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1.
Hum Mol Genet ; 31(9): 1357-1369, 2022 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-34740236

RESUMEN

Nephronophthisis-related ciliopathies (NPHP-RC) comprises a group of inherited kidney diseases, caused by mutations in genes encoding proteins localizing to primary cilia. NPHP-RC represents one of the most frequent monogenic causes of renal failure within the first three decades of life, but its molecular disease mechanisms remain unclear. Here, we identified biallelic ANKS6 mutations in two affected siblings with late-onset chronic kidney disease by whole-exome sequencing. We employed patient-derived fibroblasts generating an in vitro model to study the precise biological impact of distinct human ANKS6 mutations, completed by immunohistochemistry studies on renal biopsy samples. Functional studies using patient-derived cells showed an impaired integrity of the ciliary inversin compartment with reduced cilia length. Further analyses demonstrated that ANKS6 deficiency leads to a dysregulation of Hippo-signaling through nuclear yes-associated protein (YAP) imbalance and disrupted ciliary localization of YAP. In addition, an altered transcriptional activity of canonical Wnt target genes and altered expression of non-phosphorylated (active) ß-catenin and phosphorylated glycogen synthase kinase 3ß were observed. Upon ciliation, ANKS6 deficiency revealed a deranged subcellular localization and expression of components of the endocytic recycling compartment. Our results demonstrate that ANKS6 plays a key role in regulating the Hippo pathway, and ANKS6 deficiency is linked to dysregulation of signaling pathways. Our study provides molecular clues in understanding pathophysiological mechanisms of NPHP-RC and may offer new therapeutic targets.


Asunto(s)
Ciliopatías , Enfermedades Renales Quísticas , Enfermedades Renales Poliquísticas , Insuficiencia Renal Crónica , Cilios/patología , Ciliopatías/metabolismo , Femenino , Humanos , Enfermedades Renales Quísticas/metabolismo , Masculino , Mutación , Proteínas Nucleares , Enfermedades Renales Poliquísticas/genética
2.
Kidney Int ; 102(2): 405-420, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35643372

RESUMEN

Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD) is caused by mutations in one of at least five genes and leads to kidney failure usually in mid adulthood. Throughout the literature, variable numbers of families have been reported, where no mutation can be found and therefore termed ADTKD-not otherwise specified. Here, we aim to clarify the genetic cause of their diseases in our ADTKD registry. Sequencing for all known ADTKD genes was performed, followed by SNaPshot minisequencing for the dupC (an additional cytosine within a stretch of seven cytosines) mutation of MUC1. A virtual panel containing 560 genes reported in the context of kidney disease (nephrome) and exome sequencing were then analyzed sequentially. Variants were validated and tested for segregation. In 29 of the 45 registry families, mutations in known ADTKD genes were found, mostly in MUC1. Sixteen families could then be termed ADTKD-not otherwise specified, of which nine showed diagnostic variants in the nephrome (four in COL4A5, two in INF2 and one each in COL4A4, PAX2, SALL1 and PKD2). In the other seven families, exome sequencing analysis yielded potential disease associated variants in novel candidate genes for ADTKD; evaluated by database analyses and genome-wide association studies. For the great majority of our ADTKD registry we were able to reach a molecular genetic diagnosis. However, a small number of families are indeed affected by diseases classically described as a glomerular entity. Thus, incomplete clinical phenotyping and atypical clinical presentation may have led to the classification of ADTKD. The identified novel candidate genes by exome sequencing will require further functional validation.


Asunto(s)
Enfermedades Renales Poliquísticas , Riñón Poliquístico Autosómico Dominante , Adulto , Pruebas Genéticas , Estudio de Asociación del Genoma Completo , Humanos , Mutación , Enfermedades Renales Poliquísticas/genética , Riñón Poliquístico Autosómico Dominante/genética
3.
PLoS Genet ; 15(4): e1008088, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-31034465

RESUMEN

PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2. At the cellular level, PIK3C2A is critical for the formation of cilia and for receptor mediated endocytosis, among other biological functions. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity. Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. In particular, the considerable phenotypic overlap, yet distinct features, between this syndrome and Lowe's syndrome, which is caused by mutations in the PI-5-phosphatase OCRL, highlight the key role of PI metabolizing enzymes in specific developmental processes and demonstrate the unique non-redundant functions of each enzyme. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease.


