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PubMed1966

Gluconeogenesis in mouse-liver slices.

1. The rate of gluconeogenesis from amino acids and other known precursors in slices of mouse liver after depletion of liver glycogen by means of phlorrhizin was high with l-lactate, pyruvate, glycerol, d-glyceraldehyde, dihydroxyacetone, d-fructose, sorbitol, xylitol, alpha-glycerophosphate, alanine, proline, threonine, serine and propionate. 2. The rate was unexpectedly low or even negligible with glutamate, aspartate, other glucogenic amino acids and the intermediates of the tricarboxylic acid cycle. 3. Glutamine and asparagine gave higher rates than the corresponding amino acids but still much lower rates than kidney cortex. 4. Livers of male mice gave much lower rates than livers of female mice. Kidney cortex showed no sex difference. 5. Livers of mice fed on a low-carbohydrate diet gave the same rates as livers of mice fed on a normal diet under the test conditions, i.e. 3hr. after an injection of phlorrhizin. 6. Much carbohydrate was formed from endogenous precursors and this was accompanied by release of ammonia and urea. 7. Gluconeogenesis in well-fed mice not treated with phlorrhizin was low. 8. Maximum rates were observed 3hr. after phlorrhizin treatment. 9. Prolonged phlorrhizin treatment did not prevent extensive deposition of liver glycogen, after an initial depletion. 10. Gluconeogenesis in livers of mice fed on a high-fat diet was relatively low. 11. Livers of alloxan-diabetic mice had a high carbohydrate content after phlorrhizin treatment, and gluconeogenesis from endogenous sources was about twice as high as in normal animals. Added substrates had about the same effect in normal and diabetic livers.

Journal Article↗

PubMed1997

Expression of syntaxins in rat kidney.

Previously, we demonstrated that a putative vesicle-targeting protein, syntaxin-4, is expressed in renal collecting duct principal cells and is localized to the apical plasma membrane, suggesting a role in targeting aquaporin-2-containing vesicles to the apical plasma membrane. To investigate whether other syntaxin isoforms are present in the renal collecting duct, we determined the intrarenal localization of syntaxin-2 and -3. Reverse transcription-polymerase chain reaction (RT-PCR) experiments using total RNA extracted from kidney and various organs revealed that both syntaxin-2 and -3 are expressed in kidney cortex and medulla. RT-PCR experiments using microdissected tubules and vascular structures from the kidney revealed that syntaxin-3 mRNA, but not syntaxin-2, is expressed in collecting duct cells. Syntaxin-3 mRNA was also relatively abundant in the thick ascending limb of Henle's loop and in vasa recta. Syntaxin-2 mRNA was found chiefly in glomeruli. To investigate the localization of syntaxin-3 protein, a peptide-derived polyclonal antibody was raised in rabbits. In immunoblotting experiments, this antibody labeled a 37-kDa protein in inner medulla that was most abundant in plasma membrane-enriched subcellular fractions. Immunoperoxidase labeling of thin cryosections combined with immunogold electron microscopy showed that, in contrast to the labeling seen for syntaxin-4, syntaxin-3 labeling in medullary collecting duct was predominantly in the basolateral plasma membrane of intercalated cells. These results suggest the possibility that syntaxin-3 may be involved in selective targeting of acid-base transporters and/or in basolateral membrane remodeling in response to systemic acid-base perturbations.

Amino Acid Sequence↗

PubMed1998

Regulation of type II renal Na+-dependent inorganic phosphate transporters by 1,25-dihydroxyvitamin D3. Identification of a vitamin D-responsive element in the human NAPi-3 gene.

