Captopril inhibits the hydroosmotic effect of ADH in the cortical collecting tubule

Previous studies have shown that captopril (CP) inhibits ADH-stimulated osmotic water permeability (Pf) in the toad bladder by potentiating endogenous bradykinin (BK). The present studies examine the effect of CP on ADH-stimulated Pf in isolated, perfused rabbit cortical collecting tubules (CCT). CP (10(-4) M) reversibly inhibited Pf, stimulated by maximal concentrations of ADH (10 microU/ml). Pretreatment of CCT's with 5 microM indomethacin, however, abolished the effect of CP. Inhibition of BK production by the kallikrein inhibitors, aprotinin and benzamidine, failed to enhance Pf stimulated by submaximal concentrations of ADH (2.5 microU/ml). Since ADH exerts its effects by activation of adenylyl cyclase (AC), further experiments were performed to identify the site at which CP inhibits this cascade. CP significantly inhibited forskolin (10(-4) M) stimulated Pf; however, it had no effect on cyclic AMP (10(-5) M) stimulated Pf, suggesting that the site of action is on the catalytic subunit or one of the GTP regulatory proteins of AC. To further localize the site of CP's action, CCT's were pre-incubated with pertussis toxin (0.5 microgram/ml) to inactivate the inhibitory, guanosine triphosphate (GTP) regulatory protein, Gi. In these tubules, CP failed to inhibit the action of ADH. We conclude that CP stimulates prostaglandin production which in turn activates Gi and inhibits AC activity. We further suggest that CP stimulates PG's directly, not via BK.     

Morphometric comparison of rabbit cortical connecting tubules and collecting ducts

Connecting tubule (CNT) segments of the rabbit distal nephron were examined by scanning electron microscopy and computer-assisted morphometric analysis of transmission electron micrographs. CNT were very similar to the cortical collecting ducts (CCD) described previously. The epithelium of both segments contains two cell types, both of which can be modeled as simple cuboidal cells, and two distinct systems of extracellular channels. The lateral intercellular channels are comparable to the spaces between simple cuboidal cells but are modified by short projecting microvilli which produce a modest increase in lateral cell surface area. The basal infolded channels are best developed in the connecting tubule cells of CNT and contribute 63% of all channel-associated membranes in CNT. Total membrane areas are similar in CNT and CCD. The two segments differ only in the degree of extracellular channel dilation and the distribution of infolded membrane relative to cell height in the connecting tubule and principal cells. The relatively minor morphometric differences between CNT and CCD do not correlate well with the marked difference in transtubular volume flow induced in the two segments by ADH and an osmotic gradient.     

Postnatal maturation of the rabbit cortical collecting duct

The mature, fully differentiated cortical collecting duct plays a major role in the final renal regulation of Na⁺, K⁺ and H⁺ transport. To characterize the growth of this segment, we measured the outer diameter and the dry weight of cortical collecting ducts isolated from newborn, 1-month-old, and adult rabbits. During the 1st month of life no significant changes were observed; however, there was a 60% increase in both parameters after the 4th week of life. Growth-related accretion of K⁺ was demonstrated by showing tubular K⁺ content to increase by 60% with maturation. Concomitant with the increase in tubular size, total cell number per millimeter of tubular length rose by 30%. Approximately 50% of the observed increment in tubular size could be accounted for by cell hyperplasia, with the remaining increase resulting from cell hypertrophy. Hypertrophy of principal cells was confirmed by scanning electron microscopy, which demonstrated a doubling of the circumferential width without any change in longitudinal length. Hyperplasia was confirmed, using a fluorescent chromatin stain, by our finding of a mitotic frequency of 3/1000 cells in the neonatal mid-cortical collecting duct; the observed number of mitoses was 10-fold higher at the most cortical end (ampulla). The number of intercalated cells per millimeter of tubule length, identified by bright green fluorescence after cortical collecting ducts were stained with 6-carboxyfluorescein diacetate, was found to double during maturation, the increase being significant only after the 4th postnatal week. We conclude that maturation of the mid-cortical collecting duct results from both cellular hyperplasia and hypertrophy. It is unlikely that this segment plays a major role in regulating Na⁺, K⁺, and H⁺ transport in the neonatal kidney.     

Changes in intracellular sodium during the hydroosmotic response to vasopressin.

