Tubule Suspensions Research Articles

Glycolysis Is Not Responsible for the Tolerance of Immature Renal Tubules to Anoxia

We have previously shown that the immature tubule is tolerant of prolonged anoxia. In addition, cellular ATP is maintained at 2-fold higher levels during anoxia in the immature tubules compared with the mature tubules. The purpose of this study was: 1) to determine whether anaerobic glycolysis contributes to the tolerance to anoxia and preservation of cellular ATP in immature tubules and 2) to evaluate whether the tolerance demonstrated by immature tubules is dependent on preservation of cellular ATP. Suspensions of proximal tubules from immature (8-10 d) and mature (8-10 wk) rats were subjected to 15 and 45 min of anoxia in a standard buffer and in buffers designed to inhibit glycolysis. Lactate dehydrogenase release was used to assess plasma membrane damage, ATP levels were determined as an index of cellular energy and total lactate production was measured to evaluate glycolytic activity. After 45 min of anoxia, total lactate production was less in immature tubules (101 +/- 48 micrograms of lactate/mg of DNA) compared with mature tubules (148 +/- 36 micrograms of lactate/mg of DNA). After inhibition of glycolytic metabolism, ATP decreased to similar levels in both immature and mature tubules. However, immature tubules remained resistant to anoxic damage (lactate dehydrogenase: mature tubules 38 +/- 4%, immature tubules 29 +/- 1.0%). Therefore, enhanced glycolytic activity does not play a dominant role in the tolerance of the developing kidney to anoxia, and this tolerance is not primarily dependent on preservation of cellular ATP.

A rapid β-NADH-linked fluorescence assay for lactate dehydrogenase in cellular death

Lactate dehydrogenase (LDH) release is a common marker of cellular death. Traditionally, the fraction of LDH released has been measured using a NADH-linked UV-Vis spectrophotometric method. The limitation of this method is that samples are usually run serially and thus is time intensive. Therefore, we developed a NADH-linked LDH assay using a fluorescence plate reader that had a correlation of 0.95 with the traditional UV-Vis spectrophotometric method. Using rabbit renal proximal tubule suspensions at a concentration of 1 mg cellular protein/ml of media, the fluorescence assay can determine LDH release in 22 samples in 2 min using 12 μL of cellular homogenates and 150 μL of media. The parallel processing of samples and smaller volumes used in the fluorescence assay results in decreased analysis time and costs.

Cholinergic agonists increase phosphoinositide metabolism and cell calcium in isolated rat renal proximal tubule.

The intracellular signaling involved in cholinergic modulation of renal proximal tubule functions has not been addressed. We report that acetylcholine and carbachol increase the production of inositol phosphates and the intracellular calcium concentration in rat proximal tubule. Muscarinic M3 receptors are involved, given the inhibitory effects of selective antagonists (4-diphenylacetoxy-N-methylpiperidine > > pirenzepine > methoctramine). The muscarinic response is absent in the early part of proximal straight tubule. The response is smaller and more variable in proximal convoluted tubule segments selected at random from cortical tubule suspensions than in the early part of proximal convoluted tubule. This contrasts with the norepinephrine response, which has almost the same magnitude all along the proximal tubule. We conclude that cholinergic agonists activate muscarinic M3 receptors in proximal tubule and that there is a decreasing response gradient from the early convoluted tubule to the early straight tubule.

Protein kinase C-dependent phosphorylation of Na(+)-K(+)-ATPase alpha-subunit in rat kidney cortical tubules.

