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. 2018 May;41(5):2629-2639.
doi: 10.3892/ijmm.2018.3501. Epub 2018 Feb 16.

Early intervention with mesenchymal stem cells prevents nephropathy in diabetic rats by ameliorating the inflammatory microenvironment

Yuanmin Li  1 , Jingping Liu  1 , Guangneng Liao  1 , Jie Zhang  1 , Younan Chen  1 , Lan Li  1 , Li Li  1 , Fang Liu  2 , Bo Chen  1 , Gang Guo  1 , Chengshi Wang  1 , Lichuan Yang  2 , Jingqiu Cheng  1 , Yangrong Lu  1
Affiliations

Early intervention with mesenchymal stem cells prevents nephropathy in diabetic rats by ameliorating the inflammatory microenvironment

Yuanmin Li et al. Int J Mol Med. 2018 May.

Abstract

Diabetic nephropathy (DN) is a major complication of diabetes and represents the leading cause of end-stage renal disease. Mesenchymal stem cell (MSC) treatment has been demonstrated to be effective in DN models by reducing albuminuria and attenuating glomerular injury; however, limited in-depth understanding of the underlying mechanism and a lack of clinical trials hinders its clinical use. Additionally, most of these experimental studies were conducted on the advanced stage of nephropathy, which is difficult to reverse and consequently showed limited therapeutic efficacy. We sought to evaluate whether early intervention by MSCs has the potential to prevent DN onset and progression as well as protect kidney function when intravenously administered to rats with diabetes. Diabetes was induced in adult male SD rats by streptozotocin (STZ) injection (55 mg/kg, i.p.). The diabetic rats were injected with or without bone marrow-derived MSCs (5x106 per rat), via tail vein at 2, 4, 5 and 7 weeks after diabetes onset. Fasting blood glucose (FBG), blood urea nitrogen (BUN) and serum creatinine (Scr) levels in serum samples and glycosuria (GLU), microalbumin (MAU), and albumin to creatinine ratio (ACR) in urine samples were determined. Renal pathology and immunohistochemistry (IHC) for CD68, MCP-1, fibronectin (FN), transforming growth factor-β (TGF-β) and pro-inflammatory cytokines were also performed. Expression levels of the above factors as well as interleukin-10 (IL-10), and epidermal growth factor (EGF) were assessed by qPCR and multiplex bead-based suspension array system, respectively. Additionally, MSC tracing in vivo was performed. Ex vivo, peritoneal macrophages were co-cultured with MSCs, and expression of inflammatory cytokines was detected as well. MSC treatment profoundly suppressed renal macrophage infiltration and inflammatory cytokine secretion in diabetic rats, resulting in prominently improved kidney histology, systemic homeostasis, and animal survival, although no significant effect on hyperglycemia was observed. Engrafted MSCs were primarily localized in deteriorated areas of the kidney and immune organs 48 h after infusion. MSC treatment upregulated serum anti-inflammatory cytokines IL-10 and EGF. Ex vivo, MSCs inhibited lipopolysaccharide (LPS)-stimulated rat peritoneal macrophage activation via the downregulation of inflammatory-related cytokines such as IL-6, MCP-1, tumor necrosis factor-α (TNF-α) and IL-1β. Our results demonstrated that early intervention with MSCs prevented renal injury via immune regulation in diabetic rats, which restored the homeostasis of the immune microenvironment, contributing to the prevention of kidney dysfunction and glomerulosclerosis.

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Conflict of interest statement

