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.

Urinary CD4 + T Cells Predict Renal Relapse in ANCA-Associated Vasculitis.

SIGNIFICANCE STATEMENT: Early identification of patients at risk of renal flares in ANCA vasculitis is crucial. However, current clinical parameters have limitations in predicting renal relapse accurately. This study investigated the use of urinary CD4 + T lymphocytes as a predictive biomarker for renal flares in ANCA vasculitis. This study, including urine samples from 102 patients, found that the presence of urinary CD4 + T cells was a robust predictor of renal relapse within a 6-month time frame, with a sensitivity of 60% and a specificity of 97.8%. The diagnostic accuracy of urinary CD4 + T cells exceeded that of ANCA titers, proteinuria, and hematuria. Monitoring urinary CD4 + T lymphocytes could help assess the risk of future renal relapse, enabling early preventive measures and tailored treatment strategies. BACKGROUND: In ANCA-associated vasculitis, there is a lack of biomarkers for predicting renal relapse. Urinary T cells have been shown to differentiate active GN from remission in ANCA-associated vasculitis, but their predictive value for renal flares remains unknown. METHODS: The PRE-FLARED study was a prospective multicenter biomarker study including 102 individuals with ANCA-associated vasculitis in remission aimed to predict renal relapse by quantifying urinary CD4 + T-cell subsets using flow cytometry at baseline and monitoring clinical outcomes over a 6-month follow-up. RESULTS: Among the participants, ten experienced renal relapses, two had non-renal flares, and 90 remained in stable remission. The median baseline urinary CD4 + T-cell count was significantly higher in patients who relapsed compared with those in remission. Receiver operating characteristic curve analysis of urinary CD4 + T-cell counts showed an area under the curve value of 0.88 for predicting renal flares, outperforming ANCA titers, hematuria, and proteinuria. Using a cutoff of 490 CD4 + T cells per 100 ml urine, the sensitivity and specificity in identifying patients with future renal flares were 60% and 97.8%, respectively. In a post hoc analysis, combining urinary CD4 + T-cell counts with proteinase-3 ANCA levels suggested improved predictive performance in the PR3 + subgroup. In addition, the number of urinary CD4 + T cells showed a limited correlation with a decline in GFR and an increase in proteinuria over the follow-up period. CONCLUSIONS: This study concluded that urinary CD4 + T-cell counts could identify patients with ANCA-associated vasculitis at a substantial risk of renal relapse within 6 months. Combining these counts with ANCA levels further improved the prediction of relapse. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Urinary T Lymphocytes Predict Renal Flares in Patients With Inactive ANCA-associated Glomerulonephritis (PRE-FLARED), NCT04428398 .

Urinary eicosanoid levels in early life and risk of atopic disease in childhood.

BACKGROUND: Eicosanoids are lipid mediators including thromboxanes (TXs), prostaglandins (PGs), and leukotrienes with a pathophysiological role in established atopic disease. However, their role in the inception of disease is unclear. This study aimed to investigate the association between urinary eicosanoids in early life and development of atopic disease. METHODS: This study quantified the levels of 21 eicosanoids in urine from children from the COPSAC2010 (Copenhagen Prospective Studies on Asthma in Childhood 2010) (age 1 year, n = 450) and VDAART (Vitamin D Antenatal Asthma Reduction Trial) (age 3 years, n = 575) mother-child cohorts and analyzed the associations with development of wheeze/asthma, atopic dermatitis, and biomarkers of type-2 inflammation, applying false discovery rate of 5% (FDR5%) multiple testing correction. RESULTS: In both cohorts, analyses adjusted for environmental determinants showed that higher TXA2 eicosanoids in early life were associated with increased risk of developing atopic dermatitis (P < FDR5%) and type-2 inflammation (P < .05). In VDAART, lower PGE2 and PGI2 eicosanoids and higher isoprostanes were also associated with increased risk of atopic dermatitis (P < FDR5%). For wheeze/asthma, analyses in COPSAC2010 showed that lower isoprostanes and PGF2 eicosanoids and higher PGD2 eicosanoids at age 1 year associated with an increased risk at age 1-10 years (P < .05), whereas analyses in VDAART showed that lower PGE2 and higher TXA2 eicosanoids at age 3 years associated with an increased risk at 6 years (P < FDR5%). CONCLUSIONS: This study suggests that early life perturbations in the eicosanoid metabolism are present before the onset of atopic disease in childhood, which provides pathophysiological insight in the inception of atopic diseases.

