Rat Kidney Plasma Membranes Research Articles

The Effect of Selenium Treatment On-Diabetic-Induced Structural Variations in the Molecules of Rat Kidney Plasma Membrane

Diabetes Mellitus is a metabolic disorder affecting the great amount of world's population, in which fat, protein and carbohydrate metabolism is severely affected by deficient insulin secretion or function. In this study, the Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy was used to study diabetic kidney disease-induced structural changes, which encountered as a complication of diabetes. Furthermore, the protecting and possible therapeutic role of selenium in the course of diabetic kidney disease disclosed. The detailed spectral analysis of ATR-FTIR spectroscopy revealed that, protein and saturated lipid content of diabetic kidney plasma membrane prominently diminished. The decrease in the unsaturated lipid content indicates diabetes-induced lipid peroxidation. Nevertheless, the administration of selenium at low and medium concentrations improved the condition by changing the lipid and protein content to the normal values. The ordered structure of plasma membrane lipids due to diabetes turned back to healthy structure with the selenium treatment. The diabetes caused the decrease of membrane dynamics however; selenium treatment increased the dynamics of membrane. Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA) applied to the control, diabetic and selenium treated groups revealed clear separation of the groups with high heterogeneity in the lipid and protein spectral regions. These chemometric methods show that, low and medium dose selenium treated groups successfully segregated from diabetic group and clustered close to the control group which indicates recovery effect of selenium at these concentrations in diabetic animals. To conclude, lipid and protein structure and content of the diabetic kidney plasma membranes deteriorated, which restored after selenium administration, more preferentially at low dose. The results of the study suggest selenium treatment at appropriate dose may be related to insulin mimetic and antioxidant properties of selenium.

FT‐IR spectroscopy in diagnosis of diabetes in rat animal model

In recent years, Fourier Transform Infrared (FT-IR) spectroscopy has had an increasingly important role in the field of pathology and diagnosis of disease states. In the current study, FT-IR spectroscopy together with cluster analysis were used as a diagnostic tool in the discrimination of diabetic samples from control ones in rat kidney plasma membrane apical sides (brush-border membranes), liver microsomal membranes and Extensor digitorum longus (EDL) and Soleus (SOL) skeletal muscle tissues. A variety of alterations in the spectral parameters, such as frequency and signal intensity/area was observed in diabetic tissues and membranes compared to the control samples. Based on these spectral variations, using cluster analysis successful differentiation between diabetic and control groups was obtained in different spectral regions. The results of this current study further revealed the power and sensitivity of FT-IR spectroscopy in precise and automated diagnosis of diabetes.

Solubilization of Rat Kidney Plasma Membrane Proteins Associated with 3H-Aldosterone

The treatment of rat kidney plasma membranes with sodium dodecyl sulphate (SDS) did not essentially affect the ability of the membranes for 3H-aldosterone binding as compared with the intact plasma membranes (Ozegović et al., 1977). A gel filtration of 3H-aldosterone-kidney plasma membranes complex on Sepharose 6B yielded 2 protein and 2 3H-aldosterone peaks. The proteins which were eluted in the first peak were associated with the first 3H-aldosterone peak while the second 3H-aldosterone peak was eluted with Ve corresponding to Ve of free 3H-aldosterone. Spironolactone, a competitive antagonist of aldosterone, prevented the binding of 3H-aldosterone to the membrane proteins. The results demonstrated a high affinity of the kidney plasma membranes solubilized with SDS and a specificity of aldosterone binding to the plasma membrane proteins of higher molecular mass.

Platelet-derived growth factor stimulates the release of protein kinase A from the cell membrane.

