Non-invasive Biomarkers Research Articles

An Updated Review on Molecular Biomarkers in Diagnosis and Therapy of Colorectal Cancer.

Colorectal cancer is one of the most common cancer types worldwide. Since colorectal cancer takes time to develop, its incidence and mortality can be treated effectively if it is detected in its early stages. As a result, non-invasive or invasive biomarkers play an essential role in the early diagnosis of colorectal cancer. Many experimental studies have been carried out to assess genetic, epigenetic, or protein markers in feces, serum, and tissue. It may be possible to find biomarkers that will help with the diagnosis of colorectal cancer by identifying the genes, RNAs, and/or proteins indicative of cancer growth. Recent advancements in the molecular subtypes of colorectal cancer, DNA methylation, microRNAs, long noncoding RNAs, exosomes, and their involvement in colorectal cancer have led to the discovery of numerous new colorectal cancer biomarkers. In small-scale investigations, most biomarkers appear promising. However, large-scale clinical trials are required to validate their effectiveness before routine clinical implementation. Hence, this review focuses on small-scale investigations and results of big data analysis that may provide an overview of the biomarkers for the diagnosis, therapy, and prognosis of colorectal cancer.

Isocitrate dehydrogenase 1 upregulation in urinary extracellular vesicles from proximal tubules of type 2 diabetic rats.

Diabetic nephropathy (DN) is a major cause of chronic kidney disease. Microalbuminuria is currently the most common non-invasive biomarker for the early diagnosis of DN. However, renal structural damage may have advanced when albuminuria is detected. In this study, we sought biomarkers for early DN diagnosis through proteomic analysis of urinary extracellular vesicles (uEVs) from type 2 diabetic model rats and normal controls. Isocitrate dehydrogenase 1 (IDH1) was significantly increased in uEVs from diabetic model rats at the early stage despite minimal differences in albuminuria between the groups. Calorie restriction significantly suppressed the increase in IDH1 in uEVs and 24-hour urinary albumin excretion, suggesting that the increase in IDH1 in uEVs was associated with the progression of DN. Additionally, we investigated the origin of IDH1-containing uEVs based on their surface sugar chains. Lectin affinity enrichment and immunohistochemical staining showed that IDH1-containing uEVs were derived from proximal tubules. These findings suggest that the increase in IDH1 in uEVs reflects pathophysiological alterations in the proximal tubules and that IDH1 in uEVs may serve as a potential biomarker of DN in the proximal tubules.

Non-invasive Scores and Serum Biomarkers for Fatty Liver in the Era of Metabolic Dysfunction-associated Steatotic Liver Disease (MASLD): A Comprehensive Review From NAFLD to MAFLD and MASLD.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is rapidly increasing worldwide, making it the leading cause of liver related morbidity and mortality. Currently, liver biopsy is the gold standard for assessing individuals with steatohepatitis and fibrosis. However, its invasiveness, sampling variability, and impracticality for large-scale screening has driven the search for non-invasive methods for early diagnosis and staging. In this review, we comprehensively summarise the evidence on the diagnostic performance and limitations of existing non-invasive serum biomarkers and scores in the diagnosis and evaluation of steatosis, steatohepatitis, and fibrosis. Several non-invasive serum biomarkers and scores have been developed over the last decade, although none has successfully been able to replace liver biopsy. The introduction of new NAFLD terminology, namely metabolic dysfunction-associated fatty liver disease (MAFLD) and more recently metabolic dysfunction-associated steatotic liver disease (MASLD), has initiated a debate on the interchangeability of these terminologies. Indeed, there is a need for more research on the variability of the performance of non-invasive serum biomarkers and scores across the diagnostic entities of NAFLD, MAFLD and MASLD. There remains a significant need for finding valid and reliable non-invasive methods for early diagnosis and assessment of steatohepatitis and fibrosis to facilitate prompt risk stratification and management to prevent disease progression and complications. Further exploration of the landscape of MASLD under the newly defined disease subtypes is warranted, with the need for more robust evidence to support the use of commonly used serum scores against the new MASLD criteria and validation of previously developed scores.

Sodium signal intensity of CSF using 1H-guided 23Na-MRI as a potential noninvasive biomarker in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by cognitive decline and mnestic deficits. The pathophysiology of AD is not fully understood, which renders the development of accurate tools for early diagnosis and effective therapies exceedingly difficult. In this study, we investigated the use of 23Na-MRI to measure the relative sodium signal intensities (rSSIs) in CSF in patients with AD and healthy controls. We prospectively recruited 11 patients with biomarker-diagnosed early-stage AD, as well as 12 cognitively healthy age-matched controls. All participants underwent 23Na-MRI to measure rSSI. Statistical analyses were performed to compare CSF sodium signal intensities between groups and to evaluate the specificity and sensitivity of the rSSI in the diagnosis of AD. RSSIs in CSF were significantly higher in AD patients (mean=68.6%±7.7%) compared to healthy controls (mean=56.9%±5.5%) (p<.001). There was also a significant negative correlation between rSSI in CSF and hippocampus and amygdala volumes (r=-.54 and -.49, p<.05) as well as a positive correlation with total CSF volumes (r=.81, p<.05). Receiver operating characteristic analysis showed high diagnostic accuracy for rSSI in discriminating between AD patients and healthy controls (area under the curve=.94). Our study provides evidence that rSSI in CSF is increased in AD patients in comparison to healthy controls. rSSI may serve as a potential marker for early detection and monitoring of disease progression. Larger, longitudinal studies are needed to confirm our findings and to investigate the association between rSSI in CSF and the severity of cognitive impairment.

