Indoxyl sulfate (IS) is associated with either chronic kidney disease or renal failure, which may predict cardiovascular events via cardiorenal syndrome. The present study aimed to elucidate whether the plasma levels of IS can predict the occurrence of cardiovascular events in patients with chronic heart failure (CHF) and investigate which causes of CHF leading to cardiovascular events are highly influenced by plasma IS levels. We measured the plasma IS levels in 165 patients with CHF  admitted to our hospital in 2012, and we followed up these patients for more than 5 years (the median follow-up period: 5.3 years). We measured the plasma IS level in 165 patients with CHF, and Kaplan–Meier analyses showed that high plasma IS levels  could predict the occurrence of cardiovascular events, i.e., cardiovascular death or rehospitalization due to the worsening of CHF. The sub-analyses showed that the high IS level could predict cardiovascular events in patients with CHF due to HCM and that the plasma IS levels were closely associated with left ventricular (LV) dimension, LV systolic dysfunction, and plasma B-type natriuretic peptide levels, rather than LV diastolic dysfunction. Plasma IS level predicts cardiovascular events in patients with CHF, especially those with HCM along with cardiac dysfunction. Besides, IS may become a proper biomarker to predict cardiovascular events in patients with CHF. Uremic toxins play a substantial role in cardiovascular injury2, and indoxyl sulfate (IS) is surmised to be the most abundant and potent uremic toxin3. Several lines of evidence have shown that renal dysfunction increases IS levels4, leading not only the renal function getting affected6 but also cardiac function7. We have previously reported the increase of plasma IS levels in CHF patients with preserved renal function as well as the removal of IS using AST-120, an adsorbent of uremic toxins in the gut, restoring the left ventricular (LV) systolic and diastolic function8. This clinical observation has been confirmed in a pacing-induced HF canine model; the plasma IS levels increase along with the progression of HF, and AST-120 improves LV dysfunction9. Indeed, IS increases the expression and activation of extracellular signal-regulated kinase (ERK), P38MAP kinase, and nuclear factor kappa light chain enhancer of activated B cells (NF-kB), which may affect cardiac remodeling11. Furthermore, IS activates renin receptors12, thereby activating angiotensin receptors13. Either of these deleterious sequelae may affect cardiomyocytes, cardiac fibroblasts, and cardiac endothelial cells14 and lead to cardiovascular dysfunction. These results support the theory that plasma IS may become a novel biomarker for the prediction of cardiovascular events in patients with CHF. Hence, it is essential to investigate which causes of CHF leading to cardiovascular events are highly influenced by the plasma IS levels.In patients with chronic heart failure (CHF), dysfunctions of other organs, including kidney, liver, and intestine, rather than heart were observedTo test this theory, we retrospectively and consecutively enrolled patients with CHF who were hospitalized for the worsening of CHF, initial onset of acute HF (AHF), or precise examination in our hospital at 2012, and we followed up with these patients for more than 5 years.8. The median value of the plasma IS levels was 0.79 µg/mL, and we divided the present patients according the IS value of ≥ 0.79 (high IS level) and < 0.79 (low IS level) µg/mL. Figure The characteristics of the 165 included patients with CHF are shown in Table To investigate which causes of CHF leading to cardiovascular events are highly influenced by the plasma IS levels, we performed a sub-analysis in each group categorized by the cause of CHF, i.e., valvular disease, dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), and the others. We found that plasma IS levels especially affected the cardiovascular parameters and cardiovascular events in the patients with CHF with HCM (Supplementary Tables The major findings of this study were as follows: (1) the plasma IS levels in the patients with CHF predicted the cardiovascular events of either cardiovascular death or rehospitalization due to the worsening of HF; (2) the plasma IS level appropriately predicted the cardiovascular events in patients with CHF accompanied by HCM, whereas the other causes in the sub-analyses for the causes of CHF were not involved; and (3) the plasma IS level was closely associated with the LV systolic function in CHF patients with HCM.8, suggesting that CHF per se increases the plasma IS levels. As IS is released to urinary tubules via organic anion transporter 1, 3 (OAT1 and OAT3) and is excreted to urine via OAT415, the pathophysiology of CHF increases plasma IS level, which may be attributable to direct effect of OATs without influencing eGFR. Indeed, in the present study, eGFR did not decrease in CHF patients  may increase the plasma IS level. Our previous study revealed that the plasma IS levels increased in patients with CHF compared with control subjects with a comparable eGFR12 as well as cause angiotensin receptor activation13, which may contribute to cardiac hypertrophy and fibrosis. Furthermore, IS increases the expression of NF-kB11, enhancing the inflammations, and IS decreases the NOS activity19, which may largely contribute to the formation of CHF in the experimental and clinical studies. Ultimately, IS increases oxidative stress20, worsening the pathophysiology of HF, it decreases erythropoietin (EPO) level21 despite the cardioprotective effects of EPO22, and it decreases the expression of Klotho in kidne24. Decreasing the expression of Klotho may increase fibroblast growth factor 23 (FGF23), and the increased FGF23 level may cause cardiac hypertrophy and CHF. One or some of these factors may explain the cardiac deleterious effects of IS observed in the present study.The next issue is whether IS directly affects the tissues or cells in cardiovascular systems. Intriguingly, IS can activate the renin receptors25 and experimental27 studies, suggesting that the elevation of the plasma IS level in patients with CHF may contribute to the worsening of CHF.IS may be closely associated with the pathophysiology of CHF, and IS may worsen the severity of CHF. However, in the present study, it remained unknown whether IS is deleterious or beneficial for the severity of CHF because CHF increases the plasma BNP level, and increased BNP is beneficial for CHF due to the cardioprotective effects of BNP. However, several lines of evidence, including our earlier studies, showed that the removal of IS using AST-120 exerts beneficial effects on the pathophysiology of CHF in the clinical28. These results seem to be inconsistent with ours; however, the discrepancy may be attributable to the patients’ characteristics. Although either DCM or HCM does not necessarily provoke symptomatic CHF, the patients with CHF with DCM or HCM in the present study experienced hospitalization due to AHF, and cardiovascular events included recurrent worsening HF or cardiovascular death, which revealed that severity of CHF in DCM or HCM patients was more than that found a previous study28. The role of plasma IS as a biomarker for the severity of CHF in patients with severe DCM at the end-stage of CHF may be blunted by the other deleterious neurohumoral factors, such as tumor necrosis factor alpha (TNF-α). This may demonstrate that the sensitivity to detect the severity of HF by the plasma IS levels may be limited in patients with severe CHF.In the present study, we showed that IS plays a pivotal role in the progression of patients with CHF, especially those with HCM; besides, LV systolic dysfunction in CHF patients with HCM was found to associate with plasma IS levels. We found that RA pressure had a good correlation with plasma IS level in HCM patients although the sample size is too small to draw the definite conclusion seen in the supplemental data. The patients with HCM suffers from LV diastolic dysfunction, causing the elevation of RA pressure. The increase in RA pressure provides renal congestion and dysfunction followed by the elevation of the plasma IS levels. This is one of the scenarios that RA pressure had a correlation with plasma IS level in HCM patients in our data. However, a previous study showed that blood IS level correlated well with the severity of LV diastolic dysfunction in patients with DCMFirstly, daily dietary situation may affect the plasma IS level, because intake of the food containing tryptophan might affect the IS level. However, as we measured the blood samples after a 12-h fast, dietary situation may not have influenced the IS level in the present study.Secondly, this study was performed as a single-center study, and the differences among the hospitals should have been taken into account. However, since the diagnosis and treatment of CHF are undertaken according to the guidelines of the treatment of HF in Japan and are very similar to American College of Cardiology/American Heart Association (ACC/AHA) or European Society of Cardiology guidelines, the patients’ characteristics were found to be identical worldwide.Thirdly, the number of patients in each category of causes of CHC, including HCM and DCM, was not remarkable; therefore, further studies should be conducted to verify our findings.Lastly, although our hospital is one of high-volume centers for the HF patients, we measured the plasma IS level in the limited number of the patients because long-term follow-up is difficult. However, because of the positive results of that the plasma IS levels increase and predict clinical outcomes in patients with HF, plasma IS levels may become a biomarker of HF such as plasma BNP levels.The present study strongly suggests that the measurement of the plasma IS level in advance predicts following cardiovascular events in CHF patients, especially those CHF patients with HCM. Furthermore, we need to test the effectiveness of AST-120 for the improvement of pathophysiology of CHF.2; the patients showed a New York Heart Association functional class of either I, II or III at the discharge. Diagnosis of CHF was conducted based on the Framingham criteria15, and the patients were excluded if they had CHF with more than the middle-stage chronic kidney disease (CKD) (Stage 3 CKD)16. We sampled the blood in a stable chronic phase of CHF during hospitalization. After the discharge following the appropriate treatment of HF, we followed up with these patients for more than 5 years and searched for the cardiovascular events of either cardiovascular death or the rehospitalization due to worsening of HF.We included 165 patients who were admitted to our hospital for the treatment of worsened CHF or the initial onset of AHF from January to December 2012. The causes of CHF were categorized as valvular disease (n = 78), DCM (n = 29), HCM (n = 25), and others (n = 33) such as hypertensive heart disease (n = 12), ischemic cardiomyopathy (n = 4), amyloidosis (n = 5) and sarcoidosis (n = 2). Patients were included if they were stable for CHF and had eGFR more than 40 ml/min/1.73 m29. The Japanese-specific eGFR was calculated as follows: [194 × Serum creatinine−1.094 × Age−0.287 × ]18.Blood for the serum was collected in tubes; the serum was then separated, and blood for the plasma was collected in ethylenediaminetetraacetic acid (EDTA) tubes; the plasma was then separated and then samples for IS were frozen in plastic tubes at − 80 °C until analysis. We measured IS levels by internal-surface reversed-phase high-performance liquid chromatography (HPLC), with a HPLC system , as previously described19. A comprehensive echo-Doppler assessment was performed according to the Guidelines of American Society of Echocardiography. Whereas LV ejection fraction (EF) was obtained using the Simpson biplane method or Teichholz's formula, fractional shortening (FS) was evaluated using the following formula: FS = 100 × (LVEDD − LVESD)/LVEDD, where LVEDD is the LV end-diastolic dimension and LVESD is the LV end-systolic dimension. LV E/e′ was also measured as the ratio of peak velocity of early transmitral diastolic filling by echocardiography to early diastolic mitral annular velocity by tissue Doppler echocardiography as LV diastolic function.We measured and calculated LV systolic and diastolic dimensions according to the Guidelines of American Society of Echocardiography30. We performed a standard right heart catheterization via either the internal jugular vein or the femoral vein by using a Swan-Ganz catheter . A standard left heart catheterization was performed via either the radial artery or femoral artery by using an angled pigtail catheter .We performed right and left heart catheterizations on the study participants and prospectively collected clinical data as described beforeUsing the right and left heart catheterization data, we obtained the cardiac hemodynamic parameters, including the right atrial pressure, pulmonary artery pressure, pulmonary capillary wedge pressure and LV end-diastolic pressure.Continuous data were presented as median with interquartile range (IQR), where IQRs were presented in terms of 25th and 75th percentiles or mean ± standard deviation. Categorical data were presented as frequencies (%). Statistical significance between the groups was evaluated by One-way ANOVA or Kruskal–Wallis test. The event-free survival curves were estimated using the Kaplan–Meier method. Between-group differences in survival were assessed using the log-rank test. HRs with 95% confidence interval were calculated using Cox proportional hazards regression, including those for the subgroup analyses. The proportional hazards assumption was graphically investigated based on the Schoenfeld residuals over time. Univariate linear regression analysis was performed to assess the relationships between IS and the other variables. In the present study, all tests were two-tailed; P < 0.05 was considered statistically significant. The analyses were performed with the JMP 8.0.2 software for Windows .This study was approved by the Institutional Ethics Committee of the National Cerebral and Cardiovascular Center  and was conducted in accordance with the principles enshrined in the 2013 Declaration of Helsinki and Japanese ethical guidelines for clinical research. All the patients a signed written informed consent form.Supplementary Information.


