Jenny Szu-Chin Pan

Stat3 Activation Links a C/EBPδ to Myostatin Pathway to Stimulate Loss of Muscle Mass

Catabolic conditions like chronic kidney disease (CKD) cause loss of muscle mass by unclear mechanisms. In muscle biopsies from CKD patients, we found activated Stat3 (p-Stat3) and hypothesized that p-Stat3 initiates muscle wasting. We created mice with muscle-specific knockout (KO) that prevents activation of Stat3. In these mice, losses of body and muscle weights were suppressed in models with CKD or acute diabetes. A small-molecule that inhibits Stat3 activation produced similar responses, suggesting a potential for translation strategies. Using CCAAT/enhancer-binding protein δ (C/EBPδ) KO mice and C2C12 myotubes with knockdown of C/EBPδ or myostatin, we determined that p-Stat3 initiates muscle wasting via C/EBPδ, stimulating myostatin, a negative muscle growth regulator. C/EBPδ KO also improved survival of CKD mice. We verified that p-Stat3, C/EBPδ, and myostatin were increased in muscles of CKD patients. The pathway from p-Stat3 to C/EBPδ to myostatin and muscle wasting could identify therapeutic targets that prevent muscle wasting.

Myostatin Suppression of Akirin1 Mediates Glucocorticoid-Induced Satellite Cell Dysfunction

Glucocorticoids production is increased in many pathological conditions that are associated with muscle loss, but their role in causing muscle wasting is not fully understood. We have demonstrated a new mechanism of glucocorticoid-induced muscle atrophy: Dexamethasone (Dex) suppresses satellite cell function contributing to the development of muscle atrophy. Specifically, we found that Dex decreases satellite cell proliferation and differentiation in vitro and in vivo. The mechanism involved Dex-induced upregulation of myostatin and suppression of Akirin1, a promyogenic gene. When myostatin was inhibited in Dex-treated mice, Akirin1 expression increased as did satellite cell activity, muscle regeneration and muscle growth. In addition, silencing myostatin in myoblasts or satellite cells prevented Dex from suppressing Akirin1 expression and cellular proliferation and differentiation. Finally, overexpression of Akirin1 in myoblasts increased their expression of MyoD and myogenin and improved cellular proliferation and differentiation, theses improvements were no longer suppressed by Dex. We conclude that glucocorticoids stimulate myostatin which inhibits Akirin1 expression and the reparative functions of satellite cells. These responses attribute to muscle atrophy. Thus, inhibition of myostatin or increasing Akirin1 expression could lead to therapeutic strategies for improving satellite cell activation and enhancing muscle growth in diseases associated with increased glucocorticoid production.

Stanniocalcin-1 Inhibits Renal Ischemia/Reperfusion Injury via an AMP-Activated Protein Kinase-Dependent Pathway

AKI is associated with increased morbidity, mortality, and cost of care, and therapeutic options remain limited. Reactive oxygen species are critical for the genesis of ischemic AKI. Stanniocalcin-1 (STC1) suppresses superoxide generation through induction of uncoupling proteins (UCPs), and transgenic overexpression of STC1 inhibits reactive oxygen species and protects from ischemia/reperfusion (I/R) kidney injury. Our observations revealed high AMP-activated protein kinase (AMPK) activity in STC1 transgenic kidneys relative to wild-type (WT) kidneys; thus, we hypothesized that STC1 protects from I/R kidney injury through activation of AMPK. Baseline activity of AMPK in the kidney correlated with the expression of STCs, such that the highest activity was observed in STC1 transgenic mice followed (in decreasing order) by WT, STC1 knockout, and STC1/STC2 double-knockout mice. I/R in WT kidneys increased AMPK activity and the expression of STC1, UCP2, and sirtuin 3. Inhibition of AMPK by administration of compound C before I/R abolished the activation of AMPK, diminished the expression of UCP2 and sirtuin 3, and aggravated kidney injury but did not affect STC1 expression. Treatment of cultured HEK cells with recombinant STC1 activated AMPK and increased the expression of UCP2 and sirtuin 3, and concomitant treatment with compound C abolished these responses. STC1 knockout mice displayed high susceptibility to I/R, whereas pretreatment of STC1 transgenic mice with compound C restored the susceptibility to I/R kidney injury. These data suggest that STC1 is important for activation of AMPK in the kidney, which mediates STC1-induced expression of UCP2 and sirtuin 3 and protection from I/R.

