Henning Hagmann

Pilot Study of Extracorporeal Removal of Soluble Fms-Like Tyrosine Kinase 1 in Preeclampsia

Background— Targeted therapies to stabilize the clinical manifestations and prolong pregnancy in preeclampsia do not exist. Soluble fms-like tyrosine kinase 1 (sFlt-1), an alternatively spliced variant of the vascular endothelial growth factor receptor 1, induces a preeclampsia-like phenotype in experimental models and circulates at elevated levels in human preeclampsia. Removing sFlt-1 may benefit women with very preterm (<32 weeks) preeclampsia. Methods and Results— We first show that negatively charged dextran sulfate cellulose columns adsorb sFlt-1 in vitro. In 5 women with very preterm preeclampsia and elevated circulating sFlt-1 levels, we next demonstrate that a single dextran sulfate cellulose apheresis treatment reduces circulating sFlt-1 levels in a dose-dependent fashion. Finally, we performed multiple apheresis treatments in 3 additional women with very preterm (gestational age at admission 28, 30, and 27+4 weeks) preeclampsia and elevated circulating sFlt-1 levels. Dextran sulfate apheresis lowered circulating sFlt-1, reduced proteinuria, and stabilized blood pressure without apparent adverse events to mother and fetus. Pregnancy lasted for 15 and 19 days in women treated twice and 23 days in a woman treated 4 times. In each, there was evidence of fetal growth. Conclusions— This pilot study supports the hypothesis that extracorporeal apheresis can lower circulating sFlt-1 in very preterm preeclampsia. Further studies are warranted to determine whether this intervention safely and effectively prolongs pregnancy and improves maternal and fetal outcomes in this setting.

Podocin and MEC-2 bind cholesterol to regulate the activity of associated ion channels

The prohibitin (PHB)-domain proteins are membrane proteins that regulate a variety of biological activities, including mechanosensation, osmotic homeostasis, and cell signaling, although the mechanism of this regulation is unknown. We have studied two members of this large protein family, MEC-2, which is needed for touch sensitivity in Caenorhabditis elegans, and Podocin, a protein involved in the function of the filtration barrier in the mammalian kidney, and find that both proteins bind cholesterol. This binding requires the PHB domain (including palmitoylation sites within it) and part of the N-terminally adjacent hydrophobic domain that attaches the proteins to the inner leaflet of the plasma membrane. By binding to MEC-2 and Podocin, cholesterol associates with ion-channel complexes to which these proteins bind: DEG/ENaC channels for MEC-2 and TRPC channels for Podocin. Both the MEC-2-dependent activation of mechanosensation and the Podocin-dependent activation of TRPC channels require cholesterol. Thus, MEC-2, Podocin, and probably many other PHB-domain proteins by binding to themselves, cholesterol, and target proteins regulate the formation and function of large protein–cholesterol supercomplexes in the plasma membrane.

Removal of Soluble Fms-Like Tyrosine Kinase-1 by Dextran Sulfate Apheresis in Preeclampsia

Preeclampsia is a devastating complication of pregnancy. Soluble Fms-like tyrosine kinase-1 (sFlt-1) is an antiangiogenic protein believed to mediate the signs and symptoms of preeclampsia. We conducted an open pilot study to evaluate the safety and potential efficacy of therapeutic apheresis with a plasma-specific dextran sulfate column to remove circulating sFlt-1 in 11 pregnant women (20-38 years of age) with very preterm preeclampsia (23-32 weeks of gestation, systolic BP ≥140 mmHg or diastolic BP ≥90 mmHg, new onset protein/creatinine ratio >0.30 g/g, and sFlt-1/placental growth factor ratio >85). We evaluated the extent of sFlt-1 removal, proteinuria reduction, pregnancy continuation, and neonatal and fetal safety of apheresis after one (n=6), two (n=4), or three (n=1) apheresis treatments. Mean sFlt-1 levels were reduced by 18% (range 7%-28%) with concomitant reductions of 44% in protein/creatinine ratios. Pregnancy continued for 8 days (range 2-11) and 15 days (range 11-21) in women treated once and multiple times, respectively, compared with 3 days (range 0-14) in untreated contemporaneous preeclampsia controls (n=22). Transient maternal BP reduction during apheresis was managed by withholding pre-apheresis antihypertensive therapy, saline prehydration, and reducing blood flow through the apheresis column. Compared with infants born prematurely to untreated women with and without preeclampsia (n=22 per group), no adverse effects of apheresis were observed. In conclusion, therapeutic apheresis reduced circulating sFlt-1 and proteinuria in women with very preterm preeclampsia and appeared to prolong pregnancy without major adverse maternal or fetal consequences. A controlled trial is warranted to confirm these findings.