Asunto(s)
Enfermedades del Desarrollo Óseo/genética , Catarata/genética , Trastornos de la Motilidad Ciliar/genética , Enanismo/genética , Mutación , Fosfatidilinositol 3-Quinasas/genética , Adolescente , Adulto , Niño , Consanguinidad , Femenino , Fibroblastos/metabolismo , Humanos , Masculino , Linaje , Fenotipo , Adulto Joven
4.
Transpl Int ; 34(7): 1226-1238, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33904183

RESUMEN

Antibody-mediated rejection (AMR) is a major obstacle to long-term kidney transplantation. AMR is mostly caused by donor specific HLA antibodies, which can arise before or any time after transplantation. Incomplete donor HLA typing and unavailability of donor DNA regularly preclude the assessment of donor-specificity of circulating anti-HLA antibodies. In our centre, this problem arises in approximately 20% of all post-transplant HLA-antibody assessments. We demonstrate that this diagnostic challenge can be resolved by establishing donor renal tubular cell cultures from recipient´s urine as a source of high-quality donor DNA. DNA was then verified for genetic origin and purity by fluorescence in situ hybridization and short tandem repeat analysis. Two representative cases highlight the diagnostic value of this approach which is corroborated by analysis of ten additional patients. The latter were randomly sampled from routine clinical care patients with available donor DNA as controls. In all 12 cases, we were able to perform full HLA typing of the respective donors confirmed by cross-comparison to results from the stored 10 donor DNAs. We propose that this noninvasive diagnostic approach for HLA typing in kidney transplant patients is valuable to determine donor specificity of HLA antibodies, which is important in clinical assessment of suspected AMR.


Asunto(s)
Trasplante de Riñón , Rechazo de Injerto/diagnóstico , Antígenos HLA , Prueba de Histocompatibilidad , Humanos , Hibridación Fluorescente in Situ , Isoanticuerpos , Estudios Retrospectivos , Donantes de Tejidos
5.
Am J Transplant ; 20(5): 1410-1416, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-31814324

RESUMEN

In light of the organ shortage, there is a great responsibility to assess postmortal organs for which procurement has been consented and to increase the life span of transplanted organs. The former responsibility has moved many centers to accept extended criteria organs. The latter responsibility requires an exact diagnosis and, if possible, omission of the harmful influence on the transplant. We report the course of a kidney transplant that showed a steady decline of function over a decade, displaying numerous cysts of different sizes. Clinical workup excluded the most frequent causes of chronic transplant failure. The filed allocation documents mentioned the donor's disease of oral-facial-digital syndrome, a rare ciliopathy, which can also affect the kidney. Molecular diagnosis was performed by culturing donor tubular cells from the recipient´s urine more than 10 years after transplantation. Next-generation panel sequencing with DNA from tubular urinary cells revealed a novel truncating mutation in OFD1, which sufficiently explains the features of the kidney transplants, also found in the second kidney allograft. Despite this severe donor disease, lifesaving transplantation with good long-term outcome was enabled for 5 recipients.


