Magnesium transport in the cortical thick ascending limb of Henle''s loop of the rabbit.

Isolated cortical thick ascending limbs of Henle's loop were perfused in order to directly evaluate magnesium transport in this segment. Transepithelial potential difference was altered by varying the NaCl concentration in perfusate and bath and adding 50 microM furosemide to the perfusate. Perfusion under standard conditions with isotonic solutions resulted in a mean transepithelial potential difference of +8.8 +/- 0.7 mV and net magnesium absorption at a rate of 0.32 +/- 0.06 pmol/mm per min. Perfusion with a hypotonic solution significantly increased potential difference and the net absorptive rate of magnesium, calcium, and potassium. Conversely, reversal of the polarity of the potential difference with low NaCl bath and luminal furosemide produced net secretion of magnesium, calcium, and potassium. Parathyroid hormone in a bath concentration of 1.0 U/ml increased magnesium absorption from 0.32 +/- 0.06 to 0.63 +/- 0.06 pmol/mm per min (P less than 0.001) and calcium from 0.52 +/- 0.08 to 0.97 +/- 0.08 pmol/mm per min (P less than 0.001). Dibutyryl cyclic AMP produced similar effects on both calcium and magnesium absorption. Increasing bath calcium concentration twofold significantly inhibited net calcium absorption from 0.79 +/- 0.27 to 0.16 +/- 0.02 pmol/mm per min but magnesium transport was unaffected. Increasing bath magnesium concentration twofold significantly inhibited net magnesium absorption from 0.56 +/- 0.14 to -0.09 +/- 0.13 pmol/mm per min but had no effect upon net calcium transport. Net absorption of magnesium was significantly increased with increased concentration in the perfusate but calcium transport was unchanged. Similarly, increasing perfusate calcium concentration produced an increase in net calcium transport but did not alter magnesium transport. These data indicate that this segment of the loop of Henle is an important site for magnesium transport. Transport is influenced by luminal and bath concentration and is stimulated by parathyroid hormone and cyclic AMP. The data do not provide support for the concept of an interactive process between calcium and magnesium, and suggest that the positive transepithelial voltage is an important driving force for net reabsorption of magnesium, as well as calcium and potassium in this segment.     

Calcium and phosphate transport in isolated segments of rabbit Henle''s loop.

Calcium and phosphate transport was examined in rabbit thin descending, thin ascending, and thick ascending limbs of Henle by in vitro perfusion of isolated tubular segments. Permeability coefficients for these segments with 45Ca and 32PO4 were determined for both lumen-to-bath and bath-to-lumen directions. Both the thin descending and thin ascending limbs were found to be relatively impermeable to both 45Ca and 32PO4. In neither segment were we able to show evidence for net transport of calcium or phosphate. In contrast, the thick ascending limb of Henle showed a decrease in calcium lumen-to-bath concentration from 0.97 +/- 0.02 to 0.88 +/- 0.02 when perfused at 4.8 nl min-1. 45Ca lumen-to-bath and bath-to-lumen fluxes were 19.96 +/- 1.05 and 9.89 +/- 0.02 peq-min-1-cm-1, respectively, and the potential difference was +3.8 +/- 0.3 mV (lumen positive). The observed calcium flux ratio was significantly higher than that predicted by Ussing's equation. When ouabain was added to the bath the potential difference fell to +1.1 +/- 0.3 mV, whereas the calcium efflux was only slightly diminished (29.5 +/- 5.3-23.7 +/- 5.1 peq-cm-1-min-1). Ouabain had no effect on the influx of Ca across the thick ascending limb of Henle. There was no net transport of phosphate across the thick ascending limb. Phosphate permeability was exceedingly low bidirectionally across the thick ascending limb. Our findings indicate: (a) all segments of Henle's loop are relatively impermeable to calcium and phosphate; (b) net transport of phosphate seems to be absent in Henle's loop; (c) net calcium reabsorption, which cannot be explained by passive mechanisms, occurs in the thick ascending limb.     

