The liver injury following ischemia and reperfusion is worse in experimental knockout heterozygote mouse model for expression of connexin 43

Purpose:To evaluate that Connexin (Cx43) plays a role in lesions after hepatic ischemia/reperfusion (IR) injury.Methods:We use Cx43 deficient model (heterozygotes mice) and compared to a wild group. The groups underwent 1 hour ischemia and 24 hours reperfusion. The heterozygote genotype was confirmed by PCR. We analyzed the hepatic enzymes (AST, ALT, GGT) and histology.Results:The mice with Cx43 deficiency showed an ALT mean value of 4166 vs. 307 in the control group (p<0.001); AST mean value of 7231 vs. 471 in the control group (p<0.001); GGT mean value of 9.4 vs. 1.7 in the control group (p=0.001); histology showed necrosis and inflammation in the knockout group.Conclusions:This research demonstrated that the deficiency of Cx43 worses the prognosis for liver injury. The topic is a promising target for therapeutics advancements in liver diseases and procedures.(AU)

c-Kit immunoexpression patterns differ in melanotic and amelanotic canine oral melanomas / Análise qualitativa quanto à diferença da imunoexpressão do gene c-Kit em melanomas melânicos e amelânicos da cavidade oral em cães

Melanomas are the most common oral malignancy in dogs. Cell proliferation and connexin expression has been shown to differ in canine melanotic and amelanotic oral melanomas. This study aimed to analyze the c-Kit protein expression in melanotic and amelanotic melanomas from canine buccal cavity. A total of 34 canine buccal melanomas (19 melanotic and 15 amelanotic).were collected. The amelanotic melanomas presented faster evolution and higher incidence of metastasis than melanotic tumors. A significantly higher number of c-Kit positive cells were observed in amelanotic neoplasms. In addition, the intensity of c-Kit immunolabeling was predominantly stronger in amelanotic melanomas. These results confirm a potential role for c-Kit in canine oral melanomas with clear differences in expression patterns between the two histological types of tumor, melanotic and amelanotic. This study highlights the importance of a detailed study of c-Kit mutations in canine oral melanomas to better understand the molecular mechanisms implicated in the development of this disease(AU)

Melanomas são as mais frequentes neoplasias malignas da cavidade bucal de cães. Sabe-se que a proliferação de células e expressão de conexina diferem em melanomas melanóticos e amelanóticos da cavidade bucal de cães. Este estudo analisou a expressão da proteína c-Kit em melanomas melanóticos e amelanóticos da cavidade bucal canina. Um total de 34 melanomas bucais caninos (19 melanóticos e 15 amelanóticos) foram coletados. Os melanomas amelanóticos apresentaram evolução mais rápida e maior incidência de metástase. Foi constatado um número significativamente maior de células positivas para c-Kit em neoplasias amelanóticas. Além disso, a intensidade de imunomarcação de c-Kit foi predominantemente mais forte em melanomas amelanóticos. Estes resultados confirmam um papel potencial para c-Kit em melanomas orais caninos, com diferenças claras em padrões de expressão entre os dois tipos histológicos de tumor, melanóticos e amelanóticos. Este trabalho destaca a importância de um estudo detalhado das mutações c-Kit em melanomas orais caninos para ser possível a melhor compreensão dos mecanismos moleculares envolvidos no desenvolvimento da doença(AU)

THE ROLE OF CONNEXINS, PANNEXINS AND THEIR CHANNELS IN LIVER FIBROSIS

Embora as junções comunicantes, formadas por conexinas, tenham sido historicamente consideradas como protetoras da fisiologia tecidual, os hemicanais de conexinas e os canais de panexinas têm ganho considerável atenção como sinalizadores de processos patológicos, incluindo morte celular e inflamação. O objetivo desta tese foi investigar o papel destes dois tipos de canais, em particular os hemicanais constituídos por conexina32 (Cx32) ou conexina43 (Cx43), bem como os canais formados por panexina1 (Panx1), na doença hepática crônica. O foco foi direcionado à fibrose hepática, que é caracterizada pela formação de cicatrizes em resposta ao dano hepático, estresse oxidativo e inflamação associados à diversos tipos de doenças hepáticas crônicas. Inicialmente, verificou-se que a administração de um inibidor específico dos hemicanais de Cx43 ou um inibidor geral dos canais de conexinas em camundongos com fibrose hepática reduziu o estresse oxidativo e a inflamação no fígado, respectivamente. Adicionalmente, ambos os tratamentos aliviaram a fibrose hepática, ressaltando o envolvimento dos hemicanais de Cx43 na doença hepática crônica. Depois, a ablação genética da Cx32 revelou maior formação de cicatriz, dano hepático e estresse oxidativo em um modelo murino de fibrose hepática, apontando para um papel protetor da sinalização via Cx32 na fibrose hepática. Por fim, a ablação genética de Panx1 em dois modelos murinos de fibrose hepática mostrou resultados diferentes em relação à formação de cicatriz, danos hepáticos e inflamação, sugerindo que o envolvimento da Panx1 na fibrogênese hepática é dependente da etiologia. Coletivamente, os resultados destes estudos confirmam o papel patológico da sinalização via hemicanais de conexina e canais de panexina, e abre novas perspectivas para o tratamento clínico da fibrose hepática.

While connexin-based gap junctions have been historically considered as goal keepers of tissue physiology, connexin hemichannels and pannexin channels have lately gained considerable attention as drivers of pathological processes, including cell death and inflammation. The objective of this doctoral thesis project was to investigate the role of the latter 2 channel types, in particular hemichannels consisting of connexin32 (Cx32) and connexin43 (Cx43) as well as pannexin channels built up by pannexin1 (Panx1), in chronic liver disease. Focus was hereby put on liver fibrosis, which is characterized by scar formation in response to liver damage, oxidative stress and inflammation associated with several types of chronic liver disease. Firstly, it was found that administration of a specific Cx43 hemichannel inhibitor or a general connexin-based channel inhibitor to mice with liver fibrosis reduced hepatic oxidative stress and inflammation, respectively. In addition, both treatments alleviated liver fibrosis, thus underscoring the involvement of Cx43 hemichannels in chronic liver disease. After, genetic ablation of Cx32 in mice revealed enhanced scar formation, liver damage and oxidative stress in a mouse model of liver fibrosis, pointing to a protective role for Cx32 signaling in liver fibrosis. Finally, genetic ablation of Panx1 in 2 mouse models of liver fibrosis showed a different outcome with respect to scar formation, liver damage and inflammation, suggesting that the involvement of Panx1 in liver fibrogenesis is etiology-dependent. Collectively, the outcome of these studies confirms the pathological role of connexin hemichannel and pannexin channel signaling and opens new perspectives for the clinical treatment of liver fibrosis.

