Multiomic analysis of the Arabian camel (Camelus dromedarius) kidney reveals a role for cholesterol in water conservation.

The Arabian camel (Camelus dromedarius) is the most important livestock animal in arid and semi-arid regions and provides basic necessities to millions of people. In the current context of climate change, there is renewed interest in the mechanisms that enable camelids to survive in arid conditions. Recent investigations described genomic signatures revealing evolutionary adaptations to desert environments. We now present a comprehensive catalogue of the transcriptomes and proteomes of the dromedary kidney and describe how gene expression is modulated as a consequence of chronic dehydration and acute rehydration. Our analyses suggested an enrichment of the cholesterol biosynthetic process and an overrepresentation of categories related to ion transport. Thus, we further validated differentially expressed genes with known roles in water conservation which are affected by changes in cholesterol levels. Our datasets suggest that suppression of cholesterol biosynthesis may facilitate water retention in the kidney by indirectly facilitating the AQP2-mediated water reabsorption.

Spermine Regulates Water Balance Associated with Ca2+-Dependent Aquaporin (TrTIP2-1, TrTIP2-2 and TrPIP2-7) Expression in Plants under Water Stress.

Spermine (Spm) regulates water balance involved in water channel proteins, aquaporins (AQPs), in plants. An increase in endogenous Spm content via exogenous Spm application significantly improved cell membrane stability, photosynthesis, osmotic adjustment (OA) and water use efficiency (WUE) contributing to enhanced tolerance to water stress in white clover. Spm upregulated TrTIP2-1, TrTIP2-2 and TrPIP2-7 expressions and also increased the abundance of TIP2 and PIP2-7 proteins in white clover under water stress. Spm quickly activated intracellular Ca2+ signaling and Spm-induced TrTIP2-2 and TrPIP2-7 expressions could be blocked by Ca2+ channel blockers and the inhibitor of Ca2+-dependent protein kinase in leaves of white clover. TrSAMS in relation to Spm biosynthesis was first cloned from white clover and the TrSAMS was located in the nucleus. Transgenic Arabidopsis overexpressing the TrSAMS had significantly higher endogenous Spm content and improved cell membrane stability, photosynthesis, OA, WUE and transcript levels of AtSIP1-1, AtSIP1-2, AtTIP2-1, AtTIP2-2, AtPIP1-2, AtPIP2-1 and AtNIP2-1 than wild type in response to water stress. Current findings indicate that Spm regulates water balance via an enhancement in OA, WUE and water transport related to Ca2+-dependent AQP expression in plants under water stress.

Inner Ear Arginine Vasopressin-Vasopressin Receptor 2-Aquaporin 2 Signaling Pathway Is Involved in the Induction of Motion Sickness.

It has been identified that arginine vasopressin (AVP), vasopressin receptor 2(V2R), and the aquaporin 2 (AQP2) signaling pathway in the inner ear play important roles in hearing and balance functions through regulating the endolymph equilibrium; however, the contributions of this signaling pathway to the development of motion sickness are unclear. The present study was designed to investigate whether the activation of the AVP-V2R-AQP2 signaling pathway in the inner ear is involved in the induction of motion sickness and whether mozavaptan, a V2R antagonist, could reduce motion sickness. We found that both rotatory stimulus and intraperitoneal AVP injection induced conditioned taste aversion (a confirmed behavioral index for motion sickness) in rats and activated the AVP-V2R-AQP2 signaling pathway with a responsive V2R downregulation in the inner ears, and AVP perfusion in cultured epithelial cells from rat endolymphatic sacs induced similar changes in this pathway signaling. Vestibular training, V2R antagonist mozavaptan, or PKA inhibitor H89 blunted these changes in the V2R-AQP2 pathway signaling while reducing rotatory stimulus- or DDAVP (a V2R agonist)-induced motion sickness in rats and dogs. Therefore, our results suggest that activation of the inner ear AVP-V2R-AQP2 signaling pathway is potentially involved in the development of motion sickness; thus, mozavaptan targeting AVP V2Rs in the inner ear may provide us with a new application option to reduce motion sickness. SIGNIFICANCE STATEMENT: Motion sickness affects many people traveling or working. In the present study our results showed that activation of the inner ear arginine vasopressin-vaspopressin receptor 2 (V2R)-aquaporin 2 signaling pathway was potentially involved in the development of motion sickness and that blocking V2R with mozavaptan, a V2R antagonist, was much more effective in reducing motion sickness in both rat and dog; therefore, we demonstrated a new mechanism to underlie motion sickness and a new candidate drug to reduce motion sickness.