During vasopressin (VP)-induced water movement, toad urinary bladder epithelial cells undergo unique morphological changes. The osmolality within these responding cells remains relatively stable despite the large transcellular transport of water. We hypothesized that the hydroosmotic response to VP may be associated with a net increase in sodium either as an aid in maintaining the intracellular osmolality or as part of a Na-Ca exchange process. Changes in intracellular sodium (Nai) were monitored over time in individual hemibladders using 23Na NMR. Hemibladders were mounted as bags on glass pipets and filled with deionized water. During NMR studies, the serosal bath consisted of aerated 2.4 mM HCO3 amphibian Ringer's (pH 8.1) made up with 15% D2O containing the shift reagent, dysprosium tripolyphosphate (1 mM). This reagent allowed for visualization of Nai by shifting the extracellular Na signal; it did not affect basal or VP stimulated water flow, short-circuit current, or high energy phosphate metabolism as seen by 31P NMR. Changes in Nai were determined by integrating the area under the unshifted Na peak at each measurement and expressing differences as a ratio relative to baseline. The initial Nai signal from unstimulated hemibladders remained stable in these tissues over at least 180 minutes. Within 30 minutes of VP (20 mU/ml) exposure, however, the Nai peak increased 2.47 times above pretreatment baseline (N = 16, P less than 0.001). The Nai signal returned toward baseline values with removal of VP from the serosal bath but only after approximately 90 minutes. When change in cell shape and water movement were prevented by having isotonic sorbitol in the mucosal bath, VP produced no change in the Nai signal (N = 10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of iodide transport in the rabbit cortical collecting duct

Background Pendrin, an anion exchanger known to participate in iodide transport in the apical membrane of follicular cells of the thyroid gland, has recently been shown to exist in the apical membrane of the β- and γ-intercalated (β/γ-IC) cells of the cortical collecting duct (CCD). We examined mechanisms of iodide transport in the CCD. Methods Rabbit CCD was perfused in vitro, and lumen-to-bath flux coefficients for both ¹²⁵I⁻ (K_{I (lb)}) and ³⁶Cl⁻ (K_{Cl (lb)}) were measured simultaneously. The intracellular pH (pHi) of β/γ-IC cells in the perfused CCD was measured by microscopic fluorometory, by loading 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein tetraacetoxy methylester (BCECF-AM), a fluorescent marker for pHi. The effects on pHi of the replacement of NaCl with Na cyclamate, NaI, or NaBr in the lumen or bath were observed. Results K_{I (lb)} was comparable to or slightly higher than K_{Cl (lb)}. Both iodide and chloride in the lumen caused self- and cross-inhibitions to both fluxes. The addition of 5-nitro-2-(-3-phenylpropylamino)-benzoate (NPPB), a Cl⁻ channel inhibitor, to the bath significantly reduced K_{Cl (lb)}, but not K_{I (lb)}. Replacement of luminal fluid NaCl with Na cyclamate, NaI, or NaBr caused alkalization of pHi, no change in pHi, and slight acidification of pHi, respectively. Replacement of bath NaCl with Na cyclamate, NaI, or NaBr caused alkalization, alkalization, and acidification of pHi, respectively. Luminal NaI prevented the acidification of pHi caused by bath Na cyclamate. Conclusions The data are consistent with the model that iodide is transported via the Cl⁻/HCO₃⁻ exchanger in the apical membrane of β/γ-IC cells and exits the basolateral membrane via an electroneutral transporter that is distinct from the Cl⁻ channel. We could not, however, identify which type of β/γ-IC cell was mainly responsible.     

Sustained response to vasopressin in isolated rat cortical collecting tubule

The effect of arginine vasopressin (ADH) on water permeability and transepithelial voltage was examined in cortical collecting tubules from a specific pathogen-free line of male Sprague-Dawley rats (75-125 g body weight). Tubules were bathed in a medium resembling serum ultrafiltrate (310 mOsm/kg H2O) at 38 degrees C. Osmotic water permeability (Pf, micron/sec) was determined by the volume flow occurring with a hypo-osmotic perfusate (210-220 mOsm/kg H2O) and diffusional water permeability (Pd, micron/sec) was calculated from the lumen-to-bath flux of tritiated water using an isosmotic perfusate. In the absence of ADH, both Pf and Pd were low, 17 +/- 6 and 9.0 +/- 0.6 (SEM), respectively. ADH added to the bath at concentrations above 0.5 microunits/ml increased Pf, with a maximal response at 40 microunits/ml or greater. With 100 microunits/ml ADH, Pf and Pd were, respectively, 994 +/- 117 and 37.0 +/- 2.4. Without ADH, the transepithelial voltage was variable (range, -5.4 to +2.5 mV; mean, -1.9 +/- 0.4); however, with 100 microU/ml ADH, it hyperpolarized (lumen-negative) by 4.2 +/- 0.8 mV. In contrast to findings in the rabbit, both the hyperpolarization and the increased water permeability persisted for at least 3 hr. The higher water permeabilities are consistent with the shorter length of the cortical collecting tubule in the rat, and may reflect the importance of attaining osmotic equilibration within the cortex during maximal antidiuresis.     

