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1.
Am J Physiol Renal Physiol ; 318(3): F628-F638, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31904289

RESUMO

Excessive compensatory nephron hypertrophy (CNH) has been implicated in setting the stage for progressive nephron damage. Lack of a class III phosphatidylinositol 3-kinase (Pik3c3) inhibitor suitable for using in animals and lack of a Pik3c3-deficient animal model preclude the possibility of conclusively defining a role for Pik3c3 in CNH in previous studies. Here, we report that insertion of an Frt-flanked PGK-Neo cassette into intron 19 of the mouse Pik3c3 gene resulted in a hypomorphic allele. This allowed us to create a unique mouse model and provide the first definitive genetic evidence demonstrating whether Pik3c3 is essential for the regulation of CNH. Our results indicate that homozygous Pik3c3 hypomorphic (Pik3c3Hypo/Hypo) mice express significantly low levels of Pik3c3 than heterozygous Pik3c3 hypomorphic (Pik3c3Hypo/WT) littermates, which already express a lower level of Pik3c3 than wild-type (Pik3c3WT/WT) littermates. Interestingly, after unilateral nephrectomy (UNX), Pik3c3Hypo/Hypo mice develop a significantly lower degree of CNH than Pik3c3WT/WT mice and Pik3c3Hypo/WT mice, as revealed by measurement of kidney weight, kidney-to-body weight ratio, renal protein-to-DNA ratio, and morphometric analysis of proximal tubular and glomerular size. Mechanistically, UNX-induced mammalian target of rapamycin complex 1 (mTORC1) signaling to phosphorylation of ribosomal protein S6 (rpS6) in the remaining kidney was markedly inhibited in Pik3c3 hypomorphic mice. In conclusion, the present study reports a Pik3c3 hypomorphic mouse model and provides the first definitive evidence that Pik3c3 controls the degree of compensatory nephron hypertrophy. In addition, our signaling data provide the first definitive in vivo proof that Pik3c3 functions upstream of the mTORC1-S6 kinase 1-rpS6 pathway in the regulation of compensatory nephron hypertrophy.


Assuntos
Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Néfrons/patologia , Animais , Classe III de Fosfatidilinositol 3-Quinases/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Hipertrofia , Íntrons/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Mutagênese Insercional , Nefrectomia , Néfrons/metabolismo , Transdução de Sinais/fisiologia
2.
Curr Diabetes Rev ; 16(7): 716-732, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31951170

RESUMO

BACKGROUND: The discovery of Sodium-Glucose co-transporter-2 (SGLT2) inhibitors had rewritten the treatment of diabetes mellitus with an impressive fall in the incidence of death and associated complications. INTRODUCTION: The SGLT2 inhibitors by inhibiting the SGLT2 in the proximal nephron, helps in reducing the reabsorption of approximately 90% of the filtered glucose and increased urinary glucose excretion (UGE). METHODS: The literature related to SGLT2 inhibitors has been thoroughly explored from various available public domains and reviewed extensively for this article. Detailed and updated information related to SGLT2 inhibitors with a major focus on the recently approved Ertuglifolzin is structured in this review. RESULT: The present review is an effort to understand the management of diabetes mellitus over the past few decades with a special focus on the role of SGLT2 receptor in the causes of therapeutic and preventive strategies for diabetes mellitus. Pragmatic placement of the currently available Canagliflozin, Dapagliflozin, and Empagliflozin as oral antidiabetic agents has been done. Well accommodated stereochemistry and a high docking score of Ertugliflozin in ligand-receptor simulation studies attribute to its high potency. CONCLUSION: This review highlights the unique mechanism of SGLT2 Inhibitors coupled with pleiotropic benefits on weight and blood pressure, which make it an attractive choice of therapy to diabetic patients, not controlled by other medications.


