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The simultaneous interrogation of both lossy mode (LMR) and surface plasmon (SPR) resonances was herein exploited for the first time to devise a sensor in combination with soft molecularly imprinting of nanoparticles (nanoMIPs), specifically entailed of the selectivity towards the protein biomarker human serum transferrin (HTR). Two distinct metal-oxide bilayers, i.e. TiO 2 -ZrO 2 and ZrO 2 -TiO 2 , were used in the SPR-LMR sensing platforms. The responses to binding of the target protein HTR of both sensing configurations (TiO 2 -ZrO 2 -Au-nanoMIPs, ZrO 2 -TiO 2 -Au-nanoMIPs) showed femtomolar HTR detection, LODs of tens of fM and K Dapp  ~ 30 fM. Selectivity for HTR was demonstrated. The SPR interrogation was more efficient for the ZrO 2 -TiO 2 -Au-nanoMIPs configuration (sensitivity at low concentrations, S = 0.108 nm/fM) than for the TiO 2 -ZrO 2 -Au-nanoMIPs one (S = 0.061 nm/fM); while LMR was more efficient for TiO 2 -ZrO 2 -Au-nanoMIPs (S = 0.396 nm/fM) than for ZrO 2 -TiO 2 -Au-nanoMIPs (S = 0.177 nm/fM). The simultaneous resonance monitoring is advantageous for point of care determinations, both in terms of measurement's redundancy, that enables the cross-control of the measure and the optimization of the detection, by exploiting the individual characteristics of each resonance., (© 2023. The Author(s).)

Polymer-based surface plasmon resonance (SPR) sensors can be used to realize simple, small-size, disposable, and low-cost biosensors for application in several fields, e.g., healthcare. The performance of SPR sensors based on optical waveguides can be changed by tuning several parameters, such as the dimensions and the shape of the waveguides, the refractive index of the core, and the metal nanofilms used to excite the SPR phenomenon. In this work, in order to develop, experimentally test, and compare several polymer-based plasmonic sensors, realized by using waveguides with different core refractive indices, optical adhesives and 3D printed blocks with a trench inside have been used. In particular, the sensors are realized by filling the blocks' trenches (with two plastic optical fibers located at the end of these) with different UV-cured optical adhesives and then covering them with the same bilayer to excite the SPR phenomenon. The developed SPR sensors have been characterized by numerical and experimental results. Finally, in order to propose photonic solutions for healthcare, a comparative analysis has been reported to choose the best sensor configuration useful for developing low-cost biosensors.

In recent decades, the Surface Plasmon Resonance (SPR) phenomenon has been utilized as an underlying technique in a broad range of application fields. Herein, a new measuring strategy which harnesses the SPR technique in a way that is different from the classical methodology was explored by taking advantage of the characteristics of multimode waveguides, such as plastic optical fibers (POFs) or hetero-core fibers. The sensor systems based on this innovative sensing approach were designed, fabricated, and investigated to assess their ability to measure various physical features, such as magnetic field, temperature, force, and volume, and to realize chemical sensors. In more detail, a sensitive patch of fiber was used in series with a multimodal waveguide where the SPR took place, to alter the mode profile of the light at the input of the waveguide itself. In fact, when the changes of the physical feature of interest acted on the sensitive patch, a variation of the incident angles of the light launched in the multimodal waveguide occurred, and, as a consequence, a shift in resonance wavelength took place. The proposed approach permitted the separation of the measurand interaction zone and the SPR zone. This meant that the SPR zone could be realized only with a buffer layer and a metallic film, thus optimizing the total thickness of the layers for the best sensitivity, regardless of the measurand type. The proposed review aims to summarize the capabilities of this innovative sensing approach to realize several types of sensors for different application fields, showing the high performances obtained by exploiting a simple production process and an easy experimental setup.

Background: Dent's disease type 1 (DD1) is a rare X-linked nephropathy caused by CLCN5 mutations, characterized by proximal tubule dysfunction, including low molecular weight proteinuria (LMWP), hypercalciuria, nephrolithiasis-nephrocalcinosis, progressive chronic kidney disease (CKD) and kidney failure (KF). Current management is symptomatic and does not prevent disease progression. Here we describe the contemporary DD1 picture across Europe to highlight its unmet needs., Methods: A physician-based anonymous international e-survey supported by several European nephrology networks/societies was conducted. Questions focused on DD1 clinical features, diagnostic procedure and mutation spectra., Results: A total of 207 DD1 male patients were reported; clinical data were available for 163 with confirmed CLCN5 mutations. Proteinuria was the most common manifestation (49.1%). During follow-up, all patients showed LMWP, 66.4% nephrocalcinosis, 44.4% hypercalciuria and 26.4% nephrolithiasis. After 5.5 years, ≈50% of patients presented with renal dysfunction, 20.7% developed CKD stage ≥3 and 11.1% developed KF. At the last visit, hypercalciuria was more frequent in paediatric patients than in adults (73.4% versus 19.0%). Conversely, nephrolithiasis, nephrocalcinosis and renal dysfunction were more prominent in adults. Furthermore, CKD progressed with age. Despite no clear phenotype/genotype correlation, decreased glomerular filtration rate was more frequent in subjects with CLCN5 mutations affecting the pore or CBS domains compared with those with early-stop mutations., Conclusions: Results from this large DD1 cohort confirm previous findings and provide new insights regarding age and genotype impact on CKD progression. Our data strongly support that DD1 should be considered in male patients with CKD, nephrocalcinosis/hypercalciuria and non-nephrotic proteinuria and provide additional support for new research opportunities., (© The Author(s) 2022. Published by Oxford University Press on behalf of the ERA.)

Dent disease (DD1) is a rare tubulopathy caused by mutations in the CLCN5 gene. Glomerulosclerosis was recently reported in DD1 patients and ClC-5 protein was shown to be expressed in human podocytes. Nephrin and actin cytoskeleton play a key role for podocyte functions and podocyte endocytosis seems to be crucial for slit diaphragm regulation. The aim of this study was to analyze whether ClC-5 loss in podocytes might be a direct consequence of the glomerular damage in DD1 patients. Three DD1 kidney biopsies presenting focal global glomerulosclerosis and four control biopsies were analyzed by immunofluorescence (IF) for nephrin and podocalyxin, and by immunohistochemistry (IHC) for ClC-5. ClC-5 resulted as down-regulated in DD1 vs. control (CTRL) biopsies in both tubular and glomerular compartments (p < 0.01). A significant down-regulation of nephrin (p < 0.01) in DD1 vs. CTRL was demonstrated. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Caspase9) gene editing of CLCN5 in conditionally immortalized human podocytes was used to obtain clones with the stop codon mutation p.(R34Efs*14). We showed that ClC-5 and nephrin expression, analyzed by quantitative Reverse Transcription/Polymerase Chain Reaction (qRT/PCR) and In-Cell Western (ICW), was significantly downregulated in mutant clones compared to the wild type ones. In addition, F-actin staining with fluorescent phalloidin revealed actin derangements. Our results indicate that ClC-5 loss might alter podocyte function either through cytoskeleton disorganization or through impairment of nephrin recycling.

