miR-146b-5p has a sex-specific role in renal and cardiac pathology in a rat model of chronic kidney disease.

Chronic kidney disease presents a complex and distinct pathological landscape in men and women, yet this difference is poorly understood. microRNAs are powerful molecular regulators of pathophysiology in the kidney and other organs. We previously reported a significant upregulation of miR-146b-5p in the 5/6 nephrectomy rat model of chronic kidney disease. Here we investigated the sex-specific contribution of miR-146b-5p to renocardiac pathology by generating a novel miR-146b-/- rat and characterized the expression of miR-146b-5p in both wild-type and knockout animals. The 5/6 nephrectomy or sham surgery was performed on rats of each genotype and sex. Renal pathology was examined through gross histology, plasma and urinary analysis of electrolytes and metabolites, and by chronic blood pressure monitoring. Cardiac pathology was monitored via echocardiography and pressure-volume analysis. The miR-146b-/- rats show functional knockout of miR-146b-5p in both the kidney and heart. While 5/6 nephrectomy induced tissue hypertrophy, miR-146b-/- female rats displayed exacerbated renal hypertrophy. Additionally, miR-146b-/- female rats exhibited a marked elevation of renal fibrosis and significant renal dysfunction yet lower blood pressure and less pronounced cardiac remodeling. These phenotypic differences were not exhibited in miR-146b-/- male rats. Ovariectomy ameliorated renal pathology and abolished genotypic differences. In vitro examination of transforming growth factor-ß signaling in combination with miR-146b-5p manipulation supports a role for miR-146b-5p in mediating the protective benefit of estrogen from renal pathologies. Thus, our data highlight an important role of miR-146b-5p in modulating kidney disease progression and provide new avenues for the study of sex-specific pathology.

MiR-146a/b: a family with shared seeds and different roots.

MicroRNAs are small, noncoding, RNAs known for their powerful modulation of molecular processes, making them a major focus for studying pathological mechanisms. The human miR-146 family of microRNAs consists of two member genes, MIR146A and MIR146B These two microRNAs are located on different chromosomes and exhibit differential regulation in many cases. However, they are nearly identical in sequence, sharing a seed region, and are thus predicted to target the same set of genes. A large proportion of the microRNA (miR)-146 literature focuses on its role in regulating the innate immune response in the context of various pathologies by modulating two widely studied target genes in the toll-like receptor signaling cascade. A growing subset of the literature reports a role of miR-146 in cardiovascular and renal disease, and data suggest there is exciting potential for miR-146 as a diagnostic and therapeutic target. Nevertheless, the published literature is confounded by unclear and imprecise language concerning the specific effects of the two miR-146 family members. The present review will compare the genomic origin and regulation of miR-146a and miR-146b, discuss some approaches to overcome analytical and experimental challenges, and summarize findings in major areas of miR-146 research. Moving forward, careful evaluation of miR-146a/b specificity in analytical and experimental approaches will aid researchers in elucidating the functional relevance of differential regulation of the miR-146 family members in health and disease.

Towards a standardization of biomethane potential tests.

Production of biogas from different organic materials is a most interesting source of renewable energy. The biomethane potential (BMP) of these materials has to be determined to get insight in design parameters for anaerobic digesters. Although several norms and guidelines for BMP tests exist, inter-laboratory tests regularly show high variability of BMPs for the same substrate. A workshop was held in June 2015, in Leysin, Switzerland, with over 40 attendees from 30 laboratories around the world, to agree on common solutions to the conundrum of inconsistent BMP test results. This paper presents the consensus of the intense roundtable discussions and cross-comparison of methodologies used in respective laboratories. Compulsory elements for the validation of BMP results were defined. They include the minimal number of replicates, the request to carry out blank and positive control assays, a criterion for the test duration, details on BMP calculation, and last but not least criteria for rejection of the BMP tests. Finally, recommendations on items that strongly influence the outcome of BMP tests such as inoculum characteristics, substrate preparation, test setup, and data analysis are presented to increase the probability of obtaining validated and reproducible results.

Validity and Reliability of 2 Goniometric Mobile Apps: Device, Application, and Examiner Factors.