Na+/H+ exchanger isoform 1 activity in AQP2-expressing cells can be either proliferative or anti-proliferative depending on extracellular pH.

We have previously shown in renal cells that expression of the water channel Aquaporin-2 increases cell proliferation by a regulatory volume mechanism involving Na+/H+ exchanger isoform 2. Here, we investigated if Aquaporin-2 (AQP2) also modulates Na+/H+ exchanger isoform 1-dependent cell proliferation. We use two AQP2-expressing cortical collecting duct models: one constitutive (WT or AQP2-transfected RCCD1 cell line) and one inducible (control or vasopressin-induced mpkCCDc14 cell line). We found that Aquaporin-2 modifies Na+/H+ exchanger isoform 1 (NHE1) contribution to cell proliferation. In Aquaporin-2-expressing cells, Na+/H+ exchanger isoform 1 is anti-proliferative at physiological pH. In acid media, Na+/H+ exchanger isoform 1 contribution turned from anti-proliferative to proliferative only in AQP2-expressing cells. We also found that, in AQP2-expressing cells, NHE1-dependent proliferation changes parallel changes in stress fiber levels: at pH 7.4, Na+/H+ exchanger isoform 1 would favor stress fiber disassembly and, under acidosis, NHE1 would favor stress fiber assembly. Moreover, we found that Na+/H+ exchanger-dependent effects on proliferation linked to Aquaporin-2 relied on Transient Receptor Potential Subfamily V calcium channel activity. In conclusion, our data show that, in collecting duct cells, the water channel Aquaporin-2 modulates NHE1-dependent cell proliferation. In AQP2-expressing cells, at physiological pH, the Na+/H+ exchanger isoform 1 function is anti-proliferative and, at acidic pH, Na+/H+ exchanger isoform 1 function is proliferative. We propose that Na+/H+ exchanger isoform 1 modulates proliferation through an interplay with stress fiber formation.

Glutamine metabolism via glutaminase 1 in autosomal-dominant polycystic kidney disease.

Background: Metabolism of glutamine by glutaminase 1 (GLS1) plays a key role in tumor cell proliferation via the generation of ATP and intermediates required for macromolecular synthesis. We hypothesized that glutamine metabolism also plays a role in proliferation of autosomal-dominant polycystic kidney disease (ADPKD) cells and that inhibiting GLS1 could slow cyst growth in animal models of ADPKD. Methods: Primary normal human kidney and ADPKD human cyst-lining epithelial cells were cultured in the presence or absence of two pharmacologic inhibitors of GLS1, bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES) and CB-839, and the effect on proliferation, cyst growth in collagen and activation of downstream signaling pathways were assessed. We then determined if inhibiting GLS1 in vivo with CB-839 in the Aqp2-Cre; Pkd1fl/fl and Pkhd1-Cre; Pkd1fl/fl mouse models of ADPKD slowed cyst growth. Results: We found that an isoform of GLS1 (GLS1-GAC) is upregulated in cyst-lining epithelia in human ADPKD kidneys and in mouse models of ADPKD. Both BPTES and CB-839 blocked forskolin-induced cyst formation in vitro. Inhibiting GLS1 in vivo with CB-839 led to variable outcomes in two mouse models of ADPKD. CB-839 slowed cyst growth in Aqp2-Cre; Pkd1fl/fl mice, but not in Pkhd1-Cre; Pkd1fl/fl mice. While CB-839 inhibited mammalian target of rapamycin (mTOR) and MEK activation in Aqp2-Cre; Pkd1fl/fl, it did not in Pkhd1-Cre; Pkd1fl/fl mice. Conclusion: These findings provide support that alteration in glutamine metabolism may play a role in cyst growth. However, testing in other models of PKD and identification of the compensatory metabolic changes that bypass GLS1 inhibition will be critical to validate GLS1 as a drug target either alone or when combined with inhibitors of other metabolic pathways.