Assuntos
Diabetes Mellitus/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Néfrons/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Diabetes Mellitus/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Humanos , Hipoglicemiantes/farmacologia , Néfrons/metabolismo , Reabsorção Renal/efeitos dos fármacos , Reabsorção Renal/fisiologia , Transportador 2 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia
3.
Am J Physiol Renal Physiol ; 318(1): F183-F192, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31760771

RESUMO

Angiotensin II exacerbates oxidative stress in part by increasing superoxide (O2-) production by many renal tissues. However, whether it does so in proximal tubules and the source of O2- in this segment are unknown. Dietary fructose enhances the stimulatory effect of angiotensin II on proximal tubule Na+ reabsorption, but whether this is true for oxidative stress is unknown. We hypothesized that angiotensin II causes proximal nephron oxidative stress in part by stimulating NADPH oxidase (NOX)4-dependent O2- production and decreasing the amount of the antioxidant glutathione, and this is exacerbated by dietary fructose. We measured basal and angiotensin II-stimulated O2- production with and without inhibitors, NOX1 and NOX4 expression, and total and reduced glutathione (GSH) in proximal tubules from rats drinking either tap water (control) or 20% fructose. Angiotensin II (10 nM) increased O2- production by 113 ± 42 relative light units·mg protein-1·s-1 in controls and 401 ± 74 relative light units·mg protein-1·s-1 with 20% fructose (n = 11 for each group, P < 0.05 vs. control). Apocynin and the Nox1/4 inhibitor GKT136901 prevented angiotensin II-induced increases in both groups. NOX4 expression was not different between groups. NOX1 expression was undetectable. Angiotensin II decreased GSH by 1.8 ± 0.8 nmol/mg protein in controls and by 4.2 ± 0.9 nmol/mg protein with 20% fructose (n = 18 for each group, P < 0.047 vs. control). We conclude that 1) angiotensin II causes oxidative stress in proximal tubules by increasing O2- production by NOX4 and decreasing GSH and 2) dietary fructose enhances the ability of angiotensin II to stimulate O2- and diminish GSH, thereby exacerbating oxidative stress in this segment.


Assuntos
Angiotensina II/farmacologia , Glutationa/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Superóxidos/metabolismo , Acetofenonas/farmacologia , Animais , Antioxidantes/farmacologia , Açúcares da Dieta , Frutose , Túbulos Renais Proximais/metabolismo , Masculino , NADPH Oxidases/metabolismo , Néfrons/efeitos dos fármacos , Néfrons/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pirazóis/farmacologia , Piridonas/farmacologia , Ratos , Ratos Sprague-Dawley
4.
Nat Rev Nephrol ; 16(2): 77-98, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31554933

RESUMO

Iron is an essential element that is indispensable for life. The delicate physiological body iron balance is maintained by both systemic and cellular regulatory mechanisms. The iron-regulatory hormone hepcidin assures maintenance of adequate systemic iron levels and is regulated by circulating and stored iron levels, inflammation and erythropoiesis. The kidney has an important role in preventing iron loss from the body by means of reabsorption. Cellular iron levels are dependent on iron import, storage, utilization and export, which are mainly regulated by the iron response element-iron regulatory protein (IRE-IRP) system. In the kidney, iron transport mechanisms independent of the IRE-IRP system have been identified, suggesting additional mechanisms for iron handling in this organ. Yet, knowledge gaps on renal iron handling remain in terms of redundancy in transport mechanisms, the roles of the different tubular segments and related regulatory processes. Disturbances in cellular and systemic iron balance are recognized as causes and consequences of kidney injury. Consequently, iron metabolism has become a focus for novel therapeutic interventions for acute kidney injury and chronic kidney disease, which has fuelled interest in the molecular mechanisms of renal iron handling and renal injury, as well as the complex dynamics between systemic and local cellular iron regulation.


Assuntos
Lesão Renal Aguda/metabolismo , Proteínas Reguladoras do Ferro/metabolismo , Ferro/metabolismo , Rim/metabolismo , Insuficiência Renal Crônica/metabolismo , Eritropoese , Eritropoetina/metabolismo , Homeostase , Humanos , Inflamação/metabolismo , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/metabolismo , Mitocôndrias/metabolismo , Néfrons/metabolismo , Estresse Oxidativo
5.
Nature ; 577(7788): 121-126, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31853060