A Four-Gene Panel in Rectal Swab Samples as a Biomarker for Colorectal Cancer Screening

Background: The dysregulation of gene expression is one of the key molecular features of colorectal cancer (CRC) development. This study aimed to investigate whether such dysregulation is reflected in rectal swab specimens of CRC patients and to evaluate its potential as a non-invasive approach for screening. Methods: We compared the expression level of 14 CRC-associated genes in tumor and adjacent non-tumor tissue of CRC patients and examined the correlation of their levels in tissue with paired rectal swab specimens. The level of these 14 genes in rectal swab specimens was compared among patients with CRC or polyp and control subjects, and the diagnostic potential of each dysregulated gene and the gene panel were evaluated. Results: The expression of CXCR2, SAA, COX1, PPARδ, PPARγ, Groγ, IL8, p21, c-myc, CD44 and CSF1 was significantly higher in CRC, and there was a significant correlation in the levels of most of them between the CRC and rectal swab specimens. In the training study, we showed that CD44, IL8, CXCR2 and c-myc levels were significantly higher in the rectal swab specimens of the CRC patients. Such result was confirmed in the validation study. A panel of these four genes was developed, and ROC analysis showed that this four-gene panel could identify CRC patients with an AUC value of 0.83 and identify overall polyp and precancerous adenoma patients with AUC values of 0.6522 and 0.7322, respectively. Finally, the predictive study showed that the four-gene panel demonstrated sensitivities of 63.6%, 76.9% and 88.9% in identifying overall polyp, precancerous adenoma and CRC patients, respectively, whereas the specificity for normal subjects was 72.2%. Conclusion: The expression of CRC-associated genes in rectal swab specimens reflects the dysregulation status in colorectal tissue, and the four-gene panel is a potential non-invasive biomarker for early precancerous adenoma and CRC screening.

Type IX Collagen Turnover Is Altered in Patients with Solid Tumors

The fibrotic tumor microenvironment, characterized by its intricate extracellular matrix (ECM), consists of many collagens with diverse functions and unexplored biomarker potential. Type IX collagen is a member of the low-abundance collagen family known as the fibril-associated collagen with interrupted triple helices (FACITs) and is found mostly in cartilage. Its role in the tumor microenvironment remains unexplored. To investigate the biomarker potential of a type IX collagen in cancer, an immuno-assay was developed (PRO-C9) and technical assay performance was evaluated for the assessment of serum. PRO-C9 levels were measured in serum samples from 259 patients with various solid tumor types compared to serum levels from 73 healthy controls. PRO-C9 levels were significantly elevated in patients with solid tumors including bladder, breast, colorectal, gastric, head and neck, lung, melanoma, ovarian, pancreatic, and renal compared to levels in healthy controls (p &lt; 0.05–p &lt; 0.0001). PRO-C9 could discriminate between patients with cancer and healthy controls, with the area under the receiver operating characteristic values ranging from 0.58 to 0.86 (p &lt; 0.3–p &lt; 0.0001), indicating potential diagnostic utility. This study suggests that type IX collagen turnover is altered in patients with solid tumors and demonstrates the feasibility of using PRO-C9 as a non-invasive serum-based biomarker with relevance in multiple cancer types. Furthermore, these results underscore the potential utility of PRO-C9 to better elucidate the biology of FACITs in cancers.

Nomogram based on dual-energy CT-derived extracellular volume fraction for the prediction of microsatellite instability status in gastric cancer

