Functional characterization of novel loss-of-function mutations in the vasopressin type 2 receptor gene causing nephrogenic diabetes insipidus.

BACKGROUND: X-linked nephrogenic diabetes insipidus (NDI) is a rare polyuric disorder caused by inactivating mutations in the arginine vasopressin receptor Type 2 (AVPR2) gene. METHODS: NDI patients from six unrelated families were subjected to mutational analysis of the AVPR2 gene. In-depth in vitro characterization of novel AVPR2 mutants by a combination of functional and immunological techniques provided further insight into molecular mechanisms causing receptor dysfunction. RESULTS: Mutational analysis revealed four novel (A89P, G107R, Q174R, W208X) and three recurrent (V277A, R337X, ΔR247-G250) mutations within the AVPR2 gene. One family carried the missense mutation R337X and a 12-bp deletion (ΔR247-G250), corresponding to a fragment in the third intracellular loop (ICL3), which was not genetically linked to R337X. The functionally tested missense mutations A89P, G107R and Q174R led to reduced receptor cell surface expression in transfected COS-7 cells, most probably due to misfolding and intracellular retention, and consequently to reduction or loss of agonist-mediated cyclic adenosine monophosphate formation. Deletion of R247-G250 had no effect on receptor function in vitro. Comparison with other mammalian AVPR2 orthologs showed that this part of the ICL3 is structurally not conserved and, therefore, less relevant for receptor function. In contrast, all missense mutations (A89P, G107R, Q174R, V277A) affect receptor positions that were fully preserved during mammalian evolution. CONCLUSION: Our results provide valuable information about residues critical for AVPR2 folding, trafficking and function and proof that these mutations are responsible for causing NDI in the affected subjects.

Low-dose hydrocortisone improves shock reversal and reduces cytokine levels in early hyperdynamic septic shock.

OBJECTIVES: To investigate the effect of low-dose hydrocortisone on time to shock reversal, the cytokine profile, and its relation to adrenal function in patients with early septic shock. DESIGN: Prospective, randomized, double-blind, single-center study. SETTING: Medical intensive care unit of a university hospital. PATIENTS: Forty-one consecutive patients with early hyperdynamic septic shock. INTERVENTIONS: After inclusion and a short adrenocorticotropic hormone test, all patients were randomized to receive either low-dose hydrocortisone (50-mg bolus followed by a continuous infusion of 0.18 mg/kg body of weight/hr) or matching placebo. After shock reversal, the dose was reduced to 0.06 mg/kg/hr and afterward slowly tapered. Severity of illness was estimated using Acute Physiology and Chronic Health Evaluation II score and Sequential Organ Failure Assessment score. MEASUREMENTS AND MAIN RESULTS: Time to cessation of vasopressor support (primary end point) was significantly shorter in hydrocortisone-treated patients compared with placebo (53 hrs vs. 120 hrs, p < .02). This effect was more profound in patients with impaired adrenal reserve. Irrespective of endogenous steroid production, cytokine production was reduced in the treatment group with lower plasma levels of interleukin-6 and a diminished ex vivo lipopolysaccharide-stimulated interleukin-1 and interleukin-6 production. Interleukin-10 levels were unaltered. Adverse events were not more frequent in the treatment group. CONCLUSIONS: Treatment with low-dose hydrocortisone accelerates shock reversal in early hyperdynamic septic shock. This was accompanied by reduced production of proinflammatory cytokines, suggesting both hemodynamic and immunomodulatory effects of steroid treatment. Hemodynamic improvement seemed to be related to endogenous cortisol levels, whereas immune effects appeared to be independent of adrenal reserve.

Effects of acute psychological stress on adhesion molecules, interleukins and sex hormones: implications for coronary heart disease.

RATIONALE: Subjects with high levels of daily stress suffer from an increased risk of coronary heart disease. Increased concentrations of the intercellular adhesion molecule-1 (ICAM-1) contribute to atherosclerosis. Cell adhesion molecules may be activated by psychological stress exposure, depending on their interaction with the interleukin network, sex hormones and cortisol secretion. OBJECTIVES: To assess effects of acute psychological stress on the interaction between cell adhesion molecules, interleukins, sex hormones and cortisol in healthy male subjects. METHODS: Cell adhesion molecules, interleukin-1 beta (IL-1beta), IL-2, IL-6, sex hormones and cortisol levels of 18 healthy male physicians were measured before and after an academic oral presentation and on a control day. RESULTS: The oral presentation was perceived as a stressful event and was accompanied by a significant increase in cortisol secretion in all volunteers. Soluble ICAM-1 and IL-1beta also increased in all subjects after psychological stress exposure. The stress-associated increase in IL-2 concentrations approached statistical significance and correlated negatively with luteinizing hormone plasma levels. Estradiol concentrations correlated positively with IL-6 levels. CONCLUSIONS: Subjective ratings and the increase in cortisol plasma concentrations support the validity of the chosen stress model. Acute stress exposure was followed by an increase in IL-1beta, IL-2 and soluble ICAM-1 plasma concentrations, which can contribute to coronary heart disease and immunological disorders.