Effect of prostaglandin E2 on chloride transport across the rabbit thick ascending limb of Henle. Selective inhibitions of the medullary portion.

Prostaglandins are present in large quantities in the kidney and have been shown to directly affect transepithelial transport. The present studies were designed to examine whether prostaglandin E2 could affect chloride transport across the thick ascending limb of Henle. Isolated segments of the cortical and medullary thick ascending limb of Henle were perfused in vitro and the transepithelial voltage and net chloride flux were measured. Exposure of the medullary thick ascending limb to 2 microM prostaglandin E2 resulted in a fall in net chloride transport of 40--50% with a concomitant fall in voltage. In contrast, net chloride transport in the cortical thick ascending limb was not affected by prostaglandin E2. Under similar conditions, the medullary thick ascending limb possessed twice the capacity to transport chloride than did the cortical thick ascending limb. The results suggest that endogenous renal prostaglandins may play a modulating role in the addition of salt to the renal medullary interstitium and may, under some circumstances, by chloruretic.     

Inhibition of bicarbonate absorption by peptide hormones and cyclic adenosine monophosphate in rat medullary thick ascending limb.

In vitro microperfusion experiments were performed to examine the effects of peptide hormones on bicarbonate and ammonium transport by the medullary thick ascending limb (MTAL) of the rat. Arginine vasopressin (AVP; 2.8 X 10(-10) M in the bath) reduced bicarbonate absorption by 50% (from 7.8 to 3.7 pmol/min per mm). AVP caused a similar reduction in bicarbonate absorption in tubules perfused with 10(-4) M furosemide to inhibit net NaCl absorption. Glucagon (2 X 10(-9) M in the bath) also reduced bicarbonate absorption (from 11.7 to 7.6 pmol/min per mm). The inhibition of bicarbonate absorption could be reproduced with either exogenous 8-bromo-cAMP or forskolin. With 8-bromo-cAMP (10(-3) M) in the bath, addition of vasopressin to the bath did not significantly affect bicarbonate absorption. PTH significantly inhibited bicarbonate absorption, but the extent of inhibition was less than that observed with either AVP or glucagon. Vasopressin had no effect on net ammonium absorption in MTAL perfused and bathed with 4 mM NH4Cl. These findings indicate that: (a) vasopressin, glucagon, and PTH directly inhibit bicarbonate absorption in the MTAL of the rat; (b) this inhibition occurs independent of effects on net NaCl absorption and appears to be mediated in part by cAMP; and (c) HCO3- and NH4+ absorption can be regulated independently in the MTAL.     

Vasopressin stimulates Cl- transport in ascending thin limb of Henle's loop in hamster.

The effect of arginine vasopressin (AVP) on NaCl transport was investigated in the isolated microperfused hamster ascending thin limb of Henle's loop by measuring transepithelial voltage (Vt) and transmural 22Na+ and 36Cl- fluxes. In the presence of a transmural NaCl concentration gradient (100 mM higher in the lumen), Vt was 8.4 +/- 0.4 mV. Addition of 1 nM AVP to the basolateral solution increased Vt to 9.6 +/- 0.4 mV, which corresponds to an increase in the Cl- to Na+ permselectivity ratio (PCl/PNa) from 2.8 +/- 0.2 to 3.4 +/- 0.2. AVP at physiological concentrations increased Vt in a dose-dependent manner with an ED50 of 5 pM. AVP increased the Cl- efflux coefficient from 99.6 +/- 6.3 to 131.4 +/- 10.6 x 10(-7) cm2/s without affecting the Na+ efflux coefficient. 5-Nitro-2-(3-phenyl-propylamino)-benzoate (0.2 mM), a Cl- channel inhibitor, in the perfusate decreased the basal Cl- efflux coefficient and inhibited the AVP-induced increase in this parameter. The AVP-induced increase in Vt was not affected by [d(CH2)5(1),O-Me-Tyr2,Arg8] vasopressin, a V1 receptor antagonist, but was abolished by [d(CH2)5,D-Ile2,Ile4,Arg8] vasopressin, a V2 receptor antagonist. The selective V2 agonist dDAVP in 1 nM also increased Vt from 8.6 +/- 0.7 to 9.5 +/- 0.6 mV. Dibutyryl cAMP and forskolin both increased Vt, whereas H89, an inhibitor of cAMP-dependent protein kinase, abolished the AVP-induced increase in Vt. These results demonstrate that AVP stimulates Cl- transport in the ascending thin limb of Henle's loop by activating Cl- channels via a signal transduction cascade comprising V2 receptors, adenylate cyclase, and cAMP-dependent protein kinase. The ascending thin limb of Henle's loop thus participates in the formation of concentrated urine as one of the target renal tubular segments of AVP.     

