Regulation of PKD by the MAPK p38delta in insulin secretion and glucose homeostasis.

Dysfunction and loss of insulin-producing pancreatic beta cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38delta display improved glucose tolerance due to enhanced insulin secretion from pancreatic beta cells. Deletion of p38delta results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38delta catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38delta null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated beta cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38delta-deficient islets and glucose tolerance in p38delta null mice as well as their susceptibility to oxidative stress. In conclusion, the p38delta-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic beta cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.

Selective pharmacological inhibition of DDR1 prevents experimentally-induced glomerulonephritis in prevention and therapeutic regime.

BACKGROUND: Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase extensively implicated in diseases such as cancer, atherosclerosis and fibrosis. Multiple preclinical studies, performed using either a gene deletion or a gene silencing approaches, have shown this receptor being a major driver target of fibrosis and glomerulosclerosis. METHODS: The present study investigated the role and relevance of DDR1 in human crescentic glomerulonephritis (GN). Detailed DDR1 expression was first characterized in detail in human GN biopsies using a novel selective anti-DDR1 antibody using immunohistochemistry. Subsequently the protective role of DDR1 was investigated using a highly selective, novel, small molecule inhibitor in a nephrotoxic serum (NTS) GN model in a prophylactic regime and in the NEP25 GN mouse model using a therapeutic intervention regime. RESULTS: DDR1 expression was shown to be mainly limited to renal epithelium. In humans, DDR1 is highly induced in injured podocytes, in bridging cells expressing both parietal epithelial cell (PEC) and podocyte markers and in a subset of PECs forming the cellular crescents in human GN. Pharmacological inhibition of DDR1 in NTS improved both renal function and histological parameters. These results, obtained using a prophylactic regime, were confirmed in the NEP25 GN mouse model using a therapeutic intervention regime. Gene expression analysis of NTS showed that pharmacological blockade of DDR1 specifically reverted fibrotic and inflammatory gene networks and modulated expression of the glomerular cell gene signature, further validating DDR1 as a major mediator of cell fate in podocytes and PECs. CONCLUSIONS: Together, these results suggest that DDR1 inhibition might be an attractive and promising pharmacological intervention for the treatment of GN, predominantly by targeting the renal epithelium.

Telomerase gene therapy rescues telomere length, bone marrow aplasia, and survival in mice with aplastic anemia.

Aplastic anemia is a fatal bone marrow disorder characterized by peripheral pancytopenia and marrow hypoplasia. The disease can be hereditary or acquired and develops at any stage of life. A subgroup of the inherited form is caused by replicative impairment of hematopoietic stem and progenitor cells due to very short telomeres as a result of mutations in telomerase and other telomere components. Abnormal telomere shortening is also described in cases of acquired aplastic anemia, most likely secondary to increased turnover of bone marrow stem and progenitor cells. Here, we test the therapeutic efficacy of telomerase activation by using adeno-associated virus (AAV)9 gene therapy vectors carrying the telomerase Tert gene in 2 independent mouse models of aplastic anemia due to short telomeres (Trf1- and Tert-deficient mice). We find that a high dose of AAV9-Tert targets the bone marrow compartment, including hematopoietic stem cells. AAV9-Tert treatment after telomere attrition in bone marrow cells rescues aplastic anemia and mouse survival compared with mice treated with the empty vector. Improved survival is associated with a significant increase in telomere length in peripheral blood and bone marrow cells, as well as improved blood counts. These findings indicate that telomerase gene therapy represents a novel therapeutic strategy to treat aplastic anemia provoked or associated with short telomeres.

Growth Differentiation Factor-15 and Risk of CKD Progression.

Growth differentiation factor-15 (GDF-15) is a member of the TGF-ß cytokine superfamily that is widely expressed and may be induced in response to tissue injury. Elevations in GDF-15 may identify a novel pathway involved in loss of kidney function among patients with CKD. Among participants in the Clinical Phenotyping and Resource Biobank (C-PROBE) study and the Seattle Kidney Study (SKS), we tested whether kidney tissue expression of GDF15 mRNA correlates with circulating levels of GDF-15 and whether elevations in circulating GDF-15 are associated with decline in kidney function. In matching samples of 24 patients with CKD from the C-PROBE study, circulating GDF-15 levels significantly correlated with intrarenal GDF15 transcript levels (r=0.54, P=0.01). Among the 224 C-PROBE and 297 SKS participants, 72 (32.1%) and 94 (32.0%) patients, respectively, reached a composite end point of 30% decline in eGFR or progression to ESRD over a median of 1.8 and 2.0 years of follow up, respectively. In multivariable models, after adjusting for potential confounders, every doubling of GDF-15 level associated with a 72% higher (95% confidence interval, 1.21 to 4.45; P=0.003) and 65% higher (95% confidence interval, 1.08 to 2.50; P=0.02) risk of progression of kidney disease in C-PROBE and SKS participants, respectively. These results show that circulating GDF-15 levels strongly correlated with intrarenal expression of GDF15 and significantly associated with increased risk of CKD progression in two independent cohorts. Circulating GDF-15 may be a marker for intrarenal GDF15-related signaling pathways associated with CKD and CKD progression.

