Proteus mirabilis is a biofilm-forming agent that quickly settles on the urinary catheters and causing catheter-associated urinary tract infections. Thus, the spread of multidrug-resistant P. mirabilis isolates, with the ability to form a biofilm that carries integron, extended-spectrum β-lactamases (ESBLs), and plasmid-mediated colistin resistance genes (mcr), represents a severe threat to managing nosocomial infectious diseases. This study is aimed at surveying the prevalence of ESBL, integrase, and mcr genes of P. mirabilis, isolated from the catheter, to assess the differences in their antimicrobial susceptibility and clonal dissemination. intI1, intI2, intI3, blaTEM, blaCTX-M, blaSHV, mcr1, and mcr2) were detected by PCR. All of the isolates were characterized by repetitive sequence-based PCR.  Microtiter plate assay was adopted to measure biofilm formation. The antimicrobial susceptibility was assessed by the disk diffusion method. Antimicrobial resistance genes (P. mirabilis were isolated. All of the isolates could form a biofilm. Proteus spp. had intrinsic resistance to tetracycline (95%) and nitrofurantoin (92.5%), which explains the high resistance prevalence. The most widely resistant antibiotic was trimethoprim-sulfamethoxazole (75%). Thirty-three (82.5%) isolates were classified as multidrug resistance (MDR). The prevalence of intI1 and intI2 genes was 60% and 25%, respectively. In 6 (15%) isolates, both genes were detected. The most frequent ESBL gene detected in all of the isolates was TEMbla. Also, no detection for mcr1 and mcr2 antibiotic resistance genes was reported. Rep-PCR identified 39(GTG)5 types (G1–G39) of 40 isolates that 38 isolates had unique patterns.  From 385 collected catheters in patients admitted to the intensive care unit (ICU), 40 intI1 and blaTEM were the most prevalent genes in the integrase and ESBL gene family. High diversity was seen in the isolates with Rep-PCR. The increasing rate of MDR isolates with a high prevalence of resistance genes could be alarming and demonstrate the need for hygienic procedures to prevent the increased antibiotic resistance rate in the future. In this study, 82.5% of isolates were MDR with high antibiotic resistance to trimethoprim-sulfamethoxazole. The  Proteus mirabilis is a charming polymorphic swarming persistent colonizer bacterium that strongly correlates with catheter blockage and urinary stone development. Complicated urinary tract infections (UTIs) at an alarming rate are expanding healthcare challenges, and P. mirabilis is the pathogen that must be noticed for causing those particular catheter-associated urinary tract infections (CAUTIs). P. mirabilis is a usual cause for complicated UTIs, and it becomes intricate in patients undergoing long-term indwelling urinary catheterization who may develop CAUTIs [P. mirabilis to create crystalline biofilms, eventually leading to encrusted and blocked catheters [P. mirabilis on catheters and urolith formation in the bladder and urinary tract, which could be created by multidrug-resistant isolates. Multidrug resistance (MDR) may be mediated by resistance agents located on chromosomes or mutations in a resident gene. However, it may also expand by attaining resistance genes through horizontal transfer [p CAUTIs . Such diatheters . Likewisatheters –5. CAUTItransfer .P. mirabilis isolates were susceptible to common antibiotic classes, but recent studies in different countries have indicated that antibiotic resistance among P. mirabilis isolates is increasing. The β-lactam resistance patterns of the P. mirabilis isolates have reported the production of various classes of extended-spectrum β-lactamases (ESBLs) [These resistance genes, which are widely present on plasmids, transposons, and integrons, lead to the problem of rapid spread and treatment failure. In the past, most  (ESBLs) .The prevailing type of integrons discovered in clinical isolates is class 1 integron, which is highly associated with antibiotic resistance; therefore, they have been extensively studied . Class 2P. mirabilis [mcr) genes and is often overlooked and not screened for mcr. However, this bacterium can serve as a reservoir to transmit these genes to colistin-susceptible bacteria [The extension of ESBL indicates a severe threat to managing nosocomial infectious diseases, causing problems for remedial choices of antimicrobial applications. The prevalence of integrons and characterized gene cassettes in Gram-negative bacteria integron-associated multidrug resistance has been investigated. However, it is seldom addressed in irabilis . Colistibacteria .Various molecular methods, such as repetitive extragenic palindromic PCR (Rep-PCR), ribotyping, and pulse-field gel electrophoresis (PFGE), have been used to assess the genotypic diversity within several bacterial species .Enterobacteriaceae populations. Rep-fingerprinting uses variations in conserved intergenic palindromic DNA sequences for PCR amplification and an isolate's characterization. These DNA elements are stable, noncoding intergenic repetitive sequences scattered across the genome and act as amplification targets to produce various bands [Rep-based fingerprinting has been proved to be a fast and reliable tool to differentiate between us bands , 14.intI1, intI2, and intI3) and three ESBL genes , as well as mcr1 and mcr2, was investigated, and the comparison of integron-carrying and non-integron-carrying MDR P. mirabilis isolated from the catheter was performed to assess the differences in their antimicrobial susceptibility and clonal dissemination. The clonal relationship for finding the origin of infection of the isolates was also evaluated with Rep-PCR.In this study, the prevalence of integrase genes  from intensive care unit (ICU) patients were collected from various hospitals in Isfahan. The inclusion criteria for this study were that the collected catheters were from the patients without a primary urinary tract infection at admitting time; the minimum time of catheterization was five days.P. mirabilis was isolated from the catheters. For this purpose, we followed the procedures of Mandakhalikar et al., with some modifications [P. mirabilis suspected colonies were recultured to achieve a pure and single colony, and the ureG gene PCR was performed for genetic confirmation.ications . Brieflyμg), amoxicillin-clavulanic acid (20/10 μg), ampicillin (10 μg), nitrofurantoin (300 μg), meropenem (10 μg), cefotaxime (30 μg), ceftazidime (30 μg), ceftazidime-clavulanic acid (30/10 μg), cefixime (5 μg), aztreonam (30 μg), amikacin (30 μg), norfloxacin (10 μg), ofloxacin (5 μg), ciprofloxacin (5 μg), tetracycline (30 μg), and trimethoprim-sulfamethoxazole (1.25/23.75 μg) was determined by the disk diffusion method on Mueller-Hinton agar overnight at 37°C. The breakpoints for each antimicrobial agent were interpreted according to guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) standards [Antimicrobial resistance of the isolates to ampicillin-sulbactam (10/10 tandards . Furthertandards . In additandards .P. mirabilis isolates was diluted (1 : 100) in TSB to reach the 0.5 McFarland concentration, and 5 μl of the diluted culture was inoculated in each well of polystyrene microtiter 96-well plates . After 24 h incubation of the plates at 37°C, the medium was removed, and the wells were washed carefully with double distilled water (DDW) and fixed by adding 200 μl of ethanol (96%) for 15 minutes. Next, the medium was removed, and the plate was left to dry. The biofilms were stained with 0.1% (w/v) crystal violet for 20 minutes at 37°C. Next, the biofilm was washed three times with DDW. Then, 200 μl of ethanol–acetic acid (90 : 10) was added to each well, and optical density (OD) was measured at 590 nm with an enzyme-linked immunosorbent assay (ELISA) microtiter plate reader. Each assay was done in triplicate, and the mean absorbance ± standard deviation was calculated for all repetitions of the tests. Escherichia coli K12 and Pseudomonas aeruginosa ATCC 27853 were used as negative (weakly biofilm-forming) and positive (strong biofilm-forming) control strains, respectively [For this purpose, an overnight culture of ectively .bla genes, which coding for the blaTEM, blaSHV, blaCTX-M, and integron family  using specific primers  running buffer and subsequent exposure to UV light in the presence of a safe stain.S typing predestinated 100% [P. mirabilis. PCR condition was 3 min at 95°C; 30 cycles of 45 seconds at 94°C, 30 seconds at 50°C, and 45 seconds at 72°C; and finally, 5 min at 72°C. The PCR products were revealed by electrophoresis on a 1.5% agarose gel with 0.5x TBE at 2.5 hours with 65 voltage. This experiment was carried out three times. The graphical abstract of methods which performed in this study is shown in We followed the methods of (GTG)5-PCR fingerprinting, which was described by Gevers et al. . Analysited 100% . The banD) was calculated to varying levels of similarity index according to the formula. In the formula, N represents the number of unrelated strains evaluated, S represents the number of distinct types, and xj represents the number of strains belonging to the jth category, assuming that strains are grouped into mutually exclusive groups. A discriminatory index . The duration of patients' catheterization, which bacterial isolation was performed, was 5-15 days. Furthermore, the most bacterial isolation was from the ten days of patient catheterization (50%). Patients who had a catheter for 10 to 15 days had moderate to strong biofilm formation; in addition, 50% of patients with short catheterization (7 days) had isolates with weak biofilm development  isolates.The antimicrobial-resistant patterns of isolates are shown in P. mirabilis isolated from catheters were able to produce biofilm. Of 40 P. mirabilis, twelve isolates (30%) had strong biofilm ability (OD590 nm ≥ 2.5), twenty of them (50%) were moderate biofilm producer (1.5 ≤ OD590 nm < 2.5), and eight (20%) of them had weak biofilm (0.7 ≤ OD590 nm < 1.5) (All m < 1.5) .intI1 with 60% (24) of isolates and 25% (10) of isolates had integrase 2 (intI2), and no detection for integrase 3; also, 15% (6) of isolates had both integrase 1 and integrase 2. The most prevalent gene in the bla ESBL family was observed in blaTEM genes with 100% (40) of isolates; 82.5% (33) of isolates had blaCTX-M genes and no detection of blaSHV. Also, PCRs targeting the mcr-1 and mcr-2 genes revealed no detection for these genes. Also, it demonstrated a significant relationship among resistance to trimethoprim/sulfamethoxazole, norfloxacin, and ofloxacin with a class 2 integron (P < 0.05).The frequency of three classes of the integron family is shown in D was calculated from a constructed phylogenetic tree, and according to the formula described before, discriminatory power was calculated at 99.8%.Rep-PCR or (GTG)5-PCR fingerprints of 40 isolates generated 7 to 13 bands with the molecular size ranging from 250 bp to more than 1 kb. (GTG)5-PCR amplification identified 39(GTG)5 types (G1–G39) of 40 isolates that 38 isolates had unique patterns . D was cP. mirabilis is a common cause of complicated UTI, particularly in patients with functional or anatomical urinary tract abnormalities. Swarming motility can promote the migration of P. mirabilis from the periurethral area along the catheter surface into the urinary bladder and initiate CAUTIs in patients undergoing long-term catheterization. There are limited data about the pathogenicity and antibiotic resistance of catheterized P. mirabilis in Iran. In this study, we evaluated the molecular characteristics of P. mirabilis isolated from CAUTI. The result of the current study showed that all of the isolates had the ability of biofilm formation, being in line with other studies [ studies , 35. TheP. mirabilis over the past years [P. mirabilis