A multicentre, patient- and assessor-blinded, non-inferiority, randomised and controlled phase II trial to compare standard and torque teno virus-guided immunosuppression in kidney transplant recipients in the first year after transplantation: TTVguideIT.

BACKGROUND: Immunosuppression after kidney transplantation is mainly guided via plasma tacrolimus trough level, which cannot sufficiently predict allograft rejection and infection. The plasma load of the non-pathogenic and highly prevalent torque teno virus (TTV) is associated with the immunosuppression of its host. Non-interventional studies suggest the use of TTV load to predict allograft rejection and infection. The primary objective of the current trial is to demonstrate the safety, tolerability and preliminary efficacy of TTV-guided immunosuppression. METHODS: For this purpose, a randomised, controlled, interventional, two-arm, non-inferiority, patient- and assessor-blinded, investigator-driven phase II trial was designed. A total of 260 stable, low-immunological-risk adult recipients of a kidney graft with tacrolimus-based immunosuppression and TTV infection after month 3 post-transplantation will be recruited in 13 academic centres in six European countries. Subjects will be randomised in a 1:1 ratio (allocation concealment) to receive tacrolimus either guided by TTV load or according to the local centre standard for 9 months. The primary composite endpoint includes the occurrence of infections, biopsy-proven allograft rejection, graft loss, or death. The main secondary endpoints include estimated glomerular filtration rate, graft rejection detected by protocol biopsy at month 12 post-transplantation (including molecular microscopy), development of de novo donor-specific antibodies, health-related quality of life, and drug adherence. In parallel, a comprehensive biobank will be established including plasma, serum, urine and whole blood. The date of the first enrolment was August 2022 and the planned end is April 2025. DISCUSSION: The assessment of individual kidney transplant recipient immune function might enable clinicians to personalise immunosuppression, thereby reducing infection and rejection. Moreover, the trial might act as a proof of principle for TTV-guided immunosuppression and thus pave the way for broader clinical applications, including as guidance for immune modulators or disease-modifying agents. TRIAL REGISTRATION: EU CT-Number: 2022-500024-30-00.

Cumulative UV Exposure or a Modified SCINEXA™-Skin Aging Score Do Not Play a Substantial Role in Predicting the Risk of Developing Keratinocyte Cancers after Solid Organ Transplantation-A Case Control Study.

The risk of keratinocyte cancer is determined by intrinsic and extrinsic factors, which also influence skin aging. Few studies have linked skin aging and UV exposure with the incidence of non-melanoma skin cancer (NMSC). We evaluated signs of actinic skin damage and aging, individual UV burden, and melanocortin-1 receptor (MC1R) variants. A total of 194 organ transplant recipients (OTR) who suffered from NMSC were compared to 194 tumor-free controls matched for gender, age, type of transplanted organ, post-transplantation (TX) period, and immunosuppressive therapy. Compared with the cases, the controls scored higher in all skin aging scores and there were no differences in UV burden except for intentional whole-body UV exposure for specific UV scenarios and periods of life in favor of cases. The number of NMSCs correlated with all types of skin aging scores, the extent of intentional sun exposure, older age, longer post-TX period, shorter interval from TX to first NMSC, and specific MC1R risk groups. Multivariable models revealed a 7.5-fold risk of developing NMSC in individuals with actinic keratosis; 4.1- or 3.6-fold in those with green or blue eyes, respectively; and a 1.9-fold increased risk in the MC1R medium- + high-risk group. In the absence of skin aging contributing to NMSC development, certain MC1R risk types may identify OTR at risk for high tumor burden.

Coping with a lack of evidence: living-donor kidney transplantation in the initial phase of the SARS-CoV-2 pandemic.

Due to immunosuppressive therapy, transplant patients are more susceptible to viral and bacterial infections. A potentially deadly new virus haunted us in 2020: SARS-CoV­2, causing coronavirus disease 19 (COVID-19). We analyzed the consequences of this previously unknown risk for our living-donor transplant program in the first year of the pandemic. After the complete lockdown in spring 2020, our transplant center in Linz resumed the living-donor kidney transplantation program from June to September 2020, between the first and second waves of COVID-19 in Austria. We compared the outcomes of these living-donor kidney transplantations with the transplant outcomes of the corresponding periods of the three previous years. From June 4 to September 9, 2020, five living-donor kidney transplantations were performed. All donors and recipients were screened for COVID 19 infection by PCR testing the day before surgery. Kidney transplant recipients remained isolated in single rooms until discharge from hospital. All recipients and donors remained SARS-CoV­2 negative during the follow-up of 10 months and have been fully vaccinated to date. The number of living transplants in the studied period of 2020 was constant compared to the same months of 2017, 2018, and 2019. Living-donor kidney transplantation can be continued using testing for SARS-CoV­2 and meticulous hygienic precautions in epidemiologically favorable phases of the SARS-CoV­2 pandemic. Donors and recipients should be carefully selected and informed about risks and benefits.

