RESUMO
Inflammatory bowel disease (IBD) is associated with both impaired intestinal blood flow and increased risk of cardiovascular disease, but the functional role of perivascular nerves that control vasomotor function of mesenteric arteries (MAs) perfusing the intestine during IBD is unknown. Because perivascular sensory nerves and their transmitters calcitonin gene-related peptide (CGRP) and substance P (SP) are important mediators of both vasodilation and inflammatory responses, our objective was to identify IBD-related deficits in perivascular sensory nerve function and vascular neurotransmitter signaling. In MAs from an interleukin-10 knockout (IL-10-/-) mouse model, IBD significantly impairs electrical field stimulation (EFS)-mediated sensory vasodilation and inhibition of sympathetic vasoconstriction, despite decreased sympathetic nerve density and vasoconstriction. The MA content and EFS-mediated release of both CGRP and SP are decreased with IBD, but IBD has unique effects on each transmitter. CGRP nerve density, receptor expression, hyperpolarization, and vasodilation are preserved with IBD. In contrast, SP nerve density and receptor expression are increased, and SP hyperpolarization and vasodilation are impaired with IBD. A key finding is that blockade of SP receptors restores EFS-mediated sensory vasodilation and enhanced CGRP-mediated vasodilation in MAs from IBD but not Control mice. Together, these data suggest that an aberrant role for the perivascular sensory neurotransmitter SP and its downstream signaling in MAs underlies vascular dysfunction with IBD. We propose that with IBD, SP signaling impedes CGRP-mediated sensory vasodilation, contributing to impaired blood flow. Thus, substance P and NK1 receptors may represent an important target for treating vascular dysfunction in IBD.NEW & NOTEWORTHY Our study is the first to show that IBD causes profound impairment of sensory vasodilation and inhibition of sympathetic vasoconstriction in mesenteric arteries. This occurs alongside decreased SP-containing nerve density and increased expression of NK1 receptors for SP. In contrast, CGRP dilation, nerve density, and receptor expression are unchanged. Blocking NK1 receptors restores sensory vasodilation in MAs and increases CGRP-mediated vasodilation, indicating that SP interference with CGRP signaling may underlie impaired sensory vasodilation with IBD.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Artérias Mesentéricas/inervação , Células Receptoras Sensoriais/metabolismo , Circulação Esplâncnica , Substância P/metabolismo , Sistema Nervoso Simpático/fisiopatologia , Animais , Modelos Animais de Doenças , Feminino , Helicobacter hepaticus , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/microbiologia , Doenças Inflamatórias Intestinais/fisiopatologia , Interleucina-10/deficiência , Interleucina-10/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais , Vasoconstrição , VasodilataçãoRESUMO
Metastatic renal cell carcinoma (RCC) remains an important clinical issue; the 5-year survival rate of patients with metastasis is approximately 12%, while it is 93% in those with localized disease. There is evidence that blood cadmium and lead levels are elevated in RCC. The current studies were designed to assess the impact of cadmium and lead on the progression of RCC. The disruption of homotypic cell-cell adhesion is an essential step in epithelial-to-mesenchymal transition and tumor metastasis. Therefore, we examined the impact of cadmium and lead on the cadherin/catenin complex in Renca cells-a mouse RCC cell line. Lead, but not cadmium, induced a concentration-dependent loss of E-cadherin, while cadmium, but not lead, increased p120-catenin expression, specifically isoform 1 expression. Lead also induced a substantial increase in matrix metalloproteinase-9 levels. Both cadmium and lead significantly decreased the number of Renca cell aggregates, consistent with the disruption of the cadherin/catenin complex. Both metals enhanced wound healing in a scratch assay, and increased cell migration and invasion. These data suggest that cadmium and lead promote RCC progression.
Assuntos
Cádmio/efeitos adversos , Carcinoma de Células Renais/patologia , Agregação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Neoplasias Renais/patologia , Chumbo/efeitos adversos , Invasividade Neoplásica/patologia , Animais , Caderinas/metabolismo , Carcinoma de Células Renais/metabolismo , Cateninas/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Renais/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , delta CateninaRESUMO
The aging kidney is a marked by a number of structural and functional changes, including an increased susceptibility to acute kidney injury (AKI). Previous studies from our laboratory have shown that aging male Fischer 344 rats (24 month) are more susceptible to apoptosis-mediated injury than young counterparts. In the current studies, we examined the initial injury and early recovery phases of mercuric chloride-induced AKI. Interestingly, the aging kidney had decreased serum creatinine compared to young controls 1 day following mercuric chloride injury, but by day 4, serum creatinine was significantly elevated, suggesting that the aging kidney did not recover from injury. This conclusion is supported by the findings that serum creatinine and kidney injury molecule-1 (Kim-1) gene expression remain elevated compared to young controls at 10 days post-injury. To begin to elucidate mechanism(s) underlying dysrepair in the aging kidney, we examined the expression of Twist2, a helix-loop-helix transcription factor that may mediate renal fibrosis. Interestingly, Twist2 gene expression was elevated following injury in both young and aged rats, and Twist2 protein expression is elevated by mercuric chloride in vitro.
