Effects of ferric citrate and intravenous iron sucrose on markers of mineral, bone, and iron homeostasis in a rat model of CKD-MBD.

BACKGROUND: Anemia and chronic kidney disease-mineral and bone disorder (CKD-MBD) are common and begin early in CKD. Limited studies have concurrently compared the effects of ferric citrate (FC) versus intravenous (IV) iron on CKD-MBD and iron homeostasis in moderate CKD. METHODS: We tested the effects of 10 weeks of 2% FC versus IV iron sucrose in rats with moderate CKD (Cy/+ male rat) and untreated normal (NL) littermates. Outcomes included a comprehensive assessment of CKD-MBD, iron homeostasis and oxidative stress. RESULTS: CKD rats had azotemia, elevated phosphorus, parathyroid hormone and fibroblast growth factor-23 (FGF23). Compared with untreated CKD rats, treatment with FC led to lower plasma phosphorus, intact FGF23 and a trend (P = 0.07) toward lower C-terminal FGF23. FC and IV iron equally reduced aorta and heart calcifications to levels similar to NL animals. Compared with NL animals, CKD animals had higher bone turnover, lower trabecular volume and no difference in mineralization; these were unaffected by either iron treatment. Rats treated with IV iron had cortical and bone mechanical properties similar to NL animals. FC increased the transferrin saturation rate compared with untreated CKD and NL rats. Neither iron treatment increased oxidative stress above that of untreated CKD. CONCLUSIONS: Oral FC improved phosphorus homeostasis, some iron-related parameters and the production and cleavage of FGF23. The intermittent effect of low-dose IV iron sucrose on cardiovascular calcification and bone should be further explored in moderate-advanced CKD.

Predicting fracture healing with blood biomarkers: the potential to assess patient risk of fracture nonunion.

Fracture non-union is a significant orthopaedic problem affecting a substantial number of patients yearly. Treatment of nonunions is devastating to patients and costly to the healthcare system. Unfortunately, the diagnosis of non-union is typically made in a reactionary fashion by an orthopaedic surgeon based on clinical assessment and radiographic features several months into treatment. For this reason, investigators have been trying to develop prediction algorithms; however, these have relied on population-based approaches and lack the predictive capability necessary to make individual treatment decisions. There is also a growing body of literature focussed on identifying blood biomarkers that are associated with non-union. This review describes the research that has been done in this area. Further studies of patient-centered, precision medicine approaches will likely improve fracture non-union diagnostic/prognostic capabilities.

Regulation of reactive oxygen species in the pathogenesis of matrix vesicles induced calcification of recipient vascular smooth muscle cells.

INTRODUCTION: Increased oxidative stress is associated with vascular calcification in patients with chronic kidney disease (CKD). We have previously demonstrated that cellular-derived matrix vesicles (MV), but not media-derived MV, are endocytosed in the presence of phosphorus by recipient normal rat vascular smooth muscle cells (VSMC) and induce calcification through ERK1/2 and [Ca2+]i signaling. We hypothesized that these changes were mediated by increased reactive oxygen species (ROS) production. METHODS: MV were co-cultured with recipient VSMC in the presence of high phosphorus and ROS production and cell signaling assessed. RESULTS: The results demonstrated MV endocytosis led to increased ROS production in recipient VSMC with no increase in mitochondrial oxygen consumption or oxidative phosphorylation (OXPHOS), indicating the ROS was not from the mitochondria. The use of inhibitors demonstrated that endocytosis of these MV by VSMC led to a signaling cascade in the cytoplasm beginning with ERK1/2 signaling, then increased [Ca2+]i and stimulation of ROS production, mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)1/4. Media-derived MV did not induce this cascade, indicating endocytosis itself was not a factor. Furthermore, inhibition of either ERK1/2 activation or [Ca2+]i reduced vascular calcification. CONCLUSION: We conclude that endocytosis of pro-mineralizing MV can induce a series of signaling events in normal VSMC that culminate in generation of ROS via activation of NOX1/4. Understanding these pathways will allow the development of future targeted therapeutics.