Vitamin D is an important regulator of phosphate homeostasis. The effects of vitamin D on the expression of renal Na+-dependent inorganic phosphate (Pi) transporters (types I and II) were investigated. In vitamin D-deficient rats, the amounts of type II Na+-dependent Pi transporter (NaPi-2) protein and mRNA were decreased in the juxtamedullary kidney cortex, but not in the superficial cortex, compared with control rats. The administration of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to vitamin D-deficient rats increased the initial rate of Pi uptake as well as the amounts of NaPi-2 mRNA and protein in the juxtamedullary cortex. The transcriptional activity of a luciferase reporter plasmid containing the promoter region of the human type II Na+-dependent Pi transporter NaPi-3 gene was increased markedly by 1,25-(OH)2D3 in COS-7 cells expressing the human vitamin D receptor. A deletion and mutation analysis of the NaPi-3 gene promoter identified the vitamin D-responsive element as the sequence 5'-GGGGCAGCAAGGGCA-3' nucleotides -1977 to -1963 relative to the transcription start site. This element bound a heterodimer of the vitamin D receptor and retinoid X receptor, and it enhanced the basal transcriptional activity of the promoter of the herpes simplex virus thymidine kinase gene in an orientation-independent manner. Thus, one mechanism by which vitamin D regulates Pi homeostasis is through the modulation of the expression of type II Na+-dependent Pi transporter genes in the juxtamedullary kidney cortex.

Animals↗

PubMed1979

Glycoprotein biosynthesis in calf kidney. Glycoprotein sialyltransferase activities towards serum glycoproteins and calf Tamm-Horsfall glycoprotein.

CMP-AcNeu:glycoprotein sialyltransltransltransltransltransferase of calf kidney cortex was characterized using serum glycoproteins and Tamm-Horsfall glycoprotein, obtained from calf urine, as acceptors. Native calf Tamm-Horsfall glycoprotein showed the best acceptor properties, followed by desialylated calf fetuin and desialylated human alpha 1-acid glycoprotein exhibiting V values of, respectively, 114, 63 and 41 nmol/h per g wet wt. of kidney cortex and Km values of 0.12, 0.16 and 0.26 mM glycoprotein acceptor. Desialylated ovine submaxillary mucine appeared to be a very poor acceptor. Tamm-Horsfall glycoprotein sialyltransferase could be distinguished from serum glycoprotein sialyltransferase by competition studies. In addition the two glycoprotein sialyltransferase activities showed different distributions over the three regions of the calf kidney: the ratios of the Tamm-Horsfall to serum glycoprotein sialyltransferase activities decreased from 3.3 in the cortex to 0.8 and 0.4 in the medulla and the papilla, respectively. It was concluded that in calf kidney at least two different sialyltransferases exist. The high cortical Tamm-Horsfall glycoprotein sialyltransferases activity corresponds markedly to the origin of the urinary Tamm-Horsfall glycoprotein, namely the distal part of the kidney tubule. Inactivation of glycoprotein sialyltransferase activity by preincubation at various temperatures and during storage at 0 degree C, could be reduced by the addition of CMP-AcNeu. The possible relevance towards the in vivo sialylation of this finding is discussed.

Animals↗

PubMed1985

Effects of experimental nephrosis on basement-membrane components and enzymes of collagen biosynthesis in rat kidney.

The aim of the present study was to find out whether the basement-membrane proteins laminin and type IV collagen are involved in the development of aminonucleoside-induced nephrosis. These proteins were measured by specific radioimmunoassays in serum, urine and kidney-cortex samples, and they were localized in the glomeruli by indirect immunofluorescence. Nephrosis was induced in rats with a single intraperitoneal injection of puromycin aminonucleoside. Serum laminin concentrations, detected by a radioimmunoassay for the P2 domain of the protein, increased to reach a maximum at days 5-7, and they remained elevated until at least day 14. The increase preceded the development of proteinuria, suggesting a role for laminin in glomerular function. Concomitant with proteinuria, increasing amounts of laminin antigenicity were also found in the urine. The size of the laminin antigen in serum was estimated by gel filtration, and the serum forms were found to contain both the P1 and the P2 regions of the intact laminin molecule. On the other hand, there were no changes in the serum or urinary concentrations of type-IV-collagen-derived antigens, as detected by a radioimmunoassay for the 7S collagen domain of this protein. The total content of laminin in kidney cortex, measured after digestion of the tissue with trypsin and collagenase, was, at day 9, still comparable with normal values, and the distribution of both basement-membrane proteins in the glomeruli, studied by indirect immunofluorescence, was similar to that in the controls. The tissue damage induced by aminonucleoside, however, seems to stimulate collagen biosynthesis, as the activities of prolyl 4-hydroxylase, lysyl hydroxylase and galactosylhydroxylysyl glucosyltransferase in kidney tissue increased significantly, with maxima at days 8-10.

