RESUMEN
The role of autophagy in the kidney and many nephrological diseases has gained prominence in recent years. Much of this research has been focused on markers of autophagy that are static and reveal little about the state of this dynamic pathway. Other mechanistic investigations are limited to in vitro studies, that often provide circumstantial evidence of autophagic flux. Here we describe a method for measuring autophagic flux ex vivo that allows more direct observations to be made in situ regarding the state of autophagic flux within the renal cortex of a single animal.
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Autofagosomas/ultraestructura , Autofagia , Corteza Renal/ultraestructura , Microscopía Electrónica de Transmisión , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Regulación de la Expresión Génica , Técnicas In Vitro , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Macrólidos/farmacología , Masculino , Ratones Endogámicos C57BL , Transducción de Señal , Sirolimus/farmacología , Factores de TiempoRESUMEN
BACKGROUND/AIMS: The hanging drop technique is a well-established method used in culture of animal tissues. However, this method has not been used in adult kidney tissue culture yet. This study was to explore the feasibility of using this technique for culturing adult kidney cortex to study the time course of RNA viability in the tubules and vasculature, as well as the tissue structural integrity. METHODS: In each Petri dish with the plate covered with sterile buffer, a section of mouse renal cortex was cultured within a drop of DMEM culture medium on the inner surface of the lip facing downward. The tissue were then harvested at each specific time points for Real-time PCR analysis and histological studies. RESULTS: The results showed that the mRNA level of most Na+ related transporters and cotransporters were stably maintained within 6 hours in culture, and that the mRNA level of most receptors found in the vasculature and glomeruli were stably maintained for up to 9 days in culture. Paraffin sections of the cultured renal cortex indicated that the tubules began to lose tubular integrity after 6 hours, but the glomeruli and vasculatures were still recognizable up to 9 days in culture. CONCLUSIONS: We concluded that adult kidney tissue culture by hanging drop method can be used to study gene expressions in vasculature and glomeruli.
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Corteza Renal/citología , Técnicas de Cultivo de Tejidos/métodos , Animales , Vasos Sanguíneos , Expresión Génica , Corteza Renal/crecimiento & desarrollo , Corteza Renal/ultraestructura , Glomérulos Renales , Ratones , Factores de TiempoRESUMEN
CONTEXT: The proximal tubular epithelial cells (PTECs) are the primary target of aristolochic acids and especially vulnerable to mitochondrial injury from insults of toxic xenobiotics. OBJECTIVES: This study aimed to investigate the possible role of mitochondrial injury in Caulis Aristolochia manshuriensis (CAM)-induced aristolochic acid nephropathy (AAN). MATERIALS AND METHODS: Male Sprague-Dawley rats were gavaged with CAM extract every other week for 1, 4, 8 and 12 weeks, respectively. RESULTS: The rats in the model group showed chronic AAN as evidenced by worsening kidney function evaluated by blood urea nitrogen, creatinine and proteinuria levels, and severe tubulointerstitial injury marked by massive tubular atrophy and interstitial fibrosis in kidney tissues. Moreover, overt apoptosis and impaired regeneration of PTECs were observed in AAN rats. Furthermore, the study revealed that mitochondria in PTECs were fragmented into small, punctuate suborganelles in AAN rats. Two mitochondrial respiratory chain proteins, mitochondrial DNA (mtDNA)-encoded cytochrome c oxidase subunit Ð (COX-Ð) and nuclear DNA-encoded nicotinamide adenine dinucleotide dehydrogenase (ubiquinone)-1ß subcomplex 8 (NDUFß8), were both down-regulated after one week of CAM treatment. However, with AAN progression, NDUFß8 level restored, while COX-Ð level maintained low. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), master regulator of mitochondrial biogenesis, was significantly down-regulated at week 4 and week 8, but significantly up-regulated at week 12. In addition, mtDNA copy number reduced markedly along with AAN progression. DISCUSSION AND CONCLUSION: A rat model of chronic AAN was successfully reproduced by gavage with CAM extract. Dynamic changes of mitochondrial injury induced by CAM might contribute to the AAN progression.
