Nitric oxide (NO) is an important molecular mediator of numerous physiological processes in virtually every organ. In the kidney, NO plays prominent roles in the homeostatic regulation of glomerular, vascular, and tubular function. Differential expression and regulation of the NO synthase (NOS) gene family contribute to this diversity of action. This review explores recent advances in the molecular and cell biology of the NOS isoforms and relates these findings to functions of NO in the control of normal renal hemodynamics, the glomerular microcirculation, and renal salt excretion. Newly recognized molecular diversity of the NOS gene products, factors governing NOS isozyme gene expression and catalytic activity, and the intrarenal distribution of the NOS isoforms are examined. Physiological data regarding the complex roles of NO in the control of renal hemodynamics and the glomerular microcirculation are analyzed, and the effects of chronic NOS inhibition on glomerular function and structure are presented. The contributions of NO to renal salt excretion as well as functional and molecular biological evidence for adaptive changes in NOS isoform expression during variations in dietary salt balance are discussed. Current investigative challenges and goals for future research of renal NO biology are presented.

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Nitric oxide (NO) is a potent cell-signaling, effector, and vasodilator molecule that plays important roles in diverse biological effects in the kidney, vasculature, and many other tissues. Because of its high biological reactivity and diffusibility, multiple tiers of regulation, ranging from transcriptional to posttranslational controls, tightly control NO biosynthesis. Interactions of each of the major NO synthase (NOS) isoforms with heterologous proteins have emerged as a mechanism by which the activity, spatial distribution, and proximity of the NOS isoforms to regulatory proteins and intended targets are governed. Dimerization of the NOS isozymes, required for their activity, exhibits distinguishing features among these proteins and may serve as a regulated process and target for therapeutic intervention. An increasingly wide array of proteins, ranging from scaffolding proteins to membrane receptors, has been shown to function as NOS-binding partners. Neuronal NOS interacts via its PDZ domain with several PDZ-domain proteins. Several resident and recruited proteins of plasmalemmal caveolae, including caveolins, anchoring proteins, G protein-coupled receptors, kinases, and molecular chaperones, modulate the activity and trafficking of endothelial NOS in the endothelium. Inducible NOS (iNOS) interacts with the inhibitory molecules kalirin and NOS-associated protein 110 kDa, as well as activator proteins, the Rac GTPases. In addition, protein-protein interactions of proteins governing iNOS transcription function to specify activation or suppression of iNOS induction by cytokines. The calpain and ubiquitin-proteasome pathways are the major proteolytic systems responsible for the regulated degradation of NOS isozymes. The experimental basis for these protein-protein interactions, their functional importance, and potential implication for renal and vascular physiology and pathophysiology is reviewed.

Aldosterone is a major regulator of epithelial Na؉ absorption and acts in large part through induction of the epithelial Na ؉ channel (ENaC) gene in the renal collecting duct. We previously identified Dot1a as an aldosterone early repressed gene and a repressor of ENaC␣ transcription through mediating histone H3 Lys-79 methylation associated with the ENaC␣ promoter. Here, we report a novel aldosterone-signaling network involving AF9, Dot1a, and ENaC␣. AF9 and Dot1a interact in vitro and in vivo as evidenced in multiple assays and colocalize in the nuclei of mIMCD3 renal collecting duct cells. Overexpression of AF9 results in hypermethylation of histone H3 Lys-79 at the endogenous ENaC␣ promoter at most, but not all subregions examined, repression of endogenous ENaC␣ mRNA expression and acts synergistically with Dot1a to inhibit ENaC␣ promoter-luciferase constructs. In contrast, RNA interference-mediated knockdown of AF9 causes the opposite effects. Chromatin immunoprecipitation assays reveal that overexpressed FLAG-AF9, endogenous AF9, and Dot1a are each associated with the ENaC␣ promoter. Aldosterone negatively regulates AF9 expression at both mRNA and protein levels. Thus, Dot1a-AF9 modulates histone H3 Lys-79 methylation at the ENaC␣ promoter and represses ENaC␣ transcription in an aldosterone-sensitive manner. This mechanism appears to be more broadly applicable to other aldosterone-regulated genes because overexpression of AF9 alone or in combination with Dot1a inhibited mRNA levels of three other known aldosterone-inducible genes in mIMCD3 cells.The epithelial sodium channel (ENaC) 2 is a heteromultimeric protein composed of three partially homologous subunits (␣, ␤, and ␥) that is expressed in the apical membrane of salt-absorbing epithelia of kidney, colon, and lung where it constitutes the rate-limiting steps in active Na ϩ and fluid absorption. ENaC plays a major role in the regulation of salt homeostasis and blood pressure as evidenced by the fact that ENaC mutations are associated with genetic hypertensive and hypotensive diseases, such as Liddle's syndrome (1) and pseudohypoaldosteronism type 1 (2), and the fact that it is subject to tight and complex regulation by aldosterone. Aldosterone is a major regulator of epithelial Na ϩ absorption and acts in large part through ENaC induction in the renal collecting duct (3, 4). Aldosterone administration or hyperaldosteronism induced by a low-Na ϩ diet increases ENaC␣ gene transcription, without increasing ␤-or ␥-subunit expression (5-9), and without a separate effect on ENaC␣ mRNA turnover (10) in this segment. Although ENaC␣ synthesis is believed to be the rate-limiting step in Na ϩ channel formation in the collecting duct, only limited information exists regarding the specific mechanisms governing transcriptional regulation of this gene, in particular epigenetic mechanisms exerting such controls.Traditional models of aldosterone trans-activation of target genes, including ENaC␣, have emphasized interaction of the liganded mineralocorticoid receptor or g...

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 ENaCα 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 ENaCα promoter and derepression of ENaCα transcription. Thus, Af9 is a physiologic target of Sgk1, and Sgk1 negatively regulates the Dot1a-Af9 repressor complex that controls transcription of ENaCα and likely other aldosterone-induced genes. IntroductionThe renin-angiotensin-aldosterone system plays a major role in the control of blood pressure, extracellular fluid volume, and electrolyte balance, largely through the regulation of urinary Na + excretion. The aldosterone-sensitive distal nephron (ASDN), composed of the late distal convoluted tubule, connecting tubule, and cortical and medullary collecting ducts, is the final arbiter of renal Na + excretion. In the ASDN, transepithelial Na + absorption occurs by apical Na + entry via the epithelial Na + channel (ENaC) and basolateral Na + exit via the Na + ,K + -ATPase. ENaC, composed of 3 subunits (α, β, and γ), constitutes the rate-limiting step in this process, and changes in its activity and/or plasma membrane abundance constitute key regulatory steps. Aldosterone increases transepithelial Na + transport in the ASDN in large part through ENaCα induction in this region (1). Aldosterone increases ENaC function in 2 phases: an early phase involving upregulation of preexisting transport machinery and aldosterone-induced regulatory proteins, notably serum- and glucocorticoid-induced kinase-1 (Sgk1), which regulates the plasma membrane abundance of ENaC in part through phosphorylation of the ubiquitin ligase Nedd4-2 (2); and a delayed phase of aldosterone action involving de novo synthesis of ENaC, either from the liganded mineralocorticoid receptor directly binding hormone response elements in the ENaCα promoter to activate transcription (1) or through indirect