Biomedical subjects

M A Knepper

Publications and source records attributed to M A Knepper.

At least 37 records · Page 2Linked to original sources

PubMed2001

Downregulation of AQP1, -2, and -3 after ureteral obstruction is associated with a long-term urine-concentrating defect.

Previously, we demonstrated that 24 h of bilateral ureteral obstruction (BUO) and short-term release of BUO was associated with a decrease in the expression of aquaporin-2 (AQP2), polyuria, and a reduced urinary concentrating capacity (10). The purposes of the present study were to examine whether BUO and the long-term release of BUO (BUO-R) for 3, 14, and 30 days were associated with changes in the expression of renal AQP1, AQP2, and AQP3 and whether such changes were associated with parallel changes in urinary output and urinary concentrating capacity. Rats (n = 4-7 in each group) were kept in metabolic cages for measurements of urinary output. Kidneys were removed to determine the expression levels of AQP1, AQP2, and AQP3 by semiquantitative immunoblotting. AQP2 was downregulated after 24 h of BUO (42 +/- 3%). Downregulation of AQP2 persisted 3 (43 +/- 14%; P < 0.01) and 15 days after BUO-R (48 +/- 11%; P < 0.01) but was normalized 30 days after BUO-R. AQP3 showed a similar pattern. Moreover, AQP1 was downregulated in response to BUO (65 +/- 7%) and remained downregulated 3 days after BUO-R (41 +/- 5%), 14 days after BUO-R (57 +/- 8%), and 30 days after BUO-R (59 +/- 5%). BUO-R resulted in a significant polyuria that gradually decreased, although it remained significant at day 30. Urinary concentrating capacity remained significantly impaired when determined 3, 14, and 30 days after BUO-R in response to a 24-h period of thirst (1,712 +/- 270 vs. 2,880 +/- 91 mosmol/kgH2O at day 30, P < 0.05). In conclusion, the expression of AQP1, AQP2, and AQP3 were long-term downregulated after BUO-R, suggesting that dysregulation of aquaporins located at the proximal tubule, thin descending limb of the loop of Henle, and the collecting duct may contribute to the long-term polyuria and impairment of urinary concentrating capacity associated with obstructive nephropathy.

Animals↗

PubMed2001

Increased renal Na-K-ATPase, NCC, and beta-ENaC abundance in obese Zucker rats.

Renal sodium retention, as a result of increased abundance of sodium transporters, may play a role in the development and/or maintenance of the increased blood pressure in obesity. To address this hypothesis, we evaluated the relative abundances of renal sodium transporters in lean and obese Zucker rats at 2 and 4 mo of age by semiquantitative immunoblotting. Mean systolic blood pressure was higher in obese rats relative to lean at 3 mo, P < 0.02. Furthermore, circulating insulin levels were 6- or 13-fold higher in obese rats compared with lean at 2 or 4 mo of age, respectively. The abundances of the alpha(1)-subunit of Na-K-ATPase, the thiazide-sensitive Na-Cl cotransporter (NCC or TSC), and the beta-subunit of the epithelial sodium channel (ENaC) were all significantly increased in the obese rats' kidneys. There were no differences for the sodium hydrogen exchanger (NHE3), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2 or BSC1), the type II sodium-phosphate cotransporter (NaPi-2), or the alpha-subunit of ENaC. These selective increases could possibly increase sodium retention by the kidney and therefore could play a role in obesity-related hypertension.

Animals↗

PubMed2001

The renal thiazide-sensitive Na-Cl cotransporter as mediator of the aldosterone-escape phenomenon.