The global burden of chronic kidney disease (CKD) is rapidly increasing with aprojection of becoming the 5th most common cause of years of life lost globallyby 2040. CKD is a major cause of catastrophic health expenditure. The costs ofdialysis and transplantation consume up to 3% of the annual healthcare budget inhigh-income countries. However, the onset and progression of CKD is oftenpreventable. In 2020, the World Kidney Day campaign highlights the importance ofpreventive interventions – be it primary, secondary, or tertiary. This articlefocuses on outlining and analyzing measures that can be implemented in everycountry to promote and advance CKD prevention. Primary prevention of kidneydisease should focus on the modification of risk factors and addressingstructural abnormalities of the kidney and urinary tracts, as well as exposureto environmental risk factors and nephrotoxins. In persons with pre-existingkidney disease, secondary prevention, including blood pressure optimization andglycemic control, should be the main goal of education and clinicalinterventions. In patients with advanced CKD, management of co-morbidities suchas uremia and cardiovascular disease is a highly recommended preventativeintervention to avoid or delay dialysis or kidney transplantation. Politicalefforts are needed to proliferate the preventive approach. While nationalpolicies and strategies for non-communicable diseases might be present in acountry, specific policies directed toward education and awareness about CKDscreening, management, and treatment are often lacking. Hence, there is anurgent need to increase the awareness of preventive measures throughoutpopulations, professionals, and policy makers. Around 850 million people currently are affected by different types of kidneydisorders . Up to oImportantly, however, kidney disease can be prevented and progression to ESRD can bedelayed with appropriate access to basic diagnostics and early treatment includinglife style modifications and nutritional interventions –8. DespiAccording to the expert definitions including the Center for Disease Control andPrevention , the terde novo CKD.The incidence (new cases) and prevalence (cumulative pre-existing cases) of CKD havebeen rising worldwide . This prde novo CKDsuch as cardiovascular and atheroembolic diseases  and liver diseases . Measures to achieve effective primary prevention should focus on the two leading riskfactors for CKD including diabetes mellitus and hypertension. Evidence suggests thatan initial mechanism of injury is renal hyperfiltration with seemingly elevatedglomerular filtration rate (GFR), above normal ranges. This is often the result ofglomerular hypertension that is often seen in patients with obesity or diabetesmellitus, but it can also occur after a high dietary protein intake . Other Cde novo CKD are screeningefforts to identify and manage persons at high risk of CKD, especially those withdiabetes mellitus and hypertension. Hence, targeting primordial risk factors ofthese two conditions including metabolic syndrome and overnutrition is relevant toprimary CKD prevention as is correcting obesity (Among measures to prevent emergence of  obesity . Promoti obesity . Obesity obesity .-1·(1.73m2)-1)  orCKD stage 3B (eGFR between 45 to 60 mL·min3m2)-1) . Inthesde novo CKD, AKI events that are superimposed onpreexisting CKD may accelerate disease progression . These drugs reduce both systemic blood pressure and intraglomerularpressure by opening efferent arterioles of the glomeruli, hence, leading tolongevity of the remaining nephrons. Low protein diet appears to have a synergisticeffect on RAASi therapy . In termgression . A relatgression .In patients with advanced CKD, management of uremia and related comorbid conditionssuch as anemia, mineral and bone disorders, and cardiovascular disease is of highpriority, so that these patients can achieve the highest longevity. These measurescan be collectively referred to as “tertiary prevention” of CKD. In theseindividuals, cardiovascular disease burden is exceptionally high, especially if theyhave underlying diabetes or hypertension, while they often do not follow othertraditional profiles of cardiovascular risk such as obesity or hyperlipidemia.Indeed, in these patients, a so-called “reverse epidemiology” exists, in thathyperlipidemia and obesity appear to be protective at this advanced stage of CKD.This could be due to the overshadowing impact of the “protein-energy wasting” (PEW)that happens more frequently with worsening uremia and which is associated withweight loss and poor outcomes including cardiovascular disease and death. Whereasmany of these patients, if they survive the ravages of PEW and cardiovasculardisease, will eventually receive renal replacement therapy in the form of dialysistherapy or kidney transplantation, a new trend is emerging to maintain them longerwithout dialysis by implementing conservative management of CKD. However, in somewith additional comorbidities such as metastatic cancers, palliative measures withsupportive care can be considered.The lack of awareness of CKD around the world is one of the reasons for latepresentation of CKD in both developed and developing economies –22.The Given its asymptomatic nature, screening of CKD plays an important role in earlydetection. Consensus and positional statements have been published by theInternational Society of Nephrology (ISN) , the NatGiven that currently population screening for CKD is not recommended and it wasclaimed that it might add unintended harm to the general population being screened, there iUniversal screening of the general population would be time-consuming, expensive, andhas been shown to be not cost-effective. Unless selectively directed towardshigh-risk groups, such as the case of CKD in disadvantaged populations , accordiThe approach towards CKD early detection will include the decision for frequency ofscreening, who should perform the screening, and intervention after screening . ScreeniGiven the close links between CKD and other NCDs, it is critical that CKD advocacyefforts be aligned with existing initiatives concerning diabetes, hypertension, andcardiovascular disease, particularly in LMIC. Some countries and regions havesuccessfully introduced CKD prevention strategies as part of their NCD programs. Asan example, in 2003, a kidney health promotion program was introduced in Taiwan,with its key components including a ban on herbs containing aristolochic acid,public-awareness campaigns, patient education, funding for CKD research, and thesetting up of teams to provide integrated care . In Cuba-1·(1.73 m2)-1, rapidlydeclining kidney function, persistent proteinuria, or uncontrolled hypertension ordiabetes  had been trained using an electronic health educationplatform .The resources for nephrology care remain at critical levels in many parts of theworld. Even in Western developed countries, nephrologists are frequently in shortsupply. In a selection of European countries with similar, predominantly public,health care systems, there was a substantial variation in the nephrology workforce.Countries like Italy, Greece, and Spain reported the highest ratios, while countrieslike Ireland, Turkey, and the UK had the lowest ones . In the The causes of this shortage are multiple. Potential contributors to this variationinclude the increasing burden of CKD, erosion of nephrology practice scope by otherspecialists, lack of workforce planning in some countries relative to others, andthe development of new care delivery models . A novelSince 1994, a National Institutes of Health consensus advocated for early medicalintervention in predialysis patients. Owing to the complexity of care of CKD, it wasrecommended that patients should be referred to a multidisciplinary team consistingof nephrologist, dietitian, nurse, social worker, and health psychologist with theaim to reduce predialysis and dialysis morbidity and mortality . In MexiWhereas it is important to enhance the promotion and implementation of “Prevention”of kidney disease and kidney failure amongst healthcare professionals, it is equallyimportant to promote “Prevention” with education programs for those at risk ofkidney disease and kidney failure, and for the general population at large. It is astepwise process, from awareness, engagement, participation, empowerment, andpartnership. As highlighted above, in general, the health literacy of the generalpopulation is low. Awareness and understanding of kidney disease are inadequate.Education is key to engaging patients with kidney disease. It is the path toself-management and patient-centered care. Narva et al. found patient education isassociated with better patient outcomes .ObstaclThe internet offers a wealth of resources on education. Understanding the types ofinternet sources that CKD patients use today can help renal professionals to pointpatients in the right direction. It is important that reputable healthcareorganizations, preferably at a national level, facilitate easier access to healthinformation on their websites (Supplementary Table S1). The mode of communicationcurrently used by patients and the population at large is through the internet -websites, portals, and other social media, such as Facebook and Twitter. There arealso free apps on popular mobile devices providing education on kidney disease.There is no shortage of information on the internet. The challenge is how toeffectively “push” important healthcare information in a targeted manner, and tofacilitate users seeking information in their efforts to “pull” relevant andreliable information from the internet. It is important the “pushing” of healthinformation is targeted and specific, relevant for the condition , and is offered at the right time to the rightrecipient. It is possible with the use of information technology and informatics toprovide relevant and targeted information for patients at high risk, coupling theinformation based on diagnosis and drugs prescribed. Engagement of professionalsociety resources and patient groups is a crucial step to promote communitypartnership and patient empowerment on prevention. Additional resources may beavailable from charitable and philanthropic organizations.Given the pressing urgency pertaining to the need for increasing education andawareness on the importance of the preventive measures, we suggest the followinggoals to redirect the focus on plans and actions:Empowerment through health literacy in order to develop and support nationalcampaigns that bring public awareness to prevention of kidney disease.Population-based approaches to manage key known risks for kidney disease,such as blood pressure control and effective management of obesity anddiabetes.Implementation of the World Health Organization ‘Best Buys' approachincluding screening of at-risk populations for CKD, universal access toessential diagnostics of early CKD, availability of affordable basictechnologies and essential medicines, and task shifting from doctors tofront-line healthcare workers to more effectively target progression of CKDand other secondary preventative approaches.To that end, the motto ‘Kidney Health for Everyone, Everywhere' is more than atagline or wishful thinking. It is an imperative policy that can be successfullyachieved if policy makers, nephrologists, and healthcare professionals placeprevention and primary care for kidney disease within the context of their UniversalHealth Coverage programs.pdf].Click here to view [


