bluuebunny/crossref_metadata_2025_split huggingface dataset:
MO014DANHONG INJECTION INHIBITS LIPOPOLYSACCHARIDE-ENHANCED CELL PROLIFERATION OF RAT RENAL MESANGIAL CELLS VIA NF-ΚB SIGNALING PATHWAY
Authors: Hua Liu , Chen Lei , Wei Limin , Jiang Hongli 
Published: May 2021

Abstract

Background and Aims
Mesangioproliferative glomerulonephritis (MsPGN) is well known as one of the leading causes of end-stage renal failure (ESRD), especially in the Asian population. The principal characteristic of MsPGN is the over proliferation of glomerular mesangial cells (MCs) accompanied by expansion of the extracellular matrix (ECM). Our previous studies demonstrated that Danhong injection (DHI), is a traditional Chinese medicine injection, could improve the renal function and inhibit the MCs proliferation and ECM expansion in rats with MsPGN. However, the molecular mechanisms have not been fully elucidated. To explore the potential mechanisms of DHI in the treatment of MsPGN, we investigated the effects of DHI on lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation and its downstream inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1), inducible nitric oxide synthase (iNOS) and fibronectin (FN) protein expression in rat MCs.

Method
The rat MCs treated with different concentrations of DHI (0, 50, 100, 200, 500, 1000, and 2000 uL/L) for 12 h, then incubated with or without 100 ng/ml LPS for another 24 h. Subsequently, cell proliferation was determined by Cell Counting Kit-8 (CCK8). Furthermore, the rat MCs treated with low-dose DHI (250 uL/L), median-dose DHI (500 uL/L) and high-dose DHI (1000 uL/L) for 12 h or Ammonium pyrrolidine dithiocarbamate (PDTC) for 30 min before 24h treatment of LPS. Then the activation of NF-κB was detected by Western blot and immunofluorescence. The protein levels of ICAM-1, TGF-β1, iNOS and FN in rat MCs were detected by Western blot.

Results
The results of CCK-8 revealed that DHI significantly suppressed LPS-induced cell proliferation (shown in Fig.1). LPS stimulation resulted in a significant increment of p65 contents in nucleus and a decrement of p65 contents in cytoplasm in rat MCs compared with NC. PDTC and DHI exerted potent inhibitory effect on increasing expression of p65 in nucleus and decreasing in cytoplasm compared with LPS treatment group. The inhibitory effect on NF-κB nuclear translocation of DHI was in a dose- dependent manner (shown in Fig.2). The Western blot assay showed that the protein level of IκB-α in cytoplasm treated by LPS decreased significantly compared with that in control (shown in Fig. 3) and this decrement was significantly reversed by PDTC and DHI. In addition, the protein expression of ICAM-1, TGF-β1, iNOS and FN was also inhibited by PDTC and DHI (shown in Fig. 4).

Conclusion
DHI significantly repressed LPS-induced cell proliferation and FN expression in rat MCs through inhibiting the activation of NF-κB signaling pathway and protein expression of its downstream inflammatory mediators. The ameliorative effects of DHI on MsPGN might be associated with this inhibition effect on NF-B signaling pathway.
MO015ANALYZING THE EFFECT OF MICROPLASTIC PARTICLES ON HUMAN PODOCYTES
Authors: Yarbakht Melina , Sarau George , Kling Lasse , Müller-Deile Janina , Kotb Ahmed , Christiansen Silke , Schiffer Mario 
Published: May 2021

Abstract

Background and Aims
Despite increasing use of plastic based products and their potential health risks on the humans, very little is known about their possible accumulation in the food chain and their further long-term effect on the human health. Recently, there are increasing reports related to the potential risk of polystyrene microplastics to the human respiratory system and human intestinal epithelia cell line. In this study, we assayed the primary effect of microplastic particles on the human kidney cells. To that aim, we used human podocytes cells and four different types of plastic particles including; polyvinyl chloride (PVC), polypropylene (PP), polyamide (PA) and tyre wear particles to evaluate the effects of microplastics on the viability and morphology of human podocytes in vitro.

Method
In this study, we applied different biological methods such as, cell viability test and phalloidin staining, to assay the toxicity of particles and their further effects on the actin cytoskeleton organization in human podocytes, respectively. Furthermore, Raman imaging is used to track particle attachment on the cells and to evaluate the possible changes in the cell compartment following the particle treatment. The particle uptake by the cells and changes in cellular biological features were visualized with the use of scanning electron microscopy (SEM).

