AQP2
Aquaporin-2 (AQP-2) (ADH water channel) (Aquaporin-CD) (AQP-CD) (Collecting duct water channel protein) (WCH-CD) (Water channel protein for renal collecting duct)
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The DBA/2-FG pcy (pcy) mouse is a model of human nephronophthisis, a recessive cystic kidney disease. Renal expression of aquaporin-2 (AQP2), a water channel protein, has been shown to be altered in pcy mice. However, the relationship between the renal expression and its release in urinary extracellular vesicles (uEV-AQP2), which account for most urinary AQP2, in pcy mice has remained largely unknown. In this study, we examined age-related alterations of this relationship in pcy mice. In comparison with control mice, pcy mice after the age of 14 weeks showed defective urinary concentration ability with an increase in urinary volume. Interestingly, the release of uEV-AQP2 increased progressively up to the age of 16 weeks, but at 21 weeks the release did not significantly differ from that in control mice (i.e., a bell-shaped pattern was evident). Similar results were obtained for uEV marker proteins, including tumor susceptibility gene 101 (TSG101) protein and apoptosis-linked gene 2-interacting protein X (Alix). Immunoblot analysis revealed that renal AQP2 expression increased progressively from 11 weeks, and immunohistochemistry showed that this increase was possibly due to an increase in the number of AQP2-positive cells. Analysis of mRNAs for seven types of AQP expressed in the kidney supported this notion. These data suggest that the level of uEV-AQP2 does not simply mirror the renal expression of AQP2 and that the altered release of uEV-AQP2 in pcy mice depends on the numbers of both renal AQP2-positive cells and EVs released into the urine.
MeSH Terms
- Aging
- Animals
- Aquaporin 2
- Calcium-Binding Proteins
- DNA-Binding Proteins
- Disease Models, Animal
- Endosomal Sorting Complexes Required for Transport
- Extracellular Vesicles
- Gene Expression Regulation
- Kidney
- Kidney Diseases, Cystic
- Mice, Inbred DBA
- Mice, Mutant Strains
- RNA, Messenger
- Transcription Factors
Keywords
- Aquaporin-2
- pcy mice
- polycystic kidney disease
- urinary extracellular vesicles (exosomes)
Hypothyroid state and aging are associated with impairment in water reabsorption and changes in aquaporin water channel type 2 (AQP2). Nitric oxide (NO) is involved in AQP2 trafficking to the apical plasma membrane in medullary collecting duct cells. The purpose of this study was to investigate whether aging and hypothyroidism alter renal function, and whether medullary NO and AQP2 are implicated in maintaining water homeostasis. Sprague-Dawley rats aged 2 and 18months old were treated with 0.02% methimazole (w/v) during 28days. Renal function was examined and NO synthase (NOS) activity ([(14)C (U)]-L-arginine to [(14)C (U)]-L-citrulline assays), NOS, caveolin-1 and -3 and AQP2 protein levels were determined in medullary tissue (Western blot). Plasma membrane fraction and intracellular vesicle fraction of AQP2 were evaluated by Western blot and immunohistochemistry. A divergent response was observed in hypothyroid rats: while young rats exhibited polyuria with decreased medullary NOS activity, adult rats exhibited a decrease in urine output with increased NOS activity. AQP2 was increased with hypothyroidism, but while young rats exhibited increased AQP2 in plasma membrane, adult rats did so in the cytosolic site. Hypothyroidism contributes in a differential way to aging-induced changes in renal function, and medullary NO and AQP2 would be implicated in maintaining water homeostasis.
MeSH Terms
- Aging
- Animals
- Aquaporin 2
- Body Water
- Homeostasis
- Hypothyroidism
- Kidney
- Male
- Nitric Oxide
- Nitric Oxide Synthase
- Rats
- Rats, Sprague-Dawley
Keywords
- Aged
- Eut
- HR
- Hypo
- Intraperitoneal
- Ip
- MAP
- NO
- NOS
- Renal function
- T(4)
- TSH
- Thyroid hormones
- Water channel
- cav
- cav-1
- cav-3
- caveolin-1
- caveolin-3
- caveolins
- eNOS
- endothelial nitric oxide synthase
- euthyroid
- heart rate
- hypothyroid
- iNOS
- inducible nitric oxide synthase
- mean arterial pressure
- nNOS
- neuronal nitric oxide synthase
- nitric oxide
- nitric oxide synthase
- thyroid-stimulating hormone
- total thyroxine
The aim of this study was to evaluate the effects of N-acetylcysteine (NAC) on renal function, as well as on sodium and water transporters, in the kidneys of aged rats. Normal, 8-month-old male Wistar rats were treated (n=6) or not (n=6) with NAC (600 mg/L in drinking water) and followed for 16 months. At the end of the follow-up period, we determined inulin clearance, serum thiobarbituric acid reactive substances (TBARS), serum cholesterol, and urinary phosphate excretion. In addition, we performed immunohistochemical staining for p53 and for ED-1-positive cells (macrophages/monocytes), together with Western blotting of kidney tissue for NKCC2, aquaporin 2 (AQP2), urea transporter A1 (UT-A1) and Klotho protein. At baseline, the two groups were similar in terms of creatinine clearance, proteinuria, cholesterol, and TBARS. At the end of the follow-up period, NAC-treated rats presented greater inulin clearance and reduced proteinuria, as well as lower serum cholesterol, serum TBARS, and urinary phosphate excretion, in comparison with untreated rats. In addition, NAC-treated rats showed upregulated expression of NKCC2, AQP2, and UT-A1; elevated Klotho protein expression, low p53 expression, and few ED-1 positive cells. In conclusion, we attribute these beneficial effects of NAC (the significant improvements in inulin clearance and in the expression of NKCC2, AQP2, and UT-A1) to its ability to decrease oxidative stress, inhibit p53 expression, minimize kidney inflammation, and stimulate Klotho expression.
