Hua A. Jenny Lu

Heat Shock Protein 70 Interacts with Aquaporin-2 and Regulates Its Trafficking

The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking.

The phosphorylation state of serine 256 is dominant over that of serine 261 in the regulation of AQP2 trafficking in renal epithelial cells

Phosphorylation of serine 256 (S256) plays a critical role in vasopressin (VP)-mediated membrane accumulation of aquaporin-2 (AQP2). Recently, phosphorylation of serine 261 was also reported, raising the possibility that it has a role in AQP2 trafficking. We addressed this issue using transfected LLC-PK(1) cells that express point mutations of AQP2 S261 and S256, mimicking the phosphorylated (S to D) or dephosphorylated (S to A) states of these residues. Both AQP2 (S261A) and AQP2 (S261D) were located in the perinuclear cytoplasm without stimulation but, like wild-type AQP2, they both accumulated on the plasma membrane after 20-min exposure to VP or forskolin. Following membrane accumulation, S261A, S261D, and wild-type AQP2 reinternalization was complete over a similar time frame, between 30 and 60 min after VP washout. Using various combinations of point mutations, we showed that the phosphorylation state of S256 is dominant with respect to AQP2 behavior; AQP2 membrane accumulation and internalization were not detectably affected by the phosphorylation state of S261. Finally, blocking AQP2 endocytosis by methyl-beta-cyclodextrin caused membrane accumulation of AQP2 in cells expressing either a single S-A mutation or double mutations of S256 and S261, although as previously reported, the S256D mutation was always present at the cell surface. This suggests that constitutive recycling of AQP2 was not modified by the phosphorylation state of S261. Together, our data indicate that the phosphorylation state of AQP2 at S261 does not detectably affect regulated or constitutive trafficking of AQP2. The potential role of S261 phosphorylation/dephosphorylation in vasopressin action remains to be determined.

Phosphorylation events and the modulation of aquaporin 2 cell surface expression.

Purpose of reviewThis review highlights the role of phosphorylation in the trafficking and targeting of aquaporin 2. Current knowledge will be put into the context of modulating the cell surface expression of aquaporin 2 by vasopressin in renal epithelial cells, which is critical for regulation of urinary concentration and control of fluid and electrolyte homeostasis. Recent findingsIn addition to previously identified phosphorylation sites on aquaporin 2, new data have revealed three other serine residues in the C-terminus whose phosphorylation is altered by vasopressin. Several steps in aquaporin 2 recycling, including exocytosis and endocytosis, are coordinated by phosphorylation and dephosphorylation to regulate cell surface accumulation. Aquaporin 2 phosphorylation on serine 256 regulates aquaporin 2 association with proteins that are involved in trafficking, including hsc/hsp70 and myelin and lymphocyte-associated protein. SummaryAquaporin 2 trafficking is regulated by phosphorylation of serine 256 and other amino acid residues in its cytoplasmic domain. These events increase or decrease interaction of aquaporin 2 with key regulatory proteins to determine the cellular distribution and fate of aquaporin 2, both after vasopressin addition and under baseline conditions. Better understanding of these mechanisms may provide new therapeutic avenues for patients with X-linked nephrogenic diabetes insipidus, as well as providing basic cell biological information relevant to membrane trafficking processes in general.

Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel

The aquaporin-2 (AQP2) water channel relocates mainly to the apical plasma membrane of collecting duct principal cells after vasopressin (VP) stimulation. AQP2 transport to this membrane domain is assumed to be a direct route involving recycling of intracellular vesicles. However, basolateral plasma membrane expression of AQP2 is observed in vivo in principal cells. Here, we asked whether there is a transcytotic pathway of AQP2 trafficking between apical and basolateral membranes. We used MDCK cells in which AQP2 normally accumulates apically after VP exposure. In contrast, both site-specific biotinylation and immunofluorescence showed that AQP2 is strongly accumulated in the basolateral membrane, along with the endocytic protein clathrin, after a brief cold shock (4°C). This suggests that AQP2 may be constitutively targeted to basolateral membranes and then retrieved by clathrin-mediated endocytosis at physiological temperatures. Rab11 does not accumulate in basolateral membranes after cold shock, suggesting that the AQP2 in this location is not associated with Rab11-positive vesicles. After rewarming (37°C), basolateral AQP2 staining is diminished and it subsequently accumulates at the apical membrane in the presence of VP/forskolin, suggesting that transcytosis can be followed by apical insertion of AQP2. This process is inhibited by treatment with colchicine. Our data suggest that the cold shock procedure reveals the presence of microtubule-dependent AQP2 transcytosis, which represents an indirect pathway of apical AQP2 delivery in these cells. Furthermore, our data indicate that protein polarity data obtained from biotinylation assays, which require cells to be cooled to 4°C during the labeling procedure, should be interpreted with caution.

