Metallothioneins and renal ageing.

BACKGROUND: Human lifespan is increasing continuously and about one-third of the population >70 years of age suffers from chronic kidney disease. The pathophysiology of the loss of renal function with ageing is unclear. METHODS: We determined age-associated gene expression changes in zero-hour biopsies of deceased donor kidneys without laboratory signs of impaired renal function, defined as a last serum creatinine >0.96 mg/dL in females and >1.18 mg/dL in males, using microarray technology and the Significance Analysis of Microarrays routine. Expression changes of selected genes were confirmed by quantitative polymerase chain reaction and in situ hybridization and immunohistochemistry for localization of respective mRNA and protein. Functional aspects were examined in vitro. RESULTS: Donors were classified into three age groups (<40, 40-59 and >59 years; Groups 1, 2 and 3, respectively). In Group 3 especially, genes encoding for metallothionein (MT) isoforms were more significantly expressed when compared with Group 1; localization studies revealed predominant staining in renal proximal tubular cells. RPTEC/TERT1 cells overexpressing MT2A were less susceptible towards cadmium chloride-induced cytotoxicity and hypoxia-induced apoptosis, both models for increased generation of reactive oxygen species. CONCLUSIONS: Increased expression of MTs in the kidney with ageing might be a protective mechanism against increased oxidative stress, which is closely related to the ageing process. Our findings indicate that MTs are functionally involved in the pathophysiology of ageing-related processes.

Effect of different immunosuppressive drugs on immune cells from young and old healthy persons.

BACKGROUND: Life expectancy, as well as the average age of patients undergoing solid organ transplantation, increases constantly. Consequently, immunosuppressive therapy is no longer limited to young organ recipients. OBJECTIVE: Here, we investigate how different types of immunosuppressive therapy, namely the calcineurin inhibitors cyclosporin A and tacrolimus, as well as the mTOR inhibitor rapamycin, affect the function of immune cells in young and elderly persons. METHODS: Proliferation, cell viability, cytokine production (IL-2, IFN-γ), H2O2 production and telomere length of phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMCs) of young (n = 13; median age 27 years) and old (n = 19; median age 71 years) healthy donors were analyzed. RESULTS: The inhibition of proliferation was dampened in PBMCs from elderly donors, especially after incubation with rapamycin. All three immunosuppressive drugs inhibited the production of IL-2 equally well, whereas the production of IFN-γ was less well inhibited by rapamycin. Both calcineurin inhibitors increased H2O2 concentrations after stimulation with PHA and led to a shortening of telomeres in PBMCs from young and old individuals. Rapamycin had only minor effects on H2O2 production and telomere length. CONCLUSION: Our results demonstrate that the effects of immunosuppressive drugs on PBMCs differ between young and elderly persons. Calcineurin inhibitors compared to rapamycin have a more pronounced prosenescence effect. These data indicate that specific treatment regimens for the elderly might therefore be considered.

Lower CD28+ T cell proportions were associated with CMV-seropositivity in patients with Hashimoto's thyroiditis.

BACKGROUND: Alterations in the naive T cell subpopulations have been demonstrated in patients with T cell mediated autoimmune disorders, reminiscent of immunological changes found in the elderly during immunosenescence, including the switch from CD45RA + to CD45RO + T cells and decreased thymic function with increased compensatory proliferative mechanisms, partly associated with latent Cytomegalovirus (CMV) infection. The present study was aimed to investigate proportions of lymphocytes, their relation to CMV-seropositivity and the replicative history of CD45RA + expressing T cells in Hashimoto's thyroiditis (HT, n = 18) and healthy controls (HC, n = 70). METHODS: Proportions of peripheral T cells were investigated by flow cytometry. The replicative history was assessed by T cell receptor excision circles (TRECs) and relative telomere length (RTL). Expression of CD62L was analyzed by immunohistochemistry in thyroid sections. The role of CMV was assessed by serology, ELISPOT assay and in situ hybridization. RESULTS: Our results demonstrated a significant increase of CD28-negative T cells, associated with CMV-seropositivity in HT patients. HT showed abundant CD45RO + T cells with peripheral loss of CD62L-expressing CD8 + CD45RA + T cells, the latter mainly depending on disease duration. CD62L was expressed in thyroid lymphocyte infiltrations. The diagnosis of HT and within the HT group CMV-seropositivity were the main determinants for the loss of CD28 expression. RTL was not different between HC and HT. HT showed significantly lower TRECs in CD4 + CD45RA + T cells compared to HC. CONCLUSIONS: Patients with HT display a peripheral T cell phenotype reminiscent of findings in elderly persons or other autoimmune disorders. Whether these mechanisms are primary or secondary to the immunological alterations of autoimmune conditions should be investigated in longitudinal studies which may open research on new therapeutic regimes for treatment of HT and associated autoimmune diseases.

