Hypertension is a major risk factor of cardiovascular mortality. Mood disorders represent a growing public health problem worldwide. A complex relationship is present between mood disorders and cardiovascular diseases. However, less data is available about the level of depression and anxiety in different hypertension phenotypes. The aim of our study was to evaluate psychometric parameters in healthy controls (Cont), in patients with white-coat hypertension (WhHT), with chronic, non-resistant hypertension (non-ResHT), and with chronic, treatment-resistant hypertension (ResHT).In a cross-sectional study setup 363 patients were included with the following distribution: 82 Cont, 44 WhHT, 200 non-ResHT and 37 ResHT. The patients completed the Beck Depression Inventory (BDI) and the Hamilton Anxiety Scale (HAM-A).p < 0.05). Similarly, HAM-A points were higher in WhHT (8 (5–15)) and ResHT (10.5 (5.25–18.75)) compared with Cont (4 (1–7), p < 0.05) and also compared with non-ResHT (5 (2–10), p < 0.05). ResHT was independently associated with HAM-A scale equal or above 3 points . WhHT was independently associated with HAM-A scale equal or above 2 points  and BDI scale equal or above 5 points .BDI points were higher in WhHT (7 (3–11)) and ResHT (6 (3–11.5)) compared with Cont (3 (1–6), Our results suggest psychopathological similarities between white-coat hypertension and resistant hypertension. As recently it was demonstrated that white-coat hypertension is not a benign condition, our findings can have relevance for future interventional purposes to improve the outcome of these patients. Hypertension is considered as the leading cause of cardiovascular (CV) mortality and based on American data, it is in the second place in the list of the preventable causes of all-cause mortality as well , 2. MoodThe association between hypertension, depression and anxiety are controversially discussed in diverse studies , 7. The Anxiety was found to be associated with white-coat effect during blood pressure measurement , 12, whiThe aim of our study was to measure depression and anxiety in healthy controls, in untreated subjects with white-coat effect and in chronic hypertension with or without the presence of resistant hypertension. Based on our recent findings we hypothesized similarities between subjects with white-coat and resistant hypertension.Methods.Our present results are part of an additional analysis of a previously published cohort , includin = 82); white-coat hypertensive patients ; chronic, non-resistant hypertensive patients ; and patients with chronic, resistant hypertension . The diagnosis of resistant hypertension was always confirmed by ambulatory blood pressure monitoring (ABPM) or by home blood pressure monitoring (HBPM). The definition of non-resistant hypertension required the following criteria: chronic (the onset is longer than 3 months), treated (with the maximum use of 3 antihypertensive agents) and controlled hypertension.In the setup of a cross-sectional study, altogether 363 Caucasian patients were involved between August 2012 and January 2019, in three primary care practices in Budapest, Hungary. Four categories of the enrolled subjects were defined: healthy controls , patients with atrial fibrillation were excluded from the study. Other exclusion criteria were treated depression, anxiety, or other psychiatric conditions  and also dementia to avoid mistakes or misunderstanding of the questionnaires. The chronic, moderate use of alprazolam (< 0.5 mg/day) was not an exclusion criterion when it was added to the hypertension therapy protocol by other specialists, without the diagnosis of anxiety.Subjects were recruited into the study during the screening visit when blood pressure was measured with a validated oscillometric device (Omron M3) and written informed consent was collected. At the end of the screening visit an autoquestionnaire was handed out to the subjects including a questionnaire for the evaluation of family and personal history and also for depression. The autoquestionnaires were collected from the patients in the day of the clinical measurements.After the screening visit within the maximum of a 2-week period an appointment was scheduled for 7.00. a.m. for repeated office and/or ambulatory blood pressure measurement and also for blood sampling. Finally the evaluation of anxiety was completed in the form of an interview with the examiner.Beck Depression Inventory (BDI) was used to evaluate the severity of depression. It is one of the most widely used instruments including a 21-question multiple-choice self-report questionnaire with ratings on a four-point scale, where a higher score correlates with more severe depression [The pression .Hamilton Anxiety Scale (HAM-A) was used to study the severity of anxiety. During the evaluation the examiner reports the subject. Its scale consists 14 items, each item is scored on a scale of 0 (not present) to 4 (severe anxiety) [The anxiety) .Before the clinical measurements overnight fasting, refrain from smoking and drinking caffeine-containing beverages were required, but patients were asked to take their usual antihypertensive medication. In sitting position after 5 min of rest, two brachial blood pressure measurements were taken in 1-min interval on each arm with an oscillometric device . In the detailed analysis the mean value of the higher side of arms was further used as brachial systolic (SBP) and diastolic (DBP) blood pressures and heart rate. Pulse pressure (PP) was also calculated as SBP minus DBP. Next, untreated subjects, who had elevated office blood pressure during the screening visit, were fitted with a 24-h ABPM device . The cuff was placed on the left arm. Finally venipuncture was performed on the right arm for blood sampling. The 24-h ABPM and blood test results were discussed with the patient on the following day. In case of treated hypertensive patients with increased office blood pressure, where the diagnosis of ResHT was considered, it was confirmed within 2 weeks with HBPM or with ABPM.Descriptive data are expressed as mean ± standard deviation or median with interquartile ranges. Kolmogorov–Smirnov test was used test the normality of continuous parameters. ANOVA was applied for normally distributed parameters to compare differences between the four groups . Tukey's test was used for post-hoc analysis. Kruskal–Wallis test was applied to compare non-normally distributed parameters.To study the association of depression and anxiety with ResHT, chronic hypertensive patient groups (non-ResHT plus ResHT) were dichotomized based on the 75% quartile of the depression and anxiety scores of the patients. Next, with binary regression analysis, the association with ResHT of the patient groups reaching different BDI and HAM-A scores was studied with the adjustment for traditional CV risk factors, such as age, sex, smoking, diabetes, body mass index and total cholesterol. Finally, in the merged group of Cont plus WhHT, with binary regression analysis the association with WhHT of the patients reaching different BDI and HAM-A points was also studied with the adjustment for sex, smoking and BMI.p < 0.05 was considered as the border of significance. SPSS 22.0 for Windows was used throughout the calculations.In all analyses Demographic parameters and comorbidities, results of blood sampling, current medication and the number of the used antihypertensive medications are summarized in Table Compared with Cont, non-ResHT and ResHT patients had elevated age, higher BMI, blood glucose, uric acid and triglyceride levels, and lower HDL cholesterol. ResHT patients had decreased total and LDL cholesterol levels compared with Cont probably as a consequence of the administration of statins. BMI and eGFR were higher in WhHT compared with Cont.Hemodynamic parameters and the results of BDI and HAM-A questionnaires are summarized in Table p < 0.05). Similarly, compared with Cont, HAM-A points of WhHT and ResHT were also higher, but these two groups of patients had higher HAM-A points compared with non-ResHT as well (p < 0.05). Figure Compared with Cont SBP was higher in WhHT, non-ResHT and ResHT groups. SBP in ResHT was even higher than in non-ResHT. Compared with Cont, DBP was higher only in WhHT and ResHT. BDI points of WhHT and ResHT groups were significantly higher compared with Cont  points.Table After the adjustment of multiple variables white-coat hypertension was associated with HAM-A scale equal or above 2 points (Beta = 4.701, 95% CI 1.165–18.973, Table In our study we demonstrated that the level of depression and anxiety can vary in different hypertension phenotypes. Patients with white-coat and resistant hypertension scored higher points compared with healthy controls in BDI scale, and also compared with non-resistant hypertensive patients in HAM-A scale. In addition, almost similar threshold limits were found in respect of HAM-A scale with the independent association of white-coat and resistant hypertension.Our results are in line with our recent findings on the same cohort, where cyclothymic affective temperament points were similarly higher in white-coat and resistant hypertension compared with healthy controls and cyclothymic affective temperament points equal or above 6 were independently associated both with white-coat and resistant hypertension . TherefoHowever, it is the first study which evaluated the level of depression and anxiety in different hypertension phenotypes in one cohort, in the literature some data are already available about the similarities between white-coat and resistant hypertension. Anxiety has a pathophysiological role in both conditions as it was found to be associated with white-coat effect  and was A limitation of our study was that although standardized autoquestionnaires were used and patients with dementia were excluded, the possibility of misinterpretations or mistakes during the completion of the questionnaires could not have been totally excluded. Next, the cross-sectional design of the study limits us to make causal inferences. Furthermore, as all of participants were from Caucasian race it limits the generalizability of our results for other races. Finally, as ABPM was not performed in those patients or healthy participants who had normal office blood pressure values and did not report elevated values during home blood pressure monitoring, we were unable to diagnose masked hypertension and to analyze the level of depression and anxiety of this hypertension phenotype.Psychopathological similarities are present between white-coat and resistant hypertensive patients. In light of the recent evidence as white-coat hypertension is not a harmless condition, our findings can have relevance for future interventional purposes to improve the outcome of these patients.