PurposeTo develop and validate a nomogram based on extracellular volume (ECV) fraction derived from dual-energy CT (DECT) for preoperatively predicting microsatellite instability (MSI) status in gastric cancer (GC).Materials and methodsA total of 123 patients with GCs who underwent contrast-enhanced abdominal DECT scans were retrospectively enrolled. Patients were divided into MSI (n=41) and microsatellite stability (MSS, n=82) groups according to postoperative immunohistochemistry staining, then randomly assigned to the training (n=86) and validation cohorts (n=37). We extracted clinicopathological characteristics, CT imaging features, iodine concentrations (ICs), and normalized IC values against the aorta (nICs) in three enhanced phases. The ECV fraction derived from the iodine density map at the equilibrium phase was calculated. Univariate and multivariable logistic regression analyses were used to identify independent risk predictors for MSI status. Then, a nomogram was established, and its performance was evaluated by ROC analysis and Delong test. Its calibration performance and clinical utility were assessed by calibration curve and decision curve analysis, respectively.ResultsThe ECV fraction, tumor location, and Borrmann type were independent predictors of MSI status (all P &amp;lt; 0.05) and were used to establish the nomogram. The nomogram yielded higher AUCs of 0.826 (0.729–0.899) and 0.833 (0.675–0.935) in training and validation cohorts than single variables (P&amp;lt;0.05), with good calibration and clinical utility.ConclusionsThe nomogram based on DECT-derived ECV fraction has the potential as a noninvasive biomarker to predict MSI status in GC patients.

Rest and stress aortic flow analysis by CMR in MyoFit46

Abstract Background Aortic stiffness is a strong predictor of future cardiovascular events and all-cause mortality. Aortic stiffness has never been measured during adenosine stress perfusion before, and may provide valuable relationship about this relationship. Aim To investigate if there are differences between aortic stiffness and conditions of rest and stress perfusion, in an older age-homogenous population-based cohort. Methods 203 participants from the longitudinal 1946 Medical Research Council National Survey of Health and Development British birth cohort were included. Cardiovascular Magnetic Resonance (CMR) imaging was performed at rest and during adenosine stress perfusion. 3 established and validated biomarkers of aortic stiffness were reported - pulse wave velocity, distensibility and beta stiffness, at rest and stress perfusion. All variables were non-normally distributed, therefore the Wilcoxon signed-rank test was used to look for significant differences between rest and stress. Results Aortic stiffness increased significantly during adenosine stress perfusion, shown by all biomarkers of aortic stiffness used: pulse wave velocity (p = &amp;lt;0.001), distensibility (p = &amp;lt;0.001), and beta stiffness (p = &amp;lt;0.001). However, the aortas of participants with a higher resting aortic stiffness at baseline were less dynamic (little if any aortic stiffness change with stress) when compared to those with low resting aortic stiffness. Conclusion It is feasible to measure aortic stiffness during adenosine stress perfusion. In healthy aortas, aortic stiffness increases in the acute phase of adenosine stress suggesting a new potential mechanism for the body response to adenosine – peripheral chemoreceptor reflex activation. Stiffer arteries at baseline are less modifiable and stress aortic flow CMR has the potential to identify these persons permitting early targeted therapy. Change in aortic stiffness, measured by stress aortic flow CMR is therefore a potential non-invasive biomarker of aortic health.

Role of miR-128-3p and miR-195-5p as biomarkers of coronary artery disease in Indians: a pilot study

Coronary artery disease (CAD) imposes a significant economic burden in developing countries like India. Timely diagnosis and treatment should be prioritized to mitigate the disease. Current diagnostic tools being invasive and less specific raise the need to develop less invasive and more reliable molecular biomarkers. MicroRNAs (miRNAs) are an emerging class of molecules that can serve as a potential source of non-invasive biomarkers for CAD. The objective of this study was to determine the potential of circulatory miRNAs as diagnostic biomarkers in CAD. In this study, we have reported two microRNAs, miR-128-3p and miR-195-5p in the serum of CAD patients in Indian Population. A total of 124 subjects were recruited which included 89 angiographically proven CAD patients and 35 control subjects. Our results show a significant decrease in the levels of miR-128-3p in CAD patients while there were no significant changes in the levels of miR-195-5p. Further bioinformatics analysis revealed the potential role of miR-128-3p in cholesterol homeostasis. Altered homeostasis due to cholesterol accumulation in macrophages is the driving force behind formation of foam cells which in turn accelerates the progression of CAD. Here, we have shown that miR-128-3p increases cholesterol levels in macrophages by decreasing cholesterol efflux in-vitro.

#1933 Urinary proteomics identifying the novel biomarker associated with crescentic glomerulonephritis

