RESUMEN
Podocytes are crucially involved in blood filtration in the glomerulus. Their proper function relies on efficient insulin responsiveness. The insulin resistance of podocytes, defined as a reduction of cell sensitivity to this hormone, is the earliest pathomechanism of microalbuminuria that is observed in metabolic syndrome and diabetic nephropathy. In many tissues, this alteration is mediated by the phosphate homeostasis-controlling enzyme nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). By binding to the insulin receptor (IR), NPP1 inhibits downstream cellular signaling. Our previous research found that hyperglycemic conditions affect another protein that is involved in phosphate balance, type III sodium-dependent phosphate transporter 1 (Pit 1). In the present study, we evaluated the insulin resistance of podocytes after 24 h of incubation under hyperinsulinemic conditions. Thereafter, insulin signaling was inhibited. The formation of NPP1/IR complexes was observed at that time. A novel finding in the present study was our observation of an interaction between NPP1 and Pit 1 after the 24 h stimulation of podocytes with insulin. After downregulation of the SLC20A1 gene, which encodes Pit 1, we established insulin resistance in podocytes that were cultured under native conditions, manifested as a lack of intracellular insulin signaling and the inhibition of glucose uptake via the glucose transporter type 4. These findings suggest that Pit 1 might be a major factor that participates in the NPP1-mediated inhibition of insulin signaling.
Asunto(s)
Nefropatías Diabéticas , Resistencia a la Insulina , Podocitos , Humanos , Insulina/farmacología , Insulina/metabolismo , Podocitos/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Nefropatías Diabéticas/metabolismo , Fosfatos/metabolismo , Glucosa/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismoRESUMEN
Podocytes are sensitive to insulin, which governs the functional and structural integrity of podocytes that are essential for proper function of the glomerular filtration barrier. Lysosomes are acidic organelles that are implicated in regulation of the insulin signaling pathway. Cathepsin D (CTPD) and lysosome-associated membrane protein 1 (LAMP1) are major lysosomal proteins that reflect the functional state of lysosomes. However, the effect of insulin on lysosome activity and role of lysosomes in the regulation of insulin-dependent glucose uptake in podocytes are unknown. Our studies showed that the short-term incubation of podocytes with insulin decreased LAMP1 and CTPD mRNA levels. Insulin and bafilomycin A1 reduced both the amounts of LAMP1 and CTPD proteins and activity of CTPD, which were associated with a decrease in the fluorescence intensity of lysosomes that were labeled with LysoTracker. Bafilomycin A1 inhibited insulin-dependent endocytosis of the insulin receptor and increased the amounts of the insulin receptor and glucose transporter 4 on the cell surface of podocytes. Bafilomycin A1 also inhibited insulin-dependent glucose uptake despite an increase in the amount of glucose transporter 4 in the plasma membrane of podocytes. These results suggest that lysosomes are signaling hubs that may be involved in the coupling of insulin signaling with the regulation of glucose uptake in podocytes. The dysregulation of this mechanism can lead to the dysfunction of podocytes and development of insulin resistance.
Asunto(s)
Podocitos , Ratas , Animales , Podocitos/metabolismo , Insulina/metabolismo , Receptor de Insulina/metabolismo , Factores de Transcripción/metabolismo , Lisosomas/metabolismo , Transducción de Señal , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismoRESUMEN
The permeability of the glomerular filtration barrier (GFB) is mainly regulated by podocytes and their foot processes. Protein kinase G type Iα (PKGIα) and adenosine monophosphate-dependent kinase (AMPK) affect the contractile apparatus of podocytes and influence the permeability of the GFB. Therefore, we studied the interplay between PKGIα and AMPK in cultured rat podocytes. The glomerular permeability to albumin and transmembrane FITC-albumin flux decreased in the presence of AMPK activators and increased in the presence of PKG activators. The knockdown of PKGIα or AMPK with small-interfering RNA (siRNA) revealed a mutual interaction between PKGIα and AMPK and influenced podocyte permeability to albumin. Moreover, PKGIα siRNA activated the AMPK-dependent signaling pathway. AMPKα2 siRNA increased basal levels of phosphorylated myosin phosphate target subunit 1 and decreased the phosphorylation of myosin light chain 2. Podocytes that were treated with AMPK or PKG activators were characterized by the different organization of actin filaments within the cell. Our findings suggest that mutual interactions between PKGIα and AMPKα2 regulate the contractile apparatus and permeability of the podocyte monolayer to albumin. Understanding this newly identified molecular mechanism in podocytes provides further insights into the pathogenesis of glomerular disease and novel therapeutic targets for glomerulopathies.
