Urinary Peptides Research Articles

Discovery, validation and sequencing of urinary peptides for diagnosis of liver fibrosis—A multicentre study

BackgroundLiver fibrosis is a consequence of chronic inflammation and is associated with protein changes within the hepatocytes structure. In this study, we aimed to investigate if this is reflected by the urinary proteome and can be explored to diagnose liver fibrosis in patients with chronic liver disease.MethodsIn a multicentre combined cross-sectional and prospective diagnostic test validation study, 129 patients with varying degrees of liver fibrosis and 223 controls without liver fibrosis were recruited. Additionally, 41 patients with no liver, but kidney fibrosis were included to evaluate interference with expressions of kidney fibrosis. Urinary low molecular weight proteome was analysed by capillary electrophoresis coupled to mass spectrometry (CE-MS) and a support vector machine marker model was established by integration of peptide markers for liver fibrosis.FindingsCE-MS enabled identification of 50 urinary peptides associated with liver fibrosis. When combined into a classifier, LivFib-50, it separated patients with liver fibrosis (N = 31) from non-liver disease controls (N = 123) in cross-sectional diagnostic phase II evaluation with an area under the curve (AUC) of 0.94 (95% confidence intervals (CI): 0.89–0.97, p<0.0001). When adjusted for age, LivFib-50 demonstrated an AUC of 0.94 (95% CI: 0.89–0.97, p<0.0001) in chronic liver disease patients with (N = 19) or without (N = 17) liver fibrosis progression. In this prospective diagnostic phase III validation set, age-adjusted LivFib-50 showed 84.2% sensitivity (95% CI: 60.4–96.6) and 82.4% specificity (95% CI: 56.6–96.2) for detection of liver fibrosis. The sequence-identified peptides are mainly fragments of collagen chains, uromodulin and Na/K-transporting ATPase subunit γ. We also identified ten putative proteolytic cleavage sites, eight were specific for matrix metallopeptidases and two for cathepsins.InterpretationIn liver fibrosis, urinary peptides profiling offers potential diagnostic markers and leads to discovery of proteolytic sites that could be targets for developing anti-fibrotic therapy.

Proteomic Biomarkers to Guide Stratification for Covid‐19 Treatment: Exemplifying a Path Forward Toward Implementation?

Proteins and peptides serve as biomarkers in the context of multiple pathologies. The hypothesis that protein or peptide biomarkers may also be of value in the context of the Covid-19 pandemic appears self-evident. Proteome based biomarkers are not expected to display significant added value in the detection of viral infection but appear well suited to address a major unmet need: the prognosis of the course of disease, to guide appropriate, timely intervention. Based on similar approaches in the context of other diseases and using a CE-MS platform, urinary peptides are investigated for their value as biomarkers to assess disease progression after SARS-CoV-2 infection. The manuscript presented in this issue of Proteomics reports first results, indicating that urine peptides may be of substantial value in the assessment and prediction of severity of the Covid-19 disease course on an individual level. While the findings are not entirely surprising, the report does stand out from all others by a well-defined context-of-use, and, what is more, by presenting an already initiated validation study that may, if successful, result in immediate implementation of this proteomics-based diagnostic test. This approach should serve as positive example for the planning and execution of clinical proteomics studies.

Serum and Urinary N-Terminal Pro-brain Natriuretic Peptides as Biomarkers for Bronchopulmonary Dysplasia of Preterm Neonates.

Bronchopulmonary dysplasia (BPD) is a common cause of respiratory illness in preterm newborns with high morbidity and mortality rates. At present, there are no early prognostic biomarkers that can be used in clinical practice to predict the development of BPD. In this review, we critically appraise evidence regarding the use of serum N-terminal pro-brain natriuretic peptide (NTproBNP) levels as a biomarker for BPD in neonates. Furthermore, we summarize studies assessing the feasibility of urinary NTproBNP levels as a non-invasive method to predict BPD in preterm infants. Multiple studies reported a strong association between NTproBNP serum levels and the onset of BPD. For urinary NTproBNP there is scarce evidence showing an association with BPD. Given the promising data obtained by preliminary studies, further assessment of this biomarker in both serum and urine is needed. Standardized reference values should be defined before conducting any further clinical studies.

Urinary fetuin-A peptides as a new marker for impaired kidney function in patients with type 2 diabetes.

