bluuebunny/crossref_metadata_2025 huggingface dataset:
P0008CASE REPORT: METABOLIC ALKALOSIS RESULTING IN RESPIRATORY COMPROMISE IN A PATIENT UNDERGOING HIGH DOSE METHOTREXATE THERAPY
Authors: O'Donoghue Darragh , Truong Heather , Finnes Heidi , McDonald Jennifer , Leung Nelson
Published: June 2020

Background and Aims

High dose Methotrexate (HDMTX) is an important component of several modern oncological/haematological treatment protocols due to its central nervous system penetrance. Nephrotoxicity represents a significant adverse effect and can limit therapeutic options. Therefore, strategies to prevent this are paramount. Urinary alkalinisation and large volume resuscitation to maintain adequate hydration and urine output are the typical strategies. Urinary alkalinisation prevents tubular precipitation of methotrexate and therefore, a strict urinary pH target of 7 is maintained via a continuous bicarbonate infusion.

Method

We describe a case report, of Iatrogenic metabolic alkalosis leading to respiratory compromise in a patient receiving HDMTX from Mayo Clinic, Rochester.

Results

We present the case of a 76-year-old woman with a Diffuse Large B-Cell Lymphoma with CNS involvement who presented for elective admission for her 1st cycle of HDMTX. She received 7g of Methotrexate at dosing of 8 g/m2. She received the standard urinary alkalinisation with pre- and post-hydration. Her baseline HCO3- was 28 mEq/L. Her 48 hour MTX level was elevated at 1.2 so the urinary alkalinisation protocol was continued until <0.1 mcmol/L. On day 4, she developed frequent episodes of apnoea. Her ABG demonstrated a metabolic alkalaemia pH 7.54, pCO 53, pO2 91, HCO3 45. She was transferred to the ICU for close monitoring. Her bicarbonate infusion was discontinued and she received acetazolamide. Her bicarbonate improved to 31 after 12 hours. She had a significant improvement in her respiratory status with no further episodes of apnoea. Her bicarbonate infusion was restarted due to elevated MTX levels. She was discharged home with no further complications.

Conclusion

Iatrogenic Metabolic alkalosis leading to respiratory compromise represents a rare but important complication of urinary alkalinsation protocols for High-dose Methotrexate therapy.
P0009EVALUATION OF THE POTENTIAL FOR DRUG INTERACTIONS WITH VEVERIMER
Authors: Shao Jun , Parsell Dawn , Guttendorf Robert , Wu Yick Sen , Tsao Li , Tabakman Scott , Stasiv Yuri , Lee Angela , Biyani Kalpesh , Klaerner Gerrit
Published: June 2020

Background and Aims

Veverimer is an investigational drug being developed as an orally administered hydrochloric acid binder for the treatment of metabolic acidosis associated with chronic kidney disease (CKD). In clinical studies, treatment with veverimer safely and effectively increased serum bicarbonate and improved objective and subjective measures of physical functioning1-3. Veverimer, a free-amine polymer, is not systemically absorbed; therefore, its potential for drug-drug interactions (DDIs) is limited to those that occur in the gastrointestinal (GI) tract (i.e., direct binding or indirect effects resulting from transient increases in gastric pH). We assessed the potential for DDIs with veverimer both in vitro and in vivo in healthy subjects.

Methods

In vitro binding to veverimer was evaluated with 16 drugs of varying molecular weight and charge. In a separate study, the effect of veverimer on gastric pH was measured continuously in vivo in healthy subjects using a microelectrode pH probe placed in the gastric compartment. Human DDI studies were conducted with 4 orally administered drugs, including those that demonstrated the most in vitro binding to veverimer and those with pH-dependent solubility (furosemide, aspirin, warfarin, dabigatran).

Results

Veverimer did not bind to any of the positively charged, neutral or zwitterionic drugs tested in vitro. It bound to 3 small (MW <332 Da), negatively charged drugs (aspirin, ethacrynic acid, furosemide); these interactions were reduced or eliminated in the presence of physiologically relevant concentrations of chloride. Neither furosemide nor aspirin showed clinically meaningful changes in pharmacokinetic parameters when coadministered with veverimer in human DDI studies (Figure 1).

Veverimer increased gastric pH by ∼3.0 and 1.5 pH units in fasted and fed subjects, respectively. The increase in gastric pH was short-lived, with a peak within 1 hour after dosing and a return to baseline after ∼1.5 hours and ∼3 hours under fasting and fed conditions, respectively. The effect of veverimer on gastric pH was similar in the presence and absence of omeprazole. No clinically meaningful changes in systemic exposure, as indicated by Cmax and AUC, were observed when 3 drugs with pH-dependent solubility were coadministered with veverimer in human DDI studies (Figure 1).

Conclusions

In human DDI studies, we observed: a) no effect of veverimer on the bioavailability of drugs with physicochemical characteristics most susceptible to direct binding to the polymer; b) small, short-lived effects of veverimer on gastric pH; and c) no effect of veverimer on the bioavailability of drugs with pH-sensitive solubility. Therefore, it is concluded that there is a negligible risk of veverimer involvement in clinically significant DDIs.
P0020THE NEPHRO-PROTECTIVE ROUTE OF CGMP DOES NOT COMPENSATE NPRHOGENIC DIABETES INSIPIDUS, BOTH CONDITIONS MEDIATED BY RENAL INTEGRIN LINKED KINASE (ILK)
Authors: Griera-Merino Mercedes , García-Ayuso Diego , Gutiérrez-Calabrés Elena , Bohorquez Magro Lourdes , Campillo de Blas Sofía , Barrionuevo-González Marta , Calleros Laura , Rodríguez-Puyol Diego , Rodríguez-Puyol Manuel , De Frutos García Sergio
Published: June 2020

Background and Aims

Renal ILK transgenic deletion on mice (cKD-ILK) address basal polyuria compatible with nephrogenic diabetes insipidus (NDI), due to disrupted tubular water channel aquaporin 2 (AQP2) AQP2 expression and vesicular trafficking to the apical membrane [Cano-Peñalver JL et al. FASEB J. 2014; Mamuya FA et al. Am J Physiol Renal Physiol. 2016]. An early symptom of chronic renal diseases (CKD) is NDI. ILK depletion also have a nephron-protective effect in renal damage in mice due to the increase in cGMP [Cano-Peñalver JL et al. Mol Med. 2016; de Frutos S et al. Biochim Biophys Acta Mol Basis Dis. 2019], the latter an alternative anti-NDI mediator. Here we demonstrate that ILK-mediated regulation of AQP2, under basal or CKD contexts, does not depend on cGMP.