Calcium transport in the thick ascending limb of Henle. Heterogeneity of function in the medullary and cortical segments.

Calcium transport was studied in medullary and cortical segments of the thick ascending limb of Henle perfused in vitro. 45Ca was added to the perfusate for measuring lumen-to-bath flux (JlbCa), to the bath for measuring bath-to-lumen flux (JblCa), or to both perfusate and bath for measuring net flux (JnetCa). In the medullary segment JlbCa exceeding JblCa and the efflux:influx coefficient ratio was not different from the value predicted from the observed potential difference (PD). In the cortical segments, however, efflux:influx coefficient ratio was greater than the value predicted from the PD, suggesting that calcium transport in this segment may be active, while it is passive in the medullary segment. Furosemide, which reversibly decreases PD in both cortical and medullary segments, inhibited JlbCa only in the medullary segment. Parathyroid hormone (PTH), on the other hand, had no effect on JnetCa in the medullary segment, but it significantly augmented JnetCa in the cortical segment. These results indicate that calcium transport in the thick ascending limb is heterogeneous. In the medullary segment it is passive, inhibited by furosemide and not influenced by PTH. In the cortical segment, however, calcium transport appears to be active, not inhibited by furosemide and stimulated by PTH.     

Epidermal growth factor inhibits the hydroosmotic effect of vasopressin in the isolated perfused rabbit cortical collecting tubule.

Epidermal growth factor (EGF) is a 53-amino acid polypeptide which is a potent mitogen for cultured cells. The kidney has recently been shown to be a major site of synthesis for the EGF precursor. EGF infusions in sheep result in a diuresis and natriuresis despite a fall in GFR, suggesting a direct tubular effect. Using in vitro microperfusion of rabbit cortical collecting tubules (CCTs) at 37 degrees C, we examined the effect of EGF on the transepithelial voltage (Vt) and arginine vasopressin (AVP)-stimulated hydraulic conductivity (Lp). Pretreatment with peritubular EGF at concentrations from 10(-8) to 10(-12) M resulted in a 50% inhibition of both AVP- and 8-chlorophenythio-cyclic AMP-stimulated peak Lp. This effect was reversed by the protein kinase C inhibitor, staurosporine, but unaffected by indomethacin. CCTs with an initially negative Vt, depolarized after exposure to bath EGF. 10(-8) M EGF applied from the lumen had no effect on either Lp or Vt. Specific binding of 20 nM 125I-EGF to microdissected CCTs was also demonstrated. These results suggest that EGF can modulate both salt and water transport in the CCT via a receptor linked to protein kinase C activation.     

A1 adenosine receptors inhibit chloride transport in the shark rectal gland. Dissociation of inhibition and cyclic AMP.

