R2 = 0.4660, p < 0.01), creatinine , and urea . Furthermore, the prediction of the blood markers of kidney transplant function using the urine SERS spectra was indicated by R2 = 0.7628 (p < 0.01) for serum creatinine and R2 = 0.6539 (p < 0.01) for blood urea nitrogen. This preliminary study suggested that the urine SERS spectral analysis could be used as a convenient method for rapid assessment of kidney transplant function.Routine monitoring of kidney transplant function is required for the standard care in post-transplantation management, including frequent measurements of serum creatinine with or without kidney biopsy. However, the invasiveness of these methods with potential for clinically significant complications makes them less than ideal. The objective of this study was to develop a non-invasive tool to monitor the kidney transplant function by using Surface-Enhanced Raman Spectroscopy (SERS). Urine and blood samples were collected from kidney transplant recipients after surgery. Silver nanoparticle-based SERS spectra of the urine were measured and evaluated using partial least squires (PLS) analysis. The SERS spectra were compared with conventional chemical markers of kidney transplant function to assess its predictive ability. A total of 110 kidney transplant recipients were included in this study. PLS results showed significant correlation with urine protein ( Donor and recipient factors, surgical as well as immunologic factors may all contribute or cause the impaired graft function either immediately following the transplantation operation, termed delayed graft function, or at any other time points after transplantation5. Allograft dysfunction is typically revealed by serial laboratory tests showing rising serum creatinine (SCr), along with other potential findings including decreased urine volume, and an increase in blood pressure, pyuria and proteinuria6. Acute rejection (AR), acute tubular necrosis (ATN) and cyclosporine/tacrolimus toxicity are some of the more common etiologies of kidney transplant dysfunction9. If these issues are not properly identified and addressed, they may lead to permanent deterioration of kidney transplant function. Thus, patients with kidney transplants need routine monitoring of their kidney transplant function as a part of post-transplantation management12.Kidney transplantation represents a gold standard treatment for patients with end-stage renal disease (ESRD)13. If AR is suspected, renal transplant ultrasounds are often performed followed by transplant biopsy to confirm a pathologic diagnosis for decision of the treatment plan. The procedure of the biopsy can be uncomfortable for patients and can lead to clinically significant complications, such as bleeding or allograft loss in the most extreme cases. Therefore, a non-invasive clinical tool which accurately determines the kidney transplant function is definitely desirable from both a patient and clinician perspective.Conventional methods for evaluating the functional status of transplanted kidneys include clinical assessment of the patients based on volume status and urine output, serial SCr measurements with estimated glomerular filtration rates (eGFR), and even isotope-based examinations in rare circumstances17. The intensity of Raman scattering is proportional to the magnitude of the change in molecular polarization. Previously, the application of RS was limited because of its low sensitivity, high costs and lack of accessible, on-site analysis18. Recent technological advance of RS has brought forth a readily available, portable and relatively cheap RS device19. Surface-enhanced Raman Spectroscopy (SERS) is a subtype of RS, designed specifically to increase its sensitivity of low concentration analytes, in which the treatment of a sample with an enhancement material significantly amplifies the electromagnetic fields of adsorbate molecules that are generated by the excitation of localized surface plasmons (LSP)20. Several SERS enhancement materials have been described . These metal enhancers can increase the intensity of the Raman signal up to 104 to 106 fold21. Our previous study demonstrated that our home-made colloidal silver nanoparticles (Ag NP) are a reliable and reproducible enhancement material for SERS22. The objective of the current study was to evaluate the feasibility of using Ag NP-based SERS of patients’ urine samples to determine the kidney transplant function as compared to its conventional biochemical markers.Raman spectroscopy (RS) is a specialized and non-invasive tool that emits an incident laser light though a fluid sample, resulting in scattered photons that produce a shift in wavelength dependent on the chemical composition of the fluidg for 10 min to remove the cells and debris, and only the resultant supernatants were stored in aliquots at − 80 °C refrigerator prior to use. The levels of protein and creatinine in the urine samples were determined in the Clinical Chemistry Laboratory at Vancouver General Hospital .Both urine and blood samples were collected from kidney transplant patients in the Department of Urology of West China Hospital in Sichuan University. These samples were collected concurrently within 3 days of the kidney transplant surgery. As following a standard operating procedure, the blood samples were sent to the clinical biochemistry laboratory in the West China Hospital for SCr and blood urea nitrogen (BUN) determination. The urine samples were collected by the patients themselves by using a 50 mL sterile tube. The urine samples were then delivered to the hospital laboratory within 8 h after collection. The urine tubes were centrifuged at 1500×24. Briefly, the glassware used in the following procedures was cleaned with chromic acid lotion, and then rinsed thoroughly with milli-Q water before use. 4.5 mL of 0.1 M NaOH solution was rapidly added to 5 mL of 6 × 10−2 M hydroxylamine hydrochloride solution, and the mixture was stirred until a homogeneous suspension was formed. The resultant solution was mixed with 90 mL of 1.11 × 10−3 M AgNO3 aqueous solution at room temperature until the presence of a milky-grey color. This Ag colloid suspension was then stored in the dark before use. In order to ensure consistent shape and size of the Ag NP, all the experimental steps and processes were standardized. Once ready for use, the Ag colloids were pelleted by centrifugation at 10,000×g for 10 min. A transmission electron microscopy (TEM) image of Ag nanoparticles was taken with the sizes of the synthesized Ag NP follow a normal distribution with a mean diameter of 35 ± 5 nm  equipped with a 785 nm diode laser for Raman excitation, similar to the prototype reported previously by Zhao et al.–1 cm were extracted by subtracting the fluorescence background using the fifth-order polynomial fitting algorithm27. Partial least squares (PLS) regression with leave-one-out cross-validation (LOO-CV) of the whole spectra was used to quantify the biochemical parameters of kidney transplant function including urine protein, urine creatinine (UCr), urine urea, SCr and BUN. The concentrations of these parameters obtained from blood/urine biochemistry analysis was used as a gold standard to compare the SERS urine analysis. TIBCO STATISTICA software  was used for PLS regression analysis.The resultant SERS spectrum of the urine samples in the wavelength range of 500–1800This study ) was approved by the ethics committee at West China Hospital of Sichuan University, and all participants signed the informed consent prior to sample collection. Sichuan University and West China Hospital ethics committee confirmed that all the research activities were performed in accordance with relevant guidelines and regulations.A total of 110 kidney transplant recipients  participated in this study. The urine biochemistry results revealed that the range of urine protein was 0.05–6.83 g/L, UCr 1.03–22.49 mmol/L and urine urea 29.6–535.8 mmol/L. The biochemical results of blood tests indicated that the range of SCr was 39–1461 µmol/L and BUN 3.2–25.1 mmol/L. The demographic characteristics of patients and their urine and blood test results were summarized in Table −1, which biochemically corresponds to cytosine, urea, cholesterol, and CH3 band, respectively28. Other biochemical assignments of the SERS peaks showed in Fig. R2 > 0.67 indicated the high predictive accuracy, R2 range of 0.33–0.67 the moderate predictive accuracy, R2 range of 0.19–0.33 the low predictive accuracy and R2 < 0.19 unacceptable variables29. The urine results revealed an R2 = 0.4660 (moderate) for urine protein (p < 0.001), R2 = 0.8106 (high) for UCr (p < 0.001), and R2 = 0.7808 (high) for urine urea (p < 0.001). Figure R2 = 0.7628 (high) for SCr (p < 0.001) and R2 = 0.6539 (moderate to high) for BUN (p < 0.001). Because the low concentrations of kidney transplant function biomarkers—normal transplant function are not a concern in clinical care, we calculated the standard deviation (SD) of their high concentrations according to the borderline level. For the urine protein < 0.3 g/L, the calculated SD was 0.33 g/L, and > 0.3 g/L, the SD 0.44 g/L. For the UCr < 7 mmol/L, the calculated SD was 1.69 mmol/L, and > 7 mmol/L, the SD 1.64 mmol/L. For the urine urea < 266 mmol/L, the SD was 47.88 mmol/L, and > 266 mmol/L, the SD 29.57 mmol/L. The serum biomarkers are mainly used for the kidney transplant function in the clinical practice. For the SCr < 110 mmol/L, the SD was 87.98 mmol/L, and > 110 mmol/L, the SD 108.21 mmol/L. For the BUN < 7.7 mmol/L, the SD was 2.15, and > 7.7 mmol/L, the SD 2.32 mmol/L.The SERS spectra of Ag NP/ urine mixtures were measured under the same conditions (1 day after mixture preparation). The SERS spectra of samples revealed many dominant vibration bands, implying a tight interaction between the Ag colloid and urine molecules resulting in the signal enhancement. As shown in Fig. In clinics, post-kidney transplant function is mainly determined by monitoring the level of SCr and calculating the eGFR. This involves needle-obtained blood specimens, which can be bothersome and even traumatize some patients. There are other methods to evaluate the renal function, such as 24-h UCr clearance and nuclear medicine scans. However, these methods are rarely performed for the post-transplant care secondary to multiple factors, including logistics, expense and concerns regarding the inaccuracy when the kidney transplant function is poor. Therefore, a rapid and non-invasive tool to monitor the kidney transplant function would be a desirable alternative to these conventional means. Nowadays, non-invasive imaging methods including ultrasonography (US), computed tomography (CT), magnetic resonance imaging (MRI), and renal scintigraphy (RSG) are commonly used in clinics; however, these imaging methods have disadvantages over one or the other. US has a high inter-observer variability that makes difficulties in imaging interpretation. CT and MRI can provide the details of renal transplant anatomy and the surrounding tissue, but it is hard to measure the kidney function using these techniques. RSG, on the other hand, can shows the excretion patterns, leakage, and morphology of the kidney, it however cannot provide necessary information to differentiate between AR and ATN. Furthermore, the most concern about the nuclide seriation is low discharge rate due to the poor kidney transplant function.Urine, a product of the kidney, contains thousands of molecules that reflect the body’s homeostasis and metabolic state at any given time. Also, it represents a specimen that is readily available via non-invasive means. Of most significance for renal function and kidney health can be determined by the urine protein, UCr and urine urea measurements. For example, healthy kidneys do not allow much protein to pass through the glomerular filters, but a damaged or diseased kidney may leak a large amount of the proteins, such as albumin, from the blood into the urine. Thus, it is reasonable to assess the kidney transplant function through the urine chemical measurement.24. It has been acknowledged that the chemical composition of the urine from kidney transplant recipients is more complicated than those from the rat models and non-kidney transplant patients (e.g. kidney disease patients), as transplant patients have some remnant kidney function and take immunosuppressants routinely . The different therapeutic drugs and the activity of the remnant kidney might result in different urine composition from one patients to the other. In the present study, we successfully showed that the urine SERS spectra of a small cohort of the kidney transplant recipients are capable of predicting essential kidney biomarkers in the presence of the interference from these uncertain (remnant kidney function and therapeutic drugs) and other unknown factors. There are other studies in literature have also have assessed the abilities of RS to detect the biochemical constituent concentrations in the urine. For example, the creatinine in unaltered human urine from a calibration dataset using RS is indicated by an Root Mean Square Error (RMSE) of 0.4332 mmol/L30. In the measurement of 61 human urine samples (excluding 12 outliers) using liquid-core optical