Method

cKD-ILK and controls (WT) were treated orally with a NO donor, as precursor of cGMP formation (IDN, 300mg / Kg / day) for 24 hours. Urine volume was determined as a measure of tubular functionality. Fresh kidneys from non-treated mice were cultured ex vivo with precursors of cGMP (NO donor SNP, 1 µM or cGMP analogue 8Br-cGMP, 0.2 mM) for 30 minutes. AQP2 in the apical membrane of the tubules was quantified by immunohistochemistry. More cKD-ILK and WT were subjected for 6 weeks to a diet supplemented with 0.2% adenine (A) as CKD inducer or baseline standard diet. Urine volume and total ranal AQP2 levels were determined.

Results

Compared to WT, cGMP axis activation in vivo did not compensate the basal NDI present in cKD-ILK and the activation ex vivo did not improve the apical presence of AQP2 in cKD-ILK. Both WT and cKD-ILK subjected to CKD showed exacerbated polyuria and decreased levels of renal AQP2, but no differences were observed between groups.

Conclusion

ILK regulates AQP2, both in a basal and pathological context (CKD) independent of cGMP which otherwise is nephron-protective. Our results probably explain the co-existence of cGMP increase together with AQP2 decrease observed in the cKD-ILK kidneys.
P0022INNATE IMMUNE RESPONSES ELICITED BY TOLL-LIKE RECEPTORS ARE POSSIBLY INVOLVED IN THE PATHOGENESIS OF NEPHROTIC SYNDROME
Authors: Tanaka Eriko , Hada Ichiro , Mikami Naoaki , Yan Kunimasa
Published: June 2020

Background and Aims

Pathogenesis of idiopathic nephrotic syndrome (INS) is yet to be fully elucidated. Immunological disorders are reported to be involved in the etiology of INS. Due to the efficacy of immunosuppressant agents such as calcineurin inhibitor and rituximab in treating nephrotic syndrome, aberrant activation of the acquired immune system through T and B cells are considered to be the underlying pathogenic mechanisms of INS. Nevertheless, there is a possibility that the innate immune system plays a key role in INS pathogenesis. This study aims to investigate the involvement of innate immunity in INS pathogenesis by examining the expressions of toll-like receptors (TLRs).

Method

Kidney tissue samples from two INS patients were collected at two points of time: the first biopsy was performed during nephrosis and the second during remission. Total RNA was extracted from the kidney tissue samples, and RNA-sequencing was performed to investigate RNA expression profiles. The differences between RNA expression profiles of TLRs and molecules related to TLR pathways in the tissue samples collected during nephrosis and remission were analyzed.

Results

There was a significant decrease in RNA expression of TLR9 and TLR10 during remission compared to nephrosis: fold change in each patient was -2.12 and -2.12 for TLR9, and -2.51 and -2.09 for TLR10. RNA expression of TLR8 also decreased: fold change in each patient was -1.19 and -1.75. There were no significant changes in the RNA expression profiles of TLR1, 2, 3, 4, 5, 6, and 7. In addition, there were no differences in the RNA expression profiles of MYD88, IRAK family, and TRAF family molecules that are associated with TLR pathways. However, RNA expressions of IL6, IL1B, IL12B, and TNF, as well as the cytokines controlled by TLR8 and TLR9 pathways, which were activated during nephrosis, disappeared or decreased during remission.

Conclusion

The involvement of the innate immune system in the pathogenesis of nephrotic syndrome has been suggested in some reports. Based on the fact that the onset or recurrence of nephrosis is triggered by non-specific viral infection, it is highly possible that innate immunity is involved in the pathogenesis of nephrotic syndrome. TLRs play a key role in innate immunity as they elicit the innate immune system after detecting pathogens, induce inflammatory cytokine production, and trigger signaling pathways that activate lymphocytes via maturation of dendritic cells. Specifically, TLR8, 9, and 10 mediate pathways of the first immune response to viral infections. Our study reveals that TLRs play a pivotal role in innate immunity associated with renal tissue during the onset of nephrosis.
P0023RNA-BINDING PROTEIN HUR REGULATES HYPOXIA-INDUCED PARKIN/BNIP3L EXPRESSIONS AND MITOPHAGY IN RENAL TUBULAR CELLS
Authors: Li Chi-Yuan , Li Xin
Published: June 2020

Background and Aims

Mitochondrial dysfunction contributes to the pathophysiology of acute kidney injury (AKI). Mitophagy selectively degrades damaged mitochondria and thereby regulates cellular function and homeostasis. RNA-binding proteins (RBPs) regulate RNA processing at multiple levels and thereby control cellular function. In this study, we report that RBP, human antigen R (HuR) regulates mitophagy in renal tubular cells (HK-2) under hypoxia condition.

Method

Human kidney proximal tubular 2 (HK-2) were cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium (Gibco, Invitrogen, GmbH, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, GmbH, Germany), 1% penicillin/streptomycin (Gibco, Invitrogen, GmbH, Germany) at 37°C and 5% CO2. The cells were exposed to normoxia (37°C; 5% CO2) or hypoxia (serum and glucose deprived RPMI medium, 37°C, 1% O2) conditions. Mitophagy marker expressions (Parkin, p-Parkin, PINK1, BNIP3L, BNIP3, LC3) were determined by western blot analysis. ROS and mitochondrial membrane potential were determined using DCF-DA and JC-1 dye. Parkin and LC3 localization to mitochondria were performed by confocal laser scanning microscopy. HuR bound Parkin/BNIP3L mRNA expressions were determined by RNA-immunoprecipitation analysis.

