scholarly journals Ecto-5'-nucleotidase: localization in rat kidney by light microscopic histochemical and immunohistochemical methods.

1989 ◽  
Vol 37 (1) ◽  
pp. 39-47 ◽  
Author(s):  
T P Dawson ◽  
R Gandhi ◽  
M Le Hir ◽  
B Kaissling
Keyword(s):  
Enzyme Activity ◽  
Brush Border ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Lead Salt ◽  
Salt Precipitation ◽  
Tubular Lumen ◽  
Outer Stripe ◽  
Immunohistochemical Methods

We demonstrated the distribution pattern of ecto-5'-nucleotidase (5'-Nu) in rat kidney by enzymatic activity (lead salt precipitation) and by immunohistochemistry with a polyclonal antibody raised in rabbits. Enzyme activity was found in the brush border of the proximal tubule, highest in the P1 segments with decreasing intensity in the P2 segments and weakest in P3 segments in the medullary rays of the cortex. The P3 segments of the outer stripe showed slightly higher activity. Activity was also apparent in the intercalated cells in the connecting tubule and collecting duct, whereas all other tubular and glomerular structures were negative. Activity in peritubular and perivascular connective tissue was highest in the cortical labyrinth, weak or absent in the medullary rays of the cortex, and entirely absent in the medulla. The distribution of the antigen was fully congruent with that of the enzyme activity. With respect to the role of adenosine in regulation of renal blood flow and glomerular filtration rate, the distribution of 5'-Nu in the cortical interstitium may be particularly significant. The possibility of nucleotide cleavage at the brush-border membranes may be important for salvage of nucleotides from the tubular lumen.

Localization of PEPT1 and PEPT2 proton-coupled oligopeptide transporter mRNA and protein in rat kidney

1999 ◽  
Vol 276 (5) ◽  
pp. F658-F665 ◽  
Author(s):  
Hong Shen ◽  
David E. Smith ◽  
Tianxin Yang ◽  
Yuning G. Huang ◽  
Jürgen B. Schnermann ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Rat Kidney ◽  
Broad Band ◽  
Membrane Vesicles ◽  
Mrna Transcript ◽  
Intestinal Brush Border Membrane ◽  
Outer Stripe ◽  
Polyclonal Antisera ◽  
Pars Convoluta

To determine the renal localization of oligopeptide transporters, Northern blot analyses were performed and polyclonal antisera were generated against PEPT1 and PEPT2, the two cloned rat H+/peptide transporters. Under high-stringency conditions, a 3.0-kb mRNA transcript of rat PEPT1 was expressed primarily in superficial cortex, whereas a 3.5-kb mRNA transcript of PEPT2 was expressed primarily in deep cortex/outer stripe of outer medulla. PEPT1 antisera detected a specific band on immunoblots of renal and intestinal brush-border membrane vesicles (BBMV) with an apparent mobility of ∼90 kDa. PEPT2 antisera detected a specific broad band of ∼85 kDa in renal but not in intestinal BBMV. PEPT1 immunolocalization experiments showed detection of a brush border antigen in S1 segments of the proximal tubule and in the brush border of villi from all segments of the small intestine. In contrast, PEPT2 immunolocalization was primarily confined to the brush border of S3 segments of the proximal tubule. All other nephron segments in rat were negative for PEPT1 and PEPT2 staining. Overall, our results conclusively demonstrate that although PEPT1 is expressed in early regions of the proximal tubule (pars convoluta), PEPT2 is specific for the latter regions of proximal tubule (pars recta).

Retention of fetuin-A in renal tubular lumen protects the kidney from nephrocalcinosis in rats

2013 ◽  
Vol 304 (6) ◽  
pp. F751-F760 ◽  
Author(s):  
Isao Matsui ◽  
Takayuki Hamano ◽  
Satoshi Mikami ◽  
Kazunori Inoue ◽  
Akihiro Shimomura ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Proximal Tubules ◽  
Fetuin A ◽  
Receptor Associated Protein ◽  
Tubular Lumen ◽  
Proximal Tubular ◽  
Normal Rat ◽  
Natural Inhibitor ◽  
Renal Tubular

The serum glycoprotein fetuin-A is an important inhibitor of extraosseous calcification. The importance of fetuin-A has been confirmed in fetuin-A null mice, which develop widespread extraosseous calcification including the kidney. However, the mechanism how fetuin-A protects kidneys from nephrocalcinosis remains uncertain. Here, we demonstrate that intratubular fetuin-A plays a role in the prevention of nephrocalcinosis in the proximal tubules. Although normal rat kidney did not express mRNA for fetuin-A, we found punctate immunohistochemical staining of fetuin-A mainly in the S1 segment of the proximal tubules. The staining pattern suggested that fetuin-A passed through the slit diaphragm, traveled in the proximal tubular lumen, and was introduced into proximal tubular cells by megalin-mediated endocytosis. To test this hypothesis, we inhibited the function of megalin by intravenous injection of histidine-tagged soluble receptor-associated protein (His-sRAP), a megalin inhibitor. His-sRAP injection diminished fetuin-A staining in the proximal tubules and led to urinary excretion of fetuin-A. We further analyzed the role of fetuin-A in nephrocalcinosis. Continuous injection of parathyroid hormone (PTH) 1–34 induced nephrocalcinosis mainly in the proximal tubules in rats. His-sRAP retained fetuin-A in renal tubular lumen and thereby protected the kidneys of PTH-treated rats from calcification. Our findings suggest that tubular luminal fetuin-A works as a natural inhibitor against calcification in the proximal tubules under PTH-loaded condition.