RESUMO

Modifications of histone proteins have essential roles in normal development and human disease. Recognition of modified histones by 'reader' proteins is a key mechanism that mediates the function of histone modifications, but how the dysregulation of these readers might contribute to disease remains poorly understood. We previously identified the ENL protein as a reader of histone acetylation via its YEATS domain, linking it to the expression of cancer-driving genes in acute leukaemia1. Recurrent hotspot mutations have been found in the ENL YEATS domain in Wilms tumour2,3, the most common type of paediatric kidney cancer. Here we show, using human and mouse cells, that these mutations impair cell-fate regulation by conferring gain-of-function in chromatin recruitment and transcriptional control. ENL mutants induce gene-expression changes that favour a premalignant cell fate, and, in an assay for nephrogenesis using murine cells, result in undifferentiated structures resembling those observed in human Wilms tumour. Mechanistically, although bound to largely similar genomic loci as the wild-type protein, ENL mutants exhibit increased occupancy at a subset of targets, leading to a marked increase in the recruitment and activity of transcription elongation machinery that enforces active transcription from target loci. Furthermore, ectopically expressed ENL mutants exhibit greater self-association and form discrete and dynamic nuclear puncta that are characteristic of biomolecular hubs consisting of local high concentrations of regulatory factors. Such mutation-driven ENL self-association is functionally linked to enhanced chromatin occupancy and gene activation. Collectively, our findings show that hotspot mutations in a chromatin-reader domain drive self-reinforced recruitment, derailing normal cell-fate control during development and leading to an oncogenic outcome.


Assuntos
Linhagem da Célula , Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Mutação com Ganho de Função , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Camundongos , Néfrons/metabolismo , Néfrons/patologia , Fatores de Transcrição/química , Fatores de Transcrição/genética
6.
Am J Physiol Renal Physiol ; 317(6): F1656-F1668, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31657247

RESUMO

Angiotensin II (ANG II) raises blood pressure partly by stimulating tubular Na+ reabsorption. The effects of ANG II on tubular Na+ transporters (i.e., channels, pumps, cotransporters, and exchangers) vary between short-term and long-term exposure. To better understand the physiological impact, we used a computational model of transport along the rat nephron to predict the effects of short- and long-term ANG II-induced transporter activation on Na+ and K+ reabsorption/secretion, and to compare measured and calculated excretion rates. Three days of ANG II infusion at 200 ng·kg-1·min-1 is nonpressor, yet stimulates transporter accumulation. The increase in abundance of Na+/H+ exchanger 3 (NHE3) or activated Na+-K+-2Cl- cotransporter-2 (NKCC2-P) predicted significant reductions in urinary Na+ excretion, yet there was no observed change in urine Na+. The lack of antinatriuresis, despite Na+ transporter accumulation, was supported by Li+ and creatinine clearance measurements, leading to the conclusion that 3-day nonpressor ANG II increases transporter abundance without proportional activation. Fourteen days of ANG II infusion at 400 ng·kg-1·min-1 raises blood pressure and increases Na+ transporter abundance along the distal nephron; proximal tubule and medullary loop transporters are decreased and urine Na+ and volume output are increased, evidence for pressure natriuresis. Simulations indicate that decreases in NHE3 and NKCC2-P contribute significantly to reducing Na+ reabsorption along the nephron and to pressure natriuresis. Our results also suggest that differential regulation of medullary (decrease) and cortical (increase) NKCC2-P is important to preserve K+ while minimizing Na+ retention during ANG II infusion. Lastly, our model indicates that accumulation of active Na+-Cl- cotransporter counteracts epithelial Na+ channel-induced urinary K+ loss.


Assuntos
Angiotensina II/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Néfrons/metabolismo , Sódio/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Simulação por Computador , Creatinina/metabolismo , Canais Epiteliais de Sódio , Medula Renal/efeitos dos fármacos , Medula Renal/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Lítio/urina , Masculino , Natriurese/efeitos dos fármacos , Potássio/metabolismo , Ratos , Sódio/urina , Trocador 3 de Sódio-Hidrogênio/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/metabolismo
7.
Am J Physiol Renal Physiol ; 317(6): F1462-F1474, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31566436

