Slc20a1 and Slc20a2 regulate neuronal plasticity and cognition independently of their phosphate transport ability.

In recent years, primary familial brain calcification (PFBC), a rare neurological disease characterized by a wide spectrum of cognitive disorders, has been associated to mutations in the sodium (Na)-Phosphate (Pi) co-transporter SLC20A2. However, the functional roles of the Na-Pi co-transporters in the brain remain still largely elusive. Here we show that Slc20a1 (PiT-1) and Slc20a2 (PiT-2) are the most abundant Na-Pi co-transporters expressed in the brain and are involved in the control of hippocampal-dependent learning and memory. We reveal that Slc20a1 and Slc20a2 are differentially distributed in the hippocampus and associated with independent gene clusters, suggesting that they influence cognition by different mechanisms. Accordingly, using a combination of molecular, electrophysiological and behavioral analyses, we show that while PiT-2 favors hippocampal neuronal branching and survival, PiT-1 promotes synaptic plasticity. The latter relies on a likely Otoferlin-dependent regulation of synaptic vesicle trafficking, which impacts the GABAergic system. These results provide the first demonstration that Na-Pi co-transporters play key albeit distinct roles in the hippocampus pertaining to the control of neuronal plasticity and cognition. These findings could provide the foundation for the development of novel effective therapies for PFBC and cognitive disorders.

Usefulness and analytical performances of complement multiplex assay for measuring complement biomarkers in plasma.

INTRODUCTION: The complement system is involved in numerous diseases, through diverse mechanisms and degree of activation. With the emergence of complement targeting therapeutic, simple and accessible tools to evaluate the extent of complement activation are strongly needed. METHODS: We evaluated two multiplex panels, measuring complement activation fragments (C4a, C3a, C5a, Bb, Ba, sC5b9) and intact components or regulators (C1q, C2, C3, C4, C5, FD, FP, FH, FI). The specificity of each measurement was assessed by using complement proteins depleted sera and plasma collected from patients with complement deficiencies. Normal values distribution was estimated using 124 plasma samples from healthy donors and complement activation profile was assessed in plasma collected from 31 patients with various complement-mediated disorders. RESULTS: We observed good inter-assay variation. All tested protein deficiencies were accurately detected. We established assay-specific reference values for each analyte. Except for C3, C4 and C4a, the majority of the measurements were in good agreement with references methods or published data. CONCLUSION: Our study substantiates the utility of the Complement Multiplex assay as a tool for measuring complement activation and deficiencies. Quantifying complement cleavage fragments in patients exhibiting classical or alternative pathway activation allowed evaluating the activation state of the whole cascade.

Combining robust urine biomarkers to assess chronic kidney disease progression.

BACKGROUND: Urinary biomarkers may improve the prediction of chronic kidney disease (CKD) progression. Yet, data reporting the applicability of most commercial biomarker assays to the detection of their target analyte in urine together with an evaluation of their predictive performance are scarce. METHODS: 30 commercial assays (ELISA) were tested for their ability to quantify the target analyte in urine using strict (FDA-approved) validation criteria. In an exploratory analysis, LASSO (Least Absolute Shrinkage and Selection Operator) logistic regression analysis was used to identify potentially complementary biomarkers predicting fast CKD progression, determined as the 51CrEDTA clearance-based measured glomerular filtration rate (mGFR) decline (>10% per year) in a subsample of 229 CKD patients (mean age, 61 years; 66% men; baseline mGFR, 38 mL/min) from the NephroTest prospective cohort. FINDINGS: Among the 30 assays, directed against 24 candidate biomarkers, encompassing different pathophysiological mechanisms of CKD progression, 16 assays fulfilled the FDA-approved criteria. LASSO logistic regressions identified a combination of five biomarkers including CCL2, EGF, KIM1, NGAL, and TGF-α that improved the prediction of fast mGFR decline compared to the kidney failure risk equation variables alone: age, gender, mGFR, and albuminuria. Mean area under the curves (AUC) estimated from 100 re-samples was higher in the model with than without these biomarkers, 0.722 (95% confidence interval 0.652-0.795) vs. 0.682 (0.614-0.748), respectively. Fully-adjusted odds-ratios (95% confidence interval) for fast progression were 1.87 (1.22, 2.98), 1.86 (1.23, 2.89), 0.43 (0.25, 0.70), 1.10 (0.71, 1.83), 0.55 (0.33, 0.89), and 2.99 (1.89, 5.01) for albumin, CCL2, EGF, KIM1, NGAL, and TGF-α, respectively. INTERPRETATION: This study provides a rigorous validation of multiple assays for relevant urinary biomarkers of CKD progression which combination may improve the prediction of CKD progression. FUNDING: This work was supported by Institut National de la Santé et de la Recherche Médicale, Université de Paris, Assistance Publique Hôpitaux de Paris, Agence Nationale de la Recherche, MSDAVENIR, Pharma Research and Early Development Roche Laboratories (Basel, Switzerland), and Institut Roche de Recherche et Médecine Translationnelle (Paris, France).

