| BackgroundsDiabetic nephropathy(DN) is one of the most detrimental complications of diabetic mellitus(DM), with high morbidity and mortality. It may cause death when it develops to end-stage renal disease(ESRD). As there are no effective therapies, more and more researchers focused on cell therapy to cure DM and its complications in recent years. It is reported that bone marrow-derived mesenchymal stem cells(MSC) are capable to repair pancreas, ameliorate kidney injury, accelerate renal tubule proliferation and restore kidney function. However, insufficient MSC engrafted via intravenous injection can settle in DN kidneys. This has become one of the major barriers to the effective implementation of MSC therapy. Hence, strengthening the homing of MSC to the injured kidney is of urgent demand.Stromal cell-derived factor-1(SDF-1) and its receptor CXCR4 play a vital role in MSC migration through SDF-1/CXCR4 pathway. SDF-1 is well-known for stem cell activation, mobilization, homing and retention in injured tissues. It can decrease MSC apoptosis, increase MSC livability and enhance MSC proliferation. When tissues are injured, the localized SDF-1 concentration increases due to the cellular SDF-1 expression, which aids the mobilization and homing of MSC. Researchers found that SDF-1/CXCR4 concentration of peripheral blood lowered in DM patients, especially with multi-organ injury. Thus, up-regulation of SDF-1 in DN kidney is expected to enhance the MSC homing efficacy.Microbubbles(MBs), famous for diagnostic ultrasound contrast agents, have been developed as a carrier of genes, proteins and drugs. They can be released in a targeted manner for therapy using ultrasound irradiation. Ultrasound-targeted microbubble destruction(UTMD) also reportedly increases renal interstitial permeability and changes the microenvironment of targeted tissue. In addition, the beneficial involvement of endocrine cytokines after UTMD improves the implantation of MSC.In this study, we attempt to develop a lipid MB loaded with SDF-1 via covalent conjugation. SDF-1 is infused into circulation accompanied by MBs and released in the kidney in a targeted manner via UTMD. The up-regulation of SDF-1, combined with microenvironment changes caused by UTMD, is expected to promote the exogenously engrafted MSC homing to DN kidney and then improve the therapeutic effects. The study aims to potentiate the MSC homing to the DN kidney for DN therapy, and to provide a new approach for effective MSC-based therapy for DN repair.Objectives:1. To develop a lipid MB carrying SDF-1 which is a chemoattractant. And to evaluate its properties, biological activities in vitro and in vivo, as well as ultrasonic imaging ability as a contrast agent.2. To explore the effects of diagnostic ultrasound combining microbubbles on kidney vascular permeability in SD rats. And to study feasibility of SDF-1 up-regulation by ultrasound targeted SDF-1-loaded MBs destruction.3. To derive MSC from bone marrows of SD rats, culture and label them for engraftment. To establish early diabetic nephropathy model of SD rats.4. To study the efficacy of diagnostic ultrasound combining SDF-1-loaded microbubbles on promoting engrafted MSC homing to DN kidney.5. To study preliminary results of DN repair by ultrasound targeted SDF-1-loaded MBs destruction combining MSC engraftment.Methods:1. MSC were isolated and cultured by adherent culture method. CCK-8 assay was performed to test the cell proliferation. Flow cytometry(FCM) was applied to detect the cell cycle and the expression of cell surface marker. The morphology was evaluated by transmission electron microscope(TEM) and light microscope. Osteogenic and adipogenesis induction of MSC were performed to ensure the mutipotent differentiation. The cytoplasm, cell membrane and nucleus of MSC were labeled by green fluorescent protein lenti-virus transfection CM-Di I and DAPI, respectively.2. To develop SDF-1-loaded MBs, SDF-1 was cross-linked with COOH-PEG-COOH through covalent conjugation using EDC/sulfo-NHS as coupling agents. The morphology and distribution of MBs were detected using light microscope. The diameter and concentration were detected using a particle counting instrument. SDF-1 was labeled with fluorescein isothiocyanate(FITC) and FITC-labeled SDF-1-loaded MBs were prepared. The conjunction rate of SDF-1 was determined by FCM. The loaded SDF-1 encapsulated in MBs was quantitatively determined with an enzyme-linked immunosorbent assay(ELISA). The SDF-1 encapsulation efficiency and loading content were then calculated. Western blot was applied to detect the molecular weight of SDF-1 after cross-linked with PEG. Ultrasound imaging was performed to evaluate the contrast