Developmental expression and biological activity of gastrin-releasing peptide and its receptors in the kidney

2004 ◽  
Vol 287 (3) ◽  
pp. F578-F585 ◽  
Author(s):  
Chelsea Dumesny ◽  
Jane C. Whitley ◽  
Graham S. Baldwin ◽  
Andrew S. Giraud ◽  
Arthur Shulkes
Keyword(s):  
Rat Kidney ◽  
Regulatory Peptides ◽  
Developmental Expression ◽  
Renal Tumors ◽  
Receptor Subtype ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Gastrin Releasing Peptide ◽  
Cell Extracts ◽  
Specific Radioimmunoassay

Mammalian gastrin-releasing peptide (GRP) has a widespread distribution and multiple stimulating effects on metabolism, release of regulatory peptides, gastrointestinal and pancreatic secretions, and behavior. GRP is a potent mitogen for a number of tumor types, including colon and lung. Although GRP is known to stimulate the growth of renal tumors, little is known of its synthesis, distribution, and receptors in the developing and mature kidney. Both Northern blot analysis and RT-PCR revealed the presence of GRP mRNA in ovine kidney from midgestation through to adulthood. GRP mRNA was detected in rat kidney from embryonic day 19 to postnatal day 30 by RT-PCR. Sequence-specific radioimmunoassay demonstrated the presence of substantial amounts of fully processed amidated GRP in the ovine renal cortex and medulla. The mRNA for the major receptor subtype, GRP-R, was present in fetal and adult sheep and rat kidneys. The mRNA for the low-affinity GRP receptor, bombesin receptor subtype-3 (BRS-3), was only detected in the rat kidney. In the ovine kidney, immunohistochemistry localized GRP predominantly to the thick ascending limb of the loop of Henle. mRNAs for GRP, GRP-R, and BRS-3 were detected in the human embryonic kidney cell line HEK293, and radioimmunoassay of cell extracts and conditioned media revealed the presence of proGRP but not the amidated form. However, amidated GRP did stimulate the proliferation of these cells. These studies demonstrate that the developing and mature kidney may be previously unidentified sites of autocrine or paracrine action for GRP.

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

1995 ◽  
Vol 268 (6) ◽  
pp. F1224-F1228 ◽  
Author(s):  
P. Borensztein ◽  
M. Froissart ◽  
K. Laghmani ◽  
M. Bichara ◽  
M. Paillard
Keyword(s):  
Rat Kidney ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Medullary Thick Ascending Limb ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

Expression of Na-P(i) cotransport in rat kidney: localization by RT-PCR and immunohistochemistry

1994 ◽  
Vol 266 (5) ◽  
pp. F767-F774 ◽  
Author(s):  
M. Custer ◽  
M. Lotscher ◽  
J. Biber ◽  
H. Murer ◽  
B. Kaissling
Keyword(s):  
Transport System ◽  
Polyclonal Antibodies ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Expression Cloning ◽  
Kidney Cortex ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Rat Kidney Cortex ◽  
Proximal Tubular

We have recently identified a rat kidney cortex Na-dependent transport system for phosphate (P(i)) by expression cloning (NaP(i)-2) (S. Magagnin, A. Werner, D. Markovich, V. Sorribas, G. Stange, J. Biber, and H. Murer. Proc. Natl. Acad. Sci. USA 90: 5979, 1993). In this study we have used reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry to establish the sites of expression of the NaP(i)-2-related mRNA and protein. RT-PCR was performed with single microdissected nephron segments. From these experiments we conclude that NaP(i)-2 mRNA is predominantly expressed in the proximal tubules of superficial and deep nephrons. No NaP(i)-2 mRNA was detected in the thick ascending limb of Henle's loop; however, faint NaP(i)-2 related PCR products were also observed in collecting ducts. Expression of the NaP(i)-2 protein was examined with the use of polyclonal antibodies raised against synthetic NaP(i)-2-derived peptides. Strong specific anti-NaP(i)-2 antiserum-mediated immunofluorescence was found in the convoluted part of proximal tubules and gradually decreased along the straight part. Immunofluorescence indicated that the NaP(i)-2 protein is present in the brush border of proximal tubular cells. In addition, NaP(i)-2-specific immunofluorescence was also observed in subapical vesicles. The described distribution of the NaP(i)-2 protein is in agreement with previously described nephron sites of P(i) reabsorption in the rat kidney and therefore suggests that the NaP(i)-2 transport system represents an Na-P(i) cotransporter involved in proximal tubular apical transport of phosphate.

