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.

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.

cDNA cloning and localization of OCRL-1 in rabbit kidney

1997 ◽  
Vol 273 (5) ◽  
pp. F790-F795 ◽  
Author(s):  
Brian C. Erb ◽  
Heino Velázquez ◽  
Monique Gisser ◽  
Christine A. Shugrue ◽  
Robert F. Reilly
Keyword(s):  
Positional Cloning ◽  
Collecting Duct ◽  
Northern Analysis ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Tubular Dysfunction ◽  
Renal Tubular Dysfunction ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Glomerular Lesion

The oculocerebrorenal syndrome of Lowe (OCRL) is a hereditary multisystem disorder characterized by congenital cataracts, mental retardation, renal tubular dysfunction, and progressive renal insufficiency. Tubular abnormalities include proximal tubular dysfunction, a distal acidification defect, and a possible impairment of urinary concentrating ability. The most important renal manifestation of Lowe’s syndrome is a progressive loss of kidney function associated with a glomerular lesion that progresses to end-stage renal disease in either the third or fourth decade. The gene responsible for Lowe’s syndrome, OCRL-1, was recently identified by positional cloning, and mutations were demonstrated in many affected patients. In the present study reverse transcription-polymerase chain reaction (RT-PCR) was used to clone a partial-length cDNA encoding rabbit renal OCRL-1. There is a high degree of similarity between rabbit and human sequences, with nucleotide and amino acid identities of 92% and 97%, respectively. Northern analysis identified a 5.4-kb transcript that is expressed in both rabbit kidney cortex and medulla. Isolated nephron-segment RT-PCR showed that OCRL-1 is expressed in all segments studied: the glomerulus, proximal tubule, medullary and cortical thick ascending limb, distal convoluted tubule, connecting tubule, cortical collecting duct, and outer medullary collecting duct. Defective OCRL-1 expression in these regions may play a pathogenetic role in the renal manifestations of this syndrome.

Quantitative RT-PCR analysis of calcitonin receptor mRNAs in the rat nephron

1995 ◽  
Vol 269 (5) ◽  
pp. F702-F709 ◽  
Author(s):  
D. Firsov ◽  
A. C. Bellanger ◽  
S. Marsy ◽  
J. M. Elalouf
Keyword(s):  
Collecting Duct ◽  
Internal Standard ◽  
Pcr Analysis ◽  
Kidney Cortex ◽  
Thick Ascending Limb ◽  
Mrna Levels ◽  
Cortical Collecting Duct ◽  
Rt Pcr ◽  
Medullary Thick Ascending Limb ◽  
Henle's Loop

A quantitative assay based on the method of reverse transcription and polymerase chain reaction (RT-PCR) was developed to study the expression of calcitonin (CT) receptors in microdissected rat nephron segments. Steady-state mRNA levels of two CT-receptor spliced variants (CT1a and CT1b) were measured using a mutant cRNA as internal standard. CT1a, but not the CT1b isoform, was detected in the kidney cortex, outer medulla, and papilla. Among the tested segments, predominant expression of CT1a mRNA was found in the cortical thick ascending limb of Henle's loop (754 +/- 87 mRNA molecules/mm tubular length; n = 8). Lower expression levels were measured in the medullary thick ascending limb (460 +/- 62 molecules/mm tubule length; n = 7) and in the cortical collecting duct (327 +/- 61 molecules/mm tubule length; n = 6). A weak expression was also detected in the outer medullary collecting duct and the glomerulus. No expression was found in the proximal convoluted tubule, pars recta, and thin descending and thin ascending limb of Henle's loop. We conclude that only the CT1a-receptor mRNA is present in the rat kidney, with a significant level of expression in the cortical and medullary thick ascending limb and in the cortical collecting duct.

