Basolateral Na-H exchange in the rabbit cortical collecting tubule

We used the intracellular absorbance spectrum of the dye 4',5'-dimethyl-5- (and -6-) carboxyfluorescein (Me2CF) to measure intracellular pH (pHi) in the isolated, perfused cortical collecting tubule (CCT) of the rabbit nephron. The incident spot of light was generally 10 micron in diameter, large enough to illuminate from two to six cells. No attempt was made to distinguish principal from intercalated cells. All experiments were carried out in HCO3- -free Ringer to minimize HCO3- transport. When cells were acid-loaded by briefly exposing them to Ringer containing NH+4 and then withdrawing the NH+4, pHi spontaneously recovered from the acid load. The pHi recovery was best fit by the sum of two exponentials. When the acid loading was performed in the absence of Na+, the more rapid of the two phases of pHi recovery was absent. The remaining slow phase never returned pHi to normal and was sometimes absent. Returning Na+ to the lumen had only a slight effect on the pHi recovery. However, when Na+ was returned to the basolateral (i.e., blood-side) solution, pHi recovered rapidly and completely. The apparent Km for basolateral Na+ was 27.3 +/- 4.5 mM. The basolateral Na-dependent pHi recovery was reversibly inhibited by amiloride. We conclude that the mechanism responsible for the rapid phase of pHi recovery is an Na-H exchanger confined primarily, if not exclusively, to the basolateral membrane of the CCT.     

Aldosterone induces chloride transport in the cortical collecting tubule

The ontogenetic differentiation of transepithelial chloride transport was evaluated in the cortical collecting tubule of the rabbit kidney. Tubules from four control groups (I-IV) were studied during in vitro perfusion. I: body weight 150-280 g; II: 330-480 g; III: 530-880 g; IV: 980-1610 g. In each group, aldosterone (100 micrograms/100 g body weight/day) was given subcutaneously in three doses daily, for 6 days (IA-IVA). Transepithelial net chloride flux (pmol cm2 s1) increased by a factor of almost 3 from group I to group IV (p less than 0.01). Aldosterone induces net chloride flux by 103% (P = 0.03) in IA and by 78% (P = 0.01) in IIA; changes in groups III (21%) and IV (27%) were small. Therefore, the mineralocorticoid induces transepithelial chloride transport in cortical collecting tubule during early transport differentiation. The inducing action decreases with natural differentiation. Moreover, aldosterone alone suffices to induce the complete expression of transepithelial chloride transport in the cortical collecting tubule.     

Regulation of Na channels of the rat cortical collecting tubule by aldosterone

The activity of apical membrane Na channels in the rat cortical collecting tubule was studied during manipulation of the animals' mineralocorticoid status in vivo using a low-Na diet or the diuretic furosemide. Tubules were isolated and split open to expose the luminal membrane surface. Induction of Na channel activity was studied in cell- attached patches of the split tubules. No activity was observed with control animals on a normal diet. Channel activity could be induced by putting the animals on the low-Na diet for at least 48 h. The mean number of open channels per patch (NPo) was maximal after 1 wk on low Na. Channels were also induced within 3 h after injection of furosemide (20 mg/kg body wt per d). NPo was maximal 48 h after the first injection. In both cases, increases in NPo were primarily due to increases in the number of channels per patch (N) at a constant open probability (Po). With salt depletion or furosemide injection NPo is a saturable function of aldosterone concentration with half-maximal activity at approximately 8 nM. When animals were salt repleted after 1- 2 wk of salt depletion, both plasma aldosterone and NPo fell markedly within 6 h. NPo continued to decrease over the next 14 h, while plasma aldosterone rebounded partially. Channel activity may be dissociated from aldosterone concentrations under conditions of salt repletion.     

