RESUMEN
AIMS/HYPOTHESIS: Diabetic cardiomyopathy (DCM) is a serious and under-recognised complication of diabetes. The first sign is diastolic dysfunction, which progresses to heart failure. The pathophysiology of DCM is incompletely understood but microcirculatory changes are important. Endothelial glycocalyx (eGlx) plays multiple vital roles in the microcirculation, including in the regulation of vascular permeability, and is compromised in diabetes but has not previously been studied in the coronary microcirculation in diabetes. We hypothesised that eGlx damage in the coronary microcirculation contributes to increased microvascular permeability and hence to cardiac dysfunction. METHODS: We investigated eGlx damage and cardiomyopathy in mouse models of type 1 (streptozotocin-induced) and type 2 (db/db) diabetes. Cardiac dysfunction was determined by echocardiography. We obtained eGlx depth and coverage by transmission electron microscopy (TEM) on mouse hearts perfusion-fixed with glutaraldehyde and Alcian Blue. Perivascular oedema was assessed from TEM images by measuring the perivascular space area. Lectin-based fluorescence was developed to study eGlx in paraformaldehyde-fixed mouse and human tissues. The eGlx of human conditionally immortalised coronary microvascular endothelial cells (CMVECs) in culture was removed with eGlx-degrading enzymes before measurement of protein passage across the cell monolayer. The mechanism of eGlx damage in the diabetic heart was investigated by quantitative reverse transcription-PCR array and matrix metalloproteinase (MMP) activity assay. To directly demonstrate that eGlx damage disturbs cardiac function, isolated rat hearts were treated with enzymes in a Langendorff preparation. Angiopoietin 1 (Ang1) is known to restore eGlx and so was used to investigate whether eGlx restoration reverses diastolic dysfunction in mice with type 1 diabetes. RESULTS: In a mouse model of type 1 diabetes, diastolic dysfunction (confirmed by echocardiography) was associated with loss of eGlx from CMVECs and the development of perivascular oedema, suggesting increased microvascular permeability. We confirmed in vitro that eGlx removal increases CMVEC monolayer permeability. We identified increased MMP activity as a potential mechanism of eGlx damage and we observed loss of syndecan 4 consistent with MMP activity. In a mouse model of type 2 diabetes we found a similar loss of eGlx preceding the development of diastolic dysfunction. We used isolated rat hearts to demonstrate that eGlx damage (induced by enzymes) is sufficient to disturb cardiac function. Ang1 restored eGlx and this was associated with reduced perivascular oedema and amelioration of the diastolic dysfunction seen in mice with type 1 diabetes. CONCLUSIONS/INTERPRETATION: The association of CMVEC glycocalyx damage with diastolic dysfunction in two diabetes models suggests that it may play a pathophysiological role and the enzyme studies confirm that eGlx damage is sufficient to impair cardiac function. Ang1 rapidly restores the CMVEC glycocalyx and improves diastolic function. Our work identifies CMVEC glycocalyx damage as a potential contributor to the development of DCM and therefore as a therapeutic target.
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Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Cardiomiopatías Diabéticas , Angiopoyetina 1/metabolismo , Animales , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatías Diabéticas/metabolismo , Células Endoteliales/metabolismo , Glicocálix/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Ratones , Microcirculación , RatasRESUMEN
Elevated levels of vascular endothelial growth factor (VEGF) A are thought to cause glomerular endothelial cell (GEnC) dysfunction and albuminuria in diabetic nephropathy. We hypothesized that VEGFC could counteract these effects of VEGFA to protect the glomerular filtration barrier and reduce albuminuria. Isolated glomeruli were stimulated ex vivo with VEGFC, which reduced VEGFA- and type 2 diabetes-induced glomerular albumin solute permeability (Ps'alb). VEGFC had no detrimental effect on glomerular function in vivo when overexpression was induced locally in podocytes (podVEGFC) in otherwise healthy mice. Further, these mice had reduced glomerular VEGFA mRNA expression, yet increased glomerular VEGF receptor heterodimerization, indicating differential signaling by VEGFC. In a model of type 1 diabetes, the induction of podVEGFC overexpression reduced the development of hypertrophy, albuminuria, loss of GEnC fenestrations and protected against altered VEGF receptor expression. In addition, VEGFC protected against raised Ps'alb by endothelial glycocalyx disruption in glomeruli. In summary, VEGFC reduced the development of diabetic nephropathy, prevented VEGF receptor alterations in the diabetic glomerulus, and promoted both glomerular protection and endothelial barrier function. These important findings highlight a novel pathway for future investigation in the treatment of diabetic nephropathy.
