RESUMEN
The collagen IVα345 (Col-IVα345) scaffold, the major constituent of the glomerular basement membrane (GBM), is a critical component of the kidney glomerular filtration barrier. In Alport syndrome, affecting millions of people worldwide, over two thousand genetic variants occur in the COL4A3, COL4A4, and COL4A5 genes that encode the Col-IVα345 scaffold. Variants cause loss of scaffold, a suprastructure that tethers macromolecules, from the GBM or assembly of a defective scaffold, causing hematuria in nearly all cases, proteinuria, and often progressive kidney failure. How these variants cause proteinuria remains an enigma. In a companion paper, we found that the evolutionary emergence of the COL4A3, COL4A4, COL4A5, and COL4A6 genes coincided with kidney emergence in hagfish and shark and that the COL4A3 and COL4A4 were lost in amphibians. These findings opened an experimental window to gain insights into functionality of the Col-IVα345 scaffold. Here, using tissue staining, biochemical analysis and TEM, we characterized the scaffold chain arrangements and the morphology of the GBM of hagfish, shark, frog, and salamander. We found that α4 and α5 chains in shark GBM and α1 and α5 chains in amphibian GBM are spatially separated. Scaffolds are distinct from one another and from the mammalian Col-IVα345 scaffold, and the GBM morphologies are distinct. Our findings revealed that the evolutionary emergence of the Col-IVα345 scaffold enabled the genesis of a compact GBM that functions as an ultrafilter. Findings shed light on the conundrum, defined decades ago, whether the GBM or slit diaphragm is the primary filter.
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Colágeno Tipo IV , Membrana Basal Glomerular , Mamíferos , Animales , Anuros , Colágeno Tipo IV/clasificación , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Membrana Basal Glomerular/química , Membrana Basal Glomerular/metabolismo , Membrana Basal Glomerular/fisiología , Anguila Babosa , Mamíferos/genética , Mamíferos/metabolismo , Mamíferos/fisiología , Tiburones , Especificidad de la Especie , UrodelosRESUMEN
This commentary briefly reviews the background for the development of the horseradish peroxidase-diaminobenzidine tetrahydrochloride histochemical method originally described by Graham and Karnovsky in their citation classic, reprinted in full in this issue of Journal of Histochemistry & Cytochemistry. Some of the method's subsequent applications, including its use as a macromolecular tracer for kidney glomerular permeability and use in immunoelectron microscopy and other immunoassays, are also discussed.
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Peroxidasa de Rábano Silvestre , Histocitoquímica/métodosRESUMEN
Diseases of the glomerular basement membrane (GBM), such as Goodpasture's disease (GP) and Alport syndrome (AS), are a major cause of chronic kidney failure and an unmet medical need. Collagen IVα345 is an important architectural element of the GBM that was discovered in previous research on GP and AS. How this collagen enables GBM to function as a permselective filter and how structural defects cause renal failure remain an enigma. We found a distinctive genetic variant of collagen IVα345 in both a familial GP case and four AS kindreds that provided insights into these mechanisms. The variant is an 8-residue appendage at the C-terminus of the α3 subunit of the α345 hexamer. A knock-in mouse harboring the variant displayed GBM abnormalities and proteinuria. This pathology phenocopied AS, which pinpointed the α345 hexamer as a focal point in GBM function and dysfunction. Crystallography and assembly studies revealed underlying hexamer mechanisms, as described in Boudko et al. and Pedchenko et al. Bioactive sites on the hexamer surface were identified where pathogenic pathways of GP and AS converge and, potentially, that of diabetic nephropathy (DN). We conclude that the hexamer functions include signaling and organizing macromolecular complexes, which enable GBM assembly and function. Therapeutic modulation or replacement of α345 hexamer could therefore be a potential treatment for GBM diseases, and this knock-in mouse model is suitable for developing gene therapies.