Regulation of the Na-K pump of the rat cortical collecting tubule by aldosterone

Activities of Na channels and Na pumps were studied in the rat cortical collecting tubule (CCT) during manipulation of the animals' mineralocorticoid status in vivo using a low-Na diet, diuretics, or administration of exogenous aldosterone. Tubules were isolated and split open to expose the luminal membrane surface. Using the whole-cell patch-clamp technique, activities of the apical Na channels and the basolateral Na pumps were measured in principal cells as the currents inhibited by amiloride (10 microM) and ouabain (1 mM), respectively. Na channel current (INa) was not measurable in CCTs from control animals on a normal diet. INa was approximately 200 pA/cell in CCTs from animals on a low-Na diet or infused with aldosterone using osmotic minipumps. Currents attributable to the Na pump (Ipump) were similar in control animals and animals on a low-Na diet. Maximal currents were approximately 35 pA/cell in both groups, and decreased with hyperpolarization of the cell membrane. In contrast, administration of exogenous aldosterone increased Ipump fourfold. Coinfusion of aldosterone and amiloride in vivo through the minipumps did not affect the induction of INa but reduced the induction of Ipump by 80%. We conclude that the induction of channel activity in this tissue is a direct action of aldosterone, whereas the induction of pump activity may be a consequence of the increased Na traffic through the epithelial cells.     

Prenatal programming of rat cortical collecting tubule sodium transport

Prenatal insults have been shown to lead to elevated blood pressure in offspring when they are studied as adults. Prenatal administration of dexamethasone and dietary protein deprivation have demonstrated that there is an increase in transporter abundance for a number of nephron segments but not the subunits of the epithelial sodium channel (ENaC) in the cortical collecting duct. Recent studies have shown that aldosterone is elevated in offspring of protein-deprived mothers when studied as adults, but the physiological importance of the increase in serum aldosterone is unknown. As an indirect measure of ENaC activity, we compared the natriuretic response to benzamil in offspring of mothers who ate a low-protein diet (6%) with those who ate a normal diet (20%) for the last half of pregnancy. The natriuretic response to benzamil was greater in the 6% group (821.1 ± 161.0 μmol/24 h) compared with the 20% group (279.1 ± 137.0 μmol/24 h), consistent with greater ENaC activity in vivo (P < 0.05). In this study, we also directly studied cortical collecting tubule function from adult rats using in vitro microperfusion. There was no difference in basal or vasopressin-stimulated osmotic water permeability. However, while cortical collecting ducts of adult offspring whose mothers ate a 20% protein diet had no sodium transport (-1.9 ± 3.1 pmol·mm(-1)·min(-1)), the offspring of rats that ate a 6% protein diet during the last half of pregnancy had a net sodium flux of 10.7 ± 2.6 pmol·mm(-1)·min(-1) (P = 0.01) in tubules perfused in vitro. Sodium transport was measured using ion-selective electrodes, a novel technique allowing measurement of sodium in nanoliter quantities of fluid. Thus we directly demonstrate that there is prenatal programming of cortical collecting duct sodium transport.     

Forskolin increases osmotic water permeability of rabbit cortical collecting tubule

Summary Forskolin is a unique diterpene that may directly activate the catalytic subunit of adenylate cyclase. We therefore examined the effect of 50 m forskohn on osmotic water permeability in rabbit cortical collecting tubules perfusedin vitro. Forskolin increased net volume flux (J _v , from 0.30 to 1.22 nl/mm/min,P<0.02) in all tubules. The hydro-osmotic effect of forskolin was similar with respect to magnitude and time course to that produced by a maximal dose (250 U/ml) of arginine vasopressin. An additive effect onJ _v andL _p was not observed when maximal concentrations of forskolin and arginine vasopressin were given simultaneously. The compound d(CH₂)₅Tyr(Et) VAVP, which noncompetitively inhibits the vasopressin receptor, significantly reduced collecting tubular hydro-osmotic response to arginine vasopressin. In contrast, the hydro-osmotic response to forskolin was maintained in the presence of d(CH₂)₅ Tyr(Et)VAVP. However, the hydro-osmotic response to forskolin could be inhibited by 1.0 m guanine 5-(,-imido) triphosphate (GppNHp) and by the calmodulin inhibitor N-(6-amenohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). These results demonstrate that forskolin exerts an hydro-osmotic effect in the mammalian nephron which occurs independent of the vasopressin receptor. Guanine nucleotide regulatory proteins may modulate the osmotic water permeability effect of forskolin. Finally, calmodulin is required for full expression of the effect of forskolin to increase osmotic water flux.     

