Transcriptional profiling of native inner medullary collecting duct cells from rat kidney

Uawithya, Panapat; Pisitkun, Trairak; Ruttenberg, Brian E.; Knepper, Mark A.

doi:10.1152/physiolgenomics.00201.2007

Cited by 95 publications

(107 citation statements)

References 104 publications

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“…AKAP220, a product of the Akap11 gene, has been implicated in the modulation of cytoskeletal signaling events (23,28). Microarray analyses detect enhanced expression of the Akap11 gene in primary cultures of inner medullary collecting duct (IMCD) cells (29). In fact, Akap11 transcripts are enriched by an order of magnitude over all other AKAP transcripts in these cells.…”

Section: Resultsmentioning

confidence: 99%

AKAP220 manages apical actin networks that coordinate aquaporin-2 location and renal water reabsorption

Whiting

Ogier

Forbush

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Filtration through the kidney eliminates toxins, manages electrolyte balance, and controls water homeostasis. Reabsorption of water from the luminal fluid of the nephron occurs through aquaporin-2 (AQP2) water pores in principal cells that line the kidney-collecting duct. This vital process is impeded by formation of an "actin barrier" that obstructs the passive transit of AQP2 to the plasma membrane. Bidirectional control of AQP2 trafficking is managed by hormones and signaling enzymes. We have discovered that vasopressin-independent facets of this homeostatic mechanism are under the control of A-Kinase Anchoring Protein 220 (AKAP220; product of the Akap11 gene). CRISPR/Cas9 gene editing and imaging approaches show that loss of AKAP220 disrupts apical actin networks in organoid cultures. Similar defects are evident in tissue sections from AKAP220-KO mice. Biochemical analysis of AKAP220-null kidney extracts detected reduced levels of active RhoA GTPase, a well-known modulator of the actin cytoskeleton. Fluorescent imaging of kidney sections from these genetically modified mice revealed that RhoA and AQP2 accumulate at the apical surface of the collecting duct. Consequently, these animals are unable to appropriately dilute urine in response to overhydration. We propose that membrane-proximal signaling complexes constrained by AKAP220 impact the actin barrier dynamics and AQP2 trafficking to ensure water homeostasis. yet only approximately 1.5 L of urine is excreted. The majority of this water is reabsorbed from the luminal fluid of the nephron (1). Regulated water reabsorption in response to dehydration occurs through aquaporin-2 (AQP2) water pores in the principal cells of the collecting duct (2). This is stimulated by the hormone arginine vasopressin (AVP). Vasopressin induces PKA phosphorylation of serine 256 on AQP2 and stimulates its translocation from intracellular vesicles to the apical membranes of cells lining the collecting ducts. Reabsorption of water through the kidney preserves fluid balance and results in more concentrated urine (3, 4). Conversely, when an animal ingests excess water, decreased plasma osmolality inhibits vasopressin release, and AQP2 is recovered from the apical membrane by endocytosis. This renders the collecting duct impermeable to water, thereby diverting excess water through the ureter to the bladder.Not surprisingly, defects in AQP2 trafficking have pathophysiological outcomes. For example, nephrogenic diabetes insipidus (NDI) is associated with impaired vasopressin signaling to AQP2 (5-8). Symptoms include excessive thirst, excretion of a large volume of dilute urine, and electrolyte imbalances, including hypernatremia (1, 5). Hereditary forms of this disease appear in patients with inactivating mutations in the V2 vasopressin receptor (V2R) or AQP2 (9-12). Thus, understanding the molecular mechanisms that govern bidirectional control of AQP2 location may lead to new therapeutic approaches for the treatment of NDI.Although the enzymes and effector proteins that govern AQP2 in...

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Section: Resultsmentioning

confidence: 99%

AKAP220 manages apical actin networks that coordinate aquaporin-2 location and renal water reabsorption

Whiting

Ogier

Forbush

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…RNA-seq offers important advantages over other approaches to kidney transcriptomics, such as DNA microarrays using biochemically isolated segments 32,33 or SAGE of microdissected renal tubule segments. 2,3 Compared with biochemical isolation techniques, [34][35][36] manual dissection virtually eliminates contamination from neighboring segments.…”

