Expression of transmembrane-type protein tyrosine phosphatase mRNA along rat nephron segments

1995 ◽  
Vol 268 (6) ◽  
pp. F1102-F1108 ◽  
Author(s):  
T. Kaneko ◽  
T. Moriyama ◽  
E. Imai ◽  
Y. Akagi ◽  
M. Arai ◽  
...  
Keyword(s):  
Rank Order ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Protein Tyrosine ◽  
Nephron Segments ◽  
Medullary Collecting Duct

Protein phosphorylation on tyrosine residues is one of the main cell signaling mechanisms. Cellular phosphotyrosyl levels are regulated by the activities of protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTPase). We have previously reported cDNA cloning of several types of PTPase from rat kidney, including LRP (leukocyte common antigen-related protein; also known as the transmembrane-type tyrosine phosphatase, i.e., RPTP alpha). LRP mRNA was shown to be abundant in the kidney; however, our understanding of the functional role of LRP in the kidney is very limited. To gain keener insight into the function of LRP in the kidney, our first approach was to reveal its mRNA distribution along rat nephron segments. Large signals were found in inner medulla by Northern blot analysis. By using a reverse transcription and polymerase chain reaction assay of individual microdissected tubule segments along the nephron [proximal convoluted tubule (PCT), medullary thick ascending limb (MTAL), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD)] and glomeruli, we revealed intrarenal localization of LRP mRNA. LRP mRNA was detected in all nephron segments tested but was relatively rich in the IMCD. Rank order of the signal intensity was IMCD > PCT = OMCD > CCD > MTAL = glomeruli. Immunohistochemistry also revealed that LRP was abundant in IMCD. This pattern of expression gives rise to an interesting possibility that LRP might be involved in the specific renal tubule function, such as urinary concentrating mechanism; however, further study is required to describe the function of LRP in more detail.

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.

Microlocalization and effects of adrenomedullin in nephron segments and in mesangial cells of the rat

1997 ◽  
Vol 272 (6) ◽  
pp. F691-F697 ◽  
Author(s):  
A. Owada ◽  
H. Nonoguchi ◽  
Y. Terada ◽  
F. Marumo ◽  
K. Tomita
Keyword(s):  
Mesangial Cells ◽  
Collecting Duct ◽  
Calf Serum ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Rt Pcr ◽  
Nephron Segments ◽  
Camp Generation ◽  
Osmotic Water ◽  
Medullary Collecting Duct

We examined microlocalization of mRNA coding for adrenomedullin (AM), using reverse transcription-polymerase chain reaction (RT-PCR), and the effects of AM on adenosine 3',5'-cyclic monophosphate (cAMP) generation and water transport in microdissected rat nephron segments. We also examined intraglomerular site of the expression of AM and AM-stimulated cAMP generation in cultured rat mesangial cells (MC). RT-PCR demonstrated the signals for AM mRNA in glomerulus (Glm), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD) but not in proximal convoluted tubule (PCT) or medullary thick ascending limb (MTAL). AM (10(-7) M) stimulated cAMP generation in Glm >> CCD = IMCD > OMCD but not in PCT or MTAL, which corresponded to the results of the expression of AM mRNA. AM (10(-8) M) slightly increased osmotic water permeability by 24% in perfused terminal IMCD. Northern blot analysis revealed high expression of AM mRNA in MC. AM (10(-7) M) stimulated cAMP generation in MC both in the presence and absence of fetal calf serum, suggesting that AM-dependent cAMP generation was evident both in cycling MC and in quiescent MC. AM may work as a diuretic peptide mainly by increasing glomerular filtration rate via cAMP in MC.

Distribution of mineralocorticoid and glucocorticoid receptor mRNA along the nephron

1993 ◽  
Vol 264 (5) ◽  
pp. F781-F791 ◽  
Author(s):  
K. M. Todd-Turla ◽  
J. Schnermann ◽  
G. Fejes-Toth ◽  
A. Naray-Fejes-Toth ◽  
A. Smart ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Proximal Tubule ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Rna Extraction ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Nephron Segment ◽  
Medullary Collecting Duct ◽  
Glucocorticoid Receptor Mrna

In the present study, a competitive polymerase chain reaction (PCR) technique was used to quantitate the relative levels of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA in microdissected nephron segments from the rat kidney and of MR mRNA from isolated principal and intercalated collecting duct cells from rabbit. RNA was isolated from cells and isolated tubules, cDNA was synthesized, and receptor cDNA was coamplified by PCR with a competitive control template. beta-Actin PCR products were also obtained from each nephron segment studied, to assess variations in RNA extraction and cDNA synthesis. MR mRNA, as determined by this competitive PCR technique, was 10-fold more abundant in cortical collecting duct (CCD), outer medullary collecting duct, and inner medullary collecting duct segments than in the proximal tubule and thick ascending limb segments (P < 0.05). Both principal and beta-intercalated cells of the CCD contained detectable levels of MR mRNA, although the levels in the principal cells were threefold higher (P < 0.01). GR mRNA was twofold more abundant in glomeruli, proximal tubule, and thick ascending limb segments than in the collecting duct segments (P < 0.05). In general, the distribution pattern of MR and GR mRNA is consistent with the distribution of adrenal corticosteroid function along the nephron.

Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney

1999 ◽  
Vol 276 (6) ◽  
pp. F874-F881 ◽  
Author(s):  
Feng Wu ◽  
Frank Park ◽  
Allen W. Cowley ◽  
David L. Mattson
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Collecting Duct ◽  
Rat Kidney ◽  
No Production ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Medullary Collecting Duct ◽  
Oxide Synthase ◽  
Thin Limb

This study was designed to quantify nitric oxide synthase (NOS) activity in microdissected glomeruli (Glm), pars convoluta, pars recta, cortical collecting duct, cortical thick ascending limb, outer medullary collecting duct, medullary thick ascending limb and thin limb, inner medullary collecting duct (IMCD) and thin limb, and vasa recta (VR). Total protein from microdissected segments was incubated withl-[3H]arginine and appropriate cofactors, and thel-arginine and convertedl-citrulline were separated by reverse-phase HPLC and radiochemically quantitated. NOS activity was found to be greatest in IMCD (11.5 ± 1.0 fmol citrulline ⋅ mm−1 ⋅ h−1) and moderate in Glm (1.9 ± 0.3 fmol ⋅ glomerulus−1 ⋅ h−1) and VR (3.2 ± 0.8 fmol ⋅ mm−1 ⋅ h−1). All other renal structures studied exhibited significantly less NOS activity. The mRNA for NOS isoforms in the NOS activity-positive segments was then identified by RT-PCR. The IMCD contained mRNA for neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS), but Glm and VR only expressed the mRNA for nNOS and eNOS. These experiments demonstrate that the greatest enzymatic activity for NO production in the kidney is in the IMCD, three- to sixfold less activity is present in the Glm and VR, and minimal NOS activity is found in other segments studied.

Localization of bumetanide- and thiazide-sensitive Na-K-Cl cotransporters along the rat nephron

1996 ◽  
Vol 271 (4) ◽  
pp. F931-F939 ◽  
Author(s):  
T. Yang ◽  
Y. G. Huang ◽  
I. Singh ◽  
J. Schnermann ◽  
J. P. Briggs
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Direct Sequencing ◽  
Salt Transport ◽  
Thick Ascending Limb ◽  
Specific Primers ◽  
Nephron Segments ◽  
Medullary Thick Ascending Limb ◽  
Medullary Collecting Duct ◽  
Polymerase Chain

The present study was undertaken to investigate the mRNA localization of the two major kidney-specific Na-K-Cl transport proteins, the bumetanide-sensitive cotransporter (NKCC2 in rabbit and BSC1 in rat) and the thiazide-sensitive cotransporter (TSC). NKCC2 from rabbit and mouse has been shown to exist in three isoforms (designated A, B, and F) that differ only in a 96-bp region. The divergent region of each of the three NKCC2 isoforms was cloned from rat kidney by a polymerase chain reaction (PCR)-based strategy, and isoform-specific primers were chosen. RNA and cDNA were prepared from renal cortex and medulla and from microdissected nephron segments. Using reverse transcription (RT)-PCR, the B isoform was detected only in cortex and the F isoform only in medulla, whereas the A from was found in both. In dissected nephron segments, the B form was found exclusively in cortical thick ascending limb (CTAL) and macula densa-containing segment (MDCS), the F form only in medullary thick ascending limb (MTAL) and outer medullary collecting duct, and the A form in CTAL, MDCS, and MTAL. An additional isoform including both A and F exons was identified by direct sequencing of a 592-bp product from medulla. The AF product was found only in the medulla and was localized exclusively in MTAL. TSC mRNA was detected exclusively in the distal convoluted tubule. Differential nephron localization of NKCC2 isoforms suggests that Na-K-Cl cotransporters may differ in their transport characteristics to explain regulation of salt transport along the nephron.

