RESUMO
Progression of glomerulosclerosis is associated with loss of podocytes with subsequent glomerular tuft instability. It is thought that a diminished number of podocytes may be able to preserve tuft stability through cell hypertrophy associated with cell cycle reentry. At the same time, reentry into the cell cycle risks podocyte detachment if podocytes cross the G1/S checkpoint and undergo abortive cytokinesis. In order to study cell cycle dynamics during chronic kidney disease (CKD) development, we used a FUCCI model (fluorescence ubiquitination-based cell cycle indicator) of mice with X-linked Alport Syndrome. This model exhibits progressive CKD and expresses fluorescent reporters of cell cycle stage exclusively in podocytes. With the development of CKD, an increasing fraction of podocytes in vivo were found to be in G1 or later cell cycle stages. Podocytes in G1 and G2 were hypertrophic. Heterozygous female mice, with milder manifestations of CKD, showed G1 fraction numbers intermediate between wild-type and male Alport mice. Proteomic analysis of podocytes in different cell cycle phases showed differences in cytoskeleton reorganization and metabolic processes between G0 and G1 in disease. Additionally, in vitro experiments confirmed that damaged podocytes reentered the cell cycle comparable to podocytes in vivo. Importantly, we confirmed the upregulation of PDlim2, a highly expressed protein in podocytes in G1, in a patient with Alport Syndrome, confirming our proteomics data in the human setting. Thus, our data showed that in the Alport model of progressive CKD, podocyte cell cycle distribution is altered, suggesting that cell cycle manipulation approaches may have a role in the treatment of various progressive glomerular diseases characterized by podocytopenia.
Assuntos
Nefrite Hereditária , Podócitos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ciclo Celular , Progressão da Doença , Feminino , Humanos , Proteínas com Domínio LIM/metabolismo , Masculino , Camundongos , Proteínas dos Microfilamentos/metabolismo , Nefrite Hereditária/genética , Nefrite Hereditária/metabolismo , Podócitos/metabolismo , ProteômicaRESUMO
AIM: Scrotal ultrasounds are utilised in some primary care settings for suspected cryptorchidism, despite inaccuracies. We aim to identify the correlation between ultrasound and primary care provider (PCP) findings of undescended testicles (UDTs) as a potential source of confirmation bias. METHODS: Males referred for suspected UDT by PCPs who underwent scrotal ultrasound and paediatric urologist examination from 2014 to 2019 were included. Correlation between PCP and ultrasound findings and diagnostic accuracy were evaluated. Logistic regression was utilised to determine associations between patient factors and UDT misdiagnosis. RESULTS: Out of 145 testes, ultrasound corroborated PCPs' UDT diagnoses 87.6% of the time, 49.6% of which were confirmed as UDT by paediatric urologists. Ultrasound had a false positivity rate of 81.0% and specificity of 19.0%. Ultrasound versus paediatric urologist findings regarding testicle location were significantly different (P < 0.0001). Patients aged ≥8 years old had 5.2 times greater odds of being misdiagnosed with UDT than patients <8 years old (95% confidence interval: 1.6-16.7; P < 0.002) by PCP and ultrasound. CONCLUSION: Scrotal ultrasound highly corroborated PCPs' UDT diagnoses. Older patients were more likely to be misdiagnosed with UDT by PCP and ultrasound. As ultrasounds rarely refute PCP examinations for suspected UDTs and are highly inaccurate, confirmation bias may explain the use of ultrasound in the workup of UDT.
