Elia Ripoll

Mesenchymal Stem Cell Therapy Prevents Interstitial Fibrosis and Tubular Atrophy in a Rat Kidney Allograft Model

In solid organ transplantation, mesenchymal stem cell (MSC) therapy is strongly emerging among other cell therapies due to the positive results obtained in vitro and in vivo as an immunomodulatory agent and their potential regenerative role. We aimed at testing whether a single dose of MSCs, injected at 11 weeks after kidney transplantation for the prevention of chronic mechanisms, enhanced regeneration and provided protection against the inflammatory and fibrotic processes that finally lead to the characteristic features of chronic allograft nephropathy (CAN). Either bone marrow mononuclear cells (BMCs) injection or no-therapy (NT) were used as control treatments. A rat kidney transplantation model of CAN with 2.5 h of cold ischemia was used, and functional, histological, and molecular parameters were assessed at 12 and 24 weeks after transplantation. MSC and BMC cell therapy preserves renal function at 24 weeks and abrogates proteinuria, which is typical of this model (NT24w: 68.9 ± 26.5 mg/24 h, MSC24w: 16.6 ± 2.3 mg/24 h, BMC24w: 24.1 ± 5.3 mg/24 h, P<0.03). Only MSC-treated animals showed a reduction in interstitial fibrosis and tubular atrophy (NT24w: 2.3 ± 0.29, MSC24w: 0.4 ± 0.2, P<0.03), less T cells (NT: 39.6 ± 9.5, MSC: 8.1 ± 0.9, P<0.03) and macrophages (NT: 20.9 ± 4.7, MSC: 5.9 ± 1.7, P<0.05) infiltrating the parenchyma and lowered expression of inflammatory cytokines while increasing the expression of anti-inflammatory factors. MSCs appear to serve as a protection from injury development rather than regenerate the damaged tissue, as no differences were observed in Ki67 expression, and kidney injury molecule-1, Clusterin, NGAL, and hepatocyte growth factor expression were only up-regulated in nontreated animals. Considering the results, a single delayed MSC injection is effective for the long-term protection of kidney allografts.

Update on Controls for Isolation and Quantification Methodology of Extracellular Vesicles Derived from Adipose Tissue Mesenchymal Stem Cells

The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV. The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 μm). As a control, non-conditioned culture medium was used (control medium). To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV.

In vivo therapeutic efficacy of intra-renal CD40 silencing in a model of humoral acute rejection

The humoral branch of the immune response has an important role in acute and chronic allograft dysfunction. The CD40/CD40L costimulatory pathway is crucial in B- and T- alloresponse. Our group has developed a new small interfering RNA (siRNA) molecule against CD40 that effectively inhibits its expression. The aim of the present study was to prevent rejection in an acute vascular rejection model of kidney transplant by intra-graft gene silencing with anti-CD40 siRNA (siCD40), associated or not with sub-therapeutic rapamycin. Four groups were designed: unspecific siRNA as control; sub-therapeutic rapamycin; siCD40; and combination therapy. Long-surviving rats were found only in both siCD40-treated groups. The CD40 mRNA was overexpressed in control grafts but treatment with siCD40 decreased its expression. Recipient spleen CD40+ B-lymphocytes were reduced in both siCD40-treated groups. Moreover, CD40 silencing reduced donor-specific antibodies, graft complement deposition and immune-inflammatory mediators. The characteristic histological features of humoral rejection were not found in siCD40-treated grafts, which showed a more cellular histological pattern. Therefore, the intra-renal effective blockade of the CD40/CD40L signal reduces the graft inflammation as well as the incidence of humoral vascular acute rejection, finally changing the type of rejection from humoral to cellular.

CD40 gene silencing reduces the progression of experimental lupus nephritis modulating local milieu and systemic mechanisms.

Lupus nephritis (LN) is an autoimmune disorder in which co-stimulatory signals have been involved. Here we tested a cholesterol-conjugated-anti-CD40-siRNA in dendritic cells (DC) in vitro and in a model of LPS to check its potency and tissue distribution. Then, we report the effects of Chol-siRNA in an experimental model of mice with established lupus nephritis. Our in vitro studies in DC show a 100%intracellular delivery of Chol-siRNA, with a significant reduction in CD40 after LPS stimuli. In vivo in ICR mice, the CD40-mRNA suppressive effects of our Chol-siRNA on renal tissue were remarkably sustained over a 5 days after a single preliminary dose of Chol-siRNA. The intra-peritoneal administration of Chol-siRNA to NZB/WF1 mice resulted in a reduction of anti-DNA antibody titers, and histopathological renal scores as compared to untreated animals. The higher dose of Chol-siRNA prevented the progression of proteinuria as effectively as cyclophosphamide, whereas the lower dose was as effective as CTLA4. Chol-siRNA markedly reduced insterstitialCD3+ and plasma cell infiltrates as well as glomerular deposits of IgG and C3. Circulating soluble CD40 and activated splenic lymphocyte subsets were also strikingly reduced by Chol-siRNA. Our data show the potency of our compound for the therapeutic use of anti-CD40-siRNA in human LN and other autoimmune disorders.

