Overexpression of Circulating Soluble Nogo-B Improves Diabetic Kidney Disease by Protecting the Vasculature.

Damage to the vasculature is the primary mechanism driving chronic diabetic microvascular complications such as diabetic nephropathy, which manifests as albuminuria. Therefore, treatments that protect the diabetic vasculature have significant therapeutic potential. Soluble neurite outgrowth inhibitor-B (sNogo-B) is a circulating N-terminus isoform of full-length Nogo-B, which plays a key role in vascular remodeling following injury. However, there is currently no information on the role of sNogo-B in the context of diabetic nephropathy. We demonstrate that overexpression of sNogo-B in the circulation ameliorates diabetic kidney disease by reducing albuminuria, hyperfiltration, and abnormal angiogenesis and protecting glomerular capillary structure. Systemic sNogo-B overexpression in diabetic mice also associates with dampening vascular endothelial growth factor-A signaling and reducing endothelial nitric oxide synthase, AKT, and GSK3ß phosphorylation. Furthermore, sNogo-B prevented the impairment of tube formation, which occurred when human endothelial cells were exposed to sera from patients with diabetic kidney disease. Collectively, these studies provide the first evidence that sNogo-B protects the vasculature in diabetes and may represent a novel therapeutic target for diabetic vascular complications.

Activation of serum/glucocorticoid-induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy.

Maintaining skeletal muscle mass is essential for general health and prevention of disease progression in various neuromuscular conditions. Currently, no treatments are available to prevent progressive loss of muscle mass in any of these conditions. Hibernating mammals are protected from muscle atrophy despite prolonged periods of immobilization and starvation. Here, we describe a mechanism underlying muscle preservation and translate it to non-hibernating mammals. Although Akt has an established role in skeletal muscle homeostasis, we find that serum- and glucocorticoid-inducible kinase 1 (SGK1) regulates muscle mass maintenance via downregulation of proteolysis and autophagy as well as increased protein synthesis during hibernation. We demonstrate that SGK1 is critical for the maintenance of skeletal muscle homeostasis and function in non-hibernating mammals in normal and atrophic conditions such as starvation and immobilization. Our results identify a novel therapeutic target to combat loss of skeletal muscle mass associated with muscle degeneration and atrophy.

Heterogeneous nuclear ribonucleoprotein A2/B1 is a tissue-specific aldosterone target gene with prominent induction in the rat distal colon.

The steroid hormone aldosterone enhances transepithelial Na(+) reabsorption in tight epithelia and is crucial to achieve extracellular volume homeostasis and control of blood pressure. One of the main transport pathways regulated by aldosterone involves the epithelial Na(+) channel (ENaC), which constitutes the rate-limiting step of Na(+) reabsorption in parts of the distal nephron and the collecting duct, the distal colon, and sweat and salivary glands. Although these epithelial tissues share the same receptor for aldosterone (mineralocorticoid receptor, MR), and the same transport system (ENaC), it has become clear that the molecular mechanisms involved in the modulation of channel activity are tissue-specific. Recent evidence suggests that aldosterone controls transcription and also translation of ENaC subunits in some cell types. A possible pathway for translational regulation is binding of regulatory proteins to ENaC subunit mRNAs, such as the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). In this study, we examined whether hnRNP A2/B1 is an aldosterone-target gene in vivo. Our data show that physiological levels of aldosterone markedly induce hnRNP A2/B1 expression in an early and sustained manner in the late distal colon epithelium but not in other aldosterone-target tissues. The effect depends on MR but not on glucocorticoid receptor activity. We also demonstrate that the genomic region upstream of hnRNP A2/B1 contains aldosterone-responsive elements involved in the control of gene expression. We hypothesize that hnRNP A2/B1 is involved in the tissue-specific regulation of ENaC biosynthesis and may coordinate the response of other genes relevant for transepithelial Na(+) reabsorption by aldosterone.

Identification of permissive insertion sites for generating functional fluorescent mineralocorticoid receptors.

The mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily of transcription factors, is activated by aldosterone and mediates its natriferic action in tight epithelia. MR is also expressed in nonepithelial tissues. Importantly, it mediates the deleterious effects of inappropriately high aldosterone levels in the heart, in which it induces the development of cardiac fibrosis. Antagonism of MR in humans is useful in the treatment of severe cardiac failure and some forms of hypertension. Despite the important pathophysiological and pharmacological role of this receptor, many important questions about its cellular biology and functional roles remain unanswered. A major challenge in the study of MR is the unavailability of fully functional fluorescent derivatives of the receptor. In this study we have created a library of MR mutants with insertions of the yellow fluorescent protein in various internal locations in the receptor using a random-insertion transposon-based technique. Screening of this library using a transactivation assay allowed us to identify several fluorescent constructs that retain functionality. Detailed characterization of one of these construct showed that it induces aldosterone-target genes such as the epithelial Na(+) channel subunits and the serum and glucocorticoid-induced kinase 1 at physiological concentrations of aldosterone to an equal extent than the wild-type receptor. Furthermore, aldosterone affinity, hormone-induced nuclear translocation, DNA binding and regulation of nongenomic pathways are all indistinguishable from the wild-type receptor. This new set of fluorescent MR derivatives provides a useful tool for studying the cell biology of the receptor.

Neutrophil gelatinase-associated lipocalin is a novel mineralocorticoid target in the cardiovascular system.

Mineralocorticoid receptor (MR) activation may be deleterious to the cardiovascular system, and MR antagonists improve morbidity and mortality of patients with heart failure. However, mineralocorticoid signaling in the heart remains largely unknown. Using a pan-genomic transcriptomic analysis, we identified neutrophil gelatinase-associated lipocalin (NGAL or lipocalin 2) as a strongly induced gene in the heart of mice with conditional and targeted MR overexpression in cardiomyocytes (whereas induction was low in glucocorticoid receptor-overexpressing mice). NGAL mRNA levels were enhanced after hormonal stimulation by the MR ligand aldosterone in cultured cardiac cells and in the heart of wild-type mice. Mineralocorticoid pathological challenge induced by nephrectomy/aldosterone/salt treatment upregulated NGAL expression in the heart and aorta and its plasma levels. We show evidence for MR binding to an NGAL promoter, providing a mechanism for NGAL regulation. We propose that NGAL may be a marker of mineralocorticoid-dependent injury in the cardiovascular system in mice.

The mineralocorticoid receptor is a constitutive nuclear factor in cardiomyocytes due to hyperactive nuclear localization signals.

The mineralocorticoid receptor (MR), a member of the nuclear receptor family, mediates the action of aldosterone in target epithelia, enhancing sodium reabsorption. In addition, MR may have other physiological functions in nonepithelial tissues. Altered expression or inappropriate activation of cardiac MR is directly linked to the development of cardiac fibrosis, and MR blockade is beneficial for the treatment of heart failure. However, the physiological role, activation status, and target genes of MR in the heart are poorly known. Because ligand-free steroid receptors are typically cytoplasmic and translocate to the nucleus upon ligand binding, we examined the subcellular localization of MR under different corticosteroid levels using subcellular fractionation and immunostaining. Our results demonstrate that MR is a chromatin-bound factor in mouse left ventricle and in a cultured model of cardiomyocytes, HL-1 cells, regardless of circulating corticosteroid levels. Immunohistochemical localization of MR in human heart confirms the subcellular localization pattern. Mutation of nuclear localization signals (NLSs) demonstrates that MR constitutive nuclear localization mainly depends on the synergistic contribution of NLS0 and NLS1. Constitutive nuclear localization in HL-1 cells can be reverted by cotransfection of heat shock protein 90. Heat shock protein 90 expression levels in the mouse heart and HL-1 cells are lower than those found in other tissues, suggesting that low levels of cochaperones render MR NLSs hyperactive in cardiomyocytes. Even though MR is constitutively nuclear, corticosteroids still control the transactivation properties of the receptor in a model promoter, although other MR ligand-independent activities cannot be excluded.