MeSH Terms
- Acetylcysteine
- Age Factors
- Aging
- Animals
- Antioxidants
- Aquaporin 2
- Biomarkers
- Blotting, Western
- Cellular Senescence
- Cholesterol
- Ectodysplasins
- Glucuronidase
- Immunohistochemistry
- Inulin
- Kidney
- Male
- Membrane Transport Proteins
- Oxidative Stress
- Phosphates
- Rats
- Rats, Wistar
- Sodium-Potassium-Chloride Symporters
- Solute Carrier Family 12, Member 1
- Thiobarbituric Acid Reactive Substances
- Tumor Suppressor Protein p53
Previous studies revealed that the rat retina contains numerous membrane-located water channels, the aquaporins (AQPs). Protein expression patterns of AQP1-4, 6 and 9 were examined by immunohistochemistry. In the present study, we investigated the immunolocalization of AQP1-4, 6 and 9 during postnatal development in the rat retina and examined the effect of age on the tissue distribution of these channels. AQP1, 3, 4, 6 and 9 showed gradually increased expression in rat retinas from postnatal week 1 to week 12, and decreased in the 40-week-old rat retinas. AQP2 expression was barely seen in the first week in rat retinas and displayed a significant increase from week 1 to week 4, however no significant alteration of AQP2 was observed after 4weeks of development. AQP1 and 4 immunoreactivities were present in the inner limiting membrane (ILM), the ganglion cell layer (GCL), inner nuclear layer (INL) and retinal pigment epithelium (RPE) in the 4-, 12- and 40-week-old rat retinas. The RPE, OLM and ILM showed a remarkable expression of AQP1-4, 6 and 9 in the 4, 12 and 40-week-old rat retinas. The reduced expression of AQPs in aged rat retinas may indicate the involvement of AQPs in the pathogenesis of age-related retinal diseases.
MeSH Terms
- Aging
- Animals
- Animals, Newborn
- Aquaporins
- Gene Expression Profiling
- Gene Expression Regulation, Developmental
- Immunohistochemistry
- Rats
- Rats, Wistar
- Retina
Previously we demonstrated that neonatally induced partial unilateral ureteral obstruction (PUUO) in rats is associated with changes in the abundance of renal acid-base transporters that were paralleled by reduction in renal functions dependent on the severity and duration of obstruction. The aim of the present study was to identify whether changes in renal aquaporin abundance are age-dependent. Semiquantitative immunoblotting and immunohistochemistry were used to examine the changes in abundance of AQP1, AQP2, p-S256AQP2 (AQP2 phosphorylated at consensus site Ser(256)) and AQP3 in the kidneys of rats with neonatally induced PUUO within the first 48 h of life, and then monitored for 7 or 14 weeks. Protein abundance of AQP2 and AQP3 increased in both obstructed and non-obstructed kidneys 7 weeks after induction of neonatal PUUO (PUUO-7W). In contrast, AQP1 and AQP2 protein abundance in the obstructed kidney were reduced after 14 weeks of PUUO (PUUO-14W). Importantly, pS256-AQP2 protein abundance was reduced in obstructed kidneys of both PUUO-7W and PUUO-14W. Immunohistochemistry confirmed the persistent pS256-AQP2 downregulation in both PUUO-7W and PUUO-14W rats. The study shows that the protein abundance of AQP1, AQP2, and AQP3 in the obstructed kidney is increased in PUUO-7W, which may be a compensatory phenomenon and reduced in PUUO-14W rats suggesting a time-/age-dependent dysregulation in response to PUUO. pS256-AQP2 protein abundance is reduced consistent with obstruction-induced direct effects in the apical part of the collecting duct principal cells in response to PUUO.
