Secretion of tetraethylammonium by proximal tubules of rabbit kidneys

The secretory transport of tetraethylammonium (TEA) was investigated in perfused and nonperfused isolated S1, S2, and S3 segments of proximal tubules from rabbit kidneys. In the perfused tubules the transepithelial net secretory flux and in nonperfused tubules the TEA cellular uptake were saturable (Km = 67 microM, Vmax = 2,480 fmol X min-1 X mm-1 in perfused S2 segments), energy dependent, and inhibited by mepiperphenidol. The net secretory flux of TEA (J b leads to j TEA) at a bath TEA concentration of 40 microM differed for the three segments and decreased in the order S1 greater than S2 greater than S3. The concentration of TEA in the perfusate leaving the tubule was approximately twice as great and the intracellular TEA concentration approximately 40 times as great as that in the bath. In nonperfused segments (40 microM TEA in the incubation medium) the TEA tissue water-to-medium ratio reached 100. In the three segments the ability to accumulate TEA across the peritubular membrane, thus, was similar, but the transepithelial secretory flux differed significantly. The differences in secretory rate between the three segments presumably result from differences in the luminal membrane permeability.     

Characteristics of phosphate transport in isolated proximal tubule.

The characteristics of inorganic phosphate transport in isolated perfused proximal tubules of the rabbit were examined using radioisotopic techniques. When tubules were perfused with an ultrafiltrate of rabbit serum, the mean lumen-to-bath flux of phosphate in the convoluted segment was 6.60 +/- 1.41 (SE) pmol/mm-min with a simultaneous back-to-lumen flux of 0.45 +/- 0.08. In the straight portion of the proximal tubule, the lumen-to-bath flux was significantly lower (P less than 0.01) at 2.22 +/- 0.48 pmol/min-min with a bath-to-lumen flux of 0.31 +/- 0.05. The lumen-to-bath flux was not affected by increases in the intraluminal phosphate concentration from 2.00 +/- 0.19 to 3.12 +/- 0.34 mM or by the isohydric replacement of bicarbonate in the ambient fluids with chloride. However, phosphate absorption was completely inhibited by ouabain 10(-5) M in the bath. These data indicate that phosphate absorption in these segments occurs by a mechanism other than independent diffusion and is saturated at phosphate concentrations characteristic of normal glomerular filtrate. There is no evidence for significant phosphate transport from bath to lumen.     

The renal accumulation of urate and p-aminohippurate in the rabbit

1. During intravenous infusion of urate and p-aminohippurate (PAH), the concentration of these two substances in cortex tissue of rabbit kidney exceeded the corresponding plasma concentration.2. Following the administration of 2,4-dinitrophenol, fumarate, succinate, probenecid, and salicylate, accumulation of urate in kidney cortex and renal net secretion of urate were abolished. The accumulation and secretion of PAH were only partially inhibited after infusion of these compounds.3. The renal accumulation of urate was reduced in relation to the inhibition of urate excretion during infusion of various amounts of PAH.4. The combined data indicate a correlation between renal accumulation and excretion of urate and PAH. It appears likely that accumulation of urate and PAH in vivo is due to tubular secretion of these two substances, although in the case of urate a small contribution from tubular reabsorption of urate cannot be excluded.     

Secretion of prostaglandin E2 by rabbit proximal tubules.

This study was designed to evaluate prostaglandin secretion from bath to urine in isolated perfused rabbit proximal tubules. Active prostaglandin E2 (PGE2) secretion occurred along the entire length of the proximal tubule, but the rate of net secretion was highest in the S2 segment of the proximal straight tubule. Sixteen percent of the PGE2 secreted in the proximal straight tubule was metabolized to other products. The PGE2 cell-to-bath ratio averaged 40 and the tubule fluid-to-bath ratio averaged 3.4. These findings suggest active transport of PGE2 across the peritubular membrane and passive movement across the luminal membrane. Indomethacin, probenecid, para-aminohippurate, and ouabain partially inhibited PGE2 cell accumulation and net secretion. PGE2 entered the urine of the perfused descending limb of Henle (DLH), but at a rate two orders of magnitude below that observed in the S2 segment of the proximal tubule. No evidence of active PGE2 secretion was observed in the DLH. These results suggest that PGE2 is secreted into the urine at substantial rates by the organic anion transport system of renal proximal tubules.     

