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Characteristics of in vitro ammonia and glucose production by dog kidney cortex.

Renal cortex slices from acidotic dogs incubated with L-glutamine 1 mM at pH 7.05 produced more ammonia and glucose than slices from nonacidotic animals but no significant difference could be demonstrated at pH 7.48. At a phosphate concentration of 20-30 mM in the medium, a 20-30% increase in ammonia and a 25-40% decrease in glucose production were observed. At L-glutamine concentrations from 0 to 8 mM, a curvilinear increase in both ammonia and glucose production was noted, the effect being greater in slices from acidotic animals. D-Glutamine had little effect on ammoniagenesis. Ammonia production (1 mM L-glutamine) in vitro was 50% lower in acidotic dogs than in vivo. Slices from the remnant left kidney (4-6 wk after right nephrectomy) did not behave differently during acidosis than those from the right kidney with regard to ammonia or glucose production. In vitro ammonia and glucose production was higher in the rat than in the dog in acidotic and nonacidotic conditions when comparable concentrations of L-glutamine substrate were used.

Ammonia↗

Increase of Na/Pi-cotransport encoding mRNA in response to low Pi diet in rat kidney cortex.

Renal proximal Na/Pi-cotransport is increased in response to low dietary Pi intake. Recently, a cDNA (NaPi-2) related to the rat renal brush membrane Na/Pi-cotransporter has been cloned. In the present study, we used rats fed for 6 days with either a low Pi diet (LPD) or a high Pi diet (HPD), respectively. In parallel to an increased renal brush-border membrane Na/Pi-cotransport in LPD rats, there was also an increased content of NaPi-2 mRNA in renal cortex. After injection into Xenopus laevis oocytes, mRNA isolated from LPD rats induced a greater increase in Na/Pi-cotransport compared to mRNA from HPD rats. Hybrid depletion experiments suggested that mRNA-induced Na/Pi-cotransport is related to NaPi-2. We conclude that chronic Pi deprivation leads to an increased brush-border membrane Na/Pi-cotransport via an increase in the level of (NaPi-2) mRNA.

Animals↗

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex. Enzyme activities and some properties of glucose transport.

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of D-[3H] glucose into these vesicles was studied by a rapid filtration technique. (Na+ + K+)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of D-glucose uptake was almost unaffected by Na+ gradient (no overshoot).

Alkaline Phosphatase↗

Transcriptome-based identification of pro- and antioxidative gene expression in kidney cortex of nitric oxide-depleted rats.

Nitric oxide (NO) depletion in rats induces severe endothelial dysfunction within 4 days. Subsequently, hypertension and renal injury develop, which are ameliorated by alpha-tocopherol (VitE) cotreatment. The hypothesis of the present study was that NO synthase (NOS) inhibition induces a renal cortical antioxidative transcriptional response and invokes pro-oxidative and proinflammatory gene expression due to elimination of dampening effects of NO and enhanced oxidative stress. Male Sprague-Dawley rats received NOS inhibitor N(omega)-nitro-l-arginine (l-NNA, 500 mg/l water) for 4 (4d-LNNA), 21 (21d-LNNA), or 21 days with VitE in chow (0.7 g/kg body wt/day). Renal cortical RNA was applied to oligonucleotide rat arrays. In 4d-LNNA, 21d-LNNA, and 21d-LNNA+VitE, 120, 320, and 184 genes were differentially expressed, respectively. Genes related to glutathione and bilirubin synthesis were suppressed during 4d and 21d-LNNA and not corrected by VitE. Proteinuria, tubulointerstitial macrophages, and heme-oxygenase-1 (HO-1) expression were strongly correlated. Remarkably, pro-oxidative genes were not induced. Inflammation- and injury-related genes, including kidney injury molecule-1 and osteopontin, were unchanged at day 4, induced at 21d, and partly corrected by VitE. Superimposing HO-1 inhibition on NOS inhibition had no impact on the development of hypertension. To summarize, renal expression of genes involved in synthesis of the antioxidants glutathione and bilirubin seemed directly NO dependent, but there were no direct effects of NO depletion on pro-oxidant systems. This indicates that renal transcriptional regulation of two defense systems, glutathione and bilirubin syntheses, seems to depend upon adequate NO synthesis. Interaction between NO synthesis and heme degradation pathways for blood pressure regulation was not found.

