Independent desensitization of rat renal thick ascending limbs and collecting ducts to ADH

1989 ◽  
Vol 256 (4) ◽  
pp. F656-F663 ◽  
Author(s):  
I. Dublineau ◽  
J. M. Elalouf ◽  
P. Pradelles ◽  
C. de Rouffignac
Keyword(s):  
Collecting Duct ◽  
Synthetic Analogue ◽  
Thick Ascending Limb ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Collecting Ducts ◽  
Normal Rat ◽  
Series Of Experiments ◽  
High Doses

Recent micropuncture studies have demonstrated that administration of high doses of 1-deamino-8-D-arginine vasopressin (dDAVP), a synthetic analogue of vasopressin (AVP), causes desensitization of the thick ascending limb to AVP but may leave unaltered the effect of this hormone on the permeability to water of the collecting duct. In the present experiments, desensitization to AVP was studied by measuring adenosine 3',5'-cyclic monophosphate (cAMP) synthesis in microdissected cortical thick ascending limbs (CTAL) and cortical collecting ducts (CCD) incubated in vitro. Desensitization was induced by intramuscular injections of dDAVP (2 micrograms/day for 3 days). In a first series of experiments, performed on Brattleboro rats lacking circulating AVP, the effects of AVP on cAMP accumulation were reduced by 30% in CTAL of the rats given dDAVP, whereas in CCD no reduction was noted. Desensitization of CTAL was selective for AVP (i.e., homologous), the effects of glucagon being unaltered. In a second series of experiments, performed on Sprague-Dawley rats, a marked (up to 75% 2 h after dDAVP injection), homologous and reversible desensitization of CTAL to AVP was observed. However, here again no desensitization was obtained in CCD, indicating that in the normal rat, administration of 2 micrograms dDAVP also elicited preferential desensitization of CTAL.

Immunolocalization of ectonucleotidases along the rat nephron

2006 ◽  
Vol 290 (2) ◽  
pp. F550-F560 ◽  
Author(s):  
Renu M. Vekaria ◽  
David G. Shirley ◽  
Jean Sévigny ◽  
Robert J. Unwin
Keyword(s):  
Collecting Duct ◽  
Distal Tubule ◽  
Extracellular Nucleotides ◽  
Thick Ascending Limb ◽  
Sprague Dawley ◽  
Low Level ◽  
Amplification Method ◽  
Sprague Dawley Rats ◽  
Collecting Ducts ◽  
Calbindin D

Evidence is accumulating that extracellular nucleotides act as autocrine/paracrine agents in most tissues, including the kidneys. Several families of surface-located enzymes, collectively known as ectonucleotidases, can degrade nucleotides. Using immunohistochemistry, we have examined the segmental distribution of five ectonucleotidases along the rat nephron. Perfusion-fixed kidneys were obtained from anesthetized male Sprague-Dawley rats. Cryostat sections of cortical and medullary regions were incubated with antibodies specific to the following enzymes: ectonucleoside triphosphate diphosphohydrolase (NTPDase) 1, NTPDase2, NTPDase3, ectonucleotide pyrophosphatase phosphodiesterase 3 (NPP3), and ecto-5′-nucleotidase. Sections were then costained with Phaseolus vulgaris erythroagglutinin (for identification of proximal tubules) and antibodies against Tamm-Horsfall protein (for identification of thick ascending limb), calbindin-D28k (for identification of distal tubule), and aquaporin-2 (for identification of collecting duct). The tyramide signal amplification method was used when the ectonucleotidase and marker antibody were raised in the same species. The glomerulus expressed NTPDase1 and NPP3. The proximal tubule showed prominent expression of NPP3 and ecto-5′-nucleotidase in most, but not all, segments. NTPDase2 and NTPDase3, but not NPP3 or ecto-5′-nucleotidase, were expressed in the thick ascending limb and distal tubule. NTPDase3, with some low-level expression of ecto-5′-nucleotidase, was also found in cortical and outer medullary collecting ducts. Inner medullary collecting ducts displayed low-level staining for NTPDase1, NTPDase2, NTPDase3, and ecto-5′-nucleotidase. We conclude that these ectonucleotidases are differentially expressed along the nephron and may play a key role in activation of purinoceptors by nucleotides and nucleosides.

