RESUMEN
AIMS/HYPOTHESIS: Podocyte loss or injury is one of the earliest features observed in the pathogenesis of diabetic kidney disease (DKD), which is the leading cause of end-stage renal failure worldwide. Dysfunction in the IGF axis, including in IGF binding proteins (IGFBPs), is associated with DKD, particularly in the early stages of disease progression. The aim of this study was to investigate the potential roles of IGFBPs in the development of type 2 DKD, focusing on podocytes. METHODS: IGFBP expression was analysed in the Pima DKD cohort, alongside data from the Nephroseq database, and in ex vivo human glomeruli. Conditionally immortalised human podocytes and glomerular endothelial cells were studied in vitro, where IGFBP-1 expression was analysed using quantitative PCR and ELISAs. Cell responses to IGFBPs were investigated using migration, cell survival and adhesion assays; electrical cell-substrate impedance sensing; western blotting; and high-content automated imaging. RESULTS: Data from the Pima DKD cohort and from the Nephroseq database demonstrated a significant reduction in glomerular IGFBP-1 in the early stages of human type 2 DKD. In the glomerulus, IGFBP-1 was predominantly expressed in podocytes and controlled by phosphoinositide 3-kinase (PI3K)-forkhead box O1 (FoxO1) activity. In vitro, IGFBP-1 signalled to podocytes via ß1-integrins, resulting in increased phosphorylation of focal-adhesion kinase (FAK), increasing podocyte motility, adhesion, electrical resistance across the adhesive cell layer and cell viability. CONCLUSIONS/INTERPRETATION: This work identifies a novel role for IGFBP-1 in the regulation of podocyte function and that the glomerular expression of IGFBP-1 is reduced in the early stages of type 2 DKD, via reduced FoxO1 activity. Thus, we hypothesise that strategies to maintain glomerular IGFBP-1 levels may be beneficial in maintaining podocyte function early in DKD.
Asunto(s)
Diabetes Mellitus Tipo 2/patología , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Glomérulos Renales/metabolismo , Podocitos/metabolismo , Biopsia , Células Cultivadas , Estudios de Cohortes , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Integrina beta1/metabolismo , Riñón/metabolismo , Riñón/patología , Glomérulos Renales/patología , Podocitos/patología , Transducción de Señal/genéticaRESUMEN
Deregulation of GSK-3ß is strongly implicated in a variety of serious brain conditions, such as Alzheimer disease, bipolar disorder and schizophrenia. To understand how GSK-3ß becomes dysregulated in these conditions, it is important to understand its physiological functions in the central nervous system. In this context, GSK-3ß plays a role in the induction of NMDA receptor-dependent long-term depression (LTD) and several substrates for GSK-3ß have been identified in this form of synaptic plasticity, including KLC-2, PSD-95 and tau. Stabilization of NMDA receptors at synapses has also been shown to involve GSK-3ß, but the substrates involved are currently unknown. Recent work has identified phosphatidylinositol 4 kinase type IIα (PI4KIIα) as a neuronal GSK-3ß substrate that can potentially regulate the surface expression of AMPA receptors. In the present study, we investigated the synaptic role of PI4KIIα in organotypic rat hippocampal slices. We found that knockdown of PI4KIIα has no effect on synaptic AMPA receptor-mediated synaptic transmission but substantially reduces NMDA receptor-mediated synaptic transmission. Furthermore, the ability of the selective GSK-3 inhibitor, CT99021, to reduce the amplitude of NMDA receptor-mediated currents was occluded in shRNA-PI4KIIα transfected neurons. The effects of knocking down PI4KIIα were fully rescued by a shRNA-resistant wild-type construct, but not by a mutant construct that cannot be phosphorylated by GSK-3ß. These data suggest that GSK-3ß phosphorylates PI4KIIα to stabilize NMDA receptors at the synapse.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa , Receptores de N-Metil-D-Aspartato , Animales , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasa 3 beta , Hipocampo/metabolismo , Fosforilación , Ratas , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Insulin signaling to the glomerular podocyte via the insulin receptor (IR) is critical for kidney function. In this study we show that near-complete knockout of the closely related insulin-like growth factor 1 receptor (IGF1R) in podocytes is detrimental, resulting in albuminuria in vivo and podocyte cell death in vitro. In contrast, partial podocyte IGF1R knockdown confers protection against doxorubicin-induced podocyte injury. Proteomic analysis of cultured podocytes revealed that while near-complete loss of podocyte IGF1R results in the downregulation of mitochondrial respiratory complex I and DNA damage repair proteins, partial IGF1R inhibition promotes respiratory complex expression. This suggests that altered mitochondrial function and resistance to podocyte stress depends on the level of IGF1R suppression, the latter determining whether receptor inhibition is protective or detrimental. Our work suggests that the partial suppression of podocyte IGF1R could have therapeutic benefits in treating albuminuric kidney disease.
