RESUMEN
The role of insulin receptor (IR) activated by hyperinsulinemia in obesity-induced kidney injury is not well understood. We hypothesized that activation of kidney proximal tubule epithelial IR contributes to obesity-induced kidney injury. We administered normal-fat diet (NFD) or high-fat diet (HFD) to control and kidney proximal tubule IR-knockout (KPTIRKO) mice for 4 months. Renal cortical IR expression was decreased by 60% in male and female KPTIRKO mice. Baseline serum glucose, serum creatinine, and the ratio of urinary albumin to creatinine (ACR) were similar in KPTIRKO mice compared to those of controls. On HFD, weight gain and increase in serum cholesterol were similar in control and KPTIRKO mice; blood glucose did not change. HFD increased the following parameters in the male control mice: renal cortical contents of phosphorylated IR and Akt, matrix proteins, urinary ACR, urinary kidney injury molecule-1-to-creatinine ratio, and systolic blood pressure. Renal cortical generation of hydrogen sulfide was reduced in HFD-fed male control mice. All of these parameters were ameliorated in male KPTIRKO mice. Interestingly, female mice were resistant to HFD-induced kidney injury in both genotypes. We conclude that HFD-induced kidney injury requires renal proximal tubule IR activation in male mice.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Túbulos Renales Proximales/metabolismo , Receptor de Insulina/metabolismo , Insuficiencia Renal Crónica/etiología , Insuficiencia Renal Crónica/metabolismo , Animales , Epitelio/metabolismo , Femenino , Sulfuro de Hidrógeno/metabolismo , Resistencia a la Insulina , Corteza Renal/metabolismo , Masculino , Ratones , Ratones Noqueados , Obesidad/complicaciones , Obesidad/metabolismo , Receptor de Insulina/deficiencia , Receptor de Insulina/genética , Factores Sexuales , Transducción de SeñalRESUMEN
In vitro models for the investigation of renal vascular development are limited. We previously showed that isolated metanephric mesenchymal (MM) and ureteric bud (UB) cells grown in three-dimensional (3D) matrices formed organoids that consisted of primitive vascular structures surrounding a polarized epithelium. Here, we examined the potential of two principal effectors of vasculogenesis, vascular endothelial growth factor A (VEGF-A), and platelet-derived growth factor B chain (PDGF-BB), to stimulate MM cell differentiation. The results showed that MM cells possess angioblast characteristics by expressing phenotypic markers for endothelial and mesenchymal cells. UB cells synthesize VEGF-A and PDGF-BB proteins and RNA, whereas the MM cells express the respective cognate receptors, supporting their role in directional induction of vasculogenesis. VEGF-A stimulated proliferation of MM cells in monolayer and in 3D sponges but did not affect MM cell migration, organization, or vasculogenesis. However, PDGF-BB stimulated MM cell proliferation, migration, and vasculogenesis in monolayer and organization of the cells into primitive capillary-like assemblies in 3D sea sponge scaffolds in vitro. A role for PDGF-BB in vasculogenesis in the 3D MM/UB co-culture system was validated by direct interference with PDGF-BB or PDGF receptor-ß cell interactions to implicate PDGF-BB as a primary effector of MM cell vasculogenesis. Thus, MM cells resemble early renal angioblasts that may provide an ideal platform for the investigation of renal vasculogenesis in vitro.
Asunto(s)
Células Madre Mesenquimatosas/citología , Neovascularización Fisiológica/fisiología , Animales , Becaplermina/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Aberrant expression of microRNAs (miRs) contributes to diabetic renal complications, including renal hypertrophy and matrix protein accumulation. Reduced expression of phosphatase and tensin homolog (PTEN) by hyperglycemia contributes to these processes. We considered involvement of miR in the downregulation of PTEN. In the renal cortex of type 1 diabetic mice, we detected increased expression of miR-214 in association with decreased levels of PTEN and enhanced Akt phosphorylation and fibronectin expression. Mesangial and proximal tubular epithelial cells exposed to high glucose showed augmented expression of miR-214. Mutagenesis studies using 3'-UTR of PTEN in a reporter construct revealed PTEN as a direct target of miR-214, which controls its expression in both of these cells. Overexpression of miR-214 decreased the levels of PTEN and increased Akt activity similar to high glucose and lead to phosphorylation of its substrates glycogen synthase kinase-3ß, PRAS40, and tuberin. In contrast, quenching of miR-214 inhibited high-glucose-induced Akt activation and its substrate phosphorylation; these changes were reversed by small interfering RNAs against PTEN. Importantly, respective expression of miR-214 or anti-miR-214 increased or decreased the mammalian target of rapamycin complex 1 (mTORC1) activity induced by high glucose. Furthermore, mTORC1 activity was controlled by miR-214-targeted PTEN via Akt activation. In addition, neutralization of high-glucose-stimulated miR-214 expression significantly inhibited cell hypertrophy and expression of the matrix protein fibronectin. Finally, the anti-miR-214-induced inhibition of these processes was reversed by the expression of constitutively active Akt kinase and hyperactive mTORC1. These results uncover a significant role of miR-214 in the activation of mTORC1 that contributes to high-glucose-induced mesangial and proximal tubular cell hypertrophy and fibronectin expression.
