Generation of patterned kidney organoids that recapitulate the adult kidney collecting duct system from expandable ureteric bud progenitors.

Current kidney organoids model development and diseases of the nephron but not the contiguous epithelial network of the kidney's collecting duct (CD) system. Here, we report the generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys, or generated de novo from human pluripotent stem cells. In chemically-defined culture conditions, UB organoids generate CD organoids, with differentiated principal and intercalated cells adopting spatial assemblies reflective of the adult kidney's collecting system. Aggregating 3D-cultured nephron progenitor cells with UB organoids in vitro results in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Applying an efficient gene editing strategy to remove RET activity, we demonstrate genetically modified UB organoids can model congenital anomalies of kidney and urinary tract. Taken together, these platforms will facilitate an enhanced understanding of development, regeneration and diseases of the mammalian collecting duct system.

The Paris system of Reporting Urinary Cytology: Strengths and opportunities.

The Paris system for reporting urinary cytopathology (TPS) was created to address inherent weaknesses inherent in the practice of urinary cytopathology. While urothelial cytology has always performed well at finding high grade, genetically unstable urothelial carcinoma, it performs poorly when it comes to detecting low-grade urothelial neoplasia. TPS intends to improve the utility of urothelial cytology by focusing on what is important, high-grade urothelial carcinoma. This article is a snapshot of the current state of TPS as it heads into its second edition. Successes are described and further developments are considered.

Transplantation of human urine-derived neural progenitor cells after spinal cord injury in rats.

Spinal cord injury (SCI) is a worldwide problem and transplantation of neural progenitor cells (NPCs) represents a promising treatment strategy. Urine derived induced pluripotent stem cells (UiPSCs) which enable the generation of patient-specific NPCs, provide an invaluable source of autologous cells for future therapeutic applications after SCI. However, the fate and potential contribution of transplanted human UiPSCs-derived NPCs (UiPSC-NPCs) into injured spinal cords remain largely unknown. In this study, using a rat contusive SCI model, we evaluated the survival, migration and differentiation of UiPSC-NPCs after transplantation at subacute phase. Transplanted cells survived and migrated from the site of grafting towards the lesion epicenter. More than 25 % cells survived over 4 weeks post transplantation, with a few of them differentiated into neurons and astrocytes. Cytokine and chemokine levels within the injured spinal cord tissues were measured using multiplex immunoassays to evaluate the immune response. Pro-inflammatory factors and chemokines were significantly decreased at 3 days after UiPSC-NPCs transplantation. At 7 days post transplantation, a lower level of pro-inflammatory factor IFN-γ and a higher level of pro-inflammatory IL-2 were found in UiPSC-NPCs group than in the control. Transplantation of UiPSC-NPCs showed little effect on microglia activation at the lesion epicenter. However, the number of microglia cells at 4 mm rostral to the injury site was significantly decreased. The size of lesion cavity was reduced after transplantation of UiPSC-NPCs. In conclusions, the UiPSC-NPCs transplanted at the subacute phase of SCI showed a beneficial effect on tissue repairing.

Encapsulated three-dimensional bioprinted structure seeded with urothelial cells: a new construction technique for tissue-engineered urinary tract patch.