RESUMO

The goal of the present study was to investigate the functional implications of sexual dimorphism in the pattern of transporters along the rodent nephron as reported by Veiras et al. (J Am Soc Nephrol 28: 3504-3517, 2017). To do so, we developed sex-specific computational models of water and solute transport along the superficial nephrons from male and female rat kidneys. The models account for the sex differences in the abundance of apical and basolateral transporters, single nephron glomerular filtration rate, and tubular dimensions. Model simulations predict that ~70% and 60% of filtered Na+ is reabsorbed by the proximal tubule of male and female rat kidneys, respectively. The lower fractional Na+ reabsorption in female kidneys is due primarily to their smaller transport area, lower Na+/H+ exchanger activity, and lower claudin-2 abundance, culminating in significantly larger fractional delivery of water and Na+ to the downstream nephron segments in female kidneys. Conversely, the female distal nephron exhibits a higher abundance of key Na+ transporters, including Na+-K+-Cl- cotransporters, Na+-Cl- cotransporters, and epithelial Na+ channels. The higher abundance of transporters accounts for the enhanced water and Na+ transport along the female, relative to male, distal nephron, resulting in similar urine excretion between the sexes. Consequently, in response to a saline load, the Na+ load delivered distally is greater in female rats than male rats, overwhelming transport capacity and resulting in higher natriuresis in female rats.


Assuntos
Proteínas de Transporte/metabolismo , Néfrons/metabolismo , Animais , Claudinas/metabolismo , Feminino , Taxa de Filtração Glomerular , Túbulos Renais/metabolismo , Masculino , Modelos Biológicos , Ratos , Caracteres Sexuais , Sódio/metabolismo , Sódio/urina , Canais de Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Água/metabolismo
8.
Eur J Histochem ; 63(3)2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31544449

RESUMO

Nephron progenitors (NPs) and nephrogenesis have been extensively studied in mice and humans and have provided insights into the mechanisms of renal development, disease and possibility of NP-based therapies. However, molecular features of NPs and their derivatives in the canine fetal kidney (CFK) remain unknown. This study was focused to characterize the expression of potential markers of canine NPs and their derivatives by immuno-fluorescence and western blot analysis. Transcription factors (TFs) SIX1 and SIX2, well-characterized human NP markers, were expressed in NPs surrounding the ureteric bud in the CFK. Canine NPs also expressed ITGA8 and NCAM1, surface markers previously used to isolate NPs from the mouse and human fetal kidneys. TF, PAX2 was detected in the ureteric bud, NPs and their derivative structures such as renal vesicle and S-shaped body. This study highlights the similarities in dog, mouse and human renal development and characterizes markers to identify canine NPs and their derivatives. These results will facilitate the isolation of canine NPs and their functional characterization to develop NP-based therapies for canine renal diseases.


Assuntos
Células Epiteliais/metabolismo , Néfrons/metabolismo , Fatores de Transcrição/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Cães/embriologia , Células Epiteliais/citologia , Feminino , Imunofluorescência , Néfrons/citologia , Gravidez
9.
Commun Biol ; 2: 326, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31508501

RESUMO

The kidney's inherent complexity has made identifying cell-specific pathways challenging, particularly when temporally associating them with the dynamic pathophysiology of acute kidney injury (AKI). Here, we combine renal cell-specific luciferase reporter mice using a chemoselective luciferin to guide the acquisition of cell-specific transcriptional changes in C57BL/6 background mice. Hydrogen peroxide generation, a common mechanism of tissue damage, was tracked using a peroxy-caged-luciferin to identify optimum time points for immunoprecipitation of labeled ribosomes for RNA-sequencing. Together, these tools revealed a profound impact of AKI on mitochondrial pathways in the collecting duct. In fact, targeting the mitochondria with an antioxidant, ameliorated not only hydrogen peroxide generation, but also significantly reduced oxidative stress and the expression of the AKI biomarker, LCN2. This integrative approach of coupling physiological imaging with transcriptomics and drug testing revealed how the collecting duct responds to AKI and opens new venues for cell-specific predictive monitoring and treatment.


Assuntos
Lesão Renal Aguda/genética , Imageamento Tridimensional , Isquemia/genética , Isquemia/patologia , Transcriptoma/genética , Lesão Renal Aguda/complicações , Lesão Renal Aguda/patologia , Animais , Antioxidantes/metabolismo , Túbulos Renais Coletores/lesões , Túbulos Renais Coletores/patologia , Camundongos Endogâmicos C57BL , Néfrons/metabolismo , Néfrons/patologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia
10.
Am J Physiol Renal Physiol ; 317(4): F957-F966, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31432707