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Nefropatías Diabéticas/metabolismo , Animales , Western Blotting , Células Cultivadas , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Nefropatías Diabéticas/genética , Técnica del Anticuerpo Fluorescente , Genotipo , Humanos , Inmunoprecipitación , Podocitos/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
To investigate human glomerular structure under conditions of physiological perfusion, we have analyzed fresh and perfusion-fixed normal human glomeruli at physiological hydrostatic and oncotic pressures using serial resin section reconstruction, confocal, multiphoton, and electron microscope imaging. Afferent and efferent arterioles (21.5 ± 1.2 µm and 15.9 ± 1.2 µm diameter), recognized from vascular origins, lead into previously undescribed wider regions (43.2 ± 2.8 µm and 38.4 ± 4.9 µm diameter) we have termed vascular chambers (VCs) embedded in the mesangium of the vascular pole. Afferent VC (AVC) volume was 1.6-fold greater than efferent VC (EVC) volume. From the AVC, long nonbranching high-capacity conduit vessels ( n = 7) (Con; 15.9 ± 0.7 µm diameter) led to the glomerular edge, where branching was more frequent. Conduit vessels have fewer podocytes than filtration capillaries. VCs were confirmed in fixed and unfixed specimens with a layer of banded collagen identified in AVC walls by multiphoton and electron microscopy. Thirteen highly branched efferent first-order vessels (E1; 9.9 ± 0.4 µm diameter) converge on the EVC, draining into the efferent arteriole (15.9 ± 1.2 µm diameter). Banded collagen was scarce around EVCs. This previously undescribed branching topology does not conform to the branching of minimum energy expenditure (Murray's law), suggesting that even distribution of pressure/flow to the filtration capillaries is more important than maintaining the minimum work required for blood flow. We propose that AVCs act as plenum manifolds possibly aided by vortical flow in distributing and balancing blood flow/pressure to conduit vessels supplying glomerular lobules. These major adaptations to glomerular capillary structure could regulate hemodynamic pressure and flow in human glomerular capillaries.
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Hemodinámica , Glomérulos Renales/irrigación sanguínea , Microcirculación , Microvasos/fisiología , Circulación Renal , Humanos , Presión Hidrostática , Microscopía Confocal , Microscopía Electrónica de Transmisión , Microscopía de Fluorescencia por Excitación Multifotónica , Microvasos/ultraestructura , Modelos Biológicos , Podocitos/fisiología , Fijación del TejidoRESUMEN
Increased urinary albumin excretion is a key feature of glomerular disease but has limitations as a measure of glomerular permeability. Here we describe a novel assay to measure the apparent albumin permeability of single capillaries in glomeruli, isolated from perfused kidneys cleared of red blood cells. The rate of decline of the albumin concentration within the capillary lumen was quantified using confocal microscopy and used to calculate apparent permeability. The assay was extensively validated and provided robust, reproducible estimates of glomerular albumin permeability. These values were comparable with previous in vivo data, showing this assay could be applied to human as well as rodent glomeruli. To confirm this, we showed that targeted endothelial glycocalyx disruption resulted in increased glomerular albumin permeability in mice. Furthermore, incubation with plasma from patients with post-transplant recurrence of nephrotic syndrome increased albumin permeability in rat glomeruli compared to remission plasma. Finally, in glomeruli isolated from rats with early diabetes there was a significant increase in albumin permeability and loss of endothelial glycocalyx, both of which were ameliorated by angiopoietin-1. Thus, a glomerular permeability assay, producing physiologically relevant values with sufficient sensitivity to measure changes in glomerular permeability and independent of tubular function, was developed and validated. This assay significantly advances the ability to study biology and disease in rodent and human glomeruli.
