RESUMEN

The live attenuated human rotavirus vaccine strain RIX4414 (Rotarix®) is used worldwide to prevent severe rotavirus-induced diarrhea in infants. This strain was attenuated through the cell culture passaging of its predecessor, human strain 89-12, which resulted in multiple genomic mutations. However, the specific molecular reasons underlying its attenuation have remained elusive, primarily due to the absence of a suitable reverse genetics system enabling precise genetic manipulations. Therefore, we first completed the sequencing of its genome and then developed a reverse genetics system for the authentic RIX4414 virus. Our experimental results demonstrate that the rescued recombinant RIX4414 virus exhibits biological characteristics similar to those of the parental RIX4414 virus, both in vitro and in vivo. This novel reverse genetics system provides a powerful tool for investigating the molecular basis of RIX4414 attenuation and may facilitate the rational design of safer and more effective human rotavirus vaccines.

Asunto(s)

ADN Complementario , Genética Inversa , Infecciones por Rotavirus , Vacunas contra Rotavirus , Rotavirus , Vacunas Atenuadas , Vacunas contra Rotavirus/genética , Vacunas contra Rotavirus/inmunología , Genética Inversa/métodos , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Rotavirus/genética , Rotavirus/inmunología , Humanos , Animales , Infecciones por Rotavirus/prevención & control , Infecciones por Rotavirus/virología , ADN Complementario/genética , Genoma Viral , Ratones , Línea Celular

RESUMEN

Bone fractures represent a common health problem, particularly in an increasingly aging population. Bioresorbable magnesium (Mg) alloy-based implants offer promising alternatives to traditional metallic implants for the treatment of bone fractures because they eliminate the need for implant removal after healing. The Mg-Y-rare-earth (RE)-Zr alloy WE43, designed for orthopedic implants, has received European Conformity mark approval. However, currently, WE43 is not clinically used in certain countries possibly because of concerns related to RE metals. In this study, we investigated the use of a RE-free alloy, namely, Mg-Zn-Zr alloy (ZK30), as an implant for bone fractures. Hydrofluoric acid (HF) treatment was performed to improve the corrosion resistance of ZK30. HF-treated ZK30 (HF-ZK30) exhibited lower corrosion rate and higher biocompatibility than those of WE43 in in vitro experiments. After implanting a rod of HF-ZK30 into the fractured femoral bones of mice, HF-ZK30 held the bones and healed the fracture without deformation. Treatment results of HF-ZK30 were comparable to those of WE43, indicating the potential of HF-ZK30 as a bioresorbable and safe implant for bone repair.

Asunto(s)

Implantes Absorbibles , Aleaciones , Magnesio , Animales , Magnesio/química , Magnesio/farmacología , Aleaciones/química , Ratones , Fluoruros/química , Corrosión , Ensayo de Materiales , Fracturas Óseas/terapia , Masculino , Materiales Biocompatibles/química

RESUMEN

Vaccine development for herpes simplex virus 2 (HSV-2) has been attempted, but no vaccines are yet available. A plasmid-based reverse genetics system for Rotavirus (RV), which can cause gastroenteritis, allows the generation of recombinant RV containing foreign genes. In this study, we sought to develop simian RV (SA11) as a vector to express HSV-2 glycoprotein D (gD2) and evaluated its immunogenicity in mice. We generated the recombinant SA11-gD2 virus (rSA11-gD2) and confirmed its ability to express gD2 in vitro. The virus was orally inoculated into suckling BALB/c mice and into 8-week-old mice. Serum IgG and IgA titers against RV and gD2 were measured by ELISA. In the 8-week-old mice inoculated with rSA11-gD2, significant increases in not only antibodies against RV but also IgG against gD2 were demonstrated. In the suckling mice, antibodies against RV were induced, but gD2 antibody was not detected. Diarrhea observed after the first inoculation of rSA11-gD2 in suckling mice was similar to that induced by the parent virus. A gD2 expressing simian RV recombinant, which was orally inoculated, induced IgG against gD2. This strategy holds possibility for genital herpes vaccine development.