We have previously shown that, in oxygenated rat kidney proximal convoluted tubules (PCT), activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) directly stimulates Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity. PKC modulation of Na(+)-K(+)-ATPase activity by phosphorylation of its alpha-subunit was the postulated mechanism. The present study was therefore designed to investigate the relationship between PKC-mediated phosphorylation of the catalytic alpha-subunit and the cation transport activity of the Na(+)-K(+)-ATPase. In a suspension of rat kidney cortical tubules, activation of PKC by 10(-7) M PDBu increased the level of phosphorylation of the Na(+)-K(+)-ATPase alpha-subunit and stimulated the ouabain-sensitive 86Rb uptake by 47 and 42%, respectively. Time and dose dependence of the PDBu-induced increase in Na(+)-K(+)-ATPase activity and phosphorylation was strongly linearly correlated. The effects of PDBu on phosphorylation and activity of Na(+)-K(+)-ATPase were prevented by GF-109203X, a specific PKC inhibitor, whereas H-89, a specific PKA inhibitor, was ineffective. These results demonstrate that PKC activation induces phosphorylation of the catalytic alpha-subunit of Na(+)-K(+)-ATPase, which may participate in the stimulation of its cation transport activity in the rat PCT.

Studies on the pharmacology of the inward transport of L-DOPA in rat renal tubules.

1. The accumulation of L-DOPA in suspensions of renal tubules obtained from male Wistar rats occurred through non-saturable and saturable mechanisms. The kinetics of the saturable component of L-DOPA uptake in renal tubules was as follows: Vmax = 46.3 +/- 2.8 nmol mg-1 protein h-1 and Km = 114.4 (95% confidence limits; 83.8, 156.2) microM (n = 5). The diffusion constant (in nmol-1) of the non-saturable component for the accumulation of L-DOPA was 1.3 (1.1, 1.6; n = 8). 2. The effect of 2,4-dinitrophenol was a marked reduction in the tubular uptake of L-DOPA, with an IC50 value of 12.1 (4.0, 36.9) microM. Cocaine produced a slight (P = 0.08) decrease (22% reduction at 50 microM) in the tubular uptake of L-DOPA. Corticosterone produced a considerable inhibitory effect on the uptake of L-DOPA with an IC50 value of 11.0 (3.6, 33.5) microM. The maximal inhibitory effect of probenecid was a 24% decrease in the uptake of L-DOPA; however, the more selective organic anion transport inhibitor 4,4'-diisothiocyantostilbene-2,2'-disulphonic acid (DIDS) was, found not to affect the tubular uptake of L-DOPA. The organic cation transport inhibitor, cyanine 863, was found to produce a marked decrease in the uptake of L-DOPA (IC50 = 2.02 [1.07, 3.79]). The cyanine derivatives 1,1'-diethyl-2,2'-cyanine (decynium 22) and 1,1'-diethyl-2,4'-cyanine (decynium 24) also potently inhibited L-DOPA uptake with IC50 values (in microM) of 0.63 (0.39, 1.01) and 0.10 (0.08, 0.13), respectively, both compounds were found to be more potent than cyanine 863. 3. The inhibitory effect of decynium 24 (0.2 microM) on the tubular uptake of L-DOPA was dependent on the pH of the incubation medium; at pH = 6.5 the accumulation of L-DOPA was reduced up to 37 +/- 2% of control values, whereas at pH = 7.4 and pH = 8.2 the accumulation of L-DOPA was reduced by 56 +/- 1% and 60 +/- 6%, respectively. Cyanine 863 (2 microM), decynium 22 (1 microM) and decynium 24 (0.2 microM) were found to decrease the Vmax values for the saturable component of L-DOPA uptake without changes in Km values. 4. It is concluded that the tubular uptake of L-DOPA might be promoted through a mechanism which is dependent on the activity of the organic cation transport system.