THE ROLE OF CONNEXINS, PANNEXINS AND THEIR CHANNELS IN NON-ALCOHOLIC STEATOHEPATITIS

Embora as junções gap formadas por conexinas tenham sido consideradas, historicamente, como mantenedoras da fisiologia tecidual, recentemente os hemicanais de conexinas e os canais de panexinas ganharam atenção considerável como sinalizadores de processos patológicos, incluindo morte celular e inflamação. O objetivo desta tese de doutoramento foi investigar o papel dos dois últimos tipos de canais, em particular dos hemicanais formados por conexina32 (Cx32) ou conexina43 (Cx43), bem como dos canais de panexina, constituídos por panexina1 (Panx1), na esteatohepatite não-alcoólica (ENA). Esta doença hepática crônica é altamente prevalente em todo o mundo e é caracterizada pelo acúmulo hepatocelular de lipídios, estresse oxidativo e inflamação. Em um primeiro estudo, descobriu-se que a ENA induzida por dieta em camundongos knockout para Cx32 resulta em dano hepático, inflamação e estresse oxidativo mais pronunciados, sugerindo assim, um efeito protetor da sinalização global via Cx32. Um segundo estudo mostrou que a inibição farmacológica específica dos hemicanais de Cx32 e Cx43 alivia a manifestação clínica da ENA em camundongos, como evidenciado pela redução da inflamação e estresse oxidativo no fígado. O terceiro estudo demonstrou redução do dano hepático, inflamação e estresse oxidativo em camundongos knockout para Panx1 após indução da ENA por dieta. Coletivamente, os resultados desses estudos confirmam o papel patológico reivindicado pela sinalização via hemicanais de conexina e canais de panexina, e abre novas perspectivas para o tratamento clínico da ENA.

While connexin-based gap junctions have been historically considered as goal keepers of tissue physiology, connexin hemichannels and pannexin channels have lately gained considerable attention as drivers of pathological processes, including cell death and inflammation. The objective of this doctoral thesis project was to investigate the role of the latter 2 channel types, in particular hemichannels consisting of connexin32 (Cx32) and connexin43 (Cx43) as well as pannexin channels built up by pannexin1 (Panx1), in liver disease. Focus was hereby put on nonalcoholic steatohepatitis (NASH), which is a highly prevalent type of chronic liver disease worldwide and that is characterized by hepatocellular lipid accumulation, oxidative stress and inflammation. In a first study, it was found that diet-induced NASH in whole body Cx32-/- mice results in more pronounced liver damage, inflammation and oxidative stress, thus suggesting a protective effect of overall Cx32 signaling. A second study showed that specific pharmacological inhibition of Cx32 and Cx43 hemichannels alleviates the clinical manifestation of NASH in mice as evidenced by reduced liver inflammation and oxidative stress. The third study demonstrated reduced liver damage, inflammation and oxidative stress in whole body Panx1-/- mice upon dietbased induction of NASH. Collectively, the outcome of these studies confirms the claimed pathological role of connexin hemichannel and pannexin channel signaling, and opens new perspectives for the clinical treatment of NASH.

Gene expression and in vitro nuclear maturation in bovine cumulus oocyte complexes maturated in a medium supplemented with bovine fetal serum or bovine serum albumin / Gene expression and in vitro nuclear maturation in bovine cumulus oocyte complexes maturated in a medium supplemented with bovine fetal serum or bovine serum albumin

Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be influenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and -actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantified to obta

Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be influenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and -actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantified to obta

Gene expression and in vitro nuclear maturation in bovine cumulus oocyte complexes maturated in a medium supplemented with bovine fetal serum or bovine serum albumin

Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be infl uenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and ß-actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantifi ed to obtain the relative mRNA abundance. Part of oocytes submitted to IVM medium supplemented with FBS (G2) or BSA (G3) was stained with Hoechst 33342 to assess the nuclear maturation rate by fluorescence microscopy. The results revealed that relative abundances of HAS (P = 0.000), link protein 1 (P = 0.001), connexin 43 (P = 0.007) and ß-actin (P = 0.011) transcripts differed between COCs submitted to IVM in FBS medium (G2) and COCs not submitted to IVM (G1) or COCs maturated in BSA medium (G3). When COCs submitted to IVM in FBS or BSA media are compared, no statistically significant differences (P > 0.05) were observed in meiosis resumption (86.7% and 91.5%, respectively) or in nuclear maturation rates (56.1% and 58.5%). Discussion: HAS2 is involved in the synthesis of hyaluronic acid (HA) by cumulus cells, and plays an important role in ECM expansion and in oocyte competence development. This protein organization of the ECM, formed by the aggregation of HA and proteoglycans, depends on link protein 1; it is also produced by cumulus cells and is implicated in COC expansion. Connexin 43 belongs to a protein family that establishes gap junctions that play an important role in the cellular communication and coordinated response processes. The role of gap junctions in bovine oocytes during IVM has been associated with maturation rates and cumulus expansion; this expansion of cumulus cells is accompanied by changes in the transmembrane channels formed by connexin 43. The higher mRNA expression of the HAS2, link protein 1, connexin 43 and ß-actin genes in bovine COCs submitted to IVM in FBS medium, in comparison with COCs before IVM or COCs maturated in BSA medium may be associated with FBS constituents, which would act as transcription factors for these genes during ECM expansion. Although the results obtained allow associating the differential expression of transcripts to the presence of FBS in the IVM medium, the data reveal that meiosis resumption and nuclear maturation apparently were not influenced by the protein supplementation regimens in the IVM medium, supplemented either with FBS or BSA.

