Glutamic acid decarboxylase in tubules and glomeruli isolated from rat kidney cortex

4-Aminobutyric acid (GABA) synthesis was examined in purified glomeruli and tubules of rat kidney cortex that were incubated in the presence of [2,3-3H2]glutamate. The GABA that was formed was separated from glutamate using anion-exchange resin, and identified by means of an automatic amino acid analyser. In the renal cortex only the tubules were able to form GABA (35.0 nmol mg-1 h-1); the remaining GABA synthesis found in the glomerular preparations can most probably be attributed to a contamination by cortical tubules (9%), as shown by determination of a known tubular marker enzyme (L-gamma-glutamyltransferase). Hydroxylamine (1 mM) and ethanolamine-O-sulfate (10 mM), well-known inhibitors of cerebral GABA formation and GABA catabolism respectively, inhibited renal tubular GABA formation at 100% and 44% respectively.     

Triacylglycerol metabolism in isolated rat kidney cortex tubules

Triacylglycerol metabolism has been studied in kidney cortex tubules from starved rats, prepared by collagenase treatment. Triacylglycerol was determined by a newly developed fully enzymic method. Incubation of tubules in the absence of fatty acids led to a decrease of endogenous triacylglycerol by about 50% in 1h. Addition of albuminbound oleate or palmitate resulted in a steady increase of tissue triacylglycerol over 2h. The rate of triacylglycerol synthesis was linearly dependent on oleate concentration up to 0.8mm, reaching a saturation at higher concentrations. Triacylglycerol formation from palmitate was less than that from oleate. This difference was qualitatively the same when net synthesis was compared with incorporation of labelled fatty acids. Quantitatively, however, the difference was less with the incorporation technique. Gluconeogenic substrates, which by themselves had no effect on triacylglycerol concentrations, stimulated neutral lipid formation from fatty acids. Glucose and lysine did not have such a stimulatory effect. Inhibition of gluconeogenesis from lactate by mercaptopicolinic acid likewise inhibited triacylglycerol formation. This inhibitory effect was seen with oleate as well as with oleate plus lactate. When [2-¹⁴C]lactate was used the incorporation of label into triacylglycerol was found in the glycerol moiety exclusively. Addition of dl-β-hydroxybutyrate (5mm) to the incubation medium in the presence of oleate or oleate plus lactate led to a significant increase in triacylglycerol formation. In contrast with the gluconeogenic substrates, dl-β-hydroxybutyrate had no stimulatory effect on fatty acid uptake. The results suggest that renal triacylglycerol formation is a quantitatively important metabolic process. The finding that gluconeogenic substrates, but not glucose, increase lipid formation, indicates that the glycerol moiety is formed by glyceroneogenesis in the proximal tubules. The effect of ketone bodies seems to be caused by the sparing action of these substrates on fatty acid oxidation. The decrease of triacylglycerol in the absence of exogenous substrates confirms previous conclusions that endogenous lipids provide fatty acids for renal energy metabolism.     

Carnitine metabolism in isolated rat kidney cortex tubules

Renal carnitine metabolism was studied in isolated kidney cortex tubules from fed rats. The tubular distribution of free carnitine (C), acid-soluble short chain acylcarnitine (AcC), and total acid-soluble carnitine was measured. The content of the last-mentioned in rat cortical tubule suspensions was 2.85 +/- 0.15 nmol/mg protein, 46% representing AcC. In the absence of metabolic substrates the AcC/C ratio declined from 0.84 to 0.48 during incubation. The administration of 2mM acetoacetate or 2mM 3-hydroxybutyrate caused an increase in AcC by 45% and 51%, respectively. The rise in AcC was paralleled by a decrease in C, resulting in an increase of the tubular AcC/C ratio to 1.69 and 1.85, respectively. In the presence of 1 mM exogenous L-carnitine 35 +/- 6 nmol AcC/(mg protein X h) was formed. The addition of acetoacetate and 3-hydroxybutyrate led to a 3.5 to 3.8-fold rise in AcC formation. Other substrates which are likewise metabolized by proximal tubules were less effective. More than 90% of the formed AcC was recovered in the extracellular fluid. The results suggest that proximal renal tubule cells are the intrarenal site of carnitine acylation and may be involved in the regulation of blood and/or urinary carnitine acylation state.     

