Determination of prostaglandin E2 synthesis along rabbit nephron by enzyme immunoassay

1986 ◽  
Vol 251 (2) ◽  
pp. F238-F244 ◽  
Author(s):  
N. Farman ◽  
P. Pradelles ◽  
J. P. Bonvalet
Keyword(s):  
Arachidonic Acid ◽  
Prostaglandin E2 ◽  
Enzyme Immunoassay ◽  
Collecting Duct ◽  
Distal Tubule ◽  
Rabbit Kidney ◽  
Thick Ascending Limb ◽  
Pge2 Synthesis ◽  
Collecting Tubule ◽  
Cortical Collecting Tubule

Prostaglandin E2 (PGE2) content and synthesis have been measured in microdissected segments from the entire nephron of rabbit kidney. PGE2 was determined by an enzyme immunoassay on glomeruli or tubular segments (0.5-5 mm) either immediately after microdissection (PGE2 content) or after incubation for 15 min at 37 degrees C in the presence of arachidonic acid (PGE2 synthesis). We confirmed that collagenase used for microdissection did not modify PGE2 synthesis. A linear correlation was found between the length of tubule used in the assay and PGE2 synthesis, as well as between incubation time with arachidonic acid and PGE2 synthesis. PGE2 synthesis, expressed in picograms per millimeter tubular length per 15 min, was maximum in medullary collecting duct (517 +/- 73). High values were also found in the granular portion of distal tubule (134 +/- 22) and granular or light portion of cortical collecting tubule (199 +/- 24 and 146 +/- 10, respectively). Synthesis was lower in all other segments: 17 +/- 6 and 24 +/- 12, respectively, in convoluted and straight proximal tubule, 67 +/- 12 and 71 +/- 5, respectively, in thin descending and ascending limb, 51 +/- 9 and 23 +/- 4, respectively, in medullary and cortical thick ascending limb of Henle's loop, and 25 +/- 7 in initial distal tubule. Synthesis per glomerulus was 24 +/- 3. When the protein content of each nephron segment is taken into account, this profile was not modified, except for the thin limbs of the loop, which reached values per nanogram protein slightly higher than those of the cortical collecting tubule.(ABSTRACT TRUNCATED AT 250 WORDS)

PGE2, PGF2 alpha, 6-keto-PGF1 alpha, and TxB2 synthesis along the rabbit nephron

1987 ◽  
Vol 252 (1) ◽  
pp. F53-F59 ◽  
Author(s):  
N. Farman ◽  
P. Pradelles ◽  
J. P. Bonvalet
Keyword(s):  
Arachidonic Acid ◽  
Prostaglandin E2 ◽  
Proximal Tubule ◽  
Enzyme Immunoassay ◽  
Thick Ascending Limb ◽  
Pge2 Synthesis ◽  
Nephron Segments ◽  
Prostaglandin F2 Alpha ◽  
Collecting Tubule ◽  
Cortical Collecting Tubule

The capacity of synthesis of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 6-keto-PGF1 alpha, and thromboxane (TxB2) along the rabbit nephron was determined. A sensitive enzyme immunoassay was applied to isolated nephron segments, from the glomerulus to the terminal collecting tubule. The three prostaglandins and thromboxane (PG) were measured on the same samples after incubation in the presence of arachidonic acid. In the glomerulus, PGE2 synthesis (29.4 +/- 3.3 pg X glomerulus-1 X 30 min-1) represented 60% of the sum of the four PGs. PGF2 alpha and 6-keto-PGF1 alpha represented 22 and 17%, respectively, and TxB2 1.4%. The contribution of each PG to tubular synthesis was different, since at least 90% of PG synthesis consisted of PGE2. In the medullary collecting tubule (MCT), by far the major tubular site of PG synthesis, it was 809.6 +/- 140.8 pg X mm-1 X 30 min-1 for PGE2, 17.7 +/- 7.2 for PGF2 alpha, 8.3 +/- 1.9 for 6-keto-PGF1 alpha and 0.24 +/- 0.08 for TxB2. These relative proportions were roughly respected all along the tubule. Values were much lower in the convoluted and straight portions of the proximal tubule (proximal convoluted tubule: PGE2 8.2 +/- 2.0, PGF2 alpha 0.4 +/- 0.06, 6-keto-PGF1 alpha 0.26 +/- 0.04, TxB2 0.017) and the medullary and cortical thick ascending limb of the loop. They increased regularly along the distal structures of the tubule (light portion of the cortical collecting tubule (CCT1): PGE2 228.3 +/- 20.4, PGF2 alpha 4.34 +/- 0.6, 6-keto-PGF1 alpha 1.8 +/- 0.3, TxB2 0.22 +/- 0.07).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunolocalization of gluco- and mineralocorticoid receptors in rabbit kidney