[Diagnosis and treatment of osteoporosis in patients with chronic kidney disease : Joint guidelines of the Austrian Society for Bone and Mineral Research (ÖGKM), the Austrian Society of Physical and Rehabilitation Medicine (ÖGPMR) and the Austrian Society of Nephrology (ÖGN)]. / Diagnose und Therapie der Osteoporose bei Patienten mit chronischer Niereninsuffizienz : Gemeinsame Leitlinie der Österreichischen Gesellschaft für Knochen- und Mineralstoffwechsel (ÖGKM), der Österreichischen Gesellschaft für Physikalische Medizin und Rehabilitation (ÖGPMR) und der Österreichischen Gesellschaft für Nephrologie (ÖGN).

DEFINITION AND EPIDEMIOLOGY: Chronic kidney disease (CKD): abnormalities of kidney structure or function, present for over 3 months. Staging of CKD is based on GFR and albuminuria (not graded). Osteoporosis: compromised bone strength (low bone mass, disturbance of microarchitecture) predisposing to fracture. By definition, osteoporosis is diagnosed if the bone mineral density T­score is ≤ -2.5. Furthermore, osteoporosis is diagnosed if a low-trauma (inadequate trauma) fracture occurs, irrespective of the measured T­score (not graded). The prevalence of osteoporosis, osteoporotic fractures and CKD is increasing worldwide (not graded). PATHOPHYSIOLOGY, DIAGNOSIS AND TREATMENT OF CHRONIC KIDNEY DISEASE-MINERAL AND BONE DISORDER (CKD-MBD): Definition of CKD-MBD: a systemic disorder of mineral and bone metabolism due to CKD manifested by either one or a combination of the following: abnormalities of calcium, phosphorus, PTH, or vitamin D metabolism; renal osteodystrophy; vascular calcification (not graded). Increased, normal or decreased bone turnover can be found in renal osteodystrophy (not graded). Depending on CKD stage, routine monitoring of calcium, phosphorus, alkaline phosphatase, PTH and 25-OH-vitamin D is recommended (2C). Recommendations for treatment of CKD-MBD: Avoid hypercalcemia (1C). In cases of hyperphosphatemia, lower phosphorus towards normal range (2C). Keep PTH within or slightly above normal range (2D). Vitamin D deficiency should be avoided and treated when diagnosed (1C). DIAGNOSIS AND RISK STRATIFICATION OF OSTEOPOROSIS IN CKD: Densitometry (using dual X­ray absorptiometry, DXA): low T­score correlates with increased fracture risk across all stages of CKD (not graded). A decrease of the T­score by 1 unit approximately doubles the risk for osteoporotic fracture (not graded). A T-score ≥ -2.5 does not exclude osteoporosis (not graded). Bone mineral density of the lumbar spine measured by DXA can be increased and therefore should not be used for the diagnosis or monitoring of osteoporosis in the presence of aortic calcification, osteophytes or vertebral fracture (not graded). FRAX can be used to aid fracture risk estimation in all stages of CKD (1C). Bone turnover markers can be measured in individual cases to monitor treatment (2D). Bone biopsy may be considered in individual cases, especially in patients with CKD G5 (eGFR < 15 ml/min/1.73 m2) or CKD 5D (dialysis). SPECIFIC TREATMENT OF OSTEOPOROSIS IN PATIENTS WITH CKD: Hypocalcemia should be treated and serum calcium normalized before initiating osteoporosis therapy (1C). CKD G1-G2 (eGFR ≥ 60 ml/min/1.73 m2): treat osteoporosis as recommended for the general population (1A). CKD G3-G5D (eGFR < 60 ml/min/1.73 m2 to dialysis): treat CKD-MBD first before initiating osteoporosis treatment (2C). CKD G3 (eGFR 30-59 ml/min/1.73 m2) with PTH within normal limits and osteoporotic fracture and/or high fracture risk according to FRAX: treat osteoporosis as recommended for the general population (2B). CKD G4-5 (eGFR < 30 ml/min/1.73 m2) with osteoporotic fracture (secondary prevention): Individualized treatment of osteoporosis is recommended (2C). CKD G4-5 (eGFR < 30 ml/min/1.73 m2) and high fracture risk (e.g. FRAX score > 20% for a major osteoporotic fracture or > 5% for hip fracture) but without prevalent osteoporotic fracture (primary prevention): treatment of osteoporosis may be considered and initiated individually (2D). CKD G4-5D (eGFR < 30 ml/min/1.73 m2 to dialysis): Calcium should be measured 1-2 weeks after initiation of antiresorptive therapy (1C). PHYSICAL MEDICINE AND REHABILITATION: Resistance training prioritizing major muscle groups thrice weekly (1B). Aerobic exercise training for 40 min four times per week (1B). Coordination and balance exercises thrice weekly (1B). Flexibility exercise 3-7 times per week (1B).

