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1.
Am J Physiol Renal Physiol ; 317(6): F1450-F1461, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31566426

RESUMO

Diabetic kidney disease (DKD) is one of the leading pathological causes of decreased renal function and progression to end-stage kidney failure. To explore and characterize age-related changes in DKD and associated glomerular damage, we used a rat model of type 2 diabetic nephropathy (T2DN) at 12 wk and older than 48 wk. We compared their disease progression with control nondiabetic Wistar and diabetic Goto-Kakizaki (GK) rats. During the early stages of DKD, T2DN and GK animals revealed significant increases in blood glucose and kidney-to-body weight ratio. Both diabetic groups had significantly altered renin-angiotensin-aldosterone system function. Thereafter, during the later stages of disease progression, T2DN rats demonstrated a remarkable increase in renal damage compared with GK and Wistar rats, as indicated by renal hypertrophy, polyuria accompanied by a decrease in urine osmolarity, high cholesterol, a significant prevalence of medullary protein casts, and severe forms of glomerular injury. Urinary nephrin shedding indicated loss of the glomerular slit diaphragm, which also correlates with the dramatic elevation in albuminuria and loss of podocin staining in aged T2DN rats. Furthermore, we used scanning ion microscopy topographical analyses to detect and quantify the pathological remodeling in podocyte foot projections of isolated glomeruli from T2DN animals. In summary, T2DN rats developed renal and physiological abnormalities similar to clinical observations in human patients with DKD, including progressive glomerular damage and a significant decrease in renin-angiotensin-aldosterone system plasma levels, indicating these rats are an excellent model for studying the progression of renal damage in type 2 DKD.


Assuntos
Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/patologia , Envelhecimento , Albuminúria/etiologia , Albuminúria/prevenção & controle , Animais , Glicemia/metabolismo , Progressão da Doença , Hipertrofia , Glomérulos Renais/patologia , Masculino , Proteínas de Membrana/urina , Tamanho do Órgão , Poliúria/etiologia , Poliúria/patologia , Ratos , Ratos Wistar , Sistema Renina-Angiotensina , Desequilíbrio Hidroeletrolítico/etiologia , Desequilíbrio Hidroeletrolítico/metabolismo
2.
J Am Soc Nephrol ; 29(5): 1525-1535, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29476007

RESUMO

Background Interpreting genetic variants is one of the greatest challenges impeding analysis of rapidly increasing volumes of genomic data from patients. For example, SHROOM3 is an associated risk gene for CKD, yet causative mechanism(s) of SHROOM3 allele(s) are unknown.Methods We used our analytic pipeline that integrates genetic, computational, biochemical, CRISPR/Cas9 editing, molecular, and physiologic data to characterize coding and noncoding variants to study the human SHROOM3 risk locus for CKD.Results We identified a novel SHROOM3 transcriptional start site, which results in a shorter isoform lacking the PDZ domain and is regulated by a common noncoding sequence variant associated with CKD (rs17319721, allele frequency: 0.35). This variant disrupted allele binding to the transcription factor TCF7L2 in podocyte cell nuclear extracts and altered transcription levels of SHROOM3 in cultured cells, potentially through the loss of repressive looping between rs17319721 and the novel start site. Although common variant mechanisms are of high utility, sequencing is beginning to identify rare variants involved in disease; therefore, we used our biophysical tools to analyze an average of 112,849 individual human genome sequences for rare SHROOM3 missense variants, revealing 35 high-effect variants. The high-effect alleles include a coding variant (P1244L) previously associated with CKD (P=0.01, odds ratio=7.95; 95% CI, 1.53 to 41.46) that we find to be present in East Asian individuals at an allele frequency of 0.0027. We determined that P1244L attenuates the interaction of SHROOM3 with 14-3-3, suggesting alterations to the Hippo pathway, a known mediator of CKD.Conclusions These data demonstrate multiple new SHROOM3-dependent genetic/molecular mechanisms that likely affect CKD.


Assuntos
Proteínas dos Microfilamentos/genética , Insuficiência Renal Crônica/genética , Alelos , Animais , Núcleo Celular , Frequência do Gene , Loci Gênicos , Células HEK293 , Humanos , Camundongos , Mutação de Sentido Incorreto , Podócitos , Isoformas de Proteínas/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Transcrição Gênica , Peixe-Zebra
3.
Physiol Genomics ; 50(8): 563-579, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29727589

