Defined nephron segments were microdissected from the kidney of vitamin D-deficient rats, normal rats, and normal rats treated with la,25-dihydroxyvitamin D3 [la,25-(OH)2D3]. Tubule segments were incubated with 3H-labeled 25-hydroxyvitamin D3 and the rates of production of 3H-labeled la,25-(QH) 2D3 and 24,25-dihydroxyvitamin D3 [24, The use of defined nephron segments may be useful for stucly of the distribution and regulation of 25-hydroxyvitamin D3 hydroxylases in the kidney.It is well established that vitamin D3 must undergo sequential hydroxylations, first in the liver to 25-hydroxyvitamin D3 (25-OH-D3) and then in the kidney to either la,25-dihydroxyvitamin D3 [1a,25-(OH)2D3] or 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] before it exerts its biological actions (1). Under normal physiological conditions, the kidney is believed to be the principal site of synthesis of 1a,25-(OH)2D3 which is thought to be the primary hormonal form of vitamin D3 (1). The kidney seems also to be the major site of 24,25-(OH)2D3 production because plasma levels of 24,25-(OH)2D3 are markedly decreased in anephric patients and rats (2, 3). The activities of 25-(OH)D3 la-and 24-hydroxylase, the enzymes responsible for the production of la,25-(OH)2D3 and 24,25-(OH)2D3, respectively, are finely regulated by various ions and hormones, including calcium, phosphate, parathyroid hormone, and vitamin D; in general, there are reciprocal changes in the activities of these two enzymes (1).Recent studies have demonstrated the exclusive localization of la-hydroxylase in the proximal tubules in chicken and fetal rabbit kidneys (4, 5). However, the precise location of la-hydroxylase in the mature mammalian kidney is not known. Furthermore, the location of 24-hydroxylase in the kidney has not been studied. Determination of the precise location of both cahydroxylase and 24-hydroxylase in the kidney may provide fur, ther insight into the regulatory mechanisms of these two hydroxylase systems.Using single nephron segments microdissected from rats, we found that both la-hydroxylase and 24-hydroxylase are located only in the proximal convoluted tubules (PCT) of vitamin Ddeficient and normal rats, respectively, and that 24-hydroxylase is present in both the PCT and proximal straight tubules (PST) in normal rats treated with la,25-(OH)2D3.MATERIALS AND METHODS Animals. To create vitamin D deficiency, male Holtzman rats were fed a vitamin D-deficient diet, containing 0.45% calcium and 0.3% phosphorus, for 7-10 weeks after weaning (6). The mean (±SEM) plasma concentrations of calcium and inorganic phosphate in vitamin D-deficient rats at the time of study were 4.4 ± 0.5 and 4.8 ± 0.5 mg/100 ml, respectively. Normal male rats (Holtzman or Wistar) weighing 200-300 g were fed a laboratory rat chow; some were given daily subcutaneous injections of la,25-(OH)2D3 (25 ng/kg) for 3-10 days. Mean (±SEM) plasma concentrations of calcium and inorganic phosphate in normal rats were 9.9 ± 0.1 and 6.7 ± 0.3 mg/100 ml, respectively; in rats treated with la,25-(O...

To further evaluate the interaction between vasopressin (AVP) and prostaglandin E2 (PGE2) in the kidney, the effects of AVP and PGE2 on cell cAMP content were examined in the isolated thick ascending limb of Henle (TAL) and the cortical collecting tubule (CCT) of rat kidney. Nephron segments were incubated in the presence of phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), with 10 nM AVP and varying concentrations of PGE2 at 37 degrees C for 1-7 min, and the cAMP content was determined by radioimmunoassay. PGE2 suppressed the AVP-stimulated increase in cell cAMP in both medullary (MTAL) and cortical (CTAL) portions of the TAL in a dose-dependent manner. This inhibitory effect was evident at 0.28 nM PGE2 and maximum at 2.8-28 microM PGE2. By contrast, in the presence of MIX PGE2 did not inhibit AVP-stimulated cAMP increases in the CCT. However, in the absence of MIX, PGE2 suppressed cAMP accumulation in the CCT. These data suggest that PGE2 may suppress cell cAMP by inhibiting AVP-dependent cAMP formation in the TAL; PGE2 may suppress cAMP in the CCT by acting at a site(s) affected by MIX and not by inhibiting cAMP formation. The results show that although PGE2 may inhibit AVP-dependent cell cAMP accumulation in both TAL and CCT, the underlying cellular mechanisms may be different in these two distinct AVP-sensitive nephron segments.

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Substrate oxidation was assessed by measuring 14CO2 production from 14C-labeled substrates in proximal convoluted tubules (PCT), medullary (MTAL), and cortical (CTAL) thick ascending limb of Henle, nephron segments rich in mitochondria and characterized by active solute transport. PCT, MTAL, and CTAL were dissected from the outer cortex, outer medulla, and the medullary rays of the cortex, respectively, of collagenase-treated rat kidney slices. Tubules were incubated at 37 degrees C in 150 microliters of Krebs-Ringer-bicarbonate buffer (pH, 7.4) with 14C-labeled substrate. 14CO2 production was linear up to 4 and 2 hours in PCT and MTAL, respectively. Freeze-thawing of the tubules markedly decreased 14CO2 production, and the addition of cyanide completely abolished it. The PCT demonstrated marked 14CO2 production from labeled succinate, 2-oxoglutarate, glutamate, glutamine, and malate (approximately 10 to 45 pmoles/mm/hr) and moderate 14CO/ production from citrate (approximately 3 pmoles/ml/hr). Little 14CO2 was released from labeled glucose and lactate in PCT. These results are consistent with the existence of gluconeogenesis in this nephron segment. By contrast, MTAL and CTAL oxidized glucose, 2-oxoglutarate, lactate, glutamate, and glutamine, but not malate, succinate, and citrate. The pentose shunt pathway accounted for approximately half of the 14CO2 produced from 1-14C glucose in MTAL and CTAL. Palmitate oxidation occurred in MTAL and CTAL but minimally in PCT. The results demonstrate a distinct pattern of substrate oxidation in PCT, MTAL, and CTAL where oxidative metabolism is critical to support active solute transport.