Policistina-1 protege a los cardiomiocitos de la necrosis inducida por estrés mecánico / Policystin-1 protects cardiomyocytes from mechanical stress induced necrosis

Resumen: Antecedentes: La muerte de los cardiomiocitos es determinante en el desarrollo de patologías cardiacas posteriores al infarto del miocardio y la insuficiencia cardiaca. Las variaciones en la expresión de la familia de proteínas BCL-2 regulan vías, tanto de muerte, como de sobrevida celular. Así, BCL-2 es una proteína anti- apoptótica y NIX una proteína que induce la necrosis y/o la apoptosis celular. La Policistina-1 (PC1) es un mecanosensor vital para la función contráctil cardiaca; sin embargo, se desconoce su papel en la sobrevida de los cardiomiocitos durante el estrés mecánico. Objetivo: Determinar si PC-1 previene la muerte de los cardiomiocitos inducida por estrés mecánico y las proteínas BCL-2 y NIX. Métodos: Se utilizó cultivo de cardiomiocitos de ratas neonatas controles o deficientes en la expresión de PC1, estimulados con solución hiposmótica (HS), como modelo de estrés mecánico. Se midió la muerte por necrosis y apoptosis y los niveles de BCL-2 y NIX. Resultados: La deficiencia de la PC1 en los cardiomiocitos induce un aumento de la necrosis y los niveles proteicos de NIX en las células estimuladas con HS. El estrés mecánico induce la apoptosis basal relacionada a una disminución de BCL- 2, independiente de la expresión de la PC1. Conclusiones: La PC1 protege a los cardiomiocitos de la necrosis por estrés mecánico, lo que podría deberse en parte a su papel en la regulación de los niveles de las proteínas NIX.

Abstracts: Background: Cardiomyocytes death is a determining factor in the development of cardiac dysfunction after myocardial infarction and heart failure. The change in BCL-2 family protein expression regulates both cell death and survival pathways, whereas BCL-2 is an anti-apoptotic protein and NIX induces necrosis and/or apoptosis. Polycystin-1 (PC1) is a crucial mechanosensor for cardiac contractile function. However, its role in cardiomyocyte survival during mechanical stress is unknown. Aim: To study the relationship of PC1 with mechanical stretch-death in cardiomyocytes and the BCL-2, and NIX proteins. Methods. Controls or deficient expression of PC1 neonatal rat ventricular myocytes were stimulated with hypoosmotic solution (HS) and used as a model of mechanical stress. Necrosis or apoptosis cell death, BCL-2 and NIX protein levels were measured. Results: Deficient expression of PC1 increases cardiomyocyte necrosis and NIX protein levels in cells stimulated with HS. Mechanical stress induces basal apoptosis related to a decrease in BCL-2, independent of PC1 expression. Conclusion: PC1 protects cardiomyocytes from mechanical stress necrosis, at least in part, by regulating NIX protein levels.

Mechanism of male reproductive failure in autosomal dominant polycystic kidney disease and outcomes of assisted reproductive technology treatment / 上海交通大学学报（医学版）

Objective: To explore the potential mechanism of male reproductive failure in autosomal dominant polycystic kidney disease (ADPKD) patients and analyze the outcomes of assisted reproductive technology treatment. Methods: Next-generation sequencing was performed for genetic diagnosis of 8 ADPKD patients, who came to International Peace Maternity & Child Health Hospital, Shanghai Jiao Tong University School of Medicine, for genetic counseling. The semen of ADPKD patients and normal males who came for pre-pregnancy consultation was collected by masturbation for sperm analysis. The ultrastructure of sperm was observed by transmission electron microscopy. Outcomes of 7 patients with ADPKD who chose preimplantation genetic testing (PGT) were compared with those of 7 patients who were dystrophin (DMD) gene mutation carriers, undergoing the PGT in the same period. Results: Eight patients with ADPKD were heterozygous for polycystin 1 (PKD1) gene. Key parameters of sperm motion including progressive motility sperm percentage, curvilinear velocity, straight-line velocity, average path velocity, amplitude of lateral head displacement were much lower than those of normal semen, showing mild to severe oligozoospermia. One ADPKD patient with severe oligoathenospermia manifested bilateral seminal vesicle cysts. Transmission electron microscopy showed that the central microtubules of the sperm flagella of ADPKD patients were absent and the surrounding double microtubules were disorganized. There was no significant difference in the number of eggs, fertilization rate, cleavage rate, effective embryo rate and excellent embryo rate between the ADPKD patients and the DMD gene mutation carriers, but the ADPKD patients were prone to early abortion. Conclusion: Male reproductive failure caused by ADPKD may be related to many factors such as abnormal structure of sperm flagella and genital cysts. Further, PKD1 mutation may play a role in embryo implantation and early development.