MeSH Terms
- Age Factors
- Aging
- Animals
- Animals, Newborn
- Aquaporin 1
- Aquaporin 2
- Aquaporin 3
- Aquaporins
- Blotting, Western
- Disease Models, Animal
- Hydronephrosis
- Immunohistochemistry
- Kidney
- Male
- Phosphorylation
- Rats
- Ureteral Obstruction
Sodium-sensitive hypertension is caused by renal tubular dysfunction, leading to increased retention of sodium and water. Previous findings have suggested that single-nucleotide polymorphisms of the cytoskeletal protein, α-adducin, are associated with increased membrane expression of the Na/K pump and abnormal renal sodium transport in Milan hypertensive strain (MHS) rats and in humans. However, the possible contribution of renal aquaporins (AQPs) to water retention remains undefined in MHS rats. Kidneys from MHS rats were analyzed and compared with those from age-matched Milan normotensive strain (MNS) animals by quantitative-PCR, immunoblotting, and immunoperoxidase. Endocytosis assay was performed on renal cells stably expressing AQP4 and co-transfected either with wild-type normotensive (NT) or with mutated hypertensive (HT) α-adducin. Semiquantitative immunoblotting revealed that AQP1 abundance was significantly decreased only in HT MHS whereas AQP2 was reduced in both young pre-HT and adult-HT animals. On the other hand, AQP4 was dramatically upregulated in MHS regardless of the age. These results were confirmed by immunoperoxidase microscopy. Endocytosis assays clearly showed that the expression of mutated adducin strongly reduced the rate of constitutive AQP4 endocytosis, thereby increasing its abundance at the plasma membrane. We provide here the first evidence that AQP1, AQP2, and AQP4 are dysregulated in the kidneys of MHS animals. In particular, we provide evidence that α-adducin mutations may be responsible for AQP4 upregulation. The downregulation of AQP1 and AQP2 and the upregulation of AQP4 may be relevant for the onset and maintenance of salt-sensitive hypertension.
MeSH Terms
- Absorption
- Aging
- Animals
- Aquaporin 4
- Aquaporins
- Blood Pressure
- Calmodulin-Binding Proteins
- Disease Models, Animal
- Endocytosis
- Hypertension
- Kidney
- Male
- Polymorphism, Genetic
- Rats
- Rats, Mutant Strains
- Salt Tolerance
- Water
The study was undertaken to determine whether ageing affects kidney expression of the aquaporin-2 (AQP2) water channel in glucocorticoid-deficient rats. After adrenalectomy, 6- and 52-week-old Sprague-Dawley rats received aldosterone via osmotic minipumps (glucocorticoid-deficient rats). Aldosterone and dexamethasone were administered to control rats of the same age. An acute water load test verified impairment of water excretion in both young and aged rats with glucocorticoid deficiency, with a more serious impairment in the older rats. Despite the presence of hypoosmolality, non-suppressible release of arginine vasopressin (AVP) was particularly evident in the aged rats with glucocorticoid deficiency in comparison with the young rats. The expression levels of AQP2 mRNA and protein were lower in the aged rats, with a particularly large reduction in AQP2 protein expression. AQP2 expression levels were significantly augmented in the glucocorticoid-deficient rats compared with the controls under both basal and water-loaded conditions. Acute water loading did not suppress expression of AQP2 mRNA and protein, and the percentage increases in AQP2 mRNA and protein expression vs. the respective controls were more pronounced in the 52-week-old glucocorticoid-deficient rats compared with the 6-week-old rats. The findings indicate that upregulation of AQP2 expression is maintained dependent upon non-suppressible release of AVP in rats with glucocorticoid deficiency, and that AQP2 plays a crucial role in persistent impairment of water excretion in aged rats with glucocorticoid deficiency.
MeSH Terms
- Adrenalectomy
- Aging
- Aldosterone
- Animals
- Aquaporin 2
- Arginine Vasopressin
- Dexamethasone
- Epithelial Sodium Channels
- Glucocorticoids
- Kidney
- Male
- Osmolar Concentration
- Rats
- Rats, Sprague-Dawley
- Sodium
- Up-Regulation
- Urine
- Vasopressins
- Water
Mutations in aquaporin-2 (AQP2) that interfere with its cellular processing can produce autosomal recessive nephrogenic diabetes insipidus (NDI). Prior gene knock-in of the human NDI-causing AQP2 mutation T126M produced mutant mice that died by age 7 days. Here, we used a novel "conditional gene knock-in" strategy to generate adult, AQP2-T126M mutant mice. Mice separately heterozygous for floxed wild-type AQP2 and AQP2-T126M were bred to produce hemizygous mice, which following excision of the wild-type AQP2 gene by tamoxifen-induced Cre-recombinase gave AQP2(T126M/-) mice. AQP2(T126M/-) mice were polyuric (9-14 ml urine/day) compared to AQP2( / ) mice (1.6 ml/day) and had reduced urine osmolality (400 vs. 1800 mosmol). Kidneys of AQP2(T126M/-) mice expressed core-glycosylated AQP2-T126M protein in an endoplasmic reticulum pattern. Screening of candidate protein folding "correctors" in AQP2-T126M-transfected kidney cells showed increased AQP2-T126M plasma membrane expression with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). 17-AAG increased urine osmolality in AQP2(T126M/-) mice by >300 mosmol but had no effect in AQP2(-/-) mice. Kidneys of 17-AAG-treated AQP2(T126M/-) mice showed partial rescue of defective AQP2-T126M cellular processing. Our results establish an adult mouse model of NDI and demonstrate partial restoration of urinary concentration function by a compound currently in clinical trials for other indications.
