Influence of peritubular protein on solute absorption in the rabbit proximal tubule. A specific effect on NaCl transport.

The effect of removal of peritubular protein on the reabsorption of various solutes and water was examined in isolated rabbit proximal convoluted tubules (PCT) perfused in vitro. In 22 PCT perfused with ultrafiltrate (UF) and bathed in serum, volume absorption (Jv) was 1.44 nl/mm per min and potential difference (PD) was -3.6 mV. When these same PCT were bathed in a protein-free UF, Jv was reduced 38% without a change in PD. Simultaneous measurements of total CO2 net flux (JTCO2) and glucose efflux (JG) showed that less than 2% of the decrease in JV could be accounted for by a reduction in JTCO2 and JG, suggesting that removal of peritubular protein inhibited sodium chloride transport (JNaCl). Therefore, in eight additional PCT, JNaCl was measured, in addition to PD, Jv, JG, and JTCO2. In these PCT, the decrease in total solute transport induced by removal of bath protein was 201.7 +/- 37.5 posmol/mm per min. JG decreased slightly (9.1 +/- 3.9 posmol/mm per min); NaHCO3 transport did not change (9.2 +/- 6.6 posmol/mm per min); but JNaCl decreased markedly (160.6 +/- 35.7 posmol/mm per min). 80% of the decrease in Jv could be accounted for by a decrease in JNaCl. In 13 additional PCT perfused with simple NaCl solutions, a comparable decrease in Jv and JNaCl was observed when peritubular protein was removed without an increase in TCO2 backleak. In summary, removal of peritubular protein reduced Jv and JNacl, but did not significantly alter PD, JG, JTCO2, or TCO2 backleak. The failure to inhibit JG and JTCO2, known sodium-coupled transport processes, indicates that protein removal does not primarily affect the Na-K ATPase pump system. Furthermore, since PD and TCO2 backleak were not influenced, it is unlikely that protein removal increased the permeability of the paracellular pathway. We conclude that protein removal specifically inhibits active transcellular or passive paracellular NaCl transport.     

Lack of effect of peritubular protein on passive NaCl transport in the rabbit proximal tubule.

The effect of peritubular protein removal on passive NaCl transport was examined in the isolated rabbit proximal convoluted tubule (PCT). Three modes of passive NaCl transport were tested: (a) paracellular backflux of NaCl, (b) convective flow of NaCl through junctional complexes, and (c) anion gradient-dependent NaCl transport. The effect of peritubular protein removal on the paracellular permeability to NaCl was examined using transepithelial specific resistance. Eight PCT were perfused with ultrafiltrate (UF) and bathed in either serum or UF. Transepithelial specific resistance averaged 14.5 +/- 1.9 in the presence and 13.7 +/- 1.7 omega cm2 in the absence of peritubular protein. The effect of peritubular protein removal on the convective flow of a NaCl solution across functional complexes was examined in the absence of active transport by using colloid osmotic pressure (COP) gradients. 12 PCT were perfused with simple salt solutions in Donnan equilibrium with and without protein at 20 degrees C. A COP gradient of 60.1 and -60.1 mmHg drove only 0.06 and -0.23 nl/min, respectively. These values are approximately 10% of the value predicted for an effect of peritubular protein on NaCl solution flow (1.98 nl/min) and are approximately equal to the value predicted for pure water equilibration for the small osmotic pressure difference between solutions in Donnan equilibrium (0.17-0.18 nl/min). The effect of peritubular protein removal on the passive absorption of NaCl driven by anion concentration gradients was examined in seven PCT perfused with a high chloride solution simulating late proximal tubular fluid and bathed in either serum or UF at 20 degrees C. Volume absorption averaged 0.34 +/- 0.20 in the presence and 0.39 +/- 0.20 nl/mm min in the absence of peritubular protein. In conclusion, peritubular protein removal did not significantly affect any of the three distinct modes of passive NaCl transport tested. The lack of effect of peritubular protein removal on passive paracellular NaCl transport suggests that protein modulates an active transcellular NaCl transport process.     