Results

In this study, up-regulated mitophagy markers such as Parkin, PINK1, BNIP3 and BNIP3L protein expressions and mitophagosome formation were found in HK-2 cells under hypoxia condition. Stable HuR knockdown studies revealed that, HuR regulates mitophagy by mitophagosome (LC3 co-localization with mitochondria) and mitolysosome (mitochondria co-localization with lysosome) formation under hypoxia. Further, we show that HuR significantly regulated hypoxia-induced mitophagy by regulating Parkin/BNIP3L protein expressions and their mitochondrial localization. Bioinformatics studies showed that, HuR specifically binds to the AU-rich region in 3’UTR and CDS of Parkin and BNIP3L mRNA sequence. HuR was significantly bound to Parkin and BNIP3L mRNA and thereby regulated their mRNA stability under hypoxic conditions.

Conclusion

Altogether our data highlight on the functional importance of HuR in regulating mitophagy under hypoxic conditions in renal tubular cells through Parkin/BNIP3L expressions.
P0026METFORMIIN IMPROVES DYSFUNCTION OF MESENCHYMAL STEM CELLS ASSOCIATED WITH CHRONIC KIDNEY DISEASE VIA SENESCENCE INHIBITION
Authors: Noh Hyunjin , Yu Mi Ra , Choi Kyoungin , Hwang Dohui
Published: June 2020

Background and Aims

Mesenchymal stem cells (MSC) are promising source of cell-based regenerative therapy; however, adequate cell functionality is a critical factor for the success of autotransplantation.

Method

We investigated the effects of metformin on chronic kidney disease (CKD)-associated cellular senescence using MSC isolated from sham operated and subtotal nephrectomized mice and further explored the protective role of metformin-treated CKD MSC in renal progression.

Results

When compared to normal MSC, MSC isolated from CKD mice displayed reduced proliferation and early senescence as determined by enlarged cell morphology, increased oxidative stress, accumulation of DNA damage response marker p53 binding protein 1 (53BP1), phospho p53, p16INK4a, and β-gal expression, and decreased cyclin-dependent kinase 4 (CDK4) and cyclin D. CKD MSC exhibited activation of NFκB resulting in expression of senescence-associated secretory phenotype (SASP) factors compared to normal MSC. All of these changes were significantly prevented by metformin treatment. In vivo, metformin-treated CKD MSC attenuated inflammation and fibrosis in UUO kidney as compared to CKD MSC. Co-culture of LPS or TGF-β1-treated HK2 cells with metformin-treated CKD MSC markedly decreased LPS or TGF-β-induced tubular expression of proinflammatory markers and fibrogenesis when compared to CKD MSC suggesting paracrine action of CKD MSC enhanced by metformin treatment.

Conclusion

Our data suggest that metformin inhibits cellular senescence of CKD MSC and improves their renoprotective effects.
P0027METFORMIN INHIBITS TGF-BETA1-INDUCED FIBROTIC RESPONSE IN MOUSE INNER MEDULLARY COLLECTING DUCT CELLS IN ASSOCIATION WITH ACTIVATION OF PPARA SIGNALLING
Authors: Liu Shanying , Li Yan , Liu Dan , Liang Diefei
Published: June 2020

Background and Aims

Metformin has been recognized to inhibit renal fibrosis through protection of proximal tubular epithelial cells from inflammation and fibrotic response. However, the role of other segments of renal tubular epithelial cells in the development of renal fibrosis remains unclear. This study aimed to investigate the profibrotic effects of TGF-beta1 and the anti-fibrotic effects of metformin in mouse inner medullary collecting duct cells using mIMCD-3 cell line.

Method

Cultured mIMCD-3 cells were treated with TGF-beta 1 and metformin either alone or in combination. The morphological change of cells was inspected under an inverted microscope. The mRNA of genes associated with renal fibrosis was evaluated by real-time quantitative PCR. The protein expression of E-cadherin, Vimentin, alpha-SMA were evaluated by Western blot.

Results

The shape of mIMCD-3 cells changed from cobblestone to spindle-like after exposure to TGF-beta 1 at 10ng/mL for 72 hours, which can be inhibited by co-treatment with metformin. TGF-beta 1 remarkably increased the expression of alpha-SMA and PAI-1, while decreased E-cadherin mRNA in mIMCD-3 cells. Metformin inhibited TGF-beta 1-induced reduction of E-cadherin and upregulation of PAI-1. The protein level of E-cadherin was also decreased and alpha-SMA increased by TGF-beta 1. Metformin inhibited TGF-beta 1-induced protein expression of alpha-SMA and vimentin, and downregulation of E-cadherin. It has been reported that impaired renal PPARα signaling promoted renal fibrosis. In this study, we found that metformin upregulated basal level of PPARα mRNA. PPARα target genes including Acadvl and Cpt1a were downregulated by TGF-beta 1 in mIMCD-3 cells, which could be prevented by metformin.

Conclusion

Metformin inhibits the profibrotic effects of TGF-beta 1 probably through suppression of EMT and maintaining PPARα signaling in mouse inner medullary collecting duct cells.
P0033COMPARISON OF ANXIETY, DEPRESSION AND QUALITY OF LIFE IN PATIENTS WITH AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE: A CROSS-SECTIONAL, RANDOMIZED CONTROLLED PILOT STUDY
Authors: Ozler Tuba Elif , Alay Cemal , Cebeci Egemen , Uzun Sami , Karadag Serhat , Ozkan Oktay , Bashan Yagmur , Dag Burcu , Karaali Zeynep , Ozturk Savas
Published: June 2020

Background and Aims

Patients with autosomal dominant polycystic kidney (ADPKD), in addition to uremic signs and symptoms, the need for renal replacement therapy and the risk of transferring the disease to their children put them under physical and mental stress. This may affect the psychosocial status of patients. In this study, we compared diabetic nephropathy (DN) patients who were expected to progress rapidly with early-stage ADPKD patients to reveal anxiety, depression and quality of life.