Assuntos
Criptorquidismo , Criança , Criptorquidismo/diagnóstico por imagem , Humanos , Masculino , Exame Físico , Encaminhamento e Consulta , Escroto/diagnóstico por imagem , UltrassonografiaRESUMO
PURPOSE: To compare the prevalence of vesicoureteral reflux (VUR), febrile urinary tract infection (fUTI), and chronic kidney disease (CKD) among patients with multicystic dysplastic kidney (MCDK) diagnosed by renal scintigraphy (RS) versus follow-up renal ultrasound (RUS) alone. METHODS: This was a retrospective review of patients seen at a tertiary care center from 2010 to 2020 with MCDK diagnosed by RS or follow-up RUS. Differences in the prevalence of VUR, fUTI, and CKD by cohort were assessed using logistic regression analysis, Pearson X2 , and Fisher's Exact tests. Temporal trends in diagnostic methods used (RUS versus RUS + RS) were evaluated using the Cochran-Armitage trend test. RESULTS: One-hundred seventy-two patients were included: 50% (n = 86) underwent RUS + RS and 50% (n = 86) underwent RUS alone to diagnose MCDK. Prevalence of VUR, fUTI, and CKD did not significantly vary between groups. Among patients who had a VCUG, 4.4% had contralateral VUR (1.7% RUS + RS group; 7.4% RUS group; p = 0.19) and 14.5% had at least one fUTI (16.3% RUS + RS group; 12.8% RUS group; p = 0.52). Females were significantly more likely to have at least one fUTI (p = 0.04). Four patients (2.3%) developed CKD, all in the RUS + RS cohort (p = 0.12). Diagnosis of MCDK by RUS versus RUS + RS did not significantly vary over time (p = 0.17). CONCLUSION: Patients with unilateral MCDK confirmed by RS versus RUS alone do not significantly vary in the prevalence of VUR, fUTI, or CKD. Renal scintigraphy studies may not be necessary in unilateral MCDK diagnosis but continue to be used.
Assuntos
Rim Displásico Multicístico , Insuficiência Renal Crônica , Infecções Urinárias , Refluxo Vesicoureteral , Feminino , Humanos , Lactente , Rim/diagnóstico por imagem , Rim Displásico Multicístico/diagnóstico por imagem , Rim Displásico Multicístico/epidemiologia , Cintilografia , Insuficiência Renal Crônica/epidemiologia , Estudos Retrospectivos , Infecções Urinárias/diagnóstico por imagem , Infecções Urinárias/epidemiologia , Refluxo Vesicoureteral/diagnóstico por imagem , Refluxo Vesicoureteral/epidemiologiaRESUMO
PURPOSE: Testicular adrenal rest tumors are a well-known complication in males who have congenital adrenal hyperplasia with potential infertility in adulthood. We assessed the prevalence of testicular adrenal rest tumors in infants to young men presenting to a congenital adrenal hyperplasia Comprehensive Care Center. MATERIALS AND METHODS: A total of 35 males with congenital adrenal hyperplasia due to 21-hydroxylase deficiency underwent scrotal ultrasonography, including 7 younger than 5 years, 9 who were 5 to 12 years old and 19 who were older than 12 years. Three and 35 patients had classic and nonclassic congenital adrenal hyperplasia, respectively. Bone age x-ray or advanced bone age x-ray history, glucocorticoid dose, fludrocortisone dose, and serum 17-hydroxyprogesterone, testosterone and androstenedione levels within 3 months of ultrasound were also recorded. RESULTS: Testicular adrenal rest tumors were detected in 5 of 35 patients (14%), including 1 of 9 (11%) who were 5 to 12 years old and 4 of 19 (21%) who were older than 12 years. The tumors were not detected in any patients younger than 5 years, including 1 infant with poor hormonal control. The youngest patient with positive findings was 6.6 years old. All patients with positive findings had bilateral disease and only 1 had suspicious physical findings. The glucocorticoid dose and 17-hydroxyprogesterone did not differ between patients with vs without a testicular adrenal rest tumor. Those with a tumor were more likely to have advanced bone age x-ray results (100% vs 42%, p = 0.04) and higher fludrocortisone dose (p <0.01). All males with nonclassic congenital adrenal hyperplasia had negative tumor findings. CONCLUSIONS: Testicular adrenal rest tumors were present in young males with classic congenital adrenal hyperplasia but not in infants or toddlers. These tumors were associated with higher fludrocortisone requirements and a history of advanced bone age x-ray results. However, the tumors did not develop in all poorly controlled males. Longitudinal studies are needed to understand the individual predisposition to testicular adrenal rest tumors and the age at which to begin screening patients with congenital adrenal hyperplasia.