Bypassing Vasopressin Receptor Signaling Pathways in Nephrogenic Diabetes Insipidus

Water reabsorption in the kidney represents a critical physiological event in the maintenance of body water homeostasis. This highly regulated process relies largely on vasopressin (VP) action and on the VP-sensitive water channel (AQP2) that is expressed in principal cells of the kidney collecting duct. Defects in the VP signaling pathway and/or in AQP2 cell surface expression can lead to an inappropriate reduction in renal water reabsorption and the development of nephrogenic diabetes insipidus, a disease characterized by polyuria and polydipsia. This review focuses on the major regulatory steps that are involved in AQP2 trafficking and function. Specifically, we begin with a discussion on VP-receptor-independent mechanisms of AQP2 trafficking, with special emphasis on the nitric oxide-cyclic guanosine monophosphate signaling pathway, followed by a review of the mechanisms that govern AQP2 endocytosis and exocytosis. We then discuss emerging data illustrating roles played by the actin cytoskeleton on AQP2 trafficking, and lastly we consider elements that affect AQP2 protein expression in cells. Recent advances in each topic are summarized and are presented in the context of their potential to serve as a basis for the development of novel therapies that may ultimately improve life quality of nephrogenic diabetes insipidus patients.

Acute hypertonicity alters aquaporin-2 trafficking and induces a MAPK-dependent accumulation at the plasma membrane of renal epithelial cells.

The unique phenotype of renal medullary cells allows them to survive and functionally adapt to changes of interstitial osmolality/tonicity. We investigated the effects of acute hypertonic challenge on AQP2 (aquaporin-2) water channel trafficking. In the absence of vasopressin, hypertonicity alone induced rapid (<10 min) plasma membrane accumulation of AQP2 in rat kidney collecting duct principal cells in situ, and in several kidney epithelial lines. Confocal microscopy revealed that AQP2 also accumulated in the trans-Golgi network (TGN) following hypertonic challenge. AQP2 mutants that mimic the Ser256-phosphorylated and -nonphosphorylated state accumulated at the cell surface and TGN, respectively. Hypertonicity did not induce a change in cytosolic cAMP concentration, but inhibition of either calmodulin or cAMP-dependent protein kinase A activity blunted the hypertonicity-induced increase of AQP2 cell surface expression. Hypertonicity increased p38, ERK1/2, and JNK MAPK activity. Inhibiting MAPK activity abolished hypertonicity-induced accumulation of AQP2 at the cell surface but did not affect either vasopressin-dependent AQP2 trafficking or hypertonicity-induced AQP2 accumulation in the TGN. Finally, increased AQP2 cell surface expression induced by hypertonicity largely resulted from a reduction in endocytosis but not from an increase in exocytosis. These data indicate that acute hypertonicity profoundly alters AQP2 trafficking and that hypertonicity-induced AQP2 accumulation at the cell surface depends on MAP kinase activity. This may have important implications on adaptational processes governing transcellular water flux and/or cell survival under extreme conditions of hypertonicity.

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 β1, facilitating its turnover at focal adhesions. In vitro, disturbing the interaction between AQP2 and integrin β1 by mutating the RGD motif led to reduced endocytosis, retention of integrin β1 at the cell surface, and defective cell migration and tubulogenesis. Similarly, in vivo, AQP2-null mice exhibited significant retention of integrin β1 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.

