Synthesis of phlorizin derivatives and their inhibitory effect on the renal sodium/d-glucose cotransport system

To characterize further the Na⁺/d-glucose cotransport system in renal brush border membranes, phlorizin - a potent inhibitor of d-glucose transport - has been chemically modified without affecting the d-glucose moiety or changing the side groups that are essential for the binding of phlorizin to the Na⁺/d-glucose cotransport system. One series of chemical modifications involved the preparation of 3-nitrophlorizin and the subsequent catalytic reduction of the nitro compound to 3-aminophlorizin. From 3-aminophlorizin, 3-bromoacetamido-, 3-dansyl- and 3-azidophlorizin have been synthesized. In another approach, 3′-mercuryphlorizin was obtained by reaction of phlorizin with Hg(II) acetate. The phlorizin derivatives inhibit sodium-dependent but not sodium-independent d-glucose uptake by hog renal brush border membrane vesicles in the following order of potency: 3′-mercuryphlorizin = phlorizin > 3-aminophlorizin > 3-bromoacetamidophlorizin > 3-azidophlorizin > 3-nitrophlorizin > 3-dansylphlorizin. 3-Bromoacetamidophlorizin - a potential affinity label - also inhibits sodium-dependent but not sodium-independent phlorizin binding to brush border membranes. In addition, sodium-dependent phosphate and sodium-dependent alanine uptake are not affected by 3-bromoacetamidophlorizin. The results described above indicate that specific modifications of the phlorizin molecule at the A-ring or B-ring are possible that yield phlorizin derivatives with a high affinity and high specificity for the renal Na⁺/d-glucose cotransport system. Such compounds should be useful in future studies using affinity labeling (3-bromoacetamido- and 3-azidophlorizin) or fluorescent probes (3-dansylphlorizin).     

4-Azidophlorizin,a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes

A new phlorizin derivative ($\text{2′-O-(β-}\text{d}\text{-}\text{glucopyranosyl}\text{)-4-}\text{azidophloretin}$, 4-azidophlorizin) has been synthesized and its affinity for the d-glucose, Na⁺ co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) K′_i, the constant for fully-competitive inhibition of (Na⁺, Δψ)-dependent d-glucose uptake, (2) K′_d, the dissociation constant of 4-azido[³H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) K″_i, the constant for fully-competitive inhibition of the specific ((Na⁺, Δψ)-dependent, d-glucose protectable) high-affinity [³H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants (K′_i, K′_d and K″_i) were essentially equal and ranged between 3.2 and 5.2 μM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its K′_i values are some 15–20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244–248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.     

Structural state of the Na+ /d-glucose cotransporter in calf kidney brush-border membranes Target size analysis of Na+-dependent phlorizin binding and Na+-dependent d-glucose transport

Target sizes of the renal sodium-d-glucose cotransport system in brush-border membranes of calf kidney cortex were estimated by radiation inactivation. In brush-border vesicles irradiated at ?50°C with 1.5 MeV electron beams, sodium-dependent phlorizin binding, and Na⁺-dependent d-glucose tracer exchange decreased exponentially with increasing doses of radiation (0.4–4.4 Mrad). Inactivation of phlorizin binding was due to a reduction in the number of high-affinity phlorizin binding sites but not in their affinity. The molecular weight of the Na⁺-dependent phlorizin binding unit was estimated to be 230 000 ± 38 000. From the tracer exchange experiments a molecular weight of 345 000 ± 24 500 was calculated for the d-glucose transport unit. The validity of these target size measurements was established by concomitant measurements of two brush-border enzymes, alkaline phosphatase and γ-glutamyltransferase, whose target sizes were found to be 68 570 ± 2670 and 73 500 ± 2270, respectively. These findings provide further evidence for the assumption that the sodium-d-glucose cotransport system is a multimeric structure, in which distinct complexes are responsible for phlorizin binding and d-glucose translocation.     

