Tumor-neutrophil cross talk orchestrates the tumor microenvironment to determine the bladder cancer progression.

The immune landscape of bladder cancer progression is not fully understood, and effective therapies are lacking in advanced bladder cancer. Here, we visualized that bladder cancer cells recruited neutrophils by secreting interleukin-8 (IL-8); in turn, neutrophils played dual functions in bladder cancer, including hepatocyte growth factor (HGF) release and CCL3highPD-L1high super-immunosuppressive subset formation. Mechanistically, c-Fos was identified as the mediator of HGF up-regulating IL-8 transcription in bladder cancer cells, which was central to the positive feedback of neutrophil recruitment. Clinically, compared with serum IL-8, urine IL-8 was a better biomarker for bladder cancer prognosis and clinical benefit of immune checkpoint blockade (ICB). Additionally, targeting neutrophils or hepatocyte growth factor receptor (MET) signaling combined with ICB inhibited bladder cancer progression and boosted the antitumor effect of CD8+ T cells in mice. These findings reveal the mechanism by which tumor-neutrophil cross talk orchestrates the bladder cancer microenvironment and provide combination strategies, which may have broad impacts on patients suffering from malignancies enriched with neutrophils.

Renal Biomarkers in the Early Detection of Acute Kidney Injury After Off-Pump Coronary Artery Bypass Grafting.

BACKGROUND: Cardiac surgery-associated (CSA) acute kidney injury (AKI) is a severe postoperative complication in patients undergoing off-pump coronary artery bypass grafting (OPCAB). Early detection of postoperative CSA-AKI may be key to improving patient outcomes. This study explored the use of renal biomarkers measured immediately after surgery for the early detection of CSA-AKI in patients undergoing OPCAB.Methods and Results: In all, 111 patients who underwent OPCAB at Kumamoto University Hospital between June 2020 and October 2022 were included in this study. Urinary neutrophil gelatinase-associated lipocalin, liver-type fatty acid-binding protein, and N-acetyl-ß-D-glucosaminidase (NAG) were measured upon arrival in the intensive care unit (ICU) after surgery. AKI was diagnosed using KDIGO criteria. Of the 111 patients, 32 (28.8%) developed postoperative AKI. Regarding AKI staging, 19 (59.4%), 11 (34.4%), and 2 (6.3%) patients had Stage 1, 2, and 3 AKI, respectively. There were significant differences in chronic kidney disease, preoperative estimated glomerular filtration rate (eGFR), and NAG between the AKI and non-AKI groups. Multivariate analysis showed that preoperative eGFR (odds ratio [OR] for 5-mL/min/1.73 m2increase in eGFR 0.75; 95% confidence interval [CI] 0.63-0.89) and increasing urinary NAG concentrations at ICU admission (OR 2.44; 95% CI 1.30-4.60) were significant risk factors for CSA-AKI in OPCAB patients. CONCLUSIONS: NAG and eGFR may be valuable biomarkers for the early detection of CSA-AKI in patients undergoing OPCAB.

Acute kidney injury biomarker olfactomedin 4 predicts furosemide responsiveness.

BACKGROUND: Acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients. Olfactomedin 4 (OLFM4), a secreted glycoprotein expressed in neutrophils and stressed epithelial cells, is upregulated in loop of Henle (LOH) cells following AKI. We hypothesized that urine OLFM4 (uOLFM4) will increase in patients with AKI and may predict furosemide responsiveness. METHODS: Urine from critically ill children was collected prospectively and tested for uOLFM4 concentrations with a Luminex immunoassay. Severe AKI was defined by KDIGO (stage 2/3) serum creatinine criteria. Furosemide responsiveness was defined as > 3 mL/kg/h of urine output in the 4 h after a 1 mg/kg IV furosemide dose administered as part of standard of care. RESULTS: Fifty-seven patients contributed 178 urine samples. Irrespective of sepsis status or AKI cause, uOLFM4 concentrations were higher in patients with AKI (221 ng/mL [IQR 93-425] vs. 36 ng/mL [IQR 15-115], p = 0.007). uOLFM4 concentrations were higher in patients unresponsive to furosemide (230 ng/mL [IQR 102-534] vs. 42 ng/mL [IQR 21-161], p = 0.04). Area under the receiver operating curve for association with furosemide responsiveness was 0.75 (95% CI, 0.60-0.90). CONCLUSIONS: AKI is associated with increased uOLFM4. Higher uOLFM4 is associated with a lack of response to furosemide. Further testing is warranted to determine whether uOLFM4 could identify patients most likely to benefit from earlier escalation from diuretics to kidney replacement therapy to maintain fluid balance. A higher resolution version of the Graphical abstract is available as Supplementary information.