Quantitative scoring of progression in transplant glomerulopathy using digital pathology may be superior to Banff cg scoring.

Antibody-mediated rejection (ABMR) is a major cause of kidney allograft failure. Biopsy-based surrogate endpoints reflecting ABMR progression on sequential biopsies that predict long-term outcome offer the potential to make treatment trials for ABMR feasible. However, the Banff transplant glomerulopathy (TG) scoring system (chronic glomerular injury score [cg]) relies on relatively crude and arbitrary ordinal grades and has low inter-observer concordance that currently limits its usefulness as a surrogate endpoint for ABMR progression in clinical drug trials. Here, we describe and validate a novel quantitative method for quantifying progression of TG in ABMR. Using digital pathology in sequential biopsies from 75 patients at various stages of ABMR, we scored all capillaries in the most affected glomeruli for basement membrane duplication that were correlated with allograft function, outcome, Banff lesion scores, and gene expression. Our digital scoring reflected TG progression better than the categorical Banff cg score and correlated with Banff ABMR and chronicity lesions, but not transcript changes. In multivariate analysis, the delta change between biopsies with serum creatinine and mean percent duplicated glomerular basement membranes was significantly associated with graft loss. Neither the delta in any Banff lesion scores (including cg) nor in gene expression was associated with outcome. Receiver operating characteristic curve analysis showed that the digital pathology approach was superior to the conventional score for predicting graft failure. Thus, our digital pathology-based approach for scoring TG accurately assessed progression in TG. However, further validation as a potential surrogate endpoint in clinical trials for the treatment of ABMR is warranted.

Effect of the phosphate binder sucroferric oxyhydroxide in dialysis patients on endogenous calciprotein particles, inflammation, and vascular cells.

BACKGROUND: Calciprotein particles (CPPs), colloidal mineral-protein nanoparticles, have emerged as potential mediators of phosphate toxicity in dialysis patients, with putative links to vascular calcification, endothelial dysfunction and inflammation. We hypothesized that phosphate binder therapy with sucroferric oxyhydroxide (SO) would reduce endogenous CPP levels and attenuate pro-calcific and pro-inflammatory effects of patient serum towards human vascular cells in vitro. METHODS: This secondary analysis of a randomised controlled crossover study compared the effect of 2-week phosphate binder washout with high-dose (2000 mg/day) and low-dose (250 mg/day) SO therapy in 28 haemodialysis patients on serum CPP levels, inflammatory cytokine/chemokine arrays and human aortic smooth muscle cell (HASMC) and coronary artery endothelial cell (HCAEC) bioassays. RESULTS: In our cohort (75% male, 62 ± 12 years) high-dose SO reduced primary (amorphous) and secondary (crystalline) CPP levels {-62% [95% confidence interval (CI) -76 to -44], P < .0001 and -38% [-62 to -0.14], P < .001, respectively} compared with washout. Nine of 14 plasma cytokines/chemokines significantly decreased with high-dose SO, with consistent reductions in interleukin-6 (IL-6) and IL-8. Exposure of HASMC and HCAEC cultures to serum of SO-treated patients reduced calcification and markers of activation (IL-6, IL-8 and vascular cell adhesion protein 1) compared with washout. Serum-induced HASMC calcification and HCAEC activation was ameliorated by removal of the CPP-containing fraction from patient sera. Effects of CPP removal were confirmed in an independent cohort of chronic kidney disease patients. CONCLUSIONS: High-dose SO reduced endogenous CPP formation in dialysis patients and yielded serum with attenuated pro-calcific and inflammatory effects in vitro.

Calcification Propensity in Serum and Cardiovascular Outcome in Peripheral Artery Disease.

