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1.
Science ; 364(6436)2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30975860

RESUMO

To understand the health impact of long-duration spaceflight, one identical twin astronaut was monitored before, during, and after a 1-year mission onboard the International Space Station; his twin served as a genetically matched ground control. Longitudinal assessments identified spaceflight-specific changes, including decreased body mass, telomere elongation, genome instability, carotid artery distension and increased intima-media thickness, altered ocular structure, transcriptional and metabolic changes, DNA methylation changes in immune and oxidative stress-related pathways, gastrointestinal microbiota alterations, and some cognitive decline postflight. Although average telomere length, global gene expression, and microbiome changes returned to near preflight levels within 6 months after return to Earth, increased numbers of short telomeres were observed and expression of some genes was still disrupted. These multiomic, molecular, physiological, and behavioral datasets provide a valuable roadmap of the putative health risks for future human spaceflight.


Assuntos
Adaptação Fisiológica , Astronautas , Voo Espacial , Imunidade Adaptativa , Peso Corporal , Artérias Carótidas/diagnóstico por imagem , Espessura Intima-Media Carotídea , Dano ao DNA , Metilação de DNA , Microbioma Gastrointestinal , Instabilidade Genômica , Humanos , Masculino , Homeostase do Telômero , Fatores de Tempo , Estados Unidos , United States National Aeronautics and Space Administration
2.
Arch Biochem Biophys ; 523(2): 161-8, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-22568896

RESUMO

We showed that renal calpain 10, a mitochondrial and cytosolic Ca(2+)-regulated cysteine protease, is specifically decreased in kidneys of diabetic rats and mice, and is associated with diabetic nephropathy. The goals of this study were to examine renal calpain 10 and mitochondrial dysfunction in streptozotocin-induced hyperglycemic rats and determine the effects of siRNA-mediated knock down of renal calpain 10 on mitochondrial function. Four weeks after streptozotocin injection, calpain 10 protein and mRNA were decreased and calpain 10 substrates accumulated. We detected increased state 2 respiration in isolated renal mitochondria and increased markers of mitochondrial fission and mitophagy. All changes were prevented by daily insulin injection. Compared to scrambled siRNA, calpain 10 siRNA resulted in a marked decrease in renal calpain 10 at 2, 5 and 7 days. In concert with the loss of renal calpain 10, calpain 10 substrates accumulated, mitochondrial fusion decreased, mitochondrial fission and mitophagy increased. In summary, insulin-sensitive hyperglycemia induced loss of renal calpain 10 is correlated with renal mitochondrial dysfunction, fission and mitophagy, and specific depletion of renal calpain 10 produces similar mitochondrial defects. These results provide evidence that diabetes-induced renal mitochondrial dysfunction and renal injury may directly result from the loss of renal calpain 10.


Assuntos
Calpaína/deficiência , Calpaína/genética , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Animais , Doença Crônica , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Técnicas de Silenciamento de Genes , Hiperglicemia/induzido quimicamente , Insulina/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Estreptozocina/efeitos adversos , Fatores de Tempo
3.
Kidney Int ; 81(4): 391-400, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22012129

RESUMO

Whereas most calpains are cytosolic proteases, calpain 10 is resident in mitochondria and is important in mitochondrial homeostasis. Because calpain 10 has been implicated in type 2 diabetes, we studied its possible role in diabetes-induced renal dysfunction. We treated renal proximal tubular cells with high glucose (17 mmol/l) and found decreased mitochondrial calpain 10 mRNA and protein at 96 h compared with cells incubated with 0 or 5 mmol/l glucose or 17 mmol/l D-mannitol. High glucose increased mitochondrial calpain 10 substrates (NDUFB8 and ATP synthase ß), decreased basal and uncoupled respiration, and initiated cell apoptosis as indicated by cleaved caspase 3 and nuclear condensation. Renal calpain 10 protein and mRNA were specifically decreased in streptozotocin-induced diabetic rats with kidney dysfunction, and in diabetic ob/ob mice. In agreement with our in vitro data, the kidneys of streptozotocin-induced diabetic rats had elevated calpain 10 substrates and cleaved caspase 3. Finally, specific siRNA-induced knockdown of calpain 10 in the proximal tubules of control rats resulted in decreased renal function as evidenced by increased serum creatinine, and increased caspase 3 cleavage compared with rats receiving scrambled siRNA. Thus, the glucose-induced loss of calpain 10 in vivo results in renal cell apoptosis and organ failure through accumulation of mitochondrial calpain 10 substrates and mitochondrial dysfunction. Whether this is a major cause of the decreased renal function in diabetic nephropathy will require further studies.


