Expression Levels Of Aquaporin-2 Research Articles

The relation between autophagy modulation by intermittent fasting and aquaporin 2 expression in experimentally induced diabetic nephropathy in albino rat

Polyuria is an early sign of diabetic nephropathy (DN) that produces dehydration in diabetic patients. This could be caused by alteration of renal aquaporin 2 (AQP2) expression. This study aimed to describe the relation between autophagy modulation via intermittent fasting (IF) and renal AQP2 expression and polyuria in case of DN. We divided the rats into control, DN and IF groups. After 2 and 4 weeks of diabetes induction, blood glucose (BG), serum creatinine (Scr), urine volume, and 24 hours urine protein (UP) were examined. Diabetic nephropathy histopathological index (DNHI) was calculated to evaluate histopathological changes. Immunohistochemistry and real-time PCR were performed to measure the levels of AQP2 and the autophagy marker; LC3 in kidney tissue. DNHI was correlated to the PCR and immunoexpression of AQP2 and LC3. Intermittent fasting significantly decreased the BG, Scr, urine volume, 24 hours UP, and DNHI as compared diabetes. Diabetes significantly elevated the immunoreactivity and mRNA expression levels of AQP2 and LC3 as compared to the control. However, the IF decreased AQP2 and stimulated autophagy in cyclic fashion. Our data revealed significant positive correlations between AQP2 and LC3 at the level of immunoexpression and mRNA at 2nd weeks. Taken together, these data showed that autophagy stimulation didn’t regulate AQP2 expression in case of diabetic nephropathy, however IF decreased polyuria through improvement of glycemic state.

TNF inhibits AQP2 expression via a miR137-dependent pathway.

As miR-137 is a regulator of aquaporin (AQP)2 expression and tumor necrosis factor (TNF) inhibits the expression of several extrarenal AQPs, we tested the hypothesis that TNF inhibits AQP2 in the kidney via a miR-137-dependent mechanism. AQP2 mRNA and protein expression decreased ∼70% and 53%, respectively, in primary renal inner medullary collecting duct (IMCD) cells transfected with a miRNA mimic of mmu-miR-137, suggesting that miR-137 directly targets AQP2 mRNA in these cells. Exposure of IMCD cells for 2 h to 400 mosmol/kgH2O medium increased mmu-miR-137 mRNA expression about twofold, conditions that also increased TNF production approximately fourfold. To determine if the increase in mmu-miR-137 mRNA expression was related to the concomitant increase in TNF, IMCD cells were transfected with a lentivirus construct to silence TNF. This construct decreased mmu-miR-137 mRNA expression by ∼63%, suggesting that TNF upregulates the expression of miR-137. Levels of miR-137 also increased approximately twofold in IMCD tubules isolated from male mice given 1% NaCl in the drinking water for 3 days. Intrarenal lentivirus silencing of TNF increased AQP2 mRNA levels and protein expression concomitant with a decrease in miR-137 levels in tubules isolated from mice given NaCl. The changes in AQP2 expression levels affected the diluting ability of the kidney, which was assessed by measuring urine osmolality and urine volume, as the decrease in these parameters after renal silencing of TNF was prevented on intrarenal administration of miR-137. The study reveals a novel TNF function via a miR-137-dependent mechanism that regulates AQP2 expression and function.NEW & NOTEWORTHY An emerging intratubular tumor necrosis factor system, functioning during normotensive noninflammatory conditions, acts as a breaking mechanism that attenuates both the increases in Na+-K+-2Cl- cotransporter and aquaporin-2 induced by arginine vasopressin, thereby contributing to the regulation of electrolyte balance and blood pressure. A greater appreciation for the role of cytokines as mediators of immunophysiological responses may help reveal the relationship between the immune system and other physiological systems.

Differential expression and inhibitory effects of aquaporins on the development of adenomyosis.

