The efficacy of dupilumab as biological treatment of severe asthma and chronic rhinosinusitis with nasal polyps (CRSwNP) depends on its ability to inhibit the pathophysiologic mechanisms involved in type 2 inflammation.To assess in a large sample of subjects with severe asthma, the therapeutic impact of dupilumab in real-life, with regard to positive or negative skin prick test (SPT) and CRSwNP presence or absence.Clinical, functional, and laboratory parameters were measured at baseline and 24 weeks after the first dupilumab administration. Moreover, a comparative evaluation was carried out in relation to the presence or absence of SPT positivity and CRSwNP.Among the 127 recruited patients with severe asthma, 90 had positive SPT, while 78 reported CRSwNP. Compared with the 6 months preceding the first dupilumab injection, asthma exacerbations decreased from 4.0 (2.0-5.0) to 0.0 (0.0-0.0) (p < 0.0001), as well as the daily prednisone intake fell from 12.50 mg (0.00-25.00) to 0.00 mg (0.00-0.00) (p < 0.0001). In the same period, asthma control test (ACT) score increased from 14 (10-18) to 22 (20-24) (p < 0.0001), and sino-nasal outcome test (SNOT-22) score dropped from 55.84 ± 20.32 to 19.76 ± 12.76 (p < 0.0001). Moreover, we observed relevant increases in forced expiratory volume in one second (FEV1) from the baseline value of 2.13 L (1.62-2.81) to 2.39 L (1.89-3.06) (p < 0.0001). Fractional exhaled nitric oxide (FeNO) values decreased from 27.0 ppb (18.0-37.5) to 13.0 ppb (5.0-20.0) (p < 0.0001). These improvements were quite similar in subgroups of patients characterized by SPT negativity or positivity, and CRSwNP absence or presence. No statistically significant correlations were detected between serum IgE levels, baseline blood eosinophils or FeNO levels and dupilumab-induced changes, with the exception of FEV1 increase, which was shown to be positively correlated with FeNO values .Our results consolidate the strategic position of dupilumab in its role as an excellent therapeutic option currently available within the context of modern biological treatments of severe asthma and CRSwNP, frequently driven by type 2 airway inflammation. Dupilumab is a completely human monoclonal antibody, belonging to the IgG4 immunoglobulin class, whose mechanism of action consists of the dual antagonism of the interleukin 4 (IL-4) and 13 (IL-13) receptors .IL-4 and IL-13 play critical roles in the pathogenesis of severe type 2 asthma , 6. ChroThe efficacy of dupilumab as biological treatment of severe asthma and nasal polyposis depends on its remarkable ability to inhibit the pathophysiologic mechanisms involved in type 2 inflammation. In fact, dupilumab is an efficient dual antagonist of both IL-4 and IL-13 receptors 13 . The typDue to this powerful mechanism of action, within the context of add-on biological treatment of severe asthma and nasal polyposis dupilumab exerts remarkable therapeutic effects, well documented by several randomized controlled trials (RCTs) . In partHowever, only a few real-world studies referring to a quite low number of patients have been published so far –19. HencIn the present retrospective multicenter observational study, we recruited adult outpatients (>18 years) with severe type 2 asthma treated with dupilumab. Subjects were enrolled at the following asthma centers: Respiratory Medicine Section, University “Aldo Moro”, Bari, Italy; Allergy and Respiratory Medicine, University of Catania, Italy; Respiratory Disease Unit, University “Magna Graecia” of Catanzaro, Italy; Allergology and Clinical Immunology Unit, University of Foggia, Italy; Respiratory Disease Unit, University of Foggia, Italy; Allergy and Clinical Immunology Unit, University of Messina; Pulmonology Unit, “Monaldi” University Hospital, Naples, Italy; Pulmonology Unit, University of Palermo, Italy; Allergology and Pulmonology Unit, Provincial Outpatient Center of Palermo, Italy; Respiratory Disease Unit, University of Salerno, Italy; Division of Allergy and Clinical Immunology, University of Salerno.2-adrenergic agonists), associated with a LAMA (long-acting muscarinic receptor antagonist). Enrollment took place consecutively, and the only inclusion criteria were those needed for prescription of dupilumab. All recruited patients met the European Respiratory Society (ERS)/American Thoracic Society (ATS) criteria defining severe uncontrolled asthma /LABA , nasal polyposis, bronchiectasis, osteoporosis, anxiety, atopic dermatitis and obstructive sleep apnea syndrome (OSAS) were evaluated. Symptom control was assessed by administering to all recruited patients the asthma control test (ACT). The latter includes 5 key questions referring to the frequency of asthma symptoms and to the need of inhaled rescue medication during the previous 4 weeks . Each quThis observational study met the standards of Good Clinical Practice (GCP) and the principles of the Declaration of Helsinki. All recruited patients signed a written informed consent. Our study was also conducted in accordance with the provisions of the local Ethics Committee of Calabria Region, Italy .2-adrenergic agonists (SABA), as well as prednisone intake, ACT score, sino-nasal outcome test questionnaire (SNOT-22), the number of relapses of nasal polyposis, forced expiratory volume in one second (FEV1), forced vital capacity (FVC), mean forced expiratory flow between 25% and 75% of FVC (FEF25-75), FeNO levels, as well as blood eosinophil, basophil, and neutrophil counts were assessed at baseline and 24 weeks after the first dupilumab administration.The main purpose of this real-life study was to evaluate the efficacy of dupilumab in daily clinical practice. The number of asthma exacerbations, emergency department visits and daily inhalations of short-acting βA secondary objective was to retrospectively verify the therapeutic responses of our patients to dupilumab, in relation to SPT positivity or negativity, as well as with regard to the presence or absence of CRSwNP. The diagnosis of CRSwNP was formulated on the basis of symptoms, nasal endoscopy and computed tomography (CT) , 30. Ski1, FVC, and FEF25-75 values.Furthermore, after 6 months of adjunctive therapy with dupilumab we analyzed the possible correlations existing between the baseline concentrations of serum IgE, FeNO and blood eosinophils, and the observed changes regarding asthma exacerbations, daily consumption of prednisone and SABA, ACT score, SNOT-22 score, FEVIn addition, the occurrence of unwanted side effects was investigated on the basis of available information stored in clinical records.All data are expressed as mean ± standard deviation (SD) if normally distributed, otherwise as median values with the interquartile range (IQR). The normality of data distribution was checked using Anderson-Darling and Kolmogorov-Smirnov tests. Paired t-test and Mann-Whitney’s U-test for paired data were used to compare variables when appropriate. The latter statistical test was also used for the secondary objective of the study, i.e. the comparative evaluation of dupilumab efficacy in patients with positive or negative SPT, and with regard to the presence or absence of CRSwNP. Fisher’s test was applied to compare categorical variables. The association between baseline concentrations of type 2 inflammation biomarkers  and changes in clinical and functional parameters was assessed using linear regression analysis. In particular, the correlation index R for Spearman’s ranks was evaluated. A p-value less than 0.05 (two-tailed) was considered as statistically significant. Statistical analyses and figures were performed using Prism Version 9.4.0 software .2 (23.0-30.0). Mean baseline FEV1 was 76.56 ± 20.03% of predicted value. Among the enrolled patients, 90 (70.9%) had positive SPT for perennial and/or seasonal allergens, while 78 (61.4%) reported CRSwNP. Baseline patient characteristics are summarized in A total of 127 participants were recruited, including 63 (49.6%) women and 64 (50.4%) men, with a median age of 56.0 years (45.0-64.0), and a median body mass index (BMI) value of 26.0 Kg/m1 from the baseline value of 2.13 L (1.62-2.81) to 2.39 L (1.89-3.06) (p < 0.0001) (25-75 from 46.50% (30.75-69.00) to 63.50% (44.75-81.25) (p < 0.0001) of predicted values (Compared with the 6-month pre-treatment period (before the first injection of dupilumab), the median number of asthma exacerbations dramatically decreased from 4.0 (2.0-5.0) to 0.0 (0.0-0.0) (p < 0.0001) after 6 months of anti-IL4R/IL-13R therapy , zeroing of exacerbations and OCS (zero exacerbations and zero OCS use) , optimization of lung function (FEVOCS use) , 33, 47.1 was 0.20 L (0.00-0.48) in patients with negative SPT and 0.20 L (0.01-0.62) in subjects with positive SPT; this difference was not statistically significant (p = 0.409) (25-75 was 12.00% (3.75-19.50) in patients with negative SPT and 11.00% (0.00-29.00) in subjects with positive SPT, respectively (p = 0.827)  in patients with negative SPT and -4.00 (from -5.00 to -2.00) in subjects with positive SPT, respectively (p = 0.158)   in patients without CRSwNP, and 0.24 L (0.01-0.69) in subjects with CRSwNP; this difference was not statistically significant (p = 0.138) (25-75 was 11.00% (2.00-21.00) in patients without CRSwNP and 12.00% (1.25-29.75) in subjects with CRSwNP, respectively (p = 0.479)  in patients without CRSwNP and -4.00 (-5.00 to -2.00) in subjects with CRSwNP, respectively (p = 0.413)  subjects, 4 (3.15%) cases of conjunctivitis were reported, 2 (1.57%) injection site reactions were detected, and 1 (0.79%) patient experienced headache. All these mild side effects remitted spontaneously and did not require any specific treatment.Taken together, the results of the present multicenter real-life study, performed in patients with severe asthma and frequent nasal polyposis, show