RESUMO

Toll-like receptor 4 (TLR4) activation contributes to vascular dysfunction in pathological conditions such as hypertension and diabetes, but the role of chronic TLR4 activation on renal autoregulatory behavior is unknown. We hypothesized that subclinical TLR4 stimulation with low-dose lipopolysaccharide (LPS) infusion increases TLR4 activation and blunts renal autoregulatory behavior. We assessed afferent arteriolar autoregulatory behavior in male Sprague-Dawley rats after prolonged LPS (0.1 mg·kg-1·day-1 sq) infusion via osmotic minipump for 8 or 14 days. Some rats also received daily cotreatment with either anti-TLR4 antibody (1 µg ip), competitive antagonist peptide (CAP; 3 mg/kg ip) or tempol (2 mmol/l, drinking water) throughout the 8-day LPS treatment period. Autoregulatory behavior was assessed using the in vitro blood-perfused juxtamedullary nephron preparation. Selected physiological measures, systolic blood pressure and baseline diameters were normal and similar across groups. Pressure-dependent vasoconstriction averaged 72 ± 2% of baseline in sham rats, indicating intact autoregulatory behavior. Eight-day LPS-treated rats exhibited significantly impaired pressure-mediated vasoconstriction (96 ± 1% of baseline), whereas it was preserved in rats that received anti-TLR4 antibody (75 ± 3%), CAP (84 ± 2%), or tempol (82 ± 2%). Using a 14-day LPS (0.1 mg·kg-1·day-1 sq) intervention protocol, CAP treatment started on day 7, where autoregulatory behavior is already impaired. Systolic blood pressures were normal across all treatment groups. Fourteen-day LPS treatment retained the autoregulatory impairment (95 ± 2% of baseline). CAP intervention starting on day 7 rescued pressure-mediated vasoconstriction with diameters decreasing to 85 ± 1% of baseline. These data demonstrate that chronic subclinical TLR4 activation impairs afferent arteriolar autoregulatory behavior through mechanisms involving reactive oxygen species and major histocompatibility complex class II activation.


Assuntos
Antígenos de Histocompatibilidade Classe II/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Circulação Renal/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Masculino , Néfrons/efeitos dos fármacos , Néfrons/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Marcadores de Spin , Receptor 4 Toll-Like/antagonistas & inibidores , Vasoconstrição/efeitos dos fármacos
11.
Am J Physiol Renal Physiol ; 317(6): F1475-F1482, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31461349

RESUMO

Randall's plaque (RP; subepithelial calcification) appears to be an important precursor of kidney stone disease. However, RP cannot be noninvasively detected. The present study investigated candidate biomarkers associated with extracellular vesicles (EVs) in the urine of calcium stone formers (CSFs) with low (<5% papillary surface area) and high (≥5% papillary surface area) percentages of RP and a group of nonstone formers. RPs were quantitated via videotaping and image processing in consecutive CSFs undergoing percutaneous surgery for stone removal. Urinary EVs derived from cells of different nephron segments of CSFs (n = 64) and nonstone formers (n = 40) were quantified in biobanked cell-free urine by standardized and validated digital flow cytometer using fluorophore-conjugated antibodies. Overall, the number of EVs carrying surface monocyte chemoattractant protein (MCP)-1 and neutrophil gelatinase-associated lipocalin (NGAL) were significantly lower in CSFs compared with nonstone former controls (P < 0.05) but did not differ statistically between CSFs with low and high RPs. The number of EVs associated with osteopontin did not differ between any groups. Thus, EVs carrying MCP-1 and NGAL may directly or indirectly contribute to stone pathogenesis as evidenced by the lower of these populations of EVs in stone formers compared with nonstone formers. Validation of EV-associated MCP-1 and NGAL as noninvasive biomarkers of kidney stone pathogenesis in larger populations is warranted.


Assuntos
Oxalato de Cálcio , Cálculos Renais/metabolismo , Lipocalina-2/urina , Néfrons/metabolismo , Adulto , Biomarcadores/urina , Quimiocina CCL2/urina , Espaço Extracelular/metabolismo , Feminino , Humanos , Masculino , Osteopontina/urina
12.
Int J Mol Sci ; 20(17)2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31450703

RESUMO

Mitogen-activated protein kinases (MAPKs) are intracellular molecules regulating a wide range of cellular functions, including proliferation, differentiation, apoptosis, cytoskeleton remodeling and cytokine production. MAPK activity has been shown in normal kidney, and its over-activation has been demonstrated in several renal diseases. The extracellular signal-regulated protein kinases (ERK 1,2) signalling pathway is the first described MAPK signaling. Intensive investigations have demonstrated that it participates in the regulation of ureteric bud branching, a fundamental process in establishing final nephron number; in addition, it is also involved in the differentiation of the nephrogenic mesenchyme, indicating a key role in mammalian kidney embryonic development. In the present manuscript, we show that ERK1,2 signalling mediates several cellular functions also in mature kidney, describing its role along the nephron and demonstrating whether it contributes to the regulation of ion channels and transporters implicated in acid-base and electrolytes homeostasis.


