The microbiome and acute organ injury: focus on kidneys.

The microbiome of critically ill patients is significantly altered by both effects of the illnesses and clinical interventions provided during intensive care. Studies have shown that manipulating the microbiome can prevent or modulate complications of critical illness in experimental models and preliminary clinical trials. This review aims to discuss general concepts about the microbiome, including mechanisms of modifying acute organ dysfunction. The focus will be on the effects of microbiome modulation during experimental acute kidney injury (excluding septic AKI) and comparison with other experimental acute organ injuries commonly seen in critically ill patients.

Targeting immune cell glutamyl-prolyl-transfer RNA synthetase 1 (EPRS1) to prevent fibrosis after tubulointerstitial nephritis.

Glutamyl-prolyl-transfer RNA synthetase 1 is an enzyme that connects glutamic acid and proline to transfer RNA during protein synthesis. In this issue, a study by Kang et al. examined the role of the immune cell glutamyl-prolyl-transfer RNA synthetase 1 in toxin-induced tubulointerstitial nephritis mice. The study demonstrated that blocking glutamyl-prolyl-transfer RNA synthetase 1 may be a therapeutic target to attenuate fibrosis after toxin-induced tubulointerstitial nephritis.

Kidney double positive T cells have distinct characteristics in normal and diseased kidneys.

Multiple types of T cells have been described and assigned pathophysiologic functions in the kidneys. However, the existence and functions of TCR+CD4+CD8+ (double positive; DP) T cells are understudied in normal and diseased murine and human kidneys. We studied kidney DPT cells in mice at baseline and after ischemia reperfusion (IR) and cisplatin injury. Additionally, effects of viral infection and gut microbiota were studied. Human kidneys from patients with renal cell carcinoma were evaluated. Our results demonstrate that DPT cells expressing CD4 and CD8 co-receptors constitute a minor T cell population in mouse kidneys. DPT cells had significant Ki67 and PD1 expression, effector/central memory phenotype, proinflammatory cytokine (IFNγ, TNFα and IL-17) and metabolic marker (GLUT1, HKII, CPT1a and pS6) expression at baseline. IR, cisplatin and viral infection elevated DPT cell proportions, and induced distinct functional and metabolic changes. scRNA-seq analysis showed increased expression of Klf2 and Ccr7 and enrichment of TNFα and oxidative phosphorylation related genes in DPT cells. DPT cells constituted a minor population in both normal and cancer portion of human kidneys. In conclusion, DPT cells constitute a small population of mouse and human kidney T cells with distinct inflammatory and metabolic profile at baseline and following kidney injury.

Microbiome modulation after severe acute kidney injury accelerates functional recovery and decreases kidney fibrosis.

Targeting gut microbiota has shown promise to prevent experimental acute kidney injury (AKI). However, this has not been studied in relation to accelerating recovery and preventing fibrosis. Here, we found that modifying gut microbiota with an antibiotic administered after severe ischemic kidney injury in mice, particularly with amoxicillin, accelerated recovery. These indices of recovery included increased glomerular filtration rate, diminution of kidney fibrosis, and reduction of kidney profibrotic gene expression. Amoxicillin was found to increase stool Alistipes, Odoribacter and Stomatobaculum species while significantly depleting Holdemanella and Anaeroplasma. Specifically, amoxicillin treatment reduced kidney CD4+T cells, interleukin (IL)-17 +CD4+T cells, and tumor necrosis factor-α double negative T cells while it increased CD8+T cells and PD1+CD8+T cells. Amoxicillin also increased gut lamina propria CD4+T cells while decreasing CD8+T and IL-17+CD4+T cells. Amoxicillin did not accelerate repair in germ-free or CD8-deficient mice, demonstrating microbiome and CD8+T lymphocytes dependence for amoxicillin protective effects. However, amoxicillin remained effective in CD4-deficient mice. Fecal microbiota transplantation from amoxicillin-treated to germ-free mice reduced kidney fibrosis and increased Foxp3+CD8+T cells. Amoxicillin pre-treatment protected mice against kidney bilateral ischemia reperfusion injury but not cisplatin-induced AKI. Thus, modification of gut bacteria with amoxicillin after severe ischemic AKI is a promising novel therapeutic approach to accelerate recovery of kidney function and mitigate the progression of AKI to chronic kidney disease.

