Aquaporin-2 mRNA Levels Research Articles

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.

TNF inhibits AQP2 expression via a miR137-dependent pathway

We previously showed that production of tumor necrosis factor-alpha (TNF) by the kidney, under normotensive, non-inflammatory conditions, inhibits expression of angiotensinogen in the proximal tubule and NKCC2 isoform A in the thick ascending limb of Henle’s loop via micro-RNA (miRNA)-dependent mechanisms. As miR-137 is a regulator of aquaporin-2 (AQP2) expression and TNF inhibits the expression of several extrarenal aquaporins, we tested the hypothesis that TNF inhibits AQP2 in the kidney via a miR-137-dependent mechanism. Target seed regions of mmu-miR-137 are conserved in the 3'-untranslated region (3’-UTR) of mouse AQP2 mRNA and luciferase assays showed that mmu-miR-137 directly targeted the 3’-UTR of mouse AQP2. Moreover, AQP2 expression was decreased by approximately 70% (P<0.05) in primary renal inner medullary collecting duct (IMCD) cells transfected with a miRNA mimic of mmu-miR-137, indicating that miR-137 directly targets AQP2 mRNA in these cells. The decrease in AQP2 mRNA accumulation was accompanied by a 53% decrease (P<0.05) in AQP2 protein expression when mmu-miR-137 was overexpressed in IMCD cells. Exposure of IMCD cells for 2 hr to 400 mosmol/kg H2O medium, made by increasing the osmolality by adding NaCl, increased mmu-miR-137 mRNA expression about 2-fold (P<0.05) as determined by quantitative RT-PCR analysis. These conditions also increased TNF production approximately 2-fold (P<0.05). To determine if the increase in mmu-miR-137 mRNA expression was related to the concomitant increase in TNF, cells were transfected with either a lentivirus construct to silence TNF or a control construct. The silencing construct, EGFP-shTNF-ex4, but not U6 control, decreased mmu-miR-137 mRNA expression in the IMCD cells exposed to 400 mosmol/kg H2O medium by approximately 63% (P<0.05) indicating that TNF upregulates mmu-miR-137 mRNA expression in IMCD cells. The levels of miR-137 also increased approximately 2-fold (P<0.05) in inner medulla isolated from age- and sex-matched male or female mice given 1% NaCl in the drinking water (HS) for 3 days. Six days after intrarenal injection of lentivirus EGFP-shTNF-ex4 to silence TNF, renal AQP2 mRNA levels increased approximately 3-fold (P<0.05) in inner medullary tissue isolated from male or female mice given HS compared with their respective control groups. In addition, renal silencing of TNF in male or female mice given HS exhibited an increase in AQP2 protein expression the inner medulla compared with control group as determined by Western blot analysis. Similarly, EGFP-shTNF-ex4, but not U6, decreased miR-137 levels in the inner medulla in male or female mice given HS. Taken together, these data reveal a novel mechanism by which TNF mediates the inhibition of AQP2 via miR-137, indicating that miR-137 may be important for studying TNF-regulated effects involving the AQP2 pathway on renal water excretion and blood pressure regulation. NIH grants R01 HL133077 and HL153525 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

HDAC3 Inhibition Improved Urinary Concentrating Defect in Hypokalemia by Promoting AQP2 Transcription

Hypokalemia is a common electrolyte disturbance frequently encountered in clinical medicine. Animals with hypokalemia had a significant arginine vasopressin (AVP)‐resistant polyuria with reduced expression of aquaporin 2 (AQP2) levels in the collecting ducts of the kidneys. The precise molecular mechanisms of hypokalemia‐induced nephrogenic diabetes insipidus remain unresolved. AVP induced a marked increase in histone H3K27 acetylation (H3K27ac) across the body of the AQP2 gene, in the promoter and in upstream of the AQP2 transcriptional start site in mouse mpkCCD cell line (PMID: 29572403). The purpose of the present study was to investigate whether enhanced histone acetylation prevented decreased AQP2 expression and improved urinary concentrating defect in rats with hypokalemia. Male Wistar rats were fed potassium‐free diet with or without 4‐phenylbutyric acid (4‐PBA) or a selective inhibitor of histone deacetylase 3 (HDAC3) RGFP966 for 4 days. 4‐PBA or RGFP966 markedly increased AQP2 mRNA and protein levels in kidney inner medulla of rats with hypokalemia, which was associated with decreased urine output and increased urinary osmolality. Protein expression of H3K27ac was markedly decreased in kidney inner medulla of hypokalemic rats, which was significantly prevented by 4‐PBA or RGFP966. Expression of H3K27ac was decreased in primary cultured inner medullary collecting duct cells (IMCD) or mpkCCD cells cultured in potassium‐deprivation media, in particular, H3K27ac in the promoter region of AQP2 was markedly decreased, which was associated with reduced mRNA levels of AQP2. Protein expression of HDAC3 was significantly upregulated in both mpkCCD and IMCD cells in response to potassium deprivation; the binding of HDAC3 to the promoter of AQP2 was also dramatically increased. RGFP966 significantly increased expression of H3K27ac and AQP2 protein and enhanced the binding of H3K27ac to AQP2 in mpkCCD cells. In conclusion, HDAC3 inhibition prevented downregulation of AQP2 and improved urinary concentrating defect induced by potassium deprivation, likely through enhancing acetylation of H3K27 and transcription of AQP2.

