Urine-Derived Renal Epithelial Cells (URECs) from Transplanted Kidneys as a Promising Immunomodulatory Cell Population.

Kidney transplantation is a lifesaving procedure for patients with end-stage kidney disease (ESKD). Organs derived from donation after cardiac death (DCD) are constantly increasing; however, DCD often leads to ischaemia-reperfusion (IR) and Acute Kidney Injury (AKI) events. These phenomena increase kidney cell turnover to replace damaged cells, which are voided in urine. Urine-derived renal epithelial cells (URECs) are rarely present in the urine of healthy subjects, and their loss has been associated with several kidney disorders. The present study aimed to characterize the phenotype and potential applications of URECs voided after transplant. The results indicate that URECs are highly proliferating cells, expressing several kidney markers, including markers of kidney epithelial progenitor cells. Since the regulation of the immune response is crucial in organ transplantation and new immunoregulatory strategies are needed, UREC immunomodulatory properties were investigated. Co-culture with peripheral blood mononuclear cells (PBMCs) revealed that URECs reduced PBMC apoptosis, inhibited lymphocyte proliferation, increased T regulatory (Treg) cells and reduced T helper 1 (Th1) cells. URECs from transplanted patients represent a promising cell source for the investigation of regenerative processes occurring in kidneys, and for cell-therapy applications based on the regulation of the immune response.

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.

Renal Benefits of CO2 as a Contrast Media for EVAR Procedures: New Perspectives on 1 Year Outcomes.

BACKGROUND AND OBJECTIVES: Endovascular aneurism repair (EVAR) is a minimally invasive alternative to open surgery for the treatment of abdominal aortic aneurysm. Iodine contrast medium (ICM) is considered the gold standard, at the high price of related nephrotoxicity and allergic reactions. Carbon dioxide (CO2) has been suggested as an alternative non-nephrotoxic contrast media agent. We aimed to evaluate the safety and the renal impact of the administration of CO2, compared with ICM in EVAR procedures. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: We retrospectively reviewed data of patients who underwent EVAR at the Vascular Surgery Department of the Sant'Orsola Hospital in Bologna. Estimated glomerular filtration rate (eGFR) was evaluated before intervention, immediately after and at 12 months. RESULTS: In total, 22 patients received CO2 and low-dose ICM (CO2 Group) and 22 received standard ICM (Control Group), matched for clinical characteristics and renal function at the time of procedure. Pre and post-operative renal function values (eGFR) were compared between the two groups: in the immediate post-operative the group treated with CO2 and low-dose ICM globally showed a slight improvement in renal function (mean eGFR +5.10%±3.2), meanwhile the group treated with standard dose of ICM presented a significant worsening of renal function compared with pre-procedure values (mean eGFR -9.65%±4). Incidence of post-contrast acute kidney injury (PC-AKI) was 9% in the CO2 group vs 27% in the Control group. At 12 months, the renal impairment was significantly greater in the ICM group than in the CO2 group (mean eGFR decrease -19.2%±11.1 and -7.40%±3.5, respectively). CONCLUSIONS: Administration of either CO2 alone or along with low-dose ICM showed to be safer than full-dose ICM alone, lowering the incidence of PC-AKI in patients undergoing EVAR. Unexpectedly, our study revealed also a significant worsening of renal function in patients treated with standard dose of ICM in 1-year follow-up, introducing the concept that acute renal damage caused by ICM could elicit a chronic injury process that affect long-term renal outcomes. CLINICAL IMPACT: Evaluating the safety and the renal impact of the administration of CO2, compared to Iodinate Contrast Medium, in EVAR procedures represents a first step in order to further tayloring medical procedures on patients characteristics. Our findings can guide the clinicians and surgeons in the procedures choice, not considering only the immediate effect of ICM on renal function but also the potential long-term effects.

Vitamin D and the Kidney: Two Players, One Console.

Vitamin D belongs to the group of liposoluble steroids mainly involved in bone metabolism by modulating calcium and phosphorus absorption or reabsorption at various levels, as well as parathyroid hormone production. Recent evidence has shown the extra-bone effects of vitamin D, including glucose homeostasis, cardiovascular protection, and anti-inflammatory and antiproliferative effects. This narrative review provides an overall view of vitamin D's role in different settings, with a special focus on chronic kidney disease and kidney transplant.