Pyroglutamylated RFamide peptide (QRFP): Role in early testicular development in relation to Sertoli cell maturation in prepubertal mice.

QRFP, an orexigenic neuropeptide, binds to its cognate receptor GPR103 and regulates various biological functions. We have recently shown that QRFP and its receptor are present in mice testes and that their expression is high during early postnatal period. The present study aimed to investigate the effect of sustained high level of QRFP on Sertoli cells proliferation and differentiation and to relate these events with germ cell differentiation and lumen formation in the seminiferous tubules in mice testes during prepubertal period. QRFP was injected intraperitoneally to male mice from postnatal day 5 to 16. Morphometric analysis and various markers related to Sertoli cell maturation (WT1, p27kip1, AMH, AR and CYP19A1) and germ cell proliferation and differentiation (PCNA, GDNF and c-Kit) were evaluated. QRFP administration caused an early lumen formation in the seminiferous tubules in testis of treated mice. Further, there was a significant increase in p27kip1 expression and a marked decrease in AMH expression in QRFP-treated mice compared to controls. However, no appreciable change was noted in AR expression in treated mice. QRFP treatment also caused an increase in c-Kit expression in treated mice compared to controls, suggesting an accelerated spermatogonial differentiation in testis of QRFP-treated mice. Taken together, the present results suggest that the prolonged high level of QRFP increases Sertoli cell maturation, which, in turn, plays a contributory role in increasing the pace of germ cell differentiation and formation of lumen in the seminiferous tubules.

A Comparison of the Real-Life Clinical Effectiveness of the Leading Licensed ICS/LABA Combination Inhalers in the Treatment for COPD.

Introduction: The Fostair® 100/6 (BDP/FF) pressurized metered-dose inhaler, delivering an extrafine formulation, is licensed for asthma and COPD in the UK. However, its real-life effectiveness for COPD has not been evaluated. This study compared the clinical effectiveness of BDP/FF against other licensed ICS/LABA combination inhalers: the Seretide® Accuhaler® (FP/SAL) and the Symbicort® Turbohaler® (BUD/FF). Methods: A matched historical cohort study was conducted using records of patients with diagnostic codes for COPD from the Optimum Patient Care Research Database (OPCRD). Patients who had received BDP/FF as their first ICS/LABA were matched 1:1 with patients who had received FP/SAL or BUD/FF, resulting in two matched comparisons. Additional analysis was conducted on patients who had never had diagnostic codes for asthma. Noninferiority in terms of the proportion of patients with moderate/severe COPD exacerbations on the different inhalers in the following year was assessed. Noninferiority was achieved if the upper CI limit were ≤1.2. Results: This study included 537 and 540 patient pairs in the BDP/FF vs FP/SAL cohort and the BDP/FF vs BUD/FF cohort, respectively. The proportion of patients with COPD exacerbations in the BDP/FF group was not significantly different from either the FP/SAL (68.7% vs 70.2%, AOR 0.89, 95% CI 0.67-1.19) or BUD/FF group (68.5% vs 69.4%, AOR 0.79, 95% CI 0.58-1.08). Noninferiority of BDP/FF in preventing COPD exacerbations was fulfilled in both comparisons. In patients without asthma, BDP/FF was also noninferior to BUD/FF (proportion with COPD exacerbations, 67.8% vs 64.7%, AOR 0.79, 95% CI 0.51-1.1997). Additionally, a significantly lower proportion of patients prescribed BDP/FF had COPD exacerbations than FP/SAL (64.8% vs 73.7%, AOR 0.64 95% CI 0.43-0.96). Conclusion: Initiating ICS/LABA treatment of COPD with extrafine-formulation BDP/FF was noninferior in preventing moderate/severe exacerbations compared to FP/SAL and BUD/FF.

Pyroglutamylated RFamide peptide 43: A putative modulator of testicular steroidogenesis.

