Decreased abundance of collecting duct aquaporins in post-ischemic renal failure in rats.
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Increased urine flow is often a feature of mild to moderate acute renal failure. This study examines the possible role of dysregulation of collecting duct aquaporins as a factor in this increase. In rats, the left renal pedicle was clamped for 45 min followed by contralateral nephrectomy. Control rats were identical except that the renal pedicle was not clamped. Rats were sacrificed and the kidneys were homogenized at various time points after release of the clamp for semiquantitative immunoblotting of collecting duct aquaporins, as well as the thick ascending limb Na-K-2Cl cotransporter and the proximal tubule water channel, aquaporin-1. Urinary flow rate was significantly increased 18 h after the ischemic insult and remained increased through 72 h. Whole kidney aquaporin-2 protein abundance was 45% of controls at 18 h, 55% of controls at 36 h, and returned to normal 72 h after ischemia. Whole kidney aquaporin-3 protein abundance was 37% of controls at 18 h, 13% of controls at 36 h, and 45% of controls at 72 h. The decline in aquaporin-2 and -3 was confirmed by immunocytochemistry. Abundance of the thick ascending limb Na-K-2Cl cotransporter protein was not significantly decreased. Aquaporin-1 protein abundance was not significantly decreased at 18 h after the ischemic insult, but was significantly reduced after 36 h. Thus, the post-ischemic state is associated with decreased levels of the collecting duct aquaporins, coinciding with an increase in water excretion. It is concluded that decreased aquaporin protein abundance in collecting duct cells is a contributing factor in the increased urine flow seen in moderate post-ischernic acute renal failure. (+info)
Renal expression of aquaporins in liver cirrhosis induced by chronic common bile duct ligation in rats.
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Semiquantitative immunoblotting was used to investigate the expression levels of the four major renal aquaporins, the Na-K-2Cl cotransporter of the thick ascending limb, the type 3 Na-H exchanger, and the Na-K-ATPase in kidneys from rats with cirrhosis secondary to common bile duct ligation (CBDL). These rats had significant water retention and hyponatremia. In contrast to models of cirrhosis induced by carbon tetrachloride, aquaporin-2 expression in CBDL-induced cirrhosis was decreased. Thus, these results show that in the setting of extracellular fluid volume expansion, excessive water retention with hyponatremia can occur in the absence of increases in aquaporin-2 abundance. In addition, the expression levels of the two basolateral collecting duct aquaporins (aquaporin-3 and -4) were decreased in CBDL rats relative to sham-operated control rats. Similarly, the Na-K-2Cl cotransporter of the thick ascending limb and the type 3 Na-H exchanger showed decreases in expression. In contrast, the expression levels of aquaporin-1 and the all subunit of the Na-K-ATPase were not decreased. Thus, dysregulation of multiple water channels and ion transporters may play a role in water balance abnormalities associated with CBDL-induced cirrhosis in rats. (+info)
Clinical presentation and follow-up of 30 patients with congenital nephrogenic diabetes insipidus.
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Congenital nephrogenic diabetes insipidus is characterized by insensitivity of the distal nephron to arginine vasopressin. Clinical knowledge of this disease is based largely on case reports. For this study, data were collected on clinical presentation and during long-term follow-up of 30 male patients with congenital nephrogenic diabetes insipidus. The majority of patients (87%) were diagnosed within the first 2.5 yr of life. Main symptoms at clinical presentation were vomiting and anorexia, failure to thrive, fever, and constipation. Three older patients were diagnosed as a result of events not directly related to the disease. Except for a possibly milder phenotype in patients with a G185C mutation, no clear relationship between clinical and genetic data could be found. Most patients were on hydrochlorothiazide-amiloride treatment without significant side effects. Two patients suffered from severe hydronephrosis with a small rupture of the urinary tract after a minor trauma, and two patients experienced episodes of acute urine retention. Height SD scores for age remained below the 50th percentile in the majority of patients, whereas weight for height SD scores showed a catch-up after several years of underweight. (+info)
Kidney aquaporin-2 expression during escape from antidiuresis is not related to plasma or tissue osmolality.
