Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges.

Therapeutic genome editing: prospects and challenges

Neuropeptides are the most diverse class of chemical modulators in nervous systems They contribute to extensive modulation of circuit activity and have profound influences on animal physiology Studies on invertebrate model organisms, including the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, have enabled the genetic manipulation of peptidergic signalling, contributing to an understanding of how neuropeptides pattern the output of neural circuits to underpin behavioural adaptation Electrophysiological and pharmacological analyses of well-defined microcircuits, such as the crustacean stomatogastric ganglion, have provided detailed insights into neuropeptide functions at a cellular and circuit level These approaches can be increasingly applied in the mammalian brain by focusing on circuits with a defined and identifiable sub-population of neurons Functional analyses of neuropeptide systems have been underpinned by systematic studies to map peptidergic networks Here, we review the general principles and mechanistic insights that have emerged from these studies We also highlight some of the challenges that remain for furthering our understanding of the functional relevance of peptidergic modulation

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The long and the short of it - a perspective on peptidergic regulation of circuits and behaviour.

Background The bile acid-activated receptors, including the membrane G protein-coupled receptor TGR5 and nuclear farnesoid X receptor (FXR), have roles in kidney diseases. In this study, we investigated the role of TGR5 in renal water handling and the underlying molecular mechanisms. Methods We used tubule suspensions of inner medullary collecting duct (IMCD) cells from rat kidneys to investigate the effect of TGR5 signaling on aquaporin-2 (AQP2) expression, and examined the in vivo effects of TGR5 in mice with lithium-induced nephrogenic diabetes insipidus (NDI) and Tgr5 knockout (Tgr5 -/-) mice. Results Activation of TGR5 by lithocholic acid (LCA), an endogenous TGR5 ligand, or INT-777, a synthetic TGR5-specific agonist, induced AQP2 expression and intracellular trafficking in rat IMCD cells via a cAMP-protein kinase A signaling pathway. In mice with NDI, dietary supplementation with LCA markedly decreased urine output and increased urine osmolality, which was associated with significantly upregulated AQP2 expression in the kidney inner medulla. Supplementation with endogenous FXR agonist had no effect. In primary IMCD suspensions from lithium-treated rats, treatment with INT-767 (FXR and TGR5 dual agonist) or INT-777, but not INT-747 (FXR agonist), increased AQP2 expression. Tgr5 -/- mice exhibited an attenuated ability to concentrate urine in response to dehydration, which was associated with decreased AQP2 expression in the kidney inner medulla. In lithium-treated Tgr5 -/- mice, LCA treatment failed to prevent reduction of AQP2 expression. Conclusions TGR5 stimulation increases renal AQP2 expression and improves impaired urinary concentration in lithium-induced NDI. TGR5 is thus involved in regulating water metabolism in the kidney.

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Bile Acid G Protein-Coupled Membrane Receptor TGR5 Modulates Aquaporin 2-Mediated Water Homeostasis.

Recent evidence suggests that elevated plasma levels of Trimethylamine-N-oxide (TMAO) can prolong the duration of elevated blood pressure in rats. The purpose of this study was to investigate the plasma TMAO level in Spontaneously Hypertensive Rats (SHR) and to explore the possible relationship between TMAO and aquaporin-2 (AQP-2) in the formation of hypertension. Twelve-week-old, male Spontaneously Hypertensive rats (SHR, n = 40) and Wistar-Kyoto rats (WKY, n = 40) were accordingly grouped into SHR group and WKY group. Each group was divided randomly into four subgroups: Untreated group, TMAO group, TMAO+Tolvaptan (TMAO+TVP) group, and TVP group, respectively. Systolic blood pressure (SBP), plasma TMAO, plasma osmolality (POsm), plasma vasopressin (PAVP), and plasma AQP-2 (PAQP-2) concentration were measured, and the expression of AQP-2 in kidney medulla was detected by RT-PCR and Western blot. At 14 weeks, rats in SHR TMAO group were shown the increased plasma TMAO, POsm, PAVP, and PAQP-2 levels, while those rats in SHR TMAO+TVP group were shown the decreased plasma TMAO, POsm, and PAQP-2 levels, but an even higher PAVP (due to the blockage of TVP to V2 receptor). These findings indicate that an increase of plasma TMAO levels in SHR leads to a higher plasma osmotic pressure, triggers the regulation of the TMAO-AVP-AQP-2 axis in SHR, elicits the greater water reabsorption, and eventually leads to hypertension.

