Klotho is highly expressed in the chief sites of regulated potassium secretion, and it is stimulated by potassium intake.

Klotho regulates many pathways in the aging process, but it remains unclear how it is physiologically regulated. Because Klotho is synthesized, cleaved, and released from the kidney; activates the chief urinary K+ secretion channel (ROMK) and stimulates urinary K+ secretion, we explored if Klotho protein is regulated by dietary K+ and the potassium-regulatory hormone, Aldosterone. Klotho protein along the nephron was evaluated in humans and in wild-type (WT) mice; and in mice lacking components of Aldosterone signaling, including the Aldosterone-Synthase KO (AS-KO) and the Mineralocorticoid-Receptor KO (MR-KO) mice. We found the specific cells of the distal nephron in humans and mice that are chief sites of regulated K+ secretion have the highest Klotho protein expression along the nephron. WT mice fed K+-rich diets increased Klotho expression in these cells. AS-KO mice exhibit normal Klotho under basal conditions but could not upregulate Klotho in response to high-K+ intake in the K+-secreting cells. Similarly, MR-KO mice exhibit decreased Klotho protein expression. Together, i) Klotho is highly expressed in the key sites of regulated K+ secretion in humans and mice, ii) In mice, K+-rich diets increase Klotho expression specifically in the potassium secretory cells of the distal nephron, iii) Aldosterone signaling is required for Klotho response to high K+ intake.

Enriched Single-Nucleus RNA-Sequencing Reveals Unique Attributes of Distal Convoluted Tubule Cells.

SIGNIFICANCE STATEMENT: High-resolution single-nucleus RNA-sequencing data indicate a clear separation between primary sites of calcium and magnesium handling within distal convoluted tubule (DCT). Both DCT1 and DCT2 express Slc12a3, but these subsegments serve distinctive functions, with more abundant magnesium-handling genes along DCT1 and more calcium-handling genes along DCT2. The data also provide insight into the plasticity of the distal nephron-collecting duct junction, formed from cells of separate embryonic origins. By focusing/changing gradients of gene expression, the DCT can morph into different physiological cell states on demand. BACKGROUND: The distal convoluted tubule (DCT) comprises two subsegments, DCT1 and DCT2, with different functional and molecular characteristics. The functional and molecular distinction between these segments, however, has been controversial. METHODS: To understand the heterogeneity within the DCT population with better clarity, we enriched for DCT nuclei by using a mouse line combining "Isolation of Nuclei Tagged in specific Cell Types" and sodium chloride cotransporter-driven inducible Cre recombinase. We sorted the fluorescently labeled DCT nuclei using Fluorescence-Activated Nucleus Sorting and performed single-nucleus transcriptomics. RESULTS: Among 25,183 DCT cells, 75% were from DCT1 and 25% were from DCT2. In addition, there was a small population (<1%) enriched in proliferation-related genes, such as Top2a , Cenpp , and Mki67 . Although both DCT1 and DCT2 expressed sodium chloride cotransporter, magnesium transport genes were predominantly expressed along DCT1, whereas calcium, electrogenic sodium, and potassium transport genes were more abundant along DCT2. The transition between these two segments was gradual, with a transitional zone in which DCT1 and DCT2 cells were interspersed. The expression of the homeobox genes by DCT cells suggests that they develop along different trajectories. CONCLUSIONS: Transcriptomic analysis of an enriched rare cell population using a genetically targeted approach clarifies the function and classification of distal cells. The DCT segment is short, can be separated into two subsegments that serve distinct functions, and is speculated to derive from different origins during development.

Poly(ADP-ribose) polymerase-1 affects vasopressin-mediated AQP2 expression in collecting duct cells of the kidney.