Asunto(s)
Fallo Renal Crónico , Trasplante de Riñón , Obtención de Tejidos y Órganos , Supervivencia de Injerto , Humanos , Riñón , Fallo Renal Crónico/cirugía , Trasplante de Riñón/efectos adversos , Complicaciones Posoperatorias , Donantes de Tejidos
6.
PLoS Genet ; 13(3): e1006620, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-28267784

RESUMEN

Tubulointerstitial kidney disease is an important cause of progressive renal failure whose aetiology is incompletely understood. We analysed a large pedigree with maternally inherited tubulointerstitial kidney disease and identified a homoplasmic substitution in the control region of the mitochondrial genome (m.547A>T). While mutations in mtDNA coding sequence are a well recognised cause of disease affecting multiple organs, mutations in the control region have never been shown to cause disease. Strikingly, our patients did not have classical features of mitochondrial disease. Patient fibroblasts showed reduced levels of mitochondrial tRNAPhe, tRNALeu1 and reduced mitochondrial protein translation and respiration. Mitochondrial transfer demonstrated mitochondrial transmission of the defect and in vitro assays showed reduced activity of the heavy strand promoter. We also identified further kindreds with the same phenotype carrying a homoplasmic mutation in mitochondrial tRNAPhe (m.616T>C). Thus mutations in mitochondrial DNA can cause maternally inherited renal disease, likely mediated through reduced function of mitochondrial tRNAPhe.


Asunto(s)
ADN Mitocondrial/genética , Enfermedades Renales/genética , Túbulos Renales/patología , Mutación , Acetilglucosaminidasa/orina , Biopsia , Femenino , Fibroblastos/metabolismo , Ligamiento Genético , Humanos , Leucina/química , Masculino , Mitocondrias/metabolismo , Consumo de Oxígeno , Linaje , Fenotipo , Fenilalanina/química , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Músculo Cuádriceps/patología , ARN de Transferencia/genética
7.
J Am Soc Nephrol ; 29(9): 2298-2309, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30049680

RESUMEN

BACKGROUND: Providing the correct diagnosis for patients with tubulointerstitial kidney disease and secondary degenerative disorders, such as hypertension, remains a challenge. The autosomal dominant tubulointerstitial kidney disease (ADTKD) subtype caused by MUC1 mutations (ADTKD-MUC1) is particularly difficult to diagnose, because the mutational hotspot is a complex repeat domain, inaccessible with routine sequencing techniques. Here, we further evaluated SNaPshot minisequencing as a technique for diagnosing ADTKD-MUC1 and assessed immunodetection of the disease-associated mucin 1 frameshift protein (MUC1-fs) as a nongenetic technique. METHODS: We re-evaluated detection of MUC1 mutations by targeted repeat enrichment and SNaPshot minisequencing by haplotype reconstruction via microsatellite analysis in three independent ADTKD-MUC1 families. Additionally, we generated rabbit polyclonal antibodies against MUC1-fs and evaluated immunodetection of wild-type and mutated allele products in human kidney biopsy specimens. RESULTS: The detection of MUC1 mutations by SNaPshot minisequencing was robust. Immunostaining with our MUC1-fs antibodies and an MUC1 antibody showed that both proteins are readily detectable in human ADTKD-MUC1 kidneys, with mucin 1 localized to the apical membrane and MUC1-fs abundantly distributed throughout the cytoplasm. Notably, immunohistochemical analysis of MUC1-fs expression in clinical kidney samples facilitated reliable prediction of the disease status of individual patients. CONCLUSIONS: Diagnosing ADTKD-MUC1 by molecular genetics is possible, but it is technically demanding and labor intensive. However, immunohistochemistry on kidney biopsy specimens is feasible for nongenetic diagnosis of ADTKD-MUC1 and therefore, a valid method to select families for further diagnostics. Our data are compatible with the hypothesis that specific molecular effects of MUC1-fs underlie the pathogenesis of this disease.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad/epidemiología , Mucina-1/genética , Mutación/genética , Riñón Poliquístico Autosómico Dominante/genética , Adulto , Alelos , Animales , Biopsia con Aguja , Estudios de Cohortes , Femenino , Haplotipos , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Nefritis Intersticial/genética , Nefritis Intersticial/patología , Linaje , Riñón Poliquístico Autosómico Dominante/patología , Conejos , Estudios Retrospectivos , Medición de Riesgo , Sensibilidad y Especificidad
8.
Kidney Int ; 88(6): 1283-1292, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26200943