Abstract Background and Aims As a severe progressive acute renal injury, crescentic glomerulonephritis requires timely pathology diagnosis and treatment. New non-invasive biomarkers will be crucial when the renal biopsy is not available or unacceptable. Urinary proteomics, specifically data-independent acquisition (DIA) proteomics, might provide some potential indicators. Method Urine proteomics examination was on patients with crescentic glomerulonephritis(n = 15), chronic glomerulonephritis (n = 15), acute kidney injury (AKI) without glomerular crescentic(n = 10), and 15 healthy controls. The crescentic glomerulonephritis group is the patients with extensive glomerular crescents over 50%. We performed LC-MS/MS analysis to identify differentially expressed proteins (DEPs), Ingenuity Pathway Analysis (IPA), and the proteome map for significant pathways and crucial proteins among patients and controls. The signature proteins were validated using enzyme-linked immunosorbent assay (ELISA) analysis. Results A total of 55 population, males occupied 45.45%, with the average age being 53 ± 13 years old. A volcano plot among crescentic glomerulonephritis and control groups is in Fig. 1. IPA analysis showed that neutrophil degranulation and complement cascade were the top two pathways in crescentic glomerulonephritis but not in the healthy and nephritis groups. The activated neddylation pathway in patients with crescentic glomerulonephritis compared with AKI patients. After integrating the DEPs among three groups via the Venn plot, we observed eight DEPs significantly associated with the crescentic glomerulonephritis, among which Coagulation factor V (F5), Phospholipid transfer protein (PLTP), and Alcohol dehydrogenase 1C (ADH1C) were significant proteins. Biomarker analysis showed the area under the curve values for predicting crescentic glomerulonephritis were 0.867, 0.851, and 0.815, respectively (P &amp;lt; 0.001). Conclusion Urine proteomics holds promise in identifying the crescentic glomerulonephritis patient, thus providing a potential value as a noninvasive biomarker.

Non-Coding RNAs in HIV Infection, NeuroHIV, and Related Comorbidities

NeuroHIV affects approximately 30–60% of people living with HIV-1 (PLWH) and is characterized by varying degrees of cognitive impairments, presenting a multifaceted challenge, the underlying cause of which is chronic, low-level neuroinflammation. Such smoldering neuroinflammation is likely an outcome of lifelong reliance on antiretrovirals coupled with residual virus replication in the brains of PLWH. Despite advancements in antiretroviral therapeutics, our understanding of the molecular mechanism(s) driving inflammatory processes in the brain remains limited. Recent times have seen the emergence of non-coding RNAs (ncRNAs) as critical regulators of gene expression, underlying the neuroinflammatory processes in HIV infection, NeuroHIV, and their associated comorbidities. This review explores the role of various classes of ncRNAs and their regulatory functions implicated in HIV infection, neuropathogenesis, and related conditions. The dysregulated expression of ncRNAs is known to exacerbate the neuroinflammatory responses, thus contributing to neurocognitive impairments in PLWH. This review also discusses the diagnostic and therapeutic potential of ncRNAs in HIV infection and its comorbidities, suggesting their utility as non-invasive biomarkers and targets for modulating neuroinflammatory pathways. Understanding these regulatory roles could pave the way for novel diagnostic strategies and therapeutic interventions in the context of HIV and its comorbidities.

#3087 Phenotype of extracellular vesicles in diabetic kidney disease with albuminuria

Abstract Background and Aims Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD) and affects more than 40% of patients with type 2 diabetes mellitus (T2D). The pathogenesis of CKD is multifactorial and leads to a clinical presentation with diverse phenotypes characterised by proteinuria and/or progressive deterioration of renal function, thus urging the search for new non-invasive biomarkers that allow early diagnosis and the development of new therapeutic targets. Urinary extracellular vesicles (EVs) have emerged as an alternative, as changes in their number and composition have been described in clinical conditions associated with DM and kidney disease. EVs are able to interact with neighbouring and/or distant cells by transferring their cargo, rich in proteins, lipids or nucleic acids, from progenitor cells to recipient cells, thus participating in cell-to-cell communication processes. The aim of the present study was to analyse the different phenotypes of EVs in T2D patients with different degrees of albuminuria compared to healthy controls, as well as their correlation with the degree of albuminuria. Method A cohort of 69 subjects (49 patients with T2DM and 20 healthy volunteers) was analysed. The phenotype of EVu present between the two groups was assessed, as well as the association between the different cell lines and the presence of albuminuria. Statistical analysis was performed using Spearman correlations. Analyses were performed with R software. Results A positive correlation was observed between EVu from podocytes (PODO+) and those from macrophagocytes (R = 0.3180, p-value = 0.0082), as well as a positive association between PODO+ and neutrophil EVu, both in the whole cohort and when analysing only patients with diabetes (R = 0.3225, p-value:0.0254). When assessing the association of EVu with the degree of albuminuria, we observed a direct association with proximal tubule EVu (R: 0.3158, p-value 0.0271) both in all subjects (T2D and HV) and only in those with T2D (R = 0.4200, p-value = 0.0056). When stratifying according to the degree of albuminuria (A1, A2 and A3), we still observed a dependent association of the degree of glomerular involvement with the number of EVu of TCP (A1: mean 5.5 (SD 4.1); A2: mean 12.3 (SD 30.9); A3: mean 132.5 (SD 322.8)) p-value: 0.0609. Conclusion The results presented suggest a relevant role of TCP in the glomerular involvement of patients with DKD, in line with the emergence of new therapeutic lines such as iSGLT2. The study suggests new lines of research aimed at developing therapeutic targets that assess the cellular expression of EVu.