Sodium-glucose transport protein 2 inhibitors, commonly referred to as SGLT2i, are a group of prescription pharmaceuticals that are approved by the United States Food and Drug Administration for use with diet and exercise to lower blood glucose in adults with type 2 diabetes. Diabetes is a well-recognized major contributor to cardiovascular and renal disease burden. In addition to blood glucose control, SGLT2i have been shown to provide significant cardiovascular and renoprotective benefits in patients with and without diabetes. In this review, we describe current evidence related to the renal and cardiovascular benefits of using SGLT2i. SGLT2 inhibitors (SGLT2i) are a class of pharmaceuticals that lower blood glucose by causing glucosuria, thereby inhibiting glucose reabsorption from glomerular filtration back into circulation.3 In addition, SGLT2i have been shown to provide significant cardiovascular and renal protection in patients with and without diabetes.4 In 2013, canagliflozin was the first SGLT2i approved by the United States (US) Food and Drug Administration (FDA) for the treatment of type 2 diabetes. Since then, several more SGLT2i have been approved (1Sodium-glucose transporter 2 (SGLT2) is the major transport protein of the sodium-glucose transporter protein family and provides approximately 90% of glucose reabsorption from the kidneys.approved . Since t5 In the EMPA-REG trial, analysis of empagliflozin  as a specific component of the secondary microvascular outcome decreased progression of renal disease in patients with type 2 diabetes mellitus (T2DM) and high cardiovascular risk compared to a placebo. The end points were worsening nephropathy, seen by doubling of serum creatinine levels, and initiation of renal replacement therapy, which demonstrated a significant relative risk reduction of 44% and 55%, respectively.6Several clinical trials have demonstrated the effects of SGLT2i on kidney disease, with effects on multiple kidney outcomes including albuminuria, doubling of serum creatinine, glomerular filtration rate (GFR) decline, kidney failure, and death.2). This trial reported a relative risk reduction of 30% in progression to end-stage renal disease, doubling of serum creatinine level, or mortality from renal or cardiovascular causes.7 The CANVAS trial showed a 25% risk reduction in the progression of albuminuria and a 40% risk reduction in the composite of 40% reduction in eGFR, renal replacement therapy, or renal death with the use of canagliflozin.31 The EMPEROR-REDUCED and VERTIS-CV trials, which studied heart failure and cardiovascular outcomes with empagliflozin and ertugliflozin, noted a similar risk reduction. The absolute difference in the eGFR in the empagliflozin group was higher by 1.73 mL/min/1.73m2 than in the placebo group,33 and in the ertugliflozin48 group was higher by 2.55 mL/min/1.73 m2 than in the placebo group.The benefit of canagliflozin on the kidney was seen in the CREDENCE trial, which included patients with T2DM with macroalbuminuria  with or without albuminuria. While the primary and secondary end points had to be modified due to a change in trial sponsorship and early closure, sotagliflozin9 failed to show any significant changes in long-term dialysis, renal transplantation, the first occurrence of a sustained decrease of ≥ 50% in eGFR, or sustained eGFR of < 15 mL/min/1.73 m2.When dapagliflozin was compared with placebo in patients with chronic kidney disease (CKD) with or without diabetes, the risk of eGFR decline of ≥ 50%, progression to end-stage renal disease, and renal or cardiovascular mortality was notably lower in the treatment group.12 A summary of kidney-related outcomes from major SGLT2i trials is represented in While SGLT2i offer renoprotection, a looming question remains regarding their efficacy and safety in patients who have undergone kidney transplantation. A review of the literature shows that the antihyperglycemic effect of SGLT2i in patients with kidney transplant was comparable to the effect previously demonstrated in large randomized controlled trials in non-kidney transplant patients.14 In recent years, several clinical trials have shown robust evidence regarding the benefits of SGLT2i with respect to cardiovascular outcomes. These studies have included patients with either preexisting heart disease or those with an elevated risk for heart disease. What is striking in these studies is that across different agents in this class, they show a uniform relative risk reduction in hospitalization for heart failure, and considerable heterogeneity exists when assessing the primary cardiovascular end point, which includes death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke. Only empagliflozin15 and canagliflozin16 have shown clinically significant benefits with regard to the primary outcome. Empagliflozin is the only agent to show significant risk reduction for death from cardiovascular or any cause. Ertugliflozin shows no clinically significant benefit in any of the primary or secondary outcomes studied, but it did show a statistically significant risk of hospitalization for heart failure (9 It is well established that diabetes is a major risk factor for cardiovascular disease (CVD), which affects about a third of individuals with T2DM. Additionally, at least half of the mortality in individuals with T2DM can be attributed to CVD. failure . Sotagli18 Dapagliflozin showed similar outcomes in patients with HFrEF, with a 26% overall risk reduction.19 Although a study of sotagliflozin ended early due to lack of funding from the sponsor, it showed a 33% risk reduction with a median follow-up of only 9 months.20 Moreover, the CANVAS program reduced the overall risk of heart failure events but had no clear difference in effects on patients with HFrEF versus HFpEF. It is important to recognize that in these studies, no death benefit was noted except in the dapagliflozin study.Further trials including patients with heart failure have helped to better delineate these outcomes. Empagliflozin showed an overall risk reduction in the primary outcomes of cardiovascular death or hospitalization for heart failure of anywhere from 21% to 25% in patients with either heart failure with preserved ejection fraction (HFpEF) or with reduced ejection fraction (HFrEF).21 Additionally, Cardoso et al. noted a 14% reduction in all-cause mortality and a similar reduction in cardiovascular mortality. These findings were significantly lower in individuals treated with SGLT2i across subgroups of age, sex, race, renal function, and heart failure functional classification. Another meta-analysis by McGuire et al. studied over 46,000 patients from six trials and noted a 32% risk reduction in hospitalization for heart failure and, despite significant heterogeneity of associations with outcomes, a 15% risk reduction for cardiovascular death.22A meta-analysis of more than 20,000 patients from 15 randomized controlled trials noted a 31% reduction in hospitalization for heart failure and a 61% reduction in urgent visits for heart failure.23 The study reported a 5.8-point improvement in KCCQ-CS and an improvement of 20.1 meters in the 6MWT, both statistically significant, while noticing reduced weight, but no differences were reported in the N-terminal pro–B-type natriuretic peptide (NT-proBNP), BNP levels, or other secondary end points. The CHIEF-HF trial, which was conducted entirely virtually, assessed the primary outcome of change in the Kansas City Cardiomyopathy Questionnaire Total Symptom Score at 12 weeks in 476 participants who were randomized to either canagliflozin or placebo.24 The change in score was 4.3 points higher with canagliflozin than with placebo, independent of ejection fraction or diabetes status. While this study has its limitations due to being virtual, the improvement in the clinical outcome is significant and may be extrapolated more widely  and 6-minute walk test (6MWT) in 324 patients with HFpEF after 12 weeks of treatment with dapagliflozin.e widely .These studies indicate that SGLT2i not only improve cardiac outcomes as measured in clinical trials but likely contribute to an improved quality of life, with improved walk scores and fewer symptoms associated with heart failure.26 Adenosine locally binds to receptors, causing afferent arteriole constriction; this leads to decreases in glomerular perfusion and filtration, which otherwise cause progressive glomerular injury.28 In contrast, a study in patients with T2DM also shows that SGLT2i causes efferent arteriole dilatation, which reduces GFR.29 Proximal tubular cells require high oxygen consumption for energy and adenosine triphosphate (ATP)-dependent reabsorption of electrolytes and other organic molecules. Glucose reabsorption via SGLT2 increases in patients with T2DM due to hyperglycemia, which results in hyperfiltration and increased luminal glucose load.30 As a result, the oxygen demand of tubular cells increases, leaving the proximal tubule relatively hypoxic.31 SGLT2i decrease the reabsorption of sodium and glucose, thus reducing proximal tubular workload and overcoming the hypoxia.SGLT2i help to restore the tubulo-glomerular feedback by increasing sodium chloride transport to macula densa, which causes the release of vasoactive substances including adenosine.32 SGLT2 inhibition reduces progression of mitochondrial dysfunction and alleviates hypoxia and ATP depletion. In addition, the favorable effect of SGLT2i versus a diuretic in promoting fluid shift, especially from the renal interstitium, and decreased energy consumption by proximal tubular cells may alleviate cortical and outer medullary hypoxia.33 Alternate mechanisms of action may include increased circulating ketones,34 causing a shift from glucose to lipid oxidation that, together with an increase in the glucagon-to-insulin