The Promise of Angiogenic Markers for the Early Diagnosis and Prediction of Preeclampsia

BACKGROUND An imbalance in circulating factors that regulate blood vessel formation and health, referred to as angiogenic factors, plays a central role in the pathogenesis of preeclampsia. CONTENT Several studies have demonstrated a strong association between altered circulating angiogenic factors and preeclampsia. These factors include circulating antiangiogenic proteins such as soluble fms-like tyrosine kinase 1 and soluble endoglin and proangiogenic protein such as placental growth factor. Abnormalities in these circulating angiogenic factors are not only present during clinical disease, but also antedate clinical signs and symptoms by several weeks. These alterations are particularly prominent in patients who present with preeclamptic signs and symptoms prematurely and/or in patients with severe preeclampsia. The availability of automated platforms for the rapid measurement of circulating angiogenic proteins in blood samples has now allowed researchers and clinicians to evaluate the utility of these assays in the diagnosis of the disease, in the stratification of patients in clinical trials, or in the monitoring of therapies. In this review we highlight the various studies that have been performed, with a focus on large validation studies. SUMMARY Measurement of circulating angiogenic proteins for the diagnosis and prediction of preeclampsia is still at an early stage but is rapidly evolving. Standardization across the various automated platforms and prospective studies that demonstrate clinical utility are needed.

Intrinsic proinflammatory signaling in podocytes contributes to podocyte damage and prolonged proteinuria

Inflammation conveys the development of glomerular injury and is a major cause of progressive kidney disease. NF-κB signaling is among the most important regulators of proinflammatory signaling. Its role in podocytes, the epithelial cells at the kidney filtration barrier, is poorly understood. Here, we inhibited NF-κB signaling in podocytes by specific ablation of the NF-κB essential modulator (NEMO, IKKγ). Podocyte-specific NEMO-deficient mice (NEMO(pko)) were viable and did not show proteinuria or overt changes in kidney morphology. After induction of glomerulonephritis, both NEMO(pko) and control mice developed significant proteinuria. However, NEMO(pko) mice recovered much faster, showing rapid remission of proteinuria and restoration of podocyte morphology. Interestingly, quantification of infiltrating macrophages, T-lymphocytes, and granulocytes at day 7 revealed no significant difference between wild-type and NEMO(pko). To further investigate the underlying mechanisms, we created a stable NEMO knockdown mouse podocyte cell line. Again, no overt changes in morphology were observed. Translocation of NF-κB to the nucleus after stimulation with TNFα or IL-1 was sufficiently inhibited. Moreover, secretion of proinflammatory chemokines from podocytes after stimulation with TNFα or IL-1 was significantly reduced in NEMO-deficient podocytes and in glomerular samples obtained at day 7 after induction of nephrotoxic nephritis. Collectively, these results show that proinflammatory activity of NF-κB in podocytes aggravates proteinuria in experimental glomerulonephritis in mice. Based on these data, it may be speculated that immunosuppressive drugs may not only target professional immune cells but also podocytes directly to convey their beneficial effects in various types of glomerulonephritis.