THE ROLE OF CONNEXINS, PANNEXINS AND THEIR CHANNELS IN ACUTE LIVER FAILURE

A homeostase tecidual é controlada por uma série de mecanismos de comunicação. Ao nível intercelular, as junções gap permitem o tráfego de mensageiros pequenos e hidrofílicos, menores do que 1.5 kilodalton, entre duas células adjacentes. As junções gap são formadas por dois hemicanais, que por sua vez são compostos por seis proteínas denominadas conexinas (Cx). No fígado, os hepatócitos produzem principalmente Cx32, enquanto as células hepáticas não parenquimatosas contem predominantemente Cx43. As junções gap hepáticas são conhecidas por serem essenciais em diversas funções específicas do fígado, tais como secreção de albumina e metabolismo de xenobióticos. Na última década, tem-se tornado claro que os hemicanais de conexinas não são apenas precursores das junções gap, mas eles também podem participar da via de comunicação, contudo entre o citoplasma de células individuais e seus ambientes extracelulares. Além disso, foi descrita uma nova classe de proteínas semelhantes às conexinas, as panexinas (Panx), das quais a Panx1 é expressa no fígado. As pannexins se reúnem em uma configuração que lembra os hemicanais de conexinas e também participam da sinalização extracelular. Acredita-se que tanto os hemicanais de conexinas como os canais de pannexinas se tornam ativados preferencialmente em condições patológicas e, deste modo, promovam a morte celular e inflamação. O presente projeto de doutorado foi estabelecido para verificar se esta hipótese também é verdadeira nas doenças hepáticas, em particular na insuficiência hepática aguda. Inicialmente, o padrão de expressão das conexinas hepáticas foi totalmente caracterizado em um modelo murino de insuficiência hepática aguda induzida pelo paracetamol (APAP). Isto revelou uma mudança da Cx32 para Cx43 após a hepatotoxicidade induzida por APAP, com expressão de novo de Cx43 nos hepatócitos. Subsequentemente, a relevância funcional da expressão aumentada de Cx43 na insuficiência hepática aguda foi testada utilizando camundongos deficientes em Cx43. Verificou-se que a Cx43 tem um efeito protetor, uma vez que a lesão hepática induzida por APAP se agravou após a ablação genética da Cx43. Em paralelo, foi realizado um estudo semelhante utilizando camundongos knockout para Cx32, não mostrando diferenças significativas na extensão da hepatotoxicidade induzida pelo APAP em comparação aos animais selvagens. Uma vez que os animais deficientes em conexina não permitem diferenciar entre a comunicação via junções gap e a sinalização pelos hemicanais de conexina, uma série de experimentos seguintes baseou-se na utilização de inibidores dos hemicanais de Cx32 e Cx43, chamados de TAT-Gap24 e TAT-Gap19, respectivamente. Após os testes in vitro quanto a sua especificidade e eficiência in vitro, ambos os peptídeos foram administrados em camundongos com overdose pelo APAP. Enquanto TAT-Gap24 reduziu claramente a morte celular e inflamação, TAT-Gap19 apresentou apenas efeitos discretos sobre a lesão hepática. Além disso, o co-tratamento 12 de camundongos intoxicados pelo APAP com ambos os peptídeos revelou um efeito aditivo na redução da lesão hepática. Um estudo final foi focado na elucidação da expressão hepática e função da Panx1 na hepatotoxicidade induzida pelo APAP, utilizando o peptídeo inibidor da Panx1, 10Panx1. Verificou-se que a inibição dos canais de Panx1 minimiza as características clínicas da hepatotoxicidade desencadeada pelo APAP, em particular a relacionada com morte celular e inflamação. Em geral, este projeto de doutorado demonstra, pela primeira vez, o envolvimento dos hemicanais de conexina e canais de pannexina na insuficiência hepática aguda induzida pelo APAP, o que sugere um papel como alvos terapêuticos para fármacos.

Tissue homeostasis is controlled by a plethora of communication mechanisms. At the intercellular level, gap junctions allow the trafficking of small and hydrophilic messengers of less than 1.5 kilodalton between two adjacent cells. Gap junctions are built up by two hemichannels, which in turn are composed of six connexin (Cx) proteins. In liver, hepatocytes mainly produce Cx32, while non-parenchymal hepatic cells predominantly harbor Cx43. Hepatic gap junctions are known to underlie several liver-specific functions, such as albumin secretion and xenobiotic metabolism. In the last decade, it has become clear that the connexin hemichannels are not only precursors of gap junctions, but that they can also provide a communication pathway, albeit between the cytosol of individual cells and their extracellular environment. In addition, a novel class of connexin-like proteins has been identified, the pannexins (Panx), of which Panx1 is expressed in the liver. Pannexins gather in a configuration reminiscent of connexin hemichannels and also support extracellular signaling. Both connexin hemichannels and pannexin channels are believed to become preferably activated in pathological conditions and thereby to promote cell death and inflammation. The present doctoral project was set up to verify the hypothesis that this also holds true for liver disease, in particular acute liver failure. In a first instance, the hepatic connexin expression pattern was fully characterized in a mouse model of acute liver failure induced by acetaminophen (APAP). This revealed a switch from Cx32-to-Cx43 upon APAP-induced hepatotoxicity with de novo expression of Cx43 in hepatocytes. Subsequently, the functional relevance of enhanced Cx43 expression in acute liver failure was tested using Cx43-deficient mice. It was found that Cx43 has a protective effect, since liver APAP-induced injury became aggravated upon genetic ablation of Cx43. In parallel, a similar study was set up using Cx32 knock-out mice, showing no major differences in the extent of APAP-induced hepatotoxicity compared to wild type animals. Since connexin-deficient animals do not allow to discriminate between gap junction communication and connexin hemichannel signaling, a next set of experiments relied on the use of claimed peptide-based inhibitors of Cx32 and Cx43 hemichannels, namely TAT-Gap24 and TAT-Gap19, respectively. Following testing of their specificity and efficiency in vitro, both peptides were administered to APAPoverdosed mice. While TAT-Gap24 clearly reduced liver cell death and inflammation, TAT-Gap19 only had marginal effects on liver injury. Furthermore, co-treatment of APAPintoxicated mice with both peptides revealed an additive effect in lowering liver injury. A final study was focused on elucidating the hepatic expression and role of Panx1 in APAPinduced hepatotoxicity using the Panx1 inhibiting peptide 10Panx1. It was found that inhibition of Panx1 channels counteracts the clinical features of APAP-triggered hepatotoxicity, in particular related to cell death and inflammation. Overall, this doctoral 10 project shows for the first time the involvement of connexin hemichannels and pannexin channels in APAP-induced acute liver failure, which suggests a role as therapeutic drug targets.