To explore the potential value of three-dimensional ultrasonography (3DUS) and contrast-enhanced ultrasound (CEUS) in the diagnosis of diabetic nephropathy (DN) in Chinese diabetic patients with kidney injury.Patients with type 2 diabetes mellitus and kidney injury in our hospital were enrolled, and the clinical characteristics as well as the laboratory data of patients were collected; 3DUS and CEUS were used to evaluate the morphological structure and blood perfusion of kidneys. Eligible patients were categorized into two groups based on renal biopsy results: DN group and non-diabetic renal diseases (NDRD) group. Correlation analysis and logistic regression analysis were applied to identify the risk factors of DN development.A total of 115 patients aged from 24 to 78 years old were recruited in the experiment, of which 64 patients (55.65%) and 51 patients (44.35%) were in the DN group and NDRD group, respectively. After correction to CKD stage, BMI and right kidney volume index were retained to identify patients with DN. The ROC of the logistic regression model had an AUC of 0.703 (95% CI: 0.591–0.815) and it was higher than both indicators.3DUS has potential value in the diagnosis of diabetic nephropathy in Chinese diabetic population with kidney injury and may act as an auxiliary diagnosis for DN. More prospective and well-designed studies with larger samples are needed to verify the result. Diabetes mellitus is one of the most common metabolic diseases in the world. It is estimated that the overall prevalence rate of adult diabetes has reached 10.9% in 2013 in China [The pathogenesis, clinical manifestations, treatment, and prognosis are different between DN and NDRD. In addition, NDRD has a relatively better prognosis since it is usually treatable and even curable, whereas the renal lesions in DN are believed to be more irreversible . Renal b2 did not present microalbuminuria [Clinical practice guidelines for the disease management of type 2 diabetes recommend the screening for microalbuminuria . Howeveruminuria . Hence, Three-dimensional ultrasonography (3DUS) was invented in the early 1990s. In 3DUS, volume datasets of echo information from a large series of two-dimensional ultrasonographic images are quickly acquired and assembled by a computer . The emeFrom April 2017 to November 2018, a case control study was conducted in our hospital. Demographics and clinical data  were collected from electronic medical record (EMR) after patients were admitted to the hospital before having renal biopsy. This study was approved by the Medical Ethics Committee of Chinese PLA General Hospital (No. S2017–152-02), Beijing, China.Inclusion criteria: (1) Both male and female patients aged between 18 to 80 years old; (2) Clinically diagnosed as type 2 diabetes; (3) Renal damage (the presence of proteinuria); (4) Definite renal pathologic findings; (5) Inpatients in the department of nephrology in our hospital.The pathological types of NDRD include: membranous nephropathy, IgA nephropathy, obesity-associated nephropathy, focal glomerulosclerosis, hypertensive renal damage, and membranous proliferative glomerulonephritis.Exclusion criteria: (1) Urinary infections; (2) Other acute infections ; (3) Patients with systemic diseases ; (4) Pathological diagnosis of DN with NDRD; (5) Patients with malignant tumors; (6) Unilateral renal atrophy; (7) Unilateral or bilateral renal artery stenosis; (8) Allergic to ultrasound contrast agents.-0.411 × (0.993)*Age and Scr > 0.9 ml/dl:eGFR = 144 × (Scr/0.9)-1.209 × (0.993)*Age for male; Scr ≤ 0.7 ml/dl:eGFR = 144 × (Scr/0.7)-0.329 × (0.993)*Age and Scr > 0.7 ml/dl:eGFR = 144 × (Scr/0.7)-1.209 × (0.993)*Age for female), serum albumin, glycated hemoglobin, 24-h urine protein, presence of glomerular hematuria, and urine osmotic pressure were collected before renal biopsy.The following clinical characteristics of patients were collected: gender, age, medical history of DM, family history, body mass index (BMI), presence of hypertension, systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure, and presence of retinopathy. Laboratory parameters, including hemoglobin, serum creatinine, estimated glomerular filtration rate The renal biopsies were performed by an experienced physician, and all renal biopsy specimens were reviewed independently by two pathologists, who coordinate all disputed cases by thorough discussion. The criteria of DN diagnosis were: mesangial proliferation, diffuse capillary glomerulosclerosis, presence or absence of K-W nodules, diffuse thickening of the glomerular basement membrane (GBM), and exudative injuries such as fibrous cap or/and hyaline thrombi . PatholoA Philips EPIQ 7 high-end color doppler ultrasound instrument was used, and 3D imaging was completed through EPIQ 7. Probe × 6–1, frequency 1-6 Hz, and the instrument was equipped with QLAB image processing software.This technique has been done by doctors with 5 years working experience in ultrasonic image. The patient took lateral position during examination, the ipsilateral upper limb was placed over the head. The probe was placed on the waist to get a clear maximum section along the long axis of the kidney; the 3D mode was activated to ensure that the whole kidney was included in the image. During image acquisition, patients were asked to hold their breath. The volume data of kidneys were collected, and the DICOM images were stored in the machine. Following the same method, the volume data of each kidney was acquired for three times. All images post-processing were completed by the professional and technical personnel.3D mode was selected and activated in QLAB software. Kidney volume was measured in advanced function menu. Seven layers of cross-sections were selected to estimate the volume. After determining the length of the kidney, the software automatically calculated the volume of the kidney after manually recording 7 layers Fig. . The aveCalculation of body surface area and kidney volume index: height and body mass were measured for all subjects using the following formula: body surface area = 0.0061 × height + 0.0128 × body mass - 0.1529, and kidney volume index = kidney volume / body surface area.Siemens S2000 high-end color Doppler ultrasound, probe C6–2, frequency 2-6 Hz. The instrument was equipped with Contrast Dynamics, a post-processing software for contrast ultrasound.The patient took lateral position during examination and the ipsilateral upper limb was placed over the head. The probe was placed on the waist to get a clear maximum section along the long axis of the kidney. In this section, the parenchyma of the kidney should be parallel to the skin to minimize the sampling error caused by the position change of kidney affected by breathing. Then we started the contrast mode of the machine, activated double display, adjusted the focus position, automatically optimized the images, adjusted the settings of instrument (MI = 0.05–0.06), and reduced background gain noise. After fixing the position of the probe, the contrast agent at a dose of 0.7 ml/time was injected into the superficial vein of elbow, followed by an immediate 5 ml saline injection. In order to avoid the differences caused by different speed of injection, the time of injecting contrast agent and saline was controlled within 3 s. From the beginning of injection, the video DICOM images were stored dynamically for 3 min. During the period of corticomedullary enhancement, the patients were instructed to hold their breath, and then breathe slowly and quietly to avoid heave breathing. The kidney on the other side was examined by the same method, and the interval time between the two kidney examinations was more than 20 min.After selecting the image, we turned on Contrast Dynamics software, selected the region of interest (ROI) in the renal cortex, and kept the ROI depth of all patients consistent when sampling. Aftering turning on the image motion compensation, the time-intensity curve was automatically generated by the software. Quantitative parameters were obtained by fitting curve: peak intensity , peak time (Tp). Area under curve (AUC % s), mean transit time (MTT s). We duplicated the size of the sample and measured it three times in the cortex of the kidney Fig. .Fig. 2LSince manual selection could lead to slight inconsistence in the area size of ROI each time, we used AUC per unit area for correction. Per unit area under the curve was represented by area under curve (AUC % s)/sampling area. Slope was calculated by peak intensity  and peak time (Tp).P < 0.05 was considered to be significant.The measurement variables were expressed as mean ± standard deviation (SD) or median (IQR). Student’s t-test was used if the results of two groups were in a normal distribution; if not, Wilcoxon two-sample test would be used. The three groups were compared with the normal distribution by using one-way ANOVA. For the results which did not fit the normal distribution, Kruskal-Wallis test was used. And SNK (Student-Newman-Keuls) method was used for posterior comparisons of two groups. The categorical variables were expressed in terms of frequency (%), and the comparison between groups used the Chi-square test or the Fisher exact probability method. This study compared diabetic nephropathy (DN) with non-diabetic renal disease (NDRD) and expected to find statistically significant differences between the two groups. Since the CKD grade with stage between the DN and NDRD groups was significantly different, further screening analysis was used in order to exclude the interference