Asunto(s)
Glucemia/metabolismo , Proliferación Celular , Diabetes Mellitus Tipo 1/enzimología , Nefropatías Diabéticas/enzimología , Células Epiteliales/enzimología , Glomérulos Renales/enzimología , Túbulos Renales Proximales/enzimología , MicroARNs/metabolismo , Fosfohidrolasa PTEN/metabolismo , Regiones no Traducidas 3' , Animales , Células Cultivadas , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Modelos Animales de Enfermedad , Células Epiteliales/patología , Fibronectinas/metabolismo , Regulación Enzimológica de la Expresión Génica , Mesangio Glomerular/metabolismo , Mesangio Glomerular/patología , Hipertrofia , Glomérulos Renales/patología , Túbulos Renales Proximales/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , MicroARNs/genética , Complejos Multiproteicos/metabolismo , Fosfohidrolasa PTEN/genética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Ratas , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Transfección , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
High-glucose increases NADPH oxidase 4 (NOX4) expression, reactive oxygen species generation, and matrix protein synthesis by inhibiting AMP-activated protein kinase (AMPK) in renal cells. Because hydrogen sulfide (H2S) inhibits high glucose-induced matrix protein increase by activating AMPK in renal cells, we examined whether H2S inhibits high glucose-induced expression of NOX4 and matrix protein and whether H2S and NO pathways are integrated. High glucose increased NOX4 expression and activity at 24 h in renal proximal tubular epithelial cells, which was inhibited by sodium hydrosulfide (NaHS), a source of H2S. High glucose decreased AMPK phosphorylation and activity, which was restored by NaHS. Compound C, an AMPK inhibitor, prevented NaHS inhibition of high glucose-induced NOX4 expression. NaHS inhibition of high glucose-induced NOX4 expression was abrogated by N(ω)-nitro-l-arginine methyl ester, an inhibitor of NOS. NaHS unexpectedly augmented the expression of inducible NOS (iNOS) but not endothelial NOS. iNOS siRNA and 1400W, a selective iNOS inhibitor, abolished the ameliorative effects of NaHS on high glucose-induced NOX4 expression, reactive oxygen species generation, and, matrix laminin expression. Thus, H2S recruits iNOS to generate NO to inhibit high glucose-induced NOX4 expression, oxidative stress, and matrix protein accumulation in renal epithelial cells; the two gasotransmitters H2S and NO and their interaction may serve as therapeutic targets in diabetic kidney disease.
Asunto(s)
Células Epiteliales/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Sulfuro de Hidrógeno/farmacología , Túbulos Renales Proximales/enzimología , NADPH Oxidasas/biosíntesis , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/metabolismo , Amidinas/farmacología , Animales , Bencilaminas/farmacología , Nefropatías Diabéticas/enzimología , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/terapia , Células Epiteliales/patología , Proteínas de la Matriz Extracelular/metabolismo , Túbulos Renales Proximales/patología , Ratones , NADPH Oxidasa 4 , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacosRESUMEN
PKCßII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCßII contains the COOH-terminal hydrophobic motif site Ser-660. Whether this hydrophobic motif phosphorylation contributes to high glucose-induced mesangial cell hypertrophy has not been determined. Here we show that, in mesangial cells, high glucose increased phosphorylation of PKCßII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCßII, dominant negative PKCßII, and PKCßII hydrophobic motif phosphorylation-deficient mutant, we found that PKCßII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCßII via its phosphorylation at Ser-660 regulated phosphorylation of Akt at both catalytic loop and hydrophobic motif sites, resulting in phosphorylation and inactivation of its substrate PRAS40. Specific inhibition of mTORC2 increased mTORC1 activity and induced mesangial cell hypertrophy. In contrast, inhibition of mTORC2 decreased the phosphorylation of PKCßII and Akt, leading to inhibition of PRAS40 phosphorylation and mTORC1 activity and prevented mesangial cell hypertrophy in response to high glucose; expression of constitutively active Akt or mTORC1 restored mesangial cell hypertrophy. Moreover, constitutively active PKCßII reversed the inhibition of high glucose-stimulated Akt phosphorylation and mesangial cell hypertrophy induced by suppression of mTORC2. Finally, using renal cortexes from type 1 diabetic mice, we found that increased phosphorylation of PKCßII at Ser-660 was associated with enhanced Akt phosphorylation and mTORC1 activation. Collectively, our findings identify a signaling route connecting PI3-kinase to mTORC2 to phosphorylate PKCßII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy.
