Urinary biomarkers for renal tract malformations

Introduction: Renal tract malformations (RTMs) are congenital anomalies of the kidneys and urinary tract, which are the major cause of end-stage renal disease in children. Using immunoassay-based approaches (ELISA, western blot), individual urinary proteins including transforming growth factor β, tumor necrosis factor and monocyte attractant proteins 1 were found to be associated to RTMs. However, only mass spectrometry (MS) based methods leading to the identification of panels of protein-based markers composed of fragments of the extracellular matrix allowed the prediction of progression of RTMs and its complications. Areas covered: In this review, we summarized relevant studies identified in “Pubmed” using the keywords “urinary biomarkers” and “proteomics” and “renal tract malformations” or “hydronephrosis” or “ureteropelvic junction obstruction” or “posterior urethral valves” or “vesicoureteral reflux”. These publications represent studies on potential protein-based biomarkers, either individually or combined in panels, of RTMs in human and animal models. Expert commentary: Successful use in the clinic of these protein-based biomarkers will need to involve larger scale studies to reach sufficient power. Improved performance will potentially come from combining immunoassay- and MS-based markers

Urinary peptidomics analysis reveals proteases involved in diabetic nephropathy

Mechanisms underlying the onset and progression of nephropathy in diabetic patients are not fully elucidated. Deregulation of proteolytic systems is a known path leading to disease manifestation, therefore we hypothesized that proteases aberrantly expressed in diabetic nephropathy (DN) may be involved in the generation of DN-associated peptides in urine. We compared urinary peptide profiles of DN patients (macroalbuminuric, n = 121) to diabetic patients with no evidence of DN (normoalbuminuric, n = 118). 302 sequenced, differentially expressed peptides (adjusted p-value < 0.05) were analysed with the Proteasix tool predicting proteases potentially involved in their generation. Activity change was estimated based on the change in abundance of the investigated peptides. Predictions were correlated with transcriptomics (Nephroseq) and relevant protein expression data from the literature. This analysis yielded seventeen proteases, including multiple forms of MMPs, cathepsin D and K, kallikrein 4 and proprotein convertases. The activity of MMP-2 and MMP-9, predicted to be decreased in DN, was investigated using zymography in a DN mouse model confirming the predictions. Collectively, this proof-of-concept study links urine peptidomics to molecular changes at the tissue level, building hypotheses for further investigation in DN and providing a workflow with potential applications to other diseases

Proteomics as a quality control tool of pharmaceutical probiotic bacterial lysate products

Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87–89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots

The use of urinary proteomics in the assessment of suitability of mouse models for ageing

Ageing is a complex process characterised by a systemic and progressive deterioration of biological functions. As ageing is associated with an increased prevalence of age-related chronic disorders, understanding its underlying molecular mechanisms can pave the way for therapeutic interventions and managing complications. Animal models such as mice are commonly used in ageing research as they have a shorter lifespan in comparison to humans and are also genetically close to humans. To assess the translatability of mouse ageing to human ageing, the urinary proteome in 89 wild-type (C57BL/6) mice aged between 8–96 weeks was investigated using capillary electrophoresis coupled to mass spectrometry (CE-MS). Using age as a continuous variable, 295 peptides significantly correlated with age in mice were identified. To investigate the relevance of using mouse models in human ageing studies, a comparison was performed with a previous correlation analysis using 1227 healthy subjects. In mice and humans, a decrease in urinary excretion of fibrillar collagens and an increase of uromodulin fragments was observed with advanced age. Of the 295 peptides correlating with age, 49 had a strong homology to the respective human age-related peptides. These ortholog peptides including several collagen (N = 44) and uromodulin (N = 5) fragments were used to generate an ageing classifier that was able to discriminate the age among both wild-type mice and healthy subjects. Additionally, the ageing classifier depicted that telomerase knock-out mice were older than their chronological age. Hence, with a focus on ortholog urinary peptides mouse ageing can be translated to human ageing

Multicentric validation of proteomic biomarkers in urine specific for diabetic nephropathy

Background: Urine proteome analysis is rapidly emerging as a tool for diagnosis and prognosis in disease states. For diagnosis of diabetic nephropathy (DN), urinary proteome analysis was successfully applied in a pilot study. The validity of the previously established proteomic biomarkers with respect to the diagnostic and prognostic potential was assessed on a separate set of patients recruited at three different European centers. In this case-control study of 148 Caucasian patients with diabetes mellitus type 2 and duration >= 5 years, cases of DN were defined as albuminuria >300 mg/d and diabetic retinopathy (n = 66). Controls were matched for gender and diabetes duration (n = 82). Methodology/Principal Findings: Proteome analysis was performed blinded using high-resolution capillary electrophoresis coupled with mass spectrometry (CE-MS). Data were evaluated employing the previously developed model for DN. Upon unblinding, the model for DN showed 93.8% sensitivity and 91.4% specificity, with an AUC of 0.948 (95% CI 0.898-0.978). Of 65 previously identified peptides, 60 were significantly different between cases and controls of this study. In <10% of cases and controls classification by proteome analysis not entirely resulted in the expected clinical outcome. Analysis of patient's subsequent clinical course revealed later progression to DN in some of the false positive classified DN control patients. Conclusions: These data provide the first independent confirmation that profiling of the urinary proteome by CE-MS can adequately identify subjects with DN, supporting the generalizability of this approach. The data further establish urinary collagen fragments as biomarkers for diabetes-induced renal damage that may serve as earlier and more specific biomarkers than the currently used urinary albumin

