Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P41181 (collecting duct)
 5,183 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a procedure to detect specific mRNAs in single renal nephron segments. This approach combines microdissection, reverse transcription (RT) of the target mRNA, and amplification of the resulting cDNA using the polymerase chain reaction (PCR). After microdissection, the sample is placed in a tube where it is permeabilized and where all reactions are performed directly without the need for isolation of the RNA. Our model target was the mRNA for aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol. Its expression is modulated by changes in extracellular osmolality in the renal medulla. RT-PCR of inner medullary collecting duct (1 mm) and glomeruli (6-10) yielded a product of the predicted length (670 base pairs) defined by the PCR primers. Its identity was confirmed by a specific oligonucleotide probe that differed from the primers. RT-PCR of proximal tubules (1 mm) resulted in no aldose reductase-specific amplification product. RT-PCR is generally applicable for measuring specific gene expression in single nephron segments or small numbers of cultured cells. Utility, limitations, and refinements of this approach are discussed.
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PMID:Detection of specific mRNAs in single nephron segments by use of the polymerase chain reaction. 211 Jul 78

The renal response to changes in hydration includes variation in intracellular sorbitol, a major inner medullary osmolyte. To examine the mechanism for changes in net sorbitol production, we measured activities of enzymes regulating sorbitol production (aldose reductase) and degradation (sorbitol dehydrogenase) in untreated, water diuretic, and antidiuretic (water restriction and/or vasopressin administration) rats. Collecting duct segments dissected from collagenase-treated kidneys of Sprague-Dawley rats were divided into outer medullary and three distinct inner medullary regions. Aldose reductase activity increased during antidiuresis and decreased during diuresis. In contrast, sorbitol dehydrogenase activity was very low during antidiuresis and increased during diuresis. These changes in enzyme activity were found after 3 days, but not after 1 day, of water diuresis/antidiuresis. Enzyme activity changed only in the deepest 50% of the inner medullary collecting duct. Thus, there is coordinated regulation of aldose reductase and sorbitol dehydrogenase activities so that (a) during water diuresis, aldose reductase activity decreases while sorbitol dehydrogenase activity increases; and (b) during antidiuresis (water restriction and/or vasopressin administration), aldose reductase activity increases while sorbitol dehydrogenase activity remains low. We conclude that long-term osmoregulation in response to physiologic stimuli involves both aldose reductase and sorbitol dehydrogenase activities in rat terminal inner medullary collecting duct segments.
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PMID:Coordinated response of renal medullary enzymes regulating net sorbitol production in diuresis and antidiuresis. 212 8

Intracellular accumulation of sorbitol, generated from D-glucose via the aldose reductase pathway, is thought to play an important role in diabetic complications such as lens cataracts and neuropathy. In order to elucidate the effect of diabetes on the renal inner medulla, another sorbitol-rich tissue, male Wistar rats were treated with a single dose of streptozotocin (60 mg/kg body weight, i.p.). Six weeks later total inner medullary tissue (IM) or isolated inner medullary collecting duct (IMCD) cells were prepared. In diabetic IM tissue, sorbitol content was 1.8-fold higher than in control IM tissue (134 +/- 17 vs. 74 +/- 22 mumol/g tissue protein). Sorbitol production in both normal and diabetic IMCD cells was strongly dependent on extracellular D-glucose concentration. In normal cells, for example, sorbitol production was 90 +/- 9 mumol sorbitol/g protein x h at 45 mM D-glucose compared to 13 +/- 1 mumol/g protein x h at 5 mM. At identical D-glucose concentrations sorbitol synthesis in diabetic IMCD cells was, however, always significantly higher than in control cells (122% of control at 15 mM and 126% of control at 45 mM). In addition, aldose reductase activity in diabetic IM was found to be augmented. The maximal velocity was 4.2 times higher (97 +/- 22 U/g protein vs. 23 +/- 7 U/g protein) while the Km of the enzyme remained unchanged. Membrane permeability for sorbitol or the response to changes in extracellular osmolarity was not significantly different in diabetic IMCD cells and normal cells with correspondingly high intracellular sorbitol concentrations. Similarly the kinetic parameters of D-glucose uptake were not altered by streptozotocin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sorbitol metabolism in inner medullary collecting duct cells of diabetic rats. 252 20