Assuntos
Tumor de Resto Suprarrenal/epidemiologia , Neoplasias Testiculares/epidemiologia , Adolescente , Hiperplasia Suprarrenal Congênita/complicações , Tumor de Resto Suprarrenal/etiologia , Criança , Pré-Escolar , Estudos Transversais , Humanos , Masculino , Prevalência , Neoplasias Testiculares/etiologiaRESUMO
PURPOSE: Augmentation cystoplasty using gastrointestinal segments in children/adolescents with medically refractory neurogenic bladder is associated with significant complications. We evaluated an autologous cell seeded biodegradable scaffold (Tengion®) for bladder augmentation as an alternative to traditional enterocystoplasty in this population. MATERIALS AND METHODS: A phase II prospective study was performed in children with neurogenic bladder due to spina bifida requiring enterocystoplasty for detrusor pressure 40 cm H2O or greater despite maximum antimuscarinic medication. Following open bladder biopsy, urothelial and smooth muscle cells were grown ex vivo and seeded onto a biodegradable scaffold to form a regenerative augment as the foundation for bladder tissue regeneration. Bladder neck sling was the only concomitant surgical procedure permitted. Bladders were cycled postoperatively to promote regeneration. Primary and secondary outcomes at 12 months included change in bladder compliance, bladder capacity and safety. Long-term assessment was done with similar outcomes at 36 months. RESULTS: Compliance improved in 4 patients at 12 months and in 5 patients at 36 months, although the difference was not clinically or statistically significant. There was no clinical or statistical improvement in bladder capacity at 12 or 36 months in any patient. Adverse events occurred in all patients, and most were easily treated. Two patients had low cell growth following bladder biopsy, of whom 1 withdrew from the study and 1 underwent a second biopsy. Serious adverse events of bowel obstruction and/or bladder rupture occurred in 4 patients. CONCLUSIONS: Our autologous cell seeded biodegradable scaffold did not improve bladder compliance or capacity, and our serious adverse events surpassed an acceptable safety standard.
Assuntos
Implantes Absorvíveis , Transplante de Células , Alicerces Teciduais , Bexiga Urinaria Neurogênica/cirurgia , Bexiga Urinária/cirurgia , Adolescente , Anastomose Cirúrgica , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Músculo Liso/citologia , Estudos Prospectivos , Disrafismo Espinal/complicações , Transplante Autólogo , Bexiga Urinaria Neurogênica/etiologia , Procedimentos Cirúrgicos Urológicos/métodos , Urotélio/citologiaRESUMO
Here, we used digital spatial profiling (DSP) to describe the glomerular transcriptomic signatures that may characterize the complex molecular mechanisms underlying progressive kidney disease in Alport syndrome, focal segmental glomerulosclerosis, and membranous nephropathy. Our results revealed significant transcriptional heterogeneity among diseased glomeruli, and this analysis showed that histologically similar glomeruli manifested different transcriptional profiles. Using glomerular pathology scores to establish an axis of progression, we identified molecular pathways with progressively decreased expression in response to increasing pathology scores, including signal recognition particle-dependent cotranslational protein targeting to membrane and selenocysteine synthesis pathways. We also identified a distinct signature of upregulated and downregulated genes common to all the diseases investigated when compared with nondiseased tissue from nephrectomies. These analyses using DSP at the single-glomerulus level could help to increase insight into the pathophysiology of kidney disease and possibly the identification of biomarkers of disease progression in glomerulopathies.
Assuntos
Glomerulosclerose Segmentar e Focal , Nefrite Hereditária , Insuficiência Renal Crônica , Humanos , Transcriptoma , Glomérulos Renais/patologia , Glomerulosclerose Segmentar e Focal/patologia , Nefrite Hereditária/patologia , Insuficiência Renal Crônica/metabolismoRESUMO
The deposition of antipodocyte autoantibodies in the glomerular subepithelial space induces primary membranous nephropathy (MN), the leading cause of nephrotic syndrome worldwide. Taking advantage of the glomerulus-on-a-chip system, we modeled human primary MN induced by anti-PLA2R antibodies. Here we show that exposure of primary human podocytes expressing PLA2R to MN serum results in IgG deposition and complement activation on their surface, leading to loss of the chip permselectivity to albumin. C3a receptor (C3aR) antagonists as well as C3AR gene silencing in podocytes reduced oxidative stress induced by MN serum and prevented albumin leakage. In contrast, inhibition of the formation of the membrane-attack-complex (MAC), previously thought to play a major role in MN pathogenesis, did not affect permselectivity to albumin. In addition, treatment with a C3aR antagonist effectively prevented proteinuria in a mouse model of MN, substantiating the chip findings. In conclusion, using a combination of pathophysiologically relevant in vitro and in vivo models, we established that C3a/C3aR signaling plays a critical role in complement-mediated MN pathogenesis, indicating an alternative therapeutic target for MN.