RESUMEN

Reduced nephron number predisposes to hypertension and kidney disease. Interaction of the branching ureteric bud and surrounding mesenchymal cells determines nephron number. Since oxygen supply may be critical for intrauterine development, we tested whether hypoxia and hypoxia-inducible factor-1α (HIF-1α) influence nephrogenesis. We found that HIF-1α is required for branching of MDCK cells. In addition, culture of metanephric mouse kidneys with ureteric bud cell-specific stabilization or knockout of HIF-1α revealed a positive impact of HIF-1α on nephrogenesis. In contrast, widespread stabilization of HIF-1α in metanephric kidneys through hypoxia or HIF stabilizers impaired nephrogenesis, and pharmacological HIF inhibition enhanced nephrogenesis. Several lines of evidence suggest an inhibitory effect through the hypoxia response of mesenchymal cells. HIF-1α was expressed in mesenchymal cells during nephrogenesis. Expression of the anti-branching factors Bmp4 and Vegfa, secreted by mesenchymal cells, was increased upon HIF stabilization. The conditioned medium from hypoxic metanephric kidneys inhibited MDCK branching, which was partially rescued by Vegfa antibodies. Thus, the effect of HIF-1α on nephrogenesis appears context dependent. While HIF-1α in the ureteric bud is of importance for proper branching morphogenesis, the net effect of hypoxia-induced HIF activation in the embryonic kidney appears to be mesenchymal cell-dependent inhibition of ureter branching.

9.
Kidney Blood Press Res ; 40(5): 443-51, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26304832

RESUMEN

BACKGROUND/AIMS: Hypercalcemia can result in nephrocalcinosis/nephrolithiasis and may lead to renal failure. Idiopathic infantile hypercalcemia is caused by mutations of the CYP24A1 gene, which regulates vitamin D activity. Classically infants present with hypercalcemia. Recently, a number of individuals have been reported with late onset clinical manifestation or late diagnosis in adulthood. All these patients are believed to show hypercalciuria. METHODS: We report a 24 year old patient of healthy consanguine parents. Genetic analysis was performed by Sanger sequencing of the CYP24A1 gene in the index patient and targeted exon 2 analysis of all other family members. RESULTS: The patient was hospitalized with severe malaise during an acute EBV-infection. He showed hypercalcemia > 3mmol/l and acute, hypovolemic renal failure with profound nephrocalcinosis, but no hypercalciuria. Genetic workup revealed a homozygous loss-of-function mutation p.E143del in the CYP24A1 gene. His clinically asymptomatic brother showed nephrocalcinosis of lesser degree. Repeatedly, low parathyroid hormone levels were detected in both brothers. CONCLUSION: This family displays the highly variable phenotype of CYP24A1 biallelic mutation carriers. CYP24A1 associated disease is an important differential diagnosis for the workup and counseling of infants as well as adults with hypercalcemia since a proper genetic diagnosis may result in therapeutic consequences.


Asunto(s)
Hipercalcemia/diagnóstico , Hipercalcemia/genética , Nefrocalcinosis/diagnóstico , Nefrocalcinosis/genética , Hermanos , Vitamina D3 24-Hidroxilasa/genética , Adulto , Diagnóstico Diferencial , Humanos , Hipercalcemia/complicaciones , Masculino , Nefrocalcinosis/complicaciones , Linaje , Adulto Joven
11.
Kidney Int ; 86(3): 589-99, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24670410

