Glucocorticoids inhibit transcription and expression of the UT-A urea transporter gene

2002 ◽  
Vol 282 (5) ◽  
pp. F853-F858 ◽  
Author(s):  
Tao Peng ◽  
Jeff M. Sands ◽  
Serena M. Bagnasco
Keyword(s):  
Northern Blot Analysis ◽  
Collecting Duct ◽  
Renal Medulla ◽  
Site Directed Mutagenesis ◽  
Protein Abundance ◽  
Urea Transporter ◽  
Dexamethasone Treatment ◽  
Gel Shift Assay ◽  
Medullary Collecting Duct ◽  
Specific Factors

Dexamethasone treatment increases urea excretion and decreases urea permeability and urea transporter UT-A1 protein abundance in the inner medullary collecting duct (IMCD) of adrenalectomized rats. We examined the effect of dexamethasone treatment for 3 days on the abundance of several UT-A mRNA transcripts in rat renal medulla. By Northern blot analysis, a significant decrease in mRNA expression was observed in the inner medulla of dexamethasone-treated rats compared with controls for UT-A1 (71%), UT-A3 (75%), and UT-A3b (75%), but not for UT-A2. We then tested the effect of 100 nM dexamethasone on the activity of promoter I in the UT-A gene, using LLC-PK1-GR101 cells that express the glucocorticoid receptor. Dexamethasone significantly decreased the activity of rat UT-A promoter I (72%) but did not affect UT-A promoter II. Deletion analysis and site-directed mutagenesis demonstrated that sequences between −423 and −244 are important for this inhibition and that a 10-bp sequence at −363, which binds a nuclear protein in a gel shift assay, is necessary for basal promoter activity. The specific factors involved in repression of UT-A promoter I activity by glucocorticoids remain to be determined.

scholarly journals Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect

2010 ◽  
Vol 299 (4) ◽  
pp. F712-F719 ◽  
Author(s):  
Qi Cai ◽  
Sarah K. Nelson ◽  
Matthew R. McReynolds ◽  
Maggie Keck Diamond-Stanic ◽  
David Elliott ◽  
...  
Keyword(s):  
Er Stress ◽  
Collecting Duct ◽  
Renal Medulla ◽  
Immunogold Electron Microscopy ◽  
Protein Abundance ◽  
Wild Type ◽  
Urea Transporter ◽  
Principal Cells ◽  
Er Stress Response ◽  
Er Chaperone

Activation of V2 receptors (V2R) during antidiuresis increases the permeability of the inner medullary collecting duct to urea and water. Extracellular osmolality is elevated as the concentrating capacity of the kidney increases. Osmolality is known to contribute to the regulation of collecting duct water (aquaporin-2; AQP2) and urea transporter (UT-A1, UT-A3) regulation. AQP1KO mice are a concentrating mechanism knockout, a defect attributed to the loss of high interstitial osmolality. A V2R-specific agonist, deamino-8-d-arginine vasopressin (dDAVP), was infused into wild-type and AQP1KO mice for 7 days. UT-A1 mRNA and protein abundance were significantly increased in the medullas of wild-type and AQP1KO mice following dDAVP infusion. The mRNA and protein abundance of UT-A3, the basolateral urea transporter, was significantly increased by dDAVP in both wild-type and AQP1KO mice. Semiquantitative immunoblots revealed that dDAVP infusion induced a significant increase in the medullary expression of the endoplasmic reticulum (ER) chaperone GRP78. Immunofluorescence studies demonstrated that GRP78 expression colocalized with AQP2 in principal cells of the papillary tip of the renal medulla. Using immunohistochemistry and immunogold electron microscopy, we demonstrate that vasopressin induced a marked apical targeting of GRP78 in medullary principal cells. Urea-sensitive genes, GADD153 and ATF4 (components of the ER stress pathway), were significantly increased in AQP1KO mice by dDAVP infusion. These findings strongly support an important role of vasopressin in the activation of an ER stress response in renal collecting duct cells, in addition to its role in activating an increase in UT-A1 and UT-A3 abundance.