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Bioensayo/métodos , Capilares/metabolismo , Permeabilidad Capilar , Glomérulos Renales/irrigación sanguínea , Albúmina Sérica/metabolismo , Albuminuria/metabolismo , Albuminuria/fisiopatología , Angiopoyetina 1/farmacología , Animales , Capilares/efectos de los fármacos , Capilares/fisiopatología , Permeabilidad Capilar/efectos de los fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatología , Femenino , Glicocálix/metabolismo , Humanos , Técnicas In Vitro , Cinética , Masculino , Ratones Endogámicos C57BL , Microscopía Confocal , Síndrome Nefrótico/sangre , Síndrome Nefrótico/fisiopatología , Ratas Sprague-Dawley , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND/AIMS: Genetic cell ablation using the human diphtheria toxin receptor (hDTR) is a new strategy used for analysing cellular function. Diphtheria toxin (DT) is a cytotoxic protein that leaves mouse cells relatively unaffected, but upon binding to hDTR it ultimately leads to cell death. We used a podocyte-specific hDTR expressing (Pod-DTR) mouse to assess the anti-permeability and cyto-protective effects of the splice isoform vascular endothelial growth factor (VEGF-A165b). METHODS: The Pod-DTR mouse was crossed with a mouse that over-expressed VEGF-A165b specifically in the podocytes (Neph-VEGF-A165b). Wild type (WT), Pod-DTR, Neph-VEGF-A165b and Pod-DTR X Neph-VEGF-A165b mice were treated with several doses of DT (1, 5, 100, and 1,000 ng/g bodyweight). Urine was collected and the glomerular water permeability (LpA/Vi) was measured ex vivo after 14 days. Structural analysis and podocyte marker expression were also assessed. RESULTS: Pod-DTR mice developed an increased glomerular LpA/Vi 14 days after administration of DT (all doses), which was prevented when the mice over-expressed VEGF-A165b. No major structural abnormalities, podocyte ablation or albuminuria was observed in Pod-DTR mice, indicating this to be a mild model of podocyte disease. However, a change in expression and localisation of nephrin within the podocytes was observed, indicating disruption of the slit diaphragm in the Pod-DTR mice. This was prevented in the Pod-DTR X Neph-VEGF-A165b mice. CONCLUSION: Although only a mild model of podocyte injury, over-expression of the anti-permeability VEGF-A165b isoform in the podocytes of Pod-DTR mice had a protective effect. Therefore, this study further highlights the therapeutic potential of VEGF-A165b in glomerular disease.
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Toxina Diftérica , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/metabolismo , Glomérulos Renales , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Agua/metabolismo , Albuminuria/inducido químicamente , Albuminuria/metabolismo , Animales , Tasa de Filtración Glomerular , Enfermedades Renales/inducido químicamente , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Permeabilidad , Podocitos/metabolismo , Podocitos/patología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
KEY POINTS: Progressive depletion of all vascular endothelial growth factor A (VEGF-A) splice isoforms from the kidney results in proteinuria and increased glomerular water permeability, which are both rescued by over-expression of VEGF-A165 b only. VEGF-A165 b rescues the increase in glomerular basement membrane and podocyte slit width, as well as the decrease in sub-podocyte space coverage, produced by VEGF-A depletion. VEGF-A165 b restores the expression of platelet endothelial cell adhesion molecule in glomerular endothelial cells and glomerular capillary circumference. VEGF-A165 b has opposite effects to VEGF-A165 on the expression of genes involved in endothelial cell migration and proliferation. ABSTRACT: Chronic kidney disease is strongly associated with a decrease in the expression of vascular endothelial growth factor A (VEGF-A). However, little is known about the contribution of VEGF-A splice isoforms to kidney physiology and pathology. Previous studies suggest that the splice isoform VEGF-A165 b (resulting from alternative usage of a 3' splice site in the terminal exon) is protective for kidney function. In the present study, we show, in a quad-transgenic model, that over-expression of VEGF-A165 b alone is sufficient to rescue the increase in proteinuria, as well as glomerular water permeability, in the context of progressive depletion of all VEGF-A isoforms from the podocytes. Ultrastructural studies show that the glomerular basement membrane is thickened, podocyte slit width is increased and sub-podocyte space coverage is reduced when VEGF-A is depleted, all of which are rescued in VEGF-A165 b over-expressors. VEGF-A165 b restores the expression of platelet endothelial cell adhesion molecule-1 in glomerular endothelial cells and glomerular capillary circumference. Mechanistically, it increases VEGF receptor 2 expression both in vivo and in vitro and down-regulates genes involved in migration and proliferation of endothelial cells, otherwise up-regulated by the canonical isoform VEGF-A165 . The results of the present study indicate that manipulation of VEGF-A splice isoforms could be a novel therapeutic avenue in chronic glomerular disease.