The relation between secretion of urate and p-aminohippurate in the rabbit kidney

1. The renal excretion of urate in the rabbit was examined during infusion of other anions which are secreted by the kidney. A reduction of urate excretion occurred after administration of p-aminohippurate (PAH) and Diodrast which was proportional to the secretory rate of these compounds. On the other hand, urate was a less efficient inhibitor of PAH secretion.

2. Stop-flow experiments showed that tubular secretion of urate at maximal values for secretion of PAH and Diodrast was almost completely suppressed.

3. 2,4-Dinitrophenol, fumarate, succinate, salicylate, and probenecid depressed the excretion of urate and PAH, but the tubular secretion of urate was apparently more depressed than that of PAH by these inhibitors.

4. It is concluded that a common transport system for urate and PAH is involved in the tubular secretion of urate and PAH in the rabbit. However, the affinity of urate for the transport system appears to be smaller than that of PAH.

     

Intracellular pH in isolated rat renal papillary thin limbs of Henle's loop

Intracellular pH (pHi) was measured in isolated, nonperfused and perfused rat papillary thin limbs of Henle's loops in N-2-hydroxyethylpiperazine-N'-2-ethansulfonic acid (HEPES)- or HEPES/bicarbonate-buffered medium at pH 7.4 using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Resting pHi was about 6.7 in descending thin limbs (DTL) and about 6.9 in ascending thin limbs (ATL), even with a medium pH of 7.4. These values appeared to reflect the acid pH of the blood in the neighboring vasa recta found in vivo. The resting pHi did not differ whether or not the medium contained bicarbonate although the total buffering capacity of the tubule cells was increased in the presence of bicarbonate. In nonperfused DTL and ATL, pHi was further acidified following an NH4Cl pulse. The rate of recovery of pHi from this level to the resting pHi was reduced by Na+ removal from the bath in both DTL and ATL and by the addition of ethylisopropylamiloride (EIPA) to the bath in the presence of Na+ in DTL. The rate of recovery was not affected by Cl- removal from the bath or K+ (75 mM) or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) addition to the bath in either DTL or ATL. These results suggest that the common, amiloride-sensitive, basolateral Na+/H+ exchanger plays a role in the regulation of pHi in rat papillary DTL but that a different basolateral Na+/H+ exchanger or a luminal Na+/H+ exchanger is important in rat papillary ATL.     

Effects of inhibitors in lumen on PAH and urate transport by isolated renal tubules.

Effects of the presence of unlabeled p-aminohippurate (PAH) or urate, probenecid, and phenol red in the lumen on labeled PAH or urate transport by isolated, perfused snake (Thamnophis spp.) proximal renal tubules were studied. Net secretion of labeled urate and luminal membrane permeability to urate were unaffected by the presence of unlabeled urate (up to 0.1 mM) or probenecid (up to 1.0 mM) in lumen only. The data are compatible with movement of urate from cells to lumen during urate secretion by a simple passive process. Net secretion of labeled PAH was rapidly and reversibly depressed to about 25-35% of control when unlabeled PAH (0.05 mM), phenol red (0.05 mM), or probenecid (0.1 mM) was added to the lumen only. During maximum depression of PAH transport, luminal membrane permeability to PAH was reduced by 60-70%. The data suggest that movement of PAH from cells to lumen down an electrochemical gradient during PAH secretion occurs by a readily inhibited, mediated process.     

Urate transport in the nephron.