RESUMO

Genomic sequencing has undergone massive expansion in the past 10 yr, from a rarely used research tool into an approach that has broad applications in a clinical setting. From rare disease to cancer, genomics is transforming our knowledge of biology. The transition from targeted gene sequencing, to whole exome sequencing, to whole genome sequencing has only been made possible due to rapid advancements in technologies and informatics that have plummeted the cost per base of DNA sequencing and analysis. The tools of genomics have resolved the etiology of disease for previously undiagnosable conditions, identified cancer driver gene variants, and have impacted the understanding of pathophysiology for many diseases. However, this expansion of use has also highlighted research's current voids in knowledge. The lack of precise animal models for gene-to-function association, lack of tools for analysis of genomic structural changes, skew in populations used for genetic studies, publication biases, and the "Unknown Proteome" all contribute to voids needing filled for genomics to work in a fast-paced clinical setting. The future will hold the tools to fill in these voids, with new data sets and the continual development of new technologies allowing for expansion of genomic medicine, ushering in the days to come for precision medicine. In this review we highlight these and other points in hopes of advancing and guiding precision medicine into the future for optimal success.


Assuntos
Doença/genética , Sequenciamento do Exoma/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Análise de Sequência de DNA/métodos , Animais , Biologia Computacional/métodos , Biologia Computacional/tendências , Previsões , Genômica/tendências , Sequenciamento de Nucleotídeos em Larga Escala/tendências , Humanos , Medicina de Precisão/métodos , Medicina de Precisão/tendências , Análise de Sequência de DNA/tendências , Sequenciamento do Exoma/tendências
4.
Genome Res ; 25(1): 57-65, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25273069

RESUMO

Genome-wide association studies (GWAS) identify regions of the genome correlated with disease risk but are restricted in their ability to identify the underlying causative mechanism(s). Thus, GWAS are useful "roadmaps" that require functional analysis to establish the genetic and mechanistic structure of a particular locus. Unfortunately, direct functional testing in humans is limited, demonstrating the need for complementary approaches. Here we used an integrated approach combining zebrafish, rat, and human data to interrogate the function of an established GWAS locus (SHROOM3) lacking prior functional support for chronic kidney disease (CKD). Congenic mapping and sequence analysis in rats suggested Shroom3 was a strong positional candidate gene. Transferring a 6.1-Mb region containing the wild-type Shroom3 gene significantly improved the kidney glomerular function in FHH (fawn-hooded hypertensive) rat. The wild-type Shroom3 allele, but not the FHH Shroom3 allele, rescued glomerular defects induced by knockdown of endogenous shroom3 in zebrafish, suggesting that the FHH Shroom3 allele is defective and likely contributes to renal injury in the FHH rat. We also show for the first time that variants disrupting the actin-binding domain of SHROOM3 may cause podocyte effacement and impairment of the glomerular filtration barrier.


Assuntos
Barreira de Filtração Glomerular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Animais Congênicos , Animais Geneticamente Modificados , Clonagem Molecular , Éxons , Feminino , Loci Gênicos , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Nefropatias/genética , Masculino , Proteínas dos Microfilamentos/genética , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Plasmídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Análise de Sequência de DNA , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
5.
Physiol Genomics ; 49(9): 496-504, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28754823

RESUMO

The activity of fumarase, an enzyme in the tricarboxylic acid cycle, is lower in Dahl salt-sensitive SS rats compared with SS.13BN rats. SS.13BN rats have a Brown Norway (BN) allele of fumarase and exhibit attenuated hypertension. The SS allele of fumarase differs from the BN allele by a K481E sequence variation. It remains unknown whether higher fumarase activities can attenuate hypertension and whether the mechanism is relevant without the K481E variation. We developed SS-TgFh1 transgenic rats overexpressing fumarase on the background of the SS rat. Hypertension was attenuated in SS-TgFh1 rats. Mean arterial pressure in SS-TgFh1 rats was 20 mmHg lower than transgene-negative SS littermates after 12 days on a 4% NaCl diet. Fumarase overexpression decreased H2O2, while fumarase knockdown increased H2O2 Ectopically expressed BN form of fumarase had higher specific activity than the SS form. However, sequencing of more than a dozen rat strains indicated most rat strains including salt-insensitive Sprague-Dawley (SD) rats had the SS allele of fumarase. Despite that, total fumarase enzyme activity in the renal medulla was still higher in SD rats than in SS rats, which was associated with higher expression of fumarase in SD. H2O2 can suppress the expression of fumarase. Renal medullary interstitial administration of fumarase siRNA in SD rats resulted in higher blood pressure on the high-salt diet. These findings indicate elevation of total fumarase activity attenuates the development of hypertension and can result from a nonsynonymous sequence variation in some rat strains and higher expression in other rat strains.