fiber-based RS and multivariate statistical analysis, and the statistical error for creatinine quantification is 0.4508 mmol/L31. In Dou et al.’s study, the correlation coefficients between the concentrations of the urea and creatinine in the urine samples are presented by the intensity of the Raman peaks at 1013 and 692 cm−1 in RS, which show a R2 = 0.991 and R2 = 0.998, respectively. And the detection limits in this study are 174.93 and 13.2603 mmol/L, respectively32. Wang et al. have also investigated the SERS for the measurement of the concentration of creatinine in both artificial and human urine samples, demonstrating a correlation coefficient of r = 0.99 in the artificial urine samples over the range of 3.3946–13.6139 mmol/L, and r = 0.96 in the human urine samples over the range of 0.2263–10.1662 mmol/L33. In a follow-up study, these authors have reported R2 = 0.968 in a linear correlation of creatinine concentrations with the range from 0.442 to 15.1167 mmol/L34. Saatkamp et al. report the partial selected RS spectra to predict the urine urea and UCr concentrations with the correlation coefficient of r = 0.90 and r = 0.91, respectively35. In a separated study, they have identified the peaks specifically for the creatinine with R2 = 0.968 by using creatinine spiked in three different solutions: creatinine in water, mixture of creatinine and urea in water, and creatinine in artificial urine within physiologically relevant concentrations36. All these results may support the concept that SERS may be considered to be a reliable technique for monitoring the functions of transplanted kidneys non-invasively and rapidly.In the present study, we used SERS as a non-invasive tool to assess the kidney transplant function after surgery. Our data potentially demonstrated the feasibility of using SERS to monitor the kidney transplant function in the early post-operative period. We found a strong correlation between SERS spectra and biochemical substances in both the urine and the blood  (p < 0.01). Previously, we demonstrated that SERS spectra are able to detect or predict the renal damage in a rat model37. This is especially true for those transplant patients who regularly take immunosuppressant drugs, to whom the rate of infection and mortality are higher than the surgical patients without transplants38. To minimize the infection rates, a non-invasive method is required and should be the best choice. Moreover, for kidney transplant patients, a routine monitoring of the transplant function after the surgery is very critical for prolonging the survival of the transplants. The results from the present study indicate that SERS may provide a convenient, non-invasive and pain-free option for routine check-up of those patients. Second, SERS can detect the change of multiple substances in one sample quickly. Conventional biochemical methods such as enzyme-linked immunoreactivity method only examine one substance at a time, which is time-consuming. In technology, SERS is designed to detect the covalent bond inside the molecules which is different among different molecules. Thus, it is capable of differentiate the multiple substance at the same time. Third, potentially SERS is a more convenient, cheaper (cost-effective) and faster tool than conventional techniques in clinical biochemical laboratories. The SERS procedure can be completed as short as 1 s in the instrument, and the data can be calculated and reported immediately. The last but the least, it has great potential in other fields, such as rapid assessment of the deceased kidney donors where the baseline renal function can sometimes be in question.As compared to conventional methods, SERS has many advantages. First, it is non-invasive. By using an optical instrument, it does not cause any damage to the patient’s body and the specimens can be non-invasively and conveniently obtained. In clinic, one of the severe complications after surgery is infectionOne has to acknowledge the limitation of this preliminary study, which was mainly related to the limited number of patients (110 patients) from a single transplant center. In this small cohort, the distribution of some variables such as urine protein was not such large, the ability of Ag NP-based SERS was low in the prediction of the urine protein and perhaps the SCr.In this study, we have demonstrated the highly feasibility of using non-invasive Ag NP-based SERS of the urine to predict the kidney transplant function, indicated by the fact that the urine spectra of SERS are predictive of both the urine and blood biochemical constituents  that conventionally reflect the health of the kidney. Further researches are warranted, ideally in a larger cohort, to validate the findings from this study and to build more precise models for monitoring the kidney transplant function in patients in future.Supplementary Figure 1.


GLA deletion, not amenable to chaperone therapy, leading to profound reduction in α-galactosidase A  enzyme activity. Over a 3-year period of ERT, increasing IgG antibody titres against α-gal A were noted. After starting ERT serial urine globotriaosylceramide (Gb3) measurements showed an upward trend from 333 to 2260 μg/mmol creatinine for patient 1 and 1165 to 2260 μg/mmol creatinine for patient 2. Markedly increased levels of urine and plasma globotriaosylsphingosine (Lyso-Gb3) analogues were also found. The patients experienced recurrent infusion-associated reactions necessitating premedication and prolonged infusion times. Over the 3-year period of ERT, the patients experienced continued malaise, gastrointestinal symptoms and neuropathic pain. In addition, they had increasing anxiety related to their disease and apparent lack of response to ERT which led to a decision to ultimately stop ERT. No other approved treatment options are currently available for these patients. It is possible that the rapid development of the high antidrug neutralizing antibody (ADA) titres is related to the large GLA deletion leading to virtually absent enzyme activity. It remains unclear if their symptomatology during the period of receiving ERT is related to lack of its efficacy, the rising ADA titres, or both. These two patients highlight the need for further research into the management of antidrug antibodies and additional therapeutic approaches for FD.Enzyme replacement therapy (ERT) has been shown to stabilize certain aspects of Fabry disease (FD). However, in some patients on ERT, high antibody titres have been documented, with limited clinical improvement in systemic manifestations and often with significant adverse drug reactions. We present two related adolescent males with a 4.5 kb The development of very high antidrug antibody titres in response to ERT in two related adolescent males with FD highlight the need for other therapeutic options for patients in whom ERT or other currently approved therapies does not meet their treatment needs. Several treatment modalities are currently approved including enzyme replacement therapy (ERT) in the forms of agalsidase alfa (0.2 mg/kg intravenous (IV) every 2 weeks, Replagal™, Shire/Takeda Pharmaceutical, Lexington, MA, USA) and agalsidase-beta  and chaperone therapy with migalastat  [3 and plasma Lyso-Gb3 levels suggesting reduced action of α-gal A as reviewed by [Fabry disease (FD)  . ERT hasNJ, USA) . In CanaNJ, USA) ) and theNJ, USA) ,33,39. Tiewed by . Neutraliewed by . The assiewed by . However2Two male first cousins described below are part of a large kindred with FD. Upon confirming the FD diagnosis, both patients were enrolled in the CFDI study and the recommended schedule of baseline clinical assessments as per the CFDI protocol was followed . The patPatient 1 was diagnosed at 10 years of age. The diagnosis of FD had just been established in his maternal uncle. Patient 1 had long-standing intractable abdominal pain with nausea, cramping, diarrhea, constipation, and acroparesthesiae in his hands and feet dating back to early childhood. There was no symptomatic improvement with 6 months of treatment with ibuprofen, acetaminophen, and gabapentin. Intravenous ERT with agalsidase beta at recommended doses was initiated. While receiving ERT he required premedication with acetaminophen and antihistamines, as well as prolongation of infusion times to minimize IARs including fever and chills.Patient 2 was diagnosed at 9 years of age after the diagnosis of FD was established in his maternal uncle. He had long-standing intractable abdominal pain and acroparesthesiae, as well as heat intolerance, headaches, and tinnitus. Symptoms were unchanged after 6 months of treatment with omeprazole, loperamide, acetaminophen, and ibuprofen. He thus was started on ERT with agalsidase beta at standard dose. He also required premedication with acetaminophen and antihistamines, as well as prolongation of infusion times to minimize IARs with fever, chills and abdominal pain.After 3 years of ERT, with no improvement in symptoms of severe abdominal pain and acroparesthesiae, ERT was discontinued in both patients. They continue to experience neuropathic pain and GI symptoms. Cardiac and renal function remained normal during the 3 years of ERT and the subsequent 3 years as expected given their young age. Echocardiograms and cardiac magnetic resonance images have remained normal and there has been no evidence of proteinuria with normal eGFR.3GLA gene and α-gal A enzyme activity measured by standard fluorometric enzyme assay were performed by Mount Sinai Genetics Testing Laboratory .Sanger sequencing of the IgG antidrug antibodies (ADA) against agalsidase beta were measured in serum samples by the enzyme linked immunosorbent assay (ELISA) , and IgE antibodies were measured by fluoroenzyme immunoassay  as previously described ,39.Serum samples were evaluated for the presence of anti- antibodies as previously described . To asse3 normalized to creatinine was analyzed according to the mass spectrophotometric method developed by Auray-Blais et al. [3(C17:0) (1 μg/100 μL) and 100 μL of creatinine-D3 (40 μg/100 μL)). Thereafter, the paper disk was eluted with 4 mL of MeOH. The sample was separated by liquid chromatography using a Zorbax Bonus-RP Guard column  and an Alliance 2795 HPLC system . The total of 8 Gb3 isoforms , the Gb3(C17:0) internal standard, the creatinine and the creatinine-D3 internal standard were analyzed using the multiple reaction monitoring mode (MRM) with a Quattro Micro triple quadrupole mass spectrometer (Waters Corp.). Seven-point calibration curves were used for the quantitation of Gb3 (0–20 μg/mL) and creatinine (0–10 μmol/mL). Urine sample aliquots from an untreated Fabry male and an untreated Fabry female were used for the quality controls and analyzed with a tolerance of ±25%.Urinary Gbs et al. . Briefly3 and analogues was conducted on plasma specimens collected in EDTA tubes and stored at -20 °C. The method developed by Boutin et al. [3 and analogues in plasma. A plasma volume of 100 μL was spiked with 500 μL of the glycinated Lyso-Gb3 standard . The samples were purified by solid phase extraction using a mixed-mode strong cation exchange (MCX) cartridge and separated by ultra-performance liquid chromatography (UPLC) using a BEH C18 column  and an Acquity I-Class system (Waters Corp). Lyso-Gb3 and its 6 analogues , and the Lyso-Gb3-Gly internal standard were analyzed using the MRM mode with a Xevo TQ-S mass spectrometer (Waters Corp.). A seven-point calibration curve (0–400 nM) was used for the quantitation of Lyso-Gb3 and its analogues in plasma. Plasma sample aliquots from an untreated Fabry male and an untreated Fabry female were used for the quality controls and analyzed with a tolerance of ±20%.Analysis of plasma Lyso-Gbn et al. ,9, was u3 and analogues in urine. Urine specimens were stored at -20 °C before analysis. A urine volume of 500 μL was spiked with 500 μL of the glycinated Lyso-Gb3 standard . The samples were purified by solid phase extraction using a MCX cartridge, and separated by ultra-performance liquid chromatography (UPLC) using an Atlantis HILIC Silica column  and an Acquity I-Class system (Waters Corp). Lyso-Gb3 and its 7 analogues , and the Lyso-Gb3-Gly internal standard were analyzed using the MRM mode with a Xevo TQ-S mass spectrometer (Waters Corp.). An eleven-point calibration curve (0–700 nM) was used for the quantitation of Lyso-Gb3 and its analogues in urine. The creatinine concentration was used to normalize the Lyso-Gb3 results [The method developed by Lavoie et al. ,22 was u4GLA gene consists of 7 exons and covers 12 kilobases (kb) of genomic DNA. Results of molecular testing in this family showed a 4.5 kb genomic GLA deletion, encompassing ~37% of the gene, in all affected family members tested. At the cDNA level, the deletion begins at 533 bases upstream from the first codon of GLA and extends to nucleotide 1277 (GLAc.370-533_c.1277del4.5).The Biochemical α-gal A enzyme activity testing in patient 1 revealed very low enzyme activity in leukocytes of 0.19 nmol/h/mg protein  and plasma 0.30 nmol/h/mL . In patient 2, results showed similarly very low α-galA enzyme activity in leukocytes 1.21 nmol/h/mg protein and in plasma 0.30 nmol/h/mL.Results of serum IgG antibody titres for patients 1 and 2 in relation to ERT