Kidney tubular transcription co-activator, Yes-associated protein 1 (YAP), controls the expression of collecting duct aquaporins and water homeostasis.

Final urine volume and concentration are defined by water reabsorption through the water channel proteins aquaporin (AQP)-2, -3 and -4 in the collecting duct. However, the transcriptional regulation of these AQPs is not well understood. The Hippo/Yes-associated protein 1 (YAP) pathway plays an important role in organ size control and tissue homeostasis. When the Hippo pathway including the Mst1/Mst2 kinases is inhibited, YAP is activated and functions as a transcription co-activator. Our previous work revealed a pathological role of tubular YAP activation in chronic kidney disease, but the physiological role of YAP in the kidney remains to be established. Here, we found that tubule-specific Yap knockout mice showed increased urine output and decreased urinary osmolality. Decreases in Aqp2, -3 and -4 mRNA and protein abundance in the kidney were evident in Yap knockout mice. Analysis of Mst1/Mst2 double knockout and Mst1/Mst2/Yap triple knockout mice showed that expression of Aqp2 and Aqp4 but not Aqp3 was dependent on YAP. Furthermore, YAP was recruited to the promoters of the Aqp2 and Aqp4 genes and stimulated their transcription. Interestingly, YAP was found to interact with transcription factors GATA2, GATA3 and NFATc1. These three factors promoted Aqp2 transcription in a YAP dependent manner in collecting duct cells. These three factors also promoted Aqp4 transcription whereas only GATA2 and GATA3 enhanced Aqp3 transcription. Thus, our results suggest that YAP promotes Aqp2 and Aqp4 transcription, interacts with GATA2, GATA3 and NFATc1 to control Aqp2 expression, while Aqp-2, -3 and -4 exploit overlapping mechanisms for their baseline transcriptional regulation.

Gentamicin-Induced Acute Kidney Injury in an Animal Model Involves Programmed Necrosis of the Collecting Duct.

BACKGROUND: Gentamicin is a potent aminoglycoside antibiotic that targets gram-negative bacteria, but nephrotoxicity limits its clinical application. The cause of gentamicin-induced AKI has been attributed mainly to apoptosis of the proximal tubule cells. However, blocking apoptosis only partially attenuates gentamicin-induced AKI in animals. METHODS: Mice treated with gentamicin for 7 days developed AKI, and programmed cell death pathways were examined using pharmacologic inhibitors and in RIPK3-deficient mice. Effects in porcine and murine kidney cell lines were also examined. RESULTS: Gentamicin caused a low level of apoptosis in the proximal tubules and significant ultrastructural alterations consistent with necroptosis, occurring predominantly in the collecting ducts (CDs), including cell and organelle swelling and rupture of the cell membrane. Upregulation of the key necroptotic signaling molecules, mixed lineage kinase domain-like pseudokinase (MLKL) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), was detected in gentamicin-treated mice and in cultured renal tubule cells. In addition, gentamicin induced apical accumulation of total and phosphorylated MLKL (pMLKL) in CDs in mouse kidney. Inhibiting a necroptotic protein, RIPK1, with necrostatin-1 (Nec-1), attenuated gentamicin-induced necrosis and upregulation of MLKL and RIPK3 in mice and cultured cells. Nec-1 also alleviated kidney inflammation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, deletion of RIPK3 in the Ripk3-/- mice significantly attenuated gentamicin-induced AKI. CONCLUSIONS: A previously unrecognized role of programmed necrosis in collecting ducts in gentamicin-induced kidney injury presents a potential new therapeutic strategy to alleviate gentamicin-induced AKI through inhibiting necroptosis.

Integrin-Linked Kinase Deficiency in Collecting Duct Principal Cell Promotes Necroptosis of Principal Cell and Contributes to Kidney Inflammation and Fibrosis.

BACKGROUND: Necroptosis is a newly discovered cell death pathway that plays a critical role in AKI. The involvement of integrin-linked kinase (ILK) in necroptosis has not been studied. METHODS: We performed experiments in mice with an Ilk deletion in collecting duct (CD) principal cells (PCs), and cultured tubular epithelial cells treated with an ILK inhibitor or ILK siRNA knockdown. RESULTS: Ilk deletion in CD PCs resulted in acute tubular injury and early mortality in mice. Progressive interstitial fibrosis and inflammation associated with the activation of the canonical TGF-ß signaling cascade were detected in the kidneys of the mice lacking ILK in the CD PCs. In contrast to the minimal apoptosis detected in the animals' injured CDs, widespread necroptosis was present in ILK-deficient PCs, characterized by cell swelling, deformed mitochondria, and rupture of plasma membrane. In addition, ILK deficiency resulted in increased expression and activation of necroptotic proteins MLKL and RIPK3, and membrane translocation of MLKL in CD PCs. ILK inhibition and siRNA knockdown reduced cell survival in cultured tubular cells, concomitant with increased membrane accumulation of MLKL and/or phospho-MLKL. Administration of a necroptosis inhibitor, necrostatin-1, blocked cell death in vitro and significantly attenuated inflammation, interstitial fibrosis, and renal failure in ILK-deficient mice. CONCLUSIONS: The study demonstrates the critical involvement of ILK in necroptosis through modulation of the RIPK3 and MLKL pathway and highlights the contribution of CD PC injury to the development of inflammation and interstitial fibrosis of the kidney.

