HFE gene. Both HFE patients and Hfe‐mouse models develop a progressive accumulation of iron in the parenchymal cells of various tissues, eventually resulting in liver cirrhosis, hepatocellular carcinoma, cardiomyopathies, hypogonadism, and other pathologies. Clinical data and preclinical models have brought considerable attention to the correlation between iron overload and the development of osteoporosis in HFE/Hfe hemochromatosis. Our study critically challenges this concept. We show that systemic iron overload, at the degree present in Hfe−/− mice, does not associate with the microarchitecture impairment of long bones, thus excluding a negative effect of iron overload on bone integrity. We further reveal that Hfe actions in osteoblasts and osteoclasts are dispensable for the maintenance of bone and iron homeostasis in mice under steady‐state conditions. We conclude that, despite systemic iron overload, Hfe−/− mice present normal physiological bone homeostasis. © 2019 The Authors. JBMR Plus in published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.One of the most prevalent genetic iron overload disorders in Caucasians is caused by mutations in the  HFE gene with a carrier frequency of approximately one in eight.HFE/Hfe gene result in low production of hepcidin, a small liver‐derived peptide hormone,Disorders of iron metabolism, such as iron deficiencyHFE‐HH patients and respective disease animal models showed that HFE/Hfe actions in hepatocytes were essential to maintain appropriate hepcidin expression and systemic iron levels, establishing Hfe‐HH as the liver disease.Hfe impaired the activity of the cytoplasmic receptor‐activated small mothers against the decapentaplegic (R‐Smad) signaling pathway, resulting in low hepcidin expression.Hfe and TfR2,Hjv)Bmp2,Bmp6,Alk3, Alk2)Smad4,Studies in HFE‐HH patients; however, most patients had evidence of gonadal hormone deficiency, which is likely the primary effect of iron on reported bone loss.HFE‐hemochromatosis patients, which positively associated with increased levels of total alkaline phophatase, the presence of hypogonadism, and the severity of iron overload, comorbidities that can account for secondary osteoporosis.HFE‐HH, which was not related with hyperparathyroidism, vitamin D deficiency, hypogonadism, or cirrhosis.Hfe−/− mice proposed iron overload as the main cause of bone loss in Hfe‐HHHowever, because there is no regulated mechanism to remove the excess of iron, a progressive iron accumulation in various tissues occurs, resulting in multiple organ dysfunction and eventually failure. Liver cirrhosis, hepatocellular carcinoma, cardiomyopathies, and hypogonadism are commonly described in patients with iron‐overload disorders.Considering these data, it is surprising that iron‐depletion therapies  that are remarkably successful in preventing liver and heart complications do not fully improve bone quality.Hfe impairment, led to bone loss. Our data unequivocally showed that neither the lack of Hfe in bone cells nor the iron overload at the levels present in Hfe−/− mice, suffice to trigger bone loss, thus critically challenging the concept of iron‐mediated bone loss in Hfe‐HH.Given these studies, we aimed to dissect whether direct functions of Hfe in osteoblasts and osteoclasts were decisive for the control of bone integrity or whether iron overload, caused by Hfe−/− mice, both males and females at the ages of 11, 29, and 53 weeks, and their littermate controls (Hfe+/+), maintained on a C57BL/6 J genetic background, were used in this study. A new mouse line lacking Hfe in osteoblasts, the HfeRunx2Cre, was generated by crossing Hfeflox mice with Runx2Cre transgenic mice expressing Cre recombinase under the control of the bone‐specific distal promoter of the Runx2 geneA). Mice with hepatocyte‐ and osteoclast‐specific Hfe deletion (HfeAlfpCre(+) and HfeLysMCre(+) mutant mice, respectively) have been previously described.2 inhalation. Blood, livers, and the bones were collected. Animal experiments were approved and performed in accordance with the University Animal Care Committee and the Federal Authorities for Animal Research .Femurs and spins were collected and fixed in 4% paraformaldehyde  for 3 days and stored in 0.5% paraformaldehyde until further analysis. Distal regions of right femur and lumbar vertebra were subjected to 3D µCT analysis using SkyScan 1174 compact in vitro µCT or SkyScan 1176 in vivo high‐resolution µCT  equipped with an X‐ray tube with a voltage of 50 to 80 kV/100 μA, using a 6‐μm voxel size and 0.5‐degree rotation step. The fifth lumbar vertebras were measured at 9 µm and the rotation step was set at 1 degree. To reduce beam hardening, a 0.5‐mm aluminium filter was used. For the reconstruction of femora, the trabecular ROI was selected to extend 0.3 mm proximally to the end of the distal growth plate over 1.8 mm towards the diaphysis. Images of femora were acquired. The morphometric parameters defining trabecular bone mass, including trabecular bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N), were analyzed at the femoral midshaft according to the guidelines issued by the ASBMR Histomorphometry Nomenclature Committee.Static and dynamic histomorphometry was performed on decalcified and undecalcified femoral sections of mice.For static analysis, femurs or tibias were fixed in 4% paraformaldehyde (Merck) for 3 days, washed for 2 hours with water, and decalcified in the presence of 15% EDTA for 10 days with mild shaking at 37°C. The bones were embedded in paraffin. Osteoclast number/bone perimeter and osteoclast surface/bone surface were quantified on decalcified sections of either tibia or femur stained for tartrate‐resistant acid phosphatase (TRAP).Undecalcified femurs were first dehydrated and infiltrated with destabilized methylmetacrylate (Merck), benzoylperoxide (Merck), and nonylphenyl‐polyethyleneglycol acetate  for 14 days. Embedding in methylmetracrylate was done overnight at 4°C, and the femurs were used for dynamic analysis. Osteoblast number/bone perimeter, osteoblast surface/bone surface, and osteocyte number were quantified on toluidine blue‐stained undecalcified femoral sections. Two sections were analyzed for each mouse. Bone formation rate (BFR)/bone surface was measured on undecalcified bone sections by means of fluorochrome labeling. Osteomeasure software was used for the analysis 2O2 (Merck) for 15 min, blocked with Avidin/Biotin Blocking Kit  and incubated with secondary antibody  for 2 hours at 4°C. Afterwards, sections were incubated with horseradish peroxidase streptavidin  for 60 min at 4°C and stained with ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories) for 3 min. Counterstaining was done with Hematoxylin Gill's Formula (Vector Laboratories) for 5 min; dehydration slides were mounted with Eukitt Mounting Media . Sections were visualized under a microscope  using 400 × magnification. Sections without primary or secondary antibody were also included. Cells positive for osteocalcin stained brown.Immunohistochemical analysis of osteocalcin was performed on paraffin‐embedded femur tissues (5 µm). Briefly, sections were deparaffinized and blocked with 10% goat serum for 2 hours at 4°C, followed by incubation with monoclonal antiosteocalcin antibody  overnight at 4°C. Thereafter, sections were incubated in 0.3% HThe nonheme iron content in the liver was measured as previously described.Prussian blue staining was used to visualize iron deposition on livers and bones. Livers were paraffin‐embedded and cut in 4‐µm thick sections. Iron staining of bones was performed on both decalcified and undecalcified bones. Bones were first fixed in 4% paraformaldehyde (Merck) for 3 days and washed for 2 hours with water. Afterwards, bones were decalcified in 15% EDTA for 10 days with mild shaking at 37°C. The EDTA solution was changed every third day. Decalcified and paraffin‐embedded femurs (5‐µm sections) were mounted on polylysine‐coated slides (R. Langenbrick). Undecalcified femurs were first dehydrated and infiltrated with destabilized methylmetacrylate (Merck), benzoylperoxide (Merck), and nonylphenyl‐polyethyleneglycol acetate  for 14 days. Embedding in methylmetracrylate was done overnight at 4°C. Undecalcified and plastic‐embedded femurs were mounted on superforst slides (R. Langenbrick).All sections were stained for iron in the presence of 1% HCl and 2% potassiumhexacyanoferrate II trihydrate (Merck) for 60 min. Nucleus was counterstained with Nuclear Fast Red Solution  for 10 min and slides were mounted with Eukitt Mounting Media (R. Langenbrick). Sections were visualized under a microscope (Olympus) using 40× (liver) and 100× (bone) magnification.Quantification of iron staining in the bones was performed on undecalcified femur sections. Iron‐labeled surface per bone surface was determined using Osteomeasure software (Osteometrics).P1NP and C‐terminal collagen crosslinks telopeptide 1 (CTX‐I), markers of bone formation and resorption, respectively, were measured in plasma using ELISA kits, according to the manufacturer's instructions . Testosterone levels were measured in the plasma using an ELISA kit according to the manufacturer's instructions .HfeRunx2Cre, Hfe−/−, and HfeAlfpCre mice, 3 to 5 days old, were used. Briefly, calvarias were digested in Gibco Minimum Essential Medium Alpha  containing 0.2% collagenase A , 0.2% dispase II  at 37°C for 10 min at 700 rpm. After centrifugation at 1500 rpm for 5 min, cells were seeded in 6‐well plates. Primary cells were passaged once, and osteogenic differentiation was induced by adding 100 mg/mL ascorbic acid  and 5 mM beta‐glycerophosphate  for 6 days.Primary osteoblasts were isolated from neonatal mouse calvarias as previously described.For the determination of differentiation, after 6 days PFA‐fixed osteoblasts were stained for alkaline phosphatase (ALP) using Fast Violet B Salt  and naphtol AS‐MS phosphate alkaline solution . Images were captured using a Canon EOS 600D camera . For quantification, the Amplite Colorimetric Alkaline Phosphatase Assay Kit  was used according to the manufacturer's instructions. ALP activity was normalized by cell viability via Presto Blue Assay .HfeLysMCre mice; 1 million cells were seeded per well in 24‐well plates. Differentiation of osteoclasts was performed in α‐MEM medium containing 10% FBS and 1% penicillin/streptomycin, supplemented with 50 ng/mL RANKL  and 25 ng/mL M‐CSF  for 5 or 7 days. TRAP staining was performed using a TRAP Kit .Bone marrows were isolated from the femora of 2‐ to 3‐month‐old Gapdh using the ∆∆CT method.Total RNA was isolated from primary osteoblasts using RNeasy Mini Kit  according to the manufacturer's instructions. RNA quality and quantity were controlled using the Nanodrop 2000 system . RevertAid H Minus Reverse Transcriptase , 5× RT reaction buffer (Fermentas), random primers , and 10 mM dNTPs , were used to convert 2 µg of RNA to cDNA following the manufacturer's instructions. Quantitative real‐time PCR (qRT‐PCR) was carried out in 10 µL of reaction volume using SYBR Green I Dye  on ABI ViiA‐7 system . The mRNA abundance of the gene of interest was calculated relative to the expression of the reference gene Protein extracts were prepared from primary osteoblasts as previously described.U test were used. Statistically significant differences are indicated as p < .05 (*), p < .01 (**), and p < .005 (***) in the tables.Data were analyzed using GraphPad Prism software ; results are shown as mean ± SD. For the statistical analysis, a nonparametric distribution and the Mann‐Whitney Hfe‐HH mice