Aquaporin 2 (AQP2), AQP5 and AQP8 participate in adenomyosis (AM). Ηowever, the roles of these three molecules in AM have not been fully elucidated. In the present study, Institute of Cancer Research female mice were used to establish a model of AM. Subsequently, the endometrial tissues of the mice were observed by hematoxylin-eosin staining, and AM severity, uterus diameter, uterus index, ovary index and numbers of nodules on the uterine surface were evaluated and counted. In addition, eutopic and ectopic endometrial stromal cells (ESCs) were isolated from eutopic and ectopic endometrial samples derived from patients with AM and were then identified by immunofluorescence. The viability, and migratory and invasive ability of ESCs transfected with small interfering RNA targeting AQP5 (siAQP5) were determined by Cell Counting Κit-8, scratch wound-healing and Transwell assays, respectively. Reverse transcription-quantitative polymerase chain reaction was performed to determine the mRNA expression levels of AQP5, epithelial-mesenchymal transition (EMT)-related genes (E-and N-cadherin), matrix metalloproteinase (MMP)-2 and −9. Protein expression levels of AQP2, AQP5, AQP8, E-, N-cadherin, MMP-2 and −9 were detected by western blotting. AM severity and uterus index were higher, and there were a greater number of nodules on the uterine surface in the AM group compared with the sham group. AQP2, AQP5 and AQP8 proteins were highly expressed in eutopic and ectopic endometrium of the AM group, and AQP5 was more highly expressed than AQP2 or AQP8. In addition, the data showed that Vimentin was positively expressed in ESCs, and that siAQP5 suppressed the mRNA expression levels of AQP5, cell viability, migration, invasion, EMT and MMP-2 and −9 expression in ESCs. In conclusion, AQP2, AQP5 and AQP8 were highly expressed in eutopic and ectopic endometrium. Notably, AQP5 silencing may suppress AM by inhibiting viability, migration, invasion, EMT, and MMP-2 and −9 expression in ESCs.

Effect of ephedrine hydrochloride on regulation of body fluid metabolism and AQP1 and AQP2 in a rabbit model of mechanical ventilation

Abstract Objective Ephedrine hydrochloride (EH) is a major component from Ephedra sinica STAPF, which is used as a traditional Chinese herbal medicine. This study was designed to investigate the effect of EH on water metabolism and further explore the relevant signaling pathway of body fluid regulation in “lung governing regulation of water passage” using a rabbit model of mechanical ventilation. The molecular mechanism of the EH effect in the kidney was also investigated. Methods Rabbits were randomly divided into a control group, model group, EH group, and dexmedetomidine hydrochloride (DH) group. Urine volume was measured by the intubation method and pathologic changes in lung and renal tissue were measured by hematoxylin and eosin staining. Nitric oxide (NO) production in lung, serum, and kidney were analyzed using chemical methods. An ELISA was used to analyze angiotensin II (Ang II), antidiuretic hormone (ADH), prostaglandin E2 (PGE2), atrial natriuretic peptide (ANP), and endothelin-1 (ET-1) levels in the lung, serum, and kidney. Aquaporin-1 (AQP1) and aquaporin-2 (AQP2) mRNA and protein expression in the kidney was determined using qRT-PCR and immunohistochemistry. Results EH significantly inhibited the decrease in the total urine volume in the third and fourth stages, and displayed significant regulatory effects on NO, Ang II, ADH, PGE2, ANP, and ET-1 in serum, lung, and renal tissues compared with the model group. In the kidney, AQP1 and AQP2 mRNA and protein expression in the EH group were remarkably down-regulated compared with the model group. Conclusion EH exerted a regulatory effect on water metabolism by diffusing the lung and increased urine volume in the rabbit model, which was consistent with the decrease in kidney AQP1 and AQP2 expression levels that led to an increase in urine volume. EH could assist with DH to exert a protective effect on the clinical application of mechanical ventilation.

AQP2 is regulated by estradiol in human endometrium and is associated with spheroid attachment invitro.