of CKD grade with stage. The used methods were CKD grade group comparison, correlation analysis, and logistic multi-factor regression. The last remaining indicators were fitted with logistic model and comprehensive predictive index. The ROC and AUC of each index and logistic regression model were plotted and calculated. When the Youden index was the largest, the cutoff value of the ROC was taken. Statistical analysis was performed by using the SAS 9.2 software, both using two-sided test and n = 9); renal biopsy was not performed (n = 6); no contrast-enhanced ultrasound was performed (n = 12); patients with unilateral or bilateral renal artery stenosis (n = 4). Thus, a total of 115 patients . The proportion of patients with history of diabetes and SBP in the DN group were higher than those in the NDRD group (p < 0.05), but the BMI was lower. In the NDRD group (p = 0.010), the proportion of hematuria in the DN group was lower than that in the NDRD group . Among the included samples, the DN group and the NDRD group had significant differences in the CKD grade with stage, and the proportion of CKD stage in the DN group was higher than that in the NDRD group. The results were shown in Table One hundred forty-six cases in total with diabetes and kidney injury were preliminary included, and 31 cases were excluded due to the following reasons: pathological biopsy indicating the comorbidity of DN and NDRD (P > 0.05). The outcomes of urinary protein, plasma urea, and serum creatinine were higher in the DN group than in the NDRD group (P < 0.05), but the glomerular filtration rate in the DN group was lower than that in the NDRD group (P < 0.05). The results were shown in Table There were no significant differences between the DN group and the NDRD group in urinary creatinine and serum glucose (P > 0.05). Nevertheless, the right kidney volume index of the DN group was higher than that of the NDRD group (P = 0.021). The results were shown in Table P > 0.05). The LK PEAK, LK AUC, LK MTT, and LK Per unit area under the curve in the DN group were lower than those in the NDRD group, and the difference was statistically significant (p < 0.05). Other indicators of CEUS in both groups were similar, and the results were shown in Table There were no significant differences in right kidney volume, left kidney volume, left kidney volume index between DN group and NDRD group through the 3DUS examination .The ROC curves of DN and NDRD identified by BMI and right kidney volume index were shown in Fig. 2 had normoalbuminuria [The prevalence of DN in T2DM patients varies from place to place , which duminuria . Progresuminuria . From thThe diagnosis of renal disease is commonly based on GFR value, urinary abnormalities such as proteinuria and hematuria, and ultrasound structural kidney alterations. Ultrasound (US) with color doppler (CD) imaging  is the fCEUS has been widely accepted  and usedHence, we established a logistic regression model, in which BMI and right kidney volume index were included eventually. The AUC of the both indicators and the reference curve were statistically significant. For easy clinical use, we thus built a new method based on BMI and right kidney volume index. With respect to the significant increase in BMI in patients with DN compared with NDRD, the possible reason is that DN patients often have poor blood glucose control and abnormal glucose metabolism, leading to abnormal lipid metabolism and overweight.To our knowledge, this is the first study on the diagnostic value of 3DUS and CEUS on diabetic patients with kidney injury. However, there are still some limitations in the current study. First, the CKD grade with stage of patients in DN and NDRD groups is inconsistent, which means the severity of renal injury in DN group is relatively higher. This could possibly result in some bias in 3DUS and CEUS presentation. Additionally, the total sample size is small or study design issue, and therefore, a larger population with well designed study is needed in the future.3DUS might be used to evaluate the severity of renal pathology and may be potentially valuable for auxiliary diagnosis in DN patients.Additional file 1: Supplement Table 1. Comparison of different CKD staging Groups. Supplement Table 2. Correlation analysis between CKD staging and BMI, Right kidney volume index and LK MTT. Supplement Table 3. Comparison of BMI, Hematuria, Right kidney volume index, LK MTT in DN and NDRD patients after correcting CKD staging.


Intraclass correlations for all phases combined were 0.96, 0.92, 0.93 and 0.87 for HR, SBP, DBP and CO, respectively. Correlations for changes in CO, SBP and DBP were significant (p < 0.001) and strong (r > 0.88), with concordance rates (determined by quadrant plots) of 86%, 66% and 68%, respectively. The novel PPG-based device was accurate and valid compared to existing invasive techniques and might be used for continuous monitoring in several clinical settings following further studies.Accurate and continuous monitoring of critically ill patients is frequently achieved using invasive catheters, which is technically complex. Our purpose was to evaluate the validity and accuracy of a photoplethysmography (PPG)-based remote monitoring device compared to invasive methods of arterial line (AL) and Swan-Ganz (SG) catheters in a swine model of controlled hemorrhagic shock. Following a baseline phase, hemorrhagic shock was induced in 11 pigs by bleeding 35% of their blood volume, followed by a post-bleeding follow-up phase. Animals were monitored concomitantly by the PPG device, an AL and a SG catheter, for a median period of 447 min. Heart rate (HR), systolic and diastolic blood pressure , and cardiac output (CO) were recorded continuously. The complete data set consisted of 1312 paired observations. Correlations between the PPG-based technique and the invasive methods were significant ( Although trauma and resuscitation protocols have improved over the years, systemic organ failure still occurs in a relatively high percentage of patients suffering from acute and chronic life-threatening diseases8. Aggressive resuscitation using accurate cardiovascular monitoring can reduce tissue hypoxia, attenuate oxidative damage, prevent progression to multi-organ failure, and improve outcome in these clinical scenarios9. Ideally, the technology for measuring cardiovascular parameters should allow continuous, non-invasive, wireless, safe, inexpensive monitoring, easy to operate remotely, while being accurate and reliable.Monitoring of cardiovascular parameters, including heart rate (HR), blood pressure (BP), and cardiac output (CO), plays a vital role in the assessment, treatment, and follow-up of patients with different medical conditions12. Yet, some studies have shown that use of a pre-emptive strategy of hemodynamic monitoring including HR, BP and CO reduces morbidity and surgical mortality with the advantages and precision of the invasive measurement methods considered to outweigh their complications14. On the other hand, although any medical intervention has associated risks, it has been noted in several clinical studies that the number of life-threatening complications associated with the SG catheter is relatively high15.Over the past years, development efforts focused on methods that fulfil these requirements. Currently, the gold standard for hemodynamic monitoring of critically ill patients includes the insertion of an arterial line (AL) to measure BP and HR, and a Swan-Ganz (SG) pulmonary artery catheter to measure CO. These methods are relatively accurate and reliable, but invasive, with the potential of adverse events such as infection and damage to the arteries through which they were inserted. In addition, large retrospective and randomized controlled trials demonstrated no improvement in the mortality of patients following the use of these invasive methods compared to patients without invasive monitoringIn this study, we evaluated the monitoring capability of a new device , which measures cardiovascular parameters using a reflective photoplethysmography (PPG) sensor. This sensor allows non-invasive, cuff-less, and wireless measurements of HR, systolic blood pressure (SBP), diastolic blood pressure (DBP), and CO. The accuracy and validity of the device were determined by head-to-head comparison with readings from an AL and a SG catheter in a swine model of controlled hemorrhagic shock, an accepted model to study trauma care as described later.During all study phases, SG and AL, as well as the wearable device, provided continuous assessment of physiological parameters. Two animals did not undergo a bleeding phase due to early systemic decompensation, and therefore only baseline  data is reported for those animals. Details about the blood volume removed from each animal, and clinical data recorded during the study are presented in Table p < 0.001) and strongly correlated during all phase . Overall mean values for the invasive measurement were 65 ± 25 and 42 ± 24 mmHg for SBP and DBP, respectively. For the PPG-based device, SBP was 69 ± 24 mmHg and DBP was 46 ± 23 mmHg. There also were significant (p < 0.001) and strong correlations between methods of measurement of BP during all phases . CO values from SG (4.0 ± 17.1 l min−1) and the wearable device (4.4 ± 17.0 l min−1) were significantly (p < 0.001) correlated during each phase . Intraclass correlation coefficients (ICC) and standard error of the mean (SEM) per phase between each pair of measurements have been tested and presented in Fig. Pearson correlations and Bland–Altman plots with 95% limits of agreement (LOA) for each variable are presented in Fig. p < 0.001) and strong  and by automated devices, the necessity of using a cuff makes this measurement cumbersome and inconvenient for both patients and care providers, particularly in circumstances where beat-to-beat measurements are required or in isolated patients. While criteria exist for assessing the validity of automatic BP devices and acquiring approval by the European Society of Hypertension (ESH), American Heart Association, Food and Drug Administration and other governing bodies, criteria for cuff-less monitors such as the sensor used in this study have yet to reach widespread, general acceptance. Based on the ESH’s International Protocol for the validation of BP measuring devices26. A recent study investigating the agreement between electrocardiogram and PPG-based HR measurements reported an ICC of 0.90 and mean difference between devices of 2.7 beat/min−127. While