The kidneys "escape" from the Na-retaining effects of aldosterone when circulating levels of aldosterone are inappropriately elevated in the setting of normal or expanded extracellular fluid volume, e.g., in primary aldosteronism. Using a targeted proteomics approach, we screened renal protein extracts with rabbit polyclonal antibodies directed to each of the major Na transporters expressed along the nephron to determine whether escape from aldosterone-mediated Na retention is associated with decreased abundance of one or more of renal Na transporters. The analysis revealed that the renal abundance of the thiazide-sensitive Na-Cl cotransporter (NCC) was profoundly and selectively decreased. None of the other apical solute-coupled Na transporters displayed decreases in abundance, nor were the total abundances of the three ENaC subunits significantly altered. Immunocytochemistry showed a strong decrease in NCC labeling in distal convoluted tubules of aldosterone-escape rats with no change in the cellular distribution of NCC. Ribonuclease protection assays (RPAs) revealed that the decrease in NCC protein abundance was not associated with altered NCC mRNA abundance. Thus, the thiazide-sensitive Na-Cl cotransporter of the distal convoluted tubule appears to be the chief molecular target for regulatory processes responsible for mineralocorticoid escape, decreasing in abundance via a posttranscriptional mechanism.

Aldosterone↗

PubMed2000

Regulation of aquaporin-2 trafficking by vasopressin in the renal collecting duct. Roles of ryanodine-sensitive Ca2+ stores and calmodulin.

In the renal collecting duct, vasopressin increases osmotic water permeability (P(f)) by triggering trafficking of aquaporin-2 vesicles to the apical plasma membrane. We investigated the role of vasopressin-induced intracellular Ca(2+) mobilization in this process. In isolated inner medullary collecting ducts (IMCDs), vasopressin (0.1 nm) and 8-(4-chlorophenylthio)-cAMP (0.1 mm) elicited marked increases in [Ca(2+)](i) (fluo-4). Vasopressin-induced Ca(2+) mobilization was completely blocked by preloading with the Ca(2+) chelator BAPTA. In parallel experiments, BAPTA completely blocked the vasopressin-induced increase in P(f) without affecting adenosine 3',5'-cyclic monophosphate (cAMP) production. Previously, we demonstrated the lack of activation of the phosphoinositide-signaling pathway by vasopressin in IMCD, suggesting an inositol 1,4,5-trisphosphate-independent mechanism of Ca(2+) release. Evidence for expression of the type 1 ryanodine receptor (RyR1) in IMCD was obtained by immunofluorescence, immunoblotting, and reverse transcription-polymerase chain reaction. Ryanodine (100 microm), a ryanodine receptor antagonist, blocked the arginine vasopressin-mediated increase in P(f) and blocked vasopressin-stimulated redistribution of aquaporin-2 to the plasma membrane domain in primary cultures of IMCD cells, as assessed by immunofluorescence immunocytochemistry. Calmodulin inhibitors (W7 and trifluoperazine) blocked the P(f) response to vasopressin and the vasopressin-stimulated redistribution of aquaporin-2. The results suggest that Ca(2+) release from ryanodine-sensitive stores plays an essential role in vasopressin-mediated aquaporin-2 trafficking via a calmodulin-dependent mechanism.

Animals↗

PubMed2000

Dysregulation of renal aquaporins and Na-Cl cotransporter in CCl4-induced cirrhosis.

BACKGROUND: Severe hepatic cirrhosis is associated with abnormal renal water retention. METHODS: Semiquantitative immunoblotting was employed to investigate the abundance of the major renal aquaporins (water channels) and sodium-dependent cotransporters in kidneys from control rats and rats with cirrhosis secondary to chronic CCl4 inhalation. RESULTS: The cirrhotic rats had ascites and manifested a water excretion defect detected by a standard water-loading test. The abundance of aquaporin-1 (the major aquaporin in the proximal tubule) was increased, an effect markedly accentuated in high-density membrane fractions prepared by differential centrifugation. Differential centrifugation studies demonstrated a redistribution of aquaporin-2 from high-density to low-density membranes, compatible with increased trafficking of aquaporin-2 to the plasma membrane. The abundance of aquaporin-3, but not aquaporin-2, was increased in collecting ducts of rats with CCl4-induced cirrhosis. The Na-K-2Cl cotransporter of the thick ascending limb showed no change in abundance. However, the abundance of the thiazide-sensitive Na-Cl cotransporter of the distal convoluted tubule was markedly suppressed in cirrhotic rats, possibly contributing to a defect in urinary dilution. CONCLUSIONS: In this model of cirrhosis, the development of a defect in urinary dilution may be multifactorial, with contributions from at least four abnormalities in transporter regulation: (1) an increase in the renal abundance of aquaporin-1, (2) a cellular redistribution of aquaporin-2 in the collecting duct compatible with trafficking to the plasma membrane without an increase in total cellular aquaporin-2, (3) an increase in the renal abundance of aquaporin-3, and (4) a decrease in the abundance of the thiazide-sensitive cotransporter of the distal convoluted tubule.

