Af17 Deficiency Increases Sodium Excretion and Decreases Blood Pressure

Chen, Lihe; H, Wu; Pochynyuk, Oleh; Reisenauer, Mary Rose; Zhang, Zhijing; Huang, Le; Zaika, Oleg; Mamenko, Mykola; Zhang, Weiru; Zhou, Qiaoling; Liu, Mingyao; Xia, Yang; Zhang, Wenzheng

doi:10.1681/asn.2010121270

Cited by 24 publications

(50 citation statements)

References 47 publications

(59 reference statements)

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“…Similar findings were made using single-cell fluorescence imaging and equivalent shortcircuit current to measure ENaC activity in more physiologically relevant mouse collecting duct IMCD3 and M1 cells (31,49). In mice, deletion of Af17 led to increased dimethylation of histone H3 K79, reduced ENaC function, increased Na ϩ excretion, and decreased blood pressure (8). In contrast, inducing high levels of plasma aldosterone by a variety of methods completely compensated for Af17 deficiency with respect to sodium handling and blood pressure (8).…”

supporting

confidence: 64%

“…In mice, deletion of Af17 led to increased dimethylation of histone H3 K79, reduced ENaC function, increased Na ϩ excretion, and decreased blood pressure (8). In contrast, inducing high levels of plasma aldosterone by a variety of methods completely compensated for Af17 deficiency with respect to sodium handling and blood pressure (8). The clinical relevance of the Dot1a-Af9 pathway in regulating blood pressure is also supported by a recent clinical study.…”

mentioning

confidence: 82%

“…These assays were conducted according to our published protocols (8,30,48,54,56,57) and briefly described in Figs. 1-6.…”

Section: Methodsmentioning

confidence: 99%

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Mineralocorticoid receptor antagonizes Dot1a-Af9 complex to increaseαENaCtranscription

Zhang¹,

Zhou

Chen

et al. 2013

American Journal of Physiology-Renal Physiology

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Aldosterone is a major regulator of Na(+) absorption and acts by activating the mineralocorticoid receptor (MR) to stimulate the epithelial Na(+) channel (ENaC). MR(-/-) mice exhibited pseudohypoaldosteronism type 1 (hyponatremia, hyperkalemia, salt wasting, and high levels of aldosterone) and died around postnatal day 10. However, if and how MR regulates ENaC transcription remain incompletely understood. Our earlier work demonstrated that aldosterone activates αENaC transcription by reducing expression of Dot1a and Af9 and by impairing Dot1a-Af9 interaction. Most recently, we reported identification of a major Af9 binding site in the αENaC promoter and upregulation of αENaC mRNA expression in mouse kidneys lacking Dot1a. Despite these findings, the putative antagonism between the MR/aldosterone and Dot1a-Af9 complexes has never been addressed. The molecular defects leading to PHA-1 in MR(-/-) mice remain elusive. Here, we report that MR competes with Dot1a to bind Af9. MR/aldosterone and Dot1a-Af9 complexes mutually counterbalance ENaC mRNA expression in inner medullary collecting duct 3 (IMCD3) cells. Real-time RT-quantitative PCR revealed that 5-day-old MR(-/-) vs. MR(+/+) mice had significantly lower αENaC mRNA levels. This change was associated with an increased Af9 binding and H3 K79 hypermethylation in the αENaC promoter. Therefore, this study identified MR as a novel binding partner and regulator of Af9 and a novel mechanism coupling MR-mediated activation with relief of Dot1a-Af9-mediated repression via MR-Af9 interaction. Impaired ENaC expression due to failure to inhibit Dot1a-Af9 may play an important role in the early stages of PHA-1 (before postnatal day 8) in MR(-/-) mice.

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confidence: 64%

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confidence: 82%

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Mineralocorticoid receptor antagonizes Dot1a-Af9 complex to increaseαENaCtranscription

Zhang¹,

Zhou

Chen

et al. 2013

American Journal of Physiology-Renal Physiology

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“…Finally, our results provide the first concrete evidence for a role of Af9 as a DNA-binding protein that functions as a repressor of gene transcription. Given the roles of Af9 in cancer (22), neurodevelopmental diseases (4,5), and potentially hypertension (4,8), the present results may have broader implications.…”

Section: Discussionmentioning

confidence: 87%

An Af9 cis-element directly targets Dot1a to mediate transcriptional repression of the αENaC gene

