Claudins in the Renal Collecting Duct

The renal collecting duct fine-tunes urinary composition, and thereby, coordinates key physiological processes, such as volume/blood pressure regulation, electrolyte-free water reabsorption, and acid-base homeostasis. The collecting duct epithelium is comprised of a tight epithelial barrier resulting in a strict separation of intraluminal urine and the interstitium. Tight junctions are key players in enforcing this barrier and in regulating paracellular transport of solutes across the epithelium. The features of tight junctions across different epithelia are strongly determined by their molecular composition. Claudins are particularly important structural components of tight junctions because they confer barrier and transport properties. In the collecting duct, a specific set of claudins (Cldn-3, Cldn-4, Cldn-7, Cldn-8) is expressed, and each of these claudins has been implicated in mediating aspects of the specific properties of its tight junction. The functional disruption of individual claudins or of the overall barrier function results in defects of blood pressure and water homeostasis. In this concise review, we provide an overview of the current knowledge on the role of the collecting duct epithelial barrier and of claudins in collecting duct function and pathophysiology

Hypertonicity-affected genes are differentially expressed in clear cell renal cell carcinoma and correlate with cancer-specific survival

The heterogeneity of renal cell carcinoma (RCC) subtypes reflects the cell type of origin in the nephron, with consequences for therapy and prognosis. The transcriptional cues that determine segment-specific gene expression patterns are poorly understood. We recently showed that hypertonicity in the renal medulla regulates nephron-specific gene expression. Here, we analyzed a set of 223 genes, which were identified in the present study by RNA-Seq to be differentially expressed by hypertonicity, for the prediction of cancer-specific survival (CSS). Cluster analyses of these genes showed discrimination between tumor and non-tumor samples of clear cell RCC (ccRCC). Refinement of this gene signature to a four-gene score (OSM score) through statistical analyses enabled prediction of CSS in ccRCC patients of The Cancer Genome Atlas (TCGA) (n = 436) in univariate (HR = 4.1; 95% CI: 2.78-6.07; p = 4.39 × 10(-13)), and multivariate analyses including primary tumor (T); regional lymph node (N); distant metastasis (M); grading (G)(p = 2.3 × 10(-5)). The OSM score could be validated in an independent ccRCC study (n = 52) in univariate (HR = 1.29; 95% CI = 1.05-1.59; p = 0.011) and multivariate analyses (p = 0.016). Cell culture experiments using RCC cell lines demonstrated that the expression of the tumor suppressor ELF5 could be restored by hypertonicity. The innovation of our novel gene signature is that these genes are physiologically regulated only by hypertonicity, thereby providing the possibility to be targeted for therapy

Technologies for profiling the impact of genomic variants on transcription factor binding

Transcription factors (TFs) bind DNA in a sequence-specific manner and thereby regulate target gene expression. TF binding and its regulatory activity is highly context dependent, and is not only determined by specific cell types or differentiation stages but also relies on other regulatory mechanisms, such as DNA and chromatin modifications. Interactions between TFs and their DNA binding sites are critical mediators of phenotypic variation and play important roles in the onset of disease. A continuously growing number of studies therefore attempts to elucidate TF:DNA interactions to gain knowledge about regulatory mechanisms and disease-causing variants. Here we summarize how TF-binding characteristics and the impact of variants can be investigated, how bioinformatic tools can be used to analyze and predict TF:DNA binding, and what additional information can be obtained from the TF protein structur

Discovery of a non-canonical GRHL1 binding site using deep convolutional and recurrent neural networks

BACKGROUND: Transcription factors regulate gene expression by binding to transcription factor binding sites (TFBSs). Most models for predicting TFBSs are based on position weight matrices (PWMs), which require a specific motif to be present in the DNA sequence and do not consider interdependencies of nucleotides. Novel approaches such as Transcription Factor Flexible Models or recurrent neural networks consequently provide higher accuracies. However, it is unclear whether such approaches can uncover novel non-canonical, hitherto unexpected TFBSs relevant to human transcriptional regulation. RESULTS: In this study, we trained a convolutional recurrent neural network with HT-SELEX data for GRHL1 binding and applied it to a set of GRHL1 binding sites obtained from ChIP-Seq experiments from human cells. We identified 46 non-canonical GRHL1 binding sites, which were not found by a conventional PWM approach. Unexpectedly, some of the newly predicted binding sequences lacked the CNNG core motif, so far considered obligatory for GRHL1 binding. Using isothermal titration calorimetry, we experimentally confirmed binding between the GRHL1-DNA binding domain and predicted GRHL1 binding sites, including a non-canonical GRHL1 binding site. Mutagenesis of individual nucleotides revealed a correlation between predicted binding strength and experimentally validated binding affinity across representative sequences. This correlation was neither observed with a PWM-based nor another deep learning approach. CONCLUSIONS: Our results show that convolutional recurrent neural networks may uncover unanticipated binding sites and facilitate quantitative transcription factor binding predictions

How can patient registries facilitate guideline-based healthcare? A retrospective analysis of the CEDATA-GPGE registry for pediatric inflammatory bowel disease

