Effect of adrenalectomy on transport in the rat medullary thick ascending limb.

Previous studies in adrenalectomized (Adx) rats suggest that aldosterone may regulate ion transport in the ascending portion of Helen's loop. In order to examine directly the effect of adrenalectomy on transport, medullary thick ascending limb (Mtal) segments were isolated from Adx, Adx replaced with aldosterone (Adx + Ald, 0.5 micrograms X 100 g X body wt X d), and control Sprague-Dawley rats. Both net sodium and net chloride fluxes were significantly less in the Mtal segments from Adx rats compared with those in the control or Adx + Ald group. Physiologic levels of exogenous aldosterone increased net sodium chloride flux toward control values in the Adx + Ald group. Net potassium flux was not different among the three groups. We conclude that adrenalectomy impairs reabsorptive NaCl but not K transport in the Mtal, and that aldosterone restores this process. This reabsorptive defect may contribute to the urinary concentrating and diluting abnormality associated with adrenal insufficiency.     

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.     

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.     

Effect of acidosis on chloride transport in the cortical thick ascending limb of Henle perfused in vitro.

The present studies examined the effect of acute in vitro acidosis on chloride reabsorption in the rabbit cortical thick ascending limb of Henle (cTALH). Four protocols were used: hypercapnic acidosis; "isocapnic" peritubular acidosis (bath bicarbonate reduction to 10 mM); isocapnic luminal acidosis (luminal bicarbonate reduction to 10 mM); isocapnic peritubular acidosis in the absence of luminal potassium. Transepithelial voltage (VT) decreased during hypercapnic acidosis and increased with recovery. Chloride reabsorption (pmol X mm-1 X min-1) decreased from 50.3 +/- 8.4 to 15.7 +/- 5.6, then increased to 45.6 +/- 11.1 with recovery. Likewise, VT was decreased reversibly during isocapnic peritubular acidosis, and chloride reabsorption decreased by 60%. Chloride reabsorption was greater (28.3 +/- 3.6) when tubules were perfused at normal luminal pH than at an acidotic luminal pH (11.4 +/- 4.5; P less than 0.05). Luminal potassium removal reduced chloride transport, and acidosis had no significant additional effect. Decreased chloride reabsorption in the cTALH during acidosis could contribute to the chloruresis associated with systemic acidosis. The symmetrical nature of this effect suggests that acidosis inhibits chloride reabsorption through an effect on cytosolic pH.     

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.     

Cyclic guanosine monophosphate is the mediator of platelet-activating factor inhibition on transport by the mouse kidney thick ascending limb.

Particulate and cytosolic protein tyrosine phosphatase (PTPase) activity was measured in skeletal muscle from 15 insulin-sensitive subjects and 5 insulin-resistant nondiabetic subjects, as well as 18 subjects with non-insulin-dependent diabetes mellitus (NIDDM). Approximately 90% of total PTPase activity resided in the particulate fraction. In comparison with lean nondiabetic subjects, particulate PTPase activity was reduced 21% (P < 0.05) and 22% (P < 0.005) in obese nondiabetic and NIDDM subjects, respectively. PTPase1B protein levels were likewise decreased by 38% in NIDDM subjects (P < 0.05). During hyperinsulinemic glucose clamps, glucose disposal rates (GDR) increased approximately sixfold in lean control and twofold in NIDDM subjects, while particulate PTPase activity did not change. However, a strong positive correlation (r = 0.64, P < 0.001) existed between particulate PTPase activity and insulin-stimulated GDR. In five obese NIDDM subjects, weight loss of approximately 10% body wt resulted in a significant and corresponding increase in both particulate PTPase activity and insulin-stimulated GDR. These findings indicate that skeletal muscle particulate PTPase activity and PTPase1B protein content reflect in vivo insulin sensitivity and are reduced in insulin resistant states. We conclude that skeletal muscle PTPase activity is involved in the chronic, but not acute regulation of insulin action, and that the decreased enzyme activity may have a role in the insulin resistance of obesity and NIDDM.     

Consequences of potassium recycling in the renal medulla. Effects of ion transport by the medullary thick ascending limb of Henle''s loop.