Effects of Deletion of the Connexin 32 gene on pro and Anti-inflammatories Aspects in the Ethidium Bromide Toxic Demyelination Model

Gap junctions are cellular structures that allow transit of molecules between cells, allowing intercellular signaling and transportation. They are formed by proteins denominated connexins and represent key structures in highly complex and integrated tissues, such as the central nervous system (CNS). The present study evaluates the effects of connexin 32 (Cx32) deletion upon CNS inflammation and regeneration/repair after 1, 3, 7, 10 and 20 days after intracerebral injection of ethidium bromide in Cx32 Knock Out and normal mice. To accomplish so, Real Time PCR gene expression quantification was performed upon Tumour Necrosis Factor alpha (TNFα), Transforming Growth Factor beta 1 (TGFß1), Metalloproteinase 3 (MMP3), Metalloproteinase 9 (MMP9) and Tissue Inhibitor of Metalloproteinases 1 (TIMP1) genes. Results indicate varying differences in the expression pattern, including difference in expression of all evaluated genes in the 3 days post injection period, apex of the acute inflammation mechanisms. These results suggest that Cx32 may perform important functions on molecular, inflammatory and regenerative/repair signalling in the CNS.(AU)

Participação da conexina 32 na fisiopatogênese da doença hepática gordurosa não alcoólica em camundongos

A doença hepática gordurosa não alcoólica (DHGNA) abrange alterações desde esteatose até esteato-hepatite não alcoólica (EHNA), podendo evoluir para fibrose, cirrose e carcinoma hepatocelular. A DHGNA é considerada a doença hepática mais comum na atualidade e com prevalência mundial alarmante. Esta doença caracteriza-se, basicamente, pela deposição de triglicérides nos hepatócitos, podendo evoluir com inflamação e fibrose, e está intimamente associada com resistência à insulina (RI), diabetes mellitus tipo 2 e obesidade. Os hepatócitos representam as principais células hepáticas e se comunicam através de junções do tipo gap, formadas principalmente por conexina 32 (Cx32). Esta proteína apresenta importante função no controle da homeostase tecidual, regulando processos fisiológicos e tem sido associada como agente protetor na hepatocarcinogênese e outros processos patológicos, porem pouco se sabe sobre sua participação na DHGNA. Sendo assim, o objetivo deste trabalho foi avaliar a participação da Cx32 na fisiopatogênese da DHGNA, utilizando camundongos knockout para Cx32 (Cx32-KO) submetidos a uma dieta hiperlipídica deficiente em colina. Foram analisados dados biométricos, histopatológicos, função hepática, RI, citocinas inflamatórias, adipocinas, estresse oxidativo, peroxidação lipídica e a expressão de genes envolvidos na DHGNA. Os animais Cx32-KO apresentaram maior acumulo de triglicérides hepáticos em relação aos animais selvagens e, consequentemente, maior peso absoluto e relativo do fígado. Adicionalmente, apresentaram maior inflamação hepática demonstrado pela exacerbação da citocina TNF- e supressão da IL-10, maior dano hepatocelular indicado pelo aumento das enzimas AST e ALT, aumento da peroxidação lipídica e alterações na expressão de genes chaves na fisiopatogênese da DHGNA, como SREBP1c. No entanto, não houve diferença nos marcadores histopatológicos, RI e estresse oxidativo hepático. Por fim, os animais Cx32-KO apresentaram maior produção de leptina e adiponectina no tecido adiposo. Todos esses resultados revelam que a Cx32 pode atuar como um agente protetor ao desenvolvimento da DHGNA, sugerindo seu potencial como novo alvo terapêutico.

Nonalcoholic fatty liver disease (NAFLD) covers a spectrum of liver diseases ranging from steatosis to nonalcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis and eventually hepatocellular carcinoma. NAFLD is currently the most common liver disease in the world with an alarming prevalence worldwide. This disease is characterized by the deposition of triglycerides in hepatocytes and can develop inflammation and fibrosis. NAFLD is closely associated with insulin resistance (IR), type 2 diabetes mellitus and obesity. The gap junctions mediate intercellular communication and are critical for maintaining integral cellular processes. In hepatocytes, the major type of liver cells, the gap junctions are formed mainly by connexin 32 (Cx32). This protein is important for the control of tissue homeostasis and for physiological processesregulation. It has been linked as a protective agent in hepatocarcinogenesis and other pathological processes. However, little is known about its role on NAFLD. Thus, the aim of this study was to evaluate the participation of Cx32 in the pathogenesis of NAFLD using WT and Cx32-knockout mice (Cx32-KO) subjected to a high-fat diet deficient in choline. NAFLD was evaluated based on a battery of clinically relevant read-outs, including histopathological examination, inflammatory citokines, liver damage markers, in-depth lipid analysis, assessment of oxidative stress, insulin and glucose tolerance and lipid-related biomarkers.The Cx32-KO animals showed higher accumulation of hepatic triglycerides, increased inflammation and lipid peroxidation and increased production of leptin and adiponectin in the adipose tissue when compared with WT. Moreover, there was no difference in histopathological markers, RI and hepatic oxidative stress. Taken together, all these results show that Cx32 is a protective agent to the development of the disease, suggesting its potential as a novel therapeutic target in NAFLD

Expression of Connexins 43, 26 and 32 in normal, hyperplastic and neoplastic perianal dog glands

Connexin (Cx) expression is reportedly altered in neoplasms. This study aimed to investigate the expression of Cx43, 26 and 32 in normal and pathological canine perianal glands. Thirty perianal glands bearing pathological processes and ten normal canine perianal glands were submitted to immunohistochemistry to search for presence of Cx43, Cx26 and Cx32. Both Cx43 and Cx26 expressions were observed in normal, hyperplastic glands, and in well and moderately differentiated adenomas. However, in poorly differentiated adenomas, expressions were reduced, and they were absent in carcinomas. Cx26 was located in the cytoplasm of normal, hyperplastic perianal gland cells, and in well and moderately differentiated adenomas. Cx32 was not observed in any neoplasm neither in normal or hyperplastic glands. Our results show that Cx43 and Cx26 expressions are altered in more aggressive canine perianal gland neoplasms, and we conclude that they may be related to the perianal gland carcinogenesis process (AU)

