bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of epithelial cells from toad bladder
Authors: Rodriguez H. J. , Scholer D. W. , Purkerson M. L. , Klahr S.
Published: December 2017

The epithelial cells of the toad bladder have been isolated by brief exposure to ethylenediaminetetraacetic acid followed by treatment with collagenase, DNAse, and the application of shearing forces. This approach eliminates the need for scraping of the mucosal surface and reduces mechanical damage during harvesting of the epithelium. The method yields intact, isolated epithelial cells and few clumps. The three major types of epithelial cells described in the intact toad bladder were present in the final preparation. The cellular contents of nucleic acids and proteins (in pg/cell) were: DNA 22.5 +/- 1.1; RNA, 12.9 +/- 0.6; and protein, 192 +/- 9. The isolated cells possess rates of oxygen consumption and amino acid incorporation higher than those of epithelial sheets obtained by collagenase treatment and scraping of the intact bladder. However, the production of cyclic nucleotides in response to stimulation by vasopressin and carbachol is comparable in both preparations.
Effect of DIDS on renal tubular transport
Authors: Koschier F. J. , Stokols M. F. , Goldinger J. M. , Acara M. , Hong S. K.
Published: December 2017

Two stilbene derivates that had been used to covalently label the Cl- carrier in the erythrocyte were investigated for reactivity with the renal organic anion system. These compounds, 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and (4,4'-diisothiocyano)-dihydrostilbene-2,2'-disulfonate (H2DIDS), were found to be potent inhibitors (Ki congruent to 35 microM) of p-aminohippurate (PAH) transport in the renal cortical slice without affecting tetraethylammonium (TEA) transport or tissue viability. During renal clearance studies performed in the perfused kidney, DIDS decreased the PAH/inulin clearance ratio to congruent to 1. When the possible renal transport of [3H]H2DIDS was investigated, the renal slice transport or binding of [3H]H2DIDS reached a slice-to-medium ratio of congruent to 6, and this accumulation was decreased by probenecid. In perfused kidney experiments, the [3H]H2DIDS/inulin clearance ratio was congruent to 0.8. Since probenecid reduced this clearance ratio to congruent to 0.5, there was the possibility that H2DIDS underwent tubular secretion. In conclusion, DIDS and H2DIDS interacted with the renal organic anion transport system, which indicated that these compounds were possible probes for this transport system.
Renal perfusion and intratubular pressure during ureteral occlusion in the rat
Authors: Gaudio K. M. , Siegel N. J. , Hayslett J. P. , Kashgarian M.
Published: December 2017

To define the interrelationship between changes in total renal blood flow (TRBF) and proximal tubular hydrostatic pressure (PTP), rats were studied 2, 3, 4, 6, 12, 18, and 24 h after bilateral ureteral occlusion (BUO). In control animals, a peak rise in both TRBF (3.52 +/- 0.26 ml . min-1 . 100 g body wt-1 . kidney-1) and PTP (28.6 +/- 1.3 mmHg) occurred 3 h after BUO. At each subsequent time interval, both TRBF and PTP fell, so that by 24 h after BUO, TRBF was 1.53 +/- 0.34 and PTP was 16.5 +/- 0.6, while in sham-operated rats the values were 2.52 +/- 0.22 ml . min-1 . g body wt-1 . kidney-1 and 12.8 +/- 0.2 mmHg, respectively. In animals treated with indomethacin (10 mg/kg) at the time of BUO, the alterations in both TRBF and PTP were completely ameliorated. At 3 h, TRBF was 1.8 +/- 0.32 ml . min-1 . 100 g body wt-1 . kidney-1 and PTP was 15.3 +/- 1.0 mmHg. TRBF and PTP remained essentially unchanged at each subsequent time period. These data indicate that 1) alterations in TRBF and PTP follow a similar and parallel pattern during BUO; 2) the pattern of changes in TRBF and PTP can be eliminated by treatment with indomethacin; and 3) renal hemodynamics appear to be the dominant factor in producing these changes during the first 12 h after BUO, whereas a sustained increase in tubular hydrostatic pressure may play a primary role in decreasing TRBF 12-24 h after ureteral occlusion.
Harmaline inhibition of Na-dependent transport in renal microvillus membrane vesicles
Authors: Aronson P. S. , Bounds S. E.
Published: December 2017

The effects of the hallucinogen harmaline on D-glucose, L-alanine, and Na+ transport were studied in microvillus membrane vesicles isolated from the rabbit renal cortex. Harmaline had no effect on glucose transport in the absence of Na+, but reversibly inhibited sugar flux in the presence of NaCl. Inhibition of Na+-dependent glucose transport was inversely related to the Na+ concentrations. The hallucinogen competitively inhibited the Na+ activation of phlorizin binding to the membranes but did not inhibit phlorizin binding in the absence of Na+. Harmaline inhibited Na+-dependent alanine transport and, at higher drug concentrations, the amino acid flux in the absence of NaCl. Harmaline competitively inhibited the rate of Na+ uptake which, in the absence of glucose and alanine, is known to occur via Na+-H+ exchange. The hallucinogen trans-inhibited the efflux of glucoe and Na+ from membrane vesicles preloaded with the solutes. These findings suggest that harmaline is a direct inhibitor of microvillus membrane transport processes and acts as a competitive inhibitor of Na+ transport sites. Harmaline may therefore be a useful investigative tool for studying mechanisms of Na+-coupled transport in the luminal membrane of the proximal tubular cell.
Effect of diphosphonate treatment on phosphate transport by renal brush border vesicles
Authors: Stoll R. , Fleisch H. , Bonjour J. P.
Published: December 2017

