MicroRNAs 223-3p and 93-5p in patients with chronic kidney disease before and after renal transplantation.

Chronic kidney disease (CKD) is associated with a multifactorial dysregulation of bone and vascular calcification and closely linked to increased cardiovascular mortality and concomitant bone disease. We aimed to investigate specific microRNA (miRNA) signatures in CKD patients to find indicators for vascular calcification and/or bone mineralization changes during CKD and after kidney transplantation (KT). A miRNA array was used to investigate serum miRNA profiles in CKD patients, then selected miRNAs were quantified in a validation cohort comprising 73 patients in CKD stages 3 to 5, 67 CKD patients after KT, and 36 healthy controls. A spectrum of biochemical parameters including markers for kidney function, inflammation, glucose, and mineral metabolism was determined. The relative expression of miR-223-3p and miR-93-5p was down-regulated in patients with CKD stage 4 and 5 compared to healthy controls. This down-regulation disappeared after kidney transplantation even when lower glomerular filtration rates (eGFR) persisted. MiR-223-3p and miR-93-5p were associated with interleukin-6 (IL-6) and eGFR levels, and by trend with interleukin-8 (IL-8), C-peptide, hematocrit, and parathyroid hormone (PTH). This study contributes new knowledge of serum miRNA expression profiles in CKD, potentially reflecting pathophysiological changes of bone and calcification pathways associated with inflammation, vascular calcification, mineral and glucose metabolism. Identified miRNA signatures can contribute to future risk markers or future therapeutic targets in bone and kidney disease.

The mTOR-inhibitor rapamycin mediates proteinuria in nephrotoxic serum nephritis by activating the innate immune response.

Rapamycin (Rapa) is an immunosuppressant used to prevent rejection in recipients of renal transplants. Its clinical use is limited by de novo onset or exacerbation of preexisting proteinuria. In the present study, Rapa administration was started 14 days after induction of murine nephrotoxic serum nephritis (NTS) to study glomerular effects of this mammalian target of rapamycin (mTOR) inhibitor. Glomeruli were laser-microdissected, and real-time PCR was performed to assess effects on glomerular cells and the expression of inflammatory cytokines. Immunohistochemical stainings were performed to confirm mRNA data on the protein level. Compared with nephritic control animals, Rapa-treated mice developed significantly increased albuminuria. This was accompanied by a more prominent glomerular infiltration by CD4(+) T cells and macrophages. Glomerular mRNA expression profiling revealed increased levels of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, and the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1ß and their cognate macrophage-associated receptors CCR2 and CCR5 in the Rapa-treated animals. Furthermore, there were elevated glomerular transcription levels of the regulatory T cell phenotype transcription factor Foxp3. No differences in the glomerular expression of the podocyte marker nephrin or the endothelial cell marker CD31 were observed on the mRNA or protein level. In conclusion, our data indicate that Rapa-induced proteinuria in NTS is a result of the activation of the innate immune system rather than a direct toxicity to podocytes or glomerular endothelial cells.

The PTH (1-84)/non-PTH (1-84) ratio is a risk factor for cardiovascular events in hemodialysis patients.

BACKGROUND: We hypothesized that the PTH (1-84)/non-PTH (1-84) ratio (PTH ratio) might help to assess cardiovascular risk in hemodialysis patients. METHODS: In this prospective cohort study 70 prevalent hemodialysis patients were followed up to 4 years. The PTH ratio was determined at baseline. Primary outcomes were cardiovascular events (CVE) and all-cause mortality. Cumulative event-free survival was compared between patients with a ratio < 1 and those with a ratio > 1. The risk-association of the PTH ratio with CVE was examined using an adjusted Multiple Cox Proportional Hazards model. RESULTS: A PTH ratio > 1 was found in 34 patients (49%). During follow-up 26 patients suffered a CVE. Patients with a CVE showed a higher ratio than patients with event-free survival (p = 0.033). In patients with a ratio > 1 a significantly higher number of CVE occurred (53 vs. 22%; p = 0.013), and these patients showed a significantly shorter event-free survival (p = 0.032). In an adjusted Cox-proportional hazards model a higher PTH ratio was found to be independently associated with an elevated risk for CVE (HR = 3.2; 95% CI 1.06 - 13.63; p = 0.04). CONCLUSIONS: A higher PTH (1-84)/non-PTH (1-84) ratio is associated with an increased risk for CVE in hemodialysis patients and might therefore be useful for cardiovascular risk estimation in this population.

