Expression of the thiazide-sensitive Na-Cl cotransporter in rat and human kidney

1995 ◽  
Vol 269 (6) ◽  
pp. F900-F910 ◽  
Author(s):  
N. Obermuller ◽  
P. Bernstein ◽  
H. Velazquez ◽  
R. Reilly ◽  
D. Moser ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Human Kidney ◽  
Protein Band ◽  
Distal Region ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Nephron Segments ◽  
Collecting Tubule

An electroneutral thiazide-sensitive Na-Cl cotransport pathway (TSC) has been localized functionally to the distal convoluted tubule (DCT), although the TSC has also been detected in the connecting tubule (CNT), the cortical collecting duct, and the medullary collecting tubule as well. The present experiments were designed to localize expression of message for the TSC in rat and human kidney. A riboprobe, generated from the mouse TSC, was used for in situ hybridization. Simultaneous immunocytochemistry, using antibodies to Tamm-Horsfall protein, band 3, and the Na+/Ca2+ exchanger, permitted delineation of specific nephron segments. In rat, message for the TSC was highly expressed in DCT cells but not elsewhere. The transition from thick ascending limb to DCT was abrupt, whereas the transition to CNT was gradual. In the more distal region of rat DCT (DCT-2), which contained few intercalated cells, both TSC message and Na+/Ca2+ exchanger immunoreactivity were present. Treatment of rats with furosemide for 5 days increased expression of TSC message within the DCT but did not induce its expression elsewhere. In humans, expression of TSC message was also highest in cells of the DCT. In humans, however, expression extended well into the CNT. These experiments indicate that the TSC is expressed predominantly by DCT cells in both rat and humans, although expression extends into the CNT cells in humans. They also show that the TSC and Na+/Ca2+ exchanger are coexpressed by a subpopulation of DCT cells near the junction with the CNT.

Intrarenal and Cellular Localization of CLC-K2 Protein in the Mouse Kidney

2001 ◽  
Vol 12 (7) ◽  
pp. 1327-1334 ◽  
Author(s):  
KATSUKI KOBAYASHI ◽  
SHINICHI UCHIDA ◽  
SHUKI MIZUTANI ◽  
SEI SASAKI ◽  
FUMIAKI MARUMO
Keyword(s):  
Cellular Localization ◽  
Collecting Duct ◽  
Type A ◽  
Mouse Kidney ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Basolateral Membranes ◽  
Nephron Segments ◽  
Henle's Loop ◽  
Distal Tubules

Abstract. CLC-K2, a kidney-specific member of the CLC chloride channel family, is thought to play an important role in the transepithelial Cl- transport in the kidney. This consensus was first reached shortly after it was demonstrated that the mutations of the human CLCNKB gene resulted in Bartter's syndrome type III. To clarify the pathogenesis, the exact intrarenal and cellular localization of CLC-K2 by immunohistochemistry of the Clcnk1-/- mouse kidney were investigated by use of an anti-CLC-K antibody that recognized both CLC-K1 and CLC-K2. CLC-K2 is expressed in the thick ascending limb of Henle's loop and distal tubules, where it is localized to the basolateral membranes. The localization of CLC-K2 to these nephron segments strongly implies that CLC-K2 confers the basolateral chloride conductance in the thick ascending limb of Henle's loop and distal tubules, where Cl- is taken up by the bumetanide-sensitive Na-K-2Cl cotransporter or the thiazide-sensitive Na-Cl cotransporter at the apical membranes. CLC-K2 expression was also shown to extend into the connecting tubule in the basolateral membrane. CLC-K2 was found in basolateral membranes of the type A intercalated cells residing along the collecting duct. This localization strongly suggests that CLC-K2 confers the basolateral conductance in the type A intercalated cells where Cl- is taken up by the anion exchanger in exchange for HCO3- at the basolateral membranes. These aspects of CLC-K2 localization suggest that CLC-K2 is important in Cl- transport in the distal nephron segments.

Cortical collecting duct Na-K pump in obstructive nephropathy

1990 ◽  
Vol 258 (5) ◽  
pp. F1320-F1327 ◽  
Author(s):  
H. Kimura ◽  
S. K. Mujais
Keyword(s):  
Ureteral Obstruction ◽  
Turnover Rate ◽  
Collecting Duct ◽  
Obstructive Nephropathy ◽  
Cortical Collecting Duct ◽  
Sprague Dawley ◽  
Irreversible Damage ◽  
Sprague Dawley Rats ◽  
Collecting Tubule