Asunto(s)

Herpes Genital , Rotavirus , Animales , Ratones , Herpesvirus Humano 2/genética , Rotavirus/genética , Genética Inversa , Proteínas del Envoltorio Viral/genética , Glicoproteínas/genética , Inmunoglobulina G , Anticuerpos Antivirales

RESUMEN

BACKGROUND AND PURPOSE: High salt (HS) intake has been associated with hypertension and cognitive impairment. It is well known that the angiotensin II (Ang II)-AT1 receptor and prostaglandin E2 (PGE2)-EP1 receptor systems are involved in hypertension and neurotoxicity. However, the involvement of these systems in HS-mediated hypertension and emotional and cognitive impairments remains unclear. EXPERIMENTAL APPROACH: Mice were loaded with HS solution (2% NaCl drinking water) for 12 weeks, and blood pressure was monitored. Subsequently, effects of HS intake on emotional and cognitive function and tau phosphorylation in the prefrontal cortex (PFC) and hippocampus (HIP) were investigated. The involvement of Ang II-AT1 and PGE2-EP1 systems in HS-induced hypertension and neuronal and behavioural impairments was examined by treatment with losartan, an AT1 receptor blocker (ARB), or EP1 gene knockout. KEY RESULTS: We demonstrate that hypertension and impaired social behaviour and object recognition memory following HS intake may be associated with tau hyperphosphorylation, decreased phosphorylation of Ca2+ /calmodulin-dependent protein kinase II (CaMKII), and postsynaptic density protein 95 (PSD95) expression in the PFC and HIP of mice. These changes were blocked by pharmacological treatment with losartan or EP1 receptor gene knockout. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that the interaction of Ang II-AT1 receptor and PGE2-EP1 receptor systems could be novel therapeutic targets for hypertension-induced cognitive impairment.

Asunto(s)

Disfunción Cognitiva , Hipertensión , Ratones , Animales , Losartán/farmacología , Cloruro de Sodio , Dinoprostona/metabolismo , Angiotensina II/farmacología , Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Inhibidores de la Enzima Convertidora de Angiotensina , Hipertensión/metabolismo , Cloruro de Sodio Dietético , Receptor de Angiotensina Tipo 1/metabolismo

RESUMEN

The group A rotavirus (RVA) genome comprising 11 double-stranded RNAs encodes six structural proteins (VP1-VP4, VP6, and VP7) and six non-structural proteins (NSP1-NSP6). Among these 12 rotaviral proteins, NSP6 has been less studied as to its function. We previously prepared a recombinant NSP6-deficient RVA derived from simian strain SA11-L2 by reverse genetics, and found that the NSP6-deficient virus grew well in cell culture, although its growth was less abundant than that of the parental SA11-L2 strain. In this study, we examined the potency of a recombinant RVA incapable of NSP6 expression to cause diarrhoea in suckling mice. The suckling mice infected with the NSP6-deficient virus apparently experienced diarrhoea, although the symptom was milder and the duration of diarrhoea was shorter than in the mice infected with the authentic SA11-L2 strain. Thus, together with the results obtained for cultured cells in the previous study, it can be concluded that NSP6 is not necessarily required for replication and pathogenicity in vitro and in vivo.

Asunto(s)

Infecciones por Rotavirus , Rotavirus , Animales , Línea Celular , Células Cultivadas , Diarrea , Ratones , Rotavirus/genética