Asunto(s)
Albúminas , Proteína Quinasa Dependiente de GMP Cíclico Tipo I , Podocitos , Animales , Ratas , Adenosina Monofosfato/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Permeabilidad , Podocitos/metabolismo , Ratas Wistar , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Albúminas/metabolismoRESUMEN
Soft tissue calcification is a pathological phenomenon that often occurs in end-stage chronic kidney disease (CKD), which is caused by diabetic nephropathy, among other factors. Hyperphosphatemia present during course of CKD contributes to impairments in kidney function, particularly damages in the glomerular filtration barrier (GFB). Essential elements of the GFB include glomerular epithelial cells, called podocytes. In the present study, we found that human immortalized podocytes express messenger RNA and protein of phosphate transporters, including NaPi 2c (SLC34A3), Pit 1 (SLC20A1), and Pit 2 (SLC20A2), which are sodium-dependent and mediate intracellular phosphate (Pi) transport, and XPR1, which is responsible for extracellular Pi transport. We found that cells that were grown in a medium with a high glucose (HG) concentration (30 mM) expressed less Pit 1 and Pit 2 protein than podocytes that were cultured in a standard glucose medium (11 mM). We found that exposure of the analyzed transporters in the cell membrane of the podocyte is altered by HG conditions. We also found that the activity of tissue nonspecific alkaline phosphatase increased in HG, causing a rise in Pi generation. Additionally, HG led to a reduction of the amount of ectonucleotide pyrophosphatase/phosphodiesterase 1 in the cell membrane of podocytes. The extracellular concentration of pyrophosphate also decreased under HG conditions. These data suggest that a hyperglycemic environment enhances the production of Pi in podocytes and its retention in the extracellular space, which may induce glomerular calcification.
Asunto(s)
Calcinosis , Podocitos , Insuficiencia Renal Crónica , Calcinosis/metabolismo , Glucosa/metabolismo , Humanos , Glomérulos Renales/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Podocitos/metabolismo , Insuficiencia Renal Crónica/patología , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismoRESUMEN
Podocytes constitute the outer layer of the renal glomerular filtration barrier. Their energy requirements strongly depend on efficient oxidative respiration, which is tightly connected with mitochondrial dynamics. We hypothesized that hyperglycemia modulates energy metabolism in glomeruli and podocytes and contributes to the development of diabetic kidney disease. We found that oxygen consumption rates were severely reduced in glomeruli from diabetic rats and in human podocytes that were cultured in high glucose concentration (30 mM; HG). In these models, all of the mitochondrial respiratory parameters, including basal and maximal respiration, ATP production, and spare respiratory capacity, were significantly decreased. Podocytes that were treated with HG showed a fragmented mitochondrial network, together with a decrease in expression of the mitochondrial fusion markers MFN1, MFN2, and OPA1, and an increase in the activity of the fission marker DRP1. We showed that markers of mitochondrial biogenesis, such as PGC-1α and TFAM, decreased in HG-treated podocytes. Moreover, PINK1/parkin-dependent mitophagy was inhibited in these cells. These results provide evidence that hyperglycemia impairs mitochondrial dynamics and turnover, which may underlie the remarkable deterioration of mitochondrial respiration parameters in glomeruli and podocytes.