The erector spinae plane block (ESPB) is a novel technique used in both adult and pediatric patients. Its use in children has mostly been described in terms of perioperative pain management for various types of surgery. After its introduction, anesthesiologists began using ESPBs in various surgical settings. As adequate analgesia along with a low complication rate were reported, interest in this technique dramatically increased. Many studies in adults and children, including randomized controlled trials, have been published, resulting in the emergence of different clinical indications, with various technical and pharmacological approaches currently evident in the literature. This narrative review aims to analyze the current evidence in order to guide practitioners towards a more homogeneous approach to ESPBs in children, with a major focus on clinical applications. The ESPB is an efficient, safe, and relatively easy technique to administer. It can be applied in a wide range of surgeries, includes thoracic, abdominal, hip, and femur surgery. Its usefulness is evident in the context of enhanced recovery after surgery protocols and multimodal analgesia. Single-shot, intermittent bolus, and continuous infusion techniques have been described, and non-inferiority has been observed when compared with other locoregional techniques. Even though both the efficacy and safety of the procedure are widely accepted, current evidence is predominantly based on case reports, with very few well-designed observational studies. Consequently, the level of evidence is still poor, and more well-designed double-blind, randomized, placebo-controlled trials are needed to refine the procedure for different clinical applications in the pediatric population.

The cumulative load of genetic predisposition, early life adversity (ELA) and lifestyle shapes the prevalence of psychiatric disorders. Single nucleotide polymorphisms (SNPs) in the human FKBP5 gene were shown to modulate disease risk. To enable investigation of disease-related SNPs in behaviourally relevant context, we generated humanised mouse lines carrying either the risk (AT) or the resiliency (CG) allele of the rs1360780 locus and exposed litters of these mice to maternal separation. Behavioural and physiological aspects of their adult stress responsiveness displayed interactions of genotype, early life condition, and sex. In humanised females carrying the CG- but not the AT-allele, ELA led to altered HPA axis functioning, exploratory behaviour, and sociability. These changes correlated with differential expression of genes in the hypothalamus, where synaptic transmission, metabolism, and circadian entrainment pathways were deregulated. Our data suggest an integrative role of FKBP5 in shaping the sex-specific outcome of ELA in adulthood., (© 2022. The Author(s).)

Background: Endoscopic treatment for achalasia (POEM) is a recently introduced technique that incorporates the concepts of natural orifice transluminal surgery. Although pediatric achalasia is rare, POEM has been episodically used in children since 2012. Despite this procedure entails many implications for airway management and mechanical ventilation, evidences about anesthesiologic management are very poor. We conducted this retrospective study to pay attention on the clinical challenge for pediatric anesthesiologists. We put special emphasis on the risk in intubation maneuvers and in ventilation settings., Results: We retrieved data on children 18 years old and younger who underwent POEM in a single tertiary referral endoscopic center between 2012 and 2021. Demographics, clinical history, fasting status, anesthesia induction, airway management, anesthesia maintenance, timing of anesthesia and procedure, PONV, and pain treatment and adverse events were retrieved from the original database. Thirty-one patients (3-18 years) undergoing POEM for achalasia were analyzed. In 30 of the 31 patients, rapid sequence induction was performed. All patients manifested consequences of endoscopic CO 2 insufflation and most of them required a new ventilator approach. No life-threatening adverse events have been detected., Conclusions: POEM procedure seems to be characterized by a low-risk profile, but specials precaution must be taken. The inhalation risk is actually due to the high rate of full esophagus patients, even if the Rapid Sequence Induction was effective in preventing ab ingestis pneumonia. Mechanical ventilation may be difficult during the tunnelization step. Future prospective trials will be necessary to individuate the better choices in such a special setting., (© 2022. The Author(s).)

Plasmonic bio/chemical sensing based on optical fibers combined with molecularly imprinted nanoparticles (nanoMIPs), which are polymeric receptors prepared by a template-assisted synthesis, has been demonstrated as a powerful method to attain ultra-low detection limits, particularly when exploiting soft nanoMIPs, which are known to deform upon analyte binding. This work presents the development of a surface plasmon resonance (SPR) sensor in silica light-diffusing fibers (LDFs) functionalized with a specific nanoMIP receptor, entailed for the recognition of the protein human serum transferrin (HTR). Despite their great versatility, to date only SPR-LFDs functionalized with antibodies have been reported. Here, the innovative combination of an SPR-LFD platform and nanoMIPs led to the development of a sensor with an ultra-low limit of detection (LOD), equal to about 4 fM, and selective for its target analyte HTR. It is worth noting that the SPR-LDF-nanoMIP sensor was mounted within a specially designed 3D-printed holder yielding a measurement cell suitable for a rapid and reliable setup, and easy for the scaling up of the measurements. Moreover, the fabrication process to realize the SPR platform is minimal, requiring only a metal deposition step.

Dent disease is a rare X-linked renal tubulopathy due to CLCN5 and OCRL (DD2) mutations. OCRL mutations also cause Lowe syndrome (LS) involving the eyes, brain and kidney. DD2 is frequently described as a mild form of LS because some patients may present with extra-renal symptoms (ESs). Since DD2 is a rare disease and there are a low number of reported cases, it is still unclear whether it has a clinical picture distinct from LS. We retrospectively analyzed the phenotype and genotype of our cohort of 35 DD2 males and reviewed all published DD2 cases. We analyzed the distribution of mutations along the OCRL gene and evaluated the type and frequency of ES according to the type of mutation and localization in OCRL protein domains. The frequency of patients with at least one ES was 39%. Muscle findings are the most common ES (52%), while ocular findings are less common (11%). Analysis of the distribution of mutations revealed (1) truncating mutations map in the PH and linker domain, while missense mutations map in the 5-phosphatase domain, and only occasionally in the ASH-RhoGAP module; (2) five OCRL mutations cause both DD2 and LS phenotypes; (3) codon 318 is a DD2 mutational hot spot; (4) a correlation was found between the presence of ES and the position of the mutations along OCRL domains. DD2 is distinct from LS. The mutation site and the mutation type largely determine the DD2 phenotype.

In this work, we experimentally analyzed the effect of tapering in light-diffusing optical fibers (LDFs) when employed as surface plasmon resonance (SPR)-based sensors. Although tapering is commonly adopted to enhance the performance of plasmonic optical fiber sensors, we have demonstrated that in the case of plasmonic sensors based on LDFs, the tapering produces a significant worsening of the bulk sensitivity (roughly 60% in the worst case), against a slight decrease in the full width at half maximum (FWHM) of the SPR spectra. Furthermore, we have demonstrated that these aspects become more pronounced when the taper ratio increases. Secondly, we have established that a possible alternative exists in using the tapered LDF as a modal filter after the sensible region. In such a case, we have determined that a good trade-off between the loss in sensitivity and the FWHM decrease could be reached.

Fabry disease is an X-linked disorder due to mutations in α-galactosidase A, resulting in the accumulation of enzyme substrates and cell malfunction. Kidney involvement is frequent, affecting all native kidney cell types. Podocyte damage results in proteinuria and chronic kidney disease. End-stage kidney disease is the rule in middle-aged males and some females with the classic phenotype. In podocytes and kidney proximal tubular cells, megalin is one of the molecules involved in enzyme replacement therapy (ERT) cellular absorption. After podocyte damage, podocin concentration is decreased and contributes to progressive proteinuria. We report in a male and a female patient the decreased expression of megalin, cubilin, ClC-5 and podocin compared to controls and chronic kidney disease (CKD) biopsies. Moreover, the decrease in ClC-5, a molecule engaged in endosomal-lysosomal acidification, could also affect ERT. These findings may partially explain some of the dysfunctions described in Fabry nephropathy and could highlight possible alterations in the pharmacokinetics of the delivered enzyme., (© 2020. Italian Society of Nephrology.)