Treatment with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) given in doses of 10 mg P/kg s.c. for 7 days inhibits bone mineral retention and decreases the tubular capacity to reabsorb inorganic phosphate (Pi) in thyroparathyroidectomized (TPTX) rats. In the present work we show that pretreatment with EHDP depressed Na+-dependent Pi uptake by brush border membrane vesicles (BBMV) isolated from renal cortex of TPTX rats. The effect of EHDP was observed after feeding both high (1.2%) and low (0.2%) phosphorus diets. The EHDP-induced changes observed in vitro at the brush border level parallel the variations in the overall tubular Pi transport capacity as assessed by clearance techniques in conscious rats. Na+-dependent glucose uptake by BBMV, as well as alkaline phosphatase activity in cortical homogenates and in the BBMV were not affected by EHDP treatment. Accordingly, EHDP, given in doses that block bone mineral retention, appears to specifically affect the Na+-dependent transport of Pi across the luminal membrane of proximal tubules of renal cortex.
Effect of PTH on calcium transport across the cortical thick ascending limb of Henle's loop
Authors: Bourdeau J. E. , Burg M. B.
Published: December 2017

Cortical thick ascending limbs of Henle's loop were dissected from rabbit kidneys and perfused in vitro. Unidirectional transepithelial calcium fluxes from lumen-to-bath and bath-to-lumen were measured with 45Ca. The tubules were bathed in 150 mM sodium and perfused with 60 mM sodium to simulate conditions in the cortical thick ascending limb in vivo. During the 20-30 min preceding the addition of parathyroid hormone (PTH), net calcium absorption decreased from 0.207 to 0.084 pmol x s-1 x cm-1. After addition of synthetic bovine PTH (60-64 nM) to the bath, there was an immediate increase in calcium absorption, and by 20 min the net flux increased to 0.415 pmol x s-1 x cm-1. The increase in calcium absorption was due to an increase in the lumen-to-bath flux. Dibutyryl-cAMP or 8-BrcAMP mimicked PTH; adrenocorticotropic hormone had no effect on the calcium flux. Transepithelial voltage was unchanged after addition of PTH. We conclude that PTH increases calcium absorption across the cortical thick ascending limb, probably by stimulation of adenylate cyclase.
Localization of the nephron sites responsible for mineralocorticoid escape in rats
Authors: Kohan D. E. , Knox F. G.
Published: December 2017

Fractional delivery of sodium (FDNA) from the superficial late distal tubule is increased in deoxycorticosterone acetate-(DOCA) treated rats, which may compensate for a presumed mineralocorticoid-stimulated sodium reabsorption in the cortical collecting tubule. In the present study, late proximal (LPT) and early distal tubules (EDT) were micropunctured during volume expansion in control and DOCA-treated rats. Whereas FDNa to the LPT was similar in the two groups, FDNa to the EDT was greater in DOCA-treated than in control rats. Additionally, LPT and deep loop of Henle (LH) were micropunctured in volume-expanded control and DOCA-treated rats. FDNa to the bend of the LH was increased in DOCA-treated rats compared to controls. These findings indicate that in DOCA escape sodium reabsorption is diminished in the pars recta and/or loop of Henle of superficial nephrons and there is increased delivery to the bend of the loop of deep nephrons. We conclude that mineralocorticoid escape is most likely due to increased sodium delivery to the cortical collecting tubule from both deep and superficial nephrons.
Cholinergic modulation of water transport in the toad bladder
Authors: Arruda J. A. , Sabatini S.
Published: December 2017

The effect of carbachol on water transport by the toad bladder was studied. Carbachol caused a small increase in base-line water flow and inhibited, partially, vasopressin- (AVP) or cyclic AMP-stimulated water flow. The effect of carbachol on base-line or AVP-stimulated water flow was totally prevented by atropine, indicating that the effect of cabachol on water transport is mediated through a muscarinic receptor. Carbachol caused a significant increase in 45Ca uptake by toad bladder; this increase in calcium uptake could be prevented by atropine, pentobarbital, or lanthanum. The effect of carbachol on base-line and AVP-stimulated water flow was also prevented by pentobarbital or lanthanum, suggesting that the effect of carbachol is mediated, at least in part, by an increase in calcium uptake. The ionophore A-23187, an agent that increased 45Ca uptake, also enhanced base-line water flow and inhibited AVP-stimulated water flow. The effects of carbachol and the ionophore A-23187 on base-line water flow, AVP-stimulated water flow, and on calcium uptake were not additive, suggesting that both agents alter water transport by a similar mechanism. These data demonstrate that carbachol stimulates base-line water transport and inhibits AVP-stimulated water transport. They suggest that the alteration in water transport induced by carbachol is related to an increase in calcium uptake.
Vasopressin-stimulated water flow is decreased by thromboxane synthesis inhibition or antagonism
Authors: Burch R. M. , Knapp D. R. , Halushka P. V.
Published: December 2017

