Impact of timing administration of mesenchymal stromal cells on serum creatinine following renal ischemia/ reperfusion in rats

peer reviewedExperimental models of renal ischemia/reperfusion (I/R) have suggested protective effects of mesenchymal stromal cells (MSC) therapy. Still, param- eters of MSC injection, including volume, route and timing of cell administration, remain largely debated. Particularly, MSC infusion in mouse has been shown to be beneficial “a priori” but deleterious “a posteriori” of renal I/R injury. In order to further investigate the influence of the timing of MSC administration, we used 10-week-old Lewis rats categorized in 4 groups. Groups 1 (MSC D-7, n = 10) and 2 (MSC D + 1, n = 7) received caudal i.v. injection of MSC (1.5 9 106 in 1 ml of saline) 7 days before or 1 day after renal I/R, respectively. Control groups 3 (saline D-7, n = 6) and 4 (saline D + 1, n = 6) received equal volume of saline at similar time points. Left renal ischemia (by clamping of the renal pedicle) lasted 45 min. Right nephrectomy was simultaneously performed. Blood sample was collected from inferior vena cava at 48 h post reperfusion. MSC phenotype was confirmed by FACS analysis. In groups 1 and 3, serum creatinine (SCr) reached 1.4 ` 0.7 versus 2.4 ` 0.8 mg/dl, respectively (p < 0.05). In groups 2 and 4, SCr was 4.9 ` 0.7 versus 3.3 ` 0.9 mg/dl, respectively (p < 0.001). Furthermore, SCr levels were statistically worse when MSC were administered after renal I/R in comparison to a priori infusion (p < 0.0001). In conclusion, MSC administration 7 days prior to renal I/R attenuates kidney injury in comparison to (i) saline infusion or (ii) MSC infusion 1 day after renal I/R. Conversely, on the basis of SCr levels, MSC therapy performed after renal I/R worsens kidney injury in rats

Mesenchymal stromal cells combined with everolimus promote Treg expansion but do not synergize in a rat liver transplant rejection model

peer reviewedBackground: Mesenchymal stromal cells (MSCs) have particular properties that can be of interest in organ transplantation, including expansion of regulatory T cells (Tregs), a key factor in graft tolerance induction. The immunosuppression to be associated with MSCs has not yet been defined. Additionally, the impact of the association of everolimus with MSCs on Treg expansion and on induction of liver graft tolerance has never been studied. The aim of this study was to evaluate the effects of MSCs combined, or not, with everolimus, on Treg expansion and in a model of liver transplantation (LT) rejection in the rat. Methods: Firstly, Lewis rats received intravenous MSCs at D9 with/without subcutaneous everolimus from D0 to D14. Analysis of circulating Tregs was performed at D0, D14 and D28. Secondly, 48 h after LT with a Dark Agouti rat liver, 30 Lewis rats were randomized in 3 groups: everolimus (subcutaneous for 14 days), MSCs (intravenous injection at D2 and D9), or both everolimus and MSCs. Rejection of the liver graft was assessed by liver tests, histology and survival. Results: Individually, MSC infusion and everolimus promoted Treg expansion in rats, and everolimus had no negative impact on Treg expansion when combined with MSCs. However, in the LT model, injections of MSCs 2 and 9 days following LT were not effective at preventing acute rejection, and the combination of MSCs with everolimus failed to show any synergistic effect when compared to everolimus alone. Conclusion: Everolimus may be used in association with MSCs. However, in our model of LT in the rat, post-transplant MSC injections did not prevent acute rejection, and the association of MSCs with everolimus did not show any synergistic effect

intravenous administration of mesenchymal stream cells modulates renal lipid metabolism in rats