Asunto(s)
Hiperglucemia/metabolismo , Mitocondrias/metabolismo , Mitofagia/fisiología , Podocitos/metabolismo , Animales , Diabetes Mellitus Experimental/metabolismo , Humanos , Riñón/metabolismo , Masculino , Proteínas Quinasas/metabolismo , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Insulin plays a major role in regulating glucose homeostasis in podocytes. Protein kinase G type Iα (PKGIα) plays an important role in regulating glucose uptake in these cells. Rac1 signaling plays an essential role in the reorganization of the actin cytoskeleton and is also essential for insulin-stimulated glucose transport. The experiments were conducted using primary rat podocytes. We performed western blot analysis, evaluated small GTPases activity assays, measured radioactive glucose uptake, and performed immunofluorescence imaging to analyze the role of PKGIα-Rac1 signaling in regulating podocyte function. We also utilized a small-interfering RNA-mediated approach to determine the role of PKGIα and Rac1 in regulating glucose uptake in podocytes. The present study investigated the influence of the PKGI pathway on the insulin-dependent regulation of activity and cellular localization of small guanosine triphosphatases in podocytes. We found that the PKGIα-dependent activation of Rac1 signaling induced activation of the PAK/cofilin pathway and increased insulin-mediated glucose uptake in podocytes. The downregulation of PKGIα or Rac1 expression abolished this effect. Rac1 silencing prevented actin remodeling and GLUT4 translocation close to the cell membrane. These data provide evidence that PKGIα-dependent activation of the Rac1 signaling pathways is a novel regulator of insulin-mediated glucose uptake in cultured rat podocytes.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Insulina/farmacología , Podocitos/efectos de los fármacos , Proteína de Unión al GTP rac1/metabolismo , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Animales , Células Cultivadas , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Femenino , Podocitos/enzimología , Transporte de Proteínas , Ratas Wistar , Transducción de Señal , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rac1/genéticaRESUMEN
The protein deacetylase sirtuin 1 (SIRT1) and adenosine monophosphate-dependent protein kinase (AMPK) play important roles in the development of insulin resistance. In glomerular podocytes, crosstalk between these two enzymes may be altered under hyperglycemic conditions. SIRT1 protein levels and activity and AMPK phosphorylation decrease under hyperglycemic conditions, with concomitant inhibition of the effect of insulin on glucose uptake into these cells. Nitric oxide (NO)-dependent regulatory signaling pathways have been shown to be downregulated under diabetic conditions. The present study examined the involvement of the NO synthase (NOS)/NO pathway in the regulation of SIRT1-AMPK signaling and glucose uptake in podocytes. We examined the effects of NOS/NO pathway alterations on SIRT1/AMPK signaling and glucose uptake using pharmacological tools and a small-interfering transfection approach. We also examined the ability of the NOS/NO pathway to protect podocytes against high glucose-induced alterations of SIRT1/AMPK signaling and insulin-dependent glucose uptake. Inhibition of the NOS/NO pathway reduced SIRT1 protein levels and activity, leading to a decrease in AMPK phosphorylation and blockade of the effect of insulin on glucose uptake. Treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prevented high glucose-induced decreases in SIRT1 and AMPK activity and increased GLUT4 protein expression, thereby improving glucose uptake in podocytes. These findings suggest that inhibition of the NOS/NO pathway may result in alterations of the effects of insulin on glucose uptake in podocytes. In turn, the enhancement of NOS/NO pathway activity may prevent these deleterious effects of high glucose concentrations, thus bidirectionally stimulating the SIRT1-AMPK reciprocal activation loop.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Glucosa/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico/metabolismo , Podocitos/metabolismo , Sirtuina 1/metabolismo , Animales , Regulación hacia Abajo/fisiología , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/metabolismo , Resistencia a la Insulina/fisiología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Ratas , S-Nitroso-N-Acetilpenicilamina/farmacología , Transducción de Señal , Sirtuina 1/genéticaRESUMEN
BACKGROUND: Bladder cancer (BC) is one of the 10 most common types of cancer worldwide, with approximately 550,000 new cases each year. Early detection and appropriate diagnosis are important factors in successful treatment of the disease. MATERIAL AND METHODS: We used specific fluorogenic substrate for the quantitative determination of urine kallikrein 13 (KLK13) activity in healthy (n = 15) and BC (n = 54) patients. The proteolytic activity in individual urine samples was determined by fluorescence measurements. Then, immunoenzymatic analyses (ELISA, Western blot) were performed to confirm the presence of KLK13 in the tested samples. RESULTS: Urine samples from patients with G2 and G3 grade BC contained proteolytically active KLK13, as confirmed by kinetic analysis and immunochemical detection. KLK13 was not detected in the urine of patients with G1 grade BC. DISCUSSION: Previous clinical study reveals the KLK13 significance for BC prognosis as increased KLK13 expression was highlighted in bladder tumours compared to normal adjacent tissues. Our findings correlate to the report. KLK13 activity was confirmed in BC patients with G2 and G3 stage of disease development. CONCLUSIONS: Using specific chromogenic/fluorogenic peptides could be useful for the non-invasive disease monitoring of BC progress.
