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The ability of renal cortical slices to accumulate a monosaccharide is enhanced at temperatures below 37 degrees C. Increase in concentration gradients occurs despite a decrease in total sugar flux. The apparent explanation for this paradox is a proportionally greater inhibition of efflux.
During compensatory renal growth 45Ca2+ transport in basal-lateral plasma membrane vesicles isolated from the rat renal cortex have been investigated. Stimulation of Ca2(+)-ATPase activity was observed, without an effect of compensatory renal growth on Na+/Ca2+ exchanger activity and on passive Ca2+ permeability of the vesicles. Twelve hours following unilateral nephrectomy about 40% increase of Ca2(+)-ATPase activity above control value was observed and this effect was present until the end of the experimental period (7 days). When kinetic parameters for Ca2(+)-ATPase were studied in native membranes, an increase of Vmax was observed, whereas the Km for Ca2+ was similar in control vesicles and vesicles isolated from the remnant kidney. Depletion of endogenous calmodulin resulted in a decrease of Vmax and an increase of Km (Ca2+), while its addition reversed these parameters and increased the Hill coefficient from about 1 to about 2. Once again, only a significant increase of Vmax in vesicles isolated from the remnant kidney above the control value was observed. Finally, increase of Ca2(+)-ATPase activity during compensatory renal growth could be abolished by actinomycin D, indicating that its stimulation is due to protein synthesis.
Although selenium is taken with diet mainly as selenoamino acids, its hypoglycaemic action on hepatic gluconeogenesis has been studied with the use of inorganic selenium derivatives. The aim of the present investigation was to compare relative efficacies of inorganic and organic selenium compounds in reducing glucose synthesis in hepatocytes and renal tubules, significantly contributing to the glucose homeostasis. In contrast to hepatocytes, both selenite and methylselenocysteine inhibited renal gluconeogenesis by about 40-45% in control rabbits. Selenate did not affect this process, whereas selenomethionine inhibited gluconeogenesis by about 20% in both hepatocytes and renal tubules. In contrast to methylselenocysteine, selenite decreased intracellular ATP content, glutathione reduced/glutathione oxidized (GSH/GSSG) ratio and pyruvate carboxylase, PEPCK and FBPase activities, while methylselenocysteine diminished PEPCK activity due to elevation of intracellular 2-oxoglutarate and GSSG, inhibitors of this enzyme. Experiments in vivo indicate that in 3 of 9 alloxan-diabetic rabbits treated for 14 days with methylselenocysteine (0.182mg/kg body weight) blood glucose level was normalized, whereas in all diabetic rabbits plasma creatinine and urea levels decreased from 2.52+/-0.18 and 87.4+/-9.7 down to 1.63+/-0.11 and 39.0+/-2.8, respectively. In view of these data selenium supplementation might be beneficial for protection against diabetes-induced nephrotoxicity despite selenium accumulation in kidneys and liver.
Basolateral membrane vesicles were isolated from rat renal proximal tubules by a Percoll-centrifugation method. Transport of phosphate could be stimulated by a sodium gradient but transport of D-glucose was not or only slightly affected by sodium. The Percoll-basolateral membrane fraction showed sodium-independent trans-stimulation of phosphate by itself. To test whether sodium stimulation of phosphate transport is influenced by the crosscontamination of basolateral with brush border membranes, the basolateral membrane fraction obtained by the Percoll-method was applied to free-flow electrophoresis for further purification. Thereby a separation of basolateral from brush border marker enzymes was obtained. It was possible to correlate net sodium-dependent phosphate transport with the brush border marker enzyme activity. ATP-dependent calcium uptake and cytochalasin B sensitive, sodium-independent D-glucose uptake followed basolateral, whereas phlorizin sensitive, sodium-dependent D-glucose followed brush border marker enzymes. Sodium dependent phosphate uptake was inhibited by D-glucose, this inhibition was released by phlorizin. It is concluded that the sodium-dependent phosphate uptake present in the Percoll-basolateral membrane fraction is due to crosscontamination with brush border membranes and that phosphate translocation across the basolateral membrane is carrier-mediated and sodium-independent.