EGF Receptor Inhibition by Erlotinib Increases Aquaporin 2-Mediated Renal Water Reabsorption.

Nephrogenic diabetes insipidus (NDI) is caused by impairment of vasopressin (VP) receptor type 2 signaling. Because potential therapies for NDI that target the canonical VP/cAMP/protein kinase A pathway have so far proven ineffective, alternative strategies for modulating aquaporin 2 (AQP2) trafficking have been sought. Successful identification of compounds by our high-throughput chemical screening assay prompted us to determine whether EGF receptor (EGFR) inhibitors stimulate AQP2 trafficking and reduce urine output. Erlotinib, a selective EGFR inhibitor, enhanced AQP2 apical membrane expression in collecting duct principal cells and reduced urine volume by 45% after 5 days of treatment in mice with lithium-induced NDI. Similar to VP, erlotinib increased exocytosis and decreased endocytosis in LLC-PK1 cells, resulting in a significant increase in AQP2 membrane accumulation. Erlotinib increased phosphorylation of AQP2 at Ser-256 and Ser-269 and decreased phosphorylation at Ser-261 in a dose-dependent manner. However, unlike VP, the effect of erlotinib was independent of cAMP, cGMP, and protein kinase A. Conversely, EGF reduced VP-induced AQP2 Ser-256 phosphorylation, suggesting crosstalk between VP and EGF in AQP2 trafficking and a role of EGF in water homeostasis. These results reveal a novel pathway that contributes to the regulation of AQP2-mediated water reabsorption and suggest new potential therapeutic strategies for NDI treatment.

Stimulation of V1a receptor increases renal uric acid clearance via urate transporters: insight into pathogenesis of hypouricemia in SIADH.

BACKGROUND: Hypouricemia is pathognomonic in syndrome of inappropriate secretion of antidiuretic hormone (SIADH) but the underlying mechanism remains unclear. Based on the previous studies, we hypothesized that V1a receptor may play a principal role in inducing hypouricemia in SIADH and examined uric acid metabolism using a rat model. METHODS: Terlipressin (25 ng/h), a selective V1a agonist, was subcutaneously infused to 7-week-old male Wistar rats (n = 9). Control rats were infused with normal saline (n = 9). The rats were sacrificed to obtain kidney tissues 3 days after treatment. In addition to electrolyte metabolism, changes in expressions of the urate transporters including URAT1 (SLC22A12), GLUT9 (SLC2A9), ABCG2 and NPT1 (SLC17A1) were examined by western blotting and immunohistochemistry. RESULTS: In the terlipressin-treated rats, serum uric acid (UA) significantly decreased and the excretion of urinary UA significantly increased, resulting in marked increase in fractional excretion of UA. Although no change in the expression of URAT1, GLUT9 expression significantly decreased whereas the expressions of ABCG2 and NPT1 significantly increased in the terlipressin group. The results of immunohistochemistry corroborated with those of the western blotting. Aquaporin 2 expression did not change in the medulla, suggesting the independence of V2 receptor stimulation. CONCLUSION: Stimulation of V1a receptor induces the downregulation of GLUT9, reabsorption urate transporter, together with the upregulation of ABCG2 and NPT1, secretion urate transporters, all changes of which clearly lead to increase in renal UA clearance. Hypouricemia seen in SIADH is attributable to V1a receptor stimulation.

Defective Store-Operated Calcium Entry Causes Partial Nephrogenic Diabetes Insipidus.

