Prevalence, phenotypic characteristics and prognostic role of apparent treatment resistant hypertension in the German Chronic Kidney Disease (GCKD) study.

Treatment resistant hypertension (TRH) appears of particular relevance in patients with chronic kidney disease (CKD). However, causes and consequences of TRH in CKD patients remain incompletely understood. Therefore, we analyzed the prevalence of apparent TRH (aTRH), and phenotypic characteristics and prognosis associated with aTRH among participants of the German Chronic Kidney Disease (GCKD) study. As insufficient medication adherence has been shown to be a frequent cause of pseudoresistance, we also assessed treatment adherence. Study participants were classified as having aTRH, controlled hypertension and uncontrolled hypertension based on study visit blood pressure and self-reported medication intake. Drug adherence was assessed by comparing self-reported antihypertensive medication with detectable urinary drug metabolites measured by mass spectroscopy. Out of 4901 individuals included in this study, 38% were classified as having aTRH. Male sex, older age, lower estimated glomerular filtration rate (eGFR), higher body mass index (BMI), higher urine albumin-to-creatinine ratio (UACR) and presence of diabetes mellitus were independently associated with higher prevalence of aTRH in a multivariable adjusted regression model. Patients classified as aTRH had higher risk for major adverse cardiovascular events and worsening of kidney disease compared to patients with no aTRH after multivariate adjustment for potential confounders. There was a high agreement between self-reported medication and detectable urinary drug metabolites. In conclusion, in a cohort of Caucasian patients with moderately severe CKD, aTRH was highly prevalent and, in most cases, likely not caused by low medication adherence. Furthermore, aTRH was linked to cardio-renal endpoints, emphasizing the need for improved management.

Establishment of a novel, cell-based autotaxin assay.

Autotaxin (ATX) plays an important role in (patho-)physiological lysophosphatidic acid (LPA) signaling. Here we describe the establishment of novel cell-based ATX assay formats. ATX-mediated LPA generation is detected by using a stable LPA receptor reporter cell line. In a first assay variant, ATX-mediated LPA generation is started in the absence of cells and the reaction mix is transferred to the reporter cells after stopping the reaction (two-tube assay). In a second assay variant, ATX is added to the reporter cells expressing the known autotaxin binding partners integrin ß1, integrin ß3 and the LPA receptor 1. LPA generation is started in the presence of cells and is detected in real-time (one-tube assay). Structurally diverse ATX inhibitors with different binding modes were characterized in both cell-based assay variants and were also tested in the well-established biochemical choline release assay. ATX inhibitors displayed similar potencies, regardless if the assay was performed in the absence or presence of cells, and comparable results were obtained in all three assay formats. In summary, our novel cell-based ATX assay formats are well-suited for sensitive detection of enzyme activity as well as for the characterization of ATX inhibitors in the presence and absence of cells.

A flow cytometry approach reveals heterogeneity in conventional subsets of murine renal mononuclear phagocytes.

Mononuclear phagocytes (MNPs) participate in inflammation and repair after kidney injury, reflecting their complex nature. Dissection into refined functional subunits has been challenging and would benefit understanding of renal pathologies. Flow cytometric approaches are limited to classifications of either different MNP subsets or functional state. We sought to combine these two dimensions in one protocol that considers functional heterogeneity in each MNP subset. We identified five distinct renal MNP subsets based on a previously described strategy. In vitro polarization of bone marrow-derived macrophages (BMDM) into M1- and M2-like cells suggested functional distinction of CD86 + MHCII + CD206- and CD206 + cells. Combination of both distinction methods identified CD86 + MHCII + CD206- and CD206 + cells in all five MNP subsets, revealing their heterologous nature. Our approach revealed that MNP composition and their functional segmentation varied between different mouse models of kidney injury and, moreover, was dynamically regulated in a time-dependent manner. CD206 + cells from three analyzed MNP subsets had a higher ex vivo phagocytic capacity than CD86 + MHCII + CD206- counterparts, indicating functional uniqueness of each subset. In conclusion, our novel flow cytometric approach refines insights into renal MNP heterogeneity and therefore could benefit mechanistic understanding of renal pathology.

Self-Reported Medication Use and Urinary Drug Metabolites in the German Chronic Kidney Disease (GCKD) Study.

