In chronic kidney disease altered cardiac metabolism precedes cardiac hypertrophy.

Conduit arterial disease in chronic kidney disease (CKD) is an important cause of cardiac complications. Cardiac function in CKD has not been studied in the absence of arterial disease. In an Alport syndrome model bred not to have conduit arterial disease, mice at 225 days of life (dol) had CKD equivalent to humans with CKD stage 4-5. Parathyroid hormone (PTH) and FGF23 levels were one log order elevated, circulating sclerostin was elevated, and renal activin A was strongly induced. Aortic Ca levels were not increased, and vascular smooth muscle cell (VSMC) transdifferentiation was absent. The CKD mice were not hypertensive, and cardiac hypertrophy was absent. Freshly excised cardiac tissue respirometry (Oroboros) showed that ADP-stimulated O2 flux was diminished from 52 to 22 pmol/mg (P = 0.022). RNA-Seq of cardiac tissue from CKD mice revealed significantly decreased levels of cardiac mitochondrial oxidative phosphorylation genes. To examine the effect of activin A signaling, some Alport mice were treated with a monoclonal Ab to activin A or an isotype-matched IgG beginning at 75 days of life until euthanasia. Treatment with the activin A antibody (Ab) did not affect cardiac oxidative phosphorylation. However, the activin A antibody was active in the skeleton, disrupting the effect of CKD to stimulate osteoclast number, eroded surfaces, and the stimulation of osteoclast-driven remodeling. The data reported here show that cardiac mitochondrial respiration is impaired in CKD in the absence of conduit arterial disease. This is the first report of the direct effect of CKD on cardiac respiration.NEW & NOTEWORTHY Heart disease is an important morbidity of chronic kidney disease (CKD). Hypertension, vascular stiffness, and vascular calcification all contribute to cardiac pathophysiology. However, cardiac function in CKD devoid of vascular disease has not been studied. Here, in an animal model of human CKD without conduit arterial disease, we analyze cardiac respiration and discover that CKD directly impairs cardiac mitochondrial function by decreasing oxidative phosphorylation. Protection of cardiac oxidative phosphorylation may be a therapeutic target in CKD.

Combined effects of heavy ion exposure and simulated Lunar gravity on skeletal muscle.

BACKGROUND: The limitations to prolonged spaceflight include unloading-induced atrophy of the musculoskeletal system which may be enhanced by exposure to the space radiation environment. Previous results have concluded that partial gravity, comparable to the Lunar surface, may have detrimental effects on skeletal muscle. However, little is known if these outcomes are exacerbated by exposure to low-dose rate, high-energy radiation common to the space environment. Therefore, the present study sought to determine the impact of highly charge, high-energy (HZE) radiation on skeletal muscle when combined with partial weightbearing to simulate Lunar gravity. We hypothesized that partial unloading would compromise skeletal muscle and these effects would be exacerbated by radiation exposure. METHODS: For month old female BALB/cByJ mice were -assigned to one of 2 groups; either full weight bearing (Cage Controls, CC) or partial weight bearing equal to 1/6th bodyweight (G/6). Both groups were then divided to receive either a single whole body absorbed dose of 0.5 Gy of 300 MeV 28Si ions (RAD) or a sham treatment (SHAM). Radiation exposure experiments were performed at the NASA Space Radiation Laboratory (NSRL) located at Brookhaven National Laboratory on Day 0, followed by 21 d of CC or G/6 loading. Muscles of the hind limb were used to measure protein synthesis and other histological measures. RESULTS: Twenty-one days of Lunar gravity (G/6) resulted in lower soleus, plantaris, and gastrocnemius muscle mass. Radiation exposure did not further impact muscle mass. 28Si exposure in normal ambulatory animals (RAD+CC) did not impact gastrocnemius muscle mass when compared to SHAM+CC (p>0.05), but did affect the soleus, where mass was higher following radiation compared to SHAM (p<0.05). Mixed gastrocnemius muscle protein synthesis was lower in both unloading groups. Fiber type composition transitioned towards a faster isoform with partial unloading and was not further impacted by radiation. The combined effects of partial loading and radiation partially mitigated fiber cross-sectional area when compared to partial loading alone. Radiation and G/6 reduced the total number of myonuclei per fiber while leading to elevated BrdU content of skeletal muscle. Similarly, unloading and radiation resulted in higher collagen content of muscle when compared to controls, but the effects of combined exposure were not additive. CONCLUSIONS: The results of this study confirm that partial weightbearing causes muscle atrophy, in part due to reductions of muscle protein synthesis in the soleus and gastrocnemius as well as reduced peripheral nuclei per fiber. Additionally, we present novel data illustrating 28Si exposure reduced nuclei in muscle fibers despite higher satellite cell fusion, but did not exacerbate muscle atrophy, CSA changes, or collagen content. In conclusion, both partial loading and HZE radiation can negatively impact muscle morphology.

