Epigenetics and the control of the collecting duct epithelial sodium channel.

The apical membrane epithelial Na(+) channel subunit (ENaC) in series with the basolateral Na(+)/K(+)-adenosine triphosphatase mediates collecting duct Na(+) reabsorption. Aldosterone induces αENaC gene transcription, which appears to be rate limiting for ENaC activity in this segment. Although this response has long been assumed to be solely the result of liganded nuclear hormone receptors trans-activating αENaC, epigenetic controls of basal and aldosterone-induced transcription of αENaC in the collecting duct recently were described. These epigenetic pathways involve dynamic nuclear repressor complexes targeted to specific subregions of the αENaC promoter and consisting of the histone methyltransferase disrupter of telomeric silencing (Dot)1a together with the transcriptional factor Af9 or the nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase Sirt1, key co-regulatory proteins, including serum- and glucocorticoid-induced kinase-1 and the putative transcription factor Af17, and targeted chromatin modifications. The complexes, through the action of Dot1a, maintain chromatin associated with the αENaC promoter in a stable hypermethylated state, constraining αENaC transcription under basal conditions. Aldosterone and serum- and glucocorticoid-induced kinase-1, itself, activate αENaC transcription in large part by disrupting or diminishing the Dot1a-Af9 and Dot1a-Sirt1 complexes and their effects on chromatin. Mouse models indicate potential roles of the Dot1a pathways in renal salt excretion and hypertension.

Physical and functional interaction of Rnf2 with Af9 regulates basal and aldosterone-stimulated transcription of the α-ENaC gene in a renal collecting duct cell line.

The physical and functional interaction of Rnf2 (RING finger protein 2), a central component of the PRC (Polycomb repressive complex) 1 and Af9 (ALL1-fused gene from chromosome 9 protein), an aldosterone-sensitive transcription factor, in regulating basal and aldosterone-stimulated transcription of the α-ENaC (epithelial Na+ channel α-subunit) gene was explored in mIMCD3 CD (collecting duct) cells. Since Rnf2 lacks DNA-specific binding activity, other factors must mediate its site-specific chromatin recruitment. Rnf2 and Af9 co-localized in the nucleus and co-immunoprecipitated. A GST (glutathione transferase)-Af9 carboxy-terminal fusion protein directly interacted with in vitro translated Rnf2 in GST pull-down assays. Rnf2 knock down enhanced basal and aldosterone-stimulated α-ENaC mRNA levels and α-ENaC promoter activity. ChIP/QPCR (chromatin immunoprecipitation/quantitative PCR) assays demonstrated enrichment of Rnf2, H2AK119 (mono-ubiquitinated histone H2A lysine 119), and H3K27me3 (histone H3 lysine 27 trimethylated), a PRC2 chromatin mark, at multiple α-ENaC promoter subregions corresponding to regions of known Af9 enrichment, under basal conditions. Sequential ChIP confirmed Rnf2-Af9 co-occupancy of the α-ENaC promoter. Aldosterone provoked early and sustained depletion of Rnf2, ubiquitinated H2AK119, and trimethylated H3K27 associated with the subregions of the α-ENaC promoter. Thus, Af9 mediates site-selective physical and functional recruitment of Rnf2 to the α-ENaC promoter to constrain basal α-ENaC transcription in collecting duct cells, and aldosterone reverses this process.

Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting duct.

Aldosterone increases tubular Na(+) absorption largely by increasing α-epithelial Na(+) channel (αENaC) transcription in collecting duct principal cells. How aldosterone reprograms basal αENaC transcription to high-level activity in the collecting duct is incompletely understood. Promoter methylation, a covalent but reversible epigenetic process, has been implicated in the control of gene expression in health and disease. We investigated the role of promoter methylation/demethylation in the epigenetic control of basal and aldosterone-stimulated αENaC transcription in mIMCD3 collecting duct cells. Bisulfite treatment and sequencing analysis after treatment of the cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) identified clusters of methylated cytosines in a CpG island near the transcription start site of the αENaC promoter. 5-Aza-CdR treatment or small interfering RNA-mediated knockdown of DNMT3b or methyl-CpG-binding domain protein (MBD)-4 derepressed basal αENaC transcription, indicating that promoter methylation suppresses basal αENaC transcription. Aldosterone triggered a time-dependent decrease in 5mC and DNMT3b and a concurrent enrichment in 5-hydroxymethylcytosine (5hmC) and ten-eleven translocation (Tet)2 at the αENaC promoter, consistent with active demethylation. 5-Aza-CdR mimicked aldosterone by enhancing Sp1 binding to the αENaC promoter. We conclude that DNMT3b- and MBD4-dependent methylation of the αENaC promoter limits basal αENaC transcription, in part by limiting Sp1 binding and trans-activation. Aldosterone stimulates the dispersal of DNMT3b and recruitment of Tet2 to demethylate the αENaC promoter to induce αENaC transcription. These results disclose a novel epigenetic mechanism for the control of basal and aldosterone-induced αENaC transcription that adds to previously described epigenetic controls exerted by histone modifications.

