Renal Cortical Brush Border Membrane Research Articles

Kinetic characteristics of folate binding to rat renal brush border membrane in chronic alcoholism

The binding of folic acid to the plasma membrane is an important event for its reabsorption and conservation by renal epithelium. We studied [3H] folic acid binding to the renal cortical brush border membrane vesicles (BBMV) in rats after 12 weeks of chronic ethanol ingestion. Our results demonstrated that after chronic ethanol intake, the binding of folic acid to the membrane vesicles: (1) was decreased by a mechanism that decreased B(max) without affecting K(d), (2) was insensitive to Na+ ions in the medium (3) behaved differently to divalent cations in the medium in contrast to control group. However under such conditions there was no significant effect of ethanol on pH optimum of the process. Binding was reduced at pH less than 6 but there was no effect of ethanol on binding in pH range from 6 to 8. Increasing the osmolarity at pH 5.5 had no effect on the binding of folate to BBMV, thus confirming that the observed changes in B(max) values were due to site-specific binding in the two groups. Moreover, ethanol does not impart its effect on involvement of sulfhydryl group at the binding site of folate transport system. These findings highlight the possible mechanism of perturbed renal conservation of folate during chronic alcoholism.

Inhibition of renal Na +/H + exchange in cadmium-intoxicated rats

Chronic exposure to cadmium (Cd) results in bicarbonaturia, leading to metabolic acidosis. To elucidate the mechanism(s) by which renal bicarbonate reabsorption is inhibited, we investigated changes in renal transporters and enzymes associated with bicarbonate reabsorption in Cd-intoxicated rats. Cd intoxication was induced by subcutaneous injections of CdCl 2 (2 mg Cd/kg per day) for 3 weeks. Cd intoxication resulted in a significant reduction in V max of Na +/H + antiport with no changes in K Na in the renal cortical brush-border membrane vesicles (BBMV). Western blotting of BBM proteins and indirect immunohistochemistry in renal tissue sections, using an antibody against Na +/H + exchange-3 (NHE3), showed a diminished expression of NHE3 protein in the BBM. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that NHE3 mRNA expression was reduced in the renal cortex. The activity of carbonic anhydrase IV (CA IV) in BBM was not changed. The protein abundance of Na +-HCO 3 − cotransporter-1 (NBC1) in whole kidney membrane fractions was slightly attenuated, whereas that of the Na +-K +-ATPase α-subunit was markedly elevated in Cd-intoxicated animals. These results indicate that Cd intoxication impairs NHE3 expression in the proximal tubule, thereby reducing the capacity for bicarbonate reabsorption, leading to bicarbonaturia in an intact animal.

Atrial natriuretic peptide and endothelin-3 target renal sodium-glucose cotransporter

Atrial natriuretic peptide (ANP) and endothelin (ET) are endogenous vasoactive factors that exert potent diuretic and natriuretic actions. We have previously shown that ANP and ET-3 act through an NO pathway to inhibit the sodium-glucose cotransporter (SGLT) in the intestine [Gonzalez Bosc LV, Elustondo PA, Ortiz MC, Vidal NA. Effect of atrial natriuretic peptide on sodium-glucose cotransport in the rat small intestine. Peptides 1997; 18: 1491–5; Gonzalez Bosc LV, Majowicz MP, Ortiz MC, Vidal NA. Effects of endothelin-3 on intestinal ion transport. Peptides 2001; 22: 2069–75.]. Here we address the role of ANP and ET-3 on SGLT activity in renal proximal tubules. In rat renal cortical brush border membranes (BBV), fluorescein isothiocianate (FITC) labeling revealed a specific 72-kD peptide that exhibits increased FITC labeling in the presence of Na + and d-glucose. Using α- 14 C -methylglucose active uptake, rat BBV were shown to possess SGLT activity with an affinity constant ( K 0.5∼2.4 mM) that is consistent with the expression of the low-affinity, high-capacity SGLT2 isoform. SGLT2 activity in these preparations is dramatically inhibited by ANP and ET-3. This inhibition is independent of changes in membrane lipids and is mimicked by the cGMP analogue, 8-Br-cGMP, suggesting the involvement of cGMP/PKG pathways. These results are the first demonstration that both ANP and ET-3 inhibit rat cortical renal SGLT2 activity, and suggest a novel mechanism by which these vasoactive substances modulate hydro-saline balance at the proximal tubular nephron level.