MeSH Terms
- Aging
- Animals
- Aquaporin 2
- Base Sequence
- Cell Line
- Diabetes Insipidus, Nephrogenic
- Disease Models, Animal
- Dogs
- Gene Knock-In Techniques
- HSP90 Heat-Shock Proteins
- Mice
- Mice, Transgenic
- Mutation
To examine the effect of nitric oxide (NO) on the expression and/or localization of inner medulla collecting duct aquaporin-2 water channel (AQP2) in young and adult hemorrhaged anesthetized rats. Rats of 2 (young) and 12 mo (adult) old (n=15) were divided into: Sham animals with and without NG-nitro-l-arginine methyl ester (L-NAME) treatment (S L-NAME and S); hemorrhaged animals (20% blood loss) with and without L-NAME (H L-NAME and H). Mean arterial pressure (MAP) was continuously monitored and AQP2 expression and inmunolocalization were evaluated at 120 min after bleeding. L-NAME blunted the hypotension induced by hemorrhage at 120 min in young (106 /-2 mm Hg) and adult (103 /-4 mm Hg) rats. AQP2 expression increased after bleeding in young (from 22 to 50 densitometric units) and adult rats (from 15 to 30 densitometric units). Pretreatment with L-NAME enhanced this effect, being this rise lower in adult than young animals (young: 318%, adult: 233%). Electron microscopy showed that AQP2 labeling increased after withdrawal, being the number of gold particles smaller in adult than young animals in the inner medulla. L-NAME enhanced this effect. NOS activity decreases AQP2 expression/traffick in the inner collecting duct principal cells in response to hemorrhage and this effect is lower with aging.
MeSH Terms
- Aging
- Animals
- Aquaporin 2
- Blood Pressure
- Blotting, Western
- Enzyme Inhibitors
- Hemorrhage
- Hypovolemia
- Immunohistochemistry
- Kidney
- Male
- Microscopy, Immunoelectron
- NG-Nitroarginine Methyl Ester
- Nitric Oxide
- Nitric Oxide Synthase
- Rats
- Rats, Sprague-Dawley
- Water
Aging kidney is associated in humans and rodents with polyuria and reduced urine concentrating ability. In senescent female WAG/Rij rats, this defect is independent of arginine-vasopressin (AVP)/V(2) receptor/cAMP pathway. It has been attributed to underexpression and mistargeting of aquaporin-2 (AQP2) water channel in the inner medullary collecting duct (IMCD). We showed previously that dDAVP administration could partially correct this defect. Since AQP2 can also be regulated by AVP-independent pathways in water deprivation (WD), we investigated AQP2 and phosphorylated AQP2 (p-AQP2) regulation in thirsted adult (10 mo old) and senescent (30 mo old) female WAG/Rij rats. Following 2-day WD, urine flow rate decreased and urine osmolality increased in both groups. However, in agreement with significantly lower cortico-papillary osmotic gradient with aging, urine osmolality remained lower in senescent animals. WD induced sixfold increase of plasma AVP in all animals which, interestingly, did not result in higher papillary cAMP level. Following WD, AQP2 and p-AQP2 expression increased hugely in 10- and 30-mo-old rats and their mistargeting in old animals was corrected. Moreover, the age-related difference in AQP2 regulation was abolished after WD. To further investigate the mechanism of AQP2 underexpression with aging, AQP2 mRNA was quantified by real-time RT-PCR. In the outer medulla, preservation of AQP2 protein expression was achieved through increased AQP2 mRNA level in senescent rats. In the IMCD, no change in AQP2 mRNA was detected with aging but AQP2 protein expression was markedly lower in 30-mo-old animals. In conclusion, there is a posttranscriptional downregulation of AQP2 with aging, which is abolished by WD.
MeSH Terms
- Aging
- Animals
- Aquaporin 2
- Cyclic AMP
- Cyclic GMP
- Down-Regulation
- Female
- Kidney Concentrating Ability
- Kidney Medulla
- Osmolar Concentration
- Phosphorylation
- RNA Processing, Post-Transcriptional
- RNA, Messenger
- Rats
- Rats, Inbred Strains
- Reverse Transcriptase Polymerase Chain Reaction
- Water Deprivation
- Water-Electrolyte Balance
The limited renal concentration performance by the immature kidney traditionally is thought to be attributed to blunted renal response to arginine vasopressin (AVP) and medullary hypotonicity. The diminished AVP-dependent osmotic water permeability of the collecting duct is the result of decreased AVP binding and adenylate cyclase activation, and low expression of aquaporin-2 (AQP2) mRNA and low levels of AQP2 protein. Moreover, the immature kidney fails to establish deep cortico-papillary osmotic gradient because of structural immaturity, limited solute transport and increased medullary blood flow. Based on indirect clinical and experimental evidences this article puts forward a hypothesis that during perinatal period the abundant hyaluronan (HA) content in the renomedullary interstitium has a primary role in antagonizing water reabsorption and limiting concentration performance. Hydration-related alterations in renal HA appears to be mediated by antidiuretic hormone. The concept of HA-mediated renal water transport may imply that interfering selectively with renal HA metabolism may provide a new therapeutic approach to promote diuresis or antidiuresis, respectively, according to the elevation or reduction in renomedullary HA.
