Angiotensin II (Ang II) is an octapeptide with varying effects in different organ systems. In addition to vasoconstrictive effects in the kidney, Ang II greatly influences proximal tubule function, stimulating Na+, HCO3, and fluid reabsorption. Unlike most G-protein-coupled receptors, Ang H receptors have been identified in a polar distribution in proximal tubule epithelium at brush border and basolateral membranes. We have developed a model of proximal tubule epithelium using LLCPKC14 cells, an LLCPK clone without endogenous Ang H receptors. LLCPKC14 cells transfected with a rabbit Ang H receptor transcript express Ang II receptors at apical and basolateral membranes. In this model, Ang H receptors in either domain may display different characteristics, e.g. rates of internalization. Furthermore, apical Ang Il receptors selectively may stimulate a calcium-independent phospholipase A2 (PLA2) in contrast to basolateral Ang II receptors. Yet, the factors influencing these receptor properties as well as the determinants for membrane targeting have not yet been defined. Site-directed mutagenesis will be employed to alter regions in the rabbit Ang H receptor transcript. LLCPKC14 cells transfected with these Ang II receptor mutants will be assayed for apical vs. basolateral Ang II binding, receptor-mediated endocytosis and recycling at either surface, and signal transduction including PLA2 activity, Ang II-mediated tyrosine phosphorylation, and phosphoinositide hydrolysis. Pulse-chase studies, photoaffinity labelling and immunoblotting will be employed to determine what aspects of the Ang H receptor sequence are important for receptor trafficking and Ang II receptor-protein interactions with G-proteins and/or proteins involved in ligand-induced internalization. Finally, cells expressing Ang II receptor mutants will be assayed for Na+ transport to determine the effects of these mutations on an important physiologic process mediated by Ang II in proximal tubule epithelium. It is important to understand factors which affect proximal tubule Ang II receptors given the extent of hypertension and renal disease in our present population. Advancing our knowledge of Ang II and how it affects proximal tubule function may enhance our understanding of kidney diseases affected by Ang II, e.g. hypertensive nephropathy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08DK002420-05
Application #
2900077
Study Section
Special Emphasis Panel (SRC)
Program Officer
Rankin, Tracy L
Project Start
1996-04-15
Project End
2001-03-31
Budget Start
1999-04-01
Budget End
2001-03-31
Support Year
5
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Becker, Bryan N; Cheng, Hui-fang; Hammond, Timothy G et al. (2004) The type 1 angiotensin II receptor tail affects receptor targeting, internalization, and membrane fusion properties. Mol Pharmacol 65:362-9
Becker, B N; Jacobson, L M; Hullett, D A et al. (2002) Type 2 angiotensin II receptor expression in human renal allografts: an association with chronic allograft nephropathy. Clin Nephrol 57:19-26
Becker, B N; Becker, Y T; Pintar, T J et al. (2000) Using renal transplantation to evaluate a simple approach for predicting the impact of end-stage renal disease therapies on patient survival: observed/expected life span. Am J Kidney Dis 35:653-9
Becker, B N; Brazy, P C; Becker, Y T et al. (2000) Simultaneous pancreas-kidney transplantation reduces excess mortality in type 1 diabetic patients with end-stage renal disease. Kidney Int 57:2129-35
Burlingham, W J; O'Connell, P J; Jacobson, L M et al. (2000) Tumor necrosis factor-alpha and tumor growth factor-beta1 genotype: partial association with intragraft gene expression in two cases of long-term peripheral tolerance to a kidney transplant. Transplantation 69:1527-30
Ismail, N; Becker, B; Strzelczyk, P et al. (1999) Renal disease and hypertension in non-insulin-dependent diabetes mellitus. Kidney Int 55:1-28
Becker, B N; Kondo, S; Chen, J K et al. (1999) Tyrosine kinase inhibition affects type 1 angiotensin II receptor internalization. J Recept Signal Transduct Res 19:975-93
Becker, B N; Odorico, J S; Becker, Y T et al. (1999) Peripheral vascular disease and renal transplant artery stenosis: a reappraisal of transplant renovascular disease. Clin Transplant 13:349-55
Becker, B N; Becker, Y T; Heisey, D M et al. (1999) The impact of hypoalbuminemia in kidney-pancreas transplant recipients. Transplantation 68:72-5
Becker, B N; Yasuda, T; Kondo, S et al. (1998) Mechanical stretch/relaxation stimulates a cellular renin-angiotensin system in cultured rat mesangial cells. Exp Nephrol 6:57-66

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