Biomedical subjects

Janet D Klein

Publications and source records attributed to Janet D Klein.

At least 19 recordsLinked to original sources

PubMed2006

Vasopressin increases plasma membrane accumulation of urea transporter UT-A1 in rat inner medullary collecting ducts.

Urea transport, mediated by the urea transporter A1 (UT-A1) and/or UT-A3, is important for the production of concentrated urine. Vasopressin rapidly increases urea transport in rat terminal inner medullary collecting ducts (IMCD). A previous study showed that one mechanism for rapid regulation of urea transport is a vasopressin-induced increase in UT-A1 phosphorylation. This study tests whether vasopressin or directly activating adenylyl cyclase with forskolin also increases UT-A1 accumulation in the plasma membrane of rat IMCD. Inner medullas were harvested from rats 45 min after injection with vasopressin or vehicle. UT-A1 abundance in the plasma membrane was significantly increased in the membrane fraction after differential centrifugation and in the biotinylated protein population. Vasopressin and forskolin each increased the amount of biotinylated UT-A1 in rat IMCD suspensions that were treated ex vivo. The observed changes in the plasma membrane are specific, as the amount of biotinylated UT-A1 but not the calcium-sensing receptor was increased by forskolin. Next, whether forskolin or the V(2)-selective agonist dDAVP would increase apical membrane expression of UT-A1 in MDCK cells that were stably transfected with UT-A1 (UT-A1-MDCK cells) was tested. Forskolin and dDAVP significantly increased UT-A1 abundance in the apical membrane in UT-A1-MDCK cells. It is concluded that vasopressin and forskolin increase UT-A1 accumulation in the plasma membrane in rat IMCD and in the apical plasma membrane of UT-A1-MDCK cells. These findings suggest that vasopressin regulates urea transport by increasing UT-A1 accumulation in the plasma membrane and/or UT-A1 phosphorylation.

Animals↗

PubMed2006

Loss of N-linked glycosylation reduces urea transporter UT-A1 response to vasopressin.

The vasopressin-regulated urea transporter (UT)-A1 is a transmembrane protein with two glycosylated forms of 97 and 117 kDa; both are derived from a single 88-kDa core protein. However, the precise molecular sites and the function for UT-A1 N-glycosylation are not known. In this study, we compared Madin-Darby canine kidney cells stably expressing wild-type (WT) UT-A1 to Madin-Darby canine kidney cell lines stably expressing mutant UT-A1 lacking one (A1m1, A1m2) or both glycosylation sites (m1m2). Site-directed mutagenesis revealed that UT-A1 has two glycosylation sites at Asn-279 and -742. Urea flux is stimulated by 10 nM vasopressin (AVP) or 10 microM forskolin (FSK) in WT cells. In contrast, m1m2 cells have a delayed and significantly reduced maximal urea flux. A 15-min treatment with AVP and FSK significantly increased UT-A1 cell surface expression in WT but not in m1m2 cells, as measured by biotinylation. We confirmed this finding using immunostaining. Membrane fractionation of the plasma membrane, Golgi, and endoplasmic reticulum revealed that AVP or FSK treatment increases UT-A1 abundance in both Golgi and plasma membrane compartments in WT but not in m1m2 cells. Pulse-chase experiments showed that UT-A1 half-life is reduced in m1m2 cells compared with WT cells. Our results suggest that mutation of the N-linked glycosylation sites reduces urea flux by reducing UT-A1 half-life and decreasing its accumulation in the apical plasma membrane. In vivo, inner medullary collecting duct cells may regulate urea uptake by altering UT-A1 glycosylation in response to AVP stimulation.

Animals↗

PubMed2006

Urea transporter UT-A1 and aquaporin-2 proteins decrease in response to angiotensin II or norepinephrine-induced acute hypertension.