RESUMEN

For decades, ill-defined autosomal dominant renal diseases have been reported, which originate from tubular cells and lead to tubular atrophy and interstitial fibrosis. These diseases are clinically indistinguishable, but caused by mutations in at least four different genes: UMOD, HNF1B, REN, and, as recently described, MUC1. Affected family members show renal fibrosis in the biopsy and gradually declining renal function, with renal failure usually occurring between the third and sixth decade of life. Here we describe 10 families and define eligibility criteria to consider this type of inherited disease, as well as propose a practicable approach for diagnosis. In contrast to what the frequently used term 'Medullary Cystic Kidney Disease' implies, development of (medullary) cysts is neither an early nor a typical feature, as determined by MRI. In addition to Sanger and gene panel sequencing of the four genes, we established SNaPshot minisequencing for the predescribed cytosine duplication within a distinct repeat region of MUC1 causing a frameshift. A mutation was found in 7 of 9 families (3 in UMOD and 4 in MUC1), with one indeterminate (UMOD p.T62P). On the basis of clinical and pathological characteristics we propose the term 'Autosomal Dominant Tubulointerstitial Kidney Disease' as an improved terminology. This should enhance recognition and correct diagnosis of affected individuals, facilitate genetic counseling, and stimulate research into the underlying pathophysiology.


Asunto(s)
Aberraciones Cromosómicas , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 1 , Túbulos Renales/patología , Mucina-1/genética , Nefritis Intersticial/genética , Nefritis Intersticial/patología , Uromodulina/genética , Atrofia , Femenino , Fibrosis , Haplotipos , Humanos , Imagen por Resonancia Magnética , Masculino , Linaje , Terminología como Asunto
12.
Hypertension ; 81(9): 1857-1868, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39005223

RESUMEN

BACKGROUND: Autosomal recessive renal tubular dysgenesis is a rare, usually fatal inherited disorder of the renin-angiotensis system (RAS). Herein, we report an adolescent individual experiencing an unknown chronic kidney disease and aim to provide novel insights into disease mechanisms. METHODS: Exome sequencing for a gene panel associated with renal disease was performed. The RAS was assessed by comprehensive biochemical analysis in blood. Renin expression was determined in primary tubular cells by quantitative polymerase chain reaction and in situ hybridization on kidney biopsy samples. Allele frequencies of heterozygous and biallelic deleterious variants were determined by analysis of the Genomics England 100,000 Genomes Project. RESULTS: The patient was delivered prematurely after oligohydramnios was detected during pregnancy. Postnatally, he recovered from third-degree acute kidney injury but developed chronic kidney disease stage G3b over time. Exome sequencing revealed a previously reported pathogenic homozygous missense variant, p.(Arg375Gln), in the AGT (angiotensinogen) gene. Blood AGT concentrations were low, but plasma renin concentration and gene expression in kidney biopsy, vascular, and tubular cells revealed strong upregulation of renin. Angiotensin II and aldosterone in blood were not abnormally elevated. CONCLUSIONS: Renal tubular dysgenesis may present as chronic kidney disease with a variable phenotype, necessitating broad genetic analysis for diagnosis. Functional analysis of the RAS in a patient with AGT mutation revealed novel insights regarding compensatory upregulation of renin in vascular and tubular cells of the kidney and in plasma in response to depletion of AGT substrate as a source of Ang II (similarly observed with hepatic AGT silencing for the treatment of hypertension).


Asunto(s)
Angiotensinógeno , Humanos , Angiotensinógeno/genética , Masculino , Adolescente , Sistema Renina-Angiotensina/genética , Sistema Renina-Angiotensina/fisiología , Progresión de la Enfermedad , Renina/genética , Renina/sangre , Renina/metabolismo , Mutación Missense/genética , Secuenciación del Exoma/métodos , Femenino , Túbulos Renales Proximales/anomalías , Anomalías Urogenitales
13.
Am J Physiol Renal Physiol ; 305(5): F734-44, 2013 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-23825075