This paper summarizes the literature on the sties of urate transport, secretory and reabosrptive, in the nephron. In the animals studied thus far the bulk of urate transport occurs in the proximal tubules. Two patterns of transport have been uncovered. In one, urate is secreted and reabsorbed throughout the convoluted tubule (perhaps the pars recta as well). The direction of net transport depends on the kind of animal studied and in some instances on the experimental conditions. In the second pattern there is a strong secretory process in the pars recta, and net secretion of urate, when it occurs, is attributable to that segment. In some but not all animals it is clear that urate secretion in the proximal tubules occurs by a mechanism separate from that which secretes p-aminohippurate (PAH). One important unresolved question is: How general is the occurrence of separate secretory mechanisms for PAH and urate? Another unresolved question is: What are the magnitudes of the unidirectional fluxes of urate in the segments in which bidirectional transport occurs?     

Effect of ouabain and colloid osmotic pressure on renal tubule cell volume.

Proximal renal tubule cell volume increases in ouabain but cell swelling is limited by the tubule basement membrane (TBM) and the colloid osmotic pressure from the bath protein. We compared the effect of ouabain, external protein concentration, and TBM on cell volume of proximal convoluted (PCT), proximal straight (PST), and cortical collecting tubules (CCT). We blocked active solute transport with ouabain and evaluated cell size by measuring the outer diameter of nonperfused tubules. Proximal tubules in ouabain swelled 35-40% in isoncotic medium and 20-25% further in hyponcotic medium (0.3 g/100 ml albumin), but PCT swelled faster than PST. The CCT swelled minimally in similar mediums, indicating pronounced heterogeneity in the response of cortical nephron segments to ouabain. In the presence of ouabain, all tubules swelled extensively when we removed the TBM with collagenase. In the hyponcotic medium fluid flux across the peritubular membrane was 0.081, 0.049, and 0.030 nl/min per mm tubule length for PCT, PST, and CCT, respectively. The rates of fluid flux in PCT and PST were proportional to estimates of the respective basolateral surface areas. We suggest that differences in swelling rates between proximal segments reflect variations in surface area rather than intrinsic peritubular membrane permeability to solute and water.     

Hormonal regulation of calcium transport in thick ascending limb renal tubules

Net calcium efflux (JCanet) was compared in isolated perfused cortical and medullary segments of the thick ascending limb of Henle of the rabbit kidney. In response to the addition of calcitonin to the bathing medium, cortical segments showed no change in JCanet, whereas in medullary segments JCanet increased significantly. Similar studies substituting 8-bromo-cyclic AMP (8-BrcAMP) in concentrations of 10(-4) M or lower in the bath showed no effect on JCanet in either segment. When the concentration of 8-BrcAMP in the bath was increased to 10(-3) M, JCanet rose significantly in both segments. These results indicate heterogeneity of response to calcitonin in the cortical and medullary segments of the thick ascending limb of Henle, but a similar response of calcium transport to cAMP. Because we have previously shown that parathyroid hormone stimulates net calcium efflux in the cortical but not in the medullary segments of the thick ascending limb of Henle, the present observations suggest that cAMP may be the mediator of the actions of both calcitonin and parathyroid hormone.     

Uric acid transport in brush border membrane vesicles isolated from rabbit kidney.

The transport of uric acid was studied in brush border membrane vesicles isolated from rabbit kidney. The uptake of uric acid by the vesicles was osmotically sensitive and occurred in the absence of significant uric acid degradation. Under the conditions used to evaluate transport, urate binding to the membranes represented only 10--15% of the total uptake. The initial rate of uptake was linear over the concentration range 0.04--8 mM urate. Uptake of urage was Na+ gradient independent. It was dependent on external pH and temperature with Q10 near 3. The urate uptake was inhibited reversibly by p-chloromercuribenzoate. Probenecid, ouabain, cyclic adenosine 3',5'--monophosphate, and its dibutyryl derivative had no appreciable effects. Pyrazinoic acid and pyrazinamide stimulated urate uptake. Experiments performed with osmotically shocked vesicles demonstrated that this stimulatory effect resulted from increased binding of urate to the membranes. These results indicate that in several ways urate transport in vesicles resembles that observed with more physiologically intact preparations.     