Infection with the Zika virus (ZIKV), a member of the Flaviviridae family, can cause serious neurological disorders, most notably microcephaly in newborns. Here we investigated the innate immune response to ZIKV infection in cells of the nervous system. In human neural progenitor cells (hNPCs), a target for ZIKV infection and likely involved in ZIKV-associated neuropathology, viral infection failed to elicit an antiviral interferon (IFN) response. However, pharmacological inhibition of TLR3 partially restored this deficit. Analogous results were obtained in human iPSC-derived astrocytes, which are capable of mounting a strong antiviral cytokine response. There, ZIKV is sensed by both RIG-I and MDA5 and induces an IFN response as well as expression of pro-inflammatory cytokines such as interleukin-6 (IL-6). Upon inhibition of TLR3, also in astrocytes the antiviral cytokine response was enhanced, whereas amounts of pro-inflammatory cytokines were reduced. To study the underlying mechanism, we used human epithelial cells as an easy to manipulate model system. We found that ZIKV is sensed in these cells by RIG-I to induce a robust IFN response and by TLR3 to trigger the expression of pro-inflammatory cytokines, including IL-6. ZIKV induced upregulation of IL-6 activated the STAT3 pathway, which decreased STAT1 phosphorylation in a SOCS-3 dependent manner, thus reducing the IFN response. In conclusion, we show that TLR3 activation by ZIKV suppresses IFN responses triggered by RIG-I-like receptors. Importance Zika virus (ZIKV) has a pronounced neurotropism and infections with this virus can cause serious neurological disorders, most notably microcephaly and the Guillain-Barré syndrome. Our studies reveal that during ZIKV infection, recognition of viral RNA by TLR3 enhances the production of inflammatory cytokines and suppresses the interferon response triggered by RIG-I-like receptors (RLR) in a SOCS3-dependent manner, thus facilitating virus replication. The discovery of this crosstalk between antiviral (RLR) and inflammatory (TLR) responses may have important implications for our understanding of ZIKV-induced pathogenesis., (Copyright © 2021 Frankish and Moraes.)

Dent disease is a rare genetic proximal tubulopathy which is under-recognized. Its phenotypic heterogeneity has led to several different classifications of the same disorder, but it is now widely accepted that the triad of symptoms low-molecular-weight proteinuria, hypercalciuria and nephrocalcinosis/nephrolithiasis are pathognomonic of Dent disease. Although mutations on the CLCN5 and OCRL genes are known to cause Dent disease, no such mutations are found in about 25-35% of cases, making diagnosis more challenging. This review outlines current knowledge regarding Dent disease from another perspective. Starting from the history of Dent disease, and reviewing the clinical details of patients with and without a genetic characterization, we discuss the phenotypic and genetic heterogeneity that typifies this disease. We focus particularly on all those confounding clinical signs and symptoms that can lead to a misdiagnosis. We also try to shed light on a concealed aspect of Dent disease. Although it is a proximal tubulopathy, its misdiagnosis may lead to patients undergoing kidney biopsy. In fact, some individuals with Dent disease have high-grade proteinuria, with or without hematuria, as in the clinical setting of glomerulopathy, or chronic kidney disease of uncertain origin. Although glomerular damage is frequently documented in Dent disease patients' biopsies, there is currently no reliable evidence of renal biopsy being of either diagnostic or prognostic value. We review published histopathology reports of tubular and glomerular damage in these patients, and discuss current knowledge regarding the role of CLCN5 and OCRL genes in glomerular function.

As part of a study on triterpenoid conjugates, the dietary pentacyclic triterpenoids oleanolic ( 2a ) and ursolic acids ( 3a ) were coupled with vanillamine, and the resulting amides ( 2b and 3b , respectively) were assayed for activity on the vanilloid receptor TRPV1. Despite a structural difference limited to the location of a methyl group in their conformationally rigid pentacyclic core, oleanoloyl vanillamide dramatically outperformed ursoloyl vanillamide in terms of potency (EC 50 = 35 ± 2 nM for 2b and 5.4 ± 2.3 μM for 3b ). Using molecular docking and dynamics, this difference was translated into distinct accommodation modes at the TRPV1 vanillyl ligand pocket, suggesting a critical role of a C-H π phenyl interaction between the triterpenoid C-29 methyl and Phe591 of TRPV1. Because the molecular mechanisms underlying the activation process of transient receptor channels (TRPs) remain to be fully elucidated, the observation of spatially restricted structure-activity information is of significant relevance to identify the molecular detail of TRPV1 ligand gating.

The liverwort Lunularia cruciata was collected from the town of Acerra, in the heart of the so-called 'Land of Fires' a large area in the eastern part of Campania region of Italy affected by burning of waste and fraudulent dumping and one of the vertices of the "Italian Triangle of Death" so said for the high incidence and mortality from tumors. The data obtained from these samples were compared with samples collected in two other sites representing two different environmental conditions. The soil below the samples, and gametophytes, were collected and analyzed for the concentration of Al, As, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V. DNA damage, Reactive Oxygen Species production and localization, activity of antioxidant enzymes and presence of chelating molecules were investigated. All biomarkers provided an answer closely related to the pollution conditions at the 3 sites. We discuss the data considering the possibility of using these biological changes as environmental pollution biomarkers. Finally, it is underlined the importance of phytochelatins due to of their specificity for metal pollution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Proteinuria is a well-known risk factor, not only for renal disorders, but also for several other problems such as cardiovascular diseases and overall mortality. In the kidney, the chloride channel Cl - /H + exchanger ClC-5 encoded by the CLCN5 gene is actively involved in preventing protein loss. This action becomes evident in patients suffering from the rare proximal tubulopathy Dent disease because they carry a defective ClC-5 due to CLCN5 mutations. In fact, proteinuria is the distinctive clinical sign of Dent disease, and mainly involves the loss of low-molecular-weight proteins. The identification of CLCN5 disease-causing mutations has greatly improved our understanding of ClC-5 function and of the ClC-5-related physiological processes in the kidney. This review outlines current knowledge regarding the CLCN5 gene and its protein product, providing an update on ClC-5 function in tubular and glomerular cells, and focusing on its relationship with proteinuria and Dent disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