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Riñón/metabolismo , Proteinuria/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Línea Celular , Humanos , Riñón/patología , Ratones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Podocitos/metabolismo , Podocitos/ultraestructura , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteinuria/genética , Proteinuria/patología , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genética , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
KEY POINTS: We have developed novel techniques for paired, direct, real-time in vivo quantification of endothelial glycocalyx structure and associated microvessel permeability. Commonly used imaging and analysis techniques yield measurements of endothelial glycocalyx depth that vary by over an order of magnitude within the same vessel. The anatomical distance between maximal glycocalyx label and maximal endothelial cell plasma membrane label provides the most sensitive and reliable measure of endothelial glycocalyx depth. Sialic acid residues of the endothelial glycocalyx regulate glycocalyx structure and microvessel permeability to both water and albumin. ABSTRACT: The endothelial glycocalyx forms a continuous coat over the luminal surface of all vessels, and regulates multiple vascular functions. The contribution of individual components of the endothelial glycocalyx to one critical vascular function, microvascular permeability, remains unclear. We developed novel, real-time, paired methodologies to study the contribution of sialic acids within the endothelial glycocalyx to the structural and functional permeability properties of the same microvessel in vivo. Single perfused rat mesenteric microvessels were perfused with fluorescent endothelial cell membrane and glycocalyx labels, and imaged with confocal microscopy. A broad range of glycocalyx depth measurements (0.17-3.02 µm) were obtained with different labels, imaging techniques and analysis methods. The distance between peak cell membrane and peak glycocalyx label provided the most reliable measure of endothelial glycocalyx anatomy, correlating with paired, numerically smaller values of endothelial glycocalyx depth (0.078 ± 0.016 µm) from electron micrographs of the same portion of the same vessel. Disruption of sialic acid residues within the endothelial glycocalyx using neuraminidase perfusion decreased endothelial glycocalyx depth and increased apparent solute permeability to albumin in the same vessels in a time-dependent manner, with changes in all three true vessel wall permeability coefficients (hydraulic conductivity, reflection coefficient and diffusive solute permeability). These novel technologies expand the range of techniques that permit direct studies of the structure of the endothelial glycocalyx and dependent microvascular functions in vivo, and demonstrate that sialic acid residues within the endothelial glycocalyx are critical regulators of microvascular permeability to both water and albumin.
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Permeabilidad Capilar , Células Endoteliales/metabolismo , Glicocálix/metabolismo , Microvasos/metabolismo , Ácidos Siálicos/metabolismo , Albúminas/metabolismo , Animales , Células Endoteliales/ultraestructura , Glicocálix/ultraestructura , Masculino , Mesenterio/irrigación sanguínea , Microscopía Electrónica de Transmisión , Microvasos/ultraestructura , Ratas , Ratas Sprague-Dawley , Agua/metabolismoRESUMEN
The endothelial glycocalyx has a profound influence at the vascular wall on the transmission of shear stress, on the maintenance of a selective permeability barrier and a low hydraulic conductivity, and on attenuating firm adhesion of blood leukocytes and platelets. Major constituents of the glycocalyx, including syndecans, heparan sulphates and hyaluronan, are shed from the endothelial surface under various acute and chronic clinical conditions, the best characterized being ischaemia and hypoxia, sepsis and inflammation, atherosclerosis, diabetes, renal disease and haemorrhagic viral infections. Damage has also been detected by in vivo microscopic techniques. Matrix metalloproteases may shed syndecans and heparanase, released from activated mast cells, cleaves heparan sulphates from core proteins. According to new data, not only hyaluronidase but also the serine proteases thrombin, elastase, proteinase 3 and plasminogen, as well as cathepsin B lead to loss of hyaluronan from the endothelial surface layer, suggesting a wide array of potentially destructive conditions. Appropriately, pharmacological agents such as inhibitors of inflammation, antithrombin and inhibitors of metalloproteases display potential to attenuate shedding of the glycocalyx in various experimental models. Also, plasma components, especially albumin, stabilize the glycocalyx and contribute to the endothelial surface layer. Though symptoms of the above listed diseases and conditions correlate with sequelae expected from disturbance of the endothelial glycocalyx (oedema, inflammation, leukocyte and platelet adhesion, low reflow), therapeutic studies to prove a causal connection have yet to be designed. With respect to studies on humans, some clinical evidence exists for benefits from application of sulodexide, a preparation delivering precursors of the glycocalyx constituent heparan sulphate. At present, the simplest option for protecting the glycocalyx seems to be to ensure an adequate level of albumin. However, also in this case, definite proof of causality needs to be delivered.