RESUMEN

BACKGROUND: Cystogenesis in polycystic kidney disease (PKD) is likely accelerated by various renal insults, including crystal deposition, that activate renal tubule obstruction and dilation. We developed a capsule-based device that can be applied to cystic kidneys to restrict tubular lumen dilatation and cyst expansion. METHODS: Kidney capsule devices were designed from computed tomography images of wild-type and Cy/+ rats. Capsule devices were surgically implanted on kidneys in six surgical sessions over a period of 14 months in 7 wild-type rats of 6.5-8 weeks (3 sham operations, 2 right, 2 left) and 6 Cy/+ rats of 6.5 weeks (2 sham, 3 left, 1 bilateral). After surgery, the rats were followed for 5.4-12.4 weeks' growth and sacrificed to retrieve the kidneys. During the follow-up, serum creatinine was measured and retrieved kidneys were weighed. Histological analysis including cystic area measurement and immunohistochemistry was performed. RESULTS: Morphometric capsule devices were configured and developed by an image processing technique and produced using a 3D printer. Encapsulated Cy/+ kidneys (n = 5; mean weight 3.64 g) were consistently smaller in size (by 21-36%; p < 0.001) than unencapsulated Cy/+ kidneys (n = 7; mean weight 5.52 g). Encapsulated Cy/+ kidneys (mean %cyst area: 29.4%) showed smaller histological cystic area (by 28-58%; p < 0.001) than unencapsulated Cy/+ kidneys (mean %cyst area 48.6%). Cell proliferation and macrophages were also markedly reduced in encapsulated Cy/+ kidneys, compared to unencapsulated Cy/+ kidneys. CONCLUSIONS: We report a pilot feasibility study for the application of a novel morphometric 3D capsule device to the Cy/+ rat model showing restricted kidney volume expansion on polycystic kidney disease progression.

Asunto(s)

Quistes , Enfermedades Renales Poliquísticas , Animales , Proliferación Celular , Quistes/patología , Modelos Animales de Enfermedad , Estudios de Factibilidad , Humanos , Riñón/patología , Enfermedades Renales Poliquísticas/patología , Ratas

RESUMEN

Daily fat and sugar intake has increased in Japan, while total energy intake has decreased. However, the number of type 2 diabetes mellitus patients has increased, and this often causes renal injury characterized by autophagic vacuoles. Although many studies with comparisons of high fat or sugar versus a normal macronutrient balanced diet have been reported, there are few studies that equalized calorie intake and body weights. In the current study, AIN93M diets (CONT group) with matching energy content with lard derived high saturated fat (LARD group), soybean oil derived unsaturated fat (SOY OIL group) and sucrose (SUCROSE group) were provided to compare their effects on renal morphology in streptozotocin-injected CD-1 mice without causing obesity. The number of renal tubular vacuoles was higher in SUCROSE and slightly higher in LARD compared with CONT mice, and was higher in LARD and SUCROSE compared with SOY OIL mice. Most of those vacuoles were LAMP1-positive, a marker of lysosomal autophagy. These results suggest that despite identical energy contents, diets with high sucrose or saturated fat compared to unsaturated fat may aggravate lysosomal renal injury in a non-obese, streptozotocin-induced model of diabetes mellitus.

Asunto(s)

Diabetes Mellitus Tipo 2 , Sacarosa , Animales , Dieta , Grasas de la Dieta , Humanos , Riñón , Lisosomas , Ratones , Estreptozocina , Sacarosa/efectos adversos

RESUMEN

The anti-diuretic hormone arginine vasopressin is thought to be a detrimental factor in polycystic kidney disease (PKD). We previously reported that high water intake (HWI) reduced urine osmolality and urinary arginine vasopressin, improved renal function, and reduced the kidney/body weight ratio in PCK rats, an orthologous model of human PKD. In PKD patients, however, it is reported that HWI increases total kidney volume, urine volume, and urine sodium excretion, which could be a consequence of high salt intake. In the current study, we loaded PCK rats with high salt concurrently with HWI to determine whether this human-imitated condition exacerbates disease progression. PCK rats were assigned into 4 groups: control group (CONT: distilled water), HWI group (HWI: 5% glucose in water), HWI with 0.2% NaCl group (HWI+0.2%NaCl), and HWI with 0.45% NaCl group (HWI+0.45%NaCl). Total water intake during the experimental period was increased by 1.86-, 2.02-, and 2.42-fold in HWI, HWI+0.2%NaCl, and HWI+0.45%NaCl, and sodium intake was increased by 2.55- and 5.83-fold in HWI+0.2%NaCl and HWI+0.45%NaCl, respectively, compared with CONT. Systolic blood pressure was higher in HWI+0.2%NaCl and HWI+0.45%NaCl than in both CONT and HWI. Serum urea nitrogen, kidney/body weight ratio, cAMP, cystic area, and fibrosis index were significantly lower in HWI compared with CONT, and these ameliorative effects were not abrogated in either HWI+0.2%NaCl or HWI+0.45%NaCl. The amount of sodium excreted into the urine was increased by 2.50- and 8.38-fold in HWI+0.2%NaCl and HWI+0.45%NaCl, respectively, compared with HWI. Serum sodium levels were not different between the groups. These findings indicate that the beneficial effect of HWI against the progression of cystic kidney disease was not affected even by high salt-overload in this rodent model of PKD.