BACKGROUND: Traditional tissue engineering methods to fabricate urinary tract patch have some drawbacks such as compromised cell viability and uneven cell distribution within scaffold. In this study, we combined three-dimensional (3D) bioprinting and tissue engineering method to form a tissue-engineered urinary tract patch, which could be employed for the application on Beagles urinary tract defect mode to verify its effectiveness on urinary tract reconstruction. METHODS: Human adipose-derived stem cells (hADSCs) were dropped into smooth muscle differentiation medium to generate induced microtissues (ID-MTs), flow cytometry was utilized to detect the positive percentage for CD44, CD105, CD45, and CD34 of hADSCs. Expression of vascular endothelial growth factor A (VEGFA) and tumor necrosis factor-stimulated gene-6 (TSG-6) in hADSCs and MTs were identified by Western blotting. Then the ID-MTs were employed for 3D bioprinting. The bioprinted structure was encapsulated by transplantation into the subcutaneous tissue of nude mice for 1 week. After retrieval of the encapsulated structure, hematoxylin and eosin and Masson's trichrome staining were performed to demonstrate the morphology and reveal collagen and smooth muscle fibers, integral optical density (IOD) and area of interest were calculated for further semi-quantitative analysis. Immunofluorescent double staining of CD31 and α-smooth muscle actin (α-SMA) were used to reveal vascularization of the encapsulated structure. Immunohistochemistry was performed to evaluate the expression of interleukin-2 (IL-2), α-SMA, and smoothelin of the MTs in the implanted structure. Afterward, the encapsulated structure was seeded with human urothelial cells. Immunofluorescent staining of cytokeratins AE1/AE3 was applied to inspect the morphology of seeded encapsulated structure. RESULTS: The semi-quantitative assay showed that the relative protein expression of VEGFA was 0.355 ±â€Š0.038 in the hADSCs vs. 0.649 ±â€Š0.150 in the MTs (t = 3.291, P = 0.030), while TSG-6 expression was 0.492 ±â€Š0.092 in the hADSCs vs. 1.256 ±â€Š0.401 in the MTs (t = 3.216, P = 0.032). The semi-quantitative analysis showed that the mean IOD of IL-2 in the MT group was 7.67 ±â€Š1.26, while 12.6 ±â€Š4.79 in the hADSCs group, but semi-quantitative analysis showed that there was no statistical significance in the difference between the two groups (t = 1.724, P = 0.16). The semi-quantitative analysis showed that IOD was 71.7 ±â€Š14.2 in non-induced MTs (NI-MTs) vs. 35.7 ±â€Š11.4 in ID-MTs for collagen fibers (t = 3.428, P = 0.027) and 12.8 ±â€Š1.9 in NI-MTs vs. 30.6 ±â€Š8.9 in ID-MTs for smooth muscle fibers (t = 3.369, P = 0.028); furthermore, the mean IOD was 0.0613 ±â€Š0.0172 in ID-MTs vs. 0.0017 ±â€Š0.0009 in NI-MTs for α-SMA (t = 5.994, P = 0.027), while 0.0355 ±â€Š0.0128 in ID-MTs vs. 0.0035 ±â€Š0.0022 in NI-MTs for smoothelin (t = 4.268, P = 0.013), which indicate that 3D bioprinted structure containing ID-MTs could mimic the smooth muscle layer of native urinary tract. After encapsulation of the urinary tract patch for additional cell adhesion, urothelial cells were seeded onto the encapsulated structures, and a monolayer urothelial cell was observed. CONCLUSION: Through 3D bioprinting and tissue engineering methods, we provided a promising way to fabricate tissue-engineered urinary tract patch for further investigation.

Expression and distribution of PIEZO1 in the mouse urinary tract.

The proper function of the organs that make up the urinary tract (kidneys, ureters, bladder, and urethra) depends on their ability to sense and respond to mechanical forces, including shear stress and wall tension. However, we have limited understanding of the mechanosensors that function in these organs and the tissue sites in which these molecules are expressed. Possible candidates include stretch-activated PIEZO channels (PIEZO1 and PIEZO2), which have been implicated in mechanically regulated body functions including touch sensation, proprioception, lung inflation, and blood pressure regulation. Using reporter mice expressing a COOH-terminal fusion of Piezo1 with the sequence for the tandem-dimer Tomato gene, we found that PIEZO1 is expressed in the kidneys, ureters, bladder, and urethra as well as organs in close proximity, including the prostate, seminal vesicles and ducts, ejaculatory ducts, and the vagina. We further found that PIEZO1 expression is not limited to one cell type; it is observed in the endothelial and parietal cells of the renal corpuscle, the basolateral surfaces of many of the epithelial cells that line the urinary tract, the interstitial cells of the bladder and ureters, and populations of smooth and striated muscle cells. We propose that in the urinary tract, PIEZO1 likely functions as a mechanosensor that triggers responses to wall tension.