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Aristolochia/química , Ácidos Aristolóquicos/toxicidad , Medicamentos Herbarios Chinos/toxicidad , Mitocondrias/efectos de los fármacos , Nefritis Intersticial/inducido químicamente , Animales , Apoptosis/efectos de los fármacos , Ácidos Aristolóquicos/aislamiento & purificación , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/aislamiento & purificación , Corteza Renal/efectos de los fármacos , Corteza Renal/ultraestructura , Pruebas de Función Renal , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/ultraestructura , Masculino , Mitocondrias/ultraestructura , Nefritis Intersticial/sangre , Nefritis Intersticial/patología , Nefritis Intersticial/orina , Ratas Sprague-DawleyRESUMEN
The potential toxic effects of silver nanoparticles (AgNPs), administered by a single intratracheal instillation (i.t), was assessed in a rat model using commercial physico-chemical characterized nanosilver. Histopathological changes, overall toxic response and oxidative stress (kidney and plasma protein carbonylation), paralleled by ultrastructural observations (TEM), were evaluated to examine renal responses 7 and 28 days after i.t. application of a low AgNP dose (50 µg/rat), compared to an equivalent dose of ionic silver (7 µg AgNO3/rat). The AgNPs caused moderate renal histopathological and ultrastructural alteration, in a region-specific manner, being the cortex the most affected area. Notably, the bulk AgNO3, caused similar adverse effects with a slightly more marked extent, also triggering apoptotic phenomena. Specifically, 7 days after exposure to both AgNPs and AgNO3, dilatation of the intercapillary and peripheral Bowman's space was observed, together with glomerular shrinkage. At day 28, these effects still persisted after both treatments, accompanied by an additional injury involving the vascular component of the mesangium, with interstitial micro-hemorrhages. Neither AgNPs nor AgNO3 induced oxidative stress effects in kidneys and plasma, at either time point. The AgNP-induced moderate renal effects indicate that, despite their benefits, novel AgNPs employed in consumer products need exhaustive investigation to ensure public health safety.
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Corteza Renal/efectos de los fármacos , Riñón/efectos de los fármacos , Nanopartículas del Metal/toxicidad , Nitrato de Plata/toxicidad , Plata/toxicidad , Animales , Apoptosis/efectos de los fármacos , Proteínas Sanguíneas/metabolismo , Cápsula Glomerular/efectos de los fármacos , Humanos , Iones/toxicidad , Riñón/patología , Riñón/ultraestructura , Corteza Renal/patología , Corteza Renal/ultraestructura , Masculino , Modelos Animales , Estrés Oxidativo/efectos de los fármacos , Carbonilación Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
BACKGROUND: Ischemia/reperfusion (I/R) injury, which is commonly seen in the field of renal surgery or transplantation, is a major cause of acute renal failure (ARF). The ischemic ARF in diabetic rats is much more severe than that in the normal rats exposed to as same ischemic time. Ischemic post-conditioning (IPO) is a phenomenon by which intermittent interruptions of blood flow in the early phase of reperfusion can protect organs from I/R injury. To determine whether the renal protection effect of IPO mediates by toll-like receptor 4 (TLR4) signaling pathway in diabetic rats. METHODS: Streptozotocin-induced diabetic rats were randomly divided into three groups: sham operation group, I/R group, and IPO group. Except sham operation group, rats were subjected to 30 min of renal ischemia, both with and without treatment with IPO, then reperfusion 24 h. Light microscope and transmission electronic microscope were used to observe structural changes of renal tubule. RT-PCR was used to measure TLR4 and tumor necrosis factor-alpha (TNF-α) mRNA expression level, renal TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) protein expression was detected by Western blot. RESULTS: The results demonstrated that IPO markedly decreased renal ischemic injury caused by I/R and inhibited the proinflammatory expression levels of TLR4, TNF-α, and NF-κB, all of which up-regulated by I/R in diabetic rats. CONCLUSION: Taken together, our results suggest that proper IPO may have protective effect on the ischemic injury mediated by renal I/R, which might be associated with inhibition of TLR4 signaling pathway in diabetic rats.
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Lesión Renal Aguda/prevención & control , Diabetes Mellitus Experimental , Regulación hacia Abajo/genética , Regulación de la Expresión Génica , Poscondicionamiento Isquémico/métodos , Daño por Reperfusión/complicaciones , Receptor Toll-Like 4/genética , Lesión Renal Aguda/etiología , Lesión Renal Aguda/genética , Animales , Western Blotting , ADN/genética , Corteza Renal/ultraestructura , Masculino , Microscopía Electrónica de Transmisión , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Transducción de Señal , Receptor Toll-Like 4/biosíntesisRESUMEN
The macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is deregulated in acute kidney injury (AKI) through an unknown mechanism. In the present study, we used a previously described mouse model of ascending urinary tract infection in which uropathogenic Escherichia coli (UPEC) were transurethrally inoculated to induce kidney infections. Here, we show that urinary MIF was upregulated during AKI while MIF was abundantly expressed in the renal cortical tubules and that UPEC infection caused a decrease in tubular MIF. Infections with UPEC in vitro caused MIF release in a cell type-dependent manner, which was independent of receptor-mediated internalization, signal transduction, and transcription. Indeed, UPEC infection-induced necrotic cell death in vitro and in vivo correlated with extracellular acidification and processed MIF secretion. These data suggest that MIF is released by necrotic renal cortical tubular cells during UPEC infection.