Glucocorticoid Receptor Maintains Vasopressin‐Regulated Water Reabsorption Pathway in the Kidney Collecting Duct Cells

Aquaporin‐2 (AQP2) is a transmembrane protein expressed in the principal cells of the kidney collecting ducts, which mediate osmotic water reabsorption. AQP2 is regulated by the antidiuretic peptide hormone arginine vasopressin (AVP). When AVP binds to the vasopressin type 2 receptor (V2R) in the principal cells, it initiates a signaling network that results in 1) AQP2 trafficking to the apical plasma membrane for water transport and 2) AQP2 gene expression. In a previous study, we found that dexamethasone, a glucocorticoid receptor (GR) agonist, enhanced AVP‐induced AQP2 gene expression in the collecting duct cell model mpkCCD. Here, we showed that GR knockdown drastically decreased AVP‐induced AQP2 mRNA and protein levels, indicative of a role of GR in AVP‐induced AQP2 gene expression. We further showed that the 1,000bp region upstream to the AQP2 transcription start site was sufficient to mediate GR‐enhanced AVP‐induced promoter activity and that GR bound to the above AQP2 promoter region. RNA‐seq was used to profile the transcriptome of the GR knockdown vs. control cells. A total of 93,308 transcripts belonging to 22,025 genes were quantified. GR knockdown decreased expression of 5,266 genes and increased expression of 3,709 genes. These two gene groups respectively represent positive and negative regulators for the AVP responses. Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis showed 62 pathways associated with the positive regulators and 48 pathways associated with the negative regulators. The AVP‐regulated water reabsorption pathway was among the 62 positive pathways whose gene expression was reduced upon GR knockdown. Quantitative RT‐PCR analysis showed about 60% reduction in the V2R mRNA level upon GR knockdown. Taken together, our results implicated that GR in one hand potentiates AVP‐induced AQP2 gene expression and in the other hand maintains expression of genes involved in the AVP‐regulated water reabsorption pathway in the kidney collecting duct cells.Support or Funding InformationThis work was supported by the Ministry of Science and Technology, Taiwan (107‐2320‐B‐002‐057‐MY3).

Transcription Factor Elf3 Modulates Vasopressin-Induced Aquaporin-2 Gene Expression in Kidney Collecting Duct Cells.

Aquaporin-2 (AQP2) is a molecular water channel protein responsible for water reabsorption by the kidney collecting ducts. Many water balance disorders are associated with defects in AQP2 gene expression regulated by the peptide hormone vasopressin. Here, we studied roles of Elf3 (E26 transformation-specific (Ets)-related transcription factor 3) in AQP2 gene expression in the collecting duct cells (mpkCCD). Vasopressin increased AQP2 mRNA and protein levels without affecting AQP2 mRNA degradation, indicative of transcriptional regulation. Elf3 knockdown and overexpression, respectively, reduced and increased AQP2 gene expression under basal and vasopressin-stimulated conditions. However, the vasopressin-to-basal ratios of AQP2 gene expression levels remained constant, indicating that Elf3 does not directly mediate vasopressin response but modulates the level of AQP2 gene expression inducible by vasopressin. The Elf3-modulated AQP2 gene expression was associated with AQP2 promoter activity, in line with Elf3’s ability to bind an Ets element in the AQP2 promoter. Mutation in the Ets element reduced both basal and vasopressin-stimulated AQP2 promoter activity, again without affecting vasopressin-to-basal ratios of the AQP2 promoter activity. Lithium chloride reduced both Elf3 and AQP2 mRNA in the mpkCCD cells as well as in mouse kidney inner medulla. We conclude that Elf3 modulates AQP2 promoter activity thereby gauging vasopressin-inducible AQP2 gene expression levels. Our data provide a potential explanation to lithium-induced nephrogenic diabetes insipidus where lithium reduces Elf3 and hence AQP2 abundance.

