Renal Progenitors Derived from Urine for Personalized Diagnosis of Kidney Diseases.

BACKGROUND: Chronic kidney disease affects 10% of the world population, and it is associated with progression to end-stage kidney disease and increased morbidity and mortality. The advent of multi-omics technologies has expanded our knowledge on the complexity of kidney diseases, revealing their frequent genetic etiology, particularly in children and young subjects. Genetic heterogeneity and drug screening require patient-derived disease models to establish a correct diagnosis and evaluate new potential treatments and outcomes. SUMMARY: Patient-derived renal progenitors can be isolated from urine to set up proper disease modeling. This strategy allows to make diagnosis of genetic kidney disease in patients carrying unknown significance variants or uncover variants missed from peripheral blood analysis. Furthermore, urinary-derived tubuloids obtained from renal progenitors of patients appear to be potentially valuable for modeling kidney diseases to test ex vivo treatment efficacy or to develop new therapeutic approaches. Finally, renal progenitors derived from urine can provide insights into acute kidney injury and predict kidney function recovery and outcome. KEY MESSAGES: Renal progenitors derived from urine are a promising new noninvasive and easy-to-handle tool, which improves the rate of diagnosis and the therapeutic choice, paving the way toward a personalized healthcare.

Effect of renal tubular damage on non-cancer mortality in the general Japanese population living in cadmium non-polluted areas.

This study aimed to clarify the cause-effect relationship between renal tubular damage and non-cancer mortality in the general Japanese population. We conducted a 19-year cohort study including 1110 men and 1,03 women who lived in three cadmium-non-polluted areas in 1993 or 1994. Mortality risk ratios based on urinary ß2-microglobulin (ß2MG) and N-acetyl-ß-glucosaminidase (NAG) concentrations were estimated for specific non-cancer diseases using the Fine and Gray competing risks regression model. In men, continuous urinary NAG (+1 µg/g cre) concentrations were significantly correlated with increased mortality caused by diseases of the respiratory system (hazard ratio (HR): 1.09, 95% confidence interval (CI): 1.03-1.15). Urinary ß2MG (+100 µg/g cre) concentrations were significantly correlated with increased mortalities caused by kidney and urinary tract diseases (HR: 1.01, 95% CI: 1.00-1.03), renal diseases (HR: 1.01, 95% CI: 1.00-1.03), renal failure (HR: 1.02, 95% CI: 1.00-1.03), and external causes of mortality (HR: 1.01, 95% CI: 1.00-1.02). In women, urinary NAG (+1 µg/g cre) concentrations were significantly associated with increased mortality caused by ischemic heart diseases (HR: 1.02, 95% CI: 1.00-1.04) and kidney and urinary tract diseases (HR: 1.01, 95% CI: 1.00-1.04). Urinary ß2MG (+100 µg/g cre) concentrations were significantly correlated with increased mortality caused by cardiovascular diseases (HR: 1.01, 95%CI: 1.00-1.02), ischemic heart diseases (HR: 1.01, 95%CI: 1.00-1.02), and kidney and urinary tract diseases (HR: 1.02, 95% CI: 1.01-1.03). The present study indicates that renal tubular damage was significantly related to several non-cancer disease causes of mortality in Japan's general population living in cadmium-non-polluted areas.

Urinary angiotensinogen is associated with albuminuria in adults with sickle cell anaemia.

We explored the association of novel urinary biomarkers with albumin-creatinine ratio (ACR) in adults with sickle cell anaemia. Of 37 participants, 13 (35.2%) had persistent albuminuria (PA). Urinary levels of clusterin (p = 0.002), retinol-binding protein 4 (p = 0.008), alpha-1 microglobulin (p = 0.002) and angiotensinogen (p = 0.006) were significantly higher in participants with PA than in those without PA. Although univariate analysis showed significant associations between both alpha-1 microglobulin (p = 0.035) and angiotensinogen (p = 0.0021) with ACR, only angiotensinogen was associated with ACR in multivariable analysis (p = 0.04). Our results suggest that urinary angiotensinogen may identify sickle cell anaemia patients at risk for kidney disease.

