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1.
Am J Physiol Renal Physiol ; 320(1): F61-F73, 2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33196323

RESUMEN

Oxidative stress is a key concept in basic, translational, and clinical research to understand the pathophysiology of various disorders, including cardiovascular and renal diseases. Although attempts to directly reduce oxidative stress with redox-active substances have until now largely failed to prove clinical benefit, indirect approaches to combat oxidative stress enzymatically have gained further attention as potential therapeutic strategies. The pantetheinase Vanin-1 is expressed on kidney proximal tubular cells, and its reaction product cysteamine is described to negatively affect redox homeostasis by inhibiting the replenishment of cellular antioxidative glutathione stores. Vanin-1-deficient mice were shown to be protected against oxidative stress damage. The aim of this study was to elucidate whether pharmacological inhibition of Vanin-1 protects mice from oxidative stress-related acute or chronic kidney injury as well. By studying renal ischemia-reperfusion injury in Col4α3-/- (Alport syndrome) mice and in vitro hypoxia-reoxygenation in human proximal tubular cells we found that treatment with a selective and potent Vanin-1 inhibitor resulted in ample inhibition of enzymatic activity in vitro and in vivo. However, surrogate parameters of metabolic and redox homeostasis were only partially and insufficiently affected. Consequently, apoptosis and reactive oxygen species level in tubular cells as well as overall kidney function and fibrotic processes were not improved by Vanin-1 inhibition. We thus conclude that Vanin-1 functionality in the context of cardiovascular diseases needs further investigation and the biological relevance of pharmacological Vanin-1 inhibition for the treatment of kidney diseases remains to be proven.


Asunto(s)
Lesión Renal Aguda/prevención & control , Amidohidrolasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Nefritis Hereditaria/prevención & control , Estrés Oxidativo/efectos de los fármacos , Insuficiencia Renal Crónica/prevención & control , Daño por Reperfusión/prevención & control , Lesión Renal Aguda/enzimología , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autoantígenos/genética , Autoantígenos/metabolismo , Línea Celular , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacocinética , Fibrosis , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Túbulos Renales Proximales/enzimología , Túbulos Renales Proximales/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Nefritis Hereditaria/enzimología , Nefritis Hereditaria/genética , Nefritis Hereditaria/patología , Insuficiencia Renal Crónica/enzimología , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/patología , Daño por Reperfusión/enzimología , Daño por Reperfusión/genética , Daño por Reperfusión/patología
2.
PLoS One ; 9(6): e99083, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24915008

RESUMEN

It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.


Asunto(s)
Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Laminina/metabolismo , Nefritis Hereditaria/enzimología , Nefritis Hereditaria/etiología , Animales , Fenómenos Biomecánicos/efectos de los fármacos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Técnicas de Silenciamiento del Gen , Membrana Basal Glomerular/efectos de los fármacos , Membrana Basal Glomerular/enzimología , Membrana Basal Glomerular/patología , Membrana Basal Glomerular/ultraestructura , Proteínas I-kappa B/metabolismo , Interleucina-6/metabolismo , Cinética , Metaloproteinasas de la Matriz/metabolismo , Ratones Noqueados , Morfolinas/farmacología , Morfolinas/uso terapéutico , Inhibidor NF-kappaB alfa , Nefritis Hereditaria/tratamiento farmacológico , Nefritis Hereditaria/patología , Podocitos/enzimología , Podocitos/patología , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/metabolismo , Tetraspanina 24/metabolismo
3.
Kidney Int ; 82(10): 1061-70, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22739976

RESUMEN

Progressive elevations of fibroblastic growth factor 23 (FGF23) in chronic kidney disease may reduce serum 25-hydroxyvitamin D (25(OH)) and 1,25-dihydroxyvitamin D (1,25(OH)(2)D) levels, via stimulation of 24-hydroxylase (Cyp24a1)-mediated catabolism of these vitamin D metabolites. To test this possibility, we measured serum concentrations of 24,25-dihydroxyvitamin D (24,25(OH)(2)D), a product of Cyp24a1 hydroxylation of 25(OH)D, in the Col4a3 knockout mouse, a model of Alport syndrome-derived chronic kidney disease, and in patients with chronic kidney disease of variable severity. There was an inverse correlation between serum FGF23 and both 25(OH)D and 1,25(OH)(2)D in the mouse model, but no significant relationship was observed in the cross-sectional patient cohort. The FGF23-dependent increase in Cyp24a1 mRNA expression in the mouse kidneys was consistent with the possibility that FGF23 induces vitamin D catabolism. There was, however, a reduction in serum 24,25(OH)(2)D levels, rather than the expected elevation, in both the mice and patients with chronic kidney disease. Low 25(OH)D and elevated FGF23 and parathyroid hormone levels were correlated with the reduced serum 24,25(OH)(2)D concentrations of these patients. Thus, we failed to find support for FGF23-mediated catabolism of vitamin D metabolites in chronic kidney disease assessed by 24,25(OH)(2)D levels.