RESUMEN

The inhibitor of apoptosis protein survivin is a bifunctional molecule that regulates cellular division and survival. We have previously shown that survivin protein can be found at high concentrations in the adult kidney, particularly in the proximal tubules. Here, survivin is localized primarily at the apical membrane, a pattern that may indicate absorption of the protein. Several proteins in primary urine are internalized by megalin, an endocytosis receptor, which is in principle found in the same localization as survivin. Immunolabeling for survivin in different species confirmed survivin signal localizing to the apical membrane of the proximal tubule. Immunoelectron microscopy also showed apical localization of survivin in human kidneys. Furthermore, in polarized human primary tubular cells endogenous as well as external recombinant survivin is stored in the apical region of the cells. Costaining of survivin and megalin by immunohistochemistry and immunoelectron microscopy confirmed colocalization. Finally, by surface plasmon resonance we were able to demonstrate that survivin binds megalin and cubilin and that megalin knockout mice lose survivin through the urine. Survivin accumulates at the apical membrane of the renal tubule by reuptake, which is achieved by the endocytic receptor megalin, collaborating with cubilin. For this to occur, survivin will have to circulate in the blood and be filtered into the primary urine. It is not known at this stage what the functional role of tubular survivin is. However, a small number of experimental and clinical reports implicate that renal survivin is important for functional integrity of the kidney.


Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Túbulos Renales Proximales/fisiología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/fisiología , Proteínas Represoras/metabolismo , Animales , Células Cultivadas , Humanos , Ratones , Ratones Noqueados , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/metabolismo , Ratas , Receptores de Superficie Celular/metabolismo , Survivin
14.
Biochim Biophys Acta ; 1813(1): 1-13, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21056597

RESUMEN

Hypoxia is a common pathogenic stress, which requires adaptive activation of the Hypoxia-inducible transcription factor (HIF). In concert transcriptional HIF targets enhance oxygen availability and simultaneously reduce oxygen demand, enabling survival in a hypoxic microenvironment. Here, we describe the characterization of a new HIF-1 target gene, Rab20, which is a member of the Rab family of small GTP-binding proteins, regulating intracellular trafficking and vesicle formation. Rab20 is directly regulated by HIF-1, resulting in rapid upregulation of Rab20 mRNA as well as protein under hypoxia. Furthermore, exogenous as well as endogenous Rab20 protein colocalizes with mitochondria. Knockdown studies reveal that Rab20 is involved in hypoxia induced apoptosis. Since mitochondria play a key role in the control of cell death, we suggest that regulating mitochondrial homeostasis in hypoxia is a key function of Rab20. Furthermore, our study implicates that cellular transport pathways play a role in oxygen homeostasis. Hypoxia-induced Rab20 may influence tissue homeostasis and repair during and after hypoxic stress.


Asunto(s)
Apoptosis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/patología , Mitocondrias/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Western Blotting , Caspasas/metabolismo , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Humanos , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Unión al GTP rab/antagonistas & inhibidores , Proteínas de Unión al GTP rab/genética
15.
J Am Soc Nephrol ; 22(11): 2004-15, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21921145

RESUMEN

Hypoxia-inducible transcription factors (HIF) protect cells against oxygen deprivation, and HIF stabilization before ischemia mitigates tissue injury. Because ischemic acute kidney injury (AKI) often involves the thick ascending limb (TAL), modulation of HIF in this segment may be protective. Here, we generated mice with targeted TAL deletion of the von Hippel-Lindau protein (Vhl), which mediates HIF degradation under normoxia, using Tamm-Horsfall protein (Thp)-driven Cre expression. These mice showed strong expression of HIF-1α in TALs but no changes in kidney morphology or function under control conditions. Deficiency of Vhl in the TAL markedly attenuated proximal tubular injury and preserved TAL function following ischemia-reperfusion, which may be partially a result of enhanced expression of glycolytic enzymes and lactate metabolism. These results highlight the importance of the thick ascending limb in the pathogenesis of AKI and suggest that pharmacologically targeting the HIF system may have potential to prevent and mitigate AKI.