Insulin stimulates volume absorption in the rabbit proximal convoluted tubule.

The present in vitro microperfusion study examined whether insulin affects volume absorption (Jv) in the proximal convoluted tubule (PCT). PCT were perfused with an ultrafiltrate-like solution and were bathed in a serum-like albumin solution. Addition of a physiologic concentration of 10(-10) M insulin to the bathing solution resulted in a stimulation of Jv and a more negative transepithelial potential difference (PD). There was a progressive stimulation of the lumen negative PD and Jv with higher insulin concentrations. Maximal stimulation occurred at 10(-8) M bath insulin. The insulin-induced stimulation of volume reabsorption was also observed when glucose and amino acids were removed from the luminal perfusate. Direct examination of the effect of insulin on glucose, chloride, and bicarbonate absorption demonstrated that the transport of all these solutes was stimulated by insulin. Addition of insulin to the luminal perfusate had no affect on Jv. These data show that insulin has a direct effect to stimulate Jv in the proximal tubule.     

KCl co-transport across the basolateral membrane of rabbit renal proximal straight tubules.

Mammalian renal proximal tubules reabsorb large amounts of chloride. Mechanisms of the transcellular chloride transport are poorly understood. To determine whether KCl co-transport exists in the basolateral membrane of mammalian renal proximal tubule, isolated rabbit proximal straight tubules (S2 segment) were perfused in vitro, and intracellular activities of potassium and chloride (aKi, aCli) were measured by double-barreled ion-selective microelectrodes. aCli did not change when basolateral membrane voltage was altered by application of a direct current through perfusion pipette. aCli changes in response to bath chloride elimination were not affected by current application as well, indicating that the basolateral chloride transport is electroneutral. An increase in potassium concentration of the bath fluid from 5 to 20 mM reversibly increased aCli by 10 mM. This response of aCli to a change in the bath potassium concentration was also observed when luminal chloride was removed, or ambient sodium was totally removed. aKi significantly decreased by 5 mM when chloride was removed from the bath. These data demonstrate the existence of an electroneutral Na+-independent KCl co-transport in the basolateral membrane of the rabbit proximal tubule. Calculated electrochemical driving force was favorable for the movement of KCl from the cell to the peritubular fluid.     

Mechanism of apical and basolateral Na(+)-independent Cl-/base exchange in the rabbit superficial proximal straight tubule.

The present study was undertaken to determine the magnitude and mechanism of base transport via the apical and basolateral Na(+)-independent Cl-/base exchangers in rabbit isolated perfused superficial S2 proximal tubules. The results demonstrate that there is an apical Na(+)-independent Cl-/base exchanger on both membranes. HCO3- fails to stimulate apical Cl-/base exchange in contrast to the basolateral exchanger. Inhibition of endogenous HCO3- production does not alter the rate of apical Cl-/base exchange in Hepes-buffered solutions. Both exchangers are inhibited by H2DIDS and furosemide; however, the basolateral anion exchanger is more sensitive to these inhibitors. The results indicate that the apical and basolateral Cl-/base exchangers differ in their transport properties and are able to transport base equivalents in the absence of formate. The formate concentration in rabbit arterial serum is approximately 6 microM and in vitro tubule formate production is < 0.6 pmol/min per mm. Formate in the micromolar range stimulates Jv in a dose-dependent manner in the absence of a transepithelial Na+ and Cl- gradient and without a measurable effect on Cl(-)-induced equivalent base flux. Apical formic acid recycling cannot be an important component of any cell model, which accounts for formic acid stimulation of transcellular NaCl transport in the rabbit superficial S2 proximal tubule. We propose that transcellular NaCl transport in this nephron segment is mediated by an apical Na+/H+ exchanger in parallel with a Cl-/OH- exchanger and that the secreted H+ and OH- ions form H2O in the tubule lumen.     

Mechanism of NaCl and water reabsorption in the proximal convoluted tubule of rat kidney.