The Deubiquitylase USP4 Interacts with the Water Channel AQP2 to Modulate Its Apical Membrane Accumulation and Cellular Abundance

Aquaporin 2 (AQP2) mediates the osmotic water permeability of the kidney collecting duct in response to arginine vasopressin (VP) and is essential for body water homeostasis. VP effects on AQP2 occur via long-term alterations in AQP2 abundance and short-term changes in AQP2 localization. Several of the effects of VP on AQP2 are dependent on AQP2 phosphorylation and ubiquitylation; post-translational modifications (PTM) that modulate AQP2 subcellular distribution and function. Although several protein kinases, phosphatases, and ubiquitin E3 ligases have been implicated in AQP2 PTM, how AQP2 is deubiquitylated or the role of deubiquitylases (DUBS) in AQP2 function is unknown. Here, we report a novel role of the ubiquitin-specific protease USP4 in modulating AQP2 function. USP4 co-localized with AQP2 in the mouse kidney, and in mpkCCD14 cells USP4 and AQP2 abundance are increased by VP. AQP2 and USP4 co-immunoprecipitated from mpkCCD14 cells and mouse kidney, and in vitro, USP4 can deubiquitylate AQP2. In mpkCCD14 cells, shRNA mediated knockdown of USP4 decreased AQP2 protein abundance, whereas no changes in AQP2 mRNA levels or VP-induced cAMP production were detected. VP-induced AQP2 membrane accumulation in knockdown cells was significantly reduced, which was associated with higher levels of ubiquitylated AQP2. AQP2 protein half-life was also significantly reduced in USP4 knockdown cells. Taken together, the data suggest that USP4 is a key regulator of AQP2 deubiquitylation and that loss of USP4 leads to increased AQP2 ubiquitylation, decreased AQP2 levels, and decreased cell surface AQP2 accumulation upon VP treatment. These studies have implications for understanding body water homeostasis.

α‐actinin 4 Knockdown Reduced Vasopressin‐Induced Aquaporin‐2 Expression in the Kidney Collecting Duct Cells

Vasopressin is a pituitary peptide hormone that regulates aquaporin‐2 (AQP2) gene expression in the kidney collecting duct principal cells. Dysregulation of vasopressin‐regulated AQP2 gene expression is associated with many water balance disorders. Previously in the collecting duct cell model (mpkCCD), we identified α actinin 4, a cytoskeletal actin‐binding protein that was reported to translocate into the nucleus and acts as a transcriptional co‐activator. Here, we found that short hairpin RNA (shRNA)‐mediated α actinin 4 knockdown in the mpkCCD cells reduced vasopressin‐induced AQP2 mRNA and protein levels, suggesting a role of α actinin 4 in vasopressin‐induced AQP2 gene expression. However, vasopressin did not induce nuclear translocation of α actinin 4, suggesting a different model of α actinin 4 action. α actinin 4 was reported as a transcriptional co‐activator of the glucocorticoid receptor whose agonist dexamethasone was shown to enhance vasopressin‐induced AQP2 gene expression in the mpkCCD cells in a dose‐ and time‐dependent manner. We thus tested whether α actinin 4 interacts with glucocorticoid receptor to enhance vasopressin‐induced AQP2 gene expression. In support of our hypothesis, shRNA‐mediated glucocorticoid receptor knockdown almost blunted vasopressin‐induced AQP2 mRNA and protein expression. Our results are consistent with a role of α actinin 4 in vasopressin‐induced AQP2 gene expression involving glucocorticoid receptor.Support or Funding InformationThis work was supported by Ministry of Science and Technology, Taiwan to MJY (MOST104‐2320‐B002‐064‐MY3)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Liver X receptor β increases aquaporin 2 protein level via a posttranscriptional mechanism in renal collecting ducts.