Taking into account recent results obtained with isolated papillary collecting duct cells the metabolic pathways and membrane transport systems of collecting duct cells are reviewed. The plasma membranes contain a luminal proton AT-Pase and a contraluminal Cl-/HCO3- exchanger which are involved in proton secretion; a luminal sodium channel and a contraluminal Na+/K+-AT-Pase for sodium reabsorption; a K+ channel for potassium secretion, and a Na+/K+/Cl- cotransport system for chloride transport and/or volume regulation. The plasma membranes also possess transport systems for organic substrates and organic osmolytes. D-glucose, the main substrate of the papillary collecting duct is taken up into the cell by a sodium-independent D-glucose transport system with a Km of 1.2 mM. The plasma membrane also contains mechanisms which mediate sorbitol release into the medium. This mechanism is stimulated when cells are exposed to media with a low osmolality and inhibited when cells are exposed to media with a high osmolality. D-glucose is used as metabolic substrate in anaerobic and aerobic glycolysis and as precursor for sorbitol synthesis via the aldose reductase, which is highly enriched in papillary collecting duct cells. The cells also show gluconeogenic activity as evidenced by incorporation of labeled carbon from L-alanine into glycerol, sorbitol, and myo-inositol. Accordingly, the cells show fructose-1,6-biphosphatase activity. Sorbitol synthesis in contrast to sorbitol permeability is not affected by osmolarity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Transport mechanisms and metabolic processes in isolated cells of the collecting tubule of the kidney papilla]. 284 46

Renal papillary collecting duct cells have been postulated to adapt their intracellular osmolality to the large changes in interstitial osmolality by changing their content of 'non-perturbing' organic osmolytes such as sorbitol and myo-inositol. 13C-NMR was used in this study to elucidate the metabolic pathways leading to a synthesis of those compounds. Incubation of rabbit renal papillary tissue with [1-13C]glucose showed label scrambling mainly into sorbitol (C-1) and lactate (C-3). This result confirms activity of aldose reductase and glycolytic enzymes in renal papillary cells. Using [3-13C]alanine or [2-13C]pyruvate as carbon source, 13C-labeling of sorbitol and myo-inositol was observed, indicating that renal papillary tissue possesses, in addition, gluconeogenic activity. The latter assumption is supported by the result that in enzyme assays rabbit kidney papilla and isolated rat kidney papillary collecting duct cells show significant fructose-1,6-bisphosphatase activity.
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PMID:Pathways for organic osmolyte synthesis in rabbit renal papillary tissue, a metabolic study using 13C-labeled substrates. 290 59

Sorbitol participates in the osmoregulation of several renal cells and has also been found in isolated inner medullary collecting duct (IMCD) cells in primary culture. Therefore, osmotic regulation and distribution of sorbitol and the key enzymes of sorbitol metabolism, aldose reductase and sorbitol dehydrogenase in the renal inner medulla, were investigated in vivo under various osmotic conditions (control, diuresis, antidiuresis). In homogenates of the renal inner medulla of Wistar rats, the sorbitol content correlated with the urine osmolarity [68 +/- 12 mumol/g protein (control), 28 +/- 9 mumol/g (diuresis), 110 +/- 15 mumol/g (antidiuresis)]. Similar results were obtained for the activity of aldose reductase (sorbitol synthesis) [25 +/- 4 U/g (control), 19 +/- 3 U/g (diuresis), and 48 +/- 7 U/g (antidiuresis)]. On the contrary, the activity of sorbitol dehydrogenase (sorbitol degradation) was significantly increased to 1.26 +/- 0.42 U/g under diuretic conditions vs. control (0.84 +/- 0.14 U/g, P < 0.05). These results demonstrate the correlation between the enzymes of sorbitol synthesis and sorbitol degradation in the intact inner medulla and the urine osmolarity in vivo. Whereas the aldose reductase activity was 2.3-fold enriched in IMCD cells, the specific activity of sorbitol dehydrogenase was relatively increased in a preparation of enriched interstitial cells. This distribution was not dependent on the various diuretic conditions. These results indicate that enzymes of synthesis and of degradation of sorbitol are osmotically regulated in vivo. Therefore, the enzymatic activities of sorbitol synthesis appear to be primarily located in epithelial cells, whereas enzymatic activities of sorbitol degradation seem to be localized in interstitial cells of the renal inner medulla.
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PMID:Renal inner medullary sorbitol metabolism. 750 36