BackgroundThe hepatokine fetuin-A, released by the human liver, promotes pro-inflammatory effects of perivascular fat. The involvement of inflammation in type 2 diabetes mellitus (T2DM) can affect the kidney and contribute to the development of diabetic kidney disease. Therefore we examined the association of urinary fetuin-A protein fragments with renal damage in T2DM patients.MethodsUrinary peptides of 1491 individuals using proteome data available from the human urine proteome database were analysed. Prediction of proteases involved in urinary peptide generation was performed using the Proteasix tool.ResultsWe identified 14 different urinary protein fragments that belong to the region of the connecting peptide (amino acid 301–339) of the total fetuin-A protein. Calpains (CAPN1 and CAPN2), matrix metalloproteinase and pepsin A-3 were identified as potential proteases that were partially confirmed by previous in vitro studies. Combined fetuin-A peptides (mean of amplitudes) were significantly increased in T2DM patients with kidney disease and to a lesser extent with cardiovascular risk. Furthermore, fetuin-A peptide levels displayed a significant negative correlation with baseline estimated glomerular filtration rate (eGFR) values (r = −0.316, P < 0.0001) and with the slope (%) of eGFR per year (r = −0.096, P = 0.023). A multiple regression model including fetuin-A peptide and albuminuria resulted in a significantly improved correlation with eGFR (r = −0.354, P < 0.0001) compared with albuminuria, indicating an added value of this novel biomarker.ConclusionsThe urinary proteome analysis demonstrated the association of fetuin-A peptides with impaired kidney function in T2DM patients. Furthermore, fetuin-A peptides displayed early signs of kidney damage before albuminuria appeared and therefore can be used as markers for kidney disease detection.

Urinary proteins detected using modern proteomics intervene in early type 2 diabetic kidney disease - a pilot study.

Aim: An advanced proteomics platform for protein biomarker discovery in diabetic chronic kidney disease (DKD) was developed, validated and implemented. Materials & methods: Three Type 2 diabetes mellitus patients and three control subjects were enrolled. Urinary peptides were extracted, samples were analyzed on a hybrid LTQ-Orbitrap Velos Pro instrument. Raw data were searched using the SEQUEST algorithm and integrated into Proteome Discoverer platform. Results & discussion: Unique peptide sequences, resulted sequence coverage, scoring of peptide spectrum matches were reported to albuminuria and databases. Five proteins that can be associated with early DKD were found: apolipoprotein AI, neutrophil gelatinase-associated lipocalin, cytidine deaminase, S100-A8 and hemoglobin subunit delta. Conclusion: Urinary proteome analysis could be used to evaluate mechanisms of pathogenesis of DKD.

Urinary Proteomics Associates with COVID-19 Severity: Pilot Proof-of-Principle Data and Design of a Multicentric Diagnostic Study.

SARS-CoV-2 infection results in a mild-to-moderate disease course in most patients, allowing outpatient self-care and quarantine. However, in approx. 10% of cases a two- or three-phasic critical disease course with starting from day 7 to 10 is observed. To facilitate and plan outpatient care, biomarkers prognosing such worsening at an early stage appear of outmost importance. In this accelerated article, we report on the identification of urinary peptides significantly associated with SARS-CoV-2 infection, and the development of a multi-marker urinary peptide based test, COVID20, that may enable prognosis of critical and fatal outcomes in COVID-19 patients. COVID20 is composed of 20 endogenous peptides mainly derived from various collagen chains that enable differentiating moderate or severe disease from critical state or death with 83% sensitivity at 100% specificity. Based on the performance in this pilot study, testing in a prospective study on 1000 patients has been initiated. This article is protected by copyright. All rights reserved.

Down-Regulation of Collagen Hydroxylation in Colorectal Liver Metastasis.

Collagen is significantly upregulated in colorectal liver metastasis (CRLM) compared to liver tissue. Expression levels of specific collagen types in CRLM resemble those in colorectal cancer (CRC) and colon tissue. We investigated whether the collagen hydroxylation pattern from the primary tumor also migrates with the metastatic tumor. The degree of collagen alpha-1(I) hydroxylation in colon, CRC, liver, and CRLM tissue of the same individuals (n = 14) was studied with mass spectrometry. The degree of hydroxylation was investigated in 36 collagen alpha-1(I) peptides, covering 54% of the triple helical region. The degree of hydroxylation in liver tissue was similar to that in colon tissue. The overall degree of hydroxylation was significantly lower (9 ± 14%) in CRC tissue and also significantly lower (12 ± 22%) in CRLM tissue compared to colon. Furthermore, eleven peptides with a specific number of hydroxylations are significantly different between CRLM and liver tissue; these peptides could be candidates for the detection of CRLM. For one of these eleven peptides, a matching naturally occurring peptide in urine has been identified as being significantly different between patients suffering from CRLM and healthy controls. The hydroxylation pattern in CRLM resembles partly the pattern in liver, primary colorectal cancer and colon.