these findings may appear promising, it should be mentioned that the overall ICC for HR in this study was 0.96, the mean difference between techniques was < 1 beat/min1  for CO monitors is as high as 30%32, most devices have an error of ~ 40%29, making them unacceptable for clinical use. To the best of our knowledge this is the first scientific study to compare a high-resolution PPG technique with gold standard invasive measurements of CO. We have shown that the percentage error of the wearable device was as low as 25%, with a small bias (0.42 l/min−1) and relatively narrow LOA  before 35% of their total blood volume was withdrawn  at baseline. Arterial oxyhemoglobin saturation was continuously monitored and kept at ~ 98–100%.Animals were sedated with Xylazine , and anesthesia was induced with Ketamine . The ear vein was then cannulated for intravenous administration of a mixture of Diazepam , Ketamine , Propofol , and Tramadol  for analgesia. Cefazolin  was given prophylactically. The pigs were then intubated with a cuffed silastic endotracheal tube . Anesthesia was maintained with 2% isoflurane  in 100% oxygen, and animals were ventilated using controlled mechanical ventilation . Tidal volume was set to 10 ml/kg with a respiratory rate of 13–15 breaths per minute adjusted to an end-tidal pressure of 35 mmHg COThe following vessels were cannulated using the over-the-wire (Seldinger) technique with introducers  inserted into: the left common carotid artery for invasive blood pressure monitoring and heart rate; the right internal jugular vein for venous blood sampling; pulmonary artery catheter placement ; right femoral artery cannulation for arterial blood sampling and induction of bleeding. A urinary catheter (Tiemann Catheter 12F) was placed for collection and output monitoring. Body temperature was monitored rectally. Pulse oximeter was placed on the tail lingual or buccal surface to measure arterial oxyhemoglobin saturation. Continuous three-lead electrocardiogram monitoring was obtained using electrodes placed on the animal’s right forelimb, left forelimb, and left hind limb. At the end of the observation period, surviving animals were euthanized by an intravenous injection of KCl solution .34. Blood was withdrawn manually from the right femoral artery by using a syringe in 50 ml aliquots. The rate of bleeding was controlled to keep mean arterial pressure from dropping below 30 mmHg. When necessary, bleeding was stopped, and the animal was allowed to recover prior to the resumption of bleeding. The total bleeding time was kept between 30 and 60 min.Hemorrhage was induced by the controlled bleeding of 35% of the animal’s calculated blood volumeSBP, DBP, HR, and CO were monitored and recorded every 5 min up to the end of bleeding and every 20 min thereafter. Pulmonary artery pressures were monitored using a Datex-Ohmeda Cardiocap 5 . CO was monitored using a Vigilance II Monitor .https://mimic.physionet.org/; https://doi.org/10.1038/sdata.2016.35), and from numerous clinical studies conducted earlier by the company. The baseline calibration measurement is by a simple offset using either an approved non-invasive, cuff-based device or an invasive (AL) device as in the case of this study, with the average value of three consecutive measurements entered into the device's management application, and from that moment on it tracks changes in blood pressure. Calibration is needed once every 3 months, which increases its clinical usability. Within the context of this study, calibration was conducted only once after the animals were sedated, ventilated, and the instrumentations (including the AL and SG catheters) were inserted.PPG is commonly applied for pulse oximetry, usually transmitting light in specific red and infrared wavelengths which is absorbed by a detector on the other side of the tissue . While passing through the tissue, and upon interaction with oxy- and deoxyhemoglobin, these wavelengths show a unique absorbance pattern. The detector can measure the changing absorbance at each of the wavelengths, and by that it can determine the absorbance resulting from the pulsating arterial blood. The currently used device is based on reflective PPG Fig. , in whicn = 3; SBP: n = 1; DBP: n = 1; CO: n = 1) outlier values were excluded from the final analysis.Data obtained from the AL and SG catheter for SBP, DBP, HR, and CO were screened for outliers according to the following criteria: initially, percentage differences (%∆) between each value and the preceding and proceeding values were calculated. For each variable separately, the average standard deviation (SD) for the sum of preceding and proceeding %∆ was then obtained. Next, the individual data points (%∆) were screened, and any value for which the percentage difference from both the previous and next values was > ± 2SD was eliminated. In total, 6 out of 1312  or trends in BP and CO, four quadrant plots analyses were performed. Concordance rate was calculated as the percentage of data points lying in the upper right and lower left quadrants relative to all points. To reduce statistical noise, we implemented an exclusion zone of ± 2 mmHg and 0.5 l/min for BP and CO measurements, respectively, as suggested by Critchley et al.35 and Saugel et al.36. Statistical analyses were considered significant if p < 0.05. Data were analyzed using SPSS 23.0 .Statistical analyses were performed separately for each of the three phases  and for all phases combined. The Kolmogorov–Smirnov and Shapiro–Wilk tests were used to assess normality, as these tests are sensitive to outliers. To define the degree of correlation between the two methods (i.e. invasive vs. PPG-based), the linear regression formula was defined, Pearson’s correlation coefficient was calculated, and the hypothesis that the slope and intercept are equal to zero was tested. The level of absolute agreement between AL/SG measurements and those obtained from the wearable device for SBP, DBP, HR, and CO was evaluated using intraclass correlation coefficients (ICC) and standard error of the mean (SEM) as well as Bland–Altman plots. Results of the Bland–Altman analyses are reported as mean biases ± 95% limits of agreement (LOA). All other results are presented as means ± SD. Due to the unstable hemodynamic state imposed on the animals and to better assess the PPG device’s ability to track This study conformed to the Guide for the Care and Use of Laboratory Animals . Animal care and experimental procedures were approved by the Ethics Committee of The Hebrew University Faculty of Medicine, Jerusalem, Israel (MD-13-13751-3).


Proteinuria is a major marker of chronic kidney disease (CKD) progression and the predictor of cardiovascular mortality. The rapid development of renal failure is expected in those patients who have higher level of proteinuria however, some patients may have slow decline of renal function despite lower level of urinary protein excretion. The different mechanical (visco-elastic) and chemical properties, as well as the proteome profiles of urinary proteins might explain their tubular toxicity mechanism. Brillouin light scattering (BLS) and surface enhanced Raman scattering (SERS) spectroscopies are non-contact, laser optical-based techniques providing visco-elastic and chemical property information of probed human biofluids. We proposed to study and compare these properties of urinary proteins using BLS and SERS spectroscopies in nephrotic patient and validate hybrid BLS-SERS spectroscopy in diagnostic of urinary proteins as well as their profiling. The project ultimately aims for the development of an optical spectroscopic sensor for rapid, non-contact monitoring of urine samples from patients in clinical settings.BLS and SERS spectroscopies will be used for non-contact assessment of urinary proteins in proteinuric patients and healthy subjects and will be cross-validated by Liquid Chromatography-Mass Spectrometry (LC-MS). Participants will be followed-up during the 1 year and all adverse events such as exacerbation of proteinuria, progression of CKD, complications of nephrotic syndrome, disease relapse rate and inefficacy of treatment regimen will be registered referencing incident dates. Associations between urinary protein profiles (obtained from BLS and SERS as well as LC-MS) and adverse outcomes will be evaluated to identify most unfavored protein profiles.This prospective study is focused on the development of non-contact hybrid BLS - SERS sensing tool and its clinical deployment for diagnosis and prognosis of proteinuria. We will identify the most important types of urine proteins based on their visco-elasticity, amino-acid profile and molecular weight responsible for the most severe cases of proteinuria and progressive renal function decline. We will aim for the developed hybrid BLS - SERS sensor, as a new diagnostic & prognostic tool, to be transferred to other biomedical applications.ClinicalTrials.gov . The date of registration was March 17, 2020.The trial has been approved by  Urine generally represents the fluid biopsy of the kidney, providing an alternative to blood plasma as a potential source of disease biomarkers , 2. TherProteinuria is recognized as a major marker of chronic kidney disease (CKD) severity, the predictor of renal function decline and cardiovascular mortality –8. The mTo date, different diagnostic methods in clinical settings can be used to ascertain urinary proteins. The extent of proteinuria can be gauged using an albumin-specific dipstick, immunochemical techniques, and high-performance liquid chromatography , 13. HowMeasuring the viscosity, elasticity and chemistry of biological fluids is an important analytical tool in diagnostic, prognostic, and preventive medicine and research , 15. TheThe goal of the project is to develop and validate combined Brillouin & Surface-Enhanced Raman Scattering (SERS) Spectroscopy technique for simultaneous non-contact assessment of visco-elastic and chemical properties of human urine proteins as biomarkers of kidney disease. Systematic studies of these properties in proteins of urine samples to be taken from diseased and healthy subjects will be cross-validated by Liquid Chromatography Mass Spectrometry (LC-MS). The project ultimately aims for development of optical spectroscopic sensor for rapid, non-contact monitoring of urine samples from patients in clinical setting.This project is prospective observational validation study, including subsequently the following three stages: measurement validation and protocol