Maternal cigarette smoking and its effect on neonatal lymphocyte subpopulations and replication.

BACKGROUND: Significant immunomodulatory effects have been described as result of cigarette smoking in adults and pregnant women. However, the effect of cigarette smoking during pregnancy on the lymphocyte subpopulations in newborns has been discussed, controversially. METHODS: In a prospective birth cohort, we analyzed the peripheral lymphocyte subpopulations of smoking (SM) and non-smoking mothers (NSM) and their newborns and the replicative history of neonatal, mostly naive CD4 + CD45RA + T cells by measurements of T-cell-receptor-excision-circles (TRECs), relative telomere lengths (RTL) and the serum cytokine concentrations. RESULTS: SM had higher lymphocyte counts than NSM. Comparing SM and NSM and SM newborns with NSM newborns, no significant differences in proportions of lymphocyte subpopulations were seen. Regardless of their smoking habits, mothers had significantly lower naive T cells and higher memory and effector T cells than newborns. NSM had significantly lower percentages of CD4 + CD25++ T cells compared to their newborns, which was not significant in SM. There were no differences regarding cytokine concentrations in newborns of SM and NSM. However, NSM had significantly higher Interleukin-7 concentrations than their newborns. Regardless of smoking habits of mothers, newborns had significantly longer telomeres and higher TRECs than their mothers. Newborns of SM had significantly longer telomeres than newborns of NSM. CONCLUSIONS: Apart from higher lymphocyte counts in SM, our results did not reveal differences between lymphocyte subpopulations of SM and NSM and their newborns, respectively. Our finding of significantly longer RTL in newborns of SM may reflect potential harm on lymphocytes, such as cytogenetic damage induced by smoking.

Kidney fusion anomalies revisited: clinical and radiological analysis of 209 cases of crossed fused ectopia and horseshoe kidney.

OBJECTIVE: To analyse the morphological appearance of horseshoe kidneys (HKs) and crossed fused ectopia (CFE) and to assess the frequency and clinical significance of associated anomalies and diseases. PATIENTS AND METHODS: The findings and images of 209 patients with fused kidneys (FKs) were reviewed; in all, 244 scans from computed tomography (CT), 233 ultrasonograms and 89 micturition cysto-urethrograms, urograms, magnetic resonance images and angiograms were taken. RESULTS: HKs (found in one of 474 abdominal CT scans) and CFEs (found in one of 3078 CT scans) showed a high variability of vasculature that could not be classified. However, some generalized conclusions were possible about the renal vasculature (430 arteries in 103 kidneys). Variants of the most cephalad artery of both sides were rare. The second artery on the right had a pre-caval course. The origins of vessels located further caudal were more ventral. CFEs were anatomically different from HKs with respect to lower position, greater axial rotation, smaller pelvic width, more caudal origin, and fewer vessels, but not in accompanying anomalies. Severe anomalies or malformations were found in 23% of patients, with half of them in the urogenital system. Malformations were found considerably more often in children than in adults. There was no increased incidence of diseases such as stones or inflammation of the renal pelvis. CONCLUSION: Concomitant anomalies and diseases were equally frequent for HK and CFE, but less frequent than generally assumed. Individual cases of complex anatomical situations require special examination strategies, and CT appears to be the most reliable imaging method.