Assuntos
Glomerulonefrite Membranosa , Síndrome Nefrótica , Podócitos , Animais , Humanos , Camundongos , Albuminas , Glomerulonefrite Membranosa/genética , Glomérulos Renais/patologia , Síndrome Nefrótica/patologia , Podócitos/patologiaRESUMO
Injection of amniotic fluid stem cells ameliorates the acute phase of acute tubular necrosis in animals by promoting proliferation of injured tubular cells and decreasing apoptosis, but whether these stem cells could be of benefit in CKD is unknown. Here, we used a mouse model of Alport syndrome, Col4a5(-/-) mice, to determine whether amniotic fluid stem cells could modify the course of progressive renal fibrosis. Intracardiac administration of amniotic fluid stem cells before the onset of proteinuria delayed interstitial fibrosis and progression of glomerular sclerosis, prolonged animal survival, and ameliorated the decline in kidney function. Treated animals exhibited decreased recruitment and activation of M1-type macrophages and a higher proportion of M2-type macrophages, which promote tissue remodeling. Amniotic fluid stem cells did not differentiate into podocyte-like cells and did not stimulate production of the collagen IVa5 needed for normal formation and function of the glomerular basement membrane. Instead, the mechanism of renal protection was probably the paracrine/endocrine modulation of both profibrotic cytokine expression and recruitment of macrophages to the interstitial space. Furthermore, injected mice retained a normal number of podocytes and had better integrity of the glomerular basement membrane compared with untreated Col4a5(-/-) mice. Inhibition of the renin-angiotensin system by amniotic fluid stem cells may contribute to these beneficial effects. In conclusion, treatment with amniotic fluid stem cells may be beneficial in kidney diseases characterized by progressive renal fibrosis.
Assuntos
Rim/patologia , Nefrite Hereditária/terapia , Sistema Renina-Angiotensina/fisiologia , Transplante de Células-Tronco/métodos , Líquido Amniótico/citologia , Análise de Variância , Animais , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Fibrose/patologia , Fibrose/terapia , Imuno-Histoquímica , Rim/fisiopatologia , Testes de Função Renal , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nefrite Hereditária/patologia , Podócitos/metabolismo , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real , Estatísticas não ParamétricasRESUMO
A nephrogenic progenitor cell (NP) with cancer stem cell characteristics driving Wilms tumor (WT) using spatial transcriptomics, bulk and single cell RNA sequencing, and complementary in vitro and transplantation experiments is identified and characterized. NP from WT samples with NP from the developing human kidney is compared. Cells expressing SIX2 and CITED1 fulfill cancer stem cell criteria by reliably recapitulating WT in transplantation studies. It is shown that self-renewal versus differentiation in SIX2+CITED1+ cells is regulated by the interplay between integrins ITGß1 and ITGß4. The spatial transcriptomic analysis defines gene expression maps of SIX2+CITED1+ cells in WT samples and identifies the interactive gene networks involved in WT development. These studies define SIX2+CITED1+ cells as the nephrogenic-like cancer stem cells of WT and points to the renal developmental transcriptome changes as a possible driver in regulating WT formation and progression.