Prognostic Implications of Urinary and Plasma C-type Natriuretic Peptide in Human Acute Decompensated Heart Failure

Introduction C-type natriuretic peptide (CNP) is a cardiorenal-derived hormone that possesses potent anti-fibrotic, anti-hypertrophic, anti-apoptotic and cardiac unloading properties. While plasma and urinary CNP have been shown to be elevated in heart failure (HF) patients, limited studies have simultaneously characterized and evaluated the clinical utility of plasma and urinary CNP in acute decompensated HF (ADHF). Hypothesis We hypothesize that plasma and urinary CNP are elevated and distinctly regulated in ADHF and elevation of both are a marker of disease severity and will have additional prognostic value for adverse outcomes. Methods ADHF patients (n=208) and healthy subjects (n=109) were prospectively recruited and urinary and plasma CNP, plasma NT-proBNP, eGFR and urinary protein/creatinine were determined. The 95th percentile (%) of CNP values from healthy subjects were used to establish normal cutoffs. ADHF patients were stratified based on whether their CNP levels were considered as elevated (> 95th % of normal) or normal (≤ 95th % of normal). The adverse outcomes were all-cause rehospitalization and/or death over 3.0 (1.0, 4.9) years of follow-up. Results ADHF patients had elevated plasma and urinary CNP levels compared to healthy subjects [plasma CNP: 18.2 (10.7-30.3) vs 11.8 (9.5-15.0) pg/mL; urinary CNP: 43.1 (27.9-74.7) vs 15.8 (12.1-20.2) ng/g Cr; P Conclusions This study is the largest to date to provide novel pathophysiological insights into plasma and urinary CNP. We report for the first time that elevations of both CNP indices are associated with disease severity and have prognostic utility beyond robust clinical risk models in ADHF.

Urinary Peptides Associated Closely with Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a common disease of pregnant women, which has a higher incidence in recent years. The purpose of this study is to explore urinary biomarkers that could predict and monitor gestational diabetes mellitus (GDM). Urine samples from 30 normal pregnant women and 78 GDM patients were collected and purified by weak cationic exchange magnetic beads (MB-WCX), then analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The urinary peptide signatures of the two groups were compared by BioExplorer software. The potential ability of the differently expressed peptides to distinguish GDM patients from normal pregnant women was evaluated by receiver operating characteristic (ROC) analysis. At last, the differently expressed peptides were identified by liquid chromatography tandem mass spectrometry (LC-MS). There were four differently expressed peptides (m/z 1000.5, 1117.5, 1142.9, and 2022.9) between two groups, which were identified as fragments of urinary albumin, α2-macroglobulin, human hemopexin, and α1-microglobulin, respectively. The diagnostic efficacy of m/z 1142.9 was better than the other peptides. The area under the curve (AUC) of the m/z 1142.9 was 0.690 (95% CI: 0.583-0.796). The discovery of urinary polypeptides provides the possibility for the early prediction of GDM and the monitoring of glucose metabolism in GDM patients by a noninvasive method.

Value of Urine Peptides in Assessing Kidney and Cardiovascular Disease.

Urinary peptides gained significant attention as potential biomarkers especially in the context of kidney and cardiovascular disease. In this manuscript the recent literature since 2015 on urinary peptide investigation in human kidney and cardiovascular disease is reviewed. The technology most commonly used in this context is capillary electrophoresis coupled mass spectrometry, in part owed to the large database available and the well-defined dataspace. Several studies based on over 1000 subjects are reported in the recent past, especially examining CKD273, a classifier for assessment of chronic kidney disease based on 273 urine peptides. Interestingly, the most abundant urinary peptides are generally collagen fragments, which may have gone undetected for some time as they are typically modified via proline hydroxylation. The data available suggest that urinary peptides specifically depict inflammation and fibrosis, and may serve as a non-invasive tool to assess fibrosis, which appears to be a key driver in kidney and cardiovascular disease. The recent successful completion of the first urinary peptide guided intervention trial, PRIORITY, is expected to further spur clinical application of urinary peptidomics, aiming especially at early detection of chronic diseases, prediction of progression, and prognosis of drug response.

