Mesenchymal Stem Cells Attenuate Cisplatin-Induced Nephrotoxicity in iNOS-Dependent Manner

Abstract

Mesenchymal stem cells (MSCs) are, due to their immunomodulatory characteristics, utilized in therapy of immune-mediated diseases. We used murine model of cisplatin nephrotoxicity to explore the effects of MSCs on immune cells involved in the pathogenesis of this disease. Intraperitoneal application of MSCs significantly attenuated cisplatin nephrotoxicity, decreased inflammatory cytokines TNF-α and IL-17, and increased anti-inflammatory IL-10, IL-6, nitric oxide (NO), and kynurenine in sera of cisplatin-treated mice. MSC treatment significantly attenuated influx of leukocytes, macrophages, dendritic cells (DCs), neutrophils, CD4+ T helper (Th), and CD8+ cytotoxic T lymphocytes (CTLs) in damaged kidneys and attenuated the capacity of renal-infiltrated DCs, CD4+ Th, and CD8+ CTLs to produce TNF-α and IL-17. Similar effects were observed after intraperitoneal injection of MSC-conditioned medium (MSC-CM) indicating that MSCs exert their beneficial effects in paracrine manner. Inhibition of inducible nitric oxide synthase (iNOS) in MSC-CM resulted with increased number of TNF-α-producing DCs and IL-17-producing CTLs, decreased number of IL-10-producing tolerogenic DCs and regulatory CD4+FoxP3+ T cells, and completely diminished renoprotective effects of MSC-CM. In conclusion, MSCs, in iNOS-dependent manner, attenuated inflammation in cisplatin nephrotoxicity by reducing the influx and capacity of immune cells, particularly DCs and T lymphocytes, to produce inflammatory cytokines.

Figure 1

MSCs attenuate cisplatin-induced acute kidney injury. (a) Blood urea nitrogen (BUN) and plasma creatinine levels are evaluated. (b) Histological scores (ranging between 0 and 4) were determinated and calculated on the percentage of tubules affected (0 ≤ 10%, 1 = 10–25%, 2 = 26–50%, 3 = 51–75%, and 4 ≥ 75%). (c) Representative H&E- and PAS-stained mouse kidney. H&E staining images of kidney tissue samples are shown at the same magnifications (×200). Concentration of (d) cytokines, (e) kynurenine, and (f) NO in mice sera. (g) IL-6 and TNF-α gene expression in mouse kidneys. Values are mean ± SEM; n = 10 mice/group. ^∗p < 0.05, ^∗∗p < 0.001.

Figure 2

MSCs significantly attenuate influx of immune cells and their capacity to produce nephrotoxic and inflammatory cytokines. Total number of (a) CD45+ leukocytes, (b) CD45+CD11b+ myeloid cells, CD45+F4/80+ macrophages, CD45+CD11c+ dendritic cells, CD45+CD11b+Ly6G+ neutrophils, CD45+CD4+ T helper cells, CD45+CD8+ cytotoxic T cells, (c) TNF-α+CD11c+ dendritic cells, (d) IFN-γ+CD4+ T helper cells, and IL-17+CD4+ T helper cells in cisplatin- and cisplatin + MSC-treated mice. (e) Total number and representative flow cytometry dot plots of IFN-γ- and IL-17-producing cytotoxic CD8+ T cells. Data presented as mean ± SEM; n = 10 mice/group. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 3

MSCs reduce cisplatin-induced nephrotoxicity via soluble factors. (a) Mice were euthanized 72 h after cisplatin administration, and blood urea nitrogen (BUN) and plasma creatinine levels are measured. (b) Histological examination was performed with H&E staining. (c) H&E and PAS staining images of representative kidney tissues are shown at the same magnifications (200x). Concentration of (d) cytokines, (e) kynurenine, and (f) NO in mouse serum. (g) Expression of IL-6 and TNF-α genes in mouse kidneys. (h) Total number of renal-infiltrated CD45+ leukocytes, CD45+CD11b+ myeloid cells, CD45+F4/80+ macrophages, CD45+CD11c+ dendritic cells, CD45+CD11b+Ly6G+ neutrophils, CD45+CD4+ T helper cells, and CD45+CD8+ cytotoxic T cells. Data presented as mean ± SEM; n = 10 mice/group. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 4

MSC-CM reduces influx of inflammatory DCs and CTLs in cisplatin-induced acute kidney injury and alters their cytokine profile. Total number of (a) IL-10-producing CD45+CD11c+ DCs, CD4+CD25+FoxP3+ T regulatory cells, (b) TNF-α+CD45+CD11c+ DCs, (c) IFN-γ- and IL-17-producing CD8+ CTL cells that infiltrated kidneys of the control and experimental animals. Representative flow cytometry dot plots are shown. Values are mean ± SEM; n = 10 mice/group. ^∗p < 0.05, ^∗∗p < 0.001.

Figure 5

MSCs attenuate cisplatin-induced acute kidney injury in iNOS-dependent manner. (a) Serum levels of BUN and creatinine. (b) Histological scores. (c) Representative H&E and PAS-stained mouse kidney (magnifications ×200). (d) Serum levels of cytokines. Total numbers of (e) TNF-α-producing CD45+CD11c+ dendritic cells and IL-17-producing CD8+ cytotoxic T cells, (f) IL-10-producing CD45+CD11c+ DCs and CD4+CD25+FoxP3+ T regulatory cells. Values are mean ± SEM; n = 10 mice/group. ^∗p < 0.05, ^∗∗p < 0.001.

Figure 6

MSC-derived NO is important for activation of IDO in TNF-α-stimulated MSCs. (a) Expression of IDO and iNOS in nonstimulated and TNF-α-stimulated MSCs. (b) Concentration of kynurenine in supernatants of nonstimulated MSCs, TNF-α-stimulated MSCs, and TNF-α-stimulated MSCs cultured in the presence of L-NMMA. ^∗p < 0.05. (c) Proposed mechanism of MSC-based immunomodulation of cisplatin-induced nephrotoxicity: After cisplatin-induced kidney injury, MSCs migrate in the kidneys as a response to the inflammatory cytokines and chemokines. Under inflammatory conditions, TNF-α provokes MSCs to express iNOS and to produce NO which, in turn, increases IDO activity and augment MSC-based immunomodulation resulting with attenuated number of inflammatory TNF-α-producing DCs and IFN-γ- and IL-17-producing T cells and increased number of immunosuppressive IL-10-producing DCs and regulatory T cells in injured kidneys.