Poly(ADP-ribosyl)ation (PARylation), as a posttranslational modification mediated by poly(ADP-ribose) polymerases (PARPs) catalyzing the transfer of ADP-ribose from NAD+ molecules to acceptor proteins, involves a number of cellular processes. As mice lacking the PARP-1 gene (Parp1) produce more urine, we investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). In biotin-conjugated nicotinamide adenine dinucleotide (biotin-NAD+) pulldown and immunoprecipitation assays of poly(ADP)-ribose in mpkCCDc14 cells, immunoblots demonstrated that 1-deamino-8-D-arginine vasopressin (dDAVP) induced the PARylation of total proteins, associated with an increase in the cleavage of PARP-1 and cleaved caspase-3 expression. By inhibiting PARP-1 with siRNA, the abundance of dDAVP-induced AQP2 mRNA and protein was significantly diminished. In contrast, despite a substantial decrease in PARylation, the PARP-1 inhibitor (PJ34) had no effect on the dDAVP-induced regulation of AQP2 expression. The findings suggest that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. Bioinformatic analysis revealed that 408 proteins interact with PARP-1 in the collecting duct (CD) cells of the kidney. Among them, the signaling pathway of the vasopressin V2 receptor was identified for 49 proteins. In particular, ß-catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein. A significant decrease of ß-catenin phosphorylation (Ser552) in response to dDAVP was associated with siRNA-mediated PARP-1 knockdown. Taken together, PARP-1 is likely to play a role in vasopressin-induced AQP2 expression by interacting with ß-catenin in renal CD cells.NEW & NOTEWORTHY The poly(ADP-ribose) polymerase (PARP) family catalyzes poly(ADP-ribosylation) (PARylation), which is one of the posttranslational modifications of largely undetermined physiological significance. This study investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). The results demonstrated that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. ß-Catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein.

Single cell and spatial transcriptomics analysis of kidney double negative T lymphocytes in normal and ischemic mouse kidneys.

T cells are important in the pathogenesis of acute kidney injury (AKI), and TCR+CD4-CD8- (double negative-DN) are T cells that have regulatory properties. However, there is limited information on DN T cells compared to traditional CD4+ and CD8+ cells. To elucidate the molecular signature and spatial dynamics of DN T cells during AKI, we performed single-cell RNA sequencing (scRNA-seq) on sorted murine DN, CD4+, and CD8+ cells combined with spatial transcriptomic profiling of normal and post AKI mouse kidneys. scRNA-seq revealed distinct transcriptional profiles for DN, CD4+, and CD8+ T cells of mouse kidneys with enrichment of Kcnq5, Klrb1c, Fcer1g, and Klre1 expression in DN T cells compared to CD4+ and CD8+ T cells in normal kidney tissue. We validated the expression of these four genes in mouse kidney DN, CD4+ and CD8+ T cells using RT-PCR and Kcnq5, Klrb1, and Fcer1g genes with the NIH human kidney precision medicine project (KPMP). Spatial transcriptomics in normal and ischemic mouse kidney tissue showed a localized cluster of T cells in the outer medulla expressing DN T cell genes including Fcer1g. These results provide a template for future studies in DN T as well as CD4+ and CD8+ cells in normal and diseased kidneys.