Asunto(s)
Biomarcadores de Tumor/orina , Calicreínas/orina , Neoplasias de la Vejiga Urinaria/orina , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Calicreínas/metabolismo , Cinética , Masculino , Persona de Mediana Edad , Proteolisis , Especificidad por Sustrato , Neoplasias de la Vejiga Urinaria/diagnóstico , Adulto JovenRESUMEN
Hyperglycemic conditions (HG), at early stages of diabetic nephropathy (DN), cause a decrease in podocyte numbers and an aberration of their function as key cells for glomerular plasma filtration. Klotho protein was shown to overcome some negative effects of hyperglycemia. Klotho is also a coreceptor for fibroblast growth factor receptors (FGFRs), the signaling of which, together with a proper rate of glycolysis in podocytes, is needed for a proper function of the glomerular filtration barrier. Therefore, we measured levels of Klotho in renal tissue, serum, and urine shortly after DN induction. We investigated whether it influences levels of FGFRs, rates of glycolysis in podocytes, and albumin permeability. During hyperglycemia, the level of membrane-bound Klotho in renal tissue decreased, with an increase in the shedding of soluble Klotho, its higher presence in serum, and lower urinary excretion. The addition of Klotho increased FGFR levels, especially FGFR1/FGFR2, after their HG-induced decrease. Klotho also increased levels of glycolytic parameters of podocytes, and decreased podocytic and glomerular albumin permeability in HG. Thus, we found that the decrease in the urinary excretion of Klotho might be an early biomarker of DN and that Klotho administration may have several beneficial effects on renal function in DN.
Asunto(s)
Glucuronidasa/metabolismo , Hiperglucemia/metabolismo , Podocitos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Glucólisis , Proteínas Klotho , Masculino , Permeabilidad , Ratas WistarRESUMEN
Podocytes and their foot processes interlinked by slit diaphragms, constitute a continuous outermost layer of the glomerular capillary and seem to be crucial for maintaining the integrity of the glomerular filtration barrier. Purinergic signaling is involved in a wide range of physiological processes in the renal system, including regulating glomerular filtration. We evaluated the role of nucleotide receptors in cultured rat podocytes using non-selective P2 receptor agonists and agonists specific for the P2Y1, P2Y2, and P2Y4 receptors. The results showed that extracellular ATP evokes cAMP-dependent pathways through P2 receptors and influences remodeling of the podocyte cytoskeleton and podocyte permeability to albumin via coupling with RhoA signaling. Our findings highlight the relevance of the P2Y4 receptor in protein kinase A-mediated signal transduction to the actin cytoskeleton. We observed increased cAMP concentration and decreased RhoA activity after treatment with a P2Y4 agonist. Moreover, protein kinase A inhibitors reversed P2Y4-induced changes in RhoA activity and intracellular F-actin staining. P2Y4 stimulation resulted in enhanced AMPK phosphorylation and reduced reactive oxygen species generation. Our findings identify P2Y-PKA-RhoA signaling as the regulatory mechanism of the podocyte contractile apparatus and glomerular filtration. We describe a protection mechanism for the glomerular barrier linked to reduced oxidative stress and reestablished energy balance.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/farmacocinética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Podocitos/metabolismo , Receptores Purinérgicos P2/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Animales , Femenino , Podocitos/citología , Ratas , Ratas Wistar , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Vasodilator-stimulated phosphoprotein (VASP) is a 39-kDa protein belonging to the Ena/VASP protein family, which is involved in adhesion, migration, cell-cell interaction, and regulation of pathways connected with actin cytoskeleton remodeling. VASP is phosphorylated at Tyr39, Ser157, Ser239, Thr278, and Ser322 mainly by tyrosine kinase Abl, cAMP-dependent protein kinase, protein kinase G, AMP-activated protein kinase, and protein kinase D1, respectively. VASP phosphorylation, as a regulator of actin dynamics, may lead to impaired reorganization of the podocyte actin cytoskeleton not only by indirect interaction of VASP with actin but also by regulation of other signaling pathways. A few studies have shown that VASP participates in the development of renal diseases and mediates podocyte movement through its interaction with proteins of the slit diaphragm. VASP phosphorylation may cause reduced actin filament assembly in podocytes and mediate disturbances in regulation of filtration barrier permeability as a consequence of podocyte foot process effacement. In this paper, we describe the role of VASP in podocyte function, mainly in the context of actin dynamics and glomerular filtration barrier permeability. In addition, we discuss the involvement of VASP and its phosphorylated forms in the development of kidney diseases.