BACKGROUND: We have recently shown that QRFP and its receptor are predominantly expressed in germ cells, Sertoli cells and Leydig cells in mice testes. OBJECTIVE: The present study investigated the role of QRFP in testicular steroidogenesis in mice. MATERIALS AND METHODS: Both ex vivo and in vivo experiments were performed. For ex vivo, testicular tissues were cultured with 0, 10, 100 and 1000 nM QRFP, with or without hCG, for 6, 12 and 24 h, and media were used for testosterone assay. The hCG-stimulated testicular tissues were used for immunoblot of SF1, StAR, CYP11A1, 3ß- and 17ß-HSD. For in vivo, mice received bilateral intratesticular injection of saline or 0.3, 1 and 3nmol QRFP and were killed at 6, 12 and 24 h post-injection. Testosterone in serum was measured at above durations, while qRT-PCR of HMG-CoA synthase 1 and SR-B1 and immunoblot of steroidogenesis-related markers were performed at 24 h post-injection. RESULTS: Testosterone production under basal and hCG-stimulated conditions increased in a time-dependent manner, and QRFP supplementation to testicular culture caused an increase and a decrease in hormone production. The effect of QRFP on testosterone production under hCG-stimulated culture or in vivo conditions at 6 and 24h was similar. At 6h, testosterone production increased at 10 and 100 nM and also at 0.3 and 1nmol QRFP, while it decreased at 1000 nM and 3 nmol doses. At 24 h, testosterone level decreased at lower concentrations (10 nM and 0.3 nmol) and thereafter increased at middle (100nM and 1nmol) and higher (1000 nM and 3 nmol) concentrations under both hCG-stimulated culture and in vivo. DISCUSSION AND CONCLUSION: QRFP induced production of testosterone by modulating steroidogenic machinery at optimal doses and durations. Further, findings of in vivo study indicate that QRFP besides directly regulating testicular steroidogenesis may also have modulated other factors which act together in a holistic manner to control steroidogenesis.

Role of pyroglutamylated RFamide peptide43 in germ cell dynamics in mice testes in relation to energy metabolism.

QRFP is a neuropeptide that regulates glucose homeostasis and increases insulin sensitivity in tissues. We have previously shown that QRFP and its receptor (GPR103) are predominantly expressed in germ cells and Sertoli cells, respectively, in mice testes. In the present study, we report that QRFP caused an increase in PCNA and a decrease in p27Kip1 expressions in the testis under both in vivo and ex vivo conditions. Besides, via an in vivo study, cell cycle analysis by FACS showed an increase in 2C cells and a decrease in 1C cells. QRFP also induced expression of GDNF and phosphorylation of Akt and ERK-1/2. Together these results suggest that QRFP has a proliferative effect on germ cells in mice testes, since it caused a proportional increase in the mitotic activity and the number of spermatogonial cells. Further, observations of increased expressions of STAT-3 and Neurog3 in treated mice suggest that QRFP treatment regulates priming of undifferentiated spermatogonia to undergo differentiation, while a decrease in c-Kit expression indicate that spermatogonia at this time point are in an undifferentiated state. In addition, QRFP administration also caused an increase in intratesticular levels of glucose and lactate, and in LDH activity accompanied by increased expressions of GLUT-3 and LDH-C in the testis. Also, the phosphorylation of IR-ß and expressions of p-Akt and p-mTOR were increased under ex vivo conditions in testicular tissue. In conclusion, our findings suggest that QRFP treatment caused proliferation of germ cells independently from the hypothalamic-pituitary axis via regulation of testicular energy metabolism.

Long-term exposure of 2450 MHz electromagnetic radiation induces stress and anxiety like behavior in rats.