Cell-specific expression of amiloride-sensitive, Na(+)-conducting ion channels in the kidney

1996 ◽  
Vol 271 (4) ◽  
pp. C1303-C1315 ◽  
Author(s):  
F. Ciampolillo ◽  
D. E. McCoy ◽  
R. B. Green ◽  
K. H. Karlson ◽  
A. Dagenais ◽  
...  
Keyword(s):  
Cell Lines ◽  
Collecting Duct ◽  
Extracellular Fluid ◽  
Cation Channel ◽  
Thick Ascending Limb ◽  
Distal Nephron ◽  
Rt Pcr ◽  
Specific Expression ◽  
Nephron Segments ◽  
Medullary Collecting Duct

Amiloride-sensitive, electrogenic Na+ absorption across the distal nephron plays a vital role in regulating extracellular fluid volume and blood pressure. Recently, two amiloride-sensitive, Na(+)-conducting ion channel cDNAs were cloned. One, an epithelial Na(+)-selective channel (ENaC), is responsible for Na+ absorption throughout the distal nephron. The second, a guanosine 3',5'-cyclic monophosphate (cGMP)-inhibitable cation channel, is conductive to Na+ and Ca2+ and contributes to Na+ absorption across the inner medullary collecting duct (IMCD). As a first step toward understanding the segment-specific contributions(s) of cGMP-gated cation channels and ENaC to Na+ and Ca2+ uptake along the nephron, we used in situ reverse transcription-polymerase chain reaction (RT-PCR) hybridization, solution-phase RT-PCR, and Western blot analysis to examine the nephron and cell-specific expression of these channels in mouse kidney cell lines and/or dissected nephron segments. cGMP-gated cation channel mRNA was detected in proximal tubule, medullary thick ascending limb (mTAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and IMCD. cGMP-gated cation channel protein was detected in DCT, CCD, and IMCD cell lines. These observations suggest that hormones that modulate intracellular cGMP levels may regulate Na+, and perhaps Ca2+, uptake throughout the nephron. mRNA for alpha-mENaC, a subunit of the mouse ENaC, was detected in mTAL, DCT, CCD, OMCD, and IMCD. Coexpression of alpha-mENaC and cGMP-gated cation channel mRNAs in mTAL, DCT, CCD, OMCD, and IMCD suggests that both channels may contribute to Na+ absorption in these nephron segments.

Carbonic anhydrase II mRNA is induced in rabbit kidney cortex during chronic metabolic acidosis

1993 ◽  
Vol 265 (6) ◽  
pp. F764-F772 ◽  
Author(s):  
G. J. Schwartz ◽  
C. A. Winkler ◽  
B. J. Zavilowitz ◽  
T. Bargiello
Keyword(s):  
Metabolic Acidosis ◽  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Carbonic Anhydrase Ii ◽  
Northern Analysis ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Rt Pcr ◽  
Chronic Metabolic Acidosis ◽  
Pcr Product

Carbonic anhydrase II (CA II), the predominant isoform of carbonic anhydrase in the kidney, is believed to be localized primarily in the cytoplasm of proximal tubule and collecting duct intercalated cells. Carbonic anhydrase facilitates H+ secretion by catalyzing the formation of HCO3- from OH- in the presence of CO2. We have shown that renal cortical CA II activity is stimulated during 4-6 days of chronic metabolic acidosis [L.P. Brion, B.J. Zavilowitz, O. Rosen, and G.J. Schwartz. Am. J. Physiol. 261 (Regulatory Integrative Comp. Physiol. 30): R1204-R1213, 1991]. The purpose of these studies was to examine under similar conditions the regulation of CA II mRNA. We obtained a major portion of the rabbit CA II cDNA by reverse transcription of total RNA from rabbit kidney followed by amplification using oligonucleotide primers prepared from conserved areas in the coding regions of human, mouse, and chick CA II cDNAs in a polymerase chain reaction (RT-PCR). The 696-bp RT-PCR product was sequenced and found to be 71-86% homologous to CA II cDNAs from the other three species. The deduced amino acid sequence agreed closely (> 97%) with a previous Edman analysis of rabbit erythrocyte CA II. Northern analysis showed expression of a approximately 1.4 kb RNA, with cortex > outer medulla > inner medulla. Steady-state mRNA expression from kidney cortex of acid-treated rabbits was about twice that from controls, when normalized to the expression of beta-actin or malate dehydrogenase. The stimulation of CA II mRNA was greater after 3 days than after 5-6 days of acid treatment. (ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Carbonic anhydrase II and IV mRNA in rabbit nephron segments: stimulation during metabolic acidosis

