Segments Of Rabbit Proximal Tubule Research Articles

Properties and Regulation of Organic Cation Transport in Freshly Isolated Human Proximal Tubules

The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. Organic cation transport across the basolateral membrane of isolated collapsed proximal tubules was recorded with the fluorescent dye 4-(4-dimethylamino)styryl-N-methylpyridinium (ASP(+)). Depolarization of the cells by rising extracellular K(+) concentration to 145 mm reduced ASP(+) uptake by 20 +/- 5% (n = 15), indicating its electrogeneity. The substrates of organic cation transport tetraethylammonium (K(i) = 63 microm) and cimetidine (K(i) = 11 microm) as well as the inhibitor quinine (K(i) = 2.9 microm) reduced ASP(+) uptake concentration dependently. Maximal inhibition reached with these substances was approximately 60%. Stimulation of protein kinase C with 1,2-dioctanoyl-sn-glycerol (DOG, 1 microm) or ATP (100 microm) inhibited ASP(+) uptake by 30 +/- 3 (n = 16) and 38 +/- 13% (n = 6), respectively. The effect of DOG could be reduced with calphostin C (0.1 microm, n = 7). Activation of adenylate cyclase by forskolin (1 microm) decreased ASP(+) uptake by 29 +/- 3% (n = 10). hANP (10 nm) or 8-bromo-cGMP (100 microm) also decreased ASP(+) uptake by 17 +/- 3 (n = 9) or 32 +/- 5% (n = 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP- and the cGMP-dependent protein kinases.

PKC regulation of organic anion secretion in perfused S2 segments of rabbit proximal tubules.

To examine the role of protein kinase C (PKC) in organic anion (OA) secretion, we used epifluorescence microscopy to study steady-state transepithelial secretion of 1 microM fluorescein (FL) by isolated perfused S2 segments of rabbit renal proximal tubules. Addition of 100 nM phorbol 12-myristate 13-acetate (PMA), a known PKC activator, to the bathing medium decreased steady-state secretion of FL by approximately 30% after 25 min. This inhibition was irreversible and, indeed, increased to approximately 40% at 25 min following removal of PMA [10 microM 1,2-dioctanoyl-sn-glycerol (DOG) produced a comparable inhibition]. The inhibition produced by PMA was blocked when 100 nM of either staurosporine (ST) or bisindolylmaleimide I (BIM), both known PKC inhibitors, was added to the bath for a 20-min preexposure followed by the addition of PMA. ST or BIM alone had no significant effect on FL secretion, suggesting that the basal FL secretion rate was not under influence of PKC. Addition of 1 microM of either the peptide hormone bradykinin (BK) or the alpha(1)-receptor agonist phenylephrine (PE), both of which stimulate PKC via a ligand-receptor-PKC coupling reaction, to the bath also inhibited FL secretion by approximately 22 and approximately 27%, respectively. However, the inhibition was completely reversible after removal of BK or PE. Pretreatment of tubules with 100 nM BIM eliminated the inhibition of FL secretion produced by exposure to PE. We conclude that PKC negatively regulates the net secretion of OAs in rabbit renal proximal tubules. The data indicate that BK or catecholamines can play a physiological role in regulating OA secretion via PKC activation.

Mechanisms of action of 2,3-dimercaptopropane-1-sulfonate and the transport, disposition, and toxicity of inorganic mercury in isolated perfused segments of rabbit proximal tubules.