Effects of ET-1 on water and chloride transport in cortical collecting ducts of the rat

1993 ◽  
Vol 264 (4) ◽  
pp. F690-F696 ◽  
Author(s):  
K. Tomita ◽  
H. Nonoguchi ◽  
Y. Terada ◽  
F. Marumo
Keyword(s):  
Water Permeability ◽  
Continuous Flow ◽  
Chloride Transport ◽  
Collecting Duct ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Osmotic Water ◽  
Collecting Ducts ◽  
Medullary Collecting Duct

Endothelin-1 (ET-1) is known as a vasoconstrictor peptide. However, recent reports suggested the effects on the transport of renal tubule. We previously reported that ET-1 inhibited arginine vasopressin (AVP)-dependent adenosine 3',5'-cyclic monophosphate in rat collecting ducts. Physiologically, ET-1 reversibly and significantly inhibited AVP-stimulated water permeability in inner medullary collecting duct (IMCD). We therefore investigated the effects on water and electrolyte transport in rat cortical collecting ducts (CCD), where Na and Cl are actively reabsorbed more than in IMCD. Pathogen-free male Sprague-Dawley rats weighing 80-120 g were used after treatment with deoxycorticosterone pivalate for 1-2 wk. Isolated CCD were microperfused in vitro. The Cl concentration was measured by a continuous-flow ultra-microcolorimeter, and the raffinose concentration was measured as a volume marker by a continuous-flow ultra-microfluorometer. In the presence of 10(-9) M AVP, 10(-8) M ET-1 significantly inhibited fluid absorption (nl.mm-1 x min-1) from 0.25 +/- 0.02 to 0.15 +/- 0.05 (mean +/- SE, n = 6, P < 0.01), Cl absorption (pmol.mm-1 x min-1) from 30. 6 +/- 2.8 to 14.9 +/- 4.0 (P < 0.01), and potential difference (mV) from -5.4 +/- 1.3 to -4.0 +/- 1.2 (P < 0.01). Similar results were obtained in the lower concentration of 10(-10) M AVP and 10(-10) M ET-1. As for the osmotic water permeability (microns/s), 10(-8) M ET-1 significantly inhibited this from 320.1 +/- 50.9 to 202.1 +/- 42.2 (n = 7, P < 0.01) in the presence of 10(-9) M AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Acute calcineurin inhibition with tacrolimus increases phosphorylated UT-A1

2012 ◽  
Vol 302 (8) ◽  
pp. F998-F1004 ◽  
Author(s):  
Titilayo O. Ilori ◽  
Yanhua Wang ◽  
Mitsi A. Blount ◽  
Christopher F. Martin ◽  
Jeff M. Sands ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Membrane Association ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Collecting Ducts ◽  
Concentrate Urine ◽  
Medullary Collecting Duct