Effect of high protein diet on stone-forming propensity and bone loss in rats

High protein diets are believed to cause kidney stone formation and bone loss, but the mechanisms mediating these changes are unknown. The purpose of this study was to create an animal model of animal protein excess and to evaluate the response of kidney and bone to the dietary protein load. Rats (12 per group) were pair-fed with a high (48%) and low (12%) casein diets that were otherwise identical in their content of sodium, potassium, calcium, phosphorus, and magnesium. Compared with the low casein group, the high casein group delivered a substantial acid load during 59 days of study, since it significantly decreased urinary pH, and increased urinary ammonium, titratable acidity, and net acid excretion. Animals on high casein diet also had higher urinary volumes. On the high casein diet, urinary calcium excretion was significantly higher and urinary citrate excretion and concentration was significantly lower. On the high casein diet, urinary saturation of calcium phosphate was higher. Serum calcitriol concentration did not significantly differ between the two groups. Histomorphometric analysis of femur procured after 59 days on the diet showed marked increase in bone resorption in the high casein group. Hypocitraturia was associated with increased activity of sodium-citrate cotransporter in renal cortical brush-border membranes (BBM) in the high casein group. Both the kidney and bone contribute to the pathogenesis of hypercalciuria during high casein diet in rats. Hypocitraturia is probably renal in origin. This rat model will be useful in elucidating the mechanisms by which high protein intake increases the risk of nephrolithiasis and bone loss in human beings.

Evaluation of the fluidity and functionality of the renal cortical brush border membrane in experimental diabetes in rats

The present study was aimed at addressing the effect of hyperglycemia on the renal cortical brush border membrane. The fluidity and the functionality of the renal cortical brush border membrane have been evaluated after 6 weeks of streptozotocin-induced diabetes in rats. Lipid peroxidation and protein oxidation were first performed to confirm a state of oxidative stress. The fluidity of the brush border membrane of diabetic rats decreased significantly by 15.76%. There was an increase in the amount of early (19.39%) and advanced (42.23%) glycation end-products suggesting the accumulation of significant amount of non-enzymic glycation products at 6 weeks of diabetes. Although, the activities of both γ-glutamyl transpeptidase and alkaline phosphatase of the brush border membrane decreased, that of the latter decreased to a significant extent with an increase in K m (81%) and no change in the V max. A study of the activities of glutathione-dependent antioxidant enzymes in the renal cortical homogenates showed that the activities of glutathione peroxidase and glyoxalase II were altered significantly. Our study seems to suggest that increased free radical generation accompanied by non-enzymic glycation may be responsible for oxidative stress and an increased rigidity of the diabetic brush border membrane. Alkaline phosphatase may thus serve as a potentially useful marker of free radical induced damage to the renal cortical brush border membrane. The results also suggest that enhanced susceptibility to oxidative stress during early stages may be an important factor in the development of secondary complications of diabetes.

Cd-MT causes endocytosis of brush-border transporters in rat renal proximal tubules.