time course are plotted in 3 for patients 1 and 2 in relation to ERT time course are plotted in 3 showed an upward trend despite ERT from 333 to a peak of 2260 μg/mmol creatinine. Similarly, in patient 2, urinary Gb3 showed an upward trend from 503 to a peak of 1250 μg/mmol creatinine. In patient 1, plasma and urinary Lyso-Gb3 measured 77.3 nmol/L  and 50 pmol/mmol creatinine respectively  .Results of urinary Gbtected”)  A and B.ectively  C and D 5This report documents the persisting very high ADA response to ERT in two male adolescent FD patients receiving ERT with standard dose agalsidase beta.A key concern in the first FD ERT trials was immediate IARs. IARs were originally felt to be IgE mediated but are now known to be “anaphylactoid” (compound mediated) and IgG mediated consistent with the fact that both our patients had persisting severe IARs but remained IgE negative. Premedication protocols, usually a combination of single dose acetaminophen or a non-steroidal anti-inflammatory drug, antihistamine and corticosteroid, along with increasing infusion time, to prevent such reactions have been established successfully and are used as required . While r3 clearance in skin biopsies of patients treated with ERT and high antibody titres. A correlation between ADA levels and plasma Lyso-Gb3 has been confirmed [3 [3 levels, disease severity scores and subjective symptoms in a group of 168 patients, Lenders et al. [3 levels, as well as worsening disease severity scores in patients with serum-mediated enzyme inhibition. As well, results reported by Auray-Blais et al. [3 analogues (+16), (+34), and (+50) were positively associated with the severity of disease for the left ventricular mass index and also the Mainz Severity Score Index when adjusted for age and gender. High levels of these glycosphingolipids were also identified in children. Moreover, and similar to the results obtained for patients 1 and 2 where the analogues of Lyso-Gb3 in urine are markedly increased, a study in FD children showed that the urine Lyso-Gb3 analogues are sometimes increased greater than Lyso-Gb3 itself [To date, there remains no common ADA assay used in the care of FD patients. Assays are done via various techniques  and differ in their sensitivities and specificities which make comparisons difficult. An attempt to develop a common assay has failed . Seroconversion with ADA is reported in 73% males as opposed to 12% females treated with agalsidase beta. This is likely due to complete absence of native enzyme in males with classical FD while males with missense mutations, who express residual α-Gal A enzyme activity, are far less likely to develop antibodies . Neutralonfirmed . The samirmed [3 . Similars et al.  confirmes et al.  from a l3 itself  showed t3 and Lyso-Gb3 build up. We had previously measured the levels of urine Gb3 in “liquid” urine specimens from 33 male FD patients receiving ERT and the results ranged from 0.45 μg/mmol creatinine to 129 μg/mmol creatinine [3 isoforms analyzed on filter paper  were taken in account for the unit conversion, and the outlier values (over 1.5 times the interquartile range) were excluded.The aim of ERT is to minimize FD symptoms, prevent disease progression and Gbeatinine . These v3 levels greatly exceeded this range while receiving ERT suggesting a blunting or lack of effectiveness of ERT in these 2 patients  , as well


Plaque rupture (PR) and plaque erosion (PE) are main causes of acute myocardial infarction with different demographic and histology characteristics and need different treatment strategy. PR and PE can be identified with optical coherence tomography (OCT) accurately, but convenient and effective noninvasive markers for them are rarely found. History of diabetes mellitus (DM) was reported to be a potential predictor of PR in ST-segment elevated myocardial infarction (STEMI) patients, but the predictive value of other glucose-related variables for it is still uncertain. Present study aimed to clear the relationship between some glucose-related variables and plaque morphology in patients with STEMI.We consecutively enrolled 872 STEMI patients and divided them into PR group (n = 616) and PE group (n = 256) based on OCT diagnostic criteria. The relationship of glucose-related variables, including random plasma glucose on admission (ARPG), glycosylated hemoglobin (HbA1c), post-PCI fasting plasma glucose (PFPG), DM history, glucose variable tendency (GVT) and the acute-to-chronic glycemic ratio (A/C), to the PR risk of STEMI patients was analyzed. The correlation between the glucose-related variables and plaque morphology was analyzed meanwhile.Among the glucose-related variables, ARPG and GVT were confirmed to be independent predictors for PR after adjusting for other traditional risk factors in nondiabetic patients. The higher the ARPG level, the more PR risk the STEMI patients had. And high HbA1c and APPG were demonstrated to have a weak and positive correlation with lipid constituents and stenosis degree of culprit vessel.Compared to HbA1c, DM history, and some other glucose-related variables, ARPG and GVT were risk factors for PR in STEMI patients, especially those without DM. And high HbA1c and ARPG were positively correlated with the development of vulnerable plaque in culprit vessels.Trial registration Present study is a retrospective one and the population came from the EROSION study of our center previously. It was approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University , and all patients provided written informed consent prior to the inclusion in the study and the investigation conformed to the principles outlined in the Declaration of Helsinki. Acute myocardial infarction (AMI) is the main cause of life threat in patients with coronary heart disease (CHD). Many studies have shown the positive correlation between the bad outcome of AMI and glucose-related variables, such as serum glycosylated hemoglobin HbA1c) ,,2], the This study is a subanalysis of the EROSION study . For stuCHD risk factors of interest included age, gender, hypertension, DM history, hyperlipemia, AMI history and current smoking. All individuals had a 12-lead or 18-lead electrocardiogram measured and left ventricular ejection fraction (LVEF) was measured within 24 h of admission by an experienced practitioner using Simpson’s biplane method of disks. Blood glucose-related variables , complete blood count, electrolytes, creatinine, lipid parameters, hypersensitive C-reactive protein (hs-CRP), and maximum Cardiac Troponin I (cTnI), as well as the data of coronary angiography and OCT images, were collected.Quantitative coronary angiography (QCA) was performed using the Cardiovascular Angiography Analysis System . Culprit vessels and lesion sites were identified, and the length of the culprit lesions, reference vessel diameter, minimal lesion diameter, and degree of diameter stenosis were measured. OCT data were recorded using the C7-XR OCT intravascular imaging system . Quantitative and qualitative analyses of underlying plaques were performed by two independent operators as described in a previous study , the plaDichotomous variables were expressed as n (%) and continuous variables as mean ± SD or median , with normality of variables determined by the Kolmogorov–Smirnov test. For categorical data, Chi-squared or Fisher’s exact test was used, with comparisons of continuous data done by Student’s t test for normal variables or the Mann–Whitney test for non-Gaussian variables. Inter- and intra-observer reliability was assessed by Kappa statistics. A two-tailed P-value < 0.05 was considered statistically significant.Patients were divided into four groups according to their blood ARPG levels: non AH group [ARPG < 6.1 mmol/L (< 110 mg/dL)]; mild AH group [6.1–6.9 mmol/L 110–125 mg/dL)]; moderate AH group [7.0–11.0 mmol/L (126–198 mg/dL)] and severe AH group [> 11.0 mmol/L (> 198 mg/dL)]. PFPG levels were stratified into four groups as follow: group I [PFPG < 5.6 mmol/L (< 100 mg/dL)]; group II [5.6–6.0 mmol/L (100–110 mg/dL)]; and group III [6.1–6.9 mmol/L (110–126 mg/dL)] and group IV [> 7.0 mmol/L (> 126 mg/dL)]. Patients were also divided into two groups according to their blood HbA1c levels: low HbA1c group (HbA1c < 6.5%) and high HbA1c group (HbA1c ≥ 6.5%). GVT was defined as the ARPG-to-PFPG ratio and grouped as GVT-normal group (ARPG < 6.1 mmol/L and PFPG < 5.6 mmol/L), GVT-up group , and GVT-down group . Chronic plasma glucose (CPG) was calculated by formula (CPG (mg/dL) = 28.7 × HbA1c-46.7). A/C was defined as the ARPG-to-CPG ratio  using R.Univariate and multivariate logistic regression analyses were applied to screen the glucose-related variables that were significantly associated with the identification of plaque type. A P < 0.05 denoted statistical significance. Significant variables were subsequently included in a differential diagnostic model to evaluate the diagnosis value. Furthermore, the correlation between abnormal blood glucose and plaque morphology was analyzed with Spearman correlation. These analyses were all produced by EmpowerStats . Preliminary analysis of ROC indicated that the area under curve (AUC) of ARPG was larger than that of GVT. Combined ARPG or GVT with the risk factors  to establish combined predictive models successively, the predictive efficiency and accuracy increased significantly. Added initial TIMI flow, culprit vessel and multi-vessel lesion into the combined model, the predictive ability increased more and to over 70%   HbA1c, ARPG, GVT, A/C, and DM history were significantly and positively associated with PR risk in STEMI patients before adjustment for traditional risk factors; (2) Among the glucose-related variables, ARPG and GVT were independent predictors of PR in STEMI patients without DM rather than in those with DM; (3) The higher the ARPG levels of the STEMI patient, the greater the risk of PR; (4) High ARPG and HbA1c were positively related with plaque vulnerability; (5) The higher the ARPG levels of the STEMI patient, the higher risk of TCFA and the bigger size of lipid arc among ARPG groups.The definite DM history has immediate association with worse cardiovascular outcomes . The relPrevious studies indicated that patients with AMI caused by plaque rupture had some clinical characteristics  same as In addition, present study only indicated the association of AH with PR in STEMI patients without DM but not those with DM. Elevated ARPG in nondiabetic patients may link to impaired glucose tolerance (IGT) which was more associated with PR than normal glucose tolerance (NGT) . On the Although no association between HbA1c and plaque morphology in STEMI patients was found by multivariable logistic regression analysis after adjustment for traditional risk factors, both HbA1c and ARPG were determined to be positively correlated with degree of lumen stenosis and the lipid content in the plaque on the evidence of imaging, and HbA1c seemed have more correlation than ARPG. It provided a clinical evidence for the role chronic hyperglycemia might play in the developing of vulnerable plaque which characterized with large lipid core. It may partly explain why patients with DM had a higher prevalence of lipid-rich plaque but similar prevalence of plaque rupture and plaque erosion was also been detected in a study by Tomoyo Sugiyama . This alBesides, dynamic glucose fluctuation may be associated with PR via increasing CD14(bright)CD16(+) monocyte levels preferentially . ConsidePrevious studies also indicated that coronary plaques in CAD patients with NGT are more stable than in those with IGT and DM . HoweverThere are several limitations of this study. First, it’s a single center observational study based on retrospective design. In the present study, a total of 233/1005 cases were excluded due to poor quality of OCT image or identified as neither PR nor PE, therefore some patients with STEMI caused by PR and PE were failed to be included and those patients with STEMI caused by other reasons include calcified nodules were not studied. Second, the plasma glucose was not monitored after the acute event so that mean amplitude of glycemic excursion and stress hyperglycemia failed to be studied. Last, an important risk factor body mass index (BMI) has not been adjusted because of incomplete weiHbA1ct and heiHbA1ct record. Being a subanalysis of the EROSION study, present study was a retrospective study focus on cardiovascular specialty, and other glucose-related variables  were failed to have been collected.The findings of present study highlight the association of ARPG and GVT with plaque rupture in STEMI patients, especially in patients with admission glucose levels ≥ 7.1 mmol/l. ARPG and GVT are independent predictors for PR in STEMI patients without DM. And high HbA1c and ARPG were positively correlated with the development of vulnerable plaque in culprit vessels, the former more than the latter.Additional file 1: Table S1. Baseline characteristics between plaque rupture and plaque erosion. Table S2. Presictive models of PR established using ARPG/GVT single or combined with traditional risk factors. Table S3. The correlation between glucose-related variables and plaque morphological characteristics using Spearman correlation.