Chatbot-based serious games: A useful tool for training medical students? A randomized controlled trial.

OBJECTIVES: Chatbots, conversational agents that walk medical students (MS) though a clinical case, are serious games that seem to be appreciated by MS. Their impact on MS's performance in exams however was not yet evaluated. Chatprogress is a chatbot-based game developed at Paris Descartes University. It contains 8 pulmonology cases with step-by-step answers delivered with pedagogical comments. The CHATPROGRESS study aimed to evaluate the impact of Chatprogress on students' success rate in their end-term exams. METHODS: We conducted a post-test randomized controlled trial held on all fourth-year MS at Paris Descartes University. All MS were asked to follow the University's regular lectures, and half of them were randomly given access to Chatprogress. At the end of the term, medical students were evaluated on pulmonology, cardiology and critical care medicine. MAIN OUTCOMES MEASURES: The primary aim was to evaluate an increase in scores in the pulmonology sub-test for students who had access to Chatprogress, compared to those who didn't. Secondary aims were to evaluate an increase in scores in the overall test (Pulmonology, Cardiology and Critical care medicine test (PCC)) and to evaluate the correlation between access to Chatprogress and overall test score. Finally, students' satisfaction was assessed using a survey. RESULTS: From 10/2018 to 06/2019, 171 students had access to Chatprogress (the Gamers) and among them, 104 ended up using it (the Users). Gamers and Users were compared to 255 Controls with no access to Chatprogress. Differences in scores on the pulmonology sub-test over the academic year were significantly higher among Gamers and Users vs Controls (mean score: 12.7/20 vs 12.0/20, p = 0.0104 and mean score: 12.7/20 vs 12.0/20, p = 0.0365 respectively). This significant difference was present as well in the overall PCC test scores: (mean score: 12.5/20 vs 12.1/20, p = 0.0285 and 12.6/20 vs 12.1/20, p = 0.0355 respectively). Although no significant correlation was found between the pulmonology sub-test's scores and MS's assiduity parameters (number of finished games among the 8 proposed to Users and number of times a User finished a game), there was a trend to a better correlation when users were evaluated on a subject covered by Chatprogress. MS were also found to be fans of this teaching tool, asking for more pedagogical comments even when they got the questions right. CONCLUSION: This randomised controlled trial is the first to demonstrate a significant improvement in students' results (in both the pulmonology subtest and the overall PCC exam) when they had access to Chatbots, and even more so when they actually used it.

Nonskeletal and skeletal effects of high doses versus low doses of vitamin D3 in renal transplant recipients: Results of the VITALE (VITamin D supplementation in renAL transplant recipients) study, a randomized clinical trial.