The role of chloride concentration gradients in proximal NaCl and water reabsorption was examined in superficial proximal tubules of the rat by using perfusion and collection techniques. Reabsorptive rates (Jv), chloride concentrations, and transtubular potential difference were measured during perfusion with solutions (A) simulating an ultrafiltrate of plasma; (B) similar to (A) except that 20 meq/liter bicarbonate was replaced with acetate; (C) resembling late proximal fluid (glucose, amino acid, acetate-free, low bicarbonate, and high chloride); and (D) in which glucose and amino acids were replaced with raffinose and bicarbonate was partially replaced by poorly reabsorbable anions (cyclamate,sulfate, and methyl sulfate). In tubules perfused with solutions A and B, Jv were 2.17 and 2.7 nl mm-1 min-1 and chloride concentrations were 131.5 +/- 3.1 and 135 +/- 395 meq/liter, respectively, indicating that reabsorption is qualitatively similar to free-flow conditions and that acetate adequately replaces bicarbonate. With solution C, Jv was 2.10 nl mm-1 min-1 and potential difference was +1.5 +/- 0.2 mV, indicating that the combined presence of glucose, alanine, acetate, and bicarbonate per se is not an absolute requirement. Fluid reabsorption was virtually abolished when tubules were perfused with D solutions; Jv was not significantly different from zero despite sodium and chloride concentrations similar to plasma; chloride concentration was 110.8 +/- 0.2 meq/liter and potential difference was -0.98 mV indicating that chloride was close to electrochemical equilibrium. These results suggest the importance of the chloride gradient to proximal tubule reabsorption in regions where actively reabsorbable solutes (glucose, alanine, acetate, and bicarbonate) are lacking and provide further evidence for a passive model of NaCl and water transport.     

Interaction of chloride and bicarbonate transport across the basolateral membrane of rabbit proximal straight tubule. Evidence for sodium coupled chloride/bicarbonate exchange.

The existence of chloride/bicarbonate exchange across the basolateral membrane and its physiologic significance were examined in rabbit proximal tubules. S2 segments of the proximal straight tubule were perfused in vitro and changes in intracellular pH (pHi) and chloride activity (aCli) were monitored by double-barreled microelectrodes. Total peritubular chloride replacement with gluconate increased pHi by 0.8, and this change was inhibited by a pretreatment with an anion transport inhibitor, SITS. Peritubular bicarbonate reduction increased aCli, and most of this increase was lost when ambient sodium was totally removed. The reduction rates of pHi induced by a peritubular bicarbonate reduction or sodium removal were attenuated by 20% by withdrawal of ambient chloride. SITS application to the bath in the control condition quickly increased pHi, but did not change aCli. However, the aCli slightly decreased in response to SITS when the basolateral bicarbonate efflux was increased by reducing peritubular bicarbonate concentration. It is concluded that sodium coupled chloride/bicarbonate exchange is present in parallel with sodium-bicarbonate cotransport in the basolateral membrane of the rabbit proximal tubule, and it contributes to the basolateral bicarbonate and chloride transport.     

Effect of formate on volume reabsorption in the rabbit proximal tubule.

Studies on microvillus membrane from rabbit kidney cortex suggest that chloride absorption may occur by chloride/formate exchange with recycling of formic acid by nonionic diffusion. We tested whether this transport mechanism participates in active NaCl reabsorption in the rabbit proximal tubule. In proximal tubule S2 segments perfused with low HCO-3 solutions, the addition of formate (0.25-0.5 mM) to the lumen and the bath increased volume reabsorption (JV) by 60%; the transepithelial potential difference remained unchanged. The effect of formate on JV was completely reversible and was inhibited both by ouabain and by luminal 4,4'-diisothiocyanostilbene-2,2'-disulfonate. Formate (0.5 mM) failed to stimulate JV in early proximal convoluted tubules perfused with high HCO-3 solutions. As measured by miniature glass pH microelectrodes, this lack of formate effect on JV was related to a less extensive acidification of the tubule fluid when high HCO-3 solutions were used as perfusate. These data suggest that chloride/formate exchange with recycling of formic acid by nonionic diffusion represents a mechanism for active, electroneutral NaCl reabsorption in the proximal tubule.     