enhancement of MBs. CCK-8 assay and cell migration assay were used to detect the safety and biological activities of MBs, respectively.3. Kidneys of SD rats were sonicated by diagnostic ultrasound after MB infusion. TEM was applied to observe morphological alterations of vascular ultrastructure. Lanthanum nitrate perfusion method was used to evaluate the vascular permeability. Laser scanning confocal microscope(LSCM) was used to track target release of FITC-labeled SDF-1 using UTMD.4. Early DN rat model was established by a single intraperitoneal injection of streptozotocin(STZ). Blood glucose and urine glucose concentration were detected. Hematoxylin-eosin(HE) staining, periodic acid Schiff(PAS) staining and immunohistochemistry were performed to observe the pathological changes and SDF-1 expression in DN kidney. Normal rats and DN rats were randomized into 3 groups: Control group, UTMD group and UTMD+SDF-1 group. Comparative study of SDF-1 expression among different groups was performed by ELISA, polymerase chain reaction(PCR) and Western blot. GFP-labeled MSC were engrafted after group treatment and the number of homing MSC in kidney was calculated under LSCM.5. To evaluate the therapeutic effect of ultrasound targeted SDF-1-loaded MBs destruction combining MSC engraftment on DN kidney, rats were randomized into 5 groups: Normal group, DN group, DN+MSC group, DN+SDF-1 group and DN+ SDF- 1+ MSC group. Comparative study was carried out before treatment, 4 weeks and 12 weeks after treatment. Blood glucose, urinary albumin and urinary creatinine were detected. Urinary albumin creatinine ratio(ACR) was calculated to evaluate kidney function. Ultrasonography was performed to find out ultrasonic differences, especially blood perfusion and artery resistance index. HE staining and PAS staining were applied to observe pathological changes of pancreas and kidney. Immunohistochemistry was performed to find out differences of transforming growth factor β1(TGF-β1) expression.Results:1. MSC were successfully derived from bone marrow. Flow cytometry resulted that the MSC surface markers of CD44, CD29, CD90 were positive, and CD34, CD45 and CD11 b were negative. The MSC could differentiate into adipogenic and osteogenic cells. Approximately 83.00% of the cells stayed in G0+G1 phage of cell cycle. They were featured with strong proliferative ability and as undifferentiated cells. The cells could be transfected by GFP lenti-virus, resulting in green fluorescence in cytoplasm, dyed by CM-Di I with red fluorescence in cell membrane, and dyed by DAPI with blue fluorescence in nuclear.2. SDF-1-loaded lipid MBs were successfully prepared via covalent conjugation. They were stable at room temperature. They were milky in appearance, spherical, homogeneously distributed and well dispersed. Both the unloaded MBs and SDF-1-loaded MBs ranged from 1.5~5 mm. The mean diameter and concentration of the unloaded MBs were 1.78mm and 5~9×109/m L, respectively. The mean diameter and concentration of SDF-1-loaded MBs were 1.92 mm and 2~6×109/m L, respectively. The SDF-1 encapsulation efficiency and loading content were 79% and 15.8 mg/m L, respectively. According to the LSCM images, green fluorescent was not found in unloaded MBs, whereas, FITC-labeled SDF-1 could bind to the MBs at a binding rate of 84.8%. After cross-linked with PEG, SDF-1 molecular weight changed to 8 k D, 12 k D, 20 k D, resulting in 3 bands in Western blot assay. Under low mechanic index ultrasound(MI=0.05), the SDF-1-loaded MBs reflexed strong echoic information both in vitro and in vivo, and were capable to act as a contrast agent. CCK-8 assay showed no toxicity effect was found in SDF-1-loaded MBs solution. Cell migration assay confirmed SDF-1-loaded MBs solution could attract MSC, playing a similar role of SDF-1, and the chemoattractant could be blocked by AMD3100. The result indicated that they retained biological ability to attract MSC.3. Under TEM, part of the vessel walls of rat kidney were discontinuous and tight junction gap enlarged after UTMD. Vascular permeability increased leading to the extravasation of Lanthanum nitrate in interstitials. The increased permeability could recovered 24 hours later. LSCM showed FITC labeled SDF-1 was successfully target released in kidney.4. Three days after intraperitoneal injection of STZ, blood glucose maintained at a high level of more than 16.7 mmol/L. The DM rat model was established when the rats exhibited typical symptoms of polyphagia, polydipsia, urorrhagia and weight loss. Four weeks later, when the urinary micro albumin excretion rate was greater than 30 mg/24 h, early DN model was established. Immunohistochemistry showed weak expression of SDF-1 in DN kidney. ELISA assay revealed SDF-1 concentration was higher in DN kidney than that in normal kidney(1010.44±88.53 pg/m L VS 208.03±42.34 pg/m L,P<0.05). Immediately after UTMD, they did not increase and 6 hours later, they increased to 1257.68±103.88 pg/m L in DN kidney and 401.78±50.13 pg/m L in normal kidney. Immediately after target release of SDF-1, the concentration increased to 1750.08±131.36 pg/m L in DN kidney and 760.97±91.85 pg/m L in normal kidney. 