The mitogenic action of growth factors involves the stimulation of intracellular protein kinases. In this report we have characterized the major protein kinase released from Balb/c 3T3 and normal rat kidney plasma membranes by the action of platelet-derived growth factor (PDGF). PDGF appears to stimulate the release of approximately 10 proteins, at least one of which is a kinase capable of phosphorylating proteins on Ser or Thr (as determined by the lability of the phosphate to alkali treatment). More than 90% of the Ser/Thr kinase activity was inhibited by PKI5-22, a specific peptide inhibitor of the cAMP-dependent protein kinase (PKA). We used immunoblotting to confirm that the kinase released in response to PDGF was PKA. cAMP also stimulated the release of PKA, and the set of protein substrates phosphorylated was similar following PDGF or cAMP stimulation. Interestingly, in the presence of a cAMP analogue ((Rp)-cAMPS), cAMP could not induce dissociation of PKA from the membranes, whereas stimulation by PDGF increased the level of PKA activation. Furthermore, unlike Swiss 3T3 cells, neither Balb/c 3T3 fibroblasts nor normal rat kidney cells accumulate cAMP in response to PDGF, yet the level of PKA in the cytosol of these intact cells increases in response to PDGF. Thus, it appears as though PDGF activation of the membrane-associated form of the PKA holoenzyme occurs by a mechanism independent of an elevation in cAMP levels.

Localization of epidermal growth factor (EGF) binding sites on antiluminal plasma membrane of rat kidney: autoradiographic study using nonfiltering perfused rat kidney.

We previously demonstrated that the specific binding of EGF to the antiluminal plasma membrane was a prerequisite step for the renal uptake of EGF. In the present study, the localization of 125I-EGF binding sites on the antiluminal plasma membrane was investigated by tissue sampling and X-ray autoradiography in the nonfiltering kidney. The binding of 125I-EGF was recognized over the whole kidney and was highest in the inner medulla followed by the cortex and outer medulla. The binding of 125I-EGF in the nonfiltering kidney was completely inhibited in the presence of 20 nM unlabeled EGF, suggesting specific binding of 125I-EGF to its receptor. Further, we used a histologic tissue staining method to confirm the location of the 125I-EGF binding sites. Binding of 125I-EGF was demonstrated on the proximal straight tubules (PST), cortical collecting ducts (CCD), inner medullary collecting ducts (IMCD), and thin limb of Henle in the inner medulla (IMTLH). We found that the binding of 125I-EGF was high in the IMTLH. In addition, we determined the grain density both on the cell surface membrane and in the intracellular space of the proximal straight tubules, where the grain density on the antiluminal plasma membrane was approximately 50% that in the intracellular space at 20 min after the start of 125I-EGF perfusion, suggesting the internalization of 125I-EGF from the antiluminal plasma membrane to the intracellular compartment. In conclusion, the binding sites of 125I-EGF, which were accessible from the antiluminal side, were broadly distributed over the whole kidney and were most dense around the IMTLH.

Steroid hormone specifically binds to rat kidney plasma membrane

A high-affinity and low-capacity corticosterone specific binding was detected in the purified plasma membrane preparation from rat kidney using an in vitro steroid hormone binding assay. The specific-bound hormone was efficiently distinguished from the irreversible-bound hormone with 10 microM corticosterone. Under standardized conditions of pH 7.4 at 2 degrees C and 30 min incubation time, the binding was saturable and showed Kd = 13 +/- 3 nM and Bmax = 616 +/- 34 fmol/mg of protein. Competitive binding studies with analogue steroids indicated that corticosterone binding to kidney plasma membrane is hormone-specific. Results indicated that the possible nongenomic effects of steroids could be mediated by their interaction with plasma membrane.

Release of alkaline phosphodiesterase I from rat kidney plasma membrane produced by the phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis.

The release of plasma-membrane-bound enzymes by phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis was investigated. Among the ectoenzymes of plasma membrane tested, alkaline phosphodiesterase I was released markedly from rat kidney cortex slices, in addition to alkaline phosphatase and 5'-nucleotidase. Other membrane-bound enzymes; alanine aminopeptidase, leucine aminopeptidase, dipeptidyl peptidase, leucine aminopeptidase, dipeptidyl peptidase IV, esterase and gamma-glutamyl transpeptidase could not be liberated from the treated slices. Alkaline phosphodiesterase I was released linearly from rat kidney slices with the concentration of phosphatidylinositol-specific phospholipase C, but little enzyme was released from rat liver slices. Alkaline phosphodiesterase I separated from kidney tissue with n-butanol still retained phosphatidylinositol and was transformed into a lower molecular weight form by phosphatidylinositol-specific phospholipase C. This suggests an important function for phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membrane of rat kidney cells. The alkaline phosphodiesterase I released from rat kidney had a molecular weight of about 240,000 and an isoelectric point (pI) of 5.4. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9 and had a Km value of 0.3 mM. The enzyme was activated by Mg2+ and Ca2+, but was inhibited by EDTA. Strong inhibition took place on the addition of adenosine 5'-phosphosulfate or the nucleotide pyrophosphates, i.e., UDP-galactose and alpha, beta-methylene ATP.