Method

In our cross-sectional, randomized controlled and single-center study, 55 adult patients with early stage ADPKD with eGFR ≥ 60 ml/min/1.73 m2 and 50 patients with DN as control group were included. Both groups were applied to state trait anxiety inventory (STAI), patient health questionnaire 9 (PHQ-9), quality of life short form 36 and Beck depression inventory in order to evaluate their depression, anxiety and quality of life. Depression, anxiety and quality of life questionnaires’ results, demographic and clinical characteristics, laboratory findings of the two groups were compared.

Results

Comparison of patients’ and control groups demographic and clinical characteristics, and laboratory data were shown in Table 1. According to state trait anxiety inventory; moderate and high levels of anxiety were present in 94% patients with ADPKD and in 66% patients with DN (p<0.001). According to Beck depression inventory; depression is detected in 58.2% patients with ADPKD and in 26% patients with DN (p=0.001). When the PHQ-9 results were compared; depression is prevalent in 55.6% patients with ADPKD and in 34.7% patients with DN (p=0.037). Although ADPKD patients were at an early stage, we found anxiety and depression more frequent and severe than DN patients. While physical life quality was similar in both group; mental quality of life was lower in ADPKD patients according to quality of life short form 36 (p= 0.32 and p= 0.03 respectively) (Table 2). Independent variables affecting Beck depression inventory score; being a woman, smoking and being diagnosed with ADPKD; independent variables affecting PHQ9 score; being female and the excess of the number of medical tablets used daily; independent variables affecting personal trait anxiety score; being a woman, smoking, being diagnosed with ADPKDD, and presence of hypertension.

Conclusion

In ADPKD patients, anxiety, depression and decrease in quality of life can be seen more frequently than other uremic patients even in the early period when renal functions are preserved. This group of patients should be evaluated in this respect from the early stages of the disease.
P0034IDENTIFICATION OF FACTORS ASSOCIATED WITH PROGRESSION, PROGNOSIS AND TOLVAPTAN INDICATION IN POLYCYSTIC KIDNEY DISEASE PATIENTS
Authors: Kostopoulou Myrto , Louka Michaela , Fokas Stavros , Tigka Eirini , Drakopoulos Angelos , Gkika Vasiliki , Tsagkatakis Mihail , Tsirpanlis George
Published: June 2020

Background and Aims

The identification of possible risk factors for the progression of Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an emerging field especially after the introduction of the first disease-specific treatment. The present study aims to explore the associations between epidemiological, clinical and imagining data in a large cohort of ADPKD patients.

Method

This study was from a single outpatient clinic following patients with ADPKD. Patients were included in the study if they had a recent Magnetic Resonance Imaging (MRI) for measurement of Total Kidney Volume (TKV), a validated biomarker for disease progression. For all patients, the Mayo Clinic Imagining Category (MCIC) and the respective prediction for End Stage Renal Disease (ESRD) were calculated. Patients eligible for tolvaptan treatment (MCIC 1C, 1D, 1E, age < 55 years old and estimated-glomerular filtration rate (e-GFR) ≥ 25 ml/min) were identified. Characteristics including individual medical history, clinical and laboratory data were examined for possible associations with renal and imagining parameters using linear regression models.

Results

A total of 158 patients were included. Based on measurements of height-adjusted TKV (ht-TKV) and age, 5% of the patients were classified as 1A, 20% as 1B, 34% as 1C, 25% as 1D and 16% as 1E, MCIC. In multivariable analysis, patient’s age (p = 0.01), male sex (p < 0.001), parent’s age at which ESRD was reached (adjusted for patient age) (p < 0.001) and proteinuria (p = 0.04) were associated with ht-TKV. Parent’s age at ESRD differed significantly between the MCICs of the offspring (mean±(SD)), 70.83 (12.90) in 1A, 63.79 (11.39) in 1B, 57.32 (10.42) in 1C, 51.42 (9.18) in 1D and 47.94 (5.73) years old in 1E, (p < 0.001). Similarly, there were significant differences in the presence and the age of hypertension onset (p =0.004 and p = 0.003, respectively). In 104 patients (50 females, 54 males) who were eligible for tolvaptan treatment age at ADPKD diagnosis, age at hypertension onset and parent’s age reaching at ESRD were all significantly lower (p < 0.001 for all) when compared to non-eligible patients. Finally, factors associated with the prediction score of ESRD (e-GFR 10/ml/min) were hypertension, uric acid and the age at ESRD of the affected parent (p = 0.001, 0.02 and 0.01, respectively).

Conclusion

The age at which an affected parent had reached ESRD, as heritability estimator, was significantly associated with a worst phenotype, prognosis and tolvaptan indication. Early diagnosis of the disease, hypertension and its early onset, proteinuria and male sex are also possible risk factors for the progression of ADPKD.
P0041CHARACTERISTICS OF URINE-DERIVED RENAL EPITHELIAL CELLS (UREC) FROM CHILDREN WITH POLYCYSTIC KIDNEY DISEASE (PKD)
Authors: Ziegler Wolfgang H , Lüdiger Sarah , Mett Charlotte , Hasan Fatema , Soetje Birga , Haffner Dieter
Published: June 2020

Background and Aims

In hereditary PKD, epithelial cell defects are mostly caused by mutation of proteins of the cilia-centrosome complex. Use of primary cells from patients allows to study specific cell characteristics and altered activation of signal pathways in epithelial cell function-related culture models. In this ex vivo approach, comparison of cell properties is based on defined genetic origin and can be related to kidney function at the time of UREC collection, revealing consequences of genetic disposition and kidney-stress related changes on cell function.

Method

The study considers patients with genetically confirmed causes of PKD, autosomal recessive polycystic kidney disease (ARPKD), nephronophtisis (NPH), Bardet-Biedl syndrome (BBS), and age-matched controls with normal kidney function. UREC are grown from cellular urine sediment using selective growth conditions. Primary cells are cultured up-to 21 days (passages 2 & 3) and tested with respect to their proliferation, tubular origin and epithelial properties in 2D/3D culture conditions. To allow pathway analysis and improve control conditions, immortalized UREC lines are also included.