Effects of catecholamines on electrolyte transport in cortical collecting tubule

Summary We examined the direct effects of isoproterenol (ISO) andl-norepinephrine (NE) on electrolyte transport in isolated rabbit cortical collecting tubules (CCT) perfusedin vitro. The addition of either ISO (10^–6 m) or NE (10^–6 m) to the bath decreased transepithelial potential difference (PD), on average by 51 and 25%, respectively. These effects of ISO and NE were abolished by prior addition of the -adrenergic blocker,l-propranolol. ISO (10^–5 m) had no effect from lumen. Also, osmotic water permeability was not influenced by ISO. Ouabain and ISO had additive effects on PD. Elimination of chloride from both perfusate and bath, or addition of acetazolamide, abolished the effect of ISO on PD. Although isotopic sodium flux from lumen to bath was not influenced by ISO, chemical net chloride absorption increased from 1.1±0.4 to 2.7±0.6 peq·cm^–1·sec^–1 (n=8,p<0.005). In conclusion, both ISO and NE are capable of decreasing PD in rabbit CCT perfusedin vitro. This effect is mediated by -adrenergic receptors and is accompanied by the increase in net chloride absorption. Although the mechanism responsible for this decrease in PD with ISO is unclear, active chloride absorption, active hydrogen secretion, or membrane chloride permeability changes may account for the effects of ISO.     

Regulation of cyclic AMP metabolism in rabbit cortical collecting tubule cells by prostaglandins

Prostaglandin E1 (PGE1) at 1 nM inhibits arginine-vasopressin (AVP)-induced water reabsorption in the rabbit cortical collecting tubule (RCCT), while 100 nM PGE1, by itself, stimulates water reabsorption (Grantham, J. J., and Orloff, J. (1968) J. Clin. Invest. 47, 1154-1161). To investigate the basis for these two responses, we measured the effects of prostaglandins on cAMP metabolism in purified RCCT cells. In freshly isolated cells, PGE2, PGE1, and 16,16-dimethyl-PGE2 acting at high concentrations (0.1-10 microM) stimulated cAMP accumulation; however, one PGE2 analog, sulprostone (16-phenoxy-17,18,19,20-tetranor-PGE2 methylsulfonilamide), failed to stimulate cAMP accumulation or to antagonize PGE2-induced cAMP formation; PGD2, PGF2 alpha, and a PGI2 analog, carbacyclin (6-carbaprostaglandin I2), also failed to stimulate cAMP synthesis. These results suggest that there is a PGE-specific stimulatory receptor in RCCT cells which mediates activation of adenylate cyclase. Occupancy of this receptor would be anticipated to cause water reabsorption by the collecting tubule. At lower concentrations (0.1-100 nM) PGE2, PGE1, 16,16-dimethyl-PGE2, and, in addition, sulprostone inhibited AVP-induced cAMP accumulation by fresh RCCT cells in the presence of cAMP phosphodiesterase inhibitors. Pertussis toxin pretreatment of RCCT cells blocked the ability of both PGE2 and sulprostone to inhibit AVP-induced cAMP accumulation. In membranes prepared from RCCT cells, sulprostone prevented stimulation of adenylate cyclase by AVP. These results suggest that E-series prostaglandins (including sulprostone) can act through an inhibitory PGE receptor(s) coupled to the inhibitory guanine nucleotide regulatory protein, Gi, to block AVP-induced cAMP synthesis by RCCT cells. Occupancy of this receptor would be expected to cause inhibition of AVP-induced water reabsorption in the intact tubule. Curiously, after RCCT cells were cultured for 5-7 days, PGE2 no longer inhibited AVP-induced cAMP accumulation, but PGE2 by itself could still stimulate cAMP accumulation. In contrast to PGE2, epinephrine acting via an alpha 2-adrenergic, Gi-linked mechanism did block AVP-induced cAMP formation by cultured RCCT cells. This implies that some component of the inhibitory PGE response other than Gi is lost when RCCT cells are cultured.     

Ouabain-induced cell swelling in rabbit cortical collecting tubule: NaCl transport by principal cells