Assuntos
Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Regulação da Expressão Gênica , Proteína 2 Relacionada a Twist/genética , Fatores Etários , Animais , Biomarcadores , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Masculino , Ratos , Regeneração/genética , Fatores de Tempo , Proteína 2 Relacionada a Twist/metabolismoRESUMO
BACKGROUND: The aging kidney has a decreased ability to repair following injury. We have shown a loss in expression of α-catenin in the aging rat kidney and hypothesize that decreased α-catenin expression in tubular epithelial cells results in diminished repair capacity. METHODS: In an effort to elucidate alterations due to the loss of α-catenin, we generated NRK-52E cell lines with stable knockdown of α(E)-catenin. RESULTS: α(E)-catenin knockdown resulted in decreased wound repair due to alterations in cell migration. Analysis of gene expression in the α(E)-catenin knockdown cells demonstrated almost a complete loss of bone morphogenetic protein-7 (BMP-7) expression that was associated with decreased phospho-Smad1/5/8 staining. However, addition of exogenous BMP-7 increased phospho-Smad1/5/8, suggesting that the BMP-7 pathway remained intact in C2 cells. Given the potential role of BMP-7 in repair, we investigated its role in wound repair. Inhibition of BMP-7 decreased repair in non-targeted control cells; conversely, exogenous BMP-7 restored repair in α(E)-catenin knockdown cells to control levels. CONCLUSIONS: Taken together, the data suggests that the loss of α(E)-catenin expression and subsequent downregulation of BMP-7 is a mechanism underlying the altered migration of tubular epithelial cells that contributes to the inability of the aging kidney to repair following injury.
Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Células Epiteliais/fisiologia , Transdução de Sinais/genética , alfa Catenina/genética , alfa Catenina/metabolismo , Animais , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/farmacologia , Linhagem Celular , Movimento Celular/genética , Proliferação de Células , Regulação para Baixo , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Túbulos Renais , Masculino , Camundongos , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Proteína Smad8/metabolismo , CicatrizaçãoRESUMO
BACKGROUND: A variety of medicinal products prepared from secondary tubers of Harpagophytum procumbens subsp. procumbens (Burch.) DC.ex Meisn. (Devil's Claw) and H. zeyheri are marketed in Africa, Europe, the United States, South America and elsewhere, where they are used for inflammatory and musculoskeletal conditions such as arthritis, lower back pain, rheumatism and neuralgia, etc. While clinical studies conducted over the last twenty years support the general safety of such products, infrequent gastrointestinal disturbances (diarrhea, nausea, vomiting, abdominal pain), headache, vertigo and hypersensitivity (allergic) reactions (rash, hives and face swelling) have been documented. Sex-related differences occur in the health conditions for which Devil's Claw products are used, so it is likely that usage is similarly sex-related and so might be side effects and potential toxicities. However toxicologic studies of Devil's Claw products have been conducted primarily with male animals. To address this deficit, we report toxicological studies in female and male rats of several H. procumbens (HP) aqueous-alcohol extracts chemically analyzed by UPLC-MS. METHODS: Female and male Sprague Dawley rats were studied for one and three months in groups differing by consumption of diets without and with HP extracts at a 7-10-fold human equivalent dose (HED). Sera were analyzed for blood chemistry, and heart, liver, lung, kidney, stomach, and small and large intestine tissues were examined for histopathology. Treatment group differences for blood chemistry were analyzed by ANOVA with Dunnett's test and significant group differences for endpoints with marginal distributional properties were verified using the Kruskal-Wallis test. Group differences for histopathology were tested using Chi Square analysis. RESULTS: Significant group by sex-related differences in blood chemistry were detected in both studies. Additionally, several sex-related differences occurred between the studies. However, significant histopathology effects associated with the consumption of the extracts were not detected. CONCLUSION: Toxicologic analysis of Devil's Claw extracts cause significant sex-related effects in blood chemistry. However, in our judgement, none of the observed effects suggest serious toxicity at these doses and durations. Subsequent toxicologic and clinical studies of H. procumbens and other medicines with similar properties should explore in greater detail the basis and consequences of potential sex-related effects.