Assuntos
Calpaína/metabolismo , Nefropatias Diabéticas/metabolismo , Glucose/metabolismo , Túbulos Renais Proximais/enzimologia , Mitocôndrias/enzimologia , Animais , Apoptose , Calpaína/efeitos dos fármacos , Caspase 3/metabolismo , Células Cultivadas , Diabetes Mellitus/induzido quimicamente , Nefropatias Diabéticas/enzimologia , Regulação para Baixo , Feminino , Glucose/farmacologia , Túbulos Renais Proximais/fisiopatologia , Masculino , Camundongos , Camundongos Obesos , Mitocôndrias/fisiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Coelhos , Ratos , Ratos Sprague-Dawley , Estreptozocina
4.
Am J Physiol Renal Physiol ; 296(3): F478-86, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19144693

RESUMO

Aging is associated with abnormalities in kidney function, but the exact mechanisms are unknown. We examined calpains 1, 2, and 10 protein levels in kidneys from rats, mice, and humans of various ages and determined whether calpain 10 is required for cell viability. Calpain 10 protein expression decreased in the kidney, but not in the liver, of aging Fischer 344 rats, and this decrease was attenuated with caloric restriction. There was no change in calpains 1 or 2 levels in the kidney or liver in control and caloric-restricted aging rats. Aging mice also exhibited decreased calpain 10 protein levels. Calpain 10 protein and mRNA levels decreased linearly in human kidney samples with age in the absence of changes in calpains 1 or 2. Our laboratory previously found calpain 10 to be expressed in both the cytosol and mitochondria of rabbit renal proximal tubular cells (RPTC). Adenoviral-delivered shRNA to rabbit RPTC decreased mitochondrial calpain 10 expression below detectable levels by 3 days while cytosolic calpain 10 levels remained unchanged at 3 days and decreased to approximately 20% of control by 5 days. Knockdown of mitochondrial calpain 10 resulted in nuclear condensation and cleaved procaspase 3, markers of apoptosis. In summary, mitochondrial calpain 10 is required for cell viability and calpain 10 levels specifically decrease in aging rat, mice, and human kidney tissues when renal function decreases, suggesting that calpain 10 is required for renal function and is a biomarker of the aging kidney.


Assuntos
Envelhecimento/metabolismo , Calpaína/metabolismo , Rim/metabolismo , Adenoviridae/genética , Animais , Restrição Calórica , Calpaína/genética , Sobrevivência Celular , Células Cultivadas , Feminino , Técnicas de Silenciamento de Genes , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Coelhos , Ratos , Ratos Endogâmicos F344
5.
J Lipid Res ; 49(7): 1477-87, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18398221

RESUMO

Our previous studies showed that renal proximal tubular cells (RPTC) express Ca(2+)-independent phospholipase A(2)gamma (iPLA(2)gamma) in endoplasmic reticulum (ER) and mitochondria and that iPLA(2)gamma prevents and/or repairs lipid peroxidation induced by oxidative stress. Our present studies determined the importance of iPLA(2)gamma in mitochondrial and cell function using an iPLA(2)gamma-specific small hairpin ribonucleic acid (shRNA) adenovirus. iPLA(2)gamma expression and activity were decreased in the ER by 24 h and in the mitochondria by 48 h compared with scrambled shRNA adenovirus-treated cells. Lipid peroxidation was elevated by 2-fold at 24 h and remained elevated through 72 h in cells with decreased iPLA(2)gamma. Using electrospray ionization-mass spectrometry, primarily phosphatidylcholines and phosphatidylethanolamines were increased in iPLA(2)gamma-shRNA-treated cells. At 48 h after exposure to the iPLA(2)gamma shRNA, uncoupled oxygen consumption was inhibited by 25% and apoptosis was observed at 72 and 96 h. RPTC with decreased iPLA(2)gamma expression underwent apoptosis when exposed to a nonlethal concentration of the oxidant tert-butyl hydroperoxide (TBHP). Exposure of control cells to a nonlethal concentration of TBHP induced iPLA(2)gamma expression in RPTC. These results suggest that iPLA(2)gamma is required for the prevention and repair of basal lipid peroxidation and the maintenance of mitochondrial function and viability, providing further evidence for a cytoprotective role for iPLA(2)gamma from oxidative stress.