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.

scholarly journals Contribution of the Na+-K+-2Cl− Cotransporter (NKCC1) to Transepithelial Transport of H+, NH4+, K+, and Na+ in Rat Outer Medullary Collecting Duct

2002 ◽  
Vol 13 (4) ◽  
pp. 827-835
Author(s):  
Susan M. Wall ◽  
Michael P. Fischer
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Transepithelial Transport ◽  
In Series ◽  
Acid Secreting ◽  
Medullary Collecting Duct ◽  
Nacl Secretion

ABSTRACT. In rat kidney, the “secretory” isoform of the Na-K-Cl cotransporter, NKCC1 (BSC-2), localizes to the basolateral membrane of the α intercalated cell, the acid secreting cell of the outer medullary collecting duct (OMCD). This laboratory has reported that NKCC1 mediates Cl− uptake across the basolateral membrane in series with Cl− secretion across the apical membrane in rat OMCD. NKCC1 transports NH4+, K+, and Na+ as well as Cl−; therefore, a role for the cotransporter in the process of HCl, NH4Cl, KCl, and NaCl secretion has been suggested. Thus, it was determined if bumetanide, an inhibitor of NKCC1, alters transepithelial cation transport in rat OMCD. OMCD tubules from deoxycorticosterone pivalate (DOCP)–treated rats were perfused in vitro. Hydration of CO2, rather than NH4+, provides the principle source of H+ for net acid secretion. In HCO3−/CO2-buffered solutions, no effect of bumetanide on net K+ flux was detected. Under some conditions, bumetanide addition resulted in a small reduction in secretion of net H+ equivalents. Transepithelial Na+ flux, JNa, was −1.5 ± 1.7 pmol/mm per min, values not different from zero. However, with the application of bumetanide to the bath, JNa was +5.2 ± 1.3 pmol/mm per min (P < 0.05), which indicates net Na+ absorption. In conclusion, inhibition of NKCC1 in rat OMCD changes transepithelial movement of Na+ and Cl−. The role of NKCC1 in the secretion of net H+ equivalents is small.

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.

scholarly journals Changes of renal AQP2, ENaC, and NHE3 in experimentally induced heart failure: response to angiotensin II AT1 receptor blockade

2009 ◽  
Vol 297 (6) ◽  
pp. F1678-F1688 ◽  
Author(s):  
Sophie C. Lütken ◽  
Soo Wan Kim ◽  
Thomas Jonassen ◽  
David Marples ◽  
Mark A. Knepper ◽  
...  
Keyword(s):  
Heart Failure ◽  
Angiotensin Ii ◽  
Diastolic Pressure ◽  
Collecting Duct ◽  
Left Ventricular ◽  
Outer Medulla ◽  
Outer Stripe ◽  
Medullary Collecting Duct ◽  
Type 3

Heart failure (HF) was induced by ligation of the left anterior descending artery (LAD). Left ventricular end-diastolic pressure (LVEDP) >25 mmHg (at day 23 after LAD ligation) was the inclusion criterion. The rats were divided into three groups: sham-operated (Sham, n = 23, LVEDP: 5.6 ± 0.6 mmHg), HF ( n = 14, LVEDP: 29.4 ± 1.4 mmHg), and candesartan (1 mg·kg−1·day−1 sc)-treated HF (HF + Can, n = 9, LVEDP: 29.2 ± 1.2 mmHg). After 7 days (i.e., 29 days after LAD ligation) semiquantitative immunoblotting revealed increased abundance of inner medulla aquaporin-2 (AQP2) and AQP2 phosphorylated at Ser256 (p-AQP2) in HF. There was also markedly enhanced apical targeting of AQP2 and p-AQP2 in inner medullary collecting duct (IMCD) in HF compared with Sham rats, shown by immunocytochemistry. Candesartan treatment significantly reversed the increases in both AQP2 and p-AQP2 expression and targeting. In contrast, there were only modest changes in other collecting duct segments. Semiquantitative immunoblots revealed increased expression of type 3 Na+/H+ exchanger (NHE3) and Na+-K+-2Cl− cotransporter (NKCC2) in kidneys from HF compared with Sham rats: both effects were reversed or prevented by candesartan treatment. The protein abundance of α-epithelial sodium channel (α-ENaC) was increased while β-ENaC and γ-ENaC expression was decreased in the cortex and outer stripe of the outer medulla in HF compared with Sham rats, which was partially reversed by candesartan treatment. These findings strongly support an important role of angiotensin II in the pathophysiology of renal water and sodium retention associated with HF.

