Chronic hypokalemia enhances expression of the H(+)-K(+)-ATPase alpha 2-subunit gene in renal medulla

1996 ◽  
Vol 271 (2) ◽  
pp. F314-F321 ◽  
Author(s):  
K. Y. Ahn ◽  
K. Y. Park ◽  
K. K. Kim ◽  
B. C. Kone
Keyword(s):  
In Situ Hybridization ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Renal Medulla ◽  
Northern Analysis ◽  
Thick Ascending Limb ◽  
Subunit Gene ◽  
Enhanced Expression ◽  
Medullary Collecting Duct

Recent molecular and physiological studies suggested that at least two H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) isozymes are expressed in the rat kidney and that these ion pumps respond to changes in dietary potassium balance. We used Northern analysis and in situ hybridization to analyze the expression of mRNA encoding the "colonic" isoform of the H(+)-K(+)-ATPase alpha-subunit (HK alpha 2) in normal and potassium-deprived (2 wk) rats. Control rats exhibited low levels of HK alpha 2 mRNA in the cortical and medullary thick ascending limb, distal convoluted tubule, connecting segment, and the entire collecting duct. The potassium-deprived rats expressed approximately fivefold higher levels of HK alpha 2 mRNA in the outer and inner medulla compared with controls, as well as hypertrophy and increased in situ hybridization signal in the intercalated cells of the inner stripe of the outer medullary collecting duct and the proximal inner medullary collecting duct. In contrast, renal cortical expression of HK alpha 2 mRNA was low and comparable in the two groups. Our results suggest that enhanced expression of the HK alpha 2 subunit gene in the renal medulla contributes to potassium conservation during chronic hypokalemia.

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.

Endothelin receptor mRNA expression in renal medulla identified by in situ RT-PCR

1995 ◽  
Vol 269 (3) ◽  
pp. F449-F457 ◽  
Author(s):  
L. H. Chow ◽  
S. Subramanian ◽  
G. J. Nuovo ◽  
F. Miller ◽  
E. P. Nord
Keyword(s):  
Mrna Expression ◽  
Collecting Duct ◽  
Renal Medulla ◽  
Receptor Subtypes ◽  
Rt Pcr ◽  
Receptor Mrna ◽  
Pcr Products ◽  
Medullary Collecting Duct ◽  
Labeled Probe

Three subtypes of endothelin (ET) receptors have been identified by cDNA cloning, namely ET-RA, ET-RB, and ET-RC. In the current study the precise cellular distribution of the ET receptor subtypes in the renal medulla was explored by detecting the corresponding polymerase chain reaction (PCR)-amplified cDNAs by in situ reverse transcription (RT)-PCR. The PCR-amplified cDNAs were detected either by direct incorporation using digoxigenin-dUTP (dig-dUTP) as a nucleotide substrate in the PCR reaction or by in situ hybridization with the dig-dUTP-labeled probe. ET-RB mRNA was detected exclusively in the epithelial cells of the inner and outer medullary collecting duct. In contrast, ET-RA message was observed primarily in interstitial cells and pericytes of the vasae rectae in the outer and inner medulla. Southern blot analysis of PCR-amplified cDNAs reverse transcribed from extracted RNA of rat renal medulla confirmed the specificity of the RT-PCR products. ET-RC mRNA was not detected. We conclude that ET-RB is the major ET receptor found in rat renal medulla and is expressed exclusively on inner medullary collecting duct cells. The pattern of ET receptor mRNA expression described suggests different physiological actions for ET on the diverse cellular structures of the renal medulla.