Animals↗

PubMed2000

Aldose reductase-deficient mice develop nephrogenic diabetes insipidus.

Aldose reductase (ALR2) is thought to be involved in the pathogenesis of various diseases associated with diabetes mellitus, such as cataract, retinopathy, neuropathy, and nephropathy. However, its physiological functions are not well understood. We developed mice deficient in this enzyme and found that they had no apparent developmental or reproductive abnormality except that they drank and urinated significantly more than their wild-type littermates. These ALR2-deficient mice exhibited a partially defective urine-concentrating ability, having a phenotype resembling that of nephrogenic diabetes insipidus.

Aldehyde Reductase↗

PubMed2000

Cellular localization of P2Y(2) purinoceptor in rat renal inner medulla and lung.

Physiological and pharmacological studies have demonstrated that extracellular ATP, acting through P2Y(2) purinoceptor, modulates water permeability of renal medullary collecting duct cells and the secretion of ions, mucin, and surfactant phospholipids by respiratory epithelia. Here we provide direct molecular evidence for the expression of P2Y(2) purinoceptor in these cells. RT-PCR confirmed P2Y(2) purinoceptor mRNA expression in rat lung and kidney and demonstrated expression in renal collecting ducts. Northern analysis showed that both lung and kidney express one 3.6-kb P2Y(2) purinoceptor mRNA transcript. Immunoblots using peptide-derived polyclonal antibody to P2Y(2) purinoceptor showed that inner medullary collecting ducts (IMCD) express two distinct and specific products (47 and 105 kDa) and account for the majority of the receptor expression in inner medulla, whereas the 105-kDa form is predominant in lung. Immunoperoxidase labeling on cryosections showed localization of receptor protein in the apical and basolateral domains of IMCD principal cells and in the secretory cells (Clara cells and goblet cells) of the terminal respiratory bronchioles.

Animals↗

PubMed2000

UT-A2: a 55-kDa urea transporter in thin descending limb whose abundance is regulated by vasopressin.

The renal urea transporter gene (UT-A) produces different transcripts in the inner medullary collecting ducts (UT-A1) and thin descending limbs of Henle's loop (UT-A2), coding for distinct proteins. Peptide-directed rabbit polyclonal antibodies were used to identify the UT-A2 protein in renal medulla of mouse and rat. In the inner stripe of outer medulla, an antibody directed to the COOH terminus of UT-A recognized a membrane protein of 55 kDa. The abundance of this 55-kDa protein was strongly increased in response to chronic infusion of the vasopressin analog 1-deamino-[8-D-arginine]vasopressin (DDAVP) in Brattleboro rats, consistent with previous evidence that UT-A2 mRNA abundance is markedly increased. Immunofluorescence labeling with the COOH-terminal antibody in Brattleboro rats revealed labeling in the lower portion of descending limbs from short-looped nephrons (in the aquaporin-1-negative portion of this segment). This UT-A labeling was increased in response to DDAVP. Increased labeling was also seen in descending limbs of long-looped nephrons in the base of the inner medulla. These results indicate that UT-A2 is expressed as a 55-kDa protein in portions of the thin descending limbs of Henle's loop and that the abundance of this protein is strongly upregulated by vasopressin.

Animals↗

PubMed2000

Localization of epithelial sodium channel and aquaporin-2 in rabbit kidney cortex.