Urinary peptidome analyses for the diagnosis of chronic kidney disease in dogs

Chronic kidney disease (CKD) is clinically important in canine medicine. Current diagnostic tools lack sensitivity for detection of subclinical CKD. The aim of the present study was to evaluate urinary peptidome analysis for diagnosis of CKD in dogs. Capillary electrophoresis coupled to mass spectrometry analysis demonstrated presence of approximately 5400 peptides in dog urine. Comparison of urinary peptide abundance of dogs with and without CKD led to the identification of 133 differentially excreted peptides (adjusted P for each peptide <0.05). Sequence information was obtained for 35 of these peptides. This 35 peptide subset and the total group of 133 peptides were used to construct two predictive models of CKD which were subsequently validated by researchers masked to results in an independent cohort of 20 dogs. Both models diagnosed CKD with an area under the receiver operating characteristic (ROC) curve of 0.88 (95% confidence intervals [CI], 0.72–1.0). Most differentially excreted peptides represented fragments of collagen I, indicating possible association with fibrotic processes in CKD (similar to the equivalent human urinary peptide CKD model, CKD273). This first study of the urinary peptidome in dogs identified peptides that were associated with presence of CKD. Future studies are needed to validate the utility of this model for diagnosis and prediction of progression of canine CKD in a clinical setting

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

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

Urinary Collagen Fragments Are Significantly Altered in Diabetes: A Link to Pathophysiology

Background: The pathogenesis of diabetes mellitus (DM) is variable, comprising different inflammatory and immune responses. Proteome analysis holds the promise of delivering insight into the pathophysiological changes associated with diabetes. Recently, we identified and validated urinary proteomics biomarkers for diabetes. Based on these initial findings, we aimed to further validate urinary proteomics biomarkers specific for diabetes in general, and particularity associated with either type 1 (T1D) or type 2 diabetes (T2D). Methodology/Principal Findings: Therefore, the low-molecular-weight urinary proteome of 902 subjects from 10 different centers, 315 controls and 587 patients with T1D (n = 299) or T2D (n = 288), was analyzed using capillary-electrophoresis mass-spectrometry. The 261 urinary biomarkers (100 were sequenced) previously discovered in 205 subjects were validated in an additional 697 subjects to distinguish DM subjects (n = 382) from control subjects (n = 315) with 94% (95% CI: 92-95) accuracy in this study. To identify biomarkers that differentiate T1D from T2D, a subset of normoalbuminuric patients with T1D (n = 68) and T2D (n = 42) was employed, enabling identification of 131 biomarker candidates (40 were sequenced) differentially regulated between T1D and T2D. These biomarkers distinguished T1D from T2D in an independent validation set of normoalbuminuric patients (n = 108) with 88% (95% CI: 81-94%) accuracy, and in patients with impaired renal function (n = 369) with 85% (95% CI: 81-88%) accuracy. Specific collagen fragments were associated with diabetes and type of diabetes indicating changes in collagen turnover and extracellular matrix as one hallmark of the molecular pathophysiology of diabetes. Additional biomarkers including inflammatory processes and pro-thrombotic alterations were observed. Conclusions/Significance: These findings, based on the largest proteomic study performed to date on subjects with DM, validate the previously described biomarkers for DM, and pinpoint differences in the urinary proteome of T1D and T2D, indicating significant differences in extracellular matrix remodeling

Early detection of diabetic kidney disease by urinary proteomics and subsequent intervention with spironolactone to delay progression (PRIORITY): a prospective observational study and embedded randomised placebo-controlled trial