Chemiluminescence Immunoassay Method of Urinary Liver Fatty-acid-binding Protein as a Promising Candidate for Kidney Disease.

Liver fatty acid binding protein (L-FABP) is an intercellular lipid chaperone protein that selectively combines with unsaturated free fatty acids and transports them to mitochondria or peroxisomes. L-FABP is a promising biomarker for the early detection of renal diseases in humans. Herein a chemiluminescence method (CLIA) was demonstrated to measure the level of urinary L-FABP in the urinary samples. An anti-(L-FABP)-magnetic beads complex was prepared to capture the analyte target. Sensitivity, precision, accuracy, interference effect, high-dose hook effect of the developed assay were evaluated. Under the suitable experimental parameters, the established method have a wide linear range (0.01-10 ng/mL) and also showed a sufficiently low limit of detection of 0.0060 ng/mL. Besides, the satisfactory recoveries of the method in the urinary were ranged from 97.74%-112.32%, which was well within the requirement of clinical analysis. Furthermore, this proposed method has been successfully applied to the clinical determination of L-FABP in patients who have been diagnosed with kidney disease. The results showed that CLIA could accurately and rapidly determine the urinary level of L-FABP with high-throughput, which could be useful as a new tool to predict complications in patients with kidney disease. The clinical trial was approved by Shuyang Hospital of Traditional Chinese Medicine Ethics Committee: 20,210,202-001 at February 2, 2021.

Serum and urine profiling by high-throughput TMT-based proteomics for the investigation of renal dysfunction in canine babesiosis.

Canine babesiosis is a tick-borne disease caused by Babesia canis, with acute kidney injury as one of the common complications. In the study 8 healthy control dogs and 22 dogs with naturally occurring babesiosis were enrolled, with the aim to analyse differences in serum and urinary proteomes between healthy dogs and dogs with different degree of renal dysfunction in babesiosis using a label-based high-throughput quantitative proteomic approach. In serum, 58 proteins were found differentially abundant between healthy controls and groups of dogs with different degrees of renal dysfunction in babesiosis, while in urine there were 259 differentially abundant proteins. In addition, altered biological pathways were detected in the diseased dogs using bioinformatics tools and validation of several candidate biomarkers was performed. SIGNIFICANCE: The main aim of this comprehensive study was to perform analyses of serum and urinary proteomes of dogs with renal dysfunction in babesiosis compared to healthy dogs using, for the first time, a high-throughput proteomic method and functional enrichment analyses. Serum and urine samples of the same dogs were investigated in order to gain a more complete picture of pathologic changes taking place in renal dysfunction in babesiosis. We highlighted two putative biomarkers validated herein which could be of importance for early diagnosis of renal dysfunction in canine babesiosis, as they are easily accessible from urine and their concentration rises before the appearance of azotaemia: urinary neutrophil gelatinase-associated lipocalin (NGAL) and urinary liver-type fatty acid-binding protein (L-FABP).

Analysis of cadmium accumulation in community adults and its correlation with low-grade albuminuria.

OBJECTIVE: To investigate the effects of chronic non-occupational exposure to cadmium (Cd) on renal injury in residents living in the urban areas of China. METHODS: In this cross-sectional study, we recruited 1000 participants in Shanghai from August 2015 to August 2017 and collected data on their socio-demographic characteristics, lifetime occupation, and lifestyle factors. The urinary Cd, urinary albumin, urinary creatinine, serum creatinine, urea, and uric acid levels were tested, and 683 participants completed those measurements. RESULTS: The median urinary Cd concentration of this study population was 0.77 µg/g. The urinary Cd concentration was significantly higher in the female, older, and lower body mass index populations. There were 148 participants with dominant albuminuria who had higher urinary Cd levels than those without dominant albuminuria (0.98 vs. 0.72 µg/g; P < 0.001). Among participants without dominant albuminuria, there were 134 participants with low-grade albuminuria (13.84 ≤ ACR < 30 mg/g) and 401 participants who had normal urinary albumin excretion (ACR < 13.84 mg/g). Compared with those who had normal urinary albumin excretion, those with low-grade albuminuria had significantly higher urinary Cd levels (0.83 vs. 0.69 µg/g; P < 0.001). Among those without dominant albuminuria, participants in the highest quartile of urinary Cd were more likely to have low-grade albuminuria than those in the lowest quartile (Odd's ratio = 2.98; P < 0.001). Further adjustment for age, sex, and BMI or other potential confounding factors did not change the magnitudes of the associations. Moreover, we conducted multivariable stepwise linear regression analysis within 134 low-grade albuminuria participants and demonstrated that urinary Cd concentration (P < 0.001) were independent determinants of urine albumin after adjusting for relevant confounders. CONCLUSION: The urinary Cd levels observed in Chinese urban adults are substantial and associated with an increased risk of low-grade albuminuria. Our findings suggest that potential sources of environmental Cd exposure should be explored, and the associated renal toxicity should be studied in more detail in future.

