Targeted Delivery Of Bone Mesenchymal Tem Cells By Ultrasound Destruction Of Icrobubbles Promotes In Acute Tubular Ecrosis Model

Acute renal failure (ARF) is clinically common acute and critically illin hospitalized patients,especially in the intensive care unit. ARF mostfrequently ensues upon a toxic or ischemic insult to the kidney and ispotentially reversible, however, being often just one element of multipleorgan damage. The mortality rate in hospital acquired ARF still ranges from30to80%actually. Modern dialysis techniques had no significant impact onoverall mortality. Acute tubular necrosis (ATN) plays central roles in theprocess underlying the failure of the kidney after toxic or ischemicchallenge.In animal models, the rate of recovery depends strictly on thereplacement of damaged and/or dead epithelium with a new functioning one.Growth factors, such as insulin-like growth factor-1(IGF-1), hepatocytegrowth factor(HGF), and epidermal growth factor(EGF), have been usedconsistently to potentiate tubular regeneration in experimenta ARF andaccelerated the proliferation and migration of resident cells. However, a therapeutic trial of recombinant human IGF-1in patients with ATN failed todemonstrate efficacy. The pathophysiology of human renal tubular necrosisand repair is very complex. Regenerative factors of renal tubular repair maybe involved with the combined effects of the protein network and a singlegene product is difficult to achieve therapeutic purposes.Tissue-based stem cells have traditionally been viewed asmultipotential precursor cells that are capable of generating tissue-specificdifferentiated cells. Intravascular transplantation of BM-MSCs is apromising therapeutic approach after ATN. Some scholars reported thatMSCs homed to the kidneys with ATN, differentiated into tubular epithelialcells and promoted the recovery of morphological and functional alterations.However, some studies argue against the direct transdifferentiation of bonemarrow–derived stem cells or MSCs into the kidney. The engraftmentfrequency of bone marrow–derived stem cells was reported to be relativelysmall. The present studies demonstrate that in vivo differentiation of MSCsinto renal tubular cells may not occur at all or that it is, at most, a minorcomponent of the repair process after ischemic injury. Is there a method toimprove cell transplantation? The efficacy and targeting of MSCsengraftment should now be crucial variables determining the therapeuticvalue of MSCs transplantation. Strategies involving adhesion moleculeshave been proposed to augment the attraction and invasion of stem cells andincrease their integration into pre-treated tissue. Ultrasound targeted microbubble destruction (UTMD) has evolved as apromising tool for organ specific gene and drug delivery.Ultrasound-mediated microbubble destruction resulting from the followingbiological effects: broaden the endothelial cell gap,cytokine expressionchange, neutrophil aggregation, and inflammatory response. Interestingly,platelet-derived proinflammatory factors activated by acoustic cavitationinduce the expression of adhesion molecules (P-selectin and ICAM-1),leading to the attachment of transplanted BM-MSCs onto the endothelium.Acoustic cavitation induces the expression of adhesion molecules (VCAM-1and ICAM-1) in capillaries. Moreover, the targeting of transplanted bonemarrow-derived stem cells has been facilitated by acoustic cavitation inischemic skeletal muscle non-ischemic cardiomyopathy and acutemyocardial infarction tissue. However, no studies have been reported thatdetermine whether this method is feasible for delivering MSCs to kidneytissue in ATN. Based on these previous findings, we attempted to explorewhether acoustic cavitation augments the site-targeted engraftment of MSCsto the kidney and promotes kidney recovery in ATN in rats.The bioeffects of ultrasound in combination with microbubbles havebeen extensively studied. These studies demonstrated that inertial cavitationof microbubbles in close proximity of the capillary wall can causemicroscopic bioeffects, including microvascular leakage, capillary rupture,and local induction of cell death and inflammation. Although these phenomena can occur in several organs, the kidney appears to be especiallysensitive to the adverse side-effects of ultrasound and microbubbles. Thismay be related to the relatively high blood pressure within the capillaries ofthe glomerulus, resulting in substantial bleeding into the Bowman's spacewith subsequent loss of function of the whole nephron. Therefore, theparameters of ultrasound need to be optimized.Based on the above mentioned theoretical basis and problems, ATNwas induced by the subcutaneous injection of mercuric chloride (HgCl2) inrats and microenvironment of renal tissue induced by different intensityultrasound-mediated microbubble will be investigated, and to explore thesuitable ultrasonic irradiation condition which is suitable for MSCstransplantation will be determined in this study.4',6-diamidino-2-phenylindole (DAPI) labeled MSCs transplantation willbe performed after environment changes of renal tissue induced byacoustic cavitation. The number of MSCs was evaluated by fluorescencemicroscopy, and real-time polymerase chain reaction (RT-PCR) and westernblotting and histological examination were performed7days after MSCstransplantation.Overall,The study was composed of the following three parts. PART I: SOLATION, CULTIVATION ANDIDENTIFICATION OF RATS BONE MARROW-DERIVEDMESENCHYMAL STEM CELLS IN VITROObjective: To investigate a stable method for the isolation, purification,cultivation and phenotype analysis