Reproducibility Evaluation of Urinary Peptide Detection Using CE-MS

In recent years, capillary electrophoresis coupled to mass spectrometry (CE-MS) has been increasingly applied in clinical research especially in the context of chronic and age-associated diseases, such as chronic kidney disease, heart failure and cancer. Biomarkers identified using this technique are already used for diagnosis, prognosis and monitoring of these complex diseases, as well as patient stratification in clinical trials. CE-MS allows for a comprehensive assessment of small molecular weight proteins and peptides (<20 kDa) through the combination of the high resolution and reproducibility of CE and the distinct sensitivity of MS, in a high-throughput system. In this study we assessed CE-MS analytical performance with regards to its inter- and intra-day reproducibility, variability and efficiency in peptide detection, along with a characterization of the urinary peptidome content. To this end, CE-MS performance was evaluated based on 72 measurements of a standard urine sample (60 for inter- and 12 for intra-day assessment) analyzed during the second quarter of 2021. Analysis was performed per run, per peptide, as well as at the level of biomarker panels. The obtained datasets showed high correlation between the different runs, low variation of the ten highest average individual log2 signal intensities (coefficient of variation, CV < 10%) and very low variation of biomarker panels applied (CV close to 1%). The findings of the study support the analytical performance of CE-MS, underlining its value for clinical application.

Urinary peptidomics reveals proteases involved in idiopathic membranous nephropathy

Background Idiopathic membranous nephropathy (IMN) is a cause of nephrotic syndrome that is increasing in incidence but has unclear pathogenesis. Urinary peptidomics is a promising technology for elucidating molecular mechanisms underlying diseases. Dysregulation of the proteolytic system is implicated in various diseases. Here, we aimed to conduct urinary peptidomics to identify IMN-related proteases. Results Peptide fingerprints indicated differences in naturally produced urinary peptide components among 20 healthy individuals, 22 patients with IMN, and 15 patients with other kidney diseases. In total, 1,080 peptide-matched proteins were identified, 279 proteins differentially expressed in the urine of IMN patients were screened, and 32 proteases were predicted; 55 of the matched proteins were also differentially expressed in the kidney tissues of IMN patients, and these were mainly involved in the regulation of proteasome-, lysosome-, and actin cytoskeleton-related signaling pathways. The 32 predicted proteases showed abnormal expression in the glomeruli of IMN patients based on Gene Expression Omnibus databases. Western blot revealed abnormal expression of calpain, matrix metalloproteinase 14, and cathepsin S in kidney tissues of patients with IMN. Conclusions This work shown the calpain/matrix metalloproteinase/cathepsin axis might be dysregulated in IMN. Our study is the first to systematically explore the role of proteases in IMN by urinary peptidomics, which are expected to facilitate discovery of better biomarkers for IMN.

Urine peptidome analysis in cardiorenal syndrome reflects molecular processes

The cardiorenal syndrome (CRS) is defined as the confluence of heart-kidney dysfunction. This study investigates the molecular differences at the level of the urinary peptidome between CRS patients and controls and their association to disease pathophysiology. The urinary peptidome of CRS patients (n = 353) was matched for age and sex with controls (n = 356) at a 1:1 ratio. Changes in the CRS peptidome versus controls were identified after applying the Mann–Whitney test, followed by correction for multiple testing. Proteasix tool was applied to investigate predicted proteases involved in CRS-associated peptide generation. Overall, 559 differentially excreted urinary peptides were associated with CRS patients. Of these, 193 peptides were specifically found in CRS when comparing with heart failure and chronic kidney disease urinary peptide profiles. Proteasix predicted 18 proteases involved in > 1% of proteolytic cleavage events including multiple forms of MMPs, proprotein convertases, cathepsins and kallikrein 4. Forty-four percent of the cleavage events were produced by 3 proteases including MMP13, MMP9 and MMP2. Pathway enrichment analysis supported that ECM-related pathways, fibrosis and inflammation were represented. Collectively, our study describes the changes in urinary peptides of CRS patients and potential proteases involved in their generation, laying the basis for further validation.

SGLT2-Inhibition reverts urinary peptide changes associated with severe COVID-19: an in-silico proof-of-principle of proteomics-based drug repurposing

Severe COVID-19 is reflected by significant changes in multiple urine peptides. Based on this observation, a clinical test based on urinary peptides predicting COVID-19 severity, CoV50, was developed and registered as IVD in Germany. We have hypothesized that molecular changes displayed by CoV50, to a large degree likely reflective of endothelial damage, can be significantly reversed by specific drugs. To test this hypothesis, we have collected urinary peptide data from patients without COVID-19 prior and after drug treatment. The drugs chosen were selected based on availability of sufficient number of participants in the dataset (n>20) and potential value of drug therapies in the treatment of COVID-19 based on reports in the literature. In these participants without COVID-19, while spironolactone did not demonstrate a significant impact on CoV50 scoring, empagliflozin treatment resulted in a significant change in CoV50 scoring, indicative of a potential therapeutic benefit. The results serve as a proof-of-principle for a drug repurposing approach based on human urinary peptide signatures and support the initiation of a randomised control trial testing a potential positive effect of empagliflozin in the treatment of severe COVID-19, possibly via endothelial protective mechanisms.

A Novel Urinary Proteomics Classifier for Non-Invasive Evaluation of Interstitial Fibrosis and Tubular Atrophy in Chronic Kidney Disease

Non-invasive urinary peptide biomarkers are able to detect and predict chronic kidney disease (CKD). Moreover, specific urinary peptides enable discrimination of different CKD etiologies and offer an interesting alternative to invasive kidney biopsy, which cannot always be performed. The aim of this study was to define a urinary peptide classifier using mass spectrometry technology to predict the degree of renal interstitial fibrosis and tubular atrophy (IFTA) in CKD patients. The urinary peptide profiles of 435 patients enrolled in this study were analyzed using capillary electrophoresis coupled with mass spectrometry (CE-MS). Urine samples were collected on the day of the diagnostic kidney biopsy. The proteomics data were divided into a training (n = 200) and a test (n = 235) cohort. The fibrosis group was defined as IFTA ≥ 15% and no fibrosis as IFTA < 10%. Statistical comparison of the mass spectrometry data enabled identification of 29 urinary peptides with differential occurrence in samples with and without fibrosis. Several collagen fragments and peptide fragments of fetuin-A and others were combined into a peptidomic classifier. The classifier separated fibrosis from non-fibrosis patients in an independent test set (n = 186) with area under the curve (AUC) of 0.84 (95% CI: 0.779 to 0.889). A significant correlation of IFTA and FPP_BH29 scores could be observed Rho = 0.5, p < 0.0001. We identified a peptidomic classifier for renal fibrosis containing 29 peptide fragments corresponding to 13 different proteins. Urinary proteomics analysis can serve as a non-invasive tool to evaluate the degree of renal fibrosis, in contrast to kidney biopsy, which allows repeated measurements during the disease course.

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

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&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.

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

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.