The amiloride-sensitive epithelial sodium channel (ENaC) and the vasopressin-dependent water channel aquaporin-2 (AQP2) mediate mineralocorticoid-regulated sodium- and vasopressin-regulated water reabsorption, respectively. Distributions of ENaC and AQP2 have been shown by immunohistochemistry in rats. Functional data from rabbits suggest a different distribution pattern of these channels than in rats. We studied, by immunohistochemistry in the rabbit kidney cortex, the distributions of ENaC and AQP2, in conjunction with marker proteins for distal segments. In rabbit cortex ENaC is restricted to the connecting tubule (CNT) cells and cortical collecting duct (CCD) cells. The intracellular distribution of ENaC shifts from the apical membrane in the most upstream CNT cells to a cytoplasmic location further downstream in the CNT and in the CCD cells. AQP2 is detected in the CCD cells exclusively. The anatomic subdivisions in the rabbit distal nephron coincide exactly with distributions of apical transport systems. The differences between rabbits and rats in the distribution patterns of ENaC and AQP2 may explain functional differences in renal salt and water handling between these species.

Animals↗

PubMed2000

Decreased abundance of major Na(+) transporters in kidneys of rats with ischemia-induced acute renal failure.

Ischemia-induced acute renal failure (ARF) is known to be associated with significant impairment of tubular Na reabsorption. We examined whether temporary bilateral renal ischemia (30, 40, or 60 min) and reperfusion (1-5 days) affect the abundance of several renal Na transporters and urinary Na excretion (U(Na)V) in rats. In rats with mild ARF (30 min), immunoblotting revealed that proximal tubule type 3 Na(+)/H(+) exchanger (NHE-3) and type II Na-P(i) cotransporter (NaPi-II) were significantly decreased to 28 +/- 6 and 14 +/- 6% of sham levels, respectively, at day 1. Moreover, Na(+)-K(+)-ATPase levels were also significantly decreased (51 +/- 11%), whereas there was no significant decrease in type 1 bumetanide-sensitive cotransporter (BSC-1) and thiazide-sensitive cotransporter (TSC) levels. Consistent with reduced Na transporter abundance, fractional urinary Na excretion (FE(Na)) was significantly increased in mild ARF (30 min) and U(Na)V was unchanged, despite a marked reduction in glomerular filtration rate. Na transporter levels and renal Na handling were normalized within 5 days. Severe ischemic injury (60 min) resulted in a marked decrease in the abundance of Na(+)-K(+)-ATPase, NHE-3, NaPi-II, BSC-1, and TSC at both days 1 and 5. Consistent with this, FE(Na) was significantly increased at days 1 and 5. Intravenous K-melanocyte-stimulated hormone treatment partially prevented the ischemia-induced downregulation of renal Na transporters and reduced the high FE(Na) to control levels. We conclude that reduced levels of Na transporters along the nephron may play a critical role in the impairment of tubular Na reabsorption, and hence increased Na excretion, in ischemia-induced ARF.

Acute Kidney Injury↗

PubMed2000

Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney.

Sodium transport is increased by vasopressin in the cortical collecting ducts of rats and rabbits. Here we investigate, by quantitative immunoblotting, the effects of vasopressin on abundances of the epithelial sodium channel (ENaC) subunits (alpha, beta, and gamma) in rat kidney. Seven-day infusion of 1-deamino-[8-D-arginine]-vasopressin (dDAVP) to Brattleboro rats markedly increased whole kidney abundances of beta- and gamma-ENaC (to 238% and 288% of vehicle, respectively), whereas alpha-ENaC was more modestly, yet significantly, increased (to 142% of vehicle). Similarly, 7-day water restriction in Sprague-Dawley rats resulted in significantly increased abundances of beta- and gamma- but no significant change in alpha-ENaC. Acute administration of dDAVP (2 nmol) to Brattleboro rats resulted in modest, but significant, increases in abundance for all ENaC subunits, within 1 h. In conclusion, all three subunits of ENaC are upregulated by vasopressin with temporal and regional differences. These changes are too slow to play a major role in the short-term action of vasopressin to stimulate sodium reabsorption in the collecting duct. Long-term increases in ENaC abundance should add to the short-term regulatory mechanisms (undefined in this study) to enhance sodium transport in the renal collecting duct.