Adverse Effects of Autoclaved Diets on the Progression of Chronic Kidney Disease and Chronic Kidney Disease-Mineral Bone Disorder in Rats.

BACKGROUND: Autoclaving rodent diets is common in laboratory animals, but autoclaving increases the formation of dietary advanced glycation end-products (AGE). We studied the effect of autoclaved (AC) diet alone or in combination with a diet high in bioavailable phosphorus on biochemistries of chronic kidney disease-mineral and bone disorder (CKD-MBD), intestinal gene expression, and oxidative stress. METHODS: Male CKD rats (Cy/+) and normal littermates were fed 1 of 3 diets: AC 0.7% phosphorus grain-based diet for 28 weeks (AC); AC diet for 17 weeks followed by non-autoclaved (Non-AC) 0.7% phosphorus casein diet until 28 weeks (AC + Casein); or Non-AC diet for 16 weeks followed by a Non-AC purified diet until 30 weeks (Non-AC + Casein). RESULTS: AC diets contained ~3× higher AGEs and levels varied depending on the location within the autoclave. Rats fed the AC and AC + Casein diets had higher total AGEs and oxidative stress, irrespective of kidney function. Kidney function was more severely compromised in CKD rats fed AC or AC + Casein compared to Non-AC + Casein. There was a disease-by-diet interaction for plasma phosphorus, parathyroid hormone, and c-terminal fibroblast growth factor-23, driven by high values in the CKD rats fed the AC + Casein diet. Compared to Non-AC + Casein, AC and AC + Casein-fed groups had increased expression of receptor of AGEs and intestinal NADPH oxidase dual oxidase-2, independent of kidney function. CONCLUSIONS: Autoclaving rodent diets impacts the progression of CKD and CKD-MBD, highlighting the critical importance of standardizing diets in experiments.

Phosphate Binders and Nonphosphate Effects in the Gastrointestinal Tract.

Phosphate binders are commonly prescribed in patients with end-stage kidney disease to prevent and treat hyperphosphatemia. These binders are usually associated with gastrointestinal distress, may bind molecules other than phosphate, and may alter the gut microbiota, altogether having systemic effects unrelated to phosphate control. Sevelamer is the most studied of the available binders for nonphosphate-related effects including binding to bile acids, endotoxins, gut microbiota-derived metabolites, and advanced glycation end products. Other binders (calcium- and noncalcium-based binders) may bind vitamins, such as vitamin K and folic acid. Moreover, the relatively new iron-based phosphate binders may alter the gut microbiota, as some of the iron or organic ligands may be used by the gastrointestinal bacteria. The objective of this narrative review is to provide the current evidence for the nonphosphate effects of phosphate binders on gastrointestinal function, nutrient and molecule binding, and the gut microbiome.

Voluntary Wheel Running Has Beneficial Effects in a Rat Model of CKD-Mineral Bone Disorder (CKD-MBD).

BACKGROUND: Reduced bone and muscle health in individuals with CKD contributes to their higher rates of morbidity and mortality. METHODS: We tested the hypothesis that voluntary wheel running would improve musculoskeletal health in a CKD rat model. Rats with spontaneous progressive cystic kidney disease (Cy/+ IU) and normal littermates (NL) were given access to a voluntary running wheel or standard cage conditions for 10 weeks starting at 25 weeks of age when the rats with kidney disease had reached stage 2-3 of CKD. We then measured the effects of wheel running on serum biochemistry, tissue weight, voluntary grip strength, maximal aerobic capacity (VO2max), body composition and bone micro-CT and mechanics. RESULTS: Wheel running improved serum biochemistry with decreased creatinine, phosphorous, and parathyroid hormone in the rats with CKD. It improved muscle strength, increased time-to-fatigue (for VO2max), reduced cortical porosity and improved bone microarchitecture. The CKD rats with voluntary wheel access also had reduced kidney cystic weight and reduced left ventricular mass index. CONCLUSIONS: Voluntary wheel running resulted in multiple beneficial systemic effects in rats with CKD and improved their physical function. Studies examining exercise interventions in patients with CKD are warranted.

