Low-dose rapamycin does not impair vascular integrity and tubular regeneration after kidney transplantation in rats

mTOR inhibitors offer advantages after kidney transplantation including antiviral and antitumor activity besides facilitating low calcineurin inhibitor exposure to reduce nephrotoxicity. Concerns about adverse effects due to antiproliferative and antiangiogenic properties have limited their clinical use particularly early after transplantation. Interference with vascular endothelial growth factor (VEGF)-A, important for physiologic functioning of renal endothelial cells and tubular epithelium, has been implicated in detrimental renal effects of mTOR inhibitors. Low doses of Rapamycin (loading dose 3 mg/kg bodyweight, daily doses 1.5 mg/kg bodyweight) were administered in an allogenic rat kidney transplantation model resulting in a mean through concentration of 4.30 ng/mL. Glomerular and peritubular capillaries, tubular cell proliferation, or functional recovery from preservation/reperfusion injury were not compromised in comparison to vehicle treated animals. VEGF-A, VEGF receptor 2, and the co-receptor Neuropilin-1 were upregulated by Rapamycin within 7 days. Rat proximal tubular cells (RPTC) responded in vitro to hypoxia with increased VEGF-A and VEGF-R1 expression that was not suppressed by Rapamycin at therapeutic concentrations. Rapamycin did not impair proliferation of RPTC under hypoxic conditions. Low-dose Rapamycin early posttransplant does not negatively influence the VEGF network crucial for recovery from preservation/reperfusion injury. Enhancement of VEGF signaling peritransplant holds potential to further improve outcomes

Targeting proinflammatory cytokines ameliorates calcifying phenotype conversion of vascular progenitors under uremic conditions in vitro

Severe vascular calcification develops almost invariably in chronic kidney patients posing a substantial risk to quality of life and survival. This unmet medical need demands identification of novel therapeutic modalities. We aimed to pinpoint components of the uremic microenvironment triggering differentiation of vascular progenitors to calcifying osteoblast-like cells. In an unbiased approach, assessing the individual potency of 63 uremic retention solutes to enhance calcific phenotype conversion of vascular progenitor cells, the pro-inflammatory cytokines IL-1 beta and TNF-alpha were identified as the strongest inducers followed by FGF-2, and PTH. Pharmacologic targeting of these molecules alone or in combination additively antagonized pro-calcifying properties of sera from uremic patients. Our findings stress the importance of pro-inflammatory cytokines above other characteristic components of the uremic microenvironment as key mediators of calcifying osteoblastic differentiation in vascular progenitors. Belonging to the group of "middle-sized molecules", they are neither effectively removed by conventional dialysis nor influenced by established supportive therapies. Specific pharmacologic interventions or novel extracorporeal approaches may help preserve regenerative capacity and control vascular calcification due to uremic environment

17ß-Estradiol Regulates mTORC2 Sensitivity to Rapamycin in Adaptive Cardiac Remodeling

Adaptive cardiac remodeling is characterized by enhanced signaling of mTORC2 downstream kinase Akt. In females, 17ß-estradiol (E2), as well as Akt contribute essentially to sex-related premenopausal cardioprotection. Pharmacologic mTOR targeting with rapamycin is increasingly used for various clinical indications, yet burdened with clinical heterogeneity in therapy responses. The drug inhibits mTORC1 and less-so mTORC2. In male rodents, rapamycin decreases maladaptive cardiac hypertrophy whereas it leads to detrimental dilative cardiomyopathy in females. We hypothesized that mTOR inhibition could interfere with 17β-estradiol (E2)-mediated sexual dimorphism and adaptive cell growth and tested responses in murine female hearts and cultured female cardiomyocytes. Under physiological in vivo conditions, rapamycin compromised mTORC2 function only in female, but not in male murine hearts. In cultured female cardiomyocytes, rapamycin impaired simultaneously IGF-1 induced activation of both mTOR signaling branches, mTORC1 and mTORC2 only in presence of E2. Use of specific estrogen receptor (ER)α- and ERβ-agonists indicated involvement of both estrogen receptors (ER) in rapamycin effects on mTORC1 and mTORC2. Classical feedback mechanisms common in tumour cells with upregulation of PI3K signaling were not involved. E2 effect on Akt-pS473 downregulation by rapamycin was independent of ERK as shown by sequential mTOR and MEK-inhibition. Furthermore, regulatory mTORC2 complex defining component rictor phosphorylation at Ser1235, known to interfere with Akt-substrate binding to mTORC2, was not altered. Functionally, rapamycin significantly reduced trophic effect of E2 on cell size. In addition, cardiomyocytes with reduced Akt-pS473 under rapamycin treatment displayed decreased SERCA2A mRNA and protein expression suggesting negative functional consequences on cardiomyocyte contractility. Rictor silencing confirmed regulation of SERCA2A expression by mTORC2 in E2-cultured female cardiomyocytes. These data highlight a novel modulatory function of E2 on rapamycin effect on mTORC2 in female cardiomyocytes and regulation of SERCA2A expression by mTORC2. Conceivably, rapamycin abrogates the premenopausal “female advantage”