Asunto(s)

Enfermedades Renales Poliquísticas/dietoterapia , Cloruro de Sodio Dietético/administración & dosificación , Agua/administración & dosificación , Animales , Presión Sanguínea , Nitrógeno de la Urea Sanguínea , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Masculino , Enfermedades Renales Poliquísticas/sangre , Enfermedades Renales Poliquísticas/orina , Ratas , Ratas Sprague-Dawley , Sodio/orina , Cloruro de Sodio Dietético/efectos adversos

RESUMEN

The generation of recombinant group A rotaviruses (RVAs) entirely from cloned cDNAs has been described only for a single animal RVA strain, simian SA11-L2. We recently developed an optimized RVA reverse genetics system based on only RVA cDNAs (11-plasmid system), in which the concentration of cDNA plasmids containing the NSP2 and NSP5 genes is 3- or 5-fold increased in relation to that of the other plasmids. Based on this approach, we generated a recombinant human RVA (HuRVA)-based monoreassortant virus containing the VP4 gene of the simian SA11-L2 virus using the 11-plasmid system. In addition to this monoreassortant virus, authentic HuRVA (strain KU) was also generated with the 11-plasmid system with some modifications. Our results demonstrate that the 11-plasmid system involving just RVA cDNAs can be used for the generation of recombinant HuRVA and recombinant HuRVA-based reassortant viruses.IMPORTANCE Human group A rotavirus (HuRVA) is a leading pathogen causing severe diarrhea in young children worldwide. In this paper, we describe the generation of recombinant HuRVA (strain KU) from only 11 cloned cDNAs encoding the HuRVA genome by reverse genetics. The growth properties of the recombinant HuRVA were similar to those of the parental RVA, providing a powerful tool for better understanding of HuRVA replication and pathogenesis. Furthermore, the ability to manipulate the genome of HuRVAs "to order" will be useful for next-generation vaccine production for this medically important virus and for the engineering of clinical vectors expressing any foreign genes.

Asunto(s)

Clonación Molecular , ADN Complementario/genética , Genoma Viral , Plásmidos/genética , Rotavirus , Proteínas no Estructurales Virales , Animales , Línea Celular , Cricetinae , Humanos , Rotavirus/genética , Rotavirus/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo

RESUMEN

Autosomal dominant polycystic kidney disease (ADPKD) constitutes the most inherited kidney disease. Mutations in the PKD1 and PKD2 genes, encoding the polycystin 1 and polycystin 2 Ca2+ ion channels, respectively, result in tubular epithelial cell-derived renal cysts. Recent clinical studies demonstrate oxidative stress to be present early in ADPKD. Mitochondria comprise the primary reactive oxygen species source and also their main effector target; however, the pathophysiological role of mitochondria in ADPKD remains uncharacterized. To clarify this function, we examined the mitochondria of cyst-lining cells in ADPKD model mice (Ksp-Cre PKD1flox/flox) and rats (Han:SPRD Cy/+), demonstrating obvious tubular cell morphological abnormalities. Notably, the mitochondrial DNA copy number and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) expression were decreased in ADPKD model animal kidneys, with PGC-1α expression inversely correlated with oxidative stress levels. Consistent with these findings, human ADPKD cyst-derived cells with heterozygous and homozygous PKD1 mutation exhibited morphological and functional abnormalities, including increased mitochondrial superoxide. Furthermore, PGC-1α expression was suppressed by decreased intracellular Ca2+ levels via calcineurin, p38 mitogen-activated protein kinase (MAPK), and nitric oxide synthase deactivation. Moreover, the mitochondrion-specific antioxidant MitoQuinone (MitoQ) reduced intracellular superoxide and inhibited cyst epithelial cell proliferation through extracellular signal-related kinase/MAPK inactivation. Collectively, these results indicate that mitochondrial abnormalities facilitate cyst formation in ADPKD.