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Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Endotelio Vascular/ultraestructura , Glicocálix/ultraestructura , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Glicocálix/metabolismo , Glicosaminoglicanos/farmacología , Glicosaminoglicanos/uso terapéutico , Heparitina Sulfato/metabolismo , Humanos , Ácido Hialurónico/metabolismo , Péptido Hidrolasas/metabolismo , Insuficiencia Renal/metabolismo , Insuficiencia Renal/patología , Insuficiencia Renal/prevención & control , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control , Sepsis/metabolismo , Sepsis/patología , Sepsis/prevención & control , Albúmina Sérica/metabolismo , Sindecanos/metabolismoRESUMEN
The wall shear stress (WSS) that a moving fluid exerts on a surface affects many processes including those relating to vascular function. WSS plays an important role in normal physiology (e.g. angiogenesis) and affects the microvasculature's primary function of molecular transport. Points of fluctuating WSS show abnormalities in a number of diseases; however, there is no established technique for measuring WSS directly in physiological systems. All current methods rely on estimates obtained from measured velocity gradients in bulk flow data. In this work, we report a nanosensor that can directly measure WSS in microfluidic chambers with sub-micron spatial resolution by using a specific type of virus, the bacteriophage M13, which has been fluorescently labeled and anchored to a surface. It is demonstrated that the nanosensor can be calibrated and adapted for biological tissue, revealing WSS in micro-domains of cells that cannot be calculated accurately from bulk flow measurements. This method lends itself to a platform applicable to many applications in biology and microfluidics.
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Diabetic nephropathy is the leading cause of ESRD in high-income countries and a growing problem across the world. Vascular endothelial growth factor-A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, yet VEGF-A knockout and overexpression of angiogenic VEGF-A isoforms each worsen diabetic nephropathy. We examined the vasculoprotective effects of the VEGF-A isoform VEGF-A165b in diabetic nephropathy. Renal expression of VEGF-A165b mRNA was upregulated in diabetic individuals with well preserved kidney function, but not in those with progressive disease. Reproducing this VEGF-A165b upregulation in mouse podocytes in vivo prevented functional and histologic abnormalities in diabetic nephropathy. Biweekly systemic injections of recombinant human VEGF-A165b reduced features of diabetic nephropathy when initiated during early or advanced nephropathy in a model of type 1 diabetes and when initiated during early nephropathy in a model of type 2 diabetes. VEGF-A165b normalized glomerular permeability through phosphorylation of VEGF receptor 2 in glomerular endothelial cells, and reversed diabetes-induced damage to the glomerular endothelial glycocalyx. VEGF-A165b also improved the permeability function of isolated diabetic human glomeruli. These results show that VEGF-A165b acts via the endothelium to protect blood vessels and ameliorate diabetic nephropathy.
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Nefropatías Diabéticas/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/uso terapéutico , Albuminuria/tratamiento farmacológico , Animales , Nefropatías Diabéticas/metabolismo , Evaluación Preclínica de Medicamentos , Células Endoteliales/efectos de los fármacos , Tasa de Filtración Glomerular/efectos de los fármacos , Glicocálix/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Podocitos/metabolismo , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy.
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Empalme Alternativo , Bevacizumab , Neoplasias del Colon/metabolismo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neovascularización Patológica/metabolismo , Proteínas de Unión a Poli(A)/biosíntesis , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Animales , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Células HEK293 , Células HeLa , Xenoinjertos , Humanos , Ratones Desnudos , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Proteínas de Unión a Poli(A)/genética , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Antígeno Intracelular 1 de las Células T , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.