A bioactive injectable bulking material; a potential therapeutic approach for stress urinary incontinence.

Stress urinary incontinence (SUI) is a life changing condition, affecting 20 million women worldwide. In this study, we developed a bioactive, injectable bulking agent that consists of Permacol™ (Medtronic, Switzerland) and recombinant insulin like growth factor-1 conjugated fibrin micro-beads (fib_rIGF-1) for its bulk stability and capacity to induce muscle regeneration. Therefore, Permacol™ formulations were injected in the submucosal space of rabbit bladders. The ability of a bulking material to form a stable and muscle-inducing bulk represents for us a promising therapeutic approach to achieve a long-lasting treatment for SUI. The fib_rIGF-1 showed no adverse effect on human smooth muscle cell metabolic activity and viability in vitro based on AlamarBlue assays and Live/Dead staining. Three months after injection of fib_rIGF-1 together with Permacol™ into the rabbit bladder wall, we observed a smooth muscle tissue like formation within the injected materials. Positive staining for alpha smooth muscle actin, calponin, and caldesmon demonstrated a contractile phenotype of the newly formed smooth muscle tissue. Moreover, the fib_rIGF-1 treated group also improved the neovascularization at the injection site, confirmed by CD31 positive staining compared to bulks made of PermacolTM only. The results of this study encourage us to further develop this injectable, bioactive bulking material towards a future therapeutic approach for a minimal invasive and long-lasting treatment of SUI.

Urinary Tract Cytology.

Cytologic evaluation of the urinary tract can be diagnostically rewarding in cases of renomegaly or when discrete kidney or bladder masses are identified. Cytology can often help to distinguish between cystic, inflammatory, and neoplastic disorders. Various types of cystic and benign urinary tract lesions, diseases associated with urinary tract inflammation, and the cytologic differences between primary and metastatic neoplasms of the kidney and bladder are described. Basic sampling techniques for urinary tract cytology are also discussed.

Sex differences in lower urinary tract biology and physiology.

Females and males differ significantly in gross anatomy and physiology of the lower urinary tract, and these differences are commonly discussed in the medical and scientific literature. However, less attention is dedicated to investigating the varied development, function, and biology between females and males on a cellular level. Recognizing that cell biology is not uniform, especially in the lower urinary tract of females and males, is crucial for providing context and relevance for diverse fields of biomedical investigation. This review serves to characterize the current understanding of biological sex differences between female and male lower urinary tracts, while identifying areas for future research. First, the differences in overall cell populations are discussed in the detrusor smooth muscle, urothelium, and trigone. Second, the urethra is discussed, including anatomic discussions of the female and male urethra followed by discussions of cellular differences in the urothelial and muscular layers. The pelvic floor is then reviewed, followed by an examination of the sex differences in hormonal regulation, the urinary tract microbiome, and the reticuloendothelial system. Understanding the complex and dynamic development, anatomy, and physiology of the lower urinary tract should be contextualized by the sex differences described in this review.

Evaluation of the Paris System in atypical urinary cytology.

OBJECTIVE: Our aim was to evaluate the Paris System for reporting urinary cytology, especially in the field of atypia. METHODS: During the last year, 104 urinary cases had atypical cytology. These cases were reviewed and reclassified by three cytopathologists using the Paris criteria. Cyto-histological correlation was performed in 47 cases. Additionally, all cytology diagnoses were correlated with double immunocytochemistry for p53 and CK20 result. Interobserver consistency was also evaluated. RESULTS: Out of 104 atypical cases, 30 were classified as benign, 49 atypical and 25 suspicious for high-grade urothelial carcinoma (HGUC). Diagnostic consistency between the three observers reached 93.27%. Using the new criteria, only 47.1% of the cases remained in the atypical category. The rate of HGUC histology was 14.3%, 26.7% and 96% in the benign, atypical and suspicious for HGUC cytological categories, respectively. Immunocytochemistry positivity was observed in 25.9%, 41.8% and 80% of the cases in the three diagnostic groups. CONCLUSIONS: The Paris System for reporting urinary cytology provides clear, easy to adopt criteria, which lead to diagnostic categories with clinical significance, facilitating patient management decisions.