P > 0.05) and between those with and without arterial stiffness (cfPWV ≥ 10 m/s) (P > 0.05). After adjusting for confounders, the serum klotho concentration was not significantly associated with the peripheral or central BP (P > 0.05) and cfPWV (P > 0.05). Our data indicated that the serum klotho concentration was not associated with BP or cfPWV in the general Chinese population.Klotho, an important anti-aging protein, may be related to elevated blood pressure (BP) and arterial stiffness. We aimed to investigate associations between the serum klotho concentration and peripheral/central BP and arterial stiffness based on the carotid–femoral pulse wave velocity (cfPWV) in a Chinese population. We invited all inhabitants aged ≥ 18 years in two Dali communities for participation. The SphygmoCor system was used to record radial arterial waveforms. Aortic waveforms were derived using a generalized transfer function. The central BP was assessed by calibrating the brachial BP, which was measured using an oscillometric device. The serum klotho concentration was measured using an enzyme-linked immunosorbent assay and logarithmically transformed. Of the 716 participants (mean age: 51.9 ± 12.6 years), 467 (65.2%) were women. The median serum klotho concentration was 381.8 pg/mL. The serum klotho concentration did not significantly differ between patients with and without hypertension ( Klotho is an anti-aging gene that shortens and extends the lifespan when disrupted and overexpressed, respectively1. Klotho-deficient mice exhibit signs of accelerated aging, such as arterial stiffness, hypertension, and chronic kidney disease (CKD)1. The α-klotho protein encoded by the klotho gene is a multifunctional protein that regulates the metabolism of calcium, phosphate, and vitamin D. The following three types of α-klotho protein with potentially different functions have been identified: full-length transmembrane α-klotho, truncated soluble α-klotho, and secreted α-klotho2. Previous studies have indicated that the prevalence of hypertension and arterial stiffness increases with age4, whereas the α-klotho concentrations decrease with age5.6, salt-sensitive hypertension8, CKD9, arterial stiffness11, and cardiomyopathy12. Therefore, evaluation of circulating klotho concentrations or klotho genotypes could help identify patients at higher risk of developing age-related cardiovascular morbidities. However, previous studies on the relationship between circulating klotho and blood pressure and arterial stiffness had small sample sizes13, used a case–control design14, performed analyses only in select patients16, measured peripheral blood pressure only8, or reported conflicting results17. In a recent study in young and middle-aged swine, elevated klotho secretion was associated with increased aortic stiffness and peripheral vascular resistance with aging17. In the present population-based study, we investigated the associations between the serum klotho  concentrations and the peripheral/central blood pressure and arterial stiffness based on the carotid–femoral pulse wave velocity (cfPWV).A few studies have reported that klotho deficiency is associated with hypertensionP ≤ 0.01). Men also tended to be older and had a higher body weight, body height, body mass index, waist circumference, hip circumference, fasting levels of blood glucose, low-density lipoprotein cholesterol, triglyceride levels, peripheral systolic and diastolic blood pressures, central systolic and diastolic blood pressures, and cfPWV compared to women. The high-density lipoprotein cholesterol levels and glomerular filtration rate (P < 0.0001) were also lower in men than in women. Men and women had similar serum klotho concentrations .Of the 716 participants, 467 (65.2%), 226 (31.6%), and 76 (10.6%) were women, had hypertension, and had diabetes, respectively. Table P > 0.05). However, compared with normotensive participants, those with hypertension included fewer women and had lower glomerular filtration rates; higher rates of current smoking, alcohol intake, diabetes mellitus; older age; and higher body weight, body mass index, waist and hip circumference, total protein levels, peripheral and central blood pressures, and cfPWV (P ≤ 0.03).Table P > 0.05).The median serum klotho concentration was 382 pg/mL. The serum klotho concentration was not significantly associated with the peripheral blood pressure, central blood pressure, pulse rate, or cfPWV (P > 0.05). Further adjustments for conventional cardiovascular risk factors including sex, age, body mass index, glomerular filtration rate, hypertension, diabetes mellitus, antihypertensive treatment, current smoking, and alcohol intake did not affect these results (P > 0.05)  Table . There w5) Table .Table 3PP > 0.05)  Table . There w5) Table .Table 4PTo the best of our knowledge, this is the first study that systematically investigated the relationship between the serum klotho concentration and blood pressure/arterial stiffness using a population-based design. Our results indicated that the serum klotho concentration is not associated with peripheral or central blood pressure or indices of arterial stiffness.. reported that klotho protein absorbance (0.303 ± 0.096) was lower in the hypertensive than in the non-hypertensive group (0.489 ± 0.216)13. In a 1:1 matched case–control study (mean age: 51.7 years) of 197 Chinese patients with hypertension and 197 without hypertension, Zhou et al.14 reported that the median serum klotho concentrations were 269.67 and 313.95 ng/L (P = 0.004) in the hypertensive and normotensive groups, respectively. After adjustments for covariates including age, lifestyle, family history of hypertension, body mass index, fasting blood glucose, serum uric acid, lipid levels, and microalbumin, the risks  of hypertension in the lowest three quartile groups were 2.01 (1.08–3.71), 2.29 (1.20–4.39), and 1.55 (0.82–2.92) compared with those in the highest quartile group, respectively14.Few previous studies have indicated that a lower circulating klotho concentration was associated with higher blood pressure or was a risk factor of hypertension. Especially, in an analysis of 79 and 30 older adults with and without hypertension, respectively, Su et al14. However, our results were consistent with those of a larger population-based study18. In the InCHIANTI study of 1,203 Italian adults, the median (25th–75th percentile) plasma klotho concentration was 676 (530–819) pg/mL. Plasma klotho was correlated with age but not with systolic blood pressure18. Despite convincing experimental evidence regarding the role of klotho deficiency in the pathogenesis of hypertension and salt hypertension21, the roles of klotho in human blood pressure regulation and pathogenesis of hypertension remain largely unknown. The discrepancies between the present and previous results may be attributable to differences in the study population age13 or small sample sizes of previous studies (≤ 394 participants)14. Furthermore, it is also possible that klotho is important in regulating blood pressure only in those processes or pathologies that are associated with a very drastic reduction in its serum concentrations. Further large-scale prospective studies are required to verify the relationships between the circulating klotho levels and blood pressure changes and the risk of hypertension in different age groups and disease conditions.In contrast to the results of some previous studies, our findings did not support an association between the serum klotho concentration and blood pressure in the general population23. A previous study in 2101 participants from the KoreaN Cohort Study for Outcome in Patients With Chronic Kidney Disease (KNOW-CKD) cohort reported no significant association between the serum klotho concentration and baPWV after adjustments 16. In 109 patients with diabetic nephropathy, pulse wave velocity (PWV) was reported to increase in those with CKD but was not related to serum klotho concentration22. However, our results contradicted those reported in some small-scale studies23 and in studies that included patients undergoing hemodialysis23 or those with CKD15. In a case–control study of 130 age- and sex-matched patients undergoing hemodialysis, α-klotho concentrations were inversely associated with the aortic–brachial PWV ratio 23. In another study that included 114 patients with CKD, there were significant decreases in the serum klotho concentration in those with baPWV ≥ 1400 cm/s15.Few previous studies have investigated the relationship between circulating klotho and arterial stiffness based on baPWV or cfPWV measurements. Our finding that the serum klotho concentration was not associated with arterial stiffness in the general Chinese population is consistent with the results of most, but not all, previous studiesklotho gene were correlated with the circulating klotho levels and the susceptibility to hypertension24, salt hypertension8, and coronary artery disease25 as well as the onset of stroke26. Though we did not find a significant relationship between the serum klotho concentrations, blood pressure, and PWV, we cannot exclude the possibility that genetic polymorphisms of the klotho gene may be associated with blood pressure and arterial stiffness.A growing body of evidence has suggested that some single nucleotide polymorphisms in the 27. Klotho can function as a humoral factor and regulate nitric oxide production in the endothelium, thus, preserving endothelial permeability, calcium homeostasis in the kidneys, and inhibiting insulin-like growth factor-1 signaling28. It can also serve as a mediator for the actions of FGF23, namely urinary phosphate excretion, inhibition of calcitriol secretion, and inhibition of parathyroid hormone synthesis and secretion29. Klotho deficiency increases NADPH oxidase activity and superoxide production, collagen expression, and elastin fragmentation in the aortic media30. Furthermore, klotho deficiency causes salt-sensitive hypertension and renal damage in mice by CC chemokine receptor 2-mediated inflammation7.Although our study did not support a relationship between the serum klotho and blood pressure and arterial stiffness in middle-aged Chinese individuals, previous experimental studies have indicated that klotho deficiency affected the regulation of blood pressure and pathogenesis of arterial stiffness via promoting oxidative stress, inflammation, apoptosis, and fibroblast growth factor receptor (FGFR)/FGF23 resistance34. When compared with aging, cigarette smoking, diabetes mellitus, CKD, and other typical cardiovascular risk factors, klotho deficiency may be a relatively weaker risk factor for cardiovascular disease in humans. Further prospective follow-up studies with larger sample sizes are required to elucidate the relationships between circulating klotho, blood pressure, and arterial stiffness.Our findings clarified and complicated the understanding of the relationship between klotho and blood pressure/arterial stiffness. We measured the serum klotho concentrations, peripheral and central blood pressures, and cfPWV in a much larger population-based sample compared with previous studies, thus, increasing the possibility of identifying any significant associations. However, klotho concentration, blood pressure, and arterial stiffness were highly dependent on age and other cardiovascular risk factors, such as diabetes mellitus, cigarette smoking, CKD, and metabolic syndrome36. Second, the serum klotho concentration was estimated only once, which can be influenced by several factors and may fluctuate with time. Third, the serum was stored at – 30 ℃, and we could not exclude the possibility that the stability of serum klotho decreases with longer storing time37. Fourth, although we had very detailed demographic information of each individual, the data regarding antihypertensive medications, which might alter the serum klotho levels38, were not available. Nonetheless, the self-reported antihypertensive treatment rate was 19.6%, suggesting that the antihypertensive treatment rate was very low in this study. Further adjustment for antihypertensive treatment did not significantly change our results. Similar results were found in the sensitivity analysis among those not receiving antihypertensive treatment. Fifth, we did not