Diminished angiogenesis in the cornea of mice with heterologous deletion of Connexin 43 gene (Gja1)

Angiogenesis is involved in several physiological and pathological processes, and the proliferation of endothelial cells is a central event in the generation of new blood vessels. Gap junctions (GJ) are membrane structures that communicate adjacent cells, contribute to tissue homeostasis, and are important to the control of cell proliferation. Connexins (Cxs) are the proteins that form gap junctions. Endothelial cells may express Cx43, Cx37 and Cx40. In this study, we analyzed the effect of the heterologous deletion of the Gja1 (Cx43 gene) on the development of newly formed blood vessels (NFBV) in the mouse cornea. Heterozygous (Cx43+/-) and wild-type (Cx43+/+) mice were submitted to the silver crystal corneal cauterization model. Two parameters were quantified by image analysis 2 or 6 days after cauterization: NFBV density and length. At days 2 and 6 after corneal cauterization, Cx43+/- mice showed smaller density of NFBV as compared to Cx43+/+ mice. Therefore, deletion of one Gja1 allele (connexin-43 gene) may lead to impaired cell-cell communication in endothelial cells, diminishing angiogenesis after cauterization of the mouse cornea(AU)

Efeito da circulação extracorpórea na expressão da conexina 43 no miocárdio

As conexinas são proteínas essenciais e estão diretamente relacionadas à propagação do impulso elétrico no coração, à velocidade de condução bem como à gênese de muitas afecções cardíacas. Em face da circulação extracorpórea (CEC) ainda ser utilizada de forma inconsistente na medicina veterinária e devido aos poucos estudos observados na literatura sobre os efeitos provocados pelo emprego da CEC na expressão da conexina 43 (Cx43) no miocárdio, objetivou-se avaliá-la em 15 animais da espécie canina, distribuidos em três grupos (C, CEC-1 e CEC -2) sendo, respectivamente, antes de realizada a CEC, com 60 minutos após esta e 60 minutos de CEC seguida de 30 minutos de restauração da perfusão espontânea. Avaliou-se a Cx43 pelas técnicas de imunofluorescência, western blot e RT-PCR em tempo real no tecido muscular cardíaco de regiões correspondentes aos átrios direito (AD) e esquerdo (AE), ventrículos direito (VD) e esquerdo (VE) e septo transverso. Os resultados indicaram a presença da Cx43 em todas as regiões do miocárdio nos grupos C, CEC-1 e CEC-2. A expressão da Cx43 variou significativamente em CEC-2 no AE e VD em relação ao grupo C (p<0,05). A expressão gênica de Gja1 (gene da Cx43) não apresentou diferença significativa entre os grupos estudados. Em CEC-2 identificou-se a presença de vacuolização na túnica média de artérias de pequeno calibre do miocárdio. Concluiu-se também que a canulação da aorta bem como a instalação do circuito de CEC no modo aorto-bicaval constitui técnica exequível.

Connexins are essential proteins that are directly associated with electrical impulse propagation and speed of propagation in the heart. They also play a major role in numerous heart conditions. The use of cardiopulmonary bypass (CPB) in veterinary medicine is inconsistent and few studies describe the effect of cardiopulmonary bypass on the expression of connexin 43 (Cx43) in the myocardium. The objective of this study was to evaluate myocardial expression of Cx43. Connexin 43 of 15 dogs was assessed at 3 moments: prior to CPB (Group C); 60 minutes after CPB (Group CPB1); and 60 minutes after CPB followed by 30 minutes of spontaneous perfusion (Group CPB2). Assessment of Cx43 included immunofluorescence, western blot and RT-PCR real time of heart tissue samples from the right atrium (RA), left atrium (LA), right ventricle (LV), right ventricle (RV) and transverse septum. Our results showed the presence of Cx43 in all 5 areas of the myocardium in groups C, CPB1 and CPB2. A significant variation on the expression of Cx43 was observed when CPB2 LA and CPB2 RV were compared to group C (p<0,05). Expression of Gja1 (gene for Cx43) did not vary significantly among the study groups. Group CBP2 presented vacuolation of the tunica media of small myocardial arteries. We conclude that cannulation of the aorta and aorto-bicaval setup of the CPB circuit is feasible technique.

EXPRESSÃO DE ÁCIDO HIALURÔNICO SINTASE-2, RECEPTORES GONADOTRÓFICOS E CONEXINA 43 EM CÉLULAS DO CUMULUS DE OÓCITOS CAPRINOS

A maturação oocitária é uma importante etapa da produção in vitro de embriões, sendo dependente da qualidade do oócito, a qual determina sua competência ao desenvolvimento. A estimulação hormonal ovariana com gonadotrofinas pode influenciar a expressão gênica nas células cumulus (CCs) de complexos cumulus-oócito (CCOs) e, consequentemente, a qualidade oocitária. Adicionalmente, a maturação in vitro (MIV) dos CCOs envolve a síntese de ácido hialurônico para que haja a expansão do cumulus. Nesse evento, também ocorre a regulação da expressão de conexinas, as quais compõem as junções gap existentes entre o cumulus e o oócito. Assim, no presente trabalho, em paralelo à classificação morfológica (GI/II ou GIII) e avaliação da taxa de MIV, investigou-se a expressão dos genes que codificam a enzima ácido hialurônico sintase-2 (HAS2), os receptores gonadotróficos do hormônio folículo estimulante (FSHR) e do luteinizante (LHR) e a conexina 43 em CCs oriundas de CCOs caprinos obtidos após estimulação hormonal ovariana com múltiplas doses de FSH (MD) ou única dose de FSH/eCG (Oneshot - OS). Adicionalmente, CCOs bovinos oriundos de abatedouro foram utilizados como grupo controle. O tratamento MD produziu maior número de folículos grandes (>4 mm), CCOs com melhor aspecto morfológico (GI/II) e melhor taxa de MIV que OS. O tratamento OS produziu CCOs com CCs que apresentaram maior nível de transcritos para todos os genes analisados. Esta maior expressão gênica também foi observada em CCs de CCOs com aspecto morfológico inferior (GIII). Por outro lado, os níveis de RNAm tornaram-se mais semelhantes entre os grupos após a MIV, principalmente com o decréscimo dos transcritos para FSHR e LHR. Alguns eventos que ocorreram durante a MIV bovina, como a expansão das células, o aumento de HAS2 e decréscimo de Cx43, foram menos evidentes ou não observados na MIV caprina. Em conclusão, o tratamento OS produziu CCOs com maior nível de transcritos para HAS2, receptores gonadotróficos e conexina 43, comparado a MD. Contudo, esse maior nível de transcritos não conferiu maior competência meiótica aos oócitos e pode estar relacionado a uma regulação deficiente da expressão gênica por oócitos de menor qualidade. Finalmente, a expansão, o aumento da expressão de HAS2 e o decréscimo da síntese de Cx43, parecem ser eventos mais evidentes nas CCs de CCOs durante a MIV bovina que na caprina. Estudos adicionais são ainda necessários para investigar a importância de outras isoformas de HAS ou de conexinas em CCOs caprinos.