The consequences of K recycling and accumulation in the renal medulla were examined by measuring the effect of elevated K concentration on ion transport by the medullary thick ascending limb of Henle's loop. Perfused and bathed in vitro, thick limbs from both mouse and rabbit displayed a graded, reversible reduction of transepithelial voltage after increasing K concentration from 5 to 10, 15, or 25 mM. The effect was reproducible whether osmolality was 328 or 445 mosmol/kg H2O, and whether K replaced Na or choline. Net chloride absorption and transepithelial voltage were reduced by almost 90% when ambient K concentration was 25 mM. When either lumen or bath K was increased to 25 mM, net Na absorption was reduced. There was spontaneous net K absorption when perfusate and bath K concentration was 5 mM. Analysis of transepithelial K transfer after imposition of chemical gradients demonstrated rectification in the absorptive direction. Absorption of K by this segment provides a means to maintain high medullary interstitial concentration. Accumulation of K in the outer medulla, by reducing NaCl absorption, would increase volume flow through the loop of Henle and increase Na and water delivery to the distal nephron. K recycling thus might provide optimum conditions for K secretion by the distal nephron.     

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.     

Cellular heterogeneity of ammonium ion transport across the basolateral membrane of the hamster medullary thick ascending limb of Henle's loop.

The epithelia of the medullary thick ascending limb (MAL) consists of two cell types, high (HBC) and low basolateral conductance (LBC) cell, depending on the K+ conductance of the basolateral membrane. The NH4+ conductance distinct from the K+ conductance has been suggested to exist in the proximal tubule, MAL cell, and Xenopus oocyte. The present study was designed to examine whether there is a conductive NH4+ transport system distinct from K+ conductance in two different cell types of the hamster MAL perfused in vitro. The basolateral membrane voltage (VB) was measured by impaling cells with conventional microelectrodes. Addition of NH4+ to the bath depolarized VB in a dose-dependent manner in both cell types. The response was maintained in the absence of HCO3-. When the VB deflection elicited upon 50 mM KCl or NH4Cl in the bath (delta VBK+ or delta VBNH4+) were compared, delta VBNH4+ was almost the same as delta VBK+ in the HBC cell, whereas the former was greater than the latter in the LBC. In the HBC cell, 10 mM Ba2+ in the bath equally suppressed both delta VBK+ and delta VBNH4+, whereas in the LBC cell it suppressed delta VBK+ with a small effect on delta VBNH4+, indicating that NH4+ is transported via a pathway distinct from Ba(2+)-sensitive K+ conductance. The VB deflection by NH4+ was unaffected by addition of 0.1 mM ouabain or 10 microM 5-nitro-2-(3-phenylpropylamino)-benzoate (a Cl- channel blocker) to the bath, excluding the contribution of the Na+, K+ pump or Cl- channel. An abrupt reduction of Na+ in the bath from 200 to 20 mM did not cause any changes in VB, suggesting that a nonselective cation channel may not account for the NH4+ transport. Amiloride at 10 microM inhibited delta VBNH4+ with a higher efficacy in the LBC cell. We conclude that a rheogenic NH4+ transport system independent from the K+ conductance exists in the basolateral membrane of the LBC cell of the hamster MAL, and may play some roles in the regulation of NH4+ transport.     

Transport defects of rabbit medullary thick ascending limb cells in obstructive nephropathy.

To characterize the sodium transport defect responsible for salt wasting in obstructive nephropathy, the major sodium transporters in the medullary thick ascending limb (mTAL), the apical Na-K-2Cl cotransporter and the basolateral Na-K-ATPase, were studied in fresh suspensions of mTAL cells and outer medulla plasma membranes prepared from obstructed and untreated kidneys. Oxygen consumption (QO2) studies in intact cells revealed marked reductions in the inhibitory effects of both furosemide and ouabain on QO2 in cells from obstructed, as compared with control animals, indicating a reduction in activities of both the Na-K-2Cl cotransporter and the Na-K-ATPase. Saturable [3H]bumetanide binding was reduced in membranes isolated from obstructed kidneys, but the Kd for [3H]bumetanide was unchanged, indicating a decrease in the number of functional luminal Na-K-2Cl cotransporters in obstructed mTAL. Ouabain sensitive Na-K-ATPase activity in plasma membranes was also reduced, and immunoblots using specific monoclonal antibodies directed against the alpha and beta subunits of rabbit Na-K-ATPase showed decreased amounts of both subunits in outer medullas of obstructed kidney. A significant decrease in [3H]bumetanide binding was detected after 4 h of ureteral obstruction, whereas Na-K-ATPase activity at this time was still not different from control. We conclude that ureteral obstruction reduces the amounts of both luminal Na-K-2Cl cotransporter and basolateral Na-K-ATPase in mTAL of obstructed kidney and that these reductions contribute to the salt wasting observed after release of obstruction.     

