Giuseppina Federico

The tRNA methyltransferase Dnmt2 is required for accurate polypeptide synthesis during haematopoiesis

The Dnmt2 enzyme utilizes the catalytic mechanism of eukaryotic DNA methyltransferases to methylate several tRNAs at cytosine 38. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile, and the biological function of Dnmt2 has remained elusive. Here, we show that endochondral ossification is delayed in newborn Dnmt2‐deficient mice, which is accompanied by a reduction of the haematopoietic stem and progenitor cell population and a cell‐autonomous defect in their differentiation. RNA bisulfite sequencing revealed that Dnmt2 methylates C38 of tRNA AspGTC, GlyGCC, and ValAAC, thus preventing tRNA fragmentation. Proteomic analyses from primary bone marrow cells uncovered systematic differences in protein expression that are due to specific codon mistranslation by tRNAs lacking Dnmt2‐dependent methylation. Our observations demonstrate that Dnmt2 plays an important role in haematopoiesis and define a novel function of C38 tRNA methylation in the discrimination of near‐cognate codons, thereby ensuring accurate polypeptide synthesis.

Direct acute tubular damage contributes to Shigatoxin-mediated kidney failure

The pathogenesis and therapy of Shigatoxin 2 (Stx2)‐mediated kidney failure remain controversial. Our aim was to test whether, during an infection with Stx2‐producing E. coli (STEC), Stx2 exerts direct effects on renal tubular epithelium and thereby possibly contributes to acute renal failure. Mice represent a suitable model because they, like humans, express the Stx2‐receptor Gb3 in the tubular epithelium but, in contrast to humans, not in glomerular endothelia, and are thus free of glomerular thrombotic microangiopathy (TMA). In wild‐type mice, Stx2 caused acute tubular dysfunction with consequent electrolyte disturbance, which was most likely the cause of death. Tubule‐specific depletion of Gb3 protected the mice from acute renal failure. In vitro, Stx2 induced secretion of proinflammatory cytokines and apoptosis in human tubular epithelial cells, thus implicating a direct effect of Stx2 on the tubular epithelium. To correlate these results to human disease, kidney biopsies and outcome were analysed in patients with Stx2‐associated kidney failure (n = 11, aged 22–44 years). The majority of kidney biopsies showed different stages of an ongoing TMA; however, no glomerular complement activation could be demonstrated. All biopsies, including those without TMA, showed severe acute tubular damage. Due to these findings, patients were treated with supportive therapy without complement‐inhibiting antibodies (eculizumab) or immunoadsorption. Despite the severity of the initial disease [creatinine 6.34 (1.31–17.60) mg/dl, lactate dehydrogenase 1944 (753–2792) U/l, platelets 33 (19–124)/nl and haemoglobin 6.2 (5.2–7.8) g/dl; median (range)], all patients were discharged after 33 (range 19–43) days with no neurological symptoms and no dialysis requirement [creatinine 1.39 (range 0.84–2.86) mg/dl]. The creatinine decreased further to 0.90 (range 0.66–1.27) mg/dl after 24 months. Based on these data, one may surmise that acute tubular damage represents a separate pathophysiological mechanism, importantly contributing to Stx2‐mediated acute kidney failure. Specifically in young adults, an excellent outcome can be achieved by supportive therapy only.

Semaphorin-Plexin Signaling Controls Mitotic Spindle Orientation during Epithelial Morphogenesis and Repair

Morphogenesis, homeostasis, and regeneration of epithelial tissues rely on the accurate orientation of cell divisions, which is specified by the mitotic spindle axis. To remain in the epithelial plane, symmetrically dividing epithelial cells align their mitotic spindle axis with the plane. Here, we show that this alignment depends on epithelial cell-cell communication via semaphorin-plexin signaling. During kidney morphogenesis and repair, renal tubular epithelial cells lacking the transmembrane receptor Plexin-B2 or its semaphorin ligands fail to correctly orient the mitotic spindle, leading to severe defects in epithelial architecture and function. Analyses of a series of transgenic and knockout mice indicate that Plexin-B2 controls the cell division axis by signaling through its GTPase-activating protein (GAP) domain and Cdc42. Our data uncover semaphorin-plexin signaling as a central regulatory mechanism of mitotic spindle orientation necessary for the alignment of epithelial cell divisions with the epithelial plane.