The time course of vasopressin stimulation of water flow and immunoreactive thromboxane B2 (iTXB2) and prostaglandin E (iPGE) biosynthesis was studied in the isolated toad urinary bladder. Vasopressin (25 mU/ml) significantly stimulated iTXB2 synthesis within 8 min, synthesis reaching a maximum rate by 17 min. iPGE synthesis was significantly stimulated within 8 min, remaining unchanged for 24 min. Maximum vasopressin-stimulated water flow was reached between 16 and 24 min. 7-(1-Imidazolyl)-heptanoic acid (7IHA), a thromboxane synthetase inhibitor, inhibited both vasopressin-stimulated water flow and iTXB2 synthesis in a dose-dependent fashion, but did not affect iPGE synthesis. Vasopressin-stimulated water flow and iTXB2 synthesis were significantly correlated (r = 0.75, n = 24, P less than 0.001). 13-Azaprostanoic acid (13APA), a thromboxane antagonist, inhibited vasopressin-stimulated water flow in a dose-dependent fashion. Inhibition of arachidonic acid metabolism abolished the effects of 7IHA and 13APA on vasopressin-stimulated water flow. 7IHA and 13APA had no effect on cAMP-stimulated water flow. These results confirm that vasopressin stimulates TXA2 and PGE synthesis and support the hypothesis that TXA2 is a positive modulator of vasopressin-stimulated water flow in the toad urinary bladder.
Hypothalamic stimulation and electrolyte excretion: a micropuncture study
Authors: Silva-Netto C. R. , de Mello Aires M. , Malnic G.
Published: December 2017

The anterior part of the lateral hypothalamus was stimulated by injection of carbachol through a stereotaxically implanted cannula in the rat. Re-collection micropuncture experiments showed that this procedure, which leads to diuresis and natriuresis with only transient changes in glomerular filtration rate and renal plasma flow, reduced the TF/P inulin ratio along proximal and distal tubules without significant alteration of single nephron glomerular filtration rate in most experimental groups. Fractional proximal sodium reabsorption was significantly reduced from 0.54 +/- 0.02 to 0.34 +/- 0.05. Treatment with DOCA, vasopressin, and oxytocin caused natriuresis, but additional hypothalamic stimulation (HS) led to further reduction in TF/P inulin ratio and proximal fractional sodium reabsorption from 0.42 +/- 0.03 to 0.33 +/- 0.03. Fluid transport across proximal and distal epithelium was also studied by the split-droplet method. It was markedly reduced in both segments after HS. During hormone treatment only distal segments showed reduction of fluid transport by HS. These experiments indicate that HS caused inhibition of fluid transport in the proximal and distal tubule. This effect was only partly due to the liberation of neurohypophysial hormones, since during their administration an additional effect of HS was still observed.
Effect of hydrazine on transport in toad urinary bladder
Authors: Levine Sherman D. , Kachadorian William A. , Verna Nicholas C. , Schlondorff Detlef
Published: December 2017

Vasopressin stimulates osmotic water flow and urea permeability in the toad urinary bladder via separate cAMP-responsive mechanisms. Hydrazine (10–20 mM), added to the bladder's serosal bath, reversibly enhanced the effect of both low and saturating levels of vasopressin on osmotic water flow, without increasing urea permeability. A small increase in basal water flow was also observed. Cyclic AMP-stimulated water flow was not altered by hydrazine, but hydrazine enhanced the effect of both 8-bromo-cyclic AMP and methylisobutylxanthine. Hydrazine increased luminal membrane aggregate frequency in vasopressin-treated tissues examined by freeze-fracture electron microscopy. Hydrazine increased both basal and vasopressin-stimulated adenylate cyclase activity. We could measure no effect of hydrazine on cAMP content; however hydrazine did increase the protein kinase activity ratio (-cAMP/+cAMP) in vasopressin-treated tissues, suggesting that the kinase activity ratio is more sensitive than cAMP content as an index of cAMP-related function in the bladder. Further strengthening the relationship between kinase activation and water flow, we found that methohexital, an inhibitor of vasopressin-stimulated water flow and adenylate cyclase, also decreased the kinase activity ratio in the presence of vasopressin. These studies link closely the roles of cAMP-dependent kinase and luminal membrane aggregates to the specific mediation of vasopressin-stimulated water flow in the bladder.

antidiuretic hormone; vasopressin; Bufo marinus; methohexital; barbiturates; freeze-fracture electron microscopy; cAMP; protein kinase

Submitted on July 30, 1979

Accepted on April 10, 1980
Effect of vasopressin on renal kallikrein excretion
Authors: Fejes-TÓth Géza , Zahajszky Tibor , Filep János
Published: December 2017

In an attempt to investigate a possible interaction between vasopressin and the renal kallikrein-kinin system, renal function and urinary kallikrein excretion were monitored in trained conscious dogs and in anesthetized rats in water diuresis and in vasopressin-induced antidiuresis. Vasopressin elevated urinary kallikrein excretion in a dose-dependent way in both species, with concomitant increases in urinary osmolality and electrolyte excretion. A significant increase in kallikrein excretion was observed with a dose of vasopressin as low as 2 mU·kg-1·h-1 in the dog and 3 mU·kg-1·h-1 in the rat without a change in renal hemodynamics. In the rat vasopressin-induced changes in kallikrein excretion were positively correlated with changes in sodium and potassium excretion and negatively correlated with changes in free water clearance. It is concluded that vasopressin over its normal physiological range of concentration stimulates renal kallikrein secretion.

