Epidemiology of bacterial co-infections and risk factors in COVID-19-hospitalized patients in Spain: a nationwide study.

BACKGROUND: We performed a nationwide population-based retrospective study to describe the epidemiology of bacterial co-infections in coronavirus disease 2019 (COVID-19)-hospitalized patients in Spain in 2020. We also analyzed the risk factors for co-infection, the etiology and the impact in the outcome. METHODS: Data were obtained from records in the Minimum Basic Data Set (MBDS) of the National Surveillance System for Hospital Data in Spain, provided by the Ministry of Health and annually published with 2 years lag. COVID-19 circulated in two waves in 2020: from its introduction to 31st June and from 1st July to 31st December. The risk of developing a healthcare-associated bacterial co-infection and the risk for in-hospital and intensive care unit (ICU) mortality in co-infected patients was assessed using an adjusted logistic regression model. RESULTS: The incidence of bacterial co-infection in COVID-19 hospitalized patients was 2.3%. The main risk factors associated with bacterial co-infection were organ failure, obesity and male sex. Co-infection was associated with worse outcomes including higher in-hospital, in-ICU mortality and higher length of stay. Gram-negative bacteria caused most infections. Causative agents were similar between waves, although higher co-infections with Pseudomonas spp. were detected in the first wave and with Haemophilus influenzae and Streptococcus pneumoniae in the second. CONCLUSIONS: Co-infections are not as common as those found in other viral respiratory infections; therefore, antibiotics should be used carefully. Screening for actual co-infection to prescribe antibiotic therapy when required should be performed.

Post-fire management effects on hillslope-stream sediment connectivity in a Mediterranean forest ecosystem.

Forest fires intensify sediment transport and aggravate local and off-site consequences of soil erosion. This study evaluates the influence of post-fire measures on structural and functional sediment connectivity (SC) in five fire-affected Mediterranean catchments, which include 929 sub-catchments, by using the "aggregated index of connectivity" (AIC) at two temporal scenarios: I) immediately after the fire and before implementing post-fire practices ('Pre-man'), and II) two years after the fire ('Post-man'). The latter includes all the emergency stabilization practices, that are hillslope barriers, check-dams and afforestation. The stream system was set as the target of the computation (STR), to be representative of intense rainfall-runoff events with effective sediment delivery outside the catchments. Output normalization (AICN) allows comparing the results of the five basins between them. The sedimentological analysis is based on specific sediment yield (SSY) -measured at the check-dams installed after the fire -, and this data is used for output evaluation. Stream density and slope variables were the most influential factors on AICN-STR results at the sub-catchment scale. Post-fire hillslope treatments (barriers when built in high densities and afforestation) significantly reduced AICN-STR in comparison with untreated areas in both structural and functional approaches. Despite the presence of hillslope treatments, the higher erosive rainfall conditions resulted in higher AICN-STR values in the Post-man scenario (functional approach). A positive and good correlation was found between the measured SSY and the AICN-STR changes due to the post-fire practices and vegetation recovery, showing the good correspondence of the computation results and the real sediment dynamics of the studied catchments. Overall, AICN demonstrated to be a useful and versatile tool for post-fire management, which needs further research to optimize its applicability.

Effects of post-fire hillslope stabilisation techniques on chemical, physico-chemical and microbiological soil properties in mediterranean forest ecosystems.

Postfire restoration practices encompass those which aim to reduce negative wildfire impacts and to improve burned area rehabilitation. Contour-felled log debris (CFD) and log erosion barriers (LEB) are two techniques used worldwide on hillslopes after wildfires in order to avoid soil erosion. In this context, it is essential to evaluate how these restoration techniques can affect soil properties by increasing or decreasing wildfire impacts. The effects on several physico-chemical and biological soil parameters were here investigated by comparing three differently treated post-fire zones. Three randomly 20 × 20 m distributed plots were set up five years after wildfire in the burned and contour-felled log debris areas (CFD plots), three others in the burned and log erosion barriers area (LEB plots) and three others in the burned and unmanaged plots (BNa plots). Three more plots were set up in an unburned area close to the burned area (UB plots). The results revealed that LEB and, to a lesser extent CFD, improved postfire soil quality, which a priori favoured helped the recovery of ecosystem functions. Our results also indicate greater efficacy of LEB and CFD in retaining sediments by limiting loss of nutrients, which is considered essential to recover vegetation after a wildfire. Post-fire restoration plans should consider the use of LEB and CFD when aiming to favour ecosystem recovery processes after wildfires.

Epidemiology of fungal infection in COVID 19 in Spain during 2020 and 2021: a nationwide study.