Summary Ouabain had no effect on the volume of intercalated cells of DOCA-stimulated rabbit cortical collecting tubules, but caused principal cells to swell rapidly at an initial rate of 67% min., Principal cells swelled 133% then activated regulatory volume decrease mechanisms and shrank at an initial rate of –3%/min to a new volume 13% above control. The initial rate of ouabain swelling was completely inhibited by perfusate Na⁺ removal or reduced 95% by luminal addition of 10^–5 m amiloride. Luminal peritubular, or bilateral Cl^– removal each caused cell shrinkages of 10% and reduced the rate of ouabain swelling by 70, 85, and 99%, respectively. The presence of an apical Cl^– transport step in principal cells was confirmed by increasing luminal K⁺ from 5 to 53mm, which caused cell swelling of 22%. This volume increase was completely blocked by luminal Cl^– removal, but was unaffected by peritubular Cl^– substitution. Perfusion of CCT with 0.1mm acetazolomide, 0.1mm DPC or 0.5mm SITS caused principal cell shrinkages of 7–9% and reduced the rate of ouabain swelling by 60, 70, and 40%, respectively. The initial rate of ouabain swelling was inhibited 70% by bilateral CO₂/HCO₃ removal and 50% by whole animal acid loading. Taken together these results demounstrate that ouabain swelling is due to cellular NaCl accumulation and that Na⁺ enters the cell primarily through apical Na⁺ channels. Cellular Cl^– entry occurs at least partially through the apical membrane and may be mediated by a Cl^–/HCO ₃ ^– exchanger. Brief (45–90 sec) exposure of principal cells to ouabain is associated with a rapid inhibition of Na⁺ and/or Cl^– entry steps, whereas long-term (>5min) ouabvain exposure completely blocks one or both of these transport pathways.     

Regulation of principal cell pH by Na/H exchange in rabbit cortical collecting tubule

Summary Changes in intracellular pH (pH _i ) were measured using the pH indicator, BCECF, in principal cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morphological differences in the two major cell types in this nephron segment under transmitted light. The visual identification of the cell types was verified using emission measurements taken from single principal and intercalated cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intracellular acidosis in principal cells. In addition, the exchanger was active under basal conditions and over a wide range of pH _i . Because the exchanger was active under basal conditions we tested the hypothesis that changes in intracellular Na (Na _i ) would alter pH _i in a predictable way. Maneuvers designed to alter Na _i were without significant effects within a 10-min time frame. Specifically, addition of 100 m ouabain to increase Na _i or exposure of the tubules to 10^–5 m amiloride to decrease luminal Na entry and reduce Na _i did not have an effect on pH _i . In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pH _i . These results suggest that Na/H exchange is a major regulator of pH _i in principal cells. However, regulation of Na transport by changes in pH _i in principal cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport.This work was supported by U.S. Public Health Service grants DK27847 to L.G. Palmer and DK11489 to E.E. Windhager.     

Purinergic regulation of basal and arginine vasopressin-stimulated hydraulic conductivity in rabbit cortical collecting tubule

Summary An extracellular adenosine responsive site that stimulates adenylate cyclase activity has been identified in several tissues. There is limited information on the presence and physiologic significance of adenosine receptors in well-defined segments of the mammalian nephron. We therefore examined the effect of adenosine and selected analogues on basal hydraulic conductivity in rabbit cortical collecting tubules (CCT) perfused in vitro. Adenosine and analogues with an intact ribose moiety produced a significant, sustained increase in hydraulic conductivity. No increase in hydraulic conductivity was seen in either time control CCT's or CCT's exposed to an adenosine analogue with an altered ribose moiety. These experiments are compatible with the presence of a functional adenosine receptor which requires an intact ribose moiety and acts to increase hydraulic conductivity in the mammalian CCT.An intracellular adenosine responsive site, termed the P site, which inhibits adenylate cyclase activity, has also been described in several tissues. We therefore examined the effect of aP site agonist on hydraulic conductivity responses to arginine vasopressin, forskolin and cAMP.P site stimulation with 25 dideoxyadenosine inhibited the effect of AVP and of forskolin but not of cAMP to increase hydraulic conductivity. These results are compatible with a functionalP site in the rabbit CCT which acts at the catalytic subunit of adenylate cyclase to inhibit hydraulic conductivity. Together, these results demonstrate purinergic modulation of basal and arginine vasopressin-stimulated water flux in the mammalian collecting tubule.     

Immunocytochemical localization of cathepsin D in lysosomes of cortical collecting tubule cells of the rat kidney

Immunocytochemical localization of cathepsin D in rat renal tubules was investigated by means of indirect immunoenzyme and protein A--gold techniques. By light microscopy, fine granular staining was seen in the mesangial cells of glomeruli. Heavy reaction deposits were present in the cortical tubular segments and some of the medullary collecting tubules. The proximal tubules contained a few positive granules. Other segments were negative for cathepsin D. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were present in cytoplasmic granules and multivesicular bodies of the segment of the cortical collecting tubule. These cytoplasmic granules were presumed to be digestive vacuoles (secondary lysosomes) from their morphological profile. The proximal tubule cells contained the very weakly labeled secondary lysosomes. No specific labeling was noted in other segments of the nephron. Control experiments confirmed the specificity of the immunostaining. Quantitative analysis of the labeling density in each subcellular compartment also confirmed that the main subcellular sites for cathepsin D are the secondary lysosomes and multivesicular bodies. The labeling density in these granules of the lysosomal system varied widely with the individual granules, suggesting that there is a considerable heterogeneity of enzyme content among the granules of the lysosomal system. The prominent presence of cathepsin D in the cortical collecting tubule suggests a certain segment-specific function of this proteinase.     

