Rabbit Renal Cortex Research Articles

Sodium ion transport participates in non-neuronal acetylcholine release in the renal cortex of anesthetized rabbits.

This study examined the mechanism of release of endogenous acetylcholine (ACh) in rabbit renal cortex by applying a microdialysis technique. In anesthetized rabbits, a microdialysis probe was implanted into the renal cortex and perfused with Ringer's solution containing high potassium concentration, high sodium concentration, a Na+/K+-ATPase inhibitor (ouabain), or an epithelial Na+ channel blocker (benzamil). Dialysate samples were collected at baseline and during exposure to each agent, and ACh concentrations in the samples were measured by high-performance liquid chromatography. High potassium had no effect on renal ACh release. High sodium increased dialysate ACh concentrations significantly. Ouabain increased dialysate ACh concentration significantly. Benzamil decreased dialysate ACh concentrations significantly both at baseline and under high sodium. The finding that high potassium-induced depolarization does not increase ACh release suggests that endogenous ACh is released in renal cortex mainly by non-neuronal mechanism. Sodium ion transport may be involved in the non-neuronal ACh release.

Renal Ablation by Histotripsy—Does it Spare the Collecting System?

The feasibility of histotripsy (transcutaneous nonthermal mechanical tissue fractionation) was previously demonstrated in an in vivo rabbit renal cortex model. We explored the spectrum of histotripsy bio-effects on different tissue types in an in vitro porcine kidney model. Using an 18 element focused annular array ultrasound system we performed histotripsy treatments in 5 in vitro porcine kidneys, targeting 7 cortical volumes and 17 tissue volumes bridging the cortex, medulla and/or collecting system. Treated areas were observed using ultrasound. In 5 lesions methylene blue was infused into the collecting system to evaluate the preservation of collecting system integrity. Kidneys were sectioned and examined grossly for evidence of tissue fractionation, ie the presence of histotripsy paste, or fixed in formalin and prepared for histological analysis. Histotripsy of renal cortical tissue created tissue defects in the cortical area treated. Histotripsy targeting the renal collecting system, medulla and renal cortex resulted in tissue fractionation in the area of the cortex, intermediate damage in the medulla and minimal damage to the collecting system. There is a differential histotripsy treatment effect when comparing renal cortical tissue to renal collecting system. There is no significant architectural disruption of the renal collecting system after histotripsy. This differential effect is a notable finding that may prove useful in future planning of ablative treatments for renal tissue.

Chronic dynamic exercise increases apolipoprotein A-I expression in rabbit renal cortex as determined by proteomic technology

Objective:We have shown previously that exercise training enhances endothelium-dependent and endothelium-independent vascular relaxation in rabbit kidney. This study aimed to investigate protein expression changes in the rabbit renal cortex induced...

Identification of calcium-independent phospholipase A2γ in mitochondria and its role in mitochondrial oxidative stress

Oxidant-induced lipid peroxidation and cell death mediate pathologies associated with ischemia-reperfusion and inflammation. Our previous work in rabbit renal proximal tubular cells (RPTC) demonstrated that inhibition of Ca(2+)-independent phospholipase A(2) (iPLA(2)) potentiates oxidant-induced lipid peroxidation and necrosis, implicating iPLA(2) in phospholipid repair. This study was conducted to identify a RPTC mitochondrial PLA(2) and determine the role of PLA(2) in oxidant-induced mitochondrial dysfunction. iPLA(2) activity was detected in Percoll-purified rabbit renal cortex mitochondria (RCM) and in isolated mitochondrial inner membrane fractions from rabbit and human RCM. Immunoblot analysis and inhibitor sensitivity profiles revealed that iPLA(2)gamma is the RCM iPLA(2) activity. RCM iPLA(2) activity was enhanced in the presence of ATP and was blocked by the PKCepsilon V1-2 inhibitor. Oxidant-induced mitochondrial lipid peroxidation and swelling were accelerated by pretreatment with R-BEL, but not S-BEL. Furthermore, oxidant treatment of isolated RCM resulted in decreased iPLA(2)gamma activity. These results reveal that RCM iPLA(2) is iPLA(2)gamma, RCM iPLA(2)gamma is regulated by phosphorylation by PKCepsilon, iPLA(2)gamma protects RCM from oxidant-induced lipid peroxidation and dysfunction, and that a strategy to preserve or enhance iPLA(2)gamma activity may be of therapeutic benefit.

