Intercalated cells (IC) respond to pH changes in the blood by increasing or decreasing acid secretion in the kidney collecting duct. Dysfunction of this process results in pathophysiological disorders of different organ systems as the pH of the blood drifts away from its normal value of 7.4. The vacuolar H[+]ATPase (V-ATPase) is central to the acid/base homeostatic function of IC, but how these cells detect environmental cues that allows them to modify proton secretion appropriately remains a mystery. The existence of an acid or bicarbonate sensor in the kidney has long been suggested, but the identity of this detection system and how such a system would transmit signals to modify the acid/base transporting machinery of ICs remain to be determined. Based on work carried out in the previous funding period, we propose here that the soluble adenylate cyclase (sAC) is the much sought after renal acid/base sensor. This protein generates the second messenger cAMP upon direct stimulation by bicarbonate ions. It is, therefore, ideally suited for a bicarbonate/CO{2} sensing role in IC. We hypothesize that cAMP generated by the sAC sensor in response to acid/base cues can modify the acid secretory capacity of intercalated cells. We propose that V-ATPase and sAC are partners in a localized signaling process that modulates targeting and trafficking of the V-ATPase in specific membrane microdomains to regulate intercalated cell function, and renal proton secretion.
Our aims are: 1) To characterize the role of sAC in the regulation of V-ATPase mediated proton secretion by renal epithelial cells and 2) To determine whether V- ATPase and cytoskeletal proteins (actin, gelsolin, drebrin, nadrin and myosin VI) form a local micro-complex that regulates V-ATPase membrane accumulation and proton secretion in IC. The studies will use a multidisciplinary approach including unique animal models, isolated fluorescence-sorted intercalated cells, and cell cultures, as well as imaging technologies including static and real-time confocal microscopy to follow V- ATPase trafficking. Assays of vesicle acidification, ATPase activity and proton-selective self-referencing microelectrodes will monitor the functional expression of V-ATPase in endosomes and at the plasma membrane. Fluorescence (Forsman) resonance energy transfer (FRET) and protein-protein interaction assays will dissect whether V-ATPase subunits interact with sAC and/or cytoskeletal proteins during stimulation of proton secretion. In vitro assays, mutational analysis and phosphoproteomics will address the role of cAMP/PKA mediated V-ATPase phosphorylation in these interactions. We propose that the V-ATPase is a central partner in a localized, multi-protein complex that senses and responds to prevailing acid/base conditions by modulating the V-ATPase dependent acidification mechanism in intercalated cells.

Public Health Relevance

Maintaining the acid/base (i.e., pH) level of body fluids, including the blood, within a narrow range is critical to normal health and to the function of all cells and organ systems. The kidney plays a central role in this process by sensing and eliminating excess acid or excess base via excretion into the urine. Currently, the sensing mechanism by which the kidney detects and maintains an appropriate systemic pH balance is poorly understood. The work described here is aimed at proving that a protein called the soluble adenylate cyclase can act as this elusive sensor, and that it signals another protein called a proton pump to remove acid from the body. This work, therefore, sets out to understand the mechanisms underlying a basic physiological function that is necessary for survival. We hope to identify new protein targets for the development of new therapies and strategies to correct acid base imbalances (known as acidosis or alkalosis) in the body.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK042956-20
Application #
8250029
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Mullins, Christopher V
Project Start
1991-08-01
Project End
2013-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
20
Fiscal Year
2012
Total Cost
$366,619
Indirect Cost
$158,348
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Inoue, Yoshitaka; Yu, Yong-Ming; Kurihara, Tomohiro et al. (2016) Kidney and Liver Injuries After Major Burns in Rats Are Prevented by Resolvin D2. Crit Care Med 44:e241-52
Trepiccione, Francesco; Gerber, Simon D; Grahammer, Florian et al. (2016) Renal Atp6ap2/(Pro)renin Receptor Is Required for Normal Vacuolar H+-ATPase Function but Not for the Renin-Angiotensin System. J Am Soc Nephrol 27:3320-3330
Azroyan, Anie; Cortez-Retamozo, Virna; Bouley, Richard et al. (2015) Renal intercalated cells sense and mediate inflammation via the P2Y14 receptor. PLoS One 10:e0121419
Merkulova, Maria; Păunescu, Teodor G; Azroyan, Anie et al. (2015) Mapping the H(+) (V)-ATPase interactome: identification of proteins involved in trafficking, folding, assembly and phosphorylation. Sci Rep 5:14827
Păunescu, Teodor G; Shum, Winnie W C; Huynh, Chuong et al. (2014) High-resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa. Mol Hum Reprod 20:929-37
Roy, Jeremy W; Hill, Eric; Ruan, Ye Chun et al. (2013) Circulating aldosterone induces the apical accumulation of the proton pumping V-ATPase and increases proton secretion in clear cells in the caput epididymis. Am J Physiol Cell Physiol 305:C436-46
Breton, Sylvie; Brown, Dennis (2013) Regulation of luminal acidification by the V-ATPase. Physiology (Bethesda) 28:318-29
Rice, William L; Van Hoek, Alfred N; Păunescu, Teodor G et al. (2013) High resolution helium ion scanning microscopy of the rat kidney. PLoS One 8:e57051
Vedovelli, Luca; Rothermel, John T; Finberg, Karin E et al. (2013) 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:F522-32
Nunes, Paula; Ernandez, Thomas; Roth, Isabelle et al. (2013) Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters. Autophagy 9:550-67

Showing the most recent 10 out of 33 publications