Results
As a primary result, the cytotoxicity response of particle treatment was found to be dependent on the polymer type. As an example higher concentration of PP particle as compared to PVC, PA, and tyre wear caused a similar rate of cell mortality. Furthermore, the degree of particle attachment on the cells depended on their adhesion properties, which was higher in PA, PVC and tyre wear in comparison to PP particles. These particles remained attached to the cell surface even after two-three times of washing with PBS. Based on the phalloidin staining results, particle treatment induced cytoskeleton reorganization in podocytes in vitro. With the use of Raman imaging particle attachment was confirmed based on the fingerprint spectra related to each particle.

Conclusion
This study suggests that exposure duration and particle concentrations are two of the key factors to evaluate the toxicological effect of particles on podocytes as a highly-specialized epithelial cells in the kidney. It is supposed that two mechanisms can be related to the harmful effects of plastic particles on podocytes. First, particle attachment on the cell surface leading to limitation of nutrient uptake by the cells. Second, uptake of smaller size particles into the cells through phagocytosis. More studies are necessary to determine the direct effect of microplastics on human kidney cells.
MO018QUORUM SENSING MOLECULE INDUCES THE RECEPTOR-INTERACTING PROTEIN KINASE 1-DEPENDENT APOPTOSIS SYNERGISTICALLY WITH LIPOPOLYSACCHARIDE IN ENDOTHELIAL CELLS
Authors: Shin Jungho , Ahn Sun Hee , Oh Dong-Jin 
Published: May 2021

Abstract

Background and Aims
Endothelial dysfunction is an initial step for sepsis-associated organ failure. Bacterial quorum sensing molecules play as pathogen-associated molecular patterns, however, the impact of quorum sensing molecules on endothelial cells remains uncertain. This study investigated the molecular mechanism of quorum sensing molecule-induced cell death and the interaction with lipopolysaccharide (LPS) in human umbilical vein endothelial cells.

Method
Endothelial cells were treated with varying concentrations of N-3-oxododecanoyl-HSL (3OC12-HSL) and LPS derived from Pseudomonas aeruginosa.

Results
Treatment of 3OC12-HSL reduced cell viability in dose-dependent manners, and the combination of this molecule and LPS exacerbated cell deaths in compared to single treatment of LPS. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay revealed that there were clear increases in apoptotic death following the 3OC12-HSL treatment. Treatment of 3CO12-HSL activated the receptor-interacting protein kinase 1 (RIPK1) pathway, resulting in increases in expression of caspase 8 and caspase 3. Moreover, the combination treatment of LPS and 3OC12-HSL amplified these expressions.

Conclusion
Treatment of 3OC12-HSL reduces endothelial cell viability, and this effect is synergistical with LPS. 3OC12-HSL induces apoptotic cell death, through the activation of the RIPK1 pathway, moreover, it is amplified in combination with LPS.
MO007ANALYSIS OF CALCIUM SIGNALING IN AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE (ADPKD)
Authors: Van Giel Dorien , Decuypere Jean-Paul , Mekahli Djalila , Vennekens Rudi 
Published: May 2021

Abstract

Background and Aims
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inheritable kidney disease characterized by the development of fluid-filled cysts in all nephron segments, leading to loss of renal function. Mutations in PKD1 or PKD2, which encode polycystin-1 and polycystin-2, are the most common cause of ADPKD. The molecular mechanisms underlying cystogenesis are poorly characterized but it is postulated that disturbed calcium homeostasis is a primary event in cystogenesis. The precise molecular players that cause this disturbance are still a poorly explored area, especially in relevant human cell types. We therefore aim to characterize the profile of calcium-coupled receptors and channels in a human renal epithelial cell model, to identify which receptors and channels are present and whether their function is affected in ADPKD.

Method
Human urine-derived conditionally immortalized proximal tubule epithelial cells (ciPTECs) of ADPKD patients and healthy controls were screened for calcium-coupled GPCRs, using a GPCR agonist library on Fura-2 loaded cell populations seeded in 96-well format using the Flexstation3 (Molecular Devices). Validation of specific hits was done using single-cell measurements with a fluorescence microscope and built-in perfusion system. The expression of TRP channels and STIM/Orai proteins was determined via qPCR.

Results
From a library of 418 GPCR agonists a selective amount of calcium-coupled GPCRs was found functionally active in ciPTECs. ciPTECs from both healthy controls and ADPKD patients were found to functionally express purinergic -, histamine -, serotonin and dopamine receptors. Through qPCR we found expression of various TRP channels, including TRPML1, TRPC1/3, TRPM3/4/7, TRPV4 and TRPA1, as well as high expression of STIM1/2 and Orai1/2/3.