MeSH Terms
- Aging
- Animals
- Body Water
- Evidence-Based Medicine
- Humans
- Hyaluronic Acid
- Kidney
- Models, Biological
- Urination
- Water-Electrolyte Balance
Renal concentrating ability is known to be impaired with aging. The antidiuretic hormone AVP plays an important role in renal water excretion by regulating the membrane insertion and abundance of the water channel aquaporin-2 (AQP2); this effect is primarily mediated via the V2 subtype of the AVP receptor (V2R). This study evaluated the hypothesis that decreased renal sensitivity to AVP, with subsequent altered renal AQP2 expression, contributes to the reduced urinary concentrating ability with aging. Our results show that under baseline conditions, urine osmolality is significantly lower in aged Fischer 344 and Brown-Norway F1 hybrid (F344BN) rats despite equivalent plasma AVP concentrations as in young rats. Levels of kidney V2R mRNA expression and AQP2 abundances were also significantly decreased in aged F344BN rats, as was AQP2 immunostaining in collecting duct cells. In response to moderate water restriction, urine osmolality increased by significantly lesser amounts in aged F344BN rats compared with young rats despite similar increases in plasma AVP levels. Moderate water restriction induced equivalent relative increases in renal AQP2 abundances in all age groups but resulted in significantly lower abundances in total kidney AQP2 protein in aged compared with young F344BN rats. These results therefore demonstrate a functional impairment of renal concentrating ability in aged F344BN rats that is not due to impaired secretion of AVP but rather appears to be related to impaired responsiveness of the kidney to AVP that is secondary, at least in part, to a downregulation of renal V2R expression and AQP2 abundance.
MeSH Terms
- Aging
- Animals
- Aquaporin 2
- Aquaporins
- Arginine Vasopressin
- Dehydration
- Down-Regulation
- Gene Expression
- Immunohistochemistry
- Kidney
- Kidney Concentrating Ability
- Male
- RNA, Messenger
- Rats
- Rats, Inbred F344
- Receptors, Vasopressin
- Urine
- Water-Electrolyte Balance
Kidneys of new-born animals are resistant to arginine vasopressin (AVP). The ability of the hormone to regulate water permeability of the collecting duct can be seen from weaning period, probably due to the maturation of the intracellular signaling pathway. The purpose of the present work was to investigate the effect of V2 receptor agonist dDAVP on the water permeability of OMCD basolateral membrane in 10-, 22- and 60-day old Wistar rats. We also estimated ontogenetic gene expression of AQP2, AQP3, AQP4 and V2 receptor. Osmotic water permeability (Pf) of the basolateral membrane of microdissected OMCD was measured under control conditions and after incubation with the agonist V2 receptor desmopressin (dDAVP; 10(-7) M). Water permeability in 10- and 22-day old rats under control conditions were significantly higher than in adults. Desmopressin stimulated significant increase of this parameter in 22-day old pups (Pf = = 125 /- 4.85; Pf = 174 /- 8.2 microns/s, p < 0.001) and adult rats (Pf = 100.5 /- 7.38; Pf = 178.8 /- 9.54 microns/s, p < 0.001). Osmotic water permeability of the OMCD basolateral membrane in 10-day old rats does not depend on dDAVP (Pf = 172.5 /- 23.8; Pf = 164.8 /- 34 microns/s). With the RT-PCR, we observed a gradual increase of AQP2 and V2 receptor genes expression during postnatal ontogenesis. The gene expression of AQP3 and AQP4 remained unchanged during postnatal ontogenesis. In general, the water permeability of the OMCD basolateral membrane of rats can be stimulated by AVP since the 22nd day of postnatal life. The water permeability of the OMCD basolateral membrane under control conditions gradually decreased during postnatal development, while gene expression of AQP3 and AQP4 was unchanged. The mechanism of this decrease remains to be established.