Assuntos
Fumarato Hidratase/genética , Fumarato Hidratase/metabolismo , Variação Genética , Hipertensão/enzimologia , Hipertensão/genética , Animais , Sequência de Bases , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Dahl , Ratos Sprague-Dawley , Ratos Transgênicos , Análise de Sequência de DNA , Regulação para Cima/genética
6.
Physiol Genomics ; 48(1): 62-72, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26534937

RESUMO

A 1.37 Mbp region of chromosome 13 previously identified by exclusion mapping was consistently associated with a reduction of salt-induced hypertension in the Dahl salt-sensitive (SS) rat. This region contained five genes that were introgressed from the salt-insensitive Brown Norway (BN) rat. The goal of the present study was to further narrow that region to identify the gene(s) most likely to protect from salt-induced hypertension. The studies yielded a subcongenic SS rat strain containing a 0.71 Mbp insert from BN (26-P strain) in which salt-induced hypertension was reduced by 24 mmHg. The region contained two protein-coding genes (Astn1 and Pappa2) and a microRNA (miR-488). Pappa2 mRNA in the renal cortex of the protected 26-P was 6- to 10-fold greater than in SS fed a 0.4% NaCl diet but was reduced to levels observed in SS when fed 8.0% NaCl diet for 7 days. Compared with brain nuclei (NTS, RVLM, CVLM) and the adrenal gland, Pappa2 in the renal cortex was the only gene found to be differentially expressed between SS and 26-P and that responded to changes of salt diet. Immunohistochemistry studies found Pappa2 localized in the cytosol of the epithelial cells of the cortical thick ascending limbs. In more distal segments of the renal tubules, it was observed within tubular lumens and most notably bound to the apical membranes of the intercalated cells of collecting ducts. We conclude that we have identified a variant form of Pappa2 that can protect against salt-induced hypertension in the Dahl S rat.


Assuntos
Hipertensão/metabolismo , Proteína Plasmática A Associada à Gravidez/metabolismo , Cloreto de Sódio na Dieta/efeitos adversos , Glândulas Suprarrenais/metabolismo , Albuminúria/complicações , Albuminúria/genética , Albuminúria/fisiopatologia , Animais , Pareamento de Bases/genética , Pressão Sanguínea , Tronco Encefálico/metabolismo , Núcleo Celular/metabolismo , Cromossomos de Mamíferos/genética , Imunofluorescência , Regulação da Expressão Gênica , Genoma , Hipertensão/complicações , Hipertensão/genética , Hipertensão/fisiopatologia , Rim/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Endogâmicos Dahl , Análise de Sequência de DNA , Simportadores de Cloreto de Sódio/metabolismo
7.
Genome Res ; 23(12): 1996-2002, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24006081

RESUMO

Genome-wide association studies (GWAS) are useful for nominating candidate genes, but typically are unable to establish disease causality or differentiate between the effects of variants in linkage disequilibrium (LD). Additionally, some GWAS loci might contain multiple causative variants or genes that contribute to the overall disease susceptibility at a single locus. However, the majority of current GWAS lack the statistical power to test whether multiple causative genes underlie the same locus, prompting us to adopt an alternative approach to testing multiple GWAS genes empirically. We used gene targeting in a disease-susceptible rat model of genetic hypertension to test all six genes at the Agtrap-Plod1 locus (Agtrap, Mthfr, Clcn6, Nppa, Nppb, and Plod1) for blood pressure (BP) and renal phenotypes. This revealed that the majority of genes at this locus (five out of six) can impact hypertension by modifying BP and renal phenotypes. Mutations of Nppa, Plod1, and Mthfr increased disease susceptibility, whereas Agtrap and Clcn6 mutations decreased hypertension risk. Reanalysis of the human AGTRAP-PLOD1 locus also implied that disease-associated haplotype blocks with polygenic effects were not only possible, but rather were highly plausible. Combined, these data demonstrate for the first time that multiple modifiers of hypertension can cosegregate at a single GWAS locus.


Assuntos
Pressão Sanguínea/genética , Genes Modificadores , Hipertensão/etiologia , Hipertensão/genética , Rim/metabolismo , Locos de Características Quantitativas , Animais , Modelos Animais de Doenças , Feminino , Marcação de Genes , Predisposição Genética para Doença , Variação Genética , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Desequilíbrio de Ligação , Masculino , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , Ratos , Ratos Sprague-Dawley , Estudos Retrospectivos
8.
Physiol Genomics ; 47(5): 177-86, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25759379

