Characterization of endothelial progenitor cells (EPCs) in patients with chronic kidney disease (CKD) Immunomodulatory effects of erythropoietin (EPO)

Caratterizzazione di EPC in pazienti con malattia renale cronica. Background: Gli effetti del recettore della vitamina D (VDR)e dell'espressione dell' osteocalcina (OCN), così come VDR agonista terapia (VDRA) su circolanti cellule progenitrici endoteliali (EPCs) non è stato ancora chiarito. Metodi: sono state analizzat EPCs in 23 controlli sani e 53 pazienti sottoposti a dialisi. La percentuale di EPCs (CD34+KDR+CD133+/-CD45-) VDR+/- o OCN+/- è stata analizzata in citometria a flusso e correlata con molecole coinvolte nelle malattie. Risultati: EPCs aumentano nei pazienti con CKD trattati con VDRAs. EPCsOCN +/- in pazienti non trattati con VDRAs correlano positivamente con il calcio sierico e reticolociti, e negativamente con DKK1. La percentuale di EPCsVDR +/- correla negativamente con OPN.  EPCsOCN + nei pazienti trattati con VDRAs correla positivamente con la vitamina D. La percentuale di EPCsVDR +/- positivamente correla con IL-6. Conclusioni: la terapia VDRA influenza l'espressione VDR e di OCN su EPCs circolanti. Dal momento che l'espressione OCN può contribuire alla calcificazione vascolare, ipotizziamo un putativo effetto pro-calcificazione di VDRA. Effetti immunomodulatori di EPO. Background: Il cloruro di sodio spinge l'induzione di cellule TH17 patogeni e TH1. La correzione dell'anemia con eritropoietina (EPO) è associato ad un miglioramento della tolleranza del trapianto di rene. Prove emergenti indicano che queste osservazioni possano essere eritropoiesi-indipendente e che l'EPO presenta proprietà immunosoppressive. Metodi: esaminato gli effetti del trattamento con EPO e sale su cellule T umana, apoptosi e la produzione di INF-γ, l'effetto sulla iTh17 e iFoxP3. Le cellule sono state analizzate con l'analisi di citometria a flusso. Risultati: NaCl aumenta la proliferazione di CD4+ e CD8+, iTh17 e la produzione di INFγ. Questo effetto è prevenuto da EPO. EPO aumenta l'induzione di Foxp3. Non cambia l'apoptosi e la stabilità di FoxP3. EPO è in grado di contiene l'effetto pro infiammatorio del saleFirst project: Characterization of EPCs in patients with CKD. Background: The effects of vitamin D receptor (VDR) and osteocalcin (OCN) expression as well as VDR agonist (VDRA) therapy on circulating endothelial progenitor cells (EPCs) has not been elucidated yet. Methods: we therefore analyzed EPCs in 23 healthy controls and 53 patients undergoing dialysis. The percentage of EPCs (CD34+CD133+/-KDR+CD45-) expressing VDR or OCN were analyzed using flow cytometry and correlated with molecules involved in diseases. Results: EPCs increase in CKD patients treated with VDRAs. EPCsOCN+/- in patients untreated with VDRAs correlated positively with serum calcium and reticulocytes, and negatively with DKK1. The percentage of EPCsVDR+/- correlated negatively with OPN. EPCsOCN+ in patients treated with VDRAs correlated positively with vitamin D. The percentage of EPCsVDR+/- correlated positively with IL-6. Conclusions: our data suggest that VDRA therapy influence VDR and OCN expression on circulating EPCs. Since OC expression may contribute to vascular calcification, we hypothesize a putative pro-calcifying effect of VDRA. Second project: Immunomodulatory effects of EPO. Background: Sodium chloride drives the induction of pathogenic TH17 cells and TH1. Correction of anemia with erythropoietin (EPO) is associated with improved kidney transplant outcomes. Emerging evidence indicates that these observations may be erythropoiesis-independent and that EPO exhibits immunosuppressive properties. Methods: examined the effects of treatment with EPO and salt on human T-cell alloimmunity, the apoptosis and the production to INF-γ, the effect on iTh17 and iFoxP3. The cells were analyzed with flow cytometry analysis. Results: NaCl increases the proliferation of CD4+ and CD8+ cells, iTh17 and the production of INFγ. This effect is prevented by EPO. EPO increases the induction of Foxp3. It does not change the apoptosis and stability of FoxP3. Epo contains the inflammatory effect of NaCl. EPO in the kidney, where NaCl is high, may have a tolerance effect

Efficacy of Supra-HFR in Removing FGF23 and Cytokines: A Single Session Analysis

Background/Aim: Supra hemodiafiltration with reinfusion of the endogenous ultrafiltrate (Supra-HFR) is a dialysis technique used to improve uremic toxin removal in the range of the middle molecular weight molecules. Supra-HFR does not require the preparation and online infusion of high purity dialysis water because it allows the production of an endogenous ultrafiltrate that undergoes detoxification through an adsorbing resin. Patients and Methods: We investigated the ability of Supra-HFR to remove fibroblast growth factor 23 (FGF23), interleukin 6 (IL-6), tumor necrosis factor alpha (TNFalpha), interleukin 8 (IL-8), and transforming growth factor alpha (TGF-alpha) after a single session dialysis in nine patients affected by end stage renal disease (ESRD). The same patients underwent a single session of online hemodiafiltration (OLHDF) to evaluate possible differences in FGF23 and IL-6 levels. Results: A significant reduction in FGF23 was observed with both Supra-HFR (p=0.001) and OL-HDF. As for TNF-alpha and TGF-alpha, which were measured using Supra-HFR only, their percentage values were significantly lower at the end of dialysi

