Researchers have developed a method to design drugs that efficiently block rather than activate critical cell receptors which may help avoid adverse medication outcomes.
Department of Medicine
Breton Laboratory: Sylvie Breton, PhD
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About the Lab
The kidney and the epididymis have a common developmental origin and they, therefore, contain many comparable epithelial cell types. These cells perform similar functions, as both the epididymis and kidney must regulate luminal pH and fluid reabsorption. One of the hallmarks of our laboratory is to examine the functions of epithelial cells while they reside in their native environment. Our research has helped understand how cells work together to sense and modulate their extracellular environment. By comparing and contrasting the kidney and epididymis, we revealed novel signaling pathways that contributed to our better understanding of how specialized proton-secreting cells, the collecting duct intercalated cells and epididymal clear cells, establish and maintain an acidic luminal environment, which is essential for whole body acid/base homeostasis by the kidney, and sperm maturation and storage in the epididymis. In addition, our recent study showed novel functions for intercalated cells as sensors and mediators of sterile inflammation. We also revealed a previously unrecognized role for CFTR, whose mutations cause cystic fibrosis, as a crucial regulator of epithelial tubulogenesis and remodeling.
Renal inflammation and acute kidney injury (AKI)
Uncontrolled inflammation is one of the leading causes of acute kidney injury (AKI). Regardless of the cause of hospitalization, all patients admitted to the intensive care unit (ICU) are at a high risk of developing AKI. On a yearly basis, up to two thirds of the 5.7 million of ICU patients in the USA develop AKI. Serum creatinine, the most utilized AKI marker, becomes elevated only after most of the kidney function is lost. Consequently, the onset of AKI often remains undetected. This underscores the need for a biomarker that can detect kidney injury early, and treatment that can be administered while damage can still be prevented. How diverse organ systems communicate damage to the kidney, leading to AKI, is of great current interest. We showed that in addition to their central role in the regulation and maintenance of acid-base balance via the proton-pumping activity of the vacuolar H+-ATPase (V-ATPase), renal type A intercalated cells (A-ICs) are also sensors that mediate sterile inflammation in the kidney medulla. This resulted from our identification of high expression levels of the pro-inflammatory UDP-glucose receptor P2Y14 in A-ICs. We showed that UDP-glucose is a “danger-signal” molecule that triggers expression of pro-inflammatory chemokines in A-ICs, which attract neutrophils into the renal medulla. We are currently investigating the role of UDP-glucose as a biological mediator that initiates inter- and intra-organ crosstalk, and ultimately induces renal inflammation.
Chronic kidney disease (CKD) associated with cystic fibrosis
As cystic fibrosis (CF) patients receive better treatments that improve their life expectancy, they have now begun to develop progressive malfunction of their kidneys. Consequently, the prevalence of CKD associated with CF is now twice that of the general population. Using the epididymis as a model system, we revealed a previously unrecognized role for CFTR, the protein whose mutations cause CF, as a crucial regulator of epithelial tubulogenesis and remodeling. We showed that CFTR regulates a proliferation/differentiation switch during tubulogenesis. Through its interaction with the tight junction (TJ) protein ZO1, CFTR contributes to the retention of the transcription factor ZONAB outside the nucleus, in TJs, leading to decrease in proliferation and activation of differentiation. We are currently investigating the possibility that disruption of the CFTR/ZO1/ZONAB interaction leads to a reduced capacity of the kidney to re-differentiate after injury. Our recent findings provide a new paradigm for the etiology of diseases associated with CFTR mutations, including cystic fibrosis.
Luminal acidification and its regulation
We have identified a novel pathway for the sensing and regulation of extracellular pH via a physiological link between the bicarbonate activated soluble adenylyl cyclase (sAC) and the vacuolar H+ATPase (V-ATPase). This discovery was made using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage. In the United States, millions of couples are infertile. A significant number of these couples are affected by male fertility defects that are still unexplained. Spermatozoa acquire their ability to become motile and to fertilize an egg in the lumen of the epididymal tubule. Acidic pH and low bicarbonate concentration are involved in maintaining sperm in an immotile, dormant state during their storage period in the epididymis. We showed that proton secretion in a sub-population of epididymal epithelial cells, the clear cells, is regulated via recycling of V-ATPase, a process that is modulated by luminal pH and bicarbonate via changes in sAC activity. Recycling of V-ATPase to and from the apical membrane also occurs in renal type A intercalated cells, where systemic acid/base balance is maintained by pumping protons into the tubule lumen to remove acid from the body. We have identified PKA as a down-stream effector of sAC-dependent elevation of cAMP in both the epididymis and the kidney. We are currently investigating the purinergic regulation of luminal acidification in epididymal clear cells and renal intercalated cells.