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Moléculas de Adhesión Celular/fisiología , Enfermedades Renales/metabolismo , Proteínas de Microfilamentos/fisiología , Fosfoproteínas/fisiología , Podocitos/metabolismo , Actinas/metabolismo , Animales , Humanos , Ratones , Fosforilación , Podocitos/citología , Podocitos/patología , RatasRESUMEN
BACKGROUND/AIMS: Podocytes are dynamic polarized cells on the surface of glomerular capillaries that are an essential part of the glomerular filtration barrier. AMP-activated protein kinase (AMPK), a key regulator of glucose and fatty acid metabolism, plays a major role in obesity and type 2 diabetes. Accumulating evidence suggests that TRPC6 channels are crucial mediators of calcium transport in podocytes and are involved in regulating glomerular filtration. Here we investigated whether the AMPK-TRPC6 pathway is involved in insulin-dependent cytoskeleton reorganization and glucose uptake in cultured rat podocytes. METHODS: Western blot and immunofluorescence analysis confirmed AMPKα and TRPC6 expression, the phosphorylation of proteins associated with actin cytoskeleton reorganization (PAK, rac1, and cofilin), and the expression of insulin signaling proteins (Akt, Insulin receptor). Coimmunoprecipitation and immunofluorescence results demonstrated AMPKα/TRPC6 interaction. To ask whether TRPC6 is involved in the insulin regulation of glucose transport, we measured insulin-dependent (1, 2-3H)-deoxy-D-glucose uptake into podocytes after reducing TRPC6 activity pharmacologically and biochemically (TRPC6 siRNA). RESULTS: The results suggested a key role for the TRPC6 channel in the mediation of insulin-dependent activation of AMPKα2 and glucose uptake. Moreover, AMPK and TRPC6 activation were required to stimulate the Rac1 signaling pathway. CONCLUSION: These results suggest a potentially important new mechanism that regulates glucose transport in podocytes and that could be injurious during diabetes.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Citoesqueleto/metabolismo , Glucosa/metabolismo , Insulina/farmacología , Transducción de Señal/efectos de los fármacos , Canal Catiónico TRPC6/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/química , Actinas/metabolismo , Animales , Calcio/metabolismo , Citoesqueleto/química , Fosforilación/efectos de los fármacos , Podocitos/citología , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Canal Catiónico TRPC6/antagonistas & inhibidores , Canal Catiónico TRPC6/genética , Proteína de Unión al GTP rac1/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
Podocyte insulin sensitivity is critical for glomerular function, and the loss of appropriate insulin signaling leads to alterations and disorders featuring diabetic nephropathy. Energy-sensing pathways, such as AMP-dependent protein kinase (AMPK) and protein deacetylase SIRT1, have been shown to play an important role in insulin resistance. The absence of a stimulating effect of insulin on glucose uptake into podocytes after exposure to hyperglycemic conditions has been demonstrated to be related to a decreased level and activity of SIRT1 protein, leading to reduced AMPK phosphorylation. The present work was undertaken to investigate metformin's ability to restore the insulin responsiveness of podocytes by regulating SIRT1 and AMPK activities. Primary rat podocytes cultured with standard or high glucose concentrations for 5days were transfected with siRNAs targeting SIRT1, AMPKα1, or AMPKα2. SIRT1 activity was measured by a fluorometric method. Insulin-stimulated changes in glucose uptake were used to detect insulin resistance. Podocyte permeability was measured by a transmembrane albumin flux assay to examine podocytes functioning. Our results demonstrated that metformin activated SIRT1 and AMPK, prevented hyperglycemia-induced reduction of SIRT1 protein levels, ameliorated glucose uptake into podocytes, and decreased glomerular filtration barrier permeability. Furthermore, metformin activated AMPK in a SIRT1-independent manner, as the increase in AMPK phosphorylation after metformin treatment was not affected by SIRT1 downregulation. Therefore, the potentiating effect of metformin on insulin-resistant podocytes seemed to be dependent on AMPK, as well as SIRT1 activity, establishing multilateral effects of metformin action.
