Solute And Water Transport In Renal Epithelia

National Heart, Lung, And Blood Institute

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Abstract

A---- The Knepper Laboratory (ESBL) uses quantitative mass spectrometry (MS) and next-generation DNA sequencing (NGS), combined with computational techniques, to investigate the mechanisms of regulation of renal collecting duct function by vasopressin and other mediators. In the collecting duct epithelium, water transport is mediated by a vasopressin-regulated water channel protein, aquaporin-2 (AQP2). AQP2 is dysregulated in numerous disorders of water balance in people and animals, including those associated with polyuria (urinary tract obstruction, hypokalemia, inflammation, and lithium toxicity) and with dilutional hyponatremia (syndrome of inappropriate antidiuresis, congestive heart failure, cirrhosis). Normal regulation of AQP2 by vasopressin involves two independent regulatory mechanisms: (1) short-term regulation of AQP2 trafficking to and from the apical plasma membrane, and (2) long-term regulation of the total abundance of the AQP2 protein in the cells. Most disorders of water balance are the result of dysregulation of processes that regulate the transcription of the Aqp2 gene in collecting duct cells. Studies have focused on cellular and molecular mechanisms of vasopressin action in collecting duct cells using systems biology approaches. In addition, substantial effort has been made to share with the research community our computational tools (https://esbl.nhlbi.nih.gov/Bioinformatic%20Tools.htm) and “Omic” datasets (https://esbl.nhlbi.nih.gov/Databases/KSBP2/). B---- One point of experimental focus is on vasopressin signaling through the G-protein-coupled receptor, V2R. The experiments have used phospho-proteomics techniques to identify phosphoproteins perturbed in response to vasopressin V2 receptor occupation and to quantify specific sites of phosphorylation. These experiments demonstrated a highly selective phospho-proteomic response to vasopressin with quantifiable changes in only about 2% of detectable phosphorylation sites. Most of the upregulated phosphorylation sites were in a sequence motif compatible with activation of protein kinase A (PKA) or other basophilic kinases. To test the roles of specific protein kinases in vasopressin signaling, the ESBL is using genome editing techniques (CRISPR-Cas9) to delete candidate kinases followed by phosphoproteomic profiling to identify targets of the deleted kinases. Deletion of both PKA catalytic subunits in collecting duct cells allowed identification of multiple novel PKA substrates, including phosphoproteins whose molecular functions map to known functional effects of vasopressin. These experiments identified a number of additional protein kinases downstream from PKA that represent elements of a PKA-activated kinase cascade. PKA deletion resulted in loss of AQP2 mRNA and protein, which combined with ancillary data, showed that PKA is necessary for Aqp2 gene transcription. ESBL members have also identified phosphorylation targets in myosin light chain kinase (MLCK) in the collecting duct, carrying out phosphoproteomic analysis in MLCK CRISPR mutants. Additional projects have used CRISPR-phospoproteomics to identify targets of calmodulin-activated kinase 2-delta (CAMK2D) and Calmodulin-activated kinase kinase 2 (CAMKK2) in collecting duct cells to identify physiological roles of these protein kinases. Initial studies focusing on another protein kinase, salt-inducible protein kinase 2 (SIK2), appears to implicate this protein kinase as a negative regulator of Aqp2 gene transcription. C---- A second major point of focus is on how vasopressin regulates Aqp2 gene transcription. Studies employing various NGS techniques (RNA-Seq, ChIP-Seq, ATAC-Seq) have allowed comprehensive identification of vasopressin-regulated genes and genome-wide enhancer mapping. Identification of enhancers in the vicinity of the Aqp2 gene have provided a list of candidate transcription factors (TFs) for control of Aqp2 transcription. Among these was a so-called pioneer TF, C/EBP-beta, which was shown in ChIP-Seq experiments to bind to the predicted site in an Aqp2-vicinal enhancer in a vasopressin-dependent manner. Nuclear proteomics demonstrated C/EBP-beta nuclear translocation in response to vasopressin. To identify additional candidate transcription factors, we have developed tools for large-scale CRISPR screening. These studies have identified several transcription factors potentially involved with regulation of Aqp2 gene transcription and validation studies are in progress. Additional candidates have arisen from Bayesian integration of multiple collecting duct Omic data sets, pointing to the ATF1/CREB1/CREM family that are known to be activated through PKA-mediated phosphorylation. Although CRISPR-mediated deletion of each of these transcription factors individually did not ablate the ability of vasopressin to increase AQP2 abundance in collecting duct cells, deletion of all three together strongly inhibited this response, suggesting that ATF1, CREB1 and CREM have redundant roles. Further planned studies will use the ChIP-seq technique to identify genomic binding sites for ATF1, CREB1 and CREM. Finally, we are using large-scale CRISPR screening to identify protein kinases involved in regulation of Aqp2 gene transcription. D---- A long-term goal is to link the signaling studies described above to the studies of regulation of Aqp2 gene transcription in order to develop a comprehensive model that can explain how the kidney regulates water excretion and how this mechanism goes awry in water balance disorders. To directly identify patho-mechanisms in water balance disorders, ESBL is using RNA-Seq in single micro-dissected renal tubules to identify signaling mechanisms responsible for transcriptional changes in animal models of water balance disorders such as the syndrome of inappropriate antidiuresis (SIAD), lithium-induced nephrogenic diabetes insipidus (NDI), unilateral ureteral obstruction (UUO) and in gram-negative sepsis. To create a tool for evaluation of the role of vasopressin in these water balance disorders, ESBL members are presently creating a mouse line with inducible deletion of the Avp gene coding for arginine vasopressin, neurophysin and copeptin. D---- An additional point of focus is on data resources for sharing proteomic and transcriptomic data useful to the renal research community. While the data resources contain mainly data accrued in the conduct of studies of vasopressin action to regulate the aquaporin-2 water channel, the ESBL has recently moved resources into the use of NGS techniques and protein mass spectrometry for creation of atlas-type databases containing comprehensive transcriptomes and proteomes of each renal tubule epithelial cell type. Previously, we developed a strategy to combine fluorescence-activated cell sorting (FACS) with single-cell RNA-Seq (scRNA-Seq) to determine full transcriptomes for each of the cell types that make up the renal collecting duct epithelium. In addition, the ESBL recently completed studies using the same approach to map gene expression along the thick ascending limb (TAL) and distal convoluted tubule of the nephron. Among other observations is that there are two types of cortical TAL cells, characterized by expression of different Iroquois Homeobox TFs.