Asunto(s)
Dihidroxicolecalciferoles/sangre , Factores de Crecimiento de Fibroblastos/sangre , Nefritis Hereditaria/sangre , Insuficiencia Renal Crónica/sangre , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Anciano , Animales , Autoantígenos/genética , Biomarcadores/sangre , Distribución de Chi-Cuadrado , Colágeno Tipo IV/deficiencia , Colágeno Tipo IV/genética , Estudios Transversales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Factor-23 de Crecimiento de Fibroblastos , Humanos , Hidroxilación , Riñón/enzimología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Nefritis Hereditaria/enzimología , Hormona Paratiroidea/sangre , ARN Mensajero/metabolismo , Insuficiencia Renal Crónica/enzimología , Índice de Severidad de la Enfermedad , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Regulación hacia Arriba , Vitamina D/análogos & derivados , Vitamina D/sangre , Vitamina D3 24-Hidroxilasa
4.
Ultrastruct Pathol ; 34(4): 199-206, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20594039

RESUMEN

Trypsin digestion for 90 min revealed the alpha(5) chain of type IV collagen along the glomerular basement membrane and Bowman's capsule in paraffin-embedded renal sections of controls. In the 9 patients with the ultrastructures suggestive of Alport syndrome (AS), 8 patients were classified as X-linked dominant type due to the lack or mosaic pattern of alpha(5) chain in paraffin sections of renal biopsies by trypsin digestion, and 1 patient was classified as autosomal recessive type due to the lack of alpha(5) chain in the glomerular basement membrane only. Trypsin digestion is useful for the diagnosis of AS in paraffin-embedded renal tissue.


Asunto(s)
Antígenos/metabolismo , Colágeno Tipo IV/metabolismo , Nefritis Hereditaria/diagnóstico , Tripsina/metabolismo , Adolescente , Adulto , Antígenos/química , Biomarcadores/análisis , Biomarcadores/metabolismo , Cápsula Glomerular/ultraestructura , Niño , Preescolar , Cromosomas Humanos X , Colágeno Tipo IV/análisis , Femenino , Ligamiento Genético , Membrana Basal Glomerular/ultraestructura , Humanos , Riñón/química , Riñón/metabolismo , Riñón/patología , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Nefritis Hereditaria/enzimología , Nefritis Hereditaria/genética , Adhesión en Parafina , Tripsina/química
5.
Am J Pathol ; 172(3): 761-73, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18258846

RESUMEN

Previous work has shown that integrin alpha1-null Alport mice exhibit attenuated glomerular disease with decreased matrix accumulation and live much longer than strain-matched Alport mice. However, the mechanism underlying this observation is unknown. Here we show that glomerular gelatinase expression, specifically matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14, was significantly elevated in both integrin alpha1-null mice and integrin alpha1-null Alport mice relative to wild-type mice; however, only MMP-9 was elevated in glomeruli of Alport mice that express integrin alpha1. Similarly, cultured mesangial cells from alpha1-null mice showed elevated expression levels of all three MMPs, whereas mesangial cells from Alport mice show elevated expression levels of only MMP-9. In both glomeruli and cultured mesangial cells isolated from integrin alpha1-null mice, activation of the p38 and ERK branches of the mitogen-activated protein kinase pathway was also observed. The use of small molecule inhibitors demonstrated that the activation of the p38, but not ERK, pathway was linked to elevated MMP-2, -9, and -14 expression levels in mesangial cells from integrin alpha1-null mice. In contrast, elevated MMP-9 levels in mesangial cells from Alport mice were linked to ERK pathway activation. Blockade of gelatinase activity using a small molecule inhibitor (BAY-12-9566) ameliorated progression of proteinuria and restored the architecture of the glomerular basement membrane in alpha1 integrin-null Alport mice, suggesting that elevated gelatinase activity exacerbates glomerular disease progression in these mice.


