Biomarkers have become important tools in the diagnosis and management of cardiorenal syndrome (CRS), a complex condition characterized by dysfunction in both the cardiovascular and renal systems. Biomarkers can help identify the presence and severity of CRS, predict its progression and outcomes, and facilitate personalized treatment options. Several biomarkers, including natriuretic peptides, troponins, and inflammatory markers, have been extensively studied in CRS, and have shown promising results in improving diagnosis and prognosis. In addition, emerging biomarkers, such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin, offer potential for early detection and intervention of CRS. However, the use of biomarkers in CRS is still in its infancy, and further research is needed to establish their utility in routine clinical practice. This review highlights the role of biomarkers in the diagnosis, prognosis, and management of CRS, and discusses their potential as valuable clinical tools for personalized medicine in the future. On admission, he presents a SpO2 of 89% on room air, a heart rate of 93 beats per minute, tachypnea (26 breaths per minute), afebrile with blood pressure of 146/92 mmHg, plethoric jugular bilateral veins, and pitting edema of the abdominal wall and legs. Lung ultrasound shows 14 B-lines, right pleural effusion of 200 mL, and a Venous Excess Ultrasound Score (VExUS) is grade 3. He presents with worsening renal function with a serum creatinine level of 1.9 mg/dL on admission (baseline serum creatinine level of 1.5 mg/dL), brain natriuretic peptide (BNP) is 32,000 ng/dL, cancer antigen 125 (CA-125) is 134 mg/dL, and the spot albumin/creatinine ratio is 523 mg/g; urinary sodium, 2 h after intravenous administration of 80 mg of furosemide, is 49 mmol/L. The patient is hospitalized in order to promote rapid and effective decongestion.A 72-year-old man presents to an emergency department with dyspnea and edema that has worsened in the last 4 days; he stopped taking his usual dose of furosemide 2 weeks ago. He has chronic heart failure with reduced ejection fraction and chronic kidney disease (CKD) stage G3aA3 , is prevalent and associated with high morbidity and mortality . There aType 1 cardiorenal syndrome (CRS1) is the most fatal of all, as it involves acute cardiovascular and kidney disorders, leading to an increased risk of mortality up to five times within 28 days of onset. Furthermore, the negative impact of CRS1 continues for up to five years ,4,5. TheThe main goal is to implement a new AKI classification which includes biomarkers other than serum creatinine and urinary output. This would allow timely identification of susceptible patients and ultimately enhance patient outcomes .Although serum creatinine has its shortcomings and constraints, it remains a critical biomarker for patients with cardiovascular ailments. A prime illustration of its significance is the AKINESIS multinational cohort study, which examined 787 patients with acute heart failure (AHF) and measured five biomarkers, including brain natriuretic peptide, high sensitivity cardiac troponin I, galectin 3, serum neutrophil gelatinase-associated lipocalin, and urine neutrophil gelatinase-associated lipocalin, upon hospital admission The findings revealed that serum creatinine is the most reliable predictor of declining renal function .p ≤ 0.05) [These findings stand in contrast to other studies where a new biomarker (Cystatin C) was found to be a more effective predictor than serum creatinine. Among 262 patients with AHF, stratifying the risk of death and re-hospitalization, based on either serum Cystatin C or serum creatinine values, revealed that Cystatin C was superior at predicting which patients would develop these complications, with an area under the curve (AUC) of 0.66 compared to 0.60 for serum creatinine ( ≤ 0.05) .Albuminuria reflects damage to the kidney filtration barrier, vascular endothelial dysfunction, systemic inflammation, tubular damage, and neurohormonal activation. It has been associated with worsening fluid overload through increases in sodium and water retention and cardiac filling pressures, ultimately exacerbating heart failure . FurtherThe natriuretic peptides have utility in AKI patients. Salah et al. analyzed dynamic changes in kidney function and N-terminal pro-B-type natriuretic peptide (NT-proBNP) in 1232 patients hospitalized for AHF. They observed that a reduction in NT-proBNP of more than 30% during hospitalization was associated with worsening kidney function, but lower rates of mortality, independent of the presence of AKI . This hiNeutrophil gelatinase-associated lipocalin (NGAL) is a well-researched biomarker, but has limitations in predicting acute kidney injury during hospitalization. A study of 207 AHF patients found that plasma NGAL values >114 ng/mL were associated with a 340% increased risk of developing AKI but had an AUC of 0.67, performing poorly . SimilarHigh levels of a renin–angiotensin–aldosterone system in urine and systemically, due to the intense neurohormonal activation of CRS, may have implications in predicting AKI in AHF patients. Urinary angiotensinogen (AgtU) values were studied in a multicentric cohort of 732 AHF patients, where it was observed that elevated AgtU values were associated with a 10-fold increase in the probability of complications with AKI grades 1–2 . FurtherSerum sodium levels are closely linked to both neurohormonal activation and water balance. Thus, it is plausible that they play a crucial role in predicting declining kidney function among those with AHF. This theory is supported by the analysis of two cohorts of over 3000 patients with AHF and acute myocardial infarction (AMI), which found that hyponatremia (serum sodium < 136 mmol/L) was associated with a 90% and 50% increase in AKI risk among AHF and AMI patients, respectively .Hospitalized patients with AHF undergoing decongestion frequently develop AKI, with nearly half affected . AlthougIn cardiac surgery patients, AKI occurs in 25–50% of cases . PredictUrinary output is an important metric, and a study of 6637 patients undergoing cardiac surgery showed that those with oliguria (<400 mL/day) had a 7.6% major adverse kidney event (MAKE) within 90–180 days, compared to 4.5% of those who did not have oliguria .Rapid increases in serum creatinine in the first few hours of surgery can help predict the development of AKI in the following days. In a prospective study of 350 patients, a percentage change of >10% from the basal value was associated with up to a six-fold increase in the probability of developing AKI .Proteinuria and albuminuria, detected by urine test strips, are associated with an increased risk of acute kidney injury (AKI) in patients prior to cardiac surgery. Among 1198 evaluated patients, those with the highest proteinuria values demonstrated a three-fold increase in risk, while those with high albuminuria values had a 66% higher risk . Uric acFunctional renal reserve (FRR) is another potential biomarker that measures the kidney’s ability to increase its glomerular filtration rate in the face of physiological stress. A normal FRR increase is considered to be over 30 mL/min before the stimulus. In a cohort of 110 patients undergoing elective cardiac surgery, poor FRR (<10 mL/min) was present in all patients who developed AKI, compared to none of those with an FRR greater than 40 mL/min, with an AUC of 0.83 .Cystatin C is a highly available biomarker and one of the most commonly used . A prosp2). The study observed that patients with PO2 > 15 mmHg for more than 4.8 min during the surgery had a five-fold increase in the risk of developing AKI [In a cohort of 26 patients undergoing cardiac surgery, renal hypoxia was measured by urinary oxygen tension (POping AKI .p < 0.001) [The Nephrocheck biomarker has been extensively studied, and its usefulness demonstrated even in patients with multiple comorbidities, such as chronic heart failure, with an AUC close to 0.88 ,33. In a< 0.001) .Growth-differentiation factor-15 (GDF-15) is a promising humoral marker for risk stratification in cardiovascular disease. In 1176 patients undergoing elective cardiac surgery, elevated GDF-15 values were associated with AKI stage 3. The study reported an AUC of 0.84 .Urinary matrix metalloproteinase-7 (uMMP-7) is another novel biomarker that reflects the activity of intrarenal Wnt/b-catenin, activated in AKI models. In a multicenter cohort of >700 adults and children undergoing cardiac surgery, elevated uMMP-7 values in the first hours were associated with AKI. The study found an AUC > 0.76, outperforming other biomarkers and clinical scores .A meta-analysis evaluated the predictive capacity of different biomarkers (Kidney injury molecule 1 (KIM1), NGAL, liver-type fatty acid-binding protein (L-FABP), and Cystatin C) in the development of AKI after cardiac surgery. Biomarkers measured during surgery had an AUC < 0.75, and those measured in the postoperative period performed even worse, with an AUC < 0.72 . HoweverThe definition of CRS, known as the “five-subtype” approach, has been described in detail in One example of a biomarker of function is albumin, which not only shows glomerular injury but also endothelial dysfunction. Plasma Cystatin-C, which is not affected by muscle mass but by volume status, can differentiate AKI caused by diuretic-induced volume depletion or parenchymal injury. The Renal Optimization Strategies Evaluation–Acute Heart Failure (ROSE-HF) trial demonstrated that protocol-driven aggressive diuresis caused a decrease in eGFR using Cystatin C in 21% of participants, but it was not associated with an increase in kidney tubular injury markers . On the Biomarkers of function, damage, and cell-cycle arrest, while useful in diagnosing AKI and/or cardiac events, are challenging to implement. To improve diagnostic accuracy and discriminate AKI etiology, the 23rd Acute Disease Quality Initiative suggests combining damage and functional biomarkers to identify high-risk patients . In CRS,First-line treatment for fluid removal in HF remains loop diuretics, but there is uncertainty regarding how to administer them . SymptomNatriuretic peptide (NP) levels are of significant prognostic value in various clinical settings. These peptides are released by the heart in response to pressure and volume overload. B-Type natriuretic peptide (BNP) and N-terminal-proBNP have become essential diagnostic tools for evaluating patients who present with acute dyspnea. The NP level indicates the overall function of the heart, including systolic and diastolic function, as well as right ventricular and valvular function . When trTo effectively monitor inpatients with decompensated HF, it is reasonable to obtain an NP level upon admission and just prior to discharge, when the patient is believed to be euvolemic. These levels can aid in predicting outcomes and determining the appropriate level of post-discharge care. It may also be helpful in establishing the patient’s “dry weight” NP level, although additional diuresis after discharge may result in a lower NP level . AdditioIn the diagnosis, therapy, and prognosis of CRS, multi-biomarker strategies may be essential for optimizing clinical outcomes. The combination of biomarkers can increase their accuracy, but it is necessary to determine the optimal combinations to use .Various biomarkers that indicate function, damage, or cell-cycle arrest may predict different outcomes for CRS.p = 0.05), and was associated with a 14-fold increased risk of all-cause death or HF hospitalization in multivariate analysis [A study involving three large chronic HF trials showed that microalbuminuria (30–299 mg/g) and macroalbuminuria (≥300 mg/g) are associated with a 1.4–1.8-fold increased risk of all-cause death, cardiovascular death, or HF hospitalization ,39,40. Aanalysis . Additioanalysis .Plasma and urinary NGAL were linked with a 1.3–2-fold rise in long-term mortality risk in acute HF ,44. KIM-The patient was diagnosed with Type 1 cardiorenal syndrome, with severe vascular congestion promoting AKI. Based on their initial low urinary sodium levels, it was predicted that an increase in the dose of loop diuretics and multisegmental blockade of the nephron with thiazides, acetazolamide, and iSGLT2 would be required. Over the first 24 h, the furosemide dose was increased to 240 mg/day, and 500 mg of acetazolamide was administered orally, resulting in a daily urinary volume of 4200 mL. Over 24, 48, and 72 h, urinary sodium levels improved steadily, measuring at 233, 192, and 160 mmol/L, respectively. BNP levels decreased to 5120 ng/dL, pulmonary B-lines decreased to 2, and the VexUS score was 0. After three months, the patient reported a return to normal life, including physical activity, and held a steady adherence to their treatment routine. His kidney function matched that prior to hospitalization during his programmed clinic visit.The early and accurate identification of vulnerable patients who may develop AKI in conjunction with cardiovascular diseases is paramount to effective intervention and mitigating the risk of serious complications. Although serum creatinine has been widely used as a diagnostic method, its limitations have become increasingly evident, prompting the need to expand our diagnostic tools. The use of biomarkers in cardiorenal syndrome and acute kidney injury has revolutionized the diagnosis and timely intervention of these conditions. Biomarkers have allowed for earlier and more accurate diagnosis, improved risk stratification, and targeted therapy. Several biomarkers have been identified, validated, and incorporated into clinical practice, and their usefulness in predicting clinical outcomes is increasingly being realized. However, the interpretation of biomarkers still requires clinical judgment and additional studies are needed to further validate their utility. Nevertheless, the use of biomarkers holds tremendous promise for improving patient outcomes and reducing the burden of cardiorenal syndrome and acute kidney injury.


