Exosomes derived from human urine-derived stem cells ameliorate IL-1ß-induced intervertebral disk degeneration.

BACKGROUND: Human intervertebral disk degeneration (IVDD) is a sophisticated degenerative pathological process. A key cause of IVDD progression is nucleus pulposus cell (NPC) degeneration, which contributes to excessive endoplasmic reticulum stress in the intervertebral disk. However, the mechanisms underlying IVDD and NPC degeneration remain unclear. METHODS: We used interleukin (IL)-1ß stimulation to establish an NPC-degenerated IVDD model and investigated whether human urine-derived stem cell (USC) exosomes could prevent IL-1ß-induced NPC degeneration using western blotting, quantitative real-time polymerase chain reaction, flow cytometry, and transcriptome sequencing techniques. RESULTS: We successfully extracted and identified USCs and exosomes from human urine. IL-1ß substantially downregulated NPC viability and induced NPC degeneration while modulating the expression of SOX-9, collagen II, and aggrecan. Exosomes from USCs could rescue IL-1ß-induced NPC degeneration and restore the expression levels of SOX-9, collagen II, and aggrecan. CONCLUSIONS: USC-derived exosomes can prevent NPCs from degeneration following IL-1ß stimulation. This finding can aid the development of a potential treatment strategy for IVDD.

[Urinary cytology for the detection of urothelial lesions]. / Rôle de la cytologie urinaire dans le dépistage des lésions urothéliales.

Urine cytology is a long-used technique for the detection of high grade neoplastic urothelial lesions. Since 2016, «The Paris System¼ classification has revolutionized this field by introducing a standardized terminology widely adopted by cytopathologists and urologists. In this article, we explain this classification and discuss its impact on the clinical management of patients with urothelial lesions, as well as its role in the secondary prevention of these lesions.

La cytologie urinaire est une technique utilisée depuis longtemps dans la détection des lésions urothéliales tumorales de haut grade. Depuis 2016, la classification «The Paris System¼ a révolutionné ce domaine en introduisant une terminologie standardisée largement adoptée par les cytopathologistes et les urologues. Dans cet article, nous expliquons cette classification et discutons de son impact sur la prise en charge clinique des lésions urothéliales, ainsi que son rôle dans la prévention secondaire de ces lésions.

Exploiting urine-derived induced pluripotent stem cells for advancing precision medicine in cell therapy, disease modeling, and drug testing.

The field of regenerative medicine has witnessed remarkable advancements with the emergence of induced pluripotent stem cells (iPSCs) derived from a variety of sources. Among these, urine-derived induced pluripotent stem cells (u-iPSCs) have garnered substantial attention due to their non-invasive and patient-friendly acquisition method. This review manuscript delves into the potential and application of u-iPSCs in advancing precision medicine, particularly in the realms of drug testing, disease modeling, and cell therapy. U-iPSCs are generated through the reprogramming of somatic cells found in urine samples, offering a unique and renewable source of patient-specific pluripotent cells. Their utility in drug testing has revolutionized the pharmaceutical industry by providing personalized platforms for drug screening, toxicity assessment, and efficacy evaluation. The availability of u-iPSCs with diverse genetic backgrounds facilitates the development of tailored therapeutic approaches, minimizing adverse effects and optimizing treatment outcomes. Furthermore, u-iPSCs have demonstrated remarkable efficacy in disease modeling, allowing researchers to recapitulate patient-specific pathologies in vitro. This not only enhances our understanding of disease mechanisms but also serves as a valuable tool for drug discovery and development. In addition, u-iPSC-based disease models offer a platform for studying rare and genetically complex diseases, often underserved by traditional research methods. The versatility of u-iPSCs extends to cell therapy applications, where they hold immense promise for regenerative medicine. Their potential to differentiate into various cell types, including neurons, cardiomyocytes, and hepatocytes, enables the development of patient-specific cell replacement therapies. This personalized approach can revolutionize the treatment of degenerative diseases, organ failure, and tissue damage by minimizing immune rejection and optimizing therapeutic outcomes. However, several challenges and considerations, such as standardization of reprogramming protocols, genomic stability, and scalability, must be addressed to fully exploit u-iPSCs' potential in precision medicine. In conclusion, this review underscores the transformative impact of u-iPSCs on advancing precision medicine and highlights the future prospects and challenges in harnessing this innovative technology for improved healthcare outcomes.

Correlation between urine cytology results on the day after overnight continuous saline irrigation following transurethral resection of bladder tumor and bladder tumor recurrence.