Note:

With the Technical Assistance of Klára Peres and Edit Spitzár

water diuresis; antidiuresis; natriuresis; kinins; dog; rat

Submitted on October 8, 1979

Accepted on May 21, 1980
Failure of tubule fluid osmolarity to affect feedback regulation of glomerular filtration
Authors: Briggs J. P. , Schnermann J. , Wright F. S.
Published: December 2017

Experiments were performed in Sprague-Dawley rats in order to distinguish between sodium chloride and total solute concentration as possible luminal signals capable of eliciting tubuloglomerular feedback responses. Early proximal flow rate (VEP), an index of nephron filtration rate, was measured without perfusion of the loop of Henle and during retrograde perfusion with solutions containing 20, 35, 60 to 100 mM NaCl and varying amounts of either urea or mannitol to achieve total solute concentrations of 130, 280, or 400 mosM. Perfusion flow rate was kept constant at 20 nl/min. Perfusion with a solution containing 20 mM NaCl and made hypo-, iso-, or hypertonic with urea or mannitol caused little or no change in VEP. Perfusion with a 35 mM NaCl solution made hypo-, iso-, or hypertonic with mannitol resulted in a fall of VEP of 6-7 nl/min. When NaCl concentration was 60 mM, VEP fell by 10-14 nl/min with solutions made hypo-, iso-, or hypertonic with urea or mannitol. With 100 mM NaCl solutions made hypo-, iso-, or hypertonic with mannitol, VEP fell approximately 12 nl/min. These results indicate that feedback responses are determined by the NaCl concentration of the perfusate and that this NaCl dependency is not modified by varying perfusate osmolarity between 130 and 400 mosM with urea or mannitol as osmotic agents.
Renal medullary concentrating process: an integrative hypothesis
Authors: Bonventre J. V. , Lechene C.
Published: December 2017

The inability to demonstrate adequate active transport in the ascending thin limb of the mammalian kidney has led to the development of models of concentration with only passive transport in the inner medullary loop of Henle. These models depend on very limited solute entry into the descending thin limb, an assumption counter to much of the experimental in vivo data. In addition, these models have not incorporated the vascular-tubular relationships in the renal medulla. We hypothesize that fluid enters the inner medulla in the descending thin limb slightly hyperosmotic to the fluid leaving the inner medulla in the interstitial-vasa recta compartment. We illustrate the hypothesis with a steady-state model of the medullary concentration process with no active transport in the inner medullary loop of Henle and with tubular permeability properties and solute concentrations consistent with experimental data. A difference of 33 mosmol/kg between descending thin limb and fluid leaving the inner medulla in the interstitial-vasa recta compartment results in an increase in formative urine osmolality from 530 mosmol/kg in the collecting duct at the outer-inner medullary boundary to 2,200 mosmol/kg in the ureteral urine. The hypothesis incorporates anatomical and physiological properties of the vascular and tubular structures in the outer medulla.
Effect of urea concentration of pelvic fluid on renal concentrating ability
Authors: Bonventre J. V. , Roman R. J. , Lechene C.
Published: December 2017

The role of the pelvic urine in the renal concentration process was examined in anesthetized antidiuretic rats. The left ureter was cannulated with perfusion and collection catheters so that the composition of the fluid bathing the papilla could be altered by superperfusing solution of known composition without opening the renal pelvis, exposing the papilla, or altering intrapelvic pressure. The effect on urinary osmotic pressure (Uosmol) of papillary superperfusion with solutions of varying urea concentrations and total osmolality was examined. Papillary superperfusion with solutions containing no urea or less urea than preperfusion pelvic urine caused marked reductions in Uosmol regardless of the osmotic pressure of the solution. Superperfusion solutions with urea concentrations similar to that of preperfusion pelvic urine but lower in osmotic pressure than preperfusion urine produced no change in Uosmol. Superperfusion solutions with urea concentrations higher than that of preperfusion urine and with osmotic pressure equal to that of the preperfusion urine increased Uosmol. Finally, superperfusion of the papilla with urea solutions of greater than 2,370 mosmol/kg H2O and hypertonic to preperfusion urine decreased Uosmol. We conclude that the urea concentration of the pelvic urine plays an essential role in the urinary concentration mechanism.
Effect of pH on metabolism of alpha-ketoglutarate by renal cortical mitochondria
Authors: Tannen R. L. , Kunin A. S.
Published: December 2017

The effect of acid-base perturbations on mitochondrial alpha-ketoglutarate (alpha-KG) metabolism was quantitated by measuring the nitrogen and carbon metabolites of glutamine. alpha-KG metabolized was calculated as the difference between alpha-KG production from glutamine (glutamate deamination plus transamination) and alpha-KG accumulation in the medium. Under all experimental conditions accumulation in the medium of malate plus aspartate was altered similarly to the calculated change in alpha-KG metabolism. Mitochondria from rats with chronic acidosis were compared to pair-fed controls. Chronic acidosis resulted in increased alpha-KG production and its intramitochondrial concentration; the rate of conversion of alpha-KG to succinate was unchanged. When mitochondria from normal animals were incubated at pH 7.0, 7.4, and 7.7, the amount of alpha-KG metabolized was altered, but the magnitude and direction of the response was dependent on the concentration of glutamine (0.5, 1.0, or 5.0 mM). A low pH depressed production but stimulated the subsequent metabolism of alpha-KG, whereas an alkaline pH acted in the opposite fashion. The overall response at a given glutamine concentration depended on which effect predominated. Accordingly, chronic acidosis does not induce adaptive changes, but pH, per se, directly alters intramitochondrial alpha-KG metabolism.
Renal tubule transport of organic cations
Authors: Rennick B. R.
Published: December 2017