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Enfermedad por Anticuerpos Antimembrana Basal Glomerular/genética , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Mutación , Nefritis Hereditaria/genética , Animales , Colágeno Tipo IV/química , Ratones , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Transducción de SeñalRESUMEN
Mammalian immune systems are not mature until well after birth. However, transfer of maternal IgG to the fetus and newborn usually provides immunoprotection from infectious diseases. IgG transfer occurs before birth in humans across the placenta and continues after birth across the intestine in many mammalian species, including rodents. Transfer, which is mediated by the neonatal IgG Fc receptor, occurs by transcytosis across placental syncytiotrophoblasts and intestinal epithelium. Although maternal IgG is generally beneficial, harmful maternal allo- and autoantibodies can also be transferred to the fetus/infant, resulting in serious disease. To test this we generated transgenic mice that widely express human laminin α5 in their basement membranes. When huLAMA5 transgenic males were crossed with wild-type females, there was a maternal anti-human laminin α5 immune response. Maternal IgG alloantibody crossed the yolk sac and post-natal intestine invivo and bound in bright, linear patterns to kidney glomerular basement membranes of transgenic fetuses/neonates but not those of wild-type siblings. By postnatal day 18, most transgenic mice were proteinuric, had glomerular C3 deposits and inflammatory cell infiltrates, thickened and split glomerular basement membranes, and podocyte foot process effacement. Thus, our novel model of perinatal anti-glomerular basement membrane disease may prove useful for studying pediatric glomerulopathies, formation of the fetomaternal interface, and maternal alloimmunization.
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Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Membrana Basal Glomerular/inmunología , Inmunoglobulina G/inmunología , Laminina/inmunología , Animales , Animales Recién Nacidos , Femenino , Membrana Basal Glomerular/ultraestructura , Humanos , Inmunidad Humoral , Masculino , Ratones Transgénicos , EmbarazoRESUMEN
Alport syndrome is caused by mutations in collagen IV that alter the morphology of renal glomerular basement membrane. Mutations result in proteinuria, tubulointerstitial fibrosis, and renal failure but the pathogenic mechanisms are not fully understood. Using imaging mass spectrometry, we aimed to determine whether the spatial and/or temporal patterns of renal lipids are perturbed during the development of Alport syndrome in the mouse model. Our results show that most sulfatides are present at similar levels in both the wild-type (WT) and the Alport kidneys, with the exception of two specific sulfatide species, SulfoHex-Cer(d18:2/24:0) and SulfoHex-Cer(d18:2/16:0). In the Alport but not in WT kidneys, the levels of these species mirror the previously described abnormal laminin expression in Alport syndrome. The presence of these sulfatides in renal tubules but not in glomeruli suggests that this specific aberrant lipid pattern may be related to the development of tubulointerstitial fibrosis in Alport disease.
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Modelos Animales de Enfermedad , Túbulos Renales/metabolismo , Nefritis Hereditaria/metabolismo , Sulfoglicoesfingolípidos/metabolismo , Animales , Túbulos Renales/patología , Metabolismo de los Lípidos , Ratones , Nefritis Hereditaria/patologíaRESUMEN
Behavioral phenotyping is a crucial step in validating animal models of human disease. Most traditional behavioral analyses rely on investigator observation of animal subjects, which can be confounded by inter-observer variability, scoring consistency, and the ability to observe extremely rapid, small, or repetitive movements. Force-Plate Actimeter (FPA)-based assessments can quantify locomotor activity and detailed motor activity with an incredibly rich data stream that can reveal details of movement unobservable by the naked eye. This report describes four specific examples of FPA analysis of behavior that have been useful in specific rat or mouse models of human neurological disease, which show how FPA analysis can be used to capture and quantify specific features of the complex behavioral phenotypes of these animal models. The first example quantifies nociceptive behavior of the rat following injection of formalin into the footpad as a common model of persistent inflammatory pain. The second uses actimetry to quantify intense, rapid circling behaviors in a transgenic mouse that overexpresses human laminin α5, a basement membrane protein. The third example assesses place preference behaviors in a rat model of migraine headache modeling phonophobia and photophobia. In the fourth example, FPA analysis revealed a unique movement signature emerged with age in a digenic mutant mouse model of Tourette Syndrome. Taken together, these approaches demonstrate the power and usefulness of the FPA in the examination and quantification of minute details of motor behaviors, greatly expanding the scope and detail of behavioral phenotyping of preclinical models of human disease.