Animals↗

PubMed2000

Vasopressin V(2)-receptor-dependent regulation of AQP2 expression in Brattleboro rats.

The role of AVP-V(2) receptor (AVP-V(2)R)-dependent regulation of aquaporin-2 (AQP2) expression was evaluated in vasopressin-deficient Brattleboro (BB) rats. AQP2 levels were relatively high in BB rats (52 +/- 8% of levels in Wistar rats), and treatment with the AVP-V(2)R antagonist SR-121463A (0.8 mg/day) for 48 h was associated with 1) increased urine output (170 +/- 9%), 2), reduced AQP2 protein levels (42 +/- 10% in whole kidney and 53 +/- 8% in inner medulla), and 3) reduced AQP2 mRNA levels (36 +/- 7%). In addition, the levels of AQP2 phosphorylated in the protein kinase A (PKA) consensus site (Ser(256) of AQP2) was reduced to 3 +/- 1% of control levels. Lithium (Li) treatment of BB rats for 1 mo, known to reduce adenylyl cyclase (AC) activity, downregulated AQP2 protein levels (15 +/- 6%) and increased urine output (220%). Downregulation of AQP2 expression in response to SR-121463A or Li treatment indicates that AQP2 expression in BB rats depends in part on activation of AVP-V(2)Rs and that the signaling cascade(s) involves AC and hence cAMP. Complete water restriction of BB rats produced only a small increase in AQP2 mRNA (235 +/- 33%) and AQP2 protein (156 +/- 22%) levels. Immunoelectron microscopy confirmed the increase in AQP2 abundance but revealed no change in AQP2 apical plasma membrane labeling in response to thirsting. In conclusion, the expression and phosphorylation of AQP2 in BB rats are in part dependent on AVP-V(2)R signaling, and AVP-V(2)-mediated regulation of AQP2 trafficking and expression is effectively decoupled in BB rats, indicating differences in AVP-V(2)R-mediated regulation of AQP2 trafficking and expression.

Animals↗

PubMed2000

Long-term regulation of renal Na-dependent cotransporters and ENaC: response to altered acid-base intake.

Increased systemic acid intake is associated with an increase in apical Na/H exchange in the renal proximal tubule mediated by the type 3 Na/H exchanger (NHE3). Because NHE3 mediates both proton secretion and Na absorption, increased NHE3 activity could inappropriately perturb Na balance unless there are compensatory changes in Na handling. In this study, we use semiquantitative immunoblotting of rat kidneys to investigate whether acid loading is associated with compensatory decreases in the abundance of renal tubule Na transporters other than NHE3. Long-term (i.e., 7-day) acid loading with NH(4)Cl produced large decreases in the abundances of the thiazide-sensitive Na-Cl cotransporter (TSC/NCC) of the distal convoluted tubule and both the beta- and gamma-subunits of the amiloride-sensitive epithelial Na channel (ENaC) of the collecting duct. In addition, the renal cortical abundance of the proximal type 2 Na-dependent phosphate transporter (NaPi-2) was markedly decreased. In contrast, abundances of the bumetanide-sensitive Na-K-2Cl cotransporter of the thick ascending limb and the alpha-subunit of ENaC were unchanged. A similar profile of changes was seen with short-term (16-h) acid loading. Long-term (7-day) base loading with NaHCO(3) resulted in the opposite pattern of response with marked increases in the abundances of the beta- and gamma-subunits of ENaC and NaPi-2. These adaptations may play critical roles in the maintenance in Na balance when changes in acid-base balance occur.

Acid-Base Equilibrium↗

PubMed2000

Altered expression of renal AQPs and Na(+) transporters in rats with lithium-induced NDI.