Localization of rat CLC-K2 chloride channel mRNA in the kidney

1999 ◽  
Vol 276 (4) ◽  
pp. F552-F558 ◽  
Author(s):  
Momono Yoshikawa ◽  
Shinichi Uchida ◽  
Atsushi Yamauchi ◽  
Akiko Miyai ◽  
Yujiro Tanaka ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Chloride Channel ◽  
Rat Kidney ◽  
Water Channel ◽  
Physiological Role ◽  
Thick Ascending Limb ◽  
Nephron Segments ◽  
Henle's Loop ◽  
Collecting Ducts

To gain insight into the physiological role of a kidney-specific chloride channel, CLC-K2, the exact intrarenal localization was determined by in situ hybridization. In contrast to the inner medullary localization of CLC-K1, the signal of CLC-K2 in our in situ hybridization study was highly evident in the superficial cortex, moderate in the outer medulla, and absent in the inner medulla. To identify the nephron segments where CLC-K2 mRNA was expressed, we performed in situ hybridization of CLC-K2 and immunohistochemistry of marker proteins (Na+/Ca2+exchanger, Na+-Cl−cotransporter, aquaporin-2 water channel, and Tamm-Horsfall glycoprotein) in sequential sections of a rat kidney. Among the tubules of the superficial cortex, CLC-K2 mRNA was highly expressed in the distal convoluted tubules, connecting tubules, and cortical collecting ducts. The expression of CLC-K2 in the outer and inner medullary collecting ducts was almost absent. In contrast, a moderate signal of CLC-K2 mRNA was observed in the medullary thick ascending limb of Henle’s loop, but the signal in the cortical thick ascending limb of Henle’s loop was low. These results clearly demonstrated that CLC-K2 was not colocalized with CLC-K1 and that its localization along the nephron segments was relatively broad compared with that of CLC-K1.

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.

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.

NBCn1 is a basolateral \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Na}^{+}\mathrm{-HCO}_{3}^{-}\) \end{document} cotransporter in rat kidney inner medullary collecting ducts

2004 ◽  
Vol 286 (5) ◽  
pp. F903-F912 ◽  
Author(s):  
Jeppe Praetorius ◽  
Young-Hee Kim ◽  
Elena V. Bouzinova ◽  
Sebastian Frische ◽  
Aleksandra Rojek ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Thick Ascending Limb ◽  
Medullary Thick Ascending Limb ◽  
Collecting Ducts ◽  
Basolateral Plasma Membrane ◽  
Medullary Collecting Duct ◽  
Plasma Membrane Domain

Primary cultures of rat inner medullary collecting duct (IMCD) cells Na+ dependently import [Formula: see text] across the basolateral membrane through an undefined transport protein. We used RT-PCR, immunoblotting, and immunohistochemistry to identify candidate proteins for this basolateral [Formula: see text] cotransport. The mRNA encoding the electroneutral [Formula: see text] cotransporter NBCn1 was detected as the only [Formula: see text] cotransporter in the rat inner medulla (IM) among the five characterized Na+-dependent [Formula: see text] transporters. The mRNA of a yet uncharacterized transporter-like protein, BTR1, was also present in the IM, but its expression in microdissected tubules seemed restricted to the thin limbs of Henle's loop. Immunoblotting confirmed the presence of NBCn1 as an ∼180-kDa protein of the rat IM. Anti-NBCn1 immunolabeling was confined to the basolateral plasma membrane domain of IMCD cells in the papillary two-thirds of the IM. Consistent with the presence of NBCn1, IMCD cells possessed stilbene-insensitive, Na+- and [Formula: see text]-dependent pH recovery after acidification, as assessed by fluorescence microscopy using a pH-sensitive intracellular dye. In furosemide-induced alkalotic rats, NBCn1 protein abundance was decreased in both the IM and inner stripe of outer medulla (ISOM) as determined by immunoblotting and immunohistochemistry. In contrast, NBCn1 abundance in the IM and ISOM was unchanged in NaHCO3-loaded animals, and the NBCn1 abundance increased only in the ISOM after NH4Cl loading. In conclusion, NBCn1 is a basolateral [Formula: see text] cotransporter of IMCD cells and is differentially regulated in IMCD and medullary thick ascending limb.