Alterations in plasma membrane proteins associated with the proteolytic activation of adenylate cyclase

Treatment of intact normal rat kidney fibroblasts, or of purified NRK plasma membranes, with trypsin or papain markedly enhances adenylate cyclase activity [ATP pyrophosphatelyase (cyclizing) EC 4.6.1.1]. Limited proteolysis (25 μg/ml trypsin for 7 min) of confluent cells grown with unheated calf serum significantly increases cyclase activity, whereas similar treatment of sparse cells causes only a marginal increase in cyclic AMP formation. To determine which membrane protein(s) is altered under conditions which result in proteolytic activation of adenylate cyclase, purified plasma membranes and intact normal rat kidney cells were subjected to limited proteolysis and membrane proteins analyzed by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. Membranes prepared from intact confluent normal rat kidney cells exposed to mild trypsinization showed a decrease in proteins of 56,000, 46,000, 37,000, and 32,000 daltons. Trypsin treatment of intact, sparse cells does not activate the cyclase system and does not lead to modification of the 46,000-dalton membrane protein. Treatment of purified normal rat kidney plasma membranes results in the loss of numerous bands in the high molecular mass region (>150,000 daltons) as well as decreases membrane proteins of 56,000, 49,000, 46,000, and 23,000 daltons. Compared with trypsin, the proteolytic action of papain appears to be quite specific, causing a discernible decrease in only the 46,000-dalton protein. The correlation between modification of the 46,000-dalton membrane component and the activation of the cyclase system suggests that perhaps this protein is proteolytically modified to elicit activation of adenylate cyclase.

Partial characterization of a glutathione oxidase present in rat kidney plasma membrane fraction

The previously reported glutathione oxidizing activity of isolated renal cells was recovered in the plasma membrane fraction of a rat kidney homogenate. Glutathione disulfide and hydrogen peroxide were formed during the reaction which was dependent on the access to molecular oxygen and inhibited by KCN and EDTA, but not by NaN 3. The EDTA-inhibited activity could be restored by addition of CuSO 4, but not FeCl 3, to the plasma membrane fraction after dialysis. The results strongly suggest that a Cu-protein present in the plasma membrane is responsible for the glutathione oxidase activity of renal cells.

DIURETICS AND THE RENAL ADENYLATE CYCLASE SYSTEM

1 The relationship between the diuretic effectiveness and the effect on the renal adenylate cyclase of three diuretics, acetazolamide, frusemide and ethacrynic acid, was examined. The hypothesis that acetazolamide and parathyroid hormone (PTH), inhibit renal carbonic anhydrase by a cyclic adenosine 3',5'-monophosphate (cyclic AMP)-dependent mechanism was also tested.2In vitro, acetazolamide, frusemide and ethacrynic acid at high concentrations (10(-3)M) all produced some inhibition of basal and stimulated rat kidney plasma membrane adenylate cyclase. The effect of acetazolamide was much less than that of frusemide and ethacrynic acid. These plasma membrane effects were reproduced in studies of cyclic AMP formation in isolated kidney tubules of rats.3 Intravenous injections of acetazolamide did not change the total cyclic AMP content of the kidneys of rats killed by microwave irradiation.4 Acetazolamide produced a diuresis in the rat and a slight inhibition of the antidiuretic effect of Pitressin. Frusemide produced a diuresis and greatly reduced the antidiuretic response to Pitressin. Ethacrynic acid was ineffective as a diuretic in the rat and actually enhanced the antidiuretic response to Pitressin.5 In investigating the possible influence of diuretics and PTH on the activity and state of phosphorylation of carbonic anhydrase it was found that: there was no correlation between the ability of diuretics to inhibit carbonic anhydrase activity and to inhibit carbonic anhydrase phosphorylation; neither PTH nor cyclic AMP (in the presence of adenosine triphosphate, Mg(2+), K(+) and incubation at 37 degrees C) inhibited rat cortex homogenate carbonic anhydrase activity.6 It seems unlikely that any of the tested diuretics exerts its pharmacological effect by means of changes in kidney cyclic AMP metabolism.