Store-operated calcium entry (SOCE) is the mechanism by which extracellular signals elicit prolonged intracellular calcium elevation to drive changes in fundamental cellular processes. Here, we investigated the role of SOCE in the regulation of renal water reabsorption, using the inbred rat strain SHR-A3 as an animal model with disrupted SOCE. We found that SHR-A3, but not SHR-B2, have a novel truncating mutation in the gene encoding stromal interaction molecule 1 (STIM1), the endoplasmic reticulum calcium (Ca(2+)) sensor that triggers SOCE. Balance studies revealed increased urine volume, hypertonic plasma, polydipsia, and impaired urinary concentrating ability accompanied by elevated circulating arginine vasopressin (AVP) levels in SHR-A3 compared with SHR-B2. Isolated, split-open collecting ducts (CD) from SHR-A3 displayed decreased basal intracellular Ca(2+) levels and a major defect in SOCE. Consequently, AVP failed to induce the sustained intracellular Ca(2+) mobilization that requires SOCE in CD cells from SHR-A3. This effect decreased the abundance of aquaporin 2 and enhanced its intracellular retention, suggesting impaired sensitivity of the CD to AVP in SHR-A3. Stim1 knockdown in cultured mpkCCDc14 cells reduced SOCE and basal intracellular Ca(2+) levels and prevented AVP-induced translocation of aquaporin 2, further suggesting the effects in SHR-A3 result from the expression of truncated STIM1. Overall, these results identify a novel mechanism of nephrogenic diabetes insipidus and uncover a role of SOCE in renal water handling.

Partial nephrogenic diabetes insipidus caused by a novel AQP2 variation impairing trafficking of the aquaporin-2 water channel.

BACKGROUND: Autosomal dominant inheritance of congenital nephrogenic diabetes insipidus (CNDI) is rare and usually caused by variations in the AQP2 gene. We have investigated the genetic and molecular background underlying symptoms of diabetes insipidus (DI) in a Swedish family with autosomal dominant inheritance of the condition. METHODS: The proband and her father were subjected to water deprivation testing and direct DNA sequencing of the coding regions of the AQP2 and AVP genes. Madin-Darby canine kidney (MDCK) cells stably expressing AQP2 variant proteins were generated by lentiviral gene delivery. Localization of AQP2 variant proteins in the cells under stimulated and unstimulated conditions was analyzed by means of immunostaining and confocal laser scanning microscopy. Intracellular trafficking of AQP2 variant proteins was studied using transient expression of mutant dynamin2-K44A-GFP protein and AQP2 variant protein phosphorylation levels were assessed by Western blotting analysis. RESULTS: Clinical and genetic data suggest that the proband and her father suffer from partial nephrogenic DI due to a variation (g.4807C > T) in the AQP2 gene. The variation results in substitution of arginine-254 to tryptophan (p.R254W) in AQP2. Analysis of MDCK cells stably expressing AQP2 variant proteins revealed disabled phosphorylation, impaired trafficking and intracellular accumulation of AQP2-R254W protein. Notably, blocking of the endocytic pathway demonstrated impairment of AQP2-R254W to reach the cell surface. CONCLUSIONS: Partial CNDI in the Swedish family is caused by an AQP2 variation that seems to disable the encoded AQP2-R254W protein to reach the subapical vesicle population as well as impairing its phosphorylation at S256. The AQP2-R254W protein is thus unable to reach the plasma membrane to facilitate AVP mediated urine concentration.

Evidence of Positive Selection of Aquaporins Genes from Pontoporia blainvillei during the Evolutionary Process of Cetaceans.

BACKGROUND: Marine mammals are well adapted to their hyperosmotic environment. Several morphological and physiological adaptations for water conservation and salt excretion are known to be present in cetaceans, being responsible for regulating salt balance. However, most previous studies have focused on the unique renal physiology of marine mammals, but the molecular bases of these mechanisms remain poorly explored. Many genes have been identified to be involved in osmotic regulation, including the aquaporins. Considering that aquaporin genes were potentially subject to strong selective pressure, the aim of this study was to analyze the molecular evolution of seven aquaporin genes (AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP9) comparing the lineages of cetaceans and terrestrial mammals. RESULTS: Our results demonstrated strong positive selection in cetacean-specific lineages acting only in the gene for AQP2 (amino acids 23, 83, 107,179, 180, 181, 182), whereas no selection was observed in terrestrial mammalian lineages. We also analyzed the changes in the 3D structure of the aquaporin 2 protein. Signs of strong positive selection in AQP2 sites 179, 180, 181, and 182 were unexpectedly identified only in the baiji lineage, which was the only river dolphin examined in this study. Positive selection in aquaporins AQP1 (45), AQP4 (74), AQP7 (342, 343, 356) was detected in cetaceans and artiodactyls, suggesting that these events are not related to maintaining water and electrolyte homeostasis in seawater. CONCLUSIONS: Our results suggest that the AQP2 gene might reflect different selective pressures in maintaining water balance in cetaceans, contributing to the passage from the terrestrial environment to the aquatic. Further studies are necessary, especially those including other freshwater dolphins, who exhibit osmoregulatory mechanisms different from those of marine cetaceans for the same essential task of maintaining serum electrolyte balance.