ClC-5, the electrogenic chloride/proton exchanger strongly expressed in renal proximal tubules, belongs to the endocytic macromolecular complex responsible for albumin and low-molecular-weight protein uptake. ClC-5 was found to be overexpressed in glomeruli of glomerulonephritis and in cultured human podocytes under albumin overload. The transcriptional regulation of human ClC-5 is not fully understood. Three functional promoters of various strengths and 11 different 5' untranslated region (5'UTR) isoforms of CLCN5 messenger RNA (mRNA) were detected in the human kidney (variants 1-11). The aim of this study was to investigate the expression pattern of CLCN 5 5'UTR variants and the CLCN5 common translated region in glomerulonephritis. The 5'UTR ends and the translated region of CLCN5 mRNA were analyzed using quantitative relative real-time PCR or quantitative comparative endpoint PCR with GAPDH as housekeeping gene in 8 normal kidneys and 12 renal biopsies from patients with glomerulonephritis. The expression profile for all variants in normal and glomerulonephritis biopsies was similar, and variant 3 and alternative variant 4 were the most abundantly expressed in both sets. In glomerulonephritis biopsies, isoforms under the control of a weak promoter (variants 4, 6 and 7) showed an increased expression leading to an increase in the CLCN5 translated region, underscoring their importance in kidney pathophysiology. Since weak promoters can be turned on by different stimuli, these data support the hypothesis that proteinuria could be one of the stimuli capable of starting a signaling pathway that induces an increase in CLCN5 transcription., Competing Interests: Competing interests: None declared., (© American Federation for Medical Research 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Dent disease (DD), an X-linked renal tubulopathy, is mainly caused by loss-of-function mutations in CLCN5 (DD1) and OCRL genes. CLCN5 encodes the ClC-5 antiporter that in proximal tubules (PT) participates in the receptor-mediated endocytosis of low molecular weight proteins. Few studies have analyzed the PT expression of ClC-5 and of megalin and cubilin receptors in DD1 kidney biopsies. About 25% of DD cases lack mutations in either CLCN5 or OCRL genes (DD3), and no other disease genes have been discovered so far. Sanger sequencing was used for CLCN5 gene analysis in 158 unrelated males clinically suspected of having DD. The tubular expression of ClC-5, megalin, and cubilin was assessed by immunolabeling in 10 DD1 kidney biopsies. Whole exome sequencing (WES) was performed in eight DD3 patients. Twenty-three novel CLCN5 mutations were identified. ClC-5, megalin, and cubilin were significantly lower in DD1 than in control biopsies. The tubular expression of ClC-5 when detected was irrespective of the type of mutation. In four DD3 patients, WES revealed 12 potentially pathogenic variants in three novel genes (SLC17A1, SLC9A3, and PDZK1), and in three genes known to be associated with monogenic forms of renal proximal tubulopathies (SLC3A, LRP2, and CUBN). The supposed third Dent disease-causing gene was not discovered., Competing Interests: The authors declare no conflict of interest.

Apoptotic cell death is usually a response to the cell's microenvironment. In the kidney, apoptosis contributes to parenchymal cell loss in the course of acute and chronic renal injury, but does not trigger an inflammatory response. What distinguishes necrosis from apoptosis is the rupture of the plasma membrane, so necrotic cell death is accompanied by the release of unprocessed intracellular content, including cellular organelles, which are highly immunogenic proteins. The relative contribution of apoptosis and necrosis to injury varies, depending on the severity of the insult. Regulated cell death may result from immunologically silent apoptosis or from immunogenic necrosis. Recent advances have enhanced the most revolutionary concept of regulated necrosis. Several modalities of regulated necrosis have been described, such as necroptosis, ferroptosis, pyroptosis, and mitochondrial permeability transition-dependent regulated necrosis. We review the different modalities of apoptosis, necrosis, and regulated necrosis in kidney injury, focusing particularly on evidence implicating cell death in ectopic renal calcification. We also review the evidence for the role of cell death in kidney injury, which may pave the way for new therapeutic opportunities.

Nephrocalcinosis is a clinicopathological entity characterized by microscopic calcium crystals in the renal parenchyma, within the tubular lumen or in the interstitium. Crystal binding to tubular cells may be the cause underlying nephrocalcinosis and nephrolithiasis. Pathological circumstances, such as acute cortical necrosis, may induce healthy cells to acquire a crystal-binding phenotype. The present study aimed to investigate whether human renal proximal tubular cells (HK-2 cells) can form calcium phosphate deposits under osteogenic conditions, and whether apoptosis and/or osteogenic-like processes are involved in cell calcification. HK-2 cells were cultured in standard or osteogenic medium for 1, 5, and 15 days. Von Kossa staining and ESEM were used to analyze crystal deposition. Apoptosis was investigated, analyzing caspase activation by in-cell Western assay, membrane translocation of phosphotidylserine by annexin V-FITC/propidium iodide staining, and DNA fragmentation by TUNEL assay. qRT/PCR, immunolabeling and cytochemistry were performed to assess osteogenic activation (Runx2, Osteonectin, Osteopontin and ALP), and early genes of apoptosis (BAX, Bcl-2). HK-2 cell mineralization was successfully induced on adding osteogenic medium. Calcium phosphate deposition increased in a time-dependent manner, and calcified cell aggregates exhibited characteristic signs of apoptosis. At 15 days, calcifying HK-2 cells revealed osteogenic markers, such as Runx2, ALP, osteonectin and osteopontin. Monitoring the processes at 1, 5, and 15 days showed apoptosis starting already after 5 days of osteogenic induction, when the first small calcium phosphate crystals began to appear on areas where cell aggregates were in apoptotic conditions. The cell death process proved caspase-dependent. The importance of apoptosis was reinforced by the time-dependent increase in BAX expression, starting from day 1. These findings strongly support the hypothesis that apoptosis triggered HK-2 calcification even before any calcium phosphate crystal deposition or acquisition of an osteogenic phenotype., Competing Interests: The authors declare that they have no conflict of interest.

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Objective: The study aimed to assess image quality when using dual-energy CT (DECT) to reduce metal artifacts in subjects with knee and hip prostheses., Methods: Twenty-two knee and 10 hip prostheses were examined in 31 patients using a DECT protocol (tube voltages 100 and 140 kVp). Monoenergetic reconstructions were extrapolated at 64, 69, 88, 105, 110, 120, 140, 170, and 190 kilo-electron volts (keV) and the optimal energy was manually selected. The B60-140 and Fast DE reconstructions were made by CT. The image quality and diagnostic value were subjectively and objectively determined. Double-blind qualitative assessment was performed by two radiologists using a Likert scale. For quantitative analysis, a circular region of interest (ROI) was placed by a third radiologist within the most evident streak artifacts on every image. Another ROI was placed in surrounding tissues without artifacts as a reference., Results: The inter-reader agreement for the qualitative assessment was nearly 100%. The best overall image quality (37.8% rated "excellent") was the Fast DE Siemens reconstruction, followed by B60-140 and Opt KeV (20.5 and 10.2% rated excellent). On the other hand, DECT images at 64, 69 and 88 keV had the worse scores. The number of artifacts was significantly different between monoenergetic images. Nevertheless, because of the high number of pairwise comparisons, no differences were found in the post hoc analysis except for a trend toward statistical significance when comparing the 170 and 64 keV doses., Conclusions: DECT with specific post-processing may reduce metal artifacts and significantly enhance the image quality and diagnostic value when evaluating metallic implants.