ratio, provide increased ketone production, improved myocardial energetics, improved ionic hemostasis, altered adipokine regulation, and autophagy.In early experimental and human diabetes studies, glucose metabolic flux increases in the kidney cortex due to increased glucose uptake in proximal tubular cells, which can lead to mitochondrial dysfunction.35 It is theorized that SGLT2i may influence cardiac energy metabolism by shifting cardiac energy reliance from non-esterified fatty acids and glucose to ketones; ketones may increase the mechanical efficiency of the failing heart and are considered a “super fuel” for the failing myocardium. Another interesting hypothesis is that inhibition of Na+/H+ exchanger (NHE) 1 and NHE3 by SGLT2i in both the kidney and the heart may be a common mechanism through which these agents offer cardioprotection and renoprotection. Some experimental models and preliminary studies have shown antifibrotic properties of dapagliflozin and empagliflozin, respctively.37 It has been suggested that SGLT2i may restore the inflammatory adipokine balance, but this theory has not been confirmed. Though the exact process remains unknown, it has been observed that SGLT2i decrease inflammatory markers, including nuclear factor-kB, interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), and many other factors in investigational models of diabetes.39 Similarly, a decrease in serum tumor necrosis factor receptor 1, IL-6, urine IL-6, and MCP-1 was seen in patients with T2DM on SGLT2i.41 SGLT2i also reduced uric acid levels in patients with T2DM, thus reducing the inflammatory effects of uric acid that contribute to diabetic kidney disease.43 Joshi et al. highlighted the potential mechanisms by classifying them as conventional and novel.44 The understood conventional mechanisms can be described as diuresis and blood pressure reduction, weight loss, blood glucose control, and hemoconcentration, while the novel mechanisms comprise the improved myocardial energetics, ionic hemostasis, autophagy, and altered adipokine regulation described above.Although the causes for heterogeneity in several cardiovascular outcomes remain unexplained, a common theme across this family of hypoglycemic pharmacotherapy is its significant benefit in hospitalization for heart failure. Given that benefits to heart failure hospitalization have been noted rather early in the studies, the effects are likely not associated with the glucose-lowering properties and changes with blood pressure and cholesterol due to SGLT2i therapy, which often take a long time to yield any beneficial effects. Additionally, these benefits are noted in patients with or without diabetes and irrespective of ejection fraction and across a spectrum of renal function. SGLT2i cause glucosuria and natriuresis, which facilitate improvement in ventricular loading by reducing preload; ensuing osmotic diuresis also contributes.45 Additionally, SGLT2i administration significantly decreased triglyceride levels. No significant alteration in LDL/HDL ratio was found after SGLT2i treatment. The dapagliflozin trial demonstrated significant benefit of raising HDL cholesterol level for improving reverse cholesterol transport.46 Overall, the effects of SGLT2i on dyslipidemia in both human and animal studies are inconclusive.A meta-analysis of 48 randomized controlled trials revealed that SGLT2i significantly increased total cholesterol, low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol, and non-HDL-cholesterol.47 SGLT2i use is associated with a reduction of major adverse cardiovascular events through clinically significant weight loss.48 Compared with placebo, results of network meta-analyses show reductions of body weight for all SGLT2i treatments of about 1.5 kg to 2 kg,50 depending on the dose.52SGLT2i promote weight loss via glucosuria  and negative energy balance, leading to significant weight loss.53 Among people with prediabetes, the number needed to treat was 50 patients per year to prevent one case of new-onset diabetes (absolute risk reduction of 2 events per 100 patient-years). Several mechanisms have been proposed, including protection of pancreatic beta cells from glucose toxicity. Indirect mechanisms could include decrease in insulin resistance through weight loss and improvement of hepatic insulin sensitivity. SGLT2i may thus play a role in the primary prevention of cardiovascular and kidney disease.A prespecified pooled analysis of the DAPA-CKD and DAPA-HF trials showed that dapagliflozin reduced the incidence of new-onset diabetes by approximately one-third.Severe adverse effects of SGLT2i are rare but have been reported through clinical trials and post-marketing data, as highlighted below.54SGLT2i used as a monotherapy are all associated with a low risk of developing hypoglycemia. However, the risk is higher when SGLT2i are utilized as an add-on therapy with sulfonylurea or insulin.55 A metanalysis found that the risk of mycotic genital infections is lower when SGLT2i is combined with dipeptidyl peptidase-4 (DPP-4) inhibitors than when they are used as a monotherapy or as an adjunct to metformin.56 Necrotizing fasciitis of the perineum, called Fournier’s gangrene, is a significant adverse event, although post-marketing case reviews show a lower incidence than initially reported.57The risk for genital candidiasis increases with the development of glycosuria in poorly controlled hyperglycemia. The most common adverse event of SGLT2i as a group, compared with placebo groups, is mycotic infections, particularly among females.58 The FDA and European Medicines Agency issued safety reports on DKA risk in patients treated with SGLT2i who showed DKA-like symptoms.59 Considering the insulin-independent inhibition of glycemia, DKA may present with mildly elevated or even normal blood glucose levels, leading to delayed diagnosis and management under the term euglycemic DKA (EDKA).60 The risk of EDKA is significantly higher in the clinical settings of acute illness, fasting, perioperative states, or excess alcohol consumption.The use of SGLT2i has been associated with an increased risk of diabetic ketoacidosis (DKA). The rates of DKA in major trials were 0.1% to 0.5% over 4 to 8 years.61 However, the true risk of this complication as a causation or even a consistent correlation is not clear.62In the CANVAS clinical trial, canagliflozin showed an increased risk of toe and foot amputation. The higher risk of amputation seems inconsistent, as it was reported only in the CANVAS trial but not in empagliflozin or dapagliflozin trials, nor in the subsequent CREDENCE trial with canagliflozin.31 but other SGLT2i randomized clinical trials and meta-analyses have failed to show an increase in fractures. A recent study based on real-world, population-based Medicare data noted no increase in risk of fractures in patients treated with SGLT2i compared to patients treated with either DPP-4 or glucagon-like peptide-1 receptor agonists.63 However, it is expected to be an uncertain complication in future trials.64SGLT2i are associated with a small increase in fractures, as noted in the CANVAS study thus far,65 The mechanism may be reduced renal hyperfiltration, though an initial and transient decline in eGFR usually follows SGLT2i commencement. In summary, SGLT2i is clinically safe to use in patients with diabetes who have mild to moderate renal failure. The safety of use in patients with eGFR less than 30 is less clear.Data on using SGLT2i in severe or end-stage renal failure are scarce. Some studies have suggested that SGLT2i may prevent the development of acute kidney injury (AKI) despite adverse events related to hypovolemia.Although SGLT2i were developed as antihyperglycemic agents, a growing body of evidence demonstrates consistent reductions in risks for secondary kidney disease end points  and reductions in CVD events. Although not as robust, there are emerging data on using SGLT2i in patients who have received a kidney transplant. In addition to glycemic benefits, nonglycemic mechanisms are involved in CKD and CVD risk reductions. These effects improve glomerular hemodynamics, modify volume status, and modify local and systemic mechanisms involved in the pathogenesis of CKD and CVD. Due to significant benefits seen, use of SGLT2i in appropriate patient populations is encouraged. With the increasing use of these agents in patients with non-diabetes CVD (particularly heart failure) and CKD to prevent progression, it is of the utmost importance to identify potential significant adverse effects, such as EDKA and genitourinary infections. Although not common, these adverse effects can lead to significant morbidity and in some cases mortality. Therefore, efforts to disseminate awareness and recognition of these adverse effects is imperative since the increased use of these agents is expected to continue.Sodium-glucose transporter 2 inhibitors (SGLT2i), a group of medications that were initially approved to treat type 2 diabetes, are increasingly used for indications such as heart failure and chronic kidney disease prevention in patients without diabetes.There are significant data regarding the cardiovascular and renal benefits of these medications in patients with or without diabetes, such as decreased progression of renal disease in patients with type 2 diabetes and high cardiovascular risk, and decreased risk of cardiovascular death or hospitalization for heart failure.Significant adverse effects have been shown in clinical trials and are seen in clinical practice. Of particular concern are euglycemic diabetic ketoacidosis (DKA), genitourinary infections including severe infections, and reports of limb amputations.Clinicians need to be aware of potential adverse effects of using SGLT2i in patients who may be at high risk of infection, DKA, or limb amputation.