1. Kinetics of influx (mediated through peptide-proton cotransport) of two labelled dipeptides has been studied in apical membrane vesicles isolated from rat renal cortex. The substrates (neutral D-Phe-L-Ala and anionic D-Phe-L-Glu) have previously been shown to be transported through a single system but with different stoichiometry of proton coupling. 2. The initial rate of influx of both peptides was determined under a set of defined conditions allowing extravesicular pH, intravesicular pH, transmembrane pH and membrane potential (Em) to be varied systemically and independently. From this data the kinetic constants K(m) and Vmax were derived for each condition. Very substantial effects of pH, pH gradient and membrane potential were found; there were consistent quantitative differences when the substrates were compared. 3. Efflux of the two peptides from preloaded vesicles was also determined. At pH 5.5 (intra- and extravesicular), but not at pH 7.4, the rate constants for efflux of the two peptides were similar and addition to the extravesicular medium of unlabelled D-Phe-L-Glu (but not D-Phe-L-Ala) trans-stimulated efflux of both peptides to a similar extent; the extent of this trans-stimulation was insensitive to alterations in membrane potential. 4. A model based on a combination of classical carrier theory (the carrier being negatively charged) and of two sequential protonation steps (both to external sites predicted to be in the membrane electrical field) is described. Qualitatively this adequately accounts for all the observations made and allows for the dependence of the stoichiometry of proton-peptide coupling on the net charge carried by the substrate. Quantitatively a 50-fold greater rate of reorientation of the free carrier when unprotonated is predicted to be responsible for the coupling of proton and peptide transport. 5. Our results and the model are discussed with respect to the recently elucidated primary structure of mammalian peptide transporters.
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Estrogen replacement therapy is cardioprotective in postmenopausal women; however, the precise molecular mechanisms for this modulation are not fully elucidated. We previously showed that chronic estrogen replacement therapy reduced angiotensin-converting enzyme (ACE) activity in tissue extracts and serum with an associated reduction in plasma angiotensin II. A reverse transcriptase-polymerase chain reaction assay was developed to determine whether estrogen treatment regulates tissue ACE mRNA concentration. Total RNA was isolated from kidney cortex, kidney medulla, lung, and aorta of ovariectomized Sprague-Dawley rats after 21 days of chronic 17beta-estradiol replacement therapy (5 mg pellet per rat SC) or placebo. A marked decrease in densitometric intensity ratios of amplified ACE cDNA to elongation factor-1alpha control cDNA was observed in all tissues from placebo-treated rats compared with the estradiol-treated rats (renal cortex: 0.29+/-0.04 versus 0.14+/-0.02; renal medulla: 0. 37+/-0.04 versus 0.24+/-0.03; lung: 4.49+/-0.37 versus 2.49+/-0.59; and aorta: 0.41+/-0.04 versus 0.29+/-0.02; all P<0.05). A comparable reduction in ACE activity was detected in tissue extracts from kidney cortex, kidney medulla, and lung of hormone-treated animals. Incubation of purified rat lung ACE with 1 or 10 micromol/L 17beta-estradiol had no effect on enzyme activity. These results suggest that estrogen treatment regulates tissue ACE activity by reducing ACE mRNA concentrations. Thus, the beneficial cardiovascular effects of estrogen may be mediated in part by downregulation of ACE with a consequent reduction in the circulating levels of the vasoconstrictor angiotensin II, a decrease in the metabolism of the vasodilator bradykinin, and an increase in the production of the vasorelaxant angiotensin-(1-7).
Concentration and excretion in 24-hour urine, as well as serum concentrations of Na, K, Mg, Ca, Cl, P, uric acid and citrate were investigated in 209 calcium oxalate stone patients and 42 stone-free patients. Especially the concentration values of the urine components, except for uric acid and citrate, were found to be significantly lower for calcium oxalate stone patients. 21% of the stone patients showed hypercalciuria; hypercalciuria combined with hyperuricuria was found in only 7.1% of the cases and a solitary hyperuricuria in only 17%. As for kidney cortex, kidney papilla and muscle tissue in 10 calcium oxalate stone patients and 10 stone-free patients, the concentrations of Na, K, Ca, Mg as well as some trace elements were determined quantitatively by means of neutron activation analysis. Statistic analysis yielded a significantly lower sodium content of the kidney cortex within the stone-carrying group. Mean values of the calcium concentration in stone patients were lower for papilla and muscle tissue than in the control group. For magnesium no clear differences were found. The iron content in the papilla and muslce tissue of stone patients was significantly lower.