Animals↗

PubMedDate not supplied

Characterization of monocarboxylate transport in human kidney HK-2 cells.

The objectives of this study were to characterize the expression and function of monocarboxylate transporters (MCTs) in human kidney HK-2 cells and to compare the expression of MCTs in HK-2 cells to that found in human kidney. mRNA and protein expression of MCTs were determined by RT-PCR and Western analyses, respectively, while immunofluorescence staining was used to determine the membrane localization of MCT1. The driving force, transport kinetics, and inhibition of two MCT substrates, D-lactate and butyrate, were characterized in HK-2 cells. mRNA of MCT1, -2, -3, -4 isoforms were present in HK-2 cells and in human kidney cortex. MCT1 was present predominantly on the basal membranes of HK-2 cells. The cellular uptake of D-lactate and butyrate exhibited pH- and concentration-dependence (D-lactate, Km of 26.5 +/- 2.2 mM and Vmax of 72.0 +/- 14.5 nmol mg-1 min-1; butyrate, Km of 0.8 +/- 0.3 mM, Vmax of 29.3 +/- 2.5 nmol mg-1 min-1, and a diffusional clearance of 2.1 microL mg-1 min-1). The uptake of D-lactate and butyrate by HK-2 cells was inhibited by MCT analogues and the classical MCT inhibitors alpha-cyano-4-hydroxycinnamate, pCMB, and phloretin. The uptake of D-lactate and butyrate by HK-2 cells significantly decreased after transfection with small-interference RNA for MCT1. In summary, MCTs were present in both HK-2 cells and human kidney cortex, and HK-2 cells exhibited polarized MCT expression and pH-dependent transport of D-lactate and butyrate. Our results also support the usefulness of HK-2 cells as an in vitro model for studying monocarboxylate transport in renal proximal tubule cells.

Biological Transport↗

PubMed1990

Ouabain-insensitive, Na-ATPase activity in pure suspensions of rat kidney proximal tubules.

The present work was undertaken to evaluate the distribution of the Na-ATPase activity in the different components of the rat kidney cortex. Suspensions of glomeruli, proximal and distal tubules were prepared following a collagenase digestion of outermost kidney cortex slices and a separation on a Percoll gradient. It was found that the Na-ATPase activity is higher in the fraction enriched in proximal tubules. The fraction enriched in glomeruli and in distal tubules show also a Na-ATPase activity, but it is lower.

Alkaline Phosphatase↗

PubMed1989

Peroxisomal and mitochondrial beta-oxidation of monocarboxylyl-CoA, omega-hydroxymonocarboxylyl-CoA and dicarboxylyl-CoA esters in tissues from untreated and clofibrate-treated rats.

In control rats, long-chain monocarboxylyl-CoA, omega-hydroxymonocarboxylyl-CoA, and dicarboxylyl-CoA esters were substrates for hepatic, renal, and myocardial peroxisomal beta-oxidation. The latter enzyme system could not be detected in skeletal muscle. Clofibrate treatment resulted in an enhancement of peroxisomal beta-oxidizing capacity in various tissues. Intact mitochondria from control rat liver and kidney cortex incubated in the presence of L-carnitine were capable of oxidizing long-chain monocarboxylyl-CoAs and omega-hydroxymonocarboxylyl-CoAs but not dicarboxylyl-CoAs. However, control rat liver mitochondria permeabilized by digitonin oxidized dodecanedioyl-CoA indicating that the liver mitochondrial beta-oxidation system can act on dicarboxylyl-CoA esters even if the overall intact mitochondrial system is inactive on these substrates. Intact liver mitochondria from clofibrate-treated animals rapidly oxidized lauroyl-CoA and 12-hydroxylauroyl-CoA but not dodecanedioyl-CoA. These mitochondria were active on hexadecanedioyl-CoA and this activity amounted to 20-25% of that measured with palmitoyl-CoA and 16-hydroxypalmitoyl-CoA as substrates. No mitochondrial dicarboxylyl-CoA oxidation could be detected in kidney cortex from animals receiving clofibrate in their diet. Heart and skeletal muscle intact mitochondria from untreated and clofibrate-treated rats were capable of oxidizing each type of acyl-CoA as a substrate. Dicarboxylyl-CoA synthetase and carnitine dicarboxylyltransferase activities were detected in various tissues from untreated and clofibrate-treated rats with the exception of carnitine dodecanedioyltransferase reaction in livers from untreated and clofibrate-treated rats. In skeletal muscle, the acyl-CoA synthetase activities could be detected only in the presence of detergents.

