Introduction

Acute kidney injury (AKI) is one of the most important causes of mortality and morbidity worldwide. In clinical practice, kidney ischemia–reperfusion (I/R) is the most common cause of AKI. Limitations in the treatment have led to a search for better therapeutic options. Mesenchymal stem cell (MSC)-based therapy holds great promise for treating immune disorders and for regenerative medicine, and promising results have been reported for the application of different types of stem cells in the treatment of kidney failure [1–5, 19–21]. In addition, emerging evidence indicates that AKI in humans is closely associated with chronic kidney disease if the repair process is maladaptive [22, 23]. However, the therapeutic options are limited.

Bone marrow stem cells are an attractive therapy to promote renal tissue regeneration due to their pluripotency and ease of isolation. Using these cells also avoids the ethical ambiguities of using embryonic stem cells [4, 15, 24, 25]. Our previous studies also demonstrated that hematopoietic stem cells recruited to injured kidneys generate high levels of proangiogenic cytokines, including VEGF-A [8]. This result increased our interest in determining whether CM had beneficial effects on kidney repair.

In the present study, we obtained MSCs using typical methods and cultured these cells for four passages before use in our experiments. Light microscopy showed that these cells had typical spindle-shaped morphology and were well labeled with CMFDA. Additionally, we demonstrated that MSCs that were systemically infused 24 hours after kidney injury were selectively recruited to injured kidneys. This recruitment was associated with enhanced repair of the microvasculature and tubules, improved kidney function, increased survival, promoted the proliferation of parenchymal cells, and decreased CD68-positive macrophage infiltration and apoptotic cells. In contrast, systemic CM treatment did not have any significantly beneficial effects, even though the CM contained high levels of proangiogenic cytokines, including HGF, VEGF-A and IGF-1.

Acute ischemic injury in the kidneys primarily results in proximal tubular damage [6, 26, 27]. However, data derived from several severe AKI models and the long-term effects of ischemic injury demonstrate that capillary loss typically precedes the development of prominent renal fibrosis, the loss of capillary density and blood flow may result in poor delivery of oxygen and nutrients to the damaged area, and neoangiogenesis may be a central process in the preservation of the vascular structure and the restoration of organ function [28–31]. In this study, we demonstrated that there was a marked loss of peritubular capillaries in the injured kidneys, and that the intravenous infusion of MSCs attenuated the loss of peritubular capillaries and tubular injury and promoted cell proliferation in the kidney. These effects were associated with both the rapid recovery of kidney function and the enhanced survival of the mice.

The critical property of stem cells is that they are able to generate many or all differentiated cell types [32, 33]. Initial studies reported that bone-marrow derived stem cells can differentiate into endothelial and mesangial cells in animal models [34–36], but the number of differentiated cells was small. Recently, it was found that MSCs can produce many growth factors, suggesting that a paracrine/endocrine effect might contribute to renal protection [2, 4, 12]. Gharaibeh and colleagues have shown that the terminal differentiation capacity of implanted stem cells is not the major determinant of the cells’ regenerative potential and that the paracrine effect imparted by the transplanted cells plays a greater role in the regeneration process [37]. Zarjou and colleagues have further shown that heme oxygenase-1 enhances secretion of stromal cell-derived factor-1, VEGF-A and HGF by MSCs [38]. Many findings support a protective effect mediated in an endocrine manner, which, if true, would mean that injection of the cells themselves would not be required, and the factors that these cells secrete could be effective. The effect of CM, however, remains controversial for the moment [12, 39]. In this study we also determined the levels of HGF, VEGF-A and IGF-1, and the data showed that CM contained these factors, which have renoprotective effects after AKI. Based on these results, we hypothesized that administering the CM would protect against kidney failure, making it unnecessary to transplant stem cells and thus avoiding the risks of tumorigenesis and immunologic reactions. However, we did not observe any favorable effects in the CM group on renal function, histological alterations or cell proliferation and anti-inflammatory and anti-apoptotic effects, even though we increased the dose and repeated consecutive administration of CM. There are several possible explanations for these findings. First, the AKI injury models were induced by different methods, and we believe that the outcomes should be compared within a unique and identical model and cannot be meaningfully transposed from one model to another. Second, the microenvironment has very important effects on the production of growth factors by MSCs. Different microenvironments can stimulate stem cells to release different types and concentrations of cytokines. MSCs might secrete another set of mediators in the culture system [12]. If we want stem cells to have the same effects in vitro and in vivo, we must mimic the injury microenvironment in the culture system. In the I/R model, the loss of blood flow results in hypoxia in the tissue, and the bone marrow is also hypoxic [40, 41]. We therefore believe that the MSCs should be exposed to hypoxic conditions to mimic the in vivo environment. Some authors have performed these types of experiments [42–44]. Third, the timing of therapeutic cell delivery may be critical. Cellular populations within wounds change depending on the phases of the repair process. This change means that therapeutic cells will encounter different microenvironments at each stage of the repair process [45].

In contrast with our data, Bi and colleagues reported that administration of MSC CM was very potent in ameliorating cisplatin-induced kidney failure [12]. Comparing these two studies, there are some differences. First, the medium was harvested after 96 hours as CM but in our study was harvested after 48 hours. Second, Bi and colleagues infused 1000 μl CM twice per day for 6 days by intraperitoneal injection, and we injected 200 μl or 500 μl CM intravenously through the tail vein once per day for 7 days. Third, they gave an intraperitoneal injection of cisplatin to induce acute tubular injury, but we placed a nontraumatic microaneurysm clamp across the renal artery and vein to induce kidney I/R injury. Fourth, different mouse strains were used in these two studies (C57BI/6 compared with BALB/C). We consider that these differences account for the discrepancies in the findings at least in part. We believe the that therapeutic strategy for treatment of kidney disease with CM remains an open question, and further studies with different designs, animal models and evaluation methods are certainly required.

Conclusions

We demonstrate that systematically administered MSCs promote rapid kidney repair and reduce mortality. Our data supporting the fact that the beneficial effect seen with MSCs is probably due to the stem cells’ multipotent capacity include increased secretion of paracrine factors, improved angiogenic and anti-inflammatory activities and anti-apoptotic effects. The results of this study indicate that the MSC infusion is a promising therapeutic strategy for AKI. In the present study, we do not detect any beneficial role of CM in our animal model, indicating that MSCs play central roles in kidney repair through paracrine rather than endocrine mechanisms. We believe that considerable work with different designs and animals is still required.