Conclusion
We describe the first thorough characterization of molecular players involved in calcium signalling mechanisms in human renal epithelial cells, including the profile of calcium-coupled GPCRs and the expression of TRP channels and STIM/Orai proteins, of further interest to investigate disturbed calcium dynamics in ADPKD.
MO012ACE2 AND SARS-COV-2 INFECTION RISK: INSIGHTS FROM PATIENTS WITH TWO RARE GENETIC TUBULOPATHIES, GITELMAN‘S AND BARTTER’S SYNDROMES
Authors: Bertoldi Giovanni , Rigato Matteo , Sgarabotto Luca , Gianesello Lisa , Ravarotto Verdiana , Davis Paul A , Calò Lorenzo 
Published: May 2021

Abstract

Background and Aims
COVID-19 is spreading globally with Angiotensin Converting Enzyme (ACE)-2 serving as the entry point of SARS-CoV-2 virus. This raised concerns how ACE2 and Renin-Angiotensin (Ang)-System (RAS) are to be dealt with given their involvement in COVID-19’s morbidity and mortality. Specifically, increased ACE2 expression in response to treatment with ACE inhibitors (ACEi) and Ang II receptor blockers (ARBs) might theoretically increase COVID-19 risk by increasing SARS-CoV-2 binding sites. However, ACE2 is part of the protective counter-regulatory ACE2-Ang1-7-MasR axis, which opposes the classical ACE-AngII-AT1R regulatory axis. We used Gitelman’s and Bartter’s syndromes (GS/BS) patients, rare genetic tubulopathies, who have endogenously increased levels of ACE2, to provide more insight on these issues.

Method
128 genetically confirmed GS/BS patients, living in Lombardia, Emilia Romagna and Veneto, the Northern Italy hot spots for COVID-19, were surveyed via telephone survey regarding COVID-19.

Results
The survey found no COVID-19 infection and absence of COVID-19 symptoms in any patient. Comparison analysis with the prevalence of COVID-19 in those Regions [8.96% (95% CI 8.96-8.99% vs 0.00% (95% IC 0.00-3.62%)] showed statistical significance (p&lt;0.01).

Conclusion
The results of the study contribute to suggest that increased ACE2 does not increase risk of COVID-19 and that ACEi and ARBs by blocking excessive AT1R-mediated Ang II activation, might favour the increase of ACE2-derived Ang 1-7. The GS/BS patients’ increased ACE2 and Ang 1-7 levels and their characteristic chronic metabolic alkalosis might suggest for SARS-COV-2 a mechanism similar to that of chloroquine/hydroxychloroquine effect altering ACE2 glycosylation which resulted, in previous studies, in SARS-COV binding inhibition and block/inhibition of viral entry. Studies from our laboratory are ongoing to explore in GS/BS ACE2 glycosylation.
MO029CLINICAL CHARACTERISTICS AND PATHOGENIC GENES OF CONGENITAL SOLITARY KIDNEY WITH REPRODUCTIVE SYSTEM MALFORMATION*
Authors: HU Rongrong , Xu Lubin , Chen Minting , Chen Na , Ma Tiantian , Ji Peili , Liu Zhenzhen , Zhu Lan , Chen Limeng 
Published: May 2021

Abstract

Background and Aims
Congenital solitary kidney – one category of congenital anomalies of the kidney and urinary tract (CAKUT) may combine with other system malformations such as reproductive, cardiac, skeletal system, and so on. Our study analysed the clinical characteristics among congenital solitary kidney patients and their reproductive system malformations. And further work about probable pathogenic genes was explored.

Method
The information of CAKUT patients who were indicated by Doppler ultrasound was collected. The clinical and imaging features including reproductive system abnormalities were retrospectively reviewed in patients with congenital solitary kidney. In patients with Mayer-Rokitansky- Küster-Hauser (MRKH) syndrome, a disorder of congenital agenesis of uterus and vagina, whole exome sequencing was performed. Rare variants in CAKUT-related genes were analysed. Trio analysis was conducted to identify de novo mutations.