Mechanisms by which the dithiol chelating agent 2, 3-dimercaptopropane-1-sulfonate (DMPS) significantly alters the renal tubular transport, accumulation, and toxicity of inorganic mercury were studied in isolated perfused pars recta (S2) segments of proximal tubules of rabbits. Addition of 200 microM DMPS to the bath provided complete protection from the toxic effects of 20 microM inorganic mercury in the lumen. The protection was linked to decreased uptake and accumulation of mercury. Additional data indicated that, when DMPS and inorganic mercury were coperfused through the lumen, very little inorganic mercury was taken up from the lumen. We also obtained data indicating that DMPS is transported by the organic anion transport system and that this transport is linked to the therapeutic effects of DMPS. Interestingly, very little inorganic mercury was taken up and no cellular pathological changes were detected when inorganic mercury and DMPS were added to the bath. We also tested the hypothesis that DMPS can extract cellular mercury while being transported from the bath into the luminal compartment. Our findings showed that, when DMPS was applied to the basolateral membranes of S2 segments after they had been exposed to mercuric conjugates of glutathione of the laminal membrane, the tubular content of mercury was greatly reduced and the rates of disappearance of mercury from the lumen changed from positive values to markedly negative values. We conclude that inorganic mercury is extracted from proximal tubular cells by a transport process involving the movement of DMPS from the bathing compartment to the luminal compartment.

Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule. II. Change of Na-HCO3 cotransport stoichiometry and of response to acetazolamide.

In the preceding publication we reported that some transport properties of proximal tubules perfused in vitro differ from those of tubules perfused in vivo, and that the in vivo function can be largely recuperated by improved metabolic substrate supply and stimulation with norepinephrine (NE). Since we have previously observed that the basolateral Na-HCO3-cotransporter operates with an overall stoichiometric ratio of q approximately 3 HCO3- :1 Na+ in vivo, but with q approximately 2 HCO3- :1 Na+ in vitro and that it responds differently in both cases to acetazolamide (ACZ), we have now tested whether the cotransporter can regain its in vivo function in vitro if the incubation conditions are improved. Cell membrane potentials (Vb) and cell pH (pHi) were measured with microelectrodes and microfluorimetric techniques on isolated S2 segments of rabbit proximal tubule and the instantaneous Vb response to a 2:1 reduction of bath HCO3- or Na+ concentration was determined. (DeltaVb)HCO3 and (deltaVb)Na averaged 13.1 +/- 0.9 mV (SEM) and 6.9 +/- 0.5 mV in D-glucose-containing control HCO3-Ringer solution and decreased respectively to 10.1 +/- 0.5 mV and 3.8 +/- 0.2 mV (n = 8) after incubation in tissue culture medium and NE (10(-5) mmol/l). These data imply that q increased from 1.9 to 2.7. Concomitantly the tubules became susceptible to ACZ (1 mmol/l), which reduced (deltaVb)HCO3 in control conditions only to 94.6 +/- 1.2% but under improved incubation conditions to 64.5 +/- 2.4%. As verified in voltage divider measurements the latter reduction was not caused by activation of a basolateral K+ conductance. The results indicate that improved incubation conditions can at least partially revert cotransport function towards that of the in vivo state. The effect of ACZ may be explained if in the improved state 1 CO32- + 1 HCO3- + 1 Na+ are cotransported, in which case inhibition of carbonic anhydrase (CA) may cause a CO32-/pH disequilibrium to develop in the basal labyrinth which impedes the cotransport. Under conventional incubation conditions, however, when only 2 HCO3- + 1 Na+ are cotransported no such disequilibrium should develop irrespective of whether CA is active or inhibited.

Effect of alpha-KG in lumen on PAH transport by isolated perfused rabbit renal proximal tubules.

To determine whether dicarboxylate taken up at the luminal membrane could function in the p-aminohippurate (PAH) countertransport at the basolateral membrane, we examined the effect of adding alpha-ketoglutarate (alpha-KG) or glutarate (a nonmetabolized dicarboxylate that is countertransported for PAH at the basolateral membrane) to the luminal perfusate on net secretion of radiolabeled PAH in isolated perfused S2 segments of rabbit proximal tubules. Addition of 100 microM alpha-KG or glutarate to the luminal perfusate in tubules perfused and bathed with HEPES-buffered medium (in the absence of bicarbonate, glycine, lactate, malate, and citrate) produced a reversible twofold stimulation of net PAH transepithelial secretion. Addition of 4 mM LiCl (an inhibitor of Na-dicarboxylate transport that does not directly affect PAH transport) to the luminal perfusate along with alpha-KG eliminated stimulation of net PAH secretion. Addition of 100 microM or 1 mM alpha-KG or glutarate to the luminal perfusate in tubules perfused and bathed with bicarbonate-buffered medium containing glycine, lactate, malate, and citrate had no effect on net PAH transport from bath to lumen. These data indicate that alpha-KG (or glutarate) that enters the tubule cells via the luminal Na-dicarboxylate cotransporter can stimulate net PAH secretion, apparently via countertransport at the basolateral membrane, but only when tubules are not in an optimal metabolic state to produce intracellular alpha-KG.