Epithelial-mesenchymal crosstalk alteration in kidney fibrosis.

The incidence of chronic kidney diseases (CKD) is constantly rising, reaching epidemic proportions in the western world and leading to an enormous threat, even to modern health-care systems, in industrialized countries. Therapies of CKD have greatly improved following the introduction of drugs targeting the renin-angiotensin system (RAAS) but even this refined pharmacological approach has failed to stop progression to end-stage renal disease (ESRD) in many individuals. In vitro historical data and recent new findings have suggested that progression of renal fibrosis might occur as a result of an altered tubulo-interstitial microenvironment and, more specifically, as a result of an altered epithelial-mesenchymal crosstalk. Here we the review biological findings that support the hypothesis of an altered cellular crosstalk in an injured local tubulo-interstitial microenvironment leading to renal disease progression. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Spatial Transcriptional Mapping Reveals Site-Specific Pathways Underlying Human Atherosclerotic Plaque Rupture.

BACKGROUND: Atherosclerotic plaque ruptures, triggered by blood flow-associated biomechanical forces, cause most myocardial infarctions and strokes. OBJECTIVES: This study aims to investigate the exact location and underlying mechanisms of atherosclerotic plaque ruptures, identifying therapeutic targets against cardiovascular events. METHODS: Histology, electron microscopy, bulk and spatial RNA sequencing on human carotid plaques were studied in proximal, most stenotic, and distal regions along the longitudinal blood flow direction. Genome-wide association studies were used to examine heritability enrichment and causal relationships of atherosclerosis and stroke. Associations between top differentially expressed genes (DEGs) and preoperative and postoperative cardiovascular events were examined in a validation cohort. RESULTS: In human carotid atherosclerotic plaques, ruptures predominantly occurred in the proximal and most stenotic regions but not in the distal region. Histologic and electron microscopic examination showed that proximal and most stenotic regions exhibited features of plaque vulnerability and thrombosis. RNA sequencing identified DEGs distinguishing the proximal and most stenotic regions from the distal region which were deemed as most relevant to atherosclerosis-associated diseases as shown by heritability enrichment analyses. The identified pathways associated with the proximal rupture-prone regions were validated by spatial transcriptomics, firstly in human atherosclerosis. Of the 3 top DEGs, matrix metallopeptidase 9 emerged particularly because Mendelian randomization suggested that its high circulating levels were causally associated with atherosclerosis risk. CONCLUSIONS: Our findings show plaque site-specific transcriptional signatures associated with proximal rupture-prone regions of carotid atherosclerotic plaques. This led to the geographical mapping of novel therapeutic targets, such as matrix metallopeptidase 9, against plaque rupture.

From acute injury to chronic disease: pathophysiological hypothesis of an epithelial/mesenchymal crosstalk alteration in CKD.

Observational clinical studies link acute kidney injury to chronic kidney disease (CKD) progression. The pathophysiological mechanisms that underlie this process are currently unknown but recently published papers suggest that tubular epithelial cells and interstitial mesenchymal cells emerge as a single unit, and their integrity alteration as a whole might lead to renal fibrosis and CKD. The present article reviews the biological findings supporting the hypothesis of an altered epithelial/mesenchymal crosstalk in fibrosis development and progression toward CKD.

Current drug development challenges in chronic kidney disease (CKD)--identification of individualized determinants of renal progression and premature cardiovascular disease (CVD).

