Chronic metabolic acidosis (CMA) may occur in chronic renal insufficiency or in inherited or acquired renal tubular hydrogen or bicarbonate transport defects. CMA affects mineral homeostasis, which leads to decreased intestinal calcium absorption and negative calcium balance resulting in statural growth delay in children and rickets or osteomalacia. The mechanisms of disordered mineral metabolism in CMA, in part, alter 1,25-dihydroxyvitamin D (1,25(OH)2D3) metabolism. 1,25(OH)2D3 is produced in proximal tubule mitochondria by a monooxygenase enzyme similar to that observed in adrenal steroidogenesis. 1,25(OH)2D3 production and circulating levels are increased in response to low calcium diet (LCD);CMA reduces the expected increase in 1,25(OH)2D3 production and circulating levels during LCD. Reduced 1,25(OH)2D3 production in CMA may, in part, be caused by altered PTH-adenylate cyclase-cAMP activity, or by increased tubule Ca++. The specific interaction of PTH with its receptor in the proximal tubule will be tested by measuring cAMP production in response to varying doses of PTH added to proximal tubule membranes prepared from rats fed LCD+CMA. A more generalized defect in hormone receptor interaction in CMA will be tested by incubation of CMA tubules with L-epinephrine and measuring resultant 1,25(OH)2D3 and cAMP production. Intrinsic adenylate cyclase activity will be measured in response to forskolin, an agent which stimulates the enzyme without interaction at the hormone receptor site, and subsequent 1,25(OH)2D3 production measured. cAMP accumulation will also be measured in response to in vitro incubations in Ca++ free medium. Increased tubule Ca++ may be distributed to the cytosol or within cellular organelles, including mitochondria. The possibility that cytosol Ca++ is elevated in CMA will be tested by lowering proximal tubule cytosol Ca++ by agents that block Ca++ influx and increase Ca++ efflux when extracellular Ca++ is low, and measuring resultant 1,25(OH)2D3 production. Conversely, proximal tubule cytosol Ca++ will be lowered by agents that increase cell Ca++ efflux when extracellular Ca++ is high, and measuring subsequent 1,25(OH)2D3 production. Elevated tubule cell Ca++ may be distributed to the mitochondria in CMA. 1,25(OH)2D3 production by mitochondria as a function of Ca++ efflux in response to extramitochondrial Ca++ will be measured. In addition, 1,25(OH)2D3 production resulting from intramitochondrial C++ movement will be measured in response to setting extramitochondrial Ca++ to equal levels for LCD and LCD+CMA mitochondria, and mitochondrial Ca++ influx either prevented by ruthenium red, or allowed to occur.
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