Asunto(s)
Citoprotección/efectos de los fármacos , Glucosa/efectos adversos , Resistencia a la Insulina , Metformina/farmacología , Podocitos/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Células Cultivadas , Citoprotección/genética , Femenino , Resistencia a la Insulina/genética , Podocitos/metabolismo , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
Podocytes are dynamic polarized cells on the surface of glomerular capillaries and an essential component of the glomerular filtration barrier. Insulin increases the activation of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. In addition, accumulating evidence suggests that TRPC6 channels are crucial mediators of podocyte calcium handling and involved in the regulation of glomerular filtration. Therefore, we investigated whether TRPC6 is involved in the regulation of filtration barrier permeability by insulin via the PKGIα-dependent manner. TRPC channel inhibitor SKF96365 abolished insulin-dependent glomerular albumin permeability and transepithelial albumin flux in cultured rat podocytes. Insulin-evoked albumin permeability across podocyte monolayers was also blocked using TRPC6 siRNA. The effect of insulin on albumin permeability was mimicked by treating podocytes with TRPC channel activator (oleolyl-2-acetyl-sn-glycerol, OAG). Insulin or OAG treatment rapidly increased the superoxide generation through activation of NADH oxidase. TRPC inhibitor SKF96365 or siRNA knockdown of TRPC6 attenuated insulin-dependent increase of ROS production. Furthermore, TRPC inhibitor or downregulation of TRPC6 blocked insulin-induced rearrangement of the actin cytoskeleton and attenuated oxidative activation of PKGIα and changes in the phosphorylation of PKG target proteins MYPT1 and MLC. Moreover insulin regulated the PKGIα interaction with TRPC6 in cultured rat podocytes. Taken together, our data suggest a key role of TRPC6 channels in the mediation of insulin-dependent activation of PKGIα signaling pathways. Overall, we have identified a potentially important mechanism that may explain disturbances in filtration barrier permeability in many diseases with increased expression of TRPC6 and chronic Ca2+ overload.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Barrera de Filtración Glomerular/metabolismo , Insulina/farmacología , Transducción de Señal/efectos de los fármacos , Canales Catiónicos TRPC/metabolismo , Animales , Activación Enzimática/efectos de los fármacos , Femenino , Imidazoles/farmacología , Permeabilidad/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Diabetic nephropathy (DN) is one of the most frequent complications of diabetes. Early stages of DN are associated with hyperinsulinemia and progressive insulin resistance in insulin-sensitive cells, including podocytes. The diabetic environment induces pathological changes, especially in podocyte bioenergetics, which is tightly linked with mitochondrial dynamics. The regulatory role of insulin in mitochondrial morphology in podocytes has not been fully elucidated. Therefore, the main goal of the present study was to investigate effects of insulin on the regulation of mitochondrial dynamics and bioenergetics in human podocytes. Biochemical analyses were performed to assess oxidative phosphorylation efficiency by measuring the oxygen consumption rate (OCR) and glycolysis by measuring the extracellular acidification rate (ECAR). mRNA and protein expression were determined by real-time polymerase chain reaction and Western blot. The intracellular mitochondrial network was visualized by MitoTracker staining. All calculations were conducted using CellProfiler software. Short-term insulin exposure exerted inhibitory effects on various parameters of oxidative respiration and adenosine triphosphate production, and glycolysis flux was elevated. After a longer time of treating cells with insulin, an increase in mitochondrial size was observed, accompanied by a reduction of expression of the mitochondrial fission markers DRP1 and FIS1 and an increase in mitophagy. Overall, we identified a previously unknown role for insulin in the regulation of oxidative respiration and glycolysis and elucidated mitochondrial dynamics in human podocytes. The present results emphasize the importance of the duration of insulin stimulation for its metabolic and molecular effects, which should be considered in clinical and experimental studies of DN.
Asunto(s)
Metabolismo Energético , Glucólisis , Insulina , Mitocondrias , Dinámicas Mitocondriales , Podocitos , Podocitos/metabolismo , Podocitos/efectos de los fármacos , Humanos , Dinámicas Mitocondriales/efectos de los fármacos , Insulina/metabolismo , Insulina/farmacología , Metabolismo Energético/efectos de los fármacos , Glucólisis/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Dinaminas/metabolismo , Dinaminas/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Fosforilación Oxidativa/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Mitofagia/efectos de los fármacos , Línea CelularRESUMEN
Podocytes are crucial for regulating glomerular permeability. They have foot processes that are integral to the renal filtration barrier. Understanding their energy metabolism could shed light on the pathogenesis of filtration barrier injury. Lactate has been increasingly recognized as more than a waste product and has emerged as a significant metabolic fuel and reserve. The recent identification of lactate transporters in podocytes, the expression of which is modulated by glucose levels and lactate, highlights lactate's relevance. The present study investigated the impact of lactate on podocyte respiratory efficiency and mitochondrial dynamics. We confirmed lactate oxidation in podocytes, suggesting its role in cellular energy production. Under conditions of glucose deprivation or lactate supplementation, a significant shift was seen toward oxidative phosphorylation, reflected by an increase in the oxygen consumption rate/extracellular acidification rate ratio. Notably, lactate dehydrogenase A (LDHA) and lactate dehydrogenase B (LDHB) isoforms, which are involved in lactate conversion to pyruvate, were detected in podocytes for the first time. The presence of lactate led to higher intracellular pyruvate levels, greater LDH activity, and higher LDHB expression. Furthermore, lactate exposure increased mitochondrial DNA-to-nuclear DNA ratios and resulted in upregulation of the mitochondrial biogenesis markers peroxisome proliferator-activated receptor coactivator-1α and transcription factor A mitochondrial, regardless of glucose availability. Changes in mitochondrial size and shape were observed in lactate-exposed podocytes. These findings suggest that lactate is a pivotal energy source for podocytes, especially during energy fluctuations. Understanding lactate's role in podocyte metabolism could offer insights into renal function and pathologies that involve podocyte injury.