Animals↗

[Characteristic respiratory magnitude of isolated mitochondria of the rat kidney cortex in relation to measurement conditions].

Respiration speed, respiratory control index and ADP/O-quotient are important characteristics of mitochondria. Since their numerical values much differ in literature, the influence of the composition of the measuring medium on these parameters was established. In their value the parameters are particularly determined by the Mg2+ and the phosphate concentration as well as by the pH-value of the medium. For the investigation of the characteristics a medium of the following composition is recommended: 210 mmol/l saccharose, 10 mmol/l KCl, 10 mmol/l KH2PO4, 60 mmol/l tris(hydroxymethyl)aminomethan (pH 7.40), 0.5 mmol/l ethylene diamine tetra-acetic acid.

Adenosine Diphosphate↗

Slices and sacs: limitations on metabolic and functional studies in kidney cortex and intestine.

While all experimental models, whether based on isolated preparations in vitro or on intact animals, possess characteristic limitations, reliance on simplified systems mandates special care. On the basis of both older and new evidence, the present review emphasizes problems associated especially with the use of renal cortical slices and everted sacs of the small intestine. Although these preparations have found renewed acceptance, the significance of results obtained is still at times interpreted without due regard for physiological reality. Sacs and slices will undoubtedly continue to prove useful in the study of biological processes, but their application must be predicated upon recognition of what information they can or cannot yield.

Animals↗

[Effect of slow rates of freezing on the functional state of mitochondria from various types of tissue preparations of kidney cortex].

Changes in functional activity of mitochondria, in concentration of respiration enzymes and the state of internal membrane after slow freezing down to -5 degrees, -12 degrees, -21 degrees and -30 degrees of various tissue preparations of renal cortex (homogenates, slices, etc) were studied. The following phenomena were found: an increase in the rate of mitochondrial respiration at the second metabolic state, inhibition of electron transport most distinct during mitochondrial oxidation of alpha-ketoglutarate, a decrease in concentration of cytochromes, an increase in the internal membrane permeability. Considerable damage of mitochondria was observed at temperatures between -5 degrees and -12 degrees but the most pronounced impairment of the organelles occurred at -10 degrees -20 degrees, i. e. at the freezing point of free water in the suspension and at eutectic temperatures.

Animals↗

Heterogeneity of the beta-amino-preferring transport system in rat kidney cortex. Differential influence of glutathione oxidation.

Taurine, a naturally found beta-amino acid, is inert in rat renal cortex slices. Its active accumulation by slices is abolished by anaerobiosis, a strongly acidic media or the removal of Na+. Concentration-dependent uptake studies reveal more than one taurine carrier: the apparent Km value for uptake below 1.1 mM is 0.4 mM and the apparent Km value above 1.1 mM is 14.5 mM. Of all amino acids tested only beta-alanine, another beta-compound, inhibited uptake. The oxidizing agent diamide was used to lower the concentration of GSH in rat cortex slices. The ability to accumulate taurine by the low Km system was decreased in diamide-treated slices, but not by the high Km system. Diamide was found to greatly augment efflux of taurine taken up from lower concentrations but not from higher concentrations. GSH in the media prevented this diamide-induced inhibition of uptake and enhanced efflux at lower taurine concentrations. A possible mechanism of diamide inhibition of uptake is that intracellular GSH depletion leads to greatly enhanced efflux of taurine, thus preventing uptake.

Alanine↗

Age-related alteration of renal brush border leucine aminopeptidase in rat kidney cortex.

Renal brush border enzyme activities were significantly decreased with age. The decrease was observed in the homogenate and brush border fractions. Purified leucine aminopeptidase was significantly decreased in Vmax and Km value for leucyl-beta-naphthylamide in the old rats. Heat stability of leucine aminopeptidase from both old and young rats showed biphasic, and the enzyme from old rats was more stable at 60 degrees C than that of the young. However, other properties such as molecular weight, antigenicity, charge, and optimum pH were not significantly different between young and old rats. From these results, it is suggested that age-related alteration of leucine aminopeptidase is due to a conformational change of enzyme molecule; the conformational change might occur at the active sites of the enzyme.

Aging↗

Cleavage of des-Arg9-bradykinin by angiotensin I-converting enzyme from pig kidney cortex.