Pathological cardiac remodeling occurs early in CKD mice from unilateral urinary obstruction, and is attenuated by Enalapril.

Cardiovascular disease constitutes the leading cause of mortality in patients with chronic kidney disease (CKD) and end-stage renal disease. Despite increasing recognition of a close interplay between kidney dysfunction and cardiovascular disease, termed cardiorenal syndrome (CRS), the underlying mechanisms of CRS remain poorly understood. Here we report the development of pathological cardiac hypertrophy and fibrosis in early stage non-uremic CKD. Moderate kidney failure was induced three weeks after unilateral urinary obstruction (UUO) in mice. We observed pathological cardiac hypertrophy and increased fibrosis in UUO-induced CKD (UUO/CKD) animals. Further analysis indicated that this cardiac fibrosis was associated with increased expression of transforming growth factor ß (TGF-ß) along with significant upregulation of Smad 2/3 signaling in the heart. Moreover early treatment of UUO/CKD animals with an angiotensin-converting-enzyme inhibitor (ACE I), Enalapril, significantly attenuated cardiac fibrosis. Enalapril antagonized activation of the TGF-ß signaling pathway in the UUO/CKD heart. In summary our study demonstrates the presence of pathological cardiac hypertrophy and fibrosis in mice early in UUO-induced CKD, in association with early activation of the TGF-ß/Smad signaling pathway. We also demonstrate the beneficial effect of ACE I in alleviating this early fibrogenic process in the heart in UUO/CKD animals.

Manganese promotes intracellular accumulation of AQP2 via modulating F-actin polymerization and reduces urinary concentration in mice.

Aquaporin-2 (AQP2) is a water channel protein expressed in principal cells (PCs) of the kidney collecting ducts (CDs) and plays a critical role in mediating water reabsorption and urine concentration. AQP2 undergoes both regulated trafficking mediated by vasopressin (VP) and constitutive recycling, which is independent of VP. For both pathways, actin cytoskeletal dynamics is a key determinant of AQP2 trafficking. We report here that manganese chloride (MnCl2) is a novel and potent regulator of AQP2 trafficking in cultured cells and in the kidney. MnCl2 treatment promoted internalization and intracellular accumulation of AQP2. The effect of MnCl2 on the intracellular accumulation of AQP2 was associated with activation of RhoA and actin polymerization without modification of AQP2 phosphorylation. Although the level of total and phosphorylated AQP2 did not change, MnCl2 treatment impeded VP-induced phosphorylation of AQP2 at its serine-256, -264, and -269 residues and dephosphorylation at serine 261. In addition, MnCl2 significantly promoted F-actin polymerization along with downregulation of RhoA activity and prevented VP-induced membrane accumulation of AQP2. Finally, MnCl2 treatment in mice resulted in significant polyuria and reduced urinary concentration, likely due to intracellular relocation of AQP2 in the PCs of kidney CDs. More importantly, the reduced urinary concentration caused by MnCl2 treatment in animals was not corrected by VP. In summary, our study identified a novel effect of MnCl2 on AQP2 trafficking through modifying RhoA activity and actin polymerization and uncovered its potent impact on water diuresis in vivo.

Ultrastructural Characterization of the Glomerulopathy in Alport Mice by Helium Ion Scanning Microscopy (HIM).

The glomerulus exercises its filtration barrier function by establishing a complex filtration apparatus consisting of podocyte foot processes, glomerular basement membrane and endothelial cells. Disruption of any component of the glomerular filtration barrier leads to glomerular dysfunction, frequently manifested as proteinuria. Ultrastructural studies of the glomerulus by transmission electron microscopy (TEM) and conventional scanning electron microscopy (SEM) have been routinely used to identify and classify various glomerular diseases. Here we report the application of newly developed helium ion scanning microscopy (HIM) to examine the glomerulopathy in a Col4a3 mutant/Alport syndrome mouse model. Our study revealed unprecedented details of glomerular abnormalities in Col4a3 mutants including distorted podocyte cell bodies and disorganized primary processes. Strikingly, we observed abundant filamentous microprojections arising from podocyte cell bodies and processes, and presence of unique bridging processes that connect the primary processes and foot processes in Alport mice. Furthermore, we detected an altered glomerular endothelium with disrupted sub-endothelial integrity. More importantly, we were able to clearly visualize the complex, three-dimensional podocyte and endothelial interface by HIM. Our study demonstrates that HIM provides nanometer resolution to uncover and rediscover critical ultrastructural characteristics of the glomerulopathy in Col4a3 mutant mice.