Assuntos
Neoplasias Renais , Tumor de Wilms , Humanos , Fatores de Transcrição/genética , Tumor de Wilms/genética , Tumor de Wilms/metabolismo , Tumor de Wilms/patologia , Rim , Células-Tronco Neoplásicas/metabolismo , Neoplasias Renais/genéticaRESUMO
OBJECTIVE: To compare the perioperative parameters of paediatric patients who underwent nephrectomy via laparo-endoscopic single site (LESS) surgery (also known as single incision laparoscopic surgery or SILS) with those who underwent nephrectomy via conventional laparoscopy (LAP), robotic-assisted laparoscopy (RALN), and open surgery (OPEN). PATIENTS AND METHODS: The medical records of 69 paediatric patients at a single institution who underwent nephrectomies for non-functioning kidneys in 72 renal units (39 OPEN, 11 LAP, 11 RALN and 11 LESS) were reviewed for patient demographics and perioperative clinical parameters. RESULTS: The minimally invasive modalities in children, including LESS nephrectomy, were associated with shorter lengths of hospital stay (P < 0.001) and decreased postoperative pain medication usage (P < 0.001) than with open surgery. Similar surgical times were noted with LESS and the other minimally invasive modalities (LAP and RALN) (P= 0.056). However, the minimally invasive modalities (LESS, LAP and RALN) were associated with slightly longer surgical times when compared with open surgery (P < 0.001), which may, in part, be secondary to learning curve factors. No differences were noted among the minimally invasive modalities for postoperative pain medication usage (P= 0.354) and length of hospital stay (P= 0.86). CONCLUSIONS: The minimally invasive modalities for nephrectomy in children, including LESS nephrectomy, are associated with shorter lengths of hospital stay and decreased postoperative pain medication use when compared with open surgery. LESS nephrectomy in children is associated with similar surgical times, lengths of hospital stay and postoperative pain medication use as the other minimally invasive modalities (LAP and RALN). Slightly longer surgical times are noted with the minimally invasive modalities, including LESS nephrectomy, when compared with open surgery, which may, in part, be secondary to learning curve factors.
Assuntos
Nefropatias/cirurgia , Laparoscopia/métodos , Nefrectomia/métodos , Robótica , Adolescente , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Tempo de Internação/estatística & dados numéricos , Masculino , Nefrectomia/efeitos adversos , Dor Pós-Operatória/etiologia , Estudos Retrospectivos , Fatores de Tempo , Resultado do TratamentoRESUMO
Much attention recently has been focused on stem cell technology as a possible alternative modality of treatment of a variety of diseases. Chronic kidney disease is a serious health problem and most chronic kidney diseases share in common the presence of interstitial and glomerular fibrosis, regardless of the underlying cause. To date there are no specific therapies aimed at treating fibrosis in the kidney. In a novel effort to address the underlying pathology in kidney disease, researchers are demonstrating that stem cell therapy can attenuate fibrosis in chronic kidney disease in animal models. This review will focus on the recent developments in stem cell research and their possible implications to treat chronic kidney disease.
Assuntos
Falência Renal Crônica/cirurgia , Medicina Regenerativa/métodos , Transplante de Células-Tronco/métodos , Animais , HumanosRESUMO
Alveolar epithelial integrity is dependent upon the alveolar milieu, yet the milieu of the damaged alveolar epithelial cell type 2 (AEC2) has been little studied. Characterization of its components may offer the potential for ex vivo manipulation of stem cells to optimize their therapeutic potential. We examined the cytokine profile of AEC2 damage milieu, hypothesizing that it would promote endogenous epithelial repair while recruiting cells from other locations and instructing their engraftment and differentiation. Bronchoalveolar lavage and lung extract from hyperoxic rats represented AEC2 in vivo damage milieu, and medium from a scratch-damaged AEC2 monolayer represented in vitro damage. CINC-2 and ICAM, the major cytokines detected by proteomic cytokine array in AEC2 damage milieu, were chemoattractive to normoxic AECs and expedited in vitro wound healing, which was blocked by their respective neutralizing antibodies. The AEC2 damage milieu was also chemotactic for exogenous uncommitted human amniotic fluid stem cells (hAFSCs), increasing migration greater than 20-fold. hAFSCs attached within an in vitro AEC2 wound and expedited wound repair by contributing cytokines migration inhibitory factor and plasminogen activator inhibitor 1 to the AEC2 damage milieu, which promoted wound healing. The AEC2 damage milieu also promoted differentiation of a subpopulation of hAFSCs to express SPC, TTF-1, and ABCA3, phenotypic markers of distal alveolar epithelium. Thus, the microenvironment created by AEC2 damage not only promotes autocrine repair but also can attract uncommitted stem cells, which further augment healing through cytokine secretion and differentiation.