Recombinant human erythropoietin pretreatment attenuates acute renal tubular injury against ischemia-reperfusion by restoring transient receptor potential channel-6 expression and function in collecting ducts.

OBJECTIVE: Acute renal tubular injury is a serious complication in the postoperative period, which is associated with high mortality and increased ICU stay. We aimed to demonstrate the protective effect of rhEPO against acute tubular injury induced by ischemia-reperfusion and to explore the mechanism of canonical transient receptor potential channel-6. DESIGN: Randomized laboratory animal study. SETTINGS: Animal research laboratory. INTERVENTIONS: Male Sprague-Dawley rats were randomly divided into three groups: the sham group, the control group, and the rhEPO group. Experimental acute tubular injury was established in rats by bilateral renal arterial occlusion for 30 minutes followed by reperfusion. MEASUREMENTS AND MAIN RESULTS: Blood samples were obtained for cystatin-C and neutrophil gelatinase-associated lipocalin measurements by enzyme-linked immunosorbance assays. Seventy-two hours after reperfusion, urine samples were collected for osmolality and fractional excretion of sodium (%) assays on a chemistry analyzer. Kidneys were harvested at 24, 48, and 72 hours after reperfusion. Transient receptor potential channel-6, aquaporin-2, and Na,K-ATPase expression in collecting ducts were studied by immunofluorescence and Western blot. Coimmunoprecipitations were also performed to identify the possible signalplex relation between transient receptor potential channel-6 and aquaporin-2 or Na,K-ATPase channels. RhEPO pretreatment significantly inhibited serum cystatin-C (2 hr: 453 ± 64 µg/L vs 337 ± 28 µg/L, p < 0.01), serum neutrophil gelatinase-associated lipocalin (72 hr: 1,175 ± 107 ng/L vs 1,737 ± 402 ng/L, p < 0.05), and urinary fractional excretion of sodium (%) increase (0.9 ± 0.1 vs 2.2 ± 0.8, p < 0.05) and alleviated the decrease of urinary osmolality (1,293 ± 101 mosmol/kg H2O vs 767 ± 91 mosmol/kg H2O, p < 0.05) induced by ischemia-reperfusion injury. Meanwhile, recombinant human erythropoietin greatly improved the ischemia-reperfusion-induced attenuation of transient receptor potential channel-6 expression (48 hr: 42% ± 2% vs 67% ± 2% and 72 hr: 55% ± 2% vs 66% ± 2%), as well as aquaporin-2 and Na,K-ATPase expression in collecting ducts. Transient receptor potential channel-6 functionally interacted with Na,K-ATPase but not aquaporin-2. CONCLUSIONS: Recombinant human erythropoietin pretreatment at the dose of 5,000 IU/kg potently prevented ischemia-reperfusion-induced acute tubular injury, which might be partly attributed to the restoring the effect of transient receptor potential channel-6 expression and collecting duct function.

Integrin-linked kinase regulates tubular aquaporin-2 content and intracellular location: a link between the extracellular matrix and water reabsorption.