Sp1 trans-activates and is required for maximal aldosterone induction of the αENaC gene in collecting duct cells.

The epithelial Na+ channel (ENaC) in the distal nephron constitutes the rate-limiting step for renal sodium reabsorption. Aldosterone increases tubular sodium absorption in large part by increasing αENaC transcription in collecting duct principal cells. We previously reported that Af9 binds to +78/+92 of αENaC and recruits Dot1a to repress basal and aldosterone-sensitive αENaC transcription in mouse inner medullary collecting duct (mIMCD)3 cells. Despite this epigenetic repression, basal αENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In the present study, we identified Sp1 as one such regulator. Gel shift and antibody competition assays using a +208/+240 probe revealed DNA-Sp1-containing complexes in mIMCD3 cells. Mutation of the +222/+229 element abrogated Sp1 binding in vitro and in promoter-reporter constructs stably expressed in mIMCD3 cells. Compared with the wild-type promoter, an αENaC promoter-luciferase construct with +222/+229 mutations exhibited much lower activity and impaired trans-activation in Sp1 overexpression experiments. Conversely, Sp1 knockdown inhibited endogenous αENaC mRNA and the activity of the wild-type αENaC promoter but not the mutated construct. Aldosterone triggered Sp1 recruitment to the αENaC promoter, which was required for maximal induction of αENaC promoter activity and was blocked by spironolactone. Sequential chromatin immunoprecipitation assays and functional tests of +78/+92 and +222/+229 αENaC promoter mutants indicated that while Sp1, Dot1a, and Af9 co-occupy the αENaC promoter, the Sp1 effects are functionally independent from Dot1a and Af9. In summary, Sp1 binding to a cis-element at +222/+229 represents the first identified constitutive driver of αENaC transcription, and it contributes to maximal aldosterone trans-activation of αENaC.

Molecular regulation of Trypanosoma congolense-induced nitric oxide production in macrophages.

BALB/c mice are highly susceptible while C57BL/6 mice are relatively resistant to experimental Trypanosoma congolense infection. Several reports show that an early interferon-gamma (IFN-γ) response in infected mice is critically important for resistance via the activation of macrophages and production of nitric oxide (NO). NO is a pivotal effector molecule and possesses both cytostatic and cytolytic properties for the parasite. However, the molecular mechanisms leading to T. congolense (TC)-induced NO release from macrophages are not known. In this study, we investigated the signaling pathways induced by trypanosomes in immortalized macrophage cell lines from the highly susceptible BALB/c (BALB.BM) and relatively resistant C57Bl/6 (ANA-1) mice. We found that T. congolense whole cell extract (TC-WCE) induces significantly higher levels of NO production in IFN-γ-primed ANA-1 than BALB.BM cells, which was further confirmed in primary bone marrow-derived macrophage (BMDM) cultures. NO production was dependent on mitogen-activated protein kinase (MAPK, including p38, Erk1/2, and JNK) phosphorylation and was significantly inhibited by specific MAPK inhibitors in BALB.BM, but not in ANA-1 cells. In addition, T. congolense- and IFN-γ-induced NO production in ANA-1 and BALB.BM cells was dependent on STAT1 phosphorylation and was totally suppressed by the use of fludarabine (a specific STAT1 inhibitor). We further show that T. congolense induces differential iNOS transcriptional promoter activation in IFN-γ-primed cells, which is dependent on the activation of both GAS1 and GAS2 transcription factors in BALB.BM but only on GAS1 in ANA-1 cells. Taken together, our findings show the existence of differential signalling events that lead to NO production in macrophages from the highly susceptible and relatively resistant mice following treatment with IFN-γ and T. congolense. Understanding these pathways may help identify immunomodulatory mechanisms that regulate the outcome of infection during Trypanosome infections.