isolates, we found the highest susceptibility to ampicillin-sulbactam (95%); this may be due to differences in the types and amount of antibiotics used in different countries, geographical regions, years of study, and the fact that ampicillin-sulbactam was not used in the antibiotic test, which may explain the difference. The study also revealed a high prevalence of MDR isolates (82.5%), being consistent with the findings of one study by Alabi et al. [The survival and recovery strategy, engrossed by some microbial species in front of rough environmental situations, can form a biofilm, thereby causing antibiotic resistance boosting. The severity of the biofilm-associated antimicrobial resistance of microorganisms is higher than their planktonic form . The prest years , 37. Thest years . Althougst years  found thi et al.  so that P < 0.05). In other words, the presence of the integrase 2 gene in species could lead to resistance to antibiotics, such as trimethoprim/sulfamethoxazole, norfloxacin, and ofloxacin.This study demonstrated a significant association between resistance to trimethoprim/sulfamethoxazole, norfloxacin, and ofloxacin and a class 2 integron  typing of forty catheter isolates in this study showed 39 types, of which two isolates from one hospital were put in one GTG type (GTG8) with 100% identity, which could be due to the dissemination of the isolates from the hospital environment to patients by personnel or nurses or nosocomial infection, and other isolates were put in single GTG type, which demonstrated high diversity among isolates. Rep-typing results show high discriminatory power, being completely in agreement with the study conducted by Bedenic et al. [The constructed phylogeny tree from Rep-PCR typing effectively identified the genetic relatedness of c et al. . The stuc et al. . This isintI1 gene has been observed for one of them, which might because the gene release has not yet occurred. The high diversity of isolates in the phylogenic tree was seen in the previous study [This GTG8 type, between two isolates, has a similar resistance gene, but the lack of the us study .The limitation of this study was the lack of a modern technique, such as multilocus sequence typing (MLST) and PFGE, to determine the clonality of isolates due to limited financial sources.intI1 and blaTEM were the most prevalent genes in the integron and ESBL gene family. High diversity was observed in isolates with Rep-PCR. The outcome of persistence MDR strains under antimicrobial selective pressure in the hospital environment will increase both the chance of an advantageous mutation and the opportunity to acquire additional resistance genes. Nevertheless, the existence of the same type of isolates could be an alarming issue for healthcare providers' hygiene conditions to eliminate these infections particularly spread of resistance genes and highlight the importance of sporadically epidemiological studies and methods with high discriminatory power.In this study, 82.5% of isolates were MDR with high antibiotic resistance to trimethoprim-sulfamethoxazole. The


Hypertension remains the major modifiable risk factor of stroke recurrence. The study aimed to determine the up-to-date epidemiological features of hypertension among the survivors of ischemic stroke.Our cross-sectional study included 18,796 adults aged ≥40 years and residing in northeast China. Ischemic stroke was diagnosed according to the World Health Organization’s criteria, which requires the clinical record, computed tomography (CT) and/or magnetic resonance imaging (MRI) during the hospital stay. Hypertension was defined according to the Chinese hypertension guidelines .p < 0.001). The rates of stage 2 and above hypertension in the ischemic stroke population were 32.5 and 18.7%, and was significantly higher in rural areas than in urban areas.Of the 986 survivors of ischemic stroke, 819 (83.1%) were identified with hypertension (535 were pre-stroke hypertension and 284 were post-stroke hypertension). Among hypertensive patients, the awareness and treatment rates were 76.8 and 66.7% respectively. Only 11.0% achieved an appropriate blood pressure (< 140 mmHg and < 90 mmHg) among those who took hypertensive medications. 16.8% of treated hypertensive patients received combination therapy, and calcium channel blockers were the most frequently used anti-hypertensive medication as monotherapy. The mean systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the stroke population was 155.3 ± 22.9 mmHg and 89.2 ± 12.3 mmHg. Both SBP and DBP were higher in rural patients than in urban patients (158.5 ± 23.8 mmHg vs. 146.4 ± 17.5 mmHg and 90.3 ± 12.9 mmHg vs. 85.9 ± 10.1 mmHg, respectively; The prevalence of poorly-controlled hypertension and the high rates of blood pressures at stages 2 and above in patients with prior ischemic stroke demonstrated an alarming situation in northeast China. Stroke has emerged as one of the major causes of death and disability in the world, with more than 80% of strokes worldwide occurring in low- and middle-income countries . In ChinMore than 70% of the stroke survivors had an ischemic stroke according to our previous study , which wHypertension has long been considered the most common and important potentially modifiable risk factor for recurrent vascular events and is strongly associated with a worse prognosis . Previoun = 22,009) were eligible to participate, except those who were pregnant or had a mental disorder; in total, 18,796 (85.4%) participants ultimately completed the study  and three urban districts  were randomly selected from Liaoning Province. In total, 19 rural villages and eight communities were randomly selected from these four counties and three cities. All permanent residents   aged ≥40The prevalence time was determined as August 31, 2017, while new onset stroke cases during the survey were excluded in the calculation of stroke prevalence. Participants with a doubtful or certain history of stroke were further confirmed by well-trained neurologists in accordance with the World Health Organization recommendations. The clinical record, computed tomography (CT) and/or magnetic resonance imaging (MRI) during the hospital stay were required to make the diagnosis of ischemic stroke . The datAfter at least 5 min of rest in a seated position in the morning, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured three times at 2-min intervals using a standardized automatic electronic sphygmomanometer . Hypertension was diagnosed according to the following Chinese hypertension guidelines: a mean SBP ≥140 mmHg and/or a mean DBP ≥90 mmHg and/or self-reported use of antihypertensive medication in the past 2 weeks . In patiβ- blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers and others such as Chinese medical and vasodilators. Patients who used ≥2 drugs were defined as receiving combination therapy; those using only one drug as monotherapy.Medications were classified as thiazide-type diuretics, calcium channel blockers, 2 test for categorical variables. Pre-stroke and post-stroke hypertension were calculated according to the diagnostic time of stroke and hypertension. All statistical analyses were conducted using SPSS22.0 ; P values < 0.05 were considered statistically significant.Descriptive statistics were calculated for all variables. Continuous variables with normal distribution are reported as means and standard deviations, and compared using one-way analysis of variance. Differences between groups were compared using a χThe characteristics of the stroke population are shown in Table p < 0.001). The mean SBPs were higher in women patients than in men patients ; however, the DBP levels in the men ischemic stroke population was higher than that of women  ; however, no significant difference was found between urban and rural residents  , as shown in Table The prevalence of stages 1, 2, and 3 hypertension among hypertensive patients with ischemic stroke were 41.5, 32.5, and 18.7%, respectively. The prevalence of stages 2 and 3 hypertension were significantly higher in rural areas than in urban areas  ; however, blood pressure control was not significantly different between women and men . Although the awareness rate was similar in urban and rural residents, stroke survivors from urban areas had better control of their hypertension compared to those from rural areas .The rates of awareness and treatment were higher in women than in men  had a history of hypertension before the index stroke, 88.0% of whom were taking anti-hypertensive medicine, but the control rate was only 9.0%. Ninety-four patients (11.5%) had hypertension identified after the index stroke, the treatment and control rates were 79.8 and 12.8%, respectively Fig. .Fig. 4TAmong 546 treated patients, 83.2% of prescriptions were for one medication, 16.8% were two or more medications. The control rates among patients taking monotherapy and combination therapy were 11.2 and 9.8%, respectively. The most frequently used anti-hypertensive medication as monotherapy were calcium channel blockers (47.4%), however, the control rate among those patients was only 9.8%  . High syThe question of whether anti-hypertensive treatments significantly decrease the rate of recurrent stroke remains somewhat controversial. Anti-hypertensive medication use in patients with high baseline blood pressures have been shown to reduce all-cause and, specifically, cardiovascular mortalities; however, aggressive blood pressure reduction has also been shown to worsen outcomes in stroke patients with preexisting cardiovascular diseases , 21. A pSeveral large trials have recommended anti-hypertensive treatment for patients with a history of stroke to prevent future vascular events . PreviouMoreover, adequate blood pressure control in rural areas was worse than that in urban areas, and the prevalence of stages 2 and 3 hypertension were significantly higher in rural survivors than in urban survivors, probably due to the relatively low socioeconomic status and limited access to necessary health care in rural areas . PreviouFurthermore, we found women tended to have better control of hypertension compared to men, possibly because women were more sensitive to health education and had better compliance rates . Since aIschemic stroke remains an important healthcare challenge because of its increasing prevalence, high percentage of recurrence, and disabling sequelae . In recoThe present study comprehensively profiled the status of hypertension among ischemic stroke survivors in northeast China, providing population-based evidence for formulating comprehensive strategies for secondary stroke prevention and care in those areas. Our study has several limitations. Firstly, since it is a cross-sectional survey, we only have data on blood pressure at one point in time and further study focusing on the relationship between hypertension and long-term outcomes among the stroke population should be undertaken. Secondly, since the prevalence of hypertension was our only focus, other risk factors and comorbidities were not assessed in this study. Thirdly, our study did not include medical compliance in the present study, further implementation studies will need to account for anti-hypertensive medical adherence. Lastly, we could not analyze the duration of hypertension and time passed since stroke because of memory loss in stroke patients.Our study revealed some alarming information about hypertension among patients with prior ischemic stroke in northeast China. The high prevalence and poor control in addition to the high rate of stages 2 and above hypertension all indicate a large stroke burden in this population, especially in rural areas. Therefore, interventions tailored to improve the control of hypertension among this high-risk population should be undertaken.


