Endothelin synthesis by rabbit renal tubule cells

1991 ◽  
Vol 261 (2) ◽  
pp. F221-F226 ◽  
Author(s):  
D. E. Kohan
Keyword(s):  
High Performance ◽  
Renal Tubule ◽  
De Novo ◽  
Collecting Duct ◽  
Cell Types ◽  
Phospholipid Metabolism ◽  
Thick Ascending Limb ◽  
Medullary Collecting Duct ◽  
Tubule Cells ◽  
Endothelin 3

Endothelins regulate nephron sodium and water transport, prostaglandin E2 (PGE2) synthesis, and phospholipid metabolism. Recent studies suggest that renal tubule cells synthesize endothelins. To determine which nephron sites have such potential, endothelin production by cells derived from different nephron segments was examined. Immunoreactive endothelin 1 (ET-1) and endothelin 3 (ET-3) were measured in supernatants of cultured rabbit proximal tubule (PT), medullary thick ascending limb (MTAL), cortical collecting tubule (CCT), and inner medullary collecting duct (IMCD) cells. All cell types released immunoreactive ET-1 and ET-3. However, the amounts of endothelin produced differed as follows: IMCD greater than MTAL greater than CCT much greater than PT for ET-1 and IMCD greater than MTAL = PT = CCT for ET-3; in all cases ET-1 much greater than ET-3. To confirm de novo ET-3 synthesis, IMCD cells were labeled with [35S]cysteine, and the supernatant was immunoprecipitated with anti-ET-3 antibody. Sample and standard ET-3 eluted at identical positions on high-performance liquid chromatographs, confirming de novo synthesis of ET-3 by cultured IMCD cells. These data raise the possibility of an important functional role for nephron-derived endothelin and, in particular, endothelin produced by tubule cells in the medulla.

RT-PCR microlocalization of mRNA for guanylyl cyclase-coupled ANF receptor in rat kidney

1991 ◽  
Vol 261 (6) ◽  
pp. F1080-F1087 ◽  
Author(s):  
Y. Terada ◽  
T. Moriyama ◽  
B. M. Martin ◽  
M. A. Knepper ◽  
A. Garcia-Perez
Keyword(s):  
Guanylyl Cyclase ◽  
Renal Tubule ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Renal Tubules ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Vasa Recta ◽  
Vascular Elements ◽  
Medullary Collecting Duct

Microlocalization of mRNA coding for the guanylyl cyclase-coupled atrial natriuretic factor (ANF) receptor was carried out in the rat kidney. We used a combination of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected renal tubule segments, glomeruli, and vasa recta bundles. Relative quantitation of the resulting amplified cDNA utilized densitometry of autoradiograms from Southern blots probed with a specific 32P-labeled probe. Among renal tubule segments, the largest signal was found in the terminal inner medullary collecting duct (IMCD). Slightly smaller signals were found in the initial IMCD and in loop of Henle segments from the inner medulla. Readily detectable signals were also seen in the following segments (in descending order): cortical collecting duct, proximal convoluted tubule, medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule, and outer medullary collecting duct. Large signals were also detected in glomeruli and in vasa recta bundles from the inner stripe of the outer medulla. Based on these results, we conclude that 1) renal microlocalization of specific mRNAs coding for hormone receptors is feasible through application of the RT-PCR procedure in microdissected renal tubules and vascular elements, and 2) the gene for the guanylyl cyclase-coupled ANF receptor is broadly expressed along the nephron, raising the possibility that multiple sites of ANF action are present.

scholarly journals The co-localization of stanniocalcin protein, mRNA and kidney cell markers in the rat kidney

1998 ◽  
Vol 158 (2) ◽  
pp. 183-189 ◽  
Author(s):  
CK Wong ◽  
MA Ho ◽  
GF Wagner
Keyword(s):  
In Situ Hybridization ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Thick Ascending Limb ◽  
Mrna Levels ◽  
Glycoprotein Hormone ◽  
Anion Exchanger 1 ◽  
Medullary Collecting Duct ◽  
Tubule Cells