Chronic low-grade inflammation is considered one of the major mechanisms for the progression of diabetic kidney disease. We investigated the prognostic value of circulating soluble tumor necrosis factor receptor 2 (sTNFR2) for early nephropathy in patients with type 2 diabetes.2 were followed up for a median of 4 years. Renal outcomes were defined as a composite of either or both a >30% decline in the eGFR and/or albuminuria stage progression determined with consecutive tests.A total of 364 patients with type 2 diabetes and an estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73mSeventy-three patients developed renal composite events. Serum concentrations of sTNFR2 were strongly associated with the risk of renal function decline and progressive changes in albuminuria. Through a receiver operating characteristic curve analysis, a serum sTNFR2 level of 1.608 ng/mL was adopted as the discriminator value for predicting renal outcomes , yielding a sensitivity of 75.3% and a specificity of 51.2%. The association of sTNFR2 levels ≥1.608 ng/mL to renal outcomes was significant after adjusting for relevant variables  and remained consistent across subgroups stratified by age, sex, systolic blood pressure, eGFR, albuminuria, and the use of renin-angiotensin system blockers.Higher circulating levels of sTNFR2 are independently associated with an eGFR decline and progressive albuminuria in patients with type 2 diabetes. Although there have been improvements in diabetes management, the prevalence of diabetic kidney disease (DKD) continues to rise due to improved patient prognoses and the growing incidence of diabetes mellitus . ApproxiScreening of patients with diabetic nephropathy includes measuring their urinary albumin-creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) at least once annually . AlthougActivation of the tumor necrosis factor-α pathway has emerged as an important mechanism in the pathogenesis of DKD . Tumor n2, as determined by the Modification of Diet in Renal Disease equation, or elevated serum levels of alanine aminotransferase greater than two-fold the upper normal limit, uncontrolled cancer, or hemoglobin A1C (HbA1C) ≥9.0% were excluded. Baseline clinical parameters were registered, including age, sex, height, weight, blood pressure, and hip and waist circumferences. The following risk factors and comorbidities were evaluated: cardiovascular disease, hyperlipidemia, hypertension, retinopathy, neuropathy, and smoking status. Retinopathy was assessed by means of nonmydriatic fundus photography. Participants were diagnosed as having neuropathy by neurologists or by electrodiagnostic testing. Serum creatinine, HbA1C, lipid profiles, and urine albumin and creatinine levels were measured every 6 months in the central laboratory of the Department of Clinical Pathology and Laboratory of Taipei Veterans General Hospital. The urinary albumin-creatinine ratio was used as a measure of albuminuria. Normoalbuminuria, microalbuminuria, and macroalbuminuria were defined as a UACR <30 mg/g creatinine, 30–299 mg/g creatinine, and ≥300 mg/g creatinine, respectively. We measured serum sTNFR2 levels according to the manufacturer’s instructions . The intra-assay coefficient of variation was 2.6–4.8%, while the inter-assay coefficient of variation was 3.5–5.1%. The participants were followed until they developed renal composite events or until December 2019.Patients with type 2 diabetes mellitus, visiting the endocrinology and metabolism outpatient clinic at Taipei Veterans General Hospital between June 2014 and December 2019, were enrolled in the prospective observational cohort study. Patients with an eGFR <30 mL/min/1.73mThis prospective study was approved by the ethics committee at Taipei Veterans General Hospital. Written informed consent was obtained from each participant before entering the study. The study complied with the guidelines of the Declaration of Helsinki.The renal outcomes were a composite of sustained decreases in the eGFR and/or the progression of albuminuria stage. The sustained decreases in the eGFR were defined as a >30% reduction in the eGFR from baseline in at least two consecutive samples. The progression of albuminuria stages was defined as the transition from normoalbuminuria to microalbuminuria or macroalbuminuria, or from microalbuminuria to macroalbuminuria. We confirmed albuminuria progression if patients developed transition to a more advanced stage of albuminuria from baseline in two consecutive follow-up at 6-month intervals.The variables were expressed as percentage values for categorical data and as mean ± standard deviation for continuous data. Univariate Cox proportional analyses were used to examine the relevant variables associated with renal composite outcomes. We examined the association between serum sTNFR2 levels and renal outcomes by multivariate Cox proportional hazard models that included covariates with P <0.05 in the univariate Cox proportional analyses. The association between serum sTNFR2 levels and renal outcomes was expressed as hazard ratios (HRs) and 95% confidence intervals (95% CI). The following multivariate Cox models were built: model 1, adjusted for age and sex; model 2, adjusted for age, sex, and duration of diabetes; model 3, adjusted for retinopathy, neuropathy, systolic blood pressure, HbA1C, eGFR, UACR, RAS inhibitors, and diuretics in addition to all variables in model 2. A receiver operating characteristic curve analysis was performed, and the cut-off value of sTNFR2 for predicting renal composite events was determined on the basis of the maximum value of the Youden index. Differences between groups, which were stratified by the occurrence of renal outcomes or the cut-off value of sTNFR2, were analyzed by Pearson’s chi-squared test for categorical data and Student’s t-test for continuous variables. The survival curve for the renal composite events was illustrated using the Kaplan-Meier method, and different groups were compared using the log-rank test. A two-sided P value <0.05 was considered statistically significant. All analyses were performed with the use of SPSS Statistics version 22 .2. The baseline characteristics of the study population are summarized in A total of 364 patients were enrolled in the study. The mean age of the patients was 61.4 years, and 70.3% of the patients were men. The mean duration of diabetes was 11.7 years. One-third of the subjects had albuminuria, and the prevalence of retinopathy and neuropathy was 9.3% and 9.1%. Sixty-seven (18.4%) patients had stage 3 chronic kidney disease. The mean HbA1C was 7.1%, mean blood pressure was 114.0 mmHg, and mean body mass index (BMI) was 26.3 kg/mOver a median follow-up time of 4 years, 73 participants had a renal composite event, which is composed of 46 events of progression of albuminuria stages and 27 events of a >30% reduction in the eGFR. Among the 46 events of progressive albuminuria, 27 participants had a transition from normoalbuminuria to microalbuminuria, and 18 participants progressed from microalbuminuria to macroalbuminuria. One patient had a rapid progression from normoalbuminuria to macroalbuminuria. Patients with renal composite events had longer durations of diabetes, higher percentages of retinopathy or neuropathy, higher UACRs, and lower eGFRs at baseline. The coverage of the renin-angiotensin system (RAS) blockade was more extensive in patients with renal outcomes . CorrelaThe ideal cut-off point for sTNFR2 concentration is the point where true-positive findings and few false-positive results are detected in most patients. The optimal cut-off value of sTNFR2 levels obtained using the receiver operating characteristic curve analysis was 1.608 ng/mL, allowing for a sensitivity of 75.3% and a specificity of 51.2%. The area under the receiver operating characteristic curve of sTNFR2 was 0.63  are shown in In the univariate Cox proportional analysis, sTNFR2 concentrations ≥1.608 ng/mL were associated with higher renal event rates . A sTNFR2 concentration ≥1.608 ng/mL predicted renal events in all of the multivariate models , UACR (≥ or <30 mg/g creatinine), and the use of RAS inhibitors. The association of a sTNFR2 concentration ≥1.608 ng/mL with renal outcomes remained significant across all subgroups, including patients with an eGFR ≥60 mL/min/1.73m2 or normoalbuminuria , sex, systolic blood pressure (≥ or <140 mmHg), eGFR   was muchThe limitations of our study should be considered. First, the participants in this cohort were recruited from a veterans hospital which mainly serves male patients. It accounts for male-predominant cohort in this study. In the female participants, the borderline results of the association between sTNFR2 concentration ≥1.608 ng/mL and renal composite events may be due to relatively small sample size. Second, we estimated glomerular filtration rates from a serum creatinine-based equation, which are less accurate than estimates that are based on serum cystatin C or direct measurements of renal function. Direct measurement of the GFR may augment the strength of the association. Third, sodium/glucose co-transporter 2 (SGLT2) inhibitors and glucagon-like peptide-1 (GLP-1) analogues have renoprotective actions in patients with type 2 diabetes beyond glycemic control. Patients in our cohort rarely used sodium/glucose co-transporter 2 inhibitors or glucagon-like peptide-1 analogues because of the restricted reimbursement policies for them, owing to their high acquisition costs. Finally, the prospective observational design does not necessarily imply a causal relationship. Further studies are required to confirm that interventions to circulating sTNFR2 levels contribute to beneficial effects on DKD progression.Circulating sTNFR2 levels are associated with the progression of diabetic kidney disease in patients with type 2 diabetes independent of baseline albuminuria or renal function. Soluble tumor necrosis factor receptor 2 is a potential prognostic biomarker for the personalized management of worsening diabetic nephropathy.S1 Table(DOCX)Click here for additional data file. 31 Jan 2022