1998 ◽  
Vol 274 (2) ◽  
pp. F259-F267 ◽  
Author(s):  
Shuichi Tsuruoka ◽  
Ann M. Kittelberger ◽  
George J. Schwartz
Keyword(s):  
Metabolic Acidosis ◽  
Carbonic Anhydrase ◽  
Mrna Expression ◽  
Collecting Duct ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Rt Pcr ◽  
Chronic Metabolic Acidosis ◽  
Nephron Segments ◽  
Medullary Collecting Duct

Carbonic anhydrase (CA) facilitates renal bicarbonate reabsorption and acid excretion. Cytosolic CA II catalyzes the buffering of intracellular hydroxyl ions by CO2, whereas membrane-bound CA IV catalyzes the dehydration of carbonic acid generated from the secretion of protons. Although CA II and IV are expressed in rabbit kidney, it is not entirely clear which segments express which isoforms. It was the purpose of this study to characterize the expression of CA II and CA IV mRNAs by specific segments of the nephron using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and to determine the effect of chronic metabolic acidosis on CA expression by those segments. Individual nephron segments (usually 1–2 mm) were isolated by microdissection and subjected to RT-PCR. Amplification was performed simultaneously for CA IV, CA II, and malate dehydrogenase (MDH), a housekeeping gene. The intensities of the PCR products were quantitated by densitometry. CA IV mRNA was expressed by S1 and S2 proximal tubules and by outer medullary collecting duct from inner stripe (OMCDi) and outer stripe and initial inner medullary collecting duct (IMCDi). CA II mRNA was expressed by S1, S2, and S3 proximal tubules, thin descending limb, connecting segment (CNT), and all collecting duct segments. Acid loading induced CA IV mRNA expression in S1 and S2 proximal tubules and in OMCDi and IMCDi. CA II mRNA was induced by acidosis in all three proximal segments and nearly all distal segments beginning with CNT. No upregulation of MDH mRNA expression occurred. These adaptive increases in CA II and IV mRNAs are potentially important in the kidney’s adaptation to chronic metabolic acidosis.

scholarly journals The EP3 receptor regulates water excretion in response to high salt intake

2016 ◽  
Vol 311 (4) ◽  
pp. F822-F829 ◽  
Author(s):  
Shoujin Hao ◽  
AnnMarie DelliPizzi ◽  
Mariana Quiroz-Munoz ◽  
Houli Jiang ◽  
Nicholas R. Ferreri
Keyword(s):  
Tap Water ◽  
Collecting Duct ◽  
Urine Volume ◽  
High Salt ◽  
Whole Body ◽  
Thick Ascending Limb ◽  
Water Excretion ◽  
Water Homeostasis ◽  
Cox 2 ◽  
Ep3 Receptor

The mechanisms by which prostanoids contribute to the maintenance of whole body water homeostasis are complex and not fully understood. The present study demonstrates that an EP3-dependent feedback mechanism contributes to the regulation of water homeostasis under high-salt conditions. Rats on a normal diet and tap water were placed in metabolic cages and given either sulprostone (20 μg·kg−1·day−1) or vehicle for 3 days to activate EP3 receptors in the thick ascending limb (TAL). Treatment was continued for another 3 days in rats given either 1% NaCl in the drinking water or tap water. Sulprostone decreased expression of cyclooxygenase 2 (COX-2) expression by ∼75% in TAL tubules from rats given 1% NaCl concomitant with a ∼60% inhibition of COX-2-dependent PGE2 levels in the kidney. Urine volume increased after ingestion of 1% NaCl but was reduced ∼40% by sulprostone. In contrast, the highly selective EP3 receptor antagonist L-798106 (100 μg·kg−1·day−1), which increased COX-2 expression and renal PGE2 production, increased urine volume in rats given 1% NaCl. Sulprostone increased expression of aquaporin-2 (AQP2) in the inner medullary collecting duct plasma membrane in association with an increase in phosphorylation at Ser269 and decrease in Ser261 phosphorylation; antagonism of EP3 with L-798106 reduced AQP2 expression. Thus, although acute activation of EP3 by PGE2 in the TAL and collecting duct inhibits the Na-K-2Cl cotransporter and AQP2 activity, respectively, chronic activation of EP3 in vivo limits the extent of COX-2-derived PGE2 synthesis, thereby mitigating the inhibitory effects of PGE2 on these transporters and decreasing urine volume.