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 (TNF-alpha), 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 (OL-HDF) 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 dialysis than at the start (p=0.0028 and p=0.03, respectively). This did not change with post-dialysis rebound. Supra-HFR was found to have no effect on IL-6 and IL-8. Interestingly, the removal rate for FGF23 and IL-6 was similar to that observed with OL-HDF. CONCLUSION: Supra-HFR was not superior to OL-HDF, with suboptimal convective volume in the removal of the molecules tested, especially FGF23, which is considered a large middle molecular weight uremic toxin.

Adaptive Mechanisms of Renal Bile Acid Transporters in a Rat Model of Carbon Tetrachloride-Induced Liver Cirrhosis.

BACKGROUND: Acute kidney injury (AKI) is common in advanced liver cirrhosis, a consequence of reduced kidney perfusion due to splanchnic arterial vasodilation and intrarenal vasoconstriction. It clinically manifests as hepatorenal syndrome type 1, type 2, or as acute tubular necrosis. Beyond hemodynamic factors, an additional mechanism may be hypothesized to explain the renal dysfunction during liver cirrhosis. Recent evidence suggest that such mechanisms may be closely related to obstructive jaundice. METHODS: Given the not completely elucidated role of bile acids in kidney tissue damage, this study developed a rat model of AKI with liver cirrhosis induction by carbon tetrachloride (CCl4) inhalation for 12 weeks. Histological analyses of renal and liver biopsies were performed at sacrifice. Organic anion tubular transporter distribution and apoptosis in kidney cells were analyzed by immunohistochemistry. Circulating and urinary markers of inflammation and tubular injury were assayed in 21 treated rats over time (1, 2, 4, 8, and 12 weeks of CCl4 administration) and 5 controls. RESULTS: No renal histopathological alterations were found at sacrifice. Comparing treated rats with controls, organic anion transporters were differentially expressed and localized. High serum bile acid values were detected in cirrhotic animals, while caspase-3 staining was negative in both groups. Increased levels of serum inflammatory and urinary tubular injury biomarkers were observed during cirrhosis progression, with a peak after 4 and 8 weeks of treatment. CONCLUSIONS: These findings suggest possible adaptive tubular mechanisms for bile acid transporters in response to cirrhosis-induced AKI.

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

BACKGROUND: 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&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.

Impact of nephrotoxic drugs on urinary biomarkers of renal function in very preterm infants.

BACKGROUND: Following preterm birth, the immature kidney is exposed to several harmful conditions, with an increased risk of renal impairment. We aimed to assess urinary biomarkers of renal function in very preterm infants during early nephrotoxic treatments. METHODS: Infants ≤32 weeks' gestation and ≤1500 g were enrolled in this observational prospective study. Urine samples were collected on day 1(T1), 2-4(T2), 5-7(T3), 8-10(T4), 11-13(T5). The following urinary biomarkers were determined: osteopontin (uOPN), epidermal growth factor (uEGF), neutrophil gelatinase-associated lipocalin (uNGAL), cystatin C (uCysC). The infants were grouped according to their exposure to amikacin or ibuprofen during the study period and a between-group comparison of urinary biomarkers at each time point was performed. RESULTS: Thirty-six infants were included. Urinary CysC, uOPN, and uNGAL rose significantly during ibuprofen or amikacin treatment, while no difference was observed for uEGF. After adjustment for possible influencing factors, amikacin administration was associated with higher uCysC at T1 (p = 0.007) and T2 (p = 0.016), whereas ibuprofen increased uOPN (p = 0.001) and uNGAL concentration (p = 0.009) at T3. CONCLUSION: Nephrotoxic therapies induce molecule-specific change patterns of renal function biomarkers in treated preterm infants. Serial assessments of these biomarkers may aid to identify neonates at risk of renal impairment and to develop tailored therapeutic approaches. IMPACT: Despite the wide use of nephrotoxic therapies in neonatal settings, little is known on their effect on renal function biomarkers in preterm infants. This study describes molecule-specific change patterns of urinary biomarkers during ibuprofen and amikacin administration, suggesting underlying pathophysiological effects on renal function. Given their low analytical costs and non-invasive collection, the urinary biomarkers investigated in this study represent a promising strategy for serial monitoring of renal function in at-risk neonates and may aid the early detection of renal function impairment at different kidney levels during nephrotoxic treatments.