that dupilumab induced relevant therapeutic effects. Firstly, six months of treatment with this biologic drug cleared asthma exacerbations. This made it possible to effectively prevent any access to the emergency room, the use of short-acting bronchodilators as needed, and the intake of OCS. The latter aspect is of considerable importance as it further exceeds the efficacy data reported by the Liberty Asthma VENTURE trial . Indeed,When compared to QUEST (Q) and VENTURE (V) trials, other important differences with our real-life study regard the baseline characteristics of recruited patients. In particular, we enrolled a population of asthmatic subjects with a greater percentage of male patients , a higher number of asthma exacerbations , a greater blood eosinophil count , and especially a much higher percentage of patients presented with CRSwNP .Dupilumab significantly reduced asthma symptoms, as demonstrated by the significant increase in ACT score, which after 24 weeks of biological therapy reached and exceeded the threshold of 20, which expresses a satisfactory control of asthma symptoms, despite the baseline pre-treatment score stood at a rather low value of 14. This result confirms, in a real-life context, the data reported by the Liberty Asthma QUEST trial, in which the ACQ (Asthma Control Questionnaire) was utilized. However, compared to the ACQ test, the ACT questionnaire administered by us seems to be more appropriate to respond to the needs of practicality and easiness of completion wished by patients who refer to our severe asthma assessment centers. In fact, ACT is clearly preferred to ACQ in the few real-life studies that have recently evaluated the efficacy of dupilumab in the biological therapy of severe asthma –19.In patients with both asthma and nasal polyposis, dupilumab elicited a significant improvement in the score of SNOT-22 questionnaire. In such a group of subjects, this result was associated with a complete prevention of the relapses of nasal polyposis. These findings corroborate in a real-life setting the efficacy of dupilumab in the treatment of CRSwNP, previously demonstrated by the Liberty NP trials SINUS-24 and SINUS-52 .1, and also incremented FVC and FEF25-75. These findings indicate that dupilumab can improve lung function by increasing airway patency from the central proximal sector to the distal periphery of the respiratory tree. The clinical and functional results of the present observational study strongly suggest that clinical remission was achieved by a relevant number of our patients. Although this very important therapeutic target should be evaluated after 12 months of continuous treatment, already after 6 months we noticed that 47.24% of our patients reached the criteria of clinical remission, including significant improvements in asthma exacerbations, OCS intake, symptom control and lung function . The patients/participants provided their written informed consent to participate in this study.All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.


Body mass index (BMI) before renal denervation, 6 and 12 months after this procedure were 31.5 ± 4.2, 30.5 ± 4.4, 30.2 ± 4.0 kg/m2, (p = 0.057), respectively. Plasma adiponectin concentration before the renal denervation and 6 and 12 months after this procedure were 4.79 , 7.58 , 6.62  [µg/mL] (p = 0.007), respectively. (5) Conclusions: Plasma adiponectin concentration increases significantly after successful renal denervation in patients with resistant hypertension. Higher plasma adiponectin concentration may participate—beyond blood pressure reduction—in the cardiovascular benefits related to successful renal denervation; however’ clinical consequences of these results need further investigations.(1) Introduction: Adiponectin is synthetized by white adipose tissue and has anti-diabetic, anti-atherosclerotic, anti-thrombotic, anti-inflammatory, and cardioprotective properties. In patients with arterial hypertension, plasma concentration of adiponectin is lower than in healthy subjects. Renal denervation, i.e., percutaneous ablation of fibers from the sympathetic nervous system located in the wall of the renal arteries by radio frequency waves, is a method of resistant arterial hypertension treatment. (2) The aim of this single center, interventional, clinical study was to assess the effect of renal denervation on the plasma adiponectin concentration in patients with resistant arterial hypertension. (3) Materials and methods: 28 patients  aged 54.4 ± 9.2 years with resistant hypertension who underwent renal denervation using Simplicity catheters  were enrolled in the study. Plasma adiponectin concentration was determined using the Human Adiponectin ELISA Kit  before the renal denervation and 6 and 12 months after this procedure. (4) Results: Blood pressure (BP) values before renal denervation and 6 and 12 months after this procedure were as follows: systolic BP 190.4 ± 24.5, 160.8 ± 14.5, 155.7 ± 17.9 mmHg ( The kidneys are innervated by two types of sympathetic nervous system fibers: efferent fibers conducting impulses from the central nervous system to the kidneys and afferent fibers conducting impulses from the kidneys to the central nervous system . The conAdiponectin is one of the adipokines ,7,8,9,10The results of experimental studies indicate that activation of the sympathetic nervous system inhibits the synthesis of adiponectin by white adipose tissue . In clinTaking into account the above presented data, it is justified to conduct clinical studies assessing the impact of non-pharmacological intervention in reducing the activity of the sympathetic nervous system on plasma concentration of adiponectin.The aim of the current single-center, observational, clinical study was to evaluate the effect of ablation of sympathetic nervous system fibers in the wall of renal arteries using radio frequency waves, i.e., renal denervation, on the plasma adiponectin concentration in patients with resistant hypertension.2) and non-nephrotic range proteinuria. Thirteen patients (46%) additionally suffered from diabetes mellitus.Twenty-eight patients  aged 54.4 ± 9.2 years with resistant primary arterial hypertension were qualified for renal denervation procedure and included in this single-center, interventional, clinical study. Secondary causes of arterial hypertension in these patients were excluded by conducting careful clinical, hormonal, and radiological examinations. Among them, the diagnostic procedures of hyperthyroidism, hypercortisolemia, pheochromocytoma, and primary hyperaldosteronism were completed. Moreover, in all patients angio-CT of renal arteries was performed in order to exclude renal artery stenosis. At baseline, patients were treated with following antihypertensive drugs: β-adrenergic receptor antagonists , α-adrenergic receptor antagonists , calcium receptor antagonists , angiotensin-converting enzyme inhibitors , angiotensin-2 receptor antagonists , methyldopa , clonidine , rilmenidine , moxonidine , loop diuretics , spironolactone , thiazide diuretics , and thiazide-like diuretics . Three patients (11%) were treated with four antihypertensive drugs, 7 patients (25%) with five drugs, 13 patients (46%) with six, 3 patients (11%) with seven, one patient (4%) with eight and one patient (4%) with nine antihypertensive drugs). Left ventricular hypertrophy was found in 21 patients (75%) and hypertensive retinopathy in 22 patients (79%). Eight patients (29%) had chronic kidney disease presumably as a consequence of hypertensive kidney injury with normal eGFR . A Symplicity catheter  was used to perform all renal denervation procedures via the femoral artery, under local anesthesia. A Symplicity catheter with a 5F diameter was inserted into the lumen of the renal artery through a vascular sheath with a 6F diameter placed in the femoral artery and a catheter with a 6F diameter placed at the outlet of the renal artery. During the procedure, the catheter was connected to a radiofrequency generator. Thus, the energy of 5–8 W was emitted into the wall of the renal artery. The aim of this procedure was to damage the fibers of the sympathetic nervous system located in the renal artery adventitia. At the beginning of the procedure, the Symplicity catheter was placed in the distal part of the renal artery trunk where the first application was made. Then, under radiological control, the catheter was withdrawn approximately by 5 mm and rotated by 90°. After such displacement and rotation of the catheter tip, another application was performed. Therefore, the applications covered the entire circumference of the renal artery. Depending on the length of the trunk within one renal artery, 4–5 applications were made. In all patients, a single procedure was performed on both sides . In order to avoid an excessive increase in temperature in the lumen of the artery during the application, the catheter was rinsed continuously with physiological saline. Before and during the procedure, patients received heparin in a dose that allowed them to obtain the activated clotting time (ACT) in excess of 250 s. The time of the entire procedure was 45–60 min. In all studied subjects, no significant complications were observed during the periprocedural period. After the renal artery denervation procedure all patients were treated with acetylsalicylic acid (75 mg per day) for at least two weeks.Patients were followed-up after renal denervation for one year in the Hypertension Outpatient Clinic in the Department of Nephrology, Transplantation and Internal Medicine, Medical University of Silesia Clinic Hospital in Katowice, Poland. During this time, to prevent intraobserver variability, all patients were treated by a single physician (Dr Beata Czerwieńska) and the intention was not to change pharmacological antihypertensive