Asunto(s)
Nefropatías Diabéticas/metabolismo , Células Mesangiales/metabolismo , Complejos Multiproteicos/metabolismo , Proteína Quinasa C beta/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Secuencias de Aminoácidos , Animales , Aumento de la Célula , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Nefropatías Diabéticas/patología , Técnicas de Silenciamiento del Gen , Glucosa/toxicidad , Humanos , Hipertrofia , Immunoblotting , Inmunoprecipitación , Diana Mecanicista del Complejo 2 de la Rapamicina , Células Mesangiales/patología , Ratones , Fosforilación , ARN Interferente Pequeño , TransfecciónRESUMEN
We examined the effect of rapamycin on the life span of a mouse model of type 2 diabetes, db/db mice. At 4 months of age, male and female C57BLKSJ-lepr (db/db) mice (db/db) were placed on either a control diet, lacking rapamycin or a diet containing rapamycin and maintained on these diets over their life span. Rapamycin was found to reduce the life span of the db/db mice. The median survival of male db/db mice fed the control and rapamycin diets was 349 and 302 days, respectively, and the median survival of female db/db mice fed the control and rapamycin diets was 487 and 411 days, respectively. Adjusting for gender differences, rapamycin increased the mortality risk 1.7-fold in both male and female db/db mice. End-of-life pathological data showed that suppurative inflammation was the main cause of death in the db/db mice, which is enhanced slightly by rapamycin treatment.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Inflamación/patología , Longevidad , Sirolimus , Animales , Causas de Muerte , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/mortalidad , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/mortalidad , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/fisiopatología , Femenino , Inmunosupresores/metabolismo , Inmunosupresores/farmacología , Longevidad/efectos de los fármacos , Longevidad/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Mortalidad , Factores Sexuales , Sirolimus/metabolismo , Sirolimus/farmacología , Resultado del TratamientoRESUMEN
Diabetes-induced kidney cell injury involves an increase in matrix protein expression that is only partly alleviated by current treatment, prompting a search for new modalities. We have previously shown that hydrogen sulfide (H2S) inhibits high glucose-induced protein synthesis in kidney podocytes. We tested whether tadalafil, a phosphodiesterase 5 inhibitor used to treat erectile dysfunction, ameliorates high glucose stimulation of matrix proteins by generating H2S in podocytes. Tadalafil abrogated high glucose stimulation of global protein synthesis and matrix protein laminin γ1. Tadalafil inhibited high glucose-induced activation of mechanistic target of rapamycin complex 1 and laminin γ1 accumulation in an AMP-activated protein kinase (AMPK)-dependent manner. Tadalafil increased AMPK phosphorylation by stimulating calcium-calmodulin kinase kinase ß. Tadalafil rapidly increased the expression and activity of the H2S-generating enzyme cystathionine γ-lyase (CSE) by promoting its translation. dl-Propargylglycine, a CSE inhibitor, and siRNA against CSE inhibited tadalafil-induced AMPK phosphorylation and abrogated the tadalafil effect on high glucose stimulation of laminin γ1. In tadalafil-treated podocytes, we examined the interaction between H2S and nitric oxide (NO). N(ω)-Nitro-L-arginine methyl ester and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, inhibitors of NO synthase (NOS) and soluble guanylyl cyclase, respectively, abolished tadalafil induction of H2S and AMPK phosphorylation. Tadalafil rapidly augmented inducible NOS (iNOS) expression by increasing its mRNA, and siRNA for iNOS and 1400W, an iNOS blocker, inhibited tadalafil stimulation of CSE expression and AMPK phosphorylation. We conclude that tadalafil amelioration of high glucose stimulation of synthesis of proteins including matrix proteins in podocytes requires integration of the NO-H2S-AMPK axis leading to the inhibition of high glucose-induced mechanistic target of rapamycin complex 1 activity and mRNA translation.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Carbolinas/química , Glucosa/química , Sulfuro de Hidrógeno/química , Óxido Nítrico/química , Podocitos/metabolismo , Transducción de Señal , Animales , Calcio/química , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Riñón/citología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa 5/química , Fosforilación , Podocitos/citología , Polirribosomas/metabolismo , Ratas , Serina-Treonina Quinasas TOR/metabolismo , TadalafiloRESUMEN