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Empalme Alternativo , Amplificación de Genes , Perfilación de la Expresión Génica , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Especificidad de Anticuerpos/inmunología , Western Blotting , Línea Celular , Línea Celular Tumoral , Cartilla de ADN/genética , Humanos , Inmunoprecipitación , Ratones , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Factor A de Crecimiento Endotelial Vascular/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Vascular endothelial growth factor (VEGF)-A, a family of differentially spliced proteins produced by glomerular podocytes, maintains glomerular filtration barrier function. The expression of VEGF molecules is altered in human nephropathy. We aimed to determine the roles of the angiogenic VEGF(164) isoform, and the antiangiogenic VEGF(165)b isoform in mature, adult glomeruli in vivo using conditional, inducible transgenic overexpression systems in mice. Podocyte-specific VEGF(164) overexpression (up to 100 days) was induced by oral administration of doxycycline to adult podocin-rtTA/TetO-VEGF(164) double transgenic mice. The consequences of simultaneous overexpression of VEGF(164) and VEGF(165)b were assessed in triple-transgenic podocin-rtTA/TetO-VEGF(164)/nephrin-VEGF(165)b mice. Persistent VEGF(164) overexpression did not cause proteinuria but did increase glomerular ultrafiltration coefficient between days 3 and 7. Despite persistently increased VEGF(164) levels, glomerular ultrafiltration coefficient normalized by day 14 and remained normal up to 100 days. Decreased subpodocyte space (SPS) coverage of the glomerular capillary wall accompanied increased glomerular hydraulic conductivity in VEGF(164)-overexpressing mice. The changes in glomerular ultrafiltration coefficient and SPS coverage induced by 7 days of overexpression of VEGF(164) were not present in triple transgenic VEGF(164) and VEGF(165)b overexpressing mice. These results indicate that 1) the adult mouse glomerulus is relatively resistant to induced VEGF(164) overexpression. VEGF(164) overexpression altered glomerular permeability but did not cause proteinuria in these mature, adult animals; 2) the SPS is a dynamic VEGF-responsive modulator of glomerular function; and 3) the balance of VEGF isoforms plays a critical role in the regulation of glomerular permeability. VEGF(165)b is capable of preventing VEGF(164)-induced changes in glomerular permeability and ultrastructure in vivo.
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Agua Corporal/metabolismo , Glomérulos Renales/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Ratones , Ratones Transgénicos , Permeabilidad , Podocitos/metabolismo , Proteinuria/genética , Proteinuria/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Patients with albuminuria and CKD frequently have vascular dysfunction but the underlying mechanisms remain unclear. Because the endothelial surface layer, a meshwork of surface-bound and loosely adherent glycosaminoglycans and proteoglycans, modulates vascular function, its loss could contribute to both renal and systemic vascular dysfunction in proteinuric CKD. Using Munich-Wistar-Fromter (MWF) rats as a model of spontaneous albuminuric CKD, multiphoton fluorescence imaging and single-vessel physiology measurements revealed that old MWF rats exhibited widespread loss of the endothelial surface layer in parallel with defects in microvascular permeability to both water and albumin, in both continuous mesenteric microvessels and fenestrated glomerular microvessels. In contrast to young MWF rats, enzymatic disruption of the endothelial surface layer in old MWF rats resulted in neither additional loss of the layer nor additional changes in permeability. Intravenous injection of wheat germ agglutinin lectin and its adsorption onto the endothelial surface layer significantly improved glomerular albumin permeability. Taken together, these results suggest that widespread loss of the endothelial surface layer links albuminuric kidney disease with systemic vascular dysfunction, providing a potential therapeutic target for proteinuric kidney disease.
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Permeabilidad Capilar , Endotelio Vascular/fisiopatología , Glicocálix/metabolismo , Glomérulos Renales/fisiopatología , Proteinuria/etiología , Envejecimiento/metabolismo , Animales , Capilares/fisiopatología , Endotelio Vascular/metabolismo , Masculino , Proteinuria/fisiopatología , RatasRESUMEN
BACKGROUND: The use and timing of steroids in the management of acute tubulointerstitial nephritis (ATIN) remains debatable. AIMS: To determine the incidence and aetiology of ATIN in our unit, and to examine trends in the use of steroids and their impact on renal outcomes. METHODS: Patients with a histological diagnosis of ATIN over a 9-year period were identified and divided into steroid-treated (StG) and steroid-naïve groups (SnG). Mean change in estimated glomerular filtration rate (eGFR) was determined. RESULTS: Forty-nine patients had ATIN as their main diagnosis, 67% of cases were drug-induced, and proton pump inhibitors (PPI) were the second commonest implicated drug category. Majority (75%) of patients received steroids, and eGFR improved to a significantly greater degree in these steroid-treated patients (3.4-fold improvement vs 2.0-fold in SnG; P < 0.05, unpaired t-test). Despite comparable eGFR at presentation (StG: 11.7; SnG: 15.4), steroid-treated patients were less likely to receive dialysis, although not significantly so (OR 0.27; 95% CI 0.06-1.15, P = 0.066, chi-squared test). However, there was no significant relation between the degree of eGFR improvement and delay in starting steroids (Pearson r = -0.25, P > 0.45), and no difference in eGFR at the time of last follow-up (StG: 33 ± 3; SnG: 32 ± 7; P > 0.9, unpaired t-test). CONCLUSION: StG patients had a greater degree of improvement in renal function, but with no correlation between degree of improvement in eGFR and delay in starting steroids, and similar eGFR values at final follow-up. PPI were the second commonest drug category among drug-induced cases.