PURPOSE: To investigate the relationship between urine cytology results after overnight continuous saline irrigation (OCSI) following transurethral resection of bladder tumor (TURBT) and bladder tumor recurrence in non-muscle invasive bladder cancer (NMIBC). MATERIALS AND METHODS: A retrospective study was conducted on patients diagnosed with NMIBC between 2016 and 2020 after undergoing TURBT at our hospital. All patients received OCSI following TURBT and had urine cytology test at postoperative 1 day. Urine cytology was classified into three groups: Negative, low-grade urothelial neoplasm (LGUN)+atypical urothelial cells (AUC), and suspicious for high-grade urothelial carcinoma (SHGUC)+high-grade urothelial carcinoma (HGUC). Recurrence-free survival (RFS) in each group was compared using the Kaplan-Meier method. Univariable and multivariable Cox regression analyses were performed to evaluate independent prognostic factors. RESULTS: A total of 172 patients were included in this study. Based on urine cytology group (after OCSI), RFS did not reach the median value in the Negative group. In the LGUN+AUC group, the median RFS was 615.00 days. In the SHGUC+HGUC group, the median RFS was 377.00 days. In survival analysis, the Negative group had a longer RFS than the SHGUC+HGUC group (p=0.013). However, Cox regression analysis showed that SHGUC+HGUC was not an independent prognostic factor for recurrence. CONCLUSIONS: Urine cytology results after OCSI following TURBT in NMIBC were associated with bladder tumor recurrence. Specifically, SHGUC or HGUC in urine cytology after OCSI showed earlier recurrence than negative cases. However, further research is needed to accurately determine whether it is an independent prognostic factor.

Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation.

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.

[Practical application of the Paris system for reporting urinary cytology].

Objective: To validate the diagnostic performance of the Paris system for reporting urinary cytology (TPS). Methods: A total of 7 046 urine cytology samples from 3 402 patients collected in the Department of Pathology, Beijing Hospital, China from January 2020 to January 2022 were analyzed. 488 patients had a biopsy or resection specimen during the follow-up period of 6 months. The sensitivity, specificity, risk of malignancy (ROM) and risk of high-grade malignancy (ROHM) of the TPS were evaluated using histological diagnosis as the golden standard. Results: Among the 7 046 samples, high-grade urothelial carcinoma (HGUC) accounted for 5.7% (399/7 046), suspicious for high-grade urothelial carcinoma (SHGUC) for 3.2% (227/7 046), atypical urothelial cells (AUC) for 8.4% (593/7 046), and negative for high-grade urothelial carcinoma (NHGUC) for 72.9% (5 139/7 046) including low-grade urothelial neoplasm (LGUN) for 0.8% (59/7 046) and insufficient samples for 9.8% (688/7 046). 488 patients had a bladder biopsy or resection in the follow-up of six months, including 314 males and 174 females, aged 27 to 92 years (average, 66 years). The ROHM of TPS was 94.7% in HGUC, 83.3% in SHGUC, 41.3% in AUC and 18.8% in NHGUC. The sensitivity and specificity of urine cytology were 70.1% (169/241) and 97.0% (162/167), respectively. The negative predictive value of NHGUC was 69.2% (162/234). Conclusions: The study has shown that TPS classification has high sensitivity and specificity, high ROHM for HGUC and SHGUC, and high negative predictive value for NHGUC. The application of TPS reporting system can better interpret the clinical significance of cytology samples, improve the accuracy of urine cytopathology and ensure continuous diagnostic consistency.

[What contribution can make artificial intelligence to urinary cytology?] / Quel apport de l'intelligence artificielle en cytologie urinaire ?

Urinary cytology using the Paris system is still the method of choice for screening high-grade urothelial carcinomas. However, the use of the objective criteria described in this terminology shows a lack of inter- and intra-observer reproducibility. Moreover, if its sensitivity is excellent on instrumented urine, it remains insufficient on voided urine samples. Urinary cytology appears to be an excellent model for the application of artificial intelligence to improve performance, since the objective criteria of the Paris system are defined at cellular level, and the resulting diagnostic approach is presented in a highly "algorithmic" way. Nevertheless, there is no commercially available morphological diagnostic aid, and very few predictive devices are still undergoing clinical validation. The analysis of different systems using artificial intelligence in urinary cytology rises clear prospects for mutual contributions.

Voided urine cytology is a useful tool predicting non-muscle-invasive bladder cancer risk before surgery.