treatment which was recommended before renal denervation.Two patients died during the 12-month follow-up period and were not included in further analyses. One patient  died because of hemorrhagic stroke. The second patient  died because of complications after myocardial infarction. Therefore, the final studied group consists of 26 patients  aged 54.6 ± 8.0 years. In all patients, the following parameters were analyzed: office blood pressure, heart rate, body weight, body mass index, creatinine, fasting glucose, total cholesterol, triglycerides, CRP, adiponectin, and insulin plasma concentrations, as well as, blood concentration of glycated hemoglobin (HbA1c). The glomerular filtration rate was estimated using the short MDRD formula. Additionally, the HOMA-IR index was calculated according to the formula: fasting insulin [µU/mL] × fasting glucose [mmol/L]/22.5. In all patients, an office blood pressure measurement was performed. The above-mentioned parameters were analyzed before, 6 months, and 12 months after the renal denervation.Blood pressure measurements were taken in the doctor’s office. The room where blood pressure measurements were taken was a quiet and peaceful place with a comfortable temperature for the patient. Blood pressure measurements were performed by a physician. There were no other people in the doctor’s office during the blood pressure measurement. For at least 30 min before the doctor’s visit and blood pressure measurement, the patient did not consume a meal, caffeine, smoke cigarettes, did not undertake physical effort or exercise, and did not use medications. Blood pressure measurements were taken after at least 5 min. of rest in a sitting position. An upper arm cuff was used to measure blood pressure, the width of which was selected individually for each patient, depending on the arm’s circumference. Blood pressure was measured using an automatic, electronic (oscillometric) blood pressure monitor. During the first examination, blood pressure was measured in both arms. Subsequent measurements were completed on the arm with higher blood pressure values. If there was no difference in pressure between the arms, the following measurements were made on the arm of the side of non-dominant hand. During the measurement, the patient remained sitting, with his back resting, the elbow of the examined limb was supported, and the arm with the blood pressure monitor’s cuff was at heart level. The lower limbs were not uncrossed, and the patient’s feet were placed freely, flat on the ground. During blood pressure measurements, the patient did not talk or move. Blood pressure was measured three times during the medical visit with a 1-min. interval between measurements. The average of the last two measurements was taken into account. This method was in line with actual practice guidelines of the European Society of Hypertension . Plasma Statistical analysis was conducted using STATISTICA software. Shapiro–Wilk test was used to assess the normality of distribution. The following tests were used in statistical analysis: chi-square, Friedman’s ANOVA, and multivariable analysis. Results were presented as a mean with standard deviation (±SD), when data had parametric distribution and as a median with interquartile range .The Medical University Ethics Committee consent number is KNW/0022/KB1/22/II/12.p < 0.001 and diastolic BP: 111.7 ± 18.9, 88.9 ± 8.3, 91.2 + 10.2 mmHg, before the procedure, 6 months after, and 12 months after, respectively; p < 0.001). Heart rate in mentioned intervals decreased . The number of antihypertensive drugs was the same before ) as 6 and 12 months after renal denervation  and 6.0 , respectively).Office blood pressure (BP) decreased significantly after the renal artery denervation procedure: systolic BP , (Body weight and body mass index (BMI) decreased after the renal artery denervation procedure , .p = 0.007); before the procedure, 6 months after, and 12 months after procedure, respectively) , but there was no significant difference in the HOMA index during the follow-up period , 7.58 , 6.62  [µg/mL]; ctively) . Plasma p period .In the studied group there was no significant correlation between BMI as well as systolic/diastolic blood pressure and plasma adiponectin concentration before denervation as well as 6 and 12 months. There was also no correlation in concentration of plasma adiponectin and glucose 6 and 12 months after denervation.Two multivariable analyses were carried out. In the first, plasma adiponectin concentration after 12 months was a dependent value. Baseline plasma adiponectin concentration and glucose, body mass index, eGFR after 12 months as an independent value. It has been shown that plasma adiponectin concentration after 12 months depends only on plasma adiponectin concentration at baseline. In the second analysis, plasma adiponectin concentration after 12 months was a dependent value. Baseline plasma adiponectin concentration and change in glucose plasma concentration, body mass index, eGFR in 12 months as an independent value. It has been shown that plasma adiponectin concentration after 12 months depends also only on plasma adiponectin concentration at baseline.p = 0.9 and p = 0.1), systolic blood pressure (p = 0.6 and p = 0.5), glucose plasma concentration (p = 0.8 and p = 0.7) and eGFR (p = 0.7 and p = 0.09) at 6 and 12 months respectively.Change of plasma adiponectin concentration at 6 and 12 months in multivariable analysis did not depend on change of BMI . Because of the low number of patients treated with rilmenidine and moxonidine (only two patients) formal comparison between patients treated with rilmenidine or moxonidine and the other patients is not possible. However, it seems that the antihypertensive effect of rilmenidine and moxonidine is similar to other drugs.2) as well as higher body weight (83.7 vs. 93.6 kg) and BMI (34.3 vs. 29.3 kg/m2), in comparison to responders. Plasma adiponectin concentration increased significantly in responders during the follow-up period , 8.18 , 8.88  [µg/mL]; p = 0.03, before denervation, 6 months after and 12 months after, respectively) (p = 0.2) (p = 0.03) in comparison with low-responders , 18.8 , 18.9  [µIU/mL], p = 0.01). HOMA-IR index did not change significantly in both subgroups  and lower GFR (77.2 vs. 95.2 mL/min/1.73 mctively)  and did p = 0.2) . There wubgroups .In the present study, we were able to document the significant increase in plasma adiponectin concentration in patients with resistant hypertension after renal denervation.The relationship between renal artery denervation and plasma adiponectin concentrations has so far been investigated in only a few clinical studies. In a study by Eikelis et al. of 57 patients with resistant hypertension who underwent renal denervation procedure, it was found that three months after the procedure plasma adiponectin concentration was higher than before renal denervation . On the Heterogeneity of the above-mentioned results may be due heterogeneity of the completion of the ablation procedures. In the current study, systolic blood pressure decrease in 12 months follow-up was from 190.4 ± 24.5 to 155.7 ± 17.9 mmHg, in Mirosławska et al. study 164 ± 21 to 150 ± 18 mmHg in 2 years and in Eikelis from 168.8 ± 2.6 to 155.2 ± 3.2 mmHg in 3 months. One of the reasons for the heterogeneous antihypertensive effects of the procedure may be possible differences among the subjects in the anatomy of sympathetic nervous fibrous in renal artery adventitia. Moreover, it should be stressed that during the procedure is not possible to control directly the completeness of denervation in vivo .1-imidazoline receptors in the medulla vasomotor center and the kidneys and leads to reduction of sympathetic nerve system activity) at a dose of 1–2 mg/day. After six months of such treatment, there was a significant increase in plasma adiponectin concentration without significant changes in insulin sensitivity and body fat content [Adiponectin participates in the regulation of blood pressure through two mechanisms: by influencing the activity of the sympathetic nervous system  and by s content .Increasing the adiponectin concentration in patients with arterial hypertension may be of clinical importance. Adiponectin is an adipose tissue hormone with multidirectional beneficial effects on the circulatory system and metabolism . A meta-In the current study, patients who showed significant office blood pressure reduction (responders) after renal denervation were characterized by increased adiponectin concentration during the 12-months follow-up observation period besides no significant change in BMI . On the The major limitation of the current study is a lack of a placebo group and a low number of studied patients. On the other hand, because of its single-center study character, the procedure of denervation was performed by the same operator in one medical center, which allowed to avoid, as much as possible, bias related to the procedure. Further observation of these patients completed by single physician helped to standardize the approach to antihypertensive treatment and non-pharmacological recommendations in long-term follow-up.Results of the current study may open a discussion about the impact of adiponectin concentration and its reduction after renal denervation on the risk of cardiovascular complications in patients with resistant arterial hypertension. Additional, long-term studies are needed.Plasma adiponectin concentration increases significantly after successful renal denervation in patients with resistant hypertension. Higher plasma adiponectin concentration may participate—beyond blood pressure reduction—in the cardiovascular benefits related to successful renal denervation, however clinical consequences of these results need further investigations.