Impact of anemia on aortic pulse wave velocity in hemodialysis patients.

BACKGROUND: Recent studies indicate an increased mortality of anemic patients with renal failure when near-normal hemoglobin levels are aimed for by treatment with erythropoiesis stimulating agents. Aortic pulse wave velocity (aPWV) is a strong predictor of all-cause and cardiovascular mortality in patients with end-stage renal disease. The relationships between aPWV, hemoglobin levels and erythropoiesis stimulating agent dosage have not been evaluated to date. METHODS: In 75 patients, aPWV was measured by applanation tonometry. Associations of aPWV and a broad range of clinical, laboratory and therapeutic parameters were determined by stepwise linear regression analysis. RESULTS: aPWV was positively correlated to age (r = 0.55, p < 0.001), whereas the association with hemoglobin was significant, but negative (r = -0.31, p = 0.01). Multivariate analysis determined age (beta = 0.513, p < 0.001), mean blood pressure (beta = 0.255, p = 0.01), the presence of heart failure (beta = 0.188, p = 0.03), hemoglobin (beta = -0.226, p = 0.01), daily calcium load (beta = -0.230, p = 0.01) and the presence of diabetes mellitus (beta = 0.179, p = 0.04) to have a significant and independent influence on aPWV. CONCLUSIONS: This study demonstrates that in hemodialysis patients, aPWV is significantly but negatively associated with the serum hemoglobin concentration, even after multiple adjustments for other covariates.

Computational Drug Screening Identifies Compounds Targeting Renal Age-associated Molecular Profiles.

Aging is a major driver for chronic kidney disease (CKD) and the counterbalancing of aging processes holds promise to positively impact disease development and progression. In this study we generated a signature of renal age-associated genes (RAAGs) based on six different data sources including transcriptomics data as well as data extracted from scientific literature and dedicated databases. Protein abundance in renal tissue of the 634 identified RAAGs was studied next to the analysis of affected molecular pathways. RAAG expression profiles were furthermore analysed in a cohort of 63 CKD patients with available follow-up data to determine association with CKD progression. 23 RAAGs were identified showing concordant regulation in renal aging and CKD progression. This set was used as input to computationally screen for compounds with the potential of reversing the RAAG/CKD signature on the transcriptional level. Among the top-ranked drugs we identified atorvastatin, captopril, valsartan, and rosiglitazone, which are widely used in clinical practice for the treatment of patients with renal and cardiovascular diseases. Their positive impact on the RAAG/CKD signature could be validated in an in-vitro model of renal aging. In summary, we have (i) consolidated a set of RAAGs, (ii) determined a subset of RAAGs with concordant regulation in CKD progression, and (iii) identified a set of compounds capable of reversing the proposed RAAG/CKD signature.

Blood pressure changes after renal denervation are more pronounced in women and nondiabetic patients: findings from the Austrian Transcatheter Renal Denervation Registry.

OBJECTIVES: Three recently published sham-controlled studies proved the efficacy of renal denervation (RDN) in hypertensive patients. The study presented here analyzed a nationwide multicentre registry database to clarify which patient subgroups benefit most from radiofrequency RDN. METHODS: This is a post hoc analysis from the multicentre Austrian Transcatheter Renal Denervation Registry hosted by the Austrian Society of Hypertension. We correlated change of SBP after RDN to sex and presence/absence of comorbidities. Univariable correlation and multiple linear regression analyses were performed. RESULTS: Two hundred and ninety-one patients (43% women, median age 64 years) undergoing RDN between April 2011 and September 2014 were included in this analysis. Mean baseline ambulatory 24 h BP (systolic/diastolic) was 150 ±â€Š18/89 ±â€Š14 mmHg and mean baseline office BP was 170 ±â€Š16/94 ±â€Š14 mmHg.After RDN, mean ambulatory 24 h BP reduction was 9 ±â€Š19/6 ±â€Š16 mmHg. The following features were associated with a good response to RDN: high baseline systolic ambulatory BP, high baseline diastolic office BP, female sex, absence of diabetes mellitus, and absence of peripheral artery disease. Multivariable analysis identified female sex and absence of diabetes mellitus as strongest predictors for ambulatory BP reduction, although those groups had the lowest baseline ambulatory BP. DISCUSSION: Ambulatory BP reductions after RDN were substantially more pronounced in female and in nondiabetic patients despite lower baseline BP. It is concluded that in terms of efficacy female patients and nondiabetic patients might benefit more from RDN.

