scholarly journals Regulation of Potassium Channel Kir 1.1 (ROMK) Abundance in the Thick Ascending Limb of Henle's Loop

2001 ◽  
Vol 12 (1) ◽  
pp. 10-18
Author(s):  
CAROLYN A. ECELBARGER ◽  
GHEUN-HO KIM ◽  
MARK A. KNEPPER ◽  
JIE LIU ◽  
MARGARET TATE ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Thick Ascending Limb ◽  
Outer Medulla ◽  
Cos Cells ◽  
Selective Agonist ◽  
V2 Receptor ◽  
Counter Current

Abstract. The renal outer medullary potassium channel (ROMK) of the thick ascending limb (TAL) is a critical component of the counter-current multiplication mechanism. In this study, two new antibodies raised to ROMK were used to investigate changes in the renal abundance of ROMK with treatments known to strongly promote TAL function. These antibodies specifically recognized protein of the predicted size of 45 kD in immunoblots of rat kidney or COS cells transfected with ROMK cDNA. Infusion of 1-deamino-(8-D-arginine)-vasopressin (dDAVP), a vasopressin V2 receptor-selective agonist, for 7 d into Brattleboro rats resulted in dramatic increases in apical membrane labeling of ROMK in the TAL of dDAVP-treated rats, as assessed by immunocytochemical analyses. Using immunoblotting, a more than threefold increase in immunoreactive ROMK levels was observed in the outer medulla after dDAVP infusion. Restriction of water intake to increase vasopressin levels also significantly increased TAL ROMK immunolabeling and abundance in immunoblots. In addition, dietary Na+ levels were varied to determine whether ROMK abundance was also affected under other conditions known to alter TAL transport. Rats fed higher levels of sodium, as either NaCl or NaHCO3 (8 mEq/250 g body wt per d), exhibited significantly increased density of the 45-kD band, compared with the respective control animals. Moreover, in rats fed a low-NaCl diet (0.25 mEq/250 g body wt per d), a 50% decrease in band density for the 45-kD band was observed (relative to control rats fed 2.75 mEq/250 g body wt per d of NaCl). These results demonstrate that long-term adaptive changes in ROMK abundance occur in the TAL with stimuli that enhance transport by this segment.

Localization and regulation of the rat renal Na(+)-K(+)-2Cl- cotransporter, BSC-1

1996 ◽  
Vol 271 (3) ◽  
pp. F619-F628 ◽  
Author(s):  
C. A. Ecelbarger ◽  
J. Terris ◽  
J. R. Hoyer ◽  
S. Nielsen ◽  
J. B. Wade ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Rat Colon ◽  
Thick Ascending Limb ◽  
Outer Medulla ◽  
Water Excretion ◽  
Saline Loading ◽  
Cloned Cdna ◽  
Urinary Concentrating Ability

To investigate the role of the thick ascending limb (TAL) Na(+)-K(+)-2Cl- cotransporter in regulation of water excretion, we have prepared a peptide-derived polyclonal antibody based on the cloned cDNA sequence of the rat type 1 bumetanide-sensitive cotransporter, BSC-1 (also termed "NKCC-2"). Immunoblots revealed a single broad 161-kDa band in membrane fractions of rat renal outer medulla and cortex but not from rat colon or parotid gland. A similar protein was labeled in mouse kidney. Immunoperoxidase immunohistochemistry in rat kidney revealed labeling restricted to the medullary and cortical TAL segments. Because long-term regulation of urinary concentrating ability may depend on regulation of Na(+)-K(+)-2Cl- cotransporter abundance, we used immunoblotting to evaluate the effects of several in vivo factors on expression levels of BSC-1 protein in rat kidney outer medulla. Chronic oral saline loading with 0.16 M NaCl markedly increased BSC-1 abundance. However, long-term vasopressin infusion or thirsting of rats did not affect BSC-1 abundance. Chronic furosemide infusion caused a 9-kDa upward shift in apparent molecular mass and an apparent increase in expression level. These results support the previous identification of BSC-1 as the TAL Na(+)-K(+)-2Cl- transporter and demonstrate that the expression of this transporter is regulated.