Polycystin-2-dependent transcriptome reveals early response of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in polycystin genes, Pkd1 and Pkd2, but the underlying pathogenic mechanisms are poorly understood. To identify genes and pathways that operate downstream of polycystin-2 (PC2), a comprehensive gene expression database was created, cataloging changes in the transcriptome immediately following PC2 protein depletion. To explore cyst initiation processes, an immortalized mouse inner medullary collecting duct line was developed with the ability to knock out the Pkd2 gene conditionally. Genome-wide transcriptome profiling was performed using RNA sequencing in the cells immediately after PC2 was depleted and compared with isogenic control cells. Differentially expressed genes were identified, and a bioinformatic analysis pipeline was implemented. Altered expression of candidate cystogenic genes was validated in Pkd2 knockout mice. The expression of nearly 900 genes changed upon PC2 depletion. Differentially expressed genes were enriched for genes encoding components of the primary cilia, the canonical Wnt pathway, and MAPK signaling. Among the PC2-dependent ciliary genes, the transcription factor Glis3 was significantly downregulated. MAPK signaling formed a key node at the epicenter of PC2-dependent signaling networks. Activation of Wnt and MAPK signaling, concomitant with the downregulation of Glis3, was corroborated in Pkd2 knockout mice. The data identify a PC2 cilia-to-nucleus signaling axis and dysregulation of the Gli-similar subfamily of transcription factors as a potential initiator of cyst formation in ADPKD. The catalog of PC2-regulated genes should provide a valuable resource for future ADPKD research and new opportunities for drug development.NEW & NOTEWORTHY Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Mutations in polycystin genes cause the disease, but the underlying mechanisms of cystogenesis are unknown. To help fill this knowledge gap, we created an inducible cell model of ADPKD and assembled a catalog of genes that respond in immediate proximity to polycystin-2 depletion using transcriptomic profiling. The catalog unveils a ciliary signaling-to-nucleus axis proximal to polycystin-2 dysfunction, highlighting Glis, Wnt, and MAPK signaling.

Signaling mechanisms in renal compensatory hypertrophy revealed by multi-omics.

Loss of a kidney results in compensatory growth of the remaining kidney, a phenomenon of considerable clinical importance. However, the mechanisms involved are largely unknown. Here, we use a multi-omic approach in a unilateral nephrectomy model in male mice to identify signaling processes associated with renal compensatory hypertrophy, demonstrating that the lipid-activated transcription factor peroxisome proliferator-activated receptor alpha (PPARα) is an important determinant of proximal tubule cell size and is a likely mediator of compensatory proximal tubule hypertrophy.

Circadian gene expression in mouse renal proximal tubule.

Circadian variability in kidney function is well recognized but is often ignored as a potential confounding variable in physiological experiments. Here, we have created a data resource consisting of expression levels for mRNA transcripts in microdissected proximal tubule segments from mice as a function of the time of day. Small-sample RNA sequencing was applied to microdissected S1 proximal convoluted tubules and S2 proximal straight tubules. After stringent filtering, the data were analyzed using JTK-Cycle to detect periodicity. The data set is provided as a user-friendly webpage at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox2/. In proximal convoluted tubules, 234 transcripts varied in a circadian manner (4.0% of the total). In proximal straight tubules, 334 transcripts varied in a circadian manner (5.3%). Transcripts previously known to be associated with corticosteroid action and with increased flow were found to be overrepresented among circadian transcripts peaking during the "dark" portion of the day [zeitgeber time (ZT)14-22], corresponding to peak levels of corticosterone and glomerular filtration rate in mice. To ask whether there is a time-of-day dependence of protein abundances in the kidney, we carried out LC-MS/MS-based proteomics in whole mouse kidneys at ZT12 and ZT0. The full data set (n = 6,546 proteins) is available at https://esbl.nhlbi.nih.gov/Databases/Circadian-Proteome/. Overall, 293 proteins were differentially expressed between ZT12 and ZT0 (197 proteins greater at ZT12 and 96 proteins greater at ZT0). Among the regulated proteins, only nine proteins were found to be periodic in the RNA-sequencing analysis, suggesting a high level of posttranscriptional regulation of protein abundances.NEW & NOTEWORTHY Circadian variation in gene expression can be an important determinant in the regulation of kidney function. The authors used RNA-sequencing transcriptomics and LC-MS/MS-based proteomics to identify gene products expressed in a periodic manner. The data were used to construct user-friendly web resources.