Kidney survival in C3 glomerulopathy is significantly higher in patients with a disease chronicity score <4 and proteinuria <3.5 g/d, regardless of baseline eGFR.A faster eGFR decline in C3 glomerulopathy is associated with higher probability of kidney failure.Patients with glomerulopathy with a progressive reduction in proteinuria over time did not reach kidney failure.C3 glomerulopathy is a rare kidney disease, which makes it difficult to collect large cohorts of patients to better understand its variability. The aims of this study were to describe the clinical profiles and patterns of progression of kidney disease.2, proteinuria under/above 3.5 g/d, and histologic chronicity score under/above 4. The change in eGFR and proteinuria over time was evaluated in a subgroup with consecutive measurements of eGFR and proteinuria.This was a retrospective, observational cohort study. Patients diagnosed with C3 glomerulopathy between 1995 and 2020 were enrolled. Study population was divided into clinical profiles by combining the following predictors: eGFR under/above 30 ml/min per 1.73 m2. The median eGFR slope of patients who reached kidney failure was −6.5 ml/min per 1.73 m2 per year (interquartile range −1.6 to −17). Patients who showed a reduction in proteinuria over time did not reach kidney failure. On the basis of the rate of eGFR decline, patients were classified as faster eGFR decline (≥5 ml/min per 1.73 m2 per year), slower (<5 ml/min per 1.73 m2 per year), and those without decline. A faster eGFR decline was associated with higher probability of kidney failure.One hundred and fifteen patients with a median age of 30 years (interquartile range 19–50) were included. Patients were divided into eight clinical profiles. Kidney survival was significantly higher in patients with a chronicity score <4 and proteinuria <3.5 g/d, both in those presenting with an eGFR under/above 30 ml/min per 1.73 mKidney survival is significantly higher in patients with a chronicity score <4 and proteinuria <3.5 g/d regardless of baseline eGFR, and a faster rate of decline in eGFR is associated with higher probability of kidney failure. Both pediatric and adult populations were included. Patients with missing data on baseline kidney function, proteinuria, or outcomes were excluded.17 Patients were considered to have dense deposit disease when highly electron-dense intramembranous deposits were observed on electron microscopy and C3 glomerulonephritis when deposits did not fulfill this criterion.A diagnosis of C3G required C3 staining on immunofluorescence at least two orders of magnitude greater than any immunoglobulin staining.18 Data on eGFR, serum albumin, C3 levels, and proteinuria were collected at baseline and after 1, 3, 6, 12, 24, 60 months, and/or last follow-up. Complement genetics were performed as shown in Supplemental Methods. Plasma C3 levels were categorized as under/above 77 mg/dl, on the basis of 85% lower limit of the laboratory normal range.Baseline and follow-up data were compiled from the medical records as described elsewhere.12Kidney biopsy specimens were examined in the pathology departments of the participating hospitals. The degree of disease activity and chronicity was analyzed according to the previously published C3G histologic index.This study was approved by the Institutional Review Board of Hospital Universitario 12 de Octubre (CEI16/266) and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from participants or their parents to participate in the study.Baseline was defined as the time at which the diagnosis of C3G was made and follow-up period as the interval between kidney biopsy and last outpatient visit or kidney failure.Nephrotic syndrome was defined as a proteinuria of >3.5 g/d along with serum albumin <3 g/dl. Nephritic syndrome was the combination of hematuria, non-nephrotic proteinuria, hypertension, and kidney function impairment. Asymptomatic urinary abnormalities were defined by the presence of non-nephrotic proteinuria and/or persistent microscopic hematuria >5 erythrocytes per high power field.2, the need for dialysis or kidney transplantation. Secondary outcomes included complete remission, as an eGFR >60 ml/min per 1.73 m2 and proteinuria <0.5 g/24 hours, and partial remission, as a proteinuria reduction >50% plus stabilization/improvement in kidney function. Relapse was defined as the return of pretreatment proteinuria and/or declining kidney function after any remission.The main outcome was kidney failure, defined as an eGFR <15 ml/min per 1.73 m19 the study population was divided into different clinical profiles by combining these predictors: eGFR under/above 30, proteinuria under/above 3.5 g/d, and total chronicity score under/above 4.Since we previously found that baseline eGFR, proteinuria, and histologic total chronicity score were the main predictors of kidney failure,t test or the Mann–Whitney U test, where appropriate. Chi-squared test or Fisher exact test was used for categorical variables.This was a retrospective, longitudinal, multicenter, observational cohort study. Descriptive statistics are presented as mean±SD or median and interquartile ranges (IQR) for continuous variables and frequencies or percentages for categorical variables. Comparisons of continuous variables between two groups were assessed by using the unpaired The incidence rate was calculated as average annual incidence over the study period. The numerator included all patients diagnosed with C3G during each distinct year of the study period. The denominator was calculated based on an estimate considering the number of hospitals participating in the study and the population covered by these hospitals, assuming that all patients during the specified year were followed up for the entire year without loss to follow-up.For the evaluation of the change in eGFR and proteinuria over time, a subgroup of 85 patients who had at least four consecutive measurements of eGFR and proteinuria was used. Estimation of eGFR slope and change in proteinuria over time were analyzed with linear mixed-effects models. Negative or positive values of these parameters indicated an increase/decrease over time.Subject-specific longitudinal trajectory plots and locally weighted smoothing plots were used to represent the change of eGFR and proteinuria over follow-up.Distributions of time to kidney failure were depicted by survival curves using the Kaplan–Meier method, and the survival curves by clinical profiles were compared using the log-rank test.P value <0.05 was considered to be significant. Analyses were performed using IBM SPSS Statistics 24.0 and R software v.3.6.3.A n=21), those without alternative complement pathway genetic studies (n=20), and those with underlying monoclonal gammopathy (n=23).The total study group consisted of 115 patients after excluding patients not fulfilling diagnostic criteria for C3G . Twenty-eight (24%) were pediatric patients. Forty-one (36%) patients had a baseline eGFR<30, and 47 (41%) patients had baseline proteinuria ≥3.5 g/d. Nephrotic syndrome was the most common clinical presentation. Regarding the histologic characteristics, 46 (40%) patients had a total activity score ≥9, and 44 (38%) patients had a total chronicity score ≥4. Supplemental Table 2 presents the pathogenic variants in complement genes and acquired abnormalities in the study population.The median age was 30 years (IQR 19–50), and 51 subjects (44%) were female (Over a median follow-up of 49 months IQR 24–112), 23 (20%) patients achieved complete remission, 31 (27%) patients achieved partial remission, and 46 (40%) patients reached kidney failure. Figure 12, 23  patients with baseline eGFR ≥30: 3 (48%) had a chronicity score <4 and proteinuria <3.5g/day, 22 (30%) had a chronicity score <4 and proteinuria ≥3.5 g/d, 8 11%) had a chronicity score ≥4 and proteinuria <3.5 g/d, and 8 (11%) had a chronicity score ≥4 and proteinuria ≥3.5 g/d (Table 1% had a Of the 41 36%) patients with baseline eGFR <30: 8 20%) had a chronicity score <4 and proteinuria <3.5 g/d, 5 (12%) had a chronicity score <4 and proteinuria ≥3.5 g/d, 18 (44%) had a chronicity score ≥4 and proteinuria <3.5 g/d, and 10 (24%) had a chronicity score ≥4 and proteinuria ≥3.5 g/d —particularly urinary tract infections (10%) followed by cardiovascular events (13%), cytopenia (10%), new-onset diabetes mellitus (4%), and avascular necrosis of the hip (4%).Supplemental Table 1).For the evaluation of the slope of eGFR and change in proteinuria over follow-up time, a subgroup of 85 patients with similar characteristics to those of the overall cohort was used (2 per year (IQR −1.6 to −17). Supplemental Figure 1 shows the individual-specific longitudinal trajectories of eGFR for each treatment category, according to the development of kidney failure. A progressive increase in eGFR was observed in the group of patients not reaching kidney failure. This rise in eGFR was steeper in patients treated with corticosteroids plus MMF, whereas in the rest of therapeutic groups, the eGFR exhibited a trend toward a nonlinear quadratic pattern.Nineteen (22%) patients achieved complete remission, 29 (34%) patients achieved partial remission, and 25 (29%) patients reached kidney failure. The median eGFR slope of this latter group was -6.5 ml/min per 1.73 mSupplemental Figure 2 shows the individual-specific longitudinal trajectories of proteinuria for each treatment category, according to the development of kidney failure. Patients who showed a progressive reduction in proteinuria over time did not reach kidney failure.On the other hand, the median change in proteinuria of patients reaching kidney failure was 2.5 g/d per year (IQR 0.5–4.5). Supplemental Figure 3). In addition, when kidney survival was analyzed according to the categories of serum C3 levels at the end of follow-up, a nonsignificant trend was observed toward a better survival in patients with C3 levels ≥77 mg/dl .A trend toward a decrease in C3 levels was also observed in patients who reached kidney failure (2 per year), those with a slower eGFR decline (<5 ml/min per 1.73 m2 per year), and those without decline (i.e. positive slope in eGFR over follow-up) . Kidney survival was significantly higher in patients with a chronicity score <4 and proteinuria <3.5 g/d, regardless of eGFR at presentation. Finally, we assessed the different patterns of eGFR change over time, and as expected, a faster rate of decline in eGFR was associated with higher probability of reaching kidney failure.20 while others have not.9 Kidney failure was less likely in patients with lower disease chronicity in kidney biopsy and lower proteinuria. These findings are in line with others21 and suggest that the importance of proteinuria in C3G not only as a marker of disease severity but also as a predictor of kidney damage.23 On the other hand, the percentage of patients who reached kidney failure in the group of patients with a baseline eGFR ≥30 was higher in those with concomitant higher disease chronicity in kidney biopsy, which points toward a potential point of no return in the disease.12Eight clinical profiles of patients were identified. Among the group of patients with a baseline eGFR ≥30, those with lower disease chronicity in kidney biopsy were more likely to respond to immunosuppressive regimens and to achieve remission . Most of these patients were treated with corticosteroids plus MMF. Although the mechanism of action of this therapeutic regimen is not well understood, some studies have found beneficial effects with this regimen,The group of patients with a baseline eGFR <30 was predominantly comprised by adults. Interestingly, within this group of patients with lower disease chronicity and proteinuria, five (63%) patients achieved complete remission at the last follow-up, pointing toward a reversible kidney damage at the time of clinical