Zhang

et al. 2013

American Journal of Physiology-Renal Physiology

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The epithelial Na ϩ channel subunit-␣ (␣ENaC) of the distal nephron is essential for salt balance. We previously demonstrated that the histone methyltransferase Dot1a and its protein partner Af9 basally repress ␣ENaC transcription in mouse inner medullary collecting duct type 3 (mIMCD3) cells and link aldosterone-elicited chromatin modifications to ␣ENaC transcriptional activation. Af9 DNA-binding activity has never been demonstrated, and whether and where Af9 binds to the ␣ENaC promoter to target Dot1a are unknown. The present study sought to identify functional Af9 cis-element(s) in the Ϫ57/ϩ439 "R3" subregion of ␣ENaC, the principal site for Dot1a-Af9 interaction, in mIMCD3 cells. We also exploited connecting tubule/collecting duct-specific Dot1l-deficient mice (Dot1l AC ) to determine the impact of Dot1l inactivation on renal ␣ENaC expression in vivo. mIMCD3 cell lines expressing ␣ENaC promoter-reporter constructs harboring deletion of ϩ74/ϩ107 demonstrated greatly reduced association of Af9 and Dot1a by ChIP/qPCR. Aldosterone treatment resulted in further decrements in Af9 and Dot1a association with the ␣ENaC promoter. Gel shift and antibody competition assays using wild-type and mutant oligomers revealed Af9-containing ϩ78/ ϩ92 ␣ENaC DNA-protein complexes in nuclear extracts of mIMCD3 cells. Mutation of the ϩ78/ϩ92 element resulted in higher basal ␣ENaC promoter activity and impaired Dot1a-mediated inhibition in trans-repression assays. In agreement, mice with connecting tubule/ collecting duct-specific knockout of Dot1l exhibited greater ␣ENaC mRNA levels in kidney compared with control. Thus, we conclude that ϩ78/ϩ92 of ␣ENaC represents the primary Af9 binding site involved in recruiting Dot1a to repress basal and aldosterone-sensitive ␣ENaC transcription and that Dot1l inactivation promotes ␣ENaC mRNA expression by eliminating Dot1a-mediated repression. chromatin; transcription; collecting duct; transcription factor; gene expression THE EPITHELIAL SODIUM CHANNEL (ENaC) plays a central role in collecting duct (CD) Na ϩ reabsorption, and hence the regulation of extracellular fluid volume and blood pressure (1). ENaC is expressed in the apical membrane of salt-absorbing epithelia of kidney, distal colon, and lung, where it constitutes the rate-limiting step in active Na ϩ and fluid absorption. ENaC consists of three subunits -␣, ␤, and ␥-encoded by the Scnn1a (referred to as "␣ENaC" in this report), Scnn1b, and Scnn1c genes. ENaC is also an important molecular target of aldosterone. In the CD, aldosterone administration or hyperaldosteronism induced by a low-Na ϩ diet increases ␣ENaC gene transcription, without increasing ␤-or ␥-subunit expression or ␣ENaC mRNA turnover (14), and this response appears to be rate-limiting for ENaC activity in this segment. The physiological importance of ␣ENaC to overall salt balance is highlighted by the finding that targeted inactivation of ␣ENaC in the connecting tubule (CNT)/CD of mice results in severe renal salt wasting characteristic of a pseudohypoaldosteronism type...

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“…41 All animal studies were performed in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Texas Health Science Center at Houston Animal Welfare Committee. were assessed using VetScan i-STAT 1 (ABAXIS) according to the manufacturer's instruction.…”

Section: If Studiesmentioning

confidence: 99%

Aqp2-Expressing Cells Give Rise to Renal Intercalated Cells

H¹,

Chen²,

Zhou

et al. 2013

Journal of the American Society of Nephrology

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The mammalian collecting duct comprises principal and intercalated cells, which maintain sodium/water and acid/base balance, respectively, but the epigenetic contributors to the differentiation of these cell types remain unknown. Here, we investigated whether the histone H3 K79 methyltransferase Dot1l, which is highly expressed in principal cells, participates in this process. Taking advantage of the distribution of aquaporin 2 (Aqp2), which localizes to principal cells of the collecting duct, we developed mice lacking Dot1l in Aqp2-expressing cells (Dot1l AC ) and found that these mice had approximately 20% fewer principal cells and 13%-16% more intercalated cells than control mice. This deletion of Dot1l in principal cells abolished histone H3 K79 methylation in these cells, but unexpectedly, most intercalated cells also had undetectable di-methyl K79, suggesting that Aqp2 + cells give rise to intercalated cells. These Aqp2 + cellderived intercalated cells were present in both developing and mature kidneys. Furthermore, compared with control mice, Dot1l AC mice had 40% higher urine volume and 18% lower urine osmolarity with relatively normal electrolyte and acid-base homeostasis. In conclusion, these data suggest that Dot1l deletion facilitates the differentiation of some a-and b-intercalated cells from Aqp2-expressing progenitor cells or mature principal cells.

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Af17 Deficiency Increases Sodium Excretion and Decreases Blood Pressure

Cited by 24 publications

References 47 publications

Mineralocorticoid receptor antagonizes Dot1a-Af9 complex to increaseαENaCtranscription

Mineralocorticoid receptor antagonizes Dot1a-Af9 complex to increaseαENaCtranscription

An Af9 cis-element directly targets Dot1a to mediate transcriptional repression of the αENaC gene

Aqp2-Expressing Cells Give Rise to Renal Intercalated Cells

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