One of the clinical alterations observed in chronic renal disease (CRD) is the impaired urine concentration, known as diabetes insipidus (DI). Tubulointerstitial fibrosis of the kidney is also a pathological finding observed in CRD and involves composition of extracellular matrix (ECM). However, an association between these two events has not been elucidated. In this study, we showed that the extracellular-to-intracellular scaffold protein integrin-linked kinase (ILK) regulates expression of tubular water channel aquaporin-2 (AQP2) and its apical membrane presence in the renal tubule. Basally, polyuria and decreased urine osmolality were present in ILK conditional-knockdown (cKD-ILK) adult mice compared with nondepleted ILK littermates. No changes were observed in arginine-vasopressin (AVP) blood levels, renal receptor (V2R), or AQP3 expression. However, tubular AQP2 was decreased in expression and apical membrane presence in cKD-ILK mice, where the canonical V2R/cAMP axis activation is still functional, but independent of the absence of ILK. Thus, cKD-ILK constitutes a nephrogenic diabetes insipidus (NDI) model. AQP2 and ILK colocalize in cultured inner medullary collecting duct (mIMCD3) cells. Specific ILK siRNAs and collagen I (Col) decrease ILK and AQP2 levels and AQP2 presence on the membrane of tubular mIMCD3 cells, which impairs the capacity of the cells to transport water under hypotonic stress. The present work points to ILK as a therapeutic target in NDI.

New insights into regulated aquaporin-2 function.

PURPOSE OF REVIEW: Aquaporin-2 (AQP2) water channels in principal cells of the kidney collecting duct are essential for urine concentration. Due to application of modern technologies, progress in our understanding of AQP2 has accelerated in recent years. In this article, we highlight some of the new insights into AQP2 function that have developed recently, with particular focus on the cell biological aspects of AQP2 regulation. RECENT FINDINGS: AQP2 is subjected to a number of regulated modifications, including phosphorylation and ubiquitination, which are important for AQP2 function, cellular localization and degradation. AQP2 is likely internalized via clathrin and non-clathrin-mediated endocytosis. Regulation of AQP2 endocytosis, in addition to exocytosis, is a vital mechanism in determining overall AQP2 membrane abundance. AQP2 is associated with regulated membrane microdomains. Studies using membrane cholesterol depleting reagents, for example statins, have supported the role of membrane rafts in regulation of AQP2 trafficking. Noncanonical roles for AQP2, for example in epithelial cell migration, are emerging. SUMMARY: AQP2 function and thus urine concentration is dependent on a variety of cell signalling mechanisms, posttranslational modification and interplay between AQP2 and its lipid environment. This complexity of regulation allows fine-tuning of AQP2 function and thus body water homeostasis.

Nitric oxide and AQP2 in hypothyroid rats: a link between aging and water homeostasis.

OBJECTIVE: 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. MATERIALS/METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Role of guanine-nucleotide exchange factor Epac in renal physiology and pathophysiology.

Exchange proteins directly activated by cAMP [Epac(s)] were discovered more than a decade ago as new sensors for the second messenger cAMP. The Epac family members, including Epac1 and Epac2, are guanine nucleotide exchange factors for the Ras-like small GTPases Rap1 and Rap2, and they function independently of protein kinase A. Given the importance of cAMP in kidney homeostasis, several molecular and cellular studies using specific Epac agonists have analyzed the role and regulation of Epac proteins in renal physiology and pathophysiology. The specificity of the functions of Epac proteins may depend upon their expression and localization in the kidney as well as their abundance in the microcellular environment. This review discusses recent literature data concerning the involvement of Epac in renal tubular transport physiology and renal glomerular cells where various signaling pathways are known to be operative. In addition, the potential role of Epac in kidney disorders, such as diabetic kidney disease and ischemic kidney injury, is discussed.

Genetic ablation of aquaporin-2 in the mouse connecting tubules results in defective renal water handling.