Genetic deletion of the P2Y2 receptor offers significant resistance to development of lithium-induced polyuria accompanied by alterations in PGE2 signaling

2012 ◽  
Vol 302 (1) ◽  
pp. F70-F77 ◽  
Author(s):  
Yue Zhang ◽  
Ioana L. Pop ◽  
Noel G. Carlson ◽  
Bellamkonda K. Kishore
Keyword(s):  
Ex Vivo ◽  
Collecting Duct ◽  
Renal Medulla ◽  
Urine Osmolality ◽  
Protein Abundance ◽  
P2y2 Receptor ◽  
Dietary Regimen ◽  
Medullary Collecting Duct ◽  
Genetic Deletion ◽  
Camp Levels

Lithium (Li)-induced polyuria is due to resistance of the medullary collecting duct (mCD) to the action of arginine vasopressin (AVP), apparently mediated by increased production of PGE2. We previously reported that the P2Y2 receptor (P2Y2-R) antagonizes the action of AVP on the mCD and may play a role in Li-induced polyuria by enhancing the production of PGE2 in mCD. Hence, we hypothesized that genetic deletion of P2Y2-R should ameliorate Li-induced polyuria. Wild-type (WT) or P2Y2-R knockout (KO) mice were fed normal or Li-added diets for 14 days and euthanized. Li-induced polyuria, and decreases in urine osmolality and AQP2 protein abundance in the renal medulla, were significantly less compared with WT mice despite the lack of differences in Li intake or terminal serum or inner medullary tissue Li levels. Li-induced increased urinary excretion of PGE2 was not affected in KO mice. However, prostanoid EP3 receptor (EP3-R) protein abundance in the renal medulla of KO mice was markedly lower vs. WT mice, irrespective of the dietary regimen. The protein abundances of other EP-Rs were not altered across the groups irrespective of the dietary regimen. Ex vivo stimulation of mCD with PGE2 generated significantly more cAMP in Li-fed KO mice (130%) vs. Li-fed WT mice (100%). Taken together, these data suggest 1) genetic deletion of P2Y2-R offers significant resistance to the development of Li-induced polyuria; and 2) this resistance is apparently due to altered PGE2 signaling mediated by a marked decrease in EP3-R protein abundance in the medulla, thus attenuating the EP3-mediated decrease in cAMP levels in mCD.

Endothelin receptor mRNA expression in renal medulla identified by in situ RT-PCR

1995 ◽  
Vol 269 (3) ◽  
pp. F449-F457 ◽  
Author(s):  
L. H. Chow ◽  
S. Subramanian ◽  
G. J. Nuovo ◽  
F. Miller ◽  
E. P. Nord
Keyword(s):  
Mrna Expression ◽  
Collecting Duct ◽  
Renal Medulla ◽  
Receptor Subtypes ◽  
Rt Pcr ◽  
Receptor Mrna ◽  
Pcr Products ◽  
Medullary Collecting Duct ◽  
Labeled Probe

Three subtypes of endothelin (ET) receptors have been identified by cDNA cloning, namely ET-RA, ET-RB, and ET-RC. In the current study the precise cellular distribution of the ET receptor subtypes in the renal medulla was explored by detecting the corresponding polymerase chain reaction (PCR)-amplified cDNAs by in situ reverse transcription (RT)-PCR. The PCR-amplified cDNAs were detected either by direct incorporation using digoxigenin-dUTP (dig-dUTP) as a nucleotide substrate in the PCR reaction or by in situ hybridization with the dig-dUTP-labeled probe. ET-RB mRNA was detected exclusively in the epithelial cells of the inner and outer medullary collecting duct. In contrast, ET-RA message was observed primarily in interstitial cells and pericytes of the vasae rectae in the outer and inner medulla. Southern blot analysis of PCR-amplified cDNAs reverse transcribed from extracted RNA of rat renal medulla confirmed the specificity of the RT-PCR products. ET-RC mRNA was not detected. We conclude that ET-RB is the major ET receptor found in rat renal medulla and is expressed exclusively on inner medullary collecting duct cells. The pattern of ET receptor mRNA expression described suggests different physiological actions for ET on the diverse cellular structures of the renal medulla.

Sequential expression of NKCC2, TonEBP, aldose reductase, and urea transporter-A in developing mouse kidney

2007 ◽  
Vol 292 (1) ◽  
pp. F269-F277 ◽  
Author(s):  
Hyun-Wook Lee ◽  
Wan-Young Kim ◽  
Hyun-Kuk Song ◽  
Chul-Woo Yang ◽  
Ki-Hwan Han ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Aldose Reductase ◽  
Vascular Endothelial Cells ◽  
Collecting Duct ◽  
Renal Medulla ◽  
Thick Ascending Limb ◽  
Urea Transporter ◽  
Adult Mice ◽  
Enhancer Binding Protein ◽  
Treatment Support