Phlorizin as a probe of the small-intestinal Na+,d-glucose cotransporter. A model

(1)‘Uptake’ of phlorizin by intestinal brush border membrane vesicles is stimulated, much as that of d-glucose, by the simultaneous presence of Na_out⁺ and $\text{Δψ?0}$. However, phlorizin contrary to d-glucose, fulfills all criteria of a non-translocated ligand (i.e., of a fully competitive inhibitor) of the Na⁺,d-glucose cotransporter. (2) The stoicheiometry of Na⁺/phlorizin binding is 1, as shown by a Hill coefficient of approx. 1 in the Na_out⁺-dependence of phlorizin binding. (3) The preferred order of binding at $\text{Δψ?0}$ is Na⁺ first, phlorizin second (4) The velocity of association of phlorizin to the cotransporter, but not the velocity of its dissociation therefrom, responds to Δψ. These observations while agreeing with the effect of $\text{Δψ?0}$ on the $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of phlorizin binding in the steady-state time range, also confirm that the mobile part of the cotransporter bears a negative charge of 1. (5) A model is proposed describing the Na⁺,Δψ-dependent interaction of phlorizin with the cotransporter and agreeing with a more general model of Na⁺,d-glucose cotransport. (6) The $\text{k}{}_{\mathrm{<mtext>on</mtext>}}$, $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$ and $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ constants of phlorizin interaction with the Na⁺,d-glucose cotransporter are smaller in the kidney than in the small-intestinal brush border membrane, which results in a number of quantitative differences in the overall behaviour of the two systems.     

Carrier-mediated transfer of d-glucose in brush border vesicles derived rom rabbit renal tubules. Na+-dependent versus Na+-independent transfer

A brush border preparation from rabbit renal tubules containing a high yield of vesicles has been used to study the transfer of d-glucose through the brush border membrane. In the presence of an Na⁺ gradient across the vesicular membrane, the vesicles could concentrate d-glucose to a factor of 1.5, whereas in the absence of an Na⁺ gradient, only equilibrium with the medium was achieved. Two types of transfer could be distinguished by their requirement of Na⁺, their sensitivity to phlorizin and their pH optimum. The Na⁺-independent transfer was about 100 times less sensitive to phlorizin than the Na⁺-dependent path and exhibited a pH optimum between 7 and 8, whereas the Na⁺-dependent transfer was highest at a pH between 8 and 9.The brush border preparation could be freed of most of the contaminating material derived from the basal and lateral tubular cell membrane by a discontinuous density gradient centrifugation. It still showed both forms of transfer to a similar extent, indicating that both are located in the brush border membrane.A study of the sensitivity of d-glucose transfer to phlorizin, in the presence and absence of Na⁺ at different temperature, suggests a single carrier species functioning in two interchangeable conformational states with different affinities for phlorizin rather than two transfer systems working independently.     

Temperature dependence of solute transport and enzyme activities in hog renal brush border membrane vesicles

The temperature dependence of sodium-dependent and sodium-independent d-glucose and phosphate uptake by renal brush border membrane vesicles has been studied under tracer exchange conditions. For sodium-dependent d-glucose and phosphate uptake, discontinuities in the Arrhenius plot were observed. The apparent activation energy for both processes increased at least 4-fold with decreasing temperature. The most striking change in the slope of the Arrhenius plot occurred between 12 and 15°C. The sodium-independent uptake of d-glucose and phosphate showed a linear Arrhenius plot over the temperature range tested (35–5°C). The behavior of the transport processes was compared to the temperature dependence of typical brush border membrane enzymes. Alkaline phosphatase as intrinsic membrane protein showed a nonlinear Arrhenius plot with a transition temperature at 12.4°C. Aminopeptidase M, an extrinsic membrane protein exhibited a linear Arrhenius plot. These data indicate that the sodium-glucose and sodium-phosphate cotransport systems are intrinsic brush border membrane proteins, and that a change in membrane organization alters the activity of a variety of intrinsic membrane proteins simultaneously.     