The kidney responds to high levels of ANG II, as may occur during malignant hypertension, by increasing sodium and water excretion. To study whether kidney medullary transporters contribute to this response, rats were made hypertensive using ANG II. Within 3 days of being given ANG II, systolic blood pressure (BP) was increased (200 mmHg), vs control (130 mmHg), and remained high through day 14. Kidney inner medullary (IM) tip and base and outer medulla were analyzed for transporter protein abundance. There were significant decreases in UT-A1 urea transporter, aquaporin-2 (AQP2) water channel, and NKCC2/BSC1 Na(+)-K(+)-2Cl(-) cotransporter. To determine whether the decreases were a response to hypertension, ANG II, or an ANG II-induced increase in aldosterone, rats were given 1) norepinephrine (to increase BP) and 2) ANG II plus spironolactone (to block the mineralocorticoid receptor). Norepinephrine (7 days) increased BP, urine volume, sodium excretion, and decreased urine osmolality and UT-A1, AQP2, and NKCC2/BSC1 abundances, similar to ANG II. ANG II alone or with spironolactone yielded similar increases in BP, urine volume, and urine osmolality, and decreases in UT-A1 and AQP2 proteins in the IM tip. Plasma vasopressin was unaffected by treatment. Water diuresis did not change UT-A1 but decreased AQP2 and NKCC2/BSC1 abundances. We conclude that decreases in UT-A1, AQP2, and NKCC2/BSC1 proteins may contribute to the diuresis and natriuresis that occur following ANG II or norepinephrine-induced acute hypertension and do not appear to involve ANG II stimulation of aldosterone or thirst.

Acute Disease↗

PubMed2006

Regulation of UT-A1-mediated transepithelial urea flux in MDCK cells.

Transepithelial [(14)C]urea fluxes were measured across cultured Madin-Darby canine kidney (MDCK) cells permanently transfected to express the urea transport protein UT-A1. The urea fluxes were typically increased from a basal rate of 2 to 10 and 25 nmol.cm(-2).min(-1) in the presence of vasopressin and forskolin, respectively. Flux activation consisted of a rapid-onset component of small amplitude that leveled off within approximately 10 min and at times even decreased again, followed by a delayed, strong increase over the next 30-40 min. Forskolin activated urea transport through activation of adenylyl cyclase; dideoxyforskolin was inactive. Vasopressin activated urea transport only from the basolateral side and was blocked by OPC-31260, indicating that its action was mediated by basolateral V(2) receptors. In the presence of the phosphodiesterase inhibitor IBMX, vasopressin activated as strongly as forskolin. By itself, IBMX caused a slow increase over 50 min to approximately 5 nmol.cm(-2).min(-1). 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 300 microM) activated urea flux only when added basolaterally. IBMX augmented the activation by basolateral 8-BrcAMP. Urea flux activation by vasopressin and forskolin were only partially blocked by the protein kinase A inhibitor H-89. Even at concentrations >10 microM, urea flux after 60 min of stimulation was reduced by <50%. The rapid-onset component appeared unaffected by the presence of H-89. These data suggest that activation of transepithelial urea transport across MDCK-UT-A1 cells by forskolin and vasopressin involves cAMP as a second messenger and that it is mediated by one or more signaling pathways separate from and in addition to protein kinase A.

1-Methyl-3-isobutylxanthine↗

PubMed2006

Effects of water restriction on gene expression in mouse renal medulla: identification of 3betaHSD4 as a collecting duct protein.

To identify novel gene targets of vasopressin regulation in the renal medulla, we performed a cDNA microarray study on the inner medullary tissue of mice following a 48-h water restriction protocol. In this study, 4,625 genes of the possible approximately 12,000 genes on the array were included in the analysis, and of these 157 transcripts were increased and 63 transcripts were decreased by 1.5-fold or more. Quantitative, real-time PCR measurements confirmed the increases seen for 12 selected transcripts, and the decreases were confirmed for 7 transcripts. In addition, we measured transcript abundance for many renal collecting duct proteins that were not represented on the array; aquaporin-2 (AQP2), AQP3, Pax-8, and alpha- and beta-Na-K-ATPase subunits were all significantly increased in abundance; the beta- and gamma-subunits of ENaC and the vasopressin type 1A receptor were significantly decreased. To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting. For most of the genes examined, changes in mRNA abundances were not associated with concomitant protein abundance changes; however, AQP2 transcript abundance and protein abundance did correlate. Surprisingly, aldolase B transcript abundance was increased but protein abundance was decreased following 48 h of water restriction. Several transcripts identified by microarray were novel with respect to their expression in mouse renal medullary tissues. The steroid hormone enzyme 3beta-hydroxysteroid dehydrogenase 4 (3betaHSD4) was identified as a novel target of vasopressin regulation, and via dual labeling immunofluorescence we colocalized the expression of this protein to AQP2-expressing collecting ducts of the kidney. These studies have identified several transcripts whose abundances are regulated in mouse inner medulla in response to an increase in endogenous vasopressin levels and could play roles in the regulation of salt and water excretion.