diagnosis. Similarly, these patients were mostly treated with corticosteroids plus MMF. As expected, the probability of kidney failure was higher in those with higher disease chronicity and proteinuria. We speculate that this could be due to a more aggressive clinical phenotype or a late diagnosis of the disease.16 Thus, patients with C3G require a close follow-up after diagnosis, although the most appropriate strategy or biomarker to monitor relapses of the disease remains to be defined.Another interesting observation in our study was that a subset of patients had a clinical relapse of the disease after having achieved some degree of remission with immunosuppressive therapy. Although the overall clinical course is slowly progressive in most of the patients, disease exacerbations may also occur in the context of superimposed alternative complement activation by intercurrent factors, such as infections.11 Three different groups were distinguished: faster, lower, and no decline of eGFR over time. A large proportion of patients with a faster decline corresponded to dense deposit disease cases—although the differences did not reach statistical significance—and had been treated with an immunosuppressive regimen different than corticosteroids plus MMF. Patients without progression of kidney disease were mostly men, with nephritic syndrome as the most frequent presentation and low degree of chronicity in kidney biopsy.Finally, we analyzed the clinical patterns of eGFR change over time and confirmed the inverse relationship between eGFR change and proteinuria among different subgroups of patients with C3G on the basis of the rate of decline.24According to our results, the decline in eGFR over time is not uniformly linear in patients with C3G, and the rate of decline can vary widely between individuals. Therefore, these different patterns of progression should be considered for both clinical studies and trial recruitment. Moreover, from a clinical standpoint, they could also help to titrate the intensity of treatments or to schedule the initiation of dialysis or kidney transplantation, particularly at later stages of the disease.Taken together, our study suggests that the clinical profile of patients who could benefit most from anticomplement therapies would be those with lower degree of disease chronicity in kidney biopsy. In a subset of patients with a low eGFR at clinical presentation, the kidney function may recover—at least partially—and thus, in our opinion this sole criterion should not be used to decide whether to treat a patient with C3G with future anticomplement drugs. One might speculate that once these new anticomplement agents were prescribed, those who experienced a decrease in proteinuria or a change in the rate of decline of eGFR would be those associated with a better renal prognosis in the medium and long term, although further studies are warranted to better address this issue.This study is subject to limitations. First, owing to the observational and retrospective nature of the study, no causal relationships can be established. Second, given the retrospective nature of this study and the large period covered, some cases of C3G might not have been correctly identified during follow-up in the participating centers, which could have affected the estimated incidence rate. Third, although in this study, we were able to identify patients with C3G with poor short-term and medium-term kidney prognosis, longer follow-up may be necessary to fully capture the natural history of the disease in some patients. Despite these limitations, this study further contributes to the understanding of the disease by providing additional information for predicting outcomes in clinical settings.In conclusion, kidney survival was significantly higher in patients with a disease chronicity score <4 and proteinuria <3.5 g/d, regardless of baseline eGFR; a faster eGFR decline was associated with higher probability of kidney failure; and patients with a progressive reduction in proteinuria over time did not reach kidney failure. Nevertheless, several questions remain open for further research, and therefore, prospective studies are warranted to better elucidate them.


To compare the efficacy of intravenous Labetalol and intravenous Hydralazine in reduction of blood pressure in patients with severe pre-eclampsia.This comparative study was conducted at the Department of Obstetrics and Gynecology at Ziauddin University Hospital, Karachi from1st June 2019 to 30th June 2020. Total 208 pregnant women having severe pre-eclampsia (systolic pressure ≥160 mmHg and diastolic pressure ≥110mmHg) were included in study. Group A received I/V Labetalol. Group B received I/V Hydralazine. Efficacy of drugs was observed by reduction in blood pressure and the number of doses administered. Data was analysed using SPSS version 26.Systolic blood pressure reduction in Labetalol group was significantly lower than in hydralazine group . Diastolic blood pressure reduction was also lower in labetalol group than in hydralazine group (p= 0.03). Number of dosage of drugs in Group A  was 3.2 ±1.2 vs. Group B  was 4.4±1.4, p =0.006).The results of this study show that Labetalol is more effective as compared to Hydralazine in terms of reducing the systolic and diastolic blood pressure and number of doses (Drugs) for in patients with severe preeclampsia. Pre-eclampsia is a disorder occurring during pregnancy mostly in second trimester and if not treated, it develops to eclampsia.8Progression from mild to severe hypertension in pregnancy is not predictable and sometimes can be sudden. Pregnancies with severe hypertension with systolic blood pressure >160mmHg and diastolic blood pressure >110mmHg needs admission in hospital.13Due to limited data from the local population, the debate is still ongoing in our setting. Hence, the aim of the current study was to compare the efficacy of Labetalol and Hydralazine for the treatment of hypertension in patients with preeclampsia. As most of the previous studies reported mean reduction in blood pressure in severe pre-eclampsia, we wanted to confirm if this is true for our population. Only a few studies focused on assessing the efficacy. This study will provide current & local magnitude of efficacy in comparison of labetalol and hydralazine in addition this study will emphasize on early diagnosis in making strategies to overcome the pregnancy related complications & morbidity.st June 2019 till 30th, June 2020. Total 208 pregnant women with 20-35 years of age, gestational age ≥20weeks (determined by dating scan (9-13weeks) having severe pre-eclampsia (systolic pressure ≥160 mmHg and diastolic pressure ≥110mmHg), admitted and fulfilling the criteria were included in study. Hypertension prior to pregnancy, women with renal disease, known allergy to hydralazine/labetalol, deranged LFT'S due to any other cause, were excluded from the study.This comparative study was conducted at the Department of Obstetrics and Gynecology at Ziauddin University Hospital, Karachi during the period of 1By using WHO Sample size calculator, taking efficacy of labetalol 86.5% Two groups A and B of pregnant women were identified. The whole procedure was explained about the drugs used and procedure before taking Informed consent from the patient or her attendants. The study was approved by the ethical review committee of the institution.a). Group A was received I/V Labetalol (112 participants)b). Group B was received I/V Hydralazine (106 participants)Nonprobability sampling technique was used for sample collection. Eligible women were given verbal explanations and written informed consent forms about the study. Enrolled patient's blood pressure was recorded by using Mercury manometer. An appropriate cuff size was used for accuracy. Age, parity, and gestational age were matched to control confounding variables. Blood pressure, pulse, respiratory rate, temperature, fetal heart rate was noted. General physical  and Systemic examination were performed) to make accurate diagnosis. Protein in urine was checked by dipstick method.The patients were assigned to either of the group A or B. Group A was given 20mg bolus dose of labetalol in 2 minutes, and blood pressure was checked after 20 minutes, if not effective, then 40mg dose of labetalol was given. If still no effect was seen, then 80 mg were given every 20 minutes up to a maximum dose of 300mg and every time blood pressure was checked after 20 minutes. Group B were given I/V hydralazine 5mg in blouse dose then were repeated every 20 minutes in needed up to five doses, maximum 25mg were given. Target Blood pressure was 130/90mmHg.Throughout this period vitals were taken. Fetal hearts were closely monitored. Time taken to reduce the blood pressure up to desired level was noted on predesigned proforma. Final outcomes were measured after six hours in terms of efficacy.Information was entered in SPSS  version 240. Mean +SD and confidence interval were calculated for age, gestational age, parity, blood pressure and number of doses. Frequency and percentage were calculated for efficacy. Chi-square test with 90% confidence intervals was applied to compare the efficacy in both groups. Relative risk was calculated to estimate the magnitude of efficacy that is i.e., efficacy= 1-RR. Confounders were controlled through a stratification of age, parity, gestational age and number of doses to see the effect of these on outcome variables. Post stratification, apply chi-square test, taking P <0.05 as significant.In this study the mean age of women age was 26.6±6.0 (mean ±S. D) years. The age range was 18-45 years. In Group A , the mean age (S. D) was 27.2±6.1 years and the range were 18-45 years; In Group B , the mean age was 27.1±5.9 year with the age range was 18-45 years. Forty four percent women were belonging to ≤ 25 years of age group, as depicted in Mean, Standard deviation of Systolic blood pressure reduction in Group A  was 105.5±11.3 vs. Group B  was 115.8 ±17.1, value of ‘p’ being 0.000 which is significant. Diastolic blood pressure reduction Mean, Standard deviation in Group A  was 69.5 ±9.2 vs. Group B  was 78.9 ±11.2, value of ‘p’ being 0.003 which is statistically significantly high. Number of dosage of drugs in Group A  was 3.2 ±1.2  vs. Group B  was 4.4±1.4, the P value was more than 0.05  was significantly high. Gestational amenorrhea Mean ±Standard deviation in Group A  was 37.1±2.7 vs. Group B  was 37.7±1.7, P value 0.000 was more than 0.05 which was significant. Labetalol found more effective (54.5) as compared to Hydralazine (45.5%) in terms of reducing the systolic and diastolic blood pressure and number of doses (Drugs) for in patients with severe preeclampsia. Pre-eclampsia is one of the leading causes of maternal mortality in the postpartum period. The current study evaluated the efficacy of hydralazine for the treatment of preeclampsia as compared to labetalol. We found that both drugs were effective as antihypertensive in patients with preeclampsia however, labetalol was more efficacious than hydralazine (p<0.000). Moreover, Hydralazine required significantly higher number of doses to control blood pressure compared to labetalol . This indicates that despite the similar effectiveness of both the drugs, labetalol requires lesser dosage to treat hypertension in pregnant patients with preeclampsia hence, lesser probability of having side effects in the peripartum or postpartum period.These findings are in accordance with previous literature. In a study by Nombur , it was found that both labetalol and hydralazine were equally effective for the treatment of severe hypertension in patients with preeclampsia.In 2018, a network meta-analysis and trial sequential analysis was performed to analyse the current evidence on the most effective drugs used to treat hypertension during pregnancy.