FGF-23 and sclerostin in serum and bone of CKD patients.

AIMS: Renal osteodystrophy occurs in the early stages of chronic kidney disease (CKD) and progresses during loss of kidney function. Fibroblast growth factor (FGF)-23 and sclerostin, both produced by osteocytes, are increased in blood of patients with CKD. The aim of this study was to analyze the impact of decline in kidney function on FGF-23 and sclerostin protein expression in bone and to study their relationship with their serum levels and bone histomorphometry. MATERIALS AND METHODS: 108 patients aged 25 - 81 years (mean ± SD: 56 ± 13 years) underwent anterior iliac crest biopsies after double-tetracycline labeling. Eleven patients were CKD-2, 16 were CKD-3, 9 were CKD-4 - 5, and 64 CKD-5D. Patients were on hemodialysis for 49 ± 117 months. 18 age-matched patients without CKD were included as controls. Immunostaining was performed on undecalcified bone sections to quantify FGF-23 and sclerostin expression. Bone sections were also evaluated by histomorphometry for bone turnover, mineralization, and volume. RESULTS: FGF-23 expression in bone correlated positively with CKD stages (p < 0.001) increasing from 5.3- to 7.1-fold starting at CKD-2. No difference in FGF-23 expression was seen between trabecular and cortical bone. Sclerostin expression in bone correlated positively with CKD stages (p < 0.001) with an increase from 3.8- to 5.1-fold starting at CKD-2. This increase was progressive and significantly greater in cortical than cancellous bone. FGF-23 and sclerostin in blood and bone were strongly associated with bone turnover parameters. Expression of FGF-23 in cortical bone correlated positively with activation frequency (Ac.f) and bone formation rate (BFR/BS) (p < 0.05), while sclerostin correlated negatively with Ac.f, BFR/BS, and osteoblast and osteoclast numbers (p < 0.05). FGF-23 trabecular and cortical expressions correlated positively with cortical thickness (p < 0.001). Sclerostin bone expression correlated negatively with parameters of trabecular thickness and osteoid surface (p < 0.05). CONCLUSION: These data show a progressive increase in FGF-23 and sclerostin in blood and bone associated with decrease in kidney function. The observed relationships between bone turnover and sclerostin or FGF-23 should be considered when treatment modalities are developed for management of turnover abnormalities in CKD patients.

Treatment of bone loss in CKD5D: Better survival in patients with non-high bone turnover.

67% of CKD5D patients have low bone mass and present with high (HTO) or non-high (N-HTO) bone turnover. HTO has excessive resorption calling for anti-resorbers, while in N-HTO, anabolic therapy appears preferable. There are no data on this tailored approach. Adult CKD5D patients with dual energy X-ray absorptiometry (DXA) t-scores ≤ -1.0 were enrolled into this 12-month randomized controlled trial and stratified as HTO or N-HTO using values of parathyroid hormone (PTH), PTH-ratio, and TRAP5b. HTO patients were randomized into treatment with alendronate or controls, and N-HTO patients into teriparatide or controls. Clinical, lab, DXA, quantitative computed tomography bone mineral density (QCT BMD), and coronary artery calcifications (CAC) and aorta calcifications (AoC) MSQCT data were obtained at 0 and 12 months. Primary outcome was change (Δ) in BMD by QCT, secondary outcomes were changes in CAC (ΔCAC), in AoC (ΔAoC), and death. There were 80 HTO and 61 N-HTO patients. Median HTO baseline PTH was 664 and N-HTO 183. Bone loss improved in treated N-HTO (5.7 g/cm3 vs. -10.7) but not in HTO (0.2 g/cm3 vs. -3.5) patients. There were no differences in ΔAoC or ΔCAC between treatment groups in either arm. Across all patients in the study, ΔAoC was lower in Blacks than Whites. (3.6 vs. 8.8) The HTO ΔAoC was 5 Hounsfield Units higher than N-HTO. In N-HTO, there were 0 deaths, but 20% in HTO (p = 0.005). N-HTO patients (PTH range 138 - 337 pg/mL) had better survival and less ΔAoC than those with HTO. Teriparatide treatment significantly improved low bone mass in N-HTO patients. Blacks had less ΔAoC regardless of turnover or treatment.