We realize a nationwide population-based retrospective study to analyze the characteristics and risk factors of fungal co-infections in COVID-19 hospitalized patients as well as describe their causative agents in the Spanish population in 2020 and 2021. Data were obtained from records in the Minimum Basic Data Set of the National Surveillance System for Hospital Data in Spain, provided by the Ministry of Health, and annually published with two years lag. The assessment of the risk associated with the development of healthcare-associated fungal co-infections was assessed using an adjusted logistic regression model. The incidence of fungal co-infection in COVID-19 hospitalized patients was 1.41%. The main risk factors associated were surgery, sepsis, age, male gender, obesity, and COPD. Co-infection was associated with worse outcomes including higher in-hospital and in ICU mortality, and higher length of stay. Candida spp. and Aspergillus spp. were the microorganisms more frequent. This is the first study analyzing fungal coinfection at a national level in hospitalized patients with COVID-19 in Spanish population and one of the few studies available that demonstrate that surgery was an independent risk factor of Aspergillosis coinfection in COVID-19 patients.

Endothelial dysfunction is an early indicator of sepsis and neutrophil degranulation of septic shock in surgical patients.

BACKGROUND: Stratification of the severity of infection is currently based on the Sequential Organ Failure Assessment (SOFA) score, which is difficult to calculate outside the ICU. Biomarkers could help to stratify the severity of infection in surgical patients. METHODS: Levels of ten biomarkers indicating endothelial dysfunction, 22 indicating emergency granulopoiesis, and six denoting neutrophil degranulation were compared in three groups of patients in the first 12 h after diagnosis at three Spanish hospitals. RESULTS: There were 100 patients with infection, 95 with sepsis and 57 with septic shock. Seven biomarkers indicating endothelial dysfunction (mid-regional proadrenomedullin (MR-ProADM), syndecan 1, thrombomodulin, angiopoietin 2, endothelial cell-specific molecule 1, vascular cell adhesion molecule 1 and E-selectin) had stronger associations with sepsis than infection alone. MR-ProADM had the highest odds ratio (OR) in multivariable analysis (OR 11·53, 95 per cent c.i. 4·15 to 32·08; P = 0·006) and the best area under the curve (AUC) for detecting sepsis (0·86, 95 per cent c.i. 0·80 to 0·91; P < 0·001). In a comparison of sepsis with septic shock, two biomarkers of neutrophil degranulation, proteinase 3 (OR 8·09, 1·34 to 48·91; P = 0·028) and lipocalin 2 (OR 6·62, 2·47 to 17·77; P = 0·002), had the strongest association with septic shock, but lipocalin 2 exhibited the highest AUC (0·81, 0·73 to 0·90; P < 0·001). CONCLUSION: MR-ProADM and lipocalin 2 could be alternatives to the SOFA score in the detection of sepsis and septic shock respectively in surgical patients with infection.

ANTECEDENTES: La estratificación de la gravedad de una infección se basa actualmente en la puntuación SOFA (Sequential Organ Failure Assessment), que es difícil de calcular fuera de la unidad de cuidados intensivos. Los biomarcadores podrían ayudar a estratificar la gravedad de la infección en pacientes quirúrgicos. MÉTODOS: Se compararon las concentraciones de 10 biomarcadores que denotan disfunción endotelial, 22 que indican granulopoyesis de emergencia y 6 que expresan la degranulación de neutrófilos en tres grupos de pacientes de tres hospitales españoles (100 con infección, 95 con sepsis y 57 con shock séptico) en las primeras doce horas después del diagnóstico. RESULTADOS: Siete biomarcadores que expresan disfunción endotelial (proadrenomedulina, sindecan-1, trombomodulina, angiopoyetina-2, endocan-1, molécula de adhesión endotelial 1 y E-selectina) mostraron una fuerte asociación con la sepsis en comparación con la infección aislada. La proadrenomedulina presentó el valor más alto de la razón de oportunidades (odds ratio, OR) en el análisis multivariable (OR 11,53, i.c. del 95% 4,15-32,08, P = 0,006) y la mejor área bajo la curva para detectar sepsis (AUC 0,86, i.c. del 95% 0,80-0,91, P < 0,001). En la comparación entre sepsis y shock séptico, los biomarcadores que mostraron la asociación más estrecha con el shock séptico fueron dos biomarcadores de degranulación de neutrófilos (proteinasa-3 y lipocalina-2) (OR 8,09, i.c. del 9% 1,34-48,91, P = 0,028; OR 6.62, i.c. del 95% 2,47-17,77, P = 0,002), pero la lipocalina-2 presentó la mejor AUC (0,81, i.c. del 95% 0,73-0,90, P < 0,001). CONCLUSIÓN: la proadrenomedulina y la lipocalina-2 podrían representar alternativas a la puntuación SOFA para detectar sepsis y shock séptico en pacientes quirúrgicos con infección.

