Cells derived from young bone marrow alleviate renal aging

Abstract

Bone marrow-derived stem cells may modulate renal injury, but the effects may depend on the age of the stem cells. Here we investigated whether bone marrow from young mice attenuates renal aging in old mice. We radiated female 12-mo-old 129SvJ mice and reconstituted them with bone marrow cells (BMC) from either 8-wk-old (young-to-old) or 12-mo-old (old-to-old) male mice. Transfer of young BMC resulted in markedly decreased deposition of collagen IV in the mesangium and less β-galactosidase staining, an indicator of cell senescence. These changes paralleled reduced expression of plasminogen activator inhibitor-1 (PAI-1), PDGF-B (PDGF-B), the transdifferentiation marker fibroblast-specific protein-1 (FSP-1), and senescence-associated p16 and p21. Tubulointerstitial and glomerular cells derived from the transplanted BMC did not show β-galactosidase activity, but after 6 mo, there were more FSP-1-expressing bone marrow-derived cells in old-to-old mice compared with young-to-old mice. Young-to-old mice also exhibited higher expression of the anti-aging gene Klotho and less phosphorylation of IGF-1 receptor β. Taken together, these data suggest that young bone marrow-derived cells can alleviate renal aging in old mice. Direct parenchymal reconstitution by stem cells, paracrine effects from adjacent cells, and circulating anti-aging molecules may mediate the aging of the kidney.

Figure 1.

Reconstitution of female mice with male bone marrow. Y chromosome was detected in 80% of cells in positive control male kidneys (A) and was negative in females (B). After bone marrow transplantation (BMT), around 8% of cells were Y-chromosome positive. These bone marrow-derived cells were mostly present in mesangial areas (arrow in C) and in the interstitium (arrow in D). Some podocyte-like cells (triangle in C) and tubular epithelial cells (triangle in D) were also Y-chromosome positive. Real-time PCR showed similar amounts of Y chromosome in the kidneys of young-to-old (Y2O) and old-to-old (O2O) bone-marrow-transplant mice (E).

Figure 2.

Renal injury in aging. There was more interstitial fibrosis and glomerular mesangial expansion in old-to-old (O2O) compared with young-to-old (Y2O) bone-marrow-transplant mice (A, Masson trichrome staining, 40X). Glomerular collagen IV deposition in O2O mice increased from 15 to 18 mo and was slightly more than in sham nontransplanted age-matched controls. Young bone marrow resulted in significantly less collagen IV deposition than old bone marrow (B, 200X).

Figure 3.

Fibrosis-related factors after bone marrow transplant. PDGF-B was expressed in podocytes, mesangial, and tubular epithelial cells, as well as endothelial cells (A, arrow head, 200X). PAI-1 was expressed in podocytes, mesangial, and tubular epithelial cells (B, arrow head, 200X). PAI-1 expression was decreased at 18 mo and PDGF-B was increased at 15 mo in young-to-old (Y2O) kidneys compared with old-to-old (O2O) bone-marrow-transplant kidneys.

Figure 4.

Renal FSP-1 expression after bone marrow transplant (BMT). Fibroblastic specific protein-1 (FSP-1) was expressed in the interstitium and in some tubular epithelial and glomerular cells (A, open arrow, anti-FSP-1, 400X). Some macrophages just expressed F4/80 (brown, arrowhead), while some macrophages expressed both F4/80 and FSP-1 (brown and red, open arrow). Some tubular epithelial cells just expressed FSP-1 (red, solid arrow; B, 400X). Young-to-old (Y2O) bone-marrow-transplant mice had less FSP-1 in glomeruli and interstitium at 15 mo, and in the interstitium at 18 mo, when compared with old to old (O2O) (C).

Figure 5.

Senescence-related proteins after bone marrow transplant. Renal SA-β-gal positivity was significantly decreased in Y2O kidneys at 15 mo versus O2O kidneys (A, 100X). Cell senescence is regulated in mice by the p16/Rb pathway and ARF/p53 pathway. Mice with young bone marrow (Y2O) had less p16 and p21 mRNA expression compared with old bone marrow (O2O) (B).

Figure 6.

Renal macrophage infiltration after bone marrow transplant. Compared with aging bone marrow (O2O), mice with young bone marrow (Y2O) had less macrophage infiltration in the kidneys (A, 100X). Macrophages showed similar percentages of Y-chromosome positivity (arrow) as control male tissues in all groups, suggesting a nearly complete replacement of bone marrow cells from the donor (B, 400X).

Figure 7.

FSP-1 expression in bone marrow-derived cells. In situ hybridization for the Y chromosome (brown) with costaining for FSP-1 (red) showed that young bone marrow-derived cells (Y2O) in glomeruli expressed less FSP-1 than old bone marrow-derived cells (O2O) in aging kidneys (200X).

Figure 8.

Cell senescence and bone marrow-derived cells. The SA-β-gal-positive cells (blue) did not colocalize with the Y-chromosome-positive cells (brown), indicating that no or few bone marrow-derived cells showed senescence. Moreover, there were less β-gal-positive cells adjacent to Y-chromosome-positive cells in young-to-old (Y2O) than in old-to-old (O2O) bone-marrow-transplant kidneys (200X).

Figure 9.

Renal Klotho expression after bone marrow transplant. Klotho was expressed in distal tubules at the apical side and also in the cytoplasm (A, left panel, 200X). Compared with old bone marrow (O2O), transplantation with young bone marrow (Y2O) preserved Klotho protein in aging kidneys at age 15 mo and 18 mo (A), while IGF-1 receptor β phosphorylation decreased (B).