Activation of protein kinase C modulates dihydroxycholecalciferol synthesis in rat renal tubules.

1,25(OH)2D3, the biologically active metabolite of vitamin D, is produced from 25(OH)D3 by the renal mitochondrial 25(OH)D3 1 alpha-hydroxylase. Several studies have implicated reversible phosphorylation and a possible role for protein kinase C (PKC) in acute regulation of 1,25(OH)2D3 production. In the experiments described here, we studied 1,25(OH)2D3 production in freshly isolated rat renal tubules treated with activators and inhibitors of PKC. In this mammalian system, TPA, but not its inactive analogue 4 alpha PDD, inhibited 1,25(OH)2D3 production in a dose-dependent fashion within 20 min. The acute inhibition of 1,25(OH)2D3 production by TPA exposure was preceded by an increase in membrane associated PKC activity, which was paralleled by a decrease in cytosolic PKC activity. Pre-incubation of tubules with staurosporine, a PKC inhibitor, abolished the inhibitory effect of TPA on 1,25(OH)2D3 production. Chronic (18 h) exposure of tubules to high dose TPA resulted in down regulation of both membrane and cytosolic PKC activity and re-exposure to TPA did not affect PKC translocation or 1,25(OH)2D3 production in down regulated tubules. Our data strongly suggest that modulation of renal PKC activity may be an important mechanism for acute regulation of 1,25(OH)2D3 production.     

Metabolism of sulfolipids in isolated renal tubules from rat

Proximal-rich tubules were prepared from rat kidneys by using collagenase treatment. The isolated rat renal tubules were compared with the intact kidney on the following characteristics. (1) Composition of the sulfoglycolipid. (2) Sulfoglycolipid metabolism based on incorporation of [35S]sulfate or some properties of sulfoglycolipid metabolism, including the activities of anabolic and catabolic enzymes. The results indicated following characteristics of the isolated renal tubules in comparison to the kidney in vivo. (1) The sulfoglycolipid compositions are qualitatively similar, except that the content of glucosyl sulfatide, Gg3Cer II3-sulfate, and GM4 was slightly higher in the isolated tubules. (2) The apparent half-lives (15-55 min) of sulfoglycolipids in the isolated tubules could indicate the existence of a rapid turnover pool of these lipids. (3) The sulfotransferase and sulfatase activities related to sulfoamphiphiles in the renal tubule were similar to those reported for the whole kidney. Based on the above criteria, we conclude that the isolated rat renal tubule should be a useful metabolic system for clarification of the short-term physiological events, up to 90 min, of proximal tubular sulfoglycolipids. By using the present system, we showed that biosynthesis of the renal total sulfoglycolipid was significantly elevated in rats deprived of water for 24 h.     

Carbon flux through tricarboxylic acid cycle in rat renal tubules

Our aim was to delineate the effect(s) of chronic metabolic acidosis on renal TCA-cycle metabolism. Renal tubules isolated from control and chronically acidotic rats were incubated at pH 7.4 with either 2 mM [2,3-13C]pyruvate or [2-13C]acetate. GC-MS and/or 13C-NMR were utilized to monitor the flux of 13C through pyruvate dehydrogenase, pyruvate carboxylase and the TCA-cycle. With either, precursor acidosis was associated with significantly decreased formation of 13C-labelled citrate, malate, aspartate and alanine and increased formation of glucose, lactate and acetyl-CoA as compared with the control. The results indicate that adaptation of renal metabolism to chronic metabolic acidosis is associated with diminished flux through citrate synthetase and concomitantly increased flux through pyruvate carboxylase. The data suggest that depletion of TCA-cycle intermediates and enhanced ammoniagenesis in the kidney of chronically acidotic rats may be regulated at the site of mitochondrial citrate-condensing enzyme.     