Stanniocalcin (STC) is a glycoprotein hormone that was first discovered in fish and recently identified in mammals. STC immunoreactive (STCir) cells have been identified in rat kidney and there is also evidence that the hormone functions as a regulator of renal phosphate homeostasis. In the present study we have identified STCir cells and tubules in the rat kidney by correlative immunocytochemistry using antibodies to STC and specific antigenic markers (Tamm-Horsfall protein and anion exchanger-1). The cellular sites of STC gene expression were also identified by in situ hybridization. Correlative immunocytochemistry revealed that STCir was present in all proximal straight tubule cells, all cortical thick ascending limb cells, all distal convoluted tubule cells, and both principal and alpha-intercalated cells of the collecting duct system. On the other hand, in situ hybridization revealed that the STC gene was expressed only in cortical and medullary collecting duct cells. This suggests either that STC is being sequestered by segments that do not express the gene (making them putative targets of the hormone), or that STC mRNA levels were simply too low in these other segments to be detected by in situ hybridization.

scholarly journals Inhibition of calcium signaling in descending vasa recta endothelia by ANG II

2000 ◽  
Vol 278 (4) ◽  
pp. H1248-H1255 ◽  
Author(s):  
Thomas L. Pallone ◽  
Erik P. Silldorff ◽  
Zhong Zhang
Keyword(s):  
Collecting Duct ◽  
No Production ◽  
Vascular Bundles ◽  
Thick Ascending Limb ◽  
Ang Ii ◽  
Vasomotor Tone ◽  
Medullary Thick Ascending Limb ◽  
Vasa Recta ◽  
Medullary Collecting Duct ◽  
Peak Phase

The intracellular calcium ([Ca2+]i) response of outer medullary descending vasa recta (OMDVR) endothelia to ANG II was examined in fura 2-loaded vessels. Abluminal ANG II (10− 8 M) caused [Ca2+]i to fall in proportion to the resting [Ca2+]i ( r =0.82) of the endothelium. ANG II (10− 8 M) also inhibited both phases of the [Ca2+]i response generated by bradykinin (BK, 10− 7 M), 835 ± 201 versus 159 ± 30 nM (peak phase) and 169 ± 26 versus 103 ± 14 nM (plateau phase) (means ± SE). Luminal ANG II reduced BK (10− 7 M)-stimulated plateau [Ca2+]i from 180 ± 40 to 134 ± 22 nM without causing vasoconstriction. Abluminal ANG II added to the bath after luminal application further reduced [Ca2+]i to 113 ± 9 nM and constricted the vessels. After thapsigargin (TG) pretreatment, ANG II (10− 8 M) caused [Ca2+]i to fall from 352 ± 149 to 105 ± 37 nM. This effect occurred at a threshold ANG II concentration of 10− 10 M and was maximal at 10− 8 M. ANG II inhibited both the rate of Ca2+ entry into [Ca2+]i-depleted endothelia and the rate of Mn2+ entry into [Ca2+]i-replete endothelia. In contrast, ANG II raised [Ca2+]i in the medullary thick ascending limb and outer medullary collecting duct, increasing [Ca2+]i from baselines of 99 ± 33 and 53 ± 11 to peaks of 200 ± 47 and 65 ± 11 nM, respectively. We conclude that OMDVR endothelia are unlikely to be the source of ANG II-stimulated NO production in the medulla but that interbundle nephrons might release Ca2+-dependent vasodilators to modulate vasomotor tone in vascular bundles.

Regional and segmental localization of AE2 anion exchanger mRNA and protein in rat kidney

1995 ◽  
Vol 269 (4) ◽  
pp. F461-F468 ◽  
Author(s):  
F. C. Brosius ◽  
K. Nguyen ◽  
A. K. Stuart-Tilley ◽  
C. Haller ◽  
J. P. Briggs ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Chloride Concentration ◽  
Transepithelial Transport ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Base Exchange ◽  
Medullary Thick Ascending Limb ◽  
Nephron Segment ◽  
Medullary Collecting Duct