(15) A total of 51 studies were analysed for systematic review while 46 of these were eligible for meta-analysis. It was concluded that nifedipine, hydralazine and labetalol shared similar efficacy and safety profiles in the treatment of hypertension in pregnancy.In contrast to the above-mentioned studies, a previous study evaluating the efficacy of hydralazine and labetalol for treatment of severe hypertension in pregnancy reported the efficacy of hydralazine & labetalol was 96.2% and 86.5% respectively.16Both drugs are effective against severe hypertension in pregnancy however, in mild to moderate hypertension the doses should be carefully monitored to avoid maternal and neonatal complications. Another drug nifedipine has been used widely to treat hypertensive crisis in pregnant women with severe preeclampsia. Shi DD et al., observed that oral nifedipine was quicker in achieving blood pressure control in pregnant women as compared to intravenous labetalol. 17Despite the many strong points of the study, there are some limitations. Firstly, the patients were not followed up to record long term side effects of the drugs. Secondly, it was a single-center study. A more diversified study could highlight some significant socio-demographic and ethnic factors at play. In the current study, no short-term complications were observed. However, further longitudinal studies should be conducted to evaluate the long-term side effects of these antihypertensive drugs on pregnant women with preeclampsia.Pregnancy-induced hypertension and preeclampsia are lethal yet manageable complications of pregnancy and is a common occurrence in our setting. Adequate pharmacological intervention is necessary to lower the risk of complications in women with hypertensive diseases during pregnancy or delivery. According to our findings, labetalol, and hydralazine both give satisfactory results. However, based on lower requirement of dosage and higher efficacy rate, labetalol is considered more efficacious.The results of this study show that Labetalol is more effective as compared to Hydralazine in terms of reducing the systolic and diastolic blood pressure and number of doses (Drugs) for in patients with severe preeclampsia.


To evaluate the effect of fluorescein dye usage on renal function in patients with diabetic retinopathy (DR) and chronic kidney disease (CKD).Diabetic patients with retinopathy who were candidate for fundus fluorescein angiography (FA) were evaluated for serum creatinine and urea levels within five days prior to performing the FA. Serum creatinine levels of 1.5 mg/dl or more in males and 1.4 mg/dl or more in females were both identified as CKD and were included in the study. An increase of 0.5 mg/dl or 25% in creatinine after FA was considered as contrast-induced acute kidney injury (AKI). Estimated glomerular filtration rate (eGFR) was also calculated for all patients using a CKD-Epi formula. CKD grading was determined based on eGFR values.P = 0.475). The mean serum creatinine before and after the test was 1.89 P = 0.993). The mean eGFR before and after the test was 44.024 mL/min/1.73 m2(P = 0.875).Forty-two patients agreed to participate, of which 23 (54.8%) were male. Seventeen patients were identified with grade 3a or lower CKD, 12 with grade 3b, 11 with grade 4, and two with grade 5 CKD. Considering all grades of CKD, the mean blood urea before and after angiography was 58.48 According to the findings of this study, FA does not seem to further deteriorate kidney function in patients with diabetic associated CKD. During this time, contrast-induced nephropathy usually manifests.Fundus fluorescein angiography (FA) is a valuable method to evaluate microvascular complications in patients with diabetic retinopathy (DR). This procedure involves injection of fluorescein dye, which is a non-ionizing contrast, intravenously. Fluorescein dye is metabolized by the kidneys and excreted in the urine during the first 48–72 hr after injection.,2,3,4 Chronic kidney disease (CKD) is defined as a gradual decline in renal function which is initially subclinical. Contrast-induced acute kidney injury (AKI) is defined by an increase in serum creatinine of Considering that concomitant diabetic nephropathy is possible in patients with DR, there is a concern as to whether fluorescein usage can deteriorate the kidney function further.,6Some angiography centers require a nephrology consult, in addition to blood and urine workup, before preceding to FA in patients with CKD. Apart from the burden of cost and time on patients and healthcare system, referral of these patients for additional testing may cause a delay in diagnosis and treatment of the eye condition.Although renal side effects have been attributed to fluorescein usage in previous studies, there is still no consensus in this regard.In this study, we are going to determine whether the fluorescein can cause contrast-induced AKI in diabetic patients with CKD who are candidates for fundus FA.This prospective study was performed in the retinal imaging section of Farabi Eye Hospital from January 2019 until January 2020. The study protocol was reviewed and approved by the ethics committee of Tehran University of Medical Sciences under approval number 96014334034. Prior to recruitment, all patients were informed of the details of the study through verbal and written communication. According to the Helsinki Declaration, a written consent was also obtained from patients.The glomerular filtration rate (GFR) (ml/min/1.73 m2) was calculated for all patients based on the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.The CKD grading was determined based on the eGFR levels. According to the Kidney Disease Outcomes Quality Initiative guidelines, eGFR ofPatients with DR who were referred to the imaging section for performing FA to confirm the stage of retinopathy were routinely asked for a history of renal dysfunction. Those with a positive history were sent for blood test to determine urea and creatinine levels. If creatinine levels were The process of FA involved an injection of 2.5 ml of 10% fluorescein sodium solution (1g/5cc)  into the antecubital vein access in 5 s. For at least 10 min after the dye injection, fundus images were captured by Heidelberg retina angiography with confocal SLO . Patients were then monitored for about 15 more minutes for any complications. Blood urea and creatinine levels were retested 48–72 hr after the fluorescein injection. Serum creatinine levels, before and after contrast administration, were compared and if increased by Moreover, patients who had received contrast material for imaging during the prior two months or those with kidney failure due to another cause, final stage of renal dysfunction requiring dialysis, chronic heart failure, pregnancy and breastfeeding, history of sensitivity to the contrast agent, consumption of nonsteroidal anti-inflammatory drug, angiotensin receptor blocker, angiotensin converting enzyme inhibitor, or intravenous diuretic were all excluded from the study.t-test, and Wilcoxon singed-rank test was used to compare nonparametric variables. PData analysis was performed using the SPSS software version 18 . Alterations of the variables with normal distribution was analyzed using paired sample We included 42 patients, with the mean age of 58.1 years. Of them, 23 (54.8%) were male. Regarding the CKD grading, 17 patients were identified with grade 3a or lower CKD, 12 with grade 3b, 11 with grade 4, and two with grade 5.P = 0.475).Overall mean urea in patients before and after angiography was 58.48 P = 0.993). The mean eGFR before and after the test was 44.02423.5447 and43.850  21.8581mL/min/1.73 m2(P = 0.875) [Table 1].The mean serum creatinine levels were 1.89 Differential changes in eGFR before and after FA in all stages of CKD was not significant [Table 2].Findings in our study showed that FA does not exacerbate kidney dysfunction (AKI) in diabetic patients with an already reduced GFR (CKD). Therefore, for patients with diabetic nephropathy in this instance who are candidates for FA, nephrology consult or additional tests before and after fluorescein injection are not required.2 into three grades of severity from grade 3 to 5 CKD. None of these subgroups showed a significant alteration in eGFR following fluorescein injection.In a retrospective study by Kameda et al, patients' serum creatinine level within one month before and after FA were investigated to calculate the estimated glomerular filtration rate (eGFR). They categorized patients with eGFR of Then, the effect of FA on renal function was evaluated in these patients. However, the actual timing that they tested the serum creatinine levels before and after FA was indeterminate, especially as there were some delayed measurements after FA, which might have influenced the omission of cases with AKI. AKI definitions are based on changes up to a maximum of seven days following the presumed insult.They also included patients with other causes of renal dysfunction other than diabetes. They reported a significant change in serum creatinine levels in the high-risk group and suggested caution be applied for these patients.In another study by Chung et al, patients were divided into three categories based on serum creatinine levels before angiography; including low ( In our study, although the mean creatinine level was higher as compared to the Alemzadeh's study both before and after FA, the dose of injected fluorescein was 250 mg which was half of the dose used in their study.In another study by Alemzadeh et al on 44 diabetic patients, serum creatinine levels showed a significant increase 72 hr after FA in 20% of patients. In contrast to studies by Kameda and Chung, Alemzadeh study showed that an increase in serum creatinine and AKI can happen secondary to FA. However, their patients were not categorized in terms of kidney damage.especially as diabetes mellitus is the leading cause of propensity toward renal dysfunction.Kidney damage and increase in serum creatinine following the use of contrasts in susceptible patients has been attributed to vasoconstriction, which reduces blood flow to the medulla. Of note, renal blood flow auto regulation is defective in patients with CKD. Identifying high-risk patients and preventing the occurrence of AKI is critical,,14,15,16 However, in a recently published article with a retrospective design, fluorescein was found to play a role in the progression of nephropathy. Nevertheless, the authors were not sure of the clinical significance of their result due to two reasons. Firstly, under normal circumstances fluctuations of up to 15% are possible each time the serum creatinine level is tested. The second reason is related to the criteria used; in a patient with AKI, creatinine-based formulas are prone to overestimating the eGFR. Therefore, due to uncertainty about creatinine levels, their conclusion about the effect of the fluorescein on nephropathy is dubious.It is noteworthy that most kidney damage related to contrast agents has been due to iodinated contrast agents, while fluorescein is a non-iodinated one.Our study was a prospective study on patients with DR and CKD. We found no significant effect on renal function shortly after performing FA. We used half of the recommended dosage of fluorescein and could still obtain good-quality images of fundus angiography. Nephrology consults and further evaluation for these group of patients seem to be unnecessary.There are two major limitations for our study. Firstly, the relatively small number of cases. Secondly, we did not categorize our patients according to the multiple types of medications used by each patient; such as oral hypoglycemic agents, diuretics, or statins. However, as overall we did not observe fluorescein injection causing any AKI, lack of such data might not be affecting the results.None.None.