Vaginal estrogen therapy is associated with increased Lactobacillus in the urine of postmenopausal women with overactive bladder symptoms

Previous work has shown that the vaginal microbiome decreases in Lactobacillus predominance and becomes more diverse after menopause. It has also been shown that estrogen therapy restores Lactobacillus dominance in the vagina and that topical estrogen is associated with overactive bladder symptom improvement. We now know that the bladder contains a unique microbiome and that increased bladder microbiome diversity is associated with overactive bladder. However, there is no understanding of how quickly each pelvic floor microbiome responds to estrogen or if those changes are associated with symptom improvement. This study aimed to determine if estrogen treatment of postmenopausal women with overactive bladder decreases urobiome diversity. We analyzed data from postmenopausal participants in 2 trials (NCT02524769 and NCT02835846) who chose vaginal estrogen as the primary overactive bladder treatment and used 0.5 g of conjugated estrogen (Premarin cream; Pfizer, New York City, NY) twice weekly for 12 weeks. Baseline and 12-week follow-up data included the Overactive Bladder questionnaire, and participants provided urine samples via catheter, vaginal swabs, perineal swabs, and voided urine samples. Microbes were detected by an enhanced culture protocol. Linear mixed models were used to estimate microbiome changes over time. Urinary antimicrobial peptide activity was assessed by a bacterial growth inhibition assay and correlated with relative abundance of members of the urobiome. In this study, 12 weeks of estrogen treatment resulted in decreased microbial diversity within the vagina (Shannon, P=.047; Richness, P=.043) but not in the other niches. A significant increase in Lactobacillus was detected in the bladder (P=.037) but not in the vagina (P=.33), perineum (P=.56), or voided urine (P=.28). The change in Lactobacillus levels in the bladder was associated with modest changes in urgency incontinence symptoms (P=.02). The relative abundance of the genus Corynebacterium correlated positively with urinary antimicrobial peptide activity after estrogen treatment. Estrogen therapy may change the microbiome of different pelvic floor niches. The vagina begins to decrease in diversity, and the bladder experiences a significant increase in Lactobacillus levels; the latter is correlated with a modest improvement in the symptom severity subscale of the Overactive Bladder questionnaire.

Peptides in Plasma, Urine, and Dialysate: Toward Unravelling Renal Peptide Handling.

The peptidomes of spent hemodialysate, urine, and plasma are investigated, to shed light on peptide handling in the kidney. Fifteen plasma, 15 urine, and 13 spent hemodialysate samples are collected from age- and sex-matched subjects with chronic kidney disease. Peptide identification and quantification are performed with capillary electrophoresis-coupled mass spectrometry. A total of 6278 urinary peptides, 1743 plasma peptides, and 1727 peptides from spent hemodialysate are detected. Of these, sequences can be assigned to 1580, 419, and 352 peptides, respectively. A strong correlation in peptide abundance between urine and spent hemodialysate (p = 3×10-21 , Rho = 0.52), a moderately strong correlation between spent hemodialysate and plasma (p = 4.5×10-5 , Rho = 0.30), and no significant correlation between urine and plasma (p = 0.11, Rho = 0.094) are found. Collagen and fibrinogen alpha peptides are highly abundant in all three body fluids. In spent hemodialysate, thymosin ß4 is one of the most abundant peptides, which is shown to be negatively associated with the estimated glomerular filtration rate (Rho = -0.39, p-value = 3.9×10-81 ). The correlation of peptide abundance in these three body fluids is lower than expected, supporting the hypothesis that tubular reabsorption has a major impact on urinary peptide content. Further investigation of thymosin ß4 in hemodialysis is thus warranted.

Symptoms of gluten ingestion in patients with non-celiac gluten sensitivity: A randomized clinical trial.

Non-celiac gluten sensitivity (NCGS) is the presence of symptoms induced by gluten and relieved by a gluten-free diet (GFD) in patients without celiac disease or wheat allergy. Studies are mixed as to whether gluten is the main symptom trigger in patients with NCGS. Gluten immunogenic peptides (GIPs) in stool and urine are novel methods to monitor GFD compliance. Few studies have investigated their use in patients with NCGS. The aim of this study was to assess whether patients with NCGS have increased symptoms with gluten ingestion and to assess compliance with the GFD using stool and urine GIPs. This was a prospective, randomized, double-blinded crossover trial evaluating symptoms in patients with NCGS. Thirty patients with NCGS and 43 healthy controls were placed on a GFD. Patients received 0.5 or 2 g/d of gluten for 7 d each. The remaining weeks, they received placebo for a total of 4 wk. Symptoms were evaluated weekly using the Celiac Symptom Index (CSI). Urine and stool samples were collected weekly and measured for the detection of GIPs to detect exposure to gluten. There was no difference in symptom severity within the NCGS group whether receiving placebo or gluten (32.69 versus 31.54, P=0.64). Patients with NCGS had significantly higher CSI scores at baseline than healthy controls. Patients with NCGS were less likely to have stool and urine GIPs than healthy patients. Patients with NCGS were more adherent to the GFD based on stool and urine GIP results. Patients with NCGS had increased symptom severity at baseline compared with healthy controls. Neither group had significantly increased symptoms after ingestion of gluten.