Effect of cyclosporine, tacrolimus and sirolimus on cellular senescence in renal epithelial cells.

INTRODUCTION: In transplantation medicine calcineurin inhibitors (CNI) still represent the backbone of immunosuppressive therapy. The nephrotoxic potential of the CNI Cyclosporine A (CsA) and Tacrolimus (FK506) is well recognized and CNI not only have been linked with toxicity, but also with cellular senescence which hinders parenchymal tissue regeneration and thus may prime kidneys for subsequent insults. To minimize pathological effects on kidney grafts, alternative immunosuppressive agents like mTOR inhibitors or the T-cell co-stimulation blocker Belatacept have been introduced. METHODS: We compared the effects of CsA, FK506 and Sirolimus on the process of cellular senescence in different human renal tubule cell types (HK2, RPTEC). Telomere length (by real time PCR), DNA synthesis (by BrdU incorporation), cell viability (by Resazurin conversion), gene expression (by RT-PCR), protein (by western blotting), Immuncytochemistry and H2O2 production (by Amplex Red® conversion) were evaluated. RESULTS: DNA synthesis was significantly reduced when cells were treated with cyclosporine but not with tacrolimus and sirolimus. Resazurin conversion was not altered by all three immunosuppressive agents. The gene expression as well as protein production of the cell cycle inhibitor p21 (CDKN1A) but not p16 (CDKN2A) was significantly induced by cyclosporine compared to the other two immunosuppressive agents when determined by western blotting an immuncytochemistry. Relative telomere length was reduced and hydrogen peroxide production increased after treatment with CsA but not with FK506 or sirolimus. CONCLUSION: In summary, renal tubule cells exposed to CsA show clear signs of cellular senescence where on the contrary the second calcineurin inhibitor FK506 and the mTOR inhibitor sirolimus are not involved in such mechanisms. Chronic renal allograft dysfunction could be in part triggered by cellular senescence induced by immunosuppressive medication and the choice of drug could therefore influence long term outcome. Tacrolimus and Sirolimus are equally effective in avoiding cellular senescence compared to cyclosporine at least in parts due to a lack of induction of reactive oxygen species.

Fenestration of the Gerota's fascia as symptomatic treatment of floating kidneys.

A 16-year-old man presented with severe nephrotic syndrome complicated by massive perirenal fluid. Percutaneous drainage of fluid was performed 3 times, followed by improvement in renal function and hypertension, but perirenal fluid recurred within days. Nephrotic syndrome was unresponsive to steroid therapy. A laparoscopic bilateral fenestration of Gerota's fascia and peritoneum allowed permanent drainage of fluid into the peritoneal cavity. During the same procedure, a renal wedge biopsy was performed. Histological examination showed advanced focal glomerular sclerosis of the tip lesion variant. The glomerular disease was refractory to further treatment with cyclophosphamide, mycophenolate, and rituximab. However, perirenal fluid did not recur despite persistent nephrotic syndrome, showing that fenestration of Gerota's fascia is a successful treatment of floating kidneys in such patients.

CD34+/CD133- circulating endothelial precursor cells (CEP): characterization, senescence and in vivo application.