Acyl Coenzyme A↗

PubMed1991

Protein carboxyl methylation in kidney brush-border membranes.

Protein carboxyl methylation activity was detected in the cytosol and in purified brush-border membranes (BBM) from the kidney cortex. The protein carboxyl methyltransferase (PCMT) activity associated with the BBM was specific for endogenous membrane-bound protein substrates, while the cytosolic PCMT methylated exogenous substrates (ovalbumin and gelatin) as well as endogenous proteins. The apparent Km for S-adenosyl-L-methionine with endogenous proteins as substrates were 30 microM and 4 microM for the cytosolic and BBM enzymes, respectively. These activities were sensitive to S-adenosyl-L-homocysteine, a well known competitor of methyltransferase-catalyzed reactions, but were not affected by the presence of chymostatin and E-64, two protein methylesterase inhibitors. The activity of both cytosolic and BBM PCMT was maximal at pH 7.5, while BBM-phospholipid methylation was predominant at pH 10.0. Separation of the = methylated proteins by acidic gel electrophoresis in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride revealed distinct methyl accepting proteins in the cytosol (14, 17, 21, 27, 31, 48, 61 and 168 kDa) and in the BBM (14, 60, 66, 82, and 105 kDa). Most of the labelling was lost following electrophoresis under moderately alkaline conditions, except for a 21 kDa protein in the cytosol and a 23 kDa protein in the BBM fraction. These results suggest the existence of two distinct PCMT in the kidney cortex: a cytosolic enzyme with low selectivity and affinity, methylating endogenous and exogenous protein substrates, and a high-affinity BBM-associated methylating activity.

Animals↗

PubMed1995

Effect of streptozotocin-induced diabetes on kidney Na+/K(+)-ATPase.

The maximal capacity of low affinity ouabain binding sites in kidney medulla was found to be increased by 20 +/- 3.8% after 2 weeks, and by 35 +/- 4.5% in 4 weeks diabetes. However, in kidney cortex no similar changes could be detected. Western blot analysis of Na+/K(+)-ATPase subunits in kidney medulla indicated a significant enhancement of both the alpha 1 and beta 1 subunit in two and four weeks diabetic rats (alpha 1: 35 +/- 3.1, 51 +/- 5.8% and beta 1: 31.3 +/- 5.2 and 43.2 +/- 6.8%, respectively). However, kidney cortex showed no significant change in any condition tested. In diabetes we could detect a significant change only in the medulla in case of the b subunit mRNA transcript, which showed 1.69 +/- 0.59 and 2.89 +/- 0.81 times increased in two and four weeks diabetic state, respectively. There was no change in the alpha 1 subunit mRNA abundance. Insulin treatment of diabetic animals did not result in a complete reversal of diabetes-induced changes in ouabain binding capacity or in the amount of Na+/K(+)-ATPase alpha 1 and beta 1 subunit protein and mRNA levels. Our data indicate a good correlation between changes in low affinity ouabain binding capacity and the level of alpha 1 isoform in diabetic rats, and suggest an important role of the b subunit in the regulation of enzyme quantity.

Animals↗

PubMed1991

Distribution of tubulointerstitial nephritis antigen and evidence for multiple forms.