We observed no complete disease specificity for any of the markers tested. However, comparison among the different LSDs highlighted new applications and perspectives of the existing biomarkers. We observed elevations in glucosylceramide isoforms in the NPC and Gaucher patients relative to the controls. In NPC, there was a greater proportion of C24 isoforms, giving a specificity of 96–97% for NPC, higher than 92% for the NPC biomarker N-palmitoyl-O-phosphocholineserine ratio to lyso-sphingomyelin. We also observed significantly elevated levels of lyso-dihexosylceramide in Gaucher and Fabry disease as well as elevated lyso-globotriaosylceramide (Lyso-Gb3) in Gaucher disease and the neuronopathic forms of Mucopolysaccharidoses. In conclusion, DBS glucosylceramide isoform profiling has increased the specificity for the detection of NPC, thereby improving diagnostic accuracy. Low levels of lyso-lipids can be observed in other LSDs, which may have implications in their disease pathogenesis.Dried blood spots (DBSs) biomarkers are convenient for monitoring for specific lysosomal storage diseases (LSDs), but they could have relevance for other LSDs. To determine the specificity and utility of glycosphingolipidoses biomarkers against other LSDs, we applied a multiplexed lipid liquid chromatography tandem mass spectrometry assay to a DBS cohort of healthy controls ( In the post-COVID-19 pandemic healthcare setting, there is growing demand for remote patient management. Dried blood spots (DBSs) can offer this convenience, and DBS enzyme and biomarker assays are proving increasingly useful in the diagnosis and monitoring of patients with lysosomal storage diseases (LSDs) ,3,4. BioThe glycosphingolipids (GSLs) are lipids that accumulate in the glycosphingolipidoses, but unusually for metabolic disorders, it is the surrogate deacylated lyso-GSL equivalent lipids that have been found to be more useful as biomarkers than the accumulated GSLs themselves ,6. What We have previously described an optimised extraction protocol and ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method to measure 90 isoforms of 4 classes of GSLs from the glycosphingolipid degradation pathway  in DBS, Therefore, we aimed to determine the specificity of known biomarkers for Niemann–Pick type C (NPC), sphingomyelin (SM), and lyso-SM in an LSD disease cohort which included patients with mucopolysaccharidoses (MPS) and glycosphingolipidoses.Glucosylceramide (GlcCer) is the main ceramide monohexoside (CMH) in blood and accumulates in the tissues and circulation of those with Gaucher disease. A total of 11 GlcCer isoforms with variable fatty acid chain lengths were included in our multiplex assay. GlcCer levels were measured in four Gaucher disease (GD) patients, two with type I GD and two with type III GD. We observed a median 2-fold elevation in the total GlcCer in the GD patients in comparison with the healthy controls A; howeveThe ceramide dihexosides (CDHs), consisting of lactosylceramide (LacCer) and galabiosylceramide (Gb2), were assessed collectively, as reverse-phase chromatography does not separate structural isomers. Gb2 is a substrate of alpha-galactosidase A, the defective enzyme that results in Fabry disease (FD), and CDHs have been found previously to be elevated in FD patient urine ,12. HoweGlobotriaosylceramide (Gb3), the main GSL that accumulates in FD, was also assessed . No signFor globoside (Gb4), the molecule that cannot be broken down in Sandhoff disease (samples not available), we did not observe any significant disease group change. Certain patients within some of the disease groups did show higher levels of Gb4 than controls. Interestingly, these are the same patients who had high levels of GlcCer A.Although the total levels of each glycosphingolipid class for the various diseases proved informative, we also further evaluated the individual isoforms of GlcCer. In the controls and GD DBS, the C16:0 isoform accounts for more than 50% A. RelatiGlucosylsphingosine and galactosylsphingosine  are derived from the CMH isoforms GlcCer and galactosylceramide, respectively, and are referred to collectively as lyso-CMH. Similarly to the CDH molecules, it is not possible to separate these isomeric lyso-molecules using reverse-phase chromatography. However, given that GlcCer is the predominant CMH in blood, it is expected that the predominant lyso-CMH in the DBS will be lyso-GlcCer, known as lyso-Gb1.As expected, analysis of the DBS revealed lyso-Gb1 to be highly elevated in the GD group in comparison with control levels, showing a 41.1-median-fold change A. OveralLyso-CDH consists of lyso-lactosylceramide and lyso-galabiosylceramide, which derive from lactosylceramide and galabiosylceramide (Gb2), respectively. Both are present in blood. As mentioned previously, Gb2 is a substrate of alpha-galactosidase, and CDH accumulates in FD. Analysis of the total lyso-CDH revealed a significant increase in the median levels of lyso-CDH by 5.9- and 2.0-fold in GD and FD patients, respectively, when compared with healthy controls B.Lyso-Gb3 is a well-established diagnostic biomarker for FD ,15. As eSphingomyelin, lyso-sphingomyelin, and “lyso-sphingomyelin 509”, now identified as N-palmitoyl-O-phosphocholineserine ,17. HencOrdinarily, biomarkers for LSDs are tested against a relevant control population. We developed a multiplexed panel assay to assess the levels of known biomarkers in a cohort of patients with LSDs to better understand their specificity. Using this approach, we uncovered some novel findings regarding GSL metabolism in LSDs. In the case of the glycosphingolipidoses, typically, the glycosphingolipid substrate of the affected enzyme accumulates. However, we have shown here that the accumulation of these substrates can also occur in other disorders; e.g., whilst it is known that GlcCer accumulates in GD, some of the patients with Fabry, Hurler, and NPC disease also have increased GlcCer levels. Indeed, some of the NPC patients have GlcCer levels in the range of the Gaucher disease patients. A more detailed analysis, however, of the individual GlcCer species demonstrates that the GlcCer profile of the NPC patients is different from the controls and from those with GD. Specifically, fewer C16 GlcCer and longer-chain C24 GlcCer species are observed in NPC. GlcCer is hydrolysed to ceramide by glucocerebrosidase (GBA), of which there are two forms; one is lysosomal (GBA1) and typically is affected in GD disease, and the other (GBA2) is non-lysosomal. Previous work has shown that GBA acts as a trans-glucosidase, transferring glucose between GlcCer and cholesterol to create glucosylated cholesterol, which is elevated in GD and NPC . This isImproved specificity for NPC detection can be achieved by looking at the ratio of the C24:1 to C16 isoform of GlcCer D. HoweveWe have shown previously that changes in the ratio of GlcCer to CDH C24:2-OH occur in NPC . Levels Plasma chitotriosidase (CT) is routinely used as a surrogate marker for GD . In pracAnalysis of the samples from MPS type III patients revealed a small, although significant, increase in lyso-Gb3. This was not unique to this group of patients, with some individuals in the MPS II group also having elevated lyso-Gb3 C. All thLow levels of lyso-CDH have been observed previously in GD and NPC plasma ,25. Our In conclusion, our study demonstrates the utility of a biomarker panel approach for detecting glycosphingolipidoses and LSDs. This approach can also be used to distinguish between different subgroups. Whilst disease-specific biomarker levels can overlap with other similar disorders, the use of multiple biomarkers can help establish a diagnosis more promptly. Examination of a range of pathophysiologically relevant markers also sheds insight into the common downstream pathophysiological mechanism(s) leading to intracellular dysfunction in these disorders. Such biomarker panel strategies represent a simplified, fast, and accurate option to expedite the diagnostic process in neurometabolic and other rare disorders; they have translational potential and a role in accelerating the time to diagnosis and, ultimately, implementing targeted disease-specific treatments that are becoming increasingly available in the field.13C6 glucosyl-sphingosine, N-glycinated lyso-ceramide trihexoside, N-omega-CD3-hexadecanoyl-glucopsychosine, N-omega-CD3-hexadecanoyl-lactosylceramide, and N-omega-CD3-octadecanoyl-ceramide trihexoside further details are available in Methanol, acetonitrile, chloroform, and ethanol were from Fisher Chemicals. Formic acid and DMSO were from Sigma-Aldrich, Gillingham UK. All solvents were HPLC-grade, apart from formic acid, which was UPLC-grade. Water used was Milli-Q grade . Sphingolipid internal standards were obtained from Matreya LLC : n = 4), Fabry (n = 10 (4 on treatment)), Pompe (n = 2), MPS types I–VI ), and NPC ) patients. Diagnosis of these patients has been confirmed by genetic and biochemical enzyme testing, except in the case of NPC, where the diagnosis has been confirmed genetically. Patients were grouped according to their genetic diagnosis. MPS I patients were further stratified on the basis of their neurological assessment; i.e., Hurler patients have neurodegeneration, Hurler–Scheie patients have mild mental retardation, and individuals with Scheie disease are unaffected. In all cases, written informed consent was obtained. Samples were stored at −80 °C prior to analysis. Control blood spots (n = 10) were obtained from healthy volunteers. Further sample information is given in This study was approved by the National Research Ethics Service (NRES) in the UK . Paediatric DBS samples were from Gaucher  coupled with a Xevo TQ-S triple quadrupole mass spectrometer  was used in positive mode for the analysis. Compounds were separated on an ACQUITY UPLC 2.1 × 50 mm BEH C8 1.7-μm column . The pooled sample was injected at regular intervals during the run. For each injection, starting conditions were: 0.5 mL/min of 50% A (0.05% formic acid in Milli-Q water): 50% B (methanol). The column temperature was set to 40 °C. All UPLC–MS/MS method details have been described previously [p value < 0.05 considered significant.Mass spectrometry data were analysed using TargetLynx in MassLynx v 4.2 software (Waters Corp.). The relative abundance of each compound was obtained by ratioing its area to the corresponding internal standard area. A non-parametric Mann–Whitney test was used, and a