Transcellular active tubular transport of organic cations occurs in the proximal renal tubule in the direction of excretion. These organic cations may be primary, secondary, tertiary, or quaternary amines. Endogenous neurohumors such as choline and catecholamines and drugs such as morphine and tetraethylammonium are representative transportable organic cations. Competitive inhibition for transport is found among organic cations. Organic anions, however, do not compete for this transport. Organic cations used as drugs may interact with the transport of endogenous organic cations to alter the excretion patterns of both. Bidirectional active tubule transport can be demonstrated for choline and is accompanied by simultaneous renal metabolism of choline. Studies using vesicles prepared from luminal and antiluminal membranes of renal cortex suggest that organic cation transport occurs in both vesicle preparations. However, only the luminal vesicles showed the characteristics of carrier-mediated uphill transport.
Extrarenal potassium homeostasis
Authors: Bia M. J. , DeFronzo R. A.
Published: December 2017

Normal potassium homeostasis is regulated by both renal and extrarenal mechanisms. Although chronic potassium balance is primarily regulated by the kidneys, acute potassium tolerance is largely determined by extrarenal tissues. During the first 4-6 h following an acute potassium load, only about 50% of the potassium is excreted by the kidneys. Of the remaining 50% that is retained, over 80% is translocated into cells, and this provides the primary defense against hyperkalemia. Potassium uptake by both liver and muscle and intestinal secretion of potassium are the most important mechanisms of extrarenal potassium disposal. Several hormones, including insulin and epinephrine, have been shown to play an important role in the maintenance of normal extrarenal potassium metabolism. These hormones function by enhancing potassium uptake by liver and muscle. There is also evidence that aldosterone is necessary in the maintenance of normal extrarenal potassium tolerance. Although the major extrarenal site of action of aldosterone has not yet been clearly defined, gastrointestinal potassium secretion and muscle transport of potassium are both affected by the hormone. Evidence exists that glucocorticoids may also have an effect on extrarenal potassium homeostasis. In addition to this hormonal regulation, cellular shifts of potassium are influenced by changes in acid-base balance. Extrarenal potassium tolerance is impaired in chronic renal insufficiency. These uremia-related changes are discussed in the context of our present understanding of normal extrarenal potassium metabolism.
ADH-sensitive cAMP system in papillary collecting duct: effect of osmolality and PGE2
Authors: Edwards R. M. , Jackson B. A. , Dousa T. P.
Published: December 2017

The papillary collecting duct (PCD) is considered to be of major importance in the final elaboration of the urine, but the metabolism of cyclic adenosine 3',5'-monophosphate (cAMP) has not yet been directly studied in the PCD. Therefore, in the present study we examined the basic properties of the cAMP system in isolated PCD microdissected from rat kidney. Vasopressin (VP) caused a marked (5- to 10-fold) stimulation of adenylate cyclase (AdC) but parathyroid hormone, calcitonin, isoproterenol, and bradykinin were without effect. A gradual increase in osmolality from 200 mosM had a biphasic effect on AdC, first enhancing (at 800 mosM) then inhibiting AdC activity at 2,000 mosM. cAMP-phosphodiesterase activity was inhibited as osmolality was increased from 200 to 800 mosM and the inhibition remained constant to 2,000 mosM. Incubation of intact PCD with VP resulted in a threefold increase in cAMP levels. As the osmolality of the incubation medium ws increased from 300 to 2,000 mosM, both basal and VP-stimulated cAMP levels continued to increase. Prostaglandin E2 (PGE2) (10(-5) M) alone (in the absence of vP) caused an increase in AdC activity, but the same dose of PGE2 had no effect on AdC activity stimulated by submaximal or maximal doses of VP. PGE2 (10(-5) M) caused a small increase in cAMP levels in intact PCD. On the other hand, PGE2 inhibited VP-stimulated cAMP levels by 50%. Incubation of PCD with PGE2 had no effect on cAMP-phosphodiesterase activity. The results demonstrate that osmolality in the physiologic range has a major influence on cAMP metabolism in the PCD and document an antagonism between PGE2 and VP at the level of cAMP accumulation in the PCD.
Microfluorometric monitoring of NAD redox state in isolated perfused renal tubules
Authors: Balaban R. S. , Dennis V. W. , Mandel L. J.
Published: December 2017

We report the successful application of microfluorometric techniques to monitor the redox state of mitochondrial nicotinamide adenine dinucleotide (NAD) in conjunction with measurements of transepithelial transport in intact isolated perfused tubules from the rabbit kidney. The determination of the redox state of NAD yields important information on the delivery of substrate to, and the energy demands on, the tubular mitochondria. These studies required only minor modifications of the basic perfusion apparatus. With this technique we detected net reductions of mitochondrial NAD upon the addition of metabolic substrates (succinate), the inhibition of active ion transport (ouabain), and the inhibition of mitochondrial electron flow (cyanide). Uncoupling of oxidative phosphorylation with 1799 caused an oxidation of NAD. These changes in NAD redox state are predictable from previous studies in isolated mitochondria, intact tissues, and from experiments on cortical tubule suspensions from the rabbit cortex.
ADH-dependent nephron heterogeneity in rats with hereditary hypothalamic diabetes insipidus
Authors: Trinh-Trang-Tan M. M. , Diaz M. , Grunfeld J. P. , Bankir L.
Published: December 2017