Ontogeny of rabbit proximal tubule urea permeability.

Urea transport in the proximal tubule is passive and is dependent on the epithelial permeability. The present study examined the maturation of urea permeability (P(urea)) in in vitro perfused proximal convoluted tubules (PCT) and basolateral membrane vesicles (BLMV) from rabbit renal cortex. Urea transport was lower in neonatal than adult PCT at both 37 and 25 degrees C. The PCT P(urea) was also lower in the neonates than the adults (37 degrees C: 45.4 +/- 10.8 vs. 88.5 +/- 15.2 x 10(-6) cm/s, P < 0.05; 25 degrees C: 28.5 +/- 6.9 vs. 55.3 +/- 10.4 x 10(-6) cm/s; P < 0.05). The activation energy for PCT P(urea) was not different between the neonatal and adult groups. BLMV P(urea) was determined by measuring vesicle shrinkage, due to efflux of urea, using a stop-flow instrument. Neonatal BLMV P(urea) was not different from adult BLMV P(urea) at 37 degrees C [1.14 +/- 0.05 x 10(-6) vs. 1.25 +/- 0.05 x 10(-6) cm/s; P = not significant (NS)] or 25 degrees C (0.94 +/- 0.06 vs. 1.05 +/- 0.10 x 10(-6) cm/s; P = NS). There was no effect of 250 microM phloretin, an inhibitor of the urea transporter, on P(urea) in either adult or neonatal BLMV. The activation energy for urea diffusion was also identical in the neonatal and adult BLMV. These findings in the BLMV are in contrast to the brush-border membrane vesicles (BBMV) where we have previously demonstrated that urea transport is lower in the neonate than the adult. Urea transport is lower in the neonatal proximal tubule than the adult. This is due to a lower rate of apical membrane urea transport, whereas basolateral urea transport is the same in neonates and adults. The lower P(urea) in neonatal proximal tubules may play a role in overall urea excretion and in developing and maintaining a high medullary urea concentration and thus in the ability to concentrate the urine during renal maturation.

Properties of the Na+-H+ exchanger in renal microvillus membrane vesicles. 1980.

Am. J. Physiol. 238 (Renal Fluid Electrolyte Physiol. 7): F461-F469, 1980.-Transport of Na + and H + was evaluated in brush border membrane vesicles isolated from the rabbit renal cortex. Na + transport was assayed by a rapid filtration technique; H + transport was monitored with 5,5-dimethoxazolidine-2,4-dione by flow dialysis. Uphill Na + uptake was induced by imposition of an in > out H + gradient, and uphill H + efflux by imposition of an out > in Na + gradient, consistent with the action of a Na + -H + exchanger. The uptake of Na + was electroneutral either in the presence or absence of a H + gradient, indicating a fixed 1:1 stoichiometry for the exchange process. Na + transport was saturable and inhibited by Li + and NH 4 + but not by K + , Rb + , Cs + , or choline. Uphill H + efflux was induced by imposition of an out > in Li + gradient. Neither the uptake of Na + nor H + efflux were influenced by out > in gradients of Cl - compared to gradients of SCN - or SO 4 2- . If transport systems mediating Na + -Cl - co-transport and/or Cl - -OH - exchange are present in the microvillus membrane, their respective rates must be slow compared to the rate of Na + -H + exchange. Transport of Na + was inhibited by harmaline and amiloride, but not by acetazolamide, furosemide, or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate. We conclude that isolated renal microvillus membranes contain a tightly coupled Na + -H + exchanger that may play an important role in proximal tubular acidification.

Nephroprotective effect of betamipron on a new carbapenem, DA-1131, in rabbits.

The nephroprotective effect of betamipron (200 mg/kg) was evaluated after intravenous administration of a high dose of DA-1131 (200 mg/kg) to rabbits. Extensive tubular necrosis was observed without betamipron but the necrosis was not observed with betamipron at 8 h after intravenous administration of DA-1131 based on kidney microscopy. By treatment with betamipron, the amounts and tissue to plasma (T/P) ratios of DA-1131 in renal cortex and whole kidney decreased significantly (65-91% decrease) at both 30 min and 2 h after intravenous administration of the drug to rabbits. This indicated that the accumulation of DA-1131 in rabbit renal cortex and whole kidney was inhibited by betamipron. This resulted in significantly greater percentages of intravenous DA-1131 excreted in urine as unchanged drug, 60.9 versus 40.1%, and significantly faster renal clearance (Cl(r)) of DA-1131 (6.10 versus 3.22 mL/min/kg) by treatment with betamipron. By treatment with betamipron, the amounts and T/P ratios of DA-1131 in renal cortex and whole kidney decreased significantly from 30 min and the renal function remained intact at 8 h after intravenous administration of DA-1131. The above data suggested that the nephroprotective effect of betamipron was fast and persisted for a long period of time in rabbits.