Two types of K+ channel in thick ascending limb of rat kidney

1994 ◽  
Vol 267 (4) ◽  
pp. F599-F605 ◽  
Author(s):  
W. H. Wang
Keyword(s):  
Apical Membrane ◽  
Rat Kidney ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Inside Out

We have used the patch-clamp technique to study the apical K+ channels in the thick ascending limb (TAL) of the rat kidney. Two types of K+ channels, a low-conductance and an intermediate-conductance K+ channel, were identified in both cell-attached and inside-out patches. We confirmed the previously reported intermediate-conductance K+ channel (72 pS), which is inhibited by millimolar cell ATP, acidic pH, Ba2+, and quinidine (4). We now report a second K+ channel in apical membrane of the TAL. The slope conductance of this low-conductance K+ channel is 30 pS, and its open probability is 0.80 in cell-attached patches. This channel is not voltage dependent, and application of 2 mM ATP in the bath inhibits channel activity in inside-out patches. In addition, 250 microM glyburide, an ATP-sensitive K+ channel inhibitor, blocks channel activity, whereas the same concentration of glyburide has no inhibitory effect on the 72-pS K+ channel. Channel activity of the 30-pS K+ channel decreases rapidly upon excision of patches (channel run down). Application of 0.1 mM ATP and the catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) restores channel activity. Furthermore, addition of 0.1 mM 8-(4-chlorophenylthio)-cAMP or 50-100 pM vasopressin in the cell-attached patches increases channel activity. In conclusion, two types of K+ channels are present in the apical membrane of TAL of rat kidney, and PKA plays an important role in modulation of the low-conductance K+ channel activity.

Distribution and oligomeric association of splice forms of Na+-K+-ATPase regulatory γ-subunit in rat kidney

2002 ◽  
Vol 282 (3) ◽  
pp. F393-F407 ◽  
Author(s):  
Elena Arystarkhova ◽  
Randall K. Wetzel ◽  
Kathleen J. Sweadner
Keyword(s):  
Splice Variants ◽  
Rat Kidney ◽  
Thick Ascending Limb ◽  
Outer Medulla ◽  
Α Subunit ◽  
Proximal Tubule Cells ◽  
Γ Subunit ◽  
Outer Stripe ◽  
A Minor ◽  
Splice Forms

Renal Na+-K+-ATPase is associated with the γ-subunit (FXYD2), a single-span membrane protein that modifies ATPase properties. There are two splice variants with different amino termini, γa and γb. Both were found in the inner stripe of the outer medulla in the thick ascending limb. Coimmunoprecipitation with each other and the α-subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect Na+-K+-ATPase conformation. In the cortex, the proportion of the γb-subunit was markedly lower, and the γa-subunit predominated in isolated proximal tubule cells. By immunofluorescence, the γb-subunit was detected in the superficial cortex only in the distal convoluted tubule and connecting tubule, which are rich in Na+-K+-ATPase but comprise a minor fraction of cortex mass. In the outer stripe of the outer medulla and for a short distance in the deep cortex, the thick ascending limb predominantly expressed the γb-subunit. Because different mechanisms maintain and regulate Na+ homeostasis in different nephron segments, the splice forms of the γ-subunit may have evolved to control the renal Na+ pump through pump properties, gene expression, or both.

Sodium-dependent bicarbonate absorption by cortical thick ascending limb of rat kidney

1985 ◽  
Vol 248 (6) ◽  
pp. F821-F829 ◽  
Author(s):  
D. W. Good
Keyword(s):  
Hydrogen Exchange ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Carbonic Anhydrase Activity ◽  
Detectable Effect ◽  
Thick Ascending Limb ◽  
Bicarbonate Concentration ◽  
Sodium Dependent ◽  
Transepithelial Voltage