Assuntos
Harpagophytum/toxicidade , Extratos Vegetais/toxicidade , África , Animais , Cromatografia Líquida , Feminino , Masculino , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em TandemRESUMO
Aging is a risk factor for chronic kidney disease (CKD) and is itself associated with alterations in renal structure and function. There are no specific interventions to attenuate age-dependent renal dysfunction and the mechanism(s) responsible for these deficits have not been fully elucidated. In this study, male Fischer 344 rats, which develop age-dependent nephropathy, were feed a casein- or soy protein diet beginning at 16 mon (late life intervention) and renal structure and function was assessed at 20 mon. The soy diet did not significantly affect body weight, but was renoprotective as assessed by decreased proteinuria, increased glomerular filtration rate (GFR) and decreased urinary kidney injury molecule-1 (Kim-1). Renal fibrosis, as assessed by hydroxyproline content, was decreased by the soy diet, as were several indicators of inflammation. RNA sequencing identified several candidates for the renoprotective effects of soy, including decreased expression of Twist2, a basic helix-loop-helix transcription factor that network analysis suggest may regulate the expression of several genes associated with renal dysfunction. Twist2 expression is upregulated in the aging kidney and the unilateral ureteral obstruction of fibrosis; the expression is limited to distal tubules of mice. Taken together, these data demonstrate the renoprotective potential of soy protein, putatively by reducing inflammation and fibrosis, and identify Twist2 as a novel mediator of renal dysfunction that is targeted by soy.
RESUMO
Previous studies have shown that the aging kidney has a marked loss of α(E)-catenin in proximal tubular epithelium. α-Catenin, a key regulator of the actin cytoskeleton, interacts with a variety of actin-binding proteins. Cisplatin-induced loss of fascin2, an actin bundling protein, was observed in cells with a stable knockdown of α(E)-catenin (C2 cells), as well as in aging (24 mon), but not young (4 mon), kidney. Fascin2 co-localized with α-catenin and the actin cytoskeleton in NRK-52E cells. Knockdown of fascin2 increased the susceptibility of tubular epithelial cells to cisplatin-induced injury. Overexpression of fascin2 in C2 cells restored actin stress fibers and attenuated the increased sensitivity of C2 cells to cisplatin-induced apoptosis. Interestingly, fascin2 overexpression attenuated cisplatin-induced mitochondrial dysfunction and oxidative stress in C2 cells. These data demonstrate that fascin2, a putative target of α(E)-catenin, may play important role in preventing cisplatin-induced acute kidney injury.
Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Cisplatino/toxicidade , Proteínas dos Microfilamentos/metabolismo , Envelhecimento , Animais , Proteínas de Transporte/genética , Cateninas/genética , Cateninas/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Rim/citologia , Proteínas dos Microfilamentos/genética , Transporte Proteico , RatosRESUMO
The male Fischer 344 rat is an established model to study progressive renal dysfunction that is similar, but not identical, to chronic kidney disease (CKD) in humans. These studies were designed to assess age-dependent alterations in renal structure and function at late-life timepoints, 16-24 months. Elevations in BUN and plasma creatinine were not significant until 24 months, however, elevations in the more sensitive markers of function, plasma cystatin C and proteinuria, were detectable at 16 and 18 months, respectively. Interestingly, cystatin C levels were not corrected by caloric restriction. Urinary Kim-1, a marker of CKD, was elevated as early as 16 months. Klotho gene expression was significantly decreased at 24 months, but not at earlier timepoints. Alterations in renal structure, glomerulosclerosis and tubulointerstitial fibrosis, were noted at 16 months, with little change from 18 to 24 months. Tubulointerstitial inflammation was increased at 16 months, and remained similar from 18 to 24 months. A SEM (structural equation modeling) model of age-related renal dysfunction suggests that proteinuria is a marker of renal damage, while urinary Kim-1 is a marker of both damage and function. Taken together, these results demonstrate that age-dependent nephropathy begins as early as 16 months and progresses rapidly over the next 8 months.