Urinary Podocyte Count as a Potential Routine Laboratory Test for Glomerular Disease: A Novel Method Using Liquid-Based Cytology and Immunoenzyme Staining.

INTRODUCTION: This study investigated whether our urinary podocyte detection method using podocalyxin (PDX) and Wilms tumor 1 (WT1) immunoenzyme staining combined with liquid-based cytology can serve as a noninvasive routine laboratory test for glomerular disease. METHODS: The presence of PDX- and WT1-positive cells was investigated in 79 patients with glomerular disease and 51 patients with nonglomerular disease. RESULTS: The frequencies and numbers of PDX- and WT1-positive cells were significantly higher in the glomerular disease group than in the nonglomerular disease group. The best cutoffs for PDX- and WT1-positive cell counts for identifying patients with glomerular disease were 3.5 (sensitivity = 67.1% and specificity = 100%) and 1.2 cells/10 mL (sensitivity = 43.0% and specificity = 100%), respectively. CONCLUSION: Because our urinary podocyte detection method using PDX immunoenzyme staining can be standardized and it detected glomerular disease with high accuracy, it can likely serve as a noninvasive routine laboratory test for various glomerular diseases.

Exosomal hsa_circ_0008925 from Urine Is Related to Chronic Renal Fibrosis.

At present, there is no noninvasive biomarker of renal fibrosis. The potential diagnostic value of urinary exosome-derived circRNAs from glomerular disease patients for renal fibrosis is still uncertain. Here, we first detected the expression of hsa_circ_0008925 in TGF-ß1-cultured HK-2 cell-derived exosomes. Secondly, we collected urine samples from 95 biopsy-proven glomerular disease patients and 34 healthy controls. The expression of hsa_circ_0008925 was analyzed, and the correlation with renal function and pathological changes was calculated. The receiver operating characteristic (ROC) curve for the diagnosis of renal fibrosis was performed. The results showed that in exosomes derived from TGF-ß1-cultured HK-2 cells, the expression of hsa_circ_0008925 was increased compared with normal cultured. Further, the expression level of hsa_circ_0008925 was increased in urinary exosomes from renal fibrosis patients and correlated with serum creatinine, blood urea nitrogen (BUN), estimated glomerular filtration rate, and cystatin C. The level of hsa_circ_0008925 was furthermore correlated with the score of tubulointerstitial fibrosis (TIF) and the score of glomerular sclerosis. The ROC curve showed that hsa_circ_0008925 can diagnose renal fibrosis at a cut-off value of 0.093 with a sensitivity of 52.2% and specificity of 96.4%. In summary, we indicated that urinary exosomal hsa_circ_0008925 could be acted as a noninvasive biomarker for renal fibrosis in glomerular diseases patients.

Nephron mass determines the excretion rate of urinary extracellular vesicles.

