Preconditioning strategies for improving the survival rate and paracrine ability of mesenchymal stem cells in acute kidney injury

Abstract Acute kidney injury (AKI) is a common, severe emergency case in clinics, with high incidence, significant mortality and increased costs. Despite development in the understanding of its pathophysiology, the therapeutic choices are still confined to dialysis and renal transplantation. Considering their antiapoptotic, immunomodulatory, antioxidative and pro‐angiogenic effects, mesenchymal stem cells (MSCs) may be a promising candidate for AKI management. Based on these findings, some clinical trials have been performed, but the results are contradictory (NCT00733876, NCT01602328). The low engraftment, poor survival rate, impaired paracrine ability and delayed administration of MSCs are the four main reasons for the limited clinical efficacy. Investigators have developed a series of preconditioning strategies to improve MSC survival rates and paracrine ability. In this review, by summarizing these encouraging studies, we intend to provide a comprehensive understanding of various preconditioning strategies on AKI therapy and improve the prognosis of AKI patients by regenerative medicine.

tubular epithelial cell repair. 5 Among a variety of stem cells, mesenchymal stem cells (MSCs) have emerged as the most promising candidates for AKI therapy given their low immunogenicity, high multipotential differentiation ability, invasiveness of isolation and abundant distribution. [6][7][8] Despite the encouraging results of MSCs usage in animal models, a huge gap exists between scientific observation and clinical application. In 2017, Swaminathan et al provided a phase 2, randomized, double-blind, multicenter trial on the use of MSCs in patients with post-cardiac surgical AKI (NCT01602328). 9 After randomizing 156 adult subjects, they found that time to renal function recovery, need for dialysis, and 30-day all-cause mortality were all compatible between the two groups.
What makes MSCs lose their magic power clinically? There is growing evidence that the regenerative effect of MSCs might be mediated predominantly by paracrine action, rather than direct differentiation into target cells. [10][11][12][13] Once injected into an injured tissue, MSCs face a harsh environment, including reactive oxygen species (ROS) and anoikis, which are largely generated after AKI that may promote MSC apoptosis. [14][15][16] It is reported that more than 80%-90% of grafted cells will die within the first week after injection, 17 and the remaining 9%-19% cells may be trapped in liver, lungs and spleen. 18 Impaired MSC potency/biological activity in vivo was also reported. Silva et al concluded in their article that the limited clinical efficacy of MSCs might result from the low amount of engraftment, poor survival rate, impaired paracrine ability and delayed administration 19 (Figure 1).
To overcome this obstacle, some approaches to improve the ability of grafted MSCs have been explored in recent years. Investigators try to increase the number of injected cells, but it may be risky due to disturbance in blood flow causing embolism problems. 20 Others attempt to inject cells directly into the damaged tissue, but the invasive procedures include a high risk of haemorrhage, and the number of injected MSCs is also inaccurate, as most of the cells may escape from the injected site. 21,22 Preconditioning is a promising strategy for optimizing MSCs before their transplantation. Based on the way MSCs operate, these strategies are designed to increase the survival rate of MSCs in injured tissues, enhance their paracrine ability or help them migrate to the target tissue ( Figure 2). Previously, we have discussed those preconditioning strategies for enhancing the migratory ability of MSCs in AKI. 23 In this review, we focus on summarizing the different preconditioning strategies for increasing the MSCs survival rate or paracrine ability in AKI models. Only articles that demonstrated a clear mechanism are included in our review. We look forward a bright future in which the preconditioning strategy can be used to increase the function of MSCs and, consequently, to achieve long-term benefits of MSCs therapies in AKI patients.

SURVIVAL RATES
The low survival rate of transplanted MSCs remains an important limitation for MSC therapy. 17,24 Anoikis, ischemia, inflammation and imbalance between ROS and antioxidant are likely the major causes of cell death following transplantation. [25][26][27] Some preconditioning strategies have been proven to protect MSCs from harmful environments. These strategies include incubation with cytokines or chemical compounds, improvement of culture condition, thermosensitive hydrogel and genetic modification (Table 1).

| Incubation with cytokines or chemical compounds
Various cytokines or chemical compounds have been proven to have cell protective effects, and part of the mechanism is through the PI3K/AKT signalling pathway. AKT activation can promote cell survival, proliferation, growth and changes in cellular metabolic pathways through its numerous downstream targets. 28 27 Similarly, preconditioning with muscone, the main active ingredient of musk, also enhanced the proliferative ability of bone marrow-derived mesenchymal stem cells (BMSCs) to some degree in rats with gentamicin-induced AKI. 32 Lastly, many studies have confirmed preconditioning with insulin-like growth factor-1 (IGF-1) may enhance MSC proliferation with lower apoptosis in many other organ failure models. 33

| Thermosensitive hydrogel
After transplantation, MSCs face a harsh environment. Anoikis is very common due to the loss of anchorage-dependent attachment to the ECM. 40,41 Approaches were then explored for mimicking a cellular microenvironment more consistent with that found in vivo.
Thermosensitive hydrogel could be an excellent method for improving the microenvironment as well as enhancing the survival rate of transplanted cells. 42

| Genetic modification
As discussed above, the imbalance between ROS and antioxidants in the AKI microenvironment was regarded as the main reason for poor cell survival rate. Genetic modification to make MSCs overexpress cytokine genes or antiapoptotic genes significantly improved their survival rate in injured tissues. 46,47 Heme oxygenase-1 (HO-1), a stress-inducible enzyme that can catalyze the pro-oxidant heme into biliverdin, CO and free-iron, exerted powerful antioxidant effects. 48 pro-angiogenic activities. 65,66 Similar to the preconditioning strategies mentioned above, some methods have also been explored to enhance MSC paracrine ability after transplantation (Table 2).

| Incubation with cytokines or chemical compounds
In addition to the protective effect mentioned above, incubation with cytokines or chemical compounds also enhanced the MSC paracrine effect. Treatment of MSCs with 14S,21R-diHDHA promoted secretion of hepatocyte growth factor (HGF) and IGF-1. 29 The expression of IGF-1 and VEGF genes also revealed many fold increases after preconditioning with SNAP. 30

| Genetic modification
Compared with other preconditioning strategies, genetic modification is a more accurate way to enhance MSC paracrine ability. Overexpression of chemokine (C-X-C motif) receptor 4 (CXCR4) in BMSCs, using lentivirus vector, resulted in higher levels of BMP-7, HGF and IL-10. Significantly improved renal function, reduced ATN scoring, more PCNA tubular cells and fewer TUNEL tubular cells were also observed using in vivo studies. 76 Lipocalin 2 (Lcn2) was thought to be a cytoprotective factor against AKI due to its important role in regeneration and proliferation of tubular epithelial cells. 77  showed that these effects could be partially abolished by IKK XII (NF-κB inhibitor), indicating the role of NF-κB in regulating cell viability and migration. 89 These studies confirmed that the activation of NF-κB might be involved in the cytoprotective, migratory and paracrine processes of MSCs. In contrast, decreased NF-κB activity was observed in Rap1-/-BMSCs, which displayed more resistance to apoptosis and presented better cardioprotective effects in myocardial infarction mice than wild-type BMSCs. 90