Impact of Single Hemodialysis Treatment on immune Cell Subpopulations

: Hemodialysis (HD) is known to trigger a chronic inflammatory status, affecting the innate and acquired immune response. This study was aimed at a comparative analysis of immune cell subsets, proliferation, and apoptosis in subjects receiving chronic HD treatment with respect to a healthy control. Regardless of the dialysis filter used, we observed a reshaping of the acquired immune component both with respect to healthy patients and between the various sessions of dialysis treatment, with an impairment of CD3 cells, along with an increase in CD4 and CD8 cell populations producing pro-inflammatory factors such as IL-17 and IFN-gamma. The population of B cells, monocytes and NK cells were not impaired by the dialysis procedure. These results confirmed the high impact of the HD treatment on the patient's immune system, underlying the imbalance of T cell counterparts

A Comprehensive Phenotypic and Functional Immune Analysis Unravels Circulating Anti-Phospholipase A2 Receptor Antibody Secreting Cells in Membranous Nephropathy Patients

Introduction: Primary membranous nephropathy (MN) is characterized by the presence of antipodocyte antibodies, but studies describing phenotypic and functional abnormalities in circulating lymphocytes are limited. Methods: We analyzed 68 different B- and T-cell subsets using flow cytometry in 30 MN patients (before initiating immunosuppression) compared with 31 patients with non-immune-mediated chronic kidney disease (CKD) and 12 healthy individuals. We also measured 19 serum cytokines in MN patients and in healthy controls. Lastly, we quantified the ex vivo production of phospholipase A2 receptor (PLA2R)-specific IgG by plasmablasts (measuring antibodies in culture supernatants and by the newly developed FluoroSpot assay [AutoImmun Diagnostika, Strasberg, Germany]) and assessed the circulating antibody repertoire by phage immunoprecipitation sequencing (PhIP-Seq). Results: After adjusting for multiple testing, plasma cells and regulatory B cells (BREG) were significantly higher (P < 0.05) in MN patients compared with both control groups. The percentages of circulating plasma cells correlated with serum anti-PLA2R antibody levels (P = 0.042) and were associated with disease activity. Ex vivo-expanded PLA2R-specific IgG-producing plasmablasts generated from circulating PLA2R-specific memory B cells (mBCs) correlated with serum anti-PLA2R IgG antibodies (P < 0.001) in MN patients. Tumor necrosis factor-alpha (TNF-alpha) was the only significantly increased cytokine in MN patients (P < 0.05), whereas there was no significant difference across study groups in the autoantibody and antiviral antibody repertoire. Conclusion: This extensive phenotypic and functional immune characterization shows that autoreactive plasma cells are present in the circulation of MN patients, providing a new therapeutic target and a candidate biomarker of disease activity

An intriguing link between human leukocyte antigen G, T-regulatory cells and neutrophil gelatinase-associated lipocalin in immune tolerance induction

Not applicabl

Potential advantages of acute kidney injury management by mesenchymal stem cells

Mesenchymal stem cells are currently considered as a promising tool for therapeutic application in acute kidney injury (AKI) management. AKI is characterized by acute tubular injury with rapid loss of renal function. After AKI, inflammation, oxidative stress and excessive deposition of extracellular matrix are the molecular events that ultimately cause the end-stage renal disease. Despite numerous improvement of supportive therapy, the mortality and morbidity among patients remain high. Therefore, exploring novel therapeutic options to treat AKI is mandatory. Numerous evidence in animal models has demonstrated the capability of mesenchymal stem cells (MSCs) to restore kidney function after induced kidney injury. After infusion, MSCs engraft in the injured tissue and release soluble factors and microvesicles that promote cell survival and tissue repairing. Indeed, the main mechanism of action of MSCs in tissue regeneration is the paracrine/endocrine secretion of bioactive molecules. MSCs can be isolated from several tissues, including bone marrow, adipose tissue, and blood cord; pre-treatment procedures to improve MSCs homing and their paracrine function have been also described. This review will focus on the application of cell therapy in AKI and it will summarize preclinical studies in animal models and clinical trials currently ongoing about the use of mesenchymal stem cells after AKI