3-Hydroxysteroid Dehydrogenases↗

PubMed2006

Genetic restoration of aldose reductase to the collecting tubules restores maturation of the urine concentrating mechanism.

To investigate the underlying causes for aldose reductase deficiency-induced diabetes insipidus, we carried out studies with three genotypic groups of mice. These included wild-type mice, knockout mice, and a newly created bitransgenic line that was homozygous for both the aldose reductase null mutation and an aldose reductase knockin transgene driven by the kidney-specific cadherin promoter to direct transgene expression in the collecting tubule epithelial cells. We found that from early renal developmental stages onward, urine osmolality did not exceed 1,000 mosmol/kgH2O in aldose reductase-deficient mice. The functional defects were correlated with significant renal cellular and structural abnormalities that included cell shrinkage, apoptosis, disorganized tubular and vascular structures, and segmental atrophy. In contrast, the transgenic aldose reductase expression in the bitransgenic mice largely but incompletely rescued urine concentrating capacity and significantly improved renal cell survival, cellular morphology, and renal structures. Together, these results suggest that aldose reductase not only plays important roles in osmoregulation and medullary cell survival but may also be essential for the full maturation of the urine concentrating mechanism.

Aldehyde Reductase↗

PubMed2006

Changes in subcellular distribution of the ammonia transporter, Rhcg, in response to chronic metabolic acidosis.

The primary mechanism by which the kidneys mediate net acid excretion is through ammonia metabolism. In the current study, we examined whether chronic metabolic acidosis, which increases ammonia metabolism, alters the cell-specific and/or the subcellular expression of the ammonia transporter family member, Rhcg, in the outer medullary collecting duct in the inner stripe (OMCDi). Chronic metabolic acidosis was induced in normal SD rats by HCl ingestion for 7 days; controls were pair-fed. The subcellular distribution of Rhcg was determined using immunogold electron microscopy and morphometric analyses. In intercalated cells, acidosis increased total Rhcg, apical plasma membrane Rhcg, and the proportion of total cellular Rhcg in the apical plasma membrane. Intracellular Rhcg decreased significantly, and basolateral Rhcg was unchanged. Because apical plasma membrane length increased in parallel with apical Rhcg immunolabel, apical plasma membrane Rhcg density was unchanged. In principal cells, acidosis increased total Rhcg, apical plasma membrane Rhcg, and the proportion of total cellular Rhcg in the apical plasma membrane while decreasing the intracellular proportion. In contrast to the intercalated cell, chronic metabolic acidosis did not significantly alter apical boundary length; accordingly, apical plasma membrane Rhcg density increased. In addition, basolateral Rhcg immunolabel increased in response to chronic metabolic acidosis. These results indicate that in the rat OMCDi 1) chronic metabolic acidosis increases apical plasma membrane Rhcg in both the intercalated cell and principal cell where it may contribute to enhanced apical ammonia secretion; 2) increased apical plasma membrane Rhcg results from both increased total protein and changes in the subcellular distribution of Rhcg; 3) the mechanism of Rhcg subcellular redistribution differs in intercalated and principal cells; and 4) Rhcg may contribute to regulated basolateral ammonia transport in the principal cell.

Acidosis↗

PubMed2005

Tissue distribution of UT-A and UT-B mRNA and protein in rat.