Ultrasensitive CCL2 Detection in Urine for Diabetic Nephropathy Diagnosis Using a WS2-Based Plasmonic Biosensor.

The accurate diagnosis of diabetic nephropathy relies on achieving ultrasensitive biosensing for biomarker detection. However, existing biosensors face challenges such as poor sensitivity, complexity, time-consuming procedures, and high assay costs. To address these limitations, we report a WS2-based plasmonic biosensor for the ultrasensitive detection of biomarker candidates in clinical human urine samples associated with diabetic nephropathy. Leveraging plasmonic-based electrochemical impedance microscopy (P-EIM) imaging, we observed a remarkable charge sensitivity in monolayer WS2 single crystals. Our biosensor exhibits an exceptionally low detection limit (0.201 ag/mL) and remarkable selectivity in detecting CC chemokine ligand 2 (CCL2) protein biomarkers, outperforming conventional techniques such as ELISA. This work represents a breakthrough in traditional protein sensors, providing a direction and materials foundation for developing ultrasensitive sensors tailored to clinical applications for biomarker sensing.

Molecular-Sieving Label-Free Surface-Enhanced Raman Spectroscopy for Sensitive Detection of Trace Small-Molecule Biomarkers in Clinical Samples.

Small-molecule biomarkers are ubiquitous in biological fluids with pathological implications, but major challenges persist in their quantitative analysis directly in complex clinical samples. Herein, a molecular-sieving label-free surface-enhanced Raman spectroscopy (SERS) biosensor is reported for selective quantitative analysis of trace small-molecule trimetazidine (TMZ) in clinical samples. Our biosensor is fabricated by decorating a superhydrophobic monolayer of microporous metal-organic frameworks (MOF) shell-coated Au nanostar nanoparticles on a silicon substrate. The design strategy principally combines the hydrophobic surface-enabled physical confinement and preconcentration, MOF-assisted molecular enrichment and sieving of small molecules, and sensitive SERS detection. Our biosensor utilizes such a "molecular confinement-and-sieving" strategy to achieve a five orders-of-magnitude dynamic detection range and a limit of detection of ≈0.5 nM for TMZ detection in either urine or whole blood. We further demonstrate the applicability of our biosensing platform for longitudinal label-free SERS detection of the TMZ level directly in clinical samples in a mouse model.

Proteomics of urinary small extracellular vesicles in early diagnosis of kidney diseases in children-expectations and limitations.

The primary function of the kidneys is to maintain systemic homeostasis (disruption of renal structure and function results in multilevel impairment of body function). Kidney diseases are characterized by a chronic, progressive course and may result in the development of chronic kidney disease (CKD). Evaluation of the composition of the proteome of urinary small extracellular vesicles (sEVs) as a so-called liquid biopsy is a promising new research direction. Knowing the composition of sEV could allow localization of cellular changes in specific sections of the nephron or the interstitial tissue before fixed changes, detectable only at an advanced stage of the disease, occur. Research is currently underway on the role of sEVs in the diagnosis and monitoring of many disease entities. Reports in the literature on the subject include: diabetic nephropathy, focal glomerulosclerosis in the course of glomerulopathies, renal fibrosis of various etiologies. Studies on pediatric patients are still few, involving piloting if small groups of patients without validation studies. Here, we review the literature addressing the use of sEV for diagnosis of the most common urinary disorders in children. We evaluate the clinical utility and define limitations of markers present in sEV as potential liquid biopsy.

Comparative Analysis of Protein Quantification by the SomaScan Assay versus Orthogonal Methods in Urine from People with Diabetic Kidney Disease.