trigger bone loss, we employed three conditional mouse lines in which Hfe was specifically deleted in osteoblasts (HfeRunx2Cre), and in myeloid cells, including osteoclasts (HfeLysMCre)HfeAlfpCre).HfeAlfpCre(+) mutant mouse is a unique model of Hfe‐HH, which because of a lack of Hfe exclusively in its hepatocytes, develops iron overload and phenocopies the pathology of global Hfe−/− mice.To determine whether (1) direct functions of Hfe in the bone cells, or (2) an excess of iron at the levels present in Hfe deficiency in osteoblast (HfeRunx2Cre mice). The analysis of cancellous bone parameters, such as BV/TV, the Tb.Th, the Tb.N, and the Cs.Th, revealed no statistically significant differences; however, minor differences were detected in the Tb.Sp in the femur and the vertebra of HfeRunx2Cre(+) mutant mice when compared with sex/age‐matched HfeRunx2Cre(–) littermate controls . Moreover, the number of osteoblasts, osteoclasts, and osteocytes as well as the levels of serum P1NP and CTX‐I, markers of bone formation and bone resorption activity, respectively, were not significantly different between HfeRunx2Cre(+) mutant mice and HfeRunx2Cre(−) control littermates . In line with these observations, the differentiation capacities of primary osteoblasts, isolated from neonatal calvaria from HfeRunx2Cre(+) mutant and HfeRunx2Cre(−) control, were not significantly different as evidenced by the measurement of the levels of alkaline phosphatase . These data imply that trabecular microarchitectural organization is unaffected in HfeRunx2Cre(+) mutant mice. With respect to iron status, we show that the lack of Hfe in the osteoblasts did not affect systemic iron metabolism because the levels of iron in the blood, liver, and the femur , as well as the expression levels of major hepatic iron genes, such as hepcidin (Hamp), Bmp6, Id1, and Smad7, in HfeRunx2Cre(+) mutant mice were similar to the levels in control mice . Importantly, no overt changes in bone and iron parameters were detected in aged HfeRunx2Cre(+) mutant mice .We first assessed the bone status in mice with selective ols Fig. A–C. MorHfeflox mice with mice carrying Cre under the control of LysM promoter, which deletes in multiple myeloid lineages, including in early osteoclast lineage cells.HfeLysMCre(+) mutant mice are characterized by unaltered levels of circulating iron and show no increased iron deposition in the liver or in the femur, as revealed by Prussian blue staining for iron on liver and femur sections .C, D). Regarding bone status, no statistically significant differences in the BV/TV, the Tb.N, the Tb.Th, and the Tb.Sp in the femur and the vertebra were detected between HfeLysMCre(+) mutant and HfeLysMCre(−) control littermates . Likewise, no changes in the number of osteoblasts, osteoclasts, and osteocytes  or in the serum levels of P1NP and CTX‐I markers  were detected in HfeLysMCre(+) mutant mice when compared with sex/age‐matched HfeLysMCre(−) control littermates. Similarly, the analysis of major iron and bone parameters in aged HfeLysMCre(+) mutant mice revealed no statistically significant difference with regard to control mice .We next investigated the role of Hfe in osteoclasts in relation to bone integrity. To this end, we crossed ons Fig. A, B.10 tes Fig. E–G. Liktes Fig. H, I or ers Fig. J were dCollectively, our data imply that direct Hfe actions in osteoblasts and osteoclasts are dispensable for the maintenance of bone and iron homeostasis in mice under steady‐state conditions.HfeAlfpCre(+) mutant mice show bone loss. In contrast to HfeRunx2Cre(+) and HfeLysMCre(+) mutant mice, HfeAlfpCre(+) mutant mice develop systemic iron overload characterized by increased iron levels in the blood and the liver to an extent similar to that in mice with global Hfe‐deficiency .HfeAlfpCre(+) mutant mice . Moreover, there were no significant changes in BV/TV, or in the thickness, number, and separation of the trabeculas in the femur, whereas statistically significant changes in the Tb.Sp and Tb.N were only detected in the vertebra of HfeAlfpCre(+) mutant mice with regard to sex/age‐matched HfeAlfpCre(−) control littermates . The observed differences in Tb.Sp and Tb.N in vertebra were, however, not sufficient to result in overall changes in BV/TV . Moreover, we showed that the differentiation capacity of primary osteoblasts, isolated from HfeAlfpCre(+) mutant and control pups, was similar between cells . These data suggest that a lack of hepatic Hfe and the subsequent systemic iron overload do not affect bone integrity.Subsequently, we tested whether ncy Fig. A, B.10 ice Fig. B. Moreotes Fig. C–E. The/TV Fig. E. Moreolls Fig. F. TheseHfe mutation, which is a well‐established mouse model of Hfe‐HH. In contrast to profound iron deposition in the liver of Hfe−/− mice . Surprisingly, the analysis of bone parameters, such as BV/TV, and the number, thickness, and separation of the trabeculas, in the femur and the vertebra, revealed no statistically significant differences between Hfe−/− and control mice (Table HfeAlfpCre(+) mice and contradict the view that an excess of iron leads to bone loss in Hfe‐hemochromatosis mice.Given this unexpected result, we reconsidered the mice with a global age Fig. A. Surprce Table . These dHfe−/− mice.Hfe−/− mice. We found that iron levels were consistently increased in the blood and liver of 29‐ and 53‐week‐old Hfe−/− mice (Table A); however, no excessive iron accumulation was detected in long bones . Importantly, there was no statistically significant difference in the BV/TV, and the number, thickness, and separation of the trabeculas in the femur or in the vertebra of aged Hfe−/− mice with regard to control littermates ; (2) the BFR measurements, which were comparable between aged Hfe−/− mice and control littermates ; moreover, we showed that (3) the differentiation capacity of primary osteoblasts, isolated from iron‐overloaded Hfe−/− and Hfe+/+ control pups, was similar between cells ; and (4) the mRNA and protein levels of bone and iron markers  were comparable between Hfe‐deficient and Hfe+/+ primary osteoblasts . Of note, the mRNA expression of Sp7 was significantly lower in differentiated osteoblasts isolated from Hfe−/− pups with regard to Hfe+/+ control pups . This was, however, the only gene whose changed mRNA expression was not sufficient enough to affect the overall differentiation capacity of primary osteoblasts, as can be seen by the measurement of ALP staining and ALP intensity in Fig. C. Taken together, our findings contravene the previously proposed negative effects of iron overload on bone loss in aged Hfe−/− mice.Our findings are highly surprising as previous studies reported bone loss in ce Table A; howevnes Fig. A, B. Imes Table . Accordice Table . These oter Fig. C–F; (2)tes Fig. G; moreoHFE/Hfe‐HH varies between men and women and that in humans and in mice, the iron and the bone homeostasis are influenced by sex, diet, and genetic background,Hfe−/− mice at the ages of 11, 29, and 53 weeks, kept under the same conditions as their male counterparts. In sum, our data showed that Hfe−/− females displayed no signs of bone anomalies at any age studied despite the presence of systemic iron overload  no bone loss was present in Hfe−/− mice: young, middle‐aged, and aged male, as well as female Hfe−/− mice were all protected, despite systemic iron overload; (2) no bone loss was observed in liver‐specific Hfe‐mutant mice—which phenocopy the iron pathology of constitutive Hfe−/− mice—thus, iron overload, present in constitutive and liver‐specific Hfe mutant mice, does not disturb bone homeostasis; and finally, (3) no bone loss was present in mice lacking Hfe selectively in osteoblasts and osteoclasts, implying that Hfe actions in bone cells were not required to maintain systemic bone or iron homeostasis.Using three conditional Hfe−/− mice,Hfe−/− mice and proposed for a negative effect of iron overload on the osteoblasts.Hfe−/− mutant and control mice . Furthermore, no effect of iron overload was observed on osteoblasts, as the serum P1NP levels, number of osteoblasts, and BRF were comparable between Hfe−/− and control mice (Table Hfe−/− or from HfeAlfpCre(+) mice . Finally, the levels of iron in the diet seem to be comparable between the studies excluding iron as the major factor contributing to differences between the data. We suggest that the discrepancy between our data with regard to bone phenotype might be explained by the use of nonlittermate mice as controls in an initial report by Guggenbuhl and colleagues. From a number of studies, it is known that differences in using nonlittermates can account for a variation in bone phenotype.An initial investigation by Guggenbuhl and colleagues proposed iron overload and increased number of osteoclasts as the main cause of bone loss in 6‐month‐old male ice Fig. E, F. Fuce Figs. F, 5C. Hfe−/−mice, why then is iron still considered as the guilty one? The hypothesis that iron acts as the underlying cause of bone abnormalities in iron‐overloaded conditions has emerged from the role of free, nonbound iron, which is present when iron‐binding capacities of plasma transferrin or intracellular iron‐storage molecule ferritin are surpassed. Several in vitro and in vivo studies have shown that changes in intracellular catalytically active iron levels may play a critical role in inducing oxidative stress and triggering pathways connected with inflammatory processes, which in turn may affect bone metabolism. For example, a study by Tsay and colleagues showed that parenteral iron injections produced severe iron overload in bones and that the degree of iron overload correlated with an increase in osteoclast number, causing changes in bone microarchitecture and bone loss.Hfe‐hemochromatosis (Hfe−/− and HfeAlfpCre(+) mice), does not suffice to induce bone loss under steady‐state conditions. We suspect that the degree of iron‐loading matters, which may be a critical factor to trigger, or to contribute to, the development of osteoporosis in mice. However, there are several considerations to be taken into account when comparing Hfe‐HH and other models of acquired iron‐overload disorders. First, the etiology of iron overload in Hfe‐HH is genetic, that is caused by the lack of Hfe and consequently low hepatic hepcidin production.Hfe−/− mice. Whereas heavy iron deposits were present in the marrow and the trabeculas of iron‐injected mice,Hfe−/− mice at any age analyzed . Despite having “iron” as a common denominator, these iron disorders are profoundly different; thus, bone‐related pathologies cannot be easily compared with one another.If our study refutes any negative effect of iron overload on bone loss in zed Fig. A, B. DeHfe−/− mice, and the still limited number of clinical observations in HFE patients,HFE/Hfe‐hemochromatosis. Whether iron overload in Hfe−/− mice may act as an additional culprit when other osteoporosis triggers are present, such as vitamin D deficiency, liver cirrhosis, and endocrinological defects, is currently unknown and interesting to investigate further. Under physiological steady‐state conditions, however, systemic iron overload in Hfe‐HH mice does not suffice to cause bone loss.Based on our data from HFE‐HH patients and will aid in targeting the mechanisms of bone loss during iron‐overload pathology rather than focusing on the diversion of HFE/Hfe mutations and the iron overload itself.We are confident that our findings are of utmost relevance for the clinical care of The authors declare no conflict of interest.Supporting information.Click here for additional data file.