To determine if medium- and long-term blood glucose control as well as glycemic variability, which are known to be strong predictors of vascular complications, are associated with underlying cerebral small vessel disease (cSVD) in neurologically asymptomatic individuals with type 1 diabetes.1c) values were collected over the course of ten years before the visit including a clinical examination, biochemical sampling, and brain magnetic resonance imaging. Markers of glycemic control, measured during the visit, included HbA1c, fructosamine, and glycated albumin.A total of 189 individuals  with type 1 diabetes  were enrolled in a cross-sectional retrospective study, as part of the Finnish Diabetic Nephropathy (FinnDiane) Study. Glycated hemoglobin (HbASigns of cSVD were present in 66 (34.9%) individuals. Medium- and long-term glucose control and glycemic variability did not differ in individuals with signs of cSVD compared to those without. Further, no difference in any of the blood glucose variables and cSVD stratified for cerebral microbleeds (CMBs) or white matter hyperintensities were detected. Neither were numbers of CMBs associated with the studied glucose variables. Additionally, after dividing the studied variables into quartiles, no association with cSVD was observed.We observed no association between glycemic control and cSVD in neurologically asymptomatic individuals with type 1 diabetes. This finding was unexpected considering the large number of signs of cerebrovascular pathology in these people after two decades of chronic hyperglycemia and warrants further studies searching for underlying factors of cSVD.The online version contains supplementary material available at 10.1007/s00592-021-01821-8. High blood glucose is a major risk factor for not only microvascular complications, but also cardiovascular disease in type 1 diabetes . Cardiov1c at the time of the imaging study. This warrants further analysis of glycemic control in relation to cSVD in this type 1 diabetes cohort with more than two decades of hyperglycemia.Notably, only few of the traditional risk factors were different in type 1 diabetes individuals with and without cSVDs. Blood pressure, a well-known risk factor for cSVD , was higThe aim of this study was to retrospectively determine whether medium- or long-term blood glucose control measured by different markers were associated with cSVD in neurologically asymptomatic individuals with type 1 diabetes. Additionally, we sought to investigate whether long-term glycemic fluctuations, known to predict vascular complications in this patient group, are predictive of cSVD.This study was performed as part of the Finnish Diabetic Nephropathy (FinnDiane) Study, a nationwide multicenter study aiming to identify genetic, environmental, and clinical risk factors for micro- and macrovascular complications in type 1 diabetes . A totalAll individuals were studied at the FinnDiane Research Center (Biomedicum) and the Medical Imaging Center at Helsinki University Hospital, both in Helsinki, Finland. Clinical visits included brain MRI scans, biochemical sampling, and a thorough clinical examination. The study visits and methods have been presented in greater detail before . Briefly1c), reflecting blood glucose control during a time span of one to two months, was measured using standardized assays in a central laboratory   , 17. HbAurements . The stu1c, GA, and FA values during the visits were 8.1% (7.4–8.9%), (65.0 mmol/mol [57.0–73.0 mmol/mol]), 91.6 nM/ml (74.3–116.4 nM/ml), and 2.6 mM/l (2.4–3.0 mM/l), respectively. HbA1c-meanoverall, collected over the course of ten years before the visit , were 8.1 ± 0.9% (65.4 ± 10.3 mmol/mol)  and HbA1c vs. HbA1c-meanoverall (p < 0.001). To overcome the possibility of bias by the number of HbA1c measurements we divided the study individuals into two groups, above and below median HbA1c count. The presence of cSVD were not different between the groups .One hundred and eighty-nine individuals with type 1 diabetes were enrolled for this study, with demographics previously presented in greater detail . Brieflyp = 0.011 for CMBs and 137 p = 0.005 for WMHs). The presence of WMHs correlated also with age  and the presence of CMBs with albuminuria . The other demographic variables were not associated with CMBs or WMHs.Individuals with CMBs or WMHs had higher systolic blood pressure compared to those without CMBs or WMHs , CMBs, or WMHs in individuals with type 1 diabetes. GA and FA did not correlate with cSVD , CMBs, or WMHs in brain MRIs  , and HbA1c-ARV , did not correlate with the presence of cSVD. Similarly, no correlation was observed between glycemic variability and WMHs, CMBs, or the number of CMBs  to maintain the continuous high glucose and energy demands of the brain , 38. Mec1c measurements, reflecting long-term blood glucose levels and fluctuations, is limited and this may have an effect on the statistical power to detect differences between the groups. A larger cohort would have enabled greater statistical power. It is, however, improbable that this would markedly have changed the results considering the consistence of the observations. The strengths of this study are the standardized imaging and clinical assessment, as well as the strong phenotypic data.Our study does not go without limitations. We had serial A1c values from ten years enabling us to assess both cumulative blood glucose control and blood glucose variability. The cross-sectional retrospective nature of the study should, however, be taken into account. Our study had no data regarding short-term glucose control such as time in range (TIR) or variability measured from continuous glucose monitoring systems (CGMS), leaving this interesting topic open for future studies. The number of participants and HbAWe observed no association between medium- and long-term blood glucose control and long-term glycemic variability and cSVD in neurologically asymptomatic individuals with type 1 diabetes. This finding was unexpected considering the large number of signs of cerebrovascular pathology in these people after two decades of chronic hyperglycemia and warrants further studies searching for underlying factors of cSVD.Supplementary file1 (DOCX 21 KB)Below is the link to the electronic supplementary material.


Microbiome sequence data have been used to characterize Crohn's disease (CD) and ulcerative colitis (UC). Based on these data, we have previously identified microbiomarkers at the genus level to predict CD and CD relapse. However, microbial load was underexplored as a potential biomarker in inflammatory bowel disease (IBD). Here, we sought to study the use of fungal and bacterial loads as biomarkers to detect both CD and UC and CD and UC relapse. We analyzed the fecal fungal and bacterial loads of 294 stool samples obtained from 206 participants using real-time PCR amplification of the ITS2 region and the 16S rRNA gene, respectively. We combined the microbial data with demographic and standard laboratory data to diagnose ileal or ileocolonic CD and UC and predict disease relapse using the random forest algorithm. Fungal and bacterial loads were significantly different between healthy relatives of IBD patients and nonrelated healthy controls, between CD and UC patients in endoscopic remission, and between UC patients in relapse and non-UC individuals. Microbial load data combined with demographic and standard laboratory data improved the performance of the random forest models by 18%, reaching an average area under the receiver operating characteristic curve (AUC) of 0.842 , for IBD diagnosis and enhanced CD and UC discrimination and CD and UC relapse prediction. Our findings show that fecal fungal and bacterial loads could provide physicians with a noninvasive tool to discriminate disease subtypes or to predict disease flare in the clinical setting.IMPORTANCE Next-generation sequence data analysis has allowed a better understanding of the pathophysiology of IBD, relating microbiome composition and functions to the disease. Microbiome composition profiling may provide efficient diagnosis and prognosis tools in IBD. However, the bacterial and fungal loads of the fecal microbiota are underexplored as potential biomarkers of IBD. Ulcerative colitis (UC) patients have higher fecal fungal and bacterial loads than patients with ileal or ileocolonic CD. CD patients who relapsed harbor more-unstable fungal and bacterial loads than those of relapsed UC patients. Fecal fungal and bacterial load data improved prediction performance by 18% for IBD diagnosis based solely on clinical data and enhanced CD and UC discrimination and prediction of CD and UC relapse. Combined with existing laboratory biomarkers such as fecal calprotectin and C-reactive protein (CRP), microbial loads may improve the diagnostic accuracy of IBD and of ileal CD and UC disease activity and prediction of UC and ileal CD clinical relapse. Saccharomyces, Malassezia, and Candida  and an increased proportion of Candida albicans  are associated with IBD, without a clear distinction between Crohn’s disease (CD) and ulcerative colitis (UC), the two main forms of IBD. Furthermore, an interaction between fungi and bacteria has emerged as a potential key player in the pathophysiology of IBD  , 5 prese6–y of IBD , howeverMany studies addressing the relationship between IBD and the microbiome have focused on the analysis of bacterial composition using 16S rRNA sequencing , 8 and oHere, we performed a retrospective study using fecal fungal and bacterial loads to characterize CD and UC patients during disease progression, and we compared these data with those from two groups of healthy controls . Using random forest—a supervised learning algorithm—we also combined demographic and laboratory variables with fungal and bacterial loads to evaluate their capacity to predict IBD, IBD subtypes, and disease flare.To assess the fungal and bacterial loads  of the 294 stool samples collected from healthy controls and IBD patients, we quantified the ITS2 region  and the 16S rRNA gene  by quantification by real-time PCR (qPCR). The number of copies per gram of stool varied from 1.25E+4 to 2.06E+9 for the ITS2 region and from 1E+8 to 5.7E+13 for the 16S rRNA gene, with an average copy number of 3.84E+07 and 1.9E+12 per gram of stool, respectively, leading to an average 16S-to-ITS2 ratio of 4.95E+04 . These rn = 28) collected at two time points . The fold change of the fungal or bacterial load between the two time points showed a greater variation of former community   between the copy numbers of ITS2 and 16S rRNA, thereby suggesting that the two microbial communities