Prevalence of low bone formation in untreated patients with osteoporosis.

BACKGROUND: Osteoporosis treatment usually starts with an antiresorber and switches to an anabolic agent if it fails. It is known that suppressing bone resorption also results in reduced bone formation. In addition, patients with prior treatment with antiresorbers may have reduced response to subsequent anabolic treatment. This study determined the prevalence of low bone formation in untreated osteoporosis patients to identify patients who may not be optimally treated under the current paradigm. METHODS: This is a cross-sectional study of bone samples stored in the Kentucky Bone Registry. Included samples were from adult patients presenting for workup of osteoporosis. Exclusion criteria were other diseases or treatments affecting bone. Patients underwent iliac crest bone biopsies after tetracycline labeling for identification of bone formation. RESULTS: 107 patients met study criteria, 92 White and 5 Black women and 10 White men. Forty percent of patients (43/107) had low bone formation/bone surface (BFR/BS < 0.56 mm3/cm2/yr). Clinical and serum parameters did not differ between formation groups, except for type II diabetes, which was found exclusively in the low formation group. CONCLUSIONS: Starting treatment of osteoporotic patients with an antiresorber in all patients appears not optimal for a significant portion.

Low Turnover Renal Osteodystrophy With Abnormal Bone Quality and Vascular Calcification in Patients With Mild-to-Moderate CKD.

Introduction: Limited information is available on renal osteodystrophy (ROD) and vascular calcification (VC) during early chronic kidney disease (CKD). This study was designed to evaluate ROD and VC in 32 patients with CKD stages II to IV. Methods: Patients underwent dual-energy X-ray absorptiometry (DXA) for assessment of bone mineral density (BMD) and trabecular bone score (TBS), thoracic computed tomography for VC scoring using the Agatston method, and anterior iliac crest bone biopsy for mineralized bone histology, histomorphometry, and Fourier transform infrared spectroscopy (FTIR). Classical and novel bone markers were determined in the blood. Results: Mean estimated glomerular filtration rate (eGFR) was 44 ± 16 ml/min per 1.73 m2. Of the patients, 84% had low bone turnover. In Whites, eGFR correlated negatively with the turnover parameter activation frequency (Ac.f) (r -0.48, P = 0.019) and with parameters of bone formation. Most patients had VC (>80%) which correlated positively with levels of phosphorus, c-terminal fibroblast growth factor-23, and activin. Aortic calcifications (ACs) correlated negatively with bone formation rate (BFR) and Ac.f (rho -0.62, -0.61, P < 0.001). TBS correlated negatively with coronary calcification (rho -0.42, P = 0.019) and AC (rho -0.57, P = 0.001). These relationships remained after adjustment of age. The mineral-to-matrix ratio, an FTIR metric reflecting bone quality, was negatively related to Ac.f and positively related to AC. Conclusion: Low bone turnover and VC are predominant in early stages of CKD. This is the first study demonstrating mineral abnormalities indicating reduced bone quality in these stages of CKD.

Bone Quality and Fractures in Women With Osteoporosis Treated With Bisphosphonates for 1 to 14 Years.

Oral bisphosphonates are the primary medication for osteoporosis, but concerns exist regarding potential bone-quality changes or low-energy fractures. This cross-sectional study used artificial intelligence methods to analyze relationships among bisphosphonate treatment duration, a wide variety of bone-quality parameters, and low-energy fractures. Fourier transform infrared spectroscopy and histomorphometry quantified bone-quality parameters in 67 osteoporotic women treated with oral bisphosphonates for 1 to 14 years. Artificial intelligence methods established two models relating bisphosphonate treatment duration to bone-quality changes and to low-energy clinical fractures. The model relating bisphosphonate treatment duration to bone quality demonstrated optimal performance when treatment durations of 1 to 8 years were separated from treatment durations of 9 to 14 years. This may be due to a change in relationship of bone-quality parameters with treatment duration. This model also showed that the effects of bisphosphonate treatment duration were most highly correlated with changes in means and standard deviations of infrared spectroscopically derived mineral and matrix parameters and histomorphometric bone turnover parameters. A second model related treatment duration to bone fracture in all 22 patients who fractured while on treatment with bisphosphonates for more than 8 years. This second model showed that bisphosphonate treatment duration, not hip bone mineral density (BMD), was the most strongly correlated parameter to these low-energy bone fractures. Application of artificial intelligence enabled analysis of large quantities of structural, cellular, mineral, and matrix bone-quality parameters to determine relationships with long-term oral bisphosphonate treatment and fracture. Infrared spectroscopy provides clinically relevant bone-quality information of which bone mineral purity is among the most relevant. Nine or more years of bisphosphonate treatment was associated with abnormal bone mineral purity, matrix abnormalities, and low-energy fractures. These data justify limiting bisphosphonate treatment duration to 8 years. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Biomarkers of Bone Turnover Identify Subsets of Chronic Kidney Disease Patients at Higher Risk for Fracture.