Immunolocalization of Sphingolipid Catabolism Enzymes along the Nephron: Novel Early Urinary Biomarkers of Renal Damage.

The objective of this study was to investigate whether the activity of enzymes involved in sphingolipid catabolism could be biomarkers to predict early renal damage in streptozotocin (STZ)-induced diabetic rats and Angiotensin II (Ang II)-induced hypertension rats. Diabetic and hypertensive rats had no changes in plasma creatinine concentration. However, transmission electron microscopy (TEM) analysis showed slight ultrastructural changes in the glomeruli and tubular epithelial cells from diabetic and hypertensive rats. Our results show that the acid sphingomyelinase (aSMase) and neutral sphingomyelinase (nSMase) activity increased in the urine of diabetic rats and decreased in hypertensive rats. Only neutral ceramidase (nCDase) activity increased in the urine of diabetic rats. Furthermore, the immunofluorescence demonstrated positive staining for the nSMase, nCDase, and sphingosine kinase (SphK1) in glomerular mesangial cells, proximal tubule, ascending thin limb of the loop of Henle, thick ascending limb of Henle's loop, and principal cells of the collecting duct in the kidney. In conclusion, our results suggest that aSMase and nCDase activity in urine could be a novel predictor of early slight ultrastructural changes in the nephron, aSMase and nCDase as glomerular injury biomarkers, and nSMase as a tubular injury biomarker in diabetic and hypertensive rats.

Use of Urinary Biomarkers in Discriminating Interstitial Cystitis/Bladder Pain Syndrome from Male Lower Urinary Tract Dysfunctions.

To analyze the urinary biomarkers in men with lower urinary-tract symptoms (LUTS) and identify interstitial cystitis/bladder pain syndrome (IC/BPS) from the other lower urinary-tract dysfunctions (LUTDs) by the levels of characteristic urinary biomarkers. In total, 198 men with LUTS were prospectively enrolled and urine samples were collected before intervention or medical treatment. Videourodynamic studies were routinely performed and the LUTDs were diagnosed as having bladder-outlet obstruction (BOO) such as bladder-neck dysfunction, benign prostatic obstruction, or poor relaxation of external sphincter (PRES); and bladder dysfunction such as detrusor overactivity (DO), hypersensitive bladder (HSB), and IC/BPS. Patients suspicious of IC/BPS were further confirmed by cystoscopic hydrodistention under anesthesia. The urine samples were investigated for 11 urinary inflammatory biomarkers including eotaxin, IL-6, IL-8, CXCL10, MCP-1, MIP-1ß, RANTES, TNF-α, NGF, BDNF, and PGE2; and 3 oxidative stress biomarkers 8-OHdG, 8-isoprostane, and TAC. The urinary biomarker levels were analyzed between LUTD subgroups and IC/BPS patients. The results of this study revealed that among the patients, IC/BPS was diagnosed in 48, BOO in 66, DO in 25, HSB in 27, PRES in 15, and normal in 17. Patients with BOO had a higher detrusor pressure and BOO index than IC/BPS, whereas patients with IC/BPS, BOO, and DO had a smaller cystometric bladder capacity than the PRES and normal subgroups. Among the urinary biomarkers, patients with IC/BPS had significantly higher levels of eotaxin, MCP-1, TNF-α, 8-OHdG, and TAC than all other LUTD subgroups. By a combination of different characteristic urinary biomarkers, TNF-α, and eotaxin, either alone or in combination, had the highest sensitivity, specificity, positive predictive value, and negative predictive value to discriminate IC/BPS from patients of all other LUTD subgroups, BOO, DO, or HSB subgroups. Inflammatory biomarker MCP-1 and oxidative stress biomarkers 8-OHdG and TAC, although significantly higher in IC/BPS than normal and PRES subgroups, did not have a diagnostic value between male patients with IC/BPS and the BOO, DO, or HSB subgroups. The study concluded that using urinary TNF-α and eotaxin levels, either alone or in combination, can be used as biomarkers to discriminate patients with IC/BPS from the other LUTD subgroups in men with LUTS.