Assuntos
Apoptose/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Oxidantes/farmacologia , Fosfolipases A2 Independentes de Cálcio/metabolismo , Animais , Forma Celular , Células Cultivadas , Túbulos Renais Proximais/enzimologia , Mitocôndrias/metabolismo , Fosfolipases A2 Independentes de Cálcio/genética , Fosfolipídeos/metabolismo , Interferência de RNA , Coelhos
6.
Biochem Biophys Res Commun ; 366(1): 258-62, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18054326

RESUMO

Calpains, Ca(2+)-activated cysteine proteases, have been implicated in the progression of multiple disease states. We recently identified calpain 10 as a mitochondrial calpain that is involved in Ca(2+)-induced mitochondrial dysfunction. The goals of this study were to characterize the expression and activity of renal mitochondrial calpain 10 in rabbit, mouse, and rat. Using shRNA technology and immunoblot analysis three previously postulated splice variants of calpain 10 were identified (50, 56, and 75kDa). SLLVY-AMC zymography and immunoblot analysis was used to directly link calpeptin-sensitive calpain activity to calpain 10 splice variants. Rabbit, mouse, and rat kidney mitochondria contained 75kDa (calpain 10a), 56kDa (calpain 10c or 10d), and 50kDa (calpain 10e) splice variants. Interestingly, zymography yielded distinct bands of calpain activity containing multiple calpain 10 splice variants in all species. These results provide evidence that several previously postulated splice variants of calpain 10 are localized to the mitochondria in kidneys of rabbits, rats, and mice.


Assuntos
Calpaína/metabolismo , Expressão Gênica/fisiologia , Rim/metabolismo , Mitocôndrias/metabolismo , Animais , Camundongos , Ratos , Especificidade da Espécie
7.
Cell Commun Adhes ; 14(4): 111-23, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17957528

RESUMO

Although ischemia is associated with disruption of cadherin-mediated adhesion in renal cell lines, the impact of decreased cadherin function on the transcriptional activity of beta-catenin remains poorly defined. In these studies, we used a simulated ischemia model in normal rat kidney (NRK) cells to disrupt cadherin function. Cell viability; cadherin/catenin expression, function, and localization; and beta-catenin-mediated transcriptional activity were assessed during ischemia/reperfusion. Following 6 hr of ischemia, a decrease in the expression of E- and N-cadherin was seen that correlated with altered cell morphology indicative of decreased intercellular adhesion. While ischemia was associated with activation of glycogen synthase kinase 3 beta (GSK-3beta), this did not correlate with increased phosphorylation of beta-catenin as assessed by Western blots using phosphoryl-specific antibodies. beta-Catenin was not localized to the nucleus by immunofluorescence in ischemic NRK cells, but rather a strong perinuclear signal was seen in reperfused cells. This was consistent with the finding that neither ischemia nor reperfusion activated the transcriptional activity of beta-catenin as assessed by the TCF-optimal promoter (TOPFlash) construct. However, NRK cells possess a competent Wnt pathway, as challenge with lithium chloride elicited a ten-fold increase in luciferase activity. These results suggest that ischemia-induced disruption of cadherin/catenin complexes is not sufficient to stimulate beta-catenin transcriptional activity in NRK cells.