Nephrotoxicity in humans and experimental animals due to chronic exposure to cadmium (Cd) is manifested by defects in the reabsorptive and secretory functions of proximal tubules (PT). The main symptoms of Cd nephrotoxicity, including polyuria, phosphaturia, aminoaciduria, glucosuria, and proteinuria, suggest that various brush-border membrane (BBM) transporters are the main targets of Cd. Specific transporters may be either directly inhibited by Cd or lost from the BBM after Cd treatment, or both. We have recently proposed that Cd may impair the vesicle-dependent recycling of BBM transporters by inhibiting vacuolar H+-ATPase (V-ATPase) activity and endocytosis in PT cells (Herak-Kramberger CM, Sabolic I, and Brown D. Kidney Int 53: 1713-1726, 1998). The mechanism underlying the Cd effect was further explored in an in vivo model of experimental Cd nephrotoxicity induced by Cd-metallothionein (Cd-MT; 0.4 mg Cd/kg body mass; a single dose sc) in rats. The time-dependent redistribution of various BBM transporters was examined in this model by fluorescence and gold-labeling immunocytochemistry on tissue sections and by immunoblotting of isolated renal cortical BBM. In PT cells of Cd-MT-treated rats, we observed 1) shortening and loss of microvilli; 2) time-dependent loss of megalin, V-ATPase, aquaporin-1 (AQP1), and type 3 Na+/H+ exchanger (NHE3) from the BBM; 3) redistribution of these transporters into vesicles that were randomly scattered throughout the cell cytoplasm; and 4) redistribution of NHE3, but not megalin, into the basolateral plasma membrane. The internalization of BBM transporters was accompanied by fragmentation and loss of microtubules and by an increased abundance of alpha-tubulin monomers in PT cells. Transporter redistribution was detectable as early as 1 h after Cd-MT treatment and increased in magnitude over the next 12 h. We conclude that the early mechanism of Cd toxicity in PT cells may include a colchicine-like depolymerization of microtubules and impaired vesicle-dependent recycling of various BBM proteins. These processes may lead to a time-dependent loss of cell membrane components, resulting in reabsorptive and secretory defects that occur in Cd-induced nephrotoxicity.

The integrity of renal cortical brush-border and basolateral membrane vesicles is damaged in vitro by nephrotoxic heavy metals

Poisoning of experimental animals with cadmium (Cd), mercury (Hg), lead (Pb) or cis-diamminedichloroplatinum (cis-Pt) causes shortening and focal loss of microvilli in proximal tubule (PT) cells, thus indicating that the reduced reabsorptive surface due to damaged integrity of brush-border membrane (BBM) may contribute to the reabsorptive and secretory defects in these toxic states. In addition, in in vitro studies with isolated renal cortical BBM vesicles (BBMV), heavy metals (HM) inhibit transport of various compounds, and these data were interpreted as being a result of a direct inhibition of the respective membrane transporters. In this work we used a ΔpH-driven acridine orange fluorescence quench assay to test if various divalent cations affect in vitro the integrity of BBMV and basolateral membrane vesicles (BLMV) isolated from the rat renal cortex. In Cd-treated BBMV we found that: (a) the integrity of vesicles decreased with increasing concentrations of Cd; and (b) the loss of sealed vesicles was high at 37°C, intermediate at 25°C, and very low at 0°C. The loss of sealed BBMV was caused also by Hg, Cu, Pb and Zn (Hg⋙Cu=Cd>Pb=Zn). Cis-Pt, Al, Fe, Ba, Mg and Mn had no effect. BLMV were damaged by HM with an efficiency Hg⋙Cd=Pb=Cu, whereas other divalent cations, including Zn, were ineffective. An SH-group protector, dithiothreitol, prevented the loss of sealed vesicles in some (Hg, Pb, Cu) but not in all (Cd, Zn) cases. We conclude that the nephrotoxic HM directly damage the integrity of PT cell plasma membranes; this may cause shortening and loss of microvilli and basolateral invaginations in HM-treated experimental animals in vivo. The data also indicate that caution should be taken when effects of HM on various transports are studied in isolated membrane vesicles in vitro; an impaired transport may result from the loss of vesicle integrity, and not necessarily from the direct inhibition of a transporter.

Potassium depletion: renal membrane lipid metabolism and Na/H exchanger abundance in aged F344 x BNF1 rats.

Potassium depletion (-K) is a common electrolyte abnormality in elderly humans, occurring after diuretic use or poor oral intake. We hypothesized that aging would result in an increase in renal membrane lipid metabolism in both control and -K, and that the Na/H exchanger's protein abundance to -K would be blunted. Young and senescent non-obese male Fisher 344 x Brown-Norway F1 rats (F344 x BNF1) were fed either a normal or a K-deficient diet for 7 days. At 24-h, 32P incorporation was measured for renal cortical brush-border (BBM) and basolateral membrane (BLM) lipid metabolism. All -K animals showed a reduction in total body potassium stores, a fall in plasma aldosterone, a urinary concentrating defect, and an increase in plasma cholesterol and urine ammonium excretion (p < .001). In BBM of both age groups, -K increased phosphatidylserine, sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine concentrations, but 32P incorporation fell. In BLM of young K-depleted rats, however, only phosphatidylcholine concentration increased. In the hypokalemic aged rats, the concentration of all BLM phospholipids rose, whereas 32P incorporation fell. In both membranes, cholesterol concentration and the molar ratio of cholesterol to total phospholipid increased with -K (p < .05). Potassium depletion caused brush-border membrane NHE-3 protein abundance to rise, but only in the young rats. Neither NHE-3 nor basolateral NHE-1 protein abundance was affected in aged animals with -K. These results provide the first evidence, in non-obese aged rats, that selective age-associated modifications occur in membrane lipid metabolism and membrane transporter protein abundance during -K. That aging causes a maladaptive response in brush-border NHE-3 protein expression may have important implications for elderly humans, particularly if they are given diuretics and become potassium depleted.

