The medullary collecting duet (MCD) is the final arbiter of renal excretion of sodium chloride, potassium, urea, and hydrogen ions. Studies of whole medulla have shown that ion transport is coupled to both aerobic and anaerobic glycolysis but little else is known concerning the metabolism of either H+ secreting outer MCD cells (OMCD) or Na+ absorbing inner MCD (IMCD) cells. Recently we have isolated both OMCD and IMCD cells from the rabbit in quantities sufficient for routine biochemical analysis. These cells share morphologic, biochemical, and transport properties of MCD cells in situ. In addition, we have shown MCD cells have high rates of anaerobic glycolysis, that they depend exclusively on exogenous substrate for aerobic metabolism, and that this rate of energy generation is tightly coupled to transport. The proposed studies will examine in detail glucose metabolism in IMCD and OMCD cells and how metabolism is coupled to transport. Initially, experiments will measure the rates of glucose uptake, aerobic and anerobic glycolysis, as well as intracellular ATP/ADP levels, and ion transport activity as a function of external glucose concentration. This will establish the significance of variations in extracellular glucose concentration on collecting duct function and will provide new insights into the interrelationships of glucose uptake, metabolism, and ion transport in MCD cells. Secondly, we will measure steady-state glucose utilization, lactate generation, and oxygen consumption under conditions of stimulated and inhibited transport in MCD cells. These studies will determine the portion of cellular energy generation which supports transport under a variety of conditions, and will reveal how the cells augment and diminish energy production to meet changing demands of ion transport. Finally, we will measure incorporation of radiolabel from glucose carbons into selected intermediates and products of metabolism and analyze the data using a computer modeling technique, thus quantifying substrate flux through each component step of the cellular metabolic pathways under different ion transport conditions. This sophisticated analytical approach will be novel for renal research and will enable us to identify the sites and mechanisms of control of metabolism. MCD cell suspensions represent an ideal system for studying the coupling of transport and intermediary metabolism and the techniques proposed have all been used successfully by the authors in cell suspension studies.

Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
Greenfeld, Z; Stillman, I E; Brezis, M et al. (1997) Medullary injury in the ageing rat kidney: functional-morphometric correlations. Eur J Clin Invest 27:346-51
Stillman, I E; Brezis, M; Heyman, S N et al. (1994) Effects of salt depletion on the kidney: changes in medullary oxygenation and thick ascending limb size. J Am Soc Nephrol 4:1538-45
Brugnara, C; De Franceschi, L; Alper, S L (1993) Ca(2+)-activated K+ transport in erythrocytes. Comparison of binding and transport inhibition by scorpion toxins. J Biol Chem 268:8760-8
Heyman, S N; Stillman, I E; Brezis, M et al. (1993) Chronic amphotericin nephropathy: morphometric, electron microscopic, and functional studies. J Am Soc Nephrol 4:69-80
Heyman, S N; Brezis, M; Epstein, F H et al. (1992) Effect of glycine and hypertrophy on renal outer medullary hypoxic injury in ischemia reflow and contrast nephropathy. Am J Kidney Dis 19:578-86
Pacheco-Silva, A; Bastos, M G; Muggia, R A et al. (1992) Interleukin 2 receptor targeted fusion toxin (DAB486-IL-2) treatment blocks diabetogenic autoimmunity in non-obese diabetic mice. Eur J Immunol 22:697-702
Rauchman, M I; Wasserman, J C; Cohen, D M et al. (1992) Expression of GLUT-2 cDNA in human B lymphocytes: analysis of glucose transport using flow cytometry. Biochim Biophys Acta 1111:231-8
Heyman, S; Spokes, K; Rosen, S et al. (1992) Mechanism of glycine protection in hypoxic injury: analogies with glycine receptor. Kidney Int 42:41-5
Rosen, S; Epstein, F H; Brezis, M (1992) Determinants of intrarenal oxygenation: factors in acute renal failure. Ren Fail 14:321-5
Hallaq, H; Smith, T W; Leaf, A (1992) Modulation of dihydropyridine-sensitive calcium channels in heart cells by fish oil fatty acids. Proc Natl Acad Sci U S A 89:1760-4

Showing the most recent 10 out of 53 publications