Oral corticosteroid (OCS)-dependent severe eosinophilic asthma with chronic rhinosinusitis with nasal polyps (SEA-CRSwNP) would be a suitable phenotype for mepolizumab treatment. This study evaluated the short-term efficacy of mepolizumab treatment in OCS-dependent SEA-CRSwNP. Baseline and 24th week results  of patients who were treated for at least 24 weeks with mepolizumab were retrospectively evaluated and compared.  A total of 16 patients were enrolled in the study. Mepolizumab was discontinued in one patient due to side effects. The daily OCS dosage was reduced from baseline in all patients, and at week 24 OCS was discontinued in 40% of the patients . The number of asthma exacerbations within 24 weeks significantly decreased after beginning mepolizumab treatment , and a significant increase in ACT scores  was observed despite the decrease in daily OCS dosages. There was no significant difference in FEV1 values between baseline and week 24. Evaluation of the general symptoms of CRSwNP, as per NAS, revealed that the baseline mean NAS was 5.6 ± 4.4, and the 24th week mean NAS was 3.2 ± 3.2 (P = 0.021).  This is the first real-life study evaluating the short-term efficacy of mepolizumab treatment on OCS-dependent SEA-CRSwNP. This study demonstrates that mepolizumab is an effective and safe biologic for the treatment of this severe asthma subphenotype.  The anti-IL5 antibody mepolizumab was approved in 2015 and since then has become an established therapy for patients with severe uncontrolled eosinophilic asthma Difficult-to-treat and severe asthma in adolescent and adult patients. Diagnosis and management. The Global Initiative for Asthma 2019. [online]. Website: https://ginasthma.org/severeasthma/ . In the earliest studies, mepolizumab was administered to a nonspecific population of patients with moderate, persistent asthma and variable levels of eosinophilia. Although no significant clinical results have been reported for asthma control from the earliest studies, its effectiveness in eosinophilic asthma is now well documented . Clinical trials have shown that mepolizumab displays high clinical efficacy by reducing exacerbation rates and daily oral corticosteroid (OCS) intake and enhancing quality of life. It is apparent that careful selection of patients is required in order to achieve the best results [3–5]. Among the several different asthma phenotypes, eosinophilic inflammation occurs in more than 50% of patients with either atopic or nonatopic asthma. High eosinophil counts, in both peripheral blood and the airways, are associated with recurrent disease exacerbations and severe airflow limitation [6]. Adult-onset eosinophilic asthma is increasingly recognized as one of the most severe asthma phenotypes [7–10]. Another characteristic feature of adult-onset eosinophilic asthma is comorbid chronic rhinosinusitis with nasal polyps (CRSwNP), a feature known for many years and, in some cases, linked with aspirin and other nonsteroidal antiinflammatory drug hypersensitivities [11–13]. Concern regarding nonallergic, severe eosinophilic asthma and its associated comorbidities has increased with the awareness that this subtype is characterized by high levels of the proeosinophilic cytokine IL-5, which is mainly produced by a unique population of type 2 innate lymphoid cells [14]. We endorse an in-house classification of asthma phenotypes and a decision-making protocol which involves the first choice monoclonal antibodies (mAbs) and potential alternative mAbs in severe asthma phenotypes [11]. We prefer anti-IL5 treatment, especially in the OCS-dependent severe eosinophilic asthma with CRSwNP (SEA-CRSwNP) phenotype. To the best of our knowledge, no study evaluating the real-life efficacy of mepolizumab treatment in patients with OCS-dependent SEA-CRSwNP phenotype has been carried out to date. According to the literature, data regarding postmarketing studies that have evaluated the effects of mepolizumab in real-world settings are scarce . Thus, this short-term single-center study was carried out in a more specific group of asthmatic patients treated with add-on biologic therapy with mepolizumab.Adult patients (>18 years) with OCS-dependent SEA-CRSwNP phenotype who were treated with mepolizumab between 2018 and 2019 were retrospectively evaluated. All patients were treated with high-dose, extrafine inhaled glucocorticoids (ICS) and a long-acting β2-agonist, along with a second controller montelukast in addition to regular OCS therapy at least 6 months before mepolizumab treatment. Indications for treatment with mepolizumab were approved on the basis of the Turkish Social Security Institution Health Application Communique, according to which, mepolizumab can be administered to patients with severe eosinophilic asthma who have: a) blood eosinophil count ≥300 cells/µL  and b) controlled or uncontrolled asthma treated with regular systemic steroids for at least 6 months and/or uncontrolled asthma (approximately two attacks per year requiring systemic corticosteroids for at least 3 days) despite use of a high combination dosage of ICS  and long-acting inhaled β2 agonist for at least one year Turkish Social Security Institution Health Application Communique [online]. Website: http://www.mevzuat.gov.tr/Metin.Aspx?MevzuatKod=9.5.17229&MevzuatIliski=0&sourceXmlSearch= . However, we have reduced the criterion, and mepolizumab was employed only in patients with GINA step 5 OCS-dependent SEA-CRSwNP . Mepolizumab was administered subcutaneously at a dose of 100 mg every four weeks for at least 12 weeks. Mepolizumab was continued if there was a clinical response at week 12. Those patients who completed at least 24 weeks of treatment were included in the study.Throughout the study period parameters including ACT score, blood eosinophil count, and FEV1 were measured at baseline, week 12, and week 24 after the first injection of mepolizumab. In addition, the number of asthma exacerbations (exacerbations occurring within the previous 24 weeks) and daily intake of OCS  were also recorded, respectively, at baseline and at week 24 of mepolizumab treatment. The scores for severity of nasal polyposis on the numerical analog scale (NAS) were assessed by asking patients to indicate the severity of their CRSwNP using a 0 to 10 scale  and the irritability of the following symptoms in general: rhinorrhea, mucus in the throat, nasal blockage, and loss of smell. The NAS score for the loss of smell was evaluated separately. Changes in the severity of nasal polyposis according to NAS score from baseline to the 24th week were also recorded. In addition, the side effects of mepolizumab were assessed for all patients.Drug adherence; inhaler technique; and comorbidities including allergic rhinitis, chronic rhinosinusitis/nasal polyps, gastroesophageal reflux, obstructive sleep apnea syndrome; and trigger factors like allergens, smoking, occupational allergens and/or irritants, ACE inhibitors, and nonspecific beta-blockers which interfered with disease control were investigated for all patients. Measures taken for these comorbidities and triggers, drug compliance, and techniques were analyzed. All patients under GINA step 5 treatment with controlled or uncontrolled asthma were considered to be severely asthmatic, and OCS-dependent SEA-CRSwNP was identified as the appropriate asthma phenotype for mepolizumab treatment in these patients.Since this asthma phenotype is associated with high eosinophilia, other diseases associated with higher peripheral eosinophilia and accompanied by asthma or asthma-like symptoms, such as eosinophilic granulomatosis with polyangiitis, allergic bronchopulmonary aspergillosis, chronic eosinophilic pneumonia, hypereosinophilic syndrome, Loffler’s syndrome, and pulmonary involvement of connective tissue diseases were ruled out.An exacerbation was defined as a worsening of asthma symptoms requiring OCS at least three days a week or an increase in the OCS dose. All CRS subjects met the criteria for CRS as defined by the American Academy of Otolaryngology–Head and Neck Surgery Chronic Rhinosinusitis Task Force. The diagnosis of CRS was based on the presence of clinical symptoms  for more than 12 weeks in addition to the objective evidence of chronic inflammatory disease on sinus CT imaging or nasal endoscopy. Sinonasal involvement was assessed by paranasal sinus computerized tomography (PNCT) and nasal endoscopy [17]. CRSwNP is characterized by the occurrence for more than 12 weeks of symptoms such as nasal discharge, stuffiness, facial pressure or pain, dysfunction or loss of the sense of smell, and cough from postnasal drip and by the polypoid inflammation filling the nasal airway in the PNCT [18]. In NAS, the response to “How troublesome are your CRSwNP symptoms?” is rated from 0 to 10 .Based on placebo-controlled, phase III studies, recommendations published by the National Institute for Health and Care Excellence (NICE) define the reduction of the exacerbation rate by at least 50%, or a clinically significant reduced dose of continuous OCS, as adequate response criteria  Mepolizumab for treating severe refractory eosinophilic asthma. National Institute for Health and Care Excellence (NICE) [online]. Website: https://www.nice.org.uk/guidance/ta431 . Aspergillus specific IgE, antinuclear antibody, urinalysis, liver and renal function tests, parasite stool examination, creatine kinase, pulmonary function tests , thorax computed tomography, PNCT, and electromyography if the patients exhibited symptoms of peripheral neuropathy. The authors also requested advanced laboratory tests for eosinophilic granulomatosis with polyangiitis (EGPA), hypereosinophilic syndrome (HES), and lymphoreticular malignancy among patients who had >10% blood eosinophils . The tests included blood eosinophilia (reference range: <200 cells/mm3), C-reactive protein , erythrocyte sedimentation rate , total immunoglobulin E , The dose of methylprednisone was reduced every 4 weeks according to a predefined schedule (Table 1) if the patient had not had an exacerbation with a decrease in ACT score. In patients who were receiving a daily dose of 8 mg or more of methylprednisone at baseline, the dose of the drug was not reduced to zero without consulting endocrinology due to concern regarding withdrawal effects.All patients under follow-up at our asthma outpatient clinic provided written informed consent. Ethics approval was obtained from the Erciyes University ethics committee .Data were entered into SPSS software version 17.0 , and analyses were made using the same program. All continuous variables were presented as mean ± standard deviation (SD) due to the small sample size. For all nonparametric variables between and within groups, comparisons were made using the Mann–Whitney U-test and Wilcoxon test, respectively. P values <0.05 were considered significant in all analyses.Data from 16 patients with OCS-dependent SEA-CRSwNP who underwent treatment with mepolizumab were analyzed. All patients were classified as step 5 according to the Global Initiative for Asthma (GINA)1 and had uncontrolled asthma despite maximal therapy. The mean age of the patients was 48.6 ± 11.9 years. The mean duration of the disease and the duration of regular OCS use prior to the initiation of mepolizumab treatment were 12.9 ± 6.6 years and 5.1 ± 2.6 years, respectively. Females accounted for 81% of all the study subjects. Of the 16 patients, 14 (88%) were nonsmokers. Patient characteristics are shown in Table 2. Disease control was evaluated using ACT at baseline with a mean value of 18.2 ± 5.5. In addition, a lung function test prior to mepolizumab