Rat homolog of sulfonylurea receptor 2B determines glibenclamide sensitivity of ROMK2 in Xenopus laevisoocyte

2000 ◽  
Vol 278 (4) ◽  
pp. F659-F666 ◽  
Author(s):  
Masayuki Tanemoto ◽  
Carlos G. Vanoye ◽  
Ke Dong ◽  
Richard Welch ◽  
Takaaki Abe ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Xenopus Laevis Oocytes ◽  
Rat Tissues ◽  
Thick Ascending Limb ◽  
Sulfonylurea Receptor ◽  
Cortical Collecting Duct ◽  
Rt Pcr ◽  
Shared Identity

Recent studies showed that coexpression of Kir6.1 or Kir6.2 with the sulfonylurea receptor (SUR1, SUR2A, or SUR2B) reconstituted an inwardly rectifying, ATP-sensitive K+channel that was inhibited by glibenclamide (2, 15–17). Here we report the isolation of a rat homolog of mouse SUR2B (denoted rSUR2B) from a rat kidney cDNA library. The rSUR2B sequence contains a 4,635-bp open reading frame that encodes a 1,545-amino acid polypeptide, showing 67% shared identity with SUR1 (a pancreatic β-cell isoform) and 98% with both SUR2A (a brain isoform) and SUR2B (a vascular smooth muscle isoform). Consistent with the predicted structures of other members of the ATP-binding cassette (ABC) superfamily, the sequence of rSUR2B contains 17 putative membrane-spanning segments. Also, predicted Walker A and B consensus binding motifs, present in other ABC members, are conserved in the rSUR2B sequence. RT-PCR revealed that rSUR2B is widely expressed in various rat tissues including brain, colon, heart, kidney, liver, skeletal muscle, and spleen. The intrarenal distribution of the rSUR2B transcript was investigated using RT-PCR and Southern blot of microdissected tubules. The rSUR2B transcript was detected in proximal tubule, cortical thick ascending limb, distal collecting tubule, cortical collecting duct, and outer medullary collecting duct, but not medullary thick ascending limb. This distal distribution overlaps with that of ROMK. Coexpression of rSUR2B with ROMK2 cRNA (in 1:10 ratio) in Xenopus laevis oocytes resulted in whole cell Ba2+-sensitive K+ currents that were inhibited by glibenclamide (50% inhibition with 0.2 mM glibenclamide). In contrast, rSUR2B did not confer significant glibenclamide sensitivity to oocytes coinjected with ROMK1 or ROMK3. The interaction between ROMK2 and rSUR2B was further studied by coimmunoprecipitation of in vitro translated rSUR2B and ROMK2. In agreement with the functional data, the rSUR2B protein was coimmunoprecipitated with ROMK2 in the ROMK2-rSUR2B cotranslated samples. Our data demonstrate that ROMK2, but not ROMK1 and ROMK3, can interact with rSUR2B to confer a sulfonylurea-sensitive K+ channel, implicating SUR proteins in forming and regulating renal ATP-sensitive K+ channels. The ROMK isoform specificity of glibenclamide effects suggests that the NH2 terminus of the ROMK protein mediates rSUR2B-ROMK2 interactions.

Detection of dopamine receptor D1Asubtype-specific mRNA in rat kidney by in situ amplification

1998 ◽  
Vol 274 (1) ◽  
pp. F232-F241 ◽  
Author(s):  
Damian P. O’Connell ◽  
Anna M. Aherne ◽  
Eamon Lane ◽  
Robin A. Felder ◽  
Robert M. Carey
Keyword(s):  
Rat Kidney ◽  
Molecular Techniques ◽  
Receptor Protein ◽  
Receptor Subtype ◽  
Rt Pcr ◽  
Site Specific ◽  
Receptor Mrna ◽  
Wistar Kyoto ◽  
Specific Mrna