Analgosedation is a cornerstone therapy for mechanically ventilated patients in intensive care units (ICU). To avoid inadequate sedation and its complications, monitoring of analgosedation is of great importance. The aim of this study was to investigate whether monitoring of analgosedative drug concentrations (midazolam and sufentanil) might be beneficial to optimize analgosedation and whether drug serum concentrations correlate with the results of subjective  and objective  index) monitoring procedures.ρ).Forty-nine intubated, ventilated, and analgosedated critically ill patients treated in ICU were clinically evaluated concerning the depth of sedation using RASS Score, Ramsay Score, and BIS index twice a day. Serum concentrations of midazolam and sufentanil were determined in blood samples drawn at the same time. Clinical and laboratory data were statistically analyzed for correlations using the Spearman’s rank correlation coefficient rho (ρ = − 0.467) and Ramsay Scores (ρ = 0.476). Serum concentrations of sufentanil correlated weakly with RASS (ρ = − 0.312) and Ramsay Scores (ρ = 0.295). Correlations between BIS index and serum concentrations of midazolam (ρ = − 0.252) and sufentanil (ρ = − 0.166) were low.Average age of the population was 57.8 ± 16.0 years, 61% of the patients were males. Most frequent causes for ICU treatments were sepsis (22%), pneumonia (22%), or a combination of both (25%). Serum concentrations of midazolam correlated weakly with RASS  contains supplementary material, which is available to authorized users. Critically ill patients on intensive care units require regimens of analgosedation for several reasons such as mechanical ventilation. Finding the optimal treatment and sedation depth is often challenging because of multimorbidity and polypharmacy. High interindividual variability concerning pharmacokinetics as well as insufficient monitoring can lead to inadequate dosage of drugs, which might increase morbidity and mortality. Low states of analgosedation can cause hypercatabolism, immunosuppression, hypercoagulopathy, awareness and increased sympathetic activity, or inadvertent extubation, whereas deep sedation can be responsible for extended mechanical ventilation, higher risk of nosocomial pneumonia, increasing costs, and neuropsychological dysfunction –6. HenceTo improve individual treatment, clinical scores such as the Richmond Agitation-Sedation Scale (RASS) Score  and RamsGold standard for the assessment of sedation depth is the RASS Score in combination with physiological parameters like heart rate, blood pressure, mimic, gesture, lacrimation, and perspiration , 6. ReliThe BIS index is a unitless value ranging from 0 to 100, a value of 100 representing an adequate awake condition  as well as objective monitoring procedures (BIS-monitoring) were investigated in this study. The aim of this study was to clarify whether or not therapeutic drug monitoring is useful to assess the sedation depth in intensive care patients.This study was performed between December 2012 and December 2014 in cooperation with the ICU of the Department of Internal Medicine and the Center of Pharmacology, University Hospital of Cologne. Intubated, artificially ventilated, and analgosedated intensive care patients, who agreed to this study by themselves or through legal representatives before intubation, were included. Exclusion criteria were age < 18 years, missing patient’s consent, history of alcohol or drug abuse, history of neurological or psychiatric conditions, polytraumatization, conditions after CPR, and suspicion of hypoxic brain damage. RASS Score, Ramsay Score, BIS index, and serum concentrations of analgosedatives were measured twice a day (7:00 a.m. and 7:00 p.m.). Overall, 49 patients were included in the study, and 538 data points were determined. The maximal period under consideration was 10 days. Sepsis was defined according to the criteria by Bone et al. ./kg/h i.v.) and sufentanil (infusion rate of 0.1–1.0 μg/kg/h i.v.). According to the clinical presentation and sedation depth, the infusion rates were adapted.After induction with fentanyl, etomidate and rocuronium orotracheal intubation was performed. Analgosedation was then maintained with midazolam  or “sedated” . “Awake” patients were assessed regarding the reaction while the observer was entering the room. If the patient was considered to be “sedated,” further evaluation was made using a fixed protocol in order to cause eye-opening or a change in facial expression: observer entering room, verbal contact, light physical contact, severe physical contact by shaking patient’s shoulder, induction of light pain by pinching the back of patient’s hand, and induction of severe pain by rubbing patient’s sternum.A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative serum concentration measurements of four analgosedatives  frequently used in intensive care medicine has been previously developed and validated according to ICH Guidelines Q2 (R1) . This teρ). The value of ρ was interpreted as follows: 0 ≤ |ρ| < 0.1—no or very weak correlation; 0.1 ≤ |ρ| < 0.5—weak correlation; 0.5 ≤ |ρ| < 0.8—moderate correlation; 0.8 ≤ |ρ| ≤ 1—strong correlation. Box plots were used for graphic illustration.Statistical analysis and graphic design were performed using IBM SPSS Statistics version 22. Correlations were analyzed using Spearman’s rank correlation coefficient rho . A Ramsay Score of 6 was observed in patients with a median midazolam level of 594 ng/ml, and high intersubject variability was seen. Similar results were described by Glass et al. using the Observers’ Assessment of Alertness/Sedation Scale  [Serum concentrations of sufentanil had only a weak correlation with RASS and Ramsay Scores, whereas serum concentrations of midazolam showed a better but still weak correlation. Bremer et al.  investig= 0.746) .In this study, serum midazolam levels correlated weakly with RASS and Ramsay Scores. A Ramsay Score of 6 was associated with a median midazolam concentration clearly above 1000 ng/ml, whereas the other patients showed median midazolam concentrations lower than that.Bremer et al.  describeComedication with opioids could inhibit midazolam metabolism . MoreoveIn this study, 57% of the patients suffered at least from sepsis or acute renal failure, which led to high intersubject variability Figs.  and 2b aEthuin et al.  analyzed−1) and a large dose midazolam group (0.3 mg kg−1). After remifentanil, midazolam, and vecuronium were administered, intubation was performed, and eight blood samples were collected within 1 h before the operation. Although midazolam plasma concentrations were significantly higher in the large dose group, the authors found no differences concerning BIS index between the two groups. This indicates that there is no correlation between BIS index and serum concentrations of midazolam. In this study, BIS index and midazolam serum concentrations showed only a weak correlation (ρ = − 0.252), which stands in line with the results of Miyake et al. [Miyake et al.  investige et al. . A limite et al.  observede et al. . Severale et al. , 34. Miye et al.  found a e et al.  describee et al.  also foue et al.  showed tConclusively, BIS index will reach its limits—especially as a primary monitoring of sedation depth—when the effect of midazolam is monitored because it does not further decrease although the patient is clinically sedated and plasma concentrations are higher than needed for adequate sedation.Glass et al.  showed tThis study was a single-center study with a relatively small population of 49 intubated patients. Moreover, BIS index values are very susceptible. For example, endotracheal or oral suctioning, body hygiene procedures, passive movements, and physical contact are able to influence BIS index without changing the sedation depth necessarily , 40. To Delirium may influence the assessment of sedation. However, we did not screen our patients for delirium since Haenggi et al.  reportedCorrelations between drug serum concentrations (midazolam and sufentanil) and RASS Score, Ramsay Score, or BIS index were only weak, the results for midazolam being slightly better than those for sufentanil. This might be due to the intersubject variability, polypharmacy with drug-drug interactions, and complex metabolism, which can be altered especially in critically ill patients. Therefore, individual course of disease and patients’ comorbidity have to be taken into account. Therapeutic drug monitoring is not beneficial to determine the depth of sedation in ICU patients. Analgosedation of patients in ICUs should therefore be guided by subjective monitoring procedures.Additional file 1:Richmond Agitation Sedation Scale . (PDF 50Additional file 2:Ramsay Sedation Scale . (PDF 44Additional file 3:Monitoring the depth of sedation with BIS-monitoring . (PDF 44