Evidence of chloride/bicarbonate exchange mediating bicarbonate efflux from S3 segments of rabbit renal proximal tubule

The mechanism of HCO3- exit from rabbit renal proximal tubule S3 segments was investigated. Isolated tubules were perfused luminally and peritubularly with test solutions and cell pH (pHi), cell Cl- activity ([Cl-]i) and cell Na+ activity ([Na+]i) were measured with ion-selective microelectrodes. From the response of pHi and [Cl-]i to changes in bath Cl- or HCO3- concentrations a Cl-/HCO3- exchanger was identified in the basolateral cell membrane. It was reversibly inhibited by millimolar concentrations of the disulfonic stilbene SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid). Cell potential measurements and preliminary determinations of initial ion flux rates suggested a stoichiometry of Cl- to HCO3- flux near 1.0. The transport rate appeared to saturate already at low bath Cl- concentrations (approximately 30 mmol/l), but it was independent of bath pH in the range of 7.4-6.4. Cl-/HCO3- exchange was not directly coupled to Na+ flux although in approximately half of the experiments long-term incubation in Na(+)-free solutions indirectly inhibited the exchanger. Sudden application of SITS under control conditions revealed that the exchanger normally facilitates the exit of HCO3- from cell to interstitium at the expense of Cl- uptake into the cell. How Cl- ions recirculate towards the peritubular surface is presently not known.     

Quantitative determination of macromolecular transport rate across intestinal Peyer's patches

We used horseradish peroxidase (HRP) (mol wt, 40,000) to compare in vitro, in Ussing chambers, the rates of protein transport across segments of piglet jejunum with and without Peyer's patches. The mean HRP transport rate across intestinal segments with a patch, 25.2 +/- 4.2 SE ng . min-1 . cm-2 (22 animals), was increased threefold (P less than 0.0005) compared with control (no patch) tissue, 7.9 +/- 1.0 ng . min-1 . cm-2 (n = 29). Neither rate showed saturation with increasing concentrations of HRP; both were inhibited 75-95% by a temperature drop from 37 to 15 degrees C. Transport across patch-containing tissue was inhibited 48 +/- 6% (n = 5, P less than 0.0025) by 1 mM NaF, but NaF had no consistent effect on the transport across tissue without Peyer's patches. We conclude that HRP transport is increased across Peyer's patches. This transport is dependent on metabolism and does not involve specific receptors. These findings support the concept that the Peyer's patch serves an antigen-sampling function in the gut.     

Effects of intraluminal D-glucose and probenecid on urate absorption in the rat proximal tubule.

The in vivo microperfusion technique was employed to examine urate absorption in the proximal convoluted tubule of the rat kidney using [2-14C]urate as the marker for fractional urate absorption. With NaCl as the perfusion solution, water absorption averaged 2.53 +/- 0.16 nl.min-1.mm tubule-1, and the fractional absorption of [2-14C]urate averages 11.6 +/- 1.0%/mm tubule. The addition of D-glucose (50 mg/100 ml) enhanced water absorption to 3.62 +/- 0.19 nl.min-1.mm tubule-1, but inhibited fractional urate absorption to 6.6 +/- 1.2%/mm tubule. Phloridzin (4.4 mg/100 ml), 2-deoxy-D-glucose (45.6 mg/100 ml), and 3-O-methyl-D-glucose (53.9 mg/100 ml) also inhibited the absorption of [2-14C]urate to the same degree as did D-glucose despite differing effects on water absorption. The addition of probenecid (2.8 mg/100 ml) to the NaCl perfusion solution had no effect on water absorption but inhibited [2-14C]urate absorption to 6.4 +/- 0.6%/mm tubule. The addition of both probenecid and phloridzin further reduced [2-14C-A1urate absorption to 3.8 +/- 0.7%/mm tubule. Probenecid alone had no effect on glucose transport. These studies suggest that the presence of either certain hexose sugars, phloridzin, or probenecid in the lumen of the proximal convoluted tubule inhibits the tubular absorption of urate.     