A monoclonal antibody (A8) to a basement membrane component (TIN antigen), which is associated with autoimmune tubulointerstitial nephritis, was developed and utilized to characterize tissue distribution and properties of TIN antigen by immunofluorescence microscopy and immunoblotting. Results were confirmed with polyclonal goat anti-rabbit and human autoantibodies. TIN antigen was found in basement membranes of kidney cortex, small intestines, skin, and cornea, but was not detected in the renal medulla. Within the kidney cortex proximal tubular basement membrane (TBM) showed the strongest staining. TIN antigen was also detected in Bowman's capsule, distal TBM, peritubular capillaries, and focally in the interstitium, but not in glomerular basement membrane or mesangial matrix. Immunoblotting of SDS-extracted human, rabbit, mouse, and Brown Norway rat TBM with A8 revealed predominantly a 58 kD TIN antigen; however, other reactive components were detected in minor quantities. Bovine TBM contained components of 52 kD, 45 kD and 35 kD in varying concentrations. Immunoblotting of isolated rabbit TIN antigen revealed the major 58 kD component that was characterized previously, and minor components of 300 kD, 175 kD, 160 kD and 50 kD. TIN antigen was not detected in Lewis rat TBM by immunofluorescence or immunoblotting. These studies suggest the following: 1) TIN antigen may be synthesized as a high molecular weight glycoprotein that is processed to smaller forms; 2) it may be covalently associated with other basement membrane components; 3) the antibody reactive epitope may be present on multiple TBM components; and 4) high molecular weight forms may represent aggregates of TIN antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Thermal effects in tissues from combined simultaneous coaxial CO2 and Nd:YAG laser beams.

This study examines the depth of thermal coagulation and charring in swine liver, kidney cortex, tongue (inferior surface), skeletal muscle, inflated lung, and skin resulting from in vivo incision with simultaneous coaxial CO2 and Nd:YAG (1.064 microM) laser beams. At values of 20 w and 40 w, respectively, and at values of 30 w and 60 w, respectively, of combined CO2 and Nd:YAG laser radiation, coagulation depths determined histologically in liver were significantly greater (P less than 0.01) than in the other tissues and were significantly less in inflated lung (P less than 0.05) than in other tissues for the larger laser power settings employed. Coagulation depths achieved at 10 w and 20 w, respectively, and at 20 w and 40 w, respectively, of CO2 and Nd:YAG laser power were comparable to those obtained by other workers in liver and other relatively vascular tissues using a contact Nd:YAG laser tip. Charring depths obtained at power settings of 30 w (CO2) and 60 w (Nd:YAG) were greater in liver (P less than 0.001) than in all other tissues examined. Hemostasis during incision was achieved only for values of the ratio of CO2 to Nd:YAG laser power in the range 2-3 in the more vascular tissues, liver and kidney cortex, whereas hemostasis was achieved also in the lesser vascular tissues at higher values. These results strongly suggest the usefulness of combined simultaneous CO2 and Nd:YAG laser beams in surgery of the more vascular organs and tissues.

Animals↗

PubMed1987

Lysosomes of the renal cortex: heterogeneity and role in protein handling.

Rate sedimentation of the kidney cortical mitochondrial/lysosomal (ML) fraction yields two distinct classes of lysosomes: the large lysosomes or protein droplets and a heterogeneous broad band of smaller lysosomes. The protein droplets which are recovered as a well defined zone of high purity also sediment as a homogeneous band after equilibrium banding at a density of 1.235 g/ml in sucrose. The small lysosomes co-sediment with other subcellular organelles as a broad band, indicated by the distribution of various acid hydrolases, which exhibit subtle heterogeneity among these small lysosomes. The distribution of renin containing granules indicates that in size they represent a distinct subpopulation of small lysosomes. Further fractionation of small lysosomes by equilibrium banding separates two distinct populations at densities 1.20 (small light) and 1.235 g/ml (small dense). Comparison of lysosomal populations fractionated in these studies with the distribution of lysosomal acid hydrolases along the different segments of the nephron suggests that large and small dense lysosomes probably originate from the proximal tubule while the small light lysosomes may contain lysosomes from the distal tubule. Very small, lysosome-like organelles subfractionated from the 'microsomes' may constitute a mixture of small light lysosomes, lysosomal fragments and endocytic vesicles from a variety of cell types. Time course studies with 3H labelled Cd-thionein, following intravenous administration, suggests that uptake in the kidney cortex is very rapid and that catabolism takes place in two distinct phases: rapid breakdown starting in the endosome compartment and slower breakdown in lysosomes. From the association of labelled lysozyme (125I) and Cd-thionein (109Cd) it appears that all the different lysosomal populations identified are at some stage involved with uptake and catabolism of these two proteins.