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Infecciones por Escherichia coli/patología , Corteza Renal/patología , Túbulos Renales/patología , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Infecciones Urinarias/patología , Escherichia coli Uropatógena/fisiología , Ácidos/metabolismo , Lesión Renal Aguda/microbiología , Lesión Renal Aguda/patología , Lesión Renal Aguda/orina , Animales , Muerte Celular , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/orina , Femenino , Humanos , Corteza Renal/microbiología , Corteza Renal/ultraestructura , Túbulos Renales/microbiología , Túbulos Renales/ultraestructura , Factores Inhibidores de la Migración de Macrófagos/orina , Ratones , Ratones Endogámicos C57BL , Necrosis , Especificidad de Órganos , Transducción de Señal , Transcripción Genética , Infecciones Urinarias/microbiología , Infecciones Urinarias/orinaRESUMEN
BACKGROUND: Advances in nanotechnology have identified promising candidates for many biological and biomedical applications. Since the properties of nanoparticles (NPs) differ from that of their bulk materials, they are being increasingly exploited for medical uses and other industrial applications. The histological and the histochemical alterations in the renal tissues due to gold nanoparticles (GNPs) have not well documented and have not yet been identified. The aim of the present study was to investigate the particle-size effect of GNPs on the renal tissue in an attempt to address their potential toxicity. METHODS: A total of 70 healthy male Wistar-Kyoto rats were exposed to GNPs received 50 or 100 µl of GNPs infusion of size (10, 20 and 50 nm for 3 or 7 days) to investigate particle-size effect of GNPs on the renal tissue. Animals were randomly divided into groups, 6 GNPs-treated rats groups and one control group. Groups 1, 2 and 3 received infusion of 50 µl GNPs of size 10 nm (3 or 7 days), size 20 nm (3 or 7 days) and 50 nm (3 or 7 days), respectively; while groups 4, 5 and 6 received infusion of 100 µl GNPs of size 10 nm, size 20 nm and 50 nm, respectively. RESULTS: The histological alterations were mainly seen in the cortex and the proximal renal convoluted tubules were more affected than the distal ones. In comparison with respective control rats, exposure to GNPs doses has produced the following renal tubular alterations: cloudy swelling and renal tubular necrosis. Interstitial alterations included: intertubular blood capillaries dilatation, intertubular hemorrhage and inflammatory cell infiltrations. The glomeruli showed moderate congestion with no hypercelluraity and mesangial proliferation or basement membrane thickening. CONCLUSIONS: The induced histological alterations might be an indication of injured renal tubules due to GNPs toxicity that become unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these NPs. These alterations were size-dependent with smaller ones induced more effects and related with time exposure of GNPs. The produced histological alterations may suggest that GNPs interact with proteins and enzymes of the renal tissue interfering with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may induce stress in the renal cells to undergo atrophy and necrosis. More histomorphologcal investigations are needed to address the potential threat of GNPs as a therapeutic and diagnostic tool.
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Núcleo Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Oro/toxicidad , Riñón/efectos de los fármacos , Nanopartículas del Metal/toxicidad , Animales , Adhesión Celular , Núcleo Celular/patología , Citoplasma/patología , Oro/administración & dosificación , Oro/química , Infusiones Parenterales , Riñón/inmunología , Riñón/ultraestructura , Corteza Renal/efectos de los fármacos , Corteza Renal/inmunología , Corteza Renal/ultraestructura , Necrosis Tubular Aguda/inducido químicamente , Necrosis Tubular Aguda/patología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/inmunología , Túbulos Renales Proximales/ultraestructura , Masculino , Ensayo de Materiales , Nanopartículas del Metal/administración & dosificación , Nanopartículas del Metal/química , Nefritis Intersticial/inducido químicamente , Nefritis Intersticial/patología , Infiltración Neutrófila/efectos de los fármacos , Tamaño de la Partícula , Distribución Aleatoria , Ratas , Ratas Endogámicas WKY , Factores de TiempoRESUMEN
This study aimed to explore morphology changes in the kidneys of Dab1-/- (yotari) mice, as well as expression patterns of reelin, NOTCH2, LC3B, and cleaved caspase3 (CASP3) proteins, as potential determinants of normal kidney formation and function. We assumed that Dab1 functional inactivation may cause disorder in a wide spectrum of congenital anomalies of the kidney and urinary tract (CAKUT). Animals were sacrificed at postnatal days P4, P11, and P14. Paraffin-embedded kidney tissues were sectioned and analyzed by immunohistochemistry using specific antibodies. Kidney specimens were examined by bright-field, fluorescence, and electron microscopy. Data were analyzed by two-way ANOVA and t-tests. We noticed that yotari kidneys were smaller in size with a reduced diameter of nephron segments and thinner cortex. TEM microphotographs revealed foot process effacement in the glomeruli (G) of yotari mice, whereas aberrations in the structure of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) were not observed. A significant increase in reelin expression, NOTCH2, LC3B and cleaved CASP3 proteins was observed in the glomeruli of yotari mice. Renal hypoplasia in conjunction with foot process effacement and elevation in the expression of examined proteins in the glomeruli revealed CAKUT phenotype and loss of functional kidney tissue of yotari.