Erythropoietin Reduces Auto- and Alloantibodies by Inhibiting T Follicular Helper Cell Differentiation.

BACKGROUND: Although high-affinity IgG auto- and alloantibodies are important drivers of kidney inflammation that can result in ESKD, therapeutic approaches that effectively reduce such pathogenic antibodies remain elusive. Erythropoietin (EPO) has immunomodulatory functions, but its effects on antibody production are unknown. METHODS: We assessed the effect and underlying mechanisms of EPO/EPO receptor (EPOR) signaling on primary and secondary, T cell-dependent and T-independent antibody formation using in vitro culture systems, murine models of organ transplantation and lupus nephritis, and mice conditionally deficient for the EPOR expressed on T cells or B cells. RESULTS: In wild-type mice, recombinant EPO inhibited primary, T cell-dependent humoral immunity to model antigens and strong, polyclonal stimuli, but did not alter T-independent humoral immune responses. EPO also significantly impaired secondary humoral immunity in a potent allogeneic organ transplant model system. The effects required T cell, but not B cell, expression of the EPOR and resulted in diminished frequencies of germinal center (GC) B cells and T follicular helper cells (TFH). In vitro and in vivo experiments showed that EPO directly prevented TFH differentiation and function via a STAT5-dependent mechanism that reduces CD4+ T cell expression of Bcl6. In lupus models, EPO reduced TFH, GC B cells, and autoantibody production, and abrogated autoimmune glomerulonephritis, demonstrating clinical relevance. In vitro studies verified that EPO prevents differentiation of human TFH cells. CONCLUSIONS: Our findings newly demonstrate that EPO inhibits TFH-dependent antibody formation, an observation with potential implications for treating antibody-mediated diseases, including those of the kidney.

Toxin Removal and Inflammatory State Modulation during Online Hemodiafiltration Using Two Different Dialyzers (TRIAD2 Study).

Uremic toxins play a pathological role in atherosclerosis and represent an important risk factor in dialysis patients. Online hemodiafiltration (HDF) has been introduced to improve the clearance of middle- and large-molecular-weight solutes (>500 Da) and has been associated with reduced cardiovascular mortality compared to standard hemodialysis. This non-randomized, open-label observational study will explore the efficacy of two dialyzers currently used for online HDF, a polysulfone-based high-flux membrane, and a cellulose triacetate membrane, in hemodialysis patients with signs of middle-molecule intoxication or intradialytic hypotension. In particular, the two filters will be evaluated for their ability in uremic toxin removal and modulation of inflammatory status. Sixteen subjects in standard chronic bicarbonate hemodialysis requiring a switch to online HDF in view of their clinical status will be enrolled and divided into two treatment arms, according to the previous history of hypersensitivity to polysulfone/polyethersulfone dialysis filters and hypersensitivity to drugs or other allergens. Group A will consist of 16 patients without a previous history of hypersensitivity and will be treated with a polysulfone filter (Helixone FX100), and group B, also consisting of 16 patients, with a previous history of hypersensitivity and will be treated with asymmetric triacetate (ATA; SOLACEA 21-H) dialyzer. Each patient will be followed for a period of 24 months, with monthly assessments of circulating middle-weight toxins and protein-bound toxins, markers of inflammation and oxidative stress, lymphocyte subsets, activated lymphocytes, and monocytes, cell apoptosis, the accumulation of advanced glycation end-products (AGEs), variations in arterial stiffens measured by pulse wave velocity (PWV), and mortality rate. The in vitro effect on endothelial cells of uremic serum collected from patients treated with the two different dialyzers will also be investigated to examine the changes in angiogenesis, cell migration, differentiation, apoptosis and proliferative potential, and gene and protein expression profile. The expected results will be a better awareness of the different effects of polysulfone gold-standard membrane for online HDF and the new ATA membrane on the removal of uremic toxins removal and inflammation due to blood-membrane interaction.

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-α (TNF-α) 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.

