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, p16ink4a, 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 p16ink4a 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

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

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

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

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

Assessment of a new cell culture perfusion apparatus for in vitro chronic toxicity testing. Part I: Technical description

In vitro models for chronic toxicity, defined as a recurring exposure to compounds over a prolonged period of time, are still underrepresented in drug evaluation processes. The classical approach to cell culture is not readily suitable to long term repetitive applications. Therefore, we assessed the use of a commercially available perfusion cell culture apparatus in its applicability to chronic renal toxicity testing and describe the technical aspects of adopting the perfusion cell culture system to our purposes. It was apparent that there is a subtle dynamic difference between human renal proximal tubular cells cultured under perfusion and static conditions as illustrated by the accumulation of lactate dehydrogenase (LDH) and the secondary metabolism of resazurin to hydroresorufin, which occurred only under static conditions. The major achievement was the standardisation of the handling of this system with regard to cell cultivation, pH regulation, temperature regulation, and reproducibility of common toxicity endpoints

Assessment of a new cell culture perfusion apparatus for in vitro chronic toxicity testing. Part 1: technical description

In vitro models for chronic toxicity, defined as a recurring exposure to compounds over a prolonged period of time, are still underrepresented in drug evaluation processes. The classical approach to cell culture is not readily suitable to long term repetitive applications. Therefore, we assessed the use of a commercially available perfusion cell culture apparatus in its applicability to chronic renal toxicity testing and describe the technical aspects of adopting the perfusion cell culture system to our purposes. It was apparent that there is a subtle dynamic difference between human renal proximal tubular cells cultured under perfusion and static conditions as illustrated by the accumulation of lactate dehydrogenase (LDH) and the secondary metabolism of resazurin to hydroresorufin, which occurred only under static conditions. The major achievement was the standardisation of the handling of this system with regard to cell cultivation, pH regulation, temperature regulation, and reproducibility of common toxicity endpoints