Inhibition of 2-oxoglutarate metabolism by lithium in the isolated rat kidney cortex tubules

2-Oxoglutarate metabolism in the isolated rat kidney cortex tubules was inhibited by lithium at 5 mM concentration, and at pH increased from 7.1 to 7.6. The metabolism of pyruvate and acetylcarnitine to citrate and 2-oxoglutarate was enhanced by lithium and the increased pH value. The content of 2-oxoglutarate in the renal tubular cells was lowered by lithium but increased at elevated pH values. Both the intracellular pH value and bicarbonate ion concentrations in renal tubular cells were increased by lithium in the medium containing 2-oxoglutarate. The results obtained indicate that lithium disturbs renal metabolism by intracellular alkalization, with a simultaneous inhibition of the inflow of dicarboxylic substrates to the renal tubular cells.     

Increased cathepsin D-like activity in cortex, tubules, and glomeruli isolated from rats with experimental nephrotic syndrome.

We have examined the activity and distribution of cathepsin D (EC 3.4.23.5), a major renal lysosomal endoproteinase, in the various anatomical and functional areas of normal rat kidney. Cathepsin D-like activities (delta A280/h per mg of protein) in normal rat tissues were: cortex, 0.78 +/- 0.05, n = 37; medulla, 0.62 +/- 0.03, n = 12; papilla, 0.63 +/- 0.04, n = 12; tubules, 0.74 +/- 0.04, n = 28; glomeruli, 0.59 +/- 0.03, n = 28; and liver, 0.41 +/- 0.02, n = 28. Enzyme activity was maximal at pH 3.0-3.5 and inhibited more than 90% by pepstatin (6.7 micrograms/ml), suggesting that the enzyme is cathepsin D. In subsequent experiments we measured cathepsin D-like activity in cortex, tubules and glomeruli isolated from rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. Treated animals (15 mg of PAN/100g body wt., intraperitoneally) developed proteinuria beginning 4 days after injection and exceeding 900 mg/24h on day 9. In two separate experiments involving 52 animals we observed a significant increase in cathepsin D-like activity in cortex (+82.7%), tubules (+109.6%) and glomeruli (+54.7%) isolated from PAN-treated rats killed during marked proteinuria (day 9, mean total urinary protein excretion: 937 +/- 94 mg/24h). This increase was observed whether the activity was expressed per mg of DNA or per mg of protein. Increased cathepsin D-like activity was first observed in cortex and tubules coincident with the onset of proteinurea (day 4, mean total urinary protein excretion: 112 +/- 23 mg/24h). In contrast with the significant elevation of renal cathepsin D-like activity, the activity (nmol/h per mg of protein) of alpha-L-fucosidase (EC 3.2.1.51), a non-proteolytic enzyme, was markedly decreased in the identical samples used for the measurement of cathepsin D-like activity: cortex (-46.4%); tubules (-46.1%); and glomeruli (-38.5%). In addition to changes in renal enzyme activities, PAN-treated rats excreted large amounts of cathepsin D-like activity in their urine (beginning on day 3) compared with nearly undetectable cathepsin D-like activity in the urine from control rats. The significant increases in glomerular and tubular cathepsin D activity may reflect an important role for this enzyme in the pathophysiology associated with PAN-induced nephrotic syndrome.     

Dopamine production by isolated glomeruli and tubules from rat kidneys

To locate the sites of dopamine (D) production in rat renal cortex, we separated glomeruli and proximal tubules by sieving or centrifugation in Percoll after collagenase digestion. After centrifugation layer I contained 60-80% glomeruli and 20-40% tubule fragments, half of which did not stain with alkaline phosphatase, layer II contained 0-5% glomeruli, 10-25% tubule fragments other than proximal tubules, and 70-85% proximal tubule fragments. Layer IV contained 85-95% proximal tubules. Gluconeogenic rates were (micromoles per hour per gram wet weight) as follows: I, 4 +/- 1; II, 7 +/- 2; and IV, 16 +/- 1. Norepinephrine (NE) content was (picomoles per gram wet weight) I, 310 +/- 30; II, 540 +/- 40; IV, 195 +/- 60. D content was (picomoles per gram wet weight) I, 26 +/- 6; II, 46 +/- 13; IV, 33 +/- 7. Surgical denervation 4-6 days previously reduced the norepinephrine content of layers I and II to 35 +/- 10 (p less than 0.001) and of IV to 60 +/- 20 (p less than 0.05) and the D content of layers I and II to 13 +/- 6 and 6 +/- 6 pmol/g, respectively (p less than 0.01); D content of layer IV was unchanged. D production from 10(-7) M 3,4-dihydroxyphenylalanine (DOPA) was (nanomoles per gram per minute) I, 0.2 +/- 0.03; II, 0.7 +/- 0.1; IV, 1.0 +/- 0.04. DOPA consumption was (nanomoles per gram per minute) I, 0.6 +/- 0.1; II, 1.4 +/- 0.3; and IV, 1.8 +/- 0.2. Denervation did not change D production or DOPA consumption.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of insulin receptors from rat kidney glomeruli and tubules