In the in vitro perfused rectal gland of the dogfish shark (Squalus acanthias), the adenosine analogue 2-chloroadenosine (2Clado) completely and reversibly inhibited forskolin-stimulated chloride secretion with an IC50 of 5 nM. Other A1 receptor agonists including cyclohexyladenosine (CHA), N-ethylcarboxamideadenosine (NECA) and R-phenylisopropyl-adenosine (R-PIA) also completely inhibited forskolin stimulated chloride secretion. The "S" stereoisomer of PIA (S-PIA) was a less potent inhibitor of forskolin stimulated chloride secretion, consistent with the affinity profile of PIA stereoisomers for an A1 receptor. The adenosine receptor antagonists 8-phenyltheophylline and 8-cyclopentyltheophylline completely blocked the effect of 2Clado to inhibit forskolin-stimulated chloride secretion. When chloride secretion and tissue cyclic (c)AMP content were determined simultaneously in perfused glands, 2Clado completely inhibited secretion but only inhibited forskolin stimulated cAMP accumulation by 34-40%, indicating that the mechanism of inhibition of secretion by 2Clado is at least partially cAMP independent. Consistent with these results, A1 receptor agonists only modestly inhibited (9-15%) forskolin stimulated adenylate cyclase activity and 2Clado markedly inhibited chloride secretion stimulated by a permeant cAMP analogue, 8-chlorophenylthio cAMP (8CPT cAMP). These findings provide the first evidence for a high affinity A1 adenosine receptor that inhibits hormone stimulated ion transport in a model epithelia. A major portion of this inhibition occurs by a mechanism that is independent of the cAMP messenger system.     

Sodium Chloride and Water Transport in the Medullary Thick Ascending Limb of Henle. EVIDENCE FOR ACTIVE CHLORIDE TRANSPORT

Transport of NaCl and water was examined in the rabbit medullary thick ascending limb of Henle (ALH) by perfusing isolated segments of these nephrons in vitro. Osmotic water permeability was evaluated by perfusing tubules against imposed osmotic gradients. In these experiments the net transport of fluid remained at zero when segments of thick ALH were perfused with isotonic ultrafiltrate in a bath of rabbit serum in which the serum osmolality was increased by the addition of either 239±8 mosmol/liter of raffinose or 232±17 mosmol of NaCl indicating that the thick ascending limb of Henle is impermeant to osmotic flow of water. When these tubules were perfused at slow rates with isosmolal ultrafiltrate of same rabbit serum as used for the bath, the effluent osmolality was consistently lowered to concentrations less than the perfusate and the bath. That this decrease in collected fluid osmolality represented salt transport was demonstrated in a separate set of experiments in which it was shown that the sodium and chloride concentrations decreased to 0.79±0.02 and 0.77±0.02 respectively when compared with the perfusion fluid concentrations. In each instance the simultaneously determined transtubular potential difference (PD) revealed the lumen to be positive with the magnitude dependent on the perfusion rate. At flow rates above 2 nl·min^-1, the mean transtubular PD was stable and equal to 6.70±0.34 mv. At stop-flow conditions this PD became more positive. Ouabain and cooling reversibly decreased the magnitude of this PD. The transtubular PD remained positive, 3.3±0.2 mV, when complete substitution of Na by choline was carried out in both the perfusion fluid and the bathing media. These results are interpreted to indicate that the active transport process is primarily an electrogenic chloride mechanism. The isotopic permeability coefficient for Na was 6.27±0.38 × 10^-5 cm·s^-1 indicating that the thick ALH is approximately as permeable to Na as the proximal convoluted tubule. The chloride permeability coefficient for the thick ALH was 1.06±0.12 × 10^-5 cm·s^-1 which is significantly less than the chloride permeability of the proximal tubule.     

Hydraulic water permeability and transepithelial voltage in the isolated perfused rabbit cortical collecting tubule following acute unilateral ureteral obstruction.