Transport of d-glucose by brush border membranes isolated from the renal cortex

The transport of d-glucose by brush border membranes isolated from the rabbit renal cortex was studied. At concentrations less than 2 mM, the rate of d-glucose uptake increased linearly with the concentration of the sugar. No evidence was found for a “high-affinity” (μM) saturable site. Saturation was indicated at concentrations of d-glucose greater than 5 mM. The uptake of d-glucose was stereospecific and selectively inhibited by d-galactose and other sugars. Phlorizin inhibited the uptake of d-glucose in the presence and absence of Na⁺. The glycoside was a potent inhibitor of the efflux of d-glucose. Preloading the brush border membrane vesicles with d-glucose, but not with l-glucose, accelerated exchange diffusion of d-glucose. These results demonstrate that the uptake of d-glucose by renal brush borders represents transport into an intravesicular space rather than solely binding. The rate of d-glucose uptake was increased when the Na⁺ in the extravesicular medium was high and the membranes were preloaded with a Na⁺-free medium. The rate of d-glucose uptake was inhibited by preloading the brush border membranes with Na⁺. These results are consistent with the Na⁺ gradient hypothesis for d-glucose transport in the kidney. Thus, the presence of a Na⁺-dependent facilitated transport of d-glucose in isolated renal brush border membranes is indicated. This finding is consistent with what is known of the transport of the sugar in more physiologically intact preparations and suggests that the membranes serve as an effective model system in examining the mechanism of d-glucose transport in the kidney.     

Na+ uptake through the brush border membranes of intestine from fresh water and sea-water adapted trout (Salmo gairdneri,R.)

Summary A comparative study of the mechanisms of Na⁺ absorption through brush border membranes of enterocytes from freshwater (FW) and seawater (SW) adapted trout were carried out using purified vesicle preparations. In contrast to FW trout, SW trout were found to possess a Na⁺–K⁺–Cl^– cotransport process. This finding is regarded as a major adaptation to SW since this cotransport allows an increase of ions and water absorption. Both FW and SW trout were equipped with a Na⁺–H⁺ exchange. In FW, the intestine of the trout had both a Na⁺–Na⁺ exchange and a Na⁺ conductance which may be responsible for enterocyte Na⁺ uptake along the potential gradient.     

Na-dependent transport of tricarboxylic acid cycle intermediates by renal brush border membranes. Effects on fluorescence of a potential-sensitive cyanine dye

The effect of the transport of tricarboxylic acid cycle intermediates on the membrane potential of renal brush border vesicles was studied using fluorescence of the cyanine dye, 3,3′-dipropylthiadicarbocyanine iodide. The behavior of the dye in the preparation was established with valinomycin-induced K⁺-diffusion potentials; increases in fluorescence were associated with depolarizing conditions. Addition of 1 mM succinate or citrate to membrane/dye suspensions produced transient increases in fluorescence, indicative of a depolarizing event(s) associated with the transport of these substrates. The transient response in fluorescence was Na⁺ dependent, of greater magnitude under Na⁺-gradient as compared to Na⁺-equilibrium conditions, and was a saturable function of substrate concentration. The specificity of the fluorescence response was identical to that obtained from studies of the competitive inhibition of succinate transport by tricarboxylic acid cycle intermediates and analogs. We conclude that the major tricarboxylic acid cycle intermediates are transported via a common Na⁺-dependent transport system in renal brush border membranes.     