Chemosensory epithelial cells in the urethra: sentinels of the urinary tract.

A peculiar cell type of the respiratory and gastrointestinal epithelia, originally termed "brush cell" or "tuft cell" by electron microscopists because of its apical tuft of microvilli, utilizes the canonical bitter taste transduction cascade known from oropharyngeal taste buds to detect potential hazardous compounds, e.g. bacterial products. Upon stimulation, this cell initiates protective reflexes and local inflammatory responses through release of acetylcholine and chemokines. Guided by the understanding of these cells as sentinels, they have been newly discovered at previously unrecognized anatomical locations, including the urethra. Solitary cholinergic urethral cells express canonical taste receptors and are polymodal chemosensors for certain bitter substances, glutamate (umami) and uropathogenic Escherichia coli. Intraurethral bitter stimulation triggers cholinergic reflex activation of bladder detrusor activity, which is interpreted as cleaning flushing of the urethra. The currently known scenario suggests the presence of at least two more urethral chemosensory cell types: non-cholinergic brush cells and neuroendocrine serotonergic cells. The potential implications are enormous and far reaching, as these cells might be involved in monitoring and preventing ascending urinary tract infection and triggering of inappropriate detrusor activity. However, although appealing, this is still highly speculative, since the actual number of distinct chemosensory cell types needs to be finally clarified, as well as their embryological origin, developmental dynamics, receptor equipment, modes of signalling to adjacent nerve fibres and other cells, repertoire of chemo- and cytokines, involvement in pathogenesis of diseases and many other aspects.

A review of the Paris system for reporting urinary cytology.

After the 2013 International Congress of Cytology in Paris, consensus groups were formed to establish an international reporting system for urinary tract (UT) specimens. The recommended guidelines, known as The Paris System (TPS) for Reporting Urinary Cytology, focus on reducing the rate of unnecessary indeterminate diagnoses while maintaining the excellent performance UT cytology has for identifying high-grade urothelial carcinoma. This review highlights the major features of TPS.

Interstitial cells of Cajal and telocytes in the urinary system: facts and distribution.

Current knowledge confirms the existence of interstitial cells of Cajal (ICCs) and telocytes in the urinary system (kidneys, ureter and urinary bladder). Therefore, summarizing of available data can be helpful in understanding of pathophysiology of urological disorders. Telocytes (TCs) are a newly discovered type of cell with numerous functions, described in vertebrates (fish, reptiles, birds, mammals, including human). Despite unique characteristics, they have own differences in morphology and properties in urinary bladder and other organs of the urinary system. This review summarizes particular features of ICCs and TCs in the urinary system, emphasizing their involvement in physiological and pathophysiological processes of the urinary bladder.

Efectos de la colocación preventiva de un catéter ureteral JJ durante el tratamiento de la obstrucción ureteral con stents metálicos en modelo animal / Effects of preventive double-J stent placement in ureteral obstruction treatment with metal stents on animal model

OBJETIVOS: Evaluar el efecto aditivo de los catéteres JJ temporales conjuntamente al empleo de stents metálicos (SM) ureterales en el tratamiento de la uropatía obstructiva, con el propósito de disminuir la hiperplasia urotelial (HU) asociada a los stents. MÉTODO: Se emplearon 24 ejemplares de la especie porcina, que se sometieron a un modelo de estenosis ureteral. Transcurridas 6 semanas, la obstrucción ureteral fue confirmada mediante ecografía, ureteropielografía y ultrasonografía endoluminal. Posteriormente,los animales fueron distribuidos aleatoriamente en dos grupos homogéneos: Grupo I, donde se dispone un SM recubierto permanente y un catéter JJ durante 3 semanas. En el Grupo II, se libera el mismo tipo de SM sin catéter ureteral JJ. Los seguimientos se realizaron a las 3 semanas y a los 6 meses. RESULTADOS: La incidencia de HU fue mayor en el Grupo I, que en el Grupo II; aunque sin diferencias estadísticamente significativas. Por su parte, el Grupo II muestra significación estadística con respecto al grado de obstrucción ureteral por HU. La tasa de migración del stent metálico es similar entre ambos grupos al final del estudio (33.3%). Se muestran diferencias significativas entre los animales con infección urinaria e hiperplasia frente a los que no presentan infección pero sí hiperplasia. Existe un alto índice de correlación estadística entre la infección urinaria y el carácter obstructivo de la hiperplasia. CONCLUSIONES: La disposición de catéteres ureterales JJ no inhibe la aparición de HU asociada a los SM, sin embargo, sí reduce significativamente su carácter obstructivo a largo plazo. La infección urinaria se relaciona directamente con el desarrollo de HU y con la magnitud de esta