Circulating endothelial precursor cells (CEP) are interesting candidates for the treatment of ischemic diseases and for tumor targeting/imaging. We isolated a homogeneous population of CEP from CD34(+)/CD133(-) cells of peripheral blood that can be expanded easily on collagen-type-I coated plastic. CEP displayed a phenotype of mature endothelial cells (vWF, CD31, CD34, VEGF-R2, CD105, CD146) similar to that of cord-blood CEP and umbilical vein endothelial cells. They bound UEA-1 lectin, incorporated acetylated LDL and formed tube-like structures with capillary lumens in vitro. Weibel-Palade bodies were observed by electron microscopy. After 40-60 cell population doublings, CEP cultures underwent a terminal growth arrest, had shorter telomeres, up-regulated cell cycle inhibitory proteins, such as p21(CIP1) and stained positive for senescence-associated-beta galactosidase. During the whole expansion period CEP retained their endothelial phenotype and a normal karyotype. CEP had the capacity to home to ischemic tissue in vivo after systemic injection in nude rats. The convenient expandability, the homogenous phenotype, the functional cellular senescence program, the regular karyotype and the homing capacity to ischemic myocardium suggest autologous CEP cultures as a safe and promising tool for cell-based therapeutic approaches in targeting ischemic tissue and tumors.

Identification of Molecular Markers of Delayed Graft Function Based on the Regulation of Biological Ageing.

INTRODUCTION: Delayed graft function is a prevalent clinical problem in renal transplantation for which there is no objective system to predict occurrence in advance. It can result in a significant increase in the necessity for hospitalisation post-transplant and is a significant risk factor for other post-transplant complications. METHODOLOGY: The importance of microRNAs (miRNAs), a specific subclass of small RNA, have been clearly demonstrated to influence many pathways in health and disease. To investigate the influence of miRNAs on renal allograft performance post-transplant, the expression of a panel of miRNAs in pre-transplant renal biopsies was measured using qPCR. Expression was then related to clinical parameters and outcomes in two independent renal transplant cohorts. RESULTS: Here we demonstrate, in two independent cohorts of pre-implantation human renal allograft biopsies, that a novel pre-transplant renal performance scoring system (GRPSS), can determine the occurrence of DGF with a high sensitivity (>90%) and specificity (>60%) for donor allografts pre-transplant, using just three senescence associated microRNAs combined with donor age and type of organ donation. CONCLUSION: These results demonstrate a relationship between pre-transplant microRNA expression levels, cellular biological ageing pathways and clinical outcomes for renal transplantation. They provide for a simple, rapid quantitative molecular pre-transplant assay to determine post-transplant allograft function and scope for future intervention. Furthermore, these results demonstrate the involvement of senescence pathways in ischaemic injury during the organ transplantation process and an indication of accelerated bio-ageing as a consequence of both warm and cold ischaemia.

Effect of tissue fixatives on telomere length determination by quantitative PCR.

Telomere length is a well established marker of cellular senescence and thus biological age. Quantitative PCR allows the determination even from very low amounts of tissue by using telomere specific and single copy gene primers. Comparing a directly processed tissue sample to a 4% formaldehyde fixed one showed a significantly reduced efficiency of PCR reactions (mainly in single copy gene experiments) in a storage time-dependent manner resulting in an artificial increase in reported relative telomere length. This effect was not seen when the tissue was stored in RNA later solution. In summary, telomere length determination from formaldehyde fixed material by quantitative PCR is not a reliable method. Unfortunately therefore, many easily accessible tissue samples from pathology laboratories are unsuitable for this technique.

Influence of the delivery modus on subpopulations and replication of lymphocytes in mothers and newborns.