Due to their cardiovascular protective effect, glucagon-like peptide-1 receptor agonists (GLP-1RAs) and sodium-glucose cotransporter-2 inhibitors (SGLT2is) represent breakthrough therapies for type 2 diabetes mellitus (T2DM). In this review article, we discuss the mechanistic and clinical synergies that make the combined use of GLP-1RAs and SGLT2is appealing in patients with T2DM. Overall, the presented cumulative evidence supports the benefits of GLP-1RA plus SGLT2i combination therapy on metabolic-cardiovascular-renal disease in patients with T2DM, with a low hypoglycemia risk. Accordingly, we encourage the adoption of GLP-1RA plus SGLT2i combination therapy in patients with T2DM and established atherosclerotic cardiovascular disease (ASCVD) or multiple risk factors for ASCVD . Regarding renal effects, the evidence of SGLT2is in preventing kidney failure is more abundant than for GLP-1RAs, which showed a beneficial effect on albuminuria but not on hard kidney endpoints. Hence, in case of persistent albuminuria and/or uncontrolled metabolic risks  on SGLT2i therapy, GLP-1RAs should be considered as the preferential add-on therapy in T2DM patients with chronic kidney disease. Despite the potential clinical benefits of GLP-1RA plus SGLT2i combination therapy in patients with T2DM, several factors may delay this combination to become a common practice soon, such as reimbursement and costs associated with polypharmacy. Altogether, when administering GLP-1RA plus SGLT2i combination therapy, it is important to adopt an individualized approach to therapy taking into account individual preferences, costs and coverage, toxicity profile, consideration of kidney function and glucose-lowering efficacy, desire for weight loss, and comorbidities. Management of type 2 diabetes mellitus (T2DM) has evolved from a glucocentric to a cardiometabolic approach . ConsequOverall, both classes are indifferently recommended by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) as first-line therapy in patients with T2DM and established ASCVD or multiple ASCVD risk factors to reduce the risk of major adverse cardiovascular events (MACE), such as myocardial infarction, stroke, and cardiovascular death , 7. In sThe ADA and the EASD further recommend SGLT2is as first-line therapy in patients with T2DM and heart failure (HF) to reduce the risk of worsening HF , 7. MoreGLP-1RAs and SGLT2is act through distinct and complementary mechanisms of action to exert glycemic control and cardiovascular benefits , 9. In tOur review was based on a systematic PubMed search of randomized controlled trials (RCTs), observational studies, systematic reviews/meta-analyses, literature reviews, and case reports, published up to 2022, evaluating the combined treatment with a GLP-1RA and a SGLT2i in T2DM or prediabetes. The used search terms were: "GLP-1 receptor agonist and SGLT2 inhibitor" OR "SGLT2 inhibitor and GLP-1 receptor agonist" OR "SGLT-2 inhibitor and GLP-1 receptor agonist" OR "GLP-1 receptor agonist and SGLT-2 inhibitor" OR "GLP-1 receptor agonists as add-on to SGLT2 inhibitors" OR "GLP1 receptor agonist and SGLT-2 inhibitor" OR "GLP-1 agonist plus SGLT2 inhibitor" OR "glucagon-like peptide-1 receptor agonist and sodium-glucose cotransporter 2 inhibitor" OR "sodium-glucose cotransporter 2 inhibitor and glucagon-like peptide-1 receptor agonist" OR "SGLT2 inhibitors and GLP1-RA" OR "addition of SGLT2i to GLP-1RA" OR "SGLT-2 inhibitor therapy added to GLP-1 agonist therapy" OR "GLP-1 analogues and SGLT-2 inhibitors".GLP-1RAs and SGLT2is affect glucose metabolism differently  [The mechanisms by which GLP-1RAs and SGLT2is exert their cardiovascular and renal benefits  , 14–16. tissues) , 14, 17.tissues) . More retissues) . Further2 and absent at an eGFR < 30 mL/min/1.73 m2 [SGLT2is and GLP-1RAs exhibit clinical benefits on glycemic control, systolic blood pressure (SBP), body weight, and dyslipidemia, which may all partly contribute to the cardiovascular protection provided by these two drug classes , 19. Com/1.73 m2 .To date, no head-to-head RCTs have been performed to compare the effects of GLP-1RAs and SGLT2is on cardiovascular outcomes . NeverthMeta-analyses of CVOTs of GLP-1RAs and SGLT2is have further found that GLP-1RAs reduce the risk of stroke by up to 17%, whereas SGLT2is have no appreciable effect . The benefits of empagliflozin were consistent among patients with or without diabetes and regardless of eGFR [In terms of renal outcomes among patients with T2DM, meta-analyses of CVOTs of GLP-1RAs and SGLT2is have shown that both GLP-1RAs and SGLT2is significantly reduced the composite outcome of macroalbuminuria, worsening of eGFR or serum creatinine, end-stage renal disease, or renal death by up to 21% and 38%, respectively. However, when excluding macroalbuminuria, the effect of GLP-1RAs on kidney outcomes was no longer significant . In a meta-analysis of 5 RCTs including 21,947 participants, SGLT2is significantly reduced the risk of HHF  in patients with HF across the full spectrum of ejection fraction, including both outpatients and hospitalized patients . On the Overall, GLP-1RAs and SGLT2is reduce the risk of MACE to a similar degree, particularly in patients with established ASCVD. Moreover, SGLT2is have a more marked effect on preventing HHF and progression of CKD, whereas GLP-1RAs may reduce the risk of stroke to a greater extent . Combinehttps://clinicaltrials.gov/ct2/show/NCT05390892), which will assess the cardiorenal effects of GLP-1RA plus SGLT2i combination therapy compared to both GLP-1RA and SGLT2i monotherapy among 9,000 patients with T2DM and established ASCVD or at high ASCVD risk.The efficacy and safety of combination therapy with a GLP-1RA and a SGLT2i in patients with T2DM have been investigated in RCTs , 30–37, Despite the absence of RCTs dedicated to the study of cardiovascular and renal effects of GLP-1RA plus SGLT2i combination therapy, clinical evidence provides support for combining these two drug classes, as this has the potential to significantly reduce cardiovascular events including HHF and slow CKD progression, without the inconvenience and dangers of hypoglycemia .A meta-analysis , that inAnother meta-analysis , which iMost recently, Li et al.  pooled dOn the other hand, a 16-week single-center RCT by Ali et al.  evaluatiAmong CVOTs evaluating individual agents from the two drug classes, the combined use of GLP-1RAs and SGLT2is was rather limited. In GLP-1RA CVOTs, the prevalence of baseline SGLT2i use ranged from 0% to 15.2%, whereas in SGLT2i CVOTs, the prevalence of baseline GLP-1RA use ranged from 2.5% to 4.4% , 13.2/year; 95% CI, 0.94–2.94; p < 0.001) and exenatide alone  [A post-hoc analysis of the EXSCEL trial , which e< 0.001) .The results of the EXCEL post-hoc analysis  are compAnother post-hoc analysis of interest is that of the AMPLITUDE-O CVOT , which aOverall, these post-hoc analyses of different CVOTs support the concept that the combination of GLP-1RAs plus SGLT2is may be well-tolerated and may provide additional benefits to GLP-1RAs or SGLT2is alone in terms of cardiovascular and renal protection and mortality, without an increase in the risk of hypoglycemia , 52, 54.Accumulating real-world evidence supports the combined use of GLP-1RAs and SGLT2is to reduce the risk of MACE. Using United States insurance claims databases between April 2013 and June 2018, Dave et al. identified patients with T2DM who were already taking GLP-1RAs and who had added either SGLT2is or sulfonylurea . After 1Another real-world observational study of interest was a cohort study based on the Danish National Patient Registry, which aimed to investigate the safety of the most widely used anti-hyperglycemic dual and triple therapies for T2DM . There wIn three nested case–control studies from England and Wales, involving 440,089 patients with T2DM and without ASCVD treated with non-insulin glucose-lowering medication, combined SGLT2i and GLP-1RA therapy used for primary prevention was associated with 30% lower odds of MACE  compared with other combination regimens  . CombineLoyo and colleagues recently reviewed the United States Veterans Health Administration database, and identified 121,174 patients with T2DM and ischemic heart disease, cerebrovascular disease, or peripheral arterial disease who had been prescribed SGLT2is, GLP-1RAs, or their combination. Combined SGLT2i and GLP-1RA therapy resulted in a significantly lower risk of all-cause death, non-fatal myocardial infarction, and non-fatal stroke at 12 months than SGLT2i monotherapy  and GLP-1RA monotherapy  .Despite inherent limitations of observational data and potential for residual confounding, the results from these observational analyses support, in line with RCT findings, combining SGLT2is and GLP-1RAs to reduce cardiovascular events in patients with diabetes in routine clinical care , 43, 63.GLP-1RAs and SGLT2is are both generally well-tolerated when used individually, with a minimal risk of hypoglycemia, unless used in combination with insulin or insulin secretagogues , 9.The most common AEs of GLP-1RAs are gastrointestinal complaints, such as nausea, vomiting, constipation, and diarrhea. However, these AEs are generally mild to moderate in intensity, and resolve spontaneously within a couple of weeks of initiating therapy . A meta-Although an increased risk of pancreatitis is listed in product monographs for GLP-1RAs and caution should be exercised in individuals with a history of pancreatitis, causality has not been established , 66. Simhttps://clinicaltrials.gov/ct2/show/NCT03811561), is investigating the long-term effects of once-weekly subcutaneous semaglutide on the progression of diabetic retinopathy in individuals with T2DM.The SUSTAIN-6 CVOT , perform2, and in those taking loop diuretics [The most common AEs of SGLT2is are genital mycotic infections , 11. A miuretics . Accordiiuretics , 19. Foriuretics . Thus, iiuretics .Compared to placebo, canagliflozin was associated with an increased risk of lower limb amputations  in the CANVAS CVOT, which overall included 10,142 patients with T2DM and high cardiovascular risk . These CBecause the side effect profile of SGLT2is differs from that of GLP-1RAs, any additive negative interaction is not expected when these two classes of anti-diabetic medications are used in combination . MoreoveIn clinical studies of GLP-1RA plus SGLT2i combination therapy, the safety profile of the combination therapy was consistent with those of the individual agents, with no unexpected findings . For insIn the previously mentioned meta-analysis by Li et al. (2022)  comparinThe presented cumulative evidence supports the benefits of GLP-1RA plus SGLT2i combination therapy on metabolic-cardiovascular-renal disease in patients with T2DM. Regarding their metabolic effects, the combination of a GLP-1RA and a SGLT2i significantly reduces HbA1c level, body weight, and SBP, with an additive effect on weight loss and SBP but not on HbA1c , 57–59. In respect to their cardiovascular effects among patients with T2DM, both GLP-1RAs and SGLT2is have been shown to reduce the risk of MACE , with GLP-1RAs reducing the risk of non-fatal stroke to a greater extent , 6. Thus2, we favor the use of SGLT2is to slow kidney disease progression [In terms of renal effects, the evidence of SGLT2is in preventing kidney failure is more abundant than for GLP-1RAs, which showed a beneficial effect on albuminuria and not on hard kidney endpoints , 6, 8. Hgression . HoweverIn the setting of HF, SGLT2is reduced the risk of HHF, irrespective of ejection fraction or diabetes status , 76, 79.Despite the potential of GLP-1RA plus SGLT2i combination therapy to improve metabolic-cardiovascular-renal trajectories, several factors may delay this combination to become a common practice soon, and most T2DM patients with ASCVD or at high ASCVD risk remain inadequately managed. Indeed, CAPTURE, a multinational, cross‑sectional study  includinOverall, when administering GLP-1RA plus SGLT2i combination therapy, it is important to adopt an individualized approach to therapy taking into account individual preferences, costs and coverage, AE profile, consideration of kidney function and glucose-lowering efficacy, desire for weight loss, and comorbidities such as frailty , 22, 24.