Liver X receptors (LXRs) including LXRα and LXRβ are nuclear receptor transcription factors and play an important role in lipid and glucose metabolism. It has been previously reported that mice lacking LXRβ but not LXRα develop a severe urine concentrating defect, likely via a central mechanism. Here we provide evidence that LXRβ regulates water homeostasis through increasing aquaporin 2 (AQP2) protein levels in renal collecting ducts. LXRβ-/- mice exhibited a reduced response to desmopressin (dDAVP) stimulation, suggesting that the diabetes insipidus phenotype is of both central and nephrogenic origin. AQP2 protein abundance in the renal inner medulla was significantly reduced in LXRβ-/- mice but with little change in AQP2 mRNA levels. In vitro studies showed that AQP2 protein levels were elevated upon LXR agonist treatment in both primary cultured mouse inner medullary duct cells (mIMCD) and the mIMCD3 cell line with stably expressed AQP2. In addition, LXR agonists including TO901317 and GW3965 failed to induce AQP2 gene transcription but diminished its protein ubiquitination in primary cultured mIMCD cells, thereby inhibiting its degradation. Moreover, LXR activation-induced AQP2 protein expression was abolished by the protease inhibitor MG132 and the ubiquitination-deficient AQP2 (K270R). Taken together, the present study demonstrates that activation of LXRβ increases AQP2 protein levels in the renal collecting ducts via a posttranscriptional mechanism. As such, LXRβ represents a key regulator of body water homeostasis.

Transcription Factor Elf3 Mediates Vasopressin‐Regulated Aquaporin‐2 Expression in Renal Collecting Duct Cells

Vasopressin is a pituitary peptide hormone responsible for aquaporin‐2 (AQP2) gene expression in the kidney collecting ducts. Low levels of AQP2 gene expression are associated with a number of water balance disorders including lithium induced‐nephrogenic diabetes insipidus; however, the molecular mechanisms are not fully understood. Here, we studied the roles of the ETS‐related transcription factor Elf3 in vasopressin‐mediated AQP2 gene expression. In the mouse collecting duct cell model mpkCCD, vasopressin increased AQP2 mRNA and protein levels in a time‐dependent manner. Stable Elf3 knockdown significantly reduced basal and vasopressin‐induced increases in AQP2 mRNA and protein levels. Overexpression of Elf3 isoform 1 or 2 enhanced basal and vasopressin‐induced AQP2 promoter activity and AQP2 mRNA levels. Vasopressin did not affect Elf3 mRNA levels but induced nuclear translocation of V5‐tagged Elf3 in the mpkCCD cells. Electrophoretic mobility shift assay showed that the V5‐tagged Elf3 interacted with the AQP2 promoter DNA and that the interaction was inhibited with a competitor AQP2 promoter DNA or an antibody against the V5‐tagged Elf3. Lithium reduced Elf3 mRNA level and reduced AQP2 mRNA and protein levels in the mpkCCD cells in the presence of vasopressin. Similar observations were made in the mice that manifested polyuria and polydipsia on a lithium diet for two weeks. Taken together, our data support a role of Elf3 in vasopressin‐mediated AQP2 gene expression and provide a potential explanation to lithium induced‐nephrogenic diabetes insipidus.Support or Funding InformationThis work was supported by Ministry of Science and Technology, Taiwan to MJY (101‐2320‐B‐002‐038‐MY3) and National Taiwan University Hospital to SLL (NTUH103‐S2405).

Disruption of prostaglandin E2 receptor EP4 impairs urinary concentration via decreasing aquaporin 2 in renal collecting ducts

The antidiuretic hormone arginine vasopressin is a systemic effector in urinary concentration. However, increasing evidence suggests that other locally produced factors may also play an important role in the regulation of water reabsorption in renal collecting ducts. Recently, prostaglandin E2 (PGE2) receptor EP4 has emerged as a potential therapeutic target for the treatment of nephrogenic diabetes insipidus, but the underlying mechanism is unknown. To evaluate the role of EP4 in regulating water homeostasis, mice with renal tubule-specific knockout of EP4 (Ksp-EP4(-/-)) and collecting duct-specific knockout of EP4 (AQP2-EP4(-/-)) were generated using the Cre-loxP recombination system. Urine concentrating defect was observed in both Ksp-EP4(-/-) and AQP2-EP4(-/-) mice. Decreased aquaporin 2 (AQP2) abundance and apical membrane targeting in renal collecting ducts were evident in Ksp-EP4(-/-) mice. In vitro studies demonstrated that AQP2 mRNA and protein levels were significantly up-regulated in mouse primary inner medullary collecting duct (IMCD) cells after pharmacological activation or adenovirus-mediated overexpression of EP4 in a cAMP/cAMP-response element binding protein-dependent manner. In addition, EP4 activation or overexpression also increased AQP2 membrane accumulation in a mouse IMCD cell line (IMCD3) stably transfected with the AQP2 gene, mainly through the cAMP/protein kinase A and extracellular signal-regulated kinase pathways. In summary, the EP4 receptor in renal collecting ducts plays an important role in regulating urinary concentration under physiological conditions. The ability of EP4 to promote AQP2 membrane targeting and increase AQP2 abundance makes it a potential therapeutic target for the treatment of clinical disorders including acquired and congenital diabetes insipidus.