Interaction of [ 3H]-aldosterone with rat kidney plasma membranes

The interaction of [ 3H]-aldosterone with isolated rat kidney plasma membranes has been studied by centrifugation and gel filtration methods. The Scatchard plot analysis of data indicated the affinity of aldosterone for membrane sites, expressed as equilibrium constant K diss , to be 1.3 × 10 −8M and the concentration of binding sites ( n) 1.69 × 10 −13 mol/mg protein. The high concentration of unlabelled aldosterone present in the incubation medium, or injected into rats 45 min before killing, decreased the amount of labelled aldosterone bound to plasma membranes. Gel filtration on Sephadex G-200 and disc electrophoresis on polyacrylamide gel of solubilized [ 3H]-aldosterone-renal plasma membranes complex showed that the hormone was bound to the high molecular weight protein component of the plasma membranes. The proteinaceous nature of membrane component which bound aldosterone was determined using delipidated plasma membranes.

Myo-Inositol transport in plasma membrane of rat kidney

The myo-inositol transport system in kidney plasma mambrane preparation was investigated. myo-Inisitol uptake was more rapid than that due to non-specific uptake. Specific myo-inisitol uptake was temperature dependent and pH sensitive; the optimum was at pH 7.4. Specific myo-insitol uptake was inhibited by scyllitol and inosose-2 but not(+)-inositol, d-glucose, d-galactose or mannitol. Inhibition of myo-inositol uptake by scyllitol was of the competitive type. It showed that the transport system is stereospecific and that myo-inositol shares the transport system with scyllitol. Moreover, the specific myo-inositol uptake was inhibited by phlorizin. Counter transport of myo-inositol was demonstrated. The results indicate that myo-inositol uptake by the membrane preparation represents the entry into the intravesicular spaces rather than binding to the membrane. It was concluded that the plasma membrane of rat kidney has a cyclitol carrier system specific to myo-inositol and scyllitol.

Binding of [ 3H] phlorizin to rat kidney plasma membranes

Binding of [ 3H] phlorizin to rat kidney plasma membranes

Glycoprotein: glycosyl transferase activities of rat kidney plasma membranes.

Rat kidney plasma membrane preparations and suspensions of intact glomeruli were used to study glycoprotein: glycosyl transferase activities present on cell surfaces. Collagen: glucosyl, fetuin: galac

Properties of a synthetic plasma membrane marker: fluorescent-mercury-dextran.

Aminoethyl-Dextran T 10 (mol wt approx. 10,000) was conjugated withp-Chloromercuribenzoic acid (pCMB) and labeled with fluorescein isothiocyanate (FITC). This coupling procedure does not affect the mercurial function ofpCMB moiety of Fluorescent Mercury Dextran T 10 (FMD) since on the basis of mercury content itsK i-value for the (Na+−K+)-ATPase from rat kidney plasma membranes is identical with theK i -value of unconjugatedpCMB (3×10−6 m). FMD binds to plasma membranes if applied in vivo, which could be shown in experiments in which rat kidneys were perfused with FMD and the plasma membranes isolated after the perfusion. The membrane-FMD complex is stable during common isolation steps such as differential centrifugation, sucrose density gradient centrifugation and free-flow electrophoresis. This was shown by in vitro binding studies of FMD with isolated plasma membranes from rat kidney cortex. FMD may be removed from the plasma membranes by the addition of 1×10−4 m dithiotreitol. Since F-aminoethyl-Dextran T 10 (withoutpCMB) does not interact with the plasma membranes, it is suggested that the binding of FMD to plasma membranes may involve a Hg-SH reaction. FMD does not penetrate into rat kidney cells in contrast topCMB, but can cross capillaries. Thus, FMD seems to be suitable (a) to label luminal and contraluminal surfaces of plasma membranes of epithelial structures, and (b) to be used as a fluorescent marker for plasma membranes during succeeding isolation procedures.

The characterization of rat kidney plasma membranes prepared by zonal centrifugation.