Ureteral obstruction affects the kidney's ability to conserve water and sodium. Using the isolated perfused tubule technique, we studied cortical collecting tubules (CCT) taken from rabbits subjected to a sham operation or to 4 h of unilateral ureteral obstruction (UUO). Tubules were perfused in the presence of an osmotic gradient directed to promote water movement from lumen to bath, and volume flux (Jv), hydraulic water permeability (Lp), and transepithelial voltage (V1) were determined. In tubules from sham-operated and UUO animals, basal (before exposure to vasopressin) J, and Lp were not different from zero. After addition of 200 microU . ml-1 of arginine vasopressin (aVP) to the bath, Jv and Lp increased to 1.64 +/- 0.23 nl . mm-1 . min-1 and 127.9 +/- 19.8 cm . s-1 . atm-1 x 10(7), respectively, in tubules from sham-operated animals, but not only 0.27 +/- 0.09 nl . mm-1 . min-1 an 18.8 +/- 6.2 cm . s-1 . atm-1 . 10(7) in tubules from UUO animals. Pretreatment with desoxycorticosterone acetate (DOCA) or indomethacin in vivo did not prevent the blunted vasopressin response seen in tubules taken from UUO animals. The Jv and Lp responses to the cyclic AMP (cAMP) analogue, 8-Br-cAMP, were also diminished in tubules taken from UUO animals compared with shams. V1, measured during the basal period, was diminished in tubules from UUO kidneys (-5.0 +/- 2.1 mV) compared with shams (-21.9 +/- 4.1 mV), and pretreatment with DOCA did no prevent the effects of UUO on V1. In contrast, tubules taken from animals that received indomethacin prior to UUO developed voltages not different from voltages in tubules taken from sham-operated animals (-17.3 +/- 1.7 mV). We conclude that, although CCT from UUO animals can maintain osmotic gradients, their ability to respond to vasopressin by increasing Lp is impaired by an intrinsic defect located at a step beyond the generation of cAMP, and that prostaglandin inhibition or DOCA pretreatment do not reverse the decreased responsiveness of Lp to aVP. UUO also diminished V1, and this abnormality was prevented by previous treatment with indomethacin, suggesting that prostaglandins may mediate the effect of UUO on V1.     

Evidence for neutral transcellular NaCl transport and neutral basolateral chloride exit in the rabbit proximal convoluted tubule.

The electrical nature of active NaCl transport and the significance of a basolateral membrane chloride conductance were examined in isolated perfused rabbit proximal convoluted tubules (PCT). PCT were perfused with a high chloride solution that simulated late proximal tubular fluid and were bathed in an albumin solution that simulated rabbit serum in the control and recovery periods. The electrical nature of NaCl transport was examined by bathing the tubules in a high chloride albumin solution where there were no anion gradients. Volume reabsorption (Jv) during the control and recovery period was 0.56 and 0.51 nl/mm X min, respectively, and 0.45 nl/mm X min when the tubules were bathed in a high chloride bath. The transepithelial potential difference (PD) during the control and recovery periods averaged 2.3 mV, but decreased to 0.0 mV in the absence of anion gradients, which indicated that NaCl transport is electroneutral. Further evidence that NaCl transport is electroneutral was obtained by examining the effect of addition of 0.01 mM ouabain in PCT perfused and bathed with high chloride solutions. The Jv was 0.54 nl/mm X min in the control period and not statistically different from zero after inhibition of active transport. The PD was not different from zero in both periods. Two groups of studies examined the role of basolateral membrane Cl- conductance in NaCl transport. First, depolarizing the basolateral membrane with 2 mM bath Ba++ did not significantly affect Jv or PD. Second, the effect of the presumptive Cl- conductance inhibitor anthracene-9-CO2H was examined. Anthracene-9-CO2H did not significantly affect Jv or PD. In conclusion, these data show that NaCl transport in the PCT is electroneutral and transcellular and provide evidence against a significant role for basolateral membrane chloride conductance in the rabbit PCT.     

Endogenous adenosine is an autacoid feedback inhibitor of chloride transport in the shark rectal gland.

The present studies define the physiologic role of endogenous adenosine in the perfused shark rectal gland, a model epithelia for hormone-stimulated chloride transport. Chloride ion secretion, and venous adenosine and inosine concentrations increased in parallel in response to hormone stimulation. From a basal rate of 157 +/- 26 mu eq/h per g, chloride secretion increased to 836 +/- 96 and 2170 +/- 358 with 1 and 10 microM forskolin, venous adenosine increased from 5.0 +/- 1 to 126 +/- 29 and 896 +/- 181 nM, and inosine increased from 30 +/- 9 to 349 +/- 77 and 1719 +/- 454 nM (all P less than 0.01). Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, completely blocked the release of adenosine and inosine. Inhibition of chloride transport with bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter, or ouabain, an inhibitor of Na+/K+ ATPase activity, reduced venous adenosine and inosine to basal values. When the interaction of endogenous adenosine with extracellular receptors was prevented by adenosine deaminase, NBTI, or 8-phenyltheophylline, the chloride transport response to secretagogues increased by 1.7-2.3-fold. These studies demonstrate that endogenous adenosine is released in response to hormone-stimulated cellular work and acts at A1 adenosine receptors as a feedback inhibitor of chloride transport.     