Asunto(s)

Quistes/patología , Mitocondrias/patología , Riñón Poliquístico Autosómico Dominante/patología , Animales , Calcineurina/metabolismo , Calcio/metabolismo , Proliferación Celular/fisiología , Células Cultivadas , Quistes/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular , Humanos , Riñón/metabolismo , Riñón/patología , Ratones , Mitocondrias/metabolismo , Mutación/genética , Óxido Nítrico Sintasa/metabolismo , Riñón Poliquístico Autosómico Dominante/metabolismo , Ratas , Transducción de Señal/fisiología , Superóxidos/metabolismo , Canales Catiónicos TRPP/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth.IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.

Asunto(s)

Regulación Viral de la Expresión Génica , Genética Inversa , Infecciones por Rotavirus/virología , Rotavirus/fisiología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiología , Animales , Células Cultivadas , Cricetinae , Humanos , Sistemas de Lectura Abierta , Proteínas no Estructurales Virales/genética

RESUMEN

Cystic kidney diseases are characterized by multiple renal cysts and are the leading cause of inherited renal disease. Oxylipins are bioactive lipids derived from fatty acids formed via cyclooxygenase, lipoxygenase and cytochrome P450 activity, and are important regulators of renal health and disease. Oxylipins are altered in nephronophthisis, a type of cystic kidney disease. To further investigate and to determine whether other cystic renal diseases share these abnormalities, a targeted lipidomic analysis of renal oxylipins was performed in orthologous models of autosomal dominant polycystic kidney disease 1 (Mx1Cre+Pkd1flox/flox mouse) and 2 (Pkd2ws25/- mouse), autosomal recessive polycystic kidney disease (PCK rat) and nephronophthisis (jck/jck mouse). Kidney cyclooxygenase oxylipins were consistently higher in all diseased kidneys, even in very early stage disease. On the other hand, cytochrome P450 epoxygenase derived oxylipins were lower only in the autosomal recessive polycystic kidney disease and nephronophthisis models, while lipoxygenase and cytochrome P450 hydroxylase derived oxylipins were lower only in nephronophthisis. Sex effects on renal oxylipin alterations were observed but they did not always coincide with sex effects on disease. For oxylipins with sex effects, arachidonic acid derived oxylipins formed via cyclooxygenases and lipoxygenases were higher in females, while oxylipins from other fatty acids and via cytochrome P450 enzymes were higher in males. The consistent and unique patterns of oxylipin alterations in the different models indicates the importance of these bioactive lipids in cystic renal diseases, suggesting that pharmacological agents (e.g. cyclooxygenase inhibitors) may be useful in treating these disorders, for which effective treatment remains elusive.

Asunto(s)

Enfermedades Renales Quísticas/metabolismo , Oxilipinas/metabolismo , Caracteres Sexuales , Animales , Sistema Enzimático del Citocromo P-450 , Modelos Animales de Enfermedad , Femenino , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/patología , Masculino , Ratones , Ratones Noqueados , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo

RESUMEN

Increased intracellular cyclic AMP (cAMP) in renal tubular epithelia accelerates the progression of polycystic kidney disease (PKD). Thus, decreasing cAMP levels by an adenylyl cyclase inhibitory G protein activator is considered to be an effective approach in ameliorating PKD. In fact, pasireotide (PAS) was effective in reducing disease progression in animal models of PKD. However, hyperglycemia caused by the administration of PAS is an adverse effect in its clinical use. Whereas, co-administration of octreotide (OCT) with PAS did not increase serum glucose in normal rats. In the current study, we examined the efficacy of combined treatment with OCT and PAS in PCK rats, an autosomal recessive PKD model. Four-week-old PCK males were treated with the long-acting release type of OCT, PAS, or a combination of both (OCT/PAS) for 12 weeks. After termination, serum and renal tissue were used for analyses. Kidney weight, kidney weight per body weight, renal cyst area, renal Ki67 expression, and serum urea nitrogen were significantly decreased either in the PAS or OCT/PAS group, compared with vehicle. Renal tissue cAMP content was significantly decreased by PAS or OCT/PAS treatment, but not OCT, compared with vehicle. As a marker of cellular mTOR signaling activity, renal phospho-S6 kinase expression was significantly decreased by OCT/PAS treatment compared with vehicle, OCT, or PAS. Serum glucose was significantly increased by PAS administration, whereas no difference was shown between vehicle and OCT/PAS, possibly because serum glucagon was decreased either by the treatment of OCT alone or co-application of OCT/PAS. In conclusion, since serum glucose levels are increased by the use of PAS, its combination with OCT may reduce the risk of hyperglycemia associated with PAS monotherapy against PKD progression.