Trimethylamine-N-oxide (TMAO) increased aquaporin-2 expression in spontaneously hypertensive rats.

Aquaporin-2, a member of the aquaporin family, is an arginine vasopressin-regulated water channel expressed in the renal collecting duct, and a promising marker of the concentrating and diluting ability of the kidney. The arginine vasopressin type-2 antagonist, tolvaptan, is a new-generation diuretic; it is especially indicated in patients with decompensated heart failure refractory to conventional diuretics. However, the ideal responders to tolvaptan have not yet been identified, and non-responders experience worse clinical courses despite treatment with tolvaptan. Urine aquaporin-2 has recently been demonstrated as a promising predictor of response to tolvaptan. We here validated aquaporin-2-guided tolvaptan therapy in patients with decompensated heart failure. Long-term efficacy of tolvaptan treatment in the responders defined by aquaporin-2 needs to be validated in the future prospective study.

https://www.mdpi.com/1422-0067/17/1/105/pdf

Urine Aquaporin-2: A Promising Marker of Response to the Arginine Vasopressin Type-2 Antagonist, Tolvaptan in Patients with Congestive Heart Failure.

The kidney collecting duct is an important renal tubular segment for the regulation of body water and salt homeostasis. Water reabsorption in the collecting duct cells is regulated by arginine vasopressin (AVP) via the vasopressin V2-receptor (V2R). AVP increases the osmotic water permeability of the collecting duct cells through aquaporin-2 (AQP2) and aquaporin-3 (AQP3). AVP induces the apical targeting of AQP2 and transcription of AQP2 gene in the kidney collecting duct principal cells. The signaling transduction pathways resulting in the AQP2 trafficking to the apical plasma membrane of the collecting duct principal cells, include AQP2 phosphorylation, RhoA phosphorylation, actin depolymerization and calcium mobilization, and the changes of AQP2 protein abundance in water balance disorders have been extensively studied. These studies elucidate the underlying cellular and molecular mechanisms of body water homeostasis and provide the basis for the treatment of body water balance disorders.

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A Minireview on Vasopressin-regulated Aquaporin-2 in Kidney Collecting Duct Cells.

Sorting nexin 27 (SNX27), a PDZ (Postsynaptic density-95/Discs large/Zonula occludens 1) domain-containing protein, cooperates with a retromer complex, which regulates intracellular trafficking and the abundance of membrane proteins. Since the carboxyl terminus of aquaporin-2 (AQP2c) has a class I PDZ-interacting motif (X-T/S-X-Φ), the role of SNX27 in the regulation of AQP2 was studied. Co-immunoprecipitation assay of the rat kidney demonstrated an interaction of SNX27 with AQP2. Glutathione S-transferase (GST) pull-down assays revealed an interaction of the PDZ domain of SNX27 with AQP2c. Immunocytochemistry of HeLa cells co-transfected with FLAG-SNX27 and hemagglutinin (HA)-AQP2 also revealed co-localization throughout the cytoplasm. When the PDZ domain was deleted, punctate HA-AQP2 labeling was localized in the perinuclear region. The labeling was intensively overlaid by Lysotracker staining but not by GM130 labeling, a cis-Golgi marker. In rat kidneys and primary cultured inner medullary collecting duct cells, the subcellular redistribution of SNX27 was similar to AQP2 under 1-deamino-8-D-arginine vasopressin (dDAVP) stimulation/withdrawal. Cell surface biotinylation assay showed that dDAVP-induced AQP2 translocation to the apical plasma membrane was unaffected after SNX27 knockdown in mpkCCD cells. In contrast, the dDAVP-induced AQP2 protein abundance was significantly attenuated without changes in AQP2 mRNA expression. Moreover, the AQP2 protein abundance was markedly declined during the dDAVP withdrawal period after stimulation under SNX27 knockdown, which was inhibited by lysosome inhibitors. Autophagy was induced after SNX27 knockdown in mpkCCD cells. Lithium-induced nephrogenic diabetes insipidus in rats revealed a significant downregulation of SNX27 in the kidney inner medulla. Taken together, the PDZ domain-containing SNX27 interacts with AQP2 and depletion of SNX27 contributes to the autophagy-lysosomal degradation of AQP2.