Lack of Correlation betweenpara-Aminophenol Toxicityin Vivoandin Vitroin Female Sprague–Dawley Rats

The present study was designed to test the hypothesis thatpara-aminophenol (PAP) nephrotoxicity is due to autooxidation. We compared renal functional responses following PAP administration to female Sprague–Dawley rats and following incubation of renal proximal tubules with PAP. The concentrations of PAP selected forin vitroincubations produced cytotoxicity (for example, a decrease in oxygen consumption or adenine nucleotide concentration) in rat renal epithelial cells or rabbit proximal tubule suspensions. In rats, PAP (300 mg/kg ip) caused proximal tubular necrosis within 24 hr. Changes in renal function 24 hr following PAP administration included increased kidney weight and blood urea nitrogen concentration and decreased renal glutathione (GSH) content and adenine nucleotide concentrations. PAP did not cause hepatic damage. Within 2–4 hr following PAP administration, renal GSH content and adenine nucleotide concentrations were significantly decreased. In renal cortical slices prepared from PAP-treated rats, oxygen consumption and accumulation of organic ions (para-aminohippurate and tetraethylammonium) were significantly decreased compared with renal cortical slices prepared from control rats. In liver, GSH content was significantly decreased from 1 to 4 hr following PAP administration. In contrast to the effects of PAPin vivo,renal proximal tubules showed little evidence of injury when incubated with 0.1 or 0.5 mMPAP for up to 4 hr in the presence or absence of amino acids in the incubation medium. When tubules were incubated with 1 mMPAP for 4 hr in the presence of amino acids, GSH content, AMP concentration, and TEA uptake were significantly decreased. When amino acids were removed from the incubation medium, 1 mMPAP caused decreases in oxygen consumption and ATP concentration after 4 hr of incubation. Functional changes observed during incubation with PAPin vitrowere not consistent with functional changes observedin vivo.The discrepancy between PAP toxicityin vivoandin vitrosuggests that autooxidation is unlikely to be responsible for PAP nephrotoxicity and that nephrotoxicityin vivois primarily mediated by extrarenal bioactivation. Further, depletion of hepatic GSH content prior to changes in renal function suggests that PAP or a PAP metabolite may conjugate with hepatic GSH. These observations suggest that PAP nephrotoxicity may be mediated by PAP–GSH conjugates rather than autooxidation of PAP in the kidney.

Nitric oxide kinetics during hypoxia in proximal tubules: Effects of acidosis and glycine

In the present study, we directly monitored nitric oxide (NO) with an amperometric NO-sensor in suspensions of rat proximal tubules. Hypoxia-stimulated NO generation was characterized by an initial rise and a subsequent sustained increase which preceded cell membrane damage as assessed by lactic dehydrogenase (LDH) release. In contrast, the NO concentration remained unmeasurable in normoxic controls. Nitro-L-arginine-methyl ester (L-NAME) prevented the hypoxia-induced increase in NO in a dose dependent manner in parallel with incremental cytoprotection. The hypoxia-induced elevation in NO and the associated membrane injury were both markedly prevented by extracellular acidosis (pH 6.95). In vitro proximal tubular nitric oxide synthase (NOS) activity (3H-arginine to 3H-citrulline assay) was pH dependent with optimum activity at pH 8.0 and greatly reduced activity at acidic pH even in the presence of calcium and co-factors. However, glycine, a well recognized cytoprotective agent, did not attenuate the NO concentration during hypoxia. The present study therefore provides direct evidence that NO is generated by rat proximal tubules during hypoxia and demonstrates that the protective effect of low pH against hypoxic rat tubular injury is associated with an inhibition of this NO production.

Renal actions of angiotensin-(1-7): in vivo and in vitro studies.