Fibroblast growth factor 23 does not directly influence skeletal muscle cell proliferation and differentiation or ex vivo muscle contractility.

Skeletal muscle dysfunction accompanies the clinical disorders of chronic kidney disease (CKD) and hereditary hypophosphatemic rickets. In both disorders, fibroblast growth factor 23 (FGF23), a bone-derived hormone regulating phosphate and vitamin D metabolism, becomes chronically elevated. FGF23 has been shown to play a direct role in cardiac muscle dysfunction; however, it is unknown whether FGF23 signaling can also directly induce skeletal muscle dysfunction. We found expression of potential FGF23 receptors ( Fgfr1-4) and α-Klotho in muscles of two animal models (CD-1 and Cy/+ rat, a naturally occurring rat model of chronic kidney disease-mineral bone disorder) as well as C2C12 myoblasts and myotubes. C2C12 proliferation, myogenic gene expression, oxidative stress marker 8-OHdG, intracellular Ca2+ ([Ca2+]i), and ex vivo contractility of extensor digitorum longus (EDL) or soleus muscles were assessed after treatment with various amounts of FGF23. FGF23 (2-100 ng/ml) did not alter C2C12 proliferation, expression of myogenic genes, or oxidative stress after 24- to 72-h treatment. Acute or prolonged FGF23 treatment up to 6 days did not alter C2C12 [Ca2+]i handling, nor did acute treatment with FGF23 (9-100 ng/ml) affect EDL and soleus muscle contractility. In conclusion, although skeletal muscles express the receptors involved in FGF23-mediated signaling, in vitro FGF23 treatments failed to directly alter skeletal muscle development or function under the conditions tested. We hypothesize that other endogenous substances may be required to act in concert with FGF23 or apart from FGF23 to promote muscle dysfunction in hereditary hypophosphatemic rickets and CKD.

Matrix vesicles induce calcification of recipient vascular smooth muscle cells through multiple signaling pathways.

In patients with chronic kidney and end-stage renal diseases, the major risk factor for progression of arterial calcification is the presence of existing (baseline) calcification. Here, we tested whether calcification of arteries is extended from calcified vascular smooth muscle cells (VSMCs) to adjacent normal cells by matrix vesicle-induced alteration of cell signaling. Matrix vesicles isolated from VSMC of rats with chronic kidney disease were co-cultured with VSMCs from normal littermates. Endocytosis of vesicles by recipient cells was confirmed by confocal microscopy. The addition of cellular matrix vesicles with characteristics of exosomes and low fetuin-A content enhanced the calcification of recipient VSMC. Further, only cellular-derived matrix vesicles induced an increase in intracellular calcium ion concentration, NOX1 (NADPH oxidase) and the anti-oxidant superoxide dismutase-2 in recipient normal VSMC. The increase in intracellular calcium ion concentration was due to release from endoplasmic reticulum and partially attributed to the activation of both NOX1 and mitogen-activated protein kinase (MEK1 and Erk1/2) signaling, since inhibiting both pathways blocked the increase in intracellular calcium ion in recipient VSMC. In contrast, matrix vesicles isolated from the media had no effect on the intracellular calcium ion concentration or MEK1 signaling, and did not induce calcification. However, media matrix vesicles did increase Erk1/2, although not to the level of cellular matrix vesicles, and NOX1 expression. Blockade of NOX activity further inhibited the cellular matrix vesicle-induced accelerated calcification of recipient VSMC, suggesting a potential therapeutic role of such inhibition. Thus, addition of cellular-derived matrix vesicles from calcifying VSMC can accelerate calcification by inducing cell signaling changes and phenotypic alteration of recipient VSMC.

Intracellular calcium increases in vascular smooth muscle cells with progression of chronic kidney disease in a rat model.