To our knowledge, calibration curves or other validations for thousands of SomaScan aptamers are not publicly available. Moreover, the abundance of urine proteins obtained from these assays is not routinely validated with orthogonal methods (OMs). We report an in-depth comparison of SomaScan readout for 23 proteins in urine samples from patients with diabetic kidney disease (n = 118) vs OMs, including liquid chromatography-targeted mass spectrometry (LC-MS), ELISA, and nephelometry. Pearson correlation between urine abundance of the 23 proteins from SomaScan 3.2 vs OMs ranged from -0.58 to 0.86, with a median (interquartile ratio, [IQR]) of 0.49 (0.18, 0.53). In multivariable linear regression, the SomaScan readout for 6 of the 23 examined proteins (26%) was most strongly associated with the OM-derived abundance of the same (target) protein. For 3 of 23 (13%), the SomaScan and OM-derived abundance of each protein were significantly associated, but the SomaScan readout was more strongly associated with OM-derived abundance of one or more "off-target" proteins. For the remaining 14 proteins (61%), the SomaScan readouts were not significantly associated with the OM-derived abundance of the targeted proteins. In 6 of the latest group, the SomaScan readout was not associated with urine abundance of any of the 23 quantified proteins. To sum, over half of the SomaScan results could not be confirmed by independent orthogonal methods.

Application of Proteomics and Machine Learning Methods to Study the Pathogenesis of Diabetic Nephropathy and Screen Urinary Biomarkers.

Diabetic nephropathy (DN) has become the main cause of end-stage renal disease worldwide, causing significant health problems. Early diagnosis of the disease is quite inadequate. To screen urine biomarkers of DN and explore its potential mechanism, this study collected urine from 87 patients with type 2 diabetes mellitus (which will be classified into normal albuminuria, microalbuminuria, and macroalbuminuria groups) and 38 healthy subjects. Twelve individuals from each group were then randomly selected as the screening cohort for proteomics analysis and the rest as the validation cohort. The results showed that humoral immune response, complement activation, complement and coagulation cascades, renin-angiotensin system, and cell adhesion molecules were closely related to the progression of DN. Five overlapping proteins (KLK1, CSPG4, PLAU, SERPINA3, and ALB) were identified as potential biomarkers by machine learning methods. Among them, KLK1 and CSPG4 were positively correlated with the urinary albumin to creatinine ratio (UACR), and SERPINA3 was negatively correlated with the UACR, which were validated by enzyme-linked immunosorbent assay (ELISA). This study provides new insights into disease mechanisms and biomarkers for early diagnosis of DN.

Acute high-dose MitoQ does not increase urinary kidney injury markers in healthy adults: a randomized crossover trial.

Several human studies have used the mitochondrial antioxidant MitoQ. Recent in vitro data indicating that MitoQ may induce nephrotoxicity caused concern regarding the safety of MitoQ on the kidneys, but the doses were supraphysiological. Therefore, we sought to determine whether acute MitoQ elicits changes in urinary biomarkers associated with tubular injury in healthy adults with our hypothesis being there would be no changes. Using a randomized crossover design, 32 healthy adults (16 females and 16 males, 29 ± 11 yr old) consumed MitoQ (100-160 mg based on body mass) or placebo capsules. We obtained serum samples and a 4- to 6-h postcapsule consumption urine sample. We assessed creatinine clearance and urine kidney injury biomarkers including the chitinase 3-like-1 gene product YKL-40, kidney-injury marker-1, monocyte chemoattractant protein-1, epidermal growth factor, neutrophil gelatinase-associated lipocalin, interleukin-18, and uromodulin using multiplex assays. We used t tests, Wilcoxon tests, and Hotelling's T2 to assess global differences in urinary kidney injury markers between conditions. Acute MitoQ supplementation did not influence urine flow rate (P = 0.086, rrb = 0.39), creatinine clearance (P = 0.085, rrb = 0.42), or urinary kidney injury markers (T22,8 = 30.6, P = 0.121, univariate ps > 0.064). Using exploratory univariate analysis, MitoQ did not alter individual injury markers compared with placebo (e.g., placebo vs. MitoQ: YKL-40, 507 ± 241 vs. 442 ± 236 pg/min, P = 0.241; kidney injury molecule-1, 84.1 ± 43.2 vs. 76.2 ± 51.2 pg/min, P = 0.890; and neutrophil gelatinase-associated lipocalin, 10.8 ± 10.1 vs. 9.83 ± 8.06 ng/min, P = 0.609). In conclusion, although longer-term surveillance and data are needed in clinical populations, our findings suggest that acute high-dose MitoQ had no effect on urinary kidney injury markers in healthy adults.NEW & NOTEWORTHY We found acute high-dose mitochondria-targeted antioxidant (MitoQ) supplementation was not nephrotoxic and had no effect on markers of acute kidney injury in healthy adults. These findings can help bolster further confidence in the safety of MitoQ, particularly for future investigations seeking to examine the role of mitochondrial oxidative stress, via acute MitoQ supplementation, on various physiological outcomes.