Estradiol regulates AQP2 expression in the collecting duct: a novel inhibitory role for estrogen receptor α.

While there is evidence that sex hormones influence multiple systems involved in salt and water homeostasis, the question of whether sex hormones regulate aquaporin-2 (AQP2) and thus water handling by the collecting duct has been largely ignored. Accordingly, the present study investigated AQP2 expression, localization and renal water handling in intact and ovariectomized (OVX) female rats, with and without estradiol or progesterone replacement. OVX resulted in a significant increase in urine osmolality and increase in p256-AQP2 in the renal cortex at 7 days post-OVX, as well as induced body weight changes. Relative to OVX alone, estradiol repletion produced a significant increase in urine output, normalized urinary osmolality and reduced both total AQP2 (protein and mRNA) and p256-AQP2 expression, whereas progesterone repletion had little effect. Direct effects of estradiol on AQP2 mRNA and protein levels were further tested in vitro using the mpkCCD principal cell line. Estradiol treatment of mpkCCD cells reduced AQP2 at both the mRNA and protein level in the absence of deamino-8-d-AVP (dDAVP) and significantly blunted the dDAVP-induced increase in AQP2 at the protein level only. We determined that mpkCCD and native mouse collecting ducts express both estrogen receptor (ER)α and ERβ and that female mice lacking ERα displayed significant increases in AQP2 protein compared with wild-type littermates, implicating ERα in mediating the inhibitory effect of estradiol on AQP2 expression. These findings suggest that changes in estradiol levels, such as during menopause or following reproductive surgeries, may contribute to dysregulation of water homeostasis in women.

Estrogen increases expression of aquaporin‐2 protein in the inner medullary collecting duct (1137.3)

Previous studies of sex differences in renal water handling have focused on vasopressin and the V2 receptor rather than possible differences in their downstream target, aquaporin‐2 (AQP2). We report that inner medullary glycosylated and non‐glycosylated AQP2 protein levels are approximately 30‐40% lower in male (0.69±0.04 and 0.62±0.07 arbitrary units, a.u., n=5) compared to female C57BL/6 mice (1.00±0.08 and 1.00±0.16 a.u., n=4; P<0.05). Immunofluorescent staining and Western blot analysis revealed that freshly isolated collecting duct cells and mpk‐CCD‐clone II cells express estrogen receptor (ER) β, the ER splice variants ERα36 and ERα46, but little if any full‐length ERα66. Exposure of mpk‐CCD‐clone II cells to 10 nM estrogen for 24 h significantly increased AQP2 protein expression (2.6±0.4 vs 0.9±0.2 a.u. for vehicle, n=3; P<0.05), whereas exposure to the selective ERβ agonist DPN at 10 nM had no significant effect. Inner medullary AQP2 mRNA levels were not significantly different between male and female C57BL/6 mice (2‐ΔΔCT = 1.03±0.12 and 1.09±0.08, n=6 per group; P>0.05). Thus our data show that estrogen increases collecting duct AQP2 protein levels, potentially via a mechanism independent of gene transcription.