Developmental pattern of cystine transport in isolated rat renal tubules

Isolated renal cortical tubule fragments from rats ranging in age from less than 48 h to 15 weeks were used to examine the pattern of cystine uptake with development. Immature tubules took up cystine with a faster initial rate than mature tubules and did not reach a steady state by 60 min. By eight weeks of age, the timed uptake of cystine began to approach a steady state and between 8 and 11 weeks the uptake pattern achieved its adult form of reaching a steady state by 30 min of incubation. Analysis of the intracellular metabolism of the cystine taken up by the newborn tubules revealed that the majority had been reduced to cysteine with the formation of small amounts of reduced glutathione. Cystine entered the renal cortical tubule cell from the newborn via two saturable transport systems similar to the mature animal. The kinetic parameters of initial uptake of these two transport systems were similar in the mature and newborn animal except for a higher maximum transport velocity for the low K_m, low capacity system in the newborn. Lysine inhibited cystine uptake by newborn tubules and this inhibition appeared to occur on the low K_m, low capacity transport system similar to the adult. Cystine uptake was sodium dependent with an apparent affinity for sodium of 36 mequiv./l. From this data, the physiologic cystinuria of the immature animal does not appear to be referrable to a lower rate of influx as previously observed with the cortical slice. Other mechanisms should be sought to explain this phenomenon of immaturity.     

5-methyltetrahydrofolate is bound in intersubunit areas of rat liver folate-binding protein glycine N-methyltransferase

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. It is abundant in the liver, where it uses excess S-adenosylmethionine (AdoMet) to methylate glycine to N-methylglycine (sarcosine) and produces S-adenosylhomocysteine (AdoHcy), thereby controlling the methylating potential of the cell. GNMT also links utilization of preformed methyl groups, in the form of methionine, to their de novo synthesis, because it is inhibited by a specific form of folate, 5-methyltetrahydrofolate. Although the structure of the enzyme has been elucidated by x-ray crystallography of the apoenzyme and in the presence of the substrate, the location of the folate inhibitor in the tetrameric structure has not been identified. We report here for the first time the crystal structure of rat GNMT complexed with 5-methyltetrahydrofolate. In the GNMT-folate complex, two folate binding sites were located in the intersubunit areas of the tetramer. Each folate binding site is formed primarily by two 1-7 N-terminal regions of one pair of subunits and two 205-218 regions of the other pair of subunits. Both the pteridine and p-aminobenzoyl rings are located in the hydrophobic cavities formed by Tyr5, Leu207, and Met215 residues of all subunits. Binding experiments in solution also confirm that one GNMT tetramer binds two folate molecules. For the enzymatic reaction to take place, the N-terminal fragments of GNMT must have a significant degree of conformational freedom to provide access to the active sites. The presence of the folate in this position provides a mechanism for its inhibition.     

Acute and chronic effect of dietary phosphorus restriction on protein expression in young rat renal proximal tubules

Renal proximal tubules play a vital role in phosphorus (P) homeostasis. It is well known that dietary P restriction up-regulates the activities of 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1-OHase), an enzyme that is involved in activation of vitamin D and thereby maintaining P balance. However, the mechanism involved in such regulation is not known. In the present study, we aim to identify proteins that might be involved in the renal adaptation to dietary P restriction using a proteomic approach. Renal proximal tubules were harvested from young rats fed either normal P diet or low P diet (LPD) for 1 to 7 days. Western blotting analysis of 1-OHase and signaling proteins in insulin-like growth factor I axis indicated an increase in expression of these proteins upon dietary P restriction. Using two-dimensional electrophoresis, we found that LPD reduced the total number of protein species expressed in renal proximal tubules. Differentially expressed proteins were analyzed and located using the software Melanie III, and their identities were found using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Our results showed that beta-actin, gamma-actin, major urinary protein, phosphatidylinositol transfer protein beta isoform, and G1/S-specific cyclin D3 are up-regulated and nonspecific lipid transfer protein is down-regulated by LPD.     

Influence of folate-binding protein from bovine milk on the absorption of folate in gastrointestinal tract of rat

The absorption of [¹⁴C]pteroylglutamic acid (PteGlu) bound to the folate-binding protein of bovine milk was investigated in rat gastrointestinal tract in vivo and in situ. When bound [¹⁴C]PteGlu was given to rat intragastrically via oral intubation, a considerable amount of PteGlu was released from folate-binding protein under the acidic conditions in stomach, and it recombined with folate-binding protein in jejunum in vivo. Compared with free PteGlu, bound PteGlu was more gradually absorved in small intestine, but finally the total amount of bound PteGlu absorved was almost the same as that of free PteGlu. In all experiments in situ, bound PteGlu was only slightly absorbed in jejunum, where free PteGlu was rapidly absorved under the same conditions. On the other hand, the absorption rates of the two forms of PteGlu were almost similar to each other in ileum. These results suggest that PteGlu bound to folate-binding protein is absorbed by a manner different from that of free PteGlu in rat gastrointestinal tract.     