Stimulation by HCO 3 − of Na+ transport in rabbit gallbladder

Summary Bicarbonate presence in the bathing media doubles Na⁺ and fluid transepithelial transport and in parallel significantly increases Na⁺ and Cl^– intracellular concentrations and contents, decreases K⁺ cell concentration without changing its amount, and causes a large cell swelling. Na⁺ and Cl^– lumen-to-cell influxes are significantly enhanced, Na⁺ more so than Cl^–. The stimulation does not raise any immediate change in luminal membrane potential and cannot be due to a HCO ₃ ^– -ATPase in the brush border. The stimulation goes together with a large increase in a Na⁺-dependent H⁺ secretion into the lumen. All of these data suggests that HCO ₃ ^– both activates Na⁺–Cl^– cotransport and H⁺–Na⁺ countertransport at the luminal barrier.Thiocyanate inhibits Na⁺ and fluid transepithelial transport without affecting H⁺ secretion and HCO ₃ ^– -dependent Na⁺ influx. It reduces Na⁺ and Cl^– concentrations and contents, increases the same parameters for K⁺, causes a cell shrinking, and abolishes the lumen-to-cell Cl^– influx. It enters the cell and is accumulated in the cytoplasm with a process which is Na⁺-dependent and HCO ₃ ^– -activated. Thus, SCN^– is likely to compete for the Cl^– site on the cotransport carrier and to be slowly transferred by the cotransport system itself.     

Phenylalanine uptake in isolated renal brush border vesicles

The uptake of l-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques.Brush border microvilli but not basolateral plasma membrane vesicles take up l-phenylalanine by an Na⁺-dependent, saturable transport system. The apparent affinity of the transport system for l-phenylalanine is 6.1 mM at 100 mM Na⁺ and for Na⁺ 13 mM at 1 mM l-phenylalanine. Reduction of the Na⁺ concentration reduces the apparent affinity of the transport system for l-phenylalanine but does not alter the maximum velocity.In the presence of an electrochemical potential difference for Na⁺ across the membrane (η_Na0 >η_Na1) the brush border microvilli accumulate transiently l-phenylalanine over the concentration in the incubation medium (overshoot phenomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K⁺ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient.These results indicate that the entry of l-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na⁺ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na⁺ and on the electrical potential difference. The exit of l-phenylalanine across the basolateral plasma membranes is Na⁺-independent and probably involves facilitated diffusion.     

Small-intestinal Na+/d-glucose cotransport. Inactivation of sugar transport and phlorizin binding by thiol-group and amino-group reagents

It has previously been shown that mercurials acting from the cytoplasmic side or from within the hydrophobic part of the membrane inactivate the small intestinal Na⁺/d-glucose cotransporter by blocking essential SH-groups (Klip, A., Grinstein, S. and Semenza, G. (1979) Biochim. Biophys. Acta 558, 233–245). Another (set of) sulfhydryl(s) which are critical for phlorizin binding and sugar transport function and which may lie on the luminal side of the brush border membrane, can be blocked by DTNB and 4,4′-dithiopyridine but not by $\text{N-}\text{ethylmaleimide}$. In addition, modification of amino groups by fluorescamine, reductive methylation and (under certain conditions) DIDS also lead to inactivation of the carrier's binding and transport functions. No evidence was obtained that any of the above groups is directly involved in the binding of either Na⁺/d-glucose or phlorizin, since none of these compounds prevented inactivation of the cotransporter.     

Identification and reconstitution of a Na/K/Cl cotransporter and K channel from luminal membranes of renal red outer medulla

Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na⁺/K⁺/Cl⁻ cotransport system and a K⁺ channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of ⁸⁶Rb⁺ uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two ⁸⁶Rb⁺ fluxes. (A) A furosemide-inhibited ⁸⁶Rb⁺ flux in the absence of Na⁺ (K⁺-K⁺ exchange). This flux is stimulated by an inward Na⁺ gradient (Na⁺/K⁺ cotransport) and is inhibited also by bumetanide. (B) A Ba²⁺-inhibited ⁸⁶Rb⁺ flux, through the K⁺ channel. Luminal membranes containing the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channels, and basolateral membranes containing the Na⁺/K⁺ pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column.     