RESUMO

The renin-angiotensin system (RAS) is subject to sex-specific modulation by hormones and gene products. However, sex differences in the balance between the vasoconstrictor/proliferative ACE/ANG II/AT1 axis, and the vasodilator/antiproliferative ACE2/ANG-(1-7)/MAS axis are poorly known. Data in the rat have suggested the male-specific Y-chromosome gene Sry to contribute to balance between these two axes, but why the testis-determining gene has these functions remains unknown. A combination of in silico genetic/protein comparisons, functional luciferase assays for promoters of the human RAS, and RNA-Seq profiling in rat were used to address if regulation of Sry on the RAS is conserved in the homologous X-chromosome gene, Sox3. Both SRY and SOX3 upregulated the promoter of Angiotensinogen (AGT) and downregulated the promoters of ACE2, AT2, and MAS, likely through overlapping mechanisms. The regulation by both SRY and SOX3 on the MAS promoter indicates a cis regulation through multiple SOX binding sites. The Renin (REN) promoter is upregulated by SRY and downregulated by SOX3, likely through trans and cis mechanisms, respectively. Sry transcripts are found in all analyzed male rat tissues including the kidney, while Sox3 transcripts are found only in the brain and testis, suggesting that the primary tissue for renin production (kidney) can only be regulated by SRY and not SOX3. These results suggest that SRY regulation of the RAS is partially shared with its X-chromosome homolog SOX3, but SRY gained a sex-specific control in the kidney for the rate-limiting step of the RAS, potentially resulting in male-specific blood pressure regulation.


Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Sistema Renina-Angiotensina/genética , Fatores de Transcrição SOXB1/genética , Proteína da Região Y Determinante do Sexo/genética , Cromossomo X/genética , Cromossomo Y/genética , Sequência de Aminoácidos , Angiotensinogênio/genética , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Sequência Conservada , Cricetinae , Cricetulus , Feminino , Perfilação da Expressão Gênica , Humanos , Luciferases/metabolismo , Masculino , Dados de Sequência Molecular , Peptidil Dipeptidase A/genética , Renina/genética , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/metabolismo , Homologia de Sequência do Ácido Nucleico , Proteína da Região Y Determinante do Sexo/química , Proteína da Região Y Determinante do Sexo/metabolismo
9.
Physiol Genomics ; 46(5): 169-76, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24380873

RESUMO

Cardioprotection may be genome dependent. One example is the increased tolerance to cardiac ischemia-reperfusion (IR) in Brown Norway (BN) compared with Dahl salt-sensitive (SS) rats. By narrowing the genetic difference to chromosome 6 only, we found the consomic SS(6BN) to be similarly IR tolerant as BN. We hypothesized that better preserved mitochondrial structure and function are genetically determined and therefore critically linked to myocardial IR tolerance associated with BN chromosome 6. Langendorff-prepared BN, SS, and SS(6BN) rat hearts were subjected to IR, while corresponding controls were continuously perfused. Though largely equal in nonischemic controls, assessment of functional data and ventricular infarct size in IR experiments confirmed that BN and SS(6BN) have an equally higher tolerance to IR than SS hearts. This was complemented by equally better preserved mitochondrial structure, oxidative phosphorylation, and calcium retention capacity in BN and SS(6BN) vs. SS hearts. For the first time, our data indicate that SS(6BN) are as resistant to IR injury as BN hearts in mitochondrial and myocardial function and viability compared with SS hearts. These findings not only link myocardial and mitochondrial protection in a genetic model but also suggest that genetic information on rat chromosome 6 is critical for mitochondrial preservation and IR tolerance.


Assuntos
Mitocôndrias Cardíacas/genética , Traumatismo por Reperfusão Miocárdica/genética , Animais , Cálcio/metabolismo , Modelos Animais de Doenças , Coração/fisiologia , Masculino , Mitocôndrias Cardíacas/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Fosforilação Oxidativa , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Dahl
10.
Physiol Genomics ; 46(11): 398-410, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24714719

RESUMO

The goal of the present study was to narrow a region of chromosome 13 to only several genes and then apply unbiased statistical approaches to identify molecular networks and biological pathways relevant to blood-pressure salt sensitivity in Dahl salt-sensitive (SS) rats. The analysis of 13 overlapping subcongenic strains identified a 1.37 Mbp region on chromosome 13 that influenced the mean arterial blood pressure by at least 25 mmHg in SS rats fed a high-salt diet. DNA sequencing and analysis filled genomic gaps and provided identification of five genes in this region, Rfwd2, Fam5b, Astn1, Pappa2, and Tnr. A cross-platform normalization of transcriptome data sets obtained from our previously published Affymetrix GeneChip dataset and newly acquired RNA-seq data from renal outer medullary tissue provided 90 observations for each gene. Two Bayesian methods were used to analyze the data: 1) a linear model analysis to assess 243 biological pathways for their likelihood to discriminate blood pressure levels across experimental groups and 2) a Bayesian graphical modeling of pathways to discover genes with potential relationships to the candidate genes in this region. As none of these five genes are known to be involved in hypertension, this unbiased approach has provided useful clues to be experimentally explored. Of these five genes, Rfwd2, the gene most strongly expressed in the renal outer medulla, was notably associated with pathways that can affect blood pressure via renal transcellular Na(+) and K(+) electrochemical gradients and tubular Na(+) transport, mitochondrial TCA cycle and cell energetics, and circadian rhythms.