AE2 isoforms in rat kidney: immunohistochemical localization and regulation in response to chronic NH4Cl loading

2004 ◽  
Vol 286 (6) ◽  
pp. F1163-F1170 ◽  
Author(s):  
Sebastian Frische ◽  
Alexander S. Zolotarev ◽  
Young-Hee Kim ◽  
Jeppe Praetorius ◽  
Seth Alper ◽  
...  
Keyword(s):  
Splice Variants ◽  
Polyclonal Antibodies ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Immunohistochemical Localization ◽  
Thick Ascending Limb ◽  
Relative Distribution ◽  
Medullary Thick Ascending Limb ◽  
Medullary Collecting Duct ◽  
Antibody Specificities

Three splice variants of anion exchanger (AE)2 (AE2a, b, and c) have been described in the rat, but their relative distribution in rat kidney is not known. The purpose of this study was to describe the segmental and cellular distribution of the AE2 isoforms in the rat kidney and to evaluate whether the expression levels of these AE2 isoforms are regulated independently in response to chronic NH4Cl loading. Two polyclonal antibodies were generated, respectively, recognizing a NH2-terminal peptide unique to AE2a and an amino acid sequence common to AE2a and AE2b. Antibody specificities were tested using cells transfected separately with the AE2a, AE2b, and AE2c isoforms. Immunohistochemistry on sections of paraffin-embedded rat kidneys showed a distribution of AE2a/AE2b labeling in the kidney similar to the distribution of AE2 in the rat kidney reported previously. AE2 is highly expressed in the medullary thick ascending limb, cortical thick ascending limb (cTAL), and macula densa. The pattern of AE2a-specific labeling differed from the pattern of AE2a/AE2b labeling in that relatively more of the total immunolabel was observed in the terminal inner medullary collecting duct. NH4Cl loading (0.033 mmol NH4Cl/g body wt for 7 days) did not change the labeling of AE2 isoforms in the medulla, whereas the labeling in the cortex was intensified and included more distal parts of the cTAL. Immunoblotting confirmed upregulation of AE2a/b expression in the cortex. These results indicate that AE2a and AE2b are differentially expressed and regulated in the rat kidney. The regulation following NH4Cl loading of AE2b in the cTAL suggests a role for AE2 in transepithelial bicarbonate reabsorption in this segment.

Enhanced expression of epithelial sodium channels in the renal medulla of Dahl S rats

2011 ◽  
Vol 89 (3) ◽  
pp. 159-168 ◽  
Author(s):  
Md. Shahrier Amin ◽  
Erona Reza ◽  
Esraa El-Shahat ◽  
Hong-Wei Wang ◽  
Frédérique Tesson ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Young Male ◽  
Renal Medulla ◽  
High Salt ◽  
High Salt Diet ◽  
Salt Diet ◽  
Enhanced Expression ◽  
Mrna And Protein Expression ◽  
Medullary Collecting Duct ◽  
Apical Localization

Inner medullary collecting duct (IMCD) cells from salt-sensitive (S) Dahl rats transport twice as much Na+ as cells from salt-resistant (R) rats, possibly related to dysregulation of the renal epithelial sodium channel (ENaC). The effect of a high-salt diet on ENaC expression in the inner medulla of S versus R rats has not yet been studied. Young, male S and R rats were placed on a regular-salt (0.3%) or high-salt (8%) diet for 2 or 4 weeks. mRNA and protein expression of ENaC subunits were studied by real-time PCR and immunoblotting. Intracellular distribution of the subunits in the IMCD was evaluated by immunohistochemistry. On regular salt, the abundance of the mRNA of β and γENaC was higher in the medulla of S rats than R rats. This was associated with a greater protein abundance of 90 kDa γENaC and higher immunoreactivity for both α and γ ENaC. High salt did not affect mRNA abundance in either strain and decreased apical staining of βENaC in IMCD of R rats. In contrast, high salt did not affect the higher apical localization of αENaC and increased the apical membrane staining for β and γENaC in the IMCD of S rats. Expression of ENaC subunits is enhanced in the medulla of S vs. R rats on regular salt, and further increased on high salt. The persistent high expression of αENaC and increase in apical localization of β and γENaC may contribute to greater retention of sodium in S rats on a high-salt diet.