This study was conducted to test the hypothesis that, during renal development, the Na-K-2Cl cotransporter type 2 (NKCC2) activates the tonicity-responsive enhancer binding protein (TonEBP) transcription factor by creating medullary hypertonicity. TonEBP, in turn, drives the expression of aldose reductase (AR) and urea transporter-A (UT-A). Kidneys from 13- to19-day-old fetuses ( F13– F19), 1- to 21-day-old pups ( P1– P21), and adult mice were examined by immunohistochemistry. NKCC2 was first detected on F14 in differentiating macula densa and thick ascending limb (TAL). TonEBP was first detected on F15 in the medullary collecting duct (MCD) and surrounding endothelial cells. AR was detected in the MCD cells of the renal medulla from F15. UT-A first appeared in the descending thin limb (DTL) on F16 and in the MCD on F18. After birth, NKCC2-positive TALs disappeared gradually from the tip of the renal papilla, becoming completely undetectable in the inner medulla on P21. TonEBP shifted from the cytoplasm to the nucleus in both vascular endothelial cells and MCD cells on P1, and its abundance increased gradually afterward. Immunoreactivity for AR and UT-A in the renal medulla increased markedly after birth. Treatment of neonatal animals with furosemide dramatically reduced expression of TonEBP, AR, and UT-A1. Furosemide also prevented the disappearance of NKCC2-expressing TALs in the papilla. The sequential expression of NKCC2, TonEBP, and its targets AR and UT-A and the reduced expression TonEBP and its targets in response to furosemide treatment support the hypothesis that local hypertonicity produced by the activity of NKCC2 activates TonEBP during development.

Vasopressin receptor subtype 2 activation increases cell proliferation in the renal medulla of AQP1 null mice

2007 ◽  
Vol 293 (6) ◽  
pp. F1858-F1864 ◽  
Author(s):  
Qi Cai ◽  
Matthew R. McReynolds ◽  
Maggie Keck ◽  
Kevin A. Greer ◽  
James B. Hoying ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Collecting Duct ◽  
Renal Medulla ◽  
Nuclear Antigen ◽  
Receptor Subtype ◽  
Protein Abundance ◽  
Activating Transcription Factor 3 ◽  
Collecting Ducts ◽  
Growth Changes ◽  
Activating Transcription Factor

Aquaporin (AQP) 1 null mice have a defect in the renal concentrating gradient because of their inability to generate a hyperosmotic medullary interstitium. To determine the effect of vasopressin on renal medullary gene expression, in the absence of high local osmolarity, we infused 1-deamino-8-d-arginine vasopressin (dDAVP), a V2 receptor (V2R)-specific agonist, in AQP1 null mice for 7 days. cDNA microarray analysis was performed on the renal medullary tissue, and 5,140 genes of the possible 12,000 genes on the array were included in the analysis. In the renal medulla of AQP1 null mice, 245 transcripts were identified as increased by dDAVP infusion and 200 transcripts as decreased (1.5-fold or more). Quantitative real-time PCR measurements confirmed the increases seen for cyclin D1, early growth response gene 1, and activating transcription factor 3, genes associated with changes in cell cycle/growth. Changes in mRNA expression were correlated with changes in protein expression by semiquantitative immunoblotting; cyclin D1 and ATF3 were increased significantly in abundance following dDAVP infusion in the renal medulla of AQP1 null mice (161 and 461%, respectively). A significant increase in proliferation of medullary collecting ducts cells, following V2R activation, was identified by proliferating cell nuclear antigen immunohistochemistry; colocalization studies with AQP2 indicated that the increase in proliferation was primarily observed in principal cells of the inner medullary collecting duct (IMCD). V2R activation, via dDAVP, increased AQP2 and AQP3 protein abundance in the cortical collecting ducts of AQP1 null mice. However, V2R activation did not increase AQP2 protein abundance in the IMCD of AQP1 null mice.