Flow dependence of vasopressin-stimulated hydraulic conductivity in rabbit collecting tubule

Flow rate dependence of both electrolyte and nonelectrolyte transport in various nephron segments has been described. Prior studies have used relatively leaky epithelia in which the flow rate-dependent transport phenomena can be explained in terms of alterations in axial and radial concentration profiles. In this study, the flow rate dependence of either vasopressin or cyclic adenosine monophosphate-stimulated water flux (Jv), hydraulic conductivity (Lp), and osmotic permeability (Pf) were measured in isolated perfused rabbit cortical collecting tubules. Increasing perfusion rate from 6.0 +/- 0.4 to 20.7 +/- 1.2 nl/min results in highly significant increases in Jv (131%) and in Lp and Pf (120%). In this relatively tight epithelium, osmotic equilibrium did not occur. Although the mechanism of this effect remains to be elucidated, the present results mandate maintenance of constant flow rates when examining the perfused cortical collecting tubular response to vasopressin.     

Mineralocorticoid modulation of apical and basolateral membrane H+/OH-/HCO3- transport processes in the rabbit inner stripe of outer medullary collecting duct.

To examine the mechanism by which mineralocorticoids regulate HCO3- absorption in the rabbit inner stripe of the outer medullary collecting duct, we microfluorometrically measured intracellular pH (pHi) in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) assaying the apical and basolateral membrane H+/OH-/HCO3- transport processes in three groups of animals: those receiving chronic in vivo DOCA treatment (5 mg/kg per d x 2 wk); those with surgical adrenalectomy (ADX, [chronic x 2 wk]) on glucocorticoid replacement; and controls. Baseline pHi was not different in the three groups. Cellular volume (vol/mm) was increased 38% in DOCA tubules versus controls, but unchanged in ADX tubules versus controls. Buffer capacities (BT) were not different in the three groups. Apical membrane H+ pump activity, assayed as the Na(+)-independent pHi recovery from an acid load (NH3/NH4+ prepulse) and expressed as JH (dpHi/dt.vol/mm.BT) was increased 76% in DOCA tubules versus controls, and decreased 56% in ADX tubules versus controls. Basolateral membrane Cl-/HCO3- exchange activity assayed as the pHi response to basolateral Cl- addition was increased 73% in DOCA tubules versus controls, and decreased 44% in ADX tubules versus controls. When examined as a function of varying [Cl-], the Vmax of Cl-/HCO3- exchange activity was significantly increased in DOCA tubules (control, 72.7 +/- 15.7 pmol.mm-1.min-1 vs DOCA, 132.3 +/- 22.5 pmol.mm-1.min-1, P less than 0.02), while the K1/2 for Cl- was unchanged. Basolateral membrane Na+/H+ antiporter activity assayed as the Na(+)-dependent pHi recovery from an acid load was not changed in chronic DOCA tubules versus controls. In conclusion, the apical membrane H+ pump and basolateral membrane Cl-/HCO3- exchanger of the rabbit OMCDi are regulated in parallel without chronic alterations in pHi under the conditions of mineralocorticoid excess and deficiency. The parallel changes in these transporters accounts for the alterations in OMCDi HCO3- absorption seen under these conditions.     

Intracellular cystine loading inhibits transport in the rabbit proximal convoluted tubule.

