bluuebunny/crossref_metadata_2025_split huggingface dataset:
#1621 Quantifying ureter smooth muscle electrophysiology from calcium transient images to understand abnormal peristaltic contraction
Authors: Mahapatra Chitaranjan , Pradhan Ashish 
Published: May 2024

Abstract

Background and Aims
Abnormal peristaltic contraction of the ureter smooth muscle (USM) causes acute kidney stone episodes. Intracellular electrical activities like membrane depolarization and action potentials play important roles in modulating the USM contraction by releasing intracellular calcium from the sarcoplasmic reticulum. Therefore, an electrophysiological study will help to assess the USM cell's electrical activities and in diagnosing abnormal USM contraction. The objective of this study is to quantify the contribution of ionic currents in shaping experimental calcium transient profiles using in-silico electrophysiological modeling.

Method
The simultaneous experimental recording of action potential (AP) and intracellular calcium transient images from the mouse ureter is obtained. The single isolated USM cell model comprises several voltage-gated ion channels, such as two voltage-gated calcium (T—type, and L—type) channels, one voltage-gated fast potassium (KA) channel, one calcium-dependent large conductance potassium channel, and an HCN channel. To describe the calcium-dependent gating of Ca2+-dependent potassium channels and to update the equilibrium potential of the Ca2+ ion, the intracellular Ca2+ concentration is updated during the simulation period.

Results
Simulation of simultaneous recordings of AP and cytosolic calcium [Ca2+]i are done on a single isolated cell. The model shows [Ca2+]i as a function of synaptic input-induced AP to simulate extracted experimental data, where Ca2+ transient is recorded simultaneously during AP in mouse USM cells. Fig. 1 shows both experimental and simulation of AP (A) and Calcium transient (B) in the USM cell. In our model, the radius “r” and time constant τ of the shell influence the Ca2+ transient profile. In the USM cell model, the submembrane calcium transient occurs from a depth of 0.1 μm to a depth of 0.6 μm. We have investigated whether Ca2+ current via the L-type Ca2+ channel is responsible for the firing of APs with fast upstroke generation. The AP and calcium transients are demolished with the absence of the L-type Ca2+ channel.

Conclusion
From this study, it is found that inhibition of the L-type Ca2+ channel not only prevented AP generation, it also reduced the cytosolic Ca2+ transient. This study supports the application of L-type Ca2+ channel inhibitor as a potential drug for abnormal peristaltic contraction of the ureter smooth muscle.
#391 Calpain inhibition enhances autophagy-lysosomal pathway and ameliorates tubulointerstitial fibrosis in Nphp1-targeted models
Authors: LI Dantong , Zhang Jinglan , Ye Zhiming , Sun Liangzhong 
Published: May 2024

Abstract

Background and Aims
Nephronophthisis (NPH) belongs to a group of renal ciliopathies and is the most common genetic disease leading to renal failure in children. Mutations in Nphp1 account for the majority of NPH cases, and renal tubulointerstitial fibrosis is one of the most important pathological features. However, the molecular mechanisms of renal tubulointerstitial fibrosis remained unknown.

Method
Previously, we successfully constructed an Nphp1 knockout mice model with renal phenotype of tubulointerstitial fibrosis by CRISPR-Cas9. In this study, we used lentiviral shRNA vectors to construct Nphp1 knockdown HK2 cells. We carried out RNA sequencing in Nphp1-targeted cells and mice kidneys to explore the molecular mechanisms of renal tubulointerstitial fibrosis in NPH. Calpain (CAPN) was found to be upregulated in both models. Western blot and immunofluorescence (IF) were used to verify the expression of CAPN in Nphp1-targeted HK2 cells and mice kidneys, as well as their respective controls. We also detected autophagy activities in NPH models by western bolt, immunofluorescence, lysotracker and transmission electron microscopy (TEM). Epithelial or mesenchymal-specific markers and Masson's trichrome staining were used to detect the status of tubulointerstitial fibrosis. Furthermore, Nphp1-targeted HK2 cells and mice were treated with a CAPN inhibitor to explore the role of CAPN in autophagy-lysosomal pathway and tubulointerstitial fibrosis.

Results
CAPN was verified to be upregulated in both Nphp1-targeted HK2 cells and mice kidneys. We also discovered impaired autophagic flux in NPH models. Furthermore, the impaired autophagic flux induced tubulointerstitial fibrosis in NPH models. However, the autophagic flux was improved and the status of tubulointerstitial fibrosis was ameliorated with CAPN inhibition.