Calcium dynamics and homeostasis in a mathematical model of the principal cell of the cortical collecting tubule

Calcium (Ca) dynamics are incorporated into a mathematical model of the principal cell in the cortical collecting tubule developed earlier in Strieter et al. (1992a. Am. J Physiol. 263:F1063-1075). The Ca components are modeled after the Othmer-Tang model for IP(3)-sensitive calcium channels (1993, in Experimental and Theoretical Advances in Biological Pattern Formation, 295-319). There are IP(3)-sensitive Ca channels and ATP-driven pumps on the membrane of the endoplasmic reticulum. Calcium enters the cell passively down its electrochemical gradient. A Ca pump and Na/Ca exchange in the basolateral membrane are responsible for the extrusion of cytoplasmic calcium. Na/Ca exchange can also operate in reverse mode to transport Ca into the cell. Regulatory effects of cytoplasmic Ca on the apical Na channels are modeled after experimental data that indicate apical Na permeability varies inversely with cytoplasmic Ca concentration. Numerical results on changes in intracellular Ca caused by decreasing NaCl in the bath and the lumen are similar to those from experiments in Bourdeau and Lau (1990. Am. J Physiol. 258:F1497-1503). This match of simulation and experiment requires the synergistic action of the Na/Ca exchanger and the Ca regulated apical Na permeability. In a homogeneous medium, cytoplasmic Ca becomes oscillatory when extracellular Na is severely decreased, as observed in experiments of cultured principal cells (Koster, H., C. van Os and R. Bindels. 1993. Kidney Int.43:828-836). This essentially pathological situation arises because the hyperpolarization of membrane potential caused by Na-free medium increases Ca influx into the cell, while the Na/Ca exchanger is inactivated by the low extracellular Na and can no longer move Ca out of the cell effectively. The raising of the total amount of intracellular Ca induces oscillatory Ca movement between the cytoplasm and the endoplasmic reticulum. Ca homeostasis is investigated under the condition of severe extracellular Ca variations. As extracellular Ca is decreased, Ca regulation is greatly impaired if Ca does not regulate apical ionic transport. The simulations indicate that the Na/Ca exchanger alone has only limited regulatory capacity. The Ca regulated apical sodium or potassium permeability are essential for regulation of cytoplasmic Ca in the principal cell of the cortical collecting tubule.     

Inhibitio of Na transport by prostacyclin (PGI2) in rabbit cortical collecting tubule

The effects of prostacyclin (PGI₂) on transepithelial potential difference (PD) and sodium transport were examined in rabbit cortical collecting tubules (CCT) perfused in vitro. Addition of PGI₂ (10^?6M) to the bathing medium, which was bubbled with 95% O₂ – 5% CO₂, caused a reversible decrease in PD averaging 49±9.4 (SE)%. Maximal effect was evident between 5–10 min. After addition of PGI₂ and PD returned spontaneously towards control values within 30 min., corresponding to the rapid degradation of PGI₂. In a more alkaline bathing solution achieved by bubbling with 100% O₂, in which the degradation of PGI₂ is known to be delayed markedly, the decrease in PD by PGI₂ was continuous and dose-dependent, with half-maximal and maximal effects achieved at 10^?7 M and 10^?5 M, respectively. Neither 10^?8 M PGI₂ nor vehicle alone exerted significant effects on PD. 6-keto-PGF_1α (10^?5M), believed to be the major metabolite of PGI₂, had no effect on PD. Lumen-to-bath flux of Na decreased with PGI₂ from 9.0 to 5.6 pEq/cm/sec (n=4, p<0.005), although bath-to-lumen flux did not change significantly. In summary, PGI₂ caused a dose-dependent decrease in PD of rabbit CCT and inhibited Na absorption in this segment in vitro. These results suggest that PGI₂ may play an important role in regulating Na transport in CCT.