Immunohistochemical study of polycystin-1 in dentigerous cysts.

Background: The alterations involved in step-wise transformation of a dental follicle to dentigerous cyst (DC) is not clearly known. Primary cilium and its protein have been hypothesized to be associated with DC. Mutation of a ciliary protein, polycystin‑1 (PC1) is associated with autosomal dominant polycystic kidney disease. This study was performed to assess the immunohistochemical expression of PC1 between DC and postfunctional follicular tissue (PFFT). Materials and Methods: Thirty‑one consecutive PFFT and 15 DC formed the study group. The PFFT and DC tissues were stained with antibody against PC1. Statistical Package for Social Service was used to analyze data. Descriptive statistics and Student’s Chi‑square test were appropriately used. P ≤0.05 was taken as significant. Results: Fifteen DC (100%) and 7 (22.58%) PFFT were positive for PC1. The difference was statistically significant (P = 0.000). PC1 expression was observed in the cytoplasm with varying intensity. Discussion and Conclusion: All PC1 positive epithelial cells’ cytoplasm stained diffusely. Abnormal cytoplasmic expression of PC1 in all positive epithelial lining indicates that the PC1 probably is associated with cystic transformation.

Cyst growth, polycystins, and primary cilia in autosomal dominant polycystic kidney disease

The primary cilium of renal epithelia acts as a transducer of extracellular stimuli. Polycystin (PC)1 is the protein encoded by the PKD1 gene that is responsible for the most common and severe form of autosomal dominant polycystic kidney disease (ADPKD). PC1 forms a complex with PC2 via their respective carboxy-terminal tails. Both proteins are expressed in the primary cilia. Mutations in either gene affect the normal architecture of renal tubules, giving rise to ADPKD. PC1 has been proposed as a receptor that modulates calcium signals via the PC2 channel protein. The effect of PC1 dosage has been described as the rate-limiting modulator of cystic disease. Reduced levels of PC1 or disruption of the balance in PC1/PC2 level can lead to the clinical features of ADPKD, without complete inactivation. Recent data show that ADPKD resulting from inactivation of polycystins can be markedly slowed if structurally intact cilia are also disrupted at the same time. Despite the fact that no single model or mechanism from these has been able to describe exclusively the pathogenesis of cystic kidney disease, these findings suggest the existence of a novel cilia-dependent, cyst-promoting pathway that is normally repressed by polycystin function. The results enable us to rethink our current understanding of genetics and cilia signaling pathways of ADPKD.

Polycystin-1 C terminus cleavage and its relation with polycystin-2, two proteins involved in polycystic kidney disease / Clivaje del C-terminal de policistina-1 y su relación con policistina-2, dos proteínas involucradas en la poliquistosis renal