MeSH Terms
- Aging
- Animals
- Aquaporin 2
- Aquaporin 6
- Aquaporins
- Cell Membrane Permeability
- Deamino Arginine Vasopressin
- Female
- Kidney Medulla
- Kidney Tubules, Collecting
- Male
- Osmosis
- Rats
- Rats, Wistar
- Receptors, Vasopressin
- Renal Agents
- Water
The expression and localization of aquaporins (AQP1-AQP5), members of the water channel family, in the developing rat submandibular gland were analysed using RT-PCR, Northern blotting and immunohistochemistry to explore their relation to the development of this salivary gland. RT-PCR analysis revealed unique expression patterns of each AQP. AQP1 was expressed constitutively during prenatal development, whereas the expression of AQP5 became more intense in the course of development from embryonic day 16.5 (E16) to E20. These expression patterns concurred with the results of Northern blot analysis. AQP3 and AQP4 mRNAs in the prenatal development were not detected in Northern blots, although they were detected by RT-PCR. During postnatal development, AQP5 and AQP1 mRNAs were expressed continuously, but no message for AQP3 or AQP4 was detected. AQP2 mRNA was not detected during either prenatal or postnatal development in this tissue. Immunohistochemical studies revealed that AQP5 was first localized at the apical membrane of proacinar cells at E18, and then became clearly distributed at the apical membrane of acinar cells in accordance with the differentiation and establishment of the mature acini. In addition, some vasculature also showed immunoreactivity for AQP5. AQP1 was immunolocalized in the blood vessels, including capillaries, of the gland throughout development. These observations suggest the existence of transcriptional regulation of rat AQP5, which is one of the most probable regulators of saliva production and secretion, during the establishment of the functional submandibular salivary gland.
MeSH Terms
- Aging
- Amino Acid Sequence
- Animals
- Aquaporins
- Biological Transport, Active
- Blotting, Northern
- Body Water
- Female
- Immunohistochemistry
- Molecular Sequence Data
- Pregnancy
- RNA
- RNA, Antisense
- Rats
- Rats, Sprague-Dawley
- Reverse Transcriptase Polymerase Chain Reaction
- Sodium-Potassium-Exchanging ATPase
- Submandibular Gland
Senescent female WAG/Rij rats exhibit polyuria without obvious renal disease or defects in vasopressin plasma level or V(2) receptor mRNA expression. Normalization of urine flow rate by 1-desamino-8-d-arginine vasopressin (dDAVP) was investigated in these animals. Long-term dDAVP infusion into 30-mo-old rats reduced urine flow rate and increased urine osmolality to levels comparable to those in control 10-mo-old rats. The maximal urine osmolality in aging rat kidney was, however, lower than that in adult kidney, despite supramaximal administration of dDAVP. This improvement involved increased inner medullary osmolality and urea sequestration. This may result from upregulation of UT-A1, the vasopressin-regulated urea transporter, in initial inner medullary collecting duct (IMCD), but not in terminal IMCD, where UT-A1 remained low. Expression of UT-A2, which contributes to medullary urea recycling, was greatly increased. Regulation of IMCD aquaporin (AQP)-2 (AQP2) expression by dDAVP differed between adult and senescent rats: the low AQP2 abundance in senescent rats was normalized by dDAVP infusion, which also improved targeting of the channel; in adult rats, AQP2 expression was unaltered, suggesting that IMCD AQP2 expression is not regulated by dDAVP directly. Increased AQP3 expression in senescent rats may also be involved in improved urine-concentrating capacity owing to higher basolateral water and urea reabsorption capacity.
MeSH Terms
- Aging
- Animals
- Aquaporin 2
- Aquaporin 3
- Aquaporin 6
- Aquaporins
- Carrier Proteins
- Corticosterone
- Deamino Arginine Vasopressin
- Female
- Gene Expression
- Kidney Medulla
- Membrane Glycoproteins
- Membrane Transport Proteins
- Nitric Oxide Synthase
- Osmolar Concentration
- Polyuria
- Rats
- Rats, Inbred Strains
- Renal Agents
- Urea
- Urine
- Water-Electrolyte Balance
The mechanisms underlying the prevention of age-related polyuria by chronic food restriction were investigated in female WAG/Rij rats. The decreased osmolality of renal papilla observed in senescent rats was not corrected by food restriction. A reduced urea content in the inner medulla of senescent rats, fed ad libitum or food-restricted, was suggested by the marked decrease in expression of UT-A1 and UT-B1 urea transporters. Aquaporin-2 (AQP2) downregulation in the inner medulla of senescent rats was partially prevented by food restriction. Both AQP2 and the phosphorylated form of AQP2 (p-AQP2), the presence of which was diffuse within the cytoplasm of collecting duct principal cells in normally fed senescent rats, were preferentially targeted at the apical region of the cells in food-restricted senescent animals. Plasma vasopressin (AVP) was similar in 10- and 30-mo-old rats fed ad libitum, but was doubled in food-restricted 30-mo-old rats. This study indicates that 1) kidney aging is associated with a marked decrease in AQP2, UT-A1, and UT-B1 expression in the inner medulla and a reduced papillary osmolality; and 2) the prevention of age-related polyuria by chronic food restriction occurs through an improved recruitment of AQP2 and p-AQP2 to the apical membrane in inner medulla principal cells, permitted by increased plasma AVP concentration.