Streptozotocin diabetes induces a 4-fold increase in the maximal velocity of inner medullary aldose reductase as determined in vitro but increases sorbitol synthesis in intact inner medullary collecting duct (IMCD) cells only 1.3-fold. In order to resolve this discrepancy we investigated the importance of intracellular factors in controlling the role of cellular sorbitol synthesis. These factors include glucose concentration, sorbitol concentration, the activity of the NADPH-regenerating pentose phosphate pathway, intracellular NADP and NADPH content, and intracellular reduced (GSH) and oxidized glutathione (GSSG). It was found that the apparent Km of cellular sorbitol production for glucose was identical in control and diabetic rats (56 +/- 18 vs. 59 +/- 14 mmol/l D-glucose), whereas Vmax increased by 31% in diabetes. In inner medullary collecting duct cells of diabetic rats containing 146 +/- 5 mumol sorbitol/g protein, sorbitol synthesis was slightly lower (-15%), compared to cells which had been sorbitol-depleted prior to the experiment (87 +/- 4 mumol sorbitol/g protein). However, no inhibitory effect of sorbitol (up to 200 mmol/l) was observed on aldose reductase activity in vitro. In diabetic rats the content of NADPH was about 32% lower than in the control rats (3.8 +/- 0.3 vs. 5.6 +/- 0.4 mumol/g protein) and the ratio of NADPH/NADP was decreased from 25.6 +/- 5.1 to 8.6 +/- 1.7. In homogenates of the inner medulla the activity of 6-phospho-gluconate dehydrogenase (EC 1.1.1.43) was identical in both experimental groups, so the pentose phosphate shunt seems to be unaltered. GSH content in diabetic rats was also diminished (4.02 +/- 0.67 mumol/g protein vs. 7.41 +/- 0.5 mumol/g protein) and the GSH/GSSG ratio fell from 92.6 to 57.4. In enzyme tests in vitro an apparent Km of 7.3 +/- 1.9 mumol/l of the aldose reductase for NADPH was found; NADP acted as competitive inhibitor with an apparent K(i) of 183 +/- 31 mumol/l. Aldose reductase activity was also found to be strongly inhibited by the SH-group reagent p-chloromercurybenzoesulfonate (apparent K(i) = 0.85 x 10(-6) mol/l). Combining the results obtained on the properties of the aldose reductase in vitro and the observation made in the intact cells, the investigators suggest that the decrease in NADPH/NADP ratio, as well as changes in the redox state in the cells of diabetic animals, can play a significant role in the control of sorbitol synthesis.
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PMID:Control of sorbitol metabolism in renal inner medulla of diabetic rats: regulation by substrate, cosubstrate and products of the aldose reductase reaction. 824 Dec 88