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

The epithelial Na(+) channel subunit-α (αENaC) of the distal nephron is essential for salt balance. We previously demonstrated that the histone methyltransferase Dot1a and its protein partner Af9 basally repress αENaC transcription in mouse inner medullary collecting duct type 3 (mIMCD3) cells and link aldosterone-elicited chromatin modifications to αENaC transcriptional activation. Af9 DNA-binding activity has never been demonstrated, and whether and where Af9 binds to the αENaC promoter to target Dot1a are unknown. The present study sought to identify functional Af9 cis-element(s) in the -57/+439 "R3" subregion of αENaC, 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 αENaC expression in vivo. mIMCD3 cell lines expressing αENaC promoter-reporter constructs harboring deletion of +74/+107 demonstrated greatly reduced association of Af9 and Dot1a by ChIP/qPCR. Aldosterone treatment resulted in further decrements in Af9 and Dot1a association with the αENaC promoter. Gel shift and antibody competition assays using wild-type and mutant oligomers revealed Af9-containing +78/+92 αENaC DNA-protein complexes in nuclear extracts of mIMCD3 cells. Mutation of the +78/+92 element resulted in higher basal αENaC 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 αENaC mRNA levels in kidney compared with control. Thus, we conclude that +78/+92 of αENaC represents the primary Af9 binding site involved in recruiting Dot1a to repress basal and aldosterone-sensitive αENaC transcription and that Dot1l inactivation promotes αENaC mRNA expression by eliminating Dot1a-mediated repression.

Effects of genetic variation in H3K79 methylation regulatory genes on clinical blood pressure and blood pressure response to hydrochlorothiazide.

BACKGROUND: Nearly one-third of the United States adult population suffers from hypertension. Hydrochlorothiazide (HCTZ), one of the most commonly used medications to treat hypertension, has variable efficacy. The renal epithelial sodium channel (ENaC) provides a mechanism for fine-tuning sodium excretion, and is a major regulator of blood pressure homeostasis. DOT1L, MLLT3, SIRT1, and SGK1 encode genes in a pathway that controls methylation of the histone H3 globular domain at lysine 79 (H3K79), thereby modulating expression of the ENaCα subunit. This study aimed to determine the role of variation in these regulatory genes on blood pressure response to HCTZ, and secondarily, untreated blood pressure. METHODS: We investigated associations between genetic variations in this candidate pathway and HCTZ blood pressure response in two separate hypertensive cohorts (clinicaltrials.gov NCT00246519 and NCT00005520). In a secondary, exploratory analysis, we measured associations between these same genetic variations and untreated blood pressure. Associations were measured by linear regression, with only associations with P ≤ 0.01 in one cohort and replication by P ≤ 0.05 in the other cohort considered significant. RESULTS: In one cohort, a polymorphism in DOT1L (rs2269879) was strongly associated with greater systolic (P = 0.0002) and diastolic (P = 0.0016) blood pressure response to hydrochlorothiazide in Caucasians. However, this association was not replicated in the other cohort. When untreated blood pressure levels were analyzed, we found directionally similar associations between a polymorphism in MLLT3 (rs12350051) and greater untreated systolic (P < 0.01 in both cohorts) and diastolic (P < 0.05 in both cohorts) blood pressure levels in both cohorts. However, when further replication was attempted in a third hypertensive cohort and in smaller, normotensive samples, significant associations were not observed. CONCLUSIONS: Our data suggest polymorphisms in DOT1L, MLLT3, SIRT1, and SGK1 are not likely associated with blood pressure response to HCTZ. However, a possibility exists that rs2269879 in DOT1L could be associated with HCTZ response in Caucasians. Additionally, exploratory analyses suggest rs12350051 in MLLT3 may be associated with untreated blood pressure in African-Americans. Replication efforts are needed to verify roles for these polymorphisms in human blood pressure regulation.

CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells.

Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.

Intestinal ischemic preconditioning after ischemia/reperfusion injury in rat intestine: profiling global gene expression patterns.