Re-characterization of the Glomerulopathy in CD2AP Deficient Mice by High-Resolution Helium Ion Scanning Microscopy.

Helium ion scanning microscopy (HIM) is a novel technology that directly visualizes the cell surface ultrastructure without surface coating. Despite its very high resolution, it has not been applied extensively to study biological or pathology samples. Here we report the application of this powerful technology to examine the three-dimensional ultrastructural characteristics of proteinuric glomerulopathy in mice with CD2-associated protein (CD2AP) deficiency. HIM revealed the serial alteration of glomerular features including effacement and disorganization of the slit diaphragm, followed by foot process disappearance, flattening and fusion of major processes, and eventual transformation into a podocyte sheet as the disease progressed. The number and size of the filtration slit pores decreased. Strikingly, numerous "bleb" shaped microprojections were observed extending from podocyte processes and cell body, indicating significant membrane dynamics accompanying CD2AP deficiency. Visualizing the glomerular endothelium and podocyte-endothelium interface revealed the presence of endothelial damage, and disrupted podocyte and endothelial integrity in 6 week-old Cd2ap-KO mice. We used the HIM technology to investigate at nanometer scale resolution the ultrastructural alterations of the glomerular filtration apparatus in mice lacking the critical slit diaphragm-associated protein CD2AP, highlighting the great potential of HIM to provide new insights into the biology and (patho)physiology of glomerular diseases.

Deletion of ß1-integrin in collecting duct principal cells leads to tubular injury and renal medullary fibrosis.

The renal collecting duct (CD) contains two major cell types, intercalated (ICs) and principal cells (PCs). A previous report showed that deletion of ß1-integrin in the entire renal CD causes defective CD morphogenesis resulting in kidney dysfunction. However, subsequent deletion of ß1-integrin specifically in ICs and PCs, respectively, did not cause any morphological defects in the CDs. The discrepancy between these studies prompts us to reinvestigate the role of ß1-integrin in CD cells, specifically in the PCs. We conditionally deleted ß1-integrin in mouse CD PCs using a specific aquaporin-2 (AQP2) promoter Cre-LoxP system. The resulting mutant mice, ß-1f/fAQP2-Cre+, had lower body weight, failed to thrive, and died around 8-12 wk. Their CD tubules were dilated, and some of them contained cellular debris. Increased apoptosis and proliferation of PCs were observed in the dilated CDs. Trichrome staining and electron microscopy revealed the presence of peritubular and interstitial fibrosis that is associated with increased production of extracellular matrix proteins including collagen type IV and fibronectin, as detected by immunoblotting. Further analysis revealed a significantly increased expression of transforming growth factor-ß (TGF-ß)-induced protein, fibronectin, and TGF-ß receptor-1 mRNAs and concomitantly increased phosphorylation of SMAD-2 that indicates the activation of the TGF-ß signaling pathway. Therefore, our data reveal that normal expression of ß1-integrin in PCs is a critical determinant of CD structural and functional integrity and further support the previously reported critical role of ß1-integrin in the development and/or maintenance of the CD structure and function.

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.

Polarized Trafficking of AQP2 Revealed in Three Dimensional Epithelial Culture.

In renal collecting duct (CD) principal cells (PCs), vasopressin (VP) acts through its receptor, V2R, to increase intracellular cAMP leading to phosphorylation and apical membrane accumulation of the water channel aquaporin 2 (AQP2). The trafficking and function of basolaterally located AQP2 is, however, poorly understood. Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking. This model recapitulates the luminal architecture of the CD and bi-polarized distribution of AQP2 as seen in kidney. Without stimulation, AQP2 is located in the subapical and basolateral regions. Treatment with VP, forskolin (FK), or 8-(4-Chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate (CPT-cAMP) leads to translocation of cytosolic AQP2 to the apical membrane, but not to the basolateral membrane. Treating cells with methyl-ß-cyclodextrin (mßCD) to acutely block endocytosis causes accumulation of AQP2 on the basolateral membrane, but not on the apical membrane. Our data suggest that AQP2 may traffic differently at the apical and basolateral domains in this 3D epithelial model. In addition, application of a panel of phosphorylation specific AQP2 antibodies reveals the polarized, subcellular localization of differentially phosphorylated AQP2 at S256, S261, S264 and S269 in the 3D culture model, which is consistent with observations made in the CDs of VP treated animals, suggesting the preservation of phosphorylation dependent regulatory mechanism of AQP2 trafficking in this model. Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2. The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro.

High-throughput chemical screening identifies AG-490 as a stimulator of aquaporin 2 membrane expression and urine concentration.