Assuntos
Equilíbrio Ácido-Base , Eletrólitos/metabolismo , Sistema de Sinalização das MAP Quinases , Néfrons/metabolismo , Equilíbrio Hidroeletrolítico , Animais , Suscetibilidade a Doenças , Humanos , Túbulos Renais Coletores/metabolismo , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/metabolismo , Alça do Néfron/metabolismo
13.
Stem Cell Reports ; 13(2): 322-337, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31378669

RESUMO

Kidney formation is regulated by the balance between maintenance and differentiation of nephron progenitor cells (NPCs). Now that directed differentiation of NPCs from human induced pluripotent stem cells (iPSCs) can be achieved, maintenance and propagation of NPCs in vitro should be beneficial for regenerative medicine. Although WNT and FGF signals were previously shown to be essential for NPC propagation, the requirement for BMP/TGFß signaling remains controversial. Here we reveal that activin has superior effects to BMP7 on maintenance efficiency of human iPSC-derived NPCs. Activin expanded ITGA8+/PDGFRA-/SIX2-GFP+ NPCs by 5-fold per week at 80%-90% efficiency, and the propagated cells possessed robust capacity for nephron formation both in vitro and in vivo. The expanded cells also maintained their nephron-forming potential after freezing. Furthermore, the protocol was applicable to multiple non-GFP-tagged iPSC lines. Thus, our activin-based protocol will be applicable to a variety of research fields including disease modeling and drug screening.


Assuntos
Ativinas/farmacologia , Proteína Morfogenética Óssea 7/farmacologia , Proliferação de Células/efeitos dos fármacos , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular , Reprogramação Celular , Edição de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Néfrons/citologia , Néfrons/metabolismo , Néfrons/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Podócitos/metabolismo , Podócitos/patologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo
14.
Genet Med ; 21(12): 2755-2764, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31263215

RESUMO

PURPOSE: Haploinsufficiency of DYRK1A causes a recognizable clinical syndrome. The goal of this paper is to investigate congenital anomalies of the kidney and urinary tract (CAKUT) and genital defects (GD) in patients with DYRK1A variants. METHODS: A large database of clinical exome sequencing (ES) was queried for de novo DYRK1A variants and CAKUT/GD phenotypes were characterized. Xenopus laevis (frog) was chosen as a model organism to assess Dyrk1a's role in renal development. RESULTS: Phenotypic details and variants of 19 patients were compiled after an initial observation that one patient with a de novo pathogenic variant in DYRK1A had GD. CAKUT/GD data were available from 15 patients, 11 of whom presented with CAKUT/GD. Studies in Xenopus embryos demonstrated that knockdown of Dyrk1a, which is expressed in forming nephrons, disrupts the development of segments of embryonic nephrons, which ultimately give rise to the entire genitourinary (GU) tract. These defects could be rescued by coinjecting wild-type human DYRK1A RNA, but not with DYRK1AR205* or DYRK1AL245R RNA. CONCLUSION: Evidence supports routine GU screening of all individuals with de novo DYRK1A pathogenic variants to ensure optimized clinical management. Collectively, the reported clinical data and loss-of-function studies in Xenopus substantiate a novel role for DYRK1A in GU development.


Assuntos
Deficiência Intelectual/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Anormalidades Urogenitais/genética , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Bases de Dados Genéticas , Modelos Animais de Doenças , Exoma/genética , Feminino , Haploinsuficiência/genética , Humanos , Deficiência Intelectual/complicações , Rim/anormalidades , Rim/embriologia , Masculino , Néfrons/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sistema Urinário/embriologia , Sistema Urinário/metabolismo , Sequenciamento Completo do Exoma/métodos , Xenopus laevis/genética , Xenopus laevis/metabolismo , Adulto Jovem
15.
Dev Biol ; 454(2): 156-169, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31242448