UT-A1, the urea transporter present in the apical membrane of the inner medullary collecting duct, is crucial to the kidney's ability to concentrate urine. Phosphorylation of UT-A1 on serines 486 and 499 is important for plasma membrane trafficking. The effect of calcineurin on dephosphorylation of UT-A1 was investigated. Inner medullary collecting ducts from Sprague-Dawley rats were metabolically labeled and treated with tacrolimus to inhibit calcineurin or calyculin to inhibit protein phosphatases 1 and 2A. UT-A1 was immunoprecipitated, electrophoresed, blotted, and total UT-A1 phosphorylation was assessed by autoradiography. Total UT-A1 was determined by Western blotting. A phospho-specific antibody to pser486-UT-A1 was used to determine whether serine 486 can be hyperphosphorylated by inhibiting phosphatases. Inhibition of calcineurin showed an increase in phosphorylation per unit protein at serine 486. In contrast, inhibition of phosphatases 1 and 2A resulted in an increase in UT-A1 phosphorylation but no increase in pser486-UT-A1. In vitro perfusion of inner medullary collecting ducts showed tacrolimus-stimulated urea permeability consistent with stimulated urea transport. The location of phosphorylated UT-A1 in rats treated acutely and chronically with tacrolimus was determined using immunohistochemistry. Inner medullary collecting ducts of the acutely treated rats showed increased apical membrane association of phosphorylated UT-A1 while chronic treatment reduced membrane association of phosphorylated UT-A1. We conclude that UT-A1 may be dephosphorylated by multiple phosphatases and that the PKA-phosphorylated serine 486 is dephosphorylated by calcineurin. This is the first documentation of the role of phosphatases and the specific site of phosphorylation of UT-A1, in response to tacrolimus.

Urea transport in isolated thick ascending limbs and collecting ducts from rats

1983 ◽  
Vol 245 (5) ◽  
pp. F634-F639 ◽  
Author(s):  
M. A. Knepper
Keyword(s):  
Thick Ascending Limb ◽  
Sprague Dawley ◽  
Urea Permeability ◽  
Sprague Dawley Rats ◽  
Collecting Ducts ◽  
Outer Stripe ◽  
Passive Absorption ◽  
Urinary Concentrating Ability

The use of pathogen-free rats allows the dissection and in vitro perfusion of several rat nephron segments not previously studied. In the present experiments, net urea fluxes were measured in isolated perfused cortical and medullary thick ascending limbs and cortical collecting ducts from pathogen-free Sprague-Dawley rats. No evidence for active transport of urea was found in either cortical or medullary thick ascending limbs. Permeabilities were calculated from urea fluxes measured with 5 mM urea either in the bath or perfusate and with no urea on the opposite side of the epithelium. Permeability coefficients (cm/s X 10(-5) +/- SE) in different portions of the thick ascending limb were: inner stripe, short-looped nephrons, 0.9 +/- 0.2; inner stripe, long-looped nephrons, 0.6 +/- 0.2 (not significantly different vs. short loops); outer stripe, 1.4 +/- 0.3 (P less than 0.05 vs. inner stripe); and cortical, 1.5 +/- 0.3 (P less than 0.05 vs. inner stripe). The relatively high urea permeability of thick ascending limbs in the outer stripe of the outer medulla and medullary rays is likely to permit substantial passive absorption of urea from these segments in vivo. This will contribute to dilution of the tubule fluid in thick ascending limbs and thus indirectly enhance urinary concentrating ability. In cortical collecting ducts, the urea permeability was relatively low both in the presence of 100 microU/ml arginine vasopressin in the bath (0.5 +/- 0.1 X 10(-5) cm/s) and in its absence (0.4 +/- 0.1). These permeability values are similar to values previously measured in rabbit cortical collecting ducts.

Rosuvastatin treatment reverses impaired coronary artery vasodilation in fructose-fed, insulin-resistant rats

2004 ◽  
Vol 287 (1) ◽  
pp. R157-R160 ◽  
Author(s):  
Allison W. Miller ◽  
Christina D. Tulbert ◽  
David W. Busija
Keyword(s):  
Coronary Arteries ◽  
Fasting Insulin ◽  
Sprague Dawley ◽  
Calcium Dependent ◽  
Insulin Resistant ◽  
Functional Studies ◽  
Sprague Dawley Rats ◽  
Normal Rat ◽  
Biochemical Measurements