The mTOR inhibitor Rapamycin protects from premature cellular senescence early after experimental kidney transplantation

Interstitial fibrosis and tubular atrophy, a major cause of kidney allograft dysfunction, has been linked to premature cellular senescence. The mTOR inhibitor Rapamycin protects from senescence in experimental models, but its antiproliferative properties have raised concern early after transplantation particularly at higher doses. Its effect on senescence has not been studied in kidney transplantation, yet. Rapamycin was applied to a rat kidney transplantation model (3 mg/kg bodyweight loading dose, 1.5 mg/kg bodyweight daily dose) for 7 days. Low Rapamycin trough levels (2.1-6.8 ng/mL) prevented the accumulation of p16(INK4a) positive cells in tubules, interstitium, and glomerula. Expression of the cytokines MCP-1, IL-1 beta, and TNF-alpha, defining the proinflammatory senescence-associated secretory phenotype, was abrogated. Infiltration with monocytes/macrophages and CD8(+) T-lymphocytes was reduced and tubular function was preserved by Rapamycin. Inhibition of mTOR was not associated with impaired structural recovery, higher glucose levels, or weight loss. mTOR inhibition with low-dose Rapamycin in the immediate posttransplant period protected from premature cellular senescence without negative effects on structural and functional recovery from preservation/reperfusion damage, glucose homeostasis, and growth in a rat kidney transplantation model. Reduced senescence might maintain the renal regenerative capacity rendering resilience to future injuries resulting in protection from interstitial fibrosis and tubular atrophy

Strengthening of endogenous regeneration mechanisms by modulation the mTOR signaling network

Dank des medizinischen Fortschritts ist die Lebenserwartung in den letzten Jahrzehnten kontinuierlich gestiegen. Chronische Erkrankungen vor allem des kardiovaskulären Systems schmälern jedoch diesen Erfolg, da durch sie die Jahre in Gesundheit nicht in gleichem Maße zugenommen haben. Die Folge ist eine alternde Gesellschaft mit hoher Krankheitslast und zunehmendem Pflegebedarf. Die Stärkung endogener regenerativer Ressourcen könnte eine anhaltende Resistenz gegenüber pathogenen Stimuli bis ins hohe Alter ermöglichen, so dass der Anteil gesunder Lebensjahre zunimmt und zu mehr Lebensqualität bei geringerer Belastung des Gesundheits- und Pflegesektors führt. Die hier dargestellten Arbeiten zeigen, wie eine Modulation des mTOR-Signalnetzwerkes protektive Mechanismen auf zellulärer Ebene aktivieren kann, die vor pathologischen vorzeitigen Alterungsprozessen und altersassoziierten Erkrankungen schützen können. Insbesondere der Hemmung von mTORC1 bei gleichzeitiger Aktivierung von mTORC2 kommt hierbei eine herausragende Bedeutung zu: In humanen MSC, die als Vorläuferzellen für glatte Gefäßmuskelzellen ein relevantes Beispiel für den vaskulären Regenerationsapparat darstellen, wurde durch dieses Aktivierungsmuster des mTOR-Netzwerkes Autophagie gefördert und in der Folge zelluläre Seneszenz und Apoptose verhindert. Insgesamt resultierte diese Aktivierung protektiver Zellschicksale in einer Reduktion osteoblastärer Differenzierung und einem Schutz vor Kalzifizierung. Hieraus lässt sich das Potenzial ableiten, über eine entsprechende Beeinflussung des mTOR-Netzwerkes die endogene Regenerationsfähigkeit des Gefäßsystems durch MSC als vaskulären Progenitorzellen aufrechtzuerhalten. Außerdem stabilisierte der mTOR-Modulator Rapamycin in MSC einen kontraktilen glattmuskulären Phänotyp. Als weiteres Beispiel diente ein Nierentransplantationsmodell der Ratte. Hier zeigte Rapamycin in der unmittelbaren Posttransplantationsphase eine deutliche Reduktion zellulärer Seneszenz in multiplen Organkompartimenten ohne negative Auswirkungen auf die vaskuläre Integrität und die Rückbildung des Konservierungs-Reperfusionsschadens. Hierdurch kann die Regenerationsfähigkeit der transplantierten Niere auch gegenüber künftigen schädigenden Einflüssen erhalten bleiben, so dass sie besser vor interstitieller Fibrose und Tubulusatrophie geschützt ist. Eine gezielte Beeinflussung des mTOR-Netzwerkes durch pharmakologische und nicht-pharmakologische Maßnahmen könnte über eine Aktivierung protektiver Zellschicksale das endogene Regenerationspotenzial körpereigener Vorläuferzellen erhalten und so vor vorzeitiger Alterung in Folge pathogener Stimuli und vor degenerativen Schäden im Alter schützen. Dies könnte eine Verlängerung der Gesundheitserwartung ermöglichen und so die Bedarfssteigerung finanzieller und humaner Ressourcen trotz des demographischen Wandels hin zu einer immer älteren Gesellschaft dämpfen