Asunto(s)

Octreótido/farmacología , Riñón Poliquístico Autosómico Recesivo/tratamiento farmacológico , Somatostatina/análogos & derivados , Animales , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucagón/sangre , Corazón/efectos de los fármacos , Corazón/fisiología , Humanos , Hidrocortisona/sangre , Hiperglucemia/inducido químicamente , Insulina/sangre , Factor I del Crecimiento Similar a la Insulina/metabolismo , Antígeno Ki-67/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Riñón/fisiopatología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Octreótido/efectos adversos , Octreótido/uso terapéutico , Fosfoproteínas/metabolismo , Riñón Poliquístico Autosómico Recesivo/metabolismo , Riñón Poliquístico Autosómico Recesivo/patología , Riñón Poliquístico Autosómico Recesivo/fisiopatología , Ratas , Receptor IGF Tipo 1/sangre , Proteínas Quinasas S6 Ribosómicas/metabolismo , Somatostatina/efectos adversos , Somatostatina/farmacología , Somatostatina/uso terapéutico

RESUMEN

Human autosomal recessive polycystic kidney disease (ARPKD) produces kidneys which are massively enlarged due to multiple cysts, hypertension, and congenital hepatic fibrosis characterized by dilated bile ducts and portal hypertension. The PCK rat is an orthologous model of human ARPKD with numerous fluid-filled cysts caused by stimulated cellular proliferation in the renal tubules and hepatic bile duct epithelia, with interstitial fibrosis developed in the liver. We previously reported that a peroxisome proliferator activated receptor (PPAR)-γ full agonist ameliorated kidney and liver disease in PCK rats. Telmisartan is an angiotensin receptor blocker (ARB) used widely as an antihypertensive drug and shows partial PPAR-γ agonist activity. It also has nephroprotective activity in diabetes and renal injury and prevents the effects of drug-induced hepatotoxicity and hepatic fibrosis. In the present study, we determined whether telmisartan ameliorates progression of polycystic kidney and fibrocystic liver disease in PCK rats. Five male and 5 female PCK and normal control (+/+) rats were orally administered 3 mg/kg telmisartan or vehicle every day from 4 to 20 weeks of age. Treatment with telmisartan decreased blood pressure in both PCK and +/+ rats. Blood levels of aspartate amino transferase, alanine amino transferase and urea nitrogen were unaffected by telmisartan treatment. There was no effect on kidney disease progression, but liver weight relative to body weight, liver cystic area, hepatic fibrosis index, expression levels of Ki67 and TGF-ß, and the number of Ki67- and TGF-ß-positive interstitial cells in the liver were significantly decreased in telmisartan-treated PCK rats. Therefore, telmisartan ameliorates congenital hepatic fibrosis in ARPKD, possibly through the inhibition of signaling cascades responsible for cellular proliferation and interstitial fibrosis in PCK rats. The present results support the potential therapeutic use of ARBs for the treatment of fibrocystic liver disease in ARPKD patients.