Asunto(s)
Lesión Renal Aguda/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Asa de la Nefrona/fisiología , Uromodulina/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Lesión Renal Aguda/genética , Lesión Renal Aguda/metabolismo , Umbral Anaerobio/fisiología , Animales , Modelos Animales de Enfermedad , Eritropoyesis/fisiología , Glucólisis/fisiología , Integrasas/genética , Riñón/fisiología , Ratones , Ratones Mutantes , Nefritis/genética , Nefritis/metabolismo , Nefritis/fisiopatología , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Uromodulina/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo
16.
J Biol Chem ; 285(9): 6658-69, 2010 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-20026874

RESUMEN

Hypoxia has been shown to promote tumor metastasis and lead to therapy resistance. Recent work has demonstrated that hypoxia represses E-cadherin expression, a hallmark of epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved. Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia. In addition to the previously demonstrated LOX, we characterize LOXL2 as a direct transcriptional target of HIF-1. We demonstrate that activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Our data support a molecular pathway from hypoxia to cellular transformation. It includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.


Asunto(s)
Aminoácido Oxidorreductasas/fisiología , Cadherinas/antagonistas & inhibidores , Transformación Celular Neoplásica , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Hipoxia/metabolismo , Proteína-Lisina 6-Oxidasa/fisiología , Aminoácido Oxidorreductasas/genética , Línea Celular , Células Epiteliales , Regulación Enzimológica de la Expresión Génica , Humanos , Hipoxia/enzimología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Mesodermo/citología , Metástasis de la Neoplasia , Proteína-Lisina 6-Oxidasa/genética , ARN Mensajero/análisis , Regulación hacia Arriba/genética
17.
FASEB J ; 24(11): 4443-58, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20624928

RESUMEN

Hypoxia-inducible protein 2 (HIG2) has been implicated in canonical Wnt signaling, both as target and activator. The potential link between hypoxia and an oncogenic signaling pathway might play a pivotal role in renal clear-cell carcinoma characterized by constitutive activation of hypoxia-inducible factors (HIFs), and hence prompted us to analyze HIG2 regulation and function in detail. HIG2 was up-regulated by hypoxia and HIF inducers in all cell types and mouse organs investigated and abundantly expressed in renal clear-cell carcinomas. Promoter analyses, gel shifts, and siRNA studies revealed that HIG2 is a direct and specific target of HIF-1, but not responsive to HIF-2. Surprisingly, HIG2 was not secreted, and HIG2 overexpression neither stimulated proliferation nor activated Wnt signaling. Instead, we show that HIG2 decorates the hemimembrane of lipid droplets, whose number and size increase on hypoxic inhibition of fatty acid ß-oxidation, and colocalizes with the lipid droplet proteins adipophilin and TIP47. Normoxic overexpression of HIG2 was sufficient to increase neutral lipid deposition in HeLa cells and stimulated cytokine expression. HIG2 could be detected in atherosclerotic arteries and fatty liver disease, suggesting that this ubiquitously inducible HIF-1 target gene may play an important functional role in diseases associated with pathological lipid accumulation.


Asunto(s)
Carcinoma de Células Renales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Renales/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Carcinoma de Células Renales/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Citocinas/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Hipoxia/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/farmacología , Neoplasias Renales/patología , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Transducción de Señal , Activación Transcripcional/genética , Proteína Wnt1/metabolismo
18.
J Am Soc Nephrol ; 21(12): 2151-6, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21115615

RESUMEN

The reasons for inadequate production of erythropoietin (EPO) in patients with ESRD are poorly understood. A better understanding of EPO regulation, namely oxygen-dependent hydroxylation of the hypoxia-inducible transcription factor (HIF), may enable targeted pharmacological intervention. Here, we tested the ability of fibrotic kidneys and extrarenal tissues to produce EPO. In this phase 1 study, we used an orally active prolyl-hydroxylase inhibitor, FG-2216, to stabilize HIF independent of oxygen availability in 12 hemodialysis (HD) patients, six of whom were anephric, and in six healthy volunteers. FG-2216 increased plasma EPO levels 30.8-fold in HD patients with kidneys, 14.5-fold in anephric HD patients, and 12.7-fold in healthy volunteers. These data demonstrate that pharmacologic manipulation of the HIF system can stimulate endogenous EPO production. Furthermore, the data indicate that deranged oxygen sensing--not a loss of EPO production capacity--causes renal anemia.