Affinity labelling of the folate-binding protein in pig intestine.

A specific transport system for folate and a high-affinity folate-binding protein have been identified in pig intestinal brush-border membranes. To determine if the binding protein plays a role in folic acid (PteGlu) uptake in to the cell, the inactivation of folate binding and transport by N-hydroxysuccinimide esters of folic acid (NHS-PteGlu) was compared. In addition, the number of brush-border proteins modified by the affinity reagent was assessed. Brush-border vesicles were incubated with various concentrations of NHS-PteGlu or NHS-methotrexate. Transport and binding of [3H]PteGlu by the vesicles were measured at 37 and 4 degrees C respectively by using the vacuum-filtration technique. NHS-methotrexate and NHS-PteGlu specifically inhibited PteGlu transport. Incubating the vesicles with 1 microM-NHS-PteGlu inactivated [3H]PteGlu transport by 60% and binding by 80%. Half-maximal inhibition of both transport and binding was observed at similar concentrations of the affinity reagent (0.05 and 0.07 microM-NHS-PteGlu respectively). Treating the vesicles with radiolabelled NHS-PteGlu followed by gel electrophoresis and autoradiography revealed a specifically labelled protein with an Mr of 56,000. These results indicate that the intestinal folate-binding and transport proteins are identical and that the function of the folate-binding protein is to transport folate into the cell.     

The folate-binding protein of rat kidney. Purification, properties, and cellular distribution

Folate-binding protein (FBP) from rat kidney was isolated, and its properties and location in the kidney were determined. The particulate fraction of rat kidney homogenate was freed of its bound folate, solubilized with Triton X-100, and the FBP was purified using a combination of DEAE-cellulose and affinity chromatography. The purified protein migrated as a single band on sodium dodecyl sulfate-disc gel electrophoresis, has an isoelectric point of 5.7, contains 21.7% carbohydrate, and has an Mr of 28,500-30,000. The purified protein retained its affinities for different folate derivatives and its sensitivity to inorganic anions. Inorganic anions enhanced the binding of 5-methyltetrahydrofolate; chloride ion was the most effective, followed by Br- greater than I- greater than SO2-4. Chloride ion was also found to lower the dissociation constant of the folic acid-FBP complex at 50 degrees C by about 10-fold. This effect is thought to derive from the formation of a ternary FBP-folic acid-Cl- complex which is more stable than the binary FBP-folic acid complex. An antiserum raised against the purified protein in rabbits was used to determine the location of FBP in the kidney by immunofluorescence. Intense fluorescence staining for FBP was localized at the apices (brush border) of proximal tubules. The choroid plexus, an organ previously shown to contain FBP, also exhibited intense fluorescent staining.     

Nature and kinetic characteristics of L-DOPA uptake in rat renal proximal tubules

Summary The present study was performed with the aim to determine the kinetics and the caracteristics of cellular uptake of L-3,4-dihydroxyphenylalanine (L-DOPA) in rat renal proximal tubules. Incubation of renal tubules at 4°C in the presence of increasing concentrations of L-DOPA results in a linear and concentration-dependent accumulation of the substrate. In experiments carried out at 37°C, the accumulation of L-DOPA in renal tubules was found to be greater than that occurring at 4°C and showed a trend for saturation. The saturable component of L-DOPA uptake was derived from the total amount of L-DOPA accumulated in renal tubules at 37°C subtracted with the values obtained in experiments conducted at 4°C. The V_max and K_m values for the saturable component of L-DOPA uptake in renal tubules were, respectively, 241 ± 32 fmol µg protein^–1min^–1 and 567 ± 63 µM. Cyanine 863 (5 and 10 µM) was found to decrease the tubular uptake of L-DOPA, whereas probenecid (50 µM) did not change the rate of uptake of L-DOPA into renal tubules. The V_max and K_m values for the saturable component of L-DOPA uptake in renal tubules incubated in the presence of 10 µM cyanine 863 were, respectively, 97 ± 11 fmol µg protein^–1min^–1 and 160 ± 22 µM. It is suggested that the anionic L-DOPA may behave as an amphoteric substance, both hydroxyl groups in the aromatic ring determining the binding of the molecule to the organic cation transporter.     