Differential localization of prostaglandin E receptor subtypes in human kidney

1996 ◽  
Vol 270 (5) ◽  
pp. F912-F918 ◽  
Author(s):  
M. D. Breyer ◽  
L. Davis ◽  
H. R. Jacobson ◽  
R. M. Breyer
Keyword(s):  
In Situ Hybridization ◽  
Collecting Duct ◽  
Human Kidney ◽  
Prostaglandin E ◽  
Receptor Subtypes ◽  
Ep Receptors ◽  
Inner Medullary Collecting Duct ◽  
Ep Receptor ◽  
Medullary Collecting Duct

Four prostaglandin E2 (PGE2) receptors designated EP1, EP2, EP3, and EP4 have been pharmacologically identified, cloned, and sequenced. The present studies determined the intrarenal distribution of these EP-receptor subtypes in human kidney using in situ hybridization with riboprobes for the human EP receptors. mRNA for the phosphatidylinositol hydrolysis-coupled EP receptor was highly expressed in cortical, outer medullary, and inner medullary collecting duct. RNA for the Gi-coupled EP3 receptor was primarily expressed in the cortical and outer medullary collecting duct, as well as in the medullary thick ascending limb; however, it was absent from the inner medullary collecting duct. Expression of mRNA for EP1 and EP3 in connecting segment could not be excluded. There was no expression of the GS-coupled EP2 receptor mRNA detected in human kidney by in situ hybridization; however, mRNA for the GS-coupled EP4 receptor was highly expressed in the glomerulus. These studies demonstrate distinct regions of intrarenal expression for the different EP receptors and suggest that each receptor subtype may modulate different aspects of renal function in humans.

Low pH enhances expression of carbonic anhydrase II by cultured rat inner medullary collecting duct cells

1994 ◽  
Vol 266 (2) ◽  
pp. C508-C514 ◽  
Author(s):  
G. J. Schwartz ◽  
D. Brown ◽  
R. Mankus ◽  
E. A. Alexander ◽  
J. H. Schwartz
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Northern Analysis ◽  
Human Antibody ◽  
Inner Medullary Collecting Duct ◽  
Coding Regions ◽  
Steady State Levels ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct

Carbonic anhydrase (CA) facilitates the secretion of protons from renal epithelia by catalyzing the buffering of hydroxyl ions by CO2. We have previously found that inner medullary collecting duct (IMCD) cells cultured from rat kidney secrete protons and express CA II. Incubation of IMCD cells in acidic medium for 48 h has been shown to stimulate the secretion of protons by a protein synthesis-dependent process. To establish whether CA II might be involved in this process, IMCD cells were exposed to incubation media supplemented with 10(-7) M deoxycorticosterone acetate, pH 7.0 (acid) or pH 7.7 (control) for 48 h, and CA II mRNA and protein were quantitated. Part of the CA II cDNA was obtained by reverse transcription of total RNA from rat kidney followed by amplification using oligonucleotide primers derived from conserved areas in the coding regions of human, mouse, and chick CA II cDNAs in a polymerase chain reaction. By Northern analysis, steady-state levels of CA II mRNA from acid-incubated cells showed an increase of 80% compared with controls and 70% when expressed relative to a housekeeping mRNA, beta-actin. Western blot analysis using a human antibody to CA II showed an approximate doubling of CA II protein after acid incubation. By immunofluorescence microscopy, the domes of acid-incubated IMCD cells contained considerably more CA II-stained cells than found in control cultures. Thus incubation of IMCD cells in acid medium stimulates the expression of CA II mRNA and protein.(ABSTRACT TRUNCATED AT 250 WORDS)

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