RESUMEN
We and others have previously shown that the neuropeptide galanin modulates neurite outgrowth from adult sensory neurons via activation of the second galanin receptor; however, the intracellular signalling pathways that mediate this neuritogenic effect have yet to be elucidated. Here, we demonstrate that galanin decreases the activation state in adult sensory neurons and PC12 cells of Rho and Cdc42 GTPases, both known regulators of filopodial and growth cone motility. Consistent with this, activated levels of Rho and Cdc42 levels are increased in the dorsal root ganglion of adult galanin knockout animals compared with wildtype controls. Furthermore, galanin markedly increases the activation state of cofilin, a downstream effector of many of the small GTPases, in the cell bodies and growth cones of sensory neurons and in PC12 cells. We also demonstrate a reduction in the activation of cofilin, and alteration in growth cone motility, in cultured galanin knockout neurons compared with wildtype controls. These data provide the first evidence that galanin regulates the Rho family of GTPases and cofilin to stimulate growth cone dynamics and neurite outgrowth in sensory neurons. These findings have important therapeutic implications for the treatment of peripheral sensory neuropathies.
Asunto(s)
Factores Despolimerizantes de la Actina/fisiología , Galanina/farmacología , Neuritas/efectos de los fármacos , Células Receptoras Sensoriales/efectos de los fármacos , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Animales , Western Blotting , Activación Enzimática/efectos de los fármacos , Femenino , Ganglios Espinales/citología , Conos de Crecimiento/efectos de los fármacos , Ratones , Ratones Noqueados , Microscopía por Video , Células PC12 , Seudópodos/efectos de los fármacos , Ratas , Estimulación Química , Proteínas de Unión al GTP rac/metabolismoRESUMEN
BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) is the leading cause of acute kidney injury in children, with an associated mortality of up to 5%. The mechanisms underlying STEC-HUS and why the glomerular microvasculature is so susceptible to injury following systemic Stx infection are unclear. METHODS: Transgenic mice were engineered to express the Stx receptor (Gb3) exclusively in their kidney podocytes (Pod-Gb3) and challenged with systemic Stx. Human glomerular cell models and kidney biopsies from patients with STEC-HUS were also studied. FINDINGS: Stx-challenged Pod-Gb3 mice developed STEC-HUS. This was mediated by a reduction in podocyte vascular endothelial growth factor A (VEGF-A), which led to loss of glomerular endothelial cell (GEnC) glycocalyx, a reduction in GEnC inhibitory complement factor H binding, and local activation of the complement pathway. Early therapeutic inhibition of the terminal complement pathway with a C5 inhibitor rescued this podocyte-driven, Stx-induced HUS phenotype. CONCLUSIONS: This study potentially explains why systemic Stx exposure targets the glomerulus and supports the early use of terminal complement pathway inhibition in this devastating disease. FUNDING: This work was supported by the UK Medical Research Council (MRC) (grant nos. G0901987 and MR/K010492/1) and Kidney Research UK (grant nos. TF_007_20151127, RP42/2012, and SP/FSGS1/2013). The Mary Lyon Center is part of the MRC Harwell Institute and is funded by the MRC (A410).
Asunto(s)
Infecciones por Escherichia coli , Síndrome Hemolítico-Urémico , Enfermedades Renales , Podocitos , Escherichia coli Shiga-Toxigénica , Niño , Humanos , Ratones , Animales , Podocitos/metabolismo , Podocitos/patología , Toxina Shiga/genética , Toxina Shiga/metabolismo , Toxina Shiga/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/uso terapéutico , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/metabolismo , Síndrome Hemolítico-Urémico/tratamiento farmacológico , Síndrome Hemolítico-Urémico/metabolismo , Síndrome Hemolítico-Urémico/patología , Escherichia coli Shiga-Toxigénica/metabolismo , Activación de Complemento , Enfermedades Renales/patologíaRESUMEN
The neuropeptide galanin is widely expressed in both the central and peripheral nervous systems and is involved in many diverse biological functions. There is a substantial data set that demonstrates galanin is upregulated after injury in the DRG, spinal cord and in many brain regions where it plays a predominantly antinociceptive role in addition to being neuroprotective and pro-regenerative. To further characterise the role of galanin following nerve injury, a novel transgenic line was created using the binary transgenic tet-off system, to overexpress galanin in galaninergic tissue in a suppressible manner. The double transgenic mice express significantly more galanin in the DRG one week after sciatic nerve section (axotomy) compared to WT mice and this overexpression is suppressible upon administration of doxycycline. Phenotypic analysis revealed markedly attenuated allodynia when galanin is overexpressed and an increase in allodynia following galanin suppression. This novel transgenic line demonstrates that whether galanin expression is increased at the time of nerve injury or only after allodynia is established, the neuropeptide is able to reduce neuropathic pain behaviour. These new findings imply that administration of a galanin agonist to patients with established allodynia would be an effective treatment for neuropathic pain.