Results

UREC preparations of cohorts from ARPKD, NPH, and BBS patients, and controls were studied quantitatively to determine specific epithelial cell properties. Considering basic characteristics of each primary culture including cell morphology, expression of epithelial markers, and formation of cell-cell adhesions, the potential of UREC cells to polarize and generate liquid-filled epithelial spheroids was measured. The capacity of lumen formation in 3D culture strongly varies among individual cell preparations and appears to reflect the genetic origin of UREC cells, whereas no correlation to (remaining) kidney function (eGFR) is observed.

Conclusion

Determining specific, quantitative UREC cell properties related to genetics and kidney function, we expect to gain a better mechanistic understanding of cellular and renal epithelial defects in PKD patients. Ex vivo analysis based on UREC cells may provide options for testing of personalized pharmaceutical intervention.
P0042SPECTRUM OF RENAL INVOLVEMENT IN CHILDREN WITH HNF1B NEPHROPATHY
Authors: Papizh Svetlana , Prikhodina Larisa
Published: June 2020

Background and Aims

Hepatocyte nuclear factor 1 gene (HNF1B) encodes for HNF-1ß, a transcription factor that is expressed early in embryonic development and plays a key role for tissue-specific regulations of gene expression in various organs such as the kidneys, urogenital tract, liver, biliary ducts and pancreas. HNF1B disease (MIM 137920) is recognized to represent an autosomal dominant syndromal disorder comprising variable phenotype with renal and extrarenal manifestations. Renal phenotype in HNF1B nephropathy is highly heterogeneous. The aim of the study was to evaluate the clinical characteristics of renal involvement in children with genetically proven HNF1B nephropathy.

Method

Five children (2M/3F) from unrelated families with heterozygous HNF1β mutations and kidney disorders were examined. We evaluated serum levels of electrolytes, magnesium (Mg), phosphorus (P), uric acid, acid-base composition; urinary excretion of Ca, P, creatinine (Cr); fractional excretion (FE) of Mg, urate (Ur), Na, K; tubular maximum phosphate reabsorption per GFR (TmP/GFR), and urinary β-2 microglobulin. Molecular genetic analysis was performed using by next generation sequencing.

Results

Cortical or medullary bilateral renal cysts were detected in all five children with HNF1B nephropathy. Kidney hypoplasia, medullary nephrocalcinosis, and low-grade proteinuria (<0.5 g/m2/d) revealed in 2\5 (40%) of cases. One child had vesicoureteral reflux. All patients with HNF1B nephropathy had normal serum level of uric acid and all electrolytes, including Mg. Increased FE of Mg and urinary excretion of β-2 microglobulin was found in all cases. Increased FE of Ur, Na and K revealed in 4\5 (80%) of subjects. Decreased TmP/GFR had 3\5 (60%) of cases. At the first examination of patients aged 1.0 (IQR: 1.0; 2.0) years all of them already had decreased eGFR to CKD stage 2-3.

Conclusion

The present study demonstrates that patients with HNF1B-associated disease had wide spectrum of renal involvement. The most prevalent features of HNF1B nephropathy in our children were bilateral renal cysts, defects of proximal tubular function with wasting of electrolytes and progression to CKD stage 2-3 at early age.
P0049IDENTIFYING HUB GENES ASSOCIATED WITH CLINICAL CHARACTERISTICS IN IGA NEPHROPATHY BYWGCNA
Authors: Yang Chengyu , Xu Yan
Published: June 2020

Background and Aims

Clinically, IgA nephropathy has a variety of symptoms including paroxysmal gross hematuria, nephritic syndrome and nephrotic syndrome. This study aimed at investigating hub geneand genes modular related to IgA nephropathy clinical characteristics by using weighted gene co-expression network analysis combining clinical, microarray and network database parameters.

Method

We collected 32 human samples from the European Renal cDNA Bank and used RMA method to preprocess the raw data and utilize the limma package to obtain differentially expressed gene in renal interstitium and glomeruli. And then, we used the WGCNA package to construct the gene co-expression of differential expression genes and identify the hub genes associated with clinical characteristics in renal interstitium and glomeruli, respectively. Gene ontology enrichment analysis and KEGG analysis for hub genes which associated with clinical characteristics were performed by DAVID, and PPI information was acquired from STRING with visualization by Cytoscape.

Results

For glomeruli, 1470 genes differentially expressed between IgA nephropathy patients and healthy control, containing 10 hub genes associated with age, 8 hub genes associated with sex, 48 hub genes associated with Bp enrichd in ERK1 and ERK2 cascade and Rap1 signaling pathway, 223 hub genes associated with BMI enrich in organic acid catabolic process and fatty acid degradation pathway, 136 hub genes associated GFR enriched in immune response and PI3K-Akt signaling pathway, 82 hub genes associated with proteinuria enriched in extracellular matrix organization and PI3K-Akt signaling pathway. In tubulointerstitium, there were 480 genes differentially expressed between IgA nephropathy patients and healthy control. Among 480 DEGs, 6 hub genes associated with age, 15 hub genes associated with sex, 35 hub genes associated with Bp enrichd in positive regulation of apoptotic process, 87 hub genes associated with GFR enriched in negative regulation of macromolecule metabolic process and RNA transport, 33 hub genes associated with proteinuria enriched in regulation of apoptotic process and FoxO signaling pathway. PPI enrichment analysis shown that all hub genes sets are biologically connected cluster.

Conclusion

We made a preliminary investigation on molecular mechanisms of relationship between IgA nephropathy and clinical characteristics and identified hub genes and pathways closely related with BMI, GFR and Proteinuria in IgA nephropathy by a series of bioinformatics analysis.
P0061LOSS OF UBIQUITIN SPECIFIC PROTEASE-40 CAUSES PODOCYTE DETACHMENT AND CONTRIBUTES TO THE DISEASE PROGRESSION OF GLOMERULOSCLEROSIS
Authors: Mikami Naoaki , Tanaka Eriko , Hada Ichiro , Yan Kunimasa
Published: June 2020

Background and Aims

We reported that the ubiquitin specific protease-40 (USP40) is expressed in podocytes beyond the species and plays a crucial role in glomerulogenesis in zebrafish (Takagi H, Am J Physiol Renal Physiol 2017). However, the role of USP40 in podocytes and its functional downstream after birth is largely unknown. Recent report indicated that other USP family: USP10 regulates integrin beta1 and fibrotic wound healing in myofibroblasts (Gillespie SR, J Cell Sci 2017). The present study aimed to explore the biological role of USP40 in the acquired podocyte injury and to determine the interacting partner by focusing on podocyte detachment.