OBJECTIVE: Intestinal ischemia/reperfusion (IR) injury involves activation of inflammatory mediators, mucosal necrosis, ileus, and alteration in a variety of gene products. Ischemic preconditioning (IPC) reduced all the effects of intestinal injury seen in IR. In an effort to investigate the molecular mechanisms responsible for the protective effects afforded by IPC, we sought to characterize the global gene expression pattern in rats subjected to IPC in the setting of IR injury. METHODS: Rats were randomized into five groups: (1) Sham, (2) IPC only (3) IR, (4) Early IPC + IR (IPC --> IR), and (5) Late IPC + IR (IPC --> 24 h --> IR). At 6 h after reperfusion, ileum was harvested for total RNA isolation, pooled, and analyzed on complementary DNA (cDNA) microarrays with validation using real-time polymerase chain reaction (PCR). Significance Analysis of Microarray (SAM) software was used to determine statistically significant changes in gene expression. RESULTS: Early IPC + IR had 5,167 induced and 4 repressed genes compared with the other groups. SAM analysis revealed 474 out of 10,000 genes differentially expressed among the groups. Early and Late IPC + IR had more genes involved in redox hemostasis, the immune/inflammatory response, and apoptosis than either the IPC only or IR alone groups. CONCLUSION: The transcriptional profile suggests that IPC exerts its protective effects by regulating the gene response to injury in the intestine.

AF17 competes with AF9 for binding to Dot1a to up-regulate transcription of epithelial Na+ channel alpha.

We previously reported that Dot1a.AF9 complex represses transcription of the epithelial Na(+) channel subunit alpha (alpha-ENaC) gene in mouse inner medullary collecting duct mIMCD3 cells and mouse kidney. Aldosterone relieves this repression by down-regulating the complex through various mechanisms. Whether these mechanisms are sufficient and conserved in human cells or can be applied to other aldosterone-regulated genes remains largely unknown. Here we demonstrate that human embryonic kidney 293T cells express the three ENaC subunits and all of the ENaC transcriptional regulators examined. These cells respond to aldosterone and display benzamil-sensitive Na(+) currents, as measured by whole-cell patch clamping. We also show that AF17 and AF9 competitively bind to the same domain of Dot1a in multiple assays and have antagonistic effects on expression of an alpha-ENaC promoter-luciferase construct. Overexpression of Dot1a or AF9 decreased mRNA expression of the ENaC subunits and their transcriptional regulators and reduced benzamil-sensitive Na(+) currents. AF17 overexpression caused the opposite effects, accompanied by redirection of Dot1a from the nucleus to the cytoplasm and reduction in histone H3 K79 methylation. The nuclear export inhibitor leptomycin B blocked the effect of AF17 overexpression on H3 K79 hypomethylation. RNAi-mediated knockdown of AF17 yielded nuclear enrichment of Dot1a and histone H3 K79 hypermethylation. As with AF9, AF17 displays nuclear and cytoplasmic co-localization with Sgk1. Therefore, AF17 competes with AF9 to bind Dot1a, decreases Dot1a nuclear expression by possibly facilitating its nuclear export, and relieves Dot1a.AF9-mediated repression of alpha-ENaC and other target genes.

Sirtuin 1 functionally and physically interacts with disruptor of telomeric silencing-1 to regulate alpha-ENaC transcription in collecting duct.

Aldosterone increases renal tubular Na+ absorption in large part by increasing transcription of the epithelial Na(+) channel alpha-subunit (alpha-ENaC) expressed in the apical membrane of collecting duct principal cells. We recently reported that a complex containing the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) associates with and represses the alpha-ENaC promoter in mouse inner medullary collecting duct mIMCD3 cells, and that aldosterone acts to disrupt this complex and its inhibitory effects (Zhang, W., Xia, X., Reisenauer, M. R., Rieg, T., Lang, F., Kuhl, D., Vallon, V., and Kone, B. C. (2007) J. Clin. Invest. 117, 773-783). Here we demonstrate that the NAD(+)-dependent deacetylase sirtuin 1 (Sirt1) functionally and physically interacts with Dot1 to enhance the distributive activity of Dot1 on H3K79 methylation and thereby represses alpha-ENaC transcription in mIMCD3 cells. Sirt1 overexpression inhibited basal alpha-ENaC mRNA expression and alpha-ENaC promoter activity, surprisingly in a deacetylase-independent manner. The ability of Sirt1 to inhibit alpha-ENaC transcription was retained in a truncated Sirt1 construct expressing only its N-terminal domain. Conversely, Sirt1 knockdown enhanced alpha-ENaC mRNA levels and alpha-ENaC promoter activity, and inhibited global H3K79 methylation, particularly H3K79 trimethylation, in chromatin associated with the alpha-ENaC promoter. Sirt1 and Dot1 co-immunoprecipitated from mIMCD3 cells and colocalized in the nucleus. Sirt1 immunoprecipitated from chromatin associated with regions of the alpha-ENaC promoter known to associate with Dot1. Aldosterone inhibited Sirt1 association at two of these regions, as well as Sirt1 mRNA expression, in a coordinate manner with induction of alpha-ENaC transcription. Overexpressed Sirt1 inhibited aldosterone induction of alpha-ENaC transcription independent of effects on mineralocorticoid receptor trans-activation. These data identify Sirt1 as a novel modulator of alpha-ENaC, Dot1, and the aldosterone signaling pathway.