Angiotensin II upregulates type-1 angiotensin II receptors in renal proximal tubule.

Angiotensin II (Ang II) is an important regulator of proximal tubule salt and water reabsorption. Recent studies indicate that rabbit proximal tubule angiotensin II receptors are the type-1 (AT1R) subtype. We studied the effect of Ang II on proximal tubule receptor expression. Rabbits were treated with either angiotensin converting enzyme inhibitors or a low salt diet to modulate endogenous Ang II levels. In captopril-treated rabbits, liver and glomerular AT1R mRNA levels increased 242 +/- 125 and 141 +/- 60%, respectively (n = 6-7; P < 0.05), as determined by quantitative PCR. In contrast, proximal tubule AT1R mRNA levels decreased 40 +/- 11% (n = 6; P < 0.05). Binding of 125I Ang II to renal cortical basolateral membranes of captopril-treated rabbits decreased from 2.9 +/- 0.55 to 1.4 +/- 0.17 fmol/mg protein (n = 6; P < 0.025). In rabbits fed a sodium chloride-deficient diet for 4 wk, AT1R mRNA levels decreased 52 +/- 11% in liver and 43 +/- 7% in glomeruli (n = 4-5; P < 0.05), whereas they increased 141 +/- 85% (n = 5; P < 0.05) in proximal tubule. In basolateral membranes from rabbits on the sodium chloride-deficient diet, specific binding of 125I Ang II increased from 2.1 +/- 0.2 to 4.3 +/- 1.1 fmol/mg protein (n = 7; P < 0.05). To determine whether Ang II directly regulates expression of proximal tubule AT1 receptors, further studies were performed in cultured proximal tubule cells grown from microdissected S1 segments of rabbit proximal tubules and immortalized by transfection with a replication-defective SV40 vector. Incubation of these cells with Ang II (10(-11) to 10(-7) M) led to concentration-dependent increases in both AT1R mRNA levels and specific 125I Ang II binding. Pretreatment with pertussis toxin inhibited Ang II stimulation of AT1R mRNA. AT1R mRNA expression was decreased by either forskolin or a nonhydrolyzable cAMP analogue (dibutryl cAMP). Simultaneous Ang II administration overcame the inhibitory effect of forskolin but not dibutryl cAMP. These results indicate that proximal tubule AT1R expression is regulated by ambient Ang II levels, and Ang II increases AT1R mRNA at least in part by decreasing proximal tubule cAMP generation through a pertussis toxin-sensitive mechanism. Upregulation of proximal tubule AT1R by Ang II may be important in mediating enhanced proximal tubule sodium reabsorption in states of elevated systemic or intrarenal Ang II.

Organic osmolytes increase cytoplasmic viscosity in kidney cells.

The hypothesis was tested that accumulation of osmolytes by kidney cells grown in hyperosmolar media decreases the rotational and translational mobilities of small polar solutes in the cytosolic compartment. Rotational mobility was measured by the picosecond rotational correlation times (tau c) of 2',7'-bis(2-carboxyethyl)-5(6)carboxylfluorescein (BCECF) by multiharmonic microfluorimetry. In isolated segments of rabbit proximal tubule, thick ascending limb, and cortical collecting duct that were perfused and bathed in 300 mosM media, tau c were in the range 180-250 ps, corresponding to apparent rotational viscosities (eta r) of 1.1-1.5 cP. In cortical collecting tubule, eta r was not influenced by serosal vasopressin. In Madin-Darby canine kidney (MDCK) cells grown in 300-1,200 mosM media, eta r increased progressively by up to a factor of 1.38 +/- 0.03; measurements of tau c and macroscopic viscosity in artificial solutions containing osmolytes supported the hypothesis that the increased eta r was due to accumulation of organic osmolytes. BCECF translational mobility was measured by fluorescence photobleaching recovery using a focused 1.2-microns diameter Ar laser beam at 488 nm. Recovery half-times were 36 +/- 3 (SE) ms (n = 10) in MDCK cells grown in 300 mosM media and 62 +/- 3 ms (n = 10) when grown in 1,200 mosM media. The results suggest that accumulation of osmolytes by renal cells is associated with significantly increased cytosolic viscosity. The increased viscosity would slow enzymatic and transport processes in the cytosolic compartment.