Proton-coupled oligopeptide transport by rat renal cortical brush border membrane vesicles: a functional analysis using ACE inhibitors to determine the isoform of the transporter

We demonstrate that the angiotensin-converting enzyme inhibitors enalapril and captopril inhibit the transport of D-Phe-L-Gln into PepT1-expressing Xenopus oocytes and into rat renal cortical brush border membrane vesicles (BBMV). The kinetics of inhibition are competitive. Enalapril and captopril are not substrates for PepT2 (Boll et al., Proc. Natl. Acad. Sci. 93 (1996) 284–289). Therefore we conclude that in rat renal cortical BBMV this neutral dipeptide is transported via PepT1.

Abnormal sulfate metabolism in vitamin D-deficient rats.

To explore the possibility that vitamin D status regulates sulfate homeostasis, plasma sulfate levels, renal sulfate excretion, and the expression of the renal Na-SO4 cotransporter were evaluated in vitamin D-deficient (D-D-) rats and in D-D- rats rendered normocalcemic by either vitamin D or calcium/lactose supplementation. D-D- rats had significantly lower plasma sulfate levels than control animals (0.93+/-0.01 and 1.15+/-0.05 mM, respectively, P < 0.05), and fractional sulfate renal excretion was approximately threefold higher comparing D-D- and control rats. A decrease in renal cortical brush border membrane Na-SO4 cotransport activity, associated with a parallel decrease in both renal Na-SO4 cotransport protein and mRNA content (78+/-3 and 73+/-3% decreases, respectively, compared with control values), was also observed in D-D- rats. Vitamin D supplementation resulted in a return to normal of plasma sulfate, fractional sulfate excretion, and both renal Na-SO4 cotransport mRNA and protein. In contrast, renal sulfate excretion and renal Na-SO4 cotransport activity, protein abundance, and mRNA remained decreased in vitamin D-depleted rats fed a diet supplemented with lactose and calcium, despite that these rats were normocalcemic, and had significantly lower levels of parathyroid hormone and 25(OH)- and 1,25(OH)2-vitamin D levels than the vitamin D-supplemented groups. These results demonstrate that vitamin D modulates renal Na-SO4 sulfate cotransport and sulfate homeostasis. The ability of vitamin D status to regulate Na-SO4 cotransport appears to be a direct effect, and is not mediated by the effects of vitamin D on plasma calcium or parathyroid hormone levels. Because sulfate is required for synthesis of essential matrix components, abnormal sulfate metabolism in vitamin D-deficient animals may contribute to producing some of the abnormalities observed in rickets and osteomalacia.

Effect of acute increases in filtered [formula omitted] on renal hydrogen transporters: II. H+-ATPase

Adaptive increases in renal bicarbonate reabsorption occur in response to acute increases in filtered bicarbonate (FLHCO3). In a previous study, we showed that an increase in FLHCO3 induced by plasma volume expansion increased the Vmax for Na+/H+ exchange activity in renal cortical brush border membrane vesicles (BBMV), providing a potential mechanism for the adaptive increase in HCO3- reabsorption. The present studies were undertaken to determine whether the increase in FLHCO3 induced by plasma expansion also stimulates the other major H+ transporter in cortical BBMV, the H(+)-ATPase. H(+)-ATPase activity was assessed in BBMV obtained from hydropenic and plasma expanded Munich-Wistar rats, using a NADH-linked ATPase assay. H(+)-ATPase activity was measured as the ouabain and oligomycin-insensitive, bafilomycin A1-sensitive component of total ATPase activity. Acute plasma expansion doubled single nephron FLHCO3, and this change was associated with a 64% increase in the Vmax for H(+)-ATPase activity, with no change in apparent Km. The Vmax for H(+)-ATPase activity correlated directly with whole kidney GFR and FLHCO3 (r = 0.68 and 0.72, respectively), and with single nephron GFR and FLHCO3 (r = 0.76 and 0.80, respectively). Thus, the mechanism for the adaptive increase in proximal tubular HCO3- reabsorption that occurs in response to acute increases in FLHCO3 appears to be related to increased activity of both H(+)-ATPase and Na+/H+ exchange in the apical membrane of the proximal tubule epithelium.