Identification and characterization of urinary peptides using Q-TOF mass spectrometry in cow

Cow urine has been found to be the most effective secretion of animal origin with innumerable therapeutic values. It has potent antimicrobial activity which may be attributed to the presence of urinary peptides in it. Lack of evidence for the presence of such antimicrobial peptides (AMPs) in cow urine lead to the present study. Using a technique of membrane filtration, ion exchange chromatography, acid-urea poly-acrylamide gel electrophoresis (AU-PAGE), reverse phase high performance liquid chromatography (RP-HPLC) and ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-QTOF-MS/MS), we successfully extracted and characterized cationic peptides from the urine of healthy, normal cycling indigenous cow. We could identify two urinary peptides which were reported to contain antimicrobial properties. These identified peptides were 97 and 99 amino acids long Neutrophil defensin-4 and β-defensin-127 of defensin class. These peptides may be responsible for the innate immunity and serve as an alternative to the conventional antibiotics in the time to come.

P0360URINARY PEPTIDOMIC ANALYSIS IN PROLIFERATIVE VERSUS NON-PROLIFERATIVE LUPUS NEPHRITIS : RESULTS OF THE PEPTIDU-LUP STUDY

Abstract Background and Aims Lupus nephritis (LN) is a frequent manifestation of Systemic Lupus Erythematosus (SLE). The therapeutic strategy relies on the result of kidney biopsy, which differentiates proliferative LN (PLN) from non-proliferative LN (NPLN). The analysis of the urinary peptidome has led to the identification of prognostic biomarkers in chronic kidney disease (CKD, namely the CKD273 classifier), or, as a liquid biopsy, for the diagnosis of glomerulonephritides. We verified whether urinary peptidomics could predict the severity of renal pathological injury and damage in LN. Method Urine samples, collected before kidney biopsy, were analyzed by capillary-electrophoresis coupled to mass-spectrometry (CE-MS). Urinary peptide profiles were compared between PLN and NPLN. Predictive values for chronic pathological lesions (glomerulosclerosis and interstitial fibrosis/tubular atrophy (IF/TA)), response to therapy and development of CKD were also assessed. Clinical characteristics, routine laboratory parameters and immunological SLE markers were collected at inclusion and prospectively for at least 24 months. Results We collected 100 urinary samples from patients with LN, forming a discovery (n=67) and an independent validation (n=33) cohort. Overall there were 69 patients with PLN (class III or IV +/-V with active lesions) and 31 patients with NPLN (class II or V or chronic lesions). In the discovery cohort, the abundance of 36 urinary peptides differed between PLN and NPLN (Mann-Whitney test). However, these peptides did not resist multiple testing correction. Among them, 17 could be sequenced (fragments of collagen-I, -II, -III, apolipoprotein A-I, Fibrinogen alpha chain and Histone H2B). Of the 17 sequenced peptides, 7 were also part of the CKD273 classifier. A mathematical model combining the 36 peptides classified PLN and NPLN patients from the validation cohort with an AUC of 0.75 and sensitivity and specificity of 0.81 and 0.53, respectively. Positivity of anti-dsDNA antibodies had the best sensitivity (0.96) and sterile pyuria the best specificity (0.68) to differentiate PLN from NPLN, and including the CKD273 score did not improve the accuracy of the predictive models. No urinary peptidomics profile was identified to predict early response to therapy in patients with LN. Glomerulosclerosis was observed in 63/100 biopsies. In the discovery cohort, the abundance of 38 urinary peptides (including 22 sequenced) differed between patients with vs without glomerulosclerosis (Mann-Whitney test). Again, these peptides did not resist multiple testing correction. Of the 22 sequenced peptides, 7 (different from the peptides associated with PLN) belonged to the CKD273 classifier. A mathematical model combining the 38 peptides classified patients from the validation cohort with an AUC of 0.75 and sensitivity and specificity of 0.82 and 0.54, respectively, for the presence of glomerulosclerosis. Although the quantification of IF/TA was correlated to the pre-existing CKD273 classifier, including the CKD273 score did not improve the prediction of IF/TA when tested in combination with simple clinical parameters. Only 3 patients developed CKD after a mean follow-up of 4 years, therefore contribution of urinary peptidomics for the prediction of CKD in LN could not be evaluated. Conclusion Different urinary peptidomics signatures were identified among patients with LN, according to the presence of active proliferative lesions or chronic lesions. However, these panels did not resist multiple testing correction, and did not improve the diagnostic accuracy when combined with clinical or immunological markers. The contribution of urinary peptidomics to predict CKD in patients with LN, or as an early predictor of renal flares, could not be evaluated. Kidney biopsy remains central for the care of patients with LN.