 PONE-D-21-32495

 Soluble tumor necrosis factor receptor 2 are associated with progressive diabetic kidney disease in patients with type 2 diabetes mellitus

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Currently, your Funding Statement reads as follows:  This study was supported by research grants V104E11-004-MY2, V105C-131, V107C-201, V108C-197, V109C-179 and V110C-198 provided to L.Y.L. from Taipei Veterans General Hospital and No. 2021001 to L.H.C. from Yeezen General Hospital.Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Additional Editor Comments:Please clarify any potential overlap with your previous publication in Oct 2021 (Endocrine Practice)- what is the novelty of this publication? What is the advantage of measuring soluble TNF receptor 2 over other biomarkers?[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. 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The authors demonstrated a clear correlation between elevated levels of TNFR2 with decline in renal function . Whilst the data is presented well within the manuscript there are some concerns which the authors should address before this manuscript should be considered for publication:1. The authors published data on 284 type 2 diabetic patients investigating TNFR1 as a biomarker and predictor of renal outcomes in Endocrinology practice , Oct 2021. The authors should verify and clarify that these patients are not indeed overlapping. Futhermore, the authors need to verify that the analysis was not conducted for both markers in the same cohort and published as two seperate manuscripts.2. If they are indeed the same cohort or overlapping cohort the authors should provide a comparitive analysis in this new manuscript to determine which of the two markers are a a better and more valid biomarker of diabetic kidney disease.3. The authors have presented many tables in this current manuscript with their hazard ratios and statistical analysis. I would suggest that the most significant asssociations, eGFR and albuminuria should be presented as a figure to further strenghten the findings of this manuscript.4. Niewcaz et al published in 2012 that both TNFR1 and TNFR2 are both predictors of ESRD. The authors should clarify how their present study is adding to the previously published data. They also presented their findings in males and your cohort is primarily males. Is this a male phenomenon? You should compare males to females to answer this.5. Figure 2A and 2B should be presented in higher quality images. It is not clear or of high enough standard for publication.6. In table 3 when comparing the different models- Model 3 appears to have caused a loss in significance. The authors should address why the believe this to be the case and discuss it in their discussion.Overall, I believe the authors should first and foremost verify that this current manuscript has not overlap with their publication in Oct 2021. If it does, clarify why is has been published in this way or at the very least make it clear. I think this should be the first priority to be addressed before publication is considered.Reviewer #2: The manuscript under review investigates TNFR2 as a biomarker and a predictor of progressive renal disease in a cohort of diabetic patients. It investigates the relationship between serum TNFR2 levels and a number of parameters to conclude that serum TNFR2 levels above 1.608ng/ml are independently associated with a decline in eGFR and deteriorating albuminuria in T2D patients.The only comments I have are:1. The authors should include in their discussion other papers that have investigated this issue and how this work is different to theirs. For example there is no mention of PMID 22266663 and PMID 22266664, etc.2. The authors should elaborate how TNFR2 as a predictor of progressive kidney disease compares to other markers that are currently used. Is it an improvement? Is it an easier marker to measure?**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  3 Mar 2022Academic Editor1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.Answer to academic editor: Thanks for your kindly remind. We revise our manuscript in adherence to PLOS ONE's submission guidelines. We resubmit all required files for revision and hope that the changes made will be considered satisfactory.2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained . If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.Answer to academic editor: Thanks for your suggestion. The participants included in our study are all adults. Written informed consent was obtained from each participant before entering the study. We revise the manuscript as below:Line 105, page 8, part of “Study design and participants”, section of “Materials and methods”OriginalThis prospective study was approved by the ethics committee at Taipei Veterans General Hospital. Informed consent was obtained from all participants, and the study complied with the guidelines of the Declaration of Helsinki.RevisedThis prospective study was approved by the ethics committee at Taipei Veterans General Hospital. Written informed consent was obtained from each participant before entering the study. The study complied with the guidelines of the Declaration of Helsinki.3. Thank you for stating the following financial disclosure:This study was supported by research grants V104E11-004-MY2, V105C-131, V107C-201, V108C-197, V109C-179 and V110C-198 provided to L.Y.L. from Taipei Veterans General Hospital and No. 2021001 to L.H.C. from Yeezen General Hospital.Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed.Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.Answer to academic editor: Thanks for your suggestion. The funders in our study had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We add this information in the cover letter.Cover letterOriginalThis manuscript has not been published or presented elsewhere in part or in entirety, and is not under consideration by another journal. All study participants provided informed consent, and the study design was approved by the appropriate ethics review boards. All the authors have approved the manuscript and agree with submission to your esteemed journal. There are no conflicts of interest to declare.RevisedThis manuscript has not been published or presented elsewhere in part or in entirety, and is not under consideration by another journal. All study participants provided informed consent, and the study design was approved by the appropriate ethics review boards. All the authors have approved the manuscript and agree with submission to your esteemed journal. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There are no conflicts of interest to declare.4. Thank you for stating the following in the Acknowledgments Section of your manuscript:This study was supported by research grants V104E11-004-MY2, V105C-131, V107C-201, V108C-197, V109C-179 and V110C-198 provided to L.Y.L. from Taipei Veterans General Hospital and No. 2021001 to L.H.C. from Yeezen General Hospital.Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:This study was supported by research grants V104E11-004-MY2, V105C-131, V107C-201, V108C-197, V109C-179 and V110C-198 provided to L.Y.L. from Taipei Veterans General Hospital and No. 2021001 to L.H.C. from Yeezen General Hospital.Please include your amended statements within your cover letter; we will change the online submission form on your behalf.Answer to academic editor: Thanks for your kindly remind. Besides previous funding statement, this study was supported by research grants V111D62-002-MY3-1 from Taipei Veterans General Hospital as well. We add this information within our cover letter. The funding-related text was removed from the manuscript.Line 340, page 24, section of “Acknowledgments”OriginalThis study was supported by research grants V104E11-004-MY2, V105C-131, V107C-201, V108C-197, V109C-179 and V110C-198 provided to L.Y.L. from Taipei Veterans General Hospital and No. 2021001 to L.H.C. from Yeezen General Hospital. The authors thank the Medical Sciences & Technology Building of Taipei Veterans General Hospital for providing experimental space and facilities. The authors declare no conflicts of interest regarding the contents of this manuscript.RevisedThe authors thank the Medical Sciences & Technology Building of Taipei Veterans General Hospital for providing experimental space and facilities. The authors declare no conflicts of interest regarding the contents of this manuscript.Cover letterOriginalThis manuscript has not been published or presented elsewhere in part or in entirety, and is not under consideration by another journal. All study participants provided informed consent, and the study design was approved by the appropriate ethics review boards. All the authors have approved the manuscript and agree with submission to your esteemed journal. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There are no conflicts of interest to declare.RevisedThis manuscript has not been published or presented elsewhere in part or in entirety, and is not under consideration by another journal. All study participants provided informed consent, and the study design was approved by the appropriate ethics review boards. All the authors have approved the manuscript and agree with submission to your esteemed journal. This study was supported by research grants V104E11-004-MY2, V105C-131, V107C-201, V108C-197, V109C-179, V110C-198, and V111D62-002-MY3-1 provided to L.Y.L. from Taipei Veterans General Hospital and No. 2021001 to L.H.C. from Yeezen General Hospital. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There are no conflicts of interest to declare.5. Please clarify any potential overlap with your previous publication in Oct 2021 (Endocrine Practice)- what is the novelty of this publication? What is the advantage of measuring soluble TNF receptor 2 over other biomarkers?Answer to academic editor: Thanks for your suggestion. In our prospective observational cohort study, we started to enroll patients with type 2 diabetes mellitus who visited the endocrinology and metabolism outpatient clinic at Taipei Veterans General Hospital since June 2014. We measured serum sTNFR1 levels initially while 283 participants were enrolled in the study. Our investigation suggested that increased sTNFR1 levels were associated with renal outcomes in subjects with type 2 diabetes mellitus, which was published in Endocrine Practice . Subsequently, we measured serum sTNFR2 concentrations when 364 patients were enrolled. The participants were overlapping but more patients were recruited comparing to the previous study. Our analysis also proved that circulating levels of sTNFR2 are associated with the progression of diabetic kidney disease in patients with type 2 diabetes independent of baseline albuminuria or renal function. This cohort study is still ongoing, and more patients will be enrolled. Both serum sTNFR1 and sTNFR2 levels were measured in the 364 patients enrolled in the current study. The mean areas under the ROC curve were 0.64  for sTNFR1 and 0.63  for sTNFR2. The difference between areas under the ROC curve of sTNFR1 and sTNFR2 is not significant (p = 0.952). Both sTNFR1 and sTNFR2 are potential biomarkers in the assessment of diabetic kidney disease. The clinical methods for screening diabetic nephropathy includes annual measurement of urinary albumin-creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR). However, significant glomerular structural changes often existed when microalbuminuria becomes apparent. Patients with type 2 diabetes can also develop nonproteinuric diabetic nephropathy, and measuring UACR is an ineffective screening test for these patients. In our study, circulating sTNFR2 levels are associated with worsening albuminuria and progressive eGFR decline in patients with type 2 diabetes, including participants with eGFRs ≥60 mL/min/1.73m2 or normoalbuminuria. Our findings suggested that sTNFR2 is a biomarker to detect earlier stages of diabetic kidney disease comparing to measurement of UACR and eGFR. Measuring serum levels of sTNFR2 is a noninvasive and easily accessible method, making it a potential screening tool for diabetic nephropathy. We revise the manuscript as below:Line 247, page 19, section of “Discussion”OriginalIn our study, elevated serum sTNFR2 concentrations were associated with worsening albuminuria and progressive eGFR decline in patients with type 2 diabetes. Circulating sTNFR2 levels ≥1.608 ng/mL were a strong predictor of the progression of albuminuria and renal impairment in patients with type 2 diabetes, and the association was independent of relevant clinical covariates, including age, duration of diabetes, baseline UACR and eGFR, and the use of RAS inhibitors.RevisedIn our study, elevated serum sTNFR2 concentrations were associated with worsening albuminuria and progressive eGFR decline in patients with type 2 diabetes. Circulating sTNFR2 levels ≥1.608 ng/mL were a strong predictor of the progression of albuminuria and renal impairment in patients with type 2 diabetes, and the association was independent of relevant clinical covariates, including age, duration of diabetes, baseline UACR and eGFR, and the use of RAS inhibitors. Comparing to the current screening strategies of diabetic nephropathy as measurement of UACR and eGFR, sTNFR2 is a biomarker to detect earlier stages of diabetic kidney disease. As a noninvasive and easily accessible method, measuring serum levels of sTNFR2 is a potential screening tool for diabetic nephropathy.Line 285, page 21, section of “Discussion”OriginalWe previously reported that serum sTNFR1 levels were associated with renal outcomes in patients with type 2 diabetes [9]. The association of serum sTNFR1 levels above the cut-off value yielded by the receiver operating characteristic curve analysis with DKD progression was significant after adjusting for relevant clinical variables . Although the hazard ratios calculated for renal outcomes according to the serum sTNFR1 levels were higher than those calculated for serum sTNFR2 levels, the CIs for the hazard ratios overlapped.RevisedWe previously reported that serum sTNFR1 levels were associated with renal outcomes in patients with type 2 diabetes while 283 participants were enrolled in the same cohort in this study [9]. The association of serum sTNFR1 levels above the cut-off value yielded by the receiver operating characteristic curve analysis with DKD progression was significant after adjusting for relevant clinical variables . Although the hazard ratios calculated for renal outcomes according to the serum sTNFR1 levels were higher than those calculated for serum sTNFR2 levels, the CIs for the hazard ratios overlapped. In addition, both serum sTNFR1 and sTNFR2 levels were measured in the 364 patients enrolled in the current study. The difference between areas under the ROC curve of sTNFR1 and sTNFR2 is not significant (data not shown). Both sTNFR1 and sTNFR2 are potential biomarkers in the assessment of diabetic kidney disease. Reviewer 1Wu et al, investigated serum levels of TNFR2 in 364 type 2 diabetic patients to determine whether it was a valid biomarker for the onset of early diabetic nephropathy. The authors demonstrated a clear correlation between elevated levels of TNFR2 with decline in renal function . Whilst the data is presented well within the manuscript there are some concerns which the authors should address before this manuscript should be considered for publication:1. The authors published data on 284 type 2 diabetic patients investigating TNFR1 as a biomarker and predictor of renal outcomes in Endocrinology practice , Oct 2021. The authors should verify and clarify that these patients are not indeed overlapping. Futhermore, the authors need to verify that the analysis was not conducted for both markers in the same cohort and published as two seperate manuscripts.Answer to the reviewer: Thanks for your comments. In our prospective observational cohort study, we started to enroll patients with type 2 diabetes mellitus who visited the endocrinology and metabolism outpatient clinic at Taipei Veterans General Hospital since June 2014. We measured serum sTNFR1 levels initially while 283 participants were enrolled in the study. Our investigation suggested that increased sTNFR1 levels were associated with renal outcomes in subjects with type 2 diabetes mellitus, which was published in Endocrine Practice . Subsequently, we measured serum sTNFR2 concentrations when 364 patients were enrolled. The participants were overlapping but more patients were recruited comparing to the previous study. Our analysis also proved that circulating levels of sTNFR2 are associated with the progression of diabetic kidney disease in patients with type 2 diabetes independent of baseline albuminuria or renal function. This cohort study is still ongoing, and more patients will be enrolled. We add this information in the manuscript:Line 285, page 21, section of “Discussion”OriginalWe previously reported that serum sTNFR1 levels were associated with renal outcomes in patients with type 2 diabetes [9].RevisedWe previously reported that serum sTNFR1 levels were associated with renal outcomes in patients with type 2 diabetes while 283 participants were enrolled in the same cohort in this study [9].2. If they are indeed the same cohort or overlapping cohort the authors should provide a comparitive analysis in this new manuscript to determine which of the two markers are a a better and more valid biomarker of diabetic kidney disease.Answer to the reviewer: Thanks for your suggestion. Both serum sTNFR1 and sTNFR2 levels were measured in the 364 patients enrolled in