Asunto(s)
Regulación Enzimológica de la Expresión Génica , Integrina alfa1beta1/fisiología , Metaloproteinasas de la Matriz/genética , Células Mesangiales/enzimología , Nefritis Hereditaria/genética , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Animales , Autoantígenos/genética , Compuestos de Bifenilo , Células Cultivadas , Colágeno Tipo IV/genética , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Integrina alfa1beta1/genética , Metaloproteinasas de la Matriz/metabolismo , Células Mesangiales/metabolismo , Ratones , Ratones Noqueados , Nefritis Hereditaria/enzimología , Nefritis Hereditaria/patología , Compuestos Orgánicos/farmacología , Fenilbutiratos , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidores Tisulares de Metaloproteinasas/metabolismo
6.
Am J Pathol ; 169(1): 32-46, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16816359

RESUMEN

Alport syndrome is a glomerular basement membrane (GBM) disease caused by mutations in type IV collagen genes. A unique irregular thickening and thinning of the GBM characterizes the progressive glomerular pathology. The metabolic imbalances responsible for these GBM irregularities are not known. Here we show that macrophage metalloelastase (MMP-12) expression is >40-fold induced in glomeruli from Alport mice and is markedly induced in glomeruli of both humans and dogs with Alport syndrome. Treatment of Alport mice with MMI270 (CGS27023A), a broad spectrum MMP inhibitor that blocks MMP-12 activity, results in largely restored GBM ultrastructure and function. Treatment with BAY-129566, a broad spectrum MMP inhibitor that does not inhibit MMP-12, had no effect. We show that inhibition of CC chemokine receptor 2 (CCR2) receptor signaling with propagermanium blocks induction of MMP-12 mRNA and prevents GBM damage. CCR2 receptor is expressed in glomerular podocytes of Alport mice, suggesting MCP-1 activation of CCR2 on podocytes may underlie induction of MMP-12. These data indicate that the irregular GBM that characterizes Alport syndrome may be mediated, in part, by focal degradation of the GBM due to MMP dysregulation, in particular, MMP-12. Thus, MMP-12/CCR2 inhibitors may provide a novel and effective therapeutic stra-tegy for Alport glomerular disease.


Asunto(s)
Membrana Basal Glomerular/patología , Metaloendopeptidasas/metabolismo , Nefritis Hereditaria/enzimología , Nefritis Hereditaria/patología , Animales , Northern Blotting , Western Blotting , Inhibidores Enzimáticos/farmacología , Membrana Basal Glomerular/efectos de los fármacos , Humanos , Inmunohistoquímica , Hibridación in Situ , Metaloproteinasa 12 de la Matriz , Metaloendopeptidasas/efectos de los fármacos , Ratones , Microscopía Electrónica de Transmisión , Receptores CCR2 , Receptores de Quimiocina/efectos de los fármacos , Receptores de Quimiocina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
Am J Pathol ; 166(5): 1465-74, 2005 May.
Artículo en Inglés | MEDLINE | ID: mdl-15855646

RESUMEN

Alport syndrome results from mutations in genes encoding collagen alpha3(IV), alpha4(IV), or alpha5(IV) and is characterized by progressive glomerular disease associated with a high-frequency sensorineural hearing loss. Earlier studies of a gene knockout mouse model for Alport syndrome noted thickening of strial capillary basement membranes in the cochlea, suggesting that the stria vascularis is the primary site of cochlear pathogenesis. Here we combine a novel cochlear microdissection technique with molecular analyses to illustrate significant quantitative alterations in strial expression of mRNAs encoding matrix metalloproteinases-2, -9, -12, and -14. Gelatin zymography of extracts from the stria vascularis confirmed these findings. Treatment of Alport mice with a small molecule inhibitor of these matrix metalloproteinases exacerbated strial capillary basement membrane thickening, demonstrating that alterations in basement membrane metabolism result in matrix accumulation in the strial capillary basement membranes. This is the first demonstration of true quantitative analysis of specific mRNAs for matrix metalloproteinases in a cochlear microcompartment. Further, these data suggest that the altered basement membrane composition in Alport stria influences the expression of genes involved in basement membrane metabolism.