BACKGROUND: We sought to identify biomarkers that indicate low turnover on bone histomorphometry in chronic kidney disease (CKD) patients, and subsequently determined whether this panel identified differential risk for fractures in community-dwelling older adults. METHODS: Among CKD patients who underwent iliac crest bone biopsies and histomorphometry, we evaluated candidate biomarkers to differentiate low turnover from other bone disease. We applied this biomarker panel to 641 participants in the Health Aging and Body Composition Study (Health ABC) study with estimated glomerular filtration rate (eGFR) less than 60 mL/min/1.73 m2 who were followed for fracture. Cox proportional hazards models evaluated the association of bone mineral density (BMD) with fracture risk and determined whether biomarker-defined low bone turnover modified fracture risk at any level of BMD. RESULTS: In 39 CKD patients age 64 ± 13 years, 85% female, with mean eGFR 37 ± 14 mL/min/1.73 m2 who underwent bone biopsy, lower fibroblast growth factor (FGF)-23, higher ɑ-Klotho, and lower parathyroid hormone (PTH) indicated low bone turnover in accordance with bone histomorphometry parameters (individual area under the curve = 0.62, 0.73, and 0.55 respectively; sensitivity = 22%, specificity = 100%). In Health ABC, 641 participants with CKD were age 75 ± 3 years , 49% female, with mean eGFR 48 ± 10 mL/min/1.73 m2. For every SD lower hip BMD at baseline, there was an 8-fold higher fracture risk in individuals with biomarker-defined low turnover (hazard ratio 8.10 [95% CI, 3.40-19.30]) vs a 2-fold higher risk in the remaining individuals (hazard ratio 2.28 [95% CI, 1.69-3.08]) (Pinteraction = .082). CONCLUSIONS: In CKD patients who underwent bone biopsy, lower FGF-23, higher ɑ-Klotho, and lower PTH together had high specificity for identifying low bone turnover. When applied to older individuals with CKD, BMD was more strongly associated with fracture risk in those with biomarker-defined low turnover.

Kidney recovery in patients discharged to an acute rehabilitation facility with acute kidney injury requiring hemodialysis
.

BACKGROUND: Patients with acute kidney injury requiring renal replacement therapy (AKI-RRT) are at risk of adverse outcomes. Little is known about the incidence of AKI-RRT recovery following hospital discharge. We examine AKI-RRT recovery in hospital survivors discharged to a long-term acute care hospital (LTACH) with need of hemodialysis (HD) for AKI. MATERIALS AND METHODS: Single-center, retrospective cohort study of patients who were hospitalized (08/2015 - 04/2018), suffered from AKI-RRT, and were discharged to an affiliated LTACH with need for HD. Kidney recovery was defined as the patient being alive and no longer requiring HD. RESULTS: 41 patients were included. Mean (SD) age was 61.3 (9.7) years, 63.4% were male, and 90.2% white. At the time of discharge from LTACH, 27 (65.8%) patients had survived and had recovered kidney function (kidney recovery group), 7 had been discharged on HD, and 7 had died (no kidney recovery group, n = 14, 34.2%). In adjusted models, the presence of anemia was associated with a 91% decreased odds of kidney recovery at LTACH discharge. Each additional HD session during LTACH stay had an 18% decreased odds of kidney recovery at LTACH discharge, and each episode of intradialytic hypotension had a 20% decreased odds of kidney recovery at the end of the observation period (median follow-up of 19.0 months). CONCLUSION: Almost 2/3 of AKI-RRT patients discharged to an affiliated LTACH with ongoing HD need recovered kidney function. Anemia and the number of HD sessions and intradialytic hypotension episodes were associated with kidney recovery. Future studies should focus on developing risk-stratification tools for kidney recovery and determining best practices to promote recovery in this susceptible population.