17β‑estradiol (E2) and aquaporin 2 (AQP2) are associated with endometrial receptivity, and E2 directly regulates AQP2 expression in endometrial cancer cells. The present study aimed to investigate the role of AQP2 in embryo implantation. Normal endometrial samples were collected at the Women's Hospital (Hangzhou, China) from women seeking invitro fertilization and embryo transfer; women with endometrial abnormalities were excluded from the study. Samples were categorized into early‑mid proliferative, late proliferative, early secretory, mid‑secretory and late secretory phase groups, according to the menstrual cycle. The mRNA and protein expression levels of AQP2 were assessed in normal human endometrium in response to E2 via reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. The effects of AQP2 on spheroid attachment were assessed using an invitro co‑culture assay with small interfering (si)RNA against AQP2. The highest expression levels of AQP2 were observed in the late proliferative and mid‑secretory phases, with the lowest levels detected in the early proliferative and late secretory phases. In addition, treatment with 10‑9 or 10‑7 M E2 for 24h upregulated AQP2 in the cultured endometrium. Knockdown of AQP2 by siRNA significantly decreased JAr spheroid attachment; however, this effect was significantly reversed when AQP2 siRNA‑transfected cells were treated with 10‑7 M E2. The results of the present study suggested that AQP2 expression levels in human endometrium may be mediated by estrogen, and low AQP2 expression levels may be a potential cause of impaired uterine receptivity.

A versatile aquaporin-2 cell system for quantitative temporal expression and live cell imaging.

Aquaporin-2 (AQP2) fine tunes urine concentration in response to the antidiuretic hormone vasopressin. In addition, AQP2 has been suggested to promote cell migration and epithelial morphogenesis. A cell system allowing temporal and quantitative control of expression levels of AQP2 and phospho-mimicking mutants has been missing, as has a system allowing expression of fluorescently tagged AQP2 for time-lapse imaging. In the present study, we generated and validated a Flp-In T-REx Madin-Darby canine kidney cell system for temporal and quantitative control of AQP2 and phospho-mimicking mutants. We verified that expression levels can be temporally and quantitatively controlled and that AQP2 translocated to the plasma membrane in response to elevated cAMP, which also induced S256 phosphorylation. The phospho-mimicking mutants AQP2-S256A and AQP2-S256D localized as previously described, primarily intracellular and to the plasma membrane, respectively. Induction of AQP2 expression in combination with transient, low expression of enhanced green fluorescent protein-tagged AQP2 enabled expression without aggregation and correct translocation in response to elevated cAMP. Interestingly, time-lapse imaging revealed AQP2-containing tubulating endosomes and that tubulation significantly decreased 30 min after cAMP elevation. This was mirrored by the phospho-mimicking mutants AQP2-S256A and AQP2-S256D, where AQP2-S256A-containing endosomes tubulated, whereas AQP2-S256D-containing endosomes did not. Thus, this cell system enables a multitude of cell-based assays warranted to provide deeper insights into the mechanisms of AQP2 regulation and effects on cell migration and epithelial morphogenesis.

Estrogen nuclear receptors affect cell migration by altering sublocalization of AQP2 in glioma cell lines

Glioblastomas are capable of infiltrating into neighboring brain tissues. The prognosis of a male patient is worse than that of women. Here, we demonstrate the effects of estrogen on invasion of glioma cells via regulating estrogen nuclear receptors (ERα and ERβ) combined with aquaporin 2 (AQP2). In our study, we conclude that AQP2 was located mainly in the nuclei of the glioma cell lines and is capable of inhibiting cell invasion. According to the gene ontology analysis, out of 138 screened genes, three genes of ankyrin repeat and FYVE domain containing 1 (ANKFY1), lymphocyte transmembrane adaptor 1 (LAX1), and latent transforming growth factor beta-binding protein 1 (LTBP1) were found to be regulating the ERα and ERβ. The expression of ERα was found to be high, whereas the expression of both ERβ and AQP2 was low in glioma cells from patient tissues and glioblastoma cell lines. The expression levels of AQP2, ANKFY1, LAX1, and LTBP1 were upregulated by both ERα small interfering RNA (siRNA) and overexpression of ERβ. AQP2 inhibition of cell invasion was inversely influenced by LAX1siRNA. The luciferase report system indicated that AQP2 promoted the transcriptional activity of LAX1 and inhibited cell invasion. These data suggest that ERβ may function as AQP promoter in the nucleus to sustain cells' stability by promoting AQP production, while ERα acts as an antagonist of AQP2. The ratio between ERα and ERβ is likely to affect the distribution of AQP2 in the nucleus. Low level of ERβ reduces the inhibition of invasion of glioma cells influenced by high level of LAX1 expression, leading to an increase in the invasion ability of glioma cells.