Vitamin D sufficiency is associated with a reduced risk of fractures, diabetes mellitus, cardiovascular events, and cancers, which are frequent complications after renal transplantation. The VITALE (VITamin D supplementation in renAL transplant recipients) study is a multicenter double-blind randomized trial, including nondiabetic adult renal transplant recipients with serum 25-hydroxy vitamin D (25(OH) vitamin D) levels of <30 ng/mL, which is randomized 12 to 48 months after transplantation to receive high (100 000 IU) or low doses (12 000 IU) of cholecalciferol every 2 weeks for 2 months and then monthly for 22 months. The primary outcome was a composite endpoint, including diabetes mellitus, major cardiovascular events, cancer, and death. Of 536 inclusions (50.8 [13.7] years, 335 men), 269 and 267 inclusions were in the high-dose and low-dose groups, respectively. The serum 25(OH) vitamin D levels increased by 23 versus 6 ng/mL in the high-dose and low-dose groups, respectively (P < .0001). In the intent-to-treat analysis, 15% versus 16% of the patients in the high-dose and low-dose groups, respectively, experienced a first event of the composite endpoint (hazard ratio, 0.94 [0.60-1.48]; P = .78), whereas 1% and 4% of patients in the high-dose and low-dose groups, respectively, experienced an incident symptomatic fracture (odds ratio, 0.24 [0.07-0.86], P = .03). The incidence of adverse events was similar between the groups. After renal transplantation, high doses of cholecalciferol are safe but do not reduce extraskeletal complications (trial registration: ClinicalTrials.gov; identifier: NCT01431430).

Single-cell transcriptomics reveals age-resistant maintenance of cell identities, stem cell compartments and differentiation trajectories in long-lived naked mole-rats skin.

Naked mole-rats (NMR) are subterranean rodents characterized by an unusual longevity coupled with an unexplained resistance to aging. In the present study, we performed extensive in situ analysis and single-cell RNA-sequencing comparing young and older animals. At variance with other species, NMR exhibited a striking stability of skin compartments and cell types, which remained stable over time without aging-associated changes. Remarkably, the number of stem cells was constant throughout aging. We found three classical cellular states defining a unique keratinocyte differentiation trajectory that were not altered after pseudo-temporal reconstruction. Epidermal gene expression did not change with aging either. Langerhans cell clusters were conserved, and only a higher basal stem cell expression of Igfbp3 was found in aged animals. In accordance, NMR skin healing closure was similar in young and older animals. Altogether, these results indicate that NMR skin is characterized by peculiar genetic and cellular features, different from those previously demonstrated for mice and humans. The remarkable stability of the aging NMR skin transcriptome likely reflects unaltered homeostasis and resilience.

The sexual dimorphism of kidney growth in mice and humans.

Kidney mass and function are sexually determined, but the cellular events and the molecular mechanisms involved in this dimorphism are poorly characterized. By combining female and male mice with castration/replacement experiments, we showed that male mice exhibited kidney overgrowth from five weeks of age. This effect was organ specific, since liver and heart weight were comparable between males and females, regardless of age. Consistently, the androgen receptor was found to be expressed in the kidneys of males, but not in the liver. In growing mice, androgens led to kidney overgrowth by first inducing a burst of cell proliferation and then an increase of cell size. Remarkably, androgens were also required to maintain cell size in adults. In fact, orchiectomy resulted in smaller kidneys in a matter of few weeks. These changes paralleled the changes of the expression of ornithine decarboxylase and cyclin D1, two known mediators of kidney growth, whereas, unexpectedly, mTORC1 and Hippo pathways did not seem to be involved. Androgens also enhanced kidney autophagy, very likely by increasing transcription factor EB nuclear translocation. Functionally, the increase of tubular mass resulted in increased sodium/phosphate transport. These findings were relevant to humans. Remarkably, by studying living gender-paired kidney donors-recipients, we showed that tubular cell size increased three months after transplantation in men as compared to women, regardless of the donor gender. Thus, our results identify novel signaling pathways that may be involved in androgen-induced kidney growth and homeostasis and suggest that androgens determine kidney size after transplantation.

Effective Anti-SARS-CoV-2 Immune Response in Patients With Clonal Mast Cell Disorders.