treatment evidenced a mean FEV1 of 81% ± 30. All patients were receiving daily OCS therapy before mepolizumab treatment (a mean dose of 8.9 ± 5.2 mg of methyl-prednisolone). The mean eosinophil count at baseline was 561 ± 591 cells/µL. The mean eosinophil percentage was 5.3 ± 5.7%. The number of asthma exacerbations as well as the mean eosinophil counts decreased, and ACT results improved under daily OCS treatment before initiation of mepolizumab, in comparison to the time before regular OCS intake. There was also a nonsignificant increase in FEV1 values after starting regular daily OCS (Table 3). Mepolizumab treatment was found to be very effective in all study subjects. With regard to adverse events, only one patient showed side effects including arthralgia, and malaise occurred on the day after administration of the first two doses. The patient also had a third reaction on the day following administration, and fever, nausea, and vomiting were reported. Mepolizumab was discontinued, and this patient was excluded from the study. The patient was symptom-free during the follow-up period. Further comparisons between study subjects included only the remaining 15 patients. When comparing the change in blood eosinophil counts, daily OCS doses, and ACT scores at baseline and week 12 of mepolizumab treatment, a marked decrease in peripheral eosinophil counts  and an increase in ACT scores  were observed. The OCS dose was decreased in all of the patients; the daily OCS dosage was completely withdrawn in 3 (20%) patients, and 15 out of 15 patients (100%) were classified as treatment responders at week 12. No marked changes in FEV1 values were observed at this time point (80 ± 30.7% vs. 84 ± 26%) (Table 4).After 24 weeks of mepolizumab treatment, the decrease in blood eosinophil counts  and improvement in ACT scores  continued. The OCS dose was additionally reduced in 9 (60%) patients when compared to 12th week results, and OCS was completely withdrawn in 6 of 15 (40%) patients at week 24. A significant decrease in the 24-week exacerbation rates of pre- and postmepolizumab treatment was observed  (Table 4). Despite the decrease in daily OCS dosages, improvement in all parameters was seen at week 24 under mepolizumab treatment. Comparison of OCS dosage, number of asthma exacerbations, ACT, FEV1, and blood eosinophils at the beginning of mepolizumab treatment and at the 12th and 24th weeks is shown in Figure.During the 6-month mepolizumab treatment, demographics, baseline blood eosinophil counts, exacerbation rates, FEV1 values, and ACT scores were found to be similar regardless of whether OCS was completely withdrawn in the patients. On a 10 point NAS, the severity of CRSwNP symptoms decreased from 5.6 ± 4.4 points to 3.2 ± 3.2 points (P = 0.021), and the severity of loss of smell decreased from 4 ± 5.1 points to 2.4 ± 4.2 points (P > 0.05) after 24 weeks of mepolizumab treatment.This is the first real-life study evaluating the short-term (24 weeks) efficacy of mepolizumab treatment in a specific subphenotype of asthma. Our study showed that in patients with OCS-dependent SEA-CRSwNP phenotype, the OCS dose was decreased in all patients, and 40% of them no longer required OCS after 24 weeks of mepolizumab treatment. In addition to the decrease in daily OCS doses, a decrease in the frequency of exacerbations and an increase in ACT scores were observed. However, there were no significant differences in FEV1 values between week 24 and baseline. Both severe eosinophilic asthma and nasal polyposis are characterized by marked local eosinophilic inflammation [19]. IL-5 appears to play a key role in the pathogenesis of CRSwNP and eosinophilic asthma [19–23]. In addition, suggestions were made in the severe asthma guidelines of GINA regarding preferred biologics for the type-2 high asthma phenotype, and it was emphasized that factors determining the response to treatment should be taken into consideration .1 Therapy should be initiated with anti-IL5/anti-IL5R mAbs in patients with severe uncontrolled asthma who have a blood eosinophil count of ≥300/µL. Factors that may predict a good response to anti-IL5/IL5R biologics are defined as follows: (a) higher blood eosinophil count (strongly predictive), (b) more frequent severe exacerbations during the previous year (strongly predictive), (c) adult-onset asthma, (d) nasal polyposis, and e) maintenance with OCS at baseline. The efficacy of mepolizumab has been reported, separately, in OCS-dependent asthma, severe eosinophilic asthma, and CRSwNP . Therefore, the OCS-dependent SEA-CRSwNP phenotype appears to be the most appropriate phenotype for mepolizumab. In our study, all study subjects presented with a history of frequent exacerbations, higher eosinophil levels, OCS-dependency, and CRSwNP. For all of these reasons, we decided to initiate mepolizumab therapy for this subphenotype of asthma.One of the main endpoints of all biologics is the withdrawal of the use of OCS in severe asthma [27]. In this real-life study, all patients were prescribed with daily OCS . The OCS dose was successfully reduced in all patients, and 40% patients completely discontinued OCS after 24 weeks of mepolizumab therapy. Compared to baseline, at week 24 a significant decrease in the frequency of exacerbations and an increase in ACT scores were observed, in addition to the decrease in daily OCS doses. In the large, placebo-controlled trials, treatment with mepolizumab significantly reduced exacerbation rates and daily OCS doses [3–5]. The Steroid Reduction with Mepolizumab Study (SIRIUS) demonstrated that mepolizumab, in addition to decreasing asthma exacerbations and improving the quality of life, was also capable of significantly diminishing daily OCS intake  [3]. However, the OCS reduction (100%) and complete discontinuation (40%) rates in our study were much higher at week 24. This positive outcome could be due to the fact that the add-on therapy with mepolizumab was initiated in a specific eosinophilic asthma subphenotype that may derive a higher potential benefit from mepolizumab therapy. In another real-life study supporting the results of the present study, Pelaia et al. established that OCS could be reduced in all patients with significant reduction in exacerbations after 24 weeks of initiation of mepolizumab therapy in patients with OCS-dependent severe eosinophilic asthma [28]. The common feature of these studies is that all of the patients in both studies were OCS-dependent, and the initial eosinophil counts during OCS therapy were higher . However, nasal polyp ratios were not reported in this study. These two real-life studies have shown that the early results from mepolizumab are quite good, especially in patients with high blood eosinophilia and OCS dependency. Prioritizing these patients will be more meaningful in terms of treatment success among patients indicated for mepolizumab therapy.In the present study, the clinically relevant improvement in asthma control elicited by mepolizumab was paralleled by a prominent and sustained decrease in blood eosinophil counts. This finding confirms the results of several large-cohort and real-life studies . Undeniably, blood eosinophils are now considered a reliable biomarker for predicting and assessing the therapeutic efficacy of mepolizumab in severe eosinophilic asthma . The initial high eosinophil levels in patients in the present study were associated with a good response to mepolizumab. An increased blood eosinophil count (300 or 400 cells/μL) in asthmatic patients is associated with an increased risk of exacerbations . Therefore, one of the goals of mepolizumab treatment that should be emphasized is keeping blood eosinophils below these cut-off values in severe eosinophilic asthma [33]. In the present study, the remarkable decrease in blood eosinophil levels evoked by mepolizumab was paralleled by a reduction in the rate of asthma exacerbation and an improvement in asthma control. Contradictory reports on the effects of mepolizumab on FEV1 have been published. Some studies have indicated a modest increase in FEV1 with mepolizumab therapy , while others claim that FEV1 did not improve with the administration of mepolizumab . In the present study, no significant change in FEV1 values was noted after 12 or 24 weeks of mepolizumab treatment, compared to baseline. Yet, the important point here is that there was no deterioration in pretreatment FEV1 values, despite dosage reduction or discontinuation of OCS.The SIRIUS study, which included OCS-dependent eosinophilic asthma patients, reported the rate of nasal polyps to be 23%. In another study which included OCS-dependent eosinophilic asthma patients, this rate was 30% . However, the effect of mepolizumab on nasal polyps was not evaluated in these studies. In a randomized, double-blind, placebo-controlled study recruiting adult patients with recurrent nasal polyposis requiring surgery, patients received 750 mg intravenous mepolizumab. A significant improvement in all individual VAS symptom scores was observed in the mepolizumab group, in comparison to placebo [25]. In the present study, a significant improvement in NAS scores that assessed general CRSwNP symptoms at 24 weeks  was observed. However, there was no significant improvement when NAS was evaluated only for smell loss. We speculate that other pathways may be more dominant in the development of eosinophilic inflammation in the nasal polyposis in these subgroups (IL4/IL13 dominant type-2 inflammation) or that the dose of mepolizumab may be insufficient to reduce eosinophilic inflammation at the tissue level . The major limitations of the present study were the inclusion of a small number of patients and its retrospective design. We could not recruit a high number of patients as the study was conducted in a very specific patient cohort. Although we obtained good clinical results from mepolizumab therapy for a specific asthma subphenotype, a large-scale series is still necessary for robust results. Another limitation was that we only compared asthma exacerbation rates before and after 6 months of mepolizumab initiation. As exacerbations mainly occur during winter, assessment for at least 12 months is necessary to properly reflect the exacerbation rates [35]. However, patients who received mepolizumab for at least 6 months passed through the autumn of 2018 and winter and spring of 2019. In other words, the patients were treated with mepolizumab during the period in which the risk of asthma exacerbation is high, and the results were compared 6 months before mepolizumab initiation. The authors speculate that decreases in exacerbations during a 12-month period may be even greater.In conclusion, the authors found that subcutaneous mepolizumab administration significantly decreased blood eosinophil levels, asthma exacerbations, and daily OCS doses. These excellent therapeutic effects were associated with a marked improvement in symptoms as well as a very good short-term safety profile for the drug and its tolerability in this single-center study. Therefore, the availability of mepolizumab in daily medical practice undoubtedly represents a valuable advancement in the management of patients with OCS-dependent SEA-CRSwNP. The ideal scenario for therapeutic intervention would be specific selection criteria with a clear prediction of clinical benefits. This small study was clinically directed and highlighted the importance of patient selection for investigating the use of mepolizumab in severe asthma. Nevertheless, further extended real-life investigations are required to confirm the results obtained in our study.