measure the vitamin D and FGF23 levels. The molecular interactions of FGF23, klotho, and vitamin D coordinate to regulate the delicate phosphate levels of human body. Vitamin D can induce FGF23 and klotho synthesis to influence renal phosphate balance39. Thus, we could not exclude the possibility that vitamin D and FGF23 could affect the blood pressure regulation and pathogenesis of arterial stiffness. Sixth, the reproducibility of cfPWV measurement was not tested. Nonetheless, the cfPWV measurement was well standardized, and the same technician performed all the cfPWV measurements in this study. Thus, this limitation did not affect our results. Finally, our participants had a relatively lower profile of cardiovascular risk than those included in previous clinical studies. Future large-scale prospective studies should include participants at higher risk to explore the relationships between the serum klotho concentration, blood pressure, hypertension, and arterial stiffness.Our findings should be interpreted within the context of the strengths and limitations of this study. Our study included 716 individuals who underwent comprehensive assessments of central blood pressure and cardiovascular function. However, it presented the following limitations. First, the cross-sectional design did not allow us to make any inferences regarding causality. The current design of our study did not allow us to test the association of klotho deficiency with microalbuminuria and the predictive value for renal function declineIn conclusion, our findings demonstrated that the serum klotho concentration was not associated with peripheral/central blood pressure or arterial stiffness in the general Chinese population. Nonetheless, future prospective studies should investigate the importance of circulating klotho measurements in cardiovascular risk stratification.This cross-sectional analysis was based on data collected as part of an ongoing population study of multiple cardiovascular risk factors in Dali, Yunnan Province, China. The study participants were recruited from two communities in Dali. Between October and December 2018, we cooperated with the medical staff of the local community health service center to invite all inhabitants aged ≥ 18 years to participate in the study through notice and telephone calls. Of those invited, 764 (70%) participated. We excluded 48 individuals from our analysis because they did not have blood samples (n = 4) or arterial (n = 39) and data (n = 5). Therefore, a total of 716 participants were included in the present analysis.The Ethics Committee of Dali University approved the study protocol, and all participants provided written informed consent. All procedures were performed in accordance with the Declaration of Helsinki.40. Patients who were prescribed antihypertensive drugs were also considered to have hypertension40. A trained physician performed the anthropometric measurements. The body mass index was calculated as the body weight in kilograms divided by the body height in meters squared.After each participant had rested for at least 5 min in the sitting position, two experienced physicians measured the blood pressure five consecutive times using a mercury-based sphygmomanometer. These five readings were averaged for the final analysis. The same physicians also administered a standardized questionnaire to collect information related to the medical history, smoking habits, alcohol intake, and medications. Hypertension was defined as a peripheral systolic or diastolic blood pressure of at least 140 and 90 mmHg, respectively, while seated41. Diabetes mellitus was defined as a fasting plasma glucose level of at least 7.0 mmol/L, hemoglobin A1c level of at least 6.5%, or the use of antidiabetic agents42.Venous blood samples were drawn after overnight fasting for the measurement of plasma glucose and serum total cholesterol and other biochemical analyses. The glomerular filtration rate (GFR) was estimated using the Modification of Diet in Renal Disease Study equation44.To ensure a steady state, a trained physician performed all arterial measurements via applanation tonometry after participants had rested for 15 min in the supine position. The participants were instructed to refrain from smoking, vigorous exercise, and drinking alcohol or caffeinated beverages for at least 2 h before the examination. We used a high-fidelity SPC-301 micromanometer  interfaced with a laptop computer running SphygmoCor software v7.1  to record the arterial waveforms. The recordings were discarded when the variability in consecutive waveforms exceeded 5% or when the amplitude of the pulse wave signal was < 80 mV. We calibrated the pulse wave based on the average of two consecutive brachial blood pressure readings obtained with a participant in the supine position immediately prior to SphygmoCor recordings using a validated Omron HEM-7051 oscillometric blood pressure monitor . The SphygmoCor software calculates the aortic pulse wave from the radial signal using a validated generalized transfer function. The central systolic and diastolic blood pressures were derived from the aortic pulse wave44. Arterial stiffness was defined as a cfPWV ≥ 10 m/s45.For the cfPWV measurement, the physician recorded the right carotid and femoral waveforms (12 s each) in succession. Based on simultaneously recorded electrocardiogram data (lead 2), the time delay between the feet of the two pressure waveforms was taken as the transit time between the carotid and femoral pressure waves. The distance traveled by the pressure wave was determined based on the difference between the distances from the sternal notch to the femoral location and from the sternal notch to the carotid location. PWV was calculated as the distance traveled divided by the transit timeSerum samples were stored at − 30 °C prior to measurements. The serum klotho concentration was measured using the enzyme-linked immunosorbent assay method in accordance with manufacturer instructions . The detection range was 78.10–5000 pg/mL. The limit of the kit’s sensitivity was 50 pg/mL.t-tests and Fisher’s exact tests, respectively. We performed unadjusted and multivariate-adjusted variance analyses to investigate the associations of serum klotho concentration with the central and peripheral blood pressures and arterial stiffness. P values < 0.05 were considered statistically significant.We used SAS v9.4  for database management and statistical analyses. Normality of data was assessed using the Shapiro–Wilk test. The serum klotho concentration exhibited a non-normal distribution and was, therefore, logarithmically transformed for statistical analysis. The means and proportions were compared using Student’s


Early diagnosis is important to lower the mortality rate of acute fatty liver of pregnancy (AFLP). The Swansea criteria is commonly used to diagnose AFLP, but some terms could only be reached when symptoms and signs have progressed, or are not efficient in clinical practice. Therefore, it is necessary to select cost effective tests to simplify and facilitate early suspicion of acute fatty liver of pregnancy.This is a retrospective study of 28,800 medical records at the Second Xiangya Hospital from 2009 to 2015, including 41 patients with AFLP and 172 other diseases that could show similar symptoms to AFLP. The evaluated variables included past history of liver diseases, blood pressure, gastrointestinal symptoms, blood count, liver function test, coagulation function test and blood sugar test. The sensitivity, specificity, positive predict value and negative predict value were calculated for models in diagnosing AFLP.6/l and blood sugar< 72 mg/dl. Gastrointestinal symptoms +aminotransferase +bilirubin +bile acid +APTT/PT showed 97.6% sensitivity and 97.1% specificity to diagnose AFLP. Adding blood pressure, blood sugar or white blood cells decreased the accuracy of the statistical model.The significant variables associated with AFLP included gastrointestinal symptoms, blood pressure > 140/90 mmHg, aminotransferase> 42 IU/l, total bilirubin> 0.8 mg/dl, total bilirubin acid> 10.0 μmol/L, activated partial prothrombin time(APTT) > 34 s, prothrombin time(PT) > 14 s, white blood cells> 11 *10Application of a statistical model including maternal symptoms, biochemical and haematological parameters has high diagnostic accuracy for earlier identification of AFLP. However, this finding needs to be tested in another cohort to determine whether this statistical model has the same performance. Acute fatty liver of pregnancy (AFLP) is a rare but potentially lethal condition that typically presents during the third trimester of pregnancy. It is characterized by microvesicular fatty infiltration of the liver and can lead to multiorgan failure . Because6/l, ascites or bright liver on sonogram, aspartate aminotransferase and alanine aminotransferase > 42 IU/l, ammonia > 27.5 mg/dl (47 kmol/l),creatinine > 1.7 mg/dl (150 kmol/l),coagulopathy (prothrombin time(PT) > 14 s or activated partial thromboplastin time(APTT) > 34 s), microvesicular steatosis on liver biopsy [The Swansea criteria is commonly used to diagnose AFLP, including vomiting, abdominal pain, polydipsia/polyuria, encephalopathy, bilirubin > 0.8 mg/dl (14 kmol/l), hypoglycemia < 72 mg/dl (4 mmol/l), uric acid > 5.7 mg/dl (340 kmol/l), leukocytosis > 11 *10r biopsy . Six or This is a retrospective study in a university obstetric unit. The reference standard diagnosis of AFLP is the Swansea criteria. With approval from institutional human subject review board, we reviewed 28,800 medical records at the Second Xiangya Hospital from 2009 to 2015 and identified 41 AFLP cases diagnosed by Swansea criteria. The incidence of AFLP in our hospital (0.14%) is higher than reported , 4,whichThe significant variables associated with AFLP were selected by univariate analyses and by their biologic plausibility. The candidate variables for the diagnostic model included past history of liver diseases, blood pressure, gastrointestinal symptoms, blood count, liver function test, coagulation function test and blood sugar test. We also tried to further select markers , activated partial prothrombin time (APTT), for example) from these tests to make a more exact diagnostic model. The time of test before final diagnosis by Swansea criteria was 3 days. The past history of liver disease referred to chronic liver disease that had been diagnosed but hadn’t been cured before this pregnancy, such as viral hepatitis, autoimmune hepatitis, Wilson’s disease, excluding liver disease that had been cured before this pregnancy, such as acute viral liver disease in childhood or ICP in previous pregnancy. Risk factors that hadn’t resulted in a determined liver disease were not included in the past history of liver disease either, such as alcohol abuse.Statistical analyses were performed with STATA10.0 . Descriptive statistics included means and range for continuous variables and frequency distributions for categorical variables. Comparisons between categorical variables were tested by the use of the chi-square test. Variables were included in the model if they were statistically significant in the univariate analyses. Comparisons between normally distributed continuous variables and categorical variables were performed using Student t test and analysis of variance.6/l, blood sugar< 72 mg/dl. At the time of our test, none of the AFLP cases met 6 terms of the Swansea criteria, 19.5% of the cases met 5 terms, 75.6% met 4 terms and 4.9% met 3 terms.The demographic characteristics for study population are shown in Table In accuracy analysis, gastrointestinal symptoms +aminotransferase+ bilirubin+ bile acid +APTT/PT showed 97.6% sensitivity and 97.1% specificity to diagnose AFLP. Adding blood pressure, blood sugar or white blood cells didn’t increase the sensitivity but decrease it Table .Table 2In all of the 41 AFLP cases, 23 were delivered when our test was positive, while 18 weren’t delivered until at least 6 terms of Swansea criteria were met. Maternal mortality was significantly lower and Apgar scores significantly higher in group delivered by our test comparing to group delivered by Swansea criteria  in diagnosing microvesicular steatosis, but it is not targeted at early diagnosis. On one hand, some of its diagnostic terms