Section: Discussionmentioning

confidence: 99%

Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

Lee¹,

Chou²,

Knepper³

2015

Journal of the American Society of Nephrology

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492

480

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The function of each renal tubule segment depends on the genes expressed therein. High-throughput methods used for global profiling of gene expression in unique cell types have shown low sensitivity and high false positivity, thereby limiting the usefulness of these methods in transcriptomic research. However, deep sequencing of RNA species (RNA-seq) achieves highly sensitive and quantitative transcriptomic profiling by sequencing RNAs in a massive, parallel manner. Here, we used RNA-seq coupled with classic renal tubule microdissection to comprehensively profile gene expression in each of 14 renal tubule segments from the proximal tubule through the inner medullary collecting duct of rat kidneys. Polyadenylated mRNAs were captured by oligo-dT primers and processed into adapter-ligated cDNA libraries that were sequenced using an Illumina platform. Transcriptomes were identified to a median depth of 8261 genes in microdissected renal tubule samples (105 replicates in total) and glomeruli (5 replicates). Manual microdissection allowed a high degree of sample purity, which was evidenced by the observed distributions of well established cell-specific markers. The main product of this work is an extensive database of gene expression along the nephron provided as a publicly accessible webpage (https://helixweb.nih.gov/ESBL/Database/NephronRNAseq/index.html). The data also provide genomewide maps of alternative exon usage and polyadenylation sites in the kidney. We illustrate the use of the data by profiling transcription factor expression along the renal tubule and mapping metabolic pathways.

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“…The same analysis was also performed on several major gene categories most relevant to the renal tubular function (see Methods). The chosen gene categories were similar to those used by Uawithya et al for characterization of the inner medullary collecting duct transcriptome (26). Interestingly, this analysis revealed the existence of function-dependent temporal expression patterns for two of the gene categories, namely the genes belonging to the superfamily of solute carriers (Slc) and to the group of enzymes involved in phase I and phase II reactions of metabolism of xenobiotics or other lipophilic compounds.…”

Section: Temporal Profiling Of Distal Convoluted Tubule/connecting Tumentioning

confidence: 92%

Molecular clock is involved in predictive circadian adjustment of renal function

Zuber

Centeno

Pradervand

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

244

237

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Renal excretion of water and major electrolytes exhibits a significant circadian rhythm. This functional periodicity is believed to result, at least in part, from circadian changes in secretion/reabsorption capacities of the distal nephron and collecting ducts. Here, we studied the molecular mechanisms underlying circadian rhythms in the distal nephron segments, i.e., distal convoluted tubule (DCT) and connecting tubule (CNT) and the cortical collecting duct (CCD). Temporal expression analysis performed on microdissected mouse DCT/CNT or CCD revealed a marked circadian rhythmicity in the expression of a large number of genes crucially involved in various homeostatic functions of the kidney. This analysis also revealed that both DCT/CNT and CCD possess an intrinsic circadian timing system characterized by robust oscillations in the expression of circadian core clock genes (clock, bma11, npas2, per, cry, nr1d1) and clock-controlled Par bZip transcriptional factors dbp, hlf, and tef. The clock knockout mice or mice devoid of dbp/hlf/tef (triple knockout) exhibit significant changes in renal expression of several key regulators of water or sodium balance (vasopressin V2 receptor, aquaporin-2, aquaporin-4, ␣ENaC). Functionally, the loss of clock leads to a complex phenotype characterized by partial diabetes insipidus, dysregulation of sodium excretion rhythms, and a significant decrease in blood pressure. Collectively, this study uncovers a major role of molecular clock in renal function.circadian rhythm ͉ homeostasis ͉ renal function R ecent evidence suggests that many if not all specific physiological functions are under the control of the circadian timing system. The mammalian circadian timing system is a hierarchically organized network of molecular oscillators driven by a central pacemaker located in the suprachiasmatic nucleus (SCN) of hypothalamus. This central pacemaker functions in a self-sustained fashion, but is reset each day by exposure to environmental synchronizers, mainly the light/dark cycle. The SCN masterclock drives the rest-activity cycle, which in turn imposes the feeding pattern [reviewed in (1, 2)]. The feeding time seems to be the dominant cue for circadian rhythms in the peripheral tissues (3, 4). Central and peripheral oscillators share a similar molecular core clock based on a set of self-autonomous transcriptional/ translational feedback loops. The key molecular components of these loops are the PAS domain transcriptional factors CLOCK, BMAL1, and NPAS2 and the feedback repressors PER1, PER2, CRY1, and CRY2. The orphan nuclear receptors NR1D1 and, probably, NR1D2 form an accessory feedback loop. The core oscillators confer circadian rhythmicity on a set of output genes underlying the tissue-specific functional rhythms. Current estimates indicate that up to 10% of the cellular transcriptome may follow a circadian expression pattern (5-7). Several recent studies have also demonstrated that the transcription of only a minority of these circadian genes is driven by systemic humoral or neurona...

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Transcriptional profiling of native inner medullary collecting duct cells from rat kidney

Cited by 95 publications

References 104 publications

AKAP220 manages apical actin networks that coordinate aquaporin-2 location and renal water reabsorption

AKAP220 manages apical actin networks that coordinate aquaporin-2 location and renal water reabsorption

Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

Molecular clock is involved in predictive circadian adjustment of renal function

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