Mammalian urea transporters are facilitated membrane transport proteins belonging to two families, UT-A and UT-B. They are best known for their role of maintaining the renal inner medullary urinary concentrating gradient. Urea transporters have also been identified in tissues not typically associated with urea metabolism. The purpose of this study was to survey the major organs in rat to determine the distribution of UT-A and UT-B mRNA transcripts and protein forms and determine their cellular localization. Five kidney subregions and 17 extrarenal tissues were screened by Northern blot analysis using two UT-A and three UT-B probes and by Western blot analysis using polyclonal COOH-terminal UT-A and UT-B antibodies. Immunohistochemistry was performed on 16 extrarenal tissues using the same antibodies. In kidney, we detected mRNA transcripts and protein bands consistent with previously-identified UT-A and UT-B isoforms, as well as novel forms. We found that UT-A mRNA and protein are widely expressed in extrarenal tissues in various forms that are different from the known isoforms. We determined the cellular localization of UT-A and UT-B in these tissues. We found that both UT-A and UT-B are ubiquitously expressed as numerous tissue-specific mRNA transcripts and protein forms that are localized to cell membranes, cytoplasm, or nuclei.

Animals↗

PubMed2005

Renal expression of the ammonia transporters, Rhbg and Rhcg, in response to chronic metabolic acidosis.

Chronic metabolic acidosis induces dramatic increases in net acid excretion that are predominantly due to increases in urinary ammonia excretion. The current study examines whether this increase is associated with changes in the expression of the renal ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg). Chronic metabolic acidosis was induced in Sprague-Dawley rats by HCl ingestion for 1 wk; control animals were pair-fed. After 1 wk, metabolic acidosis had developed, and urinary ammonia excretion increased significantly. Rhcg protein expression was increased in both the outer medulla and the base of the inner medulla. Intercalated cells in the outer medullary collecting duct (OMCD) and in the inner medullary collecting duct (IMCD) in acid-loaded animals protruded into the tubule lumen and had a sharp, discrete band of apical Rhcg immunoreactivity, compared with a flatter cell profile and a broad band of apical immunolabel in control kidneys. In addition, basolateral Rhcg immunoreactivity was observed in both control and acidotic kidneys. Cortical Rhcg protein expression and immunoreactivity were not detectably altered. Rhcg mRNA expression was not significantly altered in the cortex, outer medulla, or inner medulla by chronic metabolic acidosis. Rhbg protein and mRNA expression were unchanged in the cortex, outer and inner medulla, and no changes in Rhbg immunolabel were evident in these regions. We conclude that chronic metabolic acidosis increases Rhcg protein expression in intercalated cells in the OMCD and in the IMCD, where it is likely to mediate an important role in the increased urinary ammonia excretion.

Acidosis, Renal Tubular↗

PubMed2005

Vasopressin increases urea permeability in the initial IMCD from diabetic rats.

In normal rats, vasopressin and hyperosmolality enhance urea permeability (P(urea)) in the terminal, but not in the initial inner medullary collecting duct (IMCD), a process thought to occur through the UT-A1 urea transporter. In the terminal IMCD, UT-A1 is detected as 97- and 117-kDa glycoproteins. However, in the initial IMCD, only the 97-kDa form is detected. During streptozotocin-induced diabetes mellitus, UT-A1 protein abundance is increased, and the 117-kDa UT-A1 glycoprotein appears in the initial IMCD. We hypothesize that the 117-kDa glycoprotein mediates the vasopressin- and osmolality-induced changes in P(urea). Thus, in the present study, we measured P(urea) in in vitro perfused initial IMCDs from diabetic rats by imposing a 5 mM bath-to-lumen urea gradient without any osmotic gradient. Basal P(urea) was similar in control vs. diabetic rats (3 +/- 1 vs. 5 +/- 1 x 10(-5) cm/s, n = 4, P = not significant). Vasopressin (10 nM) significantly increased P(urea) to 16 +/- 5 x 10(-5) cm/s (n = 4, P < 0.05) in diabetic but not in control rats. Forskolin (10 microM, adenylyl cyclase activator) also significantly increased P(urea) in diabetic rats. In contrast, increasing osmolality to 690 mosmol/kg H2O did not change P(urea) in diabetic rats. We conclude that initial IMCDs from diabetic rats have vasopressin- and forskolin-, but not hyperosmolality-stimulated P(urea). The appearance of vasopressin-stimulated P(urea) in initial IMCDs correlates with an increase in UT-A1 protein abundance and the appearance of the 117-kDa UT-A1 glycoprotein in this region during diabetes. This suggests that the 117-kDa UT-A1 glycoprotein is necessary for vasopressin-stimulated urea transport.