Method

We generated USP40-knockout mice (USP40KO); however, they showed no apparent kidney abnormality. Therefore, to explore the biological role of USP40 in acquired podocyte injury, we crossed USP40KO with NEP25 mice, in which selective podocyte injury can be induced by injection with an immunotoxin, LMB2. Urinary protein was analyzed until day 9 after LMB2 injection, and then kidney tissues were subjected to light microscopy, immunohistochemistry of p57, and immunofluorescence microscopy of integrin beta1. The glomeruli were isolated and subjected to immunoblot analysis of integrin beta1. The effect of USP40 knockdown on integrin beta1 expression was studied in cultured mouse podocytes. Finally, the protein binding of integrin beta1 with USP40 was determined by immunoprecipitation using cultured mouse podocytes.

Results

NEP-USP40KO mice developed earlier and higher levels of proteinuria compared with NEP mice. Light microscopy and immunohistochemistry showed higher levels of kidney injury in NEP-USP40KO mice compared with NEP mice in terms of hyalinosis (p<0.01), crescent formation (p<0.01), and cell proliferation (p<0.01) in the glomeruli, podocyte loss (p<0.05), and tubulointerstitial injury (p<0.01). Immunoblot analysis and immunofluorescence revealed decreased expression of integrin beta1 in podocytes of NEP-USP40KO mice compared with NEP mice. Gene knockdown of USP40 in cultured podocytes decreased the protein expression of integrin beta1. Finally, apparent protein binding of USP40 with integrin beta1 was observed.

Conclusion

Integrin beta1 is known to play a crucial role as the adhesion molecule that connects the podocytes with the extracellular matrix of the glomerular basement membrane. It is also known that the lack of integrin beta1 causes podocyte detachment, which is involved in the disease progression of glomerulosclerosis. Therefore, the present study suggests that USP40 may be involved in a self-defense mechanism in podocyte injury probably through interacting with the regulatory machinery of integrin beta1 in podocytes.
P0062GLUCOSYLCERAMIDE SYNTHASE INHIBITION WITH VENGLUSTAT IN CLASSIC FABRY DISEASE PATIENTS LEADS TO PROGRESSIVE REDUCTION OF ENDOTHELIAL CELL GLOBOTRIAOSYLCERAMIDE INCLUSION VOLUME
Authors: Mauer Michael , Goker-Alpan Ozlem , Germain Dominique P , Wilcox William , Hopkin Robert J , Lukina Elena , Geberhiwot Tarekegn , Deegan Patrick , Tylki-Szymanska Anna , Sensinger Charlotte , Zaher Atef , Hailman Eric , Modur Vijay , Najafian Behzad
Published: June 2020

Background and Aims

Fabry disease (FD) is a rare, X-linked, genetic disorder caused by mutations in the gene for the lysosomal enzyme alpha-galactosidase A (αGal-A). The progressive accumulation of glycosphingolipids, most notably globotriaosylceramide (GL-3), in lysosomes of a variety of organs and cell types leads, over decades, to renal, cardiovascular, and other clinical manifestations. In a phase 2 study, glucosylceramide synthase inhibition with venglustat led to a progressive reduction in plasma GL-3 to low-normal levels. Scoring by light microscopy showed a reduction in lysosomal GL-3 inclusions in skin capillary endothelial cells (EC) after 3 years of therapy, although not after 6 months. To further characterize the effect of venglustat using more precise methods, we applied unbiased stereological methods to quantitate GL-3 inclusions in skin ECs.

Methods

Skin biopsies were obtained from classic male Fabry disease patients (N = 11) at baseline and during daily treatment with venglustat in a phase 2 study (NCT02228460, NCT02489344). Images from at least 50 superficial skin capillaries were obtained using transmission electron microscopy at 7,500 X magnification. The fraction of the volume (Vv) of cytoplasm occupied by GL-3 inclusions [Vv(Inc/Endo)] in all ECs in these capillaries was estimated using point counting by a masked reader. Two-sided paired t tests were used to evaluate differences between baseline and post-treatment values at each time point.

Results

Venglustat therapy led to a significant reduction from baseline in Vv(Inc/Endo) of 21.1% after 6 months and 38.7% after 3 years of treatment (Figure).

Conclusions

Treatment with venglustat led to a reduction in GL-3 inclusion fractional volume in skin capillary ECs which was detectable after 6 months using precise quantitative methods. This was followed by further reduction over the next 2 1/2 years. We posit that, in the absence of αGal-A activity, inhibition of GL-3 production with venglustat allowed other enzymatic or non-enzymatic mechanisms to progressively reduce EC lysosomal GL-3 content, and that long-term venglustat therapy may therefore prevent or reverse progressive tissue injury in Fabry disease.
P0078INVESTIGATION THE VARIANTS AT THE BINDING SITE OF INFLAMMATORY TRANSCRIPTION FACTOR IN PATIENTS WITH ESRD
Authors: SU SUI-LUNG
Published: June 2020

Background and Aims

Inflammation is an important factor for enhancing the disease process from chronic kidney disease (CKD) to end-stage renal disease (ESRD). Nuclear factor-kappa B (NF-κB) is a transcription factor that regulates the expression of genes involved in inflammation.

We investigated the potential association with the gene polymorphism of transcription factor binding site of NF-κB in ESRD patients.

Method

We used the Taiwan Biobank database, University of California, Santa Cruz, reference genome, chromatin immunoprecipitation sequencing database to find the SNPs at potential binding sites of NF-κB. In addition, we performed a case–control study and genotyped 847 patients with ESRD and 846 healthy controls at Tri-Service-General-Hospital from 2015 to 2016. Further we used ChIP-assay and Luciferase reporter assay to identify the binding activity at different genotype.