BACKGROUND: Several studies reported that the mode of delivery may induce changes to the immune system. Our hypothesis was that the delivery mode may influence mainly the naive T cell subpopulation. AIMS: Particular focus was set on the proportions and peripheral replicative history of naive T cells and cord blood serum concentrations of IL-7, a cytokine involved in peripheral naive T cell homeostasis. STUDY DESIGN, SUBJECTS AND OUTCOME MEASURES: In a prospective cohort study, proportions of lymphocyte populations were measured in mothers and newborns delivered by spontaneous vaginal delivery (SD), vacuum extraction (VE), primary (PCS) and secondary Cesarean sections (SCS) by flow cytometry. T-cell-receptor-excision-circles (TRECs) and relative telomere lengths (RTLs) were used to estimate the replicative history of peripheral naive T cells. The cytokine profile was assessed by ELISA. RESULTS: The study demonstrated that leukocytes, neutrophils and NK cells were increased in spontaneously delivered newborns compared to PCS, whereas circulating T cells were relatively lower. TRECs and RTLs were not significantly influenced by the delivery mode. IL-2, IL-8 and IFN-γ were increased in VD. IL-7 production tends to be increased in more stress-associated delivery modes, such as VE and SCS. CONCLUSIONS: Our results demonstrate proportional changes in newborns delivered by PCS and diminished cytokine production. It has to be proven whether these alterations may be of disadvantage regarding early defense of infectious diseases. Understanding the physiological role of these changes may help to find preventive strategies for neonatal infectious risks and the development of atopy or other immune diseases.

Tacrolimus increases Nox4 expression in human renal fibroblasts and induces fibrosis-related genes by aberrant TGF-beta receptor signalling.

Chronic nephrotoxicity of immunosuppressives is one of the main limiting factors in the long-term outcome of kidney transplants, leading to tissue fibrosis and ultimate organ failure. The cytokine TGF-ß is considered a key factor in this process. In the human renal fibroblast cell line TK-173, the macrolide calcineurin inhibitor tacrolimus (FK-506) induced TGF-ß-like effects, manifested by increased expression of NAD(P)H-oxidase 4 (Nox4), transgelin, tropomyosin 1, and procollagen α1(V) mRNA after three days. The macrolide mTOR inhibitor rapamycin had similar effects, while cyclosporine A did not induce fibrose-related genes. Concentration dependence curves were sigmoid, where mRNA expression was induced already at low nanomolar levels of tacrolimus, and reached saturation at 100-300 nM. The effects were independent of extracellular TGF-ß as confirmed by the use of neutralizing antibodies, and thus most likely caused by aberrant TGF-ß receptor signaling, where binding of tacrolimus to the regulatory FKBP12 protein results in a "leaky" TGF-ß receptor. The myofibroblast marker α-smooth muscle actin was neither induced by tacrolimus nor by TGF-ß1, indicating an incomplete activation of TK-173 fibroblasts under culture conditions. Tacrolimus- and TGF-ß1-induced Nox4 protein upregulation was confirmed by Western blotting, and was accompanied by a rise in intracellular H2O2 concentration. Si-RNA mediated knock-down of Nox4 expression prevented up-regulation of procollagen α1(V) mRNA in tacrolimus-treated cells, but induced procollagen α1(V) expression in control cells. Nox4 knock-down had no significant effect on the other genes tested. TGF-ß is a key molecule in fibrosis, and the constant activation of aberrant receptor signaling by tacrolimus might contribute to the long-term development of interstitial kidney fibrosis in immunosuppressed patients. Nox4 levels possibly play a regulatory role in these processes.

Pathfinder cells provide a novel therapeutic intervention for acute kidney injury.

Pathfinder cells (PCs) are a novel class of adult-derived cells that facilitate functional repair of host tissue. We used rat PCs to demonstrate that they enable the functional mitigation of ischemia reperfusion (I/R) injury in a mouse model of renal damage. Female C57BL/6 mice were subjected to 30 min of renal ischemia and treated with intravenous (i.v.) injection of saline (control) or male rat pancreas-derived PCs in blinded experimentation. Kidney function was assessed 14 days after treatment by measuring serum creatinine (SC) levels. Kidney tissue was assessed by immunohistochemistry (IHC) for markers of cellular damage, proliferation, and senescence (TUNEL, Ki67, p16(ink4a), p21). Fluorescence in situ hybridization (FISH) was performed to determine the presence of any rat (i.e., pathfinder) cells in the mouse tissue. PC-treated animals demonstrated superior renal function at day 14 post-I/R, in comparison to saline-treated controls, as measured by SC levels (0.13 mg/dL vs. 0.23 mg/dL, p<0.001). PC-treated kidney tissue expressed significantly lower levels of p16(ink4a) in comparison to the control group (p=0.009). FISH analysis demonstrated that the overwhelming majority of repaired kidney tissue was mouse in origin. Rat PCs were only detected at a frequency of 0.02%. These data confirm that PCs have the ability to mitigate functional damage to kidney tissue following I/R injury. Kidneys of PC-treated animals showed evidence of improved function and reduced expression of damage markers. The PCs appear to act in a paracrine fashion, stimulating the host tissue to recover functionally, rather than by differentiating into renal cells. This study demonstrates that pancreatic-derived PCs from the adult rat can enable functional repair of renal damage in mice. It validates the use of PCs to regenerate damaged tissues and also offers a novel therapeutic intervention for repair of solid organ damage in situ.