Diabetic kidney disease (DKD) is a complication that strongly increases the risk of end-stage kidney disease and cardiovascular events. The identification of novel, highly sensitive, and specific early biomarkers to identify DKD patients and predict kidney function decline is a pivotal aim of translational medicine. In a previous study, after a high-throughput approach, we identified in 69 diabetic patients 5 serum mitochondrial RNAs  progressively downregulated with increasing eGFR stages. Here, we analyzed the protein serum concentrations of three well-validated biomarkers: TNFRI, TNFRII, and KIM-1. The protein biomarkers were gradually upregulated from G1 to G2 and G3 patients. All protein biomarkers correlated with creatinine, eGFR, and BUN. Performing multilogistic analyses, we found that, with respect to single protein biomarkers, the combination between (I) TNFRI or KIM-1 with each RNA transcript and (II) TNFRII with MT-ATP8, MT-ATP6, MT-COX-3, and MT-ND1 determined an outstanding improvement of the diagnostic performance of G3 versus G2 patient identification, reaching values in most cases above 0.9 or even equal to 1. The improvement of AUC values was also evaluated in normoalbuminuric or microalbuminuric patients considered separately. This study proposes a novel, promising multikind marker panel associated with kidney impairment in DKD. DKD is a microvascular complication of diabetes that affects approximately 30% of type 1 diabetes (T1D) patients and 40% of type 2 diabetes (T2D) patients . FurtherTumor necrosis factor-α (TNF-α) is an inflammatory cytokine that has emerged as being critical in the pathogenesis and progression of DKD . TNF-α cn = 24, G2: n = 25, G3: n = 20) and showed excellent diagnostic performance in G3 versus G2 patient identification. The pathogenesis of DKD is complex and involves different mechanisms, such as metabolic, fibrotic, and inflammatory pathways that crosstalk with each other. Thus, a biomarker panel consisting of several kinds of molecules could be more informative of the various dysregulated molecular mechanisms and could enhance DKD diagnostic/prognostic performance.As well as protein markers, several studies demonstrated that RNA molecules in body fluids, since they are highly stable and easily detectable , can repIn light of this evidence, the main aim of the current study is to determine the serum concentrations of TNFRI, TNFRII, and KIM-1 in the 69 T2D patient cohort and evaluate if their combination with previously identified mitochondrial RNA expression data can constitute a novel panel that allows an enhancement of the diagnostic performance of DKD. n = 24, eGFR ≥ 90 mL/min/1.73 m2; stage G2: n = 25, eGFR between 60 and 89 mL/min/1.73 m2; stage G3: n = 20, eGFR between 30 and 59 mL/min/1.73 m2). TNFRI and TNFRII were gradually upregulated, ranging from G1 to G2 and G3 stages. KIM-1 concentration was increased in G3 versus G2 and G3 versus G1 comparisons. For all three protein biomarkers, we observed a strong statistical significance in G3 versus both G1 (p < 0.0001) and G2 comparisons . The concentrations of the three protein biomarkers are represented as dot plots in p-values of analyzed protein markers.The concentrations of TNFRI, TNFRII, and KIM-1 were determined through ELISA assays in 69 sera samples of diabetic patients grouped according to eGFR stages  with eGFR stages G1 n = 13, G2 n = 13, and G3 n = 10; n = 33 microalbuminuric patients (ACR between 30 and 299 mg/g), with eGFR stage G1 n = 11, G2 = 12, and G3 = 10. In normoalbuminuric patients, TNFRI and TNFRII were upregulated in G3 versus both G1  and G2 patients , while KIM-1 was upregulated only in G2 versus G1 comparison   and G2 . The TNFRII levels were increased in G3 versus G1  and G2 versus G1 comparisons   A. In mic < 0.05) B. In order to test if the analyzed protein markers were associated with both clinical data and previously identified mitochondrial RNA markers, we performed correlation analyses. Since protein marker data followed a nonparametric distribution, Spearman’s correlation was used.p-value < 0.0001). The three proteins also had a strong correlation with blood urea nitrogen (BUN), another marker of renal function. More in detail, TNFRI and TNFRII showed a p-value < 0.0001 and KIM-1 a p-value of 0.0005. Furthermore, TNFRI, TNFRII, and KIM-1 showed a weak correlation with the albumin/creatinine ratio. Finally, both TNFRI and TNFRII levels were correlated to waist circumference (WC) and hematocrit (HCT). Only TNFRI correlated with AST levels. p-values of Spearman’s correlation analysis. As far as the clinical patients’ data are concerned, in agreement with ELISA assay data, as expected, we found a marked correlation between all the markers and serum creatinine, as well as eGFR  and TNFRII . TNFRI also correlated with MT-RNR1 . KIM-1 levels were associated only with MT-COX3 and MT-ND1  transcripts. p-values of Spearman’s correlation analysis. Concerning the correlation between mitochondrial RNA expression data and the analyzed protein markers, we observed that MT-ATP8, MT-ATP6, MT-COX3, and MT-ND1 were associated with TNFRI  markedly improved diagnostic performance. The most performant combination were the following: TNFRI + MT-COX3: AUC = 0.964, CI = 0.919–1.000, Regarding TNFRII, except for MT-RNR1, the other mitochondrial transcripts improved diagnostic power. It is important to highlight that the combination of TNFRII with MT-ATP6, MT-COX-3, or MT-ND1 expression levels reached a perfect AUC value  . p-value = 1.0 × 10−6, sensitivity 95%, and specificity 80% : p-value = 7.5 × 10−5, sensitivity = 100%, and specificity = 92.3%. Similar results were obtained in microalbuminuric patients. Indeed, the combination with all the RNA markers allowed us to obtain perfect ROC curves with AUC value = 1  or MT-ATP6 (AUC = 0.867) expression data weakly increased the diagnostic performance .n = 24 G1, n = 25 G2, n = 20 G3), independently or dependently of ACR stages. As expected, we observed a gradually increasing trend of TNFRI, TNFRII, and KIM-1 concentrations ranging from G1 to G2 and G3 stages. In agreement with these data, all three protein markers correlated with serum creatinine, as well as eGFR. The three proteins also had a strong correlation with BUN, another marker of renal function. Although these protein biomarkers have been mainly analyzed in longitudinal cohorts, some studies with cross-sectional design are in agreement with our data. For instance, Monika A Niewczas et al. observed that elevated concentrations of serum TNFRI and TNFRII levels are strongly associated with decreased renal function in nonproteinuric T1D patients [2 with respect to GFR ≥ 90 mL/min/1.73 m2, and through multivariable logistic regression analysis they demonstrated that TNFRII together with triglycerides, fibrinogen, and VCAM were associated with increased odds of having a GFR < 60 mL/min/1.73 [2) and an increased ACR (≥30 mg/gCr) [2), TNFRI levels were higher in patients with renal alteration function [DKD represents a very relevant clinical complication of diabetes. Indeed, it is the main cause of end-stage kidney disease in developed and developing countries and the most common reason for renal replacement therapy ,29. Furtpatients . J Lin emin/1.73 . Tomohit mg/gCr) . In anotfunction .n = 6 with DKD, n = 6 without DKD), 33 downregulated transcripts were identified. Among them, by real-time PCR, the downregulation of 5 mitochondrial RNAs  was validated in the same cohort of 69 patients stratified according to eGFR stage. This was the first study that highlighted the differential expression of mitochondrial RNAs in DKD. Other studies, most of them conducted in cross-sectional cohorts, reported several microRNAs dysregulated associated with ACR and/or eGFR [In a previous study conducted by our research group, we applied a high throughput transcriptomic approach to discover novel biomarkers in a nonbiased manner. After microarray analysis, conducted in 12 serum samples  involved in different pathways  strongly increased the diagnostic power for the identification of diabetic patients with functional kidney impairment. This is not surprising; indeed, the development and progression of DKD involve complex multiple pathogenic pathways such as inflammation, fibrosis, oxidative stress, and hemodynamic ; hence, Some very robust studies conducted in wide longitudinal cohorts simultaneously analyzed several protein and biochemical biomarkers specific to different pathogenic processes to establish a model that is able to improve the predictive performance toward renal function decline. In the study funded by the SUMMIT consortium, within a broad set of 207 serum protein and metabolite biomarkers measured in 154 incident cases of rapid progression of renal function decline and 153 nonprogressing, 14 biomarkers determined an improvement of the predictive performance beyond clinical covariates . In the The major limitations of our study are determined by the small sample size and the cross-sectional study design, which do not allow us to determine the progression predictive value of identified biomarker panel. However, to the best of our knowledge, this is the first study that combines data on serum protein and RNA molecule expression to establish a novel panel of biomarkers associated with kidney function impairment. These data should be validated in a larger independent external cohort. Furthermore, in order to determine if the identified panel can have a prognostic and predictive value, the model performance should be assessed in association with kidney function decline in longitudinal cohorts. Even with these limitations, this study suggests a novel outstanding performant