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Proteínas del Tejido Nervioso/genética , Fenotipo , Anomalías Urogenitales/genética , Reflujo Vesicoureteral/genética , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Genes Recesivos , Homocigoto , Corteza Renal/metabolismo , Corteza Renal/ultraestructura , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Nefronas/metabolismo , Nefronas/ultraestructura , Proteínas del Tejido Nervioso/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Proteína Reelina , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Anomalías Urogenitales/metabolismo , Anomalías Urogenitales/patología , Reflujo Vesicoureteral/metabolismo , Reflujo Vesicoureteral/patologíaRESUMEN
Diabetic nephropathy (DN) is a primary cause of endstage renal disease. Despite the beneficial effects of astragaloside IV (AS)IV on renal disease, the underlying mechanism of its protective effects against DN has not been fully determined. The aims of the present study were to assess the effects of ASIV against DN in db/db mice and to explore the mechanism of ASIV involving the NLR family pyrin domain containing 3 (NLRP3), caspase1 and interleukin (IL)1ß pathways. The 8weekold db/db mice received 40 mg/kg ASIV once a day for 12 weeks via intragastric administration. Cultured mouse podocytes were used to further confirm the underlying mechanism in vitro. ASIV effectively reduced weight gain, hyperglycemia and the serum triacylglycerol concentration in db/db mice. ASIV also reduced urinary albumin excretion, urinary albumintocreatinine ratio and creatinine clearance rate, as well as improved renal structural changes, accompanied by the upregulation of the podocyte markers podocin and synaptopodin. ASIV significantly inhibited the expression levels of NLRP3, caspase1 and IL1ß in the renal cortex, and reduced the serum levels of tumor necrosis factor (TNF)α and monocyte chemoattractant protein1. In high glucoseinduced podocytes, ASIV significantly improved the expression levels of NLRP3, procaspase1 and caspase1, and inhibited the cell viability decrease in a dosedependent manner, while NLRP3 overexpression eliminated the effect of ASIV on podocyte injury and the inhibition of the NLRP3 and caspase1 pathways. The data obtained from in vivo and in vitro experiments demonstrated that ASIV ameliorated renal functions and podocyte injury and delayed the development of DN in db/db mice via antiNLRP3 inflammasomemediated inflammation.
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Nefropatías Diabéticas/prevención & control , Inflamasomas/metabolismo , Inflamación/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Saponinas/farmacología , Triterpenos/farmacología , Animales , Caspasa 1/genética , Caspasa 1/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inflamasomas/genética , Inflamación/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Corteza Renal/ultraestructura , Masculino , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Obesidad/complicaciones , Obesidad/genética , Podocitos/citología , Podocitos/efectos de los fármacos , Podocitos/metabolismoRESUMEN
Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) play crucial roles in diabetic angiopathy. In vivo, however, the following facts remain unknown: whether COX-2 and iNOS bind, how peroxynitrite-induced nitration of COX-2 and iNOS affects their binding if they do bind and what effects of this mechanism contribute to diabetic angiopathy. This study focused on the issues above. Diabetes was induced in Wistar male rats by intraperitoneal injection of streptozotocin. As a specific scavenger of peroxynitrite, urate was used. After 13 wks of diabetes, the morphological and biochemical changes of the rats showed obvious diabetic angiopathy. There exists in vivo colocalization and binding of COX-2 and iNOS in diabetic angiopathy. The nitration level of total and co-immunoprecipitated COX-2 and iNOS increased significantly, and, simultaneously, their binding and activity increased in the diabetes group. In the diabetes + urate group, the nitration level of COX-2 and iNOS decreased and their binding reduced, consistent with their decreased activity and the attenuated pathological changes in the rat aorta and glomerulus. The results provide in vivo evidence that COX-2 and iNOS can bind in diabetic angiopathy and that peroxynitrite-induced nitration of COX-2 and iNOS promotes their binding, contributing to diabetic angiopathy.