Chronic kidney disease (CKD) and end-stage renal disease (ESRD) are currently considered as major health burdens. Notably, CKD can be regarded as an interesting clinical model of accelerated cardiovascular disease (CVD) and ageing, which offers exciting new perspectives and challenges for pharmaceutical drug development. However, during the last decades, therapeutic advances to slow down the progression of CKD and reduce CVD risk have largely failed due to several possible reasons including (i) the lack of profound understanding of the pathophysiology of chronic renal damage and its associated CVD; (ii) an inadequate characterization of molecular mechanisms of currently approved therapies such as renin-angiotensin-aldosterone-system (RAAS) blockade; (iii) the unclear biochemical property needs required for novel therapeutic approaches; (iv) the missing quantity and quality of clinical trials in the nephrology field; and, most importantly, (v) the absence of prognostic renal biomarkers that reflect the severity of the structural organ damage and predict ESRD as well as CVD mortality. There is clearly an insufficient understanding of why a significant proportion of CKD patients progress to ESRD and/or die from CVD whereas others rather remain stable. In this article, we urge renal researchers to develop novel experimental and clinical tools for rational and translational drug discovery. Identification of individualized determinants of CKD progression and/or premature CVD will enable personalized medicine and lead to novel innovative nephro- and/or cardioprotective pharmacological treatment in these high-risk patients.

Comparison of Plasma and Urine Biomarker Performance in Acute Kidney Injury.

BACKGROUND: New renal biomarkers measured in urine promise to increase specificity for risk stratification and early diagnosis of acute kidney injury (AKI) but concomitantly may be altered by urine concentration effects and chronic renal insufficiency. This study therefore directly compared the performance of AKI biomarkers in urine and plasma. METHODS: This single-center, prospective cohort study included 110 unselected adults undergoing cardiac surgery with cardiopulmonary bypass between 2009 and 2010. Plasma and/or urine concentrations of creatinine, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), liver fatty acid-binding protein (L-FABP), kidney injury molecule 1 (KIM1), and albumin as well as 15 additional biomarkers in plasma and urine were measured during the perioperative period. The primary outcome was AKI defined by AKIN serum creatinine criteria within 72 hours after surgery. RESULTS: Biomarkers in plasma showed markedly better discriminative performance for preoperative risk stratification and early postoperative (within 24h after surgery) detection of AKI than urine biomarkers. Discriminative power of urine biomarkers improved when concentrations were normalized to urinary creatinine, but urine biomarkers had still lower AUC values than plasma biomarkers. Best diagnostic performance 4h after surgery had plasma NGAL (AUC 0.83), cystatin C (0.76), MIG (0.74), and L-FAPB (0.73). Combinations of multiple biomarkers did not improve their diagnostic power. Preoperative clinical scoring systems (EuroSCORE and Cleveland Clinic Foundation Score) predicted the risk for AKI (AUC 0.76 and 0.71) and were not inferior to biomarkers. Preexisting chronic kidney disease limited the diagnostic performance of both plasma and urine biomarkers. CONCLUSIONS: In our cohort plasma biomarkers had higher discriminative power for risk stratification and early diagnosis of AKI than urine biomarkers. For preoperative risk stratification of AKI clinical models showed similar discriminative performance to biomarkers. The discriminative performance of both plasma and urine biomarkers was reduced by preexisting chronic kidney disease.

Tissue transcriptome-driven identification of epidermal growth factor as a chronic kidney disease biomarker.

Chronic kidney disease (CKD) affects 8 to 16% people worldwide, with an increasing incidence and prevalence of end-stage kidney disease (ESKD). The effective management of CKD is confounded by the inability to identify patients at high risk of progression while in early stages of CKD. To address this challenge, a renal biopsy transcriptome-driven approach was applied to develop noninvasive prognostic biomarkers for CKD progression. Expression of intrarenal transcripts was correlated with the baseline estimated glomerular filtration rate (eGFR) in 261 patients. Proteins encoded by eGFR-associated transcripts were tested in urine for association with renal tissue injury and baseline eGFR. The ability to predict CKD progression, defined as the composite of ESKD or 40% reduction of baseline eGFR, was then determined in three independent CKD cohorts. A panel of intrarenal transcripts, including epidermal growth factor (EGF), a tubule-specific protein critical for cell differentiation and regeneration, predicted eGFR. The amount of EGF protein in urine (uEGF) showed significant correlation (P < 0.001) with intrarenal EGF mRNA, interstitial fibrosis/tubular atrophy, eGFR, and rate of eGFR loss. Prediction of the composite renal end point by age, gender, eGFR, and albuminuria was significantly (P < 0.001) improved by addition of uEGF, with an increase of the C-statistic from 0.75 to 0.87. Outcome predictions were replicated in two independent CKD cohorts. Our approach identified uEGF as an independent risk predictor of CKD progression. Addition of uEGF to standard clinical parameters improved the prediction of disease events in diverse CKD populations with a wide spectrum of causes and stages.

Telomere length and telomerase activity: effect of ageing on human NK cells.