Loop of henle functional differentiation in vitro perfusion of the isolated thick ascending segment

Single segments of the loop of Henle, the cortical thick ascending limb (CTAL), were dissected from fresh renal slices (rabbit) and individually perfused in vitro. Direct analysis of transepithelial NaCl and water movement was carried out at two functional states of epithelial transport. I. Differentiating thick ascending loop of Henle (d-CTAL, day 2–10 postnatal,n _t=31); II. Mature thick ascending loop of Henle (m-CTAL, day 25–30,n _t=23). Nephron segments were dissected without collagenase. Perfusion rates were similar per unit surface area in both functional states. Perfusate was ultrafiltrate of rabbit serum or artificial solution identical to bath medium, except when osmotic hydraulic permeability or steady-state ion gradients were analyzed. Results: Osmotic hydraulic permeability (bath medium hyperosmotic) was 4.72 cm³ cm^–2 min^–1 kPa^–1 10^–8 [or 4.78±(S.D.) 1.15 cm³ cm^–2 min^–1 atm^–1 10^–6] in the d-CTAL, similar to the m-CTAL (5.19±1.21). Solute transport (ratio of collected to perfused osmolarity) was hyperosmotic in the m-CTAL (0.62±0.13), but only 0.91±0.07 in the d-CTAL. Net sodium transport across the d-CTAL was 2.13±0.37 mol cm^–1s^–110^–12, and 11.6±1.3 in the m-CTAL. Steady-state sodium concentration in the single loop lumen was 65±8 mmol·l^–1 in the m-CTAL, and 97±2.7 in the d-CTAL (bath medium: 148 mmol·l^–1). Conclusions: Differentiation of the thick ascending loop of Henle is characterized by constant osmotic hydraulic permeability. Sodium absorption, transepithelial steady-state sodium concentration gradient, and transtubular osmotic disequilibrium suggest that the electrolyte transport capacity of the single cell of the diluting segment is the major determinant of renal medullary countercurrent differentiation.     

Serum from patients with pernicious anaemia blocks gastrin stimulation of acid secretion by parietal cells.

We examined 51 sera from patients with pernicious anaemia for their capacity to block maximal gastrin stimulation of acid secretion by isolated rodent gastric parietal cells. 14C-aminopyrine accumulation was used as the index of acid secretion in vitro. Sera from patients with pernicious anaemia gave significantly (P less than 0.005) more block of maximal gastrin stimulation of acid secretion (61.7 +/- 37.8%) than sera from 10 patients with systemic lupus erythematosus (19.6 +/- 17.7%), 10 with scleroderma (34.2 +/- 22.3%), five with rheumatoid arthritis (22.4 +/- 15.6%) or 30 from healthy persons (27.4 +/- 12.8%). Maximal histamine stimulation of acid secretion was not inhibited. The blocking factor was present in serum IgG fractions, and serum and IgG fractions gave parallel dose-response and dilution curves. The serum block was abolished by absorption with gastric mucosal cells and correlated with the presence of parietal cell surface autoantibody. We conclude that serum immunoglobulin in pernicious anaemia can block gastrin stimulation of acid secretion and suggest that this block may be mediated by competition with gastrin for surface receptors on parietal cells.     