may vary independently in number of cells in order to maintain gastrointestinal (GI) tract homeostasis. Altogether, our findings confirmed previous studies showing that the fungal compartment of the gut microbiota is less abundant and less stable than the bacterial counterpart  . Interes2 . InterFaecalibacterium prausnitzii compared to nonrelated controls (n = 29) , which was due to a greater presence of fungi and a lower level of bacteria (n = 29) . Interestingly, no significant differences in the fungal and bacterial loads were found between the two groups of IBD relatives.Previous studies have reported that first-degree relatives of IBD patients present a dysbiotic gut microbial community composition and lower load of controls , 14. Ourbacteria . Althougn = 31) in remission had a higher fungal/bacterial load ratio compared to nonrelated healthy controls (n = 28)  and to CD patients in remission (n = 34)  (P = 0.0196). This difference was not observed between unaffected relatives of CD patients and their affected family members (P = 0.02), due mainly to a higher level of fungi in the former   . In cont members . Moreove 0.0076) .P = 0.0013) and bacterial  loads compared to those who remained in remission (P = 0.001), CD patients in remission (P = 0.001), CD patients in relapse (P = 0.001), and UC patients in remission (P = 0.001)  of ITS2 and 16S rRNA copies showed that both fungal and bacterial loads increased when CD patients relapsed (median fold change = 2.23) and decreased when patients remained in remission for a year (median fold change = 0.30) . In contemission . A similemission . A decre= 0.001) . This inP = 0.014). This observation strengthens the results obtained with the Spanish cohort and points to an absence of geographical differences.We validated some of these findings using another CD cohort from a prior study in Belgium  in whichP = 0.02)  were measured on available samples of IBD patients from the Spanish cohort at baseline and then at relapse or after 1 year if the patient remained in remission. Fecal calprotectin was significantly different between remission and relapse for both CD and UC patients, and CRP showed significant differences only between UC at baseline (in remission) and UC at flare   and B. ASince the utilization of immunosuppressants may impact microbial loads , we comp10.1128/mSystems.01277-20.2TABLE S1Table S1, PDF file, 0.08 MB.Characteristics of participants. Download Copyright © 2021 Sarrabayrouse et al.2021Sarrabayrouse et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Efforts channeled into developing a noninvasive test that discriminates disease subtypes or predicts disease flare have been driven by the many disadvantages of endoscopy. Endoscopy, apart from being expensive and uncomfortable for the patients, may not be decisive and therefore may delay treatment decisions. To this end, we used random forest, a supervised learning algorithm, to examine the capacity of fungal and bacterial loads to predict IBD, the two major IBD subtypes (CD and UC), and disease flare. We created several random forest models based on fungal and bacterial load data and, when available, other standard laboratory and demographic data, and we randomly split the population of interest into a training set (2/3) and a test set (1/3).n = 58) and used them as the case group. The NRHC group (n = 28) was used as the control group. For this classification and due to missing standard laboratory or clinical data as predictor variables for the NRHC subjects, we were limited to microbial data and a few demographic data, such as age, weight, height, body mass index (BMI), gender, and smoking habit. We obtained an area under the receiver operating characteristic (ROC) curve (AUC) of 0.80  (n = 29), and we improved the model with an AUC of 0.89  with the UC relatives (n = 29)  . Fungal (n = 29) . Fungal n = 65) as the case set and used the NRHC cohort (n = 28) as the control set. For these analyses, we were again limited to microbial data and a few demographic data such as age, BMI, gender, and smoking habit. The predictive variables that led to the highest AUC of 0.83 with a high sensitivity of 1 and a low specificity of 0.66  were fungal and bacterial loads, BMI, and smoking habit (n =34), whereas the AUC was 0.89  with UC (n =31)  . Splittin = 34) from UC (n = 31), we built a model based on predictor variables collected at baseline for both groups that included fungal and bacterial loads, serum hemoglobin, fecal calprotectin levels, and number of previous relapses. The AUC on the representative test set was 0.76 with a sensitivity of 0.82 and a low specificity of 0.7  (n = 31) from CD (n = 34), variables such as fungal and bacterial loads, previous number of relapses, and CRP and calprotectin levels led to the highest AUC of 0.86, sensitivity of 0.9, and specificity of 0.82  (To discriminate CD (to 0.95) . To disc.7 to 1) .n = 34), we used the data sets collected at baseline and the disease severity status after 1 year of follow up: 21 CD patients remained in remission and 13 relapsed. The most important predictor variables were bacterial load, CRP and calprotectin levels, and previous number of relapses, which allowed us to achieve the highest AUC of 0.87, sensitivity of 0.75, and specificity of 1  (n = 31), we also used the data sets collected at baseline and the disease severity status after 1 year of follow up, during which 13 UC patients remained in remission and 18 relapsed. The most important predictors were bacterial load, fungal-to-bacterial-load ratio, weight, gender, previous number of relapses, and smoking habit, for which we achieved an AUC of 0.83, sensitivity of 0.67, and specificity of 1  (To predict flare in CD patients (.6 to 1) . To pred51 to 1) .We finally evaluated the impact of standard markers such as calprotectin and CRP, measured only in IBD patients, in the predictions of relapse. For this purpose, we built two models with and without the markers. As shown in 10.1128/mSystems.01277-20.1FIG S1FIG S1, PDF file, 0.05 MB.Receiver operating characteristic (ROC) curves for the models testing calprotectin and CRP. (A) ROC curve for CD. (B) ROC curve for UC. The models either combined CRP and calprotectin with other markers such as bacterial and fungal loads  or did not take into account calprotectin and CRP . Download Copyright © 2021 Sarrabayrouse et al.2021Sarrabayrouse et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the From all the models constructed, the analyses indicated that combining microbial data with other laboratory and demographic data improved the predictions by 18% .Microbiome sequence data have been proposed to distinguish IBD from non-IBD, and CD from UC, and to predict disease relapse and led to microbiomarkers for diagnosis and prognosis , 16, 17.First, independently of health status, we estimated that the number of fungal cells accounts for 0.04% of the bacterial cells, which is very close to the 0.03% of the fecal microbiota previously proposed by Qin et al.  To the bFaecalibacterium prausnitzii than nonrelated healthy controls  and demographic data, could provide physicians with a noninvasive and highly efficient test through which to discriminate disease subtypes or predict disease flare in the hospital setting.We used fecal samples collected in two previous Spanish and Belgian IBD retrospective cohorts. The characteristics of the IBD patients are described in the work of Pascal et al.  and MachFor the Spanish cohort, inclusion criteria included a diagnosis of UC and ileal or ileocolonic CD confirmed by previous endoscopy and histology. Clinical remission for at least 3 months was diagnosed based on the validated colitis activity index (CAI) for UC and the CD activity index (CDAI) for CD, stable maintenance therapy , and a history of at least three clinical recurrences in the past 5 years. All healthy controls had no history of chronic disease. At inclusion and during the follow-up, we collected the following data: diagnostic criteria; location and behavior of CD; extension of UC; and standard laboratory and demographic data, including blood cell count, CRP, hemoglobin levels, fecal calprotectin, smoking habit, gender, height, and weight. Clinical recurrence was defined by a value of 4 or higher for the CAI and higher than 150 for the CDAI. Exclusion criteria included pregnancy or breast-feeding; severe concomitant disease involving the liver, heart, lungs, or kidneys; and treatment with antibiotics during the previous 4 weeks.For the Belgian cohort, 55 patients with active CD, who underwent an ileocecal resection, were prospectively recruited between 2011 and 2016 via the University Hospitals Leuven (Belgium) . Fecal sFor both the Spanish and Belgian cohorts, the protocols were submitted and approved by the local Ethical Committee of the University Hospital Vall d’Hebron  and of the University Hospital Gasthuisberg , respectively. All volunteers received information concerning their participation in the study and gave written informed consent.Demographic data included gender, age, height and weight, body mass index (BMI), smoking history, age at disease onset, and number of previous relapses. Standard laboratory data included blood count, serum CRP and hemoglobin levels, fecal fungal and bacterial loads, and fungal-to-bacterial load ratio. Fecal calprotectin was measured as previously described in the work of Pascal et al. .N-lauroyl sarcosine, and 500 μl of 5% N-lauroyl sarcosine. Genomic DNA was extracted by mechanical disruption of the microbial cells with beads, and nucleic acids were recovered from clear lysates by alcohol precipitation.Fecal samples collected in Spain and Belgium in the previous studies were immediately frozen by the participants in their home freezer at −20°C and later brought to the laboratory in a freezer pack, where they were stored at −80°C. Genomic DNA was extracted following the recommendations of the International Human Microbiome Standards (IHMS) . A froze−1 to 10−4, were tested in an attempt to identify the appropriate concentration of the template that would optimize the PCR amplification system, reaching a balance between the smallest amount of potential PCR inhibitors and the highest detectable amount of DNA template. The PCR product obtained from the 10−1 DNA dilution template provided the optimal PCR amplification and was then used for ligation into the pCR 2.1-TOPO cloning vector  and for bacterial transformation in the Top10F strain of Escherichia coli. After culturing the transformed bacteria, we randomly picked up 20 colonies that we recultivated in LB liquid medium to perform plasmid extraction. We