CD154-CD40 T-cell co-stimulation pathway is a key mechanism in kidney ischemia-reperfusion injury

Ischemia-reperfusion occurs in a great many clinical settings and contributes to organ failure or dysfunction. CD154-CD40 signaling in leukocyte–endothelial cell interactions or T-cell activation facilitates tissue inflammation and injury. Here we tested a siRNA anti-CD40 in rodent warm and cold ischemia models to check the therapeutic efficacy and anti-inflammatory outcome of in vivo gene silencing. In the warm ischemia model different doses were used, resulting in clear renal function improvement and a structural renoprotective effect. Renal ischemia activated the CD40 gene and protein expression, which was inhibited by intravenous siRNA administration. CD40 gene silencing improved renal inflammatory status, as seen by the reduction of CD68 and CD3 T-cell infiltrates, attenuated pro-inflammatory, and enhanced anti-inflammatory mediators. Furthermore, siRNA administration decreased a spleen pro-inflammatory monocyte subset and reduced TNFα secretion by splenic T cells. In the cold ischemia model with syngeneic and allogeneic renal transplantation, the most effective dose induced similar functional and structural renoprotective effects. Our data show the efficacy of our siRNA in modulating both the local and the systemic inflammatory milieu after an ischemic insult. Thus, CD40 silencing could emerge as a novel therapeutic strategy in solid organ transplantation.

Silencing of CD40 in vivo reduces progression of experimental atherogenesis through an NF-κB/miR-125b axis and reveals new potential mediators in the pathogenesis of atherosclerosis

BACKGROUND AND AIMS CD40/CD40L signaling exerts a critical role in the development of atherosclerosis, and microRNAs (miRNAs) are key regulators in vascular inflammation and plaque formation. In this work, we investigated mRNA/miRNA expression during progression of atherosclerotic lesions through CD40 silencing. METHODS We silenced CD40 with a specific siRNA in ApoE-/- mice and compared expression of mRNA/miRNA in ascending aorta with scrambled treated mice. RESULTS siRNA-CD40 treated mice significantly reduced the extension and severity of atherosclerotic lesions, as well as the number of F4/80+, galectin-3+ macrophages and NF-κB+ cells in the intima. Genome-wide mRNA/miRNA profiling allowed the identification of transcripts, which were significantly upregulated during atherosclerosis; among them, miR-125b and miR-30a, Xpr1, a regulator of macrophage differentiation, Taf3, a core transcription factor and the NF-κB activator Ikkβ, whereas, the NF-κB inhibitor Ikbα was downregulated during disease progression. All those changes were reversed upon CD40 silencing. Interestingly, TAF3, XPR1 and miR-125b were also overexpressed in human atherosclerotic plaques. Murine Taf3 and Xpr1 were detected in the perivascular adipose tissue (PVAT), and Taf3 also in intimal foam cells. Finally, expression of miR-125b was regulated by the CD40-NF-κB signaling axis in RAW264.7 macrophages. CONCLUSIONS CD40 silencing with a specific siRNA ameliorates progression of experimental atherosclerosis in ApoE-/- mice, and evidences a role for NF-κB, Taf3, Xpr1, and miR-125b in the pathogenesis of atherosclerosis.

Cold ischaemia, innate immunity and deterioration of the glomerular filtration barrier in antibody-mediated acute rejection