Assuntos
Caderinas/metabolismo , Isquemia/metabolismo , Rim/metabolismo , Transcrição Genética , beta Catenina/genética , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting , Células Cultivadas , Imunofluorescência , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Imunoprecipitação , Rim/citologia , Rim/efeitos dos fármacos , Luciferases/metabolismo , Regiões Promotoras Genéticas , Ratos , Transdução de Sinais , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Ativação Transcricional , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
8.
Am J Physiol Renal Physiol ; 290(1): F43-51, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16077081

RESUMO

Ischemia is a leading cause of acute renal failure (ARF), a disease associated with high morbidity and mortality. Disruption of intercellular adhesion in the proximal tubules is linked to ARF, although the molecular mechanism(s) remains unclear. Our previous studies showed that ischemia is associated with cadherin cleavage and loss in NRK cells, putatively due to a matrix metalloproteinase (MMP) (7). In the current studies, a MMP required for E-cadherin cleavage and N-cadherin loss was identified. Chemical inhibitors against a number of soluble MMPs (1, 2, 3, 8, 9) failed to completely attenuate ischemia-induced cadherin loss. Under ischemic conditions, there was an increase in active membrane-type (MT)1-MMP but a decrease in MMP-2 protein expression. Plating cells on fibronectin protected against ischemia-induced loss of cadherins and, interestingly, no increase in active MT1-MMP levels was seen in ischemic cells on fibronectin-coated dishes. In addition, L cells stably expressing E- (LE) or N-cadherin (LN), but lacking MT1-MMP expression, were resistant to ischemia-induced cadherin loss. The role of MT1-MMP in ischemia-induced cadherin loss was confirmed by either blocking MT1-MMP activity with a neutralizing antibody or expression with shRNA constructs which protected full-length E- and N-cadherin during ischemia. Using shRNA constructs to suppress MT1-MMP expression, ischemia-induced disruption of cadherin function was ablated, and cell-cell contacts were preserved. These results demonstrate that ischemia induces increased expression of active MT1-MMP and subsequent disruption of cadherin/catenin complexes, implying that MT1-MMP plays a role in ischemia-induced ARF.


Assuntos
Caderinas/metabolismo , Isquemia/metabolismo , Metaloproteinases da Matriz/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Matriz Extracelular/fisiologia , Túbulos Renais Proximais/metabolismo , Metaloproteinase 14 da Matriz , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz Associadas à Membrana , Camundongos , Dados de Sequência Molecular , Inibidores de Proteases/farmacologia , Ratos , Circulação Renal , Transfecção
9.
Am J Physiol Renal Physiol ; 289(2): F280-8, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15769936

RESUMO

Although ischemia has been shown to disrupt cell adhesion, the underlying molecular mechanism is unknown. In these studies, we adapted a model of ischemia-reperfusion to normal rat kidney (NRK) cells, examined disruption of the cadherin/catenin complex, and identified a role for matrix metalloproteinases (MMPs) in ischemia-induced cleavage of cadherins. In NRK cells, ischemia was induced by applying a thin layer of PBS solution supplemented with calcium and magnesium and a layer of mineral oil, which restricts exposure to oxygen. NRK cells exhibited extracellular 80-kDa and intracellular 40-kDa E-cadherin fragments after 4 h of ischemia, and at 6 h the expression of full-length E-cadherin decreased. While no fragments of N-cadherin, alpha-catenin, and gamma-catenin were observed at any time point, the detectable levels of these proteins decreased during ischemia. Ischemia was detected by an increase in pimonidazole adducts, as well as an increase in glucose transporter-1 protein expression. Ischemia did not decrease cell number, but there was a decrease in ATP levels. In addition, there was no evidence of cleaved caspase 3 or 9 during 6 h of ischemia. The MMP inhibitors GM-6001 and TAPI-O inhibited cleavage and/or loss of E- and N-cadherin protein expression. Tissue inhibitors of metalloproteinases (TIMP)-3 and to a lesser extent TIMP-2, but not TIMP-1, inhibit ischemic cleavage and/or loss of E- and N-cadherin. These results demonstrate that ischemia induces a selective metalloproteinase-dependent cleavage of E-cadherin and decrease in N-cadherin that are associated with a disruption of junctional contacts.


Assuntos
Caderinas/metabolismo , Isquemia/metabolismo , Rim/metabolismo , Metaloproteases/fisiologia , Circulação Renal/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting , Agregação Celular , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Rim/citologia , Rim/efeitos dos fármacos , Metaloproteases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Ratos , Inibidor Tecidual de Metaloproteinase-1/fisiologia , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Inibidor Tecidual de Metaloproteinase-3/fisiologia
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