In vivo studies were conducted in Na-replete anesthetized male Wistar rats with denervated kidneys. Intrarenal injections of angiotensin-(1-7) [ANG-(1-7) at > 1 nmol/kg produced a shallow dose-dependent decrease in renal blood flow that was mediated by the AT1-type ANG II receptor. A constant intrarenal infusion of ANG-(1-7) at 0.1 and 1 nmol.min-1.kg-1 had minimal effects on renal blood flow and blood pressure and resulted in an elevated urinary excretion of Na and water compared with the time-control saline-infused group. To determine whether ANG-(1-7) may have a direct action on tubular epithelium to inhibit Na reabsorption, we examined the effect of ANG-(1-7) on transport-dependent O2 consumption (Qo2) in fresh suspensions of rat proximal tubules in vitro. ANG-(1-7) inhibited Qo2 in a concentration-dependent fashion with a threshold concentration of approximately 100 pM. Stimulating Na-K-adenosinetriphosphatase (Na-K-ATPase) activity with nystatin caused a leftward shift of the inhibitory concentration-response curve to ANG-(1-7). The 22% inhibition of Qo2 by 1 pM ANG-(1-7) was abolished by pretreatment with 5 mM ouabain (Na-K-ATPase inhibitor), unaltered by pretreatment with 1 microM PD-123319 (AT2 receptor antagonist), partially attenuated by 1 microM losartan (AT1 receptor antagonist), and abolished by 1 microM [Sar1, Thr8]ANG II (nonselective ANG receptor antagonist). Together these findings indicate that ANG-(1-7) has biological activity in the kidney and, at nonvasoconstrictor doses, results in increased Na and water excretion in vivo. One site of action is the proximal tubule, where ANG-(1-7) can inhibit an ouabain-sensitive Na-K-ATPase exit step in cellular Na transport. This novel inhibitory action of ANG-(1-7) appears to be mediated by an AT1 receptor (minor component) and a non-AT1, non-AT2 ANG receptor (major component).

Lack of Correlation between para-Aminophenol Toxicity in Vivo and in Vitro in Female Sprague—Dawley Rats

The present study was designed to test the hypothesis that para-aminophenol (PAP) nephrotoxicity is due to autooxidation. We compared renal functional responses following PAP administration to female Sprague-Dawley rats and following incubation of renal proximal tubules with PAP. The concentrations of PAP selected for in vitro incubations produced cytotoxicity (for example, a decrease in oxygen consumption or adenine nucleotide concentration) in rat renal epithelial cells or rabbit proximal tubule suspensions. In rats, PAP (300 mg/kg i.p.) caused proximal tubular necrosis within 24 hr. Changes in renal function 24 hr following PAP administration included increased kidney weight and blood urea nitrogen concentration and decreased renal glutathione (GSH) content and adenine nucleotide concentrations. PAP did not cause hepatic damage. Within 2-4 hr following PAP administration, renal GSH content and adenine nucleotide concentrations were significantly decreased. In renal cortical slices prepared from PAP-treated rats, oxygen consumption and accumulation of organic ions (para-aminohippurate and tetraethylammonium) were significantly decreased compared with renal cortical slices prepared from control rats. In liver, GSH content was significantly decreased from 1 to 4 hr following PAP administration. In contrast to the effects of PAP in vivo, renal proximal tubules showed little evidence of injury when incubated with 0.1 or 0.5 mM PAP for up to 4 hr in the presence or absence of amino acids in the incubation medium. When tubules were incubated with 1 mM PAP for 4 hr in the presence of amino acids, GSH content, AMP concentration, and TEA uptake were significantly decreased. When amino acids were removed from the incubation medium, 1 mM PAP caused decreases in oxygen consumption and ATP concentration after 4 hr of incubation. Functional changes observed during incubation with PAP in vitro were not consistent with functional changes observed in vivo. The discrepancy between PAP toxicity in vivo and in vitro suggests that autooxidation is unlikely to be responsible for PAP nephrotoxicity and that nephrotoxicity in vivo is primarily mediated by extrarenal bioactivation. Further, depletion of hepatic GSH content prior to changes in renal function suggests that PAP or a PAP metabolite may conjugate with hepatic GSH. These observations suggest that PAP nephrotoxicity may be mediated by PAP-GSH conjugates rather than autooxidation of PAP in the kidney.

Stimulation of proximal convoluted tubule phosphate transport by epidermal growth factor: signal transduction.