High glucose-induced Akt acts as a signaling hub for mesangial cell hypertrophy and matrix expansion, which are recognized as cardinal signatures for the development of diabetic nephropathy. How mesangial cells sustain the activated state of Akt is not clearly understood. Here we show Akt-dependent phosphorylation of the transcription factor FoxO1 by high glucose. Phosphorylation-deficient, constitutively active FoxO1 inhibited the high glucose-induced phosphorylation of Akt to suppress the phosphorylation/inactivation of PRAS40 and mTORC1 activity. In contrast, dominant negative FoxO1 increased the phosphorylation of Akt, resulting in increased mTORC1 activity similar to high glucose treatment. Notably, FoxO1 regulates high glucose-induced protein synthesis, hypertrophy, and expression of fibronectin and PAI-1. High glucose paves the way for complications of diabetic nephropathy through the production of reactive oxygen species (ROS). We considered whether the FoxO1 target antioxidant enzyme catalase contributes to sustained activation of Akt. High glucose-inactivated FoxO1 decreases the expression of catalase to increase the production of ROS. Moreover, we show that catalase blocks high glucose-stimulated Akt phosphorylation to attenuate the inactivation of FoxO1 and PRAS40, resulting in the inhibition of mTORC1 and mesangial cell hypertrophy and fibronectin and PAI-1 expression. Finally, using kidney cortices from type 1 diabetic OVE26 mice, we show that increased FoxO1 phosphorylation is associated with decreased catalase expression and increased fibronectin and PAI-1 expression. Together, our results provide the first evidence for the presence of a positive feedback loop for the sustained activation of Akt involving inactivated FoxO1 and a decrease in catalase expression, leading to increased ROS and mesangial cell hypertrophy and matrix protein expression.
Asunto(s)
Retroalimentación Fisiológica/efectos de los fármacos , Factores de Transcripción Forkhead/metabolismo , Glucosa/farmacología , Células Mesangiales/efectos de los fármacos , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Catalasa/genética , Catalasa/metabolismo , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Fibronectinas/metabolismo , Expresión Génica/efectos de los fármacos , Immunoblotting , Corteza Renal/metabolismo , Corteza Renal/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Células Mesangiales/metabolismo , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Increase in protein synthesis contributes to kidney hypertrophy and matrix protein accumulation in diabetes. We have previously shown that high glucose-induced matrix protein synthesis is associated with inactivation of glycogen synthase kinase 3ß (GSK3ß) in renal cells and in the kidneys of diabetic mice. We tested whether activation of GSK3ß by sodium nitroprusside (SNP) mitigates kidney injury in diabetes. Studies in kidney-proximal tubular epithelial cells showed that SNP abrogated high glucose-induced laminin increment by stimulating GSK3ß and inhibiting Akt, mTORC1, and events in mRNA translation regulated by mTORC1 and ERK. NONOate, an NO donor, also activated GSK3ß, indicating that NO may mediate SNP stimulation of GSK3ß. SNP administered for 3 weeks to mice with streptozotocin-induced type 1 diabetes ameliorated kidney hypertrophy, accumulation of matrix proteins, and albuminuria without changing blood glucose levels. Signaling studies showed that diabetes caused inactivation of GSK3ß by activation of Src, Pyk2, Akt, and ERK; GSK3ß inhibition activated mTORC1 and downstream events in mRNA translation in the kidney cortex. These reactions were abrogated by SNP. We conclude that activation of GSK3ß by SNP ameliorates kidney injury induced by diabetes.