Cystinosis is an autosomal recessive disorder characterized by a high intracellular cystine concentration. To establish an in vitro model of this disorder and examine the mechanism of the proximal tubule transport defect seen with elevated intracellular cystine concentrations, rabbit proximal convoluted tubules (PCT) were perfused in vitro. PCTs were loaded with cystine using cystine dimethyl ester, a permeative methyl ester derivative. Bath cystine dimethyl ester (0.5 mM) reduced volume absorption (Jv) (0.67 +/- 0.07 to 0.15 +/- 0.09 nl/mm.min, P less than 0.01), bicarbonate transport (JTCO2) (47.2 +/- 4.9 to 11.1 +/- 2.8 pmol/mm.min, P less than 0.001) and glucose transport (JGLU) (34.1 +/- 1.5 to 19.7 +/- 1.5 pmol/mm.min, P less than 0.001). The methyl esters of leucine (0.5 mM), and tryptophan (0.5 and 2.0 mM) had no effect on these parameters. To examine if intracellular reduction of cystine to cysteine could contribute to the inhibition in transport, the effect of bath cysteine methyl ester on proximal tubular transport was examined. Bath cysteine methyl ester (2 but not 0.5 mM) resulted in an inhibition in Jv, JGLU, and JTCO2. Cystine dimethyl ester had no effect on mannitol or bicarbonate permeability. These data are consistent with intracellular proximal tubular cystine accumulation resulting in an inhibition of active transport.     

The role of norepinephrine in the regulation of fluid absorption in the rat proximal tubule

In order to investigate the possibility of a direct effect of adrenergic transmitter on tubular fluid absorption, we have studied the effects of norepinephrine and phenoxybenzamine on fluid absorption in the proximal convoluted tubule of the rat kidney. Net fluid absorption (Jv) was determined in the same proximal convoluted tubule before and after addition of norepinephrine or phenoxybenzamine while the tubular lumen and peritubular capillaries were simultaneously microperfused in situ. When the tubular lumen was perfused with Ringer's solution and the peritubular capillaries were perfused with albumin Ringer's solution, Jv was 2.85 +/- 0.25 nl/min X nm. Addition of 2 X 10(-6) M norepinephrine to the capillary perfusate caused at 30% increase in Jv which could be reversed by removing the adrenergic transmitter. The effect of norepinephrine was dose dependent with the maximal increase of Jv observed at a concentration of 10(-5) M. Addition of 2 X 10(-6) M phenoxybenzamine to the capillary perfusate caused a 16% decrease in Jv while the simultaneous administration of norepinephrine and phenoxybenzamine to the capillary perfusate caused a 25% decrease in Jv. On the other hand, there was no effect observed on Jv when either norepinephrine or phenoxybenzamine was added to the luminal perfusate. These results suggest that adrenergic nerves may participate in the regulation of tubular fluid absorption through the direct action of norepinephrine on alpha adrenergic receptors located on the basolateral membrane of proximal tubular cells.     

Perfusion of isolated tubules of the shark rectal gland. Electrical characteristics and response to hormones.

Both the mammalian thick ascending limb of Henle's loop and the shark rectal gland actively transport Cl against an electrochemical gradient by mechanisms involving hormone-sensitive NaCl transport. In contrast to mammalian renal tubules, individual tubules of the shark rectal gland previously have not been perfused in vitro. Using a combination of renal slice and microdissection techniques we were able to isolate and perfuse single rectal gland tubules without the use of enzyme treatment. Single tubules consistently generated lumen-negative transepithelial voltages (Vt) of -1.8 mV when perfused and bathed with identical shark Ringer's solution. The addition of cyclic AMP, vasoactive intestinal peptide (VIP), and adenosine to the bath increased Vt to -7.5, -9.0, and -4.3 mV, respectively (all P less than 0.02 compared with paired controls). Each stimulation could be reversed by addition by furosemide to the bath. The adenosine response was inhibited by theophylline, a specific inhibitor of adenosine receptors. The tubules had a low transepithelial electrical resistance of 12-26 omega X cm2 and exhibited a transepithelial permselectivity for small cations. These results indicate that tubules of the rectal gland can be perfused in vitro and have receptors for VIP and adenosine. Cyclic AMP and secretagogues hyperpolarize the membrane consistent with electrogenic chloride transport, and these effects are reversed by furosemide, an inhibitor of coupled sodium-potassium-chloride co-transport. The response of Vt to cyclic AMP and furosemide, the transepithelial electrical resistance, and the cation selective permeability of tubules are remarkably similar to measurements in perfused mammalian thick ascending limbs.     

Properties of highly purified leukotriene C4 synthase of guinea pig lung.