This study investigated the relationship between liver stiffness and carotid artery elasticity in patients with chronic viral hepatitis. We used an acoustic radiation force impulse (ARFI) technique to measure stiffness, and a radio frequency (RF) vascular quantitative ultrasound technique to measure changes in common carotid artery elasticity and vascular function.Two-hundred seventeen patients with chronic viral hepatitis caused by either hepatitis B virus (HBV) or hepatitis C virus (HCV) were enrolled. We divided the patients into two groups, one comprising 147 patients with chronic hepatitis B (CHB)  and another comprising 70 patients with chronic hepatitis C (CHC) . Additionally, 64 healthy age- and sex-matched participants  were selected as the control group. The ARFI technique was used to measure liver stiffness and the RF ultrasound technique was used to measure carotid artery elasticity parameters including intima-media thickness (IMT), pulse wave velocity (PWV), arterial wall dilation coefficient (DC), compliance coefficient (CC), sclerosis indices α and β, and augmentation index (Aix). Clinical indicators, liver stiffness, and carotid artery elasticity parameters were observed and compared between the different age groups to investigate the correlation between carotid artery elasticity parameters and liver stiffness.P < 0.001). When compared to the control group, both CHB and CHC groups showed an IMT of the same order, but had significantly higher elasticity parameters, such as α and β, as well as lower DC and CC values (P < 0.001). The PWV of the CHC group was significantly higher than that of the control group . In the CHB group, all parameters including ARFI, IMT, PWV, DC, CC, α and β, were significantly different between the two age groups (P < 0.05). Within the CHC group, all parameters including IMT, PWV, DC, α and β, were significantly different between the two age groups (P < 0.05), except for ARFI, wherein the difference was not statistically significant. The correlation analysis and stepwise multiple linear regression analysis indicated that for patients with CHB, age was an independent predictor of common carotid artery IMT . For patients with CHC, age and blood sugar were independent predictors of common carotid artery IMT .The ARFI values for the CHB and CHC groups were significantly higher than those for the control group (1.84 ± 0.52 vs. 1.04 ± 0.11 m/s; 1.86 ± 0.37 vs. 1.04 ± 0.11 m/s, respectively; Although based on ARFI and RF ultrasound, the carotid artery IMT in patients with CHB and CHC was not significantly higher than that in the control group, their functional elasticity parameters had already changed. This finding serves as a useful reference for the clinical diagnosis of vascular diseases in patients with viral hepatitis.ChiCTR1800015859 25/04/2018.ClinicalTrials:  Vascular wall elasticity is an important indicator of abnormal lipid metabolism in vascular disease, which is caused by pathogen-mediated chronic liver damage , 2. SignA total of 217 consecutive patients with chronic viral hepatitis that were treated at our hospital between December 2015 and March 2017 were enrolled. The study population comprised 147 patients with Chronic hepatitis B (CHB) and 70 patients with Chronic hepatitis C (CHC). Patients were admitted into the CHB group according to the standard chronic hepatitis B diagnostic criteria [The acoustic radiation force impulse (ARFI) technique uses the 4C1 convex probe from the Color Doppler Diagnostic Ultrasound Scanner  with a frequency of 3.0 to 4.5 MHz. During ARFI imaging, measurements were performed with the probe placed between the rib bones with the patient lying in either a dorsal decubitus or left recumbent position, taking normal breaths under a resting state or holding their breath, with displays of real-time 2-D images of the right liver lobe. When the image became clear, the operator used the cursor to locate a 5 mm × 10 mm-sized region of interest that targeted the liver parenchyma area and was free of vessels and bile duct. The measurement depth was fixed at 3 cm. When echoing was uniform within the sampling region, the operator pressed the probe button to freeze the image and display the depth and shear wave velocity (in m/s) in the region of interest. This measurement procedure was repeated 10 times for each patient. The median value of the measurements was recorded as the final result  , arterial wall dilation coefficient (DC), arterial wall compliance coefficient (CC), sclerosis indices α and β, and augmentation index (Aix) were exported as the final results   laboratory indicators including blood sugar, glycated haemoglobin, total cholesterol, triglycerides, low-density lipoprotein, high-density lipoprotein, alanine aminotransferase, aspartate aminotransferase, albumin, globulin, and platelets. (2) blood pressures including systolic and diastolic blood pressures.Statistical analysis was performed using SPSS statistical software . Metrological data were expressed as average ± standard deviation (P = 0.010) as well as higher levels of other parameters such as platelets, albumin, AST and ALT, whereas the systolic and diastolic blood pressures were similar. The CHC group demonstrated a higher blood sugar level , higher glycated haemoglobin level , and higher levels of other parameters such as AST, ALT, and platelets compared to the control group. However, there was no significant difference in the cholesterol, triglycerides, HDL, and LDL levels.General clinical data for all study participants are listed in Table P < 0.001)  and the CHC group (1.86 ± 0.37 m/s) had significantly higher liver elasticity parameters than the control group, and the inter-group differences were statistically significant  between the control group (534.08 ± 134.25 μm), the CHB group (529.56 ± 131.04 μm), and the CHC group (587.34 ± 162.70 μm). An intergroup comparison showed significant difference between the CHB and the CHC group (P = 0.032)    . The CC values were 1.290 ± 0.248, 1.054 ± 0.385, and 0.815 ± 0.378 mm2/kPa, respectively . Aix measurements showed no statistically significant differences among the 3 groups   were evidently higher than those of the CHB and the control groups, which were 6.70 ± 1.32 and 6.09 ± 0.90 m/s respectively. Differences in the intergroup comparisons were statistically significant . In the CHB group, ARFI values, as well as parameters including IMT, PWV, DC, CC, α, β, and Aix were all significantly different between the two age groups . In the CHC group, IMT, PWV, DC, α, β, and Aix were significantly different between the two age groups .All subjects were divided into two groups according to their age Tables  and 3. w2 = 0.468, F = 54.635, and P < 0.001)  and blood glucose  were independent predictors of common carotid artery IMT   and the carotid artery elasticity parameters  differed significantly between the two age groups, indicating that these parameters might be related to the time span of HBV infection. Older patients with chronic hepatitis B are likely to carry the virus for a longer period of time and, consequently, experience a higher degree of liver stiffness. This difference was not observed in the control group, which indicated that aging is not related to the natural aging and fibrosis of the liver. In a study of 459 chronic HBV carriers, liver biopsies showed that the liver tissue inflammatory activity level and degree of liver fibrosis gradually increased with age [Age is also a critical factor affecting the potential for development of arteriosclerosis , 15. In with age , which wP > 0.05). A possible explanation was that patients with CHC have a higher risk of increased blood glucose levels. Studies on using the ARFI technique to grade liver stiffness and fibrosis showed that fat in the liver is an important factor that affects the accurate measurement of the ARFI value [However, within the CHC group, the ARFI did not differ significantly between the two age groups (FI value , 18. SinThis study was limited by the fact that patients’ diagnoses were made based on clinical diagnosis primarily. Biopsy was used to obtain pathological results in only a limited number of cases. The next step is to obtain results to classify liver fibrosis into different pathological levels, to further exclude any confounding factors.In summary, using RF ultrasound and ARFI techniques to measure liver stiffness and carotid artery elasticity in patients with chronic viral hepatitis is beneficial for assessing the liver fibrosis and the structural and functional changes of the carotid artery. This serves as a reference for clinicians to monitor any vascular diseases in these patients.