Impact of empirical treatment with antifungal agents on survival of patients with candidemia.

OBJECTIVE: The objective of this study was to evaluate the impact of echinocandins and fluconazole) on mortality 7 and 30 days after candidemia onset and overall in-hospital mortality), in patients with candidemia at a Spanish tertiary hospital. METHODS: A retrospective study was conducted that enrolled all non-neutropenic adult patients diagnosed with candidemia at Hospital Clínico Universitario de Valladolid between 2007 and 2016. A total of 179 patients were evaluated, they were divided into two sub-groups: surviving patients (n = 92) and non-surviving patients (n = 87). RESULTS: The 7-day mortality was 25,1% (45), 30-day mortality was 46,9% (84), and overall in-hospital mortality was 48,6% (87). 40.8% of patients received no antifungal treatment (43.8% of surviving patients and 37.8% of non-surviving patients; p=0.15). A total of 106 (59.2%) patients were treated, of which 90 patients (50.3%) received empiric treatment. 19.6% and 47.8% of surviving patients were treated with echinocandins and fluconazole, respectively. By contrast, of non-surviving patients, 31.0% were treated with echinocandins and 47.1% received fluconazole. Survival for the first 7 days was significantly higher in treated with antifungal agents (log-rank = 0.029), however, there were not significant differences in 30-day survival. Factors linked to a significant increase in overall in-hospital mortality were age (OR 1.040), septic shock (OR 2.694) and need for mechanical ventilation > 48 h (OR 2.812). CONCLUSIONS: Patients who received antifungal treatment, regardless of whether they received fluconazole or echinocandins, had a significantly lower mortality rate after 7 days than untreated patients, although no significant differences in 30-day mortality were seen.

[Installed Capacity Perception Survey for the Training of Residents Program in Orthopedics and Care Needs in Acute and Chronic Musculoskeletal Pathology in Mexico]. / Encuesta de Percepción en Capacidad Instalada para la Formación de Médicos Residentes en Ortopedia (Traumatología) y Necesidades de la Atención en Patología Musculoesquelética Aguda y Crónica en México. Primera Parte.

INTRODUCTION: It is essential that orthopaedic resident physicians be highly proficient in all aspects, considering the balance between supply, demand, need and context. Fundamental to identify the capacity and quality installed for their training in Mexico. MATERIAL AND METHODS: Observational Study, transverse, non-probabilistic sampling-conglomerates, in two phases. The instrument has 8 domains, 57 variables and 4,867 items. 60 graduate professors of 20 states, 50 hospital sites, 22 university programs. RESULTS: 1,038 years of experience (collective intelligence), 17 years of experience/teacher (01 to 50 years). Identified: acute pathology 30 (2 to 90%), chronic pathology 30 (5 to 96%), patients 15 years, 10 (3 to 30%), patients between 15 and 65 years, 47 (2 to 78%), patients 65 years, 20 (2 to 60%), number of beds/seat 20 (2 to 510), number of clinics 3 (1 to 48), number of surgical procedures/headquarters per year at the national level, was 960 (50 to 24,650). The national average per resident doctor is 362 surgeries/year with 1,450 surgical times/year. CONCLUSIONS: The needs and resources for the training of physicians specializing in orthopedics/traumatology are highly heterogeneous, so it should be adapted to the epidemiological needs of the region of influence, in an area of epidemiological transition. 62.2% expressed not having or have bad academic and scientific infrastructure at its headquarters, more than 50% without rotation overseas and 90% without regular scientific production.

INTRODUCCIÓN: Es fundamental que los médicos residentes de ortopedia (traumatología) sean altamente competentes en todos los aspectos, considerando el equilibrio entre la oferta, demanda, necesidad y contexto. Es primordial identificar la capacidad y calidad instalada para su formación en México. MATERIAL Y MÉTODOS: Estudio observacional, transversal, muestreo no probabilístico-conglomerados, en dos fases. El instrumento tiene ocho dominios, 57 variables y 4,867 ítems. Sesenta profesores de postgrado de 20 estados, 50 sedes hospitalarias, 22 programas universitarios. RESULTADOS: 1,038 años de experiencia (inteligencia colectiva), 17 años de experiencia/profesor (01 a 50 años). Se identificó: patología aguda 30 (2 a 90%), patología crónica 30 (5 a 96%), pacientes 15 años, 10 (3 a 30%), pacientes entre 15 y 65 años, 47 (2 a 78%), pacientes 65 años, 20 (2 a 60%), número de camas/sede 20 (2 a 510), número de consultorios 3 (1 a 48), el número de procedimientos quirúrgicos/sede al año a nivel nacional fue de 960 (50 a 24,650). La media nacional por médico residente es de 362 cirugías/año con 1,450 momentos quirúrgicos/año. CONCLUSIONES: Las necesidades y recursos para la formación de médicos especialistas en ortopedia/traumatología son en alto grado heterogéneos, por lo cual se debería adaptar a las necesidades epidemiológicas de la región de influencia, en un ámbito de transición epidemiológica. Sesenta y dos punto dos por ciento expresó no tener o tener deficiente infraestructura académica y científica en su sede, más de 50% sin rotación al extranjero y 90% sin producción científica regular.

