Maintenance of acid/base and electrolyte homeostasis by the kidney and the secretion and reabsorption of ions in the gastrointestinal tract involve the concerted activity of Na/H exchangers (NHEs), Cl/HCO3 exchangers (AEs), H,K-ATPases, and other transporters. Although these transporters are known to operate in coupled systems, there are few examples of such systems that are well understood in terms of the contribution that each transporter makes to the overall process, and the ancillary mechanisms that come into play if the activity of a given transporter is perturbed. This information could be obtained more easily if animal models with defects in specific transporters were available. Thus, in aim 1 we will use embryonic stem (ES) cell/gene targeting technologies to develop mouse models in which the expression of individual transporters is ablated. We have begun experiments in which ES cells containing disruptions of the genes encoding NHE2, NHE3, NHE4, and the colon/renal H,K-ATPase are being prepared and used for blastocyst-mediated transgenesis to develop mouse lines carrying the disrupted genes. Using this approach we have obtained chimeric animals for the H,K-ATPase and NHEs and germline transmission of the mutant H,K-ATPase gene. A similar strategy will be used to prepare mice with an AE1 gene from which the mRNA encoding the kidney variant can no longer be transcribed, and mice with altered AE2 genes in which expression of either the AE2a or AE2b transcripts, but not both, has been ablated. The viability of mutant offspring will be assessed by analysis of genotype frequency, birthweight, survival and growth rate, embryonic development, and tissue histology.
In aim 2 the effects of gene disruption on renal control of acid/base and potassium homeostasis will be studied by monitoring blood gases, pH, HCO3, Na, and K under normal, acidotic, alkalotic, and K depleted conditions. The existence of compensatory changes along the nephron will be assessed morphologically by electron microscopy and functionally by measuring HCO3 flux and transporter activities in isolated tubules. The effects of gene disruption on secretion, absorption, and mucosal protection in the gastrointestinal tract will be studied by analysis of morphological changes and histopathology using both light and electron microscopy, and by analysis of gastrointestinal secretion and ion transport.
In aim 3 will clone and characterize the NHE5 Na/H exchanger and additional isoforms of the H,K-ATPase and AE families for which there is experimental evidence.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK050594-02
Application #
2377824
Study Section
General Medicine B Study Section (GMB)
Project Start
1996-03-01
Project End
2000-02-29
Budget Start
1997-03-01
Budget End
1998-02-28
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Cincinnati
Department
Genetics
Type
Schools of Medicine
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Peña-Münzenmayer, Gaspar; George, Alvin T; Shull, Gary E et al. (2016) Ae4 (Slc4a9) is an electroneutral monovalent cation-dependent Cl-/HCO3- exchanger. J Gen Physiol 147:423-36
Shawki, Ali; Engevik, Melinda A; Kim, Robert S et al. (2016) Intestinal brush-border Na+/H+ exchanger-3 drives H+-coupled iron absorption in the mouse. Am J Physiol Gastrointest Liver Physiol 311:G423-30
Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E (2016) Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine. World J Gastrointest Pathophysiol 7:138-49
Peña-Münzenmayer, Gaspar; Catalán, Marcelo A; Kondo, Yusuke et al. (2015) Ae4 (Slc4a9) Anion Exchanger Drives Cl- Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells. J Biol Chem 290:10677-88
Catalán, Marcelo A; Kondo, Yusuke; Peña-Munzenmayer, Gaspar et al. (2015) A fluid secretion pathway unmasked by acinar-specific Tmem16A gene ablation in the adult mouse salivary gland. Proc Natl Acad Sci U S A 112:2263-8
Prasad, Vikram; Chirra, Shivani; Kohli, Rohit et al. (2014) NHE1 deficiency in liver: implications for non-alcoholic fatty liver disease. Biochem Biophys Res Commun 450:1027-31
Schultheis, Patrick J; Fleming, Sheila M; Clippinger, Amy K et al. (2013) Atp13a2-deficient mice exhibit neuronal ceroid lipofuscinosis, limited ?-synuclein accumulation and age-dependent sensorimotor deficits. Hum Mol Genet 22:2067-82
Prasad, Vikram; Lorenz, John N; Miller, Marian L et al. (2013) Loss of NHE1 activity leads to reduced oxidative stress in heart and mitigates high-fat diet-induced myocardial stress. J Mol Cell Cardiol 65:33-42
Coury, Fabienne; Zenger, Serhan; Stewart, Andrew K et al. (2013) SLC4A2-mediated Cl-/HCO3- exchange activity is essential for calpain-dependent regulation of the actin cytoskeleton in osteoclasts. Proc Natl Acad Sci U S A 110:2163-8
Aihara, Eitaro; Hentz, Courtney L; Korman, Abraham M et al. (2013) In vivo epithelial wound repair requires mobilization of endogenous intracellular and extracellular calcium. J Biol Chem 288:33585-97

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