In order to directly compare the structural characteristics of renal glomerular and tubular insulin receptors, the purified isolated nephron subunits were extracted with 1% Triton X-102, fractionated by DEAE-Sephacel ion exchange column chromatography and the fractions containing insulin binding proteins were identified by the precipitation of 125I-insulin-protein complexes with polyethylene glycol (PEG). The fractions containing insulin binding proteins were pooled, incubated with 125I-insulin and covalently cross-linked with disuccinimidyl suberate, followed by chromatography of the cross-linked samples on Sepharose CL-6B. From both glomeruli and tubules, three 125I-insulin-binding complexes with molecular weights of 560 KDa, 220 KDa and 95 KDa were found. SDS-PAGE of these complexes from glomeruli and tubules under both reducing and nonreducing conditions gave similar patterns of 125I-insulin-crosslinked components, with the exception of the polypeptide pattern from the 560 KDa peak fraction which was markedly different between glomeruli and tubules with the former giving major labeled components at 170 and 68 KDa while the latter showed labeled components of 125 KDa and greater than 250 KDa. Glomerular and tubular insulin receptors, therefore, display similar subunit composition under reducing conditions, but differ in the non-reduced state, suggesting that these complexes may differ in the extent and/or nature of disulfide bonding.     

Distribution of various sites of hormonal action between microvessels, glomeruli and tubules isolated from the renal cortex of rabbits

Collagenase dispersion of rabbit kidney cortex followed by centrifugation on discontinuous sucrose gradient, allowed the simultaneous obtention of microvascular, glomerular and tubular fractions. Microscopic studies and the measurements of cellular renin activity and the muscle-specific-enzyme creatine kinase showed that these vessels were arteriolar in nature and that they contained the preglomerular arterioles. The glomerular or tubular contamination rates were assessed by means of enzymatic markers. AVP stimulated only the tubular fraction whether GTP was present or not. In the absence of GTP, beta-isoproterenol stimulated only the tubular enzyme while in the presence of GTP, the microvascular and especially the glomerular AC were also stimulated. GTP enhanced considerably the microvascular response to bPTH-(1-34) while, in the glomeruli and tubules the effects of GTP and hormones were only additive. The possible physiological significance of these results are discussed.     

The relationship between constitutive ATP release and its extracellular metabolism in isolated rat kidney glomeruli.

ATP and adenosine are important extracellular regulators of glomerular functions. In this study, ATP release from glomeruli suspension and its extracellular metabolism were investigated. Basal extraglomerular ATP concentration (1nM) increased several fold during inhibition of ecto-ATPase activity, reflecting the basal ATP release rate. Mechanical perturbation increased the amounts of ATP released from glomeruli. ATP added to glomeruli was almost completely degraded within 20 minutes. In that time, AMP was the main product of extracellular ATP metabolism. Significant accumulation of AMP was observed after 5 min (194 +/-16 microM) and 20 min (271 +/-11 microM), whereas at the same time concentration of adenosine was only 10 muM. A competitive inhibitor of ecto-5-nucleotidase alpha-beta-methylene-ADP (AOPCP), decreased extraglomerular ATP and adenosine concentration by 80% and 50%, respectively. Similarly, AMP (100 microM) also markedly reduced extraglomerular ATP accumulation, whereas IMP, its deamination product, was not effective. P1, P5-diadenosine pentaphosphate (Ap5A) - an inhibitor of ecto-adenylate kinase prevented significantly the disappearance of ATP from extraglomerular media caused by AMP. These findings demonstrate that the decrease in extracellular ATP concentration observed after addition of AOPCP or AMP is caused by the presence of ecto-adenylate kinase activity in the glomeruli. The enzyme catalyses reversible reaction 2ADP<->ATP+AMP, and a rise in the AMP concentration can lead to fall in ATP level. The present study provides evidence the extraglomerular accumulation of ATP reflects both release of ATP from glomeruli cells and its metabolism by ecto-enzymes. Our data suggest that AMP, produced from ATP in the Bowman's capsular space, might plays a dual role as a substrate for ecto-adenylate kinase and ecto-nucleotidase reactions being responsible for the regulation of intracapsular ATP and adenosine concentration. We conclude that AMP degrading and converting ecto-enzymes effectively determine the balance between ATP and adenosine concentration and thus the activation of P2 and/or adenosine receptors.     