Angiotensin-II-cleaving angiotensinase A (aminopeptidase A, E.C. 3.4.11.7, ATA) plays an important role in glomerular haemodynamics. the pathophysiology of essential arterial hypertension and the induction of vascular disorders. In order to study biochemical and immunological properties of ATA, two isoforms (I and II) of the glycoprotein were isolated for the first time from human kidney cortex. Kidney cortex homogenate, digested with bromelain, was fractionated by ammonium sulphate precipitation and subsequent hydrophobic interaction chromatography, using a fast protein liquid chromatographic (FPLC) system. By anion-exchange FPLC (Mono Q column), the isoforms of ATA were eluted in two distinct peaks and were further purified by size-exclusion FPLC and preparative polyacrylamide gel electrophoresis. Biochemical, immunological and immunohistological characterization disclosed differences in the intrarenal localization, glycosylation Michaelis constant and apparent molecular mass (native and sodium dodecyl sulphate gel electrophoresis) but similar properties in the double-immunodiffusion technique. Polyclonal rabbit antibodies, raised against ATA isoforms I and II, precipitated an analogous antigen in urine from patients with renal tubular damage.
It has been known for several years that the outwardly rectifying 30-pS chloride channel, the regulation of which has been reported to be defective in cystic fibrosis, can be activated by excision of a membrane patch from a cell. This suggested that the cytosol contains an inhibitory factor, which diffuses away after excision, thereby releasing the channel block. To test for such an inhibitor we have isolated cytosol from two epithelial cell lines, and in larger quantities from pig kidney cortex. Kidney cortex was chosen because published and unpublished evidence suggested that proximal tubular cells might also have a tonically suppressed Cl- conductance in the brush-border membrane, which is activated during isolation of membrane vesicles. The inhibitory effect of the cytosol preparations was assessed by: (a) measuring conductive Cl- fluxes on renal proximal tubular brush-border membrane vesicles preloaded with or without cytosol, and (b) recording single Cl- channel currents from excised membrane patches of nasal polyp epithelia and CFPAC-1 cells in the presence and absence of cytosol. All cytosol preparations tested were found to inhibit both conductive Cl- flux in membrane vesicles and single Cl- channels in patch-clamp experiments. In the latter case a type of flicker block was observed with a reduction of channel open probability. Stepwise dilution of the cytosol consistently reduced the inhibitory potency. Since the inhibition was preserved after boiling the cytosol for 10 min, we conclude that the inhibitor is a heat-stable substance. Whether it is identical with the postulated intracellular regulator that couples the defective function of the cystic fibrosis gene product to Cl- channel inhibition cannot be decided at present.
Concentrations of inorganic mercury (IHg), methylmercury (MeHg), and total mercury (THg) were determined for autopsy samples from 46 Japanese subjects. Two laboratories (Labs A and B) participated in Hg analyses: Lab A for THg and IHg and Lab B for THg and MeHg. Total mercury concentration values were in good agreement between the two laboratories: the averages were several hundreds of ng/g in kidney cortex, kidney medulla, and liver, and were several tens of ng/g in cerebrum, cerebellum, heart, and spleen. Inorganic mercury accumulated more in kidney and liver: its percentage THg was 81-84% in the kidney, 67% in the liver, 25% in the heart, 22% in the spleen, 20% in the cerebrum, and 14% in the cerebellum. Methylmercury levels in tissues were uniform through all organs except the liver. Approximately 80% was in the form of MeHg in the cerebrum, cerebellum, heart, and spleen, whereas the values were 33%, 15%, and 11% in the liver, kidney medulla, and kidney cortex, respectively. Age was a significant factor in increased IHg concentrations in the cerebrum and heart, decreased values of %MeHg in the cerebrum, cerebellum, and heart, and increased values of %IHg in the cerebrum and heart.