could only be reached when symptoms and signs have progressed, which might be too late for treatment. On the other hand, some of its terms are not efficient to apply in clinical practice. For example, although liver biopsy is the gold standard for diagnosis of AFLP, it is likely to cause intraperitoneal hemorrhage in the event of coagulopathy. So seldom has the diagnosis of AFLP been confirmed by liver biopsy in previous reports 0% in dia–14. In oTo simplify and facilitate early suspicion of AFLP, Goel et al. designed presence of the following three criteria for the presumptive diagnosis of AFLP: 1. late pregnancy (late 2nd or 3rd trimester), 2. Acute liver failure: jaundice with coagulopathy and/or hypoglycaemia and/or encephalopathy, 3. No other explanation for liver failure . The thiWith acceptance of the importance of early recognition and diagnosis of AFLP, maternal mortality has decreased from 85%  to 12.5%We summarized our design of diagnosis procedure as Fig. The study is restricted by some limitations. First, the case number is limited and number of every disease in the control group is generally low. Hence, we presented a composite control. Second, the study was carried out in a single hospital. The population was all Chinese who were mostly from central and southern China. So we didn’t have enough data to evaluate the influence of race and geography. Third, the test results were disclosed to the clinicians treating the women, so that clinician’s behavior may have averted diagnoses of AFLP or made them more likely. Thus, we planned to retest the model in double blind study to eliminate the influence of clinician’s behavior. We also plan to evaluate the model in multicenter study and investigate the influence of more confounders.Even though AFLP is not common, its diagnosis remains a big challenge for Obstetricians. Our study showed proper choice of test in basic prenatal care was able to diagnose AFLP. Application of a model including maternal symptoms, biochemical and haematological parameters has a high diagnostic accuracy for earlier identification of AFLP. We hope this diagnostic test of AFLP would contribute to simplify and facilitate early suspicion of AFLP. However, the finding needs to be tested in another cohort to determine whether this diagnostic model has the same performance.


Vascular remodeling is an initial step in the development of hypertension. Limb remote ischemic conditioning (LRIC) is a physiological treatment that induces endogenous protective effect during acute ischemic injury. However, the impact of long-term LRIC on hypertension, a chronic disease, is unknown. In this study, we aimed to investigate the LRIC effect on blood pressure and vascular remodeling in spontaneously hypertensive rat (SHR) model and patients with prehypertension and early-stage hypertension. LRIC of rats was performed once a day for 6-weeks. Blood pressure, vascular remodeling , inflammation  were compared among normotensive Wistar-Kyoto rats (WKY), WKY RIC group, SHR control group, and SHR RIC. Long-term LRCI treatment (twice a day for 4-weeks) was performed on patients with prehypertension or early-stage hypertension. Blood pressure and pulse wave velocity (PWV) were analyzed before and after LRIC treatment. LRIC treatment decreased blood pressure in SHR (n = 9-10). LRIC ameliorated vascular remodeling by decreasing cross-sectional area, suppressing deposition of the extracellular matrix, and hypertrophy of smooth muscle cell in conduit artery and small resistance artery (n = 7). LRIC decreased proinflammatory factors while increasing the anti-inflammatory factors in the circulation (n = 5). LRIC decreased circulating monocyte and natural killer T-cell levels (n = 5). Furthermore, LRIC treatment decreased blood pressure and improved vascular stiffness in patients (n = 20). In conclusion, long term LRIC could decrease blood pressure and ameliorate vascular remodeling via inflammation regulation. LRIC could be a preventive treatment for people with blood pressure elevation or prehypertension. Hypertension is a leading risk factor for cardiovascular, cerebrovascular, and many other diseases , especiaVascular remodeling results initially from passive physiological adaptation to blood pressure changes, then progresses into an active pathological process caused by elevated blood pressure, aging, and several other factors , 9. ThisLimb remote ischemic conditioning (LRIC) is a physiological treatment that protects against acute ischemic events and traumatic injury , 14. ChrIn this study, we aimed to address whether LRIC exhibits antihypertensive effect and if LRIC prevents or delays vascular remodeling in a chronic hypertensive pathological condition and the possible underlying mechanism of LRIC protective effect.Spontaneously hypertensive rats (SHR) were used as a genetic model of hypertension, which develop hypertension at a very young age. Before the established phase, the blood pressure of SHR increases, and vascular remodeling occurs rapidly from age 4-10 weeks . In thisThe Animal Care and Use Committee of Xuanwu Hospital, Capital Medical University, China approved all animal experiments which were conducted following the National Institutes of Health guidelines. Forty rats used in this study were purchased from the Vital River Laboratories, Beijing, China, and maintained on a 12-h light/dark cycle with unlimited access to food and water.We assigned randomly 20 WKY to WKY control (WKY-CON) and WKY + RIC groups, and 20 SHR to SHR control (SHR-CON) and SHR + RIC groups, respectively.LRIC was conducted as previously described. Rats weThe blood pressure was measured weekly, 12 hours after LRIC, with a tail-cuff system  following the manufacturer’s guidelines.The thoracic aorta, second-order mesenteric artery, and brain were harvested and fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Paraffin sections of 5 µm thickness were deparaffinized and washed, stained with hematoxylin and eosin (H&E), Sirius-red, and Verhoeff's elastic stain (VEG) . For H&ETissue slices were incubated overnight at 4°C with anti-SMA antibody . After washing with PBS, the sections were incubated for 1 h at 21 °C with the secondary antibodies. Slides were mounted with an antifade reagent containing DAPI . Fluorescence signals were detected with the Panoramic digital slide scanner , and quantified using Image J software. The α-SMA expression values were calculated from the total positive area and mean fluorescence intensity.Immunocytes in the blood were analyzed by flow cytometry. Single-cell suspensions were treated with Fc block, washed, and stained with CD3, CD4, CD8, CD45, CD161a, CD43, CD172a, and their homologous isotype-matched negative controls . Live cells were gated, and a set number of events were acquired using a Fortessa flow cytometer (BD) and analyzed.Blood from the inferior vena cava was collected into EDTA coated vacutainer tubes, followed by centrifugation at 1000g for 15 min at 21 °C. Plasma was obtained and stored at -80°C . An ELIShttps://register.clinicaltrials.gov)The Institutional Ethics Committee of Xuanwu Hospital approved this investigation. In this study, 20 pre-hypertensive or early-stage hypertensive subjects not receiving any pharmacological anti-hypertensive treatment were included. The study inclusion criteria were as follows: (1) systolic blood pressure (SBP) between 125-160 mmHg or diastolic blood pressure (DBP) between 80-100 mmHg; (2) age ≥18; (3) essential hypertension; (4) patient not receiving any anti-hypertensive medication. The exclusion criteria were: (1) patients with severe uncontrolled diabetes; (2) contraindication for remote ischemic preconditioning; (3) life expectancy less than one year; (4) patients with atrial fibrillation or other kinds of arrhythmia; (5) unwillingness for follow-up or poor compliance.  were measured following the previously described guidelines . Patientt-test. All analyses were performed with significance set at P<0.05.Data calculated using SPSS version 19.0  software were expressed as mean ± standard deviation (SD). For animal experiments, differences between groups were analyzed using one-way analysis of variance (ANOVA), and posthoc multiple comparisons were performed using Fisher’s LSD where appropriate. Two-way repeated-measures ANOVA was used for analysis of a comparison of blood pressure across time points. For the human trials, differences between groups were analyzed using two-sided paired Student’s Blood pressure in SHR increased rapidly from the age of 4-10 weeks compared to the WKY group. The mean blood pressure of the two groups was 119.8 ± 9.6 and 166.0 ± 5.7 mmHg, respectively . LRIC trHypertrophy is recognized as the primary cause during the transition from the physiological to pathological vascular remodeling in hypertension . HypertrHypertrophic remodeling causes increased peripheral resistance and decreased blood supply . The resVascular hypertrophy involves extracellular matrix production, smooth muscle cell hyperplasia, and hypertrophy . We inveSmooth muscle cell (SMC) hypertrophy and hyperplasia were reported in early-stage hypertension and play a significant role in pathological remodeling . HypertrDysregulation of circulating inflammatory factors was shown to play a central role in initiating blood pressure elevation and early-stage vascular remodeling . The effHypertrophic remodeling is initiated by vascular infiltration of inflammatory cells, especially macrophages . InflammWe investigated whether LRIC exhibits similar effects in humans by conducting a pilot study on patients with pre- and early-stage hypertension. Twenty patients completed the 4-week ischemic conditioning treatment. The results showed that a 4-week LRIC treatment was well tolerated: 5476 out of 5600 cycles were completed in total. Eleven and nine subjects completed over 90 and 80% cycles, respectively. The percentage of completed cycles for each week was 98%, 97%, 96%, and 94%, respectively. The primary reason for uncompleted cycles was intense workouts, while other reasons are, discomfort due to cuff pressure, or lack of adherence to schedule . The sysIn this study, the results indicated that SHR develops hypertension coupled with vascular remodeling as early as 4-week old, as reported previously . Long-teVascular remodeling underlies the pathological basis for all types of vascular diseases, and pathological remodeling helps establish stable hypertension . HyperteAccumulating evidence demonstrates that increased circulating pro-inflammatory factors initiate vascular remodeling pathogenesis at an early-stage, typically accompanied by a decrease in anti-inflammatory factors . InflammCXCL1 is a chemokine that promotes migration and infiltration of neutrophils and monocytes to vascular wall. The bloIL-10 is an anti-inflammatory cytokine exhibiting a protective effect on vascular dysfunction in hypertension . A signiCirculating monocyte forming macrophages entering a specific organ or tissue plays a critical role in vascular inflammation and remodeling . It is fSuccessful blood pressure management is critical to prevent cardiovascular and cerebrovascular disease. Some studies showed that sustained elevated blood pressure exceeding 125 mmHg might represent a threshold for vascular remodeling, potential hypertension, and increased risk of future cardiovascular disease and stroke . All thehttps://register.clinicaltrials.gov).However, some limitations exist in the human study. First, this is a single-arm study, and the date was not compared with the control group. It cannot exclude placebo effects, which may affect blood pressure. Instead, the data before and after ischemic conditioning were self-compared. In addition, the method used to evaluate blood pressure is not precise, and the effect on blood pressure should be explored in a longer time frame. Thus, the results from the human study are implied and are being investigated further in a randomized, double-blind placebo control study (Registration No. NCT03566654, In conclusion, long-term LRIC treatment could decrease blood pressure and attenuate vascular remodeling in SHR. LRIC exerted its protective effect by restoring the imbalance in pro- and anti-inflammatory markers. These findings describe the potential use of LRIC for the prevention of hypertension.