BACKGROUND: Polypharmacy is common among patients with CKD, but little is known about the urinary excretion of many drugs and their metabolites among patients with CKD. METHODS: To evaluate self-reported medication use in relation to urine drug metabolite levels in a large cohort of patients with CKD, the German Chronic Kidney Disease study, we ascertained self-reported use of 158 substances and 41 medication groups, and coded active ingredients according to the Anatomical Therapeutic Chemical Classification System. We used a nontargeted mass spectrometry-based approach to quantify metabolites in urine; calculated specificity, sensitivity, and accuracy of medication use and corresponding metabolite measurements; and used multivariable regression models to evaluate associations and prescription patterns. RESULTS: Among 4885 participants, there were 108 medication-drug metabolite pairs on the basis of reported medication use and 78 drug metabolites. Accuracy was excellent for measurements of 36 individual substances in which the unchanged drug was measured in urine (median, 98.5%; range, 61.1%-100%). For 66 pairs of substances and their related drug metabolites, median measurement-based specificity and sensitivity were 99.2% (range, 84.0%-100%) and 71.7% (range, 1.2%-100%), respectively. Commonly prescribed medications for hypertension and cardiovascular risk reduction-including angiotensin II receptor blockers, calcium channel blockers, and metoprolol-showed high sensitivity and specificity. Although self-reported use of prescribed analgesics (acetaminophen, ibuprofen) was <3% each, drug metabolite levels indicated higher usage (acetaminophen, 10%-26%; ibuprofen, 10%-18%). CONCLUSIONS: This comprehensive screen of associations between urine drug metabolite levels and self-reported medication use supports the use of pharmacometabolomics to assess medication adherence and prescription patterns in persons with CKD, and indicates under-reported use of medications available over the counter, such as analgesics.

The Importance of Incorporating Landscape Change for Predictions of Climate-Induced Plant Phenological Shifts.

Warming in the high Arctic is occurring at the fastest rate on the planet, raising concerns over how this global change driver will influence plant community composition, the timing of vegetation phenological events, and the wildlife that rely on them. In this region, as much as 50% of near-surface permafrost is composed of thermally sensitive ground ice that when melted produces substantial changes in topography and microbiome conditions. We take advantage of natural variations in permafrost melt to conduct a space-for-time study on Ellesmere Island in northern Canada. We demonstrate that phenological timing can be delayed in thermokarst areas when compared to stable ground, and that this change is a function of shifting species composition in these vegetation communities as well as delayed timing within species. These findings suggest that a warming climate could result in an overall broadening of blooming and leafing windows at the landscape level when these delayed timings are taken into consideration with the projected advance of phenological timings in ice-poor areas. We emphasize that the impacts of geomorphic processes on key phenological drivers are essential for enhancing our understanding of community response to climate warming in the high Arctic, with implications for ecosystem functioning and trophic interactions.

The anticancer phytochemical rocaglamide inhibits Rho GTPase activity and cancer cell migration.

Chemotherapy is one of the pillars of anti-cancer therapy. Although chemotherapeutics cause regression of the primary tumor, many chemotherapeutics are often shown to induce or accelerate metastasis formation. Moreover, metastatic tumors are largely resistant against chemotherapy. As more than 90% of cancer patients die due to metastases and not due to primary tumor formation, novel drugs are needed to overcome these shortcomings. In this study, we identified the anticancer phytochemical Rocaglamide (Roc-A) to be an inhibitor of cancer cell migration, a crucial event in metastasis formation. We show that Roc-A inhibits cellular migration and invasion independently of its anti-proliferative and cytotoxic effects in different types of human cancer cells. Mechanistically, Roc-A treatment induces F-actin-based morphological changes in membrane protrusions. Further investigation of the molecular mechanisms revealed that Roc-A inhibits the activities of the small GTPases RhoA, Rac1 and Cdc42, the master regulators of cellular migration. Taken together, our results provide evidence that Roc-A may be a lead candidate for a new class of anticancer drugs that inhibit metastasis formation.

The protein phosphatase 2A regulatory subunit B56γ mediates suppression of T cell receptor (TCR)-induced nuclear factor-κB (NF-κB) activity.

NF-κB is an important transcription factor in the immune system, and aberrant NF-κB activity contributes to malignant diseases and autoimmunity. In T cells, NF-κB is activated upon TCR stimulation, and signal transduction to NF-κB activation is triggered by a cascade of phosphorylation events. However, fine-tuning and termination of TCR signaling are only partially understood. Phosphatases oppose the role of kinases by removing phosphate moieties. The catalytic activity of the protein phosphatase PP2A has been implicated in the regulation of NF-κB. PP2A acts in trimeric complexes in which the catalytic subunit is promiscuous and the regulatory subunit confers substrate specificity. To understand and eventually target NF-κB-specific PP2A functions it is essential to define the regulatory PP2A subunit involved. So far, the regulatory PP2A subunit that mediates NF-κB suppression in T cells remained undefined. By performing a siRNA screen in Jurkat T cells harboring a NF-κB-responsive luciferase reporter, we identified the PP2A regulatory subunit B56γ as negative regulator of NF-κB in TCR signaling. B56γ was strongly up-regulated upon primary human T cell activation, and B56γ silencing induced increased IκB kinase (IKK) and IκBα phosphorylation upon TCR stimulation. B56γ silencing enhanced NF-κB activity, resulting in increased NF-κB target gene expression including the T cell cytokine IL-2. In addition, T cell proliferation was increased upon B56γ silencing. These data help to understand the physiology of PP2A function in T cells and the pathophysiology of diseases involving PP2A and NF-κB.

LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis.

DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications.

Spatial organization of the mouse genome and its role in recurrent chromosomal translocations.

The extent to which the three-dimensional organization of the genome contributes to chromosomal translocations is an important question in cancer genomics. We generated a high-resolution Hi-C spatial organization map of the G1-arrested mouse pro-B cell genome and used high-throughput genome-wide translocation sequencing to map translocations from target DNA double-strand breaks (DSBs) within it. RAG endonuclease-cleaved antigen-receptor loci are dominant translocation partners for target DSBs regardless of genomic position, reflecting high-frequency DSBs at these loci and their colocalization in a fraction of cells. To directly assess spatial proximity contributions, we normalized genomic DSBs via ionizing radiation. Under these conditions, translocations were highly enriched in cis along single chromosomes containing target DSBs and within other chromosomes and subchromosomal domains in a manner directly related to pre-existing spatial proximity. By combining two high-throughput genomic methods in a genetically tractable system, we provide a new lens for viewing cancer genomes.

The role of mechanistic factors in promoting chromosomal translocations found in lymphoid and other cancers.

Recurrent chromosomal abnormalities, especially chromosomal translocations, are strongly associated with certain subtypes of leukemia, lymphoma and solid tumors. The appearance of particular translocations or associated genomic alterations can be important indicators of disease prognosis, and in some cases, certain translocations may indicate appropriate therapy protocols. To date, most of our knowledge about chromosomal translocations has derived from characterization of the highly selected recurrent translocations found in certain cancers. Until recently, mechanisms that promote or suppress chromosomal translocations, in particular, those responsible for their initiation, have not been addressed. For translocations to occur, two distinct chromosomal loci must be broken, brought together (synapsed) and joined. Here, we discuss recent findings on processes and pathways that influence the initiation of chromosomal translocations, including the generation fo DNA double strand breaks (DSBs) by general factors or in the context of the Lymphocyte-specific V(D)J and IgH class-switch recombination processes. We also discuss the role of spatial proximity of DSBs in the interphase nucleus with respect to how DSBs on different chromosomes are justaposed for joining. In addition, we discuss the DNA DSB response and its role in recognizing and tethering chromosomal DSBs to prevent translocations, as well as potential roles of the classical and alternative DSB end-joining pathways in suppressing or promoting translocations. Finally, we discuss the potential roles of long range regulatory elements, such as the 3'IgH enhancer complex, in promoting the expression of certain translocations that are frequent in lymphomas and, thereby, contributing to their frequent appearance in tumors.

Beta1 integrin establishes endothelial cell polarity and arteriolar lumen formation via a Par3-dependent mechanism.

Maintenance of single-layered endothelium, squamous endothelial cell shape, and formation of a patent vascular lumen all require defined endothelial cell polarity. Loss of beta1 integrin (Itgb1) in nascent endothelium leads to disruption of arterial endothelial cell polarity and lumen formation. The loss of polarity is manifested as cuboidal-shaped endothelial cells with dysregulated levels and mislocalization of normally polarized cell-cell adhesion molecules, as well as decreased expression of the polarity gene Par3 (pard3). beta1 integrin and Par3 are both localized to the endothelial layer, with preferential expression of Par3 in arterial endothelium. Luminal occlusion is also exclusively noted in arteries, and is partially rescued by replacement of Par3 protein in beta1-deficient vessels. Combined, our findings demonstrate that beta1 integrin functions upstream of Par3 as part of a molecular cascade required for endothelial cell polarity and lumen formation.

Fate tracing reveals the endothelial origin of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) originate within the aortic-gonado-mesonephros (AGM) region of the midgestation embryo, but the cell type responsible for their emergence is unknown since critical hematopoietic factors are expressed in both the AGM endothelium and its underlying mesenchyme. Here we employ a temporally restricted genetic tracing strategy to selectively label the endothelium, and separately its underlying mesenchyme, during AGM development. Lineage tracing endothelium, via an inducible VE-cadherin Cre line, reveals that the endothelium is capable of HSC emergence. The endothelial progeny migrate to the fetal liver, and later to the bone marrow, and are capable of expansion, self-renewal, and multilineage hematopoietic differentiation. HSC capacity is exclusively endothelial, as ex vivo analyses demonstrate lack of VE-cadherin Cre induction in circulating and fetal liver hematopoietic populations. Moreover, AGM mesenchyme, as selectively traced via a myocardin Cre line, is incapable of hematopoiesis. Our genetic tracing strategy therefore reveals an endothelial origin of HSCs.