#1776 Distinct changes in gut microbiota of patients with kidney graft rejection

Abstract Background and Aims Kidney graft rejection still represents the major cause of graft loss in kidney transplant recipients. Of growing interest is the bidirectional relationship between gut microbiome and immune system suggesting that gut microbiota can affect allograft outcome. Method In this cross-sectional case control study, we characterized the gut microbial profile of adult renal transplant recipients with and without graft rejection in order to define a cohort-specific microbial fingerprint through 16S rRNA gene sequencing. We used very strict inclusion and exclusion criteria to address confounder of microbiota composition. Results Different relative abundances in several gut microbial taxa were detectable in control patients compared to patients with kidney allograft rejection. Alpha diversity was lower in the rejection group and beta diversity revealed dissimilarity between patients with and without kidney graft rejection (P &amp;lt; .01). When the rejection group was stratified according to different types of allograft rejection, major changes were identified between patients with chronic T cell mediated rejection (cTCMR) and controls. Changes in alpha diversity within the gut microbiome were related to the probability of cTCMR (P &amp;lt; .05). Kidney transplant patients without rejection showed significant enrichment of rather anti-inflammatory taxa whereas in the rejection group bacteria well known for their role in chronic inflammation were increased. For example, ASV 362 belonging to the genus Bacteroides and ASV 312 belonging to Tanerrelaceae were enriched in no rejection (P &amp;lt; .001 and P &amp;lt; .01) whereas ASV 365 was enriched in patients with allograft rejection (P = .04). Looking at metagenomic functions, a higher abundance of genes coding for enzymes involved in bacterial multidrug resistance and processing of short chain fatty acids was found in patients without rejection but an increase in enzymes involved in NADPH production was seen in patients with allograft rejection. Conclusion A distinct microbial fingerprint of patients with allograft rejection might serve as non-invasive biomarker in the future.

#2229 miR-186, a potential noninvasive diagnostic biomarker of Membranous nephropathy in urinary microparticles

Abstract Background and Aims Membranous nephropathy (MN) is one of the most common causes of nephrotic syndrome, and is associated with podocyte damage and proteinuria. Invasive biopsy is the gold-standard diagnostic method for this disease. The use of specific biomarkers, such as microparticles (MPs) originating from damaged podocytes released in a patient's urine, has been proposed as an early, noninvasive, and repetitive diagnostic method. According to the previous studies, MPs derived from podocytes are present in the urine of patients with different glomerulonephritis, such as diabetic nephropathy, lupus nephritis, renal injury in preeclampsia, and renovascular hypertensive disorders. The analysis of urinary microparticle-derived miRNAs from patients with kidney diseases can help detect diagnostic biomarkers. This study aimed to analyze podocyte damage-related miRNAs in MN, miR-30c, miR-135, and miR-18 in the urinary MP of MN patients to identify appropriate candidate biomarkers for MN Method Urine samples from 30 patients with membranous nephropathy and 16 healthy individuals were collected to extract podocyte MPs using ultracentrifugation. Characterization of these particles was determined using the specific protein marker of MPs, TSG-101, and the podocyte-specific marker Nephrin, by western blotting, and SEM was used to check the size and morphology. The expression levels of microRNAs extracted from podocyte MPs, miR-30c, miR-135, and miR-186, were also evaluated by real-time PCR. Results The evaluation of the characteristics of MPs showed that there were many particles with a size of more than 100 nm in the urine sample, and there were higher levels of the specific markers TSG-101 and Nephrin in the urine samples of MGN patients than in the control samples (P &amp;lt; 0.001). The expression level of miR-186 was compared between the three studied groups and based on the results of the expression of this miRNA in the group of membranous nephropathy patients compared to healthy people group, there was a significant increase in expression (P = 0.000) Conclusion This study showed that although the expression levels of miR-186 in podocyte MPs samples extracted from the urine of patients with MN were significantly changed compared to the studied controls, more studies should be performed to determine the diagnostic values.

Non-invasive myocardial tissue deformation and discoordination indices predict cardiac allograft vasculopathy in pediatric heart transplantation patients.

There is an urgent need for non-invasive imaging-based biomarkers suitable for diagnostic surveillance of cardiac allograft vasculopathy (CAV) in pediatric heart transplant (PHT) patients. The purpose of this study was to comprehensively investigate left ventricular (LV) myocardial deformation in conjunction with electromechanical discoordination in PHT. PHT patients with and without CAV were evaluated for echocardiography derived global longitudinal strain (GLS) and electromechanical discoordination indices including systolic stretch fraction (SSF) and diastolic relaxation fraction (DRF). SSF was increased in CAV(+) patients at the time of CAV diagnosis (median CAV(+) 5.0 vs. median CAV(-) 0.0, P = 0.008) and in the echocardiogram preceding the CAV diagnosis (median CAV(+) 29.0 vs. median CAV(-) 0.0, P < 0.001). DRF was also increased in the echocardiogram that preceded CAV diagnosis in CAV(+) patients (0.31 ± 0.08 vs. 0.25 ± 0.05, P = 0.008). The final model using indices 6-12 months prior to CAV diagnosis included GLS, SSF, and DRF providing AUC of 0.94 with sensitivity 98.5%, specificity 80.0%, positive predictive value 85.0%, and negative predictive value 94.1%. Systolic and diastolic electro-mechanical discoordination indices are significantly worse in PHT patients experiencing CAV. Non-invasive imaging guided surveillance using echocardiographic myocardial deformation indices can be improved by adding SSF and DRF to standard GLS measurements.