BACKGROUND: Mast cells are key players in innate immunity and the TH2 adaptive immune response. The latter counterbalances the TH1 response, which is critical for antiviral immunity. Clonal mast cell activation disorders (cMCADs, such as mastocytosis and clonal mast cell activation syndrome) are characterized by abnormal mast cell accumulation and/or activation. No data on the antiviral immune response in patients with MCADs have been published. OBJECTIVE: To study a comprehensive range of outcomes in patients with cMCAD with PCR- or serologically confirmed coronavirus disease 2019 and to characterize the specific anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune response in this setting. METHODS: Clinical follow-up and outcome data were collected prospectively over a 12-month period by members of the French Centre de Référence des Mastocytoses rare disease network. Anti-SARS-CoV-2-specific T-cell activity was measured with an ELISA, and humoral responses were evaluated by assaying circulating levels of specific IgG, IgA, and neutralizing antibodies. RESULTS: Overall, 32 patients with cMCAD were evaluated. None required noninvasive or mechanical ventilation. Two patients were admitted to hospital for oxygen and steroid therapy. The SARS-CoV-2-specific immune response was characterized in 21 of the 32 patients. Most had high counts of circulating SARS-CoV-2-specific, IFN-γ-producing T cells and high titers of neutralizing antispike IgGs. The patients frequently showed spontaneous T-cell IFN-γ production in the absence of stimulation; this production was correlated with basal circulating tryptase levels (a marker of the mast cell burden). CONCLUSIONS: Patients with cMCADs might not be at risk of severe coronavirus disease 2019, perhaps due to their spontaneous production of IFN-γ.

The naked truth: a comprehensive clarification and classification of current 'myths' in naked mole-rat biology.

The naked mole-rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled 'Surprisingly long survival of premature conclusions about naked mole-rat biology' described 28 'myths' which, those authors claimed, are a 'perpetuation of beautiful, but falsified, hypotheses' and impede our understanding of this enigmatic mammal. Here, we re-examine each of these 'myths' based on evidence published in the scientific literature. Following Braude et al., we argue that these 'myths' fall into four main categories: (i) 'myths' that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) 'myths' that are based on incomplete understanding, where more evidence is clearly needed; (iii) 'myths' where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; (iv) 'myths' where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term 'myth' is particularly inappropriate when applied to competing, evidence-based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole-rat biology and attempt to clarify some of these misconceptions.

Glomerular endothelial cell senescence drives age-related kidney disease through PAI-1.

The mechanisms underlying the development of glomerular lesions during aging are largely unknown. It has been suggested that senescence might play a role, but the pathophysiological link between senescence and lesion development remains unexplained. Here, we uncovered an unexpected role for glomerular endothelial cells during aging. In fact, we discovered a detrimental cross-talk between senescent endothelial cells and podocytes, through PAI-1. In vivo, selective inactivation of PAI-1 in endothelial cells protected glomeruli from lesion development and podocyte loss in aged mice. In vitro, blocking PAI-1 in supernatants from senescent endothelial cells prevented podocyte apoptosis. Consistently, depletion of senescent cells prevented podocyte loss in old p16 INK-ATTAC transgenic mice. Importantly, these experimental findings are relevant to humans. We showed that glomerular PAI-1 expression was predictive of poor outcomes in transplanted kidneys from elderly donors. In addition, we observed that in elderly patients, urinary PAI-1 was associated with age-related chronic kidney disease. Altogether, these results uncover a novel mechanism of kidney disease and identify PAI-1 as a promising biomarker of kidney dysfunction in allografts from elderly donors.

Is sexual harassment and psychological abuse among medical students a fatality? A 2-year study in the Paris Descartes School of Medicine.

INTRODUCTION: An observatory of sexual harassment and psychological abuse was set up at one of France's largest schools of medicine to both quantify and reduce sexual harassment or psychological abuse of medical students. METHODS: Over a 2-year period, we described the evolution of sexual harassment and psychological abuse and explored for associated factors. Moreover, a qualitative analysis using an inductive approach was performed from students' verbatim. RESULTS: 2795 responses were collected. Sexual harassment was reported in 7% and psychological abuse in 15%, at baseline, and decreased after the observatory was set up. Women had higher odds of being a victim of sexual harassment. Older students reported less often psychological abuse and being a witness of sexual harassment. Surgery departments were associated with up to 5.7-fold increased odds of sexual harassment. Surgery and pediatrics departments were associated with a 2-fold increased odds of psychological abuse. Qualitative analysis revealed four categories: humiliation, the feeling of inferiority, sexual harassment, and manifestations of violence. CONCLUSION: During clerkships, factors associated with higher odds of sexual harassment and psychological abuse were female gender, younger age, and departments of surgery. Setting up such an observatory may contribute to reduce this burden and provide a useful tool to raise awareness.

