TT treatment. Heme oxygenase (HO)-1 is a stress inducible enzyme which exert antioxidant, antiapoptotic, and immune-modulating properties. We examined whether IBM-BMT+TT could modulate the expression of HO-1 in the kidneys of db/db mice. Six-week-old db/db mice with blood glucose levels higher than 250 mg/dl were treated with IBM-BMT+TT. Six weeks later, the db/db mice showed decreased body weight, blood glucose levels and insulin, and increased plasma adiponectin levels. The upregulation of HO-1 was associated with significantly (p<0.05) increased levels of peNOS and pAKT, but decreased levels of iNOS in the kidneys of db/db mice. Plasma creatinine levels also decreased (p<0.05), and the expression of type IV collagen was improved. Thus IBM-BMT+TT unregulated the expression of HO-1, peNOS and pAKT, while decreasing iNOS levels in the kidney of db/db mice. This was associated with an improvement in renal function.

Keywords: HO-1; IBM-BMT+TT; antioxidant; diabetic nephropathy..

Figure 1

Body weight, thymus weight and the morphology of pancreas and bone marrow, and expression of insulin on the pancreata, and plasma creatinine levels and images of electron microcopy. (A) Body weights are shown (*p< 0.01). (B) Thymus weights are shown (*p<0.05). (C-J) HE staining of pancreas and bone marrow of the lean and db/db mice at each age. The islets are shown (* in Fig.C-F). The adipocytes are shown in db/db mice (arrows in Fig. H-J). (K-N) Immunochemistry staining for insulin (arrows) was performed. Scale bar =50μm in Fig. C-J, Scale bar =25μm in Fig. K-N. (O) Plasma creatinine levels of lean, (p< 0.05). (P-S) EM of kidneys. Podocyte foot fusion is shown in Fig. 3Q and R (arrows). Electron dense deposits and increased mesangium cells (*) are shown in Fig. 1R and S. Scale bar =2μm in Fig. P-R, Scale bar =10μm in Fig. S. The results are mean±SE, n=6 in each group.

Figure 1

Body weight, thymus weight and the morphology of pancreas and bone marrow, and expression of insulin on the pancreata, and plasma creatinine levels and images of electron microcopy. (A) Body weights are shown (*p< 0.01). (B) Thymus weights are shown (*p<0.05). (C-J) HE staining of pancreas and bone marrow of the lean and db/db mice at each age. The islets are shown (* in Fig.C-F). The adipocytes are shown in db/db mice (arrows in Fig. H-J). (K-N) Immunochemistry staining for insulin (arrows) was performed. Scale bar =50μm in Fig. C-J, Scale bar =25μm in Fig. K-N. (O) Plasma creatinine levels of lean, (p< 0.05). (P-S) EM of kidneys. Podocyte foot fusion is shown in Fig. 3Q and R (arrows). Electron dense deposits and increased mesangium cells (*) are shown in Fig. 1R and S. Scale bar =2μm in Fig. P-R, Scale bar =10μm in Fig. S. The results are mean±SE, n=6 in each group.

Figure 2

Body weight, blood glucose levels, plasma adiponectin, insulin. (A) Body weights are shown. (B) Blood glucose levels are shown. (C) Plasma adiponectin. (D) Plasma insulin.

Figure 3

Cell number of thymus and lymphocyte subpopulations in peripheral blood. (A) The relative percentage of total cell numbers in the thymus, which was significantly increased in the db/db mice treated with IBM-BMT+TT. (B-D) Donor-derived cells in peripheral blood, CD4⁺ cells and CD8⁺ cells are shown. The results are mean±SE, n=6 in each group.

Figure 4

Expression of HO-1, iNOS AKT, pAKT, eNOS and peNOS in the kidney, and expression of type IV collagen in the kidneys. (A-C) Western blot and densitometry analysis of HO-1, iNOS and actin proteins in kidneys of lean, non-treated db/db mice and treated db/db mice. (D and E) Western blot and densitometry analyses of AKT and pAKT and actin proteins in kidney of lean, non-treated db/db mice and treated db/db mice are shown. (F and G) Western blot and densitometry analyses of eNOS and peNOS and actin proteins in kidney of lean, non-treated db/db mice and treated db/db mice are shown. Representative immunoblots are shown. There were significant differences between non-treated and treated groups (p<0.05). (H-J) Immunochemistry staining for type IV collagen (*). (K-S) Immunochemistry staining for eNOS, pAKT and iNOS. Scale bar =25μm. (T) NO levels in the kidney.

Figure 4

Expression of HO-1, iNOS AKT, pAKT, eNOS and peNOS in the kidney, and expression of type IV collagen in the kidneys. (A-C) Western blot and densitometry analysis of HO-1, iNOS and actin proteins in kidneys of lean, non-treated db/db mice and treated db/db mice. (D and E) Western blot and densitometry analyses of AKT and pAKT and actin proteins in kidney of lean, non-treated db/db mice and treated db/db mice are shown. (F and G) Western blot and densitometry analyses of eNOS and peNOS and actin proteins in kidney of lean, non-treated db/db mice and treated db/db mice are shown. Representative immunoblots are shown. There were significant differences between non-treated and treated groups (p<0.05). (H-J) Immunochemistry staining for type IV collagen (*). (K-S) Immunochemistry staining for eNOS, pAKT and iNOS. Scale bar =25μm. (T) NO levels in the kidney.