In recent years, both molecular biological and immunohistochemical techniques, utilizing receptor subtype-specific probes and antibodies to cloned central nervous system dopamine receptors, have revealed their presence in a number of peripheral organs and tissues. Molecular techniques have been hindered by the low abundance of receptor mRNA in these sites, and reverse transcription-polymerase chain reaction (RT-PCR) has been utilized to address this problem. However, RT-PCR is most often employed on either isolated mRNA or microdissected tissue samples, thereby limiting interpretation of whole tissue distribution. The present paper describes the use of a novel self-sustained sequence replication system (3SR) to amplify a target mRNA sequence in situ within the tissue or cell of interest that is then detected with the use of an internal labeled probe, using standard nonisotopic in situ hybridization. Specifically, D1Areceptor mRNA was amplified and detected in kidney sections of Wistar-Kyoto rats (WKY). The amplified D1A receptor mRNA was localized to renal arterioles, juxtaglomerular apparatus, and both proximal and distal tubules. mRNA was colocalized to regions shown also to contain D1A receptor protein. D1A receptor mRNA was predominantly localized in the cortex. Specificity of D1A receptor mRNA detection was confirmed by appropriate localization in rat brain sections known to express D1A receptor mRNA. In addition, we confirmed the presence of renal D1A receptor mRNA by RT-PCR. We conclude that D1A receptor mRNA is expressed in a site-specific manner in the WKY kidney. The use of 3SR in situ permits elucidation of site specific mRNA localization in a manner not reported previously.

Expression and localization of prostaglandin EP3 receptor mRNA in the immature rabbit kidney

1996 ◽  
Vol 271 (1) ◽  
pp. F30-F36 ◽  
Author(s):  
M. Bonilla-Felix ◽  
W. Jiang
Keyword(s):  
Collecting Duct ◽  
Internal Standard ◽  
Developmental Expression ◽  
Rabbit Kidney ◽  
Ribonuclease Protection Assay ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Guanine Nucleotide Binding Protein ◽  
Camp Generation ◽  
Ep3 Receptor

Arginine vasopressin (AVP)-stimulated cAMP generation is decreased in the immature collecting duct (CD). This is the result of prostaglandin antagonism, most likely via the inhibitory guanine nucleotide-binding protein (Gi). The EP3-subtype prostaglandin E2 (PGE2) receptor, which is coupled to Gi, could mediate this effect. We studied the developmental expression of EP3 receptor in the rabbit kidney. Higher levels of EP3 mRNA were observed in the immature kidney using three different assays: 1) reverse transcription-polymerase chain reaction (RT-PCR) with internal standard, 2) competitive PCR, and 3) ribonuclease protection assay. The highest levels were observed at 2 wk of age. RT-PCR from isolated nephron segments detected EP3 mRNA in the medullary thick ascending limb, cortical CD (CCD), and inner medullary CD (IMCD) of adult and immature kidneys. We conclude that 1) renal expression of EP3 mRNA is increased in immature kidneys and 2) EP3 mRNA is localized in the distal nephron. This suggests that EP3 receptor may play a role in the regulation of distal tubular transport during development.

RT-PCR microlocalization of mRNA for guanylyl cyclase-coupled ANF receptor in rat kidney

1991 ◽  
Vol 261 (6) ◽  
pp. F1080-F1087 ◽  
Author(s):  
Y. Terada ◽  
T. Moriyama ◽  
B. M. Martin ◽  
M. A. Knepper ◽  
A. Garcia-Perez
Keyword(s):  
Guanylyl Cyclase ◽  
Renal Tubule ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Renal Tubules ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Vasa Recta ◽  
Vascular Elements ◽  
Medullary Collecting Duct

Microlocalization of mRNA coding for the guanylyl cyclase-coupled atrial natriuretic factor (ANF) receptor was carried out in the rat kidney. We used a combination of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected renal tubule segments, glomeruli, and vasa recta bundles. Relative quantitation of the resulting amplified cDNA utilized densitometry of autoradiograms from Southern blots probed with a specific 32P-labeled probe. Among renal tubule segments, the largest signal was found in the terminal inner medullary collecting duct (IMCD). Slightly smaller signals were found in the initial IMCD and in loop of Henle segments from the inner medulla. Readily detectable signals were also seen in the following segments (in descending order): cortical collecting duct, proximal convoluted tubule, medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule, and outer medullary collecting duct. Large signals were also detected in glomeruli and in vasa recta bundles from the inner stripe of the outer medulla. Based on these results, we conclude that 1) renal microlocalization of specific mRNAs coding for hormone receptors is feasible through application of the RT-PCR procedure in microdissected renal tubules and vascular elements, and 2) the gene for the guanylyl cyclase-coupled ANF receptor is broadly expressed along the nephron, raising the possibility that multiple sites of ANF action are present.