Antifungal treatment with echinocandins: a 10-year clinical experience.

OBJECTIVE: The number of studies evaluating the use of echinocandins, whether or not its indication meets international guidelines, in clinical practice is limited. The objective of the present study was to determine the use of echinocandins in a tertiary Spanish hospital in 10 years of clinical practice, and to evaluate its impact on prognosis. METHODS: This retrospective study involved adult nonneutropenic ill patients with suspicion of fungal invasion who started treatment with echinocandins between 2006 and 2015. RESULTS: The number of patients treated with echinocandins was 153, and candidemia was detected thereafter in 25.5%. Factors associated with in-hospital mortality in patients receiving echinocandins were: sex male, septic shock, Charlson comorbidity index, and total stay at the hospital. In-hospital mortality after 7, 30 and 90 days was 13.7%, 24.8%, and 56.8%, respectively. From patients receiving echinocandins, 98 did no show multifocal colonization, 50 had Candida score <2.5, and 49 did not meet Ostrosky-Zeichner prediction rule. A total of 19 patients did not show any of these 3 potential risk factors for candidemia. CONCLUSIONS: The use of echinocandins in 10 years of clinical practice in our tertiary hospital has been performed according to international guidelines; however, candidemia was only diagnosed thereafter in only 25.5% of cases. Furthermore, according to our results, the adequate use of echinocandins seems not to be associated with reduced mortality rates. Further studies, involving a large cohort of patients and more hospitals, are required to corroborate these results.

[Acute disseminated encephalomyelitis. Experience of a tertiary hospital in Spain]. / Encefalomielitis aguda. Experiencia de un hospital terciario español.

INTRODUCTION: To describe the epidemiological, clinical, microbiological, neuroimaging and laboratory features, treatment, and outcome in a cohort of children with acute disseminated encephalomyelitis (ADEM). PATIENTS AND METHODS: Retrospective chart review was performed of children with a diagnosis of ADEM over a 23-year period in a tertiary hospital in Spain. RESULTS: Twelve cases were identified. Ten cases (83%) occurred after 1992. Nine patients (75%) presented between April and September. The mean age was 6 years. Nine patients (75%) were male. Fifty percent of the patients had a history of infectious disease or vaccination. The most frequent nonspecific symptom was fever in 75%. The most frequent neurological manifestations were motor deficits and altered consciousness in 75%. Cerebrospinal fluid abnormalities were found in 83%. All patients had at least one brain scan and one brain magnetic resonance imaging (MRI) scan. Three patients underwent spinal MRI. The sensitivity of MRI was greater than that of the scanner in the diagnosis of ADEM. An etiologic diagnosis was made in four patients: Mycoplasma pneumoniae, beta hemolytic streptococcus group A, Epstein-Barr virus and measles-mumps-rubella vaccination. Eleven patients were treated with corticosteroids and one was treated with intravenous immunoglobulin therapy. One patient died while 75 % of the patients had a good outcome. CONCLUSIONS: ADEM is in an infrequent disease in children. The clinical features are similar to those of infectious encephalitis. Etiologic diagnosis is difficult to establish but this entity is usually preceded by an infection. The neuroimaging test of choice to establish the diagnosis is MRI. In most patients, the prognosis is good.

Intracerebroventricular infusion of aldosterone induces hypertension in rats.

