Mesenchymal stem cells transplantation ameliorates glomerular injury in streptozotocin-induced diabetic nephropathy in rats via inhibiting macrophage infiltration

https://doi.org/10.1016/j.intimp.2013.05.031Get rights and content

Highlights

  • We investigated the effect of MSC on early stage of DN and discussed the mechanism.

  • MSC dramatically ameliorated blood glucose, renal function and glomerulosclerosis.

  • MSC dramatically reduced MCP-1 expression and macrophages infiltration.

  • MSC significantly reduced the expression of IL-1β, IL-6 and TNFα.

Abstract

Mesenchymal stem cells (MSCs) treatment has been shown to be effective in diabetic nephropathy (DN). However, the mechanisms involved in the renoprotective effects of MSCs have not been clearly demonstrated. Especially, there was no study on the relationship of MSCs and macrophages in diabetic kidney. To explore the effect of MSCs on macrophages in DN, streptozotocin-induced diabetes animals received no treatment or treatment with MSCs (2 × 106, via tail vein) for two continuous weeks. Eight weeks after treatment, physical, biochemical and morphological parameters were measured. Immunohistochemistry for fibronectin (FN), CollagenI, ED-1, monocyte chemoattractant protein-1 (MCP-1) was performed. Expressions of pro-inflammatory cytokines and hepatocyte growth factor (HGF) at gene level and protein level were determined by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Blood glucose, urinary albumin excretion, creatinine clearance were significantly reduced after MSCs treatment. The glomerulosclerosis as revealed by periodic acid Schiff stain and expression of FN and CollagenI was also dramatically attenuated. Most importantly, the expression of MCP-1 and the number of infiltrated macrophages in kidney were effectively suppressed by MSCs treatment. The expression of HGF in MSCs group was up-regulated. Meanwhile, the expressions of IL-1β, IL-6 and TNFα were significantly down-regulated by MSCs treatment.

Our study suggest that MSCs treatment ameliorates DN via inhibition of MCP-1 expression by secreting HGF, thus reducing macrophages infiltration, down-regulating IL-1β, IL-6, TNFα expression in renal tissue in diabetic rats.

Introduction

Diabetic nephropathy (DN) is the most common cause of chronic kidney disease, with a significant increase in morbidity and mortality in patients with diabetes [1], [2], [3], [4]. Currently available treatments involving strict glycemic and/or blood pressure control can delay, but not eliminate the occurrence and development of DN. Therefore, it is necessary to identify new strategies that could specifically target on DN.

Recent studies have shown that chronic inflammation plays critical roles in promoting the development and progression of DN [5], [6], [7]. Macrophages are key inflammatory cells mediating kidney inflammation in DN and other types of renal disease. It has been found that macrophages accumulation is substantially increased in kidney tissue and associated with the progression of renal injury and a decline in renal function in experimental and human diabetes [8], [9], [10]. More important, several experimental studies have shown that different therapeutic strategies which can inhibit macrophage infiltration directly or indirectly can prevent the development or ameliorate renal injury in diabetes [11], [12], [13]. All these indicate that macrophage-mediated renal injury plays an important role in the development of DN.

An emerging body of studies have found that mesenchymal stem cells (MSCs) may play specific roles as immunomodulators in a wide array of disease [14], [15], [16], [17], [18], [19] due to their immunomodulatory properties including suppression of T cell proliferation [20], influencing dendritic cell (DC) maturation and function [21], [22], supression of B cell proliferation and terminal differentiation [23], etc. Recently, there is increasing evidence to suggest that the immunomodulatory effects of MSCs on macrophages is also a critical mechanism in the amelioration of inflammation-related disease, such as wounding healing, sepsis, acute myocardial infarction and renal ischemia reperfusion injury [24], [25], [26], [27]. However, the interactions between MSCs and macrophages in the process of DN are poorly understood.

Based on these findings, we hypothesized that MSCs may be effective on macrophage infiltration in kidney tissue of DN. In present study, we tested our strategy by transplanting bone marrow-derived MSCs to streptozotocin (STZ)-induced diabetic rats to explore the effect of MSCs on monocyte chemoattractant protein-1 (MCP-1) expression, macrophages infiltration and pro-inflammatory cytokines expression including IL-1β, IL-6 and TNFαin diabetic kidney.