AKI after Conditional and Kidney-Specific Knockdown of Stanniocalcin-1

Stanniocalcin-1 is an intracrine protein; it binds to the cell surface, is internalized to the mitochondria, and diminishes superoxide generation through induction of uncoupling proteins. In vitro, stanniocalcin-1 inhibits macrophages and preserves endothelial barrier function, and transgenic overexpression of stanniocalcin-1 in mice protects against ischemia-reperfusion kidney injury. We sought to determine the kidney phenotype after kidney endothelium-specific expression of stanniocalcin-1 small hairpin RNA (shRNA). We generated transgenic mice that express stanniocalcin-1 shRNA or scrambled shRNA upon removal of a floxed reporter (phosphoglycerate kinase-driven enhanced green fluorescent protein) and used ultrasound microbubbles to deliver tyrosine kinase receptor-2 promoter-driven Cre to the kidney to permit kidney endothelium-specific shRNA expression. Stanniocalcin-1 mRNA and protein were expressed throughout the kidney in wild-type mice. Delivery of tyrosine kinase receptor-2 promoter-driven Cre to stanniocalcin-1 shRNA transgenic kidneys diminished the expression of stanniocalcin-1 mRNA and protein throughout the kidneys. Stanniocalcin-1 mRNA and protein expression did not change in similarly treated scrambled shRNA transgenic kidneys, and we observed no Cre protein expression in cultured and tyrosine kinase receptor-2 promoter-driven Cre-transfected proximal tubule cells, suggesting that knockdown of stanniocalcin-1 in epithelial cells in vivo may result from stanniocalcin-1 shRNA transfer from endothelial cells to epithelial cells. Kidney-specific knockdown of stanniocalcin-1 led to severe proximal tubule injury characterized by vacuolization, decreased uncoupling of protein-2 expression, greater generation of superoxide, activation of the unfolded protein response, initiation of autophagy, cell apoptosis, and kidney failure. Our observations suggest that stanniocalcin-1 is critical for tubular epithelial survival under physiologic conditions.

Stanniocalcin-1 inhibits thrombin-induced signaling and protects from bleomycin-induced lung injury

Thrombin-induced and proteinase-activated receptor 1 (PAR1)-mediated signaling increases ROS production, activates ERK, and promotes inflammation and fibroblast proliferation in bleomycin-induced lung injury. Stanniocalcin-1 (STC1) activates anti-oxidant pathways, inhibits inflammation and provides cytoprotection; hence, we hypothesized that STC1 will inhibit thrombin/PAR1 signaling and protect from bleomycin-induced pneumonitis. We determined thrombin level and activity, thrombin-induced PAR-1-mediated signaling, superoxide generation and lung pathology after intra-tracheal administration of bleomycin to WT and STC1 Tg mice. Lungs of bleomycin-treated WT mice display: severe pneumonitis; increased generation of superoxide; vascular leak; increased thrombin protein abundance and activity; activation of ERK; greater cytokine/chemokine release and infiltration with T-cells and macrophages. Lungs of STC1 Tg mice displayed none of the above changes. Mechanistic analysis in cultured pulmonary epithelial cells (A549) suggests that STC1 inhibits thrombin-induced and PAR1-mediated ERK activation through suppression of superoxide. In conclusion, STC1 blunts bleomycin-induced rise in thrombin protein and activity, diminishes thrombin-induced signaling through PAR1 to ERK, and inhibits bleomycin-induced pneumonitis. Moreover, our study identifies a new set of cytokines/chemokines, which play a role in the pathogenesis of bleomycin-induced lung injury. These findings broaden the array of potential therapeutic targets for the treatment of lung diseases characterized by thrombin activation, oxidant stress and inflammation.