The present study investigated the signal-transduction pathway responsible for the epidermal growth factor (EGF) stimulation of phosphate transport (JPhos) in the rabbit proximal convoluted tubule (PCT). Genistein, 10(-4) M, bath and lumen, an inhibitor of EGF receptor tyrosine kinase activity, blocked the EGF effect on JPhos, consistent with a role for tyrosine kinase in the signal-transduction pathway. Both staurosporine (5 x 10(-8) M) and calphostin C (10(-8) M), inhibitors of protein kinase C, blocked the EGF stimulation of JPhos, indicating that protein kinase C is involved in EGF signaling. Intracellular calcium (Ca2+i) concentrations were measured in perfused tubules using fura PE3 to determine whether changes in Ca2+i were also part of the signaling pathway. After addition of 3 nM EGF, there was no change in Ca2+i, suggesting that stimulation of protein kinase C is not from phosphatidylinositol hydrolysis by phospholipase C-gamma. To determine whether phospholipase A2 (PLA2) is involved, the inhibitor mepacrine was used. Mepacrine (5 x 10(-5) M) had no direct effect on PCT transport but blocked the stimulatory effect of EGF on JPhos. PLA2 activity, assessed as free arachidonic acid release from proximal tubules in suspension, increased by 18.8% with 3 nM EGF. Thus the stimulation of JPhos by EGF is mediated via a signal-transduction pathway involving tyrosine kinase, protein kinase C, and PLA2.

Effect of P 2Y-Purinoceptor Stimulation on Renal Gluconeogenesis in Rats

In the freshly prepared rat renal cortical tubule suspension, the effects of ATP on intracellular free calcium mobilization and gluconeogenesis were investigated. ATP increased intracellular free calcium concentration ([Ca 2+] i) in a dose-dependent manner (10 −6 - 10 −3 M). The rank order in the potency of ATP analogs at 10 −4 M was 2-methylthio ATP>ATP≥ADP. AMP, adenosine and α,β-methylene ATP did not respond to [Ca 2+] i. These results suggest that P 2-purinoceptor in the rat kidney cortex should be specifically P 2Y subtype. Renal gluconeogenesis from pyruvate was stimulated maximally by 10 −4 M ATP(48.3 ± 13.2%). This effect was significantly inhibited by 10 −4 M suramin. Thus, the present study suggests that renal gluconeogenesis is increased via P 2Y-specific purinoceptor stimulation.

Consecutive use of hormonally defined serum-free media to establish highly differentiated human renal proximal tubule cells in primary culture.

Highly differentiated human proximal tubule (HPT) cells in primary culture were established from heterogeneous suspension of tubules prepared from the human renal cortex by an original two-step procedure. First, gluconeogenic-competent HPT cells were selected by using a hormonally defined serum-free medium without glucose or insulin; then, the selected HPT cells were grown in a medium containing a low concentration of glucose (1 mM) and insulin (0.5 micrograms/mL) but no antibiotics. HPT cells grown on plastic support formed confluent, cobblestone-like monolayers with numerous mitochondria and pinocytosis vacuoles, solitary cilia, junctional complexes, and a well-developed brush border consisting of densely packed microvilli. Compared with cell monolayers on plastic support, HPT cells grown on porous filter membranes showed better morphologic differentiation. HPT cell monolayers expressed the following differentiated functions of the proximal tubule in situ: a low-affinity, high-capacity Na(+)-dependent glucose transport system inhibited by phlorizin, a high-affinity Na(+)-dependent phosphate transport system, a basolateral organic cation uptake inhibited by mepiperphenidol, parathyroid hormone-sensitive cAMP synthesis, brush-border hydrolase activities, gluconeogenesis-associated enzymes, glutathione-S-transferases and N-acetyl-beta-D-glucosaminidase. The medium containing low glucose and insulin concentrations markedly limited the increase in glycolysis but did not prevent the falls in gluconeogenesis and brush-border hydrolase activity at any time of the culture period. Similar decreases of brush border enzyme activities were obtained for HPT cells grown either on plastic or on porous filter membrane. A thorough characterization study demonstrated that this simple and preparative experimental approach makes it possible to establish highly differentiated HPT cells in primary culture suitable for investigating human renal proximal tubular cell function.