#1940 Plasma complement activation profile in kidney transplant recipients with IgAN supports the importance of the complement alternative pathway in IgAN

Abstract Background and Aims The alternative complement and lectin pathways are key players in IgA nephropathy (IgAN). However, the precise dynamics of complement activation in IgAN remain poorly understood. Non-invasive complement biomarkers are urgently needed in view of the evolving landscape of complement-targeting therapies under evaluation in IgAN. Method We analyzed plasma complement activation biomarkers in a cohort of 87 kidney transplant recipients (KTR) with IgAN and 30 controls consisting of autosomal dominant polycystic kidney disease (ADPKD) KTR (NCT05234463, DC-2013-1990). Patients were excluded if 1) they had a biological inflammatory syndrome at the time of sampling or 2) they had experienced a cellular/humoral rejection in the year prior to or after sampling. We simultaneously measured a large panel of complement activation and regulation fragments (C3a, C5a, C4a, sC5b-9, Ba, Bb) and intact proteins (C3, C4, FI, FH, properdin) in plasma using multiplex ELISA (Quidel Ortho®, A900), nephelometry and in-house ELISA. Results There was no consumption of C3 and C4 proteins in IgAN patients. However, C3a and Bb concentrations were elevated in IgAN patients compared to ADPKD patients (161 [98-227] vs. 102 [66-172] ng/mL, p = 0.007; 3.0 [2.6-4.0] vs. 2.5 [2.0-2.8] µg/mL, p &amp;lt; 0.0001). Overall, 70/87 IgAN patients (80.5%) had elevated Bb levels. The plasma concentration of C5a was elevated in IgAN patients compared to ADPKD patients (6.3 [3.8-9.2] vs. 4.9 [2.8-6.3] ng/mL, p = 0.0274), with values within the reference range in 92.0% of IgAN patients. There was no difference in plasma levels of sC5b-9 between the two groups (p = 0.186). The combined analysis of C3a, C5a and sC5b-9 allowed us to distinguish 4 clusters of patients (Fig. 1). A total of 48 patients (55.2%) show evidence of C3 convertase activity in plasma (clusters 3 and 4), including 16 patients (18.4%) with the highest plasma concentrations of sC5b-9, C3a and Bb. In contrast, 15 patients (17.2%, cluster 1) have no (or very low) complement activation. The last group (n = 32, 36.8%, cluster 2) shows an isolated increase in sC5b-9 without evidence of plasma C3 convertase activity. Proteinuria was significantly increased in groups 3 and 4, which had the highest C3a concentrations, compared to groups 1 and 2 (p = 0.0051). Conclusion This study provides insights into the mechanisms of complement activation in IgAN, with a pivotal role for the activity and regulation of the alternative C3 pathway convertase. In addition, the combined analysis of complement biomarkers identified, for the first time, clusters of patients who may benefit from complement targeting therapies.

Quantitative Abdominal Arterial Calcification Correlates with Kidney Transplant Waitlist Mortality.

The scarcity of available organs for kidney transplantation has resulted in a substantial waiting time for patients with End Stage Kidney Disease (ESKD). This prolonged wait contributes to an increased risk of cardiovascular mortality. Calcification of large arteries is a high-risk factor in the development of cardiovascular diseases, and it is common among candidates for kidney transplant. The aim of this study is to correlate Abdominal Arterial Calcification (AAC) score value with mortality on the waitlist. We modified the coronary calcium score and used it to quantitate the AAC. We conducted a retrospective clinical study of all adult patients who were listed for kidney transplant, between 2005 and 2015, and had abdominal computed tomography scan. Patients were divided into two groups: those who died on the waiting list (DWL group) and those who survived on the waiting list (SWL group). Each 1000 increase in the AAC score value of the sum score of the abdominal aorta, bilateral common iliac, bilateral external iliac, and bilateral internal iliac was associated with increased risk of death (HR 1.034, 95%CI 1.013, 1.055) (p = 0.001). This association remained significant even after adjusting for various patient characteristics, including age, tobacco use, diabetes, coronary artery disease, and dialysis status. The study highlights the potential value of the AAC score as a noninvasive Imaging biomarker for kidney transplant waitlist patients. Incorporating the AAC scoring system into routine imaging reports could facilitate improved risk assessment and personalized care for kidney transplant candidates.