Mesenchymal stromal cells (MSC) have been shown to attenuate renal ischemia/reperfusion (I/R) injury in rodents. Still, the mechanisms of such a nephroprotection remain unclear. Here, rats were intravenously infused with MSC (1.5x10^6 cells in 1 ml saline; MSCD-7 group, n=6) or equivalent volume of saline (SD-7 group, n=6) 7 days before kidney sampling. High-throughput RNA sequencing technology was used to compare transcriptomic renal profiles, using TopHat and Cufflinks open-source software tools. A total of 494 and 256 genes were found to be significantly (q-value CD36 – a key regulator of membrane uptake of FA – was increased in MSCD-7 kidneys, with a preferential localization in proximal tubules (PT). As a whole, our data suggest that MSC infusion causes critical modifications of lipid metabolism, including (i) down-regulation of FA biosynthesis; (ii) activation of PPAR alpha pathway, and (iii) prioritization of FA as sources of energy in PT cells, which may eventually prevent lipid peroxidation and attenuate renal I/R damage

Administration of mesenchymal stromal cells before renal ischemia/reperfusion attenuates kidney injury and may modulate renal lipid metabolism in rats.

Mesenchymal stromal cells (MSC) have been demonstrated to attenuate renal ischemia/reperfusion (I/R) damage in rodent models. The mechanisms of such nephro-protection remain largely unknown. Furthermore, the optimal timing of MSC administration has been poorly investigated. Here, we compare the impact of MSC injection 7 days before (MSCD - 7) versus 1 day after (MSCD + 1) renal I/R in rats. Control groups received equivalent volumes of saline at similar time-points (SD - 7 and SD + 1). Right nephrectomy was performed, and left renal ischemia lasted 45 min. After 48-hour reperfusion, we observed significantly improved renal function parameters, reduced apoptotic index and neutrophil/macrophage infiltration in kidney parenchyma, and lower expression of tubular damage markers and pro-inflammatory cytokines in MSCD - 7 in comparison to MSCD + 1 and saline control groups. Next, comparative high-throughput RNA sequencing of MSCD - 7 vs. SD - 7 non-ischemic right kidneys highlighted significant down-regulation of fatty acid biosynthesis and up-regulation of PPAR-alpha pathway. Such a preferential regulation towards lipid catabolism was associated with decreased levels of lipid peroxidation products, i.e. malondialdehyde and 4-hydroxy-2-nonenal, in MSCD - 7 versus SD - 7 ischemic kidneys. Our findings suggest that MSC pretreatment may exert protective effects against renal I/R by modulating lipid metabolism in rats

Administration of mesenchymal stromal cells before renal ischemia/reperfusion attenuates kidney injury and modulates renal lipid metabolism in rats

Background: Mesenchymal stromal cells (MSC) have been demonstrated to attenuate renal ischemia/reperfusion (I/R) damage in rodents. The mecha- nisms of such nephroprotection remain unclear. Materials and Methods: Male Lewis rats aged of 8–10 weeks received tail i.v injection of 1.5x106 MSC in 1 mL saline (MSCD-7, n = 11) or saline alone (SD- 7, n = 6) 7 days before renal I/R. Left renal ischemia (by clamping the renal pedicle) lasted 45 min. Right nephrectomy was simultaneously performed. Blood sample was collected from inferior vena cava 48 h post reperfusion. Renal function was assessed by measuring serum creatinine (SCr) levels. Expressions of inflammatory and apoptotic markers by real-time (RT)-qPCR were comparatively quantified. High-throughput RNA sequencing was applied to MSCD-7 vs. SD-7 non-ischemic right kidneys. Relevant pathways were detected using an Over-Representation Analysis with WebGestalt, and confirmed by RT-qPCR. Results: Scr levels reached 1.4 ` 0.7 vs. 2.4 ` 0.8 mg/dL in MSCD-7 vs. SD-7 group (p < 0.05). MSC infusion significantly reduced mRNA expression of Casp3, Hsp 70, Kim-1, Mcp-1 and Il-6 and increased mRNA expression of Bcl compared to saline. Among 25 908 genes, 748 were identified as significantly differentially expressed (False Discovery Rate (FDR), <0.05) between MSCD-7 and SD-7 non-ischemic kidneys. Among the most affected metabolic pathways, renal lipid metabolism was significantly altered, with down-regulation of fatty acid biosynthesis and an up-regulation of PPARa pathway in MSCD-7 vs. SD-7 groups. By immunoblotting, PPARa and phosphorylated-PPARa were significantly increased in MSCD-7 vs. SD-7 kidneys, in both non-ischemic and ischemic conditions. Moreover, levels of malondialdehyde-derived lipid peroxidation products were decreased in MSCD-7 ischemic kidneys in comparison to SD-7 ischemic kidneys. Conclusion: MSC infusion at day 7 prior injury critically impacts renal lipid metabolism, which may condition kidney parenchyma against I/R