Dopamine causes stimulation of protein kinase C in rat renal proximal tubules by activating dopamine D 1 receptors

Although it is suggested that in the renal proximal tubules, dopamine D 1 receptor activation causes inhibition of Na +/K + ATPase via a phospholipase C and protein kinase C coupled pathway, the direct stimulation of protein kinase C by dopamine has not been reported. The present study was designed to examine the effects of dopamine and selective dopamine D 1 receptor and dopamine D 2 receptor agonists on protein kinase C activity. The renal proximal tubule suspensions were obtained from male Sprague-Dawley rats. The tubules were incubated separately with dopamine and fenoldopam in the presence or absence of dopamine D 1 receptor antagonist, SCH 23390 ([(R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine]). The protein kinase C activity was measured by using a kinase target peptide, conjugated to a fluorescent molecule in water. The amino acid sequence of this peptide is, Proline-Leucine-Serine-Arginine-Threonine-Leucine-Serine-Valine-Alanine-Alanine-Lysine (PKSRTLSVAAK). We found that dopamine and fenoldopam [6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-diol] produced concentration-dependent increases in protein kinase C activity, which was blocked by SCH 23390. However, the dopamine D 2 receptor agonist, bromocriptine [(5′α)-2-bromo-12′-hydroxy-2′-(1-methyl-ethyl)-5′-(2-methylpropyl) ergo-taman-3′,6′,18-trione] failed to stimulate protein kinase C activity at all the concentrations tested. These results provide direct evidence that dopamine stimulates protein kinase C activity via activation of dopamine D 1 receptors.

Tetrapentylammonium (TPeA): slowly dissociating inhibitor of the renal peritubular organic cation transporter

The efflux of tetraethylammonium (TEA) from suspensions of rabbit renal proximal tubules is completely blocked by 500 μM tetrapentylammonium (TPeA) in the extracellular medium. The basis of this trans-inhibition of TEA transport by TPeA was examined in tubule suspensions. At TPeA concentrations < 10 μM, efflux of TEA was reduced by ∼ 50%, whereas at concentrations > 10 μM, TPeA reduced efflux an additional 50% to produce a near complete block of TEA efflux. Increasing concentrations of TPeA from 0–500 μM were found to produce a biphasic, concentration-dependent trans-inhibition of TEA efflux from tubule suspensions suggesting that TPeA may block efflux by binding to both a high and low affinity TPeA binding site. The trans-inhibition of TEA efflux by TPeA at low concentrations (< 10 μM) may result from a slow carrier turnover when TPeA is bound to the carrier site. To determine whether the inhibitory effectiveness of TPeA was also associated with its slow dissociation from the carrier site, the effect of a 10 s preincubation with 1 μM TPeA on TEA uptake was examined. The uptake of TEA by tubules preincubated for 10 s with TPeA was reduced by ∼ 30–50% compared to control tubules not preincubated with TPeA. A 10 s preincubation with 150 μM unlabeled TEA had no effect on TEA uptake compared to control tubules not preincubated with TEA. When the 10 s preincubation with 1 μM TPeA was followed by a 10 min recovery period, TEA uptake returned to control levels, indicating that the prolonged inhibition was reversible. This prolonged inhibition of TEA uptake after a 10 s preincubation with 1 μM TPeA, as suspected, may arise from a slow dissociation of TPeA from the OC transporter following a rapid association to the binding site. TPeA inhibition of TEA uptake into tubules was competitive in nature with a K i of 1 μM. The ability of TEA to compete with TPeA for binding to the carrier suggests that the binding of TPeA to the carrier can be displaced by large concentrations of TEA. These observations suggest that the interactions of TPeA, and perhaps similarly large hydrophobic OCs, with the OC transporter are complex.