Ultramicrodetermination of vasopressin-regulated urea transporter protein in microdissected renal tubules

1997 ◽  
Vol 272 (4) ◽  
pp. F531-F537 ◽  
Author(s):  
B. K. Kishore ◽  
J. Terris ◽  
P. Fernandez-Llama ◽  
M. A. Knepper
Keyword(s):  
Collecting Duct ◽  
Enzyme Linked Immunosorbent Assay ◽  
Apical Plasma Membrane ◽  
Renal Tubules ◽  
Urea Transport ◽  
Urea Transporter ◽  
Transporter Protein ◽  
Low Protein ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct

The vasopressin-regulated urea transporter (VRUT) is a 97-kDa protein (also called “UT-1”) responsible for facilitated urea transport across the apical plasma membrane of inner medullary collecting duct (IMCD) cells. To determine the abundance of VRUT protein in collecting duct cells of the rat, we designed a sensitive fluorescence-based enzyme-linked immunosorbent assay capable of detecting <5 fmol of VRUT protein. In collecting duct segments, measurable VRUT was found in microdissected IMCD segments but not in other portions of the collecting duct. In the mid-IMCD, the measured level averaged 5.3 fmol/mm tubule length, corresponding to approximately 5 million copies of VRUT per cell. Thus VRUT is extremely abundant in the IMCD, accounting, in part, for the extremely high urea permeability of this segment. Feeding a low-protein diet (8% protein) markedly decreased urea clearance but did not alter the quantity of VRUT protein in the IMCD. Thus increased urea transport across the collecting duct with dietary protein restriction is not a consequence of increased expression of VRUT. Based on urea fluxes measured in the IMCD and our measurements of the number of copies of VRUT, we estimate a turnover number of > or = 0.3-1 x 10(5) s. In view of the large magnitude of this value and previously reported biophysical properties of urea transport in collecting ducts, we hypothesize that the VRUT may function as a channel rather than a carrier.

The vasopressin-regulated urea transporter in renal inner medullary collecting duct

1990 ◽  
Vol 259 (3) ◽  
pp. F393-F401 ◽  
Author(s):  
M. A. Knepper ◽  
R. A. Star
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Toad Urinary Bladder ◽  
Urea Transport ◽  
Urea Transporter ◽  
Transport Pathway ◽  
Inner Medullary Collecting Duct ◽  
Urea Permeability ◽  
Medullary Collecting Duct

The terminal part of the inner medullary collecting duct (terminal IMCD) is unique among collecting duct segments in part because its permeability to urea is regulated by vasopressin. The urea permeability can rise to extremely high levels (greater than 100 x 10(-5) cm/s) in response to vasopressin. Recent studies in isolated perfused IMCD segments have established that the rapid movement of urea across the tubule epithelium occurs via a specialized urea transporter, presumably an intrinsic membrane protein, present in both the apical and basolateral membranes. This urea transporter has properties similar to those of the urea transporters in mammalian erythrocytes and in toad urinary bladder, namely, inhibition by phloretin, inhibition by urea analogues, saturation kinetics in equilibrium-exchange experiments, and regulation by vasopressin. The urea transport pathway is distinct from and independent of the vasopressin-regulated water channel. The increase in transepithelial urea transport in response to vasopressin is mediated by adenosine 3',5'-cyclic monophosphate and is associated with an increase in the urea permeability of the apical membrane. However, little is known about the physical events associated with the activation or insertion of urea transporters in the apical membrane. Because of the importance of this transporter to the urinary concentrating mechanism, efforts toward understanding its molecular structure and the molecular basis of its regulation appear to be justified.

Increased expression of cyclooxygenase-1 and -2 in the diabetic rat renal medulla

2003 ◽  
Vol 285 (6) ◽  
pp. F1068-F1077 ◽  
Author(s):  
Rania Nasrallah ◽  
Anne Landry ◽  
Sonia Singh ◽  
Monika Sklepowicz ◽  
Richard L. Hébert
Keyword(s):  
Collecting Duct ◽  
Renal Medulla ◽  
Immunohistochemical Analysis ◽  
Diabetic Rats ◽  
Diabetic Rat ◽  
Fourfold Increase ◽  
Protein Levels ◽  
Cyclooxygenase 1 ◽  
Streptozotocin Diabetic Rats ◽  
Medullary Collecting Duct

Alterations in renal prostaglandins (PGs) may contribute to some of the renal manifestations in diabetes leading to nephropathy. PG production is dependent on the activity of cyclooxygenases (COX-1 AND -2) and PG synthases. Our present study investigated levels of these enzymes in streptozotocin-diabetic rats at 2, 4, 6, and 8 wk of diabetes. Immunohistochemical analysis revealed an increase in COX signal in the inner and outer medulla of diabetic rats. This was confirmed by Western blotting, showing up to a fourfold increase in both COX isoforms at 4–6 wk of diabetes. Also, Western blot analysis revealed a sixfold increase in PGE2 synthase expression in the outer medullary region of 6-wk diabetic rats but no difference in the inner medulla. In cultured rat inner medullary collecting duct (IMCD), levels of COX were increased two- to threefold in cells exposed for 4 days to 37.5 mM glucose compared with control of 17.5 mM. While no change in PGE2 synthase levels was noted, PGE2 synthesis was increased. Furthermore, levels of EP1 and EP4 mRNA were increased, as well as a twofold increase in EP4 protein levels. Future studies will determine which COX isoform is contributing to the majority of PGE2 produced in the diabetic IMCD and the significance of these findings to disturbances in IMCD function and to the progression of diabetic nephropathy.