OBJECTIVE: To determine the accuracy of voided urinary cytology (VUC) in predicting of non-muscle-invasive bladder cancer (NMIBC) risk stratification before surgery. METHODS: We prospectively collected data from all patients diagnosed with bladder cancer in our institution over 2 years. We have analyzed VUC accuracy of positive and suspicious VUC in the detection of high-risk tumors and negative and atypical VUC in the detection of low-risk tumors. To test this accuracy, we assessed sensitivity, specificity, positive (PPV) and negative predictive values (NPV), diagnostic odds ratio (DOR), and generated ROC curves (receiver operating characteristic curve). RESULTS: With 224 patients included, the positive VUC subcategory showed a specificity of 92.4% (95%CI: 83.2%-97.5%) and a PPV of 91.4 (95%CI: 81%-97.1%). DOR in this subgroup was 6.81. In the suspicious VUC, specificity was 90.9% (95%CI: 81.3%-96.6%), PPV was 88% (95%CI: 75.7%-95.5%) and DOR was 4.23. Combined analysis of positive and suspicious cytologies for detecting high-risk NMIBC showed a sensitivity of 65% (95%CI: 57.3%-73.2%) and a DOR of 9.51. Negative VUC showed high specificity in detecting low-risk (93.2% [95%CI: 87.9%-96.7%]) and a DOR of 6.90 (95%CI: 3.07-15.46). Atypical VUC was the least accurate and had rather low specificity and predictive values. CONCLUSIONS: VUC appears to be a good, inexpensive and easily available method to determine risk stratification before surgery. This can be useful in daily practice to determine which patients should receive a single instillation of MMC and to prioritize patients more likely to have a high- risk tumor.

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.

[Renal synovialosarcoma: What about pyelic cytology to make a diagnosis?] / Synovialosarcome primitif du rein : si tout était déjà dans les urines pyéliques ?

Synovialosarcoma is a malignant mesenchymal tumor of young adults that occurs in the deep soft tissues, particularly around large joints. When it occurs in more unusual sites, it could present a significant diagnostic challenge. In this case, a 19-year-old girl was treated for a pyloric mass. A pyelic urine cytology performed simultaneously with a pyloric biopsy proved to be a significant element of orientation and perfectly concordant with the histopathological aspect of the pyelic mass after nephrectomy. We report here the first case of renal synovialosarcoma documented in pyelic urine.

[The Paris System, 2022 edition: What is new?] / La version 2022 du Système de Paris : quoi de neuf ?

The second version of the Paris System for reporting urine cytology was published in 2022. It follows the first version of 2016, which was very successful and widely adopted by many cytopathologists from different countries. Thus, numerous publications using the Paris System have made possible to refine the criteria as well as discussing the limits. The diagnostic accuracy of urinary cytology is high for detection of high-grade urothelial carcinoma, but not for low-grade carcinoma where there are few cytological abnormalities. So, the chapter individualizing low-grade urothelial neoplasms was deleted; the latter were included in the category "negative for high-grade urothelial carcinoma". Indeed, the risk of malignancy is replaced by the risk of high-grade urothelial carcinoma. A new chapter has been devoted to urothelial tumors of the upper tract. Finally, the pitfalls linked to cellular degeneration are discussed for each category. The risk of high-grade malignancy associated with each category will help communication with the clinician and help patient care.

Live births from urine derived cells.

Here we report urine-derived cell (UDC) culture and subsequent use for cloning which resulted in the successful development of cloned canine pups, which have remained healthy into adulthood. Bovine UDCs were used in vitro to establish comparative differences between cell sources. UDCs were chosen as a readily available and noninvasive source for obtaining cells. We analyzed the viability of cells stored in urine over time and could consistently culture cells which had remained in urine for 48hrs. Cells were shown to be viable and capable of being transfected with plasmids. Although primarily of epithelial origin, cells were found from multiple lineages, indicating that they enter the urine from more than one source. Held in urine, at 4°C, the majority of cells maintained their membrane integrity for several days. When compared to in vitro fertilization (IVF) derived embryos or those from traditional SCNT, UDC derived embryos did not differ in total cell number or in the number of DNA breaks, measured by TUNEL stain. These results indicate that viable cells can be obtained from multiple species' urine, capable of being used to produce live offspring at a comparable rate to other cell sources, evidenced by a 25% pregnancy rate and 2 live births with no losses in the canine UDC cloning trial. This represents a noninvasive means to recover the breeding capacity of genetically important or infertile animals. Obtaining cells in this way may provide source material for human and animal studies where cells are utilized.