Body water balance is regulated via the water channel aquaporin-2 (AQP2), which is expressed in the renal connecting tubule (CNT) and collecting duct (CD). The relative roles of AQP2 in the CNT and CD are not fully understood. To study the role of AQP2 in the CNT we generated a mouse model with CNT-specific AQP2 deletion (AQP2-CNT-knockout (KO)). Confocal laser scanning microscopy and immunogold electron microscopy demonstrated an absence of AQP2 in the CNT of AQP2-CNT-KO mice. Twenty-four hour urine output was significantly increased (KO: 3.0 ± 0.3 ml (20 g body weight (BW))(-1); wild-type (WT): 1.9 ± 0.3 ml (20 g BW)(-1)) and urine osmolality decreased (KO: 1179 ± 107 mosmol kg(-1); WT: 1790 ± 146 mosmol kg(-1)) in AQP2-CNT-KO mice compared with controls. After 24 h water restriction, urine osmolality was still significantly lower in AQP2-CNT-KO mice (KO: 2087 ± 169 mosmol kg(-1); WT: 2678 ± 144 mosmol kg(-1)). A significant difference in urine osmolality between groups before desmopressin (dDAVP) (KO: 873 ± 129 mosmol kg(-1); WT: 1387 ± 163 mosmol kg(-1)) was not apparent 2 h after injection, with urine osmolality increased significantly in both groups (KO: 2944 ± 41 mosmol kg(-1); WT: 3133 ± 66 mosmol kg(-1)). Cortical kidney fractions from AQP2-CNT-KO mice had significantly reduced AQP2, with no compensatory changes in sodium potassium chloride cotransporter (NKCC2), AQP3 or AQP4. Lithium chloride treatment increased urine volume and decreased osmolality in both WT and AQP2-CNT-KO mice. After 8 days of treatment, the AQP2-CNT-KO mice still had a significantly higher urine volume and lower urine osmolality, suggesting that the CNT does not play a significant role in the pathology of lithium-induced nephrogenic diabetes insipidus. Our studies indicate that the CNT plays a role in regulating body water balance under basal conditions, but not for maximal concentration of the urine during antidiuresis.

Urine concentration in the diabetic mouse requires both urea and water transporters.

The regulation of the inner medullary collecting duct (IMCD) urea transporters (UT-A1, UT-A3) and aquaporin-2 (AQP2) and their interactions in diabetic animals is unknown. We investigated whether the urine concentrating defect in diabetic animals was a function of AQP2, the UT-As, or both transporters. UT-A1/UT-A3 knockout (UT-A1/A3 KO) mice produce dilute urine. We gave wild-type (WT) and UT-A1/A3 KO mice vasopressin via minipump for 7 days. In WT mice, vasopressin increased urine osmolality from 3,000 to 4,550 mosmol/kgH(2)O. In contrast, urine osmolality was low (800 mosmol/kgH(2)O) in the UT-A1/A3 KOs and remained low following vasopressin. Surprisingly, AQP2 protein abundance increased in UT-A1/A3 KO (114%) and WT (92%) mice. To define the role of UT-A1 and UT-A3 in the diabetic responses, WT and UT-A1/A3 KO mice were injected with streptozotocin (STZ). UT-A1/A3 KO mice showed only 40% survival at 7 days post-STZ injection compared with 70% in WT. AQP2 did not increase in the diabetic UT-A1/A3 KO mice compared with a 133% increase in WT diabetic mice. Biotinylation studies in rat IMCDs showed that membrane accumulation of UT-A1 increased by 68% in response to vasopressin in control rats but was unchanged by vasopressin in diabetic rat IMCDs. We conclude that, even with increased AQP2, UT-A1/UT-A3 is essential to optimal urine concentration. Furthermore, UT-A1 may be maximally membrane associated in diabetic rat inner medulla, making additional stimulation by vasopressin ineffective.

Aquaporin 2 promotes cell migration and epithelial morphogenesis.

The aquaporin 2 (AQP2) water channel, expressed in kidney collecting ducts, contributes critically to water homeostasis in mammals. Animals lacking or having significantly reduced levels of AQP2, however, have not only urinary concentrating abnormalities but also renal tubular defects that lead to neonatal mortality from renal failure. Here, we show that AQP2 is not only a water channel but also an integrin-binding membrane protein that promotes cell migration and epithelial morphogenesis. AQP2 expression modulates the trafficking and internalization of integrin ß1, facilitating its turnover at focal adhesions. In vitro, disturbing the interaction between AQP2 and integrin ß1 by mutating the RGD motif led to reduced endocytosis, retention of integrin ß1 at the cell surface, and defective cell migration and tubulogenesis. Similarly, in vivo, AQP2-null mice exhibited significant retention of integrin ß1 at the basolateral membrane and had tubular abnormalities. In summary, these data suggest that the water channel AQP2 interacts with integrins to promote renal epithelial cell migration, contributing to the structural and functional integrity of the mammalian kidney.