Immunosuppression and Aberrant T Cell Development in the Absence of N-Myristoylation

N-myristoylation refers to the attachment of myristic acid to the N-terminal glycine of proteins and substantially affects their intracellular targeting and functions. The thymus represents an organ with a prominent N-myristoylation activity. To elucidate the role of protein N-myristoylation for thymocyte development, we generated mice with a T cell lineage–specific deficiency in N-myristoyl transferase (Nmt)1 and 2. Depletion of Nmt activity in T cells led to a defective transmission of TCR signals, a developmental blockage of thymocytes at the transition from double-negative 3 to 4 stages, and a reduction of all the following stages. We could demonstrate that Lck and myristoylated alanine-rich C kinase substrate, two main myristoylated kinases in T cells, were mislocalized in the absence of Nmt activity. N-myristoylation was also indispensable for early and distal TCR signaling events such as CD3ζ, Zap70, and Erk activation and for release of cytokines such as IFN-γ and IL-2. As a consequence, the initiation and propagation of the TCR signaling cascade was severely impaired. Furthermore, we showed that the absence of myristoylation had an immunosuppressive effect on T cells in vivo after treatment with CpG and stimulation of the TCR with the staphylococcal enterotoxin B superantigen. Therefore, protein myristoylation is indispensable in T cell development and activation and its inhibition might offer a novel strategy to achieve immunosuppression.

Tubular Dickkopf-3 promotes the development of renal atrophy and fibrosis

Renal tubular atrophy and interstitial fibrosis are common hallmarks of etiologically different progressive chronic kidney diseases (CKD) that eventually result in organ failure. Even though these pathological manifestations constitute a major public health problem, diagnostic tests, as well as therapeutic options, are currently limited. Members of the dickkopf (DKK) family, DKK1 and -2, have been associated with inhibition of Wnt signaling and organ fibrosis. Here, we identify DKK3 as a stress-induced, tubular epithelia-derived, secreted glycoprotein that mediates kidney fibrosis. Genetic as well as antibody-mediated abrogation of DKK3 led to reduced tubular atrophy and decreased interstitial matrix accumulation in two mouse models of renal fibrosis. This was facilitated by an amplified, antifibrogenic, inflammatory T cell response and diminished canonical Wnt/β-catenin signaling in stressed tubular epithelial cells. Moreover, in humans, urinary DKK3 levels specifically correlated with the extent of tubular atrophy and interstitial fibrosis in different glomerular and tubulointerstitial diseases. In summary, our data suggest that DKK3 constitutes an immunosuppressive and a profibrotic epithelial protein that might serve as a potential therapeutic target and diagnostic marker in renal fibrosis.

Inhibition of Endothelial Notch Signaling Impairs Fatty Acid Transport and Leads to Metabolic and Vascular Remodeling of the Adult Heart

Background: Nutrients are transported through endothelial cells before being metabolized in muscle cells. However, little is known about the regulation of endothelial transport processes. Notch signaling is a critical regulator of metabolism and angiogenesis during development. Here, we studied how genetic and pharmacological manipulation of endothelial Notch signaling in adult mice affects endothelial fatty acid transport, cardiac angiogenesis, and heart function. Methods: Endothelial-specific Notch inhibition was achieved by conditional genetic inactivation of Rbp-j&kgr; in adult mice to analyze fatty acid metabolism and heart function. Wild-type mice were treated with neutralizing antibodies against the Notch ligand Delta-like 4. Fatty acid transport was studied in cultured endothelial cells and transgenic mice. Results: Treatment of wild-type mice with Delta-like 4 neutralizing antibodies for 8 weeks impaired fractional shortening and ejection fraction in the majority of mice. Inhibition of Notch signaling specifically in the endothelium of adult mice by genetic ablation of Rbp-j&kgr; caused heart hypertrophy and failure. Impaired heart function was preceded by alterations in fatty acid metabolism and an increase in cardiac blood vessel density. Endothelial Notch signaling controlled the expression of endothelial lipase, Angptl4, CD36, and Fabp4, which are all needed for fatty acid transport across the vessel wall. In endothelial-specific Rbp-j&kgr;–mutant mice, lipase activity and transendothelial transport of long-chain fatty acids to muscle cells were impaired. In turn, lipids accumulated in the plasma and liver. The attenuated supply of cardiomyocytes with long-chain fatty acids was accompanied by higher glucose uptake, increased concentration of glycolysis intermediates, and mTOR-S6K signaling. Treatment with the mTOR inhibitor rapamycin or displacing glucose as cardiac substrate by feeding a ketogenic diet prolonged the survival of endothelial-specific Rbp-j&kgr;–deficient mice. Conclusions: This study identifies Notch signaling as a novel regulator of fatty acid transport across the endothelium and as an essential repressor of angiogenesis in the adult heart. The data imply that the endothelium controls cardiomyocyte metabolism and function.