Asunto(s)
Metaloproteinasas de la Matriz/metabolismo , Nefritis Hereditaria/enzimología , Estría Vascular/enzimología , Animales , Membrana Basal/patología , Capilares/efectos de los fármacos , Capilares/patología , Sistemas de Computación , Ácidos Hidroxámicos/farmacología , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/genética , Ratones , Ratones Noqueados , Nefritis Hereditaria/patología , Inhibidores de Proteasas/farmacología , Pirazinas/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estría Vascular/efectos de los fármacos , Sulfonamidas/farmacología
9.
Am J Pathol ; 157(1): 303-11, 2000 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10880400

RESUMEN

Matrix metalloproteinases are matrix degrading enzymes implicated in many biological processes, including development and inflammation. Gelatinase B (gelB; also known as MMP-9) is expressed in the kidney and is hypothesized to be involved in basement membrane remodeling and in preventing pathogenic accumulation of extracellular matrix in the kidney. Inhibition of gelB activity in metanephric organ culture disrupts branching morphogenesis of the ureteric bud, suggesting that gelB plays a role in kidney development in vivo. We studied kidneys of gelB-deficient mice to search for developmental, histological, molecular, ultrastructural, and functional defects. Surprisingly, no differences between gelB-/- and control kidneys were detected, and renal function was normal in gelB mutants. In addition, gelB-/- embryonic kidneys developed normally in organ culture. Gelatinase B-deficient mice were bred with Col4a3-/- mice, a model for Alport syndrome, to determine whether gelB influences the progression of glomerulonephritis. This is an important question, as it has been hypothesized that proteases are involved in damaging Alport glomerular basement membrane. However, the presence or absence of gelB did not affect the rate of progression of renal disease. Thus, gelB does not have a discernible role in the normal kidney and gelB is not involved in the progression of glomerulonephritis in a mouse model of Alport syndrome.


Asunto(s)
Enfermedades Renales/patología , Riñón/embriología , Metaloproteinasa 9 de la Matriz/metabolismo , Nefritis Hereditaria/patología , Animales , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Colágeno/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genotipo , Riñón/enzimología , Riñón/ultraestructura , Enfermedades Renales/enzimología , Pruebas de Función Renal , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Noqueados , Nefritis Hereditaria/enzimología , Técnicas de Cultivo de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo
10.
Genomics ; 47(3): 350-8, 1998 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-9480748

RESUMEN

We observed a family in which two boys were diagnosed with Alport syndrome, elliptocytosis, and mental retardation and carried a large deletion of the Xq22.3-q23 region, encompassing the COL4A5 gene. This suggests the possibility of a new contiguous gene syndrome. In an attempt to characterize the genes contributing to this complex phenotype, we have isolated a gene encoding a new long-chain acyl-CoA synthetase (FACL4 or LACS4) from the region deleted in these patients. Among several ESTs identified by searching the human gene map database maintained at the National Center for Biotechnology Information, using the map position as a query, only one was deleted in the patients. RACE products containing the entire ORF were subsequently generated. Northern blot analysis showed a 5-kb mRNA expressed in several tissues except for liver and lung. Brain shows a longer transcript, possibly reflecting the use of a brain-specific upstream ATG start codon. FACL4 encodes a predicted protein product of 670 amino acids (711 in brain), with a remarkable level of conservation compared to the rat acyl-CoA synthetases ACS4 and brain-specific ACS3 protein sequences. We are investigating the possibility that the absence of this enzyme may play a role in the development of mental retardation or other signs associated with Alport syndrome in the family.


Asunto(s)
Coenzima A Ligasas/genética , Eliptocitosis Hereditaria/genética , Eliminación de Gen , Discapacidad Intelectual/genética , Nefritis Hereditaria/genética , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Preescolar , Mapeo Cromosómico , Coenzima A Ligasas/biosíntesis , Eliptocitosis Hereditaria/enzimología , Humanos , Discapacidad Intelectual/enzimología , Masculino , Datos de Secuencia Molecular , Familia de Multigenes , Nefritis Hereditaria/enzimología , Cromosoma X
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