A homozygous nonsense mutation in the cereblon ( CRBN ) gene results in autosomal recessive, nonsyndromic intellectual disability that is devoid of other phenotypic features, suggesting a critical role of CRBN in mediating learning and memory. In this study, we demonstrate that adult male Crbn knock-out ( Crbn KO ) mice exhibit deficits in hippocampal-dependent learning and memory tasks that are recapitulated by focal knock-out of Crbn in the adult dorsal hippocampus, with no changes in social or repetitive behavior. Cellular studies identify deficits in long-term potentiation at Schaffer collateral CA1 synapses. We further show that Crbn is robustly expressed in the mouse hippocampus and Crbn KO mice exhibit hyperphosphorylated levels of AMPKα (Thr172). Examination of processes downstream of AMP-activated protein kinase (AMPK) finds that Crbn KO mice have a selective impairment in mediators of the mTORC1 translation initiation pathway in parallel with lower protein levels of postsynaptic density glutamatergic proteins and higher levels of excitatory presynaptic markers in the hippocampus with no change in markers of the unfolded protein response or autophagy pathways. Acute pharmacological inhibition of AMPK activity in adult Crbn KO mice rescues learning and memory deficits and normalizes hippocampal mTORC1 activity and postsynaptic glutamatergic proteins without altering excitatory presynaptic markers. Thus, this study identifies that loss of Crbn results in learning, memory, and synaptic defects as a consequence of exaggerated AMPK activity, inhibition of mTORC1 signaling, and decreased glutamatergic synaptic proteins. Thus, Crbn KO mice serve as an ideal model of intellectual disability to further explore molecular mechanisms of learning and memory. SIGNIFICANCE STATEMENT Intellectual disability (ID) is one of the most common neurodevelopmental disorders. The cereblon ( CRBN ) gene has been linked to autosomal recessive, nonsyndromic ID, characterized by an intelligence quotient between 50 and 70 but devoid of other phenotypic features, making cereblon an ideal protein for the study of the fundamental aspects of learning and memory. Here, using the cereblon knock-out mouse model, we show that cereblon deficiency disrupts learning, memory, and synaptic function via AMP-activated protein kinase hyperactivity, downregulation of mTORC1, and dysregulation of excitatory synapses, with no changes in social or repetitive behaviors, consistent with findings in the human population. This establishes the cereblon knock-out mouse as a model of pure ID without the confounding behavioral phenotypes associated with other current models of ID., (Copyright © 2018 the authors 0270-6474/18/382781-16$15.00/0.)

Tau plays a pivotal role in the pathogenesis of neurodegenerative disorders: mutations in the gene encoding for tau (MAPT) are linked to Fronto-temporal Dementia (FTD) and hyper-phosphorylated aggregates of tau forming neurofibrillary tangles (NFTs) that constitute a pathological hallmark of Alzheimer disease (AD) and FTD. Accordingly, tau is a favored therapeutic target for the treatment of these diseases. Given the criticality of tau to dementia's pathogenesis and therapy, it is important to understand the physiological function of tau in the central nervous system. Analysis of Mapt knock out (Mapt -/- ) mice has yielded inconsistent results. Some studies have shown that tau deletion does not alter memory while others have described synaptic plasticity and memory alterations in Mapt -/- mice. To help clarifying these contrasting results, we analyzed a distinct Mapt -/- model on a B6129PF3/J genetic background. We found that tau deletion leads to aging-dependent short-term memory deficits, hyperactivity and synaptic plasticity defects. In contrast, Mapt +/- mice only showed a mild short memory deficit in the novel object recognition task. Thus, while tau is important for normal neuronal functions underlying learning and memory, partial reduction of tau expression may have fractional deleterious effects.

Nephrocalcinosis involves the deposition of microscopic crystals in the tubular lumen or interstitium. While the clinical, biochemical, and genetic aspects of the diseases causing nephrocalcinosis have been elucidated, little is known about the cellular events in this calcification process. We previously reported a phenomenon involving the spontaneous formation of Ca 2 PO 4 nodules in primary papillary renal cells from a patient with medullary nephrocalcinosis harboring a rare glial cell-derived neurotrophic factor ( GDNF ) gene variant. We also demonstrated that cultivating GDNF -silenced human kidney-2 (HK-2) cells in osteogenic conditions for 15 days triggered Ca 2 PO 4 deposits. Given the reportedly close relationship between cell death and pathological calcification, aim of the present study was to investigate whether apoptosis is involved in the calcification of GDNF -silenced HK-2 cells under osteogenic conditions. Silenced and control cells were cultured in standard and osteogenic medium for 1, 5, and 15 days, and any Ca 2 PO 4 deposition was identified by means of von Kossa staining and environmental SEM (ESEM) analyses. Based on the results of annexin V and propidium iodide (PI) analysis, and terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) assay, the silenced cells in the osteogenic medium showed a significant increase in the percentage of cells in the late phase of apoptosis and an increased Ca 2 PO 4 deposition at 15 days. The results of quantitative real-time PCR (qRT-PCR) of BAX and BCL2 , and in-cell Western analysis of caspases indicated that the cell death process was independent of caspase-3, -6, -7, and -9 activation, however. Using this model, we provide evidence of caspase-independent cell death triggering the calcification process in GDNF -silenced HK-2 cells., (© 2018 The Author(s).)

Albumin re-uptake is a receptor-mediated pathway located in renal proximal tubuli. There is increasing evidence of glomerular protein handling by podocytes, but little is known about the mechanism behind this process. In this study, we found that human podocytes in vitro are committed to internalizing albumin through a receptor-mediated mechanism even after exposure to low doses of albumin. We show that these cells express cubilin, megalin, ClC-5, amnionless and Dab2, which are partners in the tubular machinery. Exposing human podocytes to albumin overload prompted an increase in CUBILIN, AMNIONLESS and CLCN5 gene expression. Inhibiting cubilin led to a reduction in albumin uptake, highlighting its importance in this mechanism. We demonstrated that human podocytes are committed to performing endocytosis via a receptor-mediated mechanism even in the presence of low doses of albumin. We also disclosed that protein overload first acts on the expression of the cubilin-amnionless (CUBAM) complex in these cells, then involves the ClC-5 channel, providing the first evidence for a possible role of the CUBAM complex in albumin endocytosis in human podocytes.

Bardoxolone methyl (1) is the quintessential member of triterpenoid cyanoacrylates, an emerging class of bioactive compounds capable of transient covalent binding to thiols. The mechanistic basis for this unusual "pulsed reactivity" profile and the mode of its biological translation are unknown. To provide clues on these issues, a series of Δ 1 -dehydrooleanolates bearing an electron-withdrawing group at C-2 (7a-m) were prepared from oleanolic acid (3a) and comparatively investigated in terms of reactivity with thiols and bioactivity against a series of electrophile-sensitive transcription factors (Nrf2, NF-κB, STAT3). The emerging picture suggests that the triterpenoid scaffold sharply decreases the reactivity of the enone system by steric encumbrance and that only strongly electrophilic and sterically undemanding substituents such as a cyanide or a carboxylate group can re-establish Michael reactivity, albeit in a transient way for the cyanide group. In general, a substantial dissection between the thiol-trapping ability and the modulation of biological end-points sensitive to thiol alkylation was observed, highlighting the role of shape complementarity for the activity of triterpenoid thia-Michael acceptors.

TAU mutations are genetically linked to fronto-temporal dementia (FTD) and hyper-phosphorylated aggregates of Tau form neurofibrillary tangles (NFTs) that constitute a pathological hallmark of Alzheimer disease (AD) and FTD. These observations indicate that Tau has a pivotal role in the pathogenesis of neurodegenerative disorders. Tau is cleaved by caspases at Aspartate 421 , to form a Tau metabolite known as δTau; δTau is increased in AD, due to the hyper-activation of caspases in AD brains. δTau is considered a critical toxic moiety underlying neurodegeneration, which initiates and facilitates NFT formation. As Tau is a therapeutic target in neurodegeneration, it is important to rigorously determine whether δTau is a toxic Tau species that should be pharmacologically attacked. To directly address these questions, we have generated a knock-in (KI) mouse called Tau DN -that expresses a Tau mutant that cannot be cleaved by caspases. Tau DN mice present short-term memory deficits and synaptic plasticity defects. Moreover, mice carrying two mutant Tau alleles show increased total insoluble hyper-phosphorylated Tau in the forebrain. These data are in contrast with the concept that δTau is a critical toxic moiety underlying neurodegeneration, and suggest that cleavage of Tau by caspases represents a negative feedback mechanism aimed to eliminate toxic Tau species. Alternatively, it is possible that either a reduction or an increase in δTau leads to synaptic dysfunction, memory impairments and Tau pathology. Both possibilities will have to be considered when targeting caspase cleavage of Tau in AD therapy.