A novel therapeutic effect of statins on nephrogenic diabetes insipidus.

Statins competitively inhibit hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase, resulting in reduced plasma total and low-density lipoprotein cholesterol levels. Recently, it has been shown that statins exert additional ‘pleiotropic’ effects by increasing expression levels of the membrane water channels aquaporin 2 (AQP2). AQP2 is localized mainly in the kidney and plays a critical role in determining cellular water content. This additional effect is independent of cholesterol homoeostasis, and depends on depletion of mevalonate-derived intermediates of sterol synthetic pathways, i.e. farnesylpyrophosphate and geranylgeranylpyrophosphate. By up-regulating the expression levels of AQP2, statins increase water reabsorption by the kidney, thus opening up a new avenue in treating patients with nephrogenic diabetes insipidus (NDI), a hereditary disease that yet lacks high-powered and limited side effects therapy. Aspects related to water balance determined by AQP2 in the kidney, as well as standard and novel therapeutic strategies of NDI are discussed.

Salt-induced downregulation of renal aquaporins is prevented by losartan

AimsThe purpose of this study was to investigate the expression of aquaporin-1 (AQP-1) and aquaporin-2 (AQP-2) in the renal tubule of rats fed with a high-salt diet and its modulation by the AT1 receptor blocker losartan. Main methodsThe experiments were performed in four groups of rats fed for 3weeks with the following diets: regular rat chow (NS); high-salt (8% NaCl) chow (HS), NS plus losartan (NS-L) and HS plus losartan (HS-L). Losartan (40mg.kg−1) was administered in the drinking water. Systolic blood pressure (SBP) and renal function were evaluated. The intrarenal levels of angiotensin II (Ang II), TGF-β1, α-smooth muscle actin (α-SMA), endothelial nitric oxide synthase (eNOS), AQP-1 and AQP-2 were determined by immunohistochemistry. AQP-1 and AQP-2 protein levels were measured by western blot analysis. Key findingsA high-sodium diet downregulated AQP-1 and AQP-2 expression levels in the proximal tubule and collecting duct, respectively. The high-sodium diet also induced Ang II, TGF-β1 and α-SMA overexpression and decreased eNOS expression in the renal cortex and medulla. Losartan increased the diuresis and natriuresis, favoring urinary sodium concentration. Additionally, losartan prevented the profibrogenic response, decreasing Ang II, TGF-β1 and α-SMA levels and normalizing AQP-2 expression in the HS-L group. AQP-1 expression was upregulated by losartan in both the NS-L and HS-L groups. SignificanceThese results show that increased intrarenal Ang II in rats fed with a high-salt diet downregulates renal AQP-1 and AQP-2 expressions. In addition, although losartan increased diuresis and natriuresis, it prevented the downregulation of aquaporins, favoring urinary sodium concentration.

AQP2 is necessary for vasopressin- and forskolin-mediated filamentous actin depolymerization in renal epithelial cells

SummaryRemodeling of the actin cytoskeleton is required for vasopressin (VP)-induced aquaporin 2 (AQP2) trafficking. Here, we asked whether VP and forskolin (FK)-mediated F-actin depolymerization depends on AQP2 expression. Using various MDCK and LLC-PK1 cell lines with different AQP2 expression levels, we performed F-actin quantification and immunofluorescence staining after VP/FK treatment. In MDCK cells, in which AQP2 is delivered apically, VP/FK mediated F-actin depolymerization was significantly correlated with AQP2 expression levels. A decrease of apical membrane associated F-actin was observed upon VP/FK treatment in AQP2 transfected, but not in untransfected cells. There was no change in basolateral actin staining under these conditions. In LLC-PK1 cells, which deliver AQP2 basolaterally, a significant VP/FK mediated decrease in F-actin was also detected only in AQP2 transfected cells. This depolymerization response to VP/FK was significantly reduced by siRNA knockdown of AQP2. By immunofluorescence, an inverse relationship between plasma membrane AQP2 and membrane-associated F-actin was observed after VP/FK treatment again only in AQP2 transfected cells. This is the first report showing that VP/FK mediated F-actin depolymerization is dependent on AQP2 protein expression in renal epithelial cells, and that this is not dependent on the polarity of AQP2 membrane insertion.