Impact of blood source and component manufacturing on neurotrophin content and in vitro cell wound healing

none7noBackground: We evaluated neurotrophin (NF) levels and their impact on in vitro cell wound healing in eye drops from differently prepared blood sources (cord blood [CB], and peripheral blood [PB]) in the same donor, to avoid intrasubject biological variability. Materials and methods: Twenty healthy adult donor PB samples, and twenty CB samples acquired at the time of delivery were processed to obtain serum (S), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and S retrieved from PRP after activation with Ca-gluconate (PRP-R). The levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) were assessed with a Luminex xMAP (Luminex Corporation), and by using multikine kits from R&amp;D system, and were statistically analysed in the eight different preparations. The impact of S, PRP, PPP, PRP-R from both sources on a cell line responding to NF supplementation (MIO-M1, UCL Institute of Ophthalmology, London, UK) was tested with a scratch wound assay, and analysed by IncuCyte S3 equipment. Results: All the preparations from CB showed higher NF levels, except for BDNF where no difference was found as compared to PB. PRP showed higher NF levels with respect to S, PPP and PRP-R in this decreasing order. Younger donors in PB contributed with higher NF levels. The scratch assay showed different cell migration results, with a complete wound closure only recorded with the supplementation of CB-S, and a progressive reduction by using PRP, PRP-R, and PPP from both sources. Discussion: Protocols of preparation and choice of blood source determine different NF levels in the final products. The therapeutic use of a natural neurotrophin pool from blood sources might have a clinical impact in several different settings. Efforts are needed to standardise the manufacturing and the product content in order to establish and modulate the posology of the final supplementation.openValente, Sabrina; Curti, Nico; Giampieri, Enrico; Randi, Vanda; Donadei, Chiara; Buzzi, Marina; Versura, PieraValente, Sabrina; Curti, Nico; Giampieri, Enrico; Randi, Vanda; Donadei, Chiara; Buzzi, Marina; Versura, Pier

Is chronic kidney disease-mineral and bone disorder associated with the presence of endothelial progenitor cells with a calcifying phenotype?

Background: Chronic kidney disease-mineral and bone disorder (CKD-MBD) has been implicated in vascular calcification pathogenesis. CKD-MBD results in alterations in the number and function of circulating endothelial progenitor cells (EPCs), physiological regulators of angiogenesis and vessel repair, commonly defined as proangiogenic progenitor cells (PACs) by the antigen pattern CD34+CD133+KDR+CD45-and putative EPCs by the pattern CD34+CD133-KDR+CD45-. These cells might acquire a calcifying phenotype in CKD-MBD, expressing mineralization biomarkers. We investigated the expression of vitamin D receptor (VDR) and osteocalcin (OC) on EPCs of healthy individuals and haemodialysis patients, and their possible associations with circulating biomarkers of inflammation and vascular calcification. Methods: We compared EPC counts, expressing VDR or OC, in 23 healthy subjects versus 53 haemodialysis patients, 17 of them without vitamin D receptor agonist (VDRA) therapy and 35 treated with calcitriol (n = 17) or paricalcitol (n = 18). The correlations with serum levels of inflammatory and calcification indexes were also analysed. Results: All subsets expressing VDR or OC were significantly higher in haemodialysis patients compared with healthy controls, but PACs were increased only in VDRA treatment subgroup, while putative EPCs showed a similar rise also in untreated patients. In VDRA-untreated patients, OC+ PACs correlated positively with calcium levels, while in VDRA-treated patients, VDR+ PACs correlated positively with interleukin 6 levels, and OC+ PACs correlated positively 25-hydroxyvitamin D levels. Conclusions: Our data suggest that in CKD-MBD, EPCs undergo an endothelial-to-procalcific shift, representing a risk factor for vascular calcification. A link between mineral disorders and vitamin D replacement therapy emerged, with potential adverse effects for CKD patients

Erythropoietin inhibits SGK1-dependent TH17 induction and TH17-dependent kidney disease

IL-17-producing CD4(+) (Th17) cells are pathogenically linked to autoimmunity and, specifically, to autoimmune kidney disease. The newly recognized immunoregulatory functions of erythropoietin (EPO) and its predominant intrarenal source suggested that EPO physiologically regulates Th17 cell differentiation, thereby serving as a barrier to development of autoimmune kidney disease. Using in vitro studies of human and murine cells and in vivo models, we show that EPO ligation of its receptor (EPO-R) on CD4(+) T cells directly inhibits Th17 cell generation and promotes transdifferentiation of Th17 cells into IL-17 FOXP3(+)CD4(+) T cells. Mechanistically, EPO/EPO-R ligation abrogates upregulation of SGK1 gene expression and blocks p38 activity to prevent SCK1 phosphorylation, thereby inhibiting RORC-mediated transcription of IL17and IL23 receptor genes. In a murine model of Th17 cell-dependent aristolochic acid-induced interstitial kidney disease associated with reduced renal EPO production, we demonstrate that transgenic EPO overexpression or recombinant EPO (rEPO) administration limits Th17 cell formation and clinical/histological disease expression. EPO/EPO-R ligations on CD4(+) T cells abrogate, while absence of T cell-expressed EPO-R augments, Th17 cell induction and clinical/histological expression of pristane-induced glomerulonephritis (associated with decreased intrarenal EPO). rEPO prevents spontaneous glomerulonephritis and Th17 cell generation in MRL-lpr mice. Together, our findings indicate that EPO physiologically and therapeutically modulates Th17 cells to limit expression of Th17 cell-associated autoimmune kidney disease