Impact of integrating objective structured clinical examination into academic student assessment: Large-scale experience in a French medical school.

PURPOSE: Objective structured clinical examinations (OSCE) evaluate clinical reasoning, communication skills, and interpersonal behavior during medical education. In France, clinical training has long relied on bedside clinical practice in academic hospitals. The need for a simulated teaching environment has recently emerged, due to the increasing number of students admitted to medical schools, and the necessity of objectively evaluating practical skills. This study aimed at investigating the relationships between OSCE grades and current evaluation modalities. METHODS: Three-hundred seventy-nine 4th-year students of University-of-Paris Medical School participated to the first large-scale OSCE at this institution, consisting in three OSCE stations (OSCE#1-3). OSCE#1 and #2 focused on cardiovascular clinical skills and competence, whereas OSCE#3 focused on relational skills while providing explanations before planned cholecystectomy. We investigated correlations of OSCE grades with multiple choice (MCQ)-based written examinations and evaluations of clinical skills and behavior (during hospital traineeships); OSCE grade distribution; and the impact of integrating OSCE grades into the current evaluation in terms of student ranking. RESULTS: The competence-oriented OSCE#1 and OSCE#2 grades correlated only with MCQ grades (r = 0.19, P<0.001) or traineeship skill grades (r = 0.17, P = 0.001), respectively, and not with traineeship behavior grades (P>0.75). Conversely, the behavior-oriented OSCE#3 grades correlated with traineeship skill and behavior grades (r = 0.19, P<0.001, and r = 0.12, P = 0.032), but not with MCQ grades (P = 0.09). The dispersion of OSCE grades was wider than for MCQ examinations (P<0.001). When OSCE grades were integrated to the final fourth-year grade with an incremental 10%, 20% or 40% coefficient, an increasing proportion of the 379 students had a ranking variation by ±50 ranks (P<0.001). This ranking change mainly affected students among the mid-50% of ranking. CONCLUSION: This large-scale French experience showed that OSCE designed to assess a combination of clinical competence and behavioral skills, increases the discriminatory capacity of current evaluations modalities in French medical schools.

Inhibition of the replication of SARS-CoV-2 in human cells by the FDA-approved drug chlorpromazine.

INTRODUCTION: Urgent action is needed to fight the ongoing coronavirus disease 2019 (COVID-19) pandemic by reducing the number of infected cases, contagiousness and severity. Chlorpromazine (CPZ), an antipsychotic from the phenothiazine group, is known to inhibit clathrin-mediated endocytosis and has antiviral activity against severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1) and Middle East respiratory syndrome coronavirus. The aim of this in-vitro study was to test CPZ against SARS-CoV-2 in monkey and human cells. MATERIALS AND METHODS: Monkey VeroE6 cells and human alveolar basal epithelial A549-ACE2 cells were infected with SARS-CoV-2 in the presence of various concentrations of CPZ. Supernatants were harvested at day 2 and analysed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for the presence of SARS-CoV-2 RNA. Cell viability was assessed in non-infected cells. RESULTS: CPZ was found to have antiviral activity against SARS-CoV-2 in monkey VeroE6 cells, with a half maximal inhibitory concentration (IC50) of 8.2 µM, half maximal cytotoxic concentration (CC50) of 13.5 µM, and selectivity index (SI) of 1.65. In human A549-ACE2 cells, CPZ was also found to have anti-SARS-CoV-2 activity, with IC50 of 11.3 µM, CC50 of 23.1 µM and SI of 2.04. DISCUSSION: Although the measured SI values are low, the IC50 values measured in vitro may translate to CPZ dosages used in routine clinical practice because of the high biodistribution of CPZ in lungs and saliva. Also, the distribution of CPZ in brain could be of interest for treating or preventing neurological and psychiatric forms of COVID-19. CONCLUSIONS: These preclinical findings support clinical investigation of the repurposing of CPZ, a drug with mild side effects, in the treatment of patients with COVID-19.