Effects of potassium on ammonia transport by medullary thick ascending limb of the rat

Renal ammonium excretion is increased by potassium depletion and reduced by potassium loading. To determine whether changes in potassium concentration would alter ammonia transport in the medullary thick ascending limb (MAL), tubules from rats were perfused in vitro and effects of changes in K concentration within the physiological range (4-24 mM) were evaluated. Increasing K concentration from 4 to 24 mM in perfusate and bath inhibited total ammonia absorption by 50% and reduced the steady-state transepithelial NH+4 concentration gradient. The inhibition of total ammonia absorption was reversible and occurred when K replaced either Na or N-methyl-D-glucamine. Increasing K concentration in the luminal perfusate alone gave similar inhibition of total ammonia absorption. At 1-2 nl/min per mm perfusion rate, increasing K concentration in perfusion and bathing solutions had no significant effect on transepithelial voltage. With either 4 or 24 mM K in perfusate and bath, an increase in luminal perfusion rate markedly increased total ammonia absorption. Thus, both potassium concentration and luminal flow rate are important factors capable of regulating total ammonia transport by the MAL. Changes in systemic potassium balance may influence renal ammonium excretion by affecting NH+4 absorption in the MAL and altering the transfer of ammonia from loops of Henle to medullary collecting ducts.     

Luminal vasopressin modulates transport in the rabbit cortical collecting duct.

We explored the action of luminal AVP in rabbit CCD perfused in vitro at 37 degrees C. Nanomolar concentrations of luminal AVP induced a sustained hyperpolarization of transepithelial voltage (Vt) in contrast to a transient hyperpolarization caused by basolateral AVP. 10 microM basolateral ouabain abolished the latter but not the former change in Vt. Despite a sustained hyperpolarization (from -20.7 +/- 2.9 to -34.1 +/- 4.7 mV; P less than 0.01), 10 nM luminal AVP only slightly altered net Na+ and K+ fluxes (7.6% stimulation and no significant change, respectively). Instead, luminal AVP appeared to modulate an acetazolamide-sensitive electrogenic ion transport because 200 microM basolateral acetazolamide suppressed the luminal AVP-induced hyperpolarization (percentage of Vt from -50.4 +/- 10.8 to -5.1 +/- 1.4; P less than 0.005). In terms of water transport, 10 nM luminal AVP did not change hydraulic conductivity (Lp, x 10(-7) cm/atm per s) (from 3.9 +/- 0.8 to 5.0 +/- 1.2), but suppressed the increase in Lp induced by 20 pM basolateral AVP (134.9 +/- 19.2 vs. 204.3 +/- 21.1 in control; P less than 0.05). These findings demonstrate distinct luminal action of AVP, suggesting amphilateral regulation of epithelial transport by AVP in the CCD.     

Effects of osmolality on bicarbonate absorption by medullary thick ascending limb of the rat.

