Acute kidney injury associated with smoking synthetic cannabinoid.

CONTEXT AND OBJECTIVES: Synthetic cannabinoids are illegal drugs of abuse known to cause adverse neurologic and sympathomimetic effects. They are an emerging health risk: 11% of high school seniors reported smoking them during the previous 12 months. We describe the epidemiology of a toxicologic syndrome of acute kidney injury associated with synthetic cannabinoids, review the toxicologic and public health investigation of the cluster, and describe clinical implications of the cluster investigation. MATERIALS AND METHODS: Case series of nine patients affected by the toxicologic syndrome in Oregon and southwestern Washington during May-October 2012. Cases were defined as acute kidney injury (creatinine > 1.3 mg/dL) among persons aged 13-40 years without known renal disease who reported smoking synthetic cannabinoids. Toxicology laboratories used liquid chromatography and time-of-flight mass spectrometry to test clinical and product specimens for synthetic cannabinoids, their metabolites, and known nephrotoxins. Public health alerts informed clinicians, law enforcement, and the community about the cluster and the need to be alert for toxidromes associated with emerging drugs of abuse. RESULTS: Patients were males aged 15-27 years (median, 18 years), with intense nausea and flank or abdominal pain, and included two sets of siblings. Peak creatinine levels were 2.6-17.7 mg/dL (median, 6.6 mg/dL). All patients were hospitalized; one required dialysis; none died. No alternate causes of acute kidney injury or nephrotoxins were identified. Patients reported easily purchasing synthetic cannabinoids at convenience, tobacco, and adult bookstores. One clinical and 2 product samples contained evidence of a novel synthetic cannabinoid, XLR-11 ([1-(5-fluoropentyl)-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)methanone). DISCUSSION AND CONCLUSION: Whether caused by direct toxicity, genetic predisposition, or an as-yet unidentified nephrotoxin, this association between synthetic cannabinoid exposure and acute kidney injury reinforces the need for vigilance to detect new toxicologic syndromes associated with emerging drugs of abuse. Liquid chromatography and time-of-flight mass spectrometry are useful tools in determining the active ingredients in these evolving products and evaluating them for toxic contaminants.

SGK integrates insulin and mineralocorticoid regulation of epithelial sodium transport.

The epithelial Na+ channel (ENaC) constitutes the rate-limiting step for Na+ transport across tight epithelia and is the principal target of hormonal regulation, particularly by insulin and mineralocorticoids. Recently, the serine-threonine kinase (SGK) was identified as a rapidly mineralocorticoid-responsive gene, the product of which stimulates ENaC-mediated Na+ transport. Like its close relative, protein kinase B (also called Akt), SGK's kinase activity is dependent on phosphatidylinositol 3-kinase (PI3K), a key mediator of insulin signaling. In our study we show that PI3K is required for SGK-dependent stimulation of ENaC-mediated Na+ transport as well as for the production of the phosphorylated form of SGK. In A6 kidney cells, mineralocorticoid induction of the phosphorylated form of SGK preceded the increase in Na+ transport, and specific inhibition of PI3K inhibited both phosphorylation of SGK and mineralocorticoid-induced Na+ transport. Insulin both augmented SGK phosphorylation and synergized with mineralocorticoids in stimulating Na+ transport. In a Xenopus laevis oocyte coexpression assay, SGK-stimulated ENaC activity was also markedly reduced by PI3K inhibition. Finally, in vitro-translated SGK specifically interacted with the ENaC subunits expressed in Escherichia coli as glutathione S-transferase fusion proteins. These data suggest that SGK is a PI3K-dependent integrator of insulin and mineralocorticoid actions that interacts with ENaC subunits to control Na+ entry into kidney collecting duct cells.

Stimulus-transcription coupling in pheochromocytoma cells. Promoter region-specific activation of chromogranin a biosynthesis.