The oocyte maturation is an important step of in vitro embryo production, being dependent on oocyte quality, which determines their competence development. Ovarian stimulation with gonadotropins can influence gene expression in the cumulus cells (CCs) of cumulus-oocyte complexes (COCs) and, consequently, the oocyte quality. Additionally, in vitro maturation (IVM) of COCs involves hyaluronic acid synthesis for cumulus expansion. In this event, there is also the regulation of connexin expression, which make up the gap junctions existing between the cumulus and oocyte. In the present work, in parallel with the morphological classification (GI/II or GIII) and evaluation of IVM rate, we investigated the expression of genes encoding the enzyme hyaluronic acid synthase-2 (HAS2), the gonadotropic receptors of follicle stimulating hormone (FSHR) and luteinizing hormone (LHR) and connexin 43 in CCs derived from COCs goats obtained after ovarian hormonal stimulation with multiple doses of FSH (MD) or a single dose of FSH/eCG (OS). The MD treatment produced higher number of large follicles, COCs with great morphological aspects and IVM rate than OS. Additionally, bovine COCs originating from slaughterhouse were used as control group. The MD treatment produced higher number of large follicles (>4 mm), COCs with great morphological aspects and IVM rate than OS. The OS treatment produced COCs with more HAS2, FSHR, LHR and Cx43 transcripts. This gene expression pattern was also observed in CCs of COCs showing poor morphological characteristics. On the other hand, the mRNA levels becomes more similar between groups after IVM, mainly decreasing FSHR and LHR transcripts. Some events that occurred during the bovine IVM, such as the expansion of the cells, the increase of HAS2 and decrease of Cx43 were less apparent or not observed in goat IVM. In conclusion, the OS treatment produced COCs with a higher level of transcripts for HAS2, gonadotropic receptors and connexin 43, compared to MD. However, increasing in HAS2, FSHR, LHR and Cx43 expressions in goat COCs do not confer more meiotic competence to oocytes. Instead, it may be resulted from a poor regulation of gene expression in CCs by lower quality oocytes. Finally, cumulus expansion together with HAS2 upregulation and Cx43 downregulation seems to be more important IVM events for bovine than for goat COCs. Further studies are needed to investigate the importance of other HAS isoforms or connexins in goat COCs.

Expression of Connexins 43, 26 and 32 in normal, hyperplastic and neoplastic perianal dog glands

Connexin (Cx) expression is reportedly altered in neoplasms. This study aimed to investigate the expression of Cx43, 26 and 32 in normal and pathological canine perianal glands. Thirty perianal glands bearing pathological processes and ten normal canine perianal glands were submitted to immunohistochemistry to search for presence of Cx43, Cx26 and Cx32. Both Cx43 and Cx26 expressions were observed in normal, hyperplastic glands, and in well and moderately differentiated adenomas. However, in poorly differentiated adenomas, expressions were reduced, and they were absent in carcinomas. Cx26 was located in the cytoplasm of normal, hyperplastic perianal gland cells, and in well and moderately differentiated adenomas. Cx32 was not observed in any neoplasm neither in normal or hyperplastic glands. Our results show that Cx43 and Cx26 expressions are altered in more aggressive canine perianal gland neoplasms, and we conclude that they may be related to the perianal gland carcinogenesis process

Diminished angiogenesis in the cornea of mice with heterologous deletion of Connexin 43 gene (Gja1)

Angiogenesis is involved in several physiological and pathological processes, and the proliferation of endothelial cells is a central event in the generation of new blood vessels. Gap junctions (GJ) are membrane structures that communicate adjacent cells, contribute to tissue homeostasis, and are important to the control of cell proliferation. Connexins (Cxs) are the proteins that form gap junctions. Endothelial cells may express Cx43, Cx37 and Cx40. In this study, we analyzed the effect of the heterologous deletion of the Gja1 (Cx43 gene) on the development of newly formed blood vessels (NFBV) in the mouse cornea. Heterozygous (Cx43+/-) and wild-type (Cx43+/+) mice were submitted to the silver crystal corneal cauterization model. Two parameters were quantified by image analysis 2 or 6 days after cauterization: NFBV density and length. At days 2 and 6 after corneal cauterization, Cx43+/- mice showed smaller density of NFBV as compared to Cx43+/+ mice. Therefore, deletion of one Gja1 allele (connexin-43 gene) may lead to impaired cell-cell communication in endothelial cells, diminishing angiogenesis after cauterization of the mouse cornea

Expressão gênica das células do cumulus oophorus de bovinos após a vitrificação / Gene expression of bovine cumulus oophorus cells after vitrification