Diaphragm dysfunction is highly prevalent in mechanically ventilated patients. Recent work showed that changes in diaphragm shear modulus (ΔSMdi) assessed using ultrasound shear wave elastography (SWE) are strongly related to changes in Pdi (ΔPdi) in healthy subjects. The aims of this study were to investigate the relationship between ΔSMdi and ΔPdi in mechanically ventilated patients, and whether ΔSMdi is responsive to change in respiratory load when varying the ventilator settings.r) were used to investigate within-individual relationships between variables, and repeated measure correlations (R) were used for determining overall relationships between variables. Linear mixed models were used to compare breathing indices across the conditions of ventilation.A prospective, monocentric study was conducted in a 15-bed ICU. Patients were included if they met the readiness-to-wean criteria. Pdi was continuously monitored using a double-balloon feeding catheter orally introduced. The zone of apposition of the right hemidiaphragm was imaged using a linear transducer . Ultrasound recordings were performed under various pressure support settings and during a spontaneous breathing trial (SBT). A breath-by-breath analysis was performed, allowing the direct comparison between ΔPdi and ΔSMdi. Pearson’s correlation coefficients . Individual correlation displays a significant correlation in 8 patients out of 25 . Changing the condition of ventilation similarly affected ΔPdi and ΔSMdi. Patients in which ΔPdi–ΔSMdi correlation was non-significant had a faster respiratory rate as compared to that of patient with a significant ΔPdi–ΔSMdi relationship (median (Q1–Q3), 25 (18–33) vs. 21 (15–26) breaths.min−1, respectively).Thirty patients were included and 930 respiratory cycles were analyzed. Twenty-five were considered for the analysis. A significant correlation was found between ΔPdi and ΔSMdi (We demonstrate that ultrasound SWE may be a promising surrogate to Pdi in mechanically ventilated patients. Respiratory rate appears to negatively impact SMdi measurement. Technological developments are needed to generalize this method in tachypneic patients.NCT03832231. Acute respiratory failure is a common cause of admission in the intensive care unit (ICU) that can require invasive mechanical ventilation to relieve respiratory muscles work of breathing and ensure satisfactory gas exchange . HoweverThis study followed the STROBE guidelines for observational study . It was 2 > 90% or PaO2/FiO2 ≥ 150 mmHg with a fraction of inspired oxygen (FiO2) ≤ 40%, no or minimal vasopressor, and a positive end-expiratory pressure (PEEP) ≤ 8 cmH2O. Patients who were pregnant, under a legal protection measure, with a contraindication to the insertion of a gastric-esophageal probe , or with known allergies to anesthetizing were not included.Patients older than 18 years old were eligible for inclusion if they had been intubated and ventilated for at least 24 h, and failed a first spontaneous breathing trial (SBT). They could be included if they met predefined readiness-to-wean criteria on daily screening  and wereA flow sensor  connected to a spirometer  was used to continuously measure flow. Esophageal (Pes) and gastric (Pga) pressures were monitored using a double-balloon feeding catheter . The catheter was inserted through the mouth or nostril in the esophagus as demonstrated by the appearance of cardiac artifacts and appropriate negative swings of pressure during inspiration. Both balloons were inflated with 3 to 4 ml of air and connected to separated differential pressure transducers . The correct position of the esophageal balloon was confirmed by a dynamic occlusion test allowing the visualization of a corresponding deflation in esophageal pressure and airway pressure during inspiratory effort . Flow anpleura and peritoneum .The zone of apposition of the right hemidiaphragm was imaged using an ultrafast US scanner  driving a linear transducer array . The probe was placed on the mid-axillary line, vertical to the chest wall, at the 8th–11th intercostal space and the spot was skin marked. US gel was generously applied to optimize acoustic coupling and minimal pressure was applied to the transducer to limit tissue deformation and/or alteration of breathing mechanics. In this location, the diaphragm appears as a three-layered structure just superficial to the liver, consisting of a relatively non-echogenic muscular layer bounded by two echogenic lines corresponding to the diaphragm Patients were in a semi-recumbent position throughout the study. Sedations were not modified during the protocol. The study was carried out as follows: i) recordings under different conditions of pressure support ventilation, ii) recordings during a SBT.+25%), (iii) -25% pressure support with baseline PEEP (PS-25%) and (iv) baseline pressure support and zero end-expiratory pressure (PSZEEP). Each breathing condition was maintained for 10 min with 30-s acquisitions performed at 3 and 9 min within the condition.At baseline, patients were ventilated under pressure support ventilation mode. In each patient, 4 consecutive conditions of ventilation were applied in a randomized order: (i) initial ventilator settings predefined by the attending physician (PS), (ii) + 25% pressure support with baseline PEEP (PS2O). This modality of SBT, part of usual care of our ICU, reflects the work of breathing after extubation [Every patient underwent a planned 30 min SBT during which no assistance was provided from the ventilator . SMdi was reported as the median shear modulus within each ROI.Data were analyzed offline using standardized MATLAB  scripts. A rectangular region of interest (ROI) was drawn manually in the center of the diaphragm on the first frame of each recording. The ROI was replicated on subsequent frames. Diaphragm shear modulus was calculated as SMdi = ρ ⋅Vsmax) was measured before the SBT using a one-way valve allowing exhalation only [max was calculated as the difference between Pdi at functional residual capacity and maximal Pdi during the Mueller maneuver. A video showing US imaging along with the temporal evolution of flow, internal pressures, and SMdi is available in Additional file Changes in Pdi (ΔPdi), Pes (ΔPes), Pga (ΔPga) and SMdi (ΔSMdi) were computed for each respiratory cycle. The cycles were delimited by the deflations of the esophageal pressure signal and not with the flow signal since it could mask the onset of the inspiratory effort, especially when the patient has to overcome intrinsic PEEP , 30. Durion only . Pdimax Results are presented as median (Q1-Q3) for descriptive statistics. We calculated our sample size based on the expected correlation between ΔPdi and ΔSMdi of 0.7 . A requip < 0.05) ΔPdi–ΔSMdi correlation and their counterparts. Analyses were performed in the computing environment R [p < 0.05 for all tests.Pearson’s correlation coefficients were used to investigate within-individual relationships between variables. Repeated measure correlation  was used for determining overall relationships between variables . Paired onment R . Signifi2O and a PEEP level of 5 (5–5) cmH2O. A total of 3878 breathing cycles were recorded and 930 were considered for further analysis (i.e. corresponding to triplicate for each condition of ventilation). All patients tolerated each condition of the protocol.Between February 2019 and February 2020, 1087 patients were admitted within the ICU. 404 patients received invasive mechanical ventilation, 99 fulfilled the inclusion criteria leading to the enrollment of 30 patients. Twenty-five patients were considered for subsequent analysis . Regarding within-subject correlation analysis, ΔSMdi and ΔPdi exhibited a significant correlation in 8 patients and no significant correlation in 17 patients . Figure r = 0.81, p = 0.002) and in a patient with no ΔPdi–ΔSMdi correlation  during the protocol. Patients in which ΔPdi–ΔSMdi correlation was non-significant were older, had a faster respiratory rate, lower ΔPdi, lower PTPdi and lower ΔPga as compared to that of patients with significant ΔPdi–ΔSMdi correlation  failed the SBT. Reasons of failure were acute respiratory distress (5/13), neurologic impairment 4/13) and weaning induced pulmonary edema (4/13). ∆Pdi and PTPdi tended to be higher at the start of the SBT in patients who failed the SBT as compared to their counterpart. No difference was found regarding ∆SMdi between the two groups of patients. Table /13 and wThe present work provides new insights regarding the use of ultrasound shear wave elastography for the assessment of diaphragm function in patients under mechanical ventilation using a breath-by-breath analysis. First, we found that changes in diaphragm shear modulus and changes in transdiaphragmatic pressure were significantly correlated. However, when considering the relationship between diaphragm shear modulus and transdiaphragmatic pressure within individuals, the correlation was significant in only a third of patients and it was absent in the remaining patients. Second, we found no significant difference in diaphragm function and change in diaphragm shear modulus in response to changes in ventilatory conditions. Changes in transdiaphragmatic pressure and in diaphragm shear modulus significantly increased during the SBT. Third, we found no significant difference regarding SBT-induced changes in diaphragm shear modulus between patients who failed or succeeded the spontaneous breathing trial.R2 = 0.94 ± 0.05) [20) was much wider than the one in the present study (0–50 cmH2O with 62% of ΔPdi < 10 cmH2O). Therefore, the moderate relationship between ΔSMdi and ΔPdi observed in our study may be partly explained by the relatively low, albeit more physiological, range over which diaphragm effort was analyzed. Our results support this hypothesis, as patients with a significant ΔPdi–ΔSMdi correlation displayed higher ΔPdi , as compared to our previous work in healthy subjects  . In a re ± 0.05) . Our findi Table . Second,ts Table . Also, Δ0 cmH2O, ) and ii)As expected, removing PS was associated with a significant increase in diaphragm function as assessed using ΔPdi and PTPdi as repeatedly observed , 42, 43.A secondary aim of the present study was to investigate differences in diaphragm function and diaphragm shear modulus according to the outcome of a SBT. As previously reported, we observed that patients who failed the SBT had larger  ΔPdi and ΔPTPdi at the start of the SBT as compared to patients who succeeded  was found between changes in transdiaphragmatic pressure (ΔPdi) and changes in diaphragm shear modulus (ΔSMdi) can be found at the following link: https://figshare.com/s/f53dbea5b18c420a1490.Additional file 2.  Figure S2: Individual correlation coefficients between changes in transdiaphragmatic pressure (ΔPdi) and changes in diaphragm shear modulus (SMdi) can be found at the following link: https://figshare.com/s/c0f06ddf34d5a784ccb5.Additional file 3. Figure S3: Movie clip of temporal changes in esophageal pressure, gastric pressure, transdiaphragmatic pressure, and diaphragm shear modulus during pressure support ventilation in one patient with a breathing frequency of 33 breaths/min in which no correlation  was found between changes in transdiaphragmatic pressure (ΔPdi) and changes in diaphragm shear modulus (ΔSMdi) can be found at the following link: https://figshare.com/s/fb33c7701fb50c35c98d.


