Melatonin preconditioning is an effective strategy for mesenchymal stem cell‐based therapy for kidney disease

Abstract Based on multiple studies in animal models, mesenchymal stem cell (MSC)‐based therapy appears to be an innovative intervention approach with tremendous potential for the management of kidney disease. However, the clinical therapeutic effects of MSCs in either acute kidney injury (AKI) or chronic kidney disease (CKD) are still under debate. Hurdles originate from the harsh microenvironment in vivo that decreases the cell survival rate, paracrine activity and migratory capacity of MSCs after transplantation, which are believed to be the main reasons for their limited effects in clinical applications. Melatonin is traditionally regarded as a circadian rhythm‐regulated neurohormone but in recent years has been found to exhibit antioxidant and anti‐inflammatory properties. Because inflammation, oxidative stress, thermal injury, and hypoxia are abnormally activated in kidney disease, application of melatonin preconditioning to optimize the MSC response to the hostile in vivo microenvironment before transplantation is of great importance. In this review, we discuss current knowledge concerning the beneficial effects of melatonin preconditioning in MSC‐based therapy for kidney disease. By summarizing the available information and discussing the underlying mechanisms, we aim to improve the therapeutic effects of MSC‐based therapy for kidney disease and accelerate translation to clinical application.

care or dialysis, which helps little in regenerating the injured kidneys. 10 In CKD, although the application of renin-angiotensin system inhibitors (RASIs) has shown benefits in delaying renal failure, treatments are unable to induce regression of glomerulosclerosis. 11 The formation of fibrosis tissues in glomeruli and interstitial/tubules eventually leads to end-stage renal disease (ESRD) in both of these diseases. In addition to renal involvement, renal function disorder will undoubtedly affect extrarenal organs given that the kidneys are important in maintaining body homoeostasis. Pathophysiological alterations following loss of renal function, such as dysregulation of extracellular volume and electrolytes and abnormal hormone secretion, can subsequently affect multiple extrarenal organs and induce a series of severe complications. 12,13 It has been reported that even mild renal injury was relevant to the development of cardiovascular system complications. 14 The respiratory system, central nervous system, endocrine system and haematologic system, among others, may also be aggravated whether the abnormality is extensive or prolonged. 15 The high incidence and poor prognosis of kidney disease are associated with significant economic costs, accounting for more than $10 billion for the treatment of AKI and over $80 billion to care for CKD patients annually. 16,17 Exploring new interventions to delay the progression of AKI and CKD is an urgent need.
In the last 10 years, we have witnessed an explosion in MSCbased therapy for the management of AKI and CKD in multiple preclinical models. MSCs are fibroblast-like multipotent cells that possess robust self-renewal, regeneration, proliferation and multilineage differentiation ability. 18,19 Although the specific mechanisms underlying AKI and CKD are still not very clear, abnormalities in cell apoptosis or necrosis, 20 inflammation, 21,22 immunoregulation, 23 microvascular function, 24,25 oxidative stress injury 26 and the expansion of fibroblasts/myofibroblasts 11 are thought to play important roles during disease development. Numerous candidate agents targeting these abnormalities, such as Nec-1, 27 bindarit, 28 OPN-305, 29 eph-rinB2, 30 SS-31, 31 bardoxolone methyl 32 and pirfenidone, 33 have shown promising therapeutic activity in some animal and clinical kidney disease models. While pharmacologic management is often confined to a single aspect of the highly complex pathophysiological process in AKI or CKD, MSCs are able to promote kidney repair through multiple mechanisms. 34 A principal mechanism of these numerous MSC benefits resides in their paracrine/endocrine capacity. In general, it is considered that MSCs have the advantage of secreting a series of cytokines and growth factors that induce antiapoptotic, 35 antioxidative, 36 anti-inflammatory, 37 anti-fibrotic, 38 angiogenic 39 and immunomodulatory 40 activities (Figure 1). The development of MSC-based regenerative medicine may bring hope to the billions of patients who suffer from kidney disease worldwide.
In this review, we will describe the current knowledge concerning MSC-based therapy in kidney disease. We begin with the promising outcome of MSC-based therapy in animal models, and the obstacles met in attempting to translate these outcomes into clinical application. Then, the underlying reasons are analysed, and different preconditioning strategies are presented. Finally, we will discuss the beneficial effects of melatonin preconditioning on protecting MSC function after transplantation and the underlying mechanism. By summarizing the current research, we hope to provide an integral and updated view of melatonin preconditioning in MSC-based therapy for kidney disease.