Urinary extracellular vesicles (uEVs) are emerging as non-invasive biomarkers for various kidney diseases, but it is unknown how differences in nephron mass impact uEV excretion. To address this, uEV excretion was measured before and after human kidney donor nephrectomy and rat nephrectomy. In male and female donors, uEVs were quantified in cell-free spot and 24-h urine samples using nanoparticle tracking analysis (NTA), EVQuant, and CD9-time-resolved fluorescence immunoassay. Female donors had significantly lower total kidney volume (TKV) and excreted 49% fewer uEVs than male donors. uEV excretion correlated positively with estimated glomerular filtration rate (eGFR), creatinine clearance, and TKV (R's between 0.6 and 0.7). uEV excretion rate could also be predicted from spot urines after multiplying spot uEV/creatinine by 24-h urine creatinine. Donor nephrectomy reduced eGFR by 36% ± 10%, but the excretion of uEVs by only 16% (CD9+ uEVs -37%, CD9- uEVs no decrease). Donor nephrectomy increased the podocyte marker WT-1 and the proximal tubule markers NHE3, NaPi-IIa, and cubilin in uEVs two- to four-fold when correcting for the nephrectomy. In rats, the changes in GFR and kidney weight correlated with the changes in uEV excretion rate (R = 0.46 and 0.60, P < 0.01). Furthermore, the estimated degree of hypertrophy matched the change in uEV excretion rate (1.4- to 1.5-fold after uninephrectomy and four-fold after 5/6th nephrectomy). Taken together, our data show that uEV excretion depends on nephron mass, and that nephrectomy reduces uEV excretion less than expected based on nephron loss due to compensatory hypertrophy. The major implication of our findings is that a measure for nephron mass or uEV excretion rate should be included when comparing uEV biomarkers between individuals.

Urinary Biomarkers of Mycotoxin Induced Nephrotoxicity-Current Status and Expected Future Trends.

The intensifying world-wide spread of mycotoxigenic fungal species has increased the possibility of mycotoxin contamination in animal feed and the human food chain. Growing evidence shows the deleterious toxicological effects of mycotoxins from infants to adults, while large population-based screening programs are often missing to identify affected individuals. The kidney functions as the major excretory system, which makes it particularly vulnerable to nephrotoxic injury. However, few studies have attempted to screen for kidney injury biomarkers in large, mycotoxin-exposed populations. As a result, there is an urgent need to screen them with sensitive biomarkers for potential nephrotoxicity. Although a plethora of biomarkers have been tested to estimate the harmful effects of a wide spectrum of toxicants, ß2-microglobulin (ß2-MG) and N-acetyl-ß-D-glucosaminidase (NAG) are currently the dominant biomarkers employed routinely in environmental toxicology research. Nevertheless, kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) are also emerging as useful and informative markers to reveal mycotoxin induced nephrotoxicity. In this opinion article we consider the nephrotoxic effects of mycotoxins, the biomarkers available to detect and quantify the kidney injuries caused by them, and to recommend biomarkers to screen mycotoxin-exposed populations for renal damage.

Benefits and Toxicity of Disulfiram in Preclinical Models of Nephropathic Cystinosis.

Nephropathic cystinosis is a rare disease caused by mutations of the CTNS gene that encodes for cystinosin, a lysosomal cystine/H+ symporter. The disease is characterized by early-onset chronic kidney failure and progressive development of extra-renal complications related to cystine accumulation in all tissues. At the cellular level, several alterations have been demonstrated, including enhanced apoptosis, altered autophagy, defective intracellular trafficking, and cell oxidation, among others. Current therapy with cysteamine only partially reverts some of these changes, highlighting the need to develop additional treatments. Among compounds that were identified in a previous drug-repositioning study, disulfiram (DSF) was selected for in vivo studies. The cystine depleting and anti-apoptotic properties of DSF were confirmed by secondary in vitro assays and after treating Ctns-/- mice with 200 mg/kg/day of DSF for 3 months. However, at this dosage, growth impairment was observed. Long-term treatment with a lower dose (100 mg/kg/day) did not inhibit growth, but failed to reduce cystine accumulation, caused premature death, and did not prevent the development of renal lesions. In addition, DSF also caused adverse effects in cystinotic zebrafish larvae. DSF toxicity was significantly more pronounced in Ctns-/- mice and zebrafish compared to wild-type animals, suggesting higher cell toxicity of DSF in cystinotic cells.