Asunto(s)
Diabetes Mellitus Experimental/prevención & control , Glucógeno Sintasa Quinasa 3/metabolismo , Riñón/efectos de los fármacos , Nitroprusiato/farmacología , Albuminuria/prevención & control , Animales , Línea Celular Transformada , Diabetes Mellitus Experimental/enzimología , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hipertrofia/prevención & control , Immunoblotting , Riñón/enzimología , Riñón/patología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Laminina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones Endogámicos C57BL , Complejos Multiproteicos/metabolismo , Donantes de Óxido Nítrico/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Increase in matrix protein content in the kidney is a cardinal feature of diabetic kidney disease. While renal matrix protein content is increased by chronic hyperglycemia, whether it is regulated by acute elevation of glucose and insulin has not been addressed. In this study, we aimed to evaluate whether short duration of combined hyperglycemia and hyperinsulinemia, mimicking the metabolic environment of prediabetes and early type 2 diabetes, induces kidney injury. Normal rats were subjected to either saline infusion (control, n = 4) or 7 h of combined hyperglycemic-hyperinsulinemic clamp (HG+HI clamp; n = 6). During the clamp, plasma glucose and plasma insulin were maintained at about 350 mg/dl and 16 ng/ml, respectively. HG+HI clamp increased the expression of renal cortical transforming growth factor-ß (TGF-ß) and renal matrix proteins, laminin and fibronectin. This was associated with the activation of SMAD3, Akt, mammalian target of rapamycin (mTOR) complexes, and ERK signaling pathways and their downstream target events in the initiation and elongation phases of mRNA translation, an important step in protein synthesis. Additionally, HG+HI clamp provoked renal inflammation as shown by the activation of Toll-like receptor 4 (TLR4) and infiltration of CD68-positive monocytes. Urinary F2t isoprostane excretion, an index of renal oxidant stress, was increased in the HG+HI clamp rats. We conclude that even a short duration of hyperglycemia and hyperinsulinemia contributes to activation of pathways that regulate matrix protein synthesis, inflammation, and oxidative stress in the kidney. This finding could have implications for the control of short-term rises in blood glucose in diabetic individuals at risk of developing kidney disease.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Hiperglucemia/patología , Hiperinsulinismo/patología , Inflamación/patología , Riñón/patología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Glucemia , Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/metabolismo , Activación Enzimática , Fibronectinas/biosíntesis , Fibrosis , Hiperglucemia/metabolismo , Hiperinsulinismo/metabolismo , Inflamación/metabolismo , Riñón/metabolismo , Laminina/biosíntesis , Sistema de Señalización de MAP Quinasas/genética , Masculino , FN-kappa B/metabolismo , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Proteína smad3/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
We explored molecular events associated with aging-induced matrix changes in the kidney. C57BL6 mice were studied in youth, middle age, and old age. Albuminuria and serum cystatin C level (an index of glomerular filtration) increased with aging. Renal hypertrophy was evident in middle-aged and old mice and was associated with glomerulomegaly and increase in mesangial fraction occupied by extracellular matrix. Content of collagen types I and III and fibronectin was increased with aging; increment in their mRNA varied with the phase of aging. The content of ZEB1 and ZEB2, collagen type I transcription inhibitors, and their binding to the collagen type Iα2 promoter by ChIP assay also showed age-phase-specific changes. Lack of increase in mRNA and data from polysome assay suggested decreased degradation as a potential mechanism for kidney collagen type I accumulation in the middle-aged mice. These changes occurred with increment in TGFß mRNA and protein and activation of its SMAD3 pathway; SMAD3 binding to the collagen type Iα2 promoter was also increased. TGFß-regulated microRNAs (miRs) exhibited selective regulation. The renal cortical content of miR-21 and miR-200c, but not miR-192, miR-200a, or miR-200b, was increased with aging. Increased miR-21 and miR-200c contents were associated with reduced expression of their targets, Sprouty-1 and ZEB2, respectively. These data show that aging is associated with complex molecular events in the kidney that are already evident in the middle age and progress to old age. Age-phase-specific regulation of matrix protein synthesis occurs and involves matrix protein-specific transcriptional and post-transcriptional mechanisms.
Asunto(s)
Envejecimiento/genética , Matriz Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mesangio Glomerular/metabolismo , Corteza Renal/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Cistatina C/sangre , Matriz Extracelular/patología , Femenino , Tasa de Filtración Glomerular , Mesangio Glomerular/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Corteza Renal/patología , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteolisis , ARN Mensajero/biosíntesis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Albúmina Sérica/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Renal hypertrophy and accumulation of extracellular matrix proteins are among cardinal manifestations of diabetic nephropathy. TGF beta system has been implicated in the pathogenesis of these manifestations. Among signaling pathways activated in the kidney in diabetes, mTOR- (mammalian target of rapamycin-)regulated pathways are pivotal in orchestrating high glucose-induced production of ECM proteins leading to functional and structural changes in the kidney culminating in adverse outcomes. Understanding signaling pathways that influence individual matrix protein expression could lead to the development of new interventional strategies. This paper will highlight some of the diverse components of the signaling network stimulated by hyperglycemia with an emphasis on extracellular matrix protein metabolism in the kidney in diabetes.
Asunto(s)
Nefropatías Diabéticas/metabolismo , Riñón/metabolismo , Transducción de Señal/fisiología , Matriz Extracelular/metabolismo , HumanosRESUMEN
Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase ß, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase ß inhibitor, had the same effect. Renal cortical content of cystathionine ß-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.