Previously we demonstrated that arginine vasopressin (AVP) directly inhibits bicarbonate absorption (JHCO3, pmol/min per mm) in the medullary thick ascending limb (MTAL) of the rat. To determine whether changes in osmolality also may affect bicarbonate absorption, MTAL were studied in vitro with 25 mM HCO3- solutions. Control osmolality was 290 mosmol/kg H2O. In the absence of AVP, increasing osmolality to 560 in perfusate and bath by addition of 150 mM NaCl reduced JHCO3 from 13.7 to 4.5. With 2 x 10(-10) M AVP in the bath, adding 150 mM NaCl to perfusate and bath reduced JHCO3 from 6.9 to 0.6, while adding NaCl to the bath alone reduced JHCO3 from 7.1 to 0.5. Adding 150 mM NaCl to perfusate and bath caused a similar inhibition of JHCO3 in MTAL perfused with furosemide to inhibit net NaCl absorption. In the presence of AVP, adding 600 mM urea to perfusate and bath inhibited JHCO3 by 55%; adding 300 or 600 mM mannitol to perfusate and bath inhibited JHCO3 by 75%. The effects on JHCO3 were reversible and dissociable from changes in transepithelial voltage. Conclusions: (1) osmolality is a factor capable of regulating renal tubule bicarbonate absorption; (2) hypertonicity produced with NaCl, urea, or mannitol markedly inhibits bicarbonate absorption in the MTAL; (3) this inhibition occurs independent of, and is additive to, inhibition by vasopressin. Hypertonicity may shift TAL HCO3- absorption from medulla to cortex, thereby limiting delivery of bicarbonate to the medullary interstitium during antidiuresis.     

Control of sodium and potassium transport in the cortical collecting duct of the rat. Effects of bradykinin, vasopressin, and deoxycorticosterone.

Several factors interact to maintain precise control of electrolyte transport in the mammalian cortical collecting duct. We have studied the effects of deoxycorticosterone, arginine vasopressin, and bradykinin on net transepithelial sodium and potassium transport in isolated, perfused rat cortical collecting ducts. Chronic administration of deoxycorticosterone to rats increased both sodium absorption and potassium secretion above very low basal levels. Consequently, deoxycorticosterone-treated rats were used for all remaining studies. Arginine vasopressin (10(-10) M in the bath) caused a sustained fourfold increase in net sodium absorption and a sustained threefold increase in net potassium secretion. Bradykinin (10(-9) M in the bath) caused a reversible 40-50% inhibition of net sodium absorption without affecting net potassium transport or the transepithelial potential difference. In the perfusate, up to 10(-6) M bradykinin had no effect. We conclude: As in rabbits, chronic deoxycorticosterone administration to rats increases sodium absorption and potassium secretion in cortical collecting ducts perfused in vitro. Arginine vasopressin causes a reversible increase in net potassium secretion and net sodium absorption. Bradykinin in the peritubular bathing solution reversibly inhibits net sodium absorption, possibly by affecting an electroneutral sodium transport pathway.     

Effects of MK-196 and furosemide on rat medullary thick ascending limbs of Henle in vitro

The effects of the diuretic agents, MK-196 (6,7-dichloro-2-methyl-1-oxo-2-phenyl-5-indanyloxy acetic acid) and furosemide on function of medullary thick ascending limb (tALH) isolated from the rat kidney were studied. tALH were dissected from whole kidneys of male Sprague-Dawley rats which were first perfused in vitro in a recirculating system for 10 to 15 min with a solution containing collagenase. Standard techniques for dissection and perfusion of tALH were followed. Control transepithelial voltage averaged +5.5 +/- 0.5 mV (n = 25). Drug concentration causing 50% inhibition of the voltage (EC50) was determined. EC50 for both MK-196 and furosemide in rat tALH was 8 X 10(-6) M (EC50 for MK-196 in rabbit tALH ws 7 X 10(-5) M). In other experiments, net chloride transport (JCl) in rat tALH averaged 142 +/- 19 pmol.mm-1.min-1 (n = 12), a value nearly twice that reported for the rabbit. In the rat, MK-196 (2 X 10(-4) M) reduced the voltage by 79 +/- 2% and JCl by 76 +/- 13% (n = 6). The same concentration of furosemide inhibited the voltage by 78 +/- 8% and the JCl by 104 +/- 4%(n = 5). Results indicate that: 1) rat tALHs dissected from collagenase-treated kidneys have functional characteristics similar to those reported for the rabbit tALH; and 2) the rat tALH is equally sensitive to MK-196 and furosemide, drugs which act to selectively inhibit active chloride transport.     

Electrophysiological study of isolated perfused human collecting ducts: Ion dependency of the transepithelial potential difference.