Influence of substrate structure on substrate binding to the renal organic cation/H+ exchanger.

The carrier-mediated exchange of H+ for organic cations ("OC/H+ exchange") is the active step in OC secretion in renal proximal tubules. Although hydrophobicity is known to be an important criterion for binding of substrates to this transporter, the degree to which steric parameters of substrate structure influence binding to the exchanger is unclear. We examined this issue by measuring the inhibition of OC/H+ exchange produced by a group of quaternary ammonium compounds which share a common structural motif: an N1-pyridinium residue. Activity of the OC/H+ exchanger was determined by measuring transport of [14C]tetraethylammonium (TEA) in brush-border membrane vesicles (BBMV) from rabbit renal cortex. Transport was measured in the presence of a pH gradient (pHin 6.0; pHout 7.5) to maximize TEA/H+ exchange. Apparent inhibitory constants (Ki values) for each test agent were measured. The test agents included 4-phenylpyridiniums and 3-phenylpyridiniums, quinoliniums and acridiniums. The planar structure of these compounds permits a direct test of whether the presence of planar hydrophobic mass in different orientations relative to the pyridinium motif exerts a systematic effect on substrate binding to the OC/H+ exchanger. The hydrophobicity of each group of compounds was systematically varied by addition of different substituents at the quaternary nitrogen. Whereas decreases in Ki proved to be proportional to hydrophobicity, the position of the phenyl-ring substituent(s) had no effect on substrate interaction with the exchanger. The results led to the development of a preliminary quantitative structure-activity relationship (QSAR) correlating substrate hydrophobicity and substrate binding to the OC/H+ exchanger. This QSAR was used to predict the binding of 1-methyl-4-phenylpyridinium (MPP+), (+) and (-)nicotine, (+) and (-)ephedrine, quinine and quinidine to the OC/H+ exchanger. Molecular graphics representation of the 3D structures of the test agents was used to develop a working model of a hydrophobic, planar receptor surface on the OC/H+ exchanger against which substrates are suggested to interact during binding. Development of the QSAR and receptor surface model open the way to quantitative tests of the specific physical and structural determinants of substrate selectivity by the renal OC/H+ exchanger.

H-K-ATPase in the RCCT-28A rabbit cortical collecting duct cell line.

In the present study, we demonstrate that the rabbit cortical collecting duct cell line RCCT-28A possesses three distinct H-K-ATPase catalytic subunits (HKalpha). Intracellular measurements of RCCT-28A cells using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) indicated that the mechanism accounting for recovery from an acid load exhibited both K+ dependence and sensitivity to Sch-28080 characteristic of H-K-ATPases. Recovery rates were 0.022 +/- 0.005 pH units/min in the presence of K+, 0.004 +/- 0.002 in the absence of K+, and 0.002 +/- 0.002 in the presence of Sch-28080. The mRNAs encoding the HKalpha1 subunit and the H-K-ATPase beta-subunit (HKbeta) were detected by RT-PCR. In addition, two HKalpha2 species were found by RT-PCR and 5' rapid amplification of cDNA ends (5'-RACE) in the rabbit renal cortex. One was homologous to HKalpha2 cDNAs generated from other species, and the second was novel. The latter, referred to as HKalpha2c, encoded an apparent 61-residue amino-terminal extension that bore no homology to reported sequences. Antipeptide antibodies were designed on the basis of this extension, and these antibodies recognized a protein of the appropriate mass in both rabbit renal tissue samples and RCCT-28A cells. Such findings constitute very strong evidence for expression of the HKalpha2c subunit in vivo. The results suggest that the rabbit kidney and RCCT-28A cells express at least three distinct H-K-ATPases.

Beta2-glycoprotein-I (apolipoprotein H) and beta2-glycoprotein-I-phospholipid complex harbor a recognition site for the endocytic receptor megalin.