Using 13C-NMR analysis of cell extracts, enzymatic determination of metabolites and cofactors as well as enzyme assays on cell homogenates aerobic and anaerobic glycolysis, sorbitol formation by aldose reductase, the pentose phosphate shunt, and gluconeogenesis could be identified as the major pathways of D-glucose metabolism in renal inner medullary collecting ducts. In flux studies it was shown that D-glucose enters the collecting duct cells via a sodium-independent, cytochalasin- and phloretin-inhibitable transport system located at the basal-lateral cell side. At the same side sorbitol leaves the cells during regulatory volume decrease in a calcium-calmodulin-dependent fashion. From cell isolation studies it is proposed that sorbitol is taken up by adjacent (interstitial) cells, converted into fructose and then recycled to the collecting duct cells. This cycle might prevent carbohydrate wasting. Thus, IMCD cells exhibit unique aspects of carbohydrate biochemistry and physiology which enable them to function in a surrounding of low oxygen tension, low substrate supply, and extreme changes in extracellular osmolality.
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PMID:Biochemistry and physiology of carbohydrates in the renal collecting duct. 939 64

Sorbitol content was determined in porcine urinary bladder epithelial cells immediately after death of the animals and after primary culture of the cells at different osmolalities. In both instances, sorbitol content increased with urine and medium osmolality, respectively. For example, at 300 mosmol/kg the cultured cells contained 0.84 +/- 0.02 nmol/mg protein, at 600 mosmol/kg contained 21.7 +/- 0.95 nmol/mg protein, and at 900 mosmol/kg contained 59.5 +/- 2.8 nmol/mg protein. Similarly, aldose reductase activity rose from 0.27 +/- 0.04 mumol.h-1.mg protein-1 at 300 mosmol/kg to 1.81 +/- 0.16 at 600 mosmol/kg and to 3.02 +/- 0.33 at 900 mosmol/kg. These changes were, however, only observed when NaCl but not when urea was used to augment the medium osmolality, since urea equilibrated across the cell membrane. In contrast, sorbitol release from cells cultured at 900 mosmol/kg was slowest into a 900 mosmol/kg medium and fastest into a 300 mosmol/kg medium (63 +/- 16 nmol/10 min compared with 389 +/- 52 nmol/10 min). These studies demonstrate that the sorbitol content of porcine urinary bladder epithelium is regulated by changes both in sorbitol synthesis and sorbitol release. Thus the regulatory mechanisms in the urinary bladder seem to be similar to those present in the embryological related collecting duct.
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PMID:Regulation of sorbitol content in cultured porcine urinary bladder epithelial cells. 948 29

Sorbitol plays an important role in the osmoregulation of several renal cell types, especially the inner medullary collecting duct (IMCD) cells. Very little information is available concerning the expression of the enzymes of sorbitol metabolism (aldose reductase (AR) and sorbitol dehydrogenase (SDH)) on the RNA level under different osmotic conditions. We employed a RT-PCR-based strategy to investigate the regulation of mRNA coding for AR and SDH. For AR two primers (derived from the sequence of the rat eye lens) were chosen which amplify a 668-bp product. For SDH (considering the sequence of rat liver) three primers were chosen, amplifying a 367- and a 1, 068-bp fragment. Digestion with restriction enzymes and sequencing of the products clearly indicate that the specific mRNA of AR and SDH was amplified. By relative quantitative determination of the amplification products a more than 4-fold increase in mRNA for AR in IMCD cells was observed within 24 h after increasing the extracellular osmolarity from 600 to 900 mosm/l. Decreasing the osmolarity from 600 to 300 mosm/l resulted in a reduction in the mRNA level by 70%. The complete adaptation of the AR activity needed 3 (increasing osmolarity) and 6 days (decreasing osmolarity). Osmotically induced alterations in the levels of mRNA coding for SDH could not be observed. These results suggest that the adaptation of sorbitol synthesis occurs by a rapid regulation of transcription or stability of specific mRNA. For a complete synthesis or degradation of AR 3-6 days are necessary. Thus sorbitol synthesis in IMCD is more rapidly adapted to increasing osmolarities than to decreasing osmolarities.
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PMID:Rat renal expression of mRNA coding for aldose reductase and sorbitol dehydrogenase and its osmotic regulation in inner medullary collecting duct cells. 994 55


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