Effect of Cadmium on Na–PiCotransport Kinetics in Rabbit Renal Brush-Border Membrane Vesicles

Chronic exposure to cadmium impairs various renal functions, including phosphate (Pi) transport. To further investigate the mechanism of cadmium-induced alterations in renal Pitransport, kinetics of Na+-dependent Piuptake were studied in renal cortical brush-border membrane vesicles (BBMV) exposed to CdCl2in vitro.BBMVs isolated from rabbit renal outer cortex were preincubated in a buffer containing CdCl2(50 μmin most cases) for 60 min at 37°C and then tested for Piuptake at 25°C. CdCl2treatment resulted in a marked attenuation of Na+-dependent Piuptake with no changes in Na+-independent Piuptake and membrane permeability to Na+. CdMt treatment induced no changes in Pitransport. The inhibition required preincubation of vesicles with CdCl2for more than 30 min and was not reversed by extravesicular EDTA, suggesting that cadmium affects the transport system at the internal side of the membrane. Kinetic analysis indicated that two sodium ions and one phosphate ion interact with a carrier, and this stoichiometry was not altered by cadmium treatment. Cadmium treatment did not change the apparentKmfor Na+(KNa) and that for phosphate (KPi), but it markedly reduced theVmaxof the Na+-dependent Pitransport. These results indicate that exposure of proximal tubular brush border membranes to cadmium impairs the Pitransport capacity, probably by reducing the effective number of Na–Picotransporter units without altering substrate affinities of the carrier.

Human stanniocalcin inhibits renal phosphate excretion in the rat.

Stanniocalcin (STC) is a glycoprotein hormone first identified in bony fishes where it counteracts hypercalcemia by inhibiting gill calcium uptake and stimulating renal inorganic phosphate (Pi) reabsorption. Human STC (hSTC) has recently been cloned and sequenced and is highly homologous to the fish hormone at the amino acid level. The objective of this study was to examine the possible effects of hSTC on electrolyte homeostasis and renal function in the rat. Recombinant hSTC was expressed in bacteria and purified by metal-ion affinity chromatography and reverse-phase high performance liquid chromatography. Anesthetized animals were given bolus infusions of 1, 5, or 10 nmol hSTC per kilogram of body weight. Control animals received solvent alone. The most effective dosage was 5 nmol/kg, which caused significant reductions in both absolute and fractional phosphate excretion in comparison with control rats. The hSTC had no effect on the renal excretion of other ions, the glomerular filtration rate, renal blood flow, blood pressure, or plasma electrolytes (Na+, K+, Ca2+, Pi, Mg/+). The maximum effect of hSTC on phosphate excretion was observed 60-80 minutes postinjection. Lesser effects were obtained with higher and lower dosages of hormone. When renal cortical brush-border membrane vesicles were isolated from control and hormone-treated animals 80 minutes postinjection, the rate of Na+/Pi cotransport was found to be 40% higher in vesicles from hormone-treated animals (p < 0.01; 5 nmol hSTC/kg). Together, the renal clearance and membrane vesicle data indicate that hSTC participates in the renal regulation of Pi homeostasis in mammals.

Chronic metabolic acidosis increases NHE3 protein abundance in rat kidney.