Screening of serum by using a surface plasmon resonance analysis assay identified beta2-glycoprotein-I/apolipoprotein H as a plasma component binding to the renal epithelial endocytic receptor megalin. A calcium-dependent megalin-mediated beta2-glycoprotein-I endocytosis was subsequently demonstrated by ligand blotting of rabbit renal cortex and uptake analysis in megalin-expressing cells. Immunohistochemical and immunoelectron microscopic examination of kidneys and the presence of high concentrations of beta2-glycoprotein-I in urine of mice with disrupted megalin gene established that megalin is the renal clearance receptor for beta2-glycoprotein-I. A significant increase in functional affinity for purified megalin was observed when beta2-glycoprotein-I was bound to the acidic phospholipids, phosphatidylserine and cardiolipin. The binding of beta2-glycoprotein-I and beta2-glycoprotein-I- phospholipid complexes to megalin was completely blocked by receptor-associated protein. In conclusion, we have demonstrated a novel receptor recognition feature of beta2-glycoprotein-I. In addition to explaining the high urinary excretion of beta2-glycoprotein-I in patients with renal tubule failure, the data provide molecular evidence for the suggested function of beta2-glycoprotein-I as a linking molecule mediating cellular recognition of phosphatidylserine-exposing particles.

Effects of sulfate and chloride on three separate oxalate transporters reconstituted from rabbit renal cortex.

Understanding the mechanism of sulfate-dependent, oxalate-stimulated chloride reabsorption in the mammalian proximal tubule is complicated by the presence of multiple oxalate and sulfate transport pathways. Accordingly, we developed a method of reconstituting functional oxalate transport from the rabbit renal cortex so that the individual transporters might be examined. Solubilized microvillus membrane proteins were separated by hydroxyapatite chromatography and then reconstituted into proteoliposomes. Two peaks of oxalate/oxalate exchange activity were observed. Sulfate (10 mM) cis-inhibits oxalate transport in the early peak by 93% and in the later peak by 41%. In contrast, 20 mM chloride inhibits oxalate/oxalate exchange by only 32% in the early peak but inhibits oxalate exchange by 70% in the later peak. Oxalate-stimulated sulfate uptake was observed in the early fractions but not in the later fractions. These data are consistent with the recovery of the sulfate/oxalate exchanger in the early hydroxyapatite fractions and the chloride/oxalate exchanger in the later fractions. The basolateral membrane sulfate/oxalate exchanger was also reconstituted. The reconstituted basolateral and apical membrane sulfate/oxalate exchangers demonstrate nearly identical patterns of substrate specificities. However, 98% of apical sulfate/oxalate exchange activity is lost following exposure to octylglucoside at room temperature, whereas the basolateral sulfate/oxalate exchange activity was reduced 67% (P < 0.05). In conclusion, functional reconstitution of solubilized membrane proteins demonstrates that apical membrane chloride/oxalate exchange and sulfate/oxalate exchange are mediated by different transport proteins. Apical and basolateral sulfate/oxalate exchange may also represent transport on two separate exchangers.

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.

Prejunctional neuropeptide Y receptors in human kidney and atrium.

The aim of our study was to characterize functionally prejunctional neuropeptide Y (NPY) receptors in human and rabbit renal cortex, as well as in human right atrium. Segments of human atrial appendages and of human and rabbit renal cortex were preincubated with [3H]noradrenaline, superfused with Krebs-Henseleit solution and stimulated electrically in superfusion chambers. The stimulation-induced outflow of radioactivity was taken as an index of endogenous noradrenaline release. The effects of subtype-selective NPY analogs on the stimulation-induced noradrenaline release were studied. NPY, its endogenous analog, peptide YY, and its C-terminal fragment, NPY13-36, but not its analog, [Leu31,Pro34]NPY, concentration dependently (1-100 nM) inhibited [3H]noradrenaline release in all tissues studied. NPY-induced inhibition of [3H]noradrenaline release in human and rabbit kidney was abolished by pretreatment with pertussis toxin. We conclude that prejunctional inhibition of noradrenaline release in human heart and human and rabbit kidney occurs through NPY receptors of the Y2 subtype, which appear to couple to a pertussis toxin-sensitive G protein.

Renal cortical basolateral Na+/HCO3- cotransporter: IV. Characterization and localization with polyclonal antibodies.

We have previously partially purified the basolateral Na+/HCO3- cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na+/HCO3- cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na+/ HCO3- cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na+/HCO3- cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na+/HCO3- cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO3 or Na2CO3) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na+/HCO3- cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na+/HCO3- cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na+/HCO3- cotransporter protein is expressed in different regions of the kidneys and in other tissues.