Increased replication of CD4+ naive T cells and changes in T cell homeostasis in a case of acute exacerbation of juvenile idiopathic arthritis: a case comparison study.

INTRODUCTION: Juvenile idiopathic arthritis is a heterogeneous T cell-mediated autoimmune disease with symptoms of premature aging of the immune system (immunosenescence). The present work is an investigation of immunosenescence parameters, such as quantity of naive and CD28- T cells, T cell receptor excision circles, relative telomere length and alterations of peripheral T cell replication, and was performed via comparison of a case of acute exacerbation of juvenile idiopathic arthritis against six patients with juvenile idiopathic arthritis with disease remission and six age-matched healthy donors over a follow-up course of 12 months. CASE PRESENTATION: Phenotypical T cell characterization and intracellular interferon γ, tumor necrosis factor α, and interleukin 2 production were studied in peripheral blood mononuclear cells from seven patients with juvenile idiopathic arthritis and six healthy control donors, with findings determined by flow cytometry. T cell receptor excision circles and relative telomere length quantification were performed on deoxyribonucleic acid isolated from naive (CD4+CD28+CD45RA+) T cells and investigated via reverse transcription polymerase chain reaction. Ki67 expression was studied by immunohistochemistry on naive T cells. The non-parametric Mann-Whitney U test and Wilcoxon test for two independent groups of variables were used to compare healthy donors with patients with juvenile idiopathic arthritis. During follow-up, patients with juvenile idiopathic arthritis showed lower total counts of naive and CD28-expressing T cells compared to healthy donors. Acute exacerbation led to low naive and CD28+ T cell populations and elevated proportions of Ki67-expressing CD4+ naive T cells. In conditions of exacerbation, T cell receptor excision circle numbers were in the lower range in patients with juvenile idiopathic arthritis and increased after follow-up. Healthy donors showed significantly higher relative telomere lengths compared to patients with juvenile idiopathic arthritis. CONCLUSIONS: This investigation illustrates that the changes in T cell homeostasis in patients with juvenile idiopathic arthritis may be the result of several mechanisms, such as diminished thymus function and peripheral exertions to maintain the peripheral T cell pool. The results also demonstrate that hallmarks of immunosenescence such as decreased naive T cell levels and lower T cell receptor excision circle numbers can only be interpreted together with replication markers such as relative telomere length or Ki67 expression.

Pre-transplant CDKN2A expression in kidney biopsies predicts renal function and is a future component of donor scoring criteria.

CDKN2A is a proven and validated biomarker of ageing which acts as an off switch for cell proliferation. We have demonstrated previously that CDKN2A is the most robust and the strongest pre-transplant predictor of post-transplant serum creatinine when compared to "Gold Standard" clinical factors, such as cold ischaemic time and donor chronological age. This report shows that CDKN2A is better than telomere length, the most celebrated biomarker of ageing, as a predictor of post-transplant renal function. It also shows that CDKN2A is as strong a determinant of post-transplant organ function when compared to extended criteria (ECD) kidneys. A multivariate analysis model was able to predict up to 27.1% of eGFR at one year post-transplant (p = 0.008). Significantly, CDKN2A was also able to strongly predict delayed graft function. A pre-transplant donor risk classification system based on CDKN2A and ECD criteria is shown to be feasible and commendable for implementation in the near future.