Conclusion
CAPN-mediated autolysosomal pathway disorder may be an important cause of tubulointerstitial fibrosis in NPH. CAPN may be a new target for diagnosis and treatment of NPH.
#2804 Mechanisms of cAMP-induced cyst formation observed in Pkhd1-KO cells
Authors: Ziegler Wolfgang H , Hassan Fatima , Hahnenstein Susanne , Haffner Dieter 
Published: May 2024

Abstract

Background and Aims
Loss of ciliary protein function leads to altered epithelial properties in hereditary polycystic kidney diseases. To address molecular aspects of defective epithelial homeostasis, monolayered epithelial spheroids can be used to analyze lack of protein function and consequences of pharmacological intervention. Here, we employed epithelial cell clones deficient for fibrocystin/polyductin (FPC) protein function, the origin of ARPKD, to study the impact of FPC cytoplasmic domain constructs on cAMP-induced cystogenesis.

Method
We used pl-MDCK, sub-cloned principal-like cell lines, with CRISPR/Cas9-based genetic knockout (KO) of Pkhd1 / FPC, and corresponding controls. Cells were grown in matrigel to allow formation of epithelial spheroids within 3 days. Forskolin (Fsk) treatment was employed to induce cAMP-mediated cyst growth mimicking disease conditions. Measurement of proportional lumen, the ratio of lumen to spheroid area, allowed detection the enhanced water and ion transport across the barrier as characteristic for secretory epithelia. Cystogenic signals were modulated by expression of the FPC C-terminal domain constructs and/or pharmacological inhibitors, and involvement of ion channels was addressed.

Results
In Pkhd1-KO cell lines, enhanced cAMP levels resulted in massive lumen expansion of epithelial spheroids with no increase in cell number. The rise in proportional lumen was sensitive to inhibition of chloride-channels, CFTR and TMEM16A, and Src kinase. Fsk-mediated cyst induction led to activation and increased Y705 phosphorylation of STAT3. To address the impact of FPC on cystogenic signaling, expression of a membrane-bound FPC C-terminal protein domain was studied, and its processing and intracellular localization determined. Controlled expression of the FPC cytoplasmic domain in Pkhd1-KO cell lines suppressed Fsk-induced cyst formation. In addition, the FPC domain reduced activation of Src and STAT3, leading to lower STAT3-dependent transcription.

Conclusion
In FPC-deficient pl-MDCK cells, cyst formation stimulated by high cAMP levels is associated with enhanced Src/STAT3 signaling. Expression of the FPC cytoplasmic domain, leading to a gain-of-function, suppresses Src and STAT3 activity in Pkhd1-KO epithelia and limits cyst growth in vitro. Control of cystogenic Src/STAT3 signaling can be considered a physiologic function of the FPC protein.
#1389 Identification of serum lipidomic profile in nephrotic syndrome patients
Authors: Rother Nils , Rood Ilse , Bekkering Siroon , Riksen Niels , Hilbrands Luuk , Maas Rutger , Nijenhuis Tom , Duivenvoorden Raphaël 
Published: May 2024

Abstract

Background and Aims
Nephrotic syndrome describes a collection of symptoms, mainly heavy proteinuria leading to kidney function loss, that can be caused by different underlying kidney diseases. Common primary causes of nephrotic syndrome include focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD) and membranous nephropathy (MN). Nephrotic syndrome is often characterized by plasma lipid abnormalities with elevations in cholesterol, triglycerides and lipoproteins. Here we set out to generate a detailed lipidomic analysis of FSGS, MCD and MN patient's plasma to find lipid changes common to nephrotic syndrome or specific for the underlying disease.

Method
For this lipidomic study, we included patients with primary FSGS (2 cohorts with n = 5), genetic FSGS (n = 5), MCD (n = 5) and MN (n = 5) in whom serum had been collected at the time they presented at our center with active nephrotic syndrome. We compared the serum lipidome of nephrotic syndrome patients with age matched healthy controls (n = 5), and with age matched patients with familial hypercholesterolemia (n = 5) who, like patients with nephrotic syndrome, have significantly elevated lipid values. Serum was analyzed using the Shotgun Lipidomics platform by Lipotype GmbH (Dresden, Germany), as previously described [1].