A reduction or loss of plasma membrane aquaporin 2 (AQP2) in kidney principal cells due to defective vasopressin (VP) signaling through the VP receptor causes excessive urine production, i.e., diabetes insipidus. The amount of AQP2 on the plasma membrane is regulated by a balance of exocytosis and endocytosis and is the rate limiting step for water reabsorption in the collecting duct. We describe here a systematic approach using high-throughput screening (HTS) followed by in vitro and in vivo assays to discover novel compounds that enhance vasopressin-independent AQP2 membrane expression. We performed initial chemical library screening with a high-throughput exocytosis fluorescence assay using LLC-PK1 cells expressing soluble secreted yellow fluorescent protein and AQP2. Thirty-six candidate exocytosis enhancers were identified. These compounds were then rescreened in AQP2-expressing cells to determine their ability to increase AQP2 membrane accumulation. Effective drugs were then applied to kidney slices in vitro. Three compounds, AG-490, ß-lapachone, and HA14-1 increased AQP2 membrane accumulation in LLC-PK1 cells, and both AG-490 and ß-lapachone were also effective in MDCK cells and principal cells in rat kidney slices. Finally, one compound, AG-490 (an EGF receptor and JAK-2 kinase inhibitor), decreased urine volume and increased urine osmolality significantly in the first 2-4 h after a single injection into VP-deficient Brattleboro rats. In conclusion, we have developed a systematic procedure for identifying new compounds that modulate AQP2 trafficking using initial HTS followed by in vitro assays in cells and kidney slices, and concluding with in vivo testing in an animal model.

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.

Differential, phosphorylation dependent trafficking of AQP2 in LLC-PK1 cells.

The kidney maintains water homeostasis by modulating aquaporin 2 (AQP2) on the plasma membrane of collecting duct principal cells in response to vasopressin (VP). VP mediated phosphorylation of AQP2 at serine 256 is critical for this effect. However, the role of phosphorylation of other serine residues in the AQP2 C-terminus is less well understood. Here, we examined the effect of phosphorylation of S256, S261 and S269 on AQP2 trafficking and association with recycling pathway markers. We used LLC-PK1 cells expressing AQP2(S-D) or (S-A) phospho mutants and a 20°C cold block, which allows endocytosis to continue, but prevents protein exit from the trans Golgi network (TGN), inducing formation of a perinuclear AQP2 patch. AQP2-S256D persists on the plasma membrane during cold block, while wild type AQP2, AQP2-S256A, S261A, S269A and S269D are internalized and accumulate in the patch. Development of this patch, a measure of AQP2 internalization, was most rapid with AQP2-S256A, and slowest with S261A and S269D. AQP2-S269D exhibited a biphasic internalization profile with a significant amount not internalized until 150 minutes of cold block. After rewarming to 37°C, wt AQP2, AQP2-S261A and AQP2-S269D rapidly redistributed throughout the cytoplasm within 20 minutes, whereas AQP2-S256A dissipated more slowly. Colocalization of AQP2 mutants with several key vesicular markers including clathrin, HSP70/HSC70, EEA, GM130 and Rab11 revealed no major differences. Overall, our data provide evidence supporting the role of S256 and S269 in the maintenance of AQP2 at the cell surface and reveal the dynamics of internalization and recycling of differentially phosphorylated AQP2 in cell culture.

Simvastatin enhances aquaporin-2 surface expression and urinary concentration in vasopressin-deficient Brattleboro rats through modulation of Rho GTPase.

Statins are 3-hydroxyl-3-methyglutaryl-CoA reductase inhibitors that are commonly used to inhibit cholesterol biosynthesis. Emerging data have suggested that they also have "pleotropic effects," including modulating actin cytoskeleton reorganization. Here, we report an effect of simvastatin on the trafficking of aquaporin-2 (AQP2). Specifically, simvastatin induced the membrane accumulation of AQP2 in cell cultures and kidneys in situ. The effect of simvastatin was independent of protein kinase A activation and phosphorylation at AQP2-Ser(256), a critical event involved in vasopressin (VP)-regulated AQP2 trafficking. Further investigation showed that simvastatin inhibited endocytosis in parallel with downregulation of RhoA activity. Overexpression of active RhoA attenuated simvastatin's effect, suggesting the involvement of this small GTPase in simvastatin-mediated AQP2 trafficking. Finally, the effect of simvastatin on urinary concentration was investigated in VP-deficient Brattleboro rats. Simvastatin acutely (3-6 h) increased urinary concentration and decreased urine output in these animals. In summary, simvastatin regulates AQP2 trafficking in vitro and urinary concentration in vivo via events involving downregulation of Rho GTPase activity and inhibition of endocytosis. Our study provides an alternative mechanism to regulate AQP2 trafficking, bypassing the VP-vasopressin receptor signaling pathway.