1991 ◽  
Vol 260 (2) ◽  
pp. C226-C233 ◽  
Author(s):  
N. Farman ◽  
M. E. Oblin ◽  
M. Lombes ◽  
F. Delahaye ◽  
H. M. Westphal ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Distal Tubule ◽  
Interstitial Cells ◽  
Rabbit Kidney ◽  
Cell Compartments ◽  
Corticosteroid Hormones ◽  
Collecting Tubule ◽  
Show Evidence ◽  
Cortical Collecting Tubule

The localization of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) was determined in the rabbit kidney by immunohistochemistry with the use of a monoclonal, anti-GR antibody and a monoclonal, anti-idiotypic, anti-MR antibody. Immunostaining was performed on serial histological sections from normal and adrenalectomized rabbits. The specificity of immunostaining was assessed for MR by in situ competition studies with steroids and for GR by presaturation of the antibody with GR preparation. Immunostaining by both the anti-MR and the anti-GR antibodies was present in all parts of the distal nephron (beyond proximal tubule) and absent in the glomerulus and proximal tubule. The absence of staining by the anti-GR antibody in the proximal tubule suggests that the effects of glucocorticoids in this structure involve either a GR different from that of distal structures or a non-receptor mediated mechanism of action. MR immunostaining predominates in the distal and all along the collecting tubule in its cortical, medullary, and papillary portions. GR immunostaining was most abundant in the medullary ascending limb and distal tubule. Immunostaining by both antibodies was present in papillary interstitial cells and cells of the epithelium lining the papilla. Fifteen to twenty percent of the cells of the cortical collecting tubule, presumably intercalated cells, were devoid of MR and GR immunostaining. Immunostaining was present in both nuclear and cytoplasmic cell compartments. No clear difference was observed between normal and adrenalectomized rabbits. This study is the first report on renal immunolocalization of GR compared with MR. In addition, we show evidence for new targets for corticosteroid hormones such as papillary interstitial cells and papillary epithelium.

Mineralcorticoid receptors along the nephron: [3H]aldosterone binding in rabbit tubules

1981 ◽  
Vol 241 (6) ◽  
pp. F605-F611 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Thick Ascending Limb ◽  
Collecting Tubule ◽  
The Difference ◽  
Collecting Tubules ◽  
Cortical Collecting Tubule

To identify the site of mineralocorticoid action along the nephron, we measured the specific binding of [3H]aldosterone to nephron segments microdissected from aldosterone-deficient rabbits. Specific binding was defined as the difference between binding measured in the absence or in the presence of 2,000-fold excess of unlabeled hormone (in 10(-18) mol X cm tubule length-1 +/- SE). High specific binding capacity was found in the branched collecting tubule (108 +/- 4), the cortical collecting tubule (119 +/- 9), and the outer medullary collecting tubule (115 +/- 16), whereas specific binding was negligible in the proximal convoluted tubule (8 +/- 9), pars recta (2 +/- 6), medullary thick ascending limb (4 +/- 6), cortical thick ascending limb (6 +/- 2), and distal convoluted tubule (6 +/- 6). In cortical collecting tubules, Scatchard analysis of the specific [3H]aldosterone binding indicated a dissociation constant (KD) of 2.2 X 10(-9) M and a maximum number of binding sites of 157 X 10(-18) mol X cm tubule length-1. The steroid specificity was assessed from the competition of various steroids for [3H]aldosterone binding sites. Receptors from the cortical collecting tubule revealed the following sequence of affinities: aldosterone greater than DOCA greater than spironolactone greater than dexamethasone greater than 5 alpha-dihydrotestosterone = progesterone = 17 beta-estradiol, indicating that the binding sites in the collecting tubule are mineralocorticoid receptors. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity only in the collecting tubule and suggest that this nephron segment is the target site of mineralocorticoid action in the rabbit kidney.