Um dos desafios da criobiologia continua sendo o desenvolvimento de um método que proporcione a manutenção da viabilidade após a criopreservação de oócitos imaturos na espécie bovina. A vitrificação tem sido a metodologia que proporciona resultados de sobrevivência após a criopreservação mais promissores para complexos cumulus-oócito (CCOs) imaturos bovinos. Entretanto, a ação dos crioprotetores sobre as células do cumulus oophorus, no que diz respeito à regulação da expressão de genes importantes nesta fase, ainda é pouco compreendida. O objetivo do trabalho foi avaliar a expressão gênica das proteínas ácido hialurônico sintase 2 (HAS2), link protein (HAPLN1), conexina 43 (GJA1) e proteína de choque térmico HSP70-1 (HSP70-1) em células do cumulus oophorus de oócitos imaturos bovinos submetidos a exposição e/ou vitrificação na solução crioprotetora (SV) composta por 20% de etileno glicol (EG) + 20% dimetil sulfóxido (DMSO) + 0,5M de sacarose. Os CCOs foram selecionados e distribuídos em 4 grupos experimentais: G1, CCOs não submetidos a maturação in vitro (MIV); G2, CCOs submetidos à MIV; G3, CCOs maturados após a exposição à SV; e G4, CCOs maturados após a vitrificação com a SV. A MIV foi realizada em TCM 199, suplementado com soro de égua em estro, à 39oC, 5% de CO2 e máxima umidade relativa, por 22 a 24 horas. A extração do RNA das amostras de células do cumulus foi realizada pelo método do fenol-clorofórmio seguido por uma etapa de captação específica do mRNA. Após a utilização da técnica de RT-PCR para a obtenção do cDNA e amplificação dos quatro fragmentos específicos, a análise dos resultados não mostrou diferença significativa entre os grupos para a abundância relativa dos transcritos de link protein (p=0,486), HAS2 (p=0,394), conexina 43 (p=0,116) e HSP70-1 (p=0,248).

A challenge of cryobiology remains in to develop a method that provides adequate results of viability after cryopreservation of immature bovine oocytes. Vitrification has been the methodology that provides most promising survival results after cryopreservation for immature bovine cumulus-oocyte complexes (COCs). However, the action of cryoprotectants on the gene expression regulation of cumulus oophorus cells is still poorly understood. The aim of this study was to evaluate, through the RT-PCR technique, the gene expression of hyaluronic acid synthase protein 2 (HAS2), link protein, connexin 43 and heat shock protein 70 (HSP70-1) in cumulus oophorus cells of immature bovine oocytes exposed and/or vitrified in cryoprotectant solution (VS) containing 20% ethylene glycol (EG) + 20% dimethilsulfoxide (DMSO) + 0.5 M sucrose before in vitro maturation (IVM). The immature COCs were sellected, and allocated into four experimental groups: G1, COCs no submitted to IVM; G2, COCs submitted to IVM; G3, COCs exposed to VS and submitted to IVM; and G4, COCs vitrified with VS and submitted to IVM. The IVM was performed in TCM 199 supplemented with oestrus mare serum, at 39°C, under 5% of CO2 and maximum relative humidity, for 22 to 24 hours. The RNA extraction from cumulus cells samples was performed by using the phenol-chloroform followed by the specific capture of the mRNA. The mRNAs were transcribed into cDNA using RT-PCR to evaluate patterns of gene expression. The results showed no significant difference between the groups tested for the relative abundance of link protein (p=0.486), HAS2 (p=0.394), connexin 43 (p = 0.116) and HSP70-1 (p = 0.248) transcripts.

Expressão gênica das células do cumulus oophorus de bovinos após a vitrificação / Gene expression of bovine cumulus oophorus cells after vitrification

Um dos desafios da criobiologia continua sendo o desenvolvimento de um método que proporcione a manutenção da viabilidade após a criopreservação de oócitos imaturos na espécie bovina. A vitrificação tem sido a metodologia que proporciona resultados de sobrevivência após a criopreservação mais promissores para complexos cumulus-oócito (CCOs) imaturos bovinos. Entretanto, a ação dos crioprotetores sobre as células do cumulus oophorus, no que diz respeito à regulação da expressão de genes importantes nesta fase, ainda é pouco compreendida. O objetivo do trabalho foi avaliar a expressão gênica das proteínas ácido hialurônico sintase 2 (HAS2), link protein (HAPLN1), conexina 43 (GJA1) e proteína de choque térmico HSP70-1 (HSP70-1) em células do cumulus oophorus de oócitos imaturos bovinos submetidos a exposição e/ou vitrificação na solução crioprotetora (SV) composta por 20% de etileno glicol (EG) + 20% dimetil sulfóxido (DMSO) + 0,5M de sacarose. Os CCOs foram selecionados e distribuídos em 4 grupos experimentais: G1, CCOs não submetidos a maturação in vitro (MIV); G2, CCOs submetidos à MIV; G3, CCOs maturados após a exposição à SV; e G4, CCOs maturados após a vitrificação com a SV. A MIV foi realizada em TCM 199, suplementado com soro de égua em estro, à 39oC, 5% de CO2 e máxima umidade relativa, por 22 a 24 horas. A extração do RNA das amostras de células do cumulus foi realizada pelo método do fenol-clorofórmio seguido por uma etapa de captação específica do mRNA. Após a utilização da técnica de RT-PCR para a obtenção do cDNA e amplificação dos quatro fragmentos específicos, a análise dos resultados não mostrou diferença significativa entre os grupos para a abundância relativa dos transcritos de link protein (p=0,486), HAS2 (p=0,394), conexina 43 (p=0,116) e HSP70-1 (p=0,248).(AU)

A challenge of cryobiology remains in to develop a method that provides adequate results of viability after cryopreservation of immature bovine oocytes. Vitrification has been the methodology that provides most promising survival results after cryopreservation for immature bovine cumulus-oocyte complexes (COCs). However, the action of cryoprotectants on the gene expression regulation of cumulus oophorus cells is still poorly understood. The aim of this study was to evaluate, through the RT-PCR technique, the gene expression of hyaluronic acid synthase protein 2 (HAS2), link protein, connexin 43 and heat shock protein 70 (HSP70-1) in cumulus oophorus cells of immature bovine oocytes exposed and/or vitrified in cryoprotectant solution (VS) containing 20% ethylene glycol (EG) + 20% dimethilsulfoxide (DMSO) + 0.5 M sucrose before in vitro maturation (IVM). The immature COCs were sellected, and allocated into four experimental groups: G1, COCs no submitted to IVM; G2, COCs submitted to IVM; G3, COCs exposed to VS and submitted to IVM; and G4, COCs vitrified with VS and submitted to IVM. The IVM was performed in TCM 199 supplemented with oestrus mare serum, at 39°C, under 5% of CO2 and maximum relative humidity, for 22 to 24 hours. The RNA extraction from cumulus cells samples was performed by using the phenol-chloroform followed by the specific capture of the mRNA. The mRNAs were transcribed into cDNA using RT-PCR to evaluate patterns of gene expression. The results showed no significant difference between the groups tested for the relative abundance of link protein (p=0.486), HAS2 (p=0.394), connexin 43 (p = 0.116) and HSP70-1 (p = 0.248) transcripts.(AU)