Genome-Engineered mpkCCDc14 Cells as a New Resource for Studying AQP2.

mpkCCDc14 cells, a polarized epithelial cell line derived from mouse kidney cortical collecting ducts, are known to express the vasopressin V2 receptor (V2R) and aquaporin-2 (AQP2) that are responsive to vasopressin. However, a low abundance of the endogenous AQP2 protein in the absence of vasopressin and heterogeneity of AQP2 protein abundance among the cultured cells may limit the further application of the cell line in AQP2 studies. To overcome the limitation, we aimed to establish mpkCCDc14 cells constitutively expressing V2R and AQP2 via CRISPR/Cas9-mediated genome engineering technology (i.e., V2R-AQP2 cells). 3'- and 5'-Junction PCR revealed that the V2R-AQP2 expression cassette with a long insert size (~2.2 kb) was correctly integrated. Immunoblotting revealed the expression of products of integrated Aqp2 genes. Cell proliferation rate and dDAVP-induced cAMP production were not affected by the knock-in of Avpr2 and Aqp2 genes. The AQP2 protein abundance was significantly higher in V2R-AQP2 cells compared with control mpkCCDc14 cells in the absence of dDAVP and the integrated AQP2 was detected. Immunocytochemistry demonstrated that V2R-AQP2 cells exhibited more homogenous and prominent AQP2 labeling intensity in the absence of dDAVP stimulation. Moreover, prominent AQP2 immunolabeling (both AQP2 and pS256-AQP2) in the apical domain of the genome-edited cells was observed in response to dDAVP stimulation, similar to that in the unedited control mpkCCDc14 cells. Taken together, mpkCCDc14 cells constitutively expressing V2R and AQP2 via genome engineering could be exploited for AQP2 studies.

Systems Biology of the Vasopressin V2 Receptor: New Tools for Discovery of Molecular Actions of a GPCR.

Systems biology can be defined as the study of a biological process in which all of the relevant components are investigated together in parallel to discover the mechanism. Although the approach is not new, it has come to the forefront as a result of genome sequencing projects completed in the first few years of the current century. It has elements of large-scale data acquisition (chiefly next-generation sequencing-based methods and protein mass spectrometry) and large-scale data analysis (big data integration and Bayesian modeling). Here we discuss these methodologies and show how they can be applied to understand the downstream effects of GPCR signaling, specifically looking at how the neurohypophyseal peptide hormone vasopressin, working through the V2 receptor and PKA activation, regulates the water channel aquaporin-2. The emerging picture provides a detailedframework for understanding the molecular mechanisms involved in water balance disorders, pointing the way to improved treatment of both polyuric disorders and water-retention disorders causing dilutional hyponatremia.

Diesel Exhaust Particles Impair Therapeutic Effect of Human Wharton's Jelly-Derived Mesenchymal Stem Cells against Experimental Colitis through ROS/ERK/cFos Signaling Pathway.

Background and Objectives: Epidemiological investigations have shown positive correlations between increased diesel exhaust particles (DEP) in ambient air and adverse health outcomes. DEP are the major constituent of particulate atmospheric pollution and have been shown to induce proinflammatory responses both in the lung and systemically. Here, we report the effects of DEP exposure on the properties of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs), including stemness, regeneration, and immunomodulation. Methods and Results: Non-apoptotic concentrations of DEP (10 µg/ml) inhibited the migration and osteogenic differentiation capacity of WJ-MSCs. Gene expression profiling showed that DEP increased intracellular reactive oxygen species (ROS) and expression of pro-inflammatory and metabolic-process-related genes including cFos. Furthermore, WJ-MSCs cultured with DEP showed impaired suppression of T cell proliferation that was reversed by inhibition of ROS or knockdown of cFos. ERK inhibition assay revealed that DEP-induced ROS regulated cFos through activation of ERK but not NF-κB signaling. Overall, low concentrations of DEP (10 µg/ml) significantly suppressed the stemness and immunomodulatory properties of WJ-MSCs through ROS/ERK/cFos signaling pathways. Furthermore, WJ-MSCs cultured with DEP impaired the therapeutic effect of WJ-MSCs in experimental colitis mice, but was partly reversed by inhibition of ROS. Conclusions: Taken together, these results indicate that exposure to DEP enhances the expression of pro-inflammatory cytokines and immune responses through a mechanism involving the ROS/ERK/cFos pathway in WJ-MSCs, and that DEP-induced ROS damage impairs the therapeutic effect of WJ-MSCs in colitis. Our results suggest that modulation of ROS/ERK/cFos signaling pathways in WJ-MSCs might be a novel therapeutic strategy for DEP-induced diseases.