Background: Vascular smooth muscle cells (VSMCs) exhibit phenotypic plasticity, promoting vascular calcification and increasing cardiovascular risk. Changes in VSMC intracellular calcium ([Ca 2+ ] i ) are a major determinant of plasticity, but little is known about changes in [Ca 2+ ] i in chronic kidney disease (CKD). We have previously demonstrated such plasticity in aortas from our rat model of CKD and therefore sought to examine changes in [Ca 2+ ] i during CKD progression. Materials and Methods: We examined freshly isolated VSMCs from aortas of normal rats, Cy/+ rats (CKD) with early and advanced CKD, and advanced CKD rats treated without and with 3% calcium gluconate (CKD + Ca 2+ ) to lower parathyroid hormone (PTH) levels. [Ca 2+ ] i was measured with fura-2. Results: Cy/+ rats developed progressive CKD, as assessed by plasma levels of blood urea nitrogen, calcium, phosphorus, parathyroid hormone and fibroblast growth factor 23. VSMCs isolated from rats with CKD demonstrated biphasic alterations in resting [Ca 2+ ] i : VSMCs from rats with early CKD exhibited reduced resting [Ca 2+ ] i , while VSMCs from rats with advanced CKD exhibited elevated resting [Ca 2+ ] i . Caffeine-induced sarcoplasmic reticulum (SR) Ca 2+ store release was modestly increased in early CKD and was more drastically increased in advanced CKD. The advanced CKD elevation in SR Ca 2+ store release was associated with a significant increase in the activity of the sarco-endoplasmic reticulum Ca 2+ ATPase (SERCA); however, SERCA2a protein expression was decreased in advanced CKD. Following SR Ca 2+ store release, recovery of [Ca 2+ ] i in the presence of caffeine and extracellular Ca 2+ was attenuated in VSMCs from rats with advanced CKD. This impairment, together with reductions in expression of the Na + /Ca 2+ exchanger, suggest a reduction in Ca 2+ extrusion capability. Finally, store-operated Ca 2+ entry (SOCE) was assessed following SR Ca 2+ store depletion. Ca 2+ entry during recovery from caffeine-induced SR Ca 2+ store release was elevated in advanced CKD, suggesting a role for exacerbated SOCE with progressing CKD. Conclusions: With progressive CKD in the Cy/+ rat there is increased resting [Ca 2+ ] i in VSMCs due, in part, to increased SOCE and impaired calcium extrusion from the cell. Such changes may predispose VSMCs to phenotypic changes that are a prerequisite to calcification.

Calcitriol Suppression of Parathyroid Hormone Fails to Improve Skeletal Properties in an Animal Model of Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) leads to complex metabolic changes and an increased risk of fracture. Currently, calcitriol is the standard of care as it effectively suppresses parathyroid hormone (PTH) levels in CKD patients. While calcitriol and its analogs improve BMD and reduce fractures in the general population, the extension of these benefits to patients with advanced kidney disease is unclear. Here, the impact of calcitriol on the skeleton was examined in the setting of reduction in PTH. METHODS: Male Cy/+ rats, a PKD-like CKD model, were treated with either vehicle or calcitriol for 5 weeks. Their normal littermates served as controls. Animals were assessed for changes in mineral metabolism and skeletal parameters (microCT, histology, whole bone mechanics and bone quality). RESULTS: PTH levels were significantly higher (12-fold) in animals with CKD compared to normal controls. CKD animals also exhibited negative changes in bone structural and mechanical properties. Calcitriol treatment resulted in a 60% suppression of PTH levels in animals with CKD. Despite these changes, it had no impact on bone volume (cortical or cancellous), bone turnover, osteoclast number or whole bone mechanical properties. CONCLUSIONS: These data indicate that while calcitriol effectively lowered PTH in rats with CKD, it did little to prevent the negative effects of secondary hyperparathyroidism on the skeleton.

Reduced skeletal muscle function is associated with decreased fiber cross-sectional area in the Cy/+ rat model of progressive kidney disease.