Assuntos
Genoma/genética , Hipertensão/genética , Hipertensão/metabolismo , Transdução de Sinais/genética , Animais , Pressão Arterial/genética , Teorema de Bayes , Ritmo Circadiano/genética , Ciclo do Ácido Cítrico/genética , Perfilação da Expressão Gênica/métodos , Masculino , Mitocôndrias/genética , Potássio/metabolismo , Ratos , Ratos Endogâmicos Dahl , Análise de Sequência de DNA/métodos , Sódio/metabolismo , Cloreto de Sódio na Dieta/metabolismo
11.
Am J Physiol Heart Circ Physiol ; 307(8): H1103-10, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25320330

RESUMO

We previously isolated a 6.1-Mb region of SS/Mcwi (Dahl salt-sensitive) rat chromosome 12 (13.4-19.5 Mb) that significantly elevated blood pressure (BP) (Δ+34 mmHg, P < 0.001) compared with the SS-12(BN) consomic control. In the present study, we examined the role of vascular dysfunction and remodeling in hypertension risk associated with the 6.1-Mb (13.4-19.5 Mb) locus on rat chromosome 12 by reducing dietary salt, which lowered BP levels so that there were no substantial differences in BP between strains. Consequently, any observed differences in the vasculature were considered BP-independent. We also reduced the candidate region from 6.1 Mb with 133 genes to 2 Mb with 23 genes by congenic mapping. Both the 2 Mb and 6.1 Mb congenic intervals were associated with hypercontractility and decreased elasticity of resistance vasculature prior to elevations of BP, suggesting that the vascular remodeling and dysfunction likely contribute to the pathogenesis of hypertension in these congenic models. Of the 23 genes within the narrowed congenic interval, 12 were differentially expressed between the resistance vasculature of the 2 Mb congenic and SS-12(BN) consomic strains. Among these, Grifin was consistently upregulated 2.7 ± 0.6-fold (P < 0.05) and 2.0 ± 0.3-fold (P < 0.01), and Chst12 was consistently downregulated -2.8 ± 0.3-fold (P < 0.01) and -4.4 ± 0.4-fold (P < 0.00001) in the 2 Mb congenic compared with SS-12(BN) consomic under normotensive and hypertensive conditions, respectively. A syntenic region on human chromosome 7 has also been associated with BP regulation, suggesting that identification of the genetic mechanism(s) underlying cardiovascular phenotypes in this congenic strain will likely be translated to a better understanding of human hypertension.


Assuntos
Pressão Sanguínea/genética , Loci Gênicos , Hipertensão/genética , Artérias Mesentéricas/fisiopatologia , Resistência Vascular , Animais , Cromossomos/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Galectinas/genética , Galectinas/metabolismo , Hipertensão/etiologia , Hipertensão/fisiopatologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Ratos , Ratos Endogâmicos Dahl , Cloreto de Sódio na Dieta , Sulfotransferases/genética , Sulfotransferases/metabolismo
12.
FASEB J ; 27(3): 930-41, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23195032

RESUMO

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.


Assuntos
Elementos de DNA Transponíveis/genética , Inativação Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Mosaicismo , Transgenes , Animais , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/genética , Coelhos , Ratos , Ratos Sprague-Dawley
13.
Anesthesiology ; 120(4): 870-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24346177