Animals↗

PubMed2005

Regulated expression of renal and intestinal UT-B urea transporter in response to varying urea load.

Production, recycling, and elimination of urea are important to maintain nitrogen balance. Adaptation to varying loads of urea due to different protein intake or in renal failure may involve changes in urea transport and may possibly affect urea transporters. In this study, we examined the expression of the UT-B urea transporter in rats fed a low-protein diet (LPD), a high-protein diet (HPD), and a 20% urea-supplemented diet. In the kidney, UT-B protein abundance increased in the outer medulla of both LPD-fed rats and 20% urea-fed rats, without changes in the inner medulla of either group compared with controls. In HPD-fed rats, UT-B protein decreased significantly in both the outer and inner medulla. We identified expression of UT-B in the rat colon, as a 2-kb mRNA transcript and as an approximately 45-kDa protein, with apical localization in superficial colon epithelial cells. UT-B also is expressed in rat small intestine. In rat colon, UT-B protein abundance was mildly, but significantly, decreased in LPD-fed and 20% urea-fed rats. UT-B abundance also was examined in the colon of 7/8 nephrectomized, uremic rats and in HPD-fed rats and was not significantly different from that in control rats. These findings indicate that UT-B expression is regulated in response to different loads of urea, with a pattern that suggests involvement of tissue-specific regulatory mechanism in kidney and colon.

Animals↗

PubMed2004

Urea may regulate urea transporter protein abundance during osmotic diuresis.

Rats with diabetes mellitus have an increase in UT-A1 urea transporter protein abundance and absolute urea excretion, but the relative amount (percentage) of urea in total urinary solute is actually decreased due to the marked glucosuria. Urea-specific signaling pathways have been identified in mIMCD3 cells and renal medulla, suggesting the possibility that changes in the percentage or concentration of urea could be a factor that regulates UT-A1 abundance. In this study, we tested the hypothesis that an increase in a urinary solute other than urea would increase UT-A1 abundance, similar to diabetes mellitus, whereas an increase in urine urea would not. In both inner medullary base and tip, UT-A1 protein abundance increased during NaCl- or glucose-induced osmotic diuresis but not during urea-induced osmotic diuresis. Next, rats undergoing NaCl or glucose diuresis were given supplemental urea to increase the percentage of urine urea to control values. UT-A1 abundance did not increase in these urea-supplemented rats compared with control rats. Additionally, both UT-A2 and UT-B protein abundances in the outer medulla increased during urea-induced osmotic diuresis but not in NaCl or glucose diuresis. We conclude that during osmotic diuresis, UT-A1 abundance increases when the percentage of urea in total urinary solute is low and UT-A2 and UT-B abundances increase when the urea concentration in the medullary interstitium is high. These findings suggest that a reduction in urine or interstitial urea results in an increase in UT-A1 protein abundance in an attempt to restore inner medullary interstitial urea and preserve urine-concentrating ability.

Animals↗

PubMed2004

Identification and characterization of a Kidd antigen/UT-B urea transporter expressed in human colon.

We have identified a urea transporter from the mucosa of the human colon that has characteristics consistent with a Kidd antigen/UT-B urea transporter. This intestinal urea transporter encodes a 389-amino acid peptide with a sequence identical to that previously reported for the UT-B urea transporter in erythrocytes. Expression of a UT-B 2-kb mRNA transcript and of approximately 50- and approximately 98-kDa UT-B proteins is detected in human colonic mucosa by Northern and Western blot analysis. The UT-B protein is localized in the cell membrane and cytoplasm of the superficial intestinal epithelium and in the epithelial cells in the crypts. A 2-kb UT-B mRNA transcript and the UT-B protein were also identified in the intestinal cell line Caco-2. The transepithelial flux of (14)C urea was examined in Caco-2 cells growing on porous membrane support and was significantly inhibited by phloretin, 1,3-dimethylurea, and thiourea, suggesting that the transfer of urea across the Caco-2 monolayer could be mediated, at least in part, by the UT-B urea transporter. We conclude that the Kidd antigen/UT-B urea transporter is physiologically expressed in the human colon epithelium, where it could participate in the transport of urea across the colon mucosa.