MeSH Terms
- Aging
- Animals
- Aquaporin 2
- Aquaporin 6
- Aquaporins
- Carrier Proteins
- Cell Polarity
- Eating
- Female
- Kidney
- Kidney Medulla
- Kidney Tubules
- Membrane Glycoproteins
- Membrane Transport Proteins
- Osmolar Concentration
- Phosphorylation
- Polyuria
- Protein Transport
- Rats
- Urine
- Vasopressins
- Water-Electrolyte Imbalance
Fluid movement across epithelia lining portions of the male reproductive tract is important for modulating the luminal environment in which sperm mature and reside, and for increasing sperm concentration. Some regions of the male reproductive tract express aquaporin (AQP) 1 and/or AQP2, but these transmembrane water channels are not detectable in the epididymis. Therefore, we used a specific antibody to map the cellular distribution of another AQP, AQP9 (which is permeable to water and to some solutes), in the male reproductive tract. AQP9 is enriched on the apical (but not basolateral) membrane of nonciliated cells in the efferent duct and principal cells of the epididymis (rat and human) and vas deferens, where it could play a role in fluid reabsorption. Western blotting revealed a strong 30-kDa band in brush-border membrane vesicles isolated from the epididymis. AQP9 is also expressed in epithelial cells of the prostate and coagulating gland where fluid transport across the epithelium is important for secretory activity. However, it was undetectable in the seminal vesicle, suggesting that an alternative fluid transport pathway may be present in this tissue. Intracellular vesicles in epithelial cells along the reproductive tract were generally poorly stained for AQP9. Furthermore, the apical membrane distribution of AQP9 was unaffected by microtubule disruption. These data suggest that AQP9 is a constitutively inserted apical membrane protein and that its cell-surface expression is not acutely regulated by vesicular trafficking. AQP9 was detectable in the epididymis and vas deferens of 1-wk postnatal rats, but its expression was comparable with adult rats only after 3--4 wk. AQP9 could provide a route via which apical fluid and solute transport occurs in several regions of the male reproductive tract. The heterogeneous and segment-specific expression of AQP9 and other aquaporins along the male reproductive tract shown in this and in our previous studies suggests that fluid reabsorption and secretion in these tissues could be locally modulated by physiological regulation of AQP expression and/or function.
MeSH Terms
- Aging
- Animals
- Aquaporins
- Blotting, Western
- Colchicine
- Electrophoresis, Polyacrylamide Gel
- Epididymis
- Fluorescent Antibody Technique, Indirect
- Immunohistochemistry
- Kidney
- Male
- Microscopy, Immunoelectron
- Microvilli
- Rats
- Rats, Sprague-Dawley
- Vas Deferens
Despite chronic acidosis, collecting ducts in adult carbonic anhydrase II-deficient (CAD mice) are depleted of intercalated cells, including those of type A, which are acid-secreting cells. We hypothesized that this depletion could occur during postnatal development. Principal cells were identified by immunofluorescence using an antibody to rat aquaporin-2 (AQP-2), and type A intercalated cells using an antibody specific for anion exchanger (AE1). In CAD mice the proportion of AQP2-positive cells, normal at 11 days, increased progressively in the cortical (CCD) and outer medullary collecting duct (OMCD), to reach almost 100% in the OMCD in adults. The percentage of AE1-positive cells in the OMCD of CAD mice decreased by half by 6 weeks of age and further by adulthood. In controls, however, the proportion of AQP2-positive cells and that of AE1-positive cells in the OMCD remained stable after 10 days of age. AE1-positive cells accounted for the majority of intercalated cells in the OMCD. The mechanisms leading to selective postnatal cell depletion in the collecting duct in CAD mice remain to be determined.
MeSH Terms
- Aging
- Animals
- Animals, Newborn
- Antiporters
- Aquaporin 2
- Aquaporin 6
- Aquaporins
- Carbonic Anhydrases
- Chloride-Bicarbonate Antiporters
- Fluorescent Antibody Technique
- Heterozygote
- Kidney Tubules, Collecting
- Male
- Mice
- Microscopy, Fluorescence
The mechanisms underlying age-related polyuria were investigated in 10- and 30-mo-old female WAG/Rij rats. Urinary volume and osmolality were 3.9 /- 0.3 ml/24 h and 2,511 /- 54 mosmol/kgH(2)O in adult rats and 12.8 /- 0.8 ml/24 h and 1,042 /- 44 mosmol/kgH(2)O in senescent animals. Vasopressin V(2) receptor mRNA did not significantly differ between 10 and 30 mo, and [(3)H]vasopressin binding sites in membrane papilla were reduced by 30%. The cAMP content of the papilla was unchanged with age, whereas papillary osmolality was significantly lowered in senescent animals. The expression of aquaporin-1 (AQP1) and -4 was mostly unaltered from 10 to 30 mo. In contrast, aquaporin-2 (AQP2) and -3 (AQP3) expression was downregulated by 80 and 50%, respectively, and AQP2 was markedly redistributed into the intracellular compartment, in inner medulla of senescent animals, but not in renal cortex. These results indicate that age-related polyuria is associated with a downregulation of AQP2 and AQP3 expression in the medullary collecting duct, which is independent of vasopressin-mediated cAMP accumulation.