Exploratory Study Analyzing the Urinary Peptidome of T2DM Patients Suggests Changes in ECM but Also Inflammatory and Metabolic Pathways Following GLP-1R Agonist Treatment.

Type II diabetes mellitus (T2DM) accounts for approximately 90% of all diabetes mellitus cases in the world. Glucagon-like peptide-1 receptor (GLP-1R) agonists have established an increased capability to target directly or indirectly six core defects associated with T2DM, while the underlying molecular mechanisms of these pharmacological effects are not fully known. This exploratory study was conducted to analyze the effect of treatment with GLP-1R agonists on the urinary peptidome of T2DM patients. Urine samples of thirty-two T2DM patients from the PROVALID study ("A Prospective Cohort Study in Patients with T2DM for Validation of Biomarkers") collected pre- and post-treatment with GLP-1R agonist drugs were analyzed by CE-MS. In total, 70 urinary peptides were significantly affected by GLP-1R agonist treatment, generated from 26 different proteins. The downregulation of MMP proteases, based on the concordant downregulation of urinary collagen peptides, was highlighted. Treatment also resulted in the downregulation of peptides from SERPINA1, APOC3, CD99, CPSF6, CRNN, SERPINA6, HBA2, MB, VGF, PIGR, and TTR, many of which were previously found to be associated with increased insulin resistance and inflammation. The findings indicate potential molecular mechanisms of GLP-1R agonists in the context of the management of T2DM and the prevention or delaying of the progression of its associated diseases.

Validation of diagnostic nomograms based on CE-MS urinary biomarkers to detect clinically significant prostate cancer.

PURPOSE: Prostate cancer (PCa) is one of the most common cancers and one of the leading causes of death worldwide. Thus, one major issue in PCa research is to accurately distinguish between indolent and clinically significant (csPCa) to reduce overdiagnosis and overtreatment. In this study, we aim to validate the usefulness of diagnostic nomograms (DN) to detect csPCa, based on previously published urinary biomarkers. METHODS: Capillary electrophoresis/mass spectrometry was employed to validate a previously published biomarker model based on 19 urinary peptides specific for csPCa. Added value of the 19-biomarker (BM) model was assessed in diagnostic nomograms including prostate-specific antigen (PSA), PSA density and the risk calculator from the European Randomized Study of Screening. For this purpose, urine samples from 147 PCa patients were collected prior to prostate biopsy and before performing digital rectal examination (DRE). The 19-BM score was estimated via a support vector machine-based software based on the pre-defined cutoff criterion of - 0.07. DNs were subsequently developed to assess added value of integrative diagnostics. RESULTS: Independent validation of the 19-BM resulted in an 87% sensitivity and 65% specificity, with an AUC of 0.81, outperforming PSA (AUC PSA: 0.64), PSA density (AUC PSAD: 0.64) and ERSPC-3/4 risk calculator (0.67). Integration of 19-BM with the rest clinical variables into distinct DN, resulted in improved (AUC range: 0.82-0.88) but not significantly better performances over 19-BM alone. CONCLUSION: 19-BM alone or upon integration with clinical variables into DN, might be useful for detecting csPCa by decreasing the number of biopsies.

Pre-diagnosis urine exosomal RNA (ExoDx EPI score) is associated with post-prostatectomy pathology outcome.

PURPOSE: ExoDx Prostate IntelliScore (EPI) is a non-invasive urine exosome RNA-based test for risk assessment of high-grade prostate cancer. We evaluated the association of pre-biopsy test results with post-radical prostatectomy (RP) outcomes to understand the potential utility of EPI to inform invasive treatment vs active surveillance (AS) decisions. METHODS: Urine samples were collected from 2066 men scheduled for initial biopsy with PSA between 2 and 10 ng/mL, no history of prostate cancer, and ≥ 50 years across multiple clinical studies. 310 men proceeded to RP, of which 111 patients had Gleason group grade 1 (GG1) at biopsy and would have been potential candidates for AS. We compared pre-biopsy urine scores with ERSPC and PCPT multivariate risk calculator scores for men with GG1 at biopsy to post-RP pathology. RESULTS: Urine EPI scores were significantly lower in men with GG1 at biopsy than in men with > GG1 (p = 0.04), while there were no differences in multivariate risk scores used in standard clinical practice (p > 0.05). Further, EPI scores were significantly lower in men with GG1 at biopsy who remained GG1 post-RP compared to men upgraded to ≥ GG3 post-RP (p < 0.001). In contrast, none of the multiparametric risk calculators showed significant differences (p > 0.05). Men with GG1 at biopsy and EPI score < 15.6 had zero rate of upgrading to ≥ GG3 post-RP compared to 16.0% for EPI scores ≥ 15.6. CONCLUSIONS: The EPI urine biomarker outperformed the multivariate risk calculators in a homogenous risk group of pre-biopsy men. The EPI score was associated with low-risk pathology post-RP, with potential implications on informing AS decisions. TRIAL REGISTRATION: NCT02702856, NCT03031418, NCT03235687, NCT04720599.