Actions of isoproterenol in frog proximal tubules

In the present work, we compared biochemical and electrophysiological actions of isoproterenol on frog proximal tubular cells by using tubule suspensions and giant entities obtained by cell fusion. Isoproterenol (ISO) dose-dependently stimulated cAMP production in tubule suspension and depolarized the “giant cell” membrane. Both effects were triggered by β receptor occupancy, but strongly differed in their concentration-dependency, since depolarization occurred with an ISO concentration as low as 10 −12 mol/l whereas cAMP accumulation could be seen only with more than 10 −8 mol/l ISO. ISO-induced membrane depolarization was mimicked by forskolin which directly stimulated the catalytic subunit of adenylyl cyclase. In both isoproterenol- and forskolin-stimulated giant cells, membrane depolarization was accompanied by a decrease in membrane conductance, and both effects were inhibited by tetraethylammonium (TEA) and 4-aminopyridine (4-AP). On the other hand, ISO- and forskolin-induced cAMP production were not affected by TEA. The present data thus show that isopoterenol produces two independent effects in frog proximal tubule: it depolarizes the cell membrane by blocking a K + conductance and activates adenylyl cyclase.

Cross-Talk between the Na&lt;sup&gt;+&lt;/sup&gt;-K&lt;sup&gt;+&lt;/sup&gt;-ATPase and the H&lt;sup&gt;+&lt;/sup&gt;-ATPase in Proximal Tubules in Suspension

The cellular energy required for the activity of the Na(+)-K(+)-ATPase and of the H(+)-ATPase was estimated in intact proximal tubules in suspension. Both the fall in oxygen consumption (directly measured) and NADH oxidation (as estimated from exogenous substrate metabolism) were measured before and following application of ouabain (1 mM) to inhibit the sodium pump, following bafilomycin (0.1 mM) to inhibit the proton pump or following a combination of these inhibitors. The data demonstrate that the sodium pump utilizes 43% and the proton pump 19% of the phosphorylating NADH turnover of canine proximal tubules studied in vitro. However, a significant and stoichiometric stimulation of one pump was observed upon inhibition of the other. The NADH turnover related to the sodium pump increased from 308 to 402 (delta = 94) mumol.g-1 wet weight.h-1 following bafilomycin application and that of the proton pump from 136 to 230 (delta = 94) following ouabain application. This stimulation was largely abolished by inhibition of the Na+/H+ exchange occurring in either direction by amiloride or methylisobutylamiloride. It is concluded that a cross-talk occurs between the basolateral sodium pumps and the proton pumps located on the brush border membrane and/or on endosomes in proximal tubules. This cross-talk appears to be mediated by Na+/H+ exchange suggesting that both the proton pump and the Na+/H+ exchanger may contribute in a cooperative fashion to the proximal secretion of protons.

Rabbit renal proximal tubule suspensions as a model for nephrotoxicity evaluation of native or in situ metabolized beta-lactam antibiotics.

Rabbit renal proximal tubule suspensions have proved a convenient model for nephrotoxicity studies, particularly for direct nephrotoxins such as Cephaloridin (Olivier et al., 1992). In order to evaluate the ability of this model to metabolize certain drugs to nephrotoxic compounds, we used another B-lactam antibiotic, Imipenem, known to be nephrotoxic through renal metabolism via hydrolysis by dehydropeptidase I (DHP) (Birnbaum et al., 1985). The use of Cilastatin, an inhibitor of this enzyme, should confirm this metabolic pathway.

Dopamine fails to stimulate protein kinase C activity in renal proximal tubules of spontaneously hypertensive rats.