#2983 Correlation of microRNA expression with IgA nephropathy progression: preliminary results

Abstract Background and Aims IgA nephropathy (IgAN) is the most common cause of glomerulonephritis worldwide. IgAN is diagnosed based on the result of histopathological examination. The risk factors for disease progression primarily include the level of impairment of renal function at the time of diagnosis (GFR &amp;lt; 60 ml/min/1.73 m2), the presence of hypertension, daily administration of more than 1.0 g, and the occurrence of hematuria. MicroRNAs (miRNAs) are important regulators of gene expression. Their role has a significant impact on the proper functioning of the body, and disorders related to their expression can lead to the development of many pathological processes in the body. The study aimed to assess the dependence of microRNA expression on the stage of the disease in patients with IgA nephropathy. Method A study was conducted involving patients newly diagnosed with IgA nephropathy based on histopathological examination results. The expression of microRNA molecules (miR-146a, miR-155, miR-200b, miR-429) was determined in urine sediment and blood. The analysis focused on examining the relationship between microRNA expression and the stage of disease progression (proteinuria, renal parameters). Blood and urine tests were performed before the start of immunosuppressive treatment with glucocorticosteroids according to the Pozzi regimen. Results Twenty-seven patients were included in the study group- 14 women (51.9%) and 13 men (48.1%). Nephrotic-range proteinuria (&amp;gt;3000 mg/24 h) was observed in 5 patients (18.5%), while subnephrotic proteinuria was present in 22 patients (77.8%). Reserved kidney function (eGFR &amp;gt; 90) was found in 17 patients (63%). The exact distribution of results according to the stage of renal failure is given in the Table 1. Statistically significant positive correlation was found between serum creatinine concentration and miR-200b expression in urine (R = 0,398, p = 0,049). Initially, no statistically significant correlation was found between the expression of selected miRNA molecules and the degree of 24-hour proteinuria. Conclusion MiRNA molecules may be promising, non-invasive biomarkers widely used in clinical practice. Determining the viability of clinical use of microRNA molecules will require a panel of miRNAs or miRNAs used in combination with other biomarkers and clinical outcomes to improve diagnostic utility.

#683 Utility of urinary soluble CD163 as a novel biomarker for the detection of IgA nephropathy

Abstract Background and Aims IgA nephropathy (IgAN) is an immune mediated disease and a leading cause of biopsy proven glomerulonephritis (GN) worldwide. CD163 is a membrane protein localised on the surface of monocytes and macrophages. Its soluble form, sCD163, produced by enzymatic splitting of the ectodomain of inflammatory cells when activated, is found in increased concentrations in acute inflammatory diseases such as sepsis and vasculitis. Urinary sCD163 levels have also been reported to be a useful biomarker of glomerular inflammation in lupus nephritis (LN) and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Yet, few studies have evaluated the role of urinary sCD163 as a biomarker in IgAN, and comparing it across different GN subtypes. This study aims to explore the utility of urinary sCD163 as a non-invasive biomarker for IgAN, and across different immune mediated GN types. Method This is a retrospective and prospective cohort study from a single tertiary hospital. Frozen urine samples from patients with GN and healthy controls from the Asian Kidney Disease Study and Singapore Kidney Function Study respectively were utilised for the retrospective cohort. The prospective cohort consists patients with biopsy-proven IgAN recruited from May to December 2023 with informed consent taken. Urinary sCD163 was measured using enzyme-linked immunosorbent assay (ELISA). Univariate analyses were performed and urinary sCD163 values were plotted on receiver-operating characteristic (ROC) curve and the area under the curve (AUC) was calculated. This study obtained Institutional Ethics Board Approval (DSRB 2022/00698). Categorical variables were represented by n (%). Continuous variables with a parametric distribution were represented by their means ± standard deviation, non-parametric variables were expressed as their medians with interquartile ranges. Chi-square test and ANOVA were performed between categorical variables, while a student's t-test was performed for continuous variables. P-value of &amp;lt; 0.05 was taken as statistically significant. A ROC curve was performed for sCD163 values in IgAN and controls, and the AUC calculated. Results In total, urine samples of 59 subjects were analysed (IgAN n = 10; LN n = 5; healthy controls n = 44). There were 30 male subjects (50.8%) and 29 female subjects (49.2%). Median age was 41.0 (IQR 31.0-52.0) years. Median serum creatinine was 78.0 (IQR: 60.0-91.0) µmol/L. Mean urine sCD163 value was 0.32 (±0.19) ng/ml. There were no baseline differences in demographics, comorbidities and medication between groups. Prospective cohort patients with IgAN were more likely to have received steroids (p = 0.03), and there were no patients of the Malay Ethnicity in the prospective cohort (p = 0.05). Median urine protein (g/day) in IgAN was 0.66 (IQR 0.16 to 3.29)¸ 0.159 in LN (IQR 0.09 to 0.26) and 0.12 in controls (IQR 0.07 to 0.20). Using Mann-Whitney U test to compare median proteinuria between IgAN and controls showed significant difference (p = 0.001), whereas median proteinuria between IgAN and LN was not statistically significant (p = 0.08). On univariate analysis compared to healthy controls, IgAN patients had a significantly higher serum creatinine (p = 0.04), compared to LN patients against controls (p = 0.30). Urine sCD163 levels were significantly higher in IgAN patients compared to controls (0.54 ± 0.26, p = 0.02). Patients with LN do not have a significant urinary sCD163 level difference compared to controls (0.21 ± 0.20, p = 0.40), which also correlates with creatinine and degree of proteinuria. There were no other significant baseline differences against controls. On the ROC curve for IgAN patients, urine sCD163 of 0.38 ng/ml has a 70% sensitivity, and 75% specificity in distinguishing IgAN against controls (AUC 0.77; 95% CI: 0.59-0.96, p = 0.008). For LN, urine SCD163 did not distinguish between LN and controls (AUC 0.29; 95% CI: 0.000-0.58, p = 0.12). Conclusion Urine sCD163 is elevated in patients with IgAN compared to controls and LN. Urine sCD163 is useful in the diagnosis of IgAN. A urine sCD163 level of 0.38 ng/ml has a 70% sensitivity and 75% specificity in identifying IgAN against healthy subjects.