To examine whether Cl- is transported via transcellular pathways in the thin ascending limb of Henle's loop (TAL), conventional microelectrode technique was applied in isolated TAL segments of hamsters perfused in vitro. The average basolateral membrane voltage (VB) was -24.5 +/- 1.5 mV (n = 18). Ouabain (10(-4) M) had no effect on VB. Sudden reduction of basolateral Cl- concentration from 165 to 5 mmol/liter caused a large depolarizing spike (+49.1 +/- 2.7 mV, n = 18), while the transepithelial potential (VT) showed lumen positive deflection by 33.4 +/- 1.2 mV, which indicates that a large Cl- conductance exists in the basolateral membrane. Reduction of luminal Cl- concentration caused sustained depolarization of luminal cell membrane from +24.5 +/- 2.1 to -9.7 +/- 3.4 mV (n = 6), which indicates that there is also a Cl- conductance in the luminal membrane. Since we have previously shown that acidification of ambient solution suppresses the transmural Cl- permeability, we tested whether acid pH also inhibits the Cl- conductance of the basolateral membrane. When pH of the bathing fluid was lowered to 5.8, the depolarizing spike of VB and the change of VT upon sudden reduction of basolateral Cl- were almost completely abolished. From these results we conclude: (a) both the luminal and the basolateral membrane of hamster TAL segments have Cl- conductances, and (b) Cl- transport in the TAL takes place, at least in part, via a transcellular route when a transepithelial Cl- gradient is present.     

Inhibitory effects of volume expansion performed in vivo on transport in the isolated rabbit proximal tubule perfused in vitro.

To examine the renal tubular sites and mechanisms involved in the effects of hypooncotic volume expansion (VE) on renal electrolyte excretion, we performed clearance and isolated tubular perfusion studies using intact and thyroparathyroidectomized (TPTX) rabbits. We also examined the effect of VE on luminal brush border transport. In the microperfusion studies, proximal convoluted (PCT) and straight (PST) tubules were taken from rabbits without prior VE or after 30 min of 6% (body wt) VE. Acute VE increased the percentage excretion of Na, Ca, and P in TPTX animals and the percentage and absolute excretions of these ions in intact rabbits. In PST from VE animals, fluid flux (Jv) was depressed compared with Jv in PST from nonVE rabbits: Jv = 0.18 +/- 0.03, (VE) vs. 0.31 +/- 0.03 nl/mm.min, (nonVE) P less than 0.02. Phosphate transport (Jp) in the PST from VE animals was also depressed: JP = 1.58 +/- 0.10 (VE) vs. 2.62 +/- 0.47 pmol/mm.min, (nonVE) P less than 0.05. Similar results were obtained with TPTX animals. In the PCT from VE animals, Jv was decreased (0.49 +/- 0.10 (VE) vs. 0.97 +/- 0.14 nl/mm.min, (nonVE) P less than 0.02), but JP was not affected significantly. Transport inhibition was stable over approximately 90 min of perfusion. In the brush border vesicle studies, sodium-dependent phosphate transport was inhibited compared with that in control animals, at the 9-, 30-, and 60-s time points. These findings indicate that the inhibition of renal ionic transport by VE occurs in both PCT and PST and is, in part, the result of a direct effect of VE on tubular transport mechanisms.     

Axial heterogeneity of bicarbonate, chloride, and water transport in the rat proximal convoluted tubule. Effects of change in luminal flow rate and of alkalemia.