Nuclear Magnetic Resonance Metabolomic Profiling of Mouse Kidney, Urine and Serum Following Renal Ischemia/Reperfusion Injury.

Abstract BACKGROUND: Ischemia/reperfusion (I/R) is the most common cause of acute kidney injury (AKI). Its pathophysiology remains unclear. Metabolomics is dedicated to identify metabolites involved in (patho)physiological changes of integrated living systems. Here, we performed 1H-Nuclear Magnetic Resonance metabolomics using urine, serum and kidney samples from a mouse model of renal I/R. METHODS: Renal 30-min ischemia was induced in 12-week-old C57BL/6J male mice by bilaterally clamping vascular pedicles, and was followed by 6, 24 or 48-hour reperfusion (n = 12/group). Sham-operated mice were used as controls. Statistical discriminant analyses, i.e. principal component analysis and orthogonal projections to latent structures (OPLS-DA), were performed on urine, serum and kidney lysates at each time-point. Multivariate receiver operating characteristic (ROC) curves were drawn, and sensitivity and specificity were calculated from ROC confusion matrix (with averaged class probabilities across 100 cross-validations). RESULTS: Urine OPLS-DA analysis showed a net separation between I/R and sham groups, with significant variations in levels of taurine, di- and tri-methylamine, creatine and lactate. Such changes were observed as early as 6 hours post reperfusion. Major metabolome modifications occurred at 24h post reperfusion. At this time-point, correlation coefficients between urine spectra and conventional AKI biomarkers, i.e. serum creatinine and urea levels, reached 0.94 and 0.95, respectively. The area under ROC curve at 6h, 24h and 48h post surgery were 0.73, 0.98 and 0.97, respectively. Similar discriminations were found in kidney samples, with changes in levels of lactate, fatty acids, choline and taurine. By contrast, serum OPLS-DA analysis could not discriminate sham-operated from I/R-exposed animals. CONCLUSIONS: Our study demonstrates that renal I/R in mouse causes early and sustained metabolomic changes in urine and kidney composition. The most implicated pathways at 6h and 24h post reperfusion include gluconeogenesis, taurine and hypotaurine metabolism, whereas protein biosynthesis, glycolysis, and galactose and arginine metabolism are key at 48h post reperfusion