Expression and distribution of connexin 32 in rat liver with experimentally induced fibrosis / Expressão e distribuição da conexina 32 em fígados de ratos com fibrose induzida experimentalmente

The connexin 32 (Cx32) is a protein that forms the channels that promote the gap junction intercellular communication (GJIC) in the liver, allowing the diffusion of small molecules through cytosol from cell-to-cell. Hepatic fibrosis is characterized by a disruption of normal tissue architeture by cellular lesions, and may alter the GJIC. This work aimed to study the expression and distribution of Cx32 in liver fibrosis induced by the oral administration of dimethylnitrosamine in female Wistar rats. The necropsy of the rats was carried out after five weeks of drug administration. They presented a hepatic fibrosis state. Sections from livers with fibrosis and from control livers were submitted to immunohistochemical, Real Time-PCR and Western-Blot analysis to Cx32. In fibrotic livers the Cxs were diffusely scattered in the cytoplasm, contrasting with the control livers, where the Cx32 formed junction plaques at the cell membrane. Also it was found a decrease in the gene expression of Cx32 without reduction in the protein quantity when compared with controls. These results suggest that there the mechanism of intercellular communication between hepatocytes was reduced by the fibrotic process, which may predispose to the occurrence of a neoplastic process, taken in account that connexins are considered tumor suppressing genes.(AU)

A conexina 32 (Cx32) é uma proteína que constitui os canais que promovem as comunicações intercelulares via junções comunicantes (CIJC) no fígado, permitindo difusão de pequenas moléculas citoplasmáticas de uma célula à outra. A fibrose hepática caracteriza-se pela alteração da arquitetura normal do fígado e podem alterar as CIJCs. O objetivo deste trabalho foi estudar a expressão e distribuição de Cx32 na fibrose hepática. O objetivo do presente trabalho foi estudar a expressão e distribuição da Cx32 em fígados com fibrose induzida pela administração oral de dimetilnitrosamina em fêmeas de ratos Wistar. A necropsia foi realizada após cinco semanas da última administração da droga e observou-se um quadro de fibrose hepática. Amostras dos fígados com fibrose e de animais controle foram submetidas à análise imunoistoquímica, por Real Time-PCR e por Western-Blot verificando-se a presença de Cx32 difusa e dispersa no citoplasma dos fígados com fibrose. No grupo controle a Cx32 localizou-se na membrana citoplasmática com a formação de placas juncionais. O fígado com fibrose também revelou diminuição da expressão gênica de Cx32, embora sem a redução da quantidade do produto protéico, quando comparado ao grupo controle. Estes resultados sugerem que o mecanismo de comunicação intercelular entre os hepatócitos reduziu-se durante o processo fibrótico, o que pode predispor a ocorrência de processos neoplásicos, uma vez que as conexinas são consideradas genes supressores de tumores.(AU)

Compensatory kidney hypertrophy/hyperplasia after nephrectomy in mice: alterations of connexin 43 (Cx43) phosphorylated isoforms

Compensatory kidney hypertrophy/hyperplasia leads to several changes in kidney structure and function, as increased glomeruli filtration. The aim of this study was to evaluate connexin 43 in remnant mouse kidneys after unilateral nephrectomy. The right kidney was surgically removed from BALB/c mice. Groups were euthanized at 24, 48 and 72 hours, and at 7 and 30 days. Kidney sections of the reminiscent kidneys were stained with Periodic Acid/Schiff and additional slides were submitted to BrdU and Cx43 immunohistochemistry. The results demonstrated an increase in kidney weight as early as 24 hours through 30 post-nephrectomy. In addition, BrdU positive epithelial cells increased at 24 and 48 hours post-nephrectomy. Cx43 was detected in the cytoplasm and membrane of epithelial cells and vasculature. Taking into consideration the quantity, intensity and localization of Cx43 immunostaining pattern, we observed that nephrectomized mice presented lower Cx43 expression and a cytoplasmic localization after 24 hours, peaking in 48 hours. Furthermore, western blot revealed that during the first 24 and 48 hours after nephrectomy, PO (unphosphorylated) and P1 (phosphorylated) Cx43 disappeared, and the products of Cx43 degradation were reduced. On the other hand, after 72 hours the PO and P1 state reappeared and the amount of degraded peptides also increased. Seven and thirty days after nephrectomy, a higher intensity of PO and P1 state and a lower P2 (hyperphosphorylated) band were observed. In conclusion, our results suggest that Cx43 phosphorylation results in the retention of Cx43 in cytoplasm and its increased degradation during compensatory renal hyperplasia/hypertrophy.

Gap junctions and communication within the nervous system

Gap junctions are sites on the cellular membrane with intercellular channels build up by twelve protein subunits called connexins. Each connected cell contributes with a hemichannel made up by six connexins subunits. This kind of connection represents and efficient way of intercellular communication in most tissues, including the nervous system. It works as a passage for ions, secondary messenger and metabolites exchange between the cells. In a complex tissue like the nervous tissue they are particularly important because they connect the various cellular types composing a panglial syncytium that performs neuronal protection and tissue homeostasis. The expression of connexins and the intercellular communication through gap junctions are crucial to regulate vital functions as cellular motility, proliferations and survival; changes in the conformational expression of connexins may be involved in diseases as Alzheimer's disease, neoplasms, bacterial and parasitic infections, or even affect cellular groups when they occur as genetic mutations leading to functional defects of the nervous system as demyelination in the PNS (Charcot-Marie-Tooth disease), hereditary epilepsy, non-syndromic deafness and senile cataract.