Asunto(s)
L-Lactato Deshidrogenasa , Ácido Láctico , Dinámicas Mitocondriales , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Podocitos , Podocitos/metabolismo , Podocitos/patología , Animales , Ratas , Ácido Láctico/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Glucosa/metabolismo , Metabolismo Energético , Lactato Deshidrogenasa 5/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , ADN Mitocondrial/metabolismo , ADN Mitocondrial/genética , Consumo de Oxígeno , Células Cultivadas , Ácido Pirúvico/metabolismo , IsoenzimasRESUMEN
Early stages of diabetes are characterized by elevations of insulin and glucose concentrations. Both factors stimulate reactive oxygen species (ROS) production, leading to impairments in podocyte function and disruption of the glomerular filtration barrier. Podocytes were recently shown to be an important source of αKlotho (αKL) expression. Low blood Klotho concentrations are also associated with an increase in albuminuria, especially in patients with diabetes. We investigated whether ADAM10, which is known to cleave αKL, is activated in glomeruli and podocytes under diabetic conditions and the potential mechanisms by which ADAM10 mediates ROS production and disturbances of the glomerular filtration barrier. In cultured human podocytes, high glucose increased ADAM10 expression, shedding, and activity, NADPH oxidase activity, ROS production, and albumin permeability. These effects of glucose were inhibited when cells were pretreated with an ADAM10 inhibitor or transfected with short-hairpin ADAM10 (shADAM10) or after the addition soluble Klotho. We also observed increases in ADAM10 activity, NOX4 expression, NADPH oxidase activity, and ROS production in αKL-depleted podocytes. This was accompanied by an increase in albumin permeability in shKL-expressing podocytes. The protein expression and activity of ADAM10 also increased in isolated glomeruli and urine samples from diabetic rats. Altogether, these results reveal a new mechanism by which hyperglycemia in diabetes increases albumin permeability through ADAM10 activation and an increase in oxidative stress via NOX4 enzyme activation. Moreover, αKlotho downregulates ADAM10 activity and supports redox balance, consequently protecting the slit diaphragm of podocyteσ under hyperglycemic conditions.
