Sherman D. Levine

Effect of Phloretin on Water and Solute Movement in the Toad Bladder

It is generally believed that urea crosses the cell membrane through aqueous channels, and that its movement across the membrane is accelerated in the direction of net water flow (solvent drag effect). The present report presents evidence for a vasopressin-sensitive pathway for the movement of urea, other amides, and certain non-amides, which is independent of water flow. Phloretin, when present at 10(-4) M concentration in the medium bathing the luminal surface of the toad bladder, strongly inhibits the movement of urea, acetamide, and propionamide across the toad bladder, both in the absence and presence of vasopressin. The vasopressin-stimulated movement of formaldehyde and thiourea is also reduced. Osmotic water flow, on the other hand, is not affected; nor is the movement of ethanol and ethylene glycol, or the net transport of sodium. On the basis of these studies we would conclude that the movement of many, if not all, solutes across the cell membrane is independent of water flow, and that a vasopressin-sensitive carrier may be involved in the transport of certain solutes across the cell membrane.

A Saturable, Vasopressin-Sensitive Carrier for Urea and Acetamide in the Toad Bladder Epithelial Cell

The permeability of the toad bladder to a series of isotopically labeled nonelectrolytes was determined in the presence of 150 mM unlabeled acetamide. Under these conditions, overall bladder function was unimpaired, as shown by a normal response to vasopressin of short-circuit current and permeability coefficient of [(3)H]water,[(14)C]ethanol, and [(14)C]propionamide. The permeability of the bladder to isotopic acetamide and urea, however, was significantly depressed by unlabeled acetamide, in both the absence and presence of vasopressin. These experiments indicate a competition between unlabeled and isotopic species for binding sites, and show the existence of a saturable, vasopressin-sensitive carrier for urea and acetamide in the epithelial cell membrane.

De novo Kaposi's sarcoma in renal transplantation. Case report and brief review.

This report describes a de novo development of Kaposis sarcoma in a Puerto‐Rican man 9 months after a cadaveric renal transplant. Progression of the disease was observed despite local irradiation, while the patient remained immunosuppressed with prednisone and azathioprine. This was accompanied by depressed immunologic tests. Discontinuation of azathioprine and addition of chemotherapy (bleomycin and vincristine), while continuing prednisone to maintain functional survival of renal allograft, has led in this patient to regression of extensive cutaneous and suspected pulmonary Kaposis sarcoma lesions. The possible importance of a depressed immunosurveillance mechanism and activation of latent oncogenic virus by the presence of an allograft in the de novo appearance of Kaposis sarcoma in transplant recipients is briefly discussed.

Membrane pathways for water and solutes in the toad bladder: I. Independent activation of water and urea transport.

SummaryVasopressin activates a number of transport systems in the toad bladder, including the systems for water, urea, sodium, and other small solutes. Evidence from experiments with selective inhibitors indicates that these transport systems are to a large extent functionally independent. In the present study, we show that the transport systems can be separately activated. Low concentrations of vasopressin (1 mU/ml) activate urea transport with virtually no effect on water transport. This selective effect is due in part to the relatively greater inhibitory action of endogenous prostaglandins on water transport. Low concentrations of 8-bromoadenosine cyclic AMP, on the other hand, activate water, but not urea transport. In additional experiments, we found that varying the ratio of exogenous cyclic AMP to theophylline activated water or urea transport selectively. These studies support the concept of independently controlled systems for water and solute transport, and provide a basis for the study of individual luminal membrane pathways for water and solutes in the accompanying paper.

Selective inhibition of osmotic water flow by general anesthetics to toad urinary bladder.

Vasopressin increases the permeability of the total urinary bladder, an analogue of the mammalian renal collecting duct, to water and small solutes, especially the amide urea. We have observed that three general anesthetic agents of clinical importance, the gases methoxyflurane and halothane and the ultrashortacting barbiturate methohexital, reversibly inhibit vasopressin-stimulated water flow, but do not depress permeability to urea, or the the lipophilic solute diphenylhydantoin. In contrast to their effects in vasopressin-treated bladders, the anesthetics do not inhibit cyclic AMP-stimulated water flow, consistent with an effect on vasopressin-responsive adenylate cyclase. The selectivity of the anesthetic-induced depression of water flow suggests that separate adenylate cyclases and cyclic AMP pools may exist for control of water and urea permeabilities in to toad bladder. Furthermore, theophyllines usual stimulatory effect on water flow, but not its effect on urea permeability, was entirely abolished in methoxyflurane-treated bladders, suggesting that separate phosphodiesterases that control water and urea permeabilities are present as well. We conclude that the majority of water and urea transport takes place via separate pathways across the rate-limiting luminal membrane of the bladder cell, and that separate vasopressin-responsive cellular pools of cyclic AMP appear to control permeability to water and to urea.