unexplored biomarker panel for DKD.n = 24, eGFR ≥ 90 mL/min/1.73 m2; stage G2: n = 25, eGFR between 60 and 89 mL/min/1.73 m2; stage G3: n = 20, eGFR between 30 and 59 mL/min/1.73 m2). We excluded from our study patients with T1D, nondiabetic kidney disease, hepatic diseases, autoimmune disorders, cancer, and diabetes complicated with cardiovascular diseases. The biochemical/clinical data of enrolled subjects are reported in p-value < 0.05). Detailed pharmacological data of subjects grouped according to eGFR or ACR are reported in Sixty-nine diabetic patients were recruited by the Internal Medicine Unit of the Garibaldi-Nesima Hospital, University of Catania, Italy. Diabetes diagnosis was established according to the following parameters: fasting blood glucose ≥ 126 mg/dL and HbA1c ≥ 6.5% (48 mmol/mol) . AlbuminAfter one hour at room temperature, blood samples were centrifuged at 3500 rpm at 4 °C for 15 min to separate from whole blood. The serum was further centrifuged to remove eventual cell debris. The upper-layer supernatant was collected, and aliquots were stored at −80 °C until analysis .Total RNA extraction was performed from 400 μL of serum by using the miRNeasy mini kit (QIAGEN) according to the manufacturer’s instructions. RNA concentration and quality were determined through the NanoDrop One . The expression of MT-ATP8, MT-ATP6, MT-COX3, MT-ND1, and MT-RNR1 in the 69 sera of diabetic patients was determined through real-time PCR (Power SYBR Green RNA-to-CT1-Step Kit (Thermo Fisher Scientific) in a QuantStudio 5 system (Thermo Fisher Scientific). ACTB was used as a reference gene, and relative quantification data were determined through the 2−ΔΔCt method. Primer sequences were determined by using Primer Blast. MT-ATP8: forward ACAGTGAAATGCCCCAACTAAAT reverse AGGGAGGTAGGTGGTAGTTTGTG; MT-ATP6: forward ACCTTCCCTCTACACTTATCATCTT reverse CGTGCAGGTAGAGGCTTACT; MT-COX3 forward TTCACCATTTCCGACGGCAT reverse GGCGGATGAAGCAGATAGTGA; MTND1 forward CGGGCTACTACAACCCTTCG reverse AGATGTGGCGGGTTTTAGGG; MTRNR1 forward GGGTTGGTCAATTTCGTGCC reverse ACACTCTTTACGCCGGTTTCT; ACTB2 forward GAGCACAGAGCCTCGCCTTT reverse GAGCGCGGCGATATCATCA .The concentrations of TNFRI, TNFRII, and KIM-1 in the 69 sera of diabetic patients were determined through ELISA assays  according to the manufacturer’s instructions. Preliminary experiments were performed to determine the most appropriate dilution factor for each specific assay. Hence, serum was diluted at 1:35 and 1:20, respectively, for TNF-RI and TNF-RII, while dilution was not necessary for the KIM-1 assay. Concentrations were determined by interpolating data from a standard curve generating a four-parametric logistic (4-PL) curve fit as reported in the manufacturer’s instructions.To assess if clinical and ELISA assay data followed a parametric or nonparametric distribution, three different normality tests were used: the D’Agostino-Pearson omnibus test, the Shapiro–Wilk normality test, and the Kolmogorov–Smirnov normality test . The oneIn order to establish if TNFRI, TNFRII, and KIM-1 serum levels were associated with both transcript expression values and subject clinical data, Spearman’s correlation analyses were performed. The statistical analyses were performed using GraphPad Prism 6.0  ,48. ROC This study demonstrated that the simultaneous dosage of serum mitochondrial RNAs and TNFRI, TNFRII, and KIM-1 proteins can effectively strongly improve diagnostic performance for the evaluation of kidney function impairment in the context of DKD. In general, we propose a multikind marker panel consisting of molecules characterized by different biochemical natures (RNAs plus proteins) associated with kidney impairment in DKD. This approach can be applied to every aspect of translational medicine.


Hypertension is a prevalent complication of Chronic Kidney Disease (CKD) and Ambulatory Blood Pressure Monitoring (ABPM) is the gold standard for diagnosis. The aim of our study was to assess the usefulness of obtaining ABPM and to identify barriers to ABPM in this pediatric patient population.In this retrospective analysis of patients with CKD stage 3–5 who were seen in one academic medical center’s outpatient Pediatric Nephrology clinics between 2018 and 2021, we performed logistic regression to evaluate for associations between demographic factors and odds of having an ABPM.Among 96 patients included in the study, 48 patients carried a diagnosis of hypertension. 31 patients had ABPM performed with usable data. In those who had ABPM done, 21 had normotension and 10 had undertreated hypertension. Our study also showed 1 had masked hypertension and 5 had white coat hypertension or effect. We did not find a statistically significant difference in those who did or did not undergo ABPM evaluation based on gender, previous diagnosis of hypertension, distance from clinic, language preference, or racial or ethnic identity.ABPM is a useful tool in our CKD population for the diagnosis and management of hypertension. We did not identify specific barriers to ABPM in our CKD population, and there were no differences in patients who obtained ABPM when looking at specific demographic and disease characteristics. Given these findings, we recommend focusing on areas of future improvement in spheres of patient and provider education as well as better quantification using surveys to further illuminate barriers. Approximately 1% of all patients with chronic kidney disease (CKD) are children and adolescents, with CKD affecting 15-74.5 children per million globally , with a Repeated studies have shown the superiority of ambulatory blood pressure monitoring (ABPM) evaluations for BP assessment. Office BP measurement alone can misclassify BP status in over 40% of subjects, when compared to ABPM evaluation . Home BPDespite the evidence and recommendations, ABPM is not routinely obtained in the CKD population, and there have been studies done to identify barriers to obtaining ABPM. A focus-based group study on English and Spanish preferred language adults showed patients being concerned about cost of the procedure and discomfort while wearing ABPM device . AnotherThe aim of our study is to identify any additional barriers to ABPM in pediatric patients with CKD and to assess the utility of the study in this population.Retrospective chart reviews were done on all patients with CKD stage 3–5 who were seen in the Pediatric Nephrology ambulatory clinics of one academic medical center between January 2018 and December 2021. Inclusion Criteria were patients with CKD stage 3–5, age 5 or greater, and height > 120 cm. Patients on dialysis or with a history of kidney transplant were excluded. Approval was obtained from the Institutional Review Board.Office blood pressures were measured using an oscillometric device and 1–2 readings were obtained.Patients were referred by their primary nephrologists to the ABPM clinic at our center. The ABPM visit was conducted either in person or by telehealth. Office blood pressure readings were collected from the ABPM visit if in person or from the most recent nephrology visit.ABPM was performed using a Spacelab 90217 monitor. The ABPM was either placed on the non-dominant arm using an appropriately sized cuff by the pediatric nephrology nurse practitioner or by a family member with provider instruction if placement was done through telehealth. For patients who were seen by telehealth, the ABPM was mailed to their home with an included return label. BP was measured every 20 min during the day and every 30 min at night during a 24-hour period. Once ABPM was done, it was returned by mail. Usable data was defined as having at least 40 to 50 readings or 70% successful readings for a full 24-hour period.We used normative data for ambulatory BP as presented by Wuhl et al. . Using bPatients with a previous history of hypertension were considered to have well-controlled hypertension based on casual BP < 95th percentile and mean ambulatory BP < 95th percentile. Conversely, patients were considered to have under-treated hypertension if they had mean ambulatory BP ≥ 95th percentile or > 125/75.We examined the association between access to ABPM and demographic characteristics including age, gender, preferred language , race/ethnicity, physical distance from the clinic, presence of social concerns as documented by licensed clinical social worker, number of visits per year, glomerular versus non-glomerular etiology of CKD, CKD stage, and existing diagnosis of hypertension using logistic regression models.Stata 14  was used for the performance of all statistical analyses. P-values < 0.05 were considered statistically significant for all analyses.Ninety-six patients were included in this study; 54 were males and 42 were females. Forty-eight (50%) had a prior diagnosis of hypertension. ABPM was performed in 33 patients out of which 31 patients had ABPM with usable data (32%). Twenty out of those 33 patients had a diagnosis of hypertension before ABPM was performed, and 18 of these contributed usable data. Of the patients with an ABPM performed with prior diagnosis of hypertension, 9 had confirmed undertreated hypertension (50%), 5 had well-controlled hypertension (28%), 3 were diagnosed with white coat effect (16%) and 1 had masked hypertension (6%).  as being a barrier to obtaining ABPM with no association noted. In addition, our study did not identify ethnic and racial identity as being a barrier to ABPM use, in contrast to a recent study by Pagi et al. who found ABPM was performed less frequently in Black children versus White and Hispanic children . Our stuIn evaluating the specific barriers to pediatric ABPM, while we did not find specific barriers to ABPM, we have helped focus work in other areas that can help improve the gap in this important tool. There is a need for further studies to be done in the pediatric CKD populations to identify barriers to ABPM to help increase their usage. Additional exploration into patient transportation and financial insecurity, provider-based ordering and recommendation factors, and patient education and perspectives may reveal additional barriers that have not yet been described.The strength of this study is that it is one of few studies examining the prevalence of and potential barriers to ABPM in the pediatric CKD population , 21, 23.In our study, we found ABPM was a beneficial tool in diagnosing masked hypertension and white coat hypertension and therefore helpful in preventing CKD progression and extra medication burden. It also showed that approximately 50% of patients with prior diagnosis of hypertension were inadequately controlled. We did not identify any barriers to ABPM use, and we recommend focusing on areas of future improvement in spheres of patient and provider education as well as better quantification using surveys to further illuminate barriers and increase rates of ABPM monitoring. ABPM is more cost-effective than dialysis and renal transplantation and therefore should be performed routinely in CKD population to delay CKD progression and improve health outcomes.