BACKGROUND: The combination of skeletal muscle wasting and compromised function plays a role in the health decline commonly observed in chronic kidney disease (CKD) patients, but the pathophysiology of muscle mass/strength changes remains unclear. The purpose of this study was to characterize muscle properties in the Cy/+ rat model of spontaneously progressive CKD. METHODS: Leg muscle function and serum biochemistry of male Cy/+ (CKD) rats and their nonaffected littermates (NLs) were assessed in vivo at 25, 30 and 35 weeks of age. Architecture and histology of extensor digitorum longus (EDL) and soleus (SOL) muscles were assessed ex vivo at the conclusion of the experiment. We tested the hypothesis that animals with CKD have progressive loss of muscle function, and that this functional deficit is associated with loss of muscle mass and quality. RESULTS: Thirty-five-week-old CKD rats produced significantly lower maximum torque in ankle dorsiflexion and shorter time to maximum torque, and longer half relaxation time in dorsiflexion and plantarflexion compared with NL rats. Peak dorsiflexion torque (but not plantarflexion torque) in CKD remained steady from 25 to 35 weeks, while in NL rats, peak torque increased. Mass, physiologic cross-sectional area (CSA) and fiber-type (myosin heavy chain isoform) proportions of EDL and SOL were not different between CKD and NL. However, the EDL of CKD rats showed reduced CSAs in all fiber types, while only MyHC-1 fibers were decreased in area in the SOL. CONCLUSIONS: The results of this study demonstrate that muscle function progressively declines in the Cy/+ rat model of CKD. Because whole muscle mass and architecture do not vary between CKD and NL, but CKD muscles show reduction in individual fiber CSA, our data suggest that the functional decline is related to increased muscle fiber atrophy.

Low Bone Turnover in Chronic Kidney Disease Is Associated with Decreased VEGF-A Expression and Osteoblast Differentiation.

BACKGROUND: Low turnover bone (low bone formation rates (BFRs)) with decreased osteoblast number is common in patients with chronic kidney disease (CKD) and attributed to 'over-suppression' of the parathyroid hormone (PTH) despite supra-physiologic levels. An alternative hypothesis is abnormal osteoblast differentiation, resulting in low BFRs due to reduced VEGF-A. METHODS: We analyzed the expression of VEGF-A and mesenchymal stem cell (MSC) differentiation factors in freshly isolated bone marrow (BM) cells, and in BM cell-derived MSC in rats with different levels of BFRs and PTH (modulated by calcium and zoledronic acid). The regulators of VEGF in MSC were also determined. RESULTS: VEGF-A expression was reduced in the BM cells from CKD vs. normal animals (p < 0.02). In BM-derived MSC from CKD, there were decreased osteoblast transcription factors and mineralization. In CKD animals, the BM VEGF-A expression was positively correlated with BFR (r = 0.80, p < 0.001). Reducing BFRs in CKD animals led to reductions in VEGF-A expression and osteoblast transcription factors regardless of the PTH level. We therefore examined other regulators of VEGF-A and found decreased expression of hypoxia-inducible factor-1α and the master transcription factor of antioxidants nuclear factor (erythroid-derived 2)-like 2 in CKD animals with low PTH. CONCLUSION: Low BFRs in CKD are associated with a basal decrease in VEGF-A expression in BM that may be driven by altered hypoxia and oxidative stress.

Pathophysiology of Vascular Calcification.

Vascular calcification can lead to cardiovascular morbidity and mortality. The initiating factors and clinical consequences depend on the underlying disease state and location of the calcification. The pathogenesis of vascular calcification is complex and involves a transformation of vascular smooth muscle cells to an osteo/chondrocytic cell that expresses RUNX2 and produces matrix vesicles. The imbalance of promoters (such as hyperphosphatemia and hypercalcemia) and inhibitors (e.g., fetuin-A) is critical in the development of vascular calcification. The altered mineral metabolism and deficiency in inhibitors are common in patients with chronic kidney disease (CKD) and is one reason why vascular calcification is so prevalent in that population.

Pathogenesis of arrhythmias in a model of CKD.