Fast and very slow hydrolyses of des-Arg9-bradykinin and angiotensin II by angiotensin I-converting enzyme were detected by high performance liquid chromatography. The Michaelis constants of the enzyme, Km values, for des-Arg9-bradykinin and bradykinin were found to be 0.24 mM and 4.4 microM, and the maximum velocities, Vmax values (mumol . min-1 . mg protein-1) for these compounds to be 3.24 and 0.34, respectively. The enzyme also hydrolyzed Z-Gly-Pro-Gly-Gly-Pro-Ala to a tripeptide that was identified as dansyl-Gly-Pro-Ala by TLC on polyamide. These observations show that the enzyme hydrolyzes the peptides at the bond before the prolyl residue in the penultimate position.

Angiotensin II↗

Isolation of luminal and antiluminal membranes from dog kidney cortex.

Luminal (brush border) and antiluminal (basal-lateral) membranes were isolated from canine renal cortex. The enzyme marker for luminal membrane, alkaline phosphatase was enhanced 19-fold and the antiluminal enzyme marker, (Na+ + K+)-ATPase, was enhanced 22-fold in their respective membrane preparation, while the amount of cross contamination was minimal. Contamination of these preparations by enzyme markers for lysosomes, endoplasmic reticulum and mitochondria was also low. Routinely, more than 50 mg membrane protein was isolated for each membrane. Electron micrographs showed that the membranes were uniform in size, appearance, and vesicular in nature. An examination of the orientation of these membranes showed that 76.5% of the antiluminal membranes and 86% of the luminal membranes were right-side out.

Animals↗

Periodic microflow pattern measured with a new microflow probe within the rat kidney cortex.

With a newly developed microflow probe of high spatial resolution periodic changes of flow occurring within a tissue sphere of a radius of less than or equal to 50 micrometer have been observed. The frequency distribution of the periodic changes of flow was similar to the frequencies observed earlier with different techniques in the same preparation and in other organs (Basar and Weiss, 1970; Weiss and Thiemann, 1973). Preferred frequencies were: 0.02 Hz, 0.06 Hz and 0.1 Hz. Manufacture, calibration, the complete setup, and the estimation of the power of spatial resolution of the microflow probe are described. Periodic heating of the flow-sensing thermocouple at the tip of the probe (luminal diameter less than or equal to 10 micrometer) obviates the use of a reference thermocouple, renders the system (relatively) insensitive against uncontrolled changes of ambient temperature, and thus allows measurements with a very high sensitivity.

Animals↗

Evidence for existence of angiotensins I and II in mature renin granules from rat kidney cortex.

Renin granules were isolated by the combination of discontinuous and continuous Percoll density gradient centrifugation. The peak fraction containing the highest concentration of renin granules was found to contain the highest concentration of both angiotensin I and II immunoreactive substances. The identity of the immunoreactive peptides was further confirmed as angiotensin I and angiotensin II by high pressure liquid chromatography in reference to standard compounds. The coexistence of angiotensins I and II with renin indicates the formation of angiotensin II in renin granules. These findings clarify the mechanism of intracellular formation of angiotensin II as opposed to its formation in plasma and provide evidence against the internalization of angiotensin II, a hypothesis supported by the failure to detect angiotensin I in renin granules. Angiotensin II was increased by a low sodium diet while a high sodium diet did not affect its content.

Angiotensin I↗

Distribution of membrane-bound cyclic AMP-dependent protein kinase in plasma membranes of cells of the kidney cortex.

Renal cortical plasms membranes were separated by free flow electrophoresis into luminal (brush border microvilli) and contraluminal (basal-lateral membrane) fractions. These membranes were found to contain an intrinsic, self-phosphorylating system which consists of a cyclic AMP-dependent protein kinase, a phosphorprotein phosphatase and the substrate(s) of these enzymes. The kinase, but not the phosphatase, was stimulated by cyclic AMP; maximal (1.7-fold) stimulation was effected at a cyclic AMP concentration of 0.1 muM. The degree of phosphorylation of the brush borders was six times greater than that of the basal-lateral membranes in the absence of cyclic AMP and 2.3-fold greater in the presence of cyclic AMP. This preferential phosphorylation of the luminal membrane by membrane-associated protein kinase(s) may play a role in the parathyroid hormone-mediated alterations of solute reabsorption in the proximal tubule.