Assuntos
Células Epiteliais Alveolares/metabolismo , Comunicação Autócrina , Diferenciação Celular , Citocinas/metabolismo , Regeneração , Células-Tronco/metabolismo , Transportadores de Cassetes de Ligação de ATP/biossíntese , Células Epiteliais Alveolares/patologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica , Humanos , Hiperóxia/metabolismo , Hiperóxia/patologia , Masculino , Proteínas Nucleares/biossíntese , Ratos , Ratos Sprague-Dawley , Células-Tronco/patologia , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/biossínteseRESUMO
PURPOSE: Robotic assisted laparoscopic pyeloplasty is an emerging, minimally invasive alternative to open pyeloplasty in children for ureteropelvic junction obstruction. The procedure is associated with smaller incisions and shorter hospital stays. To our knowledge previous outcome analyses have not included human capital calculations, especially regarding loss of parental workdays. We compared perioperative factors in patients who underwent robotic assisted laparoscopic and open pyeloplasty at a single institution, especially in regard to human capital changes, in an institutional cost analysis. MATERIALS AND METHODS: A total of 44 patients 2 years old or older from a single institution underwent robotic assisted (37) or open (7) pyeloplasty from 2008 to 2010. We retrospectively reviewed the charts to collect demographic and perioperative data. The human capital approach was used to calculate parental productivity losses. RESULTS: Patients who underwent robotic assisted laparoscopic pyeloplasty had a significantly shorter average hospital length of stay (1.6 vs 2.8 days, p <0.05). This correlated with an average savings of lost parental wages of $90.01 and hospitalization expenses of $612.80 per patient when excluding amortized robot costs. However, cost savings were not achieved by varying length of stay when amortized costs were included. CONCLUSIONS: Robotic assisted laparoscopic pyeloplasty in children is associated with human capital gains, eg decreased lost parental wages, and lower hospitalization expenses. Future comparative outcome analyses in children should include financial factors such as human capital loss, which can be especially important for families with young children.
Assuntos
Efeitos Psicossociais da Doença , Rim/cirurgia , Laparoscopia/métodos , Robótica , Ureter/cirurgia , Obstrução Ureteral/cirurgia , Procedimentos Cirúrgicos Urológicos/métodos , Adolescente , Criança , Pré-Escolar , Custos e Análise de Custo , Feminino , Seguimentos , Humanos , Laparoscopia/economia , Tempo de Internação/economia , Masculino , Pais , Procedimentos de Cirurgia Plástica/economia , Procedimentos de Cirurgia Plástica/métodos , Estudos Retrospectivos , Fatores Socioeconômicos , Estados Unidos , Obstrução Ureteral/economia , Procedimentos Cirúrgicos Urológicos/economia , Adulto JovemRESUMO
PURPOSE OF REVIEW: Amniotic fluid, due to its contact to the fetus during development, is considered an important diagnostic tool to evaluate the health status of the fetus during pregnancy. However, amniotic fluid also contains a heterogeneous cellular population that can be safely collected by amniocentesis and easily cultured. Many different cell types have been found within amniotic fluid and currently some of them are being tested for their possible use for cellular therapy. RECENT FINDINGS: Potential of pluripotent and multipotent cells isolated from the amniotic fluid has been tested and in-vitro differentiations toward various cell types have been successfully performed. Furthermore, in-vivo studies are highlighting the benefits and mechanisms of amniotic fluid cells for therapy, with particular focus on kidney and lung diseases. SUMMARY: Amniotic fluid may represent a precious source for easily and safely retrievable cell types that may be used for regenerative medicine purposes.