Urinary protein biomarker panel predicts esophageal squamous carcinoma from control cases and other tumors.

PURPOSE: Discovery of noninvasive urinary biomarkers for the early diagnosis of esophageal squamous carcinoma (ESCC). METHODS: We conducted proteomic analyses of 499 human urine samples obtained from healthy individuals (n = 321) and ESCC (n = 83), bladder cancer (n = 17), breast cancer (n = 12), colorectal cancer (n = 16), lung cancer (n = 33) and thyroid cancer (n = 17) patients from multiple medical centers. Those samples were divided into a discovery set (n = 247) and an independent validation set (n = 157). RESULTS: Among urinary proteins identified in the comprehensive quantitative proteomics analysis, we selected a panel of three urinary biomarkers (ANXA1, S100A8, TMEM256), and established a logistic regression model in the discovery set that can correctly classify the majority of ESCC cases in the validation sets with the area under the curve (AUC) values of 0.825. This urinary biomarker panel not only discriminates ESCC patients from healthy individuals but also differentiates ESCC from other common tumors. Notably, the panel distinguishes stage I ESCC patients from healthy individuals with AUC values of 0.886. On the analysis of stage-specific biomarkers, another combination panel of protein (ANXA1, S100A8, SOD3, TMEM256) demonstrated a good AUC value of 0.792 for stage I ESCC. CONCLUSIONS: Urinary biomarker panel represents a promising auxiliary diagnostic tool for ESCC, including early-stage ESCC.

Biomarkers of Kidney Tubule Health, CKD Progression, and Acute Kidney Injury in SPRINT (Systolic Blood Pressure Intervention Trial) Participants.

RATIONALE & OBJECTIVE: The Systolic Blood Pressure Intervention Trial (SPRINT) compared the effect of intensive versus standard systolic blood pressure targets on cardiovascular morbidity and mortality. In this ancillary study, we evaluated the use of exploratory factor analysis (EFA) to combine biomarkers of kidney tubule health in urine and plasma and then study their role in longitudinal estimated glomerular filtration rate (eGFR) change and risk of acute kidney injury (AKI). STUDY DESIGN: Observational cohort nested in a clinical trial. SETTING & PARTICIPANTS: 2,351 SPRINT participants with eGFR < 60 mL/min/1.73 m2 at baseline. EXPOSURE: Levels of neutrophil gelatinase-associated lipocalin (NGAL), interleukin 18 (IL-18), chitinase-3-like protein (YKL-40), kidney injury molecule 1 (KIM-1), monocyte chemoattractant protein 1 (MCP-1), α1-microglobulin (A1M) and ß2-microglobulin (B2M), uromodulin (UMOD), fibroblast growth factor 23 (FGF-23), and intact parathyroid hormone (PTH). OUTCOME: Longitudinal changes in eGFR and risk of AKI. ANALYTICAL APPROACH: We performed EFA to capture different tubule pathophysiologic processes. We used linear mixed effects models to evaluate the association of each factor with longitudinal changes in eGFR. We evaluated the association of the tubular factors scores with AKI using Cox proportional hazards regression. RESULTS: From 10 biomarkers, EFA generated 4 factors reflecting tubule injury/repair (NGAL, IL-18, and YKL-40), tubule injury/fibrosis (KIM-1 and MCP-1), tubule reabsorption (A1M and B2M), and tubule reserve/mineral metabolism (UMOD, FGF-23, and PTH). Each 1-SD higher tubule reserve/mineral metabolism factor score was associated with a 0.58% (95% CI, 0.39%-0.67%) faster eGFR decline independent of baseline eGFR and albuminuria. Both the tubule injury/repair and tubule injury/fibrosis factors were independently associated with future risk of AKI (per 1 SD higher, HRs of 1.18 [95% CI, 1.10-1.37] and 1.23 [95% CI, 1.02-1.48], respectively). LIMITATIONS: The factors require validation in other settings. CONCLUSIONS: EFA allows parsimonious subgrouping of biomarkers into factors that are differentially associated with progressive eGFR decline and AKI. These subgroups may provide insights into the pathological processes driving adverse kidney outcomes.

