Urinary biomarkers to predict CKD: is the future in multi-marker panels?

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Urinary peptide-based classifier CKD273: towards clinical application in chronic kidney disease

Capillary electrophoresis coupled with mass spectrometry (CE-MS) has been used as a platform for discovery and validation of urinary peptides associated with chronic kidney disease (CKD). CKD affects ∼ 10% of the population, with high associated costs for treatments. A urinary proteome-based classifier (CKD273) has been discovered and validated in cross-sectional and longitudinal studies to assess and predict the progression of CKD. It has been implemented in studies employing cohorts of > 1000 patients. CKD273 is commercially available as an in vitro diagnostic test for early detection of CKD and is currently being used for patient stratification in a multicentre randomized clinical trial (PRIORITY). The validity of the CKD273 classifier has recently been evaluated applying the Oxford Evidence-Based Medicine and Southampton Oxford Retrieval Team guidelines and a letter of support for CKD273 was issued by the US Food and Drug Administration. In this article we review the current evidence published on CKD273 and the challenges associated with implementation. Definition of a possible surrogate early endpoint combined with CKD273 as a biomarker for patient stratification currently appears as the most promising strategy to enable the development of effective drugs to be used at an early time point when intervention can still be effective

PeptiCKDdb-peptide- and protein-centric database for the investigation of genesis and progression of chronic kidney disease

The peptiCKDdb is a publicly available database platform dedicated to support research in the field of chronic kidney disease (CKD) through identification of novel biomarkers and molecular features of this complex pathology. PeptiCKDdb collects peptidomics and proteomics datasets manually extracted from published studies related to CKD. Datasets from peptidomics or proteomics, human case/control studies on CKD and kidney or urine profiling were included. Data from 114 publications (studies of body fluids and kidney tissue: 26 peptidomics and 76 proteomics manuscripts on human CKD, and 12 focusing on healthy proteome profiling) are currently deposited and the content is quarterly updated. Extracted datasets include information about the experimental setup, clinical study design, discovery-validation sample sizes and list of differentially expressed proteins (P-value < 0.05). A dedicated interactive web interface, equipped with multiparametric search engine, data export and visualization tools, enables easy browsing of the data and comprehensive analysis. In conclusion, this repository might serve as a source of data for integrative analysis or a knowledgebase for scientists seeking confirmation of their findings and as such, is expected to facilitate the modeling of molecular mechanisms underlying CKD and identification of biologically relevant biomarkers.Database URL: www.peptickddb.com

Proteomic prediction and Renin angiotensin aldosterone system Inhibition prevention Of early diabetic nephRopathy in TYpe 2 diabetic patients with normoalbuminuria (PRIORITY): essential study design and rationale of a randomised clinical multicentre trial

Introduction: Diabetes mellitus affects 9% of the European population and accounts for 15% of healthcare expenditure, in particular, due to excess costs related to complications. Clinical trials aiming for earlier prevention of diabetic nephropathy by renin angiotensin system blocking treatment in normoalbumuric patients have given mixed results. This might reflect that the large fraction of normoalbuminuric patients are not at risk of progression, thereby reducing power in previous studies. A specific risk classifier based on urinary proteomics (chronic kidney disease (CKD)273) has been shown to identify normoalbuminuric diabetic patients who later progressed to overt kidney disease, and may hold the potential for selection of high-risk patients for early intervention. Combining the ability of CKD273 to identify patients at highest risk of progression with prescription of preventive aldosterone blockade only to this high-risk population will increase power. We aim to confirm performance of CKD273 in a prospective multicentre clinical trial and test the ability of spironolactone to delay progression of early diabetic nephropathy. Methods and analysis: Investigator-initiated, prospective multicentre clinical trial, with randomised double-masked placebo-controlled intervention and a prospective observational study. We aim to include 3280 type 2 diabetic participants with normoalbuminuria. The CKD273 classifier will be assessed in all participants. Participants with high-risk pattern are randomised to treatment with spironolactone 25 mg once daily, or placebo, whereas, those with low-risk pattern will be observed without intervention other than standard of care. Treatment or observational period is 3 years. The primary endpoint is development of confirmed microalbuminuria in 2 of 3 first morning voids urine samples. Ethics and dissemination: The study will be conducted under International Conference on Harmonisation – Good clinical practice (ICH-GCP) requirements, ethical principles of Declaration of Helsinki and national laws. This first new biomarker-directed intervention trial aiming at primary prevention of diabetic nephropathy may pave the way for personalised medicine approaches in treatment of diabetes complications

Visual Snow Syndrome Improves With Modulation of Resting-State Functional MRI Connectivity After Mindfulness-Based Cognitive Therapy: An Open-Label Feasibility Study