Sidium-dependent ion cotransport in steady-state Ehrlich ascites tumor cells

Summary The Ehrlich tumor cell possesses and anion-cation cotransport system which operates as a bidirectional exchanger during the physiological steady state. This cotransport system, like that associated with the volume regulatory mechanism (i.e. coupled net uptake of Cl^–+Na⁺ and/or K⁺) is Cl^–-selective and furosemide-sensitive, suggesting the same mechanism operating in two different modes. Since Na⁺ has an important function in the volume regulatory response, its role in steady-state cotransport was investigated. In the absence of Na⁺, ouabain-insensitive K⁺ and DIDS-insensitive Cl^– transport (KCl cotransport) are low and equivalent to that found in 150mm Na⁺ medium containing furosemide. Increasing the [Na⁺] results in parallel increases in K⁺ and Cl^– transport. The maximum rate of each (18 to 20 meq/(kg dry wt)·min) is reached at about 20mm Na⁺ and is maintained up to 55mm. Thus, over the range 1 to 55mm Na⁺ the stoichiometry of KCl cotransport is 11. In contrast to K⁺ and Cl^–, furosemide-sensitive Na⁺ transport is undetectable until the [Na⁺] exceeds 50mm. From 50 to 150mm Na⁺, it progressively rises to 7 meq/(kg dry wt)·min, while K⁺ and Cl^– transport decrease to 9 and 16 meq/(kg dry wt)·min, respectively. Thus, at 150mm Na⁺ the stoichiometric relationship between Cl^–, Na⁺ and K⁺ is 211. These results are consistent with the proposal that the Cl^–-dependent cation cotransport system when operating during the steady state mediates the exchange of KCl for KCl or NaCl for NaCl; the relative proportion of each determined by the extracellular [Na⁺].     

Effect of K and H on sodium/citrate cotransport in renal brush-border vesicles

The uptake of citrate by renal brush-border vesicles, prepared according to the method of Vannier, occurs by Na⁺-linked cotransport. It is ‘positive rheogenic’, i.e., stimulated by an (inside) negative, and inhibited by an (inside) positive electrical potential. The question arises whether, besides Na⁺, other ions (e.g., K⁺ and H⁺) participate in the cotransport. As to K⁺, neither an inward nor an outward directed K⁺ gradient has a significant effect on the citrate movement, but at equal concentrations of K⁺ inside and outside, equilibrium exchange of citrate, and to a smaller extent, the Na⁺-linked net uptake of citrate, are significantly stimulated. This observation is consistent with a hypothetical model in which K⁺ acts by accelerating both the empty and the fully loaded translocator. As to H⁺, citrate uptake is also stimulated by decreasing extravesicular pH, an effect previously attributed to protonization of the citrate anion in the assumption that the resulting secondary citrate anion is more acceptable to the translocator site. It was found, however, that the pH effect is still apparent if the concentration of the secondary citrate is kept constant by adjusting the total citrate concentration. This is taken as an argument against the above assumption and as being consistent with H⁺-linked cotransport. After the overshoot peak citrate exits slowly, and even after several hours does not attain equilibrium distribution, presumably owing to trapping by vesicular calcium.     

Effect of angiotensin-II on renal Na/H exchanger-NHE3 and NHE2

The purpose of the present study was to determine the effect of angiotensin II (A-II) on membrane expression of Na⁺/H⁺ exchange isoforms NHE3 and NHE2 in the rat renal cortex. A-II (500 ng/kg per min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. Arterial pressure and circulating plasma A-II level were significantly increased in A-II rats as compared to control rats. pH-dependent uptake of ²²Na⁺ study in the presence of 50 μM HOE-694 revealed that Na⁺ uptake mediated by NHE3 was increased ∼88% in the brush border membrane from renal cortex of A-II-treated rats. Western blotting showed that A-II increased NHE3 immunoreactive protein levels in the brush border membrane of the proximal tubules by 31%. Northern blotting revealed that A-II increased NHE3 mRNA abundance in the renal cortex by 42%. A-II treatment did not alter brush border NHE2 protein abundance in the renal proximal tubules. In conclusion, chronic A-II treatment increases NHE3-mediated Na⁺ uptake by stimulating NHE3 mRNA and protein content.     