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Ciclooxigenasa 2/metabolismo , Angiopatías Diabéticas/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ácido Peroxinitroso/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/enzimología , Aorta/patología , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Angiopatías Diabéticas/sangre , Angiopatías Diabéticas/patología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Inmunoprecipitación , Corteza Renal/efectos de los fármacos , Corteza Renal/enzimología , Corteza Renal/patología , Corteza Renal/ultraestructura , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/enzimología , Glomérulos Renales/patología , Glomérulos Renales/ultraestructura , Masculino , Nitrosación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Gentamicin (G) is a highly nephrotoxic aminoglucoside. It was used to experimentally induce nephrotoxicity in male Wistar rats. To find a drug capable of protecting the nephron we assayed a cardioprotector (trimetazidine, TMZ) and a hepatoprotector (N-acetyl cysteine, NAC). The rats were divided into six groups (n = 8): (A) control without drugs; (B) treated with 50 mg kg(-1) per day (i.p.) of G for 7 days; (C) diet supplemented with 20 mg kg(-1) per day of TMZ for 7 days; (D) treated with 10 mg kg(-1) per day (i.p.) of NAC for 7 days; (E) pretreated for 7 days with 20 mg kg(-1) per day of TMZ and during the following 7 days with G + TMZ; (F) pretreated for 7 days with 10 mg kg(-1) per day (i.p.) of NAC and during the following 7 days with G + NAC. Urea and creatinine as well as the excretion of urinary gamma-glutamyl transpeptidase (GGT(u)) and urinary N-acetyl-glucosaminidase (NAG(u)) were determined and structural and ultrastructural studies were carried out. Group B was used as a G-induced nephrotoxicity control. Pretreatment with TMZ (E) showed a protector effect against induced nephrotoxicity, with no biochemical or functional changes nor alterations in histoarchitecture or ultrastructure. Pretreatment with NAC (F) showed no protector effect against G-induced nephrotoxicity since no statistically significant differences were found with respect to the control group with G. We conclude that G-induced nephrotoxicity is attenuated by the cytoprotective effect of TMZ. We may infer that TMZ inhibits the reabsorption and consequently the accumulation of G in the proximal tubule cell.
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Acetilcisteína/uso terapéutico , Depuradores de Radicales Libres/uso terapéutico , Gentamicinas/efectos adversos , Enfermedades Renales/prevención & control , Nefronas/efectos de los fármacos , Trimetazidina/uso terapéutico , Acetilcisteína/administración & dosificación , Acetilglucosaminidasa/orina , Animales , Biomarcadores/sangre , Biomarcadores/orina , Creatinina/sangre , Depuradores de Radicales Libres/administración & dosificación , Corteza Renal/efectos de los fármacos , Corteza Renal/ultraestructura , Enfermedades Renales/inducido químicamente , Enfermedades Renales/patología , Masculino , Nefronas/ultraestructura , Ratas , Ratas Wistar , Trimetazidina/administración & dosificación , gamma-Glutamiltransferasa/orinaRESUMEN
Sepsis and shock states impose mitochondrial stress, and in response, adaptive mechanisms such as fission, fusion and mitophagy are induced to eliminate damaged portions of or entire dysfunctional mitochondria. The mechanisms underlying these events are being elucidated; yet a direct link between loss of mitochondrial membrane potential ΔΨm and the initiation of fission, fusion and mitophagy remains to be well characterized. The direct association between the magnitude of the ΔΨm and the capacity for mitochondria to buffer Ca2+ renders Ca2+ uniquely suited as the signal engaging these mechanisms in circumstances of mitochondrial stress that lower the ΔΨm. Herein, we show that the calcium/calmodulin-dependent protein kinase (CaMK) IV mediates an adaptive slowing in oxidative respiration that minimizes oxidative stress in the kidneys of mice subjected to either cecal ligation and puncture (CLP) sepsis or endotoxemia. CaMKIV shifts the balance towards mitochondrial fission and away from fusion by 1) directly phosphorylating an activating Serine616 on the fission protein DRP1 and 2) reducing the expression of the fusion proteins Mfn1/2 and OPA-1. CaMKIV, through its function as a direct PINK1 kinase and regulator of Parkin expression, also enables mitophagy. These data support that CaMKIV serves as a keystone linking mitochondrial stress with the adaptive mechanisms of mitochondrial fission, fusion and mitophagy that mitigate oxidative stress in the kidneys of mice responding to sepsis.