Patients with CKD have an increased risk of cardiovascular mortality from arrhythmias and sudden cardiac death. We used a rat model of CKD (Cy/+) to study potential mechanisms of increased ventricular arrhythmias. Rats with CKD showed normal ejection fraction but hypertrophic myocardium. Premature ventricular complexes occurred more frequently in CKD rats than normal rats (42% versus 11%, P=0.18). By optical mapping techniques, action potential duration (APD) at 80% of repolarization was longer in CKD rats (78±4ms) than normal rats (63±3 ms, P<0.05) at a 200-ms pacing cycle length. Calcium transient (CaT) duration was comparable. Pacing cycle length thresholds to induce CaT alternans or APD alternans were longer in CKD rats than normal rats (100±7 versus 80±3 ms and 93±6 versus 76±4 ms for CaT and APD alternans, respectively, P<0.05), suggesting increased vulnerability to ventricular arrhythmia. Ventricular fibrillation was induced in 9 of 12 CKD rats and 2 of 9 normal rats (P<0.05); early afterdepolarization occurred in two CKD rats but not normal rats. The mRNA levels of TGF-ß, microRNA-21, and sodium calcium-exchanger type 1 were upregulated, whereas the levels of microRNA-29, L-type calcium channel, sarco/endoplasmic reticulum calcium-ATPase type 2a, Kv1.4, and Kv4.3 were downregulated in CKD rats. Cardiac fibrosis was mild and not different between groups. We conclude that cardiac ion channel and calcium handling are abnormal in CKD rats, leading to increased vulnerability to early afterdepolarization, triggered activity, and ventricular arrhythmias.

Adipocyte induced arterial calcification is prevented with sodium thiosulfate.

BACKGROUND: Calcification can occur in fat in multiple clinical conditions including in the dermis, breasts and in the abdomen in calciphylaxis. All of these are more common in patients with advanced kidney disease. Clinically, hyperphosphatemia and obesity are risk factors. Thus we tested the hypothesis that adipocytes can calcify in the presence of elevated phosphorus and/or that adipocytes exposed to phosphorus can induce vascular smooth muscle cell (VSMC) calcification. METHODS: 3T3-L1 preadipocytes were induced into mature adipocytes and then treated with media containing high phosphorus. Calcification was assessed biochemically and PCR performed to determine the expression of genes for osteoblast and adipocyte differentiation. Adipocytes were also co-cultured with bovine VSMC to determine paracrine effects, and the efficacy of sodium thiosulfate was determined. RESULTS: The results demonstrated that high phosphorus induced the calcification of differentiated adipocytes with increased expression of osteopontin, the osteoblast transcription factor Runx2 and decreased expression of adipocyte transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (CEBPα), indicating that high phosphorus led to a phenotypic switch of adipocytes to an osteoblast like phenotype. Sodium thiosulfate, dose dependently decreased adipocyte calcification and inhibited adipocyte induced increase of VSMC calcification. Co-culture studies demonstrated that adipocytes facilitated VSMC calcification partially mediated by changes of secretion of leptin and vascular endothelial growth factor (VEGF) from adipocytes. CONCLUSION: High phosphorus induced calcification of mature adipocytes, and adipocytes exposed to elevated phosphorus can induce calcification of VSMC in a paracrine manner. Sodium thiosulfate inhibited this calcification and decreased the secretin of leptin and VEGF from adipocytes. These results suggest that adipocyte exposure to elevated phosphorus may be a pathogenic factor in calcification observed in the skin in calciphylaxis and other diseases.

Adverse mandibular bone effects associated with kidney disease are only partially corrected with bisphosphonate and/or calcium treatment.

