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.

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.

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.

Localization of the extracellular Ca(2+)-sensing receptor and PTH/PTHrP receptor in rat kidney

1996 ◽  
Vol 271 (4) ◽  
pp. F951-F956 ◽  
Author(s):  
D. Riccardi ◽  
W. S. Lee ◽  
K. Lee ◽  
G. V. Segre ◽  
E. M. Brown ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Physiological Role ◽  
Urinary Concentration ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Medullary Thick Ascending Limb ◽  
Parathyroid Hormone Related Protein ◽  
Pthrp Receptor

Using a strategy based on homology to the bovine parathyroid Ca(2+)-sensing receptor previously identified by us (5), we have recently isolated an extracellular, G protein-coupled Ca2+/ polyvalent cation-sensing receptor, RaKCaR (22), from rat kidney. The localization and physiological role(s) of this receptor in the kidney are not well understood. In the present study, we assessed the distribution of mRNAs for RaKCaR and the parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor along the rat nephron by in situ hybridization and reverse transcriptase-polymerase chain reaction of microdissected nephron segments. Our results show that transcripts for both receptors coexpress at glomeruli, proximal convoluted tubule, proximal straight tubule, cortical thick ascending limb, distal convoluted tubule, and cortical collecting duct. In addition, RaKCaR (but not PTH/PTHrP receptor) transcripts were found in the medullary thick ascending limb and outer medullary and inner medullary collecting ducts. These findings raise the possibility of roles for RaKCaR not only in the regulation of divalent mineral reabsorption but also in water reabsorption and urinary concentration. Taken together, our results provide new insights in understanding the effects of hypercalcemia on hormone-stimulated salt and water transport.

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.

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.

scholarly journals Inhibition of calcium signaling in descending vasa recta endothelia by ANG II

2000 ◽  
Vol 278 (4) ◽  
pp. H1248-H1255 ◽  
Author(s):  
Thomas L. Pallone ◽  
Erik P. Silldorff ◽  
Zhong Zhang
Keyword(s):  
Collecting Duct ◽  
No Production ◽  
Vascular Bundles ◽  
Thick Ascending Limb ◽  
Ang Ii ◽  
Vasomotor Tone ◽  
Medullary Thick Ascending Limb ◽  
Vasa Recta ◽  
Medullary Collecting Duct ◽  
Peak Phase

The intracellular calcium ([Ca2+]i) response of outer medullary descending vasa recta (OMDVR) endothelia to ANG II was examined in fura 2-loaded vessels. Abluminal ANG II (10− 8 M) caused [Ca2+]i to fall in proportion to the resting [Ca2+]i ( r =0.82) of the endothelium. ANG II (10− 8 M) also inhibited both phases of the [Ca2+]i response generated by bradykinin (BK, 10− 7 M), 835 ± 201 versus 159 ± 30 nM (peak phase) and 169 ± 26 versus 103 ± 14 nM (plateau phase) (means ± SE). Luminal ANG II reduced BK (10− 7 M)-stimulated plateau [Ca2+]i from 180 ± 40 to 134 ± 22 nM without causing vasoconstriction. Abluminal ANG II added to the bath after luminal application further reduced [Ca2+]i to 113 ± 9 nM and constricted the vessels. After thapsigargin (TG) pretreatment, ANG II (10− 8 M) caused [Ca2+]i to fall from 352 ± 149 to 105 ± 37 nM. This effect occurred at a threshold ANG II concentration of 10− 10 M and was maximal at 10− 8 M. ANG II inhibited both the rate of Ca2+ entry into [Ca2+]i-depleted endothelia and the rate of Mn2+ entry into [Ca2+]i-replete endothelia. In contrast, ANG II raised [Ca2+]i in the medullary thick ascending limb and outer medullary collecting duct, increasing [Ca2+]i from baselines of 99 ± 33 and 53 ± 11 to peaks of 200 ± 47 and 65 ± 11 nM, respectively. We conclude that OMDVR endothelia are unlikely to be the source of ANG II-stimulated NO production in the medulla but that interbundle nephrons might release Ca2+-dependent vasodilators to modulate vasomotor tone in vascular bundles.