Localization of cytochrome P-450 4A isoforms along the rat nephron

1998 ◽  
Vol 274 (2) ◽  
pp. F395-F404 ◽  
Author(s):  
Osamu Ito ◽  
Magdalena Alonso-Galicia ◽  
Kathleen A. Hopp ◽  
Richard J. Roman
Keyword(s):  
Collecting Duct ◽  
Female Rats ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Renal Vasculature ◽  
Nephron Segments ◽  
Male And Female Rats ◽  
Medullary Collecting Duct ◽  
Cytochrome P 450 ◽  
Preglomerular Arterioles

The expression of P-450 4A isoforms responsible for the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) was examined using the reverse transcription and polymerase chain reaction in various nephron segments and preglomerular arterioles microdissected from the kidneys of Sprague-Dawley rats. Expression of cytochrome P-450 4A1, 4A2, 4A3, and 4A8 mRNA could be detected in RNA extracted from the whole kidney. The expression of P-450 4A1, 4A3, and 4A8 mRNA was similar in the kidney of male and female rats, whereas the expression of 4A2 mRNA was fourfold greater in the kidney of male vs. female rats. At the single-nephron level, P-450 4A1 mRNA could not be detected in either preglomerular arterioles or any nephron segments. P-450 4A2 mRNA was readily detected in preglomerular arterioles, glomeruli, proximal convoluted tubule (PCT), proximal straight tubule (PST), medullary thick ascending limb (MTAL), cortical thick ascending limb (CTAL), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). P-450 4A3 mRNA was also detected in every nephron segment, but the expression of this isoform was barely detectable in preglomerular arterioles. The expression of P-450 4A8 mRNA was detected in the glomerulus, PCT, PST, CTAL, and CCD. It was not detectable in preglomerular arterioles, MTAL, OMCD, or IMCD. Immunoblot analysis using a P-450 4A antibody exhibited a strong signal for P-450 4A protein in the proximal tubule. Smaller signals were also observed in glomerulus, MTAL, and preglomerular arterioles, but no signal could be detected in the IMCD. A similar pattern of P-450 4A protein expression was seen in kidney sections immunostained with this antibody. These results indicate that the expression of P-450 4A isoforms in the kidney of rats is sex dependent and that different P-450 4A isoforms are expressed throughout various nephron segments and the renal vasculature of rats.

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.

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.

Enhanced glomerular filtration and Na+-K+-ATPase with furosemide administration

1987 ◽  
Vol 252 (5) ◽  
pp. F910-F915 ◽  
Author(s):  
P. Scherzer ◽  
H. Wald ◽  
M. M. Popovtzer
Keyword(s):  
Glomerular Filtration ◽  
Collecting Duct ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Nephron Segments ◽  
Medullary Thick Ascending Limb ◽  
Henle's Loop ◽  
Proximal Straight Tubule ◽  
X 24 ◽  
Furosemide Administration

To evaluate the effect of furosemide on kidney function, glomerular filtration rate (GFR), urinary Na excretion (UNaV), Na reabsorption (NAR), and Na+-K+-ATPase in isolated nephron segments were measured in 1) rats treated with furosemide 10 mg X 100 g-1 X 24 h-1 ip for 7 days, and 2) rats receiving an oral Na load for 12 days. In furosemide-treated rats, GFR rose from 0.61 +/- 0.03 (mean +/- SD) to 0.83 +/- 0.06 ml/min (P less than 0.01), UNaV rose from 904 +/- 71 to 1,402 +/- 85 mueq/day (P less than 0.001), and net NAR rose from 87.5 +/- 3.7 to 116.7 +/- 9.0 mueq/min (P less than 0.01). Na+-K+-ATPase remained unchanged in the proximal convoluted tubule (PCT), proximal straight tubule (PST), cortical thick ascending limb of Henle's loop (cTALH), and medullary thick ascending limb of Henle's loop (mTALH), but was increased in the distal convoluted tubule (DCT) and in cortical collecting duct (CCD) from 48.5 +/- 1.2 to 75.3 +/- 0.7 (P less than 0.001) and from 18.6 +/- 0.7 to 27.1 +/- 2.7 (P less than 0.02) X 10(-11) mol X mm-1 X min-1, respectively. In Na-loaded rats GFR rose from 0.61 +/- 0.04 to 0.86 +/- 0.03 ml/min (P less than 0.001), UNaV rose from 1,064 +/- 118 to 18,532 +/- 2,045 mueq/day (P less than 0.001), net NAR from 88.1 +/- 3.0 to 107.8 +/- 3.9 mueq/min and Na-K-ATPase in the mTALH rose from 40.3 +/- 1.4 to 56.2 +/- 2.11 (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Sign in / Sign up

Export Citation Format

Share Document