Amino Acid Sequence↗

PubMed2004

Altered expression of urea transporters in response to ureteral obstruction.

Urea plays an important role in the urinary concentrating capacity. Renal inner medullary (IM) urea transporter expression was examined in rats with bilateral (BUO) or unilateral ureteral obstruction (UUO). BUO (24 h) was associated with markedly increased plasma urea (42.4 +/- 1.0 vs. 5.2 +/- 0.2 mmol/l) and a significant decrease in expression of UT-A1 (28 +/- 8% of sham levels), UT-A3 (45 +/- 11%), and UT-B (70 +/- 8%). Immunocytochemistry confirmed downregulation of UT-A1 and UT-A3 in IM collecting duct and UT-B in the descending vasa recta. Three days after release of BUO, UT-A1, UT-A3, and UT-B remained significantly downregulated (UT-A1: 37 +/- 6%; UT-A3: 25 +/- 6%; and UT-B: 10 +/- 5% of sham levels; P < 0.05) concurrent with a persistent polyuria and a marked reduction in solute-free water reabsorption (115 +/- 11 vs. 196 +/- 8 microl.min(-1).kg(-1), P < 0.05). Moreover, 14 days after release of BUO, total UT-A1, UT-A3, and UT-B remained significantly decreased compared with sham-operated controls and urine urea remained reduced (588 +/- 43 vs. 1,150 +/- 94 mmol/l). Consistent with increased levels of plasma urea 24 h after onset of UUO (7.4 +/- 0.3 vs. 4.8 +/- 0.3 mmol/l), the protein abundance of UT-A1, UT-A3, and UT-B in IM was markedly reduced in the obstructed kidney, which was confirmed by immunocytochemistry. In the nonobstructed kidney, the expression of urea transporters did not change. In conclusion, reduced expression of UT-A1, UT-A3, and UT-B levels in both BUO and UUO rats suggests that urea transporters play important roles in the impaired urinary concentrating capacity in response to urinary tract obstruction.

Animals↗

PubMed2004

Urea transport in MDCK cells that are stably transfected with UT-A1.

Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was >1,500 Omega.cm(2). Transepithelial [(14)C]urea fluxes were measured at 37 degrees C in a HCO(3)(-)/CO(2) buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-A1-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-A1-transfected cells was UT-A1 protein expressed (as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-A1. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [(14)C]urea fluxes in the UT-A1-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial cell line stably expresses UT-A1 and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts.

Animals↗

PubMed2004

Calcineurin phosphatase activity: activation by glucocorticoids and role of intracellular calcium.

BACKGROUND: Glucocorticoids stimulate release of intracellular calcium in peripheral lymphocytes, but their effects on calcineurin phosphatase activity are unknown. METHODS: Calcineurin phosphatase activity was measured in permeabilized Jurkat T cells using a specific orthophosphate substrate. Changes in intracellular calcium were measured by FURA-2 fluorescence. Inositol triphosphate levels were measured by radioimmunoassay. Transfection with luciferase reporter plasmids linked to glucocorticoid response elements were used to evaluate glucocorticoid receptor function in Jurkat T cells. RESULTS: Dexamethasone significantly (P<0.004) increased calcineurin activity within 15 sec, peaking at 10 min (P<0.001) and returning to basal levels by 180 min. Inhibition of DNA transcription with actinomycin D failed to block calcineurin activation, but co-incubation with RU-486 completely blocked enzyme stimulation. To determine whether Jurkat T cells express active glucocorticoid receptors, cells were transfected with a luciferase reporter plasmid linked to a glucocorticoid response element (GRE). Jurkat T cells incubated with dexamethasone (10 microM) for 24 hr failed to stimulate luciferase activity, whereas cells co-transfected with a transcriptionally active glucocorticoid receptor resulted in a doubling of luciferase activity. Dexamethasone rapidly increases intracellular inositol triphosphate (IP3) and intracellular calcium within 15 sec. Cells incubated with U-73122 (a nonspecific phospholipase C [PLC] antagonist) completely blocked dexamethasone-induced activation of calcineurin, whereas U-73343 failed to block enzyme activation. Dexamethasone-induced activation of calcineurin activity stimulates dephosphorylation of the proapoptotic protein BAD and augments apoptosis through a calcineurin-dependent pathway. CONCLUSION: Dexamethasone rapidly increases calcineurin activity through a transcription-independent mechanism involving activation of phospholipase C and the release of IP3-dependent calcium stores.