Effect of vasopressin on electrical potential difference and chloride transport in mouse medullary thick ascending limb of Henle''s loop.

Medullary thick ascending limbs of Henle's loop of the Swiss-Webster mouse were perfused in vitro with an isotonic perfusate and a Ringer's bathing medium. In five studies, addition of a supramaximal concentration of synthetic arginine vasopressin (AVP) to the bathing medium resulted in an increase in electrical potential difference (PD) from 5.0 +/- 1.5 mV, lumen positive, to 10.7 +/- 1.4 mV (P < 0.001). When AVP was removed, the PD returned to 2.6 +/- 0.9 mV (P < 0.001), then increased again to 6.9 +/- 1.7 mV (P < 0.01) when AVP was added a second time. A significant, but submaximal, increase in PD of 2.3 +/- 0.6 MV (P < 0.05) was observed in five medullary thick ascending limbs when AVP was added to the bathing medium at a concentration of 10 microunits/ml. This increase was approximately one-third of the response observed at a concentration of 100 microunits/ml in the same tubule. No further increment in PD was observed in five medullary thick ascending limbs when the AVP concentration was increased from 100 to 1,000 microunits/ml. In seven thick ascendcing limbs, the effect of AVP on PD was reproduced by the addition of 8-[p-chlorophenylthio]-cyclic 3',5'-adenosine monophosphate to the bathing medium at a final concentration of 0.1 mM. AVP increased unidirectional chloride flux from lumen to bath from 29.3 +/- 3.2 to 69.8 +/- 6.2 peq/cm per s (P < 0.001) in spite of an increase in the lumen positive PD from 1.6 +/- 0.5 mV to 7.0 +/- 0.6 mV (P < 0.001). Unidirectional chloride flux from bath to lumen was not affected by AVP. In another series of experiments, net chloride flux increased from 15.6 +/- 3.0 to 41.7 +/- 5.3 peq/cm per s (P < 0.05) after addition of AVP. The effect of AVP on hydraulic water permeability (Lp) was examined by adding raffinose to the bathing medium in both the presence and the absence of AVP. The calculated Lp of 16 +/- 2 nm/s per atm in the absence of AVP, although very low, was significantly different from zero (P < 0.01). However, the Lp did not increase significantly when AVP was added to the bathing medium. These results suggest that AVP has a second site of action in the kidney to increase chloride transport by the medullary thick ascending limb in addition to its well-known effect on the water permeability of the collecting tubule. The former effect would contribute to urinary concentrating ability by increasing the axial osmotic gradient in the renal medulla.     

In vivo and in vitro studies of urinary concentrating ability in potassium-depleted rabbits.

The factors responsible for the urinary concentrating defect associated with the potassium-depleted (KD) state are uncertain. The present studies were designed to, first, determine whether a urinary concentrating defect exists in potassium-depleted rabbits and, second, to use the technique of in vitro perfusion to evaluate directly the antidiuretic hormone (ADH) responsiveness of cortical collecting tubules (CCT) in this setting. Feeding female New Zealand White rabbits a potassium-deficient diet for 2 wk caused a significant fall in plasma potassium levels in both the ad-libitum and controlled water intake groups (P less than 0.001). Muscle potassium content after 2 wk of potassium restriction fell from 45.6 +/- 0.9 to 29.0 +/- 1.2 meq/100 g fat-free dry solids (P less than 0.001). Renal papillary sodium content fell significantly from a control value of 234.6 +/- 8.0 to 182.46 +/- 10.0 meq/kg H2O after 2 wk of potassium restriction. Maximal urinary osmolality measured after 12 h of dehydration and 1.25 U pitressin IM was significantly decreased in rabbits after 2 wk of potassium restriction in both the ad-libitum and controlled water intake groups (P less than 0.001). The relationship between plasma potassium concentration and maximum urinary osmolality was significantly correlated in both the ad-libitum and controlled water intake groups, r = 0.73 and 0.68 (P less than 0.001), respectively. In addition, refeeding KD rabbits with normal chow for 1 wk resulted in normalization of both plasma potassium levels and urinary concentrating ability. CCT from control and KD rabbits were perfused in vitro at 25 degrees C. The hydraulic conductivity coefficient, Lp, was significantly reduced at all doses of ADH tested in tubules from KD rabbits when compared with control tubules. In addition, the maximal hydraulic conductivity in tubules from KD rabbits when tested with 200 microU/ml ADH at 37.5 degrees C was only 23% of control values (P less than 0.05). Furthermore, this reduced ADH responsiveness persisted when the bath potassium was elevated from 5 to 20 mM. The reflection coefficient for NaCl when compared with raffinose was 0.91 in tubules from KD animals. Thus, these data suggest that the ADH-resistant urinary concentrating defect associated with potassium depletion is due, at least in part, to a diminished responsiveness of the CCT to ADH. Therefore, further studies were designed to investigate the cellular steps involved in this abnormal response. There was no difference in the 8-para-chlorophenylthio cyclic AMP induced hydroosmotic response between CCT from KD and control rabbits. Since the cAMP-induced hydroosmotic response was similar between KD and control CCT, experiments were performed to evaluate the contribution of phosphodiesterase (PDIE) activity by using the potent PDIE inhibitor isobutylmethylxanthine (10(-4) and 10(-3)M) in the presence of ADH (200 U/ml). Although Lp was increased by PDIE inhibition in CCT from both control and KD animals, the overall hydroosmotic response in CCT from KD rabbits was still significantly reduced when compared with controls. The final experiments used forskolin to evaluate further the adenylate cyclase complex. The resulting hydroosmotic response in CCT from KD rabbits was almost identical to that obtained in controls. In conclusion, these data suggest that the decreased responsiveness of CCT from KD rabbits to ADH involves a step at or proximal to the stimulation of the catalytic subunit of adenylate cyclase, and that PDIE activity makes no contribution to this abnormal hydroosmotic response.     

