V. Gustavo Blanco, M.D., Ph.D.
Professor, Kathleen M. Osborn Chair
M.D. - Universidad Nacional de Cordoba, Argentina, 1985
Ph.D. - Instituto M. y M. Ferryra, Cordoba, Argentina, 1990
Renal biology and reproductive biology. Regulation and function of Na,K-ATPase isozymes.
Reproductive biology and renal biology. Function and regulation of Na,K-ATPase isozymes.
Laboratory Research Interest
Our laboratory studies the role of ion-transport proteins of the plasma membrane in cell function. Research is focused on the Na,K-ATPase.
The Na,K-ATPase or Na pump is an enzyme of the plasma membrane, that uses the free energy from the hydrolysis of ATP to transport cytoplasmic Na+ out of the cell in exchange for extracellular K+. The Na,K-ATPase is critical in maintaining cell volume and pH, the membrane excitability of muscle and nervous tissue, and plays a major role in the reabsorption of Na+ and water in the kidney. Na,K-ATPase consists of different isozymes, each resulting from the association of distinct molecular forms of the alpha (alpha 1, alpha 2, alpha 3 and alpha 4) and beta (beta 1, beta 2 and beta 3) subunits that constitute the enzyme. While the alpha subunit is responsible for the catalytic and transport functions of the Na,K-ATPase, the beta polypeptide participates in the correct folding and targeting of the enzyme to the plasma membrane. A third protein, the gamma subunit forms part of the Na,K-ATPase as a regulatory subunit. A schematic representation of the topology of the Na,K-ATPase subunits in the cell plasma membrane is shown below.
Studies in our laboratory are directed to understand the structure function of the Na,K-ATPase and the role of its various isozymes in cell physiology. A variety of molecular, cell biology and genetic methods are used to study these important enzymes, both in their native environment and after expression in mammalian and insect cells in culture. We have determined that different isoforms of the Na,K-ATPase have unique enzymatic properties, which suggested that Na,K-ATPase isoforms are not redundant, but rather have a specific role. While the ubiquitously expressed alpha 1 and beta 1 isoforms function as the housekeeping Na,K-ATPase in the cell, the other isozymes mediate tissue-specific roles.
These studies are important to understand how cells control water and ion balance to meet their needs under normal physiological situations and during disease, where changes in Na pump isoform expression becomes altered. At present, we have focused our interest in understanding the function of the Na,K-ATPase alpha4 isoform. We have characterized the expression and activity of alpha4 during male germ cell differentiation. Our results show that alpha4 it is only expressed in male germ cells of the testis after meiosis and that it is abundant in the sperm flagellum. The immunocytochemical photographs below show heterologous expression of Na,K-ATPase alpha4 in Sf9 insect cells using baculovirus and the presence and localization of this isoform in the native environment of the rat testes seminiferous tubules and spermatozoa.
Most recently, we have approached the study of Na,K-ATPase alpha4 by generating a series of mouse models, in which alpha4 was knocked-out or over-expressed. Sperm from mice deficient in Na,K-ATPase alpha4 have a severe reduction in sperm motility and are completely infertile both in vitro and in vivo matting assays. These results show that Na,K-ATPase alpha4 plays a critical role in male reproduction and fertility. Experiments are focused to continue elucidating the mechanisms of action and regulation of Na,K-ATPase alpha4 in sperm function and its relevance to male fertility.
Another project in the laboratory is directed to understand the role of the Na,K-ATPase in autosomal dominant polycystic kidney disease (ADPKD). ADPKD is a pathology characterized by the progressive enlargement of fluid filled cysts in the kidneys that compromise the function of the organ. The morphology of normal and cystic kidneys from a rat model of polycystic kidney disease is shown in the following figure.
We are studying the function of the Na,K-ATPase alpha1-beta1 isozyme, present in kidney, in animal models of ADPKD and in renal epithelial cells from kidneys of patients with ADPKD. In the disease, we are exploring both the enzymatic properties of the Na,K-ATPase and the function of the enzyme as a receptor and signal transducer for ouabain. Ouabain is a hormone released by the adrenal glands and normally circulating in the bloodstream of several mammals, including men.
We have found that the Na,K-ATPase of ADPKD cells exhibit an abnormally increased sensitivity to ouabain. Physiological concentrations of ouabain stimulate cystogenesis of ADPKD cells, by both accelerating the rate of growth of the cells and their capacity to secrete fluid across the apical membrane. We have characterized the pathways leading to the effects of ouabain in ADPKD cells. A scheme of these pathways is shown in the figure below. These results place ouabain as a factor capable to exacerbate ADPKD progression. Currently, we are further investigating the mechanisms by which ouabain affects ADPKD cyst formation and development.
Nguyen AN, Wallace DP, Blanco G. (2007). Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal-Regulated Kinase Activation and Cell Proliferation. J Am Soc Nephrol. 18:46-57.
Jimenez T, Sanchez G., Wertheimer E. and Blanco G (2010). Activity of the Na,K-ATPase alpha4 isoform is important for membrane potential, intracellular Ca2+, and pH to maintain motility in rat spermatozoa. Reproduction
Jimenez T, Sanchez G, McDermott JP, Nguyen AN, Kumar TR, Blanco G. (2010). Increased Expression of the Na,K-ATPase alpha4 Isoform Enhances Sperm Motility in Transgenic Mice. Biol Reprod
Jimenez T, McDermott JP, Sánchez G, Blanco G. Na,K-ATPase alpha4 isoform is essential for sperm fertility. (2010). Proc Natl Acad Sci U S A. 108(2):644-9.
Nguyen AN, Jansson K, Sanchez G, Sharma M, Reif GA, Wallace DP, Blanco G. (2011). Ouabain activates the Na,K-ATPase signalosome to induce autosomal dominant polycystic kidney disease cell proliferation. Am J Physiol Renal Physiol. 301(4): F897-906.
Jimenez T, Sánchez G and Blanco G. Activity of the Na,K-ATPase alpha4 isoform is regulated during sperm capacitation to support sperm motility. (2012) J. Androl. 33(5):1047-57.
McDermott JP, Sánchez G, Chennathukuzhi V and Blanco G. Green fluorescence protein driven by the Na,K-ATPase alpha4 isoform promoter is expressed only in male germ cells of mouse testis. (2012). J. Assisted Reprod. Genet. 29(12):1313-25.
Jansson K, Nguyen AN, Magenheimer BS, Reif GA, Aramadhaka LR, Bello-Reuss E, Wallace DP, Calvet JP, Blanco G. Endogenous concentrations of ouabain act as a cofactor to stimulate fluid secretion and cyst growth of in vitro ADPKD models via cAMP and EGFR-Src-MEK pathways. (2012). Am J Physiol Renal Physiol. 303:F982-90.
Jansson K, Magenheimer B, Calvet J and Blanco G. Over-expression of the polycystin-1 (PC1) C-tail enhances the sensitivity of M-1 cells to the effects of ouabain (2013). J. Membr. Biol. 246(7):581-90.
Blanco G. and Wallace D. Novel role of ouabain as a cystogenic factor in autosomal dominant polycystic kidney disease. Am. J. Physiol. Renal Physiol. (2013) 305(6):F797-812.