Autosomal dominant polycystic kidney disease (ADPKD), a most common genetic cause of chronic renal failure, is characterized by the progressive development and enlargement of cysts in kidneys and other organs. The cystogenic process is highly complex and involves a high proliferative rate, increased apoptosis, altered protein sorting, changed secretory characteristics, and disorganization of the extracellular matrix. ADPKD is caused by mutations in the genes encoding polycystin-1 (PC-1) or polycystin-2 (PC-2). PC-1 undergoes multiple cleavages that intervene in several signaling pathways involved in cellular proliferation and differentiation mechanisms. One of these cleavages releases the cytoplasmic C-terminal tail of PC-1. In addition, the C-terminal cytoplasmic tails of PC-1 and PC-2 interact in vitro and in vivo. The purpose of this review is to summarize recent literature that suggests that PC-1 and PC-2 may function through a common signaling pathway necessary for normal tubulogenesis. We hope that a better understanding of PC-1 and PC-2 protein function will lead to progress in diagnosis and treatment for ADPKD.

La poliquistosis renal autosómica dominante (ADPKD por sus siglas en inglés) es una causa genética muy común de falla renal crónica que se caracteriza por el progresivo desarrollo y agrandamiento de quistes en los riñones y en otros órganos. El proceso de cistogénesis comprende incrementos en la proliferación y muerte celular por apoptosis, así como alteraciones en la distribución intracelular de proteínas, el movimiento transcelular de solutos y organización de la matriz extracelular. ADPKD es causada por mutaciones en los genes que codifican para policistina-1 (PC-1) o policistina-2 (PC-2). PC-1 puede sufrir múltiples clivajes y los fragmentos generados intervienen en diferentes cascadas de señalización involucradas en mecanismos de proliferación y diferenciación celular. Uno de estos clivajes libera el extremo C-terminal citoplasmático de la PC-1. Se ha demostrado que los extremos C-terminal citoplasmático de PC-1 y PC-2 pueden interactuar tanto in vitro como in vivo. El propósito de esta revisión es resumir la literatura más reciente que sugiere que PC-1 y PC-2 pueden funcionar a través de una cascada de señalización común necesaria para la tubulogénesis normal. Creemos que una mejor comprensión de los mecanismos moleculares de acción de PC-1 y PC-2 contribuirán al progreso en el diagnóstico y tratamiento de ADPKD.

Polycystin-1 Expression in Fetal, Adult and Autosomal Dominant Polycystic Kidney

The mutation of the PKD1 gene causes autosomal dominant polycystic kidney disease (ADPKD), and the PKD1 gene encodes polycystin-1 (PC-1). PC-1 is thought to be a cell-cell/matrix adhesion receptor molecule at the cell surface that is widely expressed in the kidney. However, there are controversies about the role of PC-1 protein and its expression when using different antibodies to detect it. We used two PC-1 antibodies; C-20 (Santa Cruz, sc-10372) as the C-terminal antibody, and P-15 (Santa Cruz, sc-10307) as the N-terminal antibody. We evaluated the PC-1 expression by performing immunoblotting on the human embryonic kidney (HEK) 293 cells and the renal proximal tubular epithelial cell (RPTEC) lysates. We characterized the expression of PC-1 in the fetal, adult and polycystic kidneys tissues by performing immunohistochemistry. We confirmed the PC-1 expression in the HEK 293 cells and the RPTEC lysates, but the expression was very low. The PC-1 proteins were diffusely expressed in the tubular epithelial cells cytoplasm in the fetal and adult kidneys, and the PC-1 expression was more prominent in the proximal tubules of the fetal kidney. In the ADPKD kidney, the PC-1 proteins were heterogenously and weakly expressed in the tubular or cyst lining epithelial cells. Our data suggests that the development of the kidney may regulate the expression of PC-1, and an altered PC-1 expression may contribute to cyst formation in ADPKD.