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Movimiento/fisiología , Enfermedades del Sistema Nervioso/fisiopatología , Animales , Trastornos de Traumas Acumulados/fisiopatología , Modelos Animales de Enfermedad , Femenino , Humanos , Hiperacusia/fisiopatología , Hipercinesia/fisiopatología , Masculino , Ratones , Nocicepción/fisiología , Fotofobia/fisiopatología , RatasRESUMEN
Using a mouse model of Alport disease, Dufek et al. report that endothelial cell-derived endothelin-1 activates mesangial cells, which deposit abnormal laminin isoforms in the Alport glomerular basement membrane. This study extends findings obtained previously by this laboratory implicating mesangial cells in the early pathogenesis of Alport disease. Together with abnormalities in matrix receptor expression, cytoskeletal architecture, and proteinase secretion in podocytes, a clearer picture is emerging on the inception of proteinuria in Alport disease.
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Membrana Basal Glomerular , Nefritis Hereditaria , Animales , Endotelina-1 , Humanos , Podocitos , ProteinuriaRESUMEN
Advances in organ regeneration have been facilitated by gentle decellularization protocols that maintain distinct tissue compartments, and thereby allow seeding of blood vessels with endothelial lineages separate from populations of the parenchyma with tissue-specific cells. We hypothesized that a reconstituted vasculature could serve as a novel platform for perfusing cells derived from a different organ: thus discordance of origin between the vascular and functional cells, leading to a hybrid repurposed organ. The need for a highly vascular bed is highlighted by tissue engineering approaches that involve transplantation of just cells, as attempted for insulin production to treat human diabetes. Those pancreatic islet cells present unique challenges since large numbers are needed to allow the cell-to-cell signaling required for viability and proper function; however, increasing their number is limited by inadequate perfusion and hypoxia. As proof of principle of the repurposed organ methodology we harnessed the vasculature of a kidney scaffold while seeding the collecting system with insulin-producing cells. Pig kidneys were decellularized by sequential detergent, enzymatic and rinsing steps. Maintenance of distinct vascular and collecting system compartments was demonstrated by both fluorescent 10 micron polystyrene microspheres and cell distributions in tissue sections. Sterilized acellular scaffolds underwent seeding separately via the artery (fibroblasts or endothelioma cells) and retrograde (murine ßTC-tet cells) up the ureter. After three-day bioreactor incubation, histology confirmed separation of cells in the vasculature from those in the collecting system. ßTC-tet clusters survived in tubules, glomerular Bowman's space, demonstrated insulin immunolabeling, and thereby supported the feasibility of kidney-to-pancreas repurposing.
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Bioprótesis , Riñón/crecimiento & desarrollo , Riñones Artificiales , Páncreas Artificial , Ingeniería de Tejidos/instrumentación , Andamios del Tejido , Animales , Diseño de Equipo , Análisis de Falla de Equipo , Equipo Reutilizado , Riñón/citología , Ratones , PorcinosRESUMEN
The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex-mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex-mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG.