https://www.mdpi.com/2073-4409/9/5/1208/pdf

Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct.

Background and purpose The peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V2 receptor, which triggers a cAMP-mediated activation of a protein kinase A (PKA)-dependent signaling network. The protein kinases downstream from PKA have not been fully identified or mapped to regulated phosphoproteins. Experimental approach We carried out systems-level analysis of large-scale phosphoproteomic data quantifying vasopressin-induced changes in phosphorylation in aquaporin-2-expressing cultured collecting duct cells (mpkCCD). Quantification was done using stable isotope labeling (SILAC method). Key results 9640 phosphopeptides were quantified. Stringent statistical analysis identified significant changes in response to vasopressin in 429 of these phosphopeptides. The corresponding phosphoproteins were mapped to known vasopressin-regulated cellular processes. The vasopressin-regulated sites were classified according to the sequences surrounding the phosphorylated amino acids giving 11 groups. Among the vasopressin-regulated phosphoproteins were 25 distinct protein kinases. Among these, six plus PKA appeared to account for phosphorylation of about 81% of the 313 vasopressin-regulated phosphorylation sites. The six downstream kinases were salt-inducible kinase 2 (Sik2), cyclin-dependent kinase 18 (Cdk18/PCTAIRE-3), calmodulin-dependent kinase kinase 2 (Camkk2), protein kinase D2 (Prkd2), mitogen-activated kinase 3 (Mapk3/ERK1), and myosin light chain kinase (Mylk). Conclusion and implications In V2 receptor-mediated signaling, PKA is at the head of a complex network that includes at least 6 downstream vasopressin-regulated protein kinases that are prime targets for future study. The extensive phosphoproteomic data reported in this study is provided as a web-based data resource for future studies of G-protein coupled receptors.

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Phosphoproteomic identification of vasopressin-regulated protein kinases in collecting duct cells

Vasopressin regulates osmotic water transport in the renal collecting duct by protein kinase A (PKA)-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA-Cα) and PKA catalytic β (PKA-Cβ). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-Cα or PKA-Cβ was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4,635 phosphopeptides that were quantified, 67 phosphopeptides were significantly altered in abundance with PKA-Cα deletion, whereas 21 phosphopeptides were significantly altered in abundance with PKA-Cβ deletion. However, only four sites were changed in both. The target proteins identified in PKA-Cα-null cells were largely associated with cell membranes and membrane vesicles, whereas target proteins in PKA-Cβ-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-Cα and PKA-Cβ resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in in vivo samples showed that PKA-Cα deletion resulted in a near disappearance of AQP2 protein, whereas PKA-Cβ deletion did not decrease AQP2 abundance. We conclude that PKA-Cα and PKA-Cβ serve substantially different regulatory functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g., mediated by A-kinase anchor proteins, C-kinase anchoring proteins, or PDZ binding.

https://journals.physiology.org/doi/pdf/10.1152/ajprenal.00383.2020

Eui-Jung Park

Papers

A Minireview on Vasopressin-regulated Aquaporin-2 in Kidney Collecting Duct Cells.

Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct.

Phosphoproteomic identification of vasopressin-regulated protein kinases in collecting duct cells

Protein kinase A catalytic-α and catalytic-β proteins have nonredundant regulatory functions.

A nonbiodegradable scaffold-free cell sheet of genome-engineered mesenchymal stem cells inhibits development of acute kidney injury