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.     

Mechanism of sodium and chloride transport in the thin ascending limb of Henle.

Our previous in vitro studies have disclosed that the thin ascending limb of Henle (tALH) possesses some unique membrane characteristics. In those studies we failed to demonstrated active transport of sodium chloride by the tALH, although it was shown that the isotopic permeability to sodium and chloride was unusually high. However, we did not examine the mechanisms by which the apparent high permeation of sodium chloride occurs. Thus the purpose of the present studies was to elucidate the mechanism of sodium chloride transport across the isolated tALH of the rabbit by conducting four different types of studies: (1) comparison of the observed chloride and sodium flux ratios to those predicted by Ussing's equation under imposed salt concentration gradients; (2) kinetic evaluation of chloride and sodium fluxes; (3) examination of the effect of bromide on the kinetics of chloride transport; and (4) experiments to test for the existence of exchange diffusion of chloride. In the first set of studies the predicted and the theoretical flux ratios of sodium were identical in those experiments in which sodium chloride was added either to the perfusate or to the bath. However, the observed chloride flux ratio, lumen-to-bath/bath-to-lumen, was significantly lower than that predicted from Ussing's equation when 100 mM sodium chloride was added to the bath. In the second set of experiments the apparent isotopic permeability for sodium and for chloride was measured under varying perfusate and bath NaCl concentrations. There was no statistical change in the apparent sodium permeability coefficient when the NaCl concentration was raised by varying increments from 85.5 to 309.5 mM. However, permeation of 36Cl decrease significantly with an increase in Cl from 73.6 to 598.6 mM. These events could be explained by a two component chloride transport process consisting of simple diffusion and a saturable facilitated diffusion process with a Vmax = 3.71 neq mm-1 min-1. In the third set of studies it was shown that bromide inhibits transport of chloride and that the magnitude of inhibition is dependent on chloride concentrations. The fourth set of studies ruled out the existence of exchange diffusion. In conclusion, these studies indicate that sodium transport across tALH is by simple passive diffusion, while chloride transport across tALH involves at least two mechanisms: (1) simple passive diffusion; and (2) a specific membrane interaction process (carrier-mediated) which is competitively inhibited by bromide.     

Selective vulnerability of the medullary thick ascending limb to anoxia in the isolated perfused rat kidney.