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Galanina/metabolismo , Nociceptores/metabolismo , Animales , Axotomía , Modelos Animales de Enfermedad , Galanina/genética , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Ratones , Ratones Transgénicos , Neuralgia/metabolismo , Neuralgia/patología , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Expression of the neuropeptide galanin is up-regulated in many brain regions following nerve injury and in the basal forebrain of patients with Alzheimer's disease. We have previously demonstrated that galanin modulates hippocampal neuronal survival, although it was unclear which receptor subtype(s) mediates this effect. Here we report that the protective role played by galanin in hippocampal cultures is abolished in animals carrying a loss-of-function mutation in the second galanin receptor subtype (GalR2-MUT). Exogenous galanin stimulates the phosphorylation of the serine/threonine kinase Akt and extracellular signal-regulated kinase (ERK) in wild-type (WT) cultures by 435 +/- 5% and 278 +/- 2%, respectively. The glutamate-induced activation of Akt was abolished in cultures from galanin knockout animals, and was markedly attenuated in GalR2-MUT animals, compared with WT controls. In contrast, similar levels of glutamate-induced ERK activation were observed in both loss-of-function mutants, but were further increased in galanin over-expressing animals. Using specific inhibitors of either ERK or Akt confirms that a GalR2-dependent modulation in the activation of the Akt and ERK signalling pathways contributes to the protective effects of galanin. These findings imply that the rise in endogenous galanin observed either after brain injury or in various disease states is an adaptive response that reduces apoptosis by the activation of GalR2, and hence Akt and ERK.
Asunto(s)
Citoprotección/genética , Galanina/metabolismo , Hipocampo/metabolismo , Degeneración Nerviosa/metabolismo , Neuronas/metabolismo , Receptor de Galanina Tipo 2/metabolismo , Animales , Daño Encefálico Crónico/genética , Daño Encefálico Crónico/metabolismo , Daño Encefálico Crónico/fisiopatología , Citoprotección/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Galanina/farmacología , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/fisiopatología , Masculino , Ratones , Ratones Noqueados , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/fisiopatología , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Galanina Tipo 2/agonistas , Receptor de Galanina Tipo 2/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Expression of the neuropeptide galanin is markedly up-regulated within the adult dorsal root ganglia (DRG) following peripheral nerve injury. We have previously demonstrated that galanin knockout (Gal-KO) mice have a developmental loss of a subset of DRG neurons. Galanin also plays a trophic role in the adult animal, and the rate of peripheral nerve regeneration and neurite outgrowth is reduced in adult Gal-KO mice. Here we describe the characterization of mice with an absence of GalR2 gene transcription (GalR2-MUT) and demonstrate that they have a 15% decrease in the number of calcitonin gene-related peptide (CGRP) expressing neuronal profiles in the adult DRG, associated with marked deficits in neuropathic and inflammatory pain behaviours. Adult GalR2-MUT animals also have a one third reduction in neurite outgrowth from cultured DRG neurons that cannot be rescued by either galanin or a high-affinity GalR2/3 agonist. Galanin activates extracellular signal-regulated kinase (ERK) and Akt in adult wild-type (WT) mouse DRG. Intact adult DRG from GalR2-MUT animals have lower levels of pERK and higher levels of pAkt than are found in WT controls. These data suggest that a lack of GalR2 activation in Gal-KO and GalR2-MUT animals is responsible for the observed developmental deficits in the DRG, and the decrease in neurite outgrowth in the adult.
Asunto(s)
Conducta Animal/fisiología , Neuritas/fisiología , Neuronas Aferentes/fisiología , Dolor/genética , Dolor/psicología , Receptor de Galanina Tipo 2/genética , Receptor de Galanina Tipo 2/fisiología , Animales , Western Blotting , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Genotipo , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Traumatismos de los Nervios Periféricos , Fenotipo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/fisiología , Transcripción GenéticaRESUMEN
The neuropeptide galanin is expressed in the dorsal root ganglia (DRG) and spinal cord and is thought to be involved in the modulation of pain processing. However, its mechanisms of action are complex and poorly understood, as both facilitatory and inhibitory effects have been described. To understand further the role played by galanin in nociception, we have generated two transgenic lines that overexpress galanin in specific populations of primary afferent DRG neurons in either an inducible or constitutive manner. In the first line, a previously defined enhancer region from the galanin locus was used to target galanin to the DRG (Gal-OE). Transgene expression recapitulates the spatial endogenous galanin distribution pattern in DRG neurons and markedly overexpresses the peptide in the DRG after nerve injury but not in the uninjured state. In the second line, an enhancer region of the c-Ret gene was used to constitutively and ectopically target galanin overexpression to the DRG (Ret-OE). The expression of this second transgene does not alter significantly after nerve injury. Here, we report that intact Ret-OE, but not Gal-OE, animals have significantly elevated mechanical and thermal thresholds. After nerve damage, using a spared nerve-injury model, mechanical allodynia is attenuated markedly in both the Gal-OE and Ret-OE mice compared with WT controls. These results support an inhibitory role for galanin in the modulation of nociception both in intact animals and in neuropathic pain states.