The JAK-STAT pathway is critical in ventilator-induced diaphragm dysfunction.

Mechanical ventilation (MV) is one of the lynchpins of modern intensive-care medicine and is life saving in many critically ill patients. Continuous ventilator support, however, results in ventilation-induced diaphragm dysfunction (VIDD) that likely prolongs patients' need for MV and thereby leads to major associated complications and avoidable intensive care unit (ICU) deaths. Oxidative stress is a key pathogenic event in the development of VIDD, but its regulation remains largely undefined. We report here that the JAK-STAT pathway is activated in MV in the human diaphragm, as evidenced by significantly increased phosphorylation of JAK and STAT. Blockage of the JAK-STAT pathway by a JAK inhibitor in a rat MV model prevents diaphragm muscle contractile dysfunction (by ~85%, p < 0.01). We further demonstrate that activated STAT3 compromises mitochondrial function and induces oxidative stress in vivo, and, interestingly, that oxidative stress also activates JAK-STAT. Inhibition of JAK-STAT prevents oxidative stress-induced protein oxidation and polyubiquitination and recovers mitochondrial function in cultured muscle cells. Therefore, in ventilated diaphragm muscle, activation of JAK-STAT is critical in regulating oxidative stress and is thereby central to the downstream pathogenesis of clinical VIDD. These findings establish the molecular basis for the therapeutic promise of JAK-STAT inhibitors in ventilated ICU patients.

Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.

Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV-mediated diaphragm wasting and weakness in rats. CMV-induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.-Smith, I. J., Godinez, G. L., Singh, B. K., McCaughey, K. M., Alcantara, R. R., Gururaja, T., Ho, M. S., Nguyen, H. N., Friera, A. M., White, K. A., McLaughlin, J. R., Hansen, D., Romero, J. M., Baltgalvis, K. A., Claypool, M. D., Li, W., Lang, W., Yam, G. C., Gelman, M. S., Ding, R., Yung, S. L., Creger, D. P., Chen, Y., Singh, R., Smuder, A. J., Wiggs, M. P., Kwon, O.-S., Sollanek, K. J., Powers, S. K., Masuda, E. S., Taylor, V. C., Payan, D. G., Kinoshita, T., Kinsella, T. M. Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.

Exercise performance and peripheral vascular insufficiency improve with AMPK activation in high-fat diet-fed mice.

Intermittent claudication is a form of exercise intolerance characterized by muscle pain during walking in patients with peripheral artery disease (PAD). Endothelial cell and muscle dysfunction are thought to be important contributors to the etiology of this disease, but a lack of preclinical models that incorporate these elements and measure exercise performance as a primary end point has slowed progress in finding new treatment options for these patients. We sought to develop an animal model of peripheral vascular insufficiency in which microvascular dysfunction and exercise intolerance were defining features. We further set out to determine if pharmacological activation of 5'-AMP-activated protein kinase (AMPK) might counteract any of these functional deficits. Mice aged on a high-fat diet demonstrate many functional and molecular characteristics of PAD, including the sequential development of peripheral vascular insufficiency, increased muscle fatigability, and progressive exercise intolerance. These changes occur gradually and are associated with alterations in nitric oxide bioavailability. Treatment of animals with an AMPK activator, R118, increased voluntary wheel running activity, decreased muscle fatigability, and prevented the progressive decrease in treadmill exercise capacity. These functional performance benefits were accompanied by improved mitochondrial function, the normalization of perfusion in exercising muscle, increased nitric oxide bioavailability, and decreased circulating levels of the endogenous endothelial nitric oxide synthase inhibitor asymmetric dimethylarginine. These data suggest that aged, obese mice represent a novel model for studying exercise intolerance associated with peripheral vascular insufficiency, and pharmacological activation of AMPK may be a suitable treatment for intermittent claudication associated with PAD.

Nephrilin peptide modulates a neuroimmune stress response in rodent models of burn trauma and sepsis.

Sepsis occurs three times more often in burns than in other types of trauma, suggesting an overlap or synergy between underlying immune mechanisms in burn trauma and sepsis. Nephrilin peptide, a designed inhibitor of mTORC2, has previously been shown to modulate a neuroimmune stress response in rodent models of xenobiotic and metabolic stress. Here we investigate the effect of nephrilin peptide administration in different rodent models of burn trauma and sepsis. In a rat scald burn model, daily subcutaneous bolus injection of 4 mg/kg nephrilin significantly reduced the elevation of kidney tissue substance P, S100A9 gene expression, PMN infiltration and plasma inflammatory markers in the acute phase, while suppressing plasma CCL2 and insulin C-peptide, kidney p66shc-S36 phosphorylation and PKC-beta and CGRP in dorsal root ganglia at 14 days (chronic phase). In the mouse cecal ligation and puncture model of sepsis, nephrilin fully protected mice from mortality between surgery and day 7, compared to 67% mortality in saline-treated animals, while significantly reducing elevated CCL2 in plasma. mTORC2 may modulate important neuroimmune responses in both burn trauma and sepsis.

Mammalian target of rapamycin complex 2 regulates inflammatory response to stress.

OBJECTIVE AND DESIGN: To explore the role of mammalian target of rapamycin 2 (mTORC2) in the activation of inflammatory and oxidative responses in rodent models of acute injury and metabolic stress. MATERIAL: The impact of nephrilin, an inhibitor of mTORC2 complex, was assessed in three CD-1 mouse models of acute xenobiotic stress and in a hypertensive Dahl rat model of metabolic stress. METHODS: Animals received daily subcutaneous bolus injections of saline or 4 mg/kg nephrilin. Tissues were assayed by ELISA, gene arrays and immunohistochemical staining. RESULTS: Nephrilin significantly inhibited elevations in plasma tumor necrosis factor-alpha, kidney substance P, and CX3CR1, and urinary lipocalin-2 [urinary neutrophil gelatinase-associated lipocalin (uNGAL)] in models of acute xenobiotic stress. UCHL1 gene expression levels dropped and plasma HMGB1 levels rose in the rhabdomyolysis model. Both effects were reversed by nephrilin. The inhibitor also blocked diet-induced elevations of uNGAL and albumin-creatinine ratio (UACR) as well as kidney tissue phosphorylation of PKC-beta-2-T641 and p66shc-S36, and reduced dark ring-like staining of nuclei by anti-phos-p66shc-S36 antibody in frozen sections of diseased kidneys from hypertensive Dahl rats fed an 8 % NaCl diet for 4 weeks. CONCLUSIONS: Taken together, our results suggest a role for mTORC2 in the inflammatory-oxidative responses to stress.

A nuclear complex of rictor and insulin receptor substrate-2 is associated with albuminuria in diabetic mice.

BACKGROUND: Signaling events associated with diabetic nephropathy are not well understood. Triangulation of events triggered by unrelated bioactive peptides nephrilin and anephril, both of which inhibit albuminuria in diabetic mice, could reveal a common subset of events associated with albuminuria. METHODS: db/db mice received 20 microg/day anephril or nephrilin for 7 weeks. Streptozotocin (STZ)-treated DBA/2J mice received 2 mg/kg nephrilin for 26 days. In both studies, urine albumin and creatinine, plasma glucose, and tissue proteins were measured by enzyme-linked immunosorbent assay (ELISA). In a safety study, DBA/2J mice received 20 mg/kg nephrilin for 26 days, and tissues from these mice were fixed in formalin for histological analysis. HEK293 human kidney cells were treated with glycated hemoglobin plus 20 microg/mL nephrilin or anephril for 24 h. RESULTS: Both peptides reduced albuminuria in db/db mice compared to saline-treated animals without affecting plasma glucose or insulin. In kidney tissues, the immunoreactivities of insulin receptor substrate-2 (IRS2), phosphorylated protein kinase C (p-PKC), and serum- and glucocortocoid-inducible kinase (SGK1) were reduced. Nephrilin, but not anephril, lowered elevated SGK1 in spleens of db/db mice. In STZ-pretreated DBA/2J mice, nephrilin reduced albuminuria and the accumulation of nuclear Rictor, IRS2, and p-PKC in the kidney. In cultured kidney cells, nephrilin disrupted the association of Rictor with IRS2 and nuclear compartmentalization. Histopathological examination of tissues from mice treated with 20 mg/kg nephrilin daily for 26 days showed no significant pathology compared to saline-treated controls. CONCLUSIONS: We define a subset of signaling markers closely associated with albuminuria. Mammalian target of rapamycin complex 2 (mTORC2)::IRS complexes may play a role in the nuclear accumulation, and possible downstream transcriptional effects, of p-PKC. The activity and apparent safety of nephrilin in rodents suggest a novel intervention strategy for diabetic kidney disease.

Bioactive peptides control receptor for advanced glycated end product-induced elevation of kidney insulin receptor substrate 2 and reduce albuminuria in diabetic mice.

BACKGROUND/AIMS: Sixteen-week-old db/db mice exhibit significantly elevated blood glucose and albuminuria. Kidney mesangial cell matrix expansion and collagen IV synthesis correlate with disease progression, but the underlying mechanism is unclear. METHODS: Adaptive biochemical datasets were generated in cultured 293 kidney cells and in db/db mice. RESULTS: In animals receiving daily subcutaneous bolus injections (weeks 8-13) of 20 microg/day humanin or 40 microg/day protein kinase C (NPKC) (a PKC-beta2 inhibitor peptide), there was a significant reduction in albuminuria, insulin receptor substrate 2 (IRS-2) and phospho-Akt (Ser473) levels in kidney tissue extracts (p < 0.05 in all cases). Elevated IRS-2 (not IRS-1), altered Akt1 and selective phosphorylation of p-Akt/Ser473 and p-IRS-1/Ser307 (but not p-Akt/Thr308 or p-IRS-2/Ser731) are correlates of the receptor for advanced glycated end product activation and are linked to albuminuria in vivo, whereas in P38 peptide-treated animals, collagen IV synthesis can be uncoupled from albuminuria altogether. CONCLUSION: Taken together, our results suggest that elevated IRS-2 and altered Akt phosphorylation may be more closely tied to the cause of diabetic kidney disease in db/db mice than mesangial matrix expansion per se, though both may originate from elevated circulatory glucose, and mesangial matrix expansion may independently exacerbate kidney dysfunction.

Early adoption of new drug treatments: the role of continuing medical education and physician adaptivity.

The influence of continuing medical education (CME) on the adoption of new treatments is widely regarded as self-evident. Less well understood is how the dynamics of dissemination of new healthcare practices are influenced by the intersection of education with the adaptive characteristics of providers. We developed and validated a 43-item online instrument (eSAIL) for measuring adaptive style and used it to investigate the interplay of physician adaptivity with key components of effective medical education. Satisfactory Cronbach alpha and test-retest reliability coefficients were observed for all primary psychometric scales and a composite adaptivity scale. Discriminant, convergent, and predictive validities of eSAIL scales were consistent across all cohorts. Using an online medical education program for which data on physician behavioral change are available, we show that the rate of adoption of new drugs is driven by both psychological (adaptivity) and environmental (educational) inputs. We show for the first time that topic eminence, length of reinforcement period and physician adaptive score in the eSAIL are each proportional to early-adoption-related behavioral change. Using a simple forced-choice question, a cohort of 208 physicians was segmented into "A" (adaptive) and "C" (conservative) segments based on their eSAIL adaptivity z scores (+0.170 versus -0.234 respectively; P < 0.01). Early adoption of new drug treatments by similarly segmented physician cohorts was driven almost entirely by A-segment physicians, but only when those physicians were additionally exposed to effective medical education.