There is suggestive evidence for the direct participation of mineralocorticoids in the production of centrally mediated hypertension. Unilaterally nephrectomized Sprague-Dawley rats received a continuous infusion for 30 days using implanted osmotic minipumps with 1) artificial spinal fluid (CSF) intracerebroventricularly (icvt); 2) 0.005 micrograms aldosterone/h icvt; 3) 0.005 micrograms aldosterone/h sc; 4) 0.5 micrograms aldosterone/h sc. There was no significant difference between the groups in average weight gain (52 +/- 2 g) or organ weight to body weight, nor was urine volume increased above normal except in the group receiving the high sc dose of aldosterone. Blood pressure was significantly elevated only in those animals receiving 0.005 micrograms aldosterone/h icvt and 0.5 micrograms aldosterone/h sc. A second experiment was done using a specific spironolactone-type mineralocorticoid antagonist, prorenone. The rats were grouped as follows: 1) CSF icvt; 2) 0.005 micrograms/h aldosterone icvt; 3) 0.005 micrograms/h aldosterone plus 0.005 micrograms/h prorenone icvt; 4) 0.005 micrograms/h aldosterone plus 0.02 micrograms/h prorenone icvt; 5) 0.02 micrograms/h prorenone icvt. This study confirmed that this minute dose of aldosterone infused icvt produced a statistically significant increase in blood pressure with no increase in urine volume. Both the low, 0.005 micrograms/h, and high, 0.02 micrograms/h, doses of prorenone antagonized the pressor effect of aldosterone when infused with aldosterone into the lateral cerebral ventricle. The groups receiving 0.02 micrograms/h prorenone icvt or CSF icvt did not differ significantly in those parameters measured. A dose of aldosterone that was too small to produce changes in blood pressure when infused systemically was found to produce hypertension without polydypsia/polyuria when infused intrathecally. This pressor effect could be blocked by the concomitant infusion of a specific antagonist, prorenone. The study presented offers strong circumstantial evidence supporting a direct hypertensinogenic effect of aldosterone within the central nervous system.

19-Nordeoxycorticosterone excretion in male and female inbred salt-sensitive (S/JR) and salt-resistant (R/JR) Dahl rats.

Rats were selectively bred for susceptibility (S) and resistance (R) to the hypertensinogenic effects of excess salt intake by Dahl and further inbred to virtual homozygosity by Rapp (S/JR and R/JR). The S strain has been shown to have a mutation of the cytochrome P-450-dependent 11 beta,18-hydroxylase resulting in the enhanced production of 18-hydroxydeoxycorticosterone (18-OH-DOC) compared to that of the R strain. It is known that this enzyme is also responsible for the hydroxylation of deoxycorticosterone at the 19 position to produce 19-hydroxydeoxycorticosterone. Recently, the excretion of 19-nordeoxycorticosterone (19-nor-DOC), a potent mineralocorticoid, has been shown to be markedly increased in S/JR females compared to that in R/JR females consuming a high sodium diet. While the S/JR rat is spontaneously hypertensive, the course of the disease is greatly accelerated and exacerbated by a high sodium diet. If, indeed, 19-nor-DOC is responsible for the spontaneous hypertension in the S/JR rat, then its production should also be higher in the S/JR rat consuming a normal salt diet. Furthermore, since its production is suppressed by NaCl intake, the excretion should be even higher when not suppressed by a high sodium diet. We measured the urinary excretion of 19-nor-DOC, 18-OH-DOC, and corticosterone in male and female S/JR and R/JR rats consuming a normal sodium diet. The excretions of corticosterone and 18-OH-DOC were significantly higher by S/JR of both sexes than by R/JR, with the excretion by female rats being higher than that by male rats within the same strain. The hierarchy of excretion rates of 19-nor-DOC was: S/JR females greater than R/JR females greater than S/JR males greater than R/JR male rats. These studies indicate that while S/JR rats of both sexes develop higher blood pressures than the R/JR even on a standard salt intake, the excretion of 19-nor-DOC does not correlate well with their blood pressure elevation, since the normotensive female R/JR rat excretes significantly higher quantities of 19-nor-DOC than the hypertensive male S/JR rat. Thus, it is unclear whether 19-nor-DOC is playing a significant role in the pathogenesis of the hypertension in the S/JR rat. It also remains unknown whether the renal site of formation of 19-nor-DOC allows access to the mineralocorticoid target sites in the kidney.

A reevaluation of the mineralocorticoid and hypertensinogenic potential of 19-hydroxyandrostenedione.

Experiments were done to evaluate whether 19-hydroxyandrostenedione (19-OH-A) is a steroid which produces hypertension and amplifies the mineralocorticoid effects of aldosterone. No intrinsic mineralocorticoid or adosterone-amplifying effect was found in several bioassays using adrenalectomized rats at doses of 100 micrograms 19-OH-A and/or 0.1 microgram aldosterone or in assays in which urine electrolytes were measured daily during a 14-day continuous infusion of 100 micrograms/day 19-OH-A. In two different experiments, the twice weekly administration of 0.1-1 mg 19-OH-A in oil to intact rats' drinking water failed to produce a change in blood pressure after 6 weeks. When one kidney was removed from these rats and 0.9% saline was substituted for drinking water, the blood pressures of rats receiving 1 mg twice weekly of 19-OH-A became significantly elevated in only one of the two experiments. At the end of 10 weeks, the average 19-OH-A pressure (139 mmHg) was significantly (P less than 0.01) greater than average control pressure (114 mmHg), but significantly (P less than 0.01) less than the mean average pressure of rats receiving 5 mg deoxycorticosterone acetate (DOCA) (161 mmHg). In another experiment uninephrectomized rats received 0.5 mg 19-OH-A or 1 mg DOCA 3 times a week. After 5 weeks the pressure of those receiving 19-OH-A was 149 mmHg, significantly greater (P less than 0.01) than controls (123 mmHg), but significantly less (P less than 0.01) than that of those rats receiving DOCA (189 mmHg). Our data failed to confirm the aldosterone amplifying effect of 19-OH-A. We did obtain a small elevation of arterial pressure in unilaterally nephrectomized rats receiving saline to drink. The differences between our results and those reported for this compound are not readily apparent, but may be related to genetic differences within the Sprague-Dawley strain used in both studies and/or to diet.