Background: Early diagnosis is mandatory for the medical care of children and adolescents with pediatric-onset inflammatory bowel disease (PIBD). International guidelines ('Porto criteria') of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition recommend medical diagnostic procedures in PIBD. Since 2004, German and Austrian pediatric gastroenterologists document diagnostic and treatment data in the patient registry CEDATA-GPGE on a voluntary basis. The aim of this retrospective study was to analyze whether the registry CEDATA-GPGE reflects the Porto criteria and to what extent diagnostic measures of PIBD according to the Porto criteria are documented. Methods: Data of CEDATA-GPGE were analyzed for the period January 2014 to December 2018. Variables representing the Porto criteria for initial diagnostic were identified and categorized. The average of the number of measures documented in each category was calculated for the diagnoses CD, UC, and IBD-U. Differences between the diagnoses were tested by Chi-square test. Data on possible differences between data documented in the registry and diagnostic procedures that were actually performed were obtained via a sample survey. Results: There were 547 patients included in the analysis. The median age of patients with incident CD (n = 289) was 13.6 years (IQR: 11.2-15.2), of patients with UC (n = 212) 13.1 years (IQR: 10.4-14.8) and of patients with IBD-U (n = 46) 12.2 years (IQR: 8.6-14.7). The variables identified in the registry fully reflect the recommendations by the Porto criteria. Only the disease activity indices PUCAI and PCDAI were not directly provided by participants but calculated from obtained data. The category 'Case history' were documented for the largest part (78.0%), the category 'Imaging of the small bowel' were documented least frequently (39.1%). In patients with CD, the categories 'Imaging of the small bowel' (χ2 = 20.7, Cramer-V = 0.2, p < 0.001) and 'Puberty stage' (χ2 = 9.8, Cramer-V = 0.1, p < 0.05) were documented more often than in patients with UC and IBD-U. Conclusion: The registry fully reproduces the guideline's recommendations for the initial diagnosis of PIBD. The proportion of documented diagnostic examinations varied within the diagnostic categories and between the diagnoses. Despite technological innovations, time and personnel capacities at participating centers and study center are necessary to ensure reliable data entry and to enable researchers to derive important insights into guideline-based care

Anthelmintic Activity In Vivo Of Epiisopiloturine Against Juvenile And Adult Worms Of Schistosoma Mansoni

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Schistosomiasis is a serious disease currently estimated to affect more that 207 million people worldwide. Due to the intensive use of praziquantel, there is increasing concern about the development of drug-resistant strains. Therefore, it is necessary to search for and investigate new potential schistosomicidal compounds. This work reports the in vivo effect of the alkaloid epiisopiloturine (EPI) against adults and juvenile worms of Schistosoma mansoni. EPI was first purified its thermal behavior and theoretical solubility parameters charaterised. In the experiment, mice were treated with EPI over the 21 days post-infection with the doses of 40 and 200 mg/kg, and 45 days post-infection with single doses of 40, 100 and 300 mg/kg. The treatment with EPI at 40 mg/kg was more effective in adult worms when compared with doses of 100 and 300 mg/kg. The treatment with 40 mg/kg in adult worms reduced parasite burden significantly, lead to reduction in hepatosplenomegaly, reduced the egg burden in faeces, and decreased granuloma diameter. Scanning electron microscopy revealed morphological changes to the parasite tegument after treatment, including the loss of important features. Additionally, the in vivo treatment against juvenile with 40 mg/kg showed a reduction of the total worm burden of 50.2%. Histopathological studies were performed on liver, spleen, lung, kidney and brain and EPI was shown to have a DL50 of 8000 mg/kg. Therefore EPI shows potential to be used in schistosomiasis treatment. This is the first time that schistosomicidal in vivo activity of EPI has been reported.93Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao para a Ciencia e a Tecnologia (FCT) [PEst-C/EQB/LA0006/2011, PTDC/CTM-NAN/109877/2009]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Faculdade de Ciencias de Guarulhos FACIG/UNIESPConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Ministry of Education, Science, and Technological Development of Serbia [172008]Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CAPES [705/2009]CNPq [490585/2010-8]CNPq [40/2012, 404134/2012-2

Anthelmintic Activity In Vivo of Epiisopiloturine against Juvenile and Adult Worms of Schistosoma mansoni