To explore stimulus-transcription coupling in pheochromocytoma cells, we studied the biosynthetic response of chromogranin A, the major soluble protein co-stored and co-released with catecholamines, to chromaffin cells' physiologic nicotinic cholinergic secretory stimulation. Chromogranin A mRNA showed a time-dependent 3.87-fold response to nicotinic stimulation, and a nuclear run-off experiment indicated that the response occurred at a transcriptional level. Transfected chromogranin A promoter/luciferase reporter constructs were activated by nicotinic stimulation, in time- and dose-dependent fashions, in both rat PC12 pheochromocytoma cells and bovine chromaffin cells. Cholinergic subtype agents indicated that nicotinic stimulation was required. Promoter deletions established both positive and negative nicotinic response domains. Transfer of candidate promoter domains to a heterologous (thymidine kinase) promoter conferred region-specific nicotinic responses onto that promoter. A proximal promoter domain (from -93 to -62 base pairs) was activated in copy number- and distance-dependent fashion, and thus displayed features of a promoter element. Its activation was sufficient to account for the overall positive response to nicotine. Within this proximal region, a cAMP response element (CRE) was implicated as a major nicotinic response element, since a CRE point-gap mutation decreased nicotinic induction, transfer of CRE to a thymidine kinase promoter augmented the promoter's response to nicotine, and nicotine activated the CRE-binding protein CREB through phosphorylation at serine 133. We conclude that secretory stimulation of pheochromocytoma cells also activates the biosynthesis of the major secreted protein (chromogranin A), that the activation is transcriptional, and that a small proximal domain, including the CRE box, is, at least in part, both necessary and sufficient to account for the positive response to nicotine.

Glucocorticoid activation of chromogranin A gene expression. Identification and characterization of a novel glucocorticoid response element.

Glucocorticoids regulate catecholamine biosynthesis and storage at several sites. Chromogranin A, an abundant protein complexed with catecholamines in secretory vesicles of chromaffin cells and sympathetic axons, is also augmented by glucocorticoids. This study reports isolation of the rat chromogranin A promoter to elucidate transcriptional regulation of chromogranin A biosynthesis by glucocorticoids in neuroendocrine cells. Endogenous chromogranin A gene expression was activated up to 3.5-fold in chromaffin cells by glucocorticoid, in time-dependent fashion. Inhibition of new protein synthesis by cycloheximide did not alter the rise in chromogranin A mRNA, suggesting that glucocorticoids directly activate the chromogranin A promoter; nuclear runoff assays confirmed a 3.3-fold increased rate of initiation of new chromogranin A transcripts after glucocorticoid. Transfected rat chromogranin A promoter/luciferase reporter constructs were activated 2.6-3.1-fold by glucocorticoid, and selective agonist/antagonist studies determined that dexamethasone effects were mediated by glucocorticoid receptors. Both rat and mouse chromogranin A promoter/luciferase reporter constructs were activated by glucocorticoid. A series of promoter deletions narrowed the region of glucocorticoid action to a 93-bp section of the promoter, from position -526 to -619 bp upstream of the cap site. A 15-bp sequence ([-583 bp] 5'-ACATGAGTGTGTCCT-3' [-597 bp]) within this region showed partial homology to a glucocorticoid response element (GRE; half-site in italics) consensus sequence, and several lines of experimental evidence confirmed its function as a GRE: (a) site-directed mutation of this GRE prevented glucocorticoid activation of a chromogranin A promoter/reporter; (b) transfer of this GRE to a heterologous (thymidine kinase) promoter/reporter conferred activation by glucocorticoid, in copy number-dependent and orientation-independent fashion; and (c) electrophoretic gel mobility shifts demonstrated binding of this GRE by ligand-activated glucocorticoid receptor, though at 2.75-fold lower affinity than the glucocorticoid receptor interaction with a consensus GRE. The rat chromogranin A GRE showed functional and structural similarities to GREs in other genes proportionally regulated by glucocorticoids. We conclude that a discrete domain of the chromogranin A promoter is both necessary and sufficient to confer glucocorticoid regulation onto the gene, and that the activity of this region also explains the degree of activation of the endogenous gene by glucocorticoid.