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Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , Dinámicas Mitocondriales , Sepsis/patología , Animales , Ciego/patología , Células HEK293 , Humanos , Corteza Renal/patología , Corteza Renal/ultraestructura , Ligadura , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Mitofagia , Estrés Oxidativo , Proteínas Quinasas/metabolismo , Punciones , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Metastatic renal cell carcinoma (RCC) is highly resistant to conventional systemic treatments, including chemotherapy, radiotherapy and hormonal therapies. Previous studies have shown over-expression of EGFR is associated with high grade tumors and a worse prognosis. Recent studies suggest anticancer therapies targeting the EGFR pathway have shown promising results in clinical trials of RCC patients. Therefore, characterization of the level and localization of EGFR expression in RCC is important for target-dependent therapy. In this study, we investigated the clinical significance of cellular localization of EGFR in human normal renal cortex and RCC. RCC and adjacent normal kidney tissues of 63 patients were obtained for characterization of EGFR expression. EGFR protein expression was assessed by immunohistochemistry on a scale from 0 to 300 (percentage of positive cells x staining intensity) and Western blotting. EGFR membranous staining was significantly stronger in RCC tumors than in normal tissues (P < 0.001). In contrast, EGFR cytoplasmic staining was significantly higher in normal than in tumor tissues (P < 0.001). The levels of membranous or cytoplasmic EGFR expression in RCC tissues were not correlated with sex, tumor grade, TNM stage or overall survival (P > 0.05). These results showed abundant expression of membranous EGFR in RCC, and abundant expression of cytoplasmic EGFR in normal tissues. EGFR expression in RCC was mostly located in the cell membrane, whereas the EGFR expression in normal renal tissues was chiefly seen in cytoplasm. Our results suggest different locations of EGFR expression may be associated with human renal tumorigenesis.
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Carcinoma de Células Renales/química , Membrana Celular/química , Citoplasma/química , Receptores ErbB/análisis , Corteza Renal/química , Neoplasias Renales/química , Proteínas de la Membrana/análisis , Proteínas de Neoplasias/análisis , Anciano , Carcinoma de Células Renales/etiología , Carcinoma de Células Renales/mortalidad , Carcinoma de Células Renales/cirugía , Carcinoma de Células Renales/ultraestructura , Femenino , Humanos , Estimación de Kaplan-Meier , Corteza Renal/ultraestructura , Neoplasias Renales/etiología , Neoplasias Renales/mortalidad , Neoplasias Renales/cirugía , Neoplasias Renales/ultraestructura , Masculino , Persona de Mediana Edad , Nefrectomía , Especificidad de ÓrganosRESUMEN
The matrix of mammalian peroxisomes frequently contains crystalline inclusions. The most common inclusions are membrane associated plate-like "marginal plates" of hitherto unknown nature in renal peroxisomes and central polytubular "cores" composed of urate oxidase in hepatic peroxisomes. In bovine kidney, peroxisomes of proximal tubules exhibit peculiar angular shapes that are caused by multiple marginal plates (Zaar, K., and H.D. Fahimi. 1990. Cell Tissue Res. 260:409-414). Enriched or highly purified peroxisome preparations from this source were used to purify and characterize marginal plates. By SDS-PAGE, one major polypeptide of Mr 33,500 was observed that corresponded to the marginal plate protein. This polypeptide was identified by its enzymatic activity as well as by immunoblotting and preembedding immunocytochemistry as the isozyme B of L-alpha-hydroxyacid oxidase (EC 1.4.3.2). Morphologically, marginal plates were revealed to consist of rectangular straight-edged sheets, exhibiting a defined crystalline lattice structure. The sheets apparently are composed of a single layer of protomers which associate laterally to form a plate-like structure. As deduced from the negative staining results and the additional information of the thickness of marginal plates, each protomer seems to consist of eight subunits forming a cube-like array. The tendency of L-alpha-hydroxyacid oxidase B to self-associate in vitro (Philips, D.R., J.A. Duley, D.J. Fennell, and R.S. Holmes. 1976. Biochim. Biophys. Acta. 427:679-687) corresponds to the mode of association of cubical protomers to form the so-called marginal plates in renal peroxisomes.
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Oxidorreductasas de Alcohol/aislamiento & purificación , Microcuerpos/ultraestructura , Animales , Western Blotting , Bovinos , Fraccionamiento Celular/métodos , Inmunohistoquímica , Técnicas In Vitro , Corteza Renal/ultraestructura , Túbulos Renales Proximales/ultraestructura , Microcuerpos/enzimología , Microscopía ElectrónicaRESUMEN
The renal cortical collecting duct (CCD) consists of principal and intercalated cells. Two forms of intercalated cells, those cells involved in H+/HCO3- transport, have recently been described. H+-secreting cells are capable of apical endocytosis and have H+ATPase on the apical membrane and a basolateral Cl-/HCO3- exchanger. HCO3(-)-secreting cells bind peanut agglutinin (PNA) to apical membrane receptors and have diffuse or basolateral distribution of H+ATPase; their Cl-/HCO3- exchanger is on the apical membrane. We found that 20 h after acid feeding of rabbits, there was a fourfold increase in number of cells showing apical endocytosis and a numerically similar reduction of cells binding PNA. Incubation of CCDs at pH 7.1 for 3-5 h in vitro led to similar, albeit less pronounced, changes. Evidence to suggest internalization and degradation of the PNA binding sites included a reduction in apical binding of PNA, decrease in pH in the environment of PNA binding, and incorporation of electron-dense PNA into cytoplasmic vesicles. Such remodeling was dependent on protein synthesis. There was also functional evidence for loss of apical Cl-/HCO3- exchange on PNA-labeled cells. Finally, net HCO3- flux converted from secretion to absorption after incubation at low pH. Thus, exposure of CCDs to low pH stimulates the removal/inactivation of apical Cl-/HCO3- exchangers and the internalization of other apical membrane components. Remodeling of PNA-labeled cells may mediate the change in polarity of HCO3- flux observed in response to acid treatment.
