Mesenchymal Stem Cells Ameliorate Acute Kidney Injury Through Activation Of M2Macrophages

Background Rhabdomyolysis is characterized by the leakage ofmuscle cell contents, including electrolytes, myoglobin, and othersarcoplasmic proteins into the circulation. Acute kidney injury(AKI)associated with myoglobinuria is the most serious complication of bothtraumatic and nontraumatic rhabdomyolysis, and it may belife-threatening. The mortality of AKI is still high since there isno effective therapy even if rigorous development has been promted.Therefore, new therapy strategies focusing on pathophysiology must beinvestigated. Stem cell therapy is becoming a more viable therapy forAKI. Several studies have shown that administration of bonemarrow-derived mesenchymal stem cells(MSCs) leads to the ameliorationof AKI, but the mechanisms are still puzzling. Macrophages are the keyregulators of the innate immune system, where they can detect,phagocytose and destroy foreign antigens. Apart from tissuedestruction, it is now known that macrophages also play an importantrole in tissue homeostasis, cellular replacement and repair throughthe clearance of apoptotic cells and cellular debris. They also producemediators that downregulate inflammation and promote remodelling andregeneration. It was showed that MSCs could induce macrophage both invivo and in vitro, which mediates MSCs protection in other experimentalinflammation related organ injury. However, whether macrophageinduction was also involved in MSCs therapy in rhabdomyolysis-inducedAKI needs to be clarified. In our study, we conducted rhabdomyolysis-induced AKI model to elucidate the potentialmechanisms of MSC therapy.Methods In the first part of our study, we subjected mice torhabdomyolysis and administered MSCs (106per mouse) via tail vein.We then (1) sent the serum of rhabdomyolysis mice to detect serumcreatinine (SCr), urea nitrogen (BUN), and creatine phospho kinase (CK)(n=10).(2) performed PAS staining to evaluate tubular injury and acutetubular necrosis (ATN) score (n=4).(3) checked out serum cytokinesof inflammation.(4) stained PCNA for proliferative cells.(5)appliedlaser confocal microscopy to detect RFP-MSCs distribution in AKI mice(n=3).In the second part of our study, to clarify whether induction ofM2macrophages was involved in MSCs therapy, we first performed immuno-fluorescence to determine macrophages (F4/80) and M2macrophages(CD206) in the tissues of kidney(n=5).Then, mice kidney homogenateswere separated on polyacrylamide-SDS gel and electroblotted ontonitrocellulose membrane. The membrane was incubated with antibodiesagainst mice mannose receptor (MR; CD206) and the signals werevisualized by enhanced chemiluminescence detection. We also depletedmacrophage after MSCs therapy, and detect the result of AKI.In the third part of our study, in order to elucidate therelationship between MSCs and macrophages, we coculture macrophageswith MSCs in the transwell for72hours. Then, macrophages were stainedwith FITC-CD206, followed by intracellular cytokine staining withPE-IL-10, and subjected to flow cytometry analysis. Above all,disparate phenotype including macrophages, macrophages stimulatedwith lipopolysaccharide for2hours and macrophages cocultured withMSCs for72hours, were infused into rhabdomyolysis-induced AKI micewhich were pretreated with liposomal clodronate to achieve macrophage depletion respectively.Results In the first part of our study, we constructed stablerhabdomyolysis-induced AKI model. Our data showed that intravenouslydelived MSCs at6h after reperfusion markedly reduced BUN, Cr, andCK levels, improved tubular injury and lowered acute tubular necrosisscore. Serum proinflammatory cytokines tumor necrosis factor (TNF)-αand interleukin (IL)-6decreased and antiinflammatory cytokines IL-10increased. MSCs didn’t disperse in kidney but persisted in lung andmuscle. In the second part of our study, the data showed that MSCscould modulated macrophage phenotype to M2-polarized and reducedtissue injury. In the third part of our study, macrophages coculturewith MSCs acquired an anti-inflammatory M2phenotype characterized byan increased expression of CD206and secretory IL-10, a suppressedproduction of TNF-α and IL-10. After macrophage depletion, sham miceand mice infused with M0and M1macrophages had a more severehistological and functional injury, whereas M2macrophage-inducedtransfused mice had reduced histological and functional injury.Conclusion (1) MSCs could attenuate rhabdomyolysis-induced AKI andregulate local inflammation, mainly via modulating macrophagephenotype.(2) MSCs located in the muscle and the lung, not in thekidney.(3) M2macrophages induced by MSCs are thought tosuppress immune responses and promote tissue remodeling inrhabdomyolysis-induced AKI.