Serum bone markers in ROD patients across the spectrum of decreases in GFR: Activin A increases before all other markers
.

INTRODUCTION: Renal osteodystrophy (ROD) develops early in chronic kidney disease (CKD) and progresses with loss of kidney function. While intact parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3 (1,25D), and fibroblast growth factor-23 (FGF-23) levels are usually considered the primary abnormalities in ROD development, the role of serum activin A elevations in CKD and its relationships to ROD have not been explored. The aims of this study were to evaluate serum activin A at different CKD stages, and to establish the relationships between activin A, bone biomarkers, and bone histomorphometric parameters. MATERIALS AND METHODS: 104 patients with CKD stages 2 - 5D underwent bone biopsies. We measured in the serum activin A, BSAP, DKK1, FGF-23, α-Klotho, intact PTH, sclerostin, TRAP-5b, and 1,25D. Biochemical results were compared across CKD stages and with 19 age-matched controls with normal kidney function. RESULTS: Median activin A levels were increased in all stages of CKD compared to controls from 544 pg/mL in CKD 2 (431 - 628) to 1,135 pg/mL in CKD 5D (816 - 1,456), compared to 369 pg/mL in controls (316 - 453, p < 0.01). The increase of activin A in CKD 2 (p = 0.016) occurred before changes in the other measured biomarkers. Activin A correlated with intact PTH and FGF-23 (r = 0.65 and 0.61; p < 0.01) and with histomorphometric parameters of bone turnover (BFR/BS, Acf, ObS/BS and OcS/BS; r = 0.47 - 0.52; p < 0.01). These correlations were comparable to those found with intact PTH and FGF-23. CONCLUSION: Serum activin A levels increase starting at CKD 2 before elevations in intact PTH and FGF-23. Activin A correlates with bone turnover similar to intact PTH and FGF-23. These findings suggest a role for activin A in early development of ROD.

Value of Intraoperative Parathyroid Hormone Assay during Parathyroidectomy in Dialysis and Renal Transplant Patients with Secondary and Tertiary Hyperparathyroidism.

BACKGROUND: In dialysis and renal transplant patients with secondary and tertiary hyperparathyroidism (HPT), the value of intraoperative parathyroid hormone (ioPTH) during parathyroidectomy (PTX) and its association with long-term PTH levels are unknown. The present study aims at evaluating the relationship of ioPTH with long-term PTH levels post-PTX in dialysis and renal transplant patients in a single-center study. METHODS: The ioPTH was measured in 57 dialysis patients (33 females and 24 males) and 18 renal transplant recipients (12 males and 6 females) who underwent PTX from 2005 to 2015 for refractory HPT. Near-total PTX was performed in 56 patients and total PTX with autotransplantation in 20 patients. The PTH monitoring included 3 samples: pre-intubation, 10- and 20-min (pre-ioPTH, 10-ioPTH, and 20-ioPTH) post parathyroid gland excision. Patients were followed up for up to 5 years. RESULTS: In the dialysis group, the median (25th-75th percentile) pre-, 10-, and 20-ioPTH levels were 1,447 pg/mL (938-2,176), 143 pg/mL (78-244) and 112 pg/mL (59-153) respectively. In the renal transplant group, pre-, 10-, and 20-ioPTH levels were 273 pg/mL (180-403), 42 pg/mL (25-72), and 34 pg/mL (23-45) respectively. All patients in the transplant group had a functional kidney transplant at the time of PTX with a median serum creatinine of 1.3 mg/dL (1.2-1.7) and estimated glomerular filtration rate of 55 mL/min (40-60). The median time between renal transplant and PTX surgeries was 22 months (7-81). The last median follow-up PTH level was 66 pg/mL (15-201) in the dialysis group and 54 pg/mL (17-72) in the transplant group (p = 0.438). The mean time for last PTH post-PTX was 2.3 ± 2.0 years. In both groups, there was no significant difference between 20-ioPTH and any-time post-PTX PTH levels (p = 0.6 and p = 0.9). Nineteen patients (25%) were readmitted within 90 days because of hypocalcemia. One patient in the dialysis group was readmitted for post-PTX hematoma evacuation. No patient required repeat PTX because of recurrent HPT that was refractory to medical therapy. Only one dialysis patient required repeat PTX because the first procedure failed. CONCLUSIONS: The 20-ioPTH is a good indicator of long-term PTH levels in dialysis and renal transplant patients. Hypocalcemia is a common complication, particularly in dialysis patients, and it is the main reason for readmission after PTX. Hypoparathyroidism is a potential concern after PTX in dialysis patients.