Sp1 trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene.

The H(+)-K(+)-ATPase alpha(2) (HKalpha2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5'-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-kappaB and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the -144/-135 Sp element influences basal HKalpha2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for -154/-127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HKalpha2 gene in mIMCD3 cells in vivo. HKalpha2 minimal promoter-luciferase constructs with point mutations in the -144/-135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3, trans-activated an HKalpha2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HKalpha2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HKalpha2 promoter without altering the binding or regulatory influence of NF-kappaB p65 or CREB-1 on the proximal HKalpha2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HKalpha2 gene expression in mIMCD3 cells in vivo and in vitro.

Narrative review: evolving concepts in potassium homeostasis and hypokalemia.

Humans are intermittently exposed to large variations in potassium intake, which range from periods of fasting to ingestion of potassium-rich meals. These fluctuations would abruptly alter plasma potassium concentration if not for rapid mechanisms, primarily in skeletal muscle and the liver, that buffer the changes in plasma potassium concentration by means of transcellular potassium redistribution and feedback control of renal potassium excretion. However, buffers have capacity limits, and even robust feedback control mechanisms require that the perturbation occur before feedback can initiate corrective action. In contrast, feedforward control mechanisms sense the effect of disturbances on the system's homeostasis. This review highlights recent experimental insights into the participation of feedback and feedforward control mechanisms in potassium homeostasis. New data make clear that feedforward homeostatic responses activate when decreased potassium intake is sensed, even when plasma potassium concentration is still within the normal range and before frank hypokalemia ensues, in addition to the classic feedback activation of renal potassium conservation when plasma potassium concentration decreases. Given the clinical importance of dyskalemias in patients, these novel experimental paradigms invite renewed clinical inquiry into this important area.

Epigenetics and the control of epithelial sodium channel expression in collecting duct.

In eukaryotic nuclei, genomic DNA is compacted with histone and nonhistone proteins into a dynamic polymer termed chromatin. Reorganization of chromatin structure through histone modifications, the action of chromatin factors, or DNA methylation, can profoundly change gene expression. These epigenetic modifications allow heritable and potentially reversible changes in gene functioning to occur without altering the DNA sequence, thus extending the information potential of the genetic code. This review provides an introduction to epigenetic concepts for renal investigators and an overview of our work detailing an epigenetic pathway for aldosterone signaling and the control of epithelial Na(+) channel-alpha (ENaCalpha) subunit gene expression in the collecting duct. This new pathway involves a nuclear repressor complex, consisting of histone H3 Lys-79 methyltransferase disruptor of telomeric silencing-1a (Dot1a), ALL1 fused gene from chromosome 9 (Af9), a sequence-specific DNA-binding protein that binds the ENaCalpha promoter, and potentially other nuclear proteins. This complex regulates targeted histone H3 Lys-79 methylation of chromatin associated with the ENaCalpha promoter, thereby suppressing its transcriptional activity. Aldosterone disrupts the Dot1a-Af9 interaction by serum- and glucocorticoid-induced kinase-1 phosphorylation of Af9, and inhibits Dot1a and Af9 expression, resulting in histone H3 Lys-79 hypomethylation at specific subregions, and derepression of the ENaCalpha promoter. The Dot1a-Af9 pathway may also be involved in the control of genes implicated in renal fibrosis and hypertension.