Asunto(s)
Eritropoyetina/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/efectos de los fármacos , Fallo Renal Crónico/metabolismo , Fallo Renal Crónico/terapia , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Administración Oral , Adulto , Anciano , Anciano de 80 o más Años , Anemia/prevención & control , Eritropoyetina/sangre , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Nefrectomía , Procolágeno-Prolina Dioxigenasa/administración & dosificación , Procolágeno-Prolina Dioxigenasa/farmacocinética , Valores de Referencia , Diálisis Renal , Medición de Riesgo , Resultado del Tratamiento
19.
Science ; 374(6573): eabk0410, 2021 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-34882480

RESUMEN

Cytokinetic membrane abscission is a spatially and temporally regulated process that requires ESCRT (endosomal sorting complexes required for transport)­dependent control of membrane remodeling at the midbody, a subcellular organelle that defines the cleavage site. Alteration of ESCRT function can lead to cataract, but the underlying mechanism and its relation to cytokinesis are unclear. We found a lens-specific cytokinetic process that required PI3K-C2α (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2α), its lipid product PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate), and the PI(3,4)P2­binding ESCRT-II subunit VPS36 (vacuolar protein-sorting-associated protein 36). Loss of each of these components led to impaired cytokinesis, triggering premature senescence in the lens of fish, mice, and humans. Thus, an evolutionarily conserved pathway underlies the cell type­specific control of cytokinesis that helps to prevent early onset cataract by protecting from senescence.


Asunto(s)
Catarata/patología , Senescencia Celular , Citocinesis , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Cristalino/citología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles/metabolismo , Envejecimiento Prematuro , Animales , Evolución Biológica , Proteínas de Unión al Calcio/metabolismo , Catarata/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Humanos , Cristalino/crecimiento & desarrollo , Cristalino/metabolismo , Ratones , Mutación , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Tubulina (Proteína)/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
20.
Mol Cancer Res ; 7(1): 88-98, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19147540

RESUMEN

The mammalian target of rapamycin (mTOR) regulates cellular growth and proliferation, mainly by controlling cellular translation. Most tumors show constitutive activation of the mTOR pathway. In hypoxia, mTOR is inactivated, which is believed to be part of the program of the cell to maintain energy homeostasis. However, certain proteins are believed to be preferentially translated during hypoxia via 5' terminal oligopyrimidine tract mechanisms with controversial discussion about the involvement of the mTOR-dependent ribosomal protein S6 (rpS6). The hypoxia-inducible transcription factor (HIF) is the master regulator of hypoxic adaptation and itself strongly implicated in tumor growth. HIF is translationally regulated by mTOR. The regulatory features and the involvement of molecular oxygen itself in this regulation of HIF by mTOR are poorly understood. mTOR inhibition leads to profound attenuation of HIFalpha protein in the majority of primary and cancer cells studied. Under severe hypoxia, no influence of mTOR inhibitors was observed; thus, stimulation of HIFalpha by mTOR may only be relevant under mild hypoxia or even normoxia. HIF expression and phosphorylated rpS6 negatively correlate in experimental tumors. In cell culture, prolonged hypoxia abolishes rpS6 phosphorylation, which seems to be partly independent of the upstream p70S6 kinase. We show that hypoxic repression of rpS6 is largely dependent on HIF, implicating a negative feedback loop, which may influence cellular translational rates and metabolic homeostasis. These data implicate that the hypoxic microenvironment renders tumor cells resistant to mTOR inhibition, at least concerning hypoxic gene activation, which would add to the difficulties of other established therapeutic strategies in hypoxic cancer tissues.


Asunto(s)
Hipoxia de la Célula/genética , Factor 1 Inducible por Hipoxia/biosíntesis , Proteínas Quinasas/genética , Línea Celular Tumoral , Células HeLa , Homeostasis , Humanos , Inmunohistoquímica , Luciferasas/genética , Consumo de Oxígeno , Biosíntesis de Proteínas , Ribonucleasas , Serina-Treonina Quinasas TOR , Transfección
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