Three-dimensional architecture of nephrons in the normal and cystic kidney.

Kidney function is crucially dependent on the complex three-dimensional structure of nephrons. Any distortion of their shape may lead to kidney dysfunction. Traditional histological methods present major limitations for three-dimensional tissue reconstruction. Here, we combined tissue clearing, multi-photon microscopy and digital tracing for the reconstruction of single nephrons under physiological and pathological conditions. Sets of nephrons differing in location, shape and size according to their function were identified. Interestingly, nephrons tend to lie in planes. When this technique was applied to a model of cystic kidney disease, cysts were found to develop only in specific nephron segments. Along the same segment, cysts are contiguous within normal non-dilated tubules. Moreover, the shapes of cysts varied according to the nephron segment. Thus, our findings provide a valuable strategy for visualizing the complex structure of kidneys at the single nephron level and, more importantly, provide a basis for understanding pathological processes such as cystogenesis.

Measured glomerular filtration rate (GFR) significantly and rapidly decreases after radical cystectomy for bladder cancer.

Precise determination of glomerular filtration rate (GFR) is essential for the management of patients with muscle-invasive bladder cancer (MIBC). We aim to describe the early evolution of measured GFR (mGFR) after radical cystectomy and urinary diversion (RCUD) and to identify risk factors for GFR decline. GFR measurement using 51Cr-EDTA continuous infusion, estimated GFR (eGFR) from five published equations and renal scintigraphy with split renal function determination were performed before and 6 months after RCUD. Chronic Kidney Disease (mGFR < 60 mL/min/1.73 m2) and GFR stages were defined according to the KDIGO guidelines using mGFR. Twenty-seven patients (men 85%, median age 65, IQR 59; 68 years) were included. A total of 20 (74%) patients experienced significant mGFR decline at 6 months postoperatively. Median mGFR decreased from 84.1 pre-operatively (IQR 65.3; 97.2) to 69.9 mL/min/1.73 m2 (IQR 55.0; 77.9) 6 months after surgery (p < 0.001). Thirteen (48%) patients had a progression to a worse GFR stage. Of the 22 patients without pre-operative CKD, 5 (23%) developed post-operative CKD. Diabetes mellitus was more frequent in patients in the highest tertile of relative mGFR decline (44% vs. 11%, p = 0.02) and platinum-based adjuvant chemotherapy tended to be more frequently used in these patients (44% vs. 17%, p = 0.06). Importantly, pre-operative weight was independently and negatively associated with post-operative mGFR and with mGFR slope in multivariable analyses. In this prospective series, we demonstrated that early and significant mGFR decline occurred after RCUD and perioperative platinum-based chemotherapy, especially in patients with diabetes mellitus and overweight.

The primary cilium and lipophagy translate mechanical forces to direct metabolic adaptation of kidney epithelial cells.

Organs and cells must adapt to shear stress induced by biological fluids, but how fluid flow contributes to the execution of specific cell programs is poorly understood. Here we show that shear stress favours mitochondrial biogenesis and metabolic reprogramming to ensure energy production and cellular adaptation in kidney epithelial cells. Shear stress stimulates lipophagy, contributing to the production of fatty acids that provide mitochondrial substrates to generate ATP through ß-oxidation. This flow-induced process is dependent on the primary cilia located on the apical side of epithelial cells. The interplay between fluid flow and lipid metabolism was confirmed in vivo using a unilateral ureteral obstruction mouse model. Finally, primary cilium-dependent lipophagy and mitochondrial biogenesis are required to support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis and cytoskeletal remodelling. Our findings demonstrate how primary cilia and autophagy are involved in the translation of mechanical forces into metabolic adaptation.