Hexylaminolevulinate-mediated fluorescent urine cytology with a novel automated detection technology for screening and surveillance of bladder cancer.

OBJECTIVES: To evaluate the diagnostic performance of fluorescent voided urine cytology (FVUC) using a novel automated detection technology to screen for primary bladder cancer and for surveillance of recurrent bladder tumour. PATIENTS AND METHODS: We created a rapid, objective, automated, and high-throughput detection device for hexylaminolevulinate-mediated FVUC, named the cellular fluorescence analysis unit-II (CFAU-II). Two different cohorts were used in this study: (i) screening test for primary bladder cancer (165 patients with bladder cancer and 52 controls), and (ii) surveillance test for detecting intravesical recurrent tumour (192 patients with treated non-muscle-invasive bladder cancer and 15 with post-nephroureterectomy upper urinary tract cancer). Voided urine samples were subjected to urine analysis, conventional VUC (cVUC), and FVUC. Diagnostic performance was compared between cVUC, FVUC, and a combination of the two. RESULTS: A total of 614 urine samples were successfully collected, processed, and analysed. Comparative analysis of the screening test cohort demonstrated that the overall sensitivity of FVUC (63%, P < 0.001) and combination testing (72%, P < 0.001) was significantly higher than that of cVUC (29%). FVUC was found to be superior in most of the subgroups, especially in low-grade, Ta, and small tumours. Analysis of the surveillance test cohort showed that combination testing achieved a sensitivity of 82% and a negative predictive value of 98%, whereas those of cVUC were 39% and 96%, respectively. According to the pathological finding of recurrent tumours presenting false-negative result in the FVUC, the majority of the overlooked recurrent diseases were Ta low-grade tumours. Logistic regression analysis suggested an association between the risk of false-positive results and high density of urine white blood cells and alkaluria. CONCLUSION: The present findings clearly demonstrate that FVUC using the newly developed automation technology has superior sensitivity to cVUC for both screening for primary bladder cancer and recurrent tumour detection. It is essential to confirm the clinical usefulness of this method via further large-scale studies, in addition to ensuring its affordability and availability.

Urine soluble urokinase plasminogen activator receptor as a potential biomarker of lupus nephritis activity.

There is a lack of non-invasive biomarkers to identify lupus nephritis (LN). Soluble urokinase plasminogen activator receptor (suPAR) is a sensitive biomarker of ongoing inflammation and a potential marker of podocyte dysfunction. The aim of this study was to assess urine and plasma suPAR in LN. 14 systemic lupus erythematosus (SLE) patients with newly diagnosed LN, 8 active SLE patients (SLEDAI >8) without LN and 31 healthy individuals were enrolled. Urine and plasma samples were taken before the initiation of LN induction therapy, and monthly thereafter. Global and renal disease activity were defined using the SLEDAI-2K and the SLEDAI-2K renal domain score, respectively. suPAR concentrations were measured with the suPARnostic Flex ELISA assay. Urine and plasma suPAR levels were elevated in SLE patients with active LN compared with resolved LN and healthy controls. Urine suPAR levels were comparable to healthy controls in active SLE without LN. Urine and plasma suPAR levels were higher before than after the initiation of LN induction therapy. Prospective follow-up measurements also suggested that urine suPAR levels raised again in patients with a relapse of LN according to SLEDAI-2K renal domain score, whereas plasma suPAR levels did not correlate with renal disease activity. Urine suPAR is a promising LN activity biomarker, given its isolated elevation in urine in active LN and pronounced decrease with LN improvement.

Non-invasive urine markers for the differentiation between RCCs and oncocytoma.