Animals↗

PubMed1969

Inhibition of hepatic gluconeogenesis by ethanol.

1. Gluconeogenesis from 10mm-lactate in the perfused liver of starved rats is inhibited by ethanol. The degree of inhibition reached a maximum of 66% at 10mm-ethanol under the test conditions and decreased at higher ethanol concentrations. The concentration-dependence of the inhibition is paralleled by the concentration-dependence of the activity of alcohol dehydrogenase. The enzyme is also inhibited by ethanol concentrations above 10mm. 2. Gluconeogenesis from pyruvate is not inhibited by ethanol. 3. The degree of the inhibition of gluconeogenesis from lactate by ethanol depends on the concentration of lactate and other oxidizable substances, e.g. oleate, in the perfusion medium. 4. Ethanol also inhibits, to different degrees, gluconeogenesis from glycerol, dihydroxyacetone, proline, serine, alanine, fructose and galactose. 5. The inhibition of gluconeogenesis from lactate by ethanol is reversed by acetaldehyde. 6. Pyrazole, a specific inhibitor of alcohol dehydrogenase, also reverses the inhibition of gluconeogenesis by ethanol. 7. Gluconeogenesis in kidney cortex, where the activity of alcohol dehydrogenase is very low, is not inhibited by ethanol. 8. Kidney cortex, testis, ovary, uterus and certain tissues of the alimentary tract were the only rat tissues, apart from the liver, that showed measurable alcohol dehydrogenase activity. 9. The concentrations of pyruvate in the liver were decreased to about one-fifth by ethanol. 10. The concentration of lactate in the perfused liver was about 3mm below that of the perfusion medium 30min. after the addition of 10mm-lactate. 11. The great majority of the findings support the view that the inhibition of gluconeogensis by ethanol is caused by the alcohol dehydrogenase reaction, which decreases the [free NAD(+)]/[free NADH] ratio. The decrease lowers the concentration of pyruvate and this is the immediate cause of the inhibition of gluconeogenesis from lactate, alanine and serine: the fall in the concentration of pyruvate lowers the rate of the pyruvate carboxylase reaction, one of the rate-limiting reactions of gluconeogenesis. The cause of the inhibition of gluconeogenesis from other substrates is discussed.

Alanine↗

PubMed1995

2-[125I]iodomelatonin-binding sites in the human kidney and the effect of guanosine 5'-O-(3-thiotriphosphate).

Putative melatonin receptors in normal kidney cortical tissues of patients with transitional cell carcinoma or renal cell carcinoma were characterized using a melatonin agonist, 2-[125I]iodomelatonin, as the radioligand. 2-[125I]Iodomelatonin-binding sites in the human kidney were stable, saturable, reversible, and of high affinity. The binding affinity was 15.2 +/- 2.5 pmol/L, and the binding density was 1.79 +/- 0.19 fmol/mg protein. The unity of the Hill coefficients and linearity of the Scatchard plots suggested that 2-[125I]iodomelatonin was bound to a single class of binding sites. Pharmacological characterization showed that these binding sites were highly specific to 2-iodomelatonin, melatonin, 6-hydroxymelatonin, and 6-chloromelatonin. Guanosine 5'-O-(3-thiotriphosphate) decreased the binding affinity and density of 2-[125I]iodomelatonin-binding sites in the kidney, suggesting that these binding sites are coupled to a G-protein. The characterization of 2-[125I]iodomelatonin-binding sites in normal kidney tissues taken from patients with transitional cell carcinoma or renal cell carcinoma suggests the existence of 2-[125I]iodomelatonin-binding sites in the human kidney cortex, which is in line with the findings of 2-[125I]iodomelatonin-binding sites in kidneys of other mammals and birds. The implication of a direct melatonin action on renal function in the human is proposed.

