Mesenchymal stromal cells reset the scatter factor system and cytokine network in experimental kidney transplantation

Abstract

Background: In former studies we showed in a rat model of renal transplantation that Mesenchymal Stromal Cells (MSC) prevent acute rejection in an independent way of their endowing in the graft. In this study we investigated whether MSC operate by resetting cytokine network and Scatter Factor systems, i.e. Hepatocyte Growth Factor (HGF), Macrophage Stimulating Protein (MSP) and their receptors Met and RON, respectively.

Methods: MSC were injected into the renal artery soon after reperfusion. Controls were grafted untreated and normal rats. Rats were sacrificed 7 days after grafting. Serum and renal tissue levels of IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-10, MSP/RON, HGF/Met systems, Treg lymphocytes were investigated.

Results: In grafted untreated rats IFN-γ increased in serum and renal tissue and IL-6 rose in serum. MSC prevented both the phenomena, increased IL-10 serum levels and Treg number in the graft. Furthermore MSC increased serum and tissue HGF levels, Met tubular expression and prevented the suppression of tubular MSP/RON expression.

Conclusions: Our results demonstrate that MSC modify cytokine network to a tolerogenic setting, they suppress Th1 cells, inactivate monocytes/macrophage, recruit Tregs. In addition, MSC sustain the expression of the Scatter Factor systems expression, i.e. systems that are committed to defend survival and stimulate regeneration of tubular cells.

Figure 1

Tubular necrosis and tubular cell proliferation. Groups are defined as in Figure 8. Panel a: Columns represent the ratio between necrotic tubules number and total counted tubules (bars are SD). °p < 0.001 vs C; *p < 0.0001 vs B and C. Panel b: Boxes represent 25-75% percentile of tubular PCNA-positive cell number/HPF (bars are medians, whiskers: min to max). *p < 0.005 vs A and B.

Figure 2

Foxp3 positive cells. Foxp3 staining of representative kidney and spleen sections. Panel a shows Foxp3 positive cells in cortical tubules after 7 days from transplantation in rats of group C (Magnification X400). Panel b shows Foxp3 positive cells in medullary tubules after 7 days from transplantation in rats of group C (Magnification X 200 and X400). Panel c shows Foxp3 positive cells in spleen after 7 days from transplantation in rats of group C (Magnification X 200).

Figure 3

Serum HGF levels, HGF mRNA and HGF protein in kidney of control and allografted rats on day 7. Groups are defined as in Figure 8. Upper panel: serum HGF levels in A, B, C groups of rats. Columns are means, Bars are SD. *p < 0.005 vs A and C. Middle panel: Western blot performed with anti-HGF antibody in renal tissue of groups A, B, C. The p69-kDa band represents the α sub of active heterodimer HGF. Lower panel: HGF mRNA expression in renal tissue analyzed by RT PCR in all groups of rats. Columns indicate HGF mRNA expression normalized to the beta-actin expression and converted into fold change. °p < 0.05 vs A.

Figure 4

Met expression in kidney of control and allografted rats on day 7. Groups are defined as in Figure 8. Upper panel: Met expression in representative renal sections of control and allografted rats on day 7. Panel A: normal kidney, Panel B: untreated allograft, Panel C: MSC treated allograft (Magnification X200). Middle panel: Bars represent the medians of Met positive tubules number/HPF in all groups of rats, boxes represent the 25-75% percentile, whiskers: min to max. °p < 0.001 vs B, ^§ p < 0.005 vs A, * p < 0.005 vs C. Bottom panel: Western blot performed with anti-Met antibody in renal tissue of all groups of rats. The p170-kDa band represents pro-Met, the p140-kDa band represents mature Met β subunit.

Figure 5

MSP and RON expression in kidney of control and allografted rats on day 7. Groups are defined as in Figure 8. Panel a: MSP expression in representative renal section of normal kidney, note the strong cytoplasmic pattern of staining, panel b: MSP expression in representative renal section of untreated allograft, note the almost complete absence of MSP expression, panel c: MSP expression in representative renal section of MSC treated allograft, note the luminal pattern of MSP staining (Magnification X200). Panel d: RON expression in representative renal sections of normal kidney, panel e: RON expression in representative renal sections of untreated allograft, note the presence of RON positive inflammatory cells and the significant reduction of RON tubular staining, panel f: RON expression in representative renal sections of MSC treated allograft, note the reappearance of RON tubular staining and absence of RON positive inflammatory cells (Magnification X200). Panel g: Bars represent the medians of MSP positive tubules number/HPF in all groups of rats; Boxes represent 25-75% percentile of MSP positive tubules, whiskers: min to max) i: °p < 0.005 vs B; *p < 0.05 vs C, ^§ p < 0.01 vs A. Panel h: Bars represent the medians of RON positive tubule number/HPF in all groups, boxes represent the 25-75% percentile, whiskers: min to max. *p < 0.01 vs C, °p < 0.005 vs B and C.

Figure 6

MSP mRNA expression of control and allografted rats. Groups are defined as in Figure 8. MSP mRNA expression in renal tissue analyzed by RT PCR in all groups of rats. Columns indicate MSP mRNA expression normalized to the beta-actin expression and converted into fold change. *p < 0.05 vs A and C.

Figure 7

Semiquantitative MSPmRNA expression in PBMC, MSC in basal condition evaluated by reverse transcription (RT)–PCR that used 1 mg of RNA as the template. PBMC known for their capacity to produce MSP were used as controls. Columns indicate MSP mRNA expression normalized to the beta-actin expression and converted into fold change. *p < 0.001 vs.

Figure 8

Experimental design. We studied a kidney transplant allogeneic model (Fisher to Lewis, 20 rats). 10 allografted rats (group B) were infused into the renal artery soon after graft reperfusion only with saline. 10 allografted rats (group C) were infused into the renal artery with 3 × 10⁶MSC in 1 ml of PBS soon after graft reperfusion. Bilateral nephrectomy was performed in all rats soon before transplantation. 5 healthy rats (Lewis) were the control group (group A). No immunosuppressive therapy was administered. Blood was drawn from the caudal vein at day 0 (the day of transplantation, before the surgery), and at day 7. All the rats were sacrificed at day 7. A: healthy rats. B: allografted rats treated with saline, C: allografted rats treated with MSC.