1. Plasma membranes were isolated from a 10000g-min pellet prepared from a renal cortical homogenate in 20mm-NaHCO(3) by isopycnic centrifugation in a linear sucrose gradient in an ;A'-type zonal rotor. 2. The preparation was characterized by electron microscopy, and alkaline phosphatase, 5'-nucleotidase, l-leucine beta-naphthylamidase and l-leucine p-nitroanilidase activities were found to be selectively associated with the renal plasma membrane. 3. The preparation had a high degree of purity, as indicated by the presence of low activities of marker enzymes associated with subcellular organelles. A preliminary chemical analysis indicated that the chemical composition resembled that of plasma membranes of other tissues. 4. Plasma membranes were also prepared from tubular fragments and their enzyme contents were found to be similar to those of plasma membranes prepared from cortical homogenates. 5. l-Leucine beta-naphthylamidase, l-leucine p-nitroanilidase and 5'-nucleotidase were not enriched to the same extent as alkaline phosphatase in the preparation of plasma membranes from tubular fragments. A possible explanation for this finding is discussed.

Effect of mineralocorticoid agonists and antagonists on binding of 3H-aldosterone to adrenalectomized rat kidney plasma membranes

Binding of 3H-aldosterone to plasma membranes of adrenalectomized rat kidney has been demonstrated in vivo and in vitro . Steroids with mineralocorticoid activity, deoxycorticosterone or 9-α-fluorohydrocortisone (agonists), or aldosterone antagonists (spironolactone, progesterone) markedly reduced the binding of 3H-aldosterone to renal plasma membranes. Steroids without agonist or antagonist properties (testosterone, 17-β-estradiol) were not effective. The 3H-aldosterone-membrane complex was resistent to solubilization and subsequent chromatography employing Sephadex G-25. These experiments indicate that binding of 3H-aldosterone to adrenalectomized rat kidney plasma membranes may be involved in the cellular mode of action of aldosterone.

Characterization of the adenyl cyclase of rat kidney plasma membranes

1. 1. This paper examines the characteristics of a renal plasma membrane adenyl cyclase with respect to NaF and hormonal activation, enzyme kinetic parameters, effects of solubilization procedures on adenyl cyclase activity, and gel-filtration chromatography of a “soluble” membrane adenyl cyclase. 2. 2. Renal membrane adenyl cyclase was stimulated by NaF, vasopressin and parathyroid hormone in a concentration-dependent manner. 3. 3. A K m for ATP of 0.1 mM was obtained for both the renal cortex and medulla membrane adenyl cyclase whereas the divalent cation dependency was found to be: cortex, Mg 2+ > Mn 2+ > Co 2+; medulla, Mg 2+ = Mn 2+ > Co 2+, for sustaining adenyl cyclase activity in the presence of NaF. 4. 4. The particulate plasma membrane adenyl cyclase was solubilized by use of the detergent, Lubrol WX or by intense shear forces with a French pressure cell. Addition of NaF to the membrane suspension prior to solubilization was necessary for protection of adenyl cyclase activity. Gel-filtration chromatography with 6 % agarose gave two adenyl cyclase peaks of different specific activity and molecular size.

Characterization of Plasma Membrane Proteins in Mammalian Kidney: II. The Binding of Organic Mercurials to Rat Kidney Plasma Membrane Proteins in vivo

Abstract The binding of organic mercurial compounds to the proteins of kidney plasma membrane preparations has been examined following the administration in vivo of organic mercurial compounds labeled with 203Hg. When the kidney cells are fractionated, plasma membrane radioactivity is lower than radioactivity in the cytoplasm. The radioactivity appears to be bound to membrane protein and experiments with 14C-labeled p-chloromercuribenzoate and 3H-labeled chlormerodrin indicate that the binding 2 hours after administration is mostly that of free mercury. When plasma membranes are solubilized and chromatographed on 6% agarose gel, two major protein peaks are eluted. p-Chloromercuribenzoate label is localized primarily in the high molecular weight proteins of the initial void volume peak. Chlormerodrin, a compound with diuretic activity in the kidney, is distributed fairly evenly in the protein fractions of both column peaks. Electrophoretic data, however, suggest heterogeneity of the protein labeling by chlormerodrin. Sodium-potassium-dependent ATPase activity is found in several fractions of the first peak eluted from the agarose gel column. When ouabain, a known inhibitor of the ATPase system, is administered in vivo, it depresses the chlormerodrin radioactivity of the first peak.