A cDNA clone encoding part of the major 25000-dalton surface membrane antigen of adultSchistosoma mansoni

Immunoscreening of an adultSchistosoma mansoni cDNA expression library, using antibodies raised against purified adult worm tegumental surface membranes, identified a recombinant clone containing a 141-bp insert. Antibodies raised against the recombinant antigen bound specifically to the tegument of adult worms and immunoprecipitated the major 25000-dalton surface membrane antigen as well as a 22000-dalton nascent polypeptide generated by cell-free translation of adultS. mansoni mRNA. The mature 25000-dalton antigen was found to be precipitated by antibodies from infected mice, rats and humans.Abbreviations IPTG isopropylthio-beta-galactoside - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulphonyl fluoride - FTTC fluorescein isothiocyanate - VMS serum from mice vaccinated with highly irradiated cercariae - CMS serum from chronically infected mice - Ram rabbit anti-membrane serum - IRS infected rat serum     

Lack of effect of low [Ca2+], La3+, and pyrazinoate on urate transport by isolated,perfused snake renal tubules

Effects of low medium calcium concentration, of lanthanum, and of pyrazinoate on urate transport by isolated, perfused snake (Thamnophis spp.) distal-proximal renal tubules were studied. Removal of calcium from perfusate with 0.18 mmol/l calcium in bathing medium had no effect on net urate secretion (J _urate ^net ) or on net fluid absorption (J _v). In the presence of calcium (1.8 mmol/l), lanthanum (2.0 mmol/l) in perfusate alone, in bathing medium alone, or in both perfusate and bathing medium had no effect onJ _urate ^net . These findings suggest that urate transport, in contrast to para-aminohippurate (PAH) transport, is not sensitive to calcium entry into the cells and support the concept that urate and PAH are transported by separate mechanisms in these renal tubules. Pyrazinoate (1.0 mmol/l) in the bathing medium had no effect onJ _urate ^net orJ _v. These findings do not support the idea of a primary urate secretory process uniquely sensitive to pyrazinoate among the vertebrates.     

Fluid secretion in the nephron: Relation to renal failure.

It had been generally accepted that glomerular filtration and tubular reabsorption were the basic modes of fluid transport in mammalian nephrons. Recently, evidence was obtained to indicate that net fluid secretion may occur in mammalian nephrons as well. In the pars recta portion of proximal tubules of rabbit kidney net fluid secretion was observed in vitro in response to PAH and other aryl acids in the peritubular bathing medium. Net fluid secretion appeared to be coupled to the transcellular transport of aryl acid from bath to lumen. Serum from uremic subjects stimulated net fluid secretion in the pars recta in a manner similar to PAH. The accumulation of high levels of endogenous aryl acids may contribute to the general organ dysfunction that is a part of the uremic syndrome of advanced renal insufficiency. Futhermore, there is evidence to suggest that the fluid-secretion phenomenon in association with aryl acids may significantly affect renal excretion and morphology in slow-flow states, in patients with cystic kidney disease, and in obstructive nephropathy.     

Staphylococcus aureus and derived exotoxins induce nuclear factor kappa B-like activity in murine bone marrow macrophages.

Heat-killed gram-positive Staphylococcus aureus as well as S. aureus-derived exotoxins B and toxic shock syndrome toxin 1 can induce nuclear factor kappa B (NF-kappa B)-like activity in murine bone marrow macrophages. The induction of NF-kappa B-like activity in murine macrophages by S. aureus was as effective as induction by tumor necrosis factor alpha (TNF-alpha) or lipopolysaccharides (LPS) and was observed in macrophages derived from LPS-sensitive and LPS-resistant mice. Stimulation of macrophages with S. aureus but not with the exotoxins resulted in the accumulation of TNF-alpha in the culture medium. The induction of NF-kappa B-like activity by S. aureus, however, clearly preceded TNF-alpha secretion and was not inhibited by a neutralizing serum against TNF-alpha. In addition, pretreatment of macrophages with the protein synthesis inhibitor cycloheximide or dexamethasone, which prevented the secretion of TNF-alpha from macrophages, did not interfere with the induction of NF-kappa B-like activity by S. aureus. This findings reveal the existence of bacterial components other than LPS which can induce NF-kappa B-like activity in susceptible cells.