CONTEXT: Smartphones are being used in a variety of practice settings to measure joint range of motion (ROM). A number of factors can affect the validity of the measurements generated. However, there are no studies examining smartphone-based goniometer applications focusing on measurement variability and error arising from the electromechanical properties of the device being used. OBJECTIVE: To examine the concurrent validity and interrater reliability of 2 goniometric mobile applications (Goniometer Records, Goniometer Pro), an inclinometer, and a universal goniometer (UG). DESIGN: Nonexperimental, descriptive validation study. SETTING: University laboratory. PARTICIPANTS: 3 physical therapists having an average of 25 y of experience. MAIN OUTCOME MEASURES: Three standardized angles (acute, right, obtuse) were constructed to replicate the movement of a hinge joint in the human body. Angular changes were measured and compared across 3 raters who used 3 different devices (UG, inclinometer, and 2 goniometric apps installed on 3 different smartphones: Apple iPhone 5, LG Android, and Samsung SIII Android). Intraclass correlation coefficients (ICCs) and Bland-Altman plots were used to examine interrater reliability and concurrent validity. RESULTS: Interrater reliability for each of the smartphone apps, inclinometer and UG were excellent (ICC = .995-1.000). Concurrent validity was also good (ICC = .998-.999). Based on the Bland-Altman plots, the means of the differences between the devices were low (range = -0.4° to 1.2°). CONCLUSIONS: This study identifies the error inherent in measurement that is independent of patient factors and due to the smartphone, the installed apps, and examiner skill. Less than 2° of measurement variability was attributable to those factors alone. The data suggest that 3 smartphones with the 2 installed apps are a viable substitute for using a UG or an inclinometer when measuring angular changes that typically occur when examining ROM and demonstrate the capacity of multiple examiners to accurately use smartphone-based goniometers.

MicroRNA-21 Mediates Isoflurane-induced Cardioprotection against Ischemia-Reperfusion Injury via Akt/Nitric Oxide Synthase/Mitochondrial Permeability Transition Pore Pathway.

BACKGROUND: The role of microRNA-21 in isoflurane-induced cardioprotection is unknown. The authors addressed this issue by using microRNA-21 knockout mice and explored the underlying mechanisms. METHODS: C57BL/6 and microRNA-21 knockout mice were echocardiographically examined. Mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in vivo or ex vivo in the presence or absence of 1.0 minimum alveolar concentration of isoflurane administered before ischemia. Cardiac Akt, endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) proteins were determined by Western blot analysis. Opening of the mitochondrial permeability transition pore (mPTP) in cardiomyocytes was induced by photoexcitation-generated oxidative stress and detected by rapid dissipation of tetramethylrhodamine ethyl ester fluorescence using a confocal microscope. RESULTS: Genetic disruption of miR-21 gene did not alter phenotype of the left ventricle, baseline cardiac function, area at risk, and the ratios of phosphorylated-Akt/Akt, phosphorylated-eNOS/eNOS, and phosphorylated-nNOS/nNOS. Isoflurane decreased infarct size from 54 ± 10% in control to 36 ± 10% (P < 0.05, n = 8 mice per group), improved cardiac function after reperfusion, and increased the ratios of phosphorylated-Akt/AKT, phosphorylated-eNOS/eNOS, and phosphorylated-nNOS/nNOS in C57BL/6 mice subjected to ischemia-reperfusion injury. These beneficial effects of isoflurane were lost in microRNA-21 knockout mice. There were no significant differences in time of the mPTP opening induced by photoexcitation-generated oxidative stress in cardiomyocytes isolated between C57BL/6 and microRNA-21 knockout mice. Isoflurane significantly delayed mPTP opening in cardiomyocytes from C57BL/6 but not from microRNA-21 knockout mice. CONCLUSIONS: Isoflurane protects mouse hearts from ischemia-reperfusion injury by a microRNA-21-dependent mechanism. The Akt/NOS/mPTP pathway is involved in the microRNA-21-mediated protective effect of isoflurane.

Effects of elevation of ANP and its deficiency on cardiorenal function.

Atrial natriuretic peptide (ANP), encoded by Nppa, is a vasodilatory hormone that promotes salt excretion. Genome-wide association studies identified Nppa as a causative factor of blood pressure development, and in humans, ANP levels were suggested as an indicator of salt sensitivity. This study aimed to provide insights into the effects of ANP on cardiorenal function in salt-sensitive hypertension. To address this question, hypertension was induced in SSNPPA-/- (KO of Nppa in the Dahl salt-sensitive [SS] rat background) or SSWT (WT Dahl SS) rats by a high-salt (HS) diet challenge (4% NaCl for 21 days). Chronic infusion of ANP in SSWT rats attenuated the increase in blood pressure and cardiorenal damage. Overall, the SSNPPA-/- strain demonstrated higher blood pressure and intensified cardiac fibrosis (with no changes in ejection fraction) compared with SSWT rats. Furthermore, SSNPPA-/- rats exhibited kidney hypertrophy and higher glomerular injury scores, reduced diuresis, and lower sodium and chloride excretion than SSWT when fed a HS diet. Additionally, the activity of epithelial Na+ channel (ENaC) was found to be increased in the collecting ducts of the SSNPPA-/- rats. Taken together, these data show promise for the therapeutic benefits of ANP and ANP-increasing drugs for treating salt-sensitive hypertension.