Lactate oxidation by three segments of the rabbit proximal tubule.

Oxidation of [U14C]lactate to 14CO2 was measured in vitro, in nonperfused anatomically defined segments of rabbit proximal tubule (S1, proximal convoluted, and S2 and S3, proximal straight tubules). The rate of lactate oxidation was similar in S2 and S3 segments, and within the range of lactate oxidation rates measured in vivo. In contrast, the oxidation rate of S1 segments was significantly lower than that of S2 or S3. In proximal straight tubules, lactate oxidation was inhibited by incubation at 0 degrees C, or by application of 1 mM ouabain. To determine if the rate of transepithelial transport affected the rate of lactate oxidation, lactate oxidation was measured in proximal straight tubules after the lumen had been opened by perfusion with Ringer's containing 10 mM polyethylene glycol. No difference in lactate oxidation rate was observed between tubules with patent lumina and nonperfused tubules. These results suggest that the various segments of the renal proximal tubule have different metabolic characteristics, and that the rate of substrate oxidation is related to the activity of the Na+, K+-ATPase.

Further studies of organic base secretion by rabbit proximal tubules.

The aim of the present studies was to further characterize the mechanisms responsible for the secretion of the organic base procainamide by measuring [3H] procainamide transport in superficial (SF) and juxtamedullary (JM) S2 segments of rabbit proximal tubules perfused in vitro. When perfusate pH was changed from 7.4 to 6.2, procainamide secretion increased by 227% in SF and by 159% in JM tubules. In the presence of ouabain the acidic perfusate caused only a minimal increase in procainamide secretion. When the perfusate pH was increased, procainamide secretion was reduced. Acetazolamide, 10(-5) M in the bath, reduced procainamide secretion in both SF and JM tubules. In SF tubules amiloride, 10(-4) M in the perfusate, inhibited procainamide secretion, whereas nonradioactive procainamide, quinidine, and cimetidine did not. When ambient sodium was reduced to 10 mM, the rate of procainamide secretion was low and was not affected by amiloride in the perfusate or acetazolamide in the bath. Amiloride in the bath caused a dose-related inhibition of procainamide secretion. The rate of procainamide secretion by cortical collecting tubules was very low and was not affected by acetazolamide. The mechanisms of renal organic base secretion are complex. Some of the present data are consistent with a mechanism involving organic base-proton exchange across the apical cell membrane. However, other data are not consistent with this being the major mechanism involved.

Accumulation of [14C]urate and [3H]PAH in isolated proximal tubular segments of the rabbit kidney.

Segments of rabbit proximal tubules (S1, S2, and S3) were incubated (30 min, 37 degrees C, pH 7.4) in Ringer solution (under O2) containing [14C]urate or [3H]PAH and probenecid, pyrazinoic acid (PZA), urate, or PAH, all at 1 mM. The ratio [14C]urate (counts/min per nanoliter of tubular cell water)/(counts/min per nanoliter of incubation medium) (T/M) reached 2.92 +/- 0.31 (mean +/- SE; n = 15), 6.08 +/- 0.42 (n = 42), and 1.72 +/- 0.31 (n = 14) for S1, S2, and S3 segments, respectively. In these segments the uptake of [3H]PAH was 22.7 +/- 4.6 (n = 19), 133 +/- 18.9 (n = 24), and 66.2 +/- 15.3 (n =0). The accumulation of [14C]urate was saturated at 2 x 10(-4) M in the medium, and that of [3H]PAH at 7 x 10(-5) M in the medium. Secretory transport was saturated at the same bath PAH concentration in perfused tubules. Urate and PAH transports inhibited each other, thus suggesting a common transport mechanism mainly located in the S2 segment. Probenecid was a strong and PZA a weak inhibitor of this transport system.