Adenosine Triphosphatases↗

Mechanisms of p-aminohippurate transport by brush-border and basolateral membrane vesicles isolated from rat kidney cortex.

The uptake of [3H]-labeled p-aminohippurate by brush-border and basolateral membrane vesicles isolated from rat renal cortex has been studied by a rapid filtration technique. Some characteristics of carrier-mediated transport for p-aminohippurate were demonstrated in basolateral membrane vesicles: the uptake was inhibited by probenecid or 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS), was saturable, was stimulated by the countertransport effect, and showed discontinuity in the Arrhenius plot. In contrast, brush-border membrane vesicles failed to display saturability of p-aminohippurate uptake and stimulation by the countertransport effect, although probenecid and DIDS reduced the uptake. Furthermore, p-aminohippurate uptake by brush-border membrane vesicles was influenced more sensitively by alteration in the membrane potential compared with that by basolateral membrane vesicles.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

cDNA cloning and expression of the flavoprotein D-aspartate oxidase from bovine kidney cortex.

The isolation and sequencing of the complete cDNA coding for a d-aspartate oxidase, as well as the overexpression of the recombinant active enzyme, are reported for the first time. This 2022 bp cDNA, beside the coding portion, comprises a 5' untranslated tract and the whole 3' region including the polyadenylation signal and the poly(A) tail. The encoded protein comprises 341 amino acids, with the last three residues (-Ser-Lys-Leu) representing a peroxisomal targeting signal 1 (PTS1), hitherto unknown for this protein. The overexpression of recombinant d-aspartate oxidase was achieved in a prokaryotic system, and a soluble and active enzyme was obtained which accounted for about 10% of total bacterial protein. Comparisons with the known cDNAs for mammalian d-amino acid oxidase, another peroxisomal enzyme, are also made. The close structural and functional similarities shared by these enzymes at the protein level are not reflected at the nucleic acid level.

Amino Acid Oxidoreductases↗

Asymmetry in the transport of lactate by basolateral and brush border membranes of rat kidney cortex.

The uptake of L(+)lactate into rat renal cortical brush border (BBV) and basolateral (BLV) membrane vesicles, isolated through differential centrifugation and free flow electrophoresis, were studied using a rapid filtration technique. In contrast to the lactate transport into the BBV, that into the BLV: 1) was found to proceed only towards equilibrium, 2) showed Na+ -independent coupling of the influx of L(+)lactate and the efflux of L(+) but not to the efflux of D(-)lactate, 3) was not inhibited by D(-)lactate, 2-thiolactate or 3-phenyl-lactate, but 4) was inhibited by 3-thiolactate and alpha-hydroxybutyrate and 5) was accelerated by changes in inwardly directed ionic gradients or by increases in cation conductance both of which led to increased intravesicular positivity. The latter changes had the opposite effect on the uptake of L(+)lactate by BBV. Thus, while the L(+)lactate transport system present in BBV showed the characteristics of Na-dependent electrogenic cotransport system, that in the BLV was consistent with a carrier mediated Na-dependent, facilitated diffusion system.

Animals↗

Isolation and partial purification of dicarboxylic acid binding protein from luminal-membrane vesicles of rabbit kidney cortex.

A specific dicarboxylic acid binding protein was isolated by solubilizing highly purified renal luminal-membrane vesicles with the non-ionic detergent C12E8 , followed by affinity chromatographic procedures. SDS-polyacrylamide gel electrophoresis of the samples containing dicarboxylic acid binding protein showed a single sharp band of an apparent molecular weight of 50 000. After treatment with mercaptoethanol the protein was split in two subunits of apparent molecular weights of 35 000 and 15 000. By analytical ultracentrifugation the minimal molecular weight of the dicarboxylic acid binding protein preparation was calculated to be 54 000. Binding of the radioactive succinate and L-malate to the dicarboxylic acid binding protein preparation as studied by equilibrium dialysis showed saturation phenomenon and was specifically inhibited by addition of D-malate. The dissociation constants for succinate (0.18 mM) and L-malate (0.33 mM) calculated from the binding data agree extremely well with the apparent Km values for these organic acids found in transport studies utilizing intact luminal-membrane vesicles.

Animals↗