Background: Microalbuminuria is an early sign of kidney disease in people with diabetes and indicates increased risk of cardiovascular disease. We tested whether a urinary proteomic risk classifier (CKD273) score was associated with development of microalbuminuria and whether progression to microalbuminuria could be prevented with the mineralocorticoid receptor antagonist spironolactone. Methods: In this multicentre, prospective, observational study with embedded randomised controlled trial (PRIORITY), we recruited people with type 2 diabetes, normal urinary albumin excretion, and preserved renal function from 15 specialist centres in ten European countries. All participants (observational cohort) were tested with the CKD273 classifier and classified as high risk (CKD273 classifier score &gt;0·154) or low risk (≤0·154). Participants who were classified as high risk were entered into a randomised controlled trial and randomly assigned (1:1), by use of an interactive web-response system, to receive spironolactone 25 mg once daily or matched placebo (trial cohort). The primary endpoint was development of confirmed microalbuminuria in all individuals with available data (observational cohort). Secondary endpoints included reduction in incidence of microalbuminuria with spironolactone (trial cohort, intention-to-treat population) and association between CKD273 risk score and measures of impaired renal function based on estimated glomerular filtration rate (eGFR; observational cohort). Adverse events (particularly gynaecomastia and hyperkalaemia) and serious adverse events were recorded for the intention-to-treat population (trial cohort). This study is registered with the EU Clinical Trials Register (EudraCT 20120-004523-4) and ClinicalTrials.gov (NCT02040441) and is completed. Findings: Between March 25, 2014, and Sept 30, 2018, we enrolled and followed-up 1775 participants (observational cohort), 1559 (88%) of 1775 participants had a low-risk urinary proteomic pattern and 216 (12%) had a high-risk pattern, of whom 209 were included in the trial cohort and assigned to spironolactone (n=102) or placebo (n=107). The overall median follow-up time was 2·51 years (IQR 2·0–3·0). Progression to microalbuminuria was seen in 61 (28%) of 216 high-risk participants and 139 (9%) of 1559 low-risk participants (hazard ratio [HR] 2·48, 95% CI 1·80–3·42; p&lt;0·0001, after adjustment for baseline variables of age, sex, HbA1c, systolic blood pressure, retinopathy, urine albumin-to-creatinine ratio [UACR], and eGFR). Development of impaired renal function (eGFR &lt;60 mL/min per 1·73 m2) was seen in 48 (26%) of 184 high-risk participants and 119 (8%) of 1423 low-risk participants (HR 3·50; 95% CI 2·50–4·90, after adjustment for baseline variables). A 30% decrease in eGFR from baseline (post-hoc endpoint) was seen in 42 (19%) of 216 high-risk participants and 62 (4%) of 1559 low-risk participants (HR 5·15, 95% CI 3·41–7·76; p&lt;0·0001, after adjustment for basline eGFR and UACR). In the intention-to-treat trial cohort, development of microalbuminuria was seen in 35 (33%) of 107 in the placebo group and 26 (25%) of 102 in the spironolactone group (HR 0·81, 95% CI 0·49–1·34; p=0·41). In the safety analysis (intention-to-treat trial cohort), events of plasma potassium concentrations of more than 5·5 mmol/L were seen in 13 (13%) of 102 participants in the spironolactone group and four (4%) of 107 participants in the placebo group, and gynaecomastia was seen in three (3%) participants in the spironolactone group and none in the placebo group. One patient died in the placebo group due to a cardiac event (considered possibly related to study drug) and one patient died in the spironolactone group due to cancer, deemed unrelated to study drug. Interpretation: In people with type 2 diabetes and normoalbuminuria, a high-risk score from the urinary proteomic classifier CKD273 was associated with an increased risk of progression to microalbuminuria over a median of 2·5 years, independent of clinical characteristics. However, spironolactone did not prevent progression to microalbuminuria in high-risk patients. Funding: European Union Seventh Framework Programme

Association of kidney fibrosis with urinary peptides: a path towards non-invasive liquid biopsies?

Chronic kidney disease (CKD) is a prevalent cause of morbidity and mortality worldwide. A hallmark of CKD progression is renal fibrosis characterized by excessive accumulation of extracellular matrix (ECM) proteins. In this study, we aimed to investigate the correlation of the urinary proteome classifier CKD273 and individual urinary peptides with the degree of fibrosis. In total, 42 kidney biopsies and urine samples were examined. The percentage of fibrosis per total tissue area was assessed in Masson trichrome stained kidney tissues. The urinary proteome was analysed by capillary electrophoresis coupled to mass spectrometry. CKD273 displayed a significant and positive correlation with the degree of fibrosis (Rho = 0.430, P = 0.0044), while the routinely used parameters (glomerular filtration rate, urine albumin-to-creatinine ratio and urine protein-to-creatinine ratio) did not (Rho = -0.222; -0.137; -0.070 and P = 0.16; 0.39; 0.66, respectively). We identified seven fibrosis-associated peptides displaying a significant and negative correlation with the degree of fibrosis. All peptides were collagen fragments, suggesting that these may be causally related to the observed accumulation of ECM in the kidneys. CKD273 and specific peptides are significantly associated with kidney fibrosis; such an association could not be detected by other biomarkers for CKD. These non-invasive fibrosis-related biomarkers can potentially be implemented in future trials