Expression and cellular localization of mRNA encoding the "gastric" isoform of H(+)-K(+)-ATPase alpha-subunit in rat kidney

1995 ◽  
Vol 268 (1) ◽  
pp. F99-F109 ◽  
Author(s):  
K. Y. Ahn ◽  
B. C. Kone
Keyword(s):  
Cellular Localization ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Molecular Probes ◽  
Alpha Subunit ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Direct Demonstration ◽  
Specific Hybridization ◽  
Pcr Products

The distribution of transcripts encoding the gastric H(+)-K(+)-adenosinetriphosphatase (ATPase) alpha-subunit in the normal rat kidney was studied by reverse transcription-polymerase chain reaction (RT-PCR), combined with DNA sequence analysis and renal microdissection, and by nonradioactive in situ hybridization of fixed kidney sections using highly specific molecular probes. RT-PCR products corresponding to the gastric H(+)-K(+)-ATPase alpha-subunit were detected in the cortex, outer and inner medulla, and in isolated cortical (CCD) and inner medullary collecting ducts (IMCD). With digoxigenin-labeled cRNAs derived from the 5' and 3' ends of the gastric H(+)-K(+)-ATPase alpha-subunit cDNA, specific hybridization signal was detected prominently in all the cells of the connecting segment and CCD, the intercalated cells of the outer medullary collecting duct, the IMCD, and the renal pelvic epithelium lining the secondary pouches. Weak labeling was noted in the S3 segment of the proximal tubule, the distal convoluted tubule, and the cortical thick ascending limb of Henle. Hybridization with the sense probes produced no cellular labeling. These data provide the first direct demonstration for the expression and cellular distribution of mRNA encoding the gastric H(+)-K(+)-ATPase alpha-subunit in the normal, potassium-replete kidney, and they provide essential tools for the molecular analysis of renal acid base and potassium transport under physiological and pathophysiological conditions.

1650: Novel Gene Expression Markers in Renal Tumors Using Rt-PCR on Fixed Tissues Useful in Differentiating Histologic Subtypes

2004 ◽  
Vol 171 (4S) ◽  
pp. 436-436
Author(s):  
John A. Petros ◽  
Audry N. Schuetz ◽  
Andrew N. Young ◽  
Q. Yin Goen ◽  
So Dug Lim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Renal Tumors ◽  
Rt Pcr ◽  
Novel Gene ◽  
Histologic Subtypes

Prostanoid signaling, localization, and expression of IP receptors in rat thick ascending limb cells

1998 ◽  
Vol 275 (6) ◽  
pp. F904-F914 ◽  
Author(s):  
Richard L. Hébert ◽  
Tim O’Connor ◽  
Chris Neville ◽  
Kevin D. Burns ◽  
Odette Laneuville ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Rat Kidney ◽  
Receptor Expression ◽  
Molecular Evidence ◽  
Thick Ascending Limb ◽  
Renal Epithelial Cells ◽  
Receptor Mrna ◽  
Ip Receptor ◽  
Receptor Cdna ◽  
Nucleotide Binding Proteins

It is widely held that only one prostacyclin (IP) receptor exists that can couple to guanine stimulatory nucleotide binding proteins (Gs) leading to activation of adenyl cyclase. Although IP receptor mRNA is expressed in vascular arterial smooth muscle cells and platelets, with lower level expression in mature thymocytes, splenic lymphocytes, and megakaryocytes, there is no molecular evidence for IP receptor expression in renal epithelial cells. The purpose of the present study was to obtain molecular evidence for the expression and localization of the IP receptor and to study the signaling pathways of IP receptor in rat medullary thick ascending limb (MTAL). Biochemical studies showed that IP prostanoids do not increase cAMP in rat MTAL. However, in the presence of vasopressin, inhibition of cAMP formation by prostacyclin (PGI2) analogs is pertussis toxin sensitive and does not activate protein kinase C. In situ hybridization studies localized IP receptor mRNA expression to MTAL in the rat kidney outer medulla. The results of RT-PCR of freshly isolated RNA from MTAL, with primers specific for the mouse IP receptor cDNA, produced an amplification product of the correct predicted size that contained an expected Nco I endonuclease restriction site. We conclude that rat renal epithelial cells express the IP receptor, coupled to inhibition of cAMP production.

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