Adult↗

PubMed1992

Purification and properties of AMP-deaminase from human kidney.

AMP-deaminase from human kidney (cortex and medulla) was purified and the physicochemical properties were characterized. The enzyme from both portions of the kidney exhibited identical kinetics and regulatory properties. At optimal pH (6.6), the AMP-deaminase studied exhibited a distinctly sigmoidal substrate saturation kinetics, with the half-saturation parameter (S0.5) as high as 10 mM. ATP at 1 mM strongly activated the enzyme, decreasing S0.5 nearly 10-fold. The activating effect of ADP was less strong. Orthophosphate inhibited the enzyme, but the inhibition observed was weak (Ki approximately 16 mM) and had a pure competitive character. At pH 7.2, physiological for the kidney cortex, orthophosphate inhibition became even weaker and became partially competitive. Variations in the adenylate energy charge had potent effects on the activity of AMP-deaminase, depending on the size of the total adenine nucleotide pool examined. The results of gel filtration and SDS-PAGE indicated that human kidney AMP-deaminase is an oligomeric enzyme composed of four, probably identical, subunits weighing about 37 kDa each.

AMP Deaminase↗

PubMed1996

Localization of the thiazide sensitive Na-Cl cotransporter, rTSC1 in the rat kidney.

A thiazide sensitive Na-Cl cotransporter, rTSC1, has recently been cloned from a rat kidney cortex cDNA library. The molecular regulation and nephron localization of this protein is unknown. The purpose of this study was to examine the nephron distribution and subcellular localization of the rTSC1 protein in the rat kidney. In situ hybridization showed rTSC1 transcripts were localized to short, convoluted tubule segments in the kidney cortex. Polyclonal antibodies raised against a 110 amino acid segment from the amino terminus of rTSC1 recognized three major bands of 135, 140 and 155 kDa on Western blotting of membrane protein from cortex but not outer medulla of the rat kidney. Immunofluorescence studies using the antibody alone and in double labeling experiments with antibodies against the H+ ATPase and calbindin D28, showed intense labeling of apical membranes in the distal nephron beginning in the initial distal convoluted tubule and terminating within the connecting tubule. The intensity of labeling diminished from proximal to distal sites along the distal tubule. Ultrastructural studies by immunoelectron microscopy showed the cotransporter protein to be localized predominately on apical microvilli of the distal convoluted tubule cells. These results are consistent with rTSC1 encoding the apical thiazide sensitive Na-Cl cotransporter in the distal tubule.

Animals↗

PubMed1989

Uptake of gentamicin in foetal and newborn rats after a single intraperitoneal injection.

The aim of the present investigation has been to examine 1) the placental passage and uptake of gentamicin in foetal rat tissues and 2) uptake and organ distribution of this drug in neonatal rats with special reference to the kidney. After a single intraperitoneal injection of tritium-labelled gentamicin to the mother, we found a rapid passage of radioactivity across the rat placenta, but no pronounced tendency to accumulation in the foetus. After administration of 10 micrograms or 100 mg gentamicin per kg body weight to pregnant rats at day 11 or 13, respectively, of gestation, the maximal concentrations of the drug recovered in individual foetuses were 0.05 and 300 ng per mg wet weight. Ten and 90 min. after administration the uptake of gentamicin in the kidney cortex of the mother was 25 and 118 fold higher, respectively, than the corresponding uptake in the foetal kidney after 19 days of gestation. After a single intraperitoneal injection, gentamicin uptake in the kidney cortex of neonatal rats resembled that seen in adult animals, with a rapid initial phase followed by a relatively stable plateau, although the maximal uptake was only about 10% as large in the neonates.

Animals↗