Author Correction: Hypothermic Oxygenated New Machine Perfusion System in Liver and Kidney Transplantation of Extended Criteria Donors: First Italian Clinical Trial.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis.

Kidney glomerulosclerosis commonly progresses to end-stage kidney failure, but pathogenic mechanisms are still poorly understood. Here, we show that podocyte expression of decay-accelerating factor (DAF/CD55), a complement C3 convertase regulator, crucially controls disease in murine models of adriamycin (ADR)-induced focal and segmental glomerulosclerosis (FSGS) and streptozotocin (STZ)-induced diabetic glomerulosclerosis. ADR induces enzymatic cleavage of DAF from podocyte surfaces, leading to complement activation. C3 deficiency or prevention of C3a receptor (C3aR) signaling abrogates disease despite DAF deficiency, confirming complement dependence. Mechanistic studies show that C3a/C3aR ligations on podocytes initiate an autocrine IL-1ß/IL-1R1 signaling loop that reduces nephrin expression, causing actin cytoskeleton rearrangement. Uncoupling IL-1ß/IL-1R1 signaling prevents disease, providing a causal link. Glomeruli of patients with FSGS lack DAF and stain positive for C3d, and urinary C3a positively correlates with the degree of proteinuria. Together, our data indicate that the development and progression of glomerulosclerosis involve loss of podocyte DAF, triggering local, complement-dependent, IL-1ß-induced podocyte injury, potentially identifying new therapeutic targets.

Biomarkers of Kidney Injury in Very-low-birth-weight Preterm Infants: Influence of Maternal and Neonatal Factors.

BACKGROUND/AIM: Acute kidney injury is an important cause of mortality in very-low-birth-weight (VLBW) preterm infants. As in the general population, the detection of renal damage cannot rely on the measurement of serum creatinine, since it has been demonstrated to be a weak predictor and a delayed indicator of kidney function deterioration. However, several candidate biomarkers have failed to prove sufficient specificity and sensitivity for a routine clinical use because of the poor awareness of their biological role. This study was aimed to investigate the impact of different maternal and neonatal conditions on several renal biomarkers in VLBW preterm infants during the first week of life. PATIENTS AND METHODS: Preterm infants<32 weeks' gestation and <1500g were enrolled. We measured urinary biomarkers kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), cystatin C, epidermal growth factor (EGF) and osteopontin (OPN) on the 1st, 3rd, and 7th day after birth. RESULTS: Thirty-tree infants were included. The multivariate analysis showed a significant association between gestational age, the presence of patent ductus arteriosus, antenatal maternal hypertension and the levels of urinary biomarkers. CONCLUSION: There is a possible relation between early biomarkers of renal injury and antenatal, perinatal and post-natal characteristics in VLBW preterm infants during the first week of life.

Hypothermic Oxygenated New Machine Perfusion System in Liver and Kidney Transplantation of Extended Criteria Donors:First Italian Clinical Trial.

With the aim to explore innovative tools for organ preservation, especially in marginal organs, we hereby describe a clinical trial of ex-vivo hypothermic oxygenated perfusion (HOPE) in the field of liver (LT) and kidney transplantation (KT) from Extended Criteria Donors (ECD) after brain death. A matched-case analysis of donor and recipient variables was developed: 10 HOPE-ECD livers and kidneys (HOPE-L and HOPE-K) were matched 1:3 with livers and kidneys preserved with static cold storage (SCS-L and SCS-K). HOPE and SCS groups resulted with similar basal characteristics, both for recipients and donors. Cumulative liver and kidney graft dysfunction were 10% (HOPE L-K) vs. 31.7%, in SCS group (p = 0.05). Primary non-function was 3.3% for SCS-L vs. 0% for HOPE-L. No primary non-function was reported in HOPE-K and SCS-K. Median peak aspartate aminotransferase within 7-days post-LT was significantly higher in SCS-L when compared to HOPE-L (637 vs.344 U/L, p = 0.007). Graft survival at 1-year post-transplant was 93.3% for SCS-L vs. 100% of HOPE-L and 90% for SCS-K vs. 100% of HOPE-K. Clinical outcomes support our hypothesis of machine perfusion being a safe and effective system to reduce ischemic preservation injuries in KT and in LT.

Immunological Effects of a Single Hemodialysis Treatment.