The nephroprotective effects of Daucus carota and Eclipta prostrata against cisplatin-induced nephrotoxicity in rats.

The overuse of cisplatin (>50 mg/m2) is limited to nephrotoxicity, ototoxicity, gastrotoxicity, myelosuppression, and allergic reactions. The objective of this study was to investigate the nephroprotective effects of Daucus carota and Eclipta prostrata extracts on cisplatin-induced nephrotoxicity in Wistar albino rats. The study involved male Wistar albino rats of 8 weeks weighing 220-270 g. A single injection of 5 mg/kg was injected into the rats for nephrotoxicity. Rats were divided into four groups based on dose conentrations. Blood and urine samples of rats were collected on the 0, 7th, 14th, and 21st days for nephrological analysis. The results showed that Cis + DC/Cis + EP (600 mg/kg) significantly (p < 0.001) increased the body weight and reduced the kidney weight of cisplatin-induced nephrotoxicity in rats (p < 0.001) as compared to Cis group. The results showed that 600 mg/kg administration of Cis + DC/Cis +EP successfully (p < 0.005) improved the urine and plasmin creatinine, Na, and K level compared to the Cis group. Histopathological results confirmed that Cis + EP/Cis + DC effectively improved the renal abnormalities. It is concluded that the co-administration of Cis + EP extract showed exceptional nephroprotective effects at a dose rate of 600 mg/kg.

Biomarkers in neonates receiving potential nephrotoxic drugs.

OBJECTIVE: Novel biomarkers, such as kidney injury molecule-1 (KIM-1), cystatin, and neutrophil gelatinase-associated lipocalin (NGAL) were shown to predict acute kidney injury (AKI) earlier than serum creatinine in critically ill. We carried out the present study to evaluate these biomarkers in addition to conventional in our neonates. PATIENTS AND METHODS: We recruited 70 neonates of various gestational age groups receiving one or more potential nephrotoxic drug/s. Daily urine samples were collected for estimating KIM-1, cystatin, and NGAL. Modified neonatal kidney disease improving global outcomes (mKDIGO) classification was used in defining AKI. RESULTS: A significant trend in increased urine concentrations of KIM-1, cystatin, and NGAL were observed as we proceed from term to preterm categories. Strong positive correlation was observed between urine albumin and urine albumin creatinine ratio (ACR), and strong negative correlations between urine creatinine and urine cystatin, and between urine creatinine with urine NGAL. A moderate positive correlation was observed between urine KIM-1 and urine cystatin, between urine KIM-1 and urine NGAL, and between urine cystatin and urine NGAL; and a moderate negative correlation was observed between urine creatinine and urine KIM-1. Seven neonates met the mKDIGO criteria for AKI and ROC plot revealed that baseline KIM-1 and NGAL can significantly predict possible drug-induced AKI in neonates. CONCLUSIONS: Urine KIM-1, cystatin, and NGAL are significantly correlated with several other conventional biomarkers that reflect renal function in critically ill neonates. Baseline urine KIM-1 and NGAL concentrations can predict the AKI following potential nephrotoxic drug use in this population.

Identification of VEGF Signaling Inhibition-Induced Glomerular Injury in Rats through Site-Specific Urinary Biomarkers.

Cancer therapies targeting the vascular endothelial growth factor (VEGF) signaling pathway can lead to renal damage by disrupting the glomerular ultrafiltration apparatus. The objective of the current study was to identify sensitive biomarkers for VEGF inhibition-induced glomerular changes in rats. Male Sprague-Dawley rats were administered an experimental VEGF receptor (VEGFR) inhibitor, ABT-123, for seven days to investigate the correlation of several biomarkers with microscopic and ultrastructural changes. Glomeruli obtained by laser capture microdissection were also subjected to gene expression analysis to investigate the underlying molecular events of VEGFR inhibition in glomerulus. ABT-123 induced characteristic glomerular ultrastructural changes in rats, including fusion of podocyte foot processes, the presence of subendothelial electron-dense deposits, and swelling and loss of fenestrations in glomerular endothelium. The subtle morphological changes cannot be detected with light microscopy or by changes in standard clinical chemistry and urinalysis. However, urinary albumin increased 44-fold as early as Day three. Urinary ß2-microglobulin levels were also increased. Other urinary biomarkers that are typically associated with tubular injury were not significantly impacted. Such patterns in urinary biomarkers can provide valuable diagnostic insight to VEGF inhibition therapy-induced glomeruli injuries.