Pathways for Oxalate Transport in Rabbit Renal Microvillus Membrane Vesicles

Recent evidence suggests that apical membrane Cl--oxalate exchange plays a major role in mediating Cl- absorption in the renal proximal tubule. To sustain steady-state Cl- absorption by a mechanism of exchange for intracellular oxalate requires the presence of one or more pathways for recycling oxalate from lumen to cell. Accordingly, we evaluated the mechanisms of oxalate transport in luminal membrane vesicles isolated from the rabbit renal cortex. We found that transport of oxalate by Na+ cotransport is negligible compared to the transport of sulfate. In contrast, we demonstrated that oxalate shares the electroneutral pathway mediating Na+-independent sulfate-carbonate exchange. We also demonstrated the presence of OH--oxalate exchange (indistinguishable from H+-oxalate cotransport). The process of OH--oxalate exchange was electrogenic and partially inhibited by Cl-, indicating that it occurs, at least in part, as a mode of the Cl--oxalate exchanger described previously. An additional component of OH--oxalate exchange was insensitive to inhibition by either Cl- or sulfate, suggesting that it takes place by neither the Cl--oxalate exchanger nor the sulfate-carbonate exchanger. We conclude that multiple anion exchange mechanisms exist by which oxalate can recycle from lumen to cell to sustain Cl- absorption occurring via apical membrane Cl--oxalate exchange in the renal proximal tubule.

Cephalosporin and carbacephem nephrotoxicity: Roles of tubular cell uptake and acylating potential

Three beta-lactams, desacetylcephaloglycin, ampicillin, and loracarbef, were studied to test a hypothesis derived from retrospective analysis of previously studied cephalosporins: that beta-lactam nephrotoxicity develops in approximate proportion to tubular cell antibiotic concentrations and lactam ring reactivities. Concentrations of each beta-lactam (and inulin) in rabbit renal cortex and serum were measured at the end of 0.5-hr infusions of 100 mg antibiotic/kg body weight and 0.5 to 0.67 hr later. Total cortical AUCs (total areas under the curve of concentration and time in renal cortex) and transported cortical AUCs (total minus inulin-space beta lactam) were calculated from these measurements. Reactivities, determined by the rate constants of lactam-ring opening at pH 10, were taken from the literature. Nephrotoxicity was quantified by grades of proximal tubular cell necrosis and by serum creatinine concentrations 2 days after infusion of 100–1500 mg/kg of the antibiotics. Desacetylcephaloglycin was slightly less nephrotoxic than cephaloglycin; the AUCs, reactivities, and toxicities of these two cephalosporins fit the proposed model, particularly when allowance is made for hepatic and renal deacetylation of cephaloglycin. The very low AUCs, limited reactivity, and absence of nephrotoxicity of ampicillin also fit the model. Loracarbef had a transported AUC less than three times, and reactivity one-thirtieth, those of cefaclor, respectively. Although only at 1500 mg/kg, loracarbef was significantly more nephrotoxic than cefaclor. If the reactivity of loracarbef with its targeted bacterial proteins, which is essentially the same as that of cefaclor, is considered instead of the base hydrolysis rate constant, then loracarbef also fits the model. By the same analysis, the comparatively high in vitro stability of other carbacephems, although pharmaceutically convenient, may not limit their nephrotoxicity.

Cl- channels in basolateral renal medullary vesicles X. Cloning of a Cl- channel from rabbit outer medulla

These experiments were intended to identify candidate cDNAs which might encode basolateral membrane Cl- channels of the mTAL using a homology-based cloning strategy. We prepared a cDNA library using a 1.8 to 3.2 kb mRNA fraction from rabbit outer medulla that induces a Cl- conductance in cellular membranes of Xenopus laevis oocytes. The cDNA library was screened with two 32P-oligonucleotide probes corresponding to highly conserved sequences in other Cl- channels. We isolated two cDNAs: rbClC-Ka and rbClC-Kb. The protein sequences deduced from these two cDNAs had 99% homology. Using RT-PCR technology, cultured mouse mTAL cells were found to contain mRNA corresponding to those two cDNAs. Expression of the mRNAs corresponding to these two cDNAs was kidney-specific and was greater in rabbit renal medulla than rabbit renal cortex. Finally, by using RT-PCR technology in combination with microdissected glomeruli or tubule segments, we found mRNA for rbClC-Ka in glomeruli, proximal convoluted tubules, mTAL and cortical collecting tubules.