Results

Results of biometrics screening in the databases revealed 15 SNPs with the potential binding site of NF-κB. Genotype distributions of rs9395890 were significantly different in ESRD cases and healthy controls (P = 0.032). In the Dominant model, rs9395890 with T allele had a higher risk of ESRD (P = 0.032; odds ratio [OR] = 1.32, 95% confidence interval [CI] = 0.99–1.76). The ChIP assay reveals that around 1.49 times enrichment of NF-κB of the variant type TT when compared to that of the wild type GG in the rs9395890 (P<0.027; TT=3.20±0.16, GT=2.81±0.20, GG=1.71±0.18,) and the luciferase activity curve showed T allele was higher than G allele.

Conclusion

In conclusion, we demonstrate that rs9395890 may be associated with ESRD in the Taiwanese population.
P0079STRATEGIC ACTION FOR POLYCYSTIC KIDNEY DISEASE IN GALICIA. POPULATION APPROACH MODEL EXPORTABLE TO OTHER AUTONOMOUS COMMUNITIES
Authors: Besada Cerecedo Lara , Arhda Nisrine , Barcia de la Iglesia Ana Maria , Garcia Murias Maria , Carrera Cachaza Noa , Investigadores GalCYST Consorcio , Garcia Gonzalez Miguel
Published: June 2020

Background and Aims

The Strategic Action for Polycystic Kidney Disease (PKD) in Galicia was started in 2016, as a population strategy model (genetic cascade study) coordinated between the reference hospitals in this region, with the objective of diagnosing cost-effectively Galician families with the disease

Method

We develop panels for the simultaneous study of the currently known candidate genes, using ultrasequencing technologies (NGS). Each index case is analyzed by NGS and in the rest of the family members, the presence / absence of the eventual mutation identified is analyzed by Sanger Sequencing.Patient clinical data were collected to establish the genotype-phenotype correlation

Results

NefroCHUS has a historical record of 224 Galician families with PKD. Since January 1, 2016, 306 new PKD families have been identified from hospitals belonging to the three Galician health areas: Coruña - Ferrol (CHUAC (67 families), CHUF (17 families)), Vigo - Ourense (CHOP (44 families), CHUOU (43 families), CHUVI (32 families) and Povisa (18 families)) and Santiago-Lugo (HULA (35 families), HCB (22 families) and CHUS (28 families)), for a total record of 530 PKD families identified based on clinical criteria. Carrying out genetic studies allowed the calculation of gene mutagenicity rates for each health area: Coruña-Ferrol (58% PKD1, 36% PKD2 and 6% GANAB), Vigo-Ourense (80% PKD1, 15% PKD2 and 5 % other genes) and Santiago-Lugo (53% PKD1, 44% PKD2 and 3% GANAB). Taking into account the families of the historical record, mutagenicity rates in the Galician population are distributed as follows: 70% PKD1, 28% PKD2 and 2% other genes. So far 446 patients have been phenotypically characterized. The average age of diagnosis was 38 years, the main reason being family history (59%; n = 264). 73% of the patients presented arterial hypertension (AHT) with an average detection age of 42 years (range 6-82 years). 21% of the patients (n = 92) iniciated renal replacement therapy with a mean age of 49 years (38-80 years). The presence of liver cysts proved to be a very frequent extrarenal manifestation (67.9%; n = 303). Other manifestations to highlight are: low back pain (27%; n = 119), cystic kidney infection (7.4%; n = 33) or the presence of intracranial aneurysms (3.1%; n = 14)

Conclusion

It has been established the genetic map of the Autosomal Dominant Polycystic Kidney Disease in Galicia and at the same time a model of population approach in hereditary diseases that is exportable to other autonomous communities.
P0083HLA-DRB1*1301-DQB*0603-DRB3*0202 HAPLOTYPE IS INDEPENDENTLY ASSOCIATES WIHT POOR PROGNOSIS IN PLA2R-RELATED MEMBRANOUS NEPHROPAHTY
Authors: Le Weibo , Shi JingSong , Hou Qin
Published: June 2020

Background and Aims

The susceptibility of Idiopathic membranous nephropathy (MN) is associated with HLA alleles. We previously found almost all (98.7%) patients with PLA2R-related MN carry either or both of the HLA-DRB1*1501 and DRB3*0202 risk alleles. However, the associations of HLA alleles and renal outcome is not known.

Method

we investigated the associations between HLA alleles and renal outcome in 392 PLA2R-related MN patients.

Results

We found that HLA-DRB1*1301 and DQB1*0603, which are in high linkage disequilibrium (R2 = 1.0, D’=1.0), are associated with at least 30% eGFR decline during follow-up (HR 2.2, p<0.001). HLA-DRB1*1301 and DQB1*0603 are not a susceptible HLA allele for PLA2R-related MN. Neither HLA-DRB1*1501 nor DRB3*0202 is associated with renal outcome. After adjusting age, gender and the two susceptible HLA alleles, DRB1*1301 haplotype remains significant associated with renal outcome. Compared those patients with negative DRB1*1301, patients with positive DRB1*1301 showed a similar eGFR level at baseline, but a significant lower eGFR at the last follow up. There were 12 PLA2R-related MN patients with positive DRB1*1301, eight of them (66.7%) showed more than 30% eGFR decline during follow-up. Among the 12 patients carrying DRB1*1301, all (100%) had DRB3*0202, four (33.3%) had DRB1*1501, and none (0%) had DRB1*0301. Linkage disequilibrium analysis showed the susceptible allele DRB3*0202 was highly linked with DRB1*0301 and DRB1*1301, while HLA-DRB1*0301 was not associated with renal outcome.