BACKGROUND: Visual snow syndrome (VSS) is associated with functional connectivity (FC) dysregulation of visual networks (VNs). We hypothesized that mindfulness-based cognitive therapy, customized for visual symptoms (MBCT-vision), can treat VSS and modulate dysfunctional VNs. METHODS: An open-label feasibility study for an 8-week MBCT-vision treatment program was conducted. Primary (symptom severity; impact on daily life) and secondary (WHO-5; CORE-10) outcomes at Week 9 and Week 20 were compared with baseline. Secondary MRI outcomes in a subcohort compared resting-state functional and diffusion MRI between baseline and Week 20. RESULTS: Twenty-one participants (14 male participants, median 30 years, range 22-56 years) recruited from January 2020 to October 2021. Two (9.5%) dropped out. Self-rated symptom severity (0-10) improved: baseline (median [interquartile range (IQR)] 7 [6-8]) vs Week 9 (5.5 [3-7], P = 0.015) and Week 20 (4 [3-6], P < 0.001), respectively. Self-rated impact of symptoms on daily life (0-10) improved: baseline (6 [5-8]) vs Week 9 (4 [2-5], P = 0.003) and Week 20 (2 [1-3], P < 0.001), respectively. WHO-5 Wellbeing (0-100) improved: baseline (median [IQR] 52 [36-56]) vs Week 9 (median 64 [47-80], P = 0.001) and Week 20 (68 [48-76], P < 0.001), respectively. CORE-10 Distress (0-40) improved: baseline (15 [12-20]) vs Week 9 (12.5 [11-16.5], P = 0.003) and Week 20 (11 [10-14], P = 0.003), respectively. Within-subject fMRI analysis found reductions between baseline and Week 20, within VN-related FC in the i) left lateral occipital cortex (size = 82 mL, familywise error [FWE]-corrected P value = 0.006) and ii) left cerebellar lobules VIIb/VIII (size = 65 mL, FWE-corrected P value = 0.02), and increases within VN-related FC in the precuneus/posterior cingulate cortex (size = 69 mL, cluster-level FWE-corrected P value = 0.02). CONCLUSIONS: MBCT-vision was a feasible treatment for VSS, improved symptoms and modulated FC of VNs. This study also showed proof-of-concept for intensive mindfulness interventions in the treatment of neurological conditions

Action of Hexachlorobenzene on tumor growth and metastasis in different experimental models

Hexachlorobenzene (HCB) is a widespread organochlorine pesticide, considered a possible human carcinogen. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR). We have found that HCB activates c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways and cell migration, in an AhR-dependent manner in MDA-MB-231 breast cancer cells. The aim of this study was to investigate in vitro the effect of HCB (0.005, 0.05, 0.5, 5μM) on cell invasion and metalloproteases (MMPs) 2 and 9 activation in MDA-MB-231 cells. Furthermore, we examined in vivo the effect of HCB (0.3, 3, 30mg/kg b.w.) on tumor growth, MMP2 and MMP9 expression, and metastasis using MDA-MB-231 xenografts and two syngeneic mouse breast cancer models (spontaneous metastasis using C4-HI and lung experimental metastasis using LM3). Our results show that HCB (5μM) enhances MMP2 expression, as well as cell invasion, through AhR, c-Src/HER1 pathway and MMPs. Moreover, HCB increases MMP9 expression, secretion and activity through a HER1 and AhR-dependent mechanism, in MDA-MB-231 cells. HCB (0.3 and 3mg/kg b.w.) enhances subcutaneous tumor growth in MDA-MB-231 and C4-HI in vivo models. In vivo, using MDA-MB-231 model, the pesticide (0.3, 3 and 30mg/kg b.w.) activated c-Src, HER1, STAT5b, and ERK1/2 signaling pathways and increased MMP2 and MMP9 protein levels. Furthermore, we observed that HCB stimulated lung metastasis regardless the tumor hormone-receptor status. Our findings suggest that HCB may be a risk factor for human breast cancer progression.Fil: Pontillo, Carolina Andrea. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina;Fil: Rojas, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina;Fil: Chiappini, Florencia Ana. Consejo Nacional de Investigaciones Científicas y Técnicas . Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina; Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina;Fil: Sequeira, Gonzalo Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina;Fil: Cocca, Claudia Marcela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Físico Matemática; Argentina;Fil: Crocci, Máximo. Instituto de Inmunooncología Crescenti; Argentina;Fil: Colombo, Lucas Luis. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología; Argentina;Fil: Lanari, Claudia Lee Malvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina;Fil: Kleiman de Pisarev, Diana. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina;Fil: Randi, Andrea Silvana. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina

Hexachlorobenzene promotes angiogenesis in vivo, in a breast cancer model and neovasculogenesis in vitro, in the human microvascular endothelial cell line HMEC-1