Pretreatment with bone morphogenetic protein-7 (BMP-7) mimics ischemia preconditioning following intestinal ischemia/reperfusion injury in the intestine and liver.

Intestinal ischemia/reperfusion (I/R) injury has been shown to cause intestinal mucosal injury and adversely affect function. Ischemic preconditioning (IPC) has been shown to protect against intestinal I/R injury by reducing polymorphonuclear leukocyte infiltration, intestinal mucosal injury, and liver injury, and preserve intestinal transit. Bone morphogenetic protein 7 (BMP-7) has been shown to protect against I/R injury in the kidney and brain. Recently, microarray analysis has been used to examine the possible IPC candidate pathways. This work revealed that IPC may work through upregulation of BMP-7. The purpose of this study was to examine if pretreatment with BMP-7 would replicate the effects seen with IPC in the intestine and liver after intestinal I/R. Rats were randomized to six groups: sham, I/R (30 min of superior mesenteric artery occlusion and 6 h of R), IPC+R (three cycles of superior mesenteric artery occlusion for 4 min and R for 10 min), IPC+I/R, BMP-7+R (100 microm/kg recombinant human BMP-7), or BMP-7+I/R. A duodenal catheter was placed, and 30 min before sacrifice, fluorescein isothiocyanate-Dextran was injected. At sacrifice, dye concentrations were measured to determine intestinal transit. Ileal mucosal injury was determined by histology and myeloperoxidase activity was used as a marker of polymorphonuclear leukocyte infiltration. Serum levels of aspartate aminotransferase were measured at sacrifice to determine liver injury. Pretreatment with BMP-7 significantly improved intestinal transit and significantly decreased intestinal mucosal injury and serum aspartate aminotransferase levels, comparable to animals undergoing IPC. In conclusion, BMP-7 protected against intestinal I/R-induced intestinal and liver injury. Bone morphogenetic protein 7 may be a more logical surrogate to IPC in the prevention of injury in the setting of intestinal I/R.

Aldosterone-induced Sgk1 relieves Dot1a-Af9-mediated transcriptional repression of epithelial Na+ channel alpha.

Aldosterone plays a major role in the regulation of salt balance and the pathophysiology of cardiovascular and renal diseases. Many aldosterone-regulated genes--including that encoding the epithelial Na+ channel (ENaC), a key arbiter of Na+ transport in the kidney and other epithelia--have been identified, but the mechanisms by which the hormone modifies chromatin structure and thus transcription remain unknown. We previously described the basal repression of ENaCalpha by a complex containing the histone H3 Lys79 methyltransferase disruptor of telomeric silencing alternative splice variant a (Dot1a) and the putative transcription factor ALL1-fused gene from chromosome 9 (Af9) as well as the release of this repression by aldosterone treatment. Here we provide evidence from renal collecting duct cells and serum- and glucocorticoid-induced kinase-1 (Sgk1) WT and knockout mice that Sgk1 phosphorylated Af9, thereby impairing the Dot1a-Af9 interaction and leading to targeted histone H3 Lys79 hypomethylation at the ENaCalpha promoter and derepression of ENaCalpha transcription. Thus, Af9 is a physiologic target of Sgk1, and Sgk1 negatively regulates the Dot1a-Af9 repressor complex that controls transcription of ENaCalpha and likely other aldosterone-induced genes.

New mechanisms for transcriptional repression of ENaC And iNOS.

Gene transcription is highly regulated to ensure that specific genes are expressed at the appropriate times, places and levels in response to various genetic and environmental stimuli. Activation of some genes occurs by relief of basal repression controls, whereas termination of active transcription can involve feedback inhibition. We describe our characterization of aldosterone-triggered de-repression of the epithelial Na(+) channel-alpha subunit (ENaCalpha) gene in renal collecting duct cells in a process that involves a novel nuclear repressor complex, consisting of a histone H3 K79 methyltransferase and the putative transcription factor AF9, that regulates targeted histone H3 K79 methylation at the ENaCalpha promoter. As an example of feedback inhibition, we describe our work characterizing how the end product, nitric oxide, feedback inhibits inducible nitric oxide synthase (iNOS) gene transcription by S-nitrosylating its transactivator poly(ADP-ribose) polymerase (PARP-1) and, thereby, decreasing its ability to act at the iNOS promoter.