Group G showed a decrease in BV. All treated groups had an increase in trabecular thickness. Histology showed decreased cartilage tissue associated with increased bone in groups with improved healing, and increased fibrous tissue in poorly performing groups. Antagonism of SDF1-CXCR4 axis can boost impaired fracture healing. AMD3100 given alone was the most effective means to boost healing, whereas pretreatment with GCSF reduced healing. AMD3100 is likely mobilizing stem cells into the blood stream that home to the fracture site enhancing healing.A significant number of fractures develop nonunion. Stem cell homing is regulated through stromal cell-derived factor 1 (SDF1) and its receptor CXCR4. Stem/progenitor cell populations can be endogenously mobilized by administering growth factors with a pharmacological antagonist of CXCR4, AMD3100, which may be a means to improve fracture healing. A 1.5 mm femoral osteotomy in Wistar rats was stabilized with an external fixator. Rats were pretreated with phosphate buffered saline [PBS(P)], vascular endothelial growth factor [VEGF(V)], insulin-like growth factor-1 [IGF1(I)], or granulocyte colony stimulating factor [GCSF(G)] before AMD3100. A control group (C) did not receive growth factors or AMD3100. Bone formation after 5 weeks was analyzed. Group P had a significant increase in total bone volume (BV) (Currently ∼10% of fractures progress to delayed or nonunion with significant morbidity and economic impact. Endogenous mobilization of stem cells by pharmacological antagonism of their homing and migration receptor CXCR4 with AMD3100 experimentally reduced delayed union development. Endogenous mobilization may, therefore, translate as a low risk means to boost healing and could potentially be given as a prophylaxis to patients with fractures at risk of delayed healing or nonunion. These patients may include fragility fractures, comminuted tibial fractures, or when treating established nonunions. This approach could have promise for other conditions that may benefit from stem cell treatments. The chemokine stromal cell-derived factor 1  and its receptor CXCR4 have a key role in stem cell migration from the bone marrow stroma into the circulation and are believed to be important for homing of stem cells to a fracture site.9 Local increases in SDF1 expression have been measured in distraction osteogenesis, stress fractures, and segmental defects.10–12 Parabiotic studies have demonstrated that labeled stem cell mobilization is from the bone marrow through the peripheral circulation and these cells are able to then contribute to the fracture healing process.14Asignificant number of bone defects and fractures do not heal,15 A highly selective high-affinity competitive antagonist of the CXCR4 receptor, AMD3100,16 commercially known as Mozibil™, rapidly mobilizes high numbers of hematopoietic stem cells by blocking their interaction with SDF1 in the marrow niche.17–19 It has been shown in mice that when AMD3100 is given after pretreatment with vascular endothelial growth factor (VEGF) rather than GCSF, there is preferential mobilization of a population of MSCs and EPCs relative to hematopoietic stem cells.20Mobilization of hematopoietic stem cells is a mainstay of clinical bone marrow transplantation to treat a range of blood-related malignancies, and granulocyte colony stimulating factor (GCSF) was the first growth factor used for this purpose.21 and a single dose of AMD3100 was also shown to improve intramedullary trabecular bone reformation.22 For evaluation of diaphyseal long bone healing, Kumar and Ponnazhagan23 mobilized MSCs by pretreatment with insulin-like growth factor-1 (IGF1) followed by AMD3100, in a coapted mouse tibial segmental fracture, and showed a significant increase in bone mineral density. AMD3100 has also been evaluated after creating an “Einhorn style” mouse femoral fracture stabilized with a single intramedullary pin, and fracture healing was accelerated.24To date, a few groups have started to investigate mobilization of stem cells to augment bone healing. Critical-sized calvarial defects have shown enhanced healing with 15 daily injections of AMD3100 in mice,25 The hypothesis was that antagonism of the SDF1/CXCR4 axis using ADM3100 would improve bone healing in a delayed union fracture model, and pretreatment with IGF1 or VEGF before AMD3100 would have the greatest efficacy due to their preferential MSC mobilization.None of the aforementioned studies allow for direct comparison of the different protocols, and neither do they test the effect of endogenous mobilization in a translationally relevant delayed union model. Therefore, based on current literature, this study aimed to compare different mobilization protocols  in a biomechanically controlled, delayed union fracture model, of a rat femoral osteotomy stabilized using an external fixator.AMD3100 octahydrochloride hydrate  stock solution was prepared by dissolving 5 mg lyophilized product in 0.5 mL sterile water, then added to 4.5 mL phosphate buffered saline (PBS) to produce a 1 mg/mL injection solution, which was aliquoted and stored at −20°C until needed. Rat VEGF 165  was prepared by dissolving the lyophilized product in sterile water to make a 0.1 mg/mL stock solution and then 1 mL of stock solution was added to 4 mL of sterile PBS +0.1% bovine serum albumin (BSA)  to achieve 100 μg/mL injectable solution, which was aliquoted and stored at −20°C until needed. Recombinant human IGF1  and murine GCSF  were prepared in the same manner. Finally, PBS +0.1% BSA, “sham growth factor,” to determine the effects of AMD3100 alone was also prepared.n = 6; PBS-AMD), VEGF+AMD3100 , IGF1+AMD3100 , GCSF+AMD3100 , and non-mobilized control fracture group (n = 7). A linear type 1a micro-external fixator, with titanium blocks and carbon fiber bars was placed on the left craniolateral femur after a lateral surgical approach.25 Using a precision guide, four bicortical 1.4 mm diameter end-threaded self-tapping stainless steel pins were placed in predrilled 1.0 mm holes. Consistent proximodistal positioning was based on the distal extent of the greater trochanter. Pins were exited through separate skin incisions and the custom variable spacing fixator was attached.25A total of 12- to 14-week-old female Wistar rats (230–300 g) were randomly assigned to one of the five groups: PBS+AMD3100 , and then the skin was closed with intradermal continuous suture . Activity was unrestricted postsurgery. In two rats the surgical wounds failed to heal and were removed from the study, leaving 20 On day 5, they were given a single injection of AMD3100 at 5 mg/kg.20–24 All i.p. injections, including AMD3100 and sham PBS, were administered at a volume of 0.5 mL/100 g bodyweight based on the day 0 presurgical weight. Rats were sacrificed at 5 weeks postoperatively. All procedures were carried out with the local Animal Welfare and Ethical Review Body approval under personal and project licenses issues by the UK Home Office under the 1986 Animal Scientific Procedures Act.Twenty-four hours postsurgery, rats were given a single intraperitoneal (i.p.) injection of either VEGF, IGF1, GCSF, or PBS once daily for 4 days at 100 μg/kg.26 as follows: ununited where there was no mineralized tissue bridging between the ends of the osteotomy; uncertain where there was new bone formation; however, a radiolucent line remained between the proximal and distal segments, and not united where no gap between bone ends was visible.The left femur with the fixator in place was retrieved. To reduce microcomputed tomography (microCT) beam-hardening artifact generated from the interaction of the X-ray beam and the metallic implant, a radiolucent PEEK fixator block was connected externally to the fixator pins after careful removal of the skin with surrounding soft tissues, and then without disturbing the fracture callus the titanium block fixator was then removed. Samples were fixed in 10% buffered formaldehyde for up to 3 days. The formalin-fixed samples were wrapped in cling film to prevent dehydration and mounted into a sample holder for microCT scanning. Samples were scanned using a Bruker Skyscan 1172 microtomograph machine , at 60 kV, 167 μA with a 0.5 mm aluminum filter. A rotation step of 0.5°, without frame averaging, and an image pixel size of 4.89 μm was used. A single image capture image was taken with the image intensification “scout” before scanning, for two-dimensional (2D) radiographic assessment of the osteotomy union. Radiographic scouts were randomized and blinded to score the general impression of healing according to the AO-ASIF recommendations for long bone fractures; united, not united, or uncertainMicroCT scans were reconstructed using NRecon (Bruker) with smoothing = 2, ring artifact reduction = 12% and beam hardening artifact = 41%. Analysis was performed with CTAn (Bruker). Using the measuring tool, the center point of the osteotomy was determined and the transverse slice at that point was selected as the reference slice. The central 60% of the osteotomy gap, that is, only new bone formation within the osteotomy was analyzed. The callus was isolated using a 2D region of interest shrink wrap stretching over holes <40 pixels, despeckled <150 voxels, and then three-dimensional (3D) analysis was performed.Bones were decalcified in a 12.5% solution of ethylenediaminetetraacetic acid and sequentially dehydrated for 24 h, followed by defatting with chloroform for 48 h and then embedded into wax, with the fixator pins orthogonal to the facing surface of the block. Fixator blocks and pins were removed once the wax had set and a microtome  was used to make 5 μm thick slices. The alignment of the blocks within the microtome was altered as necessary to ensure a central sagittal slice through the femur. The position of a mid-sagittal section through the fracture gap was assessed using the fixator pin tract holes. Wax slices were mounted onto positively charged glass slides , dewaxed, and then hydrated. Samples were then stained with hematoxylin (Sigma-Aldrich) nuclear stain for 5 min. Excess stain was removed by gently washing with water for 5 min. Slides were counterstained in 1% eosin (Sigma-Aldrich) for 4 min and then washed and dehydrated in increasing concentrations of alcohol. Slides were cleaned in xylene and mounted under 40 mm coverslips using Pertex Mounting Medium .Slides were observed under a light microscope . Histomorphometric analysis using the 2.5 × objective was performed on the most central slice, using a line-intercept method with a grid scaled to the graticule and drawn using PowerPoint . The grid covered the entire visual field from top to bottom  and was centered over the osteotomy; its width was equivalent to the original 1.5 mm osteotomy. Grid “density” was 120 intersections and grid squares were 160 μm in both directions. Intersections were then scored as bone, cartilage, fibrous tissue, vascular (red blood cells seen not within tissue matrix), or void.U and Kruskal–Wallis as appropriate. Significance was set at p < 0.05 and tests were analyzed with SPSS version 24 .As the data were nonparametric, analyses included the Mann–Whitney 3, p = 0.01), compared with the untreated control (4.3 ± 3.1 μm3). Not only was the BV increased, but the overall callus tissue volume (TV) was increased compared with controls (15.3 ± 3.6 vs. 9.2 ± 6.1 μm3) (Radiographic score showed a reduction in not united (nonunion) and increases in uncertain and united (union) for all groups compared with the GCSF-AMD group. Radiographically united fractures occurred in 4/5 