Telomeres are repeats of TTAGGG sequences located at the end of eukaryotic chromosomes. They are essential for stabilisation and protection of chromosomal ends and for the regulation of cell replicative capacity. Due to the end-replication defect of DNA polymerase, telomeres shorten progressively with each cell division and telomere length may be an indicator of the replicative history of a cell. Compensatory mechanisms for the telomere loss have been identified. The most widely studied one is mediated by telomerase a ribonuclear protein-enzyme complex that synthesise telomeric repeats. In this study we have investigated whether NK cells, derived from a group of old healthy subjects, underwent the modifications of telomere length and telomerase activity observed in other sub-populations of lymphocytes with advancing age. We demonstrated that: (a) telomere shortening occurred and telomerase activity decreased in human NK cells with ageing; (b) the rate of telomere loss was different under and over 80 years of age; (c) similarly to telomere shortening, the modification of telomerase activity was particularly evident in octogenarians; (d) subjects with the most evident modifications of telomeres and telomerase were the oldest and those with increased NK cell numbers.

Different rates of telomere shortening and telomerase activity reduction in CD8 T and CD16 NK lymphocytes with ageing.

Telomeres are specialised structures located at the end of eukaryotic chromosomes, that get short during progressive cell divisions. Therefore, telomere may be an indicator of the mitotic history of a cell and it is also a determining factor for the residual cell life span. One mechanism, compensating for the telomere erosion, involves the induction of telomerase, a ribonucleoprotein-enzyme able to synthesize telomeric DNA repeats. In this study, old subjects of two consecutive decades were compared with a group of young controls to investigate whether ageing-related modifications differently affects telomere length and telomerase activity of human peripheral blood CD8 T and CD16 NK lymphocytes. Telomeres in individual cells were measured by flow-FISH and telomerase activity was determined using the TeloTAGGG telomerase PCR ELISA(PLUS) kit. Both CD8 T and NK lymphocytes showed an age-associated loss of telomeres at rates that were different between the subsets together with an age-associated reduction of telomerase activity that was progressive in CD8 and late in NK lymphocytes. We can assume that preserved innate immune response in the elderly is due to the negligible telomere shortening and the maintained telomerase expression that could allow NK cells of octogenarians to delay replicative senescence.

New renal drug development to face chronic renal disease.

INTRODUCTION: For the first time in 2012, chronic kidney diseases were added to WHO disease list because of their impact on morbidity/mortality and their substantial impact on the cost to the health care system. Although representing a clear unmet medical need and a huge business case for the pharmaceutical industry, its clinical treatment still mainly relies on drugs invented in the 1980s used for controlling blood pressure. AREAS COVERED: In this review, the authors aim to elucidate why renal drug development is feasible today. The article provides a particular focus on the treatments that target the pathways involved in inflammation, fibrosis and the core mechanisms driving the vicious cycle responsible for disease progression and organ function loss. EXPERT OPINION: Currently, it is plausible to develop effective therapeutics for renal diseases with a plethora of approaches available for their development at a preclinical and clinical level. Furthermore, the relevance of biomarkers and the use of surrogate rare disease indications as proof of mechanism for faster and/or smaller clinical development are now possible; and these developments could revolutionize the way we treat renal disease in the future.

Telomerase expression confers cardioprotection in the adult mouse heart after acute myocardial infarction.

Coronary heart disease is one of the main causes of death in the developed world, and treatment success remains modest, with high mortality rates within 1 year after myocardial infarction (MI). Thus, new therapeutic targets and effective treatments are necessary. Short telomeres are risk factors for age-associated diseases, including heart disease. Here we address the potential of telomerase (Tert) activation in prevention of heart failure after MI in adult mice. We use adeno-associated viruses for cardiac-specific Tert expression. We find that upon MI, hearts expressing Tert show attenuated cardiac dilation, improved ventricular function and smaller infarct scars concomitant with increased mouse survival by 17% compared with controls. Furthermore, Tert treatment results in elongated telomeres, increased numbers of Ki67 and pH3-positive cardiomyocytes and a gene expression switch towards a regeneration signature of neonatal mice. Our work suggests telomerase activation could be a therapeutic strategy to prevent heart failure after MI.

Targeting the epithelial cells in fibrosis: a new concept for an old disease.

Fibrosis, which affects millions of individuals worldwide, is a leading cause of organ failure. For 40 years myofibroblasts have been recognized to be the key cellular players in fibrosis. Currently, several pharmaceutical targets are under investigation that may contribute to the activation of myofibroblasts. Recent preclinical and clinical evidence suggests that other components in the fibrotic microenvironment can trigger myofibroblast activation, providing new targets for pharmaceutical intervention. Epithelial cells may represent the most promising cellular phenotype that could be exploited in the design of new anti-fibrotic medicines through their paracrine action on myofibroblasts. The present review briefly highlights this hypothesis and discusses some interesting related pharmacological targets.