Conclusion

HLA-DRB1*1301-DQB*0603-DRB3*0202 haplotype is independently associated with Poor Prognosis in PLA2R-related Membranous.
P0087TRAF3 MODULATION: A NOVEL MECHANISM OF THE ANTI-INFLAMMATORY EFFECTS OF VITAMIN D RECEPTOR AGONIST PARICALCITOL IN RENAL DISEASE
Authors: Rayego-Mateos Sandra , Morgado Pascual Jose Luis , Valdivielso José M , Sanz Ana , Bosch panadero Enrique , Rodrigues Diez Raul , Egido Jesus , Ortiz Alberto , Gonzalez Parra Emilio , Ruiz-Ortega Marta
Published: June 2020

Background and Aims

Tumor necrosis factor receptor-associated factors (TRAFs) are critical signaling adaptors downstream of pro-inflammatory receptors, involved in canonical and non-canonical nuclear factor-κB (NF-κB) activation. Vitamin D receptor agonists (VDRAs) exert beneficial effects in renal disease and possess anti-inflammatory properties, but the underlying mechanism remains unknown. Our aim was to investigate TRAF3 involvement on renal disease and its potential modulation by VDRAs.

Method

Studies were done in isolated peripheral blood mononuclear cells (PBMCs) from end-stage renal disease (ESRD) on haemodialysis (treated or not with VDRAs) patients, healthy donors or cultured renal cells. Preclinical models of renal damage were: unilateral ureteral obstruction (UUO), acute renal damage induced by Folic acid (FA) administration and TWEAK-induced renal inflammation. The effect of VDRA (paricalcitol) was tested in vitro and in vivo.

Results

In PBMCs isolated from ESRD, TRAF3 protein levels were downregulated compared to healthy controls, associated to activation of non-canonical NF-κB2 (downregulation of RelB or p52/NF-κB2 protein levels) and altered pro-inflammatory cytokine profile. Interestingly, treatment of those patients with the VDRA paricalcitol restored all the described features. In vitro experiments in PBMCs and tubular epithelial cells exposed to inflammatory stimuli showed that paricalcitol prevented TRAF3 downregulation, NF-κB2 activation and proinflammatory genes upregulation. Moreover, immunoprecipitation studies showed that paricalcitol diminished TRAF3 ubiquitination in the K48-linked chains and CIAP1-TRAF3 interaction. TRAF3 overexpression by CRIPS/cas9 technology mimics VDRAs effects. In preclinical models of renal damage paricalcitol inhibited renal NF-κB2 activation, reducing p52 and RelB nuclear accumulation and transcriptional activity associated to lower renal inflammation. In TWEAK-induced renal injury in VDR knockout mice, paricalcitol prevented TRAF3 downregulation and NF-κB2-dependent gene upregulation, suggesting a VDR-independent anti-inflammatory effect of paricalcitol.

Conclusion

The present studies identify TRAF3 downregulation as a key feature promoting inflammation in CKD identifying non-canonical NF-κB activation as a key driver of inflammation in this context. The fact that the VDR is not required for paricalcitol actions suggests novel-signaling pathways involved in the anti-inflammatory effect of paricalcitol. This may allow the design of drugs that restore TRAF3 levels but are devoid of VDR-dependent effects on mineral bone metabolism, which may limit the use of VDRAs as anti-inflammatory agents.
P0094KLOTHO DECLINE IS ASSOCIATED WITH PRO-HYPERTROPHIC SIGNALING IN MYOCARDIUM OF NEPHRECTOMIZED SPONTANEOUSELY HYPERTENSIVE RATS (PILOT SYUDY)
Authors: Bogdanova Evdokia , Dobronravov Vladimir , Semenova Natalia , Zubina Irina , Galkina Olga , Beresneva Olga , Ivanova Galina , Parastaeva Marina , Kayukov, Ivan , Smirnov Alexei
Published: June 2020

Background and Aims

Klotho deficiency is suggested to be involved in cardiac complications in chronic kidney disease (CKD) through modulation of intracellular pro-hypertrophic signaling pathways.

The aim of this study was to investigate the molecular mechanisms of myocardial remodeling in the settings of Klotho decline in experimental CKD.

Method

Systolic blood pressure (BP), myocardial mass index (MMI), serum and urinary creatinine (Cr), serum urea (Ur), inorganic phosphate (Pi), 25-hydroxyvitamin D (25OHD), parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), Klotho, myocardial Klotho, calcineurin/ NFAT and beta-catenin expression (IHC), quantitative morphometry of cardiomyocite (CM) thickness and nuclei area (hematoxylin and eosin), myocardial perivascular (PF) and interstitial fibrosis (IF), renal fibrosis (RF) (Masson’s trichrome) and Klotho expression (rKl, IHC) were analyzed in spontaneously hypertensive rats with 3/4 or 5/6 nephrectomy (Nx) and duration of experimental exposure 1 and 2 mo (Nx1, n = 9; Nx2, n = 8). Controls were sham operated (SO) animals (SO1, n = 9; SO2, n = 9).

Results

The experimental model was corresponded to the initial stages of CKD (Ur level was between 6.64 and 13.36 mmol/L, serum Cr increased less than 50% vs SO, RF did not exceed 22 % FOV). Serum concentration of Klotho and rKl expression decreased, while MMI, CM thickness increased in both Nx1 and Nx2 (Table). There were no differences in serum Pi, FGF23 and PTH levels in Nx1 vs SO (Table). In Nx2 Klotho decline was accompanied by the development of IF and PF, serum 25OHD decrease, Pi and FGF23 increase vs SO2 (Table). In multiple regression analyzes including SO and Nx animals rKl was independently associated with MMI (β = -0.38 ± 0.16, p = 0.026), and CM thickness (β = -0.64 ± 0.14, p <0.001). Myocardial fibrosis was associated with rKl level (IF: β = -0.87 ± 0.19, p = 0.001) and serum 25OHD (PF: β =-0.34 ± 0.15, p=0.032). According to pilot data of IHC, in Nx rat myocardium Klotho protein expression decreased, cytoplasmic calcineurin and nuclear NFAT in CM were observed (Figure), suggesting an increase in the activity of the calcineurin / NFAT signaling pathway. An intracellular redistribution of β-catenin from the cytoskeleton to the cytoplasm and nuclei were observed in CM of Nx rats (Figure), likely corresponding to increase in the activity of Wnt signaling.

Conclusion

Associations of CM hypertrophy, myocardial fibrosis and Klotho decline in CKD may be mediated by calcineurin / NFAT and Wnt signaling in myocardium.