Lithium (Li) treatment is often associated with nephrogenic diabetes insipidus (NDI). The changes in whole kidney expression of aquaporin-1 (AQP1), -2, and -3 as well as Na-K-ATPase, type 3 Na/H exchanger (NHE3), type 2 Na-Pi cotransporter (NaPi-2), type 1 bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1), and thiazide-sensitive Na-Cl cotransporter (TSC) were examined in rats treated with Li orally for 4 wk: protocol 1, high doses of Li (high Na(+) intake), and protocol 2, low doses of Li (identical food and normal Na(+) intake in Li-treated and control rats). Both protocols resulted in severe polyuria. Semiquantitative immunoblotting revealed that whole kidney abundance of AQP2 was dramatically reduced to 6% (protocol 1) and 27% (protocol 2) of control levels. In contrast, the abundance of AQP1 was not decreased. Immunoelectron microscopy confirmed the dramatic downregulation of AQP2 and AQP3, whereas AQP4 labeling was not reduced. Li-treated rats had a marked increase in urinary Na(+) excretion in both protocols. However, the expression of several major Na(+) transporters in the proximal tubule, loop of Henle, and distal convoluted tubule was unchanged in protocol 2, whereas in protocol 1 significantly increased NHE3 and BSC-1 expression or reduced NaPi-2 expression was associated with chronic Li treatment. In conclusion, severe downregulation of AQP2 and AQP3 appears to be important for the development of Li-induced polyuria. In contrast, the increased or unchanged expression of NHE3, BSC-1, Na-K-ATPase, and TSC indicates that these Na(+) transporters do not participate in the development of Li-induced polyuria.

Animals↗

PubMed2000

Regulation of AQP6 mRNA and protein expression in rats in response to altered acid-base or water balance.

In the rat, aquaporin-6 (AQP6) is mainly localized in intercalated cells (ICs) in collecting ducts, where it is exclusively associated with intracellular vesicles. In this study, we examined whether AQP6 protein and mRNA expression were regulated in the inner medulla or inner stripe of the outer medulla. Rats treated with dietary alkali or acid load for 7 days with a fixed daily water intake revealed appropriate changes in urine pH but unchanged urine output. AQP6 protein and mRNA abundance were increased in alkali-loaded rats (187 +/- 18 and 151 +/- 17% of control, respectively), whereas no changes were observed in acid-loaded rats. Immunohistochemistry revealed increased IC AQP6 labeling in alkali-loaded rats but not in acid-loaded rats. In contrast, administration of NH(4)Cl in the drinking water for 2 wk (free access to water) revealed a significant increase in AQP6 protein abundance (194 +/- 9% of control), but this was associated with increased water intake. Combined, this suggests that AQP6 expression was not affected by acid loading per se but rather was in response to changes in water intake. Consistent with this, water loading for 48 h was associated with increased AQP6 protein abundance, compared with thirsted rats. Moreover, rats with lithium-induced nephrogenic diabetes insipidus had a threefold increase in both AQP6 protein and mRNA expression. Overall, these results suggest that AQP6 expression in collecting duct ICs is regulated by altered acid/alkali load or water balance. Thus AQP6 may contribute to maintenance of acid-base homeostasis and water balance.

Acid-Base Equilibrium↗

PubMed1999

Aquaporin-6: An intracellular vesicle water channel protein in renal epithelia.