Annexin A5↗

PubMed2004

Aldosterone decreases UT-A1 urea transporter expression via the mineralocorticoid receptor.

Adrenalectomy in rats is associated with urinary concentrating and diluting defects. This study tested the effect of adrenal steroids on the UT-A1 urea transporter because it is involved in the urine-concentrating mechanism. Rats were adrenalectomized and given normal saline for 14 d, after which they received (1) vehicle, (2) aldosterone, or (3) spironolactone plus aldosterone. Adrenalectomy alone significantly increased UT-A1 protein in the inner medullary tip after 7 d, whereas aldosterone repletion reversed the effect. Spironolactone blocked the aldosterone-induced decrease in UT-A1, indicating that aldosterone was working via the mineralocorticoid receptor. For verifying that glucocorticoids downregulate UT-A1 protein through a different receptor, three groups of adrenalectomized rats were prepared: (1) vehicle, (2) adrenalectomy plus dexamethasone, and (3) adrenalectomy plus dexamethasone and spironolactone. Dexamethasone significantly reversed UT-A1 protein abundance increase in the inner medullary tip of adrenalectomized rats. When spironolactone was given with dexamethasone, it did not affect the dexamethasone-induced decrease in UT-A1. There was no significant change in serum vasopressin level, aquaporin 2, or Na(+)-K(+)-2Cl(-) co-transporter NKCC2/BSC1 protein abundances or UT-A1 mRNA abundance in any of the groups. In conclusion, either mineralocorticoids or glucocorticoids can downregulate UT-A1 protein. The decrease in UT-A1 does not require both steroid hormones, and each works through a different receptor.

Adrenalectomy↗

PubMed2004

Upregulation of urea transporter UT-A2 and water channels AQP2 and AQP3 in mice lacking urea transporter UT-B.

The UT-B urea transporter is the major urea transporter in red blood cells and kidney descending vasa recta. Humans and mice that lack UT-B have a mild urine-concentrating defect. Whether deletion of UT-B altered the expression of other transporter proteins involved in urinary concentration was tested. Fluorescence-based real-time reverse transcription-PCR and Northern blot analysis showed upregulation of the UT-A2 urea transporter and the aquaporin 2 (AQP2) and AQP3 water channel transcripts but no change in other urea transporters or AQP. Western blot analysis showed that UT-A2 protein abundance in the outer medulla of UT-B null mice increased to 122 +/- 6% of wild-type control. AQP2 protein abundance increased to 177 +/- 32% and 127 +/- 7% in the outer and inner medulla, respectively, of UT-B null versus wild-type mice. The abundance of UT-A1, AQP1, renal outer medullary potassium channel, and NKCC2/BSC1 proteins were not significantly different between UT-B null and wild-type mice. The increases in AQP2 and AQP3 would reduce water loss and improve concentrating ability. The lack of UT-B does not result in a change in expression of urea transporters involved in urea reabsorption from the inner medullary collecting duct (UT-A1 and UT-A3). However, UT-B null mice have a selective increase in UT-A2 protein abundance. This may be an adaptive response to the loss of UT-B, because UT-B and UT-A2 are involved in different intrarenal urea recycling pathways.

Animals↗