BACKGROUND: Recently, our group showed that Vim3 is overexpressed in tissue samples of renal oncocytomas and Mxi-2 in clear cell renal carcinoma (ccRCC). The mechanism leading to the truncation of both proteins is known and involves with two miRs, both detectable in urine. Since the analysis of miRs is time-consuming, our aim was to identify the truncated proteins in urine instead. Furthermore, urine samples from small renal masses (SRMs) (n = 45, <4 cm) were analyzed to get a pre-surgical differentiation of the cancer subtypes. METHODS: Urines were accessed from the urological biobank (n = 350). Proteins were isolated from urine samples, and Western blots were performed. Each sample was analyzed with ELISA for the expression of Vim3 and Mxi-2. A lateral flow assay was established. For the detection of SRMs, the miRs were isolated and qRT-PCR was performed. RESULTS: A significant increase of Vim3 in urines from patients with oncocytoma (n = 20) was detectable with ELISA compared to all other subtypes of RCCs (chromophobe (n = 50), papillary (n = 40), ccRCC (n = 200), and controls (n = 40) (***p < 0.0001)). Mxi-2 was predominantly overexpressed in ccRCCs (***p < 0.0001). Lateral flow assay of Vim3 and Mxi-2 shows two bands in the case of oncocytoma and ccRCC indicating the specificity of this test. For SRMs, an overexpression of miR-15a/Mxi2 was detectable in urine samples from ccRCC and chromoRCC patients. In contrast to that, miR-498/Vim3 were predominantly overexpressed in oncocytoma patients. CONCLUSION: Both proteins (Vim3 and Mxi-2) were detectable in patients' urines and can be used for the non-invasive differentiation of kidney cancers.

Candidate Genes and Proteomic Biomarkers of Serum and Urine in Medication-Overuse Headache.

Chronic headache is a topical problem of neurology, psychiatry and general practice. The medication-overuse headache (MOH) is one of the leading pathologies in the structure of chronic headache. However, early diagnosis of the MOH is challenging. We analyzed potential proteomic biomarkers of serum and urine in patients with MOH. METHODS: We searched PubMed, Springer, Scopus, Web of Science, ClinicalKey, and Google Scholar databases for English publications over the past 10 years using keywords and their combinations. RESULTS: We found and analyzed seven studies that met the search criteria for the purpose of the review, including 24 serum proteomic biomarkers and 25 urine proteomic biomarkers of MOH. Moreover, the candidate genes and locus of the studied serum (vitamin D-binding protein, lipocalin-type prostaglandin D2 synthase, apolipoprotein E, etc.) and urine proteomic biomarkers (uromodulin, alpha-1-microglobulin, zinc-alpha-2-glycoprotein, etc.) of MOH are presented in this review. CONCLUSIONS: The serum and urine proteomic biomarkers of MOH can potentially help with the identification of patients with MOH development. Due to the relevance of the problem, the authors believe that further investigation of the MOH proteomic biomarkers in different ethnic and racial groups of patients with primary headache is necessary. In addition, it is important to investigate whether medications of different drug classes influence the levels of serum and urine proteomic biomarkers.

On normalizing of urinary KIM-1 level to urine creatinine in patients with renal cell cancer.

KIM-1 (kidney injury molecule 1), a marker of acute kidney injury, is produced by epithelial cells of renal proximal tubules. Elevated KIM-1 levels in urine and plasma are associated with renal cell carcinoma (RCC). The aim of this study was to compare the significance of non-normalized uKIM-1 values and those normalized to urine creatinine, as urinary biomarkers in RCC. The uKIM-1, urine creatinine and their ratio (uKIM-1/Cre) were studied in 118 RCC patients and 58 apparently healthy subjects. The median of uKIM-1 in the healthy group was 0.71 ng/ml (1st and 3rd quartiles were 0.35 and 1.23, respectively) and in RCC patients it was 2.36 (1.43; 5.93) ng/ml. The medians of uKIM-1/Cre were 0.77 (0.49; 1.18) and 2.42 (1.41; 4.61) ng/mgCre, respectively. Stage I RCC is statistically significantly different from stages II-III and stage IV using uKIM-1/Cre values (p = 0.0056 and p = 0.0012, respectively); using uKIM-1 values significant differences occur only when comparing stages I and IV (p = 0.015). In both healthy individuals and RCC patients, uKIM-1/Cre levels were slightly lower in subgroups younger than 50 years than in subgroups older than 50 years, whereas a similar trend was observed for uKIM-1 only in patients. In healthy men and male patients, uKIM-1 levels were higher than in the corresponding groups of women (the differences were not statistically significant), but the use of uKIM-1/Cre values eliminated the gender differences. A high correlation was found between the concentrations of uKIM-1 and urine creatinine in three healthy subjects followed up for 3 weeks (Spearman's correlation coefficients were 0.758, 0.825 and 0.933, respectively). The data obtained are clear evidence of the need for normalization uKIM-1 to urine creatinine in RCC patients.

