Mesenchymal Stem Cells Protect Sepsis-associated Acute Kidney Injury

Background: Sepsis is one of the leading causes of death in the worldwide, particularlyin the developing countries, and the costs to health care systems are huge.In recentyears, Although there have been major technological advances in critical care medicineand blood purification medical, sepsis is still remain one of the most common causesof morbidity and mortality in the intensive care unit. Especially those patients withsepsis associated acute kidney injury(SA-AKI), the mortality rate even exceeds50%.Sepsis-associated acute kidney injury now is known to be an independent risk factorfor death. Its occurrence increases the complexity and costs of treatment. At present,there are no effective methods to treat or prevent SA-AKI. Therefore, new ways toprevent SA-AKI are urgently needed. Recently, Bone marrow–derived stem cells(MSC) have received increasing interest for the treatment of diseases associated withinflammation and organ injury. MSC have several advantages in that they are easilyharvested and have abundant resource, rapidly expanded in culture andnon-immunogenic, which made MSC has wide application prospect in fundamentalresearch and clinical practice.IL-17is an important inflammatory cytokine. It has been proved that IL-17takes partin the pathogenic of sepsis, and γδT cells as the predominant source of IL-17duringsepsis. However, during sepsis, the change of IL-17after MSC intervention is unclear.So the purpose of this paper is to build mice sepsis model through cecal ligation andpuncture (CLP), observe the protection effects of MSC intervention to sepsis andsepsis-associated acute kidney injury, and discuss the possible mechanism.Methods:In the first part of our study, we subjected mice through cecal ligation and puncture operation to induce sepsis and then received either normal saline or MSC (1×106cellsintravenously)3h after surgery. We then (1) observe the general status and the72hours survival rate after surgery.(2) detect serum creatinine (Cr) and blood ureanitrogen (BUN), alanine aminotransferase (ALT) and aspartate aminotransferase (AST)(n=10).(3) perform HE staining to assess the pathology injury including liver and lung(n=5). perform PAS staining to evaluate renal tubular injury and acute tubular necrosis(ATN) score (n=5).(4) test the expression of inflammatory cytokine of IL-6, IL-17,IL-10, TNF-α, INF-γ in serum by ELISA.(5)detect MSC distribution in CLP modelwith two-photon microscope technique.In the second part of our study, we use IL-17knockout mice to establish sepsis modelthrough cecal ligation and puncture operation. We then (1) observe the general statusand the72hours survival rate after surgery.(2) detect serum Cr, BUN, ALT and AST(n=10).(3) perform HE and PAS staining to assess the pathology injury including liver,lung and kidney (n=5).(4) test the infiltration of neutrophils (Ly-6G) in kidney withimmune-fluorescence technique (n=5).(5) test the expression of inflammatorycytokine of IL-6, IL-17, IL-10, TNF-α, IL-1β, INF-γ and chemokine in renal tissue byrealtime PCR.(6) test the TUNEL-postive cells in renal tissue.(7) test the expression ofCleaved caspase3in renal tissue by Western Blot.In the third part of our study, to clarify the inflammatory status about MSC in sepsisand SA-AKI, we then (1) test the infiltration of neutrophils (Ly-6G) and macrophage(F4/80) in liver and lung with immune-fluorescence technique (n=5).(2)test the ratioof αβT cell and γδT cell that secreting IL-17with flow cytometry.(3) test the mRNAexpression of IL-6, IL-1β, IL-17, IL-10, TNF-α, INF-γ, CCL2, CCL3, CXCL1,CXCL2, CXCL5in renal tissue by realtime PCR.(4) test the ratio of γδT cell secretingIL-17in renal tissue with flow cytometry.Result:In Part one, we successfully built the CLP-induced sepsis model. We find that Injecting106MSC via tail vein in3hours after surgery can improve the general status and72hsurvival rate, reduce the serum level of Cr, BUN, ALT and AST, and alleviate the pathological injury of lung, liver and kidney. and improve renal tubular necrosis score.MSC can reduce the expression of inflammatory cytokine about IL-6, IL-17, TNF-α,INF-γ in serum and raise the expression of IL-10. Intravenously delivered MSC wasmainly dispersed in lungs, spleen and celiac lymph nodes until72hours, bue not foundin liver, kidney and heart all the time.In Part two, we use IL-17gene knockout mice to make CLP model, finding the generalstatus and survival rate of knockout mice is obviously better than wide mice CLPmodel; the biochemical index and kidney pathological change are also better than widetype. Knockout IL-17can reduce the number of the infiltration of neutrophils in kidney.The mRNA express of inflammatory cytokine in renal tissue is also improvedcomparing with wide type. Compare to wild CLP model, the CLP model of IL-17knockout mice can reduce the TUNEL-postive cells and the expression of Cleavedcaspase3in renal tissue.This means knockout IL-17can obviously improve sepsis andsepsis-associated acute kidney injury.In Part three, MSC can reduce the number of the infiltration of neutrophils andmacrophage in liver, lung and kidney. MSC can reduce the percentage of γδT cell thatsecrete IL-17in spleen; and, at the same time, we find MSC treatment can reduce themRNA expression levels of IL-6, IL-1β, IL-17, TNF-α, INF-γ, CCL2, CCL3, CXCL1,CXCL2, CXCL5and improve the mRNA level of IL-10. Flow cytometry testing findsthat sepsis injury can obviously promote renal to secrete IL-17, and mainly from γδTcell. MSC can reduce IL-17secreting in local kidney, and mainly to reduce the IL-17secreted from γδT cell.Conclusion:(1) MSC can improve sepsis and associated acute kidney injury.(2) MSC can improvethe statement of inflammatory in sepsis mice.(3) In the mouse sepsis model, the IL-17mainly secreted from γδT cell.(4) MSC can reduce the percentage of γδT cell thatsecrete IL-17in spleen and kidney.(5) Knockout IL-17can protect sepsis associatedacute kidney injury. MSC maybe through inhibiting IL-17to protect sepsis andsepsis-associated acute kidney injury.