Assuntos
Líquido Amniótico/citologia , Diferenciação Celular/fisiologia , Células-Tronco Multipotentes/citologia , Regeneração/fisiologia , Medicina Regenerativa/métodos , Animais , Feminino , Humanos , GravidezRESUMO
PURPOSE: Human amniotic fluid contains multiple cell types, including pluripotent and committed progenitor cells, and fully differentiated cells. We characterized various cell populations in amniotic fluid. MATERIALS AND METHODS: Optimum culture techniques for multiple cell line passages with minimal morphological change were established. Cell line analysis and characterization were done with reverse transcriptase and real-time polymerase chain reaction. Immunoseparation was done to distinguish native progenitor cell lines and their various subpopulations. RESULTS: Endodermal and mesodermal marker expression was greatest in samples of early gestational age while ectodermal markers showed a constant rate across all samples. Pluripotent and mesenchymal cells were always present but hematopoietic cell markers were expressed only in older samples. Specific markers for lung, kidney, liver and heart progenitor cells were increasingly expressed after 18 weeks of gestation. We specifically focused on a CD24+OB-cadherin+ population that could identify uninduced metanephric mesenchyma-like cells, which in vivo are nephron precursors. The CD24+OB-cadherin+ cell line was isolated and subjected to further immunoseparation to select 5 distinct amniotic fluid kidney progenitor cell subpopulations based on E-cadherin, podocalyxin, nephrin, TRKA and PDGFRA expression, respectively. CONCLUSIONS: These subpopulations may represent different precursor cell lineages committed to specific renal cell fates. Committed progenitor cells in amniotic fluid may provide an important and novel resource of useful cells for regenerative medicine purposes.
Assuntos
Líquido Amniótico/citologia , Medicina Regenerativa/métodos , Células-Tronco , Células Cultivadas , Previsões , Humanos , Rim/fisiologia , Regeneração , Medicina Regenerativa/tendênciasRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
In this work we model the glomerular filtration barrier, the structure responsible for filtering the blood and preventing the loss of proteins, using human podocytes and glomerular endothelial cells seeded into microfluidic chips. In long-term cultures, cells maintain their morphology, form capillary-like structures and express slit diaphragm proteins. This system recapitulates functions and structure of the glomerulus, including permselectivity. When exposed to sera from patients with anti-podocyte autoantibodies, the chips show albuminuria proportional to patients' proteinuria, phenomenon not observed with sera from healthy controls or individuals with primary podocyte defects. We also show its applicability for renal disease modeling and drug testing. A total of 2000 independent chips were analyzed, supporting high reproducibility and validation of the system for high-throughput screening of therapeutic compounds. The study of the patho-physiology of the glomerulus and identification of therapeutic targets are also feasible using this chip.
Assuntos
Glomérulos Renais/metabolismo , Dispositivos Lab-On-A-Chip , Nefrite Hereditária/metabolismo , Albuminas/metabolismo , Albuminúria/tratamento farmacológico , Albuminúria/metabolismo , Células Imobilizadas/química , Células Imobilizadas/metabolismo , Células Endoteliais/química , Células Endoteliais/metabolismo , Humanos , Glomérulos Renais/química , Glomérulos Renais/efeitos dos fármacos , Masculino , Nefrite Hereditária/tratamento farmacológico , Podócitos/química , Podócitos/metabolismoRESUMO
BACKGROUND: In women, a healthy, patent vagina is important for the maintenance of a good quality of life. Apart from congenital abnormalities, such as cloacal exstrophy, intersex disorders, and an absence of the posterior two thirds of the organ, individuals may also suffer from cancer, trauma, infection, inflammation, or iatrogenic injuries leading to tissue damage and loss -- all of which require vaginal repair or replacement. Of necessity, reconstruction is often performed with nonvaginal tissue substitutes, such as segments of large intestine or skin, which are not anatomically or functionally ideal (Hendren and Atala, J Urol 1994; 152: 752; Hendren and Atala, J Pediatr Surg 1995; 30: 91). Whenever such tissue is used additional complications often ensue, such as strictures, infection, hair growth, graft shrinkage, diverticuli, and even malignancy (Filipas et al., BJU Int 2000; 85: 715; Lai and Chang, Changgeng Yi Xue Za Zhi 1999; 22: 253; Parsons et al., J Pediatr Surg 2002; 37: 629; Seccia et al., Ann Plast Surg 2002; 49: 379; Filipas, Curr Opin Urol 2001; 11: 267). METHODS: Using a rabbit model, we report here the construction of a functional vagina using autologous cells expanded from a small vaginal biopsy. RESULTS.: Six months after total vaginal replacement, radiographic analysis of rabbits implanted with the neovagina demonstrated wide, patent vaginal calibers without strictures. Histologic analysis revealed well-organized epithelial and muscle cell layers. Physiologic studies showed normal-range responses to electrical stimulation or to an adrenergic agonist. CONCLUSIONS: These data indicate that a tissue engineering approach to clinical vaginal reconstruction in women is now a realistic possibility.