Previous renal denervation (RDN) studies showed controversial results in reducing blood pressure. The aim of this study was to provide evidence supporting the effectiveness of laparoscopic-based renal denervation (L-RDN) in treating hypertension.n = 12) and sham group (n = 4). Neurogenic hypertension was generated in all dogs via carotid artery route. L-RDN was performed in the RDN group, with sham operation performed as a control. Blood pressure (BP) changes were recorded at 2, 4, 6, and 8 weeks after the procedure. Changes in serum creatinine (sCr), blood urea nitrogen (BUN) and level of norepinephrine (NE) were analyzed. Histological changes of kidney and renal arteries were also evaluated.Sixteen Beagle dogs were randomly divided into RDN group (p < 0.01). Systolic and diastolic BP of RDN group were decreased by 15.5 mmHg and 7.3 mmHg  at the eighth week after L-RDN. Invasive systolic and diastolic BP of RDN group were significantly decreased by 14.5 mmHg and 15.3 mmHg (p < 0.0001). In contrast, there was no significant decrease in blood pressure in the sham group. In addition, RDN group but not the sham group showed a significant decrease in NE levels (p < 0.001), while no significant changes in sCr and BUN were observed in both groups. Pathological examinations showed no discernible damage, tear, or dissection to the renal arteries in RND group.BP and NE levels were significantly elevated after hypertension induction (L-RDN lowered BP and NE levels in hypertensive dogs without affecting renal artery morphology and kidney function. In 2009, Krum and colleagues first reported the application of catheter-based renal denervation (RDN) in managing patients with refractory hypertension . In a suHowever, results obtained from SYMPLICITY HTN-3 trial in 2014 questioned the clinical application of RDN therapy. This clinical trial was rigorously designed, using randomized and controlled approaches with 535 patients diagnosed as refractory hypertension and including a sham group. The change in office systolic blood pressure (SBP) was monitored as the primary endpoint after the 6-month follow-up period. No significant difference in the reduction of office SBP was observed between RDN and sham groups by the end of follow-up [Despite the disappointing result from the SYMPLICITY HTN-3 trial, controversial findings from different animal and human studies of RDN therapy warrant cautious interpretation. Further, clinical trials involving a diverse population from different ethnicities, as well as real-world studies with longer follow-up periods may provide comprehensive evidence on the decision to waive or retain RDN as a potential cure for refractory hypertension. Termination of RDN trials across different countries has been discouraging further clinical studies, although carefully designed preclinical studies provide an alternative approach to delineate the effect and safety of the therapy. In this study, we conducted laparoscopic radiofrequency ablation (L-RDN) of the renal sympathetic nerve from the adventitia in a hypertensive Beagle dog model and evaluated the efficiency of the modified RDN procedure.n = 12) receiving L-RDN and the sham group (n = 4) receiving similar procedures except for radiofrequency ablation.Sixteen male or female Beagle dogs aged 10–12 months and weighing 9.4–11.6 kg were provided and housed by the Laboratory Animal Center of the Soochow University . Procedures were performed under the guidance of professional researchers in a clean laminar flow operating room. Perioperative feeding and care of the animals were carried out by professional staffs. Neurogenic hypertension was generated in all dogs, which were randomly divided into two groups, including the RDN group  was introduced. Carbon dioxide pneumoperitoneum of 10–12 mmHg was generated. Two additional 5-mm trocars were inserted through incisions in both midaxillary lines at 15 cm above the anterior iliac crest. Another two 5-mm trocars were inserted at the middle points between the laparoscope trocar and the anterior iliac crest trocars. The laparoscopic hook electrode, suction and elastic separating plier were inserted as appropriate. A combination of blunt and sharp dissection of the renal fascia was applied until the renal hilus was visualized clearly. The radiofrequency ablation catheter  was inserted through the side port of the double-channel trocar towards the renal artery and connected to a radiofrequency ablation device   was measured using a blood pressure cuff around one thigh of the hind limbs and connected to a multifunctional blood pressure monitor. NIBP were monitored before hypertension induction (defined as the baseline) and 2, 4, 6, and 8 weeks after hypertension induction. At the eighth week after hypertension induction, L-RDN and sham surgeries were performed in the RDN group and sham group, respectively, and changes in NIBP were recorded at 2, 4, 6, and 8 weeks after the procedures.To measure invasive blood pressure (IBP), animals were anesthetized and the right femoral area was disinfected. After successful puncture with a syringe, a pressure transducer  was attached for temporal IBP measurement using a PHILIPS IntelliVue X2 System. IBP was monitored before hypertension induction (defined as the baseline), at 8 weeks after hypertension induction, and at 8 weeks after RDN or sham procedures.The renal functions and norepinephrine (NE) levels before hypertension induction (defined as the baseline), at 8 weeks after hypertension induction, and at 8 weeks after RDN or sham procedures were measured at the Institute of Neuroscience and Laboratory Animal Center of Soochow University. All Beagle dogs were humanely killed by intravenous injection of potassium chloride solution (2 mg/kg) under anesthesia at 8 weeks after RDN.All experimental animals were humanely killed 8 weeks after RDN to isolate the complete renal arteries and kidneys for gross specimen examination for evidence of stenosis, dissection, and perforation in renal arteries. Tissue specimens were fixed in 4% paraformaldehyde, followed by paraffin-embedding, tissue sectioning, and hematoxylin and eosin (HE) staining using a commercial kit . Light microscopy was used to evaluate the histomorphological changes of the renal arteries in different groups.Transverse sections of the renal artery  were collected directly in glass slides  and incubated with a blocking buffer containing 10% goat serum, 1% bovine serum albumin (BSA), and 0.3% Triton X-100 for 1 h. The sections were then incubated with anti-VEGF antibody  at 4 °C overnight. After washing with PBS for 3 times, the sections were incubated with fluorescent-conjugated anti-rabbit secondary antibodies  and counterstained with DAPI for 1 h. All slides were examined under a fluorescence microscope .p-value < 0.05 was considered as statistically significant.Statistical analysis was performed using SPSS software . Continuous variables with normal and skewed distribution were expressed as means with standard deviations or medians with interquartile ranges. Categorical variables were presented as numbers and percentages. Changes in BP were expressed as mean (95% CI). Independent samples t-test and Mann-Whitney U test were used to compare the differences in continuous variables with normal and skewed distribution between RDN and sham groups. Comparison of continuous variables with normal and skewed distribution at different time points in the same group was performed using paired t-test and Mann-Whitney U test, respectively. Categorical data between the groups were compared using the Chi-square test. Homogeneity-of-variance was determined by Levene’s test of equality of variances. A two-way repeated measures ANOVA (blood pressure × time after the procedure) adjusted for baseline blood pressure was performed using the BP reduction as dependent variables. Sphericity was determined by Mauchly’s test, and interactions between variables were estimated. Variables were compared using the Greenhouse-Geisser adjustment method when sphericity was absent. Otherwise, comparisons were performed using the simple effects tests if interactions were present, or using the tests of within-subjects effects if no significant interactions were observed. Bootstrapping (1000 replications) with a simple percentile 95% CI was used to validate the results. A p < 0.0001 and p = 0.0021) (Table p < 0.0001)  Table . Consist1) Table . In cont1) Table . The ove1) Table . Similar1) Table .Table 1p = 0.002, p = 0.001). Eight weeks after the radiofrequency ablation, NE levels of the RDN group were reduced from the preoperative level of (554.6 ± 36.3) μg/L to a baseline level of (354.9 ± 59.7) μg/L (p = 0.0011). In contrast, the sham group showed no significant difference in plasma NE levels by the end of follow-up (p = 0.948) . In this study, renal denervation significantly lowered BP in patients with untreated mild to moderate hypertension. Compared with the sham group, 80 patients in the experimental group achieved average reductions in ambulatory SBP by five mmHg and in DBP by 7.7 mmHg, provided the latest evidence supporting the effectiveness of RDN therapy .Our results demonstrated adventitial ablation as an alternative pathway that allows safe and efficient ablation of the renal sympathetic nerve. Further animal studies are warranted to evaluate L-RDN in different models of hypertension before proceeding with human studies with more stringent selection criteria on specific patient groups. Quantitative assessment of the renal sympathetic nerve activity and improved devices may also facilitate future clinical studies of RDN that may hopefully provide a novel avenue in the treatment of refractory hypertension.In this study, the effectiveness of randomized RDN therapy was evaluated using a canine model of surgically induced neurogenic hypertension. Compared with sham surgery, laparoscopic renal denervation significantly lowered both systolic and diastolic BP. Meanwhile, L-RDN reduced NE levels while not altering sCr and BUN. Renal artery pathology showed no obvious vascular damage after L-RDN. In conclusion, L-RDN could be a useful treatment to optimize blood pressure control.This study modeled neurogenic hypertensive animals through the carotid artery that might not recapitulate the pathogenesis of human neurogenic hypertension. In addition, we only monitored changes in BP and circulating NE levels to evaluate the therapeutic efficacy of radiofrequency ablation due to the lack of direct assessment of renal sympathetic nerve activity in the in vivo models. In addition, our study had a small sample size and the short-term BP monitoring (i.e. 8 weeks). The main limitation is that this is just an animal experiment, and its positive results may not be achieved in human clinical treatment. Since this surgical approach is expected to treat hypertensive patients, large-scale animal studies and clinical trials are warranted to further investigate the anatomical routes for laparoscopy, energy range, duration, and equipment of radiofrequency ablation.