Impact of AMP-Activated Protein Kinase α1 Deficiency on Tissue Injury following Unilateral Ureteral Obstruction

Background AMP-activated protein kinase (Ampk) is a sensor of the cellular energy status and a powerful regulator of metabolism. Activation of Ampk was previously shown to participate in monocyte-to-fibroblast transition and matrix protein production in renal tissue. Thus, the present study explored whether the catalytic Ampkα1 isoform participates in the regulation of the renal fibrotic response following unilateral ureteral obstruction (UUO). Methods UUO was induced in gene-targeted mice lacking functional Ampkα1 (Ampkα1-/-) and in corresponding wild-type mice (Ampkα1+/+). In the obstructed kidney and, for comparison, in the non-obstructed control kidney, quantitative RT-PCR, Western blotting and immunostaining were employed to determine transcript levels and protein abundance, respectively. Results In Ampkα1+/+ mice, UUO significantly up-regulated the protein abundance of the Ampkα1 isoform, but significantly down-regulated the Ampkα2 isoform in renal tissue. Phosphorylated Ampkα protein levels were significantly increased in obstructed kidney tissue of Ampkα1+/+ mice but not of Ampkα1-/- mice. Renal expression of α-smooth muscle actin was increased following UUO, an effect again less pronounced in Ampkα1-/- mice than in Ampkα1+/+ mice. Histological analysis did not reveal a profound effect of Ampkα1 deficiency on collagen 1 protein deposition. UUO significantly increased phosphorylated and total Tgf-ß-activated kinase 1 (Tak1) protein, as well as transcript levels of Tak1-downstream targets c-Fos, Il6, Pai1 and Snai1 in Ampkα1+/+ mice, effects again significantly ameliorated in Ampkα1-/- mice. Moreover, Ampkα1 deficiency inhibited the UUO-induced mRNA expression of Cd206, a marker of M2 macrophages and of Cxcl16, a pro-fibrotic chemokine associated with myeloid fibroblast formation. The effects of Ampkα1 deficiency during UUO were, however, paralleled by increased tubular injury and apoptosis. Conclusions Renal obstruction induces an isoform shift from Ampkα2 towards Ampkα1, which contributes to the signaling involved in cell survival and fibrosis.

Dickkopf-3 Acts as a Modulator of B Cell Fate and Function

The mechanisms responsible for the generation of a mature B1 and B2 cell compartment are still poorly understood. In this study, we demonstrated that absence of Dickkopf-3 (DKK3) led to changes in the composition of the B cell compartment, which were due to an altered development and maintenance program of B cells. Development of B2 cells was impaired at the pre- and immature B cell stage, resulting in decreased numbers of follicular B cells in adult DKK3-deficient mice. Furthermore, DKK3 limited B1 cell self-maintenance in the periphery, by decreasing the survival and proliferation behavior of B1 cells. DKK3 may act via the BCR signaling pathway, as Ca2+ influx upon BCR stimulation was increased and SiglecG, a molecule shown to inhibit Calcium signaling, was downregulated in the absence of DKK3. DKK3-deficient mice exhibited altered Ab responses and an increased secretion of the cytokine IL-10. Additionally, DKK3 limited autoimmunity in a model of systemic lupus erythematosus. In summary, we identified DKK3 as a novel modulator interfering with B cell fate as well as the maintenance program of B cells, leading to changes in B cell immune responses.

Glomerulonephritis triggered by a chronically infected left ventricular assist device

