Reparative effect of mesenchymal stromal cells on endothelial cells after hypoxic and inflammatory injury

Background: The renal endothelium is a prime target for ischemia-reperfusion injury (IRI) during donation and transplantation procedures. Mesenchymal stromal cells (MSC) have been shown to ameliorate kidney function after IRI. However, whether this involves repair of the endothelium is not clear. Therefore, our objective is to study potential regenerative effects of MSC on injured endothelial cells and to identify the molecular mechanisms involved. Methods: Human umbilical vein endothelial cells (HUVEC) were submitted to hypoxia and reoxygenation and TNF-α treatment. To determine whether physical interaction or soluble factors released by MSC were responsible for the potential regenerative effects of MSC on endothelial cells, dose-response experiments were performed in co-culture and transwell conditions and with secretome-deficient MSC. Results: MSC showed increased migration and adhesion to injured HUVEC, mediated by CD29 and CD44 on the MSC membrane. MSC decreased membrane injury marker expression, oxidative stress levels, and monolayer permeability of injured HUVEC, which was observed only when allowing both physical and paracrine interaction between MSC and HUVEC. Furthermore, viable MSC in direct contact with injured HUVEC improved wound healing capacity by 45% and completely restored their angiogenic capacity. In addition, MSC exhibited an increased ability to migrate through an injured HUVEC monolayer compared to non-injured HUVEC in vitro. Conclusions: These results show that MSC have regenerative effects on injured HUVEC via a mechanism which requires both physical and paracrine interaction. The identification of specific effector molecules involved in MSC-HUVEC interaction will allow targeted modification of MSC to apply and enhance the therapeutic effects of MSC in IRI. [Figure not available: see fulltext.

A Pilot Study of Postoperative Animal Welfare as a Guidance Tool in the Development of a Kidney Autotransplantation Model With Extended Warm Ischemia

Background: This pilot study aimed to maintain acceptable animal welfare in the development of a porcine autotransplantation model with severe and incremental renal ischemic injury, a model for usage in future intervention studies. Secondary aims were to develop and test methods to collect blood and urine without the need to restrain or use sedative and avoid transportation to optimize welfare of the pig. Methods: Kidneys from 7 female pigs were subjected to incremental durations of warm ischemia (WI) 30, 45, or 75 minutes by left renal artery and vein clamping. After static cold storage, contralateral nephrectomy was performed, and the injured graft was autotransplanted and animals observed for 14 days. Animal welfare was assessed and recorded using a structured scoring sheet before and 4 days after the kidney autotransplantation. Furthermore, blood samples were drawn daily the first week and every second day the following week using a semi-central venous catheter. An ostomy bag around the genitals was tested for urine collection. Measured glomerular filtration rate was calculated using renal clearance of chromium-51-labeled ethylenediamine tetraacetic acid on day 14. Results: None of the 7 animals died during the follow-up. The animal welfare was moderately affected when applying 75 minutes of WI (n = 2), and for that reason WI was not further increased. Pigs with lower WI had no observed welfare issues. With 75 minutes of WI peak, plasma creatinine was 1486 and 1317 µmol/L, reached on day 4. Lowest glomerular filtration rate levels were observed in the pigs with 75 minutes of WI. Conclusions: WI up to 75 minutes caused the intended severely impaired renal function without significantly compromising animal welfare. Blood and urine was collected postoperatively without sedation of the pigs or use of a metabolic cage.</p

Mesenchymal Stromal Cells Are Retained in the Renal Cortex Independently of Their Metabolic State After Renal Intra-Arterial Infusion

The regenerative capacities of mesenchymal stromal cells (MSCs) make them suitable for renal regenerative therapy. The most common delivery route of MSC is through intravenous infusion, which is associated with off-target distribution. Renal intra-arterial delivery offers a targeted therapy, but limited knowledge is available regarding the fate of MSCs delivered through this route. Therefore, we studied the efficiency and tissue distribution of MSCs after renal intra-arterial delivery to a porcine renal ischemia-reperfusion model. MSCs were isolated from adipose tissue of healthy male pigs, fluorescently labeled and infused into the renal artery of female pigs. Flow cytometry allowed MSC detection and quantification in tissue and blood. In addition, quantitative polymerase chain reaction was used to trace MSCs by their Y-chromosome. During infusion, a minor number of MSCs left the kidney through the renal vein, and no MSCs were identified in arterial blood. Ischemic and healthy renal tissues were analyzed 30 min and 8 h after infusion, and 1-4 x 10(4) MSCs per gram of tissue were detected, predominantly, in the renal cortex, with a viability >70%. Confocal microscopy demonstrated mainly glomerular localization of MSCs, but they were also observed in the capillary network around tubuli. The infusion of heat-inactivated (HI) MSCs, which are metabolically inactive, through the renal artery showed that HI-MSCs were distributed in the kidney in a similar manner to regular MSCs, suggesting a passive retention mechanism. Long-term MSC survival was analyzed by Y-chromosome tracing, and demonstrated that a low percentage of the infused MSCs were present in the kidney 14 days after administration, while HI-MSCs were completely undetectable. In conclusion, renal intra-arterial MSC infusion limited off-target engraftment, leading to efficient MSC delivery to the kidney, most of them being cleared within 14 days. MSC retention was independent of the metabolic state of MSC, indicating a passive mechanism

Ex Vivo Administration of Mesenchymal Stromal Cells in Kidney Grafts Against Ischemia-reperfusion Injury-Effective Delivery Without Kidney Function Improvement Posttransplant

Background. Mesenchymal stromal cell (MSC) therapy may improve renal function after ischemia-reperfusion injury in transplantation. Ex vivo renal intraarterial administration is a targeted delivery method, avoiding the lung vasculature, a known barrier for cellular therapies. In a randomized and blinded study, we tested the feasibility and effectiveness of MSC therapy in a donation after circulatory death autotransplantation model to improve posttransplant kidney function, using an ex vivo MSC delivery method similar to the clinical standard procedure of pretransplant cold graft flush. Methods. Kidneys exposed to 75 minutes of warm ischemia and 16 hours of static cold storage were intraarterially infused ex vivo with 10 million male porcine MSCs (Tx-MSC, n = 8) or vehicle (Tx-control, n = 8). Afterwards, the kidneys were autotransplanted after contralateral nephrectomy. Biopsies an hour after reperfusion confirmed the presence of MSCs in the renal cortex. Animals were observed for 14 days. Results. Postoperatively, peak plasma creatinine was 1230 and 1274 mu mol/L (Tx-controls versus Tx-MSC, P = 0.69). During follow-up, no significant differences over time were detected between groups regarding plasma creatinine, plasma neutrophil gelatinase-associated lipocalin, or urine neutrophil gelatinase-associated lipocalin/creatinine ratio. At day 14, measured glomerular filtration rates were 40 and 44 mL/min, P = 0.66. Renal collagen content and fibrosis-related mRNA expression were increased in both groups but without significant differences between the groups. Conclusions. We demonstrated intraarterial MSC infusion to transplant kidneys as a safe and effective method to deliver MSCs to the graft. However, we could not detect any positive effects of this cell treatment within 14 days of observation

Measurement of the Mass of the Z-Boson and the Energy Calibration of Lep

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Electroweak parameters of the z0 resonance and the standard model

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