The study included 226 people: 20 healthy volunteers, for whom normative values of assessed parameters were determined, and 206 patients. Reproducibility of results obtained by two independent operators, specificity, correlation with estimated GFR (eGFR), and Cohen’s kappa were used to evaluate reliability of assessed parameters. Normative values were as follows: K ≥ 1.6, MTT ≤ 250 s, and PTT ≤ 225 s. Reproducibility of determination of K (rs = 0.99) and MTT (rs = 0.98) was significantly higher than that of PTT (rs = 0.95) (p = 0.001). Specificity was 100% for K, 81% for MTT, and 91% for PTT. Correlation of eGFR with K (rs = 0.89) was significantly higher than with PTT (rs = 0.53) and with split function (SF) (rs = 0.66) (p < 0.0001). Cohen’s kappa was κ = 0.89 for K, κ = 0.88 for MTT, and κ = 0.77 for PTT. In a group of patients where standard DRS parameters are unreliable , the use of K (the most effective among assessed parameters) changed the classification of 23/79 kidneys (29%). K enables reproducible assessment of absolute, individual kidney function without modifying routine DRS protocol. Diagnostic value of MTT and PTT is limited.This study evaluates the usefulness of parameters allowing assessment of renal function in absolute values in dynamic renal scintigraphy (DRS) with  MAX), and time of renographic curve reaching to half the peak value (T1/2) [One of the primary indications for dynamic renal scintigraphy (DRS) is the diagnosis of obstructive uro/nephropathy. Typically, this examination is assessed qualitatively  and quantitatively, using parameters such as differential renal function (split function—SF), time of the peak of renographic curve  clearance for the determination of glomerular filtration rate (GFR) or 99mTc-mercaptoacetyltriglycine (99mTc-MAG3) clearance for the determination of tubular extraction rate (TER) based on DRS study (camera-based clearance). These procedures were developed by Schlegel et al. for 131I-orthoidohippurate (131I-OIH) [99mTc-DTPA [99mTc-MAG3. These methods are more accessible than the most accurate and precise radioisotope technique for determining GFR based on a multisample clearance of 99mTc-DTPA. However, their accuracy is being questioned and they require taking into account additional information, such as precise measurement of the activity of radiopharmaceutical administered to the patient or measuring depth of kidneys in order to correct the attenuation of radiation by tissues located between the camera detector and the kidney [99mTc-ethylenedicysteine (99mTc-EC) camera-based clearance was not developed yet.Processing units of modern gamma cameras usually have software allowing determination of 31I-OIH)  and by Ge kidney . PerhapsThe aim of this study was to assess diagnostic potential of three different parameters allowing the assessment of renal function in DRS in absolute values, generated using an original software developed in our Department. These are as follows: an original parameter-uptake constant (K), proportional to the clearance function of a kidney, as well as transit time of radiopharmaceutical through the whole kidney (mean transit time—MTT) and its cortex .The study included a total of 226 people aged 18–84 years (mean: 53), divided into two groups.Group I—normative, consisted of 20 healthy volunteers (40 kidneys). In this group, ultrasound of the urinary system was performed and serum levels of urea and creatinine were determined on the same day as the DRS study. The criteria for inclusion into group I were as follows: no history of previous or ongoing urinary system diseases or other diseases that could lead to impaired renal function ; the ultrasound examination of the kidneys showed no features of congenital anomalies, urolithiasis, hydronephrosis, scars or other focal lesions (including cysts), and the results of biochemical tests (serum urea and creatinine levels as well as estimated GFR (eGFR) calculated from Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula) were within normal range.IIA—62 patients (124 kidneys) with low probability of obstructive uro/nephropathy, whose renal function was assessed as normal based on routine DRS evaluationIIB—92 patients , whose medical records had up-to-date serum creatinine results (measured no more than 2 weeks before DRS)IIC—61 patients (79 kidneys) with no or only trace function of one of the kidneys (SF < 5%) or bilateral obstructive uropathy, where SF values are unreliable .Group II consisted of 206 adult patients, who were routinely examined at our Department in years 2015–2019, selected retrospectively based on archived scintigraphic data. Patients with uncontrolled diabetes or hypertension and patients with suspicion of renovascular hypertension referred for DRS with Captopril test were excluded from the study. This group included 3 subgroups:99mTc-EC. Field of view covered both the kidneys and the heart. Image acquisition was performed in posterior view, using 128 × 128 matrix. In patients whose renographic curves in the base study remained above 30% of the peak value up to the 20th minute of the study, DRS was extended by a diuretic test performed according to the “F + 20” protocol and image acquisition was continued for additional 10 min [All volunteers and patients underwent DRS performed according to the routine protocol used in our Department. All subjects drank 0.5 L of water approximately 30 min before the examination and urinated just before radiopharmaceutical injection . DRS wasMAX, and T1/2. In group II, archived medical records were also analyzed, and patients with current serum creatinine level results were qualified to subgroup IIB.Initial evaluation of all studies, performed by the same nuclear medicine specialist, consisted of visual assessment of sequential, 2 min scintigraphic images and analysis of standard quantitative parameters—SF, TMAX > 6 min, and presence of uptake defects in the peripheral part of the kidney in the visual assessment of sequential images of uptake phase (between 2nd and 3rd minute of the study), performed by two experienced physicians (determined by consensus). At least two criteria are required, because one criterion can be present in normal kidneys . On the basis of these criteria, patients were classified into subgroups IIA and IIC (independent from subgroup IIB).Due to the retrospective character of the study and sometimes incomplete clinical data in archived documentation, further classification of kidneys was based only on the standard evaluation of scintigraphic data. Kidneys with features of obstructive uropathy or nephropathy were distinguished based on the following criteria. No decrease of the renographic curve (“cumulative curve”) or its decrease by less than 50% of the peak value after the diuretic test, in the absence of the features of obstructive nephropathy described below, was classified as a feature of obstructive uropathy. Kidneys meeting at least two of the following three criteria were classified as nephropathic: SF < 42%, TFurther analysis of scintigraphic data (post-processing) of each volunteer and patient was carried out by the same nuclear medicine specialist with an open-source program—ImageJ—using an original plug-in developed in our Department. The program automatically summed scintigraphic images in the posterior view from the base DRS study (without the diuretic test) to 60 20 s images. Each of the images was then smoothed twice with a 3 × 3 mask linear filter [On the first summed image, a region of interest (ROI) covering the left ventricle with a large margin of background was determined. Then, on summed images from the uptake phase, whole kidney ROIs were generated semiautomatically—the program plotted an area with activity higher than the threshold value, which was set manually so that the contoured area covered the whole kidney. In the next step, 3-pixel-wide ROIs of the cortical layer (parenchyma) and background were automatically generated for each of the kidneys, based on the previously plotted whole kidney ROIs .Due to poor visibility of some small and/or nephropathic kidneys on 20 s images, in some cases it was necessary to plot the whole kidney ROI by hand and manually correct automatically determined parenchymal and background ROIs.H(t), kidneys R(t), and their parenchyma Rc(t). The cardiac curve was generated from 20 pixels, with the highest activity selected automatically from the large heart ROI. Remaining curves were determined as a sum of counts in a given ROI to time.These ROIs were then used to generate time–activity curves showing the concentration of the radiopharmaceutical in the heart This post-processing was performed again, using the same procedure, by another, independent specialist for patients from subgroup IIB.R(t)—background-corrected renographic curve, H(t)—background-corrected cardiac curve, F—blood background subtraction factor, and K—uptake constant (fraction of blood activity taken up by the kidney per time unit).Uptake constant K was determined based on the Rutland space ,7. It isK represents clearance function of the kidney, although only in proportional values, hence it is a unitless parameter.According to Rutland’s theory, R(t), parenchymal Rc(t), and cardiac H(t) curves were used for deconvolution analysis. Each of the R(t) and Rc(t) curves were corrected by subtracting the background activity . MTT and PTT were determined from the curves plotted in this way, using matrix deconvolution technique.Renal The data were described by means of simple descriptive statistics of location and dispersion . Normality of distributions was verified by Shapiro–Wilk’s test. Normative values for analyzed parameters with confirmed normal distributions were assessed as mean ± 2 or 3 standard deviations in the group of healthy adults. Distributions of selected quantitative parameters, some of which were not normally distributed, were compared between different groups by nonparametric Mann–Whitney U test.2. Agreement of results was analyzed using Cohen’s kappa coefficient.Diagnostic performance of parameters of interest was evaluated by sensitivity, specificity, and inter-observer reproducibility. Relations between parameters of interest were assessed using nonparametric Spearman’s rank correlation coefficient and coefficient of determination rp ≤ 0.05.In all analyses, statistical significance was considered achieved for a value of Calculations were performed by means of Statistica v13.1 and LibreOffice v6.3 software.n = 173 kidneys) were determined by two independent operators. Correlation of results obtained by both operators was used as a measure of reproducibility of determination of each parameter. The agreement of results obtained by two independent operators  was also assessed.Parameters K, MTT, and PTT in subgroup IIB . Differences between mean values of assessed parameters in the above-mentioned groups were also analyzed.Next, in group IIB, correlation of K, PTT, and SF with estimated GFR of each kidney (single-kidney eGFR—SKeGFR) was assessed. eGFR was calculated based on the current serum creatinine level using CKD-EPI formula, recommended by the guidelines of the international work group—Kidney Disease: Improving Global Outcomes (KDIGO) . Then, iMTT was not compared with SKeGFR, because its value is influenced by both renal uptake function and the efficiency of urine outflow to the bladder. Instead, diagnostic efficacy of MTT in the diagnosis of obstructive uropathy was characterized by basic parameters of the diagnostic test—sensitivity and specificity, with diuretic test