Effect of Poria cocos on hypertonic stress‐induced water channel expression and apoptosis in renal collecting duct cells

Aquaporin‐2 (AQP2), a family of water channels, plays an important role in the urinary concentrating process and regulation of water balance in the kidney. The dried sclerotia of Poria cocos Wolf (WPC) has been used for chronic edema and nephrosis. This study was conducted to evaluate the effects of WPC on hypertonic stress (175 mM NaCl)‐induced AQP2 expression and apoptosis in murine inner medullary collecting duct cell line (mIMCD‐3). WPC significantly attenuated the hypertonicity‐induced AQP2 protein and mRNA levels and blocked the apical membrane insertion of AQP2 in a concentration‐dependent manner. Tonicity‐responsive enhancer binding protein (TonEBP) mRNA was also recovered by WPC under hypertonic stress. Furthermore, pretreatment with WPC presented the similar effect with KT5720 which was decreased hypertonic stress‐induced AQP2 expression. On the other hand, WPC significantly recovered hypertonic stress‐injured mIMCD‐3 viability and cell number. WPC markedly inhibited hypertonic stress‐induced Bax or caspase‐3 expression, whereas Bcl‐2 expression was increased. Besides, WPC attenuated the hypertonicity‐induced morphological changes in nuclei, resulting in anti‐apoptotic effect. Thus, these data suggests that WPC has beneficial effects on the therapeutic approach to renal disorder.

Protein abundance of urea transporters and aquaporin 2 change differently in nephrotic pair-fed vs. non-pair-fed rats

Salt and water retention is a hallmark of nephrotic syndrome (NS). In this study, we test for changes in the abundance of urea transporters, aquaporin 2 (AQP2), Na-K-2Cl cotransporter 2 (NKCC2), and Na-Cl cotransporter (NCC), in non-pair-fed and pair-fed nephrotic animals. Doxorubicin-injected male Sprague-Dawley rats (n = 10) were followed in metabolism cages. Urinary excretion of protein, sodium, and urea was measured periodically. Kidney inner medulla (IM), outer medulla, and cortex tissue samples were dissected and analyzed for mRNA and protein abundances. At 3 wk, all doxorubicin-treated rats developed features of NS, with a ninefold increase in urine protein excretion (from 144 ± 21 to 1,107 ± 165 mg/day; P < 0.001) and reduced urinary sodium excretion (from 0.17 to 0.12 meq/day; P < 0.001). Urine osmolalities were reduced in the nephrotic animals (1,057 ± 37, treatment vs. 1,754 ± 131, control). Unlike animals fed ad libitum, UT-A1 protein abundance was unchanged in nephrotic pair-fed rats. Glycosylated AQP2 was reduced in the IM base of both nephrotic groups. Abundances of NKCC2 and NCC were consistently reduced (71 ± 7 and 33 ± 13%, respectively) in both nephrotic pair-fed animals and animals fed ad libitum. In pair-fed nephrotic rats, we observed an increase in the cleaved form of membrane-bound γ-epithelial sodium channel (ENaC). However, α- and β-ENaC subunits were unaltered. NKCC2 and AQP2 mRNA levels were similar in treated vs. control rats. We conclude that dietary protein intake affects the response of medullary transport proteins to NS.

Lithium reduces aquaporin-2 transcription independent of prostaglandins

Vasopressin (AVP)-stimulated translocation and transcription of aquaporin-2 (AQP2) water channels in renal principal cells is essential for urine concentration. Twenty percent of patients treated with lithium develop nephrogenic diabetes insipidus (NDI), a disorder in which the kidney is unable to concentrate urine. In vivo and in mouse collecting duct (mpkCCD) cells, lithium treatment coincides with decreased AQP2 abundance and inactivation of glycogen synthase kinase (Gsk) 3β. This is paralleled in vivo by an increased renal cyclooxygenase 2 (COX-2) expression and urinary prostaglandin PGE(2) excretion. PGE(2) reduces AVP-stimulated water reabsorption, but its precise role in lithium-induced downregulation of AQP2 is unclear. Using mpkCCD cells, we here investigated whether prostaglandins contribute to lithium-induced downregulation of AQP2. In these cells, lithium application reduced AQP2 abundance, which coincided with Gsk3β inactivation and increased COX-2 expression. Inhibition of COX by indomethacin, leading to reduced PGE(2) and PGF(2α) levels, or dexamethasone-induced downregulation of COX-2 both increased AQP2 abundance, while PGE(2) addition reduced AQP2 abundance. However, lithium did not change the prostaglandin levels, and indomethacin and dexamethasone did not prevent lithium-induced AQP2 downregulation. Further analysis revealed that lithium decreased AQP2 protein abundance, mRNA levels and transcription, while PGE(2) reduced AQP2 abundance by increasing its lysosomal degradation, but not by reducing AQP2 gene transcription. In conclusion, our data reveal that in mpkCCD cells, prostaglandins decrease AQP2 protein stability by increasing its lysosomal degradation, indicating that in vivo paracrine-produced prostaglandins might have a role in lithium-induced NDI via this mechanism. However, lithium affects also AQP2 gene transcription, which is prostaglandin independent.