Sex-disaggregated analysis of acute kidney injury in hospitalized children with sickle cell anemia in Uganda.

A growing body of research is categorizing sex differences in both sickle cell anemia (SCA) and acute kidney injury (AKI); however, most of this work is being conducted in high-resource settings. Here, we evaluated risk factors and clinical parameters associated with AKI and AKI severity, stratified by sex, in a cohort of children hospitalized with SCA and vaso-occlusive pain crisis (VOC). The purpose of this study was to explore sex disparities in a high-risk, vulnerable population. This study was a secondary analysis of data collected from a cohort of Ugandan children between 2 and 18 yr of age prospectively enrolled. A total of 185 children were enrolled in the primary study; 41.6% were female and 58.4% were male, with a median age of 8.9 yr. Incident or worsening AKI (P = 0.026) occurred more frequently in female compared with male children, despite no differences in AKI on admission. Female children also had altered markers of renal function including higher creatinine levels at admission (P = 0.03), higher peak creatinine (P = 0.006), and higher urine neutrophil gelatinase-associated lipocalin (NGAL) at admission (P = 0.003) compared with male children. Female children had elevated total (P = 0.045) and conjugated bilirubin at admission (P = 0.02) compared with male children and higher rates of hematuria at admission (P = 0.004). Here, we report sex differences in AKI in children with SCA and VOC, including increased incidence and worsening of AKI in female pediatric patients, in association with an increase in biological indicators of poor renal function including creatinine, estimated glomerular filtration rate, and NGAL.NEW & NOTEWORTHY In this study, we report an increased risk of developing acute kidney injury (AKI) during hospitalization, worsening AKI, and death among females with sickle cell anemia (SCA) hospitalized with an acute pain crisis compared with males. The sex differences in AKI were not explained by socioeconomic differences, severity of pain, or disease severity among females compared with males. Together, these data suggest that female children with SCA may be at increased risk of AKI.

Altered kidney distribution and loss of ACE2 into the urine in acute kidney injury.

There are diverse pathophysiological mechanisms involved in acute kidney injury (AKI). Among them, overactivity of the renin-angiotensin system (RAS) has been described. Angiotensin-converting enzyme 2 (ACE2) is a tissue RAS enzyme expressed in the apical border of proximal tubules. Given the important role of ACE2 in the metabolism of angiotensin II, this study aimed to characterize kidney and urinary ACE2 in a mouse model of AKI. Ischemia-reperfusion injury (IRI) was induced in C57BL/6 mice by clamping of the left renal artery followed by removal of the right kidney. In kidneys harvested 48 h after IRI, immunostaining revealed a striking maldistribution of ACE2 including spillage into the tubular lumen and the presence of ACE2-positive luminal casts in the medulla. In cortical membranes, ACE2 protein and enzymatic activity were both markedly reduced (37 ± 4 vs. 100 ± 6 ACE2/ß-actin, P = 0.0004, and 96 ± 14 vs. 152 ± 6 RFU/µg protein/h, P = 0.006). In urine, full-length membrane-bound ACE2 protein (100 kDa) was markedly increased (1,120 ± 405 vs. 100 ± 46 ACE2/µg creatinine, P = 0.04), and casts stained for ACE2 were recovered in the urine sediment. In conclusion, in AKI caused by IRI, there is a marked loss of ACE2 from the apical tubular border with deposition of ACE2-positive material in the medulla and increased urinary excretion of full-length membrane-bound ACE2 protein. The deficiency of tubular ACE2 in AKI suggests that provision of this enzyme could have therapeutic applications and that its excretion in the urine may also serve as a diagnostic marker of severe proximal tubular injury.NEW & NOTEWORTHY This study provides novel insights into the distribution of kidney ACE2 in a model of AKI by IRI showing a striking detachment of apical ACE2 from proximal tubules and its loss in urine and urine sediment. The observed deficiency of kidney ACE2 protein and enzymatic activity in severe AKI suggests that administration of forms of this enzyme may mitigate AKI and that urinary ACE2 may serve as a potential biomarker for tubular injury.