Single nephron glomerular filtration rates (SNGFR) were measured by the [14C]sodium ferrocyanide infusion technique in superficial (S) and juxtamedullary nephrons (JM) of anesthetized Brattleboro rats with or without diabetes insipidus (DI and HZ, respectively). Glomerular volumes (GV) and proximal tubular lengths (PTL) were measured in the same nephrons after microdissection. Glomerular volumes were also assessed in Wistar, HZ, and DI rats in Microfil-injected kidneys. The well-known nephron heterogeneity of the mammalian kidney was absent or greatly reduced in DI compared to HZ rats. S/JM ratios for SNGFR, GV, and PTL averaged 0.71, 0.50, and 0.73 in HZ and 1.04, 0.77, and 0.90 in DI rats. This reduced nephron heterogeneity was due only to reduced dimensions and filtration rates in JM nephrons. The chronic administration of antidiuretic hormone (dDAVP or vasopressin tannate), begun at 2 wk of age and maintained until adulthood (8–10 wk), significantly decreased the S/JM ratios, i.e., restored a nearly normal nephron heterogeneity in DI rats. These results suggest that nephron heterogeneity in the rat kidney is dependent on the presence of antidiuretic hormone, and, more specifically, that ADH and/or its functional consequences can selectively induce an increase in size and filtration rate in deep nephrons.
Effect of imidazole on renal function in unilateral ureteral-obstructed rat kidneys
Authors: Strand J. C. , Edwards B. S. , Anderson M. E. , Romero J. C. , Knox F. G.
Published: December 2017

Imidazole has been proposed to reverse renal vasoconstriction following unilateral obstruction, presumably through blockade of thromboxane A2 (TXA2) synthesis. We examined this hypothesis in rats subjected to unilateral ureteral obstruction for 24 h by 1) performing renal function studies before and during imidazole infusion, and 2) measuring TXB2 and prostaglandin E2 (PGE2) in urine collected before and during imidazole infusion and the profile of products generated by metabolism of arachidonic acid with renal microsomes in vitro. Imidazole infusion was associated with only a bicarbonaturia in the postobstructed kidney; in contrast, clearance of PAH and inulin, fractional sodium excretion, and bicarbonate excretion were all increased in the contralateral kidney. In the postobstructed and contralateral kidneys, TXB2 excretion was diminished and PGE2 excretion was variable not only following imidazole infusion but after saline infusion as well. The profile of products generated by renal microsomal metabolism of arachidonic acid was similar among obstructed, contralateral, and normal kidneys. These results do not support the proposal that TXA2 is the mediator of renal vasoconstriction following unilateral ureteral obstruction.
Mineralocorticoid effects on Na-K-ATPase in individual nephron segments
Authors: Garg L. C. , Knepper M. A. , Burg M. B.
Published: December 2017

A fluorometric micromethod for the assay of Na-K-ATPase was used to determine Na-K-ATPase activity in 11 segments of the rabbit nephron. The Na-K-ATPase activity was found to be highest (greater than 100 pmol.mm1-.min-1) in proximal convoluted tubules (S1), medullary thick ascending limbs, distal convoluted tubules, and connecting tubules. There was a good correlation (r = 0.94) between Na-K-ATPase activity in different segments and net Na transport previously measured by in vitro tubule perfusion. Treatment of rabbits with deoxycorticosterone (DOCA) for 8-11 days produced increases in Na-K-ATPase activity of 100 and 500% in connecting tubules and cortical collecting ducts, respectively, without significant change in other segments. Maintenance on a low sodium diet for 12-18 days was associated with a 200% increase in Na-K-ATPase activity in cortical collecting ducts only. We conclude that the Na-K-ATPase activity is stimulated by mineralocorticoids in the cortical collecting duct in association with the previously observed changes in sodium transport.
Prostaglandin E2 and F2 alpha reduces urea reabsorption from the rat collecting duct
Authors: Roman R. J. , Lechene C.
Published: December 2017

The present study examines the possibility that prostaglandins affect the renal tubular handling of urea. Meclofenamate (1 mg.kg-1.h-1), an inhibitor of prostaglandin synthesis, decreased fractional urea clearance from 86 to 67%, increased urine osmolality, and decreased the fractional excretion of water in rats undergoing a hypertonic sodium chloride diuresis. The percentage of [14C]urea microinjected into distal convoluted tubules that was recovered in urine fell from 75 to 64% (P less than 0.01) after meclofenamate. The fraction of injected urea excreted like inulin (direct recovery) was reduced from 20 to 8% (P less than 0.0001) by meclofenamate. Addition of PGE2 (1.2 or 89 pmol) or PGF2 alpha (1.4 pmol) to the microinjectate returned the urinary recovery of the microinjected [14C]urea to the control level, but PGA2 (3 pmol) did not. Direct urea recovery was doubled by PGE2 or PGF2 alpha. These results indicate that prostaglandin E2 and F2 alpha inhibit the reabsorption of urea in the collecting duct. Prostaglandins may participate in the renal concentrating mechanism by altering the inner medullary influx of urea.
Renal ammoniagenic factor in the plasma of rats with acute metabolic acidosis
Authors: Alleyne G. A. , Barnswell J. , McFarlane-Anderson N. , Alexander J. E.
Published: December 2017