AVP-stimulated nucleotide secretion in perfused mouse medullary thick ascending limb and cortical collecting duct

2009 ◽  
Vol 297 (2) ◽  
pp. F341-F349 ◽  
Author(s):  
Elvin Odgaard ◽  
Helle A. Praetorius ◽  
Jens Leipziger
Keyword(s):  
Collecting Duct ◽  
Tubular Secretion ◽  
Hormonal Control ◽  
Similar Response ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Medullary Thick Ascending Limb ◽  
Tubular Transport ◽  
Renal Tubular Transport ◽  
Renal Tubular

Extracellular nucleotides are local, short-lived signaling molecules that inhibit renal tubular transport via both luminal and basolateral P2 receptors. Apparently, the renal epithelium itself is able to release nucleotides. The mechanism and circumstances under which nucleotide release is stimulated remain elusive. Here, we investigate the phenomenon of nucleotide secretion in intact, perfused mouse medullary thick ascending limb (mTAL) and cortical collecting duct (CCD). The nucleotide secretion was monitored by a biosensor adapted to register nucleotides in the tubular outflow. Intracellular Ca2+ concentration ([Ca2+]i) was measured simultaneously in the biosensor cells and the renal tubule with fluo 4. We were able to identify spontaneous tubular nucleotide secretion in resting perfused mTAL. In this preparation, 10 nM AVP and 1-desamino-8-d-arginine vasopressin (dDAVP) induced robust [Ca2+]i oscillations, whereas AVP in the CCD induced large, slow, and transient [Ca2+]i elevations. Importantly, we identify that AVP/dDAVP triggers tubular secretion of nucleotides in the mTAL. After addition of AVP/dDAVP, the biosensor registered bursts of nucleotides in the tubular perfusate, corresponding to a tubular nucleotide concentration of ∼0.2–0.3 μM. A very similar response was observed after AVP stimulation of CCDs. Thus AVP stimulated tubular secretion of nucleotides in a burst-like pattern with peak tubular nucleotide concentrations in the low-micromolar range. We speculate that local nucleotide signaling is an intrinsic feedback element of hormonal control of renal tubular transport.

Different mechanisms of renal Na-K-ATPase regulation by protein kinases in proximal and distal nephron

1993 ◽  
Vol 265 (3) ◽  
pp. F399-F405 ◽  
Author(s):  
T. Satoh ◽  
H. T. Cohen ◽  
A. I. Katz
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase ◽  
Collecting Duct ◽  
Common Mechanism ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Distal Nephron ◽  
Inhibitory Peptide ◽  
Medullary Thick Ascending Limb ◽  
Pump Activity

We recently reported a novel intracellular mechanism of Na-K-adenosinetriphosphatase (Na-K-ATPase) regulation in the cortical collecting duct (CCD) by agents that increase cell adenosine 3',5'-cyclic monophosphate (cAMP), which involves stimulation of protein kinase A (PKA) and phospholipase A2 (PLA2). We now determined whether this mechanism also operates in other nephron segments. In the medullary thick ascending limb (MTAL) dopamine, the DA1 agonist fenoldopam, forskolin, or dibutyryl-cAMP inhibited Na-K-ATPase activity, similar to results in CCD. In both segments this effect was blocked by 20-residue inhibitory peptide (IP20), a peptide inhibitor of PKA, but not by staurosporine, a protein kinase C (PKC) inhibitor. PKC activators phorbol 12-myristate 13-acetate, phorbol 12,13-dibutyrate, and 1,2-myristate 13-acetate, phorbol 12,13-dibutyrate, and 1,2-dioctanoylglycerol had no effect on Na-K pump activity in either CCD or MTAL. In contrast, all three PKC activators inhibited pump activity in the proximal convoluted tubule (PCT), an effect reproduced only by dopamine or by parathyroid hormone [PTH-(1-34)]. In PCT the pump inhibition by dopamine or PTH-(1-34) was abolished by staurosporine but not by IP20. The PLA2 inhibitor mepacrine prevented the effect of all agents, and arachidonic acid produced a dose-dependent pump inhibition in each of the three segments studied. We conclude that intracellular mechanisms of Na-K-ATPase regulation differ along the nephron, as they involve activation of PKA in CCD and MTAL and of PKC in PCT. These two pathways probably share a common mechanism in stimulating PLA2, arachidonic acid release, and production of eicosanoids in both the proximal and distal nephron.

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