In vitro microperfusion experiments were performed to investigate the mechanism of bicarbonate absorption in the cortical thick ascending limb of the rat. Tubules were perfused at 1.0-1.5 nl X min-1 X mm-1 and bicarbonate concentration was 25 mM in the perfusate and bath. Bicarbonate absorption rates were determined by microcalorimetry. Control tubules absorbed bicarbonate at a mean rate of 9.5 +/- 0.6 pmol X min-1 X mm-1. The limiting luminal bicarbonate concentration was approximately 5 mM for tubules perfused at slow rates with 25 mM bicarbonate in the bath. Acetazolamide (10(-4)M) in the bath reduced bicarbonate absorption by 76% without significant effect on transepithelial voltage. Removing sodium from the perfusate and bath or removing potassium from the bath reduced bicarbonate absorption and transepithelial voltage to near zero. Adding amiloride (5 X 10(-4) or 10(-3) M) to the perfusate reduced bicarbonate absorption by 60-75% without detectable effect on transepithelial voltage. Adding furosemide (10(-4)M) to the perfusate increased bicarbonate absorption significantly by 40-50% while decreasing transepithelial voltage from 17 to 1.8 mV. Thus, bicarbonate absorption by cortical thick ascending limbs requires carbonic anhydrase activity and sodium transport but is not dependent on transepithelial voltage. When considered together, the results are consistent with mediation of the bicarbonate absorption by apical membrane sodium-hydrogen exchange.

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.

Cell-specific regulation of cytosolic aspartate aminotransferase by glucocorticoids in the rat kidney

1993 ◽  
Vol 265 (5) ◽  
pp. C1298-C1305 ◽  
Author(s):  
S. Feilleux-Duche ◽  
M. Garlatti ◽  
M. Aggerbeck ◽  
M. Poyard ◽  
J. Bouguet ◽  
...  
Keyword(s):  
Aspartate Aminotransferase ◽  
Hormonal Regulation ◽  
Renal Cortex ◽  
Rat Kidney ◽  
Thick Ascending Limb ◽  
Outer Medulla ◽  
Maximal Increase ◽  
Basal Expression ◽  
Specific Increase ◽  
Specific Regulation

The basal expression and hormonal regulation of cytosolic aspartate aminotransferase (cAspAT) were investigated in the rat kidney. In adrenalectomized animals, the basal activity was highest in the renal cortex and in the inner stripe of the outer medulla (0.1-0.15 U/mg protein). The glucocorticoid analogue dexamethasone increased cAspAT activity about twofold in the cortex and in the inner stripe of the outer medulla but not in the papilla. A half-maximal increase in the activity was achieved at doses of approximately 5 micrograms/100 g body wt. The mineralocorticoid aldosterone did not modify the cAspAT activity. The cell specificity of the hormonal regulation was analyzed by in situ hybridization. In untreated adrenalectomized rats, a cAspAT cRNA probe labeled mainly the inner stripe of the outer medulla. After dexamethasone or hydrocortisone treatment, labeling was uniformly increased in this part of the medulla and was heterogeneously increased in the renal cortex. The specific increase in labeling within the cortex was shown to be confined to the distal convoluted tubule and the thick ascending limb. We conclude that, in addition to widespread basal expression, cAspAT is regulated by glucocorticoids in a highly cell-specific manner in the renal cortex. The enzyme may thus participate in the increased energy metabolism elicited by these hormones in these cells.

Regulation of sodium transporters in the thick ascending limb of rat kidney: response to angiotensin II

2003 ◽  
Vol 285 (1) ◽  
pp. F152-F165 ◽  
Author(s):  
Tae-Hwan Kwon ◽  
Jakob Nielsen ◽  
Young-Hee Kim ◽  
Mark A. Knepper ◽  
Jørgen Frøkiær ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Rat Kidney ◽  
Thick Ascending Limb ◽  
Ang Ii ◽  
Outer Medulla ◽  
Protein Protein Interaction ◽  
Sodium Transporters ◽  
Outer Strip