SARS-CoV-2-induced SIADH: a novel cause of hyponatremia

Background!#!Sensor-based monitoring allows continuous observations of patient mobilization after proximal femoral fractures. A wrist-worn motion tracker allows long-term observation that is low in interruption and constraints for subjects.!##!Objective!#!Description of steps development after hip fracture surgery on a specialized geriatric trauma ward and beyond.!##!Material and methods!#!In the explorative long-term field research study, an applicable motion tracker observed steps per day of 20 patients (80% female, mean age 85.2 years ± 7.86 years) for 10 weeks. Weekly mean values (days 1-7, 8-14 etc.) of steps per day formed the database for descriptive analysis (mean, SD, min, max, median).!##!Results!#!During observation weeks (ow) a positive development of steps took place. A mean increase factor of 1.285 (±0.351) occurred from ow 1 (M = 353.57 ± 310.15) to ow 10 (M = 2482.07 ± 1374.12). The highest increase by a factor of 1.8 could be reported from ow 2 (M = 556.27 ± 478.11) to ow 3 (M = 1024.86 ± 921.24) as well as from ow 6 (M = 1268.21 ± 880.47) to 7 (M = 2367.14 ± 1680.08). A slight decrease of steps occurred from ow 4 (M = 1208.27 ± 1210.45) to ow 5 (0.99-fold) and from ow 9 (M = 2689.98 ± 2339.71) to 10 (0.92-fold). High ranges and standard deviations in relation to the mean occurred constantly. The presence of several step development groups could be presumed.!##!Conclusion!#!Motion tracker and the variable steps per day can represent the ability to walk within an everyday environment, with a possible underestimation of < 10%. Differences regarding observation lengths and disruptions occurred. Cluster analysis should detect group attributes of different courses of development in subsequent studies

mTORC1 and mTORC2 Differentially Regulate Cell Fate Programs to Coordinate Osteoblastic Differentiation in Mesenchymal Stromal Cells

Vascular regeneration depends on intact function of progenitors of vascular smooth muscle cells such as pericytes and their circulating counterparts, mesenchymal stromal cells (MSC). Deregulated MSC differentiation and maladaptive cell fate programs associated with age and metabolic diseases may exacerbate arteriosclerosis due to excessive transformation to osteoblast-like calcifying cells. Targeting mTOR, a central controller of differentiation and cell fates, could offer novel therapeutic perspectives. In a cell culture model for osteoblastic differentiation of pluripotent human MSC we found distinct roles for mTORC1 and mTORC2 in the regulation of differentiation towards calcifying osteoblasts via cell fate programs in a temporally-controlled sequence. Activation of mTORC1 with induction of cellular senescence and apoptosis were hallmarks of transition to a calcifying phenotype. Inhibition of mTORC1 with Rapamycin elicited reciprocal activation of mTORC2, enhanced autophagy and recruited anti-apoptotic signals, conferring protection from calcification. Pharmacologic and genetic negative interference with mTORC2 function or autophagy both abolished regenerative programs but induced cellular senescence, apoptosis, and calcification. Overexpression of the mTORC2 constituent rictor revealed that enhanced mTORC2 signaling without altered mTORC1 function was sufficient to inhibit calcification. Studies in mice reproduced the in vitro effects of mTOR modulation with Rapamycin on cell fates in vascular cells in vivo. Amplification of mTORC2 signaling promotes protective cell fates including autophagy to counteract osteoblast differentiation and calcification of MSC, representing a novel mTORC2 function. Regenerative approaches aimed at modulating mTOR network activation patterns hold promise for delaying age-related vascular diseases and treatment of accelerated arteriosclerosis in chronic metabolic conditions.Peer Reviewe