P0651PROTEOMIC CHARACTERIZATION OF OBESITY-RELATED NEPHROPATHY

Abstract Background and Aims During the last years, with the increase in secondary diseases following obesity the focus on the context of abnormally high body weight and chronic kidney disease (CKD) has changed. While in individual care, CKD along with obesity and improving renal function along with weight loss can be seen frequently and trials on obesity reduction yielded promising renal results, systematic insight into the pathogenesis and epidemiology of nephropathy related to obesity without diabetes (Obesity-related nephropathy, ORN) is rather limited. This work reaches out to investigate impact of BMI on urine peptides and to characterize yet non-specified peptide patterns in urine that may be indicative of ORN and may indicate possible molecular mechanisms of ORN pathophysiology. We used urine samples from earlier projects which could be characterized for presence of diabetes, BMI, eGFR, and age. By defining such urinary peptide patterns, pathophysiology-enlightening hypotheses should be stimulated and pathways suggested to enable specific future characterization of ORN. Method In this study we investigated the association of urinary peptides with body mass index (BMI), and renal function in proteome datasets from 4015 individuals. For this study urinary proteome data stored in the Human Urine Proteome Database obtained by capillary electrophoresis coupled to mass spectrometry (CE-MS) were assessed. Results 365 urinary peptides significantly associated with BMI could be identified. The majority of these peptides were collagen fragments. In addition, most of the peptides also demonstrated a significant concordant association with eGFR in the investigated cohort with diabetes exhibiting no significant association. A classifier based on 150 urine peptides enabled distinguishing non-obese subjects with preserved kidney function from obese, non-diabetic subjects with eGFR&amp;gt;45 mL/min/1.73m in an independent cohort with an area under curve (AUC) of 0.93 (Fig.). The vast majority of significantly associated peptides are specific collagen fragments, most of them from collagen type I, and most of them (in fact all of the 10 most significant ones) are reduced with increasing BMI. Conclusion These analyses in a huge cohort of urine samples from patients with known eGFR, BMI and diabetes status showed a complex multi-level association between peptide fragments with renal risk in obesity. Of particular note, these relationships could be retrieved although the overall cohort inherited an almost negative eGFR – BMI gross correlation, presumably due to the origin of the dataset from a non-obesity-directed approach. Most peptides associated with GFR and BMI belonged to the collagen superfamily and were inversely associated with BMI but directly with eGFR. This concordant observation indicates a reduction of collagen degradation with increasing BMI and/or reduced kidney function. It is tempting to speculate that collagen homeostasis along with body mass on one hand, and kidney function on the other, are substantially interconnected. The very strict association of BMI with collagen fragments was surprising, but also indicates specificity.

P0652THYMOSIN BETA 4 AND KIDNEY FUNCTION: AN INVERSE RELATIONSHIP?