BACKGROUND In renal transplantation, cold ischaemia (CI) determines acute rejection through innate immunity among others. Acute rejection episodes are a risk factor for late allograft dysfunction and proteinuria. This implies some alteration of the glomerular filtration barrier (GFB). Besides its effects on acute rejection, we hypothesized that CI might somehow damage the GFB being directly responsible for late proteinuria. METHODS On rat kidney allografts suffering from antibody-mediated acute rejection with or without CI and compared with syngeneic grafts, we quantified the gene expression of innate and adaptive immune mediators and assessed the capillary glomerular basement membranes (CapBM) by immunostaining collagen-IV (ColIV). ColIV was also assessed in equivalent groups from a previous chronic study followed up for 24 weeks. RESULTS CI up-regulated enzymes critical in the stabilization of collagen chains, increasing ColIV deposition and thickening the CapBM. CI increased the C4d and IgG deposits within grafts, amplified innate immunity (heat shock protein 70, fibronectin, Toll-like-receptor-4 and MyD88) and synergized with alloreactivity in triggering adaptive response through CD40. CONCLUSIONS Initial CI increased the ColIV deposition in CapBM, damaging the GFB and being responsible for part of the proteinuria associated with late allograft dysfunction. This deterioration of the GFB is related to the early innate immunity activation and subsequent up-regulation of CD40 in acute rejected grafts. In chronic rejected allografts, thickened CapBM may be a consequence of an unresolved immune-inflammatory response worsened by CI.

RNAi-Based Therapy in Experimental Ischemia-Reperfusion Injury. The New Targets

BACKGROUND Ischemia reperfusion injury is an important pathophysiological process in many fields such as transplantation, stroke, atherosclerosis, trauma and myocardial infarction. Recent advances in gene silencing may help to reduce ischemic effects, targeting molecules related to this pathological process. METHODS AND RESULTS Here, we review the different silencing approaches in ischemic injury, highlighting the role of co-stimulatory molecules in renal transplantation. CONCLUSION Gene silencing appears as a new strategy to prevent the inflammation and injury associated with ischemia.

Use of mycophenolate in ANCA-associated renal vasculitis: 13 years of experience at a university hospital

INTRODUCTION Standard therapy with corticosteroids (CS) and cyclophosphamide (CYC) followed by azathioprine has been shown to improve renal and patient survival in ANCA-associated renal vasculitis (rAAV). Mycophenolate mofetil (MF) has been progressively introduced for the treatment of rAAV in the last years because of its immunosuppressive efficacy combined with a lower toxicity profile. In this study, we retrospectively analyse the results of the introduction of MF for maintenance and induction therapy in rAAV in our institution from 2001 to 2013. RESULTS We reported 67 patients treated with MF as a maintenance treatment, divided by baseline serum creatinine (>500 µmol/L: Group 1 and <500 µmol/L: Group 2) and treatment schedule. Twenty-nine of the 67 patients were also treated with MF as induction treatment, mostly in Group 2. During the follow-up (2 years after the diagnosis) creatinine levels for serum glomerular filtration rate, ANCA titres, C-reactive protein and percentage of haematuria decreased in all groups. In Group 2, parameters and also relapse rates were similar at 24 months in patients treated with CYC or MF as an induction treatment (Subgroups 2a and 2b, respectively). Median dose of MF in maintenance treatment was 1000 mg daily and prednisone dose was tapered to 10 mg daily from Month 3. After 24 months, 82% of patients remained on MF therapy, 18% had discontinued the treatment, seven of them due to medical indication and two because of gastrointestinal intolerance. The percentage of patients that started renal replacement therapy was irregular in Group 1 depending on the subgroup (25-100%), and 10% in Group 2. Adverse effects, such as neutropenia, infections and neoplasia, were more prevalent in groups treated with CYC. CONCLUSION In conclusion, in our patients with rAAV, MF demonstrated to be an effective and well-tolerated option for maintenance treatment. As an induction treatment, MF seems to be similar to CYC for patients with moderate renal failure in the diagnosis.

New approaches for the treatment of lupus nephritis in the 21st century: from the laboratory to the clinic.

Systemic lupus erythematosus is a complex autoimmune disorder affecting multiple organ systems. Glomerulonephritis leading to severe proteinuria, chronic renal failure and end-stage renal disease remains one of the most severe complications of systemic lupus erythematosus and is associated with significant morbidity and mortality. Conventional lupus nephritis (LN) treatment based on cyclophosphamide, steroids and, recently, mycophenolatemofetil has improved the outcome of the disease over the last 50 years, although failure to achieve remission or treatment resistance has been reported in 18-57% of patients. Chronic complications such as long-term toxicity dampen their ability to maintain disease remission. There is a need to develop more specific pharmacological agents for patients to provide choices that are equally effective, less toxic and have fewer complications. During the last 10 years, experimental studies based on different pathogenesis pathways of LN have provided an enormous amount of knowledge and have offered the possibility to target the disease with selective approaches. In this article, we summarize the new experimental strategies that have recently been utilized to target LN, focusing on mechanisms of action.