the current study. The mean areas under the ROC curve were 0.64  for sTNFR1 and 0.63  for sTNFR2. The difference between areas under the ROC curve of sTNFR1 and sTNFR2 is not significant (p = 0.952). Both sTNFR1 and sTNFR2 are potential biomarkers in the assessment of diabetic kidney disease. We add this information in the manuscript:Line 291, page 21, section of “Discussion”OriginalWe previously reported that serum sTNFR1 levels were associated with renal outcomes in patients with type 2 diabetes while 283 participants were enrolled in the same cohort in this study [9]. The association of serum sTNFR1 levels above the cut-off value yielded by the receiver operating characteristic curve analysis with DKD progression was significant after adjusting for relevant clinical variables . Although the hazard ratios calculated for renal outcomes according to the serum sTNFR1 levels were higher than those calculated for serum sTNFR2 levels, the CIs for the hazard ratios overlapped.RevisedWe previously reported that serum sTNFR1 levels were associated with renal outcomes in patients with type 2 diabetes while 283 participants were enrolled in the same cohort in this study [9]. The association of serum sTNFR1 levels above the cut-off value yielded by the receiver operating characteristic curve analysis with DKD progression was significant after adjusting for relevant clinical variables . Although the hazard ratios calculated for renal outcomes according to the serum sTNFR1 levels were higher than those calculated for serum sTNFR2 levels, the CIs for the hazard ratios overlapped. In addition, both serum sTNFR1 and sTNFR2 levels were measured in the 364 patients enrolled in the current study. The difference between areas under the ROC curve of sTNFR1 and sTNFR2 is not significant (data not shown). Both sTNFR1 and sTNFR2 are potential biomarkers in the assessment of diabetic kidney disease.3. The authors have presented many tables in this current manuscript with their hazard ratios and statistical analysis. I would suggest that the most significant asssociations, eGFR and albuminuria should be presented as a figure to further strenghten the findings of this manuscript.Answer to the reviewer: Thanks for your suggestion. To highlight our findings, we plot cumulative hazard curves for renal composite events in patients with type 2 diabetes based on the threshold value of soluble tumor necrosis factor receptor 2 (≥1.608 ng/mL vs. <1.608 ng/mL) in model 3. Analysis results are adjusted for age, sex, duration of diabetes, retinopathy, neuropathy, systolic blood pressure, hemoglobin A1C, estimated glomerular filtration rate, urinary albumin-creatinine ratio, renin-angiotensin system inhibitors, and diuretics. We revise the manuscript as below:Line 216, page 16, part of “Association of sTNFR2 levels ≥1.608 ng/mL with the renal outcomes”, section of “Results”OriginalIn the univariate Cox proportional analysis, sTNFR2 concentrations ≥1.608 ng/mL were associated with higher renal event rates . A sTNFR2 concentration ≥1.608 ng/mL predicted renal events in all of the multivariate models .RevisedIn the univariate Cox proportional analysis, sTNFR2 concentrations ≥1.608 ng/mL were associated with higher renal event rates . A sTNFR2 concentration ≥1.608 ng/mL predicted renal events in all of the multivariate models .Fig 3.Cumulative hazard curves for renal events  in patients with type 2 diabetes based on the threshold value of soluble tumor necrosis factor receptor 2 (≥1.608 ng/mL vs. <1.608 ng/mL). Analysis results were adjusted for age, sex, duration of diabetes, retinopathy, neuropathy, systolic blood pressure, hemoglobin A1C, estimated glomerular filtration rate, urinary albumin-creatinine ratio, renin-angiotensin system inhibitors, and diuretics.4. Niewcaz et al published in 2012 that both TNFR1 and TNFR2 are both predictors of ESRD. The authors should clarify how their present study is adding to the previously published data. They also presented their findings in males and your cohort is primarily males. Is this a male phenomenon? You should compare males to females to answer this.Answer to the reviewer: Thanks for your suggestion. Niewczas et al. demonstrated that higher serum sTNFR2 concentrations are associated with increased risks of end stage renal disease in patients with type 2 diabetes. We have cited the article as reference 11 in the second paragraph of Discussion. In our study, the renal outcomes are defined as a composite of a >30% reduction of the estimated glomerular filtration rate and/or the progression of albuminuria stages. Comparing to the previous study mainly focusing on advanced diabetic kidney disease, the findings in our study suggested that sTNFR2 is a promising predictor of the early stages of diabetic nephropathy. In our study, participants were enrolled from a veterans hospital which mainly serves male patients. It accounts for male-predominant cohort in this study. In the female participants, the association between sTNFR2 concentration ≥1.608 ng/mL and renal composite events was significant initially but became attenuated after adjustment in model 2 and model 3 . The borderline results in model 2 (p = 0.053) and model 3 (p = 0.051) may be due to relatively small sample size. Further studies are needed to confirm the association between sTNFR2 and renal outcomes among female patients with type 2 diabetes.Model 1: adjusted for age and sex.Model 2: adjusted for age, sex, and duration of diabetes.Model 3: adjusted for age, sex, duration of diabetes, retinopathy, neuropathy, systolic blood pressure, HbA1C, eGFR, UACR, RAS inhibitors, and diuretics. We revise the manuscript as below:Line 270, page 20, section of “Discussion”OriginalHigher serum sTNFR2 concentrations have been proven to be associated with increased risks of renal function decline or ESRD in patients with type 2 diabetes [10-13]. In one cohort of American Indians (Pima Indians) with type 2 diabetes, patients with macroalbuminuria whose serum sTNFR2 levels were in the highest quartile had a 88.7% cumulative incidence of ESRD, whereas those with macroalbuminuria and sTNFR2 levels in the lowest three quartiles had a 47.3% cumulative incidence of ESRD at 10 years of follow-up [12]. Higher sTNFR2 levels were associated with all-cause mortality as well [10]. The findings in our cohort suggested that sTNFR2 is a promising biomarker in the early stages of diabetic nephropathy. The association of sTNFR2 concentration ≥1.608 ng/mL with renal outcomes remained significant in all subgroups, including patients with eGFRs ≥60 mL/min/1.73m2 or normoalbuminuria.RevisedHigher serum sTNFR2 concentrations have been proven to be associated with increased risks of renal function decline or ESRD in patients with type 2 diabetes [10-13]. In one cohort of American Indians (Pima Indians) with type 2 diabetes, patients with macroalbuminuria whose serum sTNFR2 levels were in the highest quartile had a 88.7% cumulative incidence of ESRD, whereas those with macroalbuminuria and sTNFR2 levels in the lowest three quartiles had a 47.3% cumulative incidence of ESRD at 10 years of follow-up [12]. Higher sTNFR2 levels were associated with all-cause mortality as well [10]. Comparing to previous studies mainly focusing on advanced diabetic kidney disease, the findings in our cohort suggested that sTNFR2 is a promising biomarker in the early stages of diabetic nephropathy. The association of sTNFR2 concentration ≥1.608 ng/mL with renal outcomes remained significant in all subgroups, including patients with eGFRs ≥60 mL/min/1.73m2 or normoalbuminuria.Line 228, page 17, part of “Association of sTNFR2 levels ≥1.608 ng/mL with the renal outcomes”, section of “Results”OriginalWe performed a subgroup analysis based on age (younger or older than 60 years), sex, systolic blood pressure (≥ or <140 mmHg), eGFR (≥ or <60 mL/min/1.73m2), UACR (≥ or <30 mg/g creatinine), and the use of RAS inhibitors. The association of a sTNFR2 concentration ≥1.608 ng/mL with renal outcomes remained significant across all subgroups, including patients with an eGFR ≥60 mL/min/1.73m2 or normoalbuminuria (Table 3).RevisedWe performed a subgroup analysis based on age (younger or older than 60 years), sex, systolic blood pressure (≥ or <140 mmHg), eGFR (≥ or <60 mL/min/1.73m2), UACR (≥ or <30 mg/g creatinine), and the use of RAS inhibitors. The association of a sTNFR2 concentration ≥1.608 ng/mL with renal outcomes remained significant across all subgroups, including patients with an eGFR ≥60 mL/min/1.73m2 or normoalbuminuria (Table 3). In the female participants, the association between sTNFR2 concentration ≥1.608 ng/mL and renal composite events was significant initially but became attenuated after adjustment in model 2 and model 3 .Line 320, page 23, section of “Discussion”OriginalThe limitations of our study should be considered. First, the participants in this cohort were recruited from a single center, and we cannot avoid selection bias completely.RevisedThe limitations of our study should be considered. First, the participants in this cohort were recruited from a veterans hospital which mainly serves male patients. It accounts for male-predominant cohort in this study. In the female participants, the borderline results of the association