The present study examined the alterations in the cortical collecting tubule (CCT) Na-K pump that occur after unilateral ureteral obstruction and their consequences on electrolyte excretion. In male Sprague-Dawley rats, unilateral ureteral ligation led to a progressive decrease in intact CCT Na-K pump in situ turnover worsening with the duration of the obstruction: control, 20.1 +/- 0.4; obstructed kidney: 3 h, 14.6 +/- 0.3; 12 h, 12.7 +/- 0.6; 24 h, 12.8 +/- 0.5; 48 h, 11.6 +/- 0.5; and 96 h, 10.6 +/- 0.4 pmol Rb.mm-1.min-1 (all P less than 0.001 vs. control). CCT diameter increased with the duration of obstruction. Release of ureteral obstruction was associated with restitution of pump turnover rate. With 3 h of obstruction, recovery of pump in situ turnover was complete (19.7 +/- 0.4 pmol Rb.mm-1.min-1) by 24 h after release. With more prolonged obstruction (24 h) recovery was partial by 24 h postrelease (16.2 +/- 0.5 pmol Rb.mm-1.min-1) and complete (19.8 +/- 0.7 pmol Rb.mm-1.min-1) by 48 h, suggesting a delay in recovery without the occurrence of irreversible damage. The impairment in Na-K pump in situ turnover was paralleled by an impairment in the ability of the obstructed kidney to excrete an acute potassium load. This parallelism of functional and biochemical studies favors the notion that impairment of CCT Na-K pump in situ turnover contributes significantly to the abnormal potassium excretion that accompanies obstructive damage.

Immunolocalization of the Na+/Ca2+ exchanger in rabbit kidney

1993 ◽  
Vol 265 (2) ◽  
pp. F327-F332 ◽  
Author(s):  
R. F. Reilly ◽  
C. A. Shugrue ◽  
D. Lattanzi ◽  
D. Biemesderfer
Keyword(s):  
Collecting Duct ◽  
Membrane Vesicle ◽  
Immunoblot Analysis ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Intracellular Loop ◽  
Nephron Segments ◽  
Vesicle Preparation

We recently isolated a cDNA encoding a Na+/Ca2+ exchanger from rabbit kidney that was highly similar to the canine cardiac sarcolemmal Na+/Ca2+ exchanger. In the present study, we used two different antibodies to the exchanger to identify the protein and establish its cellular and subcellular localization in the kidney. The first antibody was prepared against a fusion protein consisting of 190 amino acids of the large, presumably intracellular loop of the rabbit renal exchanger fused to the maltose-binding protein. The second was a monoclonal antibody generated against the isolated purified canine cardiac sarcolemmal exchanger. To identify the Na+/Ca2+ exchanger protein, we performed immunoblot analysis against a membrane vesicle preparation from rabbit kidney cortex. Both antibodies immunoblotted proteins of 120 and 70 kDa that are known to be associated with the exchanger. Indirect immunofluorescence revealed that both antisera labeled the basolateral surface of the majority of cells in the connecting tubule (CNT). Since the phase-dense (intercalated) cells in the CNT were not stained, this suggested that the labeled cells were CNT cells. No labeling was detected in other nephron segments with the exception of occasional faint staining of the majority cell population of the cortical collecting duct. The fact that we did not detect labeling in other nephron segments is consistent with either 1) the absence of expression of the Na+/Ca2+ exchanger in these segments, 2) the expression of the exchanger in levels below the threshold of detection of the two antibodies used in this study, or 3) the exchanger in these segments is represented by a different isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Cloning and localization of a double-pore K channel, KCNK1: exclusive expression in distal nephron segments

1997 ◽  
Vol 273 (4) ◽  
pp. F663-F666 ◽  
Author(s):  
Marcelo Orias ◽  
Heino Velázquez ◽  
Freeman Tung ◽  
George Lee ◽  
Gary V. Desir
Keyword(s):  
Amino Acid ◽  
Collecting Duct ◽  
Human Kidney ◽  
Distal Tubule ◽  
Amino Acid Identity ◽  
Northern Analysis ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
K Channels

The K-selective channel, TOK1, recently identified in yeast, displays the unusual structural feature of having two putative pore regions, in contrast to all previously cloned K channels. Using the TOK1 pore regions as probes, we identified a human kidney cDNA encoding a 337-amino acid protein (hKCNK1) with four transmembrane segments and two pore regions containing the signature sequence of K channels. Amino acid identity to TOK1 is only 15% overall but 40% at the pores. Northern analysis indicates high expression of a 1.9-kb message in brain > kidney >> heart. Nephron segment localization, carried out in rabbit by reverse transcription-polymerase chain reaction, reveals that KCNK1 is expressed in cortical thick ascending limb, connecting tubule, and cortical collecting duct. It was not detected in the proximal tubule, medullary thick ascending limb, distal convoluted tubule, and glomerulus. We conclude that KCNK1 is a unique, double-pore, mammalian K channel, distantly related to the yeast channel TOK1, that is expressed in distal tubule and is a candidate to participate in renal K homeostasis.