The role of renal aquaporin 2 in the alleviation of dehydration associated with diabetic polyuria in KKAy mice

Aims Polyuria is a symptom that appears in association with diabetes mellitus. Because sustained polyuria causes serious dehydration, it is believed that the body has a compensating mechanism to alleviate dehydration. In the present study, the role of renal aquaporin 2 (AQP2) in the compensating mechanism was investigated in KKAy mice, a type 2 diabetes model. Main methods The renal AQP2 expression levels in KKAy mice aged between 5 and 24 weeks were determined using Western blotting. The hypothalamic vasopressin mRNA expression levels also were measured by real-time RT-PCR. Insulin was subcutaneously administered to 11-week-old KKAy mice twice a day for 7 days. After insulin treatment, the renal AQP2 protein expression and the hypothalamic vasopressin mRNA expression were measured. Key findings The urinary volumes of 5- and 12-week-old KKAy mice were 1.5 ± 0.3 mL and 9.5 ± 1.2 mL, respectively. The inner medullary AQP2 protein expression of 12-week-old KKAy mice was approximately 2.5-fold higher than that of 5-week-old KKAy mice. The hypothalamic vasopressin mRNA expression of 12-week-old KKAy mice was approximately twice that of 5-week-old KKAy mice. Insulin treatment in KKAy mice resulted in a significant reduction in the plasma glucose level, urinary volume, and inner medullary AQP2 protein and hypothalamic vasopressin mRNA expression. Significance The present study demonstrated that AQP2 is a renal functional molecule of vasopressin that controls urinary volume and that AQP2 in the kidney increases to alleviate dehydration due to type 2 diabetes with polyuria.

Increases in the Expression Levels of Aquaporin-2 and Aquaporin-3 in the Renal Collecting Tubules Alleviate Dehydration Associated with Polyuria in Diabetes Mellitus

Enhanced expression of renal aquaporin-2 (AQP2) has been reported when polyuria occurs in diabetic animal models. The purpose of this study was to clarify the possibility that increased AQP2 expression in the kidneys play a role as a compensatory mechanism to alleviate diabetic dehydration. Lithium carbonate (Li₂CO₃), which decreases the renal expression of AQPs, was administered to streptozotocin (STZ)-induced model mice of type I diabetes mellitus (STZ mice), to investigate the relationship between urine volume and renal AQP expression. Plasma glucose and urine glucose levels were similar between STZ mice given feed containing Li₂CO₃ for 10 d and un-treated STZ mice. Urine volume increased to 70 ml/d for the Li₂CO₃-treated STZ mice, compared to 36 ml/d for un-treated STZ mice. No changes were observed in creatinine clearance or the mRNA expression levels of sodium myo-inositol transporter and taurine transporter, which are genes associated with the regulation of osmotic pressure in the kidney, in the Li₂CO₃-treated STZ mice relative to un-treated STZ mice. Protein expression levels of AQP2 and aquaporin-3 (AQP3) of the renal inner medulla were significantly decreased in the Li₂CO₃-treated STZ mice, compared to levels in the STZ group. This study revealed that the decreased expression levels of AQP2 and AQP3 in the kidney increased the urine volume in mice without a change in urinary osmotic pressure. The results of this study suggest that the increased renal AQP2 and AQP3 expression, in the setting of polyuria, physiologically serves as a compensatory mechanism to alleviate dehydration in diabetes mellitus.

Vasopressin‐dependent upregulation of aquaporin‐2 gene expression in aged rats with glucocorticoid deficiency

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.