RU-26988--a new tool for the study of the mineralocorticoid receptor.

Aldosterone binds to two renal cytosol receptors, one with high affinity and low capacity (Type I or mineralocorticoid) and another with lower affinity but greater capacity (Type II or glucocorticoid receptor). One of the ways to study Type I receptors isolated from Type II receptors is to block the latter with a steroid which shows high affinity for the glucocorticoid receptor and low affinity for the mineralocorticoid receptor. Dexamethasone has been used widely for this purpose but previous investigations and this study have found that dexamethasone competes with [3H]aldosterone for Type I receptor binding with a relative activity of 26% that of aldosterone and therefore is less than ideal as a glucocorticoid receptor blocker. RU-26988 (11 beta, 17 beta-dihydroxy-17 alpha-pregnane-1,4,6-trien-20-yn-21-methyl-3-one) is a recently synthesized glucocorticoid that shows very low affinity for the mineralocorticoid receptor. RU-26988 competes with [3H]aldosterone and [3H]2 alpha-methyl-9 alpha-fluorocortisol, a powerful mineralocorticoid, for the cytosol receptor from renal slices of adrenalectomized rats with a relative potency of less than 0.5% in comparison to unlabeled aldosterone and 2 alpha-methyl-9 alpha-fluorocortisol. RU-26988 has over twice the ability of unlabeled dexamethasone to compete with [3H]dexamethasone for binding to the renal cytosol glucocorticoid receptor. Scatchard analysis of [3H]aldosterone binding to rat renal cytosol showed two receptors, one with a calculated dissociation constant (Kd) of 2.81 X 10(-9) M and a second with a calculated Kd of 3.33 X 10(-8) M. The addition of a 100-fold concentration of RU-26988 produced a single line with a Kd of 5.02 X 10(-10) M indicating that the specific blocking of the glucocorticoid receptors allows an accurate determination of the kinetic parameters of the mineralocorticoid receptor by itself. Scatchard analysis of [3H]2 alpha-methyl-9 alpha-fluorocortisol binding produced a straight line with a Kd of 3.7 X 10(-9) M, such as would be produced if it were binding to only one single class of receptors. However, when an excess of RU-26988 was added to block the glucocorticoid receptor, a different straight line was produced by Scatchard's analysis with a Kd of 3.78 X 10(-10) M. Whereas the explanation for this is not apparent, it may be that the much larger concentration of glucocorticoid receptors, for which 2 alpha-methyl-9 alpha-fluorocortisol also has a very great affinity, masks the binding to the mineralocorticoid receptors.

Radioimmunoassay for urinary free 19-nor-deoxycorticosterone: effects of adrenocorticotropin and sodium depletion.

A RIA for urinary free deoxycorticosterone (DOC) and 19-nor-deoxycorticosterone [21-hydroxy-19-nor-4-pregnene-3,20-dione (19-nor-DOC)] was developed and used to measure urinary free DOC and 19-nor-DOC in rats kept on normal and low sodium diets and before and after ACTH administration. The RIA required a preliminary high performance liquid chromatography purification using a Lichrosorb diol column before the eluates were assayed using highly specific antibodies. The intraassay variability was 9.8% for DOC and 10% for 19-nor-DOC; the interassay variabilities were 19% and 16%, respectively. Urinary free DOC excretion was 0.71 +/- 0.15 ng/day (mean +/- SD) in rats maintained on a normal sodium diet and 1.42 +/- 0.46 ng/day after 3 days of a sodium-restricted diet. Urinary free 19-nor-DOC was 10.5 +/- 5.5 ng/day in rats kept on the normal sodium diet and 28.6 +/- 11.5 ng/dl after 3 days of a sodium-restricted diet. ACTH increased the excretion of urinary free DOC from 0.72 +/- 0.2 to 4.62 +/- 2.3 ng/day and that of 19-nor-DOC from 5.37 +/- 2.8 to 10.15 +/- 2.5 ng/day. These data indicate that the adrenal precursor of 19-nor-DOC probably comes from both the zona fasciculata and the glomerulosa to explain the ACTH and sodium depletion responses.