Cellular and subcellular immunolocalization of ClC-5 channel in mouse kidney: colocalization with H+-ATPase

1999 ◽  
Vol 277 (6) ◽  
pp. F957-F965 ◽  
Author(s):  
Hisato Sakamoto ◽  
Yoshikazu Sado ◽  
Ichiro Naito ◽  
Tae-Hwan Kwon ◽  
Shinichi Inoue ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Collecting Duct ◽  
Type A ◽  
Mouse Kidney ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Double Labeling ◽  
Cytoplasmic Staining ◽  
Confocal Images

To determine the immunolocalization of ClC-5 in the mouse kidney, we developed a ClC-5-specific rat monoclonal antibody. Immunoblotting demonstrated an 85-kDa band of ClC-5 in the kidney and ClC-5 transfected cells. Immunocytochemistry revealed significant labeling of ClC-5 in brush-border membrane and subapical intracellular vesicles of the proximal tubule. In addition, apical and cytoplasmic staining was observed in the type A intercalated cells in the cortical collecting duct. In contrast, the staining was minimal in the outer and inner medullary collecting ducts and the thick ascending limb. Western blotting of vesicles immunoisolated by the ClC-5 antibody showed the presence of H+-ATPase, strongly indicating that these two proteins were present in the same membranes. Double labeling with antibodies against ClC-5 and H+-ATPase and analysis by confocal images showed that ClC-5 and H+-ATPase colocalized in these ClC-5-positive cells. These findings suggest that ClC-5 might be involved in the endocytosis and/or the H+ secretion in the proximal tubule cells and the cortical collecting duct type A intercalated cells in mouse kidney.

Low-NaCl diet increases H-K-ATPase in intercalated cells from rat cortical collecting duct

1998 ◽  
Vol 275 (1) ◽  
pp. F94-F102 ◽  
Author(s):  
Randi B. Silver ◽  
Han Choe ◽  
Gustavo Frindt
Keyword(s):  
Plasma Levels ◽  
Collecting Duct ◽  
Plasma Aldosterone ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Elevated Plasma ◽  
Ratiometric Fluorescence ◽  
Collecting Tubule ◽  
Acid Load ◽  
Cortical Collecting Tubule

Extracellular K+-dependent H+ extrusion after an acute acid load, an index of H/K exchange, was monitored in intercalated cells (ICs) from rat cortical collecting tubule (CCT) using ratiometric fluorescence imaging of the intracellular pH (pHi) indicator, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The hypothesis tested was that 12- to 14-day NaCl deprivation increases H-K-ATPase in rat ICs. The rate of H/K exchange in the low-NaCl ICs was double that of controls. In control ICs, H/K exchange was inhibited by Sch-28080 (10 μM). In the low-NaCl ICs, it was partially blocked by Sch-28080 or ouabain (1 mM). Simultaneous addition of both inhibitors abolished K-dependent pHirecovery. The induced H/K exchange observed with NaCl restriction was not due to elevated plasma aldosterone as evidenced by experiments on ICs from rats implanted with osmotic minipumps administering aldosterone such that plasma levels were comparable to those of NaCl-deficient rats. The results suggest that NaCl deficiency induces two isoforms of H-K-ATPase in ICs and that this effect is not mediated by elevated plasma aldosterone.

Mechanism of PGE2-induced cell swelling in distal nephron segments

1992 ◽  
Vol 263 (5) ◽  
pp. F824-F832
Author(s):  
T. Shimizu ◽  
M. Naruse ◽  
M. Takeda ◽  
M. Nakamura ◽  
K. Yoshitomi ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Cell Swelling ◽  
Rabbit Kidney ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Distal Nephron ◽  
Nephron Segments ◽  
Pg E2 ◽  
Dose Dependent ◽  
Sustained Increase

The effects of prostaglandin (PG) E2 on cell swelling were studied in isolated perfused tubules of rabbit kidney. PGE2 (1 microM) added to the bath induced cell swelling by 13.4, 7.2, and 9.6% in the connecting tubule, distal convoluted tubule, and cortical collecting duct, respectively, but it had no effect on the proximal convoluted tubule and cortical thick ascending limb. The response was dose dependent in the range of 1 nM to 1 microM. PGI2 exerted a similar effect, but PGF2 alpha had no effect. The swelling was completely blocked by basolateral Na+ removal and was attenuated by bilateral Cl- removal, suggesting that the swelling was mediated by basolateral Na+ entry in association with Cl- entry. In all segments except proximal tubule, PGE2 caused an initial transient peak followed by a sustained increase in intracellular Ca2+. Intracellular Ca2+ chelation or inhibition of Ca2+ release from intracellular stores abolished the PGE2-induced cell swelling, but extracellular Ca2+ removal did not. An inhibitor of the Na(+)-Ca2+ exchanger (3',4'-dichlorobenzamil, 100 microM) in the bath completely inhibited PGE2-induced cell swelling. Neither furosemide (1 mM) nor amiloride (1 mM) added to bath abolished the response, indicating that neither Na(+)-K(+)-2Cl- cotransport nor Na(+)-H+ exchange is involved in the action of PGE2. The swelling response to PGE2 was observed even in the presence of ouabain, indicating that the effect of PGE2 is independent of Na(+)-K(+)-adenosinetriphosphatase inhibition. Nicardipine added to bath partially inhibited the swelling response.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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