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Bicarbonatos/metabolismo , Túbulos Renales Colectores/fisiología , Túbulos Renales/fisiología , Animales , Dactinomicina/farmacología , Dieta , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Corteza Renal/fisiología , Corteza Renal/ultraestructura , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/ultraestructura , Cinética , Microscopía Electrónica , Conejos , Valores de ReferenciaRESUMEN
Intercalated epithelial cells exist in a spectrum of phenotypes; at one extreme, beta cells secrete HCO3 by an apical Cl/HCO3 exchanger and a basolateral H+ ATPase. When an immortalized beta cell line is seeded at high density it deposits in its extracellular matrix (ECM) a new protein, hensin, which can reverse the polarity of several proteins including the Cl/HCO3 exchanger (an alternately spliced form of band 3) and the proton translocating ATPase. When seeded at low density and allowed to form monolayers these polarized epithelial cells maintain the original distribution of these two proteins. Although these cells synthesize and secrete hensin, it is not retained in the ECM, but rather, hensin is present in a large number of intracellular vesicles. The apical cytoplasm of low density cells is devoid of actin, villin, and cytokeratin19. Scanning electron microscopy shows that these cells have sparse microvilli, whereas high density cells have exuberant apical surface infolding and microvilli. The apical cytoplasm of high density cells contains high levels of actin, cytokeratin19, and villin. The cell shape of these two phenotypes is different with high density cells being tall with a small cross-sectional area, whereas low density cells are low and flat. This columnarization and the remodeling of the apical cytoplasm is hensin-dependent; it can be induced by seeding low density cells on filters conditioned by high density cells and prevented by an antibody to hensin. The changes in cell shape and apical cytoskeleton are reminiscent of the processes that occur in terminal differentiation of the intestine and other epithelia. Hensin is highly expressed in the intestine and prostate (two organs where there is a continuous process of differentiation). The expression of hensin in the less differentiated crypt cells of the intestine and the basal cells of the prostate is similar to that of low density cells; i.e., abundant intracellular vesicles but no localization in the ECM. On the other hand, as in high density cells hensin is located exclusively in the ECM of the terminally differentiated absorptive villus cells and the prostatic luminal cell. These studies suggest that hensin is a critical new molecule in the terminal differentiation of intercalated cell and perhaps other epithelial cells.
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Diferenciación Celular , Citoesqueleto/ultraestructura , Proteínas de la Membrana , Receptores Inmunológicos/fisiología , Receptores de Lipoproteína , Animales , Células Epiteliales/citología , Proteínas de la Matriz Extracelular , Espacio Extracelular/metabolismo , Corteza Renal/citología , Corteza Renal/ultraestructura , Microscopía Electrónica de Rastreo , Conejos , Receptores Inmunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase BRESUMEN
Haloperidol, a typical antipsychotic, is the most commonly prescribed medication for the treatment of mental health problems such as agitation and psychosis. We attempted to determine the effects of haloperidol treatment on the kidneys of female rats. In addition, we aimed to estimate the numerical density, total number, and height of renal glomeruli and the volume and volumetric fractions of the cortex, medulla, and whole kidneys, and tried to determine whether there was a change in these stereological parameters depending on haloperidol treatment. Both the qualitative and quantitative histological features of the kidney samples were analyzed with conventional histopathological and modern stereological methods at the light microscopic level. The total number of glomeruli and numerical density of glomerulus in the haloperidol-treated groups was not changed by increasing the dose in comparison to the control group. The mean height of the glomerulus significantly increased, especially in low-dose groups. In the haloperidol-treated groups, the volumetric fractions of the cortex to the whole kidney of the rats were significantly decreased by increasing the dose. The volumetric fractions of the medulla to the whole kidney of the rats were increased significantly in parallel by the given dose. In addition, we present quantitative findings showing that haloperidol is associated with many alterations in rat kidneys. It was shown that haloperidol may lead to undesirable changes in the kidney after chronic treatment with especially high doses.