used as a reference method.In a group of patients with single functioning kidney or features of bilateral uropathy (IIC), a comparative differential analysis of obstructive uro/nephropathy was performed based on standard and assessed DRS parameters.n = 173 kidneys) were analyzed by two independent operators. Correlation coefficients and r2, used as measures of the reproducibility of determination of assessed parameters, were as follows: for K, rs = 0.99 (r2 = 0.98), for MTT, rs = 0.98 (r2 = 0.96), and for PTT, rs = 0.95 (r2 = 0.90) (p < 0.000001). Correlations of K and MTT were significantly stronger than those of PTT (p = 0.001).In order to assess reproducibility of K, MTT, and PTT, susceptibility of the method of their determination to errors resulting from the subjective factor was analyzed. Scintigraphic data of patients from subgroup IIB  of the kidneys and PTT in 113/124 (91%) of cases. .Based on these normative limits, results obtained by each operator in subgroup IIB were categorized as within normal range or below/above norm. The agreement of the results is presented in p = 0.38), while in case of MTT and PTT, the differences were at the edge of statistical significance . Differences in mean values between groups I and IIC were statistically significant for all assessed parameters—in case of K they were lower (p = 0.0002), while in case of MTT and PTT they were higher (p < 0.00001 in both cases). Group IIB was not included in this comparison, since it includes a wide range of patients both with normal and impaired kidney function.The distribution of values of assessed parameters in groups I, IIA, and IIC is presented in s = 0.89, r2 = 0.79 (p < 0.000001), rs = −0.53, r2 = 0.24 (p < 0.000001), and rs = 0.66, r2 = 0.42 (p < 0.000001), respectively. These correlations are presented in Values of K, PTT, and SF showed a significant correlation with SKeGFR in group IIB, r2 were significantly stronger than in the case of PTT (p < 0.0001) and SF (p < 0.0001).Although all correlations were statistically significant, correlation of uptake constant K with SKeGFR and its correlation coefficient rSince the reliability of K was considered to be higher than that of PTT , clinical utility of this parameter in differential diagnosis of obstructive uro/nephropathy was preliminarily assessed.Based on standard DRS criteria, described in methodology, in group IIC, no features of obstructive uro/nephropathy were found in 36 kidneys, obstructive uropathy was diagnosed in 18 kidneys, while features of obstructive nephropathy were found in 25 kidneys. Application of K changed the qualification of 23 out of 79 kidneys (29%). In 14 cases (18%), it changed the classification from nephropathy to uropathy, while in 9 cases (11%), K value suggested nephropathy with no such features found in routine DRS evaluation .Out of 362 analyzed kidneys from group II, in 108 a cumulative curve was observed in base DRS study, which in 16 cases decreased below 50% of peak value after diuretic test. The remaining 92 cases showed features of obstructive uropathy.MTT values in group II ranged from 131 s to 639 s. Sensitivity of this parameter in the diagnosis of obstructive uropathy, in relation to the reference diuretic test, was 77% (71/92 kidneys), while its specificity was 76% (204/270 kidneys).Among all kidneys without features of obstructive uropathy, in 268 out of 270 (99%) MTT was below 430 s . In the group of 16 kidneys with a cumulative curve in base study, but without obstructive uropathy, its value in 15 cases (94%) also did not exceed 430 s (except for one of the above-mentioned kidneys with MTT = 453 s). In 92 kidneys with features of obstructive uropathy, MTT was above 430 s in 20 cases (22% of the kidneys). Distribution of the MTT values is shown in 99mTc-EC—the latest radiopharmaceutical introduced to the diagnostics of the urinary system.In clinical practice, DRS is the most commonly used radioisotope technique in the diagnosis of urinary system. Despite numerous advantages, however, it has some limitations that can be eliminated by supplementing the standard evaluation of the study with additional parameters that enable an assessment of renal function in absolute values. Techniques that allow such evaluation using only scintigraphic data obtained in DRS, without the need to collect blood or urine samples or perform other additional tests, are especially valuable. Several protocols have been developed to determine renal clearance of radiopharmaceuticals in this way, and some of them have been integrated into software of modern gamma cameras. However, these camera-based techniques require acquisition of additional—nonstandard—data, such as the exact activity of the injected radiopharmaceutical (taking into account activity remaining in the syringe after administration) or measurement of the depth of the kidneys. This requires modifications of the routine DRS protocol and extends and complicates the course of the entire study. Moreover, these protocols have not yet been adapted for DRS performed with Software developed in our Department allows determination of additional quantitative parameters describing kidney function in absolute values—original parameter, uptake constant (K), mean transit time (MTT), and parenchymal transit time (PTT)—without the need of any modifications to the routine DRS protocol.99mTc-EC was based on Rutland’s theory [99mTc-EC and thus to the effective renal plasma flow (ERPF) of a given kidney. In 1985 Rehling et al. proposed a method, similar to ours, of determining iptake index (UI)—a parameter proportional to 99mTcDTPA clearance (thus GFR of a given kidney). Rehling et al. proved that UI correlates very closely with GFR value (determined with the radioisotope method based on the multisample clearance of 99mTcDTPA)—rs = 0.97 [Our method of calculating K in DRS performed with s theory . Accordit)—appears three times, so it has a decisive influence on the value of K (which is the slope of the function described by this equation). In his work, Rehilng also emphasizes the important role of the vascular background chosen to generate the cardiac curve, necessary to calculate the UI. Therefore, susceptibility of the method of generating this curve to errors resulting from the subjective factor should be minimized. For this purpose, in our work cardiac curve was generated from the sum of counts in 20 pixels, with the highest values in the large ROI covering the area of the heart. Additionally, background-correction of renographic curves was applied in this study, using automatically generated background ROIs.It should be emphasized that in the Equation (1), which is the basis of this method, cardiac curve—H was significantly lower .s = 0.99, for MTT, rs = 0.98, and for PTT, rs = 0.95. Agreement of results  obtained by independent operators was also almost perfect for K (κ = 0.89) and MTT (κ = 0.88), and strong for PTT (κ = 0.77).Automation of ROI plotting and automatic selection of pixels used to generate the cardiac curve, used in the current study, limited the influence of the subjective factor on the determination of both PTT and MTT as well as K practically exclusively to the manual selection of the threshold for semiautomatic plotting of whole kidney ROIs. As a result of such automation, we achieved very high reproducibility of determination of assessed parameters, which was for K, rDue to high variability of MTT and PTT, some authors recommend setting the limits of their normative values as mean + 3 standard deviations  . FollowiSpecificity, assessed in an independent group of patients with no abnormalities in standard DRS evaluation, was used as a measure of reliability of normative values of assessed parameters. K achieved the highest specificity (100%). A slightly lower specificity of PTT (91%) may result from cases where an automatically determined cortical ROI covered fragments of calices. K is determined from the first minutes of the study, before the radiopharmaceutical reaches the pelvicalyceal system, so the above limitation does not apply to it. MTT showed a clear tendency to generate false-positive results (specificity 81%).Moreover, it should be noted that mean values of K in the normative group I and in the group of patients with normal DRS results (IIA) did not show statistically significant differences. However, in cases of MTT and PTT, the differences between mean values in these groups were at the edge of statistical significance.s = 0.89; r2 = 0.79). This correlation was significantly stronger than in cases of PTT vs. SKeGFR and SF vs. SKeGFR.The next step in the assessment of usefulness of K and PTT was a comparison of correlations of their values with single-kidney estimated GFR (SKeGFR), which describes renal efficiency, determined for each kidney using CKD-EPI formula . Obtained values of K strongly correlated with SKeGFR (rs = 0.97) is most likely the result of the limitations of our reference method, chosen due to the retrospective nature of our research  and slightly different renal biokinetics of 99mTc-EC compared to 99mTc-DTPA. Despite the above limitations, the correlation we obtained was satisfactorily strong, which confirms the validity of the main assumptions of this method.The fact that the correlation of K vs. SKeGFR is not as close as in the case of UI determined by Rehling , which may be a source of potential errors. Hence, the reproducibility of its determination by two independent operators and agreement between obtained results, although high, were nevertheless lower than in cases of K and MTT, even despite almost complete automation of the ROI plotting process. The results of the research carried out in this study clearly indicate that the original uptake constant K is a better and more practical parameter for the assessment of renal function in DRS.Relatively low specificity of MTT in addition to the long-established and well-documented high diagnostic efficacy of base DRS study with diuretic test in the diagnosis of obstructive uropathy undermines the usefulness of this parameter. In this study, however, it was observed that obstructive uropathy is practically always present when MTT values are exceeding 430 s. This can provide useful information in situations where a diuretic test is risky , or the test was nondiagnostic . In such cases, a cumulative renographic curve in base DRS study combined with MTT value above 430 s allows for the diagnosis of obstructive uropathy with about 95% probability, even without the diuretic test.99mTc-MAG3 .It is worth noting that all studies carried out in this paper concern adults. The topic to be considered in future research is the application of K parameter in children, after determining appropriate normative values. The developed method may also be adapted for DRS performed with Original parameter-uptake constant K extends the possibilities of DRS by reproducible, quantitative assessment of the individual function of each kidney in absolute values.Determination of K does not require modification of the routine DRS protocol. Thus, it can be obtained as part of the post-processing of scintigraphic data (after conducting a standard DRS), either routinely or only in the selected clinical situations.PTT has lower diagnostic potential than K, and the methodology of its determination is more complicated, which undermines its usefulness in the diagnosis of obstructive uro/nephropathy.MTT may be useful for detection of obstructive uropathy in the rare circumstances where a diuretic test is contraindicated or its results are unequivocal.