Schistosomiasis is a serious disease currently estimated to affect more that 207 million people worldwide. Due to the intensive use of praziquantel, there is increasing concern about the development of drug-resistant strains. Therefore, it is necessary to search for and investigate new potential schistosomicidal compounds. This work reports the in vivo effect of the alkaloid epiisopiloturine (EPI) against adults and juvenile worms of Schistosoma mansoni. EPI was first purified its thermal behavior and theoretical solubility parameters charaterised. In the experiment, mice were treated with EPI over the 21 days post-infection with the doses of 40 and 200 mg/kg, and 45 days post-infection with single doses of 40, 100 and 300 mg/kg. The treatment with EPI at 40 mg/kg was more effective in adult worms when compared with doses of 100 and 300 mg/kg. The treatment with 40 mg/kg in adult worms reduced parasite burden significantly, lead to reduction in hepatosplenomegaly, reduced the egg burden in faeces, and decreased granuloma diameter. Scanning electron microscopy revealed morphological changes to the parasite tegument after treatment, including the loss of important features. Additionally, the in vivo treatment against juvenile with 40 mg/kg showed a reduction of the total worm burden of 50.2%. Histopathological studies were performed on liver, spleen, lung, kidney and brain and EPI was shown to have a DL50 of 8000 mg/kg. Therefore EPI shows potential to be used in schistosomiasis treatment. This is the first time that schistosomicidal in vivo activity of EPI has been reported

Epiisopilosine alkaloid has activity against schistosoma mansoni in mice without acute toxicity

CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSchistosomiasis is a disease caused by parasites of the genus Schistosoma, currently affecting more than 200 million people. Among the various species of this parasite that infect humans, S. mansoni is the most common. Pharmacological treatment is limited135119CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP404134/2012-22014/02282-76, 2016/18023-5, 2016/22488-3The authors are grateful to Phytobios Pesquisa Desenvolvimento e Inovação LTDA.,company of the Centroflora Group, for its support during the realization of this research. SMA is grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico

Single-cell transcriptomics reveals common epithelial response patterns in human acute kidney injury

BACKGROUND: Acute kidney injury (AKI) occurs frequently in critically ill patients and is associated with adverse outcomes. Cellular mechanisms underlying AKI and kidney cell responses to injury remain incompletely understood. METHODS: We performed single-nuclei transcriptomics, bulk transcriptomics, molecular imaging studies, and conventional histology on kidney tissues from 8 individuals with severe AKI (stage 2 or 3 according to Kidney Disease: Improving Global Outcomes (KDIGO) criteria). Specimens were obtained within 1-2 h after individuals had succumbed to critical illness associated with respiratory infections, with 4 of 8 individuals diagnosed with COVID-19. Control kidney tissues were obtained post-mortem or after nephrectomy from individuals without AKI. RESULTS: High-depth single cell-resolved gene expression data of human kidneys affected by AKI revealed enrichment of novel injury-associated cell states within the major cell types of the tubular epithelium, in particular in proximal tubules, thick ascending limbs, and distal convoluted tubules. Four distinct, hierarchically interconnected injured cell states were distinguishable and characterized by transcriptome patterns associated with oxidative stress, hypoxia, interferon response, and epithelial-to-mesenchymal transition, respectively. Transcriptome differences between individuals with AKI were driven primarily by the cell type-specific abundance of these four injury subtypes rather than by private molecular responses. AKI-associated changes in gene expression between individuals with and without COVID-19 were similar. CONCLUSIONS: The study provides an extensive resource of the cell type-specific transcriptomic responses associated with critical illness-associated AKI in humans, highlighting recurrent disease-associated signatures and inter-individual heterogeneity. Personalized molecular disease assessment in human AKI may foster the development of tailored therapies

Industrial scale isolation, structural and spectroscopic characterization of epiisopiloturine from Pilocarpus microphyllus stapf leaves: a promising alkaloid against schistosomiasis

This paper presents an industrial scale process for extraction, purification, and isolation of epiisopiloturine (EPI) (2(3H)- Furanone,dihydro-3-(hydroxyphenylmethyl)-4-[(1-methyl-1H-imidazol-4-yl)methyl]-, [3S-[3a(R*),4b]]), which is an alkaloid from jaborandi leaves (Pilocarpus microphyllus Stapf). Additionally for the first time a set of structural and spectroscopic techniques were used to characterize this alkaloid. EPI has shown schistomicidal activity against adults and young forms, as well as the reduction of the egg laying adult worms and low toxicity to mammalian cells (in vitro). At first, the extraction of EPI was done with toluene and methylene chloride to obtain a solution that was alkalinized with ammonium carbonate. The remaining solution was treated in sequence by acidification, filtration and alkalinization. These industrial procedures are necessary in order to remove impurities and subsequent application of the high performance liquid chromatography (HPLC). The HPLC was employed also to remove other alkaloids, to obtain EPI purity higher than 98%. The viability of the method was confirmed through HPLC and electrospray mass spectrometry, that yielded a pseudo molecular ion of m/z equal to 287.1 Da. EPI structure was characterized by single crystal X-ray diffraction (XRD), 1H and 13C nuclear magnetic resonance (NMR) in deuterated methanol/chloroform solution, vibrational spectroscopy and mass coupled thermal analyses. EPI molecule presents a parallel alignment of the benzene and the methyl imidazol ring separated by an interplanar spacing of 3.758 Å indicating a π-π bond interaction. The imidazole alkaloid melts at 225°C and decomposes above 230°C under air. EPI structure was used in theoretical Density Functional Theory calculations, considering the single crystal XRD data in order to simulate the NMR, infrared and Raman spectra of the molecule, and performs the signals attribution