The effect of cadmium chloride in vitro on sodium-glutamate cotransport in brush border membrane vesicles isolated from rabbit kidney.

To further elucidate the mechanism of cadmium inhibition of renal amino acid transport, brush border membrane vesicles were isolated from rabbit renal cortex and the effect of cadmium on the uptake of L-glutamate into the vesicles was investigated. Preincubation of the membranes with CdCl2 decreased sodium-dependent L-glutamate uptake at concentrations higher than 10(-6)M. In the presence of 20 mM potassium inside the vesicles a half-maximal inhibition was observed at 0.5 to 1 x 10(-4) M. Kinetic analysis revealed a strong reduction of Vmax by cadmium but only minor changes in Km for glutamate. The inhibition required preincubation of the vesicles with cadmium, was not elicited by cadmium metallothionein, and was not reversed by ethylenediaminetetraacetic acid. These findings suggest an action of cadmium at the cytoplasmic face of the brush border membrane. Furthermore, the sensitivity of the transport system for cadmium was lower in the absence of potassium. Inhibition increased in a saturable manner when intravesicular potassium was augmented, indicating that the transporter interacts with cadmium most avidly when potassium is bound to the carrier.

N-Acetyl- β- d-glucosaminidase (NAG) isoenzymes in primary cultures of rabbit kidney proximal tubule cells: a cellular model for studies on nephrotoxicity?

N-Acetyl- β- d-glucosaminidase (NAG) isoenzyme profile in primary cultures of rabbit kidney proximal tubule cells was studied. Confluent cells had high levels of NAG activity, but ion exchange chromatography showed that the NAG isoenzyme profile in cultured cells was different from that of rabbit renal cortex homogenates and freshly isolated cells. Confluent cultured cells contained an atypical acidic isoform, absent in homogenates and freshly isolated cells in which the predominant isoform is NAG-A (a heterodimer αβ). The fact that this atypical isoform was able to hydrolyse the synthetic substrate 4-methylumbelliferyl- β- N-acetylglucosaminide-6-sulphate indicated that it probably was an α-subunit homodimer. These results suggest subunit rearrangement within NAG polypeptide chains linked to down-regulation of β-subunit production in cultured rabbit proximal cells. The change in isoenzyme profile in cultured cells may make it difficult to use primary cultures of rabbit proximal tubule cells to establish correlations between in vitro and in vivo studies using NAG isoenzymes as a nephrotoxicity index, as illustrated by the effects of gentamicin.

Modulation of sodium-coupled uptake and membrane fluidity by cisplatin in renal proximal tubular cells in primary culture and brush-border membrane vesicles

The proximal tubule appears to be the main target for the adverse effects of cis-diamminedichloroplatinum (II) (cDDP). We evaluated the early effects of cDDP at concentrations (3 to 67 microM) lower that those which alter cell viability, on three apical transport systems and on the physical state of the brush border membrane (BBM) in rabbit proximal tubule (RPT) cells in primary culture. The maximal effect, corresponding to a 30% decrease in Na(+)-coupled uptake of phosphate (Pi) and alpha-methylglucopyranoside (MGP) and a twofold increase in Na(+)-coupled alanine uptake, was obtained at 17 microM (5 micrograms/ml) cDDP and occurred through a modification of their affinity. At this concentration, cDDP increased BBM fluidity and decreased the BBM cholesterol content by 28%, without increasing the permeability of tight junctions. To clarify the role of cDDP-induced increase in BBM fluidity on alterations of Na(+)-coupled uptake, these parameters were also investigated in BBM vesicles isolated from rabbit renal cortex directly exposed to cDDP. cDDP induced a concentration-dependent inhibition of Na(+)-coupled uptake of MGP, Pi and alanine in BBM vesicles from the renal cortex, associated with a decrease in protein sulfhydryl content, without modifying BBM fluidity. Our findings strongly suggest that the cDDP-induced increase in BBM fluidity in RPT cells results from an indirect mechanism, possibly an alteration of cholesterol metabolism, and did not play a major role in the cDDP-induced inhibition of Na+/Pi and Na+/glucose cotransport systems that may be mainly mediated through a direct chemical interaction with essential sulfhydryl groups of the transporters.