We have previously reported that dopamine-1 receptor-mediated activation of phospholipase C is diminished in renal cortical slices of spontaneously hypertensive rats. The present study was carried out to examine the effect of dopamine on protein kinase C (PKC), which is one of the enzymes involved in the signal-transduction pathway leading to dopamine-induced inhibition of Na+/K(+)-ATPase in the renal proximal tubule. Renal proximal tubule suspensions were obtained from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats of 10-12 weeks old. The tubules were incubated with dopamine in the presence or absence of DA-1 receptor antagonist SCH 23390. The PKC activity was measured by using a specific fluorescent peptide substrate (sequence, PKSRTLSVAAK). We found that dopamine produced a concentration-dependent increase in protein kinase C activity in the WKY rats, however, it failed to stimulate PKC activity in the SHR. Peak stimulation of 3.828 +/- 0.35 (ng/micrograms) protein in the WKY rats was observed at dopamine concentration of 1 microM, which was blocked in a concentration-dependent manner by SCH 23390 (0.25 microM). These results provide evidence that dopamine directly stimulates PKC activity via activation of DA-1 receptors in WKY rats. Furthermore, we discovered that dopamine fails to stimulate PKC activity in the SHR. This phenomenon may be responsible for the failure of dopamine to inhibit Na+/K(+)-ATPase activity in the hypertensive animals.

Immature renal tubules are resistant to prolonged anoxia

ABSTRACT: Very few data are available regarding the decreased susceptibility of the developing kidney to anoxia. Therefore, the purpose of this study was to develop an experimental system that would allow comparison of an anoxic insult in immature and mature proximal tubule segments and to investigate the hypothesis that the developing kidney is resistant to anoxia as compared with the mature kidney. Suspensions of proximal tubules from immature (age 8–10 d) and mature (8–10 wk) rats were obtained. The purity of the tubule suspension from the immature rats was documented by villin staining. A common buffer solution was developed to compare results from the immature and mature tubules. To study the response of the tubules to anoxia, we subjected the tubule suspension from both the immature and mature rats to 15, 30, 45, and 60 min of anoxia. Lactate dehydrogenase release was measured to assess plasma membrane damage, and ATP levels were determined as an index of cellular energy. After a short anoxic insult (15 or 30 min), the percentage of lactate dehydrogenase release was not significantly different from mature tubules. After prolonged anoxia (45 and 60 min) lactate dehydrogenase release continued to increase, whereas membrane integrity stabilized in the immature tubules. ATP levels decreased in both immature and mature tubules after anoxia, but the decline of ATP was greater in the mature tubules, with a plateau at 20% of basal ATP levels as compared with 40% in the immature tubules. Therefore, the developing kidney is resistant to prolonged anoxia.

Cl(-)-dependent NH4+ transport mechanisms in medullary thick ascending limb cells.

To characterize Cl(-)-dependent NH4+ transport mechanisms in renal medullary thick ascending limb (MTAL), intracellular pH (pHi) and membrane potential (PD) were monitored with use of 2',7'-bis(carboxyethyl)- 5(6)-carboxyfluorescein and 3,3'-dipropylthiadicarbocyanine, respectively, in suspensions of rat MTAL tubules in CO2-free media. Exposure of MTAL cells to 4 mM NH4Br caused, after an initial cell alkalinization due to NH3 entry, an NH4(+)-induced fall in pHi that was approximately 67% less pronounced in Cl(-)-free than in Cl(-)-containing media. The following experiments were performed in the presence of 1 microM amiloride to block the MTAL NH4+ conductance. When cells were preincubated in a Cl(-)-free gluconate medium in which K+ and Cl- conductances are greatly reduced, abrupt addition of 100 mN N-methyl-D-glucamine (NMDG)-Cl had no effect on cell PD and pHi in the absence of ammonia, but acutely acidified the cells by approximately 0.2 pH units in the presence of 4 mM NH4Br, which thus indicated nonelectrogenic (NMDG-Cl)-dependent NH4+ influx. The latter also occurred in a Cl(-)-free thiocyanate medium in which the Cl- conductance was blocked by 0.1 mM diphenylamine-2-carboxylate (DPC). An NMDG-Cl- dependent NH4(+)-induced fall in pHi was reduced approximately 33% by 10 mM Ba+, approximately 84% by 0.1 mM bumetanide, and 100% by 1.5 mM furosemide, whereas 1 mM hydrochlorothiazide had no effect; inhibition by Ba+ was observed even in the presence of 0.1 mM verapamil added to block both K+ channels and K+/NH4+ antiport.(ABSTRACT TRUNCATED AT 250 WORDS)