Alteration of mitochondria-associated membranes (MAMs) has been proposed to contribute to the pathogenesis of Alzheimer's disease (AD). We studied herein the subcellular distribution, the processing, and the protein interactome of the amyloid-β protein precursor (AβPP) and its proteolytic products in MAMs. We reveal that AβPP and its catabolites are present in MAMs in cellular models overexpressing wild type AβPP or AβPP harboring the double Swedish or London familial AD mutations, and in brains of transgenic mice model of AD. Furthermore, we evidenced that both β- and γ-secretases are present and harbor AβPP processing activities in MAMs. Interestingly, cells overexpressing APPswe show increased ER-mitochondria contact sites. We also document increased neutral lipid accumulation linked to Aβ production and reversed by inhibiting β- or γ-secretases. Using a proteomic approach, we show that AβPP and its catabolites interact with key proteins of MAMs controlling mitochondria and ER functions. These data highlight the role of AβPP processing and proteomic interactome in MAMs deregulation taking place in AD.

Background and Objectives: Dent disease is a rare X-linked disorder characterized by low molecular weight proteinuria and often considered a renal tubular disease. However, glomerulosclerosis was recently reported in several patients. Thus, Dent disease renal histopathologic features were characterized and assessed, and their association with kidney function was assessed., Design, Setting, Participants, & Measurements: Clinical renal pathology reports and slides (where available) were collected from 30 boys and men in eight countries who had undergone clinical renal biopsy between 1995 and 2014., Results: Median (25th, 75th percentiles) age at biopsy was 7.5 (5, 19) years with an eGFR of 69 (44, 94) ml/min per 1.73 m 2 and a 24-hour urine protein of 2000 (1325, 2936) mg. A repeat biopsy for steroid-resistant proteinuria was performed in 13% (four of 30) of the patients. Prominent histologic findings included focal global glomerulosclerosis in 83% (25 of 30; affecting 16%±19% glomeruli), mild segmental foot process effacement in 57% (13 of 23), focal interstitial fibrosis in 60% (18 of 30), interstitial lymphocytic infiltration in 53% (16 of 30), and tubular damage in 70% (21 of 30). Higher percentages of globally sclerotic glomeruli, foot process effacement, and interstitial inflammation were associated with lower eGFR at biopsy, whereas foot process effacement was associated with steeper annual eGFR decline., Conclusions: These associations suggest a potential role for glomerular pathology, specifically involving the podocyte, in disease progression, which deserves further study. Furthermore, Dent disease should be suspected in boys and men who have unexplained proteinuria with focal global glomerulosclerosis and segmental foot process effacement on renal biopsy., (Copyright © 2016 by the American Society of Nephrology.)

Background: Anti-pentraxin 3 (PTX3) antibodies were associated with the absence of lupus glomerulonephritis in humans., Aim: To explore the effects of anti-PTX3 antibodies in New Zealand Black/White (NZB/NZW F1) mice and their inherent mechanisms of action., Materials and Methods: 30 NZB/NZW F1 mice were subdivided into 3 groups of 10 mice each and subcutaneously injected with PTX3, alum and PBS (group 1), alum and PBS (group 2) or PBS alone (group 3), 3 times 3 weeks apart, before development of renal disease. Mice were followed until natural death. Histological analysis and immunohistochemistry were performed on harvested kidneys. Effects of anti-PTX3 antibodies on C1q binding to immobilized PTX3-anti-PTX3 immune complexes were evaluated in vitro using human SLE sera. Qualitative characterization of human IgG anti-PTX3 was performed., Results: Only group 1 mice developed anti-PTX3 antibodies. Anti-dsDNA and anti-C1q antibodies appeared significantly later and at lower levels in group 1 mice vs. controls (p < 0.0001). Proteinuria-free and overall survival were significantly increased in group 1 mice vs. controls (p < 0.05 and p = 0.03, respectively). Histopathological analysis showed that glomerular and tubular PTX3 staining and renal lesions were increased in controls compared with immunized mice. Addition of human SLE sera positive for anti-PTX3 antibodies to C1q and fixed PTX3 interfered with C1q binding to PTX3-anti-PTX3 immune complexes. Qualitative characterization of human IgG anti-PTX3 showed an increased proportion of IgG4., Conclusions: Anti-PTX3 antibodies delay lupus-like nephritis and prolong survival of NZB/NZW F1 mice. In vitro observations suggest anti-PTX3 antibodies may dampen complement activation via their Fc fragment, likely hindering renal inflammation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the NH2 terminus of the ACR, and CRL4(CRBN), which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the COOH terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4(CRBN), pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome analysis indicates that these E3 ligases are enzymatically active and ubiquitinate the ACR residues Lys(649/650/651/676/688) Deletion of Crbn reduces ubiquitination of Lys(676) suggesting that Lys(676) is physiologically ubiquitinated by CRL4(CRBN) The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mutations in BRI2/ITM2b genes cause Familial British and Danish Dementias (FBD and FDD), which are pathogenically similar to Familial Alzheimer Disease (FAD). BRI2 inhibits processing of Amyloid precursor protein (APP), a protein involved in FAD pathogenesis. Accumulation of a carboxyl-terminal APP metabolite -ß-CTF- causes memory deficits in a knock-in mouse model of FDD, called FDDKI.We have investigated further the pathogenic function of ß-CTF studying the effect of Aph1B/C deletion on FDDKI mice. This strategy is based on the evidence that deletion of Aph1B/C proteins, which are components of the γ-secretase that cleaves ß-CTF, results in stabilization of ß-CTF and a reduction of Aβ. We found that both the FDD mutation and the Aph1B/C deficiency mildly interfered with spatial long term memory, spatial working/short-term memory and long-term contextual fear memory. In addition, the Aph1BC deficiency induced deficits in long-term cued fear memory. Moreover, the two mutations have additive adverse effects as they compromise the accuracy of spatial long-term memory and induce spatial memory retention deficits in young mice. Overall, the data are consistent with a role for β-CTF in the genesis of memory deficits.

An expeditious strategy to resolve turmerone, the lipophilic anti-inflammatory principle of turmeric (Curcuma longa), into its individual bisabolane constituents (ar-, α-, and β-turmerones, 2-4, respectively) was developed. The comparative evaluation of these compounds against a series of anti-inflammatory targets (NF-κB, STAT3, Nrf2, HIF-1α) evidenced surprising differences, providing a possible explanation for the contrasting data on the activity of turmeric oil. Differences were also evidenced in the profile of more polar bisabolanes between the Indian and the Javanese samples used to obtain turmerone, and a novel hydroxylated bicyclobisabolane ketol (bicycloturmeronol, 8) was obtained from a Javanese sample of turmeric. Taken together, these data support the view that bisabolane sesquiterpenes represent an important taxonomic marker for turmeric and an interesting class of anti-inflammatory agents, whose strict structure-activity relationships are worth a systematic evaluation.