Apoptosis of human T-cells: induction by glucocorticoids or surface receptor ligation in vitro and ex vivo.

Activated T-cells are susceptible to induction of apoptosis or programmed cell death in response to ligation of several cell surface structures, including CD2, CD3, and CD95/Fas. These mechanisms may be important in the regulation of immune responses and in prevention of autoimmunity. We used flow cytometric quantitation of DNA strand breaks to detect T-cells committed to programmed cell death. Activated human peripheral blood T-lymphocytes, and freshly isolated human thymocytes underwent apoptosis when exposed to dexamethasone or to monoclonal antibodies directed at CD2 or CD3. Interleukin-2 reduced spontaneous or dexamethasone-induced apoptosis, but augmented apoptosis due to ligation of CD2. A neutralizing anti-Fas antibody reduced the amount of DNA strand breakage, not only in T-cells exposed to antibodies to CD2 or CD3, but also in dexamethasone-treated cultures. In vivo activated T-cells, from inflammatory synovial fluids, were sensitive to immediate induction of DNA strand breaks without prior in vitro activation by lectin and IL-2. Taken together, the results indicated that: 1. Human lymphocytes, like murine thymocytes, are sensitive to glucocorticoid-induced apoptosis, as well as to programmed cell death triggered through surface receptors; 2. The effects of IL-2 on T-cell apoptosis depend on the apoptotic stimulus; 3. Fas/Fas ligand interactions may be relevant for both membrane receptor and glucocorticoid-induced cell death; and 4. Induction of T-cell apoptosis may be important in therapeutic effects of glucocorticoids in human disease.

The pattern of expression of CD147/neurothelin during human T-cell ontogeny as defined by the monoclonal antibody 8D6.

A novel monoclonal antibody termed 8D6 was generated against the human leukemic T-cell line Molt 13 and was found to recognize the neurothelin structure, recently termed CD147. UM8D6 precipitated a single band from HSB2 cells of molecular weight 35 kDa nonreduced and 40 kDa reduced. CD147 is broadly expressed on human hematopoietic cells and is expressed more intensely on thymocytes than on mature peripheral blood T cells. A clear distinction was found by three-color flow cytometry between antigen density on single positive mature thymocytes subsets compared with the corresponding subset from peripheral blood. The 8D6 antibody did not have direct effects on T-cell activation or apoptosis, and the function of CD147 in development and activation of T cells is not yet clear. Nevertheless, its highly regulated expression during T-cell differentiation and the structural characteristics of this antigen suggest that it has an interesting and important role in T-cell biology.

Responsiveness of human T lymphocytes to bacterial superantigens presented by cultured rheumatoid arthritis synoviocytes.

OBJECTIVE: Type B fibroblastic synoviocytes are abundant in inflamed joints of patients with rheumatoid arthritis (RA), and can secrete cytokines and other mediators of inflammation. The aim of this study was to determine whether cell lines derived from RA type B synoviocytes could also serve as accessory cells for T lymphocyte activation. METHODS: Cells from RA synoviocyte lines, with or without preculture in interferon-gamma (IFN gamma), were cultured with purified peripheral blood T cells, in the presence or absence of superantigens or other accessory cell-dependent T cell mitogens. T cell proliferation was measured by thymidine incorporation, and synoviocyte surface markers were analyzed by flow cytometry. RESULTS: RA type B synoviocyte lines were potent accessory cells for T cell responses to bacterial superantigens or lectins, and direct cell-cell contact was required. Preculture in IFN gamma augmented synoviocyte expression of major histocompatibility complex (MHC) class II molecules and of ligands for some T cell costimulatory receptors, but synoviocyte accessory cell function was evident even in the absence of IFN gamma. Blocking studies using monoclonal antibodies supported the notion of a role CD2, CD11a/CD18 and MHC class II molecules in synoviocyte-dependent T cell activation. Monoclonal antibodies against IFN gamma, interleukin-1 beta (IL-1 beta), IL-6, IL-8, and tumor necrosis factor alpha failed to block the T cell proliferative responses, but anti-IL-2 was strongly inhibitory. CONCLUSION: Cultured RA and type B synoviocytes can perform some of the functions of professional antigen-presenting cells. If such cells have similar properties in vivo, they may be important participants in activation of immune responses, in addition to their previously described synthetic and proinflammatory roles. If RA synovial tissue T cells, like normal peripheral blood T cells, can respond to superantigens presented by synoviocytes, this interaction could be important in the pathogenesis of RA.