Assuntos
Engenharia Tecidual/métodos , Vagina/citologia , Vagina/cirurgia , Animais , Fenômenos Biomecânicos , Biópsia , Técnicas de Cultura de Células/métodos , Colágeno/metabolismo , Elastina/metabolismo , Feminino , Imuno-Histoquímica/métodos , Polímeros/química , Coelhos , Resistência à Tração , Fatores de Tempo , Engenharia Tecidual/instrumentação , Doenças Vaginais/terapiaRESUMO
OBJECTIVE: The purpose of our study was to determine whether fetal magnetic resonance imaging (MRI) provides additional information that might affect the obstetric management of pregnancies complicated by sonographically diagnosed fetal urinary tract anomalies. METHODS: Fetal MRI and sonography were used to study 39 women with suspected fetal urinary tract anomalies in the second and third trimesters of pregnancy. RESULTS: In 24 of 39 cases (61%), fetal MRI confirmed the sonographic diagnosis. In 14 cases (36%), fetal MRI modified the initial sonographic diagnosis and counseling but did not change obstetric management. In 1 case (3%), the addition of fetal MRI resulted in a substantial change in the management of the pregnancy. CONCLUSIONS: During the second and third trimesters of pregnancy, fetal MRI showed fetal urinary tract anomalies in excellent anatomic detail. Fetal MRI is a useful complementary tool in the assessment of sonographically diagnosed fetal urinary tract anomalies. In a small percentage of cases, it can have a substantial impact on obstetric management.
Assuntos
Imageamento por Ressonância Magnética/métodos , Ultrassonografia Pré-Natal/métodos , Doenças Urológicas/congênito , Doenças Urológicas/diagnóstico , Feminino , Humanos , Gravidez , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The outcome of tissue engineered organ transplants depends on the capacity of the biomaterial to promote a pro-healing response once implanted in vivo. Multiple studies, including ours, have demonstrated the possibility of using the extracellular matrix (ECM) of animal organs as platform for tissue engineering and more recently, discarded human organs have also been proposed as scaffold source. In contrast to artificial biomaterials, natural ECM has the advantage of undergoing continuous remodeling which allows adaptation to diverse conditions. It is known that natural matrices present diverse immune properties when compared to artificial biomaterials. However, how these properties compare between diseased and healthy ECM and artificial scaffolds has not yet been defined. To answer this question, we used decellularized renal ECM derived from WT mice and from mice affected by Alport Syndrome at different time-points of disease progression as a model of renal failure with extensive fibrosis. We characterized the morphology and composition of these ECMs and compared their in vitro effects on macrophage activation with that of synthetic scaffolds commonly used in the clinic (collagen type I and poly-L-(lactic) acid, PLLA). We showed that ECM derived from Alport kidneys differed in fibrous protein deposition and cytokine content when compared to ECM derived from WT kidneys. Yet, both WT and Alport renal ECM induced macrophage differentiation mainly towards a reparative (M2) phenotype, while artificial biomaterials towards an inflammatory (M1) phenotype. Anti-inflammatory properties of natural ECMs were lost when homogenized, hence three-dimensional structure of ECM seems crucial for generating an anti-inflammatory response. Together, these data support the notion that natural ECM, even if derived from diseased kidneys promote a M2 protolerogenic macrophage polarization, thus providing novel insights on the applicability of ECM obtained from discarded organs as ideal scaffold for tissue engineering.