Asunto(s)
Tasa de Filtración Glomerular/efectos de los fármacos , Riñón/efectos de los fármacos , Nefritis Intersticial/tratamiento farmacológico , Esteroides/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/efectos adversos , Biopsia , Distribución de Chi-Cuadrado , Inglaterra/epidemiología , Femenino , Humanos , Incidencia , Riñón/patología , Riñón/fisiopatología , Masculino , Persona de Mediana Edad , Nefritis Intersticial/diagnóstico , Nefritis Intersticial/epidemiología , Nefritis Intersticial/fisiopatología , Oportunidad Relativa , Inhibidores de la Bomba de Protones/efectos adversos , Recuperación de la Función , Diálisis Renal , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
Appreciation of the glomerular microcirculation as a specialized microcirculatory bed, rather than as an entirely separate entity, affords important insights into both glomerular and systemic microvascular pathophysiology. In this review we compare regulation of permeability in systemic and glomerular microcirculations, focusing particularly on the role of the endothelial glycocalyx, and consider the implications for disease processes. The luminal surface of vascular endothelium throughout the body is covered with endothelial glycocalyx, comprising surface-anchored proteoglycans, supplemented with adsorbed soluble proteoglycans, glycosaminoglycans and plasma constituents. In both continuous and fenestrated microvessels, this endothelial glycocalyx provides resistance to the transcapillary escape of water and macromolecules, acting as an integral component of the multilayered barrier provided by the walls of these microvessels (ie acting in concert with clefts or fenestrae across endothelial cell layers, basement membranes and pericytes). Dysfunction of any of these capillary wall components, including the endothelial glycocalyx, can disrupt normal microvascular permeability. Because of its ubiquitous nature, damage to the endothelial glycocalyx alters the permeability of multiple capillary beds: in the glomerulus this is clinically apparent as albuminuria. Generalized damage to the endothelial glycocalyx can therefore manifest as both albuminuria and increased systemic microvascular permeability. This triad of altered endothelial glycocalyx, albuminuria and increased systemic microvascular permeability occurs in a number of important diseases, such as diabetes, with accumulating evidence for a similar phenomenon in ischaemia-reperfusion injury and infectious disease. The detection of albuminuria therefore has implications for the function of the microcirculation as a whole. The importance of the endothelial glycocalyx for other aspects of vascular function/dysfunction, such as mechanotransduction, leukocyte-endothelial interactions and the development of atherosclerosis, indicate that alterations in the endothelial glycocalyx may also be playing a role in the dysfunction of other organs observed in these disease states.
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Albuminuria/metabolismo , Capilares/metabolismo , Permeabilidad Capilar , Endotelio Vascular/metabolismo , Glicocálix/metabolismo , Glomérulos Renales/metabolismo , Albuminuria/patología , Albuminuria/fisiopatología , Animales , Capilares/fisiopatología , Capilares/ultraestructura , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Glicocálix/ultraestructura , Humanos , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/patología , MicrocirculaciónRESUMEN
PURPOSE OF REVIEW: Three structures (glomerular endothelial fenestrae, glomerular basement membrane and podocyte interfoot process/slit diaphragms) have traditionally been considered as the major determinants of glomerular permeability. We review recent work demonstrating the functional importance of two additional layers: the endothelial surface layer (ESL) and the subpodocyte space (SPS). RECENT FINDINGS: Removing glomerular endothelial cell monolayer ESL in vitro significantly alters monolayer permeability, supporting previous in-vivo demonstrations of the importance of the ESL in determining glomerular permeability. Whether fenestral diaphragms are present to support the ESL in healthy adult glomeruli has been examined in a recent report. On the downstream side of the glomerular filtration barrier, the SPS is a recently described structure that covers approximately two-thirds of the barrier, has highly restrictive dimensions and contributes to the hydraulic resistance and ultrafiltration characteristics of the glomerulus. Different layers of the barrier have also been shown to influence the permeability characteristics of one another, either through biophysical interactions, or through the activities of ligand-receptor axes that cross the various layers of the barrier. SUMMARY: The structure and function of the glomerular filtration barrier remains an area of significant new discovery, and recent work continues to highlight the complexity of this dynamic multilayered watershed.