Molecular mechanisms of angiotensin II stimulation on aquaporin-2 expression and trafficking

ANG II plays a major role in renal water and sodium regulation. In the immortalized mouse renal collecting duct principal cells (mpkCCD(cl4)) cell line, we treated cells with ANG II and examined aquaporin-2 (AQP2) protein expression, trafficking, and mRNA levels, by immunoblotting, immunofluorescence, and RT-PCR. After 24-h incubation, ANG II-induced AQP2 protein expression was observed at the concentration of 10(-10) M and increased in a dose-dependent manner. ANG II (10(-7) M) increased AQP2 protein expression and mRNA levels at 0.5, 1, 2, 6, and 24 h. Immunofluorescence studies showed that ANG II increased the apical membrane targeting of AQP2 from 30 min to 6 h. Next, the signaling pathways underlying the ANG II-induced AQP2 expression were investigated. The PKC inhibitor Ro 31-8220 (5 × 10(-6) M) and the PKA inhibitor H89 (10(-5) M) blocked ANG II-induced AQP2 expression, respectively. Calmodulin inhibitor W-7 markedly reduced ANG II- and/or dDAVP-stimulated AQP2 expression. ANG II (10(-9) M) and/or dDAVP (10(-10) M) stimulated AQP2 protein levels and cAMP accumulation, which was completely blocked by pretreatment with the vasopressin V2 receptor (V2R) antagonist SR121463B (10(-8) M). Pretreatment with the angiotensin AT(1) receptor (AT1R) antagonist losartan (3 × 10(-6) M) blocked ANG II (10(-9) M)-stimulated AQP2 protein expression and cAMP accumulation, and partially blocked dDAVP (10(-10) M)- and dDAVP+ANG II-induced AQP2 protein expression and cAMP accumulation. In conclusion, ANG II regulates AQP2 protein, trafficking, and gene expression in renal collecting duct principal cells. ANG II-induced AQP2 expression involves cAMP, PKC, PKA, and calmodulin signaling pathways via V2 and AT(1) receptors.

Counteracting vasopressin-mediated water reabsorption by ATP, dopamine, and phorbol esters: mechanisms of action

Water homeostasis is regulated by a wide variety of hormones. When in need for water conservation, vasopressin, released from the brain, binds renal principal cells and initiates a signaling cascade resulting in the insertion of aquaporin-2 (AQP2) water channels in the apical membrane and water reabsorption. Conversely, hormones, including extracellular purines and dopamine, antagonize AVP-induced water permeability, but their mechanism of action is largely unknown, which was investigated here. Addition of these hormones to mpkCCD cells decreased total and plasma membrane abundance of AVP-induced AQP2, partly by increasing its internalization to vesicles and lysosomal degradation. This internalization was ubiquitin dependent, because the hormones increased AQP2 ubiquitination, and the plasma membrane localization of AQP2-K270R, which cannot be monoubiquitinated, was unaffected by these hormones. Both hormones also increased AQP2 phosphorylation at S261, which followed ubiquitination, but was not essential for hormone-induced AQP2 degradation. A similar process occurs in vivo, as incubation of dDAVP-treated kidney slices with both hormones also resulted in the internalization and S261 phosphorylation of AQP2. Both hormones also reduced cAMP and AQP2 mRNA levels, suggesting an additional effect on AQP2 gene transcription. Interestingly, phorbol esters only reduced AQP2 through the first pathway. Together, our results indicate that ATP and dopamine counteract AVP-induced water permeability by increasing AQP2 degradation in lysosomes, preceded by ubiquitin-dependent internalization, and by decreasing AQP2 gene transcription by reducing the AVP-induced cAMP levels.