RESUMO

BACKGROUND: Diabetes alters mitochondrial bioenergetics and consequently disrupts cardioprotective signaling. The authors investigated whether mitochondrial DNA (mtDNA) modulates anesthetic preconditioning (APC) and cardiac susceptibility to ischemia-reperfusion injury by using two strains of rats, both sharing nuclear genome of type 2 diabetes mellitus (T2DN) rats and having distinct mitochondrial genomes of Wistar and fawn-hooded hypertensive (FHH) rat strains (T2DN(mtWistar) and T2DN(mtFHH), respectively). METHODS: Myocardial infarct size was measured in Wistar, T2DN(mtWistar), and T2DN(mtFHH) rats with or without APC (1.4% isoflurane) in the presence or absence of antioxidant N-acetylcysteine. Flavoprotein fluorescence intensity, a marker of mitochondrial redox state, 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein fluorescence intensity, a marker of reactive oxygen species generation, and mitochondrial permeability transition pore opening were assessed in isolated rat ventricular cardiomyocytes with or without isoflurane (0.5 mmol/l). RESULTS: Myocardial infarct size was decreased by APC in Wistar and T2DN(mtWistar) rats (to 42 ± 6%, n = 8; and 44 ± 7%, n = 8; of risk area, respectively) compared with their respective controls (60 ± 3%, n = 6; and 59 ± 9%, n = 7), but not in T2DN(mtFHH) rats (60 ± 2%, n = 8). N-acetylcysteine applied during isoflurane treatment restored APC in T2DN(mtFHH) (39 ± 6%, n = 7; and 38 ± 5%, n = 7; 150 and 75 mg/kg N-acetylcysteine, respectively), but abolished protection in control rats (54 ± 8%, n = 6). Similar to the data on infarct size, APC delayed mitochondrial permeability transition pore opening in T2DN(mtWistar) but not in T2DN(mtFHH) cardiomyocytes. Isoflurane increased flavoprotein and 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein fluorescence intensity in all rat strains, with the greatest effect in T2DN(mtFHH) cardiomyocytes. CONCLUSION: Differences in the mitochondrial genome modulate isoflurane-induced generation of reactive oxygen species which translates into differential susceptibility to APC and ischemia-reperfusion injury in diabetic rats.


Assuntos
DNA Mitocondrial/metabolismo , Diabetes Mellitus Tipo 2/complicações , Precondicionamento Isquêmico Miocárdico/métodos , Mitocôndrias Cardíacas/metabolismo , Infarto do Miocárdio/complicações , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Acetilcisteína/metabolismo , Acetilcisteína/farmacologia , Anestésicos Inalatórios/metabolismo , Anestésicos Inalatórios/farmacologia , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Sequestradores de Radicais Livres/metabolismo , Sequestradores de Radicais Livres/farmacologia , Isoflurano/metabolismo , Isoflurano/farmacologia , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo
14.
J Am Soc Nephrol ; 24(2): 283-92, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23291471

RESUMO

We previously reported that the fawn-hooded hypertensive (FHH) rat is a natural Rab38 knockout, supported by a congenic animal (FHH.BN-Rab38) having less proteinuria than FHH animals. Because these congenic animals contain Brown Norway (BN) alleles for five other named genes; however, a causal role for Rab38 in the FHH phenotype remains uncertain. Here, we used transgenic and knockout models to validate Rab38 and to exclude other genes within the 1.5 Mb congenic region from involvement in causing the FHH phenotype. Transgenic rats homozygous for the wild-type Rab38 BN allele on the FHH background exhibited phenotypic rescue, having 43% lower proteinuria and 75% lower albuminuria than nontransgenic FHH littermates. Conversely, knockout of the Rab38 gene on the FHH.BN-Rab38 congenic line recapitulated a proteinuric phenotype indistinguishable from the FHH strain. In addition, in cultured proximal tubule LLC-PK1 cells, knockdown of Rab38 mRNA significantly decreased endocytosis of colloidal gold-coupled albumin, supporting the hypothesis that Rab38 modulates proteinuria through effects on tubular re-uptake and not by altering glomerular permeability. Taken together, these findings validate Rab38 as a gene having a causal role in determining the phenotype of the FHH rat, which models hypertension-associated renal disease. Furthermore, our data suggest that Rab38 affects urinary protein excretion via effects in the proximal tubule.


Assuntos
Hipertensão Renal/fisiopatologia , Proteinúria/fisiopatologia , Proteínas rab de Ligação ao GTP/genética , Animais , Modelos Animais de Doenças , Endocitose/fisiologia , Técnicas de Inativação de Genes , Cor de Cabelo/genética , Hipertensão Renal/genética , Hipertensão Renal/metabolismo , Rim/metabolismo , Rim/fisiopatologia , Rim/ultraestrutura , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/fisiologia , Células LLC-PK1 , Microscopia Imunoeletrônica , Proteinúria/genética , Proteinúria/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Endogâmicos BN , Ratos Transgênicos , Suínos , Proteínas rab de Ligação ao GTP/metabolismo
15.
Vet Res Commun ; 48(4): 2835-2840, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38874831