development (10–14 months), participant recruitment and evaluation (4–6 months), follow up and outcome assessment (10–14 months). Each stage consists of defined tasks, presented in Fig. The 80 patients with proteinuria will be recruited form National Scientific Medical Center, admitted to the internal medicine department. Age and gender matched 20 healthy volunteers will be enrolled from the outpatient clinics. The inclusion criteria are both male and female patients with any ethnic group, aged between 18 and 65 years old and newly diagnosed patients with glomerulonephritis and different range of proteinuria. The following exclusion criteria will be used: patients aged < 18 and > 65 years, glomerular filtration rate (GFR) < 60 ml/min, pregnant females, patients with diabetes mellitus, cancer, infectious diseases, and other life-treating comorbidities/conditions. Clinical and demographics data of the participants, medical history, comorbidities and data related to the disease will be recorded in case report forms.BLS spectroscopy Fig.  will be Raman spectroscopy Fig.  will perWe propose to construct hybrid Brillouin-Raman system, which will simultaneously assess both visco-elastic  and chemical properties at the same time from the same excitation laser source (532 nm or 780 nm). In this arrangement our Brillouin hyperfine spectrometer  system will be coupled with confocal microscope  and Nanofinder Raman spectrometer  and motorized sample stage  for 3D imaging The schematic of the proposed hybrid all-optical system is displayed in the Fig. A reversed-phase LC-MS will perform for profiling of urine protein. LC-MS system consists of a Dionex HPLC pump  and UHR-QqTOF mass spectrometer Impact II (Bruker). Protein sample will be injected through trapping column setup Acclaim PepMap100 C18 5 mm × 300 um pre-column (Thermo Scientific) and peptides separated on an analytical Acclaim Pep-Map 15 cm x 75um RSLC column (Thermo Scientific) using 75 min multistep acetonitrile gradient  at a flow rate of 0.3 ul/min. Data analysis will be performed through Mascot software using the SwissProt protein database. Furthermore, protein samples differentiated according to their molecular weights will be separately assessed by dual Raman-Brillouin hybrid system for comparative mechano-chemical characteristics and correlations.Full blood biochemistry and corresponding urinalysis will provide additional information about participant health condition, such as functional condition of liver, kidney, heart, metabolic status etc. These widely evaluated routine laboratory data will be obtained from all participants. Routine blood metabolic profile including total protein, albumin, renal and liver function tests, coagulation tests, lipid profiles as well as urinalysis, proteinuria, albumin to creatinine ratio (ACR) and protein to creatinine ratio (PCR) will be performed at the study enrollment. Control renal function tests and ACR or PCR will be performed at the end of the follow up period. All blood and urine samples from participants will be collected at the time of hospital admission or at the visits to outpatient clinic. All blood and urine samples will be stored at -80co freezers for proper preservation before further visco-elastic and chemical analysis will be performed. After last patient is enrolled to the study, all laboratory evaluations will be provided according to the study protocol.The project team is strongly committed to comply with the ethical policy. We guarantee that information provided in the frame of this project is reliable and unique. We confirm our adherence to research ethics, particularly preventing any fabrication and falsification of research data resulting in distorted research data, plagiarism and fake authorship. All work within the project will be conducted in accordance with international and national legal and ethical principles. All participants will follow ethical, health, safety and other relevant regulations. Laboratory research team and service contractors will be blinded from patients’ clinical data to assure clinically non-biased research. The knowledge acquired by this project will be completely disseminated via conventional academic channels such as international and national conference presentations and scientific publications. In order to defend intellectual property, the results of the project will be published in reputable peer-reviewed scientific journals of international standing.The primary outcome of the study is to develop and validate the Brillouin and SERS/Raman spectroscopy for non-contact urinary protein detection and protein profiling in patients with nephrotic syndrome. The secondary outcome of the study is identifying the most hazarded protein profiles associated with adverse outcomes.P-values less than 0.05 will be reported as a significant for all analyses. Mean ± standard deviation (SD) for normal and median - interquartile range (IQR) for abnormal distribution of numerical variables will be shown in all data. Categorical variables will be presented as numbers (percentage). Categorical variables will be compared using Chi-square test or Fisher’s exact test and continuous variables will be compared using Kruskal–Wallis and/or Mann–Whitney U-test, as appropriate. Comparison of repeated two or more variables will be assessed with Wilcoxon and/or Friedman test as appropriate.All statistical analyses will be performed using STATA MP Version 15  statistical package. Two-sided Obtained measurements of urinary protein by Brillouin and SERS/Raman spectroscopy will be compared with LC-MS data from same participant urine samples. Correlation between Brillouin and SERS/Raman spectroscopy parameters, LC-MS data and clinical-laboratory data of patients as well as healthy participants will be provided using Pearson’s or Spearman’s correlation as appropriate.Univariate logistic regression will be used to test the association of several risk factors with high level of proteinuria. Multivariate logistic regression analysis will be used to assess the predictors for adverse outcomes. A receiver operator characteristics curve (ROC curve) analysis will be performed to identify the sensitivity, specificity, negative and positive predictive values of cut-off levels of Brillouin and SERS/Raman spectroscopy parameters in prediction of high proteinuria. Between measurement comparison of AUC will be provided with method developed by DeLong et al. .During 1 year of follow-up, all adverse events such as exacerbation of proteinuria, progression of CKD, complications of nephrotic syndrome , disease relapse rate and inefficacy of treatment regimen will be registered with referencing incident dates. Unadjusted and adjusted regression models will be evaluated to identify the association between urinary protein profiles defined by Brillouin and SERS/Raman spectroscopy and abovementioned adverse outcomes.To the best of our knowledge, there has been no prior investigation of human urine fluids to diagnose proteinuria using by neither Brillouin nor Raman spectroscopy, nor by SERS.n is refractive index, λ is incident laser wavelength, θ is laser light scattering angle and V is a sound velocity in the probed fluid medium. Knowing V and density of medium ρ, one can evaluate longitudinal elastic bulk modulus by E = ρV2. From measured Brillouin peak’s full width at half maximum (FWHM) ∆ω, one can determine the total viscosity of the fluid Recently, dual Brillouin-Raman spectroscopy has been demonstrated as a viable diagnostic sensing tool in variety of biomedical applications –23. Brilaterials –33, inclaterials –34. Brilaterials , 29.On the other hand, Raman spectroscopy is able to provide information about chemical structure, phase and polymorphic state, crystallinity and molecular interactions of biologic samples, including urine proteins –37. It sLC-MS is able to detect all types of urinary protein (which could be missed by nephelometry immunoassay method) and sequencing the amino-acid properties as well as grades the proteins according to their molecular weight . Previous studies demonstrated that LC-MS could identify low-weight molecular urinary proteins that cannot be detectable in routine immunochemistry assay, and early discover microalbuminuria in diabetes and hypertension , 45. HowSummarizing the available relevant literature and emphasizing the scientific novelty and significance of dual Brillouin-SERS spectroscopy, we will try to study and cover following question in this proposed project: Do non-contact dual Brillouin-SERS spectroscopy provide differential and diagnostic information on mechano-chemical profile of urine and urinary protein in healthy subjects and patients with different range of proteinuria?In case of successful development of non-contact hybrid Brillouin-SERS sensing tool and its clinical deployment for diagnosis and prognosis of proteinuria, we expect its application for diagnosis and prognosis of other renal diseases of patients with abnormalities related not just to high protein loss, but also to elevated concentration of urea, creatinine, uric acid and other components. The impact of to-be-developed optical instrumentation and knowledge base is also envisioned in other areas of biomedical sciences and medicine, including biophotonic sensing of viruses, where non-invasive optical diagnostics and imaging is currently in high demand.to develop basic science foundation for measurement and analysis of visco-elastic and chemical properties of human urine and urine proteins of patients having various degree of proteinuria.to construct, optimize and test the hybrid Raman-Brillouin spectroscopy sensing system for non-contact simultaneous monitoring of visco-elastic and chemical properties of urine samples taken from healthy subjects and patients with different range of proteinuria and other kidney diseases related to elevated concentration of urea, creatinine and uric acid and cross-validate them with LC-MS.to identify the most important types of urine proteins based on their visco-elasticity, amino-acid profile and molecular weight responsible for the most severe cases of proteinuria and progressive renal function decline.to reach a roadmap for translating hybrid Brillouin-Raman sensor as a new diagnostic & prognostic tool in patients with proteinuria to a clinical setting and its potential deployment to other biomedical applications.to report research results in at least 3 international conferences and publish at least 5 papers in peer-reviewed international journals with high impact factors.to train young Kazakh medical and physical scientist in advanced inter-disciplinary areas of nephrology & biophotonics.Young scientists in Kazakhstan will be trained in important multidisciplinary areas spanning from nephrology to biomedical photonics & engineering, laser-optical instrumentation design and construction, spectroscopy, biomechanics, biorheology and biochemistry of human fluids. By the end of 3-year project we expect:


Interstitial fibrosis (IF) on kidney biopsy is one of the most potent risk factors for kidney disease progression. The furosemide stress test (FST) is a validated tool that predicts the severity of acute kidney injury  in critically ill patients. Since furosemide is secreted through the kidney tubules, the response to FST represents the tubular secretory capacity. To our knowledge there is no data on the correlation between functional tubular capacity assessed by the FST with IF on kidney biopsies from patients with chronic kidney disease (CKD). The aim of this study was to determine the association between urine output (UO), Furosemide Excreted Mass (FEM) and IF on kidney biopsies after a FST.This study included 84 patients who underwent kidney biopsy for clinical indications and a FST. The percentage of fibrosis was determined by morphometry technique and reviewed by a nephropathologist. All patients underwent a FST prior to the biopsy. Urine volume and urinary sodium were measured in addition to urine concentrations of furosemide at different times . We used an established equation to determine the FEM. Values were expressed as mean, standard deviation or percentage and Pearson Correlation.p = 0.015). Likewise, the total UO and the FEM was progressively lower with higher degrees of fibrosis. An inversely linear correlation between FEM and the degree of fibrosis  was observed.The mean age of the participants was 38 years and 44% were male. The prevalence of diabetes mellitus, hypertension and diuretic use was significantly higher with more advanced degree of fibrosis. Nephrotic syndrome and acute kidney graft dysfunction were the most frequent indications for biopsy. eGFR was inversely related to the degree of fibrosis. Subjects with the highest degree of fibrosis (grade 3) showed a significant lower UO at first hour of the FST when compared to lower degrees of fibrosis (Our findings indicate that interstitial fibrosis correlates with total urine output and FEM. Further studies are needed to determine if UO and FST could be a non-invasive tool to evaluate interstitial fibrosis.NCT02417883.ClinicalTrials.gov  Chronic kidney disease (CKD), particularly in advanced stages, is considered one of the most serious public health problems worldwide , 2.For aFor several decades the evaluation of kidney function has been based primarily on measurement glomerular filtration rate (mGFR) or on equations based either on creatinine or cystatin C where glomerular filtration rate is estimated –29.TheseWe included inpatients that were admitted to the hospital for a kidney biopsy with a clinical indication . Patients were admitted to the nephrology ward at the Instituto Nacional de Cardiologia Ignacio Chavez. Patients needed to be older than 18 years, had a creatinine eGFR ≥15 ml/min by CKD-EPI  and had The research was approved by the institutional research ethics committee and registered with the number 15–920. Written informed consent was obtained from all participants.Furosemide test was performed during the hospitalization for the kidney biopsy. Most of the FST were done 1 day after the kidney biopsy was performed in full agreement by the medical team. In order to avoid urine measurement errors in those patients who did not have a bladder catheter on the day of the test, a post-voiding bladder volume measurement was performed using pelvic sonography.All the 84 kidney biopsies were done at the bedside of the patient using ultrasound guidance (Sonosite®). For the kidney biopsies we used a Biopsy Gun (BARD Magnum®) and needles, procedures were done under local anesthesia.n = 5), 76 subjects were euvolemic and 8 subjects were hypervolemic. Fluid replacement on euvolemic subjects as given on a 1:1 cc based on the previous hourly urine output. Hypervolemic patients did not receive intravenous fluid replacement.Intravenous furosemide was administered at a dose of 1 mg /kg if subjects had not been exposed to diuretics during the previous 7 days and at a dose of 1.5 mg/kg if they have been exposed to loop diuretics within the 7 days prior to the study . MedicalThe kidney biopsy was processed by the pathology department at the Instituto Nacional de Cardiología Ignacio Chávez. All kidney biopsy tissues were considered sufficient for histopathologic evaluation. An experienced renal pathologist blinded to the FST results described the degree of interstitial fibrosis, through direct visualization of light microscopy, with emphasis on the staining of Masson’s Trichrome. In addition, fibrosis percentage was assessed by anoptic lector machine (Morphometry) using the Analyzer Olympus BX51 Microscope, image Software Image-PN- Plus 6 and Camera: VF Evolution C . This technique has been accepted as a standard as it is more reproducible . IF scorUrine Furosemide Excretion: UO was collected at every hour on the hour for 6 h and stored separately in aliquots. Furosemide analyses were determined in individual samples collected at hour 2 (0–2 h), 4 (2–4 h), 6 (4–6 h) and in a mix of all urines.Urine samples were purified using Oasis MCX Cartridge (Part#186000254 Oasis MCX 3 cc cartridge 60 mg 30 μm). The cartridge was washed with 2 ml methanol followed by 2 ml of milliQ water and loaded with the urine sample (0.5 ml adjusted to pH 6.0 with 2 M HCl and diluted with 0.5 ml of MilliQ water). Next, the cartridge was washed with 1 ml of 2% formic acid to remove acidic compounds followed by 1 ml of methanol to remove neutrals and the bound furosemide was eluted with 1 ml of 5% ammonium hydroxide in methanol. The eluted ammonium hydroxide fraction was dried in a speed-vac, reconstituted in 100ul of MilliQ water and quantified by validated HPLC test . The HPLWe estimated the furosemide excreted mass equation (FEM) expressed as percentage using the following equation:All values were expressed as means, standard deviation, and percentages. Results were expressed as average, standard deviation (SD) or as proportions as appropriate. The comparisons were made using chi  for prop2. The presence of diabetes mellitus, hypertension and use of diuretics was predominantly higher in grade III fibrosis. No episodes of hypotension or hipokalemia occurred during the study. As expected, eGFR decreased as interstitial fibrosis increased  (Table p = 0.04) in the native kidneys but not in the transplant group (p = 0.26) (Table The (FEM) was progressively lower with higher degrees of fibrosis, at hours 2 and 4 (1.6 and 0.8 for grade III fibrosis vs 6.8 and 4.0) Table . When an6) Table .Fig. 3Cp = 0.795 vs 26.2 ± 17.2 p = 0.608).We did not find a significant difference between the measurement of subjective fibrosis and morphometry , identifying that the FEM at the second hour was the measurement that best correlated with this cut point. Table .Table 4An inverse correlation between UO and the degree of fibrosis  was observed .p = .008)  but there was no significant correlation between albumin and FEM (p = 0.64). Likewise, there was no correlation between levels of proteinuria and FEM (p = 0.48). When analyzed separately, the correlation between proteinuria and FEM in native versus transplant kidneys was not different . As expected, there was a significant correlation between eGFR (CKDEPI) and FEM2 (p = 0.001) and between eGFR and fibrosis (p = < 0.001) . Results were no different between allograft and native kidneys.Since albumin is necessary for the secretion of furosemide, we evaluated the Pearson correlation between serum albumin with urine output and FEM. There was a significant correlation between serum albumin and total UO  correlated with low urine output during the same time frame. The FEM2 is conceived to function similarly to the forced exhaled volume (FEV1) in pulmonary function tests. A healthy person can expire most of the air from their lung in 1 s. Similarly, a healthy person with a full complement of functioning nephrons can rapidly eliminate furosemide into the urine. However, when tubular dysfunction is present as the case with tubular-interstitial fibrosis, the fibrosed kidney loses its capacity to efficiently move furosemide out of the plasma and into the tubular lumen. This type of functional testing may form the basis of assessing tubular function in a non-invasive fashion.We found that the furosemide excreted mass at 2 h  of 0.87 . This teThe assessment and monitoring of CKD are primarily focused on GFR and degree of proteinuria. While not demonstrated in this study, the FST may offer a non-invasive functional tool for the clinician to assess tubular function in patients with kidney disease. Nonetheless, the FST appears to offer novel information in these patients.Our study has several strengths. First, the study explores a novel concept of tubular function and its association with interstitial fibrosis in kidney biopsies. Second, the amount of fibrosis on the kidney biopsies was assessed also by morphometry removing the potential observation bias by the pathologist. Third, in addition to urine output, we measured the urine excreted mass of furosemide. Lastly, this analysis does demonstrate that the FST offers more information as compared to classic GFR assessment..Our study also has limitations. First this is a cross sectional study and we do not have data on the impact of the FST over kidney function decline over time. Second, the strength of the association among the different variables analyzed  was significant only in the subgroup of native kidneys compared to the group of renal transplantation, probably in relation to a greater renal mass and the larger sample size of the group of native kidneys Finally, although statistically significant, the correlations were not strongIn conclusion, our findings support that interstitial fibrosis correlates with FST with both total urine output and the furosemide excreted mass. This could be established as non-invasive tool to evaluate interstitial fibrosis and may offer more prognostic information over eGFR and proteinuria alone. Further longitudinal studies are needed to establish if FST is associated with kidney function decline over time.