6 hours later, they increased to 2120.23±159.75 pg/m L and 953.89±79.33 pg/m L, respectively. They were significantly higher than those in either Control group or UTMD group(P<0.05). PCR results showed no increase of SDF-1 m RNA expression was found in UTMD and UTMD+SDF-1 immediately after treatment but a significant increase was found 6 hours later(P<0.05). As Western blot showed, immediately after UTMD, no increase of SDF-1 was found in normal kidney. 6 hours later, SDF-1 expression increased. Immediately after target release of SDF-1, significant increase of SDF-1was found and 6 hours later the SDF-1 was even higher. In DN kidney, SDF-1 expression exhibited the similar trend after treatment, but was higher than that in normal kidney. 24 hours after group treatment followed by MSC engraftment, the number of GFP labeled MSC increased to 23.8±3.61 in UTMD+SDF-1group of DN kidney, much higher than that in DN group(3.6±2.07)(P<0.05).5. Blood glucose of DN rats without any treatment progressively increased from22.93±6.51 mmol/L to 30.25±4.78 mmol/L 12 weeks later, which was similar with DN+SDF-1 group. After MSC engraftment, blood glucose in both DN+MSC group and DN+SDF-1+MSC group decreased to approximately 22 mmol/L, but could not recover to normal of 4.67±1.04 mmol/L. ACR increased to 660.32±70.21 mg/mg in DN group and 620.39±50.83 mg/mg in DN+SDF-1 group 12 weeks later, higher than normal of 115~126 mg/mg. ACR in DN+SDF-1+MSC group was lower than that in DN+MSC group enhanced parenchymal echogenicity, reduced blood perfusion and increased artery resistance index(up to 0.71±0.16) 12 weeks later in DN group. No improvement was found in DN +SDF-1 group. In DN+MSC group, ultrasonic performance improved and RI reduced to 0.63±0.12. In DN+SDF-1+MSC group, ultrasonic performance was better and RI was 0.60±0.14. Pathologic examination showed a progressive destruction of pancreatic islets in DN rats. Enlarged extra cellular matrix, glomerular sclerosis, mesangial proliferation and basement membrane thickening were also found in kidney. No improvement was found in DN +SDF-1 group. In comparison, pathologic findings were better in DN+MSC group and DN+SDF-1+MSC group, without progression. For immunohistochemistry, the expression of TGF-β1 was weak in normal kidney and progressively increased in DN group and DN+SDF-1 group. As expected, the expression did not significantly increase in DN+MSC group and DN+SDF-1+MSC group. DN+SDF-1+MSC group exhibited a better suppression of TGF-β1 than DN+MSC group.Conclusions:1. Bone marrow-derived MSC can be successfully obtained by adherent culture method. The MSC were characterized with low differentiation, high self-renewal ability and multi-potent differentiation. The cytoplasm, cell membrane and nuclei can be labeled by GFP lenti-virus transfection, dyed by CM-Di I with red fluorescence, and by DAPI with blue fluorescence, respectively.2. SDF-1-loaded MBs are successfully developed through covalent conjugation. They are featured with high stability, high concentration, as well as high SDF-1 encapsulation efficiency and loading content. They can act as ultrasound contrast agents both in vitro and in vivo. The biological activity to attract MSC retains and no obvious toxicity is detected.3. Early DN rat model can be established by receiving a single intraperitoneal injection of streptozotocin.4. UTMD can enhance vascular permeability of kidney and may recover within 24 hours.5. SDF-1 can be released in the kidney in a targeted manner via UTMD. The up-regulation of SDF-1, combined with microenvironment changes caused by UTMD, promotes the exogenously engrafted MSC homing to DN kidney. The appropriate time for MSC engraftment is within 6 hours from treatment.6. MSC engraftment can repair destructed pancreatic islets, reduce blood glucose in DN rats. The kidney function can be protected by reducing TGF-β1 expression, inhibiting glomerular and renal interstitial fibrosis progression.7. Through ultrasound targeted SDF-1-loaded MBs destruction which up-regulates SDF-1 expression, MSC tropism for DN kidneys is strengthened, leading to effective DN therapy. This method provides a better repair than single MSC engraftment. |
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