Synthesis of prostaglandin E2 in different segments of isolated collecting tubules from adult and neonatal rabbits

1985 ◽  
Vol 248 (1) ◽  
pp. F134-F144 ◽  
Author(s):  
D. Schlondorff ◽  
J. A. Satriano ◽  
G. J. Schwartz
Keyword(s):  
Arachidonic Acid ◽  
Prostaglandin E2 ◽  
Feedback Loop ◽  
Pge2 Production ◽  
Large Variability ◽  
Pge2 Synthesis ◽  
Collecting Tubule ◽  
Concentrate Urine ◽  
Collecting Tubules

Prostaglandin E2 (PGE2) inhibits the action of the antidiuretic hormone (ADH) in isolated collecting tubules. A negative feedback loop has been postulated whereby ADH stimulates PGE2 synthesis. Furthermore, lysyl-bradykinin (LBK) inhibits the antidiuretic effect of ADH, probably via PGE2. Enhanced PGE2 synthesis has also been implicated as contributing to the inability to maximally concentrate urine during the neonatal period. We investigated PGE2 synthesis in microdissected cortical (CCT), medullary (MCT), and branched cortical (BCT) collecting tubules from adult and in corticomedullary collecting tubules (CT) from newborn rabbits. Isolated BCT produced significantly less PGE2 (12 +/- 2 pg X mm-1 X 20 min-1) than CCT (65 +/- 9) or MCT (76 +/- 8) from kidneys of adult rabbits. CT from newborn rabbits produced only 19 +/- 3 pg/mm, significantly less than either CCT or MCT from adults. A large variability in basal PGE2 production and hormonal response was observed from tubule to tubule. Under either basal conditions or in the presence of 2 microM arachidonic acid, LBK enhanced PGE2 synthesis in CCT and MCT from adults. ADH enhanced PGE2 production in MCT under basal conditions and in CCT in the presence of arachidonic acid. Neither LBK nor ADH stimulated PGE2 synthesis in neonatal CT. A23187 consistently stimulated PGE2 synthesis in CCT and MCT from adults and, to a lesser extent, in CT from newborn rabbits. Our results support the hypothesis that ADH and LBK enhance PGE2 synthesis in the collecting tubule. This response is, however, subject to large variations from tubule to tubule and depends on the in vitro incubation conditions.

Segmental synthesis and actions of prostaglandins along the nephron

1987 ◽  
Vol 253 (3) ◽  
pp. F377-F387 ◽  
Author(s):  
J. P. Bonvalet ◽  
P. Pradelles ◽  
N. Farman
Keyword(s):  
Renal Tubule ◽  
Thick Ascending Limb ◽  
Pge2 Synthesis ◽  
Complex Interactions ◽  
Quantitative Measurements ◽  
Medullary Thick Ascending Limb ◽  
Exogenous Substrate ◽  
Collecting Tubule ◽  
The Difference ◽  
Cortical Collecting Tubule