Chronic metabolic acidosis increases the activity of the proximal tubule apical membrane Na/H antiporter, which is encoded predominantly by the NHE3 isoform. The present studies examined the effect of chronic metabolic acidosis on apical membrane NHE3 protein abundance in rats. Rats subjected to NH4Cl in their drinking water developed a metabolic acidosis, which decreased in magnitude over 14 days. During this time, renal cortical brush-border membrane NHE3 protein abundance, assessed by Western blot, increased progressively (28% at 3 days, 59% at 7 days, and 90% at 14 days). Immunohistochemistry revealed that the acidosis-induced increase in NHE3 abundance occurred in the apical membranes of the S1 and S2 segments of the proximal tubule and the thick ascending limb. NHE3 mRNA abundance was not significantly increased in these animals, whereas phosphoenolpyruvate carboxykinase and glyceraldehyde-3-phosphate dehydrogenase mRNA abundances were significantly increased. These studies demonstrate that the increase in Na/H antiporter activity seen in metabolic acidosis involves an increase in NHE3 protein abundance, which is distributed along the proximal tubule and the thick ascending limb. In addition, these studies suggest that a component of this adaptation is unrelated to changes in NHE3 mRNA abundance.

Biochemical and functional characterization of renal cortical brush border and basolateral membranes in dogs.

The purpose of the present study is to analyze membrane fluidity, enzyme, phospholipid and fatty acid composition and cholesterol content in the brush border (BBM) and basolateral (BLM) membranes obtained from the renal cortex of normal dogs. All measurements were carried out in samples from the same kidney in order to correlate membrane fluidity with membrane composition. BBM and BLM were obtained separately by MgCl precipitation and gradient centrifugation. The order parameter of membrane fluidity was measured by 1,6-dimethyl-1,3,5-hexatriene (DPH) and 1-trimethylammoniophenyl-DPH. (TMA-DPH) steady-state polarization fluorescence. Total lipids, phospholipids and phospholipid classes, cholesterol content, and fatty acid classes were also measured. Data from BLM enzymatic activity revealed an 11-fold enrichment in Na,K-ATPase, whereas the enrichment factors for the other enzymatic markers were well below the unit, demonstrating the high purity of the preparation obtained. Similarly, BBM showed a 9 times increase in alkaline phosphatase and gamma-glutamyltranspeptidase enrichment, and values of enrichment factors for the other enzymatic markers of about 1. BBM exhibited a higher value of steady-state fluorescence anisotropy and thus a lower fluidity than BLM using either of the fluorescent probes DPH or TMA-DPH. This lower fluidity in both the central hydrophobic zone, and the fluorescent probes DPH or TMA-DPH. This lower fluidity in both the central hydrophobic zone, and the external, more hydrophilic leaflet of BBM in comparison with BLM was consistent with the findings of: (a) a higher cholesterol/protein ratio; (b) a lower phospholipid protein ratio; (c) a higher sphingomyelin/choline glycerophospholipid ratio, and (d) a lower unsaturation degree of the fatty acids.

Renal handling of carnitine in experimental vitamin C deficiency

Experimental vitamin C deficiency is associated with low carnitine concentrations in blood and some tissues, but is not due to a decreased ability of scorbutic animals to synthesize carnitine. The effect of experimental vitamin C deficiency on urinary carnitine excretion in vivo and carnitine transport into renal cortical brush-border membrane vesicles in vitro was investigated in guinea pigs fed normal and vitamin C-deficient diets for 24 days. Excretion of free and total carnitine was approximately fourfold greater in scorbutic animals as compared with normal guinea pigs during the last 6 days of the experimental regiment. The rate of carnitine transport into renal cortical brush-border membrane vesicles prepared from scorbutic animals was approximately 36% lower than the corresponding rate for vesicles prepared from normal animals. However, this effect was not specific, since rates of sodium gradient-dependent transport of glucose, lysine, and taurine (but not alanine) were also lower vesicles prepared from scorbutic animals, although the magnitude of the decrease was less than for carnitine. results are consistent with the hypothesis that carnitine depletion in vitamin C deficiency is due to decreased efficiency of carnitine reabsorption.

Phlorizin binding to renal outer cortical brush-border membranes of cadmium-injected rabbits.