Angiotensin I-converting enzyme activity in tubular fluid along the rat nephron

1997 ◽  
Vol 272 (3) ◽  
pp. F405-F409 ◽  
Author(s):  
D. E. Casarini ◽  
M. A. Boim ◽  
R. C. Stella ◽  
M. H. Krieger-Azzolini ◽  
J. E. Krieger ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Proximal Tubule ◽  
Mesangial Cells ◽  
Collecting Duct ◽  
Physiological Role ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Angiotensin I Converting Enzyme ◽  
Ace Activity

The activity of angiotensin I-converting enzyme (ACE) was determined in tubular fluid collected from several portions of the rat nephron and urine and in total and efferent arteriolar blood using hippuryl-L-His-Leu as substrate. ACE activity decreased 30% from the pre- to the postglomerular arterioles (P < 0.001), suggesting a role of the glomerulus in ACE clearance. The enzyme activity was found to be present throughout the rat nephron. However, the highest activities were found in the proximal tubule and urine (0.692 +/- 0.007 and 1.05 +/- 0.015 pmol x microl(-1) x min(-1), respectively). Compared with other segments, ACE activity decreased from the initial portion of the proximal tubule to the distal nephron and increased again in the urine. Along the proximal tubule, ACE was secreted and degraded and/or reabsorbed and then secreted again into the collecting duct; no ACE activity was found in the late distal tubule, but a high level was detected in the urine, indicating a potential physiological role in the inactivation of the kinins formed by kallikrein beyond the connecting tubules. Moreover, the possible role of mesangial cells (MC) in the decrease of intraglomerular ACE was also evaluated. The analysis of ACE gene showed that MC in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on MC function.

Contribution of the Na+-K+-2Cl−cotransporter NKCC1 to Cl− secretion in rat OMCD

2001 ◽  
Vol 280 (5) ◽  
pp. F913-F921 ◽  
Author(s):  
Susan M. Wall ◽  
Michael P. Fischer ◽  
Pramod Mehta ◽  
Kathryn A. Hassell ◽  
Stanley J. Park
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Physiological Role ◽  
Fluid Secretion ◽  
Transepithelial Potential ◽  
In Series

In rat kidney the “secretory” isoform of the Na+-K+-2Cl− cotransporter (NKCC1) localizes to the basolateral membrane of the α-intercalated cell. The purpose of this study was to determine whether rat outer medullary collecting duct (OMCD) secretes Cl− and whether transepithelial Cl− transport occurs, in part, through Cl− uptake across the basolateral membrane mediated by NKCC1 in series with Cl− efflux across the apical membrane. OMCD tubules from rats treated with deoxycorticosterone pivalate were perfused in vitro in symmetrical HCO[Formula: see text]/CO2-buffered solutions. Cl− secretion was observed in this segment, accompanied by a lumen positive transepithelial potential. Bumetanide (100 μM), when added to the bath, reduced Cl− secretion by 78%, although the lumen positive transepithelial potential and fluid flux were unchanged. Bumetanide-sensitive Cl− secretion was dependent on extracellular Na+ and either K+ or NH[Formula: see text], consistent with the ion dependency of NKCC1-mediated Cl− transport. In conclusion, OMCD tubules from deoxycorticosterone pivalate-treated rats secrete Cl−into the luminal fluid through NKCC1-mediated Cl− uptake across the basolateral membrane in series with Cl− efflux across the apical membrane. The physiological role of NKCC1-mediated Cl− uptake remains to be determined. However, the role of NKCC1 in the process of fluid secretion could not be demonstrated.

Na-K-ATPase in nephron segments of rats developing spontaneous hypertension

1985 ◽  
Vol 249 (6) ◽  
pp. F863-F869 ◽  
Author(s):  
L. C. Garg ◽  
N. Narang ◽  
S. McArdle
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Collecting Duct ◽  
Thick Ascending Limb ◽  
Spontaneously Hypertensive ◽  
Nephron Segments ◽  
Medullary Thick Ascending Limb ◽  
Abnormal Pattern ◽  
Wistar Kyoto

Na-K-ATPase activity was determined in seven specific nephron segments of 5- and 12-wk-old spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) controls. The enzyme activity in proximal convoluted tubule (PCT) and proximal straight tubule (PST) was significantly higher in 5-wk-old SHR than in WKY. However, Na-K-ATPase activity in medullary thick ascending limb (MTAL), cortical thick ascending limb (CTAL), and distal convoluted tubule (DCT) was significantly lower in 5-wk-old SHR than in WKY. There were no significant differences in the enzyme activity in PCT, PST, MTAL, CTAL, and DCT in 12-wk-old SHR and WKY. Furthermore, there were no significant differences in Na-K-ATPase activity in collecting duct segments of 5- or 12-wk-old SHR and age-matched WKY. The possible role of the abnormal pattern of Na-K-ATPase activity in PCT, PST, MTAL, CTAL, and DCT in 5-wk-old SHR in generation of hypertension in this strain remains to be determined.

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