The sites of synthesis and action of prostaglandins (PGs) along the renal tubule are examined. We focused our attention on experiments performed on well-defined nephron segments, using direct quantitative measurements of prostaglandin synthesis by radio- or enzyme-immunoassay. On the other hand, we selected, among the described effects of PGs, those obtained on precisely defined tubular segments. Among PGs, PGE2 synthesis is largely predominant all along the tubule. Its main sites of synthesis are the medullary collecting tubule and, to a lesser extent, the cortical collecting tubule and the thin limb of Henle's loop. Synthesis of PGE2 is amplified approximately tenfold in the presence of an excess exogenous substrate, arachidonic acid, compared with values measured without addition of substrate. Other eicosanoids have roughly the same distribution along the tubule as PGE2. Their rate of synthesis is, however, much less than that of PGE2, approximately 20-fold lower for PGF2 alpha and 6-keto-PGF1 alpha, and 100-fold lower for thromboxane B2 (TxB2). This contrasts with glomerular PG synthesis, where the difference between the production of PGE2 and other eicosanoids is much less marked. Most studies agree that antidiuretic hormone (ADH) and kinins augment PGE2 synthesis, whereas corticosteroids decrease it, at least in the collecting tubule. Direct effects of PGE2 have been described mainly in the medullary thick ascending limb and collecting tubule. They generally consist of a decrease in transepithelial potential difference and reabsorptive rates of water and solutes, in particular sodium and chloride. However, whatever the solute or tubular segment concerned, some studies failed to find such effects. The bulk of evidence suggests that ADH and PGs interact in kidney tubular cells. It is generally accepted that PGs antagonize the hydrosmotic effects of ADH in the collecting tubule. The mechanisms underlying these complex interactions are still under discussion: they probably involve several types of receptors and pathways for ADH action, which intervene in the modulation of both PG synthesis and cyclic nucleotides, and several types of PG receptors, either stimulatory or inhibitory to adenylate cyclase.

NEM-sensitive ATPase activity in rat nephron: effect of metabolic acidosis and alkalosis

1990 ◽  
Vol 258 (2) ◽  
pp. F297-F304 ◽  
Author(s):  
S. Sabatini ◽  
M. E. Laski ◽  
N. A. Kurtzman
Keyword(s):  
Enzyme Activity ◽  
Metabolic Acidosis ◽  
Atpase Activity ◽  
Collecting Duct ◽  
Proximal Convoluted Tubule ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Chronic Metabolic Acidosis ◽  
Collecting Tubule ◽  
Cortical Collecting Tubule

The present study was designed to quantitate the amount and to map the localization of N-ethylmaleimide (NEM)-sensitive adenosinetriphosphatase (ATPase) activity in microdissected segments of the rat nephron. After complete nephron mapping the effect of chronic metabolic acidosis and alkalosis on enzyme activity was determined. In control animals the highest enzyme activity was found in the early proximal convoluted tubule of juxtamedullary nephrons; superficial early proximal tubule as well as medullary and cortical thick ascending limbs and collecting ducts also contained substantial activity. Enzyme activity in the papillary collecting duct before entry into the ducts of Bellini was 329 +/- 93 pmol.mm-1.h-1 (n = 8); after entry, however, enzyme activity was approximately one-fourth that value (60 +/- 9 pmol.mm-1.h-1, n = 8, P less than 0.01). No NEM-sensitive ATPase activity was found in the thin limbs of the loop of Henle. Enzyme activity increased in both the medullary and cortical thick ascending limbs as well as in the cortical collecting tubule in response to NH4Cl-induced chronic metabolic acidosis; in the cortical collecting duct, metabolic acidosis increased maximum activity (Vmax) but did not change Michaelis-Menten constant (Km). In the proximal convoluted tubule, enzyme activity decreased with metabolic acidosis. Bicarbonate loading had no effect on enzyme activity except in the most distal portion of the collecting duct where it was stimulated. These results show that NEM-sensitive ATPase activity exists throughout much of the rat nephron. These data suggest that both the cortical collecting tubule and thick ascending limb are regulatory sites of distal urinary acidification during acid loading.

Effect of cAMP on prostaglandin E2 production in cultured rat inner medullary collecting tubule cells

1986 ◽  
Vol 251 (4) ◽  
pp. F671-F677 ◽  
Author(s):  
I. Teitelbaum ◽  
J. N. Mansour ◽  
T. Berl
Keyword(s):  
Arachidonic Acid ◽  
Collecting Duct ◽  
Feedback System ◽  
Ionophore A23187 ◽  
Homogeneous Population ◽  
Pge2 Synthesis ◽  
Collecting Tubule ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct ◽  
Pg E2