animals in the PBS-AMD group, whereas this was reduced for the VEGF, IGF, and GCSF pretreated groups with control animals only showing a united rate in two animals out of seven . MicroCT6.1 μm3) ; Fig. 1.p = 0.03) (The percentage bone volume (BV/TV) was not significantly increased owing to a relative proportional increase in bone and nonmineralized callus tissue. In addition, the bone structure was different in the PBS-AMD group compared with controls. Animals in the PBS-AMD group showed a significant increase in trabecular thickness (Tb.Th), 0.061 ± 0.002 μm compared with those in the control group, which had a thickness of 0.042 ± 0.003 μm ( = 0.03) ; Fig. 2.p = 0.048), but the actual TV (4.3 ± 4.7 vs. 9.2 ± 6.1 μm3) and BV (2.5 ± 2.6 vs. 4.3 ± 3.1 μm3) was reduced compared with controls. However, Tb.Th was significantly higher 0.069 ± 0.03 versus 0.042 ± 0.008 μm (p = 0.048) (p = 0.048), indicating that although GCSF-AMD group had less overall total woven bone, the bone formed was less porous and the size of each bone forming region within the fracture gap was larger than in controls.The VEGF-AMD group did not show any significant differences from the control group. Interestingly, animals in the GCSF-AMD group had a significant increase in BV/TV 63.1 ± 7.3% versus 53.8 ± 20.8% (= 0.048) , but tot3) compared with controls. BV/TV was significantly increased (p = 0.035) and the overall callus size was the same as controls (TV 9.1 ± 7.6 vs. controls 9.2 ± 6.1 μm3). There was also a significant increase in Tb.Th 0.062 ± 0.008 μm (p = 0.01) (p = 0.035). The spread of data was not significantly different.IGF1-AMD group also had an increase in BV (5.1 ± 4.2 μm = 0.01) . TotPor p = 0.033) (p = 0.003) (p = 0.043), and BV/TV (p = 0.043). All treated groups had greater bone formation than control, other than GCSF-AMD, which had a negative impact on healing. However, only PBS-AMD reached statistical significance for increased overall BV and IGF1-AMD for percentage bone (BV/TV) within the callus. Notably, all groups had significant increases in Tb.Th other than VEGF-AMD. 3D reconstructed images of the representative groups are shown in When comparing between all groups, there were significant differences in BV (= 0.033) , Tb.Th (= 0.003) , TotPor p = 0.053) and percentage fibrous tissue (p = 0.059) for the PBS-AMD compared with controls. Patterns of increased bone formation were associated with decreased cartilage in groups with improved healing, whereas the worse performing groups had an increase in fibrous tissue with decreasing bone and cartilage formation  callus compared with controls. This is similar to the findings of Toupadakis 23 Based on these studies, we hypothesized that these growth factors would have a greater influence on fracture healing than giving AMD3100 alone, which comparatively mobilizes lower levels of MSCs, EPCs, and hematopoietic stem cells. The results of this study suggest that AMD3100 is the most effective protocol to improve fracture healing, bringing into question whether total numbers of different cells types, or their relative combinations, are more important. This study shows a conclusive benefit of AMD3100, which has been comprehensively shown to exert its main interaction at the CXCR4 receptor,29 which would lead to the release of cells that could enhance repair. However, it is impossible to totally exclude other mechanisms that may improve healing not due to stem/progenitor mobilization.Prior mobilization studies have shown in mice that pretreatment with VEGF or IGF1 preferentially increased the numbers of MSCs mobilized into the peripheral circulation.28 The duration and character of the profile of cell elution into the circulatory system is probably more important and so the true kinetics of the mobilization is unknown, and hence, although VEGF or IGF1 pretreatment before AMD3100 mobilized more MSCs at 1 h postadministration, this does not mean the total number is greater. It is also clear that mobilized cells home back to the bone marrow, or indeed other tissues such as liver, spleen, or lungs and, therefore, mobilization has to be considered as a highly dynamic and complicated process.30 Therefore, the only accurate evaluation of the potential of endogenous mobilization for fracture healing is with an in vivo fracture model.An issue with measuring circulating stem/progenitor cells is that they provide only a “snapshot” of the circulating pool of cells at the predicted peak elution time of 1 h postadministration of AMD3100.24 and this study in a rat model of delayed union further identifies AMD3100 along as more effective than combining with growth factor pretreatment. This may be due to physiological elevation of growth factors after fracture, including VEGF and IGF1,31 or that the mobilization profile, including cell types, timing of mobilization, and total numbers of cells, is most beneficial with AMD3100 alone. Toupadakis et al. gave three sequential doses of AMD3100 after fracture24 and studies on the elution and pharmacokinetics of AMD3100 show that serial administration will induce a peak mobilization at 1 h post-treatment to the same level each time, suggesting that receptor/system desensitization does not occur.28 This may mean that the number of stem/progenitor cells available to home to the fracture site could be further increased, but this remains to be demonstrated as beneficial over a single dose.Three mouse studies have also shown AMD3100 alone to improve bone formation,32 AMD3100's short half-life of 0.9 h in rodents18 likely underlies the benefit of short-lived blockade early in fracture healing, as it does not persist and inhibit ongoing migration of cells into the fracture site. AMD3100 therapy deserves further evaluation as the route to clinical translation is relatively simple, being already licensed for hematopoietic stem cell mobilization.Another consideration that has not yet been addressed is the time of delivery of AMD3100, and presumably this has to be associated with SDF1 release at the fracture site and the maturity of tissue in the fracture gap. Consideration of the effect of CXCR4 blockade at the recipient fracture site also needs thought, as protracted treatment with AMD3100 throughout fracture healing or during distraction osteogenesis reduces healing.23 evaluated IGF1 with AMD3100 in a mouse model and showed a significant increase in fracture bone mineral density on DXA scan, similar to the BV/TV in this study. They also showed that IGF1 alone gave a moderate improvement in bone density, whereas AMD3100 alone did not.23 This is in contrast with this study and the differences may relate to peculiarities of their model and species. As the only other group with a significant increase in bone formation, IGF1 and AMD3100 combined may also warrant further investigation.IGF1 pretreated groups showed the development of a relatively more mineralized callus with a significant increase in percentage bone. Kumar and Ponnazhagan34 there was an expectation that VEGF would have beneficial effects.As VEGF preceding AMD3100 has previously been demonstrated in mouse models to release the largest number of MSCs, it was hypothesized that maximal mobilization of these cells would be facilitated by the administration of VEGF and this would lead to the greatest bone healing. However, pretreatment with this growth factor did not show a significant increase in healing unlike AMD3100 alone or pretreatment with IGF1. This would suggest that the differential mobilization from this combination was less beneficial than the mobilization profile from AMD3100 alone. As hypoxia and subsequent vascularization of tissues within the fracture site plays a crucial role in progressive fracture healing and VEGF is a potent angiogenesis promoter with a role in endochondral and intramembranous bone formation,35 Histomorphometric assessment of vascularization was not the objective in this study, but notably the AMD3100 group had the highest number of blood vessels, although significant differences were not detected between groups. The VEGF pretreated group had the second highest percentage vascularized tissue, but the significance of that is difficult to know. In any case, there appears no significant advantage over AMD3100 for improving bone formation.Indeed, local delivery of VEGF in rabbit mandibular defects showed increased density of bone formation, although not the quantity.Pretreatment with GCSF before AMD3100 reduced fracture healing, which has not been previously shown. Interestingly, this group had a significant increase in percentage bone, which was indicative of a much smaller overall callus that proportionally had a higher BV component compared with controls. This bone region also had increased Tb.Th and reduced porosity. All treatment groups, including GCSF-AMD, had increased Tb.Th indicating thicker woven bone formation, but in the GCSF-AMD group the smaller callus had bone present that was structurally more dense. The reduced BV may relate to the less mature chondrocytes seen histologically, indicative of delayed endochondral ossification, which may in turn be due to excessive inflammation from mobilized inflammatory cells.et al.20 showed that GCSF-AMD induced mild mobilization of MSCs and EPCs, but was principally a very effective mobilizer of hematopoietic stem cells and neutrophils. It is possible that the increased influx of neutrophils may have affected the progression of inflammation at the fracture site, preventing healing. CD34+ cells, which are a particularly well-represented population when mobilization is performed with GSCF ± AMD3100, are considered a population enriched in EPCs and hematopoietic stem cells. Transplantation of these cells has shown improved healing in several studies.37 However, this selected CD34+ population are a subset, which may explain the differences in healing seen, compared with mixed mobilized populations that include CD34+ cells. This has been borne out by studies showing a mixed GCSF mobilized mononuclear cell fraction being less efficacious than a subselected CD34+ population38 and excessive inflammation associated with the mononuclear cell population was suggested to be the cause. One study, however, has shown improved fracture healing with GCSF treatment alone, given on five consecutive days. Interestingly, their study lasted 200 days and significant differences were not seen until at least 20–30 days, with a reduction in the osteotomy gap distance. BV was significantly increased from ∼30 days, but all rats went on to nonunion.39Increased presence of hematopoietic lineage osteoclast precursors, leading to bone reduction rather than deposition is also possible. Histologically, GCSF-AMD had the lowest level of bone, cartilage, and vascular tissue, and the highest level of fibrous tissue, suggesting a pattern of reduced endochondral ossification, reduced blood supply, and fibrous tissue development. Pitchford In conclusion, AMD3100 significantly increased fracture healing in a delayed union femoral model and was superior to protocols with growth factor pretreatment. This would suggest that peak MSC mobilization protocols previously identified are not solely beneficial for fracture healing; however, further study is required. In contrast, pretreatment with GCSF, which preferentially mobilizes hematopoietic stem cells and neutrophils had a negative effect on fracture healing and should be avoided. Further evaluation of the timing, dose, and frequency of administration of AMD3100 is warranted as it potentially offers a rapid route to clinical translation.