The effect of ANG II treatment of rats for 7 days was examined with respect to the abundance and subcellular localization of key thick ascending limb (TAL) Na+ transporters. Rats were on a fixed intake of Na+ and water and treated with 0, 12.5, 25, 50 (ANG II-50), 100 (ANG II-100), and 200 (ANG II-200) ng·min-1·kg-1 ANG II (sc). Semiquantitative immunoblotting revealed that Na+/H+ exchanger 3 (NHE3) abundance in the inner stripe of the outer medulla (ISOM) of ANG II-treated rats was significantly increased: 179 ± 28 (ANG II-50, n = 5), 166 ± 23 (ANG II-100, n = 7), and 167 ± 19% (ANG II-200, n = 4) of control levels ( n = 6, P < 0.05), whereas lower doses of ANG II were ineffective. The abundance of the bumetanide-sensitive Na+-K+-2Cl- cotransporter (BSC-1) in the ISOM was also increased to 187 ± 28 (ANG II-50), 162 ± 23 (ANG II-100), and 166 ± 19% (ANG II-200) of control levels ( P < 0.05), but there were no changes in the abundance of Na+-K+-ATPase and the electroneutral Na+-HCO3 cotransporter NBCn1. Immunocytochemistry confirmed the increase in NHE3 and BSC-1 labeling in medullary TAL (mTAL). In the cortex and the outer strip of the outer medulla, NHE3 abundance was unchanged, whereas immunocytochemistry revealed markedly increased NHE3 labeling of the proximal tubule brush border, suggesting subcellular redistribution of NHE3 or differential protein-protein interaction. Despite this, ANG II-treated rats (50 ng·min-1·kg-1 for 5 days, n = 6) had a higher urinary pH compared with controls. NH4Cl loading completely blocked all effects of ANG II infusion on NHE3 and BSC-1, suggesting a potential role of pH as a mediator of these effects. In conclusion, increased abundance of NHE3 and BSC-1 in mTAL cells as well as increased NHE3 in the proximal tubule brush border may contribute to enhanced renal Na+ and HCO3 reabsorption in response to ANG II.

Chronic furosemide or hydrochlorothiazide administration increases H+-ATPase B1 subunit abundance in rat kidney

2007 ◽  
Vol 292 (6) ◽  
pp. F1701-F1709 ◽  
Author(s):  
Ki Young Na ◽  
Gheun-Ho Kim ◽  
Kwon Wook Joo ◽  
Jay Wook Lee ◽  
Hye Ryoun Jang ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Rat Kidney ◽  
Chronic Administration ◽  
Regulatory Mechanisms ◽  
Protein Abundance ◽  
Intercalated Cells ◽  
Distal Nephron ◽  
Urine Ph ◽  
Outer Medulla ◽  
Furosemide Administration

Furosemide administration stimulates distal acidification. This has been attributed to the increased lumen-negative voltage in the distal nephron, but the aspect of regulatory mechanisms of H+-ATPase has not been clear. The purpose of this study is to investigate whether chronic administration of diuretics alters the expression of H+-ATPase and whether electrogenic Na+ reabsorption is involved in this process. A 7-day infusion of furosemide or hydrochlorothiazide (HCTZ) lowered urine pH significantly. However, this effect of furosemide-induced distal acidification was not changed with amiloride-blocking electrogenic Na+ reabsorption. On immunoblotting, a polyclonal antibody against the H+-ATPase B1 subunit recognized a specific ∼56-kDa band in membrane fractions from the kidney. The protein abundance of H+-ATPase was significantly increased by furosemide and HCTZ infusion in both the cortex and outer medulla. Furosemide plus amiloride administration also increased the H+-ATPase protein abundance significantly. However, no definite subcellular redistribution of H+-ATPase was observed by furosemide ± amiloride infusion with immunohistochemistry. Chronic furosemide ± amiloride administration induced a translocation of pendrin to the apical membrane, while total protein abundance was not increased. The mRNA expression of H+-ATPase was not altered by furosemide ± amiloride infusion. We conclude that chronic administration of diuretics enhances distal acidification by increasing the abundance of H+-ATPase irrespective of electrogenic Na+ reabsorption. This upregulation of H+-ATPase in the intercalated cells may be the result of tubular hypertrophy by diuretics.