Characterization of the impact of kidney-centered radiotherapy in mice

Objective: Kidney irradiation has been associated with renal ischemic preconditioning in mice, with a potential role of cell proliferation. Here, we investigate the impact of kidney-centered irradiation on the regulatory proteins involved in the p53 pathway in mice. Methods: Adult male C57bl/6 mice were bilaterally irradiated on both kidneys at a total dose of 8 Gy. One-month post-irradiation, the renal expression of proliferating cell nuclear antigen (PCNA) and Ki67 was quantified by immunostaining. The abundance of phospho-p53 (Ser15) (p53-pS15) and murine double minute2 (MDM2), chromatin licensing and DNA replication factor 1 (CDT-1) (particularly expressed in G1 phase) and Serine 10-phosphorylated histone H3 (p-H3) (particularly expressed in G2/M phase), was assessed by immunoblotting or immunostaining. Results: Irradiated kidneys were characterized by statistically increased expression of PCNA- (p<0.001) and Ki67- (p<0.05) positive cells, as well as by an activated p53 signaling evidenced by the downregulation of MDM2 (p<0.05) and the upregulation of p53-pS15 (p<0.05)). Quantification of G2/M-phase cells by p-H3 (p<0.001) revealed a significant increase following irradiation. CDT-1 expression was unchanged in irradiated versus control kidneys. Conclusion: Kidney-centered ionizing irradiation activates the p53 pathway. The p53-mediated G2/M cell cycle arrest may participate to cell endocycling hypertrophy in conjunction with cell proliferation.Characterization of the renal ischemic preconditioning induced by kidney-centered irradiation in mic

Impact of mesenchymal stromal cells and/or everolimus on T-reg lymphocyte expansion in rats

Background: Mesenchymal stromal cells (MSC) are a promising cell-therapy in solid-organ transplantation, namely because of their immunomodulatory properties and positive impact on the expansion of T-regulator lymphocytes (Treg). The “optimal” immunosuppressive regimen to be associated with MSC has not been defined. Here, we aimed to evaluate the effects on Treg expansion of a single injection of MSC combined or not with everolimus in rats. Materials and methods: Twenty-four Lewis rats were randomly assigned to 4 groups (n=6 per group): MSC+Evero group, i.e. everolimus (0,25mg/kg/day, SC) from D0 to D14 and iv MSC (±1x106 cells) at D9; MSC group, i.e. placebo from D0 to D14 and iv MSC at D9; Evero group, i.e. everolimus from D0 to D14 and iv saline at D9; control group, i.e. placebo from D0 to D14 and iv saline at D9. T-reg blood levels were measured at D0-14-28 with flow cytometry analysis using anti-CD4,-CD25 and -FoxP3 antibodies. Results: In the two groups infused with MSC, Treg were significantly expanded at D14 and D28 (p<0.01), in comparison to D0. When compared to controls group, the “Evero” group showed a significant expansion of Treg levels at D14 but not at D28. In control, Treg levels did not significantly change compared to D0. Conclusion: A single iv MSC injection was efficient to expand T-reg blood levels. This effect was not altered by everolimus co-administration. Everolimus exposure alone promotes a transient T-reg expansion. Hence, everolimus may be regarded as a co-drug of choice in MSC-based therapy in solid-organ transplantion

Genetic deletion of DUSP3 phosphatase attenuates kidney damage and inflammation following ischemia/reperfusion in mouse

Background. Renal ischemia-reperfusion (I/R) injury represents an unavoidable event in kidney transplantation. Dual Specificity Phosphatase 3 (DUSP3, also called Vaccinia-H1 Related (VHR)) is highly expressed in endothelial cells, as well as in platelets, monocytes and macrophages. Since DUSP3 is a positive regulator of the innate immune response, its inactivation/deletion may attenuate kidney inflammation and damage caused by I/R. Methods. Ten-weeks-old C57BL/6 wild-type (WT, n=10) versus systemic knock-out (KO, n=10) mice underwent unilateral left renal ischemia for 30 minutes. Right nephrectomy was simultaneously performed. The left kidney was excised and blood sample was collected from inferior vena cava at 48h post reperfusion. Renal function was assessed upon Blood Urea Nitrogen (BUN) levels. Expressions of inflammatory and immune markers were comparatively quantified at both mRNA (real-time qPCR) and protein (immune-blotting and –staining) levels in ischemic vs. non-ischemic kidneys in DUSP3 WT vs. KO mice. Results. BUN reached 259±51 vs 78±11mg/dL in WT and KO, respectively (p0.05) of TNF and 111-fold (p0.05) of KIM-1, respectively. Conclusions. Genetic deletion of DUSP3 attenuates renal I/R-associated damage and inflammation