Peripheral artery disease (PAD) has been shown to be linked to elevated cardiovascular risk. The novel T50 test quantifies calcification propensity of serum and has been associated with cardiovascular events and mortality in patients with chronic kidney disease (CKD) and in the general population. This study investigated the association of calcification propensity measured by the T50 test in 287 patients with PAD without severe CKD. Major cardiovascular events (MACEs) including nonfatal stroke and nonfatal myocardial infarction and all-cause death (MACE + ) were evaluated after a median follow-up of 4 years and long-term cardiovascular and all-cause mortality after a median follow-up of 8.7 years by Kaplan-Meier and Cox regression analyses. Mean T50 time was 268 ± 63 minutes in the study cohort (age 69 ± 10 years, 32% women, 47% diabetes). Low T50 values that signify high calcification propensity were significantly associated with the occurrence of MACE+ (hazard ratio [HR]: 0.72; 95% confidence interval [CI]: 0.55-0.94). This association sustained multivariate adjustment for cardiovascular risk factors (CVRFs), Fontaine PAD stage, and prevalent media sclerosis (HR: 0.65; CI: 0.47-0.91). Cardiovascular mortality was significantly associated with T50 after multivariate adjustment for CVRF (HR: 0.72; CI 0.53-0.99), but not all-cause mortality (HR: 0.80; CI: 0.64-1.01). In conclusion, calcification propensity associates with MACE+ and cardiovascular mortality in patients with PAD.

Successful kidney transplantation in a patient with pre-existing chronic myeloid leukemia treated with imatinib.

Active malignancy is an absolute contraindication to kidney transplantation. As for chronic myeloid leukemia (CML), a Philadelphia chromosome-positive myeloproliferative neoplasm, the introduction of tyrosine kinase inhibitors has transformed CML from a lethal into a manageable chronic disease with a close-to-normal life expectancy. To date it is unknown whether kidney transplantation can be safely performed in patients with pre-existing CML. We describe the clinical course of a 57-year-old male patient with chronic kidney disease caused by reflux nephropathy. This patient had undergone first kidney transplantation 20 years earlier and had again been on chronic hemodialysis for 6 years when CML was diagnosed. First-line therapy with 400 mg imatinib daily was well tolerated and induced an optimal cytogenetic and molecular response 3 months after initiation. One and a half years after CML diagnosis, a second kidney transplantation from a deceased donor was performed. Immunosuppression included basiliximab, tacrolimus, mycophenolate mofetil, and corticosteroids. Currently, 2 years posttransplant, renal allograft function is stable (serum creatinine 1.09 mg/dL, estimated glomerular filtration rate 75 mL/min per 1.73 m2 ), and CML remains in deep molecular remission with imatinib. Imatinib-treated CML in deep molecular remission could be regarded as inactive malignancy and may therefore not be viewed as an absolute contraindication to kidney transplantation.

Oral Sodium Bicarbonate Supplementation Does Not Affect Serum Calcification Propensity in Patients with Chronic Kidney Disease and Chronic Metabolic Acidosis.

BACKGROUND: Cardiovascular disease is the leading cause of death in patients with chronic kidney disease (CKD) and metabolic acidosis might accelerate vascular calcification. The T50 calcification inhibition test (T50-test) is a global functional test analyzing the overall propensity of calcification in serum, and low T50-time is associated with progressive aortic stiffening and with all-cause mortality in non-dialysis CKD, dialysis, and transplant patients. Low serum bicarbonate is associated with a short T50-time and alkali supplementation could be a simple modifier of calcification propensity. The aim of this study was to investigate the short-term effect of oral sodium bicarbonate supplementation on T50-time in CKD patients. MATERIAL AND METHODS: The SoBic-study is an ongoing randomized-controlled trial in CKD-G3 and G4 patients with chronic metabolic acidosis (serum HCO3- ≤21 mmol/L), in which patients are randomized to either achieve serum HCO3- levels of 24 ± 1 mmol/L (intervention group) or 20 ± 1 mmol/L (rescue group). The effect of bicarbonate treatment on T50-time was assessed. RESULTS: The study cohort consisted of 35 (14 female) patients aged 57 (±15) years, and 18 were randomized to the intervention group. The mean T50-time was 275 (± 64) min. After 4 weeks, the mean change of T50-time was 4 (±69) min in the intervention group and 18 min (±56) in the rescue group (ß = -25; 95% CI: -71 to 22; p = 0.298). Moreover, change of serum bicarbonate in individual patients was not associated with change in T50-time, analyzed by regression analysis. Change of serum phosphate had a significant impact on change of T50-time (ß = -145; 95% CI: -237 to -52). CONCLUSION: Oral sodium bicarbonate supplementation showed no effect on T50-time in acidotic CKD patients.