Effect of uroguanylin on potassium and bicarbonate transport in rat renal tubules

The effect of uroguanylin (UGN) on K+ and H+ secretion in the renal tubules of the rat kidney was studied using in vivo stationary microperfusion. For the study of K+ secretion, a tubule was punctured to inject a column of FDC-green-colored Ringer's solution with 0.5 mmol KCl/L+/-10(-6) mol UGN/L, and oil was used to block fluid flow. K+ activity and transepithelial potential differences (PD) were measured with double microelectrodes (K+ ion-selective resin vs. reference) in the distal tubules of the same nephron. During perfusion, K+ activity rose exponentially, from 0.5 mmol/L to stationary concentration, allowing for the calculation of K+ secretion (JK). JK increased from 0.63+/-0.06 nmol.cm-2.s-1 in the control group to 0.85+/-0.06 in the UGN group (p<0.01). PD was -51.0+/-5.3 mV in the control group and -50.3+/-4.98 mV in the UGN group. In the presence of 10(-7) mol iberiotoxin/L, the UGN effect was abolished: JK was 0.37+/-0.038 nmol.cm-2.s-1 in the absence of, and 0.38+/-0.025 in the presence of, UGN, indicating its action on maxi-K channels. In another series of experiments, renal tubule acidification was studied, using a similar method: proximal and distal tubules were perfused with solutions containing 25 mmol NaHCO3/L. Acidification half-time was increased both in proximal and distal segments and, as a consequence, bicarbonate reabsorption decreased in the presence of UGN (in proximal tubules, from 2.40+/-0.26 to 1.56+/-0.21 nmol.cm-2.s-1). When the Na+/H+ exchanger was inhibited by 10(-4) mol hexamethylene amiloride (HMA)/L, the control and UGN groups were not significantly different. In the late distal tubule, after HMA, UGN significantly reduced JHCO3-, indicating an effect of UGN on H+-ATPase. These data show that UGN stimulated JK+ by acting on maxi-K channels, and decreased JHCO3- by acting on NHE3 in proximal and H+-ATPase in distal tubules.     

Characterization of primar cell cultures derived from rat renal proximal tubules

Summary Proximal tubules were prepared from rat kidney cortex by collagenase digestion and purified by percoll gradient centrifugation. Their enrichment was estimated by comparing the specific activities of various cell-specific enzymes in homogenates of renal cortex and of the isolated tubules. The tubules were cultured in a 50:50 mixture of Dulbecco’s modified Eagle’s and Ham’s F12 media supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, and prostaglandin E₁. After 2 to 3 d an extensive outgrowth of epithelial cells developed from the attached tubules. After 5 to 7 d near confluent monolayers were obtained. Hormonal responsiveness, marker enzyme activities, and transport properties were determined to further characterize the primary cultures. The cultured cells exhibited increased cyclic AMP production in response to parathyroid hormone but not calcitonin or vasopressin, consistent with the absence of cells derived from distal and collecting tubules. The cells also retained significant levels of 25-hydroxyvitamin D₃-lα-hydroxylase, alkaline phosphatase, and ψ-glytamyltranspeptidase, three enzymes that are primarily associated with the proximal tubule. The cultured epithelial cells also exhibit a Na⁺-dependent phosphate and glucose transport systems. Therefore, the cells retain many functional properties that are characteristic of proximal tubules. Thus, the primary cultures should be suitable for the study of processes that occur specifically within this segment of the rat nephron. This work was supported in part by the Veterans Administration (JBP), Washington, DC, by grant DK-37124 (NPC) from the National Institutes of Health, Bethesda, MD, and by grant BNS-86-17004 (CFL) from the National Science Foundation, Washington, DC.