These studies examined regulation of superficial proximal convoluted tubule (PCT) transport as a function of length. When single nephron glomerular filtration rate (SNGFR) increased from 28.7 +/- 0.7 nl/min in hydropenia to 41.5 +/- 0.4 nl/min in euvolemia, bicarbonate, chloride, and water reabsorption in the early (1st mm) PCT increased proportionally: from 354 +/- 21 peq/mm X min, 206 +/- 55 peq/mm X min, and 5.9 +/- 0.4 nl/mm X min to 520 +/- 12 peq/mm X min, 585 +/- 21 peq/mm X min, and 10.1 +/- 0.4 nl/mm X min, respectively. These high transport rates did not increase further, however, when SNGFR went to 51.2 +/- 0.7 or 50.7 +/- 0.6 nl/min after atrial natriuretic factor or glucagon administration. Anion and water transport rates in the late PCT were lower and exhibited less flow dependence. During chronic metabolic alkalosis, acidification was inhibited in the late but not early PCT. In conclusion, the early PCT is distinguished from the late PCT by having high-capacity, flow-responsive but saturable, anion- and water-reabsorptive processes relatively unaffected by alkalemia.     

Effects of inhibitors and substitutes for chloride in lumen on p-aminohippurate transport by isolated perfused rabbit renal proximal tubules

The transport step for p-aminohippurate (PAH) from cell to lumen across the luminal membrane of rabbit proximal tubules has not been adequately defined. To examine this process more closely, we determined the effects of possible transport inhibitors and substitutes for chloride on PAH secretion in isolated perfused S2 segments of rabbit proximal tubules. The addition of 4-acetamido-4'-isothiocyano-2,2' disulfonic stilbene (10(-4) M) to the perfusate irreversibly inhibited PAH secretion, whereas the addition of probenecid (10(-4) M) to the perfusate reversibly inhibited PAH secretion. PAH secretion was unaffected by thiocyanate replacement of chloride in the luminal perfusate, reversibly inhibited by 15 to 20% by methyl sulfate replacement, and irreversibly inhibited by isethionate replacement. Because the luminal membrane is at least as permeable to thiocyanate as to chloride, less permeable to methyl sulfate, and much less permeable to isethionate, these data suggest that the PAH transport step from cells to lumen does not require chloride in the lumen but does require a highly permeant anion. During inhibition of PAH transport from cells to lumen, PAH uptake across the basolateral membrane was also reduced, suggesting some type of feedback inhibition. The data are compatible with PAH transport across the luminal membrane by an anion exchanger, a potential-driven uniporter, both carriers, or a carrier that can function in both modes.     

Early enhancement of fluid transport in rabbit proximal straight tubules after loss of contralateral renal excretory function.

To assess the renal functional adaptation to reduced excretory capacity, we studied whole kidney and single nephron function in anesthetized volume-replete rabbits after unilateral (left kidney) nephrectomy (UNX), ureteral obstruction (UO), or ureteroperitoneostomy (UP). At 24 h, despite the absence of measurable hypertrophy of the contralateral (right) kidney, these procedures significantly increased p-aminohippurate clearance (45-54%) and inulin clearance (CIN) (64-110%) compared with sham-operated control animals. In each group, whole kidney sodium reabsorption increased in proportion to the rise in CIN. To determine whether the intrinsic transport capacity of proximal tubule segments is altered by these maneuvers, we measured fluid volume reabsorption rate (Jv) in isolated superficial proximal straight tubule (PST) segments perfused in vitro, comparing each control tubule (obtained by biopsy of the left kidney immediately before an experimental maneuver) with a corresponding tubule segment obtained 24 h or 7 d later from the contralateral kidney. Control tubule Jv in sham-24 h animals averaged 0.48 +/- 0.04 nl/(min X mm). Jv did not change significantly at 24 h or 7 d after sham maneuvers but increased significantly at 24 h after UNX [delta Jv = 0.13 +/- 0.03 nl/(min X mm)], UO [delta Jv = 0.10 +/- 0.04 nl/(min X mm)], and UP [delta Jv = 0.13 +/- 0.04 nl/(min X mm)]. Jv remained increased by similar amounts at 7 d after UNX and UO. To evaluate whether an increase in glomerular filtration rate (GFR) might be the stimulus to this augmentation in Jv values, methylprednisolone (MP) (15 mg/kg per d) was administered daily to sham-operated animals, a maneuver which induced a 73% rise in CIN by day 5. This procedure also produced a significant increase in Jv in PST at 5 d [delta Jv = 0.16 +/- 0.05 nl/(min X mm)]. The increase in Jv evident in each group at 5 or 7 d was paralleled by an equivalent change in tubule cell volume and apparent tubule luminal surface area in UNX-7d and MP-5d; no such increments in these indices, or in apparent tubule serosal surface area were evident at 24 h in any group. Thus, a 50% reduction in renal excretory function in the rabbit provokes adjustments in renal plasma flow rate and GFR in the contralateral kidney, which are evident by 24 h. The concurrent change in Jv in PST is closely related to CIN or some associated hemodynamic process, but does not appear to require an increase in tubule cell volume or apparent surface area. The ability to detect these small in vivo changes in Jv may derive from the enhanced sensitivity of paired-kidney experiments using tubule segments obtained by renal biopsy.     