Doxycycline Changes the Transcriptome Profile of mIMCD3 Renal Epithelial Cells.

Tetracycline-inducible gene expression systems have been used successfully to study gene function in vivo and in vitro renal epithelial models but the effects of the common inducing agent, doxycycline (DOX), on gene expression are not well appreciated. Here, we evaluated the DOX effects on the transcriptome of a widely used renal epithelial cell model, mIMCD3 cells, to establish a reference. Cells were grown on permeable filter supports in the absence and presence of DOX (3 or 6 days), and genome-wide transcriptome profiles were assessed using RNA-Seq. We found DOX significantly altered the transcriptome profile, changing the abundance of 1,549 transcripts at 3 days and 2,643 transcripts at 6 days. Within 3 days of treatment, DOX significantly decreased the expression of multiple signaling pathways (ERK, cAMP, and Notch) that are associated with cell proliferation and differentiation. Genes associated with cell cycle progression were subsequently downregulated in cells treated with DOX for 6 days, as were genes involved in cellular immune response processes and several cytokines and chemokines, correlating with a remarkable repression of genes encoding cell proliferation markers. The results provide new insight into responses of renal epithelial cells to DOX and a establish a resource for DOX-mediated gene expression systems.

Addition of autogenous bone chips to deproteinized bovine bone mineral does not have additional benefit in lateral ridge augmentation-A preclinical in vivo experimental study.

OBJECTIVE: To compare the outcome after extensive lateral guided bone regeneration using deproteinized bovine bone mineral (DBBM) with or without autogenous bone chips in a canine model of chronic horizontal alveolar ridge defect. MATERIALS AND METHODS: The second, third and fourth lower premolars of both sides were extracted, and the buccal bone walls were completely removed in five beagle dogs. After 4 weeks, DBBM particles mixed with autogenous bone chips at a ratio of 1:1 were grafted at one side (DBBM/Auto group), while DBBM particles alone were grafted at the contralateral side (DBBM group). The graft materials on both sides were covered by a resorbable collagen membrane and fixation pins. Microcomputed tomographic volume and histomorphometric analyses were performed at 16 weeks post-surgery. RESULTS: The ridges of both groups were recovered horizontally, but new bone formation beyond the original ridge contour at the defect site was not found. The DBBM group exhibited a larger total radiographic augmented volume and new bone volume compared with the DBBM/Auto group, but the differences were minimal (p > .05). Histologically, the regenerated area and new bone area were also slightly larger without any statistical significance in the DBBM group than in the DBBM/Auto group (p > .05). CONCLUSION: The addition of autogenous bone chips to DBBM for lateral ridge augmentation may confer no advantage over grafting DBBM alone with respect to both space maintenance and de novo bone formation in dogs.

Bayesian identification of candidate transcription factors for the regulation of Aqp2 gene expression.