Barriers to water flow in vasopressin-treated toad urinary bladder.

SummaryUnstirred layers of water complicate the measurement of water permeability across epithelia. In the toad urinary bladder, the hormone vasopressin increases the osmotic water permeability of the granular epithelial cells luminal membrane, and also leads to the appearance of aggregates of particles within this membrane. The aggregates appear to be markers for luminal membrane osmotic water permeability. This report analyzes the relationship between transbladder osmotic water flow and aggregate frequency, and demonstrates that flow across the bladder is significantly attenuated by unstirred layers of water or by structural barriers other than the luminal membrane when the luminal membrane is made permeable by vasopressin. This analysis in addition yields unique values for the permeabilities of both the luminal membrane and the barriers to water flow which lie in series with it.

Membrane pathways for water and solutes in the toad bladder: II. Reflection coefficients of the water and solute channels

SummaryUrea and water transport across the toad bladder can be separately activated by low concentrations of vasopressin or 8 Br-cAMP. Employing this method of selective activation, we have determined the reflection coefficient (σ) of urea and other small molecules under circumstances in which the bladder was transporting urea or water. An osmotic method for the determination of σ was used, in which the ability of a given solute to retard water efflux from the bladder was compared to that of raffinose (σ=1.0) or water (σ=0). When urea transport was activated (low concentration of vasopressin), σ for urea and other solutes was low, (σurea,0.08–0.39;σacetamide, 0.55; σethylene glycol, 0.60). When water transport was activated (0.1mm 8 Br-cAMP) σurea approached 1.0 σurea also approached 1.0 at high vasopressin concentrations. In a separate series of studies, σurea was determined in the presence of 2×10−5m KMnO4 in the luminal bathing medium. Under these conditions, when urea transport is selectively blocked, σurea rose from a value of 0.12 to 0.89. Thus, permanganate appears to “close” the urea transport channel. These findings indicate that the luminal membrane channels for water and solutes differ significantly in their dimensions. The solute channels, limited in number, have relatively large radii. They carry a small fraction (approximately 10%) of total water flow. The water transport channels, on the other hand, have small radii, approximately the size of a water molecule, and exclude solutes as small as urea.

Amide transport channels across toad urinary bladder

SummaryUrea and other small amides cross the toad urinary bladder by a vasopressinsensitive pathway which is independent of somotic water flow. Amide transport has characteristics of facilitated transport: saturation, mutual inhibition between amides, and selective depression by agents such as phloretin. The present studies were designed to distinguish among several types of transport including (1) movement thought a fixed selective membrane channel and (2) movement via a mobile carrier. The former wold be characterized by co-transport (acceleration of labele amide flow in the direction of net flow in the opposite direction). Mucosal to serosal (M→S) and serosal to mucosal (S→M) permeabilities of labeled amides were determined in paired bladers. Unlabeled methylurea, a particularly potent inhibitor of amide movement, was added to either the M or S bath, while osmotic water flow was eliminated by addition of ethylene glycol to the opposite bat. Co-transport of labeled methylurea and, to a lesser degree, acetamide and urea with unlabeled methylurea was observed. Co-transport of the nonamides ethylene glycol and ethanol could not be demonstrated. Methylurea did not alter water permeability or transmembrane electrical resistance. The demonstration of co-transport is consistent with the presence of ADH-sensitive amide-selective channcels rather than a mobile carrier.

Ligandin in Perfusates from Transplanted Kidneys: a Test for Tubular Necrosis

Ligandin, an intracellular organic anion-binding protein, having glutathione-S-transferase activity, was detected in concentrated perfusing solutions from 8 of 13 kidneys preserved for homotransplantation. The presence of ligandin in the perfusate correlated well with oliguric acute renal failure following transplantation. Testing the perfusate for ligandin may be useful in predicting tubular damage in renal transplants.

A Prospective Study of Urinary Ligandin in Patients at Risk of Renal Tubular Injury

The urinary excretion of ligandin, a proximal tubular enzyme and binding protein, was measured by radioimmunoassay in eight normals, six patients receiving radiocontrast media, and six patients in a critical care unit who were considered at high risk for acute renal failure. Ligandinuria was found to occur normally at rates under 5 micrograms/hr. In the patients receiving radiocontrast media, abnormal rates of ligandinuria were found in four patients. In 102 ligandin measurements in the critically ill patients, rates of ligandinuria exceeded normal only once (after contrast media exposure) despite 13 identifiable episodes of potentially nephrotoxic circumstances and two episodes of acute renal failure. Ligandinuria appears more sensitive as a marker for tubular injury from contrast media than from other renal insults.