Chloride/base exchange activity has been detected in every mammalian nephron segment in which it has been sought. However, in contrast to the Cl-/HCO3- exchanger AE1 in type A intercalated cells, localization of AE2 within the kidney has not been reported. We therefore studied AE2 expression in rat kidney. AE2 mRNA was present in cortex, outer medulla, and inner medulla. Semiquantitative polymerase chain reaction of cDNA from microdissected tubules revealed AE2 cDNA levels as follows [copies of cDNA derived per mm tubule (+/- SE)]: proximal convoluted tubule, 688 +/- 161; proximal straight tubule, 652 +/- 189; medullary thick ascending limb, 1,378 +/- 226; cortical thick ascending limb, 741 +/- 24; cortical collecting duct, 909 +/- 71; and outer medullary collecting duct, 579 +/- 132. AE2 cDNA was also amplified in thin limbs and in inner medullary collecting duct. AE2 polypeptide was detected in all kidney regions. AE2 mRNA and protein were also detected in several renal cell lines. The data are compatible with the postulated roles of AE2 in maintenance of intracellular pH and chloride concentration and with its possible participation in transepithelial transport.

Renal acid–base homeostasis

2015 ◽  
Author(s):  
Carsten A. Wagner ◽  
Olivier Devuyst
Keyword(s):  
De Novo ◽  
Morphological Changes ◽  
Collecting Duct ◽  
Ammonium Phosphate ◽  
Transport Proteins ◽  
Thick Ascending Limb ◽  
Acid Base ◽  
Major Site ◽  
Bicarbonate Reabsorption ◽  
Adaptive Mechanisms

The kidney is central to acid–base homeostasis. Major processes are reabsorption of filtered bicarbonate, de novo synthesis of bicarbonate from ammoniagenesis, and net excretion of protons. The latter requires buffers such as ammonium, phosphate, citrate and other bases binding protons (so-called titratable acids). The proximal tubule is the major site of bicarbonate reabsorption and only site of ammoniagenesis. The thick ascending limb and the distal convoluted tubule handle ammonia/ammonium and complete bicarbonate reabsorption. The collecting duct system excretes protons and ammonium, but may switch to net bicarbonate secretion. The kidney displays a great plasticity to adapt acid or bicarbonate excretion. Angiotensin II, aldosterone and endothelin are involved in regulating these processes, and they induce morphological changes along the nephron. Inborn and acquired disorders of renal acid–base handling are caused by mutations in acid–base transport proteins or by dysregulation of adaptive mechanisms.

Cell-specific expression of amiloride-sensitive, Na(+)-conducting ion channels in the kidney

1996 ◽  
Vol 271 (4) ◽  
pp. C1303-C1315 ◽  
Author(s):  
F. Ciampolillo ◽  
D. E. McCoy ◽  
R. B. Green ◽  
K. H. Karlson ◽  
A. Dagenais ◽  
...  
Keyword(s):  
Cell Lines ◽  
Collecting Duct ◽  
Extracellular Fluid ◽  
Cation Channel ◽  
Thick Ascending Limb ◽  
Distal Nephron ◽  
Rt Pcr ◽  
Specific Expression ◽  
Nephron Segments ◽  
Medullary Collecting Duct

Amiloride-sensitive, electrogenic Na+ absorption across the distal nephron plays a vital role in regulating extracellular fluid volume and blood pressure. Recently, two amiloride-sensitive, Na(+)-conducting ion channel cDNAs were cloned. One, an epithelial Na(+)-selective channel (ENaC), is responsible for Na+ absorption throughout the distal nephron. The second, a guanosine 3',5'-cyclic monophosphate (cGMP)-inhibitable cation channel, is conductive to Na+ and Ca2+ and contributes to Na+ absorption across the inner medullary collecting duct (IMCD). As a first step toward understanding the segment-specific contributions(s) of cGMP-gated cation channels and ENaC to Na+ and Ca2+ uptake along the nephron, we used in situ reverse transcription-polymerase chain reaction (RT-PCR) hybridization, solution-phase RT-PCR, and Western blot analysis to examine the nephron and cell-specific expression of these channels in mouse kidney cell lines and/or dissected nephron segments. cGMP-gated cation channel mRNA was detected in proximal tubule, medullary thick ascending limb (mTAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and IMCD. cGMP-gated cation channel protein was detected in DCT, CCD, and IMCD cell lines. These observations suggest that hormones that modulate intracellular cGMP levels may regulate Na+, and perhaps Ca2+, uptake throughout the nephron. mRNA for alpha-mENaC, a subunit of the mouse ENaC, was detected in mTAL, DCT, CCD, OMCD, and IMCD. Coexpression of alpha-mENaC and cGMP-gated cation channel mRNAs in mTAL, DCT, CCD, OMCD, and IMCD suggests that both channels may contribute to Na+ absorption in these nephron segments.