Cloning, expression, and tissue distribution of the rat nicotinamide adenine dinucleotide-dependent 11 beta-hydroxysteroid dehydrogenase.

A pcDNAI adult rat kidney complementary DNA (cDNA) library was screened using a sheep 11-hydroxysteroid dehydrogenase 2 (11 beta HSD-2) probe, and the isolated clones were sequenced. The 5'-end of the cDNA was determined by 5'-rapid amplification of cDNA ends. The rat 11 beta HSD-2 cDNA is 1864 base pair (bp) long. It consists of a 5'-untranslated region of 126 bp, an open reading frame of 1203 bp, and a 3'-untranslated region of 535 bp. The predicted protein contains 400 amino acid residues, with a calculated mol wt of 43,700. The rat 11 beta HSD-2 protein sequence is 85% homologous to human 11 beta HSD-2 and 76% to sheep 11 beta HSD-2. Expression of 11 beta HSD-2 messenger RNA by Northern blot and reverse transcription-polymerase chain reaction was high in kidney, distal colon, and adrenal and lower in the lung, hypothalamus, hippocampus, and midbrain. The rat 11 beta HSD-2 was transiently transfected into modified Chinese hamster ovary cells. Cells transfected with the 11 beta HSD-2 cDNA converted corticosterone into 11-dehydrocorticosterone. Conversion of corticosterone to 11-dehydrocorticosterone was NAD+ dependent and had a Km of 10.1 +/- 2.1 nM. In conclusion, we have cloned a rat NAD(+)-dependent 11 beta HSD with tissue distribution and kinetic characteristics suggesting that it could play a significant role in mineralocorticoid receptor selectivity.

Receptor binding and biological activity of 18-hydroxycortisol.

Recently, 18-hydroxycortisol (11 beta,17 alpha,18,21-tetrahydroxy-4-pregnene-3,20-dione) was isolated and identified from extracts of urine and adrenal incubates of patients with primary aldosteronism. The receptor-binding activity to the renal gluco- and mineralocorticoid receptors and its biological activity as a glucocorticoid and mineralocorticoid were investigated using synthetic 18-hydroxycortisol. The ability of 18-hydroxycortisol to compete with [3H]aldosterone for renal binding to the receptor was 0.13% that of unlabeled aldosterone. The addition of a specific glucocorticoid, RU-26988 (11 beta,17-dihydroxy-21-methyl-17 alpha-pregna-1,4,6-triene-20-yn-3-one) decreased the competing ability to 0.02%, indicating significant binding to the glucocorticoid receptor. The ability to compete with [3H]dexamethasone for the renal cytoplasmic glucocorticoid receptor was 0.1% that of unlabeled dexamethasone. The mineralocorticoid activity of 18-hydroxycortisol was undetectable. Its glucocorticoid activity using an in vitro bioassay based on the induction of tyrosine aminotransferase in the HTC cell was detectable at 10(-5) M, but was too low for adequate quantification. In a second in vitro glucocorticoid bioassay, inhibition of cell growth of the L929 fibroblast, 18-hydroxycortisol also showed minimal activity. In summary, it is unlikely that 18-hydroxycortisol plays a role in the metabolic syndrome in those patients who produce it in excess due to its inactivity as a gluco- or mineralocorticoid.

Renal receptor-binding activity of reduced metabolites of aldosterone: evidence for a mineralocorticoid effect outside of the classic aldosterone receptor system.

Reduced metabolites of aldosterone have been shown to have antinatriuretic and kaliuretic effects. We have studied the ability of four reduced metabolites of aldosterone to compete with [3H]aldosterone and [3H]dexamethasone for binding to the mineralocorticoid and glucocorticoid receptors of the kidney using adrenalectomized rat renal slices and cytosol, respectively, as sources of the binding proteins. 5 alpha-Dihydroaldosterone had 18.9% the ability to compete with [3H]aldosterone for binding to the cytoplasmic receptor of adrenalectomized rat renal slices in comparison to unlabeled aldosterone. Its antinatriuretic potency varied between 7-17%. Its ability to compete with [3H]dexamethasone for binding to the renal glucocorticoid receptor was only 1.9% in comparison to unlabeled dexamethasone. The relative competitive activities of 3 beta,5 alpha-tetrahydroaldosterone and 3 beta,5 beta-tetrahydroaldosterone with [3H]aldosterone to adrenalectomized rat renal slices cytosol were 1.26% and 0.05%, respectively, in comparison to unlabeled aldosterone. Their reported mineralocorticoid activities using the adrenalectomized rat bioassay (antinatriuresis) were 0.1-0.4% and 0.15%, respectively, in comparison to aldosterone. The most important aldosterone metabolite 3 alpha,5 beta-tetrahydroaldosterone showed negligible competitive activity with [3H]aldosterone or [3H]dexamethasone for the renal corticoid type I or type II receptors, respectively. However, this compound has been reported and confirmed to have weak but clear-cut mineralocorticoid activity (approximately 1/100th that of aldosterone). The mineralocorticoid activity of 3 alpha,5 beta-tetrahydroaldosterone cannot be explained by a mechanism involving the classic renal mineralocorticoid receptor. The mechanism could involve an alternative receptor system, a nonreceptor-mediated renal mechanism, or the conversion to a metabolite that would interact with classic receptors.