Insulin resistance (IR) impairs vascular responses in coronary arteries, but mechanisms of dysfunction and approaches to treatment remain unclear. We examined the ability of a new 3-hydroxy-methylglutaryl coenzyme A reductase inhibitor, rosuvastatin, to reverse reduced dilator responses in rats made IR by feeding a fructose-rich diet (FF). Sprague-Dawley rats were randomized to control (normal rat diet) or FF. After 1 wk, rats received rosuvastatin (2 mg/kg) or placebo (saline) subcutaneously for 5 wk. Biochemical measurements and in vitro functional studies of small coronary arteries were performed. Fasting insulin and triglyceride (TG) levels were markedly increased in FF-placebo rats compared with other groups. Rosuvastatin treatment of FF rats normalized TG and modestly decreased insulin levels. ACh-induced dilator responses were depressed in arteries from FF-placebo rats. This impairment was due to decreased responses via calcium-dependent K channels (KCa). Rosuvastatin treatment of FF rats completely reversed the response to ACh to normal levels. Moreover, this recovery in function was due to an improvement in vasodilation via KCa. Thus rosuvastatin treatment of IR rats normalizes coronary vascular dilator responses by improving the KCa function.

Upregulation of Na+ transporter abundances in response to chronic thiazide or loop diuretic treatment in rats

2003 ◽  
Vol 284 (1) ◽  
pp. F133-F143 ◽  
Author(s):  
Ki Young Na ◽  
Yoon Kyu Oh ◽  
Jin Suk Han ◽  
Kwon Wook Joo ◽  
Jung Sang Lee ◽  
...  
Keyword(s):  
Tap Water ◽  
Chronic Administration ◽  
Thick Ascending Limb ◽  
Sprague Dawley ◽  
Volume Depletion ◽  
Diuretic Treatment ◽  
Sprague Dawley Rats ◽  
Collecting Ducts ◽  
Site Of Action

Furosemide and hydrochlorothiazide (HCTZ) exert their diuretic actions by binding to apical Na+ transporters, viz., the Na+-K+-2Cl− cotransporter in the thick ascending limb and the Na+-Cl−cotransporter in the distal convoluted tubule, respectively. We carried out semiquantitative immunoblotting and immunohistochemistry of rat kidneys to investigate whether chronic administration of furosemide or HCTZ is associated with compensatory changes in the abundance of Na+ transporters downstream from the primary site of action. Osmotic minipumps were implanted into Sprague-Dawley rats to deliver furosemide (12 mg/day) or HCTZ (3.75 mg/day) for 7 days. To prevent volume depletion, all animals were offered tap water and a solution containing 0.8% NaCl and 0.1% KCl as drinking fluid. The diuretic/natriuretic response was quantified in response to both agents by using quantitative urine collections. Semiquantitative immunoblotting revealed that the abundances of thick ascending limb Na+-K+-2Cl− cotransporter and all three subunits of the epithelial Na+ channel (ENaC) were increased by furosemide infusion. HCTZ infusion increased the abundances of thiazide-sensitive Na+-Cl−cotransporter and β-ENaC in the cortex and β- and γ-ENaC in the outer medulla. Consistent with these results, β-ENaC immunohistochemistry showed a remarkable increase in immunoreactivity in the principal cells of collecting ducts with either diuretic treatment. These increases in the abundance of Na+transporters in response to chronic diuretic treatment may account for the generation of diuretic tolerance associated with long-term diuretic use.

cAMP-dependent fluid secretion in rat inner medullary collecting ducts

2001 ◽  
Vol 280 (6) ◽  
pp. F1019-F1029 ◽  
Author(s):  
Darren P. Wallace ◽  
Lorraine A. Rome ◽  
Lawrence P. Sullivan ◽  
Jared J. Grantham
Keyword(s):  
Fluid Secretion ◽  
Sprague Dawley ◽  
In Vitro Method ◽  
Lumen Formation ◽  
Sprague Dawley Rats ◽  
Collecting Ducts ◽  
Selective Addition ◽  
Collecting System ◽  
Kinase A