Immune disorders, involving both innate and adaptive response, are common in patients with end-stage renal disease under chronic hemodialysis. Endogenous and exogenous factors, such as uremic toxins and the extracorporeal treatment itself, alter the immune balance, leading to chronic inflammation and higher risk of cardiovascular events. Several studies have previously described the immune effects of chronic hemodialysis and the possibility to modulate inflammation through more biocompatible dialyzers and innovative techniques. On the other hand, very limited data are available on the possible immunological effects of a single hemodialysis treatment. In spite of the lacking information about the immunological reactivity related to a single session, there is evidence to indicate that mediators of innate and adaptive response, above all complement cascade and T cells, are implicated in immune system modulation during hemodialysis treatment. Expanding our understanding of these modulations represents a necessary basis to develop pro-tolerogenic strategies in specific conditions, like hemodialysis in septic patients or the last session prior to kidney transplant in candidates for receiving a graft.

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

IL-17-producing CD4+ cells (TH17) are pathogenically linked to autoimmunity including to autoimmune kidney disease. Erythropoietin's (EPO) newly recognized immunoregulatory functions and its predominant intra-renal source suggested that EPO physiologically regulates TH17 differentiation, thereby serving as a barrier to the 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 generation and promotes trans-differentiation of TH17 into IL-17-FOXP3+CD4+ T cells. Mechanistically, EPO/EPO-R ligation abrogates upregulation of SGK1 gene expression and blocks p38 activity to prevent SGK1 phosphorylation, thereby inhibiting RORC-mediated transcription of IL-17 and IL-23 receptor genes. In a murine model of TH17-dependent aristolochic acid (ArA)-induced, interstitial kidney disease associated with reduced renal EPO production, we demonstrate that transgenic EPO overexpression or recombinant EPO (rEPO) administration limits TH17 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 induction and clinical/histological expression of pristane-induced glomerulonephritis (associated with decreased intrarenal EPO). rEPO prevents spontaneous glomerulonephritis and TH17 generation in MRL-lpr mice. Together, our findings indicate that EPO physiologically and therapeutically modulate TH17 cells to limit expression of TH17-associated autoimmune kidney disease.

Impact of preformed T-cell alloreactivity by means of donor-specific and panel of reactive T cells (PRT) ELISPOT in kidney transplantation.

Donor-specific (d-sp) interferon gamma enzyme-linked immunosorbent spot (d-sp ELISPOT) and Panel of reactive T-cell (PRT) ELISPOT assays have been developed to detect alloreactive memory T (Tmem) cells in order to estimate the risk of acute rejection after kidney transplantation. Adding IL15 to the PRT assay (PRT+IL15) may uncover the presence of pathogenic alloreactive CD28-Tmem. Face-to-face comparisons of these assays have not been done yet. We performed pre-transplant d-sp ELISPOT and PRT assays (±IL15, against six B-cell lines) in 168 consecutive kidney transplant recipients and evaluated the multivariable-adjusted associations with biopsy-proven acute rejection (BPAR), de novo donor-specific antibodies (DSA), and eGFR decline over a 48-month follow-up period. D-sp ELISPOT was positive in 81 (48%) subjects, while 71 (42%) and 81 (48%) subjects displayed positive PRT and PRT+IL15, respectively. Their median [interquartile range] numerical test result was 23 [6-65], 18 [8-37], and 26 [10-45] spots/3x105 PBMCs, respectively. The number of PRT spots were weakly correlated with those of d-sp ELISPOT, but highly correlated with PRT+IL15 (rho = 0.96, P<0.001). d-sp ELISPOT, but not PRT (±IL15) was independently associated with BPAR (adjusted Odds Ratio of BPAR associated with d-sp ELISPOT positivity: 4.20 [95%CI: 1.06 to 21.73; P = 0.041]). Unlike d-sp ELISPOT, median PRT and PRT+IL15 were independently associated with higher Δ3-48month eGFR decline post-transplantation (for both assays, about -3mL/min/1.73m2 per one standard deviation unit increase in the spot number). Pre-transplant T-cell immune-monitoring using d-sp ELISPOT and PRT assays identifies kidney transplant candidates at high risk of BPAR and worse kidney allograft progression.

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.