Calcitonin Has a Vasopressin-like Effect on Aquaporin-2 Trafficking and Urinary Concentration

The most common cause of hereditary nephrogenic diabetes insipidus is a nonfunctional vasopressin (VP) receptor type 2 (V2R). Calcitonin, another ligand of G-protein-coupled receptors, has a VP-like effect on electrolytes and water reabsorption, suggesting that it may affect AQP2 trafficking. Here, calcitonin increased intracellular cAMP and stimulated the membrane accumulation of AQP2 in LLC-PK1 cells. Pharmacologic inhibition of protein kinase A (PKA) and deficiency of a critical PKA phosphorylation site on AQP2 both prevented calcitonin-induced membrane accumulation of AQP2. Fluorescence assays showed that calcitonin led to a 70% increase in exocytosis and a 20% decrease in endocytosis of AQP2. Immunostaining of rat kidney slices demonstrated that calcitonin induced a significant redistribution of AQP2 to the apical membrane of principal cells in cortical collecting ducts and connecting segments but not in the inner stripe or inner medulla. Calcitonin-treated VP-deficient Brattleboro rats had a reduced urine flow and two-fold higher urine osmolality during the first 12 hours of treatment compared with control groups. Although this VP-like effect of calcitonin diminished over the following 72 hours, the tachyphylaxis was reversible. Taken together, these data show that calcitonin induces cAMP-dependent AQP2 trafficking in cortical collecting and connecting tubules in parallel with an increase in urine concentration. This suggests that calcitonin has a potential therapeutic use in nephrogenic diabetes insipidus.

A zebrafish model of conditional targeted podocyte ablation and regeneration

Podocytes are specialized cells that contribute critically to the normal structure and function of the glomerular filtration barrier. Their depletion plays an important role in the pathogenesis of glomerulosclerosis. Here, we report generation of a genetic model of conditional podocyte ablation and regeneration in zebrafish using a bacterial nitroreductase strategy to convert a prodrug, Metronidazole, into a cytotoxic metabolite. A transgenic zebrafish line was generated that expresses a green fluorescence protein (GFP) and the nitroreductase fusion protein under the control of the podocin promoter Tg(podocin:nitroreductase-GFP). Treatment of these transgenic zebrafish with Metronidazole results in podocyte apoptosis, a loss of nephrin and podocin expression, foot process effacement, and a leaky glomerular filtration barrier. Following Metronidazole washout, proliferating cells were detected in the glomeruli of recovering transgenic fish with a restoration of nitroreductase-GFP fluorescence, nephrin and podocin expression, a reestablishment of normal foot process architecture and glomerular barrier function. Thus, our studies show that zebrafish podocytes are capable of regenerating following depletion and establish the Tg(podocin:NTR-GFP) fish as a new model to study podocyte injury and repair.

A fluorimetry-based ssYFP secretion assay to monitor vasopressin-induced exocytosis in LLC-PK1 cells expressing aquaporin-2.

Vasopressin (VP)-induced exocytosis was dissected in native and aquaporin-2 (AQP2)-expressing renal LLC-PK(1) cells by a fluorimetric exocytosis assay based on soluble secreted yellow fluorescent protein (ssYFP). YFP was targeted to the secretory pathway by addition of an 18-amino acid signal peptide from hen egg white lysozyme. Immunofluorescence labeling, together with analysis of Alexa 555-dextran internalization, revealed that ssYFP is exclusively located in the secretory pathway. Immunofluorescence and immunogold electron microscopy showed significant colocalization of ssYFP and AQP2. Fluorimetry and Western blot analysis demonstrated similar constitutive ssYFP secretion in native LLC-PK(1) and AQP2-expressing cells. In AQP2-expressing cells, a twofold increase in ssYFP secretion was observed within 15 min of VP stimulation. This transient burst of ssYFP secretion was abolished by the PKA inhibitor H-89 and was not observed in native cells. The endocytotic inhibitor methyl-beta-cyclodextrin, which also promotes membrane accumulation of AQP2, had no effect on ssYFP secretion. Although cells expressing phosphorylation-deficient AQP2-S256A showed significantly lower baseline levels of constitutive secretion, VP induced a significant increase in exocytosis. Our data indicate that 1) this assay can monitor exocytosis in cultured epithelial cells, 2) VP has an acute stimulatory effect on ssYFP secretion in AQP2-expressing, but not native, cells, and 3) phosphorylation of AQP2 at S256 may be involved in the regulation of constitutive AQP2 exocytosis and play only a minor role in the VP-induced burst. These results support the idea that, in addition to its role in reducing AQP2 endocytosis, VP increases AQP2 exocytosis.