Calcification Propensity of Serum is Independent of Excretory Renal Function.

Vascular calcification is a component of cardiovascular disease, which is leading cause of death in patients with chronic kidney disease (CKD). A functional assay (T50-test) measuring the propensity of human serum to calcify associates with mortality and cardiovascular events in CKD patients. Calcification propensity is known to increase with CKD stage. We investigated whether the T50 readout is directly dependent on excretory kidney function (eGFR) or rather explained by deranged parameters of bone and mineral metabolism in the course of CKD. T50, along with markers implicated in calcification and mineral metabolism, were measured in a cross-sectional cohort of 118 patients with CKD stage 1-5. Associations of T50 with measured parameters were analysed and partial correlations performed to test to which extent the association of T50 with eGFR can be attributed to variation of these parameters. T50 correlates with eGFR, but serum levels of phosphate and calcium largely explain this association. Phosphate, magnesium, fetuin A, albumin, bicarbonate, and serum cross-laps but not Parathyroid Hormone or Fibroblast Growth Factor 23 are associated with T50 in multivariate adjusted models. These findings indicate that T50 values depend mainly on the concentration of promoters and inhibitors of calcification in serum, but not excretory kidney function.

Low-dose calcium versus pentagastrin for stimulation of calcitonin in chronic hemodialysis patients: a pilot study.

CONTEXT: Elevated calcitonin levels occur in up to 46% of patients with chronic hemodialysis (CHD) and frequently reflect benign C-cell hyperplasia rather than medullary thyroid carcinoma. For the differential diagnosis of hypercalcitoninemia, the pentagastrin-stimulated calcitonin test was used until its availability became restricted. OBJECTIVE: This study sought to compare calcium and pentagastrin in terms of their ability to stimulate calcitonin secretion and their side effects in patients with CHD. SETTING AND DESIGN: This prospective pilot study was conducted at the chronic hemodialysis unit of the Medical University of Vienna between December 2012 and September 2013. PATIENTS: We studied six male patients with CHD with elevated basal calcitonin levels. INTERVENTION: The stimulation test was performed first with 0.5 µg/kg pentagastrin and then with 1 mg/kg calcium after a median washout period of 7 (6-9) months. MAIN OUTCOME MEASURES: We measured calcitonin, serum ionized calcium, intact PTH (iPTH), and C-terminal fibroblast growth factor 23 levels before and 2, 5, and 10 minutes after iv infusion of the stimulant and assessed the tolerability of the two substances by a questionnaire. RESULTS: Both pentagastrin and calcium significantly stimulated calcitonin secretion at 2 and 5 minutes. Partial correlation analysis revealed a strong association between calcium- and pentagastrin-stimulated calcitonin levels (r=0.875, P < .0001). Only after calcium infusion serum ionized calcium levels increased from 1.09 (0.91-1.16) mmol/l to 1.4 (1.14-1.65) mmol/l at 2 minutes (P < .01) but returned to baseline levels at 5 minutes. Moreover, calcium infusion led to a significant decrease in iPTH levels from 315 (203-723) pg/ml to 182 (121-415) pg/ml at 5 minutes (P < .05) and 171 (91-346) pg/ml at 10 minutes (P < .001). In general, calcium caused fewer and less severe side effects than pentagastrin. CONCLUSIONS: In patients with CHD, the response of calcitonin to calcium and pentagastrin was comparable, making calcium a potential substitute for pentagastrin in these patients.

Correlations and time course of FGF23 and markers of bone metabolism in maintenance hemodialysis patients.