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Haloperidol/efectos adversos , Enfermedades Renales/patología , Riñón/efectos de los fármacos , Riñón/patología , Animales , Antipsicóticos/efectos adversos , Antipsicóticos/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Haloperidol/farmacología , Inmunohistoquímica , Inyecciones Intraperitoneales , Riñón/ultraestructura , Corteza Renal/efectos de los fármacos , Corteza Renal/patología , Corteza Renal/ultraestructura , Enfermedades Renales/inducido químicamente , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/patología , Glomérulos Renales/ultraestructura , Médula Renal/efectos de los fármacos , Médula Renal/patología , Médula Renal/ultraestructura , Microscopía/métodos , Tamaño de los Órganos , Probabilidad , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Valores de ReferenciaRESUMEN
The aim of this study is to study the effect of calcium dobesilate on streptozotocin (STZ)-induced early diabetic nephrophathy (DN) in rats. All male Wistar rats were randomly divided into six groups: normal group; DN blank group; calcium dobesilate 75, 150, and 300 mg x kg(-1) groups and perindopril 0.4 mg x kg(-1) group. Blood glucose and the 24 h urinary albumin were measured dynamically during the experiment, after 8 weeks administration, the level of glycosylated hemoglobin (HbA1c) was determined, the expressions of plasminogen activator inhibitor-1 (PAI-1) and matrix metalloprotein-9 (MMP-9) in cortex of kidney were examined with immunohistochemical staining. The endothelin (ET) in plasma and kidney cortex was measured with radioimmunoassay, renal pathomorphism was observed with light and electron microscopes. Calcium dobesilate could decrease the 24 h urinary albumin and ET in plasma and kidney cortex, down-regulate the expression of PAI-1, and up-regulate MMP-9 in kidney. These findings suggested that calcium dobesilate could protect blood vessel endothelium, inhibit kidney fibrous degeneration, ameliorate renal pathological damage, and protect kidney function in many ways.
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Dobesilato de Calcio/farmacología , Diabetes Mellitus Experimental , Nefropatías Diabéticas , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Albuminuria , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Endotelinas/sangre , Endotelinas/metabolismo , Hemoglobina Glucada/metabolismo , Hemostáticos/farmacología , Corteza Renal/metabolismo , Corteza Renal/ultraestructura , Masculino , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
To map the hemodynamic responses of kidney microstructures at 7.05â¯T with improved sensitivity, a Wireless Amplified NMR Detector (WAND) with cylindrical symmetry was fabricated as an endoluminal detector that can convert externally provided wireless signal at 600.71â¯MHz into amplified MR signals at 300.33â¯MHz. When this detector was inserted inside colonic lumens to sensitively observe adjacent kidneys, it could clearly identify kidney microstructures in the renal cortex and renal medullary. Owing to the higher achievable spatial resolution, differential hemodynamic responses of kidney microstructures under different breathing conditions could be individually quantified to estimate the underlying correlation between oxygen bearing capability and local levels of oxygen unsaturation. The WAND's ability to map Blood Oxygen Level Dependent (BOLD) signal responses in heterogeneous microstructures will pave way for early-stage diagnosis of kidney diseases, without the use of contrast agents for reduced tissue retention and toxicity.
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Riñón/diagnóstico por imagen , Imagen por Resonancia Magnética/instrumentación , Imagen por Resonancia Magnética/métodos , Animales , Procesamiento de Imagen Asistido por Computador , Riñón/ultraestructura , Corteza Renal/diagnóstico por imagen , Corteza Renal/ultraestructura , Médula Renal/diagnóstico por imagen , Médula Renal/ultraestructura , Masculino , Consumo de Oxígeno , Fantasmas de Imagen , Ratas , Ratas Sprague-Dawley , Circulación Renal , Tecnología InalámbricaRESUMEN
The major pathway for HCO3- transport across the basolateral membrane of the proximal tubule cell is electrogenic Na+-HCO3- cotransport. In this study, we have determined the stoichiometry of the Na+-HCO3- cotransport system in basolateral membrane vesicles that were isolated from rabbit renal cortex by Percoll gradient centrifugation. When the membrane potential is approximated by the Nernst potential for K+, as in the presence of the K+ ionophore valinomycin, equilibrium thermodynamics predicts that the Na+-HCO3- cotransport system should come to equilibrium and mediate no net flux when (Na)i/(Na)o = [(HCO3)o/(HCO3)i]n[(K)o/(K)i]n-1, where n is the HCO3-:Na+ stoichiometry. Our experimental approach was to impose transmembrane Na+, HCO3-, and K+ gradients of varying magnitude and direction, and then to measure the net flux of Na+ over the subsequent 3-s period. In this way, we could determine the conditions for equilibrium of the transport system and thereby calculate n. The results of these experiments indicate that the value of n is greater than 2.6 and less than 3.5, consistent with a stoichiometry of 3 HCO3-:1 Na+, or a thermodynamically equivalent process. Based on reported intracellular potentials and ion activities, this value for the stoichiometry indicates that the inside-negative membrane potential is sufficient to drive HCO3- exit against the inward concentration gradients of HCO3- and Na+ that are present across the basolateral membrane of the intact proximal tubule cell under physiologic conditions.