RESUMO

Adamts18 encodes a secreted metalloprotease restricted to branch-tip progenitor pools directing the morphogenesis of multiple mammalian organs. Adamts18 was targeted to explore a potential role in branching morphogenesis. In the kidney, an arborized collecting system develops through extensive branching morphogenesis of an initial epithelial outgrowth of the mesonephric duct, the ureteric bud. Adamts18 mutants displayed a weakly penetrant phenotype: duplicated ureteric outgrowths forming enlarged, bi-lobed kidneys with an increased nephron endowment. In contrast, Adamts18 mutants showed a fully penetrant lung phenotype: epithelial growth was markedly reduced and early secondary branching scaled to the reduced length of the primary airways. Furthermore, there was a pronounced delay in the appearance of differentiated cell types in both proximal and distally positions of the developing airways. Adamts18 is closely related to Adamts16. In the kidney but not the lung, broad epithelial Adamts16 expression overlaps Adamts18 in branch tips. However, compound Adamts16/18 mutants displayed a comparable low penetrance duplicated ureteric phenotype, ruling out a possible role for Adamts16 as a functional modifier of the Adamts18 kidney phenotype. Given the predicted action of secreted Adamts18 metalloprotease, and broad expression of Adamts18 in branching organ systems, these findings suggest distinct requirements for matrix modelling in the morphogenesis of epithelial networks.


Assuntos
Proteínas ADAMTS/metabolismo , Organogênese/fisiologia , Proteínas ADAMTS/fisiologia , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Rim/citologia , Rim/embriologia , Rim/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Masculino , Metaloproteases/genética , Metaloproteases/metabolismo , Camundongos , Camundongos Knockout , Morfogênese , Néfrons/metabolismo , Técnicas de Cultura de Órgãos/métodos , Ureter/metabolismo
16.
Dev Biol ; 454(1): 44-51, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31220433

RESUMO

The zebrafish kidney regenerates after injury by development of new nephrons from resident adult kidney stem cells. Although adult kidney progenitor cells have been characterized by transplantation and single cell RNA seq, signals that stimulate new nephron formation are not known. Here we demonstrate that fibroblast growth factors and FGF signaling is rapidly induced after kidney injury and that FGF signaling is required for recruitment of progenitor cells to sites of new nephron formation. Chemical or dominant negative blockade of Fgfr1 prevented formation of nephron progenitor cell aggregates after injury and during kidney development. Implantation of FGF soaked beads induced local aggregation of lhx1a:EGFP  â€‹+ â€‹kidney progenitor cells. Our results reveal a previously unexplored role for FGF signaling in recruitment of renal progenitors to sites of new nephron formation and suggest a role for FGF signaling in maintaining cell adhesion and cell polarity in newly forming kidney epithelia.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Néfrons/metabolismo , Células-Tronco/citologia , Células-Tronco Adultas/metabolismo , Animais , Agregação Celular/fisiologia , Rim/citologia , Rim/metabolismo , Organogênese , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
17.
Gene Expr Patterns ; 33: 1-10, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31055152

RESUMO

UNC119 proteins are required for ciliary trafficking in a process called lipidated protein intraflagellar targeting (LIFT) or through vesicle transport. However, although unc119 has been studied in a variety of contexts, either organismal constraints or genetic redundancy has largely restricted their study in ciliary contexts. One possible solution for this is to use the zebrafish, however, the unc119 genes have not been well described in this species. In our study, we show in a condensed species tree that the presence of unc119 genes correlates with the presence of cilia across eukaryotes and that phylogenetic evidence suggests there are three subgroups of UNC119 proteins. Zebrafish contain all three of these subgroups: two vertebrate-specific UNC119A proteins, one vertebrate-specific UNC119B protein, and one UNC119. Expression analyses show that each of the zebrafish unc119 genes are maternally-expressed and have overlapping but distinct expression in ciliated tissues, such as the eye, pronephric duct, and spinal cord. Overall, these findings set the foundation for future studies into the use of the zebrafish to study unc119 gene knock-outs, particularly from a ciliary perspective.