Relmapirazin, a new exogenous filtration marker, and more widespread use of measured GFR.

Plasma or urinary clearance of exogenous filtration markers is required for assessment of measured glomerular filtration rate. Although multiple methods are available, none is widely used because of their complexity, each has measurement error, and standardization is limited. Recently, a study validated the plasma clearance of a new exogenous filtration marker, relmapirazin, which can be detected by its transdermal fluorescence, potentially simplifying the procedure and increasing access to measured glomerular filtration rate.

Prognostic implications of unbiased molecular categorization in kidney disease.

In this commentary, a novel approach to the reclassification of chronic kidney disease is reviewed. In the revisited study, the investigators identify 4 distinct subtypes of kidney disease derived from an unbiased self-organizing map of transcriptomic data from kidney biopsy samples. These molecular subtypes then are characterized by biologic cell processes, clinical and histopathologic features, urinary proteomics, and disease progression. The strengths and limitations of the self-organizing map approach are assessed; the prognostic, diagnostic, and therapeutic implications are considered briefly.

Deceased donor kidney function and branched chain amino acid metabolism during ex vivo normothermic perfusion.

Current kidney perfusion protocols are not optimized for addressing the ex vivo physiological and metabolic needs of the kidney. Ex vivo normothermic perfusion may be utilized to distinguish high-risk kidneys to determine suitability for transplantation. Here, we assessed the association of tissue metabolic changes with changes in a kidney injury biomarker and functional parameters in eight deceased donor kidneys deemed unsuitable for transplantation during a 12-hour ex vivo normothermic perfusion. The kidneys were grouped into good and poor performers based on blood flow and urine output. The mean age of the deceased kidney donors was 43 years with an average cold ischemia time of 37 hours. Urine output and creatinine clearance progressively increased and peaked at six hours post-perfusion among good performers. Poor performers had 71 ng/ml greater (95% confidence interval 1.5, 140) urinary neutrophil gelatinase-associated lipocalin at six hours compared to good performers corresponding to peak functional differences. Organ performance was distinguished by tissue metabolic differences in branched chain amino acid metabolism and that their tissue levels negatively correlated with urine output among all kidneys at six hours. Tissue lipid profiling showed poor performers were highlighted by the accumulation of membrane structure components including glycerolipids and sphingolipids at early perfusion time points. Thus, we showed that six hours is needed for kidney function recovery during ex vivo normothermic perfusion and that branched chain amino acid metabolism may be a major determinant of organ function and resilience.

Phosphatidylethanol Detects Occult Heavy Alcohol Use in Patients With Acute and Chronic Pancreatitis.

Given the paucity of interventions to treat pancreatitis, it is imperative to identify and intervene upon modifiable risk factors such as heavy alcohol use. Current trends indicate a concerning increase in alcohol misuse and alcohol-related disease since the onset of the coronavirus disease-2019 pandemic.1 The incidence of pancreatitis associated with alcohol misuse has increased by approximately 3% annually from 1961 to 2016.2 Alcohol recidivism may be the most important risk factor for pancreatitis recurrence and development of chronic pancreatitis in the United States.3 Early identification of alcohol misuse as a modifiable risk factor is paramount to mitigating pancreatitis-related morbidity. However, blood ethanol and urine ethyl glucuronide levels may be low in symptomatic individuals because they clear rapidly and patients may abstain from drinking in the days before their clinical presentation. Patient self-report may underestimate the quantity of alcohol intake and falsely reassure the provider that this is not a contributing factor to the presentation.4.

Rapid Fingerprinting of Urinary Volatile Metabolites and Point-of-Care Diagnosis of Phenylketonuria on a Patterned Nanorod Sensor Array with Multiplexed Surface-Enhanced Raman Scattering Readouts.