Identification of 19-hydroxydeoxycorticosterone, 19-oxo-deoxycorticosterone, and 19-oic-deoxycorticosterone as products of deoxycorticosterone metabolism by rat adrenals.

The formation of 19-hydroxydeoxycorticosterone (19,21-dihydroxy-4-pregnen-3,20-dione), 19-oxo-deoxycorticosterone (21-hydroxy-4-pregnen-3,19,20-trione), and 19-oic-deoxycorticosterone (19-oic-21-hydroxy-4-pregnen-3,20-dione) from precursor deoxycorticosterone by adrenal glands obtained from intact rats and from rats undergoing adrenal regeneration was demonstrated. These metabolites were isopolar with corresponding authentic steroid standards on thin layer chromatography, gas chromatography, and high pressure liquid chromatography. They were further characterized by either mass spectrometry or gas chromatography-mass spectrometry. Therefore, rat adrenals have the enzymes required to convert deoxycorticosterone to 19-hydroxydeoxycorticosterone, 19-oxo-deoxycorticosterone, and 19-oic-deoxycorticosterone; however, rat adrenals do not convert deoxycorticosterone or any of the oxygenated metabolites to 19-nor-deoxycorticosterone (21-hydroxy-19-nor-4-pregnen-3,20-dione). It is possible, however, that 19-nor-deoxycorticosterone is formed at peripheral sites from the oxygenated deoxycorticosterone precursors.

Mineralocorticoid and hypertensive effects of 19-nor-progesterone.

The mineralocorticoid potency of 19-nor-progesterone was evaluated by both its effect on electrolyte excretion in adrenalectomized animals and its ability to cause hypertension and electrolyte changes in mononephrectomized, salt-loaded rats. The mineralocorticoid activity, measured using an adrenalectomized rat bioassay, indicated that 19-nor-progesterone was 2.5% as potent as aldosterone but did not antagonize the effect of aldosterone when both were administered. In mononephrectomized rats, the daily administration of 1 mg/day quickly caused an enhanced consumption of 1% saline and induced severe hypertension within 3-4 weeks. Some severely hypertensive animals had marked anemia, but other did not; as a group they were found to have hypernatremia and hypokalemia. Hypertensive animals were found during life to display a relative hypothermia and, at necropsy, to have heart and kidney enlargement with severe and extensive vascular lesions in both organs, but not adrenal hypertrophy. It is concluded that 19-nor-progesterone has the characteristics of a potent mineralocorticoid and, as such, is capable of causing hypertension. It is not yet clear why this should be accompanied by hypothermia.

Aldosterone biosynthesis in the rat brain.

Messenger RNA (mRNA) for enzymes involved in adrenal steroid biosynthesis are expressed in the brain, and the coded enzymes have been shown to be active. The expression of mRNA for the cytochrome P-450 enzyme aldosterone synthase, crucial for the final step in the synthesis of aldosterone and the synthesis of aldosterone was studied in several anatomic areas of the rat brain. Expression of the mRNA for the aldosterone synthase was demonstrated by RT-PCR/Southern blot in adrenal, aorta, hypothalamus, hippocampus, amygdala, cerebrum, and cerebellum. Incubation of brain minces from intact and adrenalectomized rats demonstrated the synthesis of corticosterone and aldosterone from endogenous precursors. Incubations of brain minces with [1,2(3)H]-deoxycorticosterone, followed by extraction and three different successive TLCs, demonstrated the presence of labeled aldosterone, corticosterone, and 18-hydroxy-deoxycorticosterone. Incubation, in the presence of 10 microM cortisol or metyrapone, inhibited the synthesis of aldosterone or both aldosterone and corticosterone, respectively. These studies indicate that the rat brain has the enzymatic machinery for the synthesis of adrenal corticosteroids and is capable of synthesizing aldosterone. Aldosterone synthesized in the brain might play a paracrine role in the regulation of blood pressure.