Lens Free Holographic Imaging for Urinary Tract Infection Screening.

OBJECTIVE: The diagnosis of urinary tract infection (UTI) currently requires precise specimen collection, handling infectious human waste, controlled urine storage, and timely transportation to modern laboratory equipment for analysis. Here we investigate holographic lens free imaging (LFI) to show its promise for enabling automatic urine analysis at the patient bedside. METHODS: We introduce an LFI system capable of resolving important urine clinical biomarkers such as red blood cells, white blood cells, crystals, and casts in 2 mm thick urine phantoms. RESULTS: This approach is sensitive to the particulate concentrations relevant for detecting several clinical urine abnormalities such as hematuria and pyuria, linearly correlating to ground truth hemacytometer measurements with R 2 = 0.9941 and R 2 = 0.9973, respectively. We show that LFI can estimate E. coli concentrations of 10 3 to 10 5 cells/mL by counting individual cells, and is sensitive to concentrations of 10 5 cells/mL to 10 8 cells/mL by analyzing hologram texture. Further, LFI measurements of blood cell concentrations are relatively insensitive to changes in bacteria concentrations of over seven orders of magnitude. Lastly, LFI reveals clear differences between UTI-positive and UTI-negative urine from human patients. CONCLUSION: LFI is sensitive to clinically-relevant concentrations of bacteria, blood cells, and other sediment in large urine volumes. SIGNIFICANCE: Together, these results show promise for LFI as a tool for urine screening, potentially offering early, point-of-care detection of UTI and other pathological processes.

Genome sequencing and RNA sequencing of urinary cells reveal an intronic FBN1 variant causing aberrant splicing.

Exome sequencing and panel testing have improved diagnostic yield in genetic analysis by comprehensively detecting pathogenic variants in exonic regions. However, it is important to identify non-exonic pathogenic variants to further improve diagnostic yield. Here, we present a female proband and her father who is diagnosed with Marfan syndrome, a systemic connective tissue disorder caused by pathogenic variants in FBN1. There are also two affected individuals in the siblings of the father, indicating the genetic basis in this family. However, panel testing performed by two institutions reported no causal variants. To further explore the genetic basis of the family, we performed genome sequencing of the proband and RNA sequencing of urinary cells derived from urine samples of the proband and her father because FBN1 is strongly expressed in urinary cells though it is poorly expressed in peripheral blood mononuclear cells. Genome sequencing identified a rare intronic variant (c.5789-15G>A) in intron 47 of FBN1 (NM_000138.4), which was transmitted from her father. RNA sequencing revealed allelic imbalance (monoallelic expression) of FBN1, retention of intron 47, and fewer aberrant transcripts utilizing new acceptor sites within exon 48, which were confirmed by RT-PCR. These results highlighted urinary cells as clinically accessible tissues for RNA sequencing if disease-causing genes are not sufficiently expressed in the blood, and the usefulness of multi-omics analysis for molecular diagnosis of genetic disorders.

Prevalence of leukocyturia in a cohort of French asymptomatic aircrews.

OBJECTIVES: Sexually transmitted infections (STIs) can cause leukocyturia. We aimed to estimate the prevalence of leukocyturia in asymptomatic aircrews and the proportion of STIs in those presenting leukocyturia. METHODS: The LEUCO survey was a prospective cohort study conducted among aircrews between 14th October 2019 and 13th March 2020 at the Toulon aeromedical centre in France. All participants performed a dipstick urinalysis. Those positive for leukocyturia were offered STI screening by nucleic acid amplification test (NAAT) for Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis. RESULTS: Among the 2236 included asymptomatic participants (1912 men and 324 women), 127 (36 men and 91 women) were positive for leukocyturia. The prevalence of leukocyturia was 1.9% (1.3-2.6) in men and 28.1% (23.3-33.3) in women (p < 0.001). In men positive for leukocyturia, the NAAT positivity rate for C. trachomatis, N. gonorrhoeae, M. genitalium and T. vaginalis was 28.6% (3.7-71.0) in the age group 18-24, 20.0% (0.5-71.6) in the age group 25-34, and zero in the older age group (p 0.65). In women positive for leukocyturia it was 16.7% (4.7-37.4) in the age group 18-24, 18.2% (2.3-51.8) in the age group 25-34, and zero in the older age group (p 0.16). CONCLUSIONS: In asymptomatic individuals, leukocyturia is rare in men and more common in women. In asymptomatic adults under 35 years of age with leukocyturia, multiplex NAAT shows a high proportion of STIs and might be useful in improving STI detection.

