Effect of the Combination of Everolimus and Mesenchymal Stromal Cells on Regulatory T Cells Levels and in a Liver Transplant Rejection Model in Rats.

Introduction: Mesenchymal stromal cells (MSCs) have particular properties that are of interest in organ transplantation, including the expansion of regulatory T cells (Tregs), a key factor in transplant tolerance induction. However, the most effective immunosuppressive drug to associate with MSCs has yet to be defined. Additionally, the impact of the association of everolimus with MSCs on Treg expansion, and on the induction of liver graft tolerance, has never been studied. The aim of this study was to evaluate the effects of MSCs in combination, or not, with everolimus on Treg expansion and in a model of rejection after liver transplantation (LT) in the rat. Methods: Firstly, 24 Lewis rats were assigned to 4 groups (n=6 in each group) receiving intravenous MSCs or saline injection at day (D)9 with/without subcutaneous everolimus from D0 to D14. Analysis of circulating Tregs was performed at D0, D14 and D28. In a second set of experiment, 30 Lewis rats were randomized in 3 groups 48hours after LT with a Dark Agouti rat liver: everolimus (subcutaneous for 14 days), MSCs (intravenous injection at post-operative day 2 and 9), 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 in combination with MSCs. However, in the LT model, injections of MSCs two and nine 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.

The genetic deletion of the Dual Specificity Phosphatase 3 (DUSP3) attenuates kidney damage and inflammation following ischaemia/reperfusion injury in mouse.

AIM: Dual Specificity Phosphatase 3 (DUSP3) regulates the innate immune response, with a putative role in angiogenesis. Modulating inflammation and perfusion contributes to renal conditioning against ischaemia/reperfusion (I/R). We postulate that the functional loss of DUSP3 is associated with kidney resistance to I/R. METHODS: Ten C57BL/6 male WT and Dusp3-/- mice underwent right nephrectomy and left renal I/R (30 min/48 hours). Renal injury was assessed based on serum levels of urea (BUN) and Jablonski score. The expression of CD31 and VEGF vascular markers was quantified by RT-qPCR and immuno-staining. Renal resistivity index (RRI) was measured in vivo by Doppler ultrasound. Comparative phosphoproteomics was conducted using IMAC enrichment of phosphopeptides. Inflammatory markers were quantified at both mRNA and protein levels in ischaemic vs non-ischaemic kidneys in WT vs Dusp3-/- . RESULTS: At baseline, we located DUSP3 in renal glomeruli and endothelial cells. CD31-positive vascular network was significantly larger in Dusp3-/- kidneys compared to WT, with a lower RRI in Dusp3-/- mice. Following I/R, BUN and Jablonski score were significantly lower in Dusp3-/- vs WT mice. Phosphoproteomics highlighted a down-regulation of inflammatory pathways and up-regulation of phospho-sites involved in cell metabolism and VEGF-related angiogenesis in Dusp3-/- vs WT ischaemic kidneys. Dusp3-/- ischaemic kidneys showed decreased mRNA levels of CD11b, TNF-α, KIM-1, IL-6, IL-1ß and caspase-3 compared to controls. The numbers of PCNA-, F4-80- and CD11b-positive cells were reduced in Dusp3-/- vs WT kidneys post-I/R. CONCLUSION: Genetic inactivation of Dusp3 is associated with kidney conditioning against I/R, possibly due to attenuated inflammation and improved perfusion.

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-α 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.

Identification and pharmacological characterization of succinate receptor agonists.

BACKGROUND AND PURPOSE: The succinate receptor (formerly GPR91 or SUCNR1) is described as a metabolic sensor that may be involved in homeostasis. Notwithstanding its implication in important (patho)physiological processes, the function of succinate receptors has remained ill-defined because no pharmacological tools were available. We report on the discovery of the first family of potent synthetic agonists. EXPERIMENTAL APPROACH: We screened a library of succinate analogues and analysed their activity on succinate receptors. Also, we modelled a pharmacophore and a binding site for this receptor. New agonists were identified based on the information provided by these two approaches. Their activity was studied in various bioassays, including measurement of cAMP levels, [Ca2+ ]i mobilization, TGF-α shedding and recruitment of arrestin 3. The in vivo effects of activating succinate receptors with these new agonists was evaluated on rat BP. KEY RESULTS: We identified cis-epoxysuccinic acid and cis-1,2-cyclopropanedicarboxylic acid as agonists with an efficacy similar to that of succinic acid. Interestingly, cis-epoxysuccinic acid was 10- to 20-fold more potent than succinic acid on succinate receptors. For example, cis-epoxysuccinic acid reduced cAMP levels with a pEC50  = 5.57 ± 0.02 (EC50  = 2.7 µM), compared with succinate pEC50  = 4.54 ± 0.08 (EC50  = 29 µM). The rank order of potency of the three agonists was the same in all in vitro assays. Both cis-epoxysuccinic and cis-1,2-cyclopropanedicarboxylic acid were as potent as succinate in increasing rat BP. CONCLUSIONS AND IMPLICATIONS: We describe new agonists at succinate receptors that should facilitate further research on this understudied receptor.

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

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.

Activation of the calcium-sensing receptor before renal ischemia/reperfusion exacerbates kidney injury.

Activation of the calcium-sensing receptor (CaSR) by ischemia/reperfusion (I/R) favours apoptosis in cardiomyocytes, hepatocytes and neurons. Its role in renal I/R is unknown. We investigated the impact of pharmacological preactivation of the CaSR on kidney structure and function in a murine model of bilateral renal 30-min ischemia and 48-hour reperfusion, and in a 6-year cohort of kidney transplant recipients (KTR). C57BL/6J mice were administered daily with CaSR agonist, R-568, or with vehicle for 48 hours. Evaluation of serum urea and creatinine levels, renal histology and urine metabolome by nuclear magnetic resonance showed that R-568 was not nephrotoxic per se. Following I/R, serum urea and creatinine levels increased higher in R-568-treated animals than in controls. Jablonski's score was significantly greater in R-568-treated kidneys, which showed a higher rate of cell proliferation and apoptosis in comparison to controls. Next, we retrospectively identified 36 patients (10.7% of our cohort) who were treated by CaSR agonist, cinacalcet, at the time of kidney transplantation (KTx). After matching these to 61 KTR upon type of donor, cold ischemic time, residual diuresis, and donor age, we observed that delayed graft function, i.e. need for dialysis in the first week after KTx, occurred in 42 and 23% of cinacalcet-treated and control groups, respectively (p≤0.05). These data suggest that pharmacological preactivation of the CaSR before renal I/R exacerbates kidney injury.