OBJECTIVE: Parathyroid hormone (iPTH) and fibroblast growth factor 23 (FGF23) are elevated in secondary hyperparathyroidism. In hemodialysis, higher dialysate calcium (1.5 mmol/L) induces intradialytic suppression of iPTH, whereas its impact on FGF23 and markers of bone metabolism is unknown. We assessed the time course of FGF23 and markers of bone metabolism in relationship to dialysate calcium. DESIGN AND METHODS: In this prospective cohort study of 19 patients on maintenance hemodialysis, we measured serum calcium (sCa), inorganic phosphate (iP), blood urea nitrogen (BUN), ß2-microglobulin (ßMG), iPTH, FGF23, aminoterminal propeptide type 1 procollagen (P1NP), C-telopeptide of type I collagen for bone degradation (CTX-I), osteocalcin (OC), bone specific alkaline phosphatase (BALP), and tartrate-resistant acid phosphatase (TRAP5b) during a single hemodialysis session at baseline, 1, 2, and 3h of dialysis. The time course of measured parameters was compared according to groups of prescribed dialysate calcium of 1.25 mmol/L and 1.5 mmol/L. RESULTS: iPTH declined in the 1.5 mmol/L dialysis group as serum calcium increased whereas it tended to increase in the 1.25 mmol/L group without significant changes in serum calcium. Patients on long-term dialysate calcium of 1.5 mmol/L had significantly lower CTX-I levels and tended to lower levels of iPTH, FGF23, OC, P1NP and TRAP5b at the start of dialysis compared to those on 1.25 mmol/L. CTX-I, FGF23 and OC but not BALP, P1NP and TRAP5b decreased during dialysis independent of dialysate calcium. CONCLUSIONS: In spite of immediate effects on iPTH, dialysate calcium does not acutely affect other parameters of bone and mineral metabolism. SHORT SUMMARY: Dialysate calcium concentration is known to have both immediate and longer-term impact on parathyroid hormone levels in hemodialysis patients. Little is known about the acute impact of dialysate calcium on bone metabolism. In this cross-sectional study of prevalent hemodialysis patients, we found no evidence of immediate short-term dialysate calcium-induced changes of fibroblast growth factor 23 or anabolic and catabolic markers of bone turnover during hemodialysis. However, differences in CTX-I and to a lesser extent other parameters between groups of higher and lower dialysate calcium suggest a longer-term effect that remains to be validated.

Inverse association between bone microarchitecture assessed by HR-pQCT and coronary artery calcification in patients with end-stage renal disease.

It is a matter of debate whether vascular calcification and bone loss are simultaneously occurring but largely independent processes or whether poor bone health predisposes to vascular calcification, especially in patients with kidney disease. Here we investigated the association between the changes of microarchitecture in weight bearing bone and the extent of coronary artery calcification in patients with chronic renal failure. The bone microarchitecture of the tibia using high-resolution peripheral quantitative computed tomography (HR-pQCT), bone mineral density using dual X-ray absorptiometry (DXA) of the lumbar spine, femoral neck and distal radius as well as coronary artery calcification using multi-slice CT and reported as Agatston score were measured in 66 patients with end-stage renal disease on chronic hemodialysis. Markers of bone turnover, vitamin D status and intact parathyroid hormone (iPTH) were assessed. CAC score was found to be <100 in 39% and ≥100 in 61% of patients. The median [95% CI] total CAC score was 282 [315-2587]. By univariate analysis, significant correlations between CAC and age (R=0.52, p<0.001), weight (R=0.3, p<0.01) and serum cross laps (CTX, R=-0.39, p<0.01) were found, and parameters of bone microarchitecture were numerically but not significantly lower in patients with CAC scores ≥100. In multivariate analysis stratifying for gender and correcting for age, tibial density (Dtot) and bone volume/total volume (BV/TV) were significantly lower in patients with CAC scores ≥100 (p<0.05 for both). Low trabecular bone volume and decreased cortical bone density are associated with coronary artery calcification in dialysis patients.

Renale osteodystrophie.

The incidence of renal osteodystrophy (ROD) increases with deteriorating kidney function, affecting virtually every patient on chronic dialysis treatment. ROD can persist after kidney transplantation and may be aggravated by immunosuppressants, mainly glucocorticoids. Fracture risk, including hip fractures, is markedly elevated in patients with renal disease compared to the general population. Depending on the type of ROD, high or low bone turnover can be found. Because of poor positive and negative predictive values of serological markers of bone turnover and limited technical capabilities of various bone imaging modalities, the only reliable method to correctly classify ROD is the transiliac bone biopsy. Elevated bone turnover can be successfully treated with active vitamin D, cinacalcet, or parathyreoidectomy, but all of these therapies may lead to oversuppression of bone metabolism. Currently, no specific therapy is available for low turnover bone disease. Bisphosphonates can be a therapeutic option for selected patients after renal transplantation.

Vertical inhibition of the mTORC1/mTORC2/PI3K pathway shows synergistic effects against melanoma in vitro and in vivo.