A specific anatomical lesion sharply localized to the cells of the medullary thick ascending limbs (mTAL) and characterized by mitochondrial swelling progressing to nuclear pyknosis and cell death is elicited reproducibly in isolated rat kidneys perfused for 15 or 90 min with cell-free albumin-Ringer's medium gassed with 5% CO2, 95% O2 (O2 content, 1.5 vol/100 ml). The lesion, involving about half of mTALs, appears first in mTALs removed from vascular bundles and near the inner medulla, areas most likely to be anoxic. Hypoxic perfusion (O2 content 0.12 vol/100 ml) exaggerates the lesion, wiping out gradations of damage and extending it to all mTALs. O2-enriched perfusions using rat erythrocytes (O2 content 7.1 vol/100 ml) completely eliminates the lesion (unless gassed with carbon monoxide). Similarly, supplementation of the perfusion medium with a purified hemoglobin (O2 content 5.8 vol/100 ml) prevents mTAL injury. Perfusion with a fluorinated hydrocarbon blood substitute, Oxypherol (O2 content 4.3 vol/100 ml) also attenuates the lesion. These findings suggest that the mTAL is exquisitely susceptible to anoxic damage because of low O2 supply imposed by the medullary vascular system and the high rate of metabolism mandated by active reabsorption of sodium chloride. The vulnerability of the mTAL to anoxic injury could play a key role in the pathogenesis of ischemic renal injury.     

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.     

Intracellular Mg2+ and magnesium depletion in isolated renal thick ascending limb cells.

Magnesium reabsorption and regulation within the kidney occur principally within the cortical thick ascending limb (cTAL) cells of the loop of Henle. Fluorometry with the dye, mag-fura-2, was used to characterize intracellular Mg2+ concentration ([Mg2+]i) in single cTAL cells. Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat anti-human Tamm-Horsfall and rabbit anti-goat IgG antibodies). Basal [Mg2+]i was 0.52 +/- 0.02 mM, which was approximately 2% of the total cellular Mg. Cells cultured (16 h) in high magnesium media (5 mM) maintained basal [Mg2+]i, 0.48 +/- 0.02, in the normal range. However, cells cultured in nominally magnesium-free media possessed [Mg2+]i, 0.27 +/- 0.01 mM, which was associated with a significant increase in net Mg transport, (control, 0.19 +/- 0.03 and low Mg, 0.35 +/- 0.01 nmol.mg-1 protein.min-1) as assessed by 28Mg uptake. Mg(2+)-depleted cells were subsequently placed in high Mg solution (5 mM) and the Mg2+ refill rate was assessed by fluorescence. [Mg2+]i returned to normal basal levels, 0.53 +/- 0.03 mM, with a refill rate of 257 +/- 37 nM/s. Mg2+ entry was not changed by 5.0 mM Ca2+ or 2 mM Sr2+, Cd2+, Co2+, nor Ba2+ but was inhibited by Mn2+ approximately La3+ approximately Gd3+ approximately Zn2+ approximately Be2+ at 2 mM. Intracellular Ca2+ and 45Ca uptake was not altered by Mg depletion or Mg2+ refill, indicating that the entry is relatively specific to Mg2+. Mg2+ uptake was inhibited by nifedipine (117 +/- 20 nM/s), verapamil (165 +/- 34 nM/s), and diltiazem (194 +/- 19 nM/s) but enhanced by the dihydropyridine analogue, Bay K 8644 (366 +/- 71 nM/s). These antagonists and agonists were reversible with removal and [Mg2+]i subsequently returned to normal basal levels. Mg2+ entry rate was concentration and voltage dependent and maximally stimulated after 4 h in magnesium-free media. Cellular magnesium depletion results in increases in a Mg2+ refill rate which is dependent, in part, on de novo protein synthesis. These data provide evidence for novel Mg2+ entry pathways in cTAL cells which are specific for Mg2+ and highly regulated. These entry pathways are likely involved with renal Mg2+ homeostasis.     

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.     

Modulation of Na-K-ATPase activity in the mouse medullary thick ascending limb of Henle. Effects of mineralocorticoids and sodium.

This study investigates the effect of variations in mineralocorticoid as well as cell sodium delivery and uptake on Na-K-ATPase activity in the mouse medullary thick ascending limb of Henle (mTALH). Pharmacologic doses of the mineralocorticoid deoxycorticosterone acetate (DOCA) resulted in a 28% increase of Na-K-ATPase activity. Furosemide-induced inhibition of sodium uptake by the mTALH cell also resulted in Na-K-ATPase activity reduction (45%). Sodium deprivation did not cause a clear change in enzyme activity, either at 3 d or 2 wk, likely reflecting the result of the opposing influences of decreased sodium delivery and increased endogenous aldosterone. Finally, the behavior of Na-K-ATPase activity at 3 d of sodium deprivation in the mTALH contrasted with a 60% increase in activity observed in the cortical collecting tubule, a nephron segment known to be responsive to mineralocorticoid, and this heterogeneity of response may suggest an important role for the mTALH in maintaining salt homeostasis.