High cut-off dialysis mitigates pro-calcific effects of plasma on vascular progenitor cells

Abstract Mortality of patients with end-stage renal disease tremendously exceeds that of the general population due to excess cardiovascular morbidity. Large middle-sized molecules (LMM) including pro-inflammatory cytokines are major drivers of uremic cardiovascular toxicity and cannot be removed sufficiently by conventional high-flux (HFL) hemodialysis. We tested the ability of plasma from 19 hemodialysis patients participating in a trial comparing HFL with high cut-off (HCO) membranes facilitating removal of LMM to induce calcification in mesenchymal stromal cells (MSC) functioning as vascular progenitors. HCO dialysis favorably changed plasma composition resulting in reduced pro-calcific activity. LMM were removed more effectively by HCO dialysis including FGF23, a typical LMM we found to promote osteoblastic differentiation of MSC. Protein-bound uremic retention solutes with known cardiovascular toxicity but not LMM inhibited proliferation of MSC without direct toxicity in screening experiments. We could not attribute the effect of HCO dialysis on MSC calcification to distinct mediators. However, we found evidence of sustained reduced inflammation that might parallel other anti-calcifying mechanisms such as altered generation of extracellular vesicles. Our findings imply protection of MSC from dysfunctional differentiation by novel dialysis techniques targeted at removal of LMM. HCO dialysis might preserve their physiologic role in vascular regeneration and improve outcomes in dialysis patients

Intrinsic Deregulation of Vascular Smooth Muscle and Myofibroblast Differentiation in Mesenchymal Stromal Cells from Patients with Systemic Sclerosis.

INTRODUCTION:Obliterative vasculopathy and fibrosis are hallmarks of systemic sclerosis (SSc), a severe systemic autoimmune disease. Bone marrow-derived mesenchymal stromal cells (MSCs) from SSc patients may harbor disease-specific abnormalities. We hypothesized disturbed vascular smooth muscle cell (VSMC) differentiation with increased propensity towards myofibroblast differentiation in response to SSc-microenvironment defining growth factors and determined responsible mechanisms. METHODS:We studied responses of multipotent MSCs from SSc-patients (SSc-MSCs) and healthy controls (H-MSCs) to long-term exposure to CTGF, b-FGF, PDGF-BB or TGF-β1. Differentiation towards VSMC and myofibroblast lineages was analyzed on phenotypic, biochemical, and functional levels. Intracellular signaling studies included analysis of TGF-β receptor regulation, SMAD, AKT, ERK1/2 and autocrine loops. RESULTS:VSMC differentiation towards both, contractile and synthetic VSMC phenotypes in response to CTGF and b-FGF was disturbed in SSc-MSCs. H-MSCs and SSc-MSCs responded equally to PDGF-BB with prototypic fibroblastic differentiation. TGF-β1 initiated myofibroblast differentiation in both cell types, yet with striking phenotypic and functional differences: In relation to H-MSC-derived myofibroblasts induced by TGF-β1, those obtained from SSc-MSCs expressed more contractile proteins, migrated towards TGF-β1, had low proliferative capacity, and secreted higher amounts of collagen paralleled by reduced MMP expression. Higher levels of TGF-β receptor 1 and enhanced canonical and noncanonical TGF-β signaling in SSc-MSCs accompanied aberrant differentiation response of SSc-MSCs in comparison to H-MSCs. CONCLUSIONS:Deregulated VSMC differentiation with a shift towards myofibroblast differentiation expands the concept of disturbed endogenous regenerative capacity of MSCs from SSc patients. Disease related intrinsic hyperresponsiveness to TGF-β1 with increased collagen production may represent one responsible mechanism. Better understanding of repair barriers and harnessing beneficial differentiation processes in MSCs could widen options of autologous MSC application in SSc patients