Results
We identified 209 patients with congenital solitary kidney from July 20, 2017 to July 19, 2020 among 1160 CAKUT patients in Peking Union Medical College Hospital. There were 152 females. The average age of congenital solitary kidney patients was 35.26±18.42 years when they were diagnosed. 53.2% showed different degrees of proteinuria and hematuria. Serum creatinine elevating was proved 13.1% and 40% in women and men separately. Among 81 females who also had a gynecological ultrasound report, 88.9% combined with genital malformation, oblique vaginal septum syndrome 48.7%, the Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome 22.2%, malformed uterus 23.5%, vaginal atresia and other genital malformation 7%. Congenital heart disease, complete transposition of viscera, and scoliosis were also found in some patients with congenital solitary kidney. Furthermore, based on the whole exome data of 443 patients with MRKH syndrome, seven function-lost mutations were confirmed. And also two De nove mutations (NOTCH2 (NM_024408.3: c.703A&gt;T(p.Thr235Ser), ESRRG (NM_001243512.1:c.-169-8delT)), one homozygous patients with parents heterozygous (NM_133433.3:c.8084C&gt;T(p.Thr2695Me) were identified as possible pathogenic genes caused CAKUT.

Conclusion
We should be aware of reproductive system malformations in CAKUT patients. Whole exome sequencing may suggest common pathogenic genes between the two kinds of diseases.
MO042PREVALENCE OF FABRY DISEASE CAUSING VARIANTS IN THE UK BIOBANK
Authors: Gilchrist Mark , Casanova Francesco , Tyrrell Jessica , Fife Nicole , Young Katie , Oram Richard , Weedon Michael 
Published: May 2021

Abstract

Background and Aims
Fabry disease (FD) is an X-linked lysosomal storage disorder resulting from deficiency of the alpha-galactosidase A enzyme. This leads to the accumulation of globotriaosylceramide in multiple organ sites with prominent renal involvement, cardio and cerebrovascular disease, as well as neurological, dermatological, and gastrointestinal pathology in affected individuals. Clinical manifestations are highly heterogeneous with considerable variation depending on the specific variant and even within families with the same variant. Prevalence estimates from studies of case control cohorts range from 1 in 40,000 to 1 in 140,000. We sought to determine the prevalence of Fabry disease causing mutations in an unbiased adult population sample using the UK Biobank.

Method
UK Biobank comprises approximately 500,000 participants with extensive phenotyping and genetic data linked to clinical care records. Exome sequencing data from 200643 individuals from the UK Biobank were examined for variants in the GLA gene. Likely mutations were searched for in ClinVar and HGMD with subsequent review of supporting literature. We used ACMG guidelines to classify the pathogenicity of variants.

Results
Eighty one coding variants were identified in GLA. Nine of these variants were rare (&lt;1 in 10,000 individuals) and either protein truncating variants or previously reported to cause Fabry’s disease. Thirty seven individuals, (14 males, 22 females, 1 missing data) carried one of these GLA variants. The most common variant was the cardiac predominant phenotype causing N215S variant (18/37). 6/37 had the R363C variant, 4/37 had R356Q. The R112H, R301Q, I198T variants were each identified in 2 individuals. There were single individuals with I232T, K240fs, and E66G variants.
Two male individuals (with I232T, K240fs variants) had HES codes compatible with a known diagnosis of Fabry disease. The remaining 12/14 males did not have evidence of an existing diagnosis of Fabry disease. No female participants had HES codes indicating an existing diagnosis of Fabry disease.
The overall prevalence of Fabry disease causing variants in the UK Biobank is 1 in 5,422. The N215 variant alone has a prevalence of 1 in 11,147.

Conclusion
Reported Fabry disease causing GLA variants are far more prevalent in an unselected population sample than would be expected from the reported prevalence of Fabry disease. This may be because some of the reported variants are not pathogenic, or are predominantly later-onset causing variants with reduced penetrance. The true prevalence of Fabry disease may be substantially higher than current estimates.
MO045THE APPLICATION OF A NGS KIDNEY PANEL REVEALED KEY CHALLENGES OF PKD1-2 ANALYSIS: INTERPRETATION OF MISSENSE VARIANTS, SIGNIFICANCE OF VARIANTS IN DUPLICATED REGIONS AND HIGH ALLELIC HETEROGENEITY
Authors: Blasio Giancarlo , Zacchia Miriam , Del Vecchio Blanco Francesca , Capolongo Giovanna , Perna Alessandra , Nigro Vincenzo , Capasso Giovambattista 
Published: May 2021

Abstract

Background and Aims
Genetic testing has changed the clinical management of inherited kidney diseases patients, improving prognosis, surveillance and therapy. On the other hand, it has put geneticists and clinicians in front of new challenges, as the heterogeneity of these disorders and the high number of variants, with no clear genotype-phenotype correlation.

Method
108 patients underwent genetic analysis through a kidney focused NGS panel, named Nephroplex, containing 119 genetic loci associated with inherited kidney disorders. The study aimed to addressed the genetic landscape of cystic individuals and to analyze PKD1 and PKD2 variants in non-cystic individuals.