Renin–angiotensin system hyperactivation in autosomal-dominant polycystic kidney disease (ADPKD) patients leads to early hypertension. Cystic enlargement probably causes parenchymal hypoxia, renin secretion, and endothelial dysfunction. Sympathetic and parasympathetic balance is altered in this condition, especially during the night, also affecting blood pressure circadian rhythm. Aim of this study was to evaluate sympathetic/parasympathetic balance using heart rate variability (HRV) parameters and find a correlation between HRV and renal damage progression, as total kidney volume enlargement, in ADPKD patients.Sixteen adult ADPKD patients were enrolled in the study. Eleven patients (68.8%) were male, and the median age was 42 years (IQR 36–47.5). HRV parameters were calculated using 24 h-ECG Holter. A kidney magnetic resonance imaging (MRI) scan 3 Tesla was performed to evaluate total kidney volume (TKV) and total fibrotic volume (TFV).r = 0.595, p < 0.05) and LFday . Moreover, a statistically significant positive linear correlation exists between high frequency (HF) and TFV  or height-adjusted (ha) TFV . Finally, we found a statistically significant positive linear correlation between HFnight and TKV , ha-TKV , TFV , and ha-TFV .A statistically significant positive linear correlation was observed between length of kidneys and frequency domain parameters as low frequency (LF) (We suppose that the increase in TKV and TFV could lead to a parasympathetic tone hyperactivation, probably in response to hypoxic stress and vasoconstriction due to cystic enlargement. Autosomal-dominant polycystic kidney disease (ADPKD) is an inherited monogenic disease, with a prevalence ranging from 1 in 543 to 1 in 4000 , PMM2 3. In partWith this background, the aim of this pilot study was to assess in ADPKD patients the sympathetic/parasympathetic balance using HRV parameters and correlate it with renal damage progression, as total kidney volume enlargement..We enrolled ADPKD patients in this study. DNA samples were analyzed in all patients for PKD genetic diagnosis and mutation in the PKD1 gene was found. Fifteen patients had a family history of ADPKD, except one patient who had a de novo mutation. No patient was in dialysis treatment. Patients with diabetes were excluded a priori2  staging system), absence of cardiovascular events, blood pressure (BP) values in normal range under control with one of the following drugs: angiotensin-converting enzyme inhibitors (ACEi), angiotensin II receptor blockers (ARBs), and calcium channel blockers (CCB). The tests were performed before taking tolvaptan therapy. No patient was on beta-blocker therapy, 6 patients were on ACEi, 6 patients were on ARBs, and 11 were on CCB. No patient was on erythropoietin treatment for anemia. At the time of enrollment, medical records, physical assessment, and laboratory data were recorded in all patients. The study protocol was approved by the Local Clinical Research Ethics Committee. The study conforms to the principles outlined in the Declaration of Helsinki and a written consent by each patient enrolled was obtained.Inclusion criteria were rapidly progressive disease (as kidney function loss > 5 ml/min/year), CKD ≥ stage G3b corresponding to estimated glomerular filtration rate (eGFR) 30–44 ml/min/1.73 mAll patients were subjected to a novel MRI protocol of advanced imaging with magnet 3Tesla (T)  after positioning of the surface coil 32 channels. Kidney MRI scan 3 T, with and without contrast, was performed to evaluate total kidney volume (TKV) and total fibrotic volume (TFV). The acquisition protocol included morphological sequences, single shot T2-weighted (SSFS)  acquired on axial, sagittal, and coronal planes and Gradient Echo (GRE) T1-weighted . For the evaluation of parenchymal perfusion, ultrafast GRE T1-weighted sequences were used, acquired in the coronal plane  during administration dynamic of i.v. contrast  using a perfusion technique, with high temporal resolution of 4 s, for a total duration of about 8 min. The end of the dynamic sequence was made to coincide with the start of the administration of contrast material i.v.19. The evaluation of total perfusion volume (TPV) and TFV results from perfusional MRI after a qualitative and quantitative approach. Each parameter resulted from a post-processed slice by slice renal segmentation, respectively, in early arterial phase (first minute of perfusion) and late perfusional phase (eighth minute of perfusion). Segmentation was guided using colorimetric maps. After segmentation, software Workstation vers. 4.6 was used for three-dimensional (3D) volume rendering reconstruction, which resulted in semiquantitative estimation of parenchymal perfonded tissue and fibrotic areas. These perfusional parameters give indication of functional parenchymal areas. Perfusion volume indicates kidney areas where blood flow is preserved, which is an indirect sign of normal functional parenchyma. Fibrotic areas indicate parenchymal areas that underwent fibrotical substitution and consequent loss of function [Autonomic nervous activity was evaluated by HRV analysis during 24-h ECG recording following the recommendations of the Taskforce of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology . The tim® SPSS® Statistics 26 for Windows® software . The normality of the variables was tested using the Shapiro–Wilk method for normal distributions. All continuous variables were expressed as median and interquartile range (IQR) and categorical variables were expressed as absolute frequencies and percentages. Spearman or Pearson tests were used for bivariate correlations, as appropriate. A probability value of p < 0.05 was considered statistically significant.Data management and analysis were performed using IBMSixteen adult ADPKD patients were enrolled in this study. Eleven patients (68.8%) were male, and the median age was 42 years (IQR 36–47.5). Median serum creatinine was 1.38 mg/dl (IQR 1.2–1.61), and median eGFR was 58.1 ml/min (IQR 52–60). Median renal resistive index (RRI) was 0.65 (IQR 0.61–0.67). Table 3 (IQR 177–352.9); median length was 17.97 cm (IQR 16.41–21.39) and median stiffness was 22.13 kPa (IQR 15.8–30). These findings are summarized in Table MRI of the kidneys showed a median TKV of 2102 ml (IQR 1372.5–3155.3) and a median TFV of 298.51 cmMedian heart rate (HR) was 75.2 bpm (IQR 67.5–82.25) and median of corrected QT interval (QTc) registered was 398 ms (IQR 384–425). Moreover, median SDNN was 127.85 ms (IQR 113.9–168.25) and median RMSSD was 41.35 ms (IQR 27.2–50.5). Table r = 0.595, p < 0.05) and LFd nu . Moreover, a statistically significant positive linear correlation exists between HF nu and TFV  and height-adjusted (ha) TFV . Finally, we found a statistically significant positive linear correlation between HFn nu and TKV , ha-TKV , TFV  and ha-TFV . All linear correlations are showed in Fig. A statistically significant positive linear correlation was observed between length of kidneys and LF nu (Autonomic dysfunction is a common finding in patients with chronic kidney disease (CKD) and it is a leading cause of cardiovascular morbidity and mortality. Hyperactivation of the sympathetic system not only leads to an increased basal heart rate, but also promotes myocardial hypertrophy and fibrosis associated with increased risk for sudden cardiac death. HRV is an indirect measure of the sympathovagal interaction at the sinoatrial node and an index of cardiac neural control .In autoimmune diseases characterized by fibrous of the skin and internal organs, a parasympathetic modulation increases in relation to microcirculation dysfunction induced by Raynaud vasospasm . Thus, aThe study has some limitations. First, this is a single center study, nonrandomized, with a small cohort of patients since ADPKD is a rare disease. Second, we also have not recorded the onset of cardiovascular events. Anyway, our data show significant results which can address the search to novel early cardiovascular markers in ADPKD population, encumbered by high cardiovascular mortality. Third, the lack of follow-up cannot generalize the results in ADPKD population. Thus, considering the originality as a strength point of this pilot study, in the future, we aim to observe if the progression of renal damage could be correlated to autonomic dysfunction with large number of patients and adequate follow-up. Changes in the MRI parameters and HRV will be evaluated between two measurements  in terms of their correlations.It is advisable that future studies in this field will be multicentric and include larger populations possibly more representative of the ADPKD population, to confirm the results. We suppose that the increase in TKV and TFV could lead to a parasympathetic tone hyperactivation, probably in response to hypoxic stress and vasoconstriction due to cystic enlargement.