A 55-year-old man on the waiting list for heart transplantation with severe dilated cardiomyopathy was admitted for evaluation of low-grade fever in October, 2014. He had had a left ventricular assist device (LVAD; HeartMate II) implanted as a bridge to transplant 4 years previously. Examination was unrema rkable. Investigations showed acute kidney injury (serum creatinine increased to 354 μmol/L from 124 μmol/L 2 months previously), raised C-reactive protein, and raised procalcitonin. Urinary protein excretion was 2·2 g/day and urinalysis showed microhaematuria without dysmorphic erythrocytes or casts; previous urinalyses had been unremarkable. Blood cultures were positive for Corynebacterium jeikeium so we started intravenous linezolid 600 mg twice daily. The patient’s clinical condition continued to deteriorate so after 5 days we changed the antibiotics to daptomycin (6 mg/kg intravenously every 48 h). Further workup showed low serum complement C3 concentrations, raised PR3-ANCA, and raised rheumatoid factor concentrations, but no antiMPO-ANCA. His acute kidney injury continued to worsen so we started intermittent haemodialysis. Histological examination of a renal biopsy sample (fi gure) showed 23 glomeruli, six of which were globally sclerotic and ten that were segmentally sclerotic, cellular, and fi lled Bowman’s space circumferentially. The non-sclerotic glomerular segments had foci of increased mesangial matrix and increased mesangial cells; there was no duplication of basement membranes. Immunohistology showed positive mesangial staining for IgM, C1q, and C3; IgA and IgG were negative. Some glomerular basement membranes stained for C3 without showing humps. 80% of the cortex and outer medulla had chronic tubulointerstitial damage with interstitial matrix expansion and focal interstitial infi ltrate of mononuclear cells. Congo Red reaction was negative. Electron microscopy showed mesangial regions with increased matrix and segmental subendothelial and mesangial osmiophilic deposits (fi gure). These fi ndings indicated focal mesangial proliferative glomerulonephritis with segmental necrosis and extensive scarring (focal segmental and global glomerulosclerosis) associated with severe chronic tubulointerstitial damage in 80% of the tubulointerstitium. 18F-fl uorodeoxyglucose (18F-FDG) PET-CT showed multiple foci of increased metabolic activity at the site of the LVAD driveline insertion, the ventricular assist device pocket, and the LVAD outfl ow cannula (appendix). We made a diagnosis of focal segmental necrotising immune-complex glomerulonephritis triggered by chronic LVAD infection with C jeikeium. Blood cultures remained positive for C jeikeium despite ongoing antibiotic treatment. The patient continued to deteriorate with recurrent septicaemia and circulatory instability. At last follow-up in our centre in February, 2015, the patient asked for referral to palliative care. We report a case of immune complex glomerulonephritis with acute kidney injury RIFLE-stage L caused by a chronically infected LVAD. The pathophysiology of LVAD nephritis is similar to that of cerebral ventriculoatrial shunt nephritis, fi rst described by Black and colleagues in 1965. Shunt nephritis results from circulating bacterial antigen-antibacterial complexes, which activate the classic pathway of the complement system. Similarly our patient had low-grade fever, proteinuria, haematuria, and progressive renal failure, associated with a long-standing infection with a Gram-positive organism, low C3 concentrations, and serum positivity for PR3-ANCA. The renal histology and the long interval between surgery and onset of symptoms are also characteristic of shunt nephritis or LVAD nephritis. Dependent on the nature and extent of the glomerular injury, renal failure can recover after removal of the cerebral shunt. LVAD-specifi c infections remain among the most common causes of morbidity and mortality in patients with these devices. LVAD implantation is associated with bacteraemia as well as autoimmune eff ects such as development of cytotoxic autoantibodies. The presence of an LVAD itself causes defects in the cellular and humoral immune system leading to T-cell defects and B-cell hyperreactivity. Infection can occur at the driveline exit site, in the device pocket, or on the internal surface of the device or valves. Severe cases of LVAD-associated infection with recurrent bacteraemia usually necessitate surgical removal of the device and broad-spectrum antibiotics. The most common pathogens are Gram-positive organisms, mainly Staphylococcus and Enterococcus species, Candida Glomerulonephritis triggered by a chronically infected left ventricular assist device

The hormetic functions of Wnt pathways in tubular injury

Chronic tubulointerstitial damage with tubular epithelial atrophy and interstitial fibrosis is the hallmark of chronic kidney disease (CKD) and a predictor for progression of CKD.Several experiments have now provided evidence that the Wnt signaling pathways are significantly contributing to atrophy and fibrosis; in contrast, it also has been shown that the Wnt system fosters regenerative processes in acute tubular injury.We now have demonstrated that Dickkopf 3 (DKK3) is an agonist for canonical Wnt signaling in CKD and fosters chronic fibrosing inflammation of the tubulointerstitial compartment. Genetic- and antibody-mediated inhibition of DKK3 leads to a pronounced improvement of tubular differentiation and a reduction in fibrosis.In addition, the secreted glycoprotein DKK3 can be used as a non-invasive urinary marker for the extent of CKD in man.