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Complejo Antígeno-Anticuerpo/fisiología , Glomerulonefritis/etiología , Antígenos de Histocompatibilidad Clase I/fisiología , Receptores Fc/fisiología , Albuminuria/etiología , Albuminuria/metabolismo , Animales , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/etiología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/metabolismo , Complejo Antígeno-Anticuerpo/efectos adversos , Autoantígenos/fisiología , Colágeno Tipo IV/fisiología , Glomerulonefritis/inmunología , Glomerulonefritis/metabolismo , Células HEK293 , Humanos , Inmunoglobulina G/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Kidney glomerular basement membranes (GBMs) undergo laminin and type IV collagen isoform substitutions during glomerular development, which are believed to be required for maturation of the filtration barrier. Specifically, GBMs of earliest glomeruli contain laminin α1ß1γ1 and collagen α1α2α1(IV), whereas mature glomeruli contain laminin α5ß2γ1 and collagen α3α4α5(IV). Here, we used confocal microscopy to simultaneously evaluate expression of different laminin and collagen IV isoforms in newborn mouse GBMs. Our results show loss of laminin α1 from GBMs in early capillary loop stages and continuous linear deposition of laminin bearing the α5 chain thereafter. In contrast, collagen α1α2α1(IV) persisted in linear patterns into late capillary loop stages, when collagen α3α4α5(IV) first appeared in discontinuous, non-linear patterns. This patchy pattern for collagen α3α4α5(IV) continued into maturing glomeruli where there were lengths of linear, laminin α5-positive GBM entirely lacking either isoform of collagen IV. Relative abundance of laminin and collagen IV mRNAs in newborn and 5-week-old mouse kidneys also differed, with those encoding laminin α1, α5, ß1, ß2, and γ1, and collagen α1(IV) and α2(IV) chains all significantly declining at 5 weeks, but α3(IV) and α4(IV) were significantly upregulated. We conclude that different biosynthetic mechanisms control laminin and type IV collagen expression in developing glomeruli.
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Membrana Basal/metabolismo , Colágeno Tipo IV/metabolismo , Glomérulos Renales/citología , Glomérulos Renales/crecimiento & desarrollo , Laminina/metabolismo , Animales , Ratones , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Análisis Espacio-TemporalRESUMEN
Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated â¼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM.
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Autoantígenos/genética , Colágeno Tipo IV/genética , Integrina alfa1/metabolismo , Integrina alfa3/metabolismo , Células Mesangiales/metabolismo , Podocitos/metabolismo , Regulación hacia Arriba , Vimentina/metabolismo , Animales , Autoantígenos/metabolismo , Colágeno Tipo IV/metabolismo , Modelos Animales de Enfermedad , Membrana Basal Glomerular/metabolismo , Membrana Basal Glomerular/patología , Integrina alfa1/genética , Integrina alfa3/genética , Células Mesangiales/patología , Ratones , Ratones Noqueados , Nefritis Hereditaria/genética , Nefritis Hereditaria/metabolismo , Podocitos/patología , Vimentina/genéticaRESUMEN
This article summarizes the basic cellular and extracellular events in the development of the glomerulus and assembly of the glomerular basement membrane (GBM), paying special attention to laminin (LM) and type IV collagen. Cellular receptors for GBM proteins, including the integrins, dystroglycan, and discoidin domain receptor 1 also are discussed. Evidence is reviewed showing that the laminin isoform present in the earliest GBM, LM-111, and final isoform found in the mature GBM, LM-521, are each derived from both endothelial cells and podocytes. Although the early collagen α1α2α1(IV) similarly derives from endothelial cells and podocytes, collagen α3α4α5(IV) found in fully mature GBM is a product solely of podocytes. Genetic diseases affecting laminin and type IV collagen synthesis also are presented, with an emphasis on mutations to LAMB2 (Pierson syndrome) and COL4A3, COL4A4, and COL4A5 (Alport syndrome), and their experimental mouse models. Stress is placed on the assembly of a compositionally correct GBM for the acquisition and maintenance of glomerular barrier properties.