Studies were performed to determine whether cAMP impairs prostaglandin (PG) E2 production in a homogeneous population of cultured rat inner medullary collecting duct cells. Three structurally different cAMP analogues were shown to decrease PGE2 synthesis by 48.4% in the basal state and by 49.3% in response to the divalent cation ionophore A23187 (5 microM). Thromboxane B2 production was similarly suppressed. An increase in endogenous cAMP by forskolin also decreased PGE2 synthesis. To determine the locus of the cAMP effect we examined the response to exogenously added arachidonic acid. At a concentration of arachidonic acid (5 micrograms/ml) sufficient to render the phospholipase-dependent fraction negligible (as evidenced by the lack of a mepacrine effect), cAMP had no effect on PGE2 production, suggesting phospholipase as the site of cAMP action. Further evidence for a phospholipase-mediated mechanism derives from studies employing [5,6,8,9,11,12,14,15-3H(N)]arachidonic acid in which cAMP analogues had no effect on the rate of cellular arachidonic acid incorporation, but did impair the release of tritiated arachidonic acid in response to ionophore. These results suggest the existence of a negative feedback system that, by impairing phospholipase activity and PGE2 synthesis, could enhance the action of cAMP in the antidiuretic state.

Abnormal postpartum renal development and cystogenesis in the bcl-2 (-/-) mouse

1996 ◽  
Vol 271 (1) ◽  
pp. F184-F193 ◽  
Author(s):  
C. M. Sorenson ◽  
B. J. Padanilam ◽  
M. R. Hammerman
Keyword(s):  
Cellular Proliferation ◽  
Kidney Development ◽  
Apoptotic Cell ◽  
Collecting Duct ◽  
Distal Tubule ◽  
Renal Development ◽  
Thick Ascending Limb ◽  
Renal Hypoplasia ◽  
Cellular Origin ◽  
Bax Protein

Mice deficient for B cell leukemia/lymphoma gene 2 [bcl-2(-/-) mice] manifest congenital renal hypoplasia and develop multicystic kidney disease and renal failure postnatally. To characterize postpartum renal development, to identify the cellular origin of the cysts, and to provide insight into the role that bcl-2 deficiency plays in the cystogenic process, we examined the morphology of kidneys from bcl-2 (-/-) mice and wild-type littermates [bcl-2 (+/+)] from birth (P0) to postpartum day 28 (P28), determined whether abnormalities of cellular proliferation and apoptosis accompany cyst development, and characterized expression of the bcl-2-related protein, bax. Between P0 and P7, kidneys from bcl-2 (-/-) and bcl-2 (+/+) mice undergo a comparable increase in weight and have similar histological appearances. However, during the next 2 wk of life, weight gain in kidneys from bcl-2 (-/-) mice is reduced compared with that in kidneys from bcl-2 (+/+) animals, and cysts develop in tubules with staining characteristics of proximal tubule, distal tubule/medullary thick ascending limb of Henle's loop, and collecting duct. Unaffected glomeruli and proximal tubules in kidneys of bcl-2 (-/-) mice undergo compensatory growth. Cystogenesis is accompanied by enhanced incorporation of 5-bromo-2'-deoxyuridine in cells within cortex and medulla and apoptosis of cells within cysts and in the renal interstitium. Bax protein is expressed in the distal tubule in kidneys of bcl-2 (+/+) and bcl-2 (-/-) mice and in some, but not all cysts. We conclude that abnormal regulation of DNA synthesis and apoptosis accompany cystogenesis in bcl-2 (-/-) mice during postpartum kidney development. Continued expression of bax could enhance apoptotic cell death.0

Low Na intake suppresses expression of CYP2C23 and arachidonic acid-induced inhibition of ENaC

2006 ◽  
Vol 291 (6) ◽  
pp. F1192-F1200 ◽  
Author(s):  
Peng Sun ◽  
Dao-Hong Lin ◽  
Tong Wang ◽  
Elisa Babilonia ◽  
Zhijian Wang ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Collecting Duct ◽  
Inhibitory Effect ◽  
Thick Ascending Limb ◽  
Epoxyeicosatrienoic Acid ◽  
Distal Nephron ◽  
Na Transport ◽  
Deficient Diet ◽  
Epithelial Na Channels ◽  
Clamp Study