Effect of glycerol on gluconeogenesis in isolated rabbit kidney cortex tubules

In renal tubules isolated from fed rabbits glycerol is not utilized as a glucose precursor, probably due to the rate-limiting transfer of reducing equivalents from cytosol to mitochondria. Pyruvate and glutamate stimulated an incorporation of [14C]glycerol to glucose by 50- and 10-fold, respectively, indicating that glycerol is utilized as a gluconeogenic substrate under these conditions. Glycerol at concentration of 1.5 mM resulted in an acceleration of both glucose formation and incorporation of [14C]pyruvate and [14C]glutamate into glucose by 2- and 9-fold, respectively, while it decreased the rates of these processes from lactate as a substrate. In the presence of fructose, glycerol decreased the ATP level, limiting the rate of fructose phosphorylation and glucose synthesis. As concluded from the 'cross-over' plots, the ratios of both 3-hydroxybutyrate/acetoacetate and glycerol 3-phosphate/dihydroxyacetone phosphate, as well as from experiments performed with methylene blue and acetoacetate, the stimulatory effect of glycerol on glucose formation from pyruvate and glutamate may result from an acceleration of fluxes through the first steps of gluconeogenesis as well as glyceraldehyde-3-phosphate dehydrogenase. As inhibition by glycerol of gluconeogenesis from lactate is probably due to a marked elevation of the cytosolic NADH/NAD+ ratio resulting in a decline of flux through lactate dehydrogenase.     

Lipoxygenase activity in rat kidney glomeruli, glomerular epithelial cells, and cortical tubules

We examined the possibility that renal glomerular and cortical tubular tissue has lipoxygenase activity in addition to the well established cyclo-oxygenase pathway of arachidonic acid metabolism. Homogenized rat kidney glomeruli, in the presence of meclofenamate (33 microM) and divalent cation ionophore A23187 (3 microM), metabolized octatritiated arachidonic acid to 12-hydroxyeicosatetraenoic acid and lesser amounts of 80 and/or 9-hydroxyeicosatetraenoic acid. These products were identified by thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectroscopy. In order to rule out the synthesis of hydroxylated fatty acids by platelets and leukocytes entrapped in the glomeruli, we studied lipoxygenase products in glomerular epithelial cells after 9 days in cell culture. Homogenized glomerular epithelial cells converted octatritiated arachidonic acid to 12-hydroxyeicosatetraenoic acid solely. The lipoxygenase activity in cortical tubules was substantially less than in glomeruli and only 12-hydroxyeicosatetraenoic acid was synthesized. The production of hydroxyeicosatetraenoic acid by lipoxygenase inhibitors, nordihydroguaiaretic acid, 5,-homogenized glomeruli, glomerular epithelial cells, and cortical tubules was inhibited by three 8,11,14-eicosatetraynoic acid, and 1-phenyl-3-pyrazolidone. These data demonstrate that there is lipoxygenase activity in rat kidney glomeruli, glomerular epithelial cells and to a lesser extent cortical tubules, and may imply a role of the lipoxygenase products in the regulation of normal glomerular function and inflammatory disease of the kidney.     

Kinetic characterization of phosphofructokinase isolated from rat kidney cortex.

1. Phosphofructokinase from rat kidney cortex has been purified by affinity chromatography to a final specific activity of 15 units per mg of protein, measured at 25 degrees C and pH 8. 2. This lower spec. act., compared with that of the enzyme from other sources, shows the enzyme in proximal tubules to be less active, which would account for the main gluconeogenic role of these nephron sections. 3. The binding of fructose-6-phosphate to the enzyme is co-operative. ATP increases the Hill coefficient and produces a marked allosteric inhibition on the activity. 4. Fructose-2,6-bis-phosphate is a potent activator of the enzyme from this source. It reduces the Hill coefficient of the enzyme and the inhibition constant of ATP. A marked difference between this and the liver enzyme is that the activation is not co-operative.     