Potassium permeable channels in primary cultures of rabbit cortical collecting tubule.

Rabbit cortical collecting tubule (RCCT) primary cultures, were grown on permeable, collagen supports with 1.5 microM aldosterone. Single K+ permeable channels in principal cell apical membranes were examined. At applied patch pipette potential (Vapp) from -60 to +60 mV (cell interior with respect to pipette interior), outward currents (cell to pipette) with a unitary conductance of 8 to 10 pS were seen in cell-attached (N = 31) and excised inside-out (N = 15) patches. At resting membrane potential (Vapp = 0 mV), mean open probability (Po = 0.85 +/- 0.16) decreased by 50% with 0.75 mM luminal BaCl2 exposure. In cell-attached patches, a second type of outward current was seen only at extreme depolarization, Vapp greater than +80 mV (N = 9). Usually in the closed state (Po less than 0.0005) at no applied potential, Po for this 150 pS channel increased dramatically with depolarization and/or raising cytoplasmic Ca2+. With a calculated K+ equilibrium potential of -84 mV, excised patch reversal potentials were less than -50 mV for both the above channel types, indicating high selectivity for K+ over Na+. In cultures grown without aldosterone low conductance K+ channels were rarely observed, while mineralocorticoid status did not appear to affect high conductance K+ channel frequency. Finally, a 30 pS cation channel was found to be nonselective for K+ over Na+, and insensitive to voltage, intracellular Ca2+ or luminal Ba2+. We conclude that: 1) Principal cell apical membranes from aldosterone-stimulated, RCCT primary cultures contain (a) low conductance, Ba(2+)-inhibitable and (b) high conductance, Ca2+/voltage-dependent K+ channels; and c) nonselective cation channels. 2) The low conductance K+ channel may play an important physiologic role in native RCCT mineralocorticoid-controlled K+ secretion, while the latter two channels' functions are unknown, although similar channels have been suggested to play a role in cell volume regulation.     

Effects of in vitro aldosterone on the rabbit cortical collecting tubule

Considerable evidence indicates that the cortical collecting tubule is a target epithelium for aldosterone. Isolated perfused cortical collecting tubules from rabbits given large doses of deoxycorticosterone acetate (DOCA) for several days, or whose endogenous production of aldosterone is increased by dietary means, exhibit large lumen-negative transepithelial voltages, increased sodium (Na) absorption, and increased potassium (K) secretion compared with tubules from normal animals. However, controversy exists regarding the response of this nephron segment to acute in vitro administration of aldosterone. To address this issue we performed three groups of experiments: 1) clearance experiments on adrenalectomized rabbits to determine the minimum time required after in vivo aldosterone administration before significant changes in sodium excretion are observed; 2) microperfusion experiments on cortical collecting tubules from normal and adrenalectomized rabbits in which transepithelial voltage was measured before and after adding aldosterone to the bath; 3) microperfusion experiments on cortical collecting tubules from adrenalectomized rabbits in which transepithelial voltage, sodium and potassium flux were measured before and after in vitro exposure to aldosterone or dexamethasone. The clearance studies demonstrate that after a 2 hr latent period aldosterone produces significant antinatriuresis without change in K excretion. In vitro studies failed to reveal a steroid-induced change in the transepithelial voltage of cortical collecting tubules from either normal or adrenalectomized rabbits. However, aldosterone added in vitro to collecting tubules from adrenalectomized rabbits produced an increase in net Na absorption without a significant change in voltage or K secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mineralocorticoid effect on K+ permeability of the rabbit cortical collecting tubule

Mineralocorticoid hormones stimulate Na+ absorption and K+ secretion by the cortical collecting tubule. There is good evidence that this stimulation involves increasing luminal membrane Na+ permeability and the turnover rate (or number) of the Na+-K+ pumps. These experiments were designed to examine whether mineralocorticoid hormones also increase cell K+ permeability. Using 42K tracer measurements in tubules treated with amiloride to inhibit active Na+ and K+ transport, passive K+ permeation increased with increasing mineralocorticoid effect. Net Na+ absorption and the (passive) K+ efflux rate coefficient (KK) showed a linear relationship. The stimulatory effect was evident in vitro since 0.2 microM aldosterone added to the bath of tubules harvested from NaCl-loaded rabbits increased KK at 3 hrs while time controls showed no change. Since these tubules were also treated with amiloride, this increase in KK was not dependent on increasing Na+ absorption. The results indicate that in addition to the well-described effects of aldosterone on Na+ permeability and cell metabolism, the mineralcorticoid effect includes an increase in cellular K+ permeability.     