Phenylketonuria (PKU) is one of the most common genetic metabolic diseases, especially among newborns. Traditional clinical examination of newborn blood samples for PKU is invasive, laborious, and limited to hospitals and healthcare facilities. We reported herein a SERS-based sensor array with three thiophenolic nanoreceptors built on a patterned nanorod vertical array for rapid and inexpensive detection of characteristic volatile biomarkers indicative of PKU in the urine and accurate classification of newborn baby patients all performed on a hand-held SERS spectrophotometer. The well-ordered array was generated from the volatility-driven assembly of gold nanorods (AuNRs) into an upright and closely packed hexagonal configuration. The uniformly distributed nanowells between AuNRs offered an intense and aspect-ratio-dependent plasmonic field for the molecular enhancement of SERS outputs. The SERS-based detector was integrated into a test chip for regular monitoring of volatile phenylketone bodies in the spiked solution or patients' urine within 5 min, allowing the quantification of a wide variety of normal or abnormal metabolites at their physiologically relevant concentration range. The detection limits for common biomarkers of PKU, including phenylpyruvic acid, 4-hydroxyphenylacetic acid, and phenylacetic acid, were at a few µM and well below the diagnostic thresholds. Moreover, the volatile headspace mixtures from a given urine sample could be fingerprinted by the sensor array and discriminated using machine-learning algorithms. Ultimately, the discrimination of baby patients among 26 cases of mild and classic PKU phenotypes and 17 cases of healthy volunteers could be realized with an overall accuracy of 97%. This hand-held SERS platform plays a pivotal role in advancing healthcare applications in quick screening of neonatal PKU through a facile urinary vapor test.

Development and Evaluation of a Noninvasive Microfluidic-Based Paper Analytical Device for Leptospirosis Diagnosis.

Leptospirosis is a re-emerging infectious disease that presents a diagnostic enigma for clinicians with frequent misdiagnosis due to lack of rapid and accurate diagnostic tests, as the current methods are encumbered by inherent limitations. The development of a diagnostic sensor with a sample-in-result-out capability is pivotal for prompt diagnosis. Herein, we developed a microfluidic paper-based analytical device (spin-µPAD) featuring a sample-in-result-out fashion for the detection of Leptospira specific urinary biomarker, sph2 sphingomyelinase, crucial for noninvasive point-of-care testing. Fabrication of paper devices involved precise photolithography techniques, ensuring a high degree of reproducibility and replicability. By optimizing the device's configuration and protein components, a remarkable sensitivity and specificity was achieved for detecting leptospiral sph2 in urine, even at low concentrations down to 1.5 fg/mL, with an assay time of 15 min. Further, the spin-µPAD was validated with 20 clinical samples, suspected of leptospirosis including other febrile illnesses, and compared with gold standard microscopic agglutination test, culture, Lepto IgM ELISA, darkfield microscopy, and Leptocheck WB spot test. In contrast to commercial diagnostic tools, the spin-µPAD was noninvasive, rapid, easy to use, specific, sensitive, and cost-effective. The results highlight the potential of this innovative spin-µPAD for an efficient and dependable approach to noninvasive leptospirosis diagnosis, addressing critical needs in the realms of public health and clinical settings.

Upconversion Nanoparticle-Based Dot-Blot Immunoassay for Quantitative Biomarker Detection.

Dot-blot immunoassays are widely used for the user-friendly detection of clinical biomarkers. However, the majority of dot-blot assays have only limited sensitivity and are only used for qualitative or semiquantitative analysis. To overcome this limitation, we have employed labels based on photon-upconversion nanoparticles (UCNPs) that exhibit anti-Stokes luminescence and can be detected without optical background interference. First, the dot-blot immunoassay on a nitrocellulose membrane was optimized for the quantitative analysis of human serum albumin (HSA), resulting in a limit of detection (LOD) of 0.19 ng/mL and a signal-to-background ratio (S/B) of 722. Commercial quantum dots were used as a reference label, reaching the LOD of 4.32 ng/mL and the S/B of 3, clearly indicating the advantages of UCNPs. In addition, the potential of UCNP-based dot-blot for real sample analysis was confirmed by analyzing spiked urine samples, reaching the LOD of 0.24 ng/mL and recovery rates from 79 to 123%. Furthermore, we demonstrated the versatility and robustness of the assay by adapting it to the detection of two other clinically relevant biomarkers, prostate-specific antigen (PSA) and cardiac troponin (cTn), reaching the LODs in spiked serum of 9.4 pg/mL and 0.62 ng/mL for PSA and cTn, respectively. Finally, clinical samples of patients examined for prostate cancer were analyzed, achieving a strong correlation with the reference electrochemiluminescence immunoassay (recovery rates from 89 to 117%). The achieved results demonstrate that UCNPs are highly sensitive labels that enable the development of dot-blot immunoassays for quantitative analysis of low-abundance biomarkers.