Antibody-independent localization of the electroneutral Na+-HCO3− cotransporter NBCn1 (slc4a7) in mice

2008 ◽  
Vol 294 (2) ◽  
pp. C591-C603 ◽  
Author(s):  
Ebbe Boedtkjer ◽  
Jeppe Praetorius ◽  
Ernst-Martin Füchtbauer ◽  
Christian Aalkjaer
Keyword(s):  
Smooth Muscle ◽  
Epithelial Cells ◽  
Smooth Muscle Cells ◽  
Collecting Duct ◽  
Muscle Cells ◽  
Thick Ascending Limb ◽  
Bladder Smooth Muscle ◽  
Kidney Pelvis ◽  
Medullary Collecting Duct ◽  
Cerebellar Purkinje Cells

The expression pattern of the electroneutral Na+-HCO3−cotransporter NBCn1 (slc4a7) was investigated by β-galactosidase staining of mice with a LacZ insertion into the NBCn1 gene. This method is of particular value because it is independent of immunoreactivity. We find that the NBCn1 promoter is active in a number of tissues where NBCn1 has previously been functionally or immunohistochemically identified, including a broad range of blood vessels (vascular smooth muscle cells and endothelial cells), kidney thick ascending limb and medullary collecting duct epithelial cells, the epithelial lining of the kidney pelvis, duodenal enterocytes, choroid plexus epithelial cells, hippocampus, and retina. Kidney corpuscles, colonic mucosa, and nonvascular smooth muscle cells (from the urinary bladder, trachea, gastrointestinal wall, and uterus) were novel areas of promoter activity. Atrial but not ventricular cardiomyocytes were stained. In the brain, distinct layers of the cerebral cortex and cerebellar Purkinje cells were stained as was the dentate nucleus. No staining of skeletal muscle or cortical collecting ducts was observed. RT-PCR analyses confirmed the expression of NBCn1 and β-galactosidase in selected tissues. Disruption of the NBCn1 gene resulted in reduced NBCn1 expression, and in bladder smooth muscle cells, reduced amiloride-insensitive Na+-dependent HCO3− influx was observed. Furthermore, disruption of the NBCn1 gene resulted in a lower intracellular steady-state pH of bladder smooth muscle cells in the presence of CO2/HCO3− but not in its nominal absence. We conclude that NBCn1 has a broad expression profile, supporting previous findings based on immunoreactivity, and suggest several new tissues where NBCn1 may be important.

scholarly journals Histochemical carbonic anhydrase in rat inner medullary collecting duct.

1992 ◽  
Vol 40 (10) ◽  
pp. 1535-1545 ◽  
Author(s):  
J G Kleinman ◽  
J L Bain ◽  
C Fritsche ◽  
D A Riley
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Light And Electron Microscopy ◽  
Cell Types ◽  
Intercalated Cells ◽  
Cell Type ◽  
Variable Intensity ◽  
Inner Medullary Collecting Duct ◽  
Majority Population ◽  
Medullary Collecting Duct

Rat inner medullary collecting duct (IMCD) secretes substantial amounts of H+. However, carbonic anhydrase (CA), a concomitant of H+ secretion, has been generally reported absent in this segment. To reexamine this problem, we investigated CA and the morphological phenotypes of cells comprising the IMCD by CA histochemistry, using a modified Hansson technique with light and electron microscopy. Throughout the medulla, tubule cells exhibit histochemical CA activity. In the initial third of the inner medulla, a small proportion have features of intercalated cells and demonstrate some degree of CA activity. However, the majority population in the early portions of the IMCD appears to consist of principal cells. These also show CA staining of widely variable intensity, both among and within cells. A third cell type, previously called "IMCD cells", appears in the middle portion of the IMCD and is the only cell type present near the papilla tip. In contrast to previous reports, these "IMCD cells" have histochemical CA staining, also of highly variable intensity. These results demonstrate that stainable carbonic anhydrase to support acidification is present throughout the rat IMCD, both in intercalated cells and in some cells clearly not of this type. Therefore, the presence of CA is not specific for the intercalated cell type and suggests that other cell types may participate in acid secretion in IMCD.