RESUMO

Filaroid nematodes Setaria tundra (Issaitshikoff & Rajewskaya, 1928) and Setaria cervi (Rudolphi, 1819) are internal parasites from family Onchocercidae with occurrence in the northern hemisphere. They have a considerably wide range of final host, including many species of family Cervidae. Intermediate hosts and vectors at the same time, are represented by the several mosquito species, mostly of genus Aedes. Infection of Setaria is relatively harmless and especially in wild cervids usually pass unnoticed. Although in some cases it can induce peritonitis which might be a life threatening condition.This study was determined to reveal the presence of helminths Setaria tundra and Setaria cervi in red deer (Cervus elaphus) in Slovakia. The parasites were identified morphologically and genetically, based on the sequences of a fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. For this purpose we used partial results of our longer parasitological monitoring realized in one particular hunting area located in eastern Slovakia, near the city of Kosice. A total of 60 red deer individuals were tested, of which one was found to be infected with Setaria tundra (prevalence of 1.7%) and four were detected to be infected with Setaria cervi (prevalence 6.7%). The intensity of infection was very low, only one specimen of Setaria spp. in each positive animal.


Assuntos
Cervos , Setaria (Nematoide) , Setaríase , Animais , Cervos/parasitologia , Setaria (Nematoide)/genética , Setaria (Nematoide)/isolamento & purificação , Setaríase/parasitologia , Eslováquia , Masculino , Feminino , Complexo IV da Cadeia de Transporte de Elétrons/genética
16.
Physiol Genomics ; 45(16): 720-8, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23780848

RESUMO

Many lines of evidence demonstrate that genetic variability contributes to chronic kidney disease susceptibility in humans as well as rodent models. Little progress has been made in discovering causal kidney disease genes in humans mainly due to genetic complexity. Here, we use a minimal congenic mapping strategy in the FHH (fawn hooded hypertensive) rat to identify Sorcs1 as a novel renal disease candidate gene. We investigated the hypothesis that genetic variation in Sorcs1 influences renal disease susceptibility in both rat and human. Sorcs1 is expressed in the kidney, and knocking out this gene in a rat strain with a sensitized genome background produced increased proteinuria. In vitro knockdown of Sorcs1 in proximal tubule cells impaired protein trafficking, suggesting a mechanism for the observed proteinuria in the FHH rat. Since Sorcs1 influences renal function in the rat, we went on to test this gene in humans. We identified associations between single nucleotide polymorphisms in SORCS1 and renal function in large cohorts of European and African ancestry. The experimental data from the rat combined with association results from different ethnic groups indicates a role for SORCS1 in maintaining proper renal function.


Assuntos
Nefropatias/metabolismo , Nefropatias/fisiopatologia , Receptores de Superfície Celular/metabolismo , Animais , Transporte Biológico/genética , Transporte Biológico/fisiologia , Feminino , Genótipo , Humanos , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Nefropatias/genética , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/fisiopatologia , Masculino , Proteinúria/genética , Proteinúria/metabolismo , Proteinúria/fisiopatologia , Ratos , Receptores de Superfície Celular/genética
17.
J Biol Chem ; 287(26): 22174-82, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22544750

RESUMO

Myocardial remodeling and dysfunction are serious complications of type 2 diabetes mellitus (T2DM). Factors controlling their development are not well established. To specifically address the role of the mitochondrial genome, we developed novel conplastic rat strains, i.e. strains with the same nuclear genome but a different mitochondrial genome. The new animals were named T2DN(mtFHH) and T2DN(mtWistar), where the acronym T2DN denotes their common nuclear genome (type 2 diabetic nephropathy (T2DN) rats) and mtFHH or mtWistar the origin of their mitochondria, Fawn Hooded Hypertensive (FHH) or Wistar rats, respectively. The T2DN(mtFHH) and T2DN(mtWistar) showed a similar progression of diabetes as determined by HbA1c, cholesterol, and triglycerides with normal blood pressure, thus enabling investigation of the specific role of the mitochondrial genome in cardiac function without the confounding effects of obesity or hypertension found in other models of diabetes. Echocardiographic analysis of 12-week-old animals showed no abnormalities, but at 12 months of age the T2DN(mtFHH) showed left ventricular remodeling that was verified by histology. Decreased complex I and complex IV but not complex II activity within the electron transport chain was found only in T2DN(mtFHH), which was not explained by differences in protein content. Decreased cardiac ATP levels in T2DN(mtFHH) were in agreement with a lower ATP synthetic capacity by isolated mitochondria. Together, our data provide experimental evidence that mtDNA sequence variations have an additional role in energetic heart deficiency. The mitochondrial DNA background may explain the increased susceptibility of certain T2DM patients to develop myocardial dysfunction.