Absence of H(+)-ATPase in cortical collecting tubules of a patient with Sjogren's syndrome and distal renal tubular acidosis.

Distal urinary acidification abnormalities may arise from transepithelial voltage defects, permeability defects, or proton-secretory defects, but tests to determine the cellular mechanisms underlying secretory abnormalities have not previously been reported. A patient with Sjogren's syndrome and distal renal tubular acidosis due to a secretory defect is described, whose kidney biopsy was examined by fluorescent immunocytochemistry with an antibody to the M(r) 31,000 subunit of the mammalian kidney vacuolar H(+)-ATPase and was compared with normal human kidney. Staining with the anti-H(+)-ATPase antibody in normal human kidney was detected in the brush border microvilli and subvillar invaginations of the proximal tubule and in intercalated cells in the collecting duct. A biopsy sample from the patient was devoid of any anti-H+-ATPase staining in the intercalated cells. Staining was also absent from the proximal tubule brush border microvilli but was present in the subvillar invaginations. Although autoantibodies to normal human kidney membrane proteins were detected in the serum by immunoblot analysis, no immunocytochemical evidence for anti-intercalated cell autoantibodies was observed in the patient's serum. This report demonstrates that the basis for the proton secretory defect in some patients with distal renal tubular acidosis is likely the absence of H(+)-ATPase in the intercalated cells. It also illustrates the potential diagnostic utility of anti-H(+)-ATPase antibodies in the classification of distal renal tubular acidoses.     

Response of cortical collecting ducts from remnant kidneys to arginine vasopressin.

Chronic renal failure is associated with impaired urine concentration. Previous studies have demonstrated that cortical collecting ducts (CCD) from uremic rabbits (with remnant kidneys) have an impaired response to arginine vasopressin (AVP). To determine whether this defect is an early, integral component of compensatory renal growth by the remnant kidney, we studied the response of CCD derived from rabbits one week after 75% nephrectomy. At one week, hypertrophy and adaptation in sodium transport are fully developed, but azotemia and interstitial fibrosis are absent. The animals with remnant kidneys failed to respond normally to water deprivation and dDAVP (maximum urine osmolality 738 +/- 29.1 mOsm/kg compared to 1378 +/- 207 in sham operated). However, in isolated, perfused CCD from remnant kidneys, AVP stimulated hydraulic water permeability to the same extent as in normal CCD or CCD from sham operated animals. AVP-induced cAMP generation per mm tubule length was significantly higher in the CCD from remnant kidneys (137.4 +/- 14.5 fmol/mm) than in the control group (82.4 +/- 11.9 fmol/mm), but not different when expressed per micrograms protein. These studies demonstrate that one week after reduction in renal mass there is no defect in the response of CCD to AVP, suggesting that the mechanisms responsible for the hyposthenuria after loss of renal mass are not related to any intrinsic cellular changes that occur in CCD early during compensatory renal growth.     

Cation channels of the rabbit outer medullary collecting duct.

The outer medullary collecting duct (OMCD) produces high rates of proton secretion, and previous data show a significant role for an H+,K+-ATPase in luminal acidification. This mechanism of acidification requires proton secretion to occur in exchange for potassium absorption. Because net transport of potassium is small in the OMCD, pathways for potassium secretion would appear essential to explain the contribution of an H+,K+-ATPase under potassium-replete conditions. A significant issue is whether a potassium exit pathway (i.e., cation channel) is present at the apical and/or the basolateral membrane. In this report, using patch-clamp techniques to examine single channel conductances from the native rabbit OMCD, we show that the apical membrane has potassium permeable cation channels.     