Preparation of monoclonal antibody against LRR-WSC domain of polycystin-1 and distribution of polycystin-1 in kidney tissues and kidney cell lines / 中华检验医学杂志

Objective To prepare and identify monoclonal antibody against LRR-WSC domain of polycystin-1 and to investigate the distribution of polycystin-1 in kidney tissues and kidney cell lines. Methods BALB/c mice were immunized with fusion protein PC1-e of polycystin-1 LRR-WSC domain. The splenocytes were fused with myeloma cells by PEG 4000 and the hybridomas were selected in HAT medium. The hybridoma clones secreting antibodies against polycystin-1 LRR-WSC domain were detected by enzyme-linked immunosorbent assay ( ELISA) and cloned by limiting dilution. The specificity of anti-polycystin-1 LRR-WSC domain monoclonal antibody from hybridoma was verified by ELISA and Western blot. The distribution of polycystin-1 in tissues and cells was detected by immunohistochemical method. Results One cell line of hybridoma secreting monoclonal antibody against polycystin-1 was established. Western blot analysis showed that the monoclonal antibody reacted strongly and specifically to polycystin-1 LRR-WSC domain. Distribution of polycystin-1 in fetal kidney was localized in tubular epithelium. In normal adult kidney tissues, our study showed that polycystin-1 was mainly expressed in the medullary collecting ducts and distal convoluted tubules. Positive staining was also found in the majority of cyst-lining epithelial ceEs of cystic tissue from autosomal dominant polycystic kidney disease ( ADPKD) patients. Expressions of polycystin-1 were found in either ADPKD cyst-lining epithelia cell line and LLC-PK1, clearly plasma membrane and intracytoplasmic staining of polycystin-1 were observed. Conclusion Specific monoclonal antibody against polycystin-1 LRR-WSC domain were obtained. The antibody is important to researching the mechanism of ADPKD. The distribution of polycystin-1 in kidney tissues and cells show that polycystin-1 was important in tubular elongation and the maintenance of tubular architecture.

Preparation of monoclonal antibodies against polycystin-1 intracellular region / 第二军医大学学报

Objective: To prepare the monoclonal antibodies against polycystin 1 intracellular region and to study the distribution and expression of polycystin 1 in kidney tissues. Methods: Using the recombinant fusion protein containing polycystin 1 intracellular region as antigen, the hybrid cells secreting the monoclonal antibodies against polycystin 1 intracellular region were established by hybridoma technique. The distribution and expression of polycystin 1 in polycystic kidney, fetal kidney and adult kidney were investigated by immunohistochemical methods(standard EnVision method) with the monoclonal antibodies. Results: Four cell lines of hybrids steadily secreting the monoclonal antibodies against polycystin 1 intracellular region were established. The antibody titers were 1∶10 6. The 50th generation of these cell lines of hybrids still can secret the monoclonal antibodies and the titers remain similar. Polycystin 1 was weakly expressed in tubules and collecting ducts of normal kidney, the strong staining was seen at tubules of fetal kidney, and very strong staining of cyst lining epithelium of polycystic kidney was observed. Conclusion: The monoclonal antibodies against polycystin 1 intracellular region will be a useful tool in the studies of polycystin 1 structure and function.

The expression of extracellular matrix and polycystin-1 in autosomal dominant polycystic kidney disease / 中华肾脏病杂志

Objective To study the expression of extracellular matrix and polycystin-1 in ADPKD and their relation to cyst formation. Methods The expression of polycystin-1, fibronectin, laminin, type Ⅰ collagen, and type Ⅳ collagen were analysed in the normal kidney, fetal kidney and polycystic renal tissue by using immunohistochemical technique. Results The expression Of fibronectin, laminin, type Ⅰ collagen, and type Ⅳ collagen increased in polycystic renal tissue compared with normal kidney. The basement membrane lining cysts was markedly thickened. Type Ⅰ collagen was detected in the interstitium between cysts. Laminin, fibronectin and type Ⅳ collagen were localized in cyst basement membrane. The expression of polycystin-1 increased in polycystic renal tissue. The expression of extracellular matrix had significant correlation with the expression of polycystin-1. Conclusion The abnormal expressions of extracellular matrix and polycystin-1 exist in ADPKD. Abnormal expression of polycystin-1 may result in the alterations of extracellular matrix that is related to cyst formation.