Association of common medical comorbidities with early renal damage in the Chinese tropics with essential hypertension.

BACKGROUND: Urine albumin/creatinine ratio (UACR) is an important marker of early renal damage (ERD) caused by hypertension. Recent studies showed that blood pressure was a significant inverse association with temperature and climate. The purposes of our study were sought to explore the association of common medical comorbidities with ERD, and find independent risk factors to ERD in Chinese tropics with essential hypertension. METHODS: From January 2018 to December 2019, we assessed UACR in a total of 599 hypertensive Chinese Hainan patients. We defined ERD as a UACR between 30 mg/g and 300 mg/g. We analysed differences between qualitative variables using the chi-squared (χ2) test. We calculated correlations between UACR and age, hypertension duration (HD), systolic blood pressure (SBP), and diastolic blood pressure (DBP) using the Spearman's rho test. To determine the odds ratio (OR), we evaluated binary logistic regression models. RESULTS: Among the 599 patients, 281 (46.9%) were found to have ERD. ERD and factors related to sex, body mass index (BMI), and SBP did not differ significantly (all, p>0.05). Our main findings showed that age, HD, and DBP were associated with ERD (p<0.01, respectively). Furthermore, age ≥ 65 years, HD ≥10 years, DBP ≥ 90 mmHg, SBP ≥ 160 mmHg, and diabetes differed significantly according to ERD status (p < 0.05, respectively). In multivariate analysis using stepwise regression, age (OR = 1.468), DBP (OR = 1.853), and diabetes (OR = 2.031) were significant independent predictors of ERD. The area under the receiver operating characteristic (ROC) curve was 0.677, and the sensitivity and specificity of the optimal cut-off value were 44.5 and 81.1%, respectively. CONCLUSIONS: Common medical comorbidities are associated with ERD; age, DBP, and diabetes are independent risk factors for ERD in patients with essential hypertension who live in the Chinese tropics. Early monitoring of the UACR, as well as control of blood glucose and DBP, can effectively delay ERD.

Increasing urinary podocyte mRNA excretion and progressive podocyte loss in kidney contribute to the high risk of long-term renal disease caused by preterm birth.

Podocyte abnormalities are common mechanism driving the progression of glomerular diseases, which account for most chronic kidney diseases (CKDs). However, the role of podocyte in the mechanism of high-risk long-term CKD caused by prematurity has not been well clarified. In present study, urine samples of 86 preterm infants and 32 full-term infants were collected, and podocyte-specific podocin mRNA levels in urine pellet were applied to indicate urinary podocyte mRNA excretion. In addition, in a preterm animal rat model, preterm rats were identified by delivery 2 days early. From the age of 3 weeks-12 months, urine samples were collected to examine podocyte mRNA excretion by measuring podocyte-specific podocin mRNA levels. Kidney samples at the age of 3 weeks, 2 months, and 12 months were collected from 8, 5 and 6 preterm rats and 9, 6 and 8 full-term rats, respectively, to examine podocyte density and podocyte area by measuring the podocyte specific nuclear marker WT-1 and the podocyte specific marker synaptopodin. As results, a more than threefold increase of urinary podocyte-specific podocin mRNA excretion rate was found in preterm infants compared with full-term infants. In addition, there was negative correlation between gestational age at birth and urinary podocin mRNA excretion. In preterm rats, a reduction in the total number of differentiated podocytes in glomeruli and an increased podocyte podocin mRNA excretion rate in urine were detected at the end of kidney differentiation. Moreover, long-term follow-up data in preterm rats showed there was an increased the risk of renal disease indicated by persistent podocyte mRNA loss, proteinuria, and enlarged glomeruli. In conclusion, increasing podocyte mRNA excretion in urine and podocyte loss in kidney led by prematurity drive the progression of long-term abnormal kidney function and could potentially explain the high risk of long-term CKD in preterm infants.