The phosphatidyl inositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway has been shown to be involved in the development of melanoma. PI-103 is a kinase inhibitor blocking PI3K class IA and mTOR complex 1 and 2. Here, we studied the effect of targeting the PI3K/mTORC1/mTORC2 pathway by PI-103 and rapamycin in melanoma cells and in a melanoma mouse model. Dual targeting of PI3K and mTOR by PI-103 induced apoptosis and cell-cycle arrest, and inhibited viability of melanoma cells in vitro. Combined treatment with PI-103 and the prototypic mTORC1 inhibitor rapamycin led to the synergistic suppression of AKT and ribosomal S6 protein phosphorylation and to the induction of apoptosis. In vivo, PI-103 and rapamycin displayed only modest single-agent activity, but the combination significantly reduced the tumor growth compared with both single agents. These data show that blocking the PI3K/mTORC1/mTORC2 pathway using the combination of two distinct small-molecule inhibitors ("vertical inhibition") leads to superior efficacy against malignant melanoma in vitro and in vivo.

Mammalian target of rapamycin signaling is crucial for joint destruction in experimental arthritis and is activated in osteoclasts from patients with rheumatoid arthritis.

OBJECTIVE: Activation of the mammalian target of rapamycin (mTOR) pathway is important for immune cell activation and bone metabolism. To date, the contribution of mTOR signaling to joint inflammation and structural bone and cartilage damage is unknown. The aim of this study was to investigate the potential of inhibiting mTOR as a treatment of inflammatory arthritis. METHODS: Human tumor necrosis factor-transgenic mice in which inflammatory arthritis was developing were treated with 2 different mTOR inhibitors, sirolimus or everolimus. The effects of treatment on clinical disease activity, inflammation, and localized joint and cartilage destruction were studied. In addition, the effects of mTOR inhibition on osteoclast survival and expression of key molecules of osteoclast function were analyzed in vitro. Moreover, synovial tissue from patients with rheumatoid arthritis (RA) was assessed for activation of the mTOR pathway. RESULTS: Inhibition of mTOR by sirolimus or everolimus reduced synovial osteoclast formation and protected against local bone erosions and cartilage loss. Clinical signs of arthritis improved after mTOR inhibition, and histologic evaluation showed a decrease in synovitis. In vitro, mTOR inhibition down-regulated the expression of digestive enzymes and led to osteoclast apoptosis. Moreover, mTOR signaling was shown to be active in the synovial membrane of patients with RA, particularly in synovial osteoclasts. CONCLUSION: Signaling through mTOR is an important link between synovitis and structural damage in inflammatory arthritis. Current pharmacologic inhibitors of mTOR could be effective in protecting joints against structural damage.

mTOR inhibition by everolimus counteracts VEGF induction by sunitinib and improves anti-tumor activity against gastric cancer in vivo.

VEGF receptor blockage has been reported to increase serum VEGF. We hypothesized that mTOR inhibition by everolimus counteracts VEGF induction by sunitinib resulting in an improved anti-tumor activity of sunitinib. In vitro, sunitinib in combination with everolimus did not outperform the respective monotherapies. In vivo, monotherapies reduced tumor growth by 60%, whereas the combination of sunitinib and everolimus led to an almost complete tumor growth inhibition. This superior anti-tumor activity coincided with attenuation of VEGF peaks. In conclusion mTOR inhibition by everolimus counteracts VEGF induction by sunitinib and results in significant reduction of tumor burden and long-lasting tumor growth control.

Effects of vatalanib on tumor growth can be potentiated by mTOR blockade in vivo.

The vascular endothelial growth factor (VEGF) is a central mediator of tumor-induced angiogenesis. Everolimus, a mammalian target of rapamycin (mTOR) inhibitor, decreases VEGF-secretion of cancer cells. Vatalanib is a selective inhibitor of VEGF receptors 1-3. In the present study it was hypothesized that dual inhibition of VEGF signaling by inhibition of VEGF production and VEGF receptor signaling leads to synergistic anti-tumor effects. In vitro, effects of vatalanib and everolimus on cell proliferation, cell cycle, apoptosis and signal transduction were examined in three gastric cancer cell lines. Effects on angiogenesis were assessed using tube formation assays of cultured human umbilical vein endothelial cells (HUVECs). In vivo, the antitumor effect of compounds was studied using a gastric cancer xenograft nude mouse model. VEGF of murine origin (mVEGF) and human cancer cell-derived VEGF (hVEGF) were studied separately by specific ELISAs. Tumor vascularization and proliferation were quantified by immunohistochemistry. In vitro, everolimus but not vatalanib decreased gastric cancer proliferation without inducing apoptosis. Vatalanib abolished endothelial cell tube formation, whereas inhibition of tube formation by everolimus was incomplete. In vivo, the combination of vatalanib with everolimus was superior to single agent treatments and reduced tumor size by about 50% relative to everolimus monotherapy (p < 0.005). Pharmacodynamic analysis of VEGF plasma level showed a decrease of hVEGF by everolimus and indicated a trend towards lower mVEGF level only in the combination group. In line, there was a tendency for lower vascular density and proliferation for combination treatment. We conclude that in a preclinical model of gastric cancer the antitumor activity of vatalanib can be augmented by everolimus.