Exposure to Low-Dose X-Ray Radiation Alters Bone Progenitor Cells and Bone Microarchitecture.

Exposure to high-dose ionizing radiation during medical treatment exerts well-documented deleterious effects on bone health, reducing bone density and contributing to bone growth retardation in young patients and spontaneous fracture in postmenopausal women. However, the majority of human radiation exposures occur in a much lower dose range than that used in the radiation oncology clinic. Furthermore, very few studies have examined the effects of low-dose ionizing radiation on bone integrity and results have been inconsistent. In this study, mice were irradiated with a total-body dose of 0.17, 0.5 or 1 Gy to quantify the early (day 3 postirradiation) and delayed (day 21 postirradiation) effects of radiation on bone microarchitecture and bone marrow stromal cells (BMSCs). Female BALBc mice (4 months old) were divided into four groups: irradiated (0.17, 0.5 and 1 Gy) and sham-irradiated controls (0 Gy). Micro-computed tomography analysis of distal femur trabecular bone from animals at day 21 after exposure to 1 Gy of X-ray radiation revealed a 21% smaller bone volume (BV/TV), 22% decrease in trabecular numbers (Tb.N) and 9% greater trabecular separation (Tb.Sp) compared to sham-irradiated controls (P < 0.05). We evaluated the differentiation capacity of bone marrow stromal cells harvested at days 3 and 21 postirradiation into osteoblast and adipocyte cells. Osteoblast and adipocyte differentiation was decreased when cells were harvested at day 3 postirradiation but enhanced in cells isolated at day 21 postirradiation, suggesting a compensatory recovery process. Osteoclast differentiation was increased in 1 Gy irradiated BMSCs harvested at day 3 postirradiation, but not in those harvested at day 21 postirradiation, compared to controls. This study provides evidence of an early, radiation-induced decrease in osteoblast activity and numbers, as well as a later recovery effect after exposure to 1 Gy of X-rays, whereas osteoclastogenesis was enhanced. A better understanding of the effects of radiation on osteoprogenitor cell populations could lead to more effective therapeutic interventions that protect bone integrity for individuals exposed to low-dose ionizing radiation.

Space Environmental Factor Impacts upon Murine Colon Microbiota and Mucosal Homeostasis.

Astronaut intestinal health may be impacted by microgravity, radiation, and diet. The aim of this study was to characterize how high and low linear energy transfer (LET) radiation, microgravity, and elevated dietary iron affect colon microbiota (determined by 16S rDNA pyrosequencing) and colon function. Three independent experiments were conducted to achieve these goals: 1) fractionated low LET γ radiation (137Cs, 3 Gy, RAD), high Fe diet (IRON) (650 mg/kg diet), and a combination of low LET γ radiation and high Fe diet (IRON+RAD) in male Sprague-Dawley rats; 2) high LET 38Si particle exposure (0.050 Gy), 1/6 G partial weight bearing (PWB), and a combination of high LET38Si particle exposure and PWB in female BalbC/ByJ mice; and 3) 13 d spaceflight in female C57BL/6 mice. Low LET radiation, IRON and spaceflight increased Bacteroidetes and decreased Firmicutes. RAD and IRON+RAD increased Lactobacillales and lowered Clostridiales compared to the control (CON) and IRON treatments. Low LET radiation, IRON, and spaceflight did not significantly affect diversity or richness, or elevate pathogenic genera. Spaceflight increased Clostridiales and decreased Lactobacillales, and similar trends were observed in the experiment using a ground-based model of microgravity, suggesting altered gravity may affect colonic microbiota. Although we noted no differences in colon epithelial injury or inflammation, spaceflight elevated TGFß gene expression. Microbiota and mucosal characterization in these models is a first step in understanding the impact of the space environment on intestinal health.

Wnt16 attenuates TGFß-induced chondrogenic transformation in vascular smooth muscle.