Importance of urinary mitochondrial DNA in diagnosis and prognosis of kidney diseases.

Mitochondrial injury plays an important role in the occurrence and development of kidney diseases. However, the existing assays to determine mitochondrial function restrict our ability to understand the relationship between mitochondrial dysfunction and kidney damage. These limitations may be overcome by recent findings on urinary mitochondrial DNA (UmtDNA). Elevated UmtDNA level may serve as a surrogate biomarker of mitochondrial dysfunction, kidney damage, and progression and prognosis of kidney diseases. Herein, we review the recent research progress on UmtDNA in kidney diseases diagnosis and highlight the research areas that should be expanded in future as well as discuss the future perspectives.

Automated urine sediment analyzers underestimate the severity of hematuria in glomerular diseases.

Hematuria, either glomerular or extraglomerular, is defined as 3 or more red blood cells (RBCs)/high power field. Currently, urinalyses are commonly performed using automated urine sediment analyzers. To assess whether RBC counting by automated urine sediment analyzers is reliable for defining hematuria in glomerular disease, random specimen urinalyses of men with nephritic glomerular disease (7674 urinalyses) and bladder cancer (12,510 urinalyses) were retrospectively reviewed. Urine RBCs were counted by an automated urine sediment analyzer based on flow cytometry (UF-1000i, Sysmex Corporation) or digital image analysis (Cobas 6500, Roche Diagnostics GmbH). In about 20% of urine specimens, the specific gravity was less than 1.010, making the RBC counts unreliable. In the urine specimens with specific gravity ≥ 1.010, RBC counts measured using either UF-1000i or Cobas 6500 were well correlated with the positive grades in the dipstick blood test. However, at a trace, 1+, or higher positive dipstick tests for blood, RBC counts were graded significantly lower in glomerular disease than in bladder cancer. The findings suggest that RBC counting by UF-1000i or Cobas 6500 underestimates the severity of hematuria in glomerular disease, possibly because dysmorphic RBCs in glomerular disease are susceptible to hemolysis and/or fail to be properly recognized.

Urinary Extracellular Vesicles and Their miRNA Cargo in Patients with Fabry Nephropathy.

Current biomarkers of Fabry nephropathy lack sensitivity in detecting early kidney damage and do not predict progression of nephropathy. Urinary extracellular vesicles (uEVs) and their molecular cargo could reflect early changes in renal impairment as they are secreted by the cells lining the urinary tract. We aimed to conduct a proof-of-concept study to investigate whether analysis of uEV characteristics and expression of uEV-derived microRNAs (miRNAs) could be applicable in studies to predict the development and progression of nephropathy in Fabry disease. A total of 20 Fabry patients were divided into two groups, depending on the presence of nephropathy. Chronological urine samples collected during 10-year follow-up were used for uEVs isolation with size exclusion chromatography. Nanoparticle tracking analysis was used to determine concentration and size of uEVs. We evaluated the expression of five uEV-derived miRNAs by qPCR (miR-23a-3p, miR-29a-3p, miR-30b-5p, miR-34a-5p, miR-200a-3p). There was no difference in the concentration and size of uEVs between patients with and without nephropathy at last follow-up or longitudinally. However, we found increased expression of miR-29a-3p and miR-200a-3p in uEVs isolated from chronological samples of patients with Fabry nephropathy. This may indicate an attempt by the organism to prevent the progression of renal damage leading to end-stage renal disease as previously reported in type 1 diabetes. In addition, we found an increased expression of miR-30b-5p in the 10-year period in uEVs of patients without renal dysfunction. miR-30b-5 was reported to have a protective role in podocyte injury and may possibly be important in Fabry nephropathy. These findings indicate that uEVs and their molecular cargo could be a promising target of studies focusing on elucidation of Fabry nephropathy. Nevertheless, total concentration and size of uEVs were neither indicative of the presence nor progression of Fabry nephropathy, while the role of the analyzed miRNAs in Fabry nephropathy progression was merely indicated and needs further in-depth studies.