Long term exposure of electromagnetic radiations (EMR) from cell phones and Wi-Fi hold greater propensity to cause anxiety disorders. However, the studies investigating the effects of repeated exposure of EMR are limited. Therefore, we investigated the effects of repeated exposure of discrete frequencies of EMR in experimental animals. Male rats were exposed to EMR (900, 1800 and 2450 MHz) for 28 (1 h/day) days. Long term exposure of EMR (2450 MHz) induced anxiety like behavior. It deregulated the hypothalamic pituitary adrenal (HPA) axis in rats as observed by increase in plasma corticosterone levels apart from decreased corticotrophin releasing hormone-2 (CRH-2) and Glucocorticoid receptor (GR) expression in amygdala. Further, it impaired mitochondrial function and integrity. The expression of Bcl2 showed significant decrease while Bax and ratio of Bax: Bcl2 were increased in the mitochondria and vice versa in cytoplasm indicating altered regulation of apoptosis. EMR exposure caused release of cytochrome-c and expression of caspase-9 ensuing activation of apoptotic cell death. Additional set of experiments performed to estimate the pattern of cell death showed necrotic and apoptotic amygdalar cell death after EMR exposure. Histopathological studies also revealed a significant decrease in neuronal cells in amygdala. The above findings indicate that long-term exposure of EMR radiation (2450 MHz) acts as a stressor and induces anxiety-like behaviors with concomitant pathophysiological changes in EMR subjected rats.

Comparison of adverse events associated with different spacers used with non-extrafine beclometasone dipropionate for asthma.

Co-prescription of Aerochamber® spacer with non-extrafine beclometasone diproprionate (non-EF BDP) is common but unlicensed. We report a comparison of inhaled corticosteroid (ICS)-related adverse events between patients co-prescribed Aerochamber compared to the licensed Volumatic® spacer. We utilised two historical cohorts: questionnaire-based and electronic medical record (EMR)-based, to assess patient-reported and EMR-recorded adverse events in patients with asthma prescribed non-EF BDP. Marginal effect estimate (MEE) was calculated to determine non-inferiority of Aerochamber compared to Volumatic in terms of patient-reported oral thrush and hoarseness with margin of 0.13. Other patient-reported adverse events (sore throat, bruising, weight gain, and coughing), and EMR-recorded adverse events were also assessed. Rate of patient-reported oral adverse events were non-inferior in 385 patients prescribed Aerochamber compared to 155 patients prescribed Volumatic (27.7 vs 29.9%; MEE, -0.043; 95% CI, -0.133 to 0.047). Total patient-reported adverse events did not differ significantly between Aerochamber and Volumatic (53.3 vs 49.7% with ≥1 adverse event). The EMR-based study of 1471 matched pairs of subjects did not show significantly different number of EMR-recorded adverse events between Aerochamber and Volumatic (12.5 vs 12.8% with ≥1 adverse events). Co-prescribing Aerochamber with non-EF BDP does not increase the risk for patient-reported and EMR-recorded ICS-related adverse events compared to co-prescribing Volumatic.

Ontogeny of QRFP and its receptor expression in mouse testis during post-natal development.

Pyroglutamylated RFamide peptide (QRFP), a neuropeptide, binds to its receptor GPR103 and influences various biological functions. In the present study, expression and localization of QRFP and GPR103 in mouse testis during post-natal development were investigated. The results showed that QRFP and GPR103 and also the isoforms of the receptor, viz. GPR103A and GPR103B were expressed in mouse testis during post-natal development. Expression of QRFP and its receptor was high during the early periods of post-natal development. Immunohistochemical study demonstrated the localization of QRFP and GPR103 in both interstitial and tubular compartments of the testis throughout post-natal development. A shift in the germ cell types expressing QRFP and its receptor along the course of testicular development and also a prominent immunoreactivity of QRFP in germ cells and of GPR103 in Sertoli cells was observed. Further, the immunoreactivity of QRFP and GPR103 appeared to be stage-specific in spermatogenetically active testis. Besides in intercellular spaces, localization of QRFP was also noticed in nuclei of germ cells. In conclusion, the present results suggest potential involvement of QRFP system in the development, proliferation and differentiation of testicular cells, possibly by regulating the energy requirement for these processes.

Standardized extract of Bacopa monnieri (CDRI-08): Effect on germ cell dynamics and possible mechanisms of its beneficial action on spermatogenesis and sperm quality in male mice.