Results
The lipidomic profile of patients with nephrotic syndrome was markedly different compared to control groups (Fig. 1A). In particular, lower levels of lyso-phophatidylcholine (LPC) and higher concentrations of phosphatidylethanolamine (PE) and lyso-PE (LPE) compared to control groups were most prominent (Fig. 1B). Subspecies analysis identified LPC 16:0;0 to be highly downregulated in patients with nephrotic syndrome (Fig. 2A), whereas LPE 18:0;0 and nearly all PE subspecies were increased compared to control groups (Fig. 2B and C). The lipidomic profile was largely comparable between different underlying diseases and we could not identify lipid classes specifically different for certain diseases.

Conclusion
Our lipidomic data show that patients with nephrotic syndrome have a distinct lipid profile compared to healthy controls or other disease controls. We identified LPCs, PEs and LPEs to be changed in nephrotic syndrome. We could not identify lipids specific for certain diseases underlying nephrotic syndrome, but rather identified a general nephrotic syndrome lipid profile. This suggests that lipid changes are rather a consequence of disease manifestation in these patients with various underlying causes. Further research should focus on confirming this lipid profile in larger cohorts and unraveling the mechanisms leading to the observed lipid changes.
#896 Long-term renal outcome in childhood-onset lupus nephritis
Authors: Odone Ludovica , Peyronel Francesco , Calatroni Marta , Pala Chiara , Errichiello Carmela , Doti Federico , Delsante Marco , Conte Emanuele , Emmi Giacomo , Rossi Giovanni Maria , Moroni Gabriella Luisa , Vaglio Augusto 
Published: May 2024

Abstract

Background and Aims
Lupus nephritis (LN) is a common and severe complication of childhood-onset SLE. Little is known about the long-term renal outcome of childhood-onset LN. We investigated predictive factors of end-stage kidney disease (ESKD) and chronic kidney disease (CKD) in a paediatric cohort of patients with LN.

Method
This is a retrospective multicentre study including patients with childhood-onset (&lt;18 years) biopsy-proven LN. Clinical features were analysed at disease onset, time of kidney biopsy, 12 and 24 months after biopsy, and last follow-up. We investigated the prognostic significance of clinical, laboratory and histologic characteristics on the incidence of ESKD and CKD stage 3-5.

Results
We included 73 patients with a median age of 14 years (IQR 11.5-17) . At the time of kidney biopsy, most subjects displayed an aggressive disease: 2 (3%) children required haemodialysis, 27 (38%) had an estimated glomerular filtration rate (eGFR) &lt;60 mL/min/1.73 m2. The overall median eGFR was 70 mL/min/1.73 m2 (IQR 43-96) . Moreover, the median proteinuria was 4 g/24 h (IQR 1.35-7.41) , with 41 (59%) children displaying a nephrotic range proteinuria (i.e., &gt;3 g/24 h) . The median follow-up was 13.3 years (IQR 4.7-25.4) . A total of 10 patients (13.7%) reached ESKD, the majority of whom (12%) within 10 years from kidney biopsy; during the subsequent follow-up the incidence of ESKD stabilised (Fig. 1). At last follow-up, around 50% of patients displayed an eGFR &lt;90 mL/min/1.73 m2. A younger age at LN onset, a lower eGFR and central nervous system involvement at the time of kidney biopsy were identified as predictors of ESKD by a univariable Cox regression model (Table 1). The same features were significantly associated with the occurrence of CKD stage 3-5 at last follow-up at a univariable logistic regression analysis.

Conclusion
LN often presents with severe kidney function impairment and aggressive systemic involvement in children. In our cohort, whose follow-up was among the longest reported in the literature, 12% of patients reached ESKD within 10 years from kidney biopsy. Significant predictors of poor kidney outcome in the long term were a younger age at LN onset, a lower eGFR and the presence of neurological manifestations at the time of kidney biopsy.
#1793 Association of low serum complement C3 with reduced renal survival in ANCA associated vasculitis
Authors: Khouloud Hafi , Salem Meriem Ben , Mahmoud Nouha Ben , Gana Abir , Letaief Ahmed , Hamouda Mouna , Sabra Aloui , Salah Manel Ben , Skhiri Habib 
Published: May 2024

Abstract

Background and Aims
ANCA-associated vasculitis (AAV) is a condition that remains poorly understood. Recent data suggest the existence of a complement alternative pathway activation in the pathogenesis of this disease. And recent studies have correlated low complement levels to unfavourable outcomes. Our study aimed to determine whether low serum C3 levels could be used to estimate the severity of AAV and predict renal survival.