between sTNFR2 concentration ≥1.608 ng/mL and renal composite events may be due to relatively small sample size.5. Figure 2A and 2B should be presented in higher quality images. It is not clear or of high enough standard for publication.Answer to the reviewer: Thanks for your kindly remind. We follow the submission guidelines of PLOS ONE and upload our figure files to the Preflight Analysis and Conversion Engine (PACE). All of the figures are adjusted to meet requirements of PLOS ONE.6. In table 3 when comparing the different models- Model 3 appears to have caused a loss in significance. The authors should address why the believe this to be the case and discuss it in their discussion.Answer to the reviewer: Thanks for your comments. Chronic low-grade inflammation plays an important role in the development of diabetic kidney disease. The tumor necrosis factor-α pathway, including sTNFR2, has emerged as an important mechanism in the pathogenesis of diabetic kidney disease. Our finding suggested that circulating sTNFR2 levels ≥1.608 ng/mL were a strong predictor of the progression of albuminuria and renal impairment in patients with type 2 diabetes. However, the significance of the association between serum sTNFR2 levels and the renal outcomes disappear after adjusting several confounding factors in model 3. The failure to detect significant association may be due to inadequate sample size and duration of follow-up. Further research is needed to confirm the association of sTNFR2 with renal outcomes in diabetic patients. We revise the manuscript as below:Line 275, page 20, section of “Discussion”OriginalThe findings in our cohort suggested that sTNFR2 is a promising biomarker in the early stages of diabetic nephropathy. The association of sTNFR2 concentration ≥1.608 ng/mL with renal outcomes remained significant in all subgroups, including patients with eGFRs ≥60 mL/min/1.73m2 or normoalbuminuria.RevisedThe findings in our cohort suggested that sTNFR2 is a promising biomarker in the early stages of diabetic nephropathy. The association of sTNFR2 concentration ≥1.608 ng/mL with renal outcomes remained significant in all subgroups, including patients with eGFRs ≥60 mL/min/1.73m2 or normoalbuminuria. The significance of the association between serum sTNFR2 levels and the renal outcomes disappear after adjusting several confounding factors in model 3 (Table 2). The failure to detect significant association may be due to inadequate sample size and duration of follow-up. Further research is needed to confirm the association of sTNFR2 with renal outcomes in diabetic patients. Reviewer 2 The manuscript under review investigates TNFR2 as a biomarker and a predictor of progressive renal disease in a cohort of diabetic patients. It investigates the relationship between serum TNFR2 levels and a number of parameters to conclude that serum TNFR2 levels above 1.608ng/ml are independently associated with a decline in eGFR and deteriorating albuminuria in T2D patients. The only comments I have are:1. The authors should include in their discussion other papers that have investigated this issue and how this work is different to theirs. For example there is no mention of PMID 22266663 and PMID 22266664, etc.Answer to the reviewer: Thanks for your suggestion. Niewczas et al. demonstrated that higher serum sTNFR2 concentrations are associated with increased risks of end stage renal disease in patients with type 2 diabetes. We have cited the article as reference 11 in the second paragraph of Discussion. Gohda et al. proved that circulating sTNFR2 predicts stage 3 chronic kidney disease in patients with type 1 diabetes. In our study, the renal outcomes are defined as a composite of a >30% reduction of the estimated glomerular filtration rate and/or the progression of albuminuria stages. Comparing to previous studies mainly focusing on advanced diabetic kidney disease, the findings in our study suggested that sTNFR2 is a promising predictor of the early stages of diabetic nephropathy. We revise the manuscript as below:Line 264, page 20, section of “Discussion”OriginalHigher serum sTNFR2 concentrations have been proven to be associated with increased risks of renal function decline or ESRD in patients with type 2 diabetes [10-13]. In one cohort of American Indians (Pima Indians) with type 2 diabetes, patients with macroalbuminuria whose serum sTNFR2 levels were in the highest quartile had a 88.7% cumulative incidence of ESRD, whereas those with macroalbuminuria and sTNFR2 levels in the lowest three quartiles had a 47.3% cumulative incidence of ESRD at 10 years of follow-up [12]. Higher sTNFR2 levels were associated with all-cause mortality as well [10]. The findings in our cohort suggested that sTNFR2 is a promising biomarker in the early stages of diabetic nephropathy. The association of sTNFR2 concentration ≥1.608 ng/mL with renal outcomes remained significant in all subgroups, including patients with eGFRs ≥60 mL/min/1.73m2 or normoalbuminuria.RevisedHigher serum sTNFR2 concentrations have been proven to be associated with increased risks of renal function decline or ESRD in patients with type 2 diabetes [10-13] and chronic kidney disease in patients with type 1 diabetes [20]. In one cohort of American Indians (Pima Indians) with type 2 diabetes, patients with macroalbuminuria whose serum sTNFR2 levels were in the highest quartile had a 88.7% cumulative incidence of ESRD, whereas those with macroalbuminuria and sTNFR2 levels in the lowest three quartiles had a 47.3% cumulative incidence of ESRD at 10 years of follow-up [12]. Higher sTNFR2 levels were associated with all-cause mortality as well [10]. Comparing to previous studies mainly focusing on advanced diabetic kidney disease, the findings in our cohort suggested that sTNFR2 is a promising biomarker in the early stages of diabetic nephropathy. The association of sTNFR2 concentration ≥1.608 ng/mL with renal outcomes remained significant in all subgroups, including patients with eGFRs ≥60 mL/min/1.73m2 or normoalbuminuria.2. The authors should elaborate how TNFR2 as a predictor of progressive kidney disease compares to other markers that are currently used. Is it an improvement? Is it an easier marker to measure?Answer to the reviewer: Thanks for your comments. The clinical methods for screening diabetic nephropathy includes annual measurement of urinary albumin-creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR). However, significant glomerular structural changes often existed when microalbuminuria becomes apparent. Patients with type 2 diabetes can also develop nonproteinuric diabetic nephropathy, and measuring UACR is an ineffective screening test for these patients. In our study, circulating sTNFR2 levels are associated with worsening albuminuria and progressive eGFR decline in patients with type 2 diabetes, including participants with eGFRs ≥60 mL/min/1.73m2 or normoalbuminuria. Our findings suggested that sTNFR2 is a biomarker to detect earlier stages of diabetic kidney disease comparing to measurement of UACR and eGFR. Measuring serum levels of sTNFR2 is a noninvasive and easily accessible method, making it a potential screening tool for diabetic nephropathy. We revise the manuscript as below:Line 247, page 19, section of “Discussion”OriginalIn our study, elevated serum sTNFR2 concentrations were associated with worsening albuminuria and progressive eGFR decline in patients with type 2 diabetes. Circulating sTNFR2 levels ≥1.608 ng/mL were a strong predictor of the progression of albuminuria and renal impairment in patients with type 2 diabetes, and the association was independent of relevant clinical covariates, including age, duration of diabetes, baseline UACR and eGFR, and the use of RAS inhibitors.RevisedIn our study, elevated serum sTNFR2 concentrations were associated with worsening albuminuria and progressive eGFR decline in patients with type 2 diabetes. Circulating sTNFR2 levels ≥1.608 ng/mL were a strong predictor of the progression of albuminuria and renal impairment in patients with type 2 diabetes, and the association was independent of relevant clinical covariates, including age, duration of diabetes, baseline UACR and eGFR, and the use of RAS inhibitors. Comparing to the current screening strategies of diabetic nephropathy as measurement of UACR and eGFR, sTNFR2 is a biomarker to detect earlier stages of diabetic kidney disease. As a noninvasive and easily accessible method, measuring serum levels of sTNFR2 is a potential screening tool for diabetic nephropathy.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 29 Mar 2022Soluble tumor necrosis factor receptor 2 are associated with progressive diabetic kidney disease in patients with type 2 diabetes mellitusPONE-D-21-32495R1Dear Dr. Lin,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Whist the authors have clarified that 284 of these patients used in the current study have been used for an earlier publication. I do not believe that this current manuscript can be considered as a novel publication.Reviewer #2: No further comments. All my issues have been addressed. The revised manuscript is considerably improved.**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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