Glucocorticoid impairs growth of kidney outer medulla and accelerates loop of Henle differentiation and urinary concentrating capacity in rat kidney development

2006 ◽  
Vol 291 (4) ◽  
pp. F812-F822 ◽  
Author(s):  
Jane Stubbe ◽  
Kirsten Madsen ◽  
Finn Thomsen Nielsen ◽  
Ole Skøtt ◽  
Boye L. Jensen
Keyword(s):  
Kidney Development ◽  
Rat Kidney ◽  
Nuclear Antigen ◽  
Thick Ascending Limb ◽  
Protein Abundance ◽  
Suckling Period ◽  
Outer Medulla ◽  
Rat Pups ◽  
Urinary Concentrating Ability ◽  
And Function

In the rat, urinary concentrating ability develops progressively during the third postnatal (P) week and nearly reaches adult level at weaning ( P21) governed by a rise in circulating glucocorticoid. Elevated extracellular osmolality can lead to growth arrest of epithelial cells. We tested the hypothesis that supranormal exposure of rat pups to glucocorticoid before the endogenous surge enhances urinary concentrating ability but inhibits renomedullary cell proliferation. Proliferating-cell nuclear antigen (PCNA)-positive cells shifted from the nephrogenic zone in the first postnatal week to Tamm-Horsfall-positive thick ascending limb (TAL) cells at the corticomedullary junction at P10– 14. Renal PCNA protein abundance was stable in the suckling period and decreased 10-fold after weaning. Renal PCNA protein abundance decreased in response to dexamethasone (DEXA; 100 μg·kg−1·day−1, P8–12). Prolonged administration of DEXA ( P1-P11) reduced selectively the area and thickness of the outer medulla and the number of PCNA-positive cells. DEXA ( P8– 12) increased urinary and papillary osmolality in normohydrated and water-deprived pups and led to osmotic equilibrium between interstitium and urine, whereas apoptotic and GADD153-positive cells increased in the inner medulla. TAL-associated NaCl transporters Na-K-2Cl cotransporter, Na-K-ATPase-α1, Na/H exchanger type 3, and ROMK increased significantly at weaning and in response to DEXA. We conclude that a low level of circulating glucocorticoid is permissive for proliferation of Henle's loop and the outer medulla before weaning. A reduced papillary tonicity is a crucial factor for the reduced capacity to concentrate urine during postnatal kidney development. We speculate that supranormal exposure to glucocorticoid in the suckling period can alter kidney medullary structure and function permanently.

Immunolocalization of anion transporter Slc26a7 in mouse kidney

2006 ◽  
Vol 290 (4) ◽  
pp. F937-F945 ◽  
Author(s):  
Paul L. Dudas ◽  
SueAnn Mentone ◽  
Colin F. Greineder ◽  
Daniel Biemesderfer ◽  
Peter S. Aronson
Keyword(s):  
Proximal Tubule ◽  
Immunofluorescence Microscopy ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Mouse Kidney ◽  
Membrane Transporters ◽  
Thick Ascending Limb ◽  
Base Exchange ◽  
Proximal Tubule Cells ◽  
Anion Transporter

Previous studies have indicated that a major fraction of the filtered Cl− is reabsorbed via apical membrane Cl−/base exchange in the proximal tubule. Recent studies in Slc26a6 null mice have suggested that this transporter mediates only a portion of proximal tubule Cl−/base exchange, raising the possibility that one or more unidentified apical membrane transporters may additionally contribute. Recent studies have identified Slc26a7 as another Cl−/base exchanger expressed in the kidney. We therefore generated Slc26a7-specific polyclonal and monoclonal antibodies to examine cellular and subcellular sites of expression in mouse kidney. The specificity of each antibody was verified by immunoblotting and immunofluorescence of COS-7 cells transiently transfected with mouse Slc26a7. Immunofluorescence microscopy of mouse kidney detected the expression of Slc26a7 subapically in proximal tubule cells, and on the basolateral surface of thick ascending limb cells. Similar staining patterns were demonstrated with two antibodies shown to react with different epitopes on Slc26a7. Immunolocalization of Slc26a7 to proximal tubule and thick ascending limb was also observed in rat kidney. We conclude that Slc26a7 is expressed in the proximal tubule and thick ascending limb of the loop of Henle, and it may therefore contribute to anion transport in these nephron segments.

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