Competing interests

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Experimental design.
Figure 2
Figure 2
Assessment of renal injury in diabetic rats. Diabetic rats showed a significant increase in fasting blood glucose (FBG), serum creatinine (Scr), blood urea nitrogen (BUN) (A) as well as glycosuria (GLU), microalbumin (MAU), albumin to creatinine ratio (ACR) and urine volume (UV) (B) at 4 weeks after streptozotocin (STZ) injection. Hematoxylin and eosin (H&E) and periodicacid-Schiff (PAS)-stained sections of renal cortices at 8 weeks (C) after diabetes onset showed obvious morphologic lesions in kidney of diabetic rats, including glomerular hypertrophy, increased fractional mesangial area and tubular dilatation [(C) magnification, ×400] [*p<0.05 vs. normal group (NC)].
Figure 3
Figure 3
Characterization of bone marrow-derived mesenchymal stem cells (MSCs) isolated from SD rats. Plastic adherent cells were ex vivo expanded [(A) magnification, ×200] and differentiated into adipocytes after 14 days of culture or osteocytes after 21 days of culture [(B) magnification, ×400]. Flow cytometry showed that MSCs at passage 3 were positive for CD29 (99.9%), CD44 (99%), and negative for CD34 (0.1%) and CD45 (0.7%) (C). MSCs were ex vivo labeled with CM-Dil [(D) magnification, ×200] and showed high labeling efficiency of almost 100%. MSCs cultured with EdU ex vivo for 2 h showed >60% positive staining indicating a high proliferative activity [(E) magnification, ×200].
Figure 4
Figure 4
In vivo tracking of engrafted mesenchymal stem cells (MSCs) at 24 and 48 h. Representative images of kidney (Kid.), spleen (Spl.) and thymus (Thy.) sections from diabetic rats injected with 5×106 of CM-DiI labeled MSCs at 24 and 48 h after cell infusion. MSCs labeled with CM-DiI showed red fluorescence (arrow), and nuclei were stained by DAPI with blue fluorescence. Magnification, ×400.
Figure 5
Figure 5
Effect of mesenchymal stem cells (MSCs) on survival rate, body weight and renal function. (A) Ten-week survival rates of the 3 groups: NC group, 100%; diabetic nephropathy (DN) group, 35.7%; DN-MSC group, 81.8% (*p=0.01). Data were analyzed by log-rank test. Renal function: (B) microalbumin (MAU) (mg/l), (C) albumin to creatinine ratio (ACR) (µg/μmol) and (D) blood urea nitrogen (BUN) (mM) were measured in serum and urine in the 3 groups at 4, 5, 8 and 10 weeks after diabetes onset. Renal function was significantly improved in the MSC-treated group compared with that noted in the DN group. Data are expressed as means ± SD of evaluations from each group. [*p<0.05 vs. normal group (NC); #p<0.05 vs. DN group].
Figure 6
Figure 6
Effect of mesenchymal stem cells (MSCs) on renal histopathological changes and expression of fibronectin (FN) and transforming growth factor-β (TGF-β) at 10 weeks after diabetes onset. (A) Representative images of hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) and trichromestained sections of renal cortices from three groups of rats. Severe histologic changes were visible in the diabetic nephropathy (DN) group, including glomerular hypertrophy, increased fractional mesangial area and interstitial fibrosis. Recovery from most of the glomerular and tubular changes were observed in the MSC-treated group rats. (B) Immunohistochemical analyses of FN and TGF-β protein expression in kidney tissues of the three groups of rats. (C) Quantitative analyses of FN and TGF-β expression as measured by immunohistochemistry (IHC) (100% of normal control). Data are expressed as means ± SD of evaluations from each group [*p<0.05 vs. normal group (NC); #p<0.05 vs. DN group]. Magnification, ×400.
Figure 7
Figure 7
Effect of mesenchymal stem cells (MSCs) on renal macrophage infiltration and pro-inflammatory cytokine expression 10 weeks after diabetes onset. (A) Immunohistochemistry (IHC) analyses of ICAM-1, MCP-1 and CD68 protein expression in kidney tissues of the three groups of rats. (B) Quantitative analyses of ICAM-1, MCP-1 and CD68 expression as measured by IHC. The number of CD68+ macrophages was significantly suppressed after treatment with MSCs. (C) IHC analysis of TNF-α, interleukin-6 (IL-6) and IL-1β protein expression in kidney tissues of three groups of rats. (D) Quantitative analyses of TNF-α, IL-6 and IL-1β protein expressions as measured by IHC (100% of normal control). Data are expressed as means ± SD of evaluations from each group [*p<0.05 vs. normal group (NC); #p<0.05 vs. diabetic nephropathy (DN) group]. Magnification, ×400.
Figure. 8
Figure. 8
Effect of mesenchymal stem cells (MSCs) on pro-inflammatory, fibrotic cytokine and chemokine gene expression in kidney tissues. The mRNA expression of cytokines and fibronectin (FN) were significantly suppressed in the MSC group compared with the diabetic nephropathy (DN) group at 10 weeks after diabetes onset. All the data were normalized by control value (control was shown as 1), and are presented as means ± SD [*p<0.05 vs. normal group (NC); #p<0.05 vs. DN group].
Figure 9
Figure 9
Effect of mesenchymal stem cells (MSCs) on the serum inflammatory cytokines and growth factor expression. The levels of cytokines in the serum samples were determined by a Milliplex rat cytokine kit in the three groups of rats at 10 weeks after diabetes onset. Interleukin-1α (IL-1α), IL-1β, IL-6, interferon-γ (IFN-γ) expression lelvels were significantly suppressed in the MSC group, whereas epidermal growth factor (EGF) was increased compared with the diabetic nephropathy (DN) group. Data are expressed as means ± SD of evaluations from each group [*p<0.05 vs. normal group (NC); #p<0.05 vs. DN group].
Figure 10
Figure 10
Effect of mesenchymal stem cells (MSCs) on macrophage activation stimulated by lipopolysacharide (LPS). Primarily isolated rat peritoneal macrophages Mϕ (bright field) (A) were identified as being positive for (B) CD68 (63.9%) and F4/80 (51.2%) by flow cytometry. qPCR analyses of (C) interleukin-6 (IL-6), MCP-1, IL-1β, TNF-α and cellular immunofluorescence staining of IL-6 and MCP-1 [(D) magnification, ×200] showed that expression of inflammatory mediators secreted by LPS-stimulated macrophages were inhibited by co-culturing with MSCs at 6 h. Data are expressed as means ± SD of evaluations from each group (*p<0.05 vs. Mϕ; #p<0.05 vs. Mϕ+LPS; &p<0.05 vs. Mϕ+MSC; $p<0.05 vs. Mϕ+LPS+MSC).
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