Urine cytokines as biomarkers for diagnosing interstitial cystitis/bladder pain syndrome and mapping its clinical characteristics.

The objective of the present study was to investigate the diagnostic values of urine cytokines in patients with interstitial cystitis/bladder pain syndrome (IC/BPS) and to identify their correlations with clinical characteristics. Urine samples were collected from 127 patients with IC/BPS [European Society for the Study of Interstitial Cystitis (ESSIC) types 1 and 2] and 28 controls. Commercially available multiplex immunoassays (MILLIPLEX map kits) were used to analyze 31 targeted cytokines. Cytokine levels between patients with IC/BPS and controls were analyzed using ANOVA. Receiver-operating characteristic curves of each cytokine to distinguish IC/BPS from controls were generated for calculation of the area under the curve. Patients with IC/BPS had urine cytokine profiles that differed from those of controls. Between patients with ESSIC type 1 and 2 IC/BPS, urine cytokine profiles were also different. Among cytokines with high diagnostic values (i.e., area under the curve > 0.7) with respect to distinguish patients with ESSIC type 2 IC/BPS from controls, regulated upon activation, normal T cell expressed and presumably secreted (RANTES), macrophage inflammatory protein (MIP)-1ß, and IL-8 were of higher sensitivity, whereas macrophage chemoattractant protein (MCP)-1, chemokine (C-X-C motif) ligand 10 (CXCL10), and eotaxin-1 were of higher specificity. In multivariate logistic regression models controlling for age, sex, body mass index, and diabetes mellitus, the urine cytokines with high diagnostic values (MCP-1, RANTES, CXCL10, IL-7, and eotaxin-1) remained statistically significant in differentiating IC/BPS and controls. MCP-1, CXCL10, eotaxin-1, and RANTES were positively correlated with glomerulation grade and negatively correlated with maximal bladder capacity. In conclusion, patients with IC/BPS had urine cytokine profiles that clearly differed from those of controls. Urine cytokines might be useful as biomarkers for diagnosing IC/BPS and mapping its clinical characteristics.

Extracellular Vesicles: Cell-Derived Biomarkers of Glomerular and Tubular Injury.

Extracellular vesicles (EVs) are important mediators of intercellular communication. Since EVs are also released during pathological conditions, there has been considerable interest in their potential as sensitive biomarkers of cellular stress and/or injury. In the context of kidney disease, urinary EVs are promising indicators of glomerular and tubular damage. In the present review we discuss the role of urinary EVs in kidney health and disease. Our focus is to explore urinary large EVs (lEVs, often referred to as microparticles or microvesicles) as direct and noninvasive early biomarkers of renal injury. In this regard, studies have been demonstrating altered levels of urinary lEVs, especially podocyte-derived lEVs, preceding the decrease of renal function assessed by classical markers. In addition, we discuss the role of small EVs (sEVs, often referred to as exosomes) and their contents in kidney pathophysiology. Even though results concerning the production of sEVs during diseased conditions are varied, there has been a consensus on the importance of urinary sEV content assessment in kidney disease. These mediators, including EV-released miRNAs and mRNAs, are responsible for EV-mediated signaling in the regulation of renal cellular homeostasis, pathogenesis and regeneration. Finally, steps necessary for the validation of EVs as reliable markers will be discussed.

Urinary C3d is elevated in patients with active Lupus nephritis and a fall in its level after 3 months predicts response at 6 months on follow up.