Endogenous production of angiotensin II modulates rat proximal tubule transport.

There is evidence that angiotensin II is synthesized by the proximal tubule and secreted into the tubular lumen. This study examined the functional significance of endogenously produced angiotensin II on proximal tubule transport in male Sprague-Dawley rats. Addition of 10(-11), 10(-8), and 10(-6) M angiotensin II to the lumen of proximal convoluted tubules perfused in vivo had no effect on the rate of fluid reabsorption. The absence of an effect of exogenous luminal angiotensin II could be due to its endogenous production and luminal secretion. Luminal 10(-8) M Dup 753 (an angiotensin II receptor antagonist) resulted in a 35% decrease in proximal tubule fluid reabsorption when compared to control (Jv = 1.64 +/- 0.12 nl/mm.min vs. 2.55 +/- 0.32 nl/mm.min, P < 0.05). Similarly, luminal 10(-4) M enalaprilat, an angiotensin converting enzyme inhibitor, decreased fluid reabsorption by 40% (Jv = 1.53 +/- 0.23 nl/mm.min vs. 2.55 +/- 0.32 nl/mm.min, P < 0.05). When 10(-11) or 10(-8) M exogenous angiotensin II was added to enalaprilat (10(-4) M) in the luminal perfusate, fluid reabsorption returned to its baseline rate (Jv = 2.78 +/- 0.35 nl/mm.min). Thus, addition of exogenous angiotensin II stimulates proximal tubule transport when endogenous production is inhibited. These experiments show that endogenously produced angiotensin II modulates fluid transport in the proximal tubule independent of systemic angiotensin II.     

Bumetanide inhibition of [CO2 + HCO3]-dependent and -independent equivalent electrical flux in renal cortical thick ascending limbs

Experiments were performed on single perfused segments of cortical thick ascending limbs of Henle isolated from mouse kidney in order to evaluate the interaction of bumetanide with apical membrane [CO2 + HCO3]-dependent and -independent sodium uptake. In this nephron segment [CO2 + HCO3]-dependent apical membrane salt uptake is accomplished by parallel Na+/H+ and Cl-/HCO3- exchange whereas [CO2 + HCO3]-independent salt uptake results from NaCl cotransport. Tubules were perfused and bathed at 37 degrees C in either [CO2 + HCO3]-buffered solutions (KRB) or with 4-(2-hydroxy(ethyl)-1-piperazineethanesulfonic acid-buffered external media (KRH) at pH 7.4. The spontaneous transepithelial voltage was measured through one-half of a double-barreled perfusion pipette and bipolar d.c. current pulses were delivered through the other half in order to measure the transepithelial conductance (milliSiemens per squared centimeter). The rate of net NaCl absorption was estimated from the electrical flux (Je) and was calculated from the equivalent short-circuit current. Cumulative dose-response data over the range 10(-7) to 10(-4) M luminal bumetanide were obtained in individual tubules in both KRB and KRH solutions. Significant inhibition of transepithelial voltage and Je obtained at 5 X 10(-7) M bumetanide (in KRB) to 10(-6) M (in KRH). When the effect of [CO2 + HCO3] per se on Je was subtracted from the bumetanide-induced inhibition, the two dose-response curves converge.(ABSTRACT TRUNCATED AT 250 WORDS)