We previously demonstrated that arachidonic acid (AA) inhibits epithelial Na channels (ENaC) through the cytochrome P-450 (CYP) epoxygenase-dependent pathway ( 34 ). In the present study, we tested the hypothesis that low Na intake suppresses the expression of CYP2C23, which is mainly responsible for converting AA to epoxyeicosatrienoic acid (EET) in the kidney ( 11 ) and attenuates the AA-induced inhibition of ENaC. Immunostaining showed that CYP2C23 is expressed in the Tamm-Horsfall protein (THP)-positive and aquaporin 2 (AQP2)-positive tubules. This suggests that CYP2C23 is expressed in the thick ascending limb (TAL) and collecting duct (CD). Na restriction significantly suppressed the expression of CYP2C23 in the TAL and CD. Western blot also demonstrated that the expression of CYP2C23 in renal cortex and outer medulla diminished in rats on Na-deficient diet (Na-D) but increased in those on high-Na diet (4%). Moreover, the content of 11,12-epoxyeicosatrienoic acid (EET) decreased in the isolated cortical CD from rats on Na-D compared with those on a normal-Na diet (0.5%). Patch-clamp study showed that application of 15 μM AA inhibited the activity of ENaC by 77% in the CCD of rats on a Na-D for 3 days. However, the inhibitory effect of AA on ENaC was significantly attenuated in rats on Na-D for 14 days. Furthermore, inhibition of CYP epoxygenase with MS-PPOH increased the ENaC activity in the CCD of rats on a control Na diet. We also used microperfusion technique to examine the effect of MS-PPOH on Na transport in the distal nephron. Application of MS-PPOH significantly increased Na absorption in the distal nephron of control rats but had no significant effect on Na absorption in rats on Na-D for 14 days. We conclude that low Na intake downregulates the activity and expression of CYP2C23 and attenuates the inhibitory effect of AA on Na transport.

scholarly journals Differential Expression Patterns of Claudins, Tight Junction Membrane Proteins, in Mouse Nephron Segments

2002 ◽  
Vol 13 (4) ◽  
pp. 875-886 ◽  
Author(s):  
Yumiko Kiuchi-Saishin ◽  
Shimpei Gotoh ◽  
Mikio Furuse ◽  
Akiko Takasuga ◽  
Yasuo Tano ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Tight Junction ◽  
Polyclonal Antibodies ◽  
Collecting Duct ◽  
Expression Patterns ◽  
Distal Tubule ◽  
Northern Blotting ◽  
Thick Ascending Limb ◽  
Specific Expression ◽  
Nephron Segments

ABSTRACT. As the first step in understanding the physiologic functions of claudins (tight junction integral membrane proteins) in nephrons, the expression of claudin-1 to -16 in mouse kidneys was examined by Northern blotting. Among these claudins, only claudin-6, -9, -13, and -14 were not detectable. Claudin-5 and -15 were detected only in endothelial cells. Polyclonal antibodies specific for claudin-7 and -12 were not available. Therefore, the distributions of claudin-1, -2, -3, -4, -8, -10, -11, and -16 in nephron segments were examined with immunofluorescence microscopy. For identification of individual segments, antibodies specific for segment markers were used. Immunofluorescence microscopic analyses of serial frozen sections of mouse kidneys with polyclonal antibodies for claudins and segment markers revealed that claudins demonstrated very complicated, segment-specific, expression patterns in nephrons, i.e., claudin-1 and -2 in Bowman’s capsule, claudin-2, -10, and -11 in the proximal tubule, claudin-2 in the thin descending limb of Henle, claudin-3, -4, and -8 in the thin ascending limb of Henle, claudin-3, -10, -11, and -16 in the thick ascending limb of Henle, claudin-3 and -8 in the distal tubule, and claudin-3, -4, and -8 in the collecting duct. These segment-specific expression patterns of claudins are discussed, with special reference to the physiologic functions of tight junctions in nephrons.

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