The incidence of Acute Kidney Injury (AKI) continues to increase in the UK, with associated mortality rates remaining significant. Approximately one fifth of hospital admissions are associated with AKI and approximately a third of patients with AKI in hospital develop AKI during their time in hospital. A fifth of these cases are considered avoidable. Early risk detection remains key to decreasing AKI in hospitals, where sub-optimal care was noted for half of patients who developed AKI.Electronic anonymised data for adults admitted into the Royal Cornwall Hospitals Trust (RCHT) between 18th March and 31st December 2015 was trimmed to that collected within the first 24 h of hospitalisation. These datasets were split according to three separate time periods: data used for training the Takagi-Sugeno Fuzzy Logic Systems (FLS) and the multivariable logistic regression (MLR) models; data used for testing; and data from a later patient spell used for validation.Three fuzzy logic models and three MLR models were developed to link characteristics of patients diagnosed with a maximum stage AKI within 7 days of admission: the first models to identify any AKI Stage , the second for patterns of AKI Stage 2 or 3 , and the third to identify AKI Stage 3 . Model accuracy is expressed by area under the curve (AUC).Accuracy for each model during internal validation was: FLS I and MLR I ; FLS II  and MLR II ; FLS III and MLR III .FLS II and FLS III (and the respective MLR models) can identify with a high level of accuracy patients at high risk of developing AKI in hospital. These two models cannot be properly assessed against prior studies as this is the first attempt at quantifying the risk of developing specific Stages of AKI for a broad cohort of both medical and surgical inpatients. FLS I and MLR I performance is comparable to other existing models.The online version of this article (10.1186/s12882-019-1237-x) contains supplementary material, which is available to authorized users. Acute Kidney Injury (AKI) is emerging as an important clinical syndrome which is associated with poor clinical outcomes. AKI, previously known as Acute Renal Failure, refers to a sudden reduction in kidney function.The Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group definitions of AKI have been adopted in the UK. This classification identifies three stages of severity of AKI, based on rises in serum creatinine or reduction in urine output Table  1, 2]. , 2. 1, 2The majority of AKI is caused by sepsis and hypovolaemia. Other causes include renal tract obstruction and intrinsic renal disease. Around 1 in 5 adult hospital admissions are associated with AKI . AKI is The 2009 National Confidential Enquiry into Patient Outcome and Death (NCEPOD) AKI report found that care could have been improved for 50% of patients . Since tGiven that approximately a third of patients with AKI in hospital develop AKI during their stay in hospital, two-thirds of patients with AKI in hospital have AKI at the time of admission . So, theWe describe an attempt at predicting which patients will go on to develop AKI in hospital, and specifically which Stage of AKI. This is undertaken using methods based around fuzzy logic which are then calibrated against more conventional approaches.Three fuzzy logic systems (FLS)  and three multivariable logistic regression (MLR) models  were constructed to stratify patient risk for developing AKI (any Stage), either Stage 2 or 3 AKI, or Stage 3 AKI respectively during a stay in hospital. Each model was parameterised with data from a time period and validated using data from a later period. Data was provided by RCHT. Each FLS was distinguishable from the other due to the different sets of risk factors used in defining FLS I, II and III respectively.www.royalcornwall.nhs.uk). RCHT serves roughly 430,000 people and employs about 5000 staff with a budget of approximately £380 million. Across its three sites it admits approximately 59,000 emergencies (including maternity and births) and 9000 elective inpatients per year into 750 beds.Research presented here draws on data relating to patients admitted into RCHT in the UK , gender , type of admission Prescription of non-steroidal anti-inflammatory drugs (NSAID), prescription of angiotensin converting enzyme inhibitors (ACEI)Comorbidities: diabetes, heart failure, chronic kidney disease (CKD) and vascular disease (VD) of coronary artery and heart, not including acute myocardial infarction and associated complications. Patients with these comorbidities were identified as those presenting with known pre-existing conditions.For risk factors: platelets, white blood count (WBC), red blood count (RBC), haematocrit (HCT), haemoglobin (Hb), mean platelet volume (MPV), mean corpuscular volume (MCV), sodium (Na), potassium (K), chloride (CL-), urea, creatinine and albumin, data records available between 24 h before admission and 24 h after admission for patients undergoing tests in that time window were kept. If the data records were available for both the day before admission and the day of admission, the record from the day before admission was selected. Given the absence of the exact processing time of test results, where multiple of the same type were undertaken per patient per day, a result was selected at random from the test of the same type performed. The remaining risk factors were available on admission, which were, for each patient:Data were excluded from this study for patients under 18 years of age, for patients staying less than 48 h in hospital and for patients who developed AKI > 7 days after admission. Incomplete records were also removed.p-values< 0.25. A value of p ≤ 0.05 was considered to be statistically significant.The association between dependent and independent variables aiming to identify the most suitable risk factors to be included in each model was examined by performing univariable and multivariable analyses using SPSS version 24.0. Initially, univariable logistic regression analysis was conducted to determine the odds ratios of AKI incidence for each risk factor. Next, multiple logistic regression models based on forward stepwise selection were developed by including risk factors with univariable Takagi-Sugeno-Kang (TSK) or Sugeno fuzzy logic (FL) models based on subtractive clustering were developed using the Fuzzy Logic Toolbox within the framework of Matlab R2016a. The TSK FLS methodology was originally developed as an approach to represent a series of systems and functions by utilising input-output data , 12. TheThe Area Under the Curve (AUC) was calculated for each Receiver Operating Characteristic (ROC) Curve to evaluate the effectiveness of the models . Model pTo assess whether any loss in model accuracy might be caused by the use of TSK FL, equivalent models for FLS I, II and III were assembled using Logistic Regression . LogistiA summary of the characteristics of patients with and without AKI is shown in Additional file Additional file Patient characteristics of patients at low AKI risk, patients diagnosed with Stage 1 AKI or Stage 2 AKI, along with patients who developed Stage 3 AKI are depicted in Additional file Biochemical marker distributions and clinical characteristics are summarised in Additional file p = 0.041), RBC (p < 0.001), HTC (p = 0.001), Haemoglobin (p < 0.001), MPV (p < 0.001), MCV (p = 0.095), Sodium (p = 0.034), Potassium (p < 0.001), Chloride (p = 0.069), Urea (p < 0.001), Creatinine (p < 0.001), Albumin (p < 0.001), age (p < 0.001), gender (p = 0.027), NSAID (p = 0.009), ACEI (p = 0.037), diabetes (p = 0.009), heart failure (p < 0.001), CKD (p < 0.001) and VD  (p = 0.006) were associated with the development of any Stage AKI. Despite the fact that the p value of the type of admission (p = 0.803 > 0.25) did not fulfil the selection criteria, it was considered by the authors that it could serve as a complementary predictor despite the univariable prefiltering [p = 0.033), Urea (p = 0.007), Albumin (p < 0.001), age (p < 0.001), type of admission (p = 0.018), heart failure (p < 0.001) and CKD (p = 0.013) were retained for model development , HCT (p = 0.066), Haemoglobin (p = 0.009), MPV (p = 0.008), Potassium (p < 0.001), Urea (p < 0.001), Creatinine (p < 0.001), Albumin (p < 0.001), age (p < 0.001), NSAID (p = 0.152), ACEI (p = 0.201), diabetes (p = 0.017), heart failure (p = 0.001), CKD (p < 0.001) and VD  (p = 0.214) predicted patients diagnosed with Stage 2 or Stage 3 AKI (Table p = 0.673) was once again included in the multivariable analysis. However, following forward selection, Urea (p < 0.001), Albumin (p = 0.002) and age (p = 0.016) were identified as the most suitable risk factors , Potassium (p < 0.001), Chloride (p = 0.15), Urea (p < 0.001), Creatinine (p < 0.001), Albumin (p = 0.011) and CKD (p = 0.002) were identified as potential predictors of Stage 3 AKI using univariable analysis (Table p = 0.261) and type of admission (p = 0.340) were added to the variables identified by the univariable analysis. Following forward selection, Chloride (p = 0.001), Creatinine (p < 0.001) and Albumin (p = 0.023) were included in the model  of each FLS  Fig.  and of eFor FLS I, AUC during validation was 0.7 (95% CI: 0.64–0.77) with SN and SP holding the values of 0.62 and 0.64 respectively. Positive predictive value (PPV) 0.1 and negative predictive value (NPV) was 0.96  was 0.77 (95% CI: 0.69–0.85), SN = 0.61, SP = 0.73, PPV = 0.06 and NPV = 0.99. For the case of MLR II, the AUC dropped to 0.74 (95% CI: 0.65–0.83) versus FLS II whilst SP changed slightly to 0.72 , producing a sensitivity of 1.0 and a specificity of 0.83, with PPV = 0.08 and NPV = 1.0. Although the AUC generated by MLR III mirrored that of FLS III, values of SP and PPV dropped to 0.79 and 0.06 respectively . For Stage 2 or 3 AKI, FLS II model performance dropped comparatively , to a level lower than the AUC = 0.9 (95% CI: 0.9–0.9) and AUC = 0.87 (95% CI: 0.87–0.87) resulting from the application of a Gradient Boosting Machine algorithm forecasting patients developing Stage 2 AKI within 24 and 48 h respectively . HoweverAnother attempt at stratifying the risk of AKI development in-hospital used electronic patient data gathered 24 h either side of admission. Although categories employed to portray disease severity do not match AKI Stages used in this research and in clinical practice, an AUC of 0.75 (95% CI: 0.73–0.76) resulted . This coIn addition to forecasting AKI up to a week prior to development, our study utilises data obtained 24 h either side of admission, thereby reducing computational burden. This contrasts to an approach which uses data gathered across varying windows, up to 30 days prior to admission . AlthougOur study performs less well than that employing up to five modelling techniques using data collected within the first 24 h of a hospital stay from patients aged 60 or over  . HoweverFLS I, however, has comparable performance to all of these studies, and uses data from both medical and surgical patients and both emergency and non-emergency.According to our study data, mortality from AKI across our study period stood at 38% for Stage 3 and 17% for both AKI Stage 1 and AKI Stage 2. Given approximately 30% of cases are considered preventable, we propose that use of an accurate risk assessment tool for the development of in-hospital AKI Stage 3 could save lives. Based on the results presented in this study, the use of FLS III could evolve the potential to avoid some of these deaths.The ability of FLS III to predict patients at low risk of AKI Stage 3 is not as good as it is for those at high risk. Falsely mitigating against some conditions, e.g. Deep Vein Thrombosis, can bring about unacceptable risks, i.e. bleeds. However, we consider the potential harm to the patient from inappropriate mitigation is likely to be sufficiently outweighed by the marginal risk caused by withholding measures. So, the use of a tool such as FLS III is worthy of health