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Glomérulos Renales/anatomía & histología , Glomérulos Renales/fisiología , Animales , Endotelio/metabolismo , Endotelio/fisiología , Tasa de Filtración Glomerular , Humanos , Permeabilidad , Podocitos/metabolismo , Receptor Cross-Talk/fisiologíaRESUMEN
AIMS: In this study, we wished to determine whether angiopoietin-1 (Ang1) modified the permeability coefficients of non-inflamed, intact continuous, and fenestrated microvessels in vivo and to elucidate the underlying cellular mechanisms. METHODS AND RESULTS: Permeability coefficients were measured using the Landis-Michel technique (in frog and rat mesenteric microvessels) and an oncopressive permeability technique (in glomeruli). Ang1 decreased water permeability (L(P): hydraulic conductivity) in continuous and fenestrated microvessels and increased the retention of albumin (sigma: reflection coefficient) in continuous microvessels. Endothelial glycocalyx is common to these anatomically distinct microvascular beds, and contributes to the magnitude of both L(P) and sigma. Ang1 treatment increased the depth of endothelial glycocalyx in intact microvessels and increased the content of glycosaminoglycan of cultured microvascular endothelial cell supernatant. Ang1 also prevented the pronase-induced increase in L(P) (attributable to selective removal of endothelial glycocalyx by pronase) by restoration of glycocalyx at the endothelial cell surface. The reduction in permeability was inhibited by a cell transport inhibitor, Brefeldin. CONCLUSION: Ang1 modifies basal microvessel permeability coefficients, in keeping with previous reports demonstrating reduced solute flux in inflamed vessels. Anatomical, biochemical, and physiological evidence indicates that modification of endothelial glycocalyx is a novel mechanism of action of Ang1 that contributes to these effects.
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Angiopoyetina 1/fisiología , Permeabilidad de la Membrana Celular/fisiología , Endotelio Vascular/metabolismo , Glicocálix/metabolismo , Microvasos/metabolismo , Albúminas/metabolismo , Animales , Brefeldino A/farmacología , Endotelio Vascular/citología , Glicosaminoglicanos/metabolismo , Humanos , Glomérulos Renales/irrigación sanguínea , Microvasos/citología , Modelos Biológicos , Pronasa/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Rana temporaria , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , Agua/metabolismoRESUMEN
The glomerular filtration barrier (GFB) is generally considered to consist of three layers: fenestrated glomerular endothelium, glomerular basement membrane, and filtration slits between adjacent podocyte foot processes. Detailed anatomic examination of the GFB has revealed a novel abluminal structure, the subpodocyte space (SPS), identified as the labyrinthine space between the underside of podocyte cell body/primary processes and the foot processes. The SPS covers 50-65% of the filtration surface of the GFB, indicating that SPS may influence glomerular permeability. We have examined the contribution of the SPS to the permeability characteristics of the GFB using multiphoton microscopy techniques in isolated, perfused glomeruli and in the intact kidney in vivo. SPS were identified using this technique, with comparable dimensions to SPS examined with electron microscopy. The passage of the intermediate-weight molecule rhodamine-conjugated 10-kDa dextran, but not the low-weight molecule lucifer yellow ( approximately 450 Da), accumulated in SPS-covered regions of the GFB, compared with GFB regions not covered by SPS ("naked regions"). Net lucifer yellow flux (taken to indicate fluid flux) through identifiable SPS regions was calculated to be 66-75% of that occurring through naked regions. These observations indicate both ultrafiltration and hydraulic resistance imparted by the SPS, demonstrating the potential physiological contribution of the SPS to glomerular permeability.