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Contaminantes Atmosféricos/farmacología , Células Epiteliales/metabolismo , Proteínas de la Matriz Extracelular/biosíntesis , Glucosa/farmacología , Sulfuro de Hidrógeno/farmacología , Glomérulos Renales/metabolismo , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Edulcorantes/farmacología , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP , Proteínas Adaptadoras Transductoras de Señales , Animales , Bencimidazoles/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Liasas de Carbono-Oxígeno , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Cistationina betasintasa/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Nefropatías Diabéticas/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Factores Eucarióticos de Iniciación , Péptidos y Proteínas de Señalización Intracelular , Glomérulos Renales/citología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Complejos Multiproteicos , Naftalimidas/farmacología , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TORRESUMEN
Hyperglycemia induces a wide array of signaling pathways in the kidney that lead to hypertrophy and matrix expansion, eventually culminating in progressive kidney failure. High glucose-induced reduction of the tumor suppressor protein phosphatase and tensin homolog deleted in chromosome 10 (PTEN) contributes to renal cell hypertrophy and matrix expansion. We identified microRNA-21 (miR-21) as the molecular link between high glucose and PTEN suppression. Renal cortices from OVE26 type 1 diabetic mice showed significantly elevated levels of miR-21 associated with reduced PTEN and increased fibronectin content. In renal mesangial cells, high glucose increased the expression of miR-21, which targeted the 3'-UTR of PTEN mRNA to inhibit PTEN protein expression. Overexpression of miR-21 mimicked the action of high glucose, which included a reduction in PTEN expression and a concomitant increase in Akt phosphorylation. In contrast, expression of miR-21 Sponge, to inhibit endogenous miR-21, prevented down-regulation of PTEN and phosphorylation of Akt induced by high glucose. Interestingly, high glucose-stimulated miR-21 inactivated PRAS40, a negative regulator of TORC1. Finally, miR-21 enhanced high glucose-induced TORC1 activity, resulting in renal cell hypertrophy and fibronectin expression. Thus, our results identify a previously unrecognized function of miR-21 that is the reciprocal regulation of PTEN levels and Akt/TORC1 activity that mediate critical pathologic features of diabetic kidney disease.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Diabetes Mellitus Tipo 1/patología , Glucosa/farmacología , Riñón/metabolismo , Riñón/patología , MicroARNs/genética , Transducción de Señal/efectos de los fármacos , Animales , Secuencia de Bases , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibronectinas/genética , Humanos , Hipertrofia/inducido químicamente , Hipertrofia/genética , Hipertrofia/metabolismo , Riñón/efectos de los fármacos , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Corteza Renal/patología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/patología , Células Mesangiales/efectos de los fármacos , Células Mesangiales/metabolismo , Células Mesangiales/patología , Ratones , Fosfohidrolasa PTEN/genética , Ratas , Proteína Reguladora Asociada a mTORRESUMEN
Diabetes promotes protein synthesis to induce kidney hypertrophy and increase renal matrix proteins. Increased capacity for mRNA translation by way of ribosomal biogenesis facilitates sustained stimulation of protein synthesis. We tested the hypothesis that high glucose induces ribosomal biogenesis as indicated by an increase in rRNA synthesis in the setting of augmented protein synthesis. High glucose (30 mM) increased global protein synthesis, expression of matrix proteins, laminin γ1 and fibronectin, and rDNA transcription in glomerular epithelial cells (GECs) compared with 5 mM glucose. High glucose induced Ser388 phosphorylation of upstream binding factor (UBF), an rDNA transcription factor, along with increased phosphorylation of Erk and p70S6 kinase. Inactivation of Erk and p70S6 kinase either by their respective chemical inhibitors or by expression of their inactive mutant constructs blocked high-glucose-induced UBF phosphorylation. High glucose reduced nuclear content of p19ARF and promoted dissolution of inactive UBF-p19ARF complex. High glucose also promoted association of UBF with RPA194, a subunit of RNA polymerase I. Inhibition of Erk, p70S6 kinase, and UBF1 by transfecting GECs with their respective inactive mutants abolished laminin γ1 synthesis, protein synthesis, and rDNA transcription. Renal cortex from type 1 diabetic rats and type 2 diabetic db/db mice showed increased phosphorylation of UBF, Erk, and p70S6 kinase coinciding with renal hypertrophy and onset of matrix accumulation. Our data suggest that augmented ribosome biogenesis occurs in an UBF-dependent manner during increased protein synthesis induced by high glucose in the GECs that correlates with UBF activation and renal hypertrophy in rodents with type 1 and type 2 diabetes.