Mutations in Amyloid β Precursor Protein (APP) and in genes that regulate APP processing--such as PSEN1/2 and ITM2b/BRI2--cause familial dementia, such Familial Alzheimer disease (FAD), Familial Danish (FDD) and British (FBD) dementias. The ApoE gene is the major genetic risk factor for sporadic AD. Three major variants of ApoE exist in humans (ApoE2, ApoE3, and ApoE4), with the ApoE4 allele being strongly associated with AD. ITM2b/BRI2 is also a candidate regulatory node genes predicted to mediate the common patterns of gene expression shared by healthy ApoE4 carriers and late-onset AD patients not carrying ApoE4. This evidence provides a direct link between ITM2b/BRI2 and ApoE4. To test whether ApoE4 and pathogenic ITM2b/BRI2 interact to modulate learning and memory, we crossed a mouse carrying the ITM2b/BRI2 mutations that causes FDD knocked-in the endogenous mouse Itm2b/Bri2 gene (FDDKI mice) with human ApoE3 and ApoE4 targeted replacement mice. The resultant ApoE3, FDDKI/ApoE3, ApoE4, FDDKI/ApoE4 male mice were assessed longitudinally for learning and memory at 4, 6, 12, and 16-17 months of age. The results showed that ApoE4-carrying mice displayed spatial working/short-term memory deficits relative to ApoE3-carrying mice starting in early middle age, while long-term spatial memory of ApoE4 mice was not adversely affected even at 16-17 months, and that the FDD mutation impaired working/short-term spatial memory in ApoE3-carrying mice and produced impaired long-term spatial memory in ApoE4-carrying mice in middle age. The present results suggest that the FDD mutation may differentially affect learning and memory in ApoE4 carriers and non-carriers., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The amyloid precursor protein (APP), whose mutations cause familial Alzheimer's disease, interacts with the synaptic release machinery, suggesting a role in neurotransmission. Here we mapped this interaction to the NH2-terminal region of the APP intracellular domain. A peptide encompassing this binding domain -named JCasp- is naturally produced by a γ-secretase/caspase double-cut of APP. JCasp interferes with the APP-presynaptic proteins interaction and, if linked to a cell-penetrating peptide, reduces glutamate release in acute hippocampal slices from wild-type but not APP deficient mice, indicating that JCasp inhibits APP function.The APP-like protein-2 (APLP2) also binds the synaptic release machinery. Deletion of APP and APLP2 produces synaptic deficits similar to those caused by JCasp. Our data support the notion that APP and APLP2 facilitate transmitter release, likely through the interaction with the neurotransmitter release machinery. Given the link of APP to Alzheimer's disease, alterations of this synaptic role of APP could contribute to dementia.

Pentraxin 3 (PTX3) is an acute-phase protein involved in C1q clearance. The presence of anti-C1q and the absence of anti-PTX3 antibodies were associated with lupus glomerulonephritis (LGLN). Our aim was to assess soluble and kidney-expressed PTX3 and their relationships with anti-C1q and anti-PTX3 antibodies in LGLN. Serum PTX3, anti-C1q, anti-dsDNA, and anti-PTX3 antibodies were tested in 130 systemic lupus erythematosus (SLE) patients, 130 healthy and 127 disease controls. Twenty-nine renal biopsies from SLE patients were analyzed and PTX3 immunostaining was quantified by morphometric analysis. Parametric and nonparametric statistics were performed. PTX3 serum levels were lower in SLE versus controls, but they were correlated with proteinuria in LGLN patients (p = 0.001). LGLN patients had higher anti-C1q and lower anti-PTX3 antibody levels than those without (p < 0.0001). LGLN was more prevalent in anti-C1q(+)/anti-PTX3(-) than in anti-C1q(+)/anti-PTX3(+) patients (p < 0.001). No LGLN was observed in anti-C1q(-)/anti-PTX3(+) patients. PTX3 was expressed in glomeruli and renal interstitium. Renal PTX3 was correlated with proteinuria (p = 0.024) and interstitial fibrosis (p = 0.023). PTX3 staining and fibrosis were higher in anti-PTX3(-) than anti-PTX3(+) patients. In conclusion, PTX3 is expressed in glomeruli of LGLN patients, primarily in anti-PTX3(-) patients, where it is correlated with renal fibrosis. Anti-C1q/anti-PTX3 antibody profile seems to be useful in LGLN assessment.

Dent disease (DD) is a rare X-linked recessive renal tubulopathy characterised by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis and/or nephrolithiasis. DD is caused by mutations in both the CLCN5 and OCRL genes. CLCN5 encodes the electrogenic chloride/proton exchanger ClC-5 which is involved in the tubular reabsorption of albumin and LMW proteins, OCRL encodes the inositol polyphosphate 5-phosphatase, and was initially associated with Lowe syndrome. In approximately 25 % of patients, no CLCN5 and OCRL mutations were detected. The aim of our study was to evaluate whether calcium phosphate metabolism disorders and their clinical complications are differently distributed among DD patients with and without CLCN5 mutations. Sixty-four male subjects were studied and classified into three groups: Group I (with CLCN5 mutations), Group II (without CLCN5 mutations) and Group III (family members with the same CLCN5 mutation). LMWP, hypercalciuria and phosphaturic tubulopathy and the consequent clinical complications nephrocalcinosis, nephrolithiasis, bone disorders, and chronic kidney disease (CKD) were considered present or absent in each patient. We found that the distribution of nephrolithiasis, bone disorders and CKD differs among patients with and without CLCN5 mutations. Only in patients harbouring CLCN5 mutations was age-independent nephrolithiasis associated with hypercalciuria, suggesting that nephrolithiasis is linked to altered proximal tubular function caused by a loss of ClC-5 function, in agreement with ClC-5 KO animal models. Similarly, only in patients harbouring CLCN5 mutations was age-independent kidney failure associated with nephrocalcinosis, suggesting that kidney failure is the consequence of a ClC-5 dysfunction, as in ClC-5 KO animal models. Bone disorders are a relevant feature of DD phenotype, as patients were mainly young males and this complication occurred independently of age. The triad of symptoms, LMWP, hypercalciuria, and nephrocalcinosis, was present in almost all patients with CLCN5 mutations but not in those without CLCN5 mutations. This lack of homogeneity of clinical manifestations suggests that the difference in phenotypes between the two groups might reflect different pathophysiological mechanisms, probably depending on the diverse genes involved. Overall, our results might suggest that in patients without CLCN5 mutations several genes instead of the prospected third DD underpin patients' phenotypes.