Diabetic kidney disease (DKD) is one of the leading causes of end stage renal disease. Despite recent therapies, mortality due to DKD and resources spent on healthcare are important problems. Thus, appropriate markers are needed to predict renal outcomes. Therefore, we investigated the role of peripheral perfusion as an indicator for renal events in patients with type 2 diabetes mellitus. This retrospective cohort study included 566 patients who were admitted to Matsushita Memorial Hospital in Osaka, Japan for type 2 diabetes mellitus. Peripheral perfusion was assessed using perfusion index (PI), which represents the level of circulation through peripheral tissues and was measured on each toe using a Masimo SET Radical-7  instrument. The duration of follow up was 3.0 years. The median age of patients was 70 years (IQR range: 61–77 years) and median PI value was 2.9% (IQR range: 1.8–4.8%). Multiple logistic regression analyses showed that PI (per 1% increase) was associated with an odds ratio of composite of end-stage renal disease (ESRD) and/or doubling of serum creatinine level; n = 40 (odds ratio 0.823 [95% CI: 0.680–0.970]), and composite of ESRD, doubling of serum creatinine level, and renal death and/or cardiovascular death; n = 44 (odds ratio 0.803 [95% CI: 0.665–0.944]). The factors which were statistically significant in univariate analysis and those known to be related factors for renal event were considered simultaneously as independent variables for multiple logistic regression analysis. PI can be a novel indicator for renal events in patients with type 2 diabetes mellitus. DKD develops in approximately 40% of patients with diabetes and is one of the leading causes of end-stage renal disease5. DKD has been reported to be associated with the progression of cardiovascular diseases and increased risk of cardiovascular mortality8. Total deaths due to cardiovascular diseases and infections are highly prevalent in patients with end-stage renal disease (ESRD)9.Despite the advances in renoprotective therapies, such as renin-angiotensin system blockade and sodium-glucose cotransporter-2 inhibitors10. Indeed, normoalbuminuric renal insufficiency is common in patients with type 2 diabetes mellitus11. Interstitial changes such as chronic renal hypoxia might precede glomerular disease without albuminuria, wherein, intrarenal arteriosclerosis might be a main factor for declining eGFR. Further, the peripheral perfusion index (PI) is the ratio of pulsatile blood flow to non-pulsatile blood flow in the monitored tissue and has been shown to reflect changes in peripheral perfusion13. We have recently demonstrated that a low PI value indicates atherosclerosis in clinical care setting14 and diabetic kidney disease in the patients with type 2 disease in the cross sectional study15. However, the association of diminished PI with renal events in patients with type 2 diabetes mellitus has not been reported. Therefore, we aimed to clarify whether PI might be an indicator for renal events including decline in eGFR, cardiovascular death, and progression of albuminuria, in patients with type 2 diabetes mellitus.The progression of DKD involves glomerular hyperfiltration, albuminuria, decline in estimated glomerular filtration rate (eGFR), and ESRD. Patients with type 2 diabetes mellitus present heterogeneous process of DKDThe characteristics of 566 patients enrolled in this study are shown in Table 2. Multiple logistic regression analyses showed that PI (per 1% increase) was associated with an odds ratio of composite of ESRD and/or doubling of serum creatinine level in all patients as well as in patients with eGFR ≥ 60 ml/min/1.73 m2 (odds ratio 0.823 [95% CI: 0.680–0.970] or 0.598 [95% CI: 0.363–0.871]), and composite of ESRD, doubling of serum creatinine level, and renal death and/or cardiovascular death (odds ratio 0.803 [95% CI: 0.665–0.944] or 0.662 [95% CI: 0.458–0.891]). PI value was associated with an odds ratio of progression of albuminuria only in crude model. Moreover, multiple logistic regression analyses (Model 3) in all patients showed that baseline serum creatinine level was associated with an odds ratio of composite of ESRD and/or doubling of serum creatinine level (odds ratio 2.228 [95% CI: 0.987–5.023]), logarithm of baseline UACR was associated with an odds ratio of composite of ESRD, doubling of serum creatinine level, and renal death and/or cardiovascular death (odds ratio 1.678 [95% CI: 1.074–2.642]), and duration of diabetes, logarithm of baseline UACR or history of cardiovascular disease was associated with an odds ratio of progression of albuminuria , respectively. Multiple logistic regression analyses (Model 3) in patients with eGFR ≥ 60 ml/min/1.73 m2 showed that BMI was associated with an odds ratio of composite of ESRD and/or doubling of serum creatinine level (odds ratio 1.169 [95% CI: 1.033–1.330]) and duration of diabetes, baseline serum creatinine level (per 0.1 mg/dl increase), logarithm of baseline UACR or history of cardiovascular disease was associated with an odds ratio of progression of albuminuria , respectively.Table Net reclassification index (NRI) and integrated discrimination improvement (IDI) was derived from logistic regression models (Model 3) with and without logarithm of baseline UACR. Both NRI and IDI suggested that including baseline UACR in Model 3 results in significant improvement in performance. The NRI or IDI was estimated at 0.484 (95% CI: 0.176–0.792), 0.567 (95% CI: 0.210–0.803) and 0.167 (95% CI: −0.012–0.345) or 0.010 (95% CI: 0.0008–0.019), 0.017 (95% CI: 0.004–0.030) and 0.013 (95% CI: 0.003–0.022) in analyses for composite of ESRD and/or doubling of serum creatinine level, composite of end-stage renal disease, doubling of serum creatinine level, renal death and/or cardiovascular death and progression of albuminuria in all patients, respectively.The major finding of our study is that PI, which represents peripheral perfusion, can be a novel indicator for renal events such as composite of ESRD, doubling of serum creatinine level, renal death and/or cardiovascular death in patients with type 2 diabetes mellitus.2 or doubling of the blood creatinine level at a median duration of 15 years after diagnosis15. Patients with ESRD had high prevalence of mortality due to cardiovascular diseases and infections. The overall death rate in patients with ESRD, having creatinine levels >2 mg/dL or those receiving kidney replacement therapy was nearly 20% per year9. In spite of the development of recent therapies, large residual risks of renal events remain. Therefore, an easy and useful tool is required to improve renal outcomes in patients with type 2 diabetes mellitus. The present study suggests that PI could be a useful tool to indicate renal events in patients with type 2 diabetes mellitus.The United Kingdom Prospective Diabetes Study showed that 40% patients developed albuminuria, while 30% patients had eGFR < 60 ml/min/1.73 m15. Low PI value was more associated with eGFR < 60 mL/min per 1.73 m2 than albuminuria, which is consistent with our results. This can be explained by the mechanism and structural changes during decline of eGFR and progression of albuminuria. The initial structural change during DKD is thickening of glomerular basement membrane along with capillary and tubular basement membrane thickening18. Further, glomerular changes include loss of endothelial fenestrations, mesangial matrix expansion, and loss of podocytes17. It has been reported that albuminuria is correlated with mesangial expansion3, which is a marker for glomerular disease and an early clinical indicator of DKD. Diabetic control has been considered as an important factor for prevention of albuminuria16. Previous studies have shown high prevalence of patients with type 2 diabetes mellitus having reduced eGFR without albuminuria. Yokoyama et al.19 reported that proportion of patients with normoalbuminuria was 52% among patients with eGFR < 60 ml/min/1.73 m2. Chronic hypoxia in tubulointerstitium can explain the decline in eGFR without the presence of albuminuria. Chronic hypoxia in tubulointerstitium results in advanced tubulointerstitial lesions besides minor diabetic glomerular lesions21. Some mechanisms can explain development of chronic hypoxia in tubulointerstitium. One of the mechanisms is diminished microcirculation, such as distortion and loss of peritubular capillaries due to intrarenal arteriosclerosis26. These changes may induce a vicious cycle of regional hypoxia and decline in eGFR. Taken together, decrease in eGFR in patients without the presence of albuminuria is mainly associated with atherosclerosis. We have recently reported that low value of PI was strongly correlated with atherosclerosis14. Low value of PI can denote low peripheral perfusion resulting from atherosclerosis. Therefore, PI can be associated with decline in eGFR and not with progression of albuminuria. We have also reported that PI was associated with ankle brachial index14. Lower ankle brachial index has been associated with cardiovascular mortality27. Therefore, PI may be strongly associated with composite renal event including cardiovascular death.In our study, PI was strongly associated with decline in eGFR, composite of ESRD and/or doubling of serum creatinine level. However, multiple regression analysis suggested that PI was not associated with progression of albuminuria. Recently, we have reported the association PI and diabetic kidney disease in the cross sectional studyp = 0.0003). We postulated that PI was also affected by nutrition status. We supposed that PI could be affected by atherosclerosis. However, PI is not correlated with systolic blood pressure, which is consisted with recent report14. This could be explained by the effect of some factors such as duration of hypertension or the effect of antihypertensive medications.In our study, PI is directly correlated with total cholesterol and triglycerides. Moreover, PI is negatively correlated with age and positively correlated with BMI and serum albumin : eGFR = 194 × Cre−1.094 × age−0.287 (ml/min/1.73 m2). For women, the value of eGFR was multiplied by a correction factor of 0.739. ESRD was defined by the initiation of dialysis or eGFR < 15 ml/min/1.73 m2. The history of cardiovascular disease was defined as the patients with the history of myocardial infarction, heart failure, or stroke. Renal death was defined as death with a proximate renal cause. Death due to myocardial infarction, heart failure, or stroke was defined as cardiovascular death. Urine albumin-to-creatinine ratio (UACR) was determined using early morning urine. UACR was measured using an immunoturbidimetric assay. Microalbuminuria was defined by UACR 30 to < 300 mg/g Cr, whereas, macroalbuminuria was defined by UACR ≥ 300 mg/g Cr. Progression of albuminuria was defined as >30% increase in albuminuria, and a change from normoalbuminuria to either microalbuminuria or macroalbuminuria, or from microalbuminuria to macroalbuminuria. Patients for whom PI measurement could not be obtained were excluded from this study. Other exclusion criteria included patients with implanted cardiac pacemakers; arrhythmia, such as paroxysmal atrial fibrillation; amputations in any part of the lower extremities; and patients with malignancy or ESRD at baseline.Diabetes was diagnosed based on the report of the expert committee on the diagnosis and classification of diabetes mellitus30. After bilateral determination, the lower value of PI was considered as a representative for each subject.PI was measured using a Masimo SET Radical-7  instrument. The patients were placed in the supine position. A Masimo pulse oximeter probe was positioned on each toe and connected to Masimo SET Radical-7 machine. The patients were asked to rest for 5 min at the beginning of the procedure. PI was recorded three times: at 20 s, 40 s, and 60 s, after the 5-min rest period. The average of the three values was calculated and used as the reference value. The Masimo SET Radical-7 calculates PI as the ratio between the pulsatile and non-pulsatile components of the light reaching a light-sensitive cell of the pulse oximetry probe. The reliability and reproducibility of PI have been reported elsewhere2. Logistic regression analyses were conducted to examine the effects of PI on composite of ESRD, and/or doubling of serum creatinine level, composite of end-stage renal disease, doubling of serum creatinine level, renal death and/or cardiovascular death, and progression of albuminuria in all patients and in patients with eGFR ≥ 60 ml/min/1.73 m2. The following confounding factors were considered: age, duration of diabetes, body mass index, systolic blood pressure, hemoglobin A1c, total cholesterol and uric acid (Model 1). Model 2 was adjusted for all variables in Model 1 plus creatinine level. Model 3 was adjusted for all variables in Model 2 plus sex, logarithm of UACR, smoking status, history of cardiovascular disease, the use of renin-angiotensin system inhibitor, incretin related therapies, sodium-glucose cotransporter-2 inhibitor and statins. NRI and IDI were derived from logistic regression models (Model 3) with and without logarithm of baseline UACR to evaluate the clinical impact.The median and frequency of potential confounding variables were calculated. The relationships between PI and age, BMI, and other variables at baseline were estimated using Spearman’s rank correlation analyses. Owing to the relationship between baseline renal function and development of renal events, we also conducted analyses in a subgroup of patients with eGFR ≥ 60 ml/min/1.73 mP-value <0.05 was considered as statistically significant. The size, direction, and statistical significance of relationships were estimated by the odds ratio with 95% confidence interval (CI).All continuous variables are presented as median (interquartile range) or absolute number.