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Recent results indicate that renal escape from vasopressin-induced antidiuresis is accompanied by a marked downregulation of whole kidney aquaporin-2 (AQP-2) protein and mRNA expression. However, in those studies, the escaped animals were also markedly hypo-osmolar compared to controls as a result of water loading during antidiuresis. The present studies evaluated whether systemic or local osmolality contributes to the downregulation of AQP-2 expression in this model. In the first study, two groups of 1-deamino-[8-D-arginine]-vasopressin (dDAVP)-infused rats were water-loaded; after establishment of escape, one group was then water-restricted for 4 d to reverse the escape, whereas the other group continued daily water loading. Whole kidney AQP-2 protein was measured by Western blotting, and inner medulla AQP-2 mRNA was determined by Northern blotting. Results were compared to dDAVP-infused rats fed solid chow. After 4 d of water restriction, urine volume decreased to the same level as in the rats on solid chow; however, plasma sodium concentrations and plasma osmolality remained low. Despite maintenance of significant hypo-osmolality, rats in which escape was subsequently reversed by water restriction reestablished high dDAVP-stimulated kidney levels of AQP-2 after 4 d of water restriction. In the second study, AQP-2 expression was evaluated in different regions of kidneys from water-loaded rats undergoing escape from antidiuresis. Despite markedly different interstitial osmolalities, significant downregulation of AQP-2 expression compared to dDAVP-infused control rats was seen in the inner medulla, outer medulla, and cortex. Thus, neither systemic nor interstitial osmolality appears to appreciably be correlated with downregulation of kidney AQP-2 expression during escape from antidiuresis. These results therefore suggest that additional vasopressin- and osmolality-independent factors, likely related to the effects of extracellular fluid volume expansion, also regulate kidney AQP-2 expression in rats. (+info)
Selective V2-receptor vasopressin antagonism decreases urinary aquaporin-2 excretion in patients with chronic heart failure.
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Aquaporin-2 (AQP-2), a water channel located on the apical membrane of collecting duct cells, regulates water reabsorption under the control of vasopressin (AVP). Using an antibody directed to human AQP-2, a quantitative Western blot analysis was performed to determine the collecting duct responsiveness to an oral, nonpeptide, V2 receptor antagonist (VPA-985) in patients with chronic NYHA II and III heart failure. Standards were derived by conjugating the immunizing peptide to maleimide-activated bovine serum albumin and a standard curve was generated for each blot. Quantification of baseline steady-state AQP-2 excretion was done by collecting urine on the day before study drug administration. The next day patients received either placebo or VPA-985 at one of four different doses and urine was collected every 2 h. Thereafter, urinary AQP-2 excretion was calculated as a ratio of the urine flow and was expressed in pmol/h. During baseline, steady-state excretion did not change significantly (T0-T2, 458 +/- 44; T2-T4, 443 +/- 35; T4-T6, 422 +/- 35; T6-T8, 401 +/- 30). Compared to placebo, urinary AQP-2 excretion decreased significantly and in all groups in a dose-dependent manner during VPA-985 administration. The most impressive decrease was observed in the 250-mg group (T0-T2, 89 +/- 5; T2-T4, 50 +/- 18; T4-T6, 43 +/- 22; T6-T8, 42 +/- 23; P < 0.001 during each period compared with baseline and placebo results). VPA-985 significantly increased solute-free water clearance and urine output and significantly decreased urinary osmolality. Urinary AQP-2 excretion correlated best with solute-free water clearance during T0-T2 and T2-T4 collection, but a correlation with urinary osmolality and urinary output was also found during these periods. In conclusion, AQP-2 urinary excretion, as measured by quantitative Western analysis, is a sensitive biologic marker to assess the short-term responsiveness of the collecting duct to a V2 receptor AVP antagonist in chronic heart failure. (+info)
Regulation of aquaporin-2 expression by the alpha(2)-adrenoceptor agonist clonidine in the rat.