Three-dimensional modeling of basal cell function in pseudostratified epithelia
Many organs in the body, including those of the reproductive tract and the lungs, are comprised of a system of tubules lined by epithelial cells. The prevailing view is that so-called “basal cells” in these epithelia are never in contact with the fluid or air-filled cavity (known as the lumen). However, we showed that these cells in fact extend long, slender projections, which we named “axiopodia” that scan the lumen and modulate organ function by communicating their findings to adjacent cells. We found that basal cells act as both sensors that scan the luminal environment, and transmitters that communicate their findings to neighboring cells via the local production of nitric oxide. Using multi-photon in vivo microscopy, we made the surprising discovery that basal cell axiopodia periodically extend and retract over time. We found that axiopodia extensions and retractions follow an oscillatory pattern. This movement, which we referred to as periodic axial motility (PAM), is controlled by c-Src and MEK1/2-ERK1/2. Therapeutic inhibition of tyrosine kinase activity induces a retraction of these projections. Such unexpected cell motility may reflect a novel mechanism by which specialized epithelial cells sample the luminal environment. We have also found basal cell axiopodia in several epithelia, including the trachea, olfactory mucosa, prostate and seminal vesicles, showing that the luminal sampling property of basal cells might be a generalized phenomenon. Our research will promote new diagnostic and therapeutic strategies for diseases including male infertility, chronic obstructive airway disease, asthma and cystic fibrosis.
Azroyan A, Cortez-Retamozo V, Bouley R, Liberman R, Ruan YC, Kiselev E, Jacobson KA, Pittet MJ, Brown D, and Breton S. Renal intercalated cells sense and mediate inflammation via the P2Y14 receptor. PLoS One 10: e0121419, 2015.
Belleannee C, Da Silva N, Shum WW, Brown D, and Breton S. Role of purinergic signaling pathways in V-ATPase recruitment to the apical membrane of acidifying epididymal clear cells. Am J Physiol Cell Physiol 298: C817-830, 2010.
Breton S, and Brown D. Regulation of luminal acidification by the V-ATPase. Physiology (Bethesda) 28: 318-329, 2013.
Breton S, Ruan YC, Park YJ, and Kim B. Regulation of epithelial function, differentiation, and remodeling in the epididymis. Asian J Androl 18: 3-9, 2016.
Breton S, Smith PJ, Lui B, and Brown D. Acidification of the male reproductive tract by a proton pumping (H+)-ATPase. Nat Med 2: 470-472, 1996.
Breton S, Wiederhold T, Marshansky V, Nsumu NN, Ramesh V, and Brown D. The B1 subunit of the H+ATPase is a PDZ domain-binding protein. Colocalization with NHE-RF in renal B-intercalated cells. J Biol Chem 275: 18219-18224, 2000.
Da Silva N, Pisitkun T, Belleannee C, Miller LR, Nelson R, Knepper MA, Brown D, and Breton S. Proteomic analysis of V-ATPase-rich cells harvested from the kidney and epididymis by fluorescence-activated cell sorting. Am. J. Physiol. Cell Physiol. 298: C1326-C1342, 2010.
Da Silva N, Shum WWC, El-Annan J, Păunescu TG, McKee M, Smith PJS, Brown D, and Breton S. Relocalization of the V-ATPase B2 subunit to the apical membrane of epididymal clear cells of mice deficient in the B1 subunit. Am. J. Physiol. Cell Physiol. 293: C199-C210, 2007.
Kim B, and Breton S. The MAPK/ERK-Signaling Pathway Regulates the Expression and Distribution of Tight Junction Proteins in the Mouse Proximal Epididymis. Biol Reprod 94: 22, 2016.
Kim B, Roy J, Shum WW, Da Silva N, and Breton S. Role of testicular luminal factors on basal cell elongation and proliferation in the mouse epididymis. Biol Reprod 92: 9, 2015.