Method
Our study enrolled 26 patients diagnosed with AAV between January 2013 and January 2023. C3 levels were assessed at the time of diagnosis, and patients were categorized into two groups: those with low serum C3 levels and those with normal serum C3 levels (0.9–1.8 g/l). We examined the correlation between serum C3 levels and the severity of AAV at the time of diagnosis, as well as renal survival.

Results
The average age at diagnosis was 52.5 years, with 15 male patients (57.69%). All patients had kidney involvement, 30% of patients had respiratory symptoms (mostly due to diffuse alveolar hemorrhage) 42% had Ear Nose Throat system involvement 15% had nervous symptoms and 38% had skin signs. The mean Birmingham Vasculitis Activity Score (BVAS) at presentation was 20, and the average serum creatinine level was 360 µmol/l. The mean serum C3 and C4 levels were 108.3 and 24.5 mg/dL, respectively. Forty-two percent of the patients (eleven patients) had a low serum C3 level at presentation. Low C3 level was significantly correlated with poor renal survival (p: 0.031) and relapse after 6 months of immunosuppressive treatment (p: 0.02). Furthermore, remission at 6 months was significantly more frequent in patients with normal C3 levels.

Conclusion
The assessment of C3 level in AAV can be a valuable tool to predict the disease severity. Hypocomplementemia can predict worse renal outcome. This phenotype may confer a poor response to usual immunosuppressive approaches. Corresponding patients may greatly benefit from complement-targeting therapy.
#2056 Epigenetic profiles differ by level of kidney function in idiopathic nephrotic syndrome
Authors: Hayward Samantha , Suderman Matthew , Welsh Gavin , Saleem Moin 
Published: May 2024

Abstract

Background and Aims
DNA methylation (DNAm) is an epigenetic mechanism which can therefore induce changes in gene expression without any change to the underlying DNA sequence. Although generally quite stable, DNAm patterns in whole blood do vary across individuals, with DNAm levels associating with age and responding to environmental factors.
We aimed to investigate whether peripheral blood cell DNAm associates with level of kidney function in patients with Idiopathic Nephrotic Syndrome (INS).

Method
Peripheral blood cell DNAm values were generated using the Illumina MethylationEPIC Beadchip (&gt;850,000 CpG sites) for 293 INS patients recruited to the National Unified Renal Translational Research Enterprise–National Study of INS (NURTuRE- INS and NephroS). All patients were younger than 30 years old at INS diagnosis. An Epigenome Wide Association Study (EWAS) was performed using generalised linear models to evaluate associations between DNAm sites and estimated glomerular filtration rate (eGFR). Regions of differentially methylated sites were identified using the ‘dmrff’ R package. Sex chromosomes were excluded from the analyses and all analyses were adjusted for estimated cell type proportions, age, sex and technical variation. A Bonferroni adjusted p value of 5.88 × 10−8 was used as a significance threshold to adjust for multiple tests.

Results
Of the selected 293 INS patients, 173 (59%) were male, 213 (73%) were white and the median age at INS diagnosis was 4 years old (IQR 2-10). One hundred and forty-three (48%) patients were steroid responsive at diagnosis and the remainder were steroid resistant. Sixty-eight of the steroid resistant patients had pathogenic NS variants.
We identified 6 CpG sites associated with eGFR (p &lt; 5.88 × 10−8) in this cohort of INS patients, one replicating a previously reported association. We identified 3 differentially methylated regions, one coinciding with the transcriptional start site of an RNA binding protein which regulates alternative splicing, and another located in the body of a transcription factor regulator containing genetic variants associated with chronic kidney disease.

Conclusion
We have demonstrated variation in peripheral blood DNAm by level of kidney function in children and young adults with INS. Associations between DNAm and eGFR have previously been demonstrated in diabetic kidney disease and heterogenous chronic kidney disease cohorts, including one of the sites which we have identified in our study. Further work is underway to determine if these differences in DNAm are likely to be a cause or consequence of reduced kidney function, which will help to determine their potential clinical utility.
#1283 Rituximab in addition to plasma exchange, cyclophosphamide and steroids for treatment of anti-GBM disease
Authors: Srikantharajah Mukunthan , Cairns Tom , Pusey Charles , Prendecki Maria , Mcadoo Stephen 
Published: May 2024

Abstract

Background and Aims
Anti-GBM disease is a rare antibody-mediated vasculitis, usually treated with plasma exchange (PEX), cyclophosphamide (CYC) and steroids. There are limited data regarding the use of B-cell depleting therapies such as rituximab (RTX). We investigate the use of the addition of rituximab for the treatment of Anti-GBM disease.