This study evaluated the association between systemic arterial blood pressure andcerebral perfusion in 740 participants of the UK's largest tri-ethnic study withmeasurements of cerebral blood flow (CBF) performed using arterial spinlabelling MRI. A significant negative correlation between blood pressure, ageand CBF was observed across the patient cohort. The lowest CBF values wererecorded in the group of patients with hypertension that were prescribed withanti-hypertensive drugs, but uncontrolled on medication. These findings confirmthat hypertension is associated with reduced cerebral perfusion and highlightthe importance of blood pressure control for the benefit of maintaining brainblood flow. The effect of anti-hypertensive drugs on CBF in hypertensive patients remainsunclear. Different studies reported decreases, increases or no changes in CBF inresponse to anti-hypertensive medication.4 Two recent systematic reviewshighlighted that many of these studies were small, underpowered, and showed evidence ofa significant (moderate-to-high) risk of bias.4Hypertension affects 1.5 billion people worldwide and its prevalence increases as theglobal population ages. Hypertension is now considered one of the leading causes ofage-related cognitive impairment and is strongly associated with reduced cerebral bloodflow (CBF). with measurements of cerebral perfusion performed using arterial spin labellingMRI and quantified using a single compartment model without partial volume correction. The study cohort included white European (41.7%), first South Asian (33.7%) andAfrican Caribbean (23.5%) participants  age 71(7)years). Office blood pressure measurements were obtained on the same day when cerebralperfusion was assessed. The participants’ data were excluded from the analysis ifrecords were incomplete and/or recorded grey matter perfusion values were lower than20 ml/100 g/min. Data obtained in 690 participants were included in the final analysis.Recorded values of systolic blood pressure and patient history of hypertension were usedto stratify the study cohort into 4 groups: i) individuals with normal blood pressure; ii) patients with untreated hypertension, not receiving any anti-hypertensivedrugs ; iii) patientswith hypertension, controlled on medication with a systolic blood pressure of<130 mmHg ; and iv)patients with hypertension, prescribed with anti-hypertensive drugs, but remaininguncontrolled on medication with a systolic blood pressure of ≥130 mmHg .Here we evaluated the association between systemic arterial blood pressure and cerebralperfusion in a cohort of patients recruited in the Southall and Brent Revisited (SABRE)study, the UK's largest tri-ethnic longitudinal study involving 740 participants2 = 0.05) and white matter  was observed across the whole patient cohort  and sex, a significantnegative correlation between systolic arterial blood pressure and cerebral perfusion inboth grey individuals with hypertension, the use of anti-hypertensive medication was found to beassociated with lower grey matter CBF. In contrast, another recent study involving participants aged 50 years or older(mean age 67.5 years) showed that intensive anti-hypertensive treatment (to achievesystolic blood pressure target of <120 mmHg) was associated with increased, ratherthan decreased, perfusion in the grey and white matter. Our analysis suggests that anti-hypertensive treatment has no deleterious effecton cerebral perfusion if systemic arterial blood pressure is controlled on medicationand supports the conclusions of the meta-analysis conducted by Van Rijssel and colleagues. These findings highlight the importance of blood pressure control not only forthe purpose of reducing the risk of stroke, cardiovascular disease, heart and renalfailure, but also for the benefit of maintaining adequate cerebral blood flow andprotecting the brain from harmful effects of chronically reduced perfusion.These data confirm that high systemic arterial blood pressure is associated with reducedcerebral perfusion. The data do not support the results of a previous study thatreported no significant CBF differences between normotensive subjects and individualswith untreated hypertension.


Symptomatic or active hydrocephalus in children is linked to an elevation in intracranial pressure (ICP), which is likely to be multifactorial in origin. The CSF outflow resistance, venous sinus resistance and total cerebral blood flow are likely factors in the ICP elevation. The purpose of this paper is to define the incidence, site and significance of venous sinus stenosis and/or cerebral hyperemia in a cohort of children diagnosed with hydrocephalus at a tertiary referral hospital.The imaging database was reviewed over a 10 year period and the index MRI of all children between the ages of 4 months and 15 years, who were diagnosed with treatment naive hydrocephalus of any type (excluding secondary to tumor) and had magnetic resonance venography (MRV) and flow quantification were selected. Patients were compared with children undergoing an MRI with MRV and flow quantification who were subsequently shown to have no abnormality. The cross-sectional area and circumference of the sinuses were measured at 4 levels. The hydraulic and effective diameters were calculated. An area stenosis of 65% or greater was deemed significant. A total cerebral blood flow greater than two standard deviations above the mean for controls was taken to be abnormal.There were a total of 55 children with hydrocephalus compared to 118 age matched control MRV’s and 35 control flow quantification studies. A high grade stenosis occurred in 56% of patients but in none of the controls (p < 0.0001). The commonest site of narrowing was in the distal sigmoid sinus. Cerebral hyperemia occurred in 13% of patients but did not occur in the controls.The elevation in ICP in symptomatic hydrocephalus is multifactorial. Both high grade venous stenosis and cerebral hyperemia are common in childhood hydrocephalus. High grade stenosis was noted to be a risk factor for conservative management failure. Hyperemia was a good prognostic indicator. FRcsf is the CSF formation rate, Rout is the CSF outflow resistance and SSSp is the superior sagittal sinus pressure. The sagittal sinus pressure itself depends on Ohm’s law, i.e. the product of the outflow resistance and blood flow through the outflow plus the jugular bulb pressure [TCBF is the total blood flow leaving the capillaries to enter the venous system, Rven is the venous outflow resistance from the sagittal sinus to the jugular bulbs and CVP is the central venous pressure. Each of these 5 variables has been shown to independently be associated with active hydrocephalus in children in the literature. CSF formation rates have been noted to be increased by 2–5 times in the hydrocephalus secondary to choroid plexus papilloma [Hydrocephalus in children refers to a disorder of CSF physiology resulting in abnormal expansion of the CSF volume, typically it is associated with an increase in intracranial pressure . In the pressure . So Eq. apilloma . The CSFapilloma . Hydroceapilloma . Thus, tapilloma .out, venous sinus stenosis and an increase in total cerebral blood flow (hyperemia) are possibly more common. In 1962 Kinal described bilateral distal high grade venous outflow stenoses in 4 infants with hydrocephalus using venography following direct sinus injection through the anterior fontanel [An increase in CSF formation rate and an elevation in central venous pressure are likely to be rare associations of hydrocephalus but increased Rfontanel . Finallyfontanel . With refontanel , 12. WhaThe radiology information system at a tertiary referral hospital was retrospectively interrogated to retrieve the data from children between birth and 15 years of age who had an MRI with MR venography and flow quantification, for the investigation of any form of hydrocephalus between January 2009 and January 2019. MRV and flow quantification has been a standard component of a comprehensive MRI examination of childhood hydrocephalus over this entire period. Hydrocephalus was defined on imaging as a ventricular size index  greater than 0.3 with a disproportionate reduction in subarachnoid space width or size of sulci (to exclude atrophy) for communicating/obstructive hydrocephalus. In many instances an initiating cause of the hydrocephalus such as hemorrhage or infection was evident or there was progressive enlargement of the ventricles on subsequent imaging. If there was a question whether the ventriculomegally was due to ex-vacuo dilatation, an increase in the temporal horns of the ventricles was taken to suggest hydrocephalus . In exteAll patients were imaged on a 3.0 T superconducting magnet . In all patients, a standard brain MRI consisting of 3DT1 sagittal, T2 axial, FLAIR axial and diffusion weighted axial images was performed. An MR phase contrast flow quantification sequence was acquired with retrospective cardiac gating. The TR was 26.5 ms, TE 6.9 ms, flip angle 15º, slice thickness 5 mm, matrix 192 × 512, FOV 150 and a single excitation. The velocity encoding value was 150 cm/sec with the plane set to pass through the skull base to cross the carotid and basilar arteries . Following 4 years of age, the sinuses had largely attained full size with no significant difference in the second and third controls groups for any of the sinuses. In no control patient was the jugular bulb smaller than the distal sigmoid sinus. With regards to focal stenoses, the average area for each segment in each age control group was calculated using the effective diameter and this was compared to each individual in the control group. There were no non-significant stenoses in the youngest grouping, one 50% stenosis in the proximal sigmoid in the second group and 4 stenoses between 57% and 63% in the third group  for a total of 5/118 or 4.2%. There were no significant stenoses greater than 65% in the controls.The blood flow was also noted to peak early, being 69% higher for the 1–4 years age group compared to the 4 months–1 years age group (p = 0.006) but there was no significant difference between the older two control groups.In the 4 month–1 year hydrocephalus patients, the sagittal sinus was 1.0 mm smaller on average and the distal sigmoid sinus 3.1 mm smaller than the controls (p = 0.008 and < 0.0001 respectively), giving an average sinus area reduction of 33% and 71% respectively. For the 1–4 years age group, the distal portion of the sigmoid sinus was 2.3 mm smaller than age matched controls (p = 0.0004), giving an average area stenosis of 53%. In the 4–15 age group, the sagittal sinus was 1.3 mm smaller, transverse sinus 1.3 mm smaller and the distal sigmoid 1.9 mm smaller on average than controls , giving 35%, 30% and 41% average stenosis for these segments. With regards to individual focal stenoses, there were 6/55 or 11% of the hydrocephalus patients with stenoses between 50 and 65% and 31/55 or 56% of hydrocephalus patients with significant stenosis  and four third ventriculostomy patients  with this good result. There were two patients with a poor clinical result, Patient 15 who had obstructed hydrocephalus underwent a third ventriculostomy and continued to have early morning headaches and also developed interval papilledema. Pre-op this patient had tandem lesions with a 71% stenosis of the sagittal sinus and 79% stenosis of the distal sigmoid sinus. Following the third ventriculostomy, despite the stoma being patent, the sagittal sinus residual stenosis was 59% and the residual distal sigmoid sinus stenosis was 46%. Patient 16 had obstructed hydrocephalus and underwent third ventriculostomy. Despite the ventriculostomy being patent, the 79% stenosis of the jugular bulb was unchanged and she complained of blurred vision and daily headaches. A ventriculoperitoneal shunt was placed and the stenosis again failed to change. She continued to complain of daily headaches.In two of the hyperemic patients there was follow up to gauge the changes in blood flow with time. Patient 38 from Table This study compares the hydraulic effectiveness of varying segments of the venous outflow. Unfortunately, the differing segments have widely differing shapes in cross-section. As seen in Fig. In an infant below 1 year of age, a normal ICP averages 3.7 mmHg . In 16 iIn their review of childhood hydrocephalus Rekate and Blitz propose a dual circuit model of hydrocephalus, in which a blockage to CSF flow could be anywhere within either the CSF circuit or the venous outflow of the vascular circuit . SimilarIf an elevation in venous pressure causes both IIH and hydrocephalus, what is the difference? In a large study of 145 pediatric IIH cases, 52% showed dominant-side venous obstruction , compared with 4% of controls showing a dominant-side obstruction . These fHyperemia is defined as an increase in the amount of blood in a part, organ or tissue as a result of dilatation of the supplying arteries . In thisout as the cause of their symptoms. At 7 months of age, the CSF formation rate averages 0.1 ml/min [out is 2.9 mmHg/ml/min in infants [out would need to be 73 mmHg/ml/min. This level would only be obtained if the outflow were totally blocked by, for example acute haemorrhage or meningitis. The 4 infants with active hydrocephalus but without stenoses in the current cohort may fulfil these criteria. Indeed, in a study of 6 infants with active hydrocephalus, the Rout only averaged 7.2 mmHg/ml/min [out was not high enough in the majority of infants to elevate the ICP and another cause for their active disease must be operating. In older children, the CSF formation rate averages 0.4 ml/min [out is between 5.5 and 10 mmHg/ml/min [out ranged from 12 to 30 mmHg/ml/min [out to cause a 7 mmHg increase in ICP alone, the value would need to be above 28 mmHg/ml/min. In 6 children with active hydrocephalus, the Rout was between 9.5 and 43 [out high enough to elevate the ICP by greater than 7 mmHg. It has been shown that an elevated venous pressure in adults with IIH does not elevate the CSF outflow resistance [out on its own is relatively rare.In this study, 18/22 or 82% of the patients with active hydrocephalus had a stenosis large enough to be significant and thus symptomatic  and 6 a ventriculoperitoneal shunt (VPS). A significant venous outflow stenosis appeared to be a risk factor for the failure of conservative treatment, with 10/12 or 83% of treated children having a stenosis greater than 65%. The failure of treatment to completely abolish the stenoses appeared to be a marker of ongoing symptoms. In both of the children where surgical treatment failed to completely resolve the stenoses their symptoms remained. In contrary, an elevation in TCBF appeared to be a good prognostic indicator, with none of these 7 patients requiring surgery. The reason for this later finding may relate to a possible reduction in the blood flow, back to the normal range, occurring with development.out as its sole cause. Thus, treatments are designed to improve the Rout by either unblocking the third ventricle (ETV) or adding an additional outflow path (VPS). However, this treatment is not without morbidity. The incidence of ETV failure at 2 years is about 35% [The current hydrocephalus paradigm implies that the vast majority of children with hydrocephalus have an elevation in Rbout 35% . Shunt ibout 35%  and shunbout 35% . The conGiven descriptions of high grade stenoses in childhood hydrocephalus appear in the literature from the early 1960s , one mayThe elevation in ICP in symptomatic hydrocephalus is multifactorial. Both high grade venous stenoses and cerebral hyperemia are common in childhood hydrocephalus. High grade stenosis is noted to be a risk factor for failure of conservative management. Hyperemia is a good prognostic indicator.