of rats bone marrow-derivedmesenchymal stem cells in vitro. Methods:BM-MSCs were obtained from4-week-old rats. Bone marrow mononuclearcells were extracted from ratsfemurs and tibias bone marrow and purified via density gradientcentrifugationand then adherent culture. The expression of CD31,CD34,CD44and CD45were measured by immunohistochemistry. Flow cytometrywas used to determine the expression of CD29. Flow cytometry was used todetermine the cycle of MSCs by surface maker protein. Results:Immunohistochemical detection indicated positive expression of CD44.The expression of CD45, CD31and CD34was negative.The cellscomprised a unique phenotypic population, as visualised by flow cytometricanalysis of expressed surface antigens. These expanded MSCs wereuniformly positive for CD29(91.54%). The results from generation cycledetection showed90.36%cells in the period of G0/G1by flow cytometricanalysis.Conclusion: MSCs can be obtained by isolating rat femurs and tibiasbone marrow and purifying with adherent culture, providing a sufficient source of cells for the treatment of rat renal tubular necrosis. PART II: EFFECTS ON KIDNEYS TISSUEMICROENVIROMENT INDUCED BY ULTRASOUNDDESTRUCTION OF MICROBUBBLES IN ACUTETUBULAR NECROSIS MODELObjective: To explore the effects on kidneys tissue microenviromentinduced by acoustic cavitation with different ultrasound intensities in ratsand to determine the proper ultrasound intensity which is suitable for stemcells transplantation. Methods: ATN was induced by the subcutaneousinjection of mercuric chloride (HgCl2). Tweny eight Sprague-Dawley (SD)rats were randomly divided into the4group after the establishment of animalmodels of acute tubular necrosis. No acoustic cavitation in group1(controlgroup). One days and three days after ATN, rats in group2,3and4groupwere received pulse ultrasound (insonate for5s at1MHz and with a5s pause,totalling60s) targeted microbubble destruction focused on the lower backregion of rats with intensity of0.5W/cm2US,1.0W/cm2US and2.0W/cm2US respectively. All rats were sacrificed and the kidenys tissue washarvested4days after the last acoustic cavitation in experimental group. Real-time polymerase chain reaction (RT-PCR) and histologicalexamination were performed. Results: The results from RT-PCR revealedthat2.0W/cm2US+MB group markedly increased the level of ICAM-1andTNFα (P<0.05,3.1fold and6.3fold, respectively) compared with thecontrol group.1.0W/cm2US+MB treatment markedly increased the level ofICAM-1and TNFα (P<0.05,2.9fold and3.5fold, respectively) comparedwith the control group. The level of TNFα in2.0W/cm2US+MB group wasobviously higher than those with1.0W/cm2US+MB group (P<0.05). Theresults from Immunohistochemical examination revealed that the level ofICAM-1and TNFα in2.0W/cm2US+MB and1.0W/cm2US+MB groupwere marked increased than0.5W/cm2US+MB and the control group(P<0.05). Conclusion: Microbubble destruction by1.0W/cm2ultrasoundcan increase the level of ICAM-1of the kidney in ATN in rats and resultingin favor of the homing of stem cells with mild inflammatory response. PART Ⅲ: DELIVERY OF MSCS AFTERMICROENVIRONMENT CHANGES INDUCED BYULTRASOUND TARGETED MICROBUBBLEDESTRUCTION PROMOTE KIDNEY RECOVERY INACUTE TUBULAR NECROSIS MODELObjective: The aim of the present study was to explore whether ultrasound microbubble destruction augments site-targeted engraftment ofBM-MSCs to kidney tissue and promotes recovery of the kidney in acutetubular necrosis in rats. Methods: ATN was induced by the subcutaneousinjection of mercuric chloride (HgCl2).We performed the0.5ml infusion ofmicrobubbles via the contralateral femoral vein at days1and3after ATN.Simultaneous insonation was started at the lower back region of rats, usingan ultrasound gene transfection treatment meter [UGT]1025with thefollowing settings: insonate for5s at1MHz and1.0W/cm2with a5s pause,totalling60s. Forty Sprague-Dawley (SD) rats were randomly divided intothe following groups after the establishment of animal models of ATN injury(n=10):(1) Model group alone (control group);(2)1.0W/cm2US+MB(US/MB group), Bubble (0.5ml)+1.0W/cm2US;(3) MSCs group (ratMSCs at a concentration of2×106in1ml saline were infused via thecontralateral femoral vein at3day after ATN);(4)1.0W/cm2US+MB+MSCs group (US/MB+MSCs group), which received an infusion of ratMSCs (2×106in1ml saline) via the contralateral femoral vein1minuteafter US/MB at3day after ATN. The number of4',6-diamidino-2-phenylindole (DAPI) labelled MSCs was evaluated byfluorescence microscopy, and RT-PCR and western blotting and histologicalexamination were performed7days after MSCs transplantation. Results: Itwas observed via fluorescence microscopy that the number of DAPI labelled MSCs in the kidney for the US/MB+MSCs group was significantly morethan the MSCs group (P<0.05). The results from RT-PCR revealed that theUS/MB and US/MB+MSCs groups markedly increased the level ofICAM-1messenger ribonucleic acid (mRNA) compared with the controlgroup and the MSCs group (P<0.05). RT-PCR and Western blot analysisshowed that the expression of hepatocyte growth factor (HGF) andepidermal growth factor (EGF) in the US/MB+MSCs group were markedlyincreased compared with the all other groups (P<0.01). The extent of tubularnecrosis and dilation was significantly milder in the US/MB+MSCs group(acoustic exposure conditions:5s at1MHz and1.0W/cm2with a5s pause,totalling60s) than the all other groups (P<0.05). Conclusion: Microbubbledestruction by1.0W/cm2ultrasound can promote both the homing ofBM-MSCs to kidney tissue and the recovery of the kidney in ATN in ratswith the secretion of paracrine or autocrine of HGF and EGF.