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Colágeno Tipo IV/metabolismo , Endotelio/metabolismo , Membrana Basal Glomerular/metabolismo , Membrana Basal Glomerular/patología , Enfermedades Renales/metabolismo , Laminina/metabolismo , Podocitos/metabolismo , Animales , Colágeno Tipo IV/genética , Membrana Basal Glomerular/citología , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Enfermedades Renales/genética , Laminina/genéticaRESUMEN
Podocyte or endothelial cell VEGF-A knockout causes thrombotic microangiopathy in adult mice. To study the mechanism involved in acute and local injury caused by low podocyte VEGF-A we developed an inducible, podocyte-specific VEGF-A knockdown mouse, and we generated an immortalized podocyte cell line (VEGF(KD)) that downregulates VEGF-A upon doxycycline exposure. Tet-O-siVEGF:podocin-rtTA mice express VEGF shRNA in podocytes in a doxycycline-regulated manner, decreasing VEGF-A mRNA and VEGF-A protein levels in isolated glomeruli to ~20% of non-induced controls and urine VEGF-A to ~30% of control values a week after doxycycline induction. Induced tet-O-siVEGF:podocin-rtTA mice developed acute renal failure and proteinuria, associated with mesangiolysis and microaneurisms. Glomerular ultrastructure revealed endothelial cell swelling, GBM lamination and podocyte effacement. VEGF knockdown decreased podocyte fibronectin and glomerular endothelial alpha(V)beta(3) integrin in vivo. VEGF receptor-2 (VEGFR2) interacts with beta(3) integrin and neuropilin-1 in the kidney in vivo and in VEGF(KD) podocytes. Podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGF(KD) podocytes downregulates fibronectin and disrupts alpha(V)beta(3) integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alpha(V)beta(3) integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure.
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Técnicas de Silenciamiento del Gen , Integrina alfaVbeta3/metabolismo , Podocitos/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Doxiciclina/farmacología , Endotelio/efectos de los fármacos , Endotelio/metabolismo , Fibronectinas/metabolismo , Ratones , Modelos Animales , Neuropilina-1/metabolismo , Fenotipo , Podocitos/efectos de los fármacos , Podocitos/patología , Unión Proteica/efectos de los fármacos , Proteinuria/complicaciones , Proteinuria/metabolismo , Proteinuria/patología , Proteinuria/fisiopatología , ARN Interferente Pequeño/metabolismo , Insuficiencia Renal/complicaciones , Insuficiencia Renal/metabolismo , Insuficiencia Renal/patología , Insuficiencia Renal/fisiopatología , Transducción de Señal/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
INTRODUCTION: To address transplant organ shortage, a promising strategy is to decellularize kidneys in a manner that the scaffold retains signals for seeded pluripotent precursor cells to differentiate and recapitulate native structures: matrix-to-cell signaling followed by cell-cell and cell-matrix interactions, thereby remodeling and replacing the original matrix. This would reduce scaffold antigenicity and enable xeno-allografts. RESULTS: DAPI-labeled cells in arterial vessels and glomeruli were positive for both endothelial lineage markers, BsLB4 and VEGFR2. Rat scaffold's basement membrane demonstrated immunolabeling with anti-mouse laminin ß1. Labeling intensified over time with 14 day incubations. CONCLUSION: We provide new evidence for matrix-to-cell signaling in acellular whole organ scaffolds that induces differentiation of pluripotent precursor cells to endothelial lineage. Production of mouse basement membrane supports remodeling of host (rat)-derived scaffolds and thereby warrants further investigation as a promising approach for xenotransplantation. METHODS: We previously showed that murine embryonic stem cells arterially seeded into acellular rat whole kidney scaffolds multiply and demonstrate morphologic, immunohistochemical and gene expression evidence for differentiation. Vascular cell endothelialization was now further tested by endothelial specific BsLB4 lectin and anti-VEGFR2 (Flk1) antibodies. Remodeling of the matrix basement membranes from rat to mouse ("murinization") was assessed by a monoclonal antibody specific for mouse laminin ß1 chain.