Distribution of parathyroid hormone-sensitive adenylate cyclase in isolated rabbit renal cortex microvessels and glomeruli

The effect of the synthetic amino-terminal fragment of bovine parathyroid hormone, bPTH-(1-34), on the adenylate cyclase of microvessels and glomeruli isolated from rabbit kidney cortex was studied in the presence and absence of guanosine triphosphate (GTP). bPTH-(1-34) stimulated the vascular and glomerular adenylate cyclase in a dose-dependent manner with apparent ED50 values of 11.5 nM and 64 nM respectively, in the absence of GTP. 10(-4)M GTP greatly amplified the vascular response to bPTH-(1-34) while, in the glomeruli, both GTP and bPTH-(1-34) had only additive effects. In the presence of GTP, vascular and glomerular apparent ED50 were 190 nM and 64 nM respectively. [Nle8, Nle18, Tyr34] -bPTH-(3-34) amide, described as a PTH antagonist, inhibited the action of bPTH-(1-34) in the microvessels and to a lesser extent in the glomeruli. PTH is therefore a potent stimulator of adenylate cyclase in rabbit renal microvessels and glomeruli, and may play a role in the regulation of renal blood flow and glomerulo-tubular feedback control.     

Metabolism of arachidonic acid in isolated glomeruli from pig kidney

Arachidonic acid metabolism in isolated glomeruli from pig kidney was investigated. Arachidonic acid metabolism via cyclooxygenase was studied by three different methodological approaches: radioimmunoassay (RIA), high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). By all these techniques, the major prostaglandins (PG) formed by pig glomeruli appeared to be 6-keto-PGF1 alpha and PGF2 alpha, the former being the most abundant. RIA and GC-MS also detected lower amounts of thromboxane B2 (TxB2) and PGE2. This emphasises the similarity with human glomeruli, in which the main cyclooxygenase product has indeed been reported to be 6-keto-PGF1 alpha. The lipoxygenase activity in isolated pig glomeruli, as studied by HPLC, generated 15-HETE, 12-HETE and 5-HETE. These data demonstrate that isolated glomeruli from pig kidney possess cyclooxygenase as well as lipoxygenase activity. Since a marked functional similarity exists between human and pig kidney, the pig can be regarded as a good model for studying the influence of arachidonic acid metabolites on glomerular pathophysiology.     

Evidence from binding studies for beta 1-adrenoceptors associated with glomeruli isolated from rat kidney

The beta adrenoceptor antagonist radioligand [3H] dihydroalprenolol (DHA) has been used to characterise beta adrenoceptors in membranes prepared from rat renal glomeruli. Association of the ligand was rapid and had reached equilibrium within 10 mins at 37 degrees C. Dissociation occurred in two distinct phases, a rapidly dissociating phase (low affinity site) and a slowly dissociating phase (high affinity site). The KD value for the high affinity site calculated from the kinetic experiments was 0.8 nM. Saturation analysis of binding gave comparable values for KD (1.77 nM) and demonstrated that membranes from glomeruli had four times the density of binding sites measured in renal cortex. In all saturation studies Hill coefficients were not significantly different from unity. Binding was stereoselective with respect to the (-) isomers of isoprenaline and propranolol and the potency of the selective displacing agents betaxolol (beta 1 adrenoceptors) and ICI 118,551 (beta 2 adrenoceptors) indicated that the receptors are of the beta 1 subtype.     

Cells and intercellular contacts in glomeruli and tubules of the frog kidney

Summary By the use of thin sections and freeze-fracture replicas the glomerular and tubular structures of the kidney of the frog (Rana esculenta) were studied with special reference to intercellular junctions.In the glomerulus the filtration barrier is of very variable thickness, and frequent tight and gap junctional contacts occur between podocyte processes.Although structurally less elaborate, the proximal tubule resembles its mammalian counterpart. In the initial part the tight junctions are relatively shallow but become very broad in the mid and distal portions of the proximal tubule. The proximal tubular cells are extensively linked by gap junctions. In some animals the shapes of the cells in the proximal and distal portions of the proximal tubule were markedly different.The distal tubule consists of two segments which differ mainly in the pattern of interdigitations and the structure of the zonulae occludentes. Similarities with the tight junctional morphology of the mammalian distal tubule are striking. In the first part of the distal tubule (diluting segment) a narrow band of parallel tight junctions is found closely resembling that found in the mammalian straight distal tubule; in the more distal part of the distal tubule, however, a broad band of anastomosing tight junctional strands exists, like the zonula occludens of the mammalian convoluted distal tubule.The connecting tubule displays cellular dimorphism: its wall contains a mixture of light and dark (flask) cells. The luminal and basolateral membranes of the flask cells are covered with numerous rod-shaped particles. The tight junctions of the connecting tubule are broad and increase in depth and number of strands along its length; they are typical of a very tight epithelium.In spite of several dissimilarities with phylogenetically younger kidneys our findings suggest that many structural principles of the mammalian kidney are also represented in the kidneys of amphibians. The structural-functional relationships are discussed.