Immunolocalization of electroneutral Na-HCO3 − cotransporter in rat kidney

2000 ◽  
Vol 279 (5) ◽  
pp. F901-F909 ◽  
Author(s):  
Henrik Vorum ◽  
Tae-Hwan Kwon ◽  
Christiaan Fulton ◽  
Brian Simonsen ◽  
Inyeong Choi ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Electron Microscopic Level ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Electron Microscopic ◽  
Outer Medulla ◽  
Outer Stripe ◽  
Medullary Collecting Duct

An electroneutral Na-HCO3 − cotransporter (NBCN1) was recently cloned, and Northern blot analyses indicated its expression in rat kidney. In this study, we determined the cellular and subcellular localization of NBCN1 in the rat kidney at the light and electron microscopic level. A peptide-derived antibody was raised against the COOH-terminal amino acids of NBCN1. The affinity-purified antibody specifically recognized one band, ∼180 kDa, in rat kidney membranes. Peptide- N-glycosidase F deglycosylation reduced the band to ∼140 kDa. Immunoblotting of membrane fractions from different kidney regions demonstrated strong signals in the inner stripe of the outer medulla (ISOM), weaker signals in the outer stripe of the outer medulla and inner medulla, and no labeling in cortex. Immunocytochemistry demonstrated that NBCN1 immunolabeling was exclusively observed in the basolateral domains of thick ascending limb (TAL) cells in the outer medulla (strongest in ISOM) but not in the cortex. In addition, collecting duct intercalated cells in the ISOM and in the inner medulla also exhibited NBCN1 immunolabeling. Immunoelectron microscopy demonstrated that NBCN1 labeling was confined to the basolateral plasma membranes of TAL and collecting duct type A intercalated cells. Immunolabeling controls were negative. By using 2,7-bis-carboxyethyl-5,6-caboxyfluorescein, intracellular pH transients were measured in kidney slices from ISOM and from mid-inner medulla. The results revealed DIDS-sensitive, Na- and HCO3 −-dependent net acid extrusion only in the ISOM but not in mid-inner medulla, which is consistent with the immunolocalization of NBCN1. The localization of NBCN1 in medullary TAL cells and medullary collecting duct intercalated cells suggests that NBCN1 may be important for electroneutral basolateral HCO3 − transport in these cells.

Immunolocalization of AE2 anion exchanger in rat kidney

1997 ◽  
Vol 273 (4) ◽  
pp. F601-F614 ◽  
Author(s):  
Seth L. Alper ◽  
Alan K. Stuart-Tilley ◽  
Daniel Biemesderfer ◽  
Boris E. Shmukler ◽  
Dennis Brown
Keyword(s):  
Epithelial Cells ◽  
Anion Exchanger ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Sodium Dodecyl ◽  
Colchicine Treatment ◽  
Cell Types ◽  
Staining Intensity ◽  
Thick Ascending Limb ◽  
Renal Epithelial Cell

The cellular and subcellular localizations of the AE2 anion exchanger in rat kidney have remained elusive despite detection of moderately abundant AE2 mRNA and AE2 polypeptide in all kidney regions. In this report a simple epitope unmasking technique has allowed the immunolocalization of AE2 antigenic sites in basolateral membranes of several rat kidney tubular epithelial cells. AE2 immunostaining was faint or absent in the glomerulus and proximal tubule, present in descending and ascending thin limbs, and stronger in the medullary thick ascending limb (MTAL). A lower staining intensity was found in cortical thick ascending limbs and even less in the distal convoluted tubule. In contrast, there was an enhanced staining in the macula densa. In principal cells (PC) of the connecting segment, AE2 was undetectable but gradually increased in intensity along the collecting duct, with strongest staining in inner medullary collecting duct (IMCD) PC. A sodium dodecyl sulfate-sensitive AE2-related Golgi epitope was also detected in some interstitial and endothelial cells of the inner medulla and in epithelial cells of IMCD and MTAL. Colchicine treatment of the intact animal altered the distribution of this Golgi-associated epitope but left plasmalemmal AE2 undisturbed. Reverse transcription-polymerase chain reaction detected AE2a, AE2b, and AE2c2 but not AE2c1 transcripts in rat kidney mRNA. The results suggest a widespread occurrence of the AE2 protein in several renal epithelial cell types.

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