In recent years, immunotherapy has been postulated as one of the most effective strategies in the fight against cancer. The greatest success in this field has been achieved with the inhibition of molecules involved in slowing down the adaptive immune response by immune checkpoint inhibitors (ICIs). Despite its efficacy, ICI treatment has side effects. Regarding kidney damage, it is estimated that 4.9% of patients treated with ICIs develop renal injury. Furthermore, cancer patients who develop renal dysfunction have a worse prognosis. Current diagnostics are insufficient to predict the underlying renal injury and to identify the type of damage. Our hypothesis is that the renal injury could be subclinical, so the possibility of using new urinary biomarkers could be a useful diagnostic tool that would allow these patients to be managed in a preventive (risk biomarkers) and early (early biomarkers) way and even to clarify whether the renal damage is due to this therapy or to other factors . A prospective study to validate risk and early and differential biomarkers in patients treated with ICIs is proposed to test this hypothesis. The results derived from this study will improve the clinical practice of cancer treatment with ICIs and therefore the life expectancy and quality of life of patients. Trial Registration: ClinicalTrials.gov, NCT04902846. Advances in the knowledge of the immune response against tumours, coupled with an understanding of mechanisms of the evasion of these, have led to the development of novel, immune response-focussed treatments. The most promising immune response-focussed treatment is the inhibition of molecules involved in breaking the adaptive immune response. Examples include cytotoxic T-lymphocyte antigen-4 (CTLA-4), and programmed cell death protein-1 (PD-1), with their respective ligands. Compounds capable of blocking the action of these molecules are called immune checkpoint inhibitors (ICIs) [Owing to their mechanism of action, ICI use is limited due to their association with adverse effects derived from an autoimmune response . The mosTherapies based on combinations of ICIs have recently been developed to avoid acquired tumour treatment resistance. The use of ICIs in combination with chemotherapy has been shown to improve treatment response. However, this also increases the risk of nephrotoxic adverse events . ClinicaIn most cases, kidney damage caused by ICIs is acute tubulointerstitial nephritis (ATIN). However, other types of damage include immunoglobulin A (IgA) nephropathy, membranous nephropathy, lupus-like immune complex glomerulonephritis, C3 glomerulopathy, focal segmental glomerulosclerosis, thrombotic microangiopathy, and pauci-immune crescentic glomerulonephritis ,15. In sStrategies that focus on predicting the likely progression of kidney damage , would Patient predisposition to and risk of kidney damage should ideally be assessed prior to the commencement of ICI treatment. This would help identify and stratify patients according to their susceptibility to kidney damage. Susceptibility biomarkers have been identified in preclinical models, and their usefulness is currently being evaluated in different clinical settings ,24,25,26A new, early diagnosis that predicts elevated creatinine levels is currently being studied . This woFurthermore, several authors ,28,29 haThis study proposal has huge clinical relevance. Knowledge of novel biomarkers would enable the stratification of treatment, thus helping to avoid and minimise kidney damage at different treatment stages. Risk biomarkers would enable the prediction of individual patient risk. Furthermore, during treatment, early biomarkers would enable the detection of early lesions, and differential diagnostic biomarkers would enable the identification of the type of kidney damage, once detected.The main objective of this study is to validate biomarkers as diagnostic tools for kidney damage in ICI-treated patients, and moreover, whether biomarkers can also be a valid management tool in these patients. Pre-treatment stage: in this stage, risk biomarkers would predict individual patient risk.Phase I (1st stage): this stage is during the first stages of treatment; early biomarkers would detect early lesions.Phase II (2nd stage): this stage is during treatment; differential diagnostic biomarkers would help diagnose the type of lesion in established kidney damage.For this purpose, the following stages associated with kidney damage are proposed : Pre-treWith these study objectives, we aim to determine whether biomarkers can be a valid tool in the management of kidney damage in patients treated with ICI-based immunotherapy, responding to an unresolved diagnostic need.This prospective study will include patients undergoing ICI-based immunotherapy or combined ICI/chemotherapy treatment with platinum compounds. Patients will be recruited from two medical oncology services in Spain, Complejo Asistencial Universitario de Salamanca and Hospital Clínico Universitario de Valladolid. This study will not alter the standard clinical procedures. Patients included in the study will be classified into 2 groups. Group 1, patients treated with single or combined immunotherapy, and group 2, patients treated with immunotherapy combined with chemotherapy. The 2 groups will be further divided into 2 subgroups, controls and cases. The cases subgroup will include patients who develop acute kidney damage during their treatment, and controls will include those who do not develop kidney damage during their treatment. Acute kidney damage will be defined by elevated creatinine, according to KDIGO criteria , electroThe researcher will provide all relevant study information to the recruited patients before obtaining their consent. A patient information sheet will be provided in the local language and prepared according to ICH GCP guidelines . Prior to their inclusion in the study, all participants will sign an informed consent form, as established in the Declaration of Helsinki  and WHO Patients are required to meet the following criteria to participate in the study: have provided informed consent, are ≥18 years old, are awaiting treatment with ICI-based immunotherapy or combined ICI/chemotherapy treatment with platinum compounds, and have normal kidney function prior to treatment. Normal kidney function will be defined according to the margins established by the biochemistry service of each hospital prior to treatment.Patients with an existing disease or condition that would interfere with either participation or study evaluations will be excluded. Patients will be freely able to withdraw from the study at any time without having to provide an explanation. The study subjects will be withdrawn if a clinically relevant event occurs that affects patient safety.p-value of 0.50 was taken. This guarantees the largest sample size. A population size of 186 was obtained. It is necessary to estimate potential losses of patients for various reasons including loss of information, death, and withdrawal. As such, the sample size was increased to (N = n (1/1 − R)), where n is the number of patients without losses and R is the estimated loss (15%). Therefore, the final estimated total sample size is 220 patients, a total of 110 from each hospital. To determine the necessary study sample size, a type I error (α = 0.05), statistical power of 1-β = 0.80, and frequency of case and control occurrence will be used. Since the last parameter cannot yet be determined, a Patient data will be collected using a database that records the following variables: age, sex, family history, personal history, tumour type, antitumour treatment regimen, clinical patient characteristics, comorbidity factors, allergies, prescribed medication, occasional drugs, and follow-up data.Blood and urine samples will be collected from each patient prior to starting ICI treatment (pre-ICI) and after each ICI treatment (post-ICI)The biochemistry service of each hospital will process all patient blood samples collected. Urine samples collected at the Complejo Asistencial Universitario de Salamanca will be handled and stored in the internal hospital sample bank. Urine samples collected from the Hospital Clínico Universitario de Valladolid will be handled and stored in the hospital’s internal oncology unit. After collection, urine