OBJECTIVES: The purpose of this experimental study is to assess the additive effects of temporary JJ stent placement together with metal stents (MS) in the treatment obstructive uropathy, in order to reduce urothelial hyperplasia formation. METHODS: Twenty-four pigs were included, and an experimental model of obstructive uropathy was created. Six weeks after obstructive uropathy model induction, ureteral obstruction was confirmed using ultrasonography, ureteropyelography and endoluminal ultrasound. Afterwards, animals were randomly distributed into 2 groups. Group I underwent covered MS placement and JJ ureteral stenting for 3 weeks. Animals in Group II received the same MS without simultaneous JJ stenting. The follow-up was at 3 weeks and at 6 months. RESULTS: Incidence of urothelial hyperplasia was higher in Group I than Group II, but without statically significant differences. On the other hand, Group II showed a significantly higher degree of obstruction severity due to hyperplasia. The migration rate in both groups was 33.3% at the end of the study. Significant differences were shown on animals showing urinary tract infection (UTI) and hyperplasia against those with hyperplasia but no infection. There was a high rate of correlation between UTI and obstructive urothelial hyperplasia. CONCLUSIONS: Placement of JJ ureteral catheter does not inhibit urothelial hyperplasia associated with placement of metal mesh stents, although it significantly reduces its obstructive severity in long-term follow-up. Urinary tract infection is directly related to the development and magnitude of the urothelial hiperplasia

Nature of interstitial cells of Cajal of the upper urinary tract.

Interstitial cells of Cajal (ICCs) were discovered in the gastrointestinal tract over 100 years ago and since then numerous digestive tract pathologies involving ICCs have been described. Many researchers explored ICCs presence and function in the upper urinary tract. Currently, we know that ICCs have potential plasticity, their own spontaneous activity and that they are responsible for Ca2+ waves generation and neuromuscular transmission. ICCs are also involved in the conjugation, propagation and modulation of peristaltic waves in the upper urinary tract. Despite everything we know about ICCs, their role in the pathogenesis of the upper urinary tract abnormalities remains still unclear and results of published studies are confusing. The authors' intention was to review the scientific literature regarding ICCs and to summarise the current knowledge about their nature in the upper urinary tract.

Interstitial cells: regulators of smooth muscle function.

Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα(+) cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα(+) cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues.

Urinary tract pacemaker cells: current knowledge and insights from nonrenal pacemaker cells provide a basis for future discovery.

Coordinated ureteric peristalsis propels urine from the kidney to the bladder. Cells in the renal pelvis and ureter spontaneously generate and propagate electrical activity to control this process. Recently, c-kit tyrosine kinase and hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) were identified in the upper urinary tract. Both of these proteins are required for coordinated proximal to distal contractions in the ureter. Alterations in pacemaker cell expression are present in multiple congenital kidney diseases, suggesting a functional contribution by these cells to pathologic states. In contrast to gut and heart pacemaker cells, the developmental biology of ureteric pacemaker cells, including cell lineage and signaling mechanisms, is undefined. Here, we review pacemaker cell identify and function in the urinary pelvis and ureter and the control of pacemaker function by Hedgehog-GLI signaling. Next, we highlight current knowledge of gut and heart pacemaker cells that is likely to provide insight into developmental mechanisms that could control urinary pacemaker cells.