Generation of Induced Nephron Progenitor-like Cells from Human Urine-Derived Cells.

BACKGROUND: Regenerative medicine strategies employing nephron progenitor cells (NPCs) are a viable approach that is worthy of substantial consideration as a promising cell source for kidney diseases. However, the generation of induced nephron progenitor-like cells (iNPCs) from human somatic cells remains a major challenge. Here, we describe a novel method for generating NPCs from human urine-derived cells (UCs) that can undergo long-term expansion in a serum-free condition. RESULTS: Here, we generated iNPCs from human urine-derived cells by forced expression of the transcription factors OCT4, SOX2, KLF4, c-MYC, and SLUG, followed by exposure to a cocktail of defined small molecules. These iNPCs resembled human embryonic stem cell-derived NPCs in terms of their morphology, biological characteristics, differentiation potential, and global gene expression and underwent a long-term expansion in serum-free conditions. CONCLUSION: This study demonstrates that human iNPCs can be readily generated and expanded, which will facilitate their broad applicability in a rapid, efficient, and patient-specific manner, particularly holding the potential as a transplantable cell source for patients with kidney disease.

Combination of PD98059 and TGF-ß1 Efficiently Differentiates Human Urine-Derived Stem Cells into Smooth Muscle Cells.

Pluripotent adult stem cells have potential applications in cell therapy and tissue engineering. Urine-derived stem cells (UDSCs) differentiate into various cell types. Here, we attempted to differentiate human UDSCs (hUDSCs) into smooth muscle cells (SMCs) using transforming growth factor-beta 1 (TGF-ß1) and/or PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Both quantitative polymerase chain reaction (qPCR) and Western blot analysis showed that the expression of messenger ribonucleic acid (mRNA) and proteins for alpha-smooth muscle actin (α-SMA), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC), which are specific markers for SMCs, increased on day 9 after differentiation and again on day 14. The differentiated cells from human UDSCs (hUDSCs) with a combination of TGF-ß1 and PD98059 showed the highest expression of SMC marker proteins. Immunocytochemical staining performed to assess the molecular expression revealed CNN and α-SMA colocalizing in the cytoplasm. The cells that differentiated from hUDSCs with a combination of TGF-ß1 and PD98059 showed the strongest expression for CNN1, α-SMA, and SM-MHC. Functional testing of the differentiated cells revealed a stronger contractile capacity for the cells differentiated with a combination of PD98059 and TGF-ß1 than those differentiated with a single factor. These results suggest the combination of PD98059 and TGF-ß1 to be a more effective differentiation method and that differentiated SMCs could be used for restoring the functions of the sphincter muscle or bladder.

Suppression of collagen IV alpha-2 subunit by prolyl hydroxylase domain inhibition via hypoxia-inducible factor-1 in chronic kidney disease.

Elevation of hypoxia-inducible factor 1 protein has been shown to be protective in acute kidney injury and HIF1α enhancing drug therapies are currently in clinical trials for the treatment of anemia of chronic kidney disease. Despite its benefits, long-term HIF1 elevation seems to be associated with additional effects in the kidneys such as tubulointerstitial fibrosis. To better understand the effects of prolonged HIF1 exposure, assessment of baseline and post-therapy levels of HIF1α and other related biomarkers is essential. In this study, we assessed the effect of HIF1α enhancement using prolyl hydroxylase inhibitor (PHD-I) DMOG, on a key profibrotic marker of kidney disease. In specific, we examined the change in expression of Collagen 4 subunit A2 in cultured urinary cells of CKD patients pre and post 24-hour exposure to 1mM DMOG. Our results show that besides HIF1α enhancement, COL4A2 protein is suppressed in presence of DMOG. To determine if this effect is mediated by HIF1, we used HIF1α gene silencing in HEK293 cells and examined the effect of DMOG on protein and gene expression of COL4A2 post 24-hour exposure. We showed that silencing HIF1α reverses and amplifies the expression of COL4A2 in HEK293 cells. Our data suggest that HIF1 directly regulates the expression of COL4A2 in kidney cells and that HIF1α enhancing therapy has suppressive effects on COL4A2 that may be clinically relevant and must be considered in determining the safety and efficacy of these drugs in the treatment of anemia.