Bacopa monnieri (BM) is used in traditional medicine as nerve tonic. We have recently shown that CDRI-08, a standardized extract of BM, improves testicular functions and epididymal sperm quality in Parkes (P) mice. The aim of the present study was to investigate the effect of CDRI-08 on germ cell dynamics and mechanisms of its action on spermatogenesis and sperm quality in P mice, and to determine the chemical profile of the extract. CDRI-08 (40 and 80 mg/kg body weight) was orally administered to male mice for 28 days. Germ cell dynamics, oxidative stress parameters in testis and sperm, and expressions of nuclear factor-erythroid-2-related factor-2 (Nrf2), phosphorylated protein kinase B (p-Akt) and upstream kinases in mitogen-activated protein kinase (MAPK) pathway namely MAP2K1, MAP2K2 and MKK4 in the testis were evaluated. The treatment potentiated germ cell dynamics and improved sperm quality by enhancing antioxidant enzymes activities. The beneficial effects of CDRI-08 in the testis involve p-Akt-mediated activation of Nrf2, thereby enhancing antioxidant enzymes activities; upregulation of MAP2K1 and MAP2K2 and suppression of MKK4 are also implicated in this action. A total of 26 phytocomponents were identified in CDRI-08 by GC-MS. The results suggest that CDRI-08 also may prove useful in improving reproductive health in males.

Effect of standardized extract of Bacopa monnieri (CDRI-08) on testicular functions in adult male mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Bacopa monnieri (BM) has been used in India since the time of Rig-Veda for augmentation of learning, memory, brain health etc. AIM OF THE STUDY: The memory augmenting effect of BM is well documented. CDRI-08 is a standardized extract of Bacopa monnieri, but its effect on the male reproductive health has not been investigated. Therefore, the aim of the present study was to examine the effect of CDRI-08 administration on the male reproductive organs with special emphasis on testis in adult mice. MATERIALS AND METHODS: CDRI-08, containing at least 55% bacosides (the major constituent of BM), was investigated for its effect on testicular functions in adult Parkes (P) mice. A suspension of CDRI-08 was orally administered in doses of 40 and 80mgkg-1 body weight day-1 for 28 days and various male reproductive end points were evaluated. RESULTS: Compared to control, CDRI-08 treatment caused a significant increase (p<0.05) in spermatogenic cell density (germinal epithelial height: control, 55.03±4.22 vs 40mg, 67.15±2.65 and 80mg, 69.93±3.76; and tubular diameter: control, 206.55±2.62 vs 80mg, 253.23±12.19), PCNA index (control, 59.85±2.09 vs 40mg, 82.17±1.56 and 80mg, 84.05±3.51) and in steroidogenic indices in the testis, and in sperm viability (control, 0.67±0.010 vs 80mg, 0.80±0.04) in cauda epididymidis of the treated mice. On the other hand, however, the same treatment caused a significant decrease (p<0.05) in abnormal sperm morphology (control, 21.72±1.06 vs 40mg, 10.63±1.50 and 80mg, 15.86±0.87) in cauda epididymidis, and in lipid peroxidation level in testis of the treated mice compared to controls. CONCLUSION: The results suggest that treatment with CDRI-08 extract improves sperm quality, and spermatogenic cell density and steroidogenic indices in the testis of P mice.

Stepping down from high dose fluticasone/salmeterol to extrafine BDP/F in asthma is cost-effective.

BACKGROUND: GINA guideline recommends stepping down treatment of asthma patients where control is achieved. The aim of this analysis was to estimate the costs and health outcomes associated with step down of controlled patients on high dose fluticasone/salmeterol (FP/S 1000/100 µg daily) to either medium dose FP/S (500/100 µg) dry powder or extrafine beclometasone/formoterol (BDP/F 400/24 µg) pMDI in three European countries. METHODS: A patient-level simulation Markov model was constructed to enable the simulation of three comparative arms (FP/S 1000/100, FP/S 500/100, BDP/F 400/24). Transition probabilities and healthcare resources consumption were derived from a multinational clinical trial comparing BDP/F 400/24 µg vs. FP/S 500/100 µg as step down therapy in asthma. Direct costs and health state utilities were sourced from public source and published literature. The analysis was conducted from a health system perspective, based on six months horizon. Probabilistic sensitivity analyses were conducted. RESULTS: The ICER (Incremental Cost-Effectiveness Ratio) associated with high dose dry powder FP/S 1000/100 µg vs. extrafine BDP/F 400/24 µg was above 70,000 GBP and 200,000 €/QALY (Quality Adjusted Life Years). An ICER of 29,000 GBP/QALY and above 30,000 €/QALY was associated with medium dose dry powder FP/S 500/100 µg vs. BDP/F 400/24 µg. CONCLUSIONS: It was found that maintaining controlled patients on high dose FP/S is not cost-effective. Extrafine BDP/F 400/24 µg daily can be considered to be a cost-effective option in the countries analyzed to maintain control of asthmatic patients stepped down from high dose FP/S 1000/100 µg daily dry powder or suspension formulations.