BACKGROUND/AIMS: Patients with chronic kidney disease (CKD) have a high prevalence of periodontal disease that may predispose to tooth loss and inflammation. The goal of this study was to test the hypotheses that a genetic rat model of progressive CKD would exhibit altered oral bone properties and that treatment with either bisphosphonates or calcium could attenuate these adverse changes. METHODS: At 25 weeks of age, rats were treated with zoledronate (ZOL), calcium gluconate, or their combination for 5 or 10 weeks. Mandible bone properties were assessed using micro-computed tomography to determine bone volume (BV/TV) and cementum-enamel junction to alveolar crest distance (CEJ-AC). RESULTS: Untreated CKD animals had significantly lower BV/TV at both 30 (-5%) and 35 (-14%) weeks of age and higher CEJ-AC (+27 and 29%) compared to normal animals. CKD animals had a significantly higher parathyroid hormone (PTH) compared to normal animals, yet similar levels of C-reactive protein (CRP). ZOL treatment normalized BV/TV over the first 5 weeks but this benefit was lost by 10 weeks. Calcium treatment, alone or in combination with ZOL, was effective in normalizing BV/TV at both time points. Neither ZOL nor calcium was able to correct the higher CEJ-AC caused by CKD. Calcium, but not ZOL, significantly reduced serum PTH, while neither treatment affected CRP. CONCLUSIONS: (i) This progressive animal model of CKD shows a clear mandibular skeletal phenotype consistent with periodontitis, (ii) the periodontitis is not associated with systemic inflammation as measured by CRP, and (iii) reducing PTH has positive effects on the mandible phenotype.

Transglutaminase 2 accelerates vascular calcification in chronic kidney disease.

BACKGROUND: Transglutaminase 2 (TGM2) is a calcium-dependent enzyme that can cross-link nearly all extracellular matrix (ECM) proteins and can facilitate cell-ECM interaction through integrins. Given the importance of the ECM in vascular calcification we tested the hypothesis that increased TGM2 activity may accelerate vascular calcification in chronic kidney disease (CKD). METHODS: We utilized thoracic aortas and vascular smooth muscle cells (VSMC) from the Cy/+ rat, a model of progressive CKD that develops arterial calcification on a normal phosphorus diet, compared to normal rats. RESULTS: VSMC isolated from CKD rats had increased expression and activity of TGM2 compared to cells from normal rats. The increased calcification and expression of alkaline phosphatase activity observed in VSMC from CKD rats compared to normal was inhibited in a dose-dependent manner with the TGM inhibitors cystamine and Z006. Matrix vesicles (MV) from CKD rat VSMC also had increased TGM2 expression and the calcification of MV on type I collagen could be inhibited with cystamine and accelerated by exogenous cross-linking of fibronectin or type I collagen with TGM2. Finally, the calcification of aorta rings from CKD rats in ex vivo cultures was inhibited with TGM2 inhibitor. CONCLUSION: These data demonstrate a role of TGM2 in the pathogenesis of vascular calcification in CKD through enhancement of MV-ECM calcification.

Single-cell RNAseq provides insight into altered immune cell populations in human fracture nonunions.

Nonunion describes bone fractures that fail to heal, resulting in the fracture callus failing to fully ossify or, in atrophic cases, not forming altogether. Fracture healing is regulated, in part, by the balance of proinflammatory and anti-inflammatory processes occurring within the bone marrow and surface cell populations. We sought to further understand the role of osteoimmunology (i.e., study of the close relationship between the immune system and bone) by examining immune cell gene expression via single-cell RNA sequencing of intramedullary canal tissue obtained from human patients with femoral nonunions. Intramedullary canal tissue samples obtained by reaming were collected at the time of surgical repair for femur fracture nonunion (n = 5) or from native bone controls when harvesting autologous bone graft (n = 4). Cells within the samples were isolated and analyzed using the Chromium Single-Cell System (10x Genomics Inc.) and Illumina sequencers. Twenty-three distinct cell clusters were identified, with higher cell proportions in the nonunion samples for monocytes and CD14 + dendritic cells (DCs), and lower proportions of T cells, myelocytes, and promyelocytes in nonunion samples. Gene expression differences were identified in each of the cell clusters from cell types associated with osteoimmunology, including CD14 + DC, monocytes, T cells, promyelocytes, and myelocytes. These results provide human-derived gene profiles that can further our understanding of pathways that may be a cause or a consequence of nonunion, providing the clinical rationale to focus on specific components of osteoimmunology. Clinical significance: The novel single-cell approach may lead to clinically relevant diagnostic biomarkers during earlier stages of nonunion development and/or investigation into therapeutic options.

The Dietary Fermentable Fiber Inulin Alters the Intestinal Microbiome and Improves Chronic Kidney Disease Mineral-Bone Disorder in a Rat Model of CKD.