Everolimus (RAD001) and anti-angiogenic cyclophosphamide show long-term control of gastric cancer growth in vivo.

Metronomic dosing of cytotoxic drugs such as cyclophosphamide has shown anti-angiogenic activity, most likely by inducing hypoxia in tumors. Hypoxia leads to activation of escape mechanisms allowing tumor cell survival. This potentially limits the activity of anti-angiogenic strategies. We hypothesized that mTORC1 inhibition by everolimus (RAD001) leads to suppression of HIF-1alpha and VEGF resulting in synergistic anti-tumor activity in combination with anti-angiogenically dosed cyclophosphamide. In vitro, effects of everolimus on mTORC1 signaling, proliferation, cell cycle, HIF-1alpha expression and VEGF secretion were evaluated in two gastric cancer cell lines. In vivo, anti-tumor activity of everolimus in combination with metronomic cyclophosphamide was studied in a NCI-N87 human gastric cancer SCID mouse xenograft model. Expression of Ki-67 and HIF-1alpha, activated caspase 3, microvascular density (MVD) and tumor necrotic area assessed. Everolimus decreased proliferation and attenuated production of HIF-1alpha as well as VEGF in gastric cancer cells in vitro. In vivo, everolimus significantly inhibited tumor growth. This anti-tumor activity was linked to a significant increase in tumor necrotic area (p < 0.02) and trends for decreased proliferation, increased apoptosis, decreased HIF-1alpha and lower tumor MVD (p = n.s.). The combination of everolimus and cyclophosphamide resulted in a striking and highly significant long-term tumor growth control compared to monotherapy (p < 0.001), which was associated with a sharp increase in central tumor necrosis (p < 0.001). In conclusion, the combination of everolimus and metronomic cyclophosphamide showed synergistic anti-tumor activity. Depriving cancer cells by everolimus of factors necessary for their survival under hypoxia induced by anti-angiogenic chemotherapy appears to be a promising approach for treatment of gastric cancer.

mTOR inhibition sensitizes gastric cancer to alkylating chemotherapy in vivo.

BACKGROUND: Gastric cancer is a highly chemoresistant tumor. Previous studies suggest that cancer cells can be sensitized to standard chemotherapy, and especially alkylating agents, by inhibition of mammalian target of rapamycin (mTOR) signaling. The work presented here shows that the mTOR inhibitor everolimus, in combination with cyclophosphamide, exhibits synergistic antitumor activity in gastric cancer xenografts. MATERIALS AND METHODS: Treatment with everolimus at the minimal effective dose was studied in combination with cyclophosphamide at maximum tolerated dose in a human gastric cancer severe combined immunodeficient (SCID) mouse xenograft model. Besides tumor size, biomarker expression for proliferation (Ki-67), hypoxia (HIF-12alpha), apoptosis (activated caspase 3), angiogenesis (microvascular density, MVD) and levels of circulating endothelial progenitors (CEPs) were assessed. RESULTS: Everolimus single agent treatment significantly inhibited tumor growth relative to control and cyclophosphamide treatment (T/C 19%, p<0.01). This antitumor activity was linked to a significant decrease in tumor cell proliferation (p<0.01) and a trend towards lower tumor MVD, HIF-1alpha expression and levels of CEPs. Notably, the combination of everolimus with cyclophosphamide resulted in synergistic anti-tumor activity (T/C 9%, p<0.01). This antitumor activity coincided with a statistically significant decrease in MVD (p<0.01). Whereas treatment with everolimus was well tolerated, cyclophosphamide at maximum tolerated dose (MTD) showed significant toxicity both as monotherapy and in combination with everolimus. CONCLUSION: The antiangiogenic activity of everolimus combined with a high dose of cyclophosphamide shows synergistic antitumor activity against gastric cancer in vivo. In potential future clinical trials, the toxicity of cyclophosphamide in combination regimens with everolimus deserves careful evaluation.