The BAR domain protein Arfaptin-1 controls secretory granule biogenesis at the trans-Golgi network.

BAR domains can prevent membrane fission through their ability to shield necks of budding vesicles from fission-inducing factors. However, the physiological role of this inhibitory function and its regulation is unknown. Here we identify a checkpoint involving the BAR-domain-containing protein Arfaptin-1 that controls biogenesis of secretory granules at the trans-Golgi network (TGN). We demonstrate that protein kinase D (PKD) phosphorylates Arfaptin-1 at serine 132, which disrupts the ability of Arfaptin-1 to inhibit the activity of ADP ribosylation factor, an important component of the vesicle scission machinery. The physiological significance of this regulatory mechanism is evidenced by loss of glucose-stimulated insulin secretion due to granule scission defects in pancreatic ß cells expressing nonphosphorylatable Arfaptin-1. Accordingly, depletion of Arfaptin-1 leads to the generation of small nonfunctional secretory granules. Hence, PKD-mediated Arfaptin-1 phosphorylation is necessary to ensure biogenesis of functional transport carriers at the TGN in regulated secretion.

The N-terminal domain of ERK1 accounts for the functional differences with ERK2.

The Extracellular Regulated Kinase 1 and 2 transduce a variety of extracellular stimuli regulating processes as diverse as proliferation, differentiation and synaptic plasticity. Once activated in the cytoplasm, ERK1 and ERK2 translocate into the nucleus and interact with nuclear substrates to induce specific programs of gene expression. ERK1/2 share 85% of aminoacid identity and all known functional domains and thence they have been considered functionally equivalent until recent studies found that the ablation of either ERK1 or ERK2 causes dramatically different phenotypes. To search a molecular justification of this dichotomy we investigated whether the different functions of ERK1 and 2 might depend on the properties of their cytoplasmic-nuclear trafficking. Since in the nucleus ERK1/2 is predominantly inactivated, the maintenance of a constant level of nuclear activity requires continuous shuttling of activated protein from the cytoplasm. For this reason, different nuclear-cytoplasmic trafficking of ERK1 and 2 would cause a differential signalling capability. We have characterised the trafficking of fluorescently tagged ERK1 and ERK2 by means of time-lapse imaging in living cells. Surprisingly, we found that ERK1 shuttles between the nucleus and cytoplasm at a much slower rate than ERK2. This difference is caused by a domain of ERK1 located at its N-terminus since the progressive deletion of these residues converted the shuttling features of ERK1 into those of ERK2. Conversely, the fusion of this ERK1 sequence at the N-terminus of ERK2 slowed down its shuttling to a similar value found for ERK1. Finally, computational, biochemical and cellular studies indicated that the reduced nuclear shuttling of ERK1 causes a strong reduction of its nuclear phosphorylation compared to ERK2, leading to a reduced capability of ERK1 to carry proliferative signals to the nucleus. This mechanism significantly contributes to the differential ability of ERK1 and 2 to generate an overall signalling output.

ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially.

BACKGROUND: The mitogen-activated protein (MAP) kinases p44ERK1 and p42ERK2 are crucial components of the regulatory machinery underlying normal and malignant cell proliferation. A currently accepted model maintains that ERK1 and ERK2 are regulated similarly and contribute to intracellular signaling by phosphorylating a largely common subset of substrates, both in the cytosol and in the nucleus. RESULTS: Here, we show that ablation of ERK1 in mouse embryo fibroblasts and NIH 3T3 cells by gene targeting and RNA interference results in an enhancement of ERK2-dependent signaling and in a significant growth advantage. By contrast, knockdown of ERK2 almost completely abolishes normal and Ras-dependent cell proliferation. Ectopic expression of ERK1 but not of ERK2 in NIH 3T3 cells inhibits oncogenic Ras-mediated proliferation and colony formation. These phenotypes are independent of the kinase activity of ERK1, as expression of a catalytically inactive form of ERK1 is equally effective. Finally, ectopic expression of ERK1 but not ERK2 is sufficient to attenuate Ras-dependent tumor formation in nude mice. CONCLUSION: These results reveal an unexpected interplay between ERK1 and ERK2 in transducing Ras-dependent cell signaling and proliferation. Whereas ERK2 seems to have a positive role in controlling normal and Ras-dependent cell proliferation, ERK1 probably affects the overall signaling output of the cell by antagonizing ERK2 activity.