will be centrifuged and subsequently aliquoted and frozen at −80 °C until use.At blood sampling times, the plasma concentrations of creatinine, urea, and electrolytes and the estimated glomerular filtration rate will be analysed and issued by the biochemistry service of the respective hospitals. Analytical determinations will be carried out by The Theranostics of Renal and Cardiovascular Diseases Group of the Institute of Biomedical Research of Salamanca (IBSAL). These analyses will include the following.Urinary creatinine will be determined using a commercial colourimetric kit based on the Jaffe method , “QuantiUrinary protein excretion will be determined using the Bradford colourimetric method .For the determination of albumin and transferrin, the commercial kits “Human Albumin ELISA kit” and “Human Transferrin ELISA Kit”  will be used, respectively. To determine the GM2 ganglioside activator protein (GM2AP), Western blotting will be performed. Briefly, 21 µL of urine from each patient per sampling time will be separated by acrylamide electrophoresis. Proteins will be transferred to an Immobilon-P Transfer Membrane  and incubated with the primary antibody against GM2AP (own production). Subsequently, they will be incubated with horseradish peroxidase-conjugated secondary antibody and chemiluminescent detection  will be performed with the ChemiDoc MP imaging system .The determination of the NAG enzyme activity in urine will be carried out using a commercial colourimetric kit  following the manufacturer’s instructions. The other biomarkers will be measured using commercial kits based on the ELISA technique: “Human KIM-1 ELISA kit”  for KIM-1, and the “Human NGAL ELISA Kit 036CE”  for NGAL.A comparative statistical study will be carried out to determine whether any of the biomarkers mentioned above are related to different types of kidney damage, specifically, tubular necrosis, glomerular damage, or interstitial nephritis. In addition, two inflammatory biomarkers that can help in this differential diagnosis will be evaluated: Interleukin 9 (IL-9)  and tumour necrosis factor alpha (TNF-α) .t-test, Mann–Whitney U), ANOVA for repeated measures, and Wilcoxon test for paired data. Finally, multivariate analysis will be used to simultaneously study datasets with several variables for each patient or parameter collected. These multivariate analyses will include regression analysis, general linear models, binary logistic regression, and correspondence analysis. Significance will be defined as a p-value ≤0.05. Data will be analysed using IBM SPSS statistical software, version 25.0 . Different tests will be applied depending on the association to be studied and the type of variable. These will include normality tests , frequency comparison tests , correlation tests , comparisons of means for independent data  in urine before treatment and the appearance of kidney damage after treatment will be analysed.Objective 2: The presence of early damage biomarkers will also be evaluated during treatment, comparing the results in the different target groups.Objective 3: Finally, associations will be sought between the studied biomarkers and the etiological diagnosis of kidney damage .The study was initiated on the 1 January 2021, and the collection of samples is expected to end in 2023. We aim to complete this study in 2023.The results of this study will be communicated to both the participants and the public through a written report. Both social and communication media will be used to disseminate the study findings. The key study findings will be communicated, at the least, in a local congress addressed to individuals from the Salamanca health area. A results dissemination conference will be conducted in collaboration with the Spanish Association Against Cancer and The Association of Kidney Patients (ALCER). Here, the relevance of kidney damage in the treatment of cancer patients will be highlighted, along with the importance of detecting kidney damage in its early stages. Moreover, concepts including immunotherapy and biomarkers will be explained to patients.The diagnosis of kidney damage associated with ICIs is a challenge that must be addressed. The identification of novel biomarkers, which help avoid the requirement for biopsies, is paramount . AccordiThe study aims to identify predisposition biomarkers, capable of detecting patients susceptible to developing kidney damage prior to starting ICI treatment. Such biomarkers have been defined in preclinical models ,24,25,26Early diagnosis refers to a new type of biomarkers that anticipate creatinine elevation . As suchFinally, differential diagnosis ,28,29 isIn summary, this study aims to improve the diagnosis of kidney damage associated with ICI treatments. Such knowledge and results obtained may inform the development of novel diagnostic tools for clinicians, improving the accuracy and effectiveness of treatment plans according to individual patient needs. Ultimately, a more stratified diagnostic approach is proposed to help reduce the incidence of kidney damage and improve cancer treatment with ICIs.


Kidney diseases can cause severe morbidity, mortality, and health burden. Determining the risk factors associated with kidney damage and deterioration has become a priority for the prevention and treatment of kidney disease. This study followed 497 patients with stage 3–5 chronic kidney disease (CKD) who were treated at the ward of Taipei Veterans General Hospital from January 2006 to 2019 in Taiwan. The patients underwent 3-year-long follow-up sessions for clinical measurements, which occurred every 3 months. Three time-dependent survival models, namely the Cox proportional hazard model (Cox PHM), random survival forest (RSF), and an artificial neural network (ANN), were used to process patient demographics and laboratory data for predicting progression to renal failure, and important features for optimal prediction were evaluated. The individual prediction of CKD progression was validated using the Kaplan–Meier estimation method, based on patients’ true outcomes during and beyond the study period. The results showed that the average concordance indexes for the cross-validation of the Cox PHM, ANN, and RSF models were 0.71, 0.72, and 0.89, respectively. RSF had the best predictive performances for CKD patients within the 3 years of follow-up sessions, with a sensitivity of 0.79 and specificity of 0.88. Creatinine, age, estimated glomerular filtration rate, and urine protein to creatinine ratio were useful factors for predicting the progression of CKD patients in the RSF model. These results may be helpful for instantaneous risk prediction at each follow-up session for CKD patients. Chronic kidney disease (CKD) is a significant global health problem and a common health issue. The prevalence is increasing worldwide, amounting to more than 800 million patients . CKD is The estimated glomerular filtration rate (eGFR) is one of the most important risk factors for identifying the classification of CKD . A lowerThe prediction of CKD progression is an important task for patient care in clinical management. Machine learning (ML) methods have been used to predict the risk of CKD applications in recent years ,16,17,18The time-to-event data in CKD typically refers to the duration it takes for a specific event to occur, such as ESRD or death. By incorporating right-censored time-to-event data into the analysis, the risk factors of CKD progression can be evaluated for the varying follow-up times of patients. This allows for a more comprehensive assessment of the disease’s progression and a better understanding of the risk factors that influence the timing of events in CKD patients. Accurate risk prediction models can inform clinical decision-making and help identify patients who are at a higher risk of adverse outcomes, such as ESRD or mortality. Thus, a suitable model with a time-dependent covariate for predicting CKD progression is required. However, such a model has yet to be developed and established.In the present study, the performances of the Cox PHM, RSF, and ANN models with time-dependent variables were used to determine the risk of CKD progression over 3 years. Furthermore, the important features were evaluated as high-risk factors for determining the optimal predictive features for the models. The integration of time-dependent covariates into survival analysis using ML methods for predicting CKD progression was investigated. By characterizing and considering time-dependent covariates, the proposed methodology can predict the instantaneous risk prediction at each follow-up session for CKD patients and provide a more comprehensive understanding of the factors influencing disease progression. This novel approach has the potential to improve the precision of prognostic predictions and could assist in developing tailored interventions and treatment strategies to better manage CKD.A retrospective cohort study using de-identified pathological records was conducted in this study. The dataset was collected from November 2006 to December 2019 in a branch of the Taipei Veterans General Hospital. A total of 947 patients were collected from the National Health Insurance (NHI) CKD program, which is a clinical care and education program for patients initiated by the NHI administration under the Ministry of Health and Welfare in Taiwan. This study was approved by the Institutional Review Board of the Taipei Veterans General Hospital (No. 2020-01-024BC).In the data, the patients who had been diagnosed with stages 1 and 2 were excluded. Similarly, patients with insufficient follow-up data  were also excluded. Missing values in patients with insufficient