Assuntos
DNA Mitocondrial/genética , Complicações do Diabetes/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Cardiopatias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Ecocardiografia/métodos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Variação Genética , Teste de Tolerância a Glucose , Cardiopatias/complicações , Masculino , Microscopia Eletrônica de Transmissão/métodos , Mitocôndrias/metabolismo , Mutação , Miocárdio/patologia , Ratos , Ratos Wistar
18.
Am J Physiol Renal Physiol ; 304(5): F565-77, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23220727

RESUMO

This study examined the effect of substitution of a 2.4-megabase pair (Mbp) region of Brown Norway (BN) rat chromosome 1 (RNO1) between 258.8 and 261.2 Mbp onto the genetic background of fawn-hooded hypertensive (FHH) rats on autoregulation of renal blood flow (RBF), myogenic response of renal afferent arterioles (AF-art), K(+) channel activity in renal vascular smooth muscle cells (VSMCs), and development of proteinuria and renal injury. FHH rats exhibited poor autoregulation of RBF, while FHH.1BN congenic strains with the 2.4-Mbp BN region exhibited nearly perfect autoregulation of RBF. The diameter of AF-art from FHH rats increased in response to pressure but decreased in congenic strains containing the 2.4-Mbp BN region. Protein excretion and glomerular and interstitial damage were significantly higher in FHH rats than in congenic strains containing the 2.4-Mbp BN region. K(+) channel current was fivefold greater in VSMCs from renal arterioles of FHH rats than cells obtained from congenic strains containing the 2.4-Mbp region. Sequence analysis of the known and predicted genes in the 2.4-Mbp region of FHH rats revealed amino acid-altering variants in the exons of three genes: Add3, Rbm20, and Soc-2. Quantitative PCR studies indicated that Mxi1 and Rbm20 were differentially expressed in the renal vasculature of FHH and FHH.1BN congenic strain F. These data indicate that transfer of this 2.4-Mbp region from BN to FHH rats restores the myogenic response of AF-art and autoregulation of RBF, decreases K(+) current, and slows the progression of proteinuria and renal injury.


Assuntos
Arteríolas/fisiopatologia , Hipertensão/genética , Rim/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Circulação Renal/fisiologia , Animais , Pressão Sanguínea/fisiologia , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Rim/irrigação sanguínea , Glomérulos Renais/fisiopatologia , Masculino , Proteinúria/genética , Proteinúria/fisiopatologia , Ratos , Ratos Endogâmicos BN , Insuficiência Renal/genética , Insuficiência Renal/fisiopatologia
19.
Trends Genet ; 26(12): 510-8, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20869786

RESUMO

The rat has long been a model favored by physiologists, pharmacologists and neuroscientists. However, over the past two decades, many investigators in these fields have turned to the mouse because of its gene modification technologies and extensive genomic resources. Although the genomic resources of the rat have nearly caught up, gene targeting has lagged far behind, limiting the value of the rat for many investigators. In the past two years, advances in transposon- and zinc finger nuclease (ZFN)-mediated gene knockout as well as the establishment and culturing of embryonic and inducible pluripotent stem cells have created new opportunities for rat genetic research. Here, we provide a high-level description and the potential uses of these new technologies for investigators using the rat for biomedical research.


Assuntos
Marcação de Genes/métodos , Ratos/genética , Animais , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias , Técnicas de Inativação de Genes/métodos , Humanos , Camundongos
20.
J Am Soc Nephrol ; 23(5): 825-33, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22343117

RESUMO

The combined transfer of two renal function quantitative trait loci (QTLs), Rf-1 (rat chromosome 1) and Rf-4 (rat chromosome 14), from the Fawn-hooded hypertensive rat onto the August Copenhagen Irish genetic background significantly increases proteinuria and demonstrates an interaction between these QTLs. Because the original Rf-4 congenic region is 61.9 Mbp, it is necessary to reduce this interval to feasibly search for variants responsible for renal susceptibility in this region. Here, we generated a minimal congenic line (Rf-1a+4_a) to identify a 4.1-Mb region of the Rf-4 QTL that significantly contributes to the severity of proteinuria in the Fawn-hooded hypertensive rat. Rf-1a+4_a animals have an increased glomerular permeability to albumin without significant changes in BP, indicating that at least one genetic element in this refined region directly affects renal function. Sequence analysis revealed no variants predicted to damage protein function, implying that regulatory elements are responsible for the Rf-4 phenotype. Multiple human studies, including recent genome-wide association studies, link the homologous human region with susceptibility to renal disease, suggesting that this congenic line is an important model for studying pathways that contribute to the progression of kidney disease.


Assuntos
Nefropatias/genética , Glomérulos Renais/metabolismo , Locos de Características Quantitativas , Animais , Animais Congênicos , Pressão Sanguínea , Mapeamento Cromossômico , Estudo de Associação Genômica Ampla , Taxa de Filtração Glomerular , Humanos , Hipertensão/genética , Permeabilidade , Fenótipo , Proteinúria/genética , Ratos
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