Asunto(s)
Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide , Diabetes Mellitus Experimental , Glucuronidasa , Proteínas Klotho , Proteínas de la Membrana , Podocitos , Especies Reactivas de Oxígeno , Podocitos/metabolismo , Podocitos/efectos de los fármacos , Proteínas Klotho/metabolismo , Proteína ADAM10/metabolismo , Proteína ADAM10/genética , Especies Reactivas de Oxígeno/metabolismo , Humanos , Animales , Glucuronidasa/metabolismo , Glucuronidasa/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratas , Masculino , Diabetes Mellitus Experimental/metabolismo , NADPH Oxidasa 4/metabolismo , NADPH Oxidasa 4/genética , NADPH Oxidasas/metabolismo , Células Cultivadas , Glucosa/metabolismo , Ratas Sprague-DawleyRESUMEN
A decrease in intracellular levels of 3',5'-cyclic guanosine monophosphate (cGMP) has been implicated in the progression of diabetic nephropathy. Hyperglycemia significantly inhibits cGMP-dependent pathway activity in the kidney, leading to glomerular damage and proteinuria. The enhancement of activity of this pathway that is associated with an elevation of cGMP levels may be achieved by inhibition of the cGMP specific phosphodiesterase 5A (PDE5A) using selective inhibitors, such as tadalafil. Hyperglycemia decreased the insulin responsiveness of podocytes and impaired podocyte function. These effects were associated with lower protein amounts and activity of the protein deacetylase sirtuin 1 (SIRT1) and a decrease in the phosphorylation of adenosine monophosphate-dependent protein kinase (AMPK). We found that PDE5A protein levels increased in hyperglycemia, and PDE5A downregulation improved the insulin responsiveness of podocytes with reestablished SIRT1 expression and activity. PDE5A inhibitors potentiate nitric oxide (NO)/cGMP signaling, and NO modulates the activity and expression of SIRT1. Therefore, we investigated the effects of tadalafil on SIRT1 and AMPK in the context of improving the insulin sensitivity in podocytes and podocyte function in hyperglycemia. Our study revealed that tadalafil restored SIRT1 expression and activity and activated AMPK by increasing its phosphorylation. Tadalafil also restored stimulating effect of insulin on glucose transport in podocytes with high glucose-induced insulin resistance. Additionally, tadalafil improved the function of podocytes that were exposed to high glucose concentrations. Our results display novel mechanisms involved in the pathogenesis of glomerulopathies in diabetes, which may contribute to the development of more effective treatment strategies for diabetic nephropathy.
Asunto(s)
Nefropatías Diabéticas , Hiperglucemia , Resistencia a la Insulina , Podocitos , Humanos , Tadalafilo/farmacología , Tadalafilo/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Insulina/metabolismo , Sirtuina 1/metabolismo , Podocitos/metabolismo , Nefropatías Diabéticas/patología , Proteínas Quinasas Activadas por AMP/metabolismo , GMP Cíclico/metabolismo , Glucosa/metabolismoRESUMEN
Lactate has long been acknowledged to be a metabolic waste product, but it has more recently been found as a fuel energy source in mammalian cells. Podocytes are an important component of the glomerular filter, and their role in maintaining the structural integrity of this structure was established. These cells rely on a constant energy supply and reservoir. The utilization of alternative energy substrates to preserve energetic homeostasis is a subject of extensive research, and lactate appears to be one such candidate. Therefore, we investigated the role of lactate as an energy substrate and characterize the lactate transport system in cultured rat podocytes during sufficient and insufficient glucose supplies. The present study, for the first time, demonstrated the presence of lactate transporters in podocytes. Moreover, we observed modified the amount of these transporters in response to limited glucose availability and after l-lactate supplementation. Simultaneously, exposure to l-lactate preserved cell survival during insufficient glucose supply. Interestingly, during glucose deprivation, lactate exposure allowed the steady flow of glycolysis and prevented glycogen reserves depletion. Summarizing, podocytes utilize lactate as an energy substrate and possess a developed system that controls lactate homeostasis, suggesting that it plays an essential role in podocyte metabolism, especially during fluctuations of energy availability.
Asunto(s)
Glucosa , Podocitos , Ratas , Animales , Glucosa/metabolismo , Podocitos/metabolismo , Glucólisis/fisiología , Ácido Láctico/metabolismo , Hipoxia de la Célula/fisiología , Mamíferos/metabolismoRESUMEN
Hyperglycemia significantly decreases 3',5'-cyclic guanosine monophosphate (cGMP)-dependent pathway activity in the kidney. A well-characterized downstream signaling effector of cGMP is cGMP-dependent protein kinase G (PKG), exerting a wide range of downstream effects, including vasodilation and vascular smooth muscle cells relaxation. In podocytes that are exposed to high glucose concentrations, crosstalk between the protein deacetylase sirtuin 1 (SIRT1) and adenosine monophosphate-dependent protein kinase (AMPK) decreased, attenuating insulin responsiveness and impairing podocyte function. The present study examined the effect of enhancing cGMP-dependent pathway activity on SIRT1-AMPK crosstalk in podocytes under hyperglycemic conditions. We found that enhancing cGMP-dependent pathway activity using a cGMP analog was associated with increases in SIRT1 protein levels and activity, with a concomitant increase in the degree of AMPK phosphorylation. The beneficial effects of enhancing cGMP-dependent pathway activity on SIRT1-AMPK crosstalk also included improvements in podocyte function. Based on our findings, we postulate an important role for SIRT1-AMPK crosstalk in the regulation of albumin permeability in hyperglycemia that is strongly associated with activity of the cGMP-dependent pathway.