Plasma leakage through glomerular basement membrane ruptures triggers the proliferation of parietal epithelial cells and crescent formation in non-inflammatory glomerular injury.

Glomerular crescents are most common in rapidly progressive glomerulonephritis but also occur in non‐inflammatory chronic glomerulopathies; thus, factors other than inflammation should trigger crescent formation, eg vascular damage and plasma leakage. Here we report that Alport nephropathy in Col4A3‐deficient Sv129 mice is complicated by diffuse and global crescent formation in which proliferating parietal epithelial cells are the predominant cell type. Laminin staining and transmission and acellular scanning electron microscopy of acellular glomeruli documented disruptions and progressive disintegration of the glomerular basement membrane in Col4A3‐deficient mice. FITC‐dextran perfusion further revealed vascular leakage from glomerular capillaries into Bowmans space, further documented by fibrin deposits in the segmental crescents. Its pathogenic role was validated by showing that the fibrinolytic activity of recombinant urokinase partially prevented crescent formation. In addition, in vitro studies confirmed an additional mitogenic potential of serum on murine and human parietal epithelial cells. Furthermore, loss of parietal cell polarity and unpolarized secretion of extracellular matrix components were evident within fibrocellular crescents. Among 665 human Alport nephropathy biopsies, crescent formation was noted in 0.4%. We conclude that glomerular vascular injury and GBM breaks cause plasma leakage which triggers a wound healing programme involving the proliferation of parietal cells and their loss of polarity. This process can trigger cellular and fibrocellular crescent formation even in the absence of cellular inflammation and rupture of the Bowmans capsule. Copyright

NOX2 interacts with podocyte TRPC6 channels and contributes to their activation by diacylglycerol: essential role of podocin in formation of this complex

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathophysiology of glomerular diseases. TRPC6 channels are typically activated by diacylglycerol (DAG) during PLC-dependent transduction cascades. TRPC6 channels can also be activated by reactive oxygen species (ROS). We previously showed that podocin is required for DAG analogs to produce robust activation of TRPC6 channels in podocytes. Here we show that endogenous TRPC6 channels in immortalized podocytes reciprocally coimmunoprecipitate with the catalytic subunit of the NADPH oxidase NOX2 (gp91(phox)). The NOX2-TRPC6 interaction was not detected in cells stably expressing a short hairpin RNA targeting podocin, although NOX2 and TRPC6 were present at normal levels. Application of a membrane-permeable DAG analog [1-oleoyl-2-acetyl-sn-glycerol (OAG)] increased generation of ROS in podocytes, but this effect was not detected in podocin knockdown cells. OAG also increased steady-state surface expression of the NOX2 regulatory subunit p47(phox). In whole cell recordings, TRPC6 activation by OAG was reduced in podocytes pretreated with the NOX2 inhibitor apocynin, by the pan-NOX inhibitor diphenylene iodonium, and by tempol, a ROS quencher. Cholesterol depletion and disruption of lipid rafts by methyl-β-cyclodextrin reduced activation of podocyte TRPC6 channels by OAG and also eliminated the NOX2-TRPC6 interaction as assessed by coimmunoprecipitation. These data suggest that active NOX2 assembles with TRPC6 at podocin-organized sterol-rich raft domains and becomes catalytically active in response to DAG. The localized production of ROS contributes to TRPC6 activation by chemical stimuli such as DAG. Podocin appears to be necessary for assembly of the NOX2-TRPC6 complex in lipid rafts.

Light microscopic visualization of podocyte ultrastructure demonstrates oscillating glomerular contractions.