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Membrana Basal/metabolismo , Células Endoteliales/citología , Riñón/citología , Células Madre/citología , Andamios del Tejido , Animales , Diferenciación Celular , Linaje de la Célula , Colágeno Tipo IV/metabolismo , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Riñón/metabolismo , Laminina/metabolismo , Ratones , Ratas , Células Madre/metabolismoRESUMEN
Laminin α5 is required for kidney glomerular basement membrane (GBM) assembly, and mice with targeted deletions of the Lama5 gene fail to form glomeruli. As a tool to begin to understand factors regulating the expression of the LAMA5 gene, we generated transgenic mice carrying the human LAMA5 locus in a bacterial artificial chromosome. These mice deposited human laminin α5 protein into basement membranes in heart, liver, spleen and kidney. Here, we characterized two lines of transgenics; Line 13 expressed â¼6 times more LAMA5 than Line 25. Mice from both lines were healthy, and kidney function and morphology were normal. Examination of developing glomeruli from fetal LAMA5 transgenics showed that the human transgene was expressed at the correct stage of glomerular development, and deposited into the nascent GBM simultaneously with mouse laminin α5. Expression of human LAMA5 did not affect the timing of the mouse laminin α1-α5 isoform switch, or that for mouse laminin ß1-ß2. Immunoelectron microscopy showed that human laminin α5 originated in both glomerular endothelial cells and podocytes, known to be origins for mouse laminin α5 normally. Notably, in neonatal transgenics expressing the highest levels of human LAMA5, there was a striking reduction of mouse laminin α5 protein in kidney basement membranes compared to wildtype, and significantly lower levels of mouse Lama5 mRNA. This suggests the presence in kidney of a laminin expression monitor, which may be important for regulating the overall production of basement membrane protein.
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Laminina/metabolismo , Animales , Membrana Basal/metabolismo , Humanos , Riñón/metabolismo , Glomérulos Renales/metabolismo , Laminina/genética , Hígado/metabolismo , Ratones , Ratones Transgénicos , Miocardio/metabolismo , Bazo/metabolismoRESUMEN
Vascular endothelial growth factor, which is critical for blood vessel formation, is regulated by hypoxia inducible transcription factors (HIFs). A component of the E3 ubiquitin ligase complex, von Hippel-Lindau (VHL) facilitates oxygen-dependent polyubiquitination and proteasomal degradation of HIFalpha subunits. Hypothesizing that deletion of podocyte VHL would result in HIFalpha hyperstabilization, we crossed podocin promoter-Cre transgenic mice, which express Cre recombinase in podocytes beginning at the capillary loop stage of glomerular development, with floxed VHL mice. Vascular patterning and glomerular development appeared unaltered in progeny lacking podocyte VHL. However, urinalysis showed increased albumin excretion by 4 weeks when compared with wild-type littermates with several sever cases (>1000 microg/ml). Many glomerular ultrastructural changes were seen in mutants, including focal subendothelial delamination and widespread podocyte foot process broadening, and glomerular basement membranes (GBMs) were significantly thicker in 16-week-old mutants compared with controls. Moreover, immunoelectron microscopy showed ectopic deposition of collagen alpha1alpha2alpha1(IV) in GBM humps beneath podocytes. Significant increases in the number of Ki-67-positive mesangial cells were also found, but glomerular WT1 expression was significantly decreased, signifying podocyte death and/or de-differentiation. Indeed, expression profiling of mutant glomeruli suggested a negative regulatory feedback loop involving the HIFalpha prolyl hydroxylase, Egln3. In addition, the brain oxygen-binding protein, Neuroglobin, was induced in mutant podocytes. We conclude that podocyte VHL is required for normal maintenance of podocytes, GBM composition and ultrastructure, and glomerular barrier properties.