Ketone body transport in renal brush border membrane vesicles

Ketone body uptake by renal brush border vesicles has been investigated. Ketone bodies enter into the brush border vesicles by a carrier-mediated process. The uptake is dependent on an Na⁺ gradient ([Na⁺]outside > [Na⁺]inside) and is electroneutral. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. A pH gradient (alkaline inside) also stimulates the ketone body uptake. Acetoacetate and 3-hydroxybutyrate share the same carrier as demonstrated by the accelerated exchange diffusion and mutual inhibitory effects.     

Characterization of a Na+/glucose cotransporter cloned from rabbit small intestine

Summary The Na⁺/glucose cotransporter from rabbit intestinal brush border membranes has been cloned, sequenced, and expressed inXenopus oocytes. Injection of cloned RNA into oocytes increased Na⁺/sugar cotransport by three orders of magnitude. In this study, we have compared and contrasted the transport properties of this cloned protein expressed inXenopus oocytes with the native transporter present in rabbit intestinal brush borders. Initial rates of¹⁴C--methyl-d-glucopyranoside uptake into brush border membrane vesicles andXenopus oocytes were measured as a function of the external sodium, sugar, and phlorizin concentrations. Sugar uptake into oocytes and brush borders was Na⁺-dependent (Hill coefficient 1.5 and 1.7), phlorizin inhibitable (K _i 6 and 9 m), and saturable (-methyl-d-glucopyranosideK _m 110 and 570 m). The sugar specificity was examined by competition experiments, and in both cases the selectivity wasd-glucose>-methyl-d-glucopyranoside>d-galactose>3-O-methyl-d-glucoside. In view of the close similarity between the properties of the cloned protein expressed in oocytes and the native brush border transporter, we conclude that we have cloned the classical Na⁺/glucose cotransporter.     

Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na/K-ATPase

Background

Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na⁺/K⁺-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na⁺/K⁺-ATPase expression and activity in rats injected with Bothrops alternatus snake venom.

Methods

Male Wistar rats were injected with venom (0.8 mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na⁺/K⁺-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively.

Results

Venom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na⁺/K⁺-ATPase α₁ subunit were increased 6 h post-venom, whereas Na⁺/K⁺-ATPase activity increased 6 h and 24 h post-venom.

Conclusions

Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na⁺/K⁺-ATPase expression and activity in the early phase of renal damage.

General significance

Enhanced Na⁺/K⁺-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage.     

Interactions between Na+-dependent uptake of d-glucose,phosphate and l-alanine in rat renal brush border membrane vesicles

d-Glucose decreases phosphate reabsorption in rat proximal tubule. It is also postulated that some amino acids interact with phosphate reabsorption. To investigate the mechanism of these interactions, phosphate, d-glucose and l-alanine transport kinetics were measured in brush border membrane vesicles isolated from superficial rat kidney cortex by the calcium precipitation technique. At pH 7.4, Na⁺-dependent phosphate transport was inhibited in the presence of either d-glucose (39 mM) or l-alanine (2.4 mM). In this model, with d-glucose or with l-alanine the $\text{V}$ value of the phosphate uptake was decreased, whereas the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for the phosphate uptake was not affected. However, some inhibition of phosphate transport was observed in the presence of l-glucose, d-alanine or d-glucose after phlorizin preincubation. A 30% Na⁺-dependent l-alanine (0.1 mM) transport inhibition was observed in the presence of 5 mM phosphate. d-Glucose (1 mM) was also inhibited by 20% when 5 mM phosphate was added to incubation medium. According to several authors, in our model, d-glucose decreased the l-alanine transport and vice versa. Moreover, when the membrane potential was abolished, a clear inhibition of d-glucose by l-alanine persisted. These multiple interactions could be explained by the accelerated dissipation of the Na⁺ gradient insofar as the rate of the Na⁺ uptake was increased with d-glucose, l-alanine or phosphate and since the absence of variations in membrane potential did not suppress these inhibitions.