randomly selected five extracted plasmids and performed the ITS2 PCR as previously described. PCR results showed three ITS2 regions of different sizes inserted into the five plasmids. We tested each of them for their ability to provide a real-time PCR standard curve. Serial dilutions of the ITS2 plasmid, with concentrations ranging from 106 to 10° copies/reaction, were prepared, and the fungal ITS2 region was amplified using ITS2-fungi-sense (5′-GTG ART CAT CGA ATC TTT-3′) and ITS2-fungi-antisense (5′-GAT ATG CTT AAG TTC AGC GGG T-3′) primers. The real-time PCR amplifications were done using the ITS2 PCR program, under the following conditions: denaturation at 95°C for 2 min, followed by 40 amplification cycles at 95°C for 30 s, at 55°C for 30 s, and at 72°C for 60 s, and a final extension cycle at 72°C for 10 min. Real‐time PCR software  was used to calculate the slope and the R2 (Pearson coefficient of determination) to evaluate the standard curve efficiency of each plasmid. A satisfactory reaction should have an efficacy of between 90% and 110%, which corresponds to a slope of −3.58 to −3.10 and an R2 value between 1 and 0.90. For each plasmid, we repeated the standard curve assay three times and finally selected the plasmid HD8, which showed the greatest efficiency over the three experiments. We confirmed the reproducibility of the efficiency of the standard curve obtained with HD8 by repeating the standard curve assay several times.The internal transcribed spacer 2 (ITS2) region of fungi was amplified by PCR using the genomic DNA extracted as described above. Specific primers targeting the ITS2 gene were used for amplification: ITS2-fungi-sense (5′-GTG ART CAT CGA ATC TTT-3′) and ITS2-fungi-antisense (5′-GAT ATG CTT AAG TTC AGC GGG T-3′) . PCR was2 to 107) and fungal (from 10° to 106) copy numbers. Results were expressed in copies per gram of stool.Fungal and bacterial loads were estimated in fecal samples by quantifying the ITS2 region and 16S rRNA gene by qPCR, respectively. Amplification was carried out using a 7500 Fast real‐time PCR system . The fungal ITS2 region was amplified using ITS2-fungi-sense (5′-GTG ART CAT CGA ATC TTT-3′) and ITS2-fungi-antisense (5′-GAT ATG CTT AAG TTC AGC GGG T-3′) primers. The V4 hypervariable region of the 16S rRNA genes (290 bp) was amplified using the following primers: V4F_517_17 (5′‐GCC AGC AGC CGC GGT AA‐3′) and V4R_ 805_19 (5′‐GAC TAC CAG GGT ATC TAA T‐3′) . The PCRTo assess differences in the microbial load between groups on the basis of disease status or disease evolution, we used the nonparametric Wilcoxon signed-rank test (pairwise paired comparisons) and Mann-Whitney U test (pairwise unpaired comparisons). We considered a type I error of 5%. To evaluate the relation between clinical and/or experimental variables, we calculated nonparametric Spearman correlation index.For different disease prediction models such IBD diagnosis, CD and UC discrimination, and CD and UC relapse prediction, we used random forest, a supervised learning algorithm that randomly constructs and merges multiple decision trees on different bootstrap samples into one “forest” at the training step. To build each random forest model, we used the number of trees that provided the highest AUC (area under the receiver operating characteristic [ROC] curve). The following predictor variables were used in models that included IBD and non-IBD cohorts: age, BMI, gender, smoking status, fungal and bacterial loads, and fungal-to-bacterial load ratio. The variables of models that involved only IBD patients were as follows: age; BMI; gender; smoking habit; microbial data; height; weight; age at disease onset; number of previous relapses; smoking history; CRP, hemoglobin, and calprotectin levels; and white blood cell count.All data are incorporated into the article and its supplemental material.


KIT D816V-driven hematologic neoplasm characterized by mast cell infiltration and shortened survival. We report the results of a prespecified interim analysis of an ongoing pivotal single-arm phase 2 trial (no. NCT03580655) of avapritinib, a potent, selective KIT D816V inhibitor administered primarily at a once-daily starting dose of 200 mg in patients with AdvSM (n = 62). The primary endpoint was overall response rate (ORR). Secondary endpoints included mean baseline change in AdvSM–Symptom Assessment Form Total Symptom Score and quality of life, time to response, duration of response, progression-free survival, overall survival, changes in measures of disease burden and safety. The primary endpoint was successfully met (P = 1.6 × 10-9), with an ORR of 75%  in 32 response-evaluable patients with AdvSM who had sufficient follow-up for response assessment, including 19% with complete remission with full or partial hematologic recovery. Reductions of ≥50% from baseline in serum tryptase (93%), bone marrow mast cells (88%) and KIT D816V variant allele fraction (60%) were observed. The most frequent grade ≥3 adverse events were neutropenia (24%), thrombocytopenia (16%) and anemia (16%). Avapritinib demonstrated a high rate of clinical, morphological and molecular responses and was generally well tolerated in patients with AdvSM.Advanced systemic mastocytosis (AdvSM) is a rare,  In a prespecified interim analysis of a pivotal phase 2 trial, avapritinib, a selective KIT inhibitor, elicited robust clinical and molecular responses, was generally well tolerated and led to improved patient-reported outcomes in patients with advanced systemic mastocytosis. KIT D816V mutation in ~95% of cases. The KIT D816V mutation drives the increased proliferation and accumulation of neoplastic mast cells, leading to severe, debilitating and often unpredictable symptoms and poor quality of life (QoL)3. In AdvSM, mast cell infiltration leads to organ damage, referred to as ‘C-findings’ , with limited treatment options and poor survival6. AdvSM is comprised of three subtypes: aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN) and mast cell leukemia (MCL)2. In SM-AHN, which represents 60–70% of cases of AdvSM8, patients concurrently have both SM and another World Health Organization-defined hematologic neoplasm, usually myeloid 9. The KIT D816V mutation is frequently also present in cells comprising the AHN component9. Molecular subtyping of patients with AdvSM often reveals a heterogenous genetic landscape, with high-risk mutations in splicing factors, epigenetic regulators and transcription factors such as SRSF2, ASXL1 and RUNX1, respectively12. Treatment options are very limited, with only the multikinase inhibitor midostaurin and, more recently, the selective KIT kinase inhibitor avapritinib approved for the treatment of AdvSM.Systemic mastocytosis (SM) is a rare hematologic neoplasm that is associated with the 1. As detailed in the accompanying report13, avapritinib was investigated in patients with AdvSM in the phase 1 EXPLORER trial (no. NCT02561988)14. In this trial, avapritinib exhibited an ORR of 75% by modified (m)IWG–MRT–ECNM  criteria, including 36% with complete remission with full (CR) or partial hematologic recovery (CRh), with a median follow-up of 23 months. Responses were seen at all starting doses (30–400 mg once daily (QD)) and deepened over subsequent cycles, but occurred most rapidly at 200 mg QD and higher. Patients experienced profound reductions in objective measures of mast cell burden, including complete molecular remission of KIT D816V, reversion of mast-cell-related organ damage and improvements in symptoms. Review of safety, rapid reduction of disease burden and response rate led to selection of 200 mg as the optimal dose for patients with AdvSM.Avapritinib is a selective KIT and platelet-derived growth factor receptor-alpha (PDGFRA) kinase inhibitor with high potency for the KIT D816V and homologous PDGFRA-mutant proteins15, an ongoing, international, multicenter, open-label, single-arm, phase 2 registrational trial (no. NCT03580655) of avapritinib 200 mg QD in adult patients with a centrally confirmed diagnosis of AdvSM. Patients were enrolled into cohort 1  if they had an evaluable mIWG–MRT–ECNM C-finding  . In addition, cohort 2 included patients without an evaluable C-finding at baseline  and were therefore not evaluable for response. All enrolled patients (safety population) were included in secondary endpoints, which included changes in patient symptoms, reduction in measures of disease burden and safety.Here, we present the results of a prespecified interim analysis from the PATHFINDER trialn = 9), SM-AHN (n = 43) and MCL (n = 10)) and received avapritinib primarily at a starting dose of 200 mg QD , across cohort 1 (n = 52) and cohort 2 (n = 10) , which is associated with poor survival in SM12. The majority of patients (72%) had received previous antineoplastic therapy, including 53% with midostaurin . The median KIT D816V variant allele fraction (VAF) in peripheral blood (which rarely has circulating mast cells) was 15% . Median spleen volume was 939 ml . Baseline characteristics were generally balanced between the safety population and those included versus not included in the interim analysis efficacy population  and 56% of patients were male , with six patients (19%) achieving CRh. Ten patients (31%) achieved PR and eight (25%) had CI . Responses continued to improve over time , the confirmed ORR  was 75% (n = 62), consistent and profound reductions in measures of mast cell burden (secondary endpoint) were observed in all enrolled patients with baseline and postbaseline assessments. A reduction of ≥50% in bone marrow mast cells was observed in 88% (44/50) of patients, and 60% (30/50) had elimination of bone marrow mast cell aggregates  reduction in KIT D816V VAF in the peripheral blood in 60% (33/55) of patients; 35% (19/55) of patients achieved a VAF of <1%  of patients with SM and chronic myelomonocytic leukemia (SM-CMML), and a decrease of ≥50% in absolute eosinophil counts in 88% (14/16) of patients with eosinophilia, including all three patients with SM and chronic eosinophilic leukemia (SM-CEL)  in the interim analysis efficacy population and median overall survival (OS) in the safety population (secondary endpoints) had not been reached at the time of data cutoff. The estimated 6-, 9- and 12-month PFS rates were 91, 87 and 79%, respectively; corresponding OS rates were 94, 86 and 86%.As of the data cutoff, 52 (84%) of 62 patients were still on treatment with a median follow-up of 7.0 months (range 5.6–8.1). Reasons for treatment discontinuation included adverse events (AEs) in six patients ), disease progression as per the investigator in three patients (with one transformation to