Cardiovascular and renal diseases are closely related. Brain natriuretic peptide (BNP) and urinary albumin are established predictors for cardiac and renal morbidities, respectively. To date, no reports have investigated the combined predictive value of BNP and urinary albumin for long-term cardiovascular–renal events in patients with chronic kidney disease (CKD). The aim of this study was to investigate this theme.Four hundred eighty-three patients with CKD were enrolled into this study and followed-up for 10 years. The endpoint was cardiovascular–renal events.During the median follow-up period of 109 months, 221 patients developed cardiovascular–renal events. Log-transformed BNP and urinary albumin were identified as independent predictors for cardiovascular–renal events, with a hazard ratio of 2.59  and 2.27  for BNP and urinary albumin, respectively. For the combined variables, the group with high BNP and urinary albumin had a markedly higher risk  of cardiovascular–renal events compared with that of the group with low BNP and urinary albumin. Adding both variables to a predictive model with basic risk factors improved the C-index , net reclassification improvement , and integrated discrimination improvement  more than each of them alone.This is the first report to demonstrate that the combination of BNP and urinary albumin can stratify and improve the predictability of long-term cardiovascular–renal events in CKD patients. The aim of this study is to investigate this theme.Biomarkers are helpful in assessing these conditions. Urinary albumin, a representative urinary biomarker, is related to cardiovascular disease (CVD) and death as well as to renal prognosis.8 between February 2009 and September 2010. The inclusion criteria were as follows: (1) urinary albumin >30 mg/g Cr or estimated GFR (eGFR) <60 mL/min/1.73 m2; (2) eGFR >15 mL/min/1.73 m2; (3) a follow-up period of at least 1 year; and (4) BNP and urinary albumin were measured at enrollment.Patients were selected from the cohorts that were enrolled in our previous studies9 was used. Stroke was defined as the presence of clinical signs of focal or global disturbances in cerebral function caused by cerebrovascular damage. Progression of renal dysfunction was defined as the doubling of serum creatinine (Cr) or the initiation of renal replacement therapy.The study participants were followed-up clinically until April 2020. The primary endpoint was the composite of the occurrence of cardiovascular events and progression of renal dysfunction. Cardiovascular events were defined as HF requiring hospitalization, myocardial infarction, angina pectoris requiring a therapeutic intervention, stroke, aortic dissection, or aortic aneurysm rupture. The Framingham criteria for HFAll procedures that involved human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. This study was approved by the Ethics Committee at the Fujita Health University School of Medicine (authorized number: HM20-059). Informed consent was waived because of the retrospective nature of the study.10 Urinary albumin levels were divided into normal to mild (30 mg/g Cr), moderate (30–300 mg/g Cr), and high categories (>300 mg/g Cr) in accordance with the Kidney Disease Improving Global Outcomes (KDIGO) CKD guidelines.2 Hazard ratios (HRs) and 95% confidence intervals (CIs) for cardiovascular–renal events, cardiovascular events, or renal events were calculated for each factor using the Cox proportional hazards analysis after adjusting for age, sex, systolic blood pressure, hemoglobin, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, eGFR, history of CVD, and diabetes. To assess whether the accuracy of predicting endpoints would improve after combining BNP and urinary albumin in a baseline model using the already known risk13 score, we calculated the C-index, net reclassification improvement (NRI), and integrated discrimination improvement (IDI). The C-index, which is defined as the area under the receiver operating characteristic curve between the individual predictive probabilities for endpoints and the incidence of endpoints, was compared using the baseline model.14 The NRI is a relative indicator of how many patients showed an improvement in the predicted probability for the endpoints. The IDI indicates the average improvement in the predicted probability for the endpoints after adding variables to the baseline model.15 A p-value <0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 25.0 .Normally distributed variables are expressed as the mean±standard deviation, whereas nonparametric data are presented as the median and interquartile range. Intergroup differences were evaluated using a one-way analysis of variance, and the Kruskal–Wallis and Chi-square tests were used for continuous and categorical variables, respectively. Cumulative incidence rates of cardiovascular–renal events were estimated using the Kaplan–Meier method, and intergroup differences were compared using a log-rank test. BNP levels were divided into low (<40 pg/mL), moderate (40–100 pg/mL), and high (>100 pg/mL) categories, in accordance with the JCS 2017/JHFS 2017 Guideline on Diagnosis and Treatment of Acute and Chronic Heart Failure.2. Clinical characteristics on the basis of the BNP category are shown in Patient selection is shown in During the follow-up period of 109 (56–120) months, 221 patients (45.8%) developed cardiovascular and renal events; these included doubling of serum Cr levels in 144 patients (29.8%), induction of dialysis in 101 patients (20.9%), HF in 42 patients (8.9%), angina pectoris/myocardial infarction in 41 patients (8.5%), cerebral stroke in 48 patients (9.9%), and aortic dissection/aneurysm rupture in four patients (0.8%). On Cox multivariable analysis, log BNP  and log urinary albumin  were identified as independent predictors for cardiovascular–renal events after adjusting for sex, age, systolic blood pressure, hemoglobin, HDL-cholesterol, LDL-cholesterol, eGFR, history of CVD, and diabetes . For carP<0.0001; When patients were divided into high, moderate, and low groups on the basis of BNP and urinary albumin levels, the high BNP group had a risk of cardiovascular–renal events that was 2.41-times  higher than that of the low BNP group. A similar result was obtained on the basis of urinary albumin ; NRI, 0.497 (p<0.0001); and IDI, 0.071 (p<0.0001). When BNP and urinary albumin are compared individually, the combination of BNP and urinary albumin significantly improved the NRI  and IDI  better than each of the variables alone. Thus, these results might increase the knowledge about biomarkers for cardio-renal syndrome.16 and it is routinely used in clinical practice to diagnose or to exclude HF. BNP was classified as a Class 1 recommendation for diagnosis, severity, and prognostic evaluation in the 2017 guideline for diagnosis and treatment of acute and chronic HF.10BNP is released by the left ventricle in response to left ventricular end-diastolic wall stress,18 The following three fates are proposed for secreted BNP: (i) capture by its receptors for signal transduction or clearance; (ii) inactivation by neutral endopeptidase; or (iii) excretion from the kidney in its active form.19 In the highest urinary albumin category (300 mg/g Cr) in this study, the aHRs of cardiovascular–renal events between the BNP <40 pg/mL and 40<BNP<100 pg/mL categories were comparable. These results might be attributed to decreased excretion from the kidney that is caused by reduced renal function.Patients with CKD have higher baseline BNP levels compared with those of matched patients with normal renal function because of impaired renal clearance, chronic pressure/volume overload, and CKD-associated cardiomyopathy.20 In the consensus conference on CRS that was sponsored by the Acute Dialysis Quality Initiative (ADQI), CRS was classified into five subtypes.3 In CRS type 2, chronic HF results in CKD onset or progression. Examples of CRS type 2 include progressive CKD resulting from chronic HF in congenital or acquired heart disease or from repeated bouts of acute decompensated HF. Extended periods of chronic HF result in altered renal hemodynamics and progressive renal pathology related to albuminuria. The correlation of BNP with renal events in the present study may reflect this pathophysiology.BNP levels are also significantly elevated in patients with CRS.3 The common traditional risk factors for CKD and CVD are older age, smoking, hypertension, diabetes, and dyslipidemia.21 Other factors associated with CKD, such as anemia, albuminuria, CKD–mineral and bone disorder, inflammation, increased renin–angiotensin–aldosterone system activation, sympathetic nervous system activation, oxidative stress, and insulin resistance, are also associated with the development of CVD.13 The correlation of urinary albumin with BNP also reflects these common backgrounds.However, in CRS type 4, CKD promotes the progression of chronic HF, ventricular hypertrophy, and diastolic dysfunction and increased the risk of adverse cardiovascular events.13 which comprised age, sex, systolic blood pressure, hemoglobin, HDL-cholesterol, LDL-cholesterol, eGFR, history of CVD, and diabetes, and found that combining BNP and urinary albumin, rather than each factor alone, significantly improved the prediction of cardiovascular and renal events at 109 months better than the basic risk factors, as shown by the increase in the C-index, NRI, and IDI. Thus, measurement of both BNP and urinary albumin might improve the quality of risk management in clinical practice.We assessed the effect of adding BNP and urinary albumin to the basic risk factors,22 A drug-induced reduction in urinary albumin was also found to be renoprotective.23 On the basis of the indications for drugs that have been shown to inhibit both cardiovascular and renal events, evaluating BNP combined with albuminuria might help to improve patients’ prognosis. Monitoring the change in BNP and albuminuria will also help to evaluate the effect of drugs such as renin–angiotensin–aldosterone system inhibitors24 and sodium–glucose cotransporter-2 inhibitors.25A meta-analysis showed a decrease in the hospitalization rate due to HF, CVD, or all-cause mortality when patients underwent BNP-guided therapy.26 researched the association between plasma BNP level and renal function among Japanese patients with type 2 diabetes mellitus. In their report, the highest BNP level (≥39.2 ng/mL) was independently and positively associated with microalbuminuria and macroalbuminuria. Naganuma et al.27 investigated BNP and urinary albumin as markers for cardiovascular evaluation of kidney donors. They found that plasma BNP concentrations in kidney donors were significantly higher than those in the healthy volunteers, and among kidney donors, 16% had microalbuminuria and 4% had overt proteinuria. Wang et al.28 evaluated the incremental usefulness of ten multiple biomarkers for predicting the risk of cardiovascular events in Framingham Heart Study participants. They showed that BNP and the urinary albumin-to-creatinine ratio were the best predicted major cardiovascular events. However, there have been no reports on the significance of the combination of BNP and urinary albumin as a predictor of cardiovascular–renal events over a long period in CKD patients. Thus, the present study is novel.Several reports have been published about the relationship between BNP and urinary albumin. Furukawa et al.This study had some limitations. First, this was a single-center cohort study. Second, serum Cr levels were affected by both the eGFR and muscle mass. Third, we did not assess the serial changes in BNP and albuminuria over time.In conclusion, BNP and urinary albumin are closely associated with long-term cardiovascular–renal events in CKD patients. The present report is the first to demonstrate that the combination of BNP and urinary albumin can stratify and improve the predictability of long-term cardiovascular–renal events in CKD patients.