Aquaporin-2 (Aqp2) gene transcription is strongly regulated by vasopressin in the renal collecting duct. However, the transcription factors (TFs) responsible for the regulation of expression of Aqp2 remain largely unknown. We used Bayes' theorem to integrate several -omics data sets to stratify the 1,344 TFs present in the mouse genome with regard to probabilities of regulating Aqp2 gene transcription. Also, we carried out new RNA sequencing experiments mapping the time course of vasopressin-induced changes in the transcriptome of mpkCCD cells to identify TFs that change in tandem with Aqp2. The analysis identified 17 of 1,344 TFs that are most likely to be involved in the regulation of Aqp2 gene transcription. These TFs included eight that have been proposed in prior studies to play a role in Aqp2 regulation, viz., Cebpb, Elf1, Elf3, Ets1, Jun, Junb, Nfkb1, and Sp1. The remaining nine represent new candidates for future studies (Atf1, Irf3, Klf5, Klf6, Mef2d, Nfyb, Nr2f6, Stat3, and Nr4a1). Conspicuously absent is CREB (Creb1), which has been widely proposed to mediate vasopressin-induced regulation of Aqp2 gene transcription (Nielsen S, Frokiaer J, Marples D, Kwon TH, Agre P, Knepper MA. Physiol Rev 82: 205-244, 2002; Kortenoeven ML, Fenton RA. Biochim Biophys Acta 1840: 1533-1549, 2014; Bockenhauer D, Bichet DG. Nat Rev Nephrol 11: 576-588, 2015; Pearce D, Soundararajan R, Trimpert C, Kashlan OB, Deen PM, Kohan DE. Clin J Am Soc Nephrol 10: 135-146, 2015). Instead, another CREB-like TF, Atf1, ranked fourth among all TFs. RNA sequencing time-course experiments showed a rapid increase in Aqp2 mRNA, within 3 h of vasopressin exposure. This response was matched by an equally rapid increase in the abundance of the mRNA coding for Cebpb, which we have shown by chromatin immunoprecipitation-sequencing studies to bind downstream from the Aqp2 gene. The identified TFs provide a roadmap for future studies to understand regulation of Aqp2 gene expression.NEW & NOTEWORTHY Abetted by the advent of systems biology-based ("-omics") techniques in the 21st century, there has been a massive expansion of published data relevant to virtually every physiological question. The authors have developed a large-scale data integration approach based on the application of Bayes'' theorem. In the current work, they integrated 12 different -omics data sets to identify the transcription factors most likely to mediate vasopressin-dependent regulation of transcription of the aquaporin-2 gene.

Aquaporins implicated in the cell proliferation and the signaling pathways of cell stemness.

Aquaporins (AQPs) are water channel proteins facilitating passive transport of water and other small molecules across biomembranes. Regulation of osmotic homeostasis via AQPs is accompanied by dynamic participation of various cellular signaling pathways. Recently emerging evidence reveals that functional roles of AQPs are further extended from the osmotic regulation via water permeation into the cell proliferation and differentiation. In particular, anomalous expression of AQPs has been demonstrated in various types of cancer cells and cancer stem-like cells and it has been proposed as markers for proliferation and progression of cancer cells. Thus, a more comprehensive view on AQPs could bring a great interest in the cell stemness accompanied by the expression of AQPs. AQPs are broadly expressed across tissues and cells in a cell type- and lineage-specific manner during development via spatiotemporal transcriptional regulation. Moreover, AQPs are expressed in various adult stem cells and cells associated with a stem cell niche as well as cancer stem-like cells. However, the expression and regulatory mechanisms of AQP expression in stem cells have not been well understood. This review highlighted the AQPs expression in stem cell niches/stem cells and the involvement of AQPs in the cell proliferation and signaling pathways associated with cell stemness.

NGS-Integrator: An efficient tool for combining multiple NGS data tracks using minimum Bayes' factors.

BACKGROUND: Next-generation sequencing (NGS) is widely used for genome-wide identification and quantification of DNA elements involved in the regulation of gene transcription. Studies that generate multiple high-throughput NGS datasets require data integration methods for two general tasks: 1) generation of genome-wide data tracks representing an aggregate of multiple replicates of the same experiment; and 2) combination of tracks from different experimental types that provide complementary information regarding the location of genomic features such as enhancers. RESULTS: NGS-Integrator is a Java-based command line application, facilitating efficient integration of multiple genome-wide NGS datasets. NGS-Integrator first transforms all input data tracks using the complement of the minimum Bayes' factor so that all values are expressed in the range [0,1] representing the probability of a true signal given the background noise. Then, NGS-Integrator calculates the joint probability for every genomic position to create an integrated track. We provide examples using real NGS data generated in our laboratory and from the mouse ENCODE database. CONCLUSIONS: Our results show that NGS-Integrator is both time- and memory-efficient. Our examples show that NGS-Integrator can integrate information to facilitate downstream analyses that identify functional regulatory domains along the genome.