The effect of cadmium injection on the high-affinity phlorizin binding to renal outer cortical brush-border membranes was studied in rabbits. Animals received subcutaneous injections of CdCl2 at a dose of 3 mg Cd/kg.day for about 3 weeks. Brush-border membranes were isolated from renal outer cortex by Percoll gradient centrifugation and magnesium precipitation. Cadmium injection resulted in a marked increase in membrane cadmium content and decrease in Na(+)-dependent phlorizin binding. The latter effect was attributed to a reduction in the number of binding sites (N0) and not to changes in the dissociation constant (Kd). Similar results were observed in normal membrane vesicles directly exposed to cadmium in vitro. These results suggest that one mechanism of the cadmium-induced glycosuria is a loss of active Na(+)-glucose cotransport carrier in the proximal tubular brush-border membrane by direct interaction with free cadmium.

Transport of Inorganic Phosphate in Renal Cortical Brush-Border Membrane Vesicles of Cadmium-Intoxicated Rats

The effect of cadmium intoxication on the renal proximal tubular phosphate transport system was studied in adult male Sprague-Dawley rats. Subcutaneous injections of CdCl2 at a dose of 2 mg Cd/kg body wt per day for 2 weeks induced marked polyuria, glycosuria, proteinuria, and phosphaturia, which are characteristics of chronic cadmium intoxication. In the renal cortical brush-border membrane vesicles prepared from cadmium-intoxicated rats, the cadmium content was drastically increased and the Na(+)-dependent phosphate uptake was markedly attenuated. Similar results were obtained in normal membrane vesicles directly exposed to free cadmium. These results indicate that cadmium intoxication impairs the Na(+)-phosphate cotransport system in the proximal tubular brush-border membrane, which may lead to phosphaturia in intact animals.

Characterization of Na(+)-phosphate cotransporters in renal cortical endosomes

To determine the role of membrane recycling in proximal tubular P(i) transport, we studied the transport functions of simultaneously prepared rat renal cortical endosomal vesicles (EV) and brush-border membrane vesicles (BBMV). Initial P(i) uptake was Na+ gradient-dependent in both vesicles. Kinetic studies showed a lower apparent maximal rate (Vmax) for P(i) uptake in EV compared with BBMV (446 +/- 69 vs. 1493 +/- 117 pmol.mg protein-1 x 15 s-1, respectively; n = 4, P < 0.05), with no difference in the apparent Michaelis constant (Km). Endosomal Na(+)-P(i) cotransport was inhibited by phosphate analogues, phosphonoformic acid and arsenate, but not by the anion-exchange inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Na(+)-P(i) cotransport was electroneutral in both vesicles, with a stoichiometry of 2 for BBMV and 1 for EV. The nonpermeant sulfhydryl reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), produced a marked inhibition of Na(+)-P(i) cotransport in EV but not in BBMV, suggesting accessible sulfhydryl groups, an "inside-out" orientation in EV, and "right-side-out" orientation in BBMV. The EV and the BBMV differed significantly in their phospholipid composition and lipid fluidity. The Na(+)-P(i) cotransporter protein (NaPi-2) abundance, determined by Western blots, was five times lower in EV than in BBMV (0.25 +/- 0.05 vs. 1.36 +/- 0.20 arbitrary units). Renal cortical endosomes contain Na(+)-P(i) cotransporters, albeit at a lower density, suggesting involvement of membrane recycling in the regulation of proximal tubular P(i) transport.

Effect of thiazide on urinary citrate excretion

In order to study the effect of thiazide on urinary citrate excretion, we measured sodium dependent citrate uptake by rat renal cortical brush border membrane vesicles (BBMVs) and analyzed acid-base balance with or without thiazide treatment. 5-weeks male Wistar rats (Jcl) were housed in metabolic cages, given 0.1 mg/100 g BW/day hydrochlorothiazide (HCTZ) by a gastric tube once a day for 3 weeks (thiazide treated rats). Arterial blood was taken from aorta and BBMVs were prepared with divalent cation precipitation method. Citrate uptake was measured by a Millipore rapid membrane filtration technique. Oral treatment of HCTZ for 3 weeks showed a significant increase of urinary citrate excretion in both concentration and daily excretion. The sodium dependent citrate transport was observed in rat renal brush border membrane (BBM). But no significant effect of oral HCTZ was observed on acid base balance and sodium dependent citrate uptake in rat renal BBM. These data suggested that the stimulative effect of thiazide on citrate excretion was not via sodium dependent citrate transport in rat renal BBM.