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Aquaporin-2 (AQP-2), the major water channel responsible for water balance, has been shown to be regulated by the binding of vasopressin to V(2) vasopressin receptors in the medullary collecting duct. alpha(2)-Adrenoceptor agonists such as clonidine have been associated with an increase in free water clearance that was secondary to an inhibition of the ability of vasopressin to increase cAMP levels in the collecting ducts. This investigation focused on the possibility that this increase in free water clearance following administration of an alpha(2)-adrenoceptor agonist was associated with a reduction in medullary AQP-2 expression. In the anesthetized rat, clonidine increased urine flow rate (32+/-5 versus 137+/-16 microl/min, p<.05) and free water clearance (-58+/-6 versus 3+/-8 microl/min, p<.05) compared with the group receiving the saline vehicle infusion. The increase in free water clearance with clonidine administration was associated with a reduction in whole kidney AQP-2 mRNA levels (282+/-25 versus 216+/-11 A units, p<.05). This decrease in water reabsorption was associated with a redistribution of AQP-2 away from the luminal membrane of the medullary collecting duct to the cytosol. These effects were not secondary to changes in serum vasopressin levels, as these were similar in the vehicle control and clonidine groups (59+/-5 pg/ml versus 64+/-7 pg/ml, p = NS). The rapid redistribution of AQP-2 and the reduction in AQP-2 mRNA following clonidine administration are consistent with the hypothesis that the alpha(2) adrenoceptor regulates water excretion at least in part by effects on AQP-2. (+info)
Functional analysis of aquaporin-2 mutants associated with nephrogenic diabetes insipidus by yeast expression.
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Mutations of aquaporin-2 (AQP2) vasopressin water channel cause nephrogenic diabetes insipidus (NDI). It has been suggested that impaired routing of AQP2 mutants to the plasma membrane causes the disease; however, no determinations have been made of mutation-induced alterations of AQP2 channel water permeability. To address this issue, a series of AQP2 mutants were expressed in yeast, and the osmotic water permeability (P(f)) of the isolated vesicles was measured. Wild-type and mutant AQP2 were expressed equally well in vesicles. P(f) of the vesicles containing wild-type AQP2 was 22 times greater than that of the control, which was sensitive to mercury and weakly dependent on the temperature. P(f) measurements and mercury inhibition examinations suggested that mutants L22V and P262L are fully functional, whereas mutants N68S, R187C, and S216P are partially functional. In contrast, mutants N123D, T125M, T126M, A147T, and C181W had very low water permeability. Our results suggest that the structure between the third and fifth hydrophilic loops is critical for the functional integrity of the AQP2 water channel and that disruption of AQP2 water permeability by mutations may cause NDI. (+info)
Misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus.
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We reported that several aquaporin-2 (AQP2) point mutants that cause nephrogenic diabetes insipidus (NDI) are retained in the endoplasmic reticulum (ER) of transfected mammalian cells and degraded but can be rescued by chemical chaperones to function as plasma membrane water channels (Tamarappoo, B. K., and Verkman, A. S. (1998) J. Clin. Invest. 101, 2257-2267). To test whether mutant AQP2 proteins are misfolded, AQP2 folding was assessed by comparative detergent extractability and limited proteolysis, and AQP2 degradation kinetics was measured by label-pulse-chase and immunoprecipitation. In ER membranes from transfected CHO cells containing [(35)S]methionine-labeled AQP2, mutants T126M and A147T were remarkably detergent-resistant; for example wild-type AQP2 was >95% solubilized by 0.5% CHAPS whereas T126M was <10% solubilized. E258K, an NDI-causing AQP2 mutant which is retained in the Golgi, is highly detergent soluble like wild-type AQP2. The mutants and wild-type AQP2 were equally susceptible to digestion by trypsin, thermolysin, and proteinase K. Stopped-flow light scattering measurements indicated that T126M AQP2 at the ER was fully functional as a water channel. Pulse-chase studies indicated that the increased degradation rates for T126M (t((1)/(2)) 2.5 h) and A147T (2 h) compared with wild-type AQP2 (4 h) involve a brefeldin A-resistant, ER-dependent degradation mechanism. After growth of cells for 48 h in the chemical chaperone glycerol, AQP2 mutants T126M and A147T became properly targeted and relatively detergent-soluble. These results provide evidence that NDI-causing mutant AQP2 proteins are misfolded, but functional, and that chemical chaperones both correct the trafficking and folding defects. Strategies to facilitate protein folding might thus have therapeutic efficacy in NDI. (+info)