pathological records were replaced with multiple imputations . Data imThe characteristics of pathological records for each patient were determined during the prespecified 90-day assessment period, starting from the first clinic visit until the study endpoint date. The baseline characteristics of pathological records were defined as an initial point. The endpoint was defined as the requirement for dialysis or kidney transplantation. The patients were followed up until the endpoint date, when patients with dialysis or renal failure were established during the observation period.i period. In addition, the regression analysis was performed to determine the relationship between the predictor variables and observation period for i ≥ 3.The study investigated the time-dependent predictive risk factors of CKD patients with stage 3–5 progression. The eGFR, serum creatinine, natural logarithm-transformed urine protein to serum creatinine ratio (PCRln), and glycated hemoglobin (HbA1c) were used as time-dependent predictors for data characterization. To further characterize the differences among the risk factors of CKD, the time-dependent variability analysis To assess the high-risk factors in predictive models for progression from CKD to ESRD, the important characterized factors of CKD were selected by multivariate analysis of variance (MANOVA) and the independent chi-squared test. Randomized data subsets were used for cross-validation (K = 5). The Cox PHM, RSF, and ANN were used to determine the time-dependent prediction of CKD progression. Then, a comparison of the concordance index (C-index) and Kaplan–Meier method (KM) was used to predict the risk of progression to eventual ESRD among CKD patients with stages 3–5. The models were used to identify risk factors for predicting disease progression in CKD within 3 years. The predictor variables of Cox PHM were normalized by z-score transformation, and the value important (VIMP) of RSF was used for feature selection and prediction. The flow chart of model training and performance evaluation is shown in T is the failure time of interest, t λt |  follows ,34:(3)λLv* and split value c* were determined such that c. The CHF and survival probability t is given by [given by :(5)S^t=∏The Cox-based DNNSurv model was used. In DNNSurv model, the function pseudo survival probability for the puted by ,37:(6)S^given by :(8)C^surThe KM method was used as a survival function ed below :(9)S^ti=We utilized grid search to determine the parameters of the ANN models for hyperparameter tuning while training the model using the defined dataset. Multilayer perceptron was selected to achieve the optimal architecture. Specifically, the model was trained for 10 epochs, consisting of 3 layers with 3 neurons each, a hidden layer of size 3, a batch size of 32, momentum of 0, a learning rate of 0.02, an input layer of size 10 for predictors, an input layer and output layer of size 6 for outcomes, a sigmoid activation function, and an Adam optimizer were used for the network prediction model.The baseline characteristics and predictor variables of 497 patients were investigated from the first clinical visit to the endpoint date. Blood tests were performed during the clinical visit for biochemistry testing. In this study, the suitability of categorized and continuous variables of risk factors was assessed and compared for CKD patients with stages 3 to 5. The categorized variables included gender, hypertension, diabetes, and CVD. The continuous variables included age, systolic blood pressure (SBP), diastolic blood pressure (DBP), serum creatinine, HbA1c, PCRln, body mass index (BMI), and eGFR. The eGFR was calculated using the simplified Modification of Diet in Renal Disease equation, which was mentioned in a previous study . All basThe categorized and continuous variables of CKD patients with stages 3–5 were analyzed. The predictive performances of the three models were investigated using the C-index score and KM curves. Herein, different cut-off points  of probability in CKD progression were used to determine the sensitivity and specificity of the three models, as shown in Furthermore, the KM curves for nondialysis in CKD patients with different variables  were categorized according to the different endpoints. In The KM curves of the three models for a CKD patient without diabetes were analyzed. The probability of the three models for patients without dialysis exceeds 0.7 for endpoints of 917 days and 669 days, as shown in In addition, the KM curves of the three models for a CKD patient with diabetes were analyzed. The RSF model shows a higher prediction than other models for endpoints of 469 days and 591 days, as shown in The KM curves of the three models for a CKD patient with stage 3 were analyzed. The ANN model shows a higher predicted performance than the other models at 658 days, as shown in Moreover, the KM curves of the three models for a CKD patient with stages 4–5 were analyzed. The RSF showed a higher prediction performance than the other models at 469 days, as shown in The KM curves of the three models were analyzed for a CKD patient under 80 years old. The RSF showed a higher predicted performance than the other models at 1095 observation days (within 3 years), as shown in Furthermore, the KM curves of the three models were analyzed for a CKD patient over 81 years old. The RSF model showed a higher prediction than other models after 497 and 532 days, as shown in The RSF was constructed with 100 trees (ntree = 100) and achieved a mean prediction error rate of 0.148. The probability of nondialysis prediction for all CKD patients (N = 429) was analyzed using the three models as shown in According to the C-index values, KM curves, and boxplots for the three models, the best performance was obtained using the RSF model. The VIMP values of risk factors in CKD were analyzed for feature selection and prediction, as shown in This retrospective cohort study included CKD patients who participated in disease management programs for education purposes and the prevention of dialysis in Taiwan. The study demonstrated that PCRln and eGFR showed significant differences among CKD patients with stages 3–5, which were consistent with our previous study . AdditioIn the present study, the RSF model demonstrated the best performance, followed by the Cox PHM and ANN models, for the CKD patients who progressed within 3 years. Creatinine, age, GFR, and PCR were found to be correlated with CKD progression in the RSF model. In the Cox PHM model, CVD, along with creatinine, age, and PCR, showed higher predictability for dialysis patients. When applying the same time-dependent design, the RSF model in the present study outperformed the Cox PHM model used in our previous study, even though they utilized different risk factors . HoweverKM survival curves are commonly used to determine whether risk outcomes vary over time. Recently, the multivariable Cox PHM and KM survival curves were utilized to examine different prognostic factors in 16,752 confirmed cases of COVID-19 . Our finData characterization is a common method used to identify important features, which may enhance the credibility of the hypothesis testing in prediction models. Previous studies indicated that the methods of z-score standardization , min-maxRisk prediction based on right-censored survival data by using suitable ML methods has significant implications for CKD patient management. Based on the present results, the proposed method could be used for the risk prediction of CKD progression that allows healthcare professionals to identify individuals who may benefit from early intervention, such as timely referral for transplantation or initiation of dialysis. Additionally, risk prediction models can assist in optimizing healthcare resource allocation by targeting high-risk CKD patients for specific interventions, potentially improving patient clinical outcomes and healthcare efficiency.It is crucial to consider the trajectories of pathological indicators in the pathological progression. In this study, being superior to the conventional Cox hazard model, the RSF model was successfully developed using characterized time-dependent covariates, and the results showed good performance for CKD patients at stages 4 and 5 who progress within 3 years. The approach is suitable for personalized prediction of trajectories, even with a relatively small dataset. Creatinine, age, eGFR, and PCR were identified as useful factors for predicting the progression of CKD patients in the RSF model, as indicated by their VIMP values. On the other hand, Cox PHM showed slightly higher prediction performance than RSF for patients with stage 3 or younger ages, among those who eventually required dialysis. The present method could be utilized by physicians and care workers to assess, intervene, and treat CKD progression in a timely manner. However, this study also had several limitations. It was a retrospective cohort study with a relatively small sample size, making it challenging to increase the number of neural network layers for model training. Further studies that analyze clinical pathological records from different hospitals are necessary to ensure unbiased results, thereby increasing the amount of training data to enhance the performance of ANN for predicting progression from early- and advanced-stage CKD. Moreover, the mean age of CKD patients in the study was 80 years, with the maximum age reaching 103 years, which may limit the generalizability of the results to younger patients. Further studies should be verified in CKD patients under the age of 80 to ensure unbiased results and improve the generalization of the model for a wider population.