An integrative proteogenomics approach reveals peptides encoded by annotated lincRNA in the mouse kidney inner medulla.

Long noncoding RNAs (lncRNAs) are intracellular transcripts longer than 200 nucleotides and lack protein-coding information. A subclass of lncRNA known as long intergenic noncoding RNAs (lincRNAs) are transcribed from genomic regions that share no overlap with annotated protein-coding genes. Increasing evidence has shown that some annotated lincRNA transcripts do in fact contain open reading frames (ORFs) encoding functional short peptides in the cell. Few robust methods for lincRNA-encoded peptide identification have been reported, and the tissue-specific expression of these peptides has been largely unexplored. Here we propose an integrative workflow for lincRNA-encoded peptide discovery and test it on the mouse kidney inner medulla (IM). In brief, low molecular weight protein fractions were enriched from homogenate of IMs and trypsinized into shorter peptides, which were sequenced by high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). To curate a hypothetical lincRNA-encoded peptide database for peptide-spectrum matching following LC-MS/MS, we performed RNA-Seq on IMs, computationally removed reads overlapping with annotated protein-coding genes, and remapped the remaining reads to a database of mouse noncoding transcripts to infer lincRNA expression. Expressed lincRNAs were searched for ORFs by an existing rule-based algorithm, and translated ORFs were used for peptide-spectrum matching. Peptides identified by LC-MS/MS were further evaluated by using several quality control criteria and bioinformatics methods. We discovered three novel lincRNA-encoded peptides, which are conserved in mouse, rat, and human. The workflow can be adapted for discovery of small protein-coding genes in any species or tissue where noncoding transcriptome information is available.

Fluid-electrolyte homeostasis requires histone deacetylase function.

Histone deacetylase (HDAC) enzymes regulate transcription through epigenetic modification of chromatin structure, but their specific functions in the kidney remain elusive. We discovered that the human kidney expresses class I HDACs. Kidney medulla-specific inhibition of class I HDACs in the rat during high-salt feeding results in hypertension, polyuria, hypokalemia, and nitric oxide deficiency. Three new inducible murine models were used to determine that HDAC1 and HDAC2 in the kidney epithelium are necessary for maintaining epithelial integrity and maintaining fluid-electrolyte balance during increased dietary sodium intake. Moreover, single-nucleus RNA-sequencing determined that epithelial HDAC1 and HDAC2 are necessary for expression of many sodium or water transporters and channels. In performing a systematic review and meta-analysis of serious adverse events associated with clinical HDAC inhibitor use, we found that HDAC inhibitors increased the odds ratio of experiencing fluid-electrolyte disorders, such as hypokalemia. This study provides insight on the mechanisms of potential serious adverse events with HDAC inhibitors, which may be fatal to critically ill patients. In conclusion, kidney tubular HDACs provide a link between the environment, such as consumption of high-salt diets, and regulation of homeostatic mechanisms to remain in fluid-electrolyte balance.

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

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.

Regulation of aquaporin-2 by RNA interference.

MicroRNAs (miRNAs) are short non-coding RNAs that interact with 3'-untranslated regions of mRNAs. miRNAs modulate gene expression by regulating mRNA translation and provide novel insights into the complex regulation of protein expression and function. In this chapter, we describe the general features of RNA interference, identification of miRNAs, and interaction of miRNAs with their target genes. In particular, we have shown that several miRNAs are responsive to arginine vasopressin or aldosterone stimulation in mouse cortical collecting duct mpkCCD cells. Moreover, we identified both miR-32 and miR-137 as AQP2-targeting miRNAs using in silico analysis and also identified several target genes of miR-32 and miR-137. As the target sequences of miR-32 and miR-137 are commonly found in mRNAs of vasopressin-regulated genes, further studies regarding the interaction of miRNAs with their target genes are required to obtain comprehensive understanding of miRNA-regulated AQP2 expression in kidney collecting duct cells.