We used an unambiguous in vitro method to determine if inner medullary collecting ducts (IMCD) have intrinsic capacities to absorb and secrete solutes and fluid in an isotonic medium. IMCD1, IMCD2, and IMCD3 were dissected from kidneys of young Sprague-Dawley rats. 8-Bromo-3′,5′-cyclic monophosphate (8-BrcAMP) stimulated lumen formation and progressive dilation in all IMCD subsegments; lumen formation was greatest in IMCD1.Benzamil potentiated the rate of lumen expansion in response to 8-BrcAMP. Fluid entered tubule lumens by transcellular secretion rather than simple translocation of intracellular fluid. Secreted lumen solutes were osmometrically active. Inhibition of protein kinase A with H-89 and Rp diastereomer of adenosine 3′,5′-cyclic monophosphorothioate blocked fluid secretion. The rate of lumen expansion was reduced by the selective addition of ouabain, barium, diphenyl-2-carboxylate, bumetanide, glybenclamide, or DIDS, or reduction of extracellular Cl−. We conclude that IMCD absorb and secrete electrolytes and fluid in vitro and that secretion is accelerated by cAMP. We suggest that salt and fluid secretion by the terminal portions of the renal collecting system may have a role in modulating the composition and volume of the final urine.

Nonclinical Toxicology Assessments Support the Chronic Safety of Dapagliflozin, a First-in-Class Sodium-Glucose Cotransporter 2 Inhibitor

2013 ◽  
Vol 32 (5) ◽  
pp. 336-350 ◽  
Author(s):  
Mark Tirmenstein ◽  
Thomas E. Dorr ◽  
Evan B. Janovitz ◽  
Deborah Hagan ◽  
Lynn M. Abell ◽  
...  
Keyword(s):  
Calcium Absorption ◽  
Sprague Dawley ◽  
Clinical Dose ◽  
Mediated Effects ◽  
Sodium Glucose Cotransporter ◽  
Sprague Dawley Rats ◽  
Urinary Glucose ◽  
Patients With Diabetes ◽  
High Doses

Dapagliflozin, a first-in-class, selective inhibitor of sodium-glucose cotransporter 2 (SGLT2), promotes urinary glucose excretion to reduce hyperglycemia for the treatment of type 2 diabetes. A series of nonclinical studies were undertaken to evaluate dapagliflozin in species where it was shown to have pharmacologic activity comparable with that in humans at doses that resulted in supratherapeutic exposures. In vitro screening (>300 targets; 10 μmol/L) indicated no significant off-target activities for dapagliflozin or its primary human metabolite. Once daily, orally administered dapagliflozin was evaluated in Sprague-Dawley rats (≤6 months) and in beagle dogs (≤1 year) at exposures >5000-fold those observed at the maximum recommended human clinical dose (MRHD; 10 mg). Anticipated, pharmacologically mediated effects of glucosuria, osmotic diuresis, and mild electrolyte loss were observed, but there were no adverse effects at clinically relevant exposures, including in the kidneys or urogenital tract. The SGLT2−/− mice, which show chronic glucosuria, and dapagliflozin-treated, wild-type mice exhibited similar safety profiles. In rats but not dogs, dapagliflozin at >2000-fold MRHD exposures resulted in tissue mineralization and trabecular bone accretion. Investigative studies suggested that the effect was not relevant to human safety, since it was partially related to off-target inhibition of SGLT1, which was observed only at high doses of dapagliflozin and resulted in intestinal glucose malabsorption and increased intestinal calcium absorption. The rigorous assessment of supra- and off-target dapagliflozin pharmacology in nonclinical species allowed for a thorough evaluation of potential toxicity, providing us with confidence in its safety in patients with diabetes.

scholarly journals Effects of salt depletion on the kidney: changes in medullary oxygenation and thick ascending limb size.