OBJECTIVE: Phenotypic plasticity of vascular smooth muscle cells (VSMCs) contributes to cardiovascular disease. Chondrocyte-like transformation of VSMCs associates with vascular calcification and underlies the formation of aortic cartilaginous metaplasia induced in mice by genetic loss of matrix Gla protein (MGP). Previous microarray analysis identified a dramatic downregulation of Wnt16 in calcified MGP-null aortae, suggesting an antagonistic role for Wnt16 in the chondrogenic transformation of VSMCs. APPROACH AND RESULTS: Wnt16 is significantly downregulated in MGP-null aortae, before the histological appearance of cartilaginous metaplasia, and in primary MGP-null VSMCs. In contrast, intrinsic TGFß is activated in MGP-null VSMCs and is necessary for spontaneous chondrogenesis of these cells in high-density micromass cultures. TGFß3-induced chondrogenic transformation in wild-type VSMCs associates with Smad2/3-dependent Wnt16 downregulation, but Wnt16 does not suppress TGFß3-induced Smad activation. In addition, TGFß3 inhibits Notch signaling in wild-type VSMCs, and this pathway is downregulated in MGP-null aortae. Exogenous Wnt16 stimulates Notch activity and attenuates TGFß3-induced downregulation of Notch in wild-type VSMCs, prevents chondrogenesis in MGP-null and TGFß3-treated wild-type VSMCs, and stabilizes expression of contractile markers of differentiated VSMCs. CONCLUSIONS: We describe a novel TGFß-Wnt16-Notch signaling conduit in the chondrocyte-like transformation of VSMCs and identify endogenous TGFß activity in MGP-null VSMCs as a critical mediator of chondrogenesis. Our proposed model suggests that the activated TGFß pathway inhibits expression of Wnt16, which is a positive regulator of Notch signaling and a stabilizer of VSMC phenotype. These data advance the comprehensive mechanistic understanding of VSMC transformation and may identify a novel potential therapeutic target in vascular calcification.

FGF-23 serum levels and bone histomorphometric results in adult patients with chronic kidney disease on dialysis.

BACKGROUND: Fibroblast growth factor-23 (FGF-23) is a hormone principally produced by osteocytes/osteoblasts. In patients with chronic kidney disease (CKD), FGF-23 levels are usually elevated and can reach up to 300 - 400 times the normal range. FGF-23 is regulated by local bone-related and systemic factors, but the relationship between circulating FGF-23 concentrations and bone remodeling and mineralization in CKD has not been well characterized. In the current study, we examined the relationship between FGF-23 levels and bone histomorphometry parameters in adult patients with renal osteodystrophy. MATERIAL AND METHODS: 36 patients on dialysis (CKD-5D) underwent bone biopsies after tetracycline double labeling. Blood drawings were done at time of biopsy to determine serum levels of markers of bone and mineral metabolism. RESULTS: Patients with high bone turnover had higher values of serum FGF-23 than patients with low bone turnover. FGF-23 levels correlated with activation frequency (ρ = 0.60, p < 0.01) and bone formation rate (ρ = 0.57, p < 0.01). Normal mineralization was observed in 90% of patients with FGF-23 levels above 2,000 pg/mL. Furthermore, FGF-23 correlated negatively with mineralization lag time (ρ = -0.69, p < 0.01) and osteoid maturation time (ρ = -0.46, p < 0.05) but not with osteoid thickness (ρ = 0.08, ns). Regression analysis showed that FGF-23 was the only independent predictor of mineralization lag time. FGF-23 correlated with cancellous bone volume (ρ = 0.38, p < 0.05) but did not predict it. CONCLUSION: Circulating FGF-23 concentrations may reflect alterations in ongoing bone formation along with active mineralization, but not exclusively in bone formation or mineralization. Abnormal mineralization lag time (> 100 days) was mainly seen in patients with FGF-23 levels less than 2,000 pg/mL, while very high levels of FGF-23 are associated with normal mineralization lag time.

High LET (56)Fe ion irradiation induces tissue-specific changes in DNA methylation in the mouse.