Exposure to environmental pollutants may alter proangiogenic ability and promotes tumor growth. Hexachlorobenzene (HCB) is an organochlorine pesticide found in maternal milk and in lipid foods, and a weak ligand of the aryl hydrocarbon receptor (AhR). HCB induces migration and invasion in human breast cancer cells, as well as tumor growth and metastasis in vivo. In this study, we examined HCB action on angiogenesis in mammary carcinogenesis. HCB stimulates angiogenesis and increases vascular endothelial growth factor (VEGF) expression in a xenograft model with the human breast cancer cell line MDA-MB-231. Human microvascular endothelial cells HMEC-1 exposed to HCB (0.005, 0.05, 0.5 and 5 μM) showed an increase in cyclooxygenase-2 (COX-2) and VEGF protein expression involving AhR. In addition, we found that HCB enhances VEGF-Receptor 2 (VEGFR2) expression, and activates its downstream pathways p38 and ERK1/2. HCB induces cell migration and neovasculogenesis in a dose-dependent manner. Cells pretreatment with AhR, COX-2 and VEGFR2 selective inhibitors, suppressed these effects. In conclusion, our results show that HCB promotes angiogenesis in vivo and in vitro. HCB-induced cell migration and tubulogenesis are mediated by AhR, COX-2 and VEGFR2 in HMEC-1. These findings may help to understand the association among HCB exposure, angiogenesis and mammary carcinogenesis.Fil: Pontillo, Carolina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; ArgentinaFil: Español, Alejandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Chiappini, Florencia Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Miret, Noelia Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; ArgentinaFil: Cocca, Claudia Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Físico Matemática; ArgentinaFil: Alvarez, Laura. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; ArgentinaFil: Kleiman, Diana Leonor. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; ArgentinaFil: Sales, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Randi, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentin

Epigenetic changes induced by pesticide exposure reactivate LINE-1 retrotransposon in breast cancer and mammary epithelial cells

Expression of long interspersed nuclear element-1 (LINE-1) is reactivated during breast cancer initiation and progression. Strong ligands of aryl hydrocarbon receptor (AhR) activate LINE-1 through the transforming growth factor-β1 (TGF-β1)/Smad pathway. Studies have linked breast cancer risk with pesticide exposure, including hexachlorobenzene (HCB) and chlorpyrifos (CPF), both weak AhR ligands which promote alterations in mammary gland and tumor growth in animal models. We examined the pesticides action on LINE-1 reactivation in MDA-MB-231 breast cancer cells and NMuMG epithelial breast cells, and we evaluated the role of TGF-β1 and AhR. Results show that 0.5 μM CPF and 0.005 μM HCB reduced the methylation of the 5´-UTR of LINE-1 and increased LINE-1 mRNA expression via Smad and AhR signaling in MDA-MB-231. Besides, 5 μM CPF and 0.005 μM HCB heighten ORF1p nuclear import, the protein encoded by LINE-1, through TGF-β1/Smad and stimulate DNA double-strand breaks. Disturbingly, 5 μM CPF and 0.005 μM HCB also enhanced LINE-1 mRNA levels in NMuMG cells. CPF effect was through AhR and TGF-β1, while HCB action depends only of AhR. In addition, both pesticides increased ORF1p expression and nuclear localization. In conclusion, HCB and CPF induce LINE-1 reactivation, not only in breast cancer cells but also in epithelial mammary cells, supporting the idea that pesticide exposure could promote epigenetic changes, contributing to cell transformation and tumorigenesis in breast cancer.Fil: Miret, Noelia Victoria. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zappia, Carlos Daniel. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Altamirano, Gabriela Anahí. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pontillo, Carolina Andrea. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zárate, Lorena Vanesa. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gomez, Ayelen Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; ArgentinaFil: Lasagna, Marianela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Físico Matemática; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cocca, Claudia Marcela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Físico Matemática; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kass, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; ArgentinaFil: Monczor, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Randi, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; ArgentinaBuenos Aires Breast Cancer SymposiumBuenos AiresArgentinaAsocacion Argentina de Oncología ClinicaSociedad Argentina de Investigación ClinicaInstituto Nacional del Cance

Comparison of urine and plasma peptidome indicates selectivity in renal peptide handling

Purpose: Urine is considered to be produced predominantly as a result of plasma filtration in the kidney. However, the origin of the native peptides present in urine has never been investigated in detail. Therefore, we aimed to obtain a first insight into the origin of urinary peptides based on a side‐by‐side comprehensive analysis of the plasma and urine peptidome. Methods: Twenty‐two matched urine and plasma samples were analyzed for their peptidome using capillary electrophoresis coupled to mass spectrometry (CE‐MS; for relative quantification) and CE‐ or LC coupled to tandem mass spectrometry (CE‐ or LC‐ MS/MS; for peptide identification). The overlap and association of abundance of the different peptides present in these two body fluids were evaluated. Results: We were able to identify 561 plasma and 1461 urinary endogenous peptides. Only 90 peptides were detectable in both urine and plasma. No significant correlation was found when comparing the abundance of these common peptides, with the exception of collagen fragments. This observation was also supported when comparing published peptidome data from both plasma and urine. Conclusions and clinical relevance: Most of the plasma peptides are not detectable in urine, possibly due to tubular reabsorption. The majority of urinary peptides may in fact originate in the kidney. The notable exception is collagen fragments, which indicates potential selective exclusion of these peptides from tubular reabsorption. Experimental verification of this hypothesis is warranted