acute myeloid leukemia and two with worsening AHN) and consent withdrawal in one patient  was uncommon, at 8%. Grade ≥3 treatment-related AEs were reported in 32 (52%) patients, the most frequent of which were neutropenia (23%) and thrombocytopenia (15%). There were three (5%) deaths due to AEs , none of which were considered related to treatment. Cognitive effects  occurred in seven (11%) patients (Table n = 6), with one grade 2.The most frequent AEs are presented in Table 9 l–1) who was enrolled before the exclusion (below) of such patients. The patient was treated with avapritinib despite worsening thrombocytopenia  and developed a grade 2 subdural hematoma 2 days after a single dose of enoxaparin. The patient was not taking antiplatelet agents and had a relatively normal international normalized ratio and activated partial thromboplastin time of 1.2 and 28.1 s, respectively, before the ICB event. The patient subsequently recovered from the event and was retreated with avapritinib due to continuing benefit. However, the patient subsequently developed a grade 4 subdural hematoma despite platelets being 167 × 109 l–1, leading to treatment discontinuation.There was one (1.6%) intracranial bleeding (ICB) event , in a patient with severe thrombocytopenia at baseline  experienced AEs leading to permanent treatment discontinuation. Two (5%) patients discontinued due to a treatment-related serious AE . Dose interruptions due to AEs occurred in 34 (55%) patients, most commonly due to neutropenia (21%) and thrombocytopenia 16%)  score of only 37.8  and a high proportion of patients (31%) with a poor Eastern Cooperative Oncology Group (ECOG) performance status of 2 or 3. Mean and median Patient Global impression of Symptom Severity (PGIS) scores were 2.6 and 3.0, respectively (where 0 represents no symptoms and 4 very severe symptoms). The AdvSM–Symptom Assessment Form (SAF), an AdvSM-specific patient reported outcomes tool, showed that fatigue, abdominal pain and spots were the most severe symptoms, with a mean total symptom score (TSS) of 18.3, which was the sum of eight possible common symptoms . At baseline, patients had frequent supportive care medication use, including H1 antihistamines (58%), H2 antihistamines (39%) and corticosteroids (32%). Patients on corticosteroids remained evaluable if their dose did not exceed 20 mg dn = 51) and by 9.8 points from baseline at treatment cycle 11  , role (work or household jobs), emotional , cognitive (memory and concentration) and social  functioning domains  formed the basis of approval by the United States Food and Drug Administration for the treatment of adults with AdvSM at a recommended starting dose of 200 mg orally QD.This prespecified interim analysis of the phase 2 PATHFINDER trial demonstrated that avapritinib at a starting dose of 200 mg QD exhibited clinical benefit in patients with AdvSM, confirming findings from the phase 1 EXPLORER trial. In total, 75% of patients achieved a response regardless of AdvSM subtype, previous therapy or adverse Consistent and profound reductions in mast cell burden  and normalization of SM-related organ damage  were observed in patients across all AdvSM subtypes regardless of previous therapy. Although improvements in cytopenias were seen in some patients, including improvement of transfusion-dependent anemia in one patient, cytopenias were less likely to normalize than other C-findings, even in patients who no longer had bone marrow mast cell aggregates. This implies that persistent cytopenias were related to non-mast cell causes, such as drug effect and/or the remaining AHN component.KIT D816V VAF, consistent with broad activity against both KIT D816V-positive mast cells (which rarely circulate in the blood) and cells derived from the AHN which often harbor KIT D816V, reflecting multilineage involvement of the mutation. Some patients with SM-AHN may need AHN therapy in combination or in sequence with a KIT-inhibitor-based regimen to address the genetic and biologic heterogeneity which underpins this AdvSM subtype. However, most patients had improvement in their AHN with avapritinib alone. This activity supports exploration of avapritinib in other KIT D816V-positive hematologic malignancies.In addition, avapritinib demonstrated evidence of broader disease activity in patients with SM-AHN, which comprises the majority of AdvSM variants. For example, substantial reductions of monocytosis in 80% of patients with SM-CMML and eosinophilia in 88% patients with baseline eosinophilia or SM-CEL were observed. Overall, 60% of patients had substantial reductions in peripheral blood Patients had marked impairments in QoL at baseline due to their AdvSM symptoms yet reported rapid, durable and substantial reductions from baseline in every AdvSM symptom assessed, and improvements in QoL and functional impairment. Median global symptom severity as measured by PGIS had improved from severe to minimal by cycle 11.Avapritinib was generally well tolerated, with few discontinuations due to AEs. Overall, the safety profile at the starting dose of 200 mg QD in the PATHFINDER trial included mostly low-grade fluid retention and gastrointestinal symptoms as the most common nonhematologic AEs. Cytopenias were the most common grade ≥3 AEs and reason for dose reduction, most commonly in patients with baseline cytopenias, and 100 mg was the median daily dose after cycle 3. Cognitive effects, which are dose-related, were relatively uncommon at a starting dose of 200 mg.18, probably due to mitigation steps for severe thrombocytopenia which were implemented early in enrollment. These included exclusion of patients with platelets <50 × 109 l–1, closer monitoring of platelet counts, stricter dose modification guidelines and use of platelet transfusions and growth factors to maintain platelet count to ≥50 × 109 l–1. Dose-related, low-grade cognitive events were observed but did not result in substantial dose reductions. The optimal starting dose of 200 mg QD was confirmed in the current trial, because preliminary data generated in the EXPLORER trial suggested that this dose maintained efficacy while improving tolerability.The incidence of ICB was low (1.6%), and similar to that observed with avapritinib in patients with GIST22. Treatment with midostaurin for AdvSM can lead to challenging gastrointestinal side effects, limiting its efficacy. With avapritinib, a potent, selective inhibitor of KIT D816V, the clinical aim in AdvSM is to achieve deeper and enduring clinical, morphological and molecular responses, which could translate into extended survival with improved QoL.These data reinforce KIT D816V as a clinically validated drug target in AdvSM. While the multikinase inhibitor midostaurin is already approved for use in patients with AdvSM, there remains a substantial need for improvement in the overall rate, durability and quality of responses13.A potential limitation of this prespecified interim analysis is the short duration of follow-up (10.4 months) in a subset of trial patients, which will be addressed in long-term follow-up analyses of the study. It is anticipated that findings from long-term analyses will be consistent with those observed in the phase 1 trialKIT D816V, highlighting the potential for modification of AdvSM disease natural history.In conclusion, interim results from the current phase 2 PATHFINDER trial corroborate the promising and durable outcomes observed in the phase 1 EXPLORER trial. Avapritinib administered at a starting dose of 200 mg QD was well tolerated and led to profound reductions in disease burden, improved patient symptoms and QoL, and elicited deep molecular responses of 23 required an evaluable baseline C-finding  for ≥14 days before C1D8; and able to provide written informed consent. Patients were excluded for the following reasons: received previous treatment with avapritinib; received any cytoreductive therapy or an investigational agent <14 days and, for cladribine, interferon alpha, pegylated interferon and any antibody therapy, <28 days before obtaining screening bone marrow biopsy; received previous radiotherapy or any hematopoietic growth factor within 14 days before screening bone marrow biopsy; requires concomitant medication that is a strong inhibitor, strong inducer or moderate inducer of cytochrome P450 3A4; had a major surgical procedure within 14 days of the first dose of study drug; is a candidate for allogeneic hematopoietic stem cell transplantation for treatment of SM; eosinophilia and known positivity for the FIP1L1–PGDFRA fusion, unless the patient has demonstrated relapse or progressive disease on previous imatinib therapy; has history of another primary malignancy (within 3 years before the first dose of study drug), cerebrovascular accident or transient ischemic attacks (within 1 year before the first dose of study drug), or seizure disorder; has abnormal laboratory findings or QT interval corrected using Fridericia’s formula >480 ms; has known risk or recent history of ICB; has a primary brain malignancy or metastases to the brain; has clinically significant, uncontrolled cardiovascular disease; unless postmenopausal  or surgically sterile; is unwilling to abstain from sexual intercourse or employ highly effective contraception from the first dose of study drug and for at least 6 weeks after the last dose of study drug; pregnant or breastfeeding; hypersensitivity to avapritinib or to any of the excipients; unwilling or unable to comply with the study procedures or requirements; and participation in another interventional study.Additional inclusion criteria were as follows: patients with SM-AHN who had received previous treatment for the AHN component of disease if, in the opinion of the investigator, such therapy was appropriate; bone marrow biopsy taken within 56 days of cycle 1, day 1 (C1D1); serum tryptase levels ≥20 ng mlNCT03580655) is an ongoing, international, multicenter, open-label, single-arm, phase 2 registrational trial conducted in North America and Europe. Avapritinib was administered at a starting dose of 200 mg QD in 28-day cycles until progression, intolerance, withdrawal by the investigator or patient or death. Dose modification to as low as 25 mg QD was allowed as per prespecified criteria. Dose increases to 300 mg QD were allowed for lack of efficacy and dose interruptions for platelet counts <50 × 109 l–1 were required, although platelet transfusion and growth factor support were allowed on trial.PATHFINDER ; cromolyn sodium and other mast cell stabilizers; and omalizumab.Previous cytoreductive therapy or investigational agents were permitted if received up to 14 days before