The acute metabolic responses to metabolic acidosis in the rat include an increase in ammoniagenesis and gluconeogenesis before enzyme adaptations have taken place. In the present studies rats were made acidotic with NH4Cl or HCl. Preincubation of renal cortical slices from normal rats in plasma from acidotic rats produced an increase in ammoniagenic capacity. However, when slices were incubated directly in diluted plasma from acidotic rats, there was also an increase in ammoniagenesis and gluconeogenesis from glutamine. The ammoniagenic factor was detectable within 0.5 h of induction of acidosis, was not detectable in chronic acidosis, was not of adrenal origin, was not a protein, was dialyzable, and acted by an alpha-adrenergic-like mechanism in that its action was blocked by phentolamine but not by propranolol. Phentolamine injected into rats depressed the ammoniagenic response to acidosis. The plasma factor did not show its ammoniagenic effect in a low-sodium buffer and there was no absolute requirement for calcium. The acute response to acidosis is thus mediated by a humoral substance with the above characteristics.
Handling of luteinizing hormone-releasing hormone by renal proximal tubular segments in vitro
Authors: Stetler-Stevenson M. A. , Flouret G. , Peterson D. R.
Published: December 2017

[pyroglutamyl-3,4-3H]Luteinizing hormone-releasing hormone (LHRH) was microperfused through isolated segments of rabbit proximal straight tubules and incubated with isolated brush border microvilli from rabbit renal tubules. About 4.8% of perfused 3H label was reabsorbed into the bathing medium per millimeter of tubule length per minute, and 1% or less of perfused label was sequestered per millimeter of nephron segment. The 3H label content of the bathing medium varied linearly with perfusion time (30 min), suggesting a constant rate of reabsorption. Analysis by high performance liquid chromatography showed that the collection fluid and brush border incubation medium contained significant amounts of labeled pGlu-His, pGlu-His-Trp, and pGlu-His-Trp-Ser, as well as LHRH, while the bathing medium contained pGlu, pGlu-His, pGlu-His-Trp-Ser, a very small amount of pGlu-His-Trp, and no LHRH. These data suggest that the partial hydrolysis of [3H]LHRH to these peptide metabolites takes place in proximal tubules through contact digestion by brush border enzymes. The metabolites and/or hormone are probably reabsorbed and broken down further within the cell to produce pGlu, which becomes an additional metabolite found in the bathing medium.
Tubular handling of Pi: localization of effects of 1,25(OH)2D3 and dietary Pi in TPTX rats
Authors: Muhlbauer R. C. , Bonjour J. P. , Fleisch H.
Published: December 2017

Previous clearance studies have shown that chronic administration (26 pmol/day i.p. for 7 days) of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) decreases the tubular reabsorptive capacity for inorganic phosphate (Pi) in thyroparathyroidectomized (TPTX) rats. In the present study the tubular localization of this effect was examined by free-flow micropuncture in TPTX rats. At the mentioned dosage, 1,25(OH)2D3 inhibited net Pi reabsorption in the early portion of the proximal tubule. In addition, 1,25(OH)2D3 treatment altered the difference in Pi delivery between the distal tubule and the final urine, suggesting an inhibition of net Pi reabsorption along the terminal portion of the nephron, or, alternatively, admixture of tubular fluid with higher Pi concentration from deep nephrons. Finally, in TPTX rats the tubular localization of the effect of varying the dietary Pi content was found to be quite similar to that of 1,25(OH)2D3.
Hormonal regulation of calcium transport in thick ascending limb renal tubules
Authors: Suki W. N. , Rouse D.
Published: December 2017

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.
Functional segmentation of the mammalian nephron
Authors: Jacobson H. R.
Published: December 2017

Although each of the major experimental techniques applied to the study of renal physiology has provided its fair share of new information, the technique of in vitro microperfusion of nephron segments is notable for two major contributions. First, it has supplied a more direct and controlled means of studying epithelial transport processes, some of which already have helped us to understand certain aspects of kidney function and others of which have yet to find their application in unraveling the mysteries of the kidney. Second, in the process of delineating these transport characteristics, it has served to emphasize the epithelial specialization present in the kidney, providing functional counterparts to the already recognized anatomic heterogeneity present in the kidney. In this second role microperfusion has spawned the application of biochemical analysis of the hormonal responses of various nephron segments and contributed to the impetus for work in culturing the various cell types present in each nephron segment. This review outlines the functional characteristics of the 11 major segments of the nephron, incorporating what has been learned from some of the biochemical work on hormone response and correlating the latter with transport events.
Tubular handling of bicarbonate and chloride in the dog
Authors: Wong N. L. , Quamme G. A.
Published: December 2017

Bicarbonate reabsorption was assessed in the superficial proximal and distal tubule of the hydropenic dog. Tubule fluid bicarbonate concentration was measured by determination of total CO2 with microcalorimetry. Total CO2 declined from 21 to 14 mM along the proximal tubule, which reabsorbed 56% of the filtered bicarbonate prior to the late proximal sampling site. Absolute bicarbonate reabsorption was 124 pmol . min-1 . mm tubule length-1. Concurrent water reabsorption was 34% of the glomerular filtration rate. Tubule fluid-to-ultrafilterable chloride concentration ratio rose to 1.10 as the tubule fluid-to-blood bicarbonate ratio fell to 0.69 at the late proximal collection site. The total CO2 in distal tubule fluid was 6 mM, indicating that 94% of the filtered bicarbonate was reabsorbed prior to the distal tubule. It is concluded that bicarbonate is reabsorbed in preference to chloride in the superficial proximal tubule of the dog, which, like that of the rat, may provide a potential driving force for water and salt reabsorption.
Determination of chloride and bicarbonate permeabilities in proximal convoluted tubules
Authors: Holmberg C. , Kokko J. P. , Jacobson H. R.
Published: December 2017