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Colágeno Tipo IV/metabolismo , Globinas/metabolismo , Membrana Basal Glomerular/patología , Proteínas del Tejido Nervioso/metabolismo , Podocitos/metabolismo , Proteinuria/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Animales , Colágeno Tipo IV/genética , Femenino , Perfilación de la Expresión Génica , Globinas/genética , Membrana Basal Glomerular/citología , Membrana Basal Glomerular/metabolismo , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Proteínas del Tejido Nervioso/genética , Neuroglobina , Podocitos/citología , Proteinuria/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
The kidney collecting system develops from branching morphogenesis of the ureteric bud (UB). This process requires signaling by growth factors such as glial cell line derived neurotrophic factor (GDNF) and fibroblast growth factors (FGFs) as well as cell extracellular matrix interactions mediated by integrins. The importance of integrin signaling in UB development was investigated by deleting integrin beta1 at initiation (E10.5) and late (E18.5) stages of development. Deletion at E10.5 resulted in a severe branching morphogenesis phenotype. Deletion at E18.5 did not alter renal development but predisposed the collecting system to severe injury following ureteric obstruction. beta1 integrin was required for renal tubular epithelial cells to mediate GDNF- and FGF-dependent signaling despite normal receptor localization and activation in vitro. Aberrations in the same signaling molecules were present in the beta1-null UBs in vivo. Thus beta1 integrins can regulate organ branching morphogenesis during development by mediating growth-factor-dependent signaling in addition to their well-defined role as adhesion receptors.
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Integrina beta1/metabolismo , Túbulos Renales Colectores/embriología , Túbulos Renales Colectores/metabolismo , Uréter/embriología , Uréter/metabolismo , Animales , Adhesión Celular , Línea Celular , Movimiento Celular , Proliferación Celular , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Sustancias de Crecimiento/metabolismo , Integrina beta1/genética , Túbulos Renales Colectores/citología , Ratones , Ratones Noqueados , Ratones Transgénicos , Morfogénesis , Técnicas de Cultivo de Órganos , Transducción de SeñalRESUMEN
Data showing that the embryonic day 12 (E12) mouse kidney contains its own pool of endothelial progenitor cells is presented. Mechanisms that regulate metanephric endothelial recruitment and differentiation, including the hypoxia-inducible transcription factors and vascular endothelial growth factor/vascular endothelial growth factor receptor signaling system, are also discussed. Finally, evidence that glomerular endothelial cells contribute importantly to assembly of the glomerular basement membrane (GBM), especially the laminin component, is reviewed. Together, this forum offers insights on blood vessel development in general, and formation of the glomerular capillary in particular, which inarguably is among the most unique vascular structures in the body.
RESUMEN
Laminin and type IV collagen composition of the glomerular basement membrane changes during glomerular development and maturation. Although it is known that both glomerular endothelial cells and podocytes produce different laminin isoforms at the appropriate stages of development, the cellular origins for the different type IV collagen heterotrimers that appear during development are unknown. Here, immunoelectron microscopy demonstrated that endothelial cells, mesangial cells, and podocytes of immature glomeruli synthesize collagen alpha 1 alpha 2 alpha1(IV). However, intracellular labeling revealed that podocytes, but not endothelial or mesangial cells, contain collagen alpha 3 alpha 4 alpha 5(IV). To evaluate the origins of collagen IV further, we transplanted embryonic kidneys from Col4a3-null mutants (Alport mice) into kidneys of newborn, wildtype mice. Hybrid glomeruli within grafts containing numerous host-derived, wildtype endothelial cells never expressed collagen alpha 3 alpha 4 alpha 5(IV). Finally, confocal microscopy of glomeruli from infant Alport mice that had been dually labeled with anti-collagen alpha 5(IV) and the podocyte marker anti-GLEPP1 showed immunolabeling exclusively within podocytes. Together, these results indicate that collagen alpha 3 alpha 4 alpha 5(IV) originates solely from podocytes; therefore, glomerular Alport disease is a genetic defect that manifests specifically within this cell type.