Assuntos
Cílios/metabolismo , Sequência Conservada , Evolução Molecular , Proteínas de Peixe-Zebra/genética , Animais , Olho/metabolismo , Néfrons/metabolismo , Filogenia , Medula Espinal/metabolismo , Sintenia , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo
18.
Development ; 146(8)2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036548

RESUMO

Zebrafish kidneys use resident kidney stem cells to replace damaged tubules with new nephrons: the filtration units of the kidney. What stimulates kidney progenitor cells to form new nephrons is not known. Here, we show that wnt9a and wnt9b are induced in the injured kidney at sites where frizzled9b- and lef1-expressing progenitor cells form new nephrons. New nephron aggregates are patterned by Wnt signaling, with high canonical Wnt-signaling cells forming a single cell thick rosette that demarcates: domains of cell proliferation in the elongating nephron; and tubule fusion where the new nephron plumbs into the distal tubule and establishes blood filtrate drainage. Pharmacological blockade of canonical Wnt signaling inhibited new nephron formation after injury by inhibiting cell proliferation, and resulted in loss of polarized rosette structures in the aggregates. Mutation in frizzled9b reduced total kidney nephron number, caused defects in tubule morphology and reduced regeneration of new nephrons after injury. Our results demonstrate an essential role for Wnt/frizzled signaling in adult zebrafish kidney development and regeneration, highlighting conserved mechanisms underlying both mammalian kidney development and kidney stem cell-directed neonephrogenesis in zebrafish.


Assuntos
Rim/citologia , Rim/metabolismo , Néfrons/citologia , Néfrons/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Regeneração/fisiologia , Via de Sinalização Wnt/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
19.
Am J Physiol Regul Integr Comp Physiol ; 316(5): R640-R650, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30943054

RESUMO

Formation of the metanephric kidney requires coordinated interaction among the stroma, ureteric bud, and cap mesenchyme. The transcription factor Foxd1, a specific marker of renal stromal cells, is critical for normal kidney development. The prorenin receptor (PRR), a receptor for renin and prorenin, is also an accessory subunit of the vacuolar proton pump V-ATPase. Global loss of PRR is embryonically lethal in mice, indicating an essential role of the PRR in embryonic development. Here, we report that conditional deletion of the PRR in Foxd1+ stromal progenitors in mice (cKO) results in neonatal mortality. The kidneys of surviving mice show reduced expression of stromal markers Foxd1 and Meis1 and a marked decrease in arterial and arteriolar development with the subsequent decreased number of glomeruli, expansion of Six2+ nephron progenitors, and delay in nephron differentiation. Intrarenal arteries and arterioles in cKO mice were fewer and thinner and showed a marked decrease in the expression of renin, suggesting a central role for the PRR in the development of renin-expressing cells, which in turn are essential for the proper formation of the renal arterial tree. We conclude that stromal PRR is crucial for the appropriate differentiation of the renal arterial tree, which in turn may restrict excessive expansion of nephron progenitors to promote a coordinated and proper morphogenesis of the nephrovascular structures of the mammalian kidney.


Assuntos
Rim/crescimento & desenvolvimento , Néfrons/metabolismo , Organogênese/fisiologia , Receptores de Superfície Celular/metabolismo , Animais , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Rim/metabolismo , Camundongos Transgênicos , Renina/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
20.
Am J Physiol Renal Physiol ; 316(5): F993-F1005, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30838872

RESUMO

We have previously demonstrated that loss of miR-17~92 in nephron progenitors in a mouse model results in renal hypodysplasia and chronic kidney disease. Clinically, decreased congenital nephron endowment because of renal hypodysplasia is associated with an increased risk of hypertension and chronic kidney disease, and this is at least partly dependent on the self-renewal of nephron progenitors. Here, we present evidence for a novel molecular mechanism regulating the self-renewal of nephron progenitors and congenital nephron endowment by the highly conserved miR-17~92 cluster. Whole transcriptome sequencing revealed that nephron progenitors lacking this cluster demonstrated increased Cftr expression. We showed that one member of the cluster, miR-19b, is sufficient to repress Cftr expression in vitro and that perturbation of Cftr activity in nephron progenitors results in impaired proliferation. Together, these data suggest that miR-19b regulates Cftr expression in nephron progenitors, with this interaction playing a role in appropriate nephron progenitor self-renewal during kidney development to generate normal nephron endowment.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , MicroRNAs/metabolismo , Néfrons/metabolismo , Células-Tronco/metabolismo , Animais , Movimento Celular , Proliferação de Células , Autorrenovação Celular , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Néfrons/embriologia , Organogênese , Transdução de Sinais
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