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.

Cellular distribution of the potassium channel KCNQ1 in normal mouse kidney

2007 ◽  
Vol 292 (1) ◽  
pp. F456-F466 ◽  
Author(s):  
Wencui Zheng ◽  
Jill W. Verlander ◽  
I. Jeanette Lynch ◽  
Melanie Cash ◽  
Jiahong Shao ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Mouse Kidney ◽  
Cellular Heterogeneity ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Distal Nephron ◽  
Rt Pcr ◽  
Double Labeling ◽  
Cytoplasmic Staining ◽  
Principal Cells

Mechanisms of K+ secretion and absorption along the collecting duct are not understood fully. Because KCNQ1 participates in K+ secretion within the inner ear and stomach, distribution of KCNQ1 in mouse kidney was studied using Northern and Western analyses, RT-PCR of isolated tubules, and immunohistochemistry. Northern blots demonstrated KCNQ1 transcripts in whole kidney. RT-PCR showed KCNQ1 mRNA in isolated distal convoluted tubule (DCT), connecting segment (CNT), collecting ducts (CD), and glomeruli. Immunoblots of kidney and stomach revealed a ∼75-kDa protein, the expected mobility for KCNQ1. KCNQ1 was detected by immunohistochemistry throughout the distal nephron and CD. Thick ascending limbs exhibited weak basolateral immunolabel. In DCT and CNT cells, immunolabel was intense and basolateral, although KCNQ1 label was stronger in late than in early DCT. Initial collecting tubule and cortical CD KCNQ1 immunolabel was predominantly diffuse, but many cells exhibited discrete apical label. Double-labeling experiments demonstrated that principal cells, type B intercalated cells, and a few type A intercalated cells exhibited distinct apical KCNQ1 immunolabel. In inner medullary CD, principal cells exhibited distinct basolateral KCNQ1 immunolabel, whereas intercalated cells showed diffuse cytoplasmic staining. Thus KCNQ1 protein is widely distributed in mouse distal nephron and CD, with significant axial and cellular heterogeneity in location and intensity. These findings suggest that KCNQ1 has cell-specific roles in renal ion transport and may participate in K+ secretion and/or absorption along the thick ascending limb, DCT, connecting tubule, and CD.

Localization of connexin 30 in the luminal membrane of cells in the distal nephron

2005 ◽  
Vol 289 (6) ◽  
pp. F1304-F1312 ◽  
Author(s):  
Fiona McCulloch ◽  
Régine Chambrey ◽  
Dominique Eladari ◽  
János Peti-Peterdi
Keyword(s):  
Gap Junction ◽  
Collecting Duct ◽  
Protein A ◽  
High Salt ◽  
Rabbit Kidney ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Distal Nephron ◽  
Luminal Membrane ◽  
Junction Protein

Several isoforms of the gap junction protein connexin (Cx) have been identified in a variety of tissues that communicate intercellular signals between adjacent cells. In the kidney, Cx37, Cx40, and Cx43 are localized in the vasculature, glomerulus, and tubular segments in a punctuate pattern, typical of classic gap junction channels. We performed immunohistochemistry in the mouse, rat, and rabbit kidney to study the localization of Cx30 protein, a new member of the Cx family. The vasculature, glomerulus, and proximal nephron segments were devoid of staining in all three species. Unexpectedly, Cx30 was found throughout the luminal membrane of select cells in the distal nephron. Expression of Cx30 was highest in the rat, which also showed some diffuse cytosolic labeling, continuous from the medullary thick ascending limb to the collecting duct system, and with the highest level in the distal convoluted tubule. Labeling in the mouse and rabbit was much less, limited to intercalated cells in the connecting segment and cortical collecting duct, where the apical signal was particularly strong. A high-salt-containing diet and culture medium upregulated Cx30 expression in the rat inner medulla and in M1 cells, respectively. The distinct, continuous labeling of the luminal plasma membrane and upregulation by high salt suggest that Cx30 may function as a hemichannel involved in the regulation of salt reabsorption in the distal nephron.

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