Asunto(s)

Bencimidazoles/uso terapéutico , Benzoatos/uso terapéutico , Quistes/tratamiento farmacológico , Hepatopatías/tratamiento farmacológico , Riñón Poliquístico Autosómico Recesivo/tratamiento farmacológico , Angiotensina II/metabolismo , Animales , Bencimidazoles/farmacología , Benzoatos/farmacología , Presión Sanguínea/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quistes/patología , Quistes/fisiopatología , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Pruebas de Función Renal , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Hepatopatías/patología , Hepatopatías/fisiopatología , Pruebas de Función Hepática , Masculino , Tamaño de los Órganos/efectos de los fármacos , Riñón Poliquístico Autosómico Recesivo/patología , Riñón Poliquístico Autosómico Recesivo/fisiopatología , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1/metabolismo , Transducción de Señal/efectos de los fármacos , Telmisartán , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Polycystic kidney disease (PKD) is a hereditary disorder with abnormal cellular proliferation, fluid accumulation in numerous cysts, remodeling of extracellular matrix, inflammation, and fibrosis in the kidney and liver. The two major types of PKD show autosomal dominant (ADPKD) or autosomal recessive inheritance (ARPKD). ADPKD is one of the most common genetic diseases, with an incidence of 1:500-1,000. Approximately 50% of patients with ADPKD develop end-stage renal disease (ESRD) by the age of 60. On the other hand, ARPKD is relatively rare, with an incidence of approximately 1:20,000-40,000. ARPKD is diagnosed early in life, often prenatally. The gene products responsible for ADPKD and ARPKD distribute in primary cilia and are thought to control intercellular Ca(2+). Two types of animal model of PKD have been established: spontaneous hereditary models identified by the typical manifestations of PKD and gene-engineered models established by modification of human orthologous genes. Both types of animal models are used to study the mechanism of cystogenesis and efficacy of medical treatments. In PKD progression, critical roles of signaling pathways including MAPK, mTOR, and PPAR-γ have been discovered with these models. Therefore, experimental animal models are indispensable for investigating molecular mechanisms of PKD onset and progression as well as potential therapeutic treatments.

Asunto(s)

Modelos Animales de Enfermedad , Ratones , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/terapia , Ratas , Animales , Progresión de la Enfermedad , Marcación de Gen , Humanos , Enfermedades Renales Poliquísticas/fisiopatología , Transducción de Señal

RESUMEN

Kidneys are enlarged by aberrant proliferation of tubule epithelial cells leading to the formation of numerous cysts, nephron loss, and interstitial fibrosis in polycystic kidney disease (PKD). Pioglitazone (PIO), a PPAR-γ agonist, decreased cell proliferation, interstitial fibrosis, and inflammation, and ameliorated PKD progression in PCK rats (Am. J. Physiol.-Renal, 2011). To explore genetic mechanisms involved, changes in global gene expression were analyzed. By Gene Set Enrichment Analysis of 30655 genes, 13 of the top 20 downregulated gene ontology biological process gene sets and six of the top 20 curated gene set canonical pathways identified to be downregulated by PIOtreatment were related to cell cycle and proliferation, including EGF, PDGF and JNK pathways. Their relevant pathways were identified using the Kyoto Encyclopedia of Gene and Genomes database. Stearoyl-coenzyme A desaturase 1 is a key enzyme in fatty acid metabolism found in the top 5 genes downregulated by PIO treatment. Immunohistochemical analysis revealed that the gene product of this enzyme was highly expressed in PCK kidneys and decreased by PIO. These data show that PIO alters the expression of genes involved in cell cycle progression, cell proliferation, and fatty acid metabolism.

RESUMEN

In autosomal recessive polycystic kidney disease (ARPKD), progressive enlargement of fluid-filled cysts is due to aberrant proliferation of tubule epithelial cells and transepithelial fluid secretion leading to extensive nephron loss and interstitial fibrosis. Congenital hepatic fibrosis associated with biliary cysts/dilatations is the most common extrarenal manifestation in ARPKD and can lead to massive liver enlargement. Peroxisome proliferator-activated receptor γ (PPAR-γ), a member of the ligand-dependent nuclear receptor superfamily, is expressed in a variety of tissues, including the kidneys and liver, and plays important roles in cell proliferation, fibrosis, and inflammation. In the current study, we determined that pioglitazone (PIO), a PPAR-γ agonist, decreases polycystic kidney and liver disease progression in the polycystic kidney rat, an orthologous model of human ARPKD. Daily treatment with 10 mg/kg PIO for 16 wk decreased kidney weight (% of body weight), renal cystic area, serum urea nitrogen, and the number of Ki67-, pERK1/2-, and pS6-positive cells in the kidney. There was also a decrease in liver weight (% of body weight), liver cystic area, fibrotic index, and the number of Ki67-, pERK1/2-, pERK5-, and TGF-ß-positive cells in the liver. Taken together, these data suggest that PIO inhibits the progression of polycystic kidney and liver disease in a model of human ARPKD by inhibiting cell proliferation and fibrosis. These findings suggest that PPAR-γ agonists may have therapeutic value in the treatment of the renal and hepatic manifestations of ARPKD.