All characterized mammalian aquaporins (AQPs) are localized to plasma membranes where they function chiefly to mediate water transport across cells. Here we show that AQP6 is localized exclusively in intracellular membranes in renal epithelia. By using a polyclonal antibody to the C terminus of AQP6, immunoblots revealed a major 30-kDa band in membranes from rat renal cortex and medulla. Endoglycosidase treatment demonstrated presence of an intracellular high mannose glycan on each subunit. Sequential ultracentrifugation of rat kidney homogenates confirmed that AQP6 resides predominantly in vesicular fractions, and immunohistochemical and immunoelectron microscopic studies confirmed that >98% of AQP6 is located in intracellular membrane vesicles. In glomeruli, AQP6 is present in membrane vesicles within podocyte cell bodies and foot processes. In proximal tubules, AQP6 is also abundant in membrane vesicles within the subapical compartment of segment 2 and segment 3 cells, but was not detected in the brush border or basolateral membranes. In collecting duct, AQP6 resides in intracellular membrane vesicles in apical, mid, and basolateral cytoplasm of type A intercalated cells, but was not observed in the plasma membrane. Unlike other members of the AQP family, the unique distribution in intracellular membrane vesicles in multiple types of renal epithelia indicates that AQP6 is not simply involved in transcellular fluid absorption. Moreover, our studies predict that AQP6 participates in distinct physiological functions such as glomerular filtration, tubular endocytosis, and acid-base metabolism.

Amidohydrolases↗

PubMed1999

Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle's loop.

To investigate whether the enhancement of thick ascending limb (TAL) NaCl transport in response to long-term increases in circulating vasopressin concentration is associated with increased expression levels of the apical Na-K-2Cl cotransporter in the rat TAL, we have carried out immunoblotting and immunofluorescence studies using affinity-purified, peptide-directed antibodies. Semiquantitative immunoblotting studies demonstrated a marked increase (193% of controls) in Na-K-2Cl cotransporter band density in response to restriction of water intake to 15 ml/day for 7 days. In contrast, the expression levels of two other apical proteins of the TAL (the type 3 Na/H exchanger and Tamm-Horsfall protein) were unchanged in the outer medulla. A 7-day subcutaneous infusion of the V2 receptor-selective vasopressin analog, 1-desamino-[8-D-arginine]vasopressin (DDAVP), to Brattleboro rats also markedly increased Na-K-2Cl cotransporter expression in the outer medulla (183% of controls). Immunofluorescence localization in outer medullary tissue sections confirmed the increase in Na-K-2Cl cotransporter expression in response to DDAVP. We conclude that vasopressin strongly upregulates the expression of the Na-K-2Cl cotransporter of the TAL and that it is likely to play an important role in the long-term regulation of the countercurrent multiplication system.

Animals↗

PubMed1999

Low aquaporin-2 levels in polyuric DI +/+ severe mice with constitutively high cAMP-phosphodiesterase activity.

In the renal collecting duct, vasopressin acutely activates cAMP production, resulting in trafficking of aquaporin-2 water channels (AQP2) to the apical plasma membrane, thereby increasing water permeability. This acute response is modulated by long-term changes in AQP2 expression. Recently, a cAMP-responsive element has been identified in the AQP2 gene, raising the possibility that changes in cAMP levels may control AQP2 expression. To investigate this possibility, we determined AQP2 protein levels in a strain of mice, DI +/+ severe (DI), which have genetically high levels of cAMP-phosphodiesterase activity, and hence low cellular cAMP levels, and severe polyuria. Semiquantitative immunoblotting of membrane fractions prepared from whole kidneys revealed that AQP2 levels in DI mice were only 26 +/- 7% (+/-SE) of those in control mice (n = 10, P < 0.01). In addition, semiquantitative Northern blotting revealed a significantly lower AQP2 mRNA expression in kidneys from DI mice compared with control mice (43 +/- 6% vs. 100 +/- 10%; n = 6 in each group, P < 0.05). AQP3 levels were also reduced. The mice were polyuric and urine osmolalities were accordingly substantially lower in the DI mice than in controls (496 +/- 53 vs. 1,696 +/- 105 mosmol/kgH2O, respectively). Moreover, there was a linear correlation between urine osmolalities and AQP2 levels (P < 0.05). Immunoelectron microscopy confirmed the markedly lower expression of AQP2 in collecting duct principal cells in kidneys of DI mice and, furthermore, demonstrated that AQP2 was almost completely absent from the apical plasma membrane. Thus expression of AQP2 and AQP2 trafficking were severely impaired in DI mice. These results are consistent with the view that in vivo regulation of AQP2 expression by vasopressin is mediated by cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