Results
Following diagnostic criteria, patients were divided as cystic kidney diseases (n=36) and non-cystic kidney diseases (n=72). Among the group of cystic patients, a causative mutation was detected in 51% of cases. We found thirty-seven PKD1 and PKD2 variants in 26 out of 35 individuals. In particular, 12 variants were shown to be damaging and nine of that were reported in public database, as CLINVAR and Mayo Clinic databases. Among pathogenic variants, twelve were truncating and the remaining were missense variants. Of note, 7 out of 12 damaging PKD1 mutations were located in duplicated regions. Moreover, in three cystic patients, we found a (i) a frameshift hemizygote OFD1 mutation (ii) compound heterozygote PKHD1 variants and (iii) a frameshift MUC1 variant, framing the diagnosis of oro-facio-digital type 1, autosomal recessive polycystic kidney disease and autosomal dominant tubulointerstitial disease, respectively. Interestingly, we detected 28 PKD1-2 rare variants in 21 out of 75 adult non cystic patients (28%). The most were observed in PKD1 genes (82% vs 18% in PKD2). Eighteen of 28 variants were described in the literature as likely benign or as mutations of uncertain significance, while we found 10 novel variants. In silico analysis revealed as pathogenic a frameshift mutation located in exon 15. Of note, the great part of these variations reside into the duplicated PKD1 regions.

Conclusion
Our data showed that genetic analysis of ADPKD retains unique challenges, given the high degree of homology of PKD1 with his pseudogenes and the high allelic heterogeneity in non-cystic individuals.
MO049FUNCTIONAL IMPORTANCE OF MAPKBP1 PROTEIN DOMAINS IN NEPHRONOPHTHISIS
Authors: Hartig Christin , Schönauer Ria , Sewerin Sebastian , Ertel Anastasia , Jin Wenjun , Hantmann Elena , Saunier Sophie , Benmerah Alexandre , Halbritter Jan 
Published: May 2021

Abstract

Background and Aims
Nephronophthisis is an autosomal-recessive kidney disease that accounts for a significant proportion of end-stage renal disease (ESRD) in childhood, adolescence and early adulthood. Biallelic pathogenic variants in MAPKBP1, encoding the c-Jun N-terminale kinase (JNK)-binding protein 1, are associated with development of Nephronophthisis and subsequent chronic kidney disease (CKD) (Macia et al, AJHG, 2017). We recently characterized MAPKBP1 as microtubule-associated protein that is able to localize to centrioles and the base of primary cilia depending on dimerization via its C-terminal coiled-coil domain (Schönauer et al, Kidney Int, 2020). However, the physiological function of its N-terminal WD40 and intermediate JNK-binding domain is still poorly understood. By in vitro comparison of artificial domain deletions with known and novel patient variants, we aim at pinpointing functional consequences of pathogenic MAPKBP1 in cilia and cell cycle control.

Method
N-terminally GFP-tagged MAPKBP1 constructs with either full-domain deletions or patient-derived variants were expressed in non-ciliated HeLa and ciliated H69 cells for fluorescence microscopy studies. Furthermore, RNA-seq analysis using primary patient cells was conducted to investigate differentially regulated molecular pathways compared to healthy control individuals.

Results
Immunofluorescence microscopy revealed inappropriate intracellular localization upon single or combined deletion of any MAPKBP1 protein domain. Compared to wild type, all deletion variants showed reduced intensity at the centrosome and ciliary base. Despite preserved dimerization ability, loss of the intermediate JNK-binding domain (JBD) most effectively abolished centrosomal or ciliary targeting, whereas loss of the N-terminal WD40-domain induced strongest mitotic aberrations. Unlike wild type, both, artificial and patient-derived truncating variants were able to enter the nucleus. RNA-seq analysis using primary patient fibroblasts with varying C-terminal truncations will allow important insights into common gene expression profiles unveiling consequences of aberrant intracellular trafficking.

Conclusion
In the present work, we demonstrate that all protein domains are indispensable for appropriate MAPKBP1 intracellular localization and function. Most of clinically reported patient variants exhibiting C-terminal truncation of varying lengths resulted in comparable intracellular behavior in presence of an intact N-terminal WD40 domain. Surprisingly, deletion of the JNK-binding domain alone aggravated functional disturbances hinting at a prominent regulatory role of this protein part interdepending with dimerization. Further insights into domain-specific functions will explain molecular disease mechanisms of MAPKBP1.