economic testing for the prevention of AKI Stage 3 in hospital.Reviewing their current condition and current diagnosis;considering alternative causes of illness (particularly searching for signs of sepsis);reviewing dates of recent and/or planned iodinated contrast scans;reviewing their current medication ;examining their fluid status and assessing the need for IV fluids ; andplanning future blood tests and physiological observation monitoring.A model of mitigation would include informing the patient’s doctor, pharmacist, and nurse of their elevated risk. This would then bring forward a reassessment of the patient that includes:These measures might be considered low-cost and add minimal risk for patients to whom they are unnecessarily introduced, including those falsely identified as being at high risk. The specificity results of this study indicate that mitigating measures will be unnecessarily introduced to roughly a third of patients carrying low risk who are expected to stay more than two days in hospital. In addition, given the lack of intrusiveness as these measures are introduced to patients, this is likely to be considered acceptable.The performance of each FLS was assessed against MLR, a methodology more traditionally employed in developing risk assessment frameworks in healthcare. In all three models, the performance of FLS was at least as good as MLR, perhaps signalling that FLS could be employed more widely in health risk assessment and epidemiological research.It is hypothesised that by identifying a patient’s risk of developing AKI Stage 3 at the earliest point possible during a patient spell this will increase survival. Based on the data period within this study, the fraction of patients dying in hospital following a diagnosis of Stage 3 AKI during hospitalisation stands at 38% (25 out of 66). Assuming one fifth of these cases were preventable and FLS III would have early identified 80%, use of this risk assessment tool could have prevented approximately 4 deaths during the same hospital stay.Of 304 patients who went on to develop Stage 1 or Stage 2 AKI, 53 died. Assuming one fifth preventable and a FLS sensitivity of 60%, a further 6 lives might have been saved with early risk assessment.However, these claims should be tempered because later development of AKI can be a signature of a general end-of-life decline and greater mortality risk . NeverthAcute Kidney Injury can develop in patients in hospital with the severest type, Stage 3, having a mortality rate of between 30 and 40%. With appropriate mitigation, some cases can be avoided. We have used anonymised data taken from patients as they were admitted to hospital to create risk assessment models using Fuzzy Logic and multivariable logistic regression. The models were trained to recognise patterns in data and distinguish between those patients who went onto develop AKI any Stage, AKI Stage 2 and 3, or AKI Stage 3 and those who did not develop AKI at all. This was the first attempt at building separate models for the three currently recognised degrees of AKI severity and upon testing the models, our ability to predict AKI Stage 3 was very strong, albeit on a small number of cases. Subsequently, our view is given that AKI is preventable in some cases and any technology containing these models is designed to be deployed early on in a patient’s stay, it has the potential to save lives.Additional file 1:The additional file contains: 1) Flow diagram of the fuzzy modelling algorithm, 2) additional information regarding patient characteristics, 3) FLS membership function parameters, rules and consequent parameters, 4) MLR models’. (DOCX 74 kb)


It was demonstrated that tolvaptan get slower decline in Kidney function compared with placebo. Idiosyncratic hepatic toxicity was described in patients receiving tolvaptan, with elevations in aminotransferases levels. We describe the first case reported in the literature in which hepatic toxicity is caused by the association of amoxicillin/clavulanic acid and tolvaptan.Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary disease characterized by the presence of renal cysts. Over time the expanding cysts lead to progressive renal failure. The use of tolvaptan, a VA 41 years old woman with diagnosis of ADPKD had been in treatment with tolvaptan for 16 weeks when an elevation of liver enzyme levels was detected. She had taken autonomously amoxicillin/clavulanic acid (in doses of 825/175 mg twice a day for 7 days) about 5 weeks before.The timing of the event and the kind of hepatocellular injury could be attributed to the concomitance of medication of tolvaptan and amoxicillin/clavulanic acid.We highlight the need to careful monitor hepatic enzyme levels in order to recognize early hepatic side effects in ADPKD patients in treatment with tolvaptan and amoxicillin/clavulanic acid. ADPKD is the most common hereditary kidney disorder, accounting for about 10% of patients on dialysis or living with a renal transplant –4.For many years, the strategies employed to improve the quality of life and the patients’ life span were only based on lifestyle modifications, treatment of hypertension and renal and extra-renal complications.In the last decade, advanced knowledge in genetics and molecular pathobiology of ADPKD focused on some aberrant molecular pathways involved in the pathogenesis of the disease , 3 leadiAntidiuretic hormone arginine vasopressin promotes kidney-cyst cell proliferation and luminal fluid secretion by the up-regulation of its second messenger adenosine-3′,5′-cyclic monophosphate (cAMP) .2-receptor antagonist, has been shown to both reduce cyst burden and slow disease progression [Tolvaptan, a Vgression –8.In the Tolvaptan Efficacy and Safety in Management of Autosomal Dominant Polycystic Kidney Disease and Its Outcomes (TEMPO) 3:4 trial, tolvaptan compared with placebo reduces kidney growth and the decline of estimated glomerular filtration rate (eGFR) over a 3 years period .Adverse events were related predominantly to increased acquaresis . Elevations of liver enzyme levels were observed. More patients who received tolvaptan compared to those who received placebo 4.9% vs 1.2%) had reported levels of alanine aminotransferase (ALT) more than 3 times the upper limit of the normal range. This condition was defined as potentially clinical important. All these conditions became reversible after tolvaptan interruption or discontinuation. No persistent hepatic injury was reported [% vs 1.2%Based on the results of the TEMPO 3:4 trial, the European Medicines Agency (EMA) approved in May 2015 the use of tolvaptan (JINARC®) for ADPKD , 11.A 41 years old woman with a medical history of hypertension and diagnosis of ADPKD was eligibile for treatment with tolvaptan. She was attending the Nephrology Center of the Federico II University of Naples. The patient had an average estimated glomerular filtration rate (eGFR) of 45.2 mL/min/1.73 m2 (CKD-EPI), CKD stage 3, serum creatinine was 1.42 mg/dl.Jinarc® is to be administered twice daily in split dose regimens of 45 mg + 15 mg, 60 mg + 30 mg or 90 mg + 30 mg. The initial dose is 60 mg tolvaptan per day as a split-dose regimen of 45 mg + 15 mg. The initial dose is to be titrated upward to a split-dose regimen of 90 mg tolvaptan (60 mg + 30 mg) per day and then to a target split-dose regimen of 120 mg tolvaptan (90 mg + 30 mg) per day, if tolerated. Patients have to be maintained on the highest tolerable tolvaptan dose.According to the criteria for use of tolvaptan in ADPKD set by the Agenzia Italiana del Farmaco (AIFA), blood testing for hepatic transaminases and bilirubin is required prior to initiation of Jinarc, continuing monthly for 18 months and at regular 3-monthly intervals thereafter to mitigate the risk of significant and/or irreversible liver injury. Concurrent monitoring for symptoms is also recommended.The patient started with a split-dose regimen of 45 mg and 15 mg per day. The use of drug inhibiting the cytochrome P-450 enzyme CYP 3A4 was excluded.After 28 days, the initial dose was titrated upward to a split-dose regimen of 90 mg tolvaptan (60 mg + 30 mg) per day (week 4). Physical examination, assessment of vital signs, blood and urine tests were performed before the increase. She reported no adverse effects, with the exception of effects related to acquaresis.Dose was titrated to a target split-dose regimen of 120 mg tolvaptan (90 mg + 30 mg) per day after 28 days, according to patient tolerability and examinations (week 8).On week 12, blood testing was performed. Both transaminase levels  had increased slightly although remaining below the upper limit of the normal range . There were no alterations in the other laboratory values and in the coagulation test, bilirubin total (BT) level was 0.25 mg/dl, alkaline phosphatase (ALP) 32 U/L , albumin 4.3 g/dl, γ-glutamyltransferase (GGT) 18 UI/ml, pseudocholinesterase (CHE) 8041 UI/ml, creatinine 1.8 mg/dl (vs 1.42 mg/dl before starting tolvaptan). Test for hepatitis A, B and C were negative. An abdominal ultrasonografy revealed no biliary obstruction, cancer or masses, but only multiple liver cysts, as reported in the precedent exams. The patient reported no symptoms; therefore, no further investigation about non hepatitis viral infections was performed. The patient had neither previous history of liver disease, nor of excessive alcohol intake, heart failure, shock or sepsis. She was carefully questioned about events or drug that could have led to liver injury. She only reported the use of antibiotics for fever and pharyngitis about 5 weeks before the start of transaminase increase (the antibiotic was taken at week 11). She had taken amoxicillin/clavulanic acid in doses of 825/175 mg twice a day for 1 week, without medical prescription.As recommended, tolvaptan was immediately discontinued (week 16) .Two weeks later (week 18), the laboratory report showed an additional increase of aminotransferases: AST 172 U/L, ALT 711 U/L , followed 1 week later (week 19) by a further increase: AST 319 U/L, ALT 808 U/L. The blood test confirmed no more alterations.Aminotransferase began to decrease after 2 more weeks week 21): AST 220 U/L, ALT 565 U/L, and returned to normal levels after 2 months prior to presentation (week 24): AST 38 U/L, ALT 48 UI/L, . As a matter of fact, in this case report, liver injury classified according to laboratory parameters as acute hepatocellular injury, occurred about 3 months after the beginning of the treatment with tolvaptan and 5 weeks after the therapy with amoxicillin/clavulanic acid. Moreover, the liver disease resolved itself after the withdrawal of the medications and there is no other possible diagnosis.There is another case reported in literature that describes an elevation of AST, ALT and BT after a single dose of amoxicillin/clavulanic acid of 8 g between 2 and 3 months before liver injury . But in Furthermore in our case it is impossible to state whether the hepatotoxicity was related to tolvaptan, amoxicillin/clavulanic, or their combined use. As a matter of fact, it could just be a simple independent reaction to either tolvaptan or amoxicillin/clavulanic acid, rather than their synergy.On the other hand, we cannot exclude the causality of tolvaptan–induced hepatotoxicity, even if its readministration did not result in recurrence. Evidences show that approximately half of the subjects were able to tolerate the drug when it was reintroduced, after hepatic injury, suggesting that a form of adaptation or drug tolerance occurs. However, the other half experienced rapid ALT elevations upon re-exposure and tolvaptan treatment had to be permanently withdrawn . We deciIn conclusion, this represents the first case reporting hepatic toxicity in an ADPKD patient during the combined therapy of amoxicillin/clavulanic acid and tolvaptan; therefore, more caution should be used when prescribing amoxicillin-clavulanic acid in ADPKD patients treated with tolvaptan.