Merkulova M, Păunescu TG, Azroyan A, Marshansky V, Breton S, and Brown D. Mapping the H+(V)-ATPase interactome: identification of proteins involved in trafficking, folding, assembly and phosphorylation. Nature Scientific Reports 5: 14827, 2015.
Miller RL, Zhang P, Smith M, Beaulieu V, Păunescu TG, Brown D, Breton S, and Nelson RD. V-ATPase B1 Subunit Promoter Drives Expression of EGFP in Intercalated Cells of Kidney, Clear Cells of Epididymis and Airway Cells of Lung In Transgenic Mice. Am J Physiol Cell Physiol 288: C1134-C1144, 2005.
Pastor-Soler N, Beaulieu V, Litvin TN, Da Silva N, Chen Y, Brown D, Buck J, Levin LR, and Breton S. Bicarbonate regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling. J Biol Chem 278: 49523-49529, 2003.
Pastor-Soler N, Hallows KR, Smolak C, Gong F, Brown D, and Breton S. Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells. Am J Physiol Cell Physiol 294: C488-C494, 2008.
Păunescu TG, Da Silva N, Russo LM, McKee M, Lu HAJ, Breton S, and Brown D. Association of soluble adenylyl cyclase (sAC) with the V-ATPase in renal epithelial cells. Am. J. Physiol. Renal Physiol. 294: F130-F138, 2008.
Păunescu TG, Ljubojevic M, Russo LM, Winter C, McLaughlin MM, Wagner CA, Breton S, and Brown D. cAMP stimulates apical V-ATPase accumulation, microvillar elongation, and proton extrusion in kidney collecting duct A-intercalated cells. Am J Physiol Renal Physiol 298: F643-F654, 2010.
Păunescu TG, Shum WW, Huynh C, Lechner L, Goetze B, Brown D, and Breton S. High-resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa. Mol Hum Reprod 20: 929-937, 2014.
Roy J, Kim B, Hill E, Visconti P, Krapf D, Vinegoni C, Weissleder R, Brown D, and Breton S. Tyrosine kinase-mediated axial motility of basal cells revealed by intravital imaging. Nat Commun 7: 10666, 2016.
Ruan YC, Shum WW, Belleannee C, Da Silva N, and Breton S. ATP secretion in the male reproductive tract: essential role of CFTR. J Physiol 590: 4209-4222, 2012.
Ruan YC, Wang Y, Da Silva N, Kim B, Diao RY, Hill E, Brown D, Chan HC, and Breton S. CFTR interacts with ZO-1 to regulate tight junction assembly and epithelial differentiation via the ZONAB pathway. J. Cell Sci. 127: 4396-4408, 2014.
Shum WW, Da Silva N, McKee M, Smith PJS, Brown D, and Breton S. Transepithelial projections from basal cells are luminal sensors in pseudostratified epithelia. Cell 135: 1108-1117, 2008.
Tata PR, Mou H, Pardo-Saganta A, Zhao R, Prabhu M, Law BM, Vinarsky V, Cho JL, Breton S, Sahay A, Medoff BD, and Rajagopal J. Dedifferentiation of committed epithelial cells into stem cells in vivo. Nature 503: 218-223, 2013.
Vedovelli L, Rothermel JT, Finberg KE, Wagner CA, Azroyan A, Hill E, Breton S, Brown D, and Păunescu TG. Altered V-ATPase expression in renal intercalated cells isolated from B1-subunit deficient mice by fluorescence activated cell sorting. Am J Physiol Renal Physiol 304: F522-F532, 2013.
Vidarsson H, Westergren R, Heglind M, Blomqvist SR, Breton S, and Enerback S. The forkhead transcription factor Foxi1 is a master regulator of vacuolar H-ATPase proton pump subunits in the inner ear, kidney and epididymis. PLoS ONE 4: e4471, 2009.
Wagner CA, Finberg KE, Breton S, Marshansky V, Brown D, and Geibel JP. Renal Vacuolar H+-ATPase. Physiol. Rev. 84: 1263-1314, 2004.
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About the Nephrology Division
The Division of Nephrology at Massachusetts General Hospital is a leading provider of services for patients with kidney disease, including diagnosis and management of kidney diseases and medical management of renal transplantation.