Urea secretion by the straight segment of the proximal tubule.

Studies utilizing in vitro microperfusion were designed to examine whether urea is actively or passively transported across superficial and juxtamedullary straight segments of rabbit proximal tubules. With perfusate and bath solutions containing 1 mM urea and electrolytes similar to normal plasma, the efflux (lumen-to-bath) isotopic permeability (X 10(-5) cm s-1) of superficial segments was 1.37 +/- 0.16 and of juxtamedullary segments was 2.14 +/- 0.20. In the same tubules, the influx (bath-to-lumen) isotopic permeability was 3.70 +/- 0.35 in superficial segments and 4.75 +/- 0.37 in juxtamedullary segments. Despite net water movement in the opposite direction (0.5 nl mm-1 min-1), the influx rate was significantly higher than the efflux rate of urea in both groups. With a low perfusion rate (2 nl/min) and equivalent specific activities of [14C]urea in bath and perfusate, the collected-to-perfused ratio of [14C]urea, corrected for volume marker change, was 1.07 +/- 0.01 in superficial and 1.09 +/- 0.01 in juxtamedullary nephrons, thus indicating net secretion in both segments. In separate studies urea influx was inhibited by hypothermia (decrease from 37 degrees to 28 degrees C), by phloretin (0.1 mM in bath), by cyanide (1 mM), but not by probenecid (0.2 mM). In each case the inhibition was highly significant and reversible. These data suggest that urea is actively secreted by the straight segments of both the superficial and juxtamedullary proximal tubules. These segments may, therefore, contribute significantly to the high urea concentration found at the bend of Henle's loop by micropuncture.

Characteristics of salt and water transport in superficial and juxtamedullary straight segments of proximal tubules.

The purpose of the present studies was to characterize the nature of salt and water transport out of the superficial (SF) and juxtamedullary (JM) straight segments of rabbit proximal tubules as examined by in vitro microperfusion techniques. When the perfusate consisted of a solution simulating ultrafiltrate of plasma, there were no differences between SF and JM straight tubules in either net reabsorption of fluid (SF=0.47 nl/mm per min; JM=0.56 nl/mm per min) or in transtubular potential difference (PD) (SF=-2.1 mV; JM=-1.8 mV). Removal of glucose and alanine from the perfusate had no effect on the magnitude of the PD in either straight segment. Ouabain decreased both the net reabsorptive rates and the PD. Isosmolal replacement of NaCL by Na-cyclamate (a presumed impermeant anion) in the perfusate and the bath caused an increase in luminal negativity in both segments wheras similar substitution of NaCL by choline-CL (nontransported cation) changed the PD TO NEAR ZERO. These studies, therefore, suggest that sodium is transported out of the proximal straight tubules by an active noncoupled process that generates a PD (electrogenic process). When the perfusate consisted of a solution with a high chloride concentration (resulting from greater HCO3 than CI reabsorption in the proximal convoluted tubule), different PDs in SF and JM tubules were generated: SF=+1.6 plus or minus 0.2 mV; JM=-1.3 plus or minus 0.3 mV. This difference in PD was attributed to relative differences in Na and CI permeabilities in these two segments. Electrophysiological and isotopic estimates of the chloride to sodium permeability revealed that the SF tubule is about twice as permeant to chloride than to sodium whereas the JM tubules are approximately twice as permeant to sodium than to chloride. It is concluded that the mechanism of active sodium transport in the straight segment of proximal tubule differs from that of the convoluted segment and that both the SF and JM straight segments differ from each other with respect os sodium and chloride permeability.