NF-κB Modulates Aquaporin-2 Transcription in Renal Collecting Duct Principal Cells

Renal tubulo-interstitial inflammation is frequently associated with polyuria and urine concentration defects. This led us to investigate the effects of the major pro-inflammatory nuclear factor kappaB (NF-kappaB) pathway on aquaporin 2 (AQP2) expression by the collecting duct. Using immortalized collecting duct principal cells (mpkCCDcl4), we found that, acting independently of vasopressin, activation of NF-kappaB by lipopolysaccharide (LPS) decreased AQP2 mRNA and protein levels in a time- and dose-dependent manner but did not decrease AQP2 mRNA stability. Consistently, constitutively active IkappaB kinase beta decreased AQP2 expression. The LPS-induced decrease in AQP2 mRNA levels was confirmed in rat kidney slices and was reproduced both under conditions of elevated cAMP concentration and V(2) receptor antagonism. Computer analysis of the AQP2 promoter revealed two putative kappaB elements. Mutation of either kappaB element abolished the LPS-induced decrease of luciferase activity in cells expressing AQP2 promoter-luciferase plasmid constructs. Chromatin immunoprecipitation revealed that LPS challenge decreased p65, increased p50 and p52, and had no effect on RelB and c-Rel binding to kappaB elements of the AQP2 promoter. RNA-mediated interference silencing of p65, p50, and p52 confirmed controlled AQP2 transcription by these NF-kappaB subunits. We additionally found that hypertonicity activated NF-kappaB in mpkCCDcl4 cells, an effect that may counteract the Tonicity-responsive enhancer binding protein (TonEBP)-dependent increase in AQP2 gene transcription. Taken together, these findings indicate that NF-kappaB is an important physiological regulator of AQP2 transcription.

Gαq-dependent signaling cascades stimulate water-seeking behavior

We used the mouse nephrin promoter to express a constitutively active Galphaq [Galphaq(Q>L)] transgene in mice. As previously reported, the transgene was expressed in kidney, pancreas, and brain, and the kidney phenotype was characterized by albuminuria and reduced nephron mass. Additional studies revealed a second phenotype characterized by polyuria and polydipsia. The polyuric phenotype was not caused by abnormal glucose metabolism or hypercalcemia but was accompanied by reduced urinary concentrating ability. Additional studies found that 1) water restriction was associated with an appropriate increase in serum vasopressin levels in transgenic (TG) mice; 2) the urinary concentrating defect was not corrected by administration of desamino-d-arginine vasopressin (DDAVP); and 3) papillary length was similar in TG and non-TG mice. To examine the renal response to DDAVP at the molecular level, we monitored aquaporin 2 (AQP2) and vasopressin V2 receptor (V2R) mRNA levels in mouse kidney. Consistent with the known effects of vasopressin, administration of DDAVP caused a decrease in V2R mRNA levels and an increase in AQP2 mRNA levels in both TG and non-TG animals, suggesting an appropriate renal response to DDAVP in the TG mice. To determine whether the urine concentrating abnormality was the result of primary polydipsia, water intake by TG mice was restricted to the amount ingested by non-TG animals. After 5 days, urinary concentrating ability was similar in TG mice and non-TG littermate controls. These data are consistent with the notion that expression of the Galphaq(Q>L) transgene in the brain induced primary polydipsia in the TG mice.

Insulin potentiates AVP-induced AQP2 expression in cultured renal collecting duct principal cells

In the renal collecting duct (CD), water reabsorption depends on the presence of aquaporin-2 (AQP2) in the apical membrane of principal cells. AQP2 expression and subcellular repartition are under the control of AVP. Some pieces of experimental evidence indicate that additional hormonal factors, including insulin, may also control AQP2 expression and thereby CD water permeability. We have previously shown that AVP induces endogenous AQP2 expression in cultured mouse mpkCCD(cl4) CD principal cells (23). In the present study, we investigated the effect of insulin on AQP2 expression in mpkCCD(cl4) cells. Addition of insulin to the basal medium of cells grown on filters slightly increased AQP2 mRNA and protein expression, whereas insulin potentiated the effect of AVP. The potentiation of AVP-induced AQP2 expression by insulin was abolished by actinomycin D, a transcriptional inhibitor. Analysis of AQP2 protein expression under conditions of AVP washout and/or in the presence of chloroquine, a lysosomal degradation inhibitor, revealed that insulin did not significantly alter AQP2 protein degradation. Inhibition of ERK, p38 kinase, and phosphatidylinositol 3'-kinase (PI 3-kinase) activities prevented the insulin-induced stimulation of AQP2 expression, whereas inhibition of PKC has no effect. Taken together, our results indicate that insulin increased AQP2 protein expression mostly through increased AQP2 mRNA levels in cultured mpkCCD(cl4) cells. This effect most likely relies on increased AQP2 gene transcription in response to MAPK and PI 3-kinase activation.