Molecular cloning, structure, and expression of dopamine-beta-hydroxylase from bovine adrenal medulla.

Dopamine-beta-hydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine to norepinephrine, remains the topic of many unanswered questions. We isolated DBH cDNA clones from a bovine adrenal medulla cDNA library in the vector lambda gt10. The longest cDNA had an open reading frame encoding an entire mature DBH 578 amino acid (64,808 dalton) polypeptide chain, though lacking a portion of the signal peptide. Additional 5' clones, obtained by the polymerase chain reaction, established the sequence of a 19 amino acid signal peptide. The mature protein sequence was 84% homologous to that of human pheochromocytoma DBH, including preservation of four potential copper ligand sites (HH or HXH) and substrate binding domains. There were no hydrophobic (putative membrane spanning) domains, other than the signal peptide. All available DBH peptide and protein sequence data can be accounted for by the cDNA-deduced 578 amino acid mature protein primary structure. Prokaryotic DBH expression yielded a 65-kilodalton DBH-immunoreactive peptide that differed from eukaryotic adrenal DBH only in N-linked, endoglycosidase F-sensitive glycosylation in the latter. Southern analysis suggested one DBH gene, whereas Northern analysis suggested a single 2.6 kbp tissue-specific DBH transcript. Comparison of the DBH primary structure with other reported sequences [Protein Identification Resource (PIR), New Atlas (NEWAT)] did not indicate that DBH is a member of any known gene family. The results suggest that a single DBH gene encodes a message specifying a single DBH polypeptide chain.

Desensitization of epinephrine-initiated platelet aggregation does not alter binding to the alpha 2-adrenergic receptor or receptor coupling to adenylate cyclase.

Several investigators have shown that incubating unstirred platelets with epinephrine blunts subsequent aggregation when the platelets are stirred. Using aspirin-treated platelets, we further characterized this desensitization of alpha 2-adrenergic receptor-initiated aggregation. Desensitization occurred with a t1/2 of 3-6 min and was maximal at 20-30 min, at which time the initial rate of aggregation and its maximal extent were about half that of control platelets. When we preincubated platelets with epinephrine, and then added phentolamine to block the alpha 2-receptors, ADP-initiated aggregation occurred normally. Thus, the desensitization of epinephrine-initiated aggregation was not associated with a generalized impairment of aggregation. At concentrations too low to initiate aggregation, epinephrine is known to potentiate aggregation initiated by other agents. Clonidine also acts at alpha 2-receptors to potentiate aggregation initiated by other agents, but it does not initiate aggregation by itself. Preincubating clonidine with platelets for 30 min abolished its potentiating effect on ADP-initiated aggregation. Thus, the ability of alpha 2-receptors to both potentiate and initiate aggregation desensitizes after a brief preincubation with agonist. We performed several types of experiments to investigate the mechanism of this desensitization. Platelet alpha 2-receptors are coupled to an inhibition of adenylate cyclase. We found, however, that alpha 2-mediated inhibition of prostaglandin E1-stimulated cAMP accumulation occurred normally in desensitized platelets. Similarly, epinephrine inhibited basal adenylate cyclase activity normally in membranes prepared from desensitized platelets. In membranes prepared from desensitized platelets, epinephrine competed normally for [3H]rauwolscine binding, and this competition was modulated normally by guanine nucleotides. Thus, the properties of the alpha 2-receptors, as measured in radioligand binding experiments, were unchanged by densensitization. In conclusion, desensitization of alpha 2-adrenergic receptor-mediated aggregation occurs without change in the alpha 2-adrenergic receptors or in their coupling to an inhibition of adenylate cyclase.