The ambulatory arterial stiffness index (AASI) is an indirect measure of arterial stiffness obtained during ambulatory blood pressuring monitoring (ABPM). Its relationship to nocturnal blood pressure dipping status and major adverse cardiovascular events (MACE) are controversial and its association with vascular inflammation has not been examined. We aimed to investigate the relationship between the AASI, inflammation and nocturnal blood pressure dipping status and its association with MACE.Adults (aged 18–80 years) who underwent 24-h ABPM for the diagnosis of hypertension or its control were included. The inflammatory markers measured were the neutrophil–lymphocyte (NLR), platelet-lymphocyte (PLR) and monocyte-lymphocyte ratios (MLR). The primary MACE was a composite of cardiovascular death, acute limb ischaemia, stroke or transient ischaemic attack (TIA) or acute coronary syndrome.p < 0.0001) and correlated with the NLR  and systolic blood pressure dipping % . Overall 39 (7.7%) patients had ≥ 1 MACE which included a total of seven cardiovascular deaths and 14 non-fatal strokes/TIAs. The mean follow up was 113.7 ± 64.0 weeks. Increasing NLR, but not AASI or systolic dipping, was independently linked to MACE  and MLR to cardiovascular death or non-fatal stroke/TIA .A total of 508 patients  aged 58.8 ± 14.0 years were included; 237 (46.7%) were normal-dippers , 214 (42.1%) were non-dippers (0–10% dip) and 57 (11.2%) were reverse-dippers (< 0% dip). The AASI was significantly higher among reverse (0.56 ± 0.16) and non-dippers (0.48 ± 0.17) compared with normal dippers (0.39 ± 0.16; In conclusion AASI was associated with blood pressure dipping and chronic inflammation but not independently to MACE. The MLR and NLR were independent predictors of MACE.The online version contains supplementary material available at 10.1186/s12872-021-01946-2. Twenty four hour ambulatory blood pressure (ABP) monitoring (ABPM) represents the gold standard for the diagnosis of hypertension and assessment of its control –3. AmongLower nocturnal blood pressure dipping is thought to reflect increased arterial stiffness. Less compliant and ‘stiffer arteries’ are less distensible leading to lower relative nocturnal blood pressure and pulse pressure drop . It woulVascular inflammation is an established risk marker and key pathogenic mediator in the development of endothelial dysfunction, atherosclerosis and subsequently increasing arterial stiffness . StimulaThis study had two discrete aims. The first was to investigate the relationship between AASI, vascular inflammation (using lymphocyte ratios) and nocturnal blood pressure dipping status. The second was to examine their association with future MACE. We hypothesised that AASI would be significantly associated with blood lymphocyte ratio counts, blood pressuring dipping status and MACE.This study followed the STROBE Initiative Guidelines for cohort studies . This waKey exclusion criteria were patients with previous organ transplantation, persistent/ permanent atrial fibrillation, a calculated creatinine clearance of < 30 ml/min or known stage IV or V chronic kidney disease (CKD), pregnancy or with active cancer. Patients with severe aortic stenosis, aortic coarctation, active infection or vasculitis, on high dose steroids or who had been hospitalised within the previous week were also excluded.All tests were performed using an automatic ABPM device . An automated oscillometric cuff was placed on the non-dominant arm. Blood pressure measurements were set to minute intervals throughout a 24-h recording period. The night-time period was defined as the hours of 22:01 to 06:00 h and the day-time period as 06:01 to 22:00 h. Patients were only included if they had a minimum of 10 day-time and 5 night-time ambulatory blood pressure measures during the 24 h recording period  were shown. Comparisons of continuous data among two groups were performed using an unpaired t test and Mann–Whitney test for normal and non-normally distributed data respectively. Three group comparisons of continuous data  were performed using a One-way ANOVA and Kruskal–Wallis tests with post-tests for normally and non-normally distributed data respectively. Categorical data were examined using the Fisher’s exact tests and chi squared tests as appropriate. Correlations were investigated using Pearson and Spearman rank coefficients  as appropriate. Only notable correlations with a coefficient r ≥ 0.20 were reported.B) and 95% confidence intervals (CI), respectively. Only variables with a p value < 0.05 on univariate testing or that were considered clinically relevant with outcomes were entered into the multivariate analyses. To avoid over-fitting a forward LR (likelihood ratio) method were performed with retention set at a significance level of 0.10. Sensitivity analyses were performed to assess the robustness of the final model. A two-sided p value of < 0.05 was considered significant for all comparisons.Associations between prognostic variables and MACE were examined by univariate and multivariate Cox proportional hazards analysis with the results reported as the odds ratio . Published data has suggested an average standard deviation for the AASI of 0.06 to 0.22 among adults with and without hypertension or cardiovascular disease . Based op = 0.55).A total of 508 patients were included in this study of which 260 (51.2%) were women. The mean age was 58.8 ± 14.0 (range 18–80) years. Overall 98.6% of the population were Caucasian; 66.34% had a previous history of hypertension, 16.9% diabetes mellitus and 8.7% had suffered a previous stroke or TIA. The men (58.4 ± 13.95) and women (59.2 ± 14.1) were of similar age  patients were classed as normal-dippers, 214 (42.1%) non-dippers and 57 (11.2%) reverse-dippers , HBA1c , NLR  and systolic blood pressure . AASI inversely correlated with creatinine clearance , diastolic blood pressure  systolic blood pressure dipping % , diastolic blood pressure dipping %  and MAP blood pressure dipping % .AASI positively correlated with age . AASI values were also higher in those with, versus without respectively, a history of CAD , diabetes mellitus , previous stroke/TIA , PVD , known hypertension .The AASI was significantly greater in women versus men  patients had one or more adverse cardiovascular events which included a total of seven cardiovascular deaths and 14 non-fatal strokes/TIAs. The mean follow up  was 113.7 ± 64.0 weeks.The univariate predictors of MACE were age, history of stroke/TIA, heart failure, coronary disease, hypertension and PVD, AASI, lower systolic blood pressure dipping (%), mean arterial blood pressure dipping (%) and 24 h pulse pressure, NLR, MLR, creatinine clearance, and lower haemoglobin Table .Table 3Rp < 0.0001) (Table p = 0.009), PVD and female sex were independent predictors of future cardiovascular death or non-fatal stroke/TIA . The full database is available in the supplement  Table . PreviouThis was first study to investigate the relationship between AASI, vascular inflammation and blood pressure dipping and their association to adverse cardiovascular outcomes. We found that a reduced blood pressure dipping pattern was linked to higher AASI and measures of vascular inflammation (MLR and NLR). Systolic blood pressure dipping %, AASI, MLR and NLR were associated with MACE on univariate analyses. Increased NLRs were independently associated with MACE and MLR with cardiovascular death or non-fatal stroke/TIA.To our knowledge this is the largest and one of the few studies to investigate the relationship between systolic blood pressure dipping and vascular inflammation using lymphocyte ratios. We observed an inverse relationship between the neutrophil and monocyte-lymphocyte ratios and systolic blood pressure dipping. Higher ratios were associated with non- and reverse blood pressure dipping, which are recognised risk markers of increased cardiovascular risk . PreviouChronic vascular inflammation and the infiltration of the vascular endothelium with leukocyte subsets is one of the primary driving forces in the development of atherosclerotic cardiovascular disease . Lipid aOur interest in the AASI stems from its easy computational availability with automated results included in our standardised reports, coupled most importantly with its cited role as a potential indirect marker of arterial stiffness. All of the factors that we found to be associated with AASI and its higher levels in this study are recognised risk factors for increased arterial stiffness, with one exception. We observed that women had significantly higher AASI values than men despite their similar ages. This could relate, in part, to differences in their comorbidities and relative burden of cardiovascular risk factors . A detailed comparison was beyond the scope of this study. It is nevertheless notable that it has been reported that women exhibit greater age related increase in measures of large artery stiffness than men, supporting our findings –34.The relationship between AASI and systolic blood pressure dipping status has only been previous examined in a few studies with conflicting results , 12. We This study has a number of limitations that should be acknowledged. This was a retrospective single centre study and hence prone to potential bias. Only outcomes registered with the local hospitals could be monitored and hence we cannot exclude the possibility that some patients could have presented to another hospital with a cardiovascular event outside of our locality. However, these numbers are likely to be very small given that only local and registered patients were included and there were on-going active records available for the vast majority of patients. All 24-h ABPM were set to similar times for the determination or day-time and night-time blood pressures and its associated indices. Given the wide range of ages of patients included in this study it is likely that there would have been periods graded as effectively night-time when the patients were still very much awake and day-time when patients may have been asleep. The use of a wide fixed night-time period in this study could have led to inclusion of transition periods in the morning and evening when blood pressure changes rapidly leading to the potential misclassification of dipping status . HoweverIn conclusion, in this study we found that that AASI was significantly correlated with NLR and inversely related to systolic blood pressure dipping. Systolic blood pressure dipping, AASI, the neutrophil and monocyte-lymphocyte ratios were univariate predictors of MACE. The NLR was an independent predictor of the primary MACE, and MLR for cardiovascular death or non-fatal stroke. This data endorses the association between AASI, inflammation and cardiovascular risk.Additional file 1. The full database supplement


Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a rare hereditary disease caused by a variety of genetic mutations. Carriers of a mutation in the responsible genes are at risk of reaching end-stage kidney disease typically in middle age. The frequency of this disease is assumed to be underestimated because of a lack of disease-specific signs. Pathological findings obtained from kidney of uromodulin related ADTKD (ADTKD-UMOD) patients are regarded as non-specific and less-informative for its diagnosis. This research was undertaken to evaluate the significance of kidney biopsy in ADTKD-UMOD patients.Thirteen patients from 10 families with nine identified uromodulin (UMOD) gene mutations who underwent kidney biopsy in the past were studied. Their kidney tissues were stained with anti-UMOD antibody in addition to conventional methods such as PAS staining. When positive, the numbers of tubules with visible UMOD protein accumulations were calculated based on the total numbers of UMOD expressing tubules. Pathological findings such as tubulointerstitial fibrosis, atrophy, inflammation and glomerulosclerosis were also evaluated and analyzed.Interstitial fibrosis and tubular atrophy were present in all 13 patients. Most atrophic tubules with thickening and lamellation of tubular basement membranes showed negative UMOD staining. In all but two patients with C94F mutations, massive accumulation of UMOD proteins was observed in the renal endoplasmic reticulum. UMOD accumulations were also detectable by PAS staining as polymorphic unstructured materials in the 11 patients at frequencies of 2.6–53.4%. 80.4% of the UMOD accumulations were surrounded by halos. The detection rate of UMOD accumulations positively correlated with eGFR. Glomerulosclerosis was detected in 11/13 patients, with a frequency of 20.0 to 61.1%, while no cystic dilatations of glomeruli were detected.Massively accumulated UMOD proteins in ADTKD-UMOD kidneys are detectable not only by immunostaining using anti-UMOD antibody but also by conventional methods such as PAS staining, although their detection is not easy. These findings can provide important clues to the diagnosis of ADTKD-UMOD. Kidney biopsy in ADTKD-UMOD may be more informative than assumed previously. Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a rare genetic disease, whose characteristics include progressive kidney injury, interstitial fibrosis and tubular atrophy. Uromodulin (UMOD), Mucin-1 (MUC1), renin (REN), Hepatocyte nuclear factor 1 beta (HNF1β) and alpha subunit of the endoplasmic reticular membrane translocon (SEC61A1) are the genes responsible for ADTKD .UMOD which encodes Tamm-Horsfall protein is the first identified and one of the most common genes to cause ADTKD , 3. AutoHowever most of the clinical signs of ADTKD such as decreased glomerular filtration rate (GFR) or hyperuricemia are very common in chronic kidney disease (CKD) of other etiologies too. This makes ADTKD a condition without disease-specific manifestations, and so difficult to diagnose.Urinalysis of ADTKD patients usually shows a bland urinary sediment and no or little proteinuria which might delay early detection of ADTKD. Furthermore ADTKD is not a well-known disease even amongst nephrologists, and so a considerable number of ADTKD patients may reach end-stage kidney disease without an accurate diagnosis ever having been made.Pathological findings of kidneys in ADTKD patients are reported to be non-specific, such as interstitial fibrosis, tubular atrophy, thickening and lamellation of tubular basement membranes with normal glomeruli or glomerulosclerosis. The role of renal biopsy in the process of ADTKD diagnosis is very limited. For example in single cases without positive family histories who are suspected of having ADTKD, renal biopsy serves to show tubulointerstitial nephritis and to exclude other renal diseases .The mechanisms underlying kidney injury progression in ADTKD-UMOD are thought to be as follows. Abnormal UMOD protein encoded by mutated UMOD gene causes protein misfolding. These mutated UMOD proteins cannot exit the endoplasmic reticulum (ER) and so accumulate massively in ERs in the cytoplasm of TALH (thick ascending limb of Henle) cells. Excessively accumulated UMOD proteins trigger ER stress and tubulointerstitial inflammation and fibrosis.The abundant accumulations of UMOD proteins are visible in kidney tissue immunostaining using anti UMOD antibody, and even more clearly by immunofluorescence staining . In lighThirteen Japanese ADTKD patients from 10 families with 9 UMOD mutations who underwent kidney biopsies in the past were studied. The disease in most of them had been suspected from their clinical symptoms and extensive family histories of CKD and confirmed by genetic analysis . The diaThe severity of tubulointerstitial diseases is assessed by PAS and Masson trichrome staining. The degrees of interstitial fibrosis (ci) , tubular atrophy (ct) , and interstitial inflammation (i) were scored 0–3 according to the Banff 2015 grading system . The preThe presence and location of UMOD proteins in TALH epithelial cells were evaluated by immunofluorescence staining using anti-UMOD antibody . The staining method followed the details outlined in a previous report . To asceTo evaluate and compare the visibility of UMOD accumulations in ordinary staining in light-microscopy, we stained a series of thin-slice kidney tissues with hematoxylin and eosin (HE), PAS, periodic acid methenamine silver (PAM), and Masson trichrome in addition to anti-UMOD antibody in another ADTKD-UMOD patient with A247P mutation (case #2). In each staining method, the detection rate of accumulated UMOD was calculated based on the full field of obtained kidney tissue . Regarding all of the remaining ADTKD-UMOD patients with abnormal UMOD accumulations in immunofluorescence staining and four normal controls , the detection rate in PAS staining was calculated based on the full field of obtained kidney tissue. . The sizes of UMOD accumulations and halos around them were assessed. The method of classification: if accumulated UMOD proteins were bigger than epithelial cell nuclei: L, Smaller than nuclei: S, no halo:-, halo smaller than nuclei: +, halo bigger than nuclei: ++.p value < 0.05 was regarded as statistically significant.Correlations between all parameters: eGFR, age, scores of interstitial fibrosis, tubular atrophy, interstitial inflammation, percentage of glomerulosclerosis and detection rate of abnormal UMOD in PAS staining were tested using the Spearman’s test. A The clinical features, kidney pathology and UMOD mutations of cases are summarized in Table Five of 13 cases (38.5%) reached end-stage kidney disease requiring renal replacement therapy at an average age of 47 years . No patients had marked proteinuria or hematuria, 11/12 cases had hyperuricemia (91.7%), 4/11 cases (36.4%) had experienced gout, and 5/11 cases (45.5%) had hypertension. Small numbers of kidney cysts existed in 3/11 cases (27.3%).The pathological diagnosis made from kidney biopsy of all the patients was tubulointerstitial nephritis and/or nephrosclerosis. Table In double-immunostaining using anti-UMOD antibody and various organelle markers in an ADTKD-UMOD patient kidney (case #1), accumulated UMOD proteins were merged with increased ER proteins but not with Golgi, lysosome or secretory granules Fig. , meaningIn the series of light microscopic examinations using conventional staining methods  for another ADTKD-UMOD patient (case #2), UMOD accumulations were visible with all of the four staining methods and stained PAS positive, Trichrome blue, red in HE and PAM similar to previously reported findings , 7 Fig.. The detIn UMOD immunofluorescence staining, massive UMOD protein accumulations were detected in 11/13 cases 84.6%). Two patients: a father and a son with the same C94F mutations showed normal UMOD staining patterns throughout in highly atrophic tubules  Table . Each of7.1% TablStatistical analysis showed the detection rate of UMOD accumulations to positively correlate with eGFR, while the score of interstitial inflammation negatively correlated with age Table .Table 5Pathological characteristics of kidneys in 13 Japanese ADTKD-UMOD patients with identified UMOD mutations were evaluated. This is the first study to systematically analyze the pathology of multiple ADTKD-UMOD patients. In 11/13 patients, abnormal UMOD accumulations were detected both in immunostaining using anti-UMOD antibody and in PAS staining. It is possible that aggregated UMOD deposits are more detectable in the presence of higher GFRs suggesting that the value of kidney biopsy in ADTKD-UMOD is greater with higher GFRs. These findings may help to make the diagnosis of ADTKD-UMOD. When encountering a youngish patient suspected of having ADTKD with renal insufficiency and hyperuricemia without urinalysis abnormalities, close observation of kidney tissue obtained by biopsy would be a good option for further diagnosis when a family history of CKD is absent or unclear.In 1978, Zager et al.  and ResnThe pathological findings of ADTKD have been reported to be those of non-specific interstitial tubulopathy. Ekisi et al.  reportedIn the 2015 KDIGO consensus report, K-U Echardt et al. [However, the possibility of ADTKD-UMOD is not excluded even when a family history of CKD is absent or unclear. Bolle et al.  reportedIntracytoplasmic PAS positive deposits may be a pathological clue to suspect ADTKD-UMOD. However caution is needed. Crystalline inclusions of light chain proximal tubulopathy are also PAS positive deposits detected inside the cytosol of proximal tubular cells . BlebbedIn the present study, we could not find abnormal UMOD accumulations immunohistochemically in the kidneys of the father and son who have C94F mutation in common with clinical courses of typical ADTKD-UMOD. This result was unexpected because the transgenic mice which have the identical mouse mutation manifest UMOD accumulations in their kidneys and develop kidney disease which resembles ADTKD . This imOur study had the following limitations. First, the analyzed case numbers may not have been sufficient. Second, electron microscopic observations were not performed in our patients. Third, we could not explain why the patients with C94F UMOD mutations did not have abnormal UMOD accumulations and why most of the atrophic tubules in ADTKD-UMOD were negative for UMOD staining. Further investigations will be needed to unravel these issues in the future.This study showed that aggregated UMOD accumulations are detectable in 85% of ADTKD-UMOD patients using PAS staining light microscopy examinations, demonstrating that pathological examinations of ADTKD-UMOD kidneys provide more useful information than previously reported.Additional file 1 : Supplemental table 1. pathogenicity prediction of each UMOD variant