Severe asthma (SA) may require frequent courses or chronic use of oral corticosteroids (OCS), inducing many known side effects and complications. Therefore, it is important to identify risk factors of chronic use of OCS in SA, considering the heterogeneity of clinical and inflammatory asthma phenotypes. Another aim of the present analysis is to characterize a subpopulation of severe asthmatics, in whom blood eosinophil counts (BEC) remain elevated despite chronic OCS treatment.In a cross-sectional analysis of 982 SA patients enrolled in the Belgian Severe Asthma Registry (BSAR) between March 2009 and February 2019, we investigated the characteristics of the OCS treated patients with special attention to their inflammatory profile.3) in 44% of the OCS treated population. Multivariable logistic regression analysis showed that risk factors for chronic use of OCS in SA were late-onset asthma (i.e. age of onset > 40 yr), frequent exacerbations (i.e. ≥2 exacerbations in the previous year) and non-atopic asthma. Late-onset asthma was also a predictor for persistently high BEC in OCS treated SA patients.At enrollment, 211 (21%) SA patients were taking maintenance OCS (median dose: 8 [IQR: 5–10]) mg prednisone equivalent). BEC was high (> 400/mmThese data showed a significant association between a persistently high BEC and late-onset asthma in OCS treated SA patients. Whether it is poor compliance to treatment or corticosteroid insensitivity the reasons for this association warrants further investigation. Although severe asthma (SA) represents only 5–10% of the population with asthma . More thSA defined according to a joint statement from the European Respiratory Society and the American Thoracic Society (ERS/ATS) may require frequent or chronic use of oral corticosteroids (OCS). Indeed, OCS are prescribed as maintenance therapy in 45% of adult SA patients who remain uncontrolled despite GINA step 4–5 treatment [However, OCS are responsible for serious side effects  and comp3) despite OCS maintenance therapy.OCS effectively target elements of the T2 inflammatory pathway, suppressing blood eosinophils quite rapidly  and decrThe goals of the present analysis were to compare patients with and without OCS maintenance, and to characterize patients with high levels of blood eosinophils despite this treatment. For this purpose, we used the Belgian Severe Asthma Registry (BSAR), which is a cohort of adult severe asthmatics and aims at enhancing awareness and knowledge on the natural history of SA in order to optimize patient care .3) and low blood eosinophil counts (BEC) under OCS.In a cross-sectional analysis on SA patients enrolled in the BSAR, we compared characteristics of SA patients chronically using OCS with those SA patients who did not. Chronic OCS use was defined as daily use of OCS. Furthermore, we made a comparison of patients with high  to at least one common aeroallergen  or a positive skin prick test. An exacerbation in the previous year was defined by a course of OCS for at least 3 days for asthma worsening. Emergency room visits, hospitalizations, and intensive care unit stays were also recorded. Presence (or absence) of treatment with long-acting muscarinic antagonists (LAMA), short or long-acting ß2-agonists , anti-IgE, anti-IL5 and macrolides was recorded, as well as OCS maintenance doses.Quality of life was assessed using the self-administered Asthma Quality of Life Questionnaire (AQLQ)  and asthPatients underwent FENO measurement at a flow rate of 50 ml/s according to the ERS/ATS recommendations   followedTobacco status were recorded. An ex-smoker was defined as someone who had stopped smoking for at least 6 months.Sputum was induced almost exclusively in Liège and processed as previously reported ; 225 spuNasal polyps and sinusitis were diagnosed by Ear Nose and Throat specialists either by endoscopy or sinus CT scanner. Gastroesophageal reflux was diagnosed either upon anamnesis (symptoms of heartburn), or by the presence of oesophagitis upon gastroscopy or treatment response to PPI. Chest CT imaging was obtained in patients with an atypical presentation of SA and in case of history of smoking. Hospital Anxiety and Depression Scale (HADS) questionnaires  were useData on vocal cord dysfunction, allergic bronchopulmonary aspergillosis, Aspirin-sensitivity, eosinophilic granulomatosis with polyangiitis , occupational asthma (asthma acquired in the workplace), premenstrual asthma, and obesity were also collected.Near-fatal asthma is described as an asthma attack that require intensive care unit stay with mechanical ventilation.3, between 150 and 299/mm3, between 300 and 400/mm3, and > 400/mm3 to evaluate eosinophil levels in patients treated with OCS and the mean dose of OCS in each group.BEC were categorized in 4 groups: < 150/mm3) under OCS treatment versus those who did not.Finally, an analysis was conducted to compare patients normalizing their BEC  when not normally distributed. Categorical variables were presented as frequencies and percentages. Continuous variables were compared by t-test when normally distributed or by Wilcoxon Mann-Whitney test when non-parametric. ANOVA or Kruskal Wallis analyses were conducted in order to compare more than 2 groups (categories). Fisher exact test was used to compare qualitative variables.1%, sputum % eosinophils and neutrophils, BEC, presence of exacerbations, and hospitalizations during the last year and comorbidities such as nasal polyps, rhinosinusitis, bronchiectasis, emphysema, obesity, GERD, allergic bronchopulmonary aspergillosis, vocal cord dysfunction, aspirin-sensitivity, EGPA, psychopathy, occupational, premenstrual, or Aspergillar asthma. Chronic OCS use was used as the dependent variable. After examining for all potential predictors, the univariate association with the outcome, a stepwise backward logistic regression was conducted; initial model included variables with an association of p < 0.20. Then, the best predictive model was conducted deleting those variables that had the weakest association with the outcome (p > 0.05). This analysis (logistic regression) was repeated in order to identify predictors of BEC ≥ 400 mm3.Predicting factors of “chronic OCS use” were assessed by a logistic regression using independent variables such as atopy, gender, age, body mass index, age of asthma onset, smoking status, pack-year, and measurements of ACQ, FENO, post-bronchodilation FEVp value < 0.05 was considered statistically significant. Statistical analysis was done using STATA version 14.0 .A Between March 2009 and February 2019, 982 severe asthmatics that fulfilled the definition of SA  were enrp = 0.02) in SA patients treated with maintenance OCS and these patients significantly more often had late-onset (≥40 yr) asthma (p < 0.0001), were less atopic (p < 0.0001), and presented with higher FENO (p = 0.03) and higher exacerbation rates (p < 0.0001) compared to OCS naïve SA patients. OCS-treated SA patients also had worse asthma control (p = 0.03) and asthma-related quality of life scores (p = 0.03). Moreover, comorbidities such as emphysema (p = 0.002), bronchiectasis (p = 0.001), GERD (p = 0.007) and EGPA (p < 0.0001) were more prevalent in the chronic OCS-treated SA group.Demographic, functional and inflammatory characteristics and comorbidities of SA patients are shown in Table p = 0.2) between OCS-treated and not treated SA patients with 44% patients with high BEC (> 400/mm3) in the OCS-treated group  (Table Sp = 0.001) in SA patients treated with maintenance OCS and these patients were less atopic (p = 0.005), and presented with higher exacerbation rates (p = 0.0009) compared to OCS naïve SA patients. OCS-treated SA patients also had worse asthma control (p = 0.03). Moreover, sputum eosinophils were lower (p = 0.02) and sputum neutrophils higher (p = 0.005) in the OCS treated patients.When selecting only patients on biologics (anti-IL5 or anti-IgE)  Table S, the proThe results of the univariate logistic regression analysis showed an increased risk of using maintenance OCS in non-atopic male and between 44- and 64-years old SA patients with late-onset asthma (i.e. age of onset ≥40 yr), frequent exacerbations (i.e. ≥ 2 exacerbations in last 12 months), GERD and EGPA.p = 0.003), frequent exacerbations  and non-atopic status  were predictive of chronic use of OCS in SA.Multivariable logistic regression analysis Table  showed t3.In the population of OCS treated patients, a comparison was made between patients with BEC ≥400 and < 400 mm3) were compared to those with low BEC in Table p < 0.0001) , as well as higher eosinophil (p = 0.02) and lower neutrophil (p = 0.0004) counts in their sputum than the SA group with a low BEC. These eosinophilic patients also had a higher prevalence of nasal polyposis (p = 0.04) and a lower prevalence of anxiety and depression (p = 0.004). Moreover, patients with low BEC had slightly poorer lung function (lower FEV1 (p = 0.04) and FVC% (p = 0.009) but similar FEV1/FVC and also poorer asthma control (p = 0.04) than the group with a high BEC. These patients also had a higher prevalence of emphysema (p = 0.02).Demographic, functional and inflammatory characteristics and comorbidities of the 79 OCS-treated SA patients with high BEC ≥400/mm were com01) Fig. , higher 1%, high FENO (> 50 ppb), and increased sputum eosinophils (> 21%), but decreased risk are seen in patients with higher sputum neutrophils. Multivariable logistic regression analysis (Table p = 0.0047) was a predictor for persistently high BEC in OCS-treated SA patients. However, a high ACQ score (> 3.9) was inversely associated with high BEC (OR (IC95%): 0.17 (0.05–0.63), p = 0.0458). Indeed, compared to patients with ACQ scores lower than 3.9 (75th percentile), patients with an ACQ score higher than 3.9 (n = 31) were characterized by low BEC, impaired lung function (low FEV1) and diffusing capacity of lung for carbon monoxide (DLCO), and a higher rate of emphysema (52%) suggestive of Asthma COPD overlap.The results of the univariate logistic regression analysis showed an increased risk of being eosinophilic despite maintenance OCS in patients with late-onset asthma, high FEVis Table  showed t3) (594 (535–821)/mm3), were started on an anti-IL5 monoclonal antibody after enrolment in BSAR. We observed normalization (BEC < 400/mm3) of BEC (62 (39–91)/mm3) in all these patients after one year of anti-IL5 therapy. Also, as previously shown, the OCS dose was significantly reduced from 8 (7–10) to 4 (2–7) mg daily (p = 0.0065) after addition of the biologic therapy.In a sub-analysis, 12 patients treated with OCS and persistently elevated BEC  of these patients still showed a high BEC. The data indicated that late-onset asthma, frequent exacerbations and non-atopic status are associated with OCS maintenance use in SA. Late-onset asthma was also a predictor for persistently high BEC despite OCS.At enrollment in the registry, 21% of SA patients were taking maintenance OCS, which is a lower proportion of severe asthmatic than previously described by Chung .Our data confirms the predominance of females in the overall SA population . However3) in OCS maintenance treated severe asthmatics. Poor response of eosinophils to OCS in this subpopulation needs to be further investigated.There are different phenotypes of SA. Among these phenotypes, late-onset non allergic asthma has been found to occur after the age of 40, to be frequently associated with nasal polyps and chronic rhinosinusitis, and to be prone to exacerbations with good response to OCS . In our 3) in the OCS maintenance treated SA patients. In these patients with high BEC despite OCS maintenance, we may now offer anti-IL5 therapy to decrease the risk of exacerbation [Although OCS are known to suppress eosinophils, BEC were not different between SA patients using maintenance OCS and the ones that do not. We also noticed a high prevalence of patients with high BEC  and diffusion capacity, and higher rate of emphysema (52%). Emphysema which is associated with lower diffusion capacity, may be a cause of dyspnea, which is a symptom considered in the evaluation of asthma control. These subjects fulfill the profile of patients with Asthma COPD overlap. In contrast, as previously reported [Surprisingly, in a fairly small number of patients of this cohort of severe asthmatics, highly uncontrolled asthma was not associated with a high BEC as we could have expected. Instead, it was characterized by impaired lung function  with or without chronic OCS treatment (n = 270).