In late proximal tubules volume reabsorption linked to passive ion flows relies on the existence of differing permeability coefficients to Cl- and HCO3(-) (PCl greater than PHCO3). We measured these permeability coefficients in late segments of rabbit superficial (SFPCT) and juxtamedullary (JMPCT) proximal convoluted tubules perfused in vitro. PHCO3 and P36Cl were determined in tubules bathed in rabbit serum and perfused with a serum ultrafiltrated titrated with H2SO4 to [HCO3(-)] of 4 mM. Active transport, transepithelial voltage, and HCO3(-) reabsorption were inhibited by cooling (21 degrees C) and 10(-4) M acetazolamide. P36Cl and PHCO3 were calculated from 36Cl disappearance from and total CO2 addition to the perfusate. P36Cl in SFPCT was twice that in JMPCT but PHCO3 was the same in both segments. P36Cl exceeded PHCO3 only in SFPCT. To exclude exchange diffusion from contributing to P36Cl, additional tubules were perfused with ultrafiltrate titrated with HCl.P36Cl and simultaneously measured PCl (lumen-to-bath net chemical Cl- flux) were identical. We conclude: 1) SFPCT and JMPCT are heterogeneous with respect to Cl- permeability; 2) relative Cl--to-HCO3(-) permeabilities predict that anion gradients present in late portions of proximal tubules would support more volume reabsorption linked to passive ion flows in SF than in JMPCT; 3) no significant Cl- exchange diffusion exists in proximal tubules.
Isolation of a pure suspension of rat proximal tubules
Authors: Vinay P. , Gougoux A. , Lemieux G.
Published: December 2017

A suspension of cortical tissue fragments prepared by collagenase digestion of renal cortex obtained from fed and chronically acidotic (NH4Cl) rats was separated into four bands on a Percoll density gradient. By microscopic examination, vital staining with trypan blue, and histologic staining technique (periodic acid-Schiff) the F4 band was shown to contain only (greater than 98%) proximal tubules, whereas the F1 band was significantly enriched (70%) with distal tubules contaminated by glomeruli and short segments of proximal tubules. Intra/extracellular ratios for PAH of 15 were measured in the F4 band and of 2 in F1 band. ATP was 1.4 and 2.8 mumol/g in the F4 and F1 bands, respectively, and was stable for at least 60 min. The proximal F4 band was shown to be gluconeogenic (L-glutamine or L-lactate 2.5 mM as substrate) and to adapt to metabolic acidosis. The distal F1 band was shown to be glycolytic (glucose 2.5 mM) with no changes with acid-base status. All fractions were shown to metabolize glutamine, but the metabolic fate of this amino acid was different in proximal and distal structures. A F4/F1 activity ratio for the proximal cytoplasmic phosphoenolpyruvate carboxykinase enzyme of 2.6 and 4.3 was observed in normal and acidotic rats, respectively. In contrast, a F4/F1 ratio of 0.13 and 0.22 was observed for the distal cytoplasmic hexokinase enzyme. This preparation, therefore, allows the metabolism of a homogeneous population of proximal tubular fragments to be studied and can be used to obtain information on enzyme location within the nephron.
NaCl transport in mouse medullary thick ascending limbs. I. Functional nephron heterogeneity and ADH-stimulated NaCl cotransport
Authors: Hebert S. C. , Culpepper R. M. , Andreoli T. E.
Published: December 2017

We assessed the effects of antidiuretic hormone and cyclic adenosine monophosphate (cAMP) analogues on transepithelial voltage, Ve, and/or net chloride absorption in isolated mouse medullary (mTALH) and cortical (cTALH) thick ascending limbs of Henle; the passive NaCl permeability characteristics and electrical properties of the mTALH; and the effects of anion and cation substitutions and transport inhibitors on both basal and ADH-stimulated Ve and/or net chloride absorption in the mTALH. The data demonstrate that these two segments are functionally heterogeneous: ADH, at concentrations comparable to plasma levels seen in mammalian species during ordinary antidiuresis, and/or cAMP increase three- to fourfold the rate of NaCl absorption in the mTALH but not in the cTALH. The ion substitution and inhibitor data are consistent with the view that NaCl absorption in the mTALH depends on a secondary active transport process: NaCl entry across luminal membranes is a coupled process of indeterminate stoichiometry that is driven by the transmembrane electrochemical gradient for Na+, which is maintained by Na+-K+-ATPase. Finally, the data demonstrate that the mTALH is electrically leaky whether measured electrically, 11 omega . cm2, or isotopically, 50 omega . cm2, but essentially water impermeable; and that the mTALH is perm-selective for Na+ with respect to Cl-. The disparity between electrical resistances measured directly with respect to those calculated from tracer fluxes, together with the hybrid characteristics of mTALH junctional complexes (leaky to Na+ and Cl-; tight to water), may be reconciled by assuming that mTALH junctional complexes contain passive ion permeation pathways composed of narrow channels through which ions pass in single-file fashion.