Asunto(s)

Hepatopatías/tratamiento farmacológico , PPAR gamma/agonistas , Riñón Poliquístico Autosómico Recesivo/tratamiento farmacológico , Tiazolidinedionas/uso terapéutico , Animales , Nitrógeno de la Urea Sanguínea , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Antígeno Ki-67/análisis , Cirrosis Hepática/tratamiento farmacológico , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/análisis , Pioglitazona , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/análisis

RESUMEN

Han:SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated "Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation," "LPS/IL-1-Mediated Inhibition of RXR Function," and "Liver X Receptor (LXR)/RXR Activation." These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.

Asunto(s)

Enfermedades Renales Poliquísticas/genética , Receptores X Retinoide/fisiología , Transducción de Señal/genética , Animales , Animales Recién Nacidos , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/fisiología , Proliferación Celular , Claudinas , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Masculino , Análisis por Micromatrices , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/genética , Ratas , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Regulación hacia Arriba , Proteína del Homeodomínio PITX2

RESUMEN

Polycystic kidney disease (PKD) in Han:SPRD Cy rats is caused by a missense mutation in Anks6 (also called Pkdr1), leading to an R823W substitution in SamCystin, a protein that contains ankyrin repeats and a sterile alpha motif (SAM). The cellular function of SamCystin and the role of the Cy (R823W) mutation in cyst formation are unknown. In normal SPRD rats, SamCystin was found to be expressed in proximal tubules and glomeruli; protein expression was highest at 7 days of age and declined by ∼50-60% at 45-84 days of age. In Cy/+ and Cy/Cy kidneys, expression of SamCystin was lower than in +/+ kidneys at 3 and 7 days but became elevated at 21 days. Immunohistochemical analysis revealed that SamCystin was distributed on the brush border of proximal tubules in normal rat kidneys. In Cy/+ kidneys, there were robust SamCystin staining in cyst-lining epithelial cells and loss of apical localization, and increased number of PCNA-positive cells in cyst-lining epithelia. Verapamil, an L-type Ca(2+) channel blocker, accelerated PKD progression in this model and caused a further increase in the expression and abnormal distribution of SamCystin. We conclude that aberrant expression and mislocalization of R823W SamCystin lead to increased cell proliferation and renal cyst formation.

Asunto(s)

Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Enfermedades Renales Poliquísticas/genética , Animales , Western Blotting , Bloqueadores de los Canales de Calcio/farmacología , Proliferación Celular , Análisis Mutacional de ADN , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente , Genotipo , Inmunohistoquímica , Riñón/metabolismo , Riñón/patología , Mutación Missense/genética , Mutación Missense/fisiología , Enfermedades Renales Poliquísticas/metabolismo , Enfermedades Renales Poliquísticas/patología , Ratas , Ratas Sprague-Dawley , Verapamilo/farmacología

RESUMEN

Rotavirus is the leading pathogen for acute gastroenteritis in mammals and birds. Although the reverse genetics system has been utilized in many viruses, the system using a helper virus was developed for rotavirus in 2006. As a step for antigenic analysis of VP4 antigen of rotavirus, we prepared an infectious rotavirus with a spike protein VP4 having an antigenic mosaic by substituting one of the cross-reactive neutralization epitopes of a simian strain SA-11 with the corresponding one of a human strain DS-1. The future improvement and application of the rotavirus reverse genetics were discussed in this review.

Asunto(s)

Técnicas Genéticas/tendencias , Rotavirus/genética , Enfermedad Aguda , Animales , Proteínas de la Cápside , Epítopos , Predicción , Gastroenteritis/virología , Humanos , Infecciones por Rotavirus