Assuntos
Envelhecimento , Moléculas de Adesão Celular/urina , Cistatina C/sangue , Modelos Biológicos , Proteinúria , Insuficiência Renal Crônica , Envelhecimento/sangue , Envelhecimento/urina , Animais , Glucuronidase/metabolismo , Humanos , Proteínas Klotho , Masculino , Proteinúria/sangue , Proteinúria/urina , Ratos , Ratos Endogâmicos F344 , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/urinaRESUMO
Cisplatin is one of the most potent and widely used antitumor drugs. However, the use of cisplatin is limited by its side effect, nephrotoxicity. Evidence has shown an increased incidence and severity of acute kidney injury (AKI) in the elderly. Previous studies from our laboratory demonstrate a decrease in α(E)-catenin expression in aged kidney. In this study, we investigated whether the loss of α(E)-catenin may increase cisplatin nephrotoxicity. To study the effects of reduced α(E)-catenin, a cell line with stable knockdown of α(E)-catenin (C2 cells) was used; NT3 is nontargeted control. C2 cells exhibited a significant loss of viability as determined by MTT assay compared with NT3 cells after cisplatin challenge, but showed no difference in lactate dehydrogenase (LDH) leakage. Increased caspase 3/7 activation and PARP cleavage was observed in C2 cells after cisplatin treatment. Z-VAD, a pan-caspase inhibitor, abolished the difference in susceptibility between NT3 and C2 cells. Interestingly, the expression of α(E)-catenin was further decreased after cisplatin treatment. Furthermore, in vivo data demonstrated a significant increase in serum creatinine at 72 h after a single dose of cisplatin in 24-month-old rats, but not in 4-month-old rats. Increased expression of KIM-1 and in situ apoptosis were also detected in aged kidney after cisplatin challenge. Taken together, these data suggest that loss of α(E)-catenin increases apoptosis of tubular epithelial cells which may contribute to the increased nephrotoxicity induced by cisplatin in aged kidney.
Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Cisplatino/toxicidade , Células Epiteliais/efeitos dos fármacos , Nefropatias/induzido quimicamente , Túbulos Renais Proximais/efeitos dos fármacos , alfa Catenina/fisiologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Técnicas de Silenciamento de Genes , Nefropatias/genética , Nefropatias/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase em Tempo Real , alfa Catenina/genéticaRESUMO
The aging kidney has a decreased ability to repair following acute kidney injury. Previous studies from our laboratory have demonstrated a loss in α-catenin expression in the aging rat kidney. We hypothesize that loss of α-catenin expression in tubular epithelial cells may induce changes that result in a decreased repair capacity. In these studies, we demonstrate that decreased α-catenin protein expression is detectable as early as 20 months of age in male Fischer 344 rats. Protein loss is also observed in aged nonhuman primate kidneys, suggesting that this is not a species-specific response. In an effort to elucidate alterations due to the loss of α-catenin, we generated NRK-52E cell lines with stable knockdown of α(E)-catenin (C2 cells). Interestingly, C2 cells had decreased expression of N-cadherin, decreased cell-cell adhesion, and increased monolayer permeability. C2 had deficits in wound repair, due to alterations in cell migration. Analysis of gene expression in the migrating control cells indicated that expression of N-cadherin and N-CAM was increased during repair. In migrating C2 cells, expression of N-CAM was also increased, but the expression of N-cadherin was not upregulated. Importantly, a blocking antibody against N-cadherin inhibited repair in NRK-52E cells, suggesting an important role in repair. Taken together, these data suggest that loss of α-catenin, and the subsequent downregulation of N-cadherin expression, is a mechanism underlying the decreased migration of tubular epithelial cells that contributes to the inability of the aging kidney to repair following injury.
RESUMO
The percentage of the U.S. population over 65 is rapidly increasing, as is the incidence of chronic kidney disease (CKD). The kidney is susceptible to age-dependent alterations in structure, specifically tubulointerstitial fibrosis, that lead to CKD. Matrix metalloproteinases (MMPs) were initially characterized as extracellular matrix (ECM) proteinases; however it is clear that their biological role is much larger. We have observed increased gene expression of several MMPs in the aging kidney, including MMP-7. MMP-7 overexpression was observed starting at 16 months, and over a 500 fold up-regulation in 2 year-old animals. Overexpression of MMP-7 is not observed in age-matched, calorically restricted controls that do not develop fibrosis and renal dysfunction, suggesting a role in the pathogenesis. In order to delineate the contributions of MMP-7 to renal dysfunction, we overexpressed MMP-7 in NRK-52E cells. High-throughput sequencing of the cells revealed that two collagen genes, Col1a2 and Col3a1, were elevated in the MMP-7 overexpressing cells. These two collagen genes were also elevated in aging rat kidneys and temporally correlated with increased MMP-7 expression. Addition of exogenous MMP-7, or conditioned media from MMP-7 overexpressing cells also increased Col1A2 expression. Inhibition of PKA, src, and MAPK signaling at p38 and ERK was able to attenuate the MMP-7 up-regulation of Col1a2. Consistent with this finding, increased phosphorylation of PKA, src and ERK was seen in MMP-7 overexpressing cells and upon exogenous MMP-7 treatment of NRK-52E cells. These data suggest a novel mechanism by which MMP-7 contributes to the development of fibrosis leading to CKD.