Background: Dietary fiber is important for a healthy diet, but intake is low in CKD patients and the impact this has on the manifestations of CKD-Mineral Bone Disorder (MBD) is unknown. Methods: The Cy/+ rat with progressive CKD was fed a casein-based diet of 0.7% phosphate with 10% inulin (fermentable fiber) or cellulose (non-fermentable fiber) from 22 weeks to either 30 or 32 weeks of age (~30 and ~15 % of normal kidney function). We assessed CKD-MBD, cecal microbiota, and serum gut-derived uremic toxins. Two-way ANOVA was used to evaluate the effect of age and inulin diet, and their interaction. Results: In CKD animals, dietary inulin led to changes in microbiota alpha and beta diversity at 30 and 32 weeks, with higher relative abundance of several taxa, including Bifidobacterium and Bacteroides , and lower Lactobacillus . Inulin reduced serum levels of gut-derived uremic toxins, phosphate, and parathyroid hormone, but not fibroblast growth factor-23. Dietary inulin decreased aorta and cardiac calcification and reduced left ventricular mass index and cardiac fibrosis. Bone turnover and cortical bone parameters were improved with inulin; however, bone mechanical properties were not altered. Conclusions: The addition of the fermentable fiber inulin to the diet of CKD rats led to changes in the gut microbiota composition, lowered gut-derived uremic toxins, and improved most parameters of CKD-MBD. Future studies should assess this fiber as an additive therapy to other pharmacologic and diet interventions in CKD. Significance Statement: Dietary fiber has well established beneficial health effects. However, the impact of fermentable dietary fiber on the intestinal microbiome and CKD-MBD is poorly understood. We used an animal model of progressive CKD and demonstrated that the addition of 10% of the fermentable fiber inulin to the diet altered the intestinal microbiota and lowered circulating gut-derived uremic toxins, phosphorus, and parathyroid hormone. These changes were associated with improved cortical bone parameters, lower vascular calcification, and reduced cardiac hypertrophy, fibrosis and calcification. Taken together, dietary fermentable fiber may be a novel additive intervention to traditional therapies of CKD-MBD.

Assessing cortical bone porosity with MRI in an animal model of chronic kidney disease.

Chronic kidney disease (CKD) is characterized by secondary hyperparathyroidism and an increased risk of hip fractures predominantly related to cortical porosity. Unfortunately, bone mineral density measurements and high-resolution peripheral computed tomography (HR-pQCT) imaging have shortcomings that limit their utility in these patients. Ultrashort echo time magnetic resonance imaging (UTE-MRI) has the potential to overcome these limitations by providing an alternative assessment of cortical porosity. The goal of the current study was to determine if UTE-MRI could detect changes in porosity in an established rat model of CKD. Cy/+ rats (n = 11), an established animal model of CKD-MBD, and their normal littermates (n = 12) were imaged using microcomputed tomography (microCT) and UTE-MRI at 30 and 35 weeks of age (which approximates late-stage kidney disease in humans). Images were obtained at the distal tibia and the proximal femur. Cortical porosity was assessed using the percent porosity (Pore%) calculated from microCT imaging and the porosity index (PI) calculated from UTE-MRI. Correlations between Pore% and PI were also calculated. Cy/+ rats had higher Pore% than normal rats at both skeletal sites at 35 weeks (tibia = 7.13 % +/- 5.59 % vs. 0.51 % +/- 0.09 %, femur = 19.99 % +/- 7.72 % vs. 2.72 % +/- 0.32 %). They also had greater PI at the distal tibia at 30 weeks of age (0.47 +/- 0.06 vs. 0.40 +/- 0.08). However, Pore% and PI were only correlated in the proximal femur at 35 weeks of age (ρ = 0.929, Spearman). These microCT results are consistent with prior studies in this animal model utilizing microCT imaging. The UTE-MRI results were inconsistent, resulting in variable correlations with microCT imaging, which may be related to suboptimal bound and pore water discrimination at higher magnetic field strengths. Nevertheless, UTE-MRI may still provide an additional clinical tool to assess fracture risk without using ionizing radiation in CKD patients.