DNA methylation is an epigenetic mechanism that drives phenotype and that can be altered by environmental exposures including radiation. The majority of human radiation exposures occur in a relatively low dose range; however, the biological response to low dose radiation is poorly understood. Based on previous observations, we hypothesized that in vivo changes in DNA methylation would be observed in mice following exposure to doses of high linear energy transfer (LET) (56) Fe ion radiation between 10 and 100 cGy. We evaluated the DNA methylation status of genes for which expression can be regulated by methylation and that play significant roles in radiation responses or carcinogenic processes including apoptosis, metastasis, cell cycle regulation, and DNA repair (DAPK1, EVL, 14.3.3, p16, MGMT, and IGFBP3). We also evaluated DNA methylation of repeat elements in the genome that are typically highly methylated. No changes in liver DNA methylation were observed. Although no change in DNA methylation was observed for the repeat elements in the lungs of these same mice, significant changes were observed for the genes of interest as a direct effect and a delayed effect of irradiation 1, 7, 30, and 120 days post exposure. At delayed times, differences in methylation profiles among genes were observed. DNA methylation profiles also significantly differed based on dose, with the lowest dose frequently affecting the largest change. The results of this study are the first to demonstrate in vivo high LET radiation-induced changes in DNA methylation that are tissue and locus specific, and dose and time dependent.

Attenuation of chondrogenic transformation in vascular smooth muscle by dietary quercetin in the MGP-deficient mouse model.

RATIONALE: Cartilaginous metaplasia of vascular smooth muscle (VSM) is characteristic for arterial calcification in diabetes and uremia and in the background of genetic alterations in matrix Gla protein (MGP). A better understanding of the molecular details of this process is critical for the development of novel therapeutic approaches to VSM transformation and arterial calcification. OBJECTIVE: This study aimed to identify the effects of bioflavonoid quercetin on chondrogenic transformation and calcification of VSM in the MGP-null mouse model and upon TGF-ß3 stimulation in vitro, and to characterize the associated alterations in cell signaling. METHODS AND RESULTS: Molecular analysis revealed activation of ß-catenin signaling in cartilaginous metaplasia in Mgp-/- aortae in vivo and during chondrogenic transformation of VSMCs in vitro. Quercetin intercepted chondrogenic transformation of VSM and blocked activation of ß-catenin both in vivo and in vitro. Although dietary quercetin drastically attenuated calcifying cartilaginous metaplasia in Mgp-/- animals, approximately one-half of total vascular calcium mineral remained as depositions along elastic lamellae. CONCLUSION: Quercetin is potent in preventing VSM chondrogenic transformation caused by diverse stimuli. Combined with the demonstrated efficiency of dietary quercetin in preventing ectopic chondrogenesis in the MGP-null vasculature, these findings indicate a potentially broad therapeutic applicability of this safe for human consumption bioflavonoid in the therapy of cardiovascular conditions linked to cartilaginous metaplasia of VSM. Elastocalcinosis is a major component of MGP-null vascular disease and is controlled by a mechanism different from chondrogenic transformation of VSM and not sensitive to quercetin.

Two sides of MGP null arterial disease: chondrogenic lesions dependent on transglutaminase 2 and elastin fragmentation associated with induction of adipsin.

Mutations in matrix Gla protein (MGP) have been correlated with vascular calcification. In the mouse model, MGP null vascular disease presents as calcifying cartilaginous lesions and mineral deposition along elastin lamellae (elastocalcinosis). Here we examined the mechanisms underlying both of these manifestations. Genetic ablation of enzyme transglutaminase 2 (TG2) in Mgp(-/-) mice dramatically reduced the size of cartilaginous lesions in the aortic media, attenuated calcium accrual more than 2-fold, and doubled longevity as compared with control Mgp(-/-) animals. Nonetheless, the Mgp(-/-);Tgm2(-/-) mice still died prematurely as compared with wild-type and retained the elastocalcinosis phenotype. This pathology in Mgp(-/-) animals was developmentally preceded by extensive fragmentation of elastic lamellae and associated with elevated serine elastase activity in aortic tissue and vascular smooth muscle cells. Systematic gene expression analysis followed by an immunoprecipitation study identified adipsin as the major elastase that is induced in the Mgp(-/-) vascular smooth muscle even in the TG2 null background. These results reveal a central role for TG2 in chondrogenic transformation of vascular smooth muscle and implicate adipsin in elastin fragmentation and ensuing elastocalcinosis. The importance of elastin calcification in MGP null vascular disease is highlighted by significant residual vascular calcification and mortality in Mgp(-/-);Tgm2(-/-) mice with reduced cartilaginous lesions. Our studies identify two potential therapeutic targets in vascular calcification associated with MGP dysfunction and emphasize the need for a comprehensive approach to this multifaceted disorder.