the screening bone marrow sample or up to 28 days  before the screening marrow biopsy. Medications, including palliative and supportive care for disease-related symptoms, were permitted during the study and may include the following classes of agents: histamine receptor H1 and H2 blockers; proton pump inhibitors; osteoclast inhibitors ; leukotriene receptor antagonists; corticosteroids  had sufficient follow-up to be adjudicated for response as per mIWG–MRT–ECNM criteria.25. Other secondary endpoints included time to response (time from the start of treatment to the response according to mIWG–MRT–ECNM criteria); duration of response ; PFS ; OS (time from the start of treatment to the date of death); and changes in mast cell burden, safety and QoL using EORTC–QLQ–C30. Additional planned secondary endpoints not reported in this manuscript included pharmacokinetics of avapritinib and morphological response based on the Pure Pathological Response criteria26.The key secondary endpoint was mean change from baseline in patient-reported TSS of the AdvSM–SAF, a validated measure to assess treatment benefit in AdvSM patientsResponse according to mIWG–MRT–ECNM criteria was assessed at C1D15, C2D1, C3D1, C7D1 and every six cycles thereafter, 12 weeks after documentation of CR or PR, 4 weeks after progressive disease of the AdvSM and/or AHN components and at the end of therapy if discontinued for a reason other than progressive disease or initiation of alternative cytoreductive therapy. Patient-reported outcomes  were collected at each visit through cycle 17 and at the end of therapy (if before or at cycle 17).All patients were followed for safety (until 30 days after treatment discontinuation) and for long-term survival every 3 months. Safety assessments included determination of ECOG performance status, clinical laboratory testing, vital signs, electrocardiograms, brain imaging (magnetic resonance imaging or computerized tomography scan) and physical examinations. Treatment-emergent AEs were defined as any AE that occurred between the first dose of avapritinib through 30 days after the last dose of avapritinib, and were graded according to National Cancer Institute Common Terminology Criteria for AEs, v.5.0.19. The data cutoff date for this interim analysis was 23 June 2020, and was performed when 32 patients in cohort 1 had received six cycles of treatment and at least two postbaseline bone marrow assessments or had an end-of-study assessment at any timepoint. In the interim analysis, the null hypothesis was rejected if one-sided P < 0.00625. In the case of failure of the interim analysis, the final analysis would be tested at a one-sided alpha level of 0.02178. Two-sided 95% confidence intervals were based on the exact binomial distribution (Clopper–Pearson method). Time-to-event outcomes  were determined using the Kaplan–Meier method, and estimates were computed using Greenwood’s formula. Summary statistics are presented for time to response. Maximum percentage baseline reduction in clinicopathological measures of response  was based on patients with both baseline  and at least one baseline assessment. The mean change from baseline AdvSM–SAF TSS to C11D1 was tested against the null hypothesis of ≥0. If one-sided, one-sample t-test P < 0.025, the null hypothesis was rejected. Summary statistics and change from baseline are presented for PGIS and QoL assessment by EORTC–QLQ–C30.The null hypothesis ORR of 28% versus the alternative hypothesis, ORR of 50%, a one-sided type I error rate of 0.025 and a sample size of approximately 63 patients in cohort 1 were determined to have 93.5% power using the exact one-sample binomial test. The null hypothesis, ORR of 28%, was based on ORR as per IWG–MRT–ECNM criteria for midostaurinAll safety analyses, QoL outcomes and secondary analyses were evaluated in the safety population, comprising all enrolled patients. All statistical analyses were conducted using SAS v.9.4 or higher.The trial was designed by the sponsor (Blueprint Medicines Corporation) and trial investigators. The full protocol was approved by the institutional review board (IRB) or independent ethics committee (IEC) of each participating center: St Michael’s Hospital Research Ethics Board, Toronto, Canada; East of England – Cambridge South Research Ethics Committee, Nottingham, UK; University of Pennsylvania Office of Regulatory Affairs, PA, USA; Dana-Farber Cancer Institute, Office for Human Research Studies, MA, USA; University of Utah IRB, UT, USA; University of Michigan Medical School Institutional Review Board, MI, USA; MD Anderson Cancer Center, Office of Protocol Research, TX, USA; Stanford University, Research Compliance Office, CA, USA; Columbia University Medical Center IRB, NY, USA; Western IRB, WA, USA; Rush University Medical Center IRB, IL, USA; Washington University, Human Research Protocol Office, MO, USA; Roswell Park IRB, NY, USA; Region Syddanmark, Vejle, Denmark; Comité de protection des personnes Sud Est 1, Saint-Étienne, France; Ethikkommission II der Universität Heidelberg, Medizinische Fakultät, Mannheim, Germany; Medical Ethics Review Board, Groningen, the Netherlands; Comitato Etico Campania Sud – ASL; Napoli 3 Sud, Italy; Comitato Etico Area Vasta Centro, Florence, Italy; CEIC Hospital Universitari Pare Tau Ii – Oficina de Recerca, Barcelona, Spain; REK SØR/ØST, Oslo, Norway; and Independent Bioethics Committee for Scientific Research at the Medical University of Gdansk, Gdansk, Poland. The trial was conducted in accordance with the Declaration of Helsinki, International Conference on Harmonisation guidelines for Good Clinical Practice and local regulations. All patients provided written informed consent. Participants were not compensated, except for reimbursement of reasonable travel expenses. Each local IRB/IEC was notified based on local regulations (where required by the IRB/IEC) of all serious, unexpected adverse drug reactions involving risk to human patients. The sponsor and authors jointly collected and analyzed the data. All authors had access to all data, reviewed and provided critical input to the manuscript and made the decision to submit it for publication. All authors vouch for the validity of the trial results and adherence to the protocol.Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41591-021-01539-8.Supplementary InformationSupplementary Tables 1 and 2.Reporting Summary


Diabetic nephropathy (DN) is a chronic kidney disease that develops in patients with diabetes mellitus (DM). Renal dysfunction and persistent proteinuria are the main clinical features of DN. Podocyte injury is an important cause of persistent proteinuria and diabetic kidney disease (DKD) progression. Traditional Chinese patent medicines can improve renal function by enhancing autophagy and promoting apoptosis. Keluoxin is a Chinese patent medicine that has the effect of invigorating qi and nourishing yin, activating blood, and eliminating blood stasis. Therefore, we hypothesized that Keluoxin may have a protective effect against diabetic nephropathy in rats with type 2 DM. Rats induced with diabetes through streptozocin (STZ) injection and a high-fat and high-sugar diet were treated with Keluoxin (0.63 g/kg/day) for 8 weeks, and renal function, biochemical indicators, and histopathological changes in renal tissues were observed. Immunofluorescence staining and western blot analysis were used to detect the expression of autophagy-related proteins. The results showed that Keluoxin reduced blood glucose and lipid levels, improved renal function, and alleviated renal histopathological changes in rats with DN. The therapeutic effect was similar to that of Irbesartan (15.6 mg/kg/day). It is inferred that the mechanism works through reducing the obstruction of downstream pathways of autophagy by improving the lysosomal degradation function and alleviating podocyte injury. This study demonstrates that Keluoxin could regulate autophagy in podocytes, alleviate kidney injury in rats with DN, and have a protective effect on renal function; its mechanism can thus be a potential therapy for DN. Diabetic nephropathy (DN) is one of the most common microvascular complications in diabetes mellitus (DM); it has become the main cause of chronic kidney disease (CKD) in developed countries and is an important cause of end-stage nephropathy in China , 2. The Autophagy of cells may be the pathological basis of DN and has been widely studied in recent years . It is aLycium barbarum, Ligustrum lucidum, Leech, and Rhubarb, which are mainly used in the treatment of qi and yin deficiency and blood stasis in diabetes. Studies have shown that Astragalus membranaceus, Rhubarb, and Hirudin can reduce glomerular pathological changes by regulating autophagy and reduce mean blood glucose level, body weight, urinary albumin excretion, and serum creatinine level in rats with DN, thereby improving renal function [Ligustrum lucidum, Radix Pseudostellariae, and Lycium medlar also have certain protective effects on renal function and can reduce damage to DN podocytes. A study showed that Chinese patent medicine with the Yiqi Yangyin Huayu formula can reduce the level of urinary protein, reverse renal ultrastructural changes in rats with DN, alleviate the early onset of DN, and inhibit its progression [Traditional Chinese medicine, for example, Rhizoma Coptidis, Curcumin, Yishen capsule, Celastrol, and Hirudin, has achieved remarkable results in the treatment of DN , 18–21. function –26. Ginsfunction . Ligustrgression . Chinesen = 28) were sourced from the Chengdu Dossy Experimental Animals Company. All rats were housed in a specific pathogen-free (SPF) animal room, maintained at 24 ± 1°C with a regular 12 h light/dark cycle, and granted free access to water and food for one week before starting the experiment. The standard laboratory chow and tap water were available ad libitum. All animal experimental protocols were based on international guidelines and approved by the Ethics Committee of Chengdu Dossy Experimental Animals Company.Male Sprague Dawley rats weighing 200 ± 20 g  was intraperitoneally injected into the 21 rats in the experimental group, and the normal group was injected with the same volume of saline (pH = 4.5); after 72 h, fasting blood glucose levels were determined using blood samples from the tail vein, in all the rats. Rats with a serum glucose level ≥16.7 mmol/L for three consecutive days were deemed as representing the DM model. After two weeks, the urine over a 24 h period of rats in the experimental group was collected, and their urine microalbumin levels were found to be greater than 30 mg/24 h; the DN model in rats was thus successfully created. The rats representing the model were then randomly divided into three groups: the DN group (model group), Keluoxin group , and Irbesartan group , with seven rats in each group. Rats in the normal group and model group were administered with normal saline (10 ml/kg/day), while those in the Keluoxin group were treated with Keluoxin suspension prepared from capsules (0.63 g/kg/day), and those in the Irbesartan group were treated with Irbesartan (15.6 mg/kg/day). Rats in all the groups were observed for eight weeks. Eight weeks later, the rats were sacrificed.Spot urine and serum samples were collected just before euthanasia. Blood samples were collected from the abdominal aorta of rats under mild ether anesthesia in heparinized centrifuge tubes. The samples were immediately centrifuged at 3500 rpm to separate the plasma, extract the supernatant, and determine the relevant indicators. The appropriate kits purchased from Abcam  were used to measure fasting plasma glucose (FPG), 2 h oral glucose tolerance test (2 h OGTT) values, total cholesterol (TC), glycerin trilaurate (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). Commercial kits purchased from Bethyl Laboratories  were used to measure urine microalbumin (MAU), uric creatinine (UCr), the ratio of urine microalbumin/uric creatinine, urea, and serum creatinine (SCr) levels in rats.