Increased collecting duct urea transporter expression in Dahl salt-sensitive rats

2003 ◽  
Vol 285 (1) ◽  
pp. F143-F151 ◽  
Author(s):  
Robert A. Fenton ◽  
Chung-Lin Chou ◽  
Shana Ageloff ◽  
William Brandt ◽  
John B. Stokes ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Hydroxysteroid Dehydrogenase ◽  
Plasma Corticosterone ◽  
Urea Transporter ◽  
Rt Pcr ◽  
Collecting Ducts ◽  
Medullary Collecting Duct ◽  
Transporter Expression ◽  
Solute Transporter

Because abnormalities of inner medullary function have been proposed in Dahl salt-sensitive (DS) rats vs. salt-resistant (DR) rats, we performed transporter profiling by semiquantitative immunoblotting to determine whether specific solute transporter abundances are altered in inner medullas of DS rats vs. DR rats. Although none of the expressed Na transporters were upregulated in the inner medullas of DS rats compared with DR rats, there were marked increases in the protein abundances of the collecting duct urea transporters UT-A1 (to 212% of DR) and UT-A3 (to 223% of DR). These differences were confirmed by immunocytochemistry. Quantitative real-time RT-PCR showed higher mRNA abundance in DS rats for both UT-A1 (to 256% of DR) and UT-A3 (to 210% of DR). In isolated, perfused inner medullary collecting ducts, urea permeability was significantly greater in DS rats. Because both UT-A1 and UT-A3 are transcriptionally regulated by glucocorticoids, we measured both plasma corticosterone levels and inner medullary 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) abundances. Although the plasma corticosterone concentrations were not different between DS and DR rats, immunoblotting and immunocytochemistry revealed a marked elevation of 11β-HSD2 abundance in DS rats. Consistent with the view that an elevated 11β-HSD2 level is responsible for increased urea transporter expression in the inner medullary collecting duct, administration of the 11β-HSD2 inhibitor carbenoxolone to DS rats decreased the abundances of UT-A1 and UT-A3 to levels similar to those seen in DR rats.

Importance of medullary events in ammonium excretion: studies in acute respiratory and acute metabolic acidosis

1983 ◽  
Vol 61 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Andre Gougoux ◽  
Patrick Vinay ◽  
Guy Lemieux ◽  
Marc Goldstein ◽  
Bobby Stinebaugh ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Collecting Duct ◽  
Respiratory Acidosis ◽  
Renal Medulla ◽  
Hydrogen Ion ◽  
Ammonium Excretion ◽  
Urine Ph ◽  
Ion Secretion ◽  
Medullary Collecting Duct ◽  
Acute Metabolic Acidosis

The renal medulla can play an important role in acid excretion by modulating both hydrogen ion secretion in the medullary collecting duct and the medullary [Formula: see text]. The purpose of these experiments was to characterize the intrarenal events associated with ammonium excretion in acute acidosis. Cortical events were monitored in two ways: first, the rates of glutamine extraction and ammoniagenesis were assessed by measuring arteriovenous differences and the rate of renal blood flow; second, the biochemical response of the ammoniagenesis pathway was examined by measuring glutamate and 2-oxoglutarate, key renal cortical metabolites in this pathway. There were no significant differences noted in any of these cortical parameters between acute respiratory and metabolic acidosis. Despite a comparable twofold rise in ammonium excretion in both cases, the urine pH, [Formula: see text], and the urine minus blood [Formula: see text] difference (U-B [Formula: see text]) were lower during acute hypercapnia. In these experiments, the urine [Formula: see text] was 34 mmHg (1 mmHg = 133.322 Pa) lower than that of the blood during acute respiratory acidosis while the U-B [Formula: see text] was 5 ± 3 mmHg in acute metabolic acidosis. Thus there were significant differences in medullary events during these two conditions. Although the urine pH is critical in determining ammonium excretion in certain circumstances, these results suggest that regional variations in the medullary [Formula: see text] can modify this relationship.

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