Different effects of Cyclosporine‐A and FK506 on aquaporin‐2 expression

Immunosuppressive drugs like Cyclosporine‐A (CyA) and FK506, both calcineurin inhibitors, are used to silence the immune system after transplantation. Both drugs have often complex side effects. For example long term treatment with CyA is followed by down‐regulation of aquaporin‐2 (AQP2) at least in the rat kidney. To investigate if CyA directly influences the expression of AQP2 we used primary cultured rat inner medullary collecting duct cells (IMCD), which endogenously express AQP2 and AVP‐mediated regulation of AQP2. IMCD‐cells were treated for different time‐periods with different concentrations of CyA or FK506. We used real‐time PCR to analyze mRNA expression levels of AQP2. Treatment with 0.5 μM, 1 μM or 5 μM CyA for 30 min and for 48 h was followed by concentration and time dependent down‐regulation of AQP2 expression. In contrast treatment with FK506 (1 μM, 30 min and 48 h) showed no effect on AQP2 expression. CyA had no effect on expression and activity of the tonicity element binding protein (TonEBP), a factor involved in AQP2 mRNA expression. But CyA suppressed also the cAMP induced expression of AQP2. In conclusion our results showed that CyA directly down‐regulates the expression of AQP2 in IMCD cells. Our data indicate that the underlying mechanism is calcineurin and TonEBP independent and that the down‐regulation of AQP2 is due to a disturbance of cAMP signaling induced by CyA.

Upregulation of collecting duct aquaporin-2 by metabolic acidosis: role of vasopressin.

Metabolic acidosis is associated with alteration in fluid and electrolyte reabsorption in a number of nephron segments. However, the effects of metabolic acidosis on urine osmolality and aquaporin-2 (AQP-2) remain poorly understood. In these studies, we examined the effects of chronic metabolic acidosis on water handling by the kidney. Rats were placed in metabolic cages and subjected to water (control) or 280 mM NH(4)Cl loading for 120 h to induce metabolic acidosis. The results indicated a significant increase in urine osmolality with no change in urine volume or urinary Na(+) excretion in acid-loaded animals. This effect was independent of alteration in fluid intake or salt/Cl(-) loading. Immunoblotting and Northern hybridization studies indicated that AQP-2 protein abundance and mRNA expression levels increased significantly along the collecting duct system of NH(4)Cl-but not NaCl-loaded animals. RIA results indicated that metabolic acidosis was associated with a fourfold increase in circulating levels of vasopressin (AVP) and a significant increase in brain AVP mRNA expression levels. In conclusion, metabolic acidosis upregulates the expression levels of AQP-2 and increases urine osmolality, suggesting an adaptive increase in water reabsorption in the collecting duct. A concomitant increase in AVP synthesis and secretion likely plays an essential role in the adaptation of AQP-2 in metabolic acidosis.

Hypertonicity Is Involved in Redirecting the Aquaporin-2 Water Channel into the Basolateral, Instead of the Apical, Plasma Membrane of Renal Epithelial Cells

In renal collecting ducts, vasopressin increases the expression of and redistributes aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical membrane, leading to urine concentration. However, basolateral membrane expression of AQP2, in addition to AQP3 and AQP4, is often detected in inner medullary principal cells in vivo. Here, potential mechanisms that regulate apical versus basolateral targeting of AQP2 were examined. The lack of AQP2-4 association into heterotetramers and the complete apical expression of AQP2 when highly expressed in Madin-Darby canine kidney cells indicated that neither heterotetramerization of AQP2 with AQP3 and/or AQP4, nor high expression levels of AQP2 explained the basolateral AQP2 localization. However, long term hypertonicity, a feature of the inner medullary interstitium, resulted in an insertion of AQP2 into the basolateral membrane of Madin-Darby canine kidney cells after acute forskolin stimulation. Similarly, a marked insertion of AQP2 into the basolateral membrane of principal cells was observed in the distal inner medulla from normal rats and Brattleboro rats after acute vasopressin treatment of tissue slices that had been chronically treated with vasopressin to increase interstitial osmolality in the medulla, but not in tissues from vasopressin-deficient Brattleboro rats. These data reveal for the first time that chronic hypertonicity can program cells in vitro and in vivo to change the insertion of a protein into the basolateral membrane instead of the apical membrane.