1994 ◽  
Vol 4 (8) ◽  
pp. 1538-1545
Author(s):  
I E Stillman ◽  
M Brezis ◽  
S N Heyman ◽  
F H Epstein ◽  
K Spokes ◽  
...  
Keyword(s):  
Salt Intake ◽  
Collecting Duct ◽  
Thick Ascending Limb ◽  
Sprague Dawley ◽  
Deficient Diet ◽  
Cross Sectional ◽  
Short Loop ◽  
Sprague Dawley Rats ◽  
Salt Depletion ◽  
Urinary Concentrating Ability

Previous studies have shown that salt depletion enhances the susceptibility of the kidney to nephrotoxins (amphotericin, cyclosporine, and contrast). To study the renal response to salt depletion, Sprague-Dawley rats were fed a sodium-deficient diet (N = 12) with pair-fed controls (N = 13) for 4 wk. In addition, rats from each group underwent 24-h water deprivation studies (N = 9; four salt deprived, five normal). Plastic 1-micron horizontal sections of mid-inner stripe were examined, and cross-sectional areas of the medullary thick ascending limb (mTAL) were analyzed. The mTAL of the salt-deprived rats were smaller (P = 0.04) and showed greater variance in size (P = 0.02) than control (618 +/- 106 versus 693 +/- 50 microns2). However, mean glomerular and collecting duct cross-sectional areas were unaffected by salt intake. Cross-sectional areas of long- and short-loop mTAL were significantly different, regardless of group (518 +/- 78 versus 732 +/- 92 microns2). Maximal urinary concentrating ability was found to correlate with mTAL cross-sectional area (r = 0.85; P = 0.004) and with long-loop mTAL size (r = 0.77; P = 0.016). However, it did not significantly correlate with short loop mTAL size (r = 0.53; P = 0.14).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Treating lithium-induced nephrogenic diabetes insipidus with a COX-2 inhibitor improves polyuria via upregulation of AQP2 and NKCC2

2008 ◽  
Vol 294 (4) ◽  
pp. F702-F709 ◽  
Author(s):  
Gheun-Ho Kim ◽  
Nak Won Choi ◽  
Ju-Young Jung ◽  
Ji-Hyun Song ◽  
Chang Hwa Lee ◽  
...  
Keyword(s):  
Diabetes Insipidus ◽  
Nephrogenic Diabetes Insipidus ◽  
Collecting Duct ◽  
Urine Osmolality ◽  
Urinary Concentration ◽  
Prostaglandin E ◽  
Thick Ascending Limb ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Cox 2

Prostaglandin E2 may antagonize vasopressin-stimulated salt absorption in the thick ascending limb and water absorption in the collecting duct. Blockade of prostaglandin E2 synthesis by nonsteroidal anti-inflammatory drugs (NSAIDs) enhances urinary concentration, and these agents have antidiuretic effects in patients with nephrogenic diabetes insipidus (NDI) of different etiologies. Because renal prostaglandins are derived largely from cyclooxygenase-2 (COX-2), we hypothesized that treatment of NDI with a COX-2 inhibitor may relieve polyuria through increased expression of Na-K-2Cl cotransporter type 2 (NKCC2) in the thick ascending limb and aquaporin-2 (AQP2) in the collecting duct. To test this hypothesis, semiquantitative immunoblotting and immunohistochemistry were carried out from the kidneys of lithium-induced NDI rats with and without COX-2 inhibition. After male Sprague-Dawley rats were fed an LiCl-containing rat diet for 3 wk, the rats were randomly divided into control and experimental groups. The COX-2 inhibitor DFU (40 mg·kg−1·day−1) was orally administered to the experimental rats for an additional week. Treatment with the COX-2 inhibitor significantly relieved polyuria and raised urine osmolality. Semiquantitative immunoblotting using whole-kidney homogenates revealed that COX-2 inhibition caused significant increases in the abundance of AQP2 and NKCC2. Immunohistochemistry for AQP2 and NKCC2 confirmed the effects of COX-2 inhibition in lithium-induced NDI rats. The upregulation of AQP2 and NKCC2 in response to the COX-2 inhibitor may underlie the therapeutic mechanisms by which NSAIDs enhance antidiuresis in patients with NDI.

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