INTRODUCTION: Complement activation is central to the pathogenesis of lupus nephritis (LN). Low serum complement C3 and C4, are traditionally used as markers of lupus disease activity in general and LN in particular. In this study we prospectively measured plasma and urine C3d and C4d, degradation products of C3 and C4 corrected to creatinine in a cohort of biopsy proven LN in a longitudinal fashion for its correlation with disease activity. METHODS: Twenty eight biopsy proven active lupus nephritis (AN) were recruited along with four inactive nephritis (IN) and 10 healthy controls (HC). Plasma and urine were collected at baseline, prior to induction treatment and 3 months later. Clinical measures of disease activity, Systemic lupus erythematosus disease activity index 2000 (SLEDAI 2K), renal SLEDAI, serum C3, C4 and antibodies to ds DNA, urine protein and creatinine excretion (UP/UC) were collected. Plasma and urine C3d and C4d were measured using ELISA and normalized to spot urine creatinine value. RESULTS: Twenty eight AN of median age of 26.5 (20-31.50) years and disease duration of 3 (0.7-5) years were enrolled. The median urinary C3d/creatinine before treatment was 388.20 (48.98-1296) ng/mg which fell significantly to 62.69 (28.04-502.4) ng/mg at 3 months followup (p-0.01). The baseline values for the active renal disease was significantly different from IN group (9.9 (4.5-46.53 ng/mg) p-0.00). Treatment responders (partial and complete) at 6 months showed a significant fall in urinary C3d at 3 months whereas non responders had a non significant change in value. There was a significant correlation of urine C3d/creatinine with SLEDAI2K (r-0.433, p-0.00), renal SLEDAI (r-0.356, p-0.00), UP/UC ratio (r-0.489, p-<0.0001) but no significant correlation with C3 or C4. There was a significant fall in the median values of plasma C3d from 791.1 (516.0.00-1550.43) µg/ml to 338.52 (211.35-525.82) (p-0.00) µg/ml at the end of 3 months. The values showed a significant correlation with SLEDAI 2K, renal SLEDAI, UP/UC along with a significant negative correlation with C3 and C4. CONCLUSION: Urinary C3d/creatinine levels and plasma C3d levels can be used as biomarker of disease activity and treatment response.

Urinary metabolites identified using metabolomic analysis as potential biomarkers of nocturia in elderly men.

PURPOSE: To investigate the association between nocturia and urinary metabolites in elderly men using metabolomic analysis. METHODS: We recruited 66 men aged 65-80 years. The 3-day frequency volume chart (FCV), International Prostate Symptom Score (IPSS), and quality of life score were used to assess micturition behavior. Participants with the total IPSS > 0 and ≥ 1.5 micturition on an average for three nights were included in the nocturia group. Participants with the total IPSS < 8 and < 1.5 micturition at night were included in the control group. We conducted a comprehensive metabolomic analysis of urine samples. Metabolites were compared between the groups using an unpaired t test. A multivariable logistic regression analysis was used to determine the relationship between nocturia and these metabolites. RESULTS: The nocturia and control groups consisted of 45 and 21 men, respectively. There were no differences in the background factors between the groups except for receiving anticholinergic drug and having life style-related diseases. The FVC revealed that nocturnal urine volume, 24 h micturition frequency, and nocturnal micturition frequency were significantly higher in the nocturia group than in the control group. The metabolomic analysis revealed 16 metabolites, which were differentially expressed between the groups. The multivariate analysis showed that increased serotonin level and decreased 3-hydroxypropionic acid and 3-indoleacetonitrile levels were associated with nocturia. CONCLUSIONS: These findings suggest that abnormal urinary metabolites including serotonin, 3-hydroxypropionic acid, and 3-indoleacetonitrile are involved in the pathogenesis of nocturia in elderly men.

Urine Lactoferrin as a Potential Biomarker Reflecting the Degree of Malignancy in Urothelial Carcinoma of the Bladder.

Urothelial carcinoma of the bladder (UCB) is potentially life-threatening; therefore, we aimed to discover a novel urine biomarker for diagnosis and prognostication of UCB. This is a retrospective case-control study. Exploration of a new biomarker using urine from 20 UCB patients in the present study revealed that urinary level of lactoferrin (LF), a multifunctional glycoprotein released from neutrophils, was higher in 11 of 15 with invasive/high-grade UCB than 5 with non-invasive one, and 2 healthy adults. We therefore focused on LF and assessed the value of urine LF normalized by urine creatinine concentration (LF/Cr) using an enzyme-linked immunosorbent assay. Diagnostic performance of urine LF/Cr was examined using urine from 92 patients with primary (newly diagnosed) untreated UCB and 166 controls without UCB, including 62 patients with pyuria, and 104 subjects without pyuria consisting of 84 patients and 20 healthy adults. However, the diagnostic accuracies were accompanied by the risk of bias. In 92 primary UCB patients, both pyuria and tumor-infiltrating neutrophils (TINs) were independent predictors for urine LF/Cr. In contrast, TINs or urine LF/Cr were independent predictors for invasive histology, whereas pyuria was not. In terms of prognostication, urine LF/Cr and nodal metastasis were independent predictors of disease-specific survival in 22 patients with muscle-invasive bladder cancer, characterized by a high mortality rate, in the Cox proportional hazards model. In conclusion, urine LF/Cr linked to TINs was a predictor of both invasive histology and prognosis in UCB. Urine LF/Cr is a potential biomarker reflecting the degree of malignancy in UCB.