Medullary nephrocalcinosis is a hallmark of medullary sponge kidney (MSK). We had the opportunity to study a spontaneous calcification process in vitro by utilizing the renal cells of a patient with MSK who was heterozygous for the c.-27 + 18G>A variant in the GDNF gene encoding glial cell-derived neurotrophic factor. The cells were obtained by collagenase digestion of papillary tissues from the MSK patient and from two patients who had no MSK or nephrocalcinosis. These cells were typed by immunocytochemistry, and the presence of mineral deposits was studied using von Kossa staining, scanning electron microscopy analysis and an ALP assay. Osteoblastic lineage markers were studied using immunocytochemistry and RT-PCR. Staminality markers were also analysed using flow cytometry, magnetic cell separation technology, immunocytochemistry and RT-PCR. Starting from p2, MSK and control cells formed nodules with a behaviour similar to that of calcifying pericytes; however, Ca₂PO₄ was only found in the MSK cultures. The MSK cells had morphologies and immunophenotypes resembling those of pericytes or stromal stem cells and were positive for vimentin, ZO1, αSMA and CD146. In addition, the MSK cells expressed osteocalcin and osteonectin, indicating an osteoblast-like phenotype. In contrast to the control cells, GDNF was down-regulated in the MSK cells. Stable GDNF knockdown was established in the HK2 cell line and was found to promote Ca₂PO₄ deposition when the cells were incubated with calcifying medium by regulating the osteonectin/osteopontin ratio in favour of osteonectin. Our data indicate that the human papilla may be a perivascular niche in which pericyte/stromal-like cells can undergo osteogenic differentiation under particular conditions and suggest that GDNF down-regulation may have influenced the observed phenomenon., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Replacement of the benzylamide motif of synthetic capsaicin (nonivamide, 1c) with a tetrazole moiety was detrimental for TRPV1 binding, but unexpectedly generated a potent and non-electrophilic TRPA1 agonist (4a). Spurred by this observation and by the relatively small number of non-covalent TRPA1 ligands reported so far, the benzylamide-to-tetrazole swap was investigated in the more lipophilic and powerful vanilloids olvanil (1d), rinvanil (1e), and phenylacetylrinvanil (1f). In all cases, the replacement was detrimental for TRPV1 binding, but a clear modulation of TRPA1 activity was observed. These observations show that the capsaicinoid pharmacophore displays orthogonal structure-activity relationships for TRPV1 and TRPA1 binding, and suggest the possibility of obtaining compounds with dual TRPV1/TRPA1 modulatory properties by exploration of the chemical space around the capsaicin motif., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Amyloid Precursor Protein (APP) is a type I membrane protein that undergoes extensive processing by secretases, including BACE1. Although mutations in APP and genes that regulate processing of APP, such as PSENs and BRI2/ITM2B, cause dementias, the normal function of APP in synaptic transmission, synaptic plasticity and memory formation is poorly understood. To grasp the biochemical mechanisms underlying the function of APP in the central nervous system, it is important to first define the sub-cellular localization of APP in synapses and the synaptic interactome of APP. Using biochemical and electron microscopy approaches, we have found that APP is localized in pre-synaptic vesicles, where it is processed by Bace1. By means of a proteomic approach, we have characterized the synaptic interactome of the APP intracellular domain. We focused on this region of APP because in vivo data underline the central functional and pathological role of the intracellular domain of APP. Consistent with the expression of APP in pre-synaptic vesicles, the synaptic APP intracellular domain interactome is predominantly constituted by pre-synaptic, rather than post-synaptic, proteins. This pre-synaptic interactome of the APP intracellular domain includes proteins expressed on pre-synaptic vesicles such as the vesicular SNARE Vamp2/Vamp1 and the Ca2+ sensors Synaptotagmin-1/Synaptotagmin-2, and non-vesicular pre-synaptic proteins that regulate exocytosis, endocytosis and recycling of pre-synaptic vesicles, such as target-membrane-SNAREs (Syntaxin-1b, Syntaxin-1a, Snap25 and Snap47), Munc-18, Nsf, α/β/γ-Snaps and complexin. These data are consistent with a functional role for APP, via its carboxyl-terminal domain, in exocytosis, endocytosis and/or recycling of pre-synaptic vesicles.

The mode of inheritance of Alport syndrome (ATS) has long been controversial. In 1927, the disease was hypothesized as a dominant condition in which males were more severely affected than females. In 1990, it was considered an X-linked (XL) semidominant condition, due to COL4A5 mutations. Later on, a rare autosomal recessive (AR) form due to COL4A3/COL4A4 mutations was identified. An autosomal dominant (AD) form was testified more recently by the description of some large pedigrees but the real existence of this form is still questioned by many and its exact prevalence is unknown. The introduction of next generation sequencing (NGS) allowed us to perform an unbiased simultaneous COL4A3-COL4A4-COL4A5 analysis in 87 Italian families (273 individuals) with clinical suspicion of ATS. In 48 of them (55%), a mutation in one of the three genes was identified: the inheritance was XL semidominant in 65%, recessive in 4% and most interestingly AD in 31% (15 families). The AD form must therefore be seriously taken into account in all pedigrees with affected individuals in each generation. Furthermore, a high frequency of mutations (>50%) was shown in patients with only 1 or 2 clinical criteria, suggesting NGS as first-level analysis in cases with a clinical suspicion of ATS., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Mutations of INF2 represent the major cause of familial autosomal dominant (AD) focal segmental glomerulosclerosis (FSGS). A few patients present neurological symptoms of Charcot-Marie-Tooth (CMT) disease but the prevalence of the association has not been assessed yet., Methods: We screened 28 families with AD FSGS and identified 8 INF2 mutations in 9 families (32 patients overall), 3 of which were new. Mutations were in all cases localized in the diaphanous-inhibitory domain (DID) of the protein., Results: Clinical features associated with INF2 mutations in our patient cohort included mild proteinuria (1.55 g/L; range 1-2.5) and haematuria as a unique symptom that was recognized at a median age of 21.75 years (range 8-30). Eighteen patients developed end-stage renal disease during their third decade of life; 12 patients presented a creatinine range between 1.2 and 1.5 mg/dL and 2 were healthy at 45 and 54 years of age. CMT was diagnosed in four cases (12.5%); one of these patients presented an already known mutation on exon 2 of INF2, whereas the other patients presented the same mutation on exon 4, a region that was not previously associated with CMT., Conclusions: We confirmed the high incidence of INF2 mutations in families with AD FSGS. The clinical phenotype was mild at the onset of the disease, but evolution to ESRD was frequent. The incidence of CMT has, for the first time, been calculated here to be 12.5% of mutation carriers. Our findings support INF2 gene analysis in families in which renal failure and/or neuro-sensorial defects are inherited following an AD model., (© The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Perturbed Endoplasmic Reticulum (ER) calcium (Ca2+) homeostasis emerges as a central player in Alzheimer disease (AD). Accordingly, different studies have reported alterations of the expression and the function of Ryanodine Receptors (RyR) in human AD-affected brains, in cells expressing familial AD-linked mutations on the β amyloid precursor protein (βAPP) and presenilins (the catalytic core in γ-secretase complexes cleaving the βAPP, thereby generating amyloid β (Aβ) peptides), as well as in the brain of various transgenic AD mice models. Data converge to suggest that RyR expression and function alteration are associated to AD pathogenesis through the control of: i) βAPP processing and Aβ peptide production, ii) neuronal death; iii) synaptic function; and iv) memory and learning abilities. In this review, we document the network of evidences suggesting that RyR could play a complex dual "compensatory/protective versus pathogenic" role contributing to the setting of histopathological lesions and synaptic deficits that are associated with the disease stages. We also discuss the possible mechanisms underlying RyR expression and function alterations in AD. Finally, we review recent publications showing that drug-targeting blockade of RyR and genetic manipulation of RyR reduces Aβ production, stabilizes synaptic transmission, and prevents learning and memory deficits in various AD mouse models. Chemically-designed RyR "modulators" could therefore be envisioned as new therapeutic compounds able to delay or block the progression of AD.