| SUCCE SS FUL AT TEMP TS AT MSC-BA S ED THER APY IN ANIMAL MODEL S AND H URDLE S ME T IN CLINIC AL S E T TING S
The therapeutic effects of MSC-based therapy in kidney disease have been confirmed in multiple animal models. The first study that indicated the renotropic property and tubular regenerative potential of MSCs was conducted in a mouse model of cisplatin-induced AKI. 41 This phenomenon was further verified in many subsequent studies that utilized many other AKI models, such as ischemia/reperfusion (I/R), sepsis and glycerol models. 42 In the field of CKD, a metaanalysis that included 71 articles also demonstrated that cell-based therapy was valid for slowing the progression of CKD in preclinical settings. 43 Based on these excellent results, some clinical trials were explored.
Currently, there are 18 completed or ongoing clinical trials associated with the application of MSCs in kidney disease according to ClinicalTrials.gov ( Table 1). The first clinical trial in which the safety and efficacy of MSC therapy for AKI was evaluated was completed in 2013 (NCT00733876). MSCs were prophylactically transplanted into patients who were at high risk of developing AKI following cardiac surgery. Neither AKI nor any other adverse events occurred in the treatment group, while 20% of patients in the case-controlled group developed AKI. 44 Six autosomal dominant polycystic kidney disease (ADPKD) patients also presented tolerability to MSC therapy (NCT02166489). Moreover, a slowdown in the decline in the estimated glomerular filtration rate (eGFR) was also observed after MSC F I G U R E 1 The principal mechanism by which MSCs exert their potential beneficial effects in kidney disease. The beneficial effects of MSC-based therapy in kidney disease are reliant on the antiapoptotic, antioxidative, anti-inflammatory, anti-fibrotic, angiogenic and immunomodulatory properties of MSCs transplantation, suggesting the efficacy of this therapy. However, this trial was limited due to the lack of a control group. 45   In addition to the above-mentioned exciting results, contradictory results have been reported in other studies. A phase II, randomized, multicenter trial was terminated due to the uncertain therapeutic effects in patients with postcardiac surgical AKI (NCT01602328). 49 Packham et al aimed to assess the safety, tolerability and therapeutic effects of mesenchymal precursor cells in patients with moderate to severe diabetic nephropathy, which is a major cause of CKD. After follow-up for 12 weeks, the measured parameters, including serum creatinine, creatinine clearance, albumin-creatinine ratio, protein-creatinine ratio, HbA1c and blood pressure, were comparable in both groups (NCT01843387). 50 In the aspect of lupus nephritis (LN), the two studies mentioned above were both observational studies, which might provide insufficiently strong evidence. In a randomized double-blind, placebo-controlled trial that was published in 2017, MSC therapy did not show better therapeutic effects than the placebo (NCT01539902). 51

| MEL ATONIN PRECONDITIONING AND MSC-BA S ED THER APY FOR K IDNE Y DISE A SE
Multiple preconditioning strategies have been developed in recent years. Generally, these strategies can be sorted into four categories: incubation with cytokines or chemical compounds, hypoxia preconditioning, application of supporting materials and genetic modification. Different preconditioning strategies have unique advantages and drawbacks (Figure 2). Hypoxia preconditioning is simple and fast but faces the issues of standardization and optimization. 61 Genetic modification is a more accurate approach compared with other preconditioning strategies but presents a risk of vector toxicity and tumorigenicity. 62 The advantages of application with supporting materials are greater biocompatibility and targeting; however, the current price of these newly developed materials is still high, which may restrict their application in the clinic. 63 In addition to the above-mentioned methods, incubation of MSCs with cytokines or chemical compounds is another preconditioning strategy that has long been explored. The simple operation and safety guarantee of this technology make it a promising preconditioning strategy for clinical application, especially when the cells are incubated with certain physiological hormones. 52 However, the key challenge of this technique is to find a proper substrate.
F I G U R E 2 Advantages and drawbacks of different preconditioning strategies. Incubation with cytokines or chemical compounds, hypoxia preconditioning, application of supporting materials and genetic modification are currently the four main preconditioning strategies. These four preconditioning strategies have unique advantages and drawbacks. Currently, it is difficult to say one strategy is greater than another Melatonin is a neurohormone that is primarily secreted by the pineal gland. The physiological role of melatonin is a key regulatory molecule in circadian rhythms. 64 A low blood level of melatonin during the daytime and an increased level at night-time guarantees a sleep-wake cycle in mammals. 65 In addition to its traditional role, in recent years, melatonin has been found to take part in many other pathophysiologic processes. By possessing antioxidant and anti-inflammatory properties, 66,67 melatonin was found to be a potent free radical scavenger and present protective effects in multiple dysfunctional organs, including the kidneys. 68,69 Immune function during the course of prostate cancer therapy has also been demonstrated. 70 Based on the fact that inflammation, oxidative stress, thermal injury and hypoxia are four main factors that cause the dysfunction of injected MSCs under disease conditions, preconditioning with melatonin may become a wonderful strategy. 71 In addition, melatonin is currently applied as a dietary complement and shows little risk of genetic mutation, tumorigenicity or other major side effects. 72,73 Incubation of MSCs with melatonin prior to transplantation has been confirmed to be able to induce an enhanced therapeutic outcome in multiple animal models, including myocardial infarction, cerebral ischemia and limb ischemia models. [74][75][76] In a broad sense, it was considered that melatonin itself could efficiently serve as an antioxidant and protect MSCs from oxidation injury by biologically eliminating free radicals. 66 In addition to receptor-independent pathways, the melatonin receptors MT1 and MT2 have also been found to be highly expressed on the surface of MSCs, indicating that melatonin may regulate the fate of MSCs in a receptor-dependent manner. 77,78 Moreover, enhanced PrP C -dependent mitochondrial function, 79 Erk1/2 overexpression 80 and up-regulated phosphorylation of AMPK pathway proteins were observed after melatonin preconditioning. 81 Melatonin preconditioning can rely on various mechanisms to protect injected MSCs against premature senescence or early apoptosis after transplantation and definitely exaggerate their therapeutic effects in diseased tissues ( Figure 3). However, whether these benefits still exist in kidney disease is unclear. In the following section, we will discuss this question (Table 2).

| Application of MSCs with melatonin preconditioning to treat AKI
Sepsis-induced AKI (sepsis-AKI) is a major subtype of AKI with high morbidity and mortality. 82 To assess whether MSCs treated with me- Furthermore, their study also demonstrated the superiority of conditioned medium from melatonin-pretreated AMSCs in enhancing the proliferative, migratory, prosurvival and antiapoptotic abilities of human HK-2 cells exposed to cisplatin. 84 To assess whether these protective In terms of mechanism, the benefits of melatonin include but are not limited to its antioxidative and anti-inflammation effects.