Section snippets

Animals

Female Wistar rats weighing 200–250 g were purchased from animal experimental center of Shandong University, China. They were given free access to food and water throughout the study. Animal studies were carried out in accordance with the guidelines of the Animal Ethics Committee of the Shandong University, which are consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Diabetes model establishment

Diabetes was induced in the female Wistar rats by a single intraperitoneal

Characterization of rat MSCs

Bone marrow-derived MSCs from Wistar rats were adherent cultured with a typical fibroblast-like morphology (Fig. 1-A). They were able to differentiate into adipogenic and osteogenic cells (Fig. 1-B, C). Flow cytometric analysis showed that MSCs were positive for CD29 (99.56%), CD90 (96.26%), CD44 (88.76%), and negative for CD34 (3.80%), CD45 (1.99%), CD11b (19.03%).

MSCs tracking

MSCs transducted with GFP by lentiviral vector at the MOI of 20 resulted in a high transduction efficiency, more than 80% MSCs

Discussion

There was no doubt that MSCs played an active role in the treatment of DN, as revealed by amelioration of biochemical, physical parameters and glomerular fibrosis. In the current study, we intended to explore the mechanisms that mediate the therapeutic effects of MSCs on DN.

The diabetic animal model induced by a single intraperitoneal injection of 60 mg/kg STZ is well documented to produce hyperglycemia and insulinitis similar to human counterparts [32]. It has also been reported that 8 weeks

Conflict of interest statement

There is no conflict of interest to report.

Financial disclosure

This study was supported by the Innovation Foundation of Shandong University (No. 2009JC022).

Acknowledgements

We thank the laboratory animal center of the Second Hospital of Shandong University for the excellent rat care. We also thank Central Laboratory of the Second Hospital of Shandong University for providing the excellent experimental equipment.

References (60)

  • R. Meisel et al.

    Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2, 3-dioxygenase–mediated tryptophan degradation

    Blood

    (2004)
  • T. Morii et al.

    Association of monocyte chemoattractant protein-1 with renal tubular damage in diabetic nephropathy

    J Diabetes Complications

    (2003)
  • N. Banba et al.

    Possible relationship of monocyte chemoattractant protein-1 with diabetic nephropathy

    Kidney Int

    (2000)
  • T. Wada et al.

    Up-regulation of monocyte chemoattractant protein-1 in tubulointerstitial lesions of human diabetic nephropathy

    Kidney Int

    (2000)
  • F. Chow et al.

    Monocyte chemoattractant protein-1 promotes the development of diabetic renal injury in streptozotocin-treated mice

    Kidney Int

    (2006)
  • M. Giannopoulou et al.

    Hepatocyte growth factor exerts its anti-inflammatory action by disrupting nuclear factor-κB signaling

    Am J Pathol

    (2008)
  • H.R. Brady

    Leukocyte adhesion molecules and kidney diseases

    Kidney Int

    (1994)
  • D.L. Coleman et al.

    Interleukin-6: an autocrine regulator of mesangial cell growth

    Kidney Int

    (1992)
  • N. Koike et al.

    Induction of reactive oxygen species from isolated rat glomeruli by protein kinase C activation and TNF-α stimulation, and effects of a phosphodiesterase inhibitor

    Life Sci

    (2007)
  • A.J. Collins et al.

    US renal data system 2012 annual data report

    Am J Kidney Dis

    (2013)
  • A.J. Collins et al.

    The state of chronic kidney disease, ESRD, and morbidity and mortality in the first year of dialysis

    Clin J Am Soc Nephrol

    (2009)
  • J. Coresh et al.

    Chronic kidney disease awareness, prevalence, and trends among US adults, 1999 to 2000

    J Am Soc Nephrol

    (2005)
  • R.A. Wolfe et al.

    Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant

    N Engl J Med

    (1999)
  • J. Wada et al.

    Inflammation and the pathogenesis of diabetic nephropathy

    Clin Sci

    (2013)
  • A. Rivero et al.

    Pathogenic perspectives for the role of inflammation in diabetic nephropathy

    Clin Sci

    (2009)
  • J.F. Navarro-González et al.

    The role of inflammatory cytokines in diabetic nephropathy

    J Am Soc Nephrol

    (2008)
  • D. Nguyen et al.

    Macrophage accumulation in human progressive diabetic nephropathy

    Nephrology

    (2006)
  • F.Y. Chow et al.

    Macrophages in streptozotocin-induced diabetic nephropathy: potential role in renal fibrosis

    Nephrol Dial Transplant

    (2004)
  • A. Lim et al.

    Antibody blockade of c-fms suppresses the progression of inflammation and injury in early diabetic nephropathy in obese db/db mice

    Diabetologia

    (2009)
  • F.Y. Chow et al.

    Intercellular adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic db/db mice

    J Am Soc Nephrol

    (2005)
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