Stepping-across controlled asthmatic patients to extrafine beclometasone/formoterol combination.

BACKGROUND: Asthma management focuses on achieving and maintaining asthma control. Few studies have assessed whether complete and sustained asthma control is maintained in clinical practice after stepping-across ICS/LABA fixed combinations. Aim of this double-blind, double-dummy, randomized, parallel group, controlled study was to demonstrate clinical equivalence between equipotent doses of extrafine beclometasone/formoterol (BDP/F) pMDI and fluticasone/salmeterol (FP/S) Diskus® in maintaining lung function and asthma control. METHODS: A total of 416 asthmatic patients already controlled with FP/S 500/100 µg/day (Diskus®, pMDI or separate inhalers) were randomized to a 12-week treatment with extrafine BDP/F 400/24 µg/day pMDI or FP/S 500/100 µg/day Diskus®. Pre-dose 1-s forced expiratory volume (FEV(1)) was the primary efficacy variable; secondary variables included asthma control questionnaire (ACQ-7) and FEV(1)0-1 h area under the curve (FEV(1)AUC(0-1h)). Safety was assessed through adverse events monitoring and vital signs. RESULTS: After 12 weeks of treatment, pre-dose FEV(1) did not differ between treatments (difference between means 0.01 L; 95% CI -0.03-0.06 L) with no significant changes from baseline in both groups (p = 0.726 and p = 0.783 in BDF/F arm and FP/S, respectively). ACQ-7 score showed that control was maintained after stepping-across to extrafine BDP/F. FEV(1)AUC(0-1h) was significantly higher in BDP/F arm at the beginning (p = 0.004) and at the end of the 12-week treatment period (p = 0.019). No safety issues were reported in both groups. CONCLUSIONS: Patients previously controlled with FP/S in any device formulation can effectively step-across to extrafine BDP/F pMDI, maintaining lung function and asthma control with a 5-min onset of action.

Assessment of cardiovascular response to treadmill exercise in normal healthy Indian adolescents.

The study aims to assess the cardiovascular response to treadmill exercise test in healthy Indian adolescents. A group of 50 healthy adolescents took part in the study. Cardiovascular response was assessed by using treadmill exercise test as per Bruce protocol. Pulse rate, blood pressure and ECG were recorded before, during and after undertaking the treadmill test. Mean age and body mass index (BMI) were 18.7 +/- 0.51 yrs. and 21.4 +/- 3.44 kg/m2 respectively. Karl Pearson Correlation analysis showed highly significant negative correlation between BMI and exercise time (r = -0.598, P<0.001) and between resting DBP and Exercise Time (r = -0.424, P<0.002). While BMI and DBP showed highly significant positive correlation (r = 0.463, P<0.001). During exercise pulse and SBP rose and DBP fell. SBP rose from mean 122 to 175 (rise by 53 mm of Hg) and DBP fell from mean 78 to 65 (fall by 13 mm of Hg). One min recovery pulse was 156 indicating 22% fall from target heart rate. All the parameters returned to near resting value at 6 min recovery. In 30% students DBP showed exaggerated response i.e. rise during exercise. These students had more BMI and higher resting DBP as compared to other students, which could be the reason for exaggerated response in these participants. In ECG there were no significant ST/T changes during exercise or recovery period. This study provides normal data for small sample of healthy Indian adolescents when subjected to treadmill exercise test.