Method
Ten-year (2012-2023) single-centre retrospective cohort study of patients with anti-GBM disease treated with or without rituximab, in addition to standard care, as first-line therapy. A Cox-proportional hazards regression model incorporating age, sex, ANCA status, dialysis need at presentation, lung haemorrhage, CYC (oral or IV) and RTX treatment was used to identify disease/treatment factors associated with outcome (death, end-stage kidney disease [ESKD], infection).

Results
Forty-five patients are included; baseline demographics, disease characteristics, treatments and outcomes are summarised in Table 1.
In the Cox regression model, only increasing age was associated with risk of death (HR 1.08 [95% CI 1.02-1.17]). Age (HR 1.04 [1.01-1.08]), female sex (HR 4.93 [1.80-4.87]), and dialysis need at presentation (HR 5.28 [1.54-17.53]) were associated with ESKD-risk. Increasing age (HR 1.04 [1.00-1.09] and ANCA positivity (HR 3.08 [1.14-8.71]) were associated with risk of infection. The addition of rituximab was not significantly associated with risk of death (HR 0.82 (0.24-2.87), ESKD (HR 1.54 [0.64-3.96]) or infection 2.06 [0.67-5.71].

Conclusion
Addition of rituximab to standard treatment for anti-GBM disease was not associated with improved renal outcome or survival benefit. However, rituximab use permitted lower cumulative doses of CYC and reduction in the number of PEX, without an increased risk of infection. Rituximab may have a role as a CYC- and PEX-sparing treatment in patients with anti-GBM disease.
#1293 Endothelial calcification induced by high-phosphate is characterized by mesenchymal and osteoblastic transdifferentiation
Authors: Ciceri Paola , Artioli Luisa , Martinelli Carla , Falleni Monica , Tosi Delfina , Gianelli Umberto , Cozzolino Mario Gennaro 
Published: May 2024

Abstract

Background and Aims
Chronic kidney disease (CKD) is a prevalent condition that affects millions of people. Patients with CKD have an increased cardiovascular risk, mostly due to vascular calcification (VC). By now, it's quite clear how, in vitro and in vivo, vascular smooth muscle cells (VSMCs) actively participate to VC. In contrast to the wide studies on VSMCs, the role of endothelial cells (ECs) has been poorly investigated. It's known that ECs have a dynamic nature and following particular stimuli they can trans-differentiate and acquire several different phenotypes through the endothelial-to-mesenchymal transition (EndMT).

Methods
Human aortic ECs (HAECs) were challenged, in an in vitro calcification model, with 2, 2.5, 3, 3.5 mM Pi for up to 7 days. Early morphological changes, gene expression and cell migration were evaluated. Afterwards calcium deposition, genes and protein expression and ultrastructural changes were analyzed.

Results
High-Pi, at a concentration of 3 mM, is able to induce EC calcium deposition. Calcification is dependent on calcium influx without influencing Pit-1 expression. During the first days of high-Pi treatment ECs change their morphology from cobblestone to spindle shape acquiring motility, as demonstrated by the 24% increased closure in the scratch wound test. EndMT master genes such as SNAIL and TWIST resulted up-regulated at day 3 together with an increased protein expression of mesenchymal markers such as N-Cadherin, α-SMA, SM22α, FSP-1 and Fibronectin. Starting from day 5 high-Pi induces osteoblastic differentiation with RUNX2 and BMP2 mRNA up-regulation up to day 7, osteopontin increased protein expression and positive Alcian Blu staining. In calcified ECs mesenchymal and endothelial markers, such as α-SMA, SM22α, FSP-1 and Fibronectin or vWF progressively decrease. Finally, uremic serum is able to exacerbate in vitro calcification, compared to healthy serum characterized by an increase in both mesenchymal and osteoblastic mastergene such as TWIST and RUNX2.

Conclusions
Our data demonstrate that in vitro high-Pi induces the EndMT process and similar osteoblastic trans-differentiation suggesting a potential role of endothelium in VC. The relevancy of endothelial calcification is suggested by the calcification exacerbation induced by uremic compared to healthy sera.