Abstract Background and Aims Thymosin β4 (Tβ4) is an abundant peptide in urine and plasma, involved in multiple processes including cell morphology, wound healing, and inflammation. A major, yet not well defined role of Tβ4 was described in the context of kidney and cardiovascular disease. Knock out of Tβ4 resulted, among others, in increased albuminuria in a nephrotoxic nephritis model. In animal models it has been shown that Tβ4 can modify renal and cardiovascular disease, fibrosis and inflammation. However, most of the data are derived from animal models and the relevance of Tβ4 in human is less clear. As a first step towards investigating the potential relevance of Tβ4 in chronic kidney disease (CKD) in human, we investigated the abundance of Tβ4 in human urine. Method We investigated human urine in 2240 subjects with a wide spread of kidney function for the presence of Tβ4 using capillary-electrophoresis coupled mass spectrometry (CE-MS) and tandem mass spectrometry. In addition, we evaluated the presence of Tβ4 abundance in hemodialysis fluid of 10 subjects. Results The full length acetylated Tβ4 peptide could be identified in human urine in high abundance (molecular mass 4960.485 Da). In addition, an oxidized isoform could also be observed at high abundance. When investigating the distribution of both isoforms in control subjects and patients at different stages of CKD, a highly significant inverse association of the abundance of Tβ4 with eGFR could be observed for both, with rho -0.388 for the non-oxidized and -0.387 for the oxidized isoform, p&amp;lt;0.0001 in each case. In hemodialysate, Tβ4 is found as the most abundant peptide in 2 of the 10 analyzed subjects, on average the second most abundant peptide, with β2 microglobuline being the most abundant one. Conclusion Tβ4 is highly significantly inversely associated with kidney function, suggesting a role of Tβ4 in kidney disease and/or its complications. Based on the experimental data published from animal models, the increase in Tβ4 may reflect a potential physiological mechanism to counteract CKD and its complications. Investigation of association of Tβ4 levels with progression in CKD, cardiovascular complications and death may inform on a potentially beneficial effect of Tβ4, is currently ongoing in longitudinal datasets and will be presented.

TO006NON-INVASIVE DIAGNOSIS OF BK VIRUS-ASSOCIATED NEPHROPATHY USING URINARY PROTEOMICS IN KIDNEY ALLOGRAFT PATIENTS

Abstract Background and Aims BK virus-associated nephropathy (BKvN) is an important complication occurring after kidney transplantation. Its treatment mainly relies on lowering immune suppression. BKvN’s histological aspect can mimic T cell-mediated rejection (TCMR) and other inflammatory conditions featuring tubulitis and interstitial infiltrate. At present, the screening strategy consists of measuring urinary and blood viral loads of BKv. Upon rising BK viremia, BKvN diagnosis is established by a graft biopsy. Biopsies are invasive and are not suitable for repeated screening. Therefore, the aim of this case-control study was to establish a urinary proteome-based test for BKvN diagnosis. Method Urine was obtained from 700 allograft recipients prior to either protocol or „for cause“ biopsies during the first year of post-transplantation surveillance. Aside from normal biopsy findings (N=294), the histological diagnoses included BKvN (N=50), IFTA II-III (N=145), glomerulonephritis (N=17) and T cell-mediated, antibody-mediated, mixed or borderline rejection episodes (N=194). From all patients, relevant clinical and demographic data including Banff classification scores, HLA mismatches and the presence of donor-specific antibodies was collected at the date of sample collection and in further clinical follow-up. BKvN cases were defined as having SV40-positive immunostaining on allograft biopsy. BKvN patients were considered as cases and all other recipients were considered as controls. Patient’s urinary peptide profiles were generated using capillary electrophoresis coupled to mass spectrometry consisting of 9430 different peptides in the mass range of 0.8 to 20 kDa. Results The CE-MS peptide profiles of randomly selected 30 BKvN cases and 307 kidney allograft controls (one sample per patient) were statistically analyzed to identify the most discriminative peptide markers for BKvN. In total, there were 117 peptides detected to have significantly different urinary excretion rates between both patient groups after false discovery rate adjustment for multiple testing. Thirty-two of these peptides were selected by cross-validated variable selection to establish a Support Vector Machine-based multi-marker model. Applying the 32-peptide marker model to an independent validation cohort consisting of 20 BKvN cases and 343 controls resulted in an area under the receiver operating characteristic curve of 0.86 and a 95% confidence interval from 0.82 to 0.89 with the p-value below 0.0001 and sensitivities and specificities for BKvN diagnosis of 85 and 76 %, respectively. Most notably, distribution of the classification values (figure 1) in the different patient groups of the validation set indicated specificities of the peptide marker model for TCMR/borderline of 85% (17 of 20 true negative classifications) and for viruria/viremia of 73% (8 of 11 true negative classifications) Conclusion In conclusion, the established urinary peptide marker model might serve as a non-invasive diagnostic test for BKvN and its differentiation from TCMR and states of isolated viruria or viremia.