Cortical and outer medullary collecting duct segments were dissected from human kidneys and perfused in vitro. The transepithelial potential difference was measured and found to be lumen positive +6.8 +/- 0.6 mV (n= 20). This lumen-positive potential difference was inhibited by ouabain and furosemide but not by acetazolamide. Replacement of chloride in bath and perfusion fluids caused a reversible decrease of the potential difference to near zero. We conclude from these studies: (a) the lumen-positive potential difference is dependent upon the presence of chloride ion suggesting the existence of an active electrogenic chloride reabsorptive process in the human collecting duct and (b) it is possible to examine human renal physiology directly using in vitro microperfusion of tubule segments.     

Vasoactive intestinal peptide, forskolin, and genistein increase apical CFTR trafficking in the rectal gland of the spiny dogfish, Squalus acanthias. Acute regulation of CFTR trafficking in an intact epithelium.

Defective trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cause of cystic fibrosis. In chloride-secreting epithelia, it is well established that CFTR localizes to intracellular organelles and to apical membranes. However, it is controversial whether secretagogues regulate the trafficking of CFTR. To investigate whether acute hormonal stimulation of chloride secretion is coupled to the trafficking of CFTR, we used the intact shark rectal gland, a model tissue in which salt secretion is dynamically regulated and both chloride secretion and cellular CFTR immunofluorescence can be quantified in parallel. In rectal glands perfused under basal conditions without secretagogues, Cl- secretion was 151+/-65 microeq/h/g. Vasoactive intestinal peptide (VIP), forskolin, and genistein led to 10-, 6-, and 4-fold increases in Cl- secretion. In basal glands, quantitative confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell (7.28+/-0.35 micron). During stimulation with secretagogues, apical extension of CFTR immunofluorescence into the cell was reduced significantly to 3.24+/-0.08 micron by VIP, 4.08+/-0.13 by forskolin, and 3.19+/-0.1 by genistein (P < 0.001). Moreover, the peak intensity of CFTR fluorescence shifted towards the apical membrane (peak fluorescence 2.5+/-0.13 micron basal vs. 1.51+/-0.06, 1.77+/-0.1, and 1.38+/-0.05 for VIP, forskolin, and genistein; all P < 0.001). The increase in both Cl- secretion and apical CFTR trafficking reversed to basal values after removal of VIP. These data provide the first quantitative morphological evidence for acute hormonal regulation of CFTR trafficking in an intact epithelial tissue.     

Caffeine potentiates the renin response to furosemide in rats. Evidence for a regulatory role of endogenous adenosine

Adenosine is a potent inhibitor of renin release. It has therefore been suggested that endogenous adenosine may play a role in the regulation of renin release. Sodium-chloride transport at the level of the macula densa has been proposed as the primary source of endogenous adenosine. Evidence to support a modulatory role of adenosine on renin release in vivo is, however, limited. We therefore wanted to determine if: 1) adenosine modulates furosemide-induced renin release and 2) sodium-chloride reabsorption at the macula densa is essential for adenosine actions. To test these hypotheses, three groups of rats were pretreated either with saline or the adenosine receptor antagonists caffeine or 1,3-dipropyl-8-(p-sulfophenyl)xanthine (both at a dose of 30 mg/kg followed by 450 micrograms/kg/min). Rats then received furosemide (50 mg/kg i.v.). In the vehicle group, furosemide increased urinary volume, sodium and potassium excretion and increased plasma renin activity from 6 +/- 1 to 45 +/- 11 ngAl/ml/hr. Caffeine and 1,3-dipropyl-8-(p-sulfophenyl)xanthine potentiated the increase in plasma renin activity produced by furosemide (to 120 +/- 15 and 147 +/- 21 ng Al/ml/hr, respectively), whereas having no significant effects on urinary volume, sodium excretion or blood pressure. These results suggest that furosemide-induced renin release in vivo is restrained by endogenous adenosine. In as much as furosemide blocks sodium-chloride transport in the thick ascending limb of Henle's loop and the macula densa cells, it appears that under the conditions of this study sodium transport across these segments is not essential to initiate adenosine-mediated mechanisms.