μm sections, and stained with hematoxylin-eosin (HE), periodic acid-Schiff (PAS), and Masson. Then, all sections were observed and photographed through light microscopy. The rest of the renal tissue was frozen in liquid nitrogen at −80°C for western blot detection. After HE staining treatment, the degree of tubular stromal damage was assessed by semiquantitative, with scoring criteria being 0 (no damage), 1 (≤10%), 2 (11%∼25%), 3 (26%∼45%), 4 (46%∼75%), and 5 (>76%). At least 10 choroid boundary fields were selected for each section. The positive expression area percentage (% area) and the light density  were measured using the Image J-win64 image analysis system, and the percentage of the expression area in the fiber tissue was calculated; the statistical analysis software SPSS 22.0 was applied to perform single factor variance analysis (one-way ANOVA) on the data, the variance represented by mean ± standard difference (x ± SD).The renal tissues were fixed with 10% neutral formaldehyde, dehydrated using alcohol, embedded in paraffin, cut into 6 μm) were fixed with cold acetone for 10 min, blocked in PBS containing 10% goat serum at room temperature for 30 min, and incubated with primary antibodies against nephrin at 4°C overnight. After several PBS rinses, CyTM 2-conjugated secondary antibodies were applied for 30 min at room temperature. The nuclei were visualized using 4,6-diamidino-2-phenylindole (DAPI) staining. After several PBS rinses, the sections were mounted with an antiflu-fluorescent attenuating agent. Images were recorded using a Leica laser scanning microscope . The fluorescence intensity (intDen) and area (Area) of all the collected images were measured, and the average fluorescence intensity (mean) of each image was calculated using the Image J-win64 image analysis system; the statistical analysis software SPSS 22.0 was applied to perform single factor variance analysis (one-way ANOVA) on the data. The results of the statistical analyses were represented by mean ± standard difference (x ± SD).Renal tissues were embedded in optimum cutting temperature compounds by snap freezing in liquid nitrogen. The frozen sections  Trading Co., Ltd.), p62  Trading Co., Ltd), LC3B  Trading Co., Ltd), and nephrin  were used. Goat anti-rabbit IgG conjugated with horseradish peroxidase was used to detect the expression of primary antibodies, and protein bands were visualized using an ECL western blotting detection system .Renal cortical tissues were collected and frozen at −80°C. The concentration of extracted protein was detected using the Protein BCA Assay . Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis  and transferred onto nitrocellulose membranes . After blocking with nonfat milk, primary antibodies specific for X ± SD). Data were compared by one-way ANOVA test, followed by the SNK-q test between different groups. If there was no special explanation, P < 0.05 was considered statistically significant.SPSS 22.0 was used to analyze the experimental data. The measurement data were expressed as mean ± standard deviation (The following changes were observed in rats induced with DN after treatment with Keluoxin.P < 0.01), and their urine volume showed a significant increase (P < 0.01). Compared with the model group, the urine volumes of rats in the Keluoxin and Irbesartan groups were significantly lower (P < 0.01); their weights were slightly less, but there were no significant differences compared with the model group. As shown in P < 0.01) and the SCr level decreased significantly (P < 0.01) compared with the normal group; UCr level did not change significantly, indicating early renal injury in rats with DN. Compared to the model group, the MAU and MAU/UCR ratios in the Keluoxin group were significantly lower (P < 0.01), while the decrease in UREA levels was not statistically significant. The MAU/UCR ratio in the Irbesartan group showed a significant reduction (P < 0.01), while the decreases in the levels of UREA and MAU were not statistically significant.As shown in P < 0.05), and no significant changes in TC, LDL-C, and HDL-C were observed. Compared with the model group, the values of TC, TG, and LDL-C were lower in the Keluoxin and Irbesartan groups while those of HDL-C were higher, but the differences were not statistically significant. As shown in P < 0.01). Compared with the model group, the fasting blood glucose was significantly lower in the Keluoxin group (P < 0.01) , and the 2 h OGTT blood glucose showed improvement in the Keluoxin group, but the difference was not statistically significant. There was no significant improvement in the fasting blood glucose levels in the Irbesartan group, and although the 2 h OGTT showed improvement in this group, the improvement was not statistically significant.As shown in P < 0.01). The percentage of area under collagen expression was lower in the Keluoxin and Irbesartan groups compared to the model group (P < 0.01). As shown in P < 0.05). The percentages of the glomerular area with positive expression in the Keluoxin and Irbesartan groups were lower than that in the model group (P < 0.05). Taken together, it can be inferred that treatment with Keluoxin significantly improved the pathology and restored the glomerular structure of renal tissues.Histologically, as shown in P < 0.01) as was the ratio of LC3-ll/LC3-l (P < 0.05), while nephrin protein was significantly lower in the model group (P < 0.01). Compared with the model group, the levels of LC3-II protein in the Keluoxin and Irbesartan groups decreased significantly (P < 0.05), and the expression of nephrin protein was significantly upregulated (P < 0.01). The levels of p62 in the Keluoxin and Irbesartan groups decreased significantly . The nephrin of podocyte markers under immunofluorescence staining was downregulated in rats with DN, indicating impairment of the glomerular filtration barrier. In addition, treatment with Keluoxin and Irbesartan upregulated the expression of nephrin in the renal tissue . Compared with the model group, the nephrin protein content in the Keluoxin and Irbesartan groups was significantly higher (P < 0.05).As shown in  Figures  and 4. Aβ1 induction, activation of the renin-angiotensin-aldosterone system (RAAS), and advanced glycation end product (AGE) accumulation [DN is characterized by impairment of renal function and development of proteinuria; morphological and functional changes in podocytes are important reasons for the occurrence and development of DN –30. Podomulation . Podocytmulation . Persistmulation –36. Studmulation . This ismulation . In thismulation . After tP < 0.01) in DN-model rats, indicating that HG induces autophagy in podocytes, which is consistent with previous reports [P < 0.01), indicating that autophagy activity in podocytes was inhibited under high glucose conditions. This may have occurred due to lysosomal dysfunction, which leads to obstruction of the downstream pathways of the autophagy process. After treatment with Keluoxin and Irbesartan, the expression of LC3-II and p62 proteins decreased significantly, suggesting that obstruction of the downstream pathways of autophagy was reduced, and the autophagy activity of the podocytes was restored. Therefore, we were able to confirm the regulative effect of Keluoxin and Irbesartan on renal function in rats with DN.Obstruction of any of the autophagy processes, including autophagy induction, autophagy vacuole and lysosome fusion, and lysosome degradation of the autophagy vacuole, will affect autophagy activity . The lys reports . This ph reports . The expThus, Keluoxin can improve the autophagy function of podocytes, alleviate renal injury, and protect renal function in rats with DN. Interestingly, this study found that Keluoxin and Irbesartan had similar efficacies and could regulate kidney function as a whole in rats with DN; the side effects of traditional Chinese medicine are also low.Keluoxin can effectively improve blood glucose and blood lipid levels and renal function in rat DN models, which were established by inducing DN in rats using STZ. Keluoxin can alleviate renal histopathological changes and delay DN progression, and it has a protective effect against DN in a model of type 2 DM. This effect may be achieved by regulating autophagy in podocytes; the mechanism may be a potential therapy for DN which needs further investigation.The limitations of this study include shortage of time, energy, and research funds. The observation period of the study was short and the sample size was small. Moreover, the effect of Keluoxin on rats with DN was observed only in terms of its effects on the autophagy mechanism in podocytes, other pathological mechanisms should be undertaken in future studies, such as oxidative stress, inflammatory mechanisms, and PI3K/AKT/mTOR signaling pathways. In addition, the actual active components of Chinese medicine in the blood after metabolism in vivo are not exactly the same as those in the original medicine; treatment of podocytes in vitro with serum-containing Keluoxin needs to be undertaken in future studies. Thus, an increasing number of studies using larger samples are needed to confirm the improvement of autophagy in podocytes with Keluoxin. These limitations need to be addressed in future studies.