P0650URINE PEPTIDOME ANALYSIS IN HEART FAILURE, CHRONIC KIDNEY DISEASE AND CARDIORENAL SYNDROME TOWARDS THE DEFINITION OF DISEASE-SPECIFIC MARKERS

Abstract Background and Aims The cardiorenal syndrome (CRS) reflects the complex interplay between kidney and heart diseases, but its molecular basis remains poorly understood. Multiple studies have demonstrated the association of urinary biomarkers with both heart and kidney diseases. However, their relevance and involvement in CRS have not been investigated yet. To address this gap, a study was designed with the aim to compare urinary biomarkers specific for heart failure (HF) and chronic kidney disease (CKD) with peptides representing CRS, with the ultimate target to connect these findings towards a better understanding of CRS pathophysiology. Method A total of 3.463 urinary peptidomic datasets from patients with HF, CKD, or with both HF and CKD (CRS) as well as patients with no apparent diseases (controls) were retrieved and analyzed from the urinary peptidomics database (Latosinska A et al., Electrophoresis 2019; 40: 2294-2308). Following the matching for age, gender, heart and kidney function, differences in the abundance of urinary peptides were investigated in a cohort comprised of 390 patients with HF, 257 patients with CKD, 392 patients with CRS and 356 controls. The non-parametric Mann-Whitney U test was applied, followed by correction for multiple testing using the Benjamini-Hochberg method. To map the peptides to the protein precursor, the alignment tool Geneious (www. geneious.com) was applied, while the PeptideRanker (http://distilldeep.ucd.ie/PeptideRanker/) was used to predict probability of peptide being bioactive. Results The multiple pair-wise comparisons resulted in the identification of numerous differentially abundant peptides (p&amp;lt;0.05) between the studied conditions, including among others 176 HF-specific, 146 CKD-specific and 35 CRS-specific peptides. Among the HF-specific peptides, the majority (n=94, 53.4%) originated from collagen type I, II and III. In the case of CKD-specific peptides, 24 (16.43%) originated from alpha-1-antitrypsin, 19 (13.0%) from b2-microglobulin and 15 (10.27%) from collagen type I. For the CRS specific peptides, fragments of Ig lambda-2 chain C regions (n=4, 11.42%), collagen type III (n=4, 11.42%), secreted and transmembrane protein 1 (n=3, 8.57%) and gelsolin (n=1, 2.85%) were identified (figure: 1). Of the 176 HF-specific peptides, 94 (53.40%) were predicted as bioactive, including, among others, fragments of collagen types I (n=43, 45.74%) and III (n=21, 22.34%). In the former, peptides with the higher bioactivity scores were aligned close to the N terminus of the precursor protein, whereas in the latter, peptides were in close proximity to both N and C termini. Along the same lines, 32 (21.91%) of the 146 CKD-specific peptides were predicted as bioactive, including peptides from collagen types I and III with the highest score, as well as fragments from collagen type V and the C terminus of the b2-microglobulin and alpha-1-antitrypsin proteins. No CRS-specific peptides could be predicted as bioactive. Conclusion Specific urinary peptides significantly associated with CRS, but not with HF or CKD, could be identified. These data indicate that on a molecular level, CRS is not merely the result of a combination of HF and CKD, but may represent a distinct pathology, defined via specific proteomic changes. It is expected that interpretation of these findings in the context of existing literature as well as in vitro activity assays will help to understand their biological relevance in CRS.

P0724ASSOCIATIONS OF URINARY POLYMERIC IMMUNOGLOBULIN RECEPTOR PEPTIDES IN THE CONTEXT OF CARDIO-RENAL SYNDROM

Abstract Background and Aims The polymeric immunoglobulin receptor (pIgR) which transports immunoglobulins from the basolateral to the apical surface of epithelial cells was recently shown to be associated with kidney dysfunction. The immune defense is initiated at the apical surface where the N-terminal domain of pIgR, termed secretory component (SC), is proteolytically cleaved and released either unbound (free SC) or bound to immunoglobulins. The aim of our study was to show the association of pIgR peptides with the cardio-renal syndrome in a large cohort and to get information on how the SC will be released. Method We investigated urinary peptides of 2707 individuals available in the Human Urine Proteome Database using capillary electrophoresis coupled to mass spectrometry. Results The mean abundance of 23 different pIgR peptides correlates negatively with the estimated glomerular filtration rate (eGFR, r=-0.314, p&amp;lt;0.0001). Furthermore, pIgR peptides are significantly increased in coronary artery disease after adjustment for eGFR. We further predicted the proteases involved in urinary peptide generation using the Proteasix tool. Peptide cleavage site analysis suggests that several, and not one, proteases are involved in the generation of the SC. Conclusion In this large cohort, we could demonstrate that pIgR is associated with the cardio-renal syndrome and provide more detailed insights on how pIgR can be potentially cleaved to release the SC.