Functionally Essential Tubular Proteins Are Lost to Urine-Excreted, Large Extracellular Vesicles during Chronic Renal Insufficiency.

BACKGROUND: The 5/6 nephrectomy (5/6Nx) rat model recapitulates many elements of human CKD. Within weeks of surgery, 5/6Nx rats spontaneously exhibit proximal tubular damage, including the production of very large extracellular vesicles and brush border shedding. We hypothesized that production and elimination of these structures, termed large renal tubular extracellular vesicles (LRT-EVs), into the urine represents a pathologic mechanism by which essential tubule proteins are lost. METHODS: LRT-EVs were isolated from 5/6Nx rat urine 10 weeks after surgery. LRT-EV diameters were measured. LRT-EV proteomic analysis was performed by tandem mass spectrometry. Data are available via the ProteomeXchange Consortium with identifier PXD019207. Kidney tissue pathology was evaluated by trichrome staining, TUNEL staining, and immunohistochemistry. RESULTS: LRT-EV size and a lack of TUNEL staining in 5/6Nx rats suggest LRT-EVs to be distinct from exosomes, microvesicles, and apoptotic bodies. LRT-EVs contained many proximal tubule proteins that, upon disruption, are known to contribute to CKD pathologic hallmarks. Select proteins included aquaporin 1, 16 members of the solute carrier family, basolateral Na+/K+-ATPase subunit ATP1A1, megalin, cubilin, and sodium-glucose cotransporters (SLC5A1 and SLC5A2). Histologic analysis confirmed the presence of apical membrane proteins in LRT-EVs and brush border loss in 5/6Nx rats. CONCLUSIONS: This study provides comprehensive proteomic analysis of a previously unreported category of extracellular vesicles associated with chronic renal stress. Because LRT-EVs contain proteins responsible for essential renal functions known to be compromised in CKD, their formation and excretion may represent an underappreciated pathogenic mechanism.

Vascular endothelial growth factor regulation of endothelial nitric oxide synthase phosphorylation is involved in isoflurane cardiac preconditioning.

Aims: Previous studies indicate that nitric oxide derived from endothelial nitric oxide synthase (eNOS) serves as both trigger and mediator in anaesthetic cardiac preconditioning. The mechanisms underlying regulation of eNOS by volatile anaesthetics have not been fully understood. Therefore, this study examined the role of vascular endothelial growth factor (VEGF) in isoflurane cardiac preconditioning. Methods and results: Wistar rats underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion. Isoflurane given prior to ischaemia/reperfusion significantly decreased myocardial infarct size from 60 ± 1% in control to 40 ± 3% (n = 8 rats/group, P < 0.05). The beneficial effects of isoflurane were blocked by neutralizing antibody against VEGF (nVEGF). Coronary arterial endothelial cells (ECs) alone or together with cardiomyocytes (CMs) were subjected to hypoxia/reoxygenation injury. The expression of VEGF and eNOS was analysed by western blot, and nitric oxide was measured by ozone-based chemiluminescence. In co-cultured CMs and ECs, isoflurane administered before hypoxia/reoxygenation attenuated lactate dehydrogenase activity and increased the ratio of phosphorylated eNOS/eNOS and nitric oxide production. The protective effect of isoflurane on CMs was compromised by nVEGF and after VEGF in ECs was inhibited with hypoxia inducible factor-1α short hairpin RNA (shRNA). The negative effect of hypoxia inducible factor-1α shRNA was restored by recombinant VEGF. Conclusion: Isoflurane cardiac preconditioning is associated with VEGF regulation of phosphorylation of eNOS and nitric oxide production.

Chronic Co-Administration of Sepiapterin and L-Citrulline Ameliorates Diabetic Cardiomyopathy and Myocardial Ischemia/Reperfusion Injury in Obese Type 2 Diabetic Mice.