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Células Epiteliales/metabolismo , Glucosa/administración & dosificación , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Ribosomas/fisiología , Animales , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , ADN Polimerasa I/metabolismo , ADN Ribosómico/metabolismo , Diabetes Mellitus Experimental/metabolismo , Fibronectinas/biosíntesis , Glucosa/farmacología , Hipertrofia , Riñón/patología , Glomérulos Renales/citología , Laminina/biosíntesis , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
High glucose-induced protein synthesis in the glomerular epithelial cell (GEC) is partly dependent on reduction in phosphorylation of AMP-activated protein kinase (AMPK). We evaluated the effect of resveratrol, a phytophenol known to stimulate AMPK, on protein synthesis. Resveratrol completely inhibited high glucose stimulation of protein synthesis and synthesis of fibronectin, an important matrix protein, at 3 days. Resveratrol dose-dependently increased AMPK phosphorylation and abolished high glucose-induced reduction in its phosphorylation. We examined the effect of resveratrol on critical steps in mRNA translation, a critical event in protein synthesis. Resveratrol inhibited high glucose-induced changes in association of eIF4E with eIF4G, phosphorylation of eIF4E, eEF2, eEF2 kinase and, p70S6 kinase, indicating that it affects important events in both initiation and elongation phases of mRNA translation. Upstream regulators of AMPK in high glucose-treated GEC were explored. High glucose augmented acetylation of LKB1, the upstream kinase for AMPK, and inhibited its activity. Resveratrol prevented acetylation of LKB1 and restored its activity in high glucose-treated cells; this action did not appear to depend on SIRT1, a class III histone deacetylase. Our data show that resveratrol ameliorates protein synthesis by regulating the LKB1-AMPK axis.
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Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Glucosa/metabolismo , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Estilbenos/farmacología , Animales , Línea Celular , Ratones , Fosforilación/efectos de los fármacos , Proteínas/metabolismo , ResveratrolRESUMEN
Translation, a process of generating a peptide from the codons present in messenger RNA, can be a site of independent regulation of protein synthesis; it has not been well studied in the kidney. Translation occurs in three stages (initiation, elongation, and termination), each with its own set of regulatory factors. Mechanisms controlling translation include small inhibitory RNAs such as microRNAs, binding proteins, and signaling reactions. Role of translation in renal injury in diabetes, endoplasmic reticulum stress, acute kidney injury, and, in physiological adaptation to loss of nephrons is reviewed here. Contribution of mRNA translation to physiology and disease is not well understood. Because it is involved in such diverse areas as development and cancer, it should prove a fertile field for investigation in renal science.
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Enfermedades Renales/fisiopatología , Riñón/fisiología , Biosíntesis de Proteínas/fisiología , ARN Mensajero/biosíntesis , Animales , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/fisiopatología , Regulación de la Expresión Génica/fisiología , Humanos , Riñón/metabolismo , Enfermedades Renales/metabolismo , Transducción de SeñalRESUMEN
High glucose (30 mM) and high insulin (1 nM), pathogenic factors of type 2 diabetes, increased mRNA expression and synthesis of lamininbeta1 and fibronectin after 24 h of incubation in kidney proximal tubular epithelial (MCT) cells. We tested the hypothesis that inactivation of glycogen synthase kinase 3beta (GSK3beta) by high glucose and high insulin induces increase in synthesis of laminin beta1 via activation of eIF2Bepsilon. Both high glucose and high insulin induced Ser-9 phosphorylation and inactivation of GSK3beta at 2 h that lasted for up to 48 h. This was associated with dephosphorylation of eIF2Bepsilon and eEF2, and increase in phosphorylation of 4E-BP1 and eIF4E. Expression of the kinase-dead mutant of GSK3beta or constitutively active kinase led to increased and diminished laminin beta1 synthesis, respectively. Incubation with selective kinase inhibitors showed that high glucose- and high insulin-induced laminin beta1 synthesis and phosphorylation of GSK3beta were dependent on PI 3-kinase, Erk, and mTOR. High glucose and high insulin augmented activation of Akt, Erk, and p70S6 kinase. Dominant negative Akt, but not dominant negative p70S6 kinase, inhibited GSK3beta phosphorylation induced by high glucose and high insulin, suggesting Akt but not p70S6 kinase was upstream of GSK3beta. Status of GSK3beta was examined in vivo in renal cortex of db/db mice with type 2 diabetes at 2 weeks and 2 months of diabetes. Diabetic mice showed increased phosphorylation of renal cortical GSK3beta and decreased phosphorylation of eIF2Bepsilon, which correlated with renal hypertrophy at 2 weeks, and increased laminin beta1 and fibronectin protein content at 2 months. GSK3beta and eIF2Bepsilon play a role in augmented protein synthesis associated with high glucose- and high insulin-stimulated hypertrophy and matrix accumulation in renal disease in type 2 diabetes.