Proteomics approach to discovering non-invasive diagnostic biomarkers and understanding the pathogenesis of endometriosis: a systematic review and meta-analysis.

BACKGROUND: Endometriosis is one of the most common gynaecological diseases, yet it lacks efficient biomarkers for early detection and unravels disease mechanisms. Proteomic profiling has revealed diverse patterns of protein changes in various clinical samples. Integrating and systematically analysing proteomics data can facilitate the development of biomarkers, expediting diagnosis and providing insights for potential clinical and therapeutic applications. Hence, this systematic review and meta-analysis aimed to explore potential non-invasive diagnostic biomarkers in various biological samples and therapeutic targets for endometriosis. METHODS: Online databases, including Scopus, PubMed, Web of Science, MEDLINE, Embase via Ovid, and Google Scholar, were searched using MeSH terms. Two independent authors screened the articles, extracted the data, and assessed the methodological quality of the included studies. GO and KEGG analyses were performed to identify the pathways that were significantly enriched. Protein­protein interaction and hub gene selection analyses were also conducted to identify biomarker networks for endometriosis. RESULTS: Twenty-six observational studies with a total of 2,486 participants were included. A total of 644 differentially expressed proteins (180 upregulated and 464 downregulated) were identified from 9 studies. Proteins in peripheral blood exhibited a sensitivity and specificity of 38-100% and 59-99%, respectively, for detecting endometriosis, while proteins in urine had a sensitivity of 58-91% and specificity of 76-93%. Alpha-1-antitrypsin, albumin, and vitamin D binding proteins were significantly DEPs in both serum and urine. Complement C3 is commonly expressed in serum, menstrual blood, and cervical mucus. Additionally, S100-A8 is commonly expressed in both menstrual blood and cervical mucus. Haptoglobin is commonly detected in both serum and plasma, whereas cathepsin G is found in urine, serum, and plasma. GO and KEGG enrichment analyses revealed that proteoglycans in cancer pathways, which regulate cell-to-cell interactions, modulate the extracellular matrix, and promote the proliferation and invasion of endometrial cells, are commonly enriched in serum and urine. CONCLUSION: This comprehensive study revealed potential proteomes that were significantly differentially expressed in women with endometriosis utilizing various non-invasive clinical samples. Exploring common differentially expressed proteins in various biological samples provides insights into the diagnosis and pathophysiology of endometriosis, as well as potential clinical and therapeutic applications.

Advancing kidney transplant outcomes: the role of urinary proteomics in graft function monitoring and rejection detection.

INTRODUCTION: Kidney transplantation significantly improves the lives of those with end-stage kidney disease, offering best alternative to dialysis. However, transplant success is threatened by the acute and chronic rejection mechanisms due to complex immune responses against the new organ. AREAS COVERED: The ongoing research into biomarkers holds promise for revolutionizing the early detection and monitoring of the graft health. Liquid biopsy techniques offer a new avenue, with several diagnostic, predictive, and prognostic biomarkers showing promise in detecting and monitoring kidney diseases and an early and chronic allograft rejection. EXPERT OPINION: Evaluating the protein composition related to kidney transplant results could lead to identifying biomarkers that provide insights into the graft functionality. Non-invasive proteomic biomarkers can drastically enhance clinical outcomes and change the way how kidney transplants are evaluated for patients and physicians if they succeed in this transition. Hence, the advancement in proteomic technologies, leads toward a significant improvement in understanding of the protein markers and molecular mechanisms linked to the outcomes of kidney transplants. However, the road from discovery to the use of such proteins in clinical practice is long, with a need for continuous validation and beyond the singular research team with comprehensive infrastructure and across research groups collaboration.