BACKGROUND: Diabetic heart disease is associated with tetrahydrobiopterin oxidation and high arginase activity, leading to endothelial nitric oxide synthase dysfunction. Sepiapterin (SEP) is a tetrahydrobiopterin precursor, and L-citrulline (L-Cit) is converted to endothelial nitric oxide synthase substrate, L-arginine. Whether SEP and L-Cit are effective at reducing diabetic heart disease is not known. The present study examined the effects of SEP and L-Cit on diabetic cardiomyopathy and ischemia/reperfusion injury in obese type 2 diabetic mice. METHODS AND RESULTS: Db/db and C57BLKS/J mice at 6 to 8 weeks of age received vehicle, SEP, or L-Cit orally alone or in combination for 8 weeks. Cardiac function was evaluated with echocardiography. Db/db mice displayed hyperglycemia, obesity, and normal blood pressure and cardiac function compared with C57BLKS/J mice at 6 to 8 weeks of age. After vehicle treatment for 8 weeks, db/db mice had reduced ejection fraction, mitral E/A ratio, endothelium-dependent relaxation of coronary arteries, tetrahydrobiopterin concentrations, ratio of endothelial nitric oxide synthase dimers/monomers, and nitric oxide levels compared with vehicle-treated C57BLKS/J mice. These detrimental effects of diabetes mellitus were abrogated by co-administration of SEP and L-Cit. Myocardial infarct size was increased, and coronary flow rate and ± dP/dt were decreased during reperfusion in vehicle-treated db/db mice subjected to ischemia/reperfusion injury compared with control mice. Co-administration of SEP and L-Cit decreased infarct size and improved coronary flow rate and cardiac function in both C57BLKS/J and db/db mice. CONCLUSIONS: Co-administration of SEP and L-Cit limits diabetic cardiomyopathy and ischemia/reperfusion injury in db/db mice through a tetrahydrobiopterin/endothelial nitric oxide synthase/nitric oxide pathway.

Cardiomyocyte GTP Cyclohydrolase 1 Protects the Heart Against Diabetic Cardiomyopathy.

Diabetic cardiomyopathy increases the risk of heart failure and death. At present, there are no effective approaches to preventing its development in the clinic. Here we report that reduction of cardiac GTP cyclohydrolase 1 (GCH1) degradation by genetic and pharmacological approaches protects the heart against diabetic cardiomyopathy. Diabetic cardiomyopathy was induced in C57BL/6 wild-type mice and transgenic mice with cardiomyocyte-specific overexpression of GCH1 with streptozotocin, and control animals were given citrate buffer. We found that diabetes-induced degradation of cardiac GCH1 proteins contributed to adverse cardiac remodeling and dysfunction in C57BL/6 mice, concomitant with decreases in tetrahydrobiopterin, dimeric and phosphorylated neuronal nitric oxide synthase, sarcoplasmic reticulum Ca(2+) handling proteins, intracellular [Ca(2+)]i, and sarcoplasmic reticulum Ca(2+) content and increases in phosphorylated p-38 mitogen-activated protein kinase and superoxide production. Interestingly, GCH-1 overexpression abrogated these detrimental effects of diabetes. Furthermore, we found that MG 132, an inhibitor for 26S proteasome, preserved cardiac GCH1 proteins and ameliorated cardiac remodeling and dysfunction during diabetes. This study deepens our understanding of impaired cardiac function in diabetes, identifies GCH1 as a modulator of cardiac remodeling and function, and reveals a new therapeutic target for diabetic cardiomyopathy.

The removal of percutaneous Kirschner wires used in the stabilisation of fractures in children.

Kirschner wires are commonly used to stabilise fractures in children. There is a wide range of practice across the United Kingdom with regard to their removal, with some units routinely readmitting children for removal of wires under a general anaesthetic and others leaving the wires long and extracting them in the outpatient clinic. In a prospective study performed at The Barts and Royal London National Health Service Trust between November 1997 and December 1999, the acceptability and problems associated with the latter approach were assessed. A total of 203 percutaneous Kirschner wires were used to stabilise 119 fractures following closed reduction in 113 children. The majority of the fractures were in the upper limb and the children ranged in age from five months to fifteen years and five months. The Kirschner wires were subsequently removed in clinic without anaesthetic. Details of wire placement and complications associated with wire use and their removal were recorded. The most common complication seen was overgranulation at the entry point of the wire (5.4%). In two cases, wires had migrated below the skin surface, requiring local anaesthetic infiltration to enable retrieval. Using a visual analogue pain score, the mean pain score immediately after wire removal was 2.8 out of a maximum of 10, reducing to 0.9 after one minute. It is concluded that K-wires can be removed safely without anaesthetic in the outpatient clinic and that this is tolerated well by children.