MeSH Terms
- Aging
- Animals
- Aquaporin 2
- Aquaporin 3
- Aquaporin 6
- Aquaporins
- Binding Sites
- Body Weight
- Cell Membrane
- Cyclic AMP
- Down-Regulation
- Drinking
- Eating
- Female
- Fluorescent Antibody Technique, Indirect
- Kidney
- Kidney Medulla
- Kidney Tubules
- Osmolar Concentration
- Polyuria
- RNA, Messenger
- Rats
- Rats, Inbred Strains
- Receptors, Vasopressin
- Vasopressins
It is well known that urine-concentrating ability decreases with aging and that this decreasing ability results from a reduced sensitivity of the renal collecting duct to arginine vasopressin (AVP). AVP regulates the water channel (aquaporin-2:AQP2) through V2 receptors and increases the water permeability of the collecting duct. To elucidate the mechanism of change with aging in urine-concentrating ability, we investigated the change of V2 receptor and AQP2 mRNA expression in young (8-week-old) and older (7-month-old) rats after dehydration for 2 days. After dehydration, plasma AVP levels in older rats were higher than young rats, and urinary osmolality in older rats was lower than young rats. By Northern blot analysis, there was no significant difference between young and older rats in both V2 receptor and AQP2 mRNA expression before dehydration. After dehydration, V2 receptor mRNA expression in young and older rats decreased in the same degree, suggesting the downregulation of V2 receptors may occur in the mRNA level. Northern blot analysis and in situ hybridization histochemistry showed that AQP2 mRNA expression increased and the increased expression in older rats was less than in young rats. The present study suggests the reduced response of AQP2 mRNA expression to dehydration, observed in 7-month-old rats, might be partially responsible for the decreasing urine-concentrating ability with aging.
MeSH Terms
- Aging
- Animals
- Aquaporin 2
- Aquaporin 6
- Aquaporins
- Dehydration
- Gene Expression Regulation
- Ion Channels
- Rats
The mRNA expression and localization of the aquaporin (AQP) family in rat kidney were examined by ribonuclease protection assay and immunohistochemistry. AQP1, AQP2, AQP3, and AQP4 mRNA were hardly detectable in 16-day gestation fetuses. AQP1 mRNA was explosively expressed at 1 wk, keeping the level throughout life. AQP2 mRNA expression was apparently noticed in 18-day fetuses and was enhanced gradually with age to reach a plateau at 4 wk. AQP3 and AQP4 mRNA expression was significantly found at birth but was not changed remarkably thereafter. AQP2 protein appeared first at the apical side of collecting duct cells in 18-day fetuses. The staining intensity at the site increased with age, and basolateral staining was added in adult rats. AQP3 was distinctly demonstrated at the basolateral side of collecting duct cells after birth, and the staining intensity was almost stable throughout life. The progressive induction of AQP2 expression in the first 4 wk after birth is presumed to contribute to the maturation of urinary concentrating capacity during the kidney development.
MeSH Terms
- Aging
- Animals
- Animals, Newborn
- Embryo, Mammalian
- Embryonic and Fetal Development
- Immunohistochemistry
- Ion Channels
- Kidney
- RNA, Messenger
- Rats
- Rats, Inbred WKY
- Water
Aquaporin-1 (AQP1), located in proximal tubules (PT) and descending thin limbs of Henle (DTL), and aquaporin-2 (AQP2), located in collecting ducts (CD), are channels involved in water transport across renal tubule epithelia. Using antibodies against AQP1 and AQP2, we here show expression of AQP1 and AQP2 in normal human developing and adult kidneys and in autosomal dominant polycystic kidney disease (ADPKD). Unlike in rats, AQP1 and AQP2 are expressed early during human nephrogenesis (12-wk gestation). AQP1 was first seen in developing PT epithelia, predominantly in apical cell membranes, and, at 15 wk, was also detected in DTL. AQP2 was seen in apical cell membranes of the branching ureteric bud and CD system from 12 wk and throughout development. In adult normal kidneys, AQP1 was localized to apical and basolateral membrane domains of PT and DTL, whereas AQP2 was restricted to principal cells of CD. This distribution of AQP1 and AQP2 was also seen in early stage ADPKD, except that AQP1 was mostly located in the apical membrane region of expanded PT. In end-stage ADPKD, two-thirds of the cysts expressed either AQP1 or AQP2, but these two water channels were never colocalized in the same cyst. Western blot analysis showed maximal expression of AQP1 and AQP2 in normal adult kidneys, lower levels in fetal kidneys, and decreases associated with degree of cystic progression in ADPKD. These data 1) demonstrate specific, mutually exclusive localization of AQP1 and AQP2 in human fetal and adult kidneys; 2) show that both channels are expressed early during nephrogenesis; and 3) show that the mutual exclusivity of localization is maintained even into end-stage ADPKD.
MeSH Terms
- Aging
- Amino Acid Sequence
- Animals
- Animals, Newborn
- Aquaporin 1
- Aquaporin 2
- Aquaporin 6
- Aquaporins
- Blood Group Antigens
- Blotting, Western
- Embryonic and Fetal Development
- Humans
- Immunohistochemistry
- Ion Channels
- Kidney
- Molecular Sequence Data
- Polycystic Kidney, Autosomal Dominant
- Rats
- Reference Values
- Staining and Labeling