Podocytes, the visceral epithelial cells of the kidney glomerulus, elaborate primary and interdigitating secondary extensions to enwrap the glomerular capillaries. A hallmark of podocyte injury is the loss of unique ultrastructure and simplification of the cell shape, called foot process effacement, which is a classic feature of proteinuric kidney disease. Although several key pathways have been identified that control cytoskeletal regulation, actin dynamics, and polarity signaling, studies into the dynamic regulation of the podocyte structure have been hampered by the fact that ultrastructural analyses require electron microscopic imaging of fixed tissue. We developed a new technique that allows for visualization of podocyte foot processes using confocal laser scanning microscopy. The combination of inducible and mosaic expression of membrane-tagged fluorescent proteins in a small subset of podocytes enabled us to acquire light microscopic images of podocyte foot processes in unprecedented detail, even in living podocytes of freshly isolated glomeruli. Moreover, this technique visualized oscillatory glomerular contractions and confirmed the morphometric evaluations obtained in static electron microscopic images of podocyte processes. These data suggest that the new technique will provide an extremely powerful tool for studying the dynamics of podocyte ultrastructure.

AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis.

Following genotoxic stress, cells activate a complex signalling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumour suppressor p53 lies at the heart of this DNA damage response. However, it remains incompletely understood, which signalling molecules dictate the choice between these different cellular outcomes. Here, we identify the transcriptional regulator apoptosis‐antagonizing transcription factor (AATF)/Che‐1 as a critical regulator of the cellular outcome of the p53 response. Upon genotoxic stress, AATF is phosphorylated by the checkpoint kinase MK2. Phosphorylation results in the release of AATF from cytoplasmic MRLC3 and subsequent nuclear translocation where AATF binds to the PUMA, BAX and BAK promoter regions to repress p53‐driven expression of these pro‐apoptotic genes. In xenograft experiments, mice exhibit a dramatically enhanced response of AATF‐depleted tumours following genotoxic chemotherapy with adriamycin. The exogenous expression of a phospho‐mimicking AATF point mutant results in marked adriamycin resistance in vivo. Nuclear AATF enrichment appears to be selected for in p53‐proficient endometrial cancers. Furthermore, focal copy number gains at the AATF locus in neuroblastoma, which is known to be almost exclusively p53‐proficient, correlate with an adverse prognosis and reduced overall survival. These data identify the p38/MK2/AATF signalling module as a critical repressor of p53‐driven apoptosis and commend this pathway as a target for DNA damage‐sensitizing therapeutic regimens.

Breaking the chain at the membrane: paraoxonase 2 counteracts lipid peroxidation at the plasma membrane

Lipid peroxidation through electrophilic molecules of extracellular origin is involved in the pathogenesis of many inflammatory conditions. To counteract free radical actions at the plasma membrane, cells host a variety of antioxidative enzymes. Here we analyzed localization, membrane topology, and trafficking of PON2 a member of the paraoxonase family of 3 enzymatically active proteins (PON1–3) found to have antiatherogenic properties. Immunohistochemistry localized PON2 to the villous tip of human intestinal epithelial cells. Employing membrane preparations, surface biotinylation experiments, and mutational analyses in HEK 293T and HeLa cells, we demonstrate that PON2 is a type II transmembrane protein. A hydrophobic stretch in the N terminus was identified as single transmembrane domain of PON2. The enzymatically active domain faced the extracellular compartment, where it suppressed lipid peroxidation (P<0.05) and regulated the glucosylceramide content, as demonstrated by mass spectrometry (P<0.05). PON2 translocation to the plasma membrane was dependent on intracellular calcium responses and could be induced to >10‐fold as compared to baseline (P=0.0001) by oxidative stress. Taken together, these data identify the paraoxonase protein PON2 as a type II transmembrane protein, which is dynamically translocated to the plasma membrane in response to oxidative stress to counteract lipid peroxidation.—Hagmann, H., Kuczkowski, A., Ruehl, M., Lamkemeyer, T., Brodesser, S., Horke, S., Dryer, S., Schermer, B., Benzing, T., Brinkkoetter, P. T. Breaking the chain at the membrane: paraoxonase 2 counteracts lipid peroxidation at the plasma membrane. FASEB J. 28, 28–1769 (1779). www.fasebj.org