#2908 Renal progenitor cells an early non-invasive biomarker of silent kidney injury in Fabry disease

Abstract Background and Aims Fabry's disease (FD), a hereditary condition, is linked to the X chromosome due to a mutation in the gene encoding alpha-galactosidase A (alpha-GalA). This mutation results in a defect in the metabolism of glycosphingolipids, leading to the deposition of globotriaosylceramide (GB3) primarily in organs such as the heart, kidneys, and nervous system. Renal injury can affect any level of the structure, with the podocytes being the most commonly affected due to their low regeneration and high contact with GB3. This leads to proteinuria, and if it exceeds 0.5 g, it is associated with a rapid deterioration of renal function. Renal assessment is carried out through blood analysis, proteinuria measurement, ultrasound, and the gold standard, renal biopsy. However, there is a growing trend towards less invasive biomarkers such as lysoGB3 titration in blood and urine, podocyturia, or renal progenitor cells (RPCs) in renal biopsy material or cell culture. RPCs possess self-renewal, clonogenicity, and multidifferentiation properties, contributing to cellular remodeling. They express specific markers like CD133/CD24 and CD106 in the parietal epithelium of Bowman's capsule. It is known that GB3 expresses CD77. The proliferative response of RPCs can become dysregulated, leading to detachment and elimination in the urine. The aim of the study was to demonstrate the presence of renal progenitor cells (RPCs) in urine as a non-invasive early marker for detecting renal damage in patients with Fabry's disease (FD) and to establish a correlation between RPC presence, GB3 deposition, and their potential association with the degree of renal injury. Method It is an open observational, case-control, single-center study on the urine of patients with Fabry's disease (FD) considered as cases, patients without a history considered as healthy controls, and patients with non-FD renal disease such as Gittelman syndrome or another biopsy-proven cause as positive controls. Renal progenitor cells (RPCs) were isolated using specific markers and flow cytometry, correlating these findings with renal function and albuminuria. Results A total of 75 recent urine samples were processed, corresponding to 59 patients: 16 with Fabry's disease (FD), 11 with Gittelman syndrome, 10 with chronic kidney disease (CKD), and 22 healthy controls. The samples were divided for sediment and microalbuminuria analysis, and separately for the isolation of renal progenitor cells (RPCs). The classification and quantification of the isolated cell types were performed, establishing positivity points using compensation panels with CD3 lymphocyte markers. This was followed by the identification of RPCs through positivity for CD133+/CD24+, CD106+, and CD106– markers. Once RPCs were identified, GB3 marking was carried out using CD77+. Differences were observed among the analyzed patient groups. In healthy controls, there was minimal or no presence of renal progenitor cells (RPCs), except in two patients where isolation was repeated in three different samples, maintaining positivity. Subsequent diagnoses revealed bilateral lithiasis and previously unknown hypertension in these cases. Patients with Gittelman syndrome showed a higher quantity of RPCs compared to healthy controls, but with P &amp;gt; .05. However, clear RPCs positivity was evident in patients with Fabry's disease (FD) and chronic kidney disease (CKD) confirmed by biopsy, with P &amp;lt; .05. Additionally, differences in the number of RPCs were noted based on the degree of renal disease when correlated with the presence of proteinuria &amp;lt; 0.5 g and &amp;gt; 0.5 g, with P &amp;lt; .05. Conclusion With these results, we can conclude that renal progenitor cells (RPCs) may serve as an early biomarker for silent renal injury of various etiologies. Furthermore, if we perform labeling with CD77, we can attribute it to the deposition of GB3 in Fabry's disease (FD).