Water transport in the immature rabbit collecting duct

 Immature animals have limited ability to concentrate the urine. This is in part the result of end-organ resistance to arginine vasopressin (AVP). To characterize this response, we measured water absorption in microperfused cortical collecting ducts (iCCD) and outer medullary CD (iOMCD) derived from 2- to 12-day-old rabbits. The roles of adenosine 3′,5′-cyclic monophosphate (cAMP) and prostaglandins were investigated. Baseline osmotic water permeability (L_p, 10⁻⁷ cm/atm per s) in the iCCD (20.3±2.4) and iOMCD (19.7±5.6) was not different from mature CCD (mCCD) (14.6±3.1). After AVP, L_p in the iCCD (46.7±10.0) was significantly lower than in the mCCD (114.3±21.8). Neither stimulation with cAMP (85.6±51.3) nor inhibition of endogenous prostaglandin production with indomethacin (57.6±29.8) abolished the blunted response to AVP in the iCCD. We conclude that AVP-stimulated water transport in the iCCD is impaired. The disruption in AVP response is, at least in part, localized distal to cAMP, and is not mediated by prostaglandins. Received: 29 December 1997 / Revised: 13 April 1998 / Accepted: 14 April 1998     

Compensatory hypertrophy and adaptation in the cortical collecting duct

The cortical collecting duct (CCD) undergoes hypertrophy and functional adaptation following reduction of renal mass. The nature and mechanisms of these changes have been investigated using microperfusion of isolated CCD from rabbit remnant kidneys. By 1 week after reduction of renal mass, tubule hypertrophy and increased sodium transport are fully developed. The transport adaptations are specific or selective, since bicarbonate transport in these CCD is unchanged. Mineralocorticoids may play an important role in the hypertrophy and increased sodium transport, since plasma aldosterone increases early after reduction of renal mass. Also, adrenalectomy abolishes the changes in size and sodium transport, even with supplementation of aldosterone to unstressed physiologic levels. Epidermal growth factor also has immediate effects on CCD sodium transport; however, the direction of the effect is opposite--an inhibition of transport.     

Basolateral Na+/H+ exchange maintains potassium secretion during diminished sodium transport in the rabbit cortical collecting duct

Stimulation of the basolateral Na(+)/K(+)-ATPase in the isolated perfused rabbit cortical collecting duct by raising either bath potassium or lumen sodium increases potassium secretion, sodium absorption and their apical conductances. Here we determined the effect of stimulating Na(+)/K(+)-ATPase on potassium secretion without luminal sodium transport. Acutely raising bath potassium concentrations from 2.5 to 8.5 mM, without luminal sodium, depolarized the basolateral membrane and transepithelial voltages while increasing the transepithelial, basolateral and apical membrane conductances of principal cells. Fractional apical membrane resistance and cell pH were elevated. Net potassium secretion was maintained albeit diminished and was still enhanced by raising bath potassium, but was reduced by basolateral ethylisopropylamiloride, an inhibitor of Na(+)/H(+) exchange. Luminal iberitoxin, a specific inhibitor of the calcium-activated big-conductance potassium (BK) channel, impaired potassium secretion both in the presence and absence of luminal sodium. In contrast, iberitoxin did not affect luminal sodium transport. We conclude that basolateral Na(+)/H(+) exchange in the cortical collecting duct plays an important role in maintaining potassium secretion during compromised sodium supplies and that BK channels contribute to potassium secretion.     

Virilizing adrenal cortical carcinoma with hypertrophy of spermatic tubules in childhood.

A review of adrenal cortical carcinoma in childhood is presented, including endocrinologic aspects. Electron microscopic features are presented, as well as the finding of hypertrophied spermatic tubules. The importance of serial steroidal determinations is stressed as a "tumor marker" that may help in early detection of recurrent disease and possibly increased long-term survival.     

Fetal response to injury in the rabbit

Fetal, neonatal, and adult tissue response to a standardized injury was studied using subcutaneous wound implants, linear incisions, and punch wounds in New Zealand white rabbits. In the fetus, sutured incisions healed by primary intention without antecedent inflammation. However, neither contraction nor healing by secondary intention was seen in punch or unsutured wounds. Healing both by primary and secondary intention following inflammatory infiltration was observed uniformly in neonatal and adult rabbits. Wound implants were extensively infiltrated with collagen in the adults studied; however, no collagen was seen in fetal implants and collagen hydroxyproline content could not even be detected by high performance liquid chromatography techniques; rather, a matrix rich in hyaluronic acid was found. The fetal tissue response to injury differs from the adult, proceeding in the absence of a classical inflammatory stimulus and lacking contractile capabilities. The deposition of extracellular matrix rich in hyaluronic acid but devoid of collagen suggests that the fetal response to injury may be a process more closely resembling regeneration or growth rather than repair by scar deposition.