Retinoid signaling in progenitors controls specification and regeneration of the urothelium.

The urothelium is a multilayered epithelium that serves as a barrier between the urinary tract and blood, preventing the exchange of water and toxic substances. It consists of superficial cells specialized for synthesis and transport of uroplakins that assemble into a tough apical plaque, one or more layers of intermediate cells, and keratin 5-expressing basal cells (K5-BCs), which are considered to be progenitors in the urothelium and other specialized epithelia. Fate mapping, however, reveals that intermediate cells rather than K5-BCs are progenitors in the adult regenerating urothelium, that P cells, a transient population, are progenitors in the embryo, and that retinoids are critical in P cells and intermediate cells, respectively, for their specification during development and regeneration. These observations have important implications for tissue engineering and repair and, ultimately, may lead to treatments that prevent loss of the urothelial barrier, a major cause of voiding dysfunction and bladder pain syndrome.

Proteus mirabilis uroepithelial cell adhesin (UCA) fimbria plays a role in the colonization of the urinary tract.

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Proteus mirabilis is an opportunistic pathogen, capable of causing severe UTIs, with serious kidney damage that may even lead to death. Several virulence factors are involved in the pathogenicity of this bacterium. Among these, adherence to the uroepithelium mediated by fimbriae appears to be a significant bacterial attribute related to urovirulence. Proteus mirabilis expresses several types of fimbriae that could be involved in the pathogenesis of UTI, including uroepithelial cell adhesin (UCA). In this report, we used an uropathogenic P. mirabilis wild-type strain and an isogenic ucaA mutant unable to express UCA to study the pathogenic role of this fimbria in UTI. Ability of the mutant to adhere to desquamated uroepithelial cells and to infect mice using different experimental UTI models was significantly impaired. These results allow us to conclude that P. mirabilis UCA plays an important role in the colonization of the urinary tract.

Isolation and myogenic differentiation of mesenchymal stem cells for urologic tissue engineering.

Cell-based tissue engineering is one of the most promising areas in biotechnology for restoring tissues and organ function in the urinary tract. Current strategies for bladder tissue engineering require a competent biological scaffold that is seeded in vitro with the patient's own bladder cells. This use of autologous cells avoids graft rejection and the long-term use of immunosuppressive medications usually required after allogeneic transplantation. However, suitable bladder cells from the patient are sometimes limited or unobtainable. When suitable cells are unavailable for seeding due to bladder exstrophy, malignancy, or other reasons, the use of other cell types originating from the patient may be an alternative. A suitable alternative to autologous bladder cells could be mesenchymal stem cells (MSC). MSC reside primarily in the bone marrow, although they exist in other sites as well, including adipose tissue, peripheral and cord blood, liver tissue, and fetal tissues. Bone marrow-derived stromal cell populations contain few MSC (one MSC in 10(4)-5 × 10(7) marrow cells), with the exact number depending on the age of the patient. Despite their limited numbers, MSC possess both the ability to self-renew for extended periods of time and the potential to differentiate into several different specialized cell types under the appropriate conditions. MSC are capable of expansion and tissue-specific differentiation in vitro based on external signals and/or the environment. There are different methodologies for induction and maintenance of a differentiated cell phenotype from MSC. For example, MSC can differentiate into a smooth muscle cell (SMC) phenotype in vitro when exposed to stimuli such as conditioned medium derived from SMC cultures or specific myogenic growth factors (PDGF-BB, HGF, TGF-ß). These differential cells can migrate to a scaffold for differentiation into smooth muscle-like cells in vivo. Furthermore, stem cell-seeded scaffolds that are implanted into the bladders repopulate and reorganize the tissue rapidly, thus reducing fibrosis and restoring appropriate neural functionality.In this chapter, we describe the methods we use for the isolation of human bone marrow mesenchymal stem cells (BMSC), and demonstrate evidence of their myogenic differentiation capacity for potential use in urologic tissue engineering.