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Diabetes Mellitus Tipo 2/metabolismo , Células Epiteliales/metabolismo , Glucosa/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Hipoglucemiantes/farmacología , Insulina/farmacología , Túbulos Renales Proximales/metabolismo , Laminina/biosíntesis , Biosíntesis de Proteínas/efectos de los fármacos , Edulcorantes/farmacología , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Línea Celular Transformada , Diabetes Mellitus Tipo 2/patología , Células Epiteliales/patología , Factor 2B Eucariótico de Iniciación/genética , Factor 2B Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación , Fibronectinas/biosíntesis , Fibronectinas/genética , Glucosa/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Hipertrofia , Hipoglucemiantes/metabolismo , Insulina/metabolismo , Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Túbulos Renales Proximales/patología , Laminina/genética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Edulcorantes/metabolismo , Serina-Treonina Quinasas TOR , Factores de TiempoRESUMEN
Angiotensin II (Ang II)-induced renal injury is partly mediated by growth factors such as VEGF. We have previously shown that Ang II rapidly increases VEGF protein synthesis in proximal tubular epithelial (MCT) cells by augmenting mRNA translation, which is partly dependent on activation and binding of hnRNP K to 3' untranslated region (UTR) of VEGF mRNA. Regulation of hnRNP K activation by PKCdelta was studied in MCT cells. Transfection with a PKCdelta siRNA inhibited hnRNP K Ser302 phosphorylation and activation, and reduced Ang II stimulation of VEGF synthesis. Inhibition of PKCdelta with röttlerin also prevented binding of hnRNP K to VEGF mRNA and reduced the efficiency of VEGF mRNA translation. In db/db mice at 2 weeks of type 2 diabetes, VEGF expression was increased, which was due not to increase in transcription but to augmented translation of VEGF mRNA. Augmented VEGF expression was associated with increased binding of hnRNP K to VEGF mRNA. c-src and PKCdelta activities and hnRNP K phosphorylation on Ser302 in renal cortex of db/db mice were increased compared to control mice. We conclude: Ang II-induced VEGF mRNA translation is associated with activation of hnRNP K in MCT cells. In the signaling pathway leading to hnRNP K activation induced by Ang II, PKCdelta is downstream of c-src. PKCdelta-mediated phosphorylation of hnRNP K is required for Ang II stimulation of VEGF mRNA translation. In mice with type 2 diabetes, src and PKCdelta activation and hnRNP K phosphorylation correlate with increased VEGF mRNA translation and kidney hypertrophy. 3' UTR events are important in regulation of VEGF expression in models of renal injury.
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Angiotensina II/farmacología , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Proteína Quinasa C-delta/metabolismo , ARN Mensajero/genética , Factor A de Crecimiento Endotelial Vascular/genética , Regiones no Traducidas 3' , Animales , Línea Celular , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo K/química , Riñón/metabolismo , Riñón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Biosíntesis de Proteínas/efectos de los fármacos , Proteína Quinasa C-delta/antagonistas & inhibidores , Serina/química , Transducción de SeñalRESUMEN
High glucose and high insulin, pathogenic factors in type 2 diabetes, induce rapid synthesis of the matrix protein laminin-beta1 in renal proximal tubular epithelial cells by stimulation of initiation phase of mRNA translation. We investigated if elongation phase of translation also contributes to high glucose and high insulin induction of laminin-beta1 synthesis in proximal tubular epithelial cells. High glucose or high insulin rapidly increased activating Thr56 dephosphorylation of eEF2 and inactivating Ser366 phosphorylation of eEF2 kinase, events that facilitate elongation. Studies with inhibitors showed that PI3 kinase-Akt-mTOR-p70S6 kinase pathway controlled changes in phosphorylation of eEF2 and eEF2 kinase induced by high glucose or high insulin. Renal cortical homogenates from db/db mice in early stage of type 2 diabetes showed decrease in eEF2 phosphorylation and increment in eEF2 kinase phosphorylation in association with renal hypertrophy and glomerular and tubular increase in laminin-beta1 content. Rapamycin, an inhibitor of mTOR, abolished diabetes-induced changes in phosphorylation of eEF2, eEF2 kinase, and p70S6 kinase and ameliorated renal hypertrophy and laminin-beta1 protein content, without affecting hyperglycemia. These data show that mTOR is an attractive target for amelioration of diabetes-induced renal injury.