Remote Ischemic Preconditioning for Kidney Protection

Acutekidneyinjury (AKI) iscommonandfrequentlyassociated withsubstantialmorbidityandmortality.Elevatedserumcreatinine levels in patients after surgery are associated with poor outcomes.1,2Asmall increase inserumcreatinine inhospitalized patients is associatedwithhighermortality, longer hospitalization, andhigher cost of care.3Acute tubularnecrosis (ATN), definedasacute injury to therenal tubularepithelial cells,mayoccurfollowingsevererenal ischemiaorexposuretonephrotoxins. Preconditioning-mediated protection is a phenomenon in whichtissue,onceexposedtoaspecifictypeof insult,willbeprotected from injury during a repeated similar or sometimes dissimilar insult.4Theprotectivephenotype(preconditioning) in response to ischemia depends on a coordinated response at the genomic,molecular, cellular, and tissue levels, in whatisdescribedas“genomicreprogramming.”5Preconditioningmediatedprotectioniswelldescribedforthebrainandheart,and recentreportssuggestthat italsofunctionsforthekidneyinATN. For instance, in experimental mouse/rat models of ischemia/ reperfusion kidney injury, in which bilateral kidney pedicles (arteriesandveins)areclampedfor30to45minutes,AKIdevelopswithin24hours; invariably,kidneyfunctionrecoverswithin aweek.However, if thekidneysaresubjectedtoanequivalent insult 1or2weeksafter the initial insult,anequal increase inserum creatinine or kidney inflammationmarkers comparedwith the initial insult isnotobserved.6Resistancetorepeatedischemicepisodesaffordedby30minutesofbilateralkidneyischemiacanpersistfor12weeksafterpreconditioning,6,7whereasashorterperiod ofischemia(15minutes) ispartiallyprotectiveagainstsubsequent ischemic injury imposed8days later.Similarly, thekidneysmay displayattenuated ischemia/reperfusion injury followingnephrotoxic (eg, cisplatin) injury, andviceversa.8Amajor limitation totheclinicaluseof this ischemicpreconditioningstrategy is the need for direct access to the blood supply of the organ at risk. Recentdata suggest that ischemia inoneorgan is accompaniedwith protective changes in distant organs, a phenomenon knownas remote ischemicpreconditioning (RIPC).Application ofoneormorebrief cyclesofnonlethal ischemia/reperfusion to an organ or tissuemay protect a remote organ or tissue from a sustainedepisodeof lethal ischemia/reperfusion.Thebeneficial effectsofRIPChavebeendemonstratedindiverseorgansandtissues (lung, liver, kidney, intestine, brain, skeletal muscle) subjectedtoacuteischemia/reperfusion,9andthediscoverythatRIPC canbe inducednoninvasively by simple inflation anddeflation ofastandardbloodpressurecuffplacedona limbhas facilitated its translation into the clinical setting. ThereportbyZarbockandcolleagues inthis issueofJAMA10 examined the effects of RIPC on the rate and severity of AKI in patients undergoing cardiac surgery. In amulticenter trial, 240 patients at high risk for AKI (ClevelandClinic Foundation score ≥6)wererandomizedtoreceiveeitherRIPC(n = 120)orshamcontrol (n = 120) after inductionof anesthesia. TheRIPC involved3 cycles of 5minutes of ischemia induced by inflation of a blood pressure cuff to 200mmHgor at least 50mmgreater than systolic bloodpressure inoneupper arm, followedby5minutesof reperfusionwith the cuff deflated. The shamRIPC (control) involved3 cycles of “pseudoischemia,” consistingof 5minutesof bloodpressurecuff inflationto20mmHg,followedby5minutes of cuff deflation. All patients completed follow-up30days after surgeryand wereanalyzedaccordingto the intention-to-treatprinciple.The primary end point was the rate of AKI defined by KDIGO criteriawithinthefirst72hoursaftercardiacsurgery.11Secondaryend points included use of renal replacement therapy (RRT), duration of intensive care, occurrence ofmyocardial infarction and stroke, in-hospital and30-daymortality, andchange inAKIbiomarkers.AcutekidneyinjurywassignificantlyreducedwithRIPC (45/120; 37.5%) comparedwith control (63/120; 52.5%),withan absoluteriskreduction(ARR)of15%.FewerpatientsreceivedRRT after RIPC (7 [5.8%] vs 19 control [15.8%]; ARR, 10%) and RIPC reduced intensive care unit stay (3 vs 4 days). RIPC had no significant effect onmyocardial infarction, stroke, ormortality. Eventhoughthemechanism(s)underlyingpreconditioning remainselusive, a fewcluesabout itsnaturehaveemerged.Upregulation of inducible nitric oxide synthase and heat shock proteins7,12 as well as activation of the endoplasmic reticulum stress response13mayplaya role in the longer-termkidneyprotectionattributedto ischemicpreconditioning.Recentdatasuggestthatrecruitmentofmesenchymalstemcells iscritical forthis protection.Regenerationofproximal tubuleepitheliumafter ischemic injuryappears tobe intrinsic to thekidneyanddoesnot requiretransdifferentiationofbonemarrow–derivedstemcells.14 However, bonemarrow–derived leukocytes andcells bearingendothelialmarkers (mesenchymalcells)aredetected inthe kidney interstitium after ischemic injury.14,15 Kuo et al16 suggested that injuredkidneycells (endothelial) recruitmesenchymalstemcells throughthereleaseofpreformedvesicles(WeibelPalade bodies) packed with messenger signals that include IL-8,eotaxin3,vonWillebrandfactor,andangiopoietin2.Atrigger for the releaseof thesevesicles isuricacid, aproductofxanthine oxidase activation in the ischemic organ, through interactionwithToll-like receptors TLR2 andTLR4.Angiopoietin 2 amplifies release of Weibel-Palade bodies and plays a pivotal role in the recruitmentofmesenchymal stemcells in response touric acidandpotentiallyother“alarmsignals.”16Administration ofmesenchymal stem cells to rats protects against ischemic kidney injury, and this protection is associated with upRelated article page 2133 Opinion

Severe Nephrotoxic Nephritis following Conditional and Kidney-Specific Knockdown of Stanniocalcin-1.

Background Inflammation is the hallmark of nephrotoxic nephritis. Stanniocalcin-1 (STC1), a pro-survival factor, inhibits macrophages, stabilizes endothelial barrier function, and diminishes trans-endothelial migration of leukocytes; consistently, transgenic (Tg) overexpression of STC1 protects from nephrotoxic nephritis. Herein, we sought to determine the phenotype of nephrotoxic nephritis after conditional and kidney-specific knockdown of STC1. Methods We used Tg mice that, express either STC1 shRNA (70% knockdown of STC1 within 4d) or scrambled shRNA (control) upon delivery of Cre-expressing plasmid to the kidney using ultrasound microbubble technique. Sheep anti-mouse GBM antibody was administered 4d after shRNA activation; and mice were euthanized 10 days later for analysis. Results Serum creatinine, proteinuria, albuminuria and urine output were similar 10 days after anti-GBM delivery in both groups; however, anti-GBM antibody delivery to mice with kidney-specific knockdown of STC1 produced severe nephrotoxic nephritis, characterized by severe tubular necrosis, glomerular hyalinosis/necrosis and massive cast formation, while control mice manifested mild tubular injury and crescentic glomerulonephritis. Surprisingly, the expression of cytokines/chemokines and infiltration with T-cells and macrophages were also diminished in STC1 knockdown kidneys. Staining for sheep anti-mouse GBM antibody, deposition of mouse C3 and IgG in the kidney, and antibody response to sheep IgG were equal. Conclusions nephrotoxic nephritis after kidney-specific knockdown of STC1 is characterized by severe tubular and glomerular necrosis, possibly due to loss of STC1-mediated pro-survival factors, and we attribute the paucity of inflammation to diminished release of cytokines/chemokines/growth factors from the necrotic epithelium.

Megalin mediates plasma membrane to mitochondria cross-talk and regulates mitochondrial metabolism

Mitochondrial intracrines are extracellular signaling proteins, targeted to the mitochondria. The pathway for mitochondrial targeting of mitochondrial intracrines and actions in the mitochondria remains unknown. Megalin/LRP2 mediates the uptake of vitamins and proteins, and is critical for clearance of amyloid-β protein from the brain. Megalin mutations underlie the pathogenesis of Donnai–Barrow and Lowe syndromes, characterized by brain defects and kidney dysfunction; megalin was not previously known to reside in the mitochondria. Here, we show megalin is present in the mitochondria and associates with mitochondrial anti-oxidant proteins SIRT3 and stanniocalcin-1 (STC1). Megalin shuttles extracellularly-applied STC1, angiotensin II and TGF-β to the mitochondria through the retrograde early endosome-to-Golgi transport pathway and Rab32. Megalin knockout in cultured cells impairs glycolytic and respiratory capacities. Thus, megalin is critical for mitochondrial biology; mitochondrial intracrine signaling is a continuum of the retrograde early endosome-to-Golgi-Rab32 pathway and defects in this pathway may underlie disease processes in many systems.

Remote Ischemic Preconditioning and Postoperative Renal Dysfunction—Reply

In Reply In the demonstration we evaluated, the CDS systems did not access the entire patient record in a readily computable format. Rather, ordering clinicians entered data describing the reason each test was ordered into the CDS system. The systems varied regarding the inclusion of data pertinent to demographics, past medical history, prior imaging or other studies, symptom characterization, and social status. The ability of the CDS systems to link to and rate orders was highly dependent on the completeness and quality of the data entered